dti_2197-supplementary material-6322-1-4-20210130 drug target insights 2021 | doi: 10.33393/dti.2021.2197 | khamsai et al appendix i search terms and results of searching for corticosteroid treatments in eosinophilic meningitis. 1. search strategy in pubmed on 21 november 2019 (21 articles) #1 search meningit*[title/abstract] #2 search eosinophil*[title/abstract] #3 search parasites #4 search ((parasitic diseases) or helminthiasis or (nematode infections)) #5 search (parasit* or helminth* or nematod*) #6 search angiostrongylus cantonensis #7 search ((angiostrongylus cantonensis) or (a. cantonenesis)) #8 search (angiostrongylus cantonensis) or (a. cantonensis) #9 search (#3 or #4 or #5 or #6 or #7 or #8) #10 search (#1 and #2) #11 search (#9 and #10) #12 search "randomized controlled trial" [publication type] #13 search (random* or placebo* or trial* or groups or (controlled study)) #14 search (#12 or #13) #15 search (#11 and #14) #16 search "humans"[mesh] #17 search (#15 and #16) 2. search strategy in central on 22 november 2019 (3 articles) #1 meningit*:ti,ab,kw #2 eosinophil*:ti,ab,kw #3 parasites #4 (parasitic diseases) or helminthiasis or (nematode infections) #5 parasit* or helminth* or nematod* #6 angiostrongylus cantonensis #7 (angiostrongylus cantonensis) or (a. cantonenesis) #8 (angiostrongylus cantonensis) or (a. cantonensis) #9 #3 or #4 or #5 or #6 or #7 or #8 #10 #1 and #2 #11 #9 and #10 #12 (randomized controlled trial) or random* or placebo* or trial* or groups or (controlled study) #13 #11 and #12 #14 humans #15 #13 and #14 drug target insights 2021 | doi: 10.33393/dti.2021.2197 | khamsai et al 3. search strategy in scopus on 22 november 2019 (254 articles) title-abs-key ( meningit* ) and title-abskey ( eosinophil* or parasitic* or helminth* or nematod* or "angiostrongylu s cantonensis" or "a. cantonenesis" ) and title-abs ( "randomized controlled trial " or random* or placebo* or trial* or groups or "controlled and study" ) and all ( human* ) and srctype ( j ) 4. search strategy in cinahl plus with full-text (ebsco) on 22 november 2019 (1 article) s15 s13 and s14 s14 human s13 s11 and s12 s11 s9 and s10 s10 s1 and s2 s9 tx (s2 or s3 or s4 or s5 or s6 or s7 or s8) s8 tx (angiostrongylus cantonensis) or (a. cantonensis) s7 tx (angiostrongylus cantonensis) or (a. cantonenesis) s6 tx angiostrongylus cantonensis s5 tx parasit* or helminth* or nematod* s4 tx (parasitic diseases) or helminthiasis or (nematode infections) s3 tx parasites s2 ti eosinophil* s1 ti meningit* 5. search strategy in web of science on 22 november 2019 (9 article) #1 title: (meningit*) #2 title: (eosinophil*) #3 topic: (parasit* or helminth* or nematod*) or topic: (angiostrongylus cantonensis) or topic: (a. cantonenesis) #4 #3 or #2 #5 #4 and #1 #6 all fields: (randomized controlled trial) or all fields: (random* or placebo* or trial* or groups) or all fields: (controlled study) #7 #6 and #5 #8 topic: (human*) #9 #8 and #7 dti drug target insights 2023; 17: 1-4issn 1177-3928 | doi: 10.33393/dti.2023.2548editorial drug target insights issn 1177-3928 www.aboutscience.eu/dti © 2023 the authors. this article is published by aboutscience and licensed under creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). commercial use is not permitted and is subject to publisher’s permissions. full information is available at www.aboutscience.eu natural products & phytotherapeutics natural products & phytotherapeutics: why a new section? marcello iriti department of biomedical, surgical and dental sciences, università degli studi di milano, milan italy received: december 12, 2022 accepted: december 15, 2022 published online: january 16, 2023 corresponding author: marcello iriti department of biomedical, surgical and dental sciences università degli studi di milano milan italy marcello.iriti@polimi.it according to one of the most authoritative reports focusing on natural products as sources of new drugs, the use of natural products and their synthetic derivatives is still pivotal in the discovery of new drugs (1). indeed, among the new drugs approved (n = 1881) in the last four decades, about 25% are natural products (fig. 1a). this scenario is particularly relevant for antibacterial and anticancer agents (fig. 1b, c). this should not be surprising. since ancient times, humanity has made use of medicinal plants to heal itself, and even today, traditional medicine represents the dominant health care system in many parts of the world and for billions of people (2). this is the case of herbal medicines, the cornerstone of phytotherapy, which include, according to the world health organization (who), ‘herbs, herbal materials, herbal preparations and finished herbal products that contain, as active ingredients, parts of plants, other plant materials or combinations thereof’ (3). several famous examples could be cited, from aspirin to many anticancer drugs (tab. i). however, natural product research still suffers from some important limitations. first, the validation of traditional uses. despite hundreds (or even thousands) of preclinical (in vitro/ in vivo) studies, evidence in humans is still scanty, due to the paucity of clinical trials evaluating the real efficacy of natural products. second, the poor oral bioavailability of natural products. phytochemicals are xenobiotics metabolized, detoxified and eliminated by phase i and ii metabolizing enzymes and phase iii transporters involved in efflux mechanisms. this drawback can be bypassed by proper (nano) formulation. third, natural does not always mean safe. the safety of natural products is rarely investigated and the available information is scanty, as are the phytochemical-drug interactions with possible changes in therapeutic efficacy for some drugs with a narrow therapeutic index (4). these issues call for an evidence-based approach to be followed even for phytotherapeutics, where randomized controlled trials are at the top of the evidence-based pyramid (5). fig. 1 a) all new approved drugs by source from 1981 to 2019 (n = 1881). b) all antibacterial drugs by source from 1981 to 2019 (n = 162). c) all anticancer drugs by source from 1981 to 2019 (n = 247). categories of sources: b = biological; n = natural product; nb = natural product – botanical; nd = natural product derivative; s = synthetic; s* = synthetic (with pharmacophore from a natural product); v = vaccine. subcategory: nm = natural product mimic. adapted from newman and cragg (1). a b c https://doi.org/10.33393/dti.2023.2548 https://creativecommons.org/licenses/by-nc/4.0/legalcode natural products & phytotherapeutics: why a new section?2 © 2023 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti table i selected examples of drugs developed from medicinal plants medicinal plant drugs indications chemical structure salix spp. acetylsalicylic acid anti-inflammatory, antiaggregant catharanthus roseus vinca alkaloids (vincristine, vinblastine, vinorelbine) anticancer camptotheca acuminata camptothecin derivatives (topotecan, irinotecan) anticancer taxus brevifolia taxane derivatives (paclitaxel, docetaxel, cabazitaxel) anticancer podophyllum peltatum podophyllotoxin derivatives (etoposide, teniposide) anticancer cannabis sativa cannabinoids (tetrahydrocannabinol, cannabidiol) psychotropic cinchona spp. quinine antimalarial artemisia annua artemisinin antimalarial iriti drug target insights 2023; 17: 3 © 2023 the authors. published by aboutscience www.aboutscience.eu medicinal plant drugs indications chemical structure papaver somniferum morphine, codeine analgesic digitalis spp. glicosidi digitalici (digoxin, digitoxin) cardiotonic atropa belladonna atropine anticholinergic hyoscyamus niger hyoscyamine anticholinergic datura stramonium scopolamine anticholinergic pilocarpus jaborandi pilocarpine cholinergic colchicum autumnale colchicine antigout galanthus spp. galantamine cholinesterase inhibitor syzygium aromaticum eugenol antiseptic, anesthetic rauwolfia serpentina reserpine antihypertensive natural products & phytotherapeutics: why a new section?4 © 2023 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti not least, the combination of natural products with conventional drugs offers another area of application that should be pursued extensively. this has previously been investigated with natural products used in combination with anticancer drugs and antimicrobials. this therapeutic approach was able to (chemo)sensitize chemoresistant cancer cells, fungi and bacterial strains by inhibiting the cellular active efflux system, a conserved drug resistance mechanism that pumps xenobiotics out of the cell. the rationale for the use of natural products is based on their multitarget action mechanism of particular interest in the treatment of disorders with multistage pathogenesis. in this complex scenario, natural products still offer the best options for finding new active agents/ templates and provide the unlimited potential for discovering new structures that can lead to effective drugs in a variety of communicable and non-communicable diseases. references 1. newman dj, cragg gm. natural products as sources of new drugs over the nearly four decades from 01/1981 to 09/2019. j nat prod. 2020;83(3):770-803. crossref pubmed 2. iriti m. journal of phytomoleculs & pharmacology: ‘why a new journal?’ j phytomol pharmacol. 2022;1(1):1-2. online 3. who global report on traditional and complementary medicine 2019. world health organization; 2019. online accessed december 2022. 4. peluso i. phytomolecules-drug interactions: clinical and nutritional implications. j phytomol pharmacol. 2022;1(2):56-57. crossref 5. varoni em, lodi g, iriti m. efficacy behind activity – phytotherapeutics are not different from pharmaceuticals. pharm biol. 2015;53(3):404-406. crossref pubmed https://doi.org/10.1021/acs.jnatprod.9b01285 https://www.ncbi.nlm.nih.gov/pubmed/32162523 https://leafletpub.com/article-detail/12 https://apps.who.int/iris/handle/10665/312342 https://doi.org/10.56717/jpp.2022.v01i02.008 https://doi.org/10.3109/13880209.2014.923000 https://www.ncbi.nlm.nih.gov/pubmed/25472494 dti drug target insights 2022; 16: 69-70issn 1177-3928 | doi: 10.33393/dti.2022.2545 editorial drug target insights issn 1177-3928 www.aboutscience.eu/dti © 2022 the authors. this article is published by aboutscience and licensed under creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). commercial use is not permitted and is subject to publisher’s permissions. full information is available at www.aboutscience.eu focus on antimicrobial resistance (amr) amr research: a perspective from personal experience vijay kothari institute of science, nirma university, ahmedabad india received: december 12, 2022 accepted: december 12, 2022 published online: december 23, 2022 corresponding author: dr. vijay kothari institute of science, nirma university s-g highway, ahmedabad-382481 india vijay.kothari@nirmauni.ac.in minimum bactericidal concentration (mbc) of test compounds, that is, screening molecules/natural extracts for bactericidal activity through broth dilution assay. in my personal experience, many of these committee members are not updated with the most recent trends in amr research, for example, use of alternative model organisms (caenorhabditis elegans and zebrafish) for the study of host-pathogen interactions, and for screening a library of natural/synthetic compounds for preliminary detection of in vivo anti-pathogenic activity. such model systems also provide an excellent opportunity for detecting anti-virulence activity in test compounds and extracts (1). recently while presenting a grant proposal involving use of c. elegans as a model host, and implementing wholetranscriptome analysis of bacterial pathogen treated with certain anti-pathogenic herbal formulation for novel target identification, i had to face these naughty comments from the grant-reviewing panel: a. “instead of working with c. elegans, do experiments directly with higher animals”: despite arguing that use of simpler organisms like c. elegans at an early stage can reduce animal sacrifice at later stages, and informing the committee of few hundred papers citing c. elegans as a valid and useful model for amr research, i failed to convince the committee (or the committee failed to understand the value of c. elegans in amr research). b. “since whole genome sequence of most of the pathogenic bacteria is available, we already have sufficient targets known"!!!: while dearth of validated novel antimicrobial targets is widely accepted as one of the major hurdles in discovering new antibiotics (2), one of the committee members educated me that fullgenome sequencing of pathogens has already solved that problem, and he claimed that we need to focus more on antimicrobial surveillance. i again failed to make the committee understand that surveillance at best tells us which resistant phenotypes are more prevalent in the given geographic area, but it cannot solve the problem of finding novel targets and antibiotics. the point is that oversimplification of the amr research reducing it to simple antibacterial growth inhibition assay can do many harms. if people with such exaggerated simplistic perception of amr research happen to head some academic institute, they may do even more harm by indirectly dissuading brilliant young minds to join amr labs. antimicrobial resistance (amr) has been well recognized as a global health issue. it is a ‘slow pandemic’ with huge socioeconomic impact. with around 15 years of experience of working with antibiotic-resistant pathogenic bacteria and anti-pathogenic natural products, i believe i have developed some insight into the issue, and i consider it worth sharing with the readers the variety of experiences i had while working in the amr field. the views expressed are not claimed to be free from personal beliefs and bias, and are likely to be more relevant to researchers in the lowand middle-income countries (lmic). some of the points discussed are not exclusively relevant to amr, non-amr researchers may also correlate their experience with them, and of course, many may disagree with my observations as this is a non-diplomatic personal account! 1. finding a critical mass of people working on similar aspects of amr can be a challenge! with amr getting quite a bit of attention in scientific circles as well as the media, this statement may sound strange, but this is a reality at least for certain geographic area. when you do not have sufficient number of amr labs in your city/state, it may be difficult to find people with whom you can exchange ideas, resistant strains, protocols, etc. even finding people with most relevant expertise to act as members of research progress committees/thesis evaluation committees of your phd students becomes difficult when you do not have many of them in your near vicinity. though online meetings with experts anywhere in the world are possible, this in my opinion is never as effective as offline faceto-face interactions. 2. oversimplified perception of amr research in certain circles of scientific community: while you are presenting before grant review committees, often the committee will comprise a mix of expertise, with few of them not directly involved in wet-lab amr work. they may perceive amr research too simplistically as if it is all about determining the minimum inhibitory concentration (mic)/ https://doi.org/dti.2022.2545 https://creativecommons.org/licenses/by-nc/4.0/legalcode mailto:vijay.kothari@nirmauni.ac.in amr research: a perspective from personal experience70 © 2022 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti 3. amr surveillance vs. antibiotic discovery: while many countries have floated their national action plans to combat amr, most applicant labs are inclined towards amr surveillance. while amr surveillance is an important area of investigation, it contributes largely towards characterization of the problem and helps in identifying the priority pathogens, but the solution is arrived at only from discovery and development of novel antimicrobial compounds and formulations. inherently amr surveillance projects are guaranteed to generate some visible output because irrespective of where you source the sample from (soil, water, or clinical samples), almost all samples can be shown to contain amr genes to a more or less extent through metagenomics. on the other hand, labs pursuing identification of novel targets and/or new antibiotics cannot be sure of a visible output as the probability of negative results is quite high. i personally feel that while amr surveillance should actively be pursued by public health organizations, academic labs and university-industry partnerships should be funded more for antibiotic discovery programmes. 4. exploring natural products for anti-pathogenic activity can be tricky: while traditional medicine (tm) can offer potent leads against various diseases including antibioticresistant infections, the wholistic philosophy of tm largely mismatches with the reductionist approach of modern drug discovery programmes (3). concepts like hormesis (non-linear dose-response patterns) and ‘multiplicity of targets’ have to be understood by the researcher dealing with polyherbal formulations or multicomponent plant extracts. unfortunately, not many people can claim familiarity with both modern science as well as tm. when you present your research to an audience largely comprising either tm practitioners or modern scientists trained in reductionist approach, it is difficult to be appreciated. most tm formulations do not exert outright bactericidal effect at low concentrations, instead they may exert antivirulence effect by simultaneously affecting multiple cellular and molecular targets in susceptible pathogens. to identify such polyphasic effect, simple growth inhibition assay can never be sufficient. such widespread effects can only be grasped through ‘omics’ approach. novel antimicrobial mechanisms can be identified through novel types of assays only. training of the next generation of microbiologists needs to go beyond conventional mic determination assays. 5. amr among non-bacterial pathogens needs more attention: while resistant infections caused by bacterial pathogens are responsible for considerable morbidity and mortality, infection burden owing to fungal, protozoan, viral, and helminth infection is also heavy. for a variety of reasons, most amr research has revolved around pathogenic bacteria, and amr in non-bacterial pathogens could not get sufficient attention. since meeting the criteria of ‘selective toxicity’ is even more difficult with potential new antimicrobials against eukaryotic and viral agent of diseases, building human resource skilled in investigating such non-bacterial pathogens is urgently required. graduate courses in microbiology should be reframed to put more emphasis on eukaryotic microorganisms in theory as well as lab component of syllabus (4). finally, i thank all the contributing authors whose articles appear in this special issue, and the reviewers who devoted their precious time in the peer-review process. i enjoyed support from the publisher, particularly lucia steele, for always being responsive. happy reading to all readers! disclosures conflict of interest: the author declares no conflict of interest. financial support: this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. references 1. kothari v. adopting an anti-virulence (anti-pathogenicity) approach for dealing with the problem of antimicrobial resistance. recent patents on biotechnology. 2019; 13:252255. crossref 2. theuretzbacher u, outterson k, engel a, karlén a. the global preclinical antibacterial pipeline. nat rev microbiol. 2020 may;18(5):275-285. crossref pubmed 3. kothari v. validation of traditional medicinal practices through modern scientific approach: a case for reconsideration. current pharmacogenomics and personalized medicine. 2020;17:11-13. crossref 4. kothari v. eukaryotic microorganisms in microbiology syllabi: better representation needed. journal of applied biotechnology & bioengineering. 2016; 1:42. crossref http://dx.doi.org/10.2174/187220831304191025092737 https://doi.org/10.1038/s41579-019-0288-0 https://pubmed.ncbi.nlm.nih.gov/31745331/ http://dx.doi.org/10.2174/1875692118666191223155350 https://doi.org/10.15406/jabb.2016.01.00007 dti © 2020 the authors. this article is published by aboutscience and licensed under creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). any commercial use is not permitted and is subject to publisher’s permissions. full information is available at www.aboutscience.eu issn 1177-3928 drug target insights 2020; 14: 12-15 short communication doi: 10.33393/dti.2020.2188 paradoxical bronchoconstriction caused by β 2 -adrenoceptor agonists khadija ayed, islam latifa hadj khalifa, salma mokaddem, saloua ben khamsa jameleddine department of physiology, faculty of medicine of tunis, university of tunis el manar, tunis tunisia abstract introduction: salbutamol and terbutaline are short-acting β 2 adrenergic agonists that produce bronchial smooth muscle relaxation and are widely used in obstructive pulmonary diseases. nevertheless, their use has been the cause of a paradoxical bronchoconstriction, which is a rare and potentially serious adverse reaction. the aim of this study is to report a case of paradoxical bronchoconstriction caused by β 2 adrenergic agonists. methods: this case is about a 50-year-old asthmatic patient who describes a history of repeated acute asthma attacks after salbutamol inhalation or terbutaline nebulization. a double-blind crossover study was performed over 3 days, in order to compare the effects of each bronchodilator. forced expiratory volume in 1 second (fev 1 ), forced vital capacity (fvc), and maximal expiratory flow 25-75 (mef25-75) were measured. results: on the first day, a bronchoconstriction caused by deep and repeated inhalations was eliminated. on the second day, an airway obstruction was confirmed by a decrease in fev 1 at 40% from baseline values after nebulization of a standard dose of terbutaline. on the third day, a spirometry was performed before and after nebulization of a standard dose of ipratropium bromide, and there were no significant changes in the spirometric parameters. finally the patient was discharged with a written warning mentioning the danger of salbutamol and terbutaline use. conclusion: salbutamol and terbutaline are generally well-tolerated β 2 adrenergic agonists. nevertheless, in rare cases, these substances can cause a paradoxical bronchoconstriction. doctors must therefore remain vigilant about its side effect and possibly investigate each case. keywords: asthma, bronchoconstriction, bronchodilators, β 2 adrenergic agonists, spirometry received: september 21, 2020 accepted: september 28, 2020 published online: october 5, 2020 corresponding author: khadija ayed 12 avenue farhat hachèd soliman 8020 nabeul tunisia ayed_khadija@yahoo.fr light chain kinase hydrolysis. in addition, it promotes the exchange of calcium/sodium, which results in a decrease in intracellular calcium concentration, and stimulates the na+/k+ adenosine triphosphatase (atpase) pump. this discovery justifies the use of these substances as a pillar of the treatment of bronchial obstruction in addition to their safety and efficiency (1-5). nevertheless, use of salbutamol and terbutaline has been the cause of paradoxical bronchoconstriction, which is a rare and potentially serious adverse event requiring vigilance by treating physicians (6,7). we describe a case of paradoxical bronchoconstriction in an asthmatic patient caused by β 2 -adrenoceptor agonists objectively demonstrated with an appreciable decrease of forced expiratory volume in one second (fev 1 ) in a double blind crossover study. methods and results this case is about a 50-year-old asthmatic patient with no other past medical history and whose body mass index was 20.7 kg/m2. he described a history of repetitive acute attacks of asthma after inhalation of salbutamol. asthma was background salbutamol and terbutaline are both short-acting β 2 adrenoceptor agonists that are believed to exert their maximal therapeutic effect through bronchodilation. activated β 2 -adrenergic receptors promote their binding to a stimulating g protein called gs. this binding stimulates adenylyl cyclase, which in turn activates the intracellular cyclic adenosine monophosphate (camp) that phosphorylates several relaxation proteins. in bronchial smooth muscle, activated protein kinase inhibits both myosin and phosphoinositide ayed et al 13 © 2020 the authors. published by aboutscience diagnosed 5 years earlier, based on clinical arguments without neither atopy nor spirometric confirmation. the patient was then treated with inhaled corticosteroids and salbutamol for 3 years. during this period, the patient continued to have respiratory symptoms such as wheezing and dry cough after taking medication. in april 2016, the patient presented dramatic symptoms after taking a standard dose of terbutaline via nebulizer and developed a respiratory failure requiring intubation and hospitalization in an intensive care unit. as part of the follow-up of his disease, the patient was sent to our pulmonary function department in august 2017. spirometry and bronchodilator reversibility test were carried out by a “carefusion microlab” spirometer, and spirometry was performed by experts in respiratory functional exploration according to the latest american thoracic society (ats)/ european respiratory society (ers) recommendations. predicted values were obtained according to reference equations from the work of stocks and quanjer (8). baseline spirometry revealed a small airways obstruction, which is defined by a normal forced vital capacity (fvc), a normal fev 1 /fvc ratio, and a decrease of at least one forced expiratory flow (fef): fef50%, fef25%, or fef between 25% and 75% of the fvc (fef25-75). during bronchodilator reversibility test, the patient developed an explosive cough and dyspnea just after salbutamol inhalation (400 µg) by metered dose inhaler (mdi). a decrease in fev 1 at 25% from baseline was noticed (tab. i). an administration of ipratropium bromide via nebulizer restored basic spirometric values. in the light of this ascertainment, we studied the influence of terbutaline and ipratropium bromide on bronchial reactivity. a double-blind crossover study was performed, over 3 days; neither the patient nor the technician knew the composition of the nebulization in order to compare the effects of each bronchodilator on bronchial reactivity. an informal consent was obtained and the patient was asked to avoid using bronchodilators for at least 12 hours prior to the study. fev 1 , fvc, and fef25-75 were measured and the patient was monitored by blood oxygen saturation. on the first day, a basic spirometry was performed with eight measurements in order to eliminate bronchoconstriction due to deep inhalation during spirometry testing. no significant changes compared to baseline spirometry were found (tab. ii). on the second day, a spirometry was planned before and 15 minutes after a nebulization of a standard dose of terbutaline made up to a total volume of 5 ml with 0.9% saline solution (5 mg/unit). the patient was initially asymptomatic, but nebulization was interrupted because of important dyspnea, coughing, wheezing, and discrete cyanosis. physical examination revealed a wheezing, sinusal tachycardia at 110 per minute, and blood oxygen saturation at 85%. there was no urticaria, pruritus, or macular rash. the spirometry was performed earlier because of the reaction and revealed an airway obstruction confirmed by a decrease in fev 1 at 40% from baseline spirometry (tab. iii). on the third day, a spirometry was performed before and 30 minutes after a nebulization of a standard dose of ipratropium bromide made up to a total volume of 5 ml with 0.9% saline solution (0.5 mg/unit). there were no significant clinical or spirometric changes (tab. iv). finally, the patient was discharged with a written warning mentioning the danger of salbutamol and terbutaline use via mdi or nebulizer solutions. discussion currently, inhaled β 2 -adrenoceptor agonists are generally used in the treatment of acute asthma attacks, based on their effectiveness and relative safety. however, in rare cases, they may cause unexpected adverse effects such as paradoxical bronchoconstriction. paradoxical bronchoconstriction was recorded in this patient each time he used β 2 -adrenoceptor agonists, whether with salbutamol via mdi or terbutaline via nebulizer. many table i spirometric parameters before and after salbutamol inhalation spirometric parameters baseline spirometry after salbutamol measured % predicted measured % predicted % changing fev 1 2.36 l 74 1.76 l 55 −25 fvc 3.57 l 91 3.36 l 86 −6 fev 1 /fvc 66% – 52% – −21 fef25-75 1.34 l 36 0.68 l 18 −49 fef25-75 = fef between 25% and 75% of the fvc; fev 1 = forced expiratory volume in 1 second; fvc = forced vital capacity. table ii spirometric parameters during repeated spirometry testing fev 1 fvc fev 1 /fvc (%) baseline or 1st spirometry 2.41 l (75%) 3.27 l (83%) 73 2nd spirometry 2.36 l (74%) 3.11 l (79%) 75 3rd spirometry 2.29 l (71%) 3.15 l (80%) 73 4th spirometry 2.35 l (73%) 3.19 l (81%) 73 5th spirometry 2.30 l (71%) 3.11 l (79%) 73 6th spirometry 2.42 l (75%) 3.22 l (82%) 75 7th spirometry 2.31 l (71%) 3.20 l (81%) 72 8th spirometry 2.35 l (73%) 3.22 l (82%) 72 fev 1 = forced expiratory volume in 1 second; fvc = forced vital capacity. paradoxical bronchoconstriction caused by bronchodilators14 © 2020 the authors. published by aboutscience factors have been excluded before reaching the conclusion of paradoxical bronchoconstriction. first, in some asthma patients, deep inhalation can produce paradoxical bronchoconstriction during spirometry testing (9). this is why we performed the test on the first day with more than three measurements, but no spirometric changes were found. second, double-blind crossover study was performed to avoid psychological response to spirometry testing. in fact, psychological stress can exacerbate clinical symptoms in patients with asthma (10). third, several studies suggested that excipients (as chlorofluorocarbon propellants and surfactants) or preservatives (as benzalkonium chloride) in mdis have been incriminated in paradoxical bronchoconstriction due to a bronchial irritation (11,12). in our case, this possibility was rejected because the same response was seen after nebulization of terbutaline solution, which did not contain foreign substances as preservatives or stabilizers. in fact, excipients of our terbutaline solution were sodium chloride, sodium edetate, and hydrochloric acid. in addition, osmolarity of liquids of nebulization may induce bronchoconstriction. hyperosmolar buffers have been shown to cause histamine release from normal human basophils and mast lung cells (13,14). saline solutions that are hypotonic or hypertonic are both able to induce bronchoconstriction (15). for these reasons, we used isotonic saline in all nebulizer solutions. this bronchoconstriction was most likely related to an adverse reaction to β 2 -adrenoceptor agonists. it is, however, difficult to specify their exact action mechanism on bronchi during this paradoxical reaction. a number of questions remain to be answered with a biocellular study. all possibilities should be taken into account. it is dangerous to reject a possible cause of asthma mortality. doctors and patients should be aware that although β 2 -adrenoceptor agonists do not generally produce adverse effects, it may aggravate symptoms. this adverse reaction needs to be considered as one of the possible causes of sudden death or acute asthma attack. finally, some precautions should be taken with the use of β 2 -adrenoceptor agonists. since short-acting β 2 -adrenoceptor agonists represent the first-line treatment of acute asthma (2,3), any discrepancy between a good adherence to treatment and refractory symptoms should lead the physician to suspect paradoxical bronchoconstriction. in this case, the patient should be treated with other bronchodilators such as anticholinergics. similar reactions should lead to a new assessment including a full spirometric study with bronchial reversibility tests as well as the above-cited protocol, in order to define the causative agent. conclusion β 2 -adrenoceptor agonists are an important and well tolerated pharmacological class in the management of asthma. in rare cases, salbutamol and terbutaline may cause unusual paradoxical bronchoconstriction, whose mechanism remains unknown. therefore, physicians must be vigilant about this potentially serious side effect and investigate each case. disclosures conflict of interest: the authors declare that there is no conflict of interest. financial support: this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. table iii spirometric parameters before and after terbutaline nebulization spirometric parameters baseline spirometry after terbutaline measured % predicted measured % predicted % changing fev 1 2.36 l 74 1.41 l 44 −40 fvc 3.11 l 79 2.82 l 72 −9 fev 1 /fvc 75% – 40% – −35 fef25-75 2.22 l/s 51 0.79 l/s 18 −64 fef25-75 = fef between 25% and 75% of the fvc; fev 1 = forced expiratory volume in 1 second; fvc = forced vital capacity. table iv spirometric parameters before and after nebulization of ipratropium bromide spirometric parameters baseline spirometry after ipratropium bromide measured % predicted measured % predicted % changing fev 1 2.54 l 79 2.59 l 80 +2 fvc 3.78 l 76 3.85 l 79 +2 fev 1 /fvc 64% – 67% – +3 fef25-75 1.89 l 43 1.97 l/s 45 +4 fef25-75 = fef between 25% and 75% of the fvc; fev 1 = forced expiratory volume in 1 second; fvc = forced vital capacity. ayed et al 15 © 2020 the authors. published by aboutscience references 1. spooner lm, olin jl. paradoxical bronchoconstriction with albuterol administered by metered-dose inhaler and nebulizer solution. ann pharmacother. nov 2005;39(11):1924-1927. 2. johnson m. beta2-adrenoceptors: mechanisms of action of beta2-agonists. paediatr respir rev. mar 2001;2(1):57-62. 3. jacobson ga, hostrup m. terbutaline: level the playing field for inhaled β 2 -agonists by introducing a dosing and urine threshold. br j sports med. 26 jul 2016;bjsports-2016-096453. 4. matthew n, octavio a, ricardo mf, ian s. salbutamol or aminophylline for acute severe asthma: how to choose which one, when and why? arch dis child educ pract. dec 2014;2015(100):215-222. 5. barisione g, baroffio m, crimi e, brusasco v. beta-adrenergic agonists. pharmaceuticals. 30 mar 2010;3(4):1016-1044. 6. williams c, crossland l, finnerty j, et al. case-control study of salmeterol and near-fatal attacks of asthma. thorax. 1998;53(1): 7-13. 7. gallelli l. retrospective analysis of adverse drug reactions to bronchodilators observed in two pulmonary divisions of catanzaro, italy. pharmacol res. june 2003;47(6):493-499. 8. stocks j, quanjer ph. reference values for residual volume, functional residual capacity and total lung capacity. ats workshop on lung volume measurements. official statement of the european respiratory society. eur respir j. 1995;8: 492-506. 9. haynes jm. bronchoconstriction in response to deep inhalation during spirometry testing. respir care. 1 may 2015;60(5): e105-e109. 10. chen e, miller ge. stress and inflammation in exacerbations of asthma. brain behav immun. 2007;21(8):993-999. 11. beasley cr, rafferty p, holgate st. bronchoconstrictor properties of preservatives in ipratropium bromide (atrovent) nebuliser solution. br med j clin res ed. 1987;294(6581):1197. 12. george m, joshi sv, concepcion e, lee h. paradoxical bronchospasm from benzalkonium chloride (bac) preservative in albuterol nebulizer solution in a patient with acute severe asthma. a case report and literature review of airway effects of bac. respir med case rep. 2017;21:39-41. 13. findlay sr, dvorak am, kagey-sobotka a, lichtenstein lm. hyperosmolar triggering of histamine release from human basophils. j clin invest. 1981;67(6):1604. 14. stellato c, de crescenzo g, patella v, mastronardi p, mazzarella b, marone g. human basophil/mast cell releasability. xi. heterogeneity of the effects of contrast media on mediator release. j allergy clin immunol. 1996;97(3):838-850. 15. schoeffel re, anderson sd, altounyan re. bronchial hyperreactivity in response to inhalation of ultrasonically nebulised solutions of distilled water and saline. br med j clin res ed. 1981;283(6302):1285-1287. 9drug target insights 2016:10 a case of organizing pneumonia (op) associated with pembrolizumab paraskevi fragkou1, maria souli1, maria theochari2, christina kontopoulou3, stelios loukides4 and anna koumarianou2 1fourth department of internal medicine, infectious diseases unit, attikon university hospital, national and kapodistrian university of athens, athens, greece. 2fourth department of internal medicine, hematology-oncology unit, attikon university hospital, national and kapodistrian university of athens, athens, greece. 32nd radiology department, attikon university hospital, national and kapodistrian university of athens, athens, greece. 42nd respiratory medicine department, attikon university hospital, national and kapodistrian university of athens, athens, greece. a bstr act: until recently, chemotherapy for metastatic melanoma had disappointing results. the identification of immune checkpoints such as ctla-4 and pd-1/pd-l1 has led to the development of an array of monoclonal antibodies (mabs). these immunologic approaches against tumoral cells come with a novel kind of side effects that the clinician needs to be familiarized with. herein, we report for the first time a case of organizing pneumonia, based on imaging and cytological analyses of bronchoalveolar lavage, possibly associated with the use of pembrolizumab, an anti-pd-1 mab recently approved for the treatment of metastatic melanoma. k e y wor ds: op, organizing pneumonia, pembrolizumab, melanoma, side effect citation: fragkou et al. a case of organizing pneumonia (op) associated with pembrolizumab. drug target insights 2016:10 9–12 doi:10.4137/dti.s31565. type: case report received: february 5, 2016. resubmitted: april 14, 2016. accepted for publication: april 18, 2016. academic editor: anuj chauhan, editor in chief peer review: three peer reviewers contributed to the peer review report. reviewers’ reports totaled 454 words, excluding any confidential comments to the academic editor. funding: authors disclose no external funding sources. competing interests: authors disclose no potential conflicts of interest. copyright: © the authors, publisher and licensee libertas academica limited. this is an open-access article distributed under the terms of the creative commons cc-by-nc 3.0 license. correspondence: akoumari@yahoo.com paper subject to independent expert single-blind peer review. all editorial decisions made by independent academic editor. upon submission manuscript was subject to anti-plagiarism scanning. prior to publication all authors have given signed confirmation of agreement to article publication and compliance with all applicable ethical and legal requirements, including the accuracy of author and contributor information, disclosure of competing interests and funding sources, compliance with ethical requirements relating to human and animal study participants, and compliance with any copyright requirements of third parties. this journal is a member of the committee on publication ethics (cope). provenance: the authors were invited to submit this paper. published by libertas academica. learn more about this journal. introduction new monoclonal antibodies (mabs) targeting the immune checkpoint blockade have revolutionized the treatment of advanced melanoma patients. although these novel mab therapies can induce remarkable antitumor-specific immune responses, they have distinct side effects as compared with chemotherapy, including nonspecific immunological activation.1 to our knowledge, we report the first case of organizing pneumonia (op) in a melanoma patient under immunotherapy with pembrolizumab, a pd-1 targeting mab. the patient’s next of kin has given consent for publication of this report. case presentation a 64-year-old woman presented to our emergency department with a 10-day history of progressive dyspnea and dry cough nonresolving after a 9-day course of oral antibiotics (levofloxacin 750  mg daily). she had been diagnosed with a c-kit/braf wild-type, stage iv mucosal melanoma of the nasal cavity with involvement of cervical and mediastinal lymph nodes and metastases to the liver, brain, and bones nine months earlier. initial treatment included two cycles of dacarbazine 1000  mg/m2 and zoledronic acid 4  mg every three weeks. one month later, the patient experienced disease progression and was treated with four cycles of ipilimumab immunotherapy 3 mg/kg and zoledronic acid 4 mg q3 weeks intravenously (iv). three months later, the disease advanced further, and a second-line immunotherapeutic agent with pembrolizumab was initiated. thirteen days prior to her admission, she had received her fourth cycle of immunotherapy with pembrolizumab. her pretherapeutic thoracic ct scan was normal. on examination, she was pale and tachypneic. the chest auscultation revealed diffuse bilateral coarse crackles and areas of bronchial breathing mainly in the right middle lobe. she was afebrile, but her oxygen saturation on room air was 75%. her blood tests were normal except an erythrocyte sedimentation rate of 200  mm/hour, a white blood count of 10,970  cells/μl (differential: 83.6% neutrophils, 9.7% lymphocytes, and 6.7% monocytes), and a c-reactive protein of 173  mg/l (normal value: 0–6  mg/l). a chest radiography demonstrated patchy alveolar infiltrates mainly on the right upper, the right middle, and the left upper lobe. an initial diagnosis of a lower respiratory tract infection was made, and antibiotics were commenced, including ceftriaxone 2  g/day iv and azithromycin 500  mg/day orally. after three days of treatment, there was no improvement of signs or symptoms. subsequently, the patient underwent a high-resolution computed tomography (hrct) of the chest and fiberoptic bronchoscopy with bronchoalveolar lavage (bal). samples were submitted for staining and culture for journal name: drug target insights journal type: case report year: 2016 volume: 10 running head verso: fragkou et al running head recto: op in pembrolizumab http://www.la-press.com/drug-target-insights-journal-j23 http://www.la-press.com http://dx.doi.org/10.4137/dti.s31565 http://creativecommons.org/licenses/by-nc/3.0/ http://creativecommons.org/licenses/by-nc/3.0/ mailto:akoumari@yahoo.com http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 fragkou et al 10 drug target insights 2016:10 bacteria, fungi, mycobacteria, and nocardia, special staining for pneumocystis jirovecii, cytology, immunophenotyping, and molecular testing for viral infection. the hrct demonstrated patchy consolidation with a predominantly subpleural and peribronchial distribution, small ill-defined peribronchiolar nodules, tree-in-bud sign, and reverse halo sign, involving all lobes mainly the lower ones (fig. 1). there was no lymph nodal enlargement or pleural effusion. cytology, microbiology, and molecular testing were negative for malignant or infectious diseases. the immunophenotyping of the bal lymphocytes by flow cytometry revealed macrophage and monocyte predominance (60.7%) with a lymphocyte count of 28.7% and a cd4+/cd8+ index of 0.4. based on the imaging and clinical and bal findings that were consistent with op, therapy with corticosteroids (prednisolone 50 mg/day iv) was initiated that resulted into rapid clinical and radiographic improvement of the patient. her c-reactive protein declined to 20  mg/l within two days of steroid treatment. the treating physician assessed that op represented a grade 3 adverse event,2 most probably related to treatment with pembrolizumab, which was discontinued. three months later, the patient died of advanced metastatic disease in the brain. discussion advanced melanoma has been a challenge in oncology as the five-year survival rates were immensely low with standard chemotherapy.3 newer mab therapies targeting the immune checkpoint, ipilimumab (yervoy; bristol-myers squibb), nivolumab (opdivo; bristol-myers squibb), and pembrolizumab (keytruda; merck sharp & dohme corp) were shown to prolong progression free and overall survival compared with chemotherapy.4 pembrolizumab is a humanized igg4 mab raised against the programmed cell death protein 1 (pd-1) immune checkpoint, recently approved by the us food and drug administration and the european medicines agency for the treatment of advanced melanoma, as a result of a randomized controlled phase iii trial showing improved overall survival.5 pd-1 is a cell surface receptor that belongs to the immunoglobulin superfamily and is normally expressed on b-cells,6 t-cells,7 and macrophages.8 pd-1 and its ligands, namely, programmed cell death ligand 1 (pd-l1) and 2 (pd-l2), have been found to be abnormally expressed by tumor cells.9 these molecules play an important role in inhibiting antitumor immune responses and are responsible for the immune evasion of cancer cells by the cytotoxic effector immune cells. mab therapies against the pd-1 can reverse immune tolerance and achieve high tumor response rates and improvement of survival.10 the therapeutic effects of mabs are not fully understood, but they are mediated through direct signaling, complementdependent cellular cytotoxicity, and antibody-dependent cellular cytotoxicity.11 pemprolizumab (previously known as mk-3475 or lambrolizumab) is a humanized monoclonal igg4–kappa isotype antibody. its variable region sequences were derived from a very high-affinity mouse antihuman pd-1 antibody (dissociation constant, 28  pm) that was grafted into a human igg4 immunoglobulin with a stabilizing s228p fc alteration that does not engage fc receptors or activate complement, thus avoiding cytotoxic effects of the antibody when it binds to the pd-1 receptor of t-cells that are intended to activate against tumor cells.12 it is reasonable to assume that the mechanism of action and the resulting side effects of pembrolizumab are mediated through pd-1 direct signaling on t-cells or macrophages. cryptogenic organizing pneumonia (cop), the idiopathic form of op (formerly called bronchiolitis obliterans op), is a type of diffuse interstitial lung disease that affects the distal bronchioles, respiratory bronchioles, alveolar ducts, and alveolar walls. the primary area of injury is within the alveolar wall. since many cases of cop are secondary, it is more proper to use the term op associated with the name of the secondary cause.13 histopathologic study through lung biopsy via videoassisted thoracoscopic surgery or open thoracotomy is the optimal method for establishing the diagnosis of cop, in order to have enough tissue for the pathologist to exclude other processes. because of the low oxygen saturation level, we selected to combine the cytological profile of bal with figure 1. patient’s computed tomography of chest indicating (a) baseline ct scan before pembrolizumab therapy, (b) reverse halo sign (dotted arrow) and consolidation with air bronchogram (straight arrow) features of op following therapy of pembrolizumab, and (c) follow-up ct scan indicating complete resolution of the organized pneumonia, one month after the steroid therapy. metastatic lesion in the right middle lobe. http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 op in pembrolizumab 11drug target insights 2016:10 characteristic clinical and imaging features. a previous study has already described in detail the cytological and immunocytological profile of bal in cop, which include foamy macrophages, mast cells, plasma cells, a decreased cd4/cd8 t-cell ratio, an increase in activated t-lymphocytes based on hla-dr or interleukin-2 receptor expression and a lymphocyte increase of 20%–40% also documented in our patient with bronchiolitis obliterans. the most commonly occurring clinical findings in cop are similar to our patient’s symptoms, including nonproductive persistent cough, dyspnea, and malaise without fever.14 since the most common manifestations are nonspecific, diagnosis is often delayed (6–13 weeks), and similar to our patient, a lack of response to empiric antibiotics for community-acquired pneumonia may be the initial clue to the presence of a noninfectious, inflammatory pneumonia.15 similar to that in our patient, hrct has findings that are characteristic for cop and include peripheral bilateral patchy opacities, more frequently in the periphery and in the lower lung zone, air bronchogram, and reverse halo sign.16 once the diagnosis of cop is made, rapid clinical and imaging improvement is obtained with corticosteroid treatment, but relapses are common after stopping treatment, indicating an established immune reaction requiring long-term therapy.15 when cop is suspected in cancer patients, differential diagnoses that need to be considered include disease progression, cardiogenic edema, radiation pneumonitis, allergies, pulmonary hemorrhage, and infections that cause diffuse interstitial infiltrates in the lungs such as gram-negative or grampositive bacteria, fungi (aspergillus, candida, and p. jirovecii), parasites (toxoplasma gondii), or viruses (herpes simplex, varicella zoster, and cytomegalovirus).17 on the other hand, pneumonitis in cancer patients may hold a heterogeneous pathological background based on various antineoplastic agents including chemotherapy, such as taxanes, targeted therapy, such as everolimus, and mabs, such as rituximab, a chimeric igg1.17 in retrospective analysis of patients with non-hodgkin lymphoma treated with rituximab, 8 of 23 patients, diagnosed with cop and treated with corticosteroids, died, indicating that cop although rare may become a fatal pulmonary toxicity and the oncologists or internists must maintain a high index of suspicion to recognize this complication early. little is known regarding the clinicopathologic features of pneumonitis related to the novel immune checkpoint inhibitors. a recent study indicated the development of pneumonitis in three patients treated with nivolumab, an anti-pd-1 mab, 7–24.3 months after treatment initiation.18 two patients were radiographically found with acute interstitial pneumonia/acute respiratory distress syndrome, and one patient was with nonspecific interstitial pneumonia. two of these patients required admission in the intensive care unit, and one succumbed four weeks after diagnosis of pneumonitis. computed tomography findings differed from cop, and bal or transbronchial biopsy was not performed in these patients. another report involving a patient treated with nivolumab described an op diagnosed both radiographically and pathologically.19 similar to our case, ct of the chest showed the typical reversed halo sign and had a favorable response to corticosteroid therapy. with novel immunotherapies, the incidence of autoimmune phenomena is rising; therefore, it is important to recognize an op early in any patient receiving anti-pd-1 and presenting with symptoms of new cough or shortness of breath. the diagnosis needs to be supported by imaging and cytological investigations. although the exact causative mechanism of op by anti-pd-1 mab therapy cannot be established as yet, a t-cell or macrophage-driven effect is the most plausible explanation, and further studies are warranted to define the pathogenic mechanism underlying this possibly lethal side effect. acknowledgment the authors are obliged to professor dimitrios boumpas for critical revision of the manuscript. author contributions conceived and designed the experiments: ak. analyzed the data: ms. wrote the first draft of the manuscript: pf. contributed to the writing of the manuscript: pf, ms, mt, ck, sl, ak. agree with manuscript results and conclusions: pf, ms, mt, ck, sl, ak. jointly developed the structure and arguments for the paper: pf, ms, mt, ck, sl, ak. made critical revisions and approved final version: pf, ms, mt, ck, sl, ak. all authors reviewed and approved of the final manuscript. r efer ences 1. postow ma, callahan mk, wolchok jd. immune checkpoint blockade in cancer therapy. j clin oncol. 2015;33(17):1974–1982. 2. national cancer institute, national institutes of health, u.s. department of health and human services. common terminology criteria for adverse events (ctcae). version 4.0. bethesda, md: nih. published: may 28, 2009 (v4.03: june 14, 2010). nih publication no. 09-5410. revised june 2010. 3. mcdermott d, lebbe c, hodi fs, et al. durable benefit and the potential for long-term survival with immunotherapy in advanced melanoma. cancer treat rev. 2014;40:1056–1064. 4. eggermont am, maio m, robert c. immune checkpoint inhibitors in melanoma provide the cornerstones for curative therapies. semin oncol. 2015;42:429–435. 5. robert c, schachter j, long gv, et al. pembrolizumab versus ipilimumab in advanced melanoma. n engl j med. 2015;372:2521–2532. 6. thibult ml, mamessier e, gertner-dardenne j, et al. pd-1 is a novel regulator of human b-cell activation. int immunol. 2013;25:129–137. 7. jin ht, ahmed r, okazaki t. role of pd-1 in regulating t-cell immunity. curr top microbiol immunol. 2011;350:17–37. 8. huang x, venet f, wang yl, et al. pd-1 expression by macrophages plays a pathologic role in altering microbial clearance and the innate inflammatory response to sepsis. proc natl acad sci u s a. 2009;106:6303–6308. 9. wu p, wu d, li l, chai y, huang j. pd-l1 and survival in solid tumors: a meta-analysis. plos one. 2015;10:e0131403. 10. pardoll dm. the blockade of immune checkpoints in cancer immunotherapy. nat rev cancer. 2012;12:252–264. 11. weiner gj. rituximab: mechanism of action. semin hematol. 2010;47:115–123. http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 fragkou et al 12 drug target insights 2016:10 12. hamid o, robert c, daud a, et al. safety and tumor responses with lambrolizumab (anti-pd-1) in melanoma. n engl j med. 2013;369:134–144. 13. travis wd, costabel u, hansell dm, et al. an official american thoracic society/european respiratory society statement: update of the international multidisciplinary classification of the idiopathic interstitial pneumonias. am j respir crit care med. 2013;188:733–748. 14. barker af, bergeron a, rom wn, et al. obliterative bronchiolitis. n engl j med. 2014;370:1820–1828. 15. cordier jf. cryptogenic organising pneumonia. eur respir j. 2006;28:422–446. 16. nishimura k, itoh h. high-resolution computed tomographic features of bronchiolitis obliterans organizing pneumonia. chest. 1992;102:26s–31s. 17. kim km, kim hc, jeon kn, et al. rituximab-chop induced interstitial pneumonitis in patients with disseminated extranodal marginal zone b cell lymphoma. yonsei med j. 2008;49:155–158. 18. nishino m, sholl lm, hodi fs, et al. anti-pd-1-related pneumonitis during cancer immunotherapy. n engl j med. 2015;373:288–290. 19. nakashima k, naito t, omori s, et al. organizing pneumonia induced by nivolumab in a patient with metastatic melanoma. j thorac oncol. 2016;11(3): 432–433. http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 dti drug target insights 2022; 16: 1-5issn 1177-3928 | doi: 10.33393/dti.2022.2355case report drug target insights issn 1177-3928 www.aboutscience.eu/dti © 2022 the authors. this article is published by aboutscience and licensed under creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). commercial use is not permitted and is subject to publisher’s permissions. full information is available at www.aboutscience.eu erythrodermic psoriasis and palmoplantar hyperkeratosis successfully treated with secukinumab: a case report martino carriero dermatology unit, poliambulatorio grottaglie, grottaglie italy abstract introduction: erythrodermic psoriasis (ep) is a rare and severe form of psoriasis that affects 1% to 2.25% of patients, increasing mortality risk. to date, very few therapies have been approved for the treatment of this condition. recently, biological therapies that specifically target inflammatory cytokines have improved the management and treatment of ep. secukinumab, a human monoclonal antibody that specifically targets interleukin-17a (il-17a), has been shown to be beneficial in different psoriasis settings. methods: we report the case of a 72-year-old man affected by persistent ep and severe palmoplantar hyperkeratosis whose condition was not resolved after two rounds of treatment with prednisone and therapy with cyclosporine. results and conclusions: treatment with secukinumab significantly improved the symptoms of palmoplantar hyperkeratosis as early as the first week, with a decrease of psoriasis area and severity index (pasi) score from 60 to 10, showing almost complete remission after 1 month. consistent with the current literature, secukinumab treatment showed promising and encouraging clinical outcomes in the treatment of the patient’s ep. however, more studies are needed to clarify the il-17-dependent mechanism in the pathophysiology of ep. keywords: biologic therapy, erythrodermic psoriasis, interleukin-17, monoclonal antibody, palmoplantar hyperkeratosis, secukinumab received: november 4, 2021 accepted: february 23, 2022 published online: march 7, 2022 corresponding author: martino carriero dermatology unit poliambulatorio grottaglie via tiziano 26 grottaglie (ta), 74023 italy dott.martino.carriero@alice.it joints, nails, and other organs (2). currently, there is no cure available, and psoriasis imposes a substantial negative impact on the quality of life (qol) of patients. psoriasis can occur at any age and is most common in the age group 50-69 years. the reported country-specific prevalence of psoriasis ranges between 0.09% and 11.4%, making psoriasis a serious global problem (1), with approximately 3% of the us population and around 125 million people affected worldwide, ranging from 0.5% in asian regions to 8% of the population in norway. male and female populations are equally affected in most regions (3). psoriasis may occur in different forms, such as plaque psoriasis (characterized by dry scaly patches), which represents 80%-90% of psoriasis cases; pustular psoriasis (contains puslike fluid mainly infiltrated with white blood cells); erythrodermic psoriasis (ep, characterized by exfoliation of fine scaly skin with pain and itching); guttate psoriasis (characterized by drop-like dots); and inverse psoriasis (affects the flexure surfaces and characterized by smooth inflamed lesions) (1). the pathogenesis of psoriasis is not completely understood, and the exact mechanism remains elusive, although the literature suggests that different genetic, epigenetic, and environmental factors may be responsible for the onset of psoriasis (2). although not inherited in a mendelian fashion, a familial predisposition may be present, which significantly introduction psoriasis is an immune-mediated inflammatory disease with unknown etiology, characterized by the presence of papules and plaques of various morphology and severity over the surface of skin (1). psoriasis is classified as an inflammatory autoimmune disease in which an excessively aberrant hyperproliferation of keratinocytes is present, along with dilated, hyperplastic blood vessels and inflammatory infiltration of leukocytes predominantly into dermis (2). these features typically present as red skin patches that are itchy and scaly. as with other autoimmune diseases, psoriasis may have a systemic outcome beyond the skin, also affecting the https://doi.org/10.33393/dti.2022.2355 https://creativecommons.org/licenses/by-nc/4.0/legalcode novel biologic therapy for psoriasis management2 © 2022 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti increases the relative risk of psoriasis in families with a positive history among first-degree and second-degree relatives of patients, compared to the general population (2). epigenetic factors including dysregulated deoxyribonucleic acid (dna) methylation levels, abnormal histone modification, and micro ribonucleic acid (mirna) expressions have been recognized as crucial players in the pathophysiology of psoriasis. among other factors, excessive activation of the adaptive immune system and the interplay of immune cells and cytokines are thought to play a role in the onset of psoriasis (2,3). an initial secretion of cytokines that activate myeloid dendritic cells, from plasmacytoid dendritic cells, keratinocytes, natural killer t cells, and macrophages, is thought to initiate the first steps of the pathogenesis of psoriasis (3). erythrodermic psoriasis among the other forms of psoriasis, ep represents a rare and severe form that affects 1% to 2.25% of patients with psoriasis. the clinical characteristics of this form of psoriasis are the presence of erythematous, edematous, often exfoliative lesions that affect over 75% of the body surface area (4), and are often associated with numerous systemic symptoms such as fever, tachycardia, lymphadenopathy, arthralgia, and fatigue (5). there is a substantial risk of mortality due to transepidermal fluid and nutrient loss, and which in severe cases may lead to multiorgan failure and death (4). ep generally develops in patients with poorly controlled psoriasis and as a result of the abrupt withdrawal of systemic medications such as corticosteroids, drug reactions to medications such as lithium, and underlying systemic infections (5). for a clear and correct diagnosis of ep, two general clinical subtypes that define the disease must be considered. the first subtype is characterized by psoriatic plaques that gradually differentiate and develop into generalized erythroderma, although, overall, the plaques remain differentiable from the erythroderma. this form of ep has a stable course and a good prognosis. in contrast, the second ep subtype is more common in a psoriatic arthritis setting and plaques usually cannot be distinguishable from whole-body erythema (6). as with psoriasis, the exact pathogenesis of ep is not fully clarified. the current literature suggests the involvement of an unbalanced t-helper (th)1/th2 differentiation in favor of the th2 phenotype and its related cytokine secretion. interleukin (il)-4 and il-13, in particular, have been shown to be elevated in ep relative to both psoriasis patients and healthy controls. several studies have demonstrated that anti–tumor necrosis factor (tnf)-α agents improve ep outcomes, suggesting that tnf-α may play a role in the pathogenesis of ep (6). although difficult and challenging, the management of ep is possible through several treatment options. the us national psoriasis foundation had suggested, in 2010 in their consensus guidelines, the use of cyclosporine or infliximab as first-line therapy in unstable cases, with acitretin and methotrexate reserved for more stable cases. since then, other therapies and treatment strategies have emerged, including topical treatment with steroids and vitamin d analogues, as well as phototherapy. other studies have also demonstrated the efficacy of systemic agents, including second-generation retinoids (acitretin) and immunosuppressive drugs (methotrexate and cyclosporine), which showed complete remission and significant improvement in ep outcomes. recently, another class of drugs, called biological agents, has demonstrated high efficacy in ep management. biological therapies represent an emerging class of immunosuppressive drugs, which, thanks to their enhanced selectivity to specific cytokines, may represent a valid alternative to the canonical treatments. biological therapies include tnf-α inhibitors, il-12/23 inhibitors, and il-17 inhibitors (6). secukinumab: drug description and focus on the treatment of ep as mentioned, an unbalanced proinflammatory response and massive cytokine secretion may promote and sustain worsening symptoms of ep. besides th1 and th2, the recent discovery of the new class of t-helper cells, th17, also highlighted the possible role of the proinflammatory cytokine il-17 (secreted by th17) in the pathogenesis of ep (7). some environmental triggers, such as physical trauma, drugs, or infections, release proinflammatory cytokines, including il-23 and tnf-α. differentiation of t helper into th17 cells and the release of cytokines, such as il-17, promote keratinocyte proliferation, which, in the setting of ep, also release additional ils and chemokines (8). the homodimeric glycoprotein il-17a belongs to the il-17 family and through its receptor complex il-17ra/il-17rc binds to keratinocytes, dendritic cells, dermal fibroblast, and endothelial cells. under physiological conditions, normal levels of il-17a promote, upon binding, the proliferation of the keratinocytes necessary for healing, and protect against infectious agents. however, in psoriatic patients, levels of il-17a are elevated, and correlate with the severity of the disease. keratinocytes themselves, when stimulated, also synthesize many cytokines that can induce epidermal hyperplasia (autocrine growth factors) or neoangiogenesis (paracrine growth factors), resulting in worsening of ep and initiating a reverberating loop that perpetuates pro-proliferative and proinflammatory stimuli (9). consistently, blocking il-17a results in an improvement of psoriasis lesions, suggesting a key role in the pathogenesis of ep (8). currently, one anti-il-17a biological agent is approved for the treatment of plaque psoriasis, secukinumab, a monoclonal antibody that targets il-17a (10). secukinumab is a novel biologic agent that specifically targets il-17a. it is a fully human monoclonal antibody, and many clinical trials have demonstrated its efficacy in the treatment of plaque psoriasis, psoriatic arthritis, and ankylosing spondylitis (as) (11-14). indications approved by the european medicines agency (ema) include adult and pediatric psoriasis, psoriatic arthritis, axial spondyloarthritis (axspa), as and non-radiographic axial spondyloarthritis (nr-axspa) (15). the rationale behind the efficacy and mechanism of the action of secukinumab is a targeted approach to block the disease process at a very early stage along with a safe adverse event profile (16). by targeting il-17a, secukinumab thereby blocks its binding with il-17r and consequent expression of cytokines. blocking the il-17a pathway leads to a normalization of the inflammatory processes and thus combats epidermal carriero drug target insights 2022; 16: 3 © 2022 the authors. published by aboutscience www.aboutscience.eu hyperproliferation, t-cell infiltration, and excessive expression of pathogenic genes (16). several clinical trials have demonstrated the efficacy of secukinumab in the treatment of psoriasis (11-14). two phase iii, double-blind, 52-week trials (erasure and fixture) (17) evaluated the efficacy of secukinumab in patients with moderate-to-severe plaque psoriasis. the erasure study enrolled 738 patients, while fixture study included 1,306 patients. in both studies patients received either placebo or secukinumab subcutaneously once a week for 5 weeks, but in the fixture study patients also received etanercept (50 mg twice a week for 12 weeks, then once a week). results from the erasure study showed that a psoriasis area and severity index (pasi) 75 score at week 12 was achieved by 81.6% and 71.6% of patients administered with 300 mg and 150 mg of secukinumab, respectively, and by 4.5% of placebo recipients. corresponding results from fixture showed that 77.1% of patients administered 300 mg of secukinumab, 67% of those administered 150 mg of secukinumab, 44% of etanercept recipients, and only 4.9% of placebo patients achieved pasi 75 score at week 12 (17). another multicenter, international, retrospective, pilot study enrolled 13 ep patients who were treated with a loading dose of 300 mg of subcutaneous secukinumab at weeks 0, 1, 2, 3, and 4, followed by 300 mg every 4 weeks (18). results from this study showed that the response rate to secukinumab was 10/13 patients (77%), with a median time to clearance of 3 weeks (1.5-3 weeks). no recurrences were registered in the 52-week follow-up and a pasi score of 90 was achieved by 10/13 patients. these results demonstrated that secukinumab remains a valid and effective therapeutic option for ep (18). case presentation in this real-life clinical experience of the treatment of psoriasis with secukinumab drug, we report the case of a 72-year-old man, in general good health condition, who contacted the dermatologic outpatient clinic of the author on march 5, 2020, during the pandemic lockdown. the patient reported widespread ep with severe palmoplantar hyperkeratosis (shown in fig. 1a-c), and he was not taking any other drugs or medications. he was started on prednisone (25 mg one tablet) treatment for 8 days and at half-dose for 8 more days. in addition, topical therapy with mometasone furoate cream was prescribed. on march 16, 2020, blood chemical tests were in the normal range, and the patient continued therapy with cyclosporine (100 mg twice a day) until july 2020. the patient’s erythroderma significantly improved, but his palmoplantar hyperkeratosis persisted. due to onset of hypertensive seizures and increased creatininemia, the patient discontinued the treatment. then, after a pause of a few weeks, the patient resumed prednisone treatment in august 2020, due to the persistence of severe hyperkeratosis. however, there was no response or improvement. on september 1, 2020, the patient started secukinumab treatment at the dose of 300 mg administered by two subcutaneous injections of 150 mg each (15); significant signs of clinical resolution were observed during the induction phase, for both erythrodermic psoriasis and palmoplantar fig. 1 widespread erythrodermic psoriasis (a) and palmoplantar hyperkeratosis (b, c) at initial presentation before secukinumab treatment. (a) (b) (c) novel biologic therapy for psoriasis management4 © 2022 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti hyperkeratosis. the patient is currently under maintenance treatment with secukinumab and his general condition is good. the pasi score at the first visit was 60, and decreased to 20 after induction (shown in fig. 2a-c). at a follow-up visit, conducted at the end of february 2021, the patient showed an almost complete remission, with a pasi score of 10. as of january 2022 the patient, in perfect health conditions with normal hematochemical tests, presents a complete remission of the clinical picture. discussion and conclusions although the treatment options for ep have greatly expanded in recent years, psoriasis and its severe forms remain a major public health concern. treatment of ep with secukinumab has shown promising and encouraging clinical outcomes, and the present case report showed data consistent with other case reports from the literature. weng et al (19) reported a series of 10 cases in which secukinumab was prescribed for ep. data from those case reports showed that at week 16, 4/10 patients and 7/10 patients, respectively, were able to achieve pasi 90 and pasi 75 scores (19). in this very first and unique case for our clinical practice, the patient, affected by palmoplantar psoriasis, but who also presented a very severe form of ep, showed significant signs of clinical resolution during the induction phase, with few side effects. the initial pasi score of the patient was 60, which significantly decreased to 20 after the induction and to 10 after the follow-up visit at 6 months, suggesting complete remission of ep. however, although treatment outcomes with secukinumab are promising, additional controlled trials with extended follow-up are needed to better understand the link between il-17 inhibition and the resolution of ep. acknowledgments the author thanks enrica piras, phd, who provided editorial assistance and ray hill, an independent medical writer, who provided english-language editing prior to submission on behalf of health publishing & services srl. financial support for medical editorial assistance was provided by novartis farma, italy. the funder was not involved in the conception, drafting and writing of this article or in the decision to submit it for publication. author contributions dr. carriero: conception and design of the work, acquisition and analysis of data, draft of the work; agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. statement of ethics written informed consent for publication (including images) has been obtained from the patient. the research was conducted ethically in accordance with the declaration of helsinki. disclosure conflict of interest statement: the author has no conflicts of interest to declare. funding sources: this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability statement: all data generated or analyzed during this study are included in this article. further enquiries can be directed to the corresponding author. references 1. rajguru jp, maya d, kumar d, suri p, bhardwaj s, patel nd. update on psoriasis: a review. j family med prim care. 2020; 9(1):20-24. crossref pubmed 2. deng y, chang c, lu q. the inflammatory response in psoriasis: a comprehensive review. clin rev allergy immunol. 2016; 50(3):377-389. crossref pubmed fig. 2 improved erythrodermic (a) and palmoplantar hyperkeratosis (b, c) after secukinumab treatment. (a) (b) (c) https://doi.org/10.4103/jfmpc.jfmpc_689_19 https://www.ncbi.nlm.nih.gov/pubmed/32110559 https://doi.org/10.1007/s12016-016-8535-x https://www.ncbi.nlm.nih.gov/pubmed/27025861 carriero drug target insights 2022; 16: 5 © 2022 the authors. published by aboutscience www.aboutscience.eu 3. armstrong aw, read c. pathophysiology, clinical presentation, and treatment of psoriasis: a review. jama. 2020;323(19):19451960. crossref pubmed 4. reynolds ka, pithadia dj, lee eb, liao w, wu jj. a systematic review of treatment strategies for erythrodermic psoriasis. j dermatolog treat. 2021;32(1):49-55. crossref pubmed 5. carrasquillo oy, pabón-cartagena g, falto-aizpurua la, et al. treatment of erythrodermic psoriasis with biologics: a systematic review. j am acad dermatol. 2020;83(1):151-158. crossref pubmed 6. singh rk, lee km, ucmak d, et al. erythrodermic psoriasis: pathophysiology and current treatment perspectives. psoriasis (auckl). 2016;6:93-104. crossref pubmed 7. fitch e, harper e, skorcheva i, kurtz se, blauvelt a. pathophysiology of psoriasis: recent advances on il-23 and th17 cytokines. curr rheumatol rep. 2007;9(6):461-467. crossref pubmed 8. berg sh, balogh ea, ghamrawi ri, feldman sr. a review of secukinumab in psoriasis treatment. immunotherapy. 2021; 13(3):201-216. crossref pubmed 9. chiricozzi a, krueger jg. il-17 targeted therapies for psoriasis. expert opin investig drugs. 2013;22(8):993-1005. crossref pubmed 10. wasilewska a, winiarska m, olszewska m, rudnicka l. interleukin-17 inhibitors. a new era in treatment of psoriasis and other skin diseases. postepy dermatol alergol. 2016;33(4):247-252. crossref pubmed 11. reich k, warren rb, coates lc, di comite g. long-term efficacy and safety of secukinumab in the treatment of the multiple manifestations of psoriatic disease. j eur acad dermatol venereol. 2020;34(6):1161-1173. crossref pubmed 12. deodhar a, mease pj, mcinnes ib, et al. long-term safety of secukinumab in patients with moderate-to-severe plaque psoriasis, psoriatic arthritis, and ankylosing spondylitis: integrated pooled clinical trial and post-marketing surveillance data. arthritis res ther. 2019;21(1):111. crossref 13. baraliakos x, gossec l, pournara e, et al. secukinumab in patients with psoriatic arthritis and axial manifestations: results from the double-blind, randomised, phase 3 maximise trial. ann rheum dis. 2021;80(5):582-590. crossref pubmed 14. kiltz u, sfikakis pp, gaffney k, et al. secukinumab use in patients with moderate to severe psoriasis, psoriatic arthritis and ankylosing spondylitis in real-world setting in europe: baseline data from serena study. adv ther. 2020;37(6):2865-2883. crossref pubmed 15. cosentx-summary of product characteristics. available at: online (accessed october 2021). 16. frieder j, kivelevitch d, menter a. secukinumab: a review of the anti-il-17a biologic for the treatment of psoriasis. ther adv chronic dis. 2018;9(1):5-21. crossref pubmed 17. langley rg, elewski be, lebwohl m, et al; erasure study group; fixture study group. secukinumab in plaque psoriasis— results of two phase 3 trials. n engl j med. 2014;371(4):326338. crossref pubmed 18. damiani g, pacifico a, russo f, et al. use of secukinumab in a cohort of erythrodermic psoriatic patients: a pilot study. j clin med. 2019;8(6):770. crossref pubmed 19. weng hj, wang ts, tsai tf. clinical experience of secukinumab in the treatment of erythrodermic psoriasis: a case series. br j dermatol. 2018;178(6):1439-1440. crossref pubmed https://doi.org/10.1001/jama.2020.4006 https://www.ncbi.nlm.nih.gov/pubmed/32427307 https://doi.org/10.1080/09546634.2019.1689228 https://www.ncbi.nlm.nih.gov/pubmed/31682547 https://doi.org/10.1016/j.jaad.2020.03.073 https://www.ncbi.nlm.nih.gov/pubmed/32247872 https://doi.org/10.2147/ptt.s101232 https://www.ncbi.nlm.nih.gov/pubmed/28856115 https://doi.org/10.1007/s11926-007-0075-1 https://www.ncbi.nlm.nih.gov/pubmed/18177599 https://doi.org/10.2217/imt-2020-0195 https://www.ncbi.nlm.nih.gov/pubmed/33203276 https://doi.org/10.1517/13543784.2013.806483 https://www.ncbi.nlm.nih.gov/pubmed/23731078 https://doi.org/10.5114/ada.2016.61599 https://www.ncbi.nlm.nih.gov/pubmed/27605893 https://doi.org/10.1111/jdv.16124 https://www.ncbi.nlm.nih.gov/pubmed/31785165 https://doi.org/10.1186/s13075-019-1882-2 https://doi.org/10.1136/annrheumdis-2020-218808 https://www.ncbi.nlm.nih.gov/pubmed/33334727 https://doi.org/10.1007/s12325-020-01352-8 https://www.ncbi.nlm.nih.gov/pubmed/32378070 https://www.ema.europa.eu/en/documents/product-information/cosentyx-epar-product-information_en.pdf https://doi.org/10.1177/2040622317738910 https://www.ncbi.nlm.nih.gov/pubmed/29344327 https://doi.org/10.1056/nejmoa1314258 https://www.ncbi.nlm.nih.gov/pubmed/25007392 https://doi.org/10.3390/jcm8060770 https://www.ncbi.nlm.nih.gov/pubmed/31159169 https://doi.org/10.1111/bjd.16252 https://www.ncbi.nlm.nih.gov/pubmed/29265175 drug target insights 2016:10(s1) 15 this supplement is intended to focus on drug eluting devices. devices (eg cardiovascular stents, drug delivery biopolymers and tissue engineering devices), their bio­ compatibility, and mechanisms of transport are included within the supplement’s scope. drug target insights aims to provide researchers working in this complex, quickly developing field with online, open access to highly relevant scholarly articles by leading international researchers. in a field where the literature is ever-expanding, researchers increasingly need access to up-to-date, high quality scholarly articles on areas of specific contemporary interest. this supplement aims to address this by presenting high-quality articles that allow readers to distinguish the signal from the noise. the editor in chief hopes that through this effort, practitioners and researchers will be aided in finding answers to some of the most complex and pressing issues of our time. supplement aims and scope journal name: drug target insights journal type: editorial year: 2016 volume: 10(s1) running head verso: spadaccio et al running head recto: drug target insights introductory editorial: drug-eluting stents or drug-eluting grafts? insights from proteomic analysis §§ cristiano spadaccio current address: department of cardiothoracic surgery, golden jubilee national hospital, clydebank, glasgow, uk. university of glasgow institute of cardiovascular and medical sciences, glasgow, uk. §§ francesco nappi cardiac surgery centre cardiologique du nord de saintdenis, paris, france. §§ nawwar al-attar department of cardiothoracic surgery, golden jubilee national hospital, clydebank, glasgow, uk. §§ raffaella coccia department of biochemical sciences, sapienza university of rome, italy. §§ marzia perluigi department of biochemical sciences, sapienza university of rome, italy. §§ fabio di domenico department of biochemical sciences, sapienza university of rome, italy. current developments in drug eluting devices the rapidly expanding panorama of prosthetic replace-ment and interventional procedures for cardiovas-cular disease demands significant research for the development of devices with optimized characteristics and performance to overcome the drawbacks of the existing strategies. tissue engineering is a potent weapon in this scenario, enabling the realization of sophisticated biocompatible devices interacting with the host tissues and influencing their behavior. the fabrication and design of “smart” biomaterials, able to sense and interact with the biological milieu, represent a cornerstone in tissue engineering and regenerative medicine. this development has demolished the obsolete tenets of surgery implying reconstructing organs or part of them with artificial materials. the deeper understanding achieved by modern research has highlighted the limitations and the biological and clinical drawbacks of the long-term coexistence of a foreign material in the body, especially in the cardiovascular system.1 the need to respect the physiological reparative and remodeling processes normally occurring in nature progressively achieved significance in the current research,2,3 demonstrating the necessity to accompany the body’s physiological responses and the activities of tissue regeneration rather than pretending to replace them with “plastic” surrogates. this attention to the biology of regeneration in tissue engineering and to micro-environmental conditions at the tissue and cellular levels is expressed in the articles of this special issue of drug target insights. interesting and sophisticated approaches are proposed in this context, entailing the use of drug-delivery http://www.la-press.com/drug-target-insights-journal-j23 http://www.la-press.com spadaccio et al drug target insights 2016:10(s1)16 devices that release tailored compounds to target specific aspects of diseases or complications. in this issue, dr rapetto and colleagues extensively reviewed the role of gentamicinimpregnated collagen sponges (gicss) in preventing sternal wound infection. the use of these delivery devices allows for topical delivery of high antibiotic concentrations to the wound, reducing the complications associated with gentamycin toxicity.4 on the other side, alternative use of smart biopolymers able to release anticoagulant agents, such as heparin, to avoid early vascular graft thrombosis and failure will be presented and discussed in the same issue of the journal.5 these are not the only applications in this field, as demonstrated by other papers in this issue. indeed, at the experimental level, several research efforts have been engaged in designing scaffold-releasing factors, drugs or cytokines to improve tissue regeneration or stem cell recruitment, or to overcome prosthesis-related issues.6 the possibility to tailor a bioresorbable scaffold in order to create a microenvironment able to boost or guide tissue regeneration is an exciting area of investigation.7,8 additionally, drug-releasing devices might also allow avoidance or treatment of some of the drawbacks related to prosthetic replacement of cardiovascular structures.5,6 in the field of interventional cardiology there is a widespread use of drug-releasing stents, in particular of steroids or antiproliferative agents in order to prevent neointimal hyperplasia. interestingly, the concept of paracrine or local release of molecules with modulatory or homeostatic action is not new in biology. endothelium-mediated release of growth factors and regulatory molecules is a well-accepted natural mechanism to locally and remotely control or respond to a variety of physiological or pathological conditions, and different parts of the vascular tree might behave differently according to their biological needs. this concept has important ramifications also in the clinical side, especially when treating cardiovascular structures. release of drug from coronary stents (drug-eluting stents, des) able to influence or modify endothelial homeostasis and function is an example. more interestingly, the use of autologous non-artificial conduits in coronary artery bypass graft (cabg) surgery might be considered another intriguing system of “natural” drug delivery device. cabg might be performed using autologous saphenous vein or internal thoracic artery (ita or mammary artery), two conduits with profoundly different biological features and structure. there is a general consensus on the accelerated degeneration of venous grafts after surgery, with extremely high incidence of failure and lower patency rates in comparison to arterial grafts. this difference in angiographic patency was shown to be associated with improved clinical outcomes and rates of ischemia-free survival in patients undergoing exclusive arterial revascularization, especially in case of subjects who previously developed in-stent restenosis.9 construction of coronary graft through the use of arterial conduits, especially with internal thoracic artery (ita), is therefore advocated as desirable to ensure long-term patency and optimal clinical outcomes.10,11 the biological mechanisms underlying the poorer outcomes of venous grafts in respect to arterial ones are not well understood and still a matter of debate. reduced production of nitric oxide has been claimed as a primary factor,12 implicated in venous graft failure in relation to established risk factors for atherosclerosis;13 also, differences in thrombin receptor expression between arterial and venous grafts have been demonstrated,14 and deregulation of these receptors has been associated with in-stent restenosis.15 nitric oxide production is not reduced in arterial grafts and particularly the ita, even with severe atherosclerotic disease, and this is thought to be one of the factors in the superior outcome of these conduits.16 additionally, it has been shown that the structure of the ita itself is able to better adapt to arterial pressures and its endothelium responds to high flow rates with a higher amount of nitric oxide, providing superior reactivity to flow requirements in the coronary arteries when used as a graft in cabg.17 on this basis, ita might be considered as a “drugeluting” graft as it is able to release into the grafted myocardium nitric oxide, providing important signaling to prevent graft failure and ameliorate cardiac function. clearly, the biology underlying this process is far more complex and is not restricted to a single compound, but most probably involves a wide spectrum of molecules interacting to determine the biological effects seen both experimentally and clinically. proteomics studies allow for a comprehensive analysis and identification of the complete protein pattern of a tissue or fluid,18,19 and some studies performed using this approach showed the presence in human arterial smooth muscle of small leucine-rich proteoglycans involved in collagen fibrillogenesis, and of some non-fibrillar collagens in combination with alterations of several other proteins. this has been considered as a marker of arterial stiffness and therefore increased risk of developing atherosclerosis.20 structural proteomic studies on ita tissue showed differential expression of proteins that are implicated in cytoskeleton activity regulation,21 in the migrative capacity of vascular smooth muscle cells, extracellular matrix composition, coagulation, apoptosis, and heat shock response.22 interestingly, a proteomic analysis of the secrete of ita, known as secretome, demonstrated an increased production of gelsolin, vinculin, lamin a/c and phosphoglucomutase 5 by mammary arterial tissues. these proteins are also involved in the regulation of important intracellular mechanisms related to cell migration, ecm deposition and smooth muscle phenotype switching, which are crucial steps in atherosclerosis pathogenesis.23 the expression of specific groups of proteins in the ita is claimed to be the basis of the relative protection of this vessel from the onset and progression of atherosclerosis, and subsequently of its beneficial effects when is used as a graft for coronary artery in terms of recurrence of heart disease.22,23 from these studies, we might reliably speculate that when used in the context of cabg, the ita exerts a “paracrine activity” liberating locally and within the blood stream factors that are able to maintain a positive http://www.la-press.com/drug-target-insights-journal-j23 http://www.la-press.com drug target insights drug target insights 2016:10(s1) 17 vascular homeostasis, thereby avoiding disease recurrence in the grafted coronary and permitting high patency rate of the bypass in the long term. considering the significant difference in patency rate when conduits different from ita are used, more complex mechanisms should be underlying ita selective advantage in cabg. use of proteomics and redox-proteomics approaches to simultaneously compare the protein profile of ita, saphenous vein grafts and aorta, another tissue prone to atherosclerosis, has been advocated to better understand differences among the conduits. also, these investigations might be extended to the secretomes of these conduits in order to have a paracrine correlate to the targets found in the respective tissues. differential profiles of protein expression exclusively present in ita tissues and secrete, but not in the other vessels, have been intriguingly discovered (fig. 1) and the identification of these proteins would provide in the future precious information to elucidate reasons of ita superiority in cabg. moreover, the identification of these proteins would enable more significant clinical applications. the factors produced by the ita, and considered at the basis of the maintenance of graft patency and protection from atherosclerosis recurrence, might constitute in the future “drugs” to be administered to patients or eluted in stents or delivery devices. conversely, factors identified from saphenous tissue, which are clinically associated to poor outcomes and failure of the grafts, might represent targets for design of specific compounds with inhibitory or blocking effects. in the field of drug-eluting devices, a close observation and attention to the naturally occurring phenomena might provide us with a range of therapeutic options wider than any other drug currently used in des or tissue engineering approaches. for example, the ita naturally carries a regulated set of factors, finely modulated and intertwined, which protects against atherosclerosis and can be therefore considered the best drug-eluting device available at the moment in cardiovascular disease. in conclusion, with the increase in life expectancy and in the morbidities related to chronic diseases, smarter weapons are required to control pathology. the exciting field of drug delivery devices might provide novel strategies and open new avenues in the treatment of cardiovascular disease. in this context, scientists might need to realize that the endogenous and physiologically-occurring release of paracrine factors by the native tissues might be a “system” to better understand and to target when constructing new drug-releasing devices. r efer ences 1. spadaccio c, nappi f, al-attar n, et al. old myths, new concerns: the longterm effects of ascending aorta replacement with dacron grafts. not all that glitters is gold. j cardiovasc transl res. 2016;9(4):334–342. 2. nappi f, spadaccio c, fraldi m, et al. a composite semiresorbable armoured scaffold stabilizes pulmonary autograft after the ross operation: mr ross’s dream fulfilled. j thorac cardiovasc surg. 2016;151(1):155–164 e151. 3. spadaccio c, montagnani s, acar c, nappi f. introducing bioresorbable scaffolds into the show. a potential adjunct to resuscitate ross procedure. int j cardiol. 2015;190:50–52. 4. rapetto f, bruno vd, guida g, marsico r, chivasso p, zebele c. gentamicin impregnated collagen sponge: effectiveness in preventing sternal wound infection in high-risk cardiac surgery. drug target insights. 2016;10(suppl 1): 9–13. figure 1. comparison of proteomic profile of secreted proteins by saphenous vein graft, internal thoracic artery (ita) and aorta. http://www.la-press.com/drug-target-insights-journal-j23 http://www.la-press.com spadaccio et al drug target insights 2016:10(s1)18 5. spadaccio c, nappi f, de marco f, et al. preliminary in vivo evaluation of a hybrid armored vascular graft combining electrospinning and additive manufacturing techniques. drug target insights. 2016;10(suppl 1):1–7. 6. spadaccio c, chello m, trombetta m, rainer a, toyoda y, genovese ja. drug releasing systems in cardiovascular tissue engineering. j cell mol med. 2009;13(3):422–439. 7. rainer a, spadaccio c, sedati p, et al. electrospun hydroxyapatite-functionalized plla scaffold: potential applications in sternal bone healing. ann biomed eng. 2011;39(7):1882–1890. 8. spadaccio c, rainer a, centola m, et al. heparin-releasing scaffold for stem cells: a differentiating device for vascular aims. regen med. 2010;5(4):645–657. 9. gaudino m, cellini c, pragliola c, et al. arterial versus venous bypass grafts in patients with in-stent restenosis. circulation. 2005;112(9 suppl):i265–269. 10. hannan el, racz mj, walford g, et al. long-term outcomes of coronary-artery bypass grafting versus stent implantation. n engl j med. 2005;352(21):2174–2183. 11. al-attar n, nataf p. multiple extensive coronary artery stenting: does it compromise future surgical revascularization? curr opin cardiol. 2007;22(6):529–533. 12. harrison dg. endothelial dysfunction in the coronary microcirculation: a new clinical entity or an experimental finding? j clin invest. 1993;91(1):1–2. 13. west ne, qian h, guzik tj, et al. nitric oxide synthase (nnos) gene transfer modifies venous bypass graft remodeling: effects on vascular smooth muscle cell differentiation and superoxide production. circulation. 2001;104(13):1526–1532. 14. yang z, ruschitzka f, rabelink tj, et al. different effects of thrombin receptor activation on endothelium and smooth muscle cells of human coronary bypass vessels. implications for venous bypass graft failure. circulation. 1997;95(7): 1870–1876. 15. takamori n, azuma h, kato m, et al. high plasma heparin cofactor ii activity is associated with reduced incidence of in-stent restenosis after percutaneous coronary intervention. circulation. 2004;109(4):481–486. 16. tarr fi, sasvari m, tarr m, racz r. evidence of nitric oxide produced by the internal mammary artery graft in venous drainage of the recipient coronary artery. ann thorac surg. 2005;80(5):1728–1731. 17. luscher tf, diederich d, siebenmann r, et al. difference between endotheliumdependent relaxation in arterial and in venous coronary bypass grafts. n engl j med. 1988;319(8):462–467. 18. butterfield da, gu l, di domenico f, robinson ra. mass spectrometry and redox proteomics: applications in disease. mass spectrom rev. 2014;33(4):277–301. 19. spadaccio c, di domenico f, perluigi m, et al. serum proteomics in patients with diagnosis of abdominal aortic aneurysm. cardiovasc pathol. 2012;21(4):283–290. 20. lyck hansen m, beck hc, irmukhamedov a, jensen ps, olsen mh, rasmussen lm. proteome analysis of human arterial tissue discloses associations between the vascular content of small leucine-rich repeat proteoglycans and pulse wave velocity. arterioscler thromb vasc biol. 2015;35(8):1896–1903. 21. de la cuesta f, zubiri i, maroto as, et al. deregulation of smooth muscle cell cytoskeleton within the human atherosclerotic coronary media layer. j proteomics. 2013;82:155–165. 22. de la cuesta f, alvarez-llamas g, maroto as, et al. a proteomic focus on the alterations occurring at the human atherosclerotic coronary intima. mol cell proteomics. 2011;10(4):m110003517. 23. de la cuesta f, barderas mg, calvo e, et al. secretome analysis of atherosclerotic and non-atherosclerotic arteries reveals dynamic extracellular remodeling during pathogenesis. j proteomics. 2012;75(10):2960–2971. clinical lecturer at the institute of cardiovascular and medical science of the university of glasgow and holds a clinical position of cardiothoracic surgical fellow at the cardiothoracic surgery department of the golden jubilee national hospital. he completed his phd at university la sapienza of rome and has previously worked at the mcgowan institute for regenerative medicine at the university of pittsburgh medical center (usa), the new york presbyterian and columbia university hospital (usa), the cardiovascular surgery department of university of leuven (belgium), the campus st jan hospital osot-limburg in genk (belgium) and at the university campus biomedico of rome, italy. he now works primarily in the field of translational research and clinical in cardiovascular surgery, in tissue engineering and regenerative medicine. dr. spadaccio is the author or co-author of more than 60 pubmed-indexed published papers and has presented more than 60 abstracts and personal communications at conferences, being involved in organization of meetings and chairing of sessions as moderator. he holds editorial appointments at the journal artificial organs. cristianospadaccio@gmail.com institutional webpage http://www.gla.ac.uk/researchinstitutes/icams/ staff/?action=person&id=4edcedec8393 lead guest editor dr. cristiano spadaccio guest editors dr. nicola testa cardiovascular surgeon of italy at john paul ii foundation—catholic university in campobasso. he completed his md degree at catholic university of sacred heart in rome (2003) and cardiac surgery residency at catholic university of sacred heart in rome (2008) he has previously worked as staff cardiac surgeon in cardiovascular surgery unit—catholic university in campobasso (2008–2013) and in cardiac surgery unit “ss.annunziata hospital” chieti (2013–2014). he now works primarily in heart failure treatment and research and surgical atrial fibrillation ablation. dr. testa is the author or co-author of more than 30 published papers and has presented at more than 30 conferences. ntesta.cch@gmail.com institutional webpage http://www.fgps.it/index.php/i-dipartimenti/ malattie-cardiovascolari-e-dei-grossi-vasi/ personale http://www.la-press.com/drug-target-insights-journal-j23 http://www.la-press.com mailto:cristianospadaccio@gmail.com http://www.gla.ac.uk/researchinstitutes/icams/staff/?action=person&id=4edcedec8393 http://www.gla.ac.uk/researchinstitutes/icams/staff/?action=person&id=4edcedec8393 mailto:ntesta.cch@gmail.com http://www.fgps.it/index.php/i-dipartimenti/malattie-cardiovascolari-e-dei-grossi-vasi/personale http://www.fgps.it/index.php/i-dipartimenti/malattie-cardiovascolari-e-dei-grossi-vasi/personale http://www.fgps.it/index.php/i-dipartimenti/malattie-cardiovascolari-e-dei-grossi-vasi/personale drug target insights drug target insights 2016:10(s1) 19 dr. francesco nappi cardiovascular surgeon at centre cardiologique du nord, paris, france. he completed his md at university federico ii of naples and has previously worked as a surgeon and as a researcher in this institution. he now works primarily in cardiovascular disease and translational medicine. dr. nappi is the author or co-author of 40 published papers and has presented at over 30 conferences, and holds editorial appointments at the journal of thoracic disease. francesconappi@gmail.com dr. alberto rainer assistant professor in chemical fundamentals of technologies at università campus bio-medico di roma, italy. he completed his phd at university tor vergata of rome and has previously worked at the university of rome and triest. he now works primarily in tissue engineering, biofunctionalization of polymers and analysis of cell-to-cell interaction on micro chips. dr. rainer is the author or co-author of over 30 published papers and has presented at over 40 conferences, and holds editorial appointments at the journal artificial organs. a.rainer@unicampus.it institutional webpage http://didattica.unicampus.it/didattica/guide/ paginadocente.do;jsessionid=bb5df6e681779e9 a0d9ee809d4665e1a.jvm1a?docente_id=325 dr. vito d. bruno senior registrar in cardiac surgery at university hospitals of bristol and research associate fellow at the department of clinical science at university of bristol (uk). he completed his phd in surgery and surgical biotechnology at university of insubria, varese (italy). he has previously worked at varese university hospital (varese—italy) and cardiocentro ticino (lugano—switzerland.) he now works primarily in clinical and translational research in cardiac surgery and cardiology. dr. bruno is the author or co-author of 23 pubmed cited papers and more than 30 abstracts for national and international meetings. he is section editor at archives of medical science—civilisation disease. mddvb@bristol.ac.uk institutional webpage http://www.bris.ac.uk/clinical-sciences/ people/182709/index.html supplement title: current developments in drug eluting devices citation: spadaccio et al. current developments in drug eluting devices. drug target insights 2016:10(s1) 15–19 doi:10.4137/dti.s41240. type: editorial funding: authors disclose no external funding sources. competing interests: authors disclose no potential conflicts of interest. copyright: © the authors, publisher and licensee libertas academica limited. this is an open-access article distributed under the terms of the creative commons cc-by-nc3.0 license. correspondence: cristianospadaccio@gmail.com all authors have provided signed confirmation of their compliance with ethical and legal obligations including (but not limited to) use of any copyrighted material, compliance with icmje authorship and competing interests disclosure guidelines. http://www.la-press.com/drug-target-insights-journal-j23 http://www.la-press.com mailto:francesconappi@gmail.com mailto:a.rainer@unicampus.it http://didattica.unicampus.it/didattica/guide/paginadocente.do;jsessionid=bb5df6e681779e9a0d9ee809d4665e1a.jvm1a?docente_id=325 http://didattica.unicampus.it/didattica/guide/paginadocente.do;jsessionid=bb5df6e681779e9a0d9ee809d4665e1a.jvm1a?docente_id=325 http://didattica.unicampus.it/didattica/guide/paginadocente.do;jsessionid=bb5df6e681779e9a0d9ee809d4665e1a.jvm1a?docente_id=325 mailto:mddvb@bristol.ac.uk http://www.bris.ac.uk/clinical-sciences/people/182709/index.html http://www.bris.ac.uk/clinical-sciences/people/182709/index.html http://dx.doi.org/10.4137/dti.s41240 http://creativecommons.org/licenses/by-nc/3.0/ http://creativecommons.org/licenses/by-nc/3.0/ mailto:cristianospadaccio@gmail.com dti drug target insights 2021; 15: 21-25issn 1177-3928 | doi: 10.33393/dti.2021.2291original research article drug target insights issn 1177-3928 www.aboutscience.eu/dti © 2021 the authors. this article is published by aboutscience and licensed under creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). commercial use is not permitted and is subject to publisher’s permissions. full information is available at www.aboutscience.eu clinical factors predictive of appropriate treatment in copd: a community hospital setting sukanya tongdee1, bundit sawunyavisuth2, wattana sukeepaisarnjaroen3, watchara boonsawat3, sittichai khamsai3, kittisak sawanyawisuth3 1department of medicine, chumpae hospital, khon kaen thailand 2department of marketing, faculty of business administration and accountancy, khon kaen university, khon kaen thailand 3department of medicine, faculty of medicine, khon kaen university, khon kaen thailand abstract background: chronic obstructive pulmonary disease (copd) is a common respiratory disease. the appropriate treatment according to the global initiative for chronic obstructive lung disease (gold) guideline was 19-60%. however, there are limited data on predictors of appropriate treatment in patients with copd. this study aimed to evaluate risk factors of appropriate treatment in patients with copd according to the gold guideline in a realworld community setting. methods: this is a retrospective study conducted at a community hospital. inclusion criteria were adult patients diagnosed as copd treated at a copd clinic. the primary outcome was the appropriate treatment, defined by correct pharmacological treatment by the gold guideline according to the abcd severity assessment. clinical predictors of appropriate treatment were executed by stepwise multivariate logistic regression analysis. results: 136 patients with copd met the study criteria. of those, 100 patients had inappropriate treatment according to the gold guideline. three factors were independently associated with the appropriate treatment including number of admissions, modified medical research council (mmrc) score, and cat score. these factors had adjusted odds ratio of 3.11, 2.86, and 1.26, respectively. causes of inappropriate treatment were unavailability of long-acting muscarinic antagonist (lama) (51 patients; 79.69%), treated by inhaled corticosteroid (ics) alone (12 patients; 18.75%), and treated with only bronchodilator (1 patient; 1.56%). conclusions: appropriate copd patients’ treatment according to the gold guideline was 26.47% in community setting. factors associated with severity of copd were associated with prescribing appropriate treatments. keywords: cat, hospitalization, mmrc received: july 7, 2021 accepted: october 19, 2021 published online: november 13, 2021 corresponding authors: sittichai khamsai and kittisak sawanyawisuth 123 mitraparp road department of medicine faculty of medicine khon kaen university khon kaen, 40002 thailand sittikh@kku.ac.th and kittisak@kku.ac.th can be confirmed by evidence of incomplete irreversible airflow limitation without other causes. treatment of copd comprises both pharmacological and nonpharmacological modalities such as smoking cessation. uncontrolled copd may lead to copd exacerbations and mortality (2). a study of 73,106 patients with copd found that the mortality rate was 50% at 3.6 years after hospitalization (3), while another study found that in-hospital mortality rate was 2.6% (4). there are several factors associated with copd control such as copd severity, patient compliance, correct inhaler technique, or nonpharmacological treatment (5,6). even though patients with copd had medication adherence of 51.0%, 85 out of 549 patients or only 15.5% were under control (7). another factor that may be associated with copd symptom control is appropriately prescribed medication (6,8,9). an undertreatment according to the guideline increases risk of copd exacerbation with a coefficient of −0.179 (p < 0.001) (9). the global initiative for chronic obstructive lung disease (gold) guideline recommends introduction chronic obstructive pulmonary disease (copd) is a respiratory disease mainly caused by smoking. patients with copd suffer from several symptoms, exacerbations, or hospitalizations leading to 2.6% of disability-adjusted life years (dalys) and at least 3.2 million deaths globally (1). diagnosis of copd https:doi.org/10.33393/dti.2021.2291 https://creativecommons.org/licenses/by-nc/4.0/legalcode copd community22 © 2021 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti various pharmacological regimens based on copd severity (10). in real practice, the appropriate treatment according to the gold guideline was 19-60% (9,11,12). however, there are limited data on predictors of appropriate treatment in patients with copd. this study aimed to evaluate risk factors of appropriate treatment in patients with copd according to the gold guideline in a real-world community setting. methods this study was a retrospective study conducted at chumpae hospital, the largest community hospital in khon kaen province, khon kaen, thailand. the inclusion criteria were adult patients who were diagnosed with copd and treated at the copd clinic. the diagnosis of copd was made according to the gold guideline (10). the study period was between may and november 2019. the study protocol was approved by the institutional review board, ministry of public health, khon kaen branch, thailand (61165). eligible patients were enrolled from clinical charts and evaluated for baseline characteristics, smoking history, risk factor for copd, symptoms, chest x-ray, pulmonary function test, copd type, 6-minute walk test (6mw), history of exacerbations, history of admission, and copd assessment. history of cough was defined by the presence of cough for more than 2 weeks, while productive sputum more than 2 months was recorded. copd assessment was evaluated by using modified medical research council (mmrc), copd assessment test (cat), and copd classification by the gold guideline or abcd assessment. the primary outcome of the study was the appropriate treatment, which was defined by correct pharmacological treatment by the gold guideline to category a to d: a bronchodilator for group a; a long-acting bronchodilator (long-acting beta2-agonists: laba or long-acting muscarinic antagonist: lama) for group b; lama for group c; and lama or lama plus laba or inhaled corticosteroid (ics) plus laba for group d. treatment other than this recommendation in a particular category was defined as inappropriate treatment. the inappropriate treatment was also classified as underand overtreatment according to the recommendation for each category. note that information retrieved for the study was at the initial therapy of each patient. statistical analyses patients were categorized into two groups by appropriateness of treatment. the studied variables were compared between both groups by descriptive statistics. for numerical variables, mean and sd was reported and compared between groups by using independent t-test or wilcoxon rank sum test where appropriate. numbers and percentages of each categorical variable were reported and compared between groups by chi square test or fisher exact test where appropriate. clinical predictors of appropriate treatment were executed by stepwise multivariate logistic regression analysis. those factors with a p value of less than 0.20 by univariate logistic regression were put in the subsequent multivariate logistic regression analysis. the goodness of fit of the final model was tested by hosmer-lemeshow method. the statistical analyses were executed by the stata software (college station, texas, usa). results there were 136 patients with copd who met the study criteria. of those, 100 patients (73.53%) were with inappropriate treatment according to the gold guideline. between those with appropriate and inappropriate treatment groups, there were two significant factors in terms of baseline characters including cough and sputum production (tab. i). the appropriate treatment group had higher proportions of patients with cough and sputum production than the inappropriate treatment group (77.78% vs. 54.00%; and 80.56% vs. 60.00%, respectively). table i baseline characters of patients with chronic obstructive pulmonary diseases (copd) categorized by receiving appropriate treatment factors inappropriate n = 100 appropriate n = 36 p value mean (sd) age, years 64.51 (8.83) 63.47 (10.51) 0.566 male sex, n (%) 94 (94.00) 35 (97.22) 0.675 occupation: agricultural, n (%) 93 (93.00) 31 (86.11) 0.412 diabetes mellitus, n (%) 8 (8.00) 7 (19.44) 0.060 hypertension, n (%) 42 (42.00) 17 (47.22) 0.588 dyspnea, n (%) 100 (100.00) 37 (100.00) na cough, n (%) 54 (54.00) 28 (77.78) 0.012 sputum, n (%) 60 (60.00) 29 (80.56) 0.026 smoking history, n (%) 0.992  none 9 (9.00) 3 (8.33)  ex-smoker 72 (72.00) 26 (72.22)  current smoker 19 (19.00) 7 (19.44) mean (sd) pack-year of smoking 21.49 (15.40) 26.82 (29.24) 0.498 exposure to noxious particles, n (%) 6 (6.00) 2 (5.56) 0.999 mean (sd) bmi (kg/m2) 21.17 (3.69) 21.90 (3.95) 0.446 bmi = body mass index; na = not available. between these two groups, the appropriate treatment group had shorter 6mw test (328.05 vs. 353.49 m) and lower mmrc (1.83 vs. 0.96) than the inappropriate treatment group significantly (tab. ii). but the average cat score (15.88 vs. 7.22), average number of exacerbation (2.83 vs. 1.13 times), and average number of admissions (2.83 vs. 1.13 times) were significantly higher in the appropriate treatment group than the inappropriate treatment group (tab. ii) while the post-bronchodilator fev1/fvc was significantly lower in the appropriate treatment group than the inappropriate treatment group (53.19 vs. 57.32; p = 0.033). copd class d tongdee et al drug target insights 2021; 15: 23 © 2021 the authors. published by aboutscience www.aboutscience.eu was also found more in the appropriate treatment group than the inappropriate treatment group (100.00% vs. 3.00%). there were five factors remaining in the final model predictive of appropriate treatment in patients with copd (tab. iii). of those, three factors were independently associated with the appropriate treatment including number of admissions, mmrc score, and cat score. these factors had adjusted odds ratio of 3.11, 2.86, and 1.26, respectively. the final model had the hosmer-lemeshow chi-square of 10.72 (p = 0.218), indicating goodness of fit of the model. causes of inappropriate treatment were unavailability of lama (51 patients; 79.69%), treated by ics alone (12 patients; 18.75%), and treated with only bronchodilator (1 patient; 1.56%). categorized by copd category, overtreatment was found in categories a, b, and c, while undertreatment was reported in categories b, c, and d (tab. iv). table iv proportions of underor overtreatment by chronic obstructive airway disease category (n = 100) treatment a (n = 42) b (n = 30) c (n = 25) d (n = 3) undertreatment 0 6 (20.00) 2 (8.00) 3 (100.00) overtreatment 42 (100.00) 24 (80.00) 23 (92.00) 0 discussion this study showed that the appropriate treatment for patients with copd was 26.47%: in category d at 100.00% (tab. ii). compared with other three previous studies, this study had appropriate treatment rate comparable with the study at va hospital in the us (27.2% vs. 18.7%) and lower than two studies from tertiary hospitals. in this community hospital setting, patients with category d had highest appropriate treatment rate than others at 100.00% (tab. ii). this pattern was also found in other studies which may indicate that severe cases of copd tend to follow the gold guideline as they may have severe symptoms and required appropriate and several pharmacological therapies (10,13). this study also found another similar pattern on appropriate treatment: low appropriate treatment rate in categories a, b, and c. first, we found that inhaled corticosteroid alone was used in 12 patients or 18.75%. the study from italy also found that inhaled corticosteroid was overused despite the gold guideline that does not recommend it as shown in table v (11). but, the attending physicians believe that it is more effective. a study from sweden also found that inhaled corticosteroid was used inappropriately in 45.5% of patients with copd regardless of categories: a 33.6%; b 46.2%; table ii laboratory results and disease status of patients with chronic obstructive pulmonary diseases (copd) categorized by receiving appropriate treatment factors inappropriate n = 100 appropriate n = 36 p value cxr, n (%)  normal, n (%) 53 (53.00) 19 (52.78) 0.982  hyperinflation, n (%) 36 (36.00) 13 (36.11) 0.990 post-bronchodilator fev1, ml 66.86 (17.40) 60.30 (19.11) 0.061 post-bronchodilator fev1, % 6.02 (7.00) 6.80 (7.21) 0.602 post-bronchodilator fev1/fvc 57.32 (9.17) 53.19 (9.99) 0.033 copd type, n (%) 0.999  chronic bronchitis 3 (3.00) 1 (2.78)  emphysema 5 (5.00) 1 (2.78)  mixed 92 (92.00) 34 (94.44) mean (sd) 6mw, meters 353.49 (72.91) 328.05 (87.16) 0.222 mmrc, n (%) 0.96 (0.66) 1.83 (0.88) <0.001  0 22 (22.00) 1 (2.78)  1 62 (62.00) 13 (36.11)  2 14 (14.00) 14 (38.89)  3 2 (2.00) 7 (19.44)  4 0 1 (2.78) mean (sd) cat 7.22 (5.31) 15.88 (5.04) <0.001 exacerbation, n (%) 1.13 (2.40) 2.83 (2.09) <0.001 admission, n (%) 0.26 (0.75) 1.22 (1.17) <0.001 category, n (%) <0.001  a 42 (42.00) 0  b 30 (30.00) 0  c 25 (25.00) 0  d 3 (3.00) 36 (100.00) 6mw = 6-minute walk test; cat = copd assessment test; mmrc = modified medical research council dyspnea questionnaire; copd category by the gold guideline. table iii factors predictive of appropriate treatment in chronic obstructive pulmonary diseases (copd) treated at community hospital factors unadjusted odds ratio (95% confidence interval) adjusted odds ratio (95% confidence interval) age 0.99 (0.95, 1.03) 0.94 (0.89, 1.01) diabetes 2.77 (0.93, 8.31) 3.10 (0.46, 20.84) admission 3.73 (2.02, 6.88) 3.11 (1.39, 6.97) mmrc 4.47 (2.41, 8.30) 2.86 (1.18, 6.94) cat 1.32 (1.19, 1.47) 1.26 (1.13, 1.42) factors in the model included sex, smoking, cough, sputum, body mass index, chest x ray, 6-minute walk test, post-bronchodilator fev1, post-bronchodilator fev1/fvc, and exacerbation. cat = copd assessment test; mmrc = modified medical research council dyspnea questionnaire. copd community24 © 2021 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti c 54.8%; and d 71.0% (14). an inappropriate use of inhaled corticosteroid was also found in 50% of patients with copd in the uk (15). another limitation for community hospital in this study is lack of lama in 79.69%: it may be due to unavailability and cost of lama. not surprisingly, factors predictive for appropriate treatments were factors indicating severe copd including hospital admissions, mmrc, and cat score (tabs. ii and iii). among these three factors, admissions and mmrc had higher adjusted odds ratios than the cat score. these may imply that the two factors are slightly stronger predictors for severe copd than the cat score (9,10,13). additionally, hospitalizations may remind physicians to prescribe more proper medications for the patients as they may have more times to assess the patients than in the outpatient setting (16). this study had some limitations. first, we did not evaluate association of copd such as obstructive sleep apnea (osa) or asthma which may result in overprescription of corticosteroids (17-21). second, the study population was community hospital. the results of this study may not be applied for more complicated copd patients. second, there was no follow-up data on long-term outcomes. finally, inappropriate treatment of not using lama was due to unavailability. other causes of inappropriate treatment were treatment with only ics (18.75%) or bronchodilator alone (1.56%). conclusion appropriate treatment of patients with copd according to the gold guideline was 26.47% in community setting. factors associated with severity of copd were associated with prescribing of appropriate treatments. acknowledgments the authors would like to thank research center in back, neck other joint pain and human performance (bnojph), khon kaen university, khon kaen, thailand. disclosures conflict of interest: the authors declare that they have no conflicts of interest. financial support: none. references 1. soriano jb, abajobir aa, abate kh, et al; gbd 2015 chronic respiratory disease collaborators. global, regional, and national deaths, prevalence, disability-adjusted life years, and years lived with disability for chronic obstructive pulmonary disease and asthma, 1990-2015: a systematic analysis for the global burden of disease study 2015. lancet respir med. 2017;5(9):691-706. crossref pubmed 2. kim wj, gupta v, nishimura m, et al. identification of chronic obstructive pulmonary disease subgroups in 13 asian cities. int j tuberc lung dis. 2018;22(7):820-826. crossref pubmed 3. suissa s, dell’aniello s, ernst p. long-term natural history of chronic obstructive pulmonary disease: severe exacerbations and mortality. thorax. 2012;67(11):957-963. crossref pubmed 4. dong f, ren x, huang k, wang y, jiao j, yang t. development and validation of risk prediction model for in-hospital mortality among patients hospitalized with acute exacerbation: chronic obstructive pulmonary disease between 2015 and 2019. front med (lausanne). 2021;8:630870. crossref pubmed 5. bettoncelli g, blasi f, brusasco v, et al. the clinical and integrated management of copd. sarcoidosis vasc diffuse lung dis. 2014;31(suppl 1):3-21. crossref pubmed 6. hashimoto n, wakahara k, sakamoto k. the importance of appropriate diagnosis in the practical management of chronic obstructive pulmonary disease. diagnostics (basel). 2021;11(4): 618. crossref pubmed 7. roche n, plaza v, backer v, et al. asthma control and copd symptom burden in patients using fixed-dose combination inhalers (sprint study). npj prim care respir med. 2020;30(1):1. crossref pubmed 8. fernandes fla, cukier a, camelier aa, et al. recommendations for the pharmacological treatment of copd: questions and answers. j bras pneumol. 2017;43(4):290-301. crossref pubmed 9. foda hd, brehm a, goldsteen k, edelman nh. inverse relationship between nonadherence to original gold treatment guidelines and exacerbations of copd. int j chron obstruct pulmon dis. 2017;12:209-214. crossref pubmed 10. global initiative for chronic obstructive lung disease (gold). global strategy for the diagnosis, management and prevention of copd, online (2021, accessed 26 april 2021). 11. palmiotti ga, lacedonia d, liotino v, et al. adherence to gold guidelines in real-life copd management in the puglia region of italy. int j chron obstruct pulmon dis. 2018;13:2455-2462. crossref pubmed 12. chan kp, ko fw, chan hs, et al. adherence to a copd treatment guideline among patients in hong kong. int j chron obstruct pulmon dis. 2017;12:3371-3379. crossref pubmed table v appropriate treatment in abcd severity assessment in patients with chronic obstructive pulmonary disease study, year country setting total a b c d palmiotti, 2018 italy pulmonologists 419/693 (60.5%) 57/142 (40.1%) 110/238 (46.2%) 18/41 (43.9%) 234/272 (86.0%) foda, 2017 usa va and university hospital 164/878 (18.7%) 30/86 (34.9%) 19/379 (5.0%) 73/292 (25.0%) 42/121 (34.7%) chan, 2017 hong kong tertiary hospital 262/450 (58.2%) 1/5 (20.0%) 7/164 (1.6%) 0/8 (0%) 254/273 (56.4%) this study thailand community hospital 36/136 (26.47%) 0/42 (0%) 0/30 (0%) 0/25 (0%) 36/39 (92.31%) https://doi.org/10.1016/s2213-2600(17)30293-x https://www.ncbi.nlm.nih.gov/pubmed/28822787 https://doi.org/10.5588/ijtld.17.0524 https://www.ncbi.nlm.nih.gov/pubmed/29914609 https://doi.org/10.1136/thoraxjnl-2011-201518 https://www.ncbi.nlm.nih.gov/pubmed/22684094 https://doi.org/10.3389/fmed.2021.630870 https://www.ncbi.nlm.nih.gov/pubmed/33889584 https://doi.org/10.4081/mrm.2014.409 https://www.ncbi.nlm.nih.gov/pubmed/24820963 https://doi.org/10.3390/diagnostics11040618 https://www.ncbi.nlm.nih.gov/pubmed/33808229 https://doi.org/10.1038/s41533-019-0159-1 https://www.ncbi.nlm.nih.gov/pubmed/31911607 https://doi.org/10.1590/s1806-37562017000000153 https://www.ncbi.nlm.nih.gov/pubmed/29365005 https://doi.org/10.2147/copd.s119507 https://www.ncbi.nlm.nih.gov/pubmed/28123293 http://goldcopd.org/ https://doi.org/10.2147/copd.s157779 https://www.ncbi.nlm.nih.gov/pubmed/30147311 https://doi.org/10.2147/copd.s147070 https://www.ncbi.nlm.nih.gov/pubmed/29238182 tongdee et al drug target insights 2021; 15: 25 © 2021 the authors. published by aboutscience www.aboutscience.eu 13. ding b, small m, holmgren u. a cross-sectional survey of current treatment and symptom burden of patients with copd consulting for routine care according to gold 2014 classifications. int j chron obstruct pulmon dis. 2017;12:1527-1537. crossref pubmed 14. larsson k, ekberg-jansson a, stridsman c, hanno m, vanfleteren legw. adherence to treatment recommendations for chronic obstructive pulmonary disease – results from the swedish national airway register. int j chron obstruct pulmon dis. 2021;16:909-918. crossref pubmed 15. price d, west d, brusselle g, et al. management of copd in the uk primary-care setting: an analysis of real-life prescribing patterns. int j chron obstruct pulmon dis. 2014;9:889-904. crossref pubmed 16. ramakrishnan s, janssens w, burgel pr, et al. standardisation of clinical assessment, management and follow-up of acute hospitalised exacerbation of copd: a europe-wide consensus. int j chron obstruct pulmon dis. 2021;16:321-332. crossref pubmed 17. sawunyavisuth b. what are predictors for a continuous positive airway pressure machine purchasing in obstructive sleep apnea patients? asia pac j sci technol. 2018;23: apst-23-03-10. crossref 18. kingkaew n, antadech t. cardiovascular risk factors and 10-year cv risk scores in adults aged 30-70 years old in amnat charoen province, thailand. asia pac j sci technol. 2019;24:apst-24-04-04. online 19. jingmark s, kuhirunyaratn p, theeranut a, et al. subjective well-being and related factors among community-dwelling elderly in udon thani province, thailand. asia pac j sci technol. 2020;25:apst-25-01-09. online 20. chaiear n, nirarach k, kawamatawong t, et al. proportion of workers having work-related asthma symptoms in a cassava factory, nakhon ratchasima province, thailand. asia pac j sci technol. 2020;25:apst-25-02-08. online 21. mekov e, nuñez a, sin dd, et al. update on asthma-copd overlap (aco): a narrative review. int j chron obstruct pulmon dis. 2021;16:1783-1799. crossref pubmed https://doi.org/10.2147/copd.s133793 https://www.ncbi.nlm.nih.gov/pubmed/28579771 https://doi.org/10.2147/copd.s300299 https://www.ncbi.nlm.nih.gov/pubmed/33854309 https://doi.org/10.2147/copd.s62750 https://www.ncbi.nlm.nih.gov/pubmed/25210450 https://www.ncbi.nlm.nih.gov/pubmed/33623379 https://doi.org/10.2147/copd.s287705 https://doi.org/10.14456/apst.2018.10 https://so01.tci-thaijo.org/index.php/apst/article/view/181250 https://so01.tci-thaijo.org/index.php/apst/article/view/201240 https://so01.tci-thaijo.org/index.php/apst/article/view/239890 https://doi.org/10.2147/copd.s312560 https://www.ncbi.nlm.nih.gov/pubmed/34168440 open access full open access to this and thousands of other papers at http://www.la-press.com. drug target insights 2012:6 13–18 doi: 10.4137/dti.s9324 this article is available from http://www.la-press.com. © the author(s), publisher and licensee libertas academica ltd. this is an open access article. unrestricted non-commercial use is permitted provided the original work is properly cited. drug target insights r a p i d c o m m u n i c a t i o n drug target insights 2012:6 13 expression of luteinizing hormone receptor in the gastrointestinal tract in patients with and without dysmotility oskar hammar1, béla veress2, agneta montgomery3 and bodil ohlsson1 1department of clinical sciences, section of gastroenterology and hepatology, skåne university hospital, entrance 35, 205 02 malmö, lund university, sweden. 2department of clinical sciences, section of pathology, skåne university hospital, entrance 78, 205 02 malmö, lund university, sweden. 3department of clinical sciences, section of surgery, skåne university hospital, entrance 42, 205 02 malmö, lund university, sweden. corresponding author email: oskar.hammar@med.lu.se abstract: leuprolide is a gonadotropin-releasing hormone (gnrh) analog which has been shown to reduce symptoms in patients with irritable bowel syndrome (ibs) and chronic intestinal pseudo-obstruction (cipo). the mechanism is not known, but one hypothesis is through down-modulation of luteinizing hormone (lh) secretion, a hormone whith antagonistic effect on gastrointestinal motility. however, presence of lh receptors in the gastrointestinal tract has never been described. the aim of this study was to find one possible way of action for leuprolide by examining the presence of the lh receptor, and if present, to see whether there was different expression in patients with or without dysmotility. full-thickness biopsies from the bowel wall of patients with and without severe dysmotility were examined using immunohistochemistry staining. biopsies showed expression of lh receptors on myenteric neurons and in glial cells, neutrophils, endothelial cells and mast cells. there was no difference in expression between patient groups. keywords: luteinizing hormone (lh), gastrointestinal dysmotility, enteric neurons http://www.la-press.com http://dx.doi.org/10.4137/dti.s9324 http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 http://www.la-press.com mailto:oskar.hammar@med.lu.se hammar et al 14 drug target insights 2012:6 introduction chronic intestinal pseudo-obstruction (cipo) is a disorder affecting gastrointestinal motor activity, producing symptoms and signs resembling those of mechanical obstruction.1–3 enteric dysmotility (ed) encompasses patients with abnormal intestinal motor activity but no signs of obstruction.4 the etiologies of these two disorders are largely unknown.5 leuprolide, a gonadotropin-releasing hormone (gnrh) analog, have been used in the treatment of these diseases as well as in patients with irritable bowel syndrome (ibs).6–8 the mechanism of action behind the reduction of symptoms is not known. one hypothesis is that leuprolide continuously stimulates the hypothalamicpituitary-gonadal axis and thereby down-modulates the secretion of gonadotropines and gonadal products.9,10 these are in turn known to be neural antagonists of gastrointestinal motility11–13 and absence of these hormones could explain the improvements seen. the effect on the gastrointestinal tract demands receptors for the substance, and receptors for progesterone and estrogen have been described in the gastrointestinal tract.14,15 the luteinizing hormone (lh) receptor was first described in gonadal tissues. during the last years it has also been described in several non-gonadal tissues and on cancer cells, but its expression in the gastrointestinal tract has never been examined,16 although lh has been shown to affect intestinal motility in rat.11 to establish a possible way of action for the reported effect of gnrh analogs and lh on gastrointestinal symptoms and motility, the aim of the present study was to examine the presence of lh receptors in the gastrointestinal tract, and if present, to compare the expression in patients with and without severe gastrointestinal dysmotility. material and methods this study was performed according to the helsinki declaration and approved by the ethics committee of lund university. all patients gave their informed consent before entering the study. subjects the criterion for inclusion in the dysmotility group was the diagnosis of either cipo or ed, together with a gastrointestinal specimen available for staining of lh receptors. consecutive patients subjected to laparoscopic full-thickness biopsy at the departments of surgery or gastroenterology, skåne university hospital, malmö, between 1998 and 2009 because of severe gastrointestinal pain and dysmotility were identified and included in a retrospective manner. thorough investigation comprising radiological and/or endoscopic investigation to rule out organic disease or mechanical obstruction had been performed. gastrointestinal examination was completed with esophageal manometry, gastric emptying scintigraphy, antroduodenojejunal manometry and/or colonic transit time when appropriate. laparoscopy was performed for diagnostic purposes to exclude mechanical obstruction and to obtain a full-thickness biopsy. a previously described laparoscopy-assisted technique for taking full-thickness biopsies, preparing the biopsies and protocol for cipo analysis was used.17 in addition, patients with severe dysmotility who underwent intestinal resection (including small or large bowel, or both) within the same time-frame were included, but were not considered for laparoscopic biopsy because full-thickness intestinal wall tissue was already available for analysis. in order to set a cipo diagnosis, patients had to fulfill 3 criteria: a medical history compatible with pseudo-obstruction, documented events or chronic signs mimicking mechanical obstruction (bowel dilatation and/or air/fluid levels) and absence of mechanical obstruction or other organic cause for these symptoms and findings.1–3 the criteria for ed were documented abnormal contractile activity, but no past history of episodes, or current signs, mimicking mechanical obstruction and absence of any medication that could lead to the observed motor abnormalities.2,4 these patients represent the majority of cases of suspected cipo/ed in the most southern part of sweden. thirty-five patients fulfilled the inclusion criteria. apart from the histochemical staining for cipo analysis,17 representative sections were also available for staining of the lh receptor in 15 patients. the median age of the 15 patients (13 women) was 44 (range 18–96) years at the time of investigation. ten patients were diagnosed with ed and 5 patients with cipo. seven of the patients had been treated by opioids, and 3 had some sort of nutritional supplements. histopathological analysis revealed that inflammatory neuropathy was most http://www.la-press.com lh receptor and gastrointestinal tract drug target insights 2012:6 15 table 1. characteristics of the patients in the dysmotility group. patient characteristics number of patients number of patients with available information age, years 44 (range 18–96) 15 15 concurrent diseases* 5 15 epilepsia 1 cardiovascular diseases 1 borderline psychosis 1 diabetes mellitus 1 ehler danlos syndrome 1 gynecological problems** 8 13 endometriosis 2 extra uterine pregnancy 2 salpingo oophorectomized 2 conization 1 in vitro fertilization 2 prior surgery** 10 15 gynecological surgery 8 abdominal surgery 8 multiple prior surgery 8 employment able to work full time 3 11 partly employed 1 11 unable to work 4 11 retired 2 11 deceased 1 11 notes: *excluding gynecological diseases; **some patients have more than one gynecological problem or prior surgery. common in our cohort, either as an independent disease or in combination with myopathy (9/15 patients, 60%), whereas degenerative neuropathy or combined myoneuropathy occurred in 40% of the patients (6/15). twelve small bowel specimen and 7 large bowel specimen were available for lh receptor staining with presence of ganglia, reflecting material from resections with both small and large bowel specimens present in 4 patients. for further patient characteristics, see table 1. as controls for lh-receptor +/neurons in small bowel, sections from 6 cases of small bowel resection due to non-obliterating adenocarcinoma of the jejunum and ileum, and 2 cases of colonic carcinoma were used, median age 69 (range 53–85) years. three were women. regarding large bowel, the control group was 8 cases (5 women) with bowel resection due to diverticulosis, median age 74 (range 46–87) years. the samples were taken from areas with normal macroand microscopic appearance 10 cm above the tumor in the small bowel and from diverticulum-free normal parts of the large bowel specimen. all controls were found to have otherwise normal histology. full-thickness biopsy of the bowel full-thickness slices perpendicular to each other were cut from the specimen and embedded in paraffin for conventional, transversal sections. the remaining, larger part of the biopsy was embedded in toto for tangential sectioning. serial sections from all blocks were stained according to a protocol for cipo analysis with both classical staining (haematoxylin & eosin, pas, ps-diastase, giemsa, kresylviolet, trichrome) and immunostaining.17 apart from the histochemical staining for cipo analysis, sections were also stained for the lh receptor. the polyclonal rabbit anti-lh receptor antibody (anti-lh/cg receptor; sigma aldrich, stockholm, sweden) was applied to sections at 1:100 dilutions. as negative controls the antibodies were replaced by serum. the length of the biopsy was measured and the number of the lh receptor +/neurons per mm length of intestinal muscle in the transversal sectioning was counted and expressed as percentage of neurons both in the dysmotility group and controls. using protein g product (pgp) 9.5 staining, the percentages of labeled cells from the initial countings were verified. we found a strong correlation between the two methods, with a spearman correlation coefficient of 0.873, p = 0.000001 for the lh receptor in the small bowel. for the large bowel, the coefficient was 0.965 and p value was p = 0.000000001, for lh receptor content. statistical methods values are expressed as median and interquartile range (iqr) and range. correlations were made by spearman’s correlation test. the mann whitney u-test was used to compare differences between groups, and p , 0.05 was considered statistical significant. results the median length of counted biopsies for the small bowel was 16.00 (iqr 11.43–24.85, range 4.80–65.00) mm in the dysmotility group and 13.50 (iqr 10.25–16.75, range 10.00–35.00) mm in controls. in the large bowel, the median lenght was 30.00 (iqr 23.00–41.00, range http://www.la-press.com hammar et al 16 drug target insights 2012:6 9.00–60.40) mm in the dysmotility group and 17.5 (iqr 11.00–19.00, range 10.00–23.00) mm in controls. the number of myenteric neurons per mm in the small bowel was 6.45 (iqr 5.25–7.38, range 2.40–21.00) in the dysmotility group compared to 8.21 (iqr 5.72–9.19, range 5.34–15.23) in the control group (p = 0.43). number of neurons per mm in the large bowel was 5.50 (iqr 4.70–5.90, range 4.30–7.70) in the dysmotility group compared to 8.76 (iqr 5.77–9.08, range 5.00–10.71) in the control group (p = 0.04). all specimens in both the dysmotility group and the control group had positive lh receptors. the lh receptor was positive in cytoplasm of approximately 50% of myenteric neurons and in glial cells, neutrophils, endothelial cells and mast cells for both the dysmotility group and the controls (fig. 1). a group of submucosal neurons were labeled for lh receptors, but they were not counted as these neurons are not affected in patients suffering from dysmotility. all other cell types of the bowel wall were negative. no immunostaining was seen in the negative control sections. the percentage of labeled neurons in the dysmotility group was 42.50 (iqr 38.25–48.00, range 26.00–60.01)% in the small bowel and 50.00 (iqr 23.00–51.00, range 12.00–59.00)% in the large bowel. in controls, the median value was 47.14 (iqr 42.69–49.49, range 31.69–52.99)% and 43.40 (iqr 42.14–46.48, range 32.53–47.44)% in the small and large bowel, respectively, which was not significant different between the groups (p = 0.25 and 0.68, respectively). discussion this is to the best of our knowledge the first time lh receptor expression in the human gastrointestinal tract has been described. the results suggest no difference in the receptor expression between patients with and without gastrointestinal dysmotility, or between genders. the dysmotility group had significantly lower amount of enteric neurons per mm examined large bowel. the physiological effect of lh on gastrointestinal tract has previously only been rudimentary examined and has to be further investigated. so far, its antagonistic effect on gastrointestinal motility in rat has been described,11 which theoretically could be explained by the presence of lh receptors on myenteric neurons. further, a protective and antioxidant effect by lh in rat gastric tissue has been described, but the importance of this is unclear.14 the presence of the receptor on endothelial cells, neutrophils, mast cells and glial cells suggests circulatory, immunological and neuroprotective effects as well. luteinizing hormone has been described to exert a wide range of effects on other tissues in different species. the lh receptor is a member of a subfamily of g protein coupled receptors (gpcrs). it is well described for these receptors, that they are downregulated by continuous stimulation and up-regulated after intermittent stimulation.18 in a rat model, presence of lh receptors located in the vascular smooth muscle and endothelial cells of uterine blood vessels are hypothesized to be responsible for the reduced uterine blood flow after human chorionic gonadotropin (hcg)/lh stimulation.19 exogenous administration of lh increased the number of degranulated mast cells in the ovarian complex of mice.20 this is interesting as mast cells have been discussed in the pathophysiology of ibs and visceral hypersensitivity,21 and are expressed in an increased number in patients with severe constipation.22 glial cells which are important figure 1. control myenteric ganglion with luteinizing hormone (lh) receptor positive neurons (thick, blue arrow) and one negative neuron (arrow). glial cells within the ganglion and interstitial cells of cajal (thin arrows) are immunoreactive (lh receptor immunohistochemistry; bar: 20 µm). http://www.la-press.com lh receptor and gastrointestinal tract drug target insights 2012:6 17 to support neuronal elements, have been shown to be closely related to degranulated mast cells, and are expessed in reduced number in patients with constipation.22,23 fasting and refeeding affects the lh secretion in monkeys through cholecystokinin (cck) stimulation.24 as mutations of the lh receptor have been described in leydig cell hypoplasia,25 mutations could theoretically also affect gastrointestinal function. the gnrh analog leuprolide has been shown to reduce symptoms in disorders such as functional bowel diseases.7,8 further, leuprolide has been reported to restore motor function in the gastrointestinal tract in female ovariectomized rats and in a patient suffering from cipo.6,26 the mechanism is unclear, but one hypothesis is that leuprolide by continuous stimulation, and thereby desensitization of the pituitary gnrh receptor, down-modulates the secretion of gonadotropines and gonadal products,9,10 which are known neural antagonists of gastrointestinal motility.11–13 thus, continuous stimulation by leuprolide exerts antagonistic gnrh effects, leading to absence of lh secretion and stimulation.9,10 the present finding of lh receptors on enteric neurons gives a morphological explanation to this hypothesis, and absence of lh stimulation on the neural receptors could explain the reduction of symptoms.11 at the same time as continuous stimulation of leuprolide acetate improves patients with gastrointestinal complaints,7,8 repeated intermittent treatment with the analog buserelin in the setting of repeated in vitro fertilization (ivf) has been linked to the development of cipo in one patient.17 gonadotropinreleasing hormone and its receptor have been found on enteric neurons,17,27 why the effect evoked by gnrh analogs may be direct on enteric neurons as well, in addition to an effect on the hypothalamic-pituitarygonadal axis. the present study demonstrates the presence of lh receptors in the gastrointestinal tract, making it theoretically possible for this hormone to have a direct effect on this organ, and a possibility for gnrh analogs to affect the gasrointestinal tract. the role for regulation of expression and function of the lh receptor in the gastrointestinal tract has to be further investigated, as well as its possible involvement in the development of gastrointestinal disorders. author contributions conceived and designed the experiments: oh, bv, am, bo. analysed the data: oh, bv. wrote the first draft of the manuscript: oh, bo. contributed to the writing of the manuscript: bv, am. made critical revisions: oh, am, bo. all authors approved final version. funding crafoord foundation, bengt ihre foundation and development foundation of region skane. acknowledgments maria nilsson and annika jönsson are acknowledged for excellent technical support in performing the immunostaining. disclosures and ethics as a requirement of publication author(s) have provided to the publisher signed confirmation of compliance with legal and ethical obligations including but not limited to the following: authorship and contributorship, conflicts of interest, privacy and confidentiality and (where applicable) protection of human and animal research subjects. the authors have read and confirmed their agreement with the icmje authorship and conflict of interest criteria. the authors have also confirmed that this article is unique and not under consideration or published in any other publication, and that they have permission from rights holders to reproduce any copyrighted material. any disclosures are made in this section. the external blind peer reviewers report no conflicts of interest. references 1. de giorgio r, sarnelli g, corinaldesi r, stanghellini v. advances in our understanding of the pathology of chronic intestinal pseudo-obstruction. gut. 2004;53:1549–52. 2. stanghellini v, camilleri m, malagelada jr. chronic idiopathic intestinal pseudo-obstruction: clinical and intestinal manometric findings. gut. 1987; 28: 5–12. 3. mann sd, debinski hs, kamm ma. clinical characteristics of chronic idiopathic intestinal pseudo-obstruction in adults. gut. 1997;41:675–81. 4. wingate d, hongo m, kellow j, lindberg g, smout a. disorders of gastrointestinal motility: towards a new classification. j gastroenterol hepatol. 2002;suppl 17:s1–14. 5. stanghellini v, cogliandro rf, de giorgio r, barbara g, salvioli b, corinaldesi r. chronic intestinal pseudo-obstruction: manifestations, natural history and management. neurogastroenterol motil. 2007;19:440–52. 6. mathias jr, baskin gs, reeves-darby vg, clench mh, smith ll, calhoon jh. chronic intestinal pseudoobstruction in a patient with heart-lung transplant. therapeutic effect of leuprolide acetate. dig dis sci. 1992;37: 1761–8. http://www.la-press.com hammar et al 18 drug target insights 2012:6 7. mathias jr, clench mh, reeves-darby vg, et al. effect of leuprolide acetate in patients with moderate to severe functional bowel disease. double-blind, placebo-controlled study. dig dis sci. 1994;39:1155–62. 8. palomba s, orio f jr, manguso f, et al. leuprolide acetate treatment with and without coadministration of tibolone in premenopausal women with menstrual cycle-related irritable bowel syndrome. fertil steril. 2005;83: 1012–20. 9. rabin d, mcneil lw. pituitary and gonadal desensitization after continuous luteinizing hormone-releasing hormone infusion in normal females. j clin endocrinol metab. 1980;51:873–6. 10. hazum e, conn pm. molecular mechanism of gonadotropin releasing hormone (gnrh) action. i. the gnrh receptor. endocr rev. 1988;9: 379–86. 11. ducker te, boss jw, altug sa, et al. luteinizing hormone and human chorionic gonadotropin fragment the migrating myoelectric complex in rat small intestine. neurogastroenterol motil. 1996;8:95–100. 12. mathias jr, clench mh. relationship of reproductive hormones and neuromuscular disease of the gastrointestinal tract. dig dis. 1998;16:3–13. 13. wang f, zheng tz, li w, qu sy, he dy. action of progesterone on contractile activity of isolated gastric strips in rats. world j gastroenterol. 2003;9:775–8. 14. kumtepe y, borekci b, karaca m, salman s, hakan alp h, suleyman h. effect of acute and chronic administration of progesterone, estrogen, fsh and lh on oxidant and oxidant parameters in rat gastric tissue. chem biol interact. 2009;182:1–6. 15. schleipen b, hertrampf t, fritzemeier kh, et al. erβ-specific agonists and genistein inhibit proliferation and induce apoptosis in the large and small intestine. carcinogenesis. 2011;32:1675–83. 16. ziecik aj, kaczmarek mm, blitek a, kowalczyk ae, li x, rahman na. novel biological and possible applicable roles of lh/hcg receptor. mol cell endocrinol. 2007;269:51–60. 17. ohlsson b, veress b, janciauskiene s, montgomery a, haglund m, wallmark a. chronic intestinal pseudo-obstruction due to buserelin-induced formation of anti-gnrh antibodies. gastroenterology. 2007;132:45–51. 18. naor z. signaling by g-protein-coupled receptor (gpcr): studies on the gnrh receptor. front neuroendocrinol. 2009;30:10–29. 19. rao cv, alsip nl. use of the rat model to study hcg/lh effects on uterine blood flow. semin reprod med. 2011;19:75–85. 20. jaiswal k, krishna a. effects of hormones on the number, distribution and degranulation of mast cells in the ovarian complex of mice. acta physiol hung. 1996;84:183–90. 21. barbara g, wang b, stanghellini v, et al. mast cell-dependent excitation of visceral-nociceptive sensory neurons in irritable bowel syndrome. gastroenterology. 2007;132:26–37. 22. bassotti g, villanacci v, nascimbeni r, et al. increase of colonic mast cells in obstructed defecation and their relationship with enteric glia. dig dis sci. 2012;57:65–71. 23. bassotti g, villanacci v, maurer ca, et al. the role of glial cells and apoptosis of enteric neurones in the neuropathology of intractable slow transit constipation. gut. 2006;55:41–6. 24. schreihofer da, golden ga, cameron jl. cholecystokinin (cck)-induced stimulation of luteinizing hormone (lh) secretion in adult male rhesus monkeys: examination of the role of cck in nutritional regulation of lh secretion. endocrinology. 1993;132:1553–60. 25. kossack n, simoni m, richter-unruh a, themmen ap, gromoll j. mutations in a novel, cryptic exon of the luteinizing hormone/chorionic gonadotropin receptor gene cause male pseudohermaphroditism. plos. 2008;22:5 e88. 26. khanna r, browne rm, heiner ad, clench mh, mathias jr. leuprolide acetate affects intestinal motility in female rats before and after ovariectomy. am j physiol. 1992;262:g185–90. 27. huang w, yao b, sun l, pu r, wang l, zhang r. immunohistochemical and in situ hybridization studies of gonadotropin releasing hormone (gnrh) and its receptor in rat digestive tract. life sci. 2001;68:1727–34. http://www.la-press.com 13drug target insights 2016:10 effects and safety of linagliptin as an add-on therapy in advanced-stage diabetic nephropathy patients taking renin–angiotensin–aldosterone system blockers yuichiro ueda1, hiroki ishii1, taisuke kitano1, mitsutoshi shindo1, haruhisa miyazawa1, kiyonori ito1, keiji hirai1, yoshio kaku1, honami mori1, taro hoshino1, susumu ookawara1, masafumi kakei2, kaoru tabei3 and yoshiyuki morishita1 1division of nephrology, 2division of endocrinology and metabolism, first department of integrated medicine, saitama medical center, jichi medial university, saitama, japan. 3minamiuonuma hospital, niigata, japan. a bstr act backgrou nd: we investigated the effects and safety of linagliptin as an add-on therapy in patients with advanced-stage diabetic nephropathy (dmn) taking renin–angiotensin–aldosterone system (raas) blockers. method: twenty advanced-stage dmn patients (estimated glomerular filtration rate (egfr): 24.5 ± 13.4 ml/min/1.73 m2) taking raas blockers were administered 5 mg/day linagliptin for 52 weeks. changes in glucose and lipid metabolism and renal function were evaluated. r esults: linagliptin decreased glycosylated hemoglobin levels (from 7.32 ± 0.77% to 6.85 ± 0.87%, p , 0.05) without changing fasting blood glucose levels, and significantly decreased total cholesterol levels (from 189.6 ± 49.0 to 170.2 ± 39.2 mg/dl, p , 0.05) and low-density lipoprotein cholesterol levels (from 107.1 ± 32.4 to 90.2 ± 31.0 mg/dl, p , 0.05) without changing high-density lipoprotein cholesterol and triglyceride levels. urine protein/creatinine ratio and annual change in egfr remained unchanged. no adverse effects were observed. conclusion: linagliptin as an add-on therapy had beneficial effects on glucose and lipid metabolism without impairment of renal function, and did not have any adverse effects in this population of patients with advanced-stage dmn taking raas blockers. k e y wor ds: linagliptin, diabetic nephropathy, renin–angiotensin–aldosterone system blockers, glucose and lipid metabolism, renal function citation: ueda et al. effects and safety of linagliptin as an add-on therapy in advanced-stage diabetic nephropathy patients taking renin–angiotensin–aldosterone system blockers. drug target insights 2016:10 13–18 doi:10.4137/dti.s38339. type: original research received: may 25, 2016. resubmitted: august 11, 2016. accepted for publication: august 15, 2016. academic editor: anuj chauhan, editor in chief peer review: five peer reviewers contributed to the peer review report. reviewers’ reports totaled 910 words, excluding any confidential comments to the academic editor. funding: authors disclose no external funding sources. competing interests: authors disclose no potential conflicts of interest. copyright: © the authors, publisher and licensee libertas academica limited. this is an open-access article distributed under the terms of the creative commons cc-by-nc 3.0 license. correspondence: ymori@jichi.ac.jp paper subject to independent expert single-blind peer review. all editorial decisions made by independent academic editor. upon submission manuscript was subject to anti-plagiarism scanning. prior to publication all authors have given signed confirmation of agreement to article publication and compliance with all applicable ethical and legal requirements, including the accuracy of author and contributor information, disclosure of competing interests and funding sources, compliance with ethical requirements relating to human and animal study participants, and compliance with any copyright requirements of third parties. this journal is a member of the committee on publication ethics (cope). provenance: the authors were invited to submit this paper. published by libertas academica. learn more about this journal. introduction the prevalence of diabetic nephropathy (dmn) is increasing worldwide.1 dmn is the most common cause of end-stage renal disease.2,3 it is also a major risk factor for the development of cardiovascular disease.4 a poorly controlled blood glucose level and hypertension are the main contributors to progression to end-stage renal disease and the development of cardiovascular disease in dmn.5,6 appropriate management of blood glucose and blood pressure levels is important to improve the prognosis of patients with dmn.7–9 renin–angiotensin–aldosterone system (raas) blockers are used as first-line agents for blood pressure control in dmn patients. they have been reported to decrease blood pressure and have beneficial nephroprotective and cardioprotective effects.10–12 for blood glucose control, although many kinds of hypoglycemic agents have been developed, most cannot be used in dmn patients with decreased renal function because they have diminished elimination by the kidneys, and may cause unfavorable side effects. dipeptidyl peptidase-4 (dpp-4) inhibitors decrease blood glucose by inhibiting the degradation of glucagon-like peptide (glp-1), which enhances insulin secretion from β-cells and decreases glucagon secretion from α-cells of the pancreas.13,14 among dpp-4 inhibitors, linagliptin can be used for blood glucose control in patients with impaired renal function without any dose adjustment because it is mostly metabolized by the liver.15,16 several clinical studies have reported that linagliptin improves glucose metabolism in patients with varying degrees of renal function, either as a monotherapy or in combination with other hypoglycemic agents.17–23 linagliptin has also been reported to have beneficial effects on lipid metabolism and nephroprotective effects.22,23 this suggests that linagliptin as an add-on therapy in dmn patients taking raas blockers may show advantages in the management of dmn. journal name: drug target insights journal type: original research year: 2016 volume: 10 running head verso: ueda et al running head recto: effects and safety of linagliptin as an add-on therapy http://www.la-press.com/drug-target-insights-journal-j23 http://www.la-press.com http://dx.doi.org/10.4137/dti.s38339 http://creativecommons.org/licenses/by-nc/3.0/ http://creativecommons.org/licenses/by-nc/3.0/ mailto:ymori@jichi.ac.jp http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 ueda et al 14 drug target insights 2016:10 only a few experimental and clinical studies have been reported on the effects of linagliptin in combination with raas blockers in dmn.22,24 also, the effects and safety of linagliptin in advanced-stage dmn patients taking raas blockers have not been fully determined. in this study, we investigated the effects and safety of linagliptin as an add-on therapy in advanced-stage dmn patients taking raas blockers. participants and methods ethical considerations. this study was performed in accordance with the ethical principles contained in the declaration of helsinki and was approved by the ethics committee of saitama medical center, jichi medical university. written informed consent was obtained from all patients. patients. between march 2013 and july 2014, 30 patients were enrolled in the study. inclusion criteria were as follows: .20  years of age, suffering from type 2 dmn, dmn with an estimated glomerular filtration rate (egfr)  # 60 (ml/min/1.73  m2), urine protein/creatinine ratio (uacr) . 0.15 (g/g cr), and taking angiotensin iireceptor blockers or angiotensin-converting enzyme inhibitors. exclusion criteria were patients with type 1 diabetes mellitus or secondary diabetes mellitus, history of stroke or coronary heart disease, patients with malignancy, severe infection, urinary stones, steroid therapy, pregnant or lactating women, and patients with an allergy to linagliptin. study protocol. a diagram of the study design is shown in figure 1. the study was a 52-week, single-center, prospective study. all eligible patients were administered linagliptin 5  mg orally in the morning once daily as an add-on to existing drugs including raas blockers, hypolipidemic drugs, and anti-hyperglycemic agents but not dpp-4 inhibitors. six patients were changed from an existing dpp-4 inhibitor (vildagliptin) to 5 mg/day linagliptin. the dosage of the drugs, including raas blockers, hypolipidemic drugs, and anti-hyperglycemic agents, was not changed during the study period. changes in glucose metabolism [fasting blood glucose and glycosylated hemoglobin (hba1c)] and lipid metabolism [total cholesterol, low-density lipoprotein (ldl)-cholesterol, high-density lipoprotein (hdl)-cholesterol, and triglycerides] were evaluated at baseline and at 12, 24, 38, and 52 weeks after administration of linagliptin. changes in uacr were also measured at the same time points. the annual change in egfr (ml/min/1.73 m2/year) was evaluated before and after administration of linagliptin. patients who underwent dialysis therapy because of progression to end-stage renal disease were removed from the study because changes in renal function could not be evaluated. laboratory methods. egfr was calculated using a modified version of the modification of diet in renal disease formula of the japanese society of nephrology: egfr (ml/min/1.73  m2) = 194 × age-0.287 × serum creatinine-1.094 (multiplied by 0.739 for women).25 blood and urinary parameters were determined by the department of clinical laboratory, saitama medical center, jichi medical university. statistical analysis. data are expressed as mean ± standard deviation. repeated-measure analysis of variance was used to compare continuous data. differences with a p-value  ,  0.05 were considered statistically significant. a paired t-test was used to compare the annual egfr change before and after administration of linagliptin. results thirty patients were enrolled in the study and administered linagliptin (fig. 2). four patients were discontinued because figure 1. diagram of study design. abbreviations: wk, week; hba1c, glycosylated hemoglobin; ldl, low-density lipoprotein; hdl, high-density lipoprotein; uacr, urine protein/creatinine ratio; egfr, estimated glomerular filtration rate. http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 effects and safety of linagliptin as an add-on therapy 15drug target insights 2016:10 they progressed to end-stage renal disease and underwent hemodialysis. another two patients were discontinued because of diagnoses of colon cancer and mediastinal tumor during the study period. one patient was lost to follow-up. three patients were removed from the analysis because they were changed to different prescription drugs during the study period. twenty patients completed the study (fig. 2). the baseline characteristics of the analyzed patients who completed the study are listed in table 1. effects of linagliptin on glucose metabolism. linagliptin significantly decreased hba1c levels, but did not change fasting blood glucose levels (fig. 3 and table 2). effects of linagliptin on lipid metabolism and renal function. linagliptin significantly decreased total cholesterol and ldl-cholesterol levels (fig. 4 and table 2), but did not change hdl-cholesterol and triglyceride levels (fig. 4 and table 2); nor did it change uacr and annual egfr (fig. 5 and table 2). changes in other clinical parameters and adverse effects. other clinical and laboratory parameters were not changed by the administration of linagliptin (table 2). no adverse effects, including joint pain, hypoglycemia, severe hyperglycemia, ketosis, or electrolyte abnormalities, were observed in patients administered linagliptin during the study period. discussion in this study, linagliptin as an add-on therapy significantly decreased hba1c and total cholesterol levels in advancedstage dmn patients taking raas blockers. linagliptin administration did not change uacr and annual egfr, nor did it show any adverse effects in the patients. the results suggest that linagliptin has beneficial effects on glucose and lipid metabolism and can be used safely in such populations. linagliptin did not decrease fasting blood glucose levels. it has been reported that linagliptin decreases postprandial glucose levels rather than fasting blood glucose levels because glp-1, increased by linagliptin, is secreted from the small intestine by the stimulation of food.26 these blood glucose lowering mechanisms of linagliptin may explain the finding in the current study that linagliptin decreased hba1c levels but did not decrease fasting blood glucose levels in dmn patients. in addition to the beneficial effects of linagliptin on glucose metabolism, beneficial effects have also been reported on lipid metabolism as well as nephroprotective effects.22–24 although the detailed mechanisms have not been fully determined, linagliptin may improve lipid metabolism and show nephroprotective effects by improving endothelial functions and reducing pro-oxidative and pro-inflammatory signals and inappropriate sympathetic nervous system activation through increasing levels of glp-1 and other ligands.26 previous large-scale, double-blind clinical studies have reported that linagliptin improved glucose metabolism in dmn patients with renal impairment;21,22 however, the effects of linagliptin on lipid metabolism in this population were not studied. in the current study, linagliptin decreased total cholesterol and ldl-cholesterol levels in addition to improving glucose metabolism in advanced-stage dmn patients taking raas blockers. these results suggest that linagliptin has beneficial effects on both lipid metabolism and glucose metabolism in advanced-stage dmn patients. it should be noted that potential drug–drug interactions might have an effect on the results of the current study because the enrolled patients were on different types of drugs to control hyperglycemia and hyperlipidemia. further studies are required to elucidate the figure 2. patient flowchart. table 1. patients’ baseline characteristic. number 20 age (years) 69.7 ± 12.9 gender (male/female) 14/6 ckd stage (number) stage 3 5 stage 4 9 stage 5 6 raas blockers ace 5 arb 20 anti-hyperglycemic drugs biguanide 3 glinides 2 sulfonylurea 1 α-glucosidase inhibitors 6 thiazolidinedione 4 insulin agents 10 hypolipidemic drugs statins 11 ezetimibe 1 fenofibrate 1 abbreviations: ckd, chronic kidney disease; raas blockers, renin– angiotensin–aldosterone system blockers; ace, angiotensin-converting enzyme inhibitors; arb, angiotensin ii-receptor blockers. http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 ueda et al 16 drug target insights 2016:10 mechanisms behind the effects of linagliptin on glucose and lipid metabolism and its interactions with other drugs. it has been reported that linagliptin decreased uacr in the early to middle stages of dmn patients over the course of a 24-week study period.22 another study reported that linagliptin had little effect on renal function in dmn patients with severe renal impairment over a 1-year study period.21 in the current study, the nephroprotective effects of linagliptin were not observed over 52  weeks. these results suggest that linagliptin does not have nephroprotective effects on figure 3. changes in hba1c and fasting blood glucose (all patients, n = 20). note: *p , 0.05 vs baseline. abbreviations: hba1c, glycosylated hemoglobin; ns, not significant. table 2. changes in parameters before and after linagliptin administration. parameter at baseline at 52 weeks after linagliptin administration statistics sbp (mmhg) 141.1 ± 16.6 144.4 ± 19.8 ns dbp (mmhg) 72.5 ± 11.8 77.4 ± 14.4 ns hr (beats/min) 78.1 ± 12.6 78.2 ± 11.0 ns creatinine (mg/dl) 2.5 ± 1.0 3.6 ± 2.2 * egfr (ml/min/1.73 m2) 24.5 ± 13.2 19.5 ± 13.0 ** annual egfr change (ml/min/1.73 m2/years) -6.5 ± 14.2 -4.0 ± 3.8 ns uacr (g/g cr) 2.2 ± 2.6 1.9 ± 2.4 ns hba1c (%) 7.3 ± 0.8 6.9 ± 0.8 * blood glucose (mg/dl) 162.2 ± 54.9 158.7 ± 64.6 ns total cholesterol (mg/dl) 189.6 ± 49.0 168.47 ± 38.7 * ldl-cholesterol (mg/dl) 107.1 ± 32.4 90.2 ± 31.0 * hdl-cholesterol (mg/dl) 40.8 ± 8.4 43.6 ± 12.1 ns triglyceride (mg/dl) 225.7 ± 126.4 208.4 ± 108.0 ns total protein (g/dl) 6.9 ± 0.8 6.8 ± 0.5 ns albumin (g/dl) 3.8 ± 0.6 3.7 ± 0.5 ns sodium (mmol/l) 139.0 ± 2.7 139.5 ± 2.9 ns potassium (mmol/l) 4.6 ± 0.8 4.6 ± 0.6 ns chloride (mmol/l) 105.6 ± 4.8 107.6 ± 3.8 ns calcium (mg/dl) 9.0 ± 0.5 8.5 ± 0.8 * phosphate (mg/dl) 3.7 ± 0.7 4.3 ± 1.3 ns notes: *p , 0.05; **p , 0.01. abbreviations: sbp, systolic blood pressure; dbp, diastolic blood pressure; hr, heart rate; egfr, estimated glomerular filtration rate; ckd, chronic kidney disease; uacr, urine protein/creatinine ratio; hba1c, glycosylated hemoglobin; ldl, low-density lipoprotein; hdl, high-density lipoprotein; ns, not significant. http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 effects and safety of linagliptin as an add-on therapy 17drug target insights 2016:10 advanced-stage dmn patients over the long term. linagliptin may have nephroprotective effects at the early to middle stages of dmn, as previously reported.22 large-scale, long-term clinical studies investigating the nephroprotective effects of linagliptin at each stage of dmn are required. linagliptin did not induce any adverse effects, including joint pain, blood pressure, and electrolyte abnormalities, in the current study’s population, which suggests that linagliptin can be used safely in advanced-stage dmn patients taking raas blockers. this study had some limitations. it was a before–after study without a control group, and the number of patients was small. large-scale, double-blind trials with an appropriate control group are required to investigate the effects of linagliptin on advanced-stage dmn patients taking raas blockers. in conclusion, linagliptin as an add-on therapy significantly decreased hba1c and total cholesterol levels in this population of advanced-stage dmn patients taking raas blockers without showing any adverse effects. our results suggest that linagliptin has beneficial effects on glucose and lipid metabolism and can be used safely in such populations. acknowledgments the authors wish to thank the members of the division of nephrology and the division of endocrinology and metabolism, first department of integrated medicine, saitama medical center, jichi medical university. this study was supported by the division of nephrology, first department of integrated medicine, saitama medical center, jichi medical university. figure 4. changes in total cholesterol, ldl-cholesterol, hdl-cholesterol, and triglyceride (all patients, n = 20). note: *p , 0.05 vs baseline. abbreviations: ldl, low-density lipoprotein; hdl, high-density lipoprotein; ns, not significant. figure 5. changes in uacr and annual egfr change. abbreviations: uacr, urine protein/creatinine ratio; egfr, estimated glomerular filtration rate; ns, not significant. http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 ueda et al 18 drug target insights 2016:10 author contributions conceived and designed the experiments: hi, tk, ms, hm, ki, kh, yk, hm, th, so, mk, kt, and ym. analyzed the data: yu, so, and ym. wrote the first draft of the manuscript: yu. contributed to writing the manuscript: so. agreed with manuscript results and conclusions: hi, tk, ms, hm, ki, kh, yk, hm, th, so, mk, kt, and ym. jointly developed the structure and arguments for the paper: hi, tk, ms, hm, ki, kh, yk, hm, th, so, mk, kt, and ym. made critical revisions and approved the final version: ym. all authors reviewed and approved the final manuscript. r efer ences 1. grassmann a, gioberge s, moeller s, brown g. esrd patients in 2004: global overview of patient numbers, treatment modalities and associated trends. nephrol dial transplant. 2005;20(12):2587–2593. 2. atkins rc. the epidemiology of chronic kidney disease. kidney int suppl. 2005; 67(94):s14–s18. 3. molitch me, defronzo ra, franz mj, et al. nephropathy in diabetes. diabetes care. 2004;27(suppl 1):s79–s83. 4. preis sr, hwang sj, coady s, et al. trends in all-cause and cardiovascular disease mortality among women and men with and without diabetes mellitus in the framingham heart study, 1950 to 2005. circulation. 2009;119(13):1728–1735. 5. ohkubo y, kishikawa h, araki e, et al. intensive insulin therapy prevents the progression of diabetic microvascular complications in japanese patients with non-insulin-dependent diabetes mellitus: a randomized prospective 6-year study. diabetes res clin pract. 1995;28(2):103–117. 6. intensive blood-glucose control with sulphonylureas or insulin compared with conventional treatment and risk of complications in patients with type 2 diabetes (ukpds 33). uk prospective diabetes study (ukpds) group. lancet. 1998; 352(9131):837–853. 7. boussageon r, bejan-angoulvant t, saadatian-elahi m, et al. effect of intensive glucose lowering treatment on all cause mortality, cardiovascular death, and microvascular events in type 2 diabetes: meta-analysis of randomised controlled trials. bmj. 2011;343:d4169. 8. araki s, haneda m, koya d, et al. reduction in microalbuminuria as an integrated indicator for renal and cardiovascular risk reduction in patients with type 2 diabetes. diabetes. 2007;56(6):1727–1730. 9. heerspink hj, ninomiya t, perkovic v, et al. effects of a fixed combination of perindopril and indapamide in patients with type 2 diabetes and chronic kidney disease. eur heart j. 2010;31(23):2888–2896. 10. benedict group. the bergamo nephrologic diabetes complications trial (benedict): design and baseline characteristics. control clin trials. 2003; 24(4):442–461. 11. brenner bm, cooper me, de zeeuw d, et al. effects of losartan on renal and cardiovascular outcomes in patients with type 2 diabetes and nephropathy. n engl j med. 2001;345(12):861–869. 12. chalmers j, joshi r, kengne ap, macmahon s. blood pressure lowering with fixed combination perindopril-indapamide: key findings from advance. j hypertens suppl. 2008;26(2):s11–s15. 13. chahal h, chowdhury ta. gliptins: a new class of oral hypoglycaemic agent. q jm. 2007;100(11):671–677. 14. ahren b. dpp-4 inhibitors. best pract res clin endocrinol metab. 2007;21(4): 517–533. 15. deacon cf, holst jj. linagliptin, a xanthine-based dipeptidyl peptidase-4 inhibitor with an unusual profile for the treatment of type 2 diabetes. expert opin investig drugs. 2010;19(1):133–140. 16. graefe-mody u, friedrich c, port a, et al. effect of renal impairment on the pharmacokinetics of the dipeptidyl peptidase-4 inhibitor linagliptin. diabetes obes metab. 2011;13(10):939–946. 17. laakso m, rosenstock j, groop ph, et al. treatment with the dipeptidyl peptidase-4 inhibitor linagliptin or placebo followed by glimepiride in patients with type 2 diabetes with moderate to severe renal impairment: a 52-week, randomized, double-blind clinical trial. diabetes care. 2015;38(2):e15–e17. 18. owens dr, swallow r, dugi ka, woerle hj. efficacy and safety of linagliptin in persons with type 2 diabetes inadequately controlled by a combination of metformin and sulphonylurea: a 24-week randomized study. diabet med. 2011; 28(11):1352–1361. 19. haak t, meinicke t, jones r, weber s, von eynatten m, woerle hj. initial combination of linagliptin and metformin improves glycaemic control in type 2 diabetes: a randomized, double-blind, placebo-controlled study. diabetes obes metab. 2012;14(6):565–574. 20. schernthaner g, barnett ah, emser a, et al. safety and tolerability of linagliptin: a pooled analysis of data from randomized controlled trials in 3572 patients with type 2 diabetes mellitus. diabetes obes metab. 2012;14(5):470–478. 21. mcgill jb, sloan l, newman j, et al. long-term efficacy and safety of linagliptin in patients with type 2 diabetes and severe renal impairment: a 1-year, randomized, double-blind, placebo-controlled study. diabetes care. 2013;36(2): 237–244. 22. groop ph, cooper me, perkovic v, emser a, woerle hj, von eynatten m. linagliptin lowers albuminuria on top of recommended standard treatment in patients with type 2 diabetes and renal dysfunction. diabetes care. 2013;36(11): 3460–3468. 23. kamatani n, katoh t, sawai y, kanayama h, katada n, itoh m. comparison between the clinical efficacy of linagliptin and sitagliptin. j diabetes endocrinol. 2013;4(4):5. 24. alter ml, ott im, von websky k, et al. dpp-4 inhibition on top of angiotensin receptor blockade offers a new therapeutic approach for diabetic nephropathy. kidney blood press res. 2012;36(1):119–130. 25. matsuo s, imai e, horio m, et al. revised equations for estimated gfr from serum creatinine in japan. am j kidney dis. 2009;53(6):982–992. 26. del prato s, barnett ah, huisman h, neubacher d, woerle hj, dugi ka. effect of linagliptin monotherapy on glycaemic control and markers of beta-cell function in patients with inadequately controlled type 2 diabetes: a randomized controlled trial. diabetes obes metab. 2011;13(3):258–267. http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 dti drug target insights 2022; 16: 49-53issn 1177-3928 | doi: 10.33393/dti.2022.2481original research article drug target insights issn 1177-3928 www.aboutscience.eu/dti © 2022 the authors. this article is published by aboutscience and licensed under creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). commercial use is not permitted and is subject to publisher’s permissions. full information is available at www.aboutscience.eu focus on antimicrobial resistance (amr) success of 14-day triple and quadruple therapy for the control of helicobacter pylori infections in kohat district syed fahim shah1, sohail aziz paracha2, waheed ullah3, iqbal muhammad3, somaid iqbal3, aisha gul3, mudassir hussain3, hafiz ullah3, sadir zaman3 1department of medicine, khyber medical university, peshawar pakistan 2department of anatomy, khyber medical university, peshawar pakistan 3department of microbiology, kohat university of science and technology, kohat pakistan abstract introduction: helicobacter pylori is an important medical pathogen present in more than half of the world’s population. various treatment regimen are in use for the eradication of h. pylori, but due to the emergence of antibiotic resistance, its management is a big issue for clinicians. methods: in this study all suspected cases that had visited district headquarters hospital kohat were considered for screening of h. pylori infections. preliminary information about their age, gender, general health conditions, occupation, etc. was taken for consideration. after recording initial signs and symptoms, samples were considered for h. pylori detection using stool antigen test and endoscopy. fourteen-day proton pump inhibitor base triple and quadruple therapy were administered to each patient. results: in total (n = 178), there were high numbers of positivity in patients aged below 30 years (82; 46.06%), most of whom belonged to rural areas. conclusion: this study concludes that there were high numbers of positive patients aged below 30 years, and according to this study mel (metronidazole + esomeprazole + levofloxacin) is the most effective treatment regimen for the eradication of h. pylori. keywords: gastric pathology, helicobacter pylori, intestinal metaplasia, peptic ulcer received: august 4, 2022 accepted: november 30, 2022 published online: december 19, 2022 corresponding author: dr. waheed ullah associate professor kohat university of science and technology kohat 26000 pakistan waheedwazir@gmail.com (5,6). its invasiveness in the human stomach instigates mucosal and systemic immune responses in the infected host but it has acquired some mechanism that can evade host responses (7). h. pylori infection rates vary by geographic location, age, ethnicity and socioeconomic status of population (8). it has been documented that infection rates are higher in poor socioeconomic conditions, particularly in developing countries (9). its transmission takes place in different ways. most common routes of transmission are iatrogenic, feco-oral and person-to-person contact (10,11). along with this, contaminated food and water may be a source of infection (12). different strategies have been adopted for its treatment. commonly and most acceptable treatment therapies are triple and quadruple therapy (13,14). in recent era due to the emergence of antibiotic resistance, its success has declined. antibiotic resistance to h. pylori is considered the major cause of the eradication failure (15,16). one of the most enduring debates in the world is the optimal duration of therapy for its eradication (17). the incidence of h. pylori eradication failure and antibiotic resistance has been documented worldwide. in pakistan, the rate of h. pylori is very high due to lack of proper diagnosis of dyspepsia and the over-the-counter use of inappropriate introduction helicobacter pylori is a gram negative, microaerophilic bacteria that is very common, infecting more than half of the world’s population (1). h. pylori infection can cause gastric inflammation, peptic ulcer, intestinal metaplasia and can lead to gastric cancer (2,3). its urease activity, flagella mobility, adhesive proteins and s-shape help to colonize the human stomach and initiate infection (4). besides these, the caga and vaca genes are the major virulence factors in h. pylori, responsible for the gastric pathology. the caga gene is responsible for peptic ulcer disease and adenocarcinoma, while the vaga gene causes injury to gastric epithelium https://doi.org/10.33393/dti.2022.2481 https://creativecommons.org/licenses/by-nc/4.0/legalcode mailto:waheedwazir@gmail.com triple and quadruple therapy for the control of helicobacter pylori infections50 © 2022 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti doses of proton pump inhibitors (ppis). in our population of the district of kohat, the rate of h. pylori infection is increasing day by day, and it is yet to find factors that lead to eruption of resistance. therefore, keeping in view the existence data regarding its resistance and treatment failure of h. pylori infection, the study was conducted to determine the incidence of h. pylori infection and its response to different regimen of eradication in kohat district. the aim of the study was to recognize the best treatment regimen for the eradication of h. pylori. methods this study was conducted at the district headquarters hospital kohat and kohat university of science and technology, from april 2021 to december 2021. it was approved by the university ethical committee and written consent was taken from all patients. only positive cases were considered in this study for further analysis. patients’ data collection and risk factors all the patients were asked about the issues related to gastrointestinal problems and their socioeconomic status, and all data were recorded accordingly. a questionnaire was used as the data collection tool, and it was given after obtaining written informed consent. all the patients were also questioned about their gastric information or complaints like nausea, vomiting, epigastric pain and ballottement. sample collection from all suspected cases stool samples (10-20 g) were collected from all the patients and placed in a clean container. stool antigen test was used for the detection of h. pylori infection. blood sera were also collected from each patient. three to 5 ml of blood was taken from each patient and analyzed through an automated hematology analyzer for the complete blood count (18). stool antigen test stool antigen test was performed for the detection of h. pylori infection. about 1 g of collected sample was diluted with the buffer present in the specimen collection tube. suspending diluted sample for 2 min, 2-3 drops of the diluted specimen were added to the well and then waited for the appearance of faint line to read the result (19). treatment regimen for h. pylori eradication three different treatment regimen were used for control of h. pylori positive cases. these were mel (metronidazole + esomeprazole + levofloxacin), mrl (metronidazole + rabeprazole + levofloxacin) and melb (metronidazole + esomeprazole + levofloxacin + bismuth subcitrate) treatment strategies. in the designated study each patient received ppi base triple and bismuth quadruple treatment. all the patients were randomly assigned to the 14-day treatment comprising of esomeprazole 40 mg, rabeprazole 20 mg, metronidazole 500 mg, levofloxacin 500 mg and bismuth subcitrate. ppi and bismuth were recommended to be taken before meals while all the antibiotics were taken after meals. successful eradication was defined as negative result after reconfirmation through the stool antigen test after successful 14-day therapy (20). statistical analysis qualitative and quantitative variables are shown as percentages. the relationship between hematological parameters of h. pylori positive patients and h. pylori negative control group was evaluated using confidential interval method by which the values are calculated for each parameter that will fall between intervals. results in total there were 178 patients positive for h. pylori infection; there were 38.76% (n = 69) female and 61.23% (n = 109) male positive cases. among the total, there were high numbers of positivity (n = 82; 46.06%) in patients aged below 30 years, while the number of positive patients in group aged 30-50 years is 38.76% (n = 69) and in that of above 50 years is 15.16% (n = 27). of the 178 patients, 71.91% (n = 128) were living in the rural area while 28.08% (n = 50) were living in the urban area. according to the above results, high numbers of the patients were living in the rural area and only a small number of patients were living in the urban areas. the demographic information and characteristic of the suspected patients are shown in table i. table i demographic characteristic of positive patients demographic factors numbers percentage (%) age below 30 years 82 46.06 30-50 years 69 38.76 above 50 years 27 15.16 gender male 109 61.23 female 69 38.76 literate 40 22.47 illiterate 138 77.52 place of living rural 128 71.91 urban 50 28.08 gastrointestinal symptoms were almost similar in all the patients, but there were high numbers of patients who complained of epigastric pain and recurrent abdominal pain; all the patients showed more than one symptom as mentioned in table ii. shah et al drug target insights 2022; 16: 51 © 2022 the authors. published by aboutscience www.aboutscience.eu table ii gastrointestinal symptom of patients symptom numbers percentage (%) epigastric pain 153/178 85.95 recurrent abdominal pain 138/178 77.52 nausea 141/178 79.21 vomiting 70/178 39.32 ballottement 30/178 16.85 water brush 65/178 36.51 the hematological parameters of h. pylori patients included (n = 52) h. pylori positive patients whose hematological values were compared with h. pylori negative control group (n = 52). it showed that the hemoglobin level was low in the positive patients. comparing platelets and neutrophile, it was increased in the infected patients as shown in table iii. table iii hematological parameter parameter patients (n = 52) negative control (n = 52) hemoglobin, g/dl 12.82 ± 1.79 14.37 ± 1.06 platelet count, % 304,043 ± 8311.8 223,360 ± 3498 neutrophile 61.32 ± 5.57 59.34 ± 6.91 eosinophile 2.85 ± 0.89 3.22 ± 1.12 monocytes 2.41 ± 0.53 4.29 ± 1.16 lymphocytes 30.36 ± 7.80 27.87 ± 5.97 the above results show the eradication percentage of each given regimen in which 100 patients were given mel, out of whom 88 (88%) patients showed successful eradication (fig. 1). mrl was given to 53 patients, of whom 40 patients recovered, exhibiting 75.47% eradication percentage. the bismuth-based quadruple therapy (melb) was given to 25 patients, of whom 21 (84%) patients showed successful eradication after 14 days of therapy, as shown in table iv. table iv helicobacter pylori eradication percentage between mel, mrl and melb regimen regimen eradication (%) eradication failure mel 88/100 (88%) 12/100 (12%) mrl 40/53 (75.47%) 13/53 (24.52%) melb 21/25 (84%) 4/25 (16%) mel = metronidazole + esomeprazole + levofloxacin; mrl = metronidazole + rabeprazole + levofloxacin; melb = metronidazole + esomeprazole + levofloxacin + bismuth subcitrate discussion h. pylori infection is the most common bacterial infection in the world, infecting about half of the world’s population. this infection is more common in areas where there are poor hygienic conditions such as use of contaminated food and water. this bacterium is mainly transmitted by feco-oral route from the fecal contaminated water. the oral-oral route is also the leading cause of the infection, as few authentic studies have cultured h. pylori from the oral cavity (21); the oral-oral transmission has been examined in the eating of premasticated food, the use of the same spoon by mother and children (22). various diagnostic methods have been identified for the detection of h. pylori but the choice usually depends on the sampling and condition of the patient. in this study, the stool antigen test has been used for the diagnosis of h. pylori as stool antigen test is noninvasive and rapid for the detection of h. pylori infection (23). h. pylori analysis included 178 positive patients in this study in which most of the patients were less than 30 years of age. one of the studies in egypt included 89 asymptomatic young patients, out of whom 78 were positive for h. pylori antigen, all aged below 30 years (24). a total of 128 patients out of 178 belonged to the rural areas, exhibiting high percentage because of the poor hygienic conditions of the people living in the rural areas. most authentic studies have also shown that most of the h. pylori positive patients were from rural areas, where the environment was not hygienic. similarly, one of the studies in venezuela revealed that h. pylori in the rural population was found in 87.2% (34/39) of the patients (25). h. pylori is associated with a number of symptoms that are still in debate. in this study, all the patients complained of gastrointestinal symptoms, but there were high number of patients who complained about epigastric pain and recurrent abdominal pain. the main reason behind this is the hyperacidity during peptic ulcer. primarily, gastrin and oxyntic gland are responsible for the production of more acid during h. pylori infection that can lead to epigastric pain (26). in this study, we found abnormalities in some of the hematological parameters of h. pylori positive patients when compared to the control group. the hemoglobin level is quite lower than the control group, while the platelets and neutrophil level fig. 1 successful eradication and failure percentage between mel, mrl and melb drug combinations. mel = metronidazole + esomeprazole + levofloxacin; mrl = metronidazole + rabeprazole + levofloxacin; melb = metronidazole + esomeprazole + levofloxacin + bismuth subcitrate. triple and quadruple therapy for the control of helicobacter pylori infections52 © 2022 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti remarkably increased. this may be due to the inflammatory conditions and immune response to h. pylori. eradication of h. pylori needs combinations of drug treatment with adjuvant regimen that increase antibiotic activity and host responses. the duration of therapy also strongly affects the eradication of h. pylori. one of the studies in the united states was based on the duration of therapy of h. pylori, which shows that rac (rabeprazole, amoxicillin, and clarithromycin) treatment of 7 days and 10 days had a higher percentage of eradication than the 3-day treatment (27). similarly, a study in turkey based on levofloxacin triple therapy in which mel was given to 92 patients showed 95.5% positive response toward mel combination. in this study, each patient was given 14 days treatment of ppi base triple and quadruple therapy, which show that mel had a high percentage of eradication (88/100; 88%) followed by melb (21/25; 84%). mel and mrl are the same triple therapy with two different ppis (tab. v). the reason for changing one ppi to another was that some of the patients in either group were already using that drug with the same name, so for patient satisfaction, psychologically, we changed the drug. another reason is some of the studies showed better results with rabeprazole than esomeprazole (28). conclusion this study concludes that there were high numbers of positive patients aged below 30 years in which many patients were from rural area, and according to this study mel is the most effective treatment regimen for the eradication of h. pylori. this study recommends that clinicians may suggest mel treatment for h. pylori positive patients for complete eradication of h. pylori. disclosures financial support: this study was part of the basic research with a focus on patients’ treatment and was mainly supported and conducted at district headquarters hospital kohat, pakistan. we extend our gratitude to the department of microbiology, kohat university of science and technology, for providing lab facilities and supporting this research. conflict of interest: the authors declare that they have no conflict of interests. authors’ contribution: syed fahim shah and sohail aziz paracha conducted the experiments; somaid iqbal helped in editing the manuscript and in conducting experimentations; sadar zaman, mudassir hussain, hafeez ullah and iqbal muhammad helped in sample collection; aisha gul helped in experimentations; and waheed ullah designed the project and wrote the manuscript. references 1. hooi jky, lai wy, ng wk, et al. global prevalence of helico­ bacter pylori infection: systematic review and meta-analysis. gastroenterology. 2017;153(2):420-429. crossref pubmed 2. eshraghian a. epidemiology of helicobacter pylori infection among the healthy population in iran and countries of the eastern mediterranean region: a systematic review of prevalence and risk factors. world j gastroenterol. 2014;20(46):17618-17625. crossref pubmed 3. qadri q, rasool r, gulzar gm, naqash s, shah zah. h. pylori infection, inflammation and gastric cancer. j gastrointest cancer. 2014;45(2):126-132. crossref pubmed 4. scott dr, marcus ea, wen y, oh j, sachs g. gene expression in vivo shows that helicobacter pylori colonizes an acidic niche on the gastric surface. proc natl acad sci usa. 2007;104(17):72357240. crossref pubmed 5. al-khattaf as. helicobacter pylori virulence markers in gastroduodenal disorders. detection of cytotoxin-associated gene a and vacuolating cytotoxin-associated gene a genes in saudi patients. saudi med j. 2012;33(7):716-721. pubmed 6. ribeiro ml, godoy apo, benvengo yhb, mendonça s, pedrazzoli j jr. clinical relevance of the caga, vaca and icea genotypes of helicobacter pylori in brazilian clinical isolates. fems immunol med microbiol. 2003;36(3):181-185. crossref pubmed 7. abadi atb. strategies used by helicobacter pylori to establish persistent infection. world j gastroenterol. 2017;23(16):28702882. crossref pubmed 8. omosor ki, omosor oh, ibeh in, et al. seroprevalence of helicobacter pylori infection and risk factors among table v comparison of helicobacter pylori eradication regimen (mel, mrl and melb) patient details treatment 1 (mel) treatment 2 (mrl) treatment 3 (melb) success % failure % success % failure% success % failure% 1. male (n = 109) 57 (90.47%) 6 (9.53 = 2%) 17 (70.83%) 7 (30.43%) 20 (90.90%) 2 (9.09%) 2. female (n = 69) 31 (83.78%) 6 (16.21%) 23 (79.31%) 6 (20.68%) 1 (33.3%) 2 (66.6%) success of treatment regimen according to age 3. below 30 years (n = 82) 45 (84.90%) 8 (15.09%) 12 (63.15%) 7 (36.84%) 8 (80%) 2 (20%) 4. 30-50 years (n = 69) 31 (91.17%) 3 (8.82%) 20 (83.33%) 4 (16.66%) 9 (81.81%) 2 (18.18%) 5. after 50 years (n = 27) 12 (92.30%) 1 (7.69%) 8 (80%) 2 (20%) 4 (100%) 0 (0%) resident wise 6. rural (n = 128) 60 (86.95%) 9 (13.04%) 32 (76.19%) 10 (23.80%) 14 (82.35%) 3 (17.64%) 7. urban (n = 50) 28 (90.32%) 3 (9.67%) 8 (72.72%) 3 (27.27%) 7 (87.5%) 1 (12.5%) total 178 88/100 (88%) 12/100 (13.72%) 40/53 (75.47%) 13/53 (24.52%) 21/25 (84%) 4/25 (16%) mel = metronidazole + esomeprazole + levofloxacin; mrl = metronidazole + rabeprazole + levofloxacin; melb = metronidazole + esomeprazole + levofloxacin + bismuth subcitrate https://doi.org/10.1053/j.gastro.2017.04.022 https://www.ncbi.nlm.nih.gov/pubmed/28456631 https://doi.org/10.3748/wjg.v20.i46.17618 https://www.ncbi.nlm.nih.gov/pubmed/25516677 https://doi.org/10.1007/s12029-014-9583-1 https://www.ncbi.nlm.nih.gov/pubmed/24557546 https://doi.org/10.1073/pnas.0702300104 https://www.ncbi.nlm.nih.gov/pubmed/17438279 https://www.ncbi.nlm.nih.gov/pubmed/22821304 https://doi.org/10.1016/s0928-8244(03)00029-4 https://www.ncbi.nlm.nih.gov/pubmed/12738389 https://doi.org/10.3748/wjg.v23.i16.2870 https://www.ncbi.nlm.nih.gov/pubmed/28522905 shah et al drug target insights 2022; 16: 53 © 2022 the authors. published by aboutscience www.aboutscience.eu asymptomatic subjects in delta state, nigeria. adv microbiol. 2017;7(9):641-652. crossref 9. zamani m, ebrahimtabar f, zamani v, et al. systematic review with meta-analysis: the worldwide prevalence of helicobacter pylori infection. aliment pharmacol ther. 2018;47(7):868-876. crossref pubmed 10. brown lm. helicobacter pylori: epidemiology and routes of transmission. epidemiol rev. 2000;22(2):283-297. crossref pubmed 11. rothenbacher d, bode g, berg g, et al. helicobacter pylori among preschool children and their parents: evidence of parent-child transmission. j infect dis. 1999;179(2):398-402. crossref pubmed 12. nurgalieva zz, malaty hm, graham dy, et al. helicobacter pylori infection in kazakhstan: effect of water source and household hygiene. am j trop med hyg. 2002;67(2):201-206. crossref pubmed 13. xie y, zhu y, zhou, h, et al. furazolidone-based triple and quadruple eradication therapy for helicobacter pylori infection. world j gastroenterol. 2014;20(32):11415-11421. crossref pubmed 14. gisbert jp, romano m, gravina ag, et al. helicobacter pylori second-line rescue therapy with levofloxacinand bismuthcontaining quadruple therapy, after failure of standard triple or non-bismuth quadruple treatments. aliment pharmacol ther. 2015;41(8):768-775. crossref pubmed 15. zhong z, zhang z, wang j, et al. a retrospective study of the antibiotic-resistant phenotypes and genotypes of helicobacter pylori strains in china. am j cancer res. 2021;11(10):50275037. pubmed 16. saracino im, pavoni m, zullo a, et al. antibiotic resistance and therapy outcome in h. pylori eradication failure patients. antibiotics (basel). 2020;9(3):121. crossref pubmed 17. alruthia y, almadi ma, alqahtani s, alrasheed h, al-owairdhi m, alsohaibani f. the cost-effectiveness of sequential versus standard triple therapy for helicobacter pylori eradication in saudi arabia. saudi j gastroenterol. 2021 jul-aug;27(4):217-222. crossref pubmed 18. hsu pi, tsay fw, kao jy, et al; taiwan acid-related disease, microbiota (tard-m) consortium. tetracycline-levofloxacin versus amoxicillin-levofloxacin quadruple therapies in the second-line treatment of helicobacter pylori infection. helicobacter. 2021;26(5):e12840. crossref pubmed 19. iranikhah a, ghadir mr, sarkeshikian s, saneian h, heiari a, mahvari m. stool antigen tests for the detection of helicobacter pylori in children. iran j pediatr. 2013;23(2):138-142. pubmed 20. sun q, liang x, zheng q, et al. high efficacy of 14-day triple therapy-based, bismuth-containing quadruple therapy for initial helicobacter pylori eradication. helicobacter. 2010;15(3): 233-238. crossref pubmed 21. wang xm, yee kc, hazeki-taylor n, et al. oral helicobacter pylori, its relationship to successful eradication of gastric h. pylori and saliva culture confirmation. j physiol pharmacol. 2014;65(4):559-566. pubmed 22. fujimoto y, furusyo n, toyoda k, takeoka h, sawayama y, hayashi j. intrafamilial transmission of helicobacter pylori among the population of endemic areas in japan. helicobacter. 2007;12(2):170-176. crossref pubmed 23. kouitcheu mabeku lb, bello epesse m, fotsing s, et al. stool antigen testing, a reliable noninvasive method of assessment of helicobacter pylori infection among patients with gastroduodenal disorders in cameroon. dig dis sci. 2021;66(2):511520. crossref pubmed 24. salem oe, youssri ah, mohammad on. the prevalence of h. pylori antibodies in asymptomatic young egyptian persons. j egypt public health assoc. 1993;68(3-4):333-352. pubmed 25. contreras m, fernández-delgado m, reyes n, garcía-amado ma, rojas h, michelangeli f. helicobacter pylori infection in rural and urban dyspeptic patients from venezuela. am j trop med hyg. 2015;93(4):730-732. crossref pubmed 26. waldum hl, kleveland pm, sørdal øf. helicobacter pylori and gastric acid: an intimate and reciprocal relationship. therap adv gastroenterol. 2016;9(6):836-844. crossref pubmed 27. vakil n, lanza f, schwartz h, barth j. seven-day therapy for helicobacter pylori in the united states. aliment pharmacol ther. 2004;20(1):99-107. crossref pubmed 28. fock km, teo ek, ang tl, chua ts, ng tm, tan yl. rabeprazole vs esomeprazole in non-erosive gastro-esophageal reflux disease: a randomized, double-blind study in urban asia. world j gastroenterol. 2005;11(20):3091-3098. crossref pubmed https://doi.org/10.4236/aim.2017.79050 https://doi.org/10.1111/apt.14561 https://www.ncbi.nlm.nih.gov/pubmed/29430669 https://doi.org/10.1093/oxfordjournals.epirev.a018040 https://www.ncbi.nlm.nih.gov/pubmed/11218379 https://doi.org/10.1086/314595 https://www.ncbi.nlm.nih.gov/pubmed/9878024 https://doi.org/10.3748/wjg.v20.i32.11415 https://www.ncbi.nlm.nih.gov/pubmed/12389948 https://doi.org/10.3748/wjg.v20.i32.11415 https://pubmed.ncbi.nlm.nih.gov/25170230/ https://doi.org/10.1111/apt.13128 https://www.ncbi.nlm.nih.gov/pubmed/25703120 https://www.ncbi.nlm.nih.gov/pubmed/34765309 https://doi.org/10.3390/antibiotics9030121 https://www.ncbi.nlm.nih.gov/pubmed/32183165 https://doi.org/10.4103/sjg.sjg_536_20 https://pubmed.ncbi.nlm.nih.gov/34259193/ https://doi.org/10.1111/hel.12840 https://www.ncbi.nlm.nih.gov/pubmed/34390083 https://www.ncbi.nlm.nih.gov/pubmed/23724172 https://doi.org/10.1111/j.1523-5378.2010.00758.x https://www.ncbi.nlm.nih.gov/pubmed/20557366 https://www.ncbi.nlm.nih.gov/pubmed/25179088 https://doi.org/10.1111/j.1523-5378.2007.00488.x https://www.ncbi.nlm.nih.gov/pubmed/17309755 https://doi.org/10.1007/s10620-020-06219-0 https://www.ncbi.nlm.nih.gov/pubmed/32350723 https://www.ncbi.nlm.nih.gov/pubmed/17265652 https://doi.org/10.4269/ajtmh.15-0021 https://www.ncbi.nlm.nih.gov/pubmed/26195456 https://doi.org/10.1177/1756283x16663395 https://www.ncbi.nlm.nih.gov/pubmed/27803738 https://doi.org/10.1111/j.1365-2036.2004.02029.x https://www.ncbi.nlm.nih.gov/pubmed/15225176 https://doi.org/10.3748/wjg.v11.i20.3091 https://www.ncbi.nlm.nih.gov/pubmed/15918196 dti drug target insights 2022; 16: 6-11issn 1177-3928 | doi: 10.33393/dti.2022.2343original research article drug target insights issn 1177-3928 www.aboutscience.eu/dti © 2022 the authors. this article is published by aboutscience and licensed under creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). commercial use is not permitted and is subject to publisher’s permissions. full information is available at www.aboutscience.eu anatomical and functional responses to single brolucizumab injection in neovascular age-related macular degeneration patients not responding to antiangiogenics: a case series silvio zuccarini, fabrizio puce, alessandro crisà villa donatello hospital, department of ophthalmology, florence italy abstract introduction: neovascular age-related macular degeneration (namd) is treated with antivascular endothelial growth factor (anti-vegf) drugs. however, resistance to anti-vegf therapy is observed in some patients. brolucizumab is a new-generation anti-vegf drug for the treatment of namd, with proven efficacy in fluid resolution and long-lasting effects. methods: we report here a case series of namd patients not responding to previous anti-vegf therapy showing anatomical and functional response to a single intravitreal injection of brolucizumab. results: nine patients with namd, undergoing treatment with anti-vegf therapy (aflibercept, bevacizumab, or ranibizumab) but with either fluid persistence or frequent fluid recurrences in retinal compartments, were switched to intravitreal brolucizumab and examined 4 weeks postinjection. no signs of active disease were observed in all but one patient, with complete retinal fluid resolution in seven patients. central macular thickness and visual acuity significantly improved, and changes were sustained for up to 12 weeks in a subset of three patients. no adverse reactions were observed. conclusions: this new anti-vegf drug showed great efficacy since the first week from the injection with a significative reduction of subretinal fluid and rapid improvement of visual acuity. in conclusion, brolucizumab administered intravitreally appears to be an effective treatment in namd patients, leading to both early anatomical and functional improvements. keywords: anti-vegf, brolucizumab, case series, neovascular age-related macular degeneration (namd) received: september 24, 2021 accepted: march 16, 2022 published online: march 24, 2022 corresponding author: silvio zuccarini villa donatello hospital via attilio ragionieri, 101 50019, sesto fiorentino, firenze italy silvio.zuccarini@virgilio.it (i.e., intraretinal fluid [irf], subretinal fluid [srf], or fluid accumulating in the subretinal pigment epithelium [sub-rpe] space) are used as biomarkers for disease activity. treatment of namd is based on the use of antivascular endothelial growth factor (anti-vegf) drugs, with the objective of controlling the exudation from the vessels to minimize disease activity and consequently avoid va loss (3,4). appropriate treatment intervals maintain and sometimes improve patients’ vision in the long term (3,5-9). macular neovascularization secondary to namd requires continuous treatment because it remains active for years (10). randomized controlled trials showed that srf and/or irf is still present after two years of treatment in around 40%-50% of patients (3,11,12). long-term retrospective observational studies have also reported around 40% of patients with detectable active disease at the end of the study, after 3 and 10 years, respectively (9,13). the activity pattern of namd is often unpredictable and patients must be continuously monitored and treated to avoid permanent va loss introduction neovascular age-related macular degeneration (namd) is a chronic degenerative disease characterized by the pathological growth of vessels, which leak blood and fluids in the various retinal compartments, leading to loss of visual acuity (va) (1,2). fluids accumulating in retinal compartments https://doi.org/10.33393/dti.2022.2343 https://creativecommons.org/licenses/by-nc/4.0/legalcode zuccarini et al drug target insights 2022; 16: 7 © 2022 the authors. published by aboutscience www.aboutscience.eu (14-16), thus representing an important burden for both patients and clinicians (4,17). in clinical practice, patients are treated in a personalized way with the aim of controlling recurrences, but over time anti-vegf therapies may experience reduced effectiveness: after 5 and 10 years of treatment, 41% and 42% of patients, respectively, made at least one switch to other anti-vegfs (9,18). the poor response (with either fluid persistence or recurrences) may be due to tachyphylaxis, changes in the neovascular membrane characteristics (e.g., increased fibrosis acting like a resorption barrier), chronic changes in vessel walls, changes in the type of lesion evolving over time, or an inadequate treatment frequency (19,20). as a consequence, switching treatment becomes necessary and it is important to re-establish control of the disease. while va is not usually regained, treatment switch is consistently associated with anatomical improvement (20,21). in patients showing fluid persistence despite at least three-monthly injections with bevacizumab, switching to either aflibercept or ranibizumab showed comparable efficacy, with anatomical improvement noticeable after a single injection (22). brolucizumab 6 mg (beovu®, novartis ag, basel, switzerland), a monoclonal single-chain variable domain antibody fragment, is a new-generation anti-vegf drug approved for the treatment of namd, with proven efficacy in fluid resolution and long-lasting effects (23). brolucizumab shows great promise in reducing the risk of undertreatment and permanent va loss. results from the hawk and harrier studies (24) showed that patients treated with brolucizumab (administered at 8and 12-week intervals after three-monthly loading doses) had a significantly higher reduction in retinal central subfield thickness (cst) and absence of srf/irf at 48 and 95 weeks postinjection, compared with those treated with aflibercept (23). these data also showed that disease activity was controlled in a shorter timeframe after diagnosis when using brolucizumab compared with aflibercept (24). the use of brolucizumab in patients showing no response to other anti-vegfs offers a valuable therapeutic option to maintain disease control and va while reducing treatment burden. preliminary observations on patients switched to brolucizumab have recently been published (25,26), confirming its efficacy in controlling fluid accumulation. however, more data are needed to further characterize brolucizumab efficacy and rapidity in obtaining complete fluid resolution and the impact on functional outcomes. we report here the anatomical and functional responses observed in a group of namd patients with persistent or recurrent fluids 1 month after a single injection of brolucizumab, as assessed retrospectively in a center in italy. materials and methods study design we retrospectively analyzed data from nine patients with namd who were switched to brolucizumab 6 mg because they were not responsive to other anti-vegfs. all recruited patients were followed and treated at villa donatello hospital, florence, italy. the use of brolucizumab for treating namd was officially introduced in october 2020. patient demographic and clinical information collected at baseline (i.e., before the brolucizumab injection) included: age; gender; treatment prior to brolucizumab injection (anti-vegf used, treatment duration, number of injections, last interval between injections, time from last injection to the first brolucizumab injection); va; cst; type of lesion; and the presence of irf, srf, or sub-rpe fluid, fibrosis, subretinal fibrosis, and subretinal hyperreflective material (shrm). following the first brolucizumab injection, patients were examined at two time points (t1 = 1 ± 1 weeks and t2 = 4 ± 1 weeks) to determine the presence of anatomical and functional changes using slit-lamp ophthalmoscopy, spectral domain optical coherence tomography (sd-oct), fundus examination, and va assessment (etdrs scoring). three patients had further assessments at a longer follow-up. due to the possibility of intraocular inflammation (ioi), vasculitis, or retinal vascular occlusion, all patients were informed and instructed to report, within 24 hours of the injection, any of the following symptoms: redness, reduction of viscous, floaters, eye pain or pressure, scotoma, or blurred vision. a notification letter was sent to the local ethical committee, according to italian regulations. all patients provided written informed consent prior to receiving the brolucizumab injection. patient inclusion and exclusion criteria the study only included patients with namd not responsive anymore to other anti-vegfs (i.e., showing either fluid persistence or frequent fluid recurrences), who were first injected with brolucizumab by december 2020, and had a visit 1 month (t2 range 4±1 weeks) after the injection. patients with a maculopathy other than namd or who received brolucizumab injection after december 2020 were excluded. study outcomes the main outcome measured was the proportion of patients without srf and irf 1 month after brolucizumab injection. secondary outcomes were the proportion of patients with reduced (or absent) srf/irf 1 month after injection, the proportion of sub-rpe fluid resolution 1 month after injection in patients with sub-rpe fluid at baseline, the proportion of patients without active disease 1 month after injection, the mean time to first disease inactivation, and the mean difference of cst and va between baseline and 1-month postinjection. statistical analysis descriptive statistics were used to summarize demographic and clinical data at baseline and successive time points, as appropriate. continuous data were described using the mean, standard deviation (sd), and range, while categorical data were expressed as percentages. the difference in cst and va between baseline and the assessment at 1 month was tested using the nonparametric wilcoxon single brolucizumab intravitreal injection for namd treatment8 © 2022 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti signed-rank test, with statistical significance set at p < 0.05. the time to first disease inactivation was determined using kaplan-meier survival analysis using the two available observation time points, t1 and t2. analyses were performed using ibm® spss® version 26. results patient characteristics at baseline a total of nine patients were eligible and included in the study. none of the patients had signs of ioi at baseline or study end. patient demographics and clinical and treatment history at baseline are summarized in table i. before switching to brolucizumab, six patients were treated with a single anti-vegf: aflibercept (n = 2) or bevacizumab (n = 2) or ranibizumab (n = 2). the remaining patients switched from aflibercept to bevacizumab (n = 1), from bevacizumab to aflibercept (n = 1), and from bevacizumab to ranibizumab to aflibercept (n = 1). anatomical and functional outcomes at 1-month postinjection patients were assessed within 1 week and 1 month (mean 29.1 ± 4.6 days, range 24-35) after the first brolucizumab injection. no signs of active disease were observed in eight (88.9%) patients: in seven patients (77.8%) srf and irf were absent, while in one patient (11.1%) fluid was only partially reabsorbed. all three patients presenting with sub-rpe fluid at baseline showed complete resolution. the cst showed a significant reduction from 398.4 (97.5 sd) μm at baseline to 258.3 (32.4 sd) μm at 1 month postinjection (mean difference 131.1 μm, 95% confidence interval [ci] 46.4-215.8; related-samples wilcoxon signed-rank test p = 0.007). in parallel, va improved significantly from 54.4 (20.1 sd) letters at baseline to 72.8 (16 sd) letters at 1 month postinjection (mean difference 18.3 letters, 95% ci 6.64-30.02; related-samples wilcoxon signed-rank test p = 0.011). the estimated mean and median time to disease inactivation were 14.2 (range 5-23.4) days and 3 (range 2.03-3.97) days, respectively (shown in fig. 1). in the three patients who were further examined at 60, 62, and 92 days, respectively, from the injection, no sign of disease reactivation or fluid recurrence was observed, and the va remained stable; thus, there was no need for additional injections. as a case example, the retinal sd-oct b-scans of one of the patients at different time points following brolucizumab injection are shown in figure 2. ioi, vasculitis, or retinal vascular occlusion was not reported for any patient in the follow-up period under consideration. discussion significant anatomical improvements were observed in patients switching to brolucizumab, as expected according to the available data (25,27,28). these included a significant reduction in cst and almost complete resolution of fluids in most patients. although the most frequent timepoint used to evaluate the postinjection anatomical response is approximately 1 month, in the case of no response it would be useful to have an earlier assessment since there is a correlation between cst reduction and the time passed from the last injection (29,30). in the group of patients we described, the response to brolucizumab treatment was rapid according to anatomical improvement, which occurred on average 2 weeks after the injection. furthermore, in the subgroup of patients followed for more than 4 weeks, the improvements were stable for at least 8 to 12 weeks after injection. despite these results being obtained in non-treatment-naïve patients, they seem to be in line with those from the hawk and harrier studies, which showed that namd was controlled for at least 12 weeks in more than 50% of naïve patients injected with brolucizumab (24). such long-term effects suggest patients may be stabilized with an interval of 12 weeks between injections, thus table i baseline characteristics characteristic data age (years), mean (sd; range) 77.2 (11.6; 58-90) female gender, no. (%) 7 (77.8) clinical profile, mean (sd) va (letters) 54.4 (20.1) cst (μm) 386.4 (97.5) angiographic lesion type, no. (%) 1 4 (44.4) 2 5 (55.6) with irf 6 (66.7) with srf 7 (77.8) with sub-rpe fluid 3 (33.3) with fibrosis 3 (33.3) with subretinal fibrosis 1 (11.1) with shrm 1 (11.1) anti-vegf treatment prior to brolucizumab injection, mean (sd; min-max) treatment length (months) 9.9 (5.8; 3-18) no. of injections 5.7 (3.5; 2-12) last administration interval (months) 2 (0.7; 1-3) months from last injection to brolucizumab switch 3.1 (1.2; 2-6) reason for switching to brolucizumab, no. (%) fluid persistence 4 (44.4) frequent fluid recurrences 5 (55.6) µm = micrometers; cst = retinal central subfield thickness; irf = intraretinal fluid; max = maximum; min = minimum; no. = number; rpe = retinal pigment epithelium; sd = standard deviation; shrm = subretinal hyperreflective material; srf = subretinal fluid; va = visual acuity; vegf = vascular endothelial growth factor. zuccarini et al drug target insights 2022; 16: 9 © 2022 the authors. published by aboutscience www.aboutscience.eu constituting a significant improvement of treatment burden. this is also clearer considering that retinal fluid was not successfully controlled with injections every 8 weeks before the switch to brolucizumab. in our group of patients, we also observed a significant improvement in terms of va. this appears to contradict a recent case series of six patients who did not show any increase in va (26), and a retrospective analysis of 172 eyes, in which there was no improvement in va after one to three brolucizumab injections (28). there are two possible explanations for our observation: the low number of cases in this case series, and the fact that our patients were treated for a shorter follow-up compared with the other studies (9.9 months and at least 2 years, respectively). no inflammatory adverse events were reported in our patients, which may also be due to the small sample size and short follow-up. an independent safety review committee, analyzing inflammatory ocular adverse events in the hawk and harrier trials, observed an incidence of retinal vasculitis and retinal vascular occlusion of 3% and 2%, respectively, with a risk of severe visual loss in approximately 1 in every 200 patients (31). in this context, it is important to consider the balance between the risk of adverse events and the benefits. in our case fig. 1 kaplan-meier plot for time to disease inactivation in the nine patients. the median (50th percentile) is indicated in red. 0 0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1,0 42 86 1210 1614 2018 2422 2826 30 3432 3836 4240 c um ul at iv e in ci de nc e of in ac tiv e di se as e days from injection fig. 2 representative case of a woman, 90 years old, treated with three intravitreal injections of aflibercept during the previous 9 months. spectral domain optical coherence tomography (sd-oct) b-scans at different time points: a) baseline (before brolucizumab injection). b) follow-up 1 month after injection. c) follow-up 2 months after injection. single brolucizumab intravitreal injection for namd treatment10 © 2022 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti series, the decision to initiate brolucizumab treatment was made for patients who were not responding anymore to other anti-vegfs despite regular treatment. notwithstanding, close contact with the patient is needed during follow-up, as it is expected that adverse inflammatory events can be successfully managed if they are recognized and treated promptly. this retrospective case series has some limitations. despite the important improvements observed, the sample size was small, and the measured changes had wide ranges. to better characterize the long-term efficacy and safety profile of brolucizumab in clinical practice, patients undergoing a treatment switch to brolucizumab should be followed for longer. furthermore, naïve patients not undergoing treatment with other anti-vegfs should be recruited to verify the anatomical superiority of brolucizumab and to optimize the treatment of namd by reducing the treatment burden for both patients and clinicians (24,25). conclusion in conclusion, a single injection of brolucizumab was shown to be rapid and effective in fluid resolution, thus representing a valuable therapeutic option for controlling disease activity in namd patients. indeed, the first month after the injection was shown to be very important to predict the response to the anti-vegf drug of the single patient and brolucizumab showed a rapid and intense response. acknowledgments we thank gabriele massimetti, department of clinical and experimental medicine, university of pisa, pisa, for the statistical support. medical writing support was provided by corrado minetti on behalf of health publishing & services srl, which was funded by novartis farma spa. disclosures statement of ethics: this retrospective study was conducted in accordance with the ethical standards set forth in the 1964 declaration of helsinki and its subsequent amendments. a notification letter was sent to the local ethical committee (comitato etico area vasta centro at the careggi university hospital, florence, italy), as required by italian regulations. written informed consent for both medical treatment and publications of their data was obtained from all participants. conflict of interest: the authors have no conflicts of interest to declare. funding sources: this manuscript did not receive any funding. author contributions: study design: silvio zuccarini. data collection: fabrizio puce, alessandro crisà. all authors contributed to writing, reviewing, and editing the manuscript. data availability statement: data generated or analyzed during this study are included in this article. further enquiries can be directed to the corresponding author. references 1. grossniklaus he, green wr. choroidal neovascularization. am j ophthalmol. 2004;137(3):496-503. crossref pubmed 2. spaide rf, jaffe gj, sarraf d, et al. consensus nomenclature for reporting neovascular age-related macular degeneration data: consensus on neovascular age-related macular degeneration nomenclature study group. ophthalmology. 2020; 127(5):616-636. crossref pubmed 3. schmidt-erfurth u, waldstein sm. a paradigm shift in imaging biomarkers in neovascular age-related macular degeneration. prog retin eye res. 2016;50:1-24. crossref pubmed 4. wykoff cc, clark wl, nielsen js, brill jv, greene ls, heggen cl. optimizing anti-vegf treatment outcomes for patients with neovascular age-related macular degeneration. j manag care spec pharm. 2018 feb;24(2-a suppl):s3-s15. crossref pubmed 5. rasmussen a, brandi s, fuchs j, et al. visual outcomes in relation to time to treatment in neovascular age-related macular degeneration. acta ophthalmol. 2015;93(7):616-620. crossref pubmed 6. real jp, granero ge, de santis mo, et al. rate of vision loss in neovascular age-related macular degeneration explored. graefes arch clin exp ophthalmol. 2015;253(11):1859-1865. crossref pubmed 7. arnold jj, markey cm, kurstjens np, guymer rh. the role of sub-retinal fluid in determining treatment outcomes in patients with neovascular age-related macular degeneration—a phase iv randomised clinical trial with ranibizumab: the fluid study. bmc ophthalmol. 2016;16(1):31. crossref pubmed 8. lanzetta p, loewenstein a; vision academy steering committee. fundamental principles of an anti-vegf treatment regimen: optimal application of intravitreal anti-vascular endothelial growth factor therapy of macular diseases. graefes arch clin exp ophthalmol. 2017;255(7):1259-1273. crossref pubmed 9. gillies m, arnold j, bhandari s, et al. ten-year treatment outcomes of neovascular age-related macular degeneration from two regions. am j ophthalmol. 2020;210:116-124. crossref pubmed 10. chandra s, arpa c, menon d, et al. ten-year outcomes of antivascular endothelial growth factor therapy in neovascular age-related macular degeneration. eye (lond). 2020;34(10):1888-1896. crossref pubmed 11. heier js, brown dm, chong v, et al; view 1 and view 2 study groups. intravitreal aflibercept (vegf trap-eye) in wet agerelated macular degeneration. ophthalmology. 2012;119(12): 2537-2548. crossref pubmed 12. guymer rh, markey cm, mcallister il, et al; fluid investigators. tolerating subretinal fluid in neovascular age-related macular degeneration treated with ranibizumab using a treatand-extend regimen: fluid study 24-month results. ophthalmology. 2019;126(5):723-734. crossref pubmed 13. bhandari s, nguyen v, arnold j, et al. treatment outcomes of ranibizumab versus aflibercept for neovascular age-related macular degeneration: data from the fight retinal blindness! registry. ophthalmology. 2020;127(3):369-376. crossref pubmed 14. essex rw, nguyen v, walton r, et al; fight retinal blindness study group. treatment patterns and visual outcomes during the maintenance phase of treat-and-extend therapy for age-related macular degeneration. ophthalmology. 2016; 123(11):2393-2400. crossref pubmed 15. nguyen v, vaze a, fraser-bell s, et al; fight retinal blindness! study group. outcomes of suspending vegf inhibitors for neovascular age-related macular degeneration when lesions have been inactive for 3 months. ophthalmol retina. 2019;3(8): 623-628. crossref pubmed 16. rusakevich am, zhou b, wong tp, wykoff cc. quarterly antivascular endothelial growth factor dosing for neovascular age-related macular degeneration: real-world clinical outcomes. ophthalmic surg lasers imaging retina. 2019;50(9): e250-e256. crossref pubmed https://doi.org/10.1016/j.ajo.2003.09.042 https://www.ncbi.nlm.nih.gov/pubmed/15013874 https://doi.org/10.1016/j.ophtha.2019.11.004 https://www.ncbi.nlm.nih.gov/pubmed/31864668 https://doi.org/10.1016/j.preteyeres.2015.07.007 https://www.ncbi.nlm.nih.gov/pubmed/26307399 https://doi.org/10.18553/jmcp.2018.24.2-a.s3 https://pubmed.ncbi.nlm.nih.gov/29383980/ https://doi.org/10.1111/aos.12781 https://www.ncbi.nlm.nih.gov/pubmed/26073051 https://doi.org/10.1007/s00417-014-2885-y https://www.ncbi.nlm.nih.gov/pubmed/25491161 https://doi.org/10.1186/s12886-016-0207-3 https://www.ncbi.nlm.nih.gov/pubmed/27009515 https://doi.org/10.1007/s00417-017-3647-4 https://www.ncbi.nlm.nih.gov/pubmed/28527040 https://doi.org/10.1016/j.ajo.2019.10.007 https://www.ncbi.nlm.nih.gov/pubmed/31606444 https://doi.org/10.1038/s41433-020-0764-9 https://www.ncbi.nlm.nih.gov/pubmed/31980748 https://doi.org/10.1016/j.ophtha.2012.09.006 https://www.ncbi.nlm.nih.gov/pubmed/23084240 https://doi.org/10.1016/j.ophtha.2018.11.025 https://www.ncbi.nlm.nih.gov/pubmed/30502372 https://doi.org/10.1016/j.ophtha.2019.10.006 https://www.ncbi.nlm.nih.gov/pubmed/31757494 https://doi.org/10.1016/j.ophtha.2016.07.012 https://www.ncbi.nlm.nih.gov/pubmed/27554034 https://doi.org/10.1016/j.oret.2019.05.013 https://www.ncbi.nlm.nih.gov/pubmed/31281103 https://doi.org/10.3928/23258160-20190905-17 https://www.ncbi.nlm.nih.gov/pubmed/31589766 zuccarini et al drug target insights 2022; 16: 11 © 2022 the authors. published by aboutscience www.aboutscience.eu 17. monés j, singh rp, bandello f, souied e, liu x, gale r. undertreatment of neovascular age-related macular degeneration after 10 years of anti-vascular endothelial growth factor therapy in the real world: the need for a change of mindset. ophthalmologica. 2020;243(1):1-8. crossref pubmed 18. gillies mc, campain a, barthelmes d, et al; fight retinal blindness study group. long-term outcomes of treatment of neovascular age-related macular degeneration: data from an observational study. ophthalmology. 2015;122(9):1837-1845. crossref pubmed 19. binder s. loss of reactivity in intravitreal anti-vegf therapy: tachyphylaxis or tolerance? br j ophthalmol. 2012;96(1):1-2. crossref pubmed 20. chakravarthy u, bezlyak v, sagkriotis a, et al. effectiveness of continued ranibizumab therapy in neovascular age-related macular degeneration versus switch to aflibercept: real world evidence. ophthalmol retina. 2019;3(1):8-15.e1. crossref pubmed 21. nguyen cl, gillies mc, nguyen v, et al; fight retinal blindness! study group. characterization of poor visual outcomes of neovascular age-related macular degeneration treated with antivascular endothelial growth factor agents. ophthalmology. 2019;126(5):735-742. crossref pubmed 22. salcedo-villanueva g, feria-anzaldo e, romo-aguas jc, et al. anti-vegf treatment switch in neovascular age-related macular degeneration: a comparison of aflibercept versus ranibizumab after a single-dose switch. int ophthalmol. 2019;39(9):20232031. crossref pubmed 23. dugel pu, singh rp, koh a, et al. hawk and harrier: ninetysix-week outcomes from the phase 3 trials of brolucizumab for neovascular age-related macular degeneration. ophthalmology. 2021;128(1):89-99. crossref pubmed 24. dugel pu, koh a, ogura y, et al; hawk and harrier study investigators. hawk and harrier: phase 3, multicenter, randomized, double-masked trials of brolucizumab for neovascular age-related macular degeneration. ophthalmology. 2020;127(1):72-84. crossref pubmed 25. sharma a, kumar n, parachuri n, et al. brolucizumab-early realworld experience: brew study. eye (lond). 2021;35(4):10451047. crossref pubmed 26. avaylon j, lee s, gallemore rp. case series on initial responses to intravitreal brolucizumab in patients with recalcitrant chronic wet age-related macular degeneration. int med case rep j. 2020;13:145-152. crossref pubmed 27. seguin-greenstein s, lightman s, tomkins-netzer o. a metaanalysis of studies evaluating visual and anatomical outcomes in patients with treatment resistant neovascular age-related macular degeneration following switching to treatment with aflibercept. j ophthalmol. 2016;2016:4095852. crossref pubmed 28. enríquez ab, baumal cr, crane am, et al. early experience with brolucizumab treatment of neovascular age-related macular degeneration. jama ophthalmol. 2021;139(4):441-448. crossref pubmed 29. bontzos g, bagheri s, ioanidi l, et al. nonresponders to ranibizumab anti-vegf treatment are actually short-term responders: a prospective spectral-domain oct study. ophthalmol retina. 2020;4(12):1138-1145. crossref pubmed 30. zola m, d’alessandro e, sherif m, et al. refractory neovascular age-related macular degeneration: time-dependent changes of central retinal thickness with anti-vegf treatment. graefes arch clin exp ophthalmol. 2021 jun;259(6):1477-1486 crossref pubmed 31. monés j, srivastava sk, jaffe gj, et al. risk of inflammation, retinal vasculitis, and retinal occlusion-related events with brolucizumab: post hoc review of hawk and harrier. ophthalmology. 2021 jul;128(7):1050-1059. crossref pubmed https://doi.org/10.1159/000502747 https://www.ncbi.nlm.nih.gov/pubmed/31743912 https://doi.org/10.1016/j.ophtha.2015.05.010 https://www.ncbi.nlm.nih.gov/pubmed/26096346 https://doi.org/10.1136/bjophthalmol-2011-301236 https://www.ncbi.nlm.nih.gov/pubmed/22157632 https://doi.org/10.1016/j.oret.2018.09.001 https://www.ncbi.nlm.nih.gov/pubmed/30929819 https://doi.org/10.1016/j.ophtha.2018.11.036 https://www.ncbi.nlm.nih.gov/pubmed/30529685 https://doi.org/10.1007/s10792-018-1038-4 https://www.ncbi.nlm.nih.gov/pubmed/30386955 https://doi.org/10.1016/j.ophtha.2020.06.028 https://www.ncbi.nlm.nih.gov/pubmed/32574761 https://doi.org/10.1016/j.ophtha.2019.04.017 https://www.ncbi.nlm.nih.gov/pubmed/30986442 https://doi.org/10.1038/s41433-020-1111-x https://www.ncbi.nlm.nih.gov/pubmed/32709960 https://doi.org/10.2147/imcrj.s252260 https://www.ncbi.nlm.nih.gov/pubmed/32494207 https://doi.org/10.1155/2016/4095852 https://www.ncbi.nlm.nih.gov/pubmed/27042342 https://doi.org/10.1001/jamaophthalmol.2020.7085 https://www.ncbi.nlm.nih.gov/pubmed/33630045 https://doi.org/10.1016/j.oret.2019.11.004 https://www.ncbi.nlm.nih.gov/pubmed/31937473 https://www.ncbi.nlm.nih.gov/pubmed/31937473 https://doi.org/10.1007/s00417-020-05000-3 https://www.ncbi.nlm.nih.gov/pubmed/33245426 https://doi.org/10.1016/j.ophtha.2020.11.011 https://www.ncbi.nlm.nih.gov/pubmed/33207259 hospers et al.indd drug target insights 2007: 2 105–109 105 review correspondence: g.a.p. hospers, m.d., ph.d., dept. of medical oncology, university medical center groningen, hanzeplein 1, 9713 gz groningen, the netherlands. tel: +31 50 361 2821/1847; fax: +31 50 361 4862; email: g.a.p.hospers@int.umcg.nl please note that this article may not be used for commercial purposes. for further information please refer to the copyright statement at http://www.la-press.com/copyright.htm circulating vascular endothelial growth factor (vegf) levels in advanced stage cancer patients compared to normal controls and diabetes mellitus patients with critical ischemia yoka h. kusumanto, coby meijer, wendy dam, nanno h. mulder and geke a.p. hospers dept. of medical oncology, university of groningen and university medical center groningen, groningen, the netherlands. abstract: anti-angiogenic therapy is emerging as a valuable tool in the treatment of patients with cancer. as vegf is a central target in anti-angiogenic therapy, its levels in the circulation might be relevant in selecting tumor types or patients likely to respond to this treatment. additional vegf has been recognized as a key factor in the pathogenesis of diabetic retinopathy. recently anti-angiogenic therapy has been advocated in this situation. we measured vegf levels in whole blood in 42 patients with high grade (n = 26) and low grade (n = 16) end stage cancer, and in 28 healthy controls and 37 patients with diabetes related vascular disease. only 2/26 patients in the group of high grade cancer had signifi cantly elevated vegf levels, 1/16 in the low grade group and 1/28 in the healthy control group. in contrast, in 10/37 diabetic patients the mean vegf levels were signifi cantly elevated compared to the other groups. the mean level in these diabetic patients was signifi cantly elevated compared to the other groups. these data indicate the limitation of the use of circulating vegf levels as a potential selection criterion for anti-angiogenic therapy in cancer patients and suggest further studies into its application in the management of diabetic complications. keywords: vegf level, cancer, diabetes mellitus, critical limb ischemia introduction anti-angiogenic therapy is emerging as an important strategy in the treatment of cancer (ferrara, 2005). following extensive in vitro and preclinical testing over many decades the therapeutic implications of tumor angiogenesis are fi nally having an impact in the clinic (folkman, 1971). up till now it is impossible to predict activity of anti-angiogenic therapy for particular tumor types or individual patients. however the favorable results of vegf antibodies suggest that circulating levels of vegf might give an indication of the potential of this treatment in tumors of different grades of malignancy or even in individual patients. a profi le of elevated vegf levels in the more aggressive cancers compared to slow growing tumors and in those tumor types known to respond to vegf antibody therapy, would support further studies on vegf levels as predictive markers. increased circulating vegf levels have also been observed in patients with diabetes mellitus (chiarelli et al. 2000; valabhji et al. 2001). a variety of factors, implicated in the development of diabetic complications, have been shown to upregulate vegf expression in vitro, including high glucose concentrations and advanced glycation end-products (williams, 1997; okamoto et al. 2002). vascular proliferation is known to play a role in the development of diabetic retinopathy. it is therefore not surprising that developments in anti-angiogenic therapy in cancer have been closely followed in the fi eld of ophthalmology. preliminary evidence suggests that this treatment form either with bevacizumab or with its derivative ranibizumab is highly effective (rosenfeld, 2005a; rosenfeld, 2005b; miller, 2005; puliafi to, 2005). to further study the prevalence of elevated circulating vegf levels in cancer patients and in diabetics we measured, in a population of patients who were referred with advanced stage cancer, the incidence of increased vegf levels and compared them to values in the normal population and in a group of 106 kusumanto et al drug target insights 2007: 2 non-cancer diabetic patients known to have severe ischemic vascular disease. patients and methods forty-two patients who had incurable metastatic cancer and referred for palliative therapy were studied (median: 55 (range 19–75) years). these patients were separated in two groups, one with slow growing differentiated neuro-endocrine carcinoid tumors, comprising 16 patients, and a second group of aggressively progressive solid tumors of diverse origin, comprising 26 patients (table 1). the 6 “others” in table 1 are extragonadal germ cell tumor, metastatic insulinoom, myxoid solitary fi brous tumor, neuroendocrine pancreas tumor (no hormone producing), lung (non small cell) and testicular cancer. thirty seven diabetic patients (median: 71 (range 40–84) years of age, diabetic duration: median 16 years (range 0.5–55) hba1c median: 7.6 (range 5.8–12.2)) with end stage vascular disease of the limbs were included. in the diabetic patient group the following investigations were performed: demographic characteristics such as age and diabetic duration and body mass index but also clinical assessment of edema were included. concentrations of hemoglobin a1c (hba1c), fasting glucose, cholesterol and triglycerides, c-reactive protein (crp), creatinin, and albumin excretion ratio (two overnight urine collections) were measured. standard laboratory assays were used. fundus photographs of the retina were performed and graded as follows: no retinopathy, background retinopathy, pre-proliferative and proliferative diabetic retinopathy. ankle and toe pressures were measured according to conventional procedures using an 8 mhz parkes doppler. ankle to brachial index (abi) and toe to brachial index (tbi) were calculated as the quotient of absolute ankle and toe pressures to the simultaneously measured brachial pressure, respectively. as a control population 28 healthy (median: 29 (18–54) years) volunteers were studied and randomly recruited from the medical and laboratory personal. diabetic patients with evidence of systemic complications or systemic disease otherwise were excluded i.e. 1) proliferative eye disease, 2) history of malignancy or severe co-morbidity. the study was approved by the local human investigations committee. vegf measurements venous blood was collected in sterile tubes containing ctad (sodium citrate, theophylline, adenosine, dipyridamole, becton dickinson vacutainer systems, france, europe). blood samples were diluted with two volumes of pbs (phosphate buffered saline) and subsequently lysed by freezing and thawing twice. aliquots were stored at –80 °c. vegf levels were determined in duplicate using the quantikine human vegf enzyme-linked immunosorbent assay (elisa) (r & d systems inc. minneapolis, mn). the minimum detection level was 9.0 pg/ml in whole blood as quoted by the manufacturer. statistics the kolmogorov-smirnov test was used to confi rm the assumption of normal distribution of vegf samples in the healthy control group. the number of patients with elevated levels was compared between the groups using the mantel haenzl sqi square test. mean vegf levels were compared using the two sided student t test (unpaired). statistical signifi cance was set at p < 0.05. results and discussion circulating vegf mainly refl ects vegf derived from peripheral blood cells, including platelets and leucocytes. therefore, we used whole blood for the measurement of vegf, which contains all cell compartments, as was recommended previously (salven et al. 1999). table 1. distribution of elevated vegf levels. subjects n elevated vegf level controls 28 1 carcinoid patients 16 1 aggressive solid 26 2 tumor patients colon cancer 8 0 breast cancer 4 0 renal cancer 3 0 melanoma 5 0 others 6 2 diabetes mellitus 37 10* number of patients with elevated vegf levels (i.e. >1200 pg/ml; 95% confi dence interval in healthy controls: 157.7–1200.0 pg/ml). *: p = 0.015 by mantel haenzl chi square test. 107 vegf levels in cancer patients drug target insights 2007: 2 in the present study we found a 95% confi dence interval of values in our control population (n = 28) between 157.71 pg/ml and 1200.03 pg/ml. individual vegf levels above the upper limit of 1200 pg/ml were considered to be elevated. accordingly, in three out of 42 patients with advanced stage cancer vegf levels were elevated, one patient with carcinoid cancer, one with an aggressive no hormone producing neuro-endocrine tumor and one with lung cancer, compared to one out of 28 cases in the control group and 10 out of 37 cases in the patients with diabetes (p = 0.015, table 1). in the cancer patients there was a tendency toward higher levels in patients with aggressive solid tumors compared to controls although this did not reach signifi cance (p = 0.08, table 2). there was no difference of occurrence of high levels of whole blood vegf levels between aggressive and the more differentiated and slower growing carcinoid tumors. also vegf levels in the patients with colonic cancer, a tumor type that is accepted as an indication for angiogenic therapy did not exceed normal levels. as angiogenesis is necessary for tumor growth and the metastatic process, many attempts at quantifying this process have been made. direct measurements include determination of micro vessel density in the tumor (blann et al. 2002; blann et al. 2001; cascinu et al. 2000). however this method although effective in these studies in colorectal cancer requires tumor samples, limiting its applicability in the clinic as does immunohistochemistry on tumor cells or vessels. indirect measurements include the various proor anti angiogenic factors in blood. interestingly tien et al. (tien et al. 2006) found peripherically determined venous vegf levels to be not inferior to levels downstream in the venous blood of gastro-intestinal tumors. moreover vegf levels correlated the most closely to patients clinico pathological characteristics. a considerable number of studies have linked blood vegf levels to tumor stage and prognosis in patients with cancer. reports usually indicate that cancer patients tend to have higher levels of vegf than controls, and that levels correlate with adverse prognostic factors. subgroup analysis, for example comparing long-and short-term survivors, was suggestive for the existence of a relation of malignancy grade with vegf levels. this effect was striking in an early study in lung cancer (ohta et al. 1996). the same correlations were found in liver cancer, breast cancer and colon cancer (torimura et al. 1998; toi et al. 1995; eppenberger et al. 1998; gasparini et al. 1997; takahashi et al. 1995; cascinu et al. 2000; ishigami et al. 1998). yet, the question whether a relation can be found between vegf levels and tumor stage has been answered equivocally in various studies. in renal cell cancer such a correlation was absent, but in planocellular esophagus cancer it was striking (edgren et al. 2001; wallner et al. 2001). the same was found in cervical cancer and differentiated thyroid cancer (bachtiary et al.; tuttle et al. 2002). usually it is assumed that tumor angiogenesis under the infl uence of elevated vegf levels is the biological phenomenon involved. however the endpoint of that process, micro vessel density, was not always related to vegf levels, even when these levels had been found to have predictive clinical relevance (yudoh et al. 2001). in the latter study from yudoh et al. in sarcoma patients, local relapse, metastatic progression and short survival were predicted by high tissue levels of vegf but not with micro vessel density. in another study in bone sarcoma patients however, serum levels of vegf were elevated in contrast to tissue levels in ewing sarcoma (holzer et al. 2001). on the other hand an apparent relation between tumor burden and circulating vegf levels was shown by the rapid decrease of elevated levels after surgery for such different tumors as esophageal cancer and childhood wilms tumor (mcdonnell et al. 2001; blann et al. 2001). yet, in view of the rarity of table 2. vegf whole blood levels. controls all cancers carcinoids aggressive solid diabetes tumors mellitus vegf levels 491.7 592.6 525.6 634.1 928.9 (pg/ml) ± 275.5 ± 351.8 ± 101.9 n.s. ± 311.9 ± 443.2 p-value n.s. n.s. n.s.* 0.0001** mean vegf levels of patient groups are compared to the healthy control group by two-sided student t-test. *: p = 0.08, for trend. **: p < 0.05, considered statistically signifi cant. 108 kusumanto et al drug target insights 2007: 2 increased vegf levels found in our cancer patients it is doubtful that vegf levels will become an important guideline in the treatment with antiangiogenic drugs on an individual or tumor type oriented basis. as tissue anoxia is considered to be a major stimulus for vegf production vascular insuffi ciency could also in non-malignant disease lead to increased vegf levels (maulik et al. 2000). for this purpose we studied a group of patients with end stage diabetic vascular insuffi ciency and found signifi cantly elevated vegf levels when mean vegf levels (p = 0.001, table 2 and fig. 1) as well as numbers of patients with elevated levels were compared. although there was a trend for higher vegf levels with duration of diabetes this did not reach signifi cance (p = 0.08, data not shown). in addition we found no relation with other risk factors for diabetic complications such as, hba1c, and albumin excretion ratio (aer), but also the degree of ankle edema, renal function, lipids, retinopathy and abi/tbi were not related to circulating vegf levels (data not shown). the role of vegf in the development of diabetic vascular complications has become an increasingly intense studied subject in view of the rising prevalence of diabetes. perhaps the strongest case for vegf as a growth factor in diabetic vascular disease is proliferative diabetic retinopathy (clermont et al. 1997; malecaze et al. 1994). the results of studies on the relation with circulating vegf levels were however not unequivocal with positive correlations found in proliferative retinopathy (sydorova and lee 2005; chaturvedi, 2000). our fi ndings confi rm that vegf levels can be elevated in diabetes and that high levels are indeed common with end stage vascular disease as opposed to diabetics with healthy vessels (chiarelli et al. 2000; valabhji et al. 2001; blann et al. 2002). the common occurrence of elevated vegf levels in diabetic patients with vascular disease suggest that anti-angiogenic treatment directed at this growth factor is logical. this was already found to be successful in the treatment of diabetic retinopathy with ranibizumab, a derivative of bevacizumab. interestingly, early clinical studies in the treatment of chronic ischemic limb disease with vegf as a therapeutic agent, thus increasing endogenous vegf levels, showed benefi cial effects without causing severe adverse effects, even in diabetic patients (baumgartner et al. 1998; shyu et al. 2003). in conclusion, our study indicates that the potential of the use of circulating vegf levels as a selection criterion for anti-angiogenic therapy in cancer patients is limited. signifi cantly elevated vegf levels in end stage solid tumor patients are rare (2/26), such levels can be found also in healthy controls (1/28) and and most strikingly, in diabetic patients with ischemic vascular disease (10/37). future studies should reveal the biologic relevance 0 1000 2000 p < 0.0001 p = 0.0012 he a lthy controle ca nce r pa tie nts dia be tic pa tie nts w ho le b lo od v e g f (p g/ m l ) figure 1. mean vegf levels. p < 0.05 is considered signifi cant (unpaired student t test). 109 vegf levels in cancer patients drug target insights 2007: 2 and hence the diagnostic and therapeutic implications for the treatment of vascular complications in diabetic patients. references bachtiary, b., selzer, e., knocke, t.h. et al. 2002. serum vegf levels in patients undergoing primary radiotherapy for cervical cancer: impact on progression-free survival. cancer lett., 179:197–203. baumgartner, i., pieczek, a., manor, o. et al. 1998. constitutive expression of phvegf165 after intramuscular gene transfer promotes collateral vessel development in patients with critical limb ischemia. circulation, 97:1114–23. blann, a.d., belgore, f.m., mccollum, c.n. et al. 2002. vascular endothelial growth factor and its receptor, flt-1, in the plasma of patients with coronary or peripheral atherosclerosis, or type ii diabetes. clin. sci. (lond)., 102:187–94. blann, a.d., li, j.l., li, c. et al. 2001. increased serum vegf in 13 children with wilms’ tumour falls after surgery but rising levels predict poor prognosis. cancer lett., 173:183–6. cascinu, s., staccioli, m.p., gasparini, g. et al. 2000. expression of vascular endothelial growth factor can predict event-free survival in stage ii colon cancer. clin. cancer res., 6:2803–7. chaturvedi, n. 2000. modulation of the renin-angiotensin system and retinopathy. heart, 84 suppl 1:i29–i31. chiarelli, f., spagnoli, a., basciani, f. et al. 2000. vascular endothelial growth factor (vegf) in children, adolescents and young adults with type 1 diabetes mellitus: relation to glycaemic control and microvascular complications. diabet med., 17:650–6. clermont, a.c., aiello, l.p., mori, f. et al. 1997. vascular endothelial growth factor and severity of nonproliferative diabetic retinopathy mediate retinal hemodynamics in vivo: a potential role for vascular endothelial growth factor in the progression of nonproliferative diabetic retinopathy. am. j. ophthalmol., 124:433–46. edgren, m., lennernas, b., larsson, a. et al. 2001. angiogenic factors: vascular endothelial growth factor (vegf) and basic fi broblast growth factor (b-fgf) are not necessarily elevated in patients with advanced renal cell carcinoma. anticancer res., 21:1423–9. eppenberger, u., kueng, w., schlaeppi, j.m. et al. 1998. markers of tumor angiogenesis and proteolysis independently defi ne highand low-risk subsets of node-negative breast cancer patients. j. clin. oncol., 16:3129–36. ferrara, n. 2005. vegf as a therapeutic target in cancer. oncology, 69 suppl 3:11–6. folkman, j. 1971. tumor angiogenesis: therapeutic implications. n. engl. j. med., 285:1182–6. gasparini, g., toi, m., gion, m. et al. 1997. prognostic signifi cance of vascular endothelial growth factor protein in node-negative breast carcinoma. j. natl. cancer inst., 89:139–47. holzer, g., obermair, a., koschat, m. et al. 2001. concentration of vascular endothelial growth factor (vegf) in the serum of patients with malignant bone tumors. med. pediatr. oncol., 36:601–4. ishigami, s.i., arii, s., furutani, m. et al. 1998. predictive value of vascular endothelial growth factor (vegf) in metastasis and prognosis of human colorectal cancer. br. j. cancer. 78:1379–84. malecaze, f., clamens, s., simorre-pinatel, v. et al. 1994. detection of vascular endothelial growth factor messenger rna and vascular endothelial growth factor-like activity in proliferative diabetic retinopathy. arch. ophthalmol., 112:1476–82. maulik, n., sasaki, h., addya, s. et al. 2000. regulation of cardiomyocyte apoptosis by redox-sensitive transcription factors. febs. lett., 485:7–12. mcdonnell, c.o., harmey, j.h., bouchier-hayes, d.j. et al. 2001. effect of multimodality therapy on circulating vascular endothelial growth factor levels in patients with oesophageal cancer. br. j. surg., 88:1105–9. miller, j. 2005. marina study group. randomized, controlled phase iii study of ranibizumab (lucentis) for minimally classic or occult neovascular age-related macular degeneration. program and abstracts of the american society of retina specialists 23rd annual meeting. july 16–20, chicago, illinois. ohta, y., endo, y., tanaka, m. et al. 1996. signifi cance of vascular endothelial growth factor messenger rna expression in primary lung cancer. clin. cancer res., 2:1411–6. okamoto, t., tanaka, s., stan, a.c. et al. 2002. advanced glycation end products induce angiogenesis in vivo. microvasc. res., 63:186–95. puliafi to, c.a. 2005. lucentis for exudative amd. pharmacotherapy for macular diseases. program and abstracts of the american academy of ophthalmology 109th annual meeting, october 15–18, chicago, illinois. rosenfeld, p.j. 2005a. avastin for amd. retina 2005: changing concepts and controversies. program and abstracts of the american academy of ophthalmology 109th annual meeting. october 15–18, chicago, illinois. rosenfeld, p.j. 2005b. systemic bevacizumab (avastin) therapy for neovascular age-related macular degeneration (sana) study: 12 week outcomes. program and abstracts of the american society of retina specialists 23rd annual meeting. july 16–20, montreal, canada. salven, p., orpana, a., joensuu, h. 1999. leukocytes and platelets of patients with cancer contain high levels of vascular endothelial growth factor. clin. cancer res., 5:487–91. shyu, k.g., chang, h., wang, b.w. et al. 2003. intramuscular vascular endothelial growth factor gene therapy in patients with chronic critical leg ischemia. am. j. med., 114:85–92. sydorova, m., lee, m.s. 2005. vascular endothelial growth factor levels in vitreous and serum of patients with either proliferative diabetic retinopathy or proliferative vitreoretinopathy. ophthalmic res., 37:188–90. takahashi, y., kitadai, y., bucana, c.d. et al. 1995. expression of vascular endothelial growth factor and its receptor, kdr, correlates with vascularity, metastasis, and proliferation of human colon cancer. cancer res., 55:3964–8. tien, y.w., chang, k.j., chiu, y.f., huang, k.w., lee, p.h. 2006. comparison of angiogenic factor levels in tumor drainage and peripheral venous blood from colorectal cancer patients. ann. surg. oncol., 13:1357–63. toi, m., inada, k., suzuki, h. et al. 1995. tumor angiogenesis in breast cancer: its importance as a prognostic indicator and the association with vascular endothelial growth factor expression. breast cancer res. treat., 36:193–204. torimura, t., sata, m., ueno, t. et al. 1998. increased expression of vascular endothelial growth factor is associated with tumor progression in hepatocellular carcinoma. hum. pathol., 29:986–91. tuttle, r.m., fleisher, m., francis, g.l. et al. 2002. serum vascular endothelial growth factor levels are elevated in metastatic differentiated thyroid cancer but not increased by short-term tsh stimulation. j. clin. endocrinol metab., 87:1737–42. valabhji, j., dhanjil, s., nicolaides, a.n. et al. 2001. correlation between carotid artery distensibility and serum vascular endothelial growth factor concentrations in type 1 diabetic subjects and nondiabetic subjects. metabolism., 50:825–9. wallner, g., ciechanski, a., dabrowski, a. et al. 2001. vascular endothelial growth factor and basic fi broblast growth factor in patients with squamous cell oesophageal cancer. folia histochem cytobiol, 39 suppl 2:122–3. williams, b. 1997. factors regulating the expression of vascular permeability/vascular endothelial growth factor by human vascular tissues. diabetologia, 40 suppl 2: s118–s120. yudoh, k., kanamori, m., ohmori, k. et al. 2001. concentration of vascular endothelial growth factor in the tumour tissue as a prognostic factor of soft tissue sarcomas. br. j. cancer. 84:1610–5. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) 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2016:10(s1) gentamicin-impregnated collagen sponge: effectiveness in preventing sternal wound infection in high-risk cardiac surgery supplementary issue: current developments in drug eluting devices filippo rapetto1, vito d. bruno1, gustavo guida1, roberto marsico1, pierpaolo chivasso1 and carlo zebele2 1department of cardiac surgery, bristol heart institute, bristol, uk. 2department of cardiac surgery, citta’ di lecce hospital, lecce, italy. a bstr act: sternal wound infections represent one of the most frequent complications after cardiac surgery and are associated with high postoperative mortality. several preventive methods have been introduced, and recently, gentamicin-impregnated collagen sponges (gicss) have shown a promising effect in reducing the incidence of this type of complications. gentamicin is an aminoglycoside antibiotic that has been widely used to treat infections caused by multiresistant bacteria; despite its effectiveness, its systemic use carries a risk of toxicity. gicss appear to overcome this side effect, topically delivering high antibiotic concentrations to the wound and thus reducing the toxic-related events. although several retrospective analyses and randomized controlled trials have studied the use of gicss in cardiac surgery, conclusions regarding their efficacy in preventing sternal wound infection are inconsistent. we have reviewed the current literature focusing on high-risk patients. k e y wor ds: gentamicin, wound infection, topical drug administration supplement: current developments in drug eluting devices citation: rapetto et al. gentamicin-impregnated collagen sponge: effectiveness in preventing sternal wound infection in high-risk cardiac surgery. drug target insights 2016:10(s1) 9–13 doi:10.4137/dti.s39077. type: consise review received: february 5, 2016. resubmitted: april 5, 2016. accepted for publication: april 11, 2016. academic editor: anuj chauhan, editor in chief peer review: three peer reviewers contributed to the peer review report. reviewers’ reports totaled 333 words, excluding any confidential comments to the academic editor. funding: authors disclose no external funding sources. competing interests: authors disclose no potential conflicts of interest. copyright: © the authors, publisher and licensee libertas academica limited. this is an open-access article distributed under the terms of the creative commons cc-by-nc 3.0 license. correspondence: vitodomenicobruno@gmail.com paper subject to independent expert single-blind peer review. all editorial decisions made by independent academic editor. upon submission manuscript was subject to anti-plagiarism scanning. prior to publication all authors have given signed confirmation of agreement to article publication and compliance with all applicable ethical and legal requirements, including the accuracy of author and contributor information, disclosure of competing interests and funding sources, compliance with ethical requirements relating to human and animal study participants, and compliance with any copyright requirements of third parties. this journal is a member of the committee on publication ethics (cope). published by libertas academica. learn more about this journal. introduction sternal wound infections (swis) represent one of the most challenging postoperative complications following cardiac surgery, associated with increased hospital stay, mortality, and costs.1 it is widely acknowledged that several factors (eg, smoking, obesity, insulin-dependent diabetes, emergent surgery, prolonged operative time, reoperation, bilateral internal mammary artery (bima) harvesting, transfusions, and prolonged ventilation/intensive care unit [icu] stay) pre dispose to a higher risk of both superficial and deep swis; many of these conditions are observed with increasing frequency in the modern cardiac surgery scenario.2–4 historically, the cornerstones of prevention of swis have been preoperative skin asepsis and administration of prophylactic antimicrobial drugs and several surgical maneuvers intended to maintain sterility and achieve a stable sternal fixation.5–7 during the past few years, local administration of gentamicin through a surgically inserted collagen sponge has been gaining popularity in different surgical fields, and it has been increasingly used in cardiac surgery.8 the reported incidence of swis varies between 0.5% and 6% throughout the literature, although it is considerably higher among high-risk individuals, ranging between 12% and 20%.6 swis can be classified as super ficial swis (sswis) or deep swis (dswis), according to the extension of the infective process from the skin and subcutaneous layer to the bone and mediastinum. the centers for disease control and prevention and the national institute of clinical excellence have developed guidelines for the diagnosis and management of surgical site infections.9,10 however, currently, the assessment and treatment of swis seem to be quite heterogeneous among different centers and even among different surgeons from the same center. it is clear that dswis strongly impact on postoperative morbidity and mortality and that sswis are a common cause of prolonged hospital stay. in 2008, mauermann et al reviewed data regarding 11 series of postoperative swis, reporting an overall mortality of 13% (ranging between 6% and 30%). they also concluded that it is difficult to precisely assess the impact of swis on hospital costs due to the variability in protocols among different centers.1 since swis significantly prolong icu stay and hospitalization, they are widely known to be a major cause of increased health-care costs in cardiac surgery.1 both dswis and sswis are usually caused by gram-positive skin bacteria such as coagulasenegative staphylococci (cons) and staphylococcus aureus; in a minority of cases, they can be caused by gram-negative rods journal name: drug target insights journal type: consise review year: 2016 volume: 10(s1) running head verso: rapetto et al running head recto: effectiveness of gentamicin-impregnated collagen sponge http://www.la-press.com/drug-target-insights-journal-j23 http://www.la-press.com http://dx.doi.org/10.4137/dti.s39077 http://creativecommons.org/licenses/by-nc/3.0/ http://creativecommons.org/licenses/by-nc/3.0/ mailto:vitodomenicobruno@gmail.com http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 rapetto et al 10 drug target insights 2016:10(s1) such as escherichia coli and pseudomonas aeruginosa and fungi such as candida albicans.11–13 as shown in table 1, grampositive bacteria are isolated in 60%–80% of postoperative swis, gram-negative rods in 20%–40% of cases, and fungi in about 5% (polymicrobial isolations are frequent and occur in 10%–40% of cases).1,14,15 the emergence of multiresistant bacterial strains has led to a challenging situation: beta-lactam antibiotics have become ineffective against most cons and s. aureus clusters, and the routine use of vancomycin as a prophylactic agent is not advisable in order to avoid further antibiotic resistances.13,16 furthermore, cons have the intrinsic ability to adhere to foreign bodies (eg, sternal wires) and produce a biofilm, thus increasing their resistance to antibiotics and the probability of a chronic infection.17,18 advantages of gentamicin local delivery local administration of antibiotics such as gentamicin, tobramycin, tetracycline, minocycline, teicoplanin, and sulbactam– cefoperazone has been performed in several surgical fields.8 gentamicin has gradually become the most used molecule for this purpose due to a combination of characteristics such as broad spectrum, low cost, and favorable pharmacokinetics and pharmacodynamics when administered topically.8,19 gentamicin is an aminoglycoside antibiotic that has been widely used to treat infections caused by multiresistant bacteria (fig. 1). although its spectrum is mainly directed toward gram-negative species, gentamicin is also effective against several gram-positive strains.19 furthermore, gentamicin also shows a synergy with beta-lactam antibiotics, especially against gram-positive species such as s. aureus and cons.16 nevertheless, the principal factor limiting its systemic use is represented by its intrinsic toxicity; when administered intravenously or intramuscularly, gentamicin accumulates into renal cortex and into endolymph and perilymph of the inner ear, causing kidney injury and hearing loss.20 these drawbacks can be partially eluded by administering gentamicin locally, reducing the systemic toxicity. kidney and inner ear accumulations seem to appear when gentamicin serum concentration exceeds 10–12 mg/l, although a precise cutoff has not been established.8,21 interestingly, it has been observed that, after local administration in the sternal region, the drug serum concentration does not exceed 1  mg/l, while mediastinal fluid concentration remains above 300  mg/l for 36 hours.21 moreover, gentamicin exhibits a concentration-dependent effect, especially against gram-negative rods: this means that a high concentration of the drug circumscribed to the surgical site can lead to a bactericidal effect not only toward sensitive bacteria but also toward poorly sensitive or even resistant ones;20 an acute peak concentration in the surgical site, combined with a low serum level of the drug, is protective against the selection of resistant bacteria; in fact, prolonged high serum concentrations promote the so-called adaptive resistance to gentamicin.20 pharmacokinetics of gics surgical implants impregnated with gentamicin started to be used in the 1970s, primarily in orthopedic surgery, aiming to treat or prevent prosthetic infections. the first devices had the disadvantage that they were not made of reabsorbable materials; hence, they had to be surgically removed once the infection had been treated.22 as a consequence, biodegradable polymers such as polylactic acid, polyglycolic-polylactic acid, poly(ortho esters), and polyhydroxybutyrate-co-hydroxyvalerate were developed to carry antibiotic drugs to the surgical site.23,24 finally, since the 1980s, collagen implants began to be used in several surgical specialties for local antibiotic delivery, mainly due to collagen biocompatibility and pharmacokinetic versatility.23,24 regarding pharmacokinetics, collagen is a unique polymer because it has a complex, well-known three-dimensional structure with different hierarchical levels: primary, secondary, tertiary, and quaternary.25 the physiochemical characteristics table 1. microbiology of swis. microorganism frequency gram-positive cocci 60–80% staphylococcus aureus 40–45% coagulase-negative staphylococcus 20–35% gram-negative rods 20–40% enterobacter spp 10% pseudomonas spp 2–10% klebsiella spp 3–8% escherichia coli 5% proteus spp 2–3% fungi 5% polymicrobial 10–40% note: adapted from refs. 1,14,15. figure 1. chemical structure of gentamicin. notes: atoms are represented as spheres with conventional color coding: white represents hydrogen, gray represents carbon, blue represents nitrogen, and red represents oxygen. http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 effectiveness of gentamicin-impregnated collagen sponge 11drug target insights 2016:10(s1) of the final polymer can be modified by intervening on the molecular structure (eg, intraand intermolecular cross-links) as well as linking collagen with other polymers to obtain different drug-releasing temporal curves. hence, when collagen is used as a carrier for a drug, any structural modification can lead to different pharmacokinetic profiles.8,26,27 collagen biocompatibility and absorbability represent essential characteristics with respect to infection prevention or treatment, because they allow to avoid a further surgical procedure (which could be as well complicated by infection) to remove the drug-carrying device. gentamicin-impregnated collagen sponges (gicss) can be inserted in three different regions during sternal closure, depending on the desired primary site of action: – behind the posterior surface of the sternum, between the bone and the sternal wires; – between sternal halves; and – on the anterior surface of the sternum, under the muscular fascia. every different position of the gics corresponds to a slightly different spatial distribution of gentamicin, according to the closest sternal region.28,29 for instance, when a gics was placed posteriorly to the sternum, its effect was more evident in preventing dswi rather than sswi.30 one or two gicss per patient, corresponding to 130 and 260 mg of gentamicin, respectively, were used in most of the reviewed studies, with various combinations of the abovementioned positions. there is some evidence that collagen sponges should not be soaked in saline solution prior to use.31,32 bennett-guerrero et al conducted a multicenter randomized double-blind trial comparing high-risk cardiac surgical patients receiving a gics with patients receiving a standard sternal closure. the authors found no advantages for the study group over the control group regarding the incidence of swi up to 90 days after surgery. the gics were soaked in saline as per the study protocol, and the authors have been criticized due to this maneuver.31 gentamicin is a highly water-soluble molecule, and in vitro studies have showed that exposing a gics to saline causes the loss of 6.7%, 40.5%, and 100% of the gentamicin after 2 seconds, 1 minute, and 6 hours, respectively.32 manufacturers recommend not to soak the gics prior to use. gics use in high-risk cardiac surgery patients obesity represents one of the most important risk factors for the development of swi in cardiac surgery,15 but there are contrasting data on the effect of the gics on preventing this complication in this subgroup of patients. an important multicenter randomized controlled trial has involved 1502 high-risk patients in 48 centers (table 2).31 in this study, 1006 patients (67% of the population) were diabetic and 1137 patients (76%) were obese with a median body mass index (bmi) of 32.9 kg/m2. with a 90-day postoperative follow-up, the authors reported a comparable incidence of swis in the intention-to-treat analysis, with an incidence of dswi of 8.4% in the gics group vs 8.7% in the control group (p  =  0.83). similar results have been reported in the per-protocol analysis (8.4% vs 8.6%, p = 0.89). a further subanalysis of this study, targeting the very high-risk group and including patients who table 2. comparison of various studies that have investigated gics use in high-risk cardiac surgery patients. author type of study subgroup outcome gics no gics p-value bennett-guerrero et al31 multicenter rct, 1502 pts all pts any swi (itt) 8.4% 8.7% 0.83 any swi (pp) 8.4% 8.6% 0.89 bmi  30 any swi 8.1% 4.2% 0.07 diabetes any swi 4% 5.4% 0.52 bmi  30 + diabetes any swi 11.1% 13.8% 0.30 friberg et al16 two-centers rct, 2000 pts all pts any swi 4.3% 9% 0.001 sswi 1.9% 5.7% 0.001 dswi 2.3% 3.3% 0.20 no reoperation or early death sswi 2.7% 6.7% 0.001 dswi 2.1% 3.3% 0.088 diabetes sswi 1.67% 7.47% 0.0086 dswi 3.89% 9.77% 0.028 bmi  25 sswi 2.16% 6.45% 0.001 dswi 2.47% 4.45% 0.050 birgand et al35 single center cs, 552 pts bima + bmi  30 or id diabetes dswi 12.6% 13.8% ns abbreviations: bima, bilateral internal mammary; cs, cohort study; dswi, deep sternal wound infection; bmi, body mass index; gics, gentamicin-impregnated collagen sponge; id, insulin-dependent; itt, intention-to-treat; ns, not significant; pp, per-protocol; pts, patients; rct, randomized controlled trial; sswi, superficial sternal wound infection; swi, sternal wound infection. http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 rapetto et al 12 drug target insights 2016:10(s1) were both obese and diabetic, showed no differences between the two groups (11.1% vs 13.8%, p = 0.30). the authors concluded that the use of a gics did not reduce the 90-day swi rate.31 one of the most controversial points regarding this study was the fact that the gics was wetted in saline before application: the consequence of this may have influenced the gentamicin release,32,33 and therefore the outcome of the study would have been affected. different results were reported by a previously published randomized controlled trial,16 where the impact of obesity was specifically subanalyzed. with a cutoff of bmi    25  kg/m2 (1299 patients), the authors reported a significantly reduced incidence of sswis (2.16% vs 6.45%, p  0.001) and dswis (2.47% vs 4.45%, p  =  0.05) in the gics group. the analysis of the whole population confirmed these results only for sswis, whereas there were no differences in terms of dswis (p = 0.2); these outcomes can be interpreted as a stronger effect of gics in preventing dswi in the obese group of patients. another subgroup at high risk of swi includes patients who undergo bima harvesting during coronary artery bypass grafting (cabg), as this technique can impair sternal vascularization.34 a single-center study, published in 2012 by birgand et al (table 2), compared the incidence of dswi requiring surgery in high-risk patients defined as bima harvesting plus overweight (bmi  30 kg/m2) and/or insulin-dependent diabetes.35 the authors did not find any difference regarding the incidence of dswi between patients receiving a gics and those who did not receive it (12.6% vs 13.8%, respectively); interestingly, in that series, the probability of dswi caused by a gentamicin-resistant bacterium was higher in the gics group (21/27, 77.8%) compared with the other patients (23/56, 41.1%; p  0.01). again, as in the above-mentioned study, gics presoaking in saline solution represented a weakness. once we consider the patients who undergo an early reoperation for bleeding (or other reasons) in the immediate postoperative course, the risk of a swi is increased. in their prospective randomized trial, friberg et al16 investigated the beneficial effect of gics in 2000 patients undergoing cardiac surgery (table 2); their results showed a reduced incidence of swi at two months in the treatment group (4.3% vs 9%, rr 0.47; 95% confidence interval [ci] 0.33–0.68, p  0.001). this result appears to be even more important if we consider that the treatment group had a higher number of early reoperations for bleeding (4% vs 2.3%, p = 0.03), thus suggesting that the use of a gics is effective in reducing swi even in the presence of early resternotomy. the authors repeated their analysis, excluding the patients reoperated for bleeding (or other reasons) and those who died within two months; in this subgroup, there was still a significantly lower incidence of sswis (2.5% vs 6.7%, p = 0.001) and dswis (2.1 vs 3.3%, p = 0.088) in the gics group. in 2012, creanor et al published a meta-analysis including randomized controlled trials, which had previously investigated the use of gics in cardiac surgery.36 one of the subanalyses of this study was conducted considering high-risk patients; a statistically significant difference between treatment and control group was found with regard to dswis (odds ratio [or] 0.62, 95% ci 0.39–0.98), while no difference was found with regard to any swi (or 0.60, 95% ci 0.24–1.52).36 in a recent study published in 2014, benedetto and raja33 identified the most important risk factors for dswi after cardiac surgery. in their large analysis involving 8750 cardiac surgical patients, the authors identified several variables that can have an impact on the development of this complication: female gender, obesity, insulin-dependent diabetes, need for reexploration, isolated or combined cabg, and the use of bilateral mammary artery.33 using these variables, the authors were able to create a risk score to guide the use of gics, demonstrating that an individual assessment of dswi risk is realizable. patients were classified as low, moderate, or high risk for dswi, depending on their baseline score (table 3 and fig. 2). according to the authors’ findings, the use of gics allowed to reclassify patients with moderate predicted risk of dswi in the low-risk class. on the other hand, in high-risk patients, the observed dswi incidence was lower than expected when a gics was implanted, but the authors concluded that these patients should still be considered at higher risk.33 till date, this score appears to be the only available guide in the decisionmaking process for the use of gics in cardiac surgery. conclusion gicss represent a promising option to prevent the occurrence of swis after heart surgery. their main advantage is related to a high local concentration of gentamicin to the surgical site, combined with low serum levels of the drug, thus avoiding systemic side effects. most of the current knowledge on their use in cardiac surgery derives from underpowered studies, with different techniques of application. hence, the real clinical beneficial effects in high-risk patients undergoing cardiac surgery have not been completely established, although there seems to be a tendency toward a reduced incidence of swis with their use. this is particularly evident when the device is not soaked in saline solution, as the results appear to be negatively affected by this maneuver. table 3. score to calculate dswi predicted risk. variable points female gender 26 insulin-dependent diabetes mellitus 20 cabg (isolated or combined) 19 bima harvesting 15 need for re-exploration 51 bmi see figure 2 notes: data from ref. 33. overall score  136: low risk of dswi. overall score between 136 and 199: moderate risk of dswi. overall score  199: high risk of dswi. abbreviations: cabg, coronary artery bypass grafting; bmi, body mass index. http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 effectiveness of gentamicin-impregnated collagen sponge 13drug target insights 2016:10(s1) currently, there are no guidelines on the use of gics in cardiac surgery; the scoring system, proposed by benedetto and raja,33 seems to be the most reliable tool presently available for the indication of their use in high-risk patients. however, further prospective randomized controlled trials, particularly in high-risk patients, are needed to better clarify the impact of gics in preventing swis. author contributions conceived the concepts: fr and vdb. wrote the first draft of the manuscript: fr, vdb, and cz. contributed to the writing of the manuscript: pc, gg, and rm. agreed with manuscript results and conclusions: fr, cz, gg, rm, pc, and vdb. jointly developed the structure and arguments for the paper: fr, cz, gg, rm, pc, and vdb. made critical revisions and approved the final version: fr, cz, gg, rm, pc, and vdb. all the authors reviewed and approved the final manuscript. r efer ences 1. mauermann wj, sampathkumar p, thompson rl. sternal wound infections. best pract res clin anaesthesiol. 2008;22(3):423–436. 2. tang ghl, maganti m, weisel rd, borger ma. prevention and management of deep sternal wound infection. semin thorac cardiovasc surg. 2004;16(1):62–69. 3. mavros mn, mitsikostas pk, alexiou vg, peppas g, falagas me. gentamicin collagen sponges for the prevention of sternal wound infection: a meta-analysis of randomized controlled trials. j thorac cardiovasc surg. 2012;144(5):1235–1240. 4. abboud cs, wey sb, baltar vt. risk factors for mediastinitis after cardiac surgery. ann thorac surg. 2004;77(2):676–683. 5. lador a, nasir h, mansur n, et al. antibiotic prophylaxis in cardiac surgery: systematic review and meta-analysis. j antimicrob chemother. 2012;67(3):541–550. 6. mishra p, ashoub a, salhiyyah k, et al. role of topical application of gentamicin containing collagen implants in cardiac surgery. j cardiothorac surg. 2014; 9(1):122. 7. beckmann a, doebler k, schaefer e, koetting j, gastmeier p, graf k. sternal surgical site infection prevention—is there any room for improvement? eur j cardiothorac surg. 2011;40(2):347–351. 8. ruszczak z, friess w. collagen as a carrier for on-site delivery of antibacterial drugs. adv drug deliv rev. 2003;55(12):1679–1698. 9. horan tc, andrus m, dudeck ma. cdc/nhsn surveillance definition of health care-associated infection and criteria for specific types of infections in the acute care setting. am j infect control. 2008;36(5):309–332. 10. national institute for health and clinical excellence. nice clinical guideline 74: surgical site infection. national institute for health and clinical excellence, manchester, united kingdom; 2008:1–31. 11. gårdlund b, bitkover cy, vaage j. postoperative mediastinitis in cardiac surgery— microbiology and pathogenesis. eur j cardiothorac surg. 2002;21(5):825–830. 12. charbonneau h, maillet jm, faron m, et al. mediastinitis due to gram-negative bacteria is associated with increased mortality. clin microbiol infect. 2013;20(3): o197–o202. 13. friberg ö, dahlin l-g, källman j, kihlström e, söderquist b, svedjeholm r. collagen-gentamicin implant for prevention of sternal wound infection; longterm follow-up of effectiveness. interact cardiovasc thorac surg. 2009;9(3): 454–458. 14. lepelletier d, bourigault c, roussel jc, et al. epidemiology and prevention of surgical site infections after cardiac surgery. méd mal infect. 2013;43(10): 403–409. 15. filsoufi f, castillo jg, rahmanian pb, et al. epidemiology of deep sternal wound infection in cardiac surgery. j cardiothorac vasc anesth. 2009;23(4):488–494. 16. friberg ö, svedjeholm r, söderquist b, granfeldt h, vikerfors t, källman j. local gentamicin reduces sternal wound infections after cardiac surgery: a randomized controlled trial. ann thorac surg. 2005;79(1):153–161; discussion 161–162. 17. darouiche ro. treatment of infections associated with surgical implants. n engl j med. 2004;350(14):1422–1429. 18. olsson e, friberg ö, venizelos n, koskela a, källman j, söderquist b. coagulase-negative staphylococci isolated from sternal wound infections after cardiac surgery: attachment to and accumulation on sternal fixation stainless steel wires. apmis. 2007;115(2):142–151. 19. brunton l, chabner b, knollman b. goodman and gilman’s the pharmacological basis of therapeutics, (12th ed.). new york: mcgraw-hill. 2011; 2011. 20. pagkalis s, mantadakis e, mavros mn, ammari c, falagas me. pharmacological considerations for the proper clinical use of aminoglycosides. drugs. 2011; 71(17):2277–2294. 21. leyh rg, bartels c, sievers hh. adjuvant treatment of deep sternal wound infection with collagenous gentamycin. ann thorac surg. 1999;68(5):1648–1651. 22. wahlig h. gentamicin-pmma beads: a drug delivery system in the treatment of chronic bone and soft tissue infections. j antimicrob chemother. 1982; 10(5):463–465. 23. sendil d, gürsel i, wise dl, hasirci v. antibiotic release from biodegradable phbv microparticles. j control release. 1999;59(2):207–217. 24. türesin f, gürsel i, hasirci v. biodegradable polyhydroxyalkanoate implants for osteomyelitis therapy: in vitro antibiotic release. j biomater sci polym ed. 2001;12(2):195–207. 25. brown jc, timpl r. the collagen superfamily. int arch allergy immunol. 1995; 107(4):484–490. 26. ramshaw ja, werkmeister ja, glattauer v. collagen-based biomaterials. biotechnol genet eng rev. 1996;13:335–382. 27. rao kp. recent developments of collagen-based materials for medical applications and drug delivery systems. j biomater sci polym ed. 1995;7(7):623–645. 28. schersten h. modified prophylaxis for preventing deep sternal wound infection after cardiac surgery. apmis. 2007;115(9):1025–1028. 29. kozioł m, targońska s, stążka j, kozioł-montewka m. gentamicin-impregnated collagen sponge for preventing sternal wound infection after cardiac surgery. kardiochir torakochirurgia pol. 2014;11:21–25. 30. schimmer c, özkur m, sinha b, et al. gentamicin-collagen sponge reduces sternal wound complications after heart surgery: a controlled, prospectively randomized, double-blind study. j thorac cardiovasc surg. 2012;143(1):194–200. 31. bennett-guerrero e, ferguson tb, lin m, et al. effect of an implantable gentamicin-collagen sponge on sternal wound infections following cardiac surgery: a randomized trial. jama. 2010;304(7):755–762. 32. lovering am, sunderland j. impact of soaking gentamicin-containing collagen implants on potential antimicrobial efficacy. int j surg. 2012;10(suppl 1): s2–s4. 33. benedetto u, raja sg. scoring system to guide decision making for the use of gentamicin-impregnated collagen sponge to prevent deep sternal wound infection. j thorac cardiovasc surg. 2014;148(5):2390.e1–2396.e1. 34. toumpoulis ik, theakos n, dunning j. does bilateral internal thoracic artery harvest increase the risk of mediastinitis? interact cardiovasc thorac surg. 2007; 6(6):787–791. 35. birgand g, radu c, alkhoder s, et al. does a gentamicin-impregnated collagen sponge reduce sternal wound infections in high-risk cardiac surgery patients? interact cardiovasc thorac surg. 2013;16(2):134–141. 36. creanor s, barton a, marchbank a. effectiveness of a gentamicin impregnated collagen sponge on reducing sternal wound infections following cardiac surgery: a meta-analysis of randomised controlled trials. ann r coll surg engl. 2012;94(4):227–231. figure 2. baseline score points according to bmi (data from ref. 33). abbreviation: bmi, body mass index. http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 dti drug target insights 2022; 16: 12-16issn 1177-3928 | doi: 10.33393/dti.2022.2422original research article drug target insights issn 1177-3928 www.aboutscience.eu/dti © 2022 the authors. this article is published by aboutscience and licensed under creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). commercial use is not permitted and is subject to publisher’s permissions. full information is available at www.aboutscience.eu the association of esbl escherichia coli with mortality in patients with escherichia coli bacteremia at the emergency department pariwat phungoen1, jessada sarunyaparit1, korakot apiratwarakul1, lumyai wonglakorn2, atibordee meesing3, kittisak sawanyawisuth3 1department of emergency medicine, faculty of medicine, khon kaen university, khon kaen thailand 2department of microbiology, faculty of medicine, khon kaen university, khon kaen thailand 3department of medicine, faculty of medicine, khon kaen university, khon kaen thailand abstract background: escherichia coli is a common bloodstream infection pathogen in the emergency department (ed). patients with extended-spectrum beta-lactamase (esbl) e. coli have a higher risk of morbidity. however, there is still debate surrounding esbl e. coli-associated mortality in community, intensive care unit, and tertiary care settings. in addition, there have been few studies regarding mortality in esbl e. coli in ed settings, and results have been contradictory. methods: this was a retrospective cohort study conducted at the department of emergency medicine, faculty of medicine, khon kaen university in thailand aimed at evaluating the possible association between esbl e. coli bacteremia and mortality in the ed. the inclusion criteria were age 18 years or over, clinical presentation suspicious of infection, and positive blood culture for e. coli. predictors for mortality were analyzed by logistic regression analysis. results: during the study period, 273 patients presented at the ed with hemoculture positive for e. coli. of those, 27 (9.89%) died. five factors remained in the final model, of which plasma glucose levels, serum lactate levels, and esbl e. coli were significantly associated with 28-day mortality in the ed with adjusted odds ratios of 0.970, 1.258, and 12.885, respectively. plasma glucose of less than 113 mg/dl yielded a sensitivity of 80.95% and specificity of 64.29%, while serum lactate over 2.4 mmol/l had a sensitivity of 81.48% and specificity of 45.50%. conclusion: esbl e. coli, plasma glucose, and serum lactate levels were associated with 28-day mortality in patients with e. coli bacteremia presenting at the ed. keywords: extended-spectrum beta-lactamase-producing escherichia coli, glucose, lactate received: may 11, 2022 accepted: september 19, 2022 published online: october 17, 2022 corresponding author: kittisak sawanyawisuth department of medicine khon kaen university khon kaen 40002 thailand kittisak@kku.ac.th rate of e. coli bsi to be 9.6%. male patients aged 70 years or older are at higher risk of 30-day mortality with adjusted incidence rate ratios of 1.26 and 10.35 (3). another study found a mortality rate of 30.6% in patients infected with extendedspectrum beta-lactamase (esbl) e. coli vs 22.2% in those infected with non-esbl strains or klebsiella pneumoniae (4). the prevalence of drug-resistant gram-negative bacteria is increasing, particularly in in-hospital, intensive care unit (icu), and tertiary care settings (5-8). a study from a multispecialty hospital in india found that rates of multidrug resistant gram-negative bacteria increased from 26.16% in 2012 to 33.33% in 2014 (6). additionally, urinary tract infection patients with resistant enterobacteriaceae have been shown to be 1.447 times more likely to have severe sepsis or septic shock at presentation than those with nonresistant strains (9). data regarding the association of esbl e. coli and mortality in community, icu, and tertiary care settings have been inconclusive. two studies conducted in community settings, for example, found differences in mortality between introduction bloodstream infection (bsi) with gram-negative bacteria is common in the emergency department (ed), accounting for 39.4% of ed patients with suspected infection (1). a study from china found that escherichia coli was the most common gram-negative bsi in 3,199 patients and accounted for 34.3% of cases (2). one population-based study found the mortality https://doi.org/10.33393/dti.2022.2422 https://creativecommons.org/licenses/by-nc/4.0/legalcode mailto:kittisak@kku.ac.th phungoen et al drug target insights 2022; 16: 13 © 2022 the authors. published by aboutscience www.aboutscience.eu patients with esbl and non-esbl bacteremia (10,11), whereas a study from a tertiary care setting found comparable rates (9.7% vs 9.2%), as did a study in a teaching hospital in china (12,13). however, another study in a teaching hospital in japan found higher rate of mortality in patients with esbl strains (14), as did a study in an icu (37.5% vs 15.6%; p = 0.04) (15). this study thus aimed to evaluate if esbl e. coli bacteremia was associated with mortality in an ed setting. methods this was a retrospective cohort study conducted at the department of emergency medicine, faculty of medicine, khon kaen university in thailand as part of an ed infection project. the inclusion criteria were age 18 years or over, clinical presentation suspicious of infection, and positive blood culture for e. coli. patients who received prophylactic antibiotics, presented with cardiac arrest or symptoms related to trauma, were referred from other hospitals, or had missing clinical data were excluded. the study period was between 2016 and 2018. eligible patients were selected from the hospital database. we reviewed participants’ clinical data at the time of presentation as well as mortality data over the following 28 days. clinical data included baseline characteristics, laboratory results, and treatment. baseline characteristics reviewed were age, sex, comorbid diseases, charlson comorbidity index, physical signs, and quick sepsis related organ failure assessment (qsofa) score. laboratory results included complete blood count, chemistry, arterial blood gas, serum lactate levels, and blood culture results (for esbl e. coli positivity). the primary outcome was 28-day mortality. statistical analyses eligible patients were categorized into two groups by mortality. descriptive statistics were used to calculate differences between the two groups. predictors for mortality were analyzed using logistic regression analysis. univariate logistic analysis was used to calculate the unadjusted odds ratio with 95% confidence interval and p value for each factor. factors with a p value less than 0.05 by univariate logistic regression analysis or those that were clinically significant were subsequently subjected to stepwise, multivariate logistic regression analysis. the final model was tested for goodness of fit using the hosmer-lemeshow method. results were reported as unadjusted/adjusted odds ratios with their 95% confidence intervals. a numerical predictor for mortality as an appropriate diagnostic cutoff point was computed with its sensitivity and specificity. all statistical analyses were performed using stata version 10.1 (college station, texas, usa). results during the study period, 273 patients presented at the ed with hemoculture positive for e. coli. of those, 27 (9.89%) died. in terms of baseline characteristics and physical signs, there were 12 factors that differed significantly between those who survived and those who died (tab. i). for example, nonsurvivors had a significantly higher charlson comorbidity index (5 vs 4), respiratory rate (28 vs 24 breaths/min), and qsofa score (2 vs 1), but oxygen saturation at presentation was lower (96% vs 97%; p 0.040). qsofa scores were significantly higher in those who died than those who survived (2 vs 1; p < 0.001). with regard to laboratory tests and treatment, seven factors differed significantly between groups (tab. ii). for example, the nonsurvival group had significantly lower levels of serum bicarbonate (17 vs 21 meq/l) and plasma glucose (94 vs 131 mg/dl), higher serum lactate levels (4.5 vs 2.6 mmol/l), and a greater percentage of patients with esbl table i baseline characteristics of patients with escherichia coli bacteremia presenting at the emergency department categorized by mortality at 28 days factors survivors n = 246 nonsurvivors n = 27 p value age, years 66 (18-100) 73 (19-93) 0.161 male sex 125 (50.81) 10 (37.04) 0.224 comorbid diseases liver disease 50 (20.33) 10 (37.04) 0.053 diabetes 57 (23.85) 2 (7.41) 0.053 ckd (moderate-severe) 24 (9.76) 4 (14.81) 0.499 solid organ tumor 74 (30.08) 15 (55.56) 0.010 palliative care 4 (1.63) 4 (14.81) 0.004 leukemia 2 (0.81) 1 (3.70) 0.269 lymphoma 2 (0.81) 2 (7.41) 0.050 hypertension 90 (36.59) 7 (25.93) 0.299 hiv infection 2 (0.81) 0 0.999 cholangiocarcinoma 34 (13.82) 8 (29.63) 0.045 charlson comorbidity index 4 (0-12) 5 (1-12) <0.001 temperature, °c 38.6 (35.9-41.5) 38.2 (35.6-41.0) 0.184 pulse rate, beats/min 96 (58-190) 96 (52-148) 0.898 respiratory rate, breaths/ min 24 (18-50) 28 (18-40) 0.008 sbp, mm hg 126 (64-218) 112 (80-167) 0.003 dbp, mm hg 70 (33-112) 67 (37-95) 0.071 map, mm hg 91 (48-138) 80 (56-119) 0.009 oxygen saturation, % 97 (60-100) 96 (65-100) 0.040 gcs 15 (4-15) 15 (7-15) <0.001 sepsis score qsofa 1 (0-3) 2 (1-3) <0.001 data are presented as median (range) or number (percentage). ckd = chronic kidney disease; dbp = diastolic blood pressure; gcs = glasgow coma scale; map = mean arterial pressure; qsofa = quick sepsis related organ failure assessment; sbp = systolic blood pressure. data presented as number (percentage) unless indicated otherwise. esbl e. coli ed14 © 2022 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti e. coli (14.81% vs 2.03%) than the survival group. in addition, patients in the nonsurvival group underwent significantly more aggressive treatment (such as vasopressor treatment) and had a higher rate of icu admission. however, duration of hospital stay in the nonsurvival group was shorter (8 vs 11 days; p 0.013). five factors remained in the final model for predicting death (tab. iii). plasma glucose, serum lactate levels, and esbl e. coli were significantly associated with mortality, with adjusted odds ratios of 0.970, 1.258, and 12.885, respectively. the final model had a hosmer-lemeshow chi square of 6.73 (p = 0.565). plasma glucose of 113 mg/dl or lower yielded a sensitivity of 80.95% and specificity of 64.29%, while serum lactate level of over 2.4 mmol/l had a sensitivity of 81.48% and specificity of 45.50%. discussion the prevalence of esbl e. coli bacteremia at the ed in this study was 3.29%, which is lower than previously reported in community settings (6.7%-9.5%) (10,11,16). in addition to the difference in setting, these results may indicate differing rates among countries, as higher rates have been found in developed countries (south korea and spain). a previous report found that frequent visits to the ed increased the risk of esbl bacteremia by a factor of 9.98, including in those patients who had undergone previous antibiotic treatment. in thailand, the rate of previous antibiotic use may be lower than in some other countries. despite the inconsistency in the esbl e. coli mortality rate in other settings, this study found that patients with esbl e. coli had a 13 times higher risk of mortality than those with non-esbl strains. other factors associated with mortality in patients with e. coli infection may be personal characteristics and inappropriate antibiotic use. a report from korea found that presenting with septic shock or malignancy increased mortality risk by 26.6 and 11.9 times, respectively, while another study found that mortality rates were comparable in patients with esbl and nonesbl e. coli if antibiotics were administered appropriately (p = 0.23) (11,15). hypoglycemia has been shown to be related with higher mortality in sepsis patients and critically ill patients (17-19). although the causal relationship between hypoglycemia and mortality is not well understood, several mechanisms have been proposed including the inhibition of the physiological responses of hormones such as insulin and epinephrine, table ii laboratory results and treatment of patients with escherichia coli bacteremia presenting at the emergency department categorized by mortality at 28 days factors survivors n = 246 nonsurvivors n = 27 p value hb, g/dl 11.0 (4.6-16.0) 9.6 (4.8-13.8) 0.002 wbc, ×103/mm3 33.8 (13.0-51.7) 26.6 (14.9-41.9) 0.010 platelet, ×106 179 (4-584) 138 (13-451) 0.098 bun, mg/dl 17.9 (3.7-144.8) 27.3 (6.7-153.2) 0.009 creatinine, mg/dl 1.1 (0.4-10.4) 1.5 (0.5-11.1) 0.118 bicarbonate, meq/l 21 (7-30) 17 (7-27) <0.001 total bilirubin, mg/dl 1.4 (0.2-33.8) 2.1 (0.3-33.8) 0.251 glucose, mg/dl 131 (53-548) 94 (35-172) <0.001 pao 2 , mmhg 76 (23-512) 89 (33-253) 0.702 ph 7.44 (7.16-7.58) 7.40 (7.11-7.56) 0.194 lactate level, mmol/l 2.6 (0.5-18.3) 4.5 (1.3-17.9) 0.003 esbl e. coli 5 (2.03) 4 (14.81) 0.007 treatment mechanical ventilator 21 (8.54) 5 (18.52) 0.155 icu admission 83 (3.74) 17 (62.96) 0.005 vasopressor* 62 (25.20) 21 (77.78) <0.001 los 11 (2-56) 8 (1-54) 0.013 data are presented as median (range) or number (percentage). bun = blood urea nitrogen; esbl e. coli = extended-spectrum beta-lactamase producing escherichia coli; hb = hemoglobin; icu = intensive care unit; los = length of stay; pao 2 = partial pressure of oxygen; ph = power of hydrogen; wbc = white blood cell. *indicates that the patient received norepinephrine, adrenaline, or dopamine. table iii factors associated with a 28-day mortality in patients with escherichia coli bacteremia presenting at the emergency department factors unadjusted odds ratio (95% confidence interval) adjusted odds ratio (95% confidence interval) oxygen saturation 0.933 (0.883, 0.985) 0.934 (0.860, 1.015) hemoglobin 0.732 (0.605, 0.885) 0.856 (0.662, 1.106) plasma glucose 0.971 (0.955, 0.987) 0.970 (0.954, 0.987) serum lactate level 1.185 (1.067, 1.317) 1.258 (1.090, 1.451) esbl e. coli* 8.382 (2.103, 33.407) 12.885 (1.082, 153.338) age 1.008 (0.981, 1.036) not retained sex 0.569 (0.251, 1.293) not retained liver disease 2.306 (0.995, 5.344) not retained diabetes 0.255 (0.058, 1.111) not retained cholangiocarcinoma 2.625 (1.065, 6.469) not retained solid organ tumor 2.905 (1.297, 6.508) not retained qsofa 3.761 (1.974, 7.164) not retained dbp 0.974 (0.947, 1.001) not retained gcs 0.770 (0.630, 0.941) not retained hemoglobin 0.732 (0.605, 0.885) not retained wbc 1.014 (0.995, 1.032) not retained serum bicarbonate 0.836 (0.762, 0.918) not retained factors in the model included age, sex, liver disease, diabetes, cholangiocarcinoma, solid organ tumor, qsofa, dbp, gcs, wbc, and serum bicarbonate. dbp = diastolic blood pressure; esbl = extended-spectrum beta-lactamase; gcs = glasgow coma scale; qsofa = quick sepsis related organ failure assessment; wbc = white blood cell. phungoen et al drug target insights 2022; 16: 15 © 2022 the authors. published by aboutscience www.aboutscience.eu increased inflammatory response, and cellular damage from glucose administration (19). previous studies have also found low plasma glucose to be associated with mortality in patients with sepsis (11,20). this study found that glucose of 113 mg/dl or lower yielded a sensitivity of 80.95% compared to a previous study, in which plasma glucose of 40-69 mg/dl resulted in an adjusted odds ratio of 3.43 (95% confidence interval of 1.51, 7.82) for mortality (19,20). these results may imply that patients with e. coli bacteremia and hypoglycemia may have as high of a risk of mortality as other patients with sepsis. the different plasma glucose cutoff points in the two studies may be due to differences in study population. this study enrolled only patients with e. coli bacteremia at the ed, while the previous study included patients with sepsis, which may have been caused by various pathogens. the plasma glucose cutoff point in this study may be more specific to patients with e. coli bacteremia at the ed. as previously reported, serum lactate is an indicator for mortality in patients with infection at the ed (21-23). a previous study found that serum lactate greater than 4 mmol/l was associated with higher mortality than at 2 mmol/l (40.7% vs 2.7%) (24). in this study, we found that serum lactate over 2.4 mmol/l yielded sufficient sensitivity to predict fatality in patients with e. coli bacteremia at the ed. another study found a serum lactate cutoff point of 5.80 mmol/l in patients with necrotizing fasciitis (25). this indicates that e. coli bacteremia may be severe and that the serum lactate cutoff point may vary depending on the causative agents. although oxygen saturation and hemoglobin were significantly associated with mortality by univariate logistic regression analysis (tab. iii), they were no longer significant in the final model. these results may indicate that neither factor was a strong predictor compared with the other three. additionally, there might have been some related confounding factors. other factors included in the model that had p values of less than 0.05 by univariate analysis were not retained in the final model for the same reasons. some comorbid diseases, such as diabetes, were found to be significant predictors for mortality in a previous observational study (26). however, comorbid diseases were not significant in this study, as previously mentioned. additionally, the model used in this study differed from that in the previous study. in this study, we included clinical factors such as esbl e. coli in the model, while the previous study did not include esbl e. coli and included treatment-related factors such as peak inspiratory pressure and positive end expiratory pressure. there were some limitations to this study. first, the ed at which it was conducted was a single site at a university hospital. further prospective studies in other settings may be required to confirm the results. in addition, this was an exploratory study without validation. the final predictive model included more factors than event outcomes. there were five factors in the model with only 27 nonsurvivors, resulting in a ratio of more than 1:10. moreover, the total number of patients in the final model was 130. these limitations could have caused the model to be unbalanced or biased. however, the final model had a high goodness of fit. another limitation was that some factors were not studied such as previous antibiotic use, previous history of resistant pathogens, or special conditions (27-35). finally, mortality was defined as 28-day mortality. esbl e. coli, plasma glucose, and serum lactate levels were associated with 28-day mortality in patients with e. coli bacteremia presenting at the ed. acknowledgments the authors would like to acknowledge dr. dylan southard for editing the manuscript via the khon kaen university publication clinic (thailand). disclosures conflict of interest: the authors declare that they have no conflicts of interest. financial support: this study was funded by a grant from the khon kaen university, faculty of medicine, thailand (grant number: mn63307). references 1. chase m, klasco rs, joyce nr, donnino mw, wolfe re, shapiro ni. predictors of bacteremia in emergency department patients with suspected infection. am j emerg med. 2012;30(9): 1691-1697. crossref pubmed 2. zhu q, zhu m, li c, et al. epidemiology and microbiology of gram-negative bloodstream infections in a tertiary-care hospital in beijing, china: a 9-year retrospective study. expert rev anti infect ther. 2021;19(6):769-776. crossref pubmed 3. mackinnon mc, mcewen sa, pearl dl, et al. mortality in escherichia coli bloodstream infections: a multinational population-based cohort study. bmc infect dis. 2021;21(1):606. crossref pubmed 4. sianipar o, asmara w, dwiprahasto i, mulyono b. mortality risk of bloodstream infection caused by either escherichia coli or klebsiella pneumoniae producing extended-spectrum β-lactamase: a prospective cohort study. bmc res notes. 2019; 12(1):719. crossref pubmed 5. hawkey pm. multidrug-resistant gram-negative bacteria: a product of globalization. j hosp infect. 2015;89(4):241-247. crossref pubmed 6. nagvekar v, sawant s, amey s. prevalence of multidrug resistant gram-negative bacteria cases at admission in a multispeciality hospital. j glob antimicrob resist. 2020;22:457-461. crossref pubmed 7. morris s, cerceo e. trends, epidemiology, and management of multi-drug resistant gram-negative bacterial infections in the hospitalized setting. antibiotics (basel). 2020;9(4):e196. crossref pubmed 8. siwakoti s, subedi a, sharma a, baral r, bhattarai nr, khanal b. incidence and outcomes of multidrug-resistant gram-negative bacteria infections in intensive care unit from nepal – a prospective cohort study. antimicrob resist infect control. 2018; 7(1):114. crossref pubmed 9. lee yc, hsiao cy, hung mc, et al. bacteremic urinary tract infection caused by multidrug-resistant enterobacteriaceae are associated with severe sepsis at admission: implication for empirical therapy. medicine (baltimore). 2016;95(20):e3694. crossref pubmed 10. kang ci, song jh, chung dr, et al; korean network for study of infectious diseases (konsid). risk factors and treatment outcomes of community-onset bacteraemia caused by extendedspectrum beta-lactamase-producing escherichia coli. int j antimicrob agents. 2010;36(3):284-287. crossref pubmed https://doi.org/10.1016/j.ajem.2012.01.018 https://www.ncbi.nlm.nih.gov/pubmed/22626814 https://doi.org/10.1080/14787210.2021.1848544 https://www.ncbi.nlm.nih.gov/pubmed/33187451 https://doi.org/10.1186/s12879-021-06326-x https://www.ncbi.nlm.nih.gov/pubmed/34172003 https://doi.org/10.1186/s13104-019-4751-9 https://www.ncbi.nlm.nih.gov/pubmed/31675991 https://doi.org/10.1016/j.jhin.2015.01.008 https://www.ncbi.nlm.nih.gov/pubmed/25737092 https://doi.org/10.1016/j.jgar.2020.02.030 https://www.ncbi.nlm.nih.gov/pubmed/32165287 https://doi.org/10.3390/antibiotics9040196 https://www.ncbi.nlm.nih.gov/pubmed/32326058 https://doi.org/10.1186/s13756-018-0404-3 https://www.ncbi.nlm.nih.gov/pubmed/30275945 https://doi.org/10.1097/md.0000000000003694 https://www.ncbi.nlm.nih.gov/pubmed/27196480 https://doi.org/10.1016/j.ijantimicag.2010.05.009 https://www.ncbi.nlm.nih.gov/pubmed/20580534 esbl e. coli ed16 © 2022 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti 11. park sh, choi sm, lee dg, et al. emergence of extendedspectrum β-lactamase-producing escherichia coli as a cause of community-onset bacteremia in south korea: risk factors and clinical outcomes. microb drug resist. 2011;17(4):537-544. crossref 12. namikawa h, yamada k, fujimoto h, et al. clinical characteristics of bacteremia caused by extended-spectrum beta-lactamaseproducing escherichia coli at a tertiary hospital. intern med. 2017;56(14):1807-1815. crossref pubmed 13. xiao t, wu z, shi q, et al. a retrospective analysis of risk factors and outcomes in patients with extended-spectrum betalactamase-producing escherichia coli bloodstream infections. j glob antimicrob resist. 2019;17:147-156. crossref pubmed 14. hattori h, maeda m, nagatomo y, et al. epidemiology and risk factors for mortality in bloodstream infections: a single-center retrospective study in japan. am j infect control. 2018;46(12): e75-e79. crossref pubmed 15. haruki y, hagiya h, haruki m, sugiyama t. clinical characteristics and outcome of critically ill patients with bacteremia caused by extended-spectrum β-lactamase-producing and non-producing escherichia coli. j infect chemother. 2018;24(11):944-947. crossref pubmed 16. rodríguez-baño j, picón e, gijón p, et al; spanish network for research in infectious diseases (reipi). community-onset bacteremia due to extended-spectrum beta-lactamase-producing escherichia coli: risk factors and prognosis. clin infect dis. 2010;50(1):40-48. crossref pubmed 17. yao rq, ren c, wu gs, zhu yb, xia zf, yao ym. is intensive glucose control bad for critically ill patients? a systematic review and meta-analysis. int j biol sci. 2020;16(9):1658-1675. crossref pubmed 18. wang j, zhu ck, yu jq, tan r, yang pl. hypoglycemia and mortality in sepsis patients: a systematic review and meta-analysis. heart lung. 2021;50(6):933-940. crossref pubmed 19. park s, kim dg, suh gy, et al. mild hypoglycemia is independently associated with increased risk of mortality in patients with sepsis: a 3-year retrospective observational study. crit care. 2012;16(5):r189. crossref pubmed 20. van den berghe g, wilmer a, hermans g, et al. intensive insulin therapy in the medical icu. n engl j med. 2006;354(5):449-461. crossref pubmed 21. shapiro ni, howell md, talmor d, et al. serum lactate as a predictor of mortality in emergency department patients with infection. ann emerg med. 2005;45(5):524-528. crossref pubmed 22. vorwerk c, loryman b, coats tj, et al. prediction of mortality in adult emergency department patients with sepsis. emerg med j. 2009;26(4):254-258. crossref pubmed 23. puskarich ma, illich bm, jones ae. prognosis of emergency department patients with suspected infection and intermediate lactate levels: a systematic review. j crit care. 2014;29(3): 334-339. crossref pubmed 24. bou chebl r, el khuri c, shami a, et al. serum lactate is an independent predictor of hospital mortality in critically ill patients in the emergency department: a retrospective study. scand j trauma resusc emerg med. 2017;25(1):69. crossref pubmed 25. chang cp, fann wc, wu sr, lin cn, hsiao ct. lactate on emergency department arrival as a predictor of in-hospital mortality in necrotizing fasciitis: a retrospective study. j orthop surg res. 2019;14(1):73. crossref pubmed 26. rezoagli e, mcnicholas ba, madotto f, pham t, bellani g, laffey jg; lung safe investigators, the esicm trials group. presence of comorbidities alters management and worsens outcome of patients with acute respiratory distress syndrome: insights from the lung safe study. ann intensive care. 2022;12(1):42. crossref pubmed 27. charoentanyarak s, sawunyavisuth b, deepai s, sawanyawisuth k. a point-of-care serum lactate level and mortality in adult sepsis patients: a community hospital setting. j prim care community health. 2021;12:21501327211000233. crossref pubmed 28. jeerasuwannakul b, sawunyavisuth b, khamsai s, sawanyawisuth k. prevalence and risk factors of proteinuria in patients with type 2 diabetes mellitus. asia-pac j sci technol. 2021 jul 1; 26(04):apst-26-04-02. online (accessed may 2022). 29. soontornrungsun b, khamsai s, sawunyavisuth b, et al. obstructive sleep apnea in patients with diabetes less than 40 years of age. diabetes metab syndr. 2020;14(6):1859-1863. crossref pubmed 30. tongdee s, sawunyavisuth b, sukeepaisarnjaroen w, boonsawat w, khamsai s, sawanyawisuth k. clinical factors predictive of appropriate treatment in copd: a community hospital setting. drug target insights. 2021;15:21-25. crossref pubmed 31. namwaing p, chaisuksant s, sawadpanich r, et al. oxygen saturation is associated with recurrent primary spontaneous pneumothorax treated by intercostal chest drainage. asian j surg. 2022;45(1):431-434. crossref pubmed 32. namwaing p, chaisuksant s, sawadpanich r, et al. factors associated with duration of intercostal chest drainage in patients with primary spontaneous pneumothorax and the role of pulmonary rehabilitation. open access emerg med. 2021;13: 569-573. crossref pubmed 33. boonwang t, namwaing p, srisaphonphusitti l, et al. esports may improve cognitive skills in soccer players: a systematic review. asia-pac j sci technol. 2022;27(3). online (accessed may 2022). 34. ruangritkul p, tiamkao s, chainirun n, et al. the efficacy and safety profile of generic intravenous levetiracetam in a realworld setting. curr ther res clin exp. 2021;95:100648. crossref pubmed 35. sanlung t, sawanyawisuth k, silaruks s, et al. clinical characteristics and complications of obstructive sleep apnea in srinagarind hospital. j med assoc thai. 2020;103(1):36-39. online (accessed may 2022). https://doi.org/10.1089/mdr.2011.0072 https://doi.org/10.2169/internalmedicine.56.7702 https://www.ncbi.nlm.nih.gov/pubmed/28717075 https://doi.org/10.1016/j.jgar.2018.12.014 https://www.ncbi.nlm.nih.gov/pubmed/30634054 https://doi.org/10.1016/j.ajic.2018.06.019 https://www.ncbi.nlm.nih.gov/pubmed/30172607 https://doi.org/10.1016/j.jiac.2018.04.016 https://www.ncbi.nlm.nih.gov/pubmed/29803763 https://doi.org/10.1086/649537 https://www.ncbi.nlm.nih.gov/pubmed/19995215 https://doi.org/10.7150/ijbs.43447 https://www.ncbi.nlm.nih.gov/pubmed/32226310 https://doi.org/10.1016/j.hrtlng.2021.07.017 https://www.ncbi.nlm.nih.gov/pubmed/34433111 https://doi.org/10.1186/cc11674 https://www.ncbi.nlm.nih.gov/pubmed/23062226 https://doi.org/10.1056/nejmoa052521 https://www.ncbi.nlm.nih.gov/pubmed/16452557 https://doi.org/10.1016/j.annemergmed.2004.12.006 https://www.ncbi.nlm.nih.gov/pubmed/15855951 https://doi.org/10.1136/emj.2007.053298 https://www.ncbi.nlm.nih.gov/pubmed/19307384 https://doi.org/10.1016/j.jcrc.2013.12.017 https://www.ncbi.nlm.nih.gov/pubmed/24559577 https://doi.org/10.1186/s13049-017-0415-8 https://www.ncbi.nlm.nih.gov/pubmed/28705203 https://doi.org/10.1186/s13018-019-1108-y https://www.ncbi.nlm.nih.gov/pubmed/30841912 https://doi.org/10.1186/s13613-022-01015-7 https://www.ncbi.nlm.nih.gov/pubmed/35596885 https://doi.org/10.1177/21501327211000233 https://www.ncbi.nlm.nih.gov/pubmed/33733925 https://so01.tci-thaijo.org/index.php/apst/article/view/248718 https://doi.org/10.1016/j.dsx.2020.09.008 https://www.ncbi.nlm.nih.gov/pubmed/32992217 https://doi.org/10.33393/dti.2021.2291 https://www.ncbi.nlm.nih.gov/pubmed/34803374 https://doi.org/10.1016/j.asjsur.2021.07.021 https://www.ncbi.nlm.nih.gov/pubmed/34312054 https://doi.org/10.2147/oaem.s336637 https://www.ncbi.nlm.nih.gov/pubmed/34938130 https://so01.tci-thaijo.org/index.php/apst/article/view/257467 https://doi.org/10.1016/j.curtheres.2021.100648 https://www.ncbi.nlm.nih.gov/pubmed/34840633 http://www.jmatonline.com/index.php/jmat/article/view/10743 dti © 2020 the authors. this article is published by aboutscience and licensed under creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). any commercial use is not permitted and is subject to publisher’s permissions. full information is available at www.aboutscience.eu issn 1177-3928 drug target insights 2020; 14: 26-33 original research article doi: 10.33393/dti.2020.2103 targeting streptococcus pneumoniae udp-glucose pyrophosphorylase (ugpase): in vitro validation of a putative inhibitor monica sharma1, swati sharma1, pallab ray2, anuradha chakraborti1 1molecular genetics laboratory, department of experimental medicine and biotechnology, postgraduate institute of medical education & research (pgimer), chandigarh india 2bacteriology laboratory, department of medical microbiology, postgraduate institute of medical education & research (pgimer), chandigarh india abstract background: genome plasticity of streptococcus pneumoniae is responsible for the reduced efficacy of various antibiotics and capsular polysaccharide-based vaccines. therefore, targets independent of capsular types are sought to control the pneumococcal pathogenicity. udp-glucose pyrophosphorylase (ugpase) is one such desired candidate being responsible for the synthesis of udp-glucose, a sugar precursor in capsular biosynthesis and metabolic leloir pathway. being crucial to pneumococcal pathobiology, the effect of ugpase inhibition on virulence was evaluated in vitro. methods: a putative inhibitor, uridine diphosphate (udp), was evaluated for effective inhibitory concentration in s. pneumoniae and a549 cells, its efficacy and toxicity. the effect of udp on adherence and phagocytosis was measured in human respiratory epithelial (a549 and hep-2) and macrophage (thp1 and j774.a.1) cell lines respectively. results: a differential effective inhibitory concentration of udp for ugpase inhibition was observed in s. pneumoniae and a549 cells, that is, 5 and 100 µm respectively. udp treatments lowered percent cytotoxicity in pneumococcal-infected monolayers and didn’t exert adverse effects on viabilities. s. pneumoniae adherence to host cells decreased significantly with udp treatments. udp induced the secretion of interleukin (il)-1β, tumor necrosis factor (tnf)-α, il-6, and il-8 and increased pneumococcal phagocytosis. conclusion: our study shows udp-mediated decrease in the virulence of s. pneumoniae and demonstrates udp as an effective inhibitor of pneumococcal ugpase. keywords: capsule, galu, inhibitor, pneumococcus, udp, virulence received: february 26, 2020 accepted: july 30, 2020 published online: october 7, 2020 corresponding author: prof. anuradha chakraborti department of experimental medicine and biotechnology postgraduate institute of medical education & research (pgimer) chandigarh-160012, india superoxide14@gmail.com and non-ipds, for example, acute otitis media and sinusitis (1). pneumococcal infections are associated with significant morbidity and mortality globally, specifically in children younger than 5 years, elderly adults, and individuals with immune deficiencies. global disease burden and mortality estimates have documented 341,029 deaths in children aged below 5 years; 494,340 deaths in the elderly (aged above 70 years), and total 1,189,937 deaths among all ages due to pneumococcal infections (2-4). this huge burden of pneumococcal diseases is fueled by the rise of new capsular serotypes and spread of antimicrobial-resistant clones (5). introduction of the capsular polysaccharide (cps)based vaccines and pneumococcal conjugate vaccines had provided a little relief, but they still remain unsatisfactory due to limited serotype protection and replacement, restricted efficacy of vaccine at the mucosal surface, implementation issues, and high cost (6,7). hence, formulation of alternative preventive measures independent of serotypes introduction streptococcus pneumoniae (pneumococcus) is an important healthcare-associated pathogen being responsible for various invasive pneumococcal diseases (ipds), for example, bacteremia, pneumonia, septicemia, and meningitis, sharma et al 27 © 2020 the authors. published by aboutscience is inevitable for the better management of pneumococcal diseases. pneumococcal strains have been shown to synthesize a distinct serotype-specific polysaccharide capsule capable of dodging host immune components (8,9). the loss of capsule has been associated with rapid phagocytosis (10,11). genetic dissection of the cps/cap gene cluster encoding for cps had strongly advocated the involvement of other unlinked genes that are dispersed in chromosome in cps synthesis (12,13). one of the genes, galu, encodes for enzyme uridine diphosphate (udp)-glucose pyrophosphorylase (ugpase), and its role in the virulence of various gram-negatives and gram-positives is well studied (14-19). ugpase catalyzes the synthesis of udpglucose (udp-glc), which is an important glycosyl donor for the modification and interconversion of sugars in metabolic and capsular pathways (17,19,20). the galu mutants of pneumococcus have been shown to be acapsular despite the presence of functional cps genes (14,19). ugpase has been considered as a suitable target to control the pneumococcal virulence (17,21) as galu has been reported in all pneumococcal strains regardless of capsular types (14). also, mammalian ugpases have been evolutionarily unrelated to their prokaryotic counterparts, suggesting that its putative inhibitors would not be inimical to the host (17,21,22). the effects of ugpase inhibition in the pathogenicity of pneumococcus have not been elucidated so far despite the availability of evidence of gene mutation studies (19). a study has screened few chemical inhibitors for the inhibition of ugpase activity in a calorimetric assay from purified extracts (23), but no study is available on inhibitormediated host-pathogen interaction. therefore, for the selection of ugpase inhibitor, we modeled and analyzed the structure of s. pneumoniae ugpase using i-tasser (iterativethreading assembly refinement) server (http://zhang.bioinformatics.ku.edu/i-tasser) to delineate its tertiary structure, active site residues, and properties. in this study, we have studied the effect of ugpase inhibition on the virulence of s. pneumoniae during host-pathogen interactions and validated a putative inhibitor of pneumococcal ugpase for its inhibition potentials and efficacy in vitro. materials and methods this study was approved by the institute ethics committee (iec memo no. 87770-pg11-itrg/11384). respiratory epithelial (a549 and hep-2) and macrophage (j774.a.1 and thp1) cell lines were procured from the cell repository of national centre for cell sciences (nccs), pune, india. s. pneumoniae reference strains d39 (nctc 7466) and mtcc 655 (nctc 7465) were procured from microbial type culture collection (mtcc), imtech, chandigarh, india. a clinical blood isolate of serotype 19f was also used in the study as its occurrence has been reported frequently in ipds. clinical strain was isolated from the blood sample of a patient suffering from lower respiratory tract infection of s. pneumoniae only. cell culture a549 and thp1 cell lines were cultured and maintained in rpmi 1640, while hep-2 and j774.a.1 cell lines were cultured in minimal essential medium (mem) and dulbecco’s modified eagle’s medium (dmem), respectively, at 37°c with 5% co 2 . all the media were supplemented with 10% (v/v) heatinactivated fetal bovine serum (fbs), 0.15% (v/v) sodium bicarbonate, streptomycin (100 µg/ml), and penicillin (100 u/ml). antibiotic-free media were prepared for experiments involving bacterial infection. udp as ugpase inhibitor effective concentration of udp as inhibitor of ugpase was evaluated by ugpase assay (17) in both s. pneumoniae and a549 cells. log phase culture of s. pneumoniae and confluent cell monolayers were treated with udp at different concentrations for 1 hour at 37°c and 5% co 2 . a549 cells in phosphate-buffered saline (pbs) were lysed with three cycles of temperature-shock (5 minutes each). bacterial pellets were sonicated (3 cycles; pulse 10 seconds on, 30 seconds off) in ice bath. ugpase activity was assessed in total 1 ml reaction mixture with freshly prepared extracts (100 μl) at 25°c. reaction mixture constituted 50 mm tris-hcl buffer (ph 7.5), 16 mm mgcl 2 , 0.6 mm β-nicotinamide adenine dinucleotide phosphate (nadp), 0.6 mm udp-glucose, udp-glucose dehydrogenase (0.7 u), and phosphoglucomutase (0.07 u). reaction was initiated with the addition of sodium pyrophosphate (1.7 mm) and nadph formation was determined by measuring the increase in absorbance at 340 nm. blank consisted of reaction mixture without cell or bacterial extract. inhibitor toxicity to host cells a549 cell monolayers were treated with different concentrations of udp for 1 hour at 37°c and untreated cells were taken as reference. the effect of udp on viability of cells was evaluated using mtt assay (24). briefly, mtt solution (2.5 mg/ml; sigma) was added to monolayers and incubated for 4 hours. media containing mtt was discarded and dimethyl sulfoxide (dmso, 200 µl) was added. absorbance was recorded at 590 nm in microplate reader (bio-rad 680). expression of ugpase in host cells a549 and hep-2 monolayers were treated with udp at effective inhibiting dose for 1 hour at 37°c, 5% co 2 and untreated cells were taken as control. total protein was extracted from cell pellet by sonication (3 minutes: pulse 30 seconds on, 30 seconds off) in ice-chilled pbs and was quantitated. samples were electrophoresed in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) and separated proteins were transferred onto polyvinylidene difluoride (pvdf) membrane. membranes were processed and incubated with anti-ugp2 antibody (1:2,000, abcam) for 3 hours at 37°c followed by treatment with horseradish peroxidase (hrp)-conjugated secondary antibody (1:20,000) for 2 hours at 37°c. pierce® ecl western blotting peroxidase substrate (thermoscientific) was used for developing protein bands, which were quantitated with fluorchem m proteinsimple (bio-techne). gapdh as endogenous control was simultaneously blotted and analyzed. expression of ugpase was normalized with the value of gapdh in each sample. ugpase-mediated streptococcus pneumoniae virulence28 © 2020 the authors. published by aboutscience adherence assay efficacy of udp to inhibit the adherence of s. pneumoniae during infection with a549 and hep-2 monolayers was checked by flow cytometer (bd biosciences) (25). log phase cultures of pneumococcus were labeled with fluorescein isothiocyanate (fitc, 1.0 μg/μl) and infected onto monolayers at 100:1 multiplicity of infection (moi: bacteria/cell) in the presence and absence of inhibitor at effective dose. monolayers were incubated at 37°c at 5% co 2 for 2 hours followed by trypsinization (0.05%) and fixation with ice-cold paraformaldehyde (2%). samples were analyzed using facscan flow cytometer using cellquest 3.3 software (bd biosciences). mean fluorescence intensity (mfi) values were obtained from control samples (unlabeled host cells and bacteria). adherence was calculated from the mfi of host cell with adherent pneumococci divided by mfi of labeled pneumococci. effectiveness of inhibitor was evaluated from the adherence potential of s. pneumoniae to monolayers in the presence of inhibitor. phagocytosis assay fitc-labeled pneumococci were loaded (moi, 100:1) onto macrophage cells (thp1 and j774.a.1) in the presence and absence of udp for 1 hour at 37°c in 5% co 2 . cells were fixed with cold 2% paraformaldehyde. briefly, cells were collected, centrifuged, washed as required, and were suspended in icecold pbs. trypan blue (0.2%) was added to quench fluorescence of extracellularly attached pneumococci, just before analysis in flow cytometer (bd biosciences). mfi of fitc positive cells was calculated to assess the phagocytic activity in comparison to controls (unlabeled cells and bacteria). for opsonophagocytic assay (opa), bacterial cells (106 cfu/ ml) were labeled with fitc (1 μg/μl) after inactivation at 95°c for 5 minutes. nonviability was confirmed by overnight incubation of blood agar plates streaked with bacterial suspension (100 μl). labeled bacteria were incubated with pooled human sera (10% of 1:2 diluted in pbs) for 30 minutes at 37°c, 150 rpm. bacteria were further inoculated onto thp1 and j774.a.1 cells at moi 100:1 for 1 hour at 37°c and 5% co 2 . control tubes contained pbs or heat-inactivated (56°c for 45 minutes) pooled sera. the cells were obtained, washed, centrifuged, and fixed with cold 2% paraformaldehyde and analyzed in a flow cytometer (26). expression of postinfection cytokines a549 monolayers were incubated with effective dose of udp for 1 hour at 37°c, 5% co 2 . in another set, monolayers were treated with 10 μm mrs2578 for 30 minutes before incubation with udp (27). total ribonucleic acid (rna) was extracted from trypsinized cell pellets using trizol reagent followed by complementary deoxyribonucleic acid (cdna) synthesis using messenger rna revertaid™ first strand cdna synthesis kit (thermo scientific). expression of various cytokines (interleukin [il]-6, il-1β, il-8, tumor necrosis factor [tnf]-α) was analyzed using real-time polymerase chain reaction (pcr; roche lightcycler® 480) in reactions (10 μl) containing 50 ng cdna, 0.5 μm primer, 1x sybr mix, and deionized water. gene-specific primers were designed using primer3tool and pcrs were run in primer-specific cycling conditions and with appropriate negative controls for each reaction. statistical analysis each experiment was performed at least three times in duplicate or triplicate sets. graphpad prism 6.0 was used for statistical calculations. student’s t-test, or mann-whitney u-test or analysis of variance (anova) was applied as appropriate. a p value less than 0.05 was considered to represent a significant association. results putative inhibitor of ugpase the structure of s. pneumoniae ugpase was analyzed in silico using i-tasser server (http://zhang.bioinformatics. ku.edu/i-tasser; phd thesis). the tertiary structure, active site residues, and its substrate-binding properties were analyzed. the local conformational changes induced near the active site in response to binding of its natural substrate were analyzed. using information of the active site pocket of ugpase and literature-based evidence (11,14,17,21-23), udp was selected as a probable inhibitor of s. pneumoniae ugpase. in silico analysis showed that udp binding did not result in alteration in local conformation of enzymatic active site. the effective inhibitory concentrations of udp, its extent of inhibition, and probable toxicity to host cell lines were studied. the pneumococcal virulence and clearance of infection in response to udp were explored in vitro. effect of udp on ugpase activity ugpase activity was evaluated at different udp concentrations (0.05-100 μm) in a549 cells and s. pneumoniae d39 strain. concentration-dependent dose response curve showed that udp decreased ugpase activity in host a549 cells (fig. 1a) as well as in pneumococcus (fig. 1b). udptreated a549 cells showed a declined ugpase activity in comparison to untreated (0.0 μm) control cells (fig. 1a). ugpase activity was not significantly decreased in the cells treated with 0.1-3 μm udp. however, activity was significantly decreased at 5 μm (p = 0.009) and 100 μm (p = 0.011) udp treatments. the inhibition of ugpase activity in a549 cells was 2.8 fold higher at 100 μm udp treatment in comparison to 5 μm treatment. in s. pneumoniae d39 strain, a significant decrease in ugpase activity was observed at 2 μm udp (p = 0.02) treatment (fig. 1b). at 5 μm udp, threefold inhibition (p = 0.01) was observed in ugpase activity in comparison to 100 μm. further, effect of udp (at 5 and 100 μm) on ugpase activity was evaluated in invasive s. pneumoniae isolate (retrieved from blood sample), which showed a decrease in activity at 5 μm udp (p = 0.015) (fig. 1c). comparative analysis of udp treatment on blood isolate and reference d39 strain showed that 5 μm udp treatments for 1 hour were effective to reduce the bacterial ugpase activity (fig. 1c) by half than in its host counterpart (fig. 1a). sharma et al 29 © 2020 the authors. published by aboutscience further analysis of ugpase expression in a549 cell lysate using human anti-ugp2 antibody reconfirmed the significant decrease (p = 0.03) at 100 μm udp treatment in comparison to 5 μm treated and control (untreated) cells (fig. 1d). however, we could not analyze the ugpase expression in s. pneumoniae due to unavailability of antibody against bacterial enzymes. udp treatment and a549 cell survival the effect of udp on a549 cell viability was evaluated in a dose-dependent manner in the absence (control) and presence of s. pneumoniae infection (fig. 2). uninfected (control) cells treated with udp (0.5-100 μm) showed negligible changes in the percentage cell viability. however, a549 cells infected with pneumococci (d39 and blood isolate) showed a significant decrease in percent viability in the absence of udp treatment (p = 0.001). notably, the viability of infected a549 cells was elevated at higher udp treatments (>10 μm) in a dose-dependent manner (fig. 2a). further, a comparative analysis of udp treatments (0, 5, 100 μm) to a549 cells showed significantly improved viability at 100 μm treatment in d39 (p = 0.009) and blood isolate (p = 0.04) infected cells, while no change in cell viability was observed in uninfected (control) cells (fig. 2b). thus, udp seems safer to explore for its potential as it did not exert any cytotoxic effect on a549 cells (fig. 2), while it improved the percentage cell viability in the presence of bacterial infection. further, effect of ugpase inhibition on adherence and phagocytosis of s. pneumoniae (blood isolate and standard strains mtcc 655; a highly capsular strain and d39) was evaluated at effective 5 μm udp concentration (fig. 1). fig. 1 uridine diphosphate (udp)-glucose pyrophosphorylase (ugpase) activity in response to fractional treatment of udp in (a) a549 cells, (b) s. pneumoniae d39 strain, (c) comparative analysis of s. pneumoniae ugpase activity in response to udp treatments. the p values calculated in comparison to control (untreated). (d) western blot analysis of ugp2 in a549 cell lysate, *p < 0.05, **p < 0.01. a c b d ugpase-mediated streptococcus pneumoniae virulence30 © 2020 the authors. published by aboutscience effect of udp on s. pneumoniae adherence all s. pneumoniae strains showed a decrease in adherence to a549 (p = 0.018) and hep-2 cells (p = 0.001) in the presence of 5 μm udp (fig. 3). adherence of mtcc 655 and blood isolate was significantly (p<0.05) lowered in both the cell lines (fig. 3a and b), while d39 showed a significant (p = 0.031) decrease in hep-2 cells only (fig. 3b). adherence of blood isolate to a549 (p = 0.032) and hep-2 (p = 0.014) cells declined by threeto fivefold. effect of udp on phagocytosis of s. pneumoniae the phagocytosis of s. pneumoniae was increased in the presence of udp in both j774.a.1 (p = 0.01) and thp1 (p = 0.022) cells (fig. 4a and b respectively). phagocytosis of d39 strain was significantly higher than mtcc 655 strain (p = 0.033) in both the cell lines, which could be attributed to heavy encapsulation in the latter. udp treatment also showed an increase in phagocytosis of blood isolate by j774.a.1 (p = 0.034) and thp1 cells (p = 0.041). overall, udp-dependent fold change in phagocytosis ranged from 1.4 to 1.9 for pneumococcal strains. it is well known that host immune cells use complement factors for the clearance of s. pneumoniae, and opa of heat-inactivated s. pneumoniae was found to be enhanced in the presence of udp in both the cell lines (fig. 5a and b). interestingly, j774.a.1 cells (fig. 5a) showed marked increase in the opa of blood isolate than mtcc 655 and d39 strains. phagocytosis of mtcc 655 and d39 strains was higher in thp1 cells (fig. 5b) in the presence of udp (5 μm) and sera. real-time pcr analysis of cytokines an attempt was made to evaluate the cytokine response in a549 cells in response to udp treatment as previous studies have shown the activation of inflammatory pathway upon udp stimulation from monocytes and microglial cells (28,29). interestingly, udp treatment (5 μm) induced the secretion of a b fig. 2 effect of uridine diphosphate (udp) treatment on a549 cell survival. (a) treatment-response curve of cells infected with s. pneumoniae, (b) comparative analysis of udp treated and untreated (0 µm) cells, control: uninfected a549 cells, *p < 0.05, **p < 0.01. fig. 3 effect of uridine diphosphate (udp) treatment on adherence of s. pneumoniae by (a) a549 and (b) hep-2 cells, *p < 0.05. a b sharma et al 31 © 2020 the authors. published by aboutscience a a b b fig. 6 relative messenger ribonucleic acid (mrna) expression of cytokines induced in a549 cells, mrs2578: antagonist of uridine diphosphate (udp). fig. 4 effect of uridine diphosphate (udp, 5 µm) on phagocytosis of s. pneumoniae by (a) j774.a.1 and (b) thp1 cells, *p < 0.05, **p < 0.01. fig. 5 effect of uridine diphosphate (udp, 5 µm) on opsonophagocytosis of s. pneumoniae by (a) j774.a.1 and (b) thp1 cells, *p < 0.05, **p < 0.01, ***p < 0.001. il-1β, tnf-α, il-6, and il-8 from the a549 alveolar epithelial cells (fig. 6). an udp antagonist mrs2578 (sigma) effectively suppressed the expression of these cytokines in a549 cells. in agreement with these results, udp is shown to bind its purinergic receptor p2y6 on various cells and stimulate signaling cascade (30,31). conclusions the paradox of infections caused by s. pneumoniae lies in the availability of preventive measures such as antibiotics and vaccines, but not effective prevention from the pneumococcal diseases, especially in lowto middle-income settings. the major reason for this paradox is the genome plasticity of pneumococcus and diverse nature of cps loci, which has precluded the absolute success of vaccines (11,12,32). evidently, various virulence factors of pneumococci had been explored to understand its pathobiology and with broader ugpase-mediated streptococcus pneumoniae virulence32 © 2020 the authors. published by aboutscience prospect to yield potential targets for preventive interventions (8-11,21). elucidation of cps loci from different pneumococcal serotypes has established a crucial role of galu-encoded ugpase in capsule formation (14,17,18). ugpase-catalyzed reaction provides udp-glucose (glc), a key sugar precursor to fulfill various capsular (cps biosynthesis pathway) and metabolic (leloir pathway) needs of pneumococcus (1214,18,20,36-38). in this study, we explored the potential of ugpase in modulating s. pneumoniae pathogenicity by using udp as an ugpase inhibitor. udp proved to be an effective inhibitor of pneumococcal ugpase and mediated a significant reduction in the in vitro virulence of pneumococci. the differential concentration-based enzyme activity inhibition maxima suggests the feasibility of selective inhibition of pneumococcal ugpase (5 μm udp) without inimical effect on host. the decline in pneumococcal adherence to host cell lines also accentuates the efficacy of udp as an inhibitor. a study by zavala et al used nucleoside analogs, namely abacavir, decitabine, stavudine, and zidovudine, to show 42%-58% inhibition of ugpase, but did not elaborate on the use of high concentration (7.5 mm) of inhibitors, their toxic effects, and safety as antipneumococcal drug (23). our study establishes the noncytotoxicity of udp to host cells and boosted uptake of s. pneumoniae upon udp stimulation by macrophage cell lines. our findings are supported by previous studies on the clearance of escherichia coli from peritonitis mouse model in response to direct udp injection (33) and increased host defenses upon udp binding to p2y6 receptor (34-36). the crucial role of udp as inflammatory inducer in brain injuries has been reported in microglia and astrocytes (28). likewise, we have also found the activation of proinflammatory cytokines in alveolar epithelial cells in response to pneumococcal infection. the stimulation of cytokines by udp emphasizes its protective functional nature, which might enhance its efficacy as a potent inhibitor. the present study provides evidence to the udp-mediated reduction in the virulence of s. pneumoniae. being one of the intercellular messengers, udp acts as a protective signaling molecule and activates p2y6 receptors that are known to result in wide range of physiologic responses, such as induction of cytokines, chemokines, phagocytosis, and increase in concentration of extracellular nucleotides (33,35,37,38). the evidence of udp meddling with host-pathogen interactions has opened the vast horizon to investigate its underlying mechanisms, which would definitely shed light on its potential utility in restraining the pneumococcal virulence. the present work is a little step forward in search of potential s. pneumoniae inhibitor independent of serotypes, though the inhibitory effect of udp should be evaluated in vivo to ascertain its efficacy. our study has been limited in evaluating the cytokine response and udp-mediated behavior of p2y6 receptors, which further need a detailed investigation. mechanistic understanding of udpmediated ugpase inhibition and its in vivo validation would be crucial for translating the outcome of this study. author contributions monica sharma performed the experiments, analyzed the data, and wrote the manuscript. swati sharma helped in manuscript editing. anuradha chakraborti and monica sharma designed the study. pallab ray provided the clinical isolates for the study. anuradha chakraborti and pallab ray reviewed the study and manuscript. disclosures financial support: the authors are grateful to the indian council of medical research (icmr), new delhi, india for financial support and providing senior research fellowship (file no. 80/768/2012-ecd-1) to ms. monica sharma and funding the project. swati sharma received research fellowship from council of scientific and industrial research (csir) new delhi, india. authors disclose no other external funding sources. conflict of interest: the authors declare no conflict of interest. references 1. musher dm. streptococcus pneumoniae. in: mandell gl, bennett je, dolin r, eds. mandell, douglas, and bennett’s principles and practice of infectious diseases. 7th ed. philadelphia, pa: churchill livingstone elsevier 2010; 2623-2642. 2. troeger c, blacker b, khalil ia, et al., srm gbd 2016 lower respiratory infections collaborators. estimates of the global, regional, and national morbidity, mortality, and aetiologies of lower respiratory infections in 195 countries, 1990-2016: a systematic analysis for the global burden of disease study 2016. lancet infect dis. 2018;18:1191-1210. 3. wahl b, o’brien kl, greenbaum a, et al. burden of streptococcus pneumoniae and haemophilus influenzae type b disease in children in the era of conjugate vaccines: global, regional, and national estimates for 2000-15. lancet glob health. 2018;6:e744-e757. 4. wahl b, sharan a, deloria knoll m, et al. national, regional, and state-level burden of streptococcus pneumoniae and haemophilus influenzae type b disease in children in india: modeled estimates for 2000-15. lancet glob health. 2019;7:e735-e747. 5. kim sh, song jh, chung dr, et al. changing trends in antimicrobial resistance and serotypes of streptococcus pneumoniae isolates in asian countries: an asian network for surveillance of resistant pathogens (ansorp) study. antimicrob agents chemother. 2012;56:1418-1426. 6. azarian t, grant lr, arnold bj, et al. the impact of serotypespecific vaccination on phylodynamic parameters of streptococcus pneumoniae and the pneumococcal pan-genome. plos pathog. 2018;14:e1006966. 7. mclaughlin jm, jiang q, isturiz re, et al. effectiveness of 13 valent pneumococcal conjugate vaccine against hospitalization for community-acquired pneumonia in older us adults: a testnegative design. clin infect dis. 2018;67:1498-1506. 8. mitchell am, mitchell tj. streptococcus pneumoniae: virulence factors and variation. clin microbiol infect. 2010;16:411-418. 9. brooks lrk, mias gi. streptococcus pneumoniae’s virulence and host immunity: aging, diagnostics, and prevention. front immunol. 2018;9:1366. 10. magee ad, yother j. requirement for capsule in colonization by streptococcus pneumoniae. infect immun. 2001;69:3755-3761. 11. shainheit mg, mulé m, camilli a. the core promoter of the capsule operon of streptococcus pneumoniae is necessary for colonization and invasive disease. infect immun. 2014;82:694-705. 12. garcía e, llull d, lópez r. functional organization of the gene cluster involved in the synthesis of the pneumococcal capsule. int microbiol. 1999;2:169-176. 13. garcía e, llull d, muñoz r, mollerach m, lópez r. current trends in capsular polysaccharide biosynthesis of streptococcus pneumoniae. res microbiol. 2000;151:429-435. sharma et al 33 © 2020 the authors. published by aboutscience 14. mollerach m, lópez r, garcía e. characterization of the galu gene of streptococcus pneumoniae encoding a uridine diphosphoglucose pyrophosphorylase: a gene essential for capsular polysaccharide biosynthesis. j exp med. 1998;188:2047-2056. 15. chang hy, lee jh, deng wl, fu tf, peng hl. virulence and outer membrane properties of a galu mutant of klebsiella pneumoniae cg43. microb pathog. 1996;20:255-261. 16. priebe gp, dean cr, zaidi t, et al. the galu gene of pseudomonas aeruginosa is required for corneal infection and efficient systemic spread following pneumonia but not for infection confined to the lung. infect immun. 2004;72:4224-4232. 17. bonofiglio l, garcía e, mollerach m. biochemical characterization of the pneumococcal glucose 1-phosphate uridylyltransferase (galu) essential for capsule biosynthesis. curr microbiol. 2005;51:217-221. 18. bonofiglio l, garcía e, mollerach m. the galu gene expression in streptococcus pneumoniae. fems microbiol lett. 2012;332:47-53. 19. cools f, torfs e, vanhoutte b, et al. streptococcus pneumoniae galu gene mutation has a direct effect on biofilm growth, adherence and phagocytosis in vitro and pathogenicity in vivo. pathog dis. 2018;76. 20. frey pa. the leloir pathway: a mechanistic imperative for three enzymes to change the stereochemical configuration of a single carbon in galactose. faseb j. 1996;10:461-470. 21. alvaro berbis m, maria sanchez-puelles j, javier canada f, jimenez-barbero j. structure and function of prokaryotic udpglucose pyrophosphorylase, a drug target candidate. curr med chem. 2015;22:1687-1697. 22. mollerach m, garcía e. the galu gene of streptococcus pneumoniae that codes for a udp-glucose pyrophosphorylase is highly polymorphic and suitable for molecular typing and phylogenetic studies. gene. 2000;260:77-86. 23. zavala a, kovacec v, levín g, et al. screening assay for inhibitors of a recombinant streptococcus pneumoniae udp-glucose pyrophosphorylase. j enzyme inhib med chem. 2017;32:203-207. 24. twentyman pr, luscombe m. a study of some variables in a tetrazolium dye (mtt) based assay for cell growth and chemosensitivity. br j cancer. 1987;56:279-285. 25. hara-kaonga b, pistole tg. a dual fluorescence flow cytometric analysis of bacterial adherence to mammalian host cells. j microbiol methods. 2007;69:37-43. 26. hu bt, yu x, jones tr, et al. approach to validating an opsonophagocytic assay for streptococcus pneumoniae. clin diagn lab immunol. 2005;12:287-295. 27. kim b, jeong hk, kim jh, lee sy, jou i, joe eh. uridine 5′-diphosphate induces chemokine expression in microglia and astrocytes through activation of the p2y6 receptor. j immunol. 2011;186:3701-3709. 28. cox ma, gomes b, palmer k, et al. the pyrimidinergic p2y6 receptor mediates a novel release of proinflammatory cytokines and chemokines in monocytic cells stimulated with udp. biochem biophys res commun. 2005;330:467-473. 29. grbic dm, degagné é, larrivée jf, et al. p2y6 receptor contributes to neutrophil recruitment to inflamed intestinal mucosa by increasing cxc chemokine ligand 8 expression in an ap1-dependent manner in epithelial cells. inflamm bowel dis. 2012;18:1456-1469. 30. ginsburg-shmuel t, haas m, grbic d, et al. udp made a highly promising stable, potent, and selective p2y6-receptor agonist upon introduction of a boranophosphate moiety. bioorg med chem. 2012;20:5483-5495. 31. hao y, liang jf, chow w, cheung w, ko w. p2y6 receptor mediated proinflammatory signaling in human bronchial epithelia. plos one. 2014;9:e106235. 32. subramanian k, henriques-normark b, normark s. emerging concepts in the pathogenesis of the streptococcus pneumoniae: from nasopharyngeal colonizer to intracellular pathogen. cell microbiol. 2019;2:e13077. 33. zhang z, wang z, ren h, et al. p2y6 agonist uridine 5′-diphosphate promotes host defense against bacterial infection via monocyte chemoattractant protein-1–mediated monocytes/macrophages recruitment. j immunol. 2011;186: 5376-5387. 34. idzko m, ferrari d, eltzschig hk. nucleotide signalling during inflammation. nature. 2014;509:310-317. 35. koizumi s, shigemoto-mogami y, nasu-tada k, et al. udp acting at p2y6 receptors is a mediator of microglial phagocytosis. nature. 2007;446:1091-1095. 36. inoue k, koizumi s, kataoka a, tozaki-saitoh h, tsuda m. p2y(6)-evoked microglial phagocytosis. int rev neurobiol. 2009;85:159-163. 37. neher jj, neniskyte u, hornik t, brown gc. inhibition of udp/p2y6 purinergic signaling prevents phagocytosis of viable neurons by activated microglia in vitro and in vivo. glia. 2014;62:1463-1475. 38. zierhut m, dyckhoff s, masouris i, et al. role of purinergic signaling in experimental pneumococcal meningitis. sci rep. 2017;7:44625. untitled drug target insights 2007: 2 61–69 61 original research correspondence: maria de nazaré c. soeiro. tel: 0055 21 2598-4331; fax: 00 55 21 2260-4434; email: soeiro@ioc.fi ocruz.br please note that this article may not be used for commercial purposes. for further information please refer to the copyright statement at http://www.la-press.com/copyright.htm electrocardiographic findings in acutely and chronically t. cruzi-infected mice treated by a phenyl-substituted analogue of furamidine db569 elen m. de souza, gabriel m. oliveira and maria de nazaré c. soeiro lab. biologia celular, dubc, instituto oswaldo cruz, fiocruz, rio de janeiro, rj, brasil. abstract: aromatic diamidines have been successfully used to combat a wide range of parasites that cause important human infections. recently we reported that a n-phenyl-substituted analogue of furamidine (db569) exerts a micromolar trypanocidal activity against trypanosoma cruzi in vitro. since db569 also reduces the cardiac parasitism and increases the survival rates of t. cruzi-infected mice, our present aim was to analyze the potential protection of db569 in the development of altered cardiac electrical conduction system during acute and chronic t. cruzi infection. in our experimental model of acute infection (swiss mice inoculated with y strain of t. cruzi), the prevailing disorder observed in electrocardiogram (ecg) analyses was sinus bradycardia. this ecg alteration was reverted in acutely infected mice treated with db569. interestingly, the db569 treatment reduced signifi cantly the numbers of cd8+ t cells in the cardiac infi ltration. in addition, the noticed protection of db569 in the ecg fi ndings of acutely-infected animals was further extended to the chronic infection. our data suggest that the reversion to and further maintenance of normal ecg profi le in the db569treated infected animals may be associated with the reduced cardiac cd8+ lymphocyte infi ltration and parasitism that might be ultimately contributing to their increased survival rates. keywords: electrocardiography, aromatic diamidines, therapy, infected mice, infl ammation, heart, chagas’ disease. introduction trypanosoma cruzi is the ethiological agent of chagas’ disease, which affects over 16–18 million people in endemic areas of south and central america, leading to 50,000 deaths per year (who 2002). the disease has two phases: the acute, which appears shortly after the infection, and the chronic phase, which can develop in about one-third of the infected individuals after a silent period of years or decades called the indeterminate phase (cunha-neto et al. 2006). although the main clinical manifestations of the chagas disease include both the cardiac and the digestive alterations, the former is the most prominent: about 25% to 30% of the infected people will develop a progressive heart disease commonly displaying symptomatic ventricular arrhythmias, hypertrophy; congestive heart failure; sinus bradycardia and tachycardia; thomboembolism and sudden death (marin-neto et al. 1999; salles et al. 2003). several hypotheses have been raised to explain the chronic chagasic cardiomyopathy (ccc) including parasite persistence (golgher and gazzinelli, 2004), microvascular dysfunction (petkova et al. 2001), and imbalanced immune response with autoimmune implications (leon and engman, 2003). the sudden cardiac death is one of the most common terminal events in chagas’ disease with these patients usually presenting complex and sustained ventricular arrhythmia on ambulatory electrocardiogram (ecg) (maguire et al. 1987). the most frequent chagasic electrocardiographic abnormalities include (i) frequent sinus node dysfunction, (ii) high rate of involvement of the conduction system producing ecg alterations of the right bundle branch or left anterior hemiblock or both, (iii) high rate of ventricular arrhythmias, (iv) high frequency of atrioventricular block, (v) abnormal q waves, stsegment and t-waves and (vi) autonomic dysfunction (salles et al. 2003; garcia et al. 2005). it has been reported that most patients that present ccc display altered ecg (rocha et al. 2003). in a recent study it has been demonstrated that patients with positive serology for t. cruzi and active infection, but with normal left ventricular cineangiography, showed 100% survival at 16 years follow-up, while the 62 de souza et al drug target insights 2007: 2 presence of apical aneurysm or left ventricular systolic dysfunction was along with 60% of mortality at 4 years follow-up (dávila-spinetti et al. 2005). the available chemotherapy against t.cruzi based on nitrofurans (nifurtimox–bayer) and nitroimidazoles (benznidazole–roche) is unsatisfactory since both compounds are effective only against recent infections and have frequent toxic side effects besides the occurrence of drug resistance (reviewed in coura and de castro, 2002). recent reviews clearly point to the need of fi nding more efficient and less toxic drugs. in this respect, aromatic diamidines represent a promising class of dna-targeted anti-parasitic agents (reviewed in soeiro et al. 2005; wilson et al. 2005; werbovetz, 2006). we have recently reported the high in vitro trypanocidal activity of db569, a n-phenylsubstituted analogue of furamidine (de souza et al. 2004). we also found that the treatment of acutely infected mice with db569 signifi cantly reduced the cardiac parasitism and partially increased the survival rates (de souza et al. 2006a). since ecg alterations have also been reported in t.cruzi-infected mice (bustamante et al. 2003, 2005; zaidenberg et al. 2006) our aim in the present study was to evaluate the potential protection of db569 in the development of cardiac alterations in both acutely and chronically t. cruzi infected mice. materials and methods parasites bloodstream trypomastigotes of the y strain were harvested by heart puncture from t. cruzi-infected swiss mice at the parasitaemia peak, as described previously (meirelles et al. 1982). in vivo infection male swiss mice (25–30 g) were obtained from the fundação oswaldo cruz (fiocruz) animal facilities (rio de janeiro, brazil). mice were housed at maximum 8 per cage and kept in a conventional room at 20–24 °c under a 12/12-h light/dark cycle. the animals were provided with sterilized water and chow ad libitum. infection was performed by intraperitoneal (i.p.) injection of 104 bloodstream trypomastigotes, and age-matched uninfected mice were maintained under identical conditions. all assays were performed 3 to 4 times and all procedures were carried out in accordance with the guidelines established by the fiocruz committee of ethics for the use of animals (ceua 0099/01). experimental groups the animals were divided into the following groups: uninfected (non-infected and non-treated); untreated (infected with t. cruzi but non-treated); and treated (infected and treated with 20 mg/kg db569). for analyses in the acute infection, at least 6 mice from each group were used at each different day post infection (dpi) until the 21st dpi. after one-year post infection, the surviving t. cruziinfected animals were used as the chronic mice group. drugs and treatment schedule the synthesis of db569 has been reported previously (lansiaux et al. 2002). stock solution (200 mg) of the diamidine was prepared in dimethylsulfoxide and mice received 0.1 ml i.p. injection of 20 mg/kg/day db569, starting at 3 dpi for 10 consecutive days as described (de souza et al. 2006a). the infected and untreated mice group only received 0.1ml i.p. daily injection of phosphatebuffered saline (10 mm sodium phosphate, 0.015 m nacl, ph 7.4) (pbs) as vehicle. parasitaemia and mortality parasitaemia was individually checked by direct microscopic counting of parasites in 5 µl of blood, as described before (de souza et al. 2006a). mortality was checked daily until 30 dpi and expressed as percentage of cumulative mortality (%cm) (de souza et al. 2006a). histopathological analysis at 8, 15, 21 dpi and after one year of infection, hearts were removed, cut longitudinally, rinsed in ice-cold pbs and fi xed in millonig-rosman solution (10% formaldehyde in phosphate-buffered saline). the tissues were then dehydrated and embedded in paraffi n. sections (3 µm) stained by routine hematoxylin-eosin (he) were analyzed by light microscopy. the number of amastigote nests and of inflammatory infiltrates (more than 10 mononuclear cells) was determined in at least 30 fi elds (total magnifi cation, 40x) for each slide. the 63 electrocardiographic findings in t. cruzi-infected mice treated by db569 drug target insights 2007: 2 mean number of amastigotes’ nests or infl ammatory infi ltrates per fi eld was obtained from at least three mice per group with three sections from each mouse. flow cytometry analysis of cardiac cd4+ and cd8+ t cells to evaluate the levels of cardiac cd8+ and cd4+ t cells, three mice of each experimental group were sacrifi ced at the peak of cardiac infl ammation (15 dpi) (de souza et al. 2006a), the hearts quickly removed and their infl ammatory cells isolated for fl ow cytometry analysis as reported, with minor modifi cations (marino et al. 2004). briefl y, to isolate mononuclear cells invading the cardiac tissue, hearts were fragmented (1 to 2 mm) and submitted to seven enzymatic dissociations employing 0.2% collagenase. the recovered cells were washed in pbs, fi xed for 20 min with 1% parafomaldehyde and labeled with phycoerythrin (pe)-conjugated anti-cd4 and cy-chromeconjugated anti-cd8 mab (pharmingen/bd biosciences, san diego, ca). negative controls for specifi c labeling were prepared from isotypematched mabs (pharmingen/bd biosciences). a total of 10,000 events were acquired using a facscalibur fl ow cytometer (becton dickinson, san jose, ca, usa) equipped with cell quest software. the data analysis was performed in the winmdi software (multiple document interface flow cytometry application, v2.8, by joseph trotter, scripps research institute, san diego, ca, usa). electrocardiography ecg recording and analysis were performed in uninfected, acutely (8, 15, 21 dpi) and chronically (after 1 year post infection) t. cruzi-infected mice submitted or not to the db569 therapy, as reported (garcia et al. 2005; zeidenberg et al. 2006). briefl y, mice were placed under stable sedation with diazepan (20 mg/kg, i.p.), fi xed in the supine position, and eight-lead ecgs were recorded from 18-gauge needle electrodes subcutaneously implanted in each limb and two electrodes at precordial positions lead ii. the electrocardiographic tracing was obtained with standard lead (dipolar lead dii), recording with amplitude set to give 2 mv/1s. ecgs were recorded by using a band-pass fi ltering (bio amp—ad instruments, hastings, united kingdom) between 0.1 and 100 hz. supplementary amplifi cation and analog-digital conversion was performed with a powerlab 16s instrument (ad instruments, hastings). digital recordings (16 bit, 4 khz/channel) were analyzed with the scope (version v3.6.10) program (ad instruments). the signal-averaged ecg (saecg) was calculated by using the mouse saecg extension (version 1.2) program (ad instruments) and a templatematching algorithm. ecg parameters were evaluated in the acute and chronic chagasic mice by using the following standard criteria: (i) the heart rate was monitored by beats/minute (bpm), and (ii) the variation at p wave and pq, qrs and qt intervals were measured at milliseconds (ms). statistical analysis statistical analysis was carried out using the variance (anova), with the level of signifi cance set at p ≤ 0.05. the data are representative of 2–4 experiments run in duplicate. results db569 (fig. 1) is effective towards both the intracellular and trypomastigote forms of t. cruzi, presenting ic50 values at low-micromolar doses in vitro (de souza et al. 2004). this furamidine analogue also decreases mouse mortality and the levels of alanine aminotransferase and creatinine, indicating a protective role respectively against hepatic and renal lesions caused by the parasite infection (de souza et al. 2006a). since it signifi cantly decreases cardiac parasitism to a level similar to that found for benznidazole treatment (de souza et al. 2006a), we decided to investigate the potential protection of db569 for the development of cardiac electrophysiologic abnormalities during acute and chronic t. cruzi infection. we confi rmed our previous data showing that while the diamidine therapy slightly reduces the parasitaemia levels (fig. 2a), db569 significantly controls the cardiac parasitism, reducing in 95% the number of parasite nests (p ≤ 0.049) at the 15th dpi (fig. 2b and g), as compared to untreated animals (fig. 2b and f). in addition, the fl ow cytometry analysis revealed that db569 decreased in 40% the frequency of cardiac infi ltrating cd8+ t cells (fig. 2e), as compared to the untreated animals (fig. 2d). however, histological analysis of heart samples at the 15th dpi, which represent the peak of both cardiac parasite load as well as infl ammation in our experimental model (de souza 64 de souza et al drug target insights 2007: 2 et al. 2006a), showed non-signifi cant reduction (p ≤ 0.5) of the infl ammation (fig. 2c, h–i). interestingly, the scarce parasite’ nests found in heart tissues of drug-treated mice (fig. 2g, asterisks) were frequently associated to infl ammatory cells (fig. 2g, arrow). next we analyzed the electrocardiogram profiles in uninfected, untreated and db569-treated mice (fig. 3a). no differences among the groups were observed at the 8th dpi (fig. 3a–b). however, at 15 and 21 dpi, we noticed sinus bradycardia as the prevailing ecg disorder found in untreated mice as compared to uninfected group, as detected by low heart rates (fig. 3a,c–d). while uninfected mice exhibited values ranging from 630 to 670 beats/min (bpm), untreated and db569-treated animals, displayed decreased heart frequencies, reaching 496 and 501 bpm, respectively (fig. 3a and c). interestingly, although decreased cardiac frequencies were maintained in the untreated mice at the 21st dpi, db569treatment reverted (p ≤ 0.01) this ecg alteration, reaching values similar to the counterpartuninfected group (fig. 3a and d). as diamidine treated group showed 40% reduction of the mortality compared to non-treated mice (fig. 3e), we further investigated if the protection exerted by db569 during the acute infection would be maintained in the chronic phase. we again observed lower cardiac frequency as the prevailing electrocardiographic change found in the chronic untreated group. we also found that db569treated mice reverted the cardiac alterations found in untreated animals (p ≤ 0.01), showing ecg profi les similar to uninfected mice and indicating that they sustained normal ecg profi les (fig. 4a–b) as recovered at the 21st dpi in the acutely-infected mice. discussion the lack of an effective therapy for chagas’ disease associated to the toxicity and side effects of the available drugs (nifurtimox and beznidazole) justify the investigation of different natural as well as synthetic compounds that could substitute the current chemotherapy (coura and de castro, 2002). the well-known activity of aromatic diamidines towards many pathogens in both in vitro and in vivo studies (reviewed in soeiro et al. 2005) prompted us to verify the effect of furamidine and its n-phenyl-substitute analogue, db569. moreover, the superior activity of db569 as compared to furamidine even after short periods of in vitro treatment, without exerting toxicity towards mammalian cells (de souza et al. 2004), impelled further analysis of the db569 activity during t. cruzi infection in vivo (de souza et al. 2006a). as electrocardiographic studies give important information regarding aspects of cardiac function, representing a useful tool to measure the severity and prognosis of the chagasic cardiopathy (salles et al. 2003), here we sought to evaluate the potential protection of figure 1. structure of the db569. 65 electrocardiographic findings in t. cruzi-infected mice treated by db569 drug target insights 2007: 2 figure 2. treatment of t. cruzi-infected mice (104 y strain/mice) with db569 from 3 to 12 dpi: (a) kinetics of parasitaemia, (♦) untreated (infected and non-treated) and (■) db-treated (infected and treated with 20 mg/kg/day db569); (b) number of amastigote’ nests and (c) infl amatory infi ltration within the heart of untreated and db-treated groups at 15th dpi; cardiac cd4+ and cd8+ t cells expression measured by fl ow cytometry analysis of (d) untreated and (e) db-treated t. cruzi-infected mice at 15th dpi. the lymphocyte gate was determined according to expression of cd4+ and cd8+ in fsc × ssc dot plot in infected animals; histological patterns in heart sections at 15th dpi (f-i) of untreated (f and h) and db-treated (g and i) t. cruzi-infected mice. note the presence of parasite nests (f and g, asterisks) and the intensity of the infl ammatory process (f-i, arrow). bar = 50 µm. p indicate signifi cant differences between db-treated and untreated groups. r means the ratio between cd4+ and cd8+ cardiac cells). 66 de souza et al drug target insights 2007: 2 figure 3. electrocardiographic fi nding during acute phase of t. cruzi-infection, were verifi ed by the variation at heart rate (beats/min) and p wave and pq, qrs and qt intervals (ms) at uninfected (white), untreated (black) and db-treated (gray) groups: (a) electrocardiographic tracing at 8°dpi, 15°dpi and 21°dpi of all groups; electrocardiogram values at 8°dpi (b), 15°dpi (c) and at 21°dpi (d). cumulative mortality curve of untreated (♦) and db-treated (■) t. cruzi-infected groups accompanied until 30 dpi (e). asterisks indicate signifi cant differences of db569-treated in relation to the untreated group for electrocardiographic fi nding, p ≤ 0.01. untreated 0 2 -1 0 2 -1 0 2 -1 100 200 400 600 800 treated 0 2 -1 uninfected 0 2 -1 0 2 -1 0 2 -1 100 200 400 600 800 8°dpi 15°dpi 21°dpi a 0 100 200 300 400 500 600 700 800 p pq qrs qt freq 8 days pos-infection uninfected untreated db-20mg/kg 0 100 200 300 400 500 600 700 800 p pq qrs qt freq 15 days pos-infection uninfected untreated db-20mg/kg b c d 0 100 200 300 400 500 600 700 800 p pq qrs qt freq 21 days pos-infection uninfected untreated db-20mg/kg * 0 10 20 30 40 50 60 70 80 90 100 0 6 12 18 24 30 dpi % untreated treated e 67 electrocardiographic findings in t. cruzi-infected mice treated by db569 drug target insights 2007: 2 0 2 -1 0 2 -1 0 2 -1 100 200 400 600 800 1 year pos-infection uninfected untreated treated a 0 100 200 300 400 500 600 700 800 p pq qrs qt f req 1 year pos-infection uninf ected untreated db-20mg/kg b * figure 4. electrocardiographic fi nding at chronic phase after one year of t. cruzi infection were verifi ed by the variation at heart rate (beats/ min) and p wave and pq, qrs and qt intervals (ms) at uninfected (white), untreated (black) and db-treated (gray) groups: (a) electrocardiographic tracing and (b) electrocardiographic parameters of all groups. asterisks indicate signifi cant differences of db569-treated in relation to the untreated group, p ≤ 0.01. db569 in the development of ecg alterations in both acute and chronic murine t. cruzi infection. as previously reported, our data confi rmed that t. cruzi-infected mouse represents an interesting model for analyzing ecg alterations due to parasite infection, displaying electrocardiographic abnormalities (rivarola et al. 2005; zaidenberg et al. 2006). in our present experimental model, these abnormalities were mostly related to a remarkable decrease of the cardiac frequency, as also reported by others (bustamante et al. 2005). our results showed that db569 considerably reduced the ecg alterations in both acutely and chronically t. cruzi-infected mice similarly as described in other studies performed with different drugs (lo presti et al. 2004; rivarola et al. 2005; garcia et al. 2005; zaidenberg et al. 2006). interestingly, the db569 protection was directly related to the levels of cardiac cytotoxic t cells, since the treatment also decreased the levels of cd8+ cells found in the infl amed heart and partially recovered the cd4+/cd8+ ratio that was 0.37 and 0.50 in untreated mice and db569 treated mice, respectively. this is an interesting fi nding since it 68 de souza et al drug target insights 2007: 2 has been reported that in acutely and chronically t. cruzi-infected patients and during the murine infection, cardiac cd4+/cd8+ t cell ratio shows a predominance of cd8+ t cells suggestive of an immunologic imbalance response (higuchi et al. 1997; marino et al. 2004; fuenmayor et al. 2005; henriques-pons et al. 2005; cunha-neto et al. 2006). the parasitism control and decreased cd8+ t cells levels in the myocardium of db569-treated group was concomitantly found with an unaltered ecg profi le that might be ultimately contributing to their increased survival rates. this therapeutic effect of db569 on the bradyarrhythmia could be related to trypanocidal effect of the diamidine and/or the modulation of the host immunological response to the parasite. in fact, the regulation of immune response in t. cruzi-infected mice by other trypanocidal drugs, such as benznidazole, has already been reported (olivieri et al. 2006; romanha et al. 2002), and our previous in vitro data showed that parasite elimination was more effective after treatment of infected phagocytic cells as compared to cardiac ones (de souza et al. 2004). additionally, as db569 induces apoptosis in t. cruzi (de souza et al. 2006b), the presence of apoptotic parasites in cardiac tissues could contribute for regulation of the host infl ammatory orchestra (fadok et al. 2000; dosreis et al. 2005). although the mechanisms leading to chronic chagasic cardiomyopathy (ccc) are still unknown, the initial cardiac damage caused by the parasite seems to play a critical role for the progression of the disease (tarleton, 2001). moreover, specifi c trypanocidal treatment seems to be associated with delay in human heart disease progression (viotti et al. 2006). in addition, although accumulated data highlighted the concept that tissue damage is direct or indirectly induced by t. cruzi itself and thus the control of parasite load by a host effective infl ammatory response could represent a prerequisite to arrest the evolution of the disease, an excessive or imbalanced infl ammation (as an auto reactivity response) can also contribute to the genesis of the cardiac damage (reviewed in dutra et al. 2005). in fact, chronic chagasic patients submitted to benznidazole therapy although not parasitologically cured, presented a marked reduction in the occurrence of ecg changes and a lower frequency of deterioration in their clinical conditions (viotti et al. 1994). then, despite the genesis of ccc, the current concept suggests that parasite eradication is a requisite to arrest the evolution of the chagas disease pointing to the urgent need for the discovery and development of safe and effective therapy for the chagasic patients. in this context, our results suggest that diamidines are a potential class of compounds to be employed further in vitro and in vivo studies for chagas’ disease treatment. acknowledgments mncs and emds would like to thank conselho nacional desenvolvimento científi co e tecnológico (cnpq); fundação carlos chagas filho de amparo a pesquisa do estado do rio de janeiro (faperj) and papes iv/fiocruz for fi nancial support. the authors thank dr. vinicius cottade-almeida (lbc/ioc/fiocruz) for the careful revision. the authors express special appreciation to dr. david boykin (department of chemistry, georgia state university, atlanta, usa) for manuscript revision and for supplying the db569. references armenti, a. 2006. long-term cardiac outcomes of treating chronic chagas disease with benznidazole versus no treatment: a nonrandomized trial. ann. intern. med., 144:724–34. bustamante, j.m., rivarola, h.w. and fretes, r. et al. 2005. weekly electrocardiographic pattern in mice infected with two different trypanosoma cruzi strains. int. j. cardiol., 102:211–7. bustamante, j.m., rivarola, h.w. and fernandez, a.r. et al. 2003. trypanosoma cruzi reinfections provoke synergistic effect and cardiac beta-adrenergic receptors’ dysfunction in the acute phase of experimental chagas’ disease. exp. parasitol., 103:136–42. coura, r.j. and de castro, s.l. 2002. a critical review on chagas’ disease chemotherapy. mem. inst. oswaldo. cruz., 97:3–24. dávila-spinetti, d.f., colmenarez-mendoza, h.j. and lobo-vielma, l. 2005. mechanisms responsible for myocardial damage progression in chronic chagas disease. rev. esp. cardiol., 58:1007–09. de souza, e.m., lansiaux, a. and bailly, c. et al. 2004. phenyl substitution of furamidine markedly potentiates its anti-parasitic activity against trypanosoma cruzi and leishmania amazonensis. biochem. pharmacol., 68:593–600. de souza, e.m., oliveira, g.m. and boykin, d.w. et al. 2006a. trypanocidal activity of the phenyl-substituted analogue of furamidine db569 against trypanosoma cruzi infection in vivo. j. antimicrob chemother, 58:610–4. de souza, e.m., menna-barreto, r. and araujo-jorge, t.c. et al. 2006b. antiparasitic activity of aromatic diamidines is related to apoptosislike death in trypanosoma cruzi. parasitology, 133:75–9. dosreis, g.a., freire-de-lima, c.g. and nunes, m.p. et al. 2005. the importance of aberrant t-cell responses in chagas disease. trends parasitol, 21:237–43. dutra, w.o., rocha, m.o.c. and teixeira, m.m. 2005. the clinical immunology of human chagas disease. trends in parasitology, 21:581–7. cunha-neto, e., bilate, a.m. and kenneth, v. et al. 2006. induction of cardiac autoimmunity in chagas heart disease: a case for molecular mimicry. autoimmunity, 39:41–54. 69 electrocardiographic findings in t. cruzi-infected mice treated by db569 drug target insights 2007: 2 fadok, v.a., bratton, d.l. and rose, d.m. et al. 2000. a receptor for phosphatidylserine-specifi c clearance of apoptotic cells. nature, 405:85–90. fuenmayor, c., higuchi, m.l. and carrasco, h. et al. 2005. acute chagas’ disease: immunohistochemical characteristics of t-cell infi ltrate and its relationship with t. cruzi parasitic antigens. acta. cardiol., 60:33–7. garcia, s., ramos, c.o. and senra, j.f. et al. 2005. treatment with benznidazole during the chronic phase of experimental chagas’ disease decreases cardiac alterations. antimicrob agents chemother., 49:1521–8. golgher, d. and gazzinelli, r.t. 2004. innate and acquired immunity in the pathogenesis of chagas disease. autoimmunity, 37:399–409. henriques-pons, a., olivieri, b.p. and oliveira, g.m. et al. 2005. experimental infection with trypanosoma cruzi increases the population of cd8(+), but not cd4(+), immunoglobulin g fc receptor-positive t lymphocytes infect. immun., 73:5048–52. higuchi, m.d., ries, m.m. and aiello, v.d. et al. 1997. association of an increase in cd8+ t cells with the presence of trypanosoma cruzi antigens in chronic, human, chagasic myocarditis. am. j. trop. med. hyg., 56:485–9. lansiaux, a.f., tanious, z. and mishal, l. et al. 2002. distribution of furamidine analogues in tumor cells: targeting of the nucleus or mitochondria depending on the amidine substitution. cancer res., 62:7219–29. leon, j.s. and engman, d.m. 2003. the signifi cance of autoimmunity in the pathogenesis of chagas heart disease. front biosci., 8:315–22. lo presti, m.s., rivarola, h.w. and bustamante, j.m. et al. 2004. thioridazine treatment prevents cardiopathy in trypanosoma cruzi infected mice. int. j. antimicrob agents, 23:634–6. maguire, j.h., hoff, r. and sherlock, i. et al. 1987. cardiac morbidity and mortality due to chagas’ disease: prospective electrocardiographic study of a brazilian community. circulation, 75:1140–5. marin-neto, j.a., simoes, m.v. and sarabanda, a.v. 1999. chagas’ heart disease. arq. bras. cardiol., 72:247–80. marino, a.p., da silva, a. and dos santos, p. et al. 2004. regulated on activation, normal t cell expressed and secreted (rantes) antagonist (met-rantes) controls the early phase of trypanosoma cruzielicited myocarditis circulation, 110:1443–9. meirelles, m.n., chiari, e. and de souza, w. 1982. interaction of bloodstream, tissue culture-derived and axenic culture-derived trypomastigotes of trypanosoma cruzi with macrophages. acta trop, 39:195–203. olivieri, b.p., cotta-de-almeida, v. and araujo-jorge, t. 2002. benznidazole treatment following acute trypanosoma cruzi infection triggers cd8+ t-cell expansion and promotes resistance to reinfection. antimicrob agents chemother, 46:3790–6. olivieri, b.p., farias-de-oliveira, d.a. and araujo-jorge, t.c. et al. 2005. benznidazole therapy in trypanosoma cruzi-infected mice blocks thymic involution and apoptosis of cd4+cd8+ double-positive thymocytes. antimicrob agents chemother, 49:1981–7. olivieri, b.p., de souza, a.p. and cotta-de-almeida, v. et al. 2006. trypanosoma cruzi: alteration in the lymphoid compartments following interruption of infection by early acute benznidazole therapy in mice. exp. parasitol., 114:228–34. petkova, s.b., huang, h. and factor, s.m. et al. 2001. the role of endothelin in the pathogenesis of chagas’ disease. int. j. parasitol., 31: 499–511. rivarola, h.w., bustamante, j.m. and lo presti, s. et al. 2005. trypanosoma cruzi: chemotherapeutic effects of clomipramine in mice infected with an isolate obtained from an endemic area. exp. parasitol., 111:80–6. rocha, m.o., ribeiro, a.l. and teixeira, m.m. 2003. clinical management of chronic chagas cardiomyopathy. front biosci., 8:44–54. romanha, a.j., alves, r.o. and murta, s.m. et al. 2002. experimental chemotherapy against trypanosoma cruzi infection: essential role of endogenous interferon-gamma in mediating parasitologic cure. j. infect. dis., 186:823–8. salles, g., xavier, s. and sousa, a. et al. 2003. prognostic value of qt interval parameters for mortality risk stratifi cation in chagas’ disease: results of a long-term follow-up study. circulation, 108:305–12. soeiro, m.n., de souza, e.m. and stephens, c.e. et al. 2005. aromatic diamidines as antiparasitic agents. expert opin. investig. drugs, 14:957–72. tarleton, r.l. 2001. parasite persistence in the aetiology of chagas disease int. j. parasitol., 31:550–4. viotti, r., vigliano, c. and armenti, h. et al. 1994.treatment of chronic chagas’ disease with benznidazole: clinical and serologic evolution of patients with long-term follow-up. am. heart j., 127:151–62. viotti, r., vigliano, c. and lococo, b. et al. 2006. long-term cardiac outcomes of treating chronic chagas disease with benznidazole versus no treatment: a nonrandomized trial. ann. intern. med., 144:724–34. werbovetz, k. 2006. diamidines as antitrypanosomal, antileishmanial and antimalarial agents. curr. opin. investig. drugs., 7:147–57. wilson, w.d., nguyen, b. and tanious, f.a. et al. 2005. dications that target the dna minor groove: compound design and preparation, dna interactions, cellular distribution and biological activity. curr. med. chem. anti. cancer agents, 5:389–408. world health organization. 2002. control of chagas disease. who technical report series 905:109. zaidenberg, a., luong, t. and lirussi, d. et al. 2006. treatment of experimental chronic chagas disease with trifl uralin. basic clin. pharmacol. toxicol., 98:351–6. https://doi.org/10.1177/1177392819857089 drug target insights volume 13: 1–8 © the author(s) 2019 article reuse guidelines: sagepub.com/journals-permissions doi: 10.1177/1177392819857089 creative commons non commercial cc by-nc: this article is distributed under the terms of the creative commons attribution-noncommercial 4.0 license (http://www.creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the sage and open access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage). introduction phenylketonuria (pku) is a rare autosomal recessive condition affecting about 1 in 13 500 to 19 000 people in the united states.1 phenylketonuria is diagnosed through detection of elevated blood phenylalanine concentration.2 if untreated, pku can cause chronic intellectual, neurodevelopmental, and psychiatric disabilities. excessive phenylalanine is thought to interfere with brain growth, myelination, and neurotransmitter synthesis. most cases of pku are detected shortly after birth by newborn screening. as a result, the severe signs and symptoms of classic pku are rarely seen.2 elevated phenylalanine levels are caused by a deficiency in the enzyme, phenylalanine hydroxylase (pah).2 the spectrum of severity in untreated pku ranges from complete enzyme deficiency or classic pku (phenylalanine concentration > 1200 µmol/l), moderate pku (phenylalanine concentration = 900-1200 µmol/l), mild pku (phenylalanine concentration = 600-900 µmol/l), and mild hyperphenylalaninemia (hpa) (phenylalanine concentration = 360-600 µmol/l).3 although guidelines recommend lifelong treatment to a target blood phenylalanine level of 120 to 360 µmol/l4,5 or 120 to 600 µmol/l, (van wegberg, macdonald and ahring, 2017) therapy must be individualized to each patient. management of patients with hpa and pku should be provided by an interdisciplinary team of nutritionists, psychologists, social workers, and metabolic specialists.2 phenylketonuria is a relatively rare disease and patients may require lifelong treatment. the goal of therapy is to lower blood phenylalanine concentrations to minimize the neurocognitive and psychiatric effects of pku.4–7 the mainstay of therapy is dietary restriction of phenylalanine and supplementation with phenylalanine-free medical foods to avoid nutritional deficits.4–6 the medical foods are protein substitutes that are phenylalanine-free and fortified in tyrosine and may contain glycomacropeptides and other large neutral amino acids as the protein source.3 adherence to the restrictive diet requires planning and organization, and can be challenging for patients.4 sapropterin was food and drug administration (fda)– approved on december 13, 2007, and was the first pharmacologic therapy for treatment of pku.8 it is an oral pah cofactor a comprehensive review of pegvaliase, an enzyme substitution therapy for the treatment of phenylketonuria tasmina hydery1 and valerie azzopardi coppenrath2 1department of family medicine and community health, umass medical school—clinical pharmacy services (cps), shrewsbury, ma, usa. 2school of pharmacy—worcester/manchester, massachusetts college of pharmacy and health sciences (mcphs) university, worcester, ma, usa. abstract objective: to review the pharmacology, pharmacokinetics, efficacy, safety, and place in therapy of a phenylalanine-metabolizing enzyme indicated to reduce blood phenylalanine concentrations, pegvaliase injection. data sources: searches of medline (1946-september 1, 2018) were conducted using the terms pegvaliase and phenylalanine ammonia lyase (pal). additional data were obtained from the prescribing information, the product dossier obtained from the manufacturer, and clinicaltrials.gov. study selection and data extraction: all english language articles related to pharmacology, pharmacokinetics, efficacy, or safety of the combination therapy in human subjects were reviewed. data synthesis: pegvaliase is a pegylated pal enzyme that converts phenylalanine to ammonia and trans-cinnamic acid. blood phenylalanine levels were reduced by approximately 50% to 70% in patients receiving therapeutic doses of pegvaliase. however, most patients experienced adverse events. conclusions and relevance: the mainstay of therapy in phenylketonuria (pku) has historically consisted of dietary restriction of phenylalanine. pegvaliase injection is the first food and drug administration (fda)–approved enzyme substitution therapy for patients with pku. the therapy may be a viable option for patients with documented blood phenylalanine >600 µmol/l who have failed existing management strategies. keywords: palynziq, pegvaliase, phenylalanine ammonia lyase, pegvaliase-pqpz, phenylketonuria, pku received: may 22, 2019. accepted: may 23, 2019. type: review funding: the author(s) received no financial support for the research, authorship, and/or publication of this article. declaration of conflicting interests: the author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. corresponding author: tasmina hydery, department of family medicine and community health, umass medical school—clinical pharmacy services (cps), shrewsbury, ma 01545, usa. email: tasmina.hydery@umassmed.edu 857089dti0010.1177/1177392819857089drug target insightshydery and coppenrath review-article2019 https://uk.sagepub.com/en-gb/journals-permissions mailto:tasmina.hydery@umassmed.edu 2 drug target insights indicated to reduce blood phenylalanine concentrations in patients with hpa due to tetrahydrobiopterin (bh4)responsive pku. sapropterin is used in conjunction with a phenylalanine-restricted diet.9 the efficacy of sapropterin is based on the presence of residual pah enzymatic activity. approximately 25% to 50% of patients with pku respond to sapropterin.4 phase 3 trials for an enzyme substitution therapy for pku began in 2013 and showed promise to be effective in any patient with pku regardless of residual enzymatic activity.4 pegvaliase, fda-approved on may 24, 2018, acts as a substitute for the deficient pah enzyme.10 pegvaliase is indicated to reduce blood phenylalanine concentrations in adult patients with pku who have uncontrolled blood phenylalanine concentrations >600 µmol/l on existing management.11,12 adult patients with pku whose phenylalanine levels are not appropriately managed may experience adverse neurocognitive and psychiatric outcomes.7 the objective of this article is to review the pharmacology, pharmacokinetics, efficacy, safety, and place in therapy of pegvaliase injection. it will also provide an overview of the role of pegvaliase compared with current standard of care. data sources a search of medline (1946-september 1, 2018) was conducted using the terms pegvaliase and phenylalanine ammonia lyase (pal). the 2 terms were combined with the boolean operator or. the term pegvaliase was also searched on its own. all english language articles related to pharmacology, pharmacokinetics, efficacy, or safety of the therapy in human subjects were included. the references of included articles were searched to identify additional sources. the product dossier was obtained from the manufacturer, and additional data were obtained from the prescribing information and clinicaltrials.gov. data synthesis pharmacology pah catalyzes the irreversible conversion of phenylalanine to tyrosine. in the absence of pah in pku, dietary phenylalanine is unable to be converted to tyrosine and undergoes further processing to metabolites including l-dopa, thyroxine, dopamine, noradrenaline, adrenaline, and melanin.13 there are 2 modes of enzyme therapy through replacement of the pah enzyme or substitution with another enzyme to degrade excess phenylalanine.12 pegvaliase follows the latter category and is a pegylated pal enzyme that converts phenylalanine to ammonia and trans-cinnamic acid.8 ammonia is metabolized by the liver. trans-cinnamic acid and its final product, benzoic acid, are conjugated with glycine and excreted in the urine.13,14 pharmacokinetics due to the heterogeneity of immune response in adults with pku, the pharmacokinetics of pegvaliase exhibit high interpatient and intrapatient variability. high antibody titers correlated with higher apparent clearance. the pharmacokinetic parameters are summarized in table 1. clinical trials phase 1 clinical trial experience with pegvaliase is summarized in table 2. the first study of pegvaliase in humans was a phase 1, multicenter, open-label study in patients over the age of 18 with a diagnosis of classic pku (phenylalanine concentration >1200 µmol/l at diagnosis) and had a history of poor dietary compliance (pal-001, nct00634660). the average baseline phenylalanine concentration was 1310 µmol/l, and the average body mass index (bmi) was 26.4 kg/m2. twenty-five patients were recruited from 7 centers in the united states. patients were table 1. summary of pharmacokinetic parameters studied. pharmacokinetic parameter daily maintenance dosage pegvaliase 20 mg pegvaliase 40 mg median tmax 8 hours cmax at steady state (mean ± sd) 14.0 ± 16.3 mg/l 16.7 ± 19.5 mg/l apparent volume of distribution (mean ± sd) 26.4 ± 64.8 l 22.2 ± 19.7 l apparent clearance at steady state (mean ± sd) 0.39 ± 0.87 l/h 1.25 ± 2.46 l/h half-life (mean ± sd) 47 ± 42 hours 60 ± 45 hours steady state plasma concentrations during maintenance treatment (mean ± sd) 11.2 ± 9.0 mg/l 10.4 ± 12.7 mg/l metabolism catabolic pathways, degraded into small peptides and amino acids abbreviations: cmax, peak concentration; tmax, time to peak concentration. hydery and coppenrath 3 excluded if they had renal or hepatic dysfunction, had pregnancy or potential pregnancy, or used nicotine, drugs of abuse, or any investigational products in the 30 days prior to screening.15 twenty-five patients were assigned to 1 of 5 dosing groups in which they received a single subcutaneous injection of pegvaliase on day 1: 0.001, 0.003, 0.10, 0.03, and 0.100 mg/kg. patients were followed up for 42 days. in the patients who received the 0.1 mg/kg dose, pegvaliase significantly reduced phenylalanine concentrations (1113 to 575 mmol/l, mean reduction of 48.3%) on day 6. phenylalanine concentrations increased from day 7 to day 21 and remained relatively consistent through the end of the study.15 phase 2 the optimal dosing regimens were explored in 3 phase 2 studies of pegvaliase. pal-002 (nct00925054) was an openlabel, multicenter study of 40 adults with pku. patients entered an 8-week induction phase of various fixed and weight-based doses of pegvaliase administered weekly, followed by an 8-week titration phase during which increasing doses of pegvaliase were administered weekly with the goal of achieving reduced blood phenylalanine concentrations. the doses ranged from 0.001 to 0.1 mg/kg throughout the study. these doses were not sufficient to lower phenylalanine concentrations. table 2. summary of trials of pegvaliase for the treatment of pku in adults. phase identifier design duration dosing n results 1 nct00634660 (pal-001) ol, single dose 42 days 0.001, 0.003, 0.10, 0.03, and 0.100 mg/kg 25 48.3% reduction in phe from day 4 to 7 in the highest dosing group 2 nct00925054 (pal-002) ol, multiple dose with induction and titration phases 16 weeks 0.001-0.1 mg/kg given 5 d/ wk 40 no significant changes in phe 5% (2 patients) discontinued due to aes 2 nct01212744 (pal-004) ol, multiple dose 13 weeks 0.06-0.8 mg/kg/d given 5 d/wk 16 no significant changes in phe 6.25% (1 patient) discontinued due to aes 2 nct01560286 (165-205) ol, multiple dose with induction, titration, and maintenance phases 24 weeks 2.5 mg weekly × 4-8 weeks, then increased to a maximum of 75 mg daily 24 46% reached maintenance dose 56% reduction in phe 54% did not reach maintenance dose 8.33% (2 patients) discontinued due to aes 2 nct00924703 (pal-003) ol, multiple dose extension study extended follow-up of phase 2 studies to 264 weeks continued or increased from parent studies, up to 375 mg/wk given up to 7 d/ wk 68a 58.9% reduction in phe at 48 weeks 72.3% reduction in phe at 120 weeks 5.9% (4 patients) discontinued due to aes 3 nct01819727 (prism-1) ol, randomized multiple dose with induction, titration, and maintenance phases 24 months 2.5 mg daily titrated to either 20 or 40 mg daily 261 51.1% reduction in phe at 12 months 68.7% reduction in phe at 24 months improvement in mood and inattention scores 11% (29 patients) discontinued due to aes 3 nct01889862 (prism-2) prism-1 extension studyb part 1 ol continuation of prism-1 20 or 40 mg daily data not published part 2 r discontinuation study 8 weeks 20 mg daily, 40 mg daily, or placebo 86 no change in phe in the groups that maintained doses 62.6% increase in phe in placebo 20 mg group 56.4% increase in phe in placebo 40 mg group no patients discontinued due to aes part 3 ol, pd, pk 20 or 40 mg daily data not published part 4 ol extension ongoing 5-60 mg daily ongoing abbreviations: aes, adverse events; ol, open label; pd, pharmacodynamics; phe, phenylalanine; pk, pharmacokinetic; pku, phenylketonuria; r, randomized. apatients from the other phase 2 studies. b12 patients from the phase 2 studies, but their data are not reported. 4 drug target insights pal-004 (nct01212744) was an open-label, multicenter study of 16 adults with pku. patients were given doses of pegvaliase 5 d/wk which ranged from 0.06 to 0.8 mg/kg throughout the 13 weeks of the study. similar to pal-002, the doses studied resulted in inadequate control of blood phenylalanine levels. all patients in both studies experienced at least 1 adverse event (ae). the regimens used in pal-002 were generally well tolerated, but the higher doses in pal-004 were associated with more frequent hypersensitivity reactions (which included arthralgia, arthritis, eye inflammation, eye irritation, eye pain, joint stiffness, joint swelling, pyrexia, vision blurred, and polyarthritis) and the need for dose reduction.16 based on observations made in pal-002 and pal-004, a third phase 2 study, 165-205 (nct01560286), was conducted. this study was an open-label, multicenter study that was 24 weeks in duration. patients were eligible if they had a blood phenylalanine concentration of ⩾600 µmol/l at screening and an average concentration of ⩾600 µmol/l in the 6 months prior to the screening, and no prior treatment with sapropterin in the 4 months prior to screening. patients who were pregnant, planning to become pregnant, or breastfeeding were excluded. patients entered a 4to 8-week induction phase of 2.5 mg/wk. afterward, the dose and frequency could be increased over a minimum of 4 weeks to attain a blood phenylalanine concentration of ⩽600 µmol/l. the authors defined the “maintenance dose” as the regimen that resulted in target concentrations for 4 weeks without the need for dose adjustments. once the maintenance dose was determined, patients entered the next phase. in the maintenance phase, the maintenance dose was continued but could still be adjusted for safety or to maintain concentrations below 600 µmol/l. the maximum dose in the maintenance phase was 75 mg/d given 5 d/wk. however, the protocol was updated to change the maximum frequency to 7 d/wk. patients experiencing severe hypersensitivity adverse events (haes) had their dosing interrupted or adjusted and restarted at the same or a lower dose. another protocol change allowed patients with treatment-related acute systemic hypersensitivity events to permanently discontinue the study drug. the investigators used a modified intention-to-treat analysis consisting of participants who received at least 1 dose of the study drug and had at least 1 post-treatment blood phenylalanine level.17 twenty-four patients enrolled in the study, with 11 patients achieving maintenance dose in the first 24 weeks (group a) and 13 patients not achieving maintenance dose within 24 weeks (group b). patients had a mean age of approximately 29 years, and baseline blood phenylalanine concentrations were similar in both groups (1134.8 µmol/l in group a vs 1197.5 µmol/l in group b). however, group a was comprised mostly of women with a mean weight of 64.6 kg, whereas group b was mostly men with a mean weight of 86.4 kg. baseline total protein intake and dietary phenylalanine intake were higher in group b. mean doses of pegvaliase in the 24-week study were 64.7 mg in group a and 89.4 mg in group b. the group a patients’ mean blood phenylalanine concentration reached <600 µmol/l by week 11 of the study. the mean reduction from baseline was 627 ± 432 µmol/l (56% ± 36% reduction). furthermore, all patients in group a reached a blood phenylalanine concentration target of <120 µmol/l during the 24-week study. group a had lower anti-drug antibody responses, which was likely associated with less immune-related clearance compared with patients in group b with higher anti-drug antibody responses. ten of the 13 patients in group b continued on to the extension study, during which they achieved a blood phenylalanine concentration of ⩽600 µmol/l by week 48.17 patients in the 3 phase 2 dosing finding studies (will be referred to hereon in as “parent studies”) were invited to continue on in an extension study (pal-003, nct00924703) if they were willing to continue stable protein intake. patients taking any other injectable drug containing polyethylene glycol (peg) and patients with a history of systemic hypersensitivity events to a peg-containing product were excluded. however, patients with a previous reaction to pegvaliase could be eligible for the study on a case by case basis. doses for individuals were either continued from the parent study or increased and were adjusted throughout the study to attain or maintain phenylalanine concentrations between 60 and 600 µmol/l. in addition, doses could be adjusted to manage aes. initially, dosing ranged from 2.5 to 375 mg/wk or 0.001 to 5 mg/kg/wk, but a protocol changed limited weekly dosing to 375 mg/wk. the authors used a modified intention-totreat analysis for efficacy which included all patients who received 1 dose of pegvaliase and had at least 1 phenylalanine concentration measurement taken after treatment.17 sixty-eight of the 80 participants in the parent studies enrolled in the extension study. the baseline phenylalanine concentration was 1022.4 µmol/l. the mean daily dose of pegvaliase in the parent studies was 5.3 mg/d (sd = 6.8 mg/d), and this increased to 26.2 mg/d (sd = 17.9 mg/d) in the extension study. doses remained relatively stable from week 48 to week 120 of the study. the average duration of treatment in all of the phase 2 studies was approximately 3.4 years. mean phenylalanine concentrations decreased throughout the study by more than half (58.9%) at week 48 of treatment and by 72% at week 120 (1022.4 µmol/l at baseline, 541.6 µmol/l at week 48, and 372 µmol/l at week 120).17 approximately one-third of patients (36.8%) had their pegvaliase dosing reduced or held during the study. a small number of patients (n = 4, 5.9%) discontinued the study due to aes. all 4 patients reported resolution of the aes after discontinuing treatment. hypersensitivity adverse events were highest during early treatment in phase 2 studies and correlated with the timing of highest igm, peg igm, and peg igg antibodies.17 phase 3 pegvaliase was evaluated in a phase 3 study, prism-1 (nct01819727). in this multicenter, open-label study, patients hydery and coppenrath 5 naïve to pegvaliase were randomized to receive 1 of 2 regimens of pegvaliase. patients with a blood phenylalanine concentration of 600 µmol/l or higher for at least 6 months prior to the study were eligible. it is unclear if allocation to treatment groups was concealed. both groups received 2.5 mg once per week of pegvaliase subcutaneously for 4 weeks during the induction period. dosing and frequency were increased gradually to either 20 mg/d or to 40 mg/d. titration was performed over a period of 5 to 30 weeks. the schedule was described for the 20 mg/d group: 2.5 mg twice weekly, 10 mg weekly, 10 mg twice weekly, 10 mg 4 times weekly, 10 mg daily, and then 20 mg daily. doses were continued in the maintenance phase, which ranged from 24 to 36 weeks in duration. phenylalanine levels, safety, immunogenicity, and neuropsychiatric symptoms were assessed by trained study staff at each site using tools validated for use in pku.18 prism-1 included 261 patients: 131 in the 20 mg/d group and 130 in the 40 mg/d group. patients had a mean age of approximately 30 years at baseline and were mostly white, and approximately half of them were women. mean phenylalanine levels at baseline were 1232.7 µmol/l. the mean number of weeks to reach maintenance doses was 11.5 and 14 in the 20 mg/d and 40 mg/d groups, respectively.18 mean phenylalanine concentrations decreased by 51.1% from baseline to 564.5 µmol/l at 12 months of follow-up, and by 68.7% from baseline to 311.4 µmol/l at 24 months of follow-up. inattention and mood scores improved while on treatment with pegvaliase and correlated with a decrease in phenylalanine levels. similar to previous studies, all patients experienced at least 1 ae. however, in this study, 99% of events were mild to moderate in nature, and 96% resolved without dose reduction or interruption. this represents an improvement in the safety profile compared with previous regimens studied. the most common aes were arthralgia (70.5%), injection site reactions (62.1%), injection site erythema (47.9%), and headache (47.1%). of the aes leading to discontinuation, 2.7% were anaphylaxis, 2.7% were arthralgia, 1.1% were injection site reactions, and 0.8% were generalized rash. twelve patients (4.6%) experienced 1 or more acute systemic haes which were independently adjudicated by an allergist/immunologist. the median time after pegvaliase administration to acute systemic hae was 1.8 minutes, and all occurred within the first 50 days of dosing and were resolved quickly.18 patients enrolled in prism-1 could continue to prism-2 (nct01889862), a study to evaluate the safety and efficacy of long-term treatment. patients reaching maintenance dosing in prism-1 entered part 1 of prism-2, and continued their assigned doses, either 20 mg or 40 mg daily. part 2 was a randomized, double-blinded, placebo-controlled discontinuation study which will be described below. part 3 was an open-label pharmacodynamics and pharmacokinetic study, and part 4 is an open-label extension study which is ongoing. patients who did not reach maintenance dosing in prism-1 could enter directly in to part 4 of prism-2, in which the open-label dosing ranges from 5 to 60 mg daily.18 eighty-six patients who received pegvaliase in prism-2 part 1 enrolled in the double-blinded prism-2 part 2 discontinuation trial. an additional 9 patients were enrolled but were not included in the efficacy analysis. patients were randomized via concealed allocation to receive either their current pegvaliase dose (either 20 or 40 mg/d) or matched placebo containing either 20 mg or 40 mg of dextran 40 (22% dextran 40). the study drugs were self-administered daily for 8 weeks, rotating subcutaneous injection sites.19 the pooled active pegvaliase group phenylalanine concentrations remained stable from baseline to week 8 (503.9559 µmol/l), while levels in both placebo groups increased (563.9-1509 µmol/l in the placebo 20 mg group and 508.21164.4 µmol/l in the placebo 40 mg group). however, no significant changes in mood or inattention were observed, perhaps due to the short duration of the trial or to the tools used to measure these symptoms in the trial.19 patients in prism-1 demonstrated improvements in attention deficit hyperactivity disorder (adhd) scores at 3 months of treatment.16 overall, clinical trial experience from phase 1 to phase 3 suggests substantial reductions in blood phenylalanine concentrations and improvement in inattention and mood symptoms with the use of pegvaliase. however, these benefits must be weighed against the risk of common and sometimes serious aes. dosing and administration the package insert for palynziq recommends to obtain baseline blood phenylalanine concentrations prior to treatment initiation.11 the induction dosage for pegvaliase is 2.5 mg subcutaneously once weekly for 4 weeks under the supervision of a health care provider. the dosage should be titrated based on tolerability over at least 5 weeks to achieve a dosage of 20 mg subcutaneously once daily. therapeutic response may not be achieved until the patient is titrated to an effective maintenance dose. during treatment, patient tolerability, blood phenylalanine concentrations, dietary protein, and phenylalanine intake should be monitored. studies in adult pku patients have shown a variation in phenylalanine levels can be observed without any change in treatment.20 in clinical trials, the response to sapropterin treatment was defined as a 30% reduction in blood phenylalanine concentration from baseline.21 in contrast, therapeutic response with pegvaliase was based on a 20% reduction in blood phenylalanine concentration from baseline or a blood phenylalanine concentration ⩽600 µmol/l after 24 weeks. if not achieved, increasing the pegvaliase dose to a maximum dosage of 40 mg subcutaneously once daily can be considered. if therapeutic response has not been achieved after 16 weeks on the maximum dosage, discontinuation of pegvaliase is recommended. if 6 drug target insights patients experience blood phenylalanine concentrations <30 µmol/l during titration and maintenance, the dosage of pegvaliase may be reduced or the dietary protein and phenylalanine intake may be modified.11 for hypersensitivity reactions, premedication can be considered with an h1-receptor antagonist, h2-receptor antagonist, and/or antipyretic prior to administration based on tolerability. patients should be observed during and for at least 60 minutes after receiving pegvaliase injection. patients should also be trained on recognition of signs and symptoms of anaphylaxis, how to administer injectable epinephrine, and how to seek emergency care, if needed. if the decision is made to readminister pegvaliase after an episode of anaphylaxis, the subsequent dose should be administered under the supervision of a health care provider after titrating based on patient tolerability and therapeutic response.11 due to the risks of anaphylaxis, pegvaliase is only available through a risk evaluation and mitigation strategies (rems) program which includes prescriber and pharmacy certification requirements and patient educational enrollment. in clinical trials, most patients developed anti-peg igm and igg antibodies after treatment. the clinical effects of concomitant treatment with other pegylated products are unknown. therefore, if patients are treated in combination with other pegylated products, further monitoring for hypersensitivity reactions and anaphylaxis is recommended.11 relevance to patient care and clinical practice the risk of adverse outcomes in pku is related to disease severity, blood phenylalanine concentration, and adherence to treatment.4 the assessment of blood phenylalanine concentrations may also be indicative of patient adherence to therapy.22 poor adherence can manifest as failure to take medical foods or prescribed medications, and missing regular clinic appointments or blood phenylalanine testing.23 a presentation released by biomarin pharmaceuticals suggests there are 125 pku clinics in the united states currently managing patients through dietary counseling and/or sapropterin.24 a study of 45 pku clinics showed that non-adherence to clinic-recommended target phenylalanine concentrations increased with age. most adults had blood phenylalanine concentrations >360 µmol/l with 15% of patients aged 18 to 29 years and 20% of patients aged 30+ years reported to have concentrations >1200 µmol/l.23 given that pku can cause neurocognitive and psychiatric symptoms, there may be an unmet need for additional therapeutic approaches in adults with uncontrolled disease. dietary treatment consists of dietary restriction of phenylalanine, phenylalanine-free protein substitutes (l-amino acid mixtures), and modified low-protein foods.4,6 patients with residual enzymatic activity of pah may respond to sapropterin which increases metabolism of phenylalanine to tyrosine. safety and efficacy of sapropterin have been established in pediatric patients as young as 1 month of age.21 prior to routine treatment with sapropterin, a test should be conducted to determine if the patient is sapropterin-responsive, defined as a rapid decline in phenylalanine or increase in phenylalanine tolerance. sapropterin can provide better phenylalanine control and increase dietary phenylalanine tolerance.25 clinical guidelines have recently been updated on to address the role of pegvaliase in management of pku. the goal of pegvaliase treatment is to provide maintenance of blood phenylalanine concentrations while normalizing diet. pegvaliase can be considered for all adult patients with pku who can adhere to therapy, including requirement for monitoring of aes.26 no head-to-head trials were conducted comparing the safety and efficacy of sapropterin with pegvaliase. given patients in the clinical trials were unable to continue sapropterin in the pegvaliase trials, it is unclear whether the combination is statistically and clinically more beneficial than either treatment alone. although the package insert for pegvaliase does not specify use to be in conjunction with a phenylalanine-restricted diet, it is indicated in patients with uncontrolled blood phenylalanine concentrations “on existing management”11 which can be inferred as guideline-recommended dietary therapy and/or sapropterin pharmacotherapy. furthermore, in pegvaliase clinical trials, patients were stable in protein intake and in many instances received counseling from a dietician.15–19 however, while thomas et al observed stable total dietary protein intake, there was a decrease in medical food protein (from 26.3 to 18.4 g/d) and an increase in dietary phenylalanine intake (from 1700.2 to 2679.7 mg/d). this shift may indicate a decreased need for medical food.18 efficacy of pegvaliase without dietary therapy was not assessed in clinical trials. due to the potential shifts in dosing from changing dietary protein and phenylalanine intake, continuation of nutritional and blood phenylalanine monitoring is warranted in patients treated with pegvaliase.11,26 given only 25% to 50% of patients with pku respond to sapropterin,3 the availability of additional pharmacologic options is necessary to improve the quality of life in patients with uncontrolled pku. in 2015, the national pku alliance conducted a survey of adults and children with pku in the united states. the survey confirmed that dietary therapy is the mainstay of clinical treatment. approximately 6% of survey respondents stated they were not treating pku with medical foods or pharmacotherapy. most survey respondents (91%) stated the importance of development of new products for pku. respondents generally preferred oral therapy over injectable therapy, and home injections over injections, at a medical facility.27 pegvaliase is a novel enzyme substitution therapy which can be used in patients with more severe disease and in part meets the needs of the pku community. the subcutaneous prefilled syringe can be self-administered if patients demonstrate adequate competency.11 although an oral pegylated pal formulation has been investigated in animal studies,28 similar trials have not been conducted in humans. it is recommended that multidisciplinary teams continue involvement in hydery and coppenrath 7 care of patients with pku, including monitoring therapeutic response, adverse effects, and medication adherence. cost as of june 2018, pegvaliase has been launched. for cost considerations, the average wholesale price (awp) for pegvaliasepqpz prefilled syringes are us$585.60 which includes 3 strengths: 2.5 mg/0.5 ml, 10 mg/0.5 ml, and 20 mg/ml, whereas the awp of sapropterin 100 and 500 mg tablets are us$44.28 and us$221.40 per tablet, respectively.29,30 using these awp cost figures, the 30-day cost for a 70 kg patient at the maximum recommended sapropterin dose (20 mg/kg once daily) is us$5314 and at the maximum recommended pegvaliase-pqpz dose (40 mg once daily) is us$35 136. biomarin pharmaceuticals predicts the average annual net cost of therapy for a typical patient to be approximately us$192 000 per year based on an average of 1.5 units per patient at maintenance dosing.24 dietary treatments used in conjunction with currently available pharmacotherapy pose an additional cost burden to patients and payers. conclusions patient characteristics and preferences should be considered to help guide treatment of pku. pegvaliase reduces blood phenylalanine concentrations independent of pah activity compared with previously available therapeutic alternatives.4 in pivotal phase 3 clinical studies, treatment with pegvaliase was associated with overall safety and statistically significant improvements in blood phenylalanine concentrations.18,19 patients also had sustained reductions in blood phenylalanine concentrations that reached guideline-recommended levels. however, due to risks of anaphylaxis, pegvaliase is only available through a restricted program under rems.11 the injectable route of administration, potential risks, and factors related to insurance coverage may limit the use of pegvaliase. at the present time, pegvaliase is only fda-approved for use in adults who have uncontrolled blood phenylalanine concentrations >600 µmol/l.11 clinical guidelines have been updated to address the role of pegvaliase in management of pku.26 given the high cost, therapy may be a viable option for patients who have failed existing management strategies due to inadequate response or desire for dietary relaxation. further research should be conducted to evaluate the safety and efficacy in younger patients, those with milder forms of pku, in combination with sapropterin, and with limited dietary therapy. author contributions th and vac wrote, reviewed, and approved the final manuscript. ethical approval given there was no human participation or use of personal data involved in the writing of this review article, no ethical permission was applied for. informed consent given the writing of this review article was non-human subject research and based on the availability of published clinical trial data, written/verbal consent from patients and/or caregivers was not necessary. orcid id tasmina hydery https://orcid.org/0000-0002-7206-1668 r efer ences 1. national institutes of health consensus development panel. national institutes of health consensus development conference statement: phenylketonuria: screening and management, october 16–18, 2000. pediatrics. 2001; 108:972. 2. bodamer oa, hahn s, tepas e. overview of phenylketonuria. in: basow, ds, ed. uptodate [database on the internet]. waltham, ma: uptodate; 2018. http://www.utdol.com/utd/index.do. accessed june 19, 2018. 3. scriver cr, kaufman s. the hyperphenylalaninemias. phenylalanine hydroxylase deficiency. in: scriver cr, beaudet al, sly ws, valle d, eds. the metabolic and molecular bases of inherited disease. 8th ed. new york: mcgraw-hill; 2001:1667. 4. vockley j, andersson hc, antshel km, et al. phenylalanine hydroxylase deficiency: diagnosis and management guideline. genet med. 2014;16:188–200. doi:10.1038/gim.2013.157. 5. abadie v, berthelot j, feillet f, et al. [management of phenylketonuria and hyperphenylalaninemia: the french guidelines]. arch pediatr. 2005;12:594. doi:10.1016/j.arcped.2005.02.004. 6. van wegberg amj, macdonald a, ahring k, et al. the complete european guidelines on phenylketonuria: diagnosis and treatment. orphanet j rare dis. 2017;12:162. doi:10.1186/s13023-017-0685-2. 7. van spronsen fj, burgard p. the truth of treating patients with phenylketonuria after childhood: the need for a new guideline. j inherit metab dis. 2008;31:673679. doi:10.1007/s10545-008-0918-6. 8. biomarin announces fda approval for kuvan. biomarin. https://investors. biomarin.com/2007-12-13-biomarin-announces-fda-approval-for-kuvan. accessed september 26, 2018. 9. somaraju ur, merrin m. sapropterin dihydrochloride for phenylketonuria. cochrane database syst rev. 2015;3:cd008005. doi:10.1002/14651858. cd008005.pub4. 10. fda approves a new treatment for pku, a rare and serious genetic disease. food and drug administration. https://www.fda.gov/newsevents/newsroom/pressannouncements/ucm608835.htm. accessed september 26, 2018. 11. palynziq® [package insert]. novato, ca: biomarin pharmaceutical inc; may 2018. 12. palynziq® (pegvaliase-pqpz) formulary dossier. biomarin, data on file. 13. bell sm, wendt dj, zhang y, et al. formulation and pegylation optimization of the therapeutic pegylated phenylalanine ammonia lyase for the treatment of phenylketonuria. plos one. 2017;12:e0173269. doi:10.1371/journal. pone.0173269. 14. sarkissian cn, shao z, blain f, et al. a different approach to treatment of phenylketonuria: phenylalanine degradation with recombinant phenylalanine ammonia lyase. proc natl acad sci u s a. 1999;96:2339–2344. doi:10.1073/ pnas.96.5.2339. 15. longo n, harding co, burton bk, grange dk, et al. single-dose, subcutaneous recombinant phenylalanine ammonia lyase conjugated with polyethylene glycol in adult patients with phenylketonuria: an open-label, multicenter, phase 1 dose-escalation trial. lancet. 2014;384:37–44. doi:10.1016/s0140 -6736(13)61841. 16. thomas ja, longo n, zori r, burton bk, et al. evaluation of multiple dosing regimens in phase 2 studies of “ravpal-peg” for control of blood phenylalanine levels in adults with phenylketonuria. paper presented at: society for the study of inborn errors of metabolism (ssiem) annual symposium, lyon, france, 1–4 september 2015, p-149. 17. zori r, thomas ja, shur n, et al. induction, titration, and maintenance dosing regimen in a phase 2 study of pegvaliase for control of blood phenylalanine in adults with phenylketonuria. mol genet metab. 2018;125:217–227. doi:10.1016/j. ymgme.2018.06.010. 18. thomas j, levy h, amato s, et al. pegvaliase for the treatment of phenylketonuria: results of a long-term phase 3 clinical trial program (prism). mol genet metab. 2018;124:27–38. doi:10.1016/j.ymgme.2018.03.006. 19. harding co, amato rs, stuy m, et al. pegvaliase for the treatment of phenylketonuria: a pivotal, double-blind randomized discontinuation phase 3 https://orcid.org/0000-0002-7206-1668 http://www.utdol.com/utd/index.do https://investors.biomarin.com/2007-12-13-biomarin-announces-fda-approval-for-kuvan https://investors.biomarin.com/2007-12-13-biomarin-announces-fda-approval-for-kuvan https://www.fda.gov/newsevents/newsroom/pressannouncements/ucm608835.htm https://www.fda.gov/newsevents/newsroom/pressannouncements/ucm608835.htm 8 drug target insights clinical trial. mol genet metab. 2018;124:20-26. doi:10.1016/j.ymgme.2018 .03.003. 20. van rijn m, hoeksma m, sauer pj, modderman p, reijngoud dj, van spronsen fj. adult patients with well-controlled phenylketonuria tolerate incidental additional intake of phenylalanine. ann nutr metab. 2011;58:94–100. doi:10.1159 /000324924. 21. kuvan® [package insert]. novato, ca: biomarin pharmaceutical inc; august 2016. 22. macdonald a, van rijn m, feillet f, et al. adherence issues in inherited metabolic disorders treated by low natural protein diets. ann nutr metab. 2012;61:289– 295. doi:10.1159/000342256. 23. jurecki er, cederbaum s, kopesky j, et al. adherence to clinic recommendations among patients with phenylketonuria in the united states. mol genet metab. 2017;120:190–197. doi:10.1016/j.ymgme.2017.01.001. 24. biomarin pharmaceuticals. conference call to discuss approval of palynziq. investors.biomarin.com/download/palynziq+approval+presentation_052418. pdf. accessed september 27, 2018. 25. cunningham a, bausell h, brown m, et al. recommendations for the use of sapropterin in phenylketonuria. mol genet metab. 2012;106:269–276. doi:10.1016/j.ymgme.2012.04.004. 26. longo n, dimmock d, levy h, et al. evidenceand consensus-based recommendations for the use of pegvaliase in adults with phenylketonuria. genet med. 2018. doi:10.1038/s41436-018-0403-z. 27. brown cs, lichter-konecki u. phenylketonuria (pku): a problem solved. mol genet metab rep. 2016;6:8–12. doi:10.1016/j.ymgmr.2015.12.004. 28. sarkissian cn, kang ts, gamez a, scriver cr, stevens rc. evaluation of orally administered pegylated phenylalanine ammonia lyase in mice for the treatment of phenylketonuria. mol genet metab. 2011;104:249–254. doi:10.1016 /j.ymgme.2011.06.016. 29. pegvaliase-pqpz. in: basow ds, ed. uptodate [database on the internet]. waltham, ma: uptodate; 2018. http://www.utdol.com/utd/index.do. accessed september 26, 2018. 30. sapropterin. in: basow ds, ed. uptodate [database on the internet]. waltham, ma: uptodate; 2018. http://www.utdol.com/utd/index.do. accessed september 26, 2018. http://www.utdol.com/utd/index.do http://www.utdol.com/utd/index.do dti drug target insights 2023; 17: 45-53issn 1177-3928 | doi: 10.33393/dti.2023.2529original research article drug target insights issn 1177-3928 www.aboutscience.eu/dti © 2023 the authors. this article is published by aboutscience and licensed under creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). commercial use is not permitted and is subject to publisher’s permissions. full information is available at www.aboutscience.eu lipid profiles of people with human immunodeficiency virus with dyslipidemia after switching from efavirenz to dolutegravir supphachoke khemla¹, atibordee meesing1,2, wantin sribenjalux1,2, ploenchan chetchotisakd1 ¹division of infectious diseases and tropical medicine, department of medicine, faculty of medicine, khon kaen university, khon kaen thailand 2research and diagnostic center for emerging infectious diseases (rceid), khon kaen university, khon kaen thailand abstract introduction: human immunodeficiency virus (hiv) infection and the long-term use of antiretroviral therapy, especially efavirenz (efv)-based regimens, impact lipid profiles due to insulin resistance and lead to a higher risk of metabolic diseases. dolutegravir (dtg) is an integrase inhibitor with better lipid profiles than efv. however, data on treatment experience in thailand are limited. the primary outcome was lipid profile changes at 24 weeks after switching therapy. methods: we conducted a prospective, open-label, cohort study in people with hiv aged ≥18 years who had undergone at least 6 months of efv-based therapy, had hiv-1 ribonucleic acid levels <50 copies/ml for ≥6 months before switching, and were diagnosed with dyslipidemia or had risk factors for atherosclerosis cardiovascular disease based on modified national cholesterol education program adult treatment panel iii guidelines. results: sixty-four patients were enrolled. the mean age (standard deviation [sd]) was 48.20 ± 10.46 years, and 67.19% were male. at week 24, there were decreases from baseline in mean total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and triglycerides. however, mean body weight and waist circumference had increased. conclusions: dtg resulted in better lipid profiles after switching from efv-based therapy, suggesting that this switch could benefit patients with a high risk of cardiovascular disease. however, it is essential to note that weight gain and increased waist circumference were also observed. keywords: arv, dolutegravir, dyslipidemia, efavirenz, switching treatment received: november 18, 2022 accepted: april 11, 2023 published online: april 28, 2023 corresponding author: atibordee meesing department of medicine faculty of medicine khon kaen university khon kaen 40002 thailand atibordee@kku.ac.th long-term use of antiretroviral treatment regimens may lead to dyslipidemia, which is a significant risk factor for cardiovascular disease (2-5). previously, people with hiv in thailand were typically prescribed a firstline antiretroviral regimen that included efavirenz (efv) along with two nucleoside reverse transcriptase inhibitors (nrtis) (6). as a long-acting non-nucleoside reverse transcriptase inhibitor (nnrti), efv is an effective form of hiv treatment in clinical settings. however, there are side effects such as drug rash, hepatitis, and long-term metabolic diseases (3,4). this can raise the likelihood of developing hyperglycemia and subsequent insulin resistance while also affecting lipid metabolism. in addition, it can hinder the breakdown of fat, leading to an increase in triglycerides, very-low density lipoprotein (vldl), and low-density lipoprotein (ldl) levels, and a decrease in high-density lipoprotein (hdl) levels. this ultimately results in dyslipidemia, which can eventually cause cardiovascular disease (2). introduction people with human immunodeficiency virus (hiv) typically have the potential to live for a considerable length of time after receiving highly active antiretroviral therapy (haart). the virus triggers an inflammatory response that can result in metabolic issues such as diabetes mellitus, hypertension, and dyslipidemia. the prevalence of people with hiv with dyslipidemia is as high as 51% (1). https://doi.org/dti.2023.2529 https://doi.org/dti.2023.2522 https://creativecommons.org/licenses/by-nc/4.0/legalcode lipid profiles after switching from efavirenz to dolutegravir46 © 2023 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti the us food and drug administration approved dolutegravir (dtg) in 2013 as an integrase strand transfer inhibitor (insti)-based regimen, which works by inhibiting integrase, an enzyme that hiv needs to insert its deoxyribonucleic acid (dna) into the dna of host lymphocytes (7). it is highly effective, has few side effects compared to other drugs, and only needs to be taken once per day. however, there have been some reports of patients gaining weight after taking this drug long term (8). it is currently the first-line antiretroviral regimen administered in thailand (9). a randomized controlled trial in naive people with hiv compared the levels of lipids between an efv group and dtg group and found that the latter had less of an increase in cholesterol (10-12). a comprehensive approach is necessary for dyslipidemia management in people with hiv, which may involve lifestyle modification including controlled calories intake, exercise, and maintaining a healthy body weight or wight reduction. another approach is to choose antiretroviral drugs that do not worsen dyslipidemia, and to modify antiretroviral therapy when necessary to control lipid levels. the use of lipid-lowering agents, such as statin agents and fibrates, may also be essential to reduce the risk of cardiovascular disease. at present, there are limited data available on switching from efv to dtg in people with hiv who have dyslipidemia in thailand. the main goal of this study was to examine alterations in the lipid profile of people with hiv who have dyslipidemia, specifically at the 24-week mark following the switch from efv to dtg. secondary objectives were to evaluate the efficacy of dtg in maintaining hiv-1 ribonucleic acid (rna) levels at <50 copies/ml after 24 weeks of switching treatment, as well as its safety, tolerability, body weight, body mass index (bmi), and waist circumference. methods a prospective, open-label cohort study was conducted at srinagarind hospital, a tertiary university hospital in northeastern thailand, between april 2021 and april 2022. the patients were eligible for the study if they met all the following criteria: (1) age over 18 years, (2) having received efv-based therapy for at least 6 months, (3) hiv-1 rna <50 copies/ml for ≥6 months before switching therapies, (4) diagnosis with dyslipidemia or risk factors for atherosclerosis cardiovascular disease (ascvd) based on modified national cholesterol education program (ncep) adult treatment panel (atp) iii guidelines (13). in brief, dyslipidemia was defined as either (1) ldl-cholesterol ≥130 mg/dl with at least one of the following coronary heart disease (chd) risk factors: age >45 years if male or age >55 years if female, hypertension (blood pressure ≥140/90 mmhg or on antihypertensive medication), current cigarette smoking, or family history of premature chd and/or diabetes; (2) ldlcholesterol ≥160 mg/dl regardless of chd risk factors; or (3) previous diagnosis of dyslipidemia and on lipid-lowering drugs. exclusion criteria were pregnancy or breastfeeding, active opportunistic infections, or taking metformin >1,000 mg/day, rifampicin, st. john’s wort, antiarrhythmic drugs (e.g., dofetilide, pilsicainide), antiepileptic drugs (e.g., carbamazepine, oxcarbazepine, phenytoin, phenobarbital), or medications or supplements containing polyvalent cations (e.g., magnesium, aluminum, cation-containing antacids or laxatives, sucralfate, buffered medications). patient evaluation was performed at baseline, week 12, and week 24. data collected for each participant included age, sex, body weight, height, bmi, waist circumference, backbone regimen, chd risks, current lipid-lowering agents, duration from hiv diagnosis to enrollment, duration of first treatment with antiretroviral agents to enrollment, and duration of efv treatment to that with lipid-lowering agents. clinical laboratory testing was performed at a local laboratory. laboratory tests included hiv-1 rna, absolute cd4 cell count, %cd4, and lipid profiles including total cholesterol, ldl-cholesterol, hdl-cholesterol, and triglycerides. the safety of the studied regimens was assessed using patient interviews, medical history, physical examination, and clinical laboratory test results. study procedure upon approval to undertake the project by the human research committee at khon kaen university, the patients were screened and provided informed consent to be enrolled into this study. blood tests were obtained on the date of enrollment according to protocols. patients were changed from an efv-based to a dtg-based regimen and received dosing instructions from the investigators. patients had two follow-up appointments at 12 (±1) and 24 (±1) weeks. the study protocol was reviewed and approved by the khon kaen university center of ethics in human research (he641043). sample size calculation assuming a change in ldl-cholesterol level of 10.67, a standard deviation (sd) of ±30.37 mg/dl extrapolated from a study with 80% power, and a one-sided type 1 error of 0.05, a sample size of 64 patients was necessary. we calculated a 10% loss to follow-up, making the total required population 70 patients. statistical analysis the data were analyzed using statistical package for the social sciences (spss) version 26. categorical data were expressed as proportions, and continuous data were expressed as mean and sd, 95% confidence interval (ci), or median (range), as appropriate. the data depended on whether the distribution was normal or non-normal. comparisons between values before and after changing medications were performed using a paired dependence t-test or proportional mcnemar test, as appropriate. results a total of 64 patients with dyslipidemia were enrolled in the study at baseline, followed up on for 12 weeks, and attended study visits for 24 weeks (fig. 1). khemla et al drug target insights 2023; 17: 47 © 2023 the authors. published by aboutscience www.aboutscience.eu figure 1 panel a shows the flow diagram. panel b shows the protocols from eligible week until follow-up at week 12 and week 24. bmi = body mass index; bun = blood urea nitrogen; bw = body weight; cbc = complete blood count; cd4 = cluster of differentiation 4; cr = creatinine; fpg = fasting plasma glucose; hiv rna = human immunodeficiency virus ribonucleic acid; lft = liver function test; wc = waist circumference. the majority of patients were male (67.19%), and mean age (sd) was 48.20 ± 10.46 years. mean absolute cd4 count was 603.27 ± 237.08 cells/mm3. mean duration from diagnosis of hiv and from first antiretroviral agents until switching therapy were 103.44 ± 57.79 and 88.81 ± 44.53 months, respectively. mean body weight, height, bmi, and waist circumference were 66.0 ± 12.02 kg, 165.56 ± 8.80 cm, 23.99 ± 3.51 kg/m2, and 87.79 ± 10.82 cm, respectively. the most common nrti backbones were tenofovir disoproxil fumarate (tdf)/emtricitabine (ftc; 84.38%), followed by abacavir (abc)/lamivudine (3tc; 14.06%) and tdf/3tc (1.56%). chd risk factors were dyslipidemia (75.00%), hypertension (12.50%), and diabetes mellitus (3.13%). none of the patients were current smokers. of 64 patients, 45 (70.31%) received lipid-lowering agents for dyslipidemia before switching to a dtg-based regimen. the mean duration to initiation of lipid-lowering agents after starting efv was 35.22 ± 49.16 months. the most common lipid-lowering agents were simvastatin (34.38%), atorvastatin (21.88%), and rosuvastatin (6.25%). patient demographics and baseline characteristics are summarized in table i. at week 12 mean total cholesterol decreased significantly from baseline (−38.81 mg/dl, 95% ci −32.35 to −12.00, p < 0.001), as did ldl-cholesterol (−25.70 mg/dl, 95% ci −31.53 to −19.88, p < 0.001), hdl-cholesterol (−6.24 mg/dl, 95% ci −8.12 to −4.36, lipid profiles after switching from efavirenz to dolutegravir48 © 2023 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti p < 0.001), and triglycerides (−36.17 mg/dl, 95% ci −58.62 to −13.71, p = 0.002). mean changes in fasting lipid parameters from baseline are presented in table ii and fig. 2. there were statistically significant increases from baseline in mean body weight (0.97 kg, 95% ci 0.49 to 1.44, p < 0.001), bmi (0.32 kg/m2, 95% ci 0.16 to 0.49, p < 0.001), and waist circumference (1.53 cm, 95% ci 0.88 to 2.18, p < 0.001; table iii and fig. 3). at week 24 mean total cholesterol had decreased significantly from baseline (−32.78 mg/dl, 95% ci −41.16 to −24.39, p < 0.001), ldl-cholesterol (−21.00 mg/dl, 95% ci −28.34 to −13.65, p < 0.001), hdl-cholesterol (−4.21 mg/dl, 95% ci −6.24 to −2.18, p < 0.001), and triglycerides (−49.70 mg/dl, 95% ci −66.54 to −32.86, p < 0.001). mean changes in fasting lipid parameters from baseline are presented in table ii and fig. 2. there were significant increases from baseline in mean body weight (1.39 kg, 95% ci 0.77 to 2.01, p < 0.001), bmi (0.49 kg/m2, 95% ci 0.27 to 0.73, p < 0.001), and waist circumference (2.6 cm, 95% ci 1.53 to 3.68, p < 0.001; table iii and fig. 3). of 64 patients, 61 (95.31%) had hiv-1 rna <50 copies/ ml at week 24. the hiv-1 rna of the remaining three were 52, 67, and 62 copies/ml. nonstatistically significant changes were seen in absolute cd4 (24.09 cells/mm3, 95% ci −9.60 to 57.79, p = 0.158). mean changes in other laboratory parameters are as follows: fasting blood sugar = 0.45 mg/dl, 95% ci −5.70 to 4.79, p = 0.864, creatinine = 0.15 mg/dl, 95% ci 0.11 to 0.18, p < 0.001, and estimated glomerular filtration rate (egfr) = −9.50 ml/min/1.73 m2, 95% ci −11.97 to −7.04, p < 0.001 (table iv). discussion the use of dtg-based regimen is currently widespread as a first-line antiretroviral treatment globally, including in thailand. this study found that switching to dtg-based regimen in people with hiv with dyslipidemia resulted in improved lipid profiles. the scota study is a large observational cohort study that examined patients who switched from efv to dtg, efv to elvitegravir (evg), or efv to rilpivirine (rpv). it was found that total cholesterol significantly decreased in the efv to dtg and efv to rpv groups but not in the efv to evg group. at month 12, total cholesterol/hdl had significantly decreased in the efv to rpv group but not in the efv to dtg and efv to evg groups. the study results showed that significant reductions in triglycerides were observed only in the group that switched from efv to rpv. furthermore, the decrease in total cholesterol, ldl-cholesterol, triglycerides, and total cholesterol/hdl over 1 year was higher in patients with higher baseline levels (14). the strategy-nnrti trial examined the effects of switching from an nnrti-based regimen (efv, nvp, or rpv) combined with tdf and ftc to coformulated evg/cobicistat (c), table i demographic and baseline characteristics characters total n = 64 age, mean (sd) years 48.20 ± 10.46 male, n (%) 43 (67.19) bodyweight, mean (sd) kg 66.0 ± 12.02 height, mean (sd) cm 165.56 ± 8.80 body mass index, mean (sd) kg/m2 23.99 ± 3.51  • underweight, < 18.5, n (%) 0 (0.0)  • normal, ≥18.5 to <25, n (%) 42 (65.62)  • overweight, ≥25 to <30, n (%) 19 (29.69)  • obese, ≥30, n (%) 3 (4.69) waist circumference, mean (sd) cm 87.79 ± 10.82 backbone regimen  • tdf/ftc, n (%) 54 (84.38)  • abc/3tc, n (%) 9 (14.06)  • tdf/3tc, n (%) 1 (1.56) current cd4, mean (sd)  • absolute cd4, cells/mm3 603.27 ± 237.08  • %cd4 26.03 ± 8.22 coronary heart disease risk, n (%)  • dyslipidemia 48 (75.00)  • hypertension 8 (12.50)  • diabetes mellitus 2 (3.13)  • current smoking 0 (0.0) current lipid-lowering agent, n (%)  • none 19 (29.69)  • simvastatin 22 (34.38)  • atorvastatin 14 (21.88)  • rosuvastatin 4 (6.25)  • fenofibrate 2 (3.12)  • simvastatin plus gemfibrozil 2 (3.12)  • atorvastatin plus fenofibrate 1 (1.56) duration of hiv diagnosis to enrollment, mean (sd) months 103.44 ± 57.79 duration of the first antiretroviral agents to enrollment, mean (sd) months 88.81 ± 44.53 duration of efavirenz to lipid-lowering agents, mean (sd) months 35.22 ± 49.16 laboratory parameters  • hemoglobin, g/dl 13.84 ± 2.09  • fasting plasma glucose, mg/dl 98.88 ± 12.41  • creatinine, mg/dl 0.96 ± 0.16  • egfr, ml/min/1.73 m2 85.46 ± 22.16  • albumin, g/dl 4.76 ± 0.42  • alanine aminotransferase, u/l 36.78 ± 23.79  • aspartate aminotransferase, u/l 32.13 ± 19.66  • alkaline phosphatase, u/l 105.86 ± 31.35 3tc = lamivudine; abc = abacavir; egfr = estimated glomerular filtration rate; ftc = emtricitabine; tdf = tenofovir disoproxil fumarate; sd = standard deviation. khemla et al drug target insights 2023; 17: 49 © 2023 the authors. published by aboutscience www.aboutscience.eu table ii mean change in fasting lipid parameters from baseline lipid profiles (mg/dl) n = 64; mean (sd) week 0 week 12 week 24 change from week 0 vs. 12 p-value change from week 0 vs. 24 p-value change from week 12 vs. 24 p-value diff 95% ci diff 95% ci diff 95% ci total cholesterol 209.69 ± 38.99 170.88 ± 36.43 176.91 ± 35.14 −38.81 ± 25.86 −32.35, −12.00 <0.001 −32.78 ± 33.55 −41.16, −24.39 <0.001 6.03 ± 33.56 −2.35, 14.41 0.156 ldlcholesterol 131.88 ± 36.17 106.17 ± 31.37 110.88 ± 30.72 −25.70 ± 23.31 −31.53, −19.88 <0.001 −21.00 ± 29.41 −28.34, −13.65 <0.001 4.71 ± 26.78 −1.98, 11.39 0.165 hdlcholesterol 54.45 ± 13.56 48.20 ± 12.48 50.23 ± 13.23 −6.24 ± 7.52 −8.12, −4.36 <0.001 −4.21 ± 8.12 −6.24, −2.18 <0.001 2.03 ± 6.13 0.49, 3.56 0.010 triglycerides 181.64 ± 94.12 145.47 ± 77.75 131.94 ± 75.28 −36.17 ± 89.88 −58.62, −13.71 0.002 −49.70 ± 67.40 −66.54, −32.86 <0.001 13.53 ± 67.79 −30.46, 3.40 0.115 cholesterol/ hdl 4.00 ± 0.93 3.69 ± 1.01 3.69 ± 1.06 −0.31 ± 0.61 −0.46, −0.15 <0.001 −0.31 ± 0.85 −0.52, −0.09 0.005 −0.003 ± 0.81 −0.21, 1.9 0.973 ci = confidence interval; hdl = high-density lipoprotein; ldl = low-density lipoprotein; sd = standard deviation. figure 2 change in mean total cholesterol, ldl-cholesterol, hdl-cholesterol, triglycerides, and cholesterol/ hdl from baseline through week 24. hdl = high-density lipoprotein; ldl = low-density lipoprotein. table iii mean change in body weight, body mass index, waist circumference, and ascvd risk score n = 64; mean (sd) week 0 week 12 week 24 change from week 0 vs. 12 p-value change from week 0 vs. 24 p-value change from week 12 vs. 24 p-value diff 95% ci diff 95% ci diff 95% ci bodyweight, kg 66.00 ± 12.02 66.97 ± 12.46 67.39 ± 12.58 0.97 ± 1.88 0.49, 1.44 <0.001 1.39 ± 2.49 0.77, 2.01 <0.001 0.42 ± 1.85 −0.04, 0.88 0.073 body mass index, kg/m2 23.99 ± 3.51 24.32 ± 3.55 24.49 ± 3.67 0.32 ± 0.67 0.16, 0.49 <0.001 0.49 ± 0.92 0.27, 0.73 <0.001 0.17 ± 0.68 −0.001, 0.34 0.051 waist circumference, cm 87.79 ± 10.82 89.33 ± 11.38 90.39 ± 10.95 1.53 ± 2.60 0.88, 2.18 <0.001 2.60 ± 4.27 1.53, 3.68 <0.001 1.06 ± 3.65 0.15, 1.97 0.023 ascvd risk score* 4.56 ± 4.30 (n = 42) 3.99 ± 3.71 (n = 39) 4.69 ± 4.67 (n = 44) 0.464 ± 1.43 −0.04, 0.97 0.07 0.14 ± 1.92 −0.49, 0.77 0.66 −0.11 ± 1.48 −0.59, 0.38 0.66 ascvd = atherosclerotic cardiovascular disease; ldl = low-density lipoprotein; sd = standard deviation. *calculated score from patient age above 40 years old and ldl level above 70 mg/dl. lipid profiles after switching from efavirenz to dolutegravir50 © 2023 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti figure 3 box plot of body weight, body mass index, and waist circumference change from baseline. the horizontal line in the box interior represents the group median. the large black dot represents the group mean. (3a) body weight, (3b) body mass index, (3c) waist circumference. 3b p<0.001 p<0.001 3a p<0.001 p<0.001 3c p<0.001 p<0.001 bo dy m as s in de x, k g/ m 2 bo dy w ei gh t, k g w ai st c irc um fe re nc e, c m baseline week 12 week 24 baseline week 12 week 24 baseline week 12 week 24 tdf, or ftc or continuing the nnrti-based regimen. at 48 weeks, the only significant reduction in plasma lipid levels was observed in hdl-cholesterol levels in patients who switched to the evg/c-based regimen compared to those who continued in the nnrti-based regimen. the changes in lipid levels varied based on the type of nnrti. switching from efv to the evg/c-based regimen led to a significant decrease in total cholesterol and ldl-cholesterol and a slight decrease in hdlcholesterol compared to those who continued efv. switching from nvp or rpv to evg/c led to substantial increases in ldlcholesterol and the cholesterol/hdl ratio compared to continuing with nvp or rpv (15). however, other recent studies have shown that switching to rpv or a once-daily integrase regimen can improve lipid profiles and reduce dyslipidemia without causing virological failure (14-16). virological failure is the primary issue to consider when changing treatments for patients who are already experiencing viral suppression. the cause of viral blips, which were observed in three patients within 24 weeks of transitioning to dtg in our study, is still unknown. regimes based on instis have been associated with a low frequency of viral blips and do not appear to be linked to virologic failure. however, the occurrence of these blips may increase the clinical workload (17). therefore, these three patients must undergo further follow-up. the potential for weight gain is another significant concern when transitioning to a dtg-based regimen (18-20). our study found a substantial increase in body weight, bmi, and waist circumference after the switch. while insti-based regimens are generally recommended as the first-line treatment for hiv (21), recent studies have shown that people receiving these regimens for initial therapy may experience greater weight gain compared to those on protease inhibitors (pis) or nnrti-based regimens. for example, a cohort from brazil found that individuals on ral-based regimens had a sevenfold higher likelihood of developing obesity than those on nnrtior pi-based regimens (22). additional observational studies have indicated that insti-based regimens, particular dtg-based regimens, may be linked to more significant weight gain (23-26). the namsal study, which involved 613 people with hiv in cameroon randomized to either tdf/3tc with dtg or efv, revealed that those on the dtgbased regimen gained more weight compared to those on efv at 48 weeks, and this weight gain was most prominent in women (27). furthermore, a recent analysis of eight phase iii clinical trials, including 5,680 art-naive participants, reported that 17.3% of them had a weight gain of ≥10% from baseline, and the weight gain was greater among those taking instis (3.24 kg) than nnrtis (1.93 kg) and pis (1.72 kg) (28). female gender and african origin were factors associated with weight gain (29). the studies conducted in the asian population reported that factors such as low initial cd4 counts and starting treatment with dtg/taf/ftc were associated with weight gain (30). these findings suggest that racial diversity may influence changes in body weight among people with hiv. dtg is generally well-tolerated and appear to have less long-term adverse effects than other regimens. some khemla et al drug target insights 2023; 17: 51 © 2023 the authors. published by aboutscience www.aboutscience.eu table iv mean change in other laboratory parameters laboratory parameters n = 64; mean (sd) week 0 week 12 week 24 change from week 0 vs. 12 p-value change from week 0 vs. 24 p-value change from week 12 vs. 24 p-value diff 95% ci diff 95% ci diff 95% ci fasting blood sugar, mg/dl 98.88 ± 12.41 94.23 ± 12.61 98.42 ± 21.35 −4.64 ± 12.32 −7.72, −1.56 0.004 −0.45 ± 21.01 −5.70, 4.79 0.864 4.18 ± 17.07 −0.07, 8.45 0.054 creatinine, mg/dl 0.95 ± 0.16 1.11 ± 0.19 1.11 ± 0.19 0.15 ± 0.11 0.12, 0.18 <0.001 0.15 ± 0.13 0.11, 0.18 0.001 −0.01 ± 0.13 −0.04, 0.02 0.660 egfr, ml/min/ 1.73 m2 85.46 ± 22.16 74.92 ± 20.36 75.96 ± 20.47 −10.54 ± 8.24 −12.59, −8.47 <0.001 −9.50 ± 9.86 −11.97, −7.04 <0.001 1.03 ± 8.41 −1.07, 3.13 0.330 hemoglobin, g/dl 13.84 ± 2.09 13.94 ± 1.97 14.13 ± 2.04 0.11 ± 0.88 −0.11, 0.32 0.340 0.29 ± 0.84 0.08, 0.51 0.007 0.19 ± 0.71 0.01, 0.37 0.038 albumin, g/dl 4.76 ± 0.41 4.68 ± 0.25 4.63 ± 0.28 −0.07 ± 0.35 −0.16, 0.01 0.079 −0.12 ± 0.35 −0.21, −0.03 0.006 −0.04 ± 0.21 −0.10, 0.01 0.090 globulin, g/dl 3.05 ± 0.36 2.91 ± 0.41 3.02 ± 0.39 −0.14 ± 0.30 −0.21, −0.06 <0.001 −0.02 ± 0.29 −0.10, 0.04 0.442 0.11 ± 0.34 0.03, 0.20 0.009 alanine aminotransferase, u/l 36.78 ± 23.78 34.38 ± 27.68 31.17 ± 16.46 −2.41 ± 23.99 −8.4, 3.58 0.425 −5.61 ± 23.52 −11.48, 0.26 0.061 −3.20 ± 21.72 −8.62, 2.22 0.243 aspartate aminotransferase, u/l 32.13 ± 19.65 29.08 ± 15.17 28.61 ± 10.29 −3.04 ± 17.52 −7.42, 1.33 0.169 −3.51 ± 18.94 −8.24, 1.21 0.143 −0.46 ± 12.18 −3.51, 2.57 0.759 alkaline phosphatase, u/l 105.86 ± 31.35 86.11 ± 25.72 85.92 ± 26.25 −19.75 ± 17.17 −24.04, −15.46 <0.001 −19.93 ± 17.94 −24.42, −15.45 <0.001 −0.18 ± 10.81 −2.88, 2.51 0.890 egfr = estimated glomerular filtration rate; sd = standard deviation. patients in this cohort had elevated creatinine values and a slight decrease in egfr after switching to dtg, and these were significant compared to baseline. dtg has been found to cause a predictable, early increase in serum creatinine of approximately 10% of baseline values in treatment-naive patients and 14% in treatment-experienced patients. this increase is caused by the inhibition of tubular creatinine secretion through the organic cation transporter 2 (oct2) receptor, but it does not result in a genuine decline in the egfr (31,32). this is the first prospective cohort study to examine the consequences of switching from efv to dtg in people with hiv with dyslipidemia in thailand. our data confirm that the use of dtg is safe and adverse effects are rare in this population. there were a few limitations to this study. firstly, it was a single-arm, monocentric study, and open-label study. additionally, the sample size was relatively small. furthermore, since the follow-up duration was brief, some effects may not have been detectable yet. finally, as patients were aware when their blood lipids were high, they may have engaged in lifestyle modification, such as diet and exercise, regardless of any adjustments to their medication regimen. conclusions the study showed that switching from efv-based therapy to dtg improved lipid profiles, suggesting that this switch could benefit patients with a high risk of cardiovascular disease. however, it is essential to note that weight gain and increased waist circumference were also observed. acknowledgments the authors gratefully acknowledge those involved in this research, including associated professor piroon mootsikapun and associate professor siriluck anunnatsiri. thanks also to dr. dylan southard for editing this manuscript via the kku publication clinic (thailand). most of all, we would like to thank the patients who accepted to participate in this study. trial registration: thai clinical trials registry, tctr20221 118002. disclosures conflict of interest: the authors declare no competing interests. financial support: the research affairs division of the khon kaen university faculty of medicine funded this study. lipid profiles after switching from efavirenz to dolutegravir52 © 2023 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti cholesterol in adults (adult treatment panel iii). third report of the national cholesterol education program (ncep) expert panel on detection, evaluation, and treatment of high blood cholesterol in adults (adult treatment panel iii) final report. circulation. 2002;106(25):3143-3421. crossref pubmed 14. taramasso l, tatarelli p, ricci e, et al; cisai study group. improvement of lipid profile after switching from efavirenz or ritonavir-boosted protease inhibitors to rilpivirine or oncedaily integrase inhibitors: results from a large observational cohort study (scolta). bmc infect dis. 2018;18(1):357-364. crossref pubmed 15. pozniak a, markowitz m, mills a, et al. switching to coformulated elvitegravir, cobicistat, emtricitabine, and tenofovir versus continuation of non-nucleoside reverse transcriptase inhibitor with emtricitabine and tenofovir in virologically suppressed adults with hiv (strategy-nnrti): 48 week results of a randomised, open-label, phase 3b non-inferiority trial. lancet infect dis. 2014;14(7):590-599. crossref pubmed 16. saumoy m, sanchez-quesada jl, ordoñez-llanos j, podzamczer d. do all integrase strand transfer inhibitors have the same lipid profile? review of randomised controlled trials in naïve and switch scenarios in hiv-infected patients. j clin med. 2021;10(16):3456. crossref pubmed 17. dijkstra s, hofstra lm, mudrikova t, et al. lower incidence of hiv-1 blips was observed during integrase inhibitor-based combination antiretroviral therapy. j acquir immune defic syndr. 2022;89(5):575-582. crossref pubmed 18. koethe jr, jenkins ca, lau b, et al; north american aids cohort collaboration on research and design (na-accord). rising obesity prevalence and weight gain among adults starting antiretroviral therapy in the united states and canada. aids res hum retroviruses. 2016;32(1):50-58. crossref pubmed 19. hasse b, iff m, ledergerber b, et al; swiss hiv cohort study. obesity trends and body mass index changes after starting antiretroviral treatment: the swiss hiv cohort study. open forum infect dis. 2014;1(2):ofu040. crossref pubmed 20. eckard ar, mccomsey ga. weight gain and integrase inhibitors. curr opin infect dis. 2020;33(1):10-19. crossref pubmed 21. panel on antiretroviral guidelines for adults and adolescents guidelines for the use of antiretroviral agents in hiv-1-infected adults and adolescents. department of health and human services, 2023. available at online. accessed march 2023. 22. bakal dr, coelho le, luz pm, et al. obesity following art initiation is common and influenced by both traditional and hiv-/art-specific risk factors. j antimicrob chemother. 2018;73(8):2177-2185. crossref pubmed 23. bourgi k, rebeiro pf, turner m, et al. greater weight gain in treatment-naive persons starting dolutegravir-based antiretroviral therapy. clin infect dis. 2020;70(7):1267-1274. crossref pubmed 24. menard a, meddeb l, tissot-dupont h, et al. dolutegravir and weight gain: an unexpected bothering side effect? aids. 2017;31(10):1499-1500. crossref pubmed 25. rizzardo s, lanzafame m, lattuada e, et al. dolutegravir monotherapy and body weight gain in antiretroviral naïve patients. aids. 2019;33(10):1673-1674. crossref pubmed 26. norwood j, turner m, bofill c, et al. brief report: weight gain in persons with hiv switched from efavirenz-based to integrase strand transfer inhibitor-based regimens. j acquir immune defic syndr. 2017;76(5):527-531. crossref pubmed 27. kouanfack c, mpoudi-etame m, omgba bassega p, et al; namsal anrs 12313 study group. dolutegravir-based or lowdose efavirenz-based regimen for the treatment of hiv-1. n engl j med. 2019;381(9):816-826. crossref pubmed authors’ contributions: all authors participated in the study, contributed to manuscript preparation, and reviewed the manuscript. pc contributed to the conception and design. sk, am, and ws designed the study, analyzed data, and drafted the manuscript. references 1. so-ngern a, khan-asa b, montakantikul p, manosuthi w. dyslipidemia among thai hiv-infected adults receiving antiretroviral therapy: a hospital-based report. southeast asian j trop med public health. 2018;49(1):60-67. available at online. accessed march 2023. 2. friis-møller n, weber r, reiss p, et al; dad study group. cardiovascular disease risk factors in hiv patients – association with antiretroviral therapy. results from the dad study. aids. 2003;17(8):1179-1193. crossref pubmed 3. maggi p, bellacosa c, carito v, et al. cardiovascular risk factors in patients on long-term treatment with nevirapineor efavirenzbased regimens. j antimicrob chemother. 2011;66(4):896-900. crossref pubmed 4. rockstroh jk, lennox jl, dejesus e, et al; startmrk investigators. long-term treatment with raltegravir or efavirenz combined with tenofovir/emtricitabine for treatment-naive human immunodeficiency virus-1-infected patients: 156-week results from startmrk. clin infect dis. 2011;53(8):807-816. crossref pubmed 5. thamrongwonglert p, chetchotisakd p, anunnatsiri s, mootsikapun p. improvement of lipid profiles when switching from efavirenz to rilpivirine in hiv-infected patients with dyslipidemia. hiv clin trials. 2016;17(1):12-16. crossref pubmed 6. ruxrungtham k, puthanakit t, putacharoen o, et al. thailand national guidelines on hiv/aids treatment and prevention 2017. nonthaburi: division of aids and stis, department of disease control; 2017. available at online. accessed march 2023. 7. world health organization. update of recommendations on firstand second-line antiretroviral regimens. policy brief: world health organization; 2019. available at online. accessed march 2023. 8. taramasso l, ricci e, menzaghi b, et al; cisai study group. a cisai study group. weight gain: a possible side effect of all antiretrovirals. open forum infect dis. 2017;4(4):ofx239. crossref pubmed 9. ruxrungtham k, chokephaibulkit k, chetchotisakd p, et al. thailand national guidelines on hiv/aids treatment and prevention 2021/2022. nonthaburi: division of aids and stis, department of disease control; 2022. available at online. accessed march 2023. 10. quercia r, roberts j, martin-carpenter l, zala c. comparative changes of lipid levels in treatment-naive, hiv-1-infected adults treated with dolutegravir vs. efavirenz, raltegravir, and ritonavir-boosted darunavir-based regimens over 48 weeks. clin drug investig. 2015;35(3):211-219. crossref pubmed 11. walmsley sl, antela a, clumeck n, et al; single investigators. dolutegravir plus abacavir-lamivudine for the treatment of hiv-1 infection. n engl j med. 2013;369(19):1807-1818. crossref pubmed 12. van lunzen j, maggiolo f, arribas jr, et al. once daily dolutegravir (s/gsk1349572) in combination therapy in antiretroviral-naive adults with hiv: planned interim 48 week results from spring-1, a dose-ranging, randomised, phase 2b trial. lancet infect dis. 2012;12(2):111-118. crossref pubmed 13. national cholesterol education program (ncep) expert panel on detection, evaluation, and treatment of high blood https://doi.org/10.1161/circ.106.25.3143 https://www.ncbi.nlm.nih.gov/pubmed/12485966 https://doi.org/10.1186/s12879-018-3268-5 https://www.ncbi.nlm.nih.gov/pubmed/30064371 https://doi.org/10.1016/s1473-3099(14)70796-0 https://www.ncbi.nlm.nih.gov/pubmed/24908550 https://doi.org/10.3390/jcm10163456 https://www.ncbi.nlm.nih.gov/pubmed/34441755 https://doi.org/10.1097/qai.0000000000002898 https://www.ncbi.nlm.nih.gov/pubmed/34966148 https://doi.org/10.1089/aid.2015.0147 https://www.ncbi.nlm.nih.gov/pubmed/26352511 https://doi.org/10.1093/ofid/ofu040 https://www.ncbi.nlm.nih.gov/pubmed/25734114 https://doi.org/10.1097/qco.0000000000000616 https://www.ncbi.nlm.nih.gov/pubmed/31789693 https://clinicalinfo.hiv.gov/en/guidelines/hiv-clinical-guidelines-adult-and-adolescent-arv/whats-new https://doi.org/10.1093/jac/dky145 https://www.ncbi.nlm.nih.gov/pubmed/29722811 https://doi.org/10.1093/cid/ciz407 https://www.ncbi.nlm.nih.gov/pubmed/31100116 https://doi.org/10.1097/qad.0000000000001495 https://www.ncbi.nlm.nih.gov/pubmed/28574967 https://doi.org/10.1097/qad.0000000000002245 https://www.ncbi.nlm.nih.gov/pubmed/31305333 https://doi.org/10.1097/qai.0000000000001525 https://www.ncbi.nlm.nih.gov/pubmed/28825943 https://doi.org/10.1056/nejmoa1904340 https://www.ncbi.nlm.nih.gov/pubmed/31339676 https://www.tm.mahidol.ac.th/seameo/2018-49-1/06-70562-60.pdf https://doi.org/10.1097/00002030-200305230-00010 https://www.ncbi.nlm.nih.gov/pubmed/12819520 https://doi.org/10.1093/jac/dkq507 https://www.ncbi.nlm.nih.gov/pubmed/21393134 https://doi.org/10.1093/cid/cir510 https://www.ncbi.nlm.nih.gov/pubmed/21921224 https://doi.org/10.1080/15284336.2015.1112480 https://www.ncbi.nlm.nih.gov/pubmed/26739573 https://www.thaiaidssociety.org/wp-content/uploads/2022/02/thailand-national-guidelines-on-hiv-aids-treatment-and-prevention-2017.pdf https://apps.who.int/iris/bitstream/handle/10665/325892/who-cds-hiv-19.15-eng.pdf https://doi.org/10.1093/ofid/ofx239 https://www.ncbi.nlm.nih.gov/pubmed/29255735 https://www.thaiaidssociety.org/wp-content/uploads/2023/03/hiv-aids-guideline-2564_2565_ed2.pdf https://doi.org/10.1007/s40261-014-0266-2 https://www.ncbi.nlm.nih.gov/pubmed/25637061 https://doi.org/10.1056/nejmoa1215541 https://www.ncbi.nlm.nih.gov/pubmed/24195548 https://doi.org/10.1016/s1473-3099(11)70290-0 https://www.ncbi.nlm.nih.gov/pubmed/22018760 khemla et al drug target insights 2023; 17: 53 © 2023 the authors. published by aboutscience www.aboutscience.eu 28. sax pe, erlandson km, lake je, et al. weight gain following initiation of antiretroviral therapy: risk factors in randomized comparative clinical trials. clin infect dis. 2020;71(6):13791389. crossref pubmed 29. kanters s, renaud f, rangaraj a, et al. evidence synthesis evaluating body weight gain among people treating hiv with antiretroviral therapy – a systematic literature review and network meta-analysis. eclinicalmedicine. 2022;48:101412. crossref pubmed 30. ando n, nishijima t, mizushima d, et al. long-term weight gain after initiating combination antiretroviral therapy in treatment-naïve asian people living with human immunodeficiency virus. int j infect dis. 2021;110:21-28. crossref pubmed 31. milburn j, jones r, levy jb. renal effects of novel antiretroviral drugs. nephrol dial transplant. 2017;32(3):434-439. pubmed 32. osterholzer da, goldman m. dolutegravir: a next-generation integrase inhibitor for treatment of hiv infection. clin infect dis. 2014;59(2):265-271. crossref pubmed https://doi.org/10.1093/cid/ciz999 https://www.ncbi.nlm.nih.gov/pubmed/31606734 https://doi.org/10.1016/j.eclinm.2022.101412 https://www.ncbi.nlm.nih.gov/pubmed/35706487 https://doi.org/10.1016/j.ijid.2021.07.030 https://www.ncbi.nlm.nih.gov/pubmed/34273516 https://www.ncbi.nlm.nih.gov/pubmed/27190354 https://doi.org/10.1093/cid/ciu221 https://www.ncbi.nlm.nih.gov/pubmed/24723281 dti drug target insights 2021; 15: 26-33issn 1177-3928 | doi: 10.33393/dti.2021.2347original research article drug target insights issn 1177-3928 www.aboutscience.eu/dti © 2021 the authors. this article is published by aboutscience and licensed under creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). commercial use is not permitted and is subject to publisher’s permissions. full information is available at www.aboutscience.eu hexarelin modulates lung mechanics, inflammation, and fibrosis in acute lung injury vanessa zambelli1, laura rizzi1, paolo delvecchio1, elena bresciani1, emanuele rezoagli1, laura molteni1, ramona meanti1, maria serena cuttin2, giorgio bovo2, silvia coco1, robert j omeljaniuk3, vittorio locatelli1, giacomo bellani1, antonio torsello1 1department of medicine and surgery, university of milano-bicocca, monza italy 2asst vimercate, vimercate italy 3department of biology, lakehead university, thunder bay, ontario canada vanessa zambelli and laura rizzi contributed equally to this research. abstract introduction: acute respiratory distress syndrome (ards) is an acute form of diffuse lung injury characterized by (i) an intense inflammatory response, (ii) increased pulmonary vascular permeability, and (iii) the loss of respiratory pulmonary tissue. in this article we explore the therapeutic potential of hexarelin, a synthetic hexapeptide growth hormone secretagogue (ghs), in an experimental model of ards. hexarelin has anti-inflammatory properties and demonstrates cardiovascular-protective activities including the inhibition of cardiomyocyte apoptosis and cardiac fibrosis, both of which may involve the angiotensin-converting enzyme (ace) system. methods: in our experimental model, ards was induced by the instillation of 100 mm hcl into the right bronchus; these mice were treated with hexarelin (320 μg/kg, ip) before (pre) or after (post) hcl challenge, or with vehicle. respiratory system compliance, blood gas analysis, and differential cell counts in a selective bronchoalveolar lavage (bal) were determined 6 or 24 hours after hcl instillation. in an extended study, mice were observed for a subsequent 14 days in order to assess lung fibrosis. results: hexarelin induced a significant improvement in lung compliance and a reduction of the number of total immune cells in bal 24 hours after hcl instillation, accompanied with a lower recruitment of neutrophils compared with the vehicle group. at day 14, hexarelin-treated mice presented with less pulmonary collagen deposition compared with vehicle-treated controls. conclusions: our data suggest that hexarelin can inhibit the early phase of the inflammatory response in a murine model of hcl-induced ards, thereby blunting lung remodeling processes and fibrotic development. keywords: ards, ghs (growth hormone secretagogues), hexarelin, inflammation, lung fibrosis received: september 24, 2021 accepted: october 20, 2021 published online: november 27, 2021 corresponding author: laura rizzi school of medicine and surgery university of milano-bicocca via cadore 48 20900 monza (mb) italy laura.rizzi@unimib.it most common morphological symptom of the acute phase of ards, and is characterized by the influx of neutrophils and macrophages into alveoli, and alteration of the alveolar epithelium (5). these alterations often progress to fibrotic development accompanied by a further decrease in pulmonary compliance (6). there is no documented and approved pharmacologic treatment for ards. hexarelin is a synthetic hexapeptide that has already shown positive effects in experimental models of human pathologies such as epilepsy and cachexia (7-11). in particular, hexarelin reduced cardiac fibrosis in experimental models of myocardial infarction (12). furthermore, we previously demonstrated that the protective effects of hexarelin on the cardiovascular system could be mediated by its interaction with the angiotensin-converting enzyme (ace) system (13). modulation of ace activity associated with the reduction of angiotensin ii synthesis may also be involved with reduced fibrosis development in the lung (14). in this study we have applied our validated experimental model of unilateral acid aspiration lung injury (16) to test specific growth hormone secretagogues (ghs) as potential introduction acute respiratory distress syndrome (ards) is often underrecognized and undertreated, thereby contributing to a high mortality rate (1-3). ards constitutes an acute lung injury associated with (i) an intense inflammatory response, (ii) increased pulmonary vascular permeability, and (iii) the loss of aerated lung tissue (4). diffuse alveolar damage is the https://doi.org/10.33393/dti.2021.2347 https://creativecommons.org/licenses/by-nc/4.0/legalcode mailto:laura.rizzi@unimib.it zambelli et al drug target insights 2021; 15: 27 © 2021 the authors. published by aboutscience www.aboutscience.eu adjunctive therapeutic tools for ards. among ghs, ghrelin has demonstrated anti-inflammatory properties including inhibition of cardiac fibrosis (15). consequently, this study examined the potential utility of hexarelin in the treatment of ards in our murine model. the aim of this research was to ascertain whether hexarelin could have a potential therapeutic use to antagonize the inflammatory response and the lung fibrosis induced by unilateral acid aspiration in mice. the results of the present research demonstrate that hexarelin treatment reduces the development of lung fibrosis and further suggests that specific synthetic ghs could be developed in order to modulate lung and cardiac fibrosis such as those associated with covid-19 infections. materials and methods animals male c57/bl6j mice (23-25 g; harlan laboratories, udine, italy) were used in this experimental study. animals were housed five per cage in a limited access animal facility, with the room temperature at 20 ± 2°c and the relative humidity set at 55 ± 10%. artificial lighting provided a 12 h light/12 h dark (7 am to 7 pm) cycle. the general condition of the animals before the experiment was assessed daily. the care and husbandry of animals were in conformity with the institutional guidelines in compliance with italian and european laws and policies. the animal study was reviewed and approved by the italian ministry of health (591/2017-pr) and by the animal care unit of the university of milano-bicocca, monza, italy. in full respect of the reduction principle of the 3rs, the number of animals/groups selected was to obtain reliable results and enough biological samples to perform the analysis planned. chemicals hexarelin (his/d-2-methyl-trp/ala/trp/d-phe/lys-nh 2 ) (sigma-aldrich) was given at a dose of 320 μg/kg body weight, according to the assigned treatment group (see below). experimental protocol mice were anesthetized with ketamine (80 mg/kg, ip) (ketavet 100; intervet productions) and xylazine (4 mg/kg, ip) (rompun 2%; bayer) and orotracheally intubated. then, lung injury was induced in the right lung as described in amigoni et al (16). the experimental protocol was divided into two time points including (i) the acute ards study, in which animals were sacrificed after 6 or 24 hours following hcl instillation, and (ii) the late ards study, with sacrifice performed 14 days after hcl instillation. in the acute ards study, mice were assorted into three treatment groups: – vehicle: mice received sterile physiological saline treatment (100 µl, ip), immediately after hcl challenge; – post-hex: mice received hexarelin (320 μg/kg, ip, 100 µl), immediately after hcl challenge; – pre-hex: mice received hexarelin (320 μg/kg ip, 100 µl) 2 and 1 day before and immediately after hcl challenge. in the late ards study, mice were assorted into two treatment groups: – vehicle: mice received sterile physiological saline (100 µl, ip), immediately after hcl challenge and twice daily in the following 4 days; – hexarelin: mice received hexarelin treatment immediately after hcl challenge (entire dose, 320 μg/kg ip, 100 µl) and twice daily in the following 4 days (two half doses, 160 μg/kg ip, 100 µl). a group of healthy mice (n = 5) did not undergo any of the surgical interventions and was sacrificed (healthy mice). when, at the moment of the sacrifice, we identified that hcl instillation involved the contralateral lung (the left one) through a macroscopic lung evaluation, the animals were euthanized and excluded from analysis (approximately 2%). for some parameters (alveolar inflammatory cells count, alveolar protein content, and collagen deposition), the two lungs were analyzed separately, since the injury was induced only on the right lung. the contralateral (left) lung has been considered like an internal control. pulmonary function at the time of sacrifice, mice were anesthetized with ketamine (100 mg/kg) and xylazine (4 mg/kg), and mechanically ventilated. in order to standardize lung recruitment, a maneuver (30 cm h 2 o for 10 sec) was performed immediately after intubation. a pressure-volume (pv) curve was constructed by delivering five steps of inspiratory volume (200 µl) from functional residual capacity. for each step, the plateau pressure was recorded in order to calculate the static compliance by using a pressure transducer, which was interfaced to a powerlab (ad instruments) signal transduction unit. a mean value was calculated. after mechanical properties were measured, the chest was opened and a blood sample (0.1 ml) was withdrawn from the left ventricle and analyzed with an i-stat 1 portable analyzer to analyze oxygenation value (pao 2 ) (oxford instruments s.m., burke e burke) (16). inflammatory response an aliquot of each blood sample was used to perform peripheral leukocyte (wbcs) counts. subsequently, bronchoalveolar lavage (bal) was performed separately for each lung, by clamping alternatively the left and right bronchus. lavage was performed three times for each lung, with 600 or 400 µl of lavage solution (0.9% saline solution and protease inhibitor) respectively for the right and the left one. the bal samples obtained were then centrifuged for 10 minutes, 1500 rpm, 4°c; the supernatant was then stored at –80°c for subsequent analyses. the cell pellet was resuspended in 500 µl pbs (dulbecco’s phosphate-buffered saline; gibco). subsequently, a 100 µl aliquot was put in 200 µl of turk (acetic acid gentian violet solution; merck) for total leukocyte effects of hexarelin treatment on acute lung injury28 © 2021 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti count in a burker chamber, while another 100 µl aliquot was centrifuged by a cytospin (centrifuge, mpw-351r, mpw) and then stained with a diff-quick kit (medion diagnostics) that differentially marks the nucleus and cytoplasm, thus facilitating a differential cell count (17). protein contents in bal fluid were performed by the bca (bicinchoninic acid) method at 24 hours and 14 days. briefly, 200 µl of reagent (composed of 1:50 bca and cuso 4 ; merck) was added to the samples. a standard curve was constructed with varied concentrations of bovine serum albumin; spectrophotometric measurement was performed at 570 nm with a multilabel spectrophotometer victor3 (perkin elmer) (18). hydroxyproline (oh-pro) assay after exsanguination, a macroscopic observation of the lungs allowed identifying the localization of the acid injury; lungs were then excised and stored at −80°c. collagen content was measured with the oh-pro assay. we used the conventional method (19), which entails lung tissue homogenization and hydrolysis with 6n hcl at 120°c, followed by chloramine t and ehrlich’s solution (merck) addition to samples for the oh-proline oxidation and a colorimetric reaction. finally, absorbance was measured at 550 nm with a multilabel spectrophotometer victor3. histological analysis at sacrifice, some mice were devoted to histological analysis. briefly, lung tissue was fixed in 4% paraformaldehyde and embedded in paraffin. sections were stained with hematoxylin-eosin (h&e) (automatic stainer dako coverstainer) and masson’s trichrome (automatic stainer dako artisan link pro special staining system). morphological changes were analyzed by two experienced pathologists in a blinded fashion (light microscope leica dm 2500 and digital microimaging device leica dmd108). statistical analysis data are expressed as mean ± standard error of the mean (sem). differences in variances between treatment groups were assessed by one-way analysis of variance (anova) and tukey post hoc test. p values of less than 0.05 (two-tailed) were considered as statistically significant. in the acute ards study, anova was performed in experimental groups sacrificed at 6 hours and at 24 hours separately. statistical analysis was performed by graphpad prism (version 8.4.2). results acute ards study pulmonary function acid instillation, as expected, reduced respiratory compliance (fig. 1a); specifically, at 6 and 24 hours post instillation respiratory compliance was significantly reduced by 30% (p<0.01) and 21% (p<0.05), respectively, compared with healthy mice. mice presented no apparent adverse responses to hexarelin pretreatment or treatment regimes; moreover, hexarelin administration, in both treatment regimes, modulated acid-reduced respiratory compliance. to illustrate, at 6 hours post-instillation the beneficial effects of hexarelin were slight but not significant; by contrast, hexarelin induced a significant (p<0.05) improvement in respiratory compliance 24 hours after lung injury. by comparison, arterial oxygen partial pressure (pao 2 ) was severely and significantly decreased in all acid-treated animals (p<0.01) compared with healthy mice (fig. 1b). inflammatory response total numbers of peripheral white blood cells, an assumed index of systemic inflammation, increased significantly 6 hours after acid instillation (fig. 2a) in both vehicle (p<0.01) and hexarelin-treated (p<0.05) groups compared fig. 1 respiratory system static compliance (panel a) and oxygenation (panel b). respiratory system static compliance derived from the pressure-volume curve construction and oxygenation (pao 2 ) was measured by arterial blood from the left ventricle. a) analysis of variance (anova) in sacrifice 6 h experiment p<0.05, tukey post hoc test; *p<0.05 vs. healthy; anova in sacrifice 24 h experiment p<0.01, tukey post hoc test; *p<0.05 vs. healthy, °p<0.05 vs. post-hex, # p<0.05 vs. pre-hex. b) anova in sacrifice 6 h experiment p<0.01, tukey post hoc test; *p<0.01 vs. vehicle, post-hex and pre-hex; anova in sacrifice 24 h experiment p<0.01, tukey post hoc test; *p<0.01 vs. vehicle, posthex, and pre-hex. healthy: no surgical interventions or treatment (n = 5); vehicle: hcl instillation + vehicle treatment (n = 8); post-hex: hcl instillation + hexarelin treatment (n = 8); pre-hex: hexarelin pretreatment + hcl instillation (n = 8). zambelli et al drug target insights 2021; 15: 29 © 2021 the authors. published by aboutscience www.aboutscience.eu with healthy mice. at 24 hours post-instillation, total cell numbers decreased in all groups; in particular, cell numbers returned to normal (healthy) levels in hexarelin-treated mice. differential cell counts of bal fluid were strongly influenced both by acid instillation and by hexarelin (fig. 2b, c). in the bal of healthy mice, polymorphonuclear (pmn) cells represented 4% of total white blood cells, the balance of which (96%) were macrophages. at 6 hours post-acid instillation, there was an important increase in bal pmn; notably, pmn in the bal of the pre-hexarelin-treated group was also elevated and significantly (p = 0.010) different from those in healthy mice. acid instillation increased the differential cell count, which achieved maximum levels at 24 hours post-instillation (fig. 2b, c). at this time, hexarelin treatment and especially pretreatment modulated pmn numbers to a lesser and greater extent. curiously, macrophage numbers in hexarelin-treated mice were significantly greater compared with other groups in both the right (acid-instilled) as well as the left (acid-naive) lungs. indices of local inflammation included the total protein contents and differential cell counts of bal. acid instillation induced a very large protein extravasation into alveoli (tab. i). hexarelin treatment and pretreatment modulated acidinduced protein leakage; in fact, the bal protein contents in both hexarelin-treated groups (6 hours post-instillation) were comparable with those of healthy mice and significantly smaller (p<0.01) than those in the acid-instilled-alone group. table i local inflammation: total protein bal content in right and left lung by bca method groups right lung left lung healthy 268 ± 24 423 ± 54 sacrifice 6 h vehicle 2994 ± 496*,° 1148 ± 193 post-hex 1585 ± 316 1380 ± 262 pre-hex 1647 ±315 1013 ± 294 sacrifice 24 h vehicle 2891 ± 289** 773 ± 103 post-hex 2145 ± 266°° 616 ± 70 pre-hex 2364 ± 296# 521 ± 90 in the right lung: anova in sacrifice 6 h experiment p<0.01, tukey post hoc test; *p<0.01 vs. healthy, °p<0.05 vs. post-hex; anova in sacrifice 24 h experiment p<0.01, tukey post hoc test; **p<0.01 vs. healthy, °°p<0.01 vs. healthy, #p<0.01 vs. healthy. in the left lung: anova p = ns. healthy: no surgical interventions or treatment (n = 5); vehicle: hcl instillation + vehicle treatment (n = 8); post-hex: hcl instillation + hexarelin treatment (n = 8); pre-hex: hexarelin pretreatment + hcl instillation (n = 8). anova = analysis of variance; bal = bronchoalveolar lavage; bca = bicinchoninic acid. late ards study body weight was monitored throughout the experiment, since it is known that hexarelin stimulates food intake. as expected, mice belonging to acid-instilled groups showed a significant (p<0.01) loss of body weight 48 hours after hcl administration, compared with healthy mice (fig. 3a). fig. 2 peripheral and local inflammation: total white blood cells (a) and cell count in bronchoalveolar lavage (bal) in right (panel b) and left lung (panel c). white blood cells were collected from the arterial blood and stained with turk solution. alveolar cells were collected by performing bal and stained with diff-quik reagent solution. a) analysis of variance (anova) in sacrifice 6 h experiment p<0.01, tukey post hoc test; *p<0.01 vs. healthy, °p<0.05 vs. healthy; anova in sacrifice 24 h experiment p<0.05, tukey post hoc test; #p<0.05 vs. pre-hex. b) polymorphonuclear (pmn): anova in sacrifice 6 h experiment p = ns; anova in sacrifice 24 h experiment p<0.01, tukey post hoc test; *p = 0.01 vs. healthy, °p = 0.01 vs. pre-hex. macrophages: anova in sacrifice 6 h experiment p = 0.02, tukey post hoc test; *p = 0.01 vs. healthy; anova in sacrifice 24 h experiment p<0.01, tukey post hoc test; **p<0.01 vs. healthy, °°p<0.05 vs. vehicle, #p<0.01 vs. pre-hex. c) pmn: anova in sacrifice 6 h experiment p = ns; anova in sacrifice 24 h experiment p = 0.01, tukey post hoc test; *p<0.05 vs. healthy, °p<0.01 vs. pre-hex. macrophages: anova in sacrifice 6 h experiment p = ns; anova in sacrifice 24 h experiment p<0.01, tukey post hoc test; *p = 0.01 vs. healthy, °p<0.05 vs. pre-hex. healthy: no surgical interventions or treatment (n = 5); vehicle: hcl instillation + vehicle treatment (n = 8); post-hex: hcl instillation + hexarelin treatment (n = 8); pre-hex: hexarelin pretreatment + hcl instillation (n = 8). effects of hexarelin treatment on acute lung injury30 © 2021 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti fourteen days post-instillation body weights of mice in the vehicle group were significantly (p<0.05) smaller than those of healthy mice; hexarelin treatment preserved body weight better than vehicle treatment alone (fig. 3b). pulmonary function by 14 days post-instillation, respiratory system static compliance did not differ significantly between groups. acidtreated mice showed no different (anova, p = ns) mechanical properties (51.6 ± 3.0 and 51.0 ± 3.4 µl/mm hg in vehicle and hexarelin groups, respectively) compared with healthy mice (58.0 ± 4.5 µl/mm hg). collagen deposition in order to estimate fibrosis development at 14 days postinstillation, the collagen content of each lung was measured indirectly with the oh-pro assay. the collagen contents of both right and left lungs of the hexarelin-treated group were smaller compared with those in the vehicle-treated group; however, only the right lungs showed a statistically significant difference (p<0.05) between the hexarelinand vehicletreated groups (fig. 4a). fig. 3 body weight increment in 48 hours (panel a) and in 14 days (panel b). a) analysis of variance (anova) p<0.0001, tukey post hoc test; *p<0.01 vs. vehicle and hexarelin. b) anova p = 0.048, tukey post hoc test; *p<0.05 vs. healthy. healthy: no surgical interventions or treatment (n = 15); vehicle: hcl instillation + vehicle treatment (n = 18); hexarelin: hcl instillation + hexarelin treatment (n = 18). fig. 4 lung fibrosis: oh-proline lung content (a) and effects of hexarelin on lung histology (b) in acid-injured mice 14 days after lung injury induction. the collagen deposition was evaluated by the assessment of oh-proline content in lung-homogenized tissue with chloramine t and ehrlich’s solutions. in right lung: analysis of variance (anova) p<0.05, tukey post hoc; *p<0.05 vs. hexarelin, °p<0.05 vs. healthy. in left lung: anova p = ns. healthy: no surgical interventions or treatment (n = 10); vehicle: hcl instillation + vehicle treatment (n = 13); hexarelin: hcl instillation + hexarelin treatment (n = 13). histology: representative images are shown.tissues were prepared for masson’s trichrome staining. (b, d, f: 40× magnification; c, e, g: 400× magnification.) (b, c) healthy: no surgical interventions or treatment (n = 4); (d, e) vehicle: hcl instillation + vehicle treatment (n = 3); and (f, g) hexarelin: hcl instillation + hexarelin treatment (n = 5). areas of fibrosis (stained in green) are indicated with arrows; boxes on the 40× images indicate the locations of the 400× images. zambelli et al drug target insights 2021; 15: 31 © 2021 the authors. published by aboutscience www.aboutscience.eu histological examination in healthy mice, lung parenchyma was substantially preserved with normal lobular structure, slight bronchial ectasia, normal alveoli, and alveolar ducts (fig. 4b, c). in the vehicle-treated group, lung tissue was characterized by diffuse areas of fibrosis, especially subpleural with centrilobular extension. this fibrosis was of a “young” type, with several fibroblastic cells in deposits of collagen and with large subpleural nodules; as well, several small “fibroblastic foci” were found in adjacent parenchyma. there was also evidence of architectural distortion with marked bronchiolar dilatation forming cystic spaces resembling honeycombing-like features (fig. 4d). in a vehicle-treated (acid-instilled) lung there was evidence of a slight alveolar distortion, accompanied by many alveolar macrophages and some pmn cells (fig. 4e). in hexarelin-treated mice, by contrast, fibrosis was reduced compared to that of the vehicle-treated group; fibrotic distribution was patchy with subpleural localization prevalent. this fibrotic tissue was similar to that previously described; that is, appearing young and cellulated and accompanied by evidence of abrupt transitions from remodeled lung parenchyma to normal alveolar walls. at the center of a lobule, the presence of a fibroblastic focus is obvious (fig. 4f). almost normal alveolar walls and only some inflammatory cells were evident in lungs from hexarelin-treated mice (fig. 4g). discussion in this study, we demonstrate protective effects of hexarelin against ards in a unilateral acid aspiration lung injury model in mice. our results show that hexarelin treatment significantly (i) ameliorated respiratory system compliance, (ii) reduced protein levels in the bal, and (iii) blunted pmn infiltration compared with the vehicle-treated group; these effects eventually attenuated lung fibrosis and collagen content. to the best of our knowledge, this is the first study to evaluate the effects of hexarelin on ards in a mouse model of unilateral lung acid instillation. hexarelin, a synthetic hexapeptide, is a powerful agonist of the ghrelin receptor and, consequently, manifests both endocrine and extra-endocrine activities. these activities include positive effects on gastrointestinal-, cardiovascular-, muscular-, and nervous systems, as well as participation in regulation of energy balance (7-11). in humans, hexarelin actions are not restricted to stimulating gh release; to illustrate, acute hexarelin administration markedly increased left ventricular function in (i) normal subjects, (ii) in patients with ischemic cardiomyopathy, as well as (iii) in patients with severe gh deficiency (20). hexarelin significantly reduced indices of cardiac fibrosis in experimental models of myocardial infarction, likely through an underlying anti-inflammatory mechanism (12). the effects of subacute hexarelin treatments in rats (8) strongly suggested that hexarelin could interfere with the renin-angiotensin-aldosterone system (raas). we provided evidence in support of that hypothesis by showing that hexarelin and other synthetic ghs inhibited the activity of ace (13). the somatic form of ace is a type i membrane-anchored dipeptidyl carboxypeptidase consisting of two extracellular catalytic domains: the nand c-domains specifically catalyze conversion of angiotensin i and bradykinin, respectively (21,22). hexarelin and synthetic ghs selectively bind to and inhibit the activity of the c-domain, without being metabolized themselves (13). hexarelin inhibition of ace may account, in part, for its cardioprotective effects. to illustrate, binding sites for ghs are expressed in the heart; moreover, mrna for the specific ghs receptor (ghs-r1a) has been detected by reverse transcriptase polymerase chain reaction (rt-pcr) in the rat aorta as well as the left ventricle and left atrium (23,24). nonetheless, it is presumed that the levels of ghs-r1a expressed in these tissues are too small to be responsible for ghs cardioprotective effects. ghs cardioprotective effects are now being considered to be more related to inhibition of angiotensin ii (aii) synthesis and/or the antagonism of its receptor (at1). ace synthesizes aii, which acts through the at1 receptor to increase blood pressure and to promote fibrosis and inflammation. the vulnerability of aii to enzymatic conversion by ace2 leads to reduction of aii levels and production of angiotensin (1-7) which selectively binds to the mas receptor. the beneficial effects of ace2 activity may be both systemic as well as localized in tissues, such as the heart, kidneys, and lungs, where it antagonizes pathological changes (25). our results that demonstrate hexarelin inhibition of lung fibrosis in acid-instilled mice are in agreement with our previous results demonstrating the cardioprotective and antiinflammatory effects of hexarelin (8,26). collectively, these findings strongly suggest that hexarelin may inhibit the development of pathological fibrosis in both these organs. the lack of beneficial effect of hexarelin on oxygenation could depend on the short interval after treatment: it is possible that 24 hours is not enough to affect oxygen exchange. an impaired perfusion could also prevent an increase in oxygenation. the precise pathogenesis of fibrotic pulmonary disorder is still unclear; it could result from an excessive host inflammatory response of the lung to an infectious or noninfectious insult. a common feature is collagen accumulation that leads to destruction of alveolar structures and promotes remodeling. this purported mechanism has been proposed to play a primary role in ards (27). in the lung and other tissues, inflammation correlates with the presence of macrophages and other immune cells that can be involved in the inflammatory process and thereafter its resolution and recovery from ards (28). it has been reported that ards occurs in some severe acute respiratory syndrome (sars) patients despite a diminishing viral load, suggesting that the host immune response rather than viral infection itself could be responsible for lung damage (29). we have observed that hexarelin reduced the number of immune cells in the bal, in particular pmn, suggesting that it could partially inhibit the recruitment of pmn into the alveolar space. this is consistent with the anti-inflammatory and antifibrotic effects reported for hexarelin in the heart (12). in this study, we observed that hexarelin significantly reduced oh-proline levels, a measure of collagen deposition, in the lung treated with acid instillation. this result is effects of hexarelin treatment on acute lung injury32 © 2021 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti consistent with the ability of hexarelin to reduce the fibrotic areas in lung histology. in our histological observations, fibrosis features were similar in vehicleand hexarelin-treated groups, either for cellular type or anatomical location (subpleural origin with following centrilobular involvement, like usual interstitial pneumonia pattern) (30). the smaller amounts of fibrosis in hexarelin-treated mice could be related to a lower collagen deposition, consistent with the reduced presence of inflammatory cells. this study has some limitations that should be acknowledged. first, we did not confirm the findings in a different model of ards. second, the possible mechanisms of action of hexarelin are only speculative. third, the levels of inflammatory cytokines were not measured, which could support the results on inflammation obtained for local and peripheral inflammatory cell count. fourth, as index of lung, edema, septal thickening, and hemorrhage were not assessed in this study. in conclusion, our results suggest that hexarelin can ameliorate the static compliance of acid-injured lungs in mice, an experimental model of ards. our results also show that hexarelin can reduce lung fibrosis, a complication often reported in patients with classical and covid-19 ards. collectively, these findings suggest that hexarelin and other synthetic ghs may be successfully co-opted for use in antagonizing lung and cardiac fibrosis. acknowledgments conflict of interest: the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. funding/support: we acknowledge that this research was partially supported by the italian ministry of university and research (miur), department of excellence project premia (precision medicine approach: bringing biomarker research to clinic). dr. emanuele rezoagli was supported by the international young investigator award 2018 from the european society of intensive care medicine (esicm) with the project titled: “role of the exhaled breath condensate as non-invasive monitoring of the lung inflammation during ards: a prospective cohort study” and by the national merck sharp & dohme corporation research award 2017 from the società italiana di anestesia analgesia rianimazione e terapia intensiva (siaarti) with the project titled: “studio della concentrazione di ossido nitrico nell’esalato espiratorio come marcatore di danno polmonare acuto in pazienti adulti con ards sottoposti a ventilazione meccanica.” references 1. bellani g, laffey jg, pham t, et al; lung safe investigators; esicm trials group. epidemiology, patterns of care, and mortality for patients with acute respiratory distress syndrome in intensive care units in 50 countries. jama. 2016;315(8):788800. crossref pubmed 2. mcnicholas ba, rooney gm, laffey jg. lessons to learn from epidemiologic studies in ards. curr opin crit care. 2018;24(1): 41-48. crossref pubmed 3. heymann dl, shindo n; who scientific and technical advisory group for infectious hazards. covid-19: what is next for public health? lancet. 2020;395(10224):542-545. crossref pubmed 4. ranieri vm, rubenfeld gd, thompson bt, et al; ards definition task force. acute respiratory distress syndrome: the berlin definition. jama. 2012;307(23):2526-2533. pubmed 5. ware lb, matthay ma. the acute respiratory distress syndrome. n engl j med. 2000;342(18):1334-1349. crossref pubmed 6. bachofen m, weibel er. structural alterations of lung parenchyma in the adult respiratory distress syndrome. clin chest med. 1982;3(1):35-56. crossref pubmed 7. sibilia v, torsello a, pagani f, et al. effects of hexarelin against acid-independent and acid-dependent ulcerogens in the rat. peptides. 2004;25(12):2163-2170. crossref pubmed 8. locatelli v, rossoni g, schweiger f, et al. growth hormoneindependent cardioprotective effects of hexarelin in the rat. endocrinology. 1999;140(9):4024-4031. crossref pubmed 9. biagini g, torsello a, marinelli c, et al. beneficial effects of desacyl-ghrelin, hexarelin and ep-80317 in models of status epilepticus. eur j pharmacol. 2011;670(1):130-136. crossref pubmed 10. conte e, camerino gm, mele a, et al. growth hormone secretagogues prevent dysregulation of skeletal muscle calcium homeostasis in a rat model of cisplatin-induced cachexia. j cachexia sarcopenia muscle. 2017;8(3):386-404. crossref pubmed 11. bresciani e, pitsikas n, tamiazzo l, et al. feeding behavior during long-term hexarelin administration in young and old rats. j endocrinol invest. 2008;31(7):647-652. crossref pubmed 12. mao y, tokudome t, kishimoto i. the cardiovascular action of hexarelin. j geriatr cardiol. 2014;11(3):253-258. pubmed 13. torsello a, bresciani e, ravelli m, et al. novel domain-selective ace-inhibiting activity of synthetic growth hormone secretagogues. pharmacol res. 2012;66(4):317-324. crossref pubmed 14. deng w, deng y, deng j, et al. losartan attenuated lipopolysaccharide-induced lung injury by suppression of lectin-like oxidized low-density lipoprotein receptor-1. int j clin exp pathol. 2015;8(12):15670-6. pubmed 15. torsello a, bresciani e, rossoni g, et al. ghrelin plays a minor role in the physiological control of cardiac function in the rat. endocrinology. 2003;144(5):1787-1792. crossref pubmed 16. amigoni m, bellani g, scanziani m, et al. lung injury and recovery in a murine model of unilateral acid aspiration: functional, biochemical, and morphologic characterization. anesthesiology. 2008;108(6):1037-1046. crossref pubmed 17. amigoni m, bellani g, zambelli v, et al. unilateral acid aspiration augments the effects of ventilator lung injury in the contralateral lung. anesthesiology. 2013;119(3):642-651. crossref pubmed 18. zambelli v, bellani g, borsa r, et al. angiotensin-(1-7) improves oxygenation, while reducing cellular infiltrate and fibrosis in experimental acute respiratory distress syndrome. intensive care med exp. 2015;3(1):44. crossref pubmed 19. tager am, kradin rl, lacamera p, et al. inhibition of pulmonary fibrosis by the chemokine ip-10/cxcl10. am j respir cell mol biol. 2004;31(4):395-404. crossref pubmed 20. broglio f, benso a, valetto mr, et al. growth hormone-independent cardiotropic activities of growth hormone-releasing peptides in normal subjects, in patients with growth hormone deficiency, and in patients with idiopathic or ischemic dilated cardiomyopathy. endocrine. 2001;14(1):105-108. crossref pubmed 21. soubrier f, alhenc-gelas f, hubert c, et al. two putative active centers in human angiotensin i-converting enzyme revealed by molecular cloning. proc natl acad sci usa. 1988;85(24):93869390. crossref pubmed 22. georgiadis d, beau f, czarny b, cotton j, yiotakis a, dive v. roles of the two active sites of somatic angiotensin-converting https://doi.org/10.1001/jama.2016.0291 https://pubmed.ncbi.nlm.nih.gov/26903337 https://doi.org/10.1097/mcc.0000000000000473 https://pubmed.ncbi.nlm.nih.gov/29135617 https://doi.org/10.1016/s0140-6736(20)30374-3 https://pubmed.ncbi.nlm.nih.gov/32061313 https://pubmed.ncbi.nlm.nih.gov/22797452 https://doi.org/10.1056/nejm200005043421806 https://pubmed.ncbi.nlm.nih.gov/10793167 https://doi.org/10.1016/s0272-5231(21)00143-x https://pubmed.ncbi.nlm.nih.gov/7075161 https://doi.org/10.1016/j.peptides.2004.08.001 https://pubmed.ncbi.nlm.nih.gov/15572206 https://doi.org/10.1210/endo.140.9.6948 https://pubmed.ncbi.nlm.nih.gov/10465272 https://doi.org/10.1016/j.ejphar.2011.08.020 https://pubmed.ncbi.nlm.nih.gov/21914437 https://doi.org/10.1002/jcsm.12185 https://pubmed.ncbi.nlm.nih.gov/28294567 https://doi.org/10.1007/bf03345618 https://pubmed.ncbi.nlm.nih.gov/18787385 https://pubmed.ncbi.nlm.nih.gov/25278975 https://doi.org/10.1016/j.phrs.2012.06.006 https://pubmed.ncbi.nlm.nih.gov/22732396 https://pubmed.ncbi.nlm.nih.gov/26884836 https://doi.org/10.1210/en.2002-221048 https://pubmed.ncbi.nlm.nih.gov/12697684 https://doi.org/10.1097/aln.0b013e318173f64f https://pubmed.ncbi.nlm.nih.gov/18497604 https://doi.org/10.1097/aln.0b013e318297d487 https://pubmed.ncbi.nlm.nih.gov/23681142 https://doi.org/10.1186/s40635-015-0044-3 https://pubmed.ncbi.nlm.nih.gov/26215809 https://doi.org/10.1165/rcmb.2004-0175oc https://pubmed.ncbi.nlm.nih.gov/15205180 https://doi.org/10.1385/endo:14:1:105 https://pubmed.ncbi.nlm.nih.gov/11322491 https://doi.org/10.1073/pnas.85.24.9386 https://pubmed.ncbi.nlm.nih.gov/2849100 zambelli et al drug target insights 2021; 15: 33 © 2021 the authors. published by aboutscience www.aboutscience.eu enzyme in the cleavage of angiotensin i and bradykinin: insights from selective inhibitors. circ res. 2003;93(2):148154. crossref pubmed 23. katugampola sd, pallikaros z, davenport ap. [125i-his(9)]ghrelin, a novel radioligand for localizing ghs orphan receptors in human and rat tissue: up-regulation of receptors with athersclerosis. br j pharmacol. 2001;134(1):143-149. crossref pubmed 24. nagaya n, miyatake k, uematsu m, et al. hemodynamic, renal, and hormonal effects of ghrelin infusion in patients with chronic heart failure. j clin endocrinol metab. 2001;86(12):5854-5859. crossref pubmed 25. kuba k, imai y, ohto-nakanishi t, penninger jm. trilogy of ace2: a peptidase in the renin-angiotensin system, a sars receptor, and a partner for amino acid transporters. pharmacol ther. 2010; 128(1):119-128. crossref pubmed 26. bulgarelli i, tamiazzo l, bresciani e, et al. desacyl-ghrelin and synthetic gh-secretagogues modulate the production of inflammatory cytokines in mouse microglia cells stimulated by beta-amyloid fibrils. j neurosci res. 2009;87(12):2718-2727. crossref pubmed 27. fukuda y, ishizaki m, masuda y, kimura g, kawanami o, masugi y. the role of intraalveolar fibrosis in the process of pulmonary structural remodeling in patients with diffuse alveolar damage. am j pathol. 1987;126(1):171-182. pubmed 28. aggarwal nr, king ls, d’alessio fr. diverse macrophage populations mediate acute lung inflammation and resolution. am j physiol lung cell mol physiol. 2014;306(8):l709-l725. crossref pubmed 29. peiris js, yuen ky, osterhaus ad, stöhr k. the severe acute respiratory syndrome. n engl j med. 2003;349(25):2431-2441. crossref pubmed 30. blackwell ts, tager am, borok z, et al. future directions in idiopathic pulmonary fibrosis research. an nhlbi workshop report. am j respir crit care med. 2014;189(2):214-222. crossref pubmed https://doi.org/10.1161/01.res.0000081593.33848.fc https://pubmed.ncbi.nlm.nih.gov/12805239 https://doi.org/10.1038/sj.bjp.0704228 https://pubmed.ncbi.nlm.nih.gov/11522606 https://doi.org/10.1210/jcem.86.12.8115 https://pubmed.ncbi.nlm.nih.gov/11739451 https://doi.org/10.1016/j.pharmthera.2010.06.003 https://pubmed.ncbi.nlm.nih.gov/20599443 https://doi.org/10.1002/jnr.22088 https://pubmed.ncbi.nlm.nih.gov/19382238 https://pubmed.ncbi.nlm.nih.gov/3812636 https://doi.org/10.1152/ajplung.00341.2013 https://pubmed.ncbi.nlm.nih.gov/24508730 https://doi.org/10.1056/nejmra032498 https://pubmed.ncbi.nlm.nih.gov/14681510 https://doi.org/10.1164/rccm.201306-1141ws https://pubmed.ncbi.nlm.nih.gov/24160862 1drug target insights 2016:10 brain histamine n-methyltransferase as a possible target of treatment for methamphetamine overdose junichi kitanaka1, nobue kitanaka1, f. scott hall2, george r. uhl3 and motohiko takemura1 1department of pharmacology, hyogo college of medicine, hyogo, japan. 2department of pharmacology and experimental therapeutics, college of pharmacy and pharmaceutical sciences, university of toledo, toledo, oh, usa. 3new mexico va healthcare system/brinm, albuquerque, nm, usa. a bstr act: stereotypical behaviors induced by methamphetamine (meth) overdose are one of the overt symptoms of meth abuse, which can be easily assessed in animal models. currently, there is no successful treatment for meth overdose. there is increasing evidence that elevated levels of brain histamine can attenuate meth-induced behavioral abnormalities, which might therefore constitute a novel therapeutic treatment for meth abuse and meth overdose. in mammals, histamine n-methyltransferase (hmt) is the sole enzyme responsible for degrading histamine in the brain. metoprine, one of the most potent hmt inhibitors, can cross the blood–brain barrier and increase brain histamine levels by inhibiting hmt. consequently, this compound can be a candidate for a prototype of drugs for the treatment of meth overdose. k e y wor ds: methamphetamine, overdose, stereotyped behavior, histamine n-methyltransferase, brain histaminergic system, metoprine citation: kitanaka et al. brain histamine n-methyltransferase as a possible target of treatment for methamphetamine overdose. drug target insights 2016:10 1–7 doi:10.4137/dti.s38342. type: review received: december 23, 2015. resubmitted: january 25, 2016. accepted for publication: january 27, 2016. academic editor: anuj chauhan, editor in chief peer review: four peer reviewers contributed to the peer review report. reviewers’ reports totaled 341 words, excluding any confidential comments to the academic editor. funding: this research was supported, in part, by a grant-in-aid for researchers, hyogo college of medicine (2015 to nk) and jsps kakenhi grant number 15k08603 (to jk). the authors confirm that the funder had no influence over the study design, content of the article, or selection of this journal. competing interests: metoprine was donated by glaxosmithkline. authors disclose no other potential conflicts of interest. copyright: © the authors, publisher and licensee libertas academica limited. this is an open-access article distributed under the terms of the creative commons cc-by-nc 3.0 license. correspondence: kitanaka-hyg@umin.net paper subject to independent expert single-blind peer review. all editorial decisions made by independent academic editor. upon submission manuscript was subject to anti-plagiarism scanning. prior to publication all authors have given signed confirmation of agreement to article publication and compliance with all applicable ethical and legal requirements, including the accuracy of author and contributor information, disclosure of competing interests and funding sources, compliance with ethical requirements relating to human and animal study participants, and compliance with any copyright requirements of third parties. this journal is a member of the committee on publication ethics (cope). provenance: the authors were invited to submit this paper. published by libertas academica. learn more about this journal. journal name: drug target insights journal type: review year: 2016 volume: 10 running head verso: kitanaka et al running head recto: treatment for methamphetamine overdose introduction methamphetamine (meth; n-methyl-1-phenylpropan-2 amine) is a powerful psychomotor stimulant similar in structure to amphetamine (amph; 1-phenylpropan-2-amine). although meth is used in the treatment of attention-deficit hyperactivity disorder, narcolepsy, and severe obesity,1 the clinical utility of meth is limited by its abuse potential. meth is typically abused via intranasal, intravenous, or inhalation routes of administration, rather than orally, worldwide, including japan and the united states.2,3 meth addiction, including adverse effects associated with acute meth use and long-term effects associated with meth addiction, is a serious public health problem.4–8 currently, there are no effective treatments for meth addiction, abuse or acute overdose.7,9,10 the molecular basis of action of meth is considered to be very similar to that of amph because of their structural similarities. meth interacts with proteins that affect monoamine function, including the dopamine transporter (dat), monoamine oxidases (maos), and the vesicular monoamine transporter-2 (vmat2), inhibiting their functions in a manner similar to amph,11,12 although with somewhat different potencies on dopamine transport.13,14 meth inhibition of dat, mao, and vmat2 results in the elevation of presynaptic cytosolic da levels and the impulse-independent release of dopamine into the synaptic clefts of the dopaminergic neurons via reverse transport mediated by dat. the abnormally released dopamine then binds to preand postsynaptic dopamine d1 and d2 receptors, resulting in behavioral and psychological alterations.15 behavioral alterations in animals are augmented with repeated treatment in a dose-dependent manner (eg, sensitization).16 dopamine receptor antagonists drastically attenuate meth-induced behavioral and psychological alterations, including both acute and sensitized effects. in human beings, meth sensitization is associated with progressive development of meth-induced psychosis,17 which is improved by treatment with haloperidol,18 a classical antipsychotic that has antagonistic actions at dopamine d2 receptors, but with pronounced extrapyramidal side effects.19,20 in the search for an effective pharmacotherapy for methinduced symptoms without these adverse effects, other neuronal systems have been investigated.21–24 our research has focused on a possible involvement of brain histaminergic systems in meth actions, especially high-dose meth effects such as meth-induced stereotypy in mice. here, we will review the brain histaminergic systems, and evidence that may suggest that alterations in histaminergic function may be a possible therapeutic approach to the treatment of meth overdose associated with high meth doses, or the sensitized state associated with long-term meth use. http://www.la-press.com/drug-target-insights-journal-j23 http://www.la-press.com http://dx.doi.org/10.4137/dti.s38342 http://creativecommons.org/licenses/by-nc/3.0/ http://creativecommons.org/licenses/by-nc/3.0/ mailto:kitanaka-hyg@umin.net http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 kitanaka et al 2 drug target insights 2016:10 meth overdose: experimental procedures and behavioral effects in rodents, systemic administration of meth induces locomotor hyperactivity that is replaced by repetitive and compulsive behaviors called stereotypies at higher doses.16,25,26 for instance, a single administration of meth at doses of 0.5–2  mg/kg induces hyperlocomotion,16,27–30 while rodents exhibit stereotypy when treated with higher doses of meth (5–20  mg/kg).31–37 rodents exhibiting stereotypy after acute high doses of meth are considered to be a model for meth overdose. to evaluate meth-induced stereotypy reproducibly, tatsuta et al developed an experimental procedure using mice as follows35: test subjects are placed in a transparent acrylic box (30  ×  30  ×  35  cm) with ~25  g of fresh wood chips spread on the floor of the chamber and observed for stereotypy for one hour after drug challenge by observers unaware of the treatments. meth-induced stereotypy lasts for ~170 minutes after a 10  mg/kg i.p. injection in mice.35 the frequencies of each behavioral component of stereotypical behavior (see description of categories below) observed for two-hour postinjection are the same as the frequencies observed for one hour (two-hour observations38 vs. one-hour observations39). therefore, the period of one hour was chosen in all of our subsequent experiments. behavior is assessed at 30-second intervals, and the predominant behavior observed during each interval is recorded. since individual stereotypical behaviors are unchanged for long periods (.30 seconds) after drug treatment, it is possible to record the observations by hand. the behaviors scored are inactive (awake and inactive, or sleeping), ambulation, rearing (standing on the hind legs, with forelegs unsupported or supported on the walls), persistent locomotion, head bobbing (up-and-down movements of the head), continuous sniffing, circling, and continuous nail and/or wood chip biting or licking. ambulation, rearing, and persistent locomotion are considered to be exploratory behaviors, and the last four categories are considered stereotypies. stereotypical cage climbing40 is not observed in our experimental procedure because of the use of an acrylic test chamber without a stainless steel grid top. persistent locomotion is not classified as stereotypy because the mice scored as having persistent locomotion show horizontal locomotor activity less than or equal to that displayed by mice showing hyperlocomotion induced by 1 mg/kg meth (which is not generally defined as a stereotypy) measured by automated animex auto.41 the cumulative number of intervals within each five-minute period in which stereotypies are observed is evaluated as a time course (maximal value = 10). animal handling and care were conducted in accordance with the guide for the care and use of laboratory animals (8th edition, institute of laboratory animal resources-national research council, national academy press, 2011), and all experiments were reviewed and approved by the institutional animal research committee of hyogo college of medicine. using the experimental procedure described above, we found that a single administration of meth (5  mg/kg) induces stereotypical sniffing, while stereotypical biting is predominantly observed at 10  mg/kg meth.33,35 another group reported that a single administration of meth (20  mg/kg) induces repetitive self-injurious behavior.31,37 in line with these observations, meth-induced stereotypical biting appears to be a more severe symptom than stereotypical sniffing as an animal model of meth overdose. possible pharmacological properties of compounds that will be effective for meth overdose should (1) inhibit meth-induced stereotypical biting or (2) shift stereotypical biting to sniffing (eg, a leftward shift in the meth dose–response relationship, producing less severe stereotypies). using this approach, we investigated a possible involvement of brain histaminergic neurons in meth-induced stereotypical behavior, as a way to approach potential novel treatments for meth overdose. brain histaminergic systems: potential roles in drug addiction, drug abuse, and drug overdose histamine is a biogenic amine produced by the body and plays major roles in allergic reactions and secretion of gastric acid.42–44 it is also released by neurons that originate from the tuberomammillary nucleus of the posterior hypothalamus and project to various brain areas,45,46 suggesting that histamine has crucial roles in the central nervous system.47 brain histamine is considered to be involved in the regulation of arousal, hormone release, feeding/drinking, and pain perception.48–54 as shown in figure 1, histamine is synthesized by decarboxylation of the amino acid l-histidine in a reaction catalyzed by histidine decarboxylase (hdc), stored in mast cells, basophils, enterochromaffin-like cells, and histaminergic neurons, and released on stimulation. released histamine in turn activates histaminergic receptors, causing physiological reactions. in brain, for termination of histaminergic neurotransmission after activation of histamine receptors, histamine is transferred from the extracellular space into cytoplasm by organic cation transporter 3 and/or the equilibrative nucleoside transporter (ent4), and catabolized by the cytosolic enzyme histamine n-methyltransferase (hmt) to form n-methylhistamine, which is inactive in the histaminergic system.55,56 hmt is the sole enzyme that degrades histamine in brain,57,58 whereas diamine oxidase (dao; histaminase) catabolizes histamine in peripheral tissues.49,59 it is noted that both hmt mrna and hmt-like immunoreactivity are expressed in mouse stomach57,58 and that the urinary excretions of histamine and n t-methylhistamine are affected by food intake in human beings;60 there is a possibility that hmt might, at least in part, function in peripherally. there is evidence that some drugs of abuse (meth, ethanol, and caffeine), acting through quite different initial molecular targets, release histamine and increase endogenous histamine levels in brain.61–65 what is the role of released histamine by these drugs in drug abuse and addiction? there are two main possibilities: (1) that histamine contributes to the addictive or adverse effects associated with these drugs or (2) that histamine release acts in opposition to those effects and is part of a homeostatic counterreaction. supporting this latter idea, chandorkar and http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 treatment for methamphetamine overdose 3drug target insights 2016:10 coworkers demonstrated that intraperitoneal administration of high doses of l-histidine, a substrate for histamine synthesis (fig. 1), reduces methand apomorphine-induced stereotypical behaviors in mice, suggesting that increased levels of histamine in brain suppress abnormal behaviors associated with administration of high doses of these drugs.36,66 observation reported by ito et al support chandorkar’s perspective, finding that pretreatment with l-histidine inhibits meth-induced stereotypy and behavioral sensitization in rats, while stereotypy and behavioral sensitization are exacerbated when rats were pretreated with a-fluoromethylhistidine, an irreversible inhibitor of hdc (fig. 1) that reduces brain histamine levels.67 in line with these observations, it is likely that increasing levels of brain histamine may attenuate meth-induced behavioral effects. this is supported by the evidence that the l-histidine effects were blocked by treatment with brain-penetrating histamine h1/h2 receptor antagonists. 67 hmt: a key enzyme regulating high-dose effects of meth as described above, compounds such as l-histidine and a-fluoromethylhistidine are useful for the increase or decrease in neuronal histamine release, resulting in increasing or decreasing brain histamine levels, respectively.66–71 however, these compounds potentially alter the levels of histamines throughout the body. by contrast, inhibition of hmt activity predominantly modulates central histaminergic activity, while peripheral histaminergic activity is affected, to a lesser extent, by inhibiting an hmt activity. at present, there are no compounds that increase hmt activity. several hmt inhibitors are available for research purposes.72–74 the dimaprit analog skf 91488 (s-[4-(n,n-dimethylamino)butyl]isothiourea) is one of the most potent hmt inhibitors currently known.74 however, to inhibit hmt activity in the brain, skf 91488 needs to be administered by an intracerebroventricular route.65,75 intraperitoneal administration of skf 91488 does not appear to affect hmt activity in the brain, suggesting that the compound does not cross the brood–brain barrier.74 there are no reports of the effects of skf 91488 on rodent behavior except that by malmberg-aiello et al,75 which describes that intracerebroventricular administration of skf 91488 produces antinociceptive effects in hot plate, abdominal constriction, and paw pressure tests (table 1). these observations suggest that skf 91488 increases brain histamine levels by inhibiting an hmt activity resulting in antinociceptive effects by activating central histaminergic neurotransmission52 and that hmt inhibitors may be used to reveal important roles of central histaminergic system. however, an alternative compound would be desirable for both research and clinical applications. in contrast to the limitations of skf 9148874 for studies of central histamine function, metoprine (2,4-diamino-5(3′,4′-dichlorophenyl)-6-methylpyrimidine; formerly called bw 197u), a diaminopyrimidine derivative and potent hmt inhibitor,73 readily crosses the blood–brain barrier.76 thus, this compound can be administered systemically in order to inhibit the hmt activity in the brain. intraperitoneal administration of metoprine produces various behavioral effects, including decreases in food intake77 and increases in water consumption.78 these observations support a hypothesis that central histaminergic system may involve in the regulation of feeding/ drinking.54 studies with metoprine also suggest that brain histaminergic systems may be involved in mood and memory processes.79,80 regarding regulation of drug abuse-related phenotypes by central histaminergic systems, itoh et al81 reported that pretreatment with metoprine inhibited meth-induced hyperlocomotion in mice, suggesting that central histaminergic systems inhibit meth-induced behavioral effects. we have investigated whether metoprine could inhibit methinduced stereotypy, a high-dose behavioral effect intended to model meth overdose. pretreatment with metoprine dose figure 1. histamine synthesis and catabolism in mammals. abbreviations: adh, alcohol dehydrogenase; dao, diamine oxidase; hdc, histidine decarboxylase; hmt, histamine n-methyltransferase; mao, monoamine oxidase; sah, s-adenosylhomosysteine; sam, s-adenosylmethionine. http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 kitanaka et al 4 drug target insights 2016:10 dependently decreased meth-induced stereotypical biting, while increasing sniffing, suggesting that metoprine may ameliorate high-dose meth-induced symptoms by producing a leftward shift in meth behavioral effects (table 1).65 the inhibitory effect of metoprine on meth-induced stereotypical biting is likely to be mediated by histamine h1 (but not h2/h3) receptors located in the brain, since the metoprine effect was blocked by coadministration of metoprine with brainpenetrating histamine h1 receptor antagonists. 65 it is likely that metoprine-activated histaminergic neurotransmission via central histamine h1 receptors accounted for the attenuation of meth-induced stereotypical biting. this is supported by the evidence that (1) metoprine increased histamine levels, but decreased n t-methylhistamine levels, in the hypothalamus and (2) pretreatment with l-histidine, which increased the levels of brain histamine, also reduced the frequency of meth-induced stereotypical biting.82 iwabuchi et al83 reported that meth-induced locomotor hyperactivity and the development of behavioral sensitization were facilitated more in the histamine h1/h2 gene double knockout mice than in the wild-type mice, indicating that brain histaminergic system is negatively associated with meth action via histamine h1/h2 receptors (see also reports by munzar et al, 84,85 which described a possible involvement of histamine h3 receptors in meth-seeking behavior). in addition, pretreatment with histamine h3 receptor (autoreceptor) agonists such as (r)-a-methylhistamine, imetit, and immepip decreased hypothalamic histamine levels and increased the frequency of meth-induced stereotypical biting.86 moreover, it was noted that there was a very strong negative correlation (r = -0.918, p  ,  0.001) between the frequency of meth-induced stereotypical biting and hypothalamic histamine levels, suggesting that activation of brain histaminergic system may suppress high-dose behavioral effects of meth, and might consequently reduce high-dose effects associated with the progression to drug dependence and acute overdose.87 hmt inhibitors: candidate compounds of treatment for meth overdose no agents that modulate histaminergic system other than the hmt inhibitors and l-histidine have been reported to ameliorate symptoms of acute injections of high-dose meth, although abt-239, an antagonist selective for histamine h3 receptors, attenuates moderate doses of meth-induced locomotor hyperactivity.88 in our preliminary experiments, metoprine itself did not induce an anxiety-like behavior and memory impairments in the marble-burying test and y-maze test, respectively (s. okumura and t. sakamoto, unpublished observations). therefore, metoprine is likely to have limited side effects, although it has been associated with increases in locomotor behaviors,65,89,90 anxiogenic79 (but there is a negative finding),65 antiamnesic,80 and antinociceptive effects75 in rodents (table 1). regarding metoprine-induced locomotor hyperactivity, a dose–response effect of metoprine on general locomotion was biphasic with the greatest hyperactivity noted at a dose of 10  mg/kg of metoprine.65 the biphasic reaction to metoprine dose appears to be mediated by brain histamine-mediated effects, since histamine itself injected into the brain induces biphasic locomotor alterations as well.91,92 several types of seizures are also inhibited by metoprine (table 1).70,71,93,94 whether similar mechanisms underlie these effects and effects on meth-induced behavior is uncertain. in any case, the anticonvulsant topiramate did not affect meth-induced stereotypical biting, suggesting that the antagonism of meth-induced effects by metoprine is not something that is produced by all anticonvulsive drugs.38 another piece of evidence consistent with histaminergic modulation of systems associated with high-dose meth effects comes from studies of hdc gene knockout mice, which demonstrate tic-like stereotypical movements, which can be ameliorated by histamine repletion.95 this might table 1. effects of hmt inhibitors on rodent behaviors. hmt inhibitor effect reference feeding/drinking metoprine decrease in food intake 77 metoprine increase in water consumption 78 mood metoprine anxiogenic-like 79 memory process metoprine antiamnesic 80 pain skf 91488 antinociceptive 75 bw 301u antinociceptive 75 locomotor activity metoprine increase in locomotor activity 89 metoprine increase in number of rearing 89 metoprine increase in locomotor activity 65 metoprine increase in locomotor activity 90 seizures metoprine inhibition of audiogenic seizure 93 metoprine decrease in duration of convulsions 70 metoprine inhibition of amygdaloid kindled seizure 94 metoprine delay in the onset of seizure episodes 71 meth-induced behavior metoprine decrease in meth-induced hyperlocomotion 81 metoprine decrease in meth-induced stereotypical biting 65 skf 91488 decrease in meth-induced stereotypical biting 65 notes: metoprine = 2,4-diamino-5-(3′,4′-dichlorophenyl)-6-methylpyrimidine, skf 91488 = s-[4-(n, n-dimethylamino)butyl]isothiourea, bw 301u = 2,4 diamino-6-(2,5-dimethoxybenzyl)-5-methylpyrido[2,3-d]pyrimidine. abbreviations: hmt, histamine n-methyltransferase; meth, methamphetamine. http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 treatment for methamphetamine overdose 5drug target insights 2016:10 suggest that modulation of histaminergic function might be useful in other types of striatal dysfunctions associated with abnormal movements, or repetitive behaviors. with regard to the high-dose meth effects associated with sensitization or other adverse effects, it would appear that metoprine may be beneficial based on the model discussed here. possible treatments of metoprine with histamine h3 receptor antagonists or with modafinil for meth overdose should be evaluated in the future studies because histamine h3 receptor antagonists and modafinil increase tissue levels of histamine in the hypothalamus.96,97 it remains to be seen how metoprine will affect other meth-induced behaviors, specifically, including others more specific to addiction or meth overdose. in any case, the present data support the proposal that hmt inhibitors such as metoprine are possible candidate compounds for the treatment of meth-related conditions, including meth-induced psychosis and overdose. acknowledgments the authors are grateful to dr. tomohiro tatsuta of ibogawa hospital, hyogo, japan, for his remarkable contribution to the development of a method to evaluate stereotypy observed under meth overdose. metoprine was generously donated by glaxosmithkline (stevenage, uk). author contributions conceived and designed the experiments: jk, nk, fsh, and mt. analyzed the data: nk, jk, fsh, and mt. wrote the first draft of the manuscript: nk, jk, fsh, gru, and mt. contributed to the writing of the manuscript: jk, nk, fsh, gru, and mt. agreed with manuscript results and conclusions: jk, nk, fsh, gru, and mt. jointly developed the structure and arguments for the paper: fsh and gru. made critical revisions and approved the final version: jk, nk, fsh, gru, and mt. all the authors reviewed and approved the final manuscript. r efer ences 1. sanchez-ramos j. neurologic complications of psychomotor stimulant abuse. int rev neurobiol. 2015;120:131–160. 2. ujike h, sato m. clinical features of sensitization to methamphetamine observed in patients with methamphetamine dependence and psychosis. ann n y acad sci. 2004;1025:279–287. 3. greberman sb, wada k. social and legal factors related to drug abuse in the united states and japan. public health rep. 1994;109(6):731–737. 4. national institute on drug abuse. nida drugfacts: methamphetamine; 2014. available at: http://www.drugabuse.gov/publications/drugfacts/methamphetamine. accessed september 16, 2015. 5. sato m. a lasting vulnerability to psychosis in patients with previous methamphetamine psychosis. ann n y acad sci. 1992;654:160–170. 6. cruickshank cc, dyer kr. a review of the clinical pharmacology of methamphetamine. addiction. 2009;104(7):1085–1099. 7. brackins t, brahm nc, kissack jc. treatments for methamphetamine abuse: a literature review for the clinician. j pharm pract. 2011;24(6):541–550. 8. murray jb. psychophysiological aspects of amphetamine-methamphetamine abuse. j psychol. 1998;132(2):227–237. 9. karila l, weinstein a, aubin hj, benyamina a, reynaud m, batki sl. pharmacological approaches to methamphetamine dependence: a focused review. br j clin pharmacol. 2010;69(6):578–592. 10. cretzmeyer m, sarrazin mv, huber dl, block ri, hall ja. treatment of methamphetamine abuse: research findings and clinical directions. j subst abuse treat. 2003;24(3):267–277. 11. seiden ls, sabol ke, ricaurte ga. amphetamine: effects on catecholamine systems and behavior. annu rev pharmacol toxicol. 1993;33:639–677. 12. sulzer d. how addictive drugs disrupt presynaptic dopamine neurotransmission. neuron. 2011;69(4):628–649. 13. goodwin js, larson ga, swant j, et al. amphetamine and methamphetamine differentially affect dopamine transporters in vitro and in vivo. j biol chem. 2009; 284(5):2978–2989. 14. eshleman aj, henningsen ra, neve ka, janowsky a. release of dopamine via the human transporter. mol pharmacol. 1994;45(2):312–316. 15. shoblock jr, sullivan eb, maisonneuve im, glick sd. neurochemical and behavioral differences between d-methamphetamine and d-amphetamine in rats. psychopharmacology. 2003;165(4):359–369. 16. nishikawa t, mataga n, takashima m, toru m. behavioral sensitization and relative hyperresponsiveness of striatal and limbic dopaminergic neurons after repeated methamphetamine treatment. eur j pharmacol. 1983;88(2–3):195–203. 17. grant km, levan td, wells sm, et al. methamphetamine-associated psychosis. j neuroimmune pharmacol. 2012;7(1):113–139. 18. verachai v, rukngan w, chawanakrasaesin k, et al. treatment of methamphetamine-induced psychosis: a double-blind randomized controlled trial comparing haloperidol and quetiapine. psychopharmacology. 2014;231(16):3099–3108. 19. gao k, kemp de, ganocy sj, gajwani p, xia g, calabrese jr. antipsychoticinduced extrapyramidal side effects in bipolar disorder and schizophrenia: a systematic review. j clin psychopharmacol. 2008;28(2):203–209. 20. haddad pm, das a, keyhani s. antipsychotic drugs and extrapyramidal side effects in first episode psychosis: a systematic review of head-head comparisons. j psychopharmacol. 2012;26(5 suppl):15–26. 21. grant je, odlaug bl, kim sw. a double-blind, placebo-controlled study of n-acetyl cysteine plus naltrexone for methamphetamine dependence. eur neuropsychopharmacol. 2010;20(11):823–828. 22. whitfield tw jr, schlosburg je, wee s, et al. kappa opioid receptors in the nucleus accumbens shell mediate escalation of methamphetamine intake. j neurosci. 2015;35(10):4296–4305. 23. thorn da, winter jc, li jx. agmatine attenuates methamphetamine-induced conditioned place preference in rats. eur j pharmacol. 2012;680(1–3):69–72. 24. nguyen l, kaushal n, robson mj, matsumoto rr. sigma receptors as potential therapeutic targets for neuroprotection. eur j pharmacol. 2014;743:42–47. 25. segal ds, kuczenski r. repeated binge exposures to amphetamine and methamphetamine: behavioral and neurochemical characterization. j pharmacol exp ther. 1997;282(2):561–573. 26. berridge cw. neural substrates of psychostimulant-induced arousal. neuropsychopharmacology. 2006;31(11):2332–2340. 27. kitanaka n, kitanaka j, takemura m. behavioral sensitization and alteration in monoamine metabolism in mice after single versus repeated methamphetamine administration. eur j pharmacol. 2003;474(1):63–70. 28. kitanaka n, kitanaka j, tatsuta t, watabe k, morita y, takemura m. methamphetamine reward in mice as assessed by conditioned place preference test with supermex sensors: effect of subchronic clorgyline pretreatment. neurochem res. 2006;31(6):805–813. 29. itzhak y, martin jl. effect of riluzole and gabapentin on cocaineand methamphetamine-induced behavioral sensitization in mice. psychopharmacology. 2000;151(2–3):226–233. 30. niwa m, nitta a, mizoguchi h, et al. a novel molecule “shati” is involved in methamphetamine-induced hyperlocomotion, sensitization, and conditioned place preference. j neurosci. 2007;27(28):7604–7615. 31. mori t, ito s, kita t, narita m, suzuki t, sawaguchi t. effects of mu-, delta and kappa-opioid receptor agonists on methamphetamine-induced self-injurious behavior in mice. eur j pharmacol. 2006;532(1–2):81–87. 32. kitanaka n, kitanaka j, hall fs, et al. agmatine attenuates methamphetamineinduced hyperlocomotion and stereotyped behavior in mice. behav pharmacol. 2014; 25(2):158–165. 33. kitanaka j, kitanaka n, hall fs, et al. pretreatment with nomifensine or nomifensine analogue 4-phenyl-1,2,3,4-tetrahydroisoquinoline augments methamphetamineinduced stereotypical behavior in mice. brain res. 2012;1439:15–26. 34. horner ka, noble es, gilbert ye. methamphetamine-induced stereotypy correlates negatively with patch-enhanced prodynorphin and arc mrna expression in the rat caudate putamen: the role of mu opioid receptor activation. pharmacol biochem behav. 2010;95(4):410–421. 35. tatsuta t, kitanaka n, kitanaka j, morita y, takemura m. lobeline attenuates methamphetamine-induced stereotypy in adolescent mice. neurochem res. 2006; 31(11):1359–1369. 36. joshi vv, balsara jj, jadhav jh, chandorkar ag. effect of l-histidine and chlorcyclizine on apomorphine-induced climbing behaviour and methamphetamine stereotypy in mice. eur j pharmacol. 1981;69(4):499–502. 37. mori t, ito s, kita t, sawaguchi t. effects of dopamineand serotonin-related compounds on methamphetamine-induced self-injurious behavior in mice. j pharmacol sci. 2004;96(4):459–464. http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 http://www.drugabuse.gov/publications/drugfacts/methamphetamine http://www.drugabuse.gov/publications/drugfacts/methamphetamine kitanaka et al 6 drug target insights 2016:10 38. tatsuta t, kitanaka n, kitanaka j, morita y, takemura m. lack of effect of anticonvulsant topiramate on methamphetamine-induced stereotypy and rewarding property in mice. pharmacol biochem behav. 2007;87(1):48–55. 39. kitanaka j, kitanaka n, tatsuta t, et al. sigma1 receptor antagonists determine the behavioral pattern of the methamphetamine-induced stereotypy in mice. psychopharmacology. 2009;203(4):781–792. 40. gianutsos g, morrow g, light s, sweeney mj. dopaminergic properties of nomifensine. pharmacol biochem behav. 1982;17(5):951–954. 41. kitanaka n, kitanaka j, takemura m. inhibition of methamphetamine-induced hyperlocomotion in mice by clorgyline, a monoamine oxidase-a inhibitor, through alteration of the 5-hydroxytryptamine turnover in the striatum. neuroscience. 2005;130(2):295–308. 42. amin k. the role of mast cells in allergic inflammation. respir med. 2012;106(1): 9–14. 43. cataldi m, borriello f, granata f, annunziato l, marone g. histamine receptors and antihistamines: from discovery to clinical applications. chem immunol allergy. 2014;100:214–226. 44. huang jf, thurmond rl. the new biology of histamine receptors. curr allergy asthma rep. 2008;8(1):21–27. 45. watanabe t, taguchi y, hayashi h, et al. evidence for the presence of a histaminergic neuron system in the rat brain: an immunohistochemical analysis. neurosci lett. 1983;39(3):249–254. 46. panula p, yang hy, costa e. histamine-containing neurons in the rat hypothalamus. proc natl acad sci u s a. 1984;81(8):2572–2576. 47. prell gd, green jp. histamine as a neuroregulator. annu rev neurosci. 1986;9: 209–254. 48. brown re, stevens dr, haas hl. the physiology of brain histamine. prog neurobiol. 2001;63(6):637–672. 49. haas hl, sergeeva oa, selbach o. histamine in the nervous system. physiol rev. 2008;88(3):1183–1241. 50. roberts f, calcutt cr. histamine and the hypothalamus. neuroscience. 1983;9(4): 721–739. 51. jones be. arousal systems. front biosci. 2003;8:s438–s451. 52. nuutinen s, panula p. histamine in neurotransmission and brain diseases. adv exp med biol. 2010;709:95–107. 53. fukagawa k, sakata t, shiraishi t, et al. neuronal histamine modulates feeding behavior through h1-receptor in rat hypothalamus. am j physiol. 1989;256(3 pt 2): r605–r611. 54. wada h, inagaki n, yamatodani a, watanabe t. is the histaminergic neuron system a regulatory center for whole-brain activity? trends neurosci. 1991;14(9): 415–418. 55. ogasawara m, yamauchi k, satoh y, et al. recent advances in molecular pharmacology of the histamine systems: organic cation transporters as a histamine transporter and histamine metabolism. j pharmacol sci. 2006;101(1):24–30. 56. yoshikawa t, naganuma f, iida t, et al. molecular mechanism of histamine clearance by primary human astrocytes. glia. 2013;61(6):905–916. 57. takemura m, imamura i, mizuguchi h, fukui h, yamatodani a. tissue distribution of histamine n-methyltransferase-like immunoreactivity in rodents. life sci. 1994;54(15):1059–1071. 58. kitanaka n, kitanaka j, oue t, tada y, tanaka t, takemura m. genomic structure of the rat and mouse histamine n-methyltransferase gene. jpn j pharmacol. 2002;88(1):85–92. 59. kitanaka j, kitanaka n, tsujimura t, terada n, takemura m. expression of diamine oxidase (histaminase) in guinea-pig tissues. eur j pharmacol. 2002; 437(3):179–185. 60. imamura i, watanabe t, maeyama k, kubota a, okada a, wada h. effect of food intake on urinary excretions of histamine, n tau-methylhistamine, imidazole acetic acid and its conjugate(s) in humans and mice. j biochem. 1984;96(6): 1931–1937. 61. ito c, onodera k, sakurai e, sato m, watanabe t. the effect of methamphetamine on histamine level and histidine decarboxylase activity in the rat brain. brain res. 1996;734(1–2):98–102. 62. ito c, onodera k, yamatodani a, watanabe t, sato m. the effect of methamphetamine on histamine release in the rat hypothalamus. psychiatry clin neurosci. 1997;51(2):79–81. 63. john j, kodama t, siegel jm. caffeine promotes glutamate and histamine release in the posterior hypothalamus. am j physiol regul integr comp physiol. 2014;307(6):r704–r710. 64. subramanian n, schinzel w, mitznegg p, estler cj. influence of ethanol on histamine metabolism and release in the rat brain. ii. regions of the histaminergic pathway. pharmacology. 1980;20(1):42–45. 65. kitanaka j, kitanaka n, tatsuta t, morita y, takemura m. blockade of brain histamine metabolism alters methamphetamine-induced expression pattern of stereotypy in mice via histamine h1 receptors. neuroscience. 2007;147(3):765–777. 66. muley mp, balsara jj, chandorkar ag. effect of l-histidine pretreatment on methamphetamine induced sterotyped behaviour in rats. indian j physiol pharmacol. 1979;23(4):291–296. 67. ito c, onodera k, watanabe t, sato m. effects of histamine agents on methamphetamine-induced stereotyped behavior and behavioral sensitization in rats. psychopharmacology. 1997;130(4):362–367. 68. zawilska j, nowak jz. changes in the rat brain histamine content following metoprine and other histamine-methyltransferase (hmt) inhibitors. pol j pharmacol pharm. 1985;37(6):821–830. 69. itoh y, oishi r, nishibori m, saeki k. characterization of histamine release from the rat hypothalamus as measured by in vivo microdialysis. j neurochem. 1991;56(3):769–774. 70. yokoyama h, onodera k, maeyama k, et al. histamine levels and clonic convulsions of electrically-induced seizure in mice: the effects of alpha-fluoromethylhistidine and metoprine. naunyn schmiedebergs arch pharmacol. 1992;346(1):40–45. 71. yawata i, tanaka k, nakagawa y, watanabe y, murashima yl, nakano k. role of histaminergic neurons in development of epileptic seizures in el mice. brain res mol brain res. 2004;132(1):13–17. 72. duch ds, bacchi cj, edelstein mp, nichol ca. inhibitors of histamine metabolism in vitro and in vivo. correlations with antitrypanosomal activity. biochem pharmacol. 1984;33(9):1547–1553. 73. duch ds, bowers sw, nichol ca. elevation of brain histamine levels by diaminopyrimidine inhibitors of histamine n-methyl transferase. biochem pharmacol. 1978;27(10):1507–1509. 74. beaven ma, shaff re. inhibition of histamine methylation in vivo by the dimaprit analog, skf compound 91488. agents actions. 1979;9(5–6):455–460. 75. malmberg-aiello p, lamberti c, ipponi a, hanninen j, ghelardini c, bartolini a. effects of two histamine-n-methyltransferase inhibitors, skf 91488 and bw 301 u, in rodent antinociception. naunyn schmiedebergs arch pharmacol. 1997;355(3):354–360. 76. nichol ca, cavallito jc, woolley jl, sigel cw. lipid-soluble diaminopyrimidine inhibitors of dihydrofolate reductase. cancer treat rep. 1977;61(4):559–564. 77. lecklin a, tuomisto l, macdonald e. metoprine, an inhibitor of histamine n-methyltransferase but not catechol-o-methyltransferase, suppresses feeding in sated and in food deprived rats. methods find exp clin pharmacol. 1995;17(1):47–52. 78. lecklin a, eriksson l, leppaluoto j, tarhanen j, tuomisto l. metoprineinduced thirst and diuresis in wistar rats. acta physiol scand. 1999;165(3):325–333. 79. malmberg-aiello p, ipponi a, bartolini a, schunack w. mouse light/dark box test reveals anxiogenic-like effects by activation of histamine h1 receptors. pharmacol biochem behav. 2002;71(1–2):313–318. 80. malmberg-aiello p, ipponi a, bartolini a, schunack w. antiamnesic effect of metoprine and of selective histamine h(1) receptor agonists in a modified mouse passive avoidance test. neurosci lett. 2000;288(1):1–4. 81. itoh y, nishibori m, oishi r, saeki k. neuronal histamine inhibits methamphetamine-induced locomotor hyperactivity in mice. neurosci lett. 1984; 48(3):305–309. 82. kitanaka j, kitanaka n, tatsuta t, et al. pretreatment with l-histidine produces a shift from methamphetamine-induced stereotypical biting to persistent locomotion in mice. pharmacol biochem behav. 2010;94(3):464–470. 83. iwabuchi k, kubota y, ito c, watanabe t, yanai k. methamphetamine and brain histamine: a study using histamine-related gene knockout mice. ann n y acad sci. 2004;1025:129–134. 84. munzar p, nosal r, goldberg sr. potentiation of the discriminative-stimulus effects of methamphetamine by the histamine h3 receptor antagonist thioperamide in rats. eur j pharmacol. 1998;363(2–3):93–101. 85. munzar p, tanda g, justinova z, goldberg sr. histamine h3 receptor antagonists potentiate methamphetamine self-administration and methamphetamine induced accumbal dopamine release. neuropsychopharmacology. 2004;29(4): 705–717. 86. kitanaka j, kitanaka n, hall fs, et al. histamine h3 receptor agonists decrease hypothalamic histamine levels and increase stereotypical biting in mice challenged with methamphetamine. neurochem res. 2011;36(10):1824–1833. 87. kitanaka j, kitanaka n, takemura m. [methamphetamine-induced brain pathology and central histaminergic system] (in japanese). brain21 (nou21). 2015;18(2):83–88. 88. fox gb, esbenshade ta, pan jb, et al. pharmacological properties of abt-239 [4-(2-{2-[(2r)-2-methylpyrrolidinyl]ethyl}-benzofuran-5-yl)benzonitrile]: ii. neurophysiological characterization and broad preclinical efficacy in cognition and schizophrenia of a potent and selective histamine h3 receptor antagonist. j pharmacol exp ther. 2005;313(1):176–190. 89. sakai n, onodera k, maeyama k, yanai k, watanabe t. effects of (s)-alphafluoromethylhistidine and metoprine on locomotor activity and brain histamine content in mice. life sci. 1992;51(6):397–405. 90. samotaeva is, birioukova lm, midzyanovskaya is, kuznetsova gd, bazyan as, tuomisto l. metoprine induced behavioral modifications and brain regional histamine increase in wag/rij and wistar rats. epilepsy res. 2012;101(1–2):148–156. 91. bristow lj, bennett gw. biphasic effects of intra-accumbens histamine administration on spontaneous motor activity in the rat; a role for central histamine receptors. br j pharmacol. 1988;95(4):1292–1302. http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 treatment for methamphetamine overdose 7drug target insights 2016:10 92. chiavegatto s, nasello ag, bernardi mm. histamine and spontaneous motor activity: biphasic changes, receptors involved and participation of the striatal dopamine system. life sci. 1998;62(20):1875–1888. 93. tuomisto l, tacke u, willman a. inhibition of sound-induced convulsions by metoprine in the audiogenic seizure susceptible rat. agents actions. 1987;20(3–4): 252–254. 94. kakinoki h, ishizawa k, fukunaga m, fujii y, kamei c. the effects of histamine h3-receptor antagonists on amygdaloid kindled seizures in rats. brain res bull. 1998;46(5):461–465. 95. castellan baldan l, williams ka, gallezot jd, et al. histidine decarboxylase deficiency causes tourette syndrome: parallel findings in humans and mice. neuron. 2014;81(1):77–90. 96. arrang jm, garbarg m, schwartz jc. autoregulation of histamine release in brain by presynaptic h3-receptors. neuroscience. 1985;15(2):553–562. 97. ishizuka t, sakamoto y, sakurai t, yamatodani a. modafinil increases histamine release in the anterior hypothalamus of rats. neurosci lett. 2003;339(2):143–146. http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 dti drug target insights 2021; 15: 13-20issn 1177-3928 | doi: 10.33393/dti.2021.2342review drug target insights issn 1177-3928 www.aboutscience.eu/dti © 2021 the authors. this article is published by aboutscience and licensed under creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). commercial use is not permitted and is subject to publisher’s permissions. full information is available at www.aboutscience.eu the role of patient preferences in adherence to treatment in chronic disease: a narrative review serena losi1, cesare celeste federico berra2, riccardo fornengo3, dario pitocco4, giovanni biricolti5, marco orsini federici1 1eli lilly italy s.p.a., sesto fiorentino italy 2irccs multimedica, sesto san giovanni, milano italy 3s.s.d. di diabetologia asl to4, torino italy 4diabetes care unit fondazione policlinico universitario agostino gemelli irccs, roma italy 5eli lilly italy s.p.a., roma italy abstract adherence to prescribed medication is important to the management of all diseases, especially those of chronic nature. drug effectiveness is substantially compromised by therapy nonadherence. we reviewed the available evidences on the impact of patient preferences for therapy on adherence to a prescribed treatment in chronic diseases requiring long-term treatment. a search on pubmed retrieved 699 publications, leading to a selection of 12 publications: 6 on osteoporosis, 2 on moderate-to-severe asthma, 1 on type 1 diabetes, 1 on type 2 diabetes, 1 on kidney transplantation, and 1 on atrial fibrillation. overall, 8 studies found a positive association between patient preference and adherence to therapy, while the others found no association. in general, overall adherence was considered to be high in the published studies. the reasons for a positive association included reduced dosing frequency, route of administration, lower costs, and favorable safety profile, which is related to the diverse nature of the pathology and its type and duration of treatment. a literature review suggests that achieving good adherence and persistence to therapy requires evaluation of patient preferences. in a period of increasingly limited resources, more effort is warranted to promote better adherence to therapy, especially when patients must self-manage their disease in the long term. our results further highlight that insufficient attention has been given to the relationship between patient preference and adherence and point out the complex nature of adherence and the need for adequate patient education. more efforts are also needed to better understand the entity of cost savings for payers for specific treatments and the link with patient preference. keywords: adherence, chronic disease, preferences, therapy received: september 24, 2021 accepted: october 20, 2021 published online: november 08, 2021 corresponding author: serena losi eli lilly italia s.p.a. via a. gramsci 731/733 50019 sesto fiorentino (fi) italy losi_serena@lilly.com since disease-related symptoms are often absent, and because long-term drug effectiveness is substantially compromised by nonadherence to therapy. according to a report from the world health organization (who), adherence to long-term therapy is a problem of global magnitude and averages about 50% in developed countries (2). moreover, poor adherence is associated with poorer outcomes and increased costs of care (2). it has also been hypothesized that increasing adherence to medications may have a greater impact on a population’s health than further improvements in medical therapies (3). poor adherence decreases the effectiveness of therapy and leads to suboptimal use of resources as undertreated patients tend to develop complications and comorbidities (4,5). in type 2 diabetes and hypertension, poor adherence rates may result in poorer health outcomes and increased mortality (4-8). several studies in chronic conditions have also demonstrated that poor adherence is associated with adverse consequences in terms of risk of hospitalization and overall costs (9). for diabetes, hypercholesterolemia, and hypertension, savings in all-cause medical costs have been reported when levels of adherence are high (10). introduction patient preference to any prescribed medication or therapy is assuming an increasingly important impact in achieving clinically relevant outcomes. for example, the american diabetes association (ada) and the european association for the study of diabetes (easd) 2018 consensus report for type 2 diabetes emphasizes that patient choices and preferences are of utmost importance when choosing a therapy (1). adherence to prescribed medication is important to the management of all diseases, and especially those of a chronic nature such as diabetes, dyslipidemia, and hypertension https://doi.org/10.33393/dti.2021.2342 https://creativecommons.org/licenses/by-nc/4.0/legalcode patient preferences and adherence in chronic diseases14 © 2021 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti lack of adherence is influenced by multiple factors that include psychological issues, polypharmacy, and ease of obtaining and administering the medication, as well as patient motivation and education (11). several studies on patient preferences for a variety of therapies have suggested that increased patient preference for one therapy over another is likely to be associated with increased adherence to therapy (7,11,12). however, only a limited number of studies have measured the potential impact of patient preferences on adherence, meaning that both dimensions have been quantified in the same study. with mounting attention to overall costs of healthcare, increasing weight is being given to cost-effectiveness of any given treatment. indeed, it has been suggested that patient preferences for treatment may have significant implications for cost-effectiveness by affecting not only costs but also clinical outcomes (13). the impact of adherence and its potential importance in both clinical and economic terms can be highlighted by considering data from italy where only 31% of patients with chronic diseases refer that they are completely adherent to therapy (14). accordingly, payers should implement cost-effectiveness models that incorporate patient preferences (13). based on all these considerations, we performed a literature search and narrative review with the objective of overviewing the available evidence on the impact of patient preferences for therapy on adherence to a prescribed treatment in diseases requiring long-term treatment to help improve decision making processes for physicians and payers. focus was placed on chronic diseases, since outcomes are likely to be more linked to adherence to therapy in the long term. materials and methods overall strategy the methods, including the search strategy, were developed based on relevant literature (15-18). a narrative literature review (19) of published studies assessing compliance, adherence, or persistence and treatment preferences from 2010 (inclusive) to april 20, 2021, was conducted using the pubmed-ncbi database. the following search string was used: (“patient preference”[mesh] or preferen*[tiab]) and (adherence[tiab] or compliance[tiab] or persistence[tiab] or concordance[tiab]) and (“chronic disease”[mesh] or diabetes or copd or asthma or “chronic obstructive pulmonary disease” or hypertension or osteoporosis or “chronic disease” or “chronic condition”) with the exception of medical subject headings (mesh) terms associated with each article, to maximize the retrieval of relevant articles, keywords were searched only within the title and abstract. to increase sensitivity, the keyword “preference” was truncated to “preferen” and followed by the special operator “*” (e.g., to catch both preference and preferential). further truncation did not improve sensitivity and was associated with a drastic deterioration in specificity of articles retrieved. similar tests were carried out for all the other keywords. while the search priority was the association between patient preferences and medication adherence, associations between patient preferences and clinical efficacy were included if relevant. inclusion and exclusion criteria all studies that involved human subjects of any age with one or more chronic diseases were considered. focus was given to the centers for disease control and prevention (cdc) definition of chronic disease as those lasting more than 1 year and which require ongoing medical attention, limit activities of daily living, or both (20), with the exclusion of cancer. interventions of interest included those related to pharmacological treatment, with or without associated use of medical devices. all studies published in english language including a clear method of how patient preference and medication adherence or persistence (or efficacy) were objectively measured were included. editorials, review articles, surveys, opinion papers, letters to the editor, case reports or case studies, study protocols, and guidelines were excluded. publications describing adherence only, preferences only, or those mentioning only self-management behaviors, but not medication adherence, were also excluded, as were speculative articles where patient preference was not directly assessed. search results were imported into microsoft excel. two reviewers were responsible for data extraction. a two-part study selection process was used: title and abstract review followed by full-text review. in the first step, two reviewers separately review the title and abstract of citations from the search to determine the eligibility based on the inclusion and exclusion criteria. all articles considered relevant by either reviewer were included in the full-text evaluation where the two reviewers independently evaluated the full-text articles to determine if they met inclusion/exclusion criteria. in case of disagreement about inclusion, full-text articles were reviewed again by both reviewers and if agreement was not reached, this was resolved by consultation with an independent third reviewer. as this is a narrative review, no statistical analyses were performed. results the initial literature search on pubmed retrieved 699 publications. despite the structured query, the majority of extracted papers were not relevant to the topic of interest. analysis led to a selection of 12 publications. overall, 275 papers were excluded because they did not consider patients’ treatment or device preferences or a specific chronic disease involved; 265 due to article type (e.g., review/editorial); 71 investigated only dietary patterns, exercise, or nonpharmacological devices (e.g., mandibular advancement device or mobile apps); 56 because they explored only patient preferences without quantitative measurements of actual adherence; 12 investigated the treatment decision-making style adopted and not an actual preference for treatment; 6 because adherence of one treatment option was not assessed or close to 100% by study design (e.g., a single injection performed at study enrollment), making pointless any possible comparison with other treatments considered; and 2 because they were related to a study already included (two publications by kendler (21,22) were excluded as they refer to the one by freemantle with final results of the study (23) which was included). the selected studies, listed in table i, included 6 papers on osteoporosis, 2 on moderate-to-severe asthma, 1 on type losi et al drug target insights 2021; 15: 15 © 2021 the authors. published by aboutscience www.aboutscience.eu table i studies included on patient preference and adherence to therapy author disease main aim study design no. patients main finding eliasaf et al. 2016 (25) osteoporosis determine compliance and persistence with osteoporosis therapy among postmenopausal women and to assess attitudes regarding treatment resumption among patients on drug holiday. prospective observational study 150 compliance was high overall (80%); there was not a preferred medication among patients on drug holiday. freemantle et al. 2012 (23) osteoporosis compare treatment adherence between subcutaneous denosumab every 6 months and oral alendronate once weekly. 2-year, randomized, crossover study 250 of 198 subjects expressing treatment preference, 92.4% preferred injectable denosumab over oral alendronate. denosumab was associated with less nonadherence than alendronate (first year, 11.9% vs. 23.4%; second year, 7.5% vs. 36.5%). jarab et al. 2020 (24) osteoporosis explore factors associated with medication nonadherence in jordan. observational 296 72.3% were nonadherent; patients were less likely to adhere to the prescribed medications with each unit increase in the number of prescribed medications and if they did not have a trust in the efficacy of the medications. oral et al. 2015 (27) osteoporosis examine the level of compliance and persistence in patients with postmenopausal osteoporosis receiving daily risedronate with either fixed dosing of three different timing regimens (a: before breakfast; b: in-between meals; c: before bedtime) or with flexible dosing. randomized, crossover study 448 49.7% preferred flexible and 50.3% fixed timing; a significant difference between the flexible and fixed regimens was seen in persistence in favor of the flexible regimen. persistence was defined as the continuation of treatment at week 26. sakai et al. 2014 (26) osteoporosis evaluate the effects of once-monthly minodronate on treatment persistence and clinical parameters in outpatients previously treated with daily or weekly bisphosphonate products. multicenter, prospective, open-label, observational study 264 and 133 patients were allocated into the switch and continue groups (continue daily or weekly bisphosphonates). approximately 65% of patients were willing to switch to minodronate, with the predominant reason being “less frequent dosing more convenient.” treatment persistence was significantly higher in the switch group. persistence was assessed through kaplan-meier curves and analyzed using the log-rank test. thomasius et al. 2016 (28) osteoporosis compared the preference, acceptability, and tolerability of a reformulation of calcichew d31 500 mg/400 iu and calcichew d3 500 mg/800 iu with adcal-d32 500 mg/400 iu and kalcipos-d 500 mg/800 iu. phase iv, randomized, open-label, two-period, cross-over study 276 patients preferred calcichew d3 500/400 and calcichew d3 500/800 over comparators as it is significantly less chalky and sticky, and is easier to chew and swallow. acceptability did not affect compliance. al hayek et al. 2020 (31) type 1 diabetes compare preferences and adherence for 6-mm and 8-mm injection needles. prospective cohort study 62 6-mm needles were associated with lower pain score, higher patient adherence, greater patient satisfaction, and better glycemic control compared to 8-mm needles. (continued) patient preferences and adherence in chronic diseases16 © 2021 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti 1 diabetes, 1 on type 2 diabetes, 1 on atrial fibrillation, and 1 in patients with a stable kidney transplant. overall, 6 studies found a positive association between patient preference and adherence to therapy, while the others found no association. in general, overall adherence was considered to be high in the published studies. an exception was the study by jarab et al in which 72% of patients were nonadherent; patients were less likely to adhere to therapy with each increase in the number of medications and when they did not trust the efficacy (24). in the study by eliasaf et al, 80% of patients took their medication as directed (64% were on an oral medication, mostly bisphosphonates). however, in comparing treatments for osteoporosis, there was no preferred medication among patients on drug holiday (25). freemantle carried out a 2-year randomized, crossover trial comparing subcutaneous denosumab every 6 months to oral alendronate once weekly, and reported that denosumab was associated with less nonadherence than alendronate in both years of the trial (first year, 11.9% vs. 23.4%; second year, 7.5% vs. 36.5%) (23). this greater adherence to denosumab was likely related to the greater preference with injections every 6 months rather than an oral drug weekly. in considering dosing frequency, sakai et al similarly reported that more patients were willing to switch to a weekly bisphosphonate, rather than continuing to receive daily administration, with the predominate reason that less frequent dosing is more convenient. furthermore, they observed that treatment persistence was significantly higher in the switch group than the continue group (89.8% author disease main aim study design no. patients main finding ishii et al. 2018 (32) type 2 diabetes compare the treatment satisfaction of four classes of oral agents: dpp-4 inhibitors, α-glucosidase inhibitors, biguanides, and sulfonylureas. 12-week, randomized, controlled, open-label study 64 dpp-4 inhibitor was the most preferable option in terms of treatment satisfaction and had the highest adherence. plaza et al. 2018 (29) moderate to severe asthma assess the impact of patient satisfaction with an inhaler on adherence and health outcomes. crosssectional, observational, multicenter study 778 high specific satisfaction with an inhaler was associated with younger age, male gender, controlled asthma, high general satisfaction with treatment, high adherence to inhaler, nonsevere asthma, and no trouble with inhaler use. valero et al. 2019 (30) moderate to severe asthma compare patient satisfaction of three dry powder inhalers. register of an observational, multicenter study 328 specific satisfaction with inhaler was significantly higher with easyhaler™, as well as the percentage of patients with high satisfaction with inhaler. scores for easyhaler™ were also significantly better for items such as learning how to use, inhaler preparation, inhaler use, weight and size, and portability. there were no significant differences in asthma control or adherence between inhalers. wu et al. 2019 (33) atrial fibrillation compare persistence and outcomes of non-vitamin k antagonist oral anticoagulants (noacs) vs. warfarin. prospective cohort study 344 persistence with anticoagulants was low and dropped sharply at the third month; patients on noacs had worse persistence at 3, 6, and 12 months than those on warfarin; the main reason for anticoagulant cessation was patient preference (adverse events, costs). hugo et al. 2021 (34) kidney transplantation evaluate conversion from immediate-release tacrolimus (ir-t) to prolonged-release tacrolimus (pr-t) in stable kidney transplant recipients. 12-month, noninterventional study 183 among patients reporting a preference, 78.4% preferred pr-t. following conversion from ir-t to pr-t adherence was high and kidney function was stable over 12 months. table i (continued) losi et al drug target insights 2021; 15: 17 © 2021 the authors. published by aboutscience www.aboutscience.eu vs. 78.9%; p<0.003) (26). oral et al compared flexible to fixed dosing regimens in women receiving daily risedronate (27). while there was no difference in preference of the two regimens, a significant difference was seen, with treatment persistence favoring the flexible regimen. lastly, the trial by thomasius compared preferences of a reformulated vitamin d/calcium supplement (28). while patients preferred a formulation that was less chalky and sticky, and easier to chew and swallow, acceptability had no effect on compliance. two of the remaining studies investigated the use of inhalers in patients with moderate to severe asthma. the trial by plaza et al found that high patient satisfaction with an inhaler, independently of the medication contained within, was associated with better adherence and, accordingly, better control of asthma (29). as in the study by valero et al, patients preferred an inhaler that was easy to use and easy to learn to use (30). the study in type 1 diabetes by al hayek et al evaluated 6-mm vs. 8-mm injection needles in terms of adherence, satisfaction, and glycemic control (31). it was reported that the narrower needle was associated with greater satisfaction, better adherence, and improved glycemic control compared to the high-gauge needle. the trial by ishii et al examined treatment satisfaction of commonly used oral treatments, reporting that dpp-4 inhibitors were preferred over α-glucosidase inhibitors, biguanides, or sulfonylureas (32). dpp-4 inhibitors were also associated with better adherence to therapy vs. α-glucosidase inhibitors, biguanides, or sulfonylureas (93%, 87%, 64%, and 62%, respectively). the authors concluded that the higher treatment satisfaction of patients can motivate therapeutic adherence, likely resulting in better glycemic control (32). wu et al compared persistence and outcomes of non-vitamin k antagonist oral anticoagulants (noacs) to warfarin. patients on noacs were seen to have worse persistence at 3, 6, and 12 months than those on warfarin; the main reasons for anticoagulant discontinuation cited were related to patient preference such as adverse bleeding events and costs (33). lastly, hugo et al examined the effects of converting patients with a stable kidney graft from immediate-release tacrolimus (ir-t) to prolonged-release tacrolimus (pr-t) (34). over a period of 12 months, there was no change in renal function, adherence was high; 98% of patients referred that they were satisfied or very satisfied with the therapy, while 78% preferred pr-t. discussion herein, we performed a literature search yielding 12 publications in order to overview the available investigations on patient preferences and adherence to therapy for chronic diseases. of note, there were more studies on osteoporosis (n = 6) compared to other chronic diseases, although this may possibly be explained that during the time of these studies more costly injection therapies were beginning to replace more consolidated treatments. the majority (8/12) of the studies in the present review reported a positive association between patient preference and adherence to therapy. the reasons for a positive association included reduced dosing frequency, route and means of administration, lower costs, and a more favorable safety profile. these factors may be related to the diverse nature of the pathology and its treatment. four of the studies did not report a direct association between patient preference and adherence to treatment, although this can likely be explained by factors related to the individual study. considering the studies on osteoporosis, that by eliasaf et al reported that their study included highly motivated patients, that compliance was higher than that previously reported in the literature, and that patients on drug holiday did not have a preference for medication (25). all these factors may have had a role in the lack of significant differences. the trial by thomasius et al found that while patients clearly had a preferred formulation, acceptability did not influence compliance to therapy (28). this result could be attributed to the short-term nature of that study, which followed patients for only 30 days. oral et al, instead, observed no difference in preference of the two regimens, and thus an association between preference and adherence cannot be assessed (31). moreover, regarding the two studies on inhaler preference for moderate to severe asthma, the study by plaza et al found a positive association between inhaler satisfaction with adherence, while that by valero et al found no such association (29,30). however, those authors commented that due to the sample size in the subanalysis performed, the difference in patient satisfaction was not adequate to properly reflect differences in adherence and control of asthma. in the trial included in the present analysis on type 2 diabetes, dpp-4 inhibitors were considered to provide greater satisfaction with treatment, possibly because of the less frequent dosing and less concern over adverse events compared to other treatments; such factors likely motivate patients to better adherence to therapy and lead to superior glycemic control (32). moreover, since drug reimbursements were not completely covered by the healthcare system in which the study was carried out, the cost of dpp-4 inhibitors appeared to be a concern for some patients since management of type 2 diabetes is lifelong, thus highlighting the important role of patient preference for therapy (32). another recent study in patients with type 2 diabetes on oral treatment showed that the vast majority still prefer a daily oral simple therapy, but the second choice was for weekly injection with a ready to use device (35). in the study included in type 1 diabetes on needle preferences, a smaller gauge needle was associated with greater satisfaction in terms of injection comfort and pain as well as greater overall satisfaction. these preferences led not only to greater adherence to therapy but also to significantly fewer hypoglycemic episodes per month and to significantly lower glycated hemoglobin (7.9% vs. 8.3%) (31). in the study on atrial fibrillation by wu et al, persistence to therapy with anticoagulants was strongly influenced by costs, as well as with adverse events to treatment (33). indeed, patients prescribed noacs had worse persistence than those given warfarin and the study was carried out in china where, as noted by the authors, noacs are approximately 80 times more expensive than warfarin, which influences not only preference, but the ability to acquire the drug. in that patient preferences and adherence in chronic diseases18 © 2021 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti investigation, there was no difference in adverse events between noacs and warfarin. among the studies on osteoporosis, freemantle et al reported greater adherence and preference with a single subcutaneous injection every 6 months vs. an oral treatment once weekly (23). moreover, patients crossing over to weekly oral therapy had poorer adherence after the switch, suggesting a treatment sequence effect. the differences in adherence and preference are likely to be related to multiple factors such as frequency of administration, belief in the need for and efficacy of individual therapies, and duration of treatment. the impact of different dosing regimens in osteoporosis was explored by oral et al who reported that while there was no actual preference for a fixed or flexible dosing regimen, the latter was associated with significantly higher persistence to therapy (27). overall persistence levels with flexible dosing were 86.0% compared to 78.9% with a fixed regimen. it was speculated that alternate timing for administration of therapy might aid patients with difficulty following traditional before-breakfast dosing, thus offering an additional option that can be more easily incorporated into diverse lifestyles and needs. the importance of timing and frequency of administration can be further highlighted in the study by hugo et al in kidney transplant patients who preferred prolonged-release tacrolimus over an immediaterelease formulation (34). moreover, pr-t was also easier to remember that ir-t, with the main reasons cited for preferring the prolonged-release formulation being no need to take it in the evening and reduced pill burden. in the study by jarab et al carried out in jordan on osteoporosis, adherence was dismal, and 72% of women were nonadherent to therapy (24). similarly low rates of adherence in osteoporosis were also reported in a study from the us where nonadherents were 70% at 1 year, and 84% at 3 years (36). increased number of medications was a primary reason for nonadherence in the study by jarab et al, although it should be mentioned that patients were taking an average of five medications, three of which were for osteoporosis. this highlights the need to simplify the overall therapeutic regimen. the study also reported that lack of trust in efficacy was a major motivator for nonadherence, which stresses the need for patient education and establish a good physicianpatient relationship. the manifestation of medical and psychological complications of any disease worsens the quality of life and leads to inefficient use of resources. taken together, the consequences of poor adherence compromise the possibility that a healthcare system can fulfill the needs of patients. the problem of adherence to therapy occurs whenever self-administration of the treatment is required, regardless of the type and severity of the illness and the possibility of access to treatment. while the problem may seem simple, poor adherence is multifactorial and is related to factors related to the patient, its treatment, and the disease (2,11). for example, patients with diabetes generally have other comorbidities such as hypertension, obesity, and depression, which may contribute to a less than adequate response to therapy (37). costs also increase 2.2-3.2 times when the patient develops microand macrovascular complications that could be prevented (38). the costs of hospitalization, which include treatment of longterm complications such as heart disease, can account for more than 50% of total costs (38). thus, economic and social benefits will become substantial only if the healthcare system can achieve a greater level of efficiency in promoting adherence to self-management of chronic disease. the ongoing challenge is demonstrated from a study in italy, wherein 45% of patients with a chronic disease indicated that they did not understand their disease and were not able to self-manage it; only 31% declared that they were completely adherent to therapy and over 50% of patients said they had thought about abandoning care for their disease (14). this suggests that in order to achieve good adherence and persistence, evaluation of patient preferences is a crucial step. a study conducted in italy on preference toward different therapeutic options in injection-naïve and -non-naïve patients with type 2 diabetes clearly showed preference for simple oral therapies and with a low risk of side effects to therapy in injection-naïve subjects (39). the situation dramatically changed in patients who had already experienced injection therapy, who preferred that their therapy be administered with a ready to use device over the possibility of going back to oral therapy. moreover, when considering all the different therapeutic attributes, among all patients the most preferred option was for a weekly injectable therapy with a ready to use device, while the first oral daily therapy ranked fifth (39). the who has classified barriers to adherence into five dimensions: healthcare team/system, therapy, condition, patient, and socioeconomic-related barriers (2). better understanding of the barriers to adherence is needed to overcome them and increase therapeutic outcomes in chronic disease. in the past, less emphasis was placed on adherence, but this paradigm seems to have been gradually changing over the years; this may be related to the aging population and increased prevalence of chronic disease. considering other dimensions of the who classification, individual patient characteristics such as age and level of education may be related to adherence, in addition to factors such as costs depending on the specific setting. thus, despite the somewhat limited evidence to date, it should be assumed that patient preference has an impact on adherence. the present analysis has some limitations. firstly, we considered only a single database and it is possible that additional studies were not retrieved from the literature search. secondly, the inclusion criteria were very strict, with the result that only a small number of publications were included. lastly, four studies included considered patient satisfaction as a proxy for preference. although a higher satisfaction among treatments will likely result in a preference, this was not directly assessed in those cases. conclusion our results highlight that insufficient attention has been given to studying the direct relationship between patient preference and adherence, but seem to confirm its existence. it is hoped that this review can serve as a stimulus for further research in this little explored area, which could losi et al drug target insights 2021; 15: 19 © 2021 the authors. published by aboutscience www.aboutscience.eu help to better understand patient needs and desires, with the overarching aim of improving adherence to treatment in chronic diseases, understand the impact on total costs of treatment, and therefore achieve better outcomes. this review also stresses the complex nature of adherence, and the need for adequate patient education so that they understand the benefits of therapy for their particular condition. costs are undoubtedly important when considering any treatment for a chronic disease, and more efforts are needed to better understand the entity of cost savings for payers for specific treatments and the link with patient preference. if a patient prefers a certain treatment over another, adherence is likely to increase along with better allocation of resources. acknowledgments matteo mucchetti and patrick moore provided writing and editorial assistance for this manuscript on behalf of health publishing & services srl. disclosure dr. c. c. berra in 2019 participated in scientific boards sponsored by: eli lilly, astra zeneca, novo nordisk, boehringer, mundipharma, sanofi; conduced clinical studies sponsored by: eli lilly, novo nordisk, sofar; and participated in sponsored lectures at national conferences by: boehringer, eli lilly, sanofi, novo nordisk. dr. riccardo fornengo declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. prof. d. pitocco declares no conflict of interest. marco orsini federici, serena losi and giovanni biricolti are employees of eli lilly italia spa and minor stockholders at eli lilly & company. references 1. davies mj, d’alessio da, fradkin j, et al. management of hyperglycemia in type 2 diabetes, 2018. a consensus report by the american diabetes association (ada) and the european association for the study of diabetes (easd). diabetes care. 2018;41(12):2669-2701. crossref pubmed 2. world health organization. adherence to long-term therapies: evidence for action. online. accessed 7 july 7, 2020. 3. haynes rb, mcdonald h, garg ax, montague p. interventions for helping patients to follow prescriptions for medications. cochrane database syst rev. 2002;(2):cd000011. pubmed 4. egede le, gebregziabher m, echols c, lynch cp. longitudinal effects of medication nonadherence on glycemic control. ann pharmacother. 2014;48(5):562-570. crossref pubmed 5. martin lr, williams sl, haskard kb, dimatteo mr. the challenge of patient adherence. ther clin risk manag. 2005;1(3):189-199. pubmed 6. cramer ja. a systematic review of adherence with medications for diabetes. diabetes care. 2004;27(5):1218-1224. crossref pubmed 7. ferdinand kc, yadav k, nasser sa, et al. disparities in hypertension and cardiovascular disease in blacks: the critical role of medication adherence. j clin hypertens (greenwich). 2017;19(10): 1015-1024. crossref pubmed 8. ho pm, rumsfeld js, masoudi fa, et al. effect of medication nonadherence on hospitalization and mortality among patients with diabetes mellitus. arch intern med. 2006;166(17): 1836-1841. crossref pubmed 9. boye ks, curtis se, lage mj, garcia-perez le. associations between adherence and outcomes among older, type 2 diabetes patients: evidence from a medicare supplemental database. patient prefer adherence. 2016;10:1573-1581. crossref pubmed 10. sokol mc, mcguigan ka, verbrugge rr, epstein rs. impact of medication adherence on hospitalization risk and healthcare cost. med care. 2005;43(6):521-530. crossref pubmed 11. tiktin m, celik s, berard l. understanding adherence to medications in type 2 diabetes care and clinical trials to overcome barriers: a narrative review. curr med res opin. 2016;32(2):277-287. crossref pubmed 12. gelhorn hl, sexton cc, classi pm. patient preferences for treatment of major depressive disorder and the impact on health outcomes: a systematic review. prim care companion cns disord. 2011;13(5):pcc.11r01161. crossref pubmed 13. brazier je, dixon s, ratcliffe j. the role of patient preferences in cost-effectiveness analysis: a conflict of values? pharmacoeconomics. 2009;27(9):705-712. crossref pubmed 14. gentile s, strollo f. aderenza e semplicità: quale ruolo nella terapia con glp1-ra. collana editoriale associazione medici diabetologi. online. accessed november 10, 2020. 15. green bn, johnson cd, adams a. writing narrative literature reviews for peer-reviewed journals: secrets of the trade. j chiropr med. 2006;5(3):101-117. crossref pubmed 16. ledade sd, jain sn, darji aa, gupta vh. narrative writing: effective ways and best practices. perspect clin res. 2017;8(2): 58-62. crossref pubmed 17. pautasso m. ten simple rules for writing a literature review. plos comput biol. 2013;9(7):e1003149. crossref pubmed 18. van hoorn r, kievit w, booth a, et al. the development of pubmed search strategies for patient preferences for treatment outcomes. bmc med res methodol. 2016;16(1):88. crossref pubmed 19. siddaway ap, wood am, hedges lv. how to do a systematic review: a best practice guide for conducting and reporting narrative reviews, meta-analyses, and meta-syntheses. annu rev psychol. 2019;70(1):747-770. crossref pubmed 20. centers for disease control and prevention. about chronic diseases. online. accessed november 10, 2020. 21. kendler dl, macarios d, lillestol mj, et al. influence of patient perceptions and preferences for osteoporosis medication on adherence behavior in the denosumab adherence preference satisfaction study. menopause. 2014;21(1):25-32. crossref pubmed 22. kendler dl, mcclung mr, freemantle n, et al; daps investigators. adherence, preference, and satisfaction of postmenopausal women taking denosumab or alendronate. osteoporos int. 2011;22(6):1725-1735. crossref pubmed 23. freemantle n, satram-hoang s, tang et, et al; daps investigators. final results of the daps (denosumab adherence preference satisfaction) study: a 24-month, randomized, crossover comparison with alendronate in postmenopausal women. osteoporos int. 2012;23(1):317-326. crossref pubmed 24. jarab as, mukattash tl, hilan h. medication non-adherence in patients with osteoporosis: implications for clinical pharmacists and osteoporosis care providers. curr clin pharmacol. 2020;15(3):243-250. crossref pubmed 25. eliasaf a, amitai a, maram edry m, yosselson superstine s, rotman pikielny p. compliance, persistence, and preferences regarding osteoporosis treatment during active therapy or drug holiday. j clin pharmacol. 2016;56(11):1416-1422. crossref pubmed 26. sakai a, ikeda s, okimoto n, et al. clinical efficacy and treatment persistence of monthly minodronate for osteoporotic patients unsatisfied with, and shifted from, daily or weekly https://doi.org/10.2337/dci18-0033 https://www.ncbi.nlm.nih.gov/pubmed/30291106 https://www.who.int/chp/knowledge/publications/adherence_full_report.pdf?ua=1 https://www.ncbi.nlm.nih.gov/pubmed/12076376 https://doi.org/10.1177/1060028014526362 https://www.ncbi.nlm.nih.gov/pubmed/24586059 https://www.ncbi.nlm.nih.gov/pubmed/18360559 https://doi.org/10.2337/diacare.27.5.1218 https://www.ncbi.nlm.nih.gov/pubmed/15111553 https://doi.org/10.1111/jch.13089 https://www.ncbi.nlm.nih.gov/pubmed/28856834 https://doi.org/10.1001/archinte.166.17.1836 https://www.ncbi.nlm.nih.gov/pubmed/17000939 https://doi.org/10.2147/ppa.s107543 https://www.ncbi.nlm.nih.gov/pubmed/27574406 https://doi.org/10.1097/01.mlr.0000163641.86870.af https://www.ncbi.nlm.nih.gov/pubmed/15908846 https://doi.org/10.1185/03007995.2015.1119677 https://www.ncbi.nlm.nih.gov/pubmed/26565758 https://doi.org/10.4088/pcc.11r01161 https://www.ncbi.nlm.nih.gov/pubmed/22295273 https://doi.org/10.2165/11314840-000000000-00000 https://www.ncbi.nlm.nih.gov/pubmed/19757864 https://www.pacinimedicina.it/prodotto/aderenza-e-semplicita-quale-ruolo-nella-terapia-con-glp1-ra/ https://doi.org/10.1016/s0899-3467(07)60142-6 https://www.ncbi.nlm.nih.gov/pubmed/19674681 https://doi.org/10.4103/2229-3485.203044 https://www.ncbi.nlm.nih.gov/pubmed/28447014 https://doi.org/10.1371/journal.pcbi.1003149 https://www.ncbi.nlm.nih.gov/pubmed/23874189 https://doi.org/10.1186/s12874-016-0192-5 https://www.ncbi.nlm.nih.gov/pubmed/27473226 https://doi.org/10.1146/annurev-psych-010418-102803 https://www.ncbi.nlm.nih.gov/pubmed/30089228 https://www.cdc.gov/chronicdisease/about/index.htm https://doi.org/10.1097/gme.0b013e31828f5e5d https://www.ncbi.nlm.nih.gov/pubmed/23676636 https://doi.org/10.1007/s00198-010-1378-z https://www.ncbi.nlm.nih.gov/pubmed/20827547 https://doi.org/10.1007/s00198-011-1780-1 https://www.ncbi.nlm.nih.gov/pubmed/21927922 https://doi.org/10.2174/1574884715666200116104754 https://www.ncbi.nlm.nih.gov/pubmed/31951176 https://doi.org/10.1002/jcph.738 https://www.ncbi.nlm.nih.gov/pubmed/26999526 patient preferences and adherence in chronic diseases20 © 2021 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti bisphosphonates: the bp-musashi study. osteoporos int. 2014;25(9):2245-2253. crossref pubmed 27. oral a, lorenc r; flint-act study investigators. compliance, persistence, and preference outcomes of postmenopausal osteoporotic women receiving a flexible or fixed regimen of daily risedronate: a multicenter, prospective, parallel group study. acta orthop traumatol turc. 2015;49(1):67-74. crossref pubmed 28. thomasius f, keung nip t, ivan p. phase iv randomized preference study in patients eligible for calcium and vitamin d supplementation. curr med res opin. 2016;32(10):1623-1631. crossref pubmed 29. plaza v, giner j, calle m, et al. impact of patient satisfaction with his or her inhaler on adherence and asthma control. allergy asthma proc. 2018;39(6):437-444. crossref pubmed 30. valero a, ribó p, maíz l, et al. asthma patient satisfaction with different dry powder inhalers. expert rev respir med. 2019; 13(2):133-138. crossref pubmed 31. al hayek aa, al dawish m. evaluating the user preference and level of insulin self-administration adherence in young patients with type 1 diabetes: experience with two insulin pen needle lengths. cureus. 2020;12(6):e8673. crossref pubmed 32. ishii h, hayashino y, akai y, yabuta m, tsujii s. dipeptidyl peptidase-4 inhibitors as preferable oral hypoglycemic agents in terms of treatment satisfaction: results from a multicenter, 12-week, open label, randomized controlled study in japan (preference 4 study). j diabetes investig. 2018;9(1):137-145. crossref pubmed 33. wu s, xie s, xu y, et al. persistence and outcomes of nonvitamin k antagonist oral anticoagulants versus warfarin in patients with non-valvular atrial fibrillation. j clin nurs. 2019; 28(9-10):1839-1846. crossref pubmed 34. hugo c, weihprecht h, banas b, et al. renal function and patient-reported outcomes in stable kidney transplant patients following conversion from twice-daily immediate-release tacrolimus to once-daily prolonged-release tacrolimus: a 12-month observational study in routine clinical practice in germany (adagio). transplant proc. 2021;53(5):1484-1493. crossref pubmed 35. boye k, ross m, mody r, konig m, gelhorn h. patients’ preferences for once-daily oral versus once-weekly injectable diabetes medications: the revise study. diabetes obes metab. 2021;23(2):508-519. crossref pubmed 36. weycker d, macarios d, edelsberg j, oster g. compliance with drug therapy for postmenopausal osteoporosis. osteoporos int. 2006;17(11):1645-1652. crossref pubmed 37. ciechanowski ps, katon wj, russo je. depression and diabetes: impact of depressive symptoms on adherence, function, and costs. arch intern med. 2000;160(21):3278-3285. crossref pubmed 38. bruno g, picariello r, petrelli a, et al. direct costs in diabetic and non diabetic people: the population-based turin study, italy. nutr metab cardiovasc dis. 2012;22(8):684-690. crossref pubmed 39. marchesini g, pasqualetti p, anichini r, et al. patient preferences for treatment in type 2 diabetes: the italian discretechoice experiment analysis. acta diabetol. 2019;56(3):289-299. crossref pubmed https://doi.org/10.1007/s00198-014-2756-8 https://www.ncbi.nlm.nih.gov/pubmed/24899103 https://doi.org/10.3944/aott.2015.14.0082 https://www.ncbi.nlm.nih.gov/pubmed/25803256 https://doi.org/10.1080/03007995.2016.1202817 https://www.ncbi.nlm.nih.gov/pubmed/27322906 https://doi.org/10.2500/aap.2018.39.4183 https://www.ncbi.nlm.nih.gov/pubmed/30336791 https://doi.org/10.1080/17476348.2019.1567339 https://www.ncbi.nlm.nih.gov/pubmed/30625015 https://doi.org/10.7759/cureus.8673 https://www.ncbi.nlm.nih.gov/pubmed/32699673 https://doi.org/10.1111/jdi.12659 https://www.ncbi.nlm.nih.gov/pubmed/28296349 https://doi.org/10.1111/jocn.14797 https://www.ncbi.nlm.nih.gov/pubmed/30667111 https://doi.org/10.1016/j.transproceed.2021.01.034 https://www.ncbi.nlm.nih.gov/pubmed/33610306 https:doi.org/10.1111/dom.14244 https://www.ncbi.nlm.nih.gov/pubmed/33140575 https://doi.org/10.1007/s00198-006-0179-x https://www.ncbi.nlm.nih.gov/pubmed/16862397 https://doi.org/10.1001/archinte.160.21.3278 https://www.ncbi.nlm.nih.gov/pubmed/11088090 https://doi.org/10.1016/j.numecd.2011.04.007 https://www.ncbi.nlm.nih.gov/pubmed/21907553 https://doi.org/10.1007/s00592-018-1236-6 https://www.ncbi.nlm.nih.gov/pubmed/30306406 dti drug target insights 2023; 17: 90-91issn 1177-3928 | doi: 10.33393/dti.2023.2593case report drug target insights issn 1177-3928 www.aboutscience.eu/dti © 2023 the authors. this article is published by aboutscience and licensed under creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). commercial use is not permitted and is subject to publisher’s permissions. full information is available at www.aboutscience.eu activity of sotorasib against brain metastases from nsclc harboring kras p.g12c mutation: a case report alessandro inno1, fabiana marchetti1, matteo valerio1, niccolò giaj levra2, filippo alongi2, giovanni foti3, stefania gori1 1medical oncology, irccs ospedale sacro cuore don calabria, negrar di valpolicella (vr) italy 2advanced radiation oncology, irccs ospedale sacro cuore don calabria, negrar di valpolicella (vr) italy 3radiology, irccs ospedale sacro cuore don calabria, negrar di valpolicella (vr) italy abstract in the codebreak 100 phase 2 study, sotorasib was active for patients with metastatic non-small cell lung cancer (nsclc) harboring kirsten rat sarcoma viral oncogene homologue (kras) p.g12c mutation. however, patients with untreated and/or active brain metastases were excluded from the trial, and the activity of sotorasib in the setting of brain metastases should be further investigated. here we report the case of a kras p.g12c mutant nsclc patient with three brain metastases, of whom one was untreated and the other two had progressed after radiotherapy with symptoms requiring steroids, that responded to sotorasib. our report suggests that sotorasib may be active against untreated or progressive brain metastases, supporting further evaluation of sotorasib in this setting. keywords: brain metastases, central nervous system, kras, nsclc, sotorasib received: april 27, 2023 accepted: may 22, 2023 published online: june 20, 2023 corresponding author: alessandro inno medical oncology irccs ospedale sacro cuore don calabria via don a. sempreboni 5 37024 negrar di valpolicella (vr) italy alessandro.inno@sacrocuore.it rearrangements, target therapy achieves high intracranial response rate (4,5). however, there is still paucity of data regarding the activity of sotorasib against brain metastases from kras p.g12c mutant nsclc. in fact, although in the codebreak 100 trial approximately 20% of patients had brain metastases at baseline, patients with active untreated brain metastases were excluded from the trial. more recently, a retrospective study reported six patients with active untreated brain metastases receiving sotorasib. among four patients evaluable for response, confirmed intracranial response to sotorasib was observed in three patients, with a median duration of response of 4.1 months, and a median intracranial progression-free survival of 4.7 months (6). here we report a case of a patient with metastatic, kras p.g12c mutant nsclc with both treated and untreated active brain metastases receiving sotorasib as second-line therapy. case report in june 2017, a 72-year-old former caucasian female smoker underwent upper right lung lobectomy with regional nodal dissection for lung adenocarcinoma, stage pt2a pn1. comorbidities were: previous left nephrectomy for clear cell renal carcinoma, hypertension, type 2 diabetes, and meningioma. molecular profile of nsclc was: egfr wild type, alk negative, ros1 negative, programmed death ligand (pdl) tumor proportion score (tps) <1%, kras mutant p.g12c. at baseline staging, patient also had two synchronous intracranial metastases, in right parietal lobe and in right cerebellar hemisphere, both treated with stereotactic radiosurgery (21 gy as single fraction). the patient also received background about 25-30% of non-small cell lung cancers (nsclcs) harbor a mutation in the kirsten rat sarcoma viral oncogene homologue (kras) gene. particularly, kras p.g12c mutation is the most frequent kras mutation and it is found in approximatively 13% of nsclc (1). sotorasib, a specific inhibitor of kras p.g12c mutation, has demonstrated activity in pretreated metastatic nsclc patients. in the codebreak 100 trial, a single-group phase 2 study on 126 pretreated patients with metastatic, kras p.g12c mutant nsclc, sotorasib led to a response rate of 37.1% (95% confidence interval (ci), 28.646.2), a median progression-free survival of 6.8 months (95% ci, 5.1-8.2), and a median overall survival of 12.5 months (95% ci, 10.0-not reached), with an acceptable safety profile (2). brain metastases represent a frequent complication of nsclc. they are associated with deterioration of quality of life, poor prognosis, and low response rates to chemotherapy (3). for patients with brain metastases from oncogene addicted nsclc, such as tumors with egfr mutations or alk https://doi.org/10.33393/10.33393/dti.2023.2593 https://creativecommons.org/licenses/by-nc/4.0/legalcode mailto:alessandro.inno@sacrocuore.it inno et al drug target insights 2023; 17: 91 © 2023 the authors. published by aboutscience www.aboutscience.eu first-line chemotherapy with carboplatin plus paclitaxel for four courses, from august to october 2017. during the following surveillance program, the patient developed two lung metastases in the right middle lobe (march 2018) both treated with stereotactic radiotherapy (70 gy in 10 fractions), a further histology-proven, adenocarcinoma lung metastasis kras mutant p.g12c in upper left lobe (september 2019) also treated with stereotactic radiotherapy (60 gy in 10 fractions), and progressive right parietal and right cerebellar metastases treated with further radiation therapy, respectively 21 gy in three fractions and 27 gy in three fractions. in march 2021 the patient experienced symptomatic intracranial disease progression, with a new brain metastasis in the right frontal lobe and increase in size of the other two known metastases and appearance of surrounding edema in the right parietal lobe, requiring steroid therapy. positron emission tomography (pet)/computed tomography (ct) scan did not show extracranial disease. the patient was started on sotorasib, and the brain magnetic resonance imaging (mri) after 2 months of treatment showed stability of the cerebellar metastasis, reduction in size of the previously treated parietal right metastasis with improvement of the surrounding edema, reduction in size of the previously untreated right frontal lobe metastasis, and no appearance of new brain metastases (fig. 1). in march 2022 posterior fossa hemorrhage occurred due to bleeding of the cerebellar metastasis, which was treated with surgical evacuation of the hemorrhagic focus and metastasectomy. histology examination of the cerebellar metastasis revealed radionecrosis with no residual viable cancer tissue. treatment with sotorasib was continued and the disease remained stable until july 2022 when brain mri showed oligoprogression due to increase in size of the right frontal metastasis, which was treated with stereotactic radiotherapy (24 gy in three fractions). sotorasib was continued and, after 27 months (may 2023), treatment is still ongoing, without safety concerns, and with stable intracranial disease at brain mri and still no evidence of extracranial metastases at the pet/ct scan. conclusion we have reported a case of intracranial response to sotorasib in a patient with both pretreated and untreated symptomatic brain metastases from kras p.g12c mutant nsclc, with a duration of intracranial response of 16 months. an oligoprogressive brain metastasis was successfully managed with stereotactic radiotherapy while continuing sorafenib, with a time to treatment failure exceeding 27 months. this report supports further investigation of sotorasib in the setting of kras p.g12c mutant nsclc with untreated brain metastases. disclosures conflict of interest: the authors declare no conflict of interest. financial support: this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. authors contribution: all authors contributed equally to this manuscript. references 1. thai aa, solomon bj, sequist lv, gainor jf, heist rs. lung cancer. lancet. 2021;398(10299):535-554. crossref pubmed 2. skoulidis f, li bt, dy gk, et al. sotorasib for lung cancers with kras p.g12c mutation. n engl j med. 2021;384(25):23712381. crossref pubmed 3. inno a, di noia v, d’argento e, modena a, gori s. state of the art of chemotherapy for the treatment of central nervous system metastases from non-small cell lung cancer. transl lung cancer res. 2016;5(6):599-609. crossref pubmed 4. reungwetwattana t, nakagawa k, cho bc, et al. cns response to osimertinib versus standard epidermal growth factor receptor tyrosine kinase inhibitors in patients with untreated egfrmutated advanced non-small-cell lung cancer. j clin oncol. 2018;jco2018783118(33):jco2018783118. crossref pubmed 5. gadgeel s, peters s, mok t, et al. alectinib versus crizotinib in treatment-naive anaplastic lymphoma kinase-positive (alk+) non-small-cell lung cancer: cns efficacy results from the alex study. ann oncol. 2018;29(11):2214-2222. crossref pubmed 6. lamberti g, aizer a, ricciuti b, et al. incidence of brain metastases and preliminary evidence of intracranial activity with sotorasib in patients with krasg12c-mutant non-small-cell lung cancer. jco precis oncol. 2023;7(7):e2200621. crossref pubmed fig. 1 intracranial response to sotorasib assessed with magnetic resonance imaging. t1-weighted imaging of right parietal metastasis before (a) and after (b) 6 months of sotorasib; fluid-attenuated inversion recovery of right parietal metastasis surrounding edema before (c) and after (d) 6 months of sotorasib; t1-weighted imaging of right frontal metastasis before (e) and after (f) 6 months of sotorasib; t1-weighted imaging of right cerebellar metastasis before (g) and after (h) 6 months of sotorasib. https://doi.org/10.1016/s0140-6736(21)00312-3 https://www.ncbi.nlm.nih.gov/pubmed/34273294 https://doi.org/10.1056/nejmoa2103695 https://www.ncbi.nlm.nih.gov/pubmed/34096690 https://doi.org/10.21037/tlcr.2016.11.01 https://www.ncbi.nlm.nih.gov/pubmed/28149755 https://doi.org/10.1200/jco.2018.78.3118 https://www.ncbi.nlm.nih.gov/pubmed/30153097 https://doi.org/10.1093/annonc/mdy405 https://www.ncbi.nlm.nih.gov/pubmed/30215676 https://www.ncbi.nlm.nih.gov/pubmed/30215676 https://doi.org/10.1200/po.22.00621 https://www.ncbi.nlm.nih.gov/pubmed/36809054 dti drug target insights 2023; 17: 31-38issn 1177-3928 | doi: 10.33393/dti.2023.2510 review drug target insights issn 1177-3928 www.aboutscience.eu/dti © 2023 the authors. this article is published by aboutscience and licensed under creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). commercial use is not permitted and is subject to publisher’s permissions. full information is available at www.aboutscience.eu association between cardiovascular diseases and periodontal disease: more than what meets the eye bhavya shetty1, ibrahim fazal1, safiya fatima khan2, manjusha nambiar2, khadijathul irfana d1, rohit prasad1, akshata raj1 1department of periodontics, faculty of dental sciences, ramaiah university of applied sciences, bangalore, karnataka india 2department of periodontics, sri rajiv gandhi college of dental sciences and hospital, bangalore, karnataka india abstract cardiovascular diseases (cvds) are inflammatory diseases of coronary arteries accompanying atheroma formation that can spawn impairment and, in severe cases, death. cvds are the leading cause of death in the world. in recent decades, investigators have focused their impact on cvd by periodontal disease (pd). pd is a risk factor that can trigger the formation, maturation, and instability of atheroma in the arteries. two mechanisms have been proposed to explain this relationship: periodontopathic pathogens explicitly invade the circulation or indirectly increase systemic levels of inflammatory mediators. it has been suggested that improvement in disease state has a positive effect on others. this review summarizes evidence from epidemiological studies as well as researches focusing on potential causation channels to deliver a comprehensive representation of the relationship between pd and cvd. keywords: cardiovascular diseases, periodontal disease, periodontal therapy, risk factor, systematic review, systemic diseases received: october 25, 2022 accepted: november 10, 2022 published online: february 2, 2023 corresponding author: dr ibrahim fazal department of periodontology faculty of dental sciences m. s. ramaiah university of applied sciences bangalore 560054, karnataka india ibrahim.f.dhinda@gmail.com treatment and control of hypertension, and the widespread use of statins. periodontitis is the sixth most common disease in humans, affecting 740 million people worldwide. periodontitis is a bacterially induced chronic tissue destructive inflammation of the teeth. this periodontal microbiota causes the release of proinflammatory mediators both locally and systemically. as the paradigm of chronic infection in dental pathology, periodontal disease (pd) shares several pathogenic pathways with cvds. as a result of the low-grade state of systemic inflammation posed by periodontitis, it is considered to be strongly associated with cvds (2). there is robust association between cvd and pd. the delineating focus of the relationship has been the periodontal pathogens from the oral cavity, which directly exacerbate cvd in which chronic periodontal inflammation at the site of infection increases circulating levels of inflammatory mediators, and bacteria dispersed into the circulation provokes host inflammatory arbiter, which unswervingly alters other systemic diseases. several studies have been conducted to determine whether pd is associated with risk factors for cvd (3). c-reactive protein (crp), homocysteine, fibrinogen, highdensity lipoprotein cholesterol (hdl-c), and low-density lipoprotein cholesterol (ldl-c) have all been studied as cvd markers (4,5). from a public health standpoint, cvd is the most significant of all the systemic conditions associated with pd, accounting for high mortality rates in most countries. because multiple intervention studies, meta-analyses, and introduction cardiovascular disease (cvd) is the leading cause of death worldwide, claiming an estimated 17.9 million lives each year. cvd is an encyclopedic term for heart and blood vessel disorders. atherosclerosis is an underlying cause of cvd. atherosclerosis is a chronic vascular inflammatory condition characterized by lipid deposition (plaque) in the arterial wall (1). atherosclerotic formation and its advancement could diminish arterial blood flow and cause ischemia in tissues or organs, as well as endorse clotting. on the bright side, cardiovascular mortality has decreased. if combating infectious diseases was the public health success story of the first half of the 20th century, then the decline in mortality rates from cvd is the success story of the last four decades: a sharp decline in mortality rates, aided by rapid advances in both areas of prevention and treatment, including drastic reductions in smoking, improvements in the http://doi.org/10.33393/dti.2023.2510 https://creativecommons.org/licenses/by-nc/4.0/legalcode association between cardiovascular diseases and periodontal disease32 © 2023 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti systematic reviews have been published in this area, this review aims to answer the following questions: is there a link between cvd and periodontitis? what does the literature tell us after more than two decades of research, and why is this such a difficult question to answer? what does the bradford-hill criteria suggest about this unique association? what are our current circumstances, and what are our prospects for the future? mechanism and etiopathogenesis to explain how pd influences cvd, two mechanisms have been proposed. first, periodontopathic flora directly annex endothelial cells via a direct mechanism (6). polymerase chain reaction assays for atherosclerotic plaques support this theory. streptococcus mutans was found to be the most common bacteria in cardiovascular specimens containing thrombus tissues (78%), followed by aggregatibacter actinomycetemcomitans (7). other bacteria found in atherosclerotic lesions in coronary arteries include tannerella forsythia, prevotella intermedia, and predominantly porphyromonas gingivalis. still, it remains unclear how the existence of periodontopathogenic organisms impacts atherosclerosis intracellularly, but few pathogens, such as p. gingivalis, may induce formation of foam cells or tenacity in cells, resulting in tributary inflammation and endothelial dysfunction (8,9). the second proposed mechanism is the indirect pathway where pd causes increases in the levels of inflammatory cytokines. pd induces an inflammatory response, which results in elevation in levels of various inflammatory mediators, including interleukin 8, interleukin 6, interleukin 1 and tumor necrosis factor, which are also linked to atherosclerotic vascular disease. some can speed up the production and emission of fibrinogen and crp. moreover, bacterial lipopolysaccharides plunge the flow and elicit strong immune response (fig. 1). these elements influence atherosclerosis by acting on endothelial cells, increase the oxidative stress, and harmonize the lipid metabolism. this is confirmed by a previous study in which endothelial dysfunction was found in patients with periodontitis. to jot down, it is intelligible that due to pd, inflammation persuades which can immigrate the periodontopathic organisms or leak out the inflammatory cytokines into the circulatory system which might either lead to systemic inflammation or periodontal pathogens may end up in vascular tissues (fig. 2) which has a final ultimatum – formation, maturation, and exacerbation of atheromatous plaque. fig. 1 direct and indirect relationship between periodontal disease and cardiovascular disease. fig. 2 etiopathogenesis flowchart. shetty et al drug target insights 2023; 17: 33 © 2023 the authors. published by aboutscience www.aboutscience.eu epidemiological studies and literature review of the pd and systemic disease connection certainly, contemporary evidences have imparted useful knowledge on common biomarkers of cvd and pd, which has prognostic as well as diagnosing aptitude to crucially decrease the menace of abominable cardiac episode in an untimely manner (tab. i). cross-sectional and case-control studies genco and colleagues investigated the link between certain subgingival periodontopathogens and myocardial infarction (mi) (20), comparing 233 controls with 97 nonfatal mi patients. noninfected individuals were compared with infected individuals. for mi, odds ratio for the presence of t. forsythia was 2.99 and for p. gingivalis was 2.52. these results support the idea that specific pathogenic bacteria found in pd may also be associated with mi. arbes et al (nhanes iii) studied the relationship between pd and coronary heart disease (chd) and found that with increase in the severity of periodontitis the likelihood of suffering an mi increased. the study thus confirmed with other studies the link and displayed an undeviating robust association amidst increased severity of periodontitis and cvd (21). longitudinal studies destefano and colleagues reviewed nhanes-i data as well as a 15-year epidemiologic follow-up. they discovered periodontitis was one significant predictor of cvd in 9,760 men and women (22). these relationships were unaffected by age, gender, body mass index, education, marital status, poverty index, race, blood pressure, alcohol consumption, diabetes status, and serum cholesterol levels. beck and coworkers conducted a study of 921 men who did not have coronary artery disease at baseline. a total of 40 had stroke, 59 died of coronary artery disease, and 207 men developed coronary artery disease during an 18-year follow-up period. odds ratios for total periodontal bone loss and chd, fatal chd and stroke, cvd risk factors and age were 1.5, 2.8, and 1.9, respectively. accordingly, the odds of suffering a vascular event or chd were 0.5-2.8 times higher in individuals with radiographically proven periodontitis (23). hujoel et al conducted a longitudinal study that found no link between chronic heart disease and periodontitis (24). these authors evaluated the nhanes-i study and the results of their 21-year follow-up. it is worth noting that destefano et al (22) used the same database and discovered a link between cvd and pd in nhanes-i study 15 years later. hujoel et al adjusted extensively for potential confounders, which may have explained the lack of a relationship after adjustment (24). hujoel et al may have adjusted too heavily for factors strongly associated with infection, such as pd. it is also possible that periodontal status of subjects was significantly misclassified over time. in addition, because of treatments and extractions over time, the authors may have misclassified subjects who had pd at baseline. the misclassification being non-differential, which would have been worsened through 21-year follow-up period, could brace the null hypothesis of the research that there is no link between cvd and pd. joshipura and colleagues discovered that after controlling for other risk factors, the association between self-reported history of pd and incidence of heart disease was no longer significant (25). however, these researches merit further discussion as they were derived from a well-characterized, large longitudinal study. the majority of studies that found a correlation discovered that the amount of pd was significant. it would be impossible to quantify the extent of pd present in joshipura’s research as they answered a “yes or no” query about pds. furthermore, discrepancy based on self-reported pd is possible. the most recent longitudinal study, published by howell and colleagues (26), was a double-blind, randomized, placebo-controlled trial of beta-carotene and aspirin for the prevention of cvd and cancer in the united states in 22,071 male physicians. the study outcomes were nonfatal mi, stroke, and death from cvd. after controlling for the treatment and age (beta-carotene and aspirin), the researchers discovered a positive non-significant trend (95% confidence interval [ci], relative risk [rr] = 1.13). although most evidence from case-control and longitudinal researches advocates a link between cvd and periodontitis, the link seems to be dwindling. there is insufficient evidence to conclude that the associations are contributory. observational studies: from systematic review to meta-analysis scannapieco et al (27), in a comprehensive systematic review, concluded that there is moderate evidence of a relation between mi, cvd, atherosclerosis, and pd, but the causation was uncertain. khader et al (5), in a meta-analysis, combined two crosssectional studies and six cohort and found a lower rr of 1.15 (95% ci [1.06-1.25]). nine cohort researches compiled by janket et al (28) in a meta-analysis suggests that, for future cvd and cyclic vomiting syndrome episodes, pd is a determined risk factor and discovered that chronic periodontitis patients had a 19% surged peril for advancing such events. people under the age of 65 were at a higher risk (44%). another meta-analysis of observational studies by alessandra blaizot et al investigated the relationship between cvd and periodontitis exposure (29). researches done between 1989 and 2007 were recovered from seven databases via electronic and manual searches. the moose metaanalysis guidelines for observational studies were followed (30). there were 47 observational studies among the 215 epidemiological studies, 29 of which could be combined using meta-analysis methodology. there was increased risk (about 34%) of developing cvd in individuals having periodontitis than in individuals who are not exposed with periodontitis, where 1.34 was the rr of these seven cohort researches (p = 0.0001). this finding suggests that people with parkinson’s disease are at higher risk for developing cvd. association between cardiovascular diseases and periodontal disease34 © 2023 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti interventional studies owing to many factors, for example, financial, ethical, or methodological, the periodontal intervention and its efficacy as primary prevention for cvds such as ischemic heart disease (ihd) and death haven’t yet been studied (50). consequently, cvd proxy markers have been thoroughly investigated, and periodontal intervention shows substantial effect on these markers, as shown in table ii. there is limited affirmation on durable effect of periodontal intervention on these proxy markers. in addition, the impact of periodontal intervention on the scientific conclusions of these markers of cvd remains to be still investigated. previous research has found that rigorous periodontal intervention transiently impairs the endothelial function role, which raises levels of serum inflammatory markers, probably due to liberation of inflammatory mediators or bacterial organisms in bloodstream. nevertheless, after a few weeks of periodontal intervention the levels of inflammatory makers and periodontal parameters also seem to lower or decrease (51-54). furthermore, 6 months after periodontal treatment, carotid intimal-medial thickness is reduced. several interventions and researches have been done as shown in table ii, which has been published in the past few years, and they all brace up for the hypothesis that periodontal intervention reduces cardiovascular risk factor and thus has an impact on cvd events (55). imminent interventional studies are required toward better understanding of the association between pd and cvd, mainly the biological effects of pd on the atherogenic cascade by influencing the vascular endothelium. further enduring intervention researches are needed, ideally utilizing similar methods to assess cvd events, to determine whether the reported benefits of periodontal intervention can actually decipher the reduction in incidence of cvd. microbiological studies clinically, it is extremely strenuous to determine the causal agent of atherosclerosis. first, endothelial damage advances by masking the causative agent and progresses without symptoms. second, multiple contributing factors cause atherosclerosis, and these influencers may coexist, making it difficult to determine the causative factor (56,57). in addition, studies of interventions have shown mixed results. sometimes after periodontal intervention there is enhancement, whereas sometimes there is brief deterioration of the symptoms and sometimes there is no change. however, seven rules must be met for promoting atherosclerosis by the periodontal pathogens, which are enlisted below (50). evidence 1: systemic vascular tissues can provide a pathway for periodontal pathogens. numerous researches have demonstrated that periodontopathic bacteria could cause bacteremia by entering the systemic circulation (50,5860,62). a previous systematic review found that bacteremia after periodontal procedures could be atop 50% (63,64). table iii summarizes the prevalence of periodontopathic pathogens in systemic circulation after periodontal intervention in atheromatous lesions, with and without periodontal procedures in periodontitis patients. following periodontal procedures, periodontopathic organisms may infiltrate the circulation, being a determinant of atherosclerosis. evidence 2: affected tissues accommodate periodontal pathogens. several studies have provided sufficient evidence that dna, rna, and antigen sequencing can be used to identify different periodontal species in atheromatous lesions (65-67). analysis shows that periodontitis patients are at increased risk for emergent atherosclerosis. evidence 3: in the affected site, live periodontal pathogens will be present. this proof requires the detection of live periodontopathic bacteria. live a. actinomycetemcomitans and p. gingivalis were isolated from atherosclerotic samples in multiple studies (68,69). evidence 4: invasion of the affected cell with in vitro evidence. several in vitro researches show periodontopathic organisms can invade various types of host cells. according to many studies p. gingivalis is responsible for infiltrating the endothelial cells in studies, the significance as well as the mechanism of the specific strain type are being investigated further (70-72). evidence 5: provides indication that periodontal pathogen can encourage atherosclerosis in diseased animal model. in 2012, the european federation of periodontology and the american academy of periodontology published a review that found proof that periodontal pathogens can endorse atherosclerosis (56). p. gingivalis is shown to promote atherosclerosis in murine (73), rabbit (74), and pig models (75). furthermore, when hyperlipidemic mice were orally infected with fusobacterium nucleatum, t. forsythia, p. gingivalis, treponema denticola, and other expedient organisms from this specific class were found in aorta, atherosclerotic plaque, and oral epithelium (76,77). evidence 6: pathology is significantly less when caused by noninvasive mutants, according to in vitro and in vivo evidence where the incursion of vascular cells and tissues by bacterial strains is been investigated. noninvasive fimadeficient mutant of p. gingivalis exhibits fewer proinflammatory mediators than the invasive wild-type strain of p. gingivalis (73). evidence 7: fulfill a modified version of koch’s postulate to show that a human atheroma isolate causes disease in animal models. to do this, isolate the periodontopathogen from a human atheroma and induce atheroma formation in an animal model after inoculation. p. gingivalis were isolated from atherosclerotic samples. there is also evidence that suffused pathogenic bacteria can cause atherosclerosis. the evidence is still, however, considered incomplete as the bacterial strain utilized were not isolated from human atherosclerotic samples (68,76,77). apart from evidence 7, there are abundant researches available to support evidences 1 to 6. nonetheless, the first six proofs embrace the notion that periodontal pathogens are linked to cvd. neoteric substantiation of cvd and pd febbraio et al (88) concluded that there is an association between oral health and cvd, but causality has yet to be established. however, studies show improvements shetty et al drug target insights 2023; 17: 35 © 2023 the authors. published by aboutscience www.aboutscience.eu in cardiovascular risk factors following periodontal treatments, although with relatively short follow-up periods. rational evidence suggests that good oral health contributes to overall and heart health. the best bet is to continue to remind patients that a healthy mouth contributes to a healthy heart. fazal et al (19) concluded that non surgical periodontal therapy (nspt) lowers cardiac biomarker concentrations in patients with chronic periodontitis and may reduce the risk of cvd in the future. however, paul et al (89) suggest a contradictory result in a review that therapeutic periodontal interventions cannot be used to prevent heart disease or stroke. larvin et al (90) found an increased risk of cvd in people with pd in a systematic review and meta-analysis. males and people with severe pd are at the highest risk of developing cvd, indicating potential target populations for future public health interventions and inspection. in a cross-sectional observational single-center study, lazureanu et al (91) concluded that increasing patients’ awareness of oral healthcare measures resulted in better outcomes and improved oral-health-related quality of life. in a 13-year follow-up study, tiensripojamarn et al (92) show that severe periodontitis is linked to a higher incidence of chd, independent of existing cardiovascular risk factors. periodontitis grade b/c was linked to a higher overall cardiovascular risk, and this association was not explained by smoking confounding in participants aged 65-74 years, according to petrenya et al (93). they also recommended that patients with periodontitis, particularly those with extensive alveolar bone loss, use norrisk 2 score for cardiovascular risk assessment. individuals with a high cardiovascular risk profile should have routine periodontal examinations. in addition to adequate, evidence-based periodontal intervention, cessation of smoking and blood pressure normalization are essential in lowering cardiovascular risk in individuals with pd. what do the bradford-hill criteria imply for the relationship between cvd and pd? the canadian dental hygienists association (cdha) published a position paper that used the bradford-hill criteria to determine whether there is sufficient evidence for a causal relationship between pd and cvd (87). the bradford-hill criteria analysis found no evidence of a link between pd and cvd. although the link between pd and cvd is well established, the findings of the cdha’s recent position paper show that, while an association exists, the nature of that link is unknown, and there is insufficient evidence for that association to be causal at this time. conclusion epidemiologic researches have now substantiated that there may be a link between pd and cvd. although research continues to point to a connection between cvd and oral health, causality has not yet been established. despite the fact that the follow-up times in most studies are brief, numerous studies show an improvement of cvd risk factors after periodontal interventions. the association of good oral health and general and heart health is proved by reasonable evidence. in the coming decades, medical and dental professionals must be capable of better planning of preventive interventions as constant researches approve and account the forte of the consortium between cvd and pd. scientific data assembled thus far would seem to support the continued value of interventional periodontal therapy not just for oral health but for overall health as well. disclosures conflict of interest: the authors declare no conflict of interest. financial support: this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. authors contribution: all authors contributed equally to this manuscript. references 1. libby p, buring je, badimon l, et al. atherosclerosis. nat rev dis primers. 2019;5:56. 2. nesse w, dijkstra pu, abbas f, et al. increased prevalence of cardiovascular and autoimmune diseases in periodontitis patients: a cross-sectional study. j periodontol. 2010 nov;81(11):1622-1628. 3. sanz m, del castillo am, jepsen s, et al. periodontitis and cardiovascular diseases: consensus report. j clin periodontol. 2020 mar 1;47(3):268-288. 4. wu t, trevisan m, genco rj, dorn jp, falkner kl, sempos ct. periodontal disease and risk of cerebrovascular disease: the first national health and nutrition examination survey and its follow-up study. arch intern med. 2000;160:2749-2755. 5. khader ys, albashaireh zs, alonari ma. periodontal diseases and the risk of coronary heart and cerebrovascular diseases: a meta-analysis. j periodontol. 2004;75:1046-1053. 6. ford pj, gemmell e, chan a, et al. inflammation, heat shock proteins and periodontal pathogens in atherosclerosis: an immunohistologic study. oral microbiol immunol. 2006;21:206-211. crossref 7. nakano k, nemoto h, nomura r, et al. detection of oral bacteria in cardiovascular specimens. oral microbiol immunol. 2009;24:64-68. crossref 8. pucar a, milasin j, lekovic v, et al. correlation between atherosclerosis and periodontal putative pathogenic bacterial infections in coronary and internal mammary arteries. j periodontol. 2007;78:677-682. crossref 9. roth ga, moser b, huang sj, et al. infection with a periodontal pathogen induces procoagulant effects in human aortic endothelial cells. j thromb haemostasis. 2006;4:2256-2261. crossref 10. noack b, genco rj, trevisan m, grossi s, zambon jj, de nardin e. periodontal infections contribute to elevated systemic c-reactive protein level. j periodontol. 2001 sep;72(9):1221-1227. 11. singh n, chandel s, singh h, agrawal a, savitha an. effect of scaling & root planing on the activity of alp in gcf & serum of patients with gingivitis, chronic and aggressive periodontitis: a comparative study. j oral biol craniofac res. 2017 may 1;7(2):123-126. 12. delange n, lindsay s, lemus h, finlayson tl, kelley st, gottlieb ra. periodontal disease and its connection to systemic https://doi.org/10.1111/j.1399-302x.2006.00276.x https://doi.org/10.1111/j.1399-302x.2008.00479.x https://doi.org/10.1902/jop.2007.060062 https://doi.org/10.1111/j.1538-7836.2006.02128.x association between cardiovascular diseases and periodontal disease36 © 2023 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti biomarkers of cardiovascular disease in young american indian/alaskan natives. j periodontol. 2018 feb;89(2):219-227. 13. mohammed aa, youssef jm, metwally ss, anees mm. evaluation of the serum ceruloplasmin level before and after non-surgical periodontal therapy in patients with chronic periodontitis. stomatological dis sci. 2018 mar 14;2:3. 14. ilea a, lazăr ac, roșca d, et al. periodontitis in a group of patients with cardiovascular disease. anatomy physiol biochem int j. 2018;5(4):1-6. crossref 15. leira y, blanco j. brain natriuretic peptide serum levels in periodontitis. j periodontal res. 2018 aug;53(4):575-581. 16. ameen m, attia am, felimban a, et al. evaluation of cardiac biomarkers in smokers and non-smokers with chronic periodontitis. int j health sci. 2020 may;14(3):26. 17. gupta m, chaturvedi r, jain a. role of cardiovascular disease markers in periodontal infection: understanding the risk. indian j dent res. 2015 may 1;26(3):231. 18. boyapati r, vudathaneni v, nadella sb, ramachandran r, dhulipalla r, adurty c. mapping the link between cardiac biomarkers and chronic periodontitis: a clinico-biochemical study. j indian soc periodontol. 2020 jul;24(4):309. 19. fazal i, shetty b, yadalam u, khan sf, nambiar m. effectiveness of periodontal intervention on the levels of n-terminal probrain natriuretic peptide in chronic periodontitis patients. j circ biomark. 2022 oct 3;11:48-56. 20. genco rj, wu t, grossi s, faulkner k, zambon jj, trevisan m. periodontal microflora related to the risk of myocardial infarction: a case-control study (abstract 2811). j dent res 1999;78(special issue):457. 21. arbes sj, slade gd, beck j. association between extent of periodontal attachment loss and self-reported history of heart attack: an analysis of nhanes iii data. j dent res 1999;78:1777-1782. 22. destefano f, anda rf, kahn hs, williamson df, russell cm. dental disease and risk of coronary heart disease and mortality. bmj. 1993;306:688-691. 23. beck j, garcia r, heiss g, vokonas ps, offenbacher s. periodontal disease and cardiovascular disease. j periodontol. 1996;67:1123-1137. 24. hujoel p, drangsholt m, spiekerman c, derouen t. periodontal disease and coronary heart disease risk. jama. 2000;284: 1406-1410. 25. joshipura kj, rimm eb, douglass cw, trichopoulos d, ascherio a, willett wc. poor oral health and coronary heart disease. j dent res 1996;75:1631-1636. 26. howell th, ridker pm, ajani ua, hennekens ch, christen wg. periodontal disease and risk of subsequent cardiovascular disease in u.s. male physicians. j am coll cardiol. 2001;37:445-450. 27. scannapieco fa, bush rb, paju s. associations between periodontal disease and risk for atherosclerosis, cardiovascular disease and stroke: a systematic review. ann periodontol. 2003;8:38-53. 28. janket sj, baird ae, chuang sk, jones ja. meta-analysis of periodontal disease and risk of coronary heart disease and stroke. oral surg oral med oral pathol oral radiol endod. 2003;95:559-569. 29. blaizot a, vergnes jn, nuwwareh s, amar j, sixou m. periodontal diseases and cardiovascular events: meta-analysis of observational studies. int dent j. 2009;59:197-209. 30. stroup df, berlin ja, morton sc, et al. meta-analysis of observational studies in epidemiology: a proposal for reporting metaanalysis of observational studies in epidemiology (moose) group. jama. 2000;283:2008-2012. 31. caúla al, lira-junior r, tinoco em, fischer rg. the effect of periodontal therapy on cardiovascular risk markers: a 6-month randomized clinical trial. j clin periodontol. 2014;41:875-882. crossref 32. vidal f, cordovil i, figueredo cm, fischer rg. non-surgical periodontal treatment reduces cardiovascular risk in refractory hypertensive patients: a pilot study. j clin periodontol. 2013;40:681-687. crossref 33. bresolin ac, pronsatti mm, pasqualotto ln, et al. lipid profiles and inflammatory markers after periodontal treatment in children with congenital heart disease and at risk for atherosclerosis. vasc health risk manage. 2013;9:703-709. crossref 34. thygesen k, alpert js, jaffe as, et al. third universal definition of myocardial infarction. eur heart j. 2012;33:2551-2567. crossref 35. bokhari sa, khan aa, butt ak, et al. nonsurgical periodontal therapy reduces coronary heart disease risk markers: a randomized controlled trial. j clin periodontol. 2012;39:10651074. crossref 36. banthia r, jain p, banthia p, belludi s, parwani s, jain a. effect of phase i periodontal therapy on pro-coagulant state in chronic periodontitis patients – a clinical and haematological study. j irish dental assoc. 2013;59:183-188. 37. kiany f, hedayati a. evaluation of serum anti-cardiolipin antibodies after non-surgical periodontal treatment in chronic periodontitis patients. odontology. 2015;103:203-209. crossref 38. gupta b, sawhney a, patil n, et al. effect of surgical periodontal therapy on serum c-reactive protein levels using elisa in both chronic and aggressive periodontitis patient. j clin diagnostic res. 2015;9:zc01-zc05. crossref 39. graziani f, cei s, orlandi m, et al. acute-phase response following full-mouth versus quadrant non-surgical periodontal treatment: a randomized clinical trial. j clin periodontol. 2015;42:843-852. crossref 40. houcken w, teeuw wj, bizzarro s, et al. arterial stiffness in periodontitis patients and controls. a case-control and pilot intervention study. j hum hypertens. 2016;30:24-29. crossref 41. torumtay g, kirzioglu fy, öztürk tonguç m, kale b, calapoglu m, orhan h. effects of periodontal treatment on inflammation and oxidative stress markers in patients with metabolic syndrome. j periodontal res. 2016;51:489-498. crossref 42. siddeshappa st, nagdeve s, yeltiwar rk, parvez h, deonani s, diwan v. evaluation of various hematological parameters in patients with periodontitis after nonsurgical therapy at different intervals. j indian soc periodontol. 2016;20:180-183. crossref 43. arvanitidis e, bizzarro s, alvarez rodriguez e, loos bg, nicu ea. reduced platelet hyper-reactivity and platelet-leukocyte aggregation after periodontal therapy. thromb j. 2017;15:5. crossref 44. zhou qb, xia wh, ren j, et al. effect of intensive periodontal therapy on blood pressure and endothelial microparticles in patients with prehypertension and periodontitis: a randomized controlled trial. j periodontol. 2017;88:711-722. crossref 45. de souza ab, okawa rt, silva co, araújo mg. short-term changes on c-reactive protein (crp) levels after non-surgical periodontal treatment in systemically healthy individuals. clin oral investig. 2017;21:477-484. crossref 46. jockel-schneider y, bechtold m, haubitz i, et al. impact of anti-infective periodontal therapy on parameters of vascular health. j clin periodontol. 2018;45:354-363. crossref 47. saffi mal, rabelo-silva er, polanczyk ca, et al. periodontal therapy and endothelial function in coronary artery disease: a randomized controlled trial. oral dis. (2018) 24:1349-1357. crossref https://doi.org/10.19080/apbij.2018.05.555668 https://doi.org/10.1111/jcpe.12290 https://doi.org/10.1111/jcpe. 12110 https://doi.org/10.2147/vhrm.s52187 https://doi.org/10.1093/eurheartj/ehs184 https://doi.org/10.1111/j.1600-051x.2012.01942.x 128 https://doi.org/10.1007/s10266-014-0149-2 https://doi.org/10.7860/jcdr/2015/14680.6558 https://doi.org/10.1111/jcpe.12451 132 https://doi.org/10.1038/jhh.2015.41 https://doi.org/10.1111/jre.12328 134 https://doi.org/10.4103/0972-124x.175172 135 https://doi.org/10.1186/s12959-016-0125-x 136 https://doi.org/10.1902/jop.2017.160447 137 https://doi.org/10.1007/s00784-016-1817-0 https://doi.org/10.1111/jcpe. 12849 139 https://doi.org/10.1111/odi.12909 140 shetty et al drug target insights 2023; 17: 37 © 2023 the authors. published by aboutscience www.aboutscience.eu 48. morozumi t, yashima a, gomi k, et al. increased systemic levels of inflammatory mediators following one-stage full-mouth scaling and root planing. j periodontal res. 2018;53:536-544. crossref 49. moeintaghavi a, arab hr, moghaddam ma, shahmohammadi r, bardan by, soroush z. evaluation of effect of surgical and nonsurgical periodontal therapy on serum c-reactive protein, triglyceride, cholesterol, serum lipoproteins and fasting blood sugar in patients with severe chronic periodontitis. open dent j. 2019;13:15-21. crossref 50. herrera d, molina a, buhlin k, klinge b. periodontal diseases and association with atherosclerotic disease. periodontology 2000. 2020;83:66-89. crossref 51. tonetti ms, d’aiuto f, nibali l, et al. treatment of periodontitis and endothelial function. n engl j med. 2007;356:911-920. crossref 52. teeuw wj, slot de, susanto h, et al. treatment of periodontitis improves the atherosclerotic profile: a systematic review and meta-analysis. j clin periodontol. 2014;41:70-79. crossref 53. d’aiuto f, nibali l, parkar m, suvan j, tonetti ms. short-term effects of intensive periodontal therapy on serum inflammatory markers and cholesterol. j dent res. 2005;84:269-273. crossref 54. d’aiuto f, parkar m, nibali l, suvan j, lessem j, tonetti ms. periodontal infections cause changes in traditional and novel cardiovascular risk factors: results from a randomized controlled clinical trial. am heart j. 2006;151:977-984. crossref 55. piconi s, trabattoni d, luraghi c, et al. treatment of periodontal disease results in improvements in endothelial dysfunction and reduction of the carotid intima-media thickness. faseb j. 2009;23:1196-1204. crossref 56. reyes l, herrera d, kozarov e, roldán s, progulske-fox a. periodontal bacterial invasion and infection: contribution to atherosclerotic pathology. j clin periodontol. 2013;40(suppl.) 14:s30-s50. crossref 57. kebschull m, demmer rt, papapanou pn. “gum bug, leave my heart alone!” – epidemiologic and mechanistic evidence linking periodontal infections and atherosclerosis. j dental res. 2010;89:879-902. crossref 58. joshipura k, zevallos jc, ritchie cs. strength of evidence relating periodontal disease and atherosclerotic disease. compend contin educ dent. 2009;30:430-439. 59. ross r. atherosclerosis is an inflammatory disease. am heart j. 1999;138:s419-s420. crossref 60. ratto-tespestini a, chaparro pj, romito g, et al. comparison of independent and dependent culture methods for the detection of transient bacteremia in diabetic subjects with chronic periodontitis. biomédica revista del instituto nacional de salud. 2016;36:156-161. crossref 61. balejo rdp, cortelli jr, costa fo, et al. effects of chlorhexidine preprocedural rinse on bacteremia in periodontal patients: a randomized clinical trial. j appl oral sci. 2017;25:586-595. crossref 62. dhotre s, jahagirdar v, suryawanshi n, davane m, patil r, nagoba b. assessment of periodontitis and its role in viridans streptococcal bacteremia and infective endocarditis. indian heart j. 2018;70:225-232. crossref 63. horliana acrt, chambrone l, foz am, et al. dissemination of periodontal pathogens in the bloodstream after periodontal procedures: a systematic review. plos one. 2014;9:e98271. crossref 64. figuero e, sánchez-beltrán m, cuesta-frechoso s, et al. detection of periodontal bacteria in atheromatous plaque by nested polymerase chain reaction. j periodontol. 2011;82:14691477. crossref 65. figuero e, lindahl c, marín mj, et al. quantification of periodontal pathogens in vascular, blood, and subgingival samples from patients with peripheral arterial disease or abdominal aortic aneurysms. j periodontol. 2014;85:1182-1193. crossref 66. serra e silva filho w, casarin rc, nicolela el jr, et al. microbial diversity similarities in periodontal pockets and atheromatous plaques of cardiovascular disease patients. plos one. 2014;9:e109761. crossref 67. armingohar z, jørgensen jj, kristoffersen ak, abesha-belay e, olsen i. bacteria and bacterial dna in atherosclerotic plaque and aneurysmal wall biopsies from patients with and without periodontitis. j oral microbiol. 2014;6:1-13. crossref 68. rafferty b, jönsson d, kalachikov s, et al. impact of monocytic cells on recovery of uncultivable bacteria from atherosclerotic lesions. j internal med. 2011;270:273-280. crossref 69. kozarov ev, dorn br, shelburne ce, dunn wa jr, progulskefox a. human atherosclerotic plaque contains viable invasive actinobacillus actinomycetemcomitans and porphyromonas gingivalis. arterioscleros thromb vasc biol. 2005;25:e17-e18. crossref 70. deshpande rg, khan mb, genco ca. invasion of aortic and heart endothelial cells by porphyromonas gingivalis. infect immun. 1998;66:5337-5343. crossref 71. dorn br, dunn wa jr, progulske-fox a. invasion of human coronary artery cells by periodontal pathogens. infect immun. 1999;67:5792-5798. crossref 72. olsen i, progulske-fox a. invasion of porphyromonas gingivalis strains into vascular cells and tissue. j oral microbiol. 2015;7:28788. crossref 73. gibson fc iii, hong c, chou hh, et al. innate immune recognition of invasive bacteria accelerates atherosclerosis in apolipoprotein e-deficient mice. circulation. 2004;109:2801-2806. crossref 74. jain a, batista el jr, serhan c, stahl gl, van dyke te. role for periodontitis in the progression of lipid deposition in an animal model. infect immun. 2003;71:6012-6018. crossref 75. brodala n, merricks ep, bellinger da, et al. porphyromonas gingivalis bacteremia induces coronary and aortic atherosclerosis in normocholesterolemic and hypercholesterolemic pigs. arterioscleros thromb vasc biol. 2005;25:1446-1451. crossref 76. chukkapalli ss, velsko im, rivera-kweh mf, zheng d, lucas ar, kesavalu l. polymicrobial oral infection with four periodontal bacteria orchestrates a distinct inflammatory response and atherosclerosis in apoe null mice. plos one. 2015;10:e0143291. crossref 77. velsko im, chukkapalli ss, rivera mf, et al. active invasion of oral and aortic tissues by porphyromonas gingivalis in mice causally links periodontitis and atherosclerosis. plos one. 2014;9:e97811. crossref 78. forner l, larsen t, kilian m, holmstrup p. incidence of bacteremia after chewing, tooth brushing and scaling in individuals with periodontal inflammation. j clin periodontol. 2006;33:401-407. crossref 79. lafaurie gi, mayorga-fayad i, torres mf, et al. periodontopathic microorganisms in peripheric blood after scaling and root planing. j clin periodontol. 2007;34:873-879. crossref 80. pérez-chaparro pj, gracieux p, lafaurie gi, donnio p-y, bonnauremallet m. genotypic characterization of porphyromonas gingivalis isolated from subgingival plaque and blood sample in positive bacteremia subjects with periodontitis. j clin periodontol. 2008;35:748-753. crossref 81. castillo dm, sánchez-beltrán mc, castellanos je, et al. detection of specific periodontal microorganisms from bacteraemia samples after periodontal therapy using molecular-based diagnostics. j clin periodontol. 2011;38:418-427. crossref https://doi.org/10.1111/jre.12543 141 https://doi.org/10.2174/1874210601913010015 https://doi.org/10.1111/prd.12302 https://doi.org/10.1056/nejmoa063186 143 https://doi.org/10.1111/jcpe.12171 144 https://doi.org/10.1177/154405910508400312 145 https://doi.org/10.1016/j.ahj.2005.06.018 https://doi.org/10.1096/fj.08-119578 https://doi.org/10.1111/jcpe.12079 https://doi.org/10.1177/0022034510375281 https://doi.org/10.1016/s0002-8703(99)70266-8 https://doi.org/10.7705/biomedica.v36i1.2674 https://doi.org/10.1590/1678-7757-2017-0112 https://doi.org/10.1016/j.ihj.2017.06.019 81 https://doi.org/10.1371/journal.pone.0098271 https://doi.org/10.1902/jop.2011.100719 https://doi.org/10.1902/jop.2014.130604 https://doi.org/10.1371/journal.pone.0109761 https://doi.org/10.3402/jom.v6.23408 https://doi.org/10.1111/j.1365-2796.2011.02373.x https://doi.org/10.1161/01.atv.0000155018.67835.1a https://doi.org/10.1128/iai.66.11.5337-5343.1998 https://doi.org/10.1128/iai.67.11.5792-5798.1999 https://doi.org/10.3402/jom.v7.28788 https://doi.org/10.1161/01.cir.0000129769.17895.f0 https://doi.org/10.1128/iai.71.10.6012-6018.2003 https://doi.org/10.1161/01.atv.0000167525.69400.9c https://doi.org/10.1371/journal.pone.0143291 https://doi.org/10.1371/journal.pone.0097811 https://doi.org/10.1111/j.1600-051x.2006.00924.x 83 https://doi.org/10.1111/j.1600-051x.2007.01125.x https://doi.org/10.1111/j.1600-051x.2008.01296.x https://doi.org/10.1111/j.1600-051x.2011.01717.x association between cardiovascular diseases and periodontal disease38 © 2023 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti 82. waghmare as, vhanmane pb, savitha b, chawla rl, bagde hs. bacteremia following scaling and root planing: a clinico-microbiological study. j indian soc periodontol. 2013;17:725-730. crossref 83. sahrmann p, manz a, attin t, zbinden r, schmidlin pr. effect of application of a pvp-iodine solution before and during subgingival ultrasonic instrumentation on post-treatment bacteraemia: a randomized single-centre placebo-controlled clinical trial. j clin periodontol. 2015;42:632-639. crossref 84. marín mj, figuero e, gonzález i, et al. comparison of the detection of periodontal pathogens in bacteraemia after tooth brushing by culture and molecular techniques. med oral patol oral cir bucal. 2016;21:e276-e284. crossref 85. elkaïm r, dahan m, kocgozlu l, et al. prevalence of periodontal pathogens in subgingival lesions, atherosclerotic plaques and healthy blood vessels: a preliminary study. j periodontal res. 2008;43:224-231. crossref 86. gaetti-jardim e, marcelino sl, feitosa acr, romito ga, avilacampos mj. quantitative detection of periodontopathic bacteria in atherosclerotic plaques from coronary arteries. j med microbiol. 2009;58:1568-1575. crossref 87. lavigne se, forrest jl. an umbrella review of systematic reviews of the evidence of a causal relationship between periodontal disease and cardiovascular diseases: position paper from the canadian dental hygienists association. can j dent hyg. 2020 feb;54(1):32. 88. febbraio m, roy cb, levin l. is there a causal link between periodontitis and cardiovascular disease? a concise review of recent findings. int dent j. 2022 feb;72(1):37-51. crossref 89. paul o, arora p, mayer m, chatterjee s. inflammation in periodontal disease: possible link to vascular disease. frontiers physiol. 2021 jan 14;11:609614. 90. larvin h, kang j, aggarwal vr, pavitt s, wu j. risk of incident cardiovascular disease in people with periodontal disease: a systematic review and meta-analysis. clin exp dent res. 2021 feb;7(1):109-122. 91. lazureanu pc, popescu fg, stef l, focsa m, vaida ma, mihaila r. the influence of periodontal disease on oral health quality of life in patients with cardiovascular disease: a cross sectional observational single-center study. medicina. 2022 apr 24;58(5):584. 92. tiensripojamarn n, lertpimonchai a, tavedhikul k, et al. periodontitis is associated with cardiovascular diseases: a 13year study. j clin periodontol. 2021 mar;48(3):348-356. 93. petrenya n, hopstock la, holde ge, oscarson n, jönsson b. relationship between periodontitis and risk of cardiovascular disease: insights from the tromsø study. j periodontol. 2022 sep;93(9):1353-1365. https://doi.org/10.4103/0972-124x.124480 https://doi.org/10.1111/jcpe.12416 https://doi.org/10.4317/medoral.20842 https://doi.org/10.1111/j.1600-0765.2007.01018.x https://doi.org/10.1099/jmm.0.013383-0 https://doi.org/10.1016/j.identj.2021.07.006 dti drug target insights 2022; 16: 88-96issn 1177-3928 | doi: 10.33393/dti.2022.2522review drug target insights issn 1177-3928 www.aboutscience.eu/dti © 2022 the authors. this article is published by aboutscience and licensed under creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). commercial use is not permitted and is subject to publisher’s permissions. full information is available at www.aboutscience.eu current molecular approach for diagnosis of mrsa: a meta-narrative review sim yi xing1, lee qiao wei2, aisha abushelaibi3, kok song lai3, swee hua erin lim3, sathiya maran1 1school of pharmacy, monash university malaysia, jalan lagoon selatan, selangor malaysia 2school of science, monash university malaysia, jalan lagoon selatan, selangor malaysia 3health sciences division, abu dhabi women’s college, higher colleges of technology, abu dhabi united arab emirates swee hua erin lim and sathiya maran contributed equally to this study. abstract introduction: detection and diagnosis of methicillin-resistant staphylococcus aureus (mrsa) are important in ensuring a correct and effective treatment, further reducing its spread. a wide range of molecular approaches has been used for the diagnosis of antimicrobial resistance (amr) in mrsa. this review aims to study and appraise widely used molecular diagnostic methods for detecting mrsa. methods: this meta-narrative review was performed by searching pubmed using the following search terms: (molecular diagnosis) and (antimicrobial resistance) and (methicillin-resistant staphylococcus aureus). studies using molecular diagnostic techniques for the detection of mrsa were included, while non-english language, duplicates and non-article studies were excluded. after reviewing the libraries and a further manual search, 20 studies were included in this article. rameses publication standard for narrative reviews was used for this synthesis. results: a total of 20 full papers were reviewed and appraised in this synthesis, consisting of pcr technique (n = 7), deoxyribonucleic acid (dna) microarray (n = 1), dna sequencing (n = 2), xpert mrsa/sa bc assay (n = 2), matrix-assisted laser desorption/ionization-time of flight (maldi-tof) (n = 2), mlst (n = 4), sccmec typing (n = 1) and genecube (n = 1). discussion: different diagnostic methods used to diagnose mrsa have been studied in this review. this study concludes that pcr has been extensively used due to its higher sensitivity and cost-effectiveness in the past five years keywords: antimicrobial resistance, molecular diagnosis, mrsa received: november 17, 2022 accepted: december 31, 2022 published online: december 31, 2022 corresponding author: sathiya maran building 2, level 5, room 08 (2-5-08) monash university malaysia jalan lagoon selatan 47500 bandar sunway selangor darul ehsan malaysia sathiya.maran@monash.edu adapt to human hosts and healthcare environments, causing detrimental effects to healthcare-associated infections such as bloodstream infections (2). amr is reported as the world’s biggest 21st-century health threat, and the world health organization (who) is calling for immediate action. as amr spreads, common infections are becoming incurable. reports state that over 700,000 die yearly due to drug resistant illnesses; by 2050, the number is predicted to rise to 10 million (3). a major issue pertaining to amr is the excessive and injudicious use of antibiotics that have led to widespread resistant bacteria and dissemination of their antimicrobial resistant genes (args) (4). it is concerning that the amr rates are predicted to increase if measures are not taken. one way to overcome this is through early detection, which enables effective management, allowing efficient identification and detection of microbes such that the patient can be treated with the appropriate drug in time. introduction antimicrobial resistance (amr) is defined as changes in bacteria that result in the drug being used for its treatment becoming inefficacious (1). staphylococcus aureus is an opportunistic pathogen with a tremendous capacity to https://doi.org/dti.2022.2522 https://creativecommons.org/licenses/by-nc/4.0/legalcode yi xing et al drug target insights 2022; 16: 89 © 2022 the authors. published by aboutscience www.aboutscience.eu over the years, great leaps have been made in the diagnosis of amr and diagnostic tests are reported to be an essential tool in early diagnosis, hence it is a robust strategy against amr (4). to enhance existing approaches, this review aims to summarize new and current molecular techniques and technologies used to identify amr using a systematic meta-narrative approach, with a focus on the key benefits and drawbacks. furthermore, a critical overview of recently developed molecular approaches and an informed assessment of future direction will also be discussed. methodology study design and inclusion criteria this systematic review was carried out in a meta narrative framework. this study qualitatively appraised different molecular methods used in the recent 5 years for the diagnosis of methicillin-resistant staphylococcus aureus (mrsa). this study protocol was created according to the rameses (realist and meta-narrative evidence syntheses: evolving standards) meta-narrative review publication guidelines (5). articles that satisfied the following requirements were considered for the review: (i) original articles written in english that were published between january 2017 and may 2022, (ii) cross-sectional or cohort studies that assessed the technical performance of molecular methods (sensitivity, specificity, accuracy or concordance) for diagnosing mrsa. articles were excluded if they were: (i) case reports; (ii) review articles, commentary articles, and short communications. search strategies articles were searched using pubmed. search keywords were (((((molecular diagnosis) and (antimicrobial resistance)) not (review [publication type)) not (systematic review [publication type)) not (meta-analysis [publication type)) and (methicillin-resistant staphylococcus aureus). selection and appraisal of articles two independent reviewers (lee and sim) screened the titles and abstracts. articles with abstracts indicating the use of a molecular approach to diagnose mrsa were read in full. a final consensus was discussed between the two reviewers, and disagreements were resolved with discussion from the third reviewer (sm). endnote version 20 was used for article duplicate removal and archives. all the studies reviewed and appraised in this synthesis are summarized in table i. table i summaries of studies appraised in this review no author year country condition/patients sample study design molecular diagnosis methods reference 1 moutaouakkil et al 2022 china children diagnosed with staphylococcus aureus oai blood cultures, articular fluids, synovial tissues and/or bone fragments prospective study multiplex polymerase chain reaction (6) 2 jin et al 2022 china 1,952 mssa strains isolated from blood across 17 provinces mssa-pens isolated from invasive bsis retrospective study whole-genome sequencing (2) 3 senok et al 2021 united arab emirates 135 patients with a clinical diagnosis of severe skin and soft-tissue infections s. aureus isolates associated with ssti were tested for pvl detection n/a dna microarray assays (7) 4 reddy and whitelaw 2021 south africa 231 samples 2,822 patients with positive blood cultures exclusively showing gpcc on gram stain were included prospective study xpert mrsa/ sa bc assay (8) 5 choi et al 2021 south korea 26 children aged <15 years diagnosed with ssss involved area of the skin, the presence of nikolsky’s sign, and the status of desquamation n/a pcr (9) 6 anafo et al 2021 ghana 300 diabetes patients and 106 non-diabetic individuals anterior nasal swabs cross-sectional pcr (10) 7 verdúexpósito et al 2020 ethiopia 80 s. aureus strains isolated from human patients with sstis human samples n/a maldi-tof and pcr (11) 8 tang et al 2020 china mrse strains from the dental plaque of a normal, healthy human population dental plaque specimens n/a pcr (12) molecular diagnosis of mrsa90 © 2022 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti no author year country condition/patients sample study design molecular diagnosis methods reference 9 khawaja et al 2020 pakistan 105 samples human samples descriptive cross-sectional study pcr (13) 10 jin et al 2020 china 65-year-old healthy man with a history of leprosy isolate was obtained from the patient’s blood, and identified as an st9-mrsa strain n/a whole-genome sequencing (14) 11 geng et al 2020 china 536 neonates nasal swabs prospective surveillance study staphylococcal chromosomal cassette (and) type, spa type, mlst (15) 12 crandall et al 2020 usa 357 children with invasive s. aureus infections pleural fluid and/or blood prospective study pcr, mlst, sccmec typing (16) 13 bouza et al 2020 spain 155 adult inpatients diagnosed with skin and soft-tissue infection microbiological samples prospective study gram stain plus genexpert® mssa/mrsa ssti (17) 14 yang et al 2019 china 269 nonduplicate s. aureus clinical isolates were isolated from children steril specimens and non-steril specimen using vitek ms system n/a maldi-tof (18) 15 mutonga et al 2019 kenya 83 adult patients diagnosed with diabetic foot ulcers wound swab cultures cross-sectional study real-time pcr (19) 16 latour et al 2019 belgium 1,447 residents from nursing homes pooled sampling of nose, throat and perineum cross-sectional prevalence survey triplex pcr and mlst (20) 17 hida et al 2019 japan 263 patients suspected of having staphylococcal bacteremia fresh and frozen blood culture samples n/a genecube meca (21) 18 luo et al 2018 china 275 isolates of s. aureus, including 148 isolates from patients, 127 from ready-toeat food samples secretions, blood, phlegm, cerebrospinal fluid, transudation, urine, fresh meat, meat product, cereal products, fruits and vegetables n/a pcr, multiplex pcr (22) 19 lin et al 2018 taiwan 106 hemodialysis patients diagnosed with mrsa blood cultures retrospective study pcr and mlst (23) 20 yang et al 2017 china 104 children diagnosed with mrsa sputum, bronchioalveolar lavage fluid, skin and soft tissues, pus, secretions, secretions of omphalitis, blood, joint effusion, pleural effusion n/a mlst (24) bsi = bloodstream infection; dna = deoxyribonucleic acid; maldi-tof = matrix-assisted laser desorption/ionization-time of flight; mlst = multilocus sequence typing; mrsa = methicillin-resistant staphylococcus aureus; mrse = methicillin-resistant staphylococcus epidermidis; mssa = methicillin-sensitive staphylococcus aureus; n/a = not available; pcr = polymerase chain reaction; sccmec = staphylococcal cassette chromosome mec; spa = staphylococcal protein a. gpcc = gram positive cocci in clusters; mssa-pens = methicillin-sensitive s. aureus – penicillin-susceptible; oai = osteoarticular infections; ssss = staphylococcal scalded skin syndrome ; ssti = skin and soft tissue infections; pvl = panton valentine leukocidin results the dataset includes 20 different authors from asia (n = 13), africa (n = 5), europe (n = 1) and america (n = 1). a total of 20 studies were included in this synthesis: seven studies employed polymerase chain reaction (pcr) for diagnosing mrsa (6,9,10,12,13,19,22), one study employed deoxyribonucleic acid (dna) microarray (7), two studies used dna sequencing (2,14), xpert mrsa/sa bc assay (n = 2) (8,17), matrix-assisted laser desorption/ionization-time yi xing et al drug target insights 2022; 16: 91 © 2022 the authors. published by aboutscience www.aboutscience.eu of flight (maldi-tof; n = 2) (11,18), multilocus sequence typing (mlst; n = 4) (15,20,23,24), genecube (n = 1) (21) and staphylococcal cassette chromosome mec (sccmec) typing (n = 1) (16). figure 1 is the diagrammatic flow of the study selection and list of techniques appraised in this review. recent molecular methods for diagnosis of mrsa polymerase chain reaction pcr approaches have been commonly used for the effective diagnosis of mrsa, and the rapid emergence of mrsa has led to a series of pcr approaches that have been developed for the identification of mrsa (25). pcr approach identifies s. aureus based on a single-base-pair mismatch in the staphylococcal 16s ribosomal rna gene sequence (26). recent researchers have also cited the use of the pcr approach for meca gene detection as the gold standard method for the detection and identification of the prevalence of mrsa (27,28). in this synthesis, a total of seven studies have employed pcr for the detection and diagnosis of mrsa. a study conducted by moutaouakkil and colleagues among patients suspected of s. aureus hospitalized in pediatric orthopedic clinic reported the detection of meca using pcr (6). this study also utilized different biological samples such as blood cultures, articular fluids, synovial tissues and bone fragments for the detection of mrsa. another study showed that the fluorescence signal of realtime (rt)-pcr could display the quantity of products formed and increases exponentially, enabling a user-friendly diagnostic (29). furthermore, mutonga and colleagues (2019) have demonstrated that the sensitivity of rt-pcr for mrsa is 100% (19). multiplex pcr amplifies multiple dna sequences simultaneously, which gives an advantage over conventional pcr (30). the detection of target sequences, such as the nuc and coaa or elements necessary for methicillin resistance, such as fema, or femb, has provided the basis for pcr identification of s. aureus. it uses two pairs of primers specific to the staphylococcal nuc and meca for pcr amplification of a 280bp nuc-based fragment and a 533-bp meca-based fragment (31). tsai and colleagues (2019) reported meca gene (meca-f and meca-r) is amplified and can be used to diagnose mrsa (32). chikkala and colleagues showed that it exhibits 97% of specificity and 90% sensitivity (33). dna sequencing dna sequencing allows the detection of single nucleotide polymorphisms (snps) and known resistanceassociated genes and their variations (34). the availability of bacterial genomes in public databases facilitates the use of whole-genome sequencing for mrsa detection. it enables high-resolution characterization of antibiotic resistance (35). whole-genome sequencing has a definite edge over conventional sanger sequencing because it may produce millions of reads that are roughly 35 to 700 bp in length (36). there is growing evidence on the effectiveness of bacterial records identi�ed from databases (n = 28) records removed before screening: duplicate records removed (n = 0) records screened (n = 28) reports sought for retrieval (n = 20) reports assessed for eligibility (n = 20) studies included in this review (n = 20) pcr (n = 7)in cl u d ed sc re en in g id en ti �c at io n dna sequencing (n = 2) records excluded (n = 8) reports not retrieved (n = 0) mlst (n = 4) sccmec typing (n = 1) dna microarray (n = 1) xpert mrsa/sa bc assay (n = 2) genecube (n = 1) maldi-tof (n = 2) fig. 1 diagrammatic flow of the study selection and list of techniques appraised in this review. molecular diagnosis of mrsa92 © 2022 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti whole-genome sequencing in controlling outbreaks. wholegenome analysis, such as dna microarray, simultaneously identifies relative concentration of different nucleic acid sequence (37). it allows a bulk number of nucleic acid sequences in a mixture to be tested and analyzed. the study by jin and colleagues (2,14) used staphytype dna microarray (abbott [alere technologies gmbh], jena, germany) and the inter-array genotyping kit s. aureus (interarray gmbh, bad langensalza, germany) for the detection of mrsa. the study by senok and colleagues (2021) also reported that dna microarray exhibited 100% specificity and sensitivity (7). in a study done by ma and fellow colleagues, illumina’s nextera dna library preparation kit was used to create whole-genome sequencing libraries, which were then sequenced on an illumina miseq using the 500 cycle v2 protocol (38). xpert mrsa/sa bc assay xpert mrsa/sa blood culture is an in vitro diagnostic test for s. aureus and mrsa. the targeted dna is amplified using automated rt-pcr and fluorogenic target-specific hybridization, providing real-time detection of specific genes of mrsa and s. aureus. a study by buchan and colleagues (39) reported the use of blood cultures for the detection of staphylococcus protein a (spa) sequences, gene that encodes for methicillin resistance (meca) and sccmec. a study by reddy and colleagues has shown the performance of the xpert mrsa/sa bc assay to be 100% in specificity and sensitivity. it shows a failure rate for an interpretable result of just 1.7% (8). however, it is notable that the microbiological sampling should be of high quality to ensure rapid and accurate results, despite the significance of xpert mrsa system. maldi-tof maldi-tof mass spectrometry (ms) has become a widely used technique for the rapid and accurate identification of bacteria (40). despite the efficiency and sensitivity of malditof, this method’s limitation is that new isolates can only be detected if the spectral database contains peptide mass fingerprints (pmfs) of the type strains of specific genera/species/subspecies/strains. this method identifies microbes by comparing the pmf of unknown organisms with the pmfs deposited in the database or matching the masses of biomarkers with the proteome database. a recent study by tang and colleagues (41) reported that maldi-tof ms on intact bacteria combined with a refined analysis framework allows accurate classification of methicillin-sensitive staphylococcus aureus (mssa) and mrsa. esener and colleagues showed that maldi-tof has a sensitivity of 99.93% ± 0.25%, specificity of 95.04% ± 3.83%, and accuracy = 97.54% ± 1.91% (42). maldi-tof is low in cost, and analysis can be conducted within a short time, allowing rapid microbial resistance to be detected. latour and colleagues employed maldi biotyper database for bacterial identification of suspected colonies (20). a study by chen and colleagues has shown that mlst has been used for the past decades for mrsa epidemiological typing (43). however, it is only based on the sequences of seven house-keeping genes’ internal fragments to identify individual isolate lineages. mlst mlst is a technique that distinguishes between isolates of bacteria species by utilizing sequences of internal fragment house-keeping genes (44). the strands are sequenced on both side by using an automated dna sequencer. different sequences of house-keeping genes found in bacterial species are characterized as distinct alleles. in contrast, seven loci alleles address each isolate’s allelic profile or sequence type. hence, species isolates are unambiguously characterized by a series of seven integers which label the alleles at the seven house-keeping genes. the seven house-keeping genes used in mlst for s. aureus are the carbamate kinase (arcc), shikimate dehydrogenase (aroe), glycerol kinase (glpf), guanylate kinase (gmk), phosphate acetyltransferase (pta), triosephosphate isomerase (tpi), acetyl coenzyme a acetyltransferase (18,24,45). spa typing spa is an important gene virulence factor that allows s. aureus to avoid host immune responses (46). it codes for protein a, which is found in the cell wall of s. aureus (47). spa genes were replicated using pcr followed by dna sequencing (48). this method identifies the polymorphic x region of the protein a gene (spa). based upon repeat pattern (burp) algorithm was used, and spa types with more than five repeats were clustered into different groups, with the calculated cost between group members being less than or equal to 6 (49). spa typing is evidently reproducible and provides interchangeable information. however, a disadvantage of this method is that it requires additional targets such as sccmec, lineage-specific virulence or resistance genes or alternative polymorphic regions of the s. aureus chromosome. studies included in this synthesis employed ridom staph database and spa typer tool (http://spatyper.fortinbras.us/) (24,50,51). reports cited that spa type of t437 was more prevalent in mrsa (24). a study by luo and colleagues showed that the most prominent spa type was t030, reported to be 15.64% (43/275) (22). genecube assays genecube (toyobo co., ltd., osaka, japan) is a fully automated genetic analyzer that uses pcr to amplify a target gene (21). this tool can evaluate up to eight samples simultaneously. the target dna is amplified, and fluorescently labeled oligonucleotides are used to hybridize targets based on fluorescence intensity changes (52). data are automatically obtained on the genecube monitor after completion of the assay. the advantage of this assay is that it is time efficient and easy to prepare. genecube tests are anticipated to be clinically valuable for effectively identifying mrsa. studies have reported the sensitivity and specificity http://spatyper.fortinbras.us/ yi xing et al drug target insights 2022; 16: 93 © 2022 the authors. published by aboutscience www.aboutscience.eu of the genecube to be 100% (33). the system is accurate, rapid (52 minutes), and reliable; however, it does not detect the mecc gene (21). sccmec typing sccmec is a diagnostic method that divides sccmec elements into groups based on their structural variations (53). the mec complex, which comprises the mec gene, its regulatory genes, the meci and mecr1 genes, and several insertion sequences, confers methicillin resistance (54,55). the specific sccmec type is determined by combining the ccr gene complex and the mec gene class. sccmec typing provides valuable information about the resistance of genes to methicillin and identifies the origin of strains. a recent study by chongtrakool et al (56) typed sccmec of methicillin resistant s. aureus strains isolated in 11 asian countries. another study showed that 610 of 615 (99.2%) mrsa strains could be classified into four sccmec elements: type 3a, 370 strains; type 2a, 207; type 2b, 32; type 1b, 1 strain. this study on pandemic mrsa clones in asia reported the st59sccmeciva as the most prevalent mrsa clone (15). a study by chen and colleagues that used the web-based sccmecfinder reported that this technique is efficient for detecting mrsa (43). sccmecfinder is a web-based tool for sccmec typing using whole-genome sequences (https://cge.cbs.dtu.dk/ services/sccmecfinder/, accessed on january 11, 2023). the sccmecfinder website uses read data for whole-genome sequencing or preassembled genome/contigs to determine homology to the complete cassette in prediction of sccmec types, mec complex and j regions (57). discussion this meta-narrative review reports the commonly used molecular methods for the detection of mrsa in the past 5 years. this review has also summarized the advantages and disadvantages of each technique included in this synthesis. s. aureus is a common cause of community and hospitalacquired infection (58,59). the who has regarded it as one of the primary clinical concerns, due to the global recognition of mrsa as a public health issue and the antibiotic resistance pattern of mrsa (60). the primary issue with mrsa is the incidence of multidrug resistance, which remains high (61). the meca encodes penicillin-binding protein 2a (pbp2a), which is an enzyme responsible for crosslinking peptidoglycans in the bacterial cell wall (62). the low affinity of pbp2a for β-lactams leads to resistance to β-lactam antibiotics, including penicillins, cephalosporins (except ceftaroline and ceftobiprole) and carbapenems (63). recent reports have reported growing resistance to clindamycin and levofloxacin, necessitating an effective treatment. the virulence factor of s. aureus is multifactorial and depends on a variety of toxins, adhesion, immune evasion and other virulence characteristics (64). evaluation of the virulence factor is an effective method of predicting how these bacteria would behave in the host, enabling prediction of the onset and progression of an infection. the first stage of staphylococcal infection is when the bacterial cells connect to the host’s tissues. the surface-exposed proteins, mscramms (microbial surface components recognizing adhesive matrix molecules), are made by s. aureus, which functions to attach to one or more host extracellular matrix (ecm) components, such as laminin, elastin, fibrinogen, fibronectin and collagen (65,66). the extracellular adherence protein (eap) produced by s. aureus is a member of the serams (secretable expanded repertoire adhesive molecules) family, binds to ecm glycoproteins, including fibronectin, fibrinogen, sialoprotein and collagens (67). this protein is involved in the internalization of bacteria and the adherence of s. aureus to fibroblasts. proteases are crucial virulence factors for s. aureus and can cleave host proteins to enable mrsa cells to change from an adhesive to an invasive phenotype. early diagnostic and therapeutic intervention in patients with mrsa infection risk factors is essential (68). treatment with empiric antibiotics against mrsa should not be delayed in the event that mrsa infection is diagnosed. molecular diagnostic tests can robustly identify staphylococcal species in clinical samples, thus improving antimicrobial stewardship (69). in this review, multiple molecular methods such as pcr, dna sequencing, xpert mrsa/sa bc array, maldi-tof, mlst, spa typing and sccmec typing, have been appraised. this review summarizes that pcr technique has been widely used for the diagnosis of mrsa within the last 5 years (2017-2022). pcr technique is frequently and commonly used to detect s. aureus and it identifies a single-base-pair mismatch in the staphylococcal 16s ribosomal rna gene sequence for detection (26). pcr assay is cost and labor effective and can be conducted within a short period of time (70,71). however, studies have reported that different target genes may impact the specificity and sensitivity of pcr for diagnosis. the nuc gene has a 100% success rate (25,72). several pcr techniques such as multiplex pcr, rt-pcr and isothermal identification have been developed to identify mrsa as a result of its rapid emergence. the meca and nuc genes are being used due to their 100% sensitivity and 97% specificity respectively with a shorter turnaround time of 48 hours (73,74). the second commonly used molecular techniques are sccmec typing and mlst, respectively. over the years, the structures of novel sccmec have been identified and verified by molecular cloning and traditional sequencing (75). in a study by singh-moodley and colleagues (76), sccmec typing method was used to replace multiplex pcr and was employed to classify additional un-typeable sccmec elements based on ccr and mec gene complex combinations. however, this technique has been deemed highly complex because the sccmec region is variable and newer types are permanently being developed. another possible reason for using sccmec typing could be its potential as a benchmark for testing for the ccr gene and meca gene compared to other methods. mlst is well-established and assigns alleles at multiple house-keeping loci directly by dna sequencing. sequence type is obtained based on the alleles identified at each of the seven loci using the sa mlst database. mlst detection of mrsa is based on the sequencing of the seven housekeeping conserved genes in the bacterial chromosome (77). mlst is also widely used due to its straightforward procedure for characterizing isolates of bacterial species (78). due to molecular diagnosis of mrsa94 © 2022 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti numerous alleles in each of the seven loci, it is unlikely that two isolates will have the same allelic profile. instead, isolates with the same allelic profile can be identified as belonging to the exact clone. mlst has several advantages: (1) it uses sequence data to detect changes at the dna level; (2) it is readily reproduced and does not require specialized reagents or training; (3) it does not require high-quality genomic dna; and (4) the data generated are fully portable (79). the disadvantage of mlst is that it only uses seven genes, limiting its ability. dna microarray and xpert mrsa/sa bc assay are the least used in the last 5 years. dna microarray contains covalently immobilized probes specific for about 180 genes and 300 alleles of s. aureus (80). it allows simultaneous detection of the presence of numerous genomic loci. studies have reported that dna microarray may serve as an alternate molecular typing method, offering complementary characterization of the mrsa strains. however, this technique is labor and cost extensive and a single experiment could significantly increase the budget of the experiment. subsequently, many probe designs are based on a sequence of relatively low specificity, sensitivity and accuracy (81). conclusion this meta-narrative review has appraised and summarized molecular diagnostic methods frequently used to detect mrsa in the last 5 years (2017-2022), thus concluding that pcr technique is the most frequently used technique due to its high specificity, low cost and labor effectiveness. acknowledgment the authors would like to acknowledge the higher colleges of technology interdisciplinary research grant (interdisciplinary_212322). disclosure conflict of interest: all authors declare no conflict of interest. references 1. murray cj, ikuta ks, sharara f, et al; antimicrobial resistance collaborators. global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. lancet. 2022;399(10325): 629-655. crossref pubmed 2. jin y, zhou w, zhan q, et al. genomic epidemiology and characterisation of penicillin-sensitive staphylococcus aureus isolates from invasive bloodstream infections in china: an increasing prevalence and higher diversity in genetic typing be revealed. emerg microbes infect. 2022;11(1):326-336. crossref pubmed 3. hanson c, gabrysch s, mbaruku g, cox j, mkumbo e, manzi f, schellenberg j, ronsmans c. access to maternal health services: geographical inequalities, united republic of tanzania. bull world health organ. 2017 dec 1;95(12):810-820. crossref epub 2017 oct 31. pubmed pmcid: pmc5710083. 4. kaprou gd, bergšpica i, alexa ea, alvarez-ordóñez a, prieto m. rapid methods for antimicrobial resistance diagnostics. antibiotics (basel). 2021;10(2):209. crossref pubmed 5. wong g, greenhalgh t, westhorp g, buckingham j, pawson r. rameses publication standards: meta-narrative reviews. j adv nurs. 2013;69(5):987-1004. crossref pubmed 6. moutaouakkil k, abdellaoui h, arhoune b, et al. paediatric osteoarticular infections caused by staphylococcus aureus producing panton-valentine leucocidin in morocco: risk factors and clinical features. afr j paediatr surg. 2022;19(2):78-82. crossref pubmed 7. senok a, monecke s, nassar r, et al. lateral flow immunoassay for the detection of panton-valentine leukocidin in staphylococcus aureus from skin and soft tissue infections in the united arab emirates. front cell infect microbiol. 2021; 11:754523. crossref pubmed 8. reddy k, whitelaw a. can the xpert mrsa/sa bc assay be used as an antimicrobial stewardship tool? a prospective assay validation and descriptive impact assessment study in a south african setting. bmc infect dis. 2021;21(1):177. crossref pubmed 9. choi jh, lee h, choi eh. antimicrobial resistance and molecular analysis of staphylococcus aureus in staphylococcal scalded skin syndrome among children in korea. j korean med sci. 2021;36(3):e22. crossref pubmed 10. anafo rb, atiase y, kotey fcn, et al. methicillin-resistant staphylococcus aureus (mrsa) nasal carriage among patients with diabetes at the korle bu teaching hospital. plos one. 2021;16(9):e0257004. crossref pubmed 11. verdú-expósito c, romanyk j, cuadros-gonzález j, et al. study of susceptibility to antibiotics and molecular characterization of high virulence staphylococcus aureus strains isolated from a rural hospital in ethiopia. plos one. 2020;15(3):e0230031. crossref pubmed 12. tang b, gong t, cui y, et al. characteristics of oral methicillinresistant staphylococcus epidermidis isolated from dental plaque. int j oral sci. 2020;12(1):15. crossref pubmed 13. khawaja a, arshad f, khan i. comparison of phenotypic methods with meca gene based polymerase chain reaction for methicillin-resistant staphylococcus aureus detection. j pak med assoc. 2020;70(2):276-280. pubmed 14. jin y, yu x, chen y, et al. characterization of highly virulent community-associated methicillin-resistant staphylococcus aureus st9-sccmec xii causing bloodstream infection in china. emerg microbes infect. 2020;9(1):2526-2535. crossref pubmed 15. geng w, qi y, li w, et al. epidemiology of staphylococcus aureus in neonates on admission to a chinese neonatal intensive care unit. plos one. 2020;15(2):e0211845. crossref pubmed 16. crandall h, kapusta a, killpack j, et al. clinical and molecular epidemiology of invasive staphylococcus aureus infection in utah children; continued dominance of mssa over mrsa. plos one. 2020;15(9):e0238991. crossref pubmed 17. bouza e, onori r, semiglia-chong ma, álvarez-uría a, alcalá l, burillo a. fast track ssti management program based on a rapid molecular test (genexpert® mrsa/sa ssti) and antimicrobial stewardship. j microbiol immunol infect. 2020;53(2):328-335. crossref pubmed 18. yang x, dong f, qian s, et al. accessory gene regulator (agr) dysfunction was unusual in staphylococcus aureus isolated from chinese children. bmc microbiol. 2019;19(1):95. crossref pubmed 19. mutonga dm, mureithi mw, ngugi nn, otieno fcf. bacterial isolation and antibiotic susceptibility from diabetic foot ulcers in kenya using microbiological tests and comparison with rt-pcr in detection of s. aureus and mrsa. bmc res notes. 2019;12(1):244. crossref pubmed 20. latour k, huang td, jans b, et al. prevalence of multidrug resistant organisms in nursing homes in belgium in 2015. plos one. 2019;14(3):e0214327. crossref pubmed https://doi.org/10.1016/s0140-6736(21)02724-0 https://www.ncbi.nlm.nih.gov/pubmed/35065702 https://doi.org/10.1080/22221751.2022.2027218 https://www.ncbi.nlm.nih.gov/pubmed/34991434 https://doi.org/10.2471/blt.17.194126 https://www.ncbi.nlm.nih.gov/pubmed/29200522 https://doi.org/10.3390/antibiotics10020209 https://www.ncbi.nlm.nih.gov/pubmed/33672677 https://doi.org/10.1111/jan.12092 https://www.ncbi.nlm.nih.gov/pubmed/23356699 https://doi.org/10.4103/ajps.ajps_18_21 https://www.ncbi.nlm.nih.gov/pubmed/35017376 https://doi.org/10.3389/fcimb.2021.754523 https://www.ncbi.nlm.nih.gov/pubmed/34733796 https://doi.org/10.1186/s12879-021-05857-7 https://www.ncbi.nlm.nih.gov/pubmed/33588782 https://doi.org/10.3346/jkms.2021.36.e22 https://www.ncbi.nlm.nih.gov/pubmed/33463096 https://doi.org/10.1371/journal.pone.0257004 https://www.ncbi.nlm.nih.gov/pubmed/34534230 https://doi.org/10.1371/journal.pone.0230031 https://www.ncbi.nlm.nih.gov/pubmed/32163464 https://doi.org/10.1038/s41368-020-0079-5 https://www.ncbi.nlm.nih.gov/pubmed/32385260 https://www.ncbi.nlm.nih.gov/pubmed/32063621 https://doi.org/10.1080/22221751.2020.1848354 https://www.ncbi.nlm.nih.gov/pubmed/33174510 https://doi.org/10.1371/journal.pone.0211845 https://www.ncbi.nlm.nih.gov/pubmed/32053585 https://doi.org/10.1371/journal.pone.0238991 https://www.ncbi.nlm.nih.gov/pubmed/32946486 https://doi.org/10.1016/j.jmii.2018.07.008 https://www.ncbi.nlm.nih.gov/pubmed/30224283 https://doi.org/10.1186/s12866-019-1465-z https://www.ncbi.nlm.nih.gov/pubmed/31088356 https://doi.org/10.1186/s13104-019-4278-0 https://www.ncbi.nlm.nih.gov/pubmed/31036061 https://doi.org/10.1371/journal.pone.0214327 https://www.ncbi.nlm.nih.gov/pubmed/30921364 yi xing et al drug target insights 2022; 16: 95 © 2022 the authors. published by aboutscience www.aboutscience.eu 21. hida y, uemura k, sugimoto h, et al. evaluation of performance of the genecube assay for rapid molecular identification of staphylococcus aureus and methicillin resistance in positive blood culture medium. plos one. 2019;14(7):e0219819. crossref pubmed 22. luo k, shao f, kamara kn, et al. molecular characteristics of antimicrobial resistance and virulence determinants of staphylococcus aureus isolates derived from clinical infection and food. j clin lab anal. 2018;32(7):e22456. crossref pubmed 23. lin s-y, tu hp, chen tc, et al. association of bacterial genotypes and epidemiological features with treatment failure in hemodialysis patients with methicillin-resistant staphylococcus aureus bacteremia. plos one. 2018;13(6):e0198486. crossref pubmed 24. yang x, qian s, yao k, et al. multiresistant st59-sccmec iv-t437 clone with strong biofilm-forming capacity was identified predominantly in mrsa isolated from chinese children. bmc infect dis. 2017;17(1):733. crossref pubmed 25. liu y, zhang j, ji y. pcr-based approaches for the detection of clinical methicillin-resistant staphylococcus aureus. open microbiol j. 2016;10(1):45-56. crossref pubmed 26. saruta k, hoshina s, machida k. genetic identification of staphylococcus aureus by polymerase chain reaction using single-base-pair mismatch in 16s ribosomal rna gene. microbiol immunol. 1995;39(11):839-844. crossref pubmed 27. woods se, beiter e, drake b, engel a. the prevalence of asymptomatic methicillin-resistant staphylococcus aureus in schoolage children. east j med. 2011;16(1):18-21. 28. sahebnasagh r, saderi h, owlia p. the prevalence of resistance to methicillin in staphylococcus aureus strains isolated from patients by pcr method for detection of meca and nuc genes. iran j public health. 2014;43(1):84-92. pubmed 29. kubista m, andrade jm, bengtsson m, et al. the real-time polymerase chain reaction. mol aspects med. 2006;27(2-3): 95-125. crossref pubmed 30. markoulatos p, siafakas n, moncany m. multiplex polymerase chain reaction: a practical approach. j clin lab anal. 2002;16(1):47-51. crossref pubmed 31. barski p, piechowicz l, galiński j, kur j. rapid assay for detection of methicillin-resistant staphylococcus aureus using multiplex pcr. mol cell probes. 1996;10(6):471-475. crossref pubmed 32. tsai yh, chen ph, yu pa, chen cl, kuo lt, huang kc. a multiplex pcr assay for detection of vibrio vulnificus, aeromonas hydrophila, methicillin-resistant staphylococcus aureus, streptococcus pyogenes, and streptococcus agalactiae from the isolates of patients with necrotizing fasciitis. int j infect dis. 2019;81:73-80. crossref pubmed 33. chikkala r, ch s, divyakolu s, ratnakar ks, sritharan v. a simple sample processing protocol and multiplex pcr for direct detection of mrsa from uncultured clinical samples – a pilot study. adv infect dis. 2019;9(1):25-38. crossref 34. sanchini a. recent developments in phenotypic and molecular diagnostic methods for antimicrobial resistance detection in staphylococcus aureus: a narrative review. diagnostics (basel). 2022;12(1):208. crossref pubmed 35. gautam ss, kc r, leong kw, mac aogáin m, o’toole rf. a stepby-step beginner’s protocol for whole genome sequencing of human bacterial pathogens. j biol methods. 2019;6(1):e110. crossref pubmed 36. lakhundi s, zhang k. methicillin-resistant staphylococcus aureus: molecular characterization, evolution, and epidemio logy. clin microbiol rev. 2018;31(4):e00020-18. crossref pubmed 37. bumgarner r. overview of dna microarrays: types, applications, and their future. curr protoc mol biol. 2013 jan;chapter 22:unit 22.1. crossref 38. ma z, lasek-nesselquist e, lu j, et al. characterization of genetic changes associated with daptomycin nonsusceptibility in staphylococcus aureus. plos one. 2018;13(6):e0198366. crossref pubmed 39. buchan bw, allen s, burnham ca, et al. comparison of the nextgeneration xpert mrsa/sa bc assay and the geneohm staphsr assay to routine culture for identification of staphylococcus aureus and methicillin-resistant s. aureus in positive-blood-culture broths. j clin microbiol. 2015;53(3):804-809. crossref pubmed 40. feucherolles m, poppert s, utzinger j, becker sl. maldi-tof mass spectrometry as a diagnostic tool in human and veterinary helminthology: a systematic review. parasit vectors. 2019;12(1):245. crossref pubmed 41. tang w, ranganathan n, shahrezaei v, larrouy-maumus g. maldi-tof mass spectrometry on intact bacteria combined with a refined analysis framework allows accurate classification of mssa and mrsa. plos one. 2019;14(6):e0218951. crossref pubmed 42. esener n, maciel-guerra a, giebel k, et al. mass spectrometry and machine learning for the accurate diagnosis of benzylpenicillin and multidrug resistance of staphylococcus aureus in bovine mastitis. plos comput biol. 2021;17(6):e1009108. crossref pubmed 43. chen y, hong j, chen y, wang h, yu y, qu t. characterization of a community-acquired methicillin-resistant sequence type 338 staphylococcus aureus strain containing a staphylococcal cassette chromosome mec type v t . int j infect dis. 2020;90:181187. crossref pubmed 44. maiden mcj, bygraves ja, feil e, et al. multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms. proc natl acad sci usa. 1998;95(6):3140-3145. crossref pubmed 45. park sg, lee hs, park jy, lee h. molecular epidemiology of staphylococcus aureus in skin and soft tissue infections and bone and joint infections in korean children. j korean med sci. 2019;34(49):e315. crossref pubmed 46. votintseva aa, fung r, miller rr, et al. prevalence of staphylococcus aureus protein a (spa) mutants in the community and hospitals in oxfordshire. bmc microbiol. 2014;14(1):63. crossref pubmed 47. keener ab, thurlow lt, kang s, et al. staphylococcus aureus protein a disrupts immunity mediated by long-lived plasma cells. j immunol. 2017;198(3):1263-1273. crossref pubmed 48. goudarzi m, fazeli m, goudarzi h, azad m, seyedjavadi ss. spa typing of staphylococcus aureus strains isolated from clinical specimens of patients with nosocomial infections in tehran, iran. jundishapur j microbiol. 2016;9(7):e35685. crossref pubmed 49. strommenger b, braulke c, heuck d, et al. spa typing of staphylococcus aureus as a frontline tool in epidemiological typing. j clin microbiol. 2008;46(2):574-581. crossref pubmed 50. koreen l, ramaswamy sv, graviss ea, naidich s, musser jm, kreiswirth bn. spa typing method for discriminating among staphylococcus aureus isolates: implications for use of a single marker to detect genetic microand macrovariation. j clin microbiol. 2004;42(2):792-799. crossref pubmed 51. toleman ms, reuter s, jamrozy d, et al. prospective genomic surveillance of methicillin-resistant staphylococcus aureus (mrsa) associated with bloodstream infection, england, 1 october 2012 to 30 september 2013. euro surveill. 2019; 24(4):1800215. crossref pubmed 52. tani h, miyata r, ichikawa k, et al. universal quenching probe system: flexible, specific, and cost-effective real-time polymerase chain reaction method. anal chem. 2009;81(14):56785685. crossref pubmed https://doi.org/10.1371/journal.pone.0219819 https://www.ncbi.nlm.nih.gov/pubmed/31310615 https://doi.org/10.1002/jcla.22456 https://www.ncbi.nlm.nih.gov/pubmed/29676483 https://doi.org/10.1371/journal.pone.0198486 https://www.ncbi.nlm.nih.gov/pubmed/29864149 https://doi.org/10.1186/s12879-017-2833-7 https://www.ncbi.nlm.nih.gov/pubmed/29178841 https://doi.org/10.2174/1874285801610010045 https://www.ncbi.nlm.nih.gov/pubmed/27335617 https://doi.org/10.1111/j.1348-0421.1995.tb03280.x https://www.ncbi.nlm.nih.gov/pubmed/8657010 https://www.ncbi.nlm.nih.gov/pubmed/26060684 https://doi.org/10.1016/j.mam.2005.12.007 https://www.ncbi.nlm.nih.gov/pubmed/16460794 https://doi.org/10.1002/jcla.2058 https://www.ncbi.nlm.nih.gov/pubmed/11835531 https://doi.org/10.1006/mcpr.1996.0066 https://www.ncbi.nlm.nih.gov/pubmed/9025087 https://doi.org/10.1016/j.ijid.2019.01.037 https://www.ncbi.nlm.nih.gov/pubmed/30690211 https://doi.org/10.4236/aid.2019.91003 https://doi.org/10.3390/diagnostics12010208 https://www.ncbi.nlm.nih.gov/pubmed/35054375 https://doi.org/10.14440/jbm.2019.276 https://www.ncbi.nlm.nih.gov/pubmed/31453259 https://doi.org/10.1128/cmr.00020-18 https://www.ncbi.nlm.nih.gov/pubmed/30209034 https://doi.org/10.1002/0471142727.mb2201s101 https://doi.org/10.1371/journal.pone.0198366 https://www.ncbi.nlm.nih.gov/pubmed/29879195 https://doi.org/10.1128/jcm.03108-14 https://www.ncbi.nlm.nih.gov/pubmed/25540397 https://doi.org/10.1186/s13071-019-3493-9 https://www.ncbi.nlm.nih.gov/pubmed/31101120 https://doi.org/10.1371/journal.pone.0218951 https://www.ncbi.nlm.nih.gov/pubmed/31247021 https://doi.org/10.1371/journal.pcbi.1009108 https://www.ncbi.nlm.nih.gov/pubmed/34115749 https://doi.org/10.1016/j.ijid.2019.10.034 https://www.ncbi.nlm.nih.gov/pubmed/31682959 https://doi.org/10.1073/pnas.95.6.3140 https://www.ncbi.nlm.nih.gov/pubmed/9501229 https://doi.org/10.3346/jkms.2019.34.e315 https://www.ncbi.nlm.nih.gov/pubmed/31858755 https://doi.org/10.1186/1471-2180-14-63 https://www.ncbi.nlm.nih.gov/pubmed/24621342 https://doi.org/10.4049/jimmunol.1600093 https://www.ncbi.nlm.nih.gov/pubmed/28031339 https://doi.org/10.5812/jjm.35685 https://www.ncbi.nlm.nih.gov/pubmed/27679706 https://doi.org/10.1128/jcm.01599-07 https://www.ncbi.nlm.nih.gov/pubmed/18032612 https://doi.org/10.1128/jcm.42.2.792-799.2004 https://www.ncbi.nlm.nih.gov/pubmed/14766855 https://doi.org/10.2807/1560-7917.es.2019.24.4.1800215 https://www.ncbi.nlm.nih.gov/pubmed/30696529 https://doi.org/10.1021/ac900414u https://www.ncbi.nlm.nih.gov/pubmed/19530673 molecular diagnosis of mrsa96 © 2022 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti 53. asadollahi p, farahani nn, mirzaii m, et al. distribution of the most prevalent spa types among clinical isolates of methicillinresistant and-susceptible staphylococcus aureus around the world: a review. front microbiol. 2018;9:163. crossref pubmed 54. international working group on the classification of staphylococcal cassette chromosome elements (iwg-scc). classification of staphylococcal cassette chromosome mec (sccmec): guidelines for reporting novel sccmec elements. antimicrob agents chemother. 2009;53:4961-4967. crossref pubmed 55. ito t, hiramatsu k, tomasz a, et al; international working group on the classification of staphylococcal cassette chromosome elements (iwg-scc). guidelines for reporting novel meca gene homologues. antimicrob agents chemother. 2012;56(10):49974999. crossref pubmed 56. chongtrakool p, ito t, ma xx, et al. staphylococcal cassette chromosome mec (sccmec) typing of methicillin-resistant staphylococcus aureus strains isolated in 11 asian countries: a proposal for a new nomenclature for sccmec elements. antimicrob agents chemother. 2006;50(3):1001-1012. crossref pubmed 57. kaya h, hasman h, larsen j, et al. scc mec finder, a web-based tool for typing of staphylococcal cassette chromosome mec in staphylococcus aureus using whole-genome sequence data. msphere. 2018;3(1):e00612-e00617. crossref pubmed 58. rajput a, poudel s, tsunemoto h, et al. identifying the effect of vancomycin on health care-associated methicillin resistant staphylococcus aureus strains using bacteriological and physiological media. gigascience. 2021;10(1):giaa156. crossref pubmed 59. panwala t, gandhi p, jethwa d. inducible clindamycin resistance and mrsa amongst staphylococcus aureus isolates: a phenotypic detection. ip int j med microbiol trop dis 2020; 6(4):222–226. crossref 60. liu f, rajabi s, shi c, et al. antibacterial activity of recently approved antibiotics against methicillin-resistant staphylococcus aureus (mrsa) strains: a systematic review and meta-analysis. ann clin microbiol antimicrob. 2022;21(1):37. crossref pubmed 61. kistler jm, vroome cm, ramsey fv, ilyas am. increasing multidrug antibiotic resistance in mrsa infections of the hand: a 10-year analysis of risk factors. hand (n y). 2020;15(6): 877-881. crossref pubmed 62. kot b, piechota m, jakubczak a, et al. the prevalence of virulence determinants in methicillin-resistant staphylococcus aureus isolated from different infections in hospitalized patients in poland. sci rep. 2022;12(1):5477. crossref pubmed 63. turner na, sharma-kuinkel bk, maskarinec sa, et al. methicillinresistant staphylococcus aureus: an overview of basic and clinical research. nat rev microbiol. 2019;17(4):203-218. crossref pubmed 64. otto m. mrsa virulence and spread. cell microbiol. 2012; 14(10):1513-1521. crossref pubmed 65. moormeier de, bayles kw. staphylococcus aureus biofilm: a complex developmental organism. mol microbiol. 2017; 104(3):365-376. crossref pubmed 66. liu y, zhang j, ji y. environmental factors modulate biofilm formation by staphylococcus aureus. sci prog. 2020 jan-mar; 103(1):36850419898659. crossref pubmed 67. harraghy n, hussain m, haggar a, et al. the adhesive and immunomodulating properties of the multifunctional staphylococcus aureus protein eap. microbiology (reading). 2003;149(pt 10): 2701-2707. crossref pubmed 68. siddiqui ah, koirala j. methicillin resistant staphylococcus aureus. in: statpearls. [internet] statpearls publishing 2021. 69. tenover fc, tickler ia, le vm, dewell s, mendes re, goering rv. updating molecular diagnostics for detecting methicillin susceptible and methicillin-resistant staphylococcus aureus isolates in blood culture bottles. j clin microbiol. 2019;57 (11):e01195-e19. crossref pubmed 70. martineau f, picard fj, roy ph, ouellette m, bergeron mg. species-specific and ubiquitous-dna-based assays for rapid identification of staphylococcus aureus. j clin microbiol. 1998; 36(3):618-623. crossref pubmed 71. tübbicke a, hübner c, hübner no, wegner c, kramer a, fleßa s. cost comparison of mrsa screening and management – a decision tree analysis. bmc health serv res. 2012;12(1):438. crossref pubmed 72. brakstad og, aasbakk k, maeland ja. detection of staphylococcus aureus by polymerase chain reaction amplification of the nuc gene. j clin microbiol. 1992;30(7):1654-1660. crossref pubmed 73. chen c, zhao q, guo j, li y, chen q. identification of methicillin resistant staphylococcus aureus (mrsa) using simultaneous detection of meca, nuc, and femb by loop-mediated isothermal amplification (lamp). curr microbiol. 2017;74(8):965-971. crossref pubmed 74. zhao l, huang x, zhang t, et al. a point-of-care test device for mrsa rapid detection. j pharm biomed anal. 2022;209:114464. crossref pubmed 75. uehara y. current status of staphylococcal cassette chromosome mec (sccmec). antibiotics (basel). 2022;11(1):86. crossref pubmed 76. singh-moodley a, strasheim w, mogokotleng r, ismail h, perovic o. unconventional sccmec types and low prevalence of the panton-valentine leukocidin exotoxin in south african blood culture staphylococcus aureus surveillance isolates, 2013-2016. plos one. 2019;14(11):e0225726. crossref pubmed 77. burgold-voigt s, monecke s, simbeck a, et al. characterisation and molecular analysis of an unusual chimeric methicillin resistant staphylococcus aureus strain and its bacteriophages. front genet. 2021;12:723958-723958. crossref pubmed 78. kaku n, sasaki d, ota k, miyazaki t, yanagihara k. changing molecular epidemiology and characteristics of mrsa isolated from bloodstream infections: nationwide surveillance in japan in 2019. j antimicrob chemother. 2022;77(8):2130-2141. crossref pubmed 79. lukassen mb, saunders am, sindilariu p-d, nielsen jl. quantification of novel geosmin-producing bacteria in aquaculture systems. aquaculture. 2017;479:304-310. crossref 80. szabó j. molecular methods in epidemiology of methicillin resistant staphylococcus aureus (mrsa): advantages, disadvantages of different techniques. j med microbiol diagn. 2014; 3(3):1. crossref 81. jaksik r, iwanaszko m, rzeszowska-wolny j, kimmel m. microarray experiments and factors which affect their reliability. biol direct. 2015;10(1):46. crossref pubmed https://doi.org/10.3389/fmicb.2018.00163 https://www.ncbi.nlm.nih.gov/pubmed/29487578 https://doi.org/10.1128/aac.00579-09 https://pubmed.ncbi.nlm.nih.gov/19721075/ https://doi.org/10.1128/aac.01199-12 https://www.ncbi.nlm.nih.gov/pubmed/22869575 https://doi.org/10.1128/aac.50.3.1001-1012.2006 https://www.ncbi.nlm.nih.gov/pubmed/16495263 https://doi.org/10.1128/msphere.00612-17 https://www.ncbi.nlm.nih.gov/pubmed/29468193 https://doi.org/10.1093/gigascience/giaa156 https://www.ncbi.nlm.nih.gov/pubmed/33420779 https://doi.org/10.18231/j.ijmmtd.2020.050 https://doi.org/10.1186/s12941-022-00529-z https://www.ncbi.nlm.nih.gov/pubmed/35978400 https://doi.org/10.1177/1558944719837693 https://www.ncbi.nlm.nih.gov/pubmed/30897954 https://doi.org/10.1038/s41598-022-09517-x https://www.ncbi.nlm.nih.gov/pubmed/35361858 https://doi.org/10.1038/s41579-018-0147-4 https://www.ncbi.nlm.nih.gov/pubmed/30737488 https://doi.org/10.1111/j.1462-5822.2012.01832.x https://www.ncbi.nlm.nih.gov/pubmed/22747834 https://doi.org/10.1111/mmi.13634 https://www.ncbi.nlm.nih.gov/pubmed/28142193 https://doi.org/10.1177/0036850419898659 https://www.ncbi.nlm.nih.gov/pubmed/31902330 https://doi.org/10.1099/mic.0.26465-0 https://www.ncbi.nlm.nih.gov/pubmed/14523103 https://doi.org/10.1128/jcm.01195-19 https://www.ncbi.nlm.nih.gov/pubmed/31484703 https://doi.org/10.1128/jcm.36.3.618-623.1998 https://www.ncbi.nlm.nih.gov/pubmed/9508283 https://doi.org/10.1186/1472-6963-12-438 https://www.ncbi.nlm.nih.gov/pubmed/23198880 https://doi.org/10.1128/jcm.30.7.1654-1660.1992 https://www.ncbi.nlm.nih.gov/pubmed/1629319 https://doi.org/10.1007/s00284-017-1274-2 https://www.ncbi.nlm.nih.gov/pubmed/28573341 https://doi.org/10.1016/j.jpba.2021.114464 https://www.ncbi.nlm.nih.gov/pubmed/34915322 https://doi.org/10.3390/antibiotics11010086 https://www.ncbi.nlm.nih.gov/pubmed/35052963 https://doi.org/10.1371/journal.pone.0225726 https://www.ncbi.nlm.nih.gov/pubmed/31774877 https://doi.org/10.3389/fgene.2021.723958 https://www.ncbi.nlm.nih.gov/pubmed/34868203 https://doi.org/10.1093/jac/dkac154 https://www.ncbi.nlm.nih.gov/pubmed/35639590 https://doi.org/10.1016/j.aquaculture.2017.06.004 https://doi.org/10.4172/2161-0703.1000147 https://doi.org/10.1186/s13062-015-0077-2 https://www.ncbi.nlm.nih.gov/pubmed/26335588 untitled 9 correspondence: kunitada shimotohno, ph. d., laboratory of human tumor viruses, institute for virus research, kyoto university, 53 kawaharacho, shogoin, sakyo-ku, kyoto, 606-8507, japan. tel: +81-75-751-4000; fax: +81-75-751-3998; email: kshimoto@virus.kyoto-u.ac.jp please note that this article may not be used for commercial purposes. for further information please refer to the copyright statement at http://www.la-press.com/copyright.htm review cyclophilin and viruses: cyclophilin as a cofactor for viral infection and possible anti-viral target koichi watashi and kunitada shimotohno department of viral oncology, institute for virus research, kyoto university, kyoto, japan. abstract: cyclophilin (cyp) is a peptidyl prolyl cis/trans isomerase, catalyzing the cis-trans isomerization of proline residues in proteins. cyp plays key roles in several different aspects of cellular physiology including the immune response, transcription, mitochondrial function, cell death, and chemotaxis. in addition to these cellular events, a number of reports demonstrated that cyp plays a critical role in the life cycle of viruses, especially human immunodefi ciency virus (hiv) and hepatitis c virus (hcv). these two viruses are signifi cant causes of morbidity and mortality worldwide, but current therapies are often insuffi cient. cyp may provide a novel therapeutic target for the management and/or cure of these diseases, in particular hcv. keywords: cyclosporin, hiv, hcv, virus, replication, mptp. immunophilins and immunosuppressants cyclophilin (cyp) and fk506 binding protein (fkbp) are peptidyl-prolyl cis-trans isomerases (ppiases), enzymes that catalyze the cis-trans interconversion of peptide bonds amino terminal to proline residues (fischer et al. 1989; harding et al. 1989; takahashi, 1999; takahashi et al. 1989). cyp and fkbp are originally identifi ed as cellular factors that bind csa and fk506, respectively, both of which are immunosuppressants used clinically for the prevention of graft rejection following organ transplantation (handschumacher et al. 1984; harding et al. 1989). therefore, these ppiases are also called immunophilin. the action of ppiases leads to changes in protein conformation (takahashi, 1999), but the binding of csa and fk506 to cyp and fkbp, respectively, inhibits the activity of these enzymes (fischer et al. 1989; rosen et al. 1990; takahashi et al. 1989). however, the inhibition of ppiase activity by csa and fk506 is an insuffi cient requirement for their immunosuppressive function (bierer et al. 1990; schreiber, 1991). the csa/cyp or fk506/fkbp complex, subsequently interacts with and inhibits calcineurin (cn), a phosphatase involved in the activation of the transcription factor nf-at. proper nf-at function is essential for the generation of a productive t cell response (clipstone and crabtree, 1992; fruman et al. 1992; liu et al. 1991). in the absence of immunosuppressants, cn dephosphorylates cytoplasmic nf-at, leading to nf-at nuclear translocation and transactivation of downstream genes participating in the immune response (liu et al. 1992; mccaffrey et al. 1993). csa and fk506 prevent the dephosphorylation and subsequent nuclear translocation of nf-at leading to immunosuppression. role of cyp family members in cellular events more than 10 cyp subtypes are found in mammals (table 1). the subcellular localization of cyps varies. cypa is primarily found in the cytoplasm, while cypb, cypd, cype, and ranbp2 are distributed in the endoplasmic reticulum (er), mitochondria, nucleus, and nuclear pore, respectively. members of the cyp family play roles in a variety of cellular processes including the immune response, transcription, mitochondrial function, cell death, and chemotaxis, as described below. while a number of cyp family members have been ideitifi ed, intensive functional analysis has been performed on only a few including cypa, cypb, cypd, and cyp40. cypa is the most abundant cyp subtype found in the cells (waldmeier et al. 2003), and it is the primary factor mediating the immunosuppressive effects of csa (colgan et al. 2005). however, even in the absence of csa, cypa plays an important role in regulating the immune responses as seen in drug target insights 2007: 2 9–18 10 watashi and shimotohno cypa-defi cient mice. cypa-knockout mice have an “allergic” phenotype with increased serum igg1 and ige levels and tissue infi ltration by mononuclear cells, eosinophils, and mast cells (colgan et al. 2004), related to increased and dysregulated activity of th2 cd4+ t cells. in cypa-knockout cells, interleukin-2 tyrosine kinase (itk), a signaling molecule crucial for the development of a th2 response, is constitutively activated. itk is a member of the tec family of sh2/sh3-containing tyrosine kinases, and it participates in the signal transduction cascade leading to t cell activation. cypa can bind itk, and this negatively regulates itk activity (brazin et al. 2002). thus, cypa plays a suppressive role in the development of cd4+ t cell responses through its interaction with itk. o t h e r s t u d i e s h a v e r e p o r t e d s e v e r a l non-immune system roles for cypa. cypa interacts with apoptosis-inducing factor (aif) and promotes aif-mediated chromatinolysis during apoptosis (cande et al. 2004). additionally, cypa interacts with membrane-bound guanylate cyclase-a (gc-a), a receptor for atrial natriuretic factor (anf) (chen et al. 2004). gc-a and anf are involved in cardiovascular homeostasis, and cypa appears to function as an endogenous inhibitor of gc-a activation by competing for anf binding. further interactions of cypa with prolactin receptor (syed et al. 2003) and transcription factor yy1 (yang et al. 1995) have been observed, but the exact role of cypa in these processes remains unclear. cypa was also observed to bind dna in a zinc-dependent manner in a mouse macrophage cell line (krummrei et al. 1995). however, the best-characterized role identified for cypa is not in normal cellular physiology, but rather as co-factor during the human immunodefi ciency virus-1 (hiv-1) viral life cycle (see below). cypb was originally identifi ed as a cyp family member bearing a signal sequence leading to the er lumen or the secretory pathway (price et al. 1991), but the specifi c function of cypb is poorly understood. a yeast two-hybrid screening using cypb as a bait identified an interaction with calcium-signal modulating cyclophilin ligand (caml) (bram and crabtree, 1994). caml is located on the cytoplasmic face of the er membrane (holloway and bram, 1998). caml participates in calcium signal transduction pathway and it is essential for peripheral t cell development (tran et al. 2005). however, the importance of cypb binding to caml function remains unknown. cypb also enhances prolactin-driven cell proliferation (rycyzyn et al. 2000) and promotes the nuclear retrotranslocation of prolactin through a direct interaction. additionally, cypb potentiates prolactin-induced stat5 transactivation by promoting the dissociation of pias3, a stat5 repressor (rycyzyn and clevenger, 2002). cypb can also associate with interferon regulatory factor (irf)-3 (obata et al. 2005). extracellular cypb can bind platelets (allain et al. 1999) and table 1. human cyclophilin subtypes. protein name length genbank accession no. reference cypa 165 aa nm_021130 liu et al. 1990 cypb 216 aa nm_000942 price et al. 1991 cypc 212 aa nm_000943 friedman et al. 1991 cyp40 370 aa nm_005038 kieffer et al. 1992 cype, cyp33 301 aa nm_006112 mi et al. 1996 cypd, cypf, cyp3 207 aa nm_005729 bergsma et al. 1991 cypg, cars-cyp,srcyp 754 aa nm_004792 nestel et al. 1996 cyph, usa-cyp, snucyp-20 177 aa nm_006347 horowitz et al. 1997 ppi-l1 166 aa nm_016059 ozaki et al. 1996 ppi-l2, cyp60 520 aa nm_014337 wang et al. 1996 ppil3 165 aa nm_032472 zhou et al. 2001 ppi-l4 492 aa nm_139126 zeng et al. 2001 ppi-l5, lrr-1 414 aa nm_152329 jang et al. 2001 ranbp2 3224 aa nm_006267 yokoyama et al. 1995 drug target insights 2007: 2 11 cyclophilin and viruses this initiates a transmembranous infl ux of calcium ion, kinase activation, and platelet adhesion to collagen. accumulating evidence suggests that cyps, in particular cypa and cypb, can mediate intercellular communication similar to cytokines. cyps are secreted from cells in response to infl ammatory stimuli or oxidative stress (jin et al. 2000; seko et al. 2004; sherry et al. 1992; xu et al. 1992) and they can act as potent chemoattractants for neutrophils (sherry et al. 1992), eosinophils (xu et al. 1992), and t cells (allain et al. 2002). cypa and cypb are recognized by the cell surface receptor cd147, and cyp binding leads to erk activation and chemotaxis (pushkarsky et al. 2001; yurchenko et al. 2001; yurchenko et al. 2002). cypd plays a critical role in mitochondrial function and cell death (tanveer et al. 1996). during ischemia-induced necrosis, e.g. following a heart attack or stroke, the accumulation of calcium and increase of reactive oxygen species (ros) trigger the opening of a pore in the inner mitochondrial membrane, known as the membrane permeability transition pore (mptp) (halestrap, 1999). calcium overload and ros induce a conformational change in adenine nucleotide translocase (ant), a key component regulating the opening of mptp at the inner mitochondrial membrane. the opening of mptp leads to mitochondrial swelling, rupture of the outer membrane, and the release of small molecules (waldmeier et al. 2003). cypd is located within the matrix of the mitochondria and it binds ant facilitating its conformational change (crompton et al. 1998; woodfi eld et al. 1998). in cypd-knockout cells, necrosis induced by calcium and ros was decreased, but apoptotic cell death induced by cytokines or anticancer agents was unaffected (baines et al. 2005; nakagawa et al. 2005). cypd-knockout mice also experienced reduced cardiac injury following reperfusion after ischemia. thus, cypd is a key molecule involved in the cell death process. cyp40 regulates the activity of steroid receptors (srs) (duina et al. 1996; owens-grillo et al. 1995; ratajczak et al. 1993). srs including the glucocorticoid receptor, estrogen receptor, androgen receptor and progesterone receptor are nuclear hormone receptors that exert transcriptional activity following steroid ligand binding and nuclear translocation. in the absence of steroid ligands, srs form complexes with heat shock protein 90 (hsp90) together with the immunophilins cyp40, fkbp51, or fkbp52 in the cytoplasm. these immunophilins control sr activity by increasing receptor avidity for hormone ligands through ppiase-dependent conformational changes. upon hormone binding, this sr/hsp90/ immunophilin complex dissociates, leaving homodimeric sr, which then translocates into the nucleus to transactivate downstream genes. although there are some reports on other cyp subtypes (table 1), the precise functions and signifi cances of them are largely unknown. viruses requiring cyps as described above, cyps play essential roles in diverse cellular processes. interestingly, several viruses have evolved to use cyps during their life cycles. in particular, cyps are demonstrated to be involved in the proliferation of hiv-1 and hepatitis c virus (hcv). other viruses using cyps during their life cycle include vaccinia virus (vv), vesicular stomatitis virus (vsv), and sars-coronavirus. vaccinia virus a signifi cant role of cyp in vv replication was fi rst identifi ed through the analysis of several csa analogs. the ability of cyclosporins to suppress vv replication correlated with the inhibition of cyp function (damaso and moussatche, 1998). vv infection stabilizes cypa, leading to the accumulation of cypa (castro et al. 2003). in vv infected cells, cypa relocalizes to the peripheral region of the nucleus, colocalizing with sites of virus production. cypa is incorporated into viral particles and is located in the viral core. vesicular stomatitis virus cypa interacted with the nucleocapsid protein of vsv (bose et al. 2003), and, like vv, cypa is incorporated into vsv viral particles. although the binding and incorporation of cypa occurred beyond the virus serotypes, the functional role of cypa in the viral life cycle appears to be straindependent. inhibition of cyp activity by csa reduced primary transcription of vsv-new jersey (vsv-nj) but not vsv-indiana (vsv-ind) serotype, and cypa activity was required for the replication of vsv-nj to a greater extent than vsv-ind. the authors suggest that differential requirements of cypa are likely the results of evolutionary pressure during lineage development. drug target insights 2007: 2 12 watashi and shimotohno sars coronavirus the nucleocapsid protein (np) of severe acute respiratory syndrome coronavirus (sars-cov) binds cypa (luo et al. 2004), and another group reported that cypa is incorporated into sars-cov particles (chen et al. 2005). extracellular cypa binds cd147 on the cell surface, and treatment with a peptide that blocks cd147 binding inhibits viral infection. thus, cypa may be involved in sars-cov invasion into host cells through interaction with np and cd147, respectively. cypa and hiv-1 cypa plays an important role in the viral life cycle of hiv-1. in 1993, cypa was found to interact with hiv-1 gag (luban et al. 1993), and in 1994, cypa was reportedly incorporated into viral particles (franke et al. 1994; thali et al. 1994). a gene targeting study demonstrated that only cypa among cyp subtypes was essential for hiv-1 proliferation (braaten and luban, 2001). within the hiv-1 life cycle, cypa plays multiple roles through different interaction partners, including an early step prior to reverse transcription (braaten et al. 1996; mlynar et al. 1997; steinkasserer et al.1995). although cypa is incorporated into virions through binding to the ca domain of the gag polyprotein (franke et al. 1994; ott et al. 1995; thali et al. 1994), this incorporation is not required for viral infection. instead, target cell expressed cypa is important for productive infection and viral replication (hatziioannou et al. 2005; sokolskaja et al. 2004). it has been known for several decades that host cells express different restriction factors to prevent infection by certain retroviruses (cullen, 2003), and several recent studies have suggested that cypa modulates sensitivity to such a restriction factor early in the hiv-1 life cycle prior to reverse transcription. trim5α is a host restriction factor originally identifi ed using expression cloning that recognizes ca limiting retrovirus proliferation (stremlau et al. 2004). towers et al. showed that cypa regulates the activity of a host restriction factor (towers et al. 2003). disruption of cypa-ca binding by introducing of point mutation into ca or treating human cells with csa decreases hiv-1 infectivity. conversely, the loss of cypa-ca binding greatly enhanced hiv-1 infectivity in simian cells (berthoux et al. 2005; kootstra et al. 2003; sayah et al. 2004). from the results, the hypothesis was proposed by luban et al. that ca binding by cypa prevented normal antiviral effects mediated by trim5α during hiv-1 infection of human cells, but this same interaction mediated hiv-1 restriction in nonhuman primate cells (sokolskaja et al. 2006; luban, in press). both the mechanism of trim5α restriction of hiv-1 and the modulation of ca recognition by cypa remain unclear, and further studies are clearly needed to resolve these important issues in the hiv-1 life cycle and the host response to hiv-1 infection. cypa may be important for other aspects of hiv-1 infection. cypa interacts with cd147 (pushkarsky et al. 2001), heparans (saphire et al. 1999), vpr (zander et al. 2003), and envelope glycoprotein gp120 (endrich and gehring, 1998), although their relevances of the interactions should be further verifi ed. cypb and hcv current therapy against hcv hcv is a major causative agent of chronic liver diseases such as chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (liang et al. 1993). hcv infection is a serious health problem affecting approximately 170 million individuals worldwide (poynard et al. 2003). the current standard therapy for hcv is restricted to interferon (ifn) or pegylated-ifn either alone or in combination with ribavirin. because treatment with these agents, however, fails to produce sustained virus elimination in about one half of the patients (di bisceglie et al. 2002), alternative and effective strategies to combat against hcv are greatly needed. hcv encodes a single polypeptide that is cleaved by host and hcv-encoded protease including ns3 to generate a set of functional proteins. its genome is replicated by the hcv-encoded rna-dependent rna polymerase (rdrp) ns5b. both of these proteins, ns3 and ns5b, are essential for hcv genome replication and are possible targets for the development of anti-hcv therapeutics (di bisceglie et al. 2002). small molecule compounds targeting ns3 and ns5b have been developed, and their effi cacy has been examined in clinical trials (di bisceglie et al. 2002). in addition to these viral enzymes, host cell factors are required for viral replication, and these may provide other options for the development of novel anti-viral agents. disrupting the function of drug target insights 2007: 2 13 cyclophilin and viruses host cell derived factors is particularly appealing as the mutation rate of host proteins is much less than that of viral encoded proteins and should less give rise to drug-resistant viruses. however, while some host factors required for viral genome replication have been identifi ed, other host proteins need to be identifi ed to develop optimal anti-viral therapies with few side effects. at this time, only a limited number of host proteins have been found to be involved in hcv genome replication with biological relevance. hvap-33 is one of snare family proteins that regulate vesicle biogenesis, protein sorting, and membrane fusion. hvap-33 binds hcv ns5a and ns5b, and this interaction regulates the presence of hcv proteins in the subcellular compartment performing viral genome replication (evans et al. 2004; gao et al. 2004; tu et al. 1999). fbl2 is a member of the f-box protein family, involved in the ubiquitination pathway. fbl2 is geranylgeranylated in cells (wang et al. 2005), and associates with ns5a in a geranylgeranylation-dependent manner to regulate hcv genome replication. however, the mechanism of action of fbl2 in hcv genome replication is not known. additionally, we recently found that cypb is a cofactor for hcv replication in host cells, and this may represent a new target for anti-hcv therapeutics (watashi et al. 2005). we will fi rst discuss the role of cypb in hcv genome replication followed by the therapeutic implications of this discovery in hcv treatment. anti-hcv activity of cyclosporin we identifi ed csa as an anti-hcv agent using a hcv replicon system, a cell culture system supporting hcv genome replication (lohmann et al. 1999), and csa inhibits hcv genome replication as potently as ifnα (watashi et al. 2003). since that time, several groups have made similar observations (firpi et al. 2006; nakagawa et al. 2004; paeshuyse et al. 2006). as shown in fig.1a, cellular treatment with 1 µg/ml csa decreases hcv rna levels by approximately 1/500 (watashi et al. 2003). csa also reduces the expression of hcv-encoded proteins to undetectable levels (fig. 1b). in contrast, fk506 has no effect on the production of hcv rna or proteins (fig. 1a and b). the differences between csa and fk506 suggest that csa prevents viral genome replication independently of cn, an effector ( c o p y /p g t o ta l r n a ) a m o u n t o f h c v r n a figure. 1 csa suppresses hcv genome replication. (a) hcv rna was quantifi ed in total rna isolated from hcv replicon-bearing cells treated with various concentrations of csa, fk506, or ifnα for 7 days. the amount of hcv rna per 1 pg total rna was plotted against the concentration of csa (µg/ml), fk506 (µg/ml), or ifnα (× 100 iu/ml). (b) the expression of hcv ns5a and protein disulfi de isomerase (pdi) as a cellular protein was examined in the hcv replicon-bearing cells treated without (control) or with 100 iu/ml ifnα, 1�µg/ml csa, or 1�µg/ml fk506 for 7 days. drug target insights 2007: 2 14 watashi and shimotohno common to both csa and fk506 mediated immunosuppression. and the anti-hcv effects of csa are mediated by pathway(s) distinct from those of ifnα (watashi et al. 2003). cypb as a cellular cofactor of hcv genome replication the ability of csa to inhibit hcv genome replication correlates with the inhibition of cyp activity (watashi et al. 2005). moreover, an alternative cyp inhibitor, sanglifehrin, also decreases the levels of hcv rna. thus, the inhibition of cyp activity is essential for the anti-hcv effect of csa, and this strongly suggests that cyp plays a direct, important role in hcv genome replication. moreover, the specifi c knockdown of cypb by rnai reduced hcv rna titer, but knockdown of cypa, cypc, cype, or cyph had no effect on hcv replication activity. these data indicate that cypb plays a critical role in hcv genome replication. regulation of ns5b by cypb the effects of cypb on hcv genome replication are mediated through a direct interaction with ns5b as demonstrated in both in vitro and in cells (watashi et al. 2005) (fig. 2a). cypb do not bind any other hcv proteins involved in viral replication. ns5b binds hcv genome rna in order to function as a rdrp. cypb but not cypa promotes the rna binding activity of ns5b and stimulates hcv genome replication in cells (fig. 2a). this functional support by cypb to ns5b is essential for the effi cient replication of the hcv genome, and csa blocks the interaction of cypb with ns5b, leading to reduced rna binding (fig. 2b). figure. 2 cypb regulates the activity of ns5b. (a) in the absence of csa (normal conditions), ns5b associates with cellular cypb to effi ciently bind to the hcv genome rna and drive genome replication. (b) in the presence of csa, cypb does not interact with ns5b. free ns5b less functions, and viral genome replication is impaired in the absence of functional cypb. drug target insights 2007: 2 15 cyclophilin and viruses thus, cypb serves as a cellular cofactor for hcv genome replication. therapeutic implications of cyp inhibition for the treatment for hcv the anti-hcv activity of csa analogs correlates with their ability to inhibit cyp function (watashi et al. 2005). the dissociation of cypb and ns5b greatly reduces the extent of hcv genome replication. these observations suggest that the inhibition of cypb may represent a novel therapeutic strategy against hcv. this possibility has been examined by two reports using stronger cyp inhibitors than csa. paeshuyse et al. used the csa analog debio-025 to inhibit cyp activity (paeshuyse et al. 2006), and this compound inhibited hcv replication 10-fold more potently than csa. the authors speculated that debio-025 might be an attractive drug candidate for the treatment of individuals with hcv/hiv coinfection because csa derivatives should also inhibit hiv-1 replication. we used the non-immunosuppressive csa derivative nim811 to target cyp (goto et al. 2006; ishii et al. 2006). nim811 inhibits cyp enzymatic activity two-fold more than csa (rosenwirth et al. 1994), and this increased inhibition correlates with greater suppression of hcv genome replication than csa, especially at lower doses. cotreatment of cells with nim811 and ifnα led to a synergistic anti-hcv effect at higher doses of nim811. treatment of nim811 for three weeks eliminated hcv rna from host cells to under detectable level. because the immunosuppression in patients during a viral infection is undesirable, these non-immunosuppressive variants of csa that inhibit cyp activity are likely to offer great promise for the treatment of patients with chronic hcv infection. conclusion cyps are cellular ppiases that catalyze conformational changes in proteins, but the role(s) and substrates for this protein family in cells are not well-characterized. however, discoveries of the signifi cance of cyp in life cycles for several viruses make this protein virologically notable and present novel therapeutic anti-viral targets. further analyses of the role of cyps in viral life cycles should reveal novel functions for these proteins as well as provide mechanistic insight into possible therapeutic targets. references allain, f., durieux, s., denys, a., carpentier, m. and spik, g. 1999. cyclophilin b binding to platelets supports calcium-dependent adhesion to collagen. blood, 94:976–983. allain, f., vanpouille, c., carpentier, m., slomianny, m. c., durieux, s. and spik, g. 2002. interaction with glycosaminoglycans is required for cyclophilin b to trigger integrin-mediated adhesion of peripheral blood t lymphocytes to extracellular matrix. proc. natl. acad. sci. u.s.a., 99:2714–2719. baines, c.p., kaiser, r.a., purcell, n.h., blair, n.s., osinska, h., hambleton, m.a., brunskill, e.w., sayen, m.r., gottlieb, r.a. and dorn, g. w. et al. 2005. loss of cyclophilin d reveals a critical role for mitochondrial permeability transition in cell death. nature, 434:658–662. bergsma, d.j., eder, c., gross, m., kersten, h., sylvester, d., appelbaum, e., cusimano, d., livi, g.p., mclaughlin, m.m. and kasyan, k. et al. 1991. the cyclophilin multigene family of peptidyl-prolyl isomerases. characterization of three separate human isoforms. j. biol. chem., 266:23204–23214. berthoux, l., sebastian, s., sokolskaja, e. and luban, j. 2005. cyclophilin a is required for trim5{alpha}-mediated resistance to hiv-1 in old world monkey cells. proc. natl. acad. sci. u.s.a., 102:14849– 14853. bierer, b.e., somers, p.k., wandless, t.j., burakoff, s.j. and schreiber, s.l. 1990. probing immunosuppressant action with a nonnatural immunophilin ligand. science, 250:556–559. bose, s., mathur, m., bates, p., joshi, n. and banerjee, a.k. 2003. requirement for cyclophilin a for the replication of vesicular stomatitis virus new jersey serotype. j. gen. virol., 84:1687–1699. braaten, d., franke, e.k. and luban, j. 1996. cyclophilin a is required for an early step in the life cycle of human immunodefi ciency virus type 1 before the initiation of reverse transcription. j. virol., 70:3551–3560. braaten, d. and luban, j. 2001. cyclophilin a regulates hiv-1 infectivity, as demonstrated by gene targeting in human t cells. embo. j., 20: 1300–1309. bram, r.j. and crabtree, g.r. 1994. calcium signalling in t cells stimulated by a cyclophilin b-binding protein. nature, 371:355–358. brazin, k.n., mallis, r.j., fulton, d.b. and andreotti, a.h. 2002. regulation of the tyrosine kinase itk by the peptidyl-prolyl isomerase cyclophilin a. proc. natl. acad. sci. u.s.a., 99:1899–1904. cande, c., vahsen, n., kouranti, i., schmitt, e., daugas, e., spahr, c., luban, j., kroemer, r.t., giordanetto, f., garrido, c. et al. 2004. aif and cyclophilin a cooperate in apoptosis-associated chromatinolysis. oncogene, 23:1514–1521. castro, a.p., carvalho, t.m., moussatche, n. and damaso, c.r. 2003. redistribution of cyclophilin a to viral factories during vaccinia virus infection and its incorporation into mature particles. j .virol. 77:9052– 9068. chen, z., mi, l., xu, j., yu, j., wang, x., jiang, j., xing, j., shang, p., qian, a. and li, y. et al. 2005. function of hab18g/cd147 in invasion of host cells by severe acute respiratory syndrome coronavirus. j. infect. dis., 191:755–760. chen, z.j., vetter, m., chang, g.d., liu, s., che, d., ding, y., kim, s.s. and chang, c.h. 2004. cyclophilin a functions as an endogenous inhibitor for membrane-bound guanylate cyclase-a. hypertension, 44:963–968. clipstone, n.a. and crabtree, g.r. 1992. identifi cation of calcineurin as a key signalling enzyme in t-lymphocyte activation. nature, 357: 695–697. colgan, j., asmal, m., neagu, m., yu, b., schneidkraut, j., lee, y., sokolskaja, e., andreotti, a. and luban, j. 2004. cyclophilin a regulates tcr signal strength in cd4+ t cells via a proline-directed conformational switch in itk. immunity, 21:189–201. colgan, j., asmal, m., yu, b. and luban, j. 2005. cyclophilin a-defi cient mice are resistant to immunosuppression by cyclosporine. j. immunol., 174:6030–6038. drug target insights 2007: 2 16 watashi and shimotohno crompton, m., virji, s. and ward, j.m. 1998. cyclophilin-d binds strongly to complexes of the voltage-dependent anion channel and the adenine nucleotide translocase to form the permeability transition pore. eur. j. biochem, 258:729–735. cullen, b.r. 2003. hiv-1 infection: fooling the gatekeeper. nat. med., 9: 1112–1113. damaso, c.r. and moussatche, n. 1998. inhibition of vaccinia virus replication by cyclosporin a analogues correlates with their affi nity for cellular cyclophilins. j. gen. virol., 79 (pt 2):339–346. di bisceglie, a.m., mchutchison, j. and rice, c.m. 2002. new therapeutic strategies for hepatitis c. hepatology 35:224–231. duina, a.a., chang, h.c., marsh, j.a., lindquist, s. and gaber, r. f. 1996. a cyclophilin function in hsp90-dependent signal transduction. science, 274:1713–1715. endrich, m.m. and gehring, h. 1998. the v3 loop of human immunodefi ciency virus type-1 envelope protein is a high-affi nity ligand for immunophilins present in human blood. eur. j. biochem., 252: 441–446. evans, m.j., rice, c.m. and goff, s.p. 2004. phosphorylation of hepatitis c virus nonstructural protein 5a modulates its protein interactions and viral rna replication. proc. natl. acad. sci. u.s.a., 101: 13038–13043. firpi, r.j., zhu, h., morelli, g., abdelmalek, m.f., soldevila-pico, c., machicao, v.i., cabrera, r., reed, a.i., liu, c. and nelson, d.r. 2006. cyclosporine suppresses hepatitis c virus in vitro and increases the chance of a sustained virological response after liver transplantation. liver transpl., 12:51–57. fischer, g., wittmann-liebold, b., lang, k., kiefhaber, t. and schmid, f. x. 1989. cyclophilin and peptidyl-prolyl cis-trans isomerase are probably identical proteins. nature, 337:476–478. franke, e. k., yuan, h. e. and luban, j. 1994. specifi c incorporation of cyclophilin a into hiv-1 virions. nature, 372:359–362. friedman, j. and weissman, i. 1991. two cytoplasmic candidates for immunophilin action are revealed by affi nity for a new cyclophilin: one in the presence and one in the absence of csa. cell, 66:799–806. fruman, d.a., klee, c.b., bierer, b.e. and burakoff, s.j. 1992. calcineurin phosphatase activity in t lymphocytes is inhibited by fk 506 and cyclosporin a. proc. natl. acad. sci. u.s.a., 89:3686–3690. gao, l., aizaki, h., he, j. w. and lai, m.m. 2004. interactions between viral nonstructural proteins and host protein hvap-33 mediate the formation of hepatitis c virus rna replication complex on lipid raft. j. virol., 78:3480–3488. goto, k., watashi, k., murata, t., hishiki, t., hijikata, m. and shimotohno, k. 2006. evaluation of the anti-hepatitis c virus effects of cyclophilin inhibitors, cyclosporin a and nim811. biochem. biophys. res. commun., 343:879–884. halestrap, a.p. 1999. the mitochondrial permeability transition: its molecular mechanism and role in reperfusion injury. biochem. soc. symp., 66:181–203. handschumacher, r.e., harding, m.w., rice, j., drugge, r.j. and speicher, d.w. 1984. cyclophilin: a specifi c cytosolic binding protein for cyclosporin a. science, 226:544–547. harding, m.w., galat, a., uehling, d.e. and schreiber, s.l. 1989. a receptor for the immunosuppressant fk506 is a cis-trans peptidyl-prolyl isomerase. nature, 341:758–760. hatziioannou, t., perez-caballero, d., cowan, s. and bieniasz, p.d. 2005. cyclophilin interactions with incoming human immunodefi ciency virus type 1 capsids with opposing effects on infectivity in human cells. j. virol, 79:176–183. holloway, m.p. and bram, r.j. 1998. co-localization of calcium-modulating cyclophilin ligand with intracellular calcium pools. j. biol. chem, 273:16346–16350. horowitz, d.s., kobayashi, r. and krainer, a.r. 1997. a new cyclophilin and the human homologues of yeast prp3 and prp4 form a complex associated with u4/u6 snrnps. rna, 3:1374–1387. ishii, n., watashi, k., hishiki, t., goto, k., inoue, d., hijikata, m., wakita, t., kato, n., and shimotohno, k. 2006. diverse effects of cyclosporine on hepatitis c virus strain replication. j. virol., 80:4510–4520. jang, l.k., lee, z. h., kim, h.h., hill, j.m., kim, j.d. and kwon, b.s. 2001. a novel leucine-rich repeat protein (lrr-1): potential involvement in 4-1bb-mediated signal transduction. mol. cells., 12:304– 312. jin, z.g., melaragno, m.g., liao, d.f., yan, c., haendeler, j., suh, y.a., lambeth, j.d. and berk, b.c. 2000. cyclophilin a is a secreted growth factor induced by oxidative stress. circ. res., 87:789–796. kieffer, l.j., thalhammer, t. and handschumacher, r.e. 1992. isolation and characterization of a 40-kda cyclophilin-related protein. j. biol. chem., 267:5503–5507. kootstra, n.a., munk, c., tonnu, n., landau, n.r. and verma, i.m. 2003. abrogation of postentry restriction of hiv-1-based lentiviral vector transduction in simian cells. proc. natl. acad. sci. u.s.a., 100: 1298–1303. krummrei, u., bang, r., schmidtchen, r., brune, k. and bang, h. 1995. cyclophilin-a is a zinc-dependent dna binding protein in macrophages. febs. lett., 371:47–51. liang, t.j., jeffers, l.j., reddy, k.r., de medina, m., parker, i.t., cheinquer, h., idrovo, v., rabassa, a. and schiff, e.r. 1993. viral pathogenesis of hepatocellular carcinoma in the united states. hepatology, 18:1326–1333. liu, j., albers, m.w., chen, c.m., schreiber, s.l. and walsh, c.t. 1990. cloning, expression, and purifi cation of human cyclophilin in escherichia coli and assessment of the catalytic role of cysteines by sitedirected mutagenesis. proc. natl. acad. sci. u.s.a., 87:2304–2308. liu, j., albers, m.w., wandless, t.j., luan, s., alberg, d.g., belshaw, p. j., cohen, p., mackintosh, c., klee, c.b. and schreiber, s.l. 1992. inhibition of t cell signaling by immunophilin-ligand complexes correlates with loss of calcineurin phosphatase activity. biochemistry., 31:3896–3901. liu, j., farmer, j.d. jr., lane, w.s., friedman, j., weissman, i. and schreiber, s.l. 1991. calcineurin is a common target of cyclophilin-cyclosporin a and fkbp-fk506 complexes. cell, 66:807–815. lohmann, v., korner, f., koch, j., herian, u., theilmann, l. and bartenschlager, r. 1999. replication of subgenomic hepatitis c virus rnas in a hepatoma cell line. science, 285:110–113. luban, j., bossolt, k.l., franke, e.k., kalpana, g.v. and goff, s.p. 1993. human immunodefi ciency virus type 1 gag protein binds to cyclophilins a and b. cell., 73:1067–1078. luban, j. (in press). cyclophilin a, trim5 and resistance to hiv-1 infection. j. virol., [epub ahead of print]. luo, c., luo, h., zheng, s., gui, c., yue, l., yu, c., sun, t., he, p., chen, j., shen, j. et al. 2004. nucleocapsid protein of sars coronavirus tightly binds to human cyclophilin a. biochem. biophys. res. commun., 321:557–565. mccaffrey, p.g., perrino, b.a., soderling, t.r. and rao, a. 1993. nf-atp, a t lymphocyte dna-binding protein that is a target for calcineurin and immunosuppressive drugs. j. biol. chem., 268:3747–3752. mi, h., kops, o., zimmermann, e., jaschke, a. and tropschug, m. 1996. a nuclear rna-binding cyclophilin in human t cells. febs. lett., 398:201–205. mlynar, e., bevec, d., billich, a., rosenwirth, b. and steinkasserer, a. 1997. the non-immunosuppressive cyclosporin a analogue sdz nim 811 inhibits cyclophilin a incorporation into virions and virus replication in human immunodefi ciency virus type 1-infected primary and growth-arrested t cells. j. gen. virol., 78 (pt 4):825– 835. nakagawa, m., sakamoto, n., enomoto, n., tanabe, y., kanazawa, n., koyama, t., kurosaki, m., maekawa, s., yamashiro, t., chen, c. h. et al. 2004. specifi c inhibition of hepatitis c virus replication by cyclosporin a. biochem. biophys. res. commun., 313:42–47. drug target insights 2007: 2 17 cyclophilin and viruses nakagawa, t., shimizu, s., watanabe, t., yamaguchi, o., otsu, k., yamagata, h., inohara, h., kubo, t. and tsujimoto, y. 2005. cyclophilin d-dependent mitochondrial permeability transition regulates some necrotic but not apoptotic cell death. nature, 434:652–658. nestel, f. p., colwill, k., harper, s., pawson, t. and anderson, s. k. 1996. rs cyclophilins: identifi cation of an nk-tr1-related cyclophilin. gene., 180:151–155. obata, y., yamamoto, k., miyazaki, m., shimotohno, k., kohno, s. and matsuyama, t. 2005. role of cyclophilin b in activation of interferon regulatory factor-3. j. biol. chem., 280:18355–18360. ott, d.e., coren, l.v., johnson, d.g., sowder, r.c., 2nd, arthur, l.o. and henderson, l.e. 1995. analysis and localization of cyclophilin a found in the virions of human immunodefi ciency virus type 1 mn strain. aids. res. hum. retroviruses 11:1003–1006. owens-grillo, j.k., hoffmann, k., hutchison, k.a., yem, a.w., deibel, m.r. jr., handschumacher, r. e. and pratt, w.b. 1995. the cyclosporin a-binding immunophilin cyp-40 and the fk506-binding immunophilin hsp56 bind to a common site on hsp90 and exist in independent cytosolic heterocomplexes with the untransformed glucocorticoid receptor. j. biol. chem., 270:20479–20484. ozaki, k., fujiwara, t., kawai, a., shimizu, f., takami, s., okuno, s., takeda, s., shimada, y., nagata, m. and watanabe, t. et al. 1996. cloning, expression and chromosomal mapping of a novel cyclophilin-related gene (ppil1) from human fetal brain. cytogenet. cell. genet., 72:242–245. paeshuyse, j., kaul, a., de clercq, e., rosenwirth, b., dumont, j.m., scalfaro, p., bartenschlager, r. and neyts, j. 2006. the non-immunosuppressive cyclosporin debio-025 is a potent inhibitor of hepatitis c virus replication in vitro. hepatology, 43:761–770. poynard, t., yuen, m.f., ratziu, v. and lai, c.l. 2003. viral hepatitis c. lancet., 362:2095–2100. price, e.r., zydowsky, l.d., jin, m.j., baker, c.h., mckeon, f.d. and walsh, c.t. 1991. human cyclophilin b: a second cyclophilin gene encodes a peptidyl-prolyl isomerase with a signal sequence. proc. natl. acad. sci. u.s.a., 88:1903–1907. pushkarsky, t., zybarth, g., dubrovsky, l., yurchenko, v., tang, h., guo, h., toole, b., sherry, b. and bukrinsky, m. 2001. cd147 facilitates hiv-1 infection by interacting with virus-associated cyclophilin a. proc. natl. acad. sci. u.s.a., 98:6360–6365. ratajczak, t., carrello, a., mark, p.j., warner, b.j., simpson, r.j., moritz, r.l. and house, a.k. 1993. the cyclophilin component of the unactivated estrogen receptor contains a tetratricopeptide repeat domain and shares identity with p59 (fkbp59). j. biol. chem., 268:13187–13192. rosen, m.k., standaert, r.f., galat, a., nakatsuka, m. and schreiber, s. l. 1990. inhibition of fkbp rotamase activity by immunosuppressant fk506: twisted amide surrogate. science, 248:863–866. rosenwirth, b., billich, a., datema, r., donatsch, p., hammerschmid, f., harrison, r., hiestand, p., jaksche, h., mayer, p. and peichl, p. et al. 1994. inhibition of human immunodefi ciency virus type 1 replication by sdz nim 811, a nonimmunosuppressive cyclosporine analog. antimicrob. agents. chemother., 38:1763–1772. rycyzyn, m.a. and clevenger, c.v. 2002. the intranuclear prolactin/cyclophilin b complex as a transcriptional inducer. proc. natl. acad. sci. u.s.a., 99:6790–6795. rycyzyn, m.a., reilly, s.c., o’malley, k. and clevenger, c.v. 2000. role of cyclophilin b in prolactin signal transduction and nuclear retrotranslocation. mol. endocrinol. 14:1175–1186. saphire, a.c., bobardt, m.d. and gallay, p.a. 1999. host cyclophilin a mediates hiv-1 attachment to target cells via heparans. embo. j. 18: 6771–6785. sayah, d.m., sokolskaja, e., berthoux, l. and luban, j. 2004. cyclophilin a retrotransposition into trim5 explains owl monkey resistance to hiv-1. nature, 430:569–573. schreiber, s.l. 1991. chemistry and biology of the immunophilins and their immunosuppressive ligands. science, 251:283–287. seko, y., fujimura, t., taka, h., mineki, r., murayama, k. and nagai, r. 2004. hypoxia followed by reoxygenation induces secretion of cyclophilin a from cultured rat cardiac myocytes. biochem. biophys res. commun., 317:162–168. sherry, b., yarlett, n., strupp, a. and cerami, a. 1992. identifi cation of cyclophilin as a proinfl ammatory secretory product of lipopolysaccharide-activated macrophages. proc. natl. acad. sci. u.s.a., 89: 3511–3515. sokolskaja, e., sayah, d.m. and luban, j. 2004. target cell cyclophilin a modulates human immunodefi ciency virus type 1 infectivity. j. virol., 78:12800–12808. sokolskaja, e. and luban, j. 2006. cyclophilin a, trim5, and innate immunity to hiv-1. curr. opin. microbiol., 9:404–408. steinkasserer, a., harrison, r., billich, a., hammerschmid, f., werner, g., wolff, b., peichl, p., palfi , g., schnitzel, w. and mlynar, e. et al. 1995. mode of action of sdz nim 811, a nonimmunosuppressive cyclosporin a analog with activity against human immunodefi ciency virus type 1 (hiv-1): interference with early and late events in hiv-1 replication. j. virol., 69:814–824. stremlau, m., owens, c.m., perron, m.j., kiessling, m., autissier, p. and sodroski, j. 2004. the cytoplasmic body component trim5alpha restricts hiv-1 infection in old world monkeys. nature, 427:848–853. syed, f., rycyzyn, m.a., westgate, l. and clevenger, c.v. 2003. a novel and functional interaction between cyclophilin a and prolactin receptor. endocrine, 20:83–90. takahashi, n. 1999. pharmacodynamics of ciclosporin a (cyclosporine). clin. exp. nephrol., 3:s16–s26. takahashi, n., hayano, t. and suzuki, m. 1989. peptidyl-prolyl cis-trans isomerase is the cyclosporin a-binding protein cyclophilin. nature, 337:473–475. tanveer, a., virji, s., andreeva, l., totty, n.f., hsuan, j.j., ward, j.m. and crompton, m. 1996. involvement of cyclophilin d in the activation of a mitochondrial pore by ca2+ and oxidant stress. eur. j. biochem., 238:166–172. thali, m., bukovsky, a., kondo, e., rosenwirth, b., walsh, c.t., sodroski, j. and gottlinger, h.g. 1994. functional association of cyclophilin a with hiv-1 virions. nature, 372:363–365. towers, g.j., hatziioannou, t., cowan, s., goff, s.p., luban, j. and bieniasz, p.d. 2003. cyclophilin a modulates the sensitivity of hiv-1 to host restriction factors. nat. med., 9:1138–1143. tran, d.d., edgar, c.e., heckman, k.l., sutor, s.l., huntoon, c.j., van deursen, j., mckean, d.l. and bram, r.j. 2005. caml is a p56lck-interacting protein that is required for thymocyte development. immunity, 23:139–152. tu, h., gao, l., shi, s.t., taylor, d.r., yang, t., mircheff, a.k., wen, y., gorbalenya, a.e., hwang, s.b. and lai, m.m. 1999. hepatitis c virus rna polymerase and ns5a complex with a snare-like protein. virology, 263:30–41. waldmeier, p.c., zimmermann, k., qian, t., tintelnot-blomley, m. and lemasters, j.j. 2003. cyclophilin d as a drug target. curr. med. chem., 10:1485–1506. wang, b.b., hayenga, k.j., payan, d.g. and fisher, j.m. 1996. identifi cation of a nuclear-specifi c cyclophilin which interacts with the proteinase inhibitor eglin c. biochem. j., 314(pt 1):313–319. wang, c., gale, m., jr., keller, b. c., huang, h., brown, m.s., goldstein, j.l. and ye, j. 2005. identifi cation of fbl2 as a geranylgeranylated cellular protein required for hepatitis c virus rna replication. mol. cell., 18:425–434. watashi, k., hijikata, m., hosaka, m., yamaji, m. and shimotohno, k. 2003. cyclosporin a suppresses replication of hepatitis c virus genome in cultured hepatocytes. hepatology, 38:1282–1288. watashi, k., ishii, n., hijikata, m., inoue, d., murata, t., miyanari, y. and shimotohno, k. 2005. cyclophilin b is a functional regulator of hepatitis c virus rna polymerase. mol. cell., 19:111–122. drug target insights 2007: 2 18 watashi and shimotohno woodfi eld, k., ruck, a., brdiczka, d. and halestrap, a.p. 1998. direct demonstration of a specifi c interaction between cyclophilin-d and the adenine nucleotide translocase confi rms their role in the mitochondrial permeability transition. biochem. j., 336(pt 2):287–290. xu, q., leiva, m.c., fischkoff, s.a., handschumacher, r.e. and lyttle, c. r. 1992. leukocyte chemotactic activity of cyclophilin. j. biol. chem., 267:11968–11971. yang, w.m., inouye, c.j. and seto, e. 1995. cyclophilin a and fkbp12 interact with yy1 and alter its transcriptional activity. j. biol. chem., 270:15187–15193. yokoyama, n., hayashi, n., seki, t., pante, n., ohba, t., nishii, k., kuma, k., hayashida, t., miyata, t., aebi, u. and et al. 1995. a giant nucleopore protein that binds ran/tc4. nature, 376:184–188. yurchenko, v., o’connor, m., dai, w. w., guo, h., toole, b., sherry, b. and bukrinsky, m. 2001. cd147 is a signaling receptor for cyclophilin b. biochem. biophys. res. commun., 288:786–788. yurchenko, v., zybarth, g., o’connor, m., dai, w. w., franchin, g., hao, t., guo, h., hung, h. c., toole, b. and gallay, p. et al. 2002. active site residues of cyclophilin a are crucial for its signaling activity via cd147. j. biol. chem. 277:22959–22965. zander, k., sherman, m.p., tessmer, u., bruns, k., wray, v., prechtel, a. t., schubert, e., henklein, p., luban, j. and neidleman, j. et al. 2003. cyclophilin a interacts with hiv-1 vpr and is required for its functional expression. j. biol. chem., 278:43202–43213. zeng, l., zhou, z., xu, j., zhao, w., wang, w., huang, y., cheng, c., xu, m., xie, y. and mao, y. 2001. molecular cloning, structure and expression of a novel nuclear rna-binding cyclophilin-like gene (ppil4) from human fetal brain. cytogenet. cell. genet., 95:43–47. zhou, z., ying, k., dai, j., tang, r., wang, w., huang, y., zhao, w., xie, y. and mao, y. 2001. molecular cloning and characterization of a novel peptidylprolyl isomerase (cyclophilin)-like gene (ppil3) from human fetal brain. cytogenet. cell. genet., 92:231–236. drug target insights 2007: 2 dti © 2020 the authors. this article is published by aboutscience and licensed under creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). any commercial use is not permitted and is subject to publisher’s permissions. full information is available at www.aboutscience.eu issn 1177-3928 drug target insights 2020; 14: 16-25 review doi: 10.33393/dti.2020.2170 prevalence of multidrug-resistant and extended-spectrum beta-lactamase (esbl)-producing gram-negative bacilli: a meta-analysis report in ethiopia mengistu abayneh and teshale worku school of medical laboratory sciences, mizan-tepi university, mizan-aman ethiopia abstract multidrug-resistant (mdr) extended-spectrum beta-lactamase (esbl)-producing bacterial isolates have emerged as a global threat to human health. little is known about the overall prevalence of multidrug resistance profile and esbl-producing gram-negative bacilli (gnb) in ethiopia. therefore, this meta-analysis was performed to produce proportional estimates of multidrug resistance and esbl-producing gnb in ethiopia. a web-based search was conducted in pubmed, google scholar, research gate, scopus and other databases. articles published till 2019 on the prevalence and antimicrobial resistance profiles of esbl-producing gnb in ethiopia were included in the study. relevant data were extracted and statistical analysis was performed using comprehensive meta-analysis version 3.3.0 software. publication bias was analyzed and presented with funnel plots. in this meta-analysis, the overall proportional estimate of esbl-producing gnb was 48.9% (95% confidence interval [ci]: 0.402, 0.577). the pooled proportional estimates of esbl-producing klebsiella pneumoniae, escherichia coli and other gnb were 61.8%, 41.2% and 42.9%, respectively. regarding antimicrobial resistance profiles against selected drugs, the pooled proportional estimates of resistance against amoxicillin-clavulanic acid, trimethoprim-sulfamethoxazole, cefotaxime, ceftazidime, tetracycline, gentamicin and ciprofloxacin was 79.0%, 78.4%, 78.0%, 72.4%, 72.7%, 58.9% and 43.8%, respectively. the pooled proportional estimates of mdr isolates were found to be 82.7% (95% ci: 0.726, 0.896), which are relatively high as compared to other countries. this highlights a need for active surveillance systems which can help understand the actual epidemiology of esbl, aid in formulating national guidelines for proper screening of esbl and support developing standardized approaches for managing patients colonized with esbl. keywords: ethiopia, extended-spectrum beta-lactamase, gram negative, multidrug resistance received: june 29, 2020 accepted: september 14, 2020 published online: october 5, 2020 corresponding author: mengistu abayneh school of medical laboratory sciences mizan-tepi university mizan-aman ethiopia mengeabayneh@mtu.edu.et use could worsen the condition, increasing infections caused by such resistant strains, its emergency and transmission (1,2). for instance, in ethiopia studies show that about 36.8% of the population got antibiotics from community drug retail outlets without a prescription and 67.9% of people had discontinued the use of antibiotics once their symptoms subside (3). low educational status, dissatisfaction with healthcare services provided, pharmacy owners’ influence to maximize revenue, customer’s pressure, weak regulatory mechanism and professional conflicts of interest were found to be strong predictors of inappropriate use of antibiotic use among the community (3,4). enterobacteriaceae that produce esbl carry plasmidencoded enzymes that can efficiently hydrolyze and confer resistance to a variety of beta-lactam antibiotics, but not to carbapenems or cephamycins. besides beta-lactam class of antibiotic, esbl producers are commonly resistant to different families of antibiotics including fluoroquinolones, aminoglycosides and trimethoprim-sulfamethoxazole (sxt), which contribute to the selection and persistence of introduction the production of extended-spectrum beta-lactamase (esbl) enzymes is the main bacterial mechanism to acquire resistance to currently used broad-spectrum beta-lactam antibiotics. the infections caused by such enzyme-producing bacteria have significant potential impacts on antibiotic use and patient outcomes. especially in resource-limited countries, limited availability of drugs coupled with inappropriate abayneh and worku 17 © 2020 the authors. published by aboutscience multidrug-resistant (mdr) esbl strains and plasmids in both clinical and community settings. these enzymes are predominantly found in escherichia coli and klebsiella pneumoniae, although they are also present in other members of the gram-negative bacilli (gnb) (5-7). esbl-producing organisms play an important role in healthcare infections, increasing hospitalization time and morbidity and mortality rates (8). the presence of esbl complicates antibiotic selection, especially in patients with serious infections, such as bacteremia. the reason for this is that esbl-producing bacteria are often multiresistant to various antibiotics, an interesting feature of ctx-m (ctx stands for cefotaxime and m for munich)-producing isolates is the coresistance to various classes of antibiotics such as fluoroquinolones, aminoglycosides and co-trimoxazole due to associated resistance mechanisms, which may be either chromosomally or plasmid-encoded (8-11). the spread and the burden of esbl-producing bacteria are greater in developing countries. for instance, the major epicenters of esbl-expressing bacteria are located in asia, africa and the middle east (12-14). findings of a recent review showed that pooled prevalence of healthcare-associated infections in resource-limited settings (15.5%) was twice the average prevalence in europe (7.1%) (15). some plausible reasons for this difference include the following conditions that are prevalent in low-income countries: crowded hospitals, more extensive self-treatment and use of nonprescription antimicrobials, poorer hygiene in general and particularly in hospitals as well as less effective infection control. comprehensive data regarding esbl-producing bacteria are generally lacking in african countries, compared with the developed world. in ethiopia also, it is difficult to evaluate the spread and the burden of esbl-producing organisms, because of the limited scope of studies and lack of coordinated epidemiological surveillance systems. by combining information from all relevant studies, meta-analyses can provide more precise estimates of the effects of healthcare than those derived from the individual studies included within a review. therefore, to gain a better insight into the proportional estimates of mdr profiles and esbl-producing gnb in ethiopia, we retrieved available articles and collated the information in this study article. methods study design a descriptive meta-analysis study comprising different studies on the prevalence and antimicrobial resistance profile of esbl-producing gnb in ethiopia was conducted. data sources and search strategies a systematic and comprehensive search on available records up to december 2019 was carried out in pubmed, google scholar, research gate, scopus and other databases. the following medical subject headings (mesh) in the title or abstract, such as “clinical specimen,” “esbl,” or “extended spectrum β-lactamase” “enterobacteriaceae” or “extended-spectrum-beta-lactamase,” “gram negatives” or “antibacterial resistance,” “gram negatives” or “antimicrobial susceptibility” and “gram negatives” and “ethiopia,” were searched. in addition, bibliographies of eligible studies and other meta-analyses were manually searched carefully to identify additional relevant articles. study selection and eligibility criteria all articles related to the detection of esbl-producing gnb from clinical specimens and hospital environment samples, written in english language, possessing approved microbiological methods for pathogen detection and containing sufficient and extractable data were included in the meta-analysis. having assessed all the information from the recovered publications, online records that were available up to 2019 were considered as appropriate for eligibility assessment. all review articles and original articles conducted outside ethiopia, articles with irretrievable full texts and records with unrelated outcomes of interest were excluded during screening and eligibility assessment. screening and eligibility criteria there were no limits with regard to study type except that the study had to be a primary study. however, the analysis was made to those studies which reported sufficient information to meet outcomes of interests. some duplicates records were addressed manually due to variation in reference styles across sources. thereafter, the authors (ma and tw) independently inspected all the titles and abstracts of articles related to the study question and these were included in a group of eligible articles with their own code and with irrelevant articles being excluded. all articles in the initially selected group were further screened in a second step by reviewing the full texts and evaluated for eligibility for final inclusion (fig. 1). data extraction studies were identified by the main study name/identifier, followed by the year of publication. using a predetermined, standardized and piloted data extraction form, the authors (ma and tw) independently extracted important data related to study characteristics from each article. data extraction sheets were individually designed and pilot-tested using microsoft excel 2007. for each study meeting the review inclusion criteria, the following data, such as first author, study area, year of publication, study design, sample type, sample size, target population, isolate sources and outcomes of interests, number and common species isolated, proportion of esbl-positive strains and proportion and/or number of drug and mdr isolate and the methods used to test for esbl producers, were recorded. data were extracted and analyzed at least twice to remove any discordance. whenever there was discordance in the data extracted, a third person, a trained msc laboratory professional, played a role in checking the data and a consensus was reached by double-checking of the articles with the two authors. meta-analysis on mdr and esbl-producing gnb in ethiopia18 © 2020 the authors. published by aboutscience quality assessment of included studies the qualities of eligible studies were checked against items included in strengthening the reporting of observational studies in epidemiology (strobe) statement checklist (16). the quality scores (proportion) for each study were calculated against items of the strobe checklist adequately. there were no limits with regard to study type except that the study had to be a primary study and we did not exclude any studies based on quality. outcome measurements the first outcome measure is the proportion of esblproducing gnb from clinical specimens in ethiopia. the pooled proportion of esbl-producing gnb was calculated per bacterium isolate. the second outcome measure is the antimicrobial and mdr profiles of esbl-producing and nonesbl-producing gnb against selected antimicrobials of different categories. data processing and analysis the relevant data were analyzed using comprehensive meta-analysis version 3.3.0 software (www.meta-analysis. com). both random and fixed effects models were used to calculate the pooled proportional estimates of esbl-positive and mdr isolates. the i2 statistics was used to expresses the percentage of total variation across studies and significant heterogeneity was considered at p < 0.05 and i2 > 50%. publication bias was evaluated by using begg’s and egger’s tests and presented with funnel plots of standard error of logit event rate, and a statistical significant publication bias was considered at p < 0.05. results search and screening results and distribution of included articles a total of 39 studies were identified from several sources including pubmed, google scholar, research gate, scopus and other databases. after removing eight duplicated articles, 31 were screened and four records were removed with their title and abstract review because of irrelevance. after full-text assessment, ten articles excluded: four from other countries, three due to animal and only nonhospital environment source of sample and three outcome of interest was missing and/or insufficient. seventeen articles fulfilling the eligibility criteria were included for systematic meta-analysis (fig. 1). the eligible studies were published in the year up to 2019. the study design of all included articles was crosssectional studies. most of the studies indicated that various specimens had been utilized for screening of gnb; particularly biological fluids like blood, urine, pus, stool and cerebrospinal fluid (csf) were taken for test. hospital environment samples such as wastewater and different swab samples from hospital contact surfaces were also taken for test. with regard to sources of biological samples, most of the studies included inpatients and outpatients as their sources of samples. with regard to study areas, six (35.3%) were conducted in the southwestern parts of ethiopia, six (35.3%) in central ethiopia, four (23.5%) in northern parts of ethiopia and one (5.9%) in eastern parts of ethiopia. based on the available evidence, the maximum number of articles on this subject was published in the year 2014, which means only three articles were published in 2005, 2011 and 2012 (tab. i). the median score of published studies against strobe items was 72.2% (with a range of [50.7-79.7]). proportional estimates of esbl-positive gnb this study indicates that although different bacterial pathogens have the probability of producing esbl as their resistance mechanisms, studies on the proportion of esblproducing bacteria have not been carried out properly in all parts of ethiopia. based on the available data, the pooled proportional estimate of esbl-producing gnb in ethiopia was 0.489 (95% confidence interval [ci]: 0.402, 0.577). proportional estimates of esbl-producing k. pneumoniae were 0.618 (95% ci: 0.487, 0.734), whereas proportional estimates of esbl-producing e. coli were 0.412 (95% ci: 0.326, 0.504). the proportional estimate of other esbl-producing gnb was 0.429 (95% ci: 0.352, 0.509). overall heterogeneity was significant [i2 = 93.41%; p = 0.000] (tab. ii and fig. 2). proportional estimates of mdr profiles of bacterial isolates the bacterial isolates that showed different antimicrobial resistance profile against selected agents were extracted. accordingly, the pooled estimate indicated that 0.720 (95% a total of 34records were searched and identified frompubmed, google scholar,researchgate, scopus and other databases. id en ti fi ca ti on numbers of articles screened after 8 duplicates removed (n= 31) sc re en in g four records were removed with their title and abstract review because of irrelevance. numbers of full-text assessed articles (n= 27) e lig ib ili ty ten articles excluded due to: • four from other countries • three due to animal and only environmental source of sample • three outcome of interest was missing and/or insufficient in cl ud ed numbers of articles included in systematic meta-analysis (n=17) records identified through other sources (bibliographies) (n= 5) fig. 1 flow chart showing the selection process of articles. abayneh and worku 19 © 2020 the authors. published by aboutscience table i distribution of articles reviewed on esbl-producing clinical isolates from different regions of ethiopia first author, publication year study design study area sample types sample sources sample size total isolates total esbl positives common esbl producers esbl confirmation methode. coli k. pneumoniae other gramnegatives desta et al, 2016 (17) cs aa fecal sample/swab ip 267 295 151/295 106/235 44/58 1/2 cdt + vitek2 teklu et al, 2019 (18) cs aa urine, blood, sputum, csf, body fluid, pus and discharge ip and op 426 426 246/426 119/228 81/103 46/95 cdt moges et al, 2019 (19) cs bahir dar urine, blood, stool, pus, sputum, csf, body fluid, ear, nasal, cervical discharge ip and op 532 263 127/148 14/23 79/97 34/76 cdt legese et al, 2017 (20) cs aa blood and urine ip and op 322 33 22/28 5/6 16/19 1/8 cdt engda et al, 2018 (21) cs gondar swabs of sinks, bed, door handles, wastewater hospital env’t 384 57 57/57 20/57 24/57 13/57 hicrome esbl agar gashaw et al, 2018 (22) cs jimma urine, blood, sputum, wound/pus swab ip 118 126 51/100 19/31 16/30 18/39 ddst mulualem et al, 2012 (23) cs jimma urine, stool, sputum, wound/pus swab ip and op 359 67 24/67 24/67 na na cdt zeynudin et al, 2018 (24) cs jimma wound swabs, urine, biopsies, sputum ip and op 224 224 71/112 13/13 30/31 28/68 check-mdr microarray kits eshetie et al, 2015 (25) cs gondar urine ip and op 442 183 5/160 2/104 3/28 42 (na) chromagar beyene et al, 2011 (26) cs jimma stool, blood ip and op 1,225 113 71/113 na na 71/113 e-test abera et al, 2016 (27) cs bahir dar blood, urine, pus, csf, ear discharges, wound swab, water ip and op 757 274 127/274 73/170 36/55 17/49 ddst seid et al, 2005 (28) cs harrar sputum, urine and pus ip and op 384 57 19/57 na 19/57 na cdt abayneh et al., 2018 (29) cs jimma urine op 342 74 17/74 13/63 4/11 na ddst siraj et al, 2014 (30) cs jimma urine, sputum, blood, vaginal swabs, wound/ pus swab, eye discharge ip and op 471 112 43/112 24/85 19/27 na ddst mulisa et al, 2016 (31) cs adama urine, wound, nasal, stool, pleural fluids, ear discharge ip and op 384 133 17/68 10/35 2/8 5/25 ddst bitew (2019) (32) cs aa urine, wound, blood, csf, ear, nasal ip and op 996 153 66/135 na na 66/135 cdt beyene (2019) (33) cs aa urine, sputum, blood, vaginal, wound/pus, eye discharge ip 947 238 159/238 91/144 55/72 13/21 cdt aa = addis ababa; cdt = combination disc test; cs = cross-sectional; csf = cerebrospinal fluid; ddst = double disc synergy test; esbl = extended-spectrum beta-lactamase; e-test = epsilometric test; ip = inpatient; mdr = multidrug resistance; na = not analyzed; op = outpatient. meta-analysis on mdr and esbl-producing gnb in ethiopia20 © 2020 the authors. published by aboutscience table ii proportional estimates of esbl-producing gram-negative bacteria in different regions of ethiopia studies total proportions of esbl proportions of esbl producing e. coli proportions of esbl producing k. pneumoniae proportions of esbl producing other gns es [95% ci] weight es [95% ci] weight es [95% ci] weight es [95% ci] weight desta et al (2016) 0.51 [0.455, 0.569] 1.80 0.451 [0.389, 0.515] 2.91 0.759 [0.633, 0.852] 1.00 0.500 [0.059, 0.941] 0.45 teklu et al (2019) 0.58 [0.530, 0.624] 1.81 0.522 [0.457, 0.586] 2.91 0.786 [0.697, 0.855] 1.04 0.484 [0.386, 0.584] 3.53 moges et al (2019) 0.858 [0.792, 0.906] 1.67 0.609 [0.402, 0.782] 1.96 0.814 [0.725, 0.880] 1.03 0.447 [0.340, 0.560] 3.40 legese et al (2017) 0.786 [0.598, 0.900] 1.32 0.833 [0.369, 0.977] 0.66 0.842 [0.608, 0.948] 0.77 0.125 [0.017, 0.537] 0.72 engda et al (2018) 0.991 [0.877, 0.999] 0.39 0.351 [0.239, 0.482] 2.48 0.421 [0.301, 0.552] 1.02 0.228 [0.137, 0.354] 2.93 gashaw et al (2018) 0.510 [0.413, 0.606] 1.71 0.613 [0.435, 0.765] 2.16 0.533 [0.358, 0.701] 0.96 0.462 [0.317, 0.617] 2.90 mulualem et al (2012) 0.358 [0.253, 0.479] 1.64 0.358 [0.253, 0.479] 2.55 na na na zeynudin et al (2018) 0.634 [0.541, 0.718] 1.72 0.964 [0.616, 0.998] 0.42 0.968 [0.804, 0.995] 0.52 0.412 [0.302, 0.532] 3.31 eshetie et al (2015) 0.027 [0.011, 0.064] 1.34 0.018 [0.004, 0.069] 1.20 0.103 [0.034, 0.276] 0.78 42 (na) na beyene et al (2011) 0.628 [0.536, 0.712] 1.72 na na na 0.628 [0.536, 0.712] 3.58 abera et al (2016) 0.464 [0.405, 0.523] 1.79 0.429 [0.357, 0.505] 2.85 0.655 [0.521, 0.768] 1.01 0.415 [0.276, 0.569] 2.93 seid et al (2005) 0.333 [0.224, 0.464] 1.61 na na 0.333 [0.224, 0.464] 1.02 na na abayneh et al (2018) 0.230 [0.148, 0.339] 1.61 0.206 [0.124, 0.324] 2.36 0.364 [0.143, 0.661] 0.77 na na siraj et al (2014) 0.384 [0.299, 0.477] 1.72 0.282 [0.197, 0.387] 2.60 0.704 [0.510, 0.844] 0.92 na na mulisa et al (2016) 0.250 [0.161, 0.366] 1.61 0.286 [0.161, 0.454] 2.14 0.250 [ 0.063, 0.623] 0.64 0.200 [0.086, 0.400] 2.04 bitew (2019) 0.489 [0.406, 0.573] 2.00 na na na 0.489 [0.406, 0.573] 4.42 beyene (2019) 0.668 [0.606, 0.725] 20.5 0.632 [0.550, 0.707] 2.57 0.764 [0.653, 0.848] 1.11 0.619 [0.402, 0.797] 2.51 overall r es [95% ci] overall f es [95% ci] 0.489 [0.402, 0.577] 0.526 [0.505, 0.548] 0.412 [0.326, 0.504] 0.451 [0.422, 0.481] 0.618 [0.487, 0.734] 0.646 [0.604, 0.686] 0.429 [0.352, 0.509] 0.461 [0.424, 0.500] ci = confidence interval; esbl = extended-spectrum beta-lactamase; f = fixed; gn = gram negatives; na = not analyzed; r = random; es = event rate. ci: 0.586, 0.820) and 0.780 (95% ci: 0.615, 0.891) of the esbl-producing and non-esbl-producing gnb were found resistant to third-generation cephalosporin (ceftazidime and cefotaxime), respectively. the pooled estimate indicated that 0.589 (95% ci: 0.482, 0.688), 0.438 (95% ci: 0.347, 0.534) and 0.790 (95% ci: 0.713, 0.850) of esbl-producing and non-esbl-producing gnb isolates were resistant to gentamicin, ciprofloxacin and amoxicillin-clavulanic acid, respectively. besides, 0.784 (95% ci: 0.726, 0.832) and 0.727 (95% ci: 0.605, 0.823) of esbl-producing and non-esbl producing gnb isolates were resistant to sxt and tetracycline (tet), respectively. the pooled proportional estimates of mdr isolates were found to be 0.827 (95% ci: 0.726, 0.896) (tab. iii). abayneh and worku 21 © 2020 the authors. published by aboutscience table iii antimicrobial and multidrug resistance profile of bacterial isolates obtained from different samples in ethiopia authors total isolates total esblproducer antimicrobial resistance profiles caz ctx gnt cip sxt tet amc mdr desta et al. (2016) 295 151/295 137/150 146/150 105/150 94/150 136/150 na 134/150 150/150 teklu et al. (2019) 426 246/426 257/426 265/426 185/426 240/426 324/426 na 305/426 237/246 or 291/426* moges et al. (2019) 185 127/148 143/148 129/148 116/148 52/148 138/148 127/148 117/148 127/148 or 148/185* legese et al. (2017) 33 22/28 na 29/33 26/33 na 29/33 25/33 28/33 na engda et al. (2018) 57 57/57 57/57 57/57 11/57 25/57 37/57 na 57/57 32/57 gashaw et al. (2018) 100 51/100 63/100 60/100 68/100 48/100 79/100 90/100 94/100 28/100* mulualem et al. (2012) 67 24/67 4/67 6/67 2/67 14/67 38/67 49/67 47/67 67/67* zeynudin et al. (2018) 224 71/112 63/68 66/68 60/68 41/68 62/68 na na 67/68* eshetie et al. (2015) 183 5/183 90/160 40/160 94/160 4/160 103/160 79/160 81/160 160/183* beyene et al. (2011) 113 71/113 na na 84 1 91 45 na 78/113* abera et al. (2016) 274 127/274 na na na 106/274 174/274 na na na seid et al. (2005) 57 19/57 23/57 22/57 35/57 na 37/57 na na 41/57* abayneh et al. (2018) 74 17/74 12/17 17/17 11/17 13/17 14/17 14/17 14/17 14/17 raw weight relative weight random desta k (2016) 151 / 295 2.07 6.73 teklu ds (2019) 246 / 426 2.09 6.79 moges f (2019) 127 / 148 1.90 6.19 legese m (2017) 22 / 28 1.47 4.77 engda t (2018) 57 / 57 0.40 1.31 gashaw m (2018) 51 / 100 1.96 6.38 mulualem y (2012) 24 / 67 1.87 6.08 zeynudin a.(2018) 71 / 112 1.97 6.40 eshetie s. (2015) 5 / 160 1.48 4.81 beyene g (2011) 71 / 113 1.97 6.41 abera b (2016) 127 / 274 2.06 6.72 seid j (2005) 19 / 57 1.82 5.93 abayneh m (2018) 17 / 74 1.83 5.96 siraj sm (2014) 1.97 6.41 mulisa g (2016) 1.82 5.93 bitew a (2019) 2.00 6.51 beyene d (2019) 2.05 6.66 -0.50 0.00 0.50 1.00 overall f es [95% ci] 0.512 [0.455,0.569] 0.577 [0.530,0.624] 0.858 [0.792, 0.906] 0.991 [0.877, 0.999] 0.510 [0.413, 0.606] 0.358 [0.253, 0.479] 0.634 [0.541, 0.718] 0.031 [0.013, 0.073] 0.628 [0.536, 0.712] 0.464 [0.405, 0.523] 0.786 [0.598, 0.900] 0.333 [0.224, 0.464] 0.230 [0.148, 0.339] 0.384 [0.299, 0.477] 0.250 [0.161, 0.366] 0.489 [0.406, 0.573] 0.668 [0.606, 0.725] overall r es [95% ci] 0.489 [0.402, 0.577] heterogeneity i2 = 93.41%; p = 0.000 0.526 [0.505, 0.548] statistics for each study es [95% ci]study name esbl/total 43 / 112 17 / 68 66/135 159/238 fig. 2 proportional estimates of esbl-producing gnb in different clinical samples in ethiopia. ci = confidence interval; esbl = extended-spectrum beta-lactamase; es = event rate. (continued) meta-analysis on mdr and esbl-producing gnb in ethiopia22 © 2020 the authors. published by aboutscience authors total isolates total esblproducer antimicrobial resistance profiles caz ctx gnt cip sxt tet amc mdr siraj et al. (2014) 112 43/112 42/43 43/43 36/43 33/43 41/43 39/43 38/43 38/43 mulisa et al. (2016) 68 17/68 na na 12/17 14/17 14/17 6/17 na 17/17 bitew (2019) 135 66/135 45/135 na 32/135 47/135 86/135 92/135 100/135 110/135* beyene (2019) 238 159/238 176/238 na 117/238 137/238 195/238 191/238 148/238 225/238* overall r, es [95% ci] 1273/2487 0.72 [0.586, 0.820] 0.78 [0.615, 0.891] 0.589 [0.482, 0.688] 0.438 [0.347, 0.534] 0.784 [0.726, 0.832] 0.727 [0.605, 0.823] 0.790 [0.713, 0.850] 0.827 [0.726, 0.896] amc = ampicillin-clavulanic acid; caz = ceftazidime; ci = confidence interval; cip = ciprofloxacin; ctx = cefotaxime; gnt = gentamicin; esbl = extended-spectrum beta-lactamase; mdr = multi-drug resistance; na = not analyzed; r = resistance; sxt = trimethoprim-sulfamethoxazole; tet = tetracycline; es = event rate. *from overall isolates. laboratory methods used to estimate the proportion of esbl-producing strains according to this review, 8/17 (47.1%) of the articles reviewed used combination disk test (cdt) and 5/17 (29.4%) articles used double disk synergy test (ddst) methods alone. two (11.8%) articles used chromagar and one article used e-test to estimate esbl proportions. only one article used check-mdr ct103 microarray kits for detection and molecular characterization of the esbl genes (tab. i). publication bias funnel plots of standard error with logit event rate confirmed that there is no statistically significant evidence of publication bias on studies reporting the prevalence of esblproducing bacterial isolates from different clinical samples in ethiopia (begg’s test, p = 0.217; egger’s test, p = 0.231) (fig. 3). however, begg suggested that a nonsignificant correlation may be due to low statistical power and cannot be taken as evidence that bias is absent. discussion this is the first meta-analysis study relating the extent of the esbl-producing gnb in ethiopia. accordingly, the pooled proportional estimates of esbl-producing gnb were 48.9%, which is higher than previous estimates of meta-analysis in east africa (34) and pakistan (35), in which overall pooled proportion of esbl-producers was 42% and 40%, respectively. as compared to each country in east africa, this finding is higher than the pooled proportion of esbl-producing enterobacteriaceae in tanzania (39%) and kenya (47%) (34). the result is also higher than estimates of meta-analysis in african countries, in which the total proportion of esbl-producing isolates was 15% in 16 out of 26 studies (36). as compared to resource-rich countries, the pooled proportion of esbl-producing isolates was thus considerably higher in resource-limited countries, including our setting. for instance, the pooled global prevalence of esbl-producing enterobacteriaceae among pregnant women diagnosed with urinary tract infections (utis) is 25%, with the highest rates in africa (45%) and india (33%), followed by 15% in other asian countries, 5% (2.8%) in europe and the lowest one of 4% in south america and 3% in north america (37). this study finding is also close to data reported for china, where a nationwide survey that included 30 hospitals reported over 46% resistance due to esbl (38). however, our estimate is slightly lower than for uganda (62%) (34), ghana (49%) (39), cameroon (54%) (40) and morocco (43%) (41). with regard to the frequency of isolates, in this study, the pooled proportional estimates of esbl-producing k. pneumoniae, e. coli and other gnb were 60.3%, 39.0% and 40.9%, respectively. this is much higher than a smart study between 2009 and 2010, in which esbl prevalence among k. pneumoniae and e. coli was 38.9% and 17.6%, respectively, in europe, and 8.8% and 8.5, respectively, in -5 -4 -3 -2 -1 0 1 2 3 4 5 0.0 0.5 1.0 1.5 2.0 logit event rate st an da rd e rr or fig. 3 funnel plot depicting publication bias of studies reporting the prevalence of extended-spectrum beta-lactamase (esbl)-producing gram-negative bacilli in different clinical samples in ethiopia. table iii continued abayneh and worku 23 © 2020 the authors. published by aboutscience north america (42). moreover, our meta-analysis result is also higher than a report of in vitro activity of tigecycline and comparators against gram-negative and gram-positive organisms collected from asia-pacific during 2004-2010 and 2015, in which 24.6% and 15.8% of e. coli and k. pneumoniae were esbl producers in 2015, respectively[a: please check the clarity of this sentence.] (43). in addition, among gnb collected from intra-abdominal infections in the asia-pacific region during 2007, 42.2% and 35.8% of e. coli and klebsiella spp., respectively, were esbl positive (44). however, higher proportions of esbl-producing e. coli and k. pneumoniae were reported from india, vietnam and china (44,45). in this study, inter-study results showed a wide and statistically significant degree of variation in proportion estimates of esbl proportions (p < 0.05). there are several possible factors that may account for the variations seen in this review. the first factor is the difference in sensitivity and specificity between methods used in estimating esbl proportions. some studies reviewed estimated esbl proportions using purely phenotypic methods, while others used both phenotypic and molecular-based methods. for instance, in this study, most of the reviewed articles used cdt (17-20,23,28,32,33) and ddst (22, 27,29-31) methods alone for the detection of esbl-producing isolates, and only one study used molecular technique for investigations and characterizations of the gene encoding esbl (24). the other factor contributing to the variation in proportion estimates of esbl proportions is type of wards or units, type of specimen collected and whether patients were attending outpatient or inpatient departments. in this meta-analysis, many reviewed studies showed that esbl-producing isolates were more among inpatients than outpatients (20,24,30). in contrast, two reviewed studies showed the reverse finding (23,27). a meta-analysis study in areas of sub-saharan africa showed that pooled esbl-producing enterobacteriaceae colonization was 18%, 32% and 55% in community studies, at hospital admission and for inpatient studies, respectively (46). many reports have documented the difference in esbl proportion estimates between hospitals versus community-based surveys (23,25,27,30). however, the lack of any estimates for community-based esbl carriage in ethiopia underscores an urgent need for surveillance in the region. infection control in hospitals including hand hygiene and rational antibiotic use can be effective measures to stop further spread of the esbl-producing and mdr strains in both hospitals and communities. mdr gnb are now globally widespread and present a major challenge to modern medical practice. to date, the overall epidemiology and burden of mdr bacteria and their mechanisms of resistance have not been fully understood, especially in resource-limited countries, including ethiopia. this is the first meta-analysis study conducted to determine the pooled proportional estimates of esbl-producing gnb and mdr isolates in ethiopia. accordingly, the pooled proportional estimates of mdr isolates were found to be 82.7% (tab. iii). this finding is higher than a finding of previous meta-analysis in ethiopia, in which the pooled proportional estimates of mdr isolates were 59.7% (47); however, it was lower than a single study finding in ethiopia (33) and chile (48), in which 94.5% and 100% isolates exhibited mdr profiles. lower study finding was also reported in ethiopia, in which the overall prevalence of mdr was 69.9%, of which 81.5% was in the hospital environment, while 54.2% was in non-hospital environment samples (49). the occurrence of mdr may be linked with indiscriminate utilization of antimicrobial agents such as wrong indication, wrong duration, improper route of administration, use of leftover antibiotics from a family member and improper discontinuation of antibiotics or genetic mutation (47,50,51). a meta-analysis finding in ethiopia indicated that the pooled estimate of inappropriate antibiotic use was 49.2% and the pooled proportion of self-antibiotic prescription was 43.3% (47). thus, the frequent and inappropriate use of antibiotics in humans and animals may contribute to the recent emergence of esbl producers and mdr strains both in healthcare institutions and communities. moreover, medication required to treat esbl-producing isolates is expensive and unaffordable for the majority of the population in these settings, making these bacteria difficult to treat. a study in ethiopia concluded that mortality was significantly associated with antimicrobial resistance (52). for instance, all 11 patients with enterobacteriaceae resistant to third-generation cephalosporins died. another systematic review and meta-analysis estimated that the mortality in neonates with bloodstream infections (bsis) due to esblproducing enterobacteriaceae was 36%, as compared to 18% among all other neonates with bsi (53). it is therefore of the utmost importance to make reliable data available to guide strategies devoted to limiting the spread of particularly esblproducing pathogens and mdr strains in ethiopia. in this study, significant numbers of bacterial isolates showed different resistance profile against selected antimicrobial agents. the pooled proportional estimate of resistance against third-generation cephalosporins (cefotaxime and ceftazidime), amoxicillin-clavulanic acid, gentamicin, ciprofloxacin, sxt and tet ranges from 43.8% to 79.0%. almost similar findings were reported in ethiopia and chile, in which 45.4% to >88% isolates exhibit resistance against betalactams and non-lactam drugs (33,48). this high resistance rate might be associated with an interesting feature of esblproducing isolates as resistance mechanisms, which may be either chromosomally or plasmid-encoded, especially ctx-m that often showed feature of coresistance to various classes of antibiotics such as fluoroquinolones, aminoglycosides, and co-trimoxazole. therefore, interventions including infection control measures and restriction of low-quality and inappropriate use of antibiotics may aid in controlling the emergency and spread of esbl-producing pathogens and may actually prove cost-beneficial. as a limitation, in this study, due to the lack of sufficient records on the prevalence of esbl producers from different clinical samples in ethiopia, all primary studies, including those with very small datasets (<10 esbl-producing strains) were included. conclusion the problems related to antibiotic resistance, including mdr due to esbl, are significant in ethiopia. the scarcity of data on predictors, clinical outcomes, magnitudes and gene variants encoding resistance due to esbl-producing gnb meta-analysis on mdr and esbl-producing gnb in ethiopia24 © 2020 the authors. published by aboutscience calls for active surveillance systems, which can help understand the current epidemiology of esbl within the country. furthermore, this can aid in developing national guidelines for proper screening of esbl as well as developing standardized approaches for managing patients colonized with esblproducing gnb. disclosures conflict of interest: the authors declare that they have no competing interest. financial support: no funding was allocated for this study. availability of data and materials: all the data supporting our findings were incorporated within the manuscript, but are available from the corresponding author on reasonable request. authors’ contributions: ma and tw participated in the study design, study searching and screening and data extractions. ma analyzed the data and drafted the manuscript. tw support data analysis, read, revised and approved the final version of the manuscript. references 1. dhillon rh, clark j. esbls: a clear and present danger? hindawi publ corp. 2012;11. 2. byarugaba dk. antimicrobial resistance in developing countries springer, 2009;15-27. 3. erku da, mekuria ab, belachew sa. inappropriate use of antibiotics among communities of gondar town, ethiopia: a threat to the development of antimicrobial resistance. antimicrob resist infect control. 2017;6:112. crossref 4. gebretekle gb, serbessa mk. exploration of over the counter sales of antibiotics in community pharmacies of addis ababa, ethiopia: pharmacy professionals’ perspective. antimicrob resist infect control. 2016;5:2. crossref 5. european centre for disease prevention and control. systematic review of the effectiveness of infection control measures to prevent the transmission of extended-spectrum beta-lactamase-producing enterobacteriaceae through cross border transfer of patients. stockholm: ecdc 2014. crossref 6. morosini mi, garcía-castillo m, coque tm, et al. antibiotic co-resistance in esbl-producing enterobacteriaceae and in vitro activity of tigecycline. antimicrob agents chemother. 2006;50(8):2695-2699. 7. cantón r, novais a, valverde a, et al. prevalence and spread of extended-spectrum beta-lactamase-producing enterobacteriaceae in europe. clin microbiol infect. 2008;14 suppl 1: 144-153. 8. shamsrizi p, gladstone bp, carrara e, et al. variation of effect estimates in the analysis of mortality and length of hospital stay in patients with infections caused by bacteria-producing extended-spectrum beta-lactamases: a systematic review and meta-analysis. bmj open. 2020;10:e030266. crossref 9. paterson dl, bonomo ra. extended-spectrum β-lactamases: a clinical update. clin microbiol rev. 2005;18(4):657-686. 10. giske cg, monnet dl, cars o, carmeli y, react-action on antibiotic resistance. clinical and economic impact of common multidrug-resistant gram-negative bacilli. antimicrob agents chemother. 2008;52(3):813-821 11. bonnet r. growing group of extended-spectrum beta lactamases: the ctx-m enzymes. antimicrob agents chemother. 2004;48(1):1-14. 12. villegas m v, kattan jn, quinteros mg, casellas jm. prevalence of extended-spectrum b-lactamases in south america. clin microbiol infect. european society of clinical microbiology and infectious diseases; 2008;14:154-158. 13. hawkey pm. prevalence and clonality of extended-spectrum beta-lactamases in asia. clin microbiol infect. 2008;14:159-165. 14. bradford pa. extended-spectrum beta-lactamases in the 21st century: characterization, epidemiology, and detection of this important resistance threat. am soc microbiol. 2001;14(4):933-951. 15. tham j, melander e, walder m, edquist pj, odenholt i. prevalence of faecal esbl carriage in the community and in a hospital setting in a county of southern sweden. eur j clin microbiol infect dis. 2011;10:1159-1162 16. malta m, cardoso lo, bastos fi, magnanini mm, silva cm. strobe initiative: guidelines on reporting observational studies. saude publica. 2010;44(3):559-565. crossref 17. desta k, woldeamanuel y, azazh a, et al. high gastrointestinal colonization rate with extended-spectrum β-lactamaseproducing enterobacteriaceae in hospitalized patients: emergence of carbapenemase-producing k. pneumoniae in ethiopia. plos one. 2016;11(8):e0161685. crossref 18. teklu ds, negeri aa, legese mh, bedada tl, woldemariam hk, tullu kd. extended-spectrum beta-lactamase-production and multi-drug resistance among enterobacteriaceae isolated in addis ababa, ethiopia. antimicrob resist infect control. 2019; 8:39. crossref 19. moges f, eshetie s, abebe w, et al. high prevalence of extended-spectrum beta-lactamase-producing gram-negative pathogens from patients attending felege hiwot comprehensive specialized hospital, bahir dar, amhara region. plos one. 2019;14(4):e0215177. crossref 20. legese mh, weldearegay mg, asrat d. extended-spectrum beta-lactamaseand carbapenemase-producing enterobacteriaceae among ethiopian children. infect drug resist. 2017;10:27-34. crossref 21. engda t, moges f, gelaw a, eshete s, mekonnen f. prevalence and antimicrobial susceptibility patterns of extended spectrum betalactamase producing entrobacteriaceae in the university of gondar referral hospital environments, northwest ethiopia. bmc res notes. 2018;11:335. crossref 22. gashaw m, berhane m, bekele s, et al. emergence of high drug resistant bacterial isolates from patients with health care associated infections at jimma university medical center: a cross sectional study. antimicrob resist infect control. 2018;7:138. crossref 23. mulualem y, kasa t, mekonnen z, suleman s. occurrence of extended spectrum beta (beta)-lactamases in multi-drug resistant escherichia coli isolated from a clinical setting in jimma university specialized hospital, jimma, and southwest ethiopia. east afr j public health. 2012;9(2):58-61. pmid: 23139958. 24. zeynudin a, pritsch m, schubert s, et al. prevalence and antibiotic susceptibility pattern of ctx-m type extended-spectrum β-lactamases among clinical isolates of gram-negative bacilli in jimma, ethiopia. bmc infect dis.2018;18:524. crossref 25. eshetie s, unakal c, gelaw a, ayelign b, endris m, moges f. multidrug resistant and carbapenemase producing enterobacteriaceae among patients with urinary tract infection at referral hospital, northwest ethiopia. antimicrob resist infect control. 2015;4:12. crossref 26. beyene g, nair s, asrat d, mengistu y, engers h, wain j. multidrug resistant salmonella concord is a major cause of salmonellosis in children in ethiopia. j infect devctries. 2011;5:023033. crossref 27. abera b, kibret m, mulu w. extended-spectrum beta (β)lactamases and antibiogram in enterobacteriaceae from clinical and drinking water sources from bahir dar city, ethiopia. plos one. 2016;11(11):e0166519. crossref 28. seid j, asrat d. occurrence of extended spectrum beta-lactamse enzymes in clinical isolates of klebsiella species from https://doi.org/10.1186/s13756-017-0272-2 https://doi.org/10.1186/s13756-016-0101-z https://doi.org/10.2900/850536 https://doi.org/10.1136/bmjopen-2019-030266 https://doi.org/10.1590/s0034-89102010000300021 https://doi.org/10.1371/journal.pone.0161685 https://doi.org/10.1186/s13756-019-0488-4 https://doi.org/10.1371/journal.pone.0215177 https://doi.org/10.2147/idr.s127177 https://doi.org/10.1186/s13104-018-3443-1 https://doi.org/10.1186/s13756-018-0431-0 https://doi.org/10.1186/s12879-018-3436-7 https://doi.org/10.1186/s13756-015-0054-7 https://doi.org/10.3855/jidc.906 https://doi.org/10.1371/journal.pone.0166519 abayneh and worku 25 © 2020 the authors. published by aboutscience harar region, eastern ethiopia. acta trop. 2005;95(2);143-148. crossref 29. abayneh m, tesfaw g, abdissa a. isolation of extended-spectrum β-lactamase-(esbl) producing e. coli and k. pneumoniae from patients with community-onset urinary tract infections in jimma university specialized hospital, southwest ethiopia. can j infect dis med microbiol. 2018;2018:4846159. crossref 30. siraj sm, ali s, wondafrash b. extended-spectrum-lactamase production and antimicrobial resistance in klebsiellapneumoniae and escherichia coli among inpatients and outpatients of jimma university specialized hospital, south-west, ethiopia. afr j microbiol res. 2014;8(43):3687-3694. 31. mulisa g, selassie l, jarso g, shiferew t, zewdu a. prevalence of extended spectrum beta-lactamase producing enterobacteriaceae: a cross sectional study at adama hospital, adama, ethiopia. j emerg infect dispatho. 2016;1(1):102. 32. bitew a. high prevalence of multi-drug resistance and extended spectrum beta-lactamaseproduction in non-fermenting gramnegative bacilli in ethiopia. infect dis res treat. 2019;12:1-7. 33. beyene d, bitew a, fantew s, mihret a, evans m. multidrugresistant profile and prevalence of extended spectrum β-lactamase and carbapenemaseproduction in fermentative gram-negative bacilli recovered from patients and specimens referred to national reference laboratory, addis ababa, ethiopia. plos one. 2019;14(9):e0222911. crossref 34. sonda t, kumburu h, van zwetselaar m, et al. meta-analysis of proportion estimates of extended-spectrum-beta-lactamaseproducing enterobacteriaceae in east africa hospitals. antimicrob resist infect control.2016;5:18. crossref 35. abrar s, hussain s, khan ra, ul ain n, haider h, riaz s. prevalence of extended-spectrum-β-lactamase-producing enterobacteriaceae: first systematic meta-analysis report from pakistan. antimicrob resist infect control. 2018;7:26. crossref 36. tansarli gs, poulikakos p, kapaskelis a, falagas me. proportion of extended-spectrum b-lactamase (esbl)-producing isolates among enterobacteriaceae in africa: evaluation of the evidence—systematic review. j antimicrob chemother. 2014;69:1177-1184. crossref 37. mansouri f, sheibani h, masroor mj, afsharian m. extendedspectrum beta-lactamase (esbl)-producing enterobacteriaceae and urinary tract infections in pregnant/postpartum women: a systematic review and meta-analysis. int j clin pract. 2019;37(12). crossref 38. zhang j, zheng b, zhao l, et al. nationwide high prevalence of ctx-m and an increase of ctx-m-55 in escherichia coli isolated from patients with community-onset infections in chinese county hospitals. bmc infect dis. 2014;14:659-663. 39. obeng-nkrumah n, twum-danso k, krogfelt k, newman mj. high levels of extended-spectrum beta-lactamases in a major teaching hospital in ghana: the need for regular monitoring and evaluation of antibiotic resistance. am j trop med hyg. 2013;89:960-964. 40. magoué cl, melin p, gangoué-piéboji j, okomo assoumou m-c, boreux r, de mol p. prevalence and spread of extended-spectrum β-lactamase-producing enterobacteriaceae in ngaoundere, cameroon. clin microbiol infect. 2013;19:416-420. 41. girlich d, bouihat n, poirel l, benouda a, nordmann p. high rate of faecal carriage of extended-spectrum β-lactamase and oxa48 carbapenemase-producing enterobacteriaceae at a university hospital in morocco. clin microbiol infect. 2014;20:350-354. 42. hoban dj, lascols c, nicolle le, et al. antimicrobial susceptibility of enterobacteriaceae, including molecular characterization of extended-spectrum beta-lactamase-producing species, in urinary tract isolates from hospitalized patients in north america and europe: results from the smart study. diagn microbiol infect dis. elsevier inc. 2012;74(1):62-67. crossref 43. yang q, xu yc, kiratisin p, dowzicky mj. antimicrobial activity among gram-positive and gram-negative organisms collected from the asia-pacific region as part of the tigecycline evaluation and surveillance trial: comparison of 2015 results with previous years. diagn microbiol infect dis. 2017;89:314-323. 44. hawser sp, bouchillon sk, hoban dj, badal re, hsueh pr, paterson dl. emergence of high levels of extended-spectrum-betalactamase-producing gram-negative bacilli in the asia-pacifc region: data from the study for monitoring antimicrobial resistance trends (smart) program, 2007. antimicrob agents chemother. 2009;53:3280-3284. 45. lu pl, liu yc, toh hs, et al. epidemiology and antimicrobial susceptibility profiles of gram-negative bacteria causing urinary tract infections in the asia-pacific region: 2009-2010 results from the study for monitoring antimicrobial resistance trends (smart). int j antimicrob agents. 2012;40:s37-s43. 46. lewis jm, lester r, garner p, feasey na. gut mucosal colonisation with extended-spectrum beta-lactamase producing enterobacteriaceae in sub-saharan africa: a systematic review and meta-analysis [version 2; peerreview: 2 approved]: wellcome open res. 2020;4:160. crossref 47. muhie oa. antibiotic use and resistance pattern in ethiopia: systematic review and meta-analysis. int j microbiol. 2019, article id 2489063, 8 pages. crossref 48. pavez m, troncoso c, irma osses i, et al. high prevalence of ctx-m-1 group in esbl-producing enterobacteriaceae infection in intensive care units in southern chile. braz j infect dis. 2019;23(2):102-110. 49. moges f, endris m, belyhun y, worku w. isolation and characterization of multiple drug resistance bacterial pathogens from waste water in hospital and non-hospital environments, northwest ethiopia. bmc res notes. 2014;7:215. published 2014 apr 5. crossref 50. rodríguez-baño j, gutiérrez-gutiérrez b, machuca i, pascual a. treatment of infections caused by extended-spectrum-betalactamase-, ampc-, and carbapenemase-producing enterobacteriaceae. clin microbiol rev. 2018;31:e00079-17. crossref 51. harris p, paterson d, rogers b. facing the challenge of multidrugresistant gram-negative bacilli in australia. mja. 2015;202(5). crossref 52. seboxa t, amogne w, abebe w, et al. high mortality from blood stream infection in addis ababa, ethiopia, is due to antimicrobial resistance. plos one. 2015;10(12):e0144944. crossref 53. flokas m, karanika s, alevizakos m, mylonakis e. prevalence of esbl-producing enterobacteriaceae in pediatric blood-stream infections: a systematic review and meta-analysis. plos one. 2017;12(1). crossref https://doi.org/10.1016/j.actatropica.2005.05.009 https://doi.org/10.1155/2018/4846159 https://doi.org/10.1371/journal.pone.0222911 https://doi.org/10.1186/s13756-016-0117-4 https://doi.org/10.1186/s13756-018-0309-1 https://doi.org/10.1093/jac/dkt500 https://doi.org/10.1111/ijcp.13422 https://doi.org/10.1016/j.diagmicrobio.2012.05.024 https://doi.org/10.12688/wellcomeopenres.15514.2 https://doi.org/10.1155/2019/2489063 https://doi.org/10.1186/1756-0500-7-215 https://doi.org/10.1128/cmr.00079-17 https://doi.org/10.5694/mja14.01257 https://doi.org/10.1371/journal.pone.0144944 https://doi.org/10.1371/journal.pone.0171216 dti drug target insights 2023; 17: 54-57issn 1177-3928 | doi: 10.33393/dti.2023.2573original research article drug target insights issn 1177-3928 www.aboutscience.eu/dti © 2023 the authors. this article is published by aboutscience and licensed under creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). commercial use is not permitted and is subject to publisher’s permissions. full information is available at www.aboutscience.eu are calcium channel blockers related to lung cancer? ratchanon rattanathanoo, jarin chindaprasirt, watchara boonsawat, panita limpawattana, sittichai khamsai, kittisak sawanyawisuth department of medicine, faculty of medicine, khon kaen university, khon kaen thailand abstract background: calcium channel blocker (ccb) is a common antihypertensive agent for the treatment of hypertension. there are inconsistent data of an association of ccb and lung cancer in the literature. this study aimed to evaluate this association by a case-control design. methods: the inclusion criteria were adult patients 18 years or over, diagnosed with hypertension, lung cancer or pulmonary tuberculosis, and presenting with one of the suggestive symptoms of lung cancer. those who were pregnant or had a diagnosis of lung cancer or pulmonary tuberculosis prior to the diagnosis of hypertension were excluded. diagnosis of lung cancer was made pathologically, while tuberculosis was made by positive acid-fast bacilli on sputum examination, sputum culture positive for mycobacterium tuberculosis, or polymerase chain reaction positive for m. tuberculosis with a chest x-ray compatible with tuberculosis. cases were those diagnosed with lung cancer, while controls were those diagnosed with tuberculosis. factors associated with lung cancer were calculated by logistic regression analysis. results: there were 178 patients who met the study criteria. of those, 69 patients (38.8%) were in the case group. the lung cancer group had egfr gene mutation in 21 patients (52.5%) and adenocarcinoma was the most common cell type of lung cancer (55 patients; 79.7%). there were two factors independently associated with lung cancer including dyslipidemia and family history of lung cancer. conclusions: ccb was not associated with lung cancer in patients with hypertension but dyslipidemia and family history of lung cancer were independently associated with lung cancer in this setting. keywords: dyslipidemia, family history, risk factors received: february 7, 2023 accepted: may 1, 2023 published online: may 19, 2023 corresponding author: sittichai khamsai department of medicine, khon kaen university khon kaen 40002 thailand sittikh@kku.ac.th kittisak sawanyawisuth 123 mitraparp road, department of medicine faculty of medicine, khon kaen university khon kaen, 40002 thailand kittisak@kku.ac.th in reduction of stroke by up to 40%, and reduction in heart failure by up to 50% (3). additionally, treatment of conditions or comorbid diseases of hypertension is also crucial (4-8). antihypertensive agents are effective in blood pressure lowering including calcium channel blockers (ccbs). even though ccb is recommended as the first-line treatment for hypertension (9), it has been reported to be increasing the risk of cancer (10). several studies including a meta-analysis showed that ccb was associated with lung cancer with adjusted odds ratio of 1.13 (95% confidence interval 1.06, 1.21) (11-13), while other studies did not find this association (14,15). as there are inconsistent and inconclusive data on this issue, this study aimed to evaluate the additional association of ccb and lung cancer. methods this was a case-control study conducted at srinagarind hospital, khon kaen university, thailand. the inclusion criteria were adult patients 18 years or over, diagnosed with lung cancer or pulmonary tuberculosis, and presenting with one of the following symptoms: hemoptysis, chronic cough, constitutional symptoms, lung mass, and a checkup. those who were pregnant or with diagnosis of lung cancer introduction hypertension is a common disease, with an estimated 1.39 billion adults diagnosed with hypertension (1). generally, the prevalence of hypertension was 31.1% worldwide in 2010. the main goal of hypertension treatment is to achieve blood pressure control. a previous report found that only 23% of women and 18% of men with hypertension had a good blood pressure control globally (2). blood pressure lowering may result https://doi.org/10.33393/dti.2023.2573 https://creativecommons.org/licenses/by-nc/4.0/legalcode rattanathanoo et al drug target insights 2023; 17: 55 © 2023 the authors. published by aboutscience www.aboutscience.eu or pulmonary tuberculosis prior to the diagnosis of hypertension were excluded. diagnosis of lung cancer was made pathologically, while tuberculosis was made by positive acidfast bacilli on sputum examination, sputum culture positive for mycobacterium tuberculosis, or polymerase chain reaction (pcr) positive for m. tuberculosis with a chest x-ray compatible with tuberculosis. cases were those diagnosed with lung cancer, while controls were those diagnosed with tuberculosis. the study period was between 2007 and 2019. eligible patients were recorded for baseline characteristics, medications, and risk factors for lung cancer. baseline characteristics included age, sex, body mass index, comorbid diseases, alcohol consumption, and smoking history. risk factors of lung cancer were also recorded: family history of lung cancer, occupational exposure, or chest wall radiation. antihypertensive medications particularly ccb were retrieved as well as duration of ccb use. the primary outcome of this study was to evaluate the association of lung cancer and ccb. sample size calculation based on a probability of event in the case and control group of 0.6 and 0.3, the required sample size for case group was 49 patients with a confidence of 95% and power of 80%. the control group was enrolled for a ratio of case:control of 1:1-2. statistical analyses patients were categorized into two groups: lung cancer (cases) and tuberculosis (controls). descriptive statistics were used to compare differences between both groups: independent t-test or wilcoxon rank sum test for numerical variables and chi-square or fisher’s exact test for categorical variables. factors associated with lung cancer were calculated by logistic regression analysis. multicollinearity was checked in the logistic regression analysis. the final model was executed for goodness of fit by hosmer-lemeshow test. statistical analyses were calculated by stata software version 10.1 (college station, texas, usa). results there were 178 patients who met the study criteria. of those, 69 patients (38.8%) were in the case group or lung cancer group. the lung cancer group had tested for gene mutation in 40 patients (56.0%). egfr was the most common gene mutation (21 patients; 52.5%), followed by no mutation (15 patients; 37.5%), and alk mutation (4 patients; 10%). adenocarcinoma was the most common cell type of lung cancer (55 patients; 79.7%), followed by squamous cell carcinoma (8 patients; 11.6%). table i shows baseline characteristics and ccb treatment of the case and control groups. there were three significant factors between both groups, namely dyslipidemia, obstructive sleep apnea, and family history of lung cancer. the case group had significantly higher proportions of these three factors than the control group such as family history of lung cancer (15.9% vs. 0.9%; p < 0.001). table i baseline characteristics and ccb treatment of patients with hypertension categorized by lung cancer (case group) and tuberculosis (control group) factors cases (n = 69) controls (n = 109) p value age, years* 67 (61,71) 66 (59,71) 0.603 male 44 (63.8) 77 (70.6) 0.338 female 25 (36.2) 32 (29.4) bmi 0.676  <19 13 (18.8) 17 (15.9)  19-24.5 16 (23.2) 25 (23.4)  24.5-30 5 (7.2) 4 (3.7)  >30 35 (50.7) 61 (57.0) smoking 40 (58) 64 (58.7) 0.922 alcohol drinking 28 (40.6) 53 (48.6) 0.294 comorbidities  cvd 10 (14.5) 17(15.6) 0.841  dyslipidemia 29 (42) 27 (24.8) 0.016  dm 27 (39.1) 48 (44) 0.518  osa 5 (7.2) 1 (0.9) 0.033  ckd 10 (14.5) 23 (21.1) 0.269  ischemic stroke 6 (8.7) 2 (1.8) 0.057  autoimmune 2 (2.9) 4 (3.7) >0.999  hbv infection 2 (2.9) 3 (2.8) >0.999  hcv infection 0 5 (4.6) 0.158  hyperthyroidism 1 (1.4) 0 0.388 family history of lung cancer 11 (15.9) 1 (0.9) <0.001 occupational exposure 1 (1.4) 2 (1.8) >0.999 chest wall radiation 0 0 na ccb used 45 (65.2) 62 (56.9) 0.268 ccb duration, years* 5 (3-10) 6 (2.8-8.2) 0.642 data presented as number (percentage) except * indicated as median (1st-3rd quartile range). bmi = body mass index; ccb = calcium channel blocker; ckd = chronic kidney disease; cvd = cardiovascular disease; dm = diabetic mellitus; hbv = hepatitis b virus; hcv = hepatitis c virus; na = not available; osa = obstructive sleep apnea. there were five factors in the final model for prediction of lung cancer (tab. ii). two factors were independently associated with lung cancer including dyslipidemia and family history of lung cancer. these factors had an adjusted odds ratio (95% confidence interval) of 2.12 (1.07, 4.23) and 22.43 (2.76, 182.36), respectively. the hosmer-lemeshow chi-square was 4.88 (p = 0.559). ccb use had an adjusted odds ratio of 1.50 (95% confidence interval of 0.53, 2.01). discussion this study found that ccb was not associated with lung cancer but dyslipidemia and family history of lung cancer did. ccb and lung cancer56 © 2023 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti ccb-related cancer is still inconclusive and needs further studies to evaluate this issue (16). the possible mechanisms of ccb-related cancer include cell growth, cell proliferation, and cell apoptosis (17,18). however, some studies found that ccb may reduce risks of lung cancer as ccb may suppress lung tumorigenesis and suppression of cacna2d2, a link to non–small cell lung cancer (19,20). previous studies and meta-analyses that reported on the risk of ccb on lung cancer were mainly from database study with asymptomatic controls, while this study evaluated this association with a case-control study using controls who presented with similar symptoms of lung cancer. rotshild et al published one meta-analysis and one nested case-control study. both studies reported that ccb was associated with lung cancer with an adjusted odds ratio of 1.13 (95% confidence interval 1.06, 1.21) and a risk ratio of 1.15 (95% confidence interval 1.01-1.30) (12,13). however, there are some limitations in both studies. the case-control study did not state how lung cancer diagnosis was made and significant factors in this study, namely dyslipidemia and family history of lung cancer, were not studied (12). the results of this study were compatible with the large population cohort study in hong kong (21). among 84,116 patients with lung cancer, ccb did not increase the risk of lung cancer regardless of aspirin therapy. the hazard ratios for both settings were 0.89 (95% confidence interval of 0.73, 1.08) for ccb and 0.82 (95% confidence interval of 0.64, 1.06) for ccb plus aspirin. as data regarding ccb and lung cancer are still conflicting, further studies are required. this study found that dyslipidemia was associated with lung cancer, which was supported by a previous study from china (22). high-density lipoprotein cholesterol (hdlc) reduced the risk of non–small cell lung cancer by 77% (adjusted odds ratio of 0.233; 95% confidence interval of 0.134, 0.407). there are several biological explanations for the reduction of cancer and high hdl-c, such as suppression of signaling via lipid raft formation and reduction of viability and proliferation (23,24). regarding family history of lung cancer, several studies support this finding conducted in twins, women, and nonsmokers (25-27). this association may be explained by second-hand smokers or genetic factors such as her2 gene or egfr variant (28-32). study limitations there are some limitations in this study. first, this was a study conducted in a single, referral university hospital resulting in a small sample size. second, some factors were not completely evaluated such as genetic factors, sleep apnea, or aspirin therapy (21,33-38). similar to previous published articles, smoking history may be underestimated as smoking status may not be recorded in the medical records (12,13,21). additionally, selection bias may have existed as this study enrolled only those with pathological diagnosis of lung cancer. this may also result in small sample size. finally, some factors such as alcohol consumption may not be studied or may be missing due to retrospective design. conclusions ccb was not associated with lung cancer in patients with hypertension but dyslipidemia and family history of lung cancer were independently associated with lung cancer in this setting. disclosures conflict of interest: the authors declare that they have no conflicts of interest. financial support: this study was funded by a grant from khon kaen university, faculty of medicine, thailand (grant number: mn66003). references 1. mills kt, stefanescu a, he j. the global epidemiology of hypertension. nat rev nephrol. 2020;16(4):223-237. crossref pubmed 2. zhou b, carrillo-larco rm, danaei g, et al; ncd risk factor collaboration (ncd-risc). worldwide trends in hypertension prevalence and progress in treatment and control from 1990 to 2019: a pooled analysis of 1201 population-representative studies with 104 million participants. lancet. 2021;398(10304): 957-980. crossref pubmed 3. chobanian av, bakris gl, black hr, et al; national high blood pressure education program coordinating committee. the seventh report of the joint national committee on prevention, detection, evaluation, and treatment of high blood pressure: the jnc 7 report. jama. 2003;289(19):25602572. crossref pubmed 4. khamsai s, mahawarakorn p, limpawattana p, et al. prevalence and factors correlated with hypertension secondary from obstructive sleep apnea. multidiscip respir med. 2021;16(1): 777. crossref pubmed 5. jeerasuwannakul b, sawunyavisuth b, khamsai s, sawanyawisuth k. prevalence and risk factors of proteinuria in patients with type 2 diabetes mellitus. asia pac j sci technol. 2021;26(04):apst-2604-02: 1-5. crossref 6. manasirisuk p, chainirun n, tiamkao s, et al. efficacy of generic atorvastatin in a real-world setting. clin pharmacol. 2021;13:45-51. crossref pubmed 7. soontornrungsun b, khamsai s, sawunyavisuth b, et al. obstructive sleep apnea in patients with diabetes less than 40 years of table ii factors associated with lung cancer in patients with hypertension by logistic regression analysis factors unadjusted or 95% ci adjusted or 95% ci smoking 0.97 0.53, 1.79 1.03 0.53, 2.01 dyslipidemia 2.20 1.15, 4.20 2.12 1.07, 4.23 obstructive sleep apnea 8.44 0.96, 73.84 7.50 0.79, 71.17 family history of lung cancer 20.48 2.58, 162.61 22.43 2.76, 182.36 calcium channel blocker used 1.42 0.76, 2.67 1.50 0.53, 2.01 ci = confidence interval; or = odds ratio. https://doi.org/10.1038/s41581-019-0244-2 https://www.ncbi.nlm.nih.gov/pubmed/32024986 https://doi.org/10.1016/s0140-6736(21)01330-1 https://www.ncbi.nlm.nih.gov/pubmed/34450083 https://doi.org/10.1001/jama.289.19.2560 https://www.ncbi.nlm.nih.gov/pubmed/12748199 https://doi.org/10.4081/mrm.2021.777 https://www.ncbi.nlm.nih.gov/pubmed/34650797 https://doi.org/10.14456/apst.2021.32 https://doi.org/10.2147/cpaa.s285750 https://www.ncbi.nlm.nih.gov/pubmed/33707972 rattanathanoo et al drug target insights 2023; 17: 57 © 2023 the authors. published by aboutscience www.aboutscience.eu age. diabetes metab syndr. 2020;14(6):1859-1863. crossref pubmed 8. khamsai s, chootrakool a, limpawattana p, et al. hypertensive crisis in patients with obstructive sleep apnea-induced hypertension. bmc cardiovasc disord. 2021;21(1):310. crossref pubmed 9. james pa, oparil s, carter bl, et al. 2014 evidence-based guideline for the management of high blood pressure in adults: report from the panel members appointed to the eighth joint national committee (jnc 8). jama. 2014;311(5):507-520. crossref pubmed 10. pahor m, guralnik jm, salive me, corti mc, carbonin p, havlik rj. do calcium channel blockers increase the risk of cancer? am j hypertens. 1996;9(7):695-699. crossref pubmed 11. azoulay l, assimes tl, yin h, bartels db, schiffrin el, suissa s. long-term use of angiotensin receptor blockers and the risk of cancer. plos one. 2012;7(12):e50893. crossref pubmed 12. rotshild v, azoulay l, feldhamer i, et al. calcium channel blockers and the risk for lung cancer: a population-based nested case-control study. ann pharmacother. 2019;53(5):445-452. crossref pubmed 13. rotshild v, azoulay l, zarifeh m, et al. the risk for lung cancer incidence with calcium channel blockers: a systematic review and meta-analysis of observational studies. drug saf. 2018;41(6):555564. crossref pubmed 14. hole dj, gillis cr, mccallum ir, et al. cancer risk of hypertensive patients taking calcium antagonists. j hypertens. 1998; 16(1):119-124. crossref pubmed 15. michels kb, rosner ba, walker am, et al. calcium channel blockers, cancer incidence, and cancer mortality in a cohort of u.s. women: the nurses’ health study. cancer. 1998;83(9):20032007. crossref pubmed 16. yang r, zhang y, liao x, yao y, huang c, liu l. the relationship between anti-hypertensive drugs and cancer: anxiety to be resolved in urgent. front pharmacol. 2020;11:610157. crossref pubmed 17. mason rp. calcium channel blockers, apoptosis and cancer: is there a biologic relationship? j am coll cardiol. 1999;34(7): 1857-1866. crossref pubmed 18. jick h, jick s, derby le, vasilakis c, myers mw, meier cr. calciumchannel blockers and risk of cancer. lancet. 1997;349(9051): 525-528. crossref pubmed 19. boo h-j, min h-y, jang h-j, et al. the tobacco-specific carcinogen-operated calcium channel promotes lung tumorigenesis via igf2 exocytosis in lung epithelial cells. nat commun. 2016;7(1):12961. crossref pubmed 20. carboni gl, gao b, nishizaki m, et al. cacna2d2-mediated apoptosis in nsclc cells is associated with alterations of the intracellular calcium signaling and disruption of mitochondria membrane integrity. oncogene. 2003;22(4):615-626. crossref pubmed 21. li j, lam asm, yau sty, yiu kkl, tsoi kkf. antihypertensive treatments and risks of lung cancer: a large population-based cohort study in hong kong. bmc cancer. 2021;21(1):1202. crossref pubmed 22. hao b, yu m, sang c, bi b, chen j. dyslipidemia and non-small cell lung cancer risk in chinese population: a case-control study. lipids health dis. 2018;17(1):278. crossref pubmed 23. su f, grijalva v, navab k, et al. hdl mimetics inhibit tumor development in both induced and spontaneous mouse models of colon cancer. mol cancer ther. 2012;11(6):1311-1319. crossref pubmed 24. wolfe ar, atkinson rl, reddy jp, et al. high-density and verylow-density lipoprotein have opposing roles in regulating tumor-initiating cells and sensitivity to radiation in inflammatory breast cancer. int j radiat oncol biol phys. 2015;91(5):10721080. crossref pubmed 25. lissowska j, foretova l, dabek j, et al. family history and lung cancer risk: international multicentre case-control study in eastern and central europe and meta-analyses. cancer causes control. 2010;21(7):1091-1104. crossref pubmed 26. yin x, chan cpy, seow a, yau w-p, seow wj. association between family history and lung cancer risk among chinese women in singapore. sci rep. 2021;11(1):21862. crossref pubmed 27. kanwal m, ding x-j, cao y. familial risk for lung cancer. oncol lett. 2017;13(2):535-542. crossref pubmed 28. scragg r, glover m. parental and adolescent smoking: does the association vary with gender and ethnicity? n z med j. 2007;120(1267):u2862. pubmed 29. salber ej, macmahon b. cigarette smoking among high school students related to social class and parental smoking habits. am j public health nations health. 1961;51(12):1780-1789. crossref pubmed 30. coté ml, liu m, bonassi s, et al. increased risk of lung cancer in individuals with a family history of the disease: a pooled analysis from the international lung cancer consortium. eur j cancer. 2012;48(13):1957-1968. crossref pubmed 31. yamamoto h, higasa k, sakaguchi m, et al. novel germline mutation in the transmembrane domain of her2 in familial lung adenocarcinomas. j natl cancer inst. 2014;106(1):djt338. crossref pubmed 32. bell dw, gore i, okimoto ra, et al. inherited susceptibility to lung cancer may be associated with the t790m drug resistance mutation in egfr. nat genet. 2005;37(12):1315-1316. crossref pubmed 33. sawunyavisuth b, ngamjarus c, sawanyawisuth k. a metaanalysis to identify factors associated with cpap machine purchasing in patients with obstructive sleep apnea. biomed rep. 2022;16(6):45. crossref pubmed 34. sanlung t, sawanyawisuth k, silaruks s, et al. clinical characteristics and complications of obstructive sleep apnea in srinagarind hospital. j med assoc thai. 2020;103(1):36-39. 35. khamsai s, kachenchart s, sawunyavisuth b, et al. prevalence and risk factors of obstructive sleep apnea in hypertensive emergency. j emerg trauma shock. 2021;14(2):104-107. crossref pubmed 36. sawunyavisuth b, ngamjarus c, sawanyawisuth k. any effective intervention to improve cpap adherence in children with obstructive sleep apnea: a systematic review. glob pediatr health. 2021;8:1-8. crossref 37. sawunyavisuth b. what personal experiences of cpap use affect cpap adherence and duration of cpap use in osa patients? j med assoc thai. 2018;101(7):s245-s249. 38. kaewkes c, sawanyawisuth k, sawunyavisuth b. are symptoms of obstructive sleep apnoea related to good continuous positive airway pressure compliance? erj open res. 2020;6(3):0016902019. crossref pubmed https://doi.org/10.1016/j.dsx.2020.09.008 https://www.ncbi.nlm.nih.gov/pubmed/32992217 https://doi.org/10.1186/s12872-021-02119-x https://www.ncbi.nlm.nih.gov/pubmed/34162333 https://doi.org/10.1001/jama.2013.284427 https://www.ncbi.nlm.nih.gov/pubmed/24352797 https://doi.org/10.1016/0895-7061(96)00186-0 https://www.ncbi.nlm.nih.gov/pubmed/8806983 https://doi.org/10.1371/journal.pone.0050893 https://www.ncbi.nlm.nih.gov/pubmed/23251399 https://doi.org/10.1177/1060028018814684 https://www.ncbi.nlm.nih.gov/pubmed/30442036 https://doi.org/10.1007/s40264-018-0644-4 https://www.ncbi.nlm.nih.gov/pubmed/29484611 https://doi.org/10.1097/00004872-199816010-00017 https://www.ncbi.nlm.nih.gov/pubmed/9533425 https://doi.org/10.1002/(sici)1097-0142(19981101)83:9<2003::aid-cncr17>3.0.co;2-3 https://www.ncbi.nlm.nih.gov/pubmed/9806660 https://doi.org/10.3389/fphar.2020.610157 https://www.ncbi.nlm.nih.gov/pubmed/33381045 https://doi.org/10.1016/s0735-1097(99)00447-7 https://www.ncbi.nlm.nih.gov/pubmed/10588195 https://doi.org/10.1016/s0140-6736(97)80084-0 https://www.ncbi.nlm.nih.gov/pubmed/9048789 https://doi.org/10.1038/ncomms12961 https://www.ncbi.nlm.nih.gov/pubmed/27666821 https://doi.org/10.1038/sj.onc.1206134 https://www.ncbi.nlm.nih.gov/pubmed/12555074 https://doi.org/10.1186/s12885-021-08971-6 https://www.ncbi.nlm.nih.gov/pubmed/34763668 https://doi.org/10.1186/s12944-018-0925-z https://www.ncbi.nlm.nih.gov/pubmed/30522496 https://doi.org/10.1158/1535-7163.mct-11-0905 https://www.ncbi.nlm.nih.gov/pubmed/22416044 https://doi.org/10.1016/j.ijrobp.2014.12.039 https://www.ncbi.nlm.nih.gov/pubmed/25832697 https://doi.org/10.1007/s10552-010-9537-2 https://www.ncbi.nlm.nih.gov/pubmed/20306329 https://doi.org/10.1038/s41598-021-00929-9 https://www.ncbi.nlm.nih.gov/pubmed/34750403 https://doi.org/10.3892/ol.2016.5518 https://www.ncbi.nlm.nih.gov/pubmed/28356926 https://www.ncbi.nlm.nih.gov/pubmed/18157188 https://doi.org/10.2105/ajph.51.12.1780 https://www.ncbi.nlm.nih.gov/pubmed/14496305 https://doi.org/10.1016/j.ejca.2012.01.038 https://www.ncbi.nlm.nih.gov/pubmed/22436981 https://doi.org/10.1093/jnci/djt338 https://www.ncbi.nlm.nih.gov/pubmed/24317180 https://doi.org/10.1038/ng1671 https://www.ncbi.nlm.nih.gov/pubmed/16258541 https://doi.org/10.3892/br.2022.1528 https://www.ncbi.nlm.nih.gov/pubmed/35620316 https://doi.org/10.4103/jets.jets_47_20 https://www.ncbi.nlm.nih.gov/pubmed/34321809 https://doi.org/10.1177/2333794x211019884 https://doi.org/10.1183/23120541.00169-2019 https://www.ncbi.nlm.nih.gov/pubmed/33015148 dti drug target insights 2021; 15: 5-12issn 1177-3928 | doi: 10.33393/dti.2021.2192review drug target insights issn 1177-3928 www.aboutscience.eu/dti © 2021 the authors. this article is published by aboutscience and licensed under creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). commercial use is not permitted and is subject to publisher’s permissions. full information is available at www.aboutscience.eu tamarind (tamarindus indica l.) seed a candidate protein source with potential for combating sars-cov-2 infection in obesity ana h. de a. morais1-3, amanda f. de medeiros1, isaiane medeiros1, vanessa c. o. de lima1, anna b. s. luz1, bruna l. l. maciel2,3, thaís s. passos3 1biochemistry postgraduate biosciences center, federal university of rio grande do norte, natal brazil 2nutrition postgraduate program, center for health sciences, federal university of rio grande do norte, natal brazil 3department of nutrition, center for health sciences, federal university of rio grande do norte, natal brazil all authors contributed equally to this work. abstract introduction: obesity and coronavirus disease (covid)-19 are overlapping pandemics, and one might worsen the other. methods: this narrative review discusses one of the primary mechanisms to initiate acute respiratory distress syndrome, uncontrolled systemic inflammation in covid-19, and presents a potential candidate for adjuvant treatment. blocking the s protein binding to angiotensin-converting enzyme 2 (ace-2) and the 3c-like protease (3cl pro) is an effective strategy against severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection. results: host proteases such as furin, trypsin, and transmembrane serine protease 2 (tmprss) act in s protein activation. tamarind trypsin inhibitor (tti) shows several beneficial effects on the reduction of inflammatory markers (tumor necrosis factor α [tnf-α], leptin) and biochemical parameters (fasting glycemia, triglycerides, and very low-density lipoprotein [vldl]), in addition to improving pancreatic function and mucosal integrity in an obesity model. tti may inhibit the action of proteases that collaborate with sars-cov-2 infection and the neutrophil activity characteristic of lung injury promoted by the virus. conclusion: thus, tti may contribute to combating two severe overlapping problems with high cost and social complex implications, obesity and covid-19. keywords: 3clpro, ace-2, covid-19, furin, hne, inflammation, tmprss received: october 20, 2020 accepted: march 11, 2021 published online: april 2, 2021 corresponding author: ana h. de a. morais department of nutrition center for health sciences federal university of rio grande do norte natal, rn 59078-970 brazil aharaujomorais@gmail.com the consequences of sars-cov-2 infection range from self-limited flu to fulminant pneumonia, respiratory failure, and death (4,5). although the new coronavirus has mutations, it is still unclear whether they are related to its virulence (6), which might soon be answered through ongoing studies (7). advanced age is a risk factor that leads to the worsening of the clinical condition of the disease, placing the elderly as a vulnerable population with a high risk of death. however, severe cases also occur in middle-aged or younger people, and one of the possible contributing factors for this is the already known relationship between nutritional status and the prognosis of viral infections, which can contribute to the improvement or worsening of the disease (8-10). according to butler et al (11) and muscogiuri et al (12) broader access to a healthy and balanced diet rich in vitamins, minerals, bioactive compounds, and antioxidants is essential to assist in reducing susceptibility to sars-cov-2 infection, in addition to the complications that can occur in the long term. the regional councils of the four united nations agencies (food and agriculture organization of the united nations— fao, united nations children’s fund—unicef, world health organization—who, and world food program—wfa) issued introduction the relation between obesity and severe covid-19 coronavirus disease (covid)-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) coronavirus, is a serious threat to health systems around the world. it overlaps with obesity, another global pandemic (1-3). sars-cov-2 infection started in december 2019 and spread rapidly because its transmission occurs through the airways. https://doi.org/10.33393/dti.2021.2192 https://creativecommons.org/licenses/by-nc/4.0/legalcode protein with potential for combating sars-cov-2 infection in obesity6 © 2021 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti a joint statement on nutrition in the context of the covid-19 pandemic in asia and the pacific. the recommendations emphasized the need for continuous adoption of advice and strategies ensuring nutritional surveillance, food quality, and food security. on the other hand, the current moment demands the isolation of the world population as a strategy to contain the infection spread. isolation can increases consumers’ preference for industrialized and ultra-processed foods. this preference occurs due to the practicality, hygienic safety, longer shelf life, of industrialized and ultra-processed foods compared to fresh and unprocessed products (13). this possible trend may increase food insecurity and directly impact weight gain (13-16), increasing the risk of overweight and obesity, which can persist for an extended period (17). diet composition and obesity have a fundamental role in the coregulation of adaptive immunity (18), influencing the modulation of the immune response and, consequently, affecting the severity of respiratory diseases and other infections (9). in general, adiposity can impair the ventilation of the base of the lungs, resulting in less oxygen saturation in the blood (19). thus, in covid-19, the need for treatment in intensive care units (icus) increases, and intubations are technically more challenging in obese patients, in addition to the difficulty in obtaining a diagnosis by imaging techniques. according to the review study by johnson et al (20), obesity damages various organs, which increases the susceptibility to several diseases, such as metabolic disorders, cardiovascular diseases, cancer, and viral infections such as influenza and covid-19 (1,21). there is an intrinsic relationship between fat distribution and metabolic health status. the location of fat deposition in the body is determinant for health, and the ectopic deposition of triglycerides in the abdominal region, mainly visceral fat, causes a phenotype of high cardiometabolic risk—even for individuals who have a normal body mass index (bmi) but with a high abdominal circumference (ac)—due to unregulated cytokine secretion pathways. in addition, inflammation and increased release of circulating fatty acids is associated with visceral fat. thus, the fat distribution phenotype contributes to metabolism, leading to metabolically unhealthy individuals (22). thus, visceral obesity can be an important risk factor to increase the severity of covid-19 (23). studies with patients affected by this disease in germany and italy have shown that as the area of visceral adipose tissue increased, there was a greater need for intensive therapy, regardless of other factors, such as age, sex, and associated diseases (24,25). another study performed in china with covid-19 patients showed that higher visceral and subcutaneous adipose tissue were independent risk factors for critical illness (26). in addition, visceral adipose tissue positively regulates the expression of plasminogen activator inhibitor 1, thus generating an increased risk of developing thrombosis in patients with visceral obesity affected by covid-19 (27). thus, understanding the metabolically unhealthy individual is essential, carefully assessing the response to sars-cov vaccination (23), as obesity and impaired metabolic health generate a potentially reduced immune response, which can negatively affect the vaccine’s effectiveness (28-31). obesity is characterized by a state of low-grade chronic inflammation related to changes in immune cells, including the number and types of cells present in the inflamed tissue. at the beginning of weight gain, immune cells infiltrate adipose tissue, contributing to persistent adipose inflammation and insulin resistance. macrophages are classified into two subtypes of phenotypes, m1 proinflammatory and m2 anti-inflammatory. in eutrophic individuals, the m2 subtype is distributed throughout adipose tissue, producing interleukin (il)-10 and expressing arginase-1 for collagen synthesis, which is important for promoting tissue repair. during the progression of weight gain to obesity, the m1 subtype becomes dominant, spreading inflammation through the production of mediators, such as tumor necrosis factor α (tnf-α), il-1β, monocyte chemoattractant protein-1 (mcp1), plasminogen activator inhibitor 1 (pai-1) and reactive oxygen species (ros). as a result, m1 macrophages interrupt insulin sensitivity in adipose tissue and the liver (20). the hormone leptin overlaps when referring to the link between obesity and inflammation, since leptin synthesis is directly proportional to the amount of adipose tissue, and inflammatory cytokines stimulate this synthesis. these inflammatory cytokines, such as tnf-α, il-1β, il-6, and interferon gamma (ifn-γ), coagulation factors (fibrinogen and pai-1), acute-phase protein (c-reactive protein and amyloid serum a [saa]), white blood cell count, and chemokines are markers of inflammation. in patients with obesity, these markers are commonly elevated, and with the reduction of excess weight, there is a reduction in their plasma concentrations (32). there are also anti-inflammatory adipokines, such as il-4, il-5, and il-10, which can be observed in obesity at low concentrations. thus, an imbalance between these antiand inflammatory cytokines can induce the inflammatory response (33). considering that obesity negatively affects the immune system, there is, therefore, a clear relationship with the higher susceptibility to the more severe covid-19. this close relationship occurs due to the increased release of inflammatory cytokines by disrupting the integrity of tissues (adipose and lymphoid tissue, intestines, and lungs), which alters the activation of leukocytes, impairing the action of the immune system. as a result, there is a direct impact on the healing process, which prolongs recovery and increases the risk of evolution from respiratory infection to severe diseases with a high risk of death (34). it is important to highlight that sars-cov is responsible for coding 3c-like protease (3cl pro), a cysteine protease (35), formally known as c30 endopeptidase that is chymotrypsinlike (36). it is considered a key component in polyprotein processing (37), which synthesizes nonstructural proteins and structural proteins, such as spike (s), envelope (e), membrane (m), and nucleocapsid (n) proteins (38). thus, this protease plays an important role in the replication and transcription of viral ribonucleic acid (rna) (39). recently, hoffmann et al (40) demonstrated that the new coronavirus enters human cells through glycoprotein s, found on the surface of the virus. this glycoprotein can bind to the angiotensin-converting enzyme 2 (ace-2) located in human cells. ace-2 receptors are expressed in the intestine, kidneys, lungs, and blood vessels, becoming targets for sars-cov-2 infection. hoffmann et al (40) also morais et al drug target insights 2021; 15: 7 © 2021 the authors. published by aboutscience www.aboutscience.eu observed that the cellular transmembrane serine protease 2 (tmprss2) is needed for sars-cov-2 entrance in host cells. xu et al (41) investigated the possible routes of sars-cov-2 infection in the oral cavity mucosa, exploring the expression of ace-2 and the proportion and composition of the cells responsible for this function based on rna-seq profiles and cell transcript-independent data. the results showed that ace-2 could be expressed in the epithelial cells of the oral cavity, mainly in the tongue, oral and gingival tissues, indicating that the oral cavity mucosa can be a potential route for infection. in the respiratory system, ace-2 hydrolyzes angiotensin ii to angiotensin 1-7, with an essential role in regulating the system. when ace-1 activity is increased and ace-2 is inhibited, intact angiotensin ii acts through the angiotensin 1 (at1) or 2 (at2) receptor to exert proinflammatory responses and stimulate aldosterone secretion. as a result, these effects increase blood pressure and potentially cause hypokalemia, in addition to intensifying local vascular permeability, increasing the risk of respiratory distress syndrome. on the other hand, angiotensin 1-7 acts on the receptor pathway, leading to antiinflammatory and antifibrotic responses that would be favorable to the recovery of patients with covid-19 (42). studies also describe the importance of the renin-angiotensin system (ras) in the regulation of metabolism and the development of cardiovascular and inflammatory diseases (43-45). this system also modulates the endocrine and metabolic functions of adipocytes, hypertrophy, and hyperplasticity in obesity (46,47). obesity and its related comorbidities facilitate viral replication, increasing the risk of severe complications in sars-cov-2 infections. this increased risk occurs in obesity because the enlarged adipose tissue expresses higher levels of ace-2 and, consequently, serves as a reservoir for the virus (48). the expression of ace-2 in target tissues (lung, liver, and heart) is increased in diabetes (49), commonly seen in obese individuals. hyperglycemia and type 2 diabetes also trigger the release of inflammatory cytokines, which in cases of covid-19 can lead to higher release of cytokines (cytokine storm), generating an immune dysregulation that can lead to multiple organ failure and death (50). obesity also induces deregulated lipogenesis that promotes the high expression of ace-2 in the lungs (51). the increased leptin synthesis stimulated by inflammatory cytokines (33) can also worsen the clinical condition of obese patients with covid-19. therefore, drugs that mediate metabolic responses targeting ace-2 have been considered promising in the modulation of glucose metabolism and blood pressure control. these drugs may also prevent the entry of the new coronavirus through competitive pathways of ace (9). results can the kunitz trypsin inhibitor from tamarind seeds combat sas-cov-2 infection in obesity? considering these dysfunctions in patients with covid-19, several studies and tests with drugs, traditionally used to control these changes, whether endocrine or metabolic, appear. some adjuvant therapies for covid-19 deserve special mention, such as immunomodulatory agents, immunoglobulin therapy, corticosteroids, and anticytokines used to control endocrine and metabolic changes (9,52,53). a new mechanism related to the inhibition of the transmembrane protease serine 2, encoded by the tmprss2 gene, is an additional target for drugs for research. as already discussed, researchers have demonstrated that sarscov-2 uses the sars-cov ace-2 receptor to enter cells. the serine protease tmprss2 is also necessary for the initiation of protein s. a tmprss2 inhibitor, camostat mesylate—a synthetic serine protease inhibitor (trypsin), approved in japan for clinical use, blocked entry and could be another option treatment in covid-19 (9,40). other studies also highlight camostat mesylate for the treatment of pancreatitis (9,52) currently in phase 2 clinical trial in covid-19. furthermore, inhibition of 3cl pro can block protein s synthesis and coronavirus proliferation (39). most studies have focused mainly on small-molecule compounds from virtual screening based on a 3cl pro structure (54). several inhibitors, which can be classified as peptoids and non-peptidomimetics, have shown good inhibitory activity of this protease (39). protease inhibitors have been widely studied. one reason is that they are present in multiple forms in plants, animals, and microorganisms. in addition, protease inhibitors are natural protease regulators that are intrinsically involved in biological processes such as digestion, healing, viral replication, and the blood clotting cascade, among others, and that need precise regulation (55,56). thus, the prospect of peptides for use in biotechnology has led to a number of molecules’ discovery. its use in experimental models has exposed mammals to risks that have not yet been evaluated. although the benefits of proteins and peptides substantially outweigh the potential harmful effects, their use is not without risks. there is a balance between the ability of peptides to induce desirable effects, that is, their bioactivity, and the potential toxic effects associated with their cell-penetrating properties (57). among protease inhibitors, trypsin inhibitors are widely extracted from seeds to be purified and characterized for application in various studies (58). these molecules have shown, in recent studies, performance in different mechanisms involving the control of obesity and its related effects, such as the production of hormones related to satiety, affecting the central nervous system and the small intestine; reduction of food consumption and weight gain; improvement of the lipid profile; and reduction of the inflammatory process associated with obesity, regardless of weight loss (59). a trypsin inhibitor extracted from seeds of the tamarind fruit, tamarindus indica l., belonging to the legume family, occurring in all regions of brazil (60) has been extensively studied by our research group. the first study to evaluate the potential of the tamarind trypsin inhibitor (tti) to reduce weight gain was developed by ribeiro et al (61). in this study, male eutrophic wistar rats were fed a standard diet and received the isolated tti by gavage for 11 days at a dose of 25 mg/kg. tti administration reduced food intake in these animals. to better understand this reduction in consumption, another experiment using the same experimental model evaluated food consumption 1 h, 2 h, and 16 h after gavage with tti, in addition to serum cholecystokinin (cck), using doses of 25 and 50 mg/kg. tti protein with potential for combating sars-cov-2 infection in obesity8 © 2021 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti reduced food consumption 16 h after its administration dosedependently (61). in this study, tti did not cause changes in the liver enzymes and serum proteins of wistar rats, in addition to not affecting the histological aspects of the liver, stomach, intestine, and pancreas. in addition, tti did not cause classical deleterious effects on protein digestion and, consequently, malnutrition at the doses tested, as demonstrated by the measurement of serum proteins. obesity is a condition that leads to numerous physiological changes, and the behavior of tti in this condition needs to be assessed. for this, carvalho et al (62) used wistar rats with diet-induced obesity, assessing food consumption and other biochemical parameters, using the 25 mg/kg dose of tti, as proposed by ribeiro et al (61). in animals with obesity, tti reduced food consumption without inducing weight loss. tti reduced plasma tnf-α to undetectable concentrations, showing that the isolated inhibitor also influenced other aspects related to obesity, such as low-grade chronic inflammation (62). in the same experimental model of obesity, costa et al (63) observed that animals with obesity treated with tti decreased food intake in a similar way to eutrophic animals. the animals with obesity treated with tti showed a slight reduction in the lee index, which, although not significant, was important because of the consumption of a diet rich in ultraprocessed foods called high glycemic index and glycemic load (hgli) (64). although tti did not alter the plasma cck, it decreased the expression of the cck-1r gene and plasma leptin in animals with obesity compared to the group with obesity without treatment (63). tti is characterized as a protein isolate with high inhibitory activity for trypsin, obtained in a process that generates enrichment of this protein, but not its purification (61). therefore, it is possible that other molecules can influence the biochemical and bioactive characteristics of tti. due to the many biological functions of tti in the context of obesity, new technologies that could potentialize these functions were assessed. nanotechnology, through nanoencapsulation, can promote the protection of bioactive substances in oral administration, which exposes these molecules to digestive processes, compromising active sites essential to biological activity. in addition, nanoencapsulation provides controlled release at a specific target and further intensification of the biological effect (65). thus, tti was nanoencapsulated to increase the efficiency and stability of antitrypsin activity. the isolated and conjugated effects of chitosan and isolated whey protein on incorporation, antitrypsin activity, and tti stability at different temperatures and ph conditions were investigated. the combination of chitosan and isolated whey protein (ecw) formed nanoparticles (109 nm), promoted a reduction in the half maximal inhibitory concentration (ic50; 0.05 mg) compared to pure tti (0.21 mg), and preserved antitrypsin activity up to 80°c (35.0% [3.74]) compared to isolated agents and tti, which have no inhibitory activity. besides, the nanoparticles showed stability under different ph conditions. thus, ecw proved to be an essential strategy to improve the function and stability of tti (66). to assess the safety of administration by gavage in addition to maintaining the inhibitory activity, the encapsulated tti (ecw) was evaluated in a preclinical obesity study. costa (67) also assessed cytotoxicity using the caco-2 and ccd18co strains (human intestinal cells). the cytotoxicity assay exceeded 70% cell viability for caco-2 and ccd-18co when exposed to different concentrations of ecw. for the subacute blood toxicity of the bioactive dose of ecw, through a complete blood count, liver, and kidney function, there was an absence of subacute blood toxicity, demonstrated by the lack of toxic effects on the biochemical parameters evaluated. considering that ecw proved to be safe in the face of the parameters evaluated, its biological activity was tested after encapsulation (68). the nanoencapsulated tti was also offered by gavage in a preclinical obesity model to test whether the isolated inhibitor would maintain its modulating properties on the altered biochemical parameters of the experimental model used. the biological activity was maintained, with emphasis on ecw, which significantly reduced blood glucose, homeostatic model assessment of insulin resistance (homa-ir), and raised high-density lipoprotein cholesterol (hdl-c), in addition to possibly favoring greater protection to pancreatic tissue. the purification of these inhibitors is a necessary and critical step to define their structural characteristics and specificity of binding to other molecules. isolating these proteins from all other proteins that are present in the same biological source is difficult because trypsin inhibitors have great molecular diversity (69). medeiros et al (70) described the tti purification process, obtaining a molecule with 100% inhibition for trypsin (ptti), heat resistant and with a partially identified amino acid sequence (currently complete and with structural modeling—unpublished data), which had high homology with other trypsin inhibitors of the kunitz family. ptti also did not affect plasma cck concentrations but reduced circulating leptin concentrations in animals with obesity at a dose of 730 μg/kg. leptin is an important hormone in the energy balance, which is elevated in individuals with obesity, leading to the development of a resistance condition (70). the effect of the purified trypsin inhibitor tamarind was also evaluated in a model of metabolic changes in wistar rats with obesity and dyslipidemia. obesity was induced using the hgli diet (64). the animals treated with ptti at a dose of 730 μg/kg had significantly lower food intake than the untreated group. however, the groups did not show differences in weight gain. ptti showed great anti-inflammatory potential, reducing the relative expression of tnf-α messenger rna and positive immunostaining in adipocyte immunohistochemical analysis of obese animals, as well as plasma cytokine concentrations (71). the anti-inflammatory effects of the isolated or purified inhibitor on adipose tissue were observed regardless of weight changes, which may suggest a direct beneficial effect on this tissue that may alter its structure. this result shows the potential of this molecule since obesity is classically considered a disease that induces a low grade of chronic inflammation, causing changes in tissues, notably the intestinal mucosa (72) and adipose tissue (73). due to these promising biological effects and safety demonstrated in preclinical studies with partially purified tti, this inhibitor could be explored in alternative obesity therapies. the purification process can enhance the functional properties of a molecule, making it necessary to reassess the safety of its use. at a dose approximately thirty times lower, ptti performed the same biological activities as tti. this morais et al drug target insights 2021; 15: 9 © 2021 the authors. published by aboutscience www.aboutscience.eu result emphasizes the need to assess whether its use generated harmful effects on the liver, a target tissue in toxicity studies; pancreas, a classically affected organ by trypsin inhibitors; adipose tissue, where ptti, in previous studies, has shown anti-inflammatory effects; and intestine, a potential site of action that has not yet been studied. trypsin inhibitors have well-reported deleterious effects. evidence has shown impaired growth due to poor digestion and absorption, metabolic changes in the pancreas, such as increased enzyme secretion, hypertrophy and hyperplasia, and metabolic disturbance in the use of amino acids (74). thus, ptti was evaluated for possible toxic effects, focusing on the histopathological and stereological characteristics of organs involved in its metabolism, processing, and biological activity (liver and pancreas) and the tissues most affected by the obesity model (small intestine and visceral adipose tissue). ptti at its bioactive dose did not cause signs or symptoms of general toxicity or potential damage to the liver and pancreatic tissue of obese wistar rats. ptti also promoted a protective effect on the intestines of these animals, reducing the loss of intestinal villi, a well-characterized damage in obesity models. ptti reduced the presence of inflammatory infiltrates in perirenal (visceral) adipose tissue. therefore, its use in the tested models is safe and presents anti-inflammatory effects (unpublished data). besides the effects mentioned above, tti is a promising molecule concerning the mechanisms associated with the inhibition of genes involved in the production of ace-2, such as tmprss2 and furin (fig. 1). these genes probably act by fig. 1 obesity and associated comorbidities as risk factors for complications from sars-cov-2 infection, and hypothesis of the tti mechanism of action. obesity affects several organs, which have several responses, such as increased inflammation, changes in sensitivity and the action of hormones, dyslipidemia, and others. this metabolic deregulation favors increased expression of ace-2, which is cleaved in the cterminal segment by proteases such as tmprss2 and furin, and there is activation of the spike glycoprotein, so this process facilitates the entry of sars-cov-2 into the cells, causing viral infection. also, 3cl pro is considered a key component in polyprotein processing and plays an important role in the replication and transcription of viral rna. the tti effects in in vitro and preclinical studies show several antiobesity and anti-inflammatory effects and appear to be possible inhibitors of the proteases tmprss2, furin, and 3clpro. ace-2 = angiotensin-converting enzyme 2; cck = cholecystokinin; furin = member of the mammalian prohormone-protein convertases family; hne = human neutrophil elastase; il-1β = interleukin 1-β; lps = lipopolysaccharide; mcp-1 = monocyte chemoattractant protein 1; srebp = sterol regulatory elementbinding proteins; tg = triglyceride; tmprss2 = transmembrane serine protease 2; tnf-α = tumor necrosis factor α; tti = trypsin inhibitor from tamarind; vldl-c = very-low-density lipoprotein cholesterol; 3cl pro = 3c-like protease. protein with potential for combating sars-cov-2 infection in obesity10 © 2021 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti changing the organism’s epigenetic system responsible for the increase in ace-2 expression, a potential target for antiviral intervention by sars-cov-2. adipokines secreted by adipose tissue can also affect airway function. leptin is involved in neonatal lung development, surfactant production (75,76), and regulation of ventilatory impulse (76,77). studies have consistently demonstrated the association of high concentrations of leptin and asthma (78,79). the leptin concentration was reduced with the use of tti in animal models (63,70). several studies with trypsin inhibitors were related to obesity and its complications (59). according to fook et al. (80), tti showed selective activity, being highly effective against serine proteinases, especially against bovine trypsin and neutrophil elastase isolated from humans. the ic50 value was determined to be 55.96 μg/ml. the inhibitor also showed no cytotoxic or hemolytic activity in human blood cells. in addition, it exhibited different inhibition of the release of elastase by platelet-activating factor (paf; 44.6%) and release by n-formyl-l-methionyl-l-leucyl-phenylalanine (fmlp; 28.4%), preferentially affecting elastase release by paf stimuli. this may indicate selective inhibition in the receptors of the paf (80). the same research group in 2010 conducted another study and demonstrated that the soy inhibitor (skti) reduced lipopolysaccharide (lps)-induced acute lung injury in a preclinical model, significantly suppressing the inflammatory effects caused by elastase in a dose-dependent manner, suggesting the route of inhibition of human neutrophil elastase as a promoter of the improvement (81). several computational molecular docking studies have been carried out with some compounds to model binding interactions of various 3cl pro inhibitors and other proteases, such as tmprss2 (37,82-84). ptti-derived peptides are also shown to be strong candidates for blocking these proteases since tti is known to inhibit serine proteases such as trypsin and chymotrypsin, as previously demonstrated. conclusions therefore, trypsin inhibitors are promising alternatives, in addition to others already discussed in the scientific community, which can be used as adjuvants in covid-19, especially in obese patients. thus, the tamarind seed trypsin inhibitor may also be a preventive or adjuvant drug in the context of covid-19, especially in worsened inflammatory conditions, such as obesity. funding this study was financed in part by the coordenação de aperfeiçoamento de pessoal de nível superior—brasil (capes), finance code 001. acknowledgments the authors thank professor dr. elizeu antunes dos santos for figure editing and the federal university of rio grande do norte (ufrn), especially the pro-rectory of postgraduate and the pro-rectory of research, for all efforts dedicated to supporting the research in our institution. disclosures conflict of interest: the authors declare no conflicts of interest. financial support: this study was financed in part by the coordenação de aperfeiçoamento de pessoal de nível superior—brasil (capes), finance code 001. references 1. dietz w, santos-burgoa c. obesity and its implications for covid-19 mortality. obesity (silver spring). 2020;28(6):1005. crossref pubmed 2. jordan re, adab p, cheng kk. covid-19: risk factors for severe disease and death. bmj. 2020;368(march):m1198. crossref pubmed 3. national academies of sciences and medicine e, national academies of sciences, engineering and m. current status and response to the global obesity pandemic: proceedings of a workshop—in brief. (callahan ea, ed.). the national academies press; 2019. crossref 4. del rio c, malani pn. covid-19—new insights on a rapidly changing epidemic. jama. 2020;323(14):1339-1340. crossref pubmed 5. gupta r, ghosh a, singh ak, misra a. clinical considerations for patients with diabetes in times of covid-19 epidemic. diabetes metab syndr. 2020;14(3):211-212. crossref pubmed 6. pachetti m, marini b, benedetti f, et al. emerging sars-cov-2 mutation hot spots include a novel rna-dependent-rna polymerase variant. j transl med. 2020;18(1):179. crossref pubmed 7. yao x, ye f, zhang m, et al. in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome main point: hydroxychloroquine was found to be more potent than chloroquine at inhibiting sars-cov-2 in vit. clin infect dis. 2020;2: 1-25. 8. calder pc, waitzberg dl, klek s, martindale rg. lipids in parenteral nutrition: biological aspects. jpen j parenter enteral nutr. 2020;44(s1)(suppl 1):s21-s27. crossref pubmed 9. bornstein sr, dalan r, hopkins d, mingrone g, boehm bo. endocrine and metabolic link to coronavirus infection. nat rev endocrinol. 2020;16(6):297-298. crossref pubmed 10. ryan dh, ravussin e, heymsfield s. covid 19 and the patient with obesity—the editors speak out. obesity (silver spring). 2020;28(5):847. crossref pubmed 11. butler cc, van der velden aw, bongard e, et al. oseltamivir plus usual care versus usual care for influenza-like illness in primary care: an open-label, pragmatic, randomised controlled trial. lancet. 2020;395(10217):42-52. crossref pubmed 12. muscogiuri g, barrea l, savastano s, colao a. nutritional recommendations for covid-19 quarantine. eur j clin nutr. published online 2020:10-11. crossref 13. united nations system standing committee on nutrition— unscn. food environments in the covid-19 pandemic. unscn. published 2020. online. accessed april 29, 2020. 14. rundle ag, park y, herbstman jb, kinsey ew, wang yc. covid19-related school closings and risk of weight gain among children. obesity (silver spring). 2020;28(6):1008-1009. crossref pubmed 15. berger zd, evans ng, phelan al, silverman rd. covid-19: control measures must be equitable and inclusive. bmj. 2020;368(sept 2001):m1141. crossref https://doi.org/10.1002/oby.22818 https://www.ncbi.nlm.nih.gov/pubmed/32237206 https://doi.org/10.1136/bmj.m1198 https://www.ncbi.nlm.nih.gov/pubmed/32217618 https://doi.org/10.17226/25349 https://www.ncbi.nlm.nih.gov/pubmed/32108857 https://doi.org/10.1001/jama.2020.3072 https://doi.org/10.1016/j.dsx.2020.03.002 https://www.ncbi.nlm.nih.gov/pubmed/32172175 https://www.ncbi.nlm.nih.gov/pubmed/32321524 https://doi.org/10.1186/s12967-020-02344-6 https://doi.org/10.1002/jpen.1756 https://www.ncbi.nlm.nih.gov/pubmed/32049394 https://doi.org/10.1038/s41574-020-0353-9 https://www.ncbi.nlm.nih.gov/pubmed/32242089 https://doi.org/10.1002/oby.22808 https://www.ncbi.nlm.nih.gov/pubmed/32237212 https://doi.org/10.1016/s0140-6736(19)32982-4 https://www.ncbi.nlm.nih.gov/pubmed/31839279 https://doi.org/10.1038/s41430-020-0635-2 https://www.unscn.org/en/news-events/recent-news?idnews=2040 https://doi.org/10.1002/oby.22813 https://www.ncbi.nlm.nih.gov/pubmed/32227671 https://doi.org/10.1136/bmj.m1141 morais et al drug target insights 2021; 15: 11 © 2021 the authors. published by aboutscience www.aboutscience.eu 16. farrell p, thow am, abimbola s, faruqui n, negin j. how food insecurity could lead to obesity in lmics: when not enough is too much: a realist review of how food insecurity could lead to obesity in lowand middle-income countries. health promot int. 2018;33(5):812-826. crossref pubmed 17. kissler sm, tedijanto c, goldstein e, grad yh, lipsitch m. projecting the transmission dynamics of sars-cov-2 through the postpandemic period. science. 2020;5793(february 2019):eabb5793. crossref 18. pindjakova j, sartini c, lo re o, et al. gut dysbiosis and adaptive immune response in diet-induced obesity vs. systemic inflammation. front microbiol. 2017;8(jun):1157. crossref pubmed 19. dixon ae, peters u. the effect of obesity on lung function. expert rev respir med. 2018;12(9):755-767. crossref pubmed 20. johnson ar, milner jj, makowski l. the inflammation highway: metabolism accelerates inflammatory traffic in obesity. immunol rev. 2012;249(1):218-238. crossref pubmed 21. kassir r. risk of covid-19 for patients with obesity. obes rev. 2020;21(6):e13034. crossref pubmed 22. stefan n. causes, consequences, and treatment of metabolically unhealthy fat distribution. lancet diabetes endocrinol. 2020;8(7):616-627. crossref pubmed 23. stefan n, birkenfeld al, schulze mb. global pandemics interconnected—obesity, impaired metabolic health and covid-19. nat rev endocrinol. 2021;17(3):135-149. crossref pubmed 24. petersen a, bressem k, albrecht j, et al. the role of visceral adiposity in the severity of covid-19: highlights from a unicenter cross-sectional pilot study in germany. metabolism. 2020;110(january):154317. crossref pubmed 25. watanabe m, caruso d, tuccinardi d, et al. visceral fat shows the strongest association with the need of intensive care in patients with covid-19. metabolism. 2020;111:154319. crossref pubmed 26. yang y, ding l, zou x, et al. visceral adiposity and high intramuscular fat deposition independently predict critical illness in patients with sars-cov-2. obesity (silver spring). 2020;28(11):2040-2048. crossref pubmed 27. shimomura i, funahashi t, takahashi m, et al. enhanced expression of pai-1 in visceral fat: possible contributor to vascular disease in obesity. nat med. 1996;2(7):800-803. crossref pubmed 28. hazeldine j, lord jm. immunesenescence: a predisposing risk factor for the development of covid-19? front immunol. 2020;11(oct):573662. crossref pubmed 29. kumar h. healthy immunity: it’s all about immune regulation. int rev immunol. 2020;39(6):245-246. crossref pubmed 30. popkin bm, du s, green wd, et al. individuals with obesity and covid-19: a global perspective on the epidemiology and biological relationships. obes rev. 2020;21(11):e13128. crossref pubmed 31. ledford h. how obesity could create problems for a covid vaccine. nature. 2020;586(7830):488-489. crossref pubmed 32. esser n, legrand-poels s, piette j, scheen aj, paquot n. inflammation as a link between obesity, metabolic syndrome and type 2 diabetes. diabetes res clin pract. 2014;105(2):141-150. crossref pubmed 33. do prado wl, lofrano mc, oyama lm, et al. obesity and inflammatory adipokines: practical implications for exercise prescription. rev bras med esporte. 2009;15(5):378-383. crossref 34. morais ah de a, aquino j de s, silva-maia jk da, vale sh de l, maciel bll, passos ts. nutritional status, diet and viral respiratory infections: perspectives for sars-cov-2. br j nutr. published online august 26, 2020:1-32. crossref 35. pillaiyar t, meenakshisundaram s, manickam m. recent discovery and development of inhibitors targeting coronaviruses. drug discov today. 2020;25(4):668-688. crossref pubmed 36. rota pa, oberste ms, monroe ss, et al. characterization of a novel coronavirus associated with severe acute respiratory syndrome. science. 2003;300(5624):1394-1399. crossref 37. yu r, chen l, lan r, shen r, li p. computational screening of antagonists against the sars-cov-2 (covid-19) coronavirus by molecular docking. int j antimicrob agents. 2020;56(2):106012. crossref pubmed 38. forni d, cagliani r, clerici m, sironi m. molecular evolution of human coronavirus genomes. trends microbiol. 2017;25(1): 35-48. crossref pubmed 39. liu y, liang c, xin l, et al. the development of coronavirus 3clike protease (3clpro) inhibitors from 2010 to 2020. eur j med chem. 2020;206:112711. crossref pubmed 40. hoffmann m, kleine-weber h, schroeder s, et al. sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor. cell. published online 2020:1-10. crossref 41. xu xw, wu xx, jiang xg, et al. clinical findings in a group of patients infected with the 2019 novel coronavirus (sars-cov-2) outside of wuhan, china: retrospective case series. bmj. 2020;368(jan):m606. crossref pubmed 42. simões e silva ac, silveira kd, ferreira aj, teixeira mm. ace2, angiotensin-(1-7) and mas receptor axis in inflammation and fibrosis. br j pharmacol. 2013;169(3):477-492. crossref pubmed 43. santos ras, ferreira aj, simões e, silva ac, silva ac. recent advances in the angiotensin-converting enzyme 2-angiotensin (1-7)-mas axis. exp physiol. 2008;93(5):519-527. crossref pubmed 44. ferreira aj, santos ras, bradford cn, et al. therapeutic implications of the vasoprotective axis of the renin-angiotensin system in cardiovascular diseases. hypertension. 2010;55(2):207213. crossref pubmed 45. santos pcjl, krieger je, pereira ac. renin-angiotensin system, hypertension, and chronic kidney disease: pharmacogenetic implications. j pharmacol sci. 2012;120(2):77-88. crossref pubmed 46. shinozaki k, ayajiki k, nishio y, sugaya t, kashiwagi a, okamura t. evidence for a causal role of the renin-angiotensin system in vascular dysfunction associated with insulin resistance. hypertension. 2004;43(2 i):255-262. crossref 47. engeli s, negrel r, sharma am. physiology and pathophysiology of the adipose tissue renin-angiotensin system. hypertension. 2000;35(6):1270-1277. crossref pubmed 48. liu j, cao r, xu m, et al. hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov-2 infection in vitro. cell discov. 2020;6(1):16. crossref pubmed 49. roca-ho h, riera m, palau v, pascual j, soler mj. characterization of ace and ace2 expression within different organs of the nod mouse. int j mol sci. 2017;18(3):e563. crossref pubmed 50. mehta p, mcauley df, brown m, sanchez e, tattersall rs, manson jj. hlh across speciality collaboration, uk. covid-19: consider cytokine storm syndromes and immunosuppression. lancet. 2020;395(10229):1033-1034. crossref pubmed 51. al heialy s, hachim my, senok a, et al. regulation of angiotensin converting enzyme 2 (ace2) in obesity: implications for covid-19. biorxiv. 2020;2:2020.04.17.046938. crossref 52. sanders jm, monogue ml, jodlowski tz, cutrell jb. pharmacologic treatments for coronavirus disease 2019 (covid-19): a review. jama. 2020;323(18):1824-1836. crossref pubmed 53. mamber s, krakowka s, osborn j, et al. could unconventional immunomodulatory agents help alleviate covid-19 symptoms and severity? preprints. 2020;(april). crossref 54. he j, hu l, huang x, et al. potential of coronavirus 3c-like protease inhibitors for the development of new anti-sars-cov-2 https://doi.org/10.1093/heapro/dax026 https://www.ncbi.nlm.nih.gov/pubmed/28541498 https://doi.org/10.1126/science.abb5793 https://doi.org/10.3389/fmicb.2017.01157 https://www.ncbi.nlm.nih.gov/pubmed/28690599 https://doi.org/10.1080/17476348.2018.1506331 https://www.ncbi.nlm.nih.gov/pubmed/30056777 https://doi.org/10.1111/j.1600-065x.2012.01151.x https://www.ncbi.nlm.nih.gov/pubmed/22889225 https://doi.org/10.1111/obr.13034 https://www.ncbi.nlm.nih.gov/pubmed/32281287 https://doi.org/10.1016/s2213-8587(20)30110-8 https://www.ncbi.nlm.nih.gov/pubmed/32559477 https://doi.org/10.1038/s41574-020-00462-1 https://www.ncbi.nlm.nih.gov/pubmed/33479538 https://doi.org/10.1016/j.metabol.2020.154317 https://www.ncbi.nlm.nih.gov/pubmed/32673651 https://www.ncbi.nlm.nih.gov/pubmed/32712222 https://doi.org/10.1016/j.metabol.2020.154319 https://doi.org/10.1002/oby.22971 https://www.ncbi.nlm.nih.gov/pubmed/32677752 https://doi.org/10.1038/nm0796-800 https://www.ncbi.nlm.nih.gov/pubmed/8673927 https://doi.org/10.3389/fimmu.2020.573662 https://www.ncbi.nlm.nih.gov/pubmed/33123152 https://doi.org/10.1080/08830185.2020.1845518 https://www.ncbi.nlm.nih.gov/pubmed/33275480 https://doi.org/10.1111/obr.13128 https://www.ncbi.nlm.nih.gov/pubmed/32845580 https://doi.org/10.1038/d41586-020-02946-6 https://www.ncbi.nlm.nih.gov/pubmed/33082543 https://www.ncbi.nlm.nih.gov/pubmed/24798950 https://doi.org/10.1016/j.diabres.2014.04.006 https://doi.org/10.1590/s1517-86922009000600012 https://doi.org/10.1017/s0007114520003311 https://doi.org/10.1016/j.drudis.2020.01.015 https://www.ncbi.nlm.nih.gov/pubmed/32006468 https://doi.org/10.1126/science.1085952 https://doi.org/10.1016/j.ijantimicag.2020.106012 https://www.ncbi.nlm.nih.gov/pubmed/32389723 https://doi.org/10.1016/j.tim.2016.09.001 https://www.ncbi.nlm.nih.gov/pubmed/27743750 https://doi.org/10.1016/j.ejmech.2020.112711 https://www.ncbi.nlm.nih.gov/pubmed/32810751 https://doi.org/10.1016/j.cell.2020.02.052 https://doi.org/10.1136/bmj.m606 https://www.ncbi.nlm.nih.gov/pubmed/32075786 https://doi.org/10.1111/bph.12159 https://www.ncbi.nlm.nih.gov/pubmed/23488800 https://www.ncbi.nlm.nih.gov/pubmed/18310257 https://doi.org/10.1113/expphysiol.2008.042002 https://www.ncbi.nlm.nih.gov/pubmed/18310257 https://doi.org/10.1161/hypertensionaha.109.140145 https://www.ncbi.nlm.nih.gov/pubmed/23079502 https://doi.org/10.1254/jphs.12r03cr https://doi.org/10.1161/01.hyp.0000111136.86976.26 https://doi.org/10.1161/01.hyp.35.6.1270 https://www.ncbi.nlm.nih.gov/pubmed/10856276 https://doi.org/10.1038/s41421-020-0156-0 https://www.ncbi.nlm.nih.gov/pubmed/32194981 https://doi.org/10.3390/ijms18030563 https://www.ncbi.nlm.nih.gov/pubmed/28273875 https://doi.org/10.1016/s0140-6736(20)30628-0 https://www.ncbi.nlm.nih.gov/pubmed/32192578 https://doi.org/10.1101/2020.04.17.046938 https://doi.org/10.1001/jama.2020.6019 https://www.ncbi.nlm.nih.gov/pubmed/32282022 https://doi.org/10.20944/preprints202004.0014.v1 protein with potential for combating sars-cov-2 infection in obesity12 © 2021 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti drugs: insights from structures of protease and inhibitors. int j antimicrob agents. 2020;56(2):106055. crossref pubmed 55. farady cj, craik cs. mechanisms of macromolecular protease inhibitors. clin lymphoma. 2010;11(17):19-222341-222346. crossref 56. laskowski m jr, kato i. protein inhibitors of proteinases. annu rev biochem. 1980;49(1):593-626. crossref pubmed 57. barkia i, ketata bouaziz h, sellami boudawara t, aleya l, gargouri af, saari n. acute oral toxicity study on wistar rats fed microalgal protein hydrolysates from bellerochea malleus. environ sci pollut res int. 2020;27(16):19087-19094. crossref pubmed 58. souza dd, brandão-costa rmp, albuquerque wwc, porto alf. partial purification and characterization of a trypsin inhibitor isolated from adenanthera pavonina l. seeds. s afr j bot. 2016;104:30-34. crossref 59. cristina oliveira de lima v, piuvezam g, leal lima maciel b, heloneida de araújo morais a. trypsin inhibitors: promising candidate satietogenic proteins as complementary treatment for obesity and metabolic disorders? j enzyme inhib med chem. 2019;34(1):405-419. crossref pubmed 60. lewis gp. lista de espécies da flora do brasil. jardim botânico do rio de janeiro. crossref 61. ribeiro ja, serquiz ac, silva pf, et al. trypsin inhibitor from tamarindus indica l. seeds reduces weight gain and food consumption and increases plasmatic cholecystokinin levels. clinics (são paulo). 2015;70(2):136-143. crossref pubmed 62. carvalho fmcc, lima vcoo, costa is, et al. a trypsin inhibitor from tamarind reduces food intake and improves inflammatory status in rats with metabolic syndrome regardless of weight loss. nutrients. 2016;8(10):1-14. crossref pubmed 63. costa is, medeiros af, carvalho fmc, et al. satietogenic protein from tamarind seeds decreases food intake, leptin plasma and cck-1r gene expression in obese wistar rats. obes facts. 2018;11(6):440-453. crossref pubmed 64. luz abs, dos santos figueredo jb, salviano bdpd, et al. adipocytes and intestinal epithelium dysfunctions linking obesity to inflammation induced by high glycemic index pellet-diet in wistar rats. biosci rep. 2018;38(3):1-15. crossref pubmed 65. li s, liu l, he g, wu j. molecular targets and mechanisms of bioactive peptides against metabolic syndromes. food funct. 2018;9(1):42-52. crossref pubmed 66. de queiroz jlc, de araújo costa ro, rodrigues matias ll, et al. chitosan-whey protein nanoparticles improve encapsulation efficiency and stability of a trypsin inhibitor isolated from tamarindus indica l. food hydrocoll. 2018;84:247-256. crossref 67. costa roa. identification of safety and potential clinical application of nanoparticles loaded with a trypsin inhibitor isolated from tamarind seeds (tamarindus indica l.). dissertation. published online 2019. crossref 68. matias llr, costa roa, passos ts, et al. tamarind trypsin inhibitor in chitosan-whey protein nanoparticles reduces fasting blood glucose levels without compromising insulinemia: a preclinical study. nutrients. 2019;11(11):2770. crossref pubmed 69. santos ea, oliveira as, arajo rablo lm, ferreira a, arajo morais ah. affinity chromatography as a key tool to purify protein protease inhibitors from plants. in: affinity chromatography. intech; 2012:35. crossref 70. medeiros af, costa is, carvalho fmc, et al. biochemical characterisation of a kunitz-type inhibitor from tamarindus indica l. seeds and its efficacy in reducing plasma leptin in an experimental model of obesity. j enzyme inhib med chem. 2018;33(1):334-348. crossref pubmed 71. carvalho fmc, lima vco, costa is, et al. anti-tnf-α agent tamarind kunitz trypsin inhibitor improves lipid profile of wistar rats presenting dyslipidemia and diet-induced obesity regardless of ppar-γ induction. nutrients. 2019;11(3):e512. crossref pubmed 72. winer da, luck h, tsai s, winer s. the intestinal immune system in obesity and insulin resistance. cell metab. 2016;23(3): 413-426. crossref pubmed 73. maurizi g, della guardia l, maurizi a, poloni a. adipocytes properties and crosstalk with immune system in obesity related inflammation. j cell physiol. 2018;233(1):88-97. crossref pubmed 74. adeyemo sm, onilude aa. enzymatic reduction of antinutritional factors in fermenting soybeans by lactobacillus plantarum isolates from fermenting cereals. niger food j. 2013;31(2):84-90. crossref 75. de blasio mj, boije m, kempster sl, et al. leptin matures aspects of lung structure and function in the ovine fetus. endocrinology. 2016;157(1):395-404. crossref pubmed 76. torday js, powell fl, farmer cg, orgeig s, nielsen hc, hall aj. leptin integrates vertebrate evolution: from oxygen to the blood-gas barrier. respir physiol neurobiol. 2010;173(1) (suppl):s37-s42. crossref pubmed 77. bassi m, furuya wi, menani jv, et al. leptin into the ventrolateral medulla facilitates chemorespiratory response in leptin-deficient (ob/ob) mice. acta physiol (oxf). 2014;211(1): 240-248. crossref pubmed 78. sideleva o, dixon ae. the many faces of asthma in obesity. j cell biochem. 2014;115(3):421-426. crossref pubmed 79. sood a, ford es, camargo ca jr. association between leptin and asthma in adults. thorax. 2006;61(4):300-305. crossref pubmed 80. fook jmsll, macedo llp, moura gedd, et al. a serine proteinase inhibitor isolated from tamarindus indica seeds and its effects on the release of human neutrophil elastase. life sci. 2005;76(25):2881-2891. crossref pubmed 81. ribeiro jkc, cunha dds, fook jmsll, sales mp. new properties of the soybean trypsin inhibitor: inhibition of human neutrophil elastase and its effect on acute pulmonary injury. eur j pharmacol. 2010;644(1-3):238-244. crossref pubmed 82. thanigaimalai p, konno s, yamamoto t, et al. development of potent dipeptide-type sars-cov 3cl protease inhibitors with novel p3 scaffolds: design, synthesis, biological evaluation, and docking studies. eur j med chem. 2013;68:372-384. crossref pubmed 83. elfiky aa. ribavirin, remdesivir, sofosbuvir, galidesivir, and tenofovir against sars-cov-2 rna dependent rna polymerase (rdrp): a molecular docking study. life sci. 2020;253(february):117592. crossref pubmed 84. wu c, liu y, yang y, et al. analysis of therapeutic targets for sarscov-2 and discovery of potential drugs by computational methods. acta pharm sin b. 2020;10(5):766-788. crossref pubmed https://doi.org/10.1016/j.ijantimicag.2020.106055 https://www.ncbi.nlm.nih.gov/pubmed/32534187 https://doi.org/10.1002/cbic.201000442 https://doi.org/10.1146/annurev.bi.49.070180.003113 https://www.ncbi.nlm.nih.gov/pubmed/6996568 https://doi.org/10.1007/s11356-018-4007-6 https://www.ncbi.nlm.nih.gov/pubmed/30612348 https://doi.org/10.1016/j.sajb.2015.11.008 https://doi.org/10.1080/14756366.2018.1542387 https://www.ncbi.nlm.nih.gov/pubmed/30734596 https://doi.org/10.3109/13880209209053984 https://doi.org/10.6061/clinics/2015(02)11 https://www.ncbi.nlm.nih.gov/pubmed/25789523 https://doi.org/10.3390/nu8100544 https://www.ncbi.nlm.nih.gov/pubmed/27690087 https://doi.org/10.1159/000492733 https://www.ncbi.nlm.nih.gov/pubmed/30537704 https://doi.org/10.1042/bsr20180304 https://www.ncbi.nlm.nih.gov/pubmed/29950343 https://doi.org/10.1039/c7fo01323j https://www.ncbi.nlm.nih.gov/pubmed/29188845 https://doi.org/10.1016/j.foodhyd.2018.06.010 https://repositorio.ufrn.br/jspui/handle/123456789/27159 https://doi.org/10.3390/nu11112770 https://www.ncbi.nlm.nih.gov/pubmed/31739532 https://doi.org/10.5772/34982 https://doi.org/10.1080/14756366.2017.1419220 https://www.ncbi.nlm.nih.gov/pubmed/29322840 https://doi.org/10.3390/nu11030512 https://www.ncbi.nlm.nih.gov/pubmed/30818882 https://doi.org/10.1016/j.cmet.2016.01.003 https://www.ncbi.nlm.nih.gov/pubmed/26853748 https://doi.org/10.1002/jcp.25855 https://www.ncbi.nlm.nih.gov/pubmed/28181253 https://doi.org/10.1016/s0189-7241(15)30080-1 https://doi.org/10.1210/en.2015-1729 https://www.ncbi.nlm.nih.gov/pubmed/26479186 https://doi.org/10.1016/j.resp.2010.01.007 https://www.ncbi.nlm.nih.gov/pubmed/20096383 https://doi.org/10.1111/apha.12257 https://www.ncbi.nlm.nih.gov/pubmed/24521430 https://doi.org/10.1002/jcb.24678 https://www.ncbi.nlm.nih.gov/pubmed/24115053 https://doi.org/10.1136/thx.2004.031468 https://www.ncbi.nlm.nih.gov/pubmed/16540481 https://doi.org/10.1016/j.lfs.2004.10.053 https://www.ncbi.nlm.nih.gov/pubmed/15820500 https://doi.org/10.1016/j.ejphar.2010.06.067 https://www.ncbi.nlm.nih.gov/pubmed/20624384 https://doi.org/10.1016/j.ejmech.2013.07.037 https://www.ncbi.nlm.nih.gov/pubmed/23994330 https://doi.org/10.1016/j.lfs.2020.117592 https://www.ncbi.nlm.nih.gov/pubmed/32222463 https://doi.org/10.1016/j.apsb.2020.02.008 https://www.ncbi.nlm.nih.gov/pubmed/32292689 dti © 2020 the authors. this article is published by aboutscience and licensed under creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). any commercial use is not permitted and is subject to publisher’s permissions. full information is available at www.aboutscience.eu issn 1177-3928 drug target insights 2020; 14: 1-11 original research article doi: 10.33393/dti.2020.1548 identification of the possible therapeutic targets in the insulin-like growth factor 1 receptor pathway in a cohort of egyptian hepatocellular carcinoma complicating chronic hepatitis c type 4 nada m.k. mabrouk1, dalal m. elkaffash2,3, mona abdel-hadi1, salah-eldin abdelmoneim4, sameh saad eldeen3, gihan gewaifel6, khaled a. elella5, maher osman5, nahed baddour1 1 department of pathology, university of alexandria, alexandria egypt 2 alexandria regional center for women’s health and development, alexandria egypt 3 department of clinical and chemical pathology, university of alexandria, alexandria egypt 4 department of clinical oncology and nuclear medicine, university of alexandria, alexandria egypt 5 department of general surgery, university of alexandria, alexandria egypt 6 public health department, faculty of medicine, university of alexandria, alexandria egypt abstract background: molecular targeted drugs are the first line of treatment of advanced hepatocellular carcinoma (hcc) due to its chemoand radioresistant nature. hcc has several well-documented etiologic factors that drive hepatocarcinogenesis through different molecular pathways. currently, hepatitis c virus (hcv) is a leading cause of hcc. therefore, we included a unified cohort of hcv genotype 4-related hccs to study the expression levels of genes involved in the insulin-like growth factor 1 receptor (igf1r) pathway, which is known to be involved in all aspects of cancer growth and progression. aim: determine the gene expression patterns of igf1r pathway genes in a cohort of egyptian hcv-related hccs. correlate them with different patient/tumor characteristics. determine the activity status of involved pathways. methods: total ribonucleic acid (rna) was extracted from 32 formalin-fixed paraffin-embedded tissues of human hcv-related hccs and 6 healthy liver donors as controls. quantitative reverse transcription polymerase chain reaction (qrt-pcr) using rt2 profiler pcr array for human insulin signaling pathway was done to determine significantly upand downregulated genes with identification of most frequently coregulated genes, followed by correlation of gene expression with different patient/tumor characteristics. finally, canonical pathway analysis was performed using the ingenuity pathway analysis software. results: six genes – aebp1, akt2, c-fos, pik3r1, prkci, shc1 – were significantly overexpressed. thirteen genes – adrb3, cebpa, dusp14, ercc1, frs3, igf2, ins, irs1, jun, mtor, pik3r2, ppp1ca, rps6ka1 – were significantly underexpressed. several differentially expressed genes were related to different tumor/patient characteristics. nitric oxide and reactive oxygen species production pathway was significantly activated in the present cohort, while the growth hormone signaling pathway was inactive. conclusions: the gene expression patterns identified in this study may serve as possible therapeutic targets in hcv-related hccs. the most frequently coregulated genes may serve to guide combined molecular targeted therapies. the igf1r pathway showed evidence of inactivity in the present cohort of hcv-related hccs, so targeting this pathway in therapy may not be effective. keywords: gene expression, hepatitis c virus, hepatocellular carcinoma, molecular therapeutic targets received: january 8, 2020 accepted: january 20, 2020 published online: april 8, 2020 corresponding author: nada m.k. mabrouk department of pathology university of alexandria alexandria, egypt nada_mabrouk@hotmail.com, nada.mabrouk@alexmed.edu.eg introduction hepatocellular carcinoma (hcc) has become the fifth most common cancer in men worldwide and the ninth in women and the third leading cause of cancer-related deaths. distinct geographical variations in the incidence exist. in egypt, hcc has the highest age-standardized incidence and mortality rates (asr) from cancer in males and ranks second in females (1). most of the burden is in developing countries where 83% of cases (and deaths) occur (2). hcc has several possible therapeutic targets in igf1r pathway in egyptian hcv related hcc2 © 2020 the authors. published by aboutscience well-documented etiologic factors that drive hepatocarcinogenesis through different molecular pathways. currently, hepatitis c virus (hcv) is a leading cause of hcc. the multiplicity of etiologies of hcc and the existence of substantial molecular heterogeneity might limit the benefits of targeted approaches to hcc treatment. therefore, therapeutic vulnerabilities should be studied in homogeneous groups of hccs sharing similar drivers of the disease (3). in egypt, the estimated national prevalence of hcv is 14.7%, which is the highest in the world (4), and hccs attributable to hcv account for around 50% of cases (5). studies of the hcv genome confirmed that over 90% of egyptian hcv isolates belong to genotype 4 (6). the present study was conducted to characterize the molecular alterations in the insulin-like growth factor 1 receptor (igf1r) pathway in a unified cohort of egyptian hccs complicating chronic hcv genotype 4. the igf1r pathway was chosen as its activation plays an important role in almost every aspect of cancer development, including cell proliferation, neoplastic transformation, cancer cell survival, epithelial to mesenchymal transition (emt), and metastasis (7) and that igf1r overexpression correlated with aggressive phenotype in cancer, poor clinical outcome, and therapy resistance (8–10). aim the present work aimed to identify the gene expression of the different subcellular components of the igf1r pathway in egyptian hccs complicating chronic hcv genotype 4, followed by identification of the most frequently coregulated genes, correlation of gene expression with the different clinical and pathological patient and tumor characteristics, and determination of the activity status of involved pathways. materials two groups were included in the present study; the study group comprised 32 formalin-fixed, paraffin-embedded (ffpe) blocks from 32 egyptian hccs complicating chronic hcv genotype 4 etiology (shown as positive by hcv dna pcr using inno-lipa i and ii). twenty-four out of 32 were segmentectomy specimens, while 8 out of 32 were liver explants, and the control group comprised six core biopsies from normal liver donors. inclusion criteria were hcv genotype 4 etiology and good quantity and quality of extracted ribonucleic acid (rna) as measured by nanodrop spectrophotometer (a260:a280 ratio 1.9:2.0 indicated pure rna). exclusion criteria included positive serology for anti-hepatitis b virus surface antibody and any hcc patient who has received previous neoadjuvant chemotherapy (including transarterial chemoembolization). to accurately define our study population, further identification of other known hepatocarcinogens in the egyptian environment was performed, including: 1) hepatitis b core antigen (hbcag) positivity by immunohistochemistry (ihc) (denoting occult hepatitis b virus [hbv] infection) was found in 7/29 of our hcc cases as evidenced by cytoplasmic positivity to the antibody. 2) aflatoxin b1-dna (afb1-dna) adduct positivity by ihc was found to be present in 16/29 of our cases as evidenced by nuclear positivity to the antibody. methods the protocol of this study was approved by the ethics committee of the faculty of medicine, university of alexandria. paraffin blocks of liver biopsies were obtained from the archives of the pathology department at the faculty of medicine, alexandria university. clinical data were collected from the patients’ files including gender, age, comorbidities (diabetes mellitus and obesity), alfa-fetoprotein (afp) levels, liver function tests (alanine aminotransferase [alt], aspartate transaminase [ast], bilirubin, and albumin), hemoglobin concentration, and complete blood picture. gross pathologic examination of hcc tumors specifically addressed tumor size, multifocality, gross capsulation, gross necrosis, satellites, and bile production. histopathologic study using 5-μm-thick sections assessed pathologic tumor grade (was performed according to the edmondson and steiner grading system for hcc), histologic pattern, cell type (including giant cells and clear cells), and vascular invasion. the presence of dysplastic nodules outside hcc and pathological tumor-node-metastasis (ptnm) staging (according to the ptnm staging system of the american joint committee on cancer 8th edition [ajcc]) was also noted. real-time quantitative reverse transcription pcr preparation of the paraffin blocks and obtaining tissue sections 1) by trimming off the excess paraffin from the edges of the specimen to facilitate deparaffinization. 2) then two sections, 5 μm thick each, were cut from each block and placed in a 2 ml nuclease-free microfuge tube. total rna was extracted from ffpe tumor sections using the rneasy® ffpe kit for purification of total rna from ffpe tissue sections (qiagen, hilden, germany, cat. no.: 73504) according to the manufacturer’s instructions. its main principle is to remove all the paraffin, reverse formalin-induced cross-linking in the nucleic acid, and prevent any further degradation in the rna that is known to be induced by formalin and finally to eliminate all genomic dna. the concentration and purity of extracted rna were determined using a nanodrop nd-1000 spectrophotometer. samples were accepted when the a260/a280 ratio (nucleic acid/protein absorbance) was ≥1.9. reverse transcription of extracted rna (0.5 µg [500 ng] of rna was used for all experimental runs) to complementary dna (cdna) was done using rt2 first strand kit (qiagen, hilden, germany, cat. no.: 330401) according to the manufacturer’s instructions. rt2 sybr green rox qpcr mastermix (qiagen, hilden, germany, cat. no.: 330523) was added to the cdna. the mixture was aliquoted into the 96 wells of the commercially available rt² profiler™ pcr array human insulin signaling pathway (pahs-030za) (qiagen, mabrouk et al 3 © 2020 the authors. published by aboutscience hilden, germany, cat. no.: 330231) containing primer assays for 84 genes concerning the human insulin signaling pathway, including genes of the phosphatidylinositol-3-kinase (pi3k) and the mitogen-activated protein kinase (mapk) signaling pathways that are the two major downstream signaling pathways for igf1r signaling (11) and also genes involved in carbohydrate, lipid, and protein metabolism; transcription factors and transcription regulators; genes involved in cell proliferation, growth and differentiation (12); and five housekeeping genes (hkgs). in addition, there were one genomic dna control, three reverse transcription controls, and three positive pcr controls (the full list of genes can be found in the supplementary file). pcr is then performed using the applied biosystems® 7500 real-time pcr system (thermofisher scientific, foster city, ca, usa). finally, relative expression is determined using the δδct method using the rt2 profiler pcr array data analysis webportal (at www.sabiosciences.com/ pcrarraydataanalysis.php). two hkgs with the lowest standard deviations and most stable expression across replicates were selected for data normalization: beta-2-microglobulin (b2m) (h2) and hypoxanthine phosphoribosyl transferase 1 (hprt1) (h4). fold regulation values for each of the 84 genes in hcc cases was determined by the qiagen online data analysis webportal (at www.sabiosciences.com/pcrarraydataanalysis. php). then for each gene the percentages of cases showing upregulation, normal expression, and downregulation were determined. determination of the significantly upand downregulated genes in hcc cases using spss statistics software version 20.0 was carried out using nonparametric mannwhitney test. the most frequently coregulated genes were identified. identification of genes significantly correlated with the different patient/tumor characteristics carried out using the mann-whitney test. finally, active and inactive pathways in hcc from the present dataset were identified; an excel file containing the gene symbols, fold regulation values, and the p values for all 84 tested genes was uploaded to the ingenuity pathway analysis (ipa) software. results the demographic data of the cases included are presented in table i. gross pathological examination of hcc tumors tumor size ranged from 1.75 to 16.0 cm, with a median of 4.8 cm and a mean of 5.38 ± 3.46 cm standard deviation (sd; tab. ii). microscopic examination of hcc tumors the most frequently encountered histologic patterns are displayed in table iii. twenty-six out of 32 cases showed combined histological patterns in the following frequencies (tab. iv). frequency of different histopathological features of hcc tumors including edmonson and steiner tumor grades is shown in tables v and vi, respectively. frequency of ptnm tumor stages is shown in table vii. table i distribution of the studied cases according to demographic data (n = 32) number % gender male 24 75.0 female 8 25.0 age (years) ≤60 22 68.8 >60 10 31.3 min.–max. 47.0–67.0 mean ± standard deviation 56.06 ± 5.49 median 56.0 comorbidities 17/25 53.1 alfa-fetoprotein (≥200 ng/ml) 8/25 25.0 table ii the frequency of the gross characteristics of the tumors included in the present study gross findings number % gross capsulation (n = 32) absent present 6 26 18.8 81.3 gross necrosis (n = 32) absent present 21 11 65.6 34.4 multifocality (n = 32) absent present 4 28 12.5 87.5 satellites (n = 32) absent present 6 26 18.8 81.3 bile production (n = 32) absent present 15 17 46.9 53.1 table iii the frequency of the different histologic patterns of the hcc cases included in the present study histologic pattern of hcc (n = 32) number % trabecular absent 3 9.4 present 29 90.6 pseudoglandular absent 12 37.5 present 20 62.5 compact absent 20 62.5 present 12 37.5 hcc = hepatocellular carcinoma. possible therapeutic targets in igf1r pathway in egyptian hcv related hcc4 © 2020 the authors. published by aboutscience significantly upand downregulated genes a total of 51 deregulated genes were found in the present cohort of hccs. fourteen genes were upregulated while 37 genes were downregulated. statistically significant upregulation was observed in hcc patients, in comparison to the control group, in six genes, namely aebp1, akt2, fos, pik3r1, prkci, shc1, where shc1 was the most significantly overexpressed gene (p value: 0.001) (tab. viii, fig. 1). shc1, aebp1, akt2, and pkci were the most frequently coregulated genes among the significantly upregulated genes. on the other hand, the frequency and magnitude of the downregulation events were greater than that of upregulation events (tabs. viii and ix). thirteen genes, namely adrb3, cebpa, dusp14, ercc1, frs3, igf2, ins, irs1, jun, mtor, pik3r2, ppp1ca, and rps6ka1, were significantly underexpressed in hcc patients relative to the control group, where ppp1ca and ins were the two most significantly underexpressed genes (p value: 0.002) (tab. ix, fig. 1). ins and ppp1ca were the most frequently coregulated genes among the significantly downregulated genes. table iv the frequency of different combinations of patterns in the tumors included in the present study hccs with combined histologic pattern number (%) trabecular and pseudoglandular trabecular and compact trabecular, pseudoglandular, and compact glandular and compact 15 6 3 2 total 26 hcc = hepatocellular carcinoma. table v the frequency of giant cells, clear cells, vascular invasion, and dysplastic nodules in the hcc cases included in the present study histologic feature number % giant cells (n = 32) absent 17 53.1 present 15 46.9 clear cells (n = 32) absent 17 53.1 present 15 46.9 vascular invasion (n = 32) absent 11 34.4 present 21 65.6 dysplastic nodules outside hcc (n= 32) absent 13 40.6 present 19 59.4 hcc = hepatocellular carcinoma. table vii distribution of the studied cases according to ptnm stage (ajcc 8th edition) (n = 32) stage number % early stage (1 and 2) 12 37.5 stage i 5 15.6 stage ii 7 21.9 advanced stage (3) 20 62.5 stage iii 20 62.5 stage iv 0 0.0 ajcc = american joint committee on cancer; tnm = tumor-node-metastasis. table vi distribution of the studied cases according to edmondson and steiner grading system (n = 32) grade number % low grade (1 + 2) 14 43.8 grade i 1 3.1 grade ii 13 40.6 high grade (3 + 4) 18 56.3 grade iii 3 9.4 grade iv 15 46.9 fig. 1 bar chart representing the 6 significantly upregulated and the 13 significantly downregulated genes in the hepatocellular carcinoma group table viii the six significantly upregulated genes’ fold regulation, p values, and percentage of cases showing upregulation of these genes position gene symbol fold regulation p number % a04 aebp1 6.694 0.045* 29 90.6 a06 akt2 6.0287 0.013* 26 81.3 c01 fos 3.9608 0.045* 25 78.1 e11 pik3r1 4.1363 0.020* 25 78.1 f05 prkci 7.7467 0.006* 28 87.5 g04 shc1 11.8326 0.001* 30 93.8 p = p values for mann-whitney test for comparing between the two groups. *statistically significant at p ≤ 0.05. mabrouk et al 5 © 2020 the authors. published by aboutscience table ix the 13 significantly downregulated genes’ fold regulation, p values, and percentage of cases showing downregulation of these genes position gene symbol fold regulation p number % a03 adrb3 −11.3685 0.010* 23 71.9 b02 cebpa −5.3981 0.041* 22 68.8 b07 dusp14 −6.5717 0.016* 23 71.9 b10 ercc1 −8.7853 0.037* 22 68.8 c03 frs3 −35.5177 0.006* 27 84.4 d02 igf2 −8.6788 0.013* 25 78.1 d04 ins −31.7678 0.002* 30 93.8 d07 irs1 −8.0248 0.037* 22 68.8 d10 jun −13.0027 0.020* 23 71.9 e04 mtor −9.0478 0.045* 26 81.3 e12 pik3r2 −2.8856 0.037* 23 71.9 f03 ppp1ca −46.4755 0.002* 29 90.6 f12 rps6ka1 −5.653 0.025* 24 75.0 p = p values for mann-whitney test for comparing between the two groups. *statistically significant at p ≤ 0.05. fig. 2 scatter plot representing the normalized expression of each gene on the polymerase chain reaction array between the two groups. log base 10 of 2–δct value of each gene in the control group is plotted on the x-axis against the corresponding value in the hepatocellular carcinoma (hcc) group, which is plotted on the y-axis to visualize gene expression changes. the central boundary line indicates unchanged gene expression. the upper left section of the scatter plot (above the fold-change boundary lines) contains genes upregulated in the hcc group as compared to the control group. the lower right section of the scatter plot (below the fold-change boundary lines) contains genes downregulated in the hcc group as compared to the control group. it shows the upregulated genes in red and the downregulated genes in green. fig. 3 heat map is a color-coded representation of fold regulation expression data between hcc and control groups overlaid onto the polymerase chain reaction array plate layout. the black color represents the average magnitude of gene expression. the brightest green represents the most downregulated genes, the brightest red represents the most upregulated genes in the hcc cases compared to the normal controls. vegfa was downregulated (fold regulation of −3.2345) in 71.9% (23/32) of cases in the present study, although this did not reach statistical significance. a scatter plot and a heat map were generated on the online data analysis web portal representing the distribution of average gene expression levels (figs. 1 and 2 respectively). nonsupervised hierarchical clustering of all cases and controls according to their gene expression profile by the data analysis web portal the included cases were a homogeneous population with no outstanding clustering as evidenced by the dendrograms. this further supports the study design as it included a specific cohort of hccs: egyptian ethnicity and hcv genotype 4 etiology. correlations between the gene expression levels and demographic data in hcc patients age: there were significant differences in three gene expression levels as regards age; aebp1, akt1, and fbp1 genes were significantly overexpressed in hcc patients ≤60 years of age compared to hcc patients >60 years of age (tab. x). gender: dok1 was significantly overexpressed in female patients (p = 0.037). stage: there were no statistically significant differences detected in the gene expression levels between early ptnm stage (pt1,2) and late stage (pt3,4) hcc tumors. grade: gsk3a was significantly overexpressed in lowgrade tumors as compared to high-grade ones where it was normally expressed (p = 0.002) possible therapeutic targets in igf1r pathway in egyptian hcv related hcc6 © 2020 the authors. published by aboutscience table x differentially expressed genes between hcc cases whose age is ≤60 years and those who are >60 years of age as regards human insulin signaling pathway gene expression levels gene symbol group 1 age ≤60 years group 1 age >60 years p fold regulation fold regulation aebp1 9.6655 2.9833 0.040* akt1 2.3142 −1.394 0.024* fbp1 2.1679 −2.1895 0.007* hcc = hepatocellular carcinoma. p = p values for mann-whitney test for comparing between the two categories. *statistically significant at p ≤ 0.05. table xiii differentially expressed genes between hcc tumors with clear cells and those without as regards human insulin signaling pathway gene expression levels clear cells p absent (n = 17) present (n = 15) cebpb −2.34 −12.28 0.010* gpd1 −1.29 −2.64 0.040* hk2 −6.14 −18.17 0.050* igfbp1 1.22 −3.46 0.041* pik3r2 −1.41 −6.52 0.021* hcc = hepatocellular carcinoma. p = p values for mann-whitney test for comparing between the two categories. *statistically significant at p ≤ 0.05. tumor size: both cebpb (p = 0.027) and gpd1 (p = 0.002) were significantly downregulated in tumors >5 cm. dysplastic nodules: no statistically significant difference was detected in the gene expression levels of any genes between cases who had dysplastic nodules outside tumor mass and those who did not have any (none were <0.05). satellite nodules: aebp1 and fos genes were upregulated in the absence of satellite nodules more than double their expression level in the cases which showed satellite nodules. mapk1 gene was overexpressed in the presence of satellite nodules and normally expressed in their absence. prkcz and ucp1 genes were downregulated in the absence of satellite nodules and normally expressed in their presence (tab. xi). multifocality: aebp1 and fos genes were upregulated in the absence of multifocal masses more than double their expression level in the cases which showed multifocal masses. ldlr gene was overexpressed in the absence of multifocal masses and normally expressed in their presence (tab. xii). gross capsulation: both insl3 (p = 0.043) and irs1 (p = 0.030) genes were downregulated in the presence of gross capsulation. gross tumor necrosis: cap1 (p = 0.041), grb2 (p = 0.047), prl (p = 0.024), and tg (p = 0.025) were downregulated in the presence of gross necrosis. pik3r1 (p = 0.016) was upregulated in the absence of gross tumor necrosis. giant cells: both gsk3a (p = 0.015) and pck2 (p = 0.030) were upregulated in the absence of giant cells. ucp1 (p = 0.033) was downregulated in tumors that showed giant cells. clear cells: cebpb, hk2, gpd1, igfbp1 and pik3r2 were downregulated in all the studied cases, more so in tumors that showed clear cells than in tumors which had no clear cells (tab. xiii). vascular invasion: nos2 showed more significant downregulation in the absence of vascular invasion (p = 0.035). hbcag: rps6ka1 was more significantly underexpressed in the presence of hbcag (p = 0.034). afb1-dna adduct positivity: akt1 and mapk1 were more significantly upregulated in the presence of aflatoxin as compared to negative cases (p = 0.019, 0.017 respectively). on the other hand, rps6ka1 was more significantly underexpressed in the presence of afb1 (p = 0.046). correlations between the gene expression levels and the different clinical characteristics of the tumors in hcc patients thrombocytopenia: in patients with a low platelet count, akt1 was significantly upregulated (p = 0.038) while bcl2l1 table xi differentially expressed genes between hcc cases having satellite nodules around the tumor mass and those without, as regards human insulin signaling pathway gene expression levels satellite nodules p absent (n = 6) present (n = 26) aebp1 17.44 5.37 0.004* fos 13.21 3.00 0.007* mapk1 −1.97 2.05 0.012* prkcz −2.63 1.42 0.026* ucp1 −9.60 −1.98 0.014* hcc = hepatocellular carcinoma. p = p values for mann-whitney test for comparing between the two categories. *statistically significant at p ≤ 0.05. table xii differentially expressed genes between hcc cases having multifocal tumor masses and those with unifocal masses as regards human insulin signaling pathway gene expression levels multifocality p absent (n = 4) present (n = 28) aebp1 26.30 5.51 0.004* fos 19.81 3.15 0.003* ldlr 7.12 1.88 0.035* hcc = hepatocellular carcinoma. p = p values for mann-whitney test for comparing between the two categories. *statistically significant at p ≤ 0.05. mabrouk et al 7 © 2020 the authors. published by aboutscience was significantly downregulated (p = 0.045). vegfa was more significantly underexpressed in the presence of normal platelet numbers (p = 0.038). comorbidities: there was no statistically significant difference in the gene expression levels between patients who have comorbidities and those who do not have any. canonical pathway analysis predicted by ipa database nitric oxide and reactive oxygen species production pathway was significantly activated in the present cohort, while the growth hormone signaling pathway was significantly inactivated (fig. 4). discussion different etiologic factors result in very diverse hcc molecular profiles between different regions of the world (13,14). understanding the heterogeneity of hcc is conducive to developing personalized therapy and identifying molecular biomarkers. most published work on molecular profiling of hcc was conducted on heterogeneous groups of tumors including different etiologies and ethnic backgrounds (15–19). once hcc heterogeneity is accepted (20), studies have to be conducted on homogeneous samples to identify characteristic genetic alterations, if personalized management is the target. fig. 4 analysis of canonical pathways involved in hepatitis c virus-related hepatocellular carcinoma pathogenesis with the ingenuity pathway analysis (ipa) database using the present dataset (the 84 tested genes). the igf pathway was found to be inactive. on the other hand, the nitric oxide and reactive oxygen species production pathway were significantly activated. description: the white bars mean that half the molecules suggest an activation and half suggest inhibition, so an overall z-score of 0 (ingenuity systems). the blue bar indicates that there is evidence that the pathway is inhibited and a negative z-score. the orange bar means that there is a positive z-score indicating that there is evidence for activation of this pathway. the horizontal threshold line indicates the threshold for the overrepresentation analysis and was set at p = 0.05. only bars that are more significant than p < 0.05 are shown. the ratio line represents the ratio of the number of molecules in the present dataset that map into the pathway relative to the whole size of the pathway (ingenuity systems). the pathway analysis was generated through the use of ipa (qiagen inc., https://www.qiagenbioinformatics.com/products/ ingenuity-pathway-analysis hcc is known to be chemoand radioresistant (21); therefore, identifying therapeutic vulnerabilities for molecular targeted drugs is in dire need. the present study measured levels of messenger rna (mrna) expression of 84 insulin signaling-related genes, including genes of the pi3k and the mapk signaling pathways (11). also included are genes involved in carbohydrate, lipid and protein metabolism, transcription factors and transcription regulators, genes involved in cell proliferation, growth, and differentiation (12). of the 84-cancer pathway-focused genes in this array, upregulation was observed in 14 genes, while 37 genes appeared to be downregulated in the tumor samples, for a total of 51 differentially regulated genes. these upregulated genes may be therapeutic targets for future trials, especially those for which molecularly targeted agents are available. among the significantly upregulated genes was c-fos, a proto-oncogene which is a major nuclear target for signal transduction pathways, including mapk pathway, and is involved in the regulation of cell growth, differentiation, and transformation (22,23). a novel selective c-fos/ap-1 inhibitor t‐5224 was found to inhibit the invasion and migration of head and neck squamous cell carcinoma cells in vitro and prevented lymph node metastasis in head and neck cancer in an animal model (24). most of the significantly upregulated genes found in the present work were also found in common with prior hcc possible therapeutic targets in igf1r pathway in egyptian hcv related hcc8 © 2020 the authors. published by aboutscience and clinical trials to be conducted for agents targeting those upregulated genes to test their efficacy and their potential use in hcv-related hcc treatment. the 13 significantly downregulated genes included genes involved in carbohydrate, protein, and lipid metabolism, pi3k signaling target genes, mapk signaling target genes, insulin receptor-associated proteins, genes involved in cell cycle, cell proliferation, growth factors and receptors, insulin gene and insulin receptor-associated proteins, primary insulin signaling target genes, transcription factors, and transcription regulators (12). two of those were tumor suppressor genes. cebpa has been described as a tumor suppressor, leading to mitotic arrest through activation of p21 and repression of e2fs and cyclin-dependent kinases (50). its expression is downregulated in a number of cancers including lung (51), breast cancers (52), head and neck squamous cell carcinoma (53), and hcc. its downregulation was associated with advanced hcc stages and poor survival (54,55). recently, a modified small activating rna (sarna) retained activation of cebpa mrna and downstream targets and inhibited growth of liver cancer cell lines in vitro (56). this novel drug has been encapsulated in a liposomal formulation for liver delivery in humans and is currently in a phase i clinical trial for patients with liver cancer and represents the first human study of a sarna therapeutic (57). frs3 was significantly downregulated in our patient cohort. this is the first report of its significant downregulation in hccs. similar to our findings, frs3 expression level has been found to be downregulated in brain and lung cancer cell lines. it constitutively binds to egfr regardless of egf stimulation and inhibits egf-induced cell transformation and proliferation (58). it serves as a tumor suppressor in non-small cell lung cancer (nsclc) (59). in our work, excision repair cross-complementing rodent repair deficiency, complementation group 1 (ercc1) was significantly downregulated. it was also found to be downregulated in chinese (47) and turkish (60) hcc cohorts as well. loss of ercc1 expression in hepatocytes may be one of the leading factors for genetic instability, and thus of tumorigenesis (61). impaired nucleotide excision repair pathway, in which ercc1 plays a major role, leads to reduced dna repair capability and increases dna adduct levels and genomic instability that in turn could lead to a more malignant phenotype (62). this is not limited to hccs, but also is a finding in other types of cancer including head and neck squamous cell carcinoma (63). gene expression patterns can be used to define the suitable chemotherapeutic agent employed in any given case. ercc1 overexpression could be associated with resistance to cisplatin-based therapy in hcc, similar to what is observed in nsclc (64,65). vegfa was found to be downregulated in the present study in 71.9% of cases. several retrospective studies suggested that tumors with a vegfa amplification respond better to sorafenib, the only approved first-line treatment of advanced hcc (66), suggesting that further studies of currently used molecularly targeted therapies in egyptian hcv-related hcc patients are needed, and search for molecular biomarkers of response to therapy with prospective molecular studies; akt2, c-fos, pik3r1, prkcι, shc1 were all found to be similarly significantly upregulated (25–30). aebp1 is an exception; to the best of our knowledge, this is the first report to find this gene to be significantly and consistently (>90% of hccs) upregulated in hcc. it is a transcriptional repressor that has been studied in great detail for its role in regulating adipogenesis (31). it has also been found to be a proinflammatory mediator (32). finally, it has been shown that aebp1 induces massive obesity in mice with targeted overexpression of aebp1 in adipose tissue (33). although its exact role in tumorigenesis is not clear (34), aebp1 was found to be upregulated in many different cancers such as cervical cancer (35,36), primary glioblastoma multiforme (37), and human breast cancer cell lines (38). ladha et al found that silencing aebp1 expression and loss of its function lead to apoptosis in glioma cell lines, meaning that aebp1 has an antiapoptotic function and is an important survival factor for tumor cells (34). two studies found that directly targeting akt2 by mir302b in human hcc cells suppresses cell proliferation, invasion, and metastasis in vitro and also inhibited hcc tumor growth in vivo (25,39). a recent preclinical in vitro and in vivo trial using a highly selective allosteric pan-akt inhibitor in combination with sorafenib found that this combination yielded better outcomes and with minimal toxicity than sorafenib alone. the combination significantly reduced tumor growth, proliferation, and angiogenesis and increased apoptosis (40). the pkcι blocking agent aurothiomalate (atm) was found to negatively regulate emt and invasion of hcc in immortalized murine hepatocytes and was found to inhibit proliferation and induce apoptosis in hepg2 cells (41). also, a recent patent has been published on a novel drug targeting pkcι. it has been found to be effective at treating colon cancer cells having high level of expression of at least one apkc (42). these can potentially be tested in preclinical trials as a method for treating hccs overexpressing atypical pkcs. similarly, pkcι was upregulated in hcc in several studies involving different ethnic groups; two chinese studies (wang et al (43), du et al (44)) and an american and spanish study (chiang et al (45)). boyault et al in a french study attempted to classify hcc by unsupervised hierarchical clustering according to the tumors’ gene expression profiles by microarray; all three generated subgroups had upregulated pkcι (the hbv and non-hbv-related hccs) (46). in line with our findings, pik3r1 was found to be upregulated in hcv-related hcc subgroups (45) and in hcc patients of chinese ethnicity as well (47). in a recent study by he et al, pik3r1 transfection by small interfering rna significantly inhibited hcc cell migration and invasion (48). there are several pi3k inhibitors including both pan-pi3k and isoform-specific pi3k inhibitors that are still in early phase clinical trials (49). the strongest associations between upregulated genes in the present study were between aebp1, shc1, akt2, and prkci. possibly, these associations can serve as a guide to molecularly targeted therapy combinations to cotarget the genes that were most frequently coregulated to maximize the benefits in hcc therapy. these potential therapeutic targets and combinations open several pipelines for future preclinical mabrouk et al 9 © 2020 the authors. published by aboutscience validation of the role of vegfa expression level to predict response to therapy. one of the points of strength in our study is that we included hcc samples resulting from a single etiologic agent only (hcv genotype 4-related hcc). also, the present study unified the controls used in the analysis as we used healthy liver donors as the control group. on the other hand, in the vast majority of studies paired adjacent cirrhotic liver tissues to the hcc tumor masses were used as controls (30,45,67– 69). this complicates the interpretation of results since the gene expression patterns in the nontumor cirrhotic liver samples from different patients can vary significantly, affected by the viral infection and degree of fibrosis. thus, the tumor-specific variation in the expression patterns could not be distinguished from variation due to differences in the corresponding nontumor liver samples (70). in our cohort, two hkgs – b2m and hprt1 – were used for data normalization. in a study performed to search for appropriate hkgs to be used for data normalization in hcvinduced hcc specifically. two of the most commonly used hkgs, which were also included in the standard catalogued 5 hkgs set in the present study’s array, namely glyceraldehyde-3-phosphate dehydrogenase (gapdh) and beta-actin (actb), were found to be deregulated in hcv-induced hcc (71). thus, using them for normalization would have strong effects on the extent of differential expression of genes, leading to misinterpretation of results. this was the exact same case in our study for actb and gapdh, which were found to be unstable and had a wide sd across samples. a few weaknesses of this study that we recognize are, firstly, the fact that we were studying patients who already had developed disease at a single point in time. also, as in most studies on hcc, tumor samples have been collected during resection. it could be expected that in a more clinically advanced context with tumor progression, increased crosstalk among pathways and chromosomal instability would contribute to additional signaling deregulation. these samples do not represent the tumors of patients with advanced stage disease, who do not undergo surgery. however, as hcc diagnosis at present is not tissue based, obtaining tissue samples from late cases is not feasible at present. conclusions the significantly upand downregulated genes identified in the present study can serve as potential therapeutic targets for hcv-related hccs. in the present study, the insulin pathway genes including igf1r, igf2, ins, insr, and irs1 were found to be downregulated and the igf pathway was found to be significantly inactive, in the present work, which is in concordance with the findings of zucman-rossi et al (72). therefore, drugs targeting the igf1r pathway may not be suitable for this specific subset of hcv-related hccs. although nos2 gene expression was unchanged in our cohort, ipa revealed that nitric oxide pathway and reactive oxygen species production in hcv-related hccs were significantly activated. the gene expression pattern of each hcc tumor seems to provide a distinctive molecular portrait of that tumor, as no gene was 100% upor downregulated in the present cohort. personalized medicine mandates the knowledge of the specific genetic profile of each hcc tumor, which is currently not feasible due to costs associated especially in developing countries where most of the hcc burden lies. therefore, the present study fills a void in this respect – identifying specific morphologic and clinical patient/tumor characteristics, which significantly correlate with underlying molecular alterations. this may be used as a guide for tailoring molecularly targeted therapies. we confirm the findings of a previous study that was performed to search for appropriate hkgs to be used for data normalization in hcv-induced hcc specifically. two of the most commonly used hkgs – actb and gapdh – were found to be unstable and had a wide sd in our study and theirs; therefore these shouldn’t be used for normalization. we suggest b2m and hprt1 to be used instead, as they had more stable levels of expression. disclosures financial support: the present study was fully funded by a grant from the “alexandria regional center for women’s health and development,” alexandria, egypt (this grant covered all expenses needed for provision of all experimental kits). conflict of interest: all the authors declare no conflict of interest. references 1. international agency for research on cancer (iarc). globocan 2012: estimated cancer incidence, mortality and prevalence worldwide in 2012. france: iarc; 2012. 2. shi j, zhu l, liu s, xie wf. a meta-analysis of case-control studies on the combined effect of hepatitis b and c virus infections in causing hepatocellular carcinoma in china. br j cancer. 2005;92(3):607–612. 3. llovet jm, villanueva a, lachenmayer a, finn rs. advances in targeted therapies for hepatocellular carcinoma in the genomic era. nat rev clin oncol. 2015;12(7):408–424. 4. el-zanaty f, way a. egypt demographic and health survey 2008. cairo, egypt: ministry of health, el-zanaty and associates, and macro international; 2009. 5. maucort-boulch d, de martel c, franceschi s, plummer m. fraction and incidence of liver cancer attributable to hepatitis b and c viruses worldwide. int j cancer. 2018;142(12):2471-7. 6. ray sc, arthur rr, carella a, bukh j, thomas dl. genetic epidemiology of hepatitis c virus throughout egypt. j infect dis. 2000;182(3):698–707. 7. wang y, sun y. insulin-like growth factor receptor-1 as an anticancer target: blocking transformation and inducing apoptosis. curr cancer drug targets. 2002;2(3):191–207. 8. christopoulos pf, msaouel p, koutsilieris m. the role of the insulin-like growth factor-1 system in breast cancer. mol cancer. 2015;14(1):43. 9. papa v, gliozzo b, clark gm, et al. insulin-like growth factor-i receptors are overexpressed and predict a low risk in human breast cancer. cancer res. 1993;53(16):3736–3740. 10. jones r, campbell c, gunther e, et al. transgenic overexpression of igf-ir disrupts mammary ductal morphogenesis and induces tumor formation. oncogene. 2007;26(11):1636–1644. 11. samani aa, yakar s, leroith d, brodt p. the role of the igf system in cancer growth and metastasis: overview and recent insights. endocr rev. 2007;28(1):20–47. possible therapeutic targets in igf1r pathway in egyptian hcv related hcc10 © 2020 the authors. published by aboutscience 12. vescovo t, refolo g, vitagliano g, fimia gm, piacentini m. molecular mechanisms of hepatitis c virus–induced hepatocellular carcinoma. clin microbiol infect. 2016;22(10):853–861. 13. patel a, sun w. molecular targeted therapy in hepatocellular carcinoma: from biology to clinical practice and future. curr treat opt oncol. 2014;15(3):380–394. 14. lu l-c, hsu c-h, hsu c, cheng a-l. tumor heterogeneity in hepatocellular carcinoma: facing the challenges. liver cancer. 2016;5(2):128–138. 15. luo jh, ren b, keryanov s, et al. transcriptomic and genomic analysis of human hepatocellular carcinomas and hepatoblastomas. hepatology. 2006;44(4):1012–1024. 16. lee js, chu is, heo j, et al. classification and prediction of survival in hepatocellular carcinoma by gene expression profiling. hepatology. 2004;40(3):667–676. 17. shirota y, kaneko s, honda m, kawai hf, kobayashi k. identification of differentially expressed genes in hepatocellular carcinoma with cdna microarrays. hepatology. 2001;33(4): 832–840. 18. tackels-horne d, goodman md, williams aj, et al. identification of differentially expressed genes in hepatocellular carcinoma and metastatic liver tumors by oligonucleotide expression profiling. cancer. 2001;92(2):395–405. 19. patil ma, chua m-s, pan k-h, et al. an integrated data analysis approach to characterize genes highly expressed in hepatocellular carcinoma. oncogene. 2005;24(23):3737–3747. 20. guimei m, baddour n, elkaffash d, abdou l, taher y. gremlin in the pathogenesis of hepatocellular carcinoma complicating chronic hepatitis c: an immunohistochemical and pcr study of human liver biopsies. bmc res notes. 2012;5(1):390. 21. huynh h. molecularly targeted therapy in hepatocellular carcinoma. biochem pharmacol. 2010;80(5):550–560. 22. saez e, rutberg se, mueller e, et al. c-fos is required for malignant progression of skin tumors. cell. 1995;82(5):721–732. 23. yuen mf, wu pc, lai vch, lau jyn, lai cl. expression of c-myc, c-fos, and c-jun in hepatocellular carcinoma. cancer. 2001; 91(1):106–112. 24. kamide d, yamashita t, araki k, et al. selective activator protein-1 inhibitor t-5224 prevents lymph node metastasis in an oral cancer model. cancer sci. 2016;107(5):666–673. 25. wang l, yao j, zhang x, et al. mirna-302b suppresses human hepatocellular carcinoma by targeting akt2. mol cancer res. 2014;12(2):190–202. 26. zhang y, guo x, yang m, yu l, li z, lin n. identification of akt kinases as unfavorable prognostic factors for hepatocellular carcinoma by a combination of expression profile, interaction network analysis and clinical validation. mol biosyst. 2014;10(2):215–222. 27. kurokawa y, matoba r, takemasa i, et al. molecular features of non-b, non-c hepatocellular carcinoma: a pcr-array gene expression profiling study. j hepatol. 2003;39(6):1004–1012. 28. huang c-y, huang x-p, zhu j-y, et al. mir-128-3p suppresses hepatocellular carcinoma proliferation by regulating pik3r1 and is correlated with the prognosis of hcc patients. oncol rep. 2015;33(6):2889–2898. 29. wang jm, li q, du gs, lu jx, zou sq. significance and expression of atypical protein kinase c-iota in human hepatocellular carcinoma. j surg res. 2009;154(1):143–149. 30. chen x, cheung st, so s, et al. gene expression patterns in human liver cancers. mol biol cell. 2002;13(6):1929–1939. 31. ro h-s, roncari d. the c/ebp-binding region and adjacent sites regulate expression of the adipose p2 gene in human preadipocytes. mol cell biol. 1991;11(4):2303–2306. 32. majdalawieh a, ro hs. regulation of ikappabalpha function and nf-kappab signaling: aebp1 is a novel proinflammatory mediator in macrophages. mediators inflamm. 2010;2010:823–821. 33. zhang l, reidy sp, nicholson te, et al. the role of aebp1 in sexspecific diet-induced obesity. mol med. 2005;11(1–12):39. 34. ladha j, sinha s, bhat v, donakonda s, rao sm. identification of genomic targets of transcription factor aebp1 and its role in survival of glioma cells. mol cancer res. 2012;10(8): 1039–1051. 35. han s-j, cho yl, nam gh, kim ck, seo j-s, ahn ws. cdna microarray analysis of gene expression profiles associated with cervical cancer. cancer res treat. 2003;35(5):451–459. 36. ahn ws, bae sm, lee jm, et al. searching for pathogenic gene functions to cervical cancer. gynecol oncol. 2004;93(1):41–48. 37. reddy sp, britto r, vinnakota k, et al. novel glioblastoma markers with diagnostic and prognostic value identified through transcriptome analysis. clin cancer res. 2008;14(10):2978–2987. 38. grigoriadis a, mackay a, reis-filho js, et al. establishment of the epithelial-specific transcriptome of normal and malignant human breast cells based on mpss and array expression data. breast cancer res. 2006;8(5):r56. 39. wang l, yao j, sun h, et al. mir-302b suppresses cell invasion and metastasis by directly targeting akt2 in human hepatocellular carcinoma cells. tumor biol. 2016;37(1):847–855. 40. jilkova zm, kuyucu az, kurma k, et al. combination of akt inhibitor arq 092 and sorafenib potentiates inhibition of tumor progression in cirrhotic rat model of hepatocellular carcinoma. oncotarget. 2018;9(13):11145–11158. 41. ma c, yang y, wang j, et al. the apkcι blocking agent atm negatively regulates emt and invasion of hepatocellular carcinoma. cell death dis. 2014;5(3):e1129. 42. acevedo-duncan m. method of treating colorectal cancers using a pkc inhibitor. in: google patents; 2018. 43. wang j-m, li q, du g-s, lu j-x, zou s-q. significance and expression of atypical protein kinase c-ι in human hepatocellular carcinoma. j surg res. 2009;154(1):143–149. 44. du g-s, wang j-m, lu j-x, et al. expression of p-apkc-ι, e-cadherin, and β-catenin related to invasion and metastasis in hepatocellular carcinoma. ann surg oncol. 2009;16(6):1578–1586. 45. chiang dy, villanueva a, hoshida y, et al. focal gains of vegfa and molecular classification of hepatocellular carcinoma. cancer res. 2008;68(16):6779–6788. 46. boyault s, rickman ds, de reyniès a, et al. transcriptome classification of hcc is related to gene alterations and to new therapeutic targets. hepatology. 2007;45(1):42–52. 47. de yun f, hui z, tan y, cheng rx. effect of phosphorylation of mapk and stat3 and expression of c-fos and c-jun proteins on hepatocarcinogenesis and their clinical significance. world j gastroenterol. 2001;7(1):33–36. 48. he s, zhang j, zhang w, chen f, luo r. foxa1 inhibits hepatocellular carcinoma progression by suppressing pik3r1 expression in male patients. j exp clin cancer res. 2017;36(1):175. 49. rodon j, tabernero j. improving the armamentarium of pi3k inhibitors with isoform-selective agents: a new light in the darkness. cancer discov. 2017;7(7):666–669. 50. schuster mb, porse bt. c/ebpalpha: a tumour suppressor in multiple tissues? biochim biophys acta. 2006;1766(1):88–103. 51. costa db, li s, kocher o, et al. immunohistochemical analysis of c/ebpalpha in non-small cell lung cancer reveals frequent down-regulation in stage ii and iiia tumors: a correlative study of e3590. lung cancer (amsterdam, netherlands). 2007; 56(1):97–103. 52. gery s, tanosaki s, bose s, bose n, vadgama j, koeffler hp. down-regulation and growth inhibitory role of c/ebpalpha in breast cancer. clin cancer res. 2005;11(9):3184–3190. 53. bennett kl, hackanson b, smith lt, et al. tumor suppressor activity of ccaat/enhancer binding protein alpha is epigenetically down-regulated in head and neck squamous cell carcinoma. cancer res. 2007;67(10):4657–4664. mabrouk et al 11 © 2020 the authors. published by aboutscience 54. tseng hh, hwang yh, yeh kt, chang jg, chen yl, yu hs. reduced expression of c/ebpalpha protein in hepatocellular carcinoma is associated with advanced tumor stage and shortened patient survival. j cancer res clin oncol. 2009;135(2): 241–247. 55. tomizawa m, watanabe k, saisho h, nakagawara a, tagawa m. down-regulated expression of the ccaat/enhancer binding protein alpha and beta genes in human hepatocellular carcinoma: a possible prognostic marker. anticancer res. 2003;23(1a):351–354. 56. voutila j, reebye v, roberts tc, et al. development and mechanism of small activating rna targeting cebpa, a novel therapeutic in clinical trials for liver cancer. mol ther. 2017;25(12): 2705–2714. 57. reebye v, huang kw. gene activation of cebpa using sarna: preclinical studies of the first in human sarna drug candidate for liver cancer. 2018;37(24):3216-28. 58. huang l, watanabe m, chikamori m, et al. unique role of snt-2/frs2β/frs3 docking/adaptor protein for negative regulation in egf receptor tyrosine kinase signaling pathways. oncogene. 2006;25(49):6457. 59. iejima d, minegishi y, takenaka k, et al. frs2β, a potential prognostic gene for non-small cell lung cancer, encodes a feedback inhibitor of egf receptor family members by erk binding. oncogene. 2010;29(21):3087. 60. turhal n, bas e, er o, et al. ercc1 is not expressed in hepatocellular cancer: a turkish oncology group, gastrointestinal oncology subgroup study. j buon. 2010;15(4):794–796. 61. cheng l, spitz mr, hong wk, wei q. reduced expression levels of nucleotide excision repair genes in lung cancer: a casecontrol analysis. carcinogenesis. 2000;21(8):1527–1530. 62. simon gr, sharma s, cantor a, smith p, bepler g. ercc1 expression is a predictor of survival in resected patients with non-small cell lung cancer. chest. 2005;127(3):978–983. 63. cheng l, sturgis em, eicher sa, spitz mr, wei q. expression of nucleotide excision repair genes and the risk for squamous cell carcinoma of the head and neck. cancer. 2002;94(2):393–397. 64. ueda s, shirabe k, morita k, et al. evaluation of ercc1 expression for cisplatin sensitivity in human hepatocellular carcinoma. ann surg oncol. 2011;18(4):1204–1211. 65. fautrel a, andrieux l, musso o, boudjema k, guillouzo a, langouët s: overexpression of the two nucleotide excision repair genes ercc1 and xpc in human hepatocellular carcinoma. j hepatol. 2005;43(2):288–293. 66. lopez pm, patel p, uva p, villanueva a, llovet jm. current management of liver cancer. eur j cancer suppl. 2007;5(5):444–446. 67. xu xr, huang j, xu zg, qian bz, et al. insight into hepatocellular carcinogenesis at transcriptome level by comparing gene expression profiles of hepatocellular carcinoma with those of corresponding noncancerous liver. proc natl acad sci usa. 2001;98(26):15089–15094. 68. boyault s, rickman ds, de reynies a, et al. transcriptome classification of hcc is related to gene alterations and to new therapeutic targets. hepatology. 2007;45(1):42–52. 69. chuma m, sakamoto m, yamazaki k, et al. expression profiling in multistage hepatocarcinogenesis: identification of hsp70 as a molecular marker of early hepatocellular carcinoma. hepatology. 2003;37(1):198–207. 70. makowska z, boldanova t, adametz d, et al. gene expression analysis of biopsy samples reveals critical limitations of transcriptome-based molecular classifications of hepatocellular carcinoma. j pathol clin res. 2016;2(2):80–92. 71. waxman s, wurmbach e. de-regulation of common housekeeping genes in hepatocellular carcinoma. bmc genomics. 2007;8:243. 72. zucman-rossi j, villanueva a, nault j-c, llovet jm. genetic landscape and biomarkers of hepatocellular carcinoma. gastroenterology. 2015;149(5):1226–1239. e1224. dti drug target insights 2022; 16: 36-48issn 1177-3928 | doi: 10.33393/dti.2022.2482 original research article drug target insights issn 1177-3928 www.aboutscience.eu/dti © 2022 the authors. this article is published by aboutscience and licensed under creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). commercial use is not permitted and is subject to publisher’s permissions. full information is available at www.aboutscience.eu focus on antimicrobial resistance (amr) antimicrobial resistance surveillance system mapping in different countries ramendra pati pandey1, riya mukherjee2,3, chung-ming chang2 1centre for drug design discovery and development (c4d), srm university, sonepat, haryana india 2master & phd program in biotechnology industry, chang gung university, taoyuan city taiwan (r.o.c.) 3graduate institute of biomedical sciences, department of biotechnology, chang gung university, taoyuan city taiwan (r.o.c.) abstract objectives: excessive use of antibiotics has increased antimicrobial resistance (amr) worldwide, which is a major public concern among the countries. to control this threat proper monitoring of the antimicrobial usage with increasing rate of amr is required. moreover, alternatives for antibiotics are surveyed and are being researched for quick use in the future. thus, multisector intervention is highly encouraged for better outcomes. in this research article, six different european countries are discussed in terms of antimicrobial usage and amr in human and livestock sectors with the help of literature study and various reports published by different organizations. methods: data study has been conducted to collect data for comparison study. data sources of amr and antimicrobial usage are analyzed and both antimicrobial use and amr are compared. results: this article provides surveillance systems that are formed to keep a track on the upcoming situation of amr and the consumption of antimicrobials by humans as well as animals. the article firmly allows the readers to get broad information about the amr across six countries of europe. these annual reports have hugely helped the government to decide for alternatives and have focused in many training activities to combat the amr situation globally. conclusion: as antibiotic resistance genes persist on an interface between environment and animal and animal health, an approach is required in all three areas that stress the concept of “one approach to health.” keywords: alternative antibiotics, amr, comparative medicine, one health approach, phage therapy, surveillance received: august 9, 2022 accepted: november 4, 2022 published online: november 30, 2022 corresponding author: chung-ming chang master & ph.d. program in biotechnology industry chang gung university no. 259, wenhua 1st rd. guishan dist., taoyuan city 33302 taiwan (r.o.c.) cmchang@mail.cgu.edu.tw various other antibiotics are also becoming resistant against the microorganisms causing immense threat among the population. this global threat comprises of both commensal and pathogenic bacteria. the similarities between human and animal diseases, as well as the interactions between animals and humans who come into contact with them, have long been recognized. human and veterinary medicine diverged in the twentieth century. during the same time span, our understanding of infectious diseases and antibiotics grew dramatically. the necessity for partnerships between human health and veterinary sectors to prevent and control zoonotic illnesses and antibiotic resistance grew in the second half of the twentieth century. the notion of ecosystem health developed toward the end of the twentieth century, extending the integration and collaboration of human and animal medicine to the environment. later on, the phrase “one health” was coined to describe a holistic approach to improving human, animal, and environmental health through multidisciplinary cooperation and communication. several global plans have been established to combat the amr epidemic, including the world health organization’s (who) global action plan (gap), the new european one health action plan against amr, and the central asian and eastern european surveillance of introduction in the last few decades extensive use of antibiotics has resulted in rising cases of antimicrobial resistance (amr) against various organisms. from narrow-spectrum antibiotics, people shifted to broad-spectrum antibiotics, which eventually increased the high resistance rates. multidrugresistant (mdr) bacterial infections are rapidly emerging and spreading over the world, posing a severe threat to global healthcare. carbapenem-resistant enterobacteriaceae (cre), a type of gram-negative bacteria that has resisted all or virtually all current antibiotics, is one cause for concern. likewise, https://doi.org/dti.2022.2482 https://creativecommons.org/licenses/by-nc/4.0/legalcode pandey et al drug target insights 2022; 16: 37 © 2022 the authors. published by aboutscience www.aboutscience.eu antimicrobial resistance (caesar) network (1). surveillance and monitoring systems for antimicrobial usage (amu) and amr in humans and animals are critical for assessing and controlling global trends in antimicrobial use and antimicrobial susceptibility patterns of bacteria in various populations. in the context of a one health strategy, zoonotic and indicator microorganisms are especially important. a strategic framework for reducing infectious disease risks at the animalhuman ecosystem interface was published in 2008, adopting and promoting the one health concept. the one health approach has been supported and implemented by a wide number of national and international institutes since 2008. research on the human-animal environment interaction is critical to supporting the call for a one health approach to amr and infectious illnesses. furthermore, training and extension initiatives are critical for promoting the one health idea and facilitating its application among various stakeholders (2,3). several governments and international organizations have now included a one health approach in the amr action plans. improvements in antimicrobial use, better regulation and policy, improved surveillance, stewardship, infection control, sanitation, animal husbandry, and identifying antimicrobial alternatives are all necessary efforts. this report summarizes research and educational activity in the field of one health in western europe, with an emphasis on infectious diseases. it might act as a springboard for future collaborations and projects. materials and methods data sources we conducted a database study for collecting major characteristics of surveillance and monitoring systems on antimicrobial use and amr in cattle and people, as well as amr systems in food, in this publication. countries such as spain, germany, france, the netherlands, norway, and the united kingdom were considered for this project. the literature searches in recent times have been carried out to understand and collect the data from different gray reports and other amr databases. the database study has been conducted by searching the terms “antimicrobial resistance,” “antibiotic usage,” “one health approach” on pubmed and desired research papers or data sheets annually published by different agencies are studied for collective data required for this research article. additionally, information about one health approach in these countries was also investigated for obtaining the results. one health policy publications issued by international organizations and countries also provided background information on the one health program and european one health projects. moreover, google research on one health with its associated activities and trainings among these countries was conducted for acquiring more relevant outcomes. alternative antibiotics for resolving the issue of amr were also researched which focuses on better solution for amr and antimicrobial usage. one of the renowned projects also known as ardig (antimicrobial resistance dynamics the influence of geographic origin) together collects and gathers data related to amr and usage of antimicrobials from both human and veterinary sectors. the stipulated graph depicts the predicted deaths that will be increasing for the usage of antibiotics globally by the year 2050 (fig. 1) (4). this helps us to portray the comparison study of all the data collected for different countries along with the asian countries. in addition, all the surveillance systems for monitoring amr in different countries of europe have been explained thoroughly in this article. the primary reason for tracking the reports of european countries is because one health has gained a lot of traction throughout europe. the one health strategy is currently being promoted in europe mostly in regard to amr. many nations have adopted the one health concept in their anti-amr policies, and funding opportunities for amr research have considerably increased. in the areas of zoonotic diseases and one health, the number of national and international multidisciplinary research networks is growing (1,5,6). fig. 1 the stipulated graph depicts the predicted increase in the number of deaths for using antibiotics globally by the year 2050. x-axis denotes the continents and y-axis denotes the number of deaths by the year 2050. (from: https://www.publichea l t h p o s t .o r g /d a t a b y t e/a n t i b i o t i c resistant-bacteria/) amr surveillance system mapping in countries38 © 2022 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti surveillance strategies of amr and monitoring system data collection and data analysis different european countries have various surveillance strategies and monitoring systems for controlling the rising threat of amr. additionally, multiple organizations are coming together for joint efforts that require combating this situation. a complete summary of the data collected is provided in a tabular form for better understanding of the data gathered. france amr data related to agriculture, food, and the environment are monitored by the french agency for food, environmental, and occupational health and safety (anses). the french monitoring network for antibiotic resistance in pathogenic bacteria of animal origin (resapath) and the salmonella network are coordinated by this agency. the salmonella network is a surveillance system designed to keep nonhuman salmonella under control throughout the food chain. the investigation and surveillance of nosocomial infection network (raisin) coordinates the nosocomial infection surveillance coordination centers across the country. bmr-raisin, a private raisin module for multidrug-resistant bacteria, reports on amr data in the community. healthy animals, food, and the environment are all sampled. the resapath voluntary surveillance system compiles amr data for primary bacterial species and general isolates from sick animals from each animal sector in the annual resapath report (7). germany clinical amr data from companion and food-producing animals is collected in germany through the german veterinary monitoring system (germ-vet). amr testing in the zoonosismonitoring system (zomo) report includes data on zoonotic and commensal bacteria in various food chains, as well as amr data on salmonella from national control programs, which are also reported to the european food safety authority (efsa). antimicrobial resistance surveillance (ars) is the human national amr surveillance system. it gathers routine susceptibility data for all bacterial species from any sample site, including hospital and outpatient care facilities. the hospital infection surveillance system (kiss) is a nosocomial infection surveillance system made up of multiple sub-systems that collect amu and amr data in hospitals. surveillance of antibiotic use and resistance in intensive care units (sari) gathered data on antimicrobial sensitivity for selected pathogenic microorganisms and the creation of amu-amr on a volunteer basis (1). spain to keep track of amr, the spanish veterinary antimicrobial resistance surveillance network (vav) was formed. vav provides nonclinical data to the efsa, which is included in the agency’s annual reports. according to eu legislation, this report contains information on zoonotic infections and diseases in animals, humans, and food, as well as data on amr in select zoonotic bacteria and indicator bacteria (1,6). norway the three amr surveillance programs in norway are the norwegian surveillance system for antimicrobial drug resistance (norm), norwegian veterinary antimicrobial resistance monitoring (norm-vet), and the norwegian surveillance system for communicable diseases (msis). this annual report contains updated information on amu and amr prevalence and distribution in the human, animal, and food sectors (8,9). the netherlands the “monitoring of antimicrobial resistance and antibiotic usage in animals in the netherlands” (maran), which brings together the food and consumer product safety authority’s amr food database, is the netherlands’ amr monitoring system for animals and food. it disseminates information on foodborne pathogen resistance as well as commensal indicators from animals and food. the infectious disease surveillance information system on antibiotic resistance (isis-ar) monitors amr in key pathogens in the human sector (10). these surveillance systems are extremely helpful in tracking down the situation caused by antimicrobial use and amr. the various features of different organizations built by the agencies have successfully helped the researchers in providing the necessary data for handling the threat worldwide. in addition to strengthening the amr surveillance, numerous policies have been prepared by who and other agencies that apparently help in working with the solution of either decreasing or avoiding the amr situation. for teaching and training, surveillance and risk assessment, and research, the amr coordinating office emphasizes a one health approach. political commitment, policy formation, sustainable finance, program creation, knowledge sharing, institutional collaboration, capacity enhancement, civil society involvement, and active community participation are all part of the framework for effective one health implementation. one health is a straightforward and strong idea with complex processes. the national response to zoonoses must be revised, food safety improved, and environmental integrity guaranteed. the transformation must be driven by the senior leadership. strong, ongoing lobbying by international development partners, in particular: the fao, the oie and the who, should be shared with the leading national leadership, disseminating the evidentiary results, predicted economic benefits, and best practice globally. the interconnected sustainable development goals offer a unique opportunity for advocacy and an integrated approach to development. the effectiveness of one health implementation depends on the extent to which institutional cooperation, common planning and coordination thorough monitoring for early detection and prevention of zoonoses are achieved. the key planning, implementation and surveillance are data and science. initial efforts for rapid tracking should be performed quickly in order to create multisectoral capacity across various organizations. the theory and practice of one health should be fully integrated and visible in the educational curriculum as well as in the constant upgrading of skills for all subjects for long-term implementation. pandey et al drug target insights 2022; 16: 39 © 2022 the authors. published by aboutscience www.aboutscience.eu united kingdom in the united kingdom, the eu-harmonized surveillance system (a native uk system) collects mandatory amr data on indicator commensal escherichia coli and/or campylobacter spp. from meat and fecal content of healthy animals (chicken, beef, turkey, and pigs). there are also salmonella national control programs in the united kingdom that are hosted in the eu-harmonized surveillance system. in scotland, the scotland’s rural college veterinary services and capital diagnostics (sruc) surveillance system collects clinical isolates from animals. in england, monitoring surveillance system vet pathogens apha collects amr data from infected animals that veterinarians proactively offer for diagnostic services, covering all relevant bacteria and animal species. on the human aspect, the british society for antimicrobial chemotherapy’s (bsac) resistance surveillance program provides antibiotic resistance data from cooperating labs in the uk and ireland for a variety of clinically relevant bacteria from community-acquired respiratory illnesses. amr data are collected through the electronic communication of surveillance in scotland (ecoss) network from participating national health service (nhs) and reference laboratories in scotland (1,11). results the accomplished research revealed that various surveillance systems are actively working to follow a trail of the upcoming situation of amr and antimicrobial consumption by humans as well as animals. these surveillance systems of european countries are jointly contributing in statistically analyzing the rising situation of amr and the prominent measures taken by different organizations for implementing one health approach. moreover, various training institutes and alternative measures for preventing amr are firmly encouraged in these six european countries along with taiwan and india. data of amr solely do not arise from consuming antibiotics. there are multiple more aspects such as food habits of the humans, food chains maintained by the healthy animals, and the environment that together exhibit the importance of surveillance systems for amr as these features put up the amr issue topmost. the efsa is in charge of communication on food chain concerns. annually, the efsa and the european centre for disease prevention and control (ecdc) collect amr data on humans, food, and healthy animals from eu states and some affiliated countries (5). the european union summary report on amr in zoonotic and indicator bacteria from humans, animals, and food is prepared and published by the efsa. also, some nongovernmental organizations such as european animal health study centre (ceesa) are also contributing by researching about amr and forming relevant systems to perform the activities efficiently (6). precisely, the organization is working in monitoring the antimicrobial susceptibility of the bacterial pathogens that have the potential of causing diseases among the animals along with the foodborne pathogens in animal’s food. these organizations are not the only aide for this surveillance system; a prime system also called as the european antimicrobial resistance surveillance network (ears-net) mainly helps in the surveillance of amr data. this is an amr surveillance network established in compliance with european union and european economic area legislation. the ecdc collects amr data from eu states through ears-net and publishes the annual ears-net report. on that account it is extremely crucial to compare the percentages of antibiotic usage and amr, which will help in displaying the numbers accurately acquired from different organizations and relevant measures will be implemented for better solution. similarly, acknowledging this, two joint interagency reports for antibiotic consumption and the analysis of amr were published that clearly demonstrate the effects of using extensive antibiotics on humans and animals and the data were compared to amr reports for better understanding. this report is jointly published by the european medicines agency (ema), the efsa, and the ecdc. the ecdc and ears-net require other platforms to jointly work for this (fig. 2). every country from europe has fig. 2 different organizations jointly working together to provide data to ears-net and ecdc. amr surveillance system mapping in countries40 © 2022 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti its own surveillance system for amr that they follow, and prepared reports are further provided to efsa. the amr surveillance system is developed distinctly for humans and livestock (1). different systems of the country contribute in forming the reports, which are eventually published by efsa or ecdc (fig. 3). fig. 4 amr surveillance systems for humans of different countries reporting the data to efsa. ars = antimicrobial resistance surveillance; ears-net-es = european antimicrobial resistance surveillance network; ecoss, sgss, datastore and cosurv = the electronic communication of surveillance in scotland, second generation surveillance system; isis-ar = infectious disease surveillance information system on antibiotic resistance; norm & msis = norwegian veterinary antimicrobial resistance monitoring; onerba = national observatory of the epidemiology of bacterial antibiotic resistance. fig. 3 amr surveillance systems for livestock of different countries reporting the data to efsa. anses = the french agency for food, environmental and occupational health & safety; eu-harmonized = the eu-harmonized surveillance system; maran = monitoring of antimicrobial resistance and antibiotic usage in animals in the netherlands; norm-vet = norwegian veterinary antimicrobial resistance monitoring system; vav = the spanish veterinary antimicrobial resistance surveillance network (vav); zomo = zoonosis-monitoring system. table i amr surveillance system conducted in different countries for humans surveillance system country roles of surveillance system isis-ar the netherlands this aims at monitoring amr in major pathogens. norm and msis norway it is an amr surveillance program in norway. this annual report provides updated information on amu and amr occurrence and distribution in human beings. ars germany it is the national human medicine amr surveillance system. established by the robert koch institute, it collects routine sensitivity data from any sample site in the hospital and from ambulatory care institutions for all bacterial species. ears-net-es spain maintains the records of amr surveillance across spain. onerba france amu and amr as well as a leading amr network that collects data from a complex subsystem network is an annual french report, onerba. ecoss, sgss, datastore, and cosurv united kingdom the ecoss database gathers amr data from participating nhs laboratories and reference laboratories in scotland. electronic communication of surveillance in scotland (ecoss) (sgss) captures 98% of the national health service (nhs) laboratories across england, from routine laboratory surveillance data on infectious diseases and antimicrobial resistance. amr = antimicrobial resistance. similarly, amr surveillance system for humans is also analyzed by different organizations formed in these six european countries. figure 4 depicts the organizations that are being established for keeping the record of the amr surveillance (6). tables i and ii give details about all the aforementioned surveillance system followed by the distinct countries along with the features and roles they perform (1,7). pandey et al drug target insights 2022; 16: 41 © 2022 the authors. published by aboutscience www.aboutscience.eu table ii amr surveillance system conducted in different countries for livestock surveillance system country roles of surveillance system vav spain vav monitors the amr status throughout the country and is also responsible for monitoring animals and food. in addition, vav supplies efsa with nonclinical data. anses france anses generally monitors amr data related to food and livestock. zomo germany this report also provides data on zoonotic and commensal bacteria of the different food chains reported to efsa. maran the netherlands data on foodborne pathogens and commensal indicators from cattle and food are published in the annual report of the netherlands. norm-vet norway facilitate updated incidence and distribution information on animal amu and amr. euharmonized united kingdom mandatory amr data for meat and feces in healthy animals, using the appropriate indicator escherichia coli and/or campylobacter spp. are collected under the european harmonized supervisory system. amr = antimicrobial resistance; efsa = european food safety authority. fig. 5 amr surveillance systems for foods of different countries reporting the data to efsa. anses = the french agency for food, environmental and occupational health & safety; eu-harmonized = the eu-harmonized surveillance system; maran = monitoring of antimicrobial resistance and antibiotic usage in animals in the netherlands; norm-vet = norwegian veterinary antimicrobial resistance monitoring system; vav = the spanish veterinary antimicrobial resistance surveillance network (vav); zomo = zoonosis-monitoring system. humans and animals are exposed to amr from their food habits. thus, a surveillance system was set up especially for the food that is being consumed by both animals and humans. a thorough monitoring of the food consumed has the possibility of getting exposed to new pathogenic organisms, which could be a probable reason for pandemic, endemic, and epidemic. again, some of the organizations similar to humans and livestock are formed for keeping the track of rising amr cases from food habits (fig. 5) (1). all the aforementioned the organizations report their amr data to efsa but on the other hand, there are some organizations that do not report their amr data to efsa (tab. iii). the details of all these organizations contributing to different countries are described further (1). reported microorganisms accountable for amr in europe discussing about the surveillance systems available to control the amr and antimicrobial usage will not help the population be aware about the pathogenic disease-causing microorganisms accurately. therefore, it is very important to understand the pathogens responsible for causing amr also with the antimicrobials that are extensively used. earsnet received data from 29 countries for all eight bacterial table iii country-wise amr surveillance systems not reporting data to efsa surveillance system country hosts germ-vet germany livestock resapath france animals apha-vet pathogens united kingdom diseased animals sruc united kingdom animals peg germany human pathogens armin germany humans barda germany humans icu-kiss, op-kiss, sarikiss, mrsa-kiss germany human pathogens bsac united kingdom humans bmr-raisins france human pathogens species under observation (e. coli, klebsiella pneumoniae, pseudomonas aeruginosa, acinetobacter species, streptococcus pneumoniae, staphylococcus aureus, enterococcus faecalis, and enterococcus faecium). e. coli was the most commonly reported bacterial species (44.2%), followed by s. aureus (20.6%), k. pneumoniae (11.3%), e. faecalis (6.8%), p. aeruginosa (5.6%), s. pneumoniae (5.3%), e. faecium (4.5%), and amr surveillance system mapping in countries42 © 2022 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti acinetobacter species (4.5%) (fig. 6) (12). in 2019, more than half of e. coli isolates reported to ears-net were resistant to at least one antimicrobial group under surveillance, and more than a third of k. pneumoniae isolates were resistant to multiple antimicrobial groups. in general, resistance percentages in k. pneumoniae were higher than in e. coli. while carbapenem resistance was uncommon in e. coli, carbapenem resistance rates in k. pneumoniae were reported to be more than 10% in numerous countries. carbapenem resistance was also found in larger percentages in p. aeruginosa and acinetobacter species than in k. pneumoniae. the increase in the percentage of vancomycin-resistant e. faecium isolates in the eu/eea from 10.5% in 2015 to 18.3% in 2019 is a cause for concern. the results of antimicrobial susceptibility testing (ast) from invasive (blood or cerebrospinal fluid) isolates of eight bacterial species are provided in this article. e. coli, k. pneumoniae, p. aeruginosa, acinetobacter species, s. pneumoniae, s. aureus, e. faecalis, and e. faecium are all important bacteria for public health in europe. in 2019, the estimated national population coverage of data provided to ears-net ranged from 11% to 100%, with more than a third of the nations reporting a population coverage of 80% or above (12). pseudomonas aeruginosa although p. aeruginosa is naturally resistant to a wide range of antimicrobials, acquired resistance complicates the treatment of p. aeruginosa infections. because p. aeruginosa is still one of the most common causes of healthcare associated illness in europe, the public health consequences of amr in p. aeruginosa should not be overlooked (12). klebsiella pneumoniae due to k. pneumoniae’s great resistance, the european union is currently dealing with a significant issue. although carbapenem resistance has increased more than sevenfold since 2006, it has been more moderate in the last 5 years than in earlier eras. the who believes that novel drugs targeting third-generation cephalosporinand carbapenem resistant enterobacterales, such as k. pneumoniae and e. coli, are urgently needed. staphylococcus aureus many nations have created and implemented national methicillin-resistant staphylococcus aureus (mrsa) prevention recommendations and guidance documents, emphasizing on enhanced infection prevention and control as well as sensible antibiotic usage. despite this progress, mrsa remains a significant pathogen in europe. s. aureus is one of the most frequent bacteria that causes bloodstream infections, with a significant morbidity and fatality rate. mrsa surveillance in animals and food is currently voluntary and only carried out in a few countries. this monitoring, however, reveals an ever-changing situation, including the detection of livestock-associated mrsa (la-mrsa), healthcare-associated mrsa, and communityassociated mrsa from companion animals and/or livestock. la-mrsa has recently received increased attention as a zoonotic risk, particularly for those who work in close proximity to livestock. acinetobacter species acinetobacter species have the widest inter-country range in resistance percentages of any bacterial species under earsnet surveillance. depending on the reporting country, the percentage of isolates resistant to at least one of the antimicrobial groups under surveillance (fluoroquinolones, aminoglycosides, or carbapenems) ranged from 0% to 95.8% in 2019. because acinetobacter species is naturally resistant to many antimicrobial agents, acquired resistance complicates treatment of acinetobacter species infections. mdr acinetobacter species are a problem in the healthcare environment because they can survive for long periods of time in the environment and are notoriously difficult to eradicate once established. streptococcus pneumoniae in addition to ears-net, the enhanced surveillance program for invasive pneumococcal disease (ipd), which is also supervised by ecdc, collects additional data on ipd cases from reference laboratories across the eu/eea. the fig. 6 major species responsible for amr in europe. escherichia coli in europe, e. coli is a common cause of bloodstream infection. infections caused by antimicrobial-resistant e. coli account for the majority of amr cases in the eu. the percentages of amr reported in 2019 were substantially higher than in 2002, underlining the need for more antimicrobial stewardship and infection prevention and control activities. according to the latest data from the european surveillance of antimicrobial consumption network (esac-net), there are large inter-country variations in the use of broad-spectrum antimicrobials, indicating a need for increased antimicrobial stewardship and the potential for further antimicrobial consumption reductions (13). pandey et al drug target insights 2022; 16: 43 © 2022 the authors. published by aboutscience www.aboutscience.eu frequency of resistance to penicillin and erythromycin grew somewhat in all countries that consistently supplied antimicrobial susceptibility data, according to data from this surveillance project (13). enterococcus faecalis and enterococcus faecium there are grounds for concern that e. faecium is fast and constantly increasing in the percentage of vancomycin resistance in the eu. the ecdc study on amr’s health burden estimated that vancomycin-resistant enterococci (vre) infections and fatalities virtually doubled. a large issue for infection prevention and an important cause for dietary-related illnesses remain high levels of antimicrobial-resistant enterococci. in addition to being difficult to cure infections caused by resistant strains, enterococci are easily spread in medical settings (12). overview of the reported microorganisms resistant against the antimicrobials the above-mentioned subsequent organisms have been tried to be treated with multiple antimicrobials, which has not benefited healthcare. the initial treatment method implemented against these species was applying a single antimicrobial. later on due to nonobservance of the former antimicrobials, the healthcare sector switched to provide double antimicrobial treatment to the patients for more efficient results but to our surprise, the species were found to be successfully resistant against them. recently, a combination of antimicrobials is being applied to fight against the resistance that is acquired by the organisms but eventually extensive use of multiple antimicrobials has not only triggered amr globally but has also shown significant amounts of increase in mdr cases worldwide (tab. iv) (12-14). the number of deaths attributed to bacterial amr in 2019 has been estimated at 4.95 million based on previous research and several statistical methods. e. coli, s. aureus, k. pneumoniae, s. pneumoniae, acinetobacter baumannii, and p. aeruginosa are the top infections for mortality associated to resistance in 2019 (15). understanding the exact cost of resistance is a difficult task when trying to combat amr, especially in areas with little surveillance and scant data. high percentages of third-generation cephalosporin and carbapenem resistance in k. pneumoniae, as well as high percentages of carbapenem-resistant acinetobacter in various countries are of concern, according to a who/ecdc report from the year 2022. resistance to last-resort antibiotics like vancomycin and members of the carbapenem family is also strongly triggered. there are very few treatment choices available if these antibiotics stop working, and some of them may even be lethal if they don’t. the effectiveness of life-saving medical measures like cancer treatment and organ transplantation is likewise threatened by resistance to last-line antibiotics (16). the prevalence of mdr and xdr tuberculosis as well as resistance in gram-negative bacteria are india’s biggest worries. the community’s enterobacterales are producing extendedspectrum beta-lactamases at an alarming rate (17). one health approach and training programs regulating amr one health largely emphasizes the collaboration between human and animal health issues today, but also other disciplines should be merged, such as the environmental and social sciences. these one health training agreements are notably integrated more into veterinary schools than into medical training, as the review of one university training projects in western europe shows. moreover, multidisciplinary and global health research and training activities must be undertaken, as zoonotic illnesses and amr do not stop at national borders. increasing emergent human infectious diseases of zoonotic origin and microorganism resistance to antimicrobial medicinal products have demonstrated that there is a need for cooperation between the human, animal, and environmental sectors. increasingly, the one health concept is recognized by politicians and scientists all across the world. in this overview, research and training efforts have been assembled with the aim of focusing on infectious diseases in one health in western europe, particularly in france, spain, the netherlands, uk, germany, and norway. it can serve as a basis for future projects and partnerships. this summary indicates that one health in europe is widely recognized, as most recent educational activities are. in europe, the one health strategy in respect to amr is now being pushed. many nations have included the one health strategy in their anti-amr policy and there have been considerable increases in funding options for amr research. the number of multidisciplinary national and international research networks on zoonotic diseases and one health has grown. european institutes have researched on the topic of one health approach and many minor projects and training activities are being conducted in the countries of europe for spreading the awareness of the importance of one health approach to fight against amr and figure out a solution for it. tables 5 and 6 depict the information related to one health approach conducted or training activities performed in european countries (3,20). table iv bacterial species and the antimicrobial groups to which they are resistant (18,19) bacterial species resistant against antimicrobial groups geographical location escherichia coli resistant to beta-lactam antibiotics india, europe, usa, and taiwan staphylococcus aureus mrsa (methicillin-resistant staphylococcus aureus) india, europe, and usa klebsiella pneumoniae third-generation cephalosporin resistance, carbapenem resistance, aminoglycoside resistance, fluoroquinolone resistance india, europe pseudomonas aeruginosa carbapenem resistance, fluoroquinolone resistance, aminoglycoside resistance india, europe streptococcus pneumoniae resistant to macrolides india, europe acinetobacter species carbapenem resistance, aminoglycoside resistance, fluoroquinolone india, europe amr surveillance system mapping in countries44 © 2022 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti table v european institutes which researched on one health approach and also published article on this topic (3) country research institute topic france oie advocating the one health approach in general and in relation to rabies and rift valley fever germany freie universitate berlin publication on amr and zoonoses in the food chain such as vibrio and campylobacter united kingdom the royal veterinary college london school of hygiene and tropical medicine university of cambridge university of liverpool university of edinburgh research of amr advocating the one health concept and research of zoonoses and amr research on zoonotic diseases such as emerging zoonosis and neglected research on zoonosis such as japanese encephalitis virus and rabies norway norwegian veterinary institute eu’s horizon 2020 one health project the netherlands netherland centre for one health netherland centre for one health project spain center for veterinary health surveillance (visavet) project on one health table vi training activities conducted in the european counties on one health country institute type of training spain veterinary school of the universitat autonoma de barcelona masters on zoonoses and one health the netherlands utrecht university honours program one health, one health track united kingdom royal veterinary college (rvc)/ london school of hygiene & tropical medicine (lshtm), london royal (dick) school of veterinary sciences, edinburgh, university of bristol masters in one health honours program population medicine and one health france nantes-atlantic national college of veterinary medicine, food science and engineering, in partnership with the university of nantes’ department of medicine and the university of angers’ department of medicine master in animals in training various training activities for one health are conducted in the universities of europe. students have also paticipated n one health in recent years. some countries have one health student associations or public health veterinary organizations and networks, such as holland and the united kingdom. extension activities are part of several european research projects. an annual one health workshop and one health for next generation project is organized for instance in anticipating a global onset of novous epidemics (antigone) (3,21). one health approach regulating amr in india a national plan for controlling amr has been formed in india. the plan suggests targeting a number of critical components of amr in both the human sector and the nonhuman one, including agriculture, fishing, animal husbandry, and environment. the strategy addresses all the five main gap goals and provides a further goal of boosting india’s amr leadership. there are certain priorities that are being maintained to address all the issues. below are the main objectives of the plan: enhance awareness of amr through effective communication, training and education; enhance surveillance knowledge and evidence; reduce infection incidence by efficient infection, prevention, and control; optimize the use of antibiotics in all industries; promote amr investment, research, and innovation activities; enhance india’s amr leadership through international, national and sub-national collaborations on amr. the indian nap for amr is a well-designed global plan that incorporates all of the key gap goals and pledges to address critical antibiotic policy and regulatory problems within the “one health approach.” india’s national action plan (nap) for amr was released in april 2017 by the union ministry of health and family welfare. implementation was delayed but all parties needed a major push. failure to achieve separate funding remains the major hurdle to implement naps and/or state action plans, not just in india (21,22). the mapping of the surveillance system set up for amr in india is described in figure 7. one health approach regulating amr in taiwan taiwan’s centers for disease control (cdc) implemented the national antimicrobial stewardship program; established multi-channel monitoring of mdr organisms, hospital accreditation, and hospital infection control inspections related to antimicrobial stewardship; coordinated infection control interventions; and carried out antimicrobial control interventions in response to the growing threat posed by amr. taiwan cdc also proactively establishes relevant guidelines, e-learning materials, manual hygiene, and antimicrobial awareness campaigns to encourage everyone to reduce this condition. main objectives are: https://www.visavet.es/en/ https://www.visavet.es/en/ https://www.visavet.es/en/ pandey et al drug target insights 2022; 16: 45 © 2022 the authors. published by aboutscience www.aboutscience.eu enhanced surveillance and control of carbapenemresistant enterobacteriaceae of antimicrobial-resistant pathogens; accredit and control hospital infections; hospital inspections, the antimicrobial stewardship of all hospitals, are necessary or encouraged; offer a number of e-learning courses to improve the understanding and consciousness of health workers on antimicrobial stewardship; conduct national public and health awareness-raising campaigns; cooperate on the fight against amr with human and animal health sectors (23). thus, a comparative study has highlighted the fact that european countries as well as asian countries such as india and taiwan are equally contributing in building various agencies and organizations for combating amr by implementing various policies and many other surveillance systems, which has actively increased the implementation of one health approach. in addition, after european countries, taiwan has successfully accomplished many of their objectives which have helped the country in fighting against the amr. some of the above-mentioned strategies to prevent amr are broadly explained (figs. 8 and 9) (24). the mapping of the surveillance system set up for amr in taiwan is described further (fig. 8) (25). fig. 7 mapping of the surveillance system set up in india for controlling antimicrobial resistance (21,22) fig. 8 mapping of the surveillance system set up in taiwan for controlling antimicrobial resistance. amr surveillance system mapping in countries46 © 2022 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti discussion this article provides an overview of various surveillance systems that are formed only to keep a track on the upcoming situation of amr and the consumption of antimicrobials by humans as well as animals. the article does not provide all the details required to monitor the amr issue but firmly allows the readers to get acknowledged with the broad information about the amr across the six countries of europe along with a comparative study between taiwan and india. there are also a lot of debates on the themes of research covered by the term “one health.” any field of research, including anthropology, sociology, pedagogies, or comparative medicine, that may contribute to human, animal, or ecosystem health can be “one health.” failure to treat certain infections with currently available antibiotics is a concern for biomedicine. phage therapy as an alternative therapy against bacterial infections has been extensively investigated. although various challenges exist, bacteriophages treatment could be used in t h e future to replace antimicrobial agents with pathogens that are drug-resistant. the technique is now becoming popular as photographs are omnipresent, host-specific and harmless and can be administered with food orally. antibiotic protein in target bacteria is developed for the delivery of recombinant phages. topical treatment for open wounds or systemic infections may be performed intravenously. however, phage therapy gives rise to some serious concerns. the main thing about the host bacterium is its fine specificity. this prevents their use for acute infections as empirical therapy. the basis for their investigation was bacteriophageal lysins, the extremely specific peptidoglycan hydrolases, and was also referred to as enzybiotics. incorporated lysins represent a new therapy form that is powerful and readily available to fight amr as mdr diseases are becoming increasingly common threats (26,27). with the emerging crisis of amr, vaccine treatments are seen as a possible solution by health authorities, healthcare providers, and drug developers. the biomolecules that boost the host immune system and give immunity against infectious agents are immunotherapeutic. developments in the new technology of recombinant vaccines have been essential for reducing the use of different antibiotics for primary and secondary bacterial infection. one of the most important ways to prevent infections continues to be vaccines. increasing the internal immune system is the advantage of immunotherapeutic agents (24). the crispr case is a distinguishing adaptive immune feature in archaea and bacteria, which offers protection against invasively invading bacteriophages and provides a regularly cross-sectional breast repeat. short bacteriophages or plasmids known as spacers are inserted as a crispr array into the bacterial genome; the cas proteins use guide rnas from spacers to target the invading nucleic acid with the same sequence. phagemids from crisprcas9 could kill certain in vivo bacteria. crispr nanosized compounds can target the mec-a gene that is involved in the mrsa effectively (28). mesenchymal stem cells (mscs) have been intensively investigated for a variety of chronic diseases over several decades in order to develop a safe and promising therapeutical product. mscs show promising skills in promoting immunomodulation, tissue cure and excessive inflammation control. recently, human mscs have been shown to synthesize antimicrobial peptide (amp) factors that eradicate bacteria through several mechanisms including an inhibition of bacterial cell wall synthesis. nonbacterial effects of mscs (hucmscs) on drug-resistant clinical pathogens like e. coli, s. aureus, and k. pneumoniae have been detected (29). a number of names known as fecal microbiota transplantation are known as fecal bacteriotherapy. the fecal microbiota transplantation (fmt) process involves the transplantation, using various routes, including enema, nasogastric, nasoduodenal and colonoscopy, of a fecal suspension of commensal bacteria by a healthy individual donor into the intestinal lumen of the recipients. clinical trials have found an automotive fmt (afmt) in antibiotic-disrupted human patients that is better than probiotic therapy and that has induced a fast and almost complete recovery of gastrointestinal microbiota fig. 9 possible alternative strategies to prevent amr. pandey et al drug target insights 2022; 16: 47 © 2022 the authors. published by aboutscience www.aboutscience.eu (30). nanoparticulate materials may be used for the supply or may contain antimicrobial materials. the nanoparticles and antibiotics based on metal and metal oxides are seen as promising therapeutic candidates for future applications of biomedical science, because they have lower toxicity and improved antibacterial, antiviral, and cancer efficacy. they are of unique size, such as an increased volume-to-surface ratio, making them efficient medicine carriers and improving their solubility, compatibility, and ease of delivery (31). advancing genetic engineering and next-generation sequence have enabled scientists to develop future strategies, such as bioengineered probiotics or pharmabiotics, that can become a bacterial infection biotherapy or prophylaxis. an option against antibiotics may be bioengineered probiotics with diverse immunogenic properties. recombinant probiotics with high competence could provide a greater degree of site specificity than common drug administration regimes to produce drugs, therapeutic proteins, and gene therapy vectors (24). conclusion the regular data collected by different organizations play a vital role in monitoring the status of amr and antimicrobial usage by humans and livestock. these annual reports have highly helped the government to decide for alternatives and have focused in many training activities to combat the amr situation globally. amr prevention is linked to the one health concept. as antibiotic resistance genes persist on an interface between environment and animal health, an approach is required in all three areas that stresses the concept of “one approach to health.” finally, at any stage of life, antibiotic resistance can affect humans or animals. alternative therapies should be developed to reduce dependency on chemical therapy. as antibiotics become part of modern medicine before many decades, antibiotic effectiveness is decreasing. clinical research, microbiology, genetics and computer engineering, imaging and modeling experts should work together to develop strategies to deal with this problem and to develop new therapies. patients with normal infections should avoid unnecessary prescription and over-prescription of antibiotics and patients should be advised to follow good hygiene such as hand washing and adequate infection management measures. for the purpose of addressing the global epidemic of drug-resistant infections, an accurate evaluation of the existing and future burden of diseases caused by amr is crucial. to effectively combat an apparent rise in resistance infections, detailed and dynamic information is required. this information enables policymakers and healthcare professionals to put global amr action plans into place and allocate resources in an effective manner (32). the standard and accessibility of the supplied data affect how accurate amr results will be. the current state of the global surveillance system is unconnected and unsatisfactory (33). only 70 nations have reportedly signed up for the who’s global antimicrobial resistance surveillance system. the percentage is fewer than half of the amr rates reported (34). numerous restrictions, such as a lack of adequate global data, make it difficult to quantify amr reports. many countries lack the lab and data management capabilities needed to conduct efficient surveillance. most significantly, surveillance data or analysis cannot remedy the problem right away; they can only estimate the burden that will arise. only the creation of brand-new anti-pathogenic chemicals and herbal formulations can fix the problem. the exchange of data is also further constrained by many necessary and strict privacy concerns. more difficulties for the researchers are brought about by the lack of extensive and diverse datasets from various places, which is especially troubling in low-income countries where monitoring is essentially nonexistent. disclosures conflict of interest: the authors declare no conflict of interest. financial support: this research was supported by two industryacademia collaboration projects. vtr inc-cgu, r.o.c. project grant #scrpd1l0221 and doxabio-cgu, r.o.c. project grant #scrpd1k0131; it was additionally supported by cgu project grant #uzrpd1m0081. authors contribution: all authors contributed equally to this article. references 1. mesa varona o, chaintarli k, muller-pebody b, et al. monitoring antimicrobial resistance and drug usage in the human and livestock sector and foodborne antimicrobial resistance in six european countries. infect drug resist. 2020;13:957-993. crossref pubmed 2. ionescu gf, firoiu d, pîrvu r, et al. the potential for innovation and entrepreneurship in eu countries in the context of sustainable development. sustainability (basel). 2020;12(18):7250. crossref 3. sikkema r, koopmans m. one health training and research activities in western europe. infect ecol epidemiol. 2016;6(1): 33703. crossref pubmed 4. antibiotic use is rapidly increasing in developing countries. the lancet 2018; online. accessed august 2022. 5. the european union summary report on antimicrobial resistance in zoonotic and indicator bacteria from humans, animals and food in 2017/2018. 2020. online. accessed august 2022. 6. schrijver r, stijntjes m, rodríguez-baño j, tacconelli e, babu rajendran n, voss a. review of antimicrobial resistance surveillance programmes in livestock and meat in eu with focus on humans. clin microbiol infect. 2018;24(6):577-590. crossref pubmed 7. resapath. french surveillance network for antimicrobial resistance in bacteria from diseased animals. 2017 annual report. march 2019. online. accessed august 2022. 8. niph. norwegian institute of public health. antibiotic resistance in norway. 2014, updated 2018. online. accessed august 2022. 9. niph. norwegian institute of public health. norm og normvet: usage of antimicrobial agents and occurrence of antimicrobial resistance in norway. online. accessed august 2022. 10. national institute for public health and the environment. nethmap 2019. consumption of antimicrobial agents and antimicrobial resistance among medically important bacteria in the netherlands. online. accessed july, 2019. 11. publication office of the european union. 2013/652/eu: commission implementing decision of 12 november 2013 on the monitoring and reporting of antimicrobial resistance in zoonotic and commensal bacteria (notified under document https://doi.org/10.2147/idr.s237038 https://www.ncbi.nlm.nih.gov/pubmed/32308439 https://doi.org/10.3390/su12187250 https://doi.org/10.3402/iee.v6.33703 https://www.ncbi.nlm.nih.gov/pubmed/27906121 https://www.economist.com/graphic-detail/2018/04/02/antibiotic-use-is-rapidly-increasing-in-developing-countries https://www.efsa.europa.eu/en/efsajournal/pub/6007 https://doi.org/10.1016/j.cmi.2017.09.013 https://www.ncbi.nlm.nih.gov/pubmed/28970159 https://www.anses.fr/en/system/files/labo-ra-resapath2017en.pdf https://www.fhi.no/en/id/antimicrobial/ https://www.fhi.no/en/publ/2022/norm-og-norm-vet-usage-of-antimicrobial-agents-and-occurrence-of-antimicrob/ https://www.rivm.nl/bibliotheek/rapporten/2019-0038.pdf amr surveillance system mapping in countries48 © 2022 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti c(2013) 7145) text with eea relevance. online. accessed august 2022. 12. european centre for disease prevention and control. antimicrobial resistance in the eu/eea (ears-net). annual epidemiological report for 2019. online. accessed august 2022. 13. cassini a, högberg ld, plachouras d, et al; burden of amr collaborative group. attributable deaths and disabilityadjusted life-years caused by infections with antibiotic resistant bacteria in the eu and the european economic area in 2015: a population-level modelling analysis. lancet infect dis. 2019;19(1):56-66. crossref pubmed 14. who. 12th conference of hepa europe: “implementing healthenhancing physical activity research: from science to policy and practice”. online. accessed august 2022. 15. murray cjl, ikuta ks, sharara f, et al; antimicrobial resistance collaborators. global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. lancet. 2022;399(10325):629-655. crossref pubmed 16. online. accessed august 2022. 17. singhal t. antimicrobial resistance: the “other” pandemic!: based on 9th dr. i. c. verma excellence award for young pediatricians delivered as oration on 19th sept. 2021. indian j pediatr. 2022;89(6):600-606. crossref pubmed 18. wu d, ding y, yao k, gao w, wang y. antimicrobial resistance analysis of clinical escherichia coli isolates in neonatal ward. front pediatr. 2021;9:670470. crossref pubmed 19. chang s-m, chen j-w, tsai c-s, ko w-c, scaria j, wang j-l. antimicrobial-resistant escherichia coli distribution and whole-genome analysis of sequence type 131 escherichia coli isolates in public restrooms in taiwan. front microbiol. 2022;13:864209. crossref pubmed 20. health. ivsaisco. online. accessed august 2022. 21. singh s, charani e, devi s, et al. a road-map for addressing antimicrobial resistance in lowand middle-income countries: lessons learnt from the public private participation and codesigned antimicrobial stewardship programme in the state of kerala, india. antimicrob resist infect control. 2021;10(1):32. crossref pubmed 22. ranjalkar j, chandy sj. india’s national action plan for antimicrobial resistance – an overview of the context, status, and way ahead. j family med prim care. 2019;8(6):1828-1834. crossref pubmed 23. course. aoh. online. accessed august 2022. 24. kumar m, sarma dk, shubham s, et al. futuristic non antibiotic therapies to combat antibiotic resistance: a review. front microbiol. 2021;12:609459. crossref pubmed 25. online 26. wright rct, friman v-p, smith mcm, brockhurst ma. resistance evolution against phage combinations depends on the timing and order of exposure. mbio. 2019;10(5):1652-e19. crossref pubmed 27. vázquez r, garcía e, garcía p. phage lysins for fighting bacterial respiratory infections: a new generation of antimicrobials. front immunol. 2018;9:2252. crossref pubmed 28. pursey e, sünderhauf d, gaze wh, westra er, van houte s. crispr-cas antimicrobials: challenges and future prospects. plos pathog. 2018;14(6):e1006990. crossref pubmed 29. marx c, gardner s, harman rm, van de walle gr. the mesenchymal stromal cell secretome impairs methicillin-resistant staphylococcus aureus biofilms via cysteine protease activity in the equine model. stem cells transl med. 2020;9(7):746757. crossref pubmed 30. costello sp, hughes pa, waters o, et al. effect of fecal microbiota transplantation on 8-week remission in patients with ulcerative colitis: a randomized clinical trial. jama. 2019;321(2):156-164. crossref pubmed 31. vazquez-munoz r, lopez fd, lopez-ribot jl. bismuth nanoantibiotics display anticandidal activity and disrupt the biofilm and cell morphology of the emergent pathogenic yeast candida auris. antibiotics (basel). 2020;9(8):e461. crossref pubmed 32. schnall j, rajkhowa a, ikuta k, rao p, moore ce. surveillance and monitoring of antimicrobial resistance: limitations and lessons from the gram project. bmc med. 2019;17(1):176. crossref pubmed 33. hay si, rao pc, dolecek c, et al. measuring and mapping the global burden of antimicrobial resistance. bmc med. 2018;16(1):78. crossref pubmed 34. world health organization (who) global antimicrobial resistance surveillance system. (glass) report: early implementation 2017–2018. online. accessed august 2022. https://op.europa.eu/en/publication-detail/-/publication/83e1934f-4d39-11e3-ae03-01aa75ed71a1 https://www.ecdc.europa.eu/sites/default/files/documents/surveillance-antimicrobial-resistance-europe-2019.pdf https://doi.org/10.1016/s1473-3099(18)30605-4 https://www.ncbi.nlm.nih.gov/pubmed/30409683 https://www.euro.who.int/en/health-topics/disease-prevention/antimicrobial-resistance/surveillance/central-asian-and-european-surveillance-of-antimicrobial-resistance-caesar https://doi.org/10.1016/s0140-6736(21)02724-0 https://www.ncbi.nlm.nih.gov/pubmed/35065702 https://www.ecdc.europa.eu/en/news-events/who-and-ecdc-report-antimicrobial-resistance-remains-health-threat-europe https://doi.org/10.1007/s12098-021-04008-9 https://www.ncbi.nlm.nih.gov/pubmed/35064528 https://doi.org/10.3389/fped.2021.670470 https://www.ncbi.nlm.nih.gov/pubmed/34113589 https://doi.org/10.3389/fmicb.2022.864209 https://www.ncbi.nlm.nih.gov/pubmed/35495726 http://www.ivsa.org/standing-committees/one-health-2/scoh-projects/ https://doi.org/10.1186/s13756-020-00873-9 https://www.ncbi.nlm.nih.gov/pubmed/33573697 https://doi.org/10.4103/jfmpc.jfmpc_275_19 https://www.ncbi.nlm.nih.gov/pubmed/31334140 http://antigonefp7.eu/young-antigone-2/2016-antigone-annual-one-health-course/ https://doi.org/10.3389/fmicb.2021.609459 https://www.ncbi.nlm.nih.gov/pubmed/33574807 https://www.cdc.gov.tw/en/category/listcontent/_p6iyuu810pmdu2fctpp4g?uaid=bkm8mcw654j8je0c1u4eew https://doi.org/10.1128/mbio.01652-19 https://www.ncbi.nlm.nih.gov/pubmed/31551330 https://doi.org/10.3389/fimmu.2018.02252 https://www.ncbi.nlm.nih.gov/pubmed/30459750 https://doi.org/10.1371/journal.ppat.1006990 https://www.ncbi.nlm.nih.gov/pubmed/29902258 https://doi.org/10.1002/sctm.19-0333 https://www.ncbi.nlm.nih.gov/pubmed/32216094 https://doi.org/10.1001/jama.2018.20046 https://www.ncbi.nlm.nih.gov/pubmed/30644982 https://doi.org/10.3390/antibiotics9080461 https://www.ncbi.nlm.nih.gov/pubmed/32751405 https://doi.org/10.1186/s12916-019-1412-8 https://www.ncbi.nlm.nih.gov/pubmed/31537199 https://doi.org/10.1186/s12916-018-1073-z https://www.ncbi.nlm.nih.gov/pubmed/29860943 https://www.who.int/publications/i/item/9789241515061 dti drug target insights 2022; 16: 25-35issn 1177-3928 | doi: 10.33393/dti.2022.2476review drug target insights issn 1177-3928 www.aboutscience.eu/dti © 2022 the authors. this article is published by aboutscience and licensed under creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). commercial use is not permitted and is subject to publisher’s permissions. full information is available at www.aboutscience.eu focus on antimicrobial resistance (amr) understanding the environmental drivers of clinical azole resistance in aspergillus species pooja sen1, mukund vijay1, shweta singh2, saif hameed2, pooja vijayaraghavan1 1antimycotic and drug susceptibility laboratory, amity institute of biotechnology, amity university uttar pradesh, noida, uttar pradesh india 2amity institute of biotechnology, amity university haryana, gurugram (manesar) india abstract aspergilli are ubiquitous fungal pathogens associated with severe life-threatening infections, especially in immunocompromised patients. azoles are the first line of defence in the fight against most aspergillus-related infections. however, resistance to these therapeutic compounds has developed, which is mainly due to the existence of mutations in lanosterol 14 alpha-demethylase (cyp51a), a crucial enzyme in the pathway that produces ergosterol and is the target of azole antifungals. azole-based antifungal medications are ineffective because of infections brought on by azole-resistant aspergillus species, leading to a high fatality rate. however, resistant aspergillus isolates have also been isolated from azole-naïve patients. global agricultural practices promote the use of azole fungicides to protect crops from phytopathogens. usage of azole fungicides on a large scale has been linked to the development of resistance among aspergillus species prevalent in the environment. the infections caused by these azoleresistant aspergillus species cannot be treated by the available azole drugs, in turn leading to high morbidity and mortality rates. thus, knowledge of the environmental drivers and comprehending the genetic basis of fungal drug resistance evolution is pertinent, considering increasing numbers of patients with covid-19 infections who are sensitive to opportunistic fungal infections. this article emphasises the prevalence and underlying mechanisms of azole resistance in aspergillus species, with a focus on environmental triggers and resistance development. it also highlights the need for regular surveillance of pesticide use in agriculture, detection of triazole-resistant aspergillus species in environmental and clinical settings and development of new antifungal drugs. keywords: aspergillus, azole resistance, biofilm, cyp51a gene, environmental origin, triazoles received: july 27, 2022 accepted: october 24, 2022 published online: november 22, 2022 corresponding authors: saif hameed amity institute of biotechnology amity university haryana gurugram (manesar)-122413 india shameed@ggn.amity.edu pooja vijayaraghavan amity institute of biotechnology amity university uttar pradesh noida india vrpooja@amity.edu resistance to oxidative damage and the capacity to create proteolytic or even immunosuppressive enzymes are among aspergillus’ biological characteristics which permits growth at body temperature (1,2). aspergillus species reported as human pathogens are aspergillus fumigatus, aspergillus niger, aspergillus flavus and aspergillus nidulans. bronchitis, invasive aspergillosis (ia), and chronic pulmonary aspergillosis (cpa) are all signs of aspergillus infection. in addition, severe asthma with fungal hypersensitivity and allergic bronchopulmonary aspergillosis (abpa) are allergic symptoms of inhaled aspergillus (3). current antifungal therapy for aspergillosis falls into three main categories: polyenes, echinocandins, and azoles. of these, azoles are the drug of choice for treating aspergillus infection. in addition, they are the only orally available antifungal agents for therapy and are essential for long-term treatment (3). a. fumigatus has developed azole resistance over the past few decades because of long-term azole therapy for aspergillosis in the clinical environment (4). furthermore, azole-resistant isolates of aspergillus detected in azole-naïve (5) individuals indicate that there may be a second route for the establishment of resistance through a. fumigatus azole fungicide exposure in agro ecosystems. introduction the saprophytic genus aspergillus is universal in the environment that releases large numbers of conidiospores. after inhalation, they either reach the terminal airways or settle in large groups in the upper ventilatory system and cause sensitisation. the small size of the spores, thermotolerance, https://doi.org/10.33393/dti.2022.2476 https://creativecommons.org/licenses/by-nc/4.0/legalcode mailto:shameed@ggn.amity.edu environmental drivers of aspergillus azole resistance26 © 2022 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti global prevalence of aspergillosis aspergillosis can be either chronic or acute. most commonly, aspergillosis diseases are cpa, ia and bronchitis (6). aspergillus species are responsible for >2,00,000 cases of ia annually, as shown in table i (7). table i global burden of aspergillosis (8,9) fungal disease global burden comments asthmatic allergic bronchopulmonary aspergillosis ~4,800,000 adults only, rare in children cystic fibrosis–related allergic bronchopulmonary aspergillosis ~6675 adults only, starts from age 4 invasive aspergillosis ~3,00,000 about 10 million at risk annually chronic pulmonary aspergillosis ~3,000,000 in immunocompromised individuals, such as those with severe neutropenia, individuals who had bone marrow transplant or solid organ transplants, those with advanced acquired immunodeficiency syndrome or with chronic granulomatous illness, ia can develop (10). invasion of the lungs by aspergillus leads to tissue damage leading to sepsis and sometimes haemoptysis in later stages (11). in addition to these classic risk factors for ia, liver cirrhosis, tuberculosis, diabetes mellitus and persistent lung disease can also develop (12-15). cpa is an infectious disease that progressively damages lung tissue. this is especially true for immunocompromised people with a previous or underlying lung disease, such as tuberculosis or chronic obstructive pulmonary disease (copd). awareness of this debilitating and ultimately deadly infection is growing. it is tentatively estimated that there are 3 million cpa (chronic pulmonary aspergillosis) patients worldwide (6). among asian countries, the highest cpa burden was recorded in india (209,147) (15), followed by pakistan (55,509) (16), bangladesh (20,720) (17), nepal (6,611) (18) and sri lanka (2,886) (19). patients with abpa frequently experience uncontrolled asthma or repeated infections brought on by bronchiectasis, which progresses to lung damage, respiratory failure, and ultimately death. abpa contamination in the indian subcontinent is summarised in table ii (9,21). antifungal agents to treat aspergillosis in general, there are three different types of antifungal medications used to treat aspergillus-related illnesses: polyenes (amphotericin b), azoles (itraconazole, voriconazole and posaconazole) and echinocandins (caspofungin). polyenes polyenes are large macrolide structures with amphipathic nature (fig. 1). the oldest class of antifungal drugs are the polyenes, which include nystatin, amphotericin b and pimaricin. of these, amphotericin b is the only drug used to treat systemic infections. polyenes promote channelling in fungal membranes by interacting with sterols in cell membranes (ergosterol in fungal cells), which leads to changing the permeability of the membrane leading to the leaking of intracellular components. the major antifungal medication used to treat severe aspergillus infections is amphotericin b. however, it has detrimental side effects including fever, chills, hypotension, tachypnoea, as well as renal toxicity such as renal ischaemia, hypokalaemia, tubular acidosis and reduced erythropoiesis in the kidney (10). fig. 1 structure of polyene antifungal compounds. table ii burden of aspergillosis in the indian subcontinent country chronic pulmonary aspergillosis invasive aspergillosis allergic bronchopulmonary aspergillosis burden rate/100,000 burden rate/100,000 burden rate/100,000 india (15) 209,147 24 – – 1,380,000 114 pakistan (16) 55,509 70 10,949 5.9 94,358 51 bangladesh (17) 20,720 41 5166 5.1 90,262 56 nepal (18) 6,611 24.2 1119 4.0 9546 35 sri lanka (19) 2,886 14.4 229 1.1 10,344 49 sen et al drug target insights 2022; 16: 27 © 2022 the authors. published by aboutscience www.aboutscience.eu azoles the primary treatment includes azoles used to treat aspergillosis (20). this class of antifungals includes medicines with an azole ring (fig. 2) that stops a variety of fungi from growing (22). clotrimazole, econazole, ketoconazole, miconazole, and tioconazole are examples of two-nitrogen-atom imidazole. triazoles, in contrast, have three nitrogen atoms in the azole ring (fluconazole, itraconazole, posaconazole and voriconazole). the antifungal effects of azole compounds were first described in 1944 (23). the first compound was imidazole, followed by triazole. the first azole antifungal that could be applied topically in a therapeutic setting was chlormidazole in 1958. other imidazole-azole antifungals such as clotrimazole and miconazole were thereafter made available for topical application, and econazole was released in 1974 (24). the first drug was ketoconazole, an oral medication for systemic fungal infections, albeit its usage was constrained by its toxicity (25,26). fluconazole and itraconazole, the two most used systemic triazoles, were first made available in the united states in 1990. fluconazole and itraconazole have sufficient antifungal activity and are far less harmful than ketoconazole (27). itraconazole and fluconazole are less toxic than ketoconazole and have adequate antifungal action (27). voriconazole and posaconazole were the second-generation triazoles to be made available in the late 1990s and early 2000s. they have proven to be incredibly powerful against a. fumigatus (28) and are being utilised to manage fluconazole-resistant strains (29). triazoles under investigation include albaconazole (30), isavuconazole and pramiconazole (31). azoles work by blocking the cytochrome p-450dependent enzyme lanosterol demethylase, also known as 14α-sterol demethylase or p-450 dm , which is necessary for the formation of ergosterol, a crucial component of fungal plasma membranes (32). exposure to azoles in a. fumigatus decreases ergosterol levels and accumulates 14α-methylated sterols (33). this leads to a change in the structure and shape of the membrane, affects nutrient uptake and chitin synthesis, and inhibits fungal growth (34,35). on fungal cells, ergosterol also exhibits hormonelike characteristics that promote growth and reproduction (34). this function can also be impaired if the breakdown of ergosterol is more than 99% complete (34). triazole is indispensable for long-term treatment, because of the fact that it is the only anti-aspergillus drug that can be taken orally (36). although itraconazole is still frequently used to treat the long-established non-invasive allergic type of aspergillosis (38), voriconazole is still advised as the first-line treatment for ai (34,35,37). amphotericin b, a more lethal medication than triazoles, is the only available alternative. echinocandins the most recent addition to the family of antifungal drugs are the echinocandins, with the first example, caspofungin, entering clinical use a decade ago (39). they are lipopeptide compounds that inhibit the enzyme (1,3) betad-glucan synthase, which produces glucan, the primary building block of fungal cell walls. caspofungin (fig. 3) is non-competitively effective against a. fumigatus, a. flavus and a. terreus. it is indicated for aspergillus infections among those patients unresponsive or intolerant to other treatments. however, it has not been considered for first-line treatment (39). fig. 2 structure of azole antifungals (imidazoles; two nitrogen in the azole ring and triazoles; three nitrogen in the azole ring). environmental drivers of aspergillus azole resistance28 © 2022 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti fig. 3 structure of caspofungin antifungal. triazoles in the agricultural ecosystem plants are attacked by various pathogenic fungi that cause a variety of disease such as late blight, powdery mildew, leaf spot, blast, downy mildew, fruit rot, rust, etc. throughout the world, the usage of synthetic insecticides and pesticides is quite common to overcome crop diseases and failure. however, these can have a significant negative impact on the environment (40). thousands of tonnes of azoles are sold every year for crop protection. the advantages of azole pesticides include low cost and a diverse range of antifungal activity (41). hexaconazole, propiconazole, triadimefon and tricyclazole are some of the prominent triazole fungicides available in the international market (42). the long-term stability of azoles is a crucial characteristic. with very slight modifications to their chemical composition, several azoles can continue to function for months in agricultural habitats (soil and water). singh and dureja (43) reported that hexaconazole can remain in soil for a long time due to its hydrophobic properties. azoles are effective against several plant fungal diseases listed in table ii. the major metabolite of triadimefon, triadimenol, has a half-life in soil that varies from 110 to 375 days. the plants are sprayed several times per growing season at a dose of 100 g/ha, which is recommended to combat fungal diseases (41). there are currently 32 commercially available azole fungicides for crop protection (44). several foods have been found to contain azole residues, for example, in samples of commercially available strawberries, grapes or mint. therefore, there is evidence that large amounts of antifungal residues, especially azoles, can remain in the environment. table iii azole fungicides used in market (42-44) azole fungicides crops diseases caused by aspergillus triadimefon wheat, pea, grapes, coffee, mango, chilies, soybean bunt of wheat, powdery mildew, rust, powdery mildew, coffee rust, rust bitertanol apple, groundnut, tea, wheat, groundnut scab, rust, tikka, blister blight, karnal bunt flusilazole grapes, apple, rice, chilies powdery mildew, scab, sheath blight hexaconazole apple, rice, groundnut, mango, soybean, tea blister blight, powdery mildew rust, scab, blast, sheath blight, tikka leaf spot tebuconazole wheat, groundnut, chili, rice blast sheath blight, loose smut, flag smut, collar rot, root rot, stem rot, fruit rot, powdery mildew difenoconazole apple, groundnut, rice, chili, cumin, onion fruit rot, blight powdery mildew, sheath blight, scab, leaf spot rust, purple blotch tricyclazole paddy blast resistance to azoles developing in aspergillus clinical isolates recent years have seen a rise in the concern of azole therapy resistance in patients with aspergillus infections. a. fumigatus, which causes around 80% of invasive infections, has been found to possess the highest azole resistance (45); azole resistance has also been demonstrated in other species including a. niger, a. terreus and a. flavus (46). infections with resistant aspergillus strains lead to the effectiveness of azole antifungals, resulting in high mortality rates. azole-resistant strains of aspergillus were reported in the united states in the late 1990s (47). there have been numerous reports of infections with resistant strains in europe, particularly in the uk and the netherlands. since that time, practically every european nation has reported cases of azole resistance, including germany, ireland, italy, austria, denmark, france, sweden, portugal, spain and turkey (25,48-64). a surveillance study from the uk reported that the prevalence of azole resistance in a. fumigatus increased from 0.43% in 1998-2011 to 2.2% in 2015-2017 (65). a multicentre study conducted in taiwan found a 4% prevalence rate for a. fumigatus resistant to azoles (66). among asian countries, taiwan and china were the first to report resistance to azoles (67,68). furthermore, this study has sparked significant worries about the use of azole antifungal medications to treat ia in the future. the increase in resistance to triazoles in the clinical setting can be explained by two main phenomena: (a) an azole-resistant aspergillus strain was found after long-term treatment with azoles in patients with cavernous lung disease and aspergilloma. when susceptible aspergillus strains sen et al drug target insights 2022; 16: 29 © 2022 the authors. published by aboutscience www.aboutscience.eu acquire resistance to the pharmacological stress response of prolonged azole therapy, they develop resistance. numerous point mutations were found in the azole-resistant a. fumigatus isolate, especially at codons 54 and 220 of cyp51a (69). several resistance mechanisms associated with cyp51a have been identified in patients specifically associated with azole therapy (70). human-to-human transmission is therefore highly unlikely and spread of resistance is very rare. (b) azole fungicides are frequently employed to protect crops in agricultural settings and are structurally like medicinal triazoles. aspergillus species are found in soil together with other plant pathogens. azoles that attack plant pathogens can also affect aspergillus species found in the same ecosystem (3). fungicides used repeatedly over a long period of time can create persistent selection pressure and lead to the development of resistant aspergillus species. as a result, the environment contains aspergillus species that are azoleresistant. when these conidia are inhaled by susceptible individuals, aspergillus species become resistant to triazoles used for treatment. several cases of triazole-resistant aspergillosis in humans and animals without prior triazole treatment have been reported worldwide (71,72). resistance to triazoles in aspergillus clinical isolates is associated with the use of azole fungicides in agriculture many major agricultural fungi have developed resistance because of the site-specific mode of action and widespread application of 14α-demethylase inhibitor (dmi) fungicides to prevent post-harvest spoiling by plant-pathogenic fungus. azole overuse in agriculture may have an impact on saprophytic microbiota species as well as plant-pathogenic fungi (73). the soil provides a natural habitat for several fungi that could be harmful, including aspergillus, coccidioides, histoplasma and cryptococcus. recently, the use of azole pesticides has been identified as a significant contributing cause in the increasing prevalence of a. fumigatus isolates with a particular mechanism of resistance comprising the tr34/l98h mutation in the cyp51a gene. snelders et al.’s (74) findings that were obtained from both clinical and environmental sources exhibited crossresistance to five triazole-dmi fungicides, notably bromuconazole, propiconazole, epoxiconazole, tebuconazole and difenoconazole, support the notion that aspergillus species become resistant to triazoles as a result of environmental use of azole fungicide (74). additionally, these researchers noted that all these five dmi-triazoles have efficacy against wild-type a. fumigatus but not against the resistant tr34/ l98h a. fumigatus because of their molecular structure, which is similar to drug triazoles and when attached to the target enzyme, acquire a same conformation (74). in a related study from india, four of the five triazole dmis – bromuconazole, tebuconazole, epoxiconazole and difenoconazole – showed substantially higher mic (minimum inhibitory concentration) values with tr34/l98h-resistant a. fumigatus from environmental and clinical samples than with wild-type non-resistant isolates. these drugs are known to have performance comparable molecular structures to drug triazoles (75). in 2020, a study from india found that patients who had never taken triazole therapy had developed resistance, raising the possibility that environmental transmission may contribute to the emergence of resistance (5). most of the resistance mechanisms found in patients without prior treatment with azole are resistance mechanisms associated with tr (tandem repeats). most aspergillus isolates from the environment also possessed this resistance mechanism (tr34/l98h or tr46/y121f/t289a). norway, the netherlands, denmark, the united kingdom and india are some of the countries where all these resistant environmental isolates have been found (76). thus, retrieval of a similar resistance mechanism from environmental isolates as patient isolates supported an environmental pathway for resistance development. environmental-induced mutations in azole-resistant aspergillus isolates ergosterol is a crucial and distinct element of the fungal plasma membrane that gives the cell membrane stability and permeability. the enzyme cytochrome p450, also called sterol-14α-demethylase, converts lanosterol into ergosterol. cyp51a is a gene that codes for the cytochrome p450 enzyme. the ergosterol biosynthetic pathway is the general target of azole antifungals. triazoles hinder the cytochrome p450 enzyme from performing its lanosterol-converting role in the ergosterol biosynthetic pathway, and causes the depletion of ergosterol and the deleterious build-up of lanosterol (74). azole resistance is brought on by mutations in the cyp51a gene that change the cyp51a protein’s structure and reduce the enzymes’ affinity for azole therapies. various changes in cyp51a resulting in a pan-azole resistant phenotype have been reported in a. fumigatus isolates from environment and clinical sources worldwide. the most frequent mechanisms of resistance reported in environmental and clinical strains of a. fumigatus are mutations in the tr34/l98h and tr46/y121f/t289a genes. these changes in the cyp51a gene comprise trs within the gene’s promoter region (77). the cyp51a gene is overexpressed because of the insertion of 34 base pair (bp) trs (tr34) into the cyp51a gene’s promoter region and the substitution of the leucine 98 amino acid coding for histidine (tr34/l98h) (78). according to table iv (77), tr34/l98h is the most typical resistance mechanism found in environmental and clinical strains of a. fumigatus in many different nations. another resistance mechanism which has been proven is tr46/y121f/t289a (fig. 4) where a 46 bp tr promoter region of the cyp51a gene has substitutions of threonine 289 for alanine and tyrosine 121 for phenylalanine (tr46/y121f/t289a), resulting in an elevated resistance to voriconazole in a. fumigatus (79). one of the most frequently detected mechanisms of resistance in environmental isolates from europe are tr34/l98h and tr46/y121f/t289a, and their emergence has already been connected to the widespread use of azole-based agricultural fungicides (tebuconazole, hexaconazole and epoxiconazole). fungicide use is rising in india, where it already accounts for 19% of all pesticide use (80). even though europe is the worldwide leader in agricultural fungicide use (40%), it is followed by japan and latin america. in contrast to europe, the united environmental drivers of aspergillus azole resistance30 © 2022 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti states uses very less azoles in agriculture (http://ec.europa. eu/food/fs/se/ssc/out278_en.pdf). therefore, no tr34/l98h mutation has been reported in environmental clinical isolates in the united states, but this mutation has been found in the european setting and now also in india (77). an environmental study conducted examined a variety of soil and air samples from different regions of india. in their study, soil samples were taken from natural soils (where no azole fungicides were used). the samples tested positive for aspergillus strains, but no resistant isolates were detected. their findings were supported with those of the dutch environmental study, which discovered that none of the a. fumigatus isolates cultured in natural soil exhibited azole resistance (81). therefore, commercialised compost and samples taken from fields where fungicides are consistently treated can be the focus of environmental research to find tr34/l98h mutations in a. fumigatus isolates. in environmental samples from china (82), certain novel mutations (g448s, tr46/y121f/t289a) with 46 bp triple trs in the promoter region have been discovered. other asian nations like india, iran and kuwait have demonstrated azole resistance in environmental aspergillus strains (83,84). about 2% of aspergillus species in kuwait were found to be azole resistant, according to another study on environmental resistance (84). indian researchers have reported that environmental studies on azole resistance describe the tr46/ y121f/t289a mechanism (85), and the findings indicated that 44 out of 630 a. fumigatus isolates from soil of indoor air, paddy fields, tea gardens, cotton groves, flowerpots and hospitals were resistant and managed to retain tr34/l98h resistance (75). an environmental mechanism of resistance (tr46/y121f/1289a) in strains of a. fumigatus was also first reported in this study. in 2009, a similar mutation was discovered in a dutch patient and has also been reported in several patients from the netherlands (86). this survey, which took place between 2012 and 2013, studied 105 environmental samples taken from north indian agricultural fields. the study concluded that azole fungicide-treated agricultural soils in northern india co-occurred with tr34/l98h and tr46/y121f/t289a. the identified indian aspergillus strains were likely to be highly adaptable recombinant descendants of a cross between a native azole-sensitive strain from within india and azole-resistant strain that migrated from outside india, followed by a mutation, according to genomic analysis of the indian resistance mechanism tr34/l98h (75). reports indicated a rapid spread of this mutation in asia (83). table iv common resistance mechanisms reported in the cyp51a gene of environmental a. fumigatus geographic region/ references sample cyp51a resistance mechanisms the netherlands (81) soil unknown, f46y/ m172v/e427k france (87) dust from patients’ home h285y germany (88) soil g54a, m220i india (89) soil g54e taiwan (90) soil, air wild-type cyp51a or snps france (91) soil unknown, p216l colombia (92) soil tr46/y121f/t289a, tr34/l98h and tr53 india (77) environment tr34/l98h india (93) azole-naïve patient g54e india (5) azole-naïve patient g54r, p216l and y431c snp = single nucleotide polymorphism. multiple triazole-resistant a. fumigatus isolates with the tr/l98h genotype were found in patients with chronic respiratory disease, according to a different study that was carried out for the first time in india (75). only europe and fig. 4 various azole resistance mechanisms in aspergillus fumigatus (a) wild-type fungi in the presence of azole drug unable to make ergosterol. (b) mutations in the cyp51a region alter the structural modifications of the enzyme leading to reduce azole affinity. (c) insertion of 34 and 46 base pair in the promoter region along with point mutation in the cyp51a region causes overexpression of the gene. (d) overexpression of efflux pump genes causing a reduced intracellular accumulation of azole drug. sen et al drug target insights 2022; 16: 31 © 2022 the authors. published by aboutscience www.aboutscience.eu china have been described as having the tr/l98h mutation linked to pan-azole resistance in a. fumigatus (70,81). the two triazole-resistant a. fumigatus isolates are epidemiologically unrelated, share the same tr genotypes, and come from patients with no prior history of exposure to azoles or travel to europe, suggesting that they most likely mutated and developed resistance as a result of exposure to the environment in india. the two isolates were phylogenetically distinct from tr/l98h, which contained the 25 a. fumigatus isolates from dutch. the usage of azole fungicides in the environment may be a contributing factor in the propagation of this resistance mechanism (tr/l98h) in a. fumigatus isolates. a total of 43.7% of aspergillus isolates found in 25 agricultural soil samples were found to be resistant to azoles, according to a recent environmental study in india (94). other non-synonymous hotspot mutations in the cyp51a gene have also been discovered in azole-resistant a. fumigatus strains, in addition to the tr34/l98h and tr46/y121f/ t289a alterations. while resistance to itc (itraconazole) and pos (posaconazole) was provided by the glycine modification mutations 54 (g54) and 138 (g138), lower susceptibility to itc and pos related to the glycine 448 (g448s) (95) mutation-related resistance to vrc. methionine 220 (m220) amino acid substitution was also linked to a pronounced pattern of decreased sensitivity to triazoles (70). there have also been sporadic reports of other point mutations, including p216l, f219c, f219i, a284t, y431c, g432s and g434c (86). patients who received around 4 months (range 3 weeks to 23 months) of long-term azole treatment for persistent aspergillosis have been discovered to have the point mutations g54e/r/v and m220i/v/t/k (96). it is important to mention here that studies conducted in india, tanzania, romania and germany found g54 mutations in environmental isolates of a. fumigatus (97). an environmental study in india found an azole-resistant aspergillus species with a g54e mutation (89). this point mutation in the cyp51a gene is commonly seen in patients undergoing long-term azole therapy (89). in another study in india, a. fumigatus isolates with g54r, p216l and y431c mutations were obtained from azole corpus patients (4). the mics of many additional point mutations, including f46y, m172v, n248t, d255e and e427k, have been discovered in azole-susceptible and azole-resistant aspergillus isolates. however, this is not always restricted to clinical breaking point (75). the non-cyp51a pathway has also been linked to azole resistance in a. fumigatus. in isolates of a. fumigatus (98), voriconazole was also used to treat the link between biofilm growth and efflux pump activity to regulate homeostasis in azole resistance. additionally, aspergillus species can effectively invade and colonise the host by activating efflux pumps, specifically adenosine triphosphate (atp)binding cassette transporters and carriers of the major facilitator superfamily, to overcome the build-up of intercellular toxins (78). spread of aspergillus from the environment to hospitals aspergillus spp. is ubiquitous in the environment and cosmopolitan in nature. the main habitat of aspergillus spp. is the soil, and this saprophytic fungus has a vegetative mycelial life that develops on the decomposing matter, whether organic or vegetable, found in the soil (99). previous studies showed that the metabolic machinery in aspergillus spp. contains certain enzymes such as endo-β-glucanase, acetylxylan esterase, polygalacturonase, tannase, etc., which can easily degrade components of the plant cell wall. on the other hand, it does not contain any enzymes that can decompose plant wood (100). spread of conidia occurs by asexual sporulation, and there is airborne spread of asexual reproductive organs, or conidia. conidia mainly predominate in the air and are inhaled by individuals. it is estimated that 200 conidia are inhaled per person. however, they are stripped of pulmonary macrophages and neutrophils present in the lungs of immunocompetent humans. the clinical manifestations of aspergillus depend on the host’s immune status. they cause severe infections in immunocompromised patients with other predisposing factors and develop life-threatening aspergillosis (101). a case study examined fatal ia and found that the source of aspergillus infection was the patient’s home, which was in an agricultural area with potentially high pressure of fungicides used to protect crops. even after the patient died, household samples showed the persistence of azole-resistant strains of aspergillus spp. (63). another study tested the source of azole-resistant aspergillus spp. in a hospital environment. their samples were taken from different environments in the hospital. the assessment showed the main source of aspergillus spp. in the hospital and in the corridors (102), where the floor was decorated with tulip pots. this indicates the easy transmission of environmental aspergillus strains to hospitals and infecting patients who were mainly in immunocompromised states or in persistent drug states. therefore, it is important to identify sources of infection, whether the patient is hospitalised or a source of in-hospital contamination, due to the potential for aerosol transmission from patient to patient (103). azole-resistant aspergillus biofilms aspergillus is an opportunistic airborne pathogen capable of forming biofilms in clinical settings or in immunocompromised patients with underlying conditions leading to allergic aspergillosis or ia (104). biofilms are a community of cells strongly adherent to abiotic and biotic surfaces and surrounded by an extracellular matrix (ecm) composed of polysaccharides. the ecm acts as a protective sheet and external scaffold for adhesion and integration with the surface, and cell spreading for subsequent invasion. this protective layer becomes more sensitive to antifungal drug treatments and attacks immune cells, making them harder to fight (105). possible factors contributing to drug resistance in aspergillus spp. biofilms are: upregulation of efflux pump genes such as afumdr4, mdr1, mdr2, mdr4; induction of the hsp90 stress response pathway, which increases resistance to the antifungal drugs amphotericin b and caspofungin; by extracellular dna which reduces drug sensitivity by preventing the drug from reaching its cellular target through ecm and sister cell formation while acting as drug-tolerant cells to form new biofilms (106). environmental drivers of aspergillus azole resistance32 © 2022 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti the most important factor in ipa (invasive pulmonary aspergillosis) and aspergilloma is biofilm formation. fungal components such as drug transporters, secondary metabolites and cell wall components promote biofilm formation in host cells and are resistant to antifungal drug treatment (107). biofilm formation, which helps to penetrate the host immune system and reduce the patient’s immune competence, also contributes to increased resistance to triazoles (108). a study showed that under in vitro conditions, a. fumigatus formed multicellular biofilms of polystyrene sheets that could resist the effects of antifungal drugs (109). another study also showed an effect of itraconazole on hyphal germination and biofilm formation at an early stage, but no effect on mature biofilms, suggesting a predominance of resistant biofilms (110). biofilms in the lungs are difficult to diagnose because they occur after mature biofilms form. in the adult stage, this tissue in the lungs develops into a more complex tissue with dense ecm and limited oxygen, which encourages further growth. this makes it increasingly difficult for immune cells to recognise and influence them. it also worsens when other microbial biofilms persist and are difficult to remove with antifungal drugs, particularly in cystic fibrosis (111). therefore, a comprehensive analysis and understanding of aspergillus biofilms is required to develop new and improved antifungal targets for the treatment of complex biofilmrelated diseases (107). future directions azole resistance in environmental aspergillus spp. is a matter of grave clinical concern as transfer of resistance from environment to clinical settings is inevitable. india needs to impose strict regulatory compliance to ensure regulated usage of pesticides in agricultural fields. further studies are warranted to understand the level of transfer of resistance from the field to clinic. this can be undertaken in a state-wise study by mapping the mutations that are unique to the region. additional studies focusing on the usage of azole fungicides and the presence of azole residues in developed environments are needed, because the amounts used or quantities present are not often measured or reported. more surveillance, accurate data collection and comprehensive resistance surveillance programmes in agricultural ecosystems are needed to study the magnitude of the emerging problem of azole resistance. it will be very important to identify tipping points to ensure the agronomic use of fungicides without jeopardising their treatment goals. research at the epidemiological level can uncover geographic differences in the emergence of resistance and help identify areas with high levels of resistance. further, to overcome antifungal resistance in clinical settings, development of new antifungals with new drug target site is needed. acknowledgements authors would like to thank amity university uttar pradesh for providing the infrastructure and facilities for research. disclosures conflict of interest: the authors declare no conflict of interest. financial support: authors would like to thank csir for providing financial support in the form of senior research fellowship (09/915(0013)/2018-emr-i) to ps. authors’ contributions: ps performed literature search and drafted the manuscript; mv and ss performed literature search; sh critically analysed the manuscript; pv conceptualised the idea of review writing and critically analysed the manuscript. references 1. patterson tf, kirkpatrick wr, white m, et al. invasive aspergillosis. disease spectrum, treatment practices, and outcomes. i3 aspergillus study group medicine (baltimore). 2000;79(4): 250-260. crossref pubmed 2. lin sj, schranz j, teutsch sm. aspergillosis case-fatality rate: systematic review of the literature. clin infect dis. 2001;32(3):358-366. crossref pubmed 3. verweij pe, chowdhary a, melchers wj, meis jf. azole resistance in a. fumigatus: can we retain the clinical use of moldactive antifungal azoles? clin infect dis. 2016;62(3):362-368. crossref pubmed 4. hagiwara d, watanabe a, kamei k, goldman gh. epidemiologi cal and genomic landscape of azole resistance mechanisms in aspergillus fungi. front microbiol. 2016;7:1382. crossref pubmed 5. dabas y, xess i, bakshi s, mahapatra m, seth r. emergence of azole-resistant aspergillus fumigatus from immunocompromised hosts in india. antimicrob agents chemother. 2018;62(8):e02264-e17. crossref pubmed 6. denning dw, cadranel j, beigelman-aubry c, et al; european society for clinical microbiology and infectious diseases and european respiratory society. chronic pulmonary aspergillosis: rationale and clinical guidelines for diagnosis and management. eur respir j. 2016;47(1):45-68. crossref pubmed 7. denning dw, pleuvry a, cole dc. global burden of allergic bronchopulmonary aspergillosis with asthma and its complication chronic pulmonary aspergillosis in adults. med mycol. 2013;51(4):361-370. crossref pubmed 8. armstead j, morris j, denning dw. multi-country estimate of different manifestations of aspergillosis in cystic fibrosis. plos one. 2014;9(6):e98502. crossref pubmed 9. denning dw. the ambitious ‘95-95 by 2025’ roadmap for the diagnosis and management of fungal diseases. thorax. 2015;70(7):613-614. crossref pubmed 10. kosmidis c, denning dw. the clinical spectrum of pulmonary aspergillosis. thorax. 2015;70(3):270-277. crossref pubmed 11. mccarthy mw, walsh tj. special considerations for the diagnosis and treatment of invasive pulmonary aspergillosis. expert rev respir med. 2017;11(9):739-748. crossref pubmed 12. komase y, kunishima h, yamaguchi h, ikehara m, yamamoto t, shinagawa t. rapidly progressive invasive pulmonary aspergillosis in a diabetic man. j infect chemother. 2007;13(1):46-50. crossref pubmed 13. yan x, li m, jiang m, zou lq, luo f, jiang y. clinical characteristics of 45 patients with invasive pulmonary aspergillosis: retrospective analysis of 1711 lung cancer cases. cancer. 2009;115(21):5018-5025. crossref pubmed 14. prattes j, hoenigl m, krause r, et al. invasive aspergillosis in patients with underlying liver cirrhosis: a prospective cohort study. med mycol. 2017;55(8):803-812. crossref pubmed https://doi.org/10.1097/00005792-200007000-00006 https://www.ncbi.nlm.nih.gov/pubmed/10941354 https://doi.org/10.1086/318483 https://www.ncbi.nlm.nih.gov/pubmed/11170942 https://doi.org/10.1093/cid/civ885 https://www.ncbi.nlm.nih.gov/pubmed/26486705 https://doi.org/10.3389/fmicb.2016.01382 https://www.ncbi.nlm.nih.gov/pubmed/27708619 https://doi.org/10.1128/aac.02264-17 https://www.ncbi.nlm.nih.gov/pubmed/29891597 https://doi.org/10.1183/13993003.00583-2015 https://www.ncbi.nlm.nih.gov/pubmed/26699723 https://doi.org/10.3109/13693786.2012.738312 https://www.ncbi.nlm.nih.gov/pubmed/23210682 https://doi.org/10.1371/journal.pone.0098502 https://www.ncbi.nlm.nih.gov/pubmed/24914809 https://doi.org/10.1136/thoraxjnl-2015-207305 https://www.ncbi.nlm.nih.gov/pubmed/26024686 https://doi.org/10.1136/thoraxjnl-2014-206291 https://www.ncbi.nlm.nih.gov/pubmed/25354514 https://doi.org/10.1080/17476348.2017.1340835 https://www.ncbi.nlm.nih.gov/pubmed/28595486 https://doi.org/10.1007/s10156-006-0481-8 https://www.ncbi.nlm.nih.gov/pubmed/17334729 https://doi.org/10.1002/cncr.24559 https://www.ncbi.nlm.nih.gov/pubmed/19637340 https://doi.org/10.1093/mmy/myx011 https://www.ncbi.nlm.nih.gov/pubmed/28431001 sen et al drug target insights 2022; 16: 33 © 2022 the authors. published by aboutscience www.aboutscience.eu 15. agarwal r, denning dw, chakrabarti a. estimation of the burden of chronic and allergic pulmonary aspergillosis in india. plos one. 2014;9(12):e114745. crossref pubmed 16. jabeen k, farooqi j, mirza s, denning d, zafar a. serious fungal infections in pakistan. eur j clin microbiol infect dis. 2017;36(6):949-956. crossref pubmed 17. gugnani hc, denning dw, rahim r, sadat a, belal m, mahbub ms. burden of serious fungal infections in bangladesh. eur j clin microbiol infect dis. 2017;36(6):993-997. crossref pubmed 18. khwakhali us, denning dw. burden of serious fungal infections in nepal. mycoses. 2015;58(suppl 5):45-50. crossref pubmed 19. jayasekera pi, denning dw, perera pd, fernando a. the burden of serious fungal infections in sri lanka. mycoses. 2013;56:103 20. stevens da, moss rb, kurup vp, et al; participants in the cystic fibrosis foundation consensus conference. allergic bronchopulmonary aspergillosis in cystic fibrosis – state of the art: cystic fibrosis foundation consensus conference. clin infect dis. 2003;37(s3)(suppl 3):s225-s264. crossref pubmed 21. al-mobeireek af, al-hedaithy ss, alasali k, al-majed s, joharjy i, joharjy i; el-rab mogad. allergic bronchopulmonary mycosis in patients with asthma: period prevalence at a university hospital in saudi arabia. respir med. 2001;95(5):341-347. crossref pubmed 22. lass-flörl c. triazole antifungal agents in invasive fungal infections: a comparative review. drugs. 2011;71(18):2405-2419. crossref pubmed 23. russell pe. a century of fungicide evolution. j agric sci. 2005;143(1):11-25. crossref 24. gupta ak, sauder dn, shear nh. antifungal agents: an overview. part i. j am acad dermatol. 1994;30(5 pt 1):677-698. crossref pubmed 25. heeres j, backx lj, mostmans jh, van cutsem j. antimycotic imidazoles. part 4. synthesis and antifungal activity of ketoconazole, a new potent orally active broad-spectrum antifungal agent. j med chem. 1979;22(8):1003-1005. crossref pubmed 26. johnson lb, kauffman ca. voriconazole: a new triazole antifungal agent. clin infect dis. 2003;36(5):630-637. crossref pubmed 27. maertens ja. history of the development of azole derivatives. clin microbiol infect. 2004;10(s1)(suppl 1):1-10. crossref pubmed 28. scott lj, simpson d. voriconazole: a review of its use in the management of invasive fungal infections. drugs. 2007;67(2):269298. crossref pubmed 29. gothard p, rogers tr. voriconazole for serious fungal infections. int j clin pract. 2004;58(1):74-80. crossref pubmed 30. pasqualotto ac, thiele ko, goldani lz. novel triazole antifungal drugs: focus on isavuconazole, ravuconazole and albaconazole. curr opin investig drugs. 2010;11(2):165-174. pubmed 31. allen d, wilson d, drew r, perfect j. azole antifungals: 35 years of invasive fungal infection management. expert rev anti infect ther. 2015;13(6):787-798. crossref pubmed 32. snelders e, karawajczyk a, schaftenaar g, verweij pe, melchers wj. azole resistance profile of amino acid changes in aspergillus fumigatus cyp51a based on protein homology modeling. antimicrob agents chemother. 2010;54(6):24252430. crossref pubmed 33. georgopapadakou nh, walsh tj. antifungal agents: chemotherapeutic targets and immunologic strategies. antimicrob agents chemother. 1996;40(2):279-291. crossref pubmed 34. white tc, marr ka, bowden ra. clinical, cellular, and molecular factors that contribute to antifungal drug resistance. clin microbiol rev. 1998;11(2):382-402. crossref pubmed 35. koltin y, hitchcock ca. the search for new triazole antifungal agents. curr opin chem biol. 1997;1(2):176-182. crossref pubmed 36. maschmeyer g, haas a, cornely oa. invasive aspergillosis: epidemiology, diagnosis and management in immunocompromised patients. drugs. 2007;67(11):1567-1601. crossref pubmed 37. walsh tj, anaissie ej, denning dw, et al; infectious diseases society of america. treatment of aspergillosis: clinical practice guidelines of the infectious diseases society of america. clin infect dis. 2008;46(3):327-360. crossref pubmed 38. howard sj, pasqualotto ac, denning dw. azole resistance in allergic bronchopulmonary aspergillosis and aspergillus bronchitis. clin microbiol infect. 2010;16(6):683-688. crossref pubmed 39. mukherjee pk, sheehan d, puzniak l, schlamm h, ghannoum ma. echinocandins: are they all the same? j chemother. 2011;23(6):319-325. crossref pubmed 40. azevedo mm, faria-ramos i, cruz lc, pina-vaz c, rodrigues ag. genesis of azole antifungal resistance from agriculture to clinical settings. j agric food chem. 2015;63(34):7463-7468. crossref pubmed 41. morton v, staub t. a short history of fungicides. apsnet features. doi: 10.1094/apsnet feature-2008-0308. online 42. price cl, parker je, warrilow ag, kelly de, kelly sl. azole fungicides – understanding resistance mechanisms in agricultural fungal pathogens. pest manag sci. 2015;71(8):1054-1058. crossref pubmed 43. singh n, dureja p. persistence of hexaconazole, a triazole fungicide in soils. j environ sci health b. 2000;35(5):549-558. crossref pubmed 44. frac code list. fungal control agents sorted by cross resistance pattern and mode of action. fungicide resistance action committee; 2019. 45. messer sa, jones rn, fritsche tr. international surveillance of candida spp. and aspergillus spp.: report from the sentry antimicrobial surveillance program (2003). j clin microbiol. 2006;44(5):1782-1787. crossref pubmed 46. gomez-lopez a, garcia-effron g, mellado e, monzon a, rodriguez-tudela jl, cuenca-estrella m. in vitro activities of three licensed antifungal agents against spanish clinical isolates of aspergillus spp. antimicrob agents chemother. 2003;47(10):3085-3088. crossref pubmed 47. lelièvre l, groh m, angebault c, maherault ac, didier e, bougnoux me. azole resistant aspergillus fumigatus: an emerging problem. med mal infect. 2013;43(4):139-145. crossref pubmed 48. mellado e, garcia-effron g, alcázar-fuoli l, cuenca-estrella m, rodriguez-tudela jl. substitutions at methionine 220 in the 14α-sterol demethylase (cyp51a) of aspergillus fumigatus are responsible for resistance in vitro to azole antifungal drugs. antimicrob agents chemother. 2004;48(7): 2747-2750. crossref pubmed 49. mellado e, garcia-effron g, alcázar-fuoli l, et al. a new aspergillus fumigatus resistance mechanism conferring in vitro cross-resistance to azole antifungals involves a combination of cyp51a alterations. antimicrob agents chemother. 2007;51(6):1897-1904. crossref pubmed 50. alanio a, sitterlé e, liance m, et al. low prevalence of resistance to azoles in aspergillus fumigatus in a french cohort of patients treated for haematological malignancies. j antimicrob chemother. 2011;66(2):371-374. crossref pubmed 51. mortensen kl, jensen rh, johansen hk, et al. aspergillus species and other molds in respiratory samples from patients with cystic fibrosis: a laboratory-based study with focus on aspergillus fumigatus azole resistance. j clin microbiol. 2011;49(6):2243-2251. crossref pubmed 52. escribano p, recio s, peláez t, bouza e, guinea j. aspergillus fumigatus strains with mutations in the cyp51a gene do not https://doi.org/10.1371/journal.pone.0114745 https://www.ncbi.nlm.nih.gov/pubmed/25478929 https://doi.org/10.1007/s10096-017-2919-6 https://www.ncbi.nlm.nih.gov/pubmed/28161741 https://doi.org/10.1007/s10096-017-2921-z https://www.ncbi.nlm.nih.gov/pubmed/28161744 https://doi.org/10.1111/myc.12393 https://www.ncbi.nlm.nih.gov/pubmed/26449506 https://doi.org/10.1086/376525 https://www.ncbi.nlm.nih.gov/pubmed/12975753 https://doi.org/10.1053/rmed.2001.1047 https://www.ncbi.nlm.nih.gov/pubmed/11392574 https://doi.org/10.2165/11596540-000000000-00000 https://www.ncbi.nlm.nih.gov/pubmed/22141384 https://doi.org/10.1017/s0021859605004971 https://doi.org/10.1016/s0190-9622(08)81495-8 https://www.ncbi.nlm.nih.gov/pubmed/8176006 https://doi.org/10.1021/jm00194a023 https://www.ncbi.nlm.nih.gov/pubmed/490531 https://doi.org/10.1086/367933 https://www.ncbi.nlm.nih.gov/pubmed/12594645 https://doi.org/10.1111/j.1470-9465.2004.00841.x https://www.ncbi.nlm.nih.gov/pubmed/14748798 https://doi.org/10.2165/00003495-200767020-00009 https://www.ncbi.nlm.nih.gov/pubmed/17284090 https://doi.org/10.1111/j.1368-5031.2004.0099.x https://www.ncbi.nlm.nih.gov/pubmed/14994974 https://www.ncbi.nlm.nih.gov/pubmed/20112166 https://doi.org/10.1586/14787210.2015.1032939 https://www.ncbi.nlm.nih.gov/pubmed/25843556 https://doi.org/10.1128/aac.01599-09 https://www.ncbi.nlm.nih.gov/pubmed/20385860 https://doi.org/10.1128/aac.40.2.279 https://www.ncbi.nlm.nih.gov/pubmed/8834867 https://doi.org/10.1128/cmr.11.2.382 https://www.ncbi.nlm.nih.gov/pubmed/9564569 https://doi.org/10.1016/s1367-5931(97)80007-5 https://www.ncbi.nlm.nih.gov/pubmed/9667858 https://doi.org/10.2165/00003495-200767110-00004 https://www.ncbi.nlm.nih.gov/pubmed/17661528 https://doi.org/10.1086/525258 https://www.ncbi.nlm.nih.gov/pubmed/18177225 https://doi.org/10.1111/j.1469-0691.2009.02911.x https://www.ncbi.nlm.nih.gov/pubmed/19673966 https://doi.org/10.1179/joc.2011.23.6.319 https://www.ncbi.nlm.nih.gov/pubmed/22233814 https://doi.org/10.1021/acs.jafc.5b02728 https://www.ncbi.nlm.nih.gov/pubmed/26289797 https://www.apsnet.org/edcenter/apsnetfeatures/pages/fungicides.aspx https://doi.org/10.1002/ps.4029 https://www.ncbi.nlm.nih.gov/pubmed/25914201 https://doi.org/10.1080/03601230009373291 https://www.ncbi.nlm.nih.gov/pubmed/10968606 https://doi.org/10.1128/jcm.44.5.1782-1787.2006 https://www.ncbi.nlm.nih.gov/pubmed/16672407 https://doi.org/10.1128/aac.47.10.3085-3088.2003 https://www.ncbi.nlm.nih.gov/pubmed/14506013 https://doi.org/10.1016/j.medmal.2013.02.010 https://www.ncbi.nlm.nih.gov/pubmed/23562488 https://doi.org/10.1128/aac.48.7.2747-2750.2004 https://www.ncbi.nlm.nih.gov/pubmed/15215142 https://doi.org/10.1128/aac.01092-06 https://www.ncbi.nlm.nih.gov/pubmed/17371828 https://doi.org/10.1093/jac/dkq450 https://www.ncbi.nlm.nih.gov/pubmed/21131690 https://doi.org/10.1128/jcm.00213-11 https://www.ncbi.nlm.nih.gov/pubmed/21508152 environmental drivers of aspergillus azole resistance34 © 2022 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti always show phenotypic resistance to itraconazole, voriconazole, or posaconazole. antimicrob agents chemother. 2011;55(5):2460-2462. crossref pubmed 53. jeurissen a, cooreman s, van kerckhoven w, et al. invasive pulmonary aspergillosis due to a multi-azole resistant aspergillus fumigatus. acta clin belg. 2012;67(1):46-48. pubmed 54. alastruey-izquierdo a, mellado e, peláez t, et al; filpop study group. population-based survey of filamentous fungi and antifungal resistance in spain (filpop study). antimicrob agents chemother. 2013;57(7):3380-3387. crossref pubmed 55. steinmann j, hamprecht a, vehreschild mj, et al. emergence of azole-resistant invasive aspergillosis in hsct recipients in germany. j antimicrob chemother. 2015;70(5):1522-1526. crossref pubmed 56. van der linden jw, arendrup mc, warris a, et al. prospective multicenter international surveillance of azole resistance in aspergillus fumigatus. emerg infect dis. 2015;21(6):10411044. crossref pubmed 57. lavergne ra, morio f, favennec l, et al. first description of azole-resistant aspergillus fumigatus due to tr46/y121f/ t289a mutation in france. antimicrob agents chemother. 2015;59(7):4331-4335. crossref pubmed 58. brillowska-dąbrowska a, mroczyńska m, nawrot u, włodarczyk k, kurzyk e. examination of cyp51a and cyp51b expression level of the first polish azole resistant clinical aspergillus fumigatus isolate. acta biochim pol. 2015;62(4):837-839. crossref pubmed 59. lazzarini c, esposto mc, prigitano a, cogliati m, de lorenzis g, tortorano am. azole resistance in a. fumigatus clinical isolates from an italian culture collection. antimicrob agents chemother. 2015;60(1):682-685. crossref pubmed 60. pelaez t, monteiro mc, garcia-rubio r, bouza e, gomezlopez a, mellado e. first detection of aspergillus fumigatus azole-resistant strain due to cyp51a tr46/y121f/t289a in an azole-naive patient in spain. new microbes new infect. 2015;6:33-34. crossref pubmed 61. özmerdiven ge, ak s, ener b, et al. first determination of azole resistance in aspergillus fumigatus strains carrying the tr34/ l98h mutations in turkey. j infect chemother. 2015;21(8):581586. crossref pubmed 62. jensen rh, hagen f, astvad km, tyron a, meis jf, arendrup mc. azole-resistant aspergillus fumigatus in denmark: a laboratory-based study on resistance mechanisms and genotypes. clin microbiol infect. 2016 jun;22(6):570.e1-e9. crossref pubmed 63. lavergne ra, chouaki t, hagen f, et al. home environment as a source of life-threatening azole-resistant a. fumigatus in immunocompromised patients. clin infect dis. 2017;64(1): 76-78. crossref pubmed 64. prigitano a, esposto mc, biffi a, et al. triazole resistance in aspergillus fumigatus isolates from patients with cystic fibrosis in italy. j cyst fibros. 2017;16(1):64-69. crossref pubmed 65. abdolrasouli a, petrou ma, park h, et al. surveillance for azoleresistant aspergillus fumigatus in a centralized diagnostic mycology service, london, united kingdom, 1998-2017. front microbiol. 2018;9:2234. crossref pubmed 66. wu cj, liu wl, lai cc, et al. multicenter study of azole-resistant aspergillus fumigatus clinical isolates, taiwan. emerg infect dis. 2020;26(4):804-806. crossref pubmed 67. chen j, li h, li r, bu d, wan z. mutations in the cyp51a gene and susceptibility to itraconazole in aspergillus fumigatus serially isolated from a patient with lung aspergilloma. j antimicrob chemother. 2005;55(1):31-37. crossref pubmed 68. hsueh pr, lau yj, chuang yc, et al. antifungal susceptibilities of clinical isolates of candida species, cryptococcus neoformans, and aspergillus species from taiwan: surveillance of multicenter antimicrobial resistance in taiwan program data from 2003. antimicrob agents chemother. 2005;49(2):512517. crossref pubmed 69. bueid a, howard sj, moore cb, et al. azole antifungal resistance in aspergillus fumigatus: 2008 and 2009. j antimicrob chemother. 2010;65(10):2116-2118. crossref pubmed 70. howard sj, cerar d, anderson mj, et al. frequency and evolution of azole resistance in aspergillus fumigatus associated with treatment failure. emerg infect dis. 2009;15(7):10681076. crossref pubmed 71. meis jf, chowdhary a, rhodes jl, fisher mc, verweij pe. clinical implications of globally emerging azole resistance in aspergillus fumigatus. philos trans r soc lond b biol sci. 2016;371(1709):20150460. crossref pubmed 72. bunskoek pe, seyedmousavi s, gans sj, et al. successful treatment of azole-resistant invasive aspergillosis in a bottlenose dolphin with high-dose posaconazole. med mycol case rep. 2017;16:16-19. crossref pubmed 73. hof h. critical annotations to the use of azole antifungals for plant protection. antimicrob agents chemother. 2001;45(11): 2987-2990. crossref pubmed 74. snelders e, camps sm, karawajczyk a, et al. triazole fungicides can induce cross-resistance to medical triazoles in aspergillus fumigatus. plos one. 2012;7(3):e31801. crossref pubmed 75. chowdhary a, kathuria s, xu j, et al. clonal expansion and emergence of environmental multiple-triazole-resistant aspergillus fumigatus strains carrying the tr₃₄/l98h mutations in the cyp51a gene in india. plos one. 2012;7(12):e52871. crossref pubmed 76. mortensen kl, mellado e, lass-flörl c, rodriguez-tudela jl, johansen hk, arendrup mc. environmental study of azole-resistant aspergillus fumigatus and other aspergilli in austria, denmark, and spain. antimicrob agents chemother. 2010;54(11):4545-4549. crossref pubmed 77. chowdhary a, kathuria s, xu j, meis jf. emergence of azoleresistant aspergillus fumigatus strains due to agricultural azole use creates an increasing threat to human health. plos pathog. 2013;9(10):e1003633. crossref pubmed 78. verweij pe, mellado e, melchers wj. multiple-triazole-resistant aspergillosis. n engl j med. 2007;356(14):1481-1483. crossref pubmed 79. van der linden jw, camps sm, kampinga ga, et al. aspergillosis due to voriconazole highly resistant a. fumigatus and recovery of genetically related resistant isolates from domestic homes. clin infect dis. 2013;57(4):513520. crossref pubmed 80. thind ts. changing cover of fungicide umbrella in crop protection. indian phytopathol. 2007;60:421-433. 81. snelders e, huis in t veld ra, rijs aj, kema gh, melchers wj, verweij pe. possible environmental origin of resistance of a. fumigatus to medical triazoles. appl environ microbiol. 2009;75(12):4053-4057. crossref pubmed 82. zhang j, snelders e, zwaan bj, et al. a novel environmental azole resistance mutation in a. fumigatus and a possible role of sexual reproduction in its emergence. mbio. 2017;8(3):e00791-e17. crossref pubmed 83. badali h, vaezi a, haghani i, et al. environmental study of azole-resistant aspergillus fumigatus with tr34/l98h mutations in the cyp51a gene in iran. mycoses. 2013;56(6):659663. crossref pubmed 84. ahmad s, khan z, hagen f, meis jf. occurrence of triazoleresistant aspergillus fumigatus with tr34/l98h mutations in outdoor and hospital environment in kuwait. environ res. 2014;133:20-26. crossref pubmed https://doi.org/10.1128/aac.01358-10 https://www.ncbi.nlm.nih.gov/pubmed/21321141 https://www.ncbi.nlm.nih.gov/pubmed/22480040 https://doi.org/10.1128/aac.00383-13 https://www.ncbi.nlm.nih.gov/pubmed/23669377 https://doi.org/10.1093/jac/dku566 https://www.ncbi.nlm.nih.gov/pubmed/25630644 https://doi.org/10.3201/eid2106.140717 https://www.ncbi.nlm.nih.gov/pubmed/25988348 https://doi.org/10.1128/aac.00127-15 https://www.ncbi.nlm.nih.gov/pubmed/25918139 https://doi.org/10.18388/abp.2015_1143 https://www.ncbi.nlm.nih.gov/pubmed/26636140 https://doi.org/10.1128/aac.02234-15 https://www.ncbi.nlm.nih.gov/pubmed/26552980 https://doi.org/10.1016/j.nmni.2015.04.005 https://www.ncbi.nlm.nih.gov/pubmed/26082842 https://doi.org/10.1016/j.jiac.2015.04.012 https://www.ncbi.nlm.nih.gov/pubmed/26048062 https://doi.org/10.1016/j.cmi.2016.04.001 https://pubmed.ncbi.nlm.nih.gov/27091095/ https://doi.org/10.1093/cid/ciw664 https://www.ncbi.nlm.nih.gov/pubmed/27682064 https://doi.org/10.1016/j.jcf.2016.06.006 https://www.ncbi.nlm.nih.gov/pubmed/27356848 https://doi.org/10.3389/fmicb.2018.02234 https://www.ncbi.nlm.nih.gov/pubmed/30294314 https://doi.org/10.3201/eid2604.190840 https://www.ncbi.nlm.nih.gov/pubmed/32186508 https://doi.org/10.1093/jac/dkh507 https://www.ncbi.nlm.nih.gov/pubmed/15563516 https://doi.org/10.1128/aac.49.2.512-517.2005 https://www.ncbi.nlm.nih.gov/pubmed/15673726 https://doi.org/10.1093/jac/dkq279 https://www.ncbi.nlm.nih.gov/pubmed/20729241 https://doi.org/10.3201/eid1507.090043 https://www.ncbi.nlm.nih.gov/pubmed/19624922 https://doi.org/10.1098/rstb.2015.0460 https://pubmed.ncbi.nlm.nih.gov/28080986/ https://doi.org/10.1016/j.mmcr.2017.03.005 https://www.ncbi.nlm.nih.gov/pubmed/28409094 https://doi.org/10.1128/aac.45.11.2987-2990.2001 https://www.ncbi.nlm.nih.gov/pubmed/11600346 https://doi.org/10.1371/journal.pone.0031801 https://www.ncbi.nlm.nih.gov/pubmed/22396740 https://doi.org/10.1371/journal.pone.0052871 https://www.ncbi.nlm.nih.gov/pubmed/23285210 https://doi.org/10.1128/aac.00692-10 https://www.ncbi.nlm.nih.gov/pubmed/20805399 https://doi.org/10.1371/journal.ppat.1003633 https://www.ncbi.nlm.nih.gov/pubmed/24204249 https://doi.org/10.1056/nejmc061720 https://www.ncbi.nlm.nih.gov/pubmed/17409336 https://doi.org/10.1093/cid/cit320 https://www.ncbi.nlm.nih.gov/pubmed/23667263 https://doi.org/10.1128/aem.00231-09 https://www.ncbi.nlm.nih.gov/pubmed/19376899 https://doi.org/10.1128/mbio.00791-17 https://www.ncbi.nlm.nih.gov/pubmed/28655821 https://doi.org/10.1111/myc.12089 https://www.ncbi.nlm.nih.gov/pubmed/23668594 https://doi.org/10.1016/j.envres.2014.05.009 https://www.ncbi.nlm.nih.gov/pubmed/24906064 sen et al drug target insights 2022; 16: 35 © 2022 the authors. published by aboutscience www.aboutscience.eu 85. chowdhary a, sharma c, hagen f, meis jf. exploring azole antifungal drug resistance in aspergillus fumigatus with special reference to resistance mechanisms. future microbiol. 2014;9(5):697-711. crossref pubmed 86. van der linden jw, snelders e, kampinga ga, et al. clinical implications of azole resistance in aspergillus fumigatus, the netherlands, 2007-2009. emerg infect dis. 2011;17(10):18461854. crossref pubmed 87. dauchy c, bautin n, nseir s, et al. emergence of aspergillus fumigatus azole resistance in azole-naïve patients with chronic obstructive pulmonary disease and their homes. indoor air. 2018;28(2):298-306. crossref pubmed 88. lescar j, meyer i, akshita k, et al. aspergillus fumigatus harbouring the sole y121f mutation shows decreased susceptibility to voriconazole but maintained susceptibility to itraconazole and posaconazole. j antimicrob chemother. 2014;69(12):32443247. crossref pubmed 89. sharma c, hagen f, moroti r, meis jf, chowdhary a. triazoleresistant aspergillus fumigatus harbouring g54 mutation: is it de novo or environmentally acquired? j glob antimicrob resist. 2015;3(2):69-74. crossref pubmed 90. wang hc, huang jc, lin yh, chen yh. prevalence, mechanisms, and genetic relatedness of the human pathogenic fungus a. fumigatus sex inhibiting resistance to medical azoles in the environment of taiwan. appl environ microbiol. 2008;20:270-280. 91. jeanvoine a, rocchi s, reboux g, crini n, crini g, millon l. azoleresistant aspergillus fumigatus in sawmills of eastern france. j appl microbiol. 2017;123(1):172-184. crossref pubmed 92. alvarez-moreno c, lavergne ra, hagen f, morio f, meis jf, le pape p. azole-resistant aspergillus fumigatus harboring tr 34 / l98h, tr 46 /y121f/t289a and tr 53 mutations related to flower fields in colombia. sci rep. 2017;7(1):45631. crossref pubmed 93. chowdhary a, sharma c, kathuria s, hagen f, meis jf. prevalence and mechanism of triazole resistance in aspergillus fumigatus in a referral chest hospital in delhi, india and an update of the situation in asia. front microbiol. 2015;6:428. crossref pubmed 94. hoda s, agarwal h, ahluwalia sk, vermani m, vijayaraghavan p. antifungal resistance analysis of environmental isolates of aspergillus in north india. j pure appl microbiol. 2019;13(1): 385-392. crossref 95. mann pa, parmegiani rm, wei sq, et al. mutations in aspergillus fumigatus resulting in reduced susceptibility to posaconazole appear to be restricted to a single amino acid in the cytochrome p450 14α-demethylase. antimicrob agents chemother. 2003;47(2):577-581. crossref pubmed 96. tashiro m, izumikawa k, hirano k, et al. correlation between triazole treatment history and susceptibility in clinically isolated aspergillus fumigatus. antimicrob agents chemother. 2012;56(9):4870-4875. crossref pubmed 97. chowdhary a, sharma c, van den boom m, et al. multiazole-resistant aspergillus fumigatus in the environment in tanzania. j antimicrob chemother. 2014;69(11):2979-2983. crossref pubmed 98. rajendran r, mowat e, mcculloch e, et al. azole resistance of aspergillus fumigatus biofilms is partly associated with efflux pump activity. antimicrob agents chemother. 2011;55(5):20922097. crossref pubmed 99. tekaia f, latgé jp. aspergillus fumigatus: saprophyte or pathogen? curr opin microbiol. 2005;8(4):385-392. crossref pubmed 100. wesenberg d, kyriakides i, agathos sn. white-rot fungi and their enzymes for the treatment of industrial dye effluents. biotechnol adv. 2003;22(1-2):161-187. crossref pubmed 101. dagenais tr, keller np. pathogenesis of aspergillus fumigatus in invasive aspergillosis. clin microbiol rev. 2009;22(3): 447-465. crossref pubmed 102. godeau c, reboux g, scherer e, et al. azole-resistant aspergillus fumigatus in the hospital: surveillance from flower beds to corridors. am j infect control. 2020;48(6):702-704. crossref pubmed 103. gonzalez-jimenez i, lucio j, menéndez-fraga md, mellado e, peláez t. hospital environment as a source of azole-resistant aspergillus fumigatus strains with tr34/l98h and g448s cyp51a mutations. j fungi (basel). 2021;7(1):22. crossref pubmed 104. bowyer p, moore cb, rautemaa r, denning dw, richardson md. azole antifungal resistance today: focus on aspergillus. curr infect dis rep. 2011;13(6):485-491. crossref pubmed 105. mitchell kf, zarnowski r, andes dr. the extracellular matrix of fungal biofilms. adv exp med biol. 2016;931:21-35. crossref pubmed 106. subroto e, van neer j, valdes i, de cock h. growth of aspergillus fumigatus in biofilms in comparison to candida albicans. j fungi (basel). 2022;8(1):48. crossref pubmed 107. kaur s, singh s. biofilm formation by aspergillus fumigatus. med mycol. 2014;52(1):2-9. pubmed 108. blankenship jr, mitchell ap. how to build a biofilm: a fungal perspective. curr opin microbiol. 2006;9(6):588-594. crossref pubmed 109. mowat e, butcher j, lang s, williams c, ramage g. development of a simple model for studying the effects of antifungal agents on multicellular communities of aspergillus fumigatus. j med microbiol. 2007;56(pt 9):1205-1212. crossref pubmed 110. liu m, zheng h, zeng r, liang g, zheng n, liu w. effects of itraconazole and micafungin on aspergillus fumigatus biofilms. mycopathologia. 2021;186(3):387-397. crossref pubmed 111. morelli ka, kerkaert jd, cramer ra. aspergillus fumigatus biofilms: toward understanding how growth as a multicellular network increases antifungal resistance and disease progression. plos pathog. 2021;17(8):e1009794. crossref pubmed https://doi.org/10.2217/fmb.14.27 https://www.ncbi.nlm.nih.gov/pubmed/24957095 https://doi.org/10.3201/eid1710.110226 https://www.ncbi.nlm.nih.gov/pubmed/22000354 https://doi.org/10.1111/ina.12436 https://www.ncbi.nlm.nih.gov/pubmed/29082624 https://doi.org/10.1093/jac/dku316 https://www.ncbi.nlm.nih.gov/pubmed/25125676 https://doi.org/10.1016/j.jgar.2015.01.005 https://www.ncbi.nlm.nih.gov/pubmed/27873672 https://doi.org/10.1111/jam.13488 https://www.ncbi.nlm.nih.gov/pubmed/28497646 https://doi.org/10.1038/srep45631 https://www.ncbi.nlm.nih.gov/pubmed/28358115 https://doi.org/10.3389/fmicb.2015.00428 https://www.ncbi.nlm.nih.gov/pubmed/26005442 https://doi.org/10.22207/jpam.13.1.42 https://doi.org/10.1128/aac.47.2.577-581.2003 https://www.ncbi.nlm.nih.gov/pubmed/12543662 https://doi.org/10.1128/aac.00514-12 https://www.ncbi.nlm.nih.gov/pubmed/22751542 https://doi.org/10.1093/jac/dku259 https://www.ncbi.nlm.nih.gov/pubmed/25006238 https://doi.org/10.1128/aac.01189-10 https://www.ncbi.nlm.nih.gov/pubmed/21321135 https://doi.org/10.1016/j.mib.2005.06.017 https://www.ncbi.nlm.nih.gov/pubmed/16019255 https://doi.org/10.1016/j.biotechadv.2003.08.011 https://www.ncbi.nlm.nih.gov/pubmed/14623049 https://doi.org/10.1128/cmr.00055-08 https://www.ncbi.nlm.nih.gov/pubmed/19597008 https://doi.org/10.1016/j.ajic.2019.10.003 https://www.ncbi.nlm.nih.gov/pubmed/31753549 https://doi.org/10.3390/jof7010022 https://www.ncbi.nlm.nih.gov/pubmed/33401764 https://doi.org/10.1007/s11908-011-0218-4 https://www.ncbi.nlm.nih.gov/pubmed/21931980 https://doi.org/10.1007/5584_2016_6 https://www.ncbi.nlm.nih.gov/pubmed/27271680 https://doi.org/10.3390/jof8010048 https://www.ncbi.nlm.nih.gov/pubmed/35049988 https://www.ncbi.nlm.nih.gov/pubmed/23962172 https://doi.org/10.1016/j.mib.2006.10.003 https://www.ncbi.nlm.nih.gov/pubmed/17055772 https://doi.org/10.1099/jmm.0.47247-0 https://www.ncbi.nlm.nih.gov/pubmed/17761484 https://doi.org/10.1007/s11046-021-00534-4 https://www.ncbi.nlm.nih.gov/pubmed/33956291 https://doi.org/10.1371/journal.ppat.1009794 https://www.ncbi.nlm.nih.gov/pubmed/34437655 dti drug target insights 2023; 17: 39-44issn 1177-3928 | doi: 10.33393/dti.2023.2574 original research article drug target insights issn 1177-3928 www.aboutscience.eu/dti © 2023 the authors. this article is published by aboutscience and licensed under creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). commercial use is not permitted and is subject to publisher’s permissions. full information is available at www.aboutscience.eu treatment with levosimendan in an experimental model of early ventilator-induced diaphragmatic dysfunction vanessa zambelli1, emma j. murphy2, paolo delvecchio1, laura rizzi1, roberto fumagalli1,3, emanuele rezoagli1,4, giacomo bellani1,4 1school of medicine and surgery, university of milano-bicocca, monza italy 2life – health and bioscience research institute, midwest campus, technological university of the shannon, limerick ireland 3department of emergency medicine, asst grande ospedale metropolitano niguarda, milan italy 4 department of emergency and intensive care, fondazione irccs san gerardo dei tintori, monza italy abstract introduction: mechanical ventilation (mv) is a life-saving approach in critically ill patients. however, it may affect the diaphragmatic structure and function, beyond the lungs. levosimendan is a calcium sensitizer widely used in clinics to improve cardiac contractility in acute heart failure patients. in vitro studies have demonstrated that levosimendan increased force-generating capacity of the diaphragm in chronic obstructive pulmonary disease patients. thus the aim of this study was to evaluate the effects of levosimendan administration in an animal model of ventilator-induced diaphragmatic dysfunction (vidd) on muscle contraction and diaphragm muscle cell viability. methods: sprague-dawley rats underwent prolonged mv (5 hours). vidd+levo group received a starting bolus of levosimendan immediately after intratracheal intubation and then an intravenous infusion of levosimendan throughout the study. diaphragms were collected for ex vivo contractility measurement (with electric stimulation), histological analysis and western blot analysis. healthy rats were used as the control. results: levosimendan treatment maintained an adequate mean arterial pressure during the entire experimental protocol, preserved levels of autophagy-related proteins (lc3bi and lc3bii) and the muscular cell diameter demonstrated by histological analysis. levosimendan did not affect the diaphragmatic contraction or the levels of proteins involved in the protein degradation (atrogin). conclusions: our data suggest that levosimendan preserves muscular cell structure (cross-sectional area) and muscle autophagy after 5 hours of mv in a rat model of vidd. however, levosimendan did not improve diaphragm contractile efficiency. keywords: diaphragm contractility, levosimendan, mechanical ventilation, muscle fiber size, ventilator-induced diaphragmatic dysfunction received: february 11, 2023 accepted: march 29, 2023 published online: april 13, 2023 corresponding author: giacomo bellani & emanuele rezoagli school of medicine and surgery university of milano-bicocca via cadore 48, 20900 monza (mb) italy giacomo.bellani1@unimib.it; emanuele.rezoagli@unimib.it a rapid impairment of both lungs (i.e., ventilator-induced lung injury – vili) and diaphragm (i.e., ventilator-induced diaphragmatic dysfunction – vidd). mv can also exacerbate a preexisting vili (1-3). it is also associated with adverse effects on multiple aspects of diaphragmatic structure and function (vidd) (4). the detrimental impact of prolonged mv on the diaphragm is strictly correlated to problems in weaning patients from the ventilator (5). the incidence level can reach 35% of patients exposed to prolonged mv, with subsequent increase in morbidity and mortality (4,6,7). furthermore, body composition indexes are known to predict outcome – such as mortality and mv duration itself – in critically ill patients (8). the first evidence on the correlation between mv and vidd was published almost 35 years ago, in which a study of infant and neonates suggested that mv predisposes diaphragm fibers to atrophy (9). thenceforth introduction mechanical ventilation (mv) is a life support technique used in critically ill patients. however, prolonged mv is also associated with numerous potential complications including https://doi.org/10.33393/dti.2023.2574 https://creativecommons.org/licenses/by-nc/4.0/legalcode mailto:vanessa.zambelli@unimib.it effects of levosimendan treatment on diaphragmatic injury40 © 2023 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti numerous experimental and clinical studies documented that prolonged mv induces a rapid muscular atrophy and contractile dysfunction (10). skeletal muscle is one of the most significant muscle tissues in the body. thousands of muscle fibers are encased in connective tissue sheaths to form each skeletal muscle. the size of skeletal muscle fiber is dependent on the balance between the protein synthesis and protein degradation (11,12). animal studies have demonstrated that protein synthesis declines rapidly within the first 6 hours of mv and remains depressed during the next 12 hours (11). at the same time, the prolonged mv increases the activity of all major proteolytic systems: macroautophagy, calpains, caspases and the ubiquitin-proteasome systems. moreover prolonged mv results in oxidative damage to the diaphragm – reactive oxygen species and their derivatives have a significant impact on skeletal muscle contractile function (13). prolonged mv results in less efficient calcium activation of diaphragm fibers most likely due to oxidative modification of diaphragm contractile proteins (14). levosimendan is a calcium sensitizer, first described in 1995, that is administered to enhance cardiac contractility in patients with acute heart failure (15,16). in addition, levosimendan improves respiratory muscle function in healthy subjects and patients with chronic obstructive pulmonary disease (copd) (17), through the improvement of calcium sensitization of the contractile proteins (18). moreover, it reduces inflammation and oxidative stress (19). a recent study also demonstrated that levosimendan dampens nitrosative stress in the diaphragm of mechanically ventilated endotoxemic mice (20). thus, we administered levosimendan to an in vivo model of vidd to determine if it could alleviate the negative impacts of mv. therapeutic effects were determined by measuring muscle contraction and diaphragm muscle viability. the aim of the study was to evaluate the effect of the treatment of levosimendan in a rat model on vidd on: 1. the preservation of diaphragm muscle structure; 2. the level of autophagy activation in the diaphragm; 3. the diaphragm muscular contractile function. materials and methods animals male sprague-dawley rats (250-300 g; envigo rms s.r.l., udine, italy) were used in this study. animals were housed two per cage in a limited access animal facility, with the room temperature at 20 ± 2°c and the relative humidity set at 55 ± 10%. artificial lighting provided a 12-hour light/12-hour dark (7 am.–7 pm) cycle. the general condition of the animals before the experiment was assessed daily. the care and husbandry of animals were in conformity with the institutional guidelines in compliance with italian and european laws and policies. the animal study was reviewed and approved by the italian ministry of health (773/2018-pr) and by the animal care unit of the university of milano-bicocca, monza, italy. in full respect of the reduction principle of the 3rs (21), the number of animals/groups was selected to obtain reliable results and enough biological samples to perform the analysis planned. experimental protocol the experimental design is composed of three experimental group of rats: – healthy: non-anesthetized and spontaneously breathing rats; – vidd: anesthetized and mechanically ventilated rats without any pharmacological treatment; – vidd+levo: anesthetized and ventilated rats with levosimendan infusion. rats were anesthetized with ketamine (100 mg/kg) and xylazine (4 mg/kg), orotracheally intubated and ventilated for 5 hours (harvard inspira; tidal volume: 10 ml/kg; respiratory rate: 80/min; positive end-expiratory pressure [peep]: 2-2.5 cmh 2 o; fraction of inspired oxygen [fio 2 : 0.5]). anesthesia and paralysis were maintained throughout the experiment by infusion in the right femoral artery of propofol (13 mg/ kg/h) and ketamine (5 mg/kg/h) and in the right jugular vein of rocuronium bromide (1.5 mg/kg/h) and ringer acetate (1.8 ml/h). airway pressure and hemodynamic parameters were monitored through pressure transducers in ventilator and at the arterial catheter. a recruitment maneuver (30 cmh 2 o for 10 sec) was performed every 60 minutes, being the plateau pressure, and respiratory system static compliance was measured every hour. rats belonging to the treatment group (vidd+levo) received a dose (24 µg/kg) of levosimendan (simdax; orion pharma) into the tail vein at the beginning of the experiment and then a maintenance of the treatment was achieved by an infusion in the left jugular vein of levosimendan (0.2 µg/kg/min) (22). pulmonary function for the lung mechanical properties, a pressure to volume (pv) curve was calculated. after a recruitment maneuver, five steps of 0.5 ml inspiratory volumes (i.e., total 2.5 ml) were delivered into the lungs. for each step, the plateau pressure was recorded to calculate the static compliance. diaphragmatic contractile function diaphragmatic contractile function was measured as already described (23), briefly a diaphragm strip was mounted into a tissue bath and, through an electric stimulator, the peak tetanic tension and the force-frequency relationship were evaluated. histological analysis: muscular fibers cross-sectional area a section from the right and one from the left hemidiaphragm tissue was used for histology analysis. the cross sectional area (csa) of the muscular fiber was determined by manually tracing the cell contour on digitized zambelli et al drug target insights 2023; 17: 41 © 2023 the authors. published by aboutscience www.aboutscience.eu images from hematoxylin and eosin-stained frozen section. the csa of at least 150 fibers per diaphragm were then averaged. western blot analysis another section of the diaphragm excised at the sacrifice was immediately frozen in liquid nitrogen and stored at −80°c. the cytoplasmic extraction was prepared using an ne-per nuclear cytoplasmic extraction reagent kit (pierce, rockford, il, usa) according to the manufacturer’s instruction. equal amounts of the protein concentrations were quantified by the bicinchoninic acid assay (bca assay; pierce, rockford, il, usa), and each sample was analyzed according to standard western blotting protocols. the following antibodies were used: atrogin (ap2041; ecm biosciences, versailles, ky, usa) and lc3b (light chain 3, isoform b ii, 2775; cell signaling technology, danvers, ma, usa). the antibody α-tubulin (#2125; cell signaling, danvers, ma, usa) was used as the reference of internal control. statistical analysis data are expressed as mean ± standard error of the mean (sem). differences in variances between groups were assessed by one-way analysis of variance (anova). post hoc comparisons were performed by benjamini, krieger and yekutieli. differences in physiological variables between vidd and vidd+levo were performed by unpaired t-test. p-values < 0.05 were considered as statistically significant (two-tailed). statistical analysis was performed by graphpad prism (version 8.4.2). some histologic and western blot analyses could not be performed in some animals because of technical problems. results systemic response no rats died during the experiments. in terms of body weight, no differences were found between groups (healthy: 310 ± 6, vidd: 300 ± 6, vidd + levo: 295 ± 7 g; anova p = 0.25). the mean arterial pressure (map) was similar between the test groups at the beginning of the experiment and after 3 hours of mv. at the end of the mv, rats without levosimendan treatment showed a significant lower map versus vidd + levo group, as demonstrated in table i. the difference in map was not influenced by a difference in terms of fluid balance, since it was similar between two groups (vidd: 18.6 ± 0.6 ml vs. vidd + levo: 19.9 ± 1.3 ml, p = 0.31). regarding the respiratory function, no differences were found between ventilated groups in oxygenation nor in respiratory system static compliance. histological analysis levosimendan treatment seemed to induce a protection in muscle fiber size from cellular atrophy induced by mv. as shown in figure 1, mv affects the cross-sectional fiber area in table i mean arterial pressure, heart rate, pao 2 /fio 2 , respiratory system static compliance vidd vidd + levo p values mean arterial pressure (mm hg) start 90 ± 5 91 ± 11 0.93 3 hours 94 ± 6 97 ± 9 0.78 end 66 ± 6 89 ± 10 0.04* heart rate (/min) start 254 ± 10 285 ± 16 0.10 3 hours 329 ± 12 329 ± 6 0.95 end 306 ± 12 330 ± 9 0.15 pao 2 /fio 2 end 476 ± 48 529 ± 13 0.25 respiratory system static compliance (ml/cmh 2 o) end 0.34 ± 0.01 0.32 ± 0.01 0.38 all parameters were measured during mechanical ventilation. vidd: mv + no treatment (n = 12); vidd+levo: mv + levosimendan treatment (n = 8). mv = mechanical ventilation; vidd = ventilator-induced diaphragmatic dysfunction. *p < 0.05. fig. 1 diaphragm fiber size. cross-sectional diaphragmatic fiber size in histological slice. a) histological images, hematoxylin-eosin staining, 200×, scale bar = 200 µm. b) anova p < 0.01. benjamini, krieger and yekutieli post hoc test: p = 0.004 vs. healthy and p = 0.01 vs. vidd+levo. healthy: no surgical interventions, no mv, and no treatment (n = 8); vidd: surgical intervention + mv and no treatment (n = 10); vidd+levo: surgical intervention + mv + levosimendan treatment (n = 8). anova = analysis of variance; mv = mechanical ventilation; vidd = ventilator-induced diaphragmatic dysfunction. effects of levosimendan treatment on diaphragmatic injury42 © 2023 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti vidd animals if compared to healthy rats. vidd+levo group had fiber diameters like unventilated animals. western blot analysis western blot analysis (fig. 2) on levels of proteins involved in muscle protein degradation (atrogin) did not reveal any statistical difference between groups. proteins implicated in autophagy and autophagy-related processes (lc3bi and lc3bii) were significantly higher in the vidd group, if compared to healthy and vidd+levo groups, while these proteins did not differ in the vidd+levo group as compared with the healthy group. diaphragm contractile dysfunction after 5 hours of mv, rats that underwent ventilation (vidd and vidd+levo) showed a significant reduction in diaphragmatic contractility in response to in vitro electric stimulation, fig. 3 diaphragm contractility. force-frequency relationship (panel a) and peak tetanic tension (panel b) measured on diaphragmatic strips with electric stimulator at the end of the experiment. a) anova *p < 0.05, **p < 0.01. benjamini, krieger and yekutieli post hoc test: 10 hz p = 0.03 vs. vidd and vs. vidd+levo, 30 hz p = 0.001 vs. vidd and p = 0.004 vs. vidd+levo, 60 hz p = 0.002 vs. vidd and p = 0.005 vs. vidd+levo, 90 hz p = 0.004 vs. vidd and p = 0.01 vs. vidd+levo, 120 hz p = 0.006 vs. vidd and p = 0.01 vs. vidd+levo, 150 hz p = 0.007 vs. vidd and p = 0.002 vs. vidd+levo. b) anova p < 0.05. anova = analysis of variance; vidd = ventilatorinduced diaphragmatic dysfunction. b) anova p < 0.05. benjamini, krieger and yekutieli post-hoc test: p = 0.007 vs vidd, p = 0.01 vs vidd+levo. healthy: no surgical interventions, no mv, and no treatment (n = 8); vidd: surgical intervention + mv and no treatment (n = 12); vidd+levo: surgical intervention + mv + levosimendan treatment (n = 8). in comparison to unventilated animals. as demonstrated in figure 3a, increasing the frequency of stimulation the diaphragmatic muscle strip of ventilated rats generated less force than the healthy diaphragm. levosimendan treatment did not affect the diaphragmatic contractility. the analysis of peak tetanic tension unveiled that ventilated rats (with or without levosimendan treatment) showed a similar diaphragmatic muscular contractility (fig. 3b). discussion in this experimental model of vidd, after 5 hours of mv in a rat model of vidd as compared to control, our data suggest that the administration of levosimendan: fig. 2 western blot analysis. atrogin and lc3bii/lc3bi levels on diaphragm tissue. a) anova p = 0.21. b) anova p = 0.03. benjamini, krieger and yekutieli post hoc test: p = 0.02 vs. healthy and p = 0.04 vs. vidd+levo. healthy: no surgical interventions, no mv, and no treatment (n = 8); vidd: surgical intervention + mv and no treatment (n = 11); vidd+levo: surgical intervention + mv + levosimendan treatment (n = 8). anova = analysis of variance; mv = mechanical ventilation; vidd = ventilator-induced diaphragmatic dysfunction. zambelli et al drug target insights 2023; 17: 43 © 2023 the authors. published by aboutscience www.aboutscience.eu 1. preserves the diaphragm muscular cell structure; 2. reduces autophagy-implicated proteins of the diaphragm muscle cells; 3. does not affect diaphragm contractile function. levosimendan is a calcium-sensitizing inodilator widely used in the management of acutely decompensated chronic heart failure from over 20 years. in addition, it has been evaluated in a variety of clinical settings for both cardiac and non-cardiac disease (24,25). one possible area of investigation is respiratory muscle dysfunction, because, in addition to cellular atrophy, reduced calcium sensitivity of contraction causes respiratory muscle weakness (26). in vitro studies on isolated muscle fibers showed that levosimendan increased force-generating capacity of diaphragm from copd and non-copd patients by improving calcium sensitivity of force generation (17). clinical studies have produced mixed results, the same investigators discovered that levosimendan increased in vivo diaphragmatic contractile efficiency in healthy participants (27), while it had no effect on diaphragm function in intensive care unit (icu) patients (28). the role of levosimendan as potential therapeutic intervention of diaphragm dysfunction is an open field of current pharmacological research for vidd in addition to the one for vili because of the need of mv in the presence of severe respiratory failure (3,29). in this study we evaluated the effects of levosimendan treatment in preclinical in vivo rodent model of vidd. we have previously demonstrated that the model resembles the main features of diaphragmatic dysfunction (22). as levosimendan has a short half-life of 1 hour, a bolus of 24 µg/ kg was immediately administered after orotracheal intubation and mv. it is well known that the use of a bolus dose should be avoided due to the risk of hypotension (30), but the animals in this study were healthy at the beginning of the study with physiological hemodynamics, as shown in table i. in our experimental design, levosimendan treatment, rather than inducing hypotension, maintained higher levels of map as compared to untreated animals at the end of experiment (tab. i). the diaphragmatic contraction was evaluated by in vitro electric stimulation after 5 hours of mv: both ventilated groups showed a significant reduction in muscular force compared to healthy control rats (fig. 3a, b). levosimendan treatment did not improve diaphragm contraction, maybe due to the bolus dose used in the study: 24 vs. 40 µg/kg, which was used in similar investigations (27). however, histological analysis of muscular diaphragmatic fiber revealed that levosimendan administration preserved significant cellular diameter from atrophy. prolonged mv led to a reduction in cross-sectional muscular fiber area of 16% (fig. 1), whereas levosimendan-treated rats showed conserved cellular structure when compared to the vidd group. the levels of atrogin, a protein belonging to the ubiquitin-proteasome system of proteolysis, did not differ between groups (fig. 2), whereas microtubule-associated protein light chain 3 i and ii (lc3bi and ii), autophagosome marker, were significantly modified by mv. it has been previously reported that prolonged mv can lead to upregulation of atrogin and lc3b (4,31). we hypothesize that, in our experimental protocol, the hours of mv should influence the protein expression, maybe 5 hours mv were not sufficient to stimulate atrogin production. this study has some limitations that should be acknowledged. first, the duration of mv (5 hours) potentially did not allow enough time to evaluate all the parameters (such as atrogin levels). however, the purpose of this study was to determine the early effects of levosimendan. early administration ensured that possible side effects on hemodynamics were avoided. it has been previously demonstrated that mv can have detrimental effects on muscle fibers. therefore, future studies will identify more specifically the different types of muscle fibers after mv with and without treatment. second, we decided to administer the dose of levosimendan used routinely in the clinical setting (decompensated heart failure). we cannot exclude that the use of a higher dose of levosimendan might provide a benefit in terms of diaphragmatic contraction. a dose-response study may be a useful potential step to further explore the role of levosimendan during vidd. third, during the mv period, all rats received a continuous infusion of rocuronium bromide that may probably affect the diaphragmatic muscle contraction that was assessed ex vivo after animal euthanasia. indeed, it is known that, simultaneously with mv, some drugs – such as neuromuscular blocking agents – may worsen the diaphragm dysfunction, albeit data on their effects on diaphragm are not debated (32-35). in conclusion, in an experimental animal model of vidd, levosimendan prevented autophagy allowing to preserve diaphragm muscular cellular structure as compared with vidd untreated group. however, levosimendan did not improve the diaphragm contractile efficiency. disclosures conflict of interest: the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. funding/support: we acknowledge that this research was partially supported by the italian ministry of university and research (miur), department of excellence project premia (precision medicine approach: bringing biomarker research to clinic). dr. emanuele rezoagli was supported by the bicocca starting grant 2020 from the university of milano-bicocca with the project titled: “functional residual capacity assessment using a wash-in/washout technique based on a fast mainstream o2 sensor with nanofluorescent geometry for severe lung injury (fast) – covid and beyond” and by the international young investigator award 2018 from the european society of intensive care medicine (esicm) with the project titled: “role of the exhaled breath condensate as non-invasive monitoring of the lung inflammation during ards: a prospective cohort study.” references 1. dreyfuss d, saumon g. ventilator-induced lung injury: lessons from experimental studies. am j respir crit care med. 1998;157(1):294-323. crossref pubmed 2. rezoagli e, laffey jg, bellani g. monitoring lung injury severity and ventilation intensity during mechanical ventilation. semin respir crit care med. 2022;43(3):346-368. crossref pubmed https://doi.org/10.1164/ajrccm.157.1.9604014 https://www.ncbi.nlm.nih.gov/pubmed/9445314 https://doi.org/10.1055/s-0042-1748917 https://www.ncbi.nlm.nih.gov/pubmed/35896391 effects of levosimendan treatment on diaphragmatic injury44 © 2023 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti 3. horie s, mcnicholas b, rezoagli e, et al. emerging pharmacological therapies for ards: covid-19 and beyond. intensive care med. 2020;46(12):2265-2283. crossref pubmed 4. powers sk, wiggs mp, sollanek kj, smuder aj. ventilatorinduced diaphragm dysfunction: cause and effect. am j physiol regul integr comp physiol. 2013;305(5):r464-r477. crossref pubmed 5. gatti s, abbruzzese c, ippolito d, et al. ultrasound versus computed tomography for diaphragmatic thickness and skeletal muscle index during mechanical ventilation. diagnostics (basel). 2022;12(11):2890. crossref pubmed 6. vassilakopoulos t, petrof bj. ventilator-induced diaphragmatic dysfunction. am j respir crit care med. 2004;169(3):336-341. crossref pubmed 7. pham t, heunks l, bellani g, et al. weaning from mechanical ventilation in intensive care units across 50 countries (wean safe): a multicentre, prospective, observational cohort study. lancet respir med. 2023 jan 20:s2213-2600(22)00449-0. crossref pubmed. epub ahead of print. 8. giani m, rezoagli e, grassi a, et al. low skeletal muscle index and myosteatosis as predictors of mortality in critically ill surgical patients. nutrition. 2022;101:111687. crossref pubmed 9. knisely as, leal sm, singer db. abnormalities of diaphragmatic muscle in neonates with ventilated lungs. j pediatr. 1988;113(6):1074-1077. crossref pubmed 10. powers sk, kavazis an, levine s. prolonged mechanical ventilation alters diaphragmatic structure and function. crit care med. 2009;37(10)(suppl):s347-s353. crossref pubmed 11. shanely ra, van gammeren d, deruisseau kc, et al. mechanical ventilation depresses protein synthesis in the rat diaphragm. am j respir crit care med. 2004;170(9):994-999. crossref pubmed 12. tobin mj, laghi f, jubran a. narrative review: ventilatorinduced respiratory muscle weakness. ann intern med. 2010;153(4):240-245. crossref pubmed 13. powers sk, jackson mj. exercise-induced oxidative stress: cellular mechanisms and impact on muscle force production. physiol rev. 2008;88(4):1243-1276. crossref pubmed 14. andrade fh, reid mb, westerblad h. contractile response of skeletal muscle to low peroxide concentrations: myofibrillar calcium sensitivity as a likely target for redox-modulation. faseb j. 2001;15(2):309-311. crossref pubmed 15. pollesello p, ovaska m, kaivola j, et al. binding of a new ca2+ sensitizer, levosimendan, to recombinant human cardiac troponin c. a molecular modelling, fluorescence probe, and proton nuclear magnetic resonance study. j biol chem. 1994;269(46):28584-28590. erratum in: j biol chem. 1995 feb 10;270. 6.: 2880. crossref pubmed 16. haikala h, kaivola j, nissinen e, wall p, levijoki j, lindén ib. cardiac troponin c as a target protein for a novel calcium sensitizing drug, levosimendan. j mol cell cardiol. 1995;27(9):18591866. crossref pubmed 17. van hees hw, dekhuijzen pn, heunks lm. levosimendan enhances force generation of diaphragm muscle from patients with chronic obstructive pulmonary disease. am j respir crit care med. 2009;179(1):41-47. crossref pubmed 18. follath f, cleland jgf, just h, et al; steering committee and investigators of the levosimendan infusion versus dobutamine (lido) study. efficacy and safety of intravenous levosimendan compared with dobutamine in severe low-output heart failure (the lido study): a randomised double-blind trial. lancet. 2002;360(9328):196-202. crossref pubmed 19. sareila o, korhonen r, auvinen h, et al. effects of levoand dextrosimendan on nf-kappab-mediated transcription, inos expression and no production in response to inflammatory stimuli. br j pharmacol. 2008;155(6):884-895. crossref pubmed 20. schellekens wj, van hees hw, linkels m, et al. levosimendan affects oxidative and inflammatory pathways in the diaphragm of ventilated endotoxemic mice. crit care. 2015;19(1):69. crossref pubmed 21. díaz l, zambrano e, flores me, et al. ethical considerations in animal research: the principle of 3r’s. rev invest clin. 2020;73(4):199-209. crossref pubmed 22. innes ca, wagstaff aj. levosimendan: a review of its use in the management of acute decompensated heart failure. drugs. 2003;63(23):2651-2671. crossref pubmed 23. zambelli v, sigurtà a, rizzi l, et al. angiotensin-(1-7) exerts a protective action in a rat model of ventilator-induced diaphragmatic dysfunction. intensive care med exp. 2019;7(1):8. crossref pubmed 24. papp z, agostoni p, alvarez j, et al. levosimendan efficacy and safety: 20 years of simdax in clinical use. j cardiovasc pharmacol. 2020;76(1):4-22. crossref pubmed 25. girardis m, bettex d, bojan m, et al. levosimendan in intensive care and emergency medicine: literature update and expert recommendations for optimal efficacy and safety. j anesth analg crit care. 2022;2(1):1-22. crossref 26. hooijman pe, beishuizen a, de waard mc, et al. diaphragm fiber strength is reduced in critically ill patients and restored by a troponin activator. am j respir crit care med. 2014;189(7):863-865. crossref pubmed 27. doorduin j, sinderby ca, beck j, et al. the calcium sensitizer levosimendan improves human diaphragm function. am j respir crit care med. 2012;185(1):90-95. crossref pubmed 28. roesthuis l, van der hoeven h, sinderby c, et al. effects of levosimendan on respiratory muscle function in patients weaning from mechanical ventilation. intensive care med. 2019;45(10):1372-1381. crossref pubmed 29. zambelli v, rizzi l, delvecchio p, et al. hexarelin modulates lung mechanics, inflammation, and fibrosis in acute lung injury. drug target insights. 2021;15:26-33. crossref pubmed 30. nieminen ms, buerke m, cohen-solál a, et al. the role of levosimendan in acute heart failure complicating acute coronary syndrome: a review and expert consensus opinion. int j cardiol. 2016;218:150-157. crossref pubmed 31. schellekens wj, van hees hw, vaneker m, et al. toll-like receptor 4 signaling in ventilator-induced diaphragm atrophy. anesthesiology. 2012;117(2):329-338. crossref pubmed 32. dres m, demoule a. diaphragm dysfunction during weaning from mechanical ventilation: an underestimated phenomenon with clinical implications. crit care. 2018;22(1):73. crossref pubmed 33. testelmans d, maes k, wouters p, et al. rocuronium exacerbates mechanical ventilation-induced diaphragm dysfunction in rats. crit care med. 2006;34(12):3018-3023. crossref pubmed 34. testelmans d, maes k, wouters p, powers sk, decramer m, gayan-ramirez g. infusions of rocuronium and cisatracurium exert different effects on rat diaphragm function. intensive care med. 2007;33(5):872-879. crossref pubmed 35. hraiech s, forel jm, papazian l. the role of neuromuscular blockers in ards: benefits and risks. curr opin crit care. 2012;18(5):495-502. crossref pubmed https://doi.org/10.1007/s00134-020-06141-z https://www.ncbi.nlm.nih.gov/pubmed/32654006 https://doi.org/10.1152/ajpregu.00231.2013 https://www.ncbi.nlm.nih.gov/pubmed/23842681 https://doi.org/10.3390/diagnostics12112890 https://www.ncbi.nlm.nih.gov/pubmed/36428947 https://doi.org/10.1164/rccm.200304-489cp https://www.ncbi.nlm.nih.gov/pubmed/14739134 https://doi.org/10.1016/s2213-2600(22)00449-0 https://www.ncbi.nlm.nih.gov/pubmed/36693401 https://doi.org/10.1016/j.nut.2022.111687 https://www.ncbi.nlm.nih.gov/pubmed/35700589 https://doi.org/10.1016/s0022-3476(88)80585-7 https://www.ncbi.nlm.nih.gov/pubmed/3142983 https://doi.org/10.1097/ccm.0b013e3181b6e760 https://www.ncbi.nlm.nih.gov/pubmed/20046120 https://doi.org/10.1164/rccm.200304-575oc https://www.ncbi.nlm.nih.gov/pubmed/15297271 https://doi.org/10.7326/0003-4819-153-4-201008170-00006 https://www.ncbi.nlm.nih.gov/pubmed/20713792 https://doi.org/10.1152/physrev.00031.2007 https://www.ncbi.nlm.nih.gov/pubmed/18923182 https://doi.org/10.1096/fj.00-0507fje https://www.ncbi.nlm.nih.gov/pubmed/11156946 https://doi.org/10.1016/s0021-9258(19)61945-9 https://www.ncbi.nlm.nih.gov/pubmed/7961805 https://doi.org/10.1016/0022-2828(95)90009-8 https://www.ncbi.nlm.nih.gov/pubmed/8523447 https://doi.org/10.1164/rccm.200805-732oc https://www.ncbi.nlm.nih.gov/pubmed/18990676 https://doi.org/10.1016/s0140-6736(02)09455-2 https://www.ncbi.nlm.nih.gov/pubmed/12133653 https://doi.org/10.1038/bjp.2008.328 https://www.ncbi.nlm.nih.gov/pubmed/19002103 https://doi.org/10.1186/s13054-015-0798-8 https://www.ncbi.nlm.nih.gov/pubmed/25888356 https://doi.org/10.24875/ric.20000380 https://www.ncbi.nlm.nih.gov/pubmed/33090120 https://doi.org/10.2165/00003495-200363230-00009 https://www.ncbi.nlm.nih.gov/pubmed/14636085 https://doi.org/10.1186/s40635-018-0218-x https://www.ncbi.nlm.nih.gov/pubmed/30659381 https://doi.org/10.1097/fjc.0000000000000859 https://www.ncbi.nlm.nih.gov/pubmed/32639325 https://doi.org/10.1186/s44158-021-00030-7 https://doi.org/10.1164/rccm.201312-2260le https://www.ncbi.nlm.nih.gov/pubmed/24684359 https://doi.org/10.1164/rccm.201107-1268oc https://www.ncbi.nlm.nih.gov/pubmed/21960535 https://doi.org/10.1007/s00134-019-05767-y https://www.ncbi.nlm.nih.gov/pubmed/31576436 https://doi.org/10.33393/dti.2021.2347 https://www.ncbi.nlm.nih.gov/pubmed/34871336 https://doi.org/10.1016/j.ijcard.2016.05.009 https://www.ncbi.nlm.nih.gov/pubmed/27232927 https://doi.org/10.1097/aln.0b013e3182608cc0 https://www.ncbi.nlm.nih.gov/pubmed/22722577 https://doi.org/10.1186/s13054-018-1992-2 https://www.ncbi.nlm.nih.gov/pubmed/29558983 https://doi.org/10.1097/01.ccm.0000245783.28478.ad https://www.ncbi.nlm.nih.gov/pubmed/17012910 https://doi.org/10.1007/s00134-007-0584-4 https://www.ncbi.nlm.nih.gov/pubmed/17361387 https://doi.org/10.1097/mcc.0b013e328357efe1 https://www.ncbi.nlm.nih.gov/pubmed/22941207 https://doi.org/10.1177/1177392817737515 drug target insights volume 11: 1–3 © the author(s) 2017 reprints and permissions: sagepub.co.uk/journalspermissions.nav doi: 10.1177/1177392817737515 creative commons non commercial cc by-nc: this article is distributed under the terms of the creative commons attribution-noncommercial 4.0 license (http://www.creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the sage and open access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage). background and introduction in march 2017, ocrelizumab (ocr) was approved by the food and drug administration (fda) for the treatment of relapsing forms of multiple sclerosis (ms) and primary-progressive multiple sclerosis. it is a humanized anti-cd20 monoclonal antibody (mab) molecule that leads the mab revolution in the treatment of ms. to understand ocr and its pharmacodynamics, a closer look at rituximab (rtx) helps one to decode ocr dosing. published literature suggests that rtx is a chimeric (human/ murine) mab directed against the human cd20 molecule1 and promotes cytotoxicity and apoptosis. it was approved by the fda for the treatment of rheumatoid arthritis (ra) in 2006 and was the first therapeutic b-cell–depleting chimeric mab to be used in ms with success. diseases such as ms, ra, neuromyelitis optica/neuromyelitis optica spectrum disorder (nmo/ nmosd), systemic lupus erythematosus, peripheral neuropathies, antimyelin-associated glycoprotein neuropathy, chronic inflammatory demyelinating polyneuropathy, subacute ataxic neuropathy without paraproteinemia, myasthenia gravis, opsoclonus-myoclonus syndrome, and inflammatory myopathies have been treated using anti-cd20 mabs. both ocr and rtx bind to an extracellular cd20 epitope on b cells whose binding site overlaps between each drug. following cd19 cell counts as a surrogate marker for cd20 cells in the peripheral blood in patients with ra, nmosd, and ms on rtx therapy helps us understand how the dosing of ocr dosing may be optimized in the treatment of ms. in general, rtx treatment produces a rapid depletion of cd20 cells from the circulation but does not directly target pro-b cells, their precursors, or plasma cells.2–3 as rtx interferes with flow cytometric analysis of cd20 cells, cd19 cells, which carry a similar expression profile, are typically used as surrogate markers to schedule reinfusion based on cd19 cell counts. it is thought that rtx binding to cd20 enables cells to mediate trogocytosis or “shaving” causing internalization of the rtx-cd20 complex and accompanying cell membrane through an fcγ receptor–dependent mechanism4–5—this process is thought to interfere with the flow cytometric analysis of cd20 cells, and therefore, cd19 cell counts serve as the surrogate marker to monitor treatment efficiency of anti-cd20 cell therapies. the depth of b-cell depletion is variable among patients, but restoration of the b-cell repertoire takes between 9 and 12 months from the last perfusion of rtx.6 in patients with ra, treatment with rtx induces depletion of peripheral b lymphocytes, with many patients demonstrating near complete depletion (cd19 counts are <20 cells/μl or below the lower limit of quantification) within 2 weeks after receiving the first dose of the drug. some patients show peripheral b-cell depletion that lasts for at least 6 months. up to ~4% of patients with ra had prolonged peripheral b-cell depletion lasting more than 3 years after a single course of rtx. equally important, some patients may need more infusions than a 6-month re-administration schedule. the reconstitution of peripheral blood b cells after rtx therapy in patients with ra was noted after a mean of 8 months posttreatment.7 anti-cd20 cell therapies in multiple sclerosis—a fixed dosing schedule for ocrelizumab is overkill jagannadha avasarala1,2 1department of medicine, division of neurology, usc school of medicine greenville, greenville, sc, usa. 2department of internal medicine, division of neurology, usc school of medicine and umg-neuroscience associates, greenville, sc, usa. abstract: anti-cd 20 therapies have found significant uses in multiple sclerosis (ms). based singularly on the accumulated evidence with the use of rituximab (rtx; rituxan, genentech, and biogen) in neuroimmunological diseases, ocrelizumab (ocr; ocrevus, genentech) was developed as a treatment option for ms and selectively targets cd20 b cells, a cell surface antigen found on pre-b cells, mature, and memory b cells, but not on lymphoid stem cells and plasma cells. on the basis of indirect evidence, elimination of the antigen-presenting capabilities and antigen nonspecific immune functions of b cells appear to be central to the therapeutic efficacy of anti-cd20 b-cell therapies. an important question is this—why does the drug need to be dosed at fixed intervals and not based on a measurable endpoint, such as tracking peripheral cd20 cell counts? there is minimal scientific validity in infusing the drug every 6 months particularly if cd20 cell counts are negligible in the peripheral blood. in this analysis, a case is made for following cd19 cell populations as a surrogate for cd20 cells on a monthly basis to guide ocr redosing parameters and does not follow a scheduled dosing parameter. keywords: ocrelizumab, multiple sclerosis, cd19/20 cells, anti-cd therapies, rituxan, dosing schedules, ocrevus, neuromyelitis optica spectrum disorder received: june 30, 2017. accepted: september 4, 2017. peer review: two peer reviewers contributed to the peer review report. reviewers’ reports totaled 230 words, excluding any confidential comments to the academic editor. type: rapid communication funding: the author(s) received no financial support for the research, authorship, and/or publication of this article. declaration of conflicting interests: the author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. corresponding author: jagannadha avasarala, umg-neuroscience associates, greenville, sc 29615, usa. email: javasarala@ghs.org 737515dti0010.1177/1177392817737515drug target insightsavasarala research-article2017 https://uk.sagepub.com/en-gb/journals-permissions mailto:javasarala@ghs.org 2 drug target insights in a study involving 107 patients with ms, 105 (98.1%) had at least 1 follow-up cd20 count after the first rtx administration and follow-up levels occurred at an average of 138.3 ± 121.4 days apart.8 the cd20 counts of patients who received 1000 mg with a concurrent 1000 mg dose 2 weeks later were above 0 by 6 months in 20% of patients, whereas 3% of patients had a cd20 count above 2% of baseline numbers. of patients receiving a single rtx 1000 mg dose, 20% had a cd20 count above 0 by 6 months, and 5% of patients had >2% by 6 months. this small, observational study in one ms care clinic clearly showed how widely different the repopulation of cd20 counts were, suggesting that a fixed time scheduling is perhaps not optimal. given the wide spectrum of unpredictable b-cell suppression, it is impossible to predict how each individual patient would respond to rtx and what the optimal dosing interval ought to be in each patient. a more obvious question is whether scheduled rtx infusions are required when cd20 counts are negligible—what is the target cell when counts are less than 20 cells/μl and what is the rationale for reinfusion? there are no hard data to support this treatment regimen. in another disease model, the treatment of nmo/nmosd with rtx tightly scheduled every 6 to 9 months to prevent relapses was not globally successful either9–11 casting doubt on the theory that treatment protocols should follow a scheduled dosing pattern. in addition, greenberg and colleagues9 retrospectively reviewed rtx dosing in an nmo clinical cohort and concluded that patients should be redosed prior to evidence of b-cell reconstitution by cd20 counts which is probably optimal and individualized. pellkofer and colleagues also reviewed rtx experience in patients with nmo, and based on their results concluded that a fixed dosing schedule every 69 months was advisable.11 studies have also shown that drugs such as rtx also deplete anti-cd20 t cells demonstrating that peripheral depletion of all cd20 cells contributes to suppression of disease.12 taken together, studies in ra, ms, and nmosd have demonstrated why a fixed dosing schedule with rtx may not be optimal. in the case of rtx, the package insert clearly notes that redosing for patients with ra is based on (1) clinical evaluation or (2) every 24 weeks. however, no such options exist for the use of ocr in patients with ms and dosing schedules are fixed. discussion the scheduled dosing of ocr for both forms of ms is slated at 6-month intervals. because ocr avidly targets cd20 cell populations and depletes them and as their numbers can be monitored by peripheral blood counts of cd19 cells, it remains poorly understood why ocr needs to be reinfused at scheduled intervals regardless of cd20 cell counts. in addition, in up to 1% (12/1311) of all patients with ms in clinical trials, relapsing and primary-progressive types, antidrug antibodies (adas), and particularly neutralizing antibodies appeared in 2 patients, clearing ocr faster and rendering b-cell repletion quicker. this is one other reason why following cd20 cells are prudent. additional validity and strength of my analysis come from the original data submitted to the center for drug evaluation and research as shown in figures 1 and 2 which depict cd19 cell populations in clinical trials at <20 cells/μl, the lower limit of quantification, at 24 weeks postinfusion. these results are derived from the clinical pharmacology and biopharmaceutics review (application no. 761053orig1s000), the document that was originally submitted to the fda for evaluation and approval of ocrevus. finally, in the package insert for ocr, one of the statements warns not to administer subsequent doses if the separation between doses is not at least 5 months. this statement is pithy but ignores the fact that repopulation of cd20 cells could also remain undetectable at 6 months postinfusion. hence, to correctly assess the need to reinfuse, following monthly cd19 cell counts is a small price to pay both in the scientific and literal sense. in addition, data on long-term ocr therapy are lacking and concern regarding prolonged b-cell depletion remains; these could come to light in postmarketing data. figure 1. median peripheral blood b-cell profiles following intravenous ocrelizumab (ocr) administration in subjects with rheumatoid arthritis in study act2847g. avasarala 3 specifically, ocr is an expensive biologic that promises to deliver clinical benefit. however, the long-term safety of repeated ocr treatment remains unknown and there is no scientific validity to giving the drug when cd20 cells are nonexistent in the periphery at counts below 20 cells/μl. any effective treatment strategy that aims to minimize unnecessary patient exposure to the drug helps with patient safety and allows for significant cost savings to the patient and third-party payers. therefore, the following recommendations are suggested. (1) if the disease activity stabilizes both clinically and from a radiological perspective, less frequent retreatment might be sufficient to prevent relapses, although the correlation between clinical/radiological criteria to disease activity is not a linear relationship and therefore must be individualized based on monthly cd20 cell counts by monitoring cd19 cells. (2) alternatively, cd20 cell counts must be monitored monthly on a routine basis irrespective of clinical or radiological status and reinfusion of the drug carried out after the cell population rebounds to ≥20 cells/μl; this holds true also for patients who develop adas that can neutralize ocr activityin which case the cd19 cell count would repopulate. author contributions ja: idea/conceptualization, data collection/collation and analysis, as well as write-up of the manuscript and submission. r efer ences 1. browning jl. b cells move to centre stage: novel opportunities for autoimmune disease treatment. nat rev drug discov. 2006;5:564–576. 2. edwards jc, cambridge g. prospects for b-cell-targeted therapy in autoimmune disease. rheumatol. (oxford). 2005;44:151–156. 3. hoyer bf, manz ra, radbruch a, hiepe f. long-lived plasma cells and their contribution to autoimmunity. ann n y acad sci. 2005;1050:124–133. 4. beum pv, kennedy ad, williams me, lindofer ma, taylor rp. the shaving reaction: rituximab/cd20 complexes are removed from mantle cell lymphoma and chronic lymphocytic leukemia cells by thp-1 monocytes. j immunol. 2006;176:2600–2609. 5. pedersen ae, jungersen mb, pedersen cd. monocytes mediate shaving of b-cell-bound anti-cd20 antibodies. immunology. 2011;133:239–245. 6. dass s, rawstron ac, vital em, henshaw k, mcgonagle d, emery p. highly sensitive b cell analysis predicts response to rituximab therapy in rheumatoid arthritis. arthritis rheum. 2008;58:2993–2999. 7. leandro mj, cambridge g, ehrenstein mr, edwards jc. reconstitution of peripheral blood b cells after depletion with rituximab in patients with rheumatoid arthritis. arthritis rheum. 2006;54:613–620. 8. barra me, soni d, huy vo k, chitnis t, stankiewicz jm. experience with long-term rituximab use in a multiple sclerosis clinic [published online ahead of print october 9, 2016]. mult scler j exp transl and clin. doi:10.1177/ 2055217316672100. 9. jacob a, weinshenker bg, violich i, et al. treatment of neuromyelitis optica with rituximab: retrospective analysis of 25 patients. arch neurol. 2008;65: 1443–1448. 10. greenberg bm, graves d, remington g, et al. rituximab dosing and monitoring strategies in neuromyelitis optica patients: creating strategies for therapeutic success. mult scler j. 2012;18:1022–1026. 11. pellkofer hl, krumbholz m, berthele a, et al. long term follow-up of patients with neuromyelitis optica after repeated therapy with rituximab. neurol. 2011;76:1310–1315. 12. palanichamy a, jahn s, nickles d, et al. rituximab efficiently depletes increased cd20-expressing t cells in multiple sclerosis patients. j of immunol. 2014;193:580–586. figure 2. median b-cell count in study wa25046 (primary-progressive multiple sclerosis). ocr indicates ocrelizumab. dti drug target insights 2023; 17: 70-77issn 1177-3928 | doi: 10.33393/dti.2022.2583review drug target insights issn 1177-3928 www.aboutscience.eu/dti © 2023 the authors. this article is published by aboutscience and licensed under creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). commercial use is not permitted and is subject to publisher’s permissions. full information is available at www.aboutscience.eu licorice as a herbal extract in periodontal therapy safiya fatima khan1, bhavya shetty1, ibrahim fazal1, asim mustafa khan2, faheem muzaffar mir3, muhamood moothedath4, vj reshma2, muhaseena muhamood2 1department of periodontology, faculty of dental sciences, ramaiah university of applied sciences, bangalore india 2department of biomedical dental sciences, college of dentistry, imam abdulrahman bin faisal university, dammam saudi arabia 3department of preventive dental sciences, college of dentistry, imam abdulrahman bin faisal university, dammam saudi arabia 4department of oral and dental health, college of applied health sciences in arrass, qassim university, buraidah saudi arabia abstract periodontal disease is caused by specific pathogens which results in inflammation of the tooth-supporting structures and subsequently causes the continued breakdown of alveolar bone and periodontal ligament. licorice (glycyrrhiza glabra) is a perennial herb with substantial medicinal value. licorice extract is derived from dried, unpeeled stolons and roots of glycyrrhiza uralensis and g. glabra. the bioactive ingredients in licorice extract such as glycyrrhizin, licoricidin, glabridin, licochalcone a, and licorisoflavan a have anti-inflammatory, antimicrobial, and anti-adherence effects that are beneficial against periodontal disease. since periodontal disease has a complex etiology that includes the host response and microorganisms, licorice phytochemicals offer a therapeutic advantage due to their dual functionality. the aim of this review was to enumerate the bioactive compounds present in herbal licorice extract and to elucidate the beneficial effects of licorice and its derivatives in periodontal therapy. literature review and clinical trials evaluating the effect of licorice on periodontopathogens and periodontal disease are included in this article. keywords: gingivitis, glycyrrhiza glabra, herbal therapy, periodontitis received: march 20, 2023 accepted: may 18, 2023 published online: june 5, 2023 corresponding author: safiya fatima khan department of periodontology faculty of dental sciences ramaiah university of applied sciences new bel road, msr nagar bangalore 560054 india safisupernova@gmail.com which is the over-production of mediators of inflammation such as cytokines, prostaglandins, and matrix metalloproteinases (mmps), which regulate the continuance of periodontal disease (3). periodontal therapy primarily includes scaling and root planing to eliminate the local factors, that is, plaque and calculus, and to maintain satisfactory oral hygiene. since periodontal disease is known to be an inflammatory condition with a microbial etiology, the adjunctive use of locally applied or systemic administration of antimicrobials and/or host response-modulating medications has been suggested. conventional synthetic agents such as chlorhexidine products which are used therapeutically and prophylactically in dentistry have some disadvantages such as altered taste sensation, tooth staining, and resistance to bacteria, which limit their usage over a long term (4). therefore, innovative strategies need to be developed against periodontal diseases, such as exploring the extensively available medicinal plants. the active ingredients in medicinal plants restore health, with maximum efficiency and minimal side effects. herbal extracts incorporated into medications have been found to be safe and efficacious for the treatment of several oral health conditions such as gingival bleeding, dental caries, halitosis, and mouth ulcers. the extracts obtained from aloe vera, green tea plant, neem, tulsi, propolis, rosemary, meswak, turmeric, chamomile, tea tree oil, peppermint oil, cranberry, clove, ginger, etc. have been used commonly for introduction periodontal disease is caused by specific pathogens which results in inflammation of the tooth-supporting structures and subsequently causes the continued breakdown of alveolar bone and periodontal ligament. the pathogenesis of periodontal diseases involves two major causative factors. the first is the microbial factor, that is, the presence of increased levels of periodontopathogenic bacteria in subgingival tissues, which causes periodontal destruction by producing proteinases and toxins (1,2). the pathogens associated with periodontitis are porphyromonas gingiva­ lis, treponema denticola, aggregatibacter actinomycetem­ comitans, and tannerella forsythia. the other factor is the immune response of the host to the periodontal pathogens, https://doi.org/10.33393/dti.2023.2583 https://creativecommons.org/licenses/by-nc/4.0/legalcode khan et al drug target insights 2023; 17: 71 © 2023 the authors. published by aboutscience www.aboutscience.eu the prevention and treatment of different oral diseases. herbal extracts contain phytochemicals that are responsible for the desired anti-inflammatory and antimicrobial effects (5). herbal formulations are gaining widespread attention as they do not contain artificial preservatives, alcohol, colors, or flavors, which are commonly found in other drugstore products. one such herb with medicinal properties is licorice (glycyrrhiza glabra). licorice, synonym being sweet wood, is found in the mediterranean region and in a few regions of asia. licorice is a perennial herb that holds a sweet taste and has widespread pharmacological effects on human beings. licorice extract is derived from the dried, unpeeled stolons and roots of g lycyrrhiza uralensis and g. glabra. however, limited information is available on the use of this herb in periodontal therapy. the mechanism of action by which licorice works against periodontal diseases has not been elucidated in previous studies. the aim of this review was to enumerate the bioactive compounds present in herbal licorice extract and to elucidate the beneficial effects of licorice and its derivatives in periodontal therapy. methodology the literature search was performed using three databases which included pubmed, google scholar, and cochrane, in addition to searching reference lists of original and review articles. the combination of the following keywords was used to search for relevant articles: “licorice,” “glycyrrhiza glabra,” “periodontal therapy,” and “periodontal disease.” relevant studies published between 1985 and 2022 were selected. only articles in english language were considered, and unpublished data were not sought. two reviewers obtained information on the quality and characteristics of the included studies. components in licorice extract licorice is a potential source of natural anti-inflammatory agents. its major active component is glycyrrhetinic acid (ga) that is derived from licorice root extract. major phytochemicals found in licorice are shown in table i (fig. 1) (6). safe usage of licorice the food and drug administration (fda) has labeled licorice as “generally recognized as safe.” it has been suggested to be safe when used in minimal quantities by people who are not allergic to glycyrrhizin (7,8). intake of excessive quantity, that is, over 200 mg, of licorice may cause hypertension, hypokalemia, rhabdomyolysis, respiratory impairment, muscle paralysis, hyperparathyroidism, acute renal failure, and encephalopathy (9). according to the world health organization (who), 100 mg/day of licorice can be used safely without adverse effects. a potential risk of excessive bleeding may be seen in patients using medications for anti-clotting for cerebrovascular or cardiovascular diseases in conjunction with licorice-containing herbal medications due to its antiplatelet and anticoagulant effects (10). table i important phytochemicals in licorice root group bioactive compounds aurones licoagroaurone benzofurans licocoumarone chalcones isoliquiritigenin, licochalcone a coumarins glycerol, glabrocoumarone, glycocoumarin, licofuranocoumarin, glabrocoumarin flavonoids glabrol, liquiritigenin isoflavonoids glabridin, glabrone, licoricidin, licoisoflavones a and b, licorisoflavan a pterocarpenes glycyrrhizol a saponins glycyrrhizin, glycyrrhizic acid, 18β-glycyrrhetinic acid, liquiritic acid, glabrolide stilbenes gancaonin g fig. 1 chemical structure of glycyrrhetinic acid. effects of licorice licorice constituents have shown antimicrobial (11), antiviral (12), anti-inflammatory (13), antidiabetic, antitumor, immunoregulatory (14), sedative (15), antidepressive (16), estrogenic (17,18), antioxidant (19), hepatoprotective (20), neuroprotective activities (21), and skin effects (22). the active constituents of licorice extract have a potential role on the oral microorganisms as well as the host response involved in orodental diseases like periodontitis, dental caries, recurrent aphthous ulcers, and candidiasis. antiviral effects – licorice extracts inhibit the growth of viruses such as herpes simplex, influenza virus, and vesicular stomatitis virus. glycyrrhizin prevents the replication of viruses and interferes with viral binding (12). antidiabetic activity – glycycoumarin, glycerin, etc., present in g. glabra extracts lower blood glucose level by binding to peroxisome proliferator-activated receptor (ppar) gamma. glabridin helps in efficient glucose utilization and prevents glucose intolerance by translocation of glut-4 (14). licorice in periodontal therapy72 © 2023 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti antitumor activity – 18-β-ga and glycyrrhizic acids induce mitochondrial permeability transition causing tumor cell apoptosis (14). immunoregulatory effect – glycyrrhiza extracts stimulate the immune system by production of macrophages and lymphocytes, and increasing the phagocytic capacity of neutrophils. it prevented the accumulation of immune complexes involved in autoimmune diseases such as systemic lupus erythematosus (14). sedative effect – glabridin shows sedative and hypotonic effects by positively modulating the gamma-aminobutyric acid (gaba) receptors (15). antidepressive effect – licorice shows antidepressive effects by inhibition of monoamine oxidase and increasing epinephrine and dopamine levels in the brain (16). estrogenic effect – licorice extracts show estrogenic activity through uterine retention and vaginal opening. isoflavones present in licorice can influence sexual development, impair estrus cycling, and alter the proper functioning of the ovarian, hypothalamus, and pituitary glands. glabridin can be used as a treatment for menopausal symptoms (17,18). hepatoprotective activities – glycyrrhizin has shown improved liver histology and reduced serum aminotransferases. it shows hepatoprotective effect against ccl4-induced oxidative stress, prevents oxidative and hepatic damage caused due to aflatoxin, and improves liver function (20). neuroprotective activities – licorice has an antioxidant activity that can reduce brain damage by eliminating or utilizing the free radicals and improving neural function and memory (21). skin effects – licorice is popular in treating dermatitis, pruritus, cysts, and eczema. it is also used for cosmetic formulation as a depigmenting agent to inhibit the tyrosinase enzyme (22). mechanism of action the beneficial effects of licorice can be due to various mechanisms. antimicrobial activity microbial growth is selectively inhibited by the isoprenoid phenols present in g. glabra. the presence of secondary metabolites, such as alkaloids, saponins, flavonoids, and alkaloids, is responsible for the antibacterial activity (23,24). the reduction in bacterial gene expression, decrease in growth of bacteria, and inhibition of production of bacterial toxins are suggested as the mechanism behind this (24,25). licorice extract showed antimicrobial effects against p. gingivalis with minimum inhibitory concentration (mbc) and minimum bactericidal concentration (mic) of 25 and 62.5 μg/ml, respectively (26). glycyrrhizol a showed a strong antibacterial effect against streptococcus mutans with mic of 1 μg/ml. ga at an appropriate concentration has good efficacy against isolated periodontopathogenic and capnophilic bacteria (27). the mics of ga were 8, 16, and 8 mg/l for a. actino­ mycetemcomitans, eikenella corrodens, and capnocytophaga, respectively, and the mbc was 16 mg/l for all species (28). antioxidant activity licorice phytochemicals exhibit significant antioxidant activity. licorice hinders the synthesis of reactive oxygen species (ros) by neutrophils at the site of inflammation. g. glabra contains licochalcones b and d, which show powerful scavenging activity on dpph (2,2-diphenyl-1-picryl-hydrazyl-hydrate) radical and also have the ability to prevent the peroxidation of microsomal lipids (29,30). anti‐inflammatory activity ga activates signaling of glucocorticoid receptors since its chemical structure resembles the glucocorticoids, and it also inhibits the classical complement pathway, both of which are responsible for its anti-inflammatory properties. preclinical studies have also shown that licorice inhibits synthesis of prostaglandins and cyclooxygenase activity, and also indirectly inhibits aggregation of platelets as well as the components of the inflammatory cascade (31). licorice extract prevents the phosphorylation of proteins involved in intracellular signaling of macrophages, such as the transcription factors, nuclear factor-kappa b, and activator protein (ap) 1, which play an important role in the pathways of inflammatory signaling (32). licorice in periodontal disease the bioactive ingredients in licorice like glycyrrhizin, glabridin, licorisoflavan a, licochalcone a, and licoricidin are effective against periodontal disease. table ii summarizes the studies which suggest the potential use of licorice in periodontal therapy. these ingredients exhibit antimicrobial, anti-inflammatory, and anti-adherence effects (fig. 2). since the etiology of periodontal diseases is complex involving the periodontal pathogens and host immune response, dual functionality compounds such as the phytochemicals in licorice offer therapeutic superiority. phytochemicals are structurally distinct from the conventional microbial-derived antibiotics, and so are advantageous as antimicrobials. the phytochemicals act against different bacterial strains by inhibiting the efflux pumps, inhibiting the cell wall biosynthesis by interacting with the cell membrane, and by inhibition of enzymes such as dihydrofolate reductase, urease, and sortase a (51). thus, their mechanisms of action are different from classic substances and against which microbial resistance does not develop. the ability of licorice to inhibit the formation of dental plaque enhances its significance in the treatment of periodontal disease. at high concentrations of licorice extract (5%-10%), there was a slight reduction in bacterial growth and formation of plaque was completely inhibited. no effect was seen on either adherence or growth with lower concentrations. at high concentrations (0.5%-1%) of its pure active component, that is, glycyrrhizin, there was a complete inhibition in the adherence, whereas partial inhibition was observed at lower concentrations. the surface activity of glycyrrhizin could be responsible for its inhibitory effect, as khan et al drug target insights 2023; 17: 73 © 2023 the authors. published by aboutscience www.aboutscience.eu table ii list of studies suggesting the potential use of licorice in periodontal therapy authors study design findings sharma et al 2022 (33) randomized controlled trial both licorice and chlorhexidine mouthwash inhibited the accumulation of plaque and inflammation of the gingiva. the herbal mouthwash was shown to be effective as a self-care treatment since chemical formulations are associated with adverse effects with long-term usage. madan et al 2019 (34) randomized clinical trial bleeding of the gingiva, probing pocket depth, and attachment loss were significantly decreased in patients using glycyrrhiza glabra gum paint in 10% concentration. it can be used for longer periods to prevent and treat periodontal disease as they do not have any side effects, and thus, it is also effective as an alternative for synthetic agents. takamori et al 2018 (35) animal study loss of attachment, immune complex formation, and inflammatory cell infiltration were greater in the lipopolysaccharide (lps) group than in the control, and were completely reduced in the glycyrrhetinic acid (ga) groups. increased alveolar bone destruction was seen in the lps group than in the ga or control groups. hence, in the experimentally induced periodontitis model in rats, ga had the ability to reduce periodontal destruction. suwannakul and chaibenjawong 2017 (26) in vitro study licorice extract showed antimicrobial effects against porphyromonas gingivalis with mbc and mic of 25 and 62.5 μg/ml respectively. it also reduced the quantity of biofilm and the activities of argand kgp-proteases. salehi et al 2017 (36) double-blind clinical trial mucoadhesive tablets containing licorice extract can relieve pain, decrease the diameter of the ulcer and the inflammation around it, and improve the recovery in aphthous stomatitis. jain et al 2017 (37) randomized clinical trial licorice mouthwash reduced the accumulation of plaque and inflammation of gingiva, without any tooth discoloration or unpleasant taste sensation. shivprasad et al 2017 (38) randomized controlled trial subgingivally delivered licorice as an adjunctive treatment modality to scaling and root planing showed clinical and microbiological benefits in periodontal therapy, with a reduction in the prevalence of p. gingivalis. ali and mohammed 2016 (39) comparative human study licorice extract based mouthwash inhibits plaque formation and inflammation of gingiva without any adverse effects. therefore, it can be used as an adjunctive to scaling and root planing in periodontal treatment. hamdon et al 2014 (40) human study licorice extract showed antibacterial effects against aggregatibacter actinomycetemcomitans, based on antimicrobial sensitivity tests. the antibacterial effect was greater against planktonic cells as compared to the cells within the biofilm. it produced an inhibition zone similar to tetracyclines with a concentration of 250 μg. kim et al 2013 (41) invitro study 18α-ga is effective in the treatment of vascular diseases caused by p. gingivalis. it reduces vascular permeability induced by lps by inhibiting il-8 production from the endothelium. farhad et al 2013 (42) experimental human study a significant reduction of mmp-8 concentration was seen in both licorice and doxycycline groups than in the placebo group. the licorice group showed better reduction of mmp-8 concentration than doxycycline group, which was not statistically significant. hence, licorice extract can be as potent as antibiotics such as doxycycline to treat periodontal diseases by preventing the mmp production by host cells. kim et al 2012 (43) in vitro study glabridin inhibits activation of signaling molecules induced by rankl and other transcription factors of osteoclast precursors, and so it can be used to inhibit osteoclastogenesis. zhu et al 2012 (44) animal study isoliquiritigenin (isl) inhibits osteoclastogenesis induced by rankl and bone loss by various signaling pathways. hence, it has the potential to be used as a therapeutic or preventive agent for the treatment of lytic bone diseases. feldman et al 2012 (45) in vitro study licochalcone a inhibits the two primary causative factors of periodontitis, i.e., formation of biofilm with p. gingivalis and the immune response of the host. la et al 2011 (46) in vitro study licoricidin (lc) and licorisoflavan a (lia) inhibited the production of il-6, mmp-7, -8, and -9 in macrophages. they can be used to treat mmp and cytokine-mediated conditions such as periodontal disease, and are potent host-modulating agents. bodet et al 2008 (32) in vitro study licorice extract showed anti-inflammatory effects by reducing the il-1b, -6, -8 and tnf-α responses of macrophages induced by lps. it is a potential therapeutic agent to prevent or treat the tissue destruction caused due to periodontal disease. wittschier et al 2006 (47) in vitro study the polysaccharides in g. glabra inhibit bacterial adhesion and thus can be potential therapeutic agents against bacterial infection. he et al 2006 (27) in vitro study glycyrrhizol b and gancaonin g showed moderate antibacterial effect against streptococcus mutans, while glycyrrhizol a showed strong antibacterial effect with mic of 1 μg/ml. hence, the roots of glycyrrhiza uralensis contain isoflavones which exhibit antibacterial effects. licorice in periodontal therapy74 © 2023 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti authors study design findings choi 2005 (48) animal study glabridin, an estrogenic plant product, stimulates the in vitro formation of bone in cultured osteoblasts. thus, glabridin can be a potent agent in the management of osteoporosis. salari et al 2003 (49) human study enoxolone with the mentioned concentrations is effective against isolated periodontopathogenic and capnophilic bacteria. its mics were 8 µg/ml for a. actinomycetemcomitans and capnocytophaga species, and 16 µg/ml for eikenella corrodens. the mbc was also 16 µg/ml for all the microorganisms. salari and kadkhoda 2003 (28) in vitro study ga at an appropriate concentration has good efficacy against isolated periodontopathogenic and capnophilic bacteria. the mics of ga were 8, 16, and 8 mg/l for a. actinomycetemcomitans, eikenella corrodens and capnocytophaga, respectively, and the mbc was 16 mg/l for all species. saeedi et al 2003 (50) randomized, controlled trial edema, erythema, and itching were more effectively reduced with 2% licorice topical gel than with 1% gel in 2 weeks. il = interleukin; mbc = minimum inhibitory concentration; mic = minimum bactericidal concentration; mmp = matrix metalloproteinase; tnf = tumor necrosis factor. fig. 2 mechanism of action of licorice in periodontal therapy. bacterial adherence and growth are known to be affected by surfactants. bacterial adherence can also be inhibited by the adsorption of glycyrrhizin onto smooth surfaces. licorice extract shows minimal antibacterial activity along with its effect on inhibition of plaque. glycyrrhizin as a vehicle can be effective for topical agents used orally due to its sweetness, good dispersing properties, and the ability to remain stable in the form of aqueous gels. this suggests that the balance of the oral microbial flora will not be affected on using glycyrrhizin as an oral medication (52). phytochemicals of g. glabra reduce bacterial growth and inhibit the mediators of inflammation at the infection site. it also inhibits the activity of osteoclasts responsible for destruction of alveolar bone in periodontal disease and promotes the formation of bone by stimulating osteoblastogenesis. high amounts of inflammatory markers like interleukin (il)-1β, il-2, il-6, il-8, tumor necrosis factor (tnf)-α, and rankl are present in patients with periodontal disease. licorice extract showed potent anti-inflammatory properties by inhibiting these proinflammatory mediators stimulated by lipopolysaccharide (lps) from a. actinomycetemcomitans and p. gingivalis (32). resorption of alveolar bone is an important feature of periodontitis. the differentiation, activation, and survival of khan et al drug target insights 2023; 17: 75 © 2023 the authors. published by aboutscience www.aboutscience.eu osteoclasts are regulated by rankl leading to bone resorption. glabridin can be used to inhibit osteoclastogenesis by preventing the activation of signaling molecules induced by rankl and subsequent transcription factors for osteoclast precursors, suggesting its therapeutic potential (34). recommendations for future research though the use of herbals for medicinal purpose is traced back to several centuries, it is only in recent evidence-based era that methodical and systematic approaches to study their properties have been reinstituted. this has sparked a wide interest for their application in all healthcare specialties including periodontal therapy. the beneficial phytochemicals in licorice must be studied so as to incorporate these herbal extracts in oral care products that may be useful in dental therapy. in vitro studies have shown the capability of licorice and its bioactive components in periodontal therapy; however, most of the clinical studies have limitations pertaining to the design of the study and the number of participants included in the study, which makes them statistically insignificant. thus, further clinical studies need to be carried out to investigate the oral care products containing licorice extracts, in the forms of toothpaste, mouthwash chewing gum, and gel to be able to validate its beneficial effects. the local application of these bioactive compounds would be more suitable. for example, the local application of a licorice-based gel into sites with periodontal disease permits the bioactive ingredients to be released slowly, which will act locally on the periodontal pathogens and the host immune response, the two contributory factors in the destruction of periodontal tissues. further research focusing on the in vivo anti-inflammatory/antimicrobial effects of licorice is required on larger sample sizes to better understand its specific role in the management of periodontitis. clinical trials evaluating the effect of licorice extract on periodontopathogens and inflammatory cytokines would be recommended. conclusion the usage of herbal agents for the treatment of periodontal disease is considered as an intriguing alternative to conventional antibiotics due to their lesser negative effects and to overcome drug resistance during treatment. licorice extracts exhibit a wide range of biological effects such as antiinflammatory, antioxidant, and antimicrobial activities. it has the ability to prevent the release of proinflammatory mediators and mmps from host cells, and hence it is a potent agent for periodontal therapy. the bioactive ingredients present in this herb help in reducing loss of alveolar bone, which is commonly associated with periodontal disease. it should also be emphasized that no adverse effects have been seen with the use of licorice extracts. hence, local application of licoricecontaining agents into the diseased periodontal sites can be beneficial, which would act locally on periodontopathogens and the host inflammatory response. disclosures conflict of interest: the authors declare no conflict of interest. financial support: this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. references 1. o’brien-simpson nm, veith pd, dashper sg, reynolds ec. antigens of bacteria associated with periodontitis. perio 2000. 2004;35(1):101-134. crossref pubmed 2. feng z, weinberg a. role of bacteria in health and disease of periodontal tissues. perio 2000. 2006;40(1):50-76. crossref pubmed 3. garlet gp. destructive and protective roles of cytokines in periodontitis: a re-appraisal from host defense and tissue destruction viewpoints. j dent res. 2010;89(12):1349-1363. crossref pubmed 4. poppolo deus f, ouanounou a. chlorhexidine in dentistry: pharmacology, uses, and adverse effects. int dent j. 2022;72(3):269-277. crossref pubmed 5. şener b, kiliç m. herbal extracts used in dental disorders. biomed j sci tech res. 2019;19(1):14107-14111. crossref 6. sidhu p, shankargouda s, rath a, hesarghatta ramamurthy p, fernandes b, kumar singh a. therapeutic benefits of liquorice in dentistry. j ayurveda integr med. 2020;11(1):82-88. crossref pubmed 7. isbrucker ra, burdock ga. risk and safety assessment on the consumption of licorice root (glycyrrhiza sp.), its extract and powder as a food ingredient, with emphasis on the pharmacology and toxicology of glycyrrhizin. regul toxicol pharmacol. 2006;46(3):167-192. crossref pubmed 8. messier c, epifano f, genovese s, grenier d. licorice and its potential beneficial effects in common oro-dental diseases. oral dis. 2012;18(1):32-39. crossref pubmed 9. 9. yasue h, itoh t, mizuno y, harada e. severe hypokalemia, rhabdomyolysis, muscle paralysis, and respiratory impairment in a hypertensive patient taking herbal medicines containing licorice. intern med. 2007;46(9):575-578. crossref pubmed 10. 1tsai hh, lin hw, lu yh, chen yl, mahady gb. a review of potential harmful interactions between anticoagulant/antiplatelet agents and chinese herbal medicines. plos one. 2013;8(5):e64255. crossref pubmed 11. long dr, mead j, hendricks jm, hardy me, voyich jm. 18β glycyrrhetinic acid inhibits methicillin-resistant staphylococcus aureus survival and attenuates virulence gene expression. antimicrob agents chemother. 2013;57(1):241-247. crossref pubmed 12. feng yeh c, wang kc, chiang lc, shieh de, yen mh, san chang j. water extract of licorice had anti-viral activity against human respiratory syncytial virus in human respiratory tract cell lines. j ethnopharmacol. 2013;148(2):466-473. crossref pubmed 13. chandrasekaran cv, deepak hb, thiyagarajan p, et al. dual inhibitory effect of glycyrrhiza glabra (gutgard™) on cox and lox products. phytomedicine. 2011;18(4):278-284. crossref pubmed 14. li s, zhu jh, cao lp, et al. growth inhibitory in vitro effects of glycyrrhizic acid in u251 glioblastoma cell line. neurol sci. 2014;35(7):1115-1120. crossref pubmed 15. jin z, kim s, cho s, kim ih, han d, jin yh. potentiating effect of glabridin on gabaa receptor-mediated responses in dorsal raphe neurons. planta med. 2013;79(15):1408-1412. crossref pubmed https://doi.org/10.1111/j.0906-6713.2004.003559.x https://www.ncbi.nlm.nih.gov/pubmed/15107060 https://doi.org/10.1111/j.1600-0757.2005.00148.x https://www.ncbi.nlm.nih.gov/pubmed/16398685 https://doi.org/10.1177/0022034510376402 https://www.ncbi.nlm.nih.gov/pubmed/20739705 https://doi.org/10.1016/j.identj.2022.01.005 https://www.ncbi.nlm.nih.gov/pubmed/35287956 https://doi.org/10.26717/bjstr.2019.19.003254 https://doi.org/10.1016/j.jaim.2017.12.004 https://www.ncbi.nlm.nih.gov/pubmed/30391123 https://doi.org/10.1016/j.yrtph.2006.06.002 https://www.ncbi.nlm.nih.gov/pubmed/16884839 https://doi.org/10.1111/j.1601-0825.2011.01842.x https://www.ncbi.nlm.nih.gov/pubmed/21851508 https://doi.org/10.2169/internalmedicine.46.6316 https://www.ncbi.nlm.nih.gov/pubmed/17473492 https://doi.org/10.1371/journal.pone.0064255 https://www.ncbi.nlm.nih.gov/pubmed/23671711 https://doi.org/10.1128/aac.01023-12 https://www.ncbi.nlm.nih.gov/pubmed/23114775 https://doi.org/10.1016/j.jep.2013.04.040 https://www.ncbi.nlm.nih.gov/pubmed/23643542 https://doi.org/10.1016/j.phymed.2010.08.001 https://www.ncbi.nlm.nih.gov/pubmed/20864324 https://doi.org/10.1007/s10072-014-1661-4 https://www.ncbi.nlm.nih.gov/pubmed/24514918 https://doi.org/10.1055/s-0033-1350698 https://www.ncbi.nlm.nih.gov/pubmed/23975867 licorice in periodontal therapy76 © 2023 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti 16. dhingra d, sharma a. antidepressant-like activity of glycyrrhiza glabra l. in mouse models of immobility tests. prog neuropsychopharmacol biol psychiatry. 2006;30(3):449-454. crossref pubmed 17. sharma g, kar s, palit s, das pk. 18β-glycyrrhetinic acid in duces apoptosis through modulation of akt/foxo3a/bim pathway in human breast cancer mcf-7 cells. j cell physiol. 2012;227(5):1923-1931. crossref pubmed 18. su wei poh m, voon chen yong p, viseswaran n, chia yy. estrogenicity of glabridin in ishikawa cells. plos one. 2015;10(3):e0121382. crossref pubmed 19. singh v, pal a, darokar mp. a polyphenolic flavonoid glabridin: oxidative stress response in multidrug-resistant staphylococcus aureus. free radic biol med. 2015;87:48-57. crossref pubmed 20. sharifzadeh m, shamsa f, shiran s, et al. a time course analysis of systemic administration of aqueous licorice extract on spatial memory retention in rats. planta med. 2008;74(5):485490. crossref pubmed 21. michel he, tadros mg, abdel-naim ab, khalifa ae. prepulse inhibition (ppi) disrupting effects of glycyrrhiza glabra extract in mice: a possible role of monoamines. neurosci lett. 2013; 544:110-114. crossref pubmed 22. halder rm, richards gm. topical agents used in the management of hyperpigmentation. skin therapy lett. 2004;9(6):1-3. pubmed 23. fukui h, goto k, tabata m. two antimicrobial flavanones from the leaves of glycyrrhiza glabra. chem pharm bull (tokyo). 1988;36(10):4174-4176. crossref pubmed 24. wang l, yang r, yuan b, liu y, liu c. the antiviral and antimicrobial activities of licorice, a widely-used chinese herb. acta pharm sin b. 2015;5(4):310-315. crossref pubmed 25. gupta vk, fatima a, faridi u, et al. antimicrobial potential of glycyrrhiza glabra roots. j ethnopharmacol. 2008;116(2): 377-380. crossref pubmed 26. suwannakul s, chaibenjawong p. antibacterial activities of glycyrrhiza glabra linn. (licorice) root extract against por­ phyromonas gingivalis and its inhibitory effects. j dent indones. 2017;24(3):85-92. crossref 27. he j, chen l, heber d, shi w, lu qy. antibacterial compounds from glycyrrhiza uralensis. j nat prod. 2006;69(1):121-124. crossref pubmed 28. salari mh, kadkhoda z. in vitro antibacterial effects of glycyrrhetinic acid on periodontopathogenic and capnophilic bacteria isolated from adult periodontitis. clin microbiol infect. 2003;9(9):987-988. crossref pubmed 29. biondi dm, rocco c, ruberto g. new dihydrostilbene derivatives from the leaves of glycyrrhiza glabra and evaluation of their antioxidant activity. j nat prod. 2003;66(4):477-480. crossref pubmed 30. sharma v, katiyar a, agrawal rc. glycyrrhiza glabra: chemistry and pharmacological activity. sweeteners; 2018:87-100. crossref 31. hasan mk, ara i, mondal msa, kabir y. phytochemistry, pharmacological activity, and potential health benefits of glycyrrhiza glabra. heliyon. 2021;7(6):e07240. crossref pubmed 32. bodet c, la vd, gafner s, bergeron c, grenier d. a licorice extract reduces lipopolysaccharide-induced proinflammatory cytokine secretion by macrophages and whole blood. j periodontol. 2008;79(9):1752-1761. crossref pubmed 33. sharma s, sogi gm, saini v, chakraborty t, sudan j. effect of liquorice (root extract) mouth rinse on dental plaque and gingivitis a randomized controlled clinical trial. j indian soc periodontol. 2022;26(1):51-57. crossref pubmed 34. madan s, kashyap s, mathur g. glycyrrhiza glabra: an efficient medicinal plant for control of periodontitis – a randomized clinical trial. j int clin dent res organ. 2019;11(1):32-35. crossref 35. takamori a, yoshinaga y, ukai t, et al. topical application of glycyrrhetinic acid in the gingival sulcus inhibits attachment loss in lipopolysaccharide-induced experimental periodontitis in rats. j periodontal res. 2018;53(3):422-429. crossref pubmed 36. salehi m, saeedi m, ehsani h, et al. analyzing glycyrrhiza glabra (licorice) extract efficacy in recurrent aphthous stomatitis recovery. j res med dent sci. 2018;6(1):68-75. online 37. jain p, sontakke p, walia s, yadav p, biswas g, kaur d. assessment of the efficacy of licorice versus 0.2% chlorhexidine oral rinse on plaque-induced gingivitis: a randomized clinical trial. indian j oral health res. 2017;3(1):15-18. crossref 38. shivprasad bm, sonali c, navnita s, shilpa s, sruthi kn, savita s. liquorice as an adjunct to scaling and root planing in the treatment of chronic periodontitis: a clinico-microbiological study. int j sci res. 2017;6(7):73-76. crossref 39. ali a, mohammed r. the iraqi method of natural liquorice as a mouth rinse and its effect in patient with chronic periodontitis. iraqi dent j. 2016;38(1):43-47. crossref 40. hamdon sm, ghada y, rahman a. glycyrrhiza glabara as antibacterial agent on biofilm and planktonic cell of aggre­ gatibacter actinomycetemcomitans. int j dent sci res. 2014; 2:42-46. crossref 41. kim sr, jeon hj, park hj, et al. glycyrrhetinic acid inhibits porphyromonas gingivalis lipopolysaccharide-induced vascular permeability via the suppression of interleukin-8. inflamm res. 2013;62(2):145-154. crossref pubmed 42. farhad sz, aminzadeh a, mafi m, barekatain m, naghney m, ghafari mr. the effect of adjunctive low-dose doxycycline and licorice therapy on gingival crevicular fluid matrix metalloproteinase-8 levels in chronic periodontitis. dent res j (isfahan). 2013;10(5):624-629. pubmed 43. kim hs, suh ks, sul d, kim bj, lee sk, jung ww. the inhibitory effect and the molecular mechanism of glabridin on ranklinduced osteoclastogenesis in raw264.7 cells. int j mol med. 2012;29(2):169-177. crossref pubmed 44. zhu l, wei h, wu y, et al. licorice isoliquiritigenin suppresses rankl-induced osteoclastogenesis in vitro and prevents inflammatory bone loss in vivo. int j biochem cell biol. 2012;44(7):1139-1152. crossref pubmed 45. feldman m, grenier d. cranberry proanthocyanidins act in synergy with licochalcone a to reduce porphyromonas gingivalis growth and virulence properties, and to suppress cytokine secretion by macrophages. j appl microbiol. 2012;113(2):438447. crossref pubmed 46. la vd, tanabe s, bergeron c, gafner s, grenier d. modulation of matrix metalloproteinase and cytokine production by licorice isolates licoricidin and licorisoflavan a: potential therapeutic approach for periodontitis. j periodontol. 2011;82(1):122-128. crossref pubmed 47. wittschier n, faller g, beikler t, stratmann u, hensel a. polysaccharides from glycyrrhiza glabra l. exert significant anti-adhesive effects against helicobacter pylori and porphyromonas gingivalis. planta med. 2006;72(11):238. crossref 48. choi em. the licorice root derived isoflavan glabridin increases the function of osteoblastic mc3t3-e1 cells. biochem pharmacol. 2005;70(3):363-368. crossref pubmed 49. salari mh, sohrabi n, kadkhoda z, khalili mb. antibacterial effects of enoxolone on periodontopathogenic and capnophilic https://doi.org/10.1016/j.pnpbp.2005.11.019 https://www.ncbi.nlm.nih.gov/pubmed/16443316 https://doi.org/10.1002/jcp.22920 https://www.ncbi.nlm.nih.gov/pubmed/21732363 https://doi.org/10.1371/journal.pone.0121382 https://www.ncbi.nlm.nih.gov/pubmed/25816349 https://doi.org/10.1016/j.freeradbiomed.2015.06.016 https://www.ncbi.nlm.nih.gov/pubmed/26117328 https://doi.org/10.1055/s-2008-1074494 https://www.ncbi.nlm.nih.gov/pubmed/18404595 https://doi.org/10.1016/j.neulet.2013.03.055 https://www.ncbi.nlm.nih.gov/pubmed/23583589 https://www.ncbi.nlm.nih.gov/pubmed/15334278 https://doi.org/10.1248/cpb.36.4174 https://www.ncbi.nlm.nih.gov/pubmed/3245990 https://doi.org/10.1016/j.apsb.2015.05.005 https://www.ncbi.nlm.nih.gov/pubmed/26579460 https://doi.org/10.1016/j.jep.2007.11.037 https://www.ncbi.nlm.nih.gov/pubmed/18182260 https://doi.org/10.14693/jdi.v24i3.1075 https://doi.org/10.1021/np058069d https://www.ncbi.nlm.nih.gov/pubmed/16441081 https://doi.org/10.1046/j.1469-0691.2003.00721.x https://www.ncbi.nlm.nih.gov/pubmed/14616694 https://doi.org/10.1021/np020365s https://www.ncbi.nlm.nih.gov/pubmed/12713396 https://doi.org/10.1007/978-3-319-27027-2_21 https://doi.org/10.1016/j.heliyon.2021.e07240 https://www.ncbi.nlm.nih.gov/pubmed/34189299 https://doi.org/10.1902/jop.2008.080052 https://www.ncbi.nlm.nih.gov/pubmed/18771378 https://doi.org/10.4103/jisp.jisp_517_20 https://www.ncbi.nlm.nih.gov/pubmed/35136317 https://doi.org/10.4103/jicdro.jicdro_7_19 https://doi.org/10.1111/jre.12529 https://www.ncbi.nlm.nih.gov/pubmed/29446076 https://www.jrmds.in/abstract/analyzing-glycyrrhiza-glabra-licorice-extract-efficacy-in-recurrent-aphthous-stomatitis-recovery-1736.html https://doi.org/10.4103/ijohr.ijohr_18_17 https://doi.org/10.36106/ijsr https://doi.org/10.26477/idj.v38i1.72 https://doi.org/10.12691/ijdsr-2-2-4 https://doi.org/10.1007/s00011-012-0560-5 https://www.ncbi.nlm.nih.gov/pubmed/23064654 https://www.ncbi.nlm.nih.gov/pubmed/24348620 https://doi.org/10.3892/ijmm.2011.822 https://www.ncbi.nlm.nih.gov/pubmed/22038020 https://doi.org/10.1016/j.biocel.2012.04.003 https://www.ncbi.nlm.nih.gov/pubmed/22521613 https://doi.org/10.1111/j.1365-2672.2012.05329.x https://www.ncbi.nlm.nih.gov/pubmed/22563853 https://doi.org/10.1902/jop.2010.100342 https://www.ncbi.nlm.nih.gov/pubmed/20722535 https://doi.org/10.1055/s-2006-950038 https://doi.org/10.1016/j.bcp.2005.04.019 https://www.ncbi.nlm.nih.gov/pubmed/15922308 khan et al drug target insights 2023; 17: 77 © 2023 the authors. published by aboutscience www.aboutscience.eu bacteria isolated from specimens of periodontitis patients. iran biomed j. 2003;7(1):39-42. 50. saeedi m, morteza-semnani k, ghoreishi mr. the treatment of atopic dermatitis with licorice gel. j dermatolog treat. 2003;14(3):153-157. crossref pubmed 51. khameneh b, iranshahy m, soheili v, fazly bazzaz bs. review on plant antimicrobials: a mechanistic viewpoint. antimicrob resist infect control. 2019;8(118):118. crossref pubmed 52. segal r, pisanty s, wormser r, azaz e, sela mn. anticariogenic activity of licorice and glycyrrhizine i: inhibition of in vitro plaque formation by streptococcus mutans. j pharm sci. 1985;74(1):79-81. crossref pubmed https://doi.org/10.1080/09546630310014369 https://www.ncbi.nlm.nih.gov/pubmed/14522625 https://doi.org/10.1186/s13756-019-0559-6 https://www.ncbi.nlm.nih.gov/pubmed/31346459 https://doi.org/10.1002/jps.2600740121 https://www.ncbi.nlm.nih.gov/pubmed/3981425 https://doi.org/10.1177/1177392818785136 drug target insights volume 12: 1–2 © the author(s) 2018 reprints and permissions: sagepub.co.uk/journalspermissions.nav doi: 10.1177/1177392818785136 creative commons non commercial cc by-nc: this article is distributed under the terms of the creative commons attribution-noncommercial 4.0 license (http://www.creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the sage and open access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage). during the drug review process, as noted in the center for drug evaluation and research (cder, https://www.accessdata.fda.gov/scripts/cder/daf/index.cfm), one of the reviewers found that the drug caused microglial aggregates throughout the brain and spinal cord of study animals (cynomolgus monkeys), albeit a nonclinical finding, and recommended nonapproval for clinical use. while the biological significance of this finding, particularly as it relates to the dosing (7-fold safety margin at the 150-mg dose), at which the drug was studied remains unclear, it must be noted that microglial aggregates did not cause neuronal degeneration, axonal fragmentation, or demyelination. the drug was subsequently approved. the black-box warning for zinbryta package insert did note that, across all clinical trials, serious drug-related hepatic injury occurred in 1.7% of zinbryta-treated patients. furthermore, 5% of patients on zinbryta developed serious immune-mediated disorders including skin reactions and lymphadenopathy. across all clinical studies, immune-mediated disorders occurred in 28% of patients on zinbryta, including skin reactions and lymphadenopathy. some patients required invasive procedures for diagnosis and some patients did not improve even after stopping zinbryta. curiously, no cases of inflammatory encephalitis or meningoencephalitis were noted, however, but a safety and tolerability study did mention drug reaction with eosinophilia and systemic symptoms or dress syndrome as a complication.1 however, dress syndrome which is a purely clinical event will only be recognized if clinicians are alert to the possibility and are trained to recognize such events and not relegate them to an “ms relapse.” as there is no established method to revisit data sets from clinical trials unless drug companies themselves put out such information in the face of drug being pulled from the market, that particular piece of information will forever be lost. the ema document published on march 6, 2018, notes that 4 patients developed skin rash and involvement of other organs including eosinophilia, whereas 5 other patients developed multi-organ failure probably related to immune-mediated phenomena. specifically, none of these cases were initially identified as secondary to side effects of the drug; later, they were recognized as (dress) a conclusion that could have major ramifications on safety, and how data are interpreted by clinicians both in the clinical trials and developmental stages of the drug as well as in phase 4 use of the drug after approval. it is equally strange that no cases of encephalitis or meningoencephalitis were noted in the clinical trials and whether signs and symptoms were erroneously missed or misclassified as clinical worsening of ms remains a worry. if this is the case, the signs are ominous for future drug development strategies. in general, the phenomenon of “ms relapse” remains a major concern even in routine clinical assessment of patients with ms on medications. patients are unlikely to be told that their findings are “drug related” and more likely told that their disease is worsening. it is important to note that almost all the cases described by the ema led to pulling zinbryta off the market, and the initial assessment was largely attributed to “worsening ms disease.” the mishap with zinbryta ought to be a warning call to all clinicians to revisit definitions of what represents an ms relapse or clinical worsening and when to seek alternative explanations for “ms relapse.” dress syndrome and daclizumab failure—were potentially dangerous signs missed in clinical trials? jagannadha avasarala1,2 1department of internal medicine/division of neurology, school of medicine, university of south carolina, greenville, sc, usa. 2greenville health system, greenville, sc, usa abstract: the us food and drug administration (fda) approved zinbryta, an interleukin-2 receptor blocking antibody (daclizumab; biogen and abbvie) for the treatment of adults with relapsing forms of multiple sclerosis (ms) in may, 2016. it was also approved by the european union in july, 2016. zinbryta is a long-acting, self-administered monthly injection that was branded as a new ms drug for patients who needed a “new option for treatment.” it blocks interleukin-2 receptor alpha (cd25) and modulates t-cell expansion. the drug was withdrawn from the market in march, 2018 following 12 reports from germany (9), united states (2), and spain (1) following the development of “inflammatory encephalitis and meningoencephalitis” in patients on zinbryta. although cases of hepatotoxicity made news with zinbryta earlier along this drug’s postmarketing journey in the treatment of patients with ms, the european medicines agency (ema) ordered a review of the risks of hepatotoxicity with zinbryta use june, 2017; this analysis will focus on the pharmacovigilance data concerning the central nervous system (cns) complications. the details of the cns complications have been elucidated by ema. every drug failure provides an opportunity for learning, but it is also noteworthy that no fda-approved ms drug in modern times has met with such an untimely, sudden, and inglorious exit. this should serve as a cautionary tale for all clinicians who use “newer ms drugs” that have mushroomed in recent memory following a flurry of recent fda approvals. keywords: daclizumab, dress syndrome, encephalitis, meningoencephalitis, cns toxicity, drug trials received: march 27, 2018. accepted: june 4, 2018. type: perspective funding: the author(s) received no financial support for the research, authorship, and/or publication of this article. declaration of conflicting interests: the author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. corresponding author: jagannadha avasarala, greenville health system, 200 b patewood drive, bldg b, greenville, sc 29615, usa. email: javasarala@ghs.org 785136dti0010.1177/1177392818785136drug target insightsavasarala research-article2018 https://uk.sagepub.com/en-gb/journals-permissions https://www.accessdata.fda.gov/scripts/cder/daf/index.cfm https://www.accessdata.fda.gov/scripts/cder/daf/index.cfm mailto:javasarala@ghs.org 2 drug target insights the case report (n = 12) concerning the central nervous system (cns) complications resulting from zinbryta use in patients with ms was reported by the pharmacovigilance risk assessment committee (prac) of the ema, which include 9 from germany, 2 from the united states, and 1 from spain, and published on march 6, 2018 (article 20 of regulation ec # 726/2004, pharmacovigilance data). the predominant clinical finding in most of the cases that led to the cns complications was from dress syndrome (cases 1, 2, 6, 12, and probably case 3), whereas some cases had anti-nmda encephalitis. the zinbryta dosing for the cases labeled 1 through 5 included the following: 2, 4, 2, 2, and 8, respectively (doses given to each patient prior to clinical worsening attributed to dress syndrome). the pertinent findings for this case cluster included the following—exanthematous skin rash, fever, altered mental status, peripheral eosinophilia (9.3% that increased to 25.5% in case 1, 11.4% for case 2), and “ms relapse” characterized by clinical and radiological worsening. brain biopsies, where performed, showed tand b-cell infiltration, as well as plasma cell and eosinophilic granulocytes. in all, at least 3 patients died, in the reported cohort. dress syndrome or drug reaction with eosinophilia and systemic symptoms is a life-threatening disease with cutaneous manifestations and internal organ involvement2; it carries a mortality rate of approximately 10%. the time of symptom onset varies, following drug exposure, and can be between 2 and 8 weeks. the incidence is unclear and overall population risk varies between 1 in 1000 and 1 in 10 000 drug exposures.3,4 in general, dress syndrome is probably missed or overlooked owing to its varied presentation and a lack of understanding of its manifestations among physicians. typical features of dress syndrome (figure 1) include fever, widespread cutaneous lesions, eosinophilia,5 and atypical lymphocytosis,6 as well as hepatic injury,2 lymphadenopathy, and renal failure.2 additional organ involvement includes lung,2 cardiac,7 and cns involvement characterized as meningitis or encephalitis.8 therefore, familiarity with the clinical features help clinicians pinpoint the diagnosis, and further worsening of symptoms can be mitigated by discontinuation of the offending agent. sometimes this may be difficult if multiple drugs are used but understanding of triggers, time course, and clinical manifestations including dermal, internal organ, and laboratory abnormalities can help nail the diagnosis. viral reactivation, particularly hhv-6 reactivation, is thought to play an important role in dress syndrome, as seen in valproic acid use, for example.9 in addition, whether the patients who developed dress syndrome in the cohort of patients with ms also had other increased risk factors such as hla class ii alleles would be worth exploring. many studies have reported dress syndrome patients with genetic predisposition linked to specific drugs and one wonders if zinbryta use, dress syndrome, and hla class ii alleles were linked as well. author contributions ja is the sole author, therefore, the concept, analysis, layout and presentation is all done by ja. r efer ences 1. giovannoni g, kappos l, gold r, et al. safety and tolerability profile of daclizumab in patients with relapsing-remitting multiple sclerosis: an integrated analysis of clinical studies. mult scler relat dis. 2016;9:36–46. 2. chen yc, chiu hc, chu cy. drug reaction with eosinophilia and systemic symptoms. a retrospective study of 60 cases. arch dermatol. 2010;146:1373–1379. 3. chiou cc, yang lc, hung si, et al. clinicopathological features and prognosis of drug rash with eosinophilia and systemic symptoms: a study of 30 cases in taiwan. j eur acad dermatol venereol. 2008;22:1044–1049. 4. shiohara t, kano y. review of drug-induced hypersensitivity: special emphasis on drug-induced hypersensitivity syndrome. expert dermatol. 2012;7:539–547. 5. cacoub p, musette p, descamps v, et al. the dress syndrome: a literature review. am j med. 2011;124:588–597. 6. kardaun sh, sekula p, valeyrie-allanore l, et al. drug reaction with eosinophilia and systemic symptoms (dress): an original multisystem adverse drug reaction. results from the prospective regiscar study. br j dermatol. 2013;169:1071–1080. 7. bourgeois gp, cafardi ja, groysman v, pamboukian sv, kirklin jk, andea aa. fulminant myocarditis as a late sequela of dress: two cases. j am acad dermatol. 2011;65:889–890. 8. husain z, reddy by, schwartz ra. dress syndrome part 1. clinical perspectives. j am acad dermatol. 2013;68:693e1-639e14. 9. mardivirin l, lacroix ad, descamps v, ranger-rogez s. augmentation de la replication in vitro de i’herpesvirus humain 6 en presence de valproate de sodium. virologie. 2007;11:1–3. figure 1. salient features of dress syndrome, typically seen in the context of fever and skin rash. https://doi.org/10.1177/1177392819861987 drug target insights volume 13: 1–2 © the author(s) 2019 article reuse guidelines: sagepub.com/journals-permissions doi: 10.1177/1177392819861987 creative commons non commercial cc by-nc: this article is distributed under the terms of the creative commons attribution-noncommercial 4.0 license (http://www.creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the sage and open access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage). venous thromboembolism (vte), that includes deep vein thrombosis (dvt) and pulmonary embolism (pe), is a common and potentially fatal disease. the cause of vte includes cancer, systemic, surgical, and medication-related conditions and its treatment is based on anticoagulation.1 we present the case of a 43-year-old man with a history of severe extrinsic allergic asthma treated with once-monthly omalizumab (600 mg) for the last 15 months. he was not receiving other treatments. he presented to the emergency room with a 2-week history of right lower limb pain and chest pleuritic pain. on admission, blood pressure was 160/94 mm hg, heart rate was 93 bpm, and basal oxygen saturation was 98%. the remainder of physical examination was normal and no signs of dvt were found. c-reactive protein was 3.2 mg/dl (normal < 0.5 mg/dl) and d-dimer was reported as 5.113 ng/ ml (normal < 250). the ct pulmonary angiography showed bilateral pe with right-sided pulmonary infarction and ultrasound of right lower limb confirmed distal dvt. he was admitted and low molecular weight heparin was started. pulmonary embolism severity index (pesi) score was 53 points (class i mortality risk). echocardiogram showed mild dilation of the right ventricle, the right-to-left ventricle diameter ratio was 0.85 (normal), with normal systolic function and normal tricuspid annular plane systolic excursion (22 mm). the patient’s hospital stay was uneventful and he was discharged 4 days later under treatment with rivaroxaban. treatment with omalizumab was stopped. no known risk factors were identified: his body mass index was normal (24 kg/m2) and he denied any history of smoking; he had not recent history of immobilization or prolonged travel; no family history of vte was present. a colonoscopy was normal, and prostate-specific antigen was within normal range (0.41 µg/l). thrombophilia testing including antithrombin, homocysteine, protein c and s, antiphospholipid antibodies, factor v leiden, and prothrombin mutation was performed 3 months after the vte event, and it was normal. anticoagulation was maintained for 6 months. c-reactive protein was 0.3 mg/dl and d-dimer was 90 ng/ml after 3 months of the vte episode. the naranjo adverse drug reaction (adr) probability scale classifies this as a probable adr (score of 6: there are previous conclusive reports on this reaction (+1), the adverse even appeared after the suspected drug was administered (+2), it improved after the drug was discontinued (+1), there were not alternative causes (+2)).2 omalizumab is a humanized monoclonal anti-ige antibody indicated for the treatment of persistent moderate-to-severe asthma and certain chronic refractory urticaria. it has shown reduction in severity, exacerbation of symptoms, and the use of corticosteroids and improvement in quality of life. omalizumab plays a major role in chronic allergic inflammatory processes acting both innate and humoral immunity. studies have shown a link between d-dimer levels and symptomatic load in chronic urticaria, and both features improve in patients taking omalizumab as a provoking factor for venous thromboembolism crhistian-mario oblitas1, francisco galeano-valle1,2 , laura vela-de la cruz1, jorge del toro-cervera1,2 and pablo demelo-rodríguez1,2 1venous thromboembolism unit, hospital general universitario gregorio marañón, madrid, spain. 2instituto de investigación sanitaria gregorio marañón (iisgm), madrid, spain. abstract a 43-year-old man with a history of severe extrinsic allergic asthma treated with once-monthly omalizumab (600 mg) for the last 15 months. he presented to the emergency room with a 2-week history of right lower limb pain and chest pleuritic pain. computed tomography pulmonary angiography showed bilateral pulmonary embolism with right-sided pulmonary infarction and ultrasound of right lower limb confirmed distal deep vein thrombosis. no other known risk factors were identified. treatment with omalizumab was stopped during hospitalization. the naranjo adverse drug reaction (adr) probability scale classifies this as a probable adr (score of 6). omalizumab is a humanized monoclonal anti-ige antibody indicated for the treatment of persistent moderate-to-severe asthma and certain chronic refractory urticaria. the excels study (the epidemiologic study of xolair (omalizumab): evaluating clinical effectiveness and long-term safety in patients with moderate-to-severe asthma), a postmarketing observational cohort study to assess clinical safety profile of omalizumab, showed a significant increase in venous thromboembolism. in conclusion, omalizumab has been associated with arterial and venous thromboembolic events, although the evidence is not definitive. keywords: omalizumab, venous thromboembolism received: may 31, 2019. accepted: june 11, 2019. type: letter to the editor funding: the author(s) received no financial support for the research, authorship, and/or publication of this article. declaration of conflicting interests: the author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. corresponding author: pablo demelo-rodríguez, venous thromboembolism unit, hospital general universitario gregorio marañón, c/. doctor esquerdo, 46, 28007 madrid, spain. email: pbdemelo@hotmail.com 861987dti0010.1177/1177392819861987drug target insightsoblitas et al letter2019 https://uk.sagepub.com/en-gb/journals-permissions mailto:pbdemelo@hotmail.com 2 drug target insights omalizumab.3,4 the efficacy of omalizumab in these disorders has been evaluated by observing symptomatic improvement and decreased levels of d-dimer, by mechanisms not well known. it has been suggested that this effects could simply reflect the reduction of the general inflammatory state of the disease. in this context, active chronic urticaria and persistent allergic asthma are associated with a procoagulant state by activating the extrinsic pathway of coagulation in relation to high levels of circulatory ige, molecule which is blocked by omalizumab reducing levels of d-dimer. paradox ically, it does not result in decreasing the risk of vte.4,5 the d-dimer levels are considered to be useful for the diagnosis of thrombosis, and they can be clinically used due to its high negative predictive value. hence, the decrease in d-dimer levels could mask its diagnostic predictive value in cases of vte in patients receiving omalizumab. recently, some articles reporting the association of omalizumab with pulmonary vein thrombosis6 and some studies evaluating the long-term use of omalizumab have been published. the most important study to date is the excels study, a postmarketing 5-year follow-up observational cohort study to assess clinical safety profile of omalizumab (7857 patients were included). this study, primarily designed for the detection of malignancies, showed an increased risk in vte (crude incidence rate = 3.2 [95% ci = 2.4-4.3] vs 1.5 [95% ci = 0.82.5] per 1000 person-years). the incidence rate of primary neoplasms was similar in both groups.7 consequently, the us food and drug administration8 published a drug safety communication on september 26, 2014, describing slightly higher risk of blood clots in the lungs and veins, in addition to heart and brain adverse events. however, conflicting results of other recent studies have been published. a letter reported results from a pooled analysis of cardiovascular events from 25 randomized controlled trials and 2 extension studies of omalizumab. this analysis did not find any differences in the incidence of vte between omalizumab or placebo.9 there are no specific recommendations for patients who suffer a vte event under treatment with omalizumab, but if we consider it as a temporary risk factor for vte, we suggest drug withdrawal and anticoagulation for at least 6 months in the absence of other risk factors.10 because omalizumab is administered in a health care setting, it may not be listed on the patient’s home medication list, and therefore, physicians and pharmacists should evaluate clinicadministered medication when evaluating vte risk factors. in conclusion, omalizumab has been associated with arterial and venous thromboembolic events, although the evidence is not definitive. it has been associated with decrease in d-dimer levels that could mask its diagnostic predictive value. we point out the need for studies that clarify the role of omalizumab in the setting of vte. author contributions all authors contributed equally to the colletion of data, analysis and interpretation, manuscript writing and final approval of manuscript. informed consent we obtained the patient’s written informed consent before the submission of the letter. orcid id francisco galeano-valle https://orcid.org/0000-0003-1321 -6866 r efer ences 1. tritschler t, kraaijpoel n, le gal g, wells ps. venous thromboembolism: advances in diagnosis and treatment. jama. 2018;320:1583–1594. 2. naranjo ca, busto u, sellers em, sandor p, ruiz i, roberts ea. a method for estimating the probability of adverse drug reactions. clin pharmacol ther. 1981;30:239–245. 3. d’amato g, stanziola a, sanduzzi a, et al. treating severe allergic asthma with anti-ige monoclonal antibody (omalizumab): a review. multidiscip respir med. 2014;9:23. 4. asero r. serial d-dimer plasma levels in a patient with chronic spontaneous urticaria developing resistance to omalizumab. clin exp dermatol. 2017;42:667–669. 5. yalcin ad, celik b, gumuslu s. d-dimer levels decreased in severe allergic asthma and chronic urticaria patients with the omalizumab treatment. expert opin biol ther. 2014;14:283–286. 6. narukonda s, vinod nr, joshi m. a case of pulmonary vein thrombosis associated with treatment of omalizumab. j investig med high impact case rep. 2017;5:2324709617724176. 7. iribarren c, rahmaoui a, long aa, et al. cardiovascular and cerebrovascular events among patients receiving omalizumab: results from excels, a prospective cohort study in moderate to severe asthma. j allergy clin immunol. 2017;139:1489.e5–1495.e5. 8. us food and drug administration. fda drug safety communication: fda approves label changes for asthma drug xolair (omalizumab), including describing slightly higher risk of heart and brain adverse events. website. https://www.fda .gov/drugs/drugsafety/ucm414911.htm. updated september, 2014. accessed january 22, 2019. 9. iribarren c, rothman kj, bradley ms, carrigan g, eisner md, chen h. cardiovascular and cerebrovascular events among patients receiving omalizumab: pooled analysis of patient-level data from 25 randomized, double-blind, placebocontrolled clinical trials. j allergy clin immunol. 2017;139:1678–1680. 10. peñaloza-martínez e, demelo-rodríguez p, proietti m, et al. update on extended treatment for venous thromboembolism. ann med. 2018;50:666–674. https://orcid.org/0000-0003-1321-6866 https://orcid.org/0000-0003-1321-6866 https://www.fda.gov/drugs/drugsafety/ucm414911.htm https://www.fda.gov/drugs/drugsafety/ucm414911.htm dti © 2020 the authors. this article is published by aboutscience and licensed under creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). any commercial use is not permitted and is subject to publisher’s permissions. full information is available at www.aboutscience.eu issn 1177-3928 drug target insights 2020; 14: 34-47 review doi: 10.33393/dti.2020.2185 interaction of drugs with lipid raft membrane domains as a possible target hironori tsuchiya1, maki mizogami2 1asahi university school of dentistry, mizuho, gifu japan 2department of anesthesiology, kizawa memorial hospital, minokamo, gifu japan abstract introduction: plasma membranes are not the homogeneous bilayers of uniformly distributed lipids but the lipid complex with laterally separated lipid raft membrane domains, which provide receptor, ion channel and enzyme proteins with a platform. the aim of this article is to review the mechanistic interaction of drugs with membrane lipid rafts and address the question whether drugs induce physicochemical changes in raft-constituting and raftsurrounding membranes. methods: literature searches of pubmed/medline and google scholar databases from 2000 to 2020 were conducted to include articles published in english in internationally recognized journals. collected articles were independently reviewed by title, abstract and text for relevance. results: the literature search indicated that pharmacologically diverse drugs interact with raft model membranes and cellular membrane lipid rafts. they could physicochemically modify functional protein-localizing membrane lipid rafts and the membranes surrounding such domains, affecting the raft organizational integrity with the resultant exhibition of pharmacological activity. raft-acting drugs were characterized as ones to decrease membrane fluidity, induce liquid-ordered phase or order plasma membranes, leading to lipid raft formation; and ones to increase membrane fluidity, induce liquid-disordered phase or reduce phase transition temperature, leading to lipid raft disruption. conclusion: targeting lipid raft membrane domains would open a new way for drug design and development. since angiotensin-converting enzyme 2 receptors which are a cell-specific target of and responsible for the cellular entry of novel coronavirus are localized in lipid rafts, agents that specifically disrupt the relevant rafts may be a drug against coronavirus disease 2019. keywords: drug target, fluidity, lipid raft, membrane domain, membrane interaction received: september 17, 2020 accepted: november 11, 2020 published online: december 22, 2020 corresponding author: hironori tsuchiya asahi university school of dentistry 1851 hozumi, mizuho, gifu 501-0296 japan tsuchi-hiroki16@dent.asahi-u.ac.jp of the membrane structures (2), whereas caveolae are a subset of lipid rafts and organizationally maintained by characteristic protein caveolins (3). lipid rafts in a liquid-ordered (l o ) phase coexist with the bulk of membranes in a liquid disordered (l d ) phase (4). lipid raft membrane domains play an important role in cellular signal transduction and trafficking by compartmentalizing membranes and providing functional membrane proteins with a platform (4-7). pharmacologically relevant receptors, ion channels and enzymes are localized or cluster in membrane lipid rafts and caveolae (8-11). given the localization of receptors, ion channels and enzymes in membrane lipid rafts, the mode of drug action is first interpretable in a simple manner of receptor/channel/ enzyme and ligand interaction as known in the conventional mechanistic theory. the second possibility is that drugs may act on membrane lipids to affect the organizational integrity of lipid rafts, resulting in modulation of the activity of receptors, ion channels and enzymes embedded in membrane domains. it is of much interest to know whether drugs interact introduction since singer and nicolson proposed a fluid mosaic model, the concept of membrane organization has progressively changed, that is, plasma membranes are not the homogeneous bilayers of uniformly distributed lipids but the lipid complex with laterally separated membrane domains such as lipid rafts and caveolae (1). lipid rafts are small (10-200 nm), heterogeneous, dynamic, and cholesteroland sphingolipidenriched membrane domains that are distinct from the rest tsuchiya and mizogami 35 © 2020 the authors. published by aboutscience preferentially with lipid rafts compared with non-raft overall membrane lipid bilayers and whether such interaction at a membrane lipid level is linked to pharmacological and cytotoxic effects of drugs. while cholesterol is essential to raft and caveola formation, the regulatory effects of membrane domains on receptors and ion channels were confirmed by depleting cholesterol in plasma membranes (12-15). the purpose of the present study is to review the interaction of drugs with membrane lipid rafts and the membranes surrounding such domains by searching scientific articles from a mechanistic point of view in order to gain new insights into a drug target. since various proteins embedded in membranes are functionally modulated by membrane fluidity, order and phase transition, the focus of our review is on addressing the question whether drugs modify the physicochemical properties of raft-constituting and raft-surrounding membranes to affect the formation, stability and integrity of lipid raft membrane domains. methods the present review is based on articles that were retrieved from pubmed/medline and google scholar by searching databases from 2000 to 2020. the publications earlier than 2000 were exceptionally cited if they are essential to advancing the discussion. research papers published in english in internationally recognized journals and online journals were preferred, but review articles were additionally used to deepen understanding of the concept of plasma membranes and the mode of drug action. for reviewing as diverse drugs as possible without confining to a specific class of drug, the literature searches were carried out using the following terms or combinations thereof: “lipid raft,” “caveola,” “membrane domain,” “membrane interaction,” “fluidity,” “receptor,” “channel” and “enzyme.” collected articles were independently reviewed by title, abstract and text for relevance with preference to more recent publications. results and discussion drug and raft interaction methodology since the methodology of drug and membrane raft interaction is essential to facilitate readers’ understanding of individual studies, representative experiments are mentioned as follows. in in vitro experiments, drugs are subjected to the reaction with raft model (raft-like) membranes or liposomes that mimic the lipid composition and property of lipid raft micro domains (16,17). ternary lipid membranes are used as a raft model, which is frequently prepared with an equimolar mixture of 1-palmitoyl-2-oleoylphosphatidylcholine (popc), sphingomyelin (sm) and cholesterol (18), in which cholesterol functions as a spacer between sphingolipid hydrocarbon chains and as a glue to keep the raft assembly together (19). such raft model membranes have the advantage that the membrane effects of drugs can be determined more easily than in vivo experiments (20). lipid rafts isolated from cells are also used experimentally. since lipid rafts are relatively insoluble in cold non-ionic detergents, cells are treated with triton x-100 and membrane lipid rafts are fractionated by sucrose density gradient centrifugation (sdgc) (21). in in vivo experiments, human and animal subjects are treated with drugs, followed by sdgc to isolate cellular membrane lipid rafts. cholesterol is not only a critical determinant for membrane fluidity but also an essential component to form the l o membrane domains. cellular cholesterol contents are manipulated by treating animals with cholesterol metabolic inhibitors, culturing cells in cholesterol-deficient media and using cholesterol-depleting agents. methyl-βcyclodextrin (mbc), to form a 2:1 complex with cholesterol (22), is most widely used for cholesterol depletion (23). drug-induced physicochemical or biophysical changes in raft model membranes and membrane lipid rafts are determined by fluorescence polarization (fp) or anisotropy (fa), differential scanning calorimetry (dsc), nuclear magnetic resonance (nmr) spectroscopy, neutron diffraction (nd), xray diffraction (xd) and their complementary combination. general anesthetics general anesthetics and their related sedatives, anxiolytics and adjuncts act on inhibitory γ-aminobutyric acid type a (gaba a ) receptors and excitatory n-methyl-daspartate (nmda) receptors (24). intravenous and inhalational anesthetics are a positive allosteric modulator or a direct activator of gaba a receptors to enhance their inhibitory functions, inducing general anesthesia, sedation, anxiolysis and convulsion cessation (25). inhalational anesthetics are also a non-competitive antagonist of nmda receptors to reduce neuronal excitation, producing analgesic, sedative and anesthesia-maintaining effects (26,27). these anesthesiarelevant gaba a receptors and nmda receptors are associated with lipid raft membrane domains (28,29). results of the literature search indicated that general anesthetics interact with membrane lipid rafts and membranes as shown in table i. intravenous anesthetic propofol fp experiments demonstrated that propofol structurespecifically interacts with binary liposomal membranes prepared with 80 mol% popc and 20 mol% cholesterol (30) and quinary liposomal membranes prepared with 55 mol% phospholipids (popc, sm, 1-palmitoyl-2-oleoylphosphatidylethanolamine (pope) and 1-palmitoyl-2-oleoylphosphatidylserine (pops)) and 45 mol% cholesterol (31), resulting in an increase of membrane fluidity at clinically relevant 0.125-10 μm. l o and l d phase equilibrium is present in giant plasma membrane vesicles (gpmvs) isolated from rat basophil leukemia cells, which are used as a model of membrane heterogeneity for lipid rafts. gray et al treated gpmvs with propofol and its structural analogs to examine their effects on liquid-liquid transition by analyzing the lateral distribution of fluorescent probe dii-c 12 microscopically (32). propofol reduced the critical transition temperature at 2.5-10 μm, but not 2,6-di-tert-butylphenol without the anesthetic activity at the same concentrations. therefore, propofol is considered interaction of drugs with lipid rafts36 © 2020 the authors. published by aboutscience to decrease the magnitude of membrane heterogeneity structure-specifically, affecting receptor and ion channel proteins sensitive to raft heterogeneity. while propofol is known to produce bronchodilatation, the airway relaxation involves a decrease of ca2+ concentrations in airway smooth muscle cells that are regulated by caveolae. by exposing human airway smooth muscle cells to propofol at 10 and 30 μm, grim et al found that propofol increases in membrane caveolae and reduces the intracellular ca2+ concentration response to 10 μm histamine (33). they also suggested that propofol may induce caveolar disruption and caveolin-1 expression decrease. inhalational anesthetics patel et al investigated the membrane effects of isoflurane using different membrane systems such as popc/cholesterol liposomal membranes, erythrocyte ghosts and brain endothelial cell-mimetic membranes (34). fa measurements indicated that isoflurane increases the membrane fluidity at 1 and 5 mm. turkyilmaz et al prepared large unilamellar vesicles (luvs) with 1,2-dipalmitoylphosphatidylcholine (dppc) and cholesterol to be 2.5 mol% or 37.5 mol% cholesterol containing dppc membranes to verify the membrane effects of inhalational anesthetics (35). isoflurane and halothane weakened and strengthened the sterol-phospholipid association in cholesterol-rich l o phase membranes and in cholesterol-poor l d phase membranes, respectively, at 2.5-12 mm. in nd and xd experiments of weinrich et al, halothane was subjected to the reaction with multilayer membranes that were prepared with an equimolar mixture of dppc and 1,2-dilauroylphosphatidylcholine (dlpc) to form distinct dppc-rich ordered and dlpc-rich fluid phase (36). halothane reduced the transition temperature by about 5°c at 1.5 mol% corresponding to about twice the minimum alveolar concentration (mac) for human anesthesia, but not non-anesthetic table i interaction of general anesthetics with lipid raft membrane domains and membranes drug class drug membrane induced membrane modification reference intravenous anesthetic propofol (0.125-1.0 μm) binary liposomal membranes (80 mol% popc and 20 mol% cholesterol) increased membrane fluidity 30 intravenous anesthetic propofol (10 μm) quinary liposomal membranes (55 mol% phospholipids (popc, sm, pope and pops) and 45 mol% cholesterol) increased membrane fluidity 31 intravenous anesthetic propofol (2.5-10 μm) gpmvs isolated from rat basophil leukemia cells reduced the critical transition temperature structure-specifically 32 intravenous anesthetic propofol (10 and 30 μm) human airway smooth muscle cell membranes reduced the intracellular ca2+ concentration responses to 10 μm histamine, disrupted caveolae and decreased caveolin-1 expression 33 inhalational anesthetic isoflurane (1 and 5 mm) popc/cholesterol liposomal membranes, erythrocyte ghosts and brain endothelial cell-mimetic membranes increased membrane fluidity 34 inhalational anesthetic isoflurane (2.5-12 mm) luvs (62.5 mol% dppc and 37.5 mol% cholesterol) weakened the sterol-phospholipid association in cholesterol-rich l o phase membranes 35 inhalational anesthetic halothane (1.5 mol%) multilayer membranes (dppc and dlpc, 1:1 molar ratio) reduced the transition temperature by about 5°c 36 inhalational anesthetic xenon (4.6-fold mac) nitrous oxide (4.6-fold mac) halothane (threeto fivefold mac) isoflurane (three to fivefold mac) raft model membranes (dopc, sm and cholesterol, 1:1:0.2 molar ratio) increased the l d phase decreased the relative intensity of l o to l d phase 37 barbiturate rats injected with sodium pentobarbital (50 mg/kg, i.p.) lipid rafts isolated from rat brains 15 minutes after drug injection reduced the transition temperature 38 dlpc = 1,2-dilauroylphosphatidylcholine; dopc = 1,2-dioleoylphosphatidylcholine; dppc = 1,2-dipalmitoylphosphatidylcholine; gpmv = giant plasma membrane vesicle; luv = large unilamellar vesicle; mac = minimum alveolar concentration; popc = 1-palmitoyl-2-oleoylphosphatidylcholine; pope = 1-palmitoyl2-oleoylphosphatidylethanolamine; pops = 1-palmitoyl-2-oleoylphosphatidylserine; sm = sphingomyelin. tsuchiya and mizogami 37 © 2020 the authors. published by aboutscience 1,2-dichlorohexafluorocyclobutane even at fivefold mac. weinrich and worcester determined the effects of different anesthetics on liquid phase distribution in raft model membranes prepared with 1,2-dioleoylphosphatidylcholine (dopc), sm and cholesterol (1:1:0.2 molar ratio) by nd and xd analysis (37). xenon and nitrous oxide increased the l d phase at 4.6-fold mac, and halothane and isoflurane decreased the relative intensity of l o to l d phase at threeto fivefold mac. barbiturate pentobarbital is intravenously and intraperitoneally administered especially in veterinary anesthesia or sedation. sierra-valdez et al characterized the in vivo effects of pentobarbital on rat brain lipid rafts, which were isolated 15 min after injecting rats with sodium pentobarbital at 50 mg/kg intraperitoneally (38). dsc analysis revealed that pentobarbital reduces the transition temperature from l o to l d phase. membranous sodium channel blocker local anesthetics local anesthetics reversibly block voltage-gated sodium (nav) channels that are responsible for the initiation and propagation of action potentials in excitable cells, inhibiting sensory and motor functions (39). among nine distinct nav channels (nav1.1 to nav1.9) cloned from mammals, nav1.8 channel plays a crucial role in pain transmission and this isoform is implicated as a site of action for anesthetic and analgesic drugs. while nav channels are present in caveolae-type and non-caveolae-type lipid rafts, nav1.8 channel clustering in such membrane domains is essential to the propagation of action potentials in nociceptive axons (40,41). nav1.8 channels are associated with lipid rafts in rat dorsal root ganglionic neurons, but cholesterol depletion induces dissociation between nav1.8 channels and lipid rafts (42). results of the literature search on the interaction of local anesthetics with membrane lipid rafts and membranes are shown in table ii. kamata et al incubated human erythrocytes with lidocaine at 18.4 mm and prepared erythrocyte ghosts, followed by sdgc fractionation and immunoblotting analysis for flotillin-1 (caveolae-associated integral membrane protein) that is assumed to stabilize lipid rafts (43). lidocaine reversibly disrupted erythrocyte membrane lipid rafts and abolished flotillin-1 in lipid rafts together with depleting cholesterol. bandeiras et al treated luvs prepared with popc, sm and cholesterol (1:1:1 molar ratio) with tetracaine and lidocaine, and then evaluated their membrane effects by dsc and phosphorus nmr spectroscopy (44). tetracaine and lidocaine increased the fluidity of raft-like membranes at 25 and 69 mm, table ii interaction of membranous sodium channel blocker local anesthetics with lipid raft membrane domains and membranes drug class drug membrane induced membrane modification reference local anesthetic lidocaine (18.4 mm) human erythrocyte membranes disrupted membrane rafts reversely and abolished flotillin-1 in lipid rafts 43 local anesthetic tetracaine (25 mm) lidocaine (69 mm) luvs (popc, sm and cholesterol, 1:1:1 molar ratio) increased the fluidity of raft-like membranes 44 local anesthetic dibucaine (0.05 and 0.2 mm) raft-like membranes (popc, dppc and cholesterol, 2:1:1 molar ratio) reduced the miscibility temperature of l o and l d phase separation 45 local anesthetic lidocaine (10-20 mol%) tetracaine (10-20 mol%) raft-like membranes (popc, dppc and cholesterol, 2:2:1 molar ratio) reduced the miscibility temperature of l o and l d phase separation and decreased the line tension at l o /l d phase boundary 46 local anesthetic dibucaine (0.2 mm) tetracaine (0.2 mm) luvs (popc, sm and cholesterol, 16:43:41 molar ratio) increased the fluidity of l o phase membranes, but not l d phase membranes 47 local anesthetic lidocaine (50-200 μm) bupivacaine (50-200 μm) ropivacaine (50-200 μm) prilocaine (50-200 μm) suvs (dopc, pope, sm, cb and cholesterol, 16.7:16.7:16.7:16.7:33.3; dopc, sm and cholesterol, 33.3:33.3:33.3; and dopc, pope, pops, sm and cholesterol, 5:5:10:40:40 molar ratio) increased the membrane fluidity with the relative potency being bupivacaine > ropivacaine > lidocaine > prilocaine more effective in interacting with the reference biomimetic membranes than the raft model membranes 49 local anesthetic bupivacaine enantiomers (5-50 μm) suvs (popc, pope, pops, popi, sm, cardiolipin and cholesterol, 25:16:3:3:3:10:40 molar ratio) increased the fluidity of biomimetic membranes with the relative potency being r(+)-bupivacaine > racemic bupivacaine > s(–)-bupivacaine 50 cb = cerebroside; dopc = 1,2-dioleoylphosphatidylcholine; dppc = 1,2-dipalmitoylphosphatidylcholine; luv = large unilamellar vesicle; popc = 1-palmitoyl2-oleoylphosphatidylcholine; pope = 1-palmitoyl-2-oleoylphosphatidylethanolamine; popi = 1-palmitoyl-2-oleoylphosphatidylinositol; pops = 1-palmitoyl2-oleoylphosphatidylserine; sm = sphingomyelin; suv = small unilamellar vesicle. interaction of drugs with lipid rafts38 © 2020 the authors. published by aboutscience respectively. yoshida et al prepared lipid bilayer membranes with dopc, dppc and cholesterol (2:1:1 molar ratio) to be laterally separated into l o and l d phase together with labeling the membranes with fluorescent probe rhodamine dhpe (dihexadecanoyl-sn-glycero-3-phosphoethanolamine) (45). after treating the membrane preparations with dibucaine at 0.05 and 0.2 mm, they observed the raft-like membrane domains by fluorescence microscopy at 20-40°c to determine changes in miscibility temperature of the l o and l d phase separation and in line tension at the l o /l d phase boundary. dibucaine reduced the miscibility temperature, which was accompanied by the line tension decrease. dibucaine also made the l o domains smaller at 25°c, although most membranes were present without such raft-like domains at above 25°c. in a similar microscopic experiment using liposomes prepared with dopc, dppc and cholesterol (2:2:1 molar ratio), lidocaine and tetracaine reduced the miscibility temperature of ternary membranes at 10-20 mol% relative to liposomal lipids, but not binary membranes without cholesterol (46). both local anesthetics also decreased the line tension at the l o /l d phase boundary. kinoshita et al performed fa experiments to reveal the effects of local anesthetics on raft-like l o /non-raft l d phase membranes by using luvs that were prepared with dopc, sm and cholesterol (16:43:41 and 65:16:19 in molar ratio for l o phase and l d phase, respectively) (47). dibucaine disordered the lipid packing or increased the fluidity of l o phase membranes at 0.2 mm more potently than tetracaine, whereas dibucaine and tetracaine showed no significant effects on l d phase membranes. however, these studies (43-47) used drug concentrations much higher than clinically and experimentally relevant ones (48) and the tested dibucaine and tetracaine are not widely used in clinical anesthesia. tsuchiya et al prepared small unilamellar vesicles (suvs) with dopc, pope, sm, cerebroside (cb) and cholesterol (16.7:16.7:16.7:16.7:33.3 molar ratio); dopc, sm and cholesterol (33.3:33.3:33.3 molar ratio); and dopc, pope, pops, sm and cholesterol (5:5:10:40:40 molar ratio) for raft model membranes, and popc, pope, pops, 1-palmitoyl2-oleoylphosphatidylinositol (popi), sm, cardiolipin and cholesterol (25:16:3:3:3:10:40 molar ratio) for reference biomimetic membranes (49,50). they treated these membrane preparations with lidocaine, bupivacaine, ropivacaine and prilocaine at anesthetic and cardiotoxic concentrations, followed by fp measurements. all the tested anesthetics interacted with raft model and biomimetic membranes to increase the membrane fluidity at 50-200 μm with the relative potency being bupivacaine > ropivacaine > lidocaine > prilocaine (49). they were more effective in interacting with the reference membranes than the raft membranes. biomimetic membranes showed different interactivity with the relative potency being r(+)-bupivacaine > racemic bupivacaine > s(–)-bupivacaine at 5-50 μm, being consistent with the rank order of their anesthetic and cardiotoxic effects (50). however, raft model membranes did not exhibit significant enantioselectivity as the reference biomimetic membranes. these results may suggest that lipid rafts are less likely to contribute at least to the enantioselective effects of local anesthetics. membranous receptorand enzyme-acting drugs results of the literature search on the interaction of receptor-acting adrenergic and opioid drugs and enzyme acting anti-inflammatory drugs with membrane lipid rafts and membranes are shown in table iii. beta-adrenergic blockers beta-blockers are perioperatively used to reduce the risk of myocardial ischemia, arrhythmia and cardiac morbidity during anesthesia. lipid raft/caveola domains encompass β 2 adrenergic receptors, but not β 1 -adrenergic receptors for signal transduction (12,51). mizogami et al prepared suvs with popc, sm, pope, cb and cholesterol (1:1:1:1:2 molar ratio) to compare the membrane effects between different β-blockers at 0.2 and 1 mm by measuring fp (52). nonselective propranolol most potently increased the fluidity of raft model membranes, followed by alprenolol and oxprenolol, but not β 1 -selective atenolol, metoprolol and esmolol. in a similar fp study using suvs prepared with 33.3 mol% cholesterol and 66.7 mol% phospholipids consisting of equimolar dopc, sm, pope and cb, nonselective propranolol and alprenolol increased the fluidity of raft model membranes at 20-200 μm, whereas β 1 -selective landiolol and esmolol were not effective even at 200 μm (53). nonselective β-blockers could reduce the activity of β 2 -adrenergic receptors by fluidizing the membrane lipid rafts together with antagonizing β 1 -adrenergic receptors by interacting with β 1 -adrenergic receptor proteins, producing nonselective blockade of β-adrenergic receptors. in contrast, selective β 1 -blockers do not affect β 2 -adrenergic receptors through interaction with lipid rafts, thereby enhancing the selectivity for β 1 -adrenergic receptors. beta-blockers, particularly β 1 -selective agents, have been used for treating hypertension (54). although the altered vascular signaling processes are implicated in hypertension, whether lipid rafts/caveolae are responsible for such pathogenic events remains unclear (55), so no significant interaction between antihypertensive drugs and lipid rafts was found in the literature. alpha-adrenergic agonists alpha 2 -agonists with the sedative, analgesic, anestheticsparing and sympatholytic activity are used as an adjuvant for anesthesia. mizogami and tsuchiya performed fp experiments to investigate their effects on suvs that were prepared with 33.3 mol% cholesterol and 66.7 mol% phospholipids (consisting of equimolar dopc, sm, pope and cb) to be raft model membranes and with cholesterol and phospholipids of different compositions to be neuro-mimetic and cardiomyocyte-mimetic membranes (56). dexmedetomidine interacted with the non-raft membranes to increase their fluidity most potently at 5-200 μm, followed by levomedetomidine and clonidine. however, these α 2 -agonists exerted much weaker effects on the raft model membranes so that dexmedetomidine and levomedetomidine did not show large difference in membrane interactivity despite being significantly different in sedative activity between medetomidine enantiomers. tsuchiya and mizogami 39 © 2020 the authors. published by aboutscience table iii interaction of membranous receptorand enzyme-acting drugs with lipid raft membrane domains and membranes drug class drug membrane induced membrane modification reference adrenergic receptor-acting drug nonselective β-blockers (0.2 and 1 mm) selective β 1 -blockers (0.2 and 1 mm) suvs (popc, sm, pope, cb and cholesterol, 1:1:1:1:2 molar ratio) nonselective propranolol most potently increased the membrane fluidity, followed by alprenolol and oxprenolol, but not β 1 -selective atenolol, metoprolol and esmolol 52 adrenergic receptor-acting drug nonselective β-blockers (20-200 μm) selective β 1 -blockers (20-200 μm) suvs (33.3 mol% cholesterol and 66.7 mol% phospholipids of equimolar dopc, sm, pope and cb) nonselective propranolol and alprenolol increased the membrane fluidity, but not β 1 -selective landiolol and esmolol 53 adrenergic receptor-acting drug alpha 2 -agonists (5-200 μm) suvs (33.3 mol% cholesterol and 66.7 mol% phospholipids (dopc, sm, pope and cb)) dexmedetomidine increased the fluidity of non-raft membranes most potently, followed by levomedetomidine and clonidine, although the effects on raft model membranes were much weaker without showing large difference between medetomidine enantiomers 56 opioid receptoracting drug rats injected with morphine (25 mg/kg, i.p.) rats injected with naloxone (2 mg/kg, i.p.) morphine (10 nm and 10 μm) naloxone (1 nm) hippocampus and caudate membranes hippocampus and caudate membranes rat brain membrane preparations rat brain membrane preparations increased the membrane fluidity decreased the membrane fluidity increased the membrane fluidity reversed the membrane-fluidizing effects of 10 nm morphine 59 opioid receptoracting drug codeine (0.1 m) n-methylcodeine (0.1 m) dppc mlvs reduced the phase transition temperature 60 opioid receptoracting drug etorphine (10 nm) mice injected with etorphine (5 μg/kg, s.c.) human embryonic kidney cells expressing μ-receptors hippocampi isolated after drug injection translated μ-receptors from lipid rafts to non-raft regions 61 cyclooxygenaseacting antiinflammatory drug aspirin (3 mm) dppc bilayer membranes containing 32.5 mol% cholesterol increased the membrane fluidity disrupted the membrane organization and prevented raft formation 63 cyclooxygenaseacting antiinflammatory drug aspirin (10 mol%) mlvs (70 mol% dmpc and 30 mol% cholesterol) bound to raft-like l o phase domains and disturbed their organization 64 cyclooxygenaseacting antiinflammatory drug indomethacin (5 μm) naproxen (25 μm) aspirin (50 μm) ibuprofen (150 μm) baby hamster kidney cells affected the organization of raft-like ordered lipid and protein membrane nanoclusters 65 cb = cerebroside; dmpc = 1,2-dimyristoylphosphatidylcholine; dopc = 1,2-dioleoylphosphatidylcholine; dppc = 1,2-dipalmitoylphosphatidylcholine; mlv = multilamellar vesicle; popc = 1-palmitoyl-2-oleoylphosphatidylcholine; pope = 1-palmitoyl-2-oleoylphosphatidylethanolamine; sm = sphingomyelin; suv = small unilamellar vesicle. interaction of drugs with lipid rafts40 © 2020 the authors. published by aboutscience the mechanistic relevance of lipid rafts to the enantioselective effects of α 2 -agonists is inconclusive as morris et al reported that α 1 -adrenergic receptors, but not α 2 -adrenergic receptors, occupy membrane lipid rafts (57). opioid analgesics morphine and its related drugs act on inhibitory opioid receptors of μ, κ and δ subtypes expressed in nociceptive neuronal circuits. mu-receptors responsible for the effects of opioid analgesics and antagonists are located within lipid raft/caveola membrane domains (58). heron et al performed in vivo experiments to inject rats with opioids intraperitoneally and in vitro experiments to subject membranes prepared from rat brains to the reaction with opioids, followed by fp measurements (59). morphine increased the fluidity of hippocampus and caudate membranes from rats injected at 25 mg/kg (i.p.) and the fluidity of the membrane preparations at 10 nm and 10 μm. in contrast, opioid antagonist naloxone decreased the membrane fluidity of the same brain regions at 2 mg/kg (i.p.) and reversed the in vitro membrane-fluidizing effect of 10 nm morphine at 1 nm. budai et al evaluated the effects of different opioids on dppc multilamellar vesicles (mlvs) by dsc and electron paramagnetic resonance (epr) spectroscopy (60). codeine and n-methylcodeine reduced the phase transition temperature of dppc membranes at 0.1 m. zheng et al treated hek (human embryonic kidney) 293 cells expressing μ-receptors with or subcutaneously injected mice with opioid agonists (61). sdgc cell fractions and hippocampus isolates demonstrated that etorphine of 10 nm and 5 μg/kg (s.c.) translocate μ-receptors from lipid rafts to non-raft regions as well as cholesterol-depleting mbc. anti-inflammatory drugs non-steroidal anti-inflammatory drugs are considered to exert therapeutic and adverse effects by inhibiting cyclooxygenase (cox)-2 and cox-1, respectively. cox-2 is localized in lipid raft/caveola membrane domains and associated with caveolin-1 (62). alsop et al studied the effects of aspirin on different dppc/cholesterol bilayer membrane systems by langmuirblodgett, dsc and nd experiments (63). aspirin (3 mm) increased the membrane fluidity of dppc membranes containing 32.5 mol% cholesterol, disrupted the membrane organization and prevented the formation of l o phase lipid rafts. in the following neutron scattering experiments and molecular dynamics simulations, they prepared mlvs with 70 mol% 1,2-dimyristoylphosphatidylcholine (dmpc) and 30 mol% cholesterol to study the membrane effect of aspirin (64). aspirin bound to raft-like l o phase domains and disrupted their organization at 10 mol%. zhou et al reported that 5 μm indomethacin, 25 μm naproxen, 50 μm aspirin and 150 μm ibuprofen acted on bhk (baby hamster kidney) cells to affect the organization of raft-like ordered lipid and protein membrane nanoclusters by interacting with plasma membranes (65). anticancer drugs in addition to conventional mechanistic effects, anticancer drugs exhibit apoptosis-inducing activity. lipid rafts contribute to induction of the apoptosis selective for cancer cells (66). alkylphospholipids, platinum(ii) complex and antibiotics are presumed to act on lipid rafts as a membrane gateway to induce apoptosis (67). alves et al recently published an excellent review on the biophysics of cancer cells and the relevance of drug and membrane interaction to cancer therapy (68). results of the literature search on the interaction of anticancer drugs with membrane lipid rafts and membranes are shown in table iv. alkylphospholipids ausili et al treated mlvs prepared with popc, sm and cholesterol (1:1:1 molar ratio) with edelfosine at 10-20 mol% relative to membrane lipids, followed by dsc, xd and nmr analysis (69). edelfosine altered the raft organization and induced the appearance of a sharp phase transition at 20 mol%, suggesting a fluidity increase in membrane lipid rafts. when incubating with human acute t-cell leukemia (jurkat t) cells, edelfosine colocalized in lipid rafts at concentrations higher than 20 mol%. 10-(octyloxy) decyl-2-(trimethylammonium) ethyl phosphate (odpc) with the cytotoxic activity against cancer cell lines inhibits the proliferation of leukemia cells by inducing apoptosis. gomide et al prepared giant unilamellar vesicles (guvs) with dopc, sm and cholesterol (1:1:1 molar ratio) to examine the effects of perifosine and odpc on lipid rafts (70). in fluorescence microscopic observations, perifosine and odpc disrupted membrane raft domains in guvs so that the domains disappeared in less than 1 min after treatment at 100 μm. castro et al treated mlvs or unilamellar vesicles (ulvs) prepared with popc, n-palmitoyl-sm and cholesterol (1:1:1 molar ratio) with anticancer alkylphospholipids, followed by fa measurements (71). edelfosine and miltefosine were demonstrated to increase the fluidity of raft model membranes at 5-10 mol% relative to membrane lipids. wnętrzak et al studied the effects of synthetic phospholipid analog erucylphosphocholine on raft-mimic langmuir monolayers composed of sm and cholesterol (2:1 molar ratio) (72). erucylphosphocholine increased the membrane raft fluidity at higher than 0.3 mol% relative to membrane lipids and weakened the interaction between cholesterol and sm. in a thermodynamic study using the same langmuir monolayers, anticancer 2-hydroxyoleic acid increased the membrane fluidity of raft-mimic monolayers at higher than 0.1 mol% relative to membrane lipids (73). cisplatin cisplatin acts on plasma membranes to trigger the fas death receptor pathway at a membrane level (74). lacour et al treated human colon carcinoma (ht29) cells (7 × 105 cells) with cisplatin at 5 μg/ml for 0.25-4 hours (75). the cells were subjected to 12-dsa (12-doxylstearic acid) spin labeling followed by epr spectroscopic analysis or cell lysis with triton x-100 followed by sdgc fractionation and immunoblot tsuchiya and mizogami 41 © 2020 the authors. published by aboutscience table iv interaction of anticancer drugs with lipid raft membrane domains and membranes drug class drug membrane induced membrane modification reference alkylphospholipid edelfosine (≥20 mol%) mlvs (popc, sm and cholesterol, 1:1:1, molar ratio) human acute t-cell leukemia cells increased the fluidity of lipid rafts colocalized in membrane lipid rafts 69 alkylphospholipid perifosine (100 μm) odpc (100 μm) guvs (dopc, sm and cholesterol, 1:1:1 molar ratio) disrupted membrane raft domains 70 alkylphospholipid edelfosine (5-10 mol%) miltefosine (5-10 mol%) mlvs or ulvs (popc, n-palmitoyl-sm and cholesterol, 1:1:1 molar ratio) increased the fluidity of raft model membranes 71 alkylphospholipid erucylphosphocholine (≥0.3 mol%) raft-mimic langmuir monolayers (sm and cholesterol, 2:1 molar ratio) increased the membrane raft fluidity and weakened the interaction between cholesterol and sm 72 alkylphospholipid 2-hydroxyoleic acid (≥0.1 mol%) raft-mimic langmuir monolayers (sm and cholesterol, 2:1 molar ratio) increased the membrane raft fluidity 73 platinum(ii) complex cisplatin (5 μg/ml) human colon carcinoma cells increased the membrane fluidity, which was inhibited by 10 μg/ml nystatin pretreatment translocated cd95 into lipid rafts, which was prevented by 10 μg/ml nystatin pretreatment 75 platinum(ii) complex cisplatin (25 μm) human colon carcinoma cells increased the membrane raft fluidity and induced apoptosis, which was inhibited by cholesterol (30 μg/ml) and monosialoganglioside-1 (80 μm) 76 antibiotic azithromycin (132 μm) suvs (dopc, sm and cholesterol, 1:1:1 molar ratio) increased the fluidity of raft-like membranes 77 antibiotic daunorubicin (40-75 μm) luvs (dmpc, sm and cholesterol, 7:1.5:1.5 molar ratio) decreased the fluidity of raft-like membranes 78 antibiotic doxorubicin (40-75 μm) luvs (dmpc and sm, 8:2 molar ratio or dmpc, sm and cholesterol, 7:1.5:1.5 molar ratio) increased the fluidity of binary membranes, but not ternary membranes 79 dmpc = 1,2-dimyristoylphosphatidylcholine; dopc = 1,2-dioleoylphosphatidylcholine; guv = giant unilamellar vesicle; luv = large unilamellar vesicle; mlv = multilamellar vesicle; odpc = 10-(octyloxy) decyl-2-(trimethylammonium) ethyl phosphate; popc = 1-palmitoyl-2-oleoylphosphatidylcholine; sm = sphingomyelin; suv = small unilamellar vesicle; ulv = unilamellar vesicle. analysis. cisplatin increased the fluidity of plasma membranes as soon as 0.25 hours after the treatment, although its membrane effect was inhibited by pretreating with cholesterol sequestering nystatin at 10 μg/ml. the cell exposure to cisplatin for 4 hours induced the translocation of cd95 (cluster of differentiation 95 known as fas receptor) into lipid rafts, which was prevented by nystatin pretreated at 10 μg/ ml. rebillard et al treated human colon carcinoma (ht29) cells growing in the exponential phase with cisplatin at 25 μm for 1-72 hours (76). they isolated lipid rafts by sdgc and performed epr spectroscopic analysis after 12-dsa spin labeling. cisplatin treatment for 1 hour increased membrane raft fluidity and that for 72 hours induced apoptosis. such effects were inhibited by membrane-stabilizing cholesterol (30 μg/ml) and monosialoganglioside-1 (80 μm). anticancer antibiotics berquand et al treated suvs prepared with dopc, sm and cholesterol (1:1:1 molar ratio) with macrolide antibiotic azithromycin (77). fp analysis revealed that azithromycin increases the fluidity of a hydrophobic region of raft-like membranes at 132 μm. in fa experiments of alves et al (78), anthracycline antibiotic daunorubicin (40-75 μm) decreased the fluidity of raft-like membranes of luvs prepared with dmpc, sm and cholesterol (7:1.5:1.5 molar ratio), while this antibiotic was more effective in decreasing the membrane fluidity of luvs prepared without cholesterol. alves et al also investigated the effects of doxorubicin on luvs prepared with dmpc and sm (8:2 molar ratio) or with dmpc, sm and cholesterol (7:1.5:1.5 molar ratio) by measuring fa (79). doxorubicin increased the fluidity of binary membranes at 40-75 μm, but not raft-like ternary membranes containing cholesterol. phytochemicals a variety of phytochemicals (bioactive components in plants) such as flavonoids exhibit a broad spectrum of pharmacological activity including antioxidant, antitumor, interaction of drugs with lipid rafts42 © 2020 the authors. published by aboutscience anti-inflammatory, analgesic, antimicrobial, cardioprotective, anti-allergic and antiplatelet ones. many of them with the amphiphilic structure share the property to interact with artificial and biological membranes. the membrane interactivity of phytochemicals was recently reviewed by tsuchiya (80), especially the interaction of flavonoids with lipid rafts by tarahovsky et al (81) and their induced changes in membrane fluidity by selvaraj et al (82). results of the literature search on the interaction of phytochemicals with membrane lipid rafts and membranes are shown in table v. table v interaction of phytochemicals with lipid raft membrane domains and membranes drug class drug membrane induced membrane modification reference flavonoid quercetin (10 μm) egcg (10 μm) cyanidin (10 μm) suvs (phospholipids (popc and sm) and cholesterol by varying the composition 55-80 mol% and 20-45 mol%) quercetin decreased the membrane fluidity most potently, followed by cyanidin and egcg 84 flavonoid quercetin (30 μm) human colon cancer cells (ht-29, sw-620 and caco-2) enhanced trail efficacy to induce apoptosis by accumulating death receptors in membrane lipid rafts 85 flavonoid quercetin (10 and 100 μm) luteolin (10 and 100 μm) mouse macrophages suppressed the accumulation of lipid rafts to inhibit tnf-α production 86 flavonoid quercetin (2-16 μm) suvs (dmpc plus 20 or 33 mol% cholesterol) increased the fluidity of raft model membranes 87 flavonoid egcg (5-100 μm) suvs (5 mol% cholesterol and 95 mol% popc or dopc) decreased the fluidity of binary membranes 88 flavonoid egcg (5-20 μg/ml) human colon carcinoma cells reduced the membrane resistance to triton x-100 by decreasing ordered membrane domains 89 flavonoid egcg (5 μm) human prostate cancer cells inhibited diic 16 accumulation in lipid ordered domains and disrupted lipid rafts 90 flavonoid egcg (5-20 μm) human multiple myeloma cells induced lipid raft clustering and apoptotic cell death 91 flavonoid dimeric procyanidin (0.05-1 μg/ml) human acute t-cell leukemia cells increased the membrane fluidity 92 flavonoid hexameric procyanidin (10 μm) human colon cancer cells decreased the membrane fluidity, although the membrane interactivity was lost by mbc (2.5 mm) prevented the lipid raft disruption induced by mbc or deoxycholate 93 stilbenoid resveratrol (10-80 μm) luvs (egg phosphatidylcholine, sm and cholesterol, 1:1:1 molar ratio) formed the ordered membrane domains and enhanced the membrane resistance to triton x-100 94 anthraquinonoid emodin (1-5 mol%) aloin (1-5 mol%) mlvs composed of dmpc reduced the phase transition temperature 95 anthraquinonoid emodin (10-50 μg/ml) human umbilical vein endothelial cells disrupted lipid rafts 96 terpenoid ginsenosides rb2, rc, rd, re, rf, rg1, rg2 and rh2 (50 μm) hela cells increased the membrane fluidity reduced the raft-marker protein concentration in lipid rafts 98 terpenoid saikosaponin a (3-12 μm) mouse macrophages inhibited lps-induced cytokine expression and toll-like receptor localization in lipid rafts, and reduced membrane cholesterol levels 99 dmpc = 1,2-dimyristoylphosphatidylcholine; dopc = 1,2-dioleoylphosphatidylcholine; egcg = (–)-epigallocatechin-3-gallate; lps = lipopolysaccharide; luv = large unilamellar vesicle; mbc = methyl-β-cyclodextrin; mlv = multilamellar vesicle; popc = 1-palmitoyl-2-oleoylphosphatidylcholine; sm = sphingomyelin; suv = small unilamellar vesicle; tnf = tumor necrosis factor; trail = tnf-related apoptosis-inducing ligand. tsuchiya and mizogami 43 © 2020 the authors. published by aboutscience flavonoids considering the distribution and accumulation in lipid bilayers, representative flavonoid quercetin and (–)-epigallocatechin-3-gallate (egcg) possibly alter membrane fluidity and order, making or breaking raft-like domains (83). tsuchiya and mizogami compared the effects of different flavonoids on suvs that were prepared with phospholipids (popc and sm) and cholesterol by varying their compositions 55-80 mol% and 20-45 mol%, respectively (84). fp data indicated that quercetin interacts preferentially with the hydrophobic region of membranes to decrease the fluidity at 10 μm most potently, followed by cyanidin and egcg. psahoulia et al investigated the mechanism underlying an apoptosis enhancing effect of quercetin by treating human colon cancer cells (ht-29, sw-620 and caco-2) with quercetin at 30 μm (85). while tumor necrosis factor (tnf)-related apoptosisinducing ligand (trail) contributes to apoptosis induction, quercetin enhanced the trail efficacy to induce apoptosis by accumulating death receptors in membrane lipid rafts. in a cell culture study of kaneko et al, quercetin and luteolin suppressed the accumulation of lipid rafts at 10 and 100 μm to inhibit tnf-α production in mouse macrophages (86). they also suggested that these flavonoids change membrane fluidity. ionescu et al prepared suvs with dmpc plus 20 or 33 mol% cholesterol to form the l o phase and examine the membrane effect of quercetin (87). fp measurements showed that quercetin increases the fluidity of raft model membranes at 2-16 μm. tsuchiya treated suvs consisting of 5 mol% cholesterol and 95 mol% popc or dopc with several catechins, followed by fp measurements (88). of the tested catechins, egcg most potently interacted with binary membranes to decrease their fluidity at 5-100 μm. adachi et al stained human colon carcinoma (ht29) cells with fluorescent diic 16 (1,1’-dihexadecyl3,3,3’,3’-tetramethylindocarbocyanine perchlorate) that is preferentially incorporated into the ordered membranes, and then treated the cells with egcg at 5-20 μg/ml to analyze its membrane effects by fluorescent confocal microscopy (89). egcg reduced the membrane resistance to triton x-100 at as little as 5 μg/ml, possibly by decreasing the content of ordered membrane domains. duhon et al exposed human prostate cancer (du145) cells to diic 16 in the presence or absence of 5 μm egcg, followed by fluorescence microscopic analysis (90). egcg inhibited the accumulation of diic 16 in lipid-ordered domains and disrupted lipid rafts. tsukamoto et al treated human multiple myeloma (u266) cells with egcg at 5-20 μm for 3 hours and at 10 μm for 1-3 hours (91). fluorescence resonance energy transfer and fluorescence microscopic assays indicated that egcg doseand time-dependently induces lipid raft clustering and apoptotic cell death. procyanidins contained in fruits and vegetables are oligomeric flavonoids with the anticancer activity. verstraeten et al treated human acute t-cell leukemia (jurkat t) cells (6 × 104 cells) with cocoa procyanidins and measured fp (92). dimeric procyanidin increased the fluidity of plasma membranes in a concentration-dependent manner at 0.05-1 μg/ ml. in the following experiment, they incubated human colon cancer (caco-2) cells with 10 μm hexameric procyanidin in the absence or presence of 2.5 mm mbc (93). in contrast to dimeric procyanidin, hexameric procyanidin decreased the fluidity of plasma membranes, although its membrane interactivity was lost by cholesterol-depleting mbc. this procyanidin also prevented lipid raft disruption induced by mbc or deoxycholate (cholesterol depletion/redistribution). stilbenoids resveratrol present in grape skins and seeds has anticancer, antioxidant and cardioprotective property. neves et al treated luvs prepared with egg phosphatidylcholine, sm and cholesterol (1:1:1 molar ratio) with 10-80 μm resveratrol to investigate the effects on raft model membranes by three different methods (94). resveratrol induced the phase separation and formed the ordered membrane domains at concentrations higher than 10 μm. such effects were more pronounced in the presence of cholesterol and sm. resveratrol was also effective at 80 μm in enhancing the membrane resistance to triton x-100. anthraquinonoids pharmacological effects of aloe are attributed to anthraquinonoid component emodin and aloin (barbaloin). dsc experiments of alves et al demonstrated that emodin interacts with mlvs composed of dmpc to reduce the phase transition temperature at 1-5 mol% more potently than aloin (95). meng et al investigated the mechanism underlying a vascular anti-inflammatory effect of aloe by treating human umbilical vein endothelial cells grown to approximately 90% confluence with emodin (96). emodin (10-50 μg/ml) inhibited the expression of proinflammatory cytokines and chemokines induced by 0.1 μg/ml lipopolysaccharide (lps). similar to cholesterol-depleting mbc (5-12.5 mm), emodin (10-50 μg/ml) disrupted lipid rafts that are relevant to the cell activation by lps. lipid raft disruption associated with integrin signaling pathway is also responsible for the inhibitory effects of emodin on tumor cell adhesion and spreading (97). terpenoids triterpenoid glycosides from panax ginseng and triterpenoid saponin derivatives from radix bupleuri have anti inflammatory and anticancer activity. yi et al treated hela cells with different ginsenosides at 50 μm and stained the cells with carboxy laurdan, followed by fluorescence microscopy and generalized polarization imaging (98). ginsenosides rb2, rc, rd, re, rf, rg1, rg2 and rh2 increased the fluidity of plasma membranes as well as cholesterol-depleting mbc (10 mm). when fractionating the hela cells by sdgc, ginsenoside rh2 and mbc reduced the concentration of raft-marker proteins in the raft fraction, indicating that they disrupt lipid rafts. these effects of ginsenoside rh2 were reversed by cholesterol overloading (20 μg/ml). in a cell culture study of wei et al (99), 3-12 μm saikosaponin a inhibited the expression of cytokines in primary mouse macrophages stimulated by 0.1 μg/ml lps. such inhibitory effects were attenuated by replenishment of 84 μg/ml cholesterol, while 3-12 μm saikosaponin a reduced cholesterol levels in macrophage membranes. saikosaponin a (3-12 μm) and mbc (10 mm) also inhibited the lps-induced interaction of drugs with lipid rafts44 © 2020 the authors. published by aboutscience localization in lipid rafts of toll-like receptors that play a crucial role in the innate immune system. conclusions results of the literature search indicate that different classes of drugs interact with raft model membranes and cellular membrane lipid rafts in addition to interacting directly with membrane receptors, ion channels and enzymes. they could physicochemically modify membrane lipid rafts to be a platform for functional proteins and the membranes surrounding such raft domains, affecting the organizational integrity of lipid rafts with the subsequent alteration of receptor, channel and enzyme activity, thereby producing pharmacological effects. with respect to the induced membrane modification, raft-acting drugs are characterized as ones to decrease membrane fluidity, induce l o phase or order plasma membranes, leading to lipid raft formation; and ones to increase membrane fluidity, induce l d phase or reduce phase transition temperature, leading to lipid raft disruption. targeting lipid raft membrane domains would open a new way for drug design and development. given the critical role of lipid rafts/caveolae in cellular signal transduction, odontology may be the promising field to which a raft-targeting concept is applied. anticancer drugs interact with membrane lipid rafts to affect their physicochemical property and organizational integrity in association with apoptosis induction. lipid raft membrane domains are responsible for cancer cell adhesion and migration, and the levels of cholesterol-rich lipid rafts are elevated in cancer cells compared with normal counterparts (100,101). while phytochemicals interact with membrane lipid rafts and regulate raft formation (83,85), such interactivity is responsible for their diverse bioactivities including apoptosis induction. among raft-targeting compounds, alkylphospholipids and flavonoids could be a novel type of anticancer drug. since an outbreak of atypical pneumonia was first reported in wuhan (china) in december 2019, novel coronavirus or severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infections have spread worldwide, causing a global pandemic of coronavirus disease 2019 (covid-19). sars-cov-2 spike proteins have a strong binding affinity to human angiotensin-converting enzyme 2 (ace2) (102). host cell ace2 receptors, which are a cell-specific target of and responsible for the cellular entry of sars-cov-2, are localized in lipid rafts (103). cholesterol-rich membrane domains are essential for the spike proteins to interact with ace2 receptors efficiently (104) and cellular cholesterol levels are closely associated with covid-19 lethality (105). agents that specifically disrupt ace2-localizing lipid rafts and deplete raft cholesterol may be a drug to reduce sars-cov-2 infectivity and covid-19 severity. abbreviations l o , liquid-ordered; l d , liquid-disordered; popc, 1-palmitoyl-2-oleoylphosphatidylcholine; sm, sphingomyelin; sdgc, sucrose density gradient centrifugation; mbc, methyl-βcyclodextrin; fp, fluorescence polarization; fa, fluorescence anisotropy; dsc, differential scanning calorimetry; nmr, nuclear magnetic resonance; nd, neutron diffraction; xd, xray diffraction; gaba a , γ-aminobutyric acid type a; nmda, n-methyl-d-aspartate; pope, 1-palmitoyl-2-oleoylphosphatidylethanolamine; pops, 1-palmitoyl-2-oleoylphosphatidylserine; gpmv, giant plasma membrane vesicle; luv, large unilamellar vesicle; dppc, 1,2-dipalmitoylphosphatidylcholine; dlpc, 1,2-dilauroylphosphatidylcholine; mac, minimum alveolar concentration; dopc, 1,2-dioleoylphosphatidylcholine; nav, voltage-gated sodium; suv, small unilamellar vesicle; cb, cerebroside; popi, 1-palmitoyl-2-oleoylphosphatidylinositol; mlv, multilamellar vesicle; epr, electron paramagnetic resonance; cox, cyclooxygenase; dmpc, 1,2-dimyristoylphosphatidylcholine; odpc, 10-(octyloxy) decyl-2-(trimethylammonium) ethyl phosphate; guv, giant unilamellar vesicle; ulv, unilamellar vesicle; egcg, (–)epigallocatechin-3-gallate; tnf, tumor necrosis factor; trail, tnf-related apoptosis-inducing ligand; lps, lipopolysaccharide; sars-cov-2, severe acute respiratory syndrome coronavirus 2; covid-19, coronavirus disease 2019; ace2, angiotensin-converting enzyme 2. author contributions ht designed and conducted the present study and prepared the first draft of the manuscript. ht and mm did literature search, information analysis and manuscript preparation. both authors reviewed and approved the final manuscript. disclosures conflict of interest: authors disclose no potential conflicts of interest. financial support: this study was supported by jsps kakenhi grant number 20k10152 and jsps kakenhi grant number 17k11924. references 1. kusumi a, fujiwara tk, chadda r, et al. dynamic organizing principles of the plasma membrane that regulate signal transduction: commemorating the fortieth anniversary of singer and nicolson’s fluid-mosaic model. annu rev cell dev biol. 2012;28(1):215-250. crossref medline 2. pike lj. rafts defined: a report on the keystone symposium on lipid rafts and cell function. j lipid res. 2006;47(7):1597-1598. crossref medline 3. hanzal-bayer mf, hancock jf. lipid rafts and membrane traffic. febs lett. 2007;581(11):2098-2104. crossref medline 4. mcmullen t, lewis r, mcelhany r. cholesterol-phospholipid interactions, the liquid-ordered phase and lipid rafts in model and biological membranes. curr opin colloid interface sci. 2004;8(6):459-468. crossref 5. simons k, toomre d. lipid rafts and signal transduction. nat rev mol cell biol. 2000;1(1):31-39. crossref medline 6. laude aj, prior ia. plasma membrane microdomains: organization, function and trafficking. mol membr biol. 2004;21(3):193205. crossref medline 7. george ks, wu s. lipid raft: a floating island of death or survival. toxicol appl pharmacol. 2012;259(3):311-319. crossref medline 8. o’connell km, martens jr, tamkun mm. localization of ion channels to lipid raft domains within the cardiovascular system. trends cardiovasc med. 2004;14(2):37-42. crossref medline https://doi.org/10.1146/annurev-cellbio-100809-151736 https://www.ncbi.nlm.nih.gov/pubmed/22905956 https://doi.org/10.1194/jlr.e600002-jlr200 https://www.ncbi.nlm.nih.gov/pubmed/16645198 https://doi.org/10.1016/j.febslet.2007.03.019 https://www.ncbi.nlm.nih.gov/pubmed/17382322 https://doi.org/10.1016/j.cocis.2004.01.007 https://doi.org/10.1038/35036052 https://www.ncbi.nlm.nih.gov/pubmed/11413487 https://doi.org/10.1080/09687680410001700517 https://www.ncbi.nlm.nih.gov/pubmed/15204627 https://doi.org/10.1016/j.taap.2012.01.007 https://www.ncbi.nlm.nih.gov/pubmed/22289360 https://doi.org/10.1016/j.tcm.2003.10.002 https://www.ncbi.nlm.nih.gov/pubmed/15030787 tsuchiya and mizogami 45 © 2020 the authors. published by aboutscience 9. maguy a, hebert te, nattel s. involvement of lipid rafts and caveolae in cardiac ion channel function. cardiovasc res. 2006;69(4):798-807. crossref medline 10. dalskov sm, immerdal l, niels-christiansen ll, hansen gh, schousboe a, danielsen em. lipid raft localization of gaba a receptor and na+, k+-atpase in discrete microdomain clusters in rat cerebellar granule cells. neurochem int. 2005;46(6):489499. crossref medline 11. patel hh, murray f, insel pa. g-protein-coupled receptor-signaling components in membrane raft and caveolae microdomains. handb exp pharmacol. 2008;186(186):167-184. crossref medline 12. xiang y, rybin vo, steinberg sf, kobilka b. caveolar localization dictates physiologic signaling of β 2 -adrenoceptors in neonatal cardiac myocytes. j biol chem. 2002;277(37):34280-34286. crossref medline 13. pottosin ii, valencia-cruz g, bonales-alatorre e, shabala sn, dobrovinskaya or. methyl-β-cyclodextrin reversibly alters the gating of lipid rafts-associated kv1.3 channels in jurkat t lymphocytes. pflugers arch. 2007;454(2):235-244. crossref medline 14. levitt es, clark mj, jenkins pm, martens jr, traynor jr. differential effect of membrane cholesterol removal on μand δ-opioid receptors: a parallel comparison of acute and chronic signaling to adenylyl cyclase. j biol chem. 2009;284(33):2210822122. crossref medline 15. ding xq, fitzgerald jb, matveev av, mcclellan me, elliott mh. functional activity of photoreceptor cyclic nucleotide-gated channels is dependent on the integrity of cholesteroland sphingolipid-enriched membrane domains. biochemistry. 2008;47(12):3677-3687. crossref medline 16. gandhavadi m, allende d, vidal a, simon sa, mcintosh tj. structure, composition, and peptide binding properties of detergent soluble bilayers and detergent resistant rafts. biophys j. 2002;82(3):1469-1482. crossref medline 17. koumanov ks, tessier c, momchilova ab, rainteau d, wolf c, quinn pj. comparative lipid analysis and structure of detergentresistant membrane raft fractions isolated from human and ruminant erythrocytes. arch biochem biophys. 2005;434(1): 150-158. crossref medline 18. holland gp, mcintyre sk, alam tm. distinguishing individual lipid headgroup mobility and phase transitions in raft-forming lipid mixtures with 31p mas nmr. biophys j. 2006;90(11):42484260. crossref medline 19. simons k, ehehalt r. cholesterol, lipid rafts, and disease. j clin invest. 2002;110(5):597-603. crossref medline 20. jacobson k, mouritsen og, anderson rg. lipid rafts: at a crossroad between cell biology and physics. nat cell biol. 2007;9(1):7-14. crossref medline 21. boesze-battaglia k. isolation of membrane rafts and signaling complexes. methods mol biol. 2006;332:169-179. crossref medline 22. loftsson t, magnúsdóttir a, másson m, sigurjónsdóttir jf. selfassociation and cyclodextrin solubilization of drugs. j pharm sci. 2002;91(11):2307-2316. crossref medline 23. mahammad s, parmryd i. cholesterol depletion using methylβ-cyclodextrin. methods mol biol. 2015;1232:91-102. crossref medline 24. chau pl. new insights into the molecular mechanisms of general anaesthetics. br j pharmacol. 2010;161(2):288-307. crossref medline 25. olsen rw, li gd. gaba a receptors as molecular targets of general anesthetics: identification of binding sites provides clues to allosteric modulation. can j anaesth. 2011;58(2):206-215. crossref medline 26. yamakura t, harris ra. effects of gaseous anesthetics nitrous oxide and xenon on ligand-gated ion channels. comparison with isoflurane and ethanol. anesthesiology. 2000;93(4):10951101. crossref medline 27. daniell lc. effect of anesthetic and convulsant barbiturates on n-methyl-d-aspartate receptor-mediated calcium flux in brain membrane vesicles. pharmacology. 1994;49(5):296-307. crossref medline 28. li x, serwanski dr, miralles cp, bahr ba, de blas al. two pools of triton x-100-insoluble gaba a receptors are present in the brain, one associated to lipid rafts and another one to the postsynaptic gabaergic complex. j neurochem. 2007;102(4):13291345. crossref medline 29. parat mo. could endothelial caveolae be the target of general anaesthetics? br j anaesth. 2006;96(5):547-550. crossref medline 30. tsuchiya h. structure-specific membrane-fluidizing effect of propofol. clin exp pharmacol physiol. 2001;28(4):292-299. crossref medline 31. tsuchiya h, ueno t, tanaka t, matsuura n, mizogami m. comparative study on determination of antioxidant and membrane activities of propofol and its related compounds. eur j pharm sci. 2010;39(1-3):97-102. crossref medline 32. gray e, karslake j, machta bb, veatch sl. liquid general anesthetics lower critical temperatures in plasma membrane vesicles. biophys j. 2013;105(12):2751-2759. crossref medline 33. grim kj, abcejo aj, barnes a, et al. caveolae and propofol effects on airway smooth muscle. br j anaesth. 2012;109(3):444453. crossref medline 34. patel j, chowdhury ea, noorani b, bickel u, huang j. isoflurane increases cell membrane fluidity significantly at clinical concentrations. biochim biophys acta biomembr. 2020;1862(2):183140. crossref medline 35. turkyilmaz s, almeida pf, regen sl. effects of isoflurane, halothane, and chloroform on the interactions and lateral organization of lipids in the liquid-ordered phase. langmuir. 2011;27(23):14380-14385. crossref medline 36. weinrich m, nanda h, worcester dl, majkrzak cf, maranville bb, bezrukov sm. halothane changes the domain structure of a binary lipid membrane. langmuir. 2012;28(10):4723-4728. crossref medline 37. weinrich m, worcester dl. xenon and other volatile anesthetics change domain structure in model lipid raft membranes. j phys chem b. 2013;117(50):16141-16147. crossref medline 38. sierra-valdez fj, ruiz-suárez jc, delint-ramirez i. pentobarbital modifies the lipid raft-protein interaction: a first clue about the anesthesia mechanism on nmda and gaba a receptors. biochim biophys acta. 2016;1858(11):2603-2610. crossref medline 39. fozzard ha, lee pj, lipkind gm. mechanism of local anesthetic drug action on voltage-gated sodium channels. curr pharm des. 2005;11(21):2671-2686. crossref medline 40. pristerá a, okuse k. building excitable membranes: lipid rafts and multiple controls on trafficking of electrogenic molecules. neuroscientist. 2012;18(1):70-81. crossref medline 41. bao l. trafficking regulates the subcellular distribution of voltage-gated sodium channels in primary sensory neurons. mol pain. 2015;11:61. crossref medline 42. pristerà a, baker md, okuse k. association between tetrodotoxin resistant channels and lipid rafts regulates sensory neuron excitability. plos one. 2012;7(8):e40079. crossref medline 43. kamata k, manno s, ozaki m, takakuwa y. functional evidence for presence of lipid rafts in erythrocyte membranes: gsα in rafts is essential for signal transduction. am j hematol. 2008;83(5):371-375. crossref medline 44. bandeiras c, serro ap, luzyanin k, fernandes a, saramago b. anesthetics interacting with lipid rafts. eur j pharm sci. 2013;48(1-2):153-165. crossref medline https://doi.org/10.1016/j.cardiores.2005.11.013 https://www.ncbi.nlm.nih.gov/pubmed/16405931 https://doi.org/10.1016/j.neuint.2004.11.010 https://doi.org/10.1016/j.neuint.2004.11.010 https://doi.org/10.1007/978-3-540-72843-6_7 https://www.ncbi.nlm.nih.gov/pubmed/18491052 https://doi.org/10.1074/jbc.m201644200 https://www.ncbi.nlm.nih.gov/pubmed/12097322 https://doi.org/10.1007/s00424-007-0208-4 https://www.ncbi.nlm.nih.gov/pubmed/17242956 https://doi.org/10.1074/jbc.m109.030411 https://www.ncbi.nlm.nih.gov/pubmed/19520863 https://doi.org/10.1021/bi7019645 https://www.ncbi.nlm.nih.gov/pubmed/18303857 https://doi.org/10.1016/s0006-3495(02)75501-x https://www.ncbi.nlm.nih.gov/pubmed/11867462 https://doi.org/10.1016/j.abb.2004.10.025 https://www.ncbi.nlm.nih.gov/pubmed/15629118 https://doi.org/10.1529/biophysj.105.077289 https://www.ncbi.nlm.nih.gov/pubmed/16533851 https://doi.org/10.1172/jci0216390 https://www.ncbi.nlm.nih.gov/pubmed/12208858 https://doi.org/10.1038/ncb0107-7 https://www.ncbi.nlm.nih.gov/pubmed/17199125 https://doi.org/10.1385/1-59745-048-0:167 https://www.ncbi.nlm.nih.gov/pubmed/16878692 https://doi.org/10.1002/jps.10226 https://www.ncbi.nlm.nih.gov/pubmed/12379916 https://doi.org/10.1007/978-1-4939-1752-5_8 https://www.ncbi.nlm.nih.gov/pubmed/25331130 https://doi.org/10.1111/j.1476-5381.2010.00891.x https://www.ncbi.nlm.nih.gov/pubmed/20735416 https://doi.org/10.1007/s12630-010-9429-7 https://www.ncbi.nlm.nih.gov/pubmed/21194017 https://doi.org/10.1097/00000542-200010000-00034 https://www.ncbi.nlm.nih.gov/pubmed/11020766 https://doi.org/10.1159/000139246 https://www.ncbi.nlm.nih.gov/pubmed/7862741 https://doi.org/10.1111/j.1471-4159.2007.04635.x https://www.ncbi.nlm.nih.gov/pubmed/17663755 https://doi.org/10.1093/bja/ael079 https://www.ncbi.nlm.nih.gov/pubmed/16600902 https://doi.org/10.1046/j.1440-1681.2001.03441.x https://www.ncbi.nlm.nih.gov/pubmed/11251643 https://doi.org/10.1016/j.ejps.2009.11.001 https://www.ncbi.nlm.nih.gov/pubmed/19897032 https://doi.org/10.1016/j.bpj.2013.11.005 https://www.ncbi.nlm.nih.gov/pubmed/24359747 https://doi.org/10.1093/bja/aes130 https://www.ncbi.nlm.nih.gov/pubmed/22542538 https://doi.org/10.1016/j.bbamem.2019.183140 https://www.ncbi.nlm.nih.gov/pubmed/31790694 https://doi.org/10.1021/la2035278 https://www.ncbi.nlm.nih.gov/pubmed/21995557 https://doi.org/10.1021/la204317k https://www.ncbi.nlm.nih.gov/pubmed/22352350 https://doi.org/10.1021/jp411261g https://www.ncbi.nlm.nih.gov/pubmed/24299622 https://doi.org/10.1016/j.bbamem.2016.07.011 https://www.ncbi.nlm.nih.gov/pubmed/27457704 https://doi.org/10.2174/1381612054546833 https://www.ncbi.nlm.nih.gov/pubmed/16101448 https://doi.org/10.1177/1073858410393977 https://www.ncbi.nlm.nih.gov/pubmed/21518816 https://doi.org/10.1186/s12990-015-0065-7 https://www.ncbi.nlm.nih.gov/pubmed/26423360 https://doi.org/10.1371/journal.pone.0040079 https://www.ncbi.nlm.nih.gov/pubmed/22870192 https://doi.org/10.1002/ajh.21126 https://www.ncbi.nlm.nih.gov/pubmed/18181202 https://doi.org/10.1016/j.ejps.2012.10.023 https://www.ncbi.nlm.nih.gov/pubmed/23142844 interaction of drugs with lipid rafts46 © 2020 the authors. published by aboutscience 45. yoshida k, takashima a, nishio i. effect of dibucaine hydrochloride on raft-like lipid domains in model membrane system. medchemcomm. 2015;6(8):1444-1451. crossref 46. sugahara k, shimokawa n, takagi m. thermal stability of phaseseparated domains in multicomponent lipid membranes with local anesthetics. membranes (basel). 2017;7(3):33. crossref medline 47. kinoshita m, chitose t, matsumori n. mechanism of local anesthetic-induced disruption of raft-like ordered membrane domains. biochim biophys acta, gen subj. 2019;1863(9):13811389. crossref medline 48. groban l, deal dd, vernon jc, james rl, butterworth j. cardiac resuscitation after incremental overdosage with lidocaine, bupivacaine, levobupivacaine, and ropivacaine in anesthetized dogs. anesth analg. 2001;92(1):37-43. crossref medline 49. tsuchiya h, ueno t, mizogami m, takakura k. do local anesthetics interact preferentially with membrane lipid rafts? comparative interactivities with raft-like membranes. j anesth. 2010;24(4):639-642. crossref medline 50. tsuchiya h, mizogami m. r(+)-, rac-, and s(-)-bupivacaine stereostructure-specifically interact with membrane lipids at cardiotoxically relevant concentrations. anesth analg. 2012;114(2): 310-312. crossref medline 51. pontier sm, percherancier y, galandrin s, breit a, galés c, bouvier m. cholesterol-dependent separation of the β 2 -adrenergic receptor from its partners determines signaling efficacy: insight into nanoscale organization of signal transduction. j biol chem. 2008;283(36):24659-24672. crossref medline 52. mizogami m, takakura k, tsuchiya h. the interactivities with lipid membranes differentially characterize selective and nonselective β 1 -blockers. eur j anaesthesiol. 2010;27(9):829-834. crossref medline 53. tsuchiya h, mizogami m. characteristic interactivity of landiolol, an ultra-short-acting highly selective β 1 -blocker, with biomimetic membranes: comparisons with β 1 -selective esmolol and non-selective propranolol and alprenolol. front pharmacol. 2013;4:150. crossref medline 54. prichard bn, cruickshank jm, graham br. beta-adrenergic blocking drugs in the treatment of hypertension. blood press. 2001;10(5-6):366-386. crossref medline 55. callera ge, montezano ac, yogi a, tostes rc, touyz rm. vascular signaling through cholesterol-rich domains: implications in hypertension. curr opin nephrol hypertens. 2007;16(2):90104. crossref medline 56. mizogami m, tsuchiya h. membrane interactivity of anesthetic adjuvant dexmedetomidine discriminable from clonidine and enantiomeric levomedetomidine. j adv med med res. 2019;29:1-15. crossref 57. morris dp, lei b, wu yx, michelotti ga, schwinn da. the α 1a adrenergic receptor occupies membrane rafts with its g protein effectors but internalizes via clathrin-coated pits. j biol chem. 2008;283(5):2973-2985. crossref medline 58. huang p, xu w, yoon si, et al. agonist treatment did not affect association of mu opioid receptors with lipid rafts and cholesterol reduction had opposite effects on the receptor-mediated signaling in rat brain and cho cells. brain res. 2007;1184:4656. crossref medline 59. heron ds, shinitzky m, zamir n, samuel d. adaptive modulations of brain membrane lipid fluidity in drug addiction and denervation supersensitivity. biochem pharmacol. 1982;31(14):2435-2438. crossref medline 60. budai m, szabó z, szogyi m, gróf p. molecular interactions between dppc and morphine derivatives: a dsc and epr study. int j pharm. 2003;250:239–250. crossref 61. zheng h, chu j, qiu y, loh hh, law py. agonist-selective signaling is determined by the receptor location within the membrane domains. proc natl acad sci usa. 2008;105(27):9421-9426. crossref medline 62. liou jy, deng wg, gilroy dw, shyue sk, wu kk. colocalization and interaction of cyclooxygenase-2 with caveolin-1 in human fibroblasts. j biol chem. 2001;276(37):34975-34982. crossref medline 63. alsop rj, toppozini l, marquardt d, kučerka n, harroun ta, rheinstädter mc. aspirin inhibits formation of cholesterol rafts in fluid lipid membranes. biochim biophys acta. 2015;1848(3):805-812. crossref medline 64. alsop rj, himbert s, dhaliwal a, schmalzl k, rheinstädter mc. aspirin locally disrupts the liquid-ordered phase. r soc open sci. 2018;5(2):171710. crossref medline 65. zhou y, cho kj, plowman sj, hancock jf. nonsteroidal antiinflammatory drugs alter the spatiotemporal organization of ras proteins on the plasma membrane. j biol chem. 2012;287(20):16586-16595. crossref medline 66. alves acs, dias ra, kagami lp, et al. beyond the “lock and key” paradigm: targeting lipid rafts to induce the selective apoptosis of cancer cells. curr med chem. 2018;25(18):2082-2104. crossref medline 67. van der luit ah, vink sr, klarenbeek jb, et al. a new class of anticancer alkylphospholipids uses lipid rafts as membrane gateways to induce apoptosis in lymphoma cells. mol cancer ther. 2007;6(8):2337-2345. crossref medline 68. alves ac, ribeiro d, nunes c, reis s. biophysics in cancer: the relevance of drug-membrane interaction studies. biochim biophys acta. 2016;1858(9):2231-2244. crossref medline 69. ausili a, torrecillas a, aranda fj, et al. edelfosine is incorporated into rafts and alters their organization. j phys chem b. 2008;112(37):11643-11654. crossref medline 70. gomide ab, thomé ch, dos santos ga, et al. disrupting membrane raft domains by alkylphospholipids. biochim biophys acta. 2013;1828(5):1384-1389. crossref medline 71. castro bm, fedorov a, hornillos v, et al. edelfosine and miltefosine effects on lipid raft properties: membrane biophysics in cell death by antitumor lipids. j phys chem b. 2013;117(26):79297940. crossref medline 72. wnętrzak a, łątka k, makyła-juzak k, zemla j, dynarowicz-łątka p. the influence of an antitumor lipid erucylphosphocholine on artificial lipid raft system modeled as langmuir monolayer. mol membr biol. 2015;32(5-8):189-197. crossref medline 73. węder k, mach m, hąc-wydro k, wydro p. studies on the interactions of anticancer drug minerval with membrane lipids in binary and ternary langmuir monolayers. biochim biophys acta biomembr. 2018;1860(11):2329-2336. crossref medline 74. rebillard a, lagadic-gossmann d, dimanche-boitrel mt. cisplatin cytotoxicity: dna and plasma membrane targets. curr med chem. 2008;15(26):2656-2663. crossref medline 75. lacour s, hammann a, grazide s, et al. cisplatin-induced cd95 redistribution into membrane lipid rafts of ht29 human colon cancer cells. cancer res. 2004;64(10):3593-3598. crossref medline 76. rebillard a, tekpli x, meurette o, et al. cisplatin-induced apoptosis involves membrane fluidification via inhibition of nhe1 in human colon cancer cells. cancer res. 2007;67(16):7865-7874. crossref medline 77. berquand a, fa n, dufrêne yf, mingeot-leclercq mp. interaction of the macrolide antibiotic azithromycin with lipid bilayers: effect on membrane organization, fluidity, and permeability. pharm res. 2005;22(3):465-475. crossref medline 78. alves ac, ribeiro d, horta m, lima jlfc, nunes c, reis s. a biophysical approach to daunorubicin interaction with model membranes: relevance for the drug’s biological activity. j r soc interface. 2017;14(133):20170408. crossref medline 79. alves ac, magarkar a, horta m, et al. influence of doxorubicin on model cell membrane properties: insights from in vitro and in silico studies. sci rep. 2017;7(1):6343. crossref medline https://doi.org/10.1039/c5md00108k https://doi.org/10.3390/membranes7030033 https://www.ncbi.nlm.nih.gov/pubmed/28661445 https://doi.org/10.1016/j.bbagen.2019.06.008 https://www.ncbi.nlm.nih.gov/pubmed/31207252 https://doi.org/10.1097/00000539-200101000-00008 https://www.ncbi.nlm.nih.gov/pubmed/11133597 https://doi.org/10.1007/s00540-010-0943-0 https://www.ncbi.nlm.nih.gov/pubmed/20414686 https://doi.org/10.1213/ane.0b013e31823ed410 https://www.ncbi.nlm.nih.gov/pubmed/22156330 https://doi.org/10.1074/jbc.m800778200 https://www.ncbi.nlm.nih.gov/pubmed/18566454 https://doi.org/10.1097/eja.0b013e32833bf5e4 https://www.ncbi.nlm.nih.gov/pubmed/20601889 https://doi.org/10.3389/fphar.2013.00150 https://www.ncbi.nlm.nih.gov/pubmed/24339816 https://doi.org/10.1080/080370501753400665 https://www.ncbi.nlm.nih.gov/pubmed/11822540 https://doi.org/10.1097/mnh.0b013e328040bfbd https://www.ncbi.nlm.nih.gov/pubmed/17293683 https://doi.org/10.9734/jammr/2019/v29i1130142 https://doi.org/10.1074/jbc.m705795200 https://www.ncbi.nlm.nih.gov/pubmed/18048357 https://doi.org/10.1016/j.brainres.2007.09.096 https://www.ncbi.nlm.nih.gov/pubmed/17980352 https://doi.org/10.1016/0006-2952(82)90543-3 https://www.ncbi.nlm.nih.gov/pubmed/6889866 https://doi.org/10.1016/s0378-5173(02)00560-4 https://doi.org/10.1073/pnas.0802253105 https://www.ncbi.nlm.nih.gov/pubmed/18599439 https://doi.org/10.1074/jbc.m105946200 https://www.ncbi.nlm.nih.gov/pubmed/11432874 https://doi.org/10.1016/j.bbamem.2014.11.023 https://www.ncbi.nlm.nih.gov/pubmed/25475646 https://doi.org/10.1098/rsos.171710 https://www.ncbi.nlm.nih.gov/pubmed/29515878 https://doi.org/10.1074/jbc.m112.348490 https://www.ncbi.nlm.nih.gov/pubmed/22433858 https://doi.org/10.2174/0929867325666180111100601 https://www.ncbi.nlm.nih.gov/pubmed/29332565 https://doi.org/10.1158/1535-7163.mct-07-0202 https://www.ncbi.nlm.nih.gov/pubmed/17699729 https://doi.org/10.1016/j.bbamem.2016.06.025 https://www.ncbi.nlm.nih.gov/pubmed/27368477 https://doi.org/10.1021/jp802165n https://www.ncbi.nlm.nih.gov/pubmed/18712919 https://doi.org/10.1016/j.bbamem.2013.01.017 https://www.ncbi.nlm.nih.gov/pubmed/23376656 https://doi.org/10.1021/jp401407d https://www.ncbi.nlm.nih.gov/pubmed/23738749 https://doi.org/10.3109/09687688.2015.1125537 https://www.ncbi.nlm.nih.gov/pubmed/26911703 https://doi.org/10.1016/j.bbamem.2018.05.019 https://www.ncbi.nlm.nih.gov/pubmed/29864405 https://doi.org/10.2174/092986708786242903 https://www.ncbi.nlm.nih.gov/pubmed/18991628 https://doi.org/10.1158/0008-5472.can-03-2787 https://www.ncbi.nlm.nih.gov/pubmed/15150117 https://doi.org/10.1158/0008-5472.can-07-0353 https://www.ncbi.nlm.nih.gov/pubmed/17699793 https://doi.org/10.1007/s11095-004-1885-8 https://www.ncbi.nlm.nih.gov/pubmed/15835753 https://doi.org/10.1098/rsif.2017.0408 https://www.ncbi.nlm.nih.gov/pubmed/28855387 https://doi.org/10.1038/s41598-017-06445-z https://www.ncbi.nlm.nih.gov/pubmed/28740256 tsuchiya and mizogami 47 © 2020 the authors. published by aboutscience 80. tsuchiya h. membrane interactions of phytochemicals as their molecular mechanism applicable to the discovery of drug leads from plants. molecules. 2015;20(10):18923-18966. crossref medline 81. tarahovsky ys, kim ya, yagolnik ea, muzafarov en. flavonoidmembrane interactions: involvement of flavonoid-metal complexes in raft signaling. biochim biophys acta. 2014;1838(5):12351246. crossref medline 82. selvaraj s, krishnaswamy s, devashya v, sethuraman s, krishnan um. influence of membrane lipid composition on flavonoidmembrane interactions: implications on their biological activity. prog lipid res. 2015;58:1-13. crossref medline 83. tarahovsky ys, muzafarov en, kim ya. rafts making and rafts braking: how plant flavonoids may control membrane heterogeneity. mol cell biochem. 2008;314(1-2):65-71. crossref medline 84. tsuchiya h, mizogami m. plant components exhibit pharmacological activities and drug interactions by acting on lipid membranes. pharmacog commun. 2012;2:58–71. online 85. psahoulia fh, drosopoulos kg, doubravska l, andera l, pintzas a. quercetin enhances trail-mediated apoptosis in colon cancer cells by inducing the accumulation of death receptors in lipid rafts. mol cancer ther. 2007;6(9):2591-2599. crossref medline 86. kaneko m, takimoto h, sugiyama t, seki y, kawaguchi k, kumazawa y. suppressive effects of the flavonoids quercetin and luteolin on the accumulation of lipid rafts after signal transduction via receptors. immunopharmacol immunotoxicol. 2008;30(4):867-882. crossref medline 87. ionescu d, margină d, ilie m, iftime a, ganea c. quercetin and epigallocatechin-3-gallate effect on the anisotropy of model membranes with cholesterol. food chem toxicol. 2013;61:94100. crossref medline 88. tsuchiya h. stereospecificity in membrane effects of catechins. chem biol interact. 2001;134(1):41-54. crossref medline 89. adachi s, nagao t, ingolfsson hi, et al. the inhibitory effect of (-)-epigallocatechin gallate on activation of the epidermal growth factor receptor is associated with altered lipid order in ht29 colon cancer cells. cancer res. 2007;67(13):6493-6501. crossref medline 90. duhon d, bigelow rl, coleman dt, et al. the polyphenol epigallocatechin-3-gallate affects lipid rafts to block activation of the c-met receptor in prostate cancer cells. mol carcinog. 2010;49(8):739-749. crossref medline 91. tsukamoto s, hirotsu k, kumazoe m, et al. green tea polyphenol egcg induces lipid-raft clustering and apoptotic cell death by activating protein kinase cδ and acid sphingomyelinase through a 67 kda laminin receptor in multiple myeloma cells. biochem j. 2012;443(2):525-534. crossref medline 92. verstraeten sv, oteiza pi, fraga cg. membrane effects of cocoa procyanidins in liposomes and jurkat t cells. biol res. 2004;37(2):293-300. crossref medline 93. verstraeten sv, jaggers gk, fraga cg, oteiza pi. procyanidins can interact with caco-2 cell membrane lipid rafts: involvement of cholesterol. biochim biophys acta. 2013;1828(11):26462653. crossref medline 94. neves ar, nunes c, reis s. resveratrol induces ordered domains formation in biomembranes: implication for its pleiotropic action. biochim biophys acta. 2016;1858(1):12-18. crossref medline 95. alves ds, pérez-fons l, estepa a, micol v. membrane-related effects underlying the biological activity of the anthraquinones emodin and barbaloin. biochem pharmacol. 2004;68(3):549561. crossref medline 96. meng g, liu y, lou c, yang h. emodin suppresses lipopolysaccharide-induced pro-inflammatory responses and nf-κb activation by disrupting lipid rafts in cd14-negative endothelial cells. br j pharmacol. 2010;161(7):1628-1644. crossref medline 97. huang q, shen hm, shui g, wenk mr, ong cn. emodin inhibits tumor cell adhesion through disruption of the membrane lipid raft-associated integrin signaling pathway. cancer res. 2006;66(11):5807-5815. crossref medline 98. yi js, choo hj, cho br, et al. ginsenoside rh2 induces ligandindependent fas activation via lipid raft disruption. biochem biophys res commun. 2009;385(2):154-159. crossref medline 99. wei z, wang j, shi m, liu w, yang z, fu y. saikosaponin a inhibits lps-induced inflammatory response by inducing liver x receptor alpha activation in primary mouse macrophages. oncotarget. 2016;7(31):48995-49007. crossref medline 100. murai t. the role of lipid rafts in cancer cell adhesion and migration. int j cell biol. 2012;2012:763283. crossref medline 101. li yc, park mj, ye sk, kim cw, kim yn. elevated levels of cholesterol-rich lipid rafts in cancer cells are correlated with apoptosis sensitivity induced by cholesterol-depleting agents. am j pathol. 2006;168(4):1107-1118. crossref medline 102. zhang h, penninger jm, li y, zhong n, slutsky as. angiotensinconverting enzyme 2 (ace2) as a sars-cov-2 receptor: molecular mechanisms and potential therapeutic target. intensive care med. 2020;46(4):586-590. crossref medline 103. lu y, liu dx, tam jp. lipid rafts are involved in sars-cov entry into vero e6 cells. biochem biophys res commun. 2008;369(2):344349. crossref medline 104. glende j, schwegmann-wessels c, al-falah m, et al. importance of cholesterol-rich membrane microdomains in the interaction of the s protein of sars-coronavirus with the cellular receptor angiotensin-converting enzyme 2. virology. 2008;381(2):215221. crossref medline 105. wang h, yuan z, pavel ma, hobson r, hansen sb. the role of high cholesterol in age-related covid19 lethality. biorxiv. 2020.05.09.086249. crossref https://doi.org/10.3390/molecules201018923 https://www.ncbi.nlm.nih.gov/pubmed/26501254 https://doi.org/10.1016/j.bbamem.2014.01.021 https://www.ncbi.nlm.nih.gov/pubmed/24472512 https://doi.org/10.1016/j.plipres.2014.11.002 https://www.ncbi.nlm.nih.gov/pubmed/25479162 https://doi.org/10.1007/s11010-008-9766-9 https://www.ncbi.nlm.nih.gov/pubmed/18414995 http://phcogfirst.com/article/869 https://doi.org/10.1158/1535-7163.mct-07-0001 https://www.ncbi.nlm.nih.gov/pubmed/17876056 https://doi.org/10.1080/08923970802135690 https://www.ncbi.nlm.nih.gov/pubmed/18720166 https://doi.org/10.1016/j.fct.2013.03.007 https://www.ncbi.nlm.nih.gov/pubmed/23523830 https://doi.org/10.1016/s0009-2797(00)00308-2 https://www.ncbi.nlm.nih.gov/pubmed/11248221 https://doi.org/10.1158/0008-5472.can-07-0411 https://www.ncbi.nlm.nih.gov/pubmed/17616711 https://doi.org/10.1002/mc.20649 https://www.ncbi.nlm.nih.gov/pubmed/20623641 https://doi.org/10.1042/bj20111837 https://www.ncbi.nlm.nih.gov/pubmed/22257159 https://doi.org/10.4067/s0716-97602004000200016 https://www.ncbi.nlm.nih.gov/pubmed/15455659 https://doi.org/10.1016/j.bbamem.2013.07.023 https://www.ncbi.nlm.nih.gov/pubmed/23899501 https://doi.org/10.1016/j.bbamem.2015.10.005 https://www.ncbi.nlm.nih.gov/pubmed/26456556 https://doi.org/10.1016/j.bcp.2004.04.012 https://www.ncbi.nlm.nih.gov/pubmed/15242821 https://doi.org/10.1111/j.1476-5381.2010.00993.x https://www.ncbi.nlm.nih.gov/pubmed/20726986 https://doi.org/10.1158/0008-5472.can-06-0077 https://www.ncbi.nlm.nih.gov/pubmed/16740720 https://doi.org/10.1016/j.bbrc.2009.05.028 https://www.ncbi.nlm.nih.gov/pubmed/19445898 https://doi.org/10.18632/oncotarget.9863 https://www.ncbi.nlm.nih.gov/pubmed/27285988 https://doi.org/10.1155/2012/763283 https://www.ncbi.nlm.nih.gov/pubmed/22253629 https://doi.org/10.2353/ajpath.2006.050959 https://www.ncbi.nlm.nih.gov/pubmed/16565487 https://doi.org/10.1007/s00134-020-05985-9 https://www.ncbi.nlm.nih.gov/pubmed/32125455 https://doi.org/10.1016/j.bbrc.2008.02.023 https://www.ncbi.nlm.nih.gov/pubmed/18279660 https://doi.org/10.1016/j.virol.2008.08.026 https://www.ncbi.nlm.nih.gov/pubmed/18814896 https://doi.org/10.1101/2020.05.09.086249 39drug target insights 2014:8 open access: full open access to this and thousands of other papers at http://www.la-press.com. drug target insights febuxostat for hyperuricemia in patients with advanced chronic kidney disease tetsu akimoto1,2, yoshiyuki morishita1,3,4, chiharu ito1,3,4, osamu iimura3, sadao tsunematsu4, yuko watanabe1, eiji kusano1 and daisuke nagata1 1division of nephrology, department of internal medicine, jichi medical university, tochigi, japan. 2green town clinic, tochigi, japan. 3kumakura clinic, tochigi, japan. 4yuki clinic, ibaraki, japan. a bstr act: febuxostat is a nonpurine xanthine oxidase (xo) inhibitor, which recently received marketing approval. however, information regarding the experience with this agent among advanced chronic kidney disease (ckd) patients is limited. in the current study, we investigated the effects of oral febuxostat in patients with advanced ckd with asymptomatic hyperuricemia. we demonstrated, for the first time, that not only the serum levels of uric acid (ua) but also those of 8-hydroxydeoxyguanosine, an oxidative stress marker, were significantly reduced after six months of febuxostat treatment, with no adverse events. these results encouraged us to pursue further investigations regarding the clinical impact of lowering the serum ua levels with febuxostat in advanced ckd patients in terms of concomitantly reducing oxidative stress via the blockade of xo. more detailed studies with a larger number of subjects and assessments of the effects of multiple factors affecting hyperuricemia, such as age, sex, and dietary habits, would shed light on the therapeutic challenges of treating asymptomatic hyperuricemia in patients with various stages of ckd. k e y wor ds: febuxostat, chronic kidney disease, hemodialysis, uric acid, oxidative stress citation: akimoto et al. febuxostat for hyperuricemia in patients with advanced chronic kidney disease. drug target insights 2014:8 39–43 doi:10.4137/dti.s16524. received: april 28, 2014. resubmitted: june 27, 2014. accepted for publication: july 1, 2014. academic editor: anuj chauhan, editor in chief type: rapid communication funding: authors disclose no funding sources. competing interests: authors disclose no potential conflicts of interest. copyright: © the authors, publisher and licensee libertas academica limited. this is an open-access article distributed under the terms of the creative commons cc-by-nc 3.0 license. correspondence: tetsu-a@jichi.ac.jp this paper was subject to independent, expert peer review by a minimum of two blind peer reviewers. all editorial decisions were made by the independent academic editor. all authors have provided signed confirmation of their compliance with ethical and legal obligations including (but not limited to) use of any copyrighted material, compliance with icmje authorship and competing interests disclosure guidelines and, where applicable, compliance with legal and ethical guidelines on human and animal research participants. introduction hyperuricemia, defined as a serum urate level exceeding the limit of solubility, mirrors supersaturation of the extracellular fluid with urate, and predisposes affected subjects to gout, which is characterized by the tissue deposition of monosodium urate crystals, although it is a necessary but not a substantial factor for the development of the disease.1 the current uratelowering strategies include reducing the urate production with xanthine oxidase (xo) inhibitors and accelerating the urinary excretion of uric acid (ua) with uricosuric agents.2,3 uricosuric agents, such as probenecid and benzbromarone, may have limited effectiveness in patients with reduced renal function.3,4 the purine analog xo inhibitor, allopurinol, has remained widely prescribed for the treatment of hyperuricemia, but requires dose adjustment in subjects with renal impairment, which may lead to a reduced benefit.2,3,5,6 febuxostat, a nonpurine xo inhibitor that recently received marketing approval, has been focused on as an alternative option for the treatment of hyperuricemia in patients with chronic kidney disease (ckd) because it undergoes hepatic metabolism and may require less dose adjustment in association with the renal function.6,7  moreover, several lines of evidence have focused on the blockade of xo activity as a potential therapeutic strategy for various other kinds of oxidative stress-mediated tissue and vascular injuries.8,9  however, information regarding the experience with this therapeutic agent among patients with advanced ckd is limited.7 in this regard, the current study investigated the effects of febuxostat http://www.la-press.com/drug-target-insights-journal-j23 http://www.la-press.com http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 http://dx.doi.org/10.4137/dti.s16524 http://creativecommons.org/licenses/by-nc/3.0/ http://creativecommons.org/licenses/by-nc/3.0/ mailto:tetsu-a@jichi.ac.jp akimoto et al 40 drug target insights 2014:8 in patients with advanced ckd with hyperuricemia in terms of the reduction of the serum ua levels and the longitudinal changes in several serum indicators for oxidative stress. materials and methods seventeen patients on chronic hemodialysis (hd) treatment who had serum ua levels above 8.0  mg/dl and who were not receiving anti-hyperuricemic agents participated in the study. all subjects had oliguria or anuria. the subjects had to be in stable condition, and they had no history of active liver diseases or any other significant medical status, no change in diuretics or steroid therapy within one month of study enrollment and were not chronic users of any nonsteroidal anti-inflammatory drugs. the usual medications, such as anti-hypertensive agents, erythropoietin, and phosphate binders, were continued during the study period. sex was not considered. the exclusion criteria were as follows: age 20 years or 90 years, type i diabetes mellitus or type ii diabetes mellitus with poor glucose control (glycosylated hemoglobin 9% at the start of the observation period), treatment with mercaptopurine hydrate or azathiopurine, pregnancy, and any medical or surgical conditions that made patients unsuitable for this study as judged by the attending physician. all patients were assigned to oral febuxostat and entered the six-month treatment period from july through august 2012, during which they initially received febuxostat 10 mg orally once daily in the morning. the target serum ua level was 6.0 mg/dl, and the dose of febuxostat was titrated or increased up to a maximum of 40 mg/day. the blood pressure (bp) was measured before all hd sessions, and the data regarding the systolic bp and diastolic bp were the average of each value on the last hd day of the week. the blood samples were obtained from vascular access, including arteriovenous fistulas and arteriovenous grafts, before hd sessions. the hemoglobin (hb), hematocrit (hct), platelet count (plt), serum levels of ua, blood urea nitrogen (bun), creatinine (cr), sodium (na), chloride (cl), potassium (k), calcium (ca), inorganic phosphate (pi), aspartate aminotransferase (ast), alanine aminotransferase (alt), and lactate dehydrogenase (ldh) were measured at baseline (week 0) and every four weeks during the observation period. the serum levels of 8-hydroxydeoxyguanosine (8-ohdg), 3-nitrotyrosine-modified proteins (3-nt), and protein carbonyls were determined at baseline and at weeks 4, 12, and 24 during the treatment period. the serum levels of 8-ohdg were measured by an enzyme-linked immunosorbent assay (elisa) as described previously.10  the elisa method was also used to measure the serum levels of 3-nt (japan institute for the control of aging, nikken seil co, shizuoka, japan) and protein carbonyls (biocell co, auckland, new zealand). this study was performed in accordance with the declaration of helsinki and was approved by the medical ethics committee of jichi medical university, and all patients included in the present study provided their informed consent. the data were expressed either as the number of participants or as the percentage (%) of the study population. the remaining data were expressed as the means ± standard deviation (sd), or as medians and interquartile ranges (ir) for variables with a skewed distribution. a repeated measures analysis of variance combined with fisher’s protected least significant difference test for normal distributions and the kruskal– wallis test with dunn’s method for skewed distributions were used to compare the time course data, when appropriate. values of p  0.05 were considered to be statistically significant. the statistical analyses were performed using the sigmaplot 12 software program for windows (systat software, inc., san jose, ca) unless otherwise stated. results and discussion the demographic profiles of the 17  patients included in the present study are summarized in table 1. no subjects had a history of gouty attacks. the patients had been treated with chronic hd for a median of four years. the causes of advanced ckd included diabetic nephropathy, chronic glomerulonephritis, hypertensive nephrosclerosis, and polycystic kidney disease. all subjects were on the optimum tolerated medical management. febuxostat lowered the serum ua levels (8.9 ± 1.0 at baseline) significantly from one month after the initiation of the treatment (fig. 1), and the target serum ua level (6.0  mg/dl) was achieved in 12  patients (70.5%) after one month of treatment, compared to 13 (76.4%) and 14 (82.3%) patients after three and six months of treatment, respectively. after six months, 16  subjects were still under the treatment with oral febuxostat (10  mg/day), and there was table 1. demographic profiles of the patients at the start of the study. demographic characteristics age (years) 64 ± 10 sex (male/female) 15/2 hd duration (years) 6.4 ± 5.7 underlying causes of ckd, n (%) diabetic nephropathy 9 (53) chronic glomerulonephritis 3 (18) hypertensive nephrosclerosis 3 (18) polycystic kidney disease 2 (12) medications, n (%) calcium channel antagonist(s) 7 (41) angiotensin-converting-enzyme inhibitor 1 (6) angiotensin receptor blocker(s) 7 (41) renin inhibitor 1 (6) other anti-hypertensive agent(s) 4 (24) loop diuretic(s) 3 (18) http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 febuxostat and advanced chronic kidney disease patients 41drug target insights 2014:8 one subject who was being treated with a reduced dose of 5 mg/day. also, only two patients were treated with the agent at a dose of above 10 mg/day at that point, one with 20 mg/day and one with 40  mg/day. all doses of febuxostat were well tolerated by the patients with no withdrawals because of side effects or allergic reactions. although there was a significant increase in the systolic bp values obtained at five and six months compared to those observed at baseline, no significant changes in diastolic bp, hb, hct, plt, bun, or the serum levels of cr, na, cl, k, ca, pi, ast, alt, or ldh were noted during the observation period (table 2). there were similar trends in the serum levels of protein carbonyls and 3-nt, while the serum 8-ohdg levels measured after six months were significantly lower than those at the baseline (table 3). no patients experienced symptoms of gouty arthritis, including joint pain, swelling, or redness,2 during the observation period. the treatment of patients with asymptomatic hyperuricemia (a serum ua level higher than 8  mg/dl) with urate lowering agents has been recommended and applied in japan,7,11 while the appropriate dose of febuxostat among subjects with advanced ckd has not yet been established. the current observations suggest that even relatively low doses of febuxostat, which is approved at a dose of 40  to 60  mg/day as the standard dose for the treatment of hyperuricemia with or without gouty arthritis in japan, may also work effectively among chronic hd patients for reducing the serum ua to a level that has been arbitrarily proposed as a therapeutic target for hyperuricemia.7,11,12 the validity of the indications for uratelowering agents among overall subjects with asymptomatic after the treatment with oral febuxostat (month(s)) baseline 1 4 6 8 * * * * * * * * * * * * 10 12 2 3 4 5 6 u a (m g/ dl ) figure 1. the serum ua levels before and after the initiation of the treatment with oral febuxostat. after six months of febuxostat treatment, the serum ua levels were significantly decreased from those at baseline. all patients were entered into the six-month treatment period from july through august 2012. note that the decrease in the serum ua levels was already significant after one month treatment with febuxostat. notes: n = 17, **p  0.01 versus baseline. ta b le 2 . c ha ng es in c lin ic al p ar am et er s du rin g th e ob se rv at io n pe rio d. a f t e r t h e in it ia t io n o f o r a l fe b u x o s ta t t r e a t m e n t p v a lu e b a s e l in e 1 m o n t h 2 m o n t h s 3 m o n t h s 4 m o n t h s 5 m o n t h s 6 m o n t h s s ys to lic b p (m m h g) 14 1 ± 20 13 7 ± 16 14 1 ± 29 14 1 ± 16 14 2 ± 21 14 8 ± 21 * 14 9 ± 19 *  0. 00 1 d ia st ol ic b p (m m h g) 74 ± 1 1 73 ± 9 73 ± 1 0 76 ± 1 0 76 ± 1 2 74 ± 1 1 73 ± 1 1 0. 18 6 h b (g /d l) 10 .7 ± 1 .0 10 .8 ± 1 .0 10 .9 ± 1 .1 10 .8 ± 1 .6 10 .9 ± 1 .3 10 .9 ± 1 .3 10 .6 ± 1 .0 0. 98 9 h ct ( % ) 33 .9 ± 3 .1 33 .9 ± 3 .2 33 .5 ± 3 .4 34 .1 ± 3 .4 34 .0 ± 4 .3 34 .0 ± 3 .6 33 .1 ± 2 .9 0. 97 6 p lt (× 10 4 / μ l) 16 .8 ( ir : 1 4. 2– 22 .7 ) 15 .6 ( ir : 1 3. 6 –2 5. 1) 17 .8 ( ir : 1 4. 2– 24 .6 ) 18 .4 ( ir : 1 4. 9 –2 5. 2) 17 .7 ( ir : 1 2. 8 –2 3. 1) 16 .7 ( ir : 1 2. 8 –2 1. 6) 18 .3 ( ir : 1 1. 7– 21 .8 ) 0. 88 7 b u n (m g/ dl ) 62 .1 ± 1 1. 7 64 .8 ± 1 8. 6 65 .1 ± 1 5. 8 63 .2 ± 1 4. 1 64 .7 ± 1 9. 1 67 .0 ± 2 1. 4 69 .9 ± 1 6. 5 0. 88 6 c r (m g/ dl ) 11 .4 ± 1 .9 11 .8 ± 2 .0 11 .8 ± 1 .9 11 .8 ± 2 .3 11 .9 ± 2 .2 11 .5 ± 2 .2 11 .5 ± 2 .2 0. 99 1 n a (m m ol /l) 13 8 ± 4 13 9 ± 4 14 0 ± 4 13 9 ± 4 13 9 ± 4 13 9 ± 3 13 8 ± 2 0. 80 5 k (m m ol /l) 4. 9 ± 0. 9 4. 9 ± 0. 8 5. 0 ± 1. 0 5. 0 ± 0. 9 4. 9 ± 0. 7 5. 0 ± 0. 8 5. 0 ± 0. 9 0. 99 9 c l ( m m ol /l) 10 2 ± 5 10 3 ± 4 10 3 ± 4 10 3 ± 4 10 3 ± 5 10 2 ± 4 10 3 ± 3 0. 93 c a (m g/ dl ) 8. 8 ± 0. 8 9. 0 ± 0. 9 8. 8 ± 0. 6 8. 8 ± 0. 6 8. 9 ± 0. 7 9. 1 ± 0. 8 9. 0 ± 0. 9 0. 85 2 p i ( m g/ dl ) 4. 5 ± 1. 3 5. 1 ± 1. 2 5. 3 ± 1. 4 5. 0 ± 0. 9 5. 1 ± 1. 5 5. 6 ± 1. 5 4. 9 ± 1. 0 0. 29 6 a s t ( u /l) 16 .5 ± 1 5. 8 18 .0 ± 1 5. 4 15 .0 ± 6 .7 15 .0 ± 6 .4 17 .2 ± 7 .5 18 .4 ± 8 .4 18 .3 ± 1 3. 6 0. 93 8 a lt ( u /l) 17 .9 ± 1 7. 6 18 .8 ± 1 6. 7 15 .2 ± 9 .4 14 .4 ± 8 .3 14 .0 ± 8 .0 17 .5 ± 1 0. 4 19 .4 ± 1 8. 2 0. 85 7 ld h ( u /l) 17 9 ± 40 18 9 ± 39 17 9 ± 28 17 8 ± 26 18 0 ± 30 19 1 ± 45 18 8 ± 48 0. 92 3 n o te : * p  0 .0 5 ve rs us b as el in e. http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 akimoto et al 42 drug target insights 2014:8 table 3. the serum levels of several oxidative stress markers before and after the initiation of the treatment with oral febuxostat. after the initiation of oral febuxostat treatment p value baseline 1 month 3 months 6 months protein carbonyls (nmol/mg protein) 0.07 (ir: 0.03–0.12) 0.10 (ir: 0.06–0.14) 0.07 (ir: 0.06–0.15) 0.05 (ir: 0.03–0.11) 0.329 3-nt (nm) 30.4 (ir: 25.2–40.4) 33.2 (ir: 25.5–42.2) 37.3 (ir: 25.3–45.3) 28.7 (ir: 24.7–43.9) 0.928 8-ohdg (ng/ml) 1.64 (ir: 1.24–2.95) 2.51 (ir: 1.24–4.74) 1.05 (ir: 0.84–1.87) 0.54 (ir: 0.204–1.16)* 0.002 note: *p  0.05 versus baseline. hyperuricemia remains to be delineated, and clinicians should bear in mind that the prevalence of refractory gout and/or gouty tophi is much lower in japan in comparison to that in the united states and europe, where negative opinions regarding pharmaceutical interventions predominate.11–14 one may argue that the clinical benefit of using urate-lowering agents requires careful evaluation, especially in subjects on chronic hd treatment, since an incipient gouty attack is quite rare in hyperuricemic long-term hd patients, and the frequency of gouty arthritis has been shown to decrease after the initiation of a periodic hd program in advanced ckd subjects.15,16  moreover, a significant association between higher serum ua levels and lower mortality, which may be dependent on the favorable nutritional status, has been demonstrated in the hd population.17  otherwise, it may be necessary to focus on the fact that some of the reactive oxygen species are produced as a by-product of urate formation through a xodependent pathway and the pharmacological nature of febuxostat, which is characterized by a higher bioavailability and a more potent blockade of xo activity than the traditional xo inhibitor allopurinol.1,2,8,18  indeed, the superior potency of febuxostat to allopurinol for the inhibition of reactive oxygen synthesis has been demonstrated in several reports,19,20 and the serum ua levels could be used as a surrogate indicator of the xo activity. in the current study, 8-ohdg, 3-nt, and protein carbonyls, which have been included in the list of the most common oxidative stress biomarkers in various settings,20,21 were used as the indices of the condition of the present patients. numerous processes other than the xo-mediated pathway have been implicated in the oxidative stress among the subjects with ckd.21,22  in addition, it has been shown that chronic hd treatment also leads to excessive radical production and impairment of the anti-oxidant capacity.23  despite the lack of qualitative information regarding the significance of individual oxidative stress markers,24 we feel that it is reasonable to consider that 8-ohdg, but not 3-nt and protein carbonyls, may help to detect the inhibitory effects of febuxostat on xo-mediated oxidative stress among the chronic hd patients. although the precise role of febuxostat in reducing the serum levels of 8-ohdg remains to be delineated, our findings suggest that the generation of 8-ohdg may be more dependent on xo than that of 3-nt or protein carbonyls. on the other hand, it has been proposed that the xo inhibitors may be utilized as adjunctive anti-hypertensive agents in some subsets of hyperuricemic and hypertensive subjects associated with or without advanced ckd,25,26 while we found that the systolic bp levels after five and six months of treatment with febuxostat were higher than those at baseline, despite the titration of anti-hypertensive agents corresponding to the context. we have no explanation for this discrepancy; however, a seasonal bias may have been involved. indeed, it has been reported that the systolic bp shows seasonal changes in chronic hd patients, with peak bp noted in the winter.27 finally, the number of patients included in the present series was quite small, thus implying that this study may be statistically underpowered or that the clinical parameters may have been overestimated. as such, our findings should be interpreted with caution. nevertheless, our results encourage us to pursue further investigations regarding the clinical impact of lowering the serum ua level with febuxostat in chronic hd patients in terms of determining the appropriate dose of the agent as well as concomitantly reducing oxidative stress by blocking xo. indeed, despite the comparable serum levels of low-density lipoprotein (ldl) between baseline and week 24, we found that the serum levels of the oxidative form of ldl, another oxidative stress marker,20 at week 24 were significantly decreased compared to those observed at baseline among the 12 subjects who led us to determine the serum levels of these parameters (data not shown). obviously, more detailed studies with a larger number of subjects and assessments of the effects of multiple factors affecting hyperuricemia, such as age, sex, and dietary habits,2,14 would shed light on the therapeutic challenges of treating asymptomatic hyperuricemia in patients with various stages of ckd.28 author contributions ta drafted the manuscript. ym, ci, oi, st, and yw made contributions to the acquisition of the clinical data. ek and dn provided a detailed review of the contents and structure of the manuscript, resulting in significant changes to the original document. all authors have read and approved the final manuscript. http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 febuxostat and advanced chronic kidney disease patients 43drug target insights 2014:8 r efer ences 1. gaffo al, saag kg. febuxostat: the evidence for its use in the treatment of hyperuricemia and gout. core evid. 2009;4:25–36. 2. becker ma, schumacher hr jr, wortmann rl, et  al. febuxostat compared with allopurinol in patients with hyperuricemia and gout. n engl j med. 2005; 353(23):2450–2461. 3. whelton a, macdonald pa, zhao l, hunt b, gunawardhana l. renal function in gout: long-term treatment effects of febuxostat. j clin rheumatol. 2011;17(1):7–13. 4. fujimori s, ooyama k, ooyama h, moromizato h. efficacy of benzbromarone in hyperuricemic patients associated with chronic kidney disease. nucleosides nucleotides nucleic acids. 2011;30(12):1035–1038. 5. dalbeth n, kumar s, stamp l, gow p. dose adjustment of allopurinol according to creatinine clearance does not provide adequate control of hyperuricemia in patients with gout. j rheumatol. 2006;33(8):1646–1650. 6. hosoya t, ono i. a repeated oral administration study of febuxostat (tmx-67), a non-purine-selective inhibitor of xanthine oxidase, in patients with impaired renal function in japan: pharmacokinetic and pharmacodynamic study. j clin rheumatol. 2011;17(4 suppl 2):s27–s34. 7. horikoshi r, akimoto t, inoue m, morishita y, kusano e. febuxostat for hyperuricemia: experience with patients on chronic hemodialysis treatment. clin exp nephrol. 2013;17(1):149–150. 8. george j, carr e, davies j, belch jj, struthers a. high-dose allopurinol improves endothelial function by profoundly reducing vascular oxidative stress and not by lowering uric acid. circulation. 2006;114(23):2508–2516. 9. tsuda h, kawada n, kaimori jy, et al. febuxostat suppressed renal ischemiareperfusion injury via reduced oxidative stress. biochem biophys res commun. 2012;427(2):266–272. 10. morishita y, watanabe m, hirahara i, akimoto t, muto s, kusano e. level of 8-ohdg in drained dialysate appears to be a marker of peritoneal damage in peritoneal dialysis. int j nephrol renovasc dis. 2012;5:9–14. 11. yamanaka h. japanese society of gout and nucleic acid metabolism. japanese guideline for the management of hyperuricemia and gout: second edition. nucleosides nucleotides nucleic acids. 2011;30(12):1018–1029. 12. el-zawawy h, mandell bf. managing gout: how is it different in patients with chronic kidney disease? cleve clin j med. 2010;77(12):919–928. 13. zhang w, doherty m, bardin t, et  al. eular evidence based recommendations for gout. part ii: management. report of a task force of the eular standing committee for international clinical studies including therapeutics (escisit). ann rheum dis. 2006;65:1312–1324. 14. smith eu, díaz-torné c, perez-ruiz f, march lm. epidemiology of gout: an update. best pract res clin rheumatol. 2010;24(6):811–827. 15. ifudu o, tan cc, dulin al, delano bg, friedman ea. gouty arthritis in end-stage renal disease: clinical course and rarity of new cases. am j kidney dis. 1994;23(3):347–351. 16. ohno i, ichida k, okabe h, et al. frequency of gouty arthritis in patients with end-stage renal disease in japan. intern med. 2005;44(7):706–709. 17. latif w, karaboyas a, tong l, et  al. uric acid levels and all-cause and cardiovascular mortality in the hemodialysis population. clin j am soc nephrol. 2011;6(10):2470–2477. 18. george j, struthers a. the role of urate and xanthine oxidase in vascular oxidative stress: future directions. ther clin risk manag. 2009;5:799–803. 19. malik uz, hundley nj, romero g, et al. febuxostat inhibition of endothelialbound xo: implication for targeting vascular ros production. free radic biol med. 2011;51(1):179–184. 20. sezai a, soma m, nakata k, et  al. comparison of febuxostat and allopurinol for hyperuricemia in cardiac surgery patients (nu-flash trial). circ j. 2013; 77(8):2043–2049. 21. himmelfarb j. uremic toxicity, oxidative stress, and hemodialysis as renal replacement therapy. semin dial. 2009;22(6):636–643. 22. griffiths hr, dias ih, willetts rs, devitt a. redox regulation of protein damage in plasma. redox biol. 2014;2:430–435. 23. massy za, stenvinkel p, drueke tb. the role of oxidative stress in chronic kidney disease. semin dial. 2009;22(4):405–408. 24. halliwell b, whiteman m. measuring reactive species and oxidative damage in vivo and in cell culture: how should you do it and what do the results mean? br j pharmacol. 2004;142(2):231–255. 25. jalalzadeh m, nurcheshmeh z, mohammadi r, mousavinasab n, ghadiani mh. the effect of allopurinol on lowering blood pressure in hemodialysis patients with hyperuricemia. j res med sci. 2012;17(11):1039–1046. 26. agarwal v, hans n, messerli fh. effect of allopurinol on blood pressure: a systematic review and meta-analysis. j clin hypertens (greenwich). 2013;15(6): 435–442. 27. argilés a, mourad g, mion c. seasonal changes in blood pressure in patients with end-stage renal disease treated with hemodialysis. n engl j med. 1998; 339(19):1364–1370. 28. murea m. advanced kidney failure and hyperuricemia. adv chronic kidney dis. 2012;19(6):419–424. http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 https://doi.org/10.1177/1177392818782899 drug target insights volume 12: 1–5 © the author(s) 2018 reprints and permissions: sagepub.co.uk/journalspermissions.nav doi: 10.1177/1177392818782899 creative commons non commercial cc by-nc: this article is distributed under the terms of the creative commons attribution-noncommercial 4.0 license (http://www.creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the sage and open access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage). introduction the administration of acyclovir antiviral therapy has been a treatment option for varicella zoster virus infection.1,2 valacyclovir is the valyl ester of acyclovir and after its oral administration it undergoes first-pass intestinal and/or hepatic metabolism to produce active-moiety acyclovir and l-valine at a high bioavailability that is several times greater than that obtained from oral acyclovir.1,3 both agents are well tolerated in the ordinary clinical settings; however, it is not surprising that acyclovir and oral valacyclovir share an adverse event profile that is both qualitatively and quantitatively similar.1 subjects with chronic renal insufficiency are susceptible to adverse events characterized by neuropsychiatric manifestations.3,4 as such, dosing adjustments proportionate to renal impairment may be required.1,2 in this report, we describe our experience with a case of valacyclovir neurotoxicity accompanied by acute kidney injury (aki) in an aged female patient who barely appeared to have acceptable renal function to receive the agent at the standard dosage for the treatment of herpes zoster (hz). several management concerns that emerged in the current case are also discussed. case report a 66-year-old woman with a history of hypertension and hyperlipidemia was referred and admitted to our hospital in the middle of november 2015 due to coma accompanied by aki. six years earlier, she had been found to have these diseases and had been treated with atorvastatin as well as antihypertensive agents, including amlodipine, olmesartan medoxomil, and carvedilol. although the renal parameters had not been monitored on a regular basis, her serum creatinine (scr) level slightly increased from 0.58 mg/dl in july 2013 to 0.69 mg/dl in september 2015, indicating declines in the estimated cr clearance (ecrcl) determined by cockcroft-gault formula5 and the estimated glomerular filtration rate (egfr) based on the revised japanese equation6 from 72.7 to 59.5 ml/min and 78.9 to 64.7 ml/min/1.73 m2, respectively. eight days before this admission, she had noticed a maculopapular rash over the left ear and been empirically treated with oral cefcapene pivoxil hydrochloride 300 mg/day combined with topical gentamicin by her general practitioner. three days later, the rash had progressed to clusters of clear vesicles. she was then diagnosed with left trigeminal hz and subjected to oral valacyclovir 1 g three times a day. on the fifth day of the valacyclovir treatment, the patient started to exhibit mild dysarthria characterized by slurred speech followed by a progressive deterioration of consciousness. the next day, she was brought to another emergency hospital by her son. a physical examination revealed her to be stuporous and afebrile without any facial drooping, tongue deviation, hemiparesis, or quadriparesis. diagnostic brain computed tomography and magnetic resonance imaging revealed no remarkable abnormalities, whereas a laboratory analysis revealed the elevated levels of scr of 7.44 mg/dl and valacyclovir neurotoxicity and nephrotoxicity in an elderly patient complicated by hyponatremia takuya murakami, tetsu akimoto, mari okada, erika hishida, taro sugase, atsushi miki, marina kohara, hiromichi yoshizawa, takahiro masuda, takahisa kobayashi, osamu saito, shigeaki muto and daisuke nagata division of nephrology, department of internal medicine, jichi medical university, shimotsuke, japan. abstract: a 66-year-old women with no history of renal disease was admitted due to a coma and acute kidney injury with a serum creatinine level of 7.44 mg/dl which were ascribed to valacyclovir neurotoxicity and nephrotoxicity, respectively. she had received valacyclovir at a standard dosage for the treatment of herpes zoster and was finally discharged, having fully returned to her normal baseline mental status with a recovered serum creatinine level of 0.68 mg/dl. we feel that awareness of this pathology remains a challenge for physicians and therefore strongly recommend the further accumulation of experiences similar to our own. our experience underscores the pitfalls of administering valacyclovir to elderly patients who barely appear to have a favorable renal function. several concerns regarding the therapeutic management, including blood purification strategies, that emerged in this case are also discussed. keywords: valacyclovir neurotoxicity, acute kidney injury, hemodialysis, hyponatremia, osmotic demyelination syndrome received: january 28, 2018. accepted: may 16, 2018. type: case report funding: the author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: this study was supported in part by a grant-in-aid for research on advanced chronic kidney disease, practical research project for renal diseases from the japan agency for medical research and development (amed), and by a grant for private university research branding project from the ministry of education, science and culture, japan. declaration of conflicting interests: the author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. corresponding author: tetsu akimoto, division of nephrology, department of internal medicine, jichi medical university, 3311-1 yakushiji, shimotsuke-shi 329-0498, tochigi, japan. email: tetsu-a@jichi.ac.jp 782899dti0010.1177/1177392818782899drug target insightsmurakami et al case-report2018 https://uk.sagepub.com/en-gb/journals-permissions mailto:tetsu-a@jichi.ac.jp 2 drug target insights serum potassium of 6.2 mmol/l. she was empirically given 10 mg of furosemide as well as 10 ml of 8.5% calcium gluconate intravenously and then transferred and admitted to our hospital for further work-up. on admission, the patient had a blood pressure of 191/105 mm hg with a pulse of 101 beats/min. she was a wellnourished woman of 152 cm in height and 47.4 kg in weight. her consciousness level was e3v4m6 on the glasgow coma scale (gcs). renal sonography on both kidneys revealed preservation of the size with normal renal cortex echogenicity. a laboratory examination revealed the following results: hemoglobin, 11.9 g/dl; hematocrit, 36.9%; platelet count, 15.9 × 104/μl; blood urea nitrogen, 53 mg/dl; scr, 7.27 mg/dl; uric acid, 8.8 mg/dl; total protein, 6.4 g/dl; serum albumin, 3.3 g/dl; sodium, 122 mmol/l; potassium, 5.9 mmol/l; chloride, 92 mmol/l; ca, 8.3 mg/dl; phosphorus, 5.0 mg/dl; c3, 112 mg/dl; c4, 25 mg/dl; immunoglobulin (ig) g, 1246 mg/ dl; iga, 239 mg/dl; and igm, 73 mg/dl. a serological study revealed an increased level of c-reactive protein of 3.55 mg/dl, whereas the patient was negative for antimyeloperoxidase antineutrophil cytoplasmic antibody (anca), antiproteinase 3-anca, anti-glomerular basement membrane antibodies, and anti–double-stranded dna antibodies. no serum thyroidstimulating hormone abnormalities were found. a lumbar puncture revealed a protein concentration of 54 mg/dl and glucose concentration of 91 mg/dl, whereas gram staining failed to show microorganisms, and cultures of blood and cerebrospinal fluid (csf) were also negative. although we did not perform a polymerase chain reaction to screen the patient’s csf for hz virus dna, the serum and csf acyclovir levels at this point were 26.9 and 6.25 μg/ml, respectively; as such, valacyclovir-related pathologies were strongly suspected to be implicated in the patient’s neuropsychiatric manifestations. all medications were discontinued, and she was subjected to a single session of hemofiltration (hf) for the management of anuric aki accompanied by hyperkalemia and hyponatremia when her neurological status further worsened (e1v1m4 on gcs). a temporary dialysis catheter was placed in the right jugular vein and a foley catheter was inserted in the bladder to monitor the urine output. then, hf was initiated with a high-flux hemodialyzer (fb-70u, cellulose triacetate membrane, 0.7 m2; nipro co., osaka, japan). the rates of blood flow and ultrafiltration were set at 120 ml/min and 1000 ml/h, respectively. the hf replacement fluid was the standard formulation of sublood®-bsg (fuso pharmaceutical industries ltd., osaka, japan) administered after filter at 1000 ml/h. after 3 hours, the patient developed respiratory distress due to aspiration and was intubated for airway protection, and then she was placed on mechanical ventilation when an urgent laboratory analysis revealed that her serum levels of potassium and sodium were 4.1 and 124 mmol/l, respectively. hf was ceased, and she was then started on intravenous 3% hypertonic saline of varying doses to correct the reduced serum sodium level at a recommended rate according to the therapeutic guidance.7 on the following day, there was a slight improvement in the patient’s neurological status (e2vtm4), and the serum sodium level increased to 131 mmol/l when the patient underwent a 3-hour session of hemoperfusion (hp) using an activated charcoal cartridge (hemosorba chs-350®; asahi medical co., tokyo, japan), and it further increased to 139 mmol/l at 14 hours later. after an initial hemodialysis (hd) session, her urine output began to increase (figure 1) along with gradual improvements in her consciousness from e3vtm6 on hospital day 3 to e4vtm6 on hospital day 5, whereas the serum levels of figure 1. serial changes in the scr, serum sodium levels, and urine volume during the observation period. hf and hp were performed on hospital days 1 and 2, respectively, whereas the patient received the hd treatment on hospital days 3 and 5. the administration of intravenous hypertonic saline was ceased after confirming that the patient’s serum sodium level had been corrected to 139 mmol/l on hospital day 3. murakami et al 3 acyclovir steadily decreased during the observation period (table 1). the patient was eventually extubated on hospital day 7 with a favorable neurological recovery (e4v5m6), and medications including statins and antihypertensive agents were resumed thereafter. she was discharged on hospital day 16, having fully returned to her normal baseline mental status with a recovered scr level of 0.68 mg/dl corresponding to an ecrcl of 60.4 ml/min and egfr of 65.7 ml/min/1.73 m2 at the time, the results of a urine sediment examination were unremarkable, and a urine dipstick analysis was negative for protein and occult blood. discussion the clinical scenario of the present case, which was characterized by a set of conditions including neurotoxicity and aki in an hz patient receiving valacyclovir may be too common to be described in the literature. indeed, there are numerous reports of nephrotoxic and neuropsychiatric side effects in acyclovirtreated patients, whereas aki and various neurological manifestations are occasionally observed as characteristic phenotypes of valacyclovir toxicity.3,4,8–11 however, the significance of this report should be carefully evaluated to investigate the impact of concurrent hyponatremia on the therapeutic decisions in the present case. furthermore, our experience underscores the pitfalls associated with valacyclovir treatment for hz, implying its limited feasibility and safety, especially in elderly patients. the withdrawal or discontinuation of the offending agent is the mainstay treatment for acyclovir or valacyclovir-mediated poisoning; however, there are several anecdotal reports suggesting that hd may play a role in the rapid reduction in serum acyclovir levels, thereby accelerating the recovery from renal and/or neurological disorders.4,8,11–14 given the chemical and pharmacokinetic characteristics of acyclovir, including its small molecular weight (225 da), limited protein-binding rate, low steady-state volume of distribution, and high water solubility, it can be readily removed with extracorporeal procedures, including hd, hf, and hemodiafiltration, with a high elimination rate in hd.4,8,12,15 in the present case, the concurrent hyponatremia precluded us from promptly performing hd as the procedure to remedy volume overload, azotemia, and abnormal serum electrolyte levels results in the rapid correction of the serum sodium level and predisposes patients to a risk of osmotic demyelination syndrome (ods).16 in this context, we decided to perform hf, which may have a lower magnitude of sodium exchange than hd,17 as an initial mode of blood purification with the aim of correcting the hyperkalemia associated with aki as well as active acyclovir removal, and it was not until the confirmation of successfully corrected serum sodium levels that hd was finally applied without any hesitation. a lack of information regarding the therapeutic benefit of hf in cases with valacyclovir or acyclovir toxicity18 also encouraged us to employ hp empirically as an adjunct treatment for the disease after confirming the normalization of the patient’s serum potassium levels, although acyclovir is not necessarily listed as an agent that can be removed by this procedure.19 given our experience with the current patient, we believe that hf and/or hp are viable therapeutic options in patients with hyponatremia who are at risk of developing a rate of sodium correction with hd that far exceeds the safe recommendation, although it does not allow us to objectively determine the clinical impact of each procedure on this pathology. despite these promising outcomes, our policy regarding tailored approaches to selecting and scheduling blood purification modalities in the present patient may not be appropriate because azotemia and/or uremia may protect against the demyelination that results from the rapid correction of hyponatremia during hd.20-22 however, we believe that it is necessary to take a proactive approach before serious events become apparent. of note, it has been reported anecdotally that uremia did not provide full protection against dialysis-related ods.23 the reason why our patient manifested hyponatremia, which is one of the most common electrolytes disorders encountered in the ordinary clinical setting,7 may be another point that needs to be addressed carefully. although this abnormality may occasionally be seen,24 and can arise in a wide range of diseases, a detailed medical record review, a thorough clinical interview, the patient’s clinical manifestations, and the prompt and timely resolution without any recurrence during the observation period, hampered our identification of disease states other than the renal failure.7 whether there is any association between valacyclovir toxicity and hyponatremia remains inconclusive.25 the hyponatremia may have acted as a confounder of the table 1. changes in the serum acyclovir levels just before and after the blood purification procedures. hospital day mode of blood purification serum acyclovir level, μg/ml before the procedure after the procedure 1 hf 26.9 na 2 hp 15.88 na 3 hd 4.72 1.95 5 hd na 0.43 abbreviations: na: not available. 4 drug target insights valacyclovir-mediated depression of the patient’s consciousness as the clinical manifestations of hyponatremia are primarily neurological26; however, it is difficult to determine the precise contributions of each factor. valacyclovir or acyclovir neurotoxicity commonly develops in patients with an impaired renal function, implying a role of a declined renal clearance–mediated accumulation of acyclovir and/or its metabolites in the bloodstream.3,4 given that the renal clearance of acyclovir absolutely exceeds that of creatinine, indicating that renal tubular secretion is actively involved in the elimination of the agent,1 it is not surprising that acyclovir can induce renal failure through precipitation within the tubular lumen, which leads to obstructive nephropathy.2,8 in rare instances, interstitial nephritis and direct tubular necrosis from acyclovir may also result in renal insufficiency.2,3,8 alternatively, or in addition, the gfr may decline due to an acyclovir-mediated increase in the total renal vascular resistance with a reduction in the renal plasma flow.2,11,27 the reason why adverse renal events were induced in our patient remains to be clarified. the patient’s prompt recovery from aki does not support the concurrent presence of primary and/or secondary renal parenchymal damage. instead, we believe that the diminished kidney reserve resulting from age-dependent functional and morphological changes with a moderate reduction in the gfr at baseline was likely to have played a major role, thereby making our patient vulnerable to acute stress from the agent and more likely to develop clinically relevant aki.28 unfavorably modulated renal hemodynamics induced by the concurrent use of angiotensin ii receptor antagonists might also lay the groundwork for the development of this disease.11,28 otherwise, our patient might have received an inappropriately high dose of valacyclovir as determining ecrcl with scr levels measured by enzymatic but not by jaffe assays, which are not common in japan, without standardization can overestimate actual gfr, although the optimum strategy for solving this problem remains to be delineated.5,29,30 the pathogenesis of valacyclovir and/or acyclovir neurotoxicity is still a matter of debate. although a specific therapeutic range has not been equivocally established, these values overlap with the reported trough levels, ranging from 0.5 to 3 μg/ml,31 whereas peak concentrations of approximately 5.6 μg/ml and trough levels of <2 μg/ml may be efficacious and below the concentrations at which acyclovir displays apparent neurotoxicity.1,11,32 acyclovir slowly crosses the blood-brain barrier and is then actively transported out of the csf compartment.33 however, the impact of the acyclovir on the pathogenesis of neurotoxicity remains to be determined because its serum levels are not necessarily above the therapeutic range in patients presenting with neuropsychiatric manifestations.4,34 rather, we may need to focus on 9-carboxymethoxymethylguanine (cmmg), a major metabolite of acyclovir, as a candidate offender as it has been regarded as a significant reliable predictor of developing neuropsychiatric events during acyclovir treatment.4,35 not surprisingly, the determination of serum levels of cmmg may also be a useful diagnostic tool for valacyclovir and/or acyclovir neurotoxicity,35 although such a procedure was not applicable in the current patient. the cumulative number of publications on these topics has been steadily growing2–4,8–15; however, we are of the opinion that awareness of this pathology remains limited and that an early diagnosis remains a challenge for the physicians in the ordinary clinical settings. needless to say, it is important to consider how to prevent the adverse events associated with valacyclovir and/or acyclovir that were described in this study. some possible approaches include establishing a euvolemic state before the administration of the medication, slower drug infusion, dose adjustment according to the renal function, and avoiding the use of other nephrotoxic agents.2,36 the published literature lacks information on the safety of substitution with other antiviral agents; however, it has been proposed anecdotally that famciclovir may be a candidate for substitution—at least in patients who develop nephrotoxicity.37,38 however, famciclovir treatment may still be associated with some risk of neuropsychiatric events.39 finally, we strongly recommend the accumulation of more cases similar to our own, thereby allowing us to clarify the nature of adverse valacyclovir-mediated reactions more precisely as well as establish an optimum management strategy, especially regarding appropriate valacyclovir dosing regimens in elderly patients with hz who barely appear to have a favorable renal function. author contributions tmurakami and ta drafted the manuscript. mo, eh, ts, am, mk, hy, and tmasuda made contributions to the acquisition of the clinical data. tk, os, sm, and dn provided a detailed review of the contents and structure of the manuscript, resulting in significant changes to the original document. all authors have read and approved the final manuscript. disclosure and ethics as a requirement of publication, the authors have provided the publisher with a signed confirmation of compliance with legal and ethical obligations, including but not limited to the following: authorship and contributorship, conflicts of interest, privacy and confidentiality, and (where applicable) protection of human and animal research subjects. the authors have read and confirmed their agreement with the icmje authorship and conflict of interest criteria. the authors have also confirmed that this article is unique and that it is not been published nor is it under consideration by any other publication, and that they have permission from the rights holders to reproduce any copyrighted material. the external blind peer reviewers report no conflicts of interest in association with this study. r efer ences 1. ormrod d, goa k. valaciclovir: a review of its use in the management of herpes zoster. drugs. 2000;59:1317–1340. 2. perazella ma. crystal-induced acute renal failure. am j med. 1999;106: 459–465. murakami et al 5 3. asahi t, tsutsui m, wakasugi m, et al. valacyclovir neurotoxicity: clinical experience and review of the literature. eur j neurol. 2009;16:457–460. 4. yang hh, hsiao yp, shih hc, yang jh. acyclovir-induced neuropsychosis successfully recovered after immediate hemodialysis in an end-stage renal disease patient. int j dermatol. 2007;46:883–884. 5. cockcroft dw, gault mh. prediction of creatinine clearance from serum creatinine. nephron. 1976;16(1):31–41. 6. matsuo s, imai e, horio m, et al. revised equations for estimated gfr from serum creatinine in japan. am j kidney dis. 2009;53(6):982–992. 7. verbalis jg, goldsmith sr, greenberg a, schrier rw, sterns rh. hyponatremia treatment guidelines 2007: expert panel recommendations. am j med. 2007; 120(11 suppl 1):s1–21. 8. krieble bf, rudy dw, glick mr, clayman md. case report: acyclovir neurotoxicity and nephrotoxicity—the role for hemodialysis. am j med sci. 1993;305:36–39. 9. sodhi pk, ratan sk. a case of chronic renal dysfunction following treatment with oral acyclovir. scand j infect dis. 2003; 35(10):770–772. 10. carlon r, possamai c, corbanese u. acute renal failure and severe neurotoxicity following valacyclovir. intensive care med. 2005; 31(11):1593. 11. sugimoto t, yasuda m, sakaguchi m, et al. oliguric acute renal failure following oral valacyclovir therapy. q jm. 2008; 101(2):164–166. 12. leikin jb, shicker l, orlowski j, blair at, mcallister k. hemodialysis removal of acyclovir. vet hum toxicol. 1995;37:233–234. 13. hsu cc, lai ti, lien wc, chen wj, fang cc. emergent hemodialysis for acyclovir toxicity. am j emerg med. 2005;23:899–900. 14. kambhampati g, pakkivenkata u, kazory a. valacyclovir neurotoxicity can be effectively managed by hemodialysis. eur j neurol. 2011;18:e33. 15. bleyzac n, barou p, massenavette b, et al. assessment of acyclovir intraindividual pharmacokinetic variability during continuous hemofiltration, continuous hemodiafiltration, and continuous hemodialysis. ther drug monit. 1999;21: 520–525. 16. yessayan l, yee j, frinak s, szamosfalvi b. treatment of severe hyponatremia in patients with kidney failure: role of continuous venovenous hemofiltration with low-sodium replacement fluid. am j kidney dis. 2014;64:305–310. 17. di filippo s, manzoni c, andrulli s, tentori f, locatelli f. sodium removal during pre-dilution haemofiltration. nephrol dial transplant. 2003;18:vii31–vii36. 18. meng jb, zheng x, zhang g, fang q. oral acyclovir induced acute renal failure. world j emerg med. 2011;2:310–313. 19. winchester jf, harboud nb, charen e, ghannoum m. use of dialysis and hemoperfusion in the treatment of poisoning. in: daugirdas jt, blake pg, ing ts, eds. handbook of dialysis, 5th ed. philadelphia, pa: wolters kluwer; 2015:368–390. 20. oo tn, smith cl, swan sk. does uremia protect against the demyelination associated with correction of hyponatremia during hemodialysis? a case report and literature review. semin dial. 2003;16:68–71. 21. sirota jc, berl t. is osmotic demyelination a concern dialyzing hyponatremic patients? semin dial. 2011;24:407–409. 22. dhrolia mf, akhtar sf, ahmed e, naqvi a, rizvi a. azotemia protects the brain from osmotic demyelination on rapid correction of hyponatremia. saudi j kidney dis transpl. 2014;25:558–566. 23. huang w y, weng wc, peng ti, ro ls, yang cw, chen kh. central pontine and extrapontine myelinolysis after rapid correction of hyponatremia by hemodialysis in a uremic patient. ren fail. 2007;29:635–638. 24. ferreira m, vega c, rivas b, selgas r. acute renal failure and severe neurotoxicity after unintentional overdose of valacyclovir in a geriatric population: a case report. nefrología. 2018;38:323–325. 25. cortejoso l, gómez-antúnez m, muiño-míguez a, durán-garcía me, sanjurjo-sáez m. acyclovir and hyponatremia: a case report. am j ther. 2014;21:e151–e153. 26. sterns rh. disorders of plasma sodium—causes, consequences, and correction. n engl j med. 2015;372:55–65. 27. dos santos mde f, dos santos of, boim ma, et al. nephrotoxicity of acyclovir and ganciclovir in rats: evaluation of glomerular hemodynamics. j am soc nephrol. 1997;8:361–367. 28. coca sg. acute kidney injury in elderly persons. am j kidney dis. 2010;56: 122–131. 29. ando y, minami h, saka h, ando m, sakai s, shimokata k. adjustment of creatinine clearance improves accuracy of calvert’s formula for carboplatin dosing. br j cancer. 1997;76:1067–1071. 30. delanaye p, cavalier e, pottel h. serum creatinine: not so simple! nephron. 2017;136:302–308. 31. adair jc, gold m, bond re. acyclovir neurotoxicity: clinical experience and review of the literature. south med j. 1994;87:1227–1231. 32. collins a, krieff d, smith c, singer c. neuropsychiatric toxicity in a patient undergoing hemodialysis and receiving treatment with oral acyclovir. clin infect dis. 1996;22:187–188. 33. pouplin t, pouplin jn, van toi p, et al. valacyclovir for herpes simplex encephalitis. antimicrob agents chemother. 2011;55:3624–3626. 34. de knegt rj, van der pijl h, van es la. acyclovir-associated encephalopathy, lack of relationship between acyclovir levels and symptoms. nephrol dial transplant. 1995;10:1775–1777. 35. helldén a, odar-cederlöf i, diener p, et al. high serum concentrations of the acyclovir main metabolite 9-carboxymethoxymethylguanine in renal failure patients with acyclovir-related neuropsychiatric side effects: an observational study. nephrol dial transplant. 2003;18:1135–1141. 36. fleischer r, johnson m. acyclovir nephrotoxicity: a case report highlighting the importance of prevention, detection, and treatment of acyclovir-induced nephropathy. case rep med. 2010;2010:602783. 37. htwe th, bergman s, koirala j. famciclovir substitution for patients with acyclovir-associated renal toxicity. j infect. 2008;57:266–268. 38. yoon h, rhew ky. famciclovir as an antiviral agent for a patient with acute renal failure. int j clin pharm. 2013;35:173–175. 39. gales bj, gales ma. confusion and bradykinesia associated with famciclovir therapy for herpes zoster. am j health syst pharm. 1996;53:1454, 1456. 29drug target insights 2015:9 management of diabetes associated with nephrotic syndrome: therapeutic potential of dapagliflozin for protracted volume retention toshimi imai, tetsu akimoto, chiharu ito, takahiro masuda and daisuke nagata division of nephrology, department of internal medicine, jichi medical university, shimotsuke-shi, tochigi, japan. a bstr act: a 48-year-old female was admitted to our hospital presenting with a chief complaint of progressive swelling because of diabetic nephrotic syndrome. dapagliflozin seemed to play a role in accelerating the patient’s urinary sodium excretion as well as reducing gross fluid retention despite the fact that her nephrotic condition was resistant to furosemide. our experience emphasizes a potential novel approach to overcoming loop diuretic resistance using this agent among some subsets of type 2 diabetic subjects complicated with severe volume accumulation. we believe that combination treatment consisting of dapagliflozin and furosemide may produce diuretic synergy via sequential nephron blockade. the accumulation of more experience with additional cases similar to ours requires continuous and careful attention. k e y wor ds: nephrotic syndrome, sglt-2, dapagliflozin, diabetes, furosemide citation: imai et al. management of diabetes associated with nephrotic syndrome: therapeutic potential of dapagliflozin for protracted volume retention. drug target insights 2015:9 29–31 doi:10.4137/dti.s31710. type: case report received: july 13, 2015. resubmitted: september 10, 2015. accepted for publication: september 14, 2015. academic editor: anuj chauhan, editor in chief peer review: three peer reviewers contributed to the peer review report. reviewers’ reports totaled 358 words, excluding any confidential comments to the academic editor. funding: authors disclose no funding sources. competing interests: authors disclose no potential conflicts of interest. copyright: © the authors, publisher and licensee libertas academica limited. this is an open-access article distributed under the terms of the creative commons cc-by-nc 3.0 license. correspondence: r0609ti@jichi.ac.jp paper subject to independent expert blind peer review. all editorial decisions made by independent academic editor. upon submission manuscript was subject to antiplagiarism scanning. prior to publication all authors have given signed confirmation of agreement to article publication and compliance with all applicable ethical and legal requirements, including the accuracy of author and contributor information, disclosure of competing interests and funding sources, compliance with ethical requirements relating to human and animal study participants, and compliance with any copyright requirements of third parties. this journal is a member of the committee on publication ethics (cope). published by libertas academica. learn more about this journal. introduction dapagliflozin, a sodium–glucose cotransporter 2 (sglt-2) inhibitor, which recently received marketing approval as a novel therapeutic option for type 2 diabetes, promotes glycosuria via an osmotic diuretic effect.1 in this report, we describe our serendipitous experience with a case of type 2 diabetes accompanied by nephrotic syndrome in which dapagliflozin seemed to play a role in controlling diuretic-resistant fluid retention. case report a 48-year-old female was admitted to our hospital presenting with a complaint of progressive swelling of her legs. she had gained ~20  kg in the past four months. at 35 years of age, she was found to have type 2 diabetes with a hemoglobin a1c (hba1c) level of 9.4%, for which she had received sporadic medical care. two months before admission, when she was found to have hypertension and hypercholesterolemia as well as uncontrolled diabetes with a serum hba1c level of 7.1%, treatment with furosemide at a dose of 60  mg/day combined with alogliptin at a dose of 25 mg/day, irbesartan at a dose of 100 mg/day, amlodipine at a dose of 10 mg/day, and rosuvastatin at a dose of 2.5  mg/day was started; however, her generalized edema persisted and subsequently worsened. therefore, she was referred and admitted for a further workup. she neither smoked nor drank alcohol and denied using any drugs. a physical examination completed on admission revealed that the patient’s face was swollen, with significant edema noted in the upper and lower extremities. her blood pressure (bp) was 153/87 mmhg, her pulse was 90 beats/minute, and her temperature was 36.0°c. although the oxygen saturation was 98% while she breathed ambient air, the presence of bilateral pleural effusion and ascites was confirmed on chest x-ray and/or computed tomography scans. no findings suggestive of heart failure were observed on echocardiography. a laboratory evaluation revealed the following results: hb, 9.5 g/dl; platelet count, 33.6 × 104/μl; total protein, 5.5 g/dl; serum albumin, 2.0  g/dl; blood urea nitrogen, 14.5  mg/dl; creatinine (cr), 1.01  mg/dl; sodium, 141  mmol/l; potassium, 4.0  mmol/l; chloride, 105.1 mmol/l; aspartate aminotransferase, 20 u/l; alanine aminotransferase, 9  u/l; fasting plasma glucose, 180 mg/dl; hba1c, 6.6%; and c-reactive protein, 0.3 mg/dl. the patient’s urine contained 7.0  g of protein in a 24-hour specimen, and the sediment contained five to nine red blood cells per high-power field. the cr clearance was 29.0 ml/minute. an ophthalmologic analysis revealed severe nonproliferative diabetic retinopathy. based on the clinical picture and laboratory findings, the patient was thus diagnosed as having nephrotic syndrome due to diabetic nephropathy. despite treatment with an increased dose of furosemide, given both orally and intravenously, her body weight and daily urine volume remained almost constant, keeping her grossly edematous. she then received journal name: drug target insights journal type: case report year: 2015 volume: 9 running head verso: imai et al running head recto: management of diabetes associated with nephrotic syndrome http://www.la-press.com/drug-target-insights-journal-j23 http://www.la-press.com http://dx.doi.org/10.4137/dti.s31710 http://creativecommons.org/licenses/by-nc/3.0/ http://creativecommons.org/licenses/by-nc/3.0/ mailto:r0609ti@jichi.ac.jp http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 imai et al 30 drug target insights 2015:9 treatment with oral dapagliflozin at a dose of 5  mg/day on hospital day 5, which resulted in a remarkable increase in her urine volume as well as the amount of urinary excreted sodium and glucose, with the gradual disappearance of the generalized edema, despite the almost constant levels of her systolic and diastolic bp during the observation period (fig.  1). finally, her body weight settled at around 65  kg under the treatment with sodium and fluid restriction and the same dose of dapagliflozin combined with oral furosemide (220 mg/day). at three months of follow-up, she is currently doing well with an hba1c level of 6.1% despite protracted nephrotic-range proteinuria at approximately 5 g/day. discussion nephrologists occasionally face difficulties in controlling fluid retention despite the availability of various types of diuretic agents.2,3 the pathogenic processes responsible for diuretic resistance are multifactorial, and the albumin present in the luminal content as a result of the disease process is capable of binding the diuretic drug and thereby eliciting resistance in subjects with nephrotic syndrome.4 consequently, one may argue that some of the clinical manifestations and therapeutic conundrums observed in our patient are too common to be described in the literature; however, the clinical significance of the current report should be evaluated carefully in terms of assessing the therapeutic potential of dapagliflozin for the management of refractory edema in the cases of diabetic nephrotic syndrome. comprehensive insight into the role of sglt-2, which is located mainly in the brush border membrane of the early proximal tubule,5 in glucose handling within the kidney has led to the development of selective orally available sodium– glucose transport inhibitors as a means of regulating the serum glucose level.1,6 recent clinical trials of these agents have demonstrated favorable safety profiles, whereas neither major hypoglycemic events nor adverse changes in the renal function were reported.7,8 on the other hand, there are several observations suggesting that the promising benefits of these drugs may extend beyond glycemic control. the blockade of sglt-2 has been shown to result in glycosuria-mediated calorie wasting, leading to weight loss.7 such treatment may also lead to reduced sodium reabsorption in the proximal tubule, thereby accelerating sodium excretion.9 although too few studies have reported the cases of urinary sodium excretion to allow for a substantial analysis,10–12 the association between the use of sglt-2 inhibitors and the significant decreases in bp demonstrated in several recent studies should encourage researchers to pursue further investigations regarding the diuretic-like figure 1. clinical course. on hospital day 12, intravenous furosemide was terminated, while oral furosemide was continued with an increased dose of 300 mg/day, and the patient was discharged on hospital day 16. note that the levels of bp and serum cr (scr) were almost constant during the observation period despite prominent elevation of the daily urine output after the commencement of oral dapagliflozin. http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 management of diabetes associated with nephrotic syndrome 31drug target insights 2015:9 antihypertensive actions of these medications, which should be linked to natriuresis as well as glucose-mediated osmotic diuresis.1,9,13 at present, we have no idea why we failed to confirm such an effect in the present patient; however, it may be reasonable to consider that her volume overload status might have masked the diuresis-dependent antihypertensive properties.14 otherwise, what subtypes of diabetic subjects are vulnerable to these kinds of agents also needs to be assessed in more detail. a case of volume depletion and prerenal azotemia that required rehydration and the withdrawal of angiotensin-converting inhibitor and diuretic treatment was recently reported in a phase ii trial of dapagliflozin.15 not surprisingly, dapagliflozin was effective and well tolerated as an adequate therapeutic option for modulating the present patient’s level of glycemic control. our experience with this agent in the current patient rather emphasizes a potential approach for overcoming loop diuretic resistance among some subsets of type 2 diabetic subjects complicated with severe volume accumulation. indeed, dapagliflozin seemed to play a role in accelerating urinary sodium excretion as well as reducing the considerable fluid retention in the current patient despite the fact that her nephrotic condition was resistant to furosemide. the fact that the natriuretic effect of dapagliflozin did not persist may be ascribed to the establishment of a new steady state, in which the patient’s depleted volume status and increased activity of the renin angiotensin system stimulated renal sodium uptake through alternate pathways and compensated for the natriuretic effect of the agent.6,16 finally, we believe that combination treatment consisting of dapagliflozin and furosemide may have produced diuretic synergy via the sequential blockade of solute reabsorption at the proximal tubules and at the thick ascending limb of the loop of henle in our patient. the lack of longitudinal data regarding the gross daily urine output, as well as the amount of urinary excreted sodium after the commencement of oral dapagliflozin treatment in the previous studies, precludes us from evaluating the validity of our findings.10–12 nevertheless, list et al, demonstrated that the administration of dapagliflozin in treatment-naive patients with type 2 diabetes resulted in an increased urinary output of between 107 ml/day and 470 ml/day, equating to ~0.3–1.5 additional voids per day.1,17 this highlights the need for further investigation regarding the impact of the diuretic properties of this agent on the overall management of diabetic patients.9 obviously, the accumulation of more experience with additional cases similar to ours requires continuous and careful attention, and such a strategy would aid in the establishment of a novel approach to treating fluid retention as well as investigating the therapeutic impact of sglt-2 inhibitors in diabetic patients with nephrotic syndrome. author contributions drafted the manuscript: ti and ta. made contributions to the acquisition of the clinical data: ci and tm. provided a detailed review of the contents and structure of the manuscript, resulting in significant changes to the original document: dn. all authors have read and approved the final manuscript. r efer ences 1. list jf, woo v, morales e, tang w, fiedorek ft. sodium-glucose cotransport inhibition with dapagliflozin in type 2 diabetes. diabetes care. 2009;32(4): 650–657. 2. onoyama k, kumagai h, fujishima m. hemodynamic and volume changes by ultrafiltration in refractory edema of diabetic nephrotic syndrome with severe renal insufficiency. clin nephrol. 1987;27(1):21–25. 3. davenport a. ultrafiltration in diuretic-resistant volume overload in nephrotic syndrome and patients with ascites due to chronic liver disease. cardiology. 2001; 96(3–4):190–195. 4. brater dc. diuretic resistance: mechanisms and therapeutic strategies. cardiology. 1994;84(suppl 2):57–67. 5. vallon v, platt ka, cunard r, et al. sglt2 mediates glucose reabsorption in the early proximal tubule. j am soc nephrol. 2011;22(1):104–112. 6. gallo la, wright em, vallon v. probing sglt2 as a therapeutic target for diabetes: basic physiology and consequences. diab vasc dis res. 2015;12(2):78–89. 7. musso g, gambino r, cassader m, pagano g. a novel approach to control hyperglycemia in type 2 diabetes: sodium glucose co-transport (sglt) inhibitors: systematic review and meta-analysis of randomized trials. ann med. 2012;44(4): 375–393. 8. shah nk, deeb we, choksi r, epstein bj. dapagliflozin: a novel sodiumglucose cotransporter type 2 inhibitor for the treatment of type 2 diabetes mellitus. pharmacotherapy. 2012;32(1):80–94. 9. lambers heerspink hj, de zeeuw d, wie l, leslie b, list j. dapagliflozin a glucose-regulating drug with diuretic properties in subjects with type 2 diabetes. diabetes obes metab. 2013;15(9):853–862. 10. komoroski b, vachharajani n, feng y, li l, kornhauser d, pfister m. dapagliflozin, a novel, selective sglt2 inhibitor, improved glycemic control over 2 weeks in patients with type 2 diabetes mellitus. clin pharmacol ther. 2009;85(5): 513–519. 11. devineni d, morrow l, hompesch m, et al. canagliflozin improves glycaemic control over 28 days in subjects with type 2 diabetes not optimally controlled on insulin. diabetes obes metab. 2012;14(6):539–545. 12. devineni d, vaccaro n, polidori d, rusch s, wajs e. effects of hydrochlorothiazide on the pharmacokinetics, pharmacodynamics, and tolerability of canagliflozin, a sodium glucose co-transporter 2 inhibitor, in healthy participants. clin ther. 2014;36(5):698–710. 13. baker wl, smyth lr, riche dm, bourret em, chamberlin kw, white wb. effects of sodium-glucose co-transporter 2 inhibitors on blood pressure: a systematic review and meta-analysis. j am soc hypertens. 2014;8(4):262–275. 14. meltzer ji, keim hj, laragh jh, sealey je, jan km, chien s. nephrotic syndrome: vasoconstriction and hypervolemic types indicated by renin-sodium profiling. ann intern med. 1979;91(5):688–696. 15. wilding jp, norwood p, t’ joen c, bastien a, list jf, fiedorek ft. a study of dapagliflozin in patients with type 2 diabetes receiving high doses of insulin plus insulin sensitizers: applicability of a novel insulin-independent treatment. diabetes care. 2009;32(9):1656–1662. 16. thomas mc, jandeleit-dahm k, bonnet f. beyond glycosuria: exploring the intrarenal effects of sglt-2 inhibition in diabetes. diabetes metab. 2014;40 (6 suppl 1):s17–s22. 17. list jf, whaley jm. glucose dynamics and mechanistic implications of sglt2 inhibitors in animals and humans. kidney int. 2011;79(suppl 120):s20–s27. http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 drug target insights 2011:5 33–41 doi: 10.4137/dti.s7527 this article is available from http://www.la-press.com. © the author(s), publisher and licensee libertas academica ltd. this is an open access article. unrestricted non-commercial use is permitted provided the original work is properly cited. open access full open access to this and thousands of other papers at http://www.la-press.com. drug target insights o r i g i n a l r e s e a r c h drug target insights 2011:5 33 predictors of virologic failure in hiv/aids patients treated with highly active antiretroviral therapy in brasília, brazil during 2002–2008 edson josé monteiro bello1, amabel fernandes correia2, josé ricardo pio marins3, edgar merchan-hamann4 and luis isamu barros kanzaki5 1m.sc. in health sciences, nucleus of virology, 2 m.sc in health sciences nucleus of parasitology, central public health laboratory, brasilia, df, brazil, cep 70830-010. 3 ph.d secretary of state for health, federal district, central public health laboratory, federal district, brasília, df, brazil, cep 70058-900. 4 ph.d departament of public health, faculty of health sciences, 5d.sc. laboratory of bioprospection, university of brasilia, brasília, df, brazil, cep 70910-900. corresponding author email: kanzaki@unb.br abstract: little data exists concerning the efficacy of the antiretroviral therapy in the federal district in brazil, therefore in order to improve hiv/aids patients’ therapy and to pinpoint hot spots in the treatment, this research work was conducted. of 139 hiv/aids patients submitted to the highly active antiretroviral therapy, 12.2% failed virologically. the significant associated factors related to unresponsiveness to the lentiviral treatment were: patients’ place of origin (or = 3.28; ic95% = 1.0–9.73; p = 0.032) and mycobacterium tuberculosis infection (rr = 2.90; ic95% = 1.19–7.02; p = 0.019). in the logistic regression analysis, the remaining variables in the model were: patients’ birthplace (or = 3.28; ic95% = 1.10–9.73; p = 0.032) and tuberculosis comorbidity (or = 3.82; ic95% = 1.19–12.22; p = 0.024). the patients enrolled in this survey had an 88.0% therapeutic success rate for the maximum period of one year of treatment, predicting that t cd4+ low values and elevated viral loads at pretreatment should be particularly considered in tuberculosis coinfection, besides the availability of new antiretroviral drugs displaying optimal activity both in viral suppression and immunological reconstitution. keywords: antiretroviral therapy, tuberculosis coinfection, brasilia, epidemiology http://dx.doi.org/10.4137/dti.s7527 http://www.la-press.com http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 http://www.la-press.com mailto:kanzaki@unb.br bello et al 34 drug target insights 2011:5 introduction plasmatic viral load strongly predicts t cd4+ cell count decline, aids progression and death. anyway, disease prognosis in hiv infected subjects is more rigorously defined by the combination of plasmatic viral load quantification and t cd4+ cell count.1 it is internationally noted that the main predictive factors for failure of antiretroviral therapy are t cd4+ cells low count and elevated viral load before commencing treatment.2 the efficacy of the highly active antiretroviral therapy (haart) and the pattern of therapy management could be evaluated based on plasmatic rna viral load assessement. in hiv/aids treatment the response is considered successful when the hiv rna levels remain undetectable by certified commercial assays, notwithstanding in a variable proportion of haart submitted patients, the viral replication and evolution goes on which could eventually contribute to the development of antiretroviral drug resistance and therapeutic failure.3 unsuccessful antiretroviral therapy could occur due to virologic and immunologic failure and clinical manifestations that can appear during the course of hiv infection. the identification of therapeutic failure is based on patient follow-up during treatment, taking into account the initial level of t cd4+, plasmatic hiv rna load and the patient clinical evolution. also, many factors could be related to therapeutic failure such as low treatment adhesion, insufficient drug dosage, mal-absorption, hiv antiretroviral resistance and drug interactions that could reduce the efficacy and hiv resistance to antiretroviral medications. patients that failed to respond to antiretroviral therapy confirmed by the genotype resistant assay will be guided to choose new drugs. this choice is based on the knowledge of previous treatments and yet the reason to remove a drug from the antiretroviral regimen is mainly justified by the in vitro antiretroviral resistance profile. the antiretroviral therapeutic suspension as an alternative therapy is based on the reemergence of the initial virus population before treatment initiation, which would be susceptible to the antiretroviral drugs earlier prescribed, so this proposal is presently under evaluation. the essential objective of treatment in patients presenting therapeutic failure is to maintain t cd4+ acceptable cell population density, whilst new therapeutic options are awaited, contrary to the priority aim to reach and keep up an undetectable viral load.4 according to moore et al,5 virological failure is characterized by viral load higher than 400 copies/ml after 48 weeks of initial treatment or among subjects that had complete viral suppression, but later on the viral load will recrudesce. the viral failure precedes the immunologic failure which is defined by a decline of more than 25.0% in the subsequent count of t cd4+ lymphocytes or regresion to the initial t cd4+ cell count before treatment. these conditions are mainly suggestive of immunologic failure but laboratory analysis confirmation is mandatory.6 cozzi-lepri et al7 concluded that patients remaining in haart failure presented viral load slowly progressing during the next 12 months, differently to t cd4+ cell count which remained stable. the impact of haart on t cd4+ cell count and viral load of hiv infected patients has been demonstrated to improve the patient’s immunological status and it also diminished the viral load, interrupting the aids progression.8 however there are crucial limitations of haart regimen, as it does not eradicate viral infection despite long and permanent antiretroviral therapy. consequently, a significant number of these patients on haart therapy develop viral resistance to the drugs besides diverse side effects including metabolic disorders. therefore new approaches are necessary to control and/or eradicate hiv infection.9,10 this research work aimed to analyze t cd4+ cell density and viral load response in hiv infected patients undergoing different therapeutic regimens which failed virologically, and also the associated factors to it during 2002–2008 in brasilia, federal district, brazil. material and methods patients a cohort study was conducted in the health center number 01 attached to the secretary of health, federal district (ses/df) (centro de saúde número 01, secretaria de saúde do distrito federal) which included 139 hiv-1 infected patients. the patients had clinical and laboratory diagnosis as defined by the 1993 aids clinical course criteria and by the us center for disease control (cdc)11 and also by the recommendations of the brazilian consensus of antiretroviral therapy in infected adults and adolescents by the ministry of health, secretary of http://www.la-press.com antiretroviral treatment in the federal district, brazil, federal district drug target insights 2011:5 35 health surveillance, brazilian national program of sexually transmitted diseases and aids.12 patients selected for the study were identified in the records of logistic system of the ministry of health, siclom13 utilized by the hospital dia, ses/df. the plasmatic hiv rna load was quantified by the bdna system 340 presenting sensitivity of less than 50 copies/ml. the t cd4+ helper lymphocytes’ count was carried out by automatized flow cytometry utilizing the facscalibur count® system. the sampling utilized for patient selection was by convenience whose inclusion criteria were: (a) age higher than 18 years old and of both sexes; (b) hiv infection diagnosis defined by the brazillian ministry of health standardized patterns; (c) detectable viral load previous to haart beginning .400 copies/ml; (d) t cd4+ cell count ,500 cells/mm3 before haart regimen initiation; (e) present laboratory analysis for viral load and t cd4+ count between 6 and 12 months in order to compare the former ones with those of baseline; (f) patients have had clinical and immunological follow up during 3 months intervals. if undetectable viral load of 50 copies/ml was not sustained during 24 weeks of treatment or higher than 400 copies/ml after 48 weeks treatment, it was considered virological failure to the initial antiretroviral therapeutic regimen.5 immunological failure was considered by the declining of t cd4+ cell counting $ 25.0% of the absolute values6 or by returning to the t cd4+ cell count initial values before antiretroviral therapy initiation. the viral load previous to haart regimen initiation and t cd4+ cell count were evaluated as possible predictors of viral failure leading us to establish the cut off values to viral load . 100.000 copies/ml or #100.000 copies/ml as also t cd4+ . 200 cells/mm3 or #200 cells/mm3. the antiretroviral therapy regimen were: (a) double with two nucleoside reverse transcriptase inhibitor and non nucleoside reverse transcriptase inhibitor; (b) triple with two nucleoside reverse transcriptase inhibitor and one protease inhibitor and (c) three nucleoside reverse transcriptase inhibitor, according to cdc classification and the brazilian ministry of health guidelines as previously described. in the population here studied, the chosen antiretroviral regimen was decided by the physician in charge without interference by the authors of this research work. the criteria for the diagnosis of hepatitis b and c virus infection followed the guidelines established by the manual of viral hepatitis consultancy, secretary of health surveillance, department of epidemiological surveillance, brasília, ministry of health14 and also for the tuberculosis diagnosis, the technical manual for tuberculosis control, notebook of basic attention, secretary of health politics, department of basic attention, brasília. ministry of health.15 the exclusion criteria for this research were: (a) be pregnant or minor under 18 years old; (b) be prescribed with medications which could metabolically interact with antiretroviral drugs, including phytotherapics; (c) have had last clinical appointment in a period longer than 6 months which was considered lost to follow-up. the analysis of baseline characteristics (initial data previous to initiation of treatment referring to t cd4+ cell count and viral load) included frequency tables of categorical variables and their descriptive statistics (median, arithmetic mean and standard deviation) as also as continuous variables. in order to measure the dependent association between two categorial variables it was utilized the chi-square test (x2) or when necessary the exact fisher test. to compare before and after results the macnemar test was applied. for the analysis of logistic regression and to predict the event of virological failure16 (depedent variable), the wald method was applied. the association measurement calculated from the logistic model is the adjusted odds ratio (or). the statistical analysis was carried out utilizing spss 17.0. the statistical association was considered when p , 0.05. the patients’ data were confidential and consentment to utilize information were obtained at the health center number 01 direction according to the federal district secretary (ses/df) of health agreement. the research work protocol was approved by the federal district secretary of health researh ethics committee, foundation of teaching and research in health sciences (fepecs). results and discussion initially, 165 patients were eligible to participate in the study for starting the treatment in the period 2002–2008. however, only 139 patients met the inclusion criteria. among them (table 1a and b), http://www.la-press.com bello et al 36 drug target insights 2011:5 males predominated (74.1%). ages ranged from 20–65 years old (mean = 39.7 years, median = 40.0). there was a slight predominance of patients in the range of 41–65 years old (51.1%) compared to younger patients. as for race/ethnical background, there was a predominance of mestizos (54.6%). regarding to the marital status, there was a predominance of single people (48.2%). high school education (25.9%) predominated over other categories. people living in taguatinga (13.7%), a satellite city located in the surrounding areas of brasília, represented the majority of the patients enrolled in this study. the place of birth showed that patients predominated from the midwest (40.3%) and the northeast of brazil (34.5%). hetrosexual relationships predominated (51.8%), followed by the homosexual relationships (23.7%). the initial viral load ranged between 1.643 copies/ml to 500.000 copies/ml (mean = 180.721 copies/ml; arithmetic mean = 126.417 copies/ml) presenting more than half of the patients (55.4%) cell density higher than 100.000 copies/ml (table 2). after antiretroviral therapy, the initial profile changed substantially more than 70% of the patients had viral load lower than 50 copies/ml. in reference to the initial t cd4+ cell count, 68.3% of the patients were in the range of 0–200 cells/mm3. after treatment a relevant change was observed in the patients profile concerning t cd4+ cell counts, remaining less than 37% of the cases in this stage. in the following cell density interval, there was an expressive increment in the number of cases, representing more than 43.0% of the patients (table 2). initially the most utilized antiretroviral regimen included two drugs, the nucleoside reverse transcriptase inhibitor and non nucleoside reverse transcriptase inhibitor in 71.2% of the cases and the remaining 28.1% included two nucleoside reverse transcriptase inhibitor and one protease inhibitor. during the course of the treatment, the presented regimen distribution slightly changed to 66.9% to the first drug combination and table 1b. distribution of socioeconomic and demographic profile of patients attended in the health center no. 1 (hospita dia) during 2002–2008. variable category n % residence pilot plan 29 20.9 guara 16 11.5 ceilandia 17 12.2 taguatinga 19 13.7 bandeirante 9 6.5 gama 14 10.0 sobradinho 3 2.2 planaltina 4 2.9 san sebastian 7 5.0 paranoa 2 1.4 around brasília 19 13.7 birthplace north 4 2.9 northeast 48 34.5 south 4 2.9 southeast 25 18.0 midwest 56 40.3 african continent 2 1.4 exposure category accident at work 1 0.7 bisexual 23 16.7 heterosexual 72 51.8 homosexual 33 23.7 blood transfusion 1 0.7 udi 9 6.6 total 139 100 source: siclom (ms-brazil). abbreviation: udi, user injecting drug. table 1a. distribution of socioeconomic and demographic profiles of patients attended at health center no. 1 (hospital dia) during 2002–2008. variable category n % sex male 103 74.1 female 36 25.9 age 20–40 68 48.9 41–65 71 51.1 race/ethnical background white 60 43.2 mestizo 76 54.6 black 3 2.2 marital status single 67 48.2 married 41 29.5 stable 18 12.9 separate 10 7.2 widow 3 2.2 education incomplete elementary schooling 28 20.1 complete elementary schooling 24 17.3 middle incomplete schooling 19 7.2 middle complete schooling 36 25.9 incomplete higher schooling 4 2.9 complete college schooling 27 19.4 illiterate 9 6.5 not informed 1 0.7 total 139 100.0 source: siclom (ms-brazil). http://www.la-press.com antiretroviral treatment in the federal district, brazil, federal district drug target insights 2011:5 37 32.4% to the second one, there remaining only one utilizing three nucleoside reverse transcriptase inhibitor (table 2). virologic failure was 12.2% (table 3), or antiretroviral treatment success was 88.8%, exceeding the results obtained by johnson and way of 80%.17 among the socioeconomic and demographic variables, just the birthplace and the fact that originating in the midwest region which includes the federal district, was associated with the occurrence of virologic failure in the study. cole et al18 stated that hiv infection is sufficiently widespread, suggesting that it is a multidimensional epidemic, with demographic, residential, social, biological and behavioral significance. perhaps this is a natural social structural marker as the study was conducted in the midwest region compared to those migrating people from other regions to the federal district. there was a trend toward a higher incidence of virologic failure being male, over 40 years old, mestizo, unmarried or separate and also heterossexual exposure category. however, there was not any statistical significance of such associations. it was found that only the birthplace presented statistical significance. patients from the midwestern had 2.7 times greater risk of virologic failure than those from other regions of brazil, especially because the study was conducted in brasilia, which is part of this region. in this study, the initial viral load is not statistically associated and significant to the occurrence of virologic failure. however, there is the presence of a higher incidence of virologic failure among those who had viral load greater than 100.000 copies/ml after the introduction of haart. the virologic target for patients on haart is to achieve viral load plasma levels below 50 copies/ml when two or more potent drugs are used. these results highlight the importance of compliance with primary success and reinforces the need to work on the accession of such patients. some authors19 interpret and agree that those with viral load higher than the baseline level of 100.000 copies/ml had a slower pace to achieve viral suppression. however, a potent and well tolerated prophylactic regimen with haart can improve cd4+ t cell count at the beginning, during and after treatment. kantor et al2 concluded table 2. viral load and t cd4+ cell counts distribution in 139 patients submitted to antiretroviral therapy in the health center no. 1 (hospital dia) during 2002–2008. variable category n % initial viral load (treatment-naive) ,50 copies/ml 0 0 50.1–400 copies/ml 0 0 400.1–10,000 copies/ml 5 3.6 10,000.1–100,000 copies/ml 57 41.0 100,0000.1–500,000 copies/ml 77 55.4 viral load after haart ,50 copies/ml 99 71.2 50.1–400 copies/ml 20 14.4 400.1–10,000 copies/ml 10 7.2 10,000.1–100,000 copies/ml 8 5.8 100,000–500,000 copies/ml 2 1.4 initial t cd4+ cell counts (treatment-naive) 0–200 cells/mm3 96 68.4 200.1–350 cells/mm3 37 26.6 351–500 cells/ 7 5.0 above de 500 cells/mm3 0 0 t cd4+ cell counts after haart 0–200 cells/mm3 51 36.7 200.1–350 cells/mm3 60 43.2 351–500 cells/mm3 21 15.1 above de 500 cells/mm3 7 5.0 haart 2 itrn + 1 itrnn 99 71.2 2 itrn + 1 ip 39 28.1 3 itrn 1 0.7 139 100 abbreviations: haart, highly active antiretroviral therapy; nrti, nucleoside reverse transcriptase inhibitor; nnrti, non-nucleoside reverse transcriptase inhibitor. http://www.la-press.com bello et al 38 drug target insights 2011:5 that plasma viral load strongly predicts the rate of cd4+ lymphocyte count decrease, progression to aids and death. but the prognosis for people infected with hiv is more strictly defined by the combination of plasma viral load measurement and cd4+ lymphocytes’ counts. some patients with viral load higher than 100.000 copies/ml remained in treatment failure after haart regimen in contrast to t cd4+ lymphocyte counts decrease at a slower pace, leaving some in virologic failure, probably due to the emergence of hiv resistant strains. cozzi-lepri et al7 advocate that patients who remained in haart regimen failure, the viral load in the next twelve months was growing at a relatively slow pace, the cd4+ t cell count was stable and the time course of viral replication in patients with virological failure table 3. distribution of variables, viral load, t cd4+ cell counts, haart and comorbidity in relation to the significance of virologic failure in patients treated at the health centre no. 1 (hospital dia) during 2002–2008. variable category vf n ci% rr ci95% p sex male 103 14 13.6 1.63 0.49–5.35 0.407 female 36 3 8.3 age 41–65 60 10 16.7 1.88 0.76–4.75 0.164 20–40 79 7 8.9 race mestizo 79 10 12.7 1.08 0.43–2.68 0.860 white 60 7 11.7 marital status single/separate 80 11 13.8 1.35 0.53–3.44 0.524 married/stable union 59 6 10.2 education nf incomplete/illiterate 38 6 15.8 1.45 0.57–3.64 0.432 other school levels 101 11 10.9 residence brasilia suroundings 19 3 15.8 1.35 0.42–4.27 0.610 pilot plan/satellite city 120 14 11.7 birthplace midwest 56 11 19.6 2.71 1.06–6.92 0.028 north, northeast, south, southeast 83 6 7.2 exposury category heterosexual 83 11 13.3 1.23 0.48–3.15 0.654 homosexual 56 6 10.7 abbreviations: n, patients number; vf, virological failure; ci, incidence coefficient; rr, relative risk; ic95%, interval confidence; p, significance: nf, uninformed. table 4. distribution of association of viral load, t cd4+ cell count, haart and comorbidities in terms of the significance of virologic failure in patients treated at the health centre no. 1 during the years 2002 to 2008. variable category n vf ic% rr ic95% p viral load after haart .100.000 77 10 13.0 1.15 0.46–2.84 0.762 #100.000 62 7 11.3 t cd4+ cell after haart .200 95 8 10.5 0.66 0.27–1.63 0.368 #200 44 9 15.9 haart 2 itrn + 1 itrnn 100 12 12.0 0.93 0.35–2.48 0.804 2 itrnn + 1 ip 39 5 12.8 hcv (hcv-anti) positive 10 1 10.0 0.80 0.11–3.47 0.823 negative 129 16 12.4 hbv (hbsag) positive 31 3 9.7 0.74 0.22–2.43 0.623 negative 108 14 13.9 tb (baar) positive 22 6 27.3 2.90 1.19–7.02 0.019 negative 117 11 10.3 total 139 17 abbreviations: haart, antiretroviral therapy; t cd4+, lymphocyte count; n, number of patients; vf, virologic failure values; ci, incidence rate; rr, relative risk; ic95%, confidence interval; p, significance; hcv, antibody to the hepatitis “c”virus; hbv, antibody against hepatitis “b” virus antigens; tb, tuberculosis; baar, alcohol acid resistant bacilli. http://www.la-press.com antiretroviral treatment in the federal district, brazil, federal district drug target insights 2011:5 39 had not been fully elucidated. asjo and langeland20 suggest that the lack of complete suppression of viral replication allows the continued development of hiv variants with different degrees of resistance. it results not only in treatment failure, but it also increases the risk of hiv primary resistance and dissemination. patients with initial cd4+ t cell count # 200 cells/mm3 had a tendency to virologic failure, however, there was not any statistical significance. tuberculosis coinfection was associated with virologic failure demonstrated with statistical significance, p = 0.019. patients with a previous diagnosis of tuberculosis infection had 2.9 times great risk of virologic failure. it is observed that the t cd4+ cell count baseline above 200 cells/mm3 was not statistically associated with virological failure, but the incidence of failure in this group was noticeably lower than in those with initial t cd4+ less than 200 cells/mm3. studies conducted by skowron et al21 demonstrated that t cd4+ cell count is a better predictor of viral suppression. however, in order to achieve viral load suppression to undetectable levels, it is necessary to have an optimal response of t cd4+ cell count in patients under antiretroviral therapy. piliero22 comments that the maximum suppression of viral replication remains the primary goal of therapy after haart regimen, therefore t cd4+ cell count and hiv plasma viral load values are the prognostic markers for treatment success after four, eight or twelve weeks post treatment, as the changes are predictive of favorable longterm success in six months or more. according to moreno et al23 in multifailed patients, at least two active drugs can not be used, the therapeutic scheme should be kept in use until new drugs become available, assuming that there is an immunologic and clinical stability in order to avoid the use of a drug from a common chemical group which usually leads to a rapid viral resistance development, further limiting future treatment options. geretti et al24 stated that patients in first haart line who maintained consistently undetectable plasma viral load for a year, had a low risk of virologic failure. in this study, the tuberculosis comorbidity had significant influence on the occurrence of virologic failure. special attention should be given to early tuberculosis diagnosis during the early haart table 5. logistic regresion in patients attended in the health center no. 1 (hospital dia) from 2002 to 2008. step variable β wald test p-value odds ratio ic95% step 1 gender 1.142 1.718 0.182 3.132 0.586–16.746 age 0.894 1.906 0.167 2.444 0.687–8.692 skin colour 0.059 0.007 0.932 1.061 0.274–4.107 marital status 0.512 0.588 0.443 1.668 0.451–6.170 education 0.745 1.129 0.288 2.106 0.533–8.319 zone 0.149 0.034 0.854 1.160 0.237–5.683 birthplace 1.240 3.656 0.056 3.454 0.969–12.310 expsosure category 0.580 0.475 0.491 1.786 0.343–9.301 viral load after haart 21.291 0.000 1.000 0.000 0.000–0.000 t cd4+ after haart 0.451 0.417 0.518 1.569 0.400–6.156 haart -0.112 0.023 0.880 0.894 0.207–3.851 hbv (hbsag) -0,539 0,210 0,647 0,583 0,058–5,859 hcv (anti hcv) -0.356 0.191 0.662 0.701 0.142–3.450 tb (b.a.a.r.) 1.107 2.536 0.111 3.025 0.775–11.810 constant -48.150 0.000 1.000 0.000 0.000–0.000 step 14 age 0.812 2.125 0.145 2.252 0.756–6.707 birthplace 1.308 5.247 0.022 3.698 1.208–11.322 tb (b.a.a.r.) 1.311 4.786 0.029 3.710 1.146–12.008 constant -3.508 4.353 0.037 0.030 0.000–0.000 paso 15 birthplace 1.188 4.584 0.032 3.280 1.106–9.731 tb (b.a.a.r.) 1.340 5.098 0.024 3.820 1.194–12.229 constant -2.173 2.567 0.109 0.114 0.000–0.000 abbreviations: β, maximum likehood values; haart, antiretroviral therapy; cd4+, lymphocyte count; ic95%, confidence interval; hcv, hepatitis c virus; anti hcv, antibody to the hepatitis “c” virus; hbv, hepatitis b virus; hbsag, hepatitis b virus surface antigen; tb, tuberculosis; b.a.a.r, alcohol acid resistant bacilli. http://www.la-press.com bello et al 40 drug target insights 2011:5 regimen prescription. studies reported by bekker et al25 and von reyn et al26 showed that the mycobacterial disease was a major contributor to hiv mortality. in addition, the infection increases the risk of latent tuberculosis reactivation, a new infection progression or re-infection to active disease, increasing the risk of the emergence of hiv resistant strains to the usual antiretroviral therapy. tuberculosis also accelerates the course of hiv induced disease by activating viral replication and accentuating the decline of t cd4+ cells. in the logistic regresion analysis the best model to explain the event of virological failure is the one that includes variables such as origin and infection by m. tuberculosis, yielding or values of 3.2 and 3.8 respectively. conclusion despite the enormous progress as a result of the impact of haart-related morbidity and mortality in patients with hiv/aids continue and will continue to fail in the face of the therapeutic classes of haart currently available. the presented results reinforce the importance of monitoring the biological markers t cd4+ cells and viral load in patients living with and without aids, both to ensure that viral replication is under control and to reassure the maintenance of immune reconstitution compatible with life, and to predict the risk of developing resistance and therapeutic failures in the course of the treatment of hiv-infected people. it should be noted that the sample size limited the analysis of this study, as the retrospective information taken from the medical records, excluded some data from the laboratory and the clinical follow up. the sample obtained in this way may have had a limited ability to highlight the predictive differences of risk for both t cd4+ cell count and viral load in naive patients. it is possible that working with a larger cohort of patients and longer follow-up of these predictors probably would show more consistent evidence that is statistically supported. initial treatment with any nnrti-based regimen or an ip, but not both, is a good strategy for managing the long-term antiretroviral treatment in naive hiv patients. moreover, the recent availability of new antiretroviral agents for the treatment of hiv has increased treatment options and improved durability, tolerability and efficacy in the long-term haart regimen. other limitations that should be highlighted refer to the possibility of verifying the association of hbv and hcv coinfections which do not have evolutionary studies of these hiv comorbidities. it is possible that if we had a homogeneous group of patients already in advanced stages of these pathologies, the risk of virologic failure would be high. the sociodemographic data were collected from medical records and not through interviews, this fact may also have skewed the results of the analysis of association between these variables and treatment failure. however, this service data and variables accounted are limitations imposed by real situations in nosocomial institutions in the federal district in brazil. it is of great relevance to the findings in this study considering the limitations and difficulties of conducting studies in routine service, as it can be concluded that even in the non-ideal conditions of a clinical trial, patients treated in this unit had virological success of 12.2% up to one year of treatment, a fact that can predict the durability of the first scheme which is better than expected in many other international centers.17 also tuberculosis infection should be traced as a priority as early as possible. as the low values of t cd4+ cell counts and high viral load pre-treatment should be considered to implement the prescribed antiretroviral therapy, especially when new classes are available and have superior performance, as much viral suppression and immune reconstitution could be achieved. disclosures author(s) have provided signed confirmations to the publisher of their compliance with all applicable legal and ethical obligations in respect to declaration of conflicts of interest, funding, authorship and contributorship, and compliance with ethical requirements in respect to treatment of human and animal test subjects. if this article contains identifiable human subject(s) author(s) were required to supply signed patient consent prior to publication. author(s) have confirmed that the published article is unique and not under consideration nor published by any other publication and that they have consent to reproduce any copyrighted material. the peer reviewers declared no conflicts of interest. http://www.la-press.com publish with libertas academica and every scientist working in your field can read your article “i would like to say that this is the most author-friendly editing process i have experienced in over 150 publications. thank you most sincerely.” “the communication between your staff and me has been terrific. whenever progress is made with the manuscript, i receive notice. quite honestly, i’ve never had such complete communication with a journal.” “la is different, and hopefully represents a kind of scientific publication machinery that removes the hurdles from free flow of scientific thought.” your paper will be: • available to your entire community free of charge • fairly and quickly peer reviewed • yours! you retain copyright http://www.la-press.com antiretroviral treatment in the federal district, brazil, federal district drug target insights 2011:5 41 references 1. mellors jw, munoz a, giorgi jv, margolick jb, tassoni cj, gupta p. plasma viral load and cd4+ lymphocytes as prognostic markers of hiv-1 infection. ann intern med. jun 15, 1997;126(12):946–54. 2. kantor r, diero l, delong a, kamle l, muyonga s, mambo f. misclassification of first-line antiretroviral treatment failure based on immunological monitoring of hiv infection in resource-limited settings. clin infect dis. aug 1, 2009;49(3):454–62. 3. pasternak ao, adema kw, bakker m, jurriaans s, berkhout b, cornelissen m. highly sensitive methods based on seminested real-time reverse transcription-pcr for quantitation of human immunodeficiency virus type 1 unspliced and multiply spliced rna and proviral dna. j clin microbiol. jul 2008;46(7):2206–11. 4. pinganaud c, goujard c. management of antiretroviral treatment in hiv-infected patients exhibiting virologic failure. presse med. jun 22, 2002;31(22):1034–40. 5. gallant je. drug resistance after failure of initial antiretroviral therapy in resource-limited countries. clin infect dis. feb 1, 2007;44(3):453–5. 6. turner bj, hecht fm, ismail rb. cd4+ t-lymphocyte measures in the treatment of individuals infected with human immunodeficiency virus type 1. a review for clinical practitioners. arch intern med. jul 25, 1994;154(14):1561–73. 7. cozzi-lepri a, phillips an, miller v, katlama c, ledergerber b, vella s. changes in viral load in people with virological failure who remain on the same haart regimen. antivir ther. apr 2003;8(2):127–36. 8. resino s, bellon jm, ramos jt, resino r, gurbindo md, mellado mj. impact of highly active antiretroviral therapy on cd4+ t cells and viral load of children with aids: a population-based study. aids res hum retroviruses. sep 2004;20(9):927–31. 9. battegay m, fehr j, fluckiger u, elzi l. antiretroviral therapy of late presenters with advanced hiv disease. j antimicrob chemother. jul 2008; 62(1):41–4. 10. melchior r, nemes mi, alencar tm, buchalla cm. challenges of treatment adherence by people living with hiv/aids in brazil. rev saude publica. dec 2007;41 suppl 2:87–93. 11. cdc revises hiv classification system, aids definition. w v med j. feb 1993;89(2):74. 12. brazil moh, secretariat of health surveillance, national std and aids. recommendations for antiretroviral therapy in adults and adolescents infected with hiv. 2008. available: http://bvsms.saude.gov.br/bvs/ publicacoes/recomendacao_terapia.pdf access: 28/05/2011. 13. brazil mohssoh, national std aids. quick reference for the arv drug dispensing siclom. 2007. available: http://siclom.aids.gov.br access: 28.05.2011. 14. brazil npfpacovh. national program for prevention and control of viral hepatitis. 2005. national program for prevention and control of viral hepatitis http://bvsms.saude.gov.br/bvs/politicas/hepatites_aconselhamento.pdf access: 28.05.2011. 15. brazil moh, department of health policy, department of primary care. technical manual for the control of tuberculosis. 2002. available to: http:// bvsms.saude.gov.br/bvs/publicacoes/guia_controle_tuberculose.pdf access: 28.05.2011. 16. faucher jf, challier b, chirouze c, drobacheff c, fischer p, lang jm. predictive factors of virological response to primary antiretroviral treatment. presse med. mar 13, 2004;33(5):310–5. 17. johnson k, way a. risk factors for hiv infection in a national adult population: evidence from the 2003 kenya demographic and health survey. j acquir immune defic syndr. aug 15, 2006;42(5):627–36. 18. cole sr, hernan ma, anastos k, jamieson bd, robins jm. determining the effect of highly active antiretroviral therapy on changes in human immunodeficiency virus type 1 rna viral load using a marginal structural leftcensored mean model. am j epidemiol. jul 15, 2007;166(2):219–27. 19. phillips an, staszewski s, weber r, kirk o, francioli p, miller v. hiv viral load response to antiretroviral therapy according to the baseline cd4 cell count and viral load. jama. nov 28, 2001;286(20):2560–7. 20. asjo b, langeland n. drug resistance in hiv infection. tidsskr nor laegeforen. nov 20, 2008;128(22):2593–6. 21. skowron g, street jc, obee em. baseline cd4(+) cell count, not viral load, correlates with virologic suppression induced by potent antiretroviral therapy. j acquir immune defic syndr. dec 1, 2001;28(4):313–9. 22. piliero pj. early factors in successful anti-hiv treatment. j int assoc physicians aids care (chic ill). jan–mar 2003;2(1):10–20. 23. moreno cuerda vj, rubio garcia r, morales conejo m. new therapeutic options in protracted hiv-infected patients with virological failure. med clin (barc). jan 26, 2008;130(2):66–70. 24. geretti am, smith c, haberl a, garcia-diaz a, nebbia g, johnson m. determinants of virological failure after successful viral load suppression in first-line highly active antiretroviral therapy. antivir ther. 2008;13(7): 927–36. 25. bekker lg, orrell c, reader l, matoti k, cohen k, martell r. antiretroviral therapy in a community clinic—early lessons from a pilot project. s afr med j. jun 2003;93(6):458–62. 26. von reyn cf, kimambo s, mtei l, et al. disseminated tuberculosis in human immunodeficiency virus infection: ineffective immunity, polyclonal disease and high mortality. int j tuberc lung dis. aug 2011;15(8):1087–92. http://www.la-press.com http://www.la-press.com http://bvsms.saude.gov.br/bvs/publicacoes/recomendacao_terapia.pdf http://bvsms.saude.gov.br/bvs/publicacoes/recomendacao_terapia.pdf http://siclom.aids.gov.br http://bvsms.saude.gov.br/bvs/politicas/hepatites_aconselhamento.pdf http://bvsms.saude.gov.br/bvs/publicacoes/guia_controle_tuberculose.pdf http://bvsms.saude.gov.br/bvs/publicacoes/guia_controle_tuberculose.pdf https://doi.org/10.1177/1177392817728725 drug target insights volume 11: 1–8 © the author(s) 2017 reprints and permissions: sagepub.co.uk/journalspermissions.nav doi: 10.1177/1177392817728725 creative commons non commercial cc by-nc: this article is distributed under the terms of the creative commons attribution-noncommercial 4.0 license (http://www.creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the sage and open access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage). introduction based on the latest world health organization (who) estimates of december 2016, there were 212 million cases of malaria in 2015 and 429 000 deaths, with sub-saharan africa accounting for 90% of all malaria deaths in the world.1 this staggering number of deaths resulting from malaria is associated with the continuous spread of plasmodium falciparum resistance to available antimalarial drugs, posing severe threats to existing malaria control programs. the lack of a deployable malaria vaccine coupled with increasing cases of drug-resistant infections makes the search for new antimalarials imperative.2,3 owing to the high cost of malaria treatment in developing countries in general and nigeria in particular, many have resulted to consulting traditional herbalists or the use of medicinal plants for treatment of infection.4 for thousands of years, in various parts of the world, traditional herbal medicines have been used to treat malaria.5–9 two of the most important antimalarial drugs in use, artemisinin and quinine, were either derived directly from plants or synthesized using chemical structures of plant-derived compounds as templates.5,9,10 most of the people in malaria endemic countries still depend on traditional herbal remedies, which could be linked to limited availability as well as unaffordability of existing pharmaceutical drugs.11 blighia sapida, also referred to as ackee or ackee apple, is a medicinal plant of the family sapindaceae that naturally exists in the forests of most west african countries, where the fruits are used for several purposes, but rarely eaten.12 when mixed with water, the green fruits of this plant produce lather and are used for laundry, in addition to its seeds being used for making soap due to its high oil content.13 some parts of this plant also serve medicinal purposes, such as the juice from the leaf is traditionally used to treat conjunctivitis,12 whereas its twiggy leaves, once beaten into a pulp, are applied to the forehead for treating migraine or headaches.14 for over 20 years, the centre for scientific research into plant medicine (csrpm) in ghana has used this plant for the treatment of diarrhea.15 the antidiarrheal activity of the stem bark of b sapida has also been reported,16 including reports on its traditional use for the treatment of dysentery, yellow fever, burns, wounds, or conjunctivitis, etc.14 murine experiments have also demonstrated its utility in treating pancreatic β-cell dysfunction,17 elevated blood glucose, dyslipidemia, and oxidative stress in alloxan-induced diabetic rats.18 in view of the significant challenges imposed by drug resistance, traditional plant extracts continue to be promising sources of new antimalarial compounds.9 the use of traditional medicines for the treatment of malaria and other diseases continues to be a growing practice among many african families, despite the availability of orthodox method of treatment.19 in addition, the short supply of artemisinin compounds leading to alternative ethanol extract of blighia sapida stem bark show remarkable prophylactic activity in experimental plasmodium berghei–infected mice olayinka o otegbade1, johnson a ojo1, dolapo i adefokun2, oyindamola o abiodun3, bolaji n thomas4 and olusola ojurongbe1 1department of medical microbiology & parasitology, ladoke akintola university of technology, osogbo, nigeria. 2department of pharmacology & therapeutics, ladoke akintola university of technology, osogbo, nigeria. 3department of pharmacology & therapeutics, university of ibadan, ibadan, nigeria. 4department of biomedical sciences, college of health sciences and technology, rochester institute of technology, rochester, ny, usa. abstract: this work explores the antiplasmodial potential of ethanol extract of blighia sapida (lin. sapindaceae) in chloroquine (cq)resistant plasmodium berghei (anka strain)–infected mice. chloroquine-resistant (anka) strain of p berghei was inoculated intraperitoneally into swiss albino mice. mice were treated orally for 4 consecutive days, before and after inoculation (prophylactic, suppressive, and curative models) with graded doses of the plant extracts with artemether-lumefantrine (coartem) as control. prophylactically, the extract showed a remarkable activity in the chemosuppression of p berghei parasites (p < .01) ranging from 57% to 36.5% at doses of 200 to 800 mg/kg, respectively, whereas coartem (10 mg/kg) produced 62.1% chemosuppression. no significant chemosuppression was observed in the curative and suppressive models. the plant extract appeared to be safe at the highest dose tested (5000 mg/kg) for acute toxicity, with no adverse effect on the different organs. the plant extract possesses prophylactic antimalarial activity, which supports its use in the prevention of malaria. keywords: plasmodium berghei, blighia sapida, chemosuppression, curative, prophylaxis, efficacy and safety received: june 7, 2017. accepted: august 8, 2017. peer review: two peer reviewers contributed to the peer review report. reviewers’ reports totaled 428 words, excluding any confidential comments to the academic editor. type: original research funding: the author(s) received no financial support for the research, authorship, and/or publication of this article. declaration of conflicting interests: the author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. corresponding author: olusola ojurongbe, department of medical microbiology & parasitology, ladoke akintola university of technology, pmb, 4400 osogbo, nigeria. email: oojurongbe@lautech.edu.ng 728725dti0010.1177/1177392817728725drug target insightsotegbade et al research-article2017 https://uk.sagepub.com/en-gb/journals-permissions mailto:oojurongbe@lautech.edu.ng 2 drug target insights production methods,9 as well as the desire for low-cost drug delivery strategies,7 has led to significant research efforts to decipher alternative plant products that could serve as effective antimalarials, especially in sub-saharan africa. in this report, the antiplasmodial effect of ethanolic extract of b sapida stem bark in vivo is reported, using the suppressive, curative, and prophylactic model. materials and methods plant collection, authentication, and extract preparation the stem bark of b sapida was collected in december 2014 from ipetumodu in the south west of nigeria. plant material was identified in the herbarium of the botany department of obafemi awolowo university, ile-ife, osun state nigeria with voucher number ife-17629 by mr ga ademoriyo. the stem bark was air-dried at room temperature and powdered. about 50 g of the powdered plant was macerated in 500 ml of 99% methanol (1:10 w/v ) for 48 hours and then filtered. with the aid of a rotatory evaporator, filtrate was concentrated to dryness and the residue obtained was stored until future use. ethical consideration experimental procedures and protocols used in this study were in conformity with national institute of health’s recommendations in guide for the care and use of laboratory animals and in accordance with the principles of helsinki declaration. parasite/infection chloroquine-resistant p berghei anka, obtained from institute for medical research and training (imrat), university college hospital, ibadan, nigeria, was used to evaluate the antimalarial activity of b sapida in this study. groups of swiss albino mice (5 per cage) were infected intraperitoneally with 1 × 107 parasitized erythrocytes from an infected donor mouse. the day of infection was defined as day 0 (d0) and subsequent days d1, d2, etc. antimalarial medication standard coartem (artemether and lumefantrine), used for treating malaria (mekophar chemical pharmaceutical jointstock co, ho chi minh city, vietnam) and obtained from a pharmacy, was used as reference for the antimalarial screening in this study. acute toxicity test (assessment of minimum lethal dose) the assessment of minimum lethal dose was done in 2 phases, following published methods.20 in phase 1, groups of 3 mice in each group were given oral doses of 10, 100, and 1000 mg/kg body weight of b sapida ethanolic extract, respectively, and observed for 24 hours for mortality. in phase 2, 3 mice in each group groups were orally administered with 1600, 2900, and 5000 mg/kg body weight of the extract, respectively, and monitored for 24 hours, observing for mortality. the phase 2 study was conducted because we observed no deaths among animals in phase 1 study. the lethal dose and the penultimate dose to the lethal dose would indicate the value of the ld50.21,22 in vivo antiplasmodial determination swiss albino mice (weight: 18-25 g), obtained from the animal house, department of pharmacology, university of ibadan, kept according to institutional animal care and use committee (iacuc) standards, were allowed to acclimatize to the new environment for a week before study initiation. each mouse was inoculated with 0.2 ml of infected blood containing about 1 × 107 dose of p berghei berghei (about 16.6%) from a donor mouse. each mouse was inoculated on day 0 (d0) (intraperitoneally) for the suppressive and curative model and on the fifth day (d4) for the prophylactic model. suppressive test (4-day test) the 4-day suppressive test was conducted according to the method of peters (1975). in this, 25 mice distributed into 5 groups of 5 mice each were infected with 1 × 107 of parasitized red blood cells on day 0. animals in group 1 received normal saline, those in groups 2 to 4 received 200, 400, and 800 mg/kg of ethanol extract of b sapida test extract, whereas those in group 5 (positive control) received 10 mg/kg of the standard drug coartem. treatment was administered orally and continued daily for the next 3 days for the group that received coartem or 4 consecutive days for extract-treated group. on day 4, blood samples were collected from the caudal vein and stained with 10% giemsa stain. thereafter, the number of the parasitized cells was estimated under the microscope using ×100 objective.23 the numbers of infected erythrocytes were counted until 1000 erythrocytes were achieved. the average percentage suppression of parasitemia was calculated in comparison with controls as follows: % % % suppression parasitemia in negative control parasitemia in t = _ eest group parasitemia in negative control% 100× prophylactic test the prophylactic activity of the extract was performed as described previously.24 in this, swiss albino mice were randomized into 5 groups of 5 mice each: group 1 was treated with normal saline only, groups 2 to 4 were treated with 200, 400, and 800 mg/kg/d of the ethanol extract of b sapida, and group otegbade et al 3 5 served as positive control (treated with 10 mg/kg/d of the standard drug coartem). treatment started on day 0 and continued for 4 consecutive days, by which mice were inoculated intraperitoneally with 1 × 107 of the parasite. on day 7 (72 hours after inoculation), blood smears made from each mouse were stained with giemsa, number of the parasitized cells was estimated, and the percentage suppression was evaluated. curative test this was also performed with standard protocol,24 as previously described. twenty-five mice distributed into 5 groups of 5 were infected intraperitoneally on the first day (day 0) with 0.2 ml of parasite inoculum. after 72 hours of infection, all animals were treated with different regimens: group 1 received normal saline only; groups 2 to 4 received 200, 400, and 800 mg/ kg body weight of plant extract, respectively; whereas group 5 received 10 mg/kg body weight of the standard antimalarial drug (coartem). treatment was continued for 5 consecutive days, blood smears were made and microscopically examined to monitor the degree of parasitemia as well as estimating the number of parasitized cells. histologic procedures these were performed with hematoxylin and eosin procedures.25 briefly, organs such as liver, spleen, kidney, and testes from each animal were fixed in 10% formal saline, grossed and cut longitudinally into pieces of 4 mm in thickness, and processed for histologic study using standard procedure. microtome sections were dried on a hot plate and later stained with hematoxylin and eosin stains for examination with a light microscope. data analysis percentage parasitemia and mean chemosuppression were determined using 1-way analysis of variance; studentnewman-keuls test was used for analysis and comparing of results at 95% confidence interval; p < .05 was set for statistical significance. results about 11.10 g of brown crude extract was obtained from the extraction procedure, a percentage yield of 22%. the acute toxicity test revealed that the extract appeared to be nontoxic at the highest dose (5000 mg/kg) tested. no toxic symptoms or mortality was observed in any of the 3 animals in 3 groups both in phase 1 and phase 2 acute toxicity test study. all animals were still alive at the end of the acute toxicity test, with ld50 value greater than 5000 mg/kg body weight, showing the extract to be nontoxic. histology sections of the liver, spleen, kidney, and testes reveal normal tissue morphology at all doses, except at 5000 mg/kg (black ring), where we observed a mild perivascular infiltration of liver inflammatory cells (figure 1); otherwise, hepatocytes appeared normal. suppressive effect of b sapida ethanolic extract on p berghei in the suppressive test, mean parasitemia on day 4 after infection ranged from 14.20 ± 1.17 to 18.96 ± 0.60 for 200 and 800 mg/kg, respectively (figure 2). the value for control was 19.02 ± 1.20, whereas for coartem was 8.38 ± 0.39. the percentage chemosuppression of the extract ranged from 6.8% to 0.3% for 200 and 800 mg/kg, respectively, whereas that for standard antimalarial coartem was 55.9%. percentage chemosuppression showed increases with decrease in dose levels of the extract, indicating that maximal chemosuppression could be reached at the lowest dose, although intangible. percentage chemosuppression in coartem was very much higher than in control and all the doses of the extract, revealing that extract had little or no suppressive effect at all doses. percentage parasitemia in control, compared with 400 and 800 mg/kg, was not statistically significant (p > .05), whereas that of 200 mg/kg compared with control, 400 and 800 mg/kg, was statistically significant (p < .01) (figure 2). percentage parasitemia in coartem was much lower than in extract at the highest dose, showing statistical significance (p < .001), but percentage parasitemia of the lowest dose of the extract was slightly higher in comparison with coartem, with the difference not significant (figure 2). prophylactic effect of b sapida ethanolic extract on p berghei the percentage parasitemia in mice infected and treated with 200, 400, and 800 mg/kg was 8.22 ± 0.32, 9.7 ± 0.31, and 12.12 ± 0.54, respectively (figure 3). untreated saline group had percentage parasitemia of 19.10 ± 0.33, whereas that of coartem-treated positive control was 7.24 ± 0.91. percentage chemosuppression of parasites in mice treated with 200, 400, figure 1. photomicrographs of liver and kidney at the end of acute toxicity assessment. (a) normal liver morphology. (b) (black ring) mild perivascular infiltration of the liver inflammatory cells after treatment with 5000 mg/kg of the extract. (c) normal kidney morphology. (d) kidney remains normal after treatment with 5000 mg/kg of the extract. haematoxylin and eosin, 400x original magnification. 4 drug target insights and 800 mg/kg was 57%, 50.8%, and 36.5%, respectively, whereas that of coartem was 62.1%. unlike our observation in the suppressive model, percentage parasitemia decreased with a decrease in extract dose levels, showing that a tangible optimal figure 2. suppressive effect of methanol extract of bark of blighia sapida on plasmodium berghei. bars are expressed as mean ± sem (n = 5); ***p < .001, coartem versus normal saline (ns); **p < .01, 200 mg/kg versus normal saline. figure 3. prophylactic effect of ethanol extract of blighia sapida on plasmodium berghei in mice. bars are expressed as mean ± sem (n = 5); ***p < .001, test and control versus normal saline (ns). otegbade et al 5 chemosuppression can be obtained at the lowest dose in this model and is statistically significant at all doses when compared with nontreated group (p < .001; figure 3). the percentage of the extract at the lowest dose (200 mg/kg) was comparable with that of coartem, although coartem exhibited a highly significant (p < .001) decrease in percentage parasitemia compared with negative control and 800 mg/kg. curative effect of b sapida ethanolic extract on p berghei figure 4 shows curative effect of the extract at different graded doses. unlike the suppressive and prophylactic models, none of the extract doses tested showed significant activity. the lowest dose (200 mg/kg) that had shown significant activity in other models showed no activity in our model. for day 3, the values for coartem versus control versus 200 mg/kg of the extract was 1.74 ± 0.29 versus 7.58 ± 0.44 versus 7.18 ± 0.82, with similar findings on day 7 (21.48 ± 0.68, 17.00 ± 0.58, and 2.06 ± 0.39 for control), 200 mg/kg of extract and coartem, respectively, revealing the extract had no significant activity against the parasite when compared with control. effect of malaria on weight of malaria-induced mice the weights of all test animals decreased 24 hours after infection, with this decrease continuing among the suppressive, curative, and untreated groups 72 hours after treatment. however, by day 5 (21.40 ± 1.20), this progressive weight loss was turned around for the prophylactic group when compared with the weights obtained at the same dose on day 0 (20.78 ± 0.92) and day 7 (21.70 ± 1.08) (table 1). effect of b sapida ethanolic extract on organs of p berghei–infected mice the kidney, liver, and spleen of the mice revealed normal tissue architecture after treatment, demonstrating that this extract appears safe on the major organs of the mice (slides not shown). figure 4. curative effect of ethanol extract of blighia sapida on plasmodium berghei in mice. ns indicates normal saline. table 1. weights of mice for prophylactic model. dose, mg/kg day 0 day 1 day 2 day 3 day 4 day 5 day 6 day 7 ns 21.40 ± 1.02 21.85 ± 0.99 22.08 ± 1.0 21.94 ± 0.96 22.28 ± 1.10 21.90 ± 0.93 19.45 ± 0.83 17.02 ± 0.75 200 20.78 ± 0.92 20.16 ± 0.94 21.02 ± 1.02 20.24 ± 1.10 20.96 ± 1.03 21.40 ± 1.20 20.16 ± 1.00 21.70 ± 1.08 400 17.28 ± 0.44 17.20 ± 0.39 17.46 ± 0.54 17.36 ± 0.51 17.62 ± 0.41 18.00 ± 0.47 17.32 ± 0.43 16.96 ± 0.40 800 22.28 ± 1.21 22.20 ± 1.40 22.50 ± 1.47 21.64 ± 1.33 22.12 ± 1.34 22.80 ± 1.52 21.90 ± 1.36 22.68 ± 1.44 coartem 19.58 ± 1.00 19.88 ± 0.90 20.14 ± 0.78 19.78 ± 0.70 19.58 ± 0.70 20.12 ± 0.75 19.52 ± 0.63 20.42 ± 0.51 abbreviation: ns, normal saline. the baseline weights were taken before inoculation and subsequent weights taken before, during and after treatments. data are expressed as mean ± sem. values do not indicate statistical significance to one another. 6 drug target insights survival time of mice in the 3 models the survival time for mice in the prophylactic group was longer than those in suppressive and curative groups (figure 5). mice in prophylactic group started dying on day 9, whereas most were still alive at day 20 and beyond. however, animals in curative group started dying on day 5 and were all dead by day 9, whereas those in the suppressive group started dying on day 5 with 98% dead by day 11. these results clearly demonstrate the prophylactic effect of this extract, at different doses, on malariainfected animal. discussion despite the many and varying efforts at malaria control, this disease is still a global threat of enormous proportion and a significant contributor to health and economic inequities in endemic countries. inhabitants of these countries, under the scourge of disease, face the conundrum of finding appropriate, effective, and cheap means to protect themselves against infection, whereas the parasite, however, has shown an increasing resistance to available antimalarial drugs. this resistance is also favored by population movement and human migration leading to the introduction of resistant parasite to areas previously free of drug resistance. the possibility of synthesizing new antimalarials from plants has become a major policy goal in the control arena, becoming more urgent, in light of the limited number of antimalarials in development, the potential of parasite resistance to the only available therapeutic drugs (artemisinin and its derivatives), and the poor solubility of artemisinin. these are some of the factors driving the design of alternative methods of artemisinin delivery that could potentially slow the process of resistance evolution.10,26,27 we demonstrate that b sapida investigated in this study possesses antimalarial properties that deserve further analysis and could become a cheap, readily available antimalarial in developing countries. this study shows that b sapida has remarkable activity (almost equivalent with coartem), when used as a prophylactic agent in chloroquine-resistant p berghei–infected mice, with the 7-day assay leading to a significant parasitemia suppression. maximum activity was recorded at the lowest dose (200 mg/kg), with a decrease with increasing dosage, implying that the extract had no pronounced activity at higher doses, and that the small dose suffices to produce significant antimalarial activity. this observation concurs with previous result, where the minimum inhibitory activity shown by the methanolic extract of b sapida against staphylococcus aureus started at a dose of 200 mg/ml, but further increase in activity was seen as the dose was decreased to 100 mg/ml, suggesting that the lower the dose, the higher the activity displayed.28 it is worth noting that b sapida extract exhibited the most active antiplasmodial properties at the lowest dosage tested. this dosedependent activity potentially shows that response at low dose is more effective than response inhibition at high dose.29 our results show that b sapida extracts will serve as excellent antimalarial agent in the prophylactic model of treatment. the absence of death after administering 5000 mg/kg body weight of b sapida extract in the acute toxicity test suggests that the extract appears to be nontoxic, an observation supported by the toxicity scale principle, which states that any chemical showing an ld50 greater than 5000 mg/kg is practically nontoxic.30 in this report, the acute toxicity test conducted with the extracts demonstrate their safety because the highest dose caused no death. interestingly, the highest dose used to treat parasite-infected mice from which antimalarial activity was elicited was much lower than the highest acute dose, thus making the extract quite selective and safe. loss of appetite, ultimately leading to weight loss, is one of the characteristics of malaria infection.31 we observed weight loss in p berghei–infected animals after infection, with the initial weight loss recorded reversed after 5 days of treatment with the extracts in the prophylactic group, but not in the curative and suppressive groups, which showed progressive weight loss. this result in the suppressive and curative groups agrees with earlier report of continuous weight loss among treated animals compared with nontreated animals32 but disagrees with our previous study using extracts from russelia equisetiformis, which showed a reversal in body weight after treatment.3 however, the prophylactic group in this study where we observed slight increase after treatment agrees with our previous study. the survival time for mice in the prophylactic group extended beyond day 20, whereas those in the curative model did not survive beyond day 9. about 98% of the mice in the suppressive model were dead by day 15, with only 2% surviving till day 20 but not beyond, further confirming the prophylactic activity of b sapida ethanol extract. although there have been some reports of b sapida poisoning in people who consumed the fruit (ackee) in particular,33–37 such poisonous effect was not observed with the stem bark used in our study. histologic examination of the liver, kidney, and spleen showed no toxicity to the organs after treatment with the at all doses used, agreeing with the results figure 5. graph showing survival time of mice in the 3 models. otegbade et al 7 of a different study using blighia unijugata, which showed no toxicity to the liver and kidney when administered in a dosedependent fashion to wistar rats more than 4 weeks.38 histology of the testes reveals that extract has no toxic effect on male animals and as such not inhibitory to fertility. the difference in the reported toxicity may be due to the part of the plant examined. the seed is toxic, whereas the stem bark appears safe. conclusively, our results suggest that the ethanol bark extract of b sapida show some intrinsic antimalarial activities by its prophylactic ability against chloroquine-resistant p berghei parasites. this performance can surely be improved on in future studies if the crude extract is purified and the active substituents are identified and the doses further fractionated. the extracts have considerably low toxicities in experimental mice, this result supporting the traditional use of this plant for the prophylactic treatment of malaria only. acknowledgements the authors are grateful to dr go gbotosho, malaria research laboratory, institute for medical research and training (imrat), university college hospital, ibadan, nigeria, for providing the malaria parasite used for the study, also to mrs sakirat braimah for technical assistance. author contributions oo conceived and designed the experiments. ooo, jao, and dia performed the laboratory work. jao and dia analyzed data. ooo wrote first draft of manuscript. jao, dia, and bnt contributed to writing of manuscript. bnt, ooa, and oo made critical revisions and approved final version. all authors reviewed and approved the final manuscript. disclosures and ethics the authors also confirmed that this article is unique and not under consideration or published in any other journal. experimental procedures and protocols are in conformity with national institute of health’s recommendations in guide for the care and use of laboratory animals and the principles of the declaration of helsinki. r efer ences 1. who. who—world malaria report 2015. who; http://www.who.int/malaria/publications/world-malaria-report-2015/report/en/. published 2015. accessed february 5, 2017. 2. ogungbamigbe to, ojurongbe o, ogunro ps, et al. chloroquine resistant plasmodium falciparum malaria in osogbo nigeria: efficacy of amodiaquine + sulfadoxine-pyrimethamine and chloroquine + chlorpheniramine for treatment. mem inst oswaldo cruz. 2008;103:79–84. 3. ojurongbe o, ojo ja, adefokun di, abiodun oo, odewale g, awe eo. in vivo antimalarial activities of russelia equisetiformis in plasmodium berghei infected mice. indian j pharm sci. 2015;77:504–510. 4. awe eo, adeloye a, idowu t, olajide oa, makinde j. antinociceptive effect of russelia equisetiformis leave extracts: identification of its active constituents. phytomedicine. 2008;15:301–305. 5. basco lk, mitaku s, skaltsounis al, et al. in vitro activities of furoquinoline and acridone alkaloids against plasmodium falciparum. antimicrob agents chemother. 1994;38:1169–1171. 6. ekué mrm, sinsin b, eyog-matig o, finkeldey r. uses, traditional management, perception of variation and preferences in ackee (blighia sapida k.d. koenig) fruit traits in benin: implications for domestication and conservation. j ethnobiol ethnomedicine. 2010;6:12. 7. weathers pj, arsenault pr, covello ps, mcmickle a, teoh kh, reed dw. artemisinin production in artemisia annua: studies in planta and results of a novel delivery method for treating malaria and other neglected diseases. phytochem rev. 2011;10:173–183. 8. elfawal ma, towler mj, reich ng, et al. dried whole plant artemisia annua as an antimalarial therapy. plos one. 2012;7:e52746. 9. weathers pj, elfawal ma, towler mj, golenbock d, weathers pj, rich sm. pharmacokinetics of artemisinin delivered by oral consumption of artemisia annua dried leaves in healthy vs. plasmodium chabaudi-infected mice. j ethnopharmacol. 2014;153:732–736. 10. desrosiers mr, weathers pj. effect of leaf digestion and artemisinin solubility for use in oral consumption of dried artemisia annua leaves to treat malaria. j ethnopharmacol. 2016;190:313–318. 11. zirihi gn, mambu l, guédé-guina f, bodo b, grellier p. in vitro antiplasmodial activity and cytotoxicity of 33 west african plants used for treatment of malaria. j ethnopharmacol. 2005;98:281–285. 12. rashford j. those that do not smile will kill me: the ethnobotany of the ackee in jamaica. econ bot. 2001;55:190–211. 13. aderinola o, farinu g, akinlade j, et al. nutritional potential of blighia sapida k konig (ackee ackee) leaves as a dry season feed resource for west african dwarf goats in the derived savanna zone of nigeria. livest res rural dev. 2007;19. http://www.lrrd.org/lrrd19/6/ader19078.htm. 14. etukudo i. ethnobotany: conventional and traditional uses of plants. uyo, nigeria: the verdict press; 2003. 15. abbiw d. useful plants of ghana. london: intermediate technology publications & the royal botanic gardens kew; 1990. 16. antwi s, martey o, donkor k, nii-ayitey okine lk. anti-diarrhoeal activity of blighia sapida (sapindaceae) in rats and mice. j pharmacol toxicol. 2009;4:117–125. 17. ojo oa, ajiboye bo, ojo ab, oyinloye be, imiere od, adeyonu o. ameliorative potential of blighia sapida k.d. koenig bark against pancreatic βcell dysfunction in alloxan-induced diabetic rats [published online ahead of print 17 march 2017]. j complement integr med. epub ahead of print doi:10.1515/ jcim-2016-0145. 18. oloyede ob, ajiboye to, abdussalam af, et al. blighia sapida leaves halt elevated blood glucose, dyslipidemia and oxidative stress in alloxan-induced diabetic rats. j ethnopharmacol. 2014;157:309–319. 19. maroyi a. traditional use of medicinal plants in south-central zimbabwe: review and perspectives. j ethnobiol ethnomedicine. 2013;9:31. 20. lorke d. a new approach to practical acute toxicity testing. arch toxicol. 1983;54:275–287. 21. matsumura f. toxicology of insecticides. springer; 1975. http://www.springer. com/us/book/9781461344124. accessed may 8, 2017. 22. corbett j, wright k, baillie a. the biochemical mode of action of pesticides. 2nd ed. london and new york: academic press; 1984. 23. ljungström i, perlmann h, schlichtherle m, et al. method in malaria research. manassas, va: mr4/atcc; 2004. 24. peters w. the chemotherapy of rodent malaria, x xii. the value of drug-resistant strains of p. berghei in screening for blood schizontocidal activity. ann trop med parasitol. 1975;69:155–171. 25. layton c, bancroft jd. bancroft’s theory and practice of histological techniques, expert consult: online and print. 7th ed. london, england: elsevier health sciences; 2013. 26. van der kooy f, verpoorte r. the content of artemisinin in the artemisia annua tea infusion. planta med. 2011;77:1754–1756. 27. suberu jo, gorka ap, jacobs l, et al. anti-plasmodial polyvalent interactions in artemisia annua l. aqueous extract–possible synergistic and resistance mechanisms. plos one. 2013;8: e80790. 28. ubulom pm, udobi ce, akpabio e, et al. antimicrobial activities of leaf and stem bark extracts of blighia sapida. j plant stud. 2013;2:47. 29. calabrese ej, baldwin la. the hormetic dose-response model is more common than the threshold model in toxicology. toxicol sci off j soc toxicol. 2003;71:246–250. 30. sandu rb, tarţău l, miron a, zagnat m, ghiciuc cm, lupuşoru ce. experimental researches on acute toxicity of a bidens tripartita extract in mice— preliminary investigations. rev med chir soc med nat iasi. 2012;116:1230–1234. 31. dikasso d, makonnen e, debella a, et al. anti-malarial activity of withania somnifera l. dunal extracts in mice. ethiop med j. 2006;44:279–285. 32. chinchilla m, guerrero om, abarca g, et al. an in vivo model to study the anti-malaric capacity of plant extracts. rev biol trop. 1998;46:35–39. 33. meda ha, diallo b, buchet jp, et al. epidemic of fatal encephalopathy in preschool children in burkina faso and consumption of unripe ackee (blighia sapida) fruit. lancet. 1999;353:536–540. http://www.who.int/malaria/publications/world-malaria-report-2015/report/en/ http://www.who.int/malaria/publications/world-malaria-report-2015/report/en/ http://www.lrrd.org/lrrd19/6/ader19078.htm http://www.springer.com/us/book/9781461344124 http://www.springer.com/us/book/9781461344124 8 drug target insights 34. joskow r, belson m, vesper h, et al. ackee fruit poisoning: an outbreak investigation in haiti 2000-2001, and review of the literature. clin toxicol (phil). 2006;44:267–273. 35. moya j. ackee (blighia sapida) poisoning in the northern province, haiti, 2001. epidemiol bull. 2001(22):8–9. 36. katibi os, olaosebikan r, abdulkadir mb, ogunkunle to, ibraheem rm, murtala r. ackee fruit poisoning in eight siblings: implications for public health awareness. am j trop med hyg. 2015;93:1122–1123. 37. gaillard y, carlier j, berscht m, et al. fatal intoxication due to ackee (blighia sapida) in suriname and french guyana. gc-ms detection and quantification of hypoglycin-a. forensic sci int. 2011;206:e103–e107. 38. aquaisua an, bassey rb, ikpeme bm, et al. effect of crude extracts of blighia unijugata on histology of the liver and kidney of adult wistar rats. int res j pharm pharmacol. 2011;1:17–22. http://www.interesjournals.org/irjpp/april-2011-vol1-i s s u e -2 /e ff e c tofc r u d e e x t r a c t sof-bl i g h i a-u n ij u g at aon-h i s tol o g y of-the-liver-and-kidney-of-adult-wistar-rats. accessed may 8, 2017. http://www.interesjournals.org/irjpp/april-2011-vol-1-issue-2/effect-of-crude-extracts-of-blighia-unijugata-on-histology-of-the-liver-and-kidney-of-adult-wistar-rats http://www.interesjournals.org/irjpp/april-2011-vol-1-issue-2/effect-of-crude-extracts-of-blighia-unijugata-on-histology-of-the-liver-and-kidney-of-adult-wistar-rats http://www.interesjournals.org/irjpp/april-2011-vol-1-issue-2/effect-of-crude-extracts-of-blighia-unijugata-on-histology-of-the-liver-and-kidney-of-adult-wistar-rats drug target insights 2013:7 1–8 doi: 10.4137/dti.s10837 this article is available from http://www.la-press.com. © the author(s), publisher and licensee libertas academica ltd. this is an open access article. unrestricted non-commercial use is permitted provided the original work is properly cited. open access full open access to this and thousands of other papers at http://www.la-press.com. drug target insights r a p i d c o m m u n i c a t i o n drug target insights 2013:7 1 antibodies against gonadotropin-releasing hormone in patients with posterior laryngitis hillevi pendleton1, ragnar alm2, gunilla nordin fredrikson2,3 and bodil ohlsson4 1department of clinical sciences, division of otorhinolaryngology, skåne university hospital, malmö, lund university, lund, sweden. 2department of clinical sciences, experimental cardiovascular research unit, skåne university hospital, malmö, lund university. 3faculty of health and society, malmö university, sweden. 4department of clinical sciences, division of gastroenterology, skåne university hospital, malmö, lund university, lund, sweden. corresponding author email: bodil.ohlsson@med.lu.se abstract: patients with functional gastrointestinal disorders express antibodies against gonadotropin-releasing hormone (gnrh) in serum. one common cause of posterior laryngitis (pl) is extra-esophageal reflux, but a functional etiology has also been suggested. the aim of this study was to scrutinize patients with pl with regard to the presence of gnrh antibodies and to examine the association between antibodies and symptoms and reflux. consecutive pl patients were included after examination. serum was analyzed for the presence of antibodies using an enzyme-linked immunosorbent assay (elisa) method and expressed as relative units (ru). two ageand gender-matched healthy subjects per case served as controls. the prevalence of igm gnrh antibodies in patients was 35% compared with 28% in controls (p = 0.06), with higher levels in patients (0.8 (0.3–2.2) ru) than in controls (0.2 (0.1–0.6) ru) (p = 0.007). the corresponding igg antibody prevalences were 43% and 4%, respectively (p = 0.001), with no difference in levels (p = 0.70). there was no association between antibodies and clinical findings. keywords: gonadotropin-releasing hormone, posterior laryngitis, functional disorders, acid reflux http://dx.doi.org/10.4137/dti.s10837 http://www.la-press.com http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 http://www.la-press.com mailto:bodil.ohlsson@med.lu.se pendleton et al 2 drug target insights 2013:7 introduction reflux of stomach contents into the esophagus is the most common cause of severe symptoms from the upper gastrointestinal tract. although the exact mechanism is not known, reflux is associated with esophageal dysmotility in 40% to 50% of cases and reduced pressure of the lower esophageal sphincter (les).1–3 reflux leads to heartburn, acid regurgitation, and esophagitis,2,3 but reflux may also lead to more proximal signs such as posterior laryngitis (pl). pl is characterized by an inflammatory response of the posterior part of the glottic region causing symptoms such as chronic cough, hoarseness, globus, excessive throat clearing, voice fatigue, and throat pain.4 posterior laryngitis has been assumed to often depend on acid reflux, why proton pump inhibitors (ppi) is the choice of treatment.5,6 however, pl may be caused also by other mechanisms, and similar findings of the glottic region as in pl are described in healthy individuals without the typical symptoms of pl.7,8 gonadotropin-releasing hormone (gnrh) is best known to bind to specific receptors on the pituitary, controlling the secretion of the sex hormones.9,10 its presence and function as a neurotransmitter in the enteric nervous system (ens) has recently been described.11 antibodies against gnrh have been observed in patients with primary functional bowel disorders such as irritable bowel syndrome (ibs), in patients with functional bowel symptoms secondary to primary sjögren´s syndrome, as well as in patients affected by unclear dysmotility disorders. patients with organic gastrointestinal diseases such as inflammatory bowel disease, celiac disease, and scleroderma do not express antibodies.12,13 this raises the hypothesis that patients with different functional disorders, which often are associated and show an overrepresentation in women,14,15 may express gnrh antibodies as a common feature. we have recently shown how the majority of patients suffering from pl are women who are not improved by ppi treatment and express low health-related quality of life.16 this is in accordance with the hypothesis of a functional etiology of proximal gastrointestinal and extra-esophageal complaints.8,17 the presence of gnrh antibodies in patients suffering from pl has not been investigated. the aim of the present study was to scrutinize consecutive patients with pl for the prevalence of acid reflux and the presence of gnrh antibodies and to examine the association between antibodies and other measured parameters. materials and methods this study was performed according to the helsinki declaration and was approved by the regional ethics review board of lund university. all subjects gave written informed consent before entering the study. subjects and methods consecutive patients over 18 years of age from the division of otorhinolaryngology, skåne university hospital, malmö, sweden, were invited from june 1, 2007 through may 31, 2011, to participate in the study if the diagnosis pl was made by fiber laryngoscopy. the diagnosis criteria for pl and, thereby, the criteria for inclusion in the study were thickening and/or edema of the posterior part of the glottic region in combination with one or several of the following symptoms: globus, hoarseness, excessive throat clearing, excessive phlegm, acid regurgitation/reflux, heartburn, coughing, voice fatigue, breathing difficulties. or a feeling of cramp in the throat. these symptoms were chosen as they are the most frequently described symptoms in this group of patients.4 when seeing the ear, nose, and throat specialist, the patients were asked about the symptoms mentioned above, and perceived symptoms were registered in their medical records. in addition, information such as age, duration of symptoms, drug treatments, concomitant diseases, and body mass index (bmi) were registered. all patients were referred for 24-hour, single probe, ph monitoring in the proximal part of the esophagus and esophagogastroduodenoscopy (egd) to examine the upper gastrointestinal tract. blood samples for the analysis of anti-gnrh antibodies were collected at the time of the egd. exclusion criteria were pregnancy, mental illness, and serious illness such as severe heart, lung, liver, and kidney disease. patients with hepatitis b, hepatitis c, or hiv/aids were not included. thirty-seven of the 60 patients invited agreed to give blood samples for the analysis of anti-gnrh antibodies and were included in the study. five of these 37 patients could not tolerate the catheter used for 24-hour ph monitoring. all patients included tolerated the egd. http://www.la-press.com gnrh antibodies and posterior laryngitis drug target insights 2013:7 3 ambulatory 24-hour ph monitoring all participants were instructed to discontinue proton pump inhibiting therapy seven days before monitoring and to discontinue other acid inhibitors 16 hours before monitoring. participants were asked to avoid acidic beverages such as juice during the 24-hour period of ph monitoring and to fast 4 hours before the catheter was introduced. the positioning of the catheter was done in the diagnostic center of imaging and functional medicine with the aid of fluoroscopy (philips multidiagnost eleva, ca, usa). the catheter was introduced through the nose and under fluoroscopic control positioned in the proximal part of the esophagus, 5 cm below the upper esophageal sphincter. esophageal ph monitoring was performed using an antimony ph electrode with an internal reference electrode (versaflex, sierra scientific instruments, los angeles, ca, usa). before each study, the ph-probe was calibrated in buffer solutions of ph 7 and ph 1. an episode of acid reflux was defined as a decrease in esophageal ph below 4 for more than 10 seconds. previously established upper limits of normal acid exposure in clinical studies, with ph , 4 for 1% of total time, were used in the analysis of the data.18,19 the data were stored on a portable digital recorder (digitrapper ph400, synectics medical, stockholm, sweden). data were analyzed with the aid of commercially available software (polygram net, synmed medical, stockholm, sweden). measurement of human antibodies against gonadotropin-releasing hormone blood samples were drawn from patients and the serum was separated and kept frozen at -20 °c until analyzed. analysis of anti-gnrh antibodies was carried out by an enzyme-linked immunosorbent assay (elisa) method slightly modified on the basis of the results described in previous studies.11,12 the wells of microtiter plates were coated with human gnrh (l7134, sigma, st louis, mo, usa) for an overnight incubation at 4 °c, and, thereafter, the plastic wells were blocked with 0.5% fish gel solution (g7765, sigma, st louis, mo, usa) in phosphate buffered saline (pbs) containing 0.05% tween-20 (pbs-t). serial dilutions of patient serum (1/100, 1/500 and 1/2500 in pbs-t) were then added to the plates and incubated for 2 hours at room temperature and overnight at 4 °c. after rinsing with pbs-t, deposition of autoantibodies directed to gnrh was detected using biotinylated rabbit antihuman igm (673211, mp biomedicals, solon, oh, usa) or igg antibodies (ab7159, abcam, cambridge, ma, usa) appropriately diluted in pbs-t. after another incubation for 2 hours at room temperature, the plates were washed, and the bound, biotinylated antibodies detected by alkaline phosphatase-conjugated streptavidin (405211, biolegend, san diego, ca, usa) and incubated for 1 hour at room temperature. to develop a color reaction, a phosphatase substrate kit (37620, pierce, rockford, ill, usa) was used. the absorbance at 405 nm was measured after 2 hours of incubation at room temperature. a plasma pool from healthy blood donors was included on each elisa-plate for measurements of the variation. the plasma pool was used for the calculation of the intraassay and interassay coefficient of variations, which were 11.5% and 16.1%, respectively, for igm and 11.5% and 25.4%, respectively, for igg. antibody levels are presented as relative units (ru) (absorbance values after subtraction of background levels and multiplied by 100). relative units over 0 were considered as a positive antibody level.13 the controls were chosen from a cohort of healthy blood donors previously described in detail.13 over a period of five months (october 1996–february 1997), blood donors were offered antibody screening for gastrointestinal diseases. to be able to include all blood donors in malmö, sera from male donors were collected over a 3-month period and from female donors over a 4-month period (in accordance with their regular donation intervals). a total of 1970 donors were included. during this period, 2135 blood donations took place, which means that at least 92% of donors agreed to be included. from this sample cohort, 50 men and 50 women from each 10-year age span period, between 20 and 70 years, were randomly included. as few blood donors are over the age of 60 years, only 16 women and 40 men were included in the age group 60 to 70 years. in total, 456 controls were examined during the same time period as the patients. from this cohort, two ageand gender-matched controls were randomly extracted for each patient in this study. statistical analyses the data were analyzed using the statistical software package spss for windows, release 19.0 http://www.la-press.com pendleton et al 4 drug target insights 2013:7 (ibm corporation, armonk, ny, usa). values are expressed as median and interquartile range (iqr). group-wise differences were tested by using the mann whitney u test or fisher exact test. correlations were calculated by the spearman test. results where p , 0.05 were considered statistically significant. results patient characteristics thirty-seven patients with verified pl (20 women) with a mean age of 56 (range 40 to 69) years were included (table 1). esophagogastroduodenoscopy found no ulcerations or tumors, but 7 patients suffered from esophagitis, 10, from barrett´s esophagus, 13, from hiatal hernia, and 12 (of 32 examined), from proximal esophageal reflux. the most common symptoms present were globus (65%), excessive phlegm (46%), and hoarseness (32%) (table 2). apart from symptoms associated with pl, 7 patients also described dysphagia. antibodies against gonadotropinreleasing hormone the prevalence of igm antibodies against gnrh in patients was 35% compared with 28% in controls (p = 0.06), and the antibody level was significantly higher in the patients (p = 0.007) (fig. 1a and table 3). the prevalence of igg was 43% in patients and 4% in the controls (p = 0.001), with no difference in the level of antibodies between the patients and controls (p = 0.70) (fig. 1b and table 3). there was no association between the expression of igm and igg antibodies (p = 0.79), but one or both of these antibodies were found in 24 of 37 (65%) patients compared with 24 of 74 (32%) controls (p = 0.002). neither was there any association between the presence of antibodies and symptoms, duration of symptoms, esophageal diseases or bmi (data not shown). the level of antibody titer did not differ between those who had symptoms and those who did not. there was no correlation between number of symptoms and presence or levels of antibodies (data not shown). discussion the present study showed that patients with pl had few organic findings on 24-hours ph monitoring and egd examination. thirty-eight percent had pathological proximal acid reflux, and 46% had signs of distal acid reflux. the majority, 65%, expressed antibodies against gnrh in serum compared with 32% in controls. gonadotropin-releasing hormone is secreted by the hypothalamus, and its most important effect is on the pituitary, stimulating gonadotropin synthesis and secretion. it is a crucial neuropeptide in reproductive physiology and sexual behaviour.9 peripherally, gnrh and gnrh receptors have been found in the rat myenteric plexus and the intestinal epithelium.20,21 we have recently described the expression of gnrh in human myenteric neurons.11 the effect on the ens is not completely evaluated, but gnrh has been shown to inhibit the release of gastric secretion and gastrin release in dogs,22 to stimulate motor function in the gastrointestinal tract in female rats,23 and to restore motor function in a patient suffering from chronic intestinal pseudo-obstruction.24 although it has now been described in several studies that patients with ibs and dysmotility express gnrh antibodies,11–13 the effects of gnrh table 1. characteristics of patients with posterior laryngitis. patients (n = 37) age (years) 56.0 (40.5–69.0) gender (female/male) (n) 20/17 duration of the disease (months) 6.0 (2.0–15.0) bmi (kg/m2)* 25.8 (21.6–28.1) proximal reflux (n, %) 12 (38) notes: *missing values for 13 patients. values are given as median (interquartile ranges). abbreviation: bmi, body mass index. table 2. the prevalence of various symptoms in patients with posterior laryngitis (n = 37). symptomsa number and percentage of patients globus 24 (65) hoarseness 12 (32) excessive throat clearing 9 (24) excessive phlegm 17 (46) acid regurgitation/reflux 7 (19) heartburn 8 (21) coughing 10 (28) voice fatigue 11 (30) breathing difficulties 1 (3) feeling of cramp in the throat 0 (0) note: amore than one symptom for each patient was registered. http://www.la-press.com gnrh antibodies and posterior laryngitis drug target insights 2013:7 5 0.10 0.20 0.30 0.50 0.60 0.80 0.90 1.20 1.30 1.90 2.00 2.70 12.90 29.30 igm levels (ru) group 1.00 2.00 figure 1 the level of igm and igg antibodies expressed as relative units (ru). (a) group 1 = patients (13 of 37), group 2 = controls (21 of 74). (b) group 1 = patients (16 of 37), group 2 = controls (4 of 74). 0.10 0.20 0.50 0.60 0.70 0.900.80 1.10 1.60 1.80 2.601.00 igg levels (ru) group 1.00 2.00 and/or its antibodies on the normal physiology of the gastrointestinal tract, as well as on pathological processes and symptom development, remain to be determined. we do not know whether there is a difference between the expression of igm and igg antibodies in these patients. some chronic inflammatory diseases present themselves with igm antibodies instead of igg antibodies. probably, the expression of igm antibodies is long-standing in this entity, as the patients had been sick for several years before inclusion in the study. many factors, such as rising obesity rates, greater consumption of medications affecting esophageal function, and potentially changing prevalence rates of helicobacter pylori infection, have been discussed as the etiology of reflux.25 although pl is considered to http://www.la-press.com pendleton et al 6 drug target insights 2013:7 depend on acid reflux,4 only 12 of 32 (38%) patients had a pathological 24-hour ph monitoring as proof of proximal acid reflux, and 17 of 37 (46%) showed signs of distal acid reflux. furthermore, we have recently shown that 63% of patients with pl still have symptoms after ppi treatment,16 showing the need to consider other etiologies than acid reflux as a cause of pl. factors such as pepsin, bile, infections, allergy, and smoking have been evaluated and discussed.7,8 nobody has examined whether signs of functional disorders are present in this patient group, despite that the disease is characterized by female predominance as in other functional disorders.4,14,15 the hypothesis that the symptoms of pl might partly be functional has been raised.8,17 this led us to perform the present study. accordingly, 65% of patients with pl expressed either igm or igg antibodies in serum and had higher antibody levels than controls, as found in functional gastrointestinal disorders previously.12,13 no association between signs of reflux at egd or ph monitoring and antibodies was found. neither was there any association between symptoms and antibody level. it is a well-known phenomenon that there is a weak or absent correlation between objective signs and subjective symptoms from the gastrointestinal tract. the reason is unclear, but may depend on different central processing of visceral, afferent information in healthy controls and patients suffering from functional bowel diseases.26 psychological factors have a great impact in the pathophysiology of visceral hyperalgesi.27,28 the presence of antibodies observed in this study strengthens the hypothesis that pl may be a functional disorder in a subgroup of patients presenting themselves with laryngitic inflammation and symptoms, as gnrh antibodies are described in patients with functional disorders without any association between antibodies and symptoms and signs.11–13 gonadotropin-releasing hormone and its receptors are present both centrally and peripherally, and deregulation of this peptide and/or its receptor may be involved in the pathophysiology of functional disorders. the controls used were healthy blood donors. however, functional disorders are not exclusion criteria for blood donors and are very common in the general population,14,15 and the controls were not asked about symptoms. thus, among the controls may be hidden some persons with functional disorders as well, explaining some of the antibodies in this group. if only persons who had denied all types of functional disorders had been used as controls, the differences between controls and patients might have been greater. one of the limitations of this study is the small sample size. however, it is necessary to perform small pilot studies before testing our hypothesis in larger cohorts of hundreds of patients. in future studies, it seems that it would be more relevant to focus on functional etiologies to pl and not only focus on acid reflux, which seems to be a lesser problem among these patients. another important task is to elucidate whether the presence of antibodies is a primary or a secondary phenomenon, which may have an impact on associations. the test method also needs to be further evaluated in relation to the optimal cutoff level. in our elisa, we considered all relative antibody units above 0 as positive expression. if a higher baseline level of antibodies present in serum had been regarded as positive expression, more controls than patients would have been below this level. when more patients have been studied, the cutoff level for positive expression should be redefined. conclusions patients with pl express igg antibodies against gnrh in serum in higher prevalence, whereas the level of igm antibodies is increased compared to controls. table 3. prevalence and levels of antibodies against gonadotropin-releasing hormone in patients and controls. igg n (%) p value igg (ru) p value igm n (%) p value igm (ru) p value posterior laryngitis (37) 16 (43) 0.001 0.7 (0.2–1.0) 0.697 13 (35) 0.062 0.8 (0.3–2.2) 0.007 matched controls (74) 3 (4) 0.6 (0.6–) 21 (28) 0.2 (0.1–0.6) notes: antibody levels are presented as relative units (ru), median (interquartile range). (n) = number of subjects. mann whitney u test. p , 0.05 is considered statistical significance. http://www.la-press.com gnrh antibodies and posterior laryngitis drug target insights 2013:7 7 only one-third of the patients had objective signs of proximal acid reflux in the esophagus. no associations between antibody expression and other parameters were found. a subgroup of patients with pl may suffer from functional disease, when no other etiology is found. acknowledgements we thank ola thorsson, department of clinical sciences, nuclear medicine, diagnostic centre of imaging and functional medicine, skåne university hospital, malmö, klas sjöberg who collected samples from healthy blood donors, and peter höglund, region skånes kompetens centrum (rskc) for assistance with the statistical calculations. funding this study was sponsored by grants from the crafoord and bengt ihre foundations and from the development foundation of region skåne. competing interests author(s) disclose no potential conflicts of interest. author contributions all authors participated in the design of the study. collected the blood samples and data from the division of otorhinolaryngology: hp. performed the elisa analyses: ra and gnf. contributed to the statistical analyses and wrote the manuscript: hp and bo. supported the study financially (crafoordand bengt ihre foundations, and development foundations of region skane): bo. all authors contributed to the manuscript with constructive criticism and read and approved the final manuscript. disclosures and ethics as a requirement of publication author(s) have provided to the publisher signed confirmation of compliance with legal and ethical obligations including but not limited to the following: authorship and contributorship, conflicts of interest, privacy and confidentiality and (where applicable) protection of human and animal research subjects. the authors have read and confirmed their agreement with the icmje authorship and conflict of interest criteria. the authors have also confirmed that this article is unique and not under consideration or published in any other publication, and that they have permission from rights holders to reproduce any copyrighted material. any disclosures are made in this section. the external blind peer reviewers report no conflicts of interest. references 1. kahrilas pj. anatomy and physiology of the gastroesophageal junction. gastroenterol clin north am. 1997;26:467–86. 2. diener u, patti mg, molena d, fisichella pm, way lw. esophageal dysmotility and gastroesophageal reflux disease. j gastrointest surg. 2001;5:260–5. 3. ho s-c, chang c-s, wu c-y, chen g-h. ineffective esophageal motility is a primary motility disorder in gastroesophageal reflux disease. dig dis sci. 2002;47:652–6. 4. koufman ja. the otolaryngologic manifestations of gastroesophageal reflux disease (gerd): a clinical investigation of 225 patients using ambulatory 24-hour ph monitoring and an experimental investigation of the role of acid and pepsin in the development of laryngeal injury. laryngoscope. 1991;101:1–78. 5. vaezi mf, richter je, stasney cr, et al. treatment of chronic posterior laryngitis with esomeprazole. laryngoscope. 2006;116:254–60. 6. toros sz, toros ab, yuksel od, ozel l, akkaynak c, naiboglu b. association of laryngopharyngeal manifestations and gastroesophageal reflux. eur arch otorhinolaryngol. 2009;266:403–9. 7. pearson jp, parikh s, orlando rc, et al. review article: reflux and its consequences—the laryngeal, pulmonary and oesophageal manifestations. conference held in conjunction with the 9th international symposium on human pepsin (ishp) kingston-upon-hull, uk, apr 21–23, 2010. aliment pharmacol ther. 2011;33(suppl 1):1–71. 8. kotby mn, hassan o, el-makhzangy am, farahat m, milad p. gastroesophageal reflux/laryngopharyngeal reflux disease: a critical analysis of the literature. eur arch otorhinolaryngol. 2010;267:171–9. 9. maeda k, ohkura s, uenoyama y, et al. neurobiological mechanisms underlying gnrh pulse generation by the hypothalamus. brain res. 2010; 1364:103–15. 10. hazum e, conn pm. molecular mechanism of gonadotropin releasing hormone (gnrh) action, i. the gnrh receptor. endocr rev. 1988;9: 379–86. 11. ohlsson b, veress b, ekblad e, montgomery a, janciauskiene s. antibodies against gonadotropin-releasing hormone (gnrh) and destruction of enteric neurons in 3 patients suffering from gastrointestinal dysfunction. bmc gastroenterol. 2010;10:48. 12. ohlsson b, scheja a, janciauskiene s, mandl t. functional bowel symptoms and gnrh antibodies: common findings in patients with primary sjögren’s syndrome but not in systemic sclerosis. scand j rheumatol. 2009; 23:1–2. 13. ohlsson b, sjöberg k, alm r, nordin fredrikson g. patients with irritable bowel syndrome and dysmotility express antibodies against gonadotropinreleasing hormone in serum. neurogastroenterol motil. 2011;23:1000–6. 14. simrén m, abrahamsson h, svedlund j, björnsson es. quality of life in patients with irritable bowel syndrome seen in referral centers versus primary care: the impact of gender and predominant bowel pattern. scand j gastroenterol. 2001;36:545–52. 15. north cs, downs d, clouse re, et al. the presentation of irritable bowel syndrome in the context of somatization disorder. clin gastroenterol hepatol. 2004;2:787–95. 16. pendleton h, ahlner-elmqvist m, jannert m, ohlsson b. posterior laryngitis: a study of persisting symptoms and health-related quality of life. eur arch otorhinolaryngol. 2012. [epub ahead of print.] 17. kahrilas pj, hughes n, howden cw. response of unexplained chest pain to proton pump inhibitor treatment in patients with and without objective evidence of gastro-oesophageal reflux disease. gut. 2011;60: 1473–8. http://www.la-press.com pendleton et al 8 drug target insights 2013:7 18. dobhan r, castell do. normal and abnormal proximal esophageal acid exposure: results of ambulatory dual-probe ph monitoring. am j gastroenterol. 1993;88:25–9. 19. postma gn. ambulatory ph monitoring methodology. ann otol rhinol laryngol suppl. 2000;184:10–4. 20. ho js, nagle gt, mathias jr, et al. presence of gonadotropin-releasing hormone (gnrh) receptor mrna in rat myenteric plexus cells. comp biochem physiol b biochem mol biol. 1996;113:817–21. 21. huang w, yao b, sun l, pu r, wang l, zhang r. immunohistochemical and in situ hybridization studies of gonadotropin releasing hormone (gnrh) and its receptor in rat digestive tract. life sci. 2001;68:1727–34. 22. soldani g, del tacca mm, bambini g, et al. effects of gonadotropin releasing hormone (gnrh) on gastric secretion and gastrin release in the dog. j endocrinol invest. 1982;5:393–6. 23. khanna r, browne rm, heiner ad, clench mh, mathias jr. leuprolide acetate affects intestinal motility in female rats before and after ovariectomy. am j physiol. 1992;262(1 pt 1):g185–90. 24. mathias jr, baskin gs, reeves-darby vg, clench mh, smith ll, calhoon jh. chronic intestinal pseudoobstruction in a patient with heartlung transplant. therapeutic effect of leuprolide acetate. dig dis sci. 1992;37: 1761–8. 25. pandolfino je, kwiatek ma, kahrilas pj. the pathophysiologic basis for epidemiologic trends in gastroesophageal reflux disease. gastroenterol clin north am. 2008;37:827–43. 26. ringel y, drossman da, leserman jl, et al. effect of abuse history on pain reports and brain responses to aversive visceral stimulation: an fmri study. gastroenterology. 2008;134:396–404. 27. gregory lj, yágüez l, williams sc, et al. cognitive modulation of cerebral processing of human oesophageal sensation using functional magnetic resonance imaging. gut. 2003;52:1671–7. 28. elsenbruch s, rosenberger c, enck p, forsting m, schedlowski m, gizewski er. affective disturbances modulate the neural processing of visceral pain stimuli in irritable bowel syndrome: an fmri study. gut. 2010;59:489–95. http://www.la-press.com drug target insights 2012:6 1–11 doi: 10.4137/dti.s9442 this article is available from http://www.la-press.com. © the author(s), publisher and licensee libertas academica ltd. this is an open access article. unrestricted non-commercial use is permitted provided the original work is properly cited. open access full open access to this and thousands of other papers at http://www.la-press.com. drug target insights o r i g i n a l r e s e a r c h drug target insights 2012:6 1 resveratrol targeting of carcinogen-induced brain endothelial cell inflammation biomarkers mmp-9 and cox-2 is sirt1-independent borhane annabi1, simon lord-dufour2, amélie vézina1 and richard béliveau2 1laboratoire d’oncologie moléculaire, 2laboratoire de médecine moléculaire, centre de recherche biomed, université du québec à montréal, quebec, canada. corresponding author email: annabi.borhane@uqam.ca abstract: the occurrence of a functional relationship between the release of metalloproteinases (mmps) and the expression of cyclooxygenase (cox)-2, two inducible pro-inflammatory biomarkers with important pro-angiogenic effects, has recently been inferred. while brain endothelial cells play an essential role as structural and functional components of the blood-brain barrier (bbb), increased bbb breakdown is thought to be linked to neuroinflammation. chemopreventive mechanisms targeting both mmps and cox-2 however remain poorly investigated. in this study, we evaluated the pharmacological targeting of sirt1 by the diet-derived and antiinflammatory polyphenol resveratrol. total rna, cell lysates, and conditioned culture media from human brain microvascular endothelial cells (hbmec) were analyzed using qrt-pcr, immunoblotting, and zymography respectively. tissue scan microarray analysis of grade i–iv brain tumours cdna revealed increased gene expression of sirt-1 from grade i–iii but surprisingly not in grade iv brain tumours. hbmec were treated with a combination of resveratrol and phorbol 12-myristate 13-acetate (pma), a carcinogen known to increase mmp-9 and cox-2 through nf-κb. we found that resveratrol efficiently reversed the pma-induced mmp-9 secretion and cox-2 expression. gene silencing of sirt1, a critical modulator of angiogenesis and putative target of resveratrol, did not lead to significant reversal of mmp-9 and cox-2 inhibition. decreased resveratrol inhibitory potential of carcinogen-induced iκb phosphorylation in sisirt1-transfected hbmec was however observed. our results suggest that resveratrol may prevent bbb disruption during neuroinflammation by inhibiting mmp-9 and cox-2 and act as a pharmacological nf-κb signal transduction inhibitor independent of sirt1. keywords: angiogenesis, inflammation, resveratrol, brain endothelial cells, mmp-9, cox-2, sirt1 http://dx.doi.org/10.4137/dti.s9442 http://www.la-press.com http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 http://www.la-press.com mailto:annabi.borhane@uqam.ca annabi et al 2 drug target insights 2012:6 introduction tumour-associated angiogenesis, a fundamental process in tumour growth and metastasis, consists of recruiting ec toward an angiogenic stimulus.1 the cells subsequently proliferate and differentiate to form endothelial tubes and capillary-like structures in order to deliver nutrients and oxygen to the tumour and to remove the products of its metabolism. in recent years, several pathways have, in addition to stimulation of tumor angiogenesis, been suggested to contribute to the cell metabolic adaptations required for carcinogenesis, which include decreased tumoural apoptosis, increased invasion and metastasis, immune suppression and tumourassociated inflammation.2,3 an interesting link between overexpression of pro-inflammatory markers such as matrix metalloproteinase-9 (mmp-9) and cyclooxygenase (cox)-2, and tumour angiogenesis was recently described as one such metabolic adaptative phenotype.4–6 while flavonoids and related polyphenolic compounds, such as resveratrol, have demonstrated significant antiinflammatory activity, predominantly, through the inhibition of nf-κb signaling,7 their potential therapeutic application upon the cerebral vascular compartment in neuroinflammation still remains poorly documented. while human brain microvascular endothelial cells (hbmec) play an essential role as structural and functional components of the blood-brain barrier (bbb), its disruption by mmp-9 is believed to favor tumor invasion.8,9 recent studies in fact delineated a unique brain endothelial phenotype in which mmp-9 secretion by hbmec was increased upon treatment with the tumor-promoting agent phorbol 12-myristate 13-acetate (pma).10,11 inhibition of mmp-9 secretion was demonstrated to reduce both in vitro invasion and angiogenesis in human microvascular ec.12 among the signalling pathways involved, nf-κb signalling was the one that enabled the joint control of both mmp-9 and cox-2 inflammation marker expression.13,14 recent studies point to sirt1, the most widely investigated and best known sirtuin, as a key regulator of vascular endothelial homeostasis controlling angiogenesis, vascular tone and endothelial dysfunction.15 sirtuins are a family of conserved proteins with deacetylase and adp-ribosyltransferase activity encoded in humans by seven genes (sirt1-7).16 in cancer cells, conflicting reports regarding the expression of sirt1 show either up-regulation17,18 or downregulation19 of sirt1. sirt1 may thus play a critical role in tumor progression, and drug resistance. while sirt1 inhibitors have shown promising anticancer effects in animal models of cancer, its implication in a brain ec model has yet to be documented. more importantly, the expression of sirt1 in the brain ec compartment as well as impact of its pharmacological targeting is still unknown. given that sirt1-independent mechanisms were recently reported in the action of resveratrol,20 this current study therefore focuses on resveratrol as a potential signal transduction inhibitor of carcinogen-mediated induction of the nf-κb pathway in a human brain ec model and questions the involvement of sirt1. materials and methods reagents and cell culture bovine serum albumin (bsa) was purchased from myclone laboratories (logan, ut). electrophoresis reagents were purchased from bio-rad (mississauga, on). the enhanced chemiluminescence (ecl) reagents were from perkin elmer (waltham, ma). micro bicinchoninic acid protein assay reagents were from pierce (rockford, il). the polyclonal antibodies against iκb and phospho-iκb were purchased from cell signaling (danvers, ma). the monoclonal antibody against gapdh was from advanced immunochemical inc. (long beach, ca). horseradish peroxidase-conjugated donkey anti-rabbit and anti-mouse igg secondary antibodies were from jackson immunoresearch laboratories (west grove, pa). sodium dodecylsulfate (sds) and all other reagents were from sigma-aldrich canada. human brain microvascular endothelial cells (hbmec) were characterized and generously provided by dr. kwang sik kim of the johns hopkins university school of medicine (baltimore, md).21,22 cells were cultured at 37 °c under a humidified atmosphere containing 5% co2. all experiments were performed using passages 3 to 28. gelatin zymography and immunoblotting procedures proteins from control and treated cells were separated by sds-page, and electrotransferred to polyvinylidene difluoride membranes as previously described.21 gelatin zymography was used to assess the extent of http://www.la-press.com sirt1 expression in inflammatory cerebral endothelium drug target insights 2012:6 3 prommp-9 activity as previously described.23 briefly, an aliquot (20 µl) of the culture medium was subjected to sds-page in a gel containing 0.1 mg/ml gelatin. the gels were then incubated in 2.5% triton x-100 and rinsed in nanopure distilled h2o. gels were further incubated at 37 °c for 20 hrs in 20 mm nacl, 5 mm cacl2, 0.02% brij-35, 50 mm tris-hcl buffer, ph 7.6, then stained with 0.1% coomassie brilliant blue r-250 and destained in 10% acetic acid, 30% methanol in h2o. gelatinolytic activity was detected as unstained bands on a blue background. total rna isolation, cdna synthesis and real-time quantitative rt-pcr total rna was extracted from cell monolayers using trizol reagent (life technologies, gaithersburg, md). for cdna synthesis, 2 µg of total rna were reversetranscribed using a high capacity cdna reverse transcription kit (applied biosystems, foster city, ca). cdna was stored at -80 °c prior to pcr. gene expression was quantified by real-time quantitative pcr using iq sybr green supermix (bio-rad, hercules, ca). dna amplification was carried out using an icycler iq5 (bio-rad, hercules, ca) and product detection was performed by measuring binding of the fluorescent dye sybr green i to double-stranded dna. the quantitect primer sets were provided by qiagen (valencia, ca): mmp-9 (qt00040040), cox-2 (qt00040586), β-actin (qt01136772). gapdh primer sets were synthesized by biocorp (dollard-des-ormeaux, qc) with the following sequences: forward ccatcaccatcttccaggag and reverse cctgcttcaccaccttcttg. the relative quantities of target gene mrna compared against two internal controls, gapdh and β-actin mrna, were measured by following a ∆ct method employing an amplification plot (fluorescence signal vs. cycle number). the difference (∆ct) between the mean values in the triplicate samples of target gene and those of gapdh and β-actin mrnas were calculated by iq5 optical system software version 2.0 (biorad, hercules, ca) and the relative quantified value (rqv) was expressed as 2-∆ct. tissuescan cdna arrays of grade i-iv brain tumour tissues tissuescan™ cancer and normal tissue cdna arrays were purchased from origene (rockville, md), covered 43 clinical samples of the brain cancer four stages and normal tissues, and were used to analyze differential sirt1 gene expression according to the manufacturer’s recommendation. tissue cdnas of each array are synthesized from high quality total rnas of pathologist-verified tissues, normalized and validated with β-actin in two sequential qpcr analyses, and provided with clinical information for 2 normal brain, 18 who grade i, 11 who grade ii, 10 who grade iii, and 2 who grade iv brain tumours. transfection method and rna interference cells were transiently transfected with 20 nm sirna (qiagen) against sirt1 (hs_sirt1_1 flexitube sirna, si00098434) or scrambled sequences (allstar negative control sirna, 1027281) using lipofectamine 2000 (invitrogen, on). specific gene knockdown was evaluated by qrt-pcr as described above. small interfering rna and mismatch sirna were synthesized by qiagen (valencia, ca) and annealed to form duplexes. statistical data analysis data statistical significance (p values ,0.05) was assessed using student’s unpaired t-test from three or more independent experiments. results sirt1 gene expression profiling in grade i-iv brain tumour tissues fourthy-eight clinical tissue samples were first analyzed for sirt1 gene expression profil using tissuescan™ cancer and normal tissue cdna arrays (origene, rockville, md) from 43 clinical samples covering brain cancer four stages and normal tissues as described in the methods section. we found that sirt1 gene expression levels increased from grade i to grade iii brain tumour tissues (fig. 1, black bars), but not in grade iv as compared to normal brain tissue (fig. 1, white bars). this suggests that discrepancy found in the literature regarding sirt1 expression in tissue samples may be explained by the invasive stage status of a given tumour. given that tumour samples are composed of a heterogeneous cell composition including http://www.la-press.com annabi et al 4 drug target insights 2012:6 cancer, inflammatory, as well as endothelial cells, we decided to focus our study on the sirt1 contribution within a carcinogen-treated brainassociated endothelial cell compartment. resveratrol dose-dependent inhibition of carcinogen-induced cox-2 gene and protein expression various molecular mechanisms mediate inflammatory processes and angiogenesis, one of which is reflected by increased expression of the inflammatory biomarker cox-2.7 in order to investigate the effect of resveratrol on hbmec-associated inflammation, we tested the effects of resveratrol on pma-induced cell signaling in hbmec by western blotting. cells were therefore treated with 1 µm of pma in the presence of increasing concentrations of resveratrol for 18h and cox-2 expression was evaluated in cell lysates by western blotting (fig. 2a). while 100 µm resveratrol was without effect on cox-2 basal expression, it significantly inhibited pma-induced cox-2 protein expression (ic50 6.1 ± 2.2 µm, fig. 2b) and gene (fig. 2c) expression. resveratrol inhibition of carcinogeninduced mmp-9 gene expression and protein secretion among the secreted enzymes involved in ecm degradation, matrix metalloproteinases (mmp) are well-documented as being involved in ec migration and tubulogenesis.10,24 mmp-9, an enzyme involved in the degradation of the extracellular matrix (ecm), is secreted by a variety of cells and its presence was shown to be increased upon carcinogen promoting agents such as the phorbol ester pma.25–27 hbmec were first treated for 18 h with pma. gelatin zymography was then used to measure prommp-9 levels, which were significantly increased upon pma treatment in comparison to vehicle-treated cells (fig. 3a). addition of increasing concentrations of resveratrol to pmatreated cells resulted in a dose-dependent inhibition of mmp-9 activity (ic50 9.1 ± 0.8 µm, fig. 3b and c). it was found that pma also increased mmp-9 gene expression while the presence of resveratrol inhibited this increase suggesting transcriptional regulation of the mmp-9 gene (fig. 3d). carcinogen-induced iκb phosphorylation is inhibited by resveratrol among mmp-9 expression regulators, the nuclear factor-kappab (nf-κb) signalling pathway has been demonstrated to link cancer to inflammatory diseases.28 we therefore assessed whether this signalling was activated upon pma treatment and whether it was reflected in ikappab (iκb) degradation. hbmec were serum-starved then treated with 1 µm pma for up to 30 minutes, lysates were isolated and iκb phosphorylation was assessed through western blotting (fig. 4a, upper panel). pma signalling led to the phosphorylation of iκb peaking at 15 minutes.29 resveratrol effect on pma-mediated phosphorylation of iκb was next assessed in order to demonstrate whether this mechanism contributes to the anti-cox-2 inhibitory activities of resveratrol. preincubation with 30 µm resveratrol followed by a 30 min pma treatment led to diminished iκb phosphorylation as demonstrated by the decreased ratios of phosphorylated iκb over gapdh expression (fig. 4b). 20 ∗ ∗ ∗ 10 15 s ir t1 g e n e e x p re s s io n (a rb . le v e ls ) 5 0 nb i who tumour grade ii iii iv figure 1. sirt1 gene expression profiling in grade i-iv brain tumour tissues. tissuescan™ cancer and normal tissue cdna arrays from 43 clinical samples covering brain cancer four stages and normal brain tissues were used to analyze differential sirt1 gene expression. tissue cdnas of each array are synthesized from high quality total rnas of pathologistverified tissues, normalized and validated with β-actin and provided with clinical information for 2 normal brain, 18 who grade i, 11 who grade ii, 10 who grade iii, and 2 who grade iv brain tumours. abbreviation: nb, normal brain tissue. http://www.la-press.com sirt1 expression in inflammatory cerebral endothelium drug target insights 2012:6 5 sirt1 contributes to the resveratrol inhibitory effect of pma-induced iκb phosphorylation sirt1 is, among the sirtuins family, documented to be targeted by resveratrol. in order to assess the contribution of sirt1 in the resveratrol inhibition of nf-κb, hbmec were next transiently transfected with a scrambled sirna sequence (mock) or an sirna designed to downregulate sirt1 (sisirt1) gene expression. preincubation with 30 µm resveratrol followed by a 30 min pma treatment was performed c b a vehicle pma (1 µm) resveratrol (µm) resveratrol vehicle pma resveratrol (µm) cox-2 ns gapdh 0 0 5 10 25 50 100100 100 80 60 40 20 0 0 20 40 60 80 ∗ 100 120 100 80 60 40 20 0 − + − + 120 c o x -2 g e n e e x p re s s io n (x -f o ld o v e r c tr l) c o x -2 /g a p d h ( % o f p m a ) figure 2. resveratrol dose-dependent inhibition of carcinogen-induced cox-2 protein and gene expression. (a) hbmec were serum-starved in the presence of various concentrations of resveratrol and in combination with vehicle or 1 µm pma for 18 hours. lysates were isolated, electrophoresed via sds-page, and immunodetection of cox-2 and gapdh performed as described in the methods section (ns, non specific immunoreactivity). (b) scanning densitometry of cox-2 expression was only performed in pma-treated cells since no cox-2 was detectable in vehicle-treated hbmec. densitometric data of a representative blot is shown out of three independent experiments. (c) total rna isolation, rt-pcr, and qpcr were performed as described in the methods section to assess cox-2 gene expression in the above-described conditions. (pma = 1 µm; resveratrol = 30 µm). notes: data are representative of three independent qpcr experiments. probability values of less than 0.05 were considered significant, and an asterisk (*) identifies such significance to the respective pma treatment. http://www.la-press.com annabi et al 6 drug target insights 2012:6 in mock and in sisirt1-transfected cells. lysates were isolated, and gapdh or iκb phosphorylation assessed through western blotting (fig. 5a, upper panel). while ∼90% inhibition in sirt1 gene expression was observed in sisirt1-transfected cells (fig. 5b), we found that resveratrol inhibitory potential was significantly diminished in those cells which had sirt1 expression silenced (fig. 5c, closed circles) in comparison to mock-transfected cells (fig. 5c, open circles). pma treatment by itself did not affect sirt1 gene expression (not shown). this suggests that sirt1 is indeed important in the inhibitory potential of resveratrol against pma-mediated nf-κb signaling pathway. sirt1-independent inhibition by resveratrol of pma-induced cox-2 expression and of pma-induced mmp-9 secretion whether sirt1 serves as an intermediate in the resveratrol inhibitory potential was next investigated on pma-treated cells for cox-2 and mmp-9 expression. as described above, serum-starved mock and sisirt1-transfected hbmec were treated with pma, resveratrol or a combination of both agents. while resveratrol efficiently inhibited both pma-induced cox-2 (fig. 6a) and mmp-9 (fig. 6b), silencing of sirt1 did not lead to any reversal of effect as resveratrol still efficiently inhibited cox-2 and mmp-9. this suggests that resveratrol inhibition of pma-induced cox-2 and mmp-9 is a sirt1 independent event. discussion the adaptive mechanisms responsible for ec metabolic adaptation and survival under procarcinogenic stimulation, or as encountered within a tumour microenvironment, still remain poorly documented. while interest has been manifested towards cancer therapies that jointly target ec angiogenic and inflammatory phenotypes, the design, synthesis and evaluation of flavonoid derivatives that target neurodegenerative disorders have accordingly paved the road to strategies leading to decreased extracellular matrix (ecm) degradation and inflammation processes.30 the objectives of this current study were first to trigger in vitro pro-carcinogenic stimulation of a brain microvascular ec model using pma in combination with resveratrol, and assess impact on inflammation biomarkers mmp-9 and cox-2 expression. secondly, given that resveratrol is a well documented pharmacological agonist of sirt1, pma mmp-9 mmp-9 0 10 20 2510500 a c d b 0.1 0.3 1 50 30 40 50 120 100 80 60 40 20 0 30 * 25 20 15 10 5 0 m m p -9 ( % o f p m a ) m m p -9 g e n e e x p re s s io n (x -f o ld o v e r c tr l) pma (µm) vehicle resveratrol (µm) resveratrol (µm) resveratrol− + − + figure 3. resveratrol inhibition of carcinogen-induced mmp-9 gene expression and protein secretion. hbmec were serum-starved in the presence of (a) various pma concentrations for 18 hours, or (b) a combination of 1 µm pma with increasing resveratrol concentrations. conditioned media were then harvested and gelatin zymography was performed in order to detect pma-induced prommp-9 and hydrolytic activity as described in the methods section. (c) scanning densitometry was used to quantify the extent of prommp-9 gelatinolytic activity in the combined pma and resveratrol treatments. data shown is representative of two independent experiments. (d) total rna isolation and qrt-pcr were performed as described in the methods section to assess mmp-9 gene expression in the above-described conditions. (pma = 1 µm; resveratrol = 30 µm). notes: data are representative of three independent qpcr experiments. probability values of less than 0.05 were considered significant, and an asterisk (*) identifies such significance to the respective pma treatment. http://www.la-press.com sirt1 expression in inflammatory cerebral endothelium drug target insights 2012:6 7 but that conflicting data regarding sirt1 expression in glioblastoma has been reported,17–19 we specifically explored its contribution to the resveratrol anti-pma pharmacological properties. our first objective was confirmed by the increases in inflammatory biomarkers mmp-9 and cox-2 expression upon carcinogenic stimulation in hbmec. as such, the calculated ic50 concentrations of resveratrol inhibitory effects extracted from our in vitro model closely approximate those actual concentrations assessed in the plasma ranging between 1.2–2.6 µm.31,32 both biomarkers expression was inhibited by resveratrol and this required inhibition of the nf-κb signaling pathway as validated by reduced iκb phosphorylation. inhibition of nf-κb (p65 subunit) translocation to the nucleus by resveratrol is a mechanism that has already been inferred in an experimental hepatocarcinogenesis model.33 our data thus indirectly support this nuclear translocation effect since iκb phosphorylation, which ultimately releases p65 and p50 in order to allow nuclear translocation, was found inhibited by resveratrol in our model (fig. 4b). although not the scope of this study, chip assays may further help address nf-κb capacity to bind cox-2 and mmp-9 promoter regions. given that the involvement of sirt1still remains undefined, at least within the carcinogenic context we report herein, our second objective was evaluated and confirmed a sirt1-independent, but efficient, targeting by resveratrol of the pma-induced nf-κb signaling pathway that ultimately leads to reduced 120 100 80 ∗ ∗ ∗ 60 40 20 0 0 5 10 15 resveratrol r e sv e ra tr o l vehicle v e h ic le time (min) p lκ b /g a p d h ( a rb . u n it s ) 20 25 30 0 a b 2 5 10 time of pma treatment (min) 15 20 25 30 plκb gapdh plκb gapdh figure 4. carcinogen-induced iκb phosphorylation is inhibited by resveratrol. (a) hbmec were serum-starved for 30 minutes in the presence or not of 30 µm resveratrol, then treated with 1 µm pma for the indicated time. lysates were isolated, electrophoresed via sds-page and immunodetection of phosphorylated iκb (p-iκb) and gapdh proteins was performed as described in the methods section. (b) quantification was performed by scanning densitometry of the autoradiograms. notes: data were expressed as the percent of basal phospho-iκb/gapdh ratios in vehicle (open circles) and resveratrol pre-treated cells (closed circles). densitometric data of a representative blot out of three is shown. http://www.la-press.com annabi et al 8 drug target insights 2012:6 mmp-9 and cox-2 expression. our current data do not support sirt1 requirements in our brain ec model suggesting that cell specificity may dictate resveratrol efficacy in inhibiting mmp-9 in neuroinflammation or brain tumor angiogenesis. in accordance with our current study, the presence or absence of sirt1, as assessed through the use of sirt1-null animals, had no effect on incidence and tumor load induced by a two-stage carcinogenesis protocol.42 in fact, only part of the chemopreventive effect of resveratrol was attributed to sirt1.34 moreover, while most biological effects of sirtuins have so far been attributed to their enzymatic activity, opposing effects of sirtuins on neuronal survival have also been reported suggestive of sirt1-mediated neuroprotection independent of deacetylase activity.35 although resveratrol is a potent pharmacological agonist of sirt1,36 and expected b a s ir t1 g e n e e x p re s s io n (% o f m o c k ) r e s v e ra tr o l in h ib it o ry p o te n c y ( a rb . u n it s ) resveratrol pre-treatmentvehicle pre-treatment c 0 2 120 100 80 60 40 20 0 120 100 80 60 40 20 0 mock mock gapdh plκb gapdh plκb sisirt1 m o ck si s ir t1 sisirt1 time (min) 5 10 15 20 25 30 0 5 10 15 20 25 30 0 2 5 10 15 20 25 30 (min) figure 5. sirt1 contributes to the resveratrol inhibitory effect of pma-induced ikb phosphorylation. hbmec were transiently transfected with a scrambled sirna sequence (mock) or a sirna designed to downregulate sirt1 (sisirt1) as described in the methods section. (a) mock and sisirt1-transfected hbmec were then serum-starved for 30 minutes in the presence or not of 50 µm resveratrol, then treated with 1 µm pma for the indicated time. lysates were isolated, electrophoresed via sds-page and immunodetection of phosphorylated iκb (p-iκb) and gapdh proteins was performed as described in the methods section. (b) total rna isolation, rt-pcr, and qpcr were performed as described in the methods section to assess sirt1 gene expression in the mock and sisirt1-transfected hbmec. (c) quantification was performed by scanning densitometry of the autoradiograms obtained in (a). notes: data were expressed as the percent of basal phospho-iκb/gapdh ratios in vehicle (open circles) and resveratrol pre-treated cells (closed circles). densitometric data of a representative blot out of three is shown. to induce deacetylation of known sirt1 substrates such as nf-κb, pgc-1α, and p53,37,38 one may now have to consider alternate mechanisms of resveratrol to those mediated through sirt1. among those mechanisms, several flavonoids have been reported to interfere with the inducible nitric-oxide synthase activity, as well as of both the cox and 5-lipoxygenase pathways.39,40 interestingly, sirt1 is a putative suppressive regulator found in the mmp-9 gene promoter,41 and activation of sirt1 by resveratrol was found to be required for inhibition of pma-induced mmp-9 expression.42,43 generalized interpretation, in part due to the occurrence of multiple in vitro cell culture models, has put forward large number of mechanisms of action attributed to flavonoids commonly found in fruits, vegetables, wine, or tea as their metabolites also can act http://www.la-press.com sirt1 expression in inflammatory cerebral endothelium drug target insights 2012:6 9 as potent antioxidants and free radical scavengers.21,44 whether the inhibitory effects observed in vitro are due to resveratrol metabolites has therefore to be promptly considered and carefully addressed. as such, oral bioavailability of resveratrol has been reported to be low because it is rapidly metabolized in intestines and liver into conjugated forms such as glucuronate and sulfonate.45 in mammals, less than 5% of the oral dose is being observed as free resveratrol in blood plasma.45–47 the most abundant resveratrol metabolites being trans-resveratrol-3-o-glucuronide and transresveratrol-3-sulfate,48 the impact of glucuronides and of these sulfate conjugates will also ultimately need further study. in summary, the present study has confirmed resveratrol as a signal transduction inhibitor against carcinogen-mediated induction of cox-2 and mmp-9. moreover, evidence that the nf-κb pathway may be inhibited through the targeting of iκk phosphorylation capacity that ultimately may reduce both the acquisition of a pro-inflammatory phenotype, as reflected by decreased cox-2 expression, and the acquisition of pro-angiogenic phenotype, as reflected by a decrease in mmp-9. our results further discriminate the role of sirt1 in the anti-pma action of resveratrol suggesting alternative intracellular targeting. whether other sirtuin members are involved remains to be investigated. bbb disruption during neuroinflammation may, in light of our results, be pharmacologically reduced by a specific class of flavonoids acting as nf-κb signal transduction inhibitors. list of abbreviations bbb, blood-brain barrier; cox, cyclooxygenase; ec, endothelial cells; er, endoplasmic reticulum; mmp-9, matrix metalloproteinase-9; nf-κb, nuclear factor-kappa b; pma, phorbol 12-myristate 13-acetate. authors’ contributions ba has conceived, designed, analyzed and interpreted the data of this study. sld has acquired, analyzed, and was involved in drafting the manuscript. av has acquired and analyzed the data. rb has conceived, designed, supported financially this study, and was involved in drafting the manuscript. all authors read and approved the final version of this manuscript. competing interests ba holds a canada research chair in molecular and metabolic oncology from the canadian institutes of health research (cihr). rb holds an institutional uqam research chair in cancer prevention and treatment. funding this study was funded by grants from the natural sciences and engineering research council of canada (nserc) to rb. disclosures and ethics as a requirement of publication author(s) have provided to the publisher signed confirmation of compliance with legal and ethical obligations including but not limited to the following: authorship and contributorship, conflicts of interest, privacy and confidentiality and (where applicable) protection of human and animal research subjects. the authors have read and confirmed their agreement with the icmje authorship and conflict of interest criteria. the authors have also confirmed that this article is unique and not under consideration or published in any other publication, and that they have permission from rights holders to reproduce any copyrighted material. any disclosures are made in this section. mock − − − + + − + − + − + + − − + + − − − + + − + − + − + + − − + + a b sisirt1 pma resveratrol pma resveratrol cox-2 prommp-9 gapdh mock sisirt1 figure 6. sirt1-independent inhibition by resveratrol of pma-induced cox-2 expression and of pma-induced mmp-9 secretion. mock or sisirt1-transfected hbmec were serum-starved in the presence of 1 µm pma, 30 µm resveratrol, or a combination of both agents for 18 hours. (a) lysates were isolated, electrophoresed via sds-page, and immunodetection of cox-2 and gapdh performed as described in the methods section. (b) conditioned media were also harvested and gelatin zymography was performed in order to detect pma-induced prommp-9 and hydrolytic activity as described in the methods section. http://www.la-press.com annabi et al 10 drug target insights 2012:6 the external blind peer reviewers report no conflicts of interest. references 1. hanahan d, folkman j. patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. cell. 1996;86:353–64. 2. rojas a, figueroa h, morales e. fueling inflammation at tumor microenvironment: the role of multiligand/rage axis. carcinogenesis. 2010; 31:334–41. 3. hayes a. cancer, cyclo-oxygenase and nonsteroidal anti-inflammatory drugs—can we combine all three? vet comp oncol. 2007;5:1–13. 4. costa c, soares r, reis-filho js, leitão d, amendoeira i, schmitt fc. cyclo-oxygenase 2 expression is associated with angiogenesis and lymph node metastasis in human breast cancer. j clin pathol. 2002;55:429–34. 5. fournier ls, novikov v, lucidi v, et al. mr monitoring of cyclooxygenase-2 inhibition of angiogenesis in a human breast cancer model in rats. radiology. 2007;243:105–11. 6. müller-decker k, fürstenberger g. the cyclooxygenase-2-mediated prostaglandin signaling is causally related to epithelial carcinogenesis. mol carcinog. 2007;46:705–10. 7. neergheen vs, bahorun t, taylor ew, jen ls, aruoma oi. targeting specific cell signaling transduction pathways by dietary and medicinal phytochemicals in cancer chemoprevention. toxicology. 2010;278:229–41. 8. lakka ss, gondi cs, rao js. proteases and glioma angiogenesis. brain pathol. 2005;15:327–41. 9. bonoiu a, mahajan sd, ye l, et al. mmp-9 gene silencing by a quantum dot-sirna nanoplex delivery to maintain the integrity of the blood brain barrier. brain res. 2009;1282:142–55. 10. annabi b, rojas-sutterlin s, laroche m, lachambre mp, moumdjian r, béliveau r. the diet-derived sulforaphane inhibits matrix metalloproteinase9-activated human brain microvascular endothelial cell migration and tubulogenesis. mol nutr food res. 2008;52:692–700. 11. roomi mw, monterrey jc, kalinovsky t, rath m, niedzwiecki a. distinct patterns of matrix metalloproteinase-2 and -9 expression in normal human cell lines. oncol rep. 2009;21:821–6. 12. jadhav u, chigurupati s, lakka ss, mohanam s. inhibition of matrix metalloproteinase-9 reduces in vitro invasion and angiogenesis in human microvascular endothelial cells. int j oncol. 2004;25:1407–14. 13. luqman s, pezzuto jm. nfkappab: a promising target for natural products in cancer chemoprevention. phytother res. 2010;24:949–63. 14. tergaonkar v. nfkappab pathway: a good signaling paradigm and therapeutic target. int j biochem cell biol. 2006;38:1647–53. 15. potente m, dimmeler s. emerging roles of sirt1 in vascular endothelial homeostasis. cell cycle. 2008;7:2117–22. 16. schlicker c, boanca g, lakshminarasimhan m, steegborn c. structurebased development of novel sirtuin inhibitors. aging (albany ny). 2011;3: 852–72. 17. liu g, yuan x, zeng z, et al. analysis of gene expression and chemoresistance of cd133+ cancer stem cells in glioblastoma. mol cancer. 2006;5:67. 18. chang cj, hsu cc, yung mc, et al. enhanced radiosensitivity and radiation-induced apoptosis in glioma cd133-positive cells by knockdown of sirt1 expression. biochem biophys res commun. 2009;380:236–42. 19. wang rh, sengupta k, li c, et al. impaired dna damage response, genome instability, and tumorigenesis in sirt1 mutant mice. cancer cell. 2008;14:312–23. 20. centeno-baez c, dallaire p, marette a. resveratrol inhibition of inducible nitric oxide synthase in skeletal muscle involves ampk but not sirt1. am j physiol endocrinol metab. 2011;301:e922–30. 21. tahanian e, peiro s, annabi b. low intracellular atp levels exacerbate carcinogen-induced inflammatory stress response and inhibit in vitro tubulogenesis in human brain endothelial cells. j inflamm res. 2011;4:1–10. 22. greiffenberg l, goebel w, kim ks, et al. interaction of listeria monocytogenes with human brain microvascular endothelial cells: inlb-dependent invasion, long-term intracellular growth, and spread from macrophages to endothelial cells. infect immun. 1998;66:5260–7. 23. sina a, lord-dufour s, annabi b. cell-based evidence for aminopeptidase n/cd13 inhibitor actinonin targeting of mt1-mmp-mediated prommp-2 activation. cancer lett. 2009;279:171–6. 24. abécassis i, olofsson b, schmid m, zalcman g, karniguian a. rhoa induces mmp-9 expression at cd44 lamellipodial focal complexes and promotes hmec-1 cell invasion. exp cell res. 2003;291:363–76. 25. annabi b, currie jc, moghrabi a, béliveau r. inhibition of hur and mmp-9 expression in macrophage-differentiated hl-60 myeloid leukemia cells by green tea polyphenol egcg. leuk res. 2007;31:1277–84. 26. tahanian e, lord-dufour s, das a, khosla c, roy r, annabi b. inhibition of tubulogenesis and of carcinogen-mediated signaling in brain endothelial cells highlight the antiangiogenic properties of a mumbaistatin analog. chem biol drug des. 2010;75:481–8. 27. annabi b, vaillancourt-jean e, weil ag, béliveau r. pharmacological targeting of β-adrenergic receptor functions abrogates nf-κb signaling and mmp-9 secretion in medulloblastoma cells. onco targets ther. 2010;3: 219–26. 28. dong j, jimi e, zeiss c, hayden ms, ghosh s. constitutively active nfkappab triggers systemic tnfalpha-dependent inflammation and localized tnfalpha-independent inflammatory disease. genes dev. 2010;24: 1709–17. 29. solt la, may mj. the ikappab kinase complex: master regulator of nfkappab signalling. immunol res. 2008;42:3–18. 30. craggs l, kalaria rn. revisiting dietary antioxidants, neurodegeneration and dementia. neuroreport. 2011;22:1–3. 31. boocock dj, patel kr, faust ge, et al. quantitation of trans-resveratrol and detection of its metabolites in human plasma and urine by high performance liquid chromatography. j chromatogr b analyt technol biomed life sci. 2007;848:182–7. 32. meng x, maliakal p, lu h, lee mj, yang cs. urinary and plasma levels of resveratrol and quercetin in humans, mice, and rats after ingestion of pure compounds and grape juice. j agric food chem. 2004;52:935–42. 33. bishayee a, waghray a, barnes kf, et al. suppression of the inflammatory cascade is implicated in resveratrol chemoprevention of experimental hepatocarcinogenesis. pharm res. 2010;27:1080–91. 34. boily g, he xh, pearce b, jardine k, mcburney mw. sirt1-null mice develop tumors at normal rates but are poorly protected by resveratrol. oncogene. 2009;28:2882–9283. 35. pfister ja, ma c, morrison be, d’mello sr. opposing effects of sirtuins on neuronal survival: sirt1-mediated neuroprotection is independent of its deacetylase activity. plos one. 2008;3:e4090. 36. howitz kt, bitterman kj, cohen hy, et al. small molecule activators of sirtuins extend saccharomyces cerevisiae lifespan. nature. 2003;425: 191–6. 37. knutson md, leeuwenburgh c. resveratrol and novel potent activators of sirt1: effects on aging and age-related diseases. nutr rev. 2008;6:591–6. 38. anekonda ts. resveratrol--a boon for treating alzheimer’s disease? brain res rev. 2006;52:316–236. 39. van acker sa, tromp mn, haenen gr, van der vijgh wj, bast a. flavonoids as scavengers of nitric oxide radical. biochem biophys res commun. 1995;214:755–9. 40. laughton mj, evans pj, moroney ma, hoult jr, halliwell b. inhibition of mammalian 5-lipoxygenase and cyclo-oxygenase by flavonoids and phenolic dietary additives. relationship to antioxidant activity and to iron ion-reducing ability. biochem pharmacol. 1991;42:1673–81. 41. nakamaru y, vuppusetty c, wada h, et al. a protein deacetylase sirt1 is a negative regulator of metalloproteinase-9. faseb j. 2009;23:2810–9. 42. woo jh, lim jh, kim yh, et al. resveratrol inhibits phorbol myristate acetate-induced matrix metalloproteinase-9 expression by inhibiting jnk and pkc delta signal transduction. oncogene. 2004;23:1845–53. 43. gao z, ye j. inhibition of transcriptional activity of c-jun by sirt1. biochem biophys res commun. 2008;376:793–6. 44. in: rice-evans c, packer l, editors: flavonoids in health and disease (second ed.), marcel dekker inc, new york/basel (2003). 45. walle t, hsieh f, delegge mh, oatis je jr, walle uk. high absorption but very low bioavailability of oral resveratrol in humans. drug metab dispos. 2004;32:1377–82. http://www.la-press.com sirt1 expression in inflammatory cerebral endothelium drug target insights 2012:6 11 46. marier jf, vachon p, gritsas a, zhang j, moreau jp, ducharme mp. metabolism and disposition of resveratrol in rats: extent of absorption, glucuronidation, and enterohepatic recirculation evidenced by a linked-rat model. j pharmacol exp ther. 2002;302:369–73. 47. abd el-mohsen m, bayele h, kuhnle g, et al. distribution of [3h]transresveratrol in rat tissues following oral administration. br j nutr. 2006;96: 62–70. 48. yu c, shin yg, chow a, et al. human, rat, and mouse metabolism of resveratrol. pharm res. 2002;19:1907–14. http://www.la-press.com tsen et al.indd drug target insights 2008:3 31–36 31 rapid communication correspondence: shaw-wei d. tsen, department of pathology, johns hopkins medical institutions, baltimore, md, u.s.a., email: tsen@jhu.edu. copyright in this article, its metadata, and any supplementary data is held by its author or authors. it is published under the creative commons attribution by licence. for further information go to: http://creativecommons.org/licenses/by/3.0/. evidence of a novel gene from aeromonas hydrophila encoding a putative siderophore receptor involved in bacterial growth and survival shaw-wei d. tsen department of pathology, johns hopkins medical institutions, baltimore, md u.s.a. abstract: the pathogenic bacterium aeromonas hydrophila has been shown to exclusively utilize a ligand exchange mechanism for siderophore-mediated iron uptake, with a single nonspecifi c siderophore receptor facilitating iron exchange. however, the genes involved in this process, including the gene encoding the nonspecifi c receptor, are unknown. here we identify and characterize a novel gene, nsr1, from a. hydrophila that encodes a putative protein with high homology and signifi cant predicted structural similarities to the fhua protein and other known ferric-siderophore receptors. this protein appears to localize on the cell membrane and is likely to be the receptor involved in the ligand exchange siderophore-mediated iron uptake mechanism of a. hydrophila. it is expected that this information may lead to the development of new antibiotics targeting either nsr1 or its gene product for use in controlling a. hydrophila infection. keywords: ferric iron, iron uptake, bacterial virulence, pathogenicity, infection introduction iron is necessary for many of the critical biochemical processes in bacterial growth and metabolism. although iron is a relatively abundant element, its bioavailability is severely limited due to its low solubility (apostol et al. 2005). to overcome this problem, bacteria secrete low molecular weight fe(iii)chelating compounds known as siderophores to assist in iron acquisition. these siderophores form complexes with extracellular iron and are generally transported into the bacterial periplasm via membrane transport proteins (stintzi et al. 2000). the ability of bacteria to scavenge iron sources from the environment has been shown to be a signifi cant factor in bacterial survival, as well as in bacterial pathogenicity (payne and finkelstein, 1978). therefore, siderophore-mediated iron acquisition systems play a central role in the progression of bacterial infection (stintzi et al. 2000; wooldridge and williams, 1993). recently, members of the genus aeromonas have drawn increased interest as human pathogens (janda and abbott, 1998). in particular, the bacterium aeromonas hydrophila has been reported to cause a multitude of human diseases, including wound infections, septicemia, and diarrhea (agger et al. 1985). iron transport in a. hydrophila has been shown to occur via a single membrane-bound siderophore receptor that is able to recognize a broad range of siderophores, utilizing a ligand exchange uptake mechanism (stintzi et al. 2000). however, the genes involved in this iron uptake mechanism have not been reported or characterized to date. in the current paper we identify and characterize a novel gene from a. hydrophila, nsr1, encoding a putative membrane receptor protein likely involved in siderophore-mediated iron uptake. the gene product exhibits a high degree of homology to the fhua protein, which encodes an outer membraneassociated ferric-siderophore receptor in many bacteria (coulton et al. 1986). further investigation into the structure and function of the protein product of nsr1 may elucidate the iron acquisition mechanism of a. hydrophila and may provide insight into the pathogenesis of a. hydrophila infection in humans. materials and methods data mining the tigr database (www.tigr.org) was mined for genomic data, and the novel gene sequence was derived from the unfi nished genome of a. hydrophila, contig. 1047085923793 (5'-ggccttctgt… http://creativecommons.org/licenses/by/3.0/ http://creativecommons.org/licenses/by/3.0/ 32 tsen drug target insights 2008:3 gttgcgagc-3'), by selection of the sequence encoding the longest observed translated open reading frame within the contig (sixframe program, biology workbench, san diego, ca, http://workbench.sdsc.edu). this yielded a 2106-bp dna sequence encoding the putative protein (fig. 1). sequence/product analysis database comparisons to known nucleotide sequences and proteins were made by using the rapid sequence database query programs blastp, blastn, and tblastn (genbank, national center for biotechnology information, bethesda, md, http://www.ncbi.nlm.nih.gov/blast). protein crystal structure prediction and hydropathy plots were carried out using publicly available online programs including the 3d-jigsaw protein comparative modeling server (http://www.bmm. icnet.uk/servers/3djigsaw), the expasy proteomics server (www.expasy.org), and biology workbench. results and discussion dna sequence analysis of nsr1 the nucleotide sequence of nsr1 was compared to known dna sequences in the genbank database with blastn (genbank, ncbi) and no signifi cant similarities were found. this is not surprising, given the generally high mutation rate in bacteria and the evolutionary time span of iron uptake mechanisms which were crucial for survival in the earliest known organisms. the nucleotide sequence of nsr1 contains a high gc content (61.8%) characteristic of gene-containing genomic regions (nastats, biology workbench). characterization of the nsr1 gene product the dna sequence of nsr1 was translated and found to contain an open reading frame of 702 amino acids encoding the putative protein (sixframe program, biology workbench). the molecular mass of the resulting protein was predicted to be ∼77.4 kda (expasy, swiss institute of bioinformatics, canada). a kyte-doolittle hydropathy plot indicates the presence of numerous potential membranespanning regions in the predicted polypeptide, suggesting that the putative protein may be localized on the cell membrane (kyte and doolittle, 1982) (fig. 2). this is supported by the high percentage of hydrophobic residues (%lvifm = 23.8%) present in the sequence (saps program, biology workbench). individual amino acid composition of the polypeptide includes a high percentage of glycine (9.5%) and leucine (9.6%) residues (aastats program, biology workbench), and further analysis of the polypeptide revealed alternating glycine-rich and leucine-rich regions. this observed pattern of alternating hydrophilic and hydrophobic regions is also seen in other bacterial ferric-siderophore receptors (newton et al. 1997); hydrophilic regions constitute surface loops and may facilitate siderophore binding in the extracellular compartment, while hydrophobic regions anchor the protein to the cell membrane. the protein is predicted to be slightly negatively charged in general (%kr—ed = −3.4%); however, no charge clusters were predicted (saps program, biology workbench). protein pi was estimated to be ∼4.94, in agreement with the environment necessary for the acidifi cation process and removal of iron within the barrel of the receptor in a ligand exchange iron uptake mechanism (stintzi et al. 2000). the homology of the putative protein to currently known proteins was characterized with blastp (genbank, ncbi). the fhua protein and similar iron uptake proteins from various bacteria were obtained and aligned using the algorithm of thompson et al. (thompson et al. 1994) (clustalw program, biology workbench). in the multiple sequence alignment (fig. 3), the conserved residues are distributed along the entire length of the polypeptide, suggesting a strong evolutionary pressure to preserve amino acids at specifi c positions for structural and/or functional reasons. ferric-siderophore receptors in general are characterized by evolutionarily conserved surface loops and anti-parallel β-barrels; the former is required for interaction with iron-loaded siderophores in the extracellular compartment, and the latter constitutes the iron removal acidifi cation site within the receptor (stintzi et al. 2000; newton et al. 1997). the predicted crystal structure of the putative protein (fig. 4) was modeled using the 3djigsaw protein comparative modeling server (bates et al. 2001; bates and sternberg, 1999; contrerasmoreira and bates, 2002) and contains an 33 evidence of a novel gene from aeromonas hydrophila drug target insights 2008:3 figure 1. dna sequence of nsr1 and its translated amino acid sequence. only those nucleotides encoding the predicted polypeptide are numbered. 34 tsen drug target insights 2008:3 anti-parallel β-barrel and a globular domain that forms surface loops and folds down into the β-barrel—two features thought to be common to all ferric-siderophore receptors (stintzi et al. 2000). the interior of the β-barrel represents the site where iron is removed from iron-loaded siderophores, and the n-terminal domain predominantly consists of surface loops that appear to be hydrophilic in nature and that may facilitate binding of hydrophilic ferric-siderophores to the receptor. a. hydrophila has been shown to acquire iron via a ligand exchange model (stintzi et al. 2000), in which a siderophore is initially bound to the receptor as a siderophore-receptor complex. when a second, iron-loaded siderophore is in close proximity of the receptor, iron is likely removed from the second siderophore via a ph gradient within the barrel of the receptor (stintzi et al. 2000). this results in a protonation/deprotonation reaction, and the iron is donated to the initial siderophore which translocates from the membrane to the periplasmic space. the second siderophore then binds to the receptor, replacing the initial siderophore. in the predicted structure of the nsr1 gene product, the interior of the protein is enclosed by β-sheets and is likely to be a suitable environment for local acidifi cation. the hydrophilic regions probably extend into the extracellular space to interact with ferric-siderophores, as is seen with many bacterial siderophore uptake proteins such as fepa, the enterobactin receptor expressed by e. coli (newton et al. 1997). bacteria frequently possess feedback systems that upregulate or downregulate the expression of certain iron uptake proteins, including the energy transducing protein tonb, in response to environmental iron levels (beddek et al. 2004). however, expression of the fhua gene has been shown to be unaffected by iron conditions (mikael et al. 2003). based upon the similarities between the protein products of nsr1 and fhua, it is possible that expression of nsr1 may also be unaffected in the presence or absence of iron. in a. hydrophila, and in other bacteria, iron-dependent regulation of iron uptake occurs at the level of siderophore biosynthesis rather than at the level of siderophore receptor production (stintzi et al. 2000; venturi et al. 1995). therefore it appears to be advantageous for a. hydrophila to express nsr1 constitutively to maintain a constant means of iron acquisition. further studies are warranted to clarify the irondependent (and potentially iron-independent) mechanisms by which nsr1 is regulated. the presence of a unique nsr1-driven siderophore system has implications for the development of drugs to control a. hydrophila. since the utilization of specifi c siderophore systems are often confi ned to certain bacteria, and because siderophores often play a critical role in bacterial growth, survival and virulence, targeting siderophore-mediated iron uptake is an attractive approach to antibacterial drug development. siderophore-antibiotic conjugates, called sideromycins, have been shown to be highly effective at entering bacteria by exploiting natural figure 2. hydropathy plot of the predicted polypeptide of nsr1, obtained using the kyte-doolittle algorithm and hydropathy values (kyte and doolittle, 1982). alternating regions of hydrophilicity and hydrophobicity are observed. 35 evidence of a novel gene from aeromonas hydrophila drug target insights 2008:3 siderophore uptake mechanisms to traverse bacterial cell membranes (for a review, see miethke and marahiel, 2007). this could provide a method for specifi c and effi cient drug targeting to a. hydrophila. on the other hand, drugs that inhibit expression of nsr1 or interfere with siderophore pathways involving nsr1 could conceivably suppress bacterial multiplication by blocking iron metabolism. these and other strategies may provide a basis for therapeutic intervention for the control of a. hydrophila infection. conclusions the iron uptake strategy of a. hydrophila has previously been described as a ligand exchange mechanism utilizing a single nonspecific siderophore receptor (stintzi et al. 2000). here we report the identifi cation of a novel gene from a. hydrophila potentially encoding the abovementioned ligand exchange siderophore receptor. the putative protein product of nsr1 bears high amino acid sequence identity and similarity to several known siderophore receptors. in addition, the predicted secondary structure of the protein features two distinct functional domains that are thought to be common to all ferric-siderophore receptors (stintzi et al. 2000). thus nsr1 likely encodes a receptor protein involved in siderophoremediated iron transport. as a. hydrophila has been shown to exclusively utilize a single ferricsiderophore receptor for iron transport, the gene product of nsr1 is an excellent candidate for the receptor. further investigation and molecular characterization of the protein product of nsr1 may figure 3. amino acid sequence alignment of the putative a. hydrophila siderophore receptor (here denoted ahyd) with known ferric-siderophore receptors from various bacteria: vpar, vibrio parahaemolyticus ferrichrome receptor fhua (genbank bad06905); csal, chromohalobacter salexigens tonb-dependent siderophore receptor (genbank yp_573101); and aple, actinobacillus pleuropneumoniae ferric hydroxamate receptor fhua (genbank dq249800). single fully conserved residues (*) are highlighted, and strong conserved groups (:) and weak conserved groups (.) are indicated. 36 tsen drug target insights 2008:3 contribute to the elucidation of the iron uptake mechanism of a. hydrophila and may lead to the development of new antibiotics targeting either nsr1 or its product for use in controlling a. hydrophila infection. acknowledgements the author would like to thank nicholas fitzkee for his important contributions to the implementation of the computer programs used in this study and for his helpful and informative discussions. references agger, w.a., mccormick, j.d. and gurwith, m.j. 1985. clinical and microbiological features of aeromonas hydrophila-associated diarrhea. j. clin. microbiol., 21:909–13. apostol, m., baret, p., serratrice, g., desbrières, j., putaux, j.l., stèbè, m.j., expert, d. and pierre, j.l. 2005. self-assembly of an amphiphilic iron(iii) chelator: mimicking iron acquisition in marine bacteria. angew. chem., 117:2636–8. bates, p.a., kelley, l.a., maccallum, r.m. and sternberg, m.j.e. 2001. enhancement of protein modeling by human intervention in applying the automatic programs 3d-jigsaw and 3dpssm. proteins: structure, function and genetics, (suppl 5):39–46. bates, p.a. and sternberg, m.j.e. 1999. model building by comparison at casp3: using expert knowledge and computer automation. proteins: structure, function and genetics, (suppl 3):47–54. beddek, a.j., sheehan, b.j., bossé, j.t., rycroft, a.n., kroll, j.s. and langford, p.r. 2004. two tonb systems in actinobacillus pleuropneumoniae: their roles in iron acquisition and virulence. infect. immun., 72:701–8. contreras-moreira, b. and bates, p.a. 2002. domain fi shing: a fi rst step in protein comparative modeling. bioinformatics, 18:1141–2. coulton, j.w., mason, p., cameron, d.r., carmel, g., jean, r. and rode, h.n. 1986. protein fusions of β-galactosidase to the ferrichrome-iron receptor of escherichia coli. j. bacteriol., 165:181–92. janda, j.m. and abbott, s.l. 1998. evolving concepts regarding the genus aeromonas: an expanding panorama of species, disease presentations, and unanswered questions. clin. infect. dis., 2:332–44. kyte, j. and doolittle, r.f. 1982. a simple method for displaying the hydropathic character of a protein. j. mol. biol., 157:105–32. miethke, m. and marahiel, m.a. 2007. siderophore-based iron acquisition and pathogen control. microbiol. mol. biol. rev., 71:413–51. mikael, l.g., srikumar, r., coulton, j.w. and jacques, m. 2003. fhua of actinobacillus pleuropneumoniae encodes a ferrichrome receptor but is not regulated by iron. infect. immun., 71:2911–5. newton, s.m.c., allen, j.s., cao, z., qi, z., jian, x., sprencel, c., igo, j.d., foster, s.b., payne, m.a. and klebba, p.e. 1997. double mutagenesis of a positive charge cluster in the ligand binding site of the ferric enterobactin receptor, fepa. proc. natl. acad. sci., 94:4560–5. payne, s.m. and finkelstein, r.a. 1978. the critical role of iron in hostbacterial interactions. j. clin. invest., 61:1428–40. stintzi, a., barnes, c., xu, j. and raymond, k.n. 2000. microbial iron transport via a siderophore shuttle: a membrane ion transport paradigm. proc. natl. acad. sci., 97:10691–6. thompson, j.d., higgins, d.g. and gibson, t.j. 1994. clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specifi c gap penalties and weight matrix choice. nucleic acids res., 22:4673–80. venturi, v., ottevanger, c., bracke, m. and weisbeek, p. 1995. iron regulation of siderophore biosynthesis and transport in pseudomonas putida wcs358: involvement of a transcriptional activator and of the fur protein. mol. microbiol., 15:1081–93. wooldridge, k.g. and williams, p.h. 1993. iron uptake mechanisms of pathogenic bacteria. fems microbiol. rev., 12:325. figure 4. predicted crystal structure of the gene product of nsr1 (bates et al. 2001; bates and sternberg, 1999; contreras-moreira and bates, 2002) (3d-jigsaw protein comparative modeling server, uk). the structure bears striking similarity to all other currently known ferric siderophore receptors. the putative protein is comprised of an antiparallel β-barrel enclosing the iron removal site within the receptor, as well as an n-terminal globular domain that forms surface loops and folds down into the interior of the 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2014:8 open access: full open access to this and thousands of other papers at http://www.la-press.com. drug target insights effects of zaprinast and rolipram on olfactory and visual memory in the social transmission of food preference and novel object recognition tests in mice furuzan akar1, oguz mutlu1, ipek k. celikyurt1, emine bektas1, mehmet h. tanyeri2, guner ulak1, pelin tanyeri3 and faruk erden1 1medical faculty, department of pharmacology, kocaeli university, kocaeli, turkey. 2department of urology, yenikent government hospital, sakarya, turkey. 3faculty of medicine, department of pharmacology, sakarya university, sakarya, turkey. a bstr act: the role of phosphodiesterase (pde) inhibitors in central nervous system has been investigated and shown to stimulate neuronal functions and increase neurogenesis in alzheimer patients. the aim of this study is to investigate effect of pde5 inhibitor zaprinast and pde4 inhibitor rolipram on visual memory in novel object recognition (nor) test, on olfactory memory in social transmission of food preference (stfp) test, and also on locomotion and anxiety in open field test in naive mice. male balb-c mice were treated intraperitoneally (i.p.) with zaprinast (3 and 10 mg/kg), rolipram (0.05 and 0.1 mg/kg), or physiological saline. zaprinast (10 mg/kg) significantly increased cued/non-cued food eaten compared to control group, while rolipram had a partial effect on retention trial of stfp test. zaprinast (10 mg/kg) and rolipram (0.05 and 0.1 mg/kg) significantly increased ratio index (ri) compared to control group in retention trial of nor test. there was no significant effect of zaprinast and rolipram on total distance moved, speed, and center zone duration in open field test. results of this study revealed that both zaprinast and rolipram enhanced visual memory in nor test, however zaprinast exerted a significant memory-enhancing effect compared to rolipram in stfp test in mice. k e y wor ds: zaprinast, rolipram, memory, open field test, mice citation: akar et al. effects of zaprinast and rolipram on olfactory and visual memory in the social transmission of food preference and novel object recognition tests in mice. drug target insights 2014:8 23–29 doi:10.4137/dti.s14813. received: february 11, 2014. resubmitted: march 31, 2014. accepted for publication: april 4, 2014. academic editor: anuj chauhan, editor in chief type: original research funding: authors disclose no funding sources. competing interests: authors disclose no potential conflicts of interest. copyright: © the authors, publisher and licensee libertas academica limited. this is an open-access article distributed under the terms of the creative commons cc-by-nc 3.0 license. correspondence: oguzmutlu80@hotmail.com introduction accumulating evidence indicates that the inhibition of phosphodiesterase (pde) activity may consist of a particularly interesting mechanism for memory enhancement.1,2 this is related to the substrates of pdes: cyclic adenosine monophosphate (camp) and cyclic guanosine monophosphate (cgmp). both cyclic nucleotides play an important role in intracellular signaling3,4 and in processes of neuroplasticity, such as longterm potentiation (ltp). inhibitors of pdes are expected to increase camp and/or cgmp levels in neurons and hence might improve memory. the underlying mechanism for pde4 inhibitors for their cognitive-enhancing effects may involve modulation of activity within the camp/protein kinase a (pka)/camp response element-binding (creb) protein pathway. the prototypical pde4 inhibitor most widely used in cognition studies is rolipram. it possesses significant brain penetration and has a halflife of one to three hours.5 in vitro studies showed that camp levels increased in hippocampal slices treated with rolipram.6 rolipram attenuated deficits in spatial and non-spatial shortterm memory and working memory in several behavioral tasks.7 rolipram reversed the disruption in reference memory and/or working memory by the glutamate antagonist mk-801 in a radial-arm maze and reversed the effects of mk-801 in a passive avoidance task.8 rolipram or ht0712 treatment of mice 20 minutes before training in the object recognition task http://www.la-press.com http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 http://dx.doi.org/10.4137/dti.s14813 http://creativecommons.org/licenses/by-nc/3.0/ http://creativecommons.org/licenses/by-nc/3.0/ mailto:oguzmutlu80@hotmail.com akar et al 24 drug target insights 2014:8 improved retention performance 24 hours later.9 similarly, the retention performance 24 hours after contextual fear learning was improved in mice treated with rolipram 30 minutes before training.10 pde4 inhibition can reverse memory performance in different mouse disease models,9,11 providing strong support that this treatment approach may be effective in alzheimer’s disease. in 1997, pde5 inhibition was first described to improve memory processes.12 pde5 inhibitors (pde5-i) such as sildenafil and vardenafil have not only been shown to be effective in the treatment of erectile dysfunction but are also candidate drugs for cognition enhancement. for instance, the specific pde5-is sildenafil and vardenafil have been shown to improve object recognition memory when injected immediately following the first trial.13 pde5-is are assumed to improve early processes of memory consolidation via either a presynaptic or postsynaptic mechanism. the presynaptic mechanism acts through the nitric oxide (no)–cgmp signaling pathway and the postsynaptic mechanism through the cgmp/protein kinase g (pkg)/creb protein signaling pathway.1 zaprinast was used to inhibit pde5 and improved the long-tem memory (ltm) performance of rats in the object recognition task when given immediately after training at a dose of 10 mg/kg (intraperitoneally, i.p.).12 zaprinast also inhibits pde1, 9, 10, and 11. previous studies also showed that zaprinast reversed the object memory deficits induced by the nos inhibitor 7nitroindazole in rats in the object recognition task.12 however, zaprinast was unable to reverse memory deficits in aged rats in this task.14 animal studies indicate that pde5 inhibitors have the potential to improve early consolidation processes of long-term memory, though this may exclude spatial information. this memory improvement is speculatively mediated by elevations in central cgmp levels. as a result of our literature search, we found no study investigating the effects of zaprinast and rolipram on memory in the novel object recognition (nor) test and social transmission of food preference (stfp) test. the aim of this study is to investigate the effect of pde5 inhibitor zaprinast and pde4 inhibitor rolipram on hippocampal-dependent visual memory in the nor, on hippocampal-dependent olfactory memory in the stfp, and also on locomotion and anxiety in the open-field test in naive mice. methods animals. a total of 90 male inbred balb/c byj mice (mam tubi̇tak, gebze, kocaeli, turkey) aged seven weeks upon arrival to the laboratory were used in this study. animals (four to five per cage) were kept in the laboratory at 21 ± 1.5°c with 60% relative humidity under a 12-h light/dark cycle (light on at 8.00 p.m.) for two weeks before experimentation. all animals received food and water ad libitum. all procedures described in this paper were conducted in accordance with the european community council directive for the ethical treatment of animals (86/609/eec) and with the ethical approval of the kocaeli university ethics committee (number: aek 9/4-2010, kocaeli, turkey). stfp test. hippocampus-dependent non-spatial olfactory memory15 was studied using the stfp task. in this test, mice are required to remember the scent of food smelled on the muzzle of a demonstrator mouse 24 hours earlier. when offered a choice of the flavored food eaten by the demonstrator mouse (cued food), or another novel-flavored food, mice with normal olfactory memory will eat a greater proportion of the familiar cued food than of the novel food. the experiment was conducted in three phases: (i) habituation to flavored food, (ii) interaction between “demonstrator” and “observer” mice, and (iii) test of the food preference in the “observer” mice.15 mice were housed at a ratio of three to four observer mice to one demonstrator. in the habituation phase, a demonstrator mouse was chosen from each cage. demonstrators were housed singly in a cage separate from the colony for three hours with free access to water but not food. at the end of three hours, each demonstrator was allowed to eat powdered ground chow scented with either cinnamon (1%, w/w) or cocoa (2%, w/w).15 half of the demonstrators received cocoa-flavored food, and the other half received cinnamon-flavored food. the demonstrators were allowed to eat the flavored food for two hours. the pellets were weighed before and after presentation to the demonstrators. the criterion for inclusion in the experiment was consumption of 0.2 g. all demonstrator mice tested met this criterion. each demonstrator was then placed back with its observer cagemates for 30 minutes.15 observers interacted with the demonstrator including sniffing the scent around the muzzle and on the breath of the demonstrator mouse.15 after the interaction period, the demonstrator mouse was removed from the interaction cage and returned to its individual cage. in the final phase of the experiment, the food preference of the observer mice was tested 24 hours after the end of the interaction with the demonstrator. five hours before the preference test, observer mice were caged individually with free access to water and food, in the same room where the interaction was performed. three hours before the preference test, the food was removed from the observer’s cage. the two-hour preference test consisted of presenting each observer with a pair of weighed food pellets in the individual cage. one pellet contained the flavor of food eaten by the demonstrator (cued); the other contained the novel flavor of the pair (novel). thus, half of the observers were tested with the cinnamon-flavored cued food eaten by their demonstrator versus the novel cocoaflavored food and the other half were tested with the cocoaflavored cued food eaten by their demonstrator versus the novel cinnamon-flavored food.15 after two hours, both food pellets were removed and weighed to quantify the food preference of the observer mice. the ratio of the weight of the cued food eaten and the total weight of food eaten was used as a measure of food preference.15 http://www.la-press.com effects of zaprinast and rolipram on olfactory and visual memory 25drug target insights 2014:8 nor test. the protocol according to ennaceur and delacour16 was adjusted. the apparatus consisted of a circular open field 40  cm in diameter and 30  cm in height made of pvc with a black and white-striped cardboard pattern (30 × 20 cm) nailed on one of the walls. the floor was divided into six peripheral sections and one central section of the same dimension. the apparatus was placed in a sound-isolated room.16 a light bulb provided a constant illumination of about 100 lux above the central section. the nor task procedure consisted of three trials: habituation, training, and retention trials. each mouse was individually habituated to the apparatus, with five minutes of exploration in the absence of objects (habituation trial).16 a mouse was always placed at the center section of the apparatus. in all, 30 minutes after the habituation trial, the mouse was placed in the apparatus for the first trial (t1) and two identical objects (moon or butterfly) were placed in a symmetrical position 10 cm above the side wall.16 a mouse could not displace the objects (they were sticked to the wall of the open field by patafix). the order of objects used per subject per trial was determined randomly. all combinations and locations of objects were used in a balanced manner to reduce potential biases because of preferences for particular locations or objects. a mouse was then placed in the central section of the box and the total time spent in exploring the two objects was recorded for five minutes by the experimenter. to avoid the presence of olfactory trails, the apparatus after each trial was thoroughly cleaned. exploration of an object was defined as directing the nose to the object at a distance of maximum 1 cm and/or touching it with the nose. after the first exploration period, the mouse was put back in its home cage. subsequently, after a predetermined retention interval (intertrial interval of one hour), the mouse was again placed in the apparatus for the second trial (t2, choice phase), but now with two dissimilar objects, a familiar one (the reference) and a new one.16 the object not used in the acquisition trial was used as the novel object in the recognition trial. the animals were then allowed to explore freely for five minutes, and the time spent exploring each object was recorded. if recognition memory was intact, the mice were expected to spend more time exploring the novel object.16 a ratio index (ri) was calculated as the time spent exploring the new object (n ) divided by the total time exploring the objects (n + r) multiplied by 100. higher ri is considered to reflect greater memory retention.16 open field test. treatment effects on animal locomotor activity were measured using the open field test. this test is also used to examine anxiety-like behaviors and is used to evaluate anxiolytic treatment.17 this experiment was performed as previously described.18 briefly, the testing apparatus consisted of a wooden box (33 cm × 33 cm × 30 cm) with an indirect red light. an animal was placed in the center of test box, and total distance moved throughout the area, speed of animals, and time spent in center zone were recorded using ethovision xt (noldus) for five minutes. drug administration. zaprinast and rolipram were purchased from sigma chemical company (sigma, st. louis, mo) and were dissolved in saline supplemented with small amounts of dmso. all drugs were freshly prepared and administered in a volume of 0.1  ml/10  g body weight. the control groups received the same volume of vehicle. zaprinast (3 and 10 mg/kg) and rolipram (0.05 and 0.1 mg/kg) or vehicle were administered i.p. 60 and 30 minutes, respectively, before the retention sessions of nor and stfp tests and before the open field test. six animals were in each group. the effective dose of each drug was selected according to previous behavioral and neurochemical studies.19 statistics. a one-way analysis of variance (anova) post hoc tukey’s test was used to analyze the ri of the animals in the nor test: total distance moved, speed, and time spent in the center zone in the open field test. the kruskal–wallis post hoc dunn’s test was used to analyze the cued food/total food% eaten and total food consumption in the stfp test. the data are expressed as mean ± sem values. statistical significance was set at p  0.05. results effects of zaprinast and rolipram on olfactory memory in the stfp test. when zaprinast (3 and 10  mg/kg) and rolipram (0.05 and 0.1 mg/kg) were administered before the retention session of stfp test, there was a significant difference among the groups when the percentage of cued food per total food eaten was evaluated (h = 10.38, p = 0.03; fig. 1a). zaprinast (10  mg/kg) significantly increased percentage of cued food per total food eaten compared to the control group (p  0.05; fig. 1a), whereas rolipram had no significant effect. when the total food consumption in the stfp test was evaluated, there was no significant effect of drugs compared to control group (h = 3.28, p = 0.51; fig. 1b). effects of zaprinast and rolipram on v isual memor y in the nor test. when zaprinast (3 and 10  mg/kg) and rolipram (0.05 and 0.01  mg/kg) were administered before the retention session of nor test, there was a significant difference among the groups when the ri was evaluated [f (4, 29) = 5.94, p = 0.0017; fig. 2]. zaprinast (10 mg/kg) (p  0.001) and rolipram (0.05 and 0.1 mg/kg) (p  0.05) significantly increased the ri compared to the control group (fig. 2). effects of zaprinast and rolipram on locomotion and anxiety in the open field test. there was no significant difference between zaprinast (3 and 10  mg/kg), rolipram (0.05 and 0.01 mg/kg), and control groups about the total distance moved [f(4, 29)  =  0.90; p    0.05], speed [f(4, 29)  =  0.70; p  0.05], and center zone duration [f(4, 29) = 0.73; p  0.05] in the open field test evaluation (fig. 3). discussion this study revealed that both pde5 inhibitor zaprinast (10 mg/kg) and pde4 inhibitor rolipram (0.05 and 0.1 mg/kg) http://www.la-press.com akar et al 26 drug target insights 2014:8 increased the ri in the nor test. concerning the stfp test, zaprinast (10 mg/kg) enhanced percentage of cued/non-cued food eaten, while rolipram had a partial effect, although it did not reach a significant level. both zaprinast and rolipram had no significant effect on total food consumption in the stfp test. both drugs had no significant effect on total distance moved, speed and center zone duration in the open field test. pde enzymes may be involved in the etiology of a number of cns diseases, including alzheimer’s disease, schizophrenia, and affective disorders, and have recently been proposed as potential targets for therapeutic intervention.20,21 in addition, pdes may be targeted for the cognitive enhancement, and inhibitors of pdes have proven to be useful experimental tools in exploring mechanisms of learning and memory.1,22 selective pde inhibitors of at least five types and inhibitors of pde2,23 pde4,24 pde5,25 and pde926 have all been shown to enhance memory in different behavioral paradigms and in different species.19 these memory enhancements may be related to the subsequent increases in intracellular cgmp and/or camp levels after pde inhibition, particularly, as both cgmp and camp are important intracellular second messenger molecules that have been observed in consolidation processes.27 interestingly, selective pde inhibitor treatments are in line with the sequence of molecular changes taking place in the hippocampus during memory consolidation, as recently described by izquierdo et al.28 possible underlying mechanisms of action for memory enhancement after pde inhibition are closely related to electrophysiological theories of learning and memory. thus, the camp/pka/creb pathway as well as the cgmp/pkg/creb pathway are key candidates in providing the biochemical substrate of long-term memory effects. the activation of both pathways may lead to creb phosphorylation and, consequently, de novo protein synthesis. the involvement of campand cgmp-mediated signaling in learning and memory is well known, and both have been posited to be involved in hippocampal ltp formation.29 more recently, it has been suggested that hippocampal cgmp can enhance postsynaptic camp and pka30 or regulate ltp via presynaptic cgmp and pkg.31 ample evidence supports a role for the camp/pka/ creb pathway in learning and memory processes.32 pde4 inhibitors specif ically inhibit ca mp, and the underlying mechanism for their cognitive-enhancing effects may involve modulation of activity within the camp/pka/creb pathway.33 the prototypical pde4 inhibitor most widely used in cognition studies is rolipram. it possesses significant brain penetration and has a half-life of one to three hours.5 in vitro studies showed that camp levels increased in hippocampal stfp test 0 20 40 60 80 100 120 drugs (mg/kg) c u ed f o o d /t o ta l f o o d e at en a control zap 3 zap 10 rol 0.05 rol 0.1 * stfp te st 0 0,2 0,4 0,6 0,8 1 1,2 1,4 1,6 1,8 2 drugs (m g/kg) t o ta l f o o d c o n su m p ti o n control zap 3 zap 10 rol 0.05 rol 0.1 b figure 1. effect of zaprinast (3 and 10 mg/kg) and rolipram (0.05 and 0.1 mg/kg) (n = 6) on (a) percentage of cued per non-cued food eaten and (b) total food consumption (in which zaprinast and rolipram were administered 60 and 30 minutes, respectively, before the retention trial) in the stfp test in mice. the data are expressed as the mean ± sem values of animals. note: *p  0.05 compared to control group. nor test 0 20 40 60 80 100 120 drugs r at io in de x control zap 3 zap 10 rol 0.05 rol 0.1 * * * * figure 2. effect of zaprinast (3 and 10 mg/kg) and rolipram (0.05 and 0.1 mg/kg) on ri (n = 6) (in which zaprinast and rolipram were administered 60 and 30 minutes, respectively, before the retention trial) in the nor test in mice. the data are expressed as the mean ± sem values of animals. notes: *p  0.05, **p  0.001 compared to control group. http://www.la-press.com effects of zaprinast and rolipram on olfactory and visual memory 27drug target insights 2014:8 slices treated with rolipram.6 studies have shown that rolipram produces memory-enhancing effects in a number of models and has antidepressant-like activity in both preclinical34 and clinical models.35 in 1997, it was first described that pde5 inhibition improves memory processes.12 zaprinast was used to inhibit pde5 and when given immediately after training at a dose of 10 mg/kg (i.p.), improved the ltm performance of rats in the object recognition task.12 however, zaprinast also inhibits pde1, 9, 10, and 11. the memory-improving effects of pde5 inhibitors may also, or alternatively, be related to an increased blood flow and, consequently, increased glucose metabolism, as pde5 inhibitors are known to result in vasodilatation, most likely via cgmp.36,37 a decrease in blood flow generally results in a decrease in blood pressure. it was observed that a dose of 10  mg/kg zaprinast administration (i.p.) slightly increased the mean arterial blood pressure in conscious rats from one to four hours, after which recovery occurred.12 this had been observed before, and the mechanism by which zaprinast elevates mean arterial blood pressure is not clear.36 yet, a depressor response after systemic administration of zaprinast has been observed at doses above 10 mg/kg.36,37 as the doses of zaprinast we used in our study have no effect on mean arterial blood pressure,36,37 it is unlikely that effects on peripheral blood pressure after zaprinast treatment contributed to its memory improvement. the nor test for rodents was formulated by ennaceur and delacour16 to measure the spontaneous exploratory activity toward a novel object and a familiar object. this test does not involve rule learning or reinforcement and is thought to evaluate working and visual memory. this test has many useful applications to study the neurobiological mechanisms of learning and memory. in the nor test, the failure to discriminate between familiar and novel objects can be related to either impaired memory of the object or the inability to use spatial information. occasionally, the effect might be unrelated to an amnesic action, as mice display an inhibition of locomotion. however, such an explanation does not apply to zaprinast and rolipram at the doses used because they do not affect locomotion and anxiety in the open field test in our study. therefore, the performance observed at the nor test can be attributed to memory-enhancing effects in the nor test. hall38 originally described the open field test for the study of rat emotion. the procedure consists of placing an animal in an unknown environment from which escape is prevented by the surrounding walls.39 the open field test is a very common procedure used in animal psychology.18 rodents naturally prefer periphery of the apparatus to the middle area of the open field. treatments that increase time spent in central area without impairment of locomotion are deemed anxiolytic like, whereas treatments that decrease these variables produce anxiogenic effects. open field test 0 100 200 300 400 500 600 700 800 900 drugs to ta l d is ta nc e m ov ed (c m ) control zap 3 zap 10 rol 0.05 rol 0.1 a open field test 0 0,5 1 1,5 2 2,5 3 3,5 drugs s pe ed (c m /s ) control zap 3 zap 10 rol 0.05 rol 0.1 b open field test 0 2 4 6 8 10 12 14 16 18 drugs c en te r zo ne d ur at io n (s ) control zap 3 zap 10 rol 0.05 rol 0.1 c figure 3. effect of zaprinast (3 and 10 mg/kg) and rolipram (0.05 and 0.1 mg/kg) (n = 6) administration on locomotion and anxiety in the open field test. drugs were injected 60 and 30 minutes, respectively, before testing. the data are expressed as the mean ± sem values. (a) total distance moved, (b) speed, and (c) center zone duration in the open field test. http://www.la-press.com akar et al 28 drug target insights 2014:8 there are controversial results for the effects of no on anxiety. in the following studies, nos inhibitors were known to possess anxiolytic effects,40 whereas no donors had anxiogenic effects.41 because inhibition of pde had an opposite action compared to nos inhibitors, they increase the formation of no and can be expected to exert anxiogenic effects. in our study, both zaprinast and rolipram did not change center zone duration conferring no significant effect on anxiety. stfp is a hippocampal-dependent olfactory memory test.15 in figure 1a, we found statistically significant difference for 10  mg/kg zaprinast, showing that zaprinast at this dose increased percentage of cued food per non-cued food eaten ie enhancement of olfactory memory. figure 1b reflects total food consumption, and zaprinast at this dose failed to affect this parameter ie zaprinast had no nonspecific effects on olfactory memory; it affected only cued food consumption. in this study, in the hippocampal-dependent nor test, both zaprinast and rolipram enhanced visual memory; only zaprinast, at the higher dose, increased olfactory memory in the stfp test, and rolipram had a partial effect. different explanations of this discrepancy can be proposed. olfactory learning has no spatial components, as in the nor test. however, it could be that distinct brain processes underlie spatial visual and olfactory memory formation. there are also findings claiming that no does not affect the retrieval of olfactory memory in adult sheep42 and that no release is involved in the acquisition phase in an olfactory recognition test but does not affect post-acquisition recall.43 in conclusion, the present study demonstrates that both the pde5 inhibitor zaprinast and the pde4 inhibitor rolipram enhanced visual memory in the nor test, whereas only zaprinast significantly enhanced olfactory memory in the stfp test. both zaprinast and rolipram had no significant effect on locomotion and anxiety in the open field test. future studies using different pde inhibitors with different cognition methods can be performed to support our findings. author contributions fa, om, ikc, and mht conceived and designed the experiments. om, eb, gu, and pt analyzed the data. fa, om, and gu wrote the first draft of the manuscript. mht, gu, and fe contributed to the writing of the manuscript. fa, om, ikc, eb, mht, gu, pt, and fe agreed with manuscript results and conclusions. fa, eb, gu, pt, and fe jointly developed the structure and arguments for the paper. fa, ikc, and eb made critical revisions and approved the final version. all authors reviewed and approved the final manuscript. disclosures and ethics this paper was subject to independent, expert peer review by a minimum of two blind peer reviewers. all editorial decisions were made by the independent academic editor. all authors have provided signed confirmation of their compliance with ethical and legal obligations including (but not limited to) use of any copyrighted material, compliance with icmje authorship and competing interests disclosure guidelines and, where applicable, compliance with legal and ethical guidelines on human and animal research participants. r efer ences 1. prickaerts j, sik a, van staveren wc, et al. phosphodiesterase type 5 inhibition improves early memory consolidation of object information. neurochem int. 2004;45:915–928. 2. fedele e, raiteri m. in vivo studies of the cerebral glutamate receptor/no/ cgmp pathway. prog neurobiol. 1999;58:89–120. 3. son h, lu yf, zhuo m, arancio o, kandel er, hawkins rd. the specific role of cgmp in hippocampal ltp. learn mem. 1998;5:231–245. 4. bailey ch, bartsch d, kandel er. toward a molecular definition of long-term memory storage. proc natl acad sci u s a. 1996;93:13445–13452. 5. krause w, kuhne g. pharmacokinetics of rolipram in the rhesus and cynomolgus monkeys, the rat and the rabbit. studies on species differences. xenobiotica. 1988;18:561–571. 6. van staveren wc, markerink-van ittersum m, steinbusch hw, de vente j. the effects of phosphodiesterase inhibition on cyclic gmp and cyclic amp accumulation in the hippocampus of the rat. brain res. 2001;888:275–286. 7. zhang ht, o’donnell jm. effects of rolipram on scopolamine-induced impairment of working and reference memory in the radial-arm maze tests in rats. psychopharmacology. 2000;150:311–6. 8. zhang ht, huang y, suvarna nu, et al. effects of the novel pde4 inhibitors mem1018 and mem1091 in models of memory and antidepressant sensitivity. abstr soc neurosci. 2002;684:12. 9. bourtchouladze r, lidge r, catapano r, et al. a mouse model of rubinsteintaybi syndrome: defective long-term memory is ameliorated by inhibitors of phosphodiesterase 4. proc natl acad sci u s a. 2003;100:10518–10522. 10. barad m, bourtchouladze r, winder dg, golan h, kandel e. rolipram, a type iv-specific phosphodiesterase inhibitor, facilitates the establishment of long-lasting long-term potentiation and improves memory. proc natl acad sci u s a. 1998;95:15020–15025. 11. comery ta, martone rl, aschmies s, et al. acute secretase inhibition improves cognition in the tg2576 mouse model of alzheimer’s disease. abstr soc neurosci. 2003;525:21. 12. prickaerts j, steinbusch hwm, smits jfm, de vente j. possible role of nitric oxide-cyclic gmp pathway in object recognition memory: effects of 7-nitroindazole and zaprinast. eur j pharmacol. 1997;337:125–136. 13. prickaerts j, van staveren wc, sik a, et al. effects of two selective phosphodiesterase type 5 inhibitors, sildenafil and vardenafil, on object recognition memory and hippocampal cyclic gmp levels in the rat. neuroscience. 2002;113:351–361. 14. domek-lopacinska k, strosznajder jb. the effect of selective inhibition of cyclic gmp hydrolyzing phosphodiesterases 2 and 5 on learning and memory processes and nitric oxide synthase activity in brain during aging. brain res. 2008;1216: 68–77. 15. bunsey m, eichenbaum h. selective damage to the hippocampal region blocks long-term retention of a natural and nonspatial stimulus-stimulus association. hippocampus. 1995;5:546–556. 16. ennaceur a, delacour j. a new one-trial test for neurobiological studies of memory in rats. 1. behavioral data. behav brain res. 1988;31:47–59. 17. prut l, belzung c. the open field as a paradigm to measure the effects of drugs on anxiety-like behaviors: a review. eur j pharmacol. 2003;463:3–33. 18. belzung c. measuring exploratory behavior. in: crusio we, gerlai rt eds. handbook of molecular genetic techniques for brain and behavior research (techniques in the behavioral and neural sciences). amsterdam: elsevier; 1999: 739–749. 19. reneerkens oa, rutten k, steinbusch hw, blokland a, prickaerts j. selective phosphodiesterase inhibitors: a promising target for cognition enhancement. psychopharmacology (berl). 2009;202:419–443. 20. halene tb, siegel sj. pde inhibitors in psychiatry—future options for dementia, depression and schizophrenia? drug discov today. 2007;12:870–878. 21. menniti fs, chappie ta, humphrey jm, schmidt cj. phosphodiesterase 10a inhibitors: a novel approach to the treatment of the symptoms of schizophrenia. curr opin investig drugs. 2007;8:54–59. 22. blokland a, schreiber r, prickaerts j. improving memory: a role for phosphodiesterases. curr pharm des. 2006;12:2511–2523. 23. rutten k, prickaerts j, hendrix m, van der staay fj, sik a, blokland a. timedependent involvement of camp and cgmp in consolidation of object memory: studies using selective phosphodiesterase type 2, 4 and 5 inhibitors. eur j pharmacol. 2007;558:107–112. 24. rutten k, prickaerts j, blokland a. rolipram reverses scopolamine-induced and time-dependent memory deficits in object recognition by different mechanisms of action. neurobiol learn mem. 2006;85:132–138. 25. devan bd, sierra-mercado d jr, jimenez m, et al. phosphodiesterase inhibition by sildenafil citrate attenuates the learning impairment induced by blockade of cholinergic muscarinic receptors in rats. pharmacol biochem behav. 2004;79:691–699. http://www.la-press.com effects of zaprinast and rolipram on olfactory and visual memory 29drug target insights 2014:8 26. van der staay fj, rutten k, bärfacker l, et al. the novel selective pde9 inhibitor bay 73-6691 improves learning and memory in rodents. neuropharmacology. 2008;55:908–918. 27. izquierdo la, barros dm, vianna mr, et al. molecular pharmacological dissection of shortand long-term memory. cell mol neurobiol. 2002;22:269–287. 28. izquierdo i, bevilaqua lr, rossato ji, bonini js, medina jh, cammarota m. different molecular cascades in different sites of the brain control memory consolidation. trends neurosci. 2006;29:496–505. 29. frey u, huang y y, kandel er. effects of camp simulate a late stage of ltp in hippocampal ca1 neurons. science. 1993;260(5114):1661–1664. 30. matsumoto y, unoki s, aonuma h, mizunami m. critical role of nitric oxidecgmp cascade in the formation of camp-dependent long-term memory. learn mem. 2006;13(1):35–44. 31. zhuo m, hu y, schultz c, kandel er, hawkins rd. role of guanylyl cyclase and cgmp-dependent protein kinase in long-term potentiation. nature. 1994; 368(6472):635–639. 32. tully t, bourtchouladze r, scott r, tallman j. targeting the creb pathway for memory enhancers. nat rev drug discov. 2003;2:267–277. 33. nagakura a, niimura m, takeo s. effects of a phosphodiesterase iv inhibitor rolipram on microsphere embolism-induced defects in memory function and cerebral cyclic amp signal transduction system in rats. br j pharmacol. 2002;135: 1783–1793. 34. itoh t, abe k, tokumura m, horiuchi m, inoue o, ibii n. different regulation of adenylyl cyclase and rolipram sensitive phosphodiesterase activity on the frontal cortex and hippocampus in learned helpnessness rats. brain res. 2003; 991(1–2):142–149. 35. scott ai, perini af, shering pa, whalley jl. in-patient major depression: is rolipram as effective as amitriptyline. eur j clin pharmacol. 1991;40(2):127–129. 36. dundore rl, clas dm, wheeler lt, et al. zaprinast increases cyclic gmp levels in plasma and in aortic tissue of rats. eur j pharmacol. 1993;249:293–297. 37. dundore rl, habeeb pg, pratt pf, becker lt, clas dm, buchholz ra. differential hemodynamic responses to selective inhibitors of cyclic nucleotide phosphodiesterases in conscious rats. j cardiovasc pharmacol. 1992;19:937–944. 38. hall cs. emotional behavior in the rat: i. defecation and urination as measures of individual differences in emotionality. j comp psychol. 1934;18:385–403. 39. walsh rn, cummins ra. the open field test: a critical review. psychol bull. 1976;83:481–504. 40. yıldız f, ulak g, erden bf, gacar n. anxiolytic-like effects of 7-nitroindazole in the rat plus-maze test. pharmacol biochem behav. 2000;65(2):199–202. 41. caton pw, tousman sa, quock rm. involvement of nitric oxide in nitrous oxide anxiolysis in the elevated plus-maze. pharmacol biochem behav. 1994;48: 689–692. 42. kendrick km, guevara-guzman r, zorrilla j, et al. formation of olfactory memories mediated by nitric oxide. nature. 1997;388:670–674. 43. sanchez-andrade g, james bm, kendrick km. neural encoding of olfactory recognition memory-review. j reprod dev. 2005;51:547–558. http://www.la-press.com 45drug target insights 2014:8 open access: full open access to this and thousands of other papers at http://www.la-press.com. drug target insights the expression of serum antibodies against gonadotropin-releasing hormone (gnrh1), progonadoliberin-2, luteinizing hormone (lh), and related receptors in patients with gastrointestinal dysfunction or diabetes mellitus bodil roth1, kerstin berntorp2 and bodil ohlsson1 department of clinical sciences, 1section of internal medicine and 2section of endocrinology, skåne university hospital, malmö, sweden. lund university, lund, sweden. a bstr act: gonadotropin-releasing hormone (gnrh) 1 and 2 and luteinizing hormone (lh) receptors have been described in the gastrointestinal tract. we have previously demonstrated antibodies in serum against gnrh1 in patients with gastrointestinal dysfunction and diabetes mellitus, and antibodies against gnrh receptor, lh, and lh receptor in patients with infertility. the aim of this study was to search for the expression of serum antibodies against gnrh1 with an improved enzyme-linked immune sorbent assay (elisa), and antibodies against progonadoliberin-2, gnrh2, gnrh receptor, lh, and lh receptor with newly developed elisas, in patients with gastrointestinal dysfunction or diabetes mellitus. healthy blood donors served as controls. medical records were scrutinized. our conclusion was that igm antibodies against gnrh1, progonadoliberin-2, and/or gnrh receptors were more prevalent in patients with functional gastrointestinal disorders, gastrointestinal dysmotility, and/or diabetes mellitus, whereas igg antibodies against these peptides, and lhand lh receptor antibodies, were expressed in the same magnitude as in controls. k e y wor ds: antibodies, diabetes mellitus, gastrointestinal dysmotility, gonadotropin-releasing hormone (gnrh), luteinizing hormone (lh), progonadoliberin-2 citation: roth et al. the expression of serum antibodies against gonadotropin-releasing hormone (gnrh1), progonadoliberin-2, luteinizing hormone (lh), and related receptors in patients with gastrointestinal dysfunction or diabetes mellitus. drug target insights 2014:8 45–50 doi:10.4137/dti.s19352. received: august 13, 2014. resubmitted: september 19, 2014. accepted for publication: october 8, 2014. academic editor: anuj chauhan, editor in chief type: original research funding: grants from the bengt ihre foundation, the king gustaf v:s and queen victoria free mason’s foundation, albert påhlsson foundation, and the development foundation of region skåne sponsored this study. the authors confirm that the funder had no influence over the study design, content of the article, or selection of this journal. competing interests: authors disclose no potential conflicts of interest. copyright: © the authors, publisher and licensee libertas academica limited. this is an open-access article distributed under the terms of the creative commons cc-by-nc 3.0 license. correspondence: bodil.ohlsson@med.lu.se paper subject to independent expert blind peer review by minimum of two reviewers. all editorial decisions made by independent academic editor. upon submission manuscript was subject to anti-plagiarism scanning. prior to publication all authors have given signed confirmation of agreement to article publication and compliance with all applicable ethical and legal requirements, including the accuracy of author and contributor information, disclosure of competing interests and funding sources, compliance with ethical requirements relating to human and animal study participants, and compliance with any copyright requirements of third parties. this journal is a member of the committee on publication ethics (cope). introduction irritable bowel syndrome (ibs) is a common gastrointestinal disorder, occurring in 10–15% of the population. the etiology is unknown, and no organic, pathognomonic changes have been established so far.1 relatively severe gastrointestinal dysmotility is often found as a complication in diabetes mellitus and rheumatologic and neurologic diseases.2,3 as women are affected by these disorders more often than men, hormonal influences on the gastrointestinal tract have been assumed.4 we have recently described the expression of gonadotropinreleasing hormone (gnrh, 1 and 2) and receptors for luteinizing hormone (lh) in the human enteric nervous system (ens).5–8 the effects of these peptides are not known, but a few studies have described an influence by gnrh and lh on the migrating motor complex (mmc).9–11 we have developed an in-house enzyme-linked immune sorbent assay (elisa) to describe the presence of serum antibodies against gnrh1 in patients with ibs, dysmotility, and diabetes mellitus, but not in inflammatory bowel disease (ibd) or celiac disease,12,13 as well as in patients with functional gastrointestinal complaints in association with primary sjögren’s syndrome.3,14 the pathophysiological role of these antibodies is unknown. the original http://www.la-press.com/drug-target-insights-journal-j23 http://www.la-press.com http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 http://dx.doi.org/10.4137/dti.s19352 http://creativecommons.org/licenses/by-nc/3.0/ http://creativecommons.org/licenses/by-nc/3.0/ mailto:bodil.ohlsson@med.lu.se roth et al 46 drug target insights 2014:8 elisa was associated with a high degree of background signaling.3,5,6,12,13 the method has now been further improved and we have also developed elisas for the gnrh receptor, lh, and lh receptor.14–16 furthermore, we have developed an elisa for measurement of antibodies against gnrh2 and its precursor, progonadoliberin-2, as these peptides are abundantly expressed in the ens,8,17 and could hypothetically be more important for gastrointestinal function than gnrh1. the aim of the present study was to search for the expression of antibodies against gnrh1, progonadoliberin-2, gnrh2, gnrh receptor, lh, and lh receptor with new, sensitive elisas, in a population of patients with symptoms and signs of functional gastrointestinal disorders or gastrointestinal dysmotility, and/or diabetes mellitus, and compare these to healthy blood donors, to confirm the presence of autoantibodies in these patients. materials and methods the study was performed in accordance with the helsinki declaration and after approval by the ethics review board of lund university. consecutive patients visiting the out-patient clinic of the departments of gastroenterology or endocrinology, skåne university hospital, malmö, were included. all patients and blood donors had given their written, informed consent to participate in the study. patients suffering from ibs or gastrointestinal dysmotility. consecutive patients suffering from gastrointestinal symptoms suggesting ibs or dysmotility over a 2-year period (2003–2004) were investigated depending on the severity of the symptoms.12 they underwent extensive examinations to exclude organic gastrointestinal disorders, eg endoscopy, radiologic examination, and abdominal ultrasound. this had been performed repeatedly for several years in some cases. routine blood and urine samples were analyzed. the gastrointestinal examination was then further complemented in order to evaluate gastrointestinal motility by manometry, gastric transit time, and full-thickness biopsies of the bowel wall, depending on the clinical picture.5,18–22 patients with functional sub-occlusion without mechanical obstruction, but abnormality in the antroduodenal manometry, were diagnosed as having chronic intestinal pseudo-obstruction (cipo).23 the criterion for enteric dysmotility (ed) was abnormality in the small bowel manometry, without sub-occlusion episodes.24 the diagnosis idiopathic gastroparesis was set when delayed gastric emptying rate was the only finding, without engagement of other segments. patients with gastrointestinal symptoms fulfilling the rome-iii criteria, with no abnormal pattern on antroduodenal manometry, were diagnosed as having ibs.1 patients were invited to participate in the study when organic, gastrointestinal diseases were excluded, and one of the above-mentioned diagnoses was set. patients suffering from diabetes mellitus. consecutive patients suffering from gastrointestinal complaints in addition to diabetes mellitus were examined during 2003–2004.2,12 the patients were examined by gastroduodenoscopy, esophageal manometry, and/or gastric emptying scintigraphy to evaluate the gastrointestinal motility.18–20,22 patients diagnosed as suffering from diabetes-related dysmotility, or who fulfilled the criteria for ibs, were invited to participate in the study. in addition, consecutive patients with diabetes mellitus, independent of the presence or absence of gastrointestinal symptoms, were considered for inclusion for a further 2-year period. inclusion criteria for the study were age 18 years and diabetes mellitus. the first patient who fulfilled the criteria at each consultation at the section of endocrinology, skåne university hospital, malmö, january 2008–february 2010, was asked to take part in the study when visiting the clinic for routine follow-up. exclusion criteria were renal failure requiring dialysis and severe cardiac morbidity. examinations of esophageal motility and gastric emptying were performed.18–20,22 for further description of the patients, see berntorp et al.13 at the time of inclusion, all patients with diabetes mellitus completed a questionnaire concerning symptoms related to disturbances of the gastrointestinal tract (“loss of appetite, swallowing disturbances, meal-related cough, early satiety, nausea, vomiting, weight loss, abdominal fullness, bloating, regurgitation, constipation, diarrhea, evacuation incontinence, symptomatic postprandial hypoglycemia, and postprandial perspiration”), which had previously been used for these patients.2,25,26 controls. blood samples from 200 consecutive, healthy blood donors (100 women), mean age of 42 ± 13 years, were collected at skåne university hospital, malmö, and provided a control group for the antibody analyses. study design. the patients and controls gave a blood sample, which consisted of 5.0  ml blood drawn into ssttubes (366566, bd vacutainer, plymouth, uk) containing gel and a coagulation activator. after the tubes had been turned 10 times, they were centrifuged for 15  minutes at 1,500 g. serum was separated, measured into aliquots, and frozen at -20°c and analyzed for the expression of antibodies. medical records were reviewed concerning duration of gastrointestinal symptoms, co-existing diseases, therapy treatments, hereditary factors, and routine laboratory analyses. measurement of antibodies. analyses of antibodies (igm and igg) against gnrh1, lh, and their receptors were carried out by elisas developed in-house as described previously.15,16 serum was chosen because heparin and citrate can affect the analyses and because serum had been taken from the control group. the method has been improved from the original elisa for gnrh antibodies,3,5,6,12,13 and we now use gnrh conjugated to ovalbumin (ova) (90215.02, innovagen, lund, sweden) with 16  mol gnrh/mol ova instead of a small peptide, ie 10 amino acids, as antigen. this provides a more stable and efficient analysis. another improvement is the calculation of relative units (ru) from a http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 the expression of serum antibodies against gnrh1 47drug target insights 2014:8 constructed standard curve generated from 200 blood donors, while in earlier studies3,5,6,12,13 any value above 0 was considered positive. the absorbance was measured at 405  nm after 30 minutes (gnrh1, gnrh receptor, lh receptor, and lh igm) or 60  minutes (lh igg) of incubation at room temperature (rt). the grades of anti-lh-, anti-gnrh1-, and anti-gnrh receptor antibodies were calculated as ru based on each standard curve. to construct standard curves, rabbit anti-human lh antibodies (mbs535386, mybiosource, san diego, ca, usa) were diluted from 1:3,000 to 1:192,000, mouse anti-human gnrh antibodies (ab62432, abcam, cambridge, usa) were diluted from 1:2,000 to 1:32,000, and rabbit anti-human gnrh receptor antibodies (90217.09, innovagen) were diluted from 1:8,000 to 1:128,000. antilh receptor antibodies were calculated as absorbance values multiplied by 1,000. in both cases, the background values of each sample were subtracted before calculation. the cut-off value for the presence of antibodies in healthy blood donors was set as ru 97.5th percentile. the intra-assay correlation coefficient of variation (cv) of gnrh and gnrh receptor igm antibodies was 10% and 8%, respectively (n = 6), and the inter-assay cv was 11% and 6%, respectively (n = 12). because of the lack of positive serum, no intra-assay or inter-assay cv of igg antibodies was calculated. the intra-assay cv of lh igg and lh igm was 5.6% and 9.2%, respectively (n = 8), and the inter-assay cv was 7.7% and 6.1%, respectively (n = 17). because of the lack of an appropriate commercial antibody, no intra-assay or inter-assay cv of lh receptor was calculated. since antibodies against lh and lh receptors were not expressed in patients with diabetes mellitus, these analyses were omitted in our ibs and dysmotility patients. to perform competitive elisa, sera from patients with antibodies above the cut-off level were incubated with 0.5% bovine serum albumin (bsa) in phosphate-buffered saline (pbs)-t with various amounts of gnrh1, gnrh receptor, or lh receptor, all three both conjugated and unconjugated with an ova peptide (innovagen) or lh (mbs537383, mybiosource), ie 50, 100, or 200 ng/100 μl, 30 minutes prior to the application to the microtiter plates. a new in-house elisa was set up for the analysis of igm antibodies against progonadoliberin-2, the precursor of gnrh2.17 the microtiter plates (456537, nunc, roskilde, denmark) were provided with a layer of recombinant progonadoliberin-2 (mbs1014236, mybiosource) in pbs or pbs only (an internal blank). after an overnight incubation at 4°c, the plates were washed three times with pbs-t and thereafter blocked with 0.5% bsa (a7030, sigma, st louis, usa) in pbs-t. sera from patients and healthy blood donors diluted to 1:400, or igg antibodies against human gnrh2 raised in rabbits (mbs6004097, mybiosource) in serial dilution (to provide a standard curve) with bsa in pbs-t, were added to the plates in triplicate (two wells coated with progonadoliberin-2 and one well coated with pbs) and incubated at rt for 2 hours. the washing procedure was repeated, and the deposition of autoantibodies directed to progonadoliberin-2 was detected using biotinylated, rabbit anti-human igm (673211, mp biomedicals, santa ana, california, usa), or goat anti-rabbit igg (b7389, sigma), diluted in pbs-t. a phosphatase substrate kit (37620, pierce, rockford, il, usa) was used to develop a color reaction. the absorbance was measured at 405  mm after 30  minutes of incubation at rt. antibody levels are presented as ru (values after subtracted background), and the concentration in each doublet is interpolated from the standard curve. the cut-off value to define the expression of antibodies in the healthy blood donors was set to ru 97.5th percentile. the intra-assay cv was 5.7% (n  =  8) and inter-assay cv was 8.3% (n  =  8). as igg antibodies against gnrh1 or its receptor have not been shown to be of importance in any previous study, those were not analyzed for progonadoliberin-2. statistical methods. data are described as mean ± standard deviation (sd) or median (interquartile range [iqr]). fischer’s exact test was used for dichotomous variables and the mann–whitney u-test for continuous variables. p  0.05 was considered as statistically significant. results patients suffering from ibs or dysmotility. forty-five patients (37 women [82%]), mean age 43  ±  17  years, were included. the mean duration of the gastrointestinal symptoms was 16 ± 15 years. twenty-five patients were diagnosed as having ibs, 11 of these patients had the alternating type of ibs (ibs-a), eight had constipation-predominant ibs (ibs-c), and six had diarrhea-predominant ibs (ibs-d). twelve patients suffered from ed, five from cipo, and three from idiopathic gastroparesis (table 1). apart from the gastrointestinal diagnosis, five patients had current or recent treatment for depression, and two cases of each of the following diseases were noted: asthma bronchialis, table 1. the expression of igm antibodies in patients with ibs or dysmotility. gnrh1 n (%) progonadoliberin-2 n (%) gnrh receptor n (%) ibs-a (n = 11) 4 (36) 3 (27) 2 (18) ibs-c (n = 8) 1 (12) ibs-d (n = 6) ed (n = 12) 2 (17) 1 (8) cipo (n = 5) 1 (2) gastroparesis (n = 3) note: the cut-off value to define the expression of antibodies in the healthy blood donors was set to relative unit (ru) 97.5th percentile. abbreviations: cipo, chronic intestinal pseudo-obstruction; ed, enteric dysmotility; gnrh, gonadotropin-releasing hormone; ibs-a , alternating type of ibs; ibs-c, constipation-predominant ibs; ibs-d, diarrhea-predominant ibs; n, number. http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 roth et al 48 drug target insights 2014:8 endometriosis, hypertension, hypothyroidism, and rheumatologic diseases. other sporadic diseases were migraine, panic attacks, parkinson’s disease, and thyrotoxicosis. all routine blood samples were within the reference values (data not shown). patients suffering from diabetes mellitus and gastrointestinal complaints. nineteen patients with diabetes mellitus and gastrointestinal complaints (10 women [53%]), mean age 50 ± 11 years, were included. the mean duration of diabetes mellitus was 31 ± 12 years, and of gastrointestinal complaints was 5 ± 4 years. seventeen patients had type 1 diabetes and two had type 2 diabetes. all were on insulin treatment and were under acceptable metabolic control (hba1c: 64.9 ± 10.1 mmol/mol). all 19 patients were examined using gastric emptying scintigraphy, and 12 using esophageal manometry. gastroparesis was found in 10 patients and esophageal dysmotility in eight patients. six of the patients had abnormal findings in both examinations, with no correlation between the abnormalities in the two organs. most of the patients also suffered from secondary complications of diabetes mellitus, such as retinopathy (79%), autonomic neuropathy (53%), and peripheral neuropathy (47%). as some of the patients with diabetes mellitus and gastrointestinal complaints had no pathological changes as found in the examinations (n  =  3), they were diagnosed as ibs patients. the most common symptoms were abdominal fullness (95%), bloating (79%), early satiety (68%), and constipation (68%). four of the patients had hypertension, two had hypothyroditis, two had chronic pancreatitis, and one had atrophic gastritis, depression, hypopituitarism, and rheumatoid arthritis, respectively. patients suffering from diabetes mellitus. forty patients with diabetes mellitus (27 women [68%]), mean age 51 ± 13 years, were included in the study. the patients were insulintreated (type 1 diabetes: 37 patients [92%]) and were under acceptable metabolic control (hba1c: 65.2 ± 9.5 mmol/mol). the duration of diabetes mellitus was 26 ± 13 years. esophageal dysmotility was more often found (60%) than gastroparesis (20%). twelve percent had dysmotility in both esophagus and the stomach. the most common diabetes complication was retinopathy (65%), followed by peripheral neuropathy (48%) and angiopathy (28%). although the patients were included consecutively, regardless of gastrointestinal symptoms, the majority reported symptoms related to food intake and the gastrointestinal tract. the most common symptoms were bloating (48%), abdominal fullness (40%), and early satiety (35%). apart from diabetes mellitus, three patients suffered from hypothyroidism and one from addison’s disease, atoxic adenoma, celiac disease, pernicious anemia, sjögren’s syndrome, thyrotoxicosis, and vitiligo, respectively. serum antibodies. the distribution of antibodies among subgroups of ibs or dysmotility patients is shown in table 1. when calculated as a whole group, they had a higher prevalence of igm antibodies against gnrh1 and progonadoliberin-2, and a tendency to a higher prevalence of igm antibodies against gnrh receptor, than healthy controls (table 2). the expression of igg antibodies against gnrh1 and gnrh receptor did not differ from that of the controls (p = 1.000 and p = 0.625, respectively). the expression of any of the antibodies was not associated with age (p = 0.311), duration of symptoms (p = 0.743), or subgroup of patients (p = 0.353). when comparing patients with ibs and dysmotility, the prevalence of antibodies was equal (p = 1.000). patients with gastrointestinal complaints related to diabetes mellitus only expressed igm antibodies against gnrh1, prevalence of which tended to be significantly elevated compared with controls (table 2). consecutive patients with diabetes mellitus had a higher prevalence of igm antibodies against progonadoliberin-2 than controls (table 2), whereas the expression of igm and igg antibodies against gnrh1 was equal (p = 1.000 and p = 1.000, respectively). except for a tendency toward a shorter half-time of gastric emptying rate in patients with antibodies against progonadoliberin-2 (27.0 [26.0–50.5] and 52.5 [36.2–71.438], respectively, p  =  0.056), no other clinical associations could be found. none of the patients with diabetes mellitus showed antibodies against the gnrh receptor, lh, or lh receptor. the elisa for the analysis of gnrh2 was unstable, and for this reason these data are not shown. the distribution of antibodies was equal in men and women among controls and patients with diabetes mellitus. in patients with ibs and dysmotility, all antibodies were expressed in women. table 2. the expression of igm antibodies in patients with ibs, dysmotility, and/or diabetes mellitus. gnrh1 n (%) p-value progonadoliberin-2 n (%) p-value gnrh receptor n (%) p-value ibs/dysmotility (n = 45) 6 (13) 0.007 4 (9) 0.040 4 (9) 0.087 diabetes mellitus with gi complaints (n = 19) 2 (11) 0.088 0 1.000 0 1.000 consecutive patients with diabetes mellitus (n = 40) 1 (2) 1.000 5 (12) 0.008 0 1.000 notes: fischer’s exact test was used for the comparisons between the prevalence of antibodies in the different groups and healthy controls. the cut-off value to define the expression of antibodies in the healthy blood donors was set to relative unit (ru) 97.5th percentile. p  0.05 was considered as statistically significant. abbreviations: gi, gastrointestinal; gnrh, gonadotropin-releasing hormone; n, number. http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 the expression of serum antibodies against gnrh1 49drug target insights 2014:8 discussion the major finding in our present study was that the expression in serum of igm antibodies against gnrh1 was higher in patients with ibs or dysmotility, and tended to be higher in patients with gastrointestinal complaints related to diabetes mellitus, compared to controls. the prevalence of igm antibodies against progonadoliberin-2 was higher in patients with ibs or dysmotility and in consecutive patients with diabetes mellitus, and the prevalence of igm antibodies against the gnrh receptor tended to be higher in patients with ibs or dysmotility, compared to controls. although these three diseases are classified as different entities, they have gastrointestinal pain and dysfunction in common. the difference in antibody prevalence between the two diabetes cohorts may be because of the fact that the patients with gastrointestinal complaints had a more severe gastrointestinal disorder with gastroparesis in 53% of the patients, compared to 20% in consecutive diabetes patients, and a higher prevalence of gastrointestinal symptoms. the present study confirms previous results that igm antibodies against gnrh1 and gnrh receptors are present in a subgroup of patients suffering from functional disorders and dysmotility,3,12,14 as well as in patients with diabetes mellitus.12,13 in the present elisa, a titer above the 97.5th percentile of the standard curves in controls was considered positive. in the former elisa, we classified all antibody levels above 0 in absorbance, after subtraction of background levels, as positive, and therefore the prevalence was lower in both controls and patients.3,12,13 we have previously reported gastrointestinal complications and expression of gnrh antibodies in serum and a reduced number of gnrh-containing enteric neurons in patients after treatment with gnrh analogs.5,6,27 however, the patients who expressed antibodies against gnrh1 in the present study cohort have not been treated by any gnrh analogs. both gnrh1 and gnrh2 are expressed in enteric neurons of the human ens.5,6,8,27 the antibodies may represent a primary, autoimmune disease rendering neuronal damage and gastrointestinal complaints. another possibility is that the antibodies are secondary to all kinds of damage of the ens exposing gnrh to immune-reactive cells.28 our hypothesis is, taking all studies in this field into consideration,3,5,6,12–14,16,27,29 that the antibodies are secondary to the neuronal damage in a subgroup of patients, and not causal. full-thickness biopsies are not considered in patients with ibs. thus, we cannot examine the expression of gnrh in the bowel wall in these patients. however, as gnrh is expressed in enteric neurons, and the number of gnrh-containing enteric neurons is reduced in patients suffering from dysmotility with serum antibodies against gnrh and progonadoliberin-2,5,6,8,27 we postulate that neuronal damage exposes gnrh and progonadoliberin-2 to immune-presenting cells, resulting in antibody formation. the antibodies could thus be markers of enteric neuron damage. we know from animal trials that in spite of a marked loss of 50% of enteric neurons after treatment with the gnrh analog buserelin, the gastrointestinal function is well preserved with unaffected weight and healthy rats.29 thus, a subgroup of ibs patients may have enteric neuropathy, which remains undiscovered by non-sensitive, clinical examinations. furthermore, we know neither the time relation in the development of symptomatology and antibodies nor the difference between the formation of antibodies against gnrh1 and progonadoliberin-2. it could possibly reflect a difference in time, one antibody appearing early in the disease process and one appearing later. all blood samples were collected several years after the debut of gastrointestinal complaints, which can influence the results, as the titer declines with time.5 no organic changes exist that are pathognomonic for ibs, although inflammatory mediators have been discussed in recent years,30 and active immune responses have been found in subpopulations of ibs patients.31 antibodies against gnrh and its receptor are the first specific, organic parameter found so far, and could represent a subpopulation of ibs. that only female patients with ibs and dysmotility expressed antibodies may depend on the female predominance in this group, and does not exclude the possibility that male patients may express antibodies as well. ibs is associated with affective disturbances and psychiatric disorders.4 functional magnetic resonance imaging (f mri) reveals significant differences in the neural processing of pain between ibs patients and controls, further underlining the importance of central mechanisms in the pathophysiology of visceral hypersensitivity in these patients.32,33 both diabetes mellitus and primary sjögren’s syndrome are associated with autonomic neuropathy and gastrointestinal complaints.2,14,25,26 since gnrh1 and gnrh2 are found in both the central and peripheral nervous systems,8,17,34 we do not know whether the antibody formation originates from a central or a peripheral neural injury. the association between autonomic neuropathy and lowered body mass index (bmi), and the occurrence of gnrh antibodies in the initial elisa,13 could not be confirmed. the present and former small studies do not allow a search for clinical associations with the antibody occurrence.3,14 the effect of gnrh and lh on the ens is not thoroughly evaluated, but seems to affect motility and secretion.9–11,35 no patient with diabetes mellitus in the present study, or primary sjögren’s syndrome in a previous study,14 expressed antibodies against lh or its receptor, as has previously been described in patients with infertility.16 autoantibodies against folliclestimulating hormone (fsh), lh, and ovarian factors have also been found by others in infertile women, and could indicate an autoimmune disorder targeting the ovary.36 this suggests that antibodies against lh or its receptor are more frequent in gynecological disorders than in gastrointestinal disorders. nevertheless, the presence of lh receptors in genital organs and the gastrointestinal tract7,8,34 could be a plausible explanation for the observed connection between dysfunction of the digestive tract and diseases of the genital organs in women.37–39 http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 roth et al 50 drug target insights 2014:8 in conclusion, we confirm previous results that igm antibodies against gnrh1 are elevated in serum in patients with ibs, dysmotility, and/or diabetes mellitus compared to controls. furthermore, also igm antibodies against progonadoliberin-2 and gnrh receptors are elevated in these patients, whereas igg antibodies against these peptides, or antibodies against lh or lh receptor, are not present. it remains to explain the mechanisms behind the antibody formation and to examine whether the gastrointestinal complaints, the autonomic neuropathy, or the psychological factors are associated with the formation. abbreviations cipo, chronic intestinal pseudo-obstruction; ed, enteric dysmotility; ens, enteric nervous system; gnrh, gonadotropin-releasing hormone; ibs, irritable bowel syndrome; ivf, in vitro fertilization; lh, luteinizing hormone. author contributions conceived and designed the experiments: br, kb, and bo. analyzed the data: br and bo. wrote the first draft of the manuscript: bo. contributed to the writing of the manuscript: br and kb. agree with manuscript results and conclusions: br, kb, and bo. jointly developed the structure and arguments for the paper: br, kb, and bo. made critical revisions and approved final version: br, kb, and bo. all authors reviewed and approved of the final manuscript. r efer ences 1. longstreth gf, thompson wg, chey wd, houghton la, mearin f, spiller rc. functional bowel disorders. gastroenterology. 2006;130:1480–1491. 2. ohlsson b, melander o, thorson o, olsson r, ekberg o, sundkvist g. oesophageal dysmotility, delayed gastric emptying and autonomic neuropathy correlate to disturbed glucose homeostasis. diabetologia. 2006;49:2010–2014. 3. ohlsson b, scheja a, janciauskiene s, mandl t. gnrh-antibodies and gastrointestinal complaints in patients with sjögren’s syndrome and progressive systemic sclerosis. scand j rheumatol. 2009;23:1–2. 4. canavan c, west j, card t. the epidemiology of irritable bowel syndrome. clin epidemiol. 2014;6:71–80. 5. ohlsson b, veréss b, janciauskiene s, montgomery a, haglund m, wallmark a. chronic intestinal pseudo-obstruction due to buserelin-induced formation of anti-gnrh antibodies. gastroenterology. 2007;132:45–51. 6. hammar o, ohlsson b, veress b, nordin fredrikson g, alm r, montgomery a. depletion of enteric gonadotropin-releasing hormone is found in a few patients suffering from severe gastrointestinal dysmotility. scand j gastroenterol. 2012; 47:1165–1173. 7. hammar o, veress b, montgomery a, ohlsson b. expression of luteinizing hormone receptor in the gastrointestinal tract in patients with and without dysmotility. dti. 2012;6:13–18. 8. sand e, bergvall m, ekblad e, d’amato m, ohlsson b. expression and distribution of gnrh, lh, and fsh and their receptors in gastrointestinal tract of man and rat. regul pept. 2013;10:24–28. 9. khanna r, browne rm, heiner ad, clench mh, mathias jr. leuprolide acetate affects intestinal motility in female rats before and after ovariectomy. am j physiol. 1992;262:g185–g190. 10. mathias jr, baskin gs, reeves-darby vg, clench mh, smith ll, calhoon jh. chronic intestinal pseudoobstruction in a patient with heart-lung transplant. therapeutic effect of leuprolide acetate. dig dis sci. 1992;37:1761–1768. 11. ducker te, boss jw, altug sa, et al. luteinizing hormone and human chorionic gonadotropin fragment the migrating myoelectric complex in rat small intestine. neurogastroenterol motil. 1996;8:95–100. 12. ohlsson b, sjöberg k, alm r, nordin fredrikson g. patients with irritable bowel syndrome and dysmotility express antibodies against gonadotropinreleasing hormone in serum. neurogastroenterol motil. 2011;23:1000–1006. 13. berntorp k, frid a, alm r, fredrikson gn, sjöberg k, ohlsson b. antibodies against gonadotropin-releasing hormone (gnrh) in patients with diabetes mellitus is associated with lower body weight and autonomic neuropathy. bmc res notes. 2013;6:329. 14. mandl t, roth b, ohlsson b. antibodies against gnrh and its receptor in patients with primary sjögren’s syndrome. scand j rheumatol. 2014;43:338–348. 15. roth b, ohlsson b. gastrointestinal symptoms and psychological well-being in patients with microscopic colitis. scand j gastroenterol. 2013;48:27–34. 16. hammar o, roth b, bengtsson m, mandl t, ohlsson b. autoantibodies and gastrointestinal symptoms in infertile women in relation to in vitro fertilization. bmc pregnancy childbirth. 2013;13:201. 17. pawson aj, morgan k, maudsley sr, millar rp. type ii gonadotrophinreleasing hormone (gnrh-ii) in reproductive biology. reproduction. 2003;126: 271–278. 18. collins pj, horowitz m, cook dj, harding pe, shearman dj. gastric emptying in normal subjects: a reproducible technique using a single scintillation camera and computer system. gut. 1983;24:1117–1125. 19. hanson m, lilja b. gastric emptying in smokers. scand j gastroenterol. 1987;22: 1102–1104. 20. castell ja, dalton cb, castell do. on-line computer analysis of human lower esophageal sphincter relaxation. am j physiol. 1988;255:g794–g799. 21. abrahamsson h, antov s, bosaeus i. gastrointestinal and colonic segmental transit time evaluated by a single abdominal x-ray in healthy subjects and constipated patients. scand j gastroenterol. 1988;23:72–80. 22. spechler sj, castell do. classification of oesophageal motility abnormalities. gut. 2001;49:145–151. 23. de giorgio r, sarnelli g, corinaldesi r, stanghellini v. advances in our understanding of the pathology of chronic intestinal pseudo-obstruction. gut. 2004;53: 1549–1552. 24. wingate d, hongo m, kellow j, lindberg g, smout a. disorders of gastrointestinal motility: towards a new classification. j gastroenterol hepatol. 2002; 17(suppl):s1–s14. 25. faraj j, melander o, sundkvist g, et al. oesophageal dysmotility, delayed gastric emptying and gastrointestinal symptoms in patients with diabetes mellitus. diabet med. 2007;24:1235–1239. 26. gustafsson rj, littorin b, berntorp k, et al. esophageal dysmotility is more common than gastroparesis in diabetes mellitus and is associated with retinopathy. rev diabet stud. 2011;8:258–265. 27. cordeddu l, bergvall m, sand m, roth b, papadaki e, li l, d’amato m, ohlsson b. severe, gastrointestinal dysmotility developed after treatment with gonadotropin-releasing hormone analogs. scand j gastroenterol. in press 2014. 28. klooker tk, braak b, koopman ke, et al. the mast cell stabiliser ketotifen decreases visceral hypersensitivity and improves intestinal symptoms in patients with irritable bowel syndrome. gut. 2010;59:1213–1221. 29. sand e, voss u, hammar o, et al. gonadotropin-releasing hormone analog buserelin causes neuronal loss in rat gastrointestinal tract. cell tissue res. 2013;351: 521–534. 30. de giorgio r, barbara g. is irritable bowel syndrome an inflammatory disorder? curr gastroenterol rep. 2008;10:385–390. 31. öhman l, simrén m. pathogenesis of ibs: role of inflammation, immunity and neuroimmune interactions. nat rev gastroenterol hepatol. 2010;7:163–173. 32. elsenbruch s, rosenberger c, enck p, forsting m, schedlowski m, gizewski er. affective disturbances modulate the neural processing of visceral pain stimuli in irritable bowel syndrome: an f mri study. gut. 2010;59:489–495. 33. larsson mb, tillisch k, craig ad, et al. brain responses to visceral stimuli reflect visceral sensitivity thresholds in patients with irritable bowel syndrome. gastroenterology. 2012;142:463.e–472.e. 34. naor z. signaling by g-protein-coupled receptor (gpcr): studies on the gnrh receptor. front neuroendocrinol. 2009;30:10–29. 35. soldani g, del tacca mm, bambini g, et al. effects of gonadotropin-releasing hormone (gnrh) on gastric secretion and gastrin release in the dog. drug metab dispos. 1993;21:818–822. 36. meyer wr, lavy g, decherney ah, visintin i, economy k, luborsky jl. evidence of gonadal and gonadotropin antibodies in women with a suboptimal ovarian response to exogenous gonadotropin. obstet gynecol. 1990;75:795–799. 37. wald a, van thiel dh, hoechstetter l, et al. gastrointestinal transit: the effect of the menstrual cycle. gastroenterology. 1981;80:1497–1500. 38. mathias jr, franklin r, quast dc, et al. relation of endometriosis and neuromuscular disease of the gastrointestinal tract: new insights. fertil steril. 1998;70:81–88. 39. gonenne j, esfandyari t, camilleri m, et al. effect of female sex hormone supplementation and withdrawal on gastrointestinal and colonic transit in postmenopausal women. neurogastroenterol motil. 2006;18:911–918. http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 https://doi.org/10.1177/1177392817701726 creative commons non commercial cc by-nc: this article is distributed under the terms of the creative commons attribution-noncommercial 4.0 license (http://www.creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the sage and open access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage). drug target insights volume 11: 1–14 © the author(s) 2017 reprints and permissions: sagepub.co.uk/journalspermissions.nav doi: 10.1177/1177392817701726 introduction dengue is a mosquito-borne infection found in the tropical and subtropical regions. in recent years, the transmission has increased in urban and semiurban areas and has become a major health problem of the international community. approximately 2.5 billion people are at risk of infection, of which almost 40% of them live in tropical or subtropical countries such as africa, southeast asia, the americas, and the pacific.1–3 this disease is caused by dengue virus (denv ) vector or carrier through female aedes mosquitoes, such as aedes albopictus (stegomyia albopicta) and aedes aegypti (stegomyia aegypti). most of the denv infections are asymptomatic; however, some of them showed symptoms of mild fever, which is also known as dengue fever (df), whereas few patients showed more acute severity, such as thrombocytopenia and ruptured blood capillaries, which is commonly called dengue hemorrhagic fever. in less severe cases, patients may experience shock hypovolemia, often called dengue shock syndrome, with the level of mortality being 5% to 10% per case.4 although most people could recover from this disease within a period of 2 to 3 weeks, there are neither specific treatments nor available drugs for df disease until now. dengue virus is a single-stranded rna virus which belongs to the flavivirus genus within the flaviviridae family, which also consists of several other pathogenic viruses such as west nile virus, tick-borne encephalitis virus, and yellow fever virus.3,5,6 it is composed of 5 serotypes, namely, denv-1, denv-2, denv-3, denv-4, and denv-5.7–10 this virus consists of 3 structural and 7 nonstructural (ns) proteins. the structural proteins comprise envelope protein (e), protein premembrane (prm), and protein core/capsid (c), whereas the ns modification of s-adenosyl-l-homocysteine as inhibitor of nonstructural protein 5 methyltransferase dengue virus through molecular docking and molecular dynamics simulation usman sumo friend tambunan1, mochammad arfin fardiansyah nasution1, fauziah azhima1, arli aditya parikesit1, erwin prasetya toepak1, syarifuddin idrus2 and djati kerami3 1bioinformatics research group, department of chemistry, faculty of mathematics and natural science, university of indonesia, depok, indonesia. 2industrial standardization laboratory, ministry of industrial affair, ambon, indonesia. 3mathematics computation research group, department of mathematics, faculty of mathematics and natural science, university of indonesia, depok, indonesia. abstract: dengue fever is still a major threat worldwide, approximately threatening two-fifths of the world’s population in tropical and subtropical countries. nonstructural protein 5 (ns5) methyltransferase enzyme plays a vital role in the process of messenger rna capping of dengue by transferring methyl groups from s-adenosyl-l-methionine to n7 atom of the guanine bases of rna and the rna ribose group of 2′oh, resulting in s-adenosyl-l-homocysteine (sah). the modification of sah compound was screened using molecular docking and molecular dynamics simulation, along with computational adme-tox (absorption, distribution, metabolism, excretion, and toxicity) test. the 2 simulations were performed using molecular operating environment (moe) 2008.10 software, whereas the adme-tox test was performed using various software. the modification of sah compound was done using several functional groups that possess different polarities and properties, resulting in 3460 ligands to be docked. after conducting docking simulation, we earned 3 best ligands (sah-m331, sah-m2696, and sah-m1356) based on δgbinding and molecular interactions, which show better results than the standard ligands. moreover, the results of molecular dynamics simulation show that the best ligands are still able to maintain the active site residue interaction with the binding site until the end of the simulation. after a series of molecular docking and molecular dynamics simulation were performed, we concluded that sah-m1356 ligand is the most potential sah-based compound to inhibit ns5 methyltransferase enzyme for treating dengue fever. keywords: s-adenosyl-l-homocysteine (sah), dengue virus (denv), ns5 methyltransferase, drug design, molecular docking, molecular dynamics received: december 7, 2016. accepted: march 1, 2017. peer review: six peer reviewers contributed to the peer review report. reviewers’ reports totaled 1093 words, excluding any confidential comments to the academic editor. type: original research funding: the author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: publication of this article was funded by the directorate of research and community engagement of universitas indonesia (drpm ui) through penelitian unggulan perguruan tinggi (pupt) 2016 (grant number: 1713/un2.r12/hkp.05.00/2016). declaration of conflicting interests: the author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. corresponding author: usman sumo friend tambunan, bioinformatics research group, department of chemistry, faculty of mathematics and natural science, university of indonesia, 16424 depok, indonesia. email: usman@ui.ac.id 701726dti0010.1177/1177392817701726drug target insightstambunan et al research-article2017 http://www.creativecommons.org/licenses/by-nc/4.0/ https://uk.sagepub.com/en-gb/journals-permissions mailto:usman@ui.ac.id 2 drug target insights protein consists of ns1, ns2a, ns2b, ns3, ns4a, ns4b, and ns5. the order of the highest immunogenicity to stimulate antibody production among structural protein is protein e, followed by protein prm and c. nonstructural enzymes such as ns3 protease with cofactor ns2b, ns3 helicase/nucleoside triphosphatase/rna 5′ triphosphatase, ns5 methyltransferase, and ns5 rna–dependent rna polymerase are known to have an imperative role in the replication of denv.11,12 the ns5 protein, which is known as the largest protein encoded by the flavivirus genome (contains a total of 900 amino acid residues), plays a crucial role in the replication process of viral life cycle.13,14 this protein possessed 3 conserved binding sites: s-adenosylmethionine (sam)/s-adenosyl-lhomocysteine (sah) binding site, guanosine triphosphate– binding site, and rna-binding site.15 the ns5 protein is responsible for 2 methylation activities: guanine n7 and nucleoside 2′-o-methylation, with the former occurring before the latter. these processes convert sam, which acts as the methyl donor in both processes, into sah.16,17 recent studies found that the inhibition of n7 methylation is more lethal to denv than 2′-o-methylation, as inhibition of n7 methylation can greatly reduce the replication activity of denv.18 therefore, the inhibition of n7 methylation process, which can be induced by targeting the sam/sah-binding site of ns5 methyltransferase, can become a promising target for the discovery of new denv drugs. drug design and discovery is a process that involves many disciplines, such as medicinal chemistry, pharmacology, biochemistry, and computational biology. previously, researchers conducted a trial-and-error method to find compounds that can act as inhibitors or potentially be developed as a lead drug, which is time-consuming and cost-intensive. today, with the help of computers, drug design can be more efficient.19,20 in the process of developing new drug molecules, virtual screening (vs) is a powerful tool that can be significantly involved as part of the computer-aided drug design and development (caddd) method. virtual screening can be used to assess whether known compounds, which are retained from any source of a molecular database, are possible to become lead compounds for a specific target.21 one method that is frequently used together with the vs process is molecular docking simulation; this simulation is often used to predict the small molecule’s binding orientation of the targeted protein in the hope of obtaining potential drug candidates based on the predicted binding affinities and activity.22–24 moreover, the caddd process is frequently followed by molecular dynamics simulation. this simulation is useful to predict the complex stability of the ligand and its target protein, although the whole process itself is computationally expensive and time-consuming.25,26 several studies related to the inhibition of ns3 and ns5 proteins, either in silico or in vitro, have been conducted earlier.13,16,27–36 the modification of certain chemical compounds, supported by the advancement of technology in bioinformatics and medicinal chemistry fields, can be done to find the structure that can be used as a new, potential antiviral drug candidate. in this study, we tried to prove that the modified compound of sah can inhibit the ns5 methyltransferase enzyme of denv using molecular docking and molecular dynamics simulation. the computational adme-tox (absorption, distribution, metabolism, excretion, and toxicity) test would be conducted as well to eliminate further the remaining potential ligand. materials and methods the methodology of this experiment has been further modified based on our established research pipeline.37–39 in this research, we used several kinds of offline and online software. molecular operating environment (moe) 2008.10,40,41 acd/labs chemsketch 12.01, perkinelmer chembiodraw 13.0, vegazz 3.0.5, caesa (computer-assisted estimation of synthetic accessibility, v2.4; symbiosis inc, lanham, md, usa), toxtree 2.5.0,42 and osiris property explorer43 were the offline and online software that were used to conduct this research, respectively. preparation of the standard ligands and the modif ied sah ligands in this study, the modified sah ligands and the standards were drawn in 2-dimensional format using acd/labs chemsketch 12.01 software, which later can be saved in .mol (mdl format molfile). all these ligands were further optimized using the vegazz software before they were entered into the moe database. in this software, optimization of several ligands occurred, such as geometry optimization and 3-dimensional (3d) structure-energy minimization. all ligands were optimized using mmff94x force field with root mean square (rms) gradient of 0.001. the other parameters were adapted to the existing default of moe 2008.10 software. the optimized ligands were later stored in .mdb database format. preparation of the ns5 methyltransferase denv the sequence data of ns5 methyltransferase enzyme were searched through the national center for biotechnology information (ncbi) database.44 the sequence was stored in fasta format. the entire sequence was further input into clustalw2 server (http://www.ebi.ac.uk/tools/clustalw2). on this server, the multiple sequence alignment (msa) was performed between ns5 methyltransferase enzyme sequences, which resulted in the most viable ns5 methyltransferase enzyme sequence for the 3d structure template, based on its highest score. the 3d structure of this enzyme can be later downloaded from the research collaboratory for structural bioinformatics protein data bank (rcsb pdb) through the http://www.rcsb.org/pdb/home/home.do. http://www.ebi.ac.uk/tools/clustalw2 http://www.rcsb.org/pdb/home/home.do tambunan et al 3 the downloaded pdb data were then opened with moe 2008.10 software for further optimization. the optimization process of ns5 methyltransferase protein begins by removing the water molecules and metal atoms, followed by protonation of the protein using “protonate 3d” default protocol. then, the energy minimization of the protein was done by selecting amber99 force field in the gas phase solvation. the rms gradient of 0.05 kcal/mol ǻ was selected, whereas the rest of parameters used the standard defaults that already exist. then, the minimized ns5 protein structure was saved in .moe format. molecular docking simulation of ns5 methyltransferase and modif ied sah ligands the process of molecular docking simulation between enzyme and ligand begins by opening the previously optimized protein structure in .moe format and the ligand database in .mdb format. after choosing the selected amino acid residues using “sitefinder” feature, the docking process was conducted by selecting compute → simulations → dock feature. the docking area was chosen based on the “sitefinder” result, whereas the “triangle matcher” placement method and london dg scoring function were selected during the docking process. the docking results were further validated with postplacement refinement, using the gbvi/wsa dg method. the docking simulation was conducted twice, first with 30 times and second with 100 times of placement retention process. the other parameters were adjusted based on a default setting. the docking results were stored in .mdb format. the best enzyme-ligand complex was selected based on its free binding energy value, inhibition constants, and molecular interactions. computational adme-tox and synthetic accessibility prediction in this study, the remaining ligand candidates were screened through their adme-tox properties using various software, namely, osiris property explorer (www.organic-chemistry. org), toxtree (toxtree.sourceforge.net), and adme-tox (http:// ilab.acdlabs.com). these software analyzed the druglikeness properties, prediction of adme-tox properties, and carcinogenicity/mutagenicity prognosis of the ligands, respectively. furthermore, the best ligands which have the best binding affinity to the ns5 methyltransferase protein would be checked for their synthetic accessibilities using caesa software. molecular dynamics simulation in this study, the molecular dynamics simulation was performed using moe 2008.10 software. the preparation begins with the ns5 methyltransferase-ligand preparation, which involves energy minimization and geometry optimization. the parameters set in the energy minimization step were almost similar to those in the molecular docking simulation; the only difference is the generalized born solvation model which was set in this step. this simulation consists of 3 major steps: initialization, equilibration, and production. the simulation was conducted with nosé-poincaré-anderson (npa) algorithm and canonical ensemble (nvt). in the initialization step, the simulation was performed for 100 ps at 300 k, whereas in the equilibration step, the simulation was conducted at 300 k, gradually increasing to 310 k for 100 ps. finally, the production step was implemented for 5000 ps (5 ns). the simulation results were later analyzed for determining the ns5 methyltransferase-ligand complex stabilities. results and discussion preparation of the standard ligands and the modif ied sah ligands the molecular structure of sah compound consists of 5 functional groups, including 2 amines, 2 alcohols, and a carboxylic group. these groups can be modified by other functional groups to alter its polarity or enhance its druglikeness. however, some groups, such as anhydride, epoxide, acyl halide, thiol, thiourea, aliphatic imine, and acrylamide, are potentially toxic and harmful because they are likely to undergo metabolic activation.45,46 the ns5 methyltransferase enzyme has 2 binding sites that are connected by a y-shaped slit. the first one is the sam-binding (methyl group donor) site, whereas the second is the rna-cap–binding site.47 in this study, the modification of the sah ligand was conducted based on the polarity and shape of the sam-binding site (figure 1). the sam-binding site itself consists of 14 amino acids (ser56, lys61, cys82, gly86, trp87, thr104, lys105, asp131, val132, phe133, asp146, ile147, lys181, and glu217) that could be selected as a drug target for ns5 methyltransferase.48 therefore, the modification of sah ligand should fit with the sam-binding site. the functional groups used in the sah modification are alcohols, esters, aldehydes, ketones, amides, amines, and carboxylic acids. these groups were chosen because they are commonly found in the marketed drugs and used in the drug design and development studies.49 based on the molecular structure, the sah modification took place at 5 different functional group sites that are mentioned earlier in this article (figure 2). before the sah modification was done and drawn, we made a list of the modification of the respective functional group to simplify the drawing process, and so, there are no identical compounds in the resulting 3460 sah-based ligands. also, 3 standard ligands were drawn: sah, sam, and ctwyc ligand (a cyclic peptide compound); the former 2 compounds were used as a natural substrate of ns5 methyltransferase enzyme, whereas the latter compound was the best ligand from our previous studies.50 each ligand was drawn using chemsketch acd/labs software and stored in mdl molfiles (v2000) (.mol) format. then, all ligands were http://www.organic-chemistry.org http://www.organic-chemistry.org http://ilab.acdlabs.com http://ilab.acdlabs.com 4 drug target insights optimized using the vegazz software before the docking process proceeded. the ligand optimization was further performed using moe 2008.10 software; this optimization includes geometry optimization and energy minimization. furthermore, the optimization of the ligand was done using mmff94x force field; this was done because this force field is frequently used to optimize the small-molecule structures that can be used later for molecular docking purpose. then, the “partial charge” protocol was conducted as well to nullify the binding energy that occurs outside the system, as well as balancing the charge’s system so that the docking process can be run smoothly. afterward, energy minimization was done to eliminate the undesirable interaction in the ligand structure, such as unfit, yet unlikely van der waals forces, and to minimize the steric effect that may occur in the system. the minimization was done at an rms gradient of 0.001 kcal/å.51 in general, the aim of ligand optimization itself is to eliminate any bad contact in the system and to generate the actual geometry of structure that may occur in nature. preparation of the ns5 methyltransferase denv searching of ns5 methyltransferase enzyme sequences. the ns5 methyltransferase enzyme sequences are searchable at ncbi web site, with “dengue methyltransferase” as the keywords in “protein sequences” selection. in this step, we obtained thousands of sequences that match the keywords, but we further eliminated these sequences with only 12 sequences left because we wanted to use “chain a dengue methyltransferase” as this sequence has the crystal structure in rcsb pdb database and is widely used in the dengue research field. these 12 sequences were saved in fasta format so that it can later be used for the msa step. multiple sequence alignment the aim of the msa step is to align all the leftover sequences to obtain the best sequence that will be used in the later process. this alignment was done using clustalw software via european molecular biology laboratory (embl) european bioinformatics institute web site (http://www.ebi.ac.uk). based on the result of alignment, we obtained 7 pdb sequences that have high similarity among the 12 sequences, which were 2p3l, 2p40, 2p3o, 2p41, 2p3q, 2p1d, and 1l9k. of these sequences, we chose 2p4152 because this pdb has the lowest resolution (at 0.18 nm). in addition, the 2p41 pdb structure has sah ligand as well, which can be used later for molecular docking simulation purposes. the structure optimization of the ns5 methyltransferase enzyme similar to the ligand optimization process, the optimization of the ns5 methyltransferase enzyme was done using moe 2008.10 software. in this particular step, we first removed any water molecules and sulfate ions ( )so4 2− that lie in the pdb structure. then, we selected the active site residues of the enzyme. figure 1. the 3-dimensional structure of ns5 methyltransferase enzyme (protein data bank id: 2p41) and the s-adenosylmethionine/s-adenosyl-lhomocysteine-binding site. figure 2. the place of functional group modification of s-adenosyl-lhomocysteine; the modification was conducted by changing the respective functional group into another, such as alcohol, carboxylic acid, and amine, resulting in 3460 ligands in the process. http://www.ebi.ac.uk tambunan et al 5 next is the “3d protonate step,” as well as “partial charge” and “energy minimization” steps. the 3d protonate step aims to add the hydrogen atoms that are likely missing in the pdb structure. then, the partial charge step was followed by an mmff94x method. finally, the energy minimization step was conducted in the gas phase solvation model. furthermore, the minimization force field was set into mmff94x; this force field is widely used in the field of computational biology for peptides, proteins, dna, and drug-like molecules.53 also, the rms gradient was set to 0.05 kcal/å, which is suitable for protein. molecular docking simulation of ns5 methyltransferase enzyme and sah-based ligands molecular docking simulation is a computational method that is used to describe the molecular interaction between a molecule (ligand) and its receptor, either a protein or an enzyme. the purpose of this simulation is to predict the ligand activity toward its receptor and to filter any compounds that can be used to interact with the receptor to become the potential inhibitors of the protein/enzyme.22,54 in this study, the molecular docking simulation was performed using moe 2008.10 software. the docking parameter involves placement, rescoring, retain, and refinement. we used triangle matcher as the placement parameter; this parameter serves to show a random movement of the ligand, based on charge and spatial fit, in the active site of the enzyme to produce the optimal bond orientation.55 also, this parameter is better than the “alpha matcher” because it can produce the pose/conformation which is more accurate and systematic.56 furthermore, london dg was used as the rescoring parameter. it is used to measure the biological activity of the ligand and the target protein by calculating the binding energies based on their molecular interaction from each pose/conformation that is generated in the software.57 the determination of the complex structure was done by selecting the smallest gibbs free binding energy (δgbinding) from the docking simulation result. the most negative δgbinding result indicates that the ligand conformation is the most stable, yet favorable complex conformation above all. from 3460 ligands that we tested in this simulation, we selected 98 ligands that have the lowest δgbinding from the standards. afterward, we ran another docking process to validate the docking results that we obtained previously. this led to 3 ligands that have the lowest δgbinding among others. table 1 shows the δgbinding and inhibition constant (pki) of the 3 best ligands, as well as 3 standard ligands that we tested in this study (figure 3). besides gibbs free binding energy and the inhibition constants, the molecular interaction of the enzyme and the ligand can also be observed. this molecular interaction includes hydrogen bonds, which are defined as the intermolecular force that occurs between an electronegative atom and hydrogen atom that covalently binds to high electronegativity atom.58 based on the docking results between the modified sah ligands with the active site of ns5 methyltransferase enzyme, the molecular interaction is as shown in table 2. in table 2, the red-colored letters show the sam-binding site residues. it can be seen that ligand sah-m331 has formed 4 hydrogen bonding interactions with the active site of ns5 methyltransferase enzyme, of which 1 interaction occurs in the pi-pi cation interactions with trp87. moreover, 6 hydrogen bonds, with 4 of them occurring in the active site of ns5 methyltransferase enzyme, occur in the ligand sah-2696. finally, the interaction between sah-1356 and ns5 methyltransferase enzyme has generated 4 hydrogen bonds, with 3 of them occurring in the active site (figure 4). this result shows that the modified sahbased ligands have a better binding affinity regarding the hydrogen bonding than the sah, sam, and ctwyc ligands, which has 1, 2, and 3 hydrogen bonds in the active site, respectively. the molecular interaction between ns5 methyltransferase enzyme and the modified sah-based ligands, as well as the standard ligands, is shown in figures 4 and 5, respectively. in silico adme-tox prediction predicting adme-tox properties by adme-tox software. the process of finding a suitable, yet effective, drug for a disease can be considered by their adme-tox (absorption, distribution, table 1. δgbinding and pki value from the docking results. ligand δgbinding (kcal/mol) pki sah-m331 −25.6660 18.6972 sah-m2696 −22.3764 16.3008 sah-m1356 −21.0147 15.3088 saha −17.5047 13.0172 sama −17.8713 13.0189 ctwyca −20.6330 15.0317 abbreviations: sah, s-adenosyl-l-homocysteine; sam, s-adenosylmethionine. astandard ligand. table 2. ligand interaction with the sam-binding site of ns5 methyltransferase enzyme. ligand hydrogen bond interaction site sah-m331 trp87, asp146, lys105, lys181 sah-m2696 asp146, lys181, lys61, trp87, gly81 sah-m1356 lys105, his110, glu149, lys61, asp146 saha arg160, lys105, glu149 sama asp 146, lys 61, glu111, his110 ctwyca lys181, asp146, lys61, arg57 abbreviations: sah, s-adenosyl-l-homocysteine; sam, s-adenosylmethionine. astandard ligand. red color indicates the active site of ns5 methyltransferase enzyme contact. 6 drug target insights metabolism, excretion, and toxicity) properties. any drug candidates that do not meet the adme-tox criteria will be eliminated to suppress the failure possibility in clinical trials. nowadays, the adme-tox prediction can be easily made with the help of computers using in silico method; one of the software that is frequently used in the analysis is admetox, which is an online software available in acd/labs (via https://ilab.acdlabs.com/ilab2/).59 through this software, we can predict the toxicity properties of a compound by comparing them with each fragment’s properties that are stored in the database, including oral bioavailability, passive absorption, health effect, toxicity probability, and active transport. a bioactive molecule which has a high oral bioavailability is often considered and can be developed further as therapeutic agents for many diseases.60 the adme-tox prediction using adme-tox software is shown in table 3. the numbers which are listed in the table show the toxicity probability; if the score is less than 0.7, then we assumed that the compound is safe for the selected organ/organ system. based on table 3, all of the 3 standard ligands have low oral bioavailability, yet they have weak active transport probability. however, the health effect prediction of these ligands shows a real promise, as they have great results compared with their standard ligands. overall, this test revealed sah-m1356 as the best ligand, whereas sah-m331 and sah-m2696 ligands show less favorable property than the former ligand but are still better than the standard ligands. carcinogenicity and mutagenicity predicting test by toxtree software. the next in silico analysis is to determine the carcinogenicity and mutagenicity probabilities of a compound. figure 3. the chemical structures of the standards and the best ligands from molecular docking simulation. https://ilab.acdlabs.com/ilab2/ tambunan et al 7 figure 4. molecular interaction and 3-dimensional visualization of binding site of sah-based ligands, namely, sah-m331 (top), sah-m2696 (middle), and sah-m1356 (bottom). sah indicates s-adenosyl-l-homocysteine. 8 drug target insights figure 5. molecular interaction and 3-dimensional visualization of binding site of standard ligands, namely, sah (top), sam (middle), and ctwyc (bottom). sah indicates s-adenosyl-l-homocysteine; sam, s-adenosylmethionine. tambunan et al 9 therefore, the toxtree (v2.5.0) software was used.42 this software predicts the probabilities based on the benigni/ bossa rule for carcinogenicity and mutagenicity compounds, which involves searching the structural alerts of a f ragment that could potentially contain either mutagenic or carcinogenic property in the compound.61 the carcinogenic properties of a compound can fall into 3 categories: genotoxic, nongenotoxic, and quantitative structure activity relationship (qsar)–based carcinogens, whereas the mutagenic feature of a compound can be determined by testing it against salmonella typhimurium. the results f rom this trial are shown in table 4. it can be seen from table 4 that sam and ctwyc peptide ligands have good properties, which did not show any carcinogenicity or mutagenicity probability. however, the sah ligand showed positive results in genotoxic carcinogens. interestingly, sah-m331 and sah-m2696 ligands are indicated to have same properties as well. in this test, once again, the sah-m1356 ligand proves to be the best ligand for showing no carcinogenic or mutagenic possibility. druglikeness prediction by osiris property explorer software. the toxicity and druglikeness analyses can be performed further using osiris property explorer software (www.organic-chemistry.org).43,62 the analysis using this software is similar to toxtree software, such as mutagenic and carcinogenic analysis. however, the osiris property explorer also offers irritant, reproductive harmless, and druglikeness analysis, which is more useful and beneficial than the later software regarding drug design and discovery field.63 druglikeness is related to the lipinski rule (also known as lipinski rule of five, ro5). this rule helps us to distinguish between drug-like and non–drug-like molecules by predicting its oral bioavailability through its molecular properties.64 in general, this rule states that the probability of a compound to be absorbed into the human body via lipid bilayer is high if it satisfies two or more criteria below: •• the molecular weight of the compound is less than 500 g/mol; •• has high lipophilicity (logp less than 5.0); table 3. the absorption, distribution, metabolism, excretion, and toxicity (adme-tox) property prediction by adme-tox software. parameters/ligands sah-m331 sah-m2696 sah-m1356 saha sama ctwyca oral bioavailability less than 30% less than 30% less than 30% less than 30% less than 30% less than 30% passive absorption poor poor poor poor poor poor health effects blood 0.78 0.83 0.67 0.79 0.10 0.99 cardiovascular 0.01 0.39 0.21 0.37 0.48 0.67 gastrointestinal 0.93 0.70 0.78 0.99 0.98 0.76 kidney 0.69 0.79 0.16 0.23 0.81 0.89 liver 0.74 0.58 0.58 0.66 0.83 1 lungs 0.59 0.39 0.12 0.18 0.51 0.07 active transport pep t1 not transported not transported not transported not transported not transported not transported asbt not transported not transported not transported not transported not transported not transported abbreviations: sah, s-adenosyl-l-homocysteine; sam, s-adenosylmethionine. astandard ligand. table 4. carcinogenicity and mutagenicity prediction by toxtree software. parameters/ligands sah-m331 sah-m2696 sah-m1356 saha sama ctwyca negative for genotoxic carcinogenicity no no yes no yes yes negative for nongenotoxic carcinogenicity yes yes yes yes yes yes potential salmonella typhimurium ta100 mutagen based on qsar no no no no no no potential carcinogen based on qsar no no no no no no abbreviations: sah, s-adenosyl-l-homocysteine; sam, s-adenosylmethionine; qsar, quantitative structure activity relationship. astandard ligand. http://www.organic-chemistry.org 10 drug target insights •• has less than 5 hydrogen bond donors; •• has no more than 10 hydrogen bond acceptors. furthermore, the determination of drug score parameter in the osiris property explorer is based on the calculation of the lipinski rule of five, the veber rule, and the mutagenic, tumorigenic, irritant, and reproductive effect probabilities.60,65 the result of this analysis is shown in table 5. the analysis shown in table 5 indicates that the standard ligands have good drug results, such as no risk of being mutagenic, tumorigenic, and other health effects, although the druglikeness and the drug score of the standard ligands showed poor results. furthermore, sah-m2696 and sah-m1356 ligands had no mutagenic, tumorigenic, irritant, and reproductive effect, although the former ligand has a good value of druglikeness and drug score than the latter. the sah-m331, however, reveals to be an irritant, thus decreasing its druglikeness and drug score. synthetic accessibility prediction. the synthetic accessibility prediction is used to analyze whether any modification of a compound can be synthesized in a wet lab or not. this prediction can be made using several kinds of software. in this study, we used caesa software to analyze the synthetic accessibility, residual complexity, and starting material of a compound based on databases such as acros, sigma-aldrich, and lancaster. the result of the analysis can be shown as a percentage; the greater the percentage, the bigger the chances for the compound to be synthesized. the result of synthetic accessibility prediction is shown in table 6. the above data indicate that the sah-m2696 ligand has the highest possibility to be synthesized rather than other ligands; this is because it has the highest percentage (at 58%) in synthetic accessibility, far greater than sah-m331 and sahm1356 (at 38% and 42%, respectively). molecular dynamics simulation of ns5 methyltransferase enzyme and sah-based ligands molecular dynamics simulation is used to predict the stability, movement, and conformation of protein-ligand complex that is observed over comparable time periods.66 thus, this simulation allows the enzyme-ligand complex to apply the induced-fit model and makes it more accurate and reliable than the docking simulation.25 in this study, the molecular dynamics simulation was done using moe 2008.10 software. the npa algorithm was applied in this simulation, with the amber94 force field, nvt canonical ensemble, at a temperature of 310 and 312 k. furthermore, this simulation was completely done using implicit solvent models. in general, this simulation can be divided into 3 main phases: initialization, equilibration, and production. first, the initialization stage was conducted to determine the state of the system, such as atomic coordinates, velocity, and potential energy. then, the equilibration stage was conducted to provide a relaxed state on the complex as a result of restraint when the system is heated.58 finally, the production stage was done to generate a trajectory of a simulation that showed the conformation changes of the complex in forms of atom coordinates in the simulation period.67 the production stage in this study was table 5. druglikeness property prediction by osiris property explorer software. parameters/ligands sah-m331 sah-m2696 sah-m1356 saha sama ctwyca mutagenic no no no no no no tumorigenic no no no no no no irritant low no no no no no reproductive effect no no no no no no druglikeness −1.12 0.89 −4.91 −7.18 −7.41 −1.8 drug score 0.42 0.74 0.42 0.43 0.41 0.27 abbreviations: sah, s-adenosyl-l-homocysteine; sam, s-adenosylmethionine. astandard ligand. red color indicates the unfavorable drug properties of a compound. table 6. synthetic accessibility prediction of the selected ligands. parameters/ligands sah-m331 sah-m2696 sah-m1356 synthetic accessibility 38% 58% 42% residual complexity 56% 36% 53% starting materials 3% 42% 23% abbreviation: sah, s-adenosyl-l-homocysteine. this test was done using computer-assisted estimation of synthetic accessibility software. tambunan et al 11 conducted in 5 ns. the root-mean-square deviation (rmsd) value and molecular interaction of the complex from this simulation should be checked to determine the ligand-ns5 methyltransferase enzyme. the purpose of rmsd value is to describe the conformational changes between 2 atomic coordinates during molecular dynamics simulation. therefore, we can decide which ligandenzyme complex has better stability. as the temperature increases, the molecular interaction between the enzyme and the ligand should change, but not in an insignificant manner. hopefully, the presence of the modification of sah ligand can inhibit the action of the active denv at fever temperature (around 39°c or 312 k). as shown in figure 6, the ligand sah-m1356 tends to have the most stable complex with ns5 methyltransferase enzyme; this is because the complex of sah-m1356 ligand and ns5 methyltransferase enzyme has the smallest rmsd value at 312 k, indicating that the complex would be stably formed and the capping of new rna of denv would unlikely happen. in addition, the rmsd value of sah-m331 complex at different temperatures was very different. although at 310 k the complex seems to have low rmsd value (at 1.5-1.8 å), the complex tends to have high rmsd value (at 3.5-4.5 å) at 312 k. moreover, the ligand sah-m2696 has the highest rmsd value, compared with the other 2 ligands (3.5-4.0 å and 4.05.0 å at 310 and 312 k, respectively), suggesting that the ligand modification of sah-m2696 is more active and forms less stable ligand-enzyme complex than the other ligands. furthermore, the molecular interaction between the ligand-enzyme complexes between the different molecular dynamics simulation stages was also evaluated. in this study, we compared the molecular interaction, including hydrogen bond and residue contact, before and after molecular dynamics simulation was performed. as shown in table 7, the molecular interaction between the ligand modification of sah-m331, sah-m2696, and sah-m1356 still occurs with their respective enzymes at 2 different temperatures. however, we can also see that the molecular interaction of figure 6. the rmsd curve of sah-m331 ligand complex (top), sah-m2696 (middle), and sah-m1356 (bottom). rmsd indicates root-mean-square deviation; sah, s-adenosyl-l-homocysteine. 12 drug target insights figure 7. molecular interaction and 2-dimensional visualization of sah-based ligands after molecular dynamics simulation at 310 k (left side) and 312 k (right side), namely, sah-m331 (top), sah-m2696 (middle), and sah-m1356 (bottom). sah indicates s-adenosyl-l-homocysteine. table 7. comparison of molecular interaction before (after docking) and after dynamics simulation. simulation time/ ligands sah-m331 sah-m2696 sah-m1356 docking simulation trp87, asp146, lys105, lys181, asp146 asp146, lys181, lys61, asp146, trp87, gly81, asp146, trp87 lys105, his110, glu149, lys61, asp146 molecular dynamics simulation (310 k) asp79, asp79, gly81, gly81, asp146, asp146, asp146, arg57, lys61, arg160, lys181, tyr219, asp146, asp146, his110, trp87, trp87, trp87 asp79, asp79, asp79, leu80, glu111, asp146, asp146, asp146, glu149, glu149, lys61, trp87, lys181, asp79, glu111, asp146, asp146, lys61, trp87 asp146, gly148, lys42, arg57, arg57, lys61, trp87, his110, arg163, arg163, lys181 molecular dynamics simulation (312 k) gly81, glu111, asp146, asp146, asp146, gly148, lys105, lys105, arg160, arg163, arg163, asp146, glu149, trp87, arg163 ser59, asp79, gly106, glu111, glu111, leu144, asp146, asp 46, asp146, lys22, his110, asp146, lys22 glu111, asp146, gly148, arg163, arg163, arg212, arg212, lys105 abbreviation: sah, s-adenosyl-l-homocysteine. red color indicates the active site of ns5 methyltransferase enzyme contact. tambunan et al 13 these ligands, at various temperatures and stages, has significantly changed as well. after molecular dynamics simulation at 310 k, the sah-m331 complex tends to have better stability; this is shown from the 18 interactions that occur in the complex, including 5 interactions with asp146 and 2 interactions with lys181. moreover, the arene-cation interaction between nh3 + also happens with the benzene group of trp87. then, the sah-m2696 complex shows similar stability when 4 interactions with asp146 and lys61 occur. furthermore, the molecular interaction of sah-m1356 complex is interestingly changed from its docking interaction when the compound binds well with asp146, lys61, trp87, and lys161. during simulation at 312 k, the ligand modification of sah-m331, sah-m2696, and sahm1356 still undergoes its molecular interaction with their enzymes, forming 15, 13, and 8 interactions, respectively. therefore, we concluded that the molecular dynamics simulation increases the stability of the complex, and the molecular interactions occur more often because the enzymes and ligands are produced under flexible conditions, thus making the complex stable. the molecular interaction of sahm331, sah-m2696, and sah-m1356 with ns5 methyltransferase enzymes after molecular dynamics simulation at 310 and 312 k is shown in figure 7. conclusions in this study, 3460 modified sah-based ligands were created and tested against ns5 methyltransferase inhibitors through molecular docking and molecular dynamics simulation, along with computational adme-tox test. through all the simulations that we have done, we concluded that sah-m1356 is the most potential sah-based ligand to inhibit ns5 methyltransferase. this ligand was chosen because it has lower gibbs free binding energy and higher inhibition constant than our standard ligands (sah, sam, and ctwyc). furthermore, this ligand also has good adme-tox properties, such as low health risk effect, no carcinogenicity and mutagenicity risk, and good drug-like properties, from the computational adme-tox results. the molecular dynamics results also confirmed that sah-m1356 ligand has stable molecular interaction and rmsd score and indicated that the sah-m1356 and ns5 methyltransferase complex can be likely formed. therefore, to prove the computational results from our research, we highly recommended synthesizing this compound to further determine its bioactivity and inhibitory activity against denv infection by wet lab experiments. acknowledgements the authors would like to thank ratih dyah puspitasari for helping us to proofread the final manuscript. author contributions usf t and aap designed the whole study and supervised this research. dk and si created the general pipeline of this study and gave more relevant materials for the paper. mafn and ept helped in writing the paper. fa conducted the technical and experimental details. all authors approved the final manuscript. r efer ences 1. world health organization (who) and the special programme for research and training in tropical diseases (tdr). dengue: guidelines for diagnosis, treatment, prevention, and control. 2009:147. 2. world health organization (who). fact sheet: dengue and severe dengue. january 2012:1–4. 3. guzman mg, halstead sb, artsob h, et al. dengue: a continuing global threat. nat rev microbiol. 2010;8:s7–s16. 4. tomlinson sm, malmstrom rd, watowich sj. new approaches to structurebased discovery of dengue protease inhibitors. infect disord drug targets. 2009;9:327–343. 5. kuno g, chang g, tsuchiya k, karabatsos n, cropp c. phylogeny of the genus flavivirus. j virol. 1998;72:73–83. 6. nitsche c, holloway s, schirmeister t, klein cd. biochemistry and medicinal chemistry of the dengue virus protease. chem rev. 2014;114: 11348–11381. 7. mukhopadhyay s, kuhn rj, rossmann mg. a structural perspective of the flavivirus life cycle. nat rev microbiol. 2005;3:13–22. 8. amarilla aa, de almeida ft, jorge dm, et al. genetic diversity of the e protein of dengue type 3 virus. virol j. 2009;6:113. 9. vazquez y, pupo-antúnez m, vazquez sv, et al. monoclonal antibody to dengue capsid protein: its application in dengue studies. mabs. 2009;1:157–162. 10. mustafa ms, rasotgi v, jain s, gupta v. discovery of fifth serotype of dengue virus (denv-5): a new public health dilemma in dengue control. med j armed forces india. 2015;71:67–70. 11. khan am, miotto o, nascimento ejm, et al. conservation and variability of dengue virus proteins: implications for vaccine design. plos negl trop dis. 2008;2:e272. 12. halstead sb. dengue virus-mosquito interactions. annu rev entomol. 2008;53:273–291. 13. lim sp, noble cg, shi p-y. the dengue virus ns5 protein as a target for drug discovery. antiviral res. 2015;119:57–67. 14. perera r, kuhn rj. structural proteomics of dengue virus. curr opin microbiol. 2008;11:369–377. 15. dong h, zhang b, shi py. flavivirus methyltransferase: a novel antiviral target. antiviral res. 2008;80:1–10. 16. noble cg, chen yl, dong h, et al. strategies for development of dengue virus inhibitors. antiviral res. 2010;85:450–462. 17. podvinec m, lim sp, schmidt t, et al. novel inhibitors of dengue virus methyltransferase: discovery by in vitro-driven virtual screening on a desktop computer grid. j med chem. 2010;53:1483–1495. 18. dong h, chang dc, xie x, et al. biochemical and genetic characterization of dengue virus methyltransferase. virology. 2010;405:568–578. 19. schneider g, fechner u. computer-based de novo design of drug-like molecules. nat rev drug discov. 2005;4:649–663. 20. kapetanovic im. computer-aided drug discovery and development (caddd): in silico-chemico-biological approach. chem biol interact. 2008;171:165–176. 21. patrick gl. introduction of medicinal chemistry. 5th ed. oxford, uk: oxford university press; 2013. 22. kitchen db, decornez h, furr jr, bajorath j. docking and scoring in virtual screening for drug discovery: methods and applications. nat rev drug discov. 2004;3:935–949. 23. kolb p, ferreira rs, irwin jj, shoichet bk. docking and chemoinformatic screens for new ligands and targets. curr opin biotechnol. 2009;20:429–436. 24. walker sd, mceldowney s. molecular docking: a potential tool to aid ecotoxicity testing in environmental risk assessment of pharmaceuticals. chemosphere. 2013;93:2568–2577. 25. alonso h, bliznyuk aa, gready je. combining docking and molecular dynamic simulations in drug design. med res rev. 2006;26:531–568. 26. zhao h, caflisch a. molecular dynamics in drug design. eur j med chem. 2014;91:4–14. 27. chung ky, dong h, chao at, shi py, lescar j, lim sp. higher catalytic efficiency of n-7-methylation is responsible for processive n-7 and 2′-o methyltransferase activity in dengue virus. virology. 2010;402:52–60. 28. lim sp, sonntag ls, noble c, et al. small molecule inhibitors that selectively block dengue virus methyltransferase. j biol chem. 2011;286:6233–6240. 29. stahla-beek hj, april dg, saeedi bj, et al. identification of a novel antiviral inhibitor of the flavivirus guanylyltransferase enzyme. j virol. 2012;86: 8730–8739. 14 drug target insights 30. tambunan usf, zahroh h, utomo bb, parikesit aa. screening of commercial cyclic peptide as inhibitor ns5 methyltransferase of dengue virus through molecular docking and molecular dynamics simulation. bioinformation. 2014;10:23–27. 31. de sousa lrf, wu h, nebo l, et al. flavonoids as noncompetitive inhibitors of dengue virus ns2b-ns3 protease: inhibition kinetics and docking studies. bioorganic med chem. 2015;23:466–470. 32. hassandarvish p, rothan ha, rezaei s, et al. in silico study on baicalein and baicalin as inhibitors of dengue virus replication. rsc adv. 2016;6:31235–31247. 33. deng j, li n, liu h, et al. discovery of novel small molecule inhibitors of dengue viral ns2b-ns3 protease using virtual screening and scaffold hopping. j med chem. 2012;55:6278–6293. 34. mukhametov a, newhouse ei, aziz na, saito ja, alam m. allosteric pocket of the dengue virus (serotype 2) ns2b/ns3 protease: in silico ligand screening and molecular dynamics studies of inhibition. j mol graph model. 2014;52:103–113. 35. xu s, li h, shao x, et al. critical effect of peptide cyclization on the potency of peptide inhibitors against dengue virus ns2b-ns3 protease. j med chem. 2012;55:6881–6887. 36. liu hh, wu r, sun y, et al. identification of novel thiadiazoloacrylamide analogues as inhibitors of dengue-2 virus ns2b/ns3 protease. bioorganic med chem. 2014;22:6344–6352. 37. tambunan usf, bramantya n, parikesit aa. in silico modification of suberoylanilide hydroxamic acid (saha) as potential inhibitor for class ii histone deacetylase (hdac). bmc bioinformatics. 2011;12:s23. 38. bakri r, parikesit aa, satriyanto cp, kerami d, tambunan usf. utilization of boron compounds for the modification of suberoyl anilide hydroxamic acid as inhibitor of histone deacetylase class ii homo sapiens. adv bioinformatics. 2014;7:1–10. 39. tambunan usf, parikesit aa, ghifari as, satriyanto cp. in silico identification of 2-oxo-1,3-thiazolidine derivatives as novel inhibitors candidate of class ii histone deacetylase (hdac) in cervical cancer treatment. arab j chem. 2015. doi:10.1016/j.arabjc.2015.07.010. 40. molecular operating environment (moe), 2008.10; chemical computing group inc., 1010 sherbooke st. west, suite #910, montreal, qc, canada, h3a 2r7, 2016. 41. vilar s, cozza g, moro s. medicinal chemistry and the molecular operating environment (moe): application of qsar and molecular docking to drug discovery. curr top med chem. 2008;8:1555–1572. 42. toxtree—toxic hazard estimation by decision tree approach. http://toxtree. sourceforge.net/. accessed may 26, 2015. 43. sander t, freyss j, von korff m, reich jr, rufener c. osiris, an entirely inhouse developed drug discovery informatics system. j chem inf model. 2009;49:232–246. 44. national center for biotechnology information. refseq: ncbi reference sequence database. https://www.ncbi.nlm.nih.gov/refseq/. accessed january 18, 2017. 45. ansel hc. pengantar bentuk sediaan farmasi. 1st ed. jakarta, indonesia: universitas indonesia press; 2005. 46. hakimelahi gh, khodarahmi ga. the identification of toxicophores for the prediction of mutagenicity, hepatotoxicity and cardiotoxicity. j iran chem soc. 2005;2:244–267. 47. lim sp, wen d, yap tl, yan ck, lescar j, vasudevan sg. a scintillation proximity assay for dengue virus ns5 2′-o-methyltransferase-kinetic and inhibition analyses. antiviral res. 2008;80:360–369. 48. idrus s, tambunan u, zubaidi aa. designing cyclopentapeptide inhibitor as potential antiviral drug for dengue virus ns5 methyltransferase. bioinformation. 2012;8:348–352. 49. ertl p. cheminformatics analysis of organic substituents: identification of the most common substituents, calculation of substituent properties, and automatic identification of drug-like bioisosteric groups. cheminform. 2003;34:374–380. 50. tambunan usf, alamudi s. designing cyclic peptide inhibitor of dengue virus ns3-ns2b protease by using molecular docking approach. bioinformation. 2010;5:250–254. 51. singh s, malik bk, sharma dk. molecular drug targets and structure based drug design: a holistic approach. bioinformation. 2006;1:314–320. 52. egloff mp, decroly e, malet h, et al. structural and functional analysis of methylation and 5′-rna sequence requirements of short capped rnas by the methyltransferase domain of dengue virus ns5. j mol biol. 2007;372:723–736. 53. panigrahi sk, desiraju gr. strong and weak hydrogen bonds in the protein-ligand interface. proteins. 2007;67:128–141. 54. teodoro ml, phillips j, kavraki le. molecular docking: a problem with thousands of degrees of freedom. ieee int conf robot. 2001;1:960–965. 55. cook it, leyh ts, kadlubar sa, falany cn. structural rearrangement of sult2a1: effects on dehydroepiandrosterone and raloxifene sulfation. horm mol biol clin investig. 2010;1:81–87. 56. manavalan b, murugapiran sk, lee g, choi s. molecular modeling of the reductase domain to elucidate the reaction mechanism of reduction of peptidyl thioester into its corresponding alcohol in non-ribosomal peptide synthetases. bmc struct biol. 2010;10:1. 57. nylander e. dockcontrol: a new integrated software for design of experiments and molecular docking: application to hiv-protease inhibitors. http://www8.cs.umu. se/education/examina/rapporter/evanylander.pdf. published 2007. 58. nurbaiti s, nagao h, saito h, hertadi r, martoprawiro ma. akhmaloka. domain motions of klenow-like dna polymerase i itb-1 in the absence of substrate. int j integr biol. 2010;9:104–110. 59. acd/i-lab 2.0. https://ilab.acdlabs.com/ilab2/index.php. accessed may 26, 2015. 60. veber df, johnson sr, cheng h, smith br, ward kw, kopple kd. molecular properties that influence the oral bioavailability of drug candidates. j med chem. 2002;45:2615–2623. 61. benigni r, bossa c, jeliazkova n, netzeva t, worth a. the benigni/bossa rulebase for mutagenicity and carcinogenicity—a module of toxtree. https:// eurl-ecvam.jrc.ec.europa.eu/laboratories-research/predictive_toxicology/doc/ eur_23241_en.pdf. published 2008. 62. osiris property explorer. http://www.organic-chemistry.org/prog/peo/. accessed may 26, 2015. 63. jarrahpour a, motamedifar m, zarei m, et al. petra, osiris, and molinspiration together as a guide in drug design: predictions and correlation structure/antibacterial activity relationships of new n-sulfonyl monocyclic β-lactams. phosphorus sulfur silicon relat elem. 2010;185:491–497. 64. lipinski ca. drug-like properties and the causes of poor solubility and poor permeability. j pharmacol toxicol methods. 2000;44:235–249. 65. lipinski ca, lombardo f, dominy bw, feeney pj. experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. adv drug deliv rev. 1997;23:3–25. 66. wagner s, steinmetz m, bode a, müller mm. high performance computing in science and engineering, garching/munich 2009 (transactions of the fourth joint hlrb and konwihr review and results workshop, dec. 8–9, 2009, leibniz supercomputing centre, garching/munich, germany). berlin, germany: springer; 2010. 67. sharma rd, lynn am, sharma pk, rajnee jawaid s. high temperature unfolding of bacillus anthracis amidase-03 by molecular dynamics simulations. bioinformation. 2009;3:430–434. http://toxtree.sourceforge.net/ http://toxtree.sourceforge.net/ https://www.ncbi.nlm.nih.gov/refseq/ http://www8.cs.umu.se/education/examina/rapporter/evanylander.pdf http://www8.cs.umu.se/education/examina/rapporter/evanylander.pdf https://ilab.acdlabs.com/ilab2/index.php https://eurl-ecvam.jrc.ec.europa.eu/laboratories-research/predictive_toxicology/doc/eur_23241_en.pdf https://eurl-ecvam.jrc.ec.europa.eu/laboratories-research/predictive_toxicology/doc/eur_23241_en.pdf https://eurl-ecvam.jrc.ec.europa.eu/laboratories-research/predictive_toxicology/doc/eur_23241_en.pdf http://http://www.organic-chemistry.org/prog/peo/ https://doi.org/10.1177/1177392819861114 drug target insights volume 13: 1–7 © the author(s) 2019 article reuse guidelines: sagepub.com/journals-permissions doi: 10.1177/1177392819861114 creative commons non commercial cc by-nc: this article is distributed under the terms of the creative commons attribution-noncommercial 4.0 license (http://www.creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the sage and open access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage). introduction atrial fibrillation (af) is the most common sustained cardiac arrhythmia, its prevalence increases dramatically with age and this prevalence is likely to increase in the next 50 years.1,2 moreover, af is associated with significant mortality and morbidity, and one in five of all strokes is attributed to af.3 management of af is a multifaceted strategy: control of underlying heart disease and risk factors, reversal of arrhythmia (rhythm control), controlling the rate (rate control), along with prevention of associated risk of thrombo-embolic events, heart failure, and cardiac ischemia.3,4 rate or rhythm control often constitutes a dilemma in the management of af,5 and although clinical trials showed no major differences in outcomes in rhythm or rate control strategies, rhythm control is advised in recent-onset af, highly symptomatic patients, young and active individuals.6 moreover, rhythm control provides better clinical benefit when compared with rate control; also it decreases the risk of progression to permanent af.7 rhythm control can be achieved either by electrical or pharmacological cardioversion. however, acute cardioversion is only one of the strategies for rhythm control, and long-term anti-arrhythmic drug (aad) therapy and catheter ablation play critical roles in this perspective. vernakalant is a relatively novel aad, available in many parts of the world; it has been approved for pharmacological cardioversion of recent-onset af (⩽7 days) and early (⩽3 days) post cardiac surgery af.8,9 this review focuses on the role and benefit of vernakalant in the management recent-onset af, on the light of the current available scientific and medical literature. methodology through a medline/pubmed research, we used separately the terms “vernakalant,” “rsd1235,” “atrial fibrillation.” the search started from 2000, selected articles mainly address the clinical use, benefit, and safety rather than the pure pharmacodynamic effect. editorial reports were excluded, and we retained 51 articles found to be relevant for the study. background the increasing prevalence of af in the general population, along with the increase in af complications, makes the burden of af significantly heavy as a medical condition, also as a socio-economical issue given the cost associated with management, prevention, and complications3,4 moreover, despite all recent therapeutic efforts, there appears to be in most cases, an inevitable progression from paroxysmal to persistent and then to permanent form.3 there is still some controversy regarding rate vs rhythm control in af, and a wait-and-see approach with rate control medication may be adopted for patients with recent-onset symptomatic af in the emergency department, especially that recent-onset af resolves spontaneously within 24 h in more than 70% of the cases.10 moreover, the 2016 esc guidelines stated that rhythm control should be considered in patients who remain symptomatic despite rate control approach.3 however, there is a clinical trend or opinion that rhythm control is better to prevent atrial remodeling and progression from paroxysmal or persistent to permanent af.6,7 rhythm control may be achieved via either electrical or pharmacological cardioversion; in this regard, physician preference plays a role in the vernakalant in atrial fibrillation: a relatively new weapon in the armamentarium against an old enemy antoine kossaify electrophysiology unit, cardiology division, holy spirit university of kaslik (usek) and university hospital notre dame des secours, byblos, lebanon. abstract: atrial fibrillation is the most common sustained cardiac arrhythmia, and its prevalence is increasing with age; also it is associated with significant morbidity and mortality. rhythm control is advised in recent-onset atrial fibrillation, and in highly symptomatic patients, also in young and active individuals. moreover, rhythm control is associated with lower incidence of progression to permanent atrial fibrillation. vernakalant is a relatively new anti-arrhythmic drug that showed efficacy and safety in recent-onset atrial fibrillation. vernakalant is indicated in atrial fibrillation (⩽7 days) in patients with no heart disease (class i, level a) or in patients with mild or moderate structural heart disease (class iib, level b). moreover, vernakalant may be considered for recent-onset atrial fibrillation (⩽3 days) post cardiac surgery (class iib, level b). although it is mainly indicated in patients with recent-onset atrial fibrillation and with no structural heart disease, it can be given in moderate stable cardiac disease as alternative to amiodarone. similarly to electrical cardioversion, pharmacological cardioversion requires a minimal evaluation and cardioversion should be included in a comprehensive management strategy for better outcome. keywords: vernakalant, atrial, fibrillation, pharmaceutical, cardioversion received: april 23, 2019. accepted: june 3, 2019. type: review funding: the author(s) received no financial support for the research, authorship, and/or publication of this article. declaration of conflicting interests: the author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. corresponding author: antoine kossaify, electrophysiology unit, cardiology division, holy spirit university of kaslik (usek) and university hospital notre dame des secours, byblos, p.o box 3, zip 12345; jbeil, lebanon. email: antoinekossaify@yahoo.com 861114dti0010.1177/1177392819861114drug target insightskossaify review-article2019 https://uk.sagepub.com/en-gb/journals-permissions mailto:antoinekossaify@yahoo.com 2 drug target insights decision process, and this decision varies depending on previous experience, local tradition, and regulations. of note, pharmacological cardioversion is preferred as a first-line approach in patients who tolerate their arrhythmia especially when no hemodynamic compromise is present.3 the advantages of pharmacological cardioversion are that there is no need for general anesthesia or conscious sedation with fasting, along with potentially lower psychological impact related to electrical cardioversion and arguably a lower risk of immediate recurrence; the lower risk of immediate recurrence is probably related to better aad loading ordinarily implemented in pharmacological cardioversion, providing a significant efficacy immediately and for the subsequent hours and days following cardioversion.9 pharmacological technique using the “pill-in-the-pocket” (ie, flecainide, propafenone, sotalol) as a prompt method to terminate paroxysmal af is debated. use and success of the “pill-in-the-pocket” technique depends on the context, urgency of the situation, patient compliance, physician experience, availability of the drug, underlying heart disease, and so on.11 in this respect, there is currently insufficient evidence to support a recommendation for the use of the “pill-in-the-pocket” strategy in patients with paroxysmal af.11 moreover, in the setting of paroxysmal af, many aads have a slow onset of action (ie, amiodarone, beta-blockers) or may have some restrictions for use in patients with underlying heart disease (class 1 aad). in view of this, the development of new and effective aad to manage patients with paroxysmal af was sought.9,11 vernakalant was first used in clinical practice in 2004 by craft investigators,12 under the investigational product name of rsd1235, and the authors concluded that rsd1235 is a new atrial-selective aad, which is efficacious and safe for converting recent-onset af to sinus rhythm. later on in 2007, fedida13 reported on rsd1235 using the term vernakalant for the first time, which was presented as a novel atrial-selective antifibrillatory agent, with the electrophysiological properties targeting potassium channels that are selectively present in human atria; also they reported that vernakalant allows a safe and rapid conversion of acute af back to sinus rhythm. the 2010 guidelines for the management of af stated that vernakalant has recently been recommended for approval for rapid cardioversion of recent-onset af to sinus rhythm in adults.14 the 2016 esc guidelines for the management of af classified af in five categories: first diagnosed, paroxysmal, persistent, long-standing persistent, and permanent; also the 2016 esc guidelines stated that vernakalant should be considered for pharmacological cardioversion of recent-onset af, in patients with no history of ischemic or structural heart disease.3 electrophysiological properties of vernakalant normal atria have a resting membrane potential of (–70) to (–80) mv, about 10 mv more positive than that of the ventricles. during af, atria fail to fully repolarize, and therefore, the difference in resting membrane between atria and ventricles increases, and this accentuated difference is thought to play a part in vernakalant selectivity to atria as opposed to normal ventricles.15 vernakalant is a relatively new aad, and its antiarrhythmic activity is mainly correlated to the blocking property of the sodium channels (ina).15 moreover, vernakalant action varies with heart rate and with baseline membrane potential; at low rates and at negative membrane potential, vernakalant has a relatively weak blocking activity of the ina.13 as the heart rate increases, the affinity of vernakalant for ina increases and leads to greater ina blockade and fast onset of action, and this phenomenon explains why vernakalant is not efficient as preventive therapy.15,16 interestingly, vernakalant demonstrates a quick offset of binding once heart rate slows and when ina blockade in no longer required.15 moreover, vernakalant is able to block certain potassium channels, ikur (atrial-selective potassium channels), involved in atrial repolarization along with other potassium channels (ikach), resulting in prolonged action potential plateau; similarly, vernakalant blocks potassium channels ito, involved more with atrial than ventricular ref ractoriness.13,17 herg is a gene that codes for potassium channel known for its contribution to repolarization through ikr (“rapid” delayed rectifier current). when this channel is inhibited or compromised, it can result in a long qt interval.17 vernakalant is known to partially blocks the herg channel (underlying channel of ikr); this may prolong qt interval; however, this blockade occurs at minimal level and usually has no conclusive clinical effect at target vernakalant concentrations.15 all these properties result in clinical and electrophysiological effects of vernakalant, with prolongation of atrial refractory period, slowing atrio-ventricular (av ) nodal conduction, but without effect on av nodal refractoriness or on ventricular cells;18 moreover, qt interval and ventricular effective refractory period do not significantly change at target dosage. pharmacokinetics of vernakalant when vernakalant hydrochloride is infused at a dose of 0.1 to 5 mg/kg, intravenously over 10 min, the maximum plasma concentration (cmax) reaches 0.08 to 4 µg/ml and increases linearly with dose, the average cmax reaches 3.9 μg/ml after a single 10-min infusion of 3 mg/kg, and the average cmax is 4.3 μg/ml after 15 min of a second infusion of 2 mg/kg.19 moreover, the dose-normalized values for the area under the concentration-time curve (auc) do not differ significantly among doses, although the cmax is dose proportional. vernakalant half-life (t½) is between 2 and 4 h according to cytochrome p450 enzyme metabolizing activity.19 of note, the auc between 0 and 90 min after infusion of vernakalant is estimated to be 15% higher in cyp2d6 poor metabolizers than extensive metabolizers, with age and serum kossaify 3 creatinine having smaller influences on exposure; therefore, dose adjustments based on patient characteristics (concomitant drugs, renal function, age, etc) are unnecessary for intravenous vernakalant.20,21 in this regard, there is no dosage adjustment required in patient with renal or hepatic failure, or in the elderly; however, there is currently not enough data to recommend use of vernakalant in patients below 18 years old.21 the drug is extensively and rapidly distributed in the body after intravenous infusion, with a serum free fraction of 53% to 63% at concentration range of 1 to 5 μg/ml.20 vernakalant is not highly protein bound and accordingly, there is no significant competition between vernakalant and other highly protein-bound drugs such as amiodarone, warfarin, propranolol, diltiazem, and verapamil.21,22 clinical use of vernakalant since 2004, vernakalant (rsd1235) was presented by the craft investigators as a new atrial-selective aad, efficacious and safe for converting recent-onset af to sinus rhythm.12 later on, vernakalant has been tested in three placebo controlled trials (act i, act ii, and act iii) and was more effective than placebo for the rapid conversion of recent-onset af, and without significant adverse events.23–25 further studies showed that vernakalant is efficacious in converting recentonset (⩽7 days) af; also it is efficacious for recent-onset af (⩽3 days) occurring following heart surgery.26,27 in the avro study, vernakalant demonstrated superior efficacy compared with amiodarone for acute conversion of recent-onset af, and success rate was 51.7%, with a median time of conversion of 11 min; moreover, there were no significant side effects, also there were no cases of ventricular arrhythmia.28 table 1 illustrates key studies addressing vernakalant. the 2010 guidelines for the management of af recommended vernakalant for approval for rapid cardioversion of recent-onset af to sinus rhythm in adults.14 the 2012 focused update of the 2010 esc guidelines stated that vernakalant should be considered for recent-onset af (⩽7 days) in patients with mild or moderate structural heart disease (class iib, level b); also vernakalant may be considered for recent-onset af post cardiac surgery (⩽3 days) (class iib, level b).34 the 2016 esc guidelines for the management of af3 recommended vernakalant for pharmacological cardioversion (rhythm control) of recent-onset af, in patients with no history of ischemic or structural heart disease (recommendation: class i, level a). according to the same study,3 vernakalant may be considered as alternative to amiodarone in patients with stable ischemic and/or moderate structural heart disease, including mild to moderate heart failure (class ii, level b). figure 1 illustrates these clinical scenario. in a randomized controlled trial including 100 patients with recent-onset af, vernakalant was superior to ibutilide in converting af to sinus rhythm (shorter time to conversion to sinus rhythm and higher conversion rate at 90 min).30 of note, vernakalant is significantly more expensive when compared with ibutilide; however, fewer side effects and more rapid restoration of sinus rhythm are observed with vernakalant, reducing the overall cost of hospitalization in recent-onset af patients.35 in a meta-analysis conducted by bash et al, the efficacy of cardioversion of recent-onset af by vernakalant and by “comparators” (propafenone and flecainide) was studied, and the authors concluded that all these aads are efficient for rapid restoration of sinus rhythm; however, there was no mention regarding the time to conversion observed with each drug.36 when compared head-to-head with flecainide in patients with recent-onset af, vernakalant was more effective in cardio version to sinus rhythm; moreover, vernakalant allowed faster restoration of sinus rhythm, and therefore, patients can be discharged earlier from the emergency department.33,37 similarly, when compared head-to-head with propafenone in patients with recent-onset af, vernakalant showed higher efficacy and a shorter time to conversion of af to sinus rhythm and was associated with shorter hospital stay.29 vernakalant is usually a well-tolerated drug and most common side effects include paresthesia, dysgeusia, dizziness, sneezing, and nausea and these effects are probably related to the inhibition of the sodium channels in the central nervous system; also most of these side effects are mild and transient.38 of note, de riva-silva et al39 reported a case of 1:1 av conduction atrial flutter after vernakalant administration for af conversion; however, guerra et al38 reported that no cases of significant arrhythmia or hemodynamic dysfunction are generally observed when cautions and contraindications are respected. vernakalant is contraindicated in patients with hypersensitivity to the drug, high-grade av block which is not backed up by pacemaker, hypotension as defined by systolic blood pressure less than 100 mm hg, recent (<30 days) acute coronary syndrome, heart failure with nyha class iii and iv, severe aortic stenosis, and long qt interval; also vernakalant is contraindicated within 4 h after use of amiodarone (oral or iv ).34,38 vernakalant did not restore sinus rhythm in patients with atrial flutter in act ii and act iii trials,24,25 and only a reduced mean ventricular response rate was mostly observed in the studied patients. vernakalant has rate related blocking effects on sodium channels (ina), which is the probable explanation for the lack of efficacy on atrial flutter patients; of note, no cases of 1:1 av conduction with rapid ventricular response are observed when vernakalant is used in atrial flutter.40 oral vernakalant (150, 300, and 500 mg) was studied in a single phase iib trial as a prophylactic drug for sinus rhythm maintenance after direct current cardioversion;41 the 150 and 300 mg doses did not show superiority against placebo, and 4 drug target insights table 1. main studies addressing vernakalant. study/author/design/objective date patients conversion rate results regarding vernakalant craft12 multi-centered, randomized, doubleblinded (vkl vs placebo) 2004 56 pts recent-onset af 53% rsd1235: efficacious and safe for converting recent-onset af act i23 randomized, double-blind, placebocontrolled 2008 336 pts recent-onset af 51.7% rapid conversion of short-duration af act ii24 randomized, double-blind, placebocontrolled 2009 100 pts af or afl post cardiac surgery 47% safe and effective in the rapid conversion of af post cardiac surgery act iii25 randomized, double-blind, placebocontrolled 2010 138 pts af or afl of recent onset 51.2% rapid and efficient for conversion of short-duration af stiell et al26 (act iv) multicenter, open-label study 2010 236 pts recent-onset af 50.9% vkl rapidly converted recent-onset af to sr, was well tolerated avro28 randomized, double-blind, activecontrolled with amd 2011 254 pts recent-onset af 51.7% vkl vs 5.2% amd vkl was superior to amd for acute conversion of recent-onset af conde et al29 prospective trial 2013 36 pts recent-onset af 93% vkl vs 78% propafenone vkl was superior to propafenone simon et al30 randomized controlled trial 2017 100 pts recent-onset af 69% vkl vs 43% ibutilide vkl was superior to ibutilide carbajosa dalmau et al31 prospective multicenter 2017 165 pts recent-onset af 77.6% vkl is effective and safe for restoring sr in the emergency department akel and lafferty32 meta-analysis 2018 1421 pts recent-onset af na vkl is effective for rapid conversion of af pohjantähti-maaroos et al33 monocentric, retrospective 2019 200 pts recent-onset af 67% with vkl vs 46% flecainide vkl was more effective and faster than flecainide in cardioversion of af abbreviations: afl, trial flutter; amd, amiodarone; na, non available; pts, patients; sr, sinus rhythm; vkl, vernakalant. figure 1. clinical scenario and management strategy according to each case. class and level of evidence are represented between parentheses. af indicates atrial fibrillation. kossaify 5 only the 500 mg dose showed a slight superiority in preventing recurrence of af at 3 months. nevertheless, in march 2012, cardiome and merck announced the discontinuation of further research on oral vernakalant.38 vernakalant was approved in 2010 by the european union for cardioversion of af which was less than 7 days in duration, or for post-operative af less than 3 days in duration.9 vernakalant is still not approved by the united states food and drug administration, namely after the study “act v” was discontinued following the death of a single patient, and therefore, further safety data and protocol revision regarding drug administration were required for approval by the food and drug administration.9,38 opinions, clinical implications a lot of progress has occurred in rhythm control strategies for af, especially with the development of ablation procedures; however, pharmacological cardioversion should be preserved as first and integrated approach for the management of af. vernakalant showed a superior efficacy to amiodarone and to other aad for rapid conversion of recent-onset af;29,33,36,37,42 although it can be given in patients with mild to moderate cardiovascular conditions, the absence of structural heart disease is associated with greater conversion rate to sinus rhythm.43 vernakalant is a relatively safe drug and manolis et al44 reported successful utilization of intravenous vernakalant for af conversion in the regular ward under only bedside monitoring. anticoagulation should be started promptly in all patients presenting with af, not only in those presenting with af lasting since more than 48 h.45–48 moreover, cardioversion should not be attempted in those presenting with af lasting since more than 48 h before effective anticoagulation has been established for at least 3 weeks (except for patients with hemodynamic compromise requiring immediate cardioversion). however, if cardioversion needs to be performed sooner, then transesophageal echo guidance is recommended.49 clinical risk scores for stroke and systemic embolism including the cha2ds2-vasc score should be implemented more frequently in real practice.3,47 in this regard, a patient presenting to the emergency department needs a minimum workup to ensure that it is a recent-onset af, and this is required for evaluating potential underlying cardiopathy, and for assessment of the relevance of vernakalant use according to the estimated af duration. the new af guidelines3 classify a first time diagnosed af as “first diagnosed af,” which is defined as af that has not been diagnosed before, irrespective of its duration or severity. accordingly, patients presenting to emergency room for a first episode of af are classified as “first diagnosed af,” and this af may be otherwise paroxysmal or even persistent. in other terms, a patient may present to the emergency department with af only when symptoms become severe; however, he may be asymptomatic or pauci-symptomatic with an af lasting since days, weeks, or months, especially if the af is intermittent. before using vernakalant, patients should be adequately hydrated; the current dosage recommendation consists of a first infusion 3 mg/kg over 10 minutes, then a monitoring period of 15 minutes, and if af persists, a second infusion of 2 mg/kg over 10 minutes with a second monitoring period of 15 minutes, the maximal dose over 24 hours being 5 mg/kg3. interestingly, electrical cardioversion is still feasible after administration of vernakalant, and it enhances restoration of sinus rhythm as integrated approach for af; however, its use is recommended only 120 min after vernakalant administration; therefore, vernakalant may be considered as a useful agent for facilitated electrical cardioversion in resistant and recent-onset af;50 figure 2 illustrates this scenario. of note, vernakalant is indicated for recent-onset af; nonetheless, the indication is not extended to atrial flutter.3 carbajosa dalmau et al31 showed that af duration of less than 12 h was significantly associated with greater effectiveness in the hospital emergency department. finally, cost-effectiveness studies of vernakalant applied in “real world” remain to be figure 2. image showing the infusion scenario with dosage and monitoring time, ending with electrical cardioversion if af persists. af indicates atrial fibrillation. 6 drug target insights evaluated; moreover, caution about safety and use of vernakalant within specific patients’ subgroups must be considered.32,51 conclusions af is the most common arrhythmia in patients admitted to emergency departments or hospitalized in intensive care units; also it is associated with increased morbidity and mortality. the increasing incidence of af has prompted researches to find new therapeutic alternatives for such common and refractory arrhythmia. vernakalant, a relatively new aad with atrial-selective anti-arrhythmic activity, is currently approved in the european union and in many other countries for pharmacological cardioversion of recent-onset af. of note, the drug is still not approved in the united states; also multicentric cost effectiveness studies evaluating vernakalant in “real world” are still missing. nevertheless, current medical literature showed that vernakalant is safe and efficacious as anti-arrhythmic agent for terminating recent-onset af. author contributions conceived the concepts: ak. analyzed the data: ak. wrote the first draft of the manuscript: ak. contributed to the writing of the manuscript: ak. agree with manuscript results and conclusions: ak. developed the structure and arguments for the paper: ak. made critical revisions and approved final version: ak. orcid id antoine kossaify https://orcid.org/0000-0002-7104-1323 r efer ences 1. stewart s, hart cl, hole dj, mcmurray jj. population prevalence, incidence, and predictors of atrial fibrillation in the renfrew/paisley study. heart. 2001;86:516–521. 2. naccarelli gv, varker h, lin j, schulman kl. increasing prevalence of atrial fibrillation and flutter in the united states. am j cardiol. 2009;104:1534–1539. 3. kirchhof p, benussi s, kotecha d, et al. 2016 esc guidelines for the management of atrial fibrillation developed in collaboration with eacts. europace. 2016;18:1609–1678. 4. anderson jl, halperin jl, albert nm, et al. management of patients with atrial fibrillation. j am coll cardiol. 2013;61:1935–1944. 5. manolis as. rhythm or rate control management of atrial fibrillation: an overrated dilemma. hellenic j cardiol. 2015;56:495–500. 6. camm aj, savelieva i. atrial fibrillation: the rate versus rhythm management controversy. j r coll physicians edinb. 2012;42:23–34. 7. camm aj, breithardt g, crijns h, et al. real-life observations of clinical outcomes with rhythm-and rate-control therapies for atrial fibrillation recordaf. j am coll cardiol. 2011;58:493–501. 8. savelieva i, graydon r, camm aj. pharmacological cardioversion of atrial fibrillation with vernakalant: evidence in support of the esc guidelines. europace. 2014;16:162–173. 9. camm aj. the vernakalant story: how did it come to approval in europe and what is the delay in the u.s.a. curr cardiol rev. 2014;10:309–314. 10. dudink e, essers b, holvoet w, et al. acute cardioversion vs a wait-and-see approach for recent-onset symptomatic atrial fibrillation in the emergency department: rationale and design of the randomized acwas trial. am heart j. 2017;183:49–53. 11. saborido cm, hockenhull j, bagust a, boland a, dickson r, todd d. systematic review and cost-effectiveness evaluation of “pill-in-the-pocket” strategy for paroxysmal atrial fibrillation compared to episodic in-hospital treatment or continuous antiarrhythmic drug therapy. health technol assess. 2010;14:iii– iv, 1–75. 12. roy d, rowe bh, stiell ig, et al. a randomized, controlled trial of rsd1235, a novel anti-arrhythmic agent, in the treatment of recent onset atrial fibrillation. j am coll cardiol. 2004:2355–2361. 13. fedida d. vernakalant (rsd1235): a novel, atrial-selective antifibrillatory agent. expert opin investig drugs. 2007;16:519–532. 14. camm aj, kirchhof p, lip gy, et al. guidelines for the management of atrial fibrillation: the task force for the management of atrial fibrillation of the european society of cardiology (esc). eur heart j. 2010;31:2369–2429. 15. finnin m. vernakalant: a novel agent for the termination of atrial fibrillation. am j health syst pharm. 2010;67:1157–1164. 16. wettwer e, christ t, endig s, et al. the new antiarrhythmic drug vernakalant: ex vivo study of human atrial tissue from sinus rhythm and chronic atrial fibrillation. cardiovasc res. 2013;98:145–154. 17. heidbuchel h, vereecke j, carmeliet e. three different potassium channels in human atrium. circ res. 1990;66:1277–1286. 18. dorian p, pinter a, mangat i, korley v, cvitkovic ss, beatch gn. the effect of vernakalant (rsd1235), an investigational antiarrhythmic agent, on atrial electrophysiology in humans. j cardiovasc pharmacol. 2007;50:35–40. 19. mao zl, wheeler jj, clohs l, et al. pharmacokinetics of novel atrial-selective antiarrhythmic agent vernakalant hydrochloride injection (rsd1235): influence of cyp2d6 expression on other factors. j clin pharmacol. 2009;49:17–29. 20. mao zl, townsend rw, gao y, wheeler jj, kastrissios h, keirns j. population pharmacokinetics of vernakalant hydrochloride injection (rsd1235) in patients with atrial fibrillation or atrial flutter. j clin pharmacol. 2012;52: 1042–1053. 21. mao zl, wheeler jj, townsend r, gao y, kshirsagar s, keirns jj. population pharmacokinetic-pharmacodynamic analysis of vernakalant hydrochloride injection (rsd1235) in atrial fibrillation or atrial flutter. j pharmacokinet pharmacodyn. 2011;38:541–562. 22. cialdella p, pedicino d, santangeli p. novel agents for the acute conversion of atrial fibrillation: focus on vernakalant. recent pat cardiovasc drug discov. 2011;6:1–8. 23. roy d, pratt cm, torp-pedersen c, et al. vernakalant hydrochloride for rapid conversion of atrial fibrillation: a phase 3, randomized, placebo-controlled trial. circulation. 2008;117:1518–1525. 24. kowey pr, dorian p, mitchell lb, et al; atrial arrhythmia conversion trial investigators. vernakalant hydrochloride for the rapid conversion of atrial fibrillation after cardiac surgery: a randomized, double-blind, placebo-controlled trial. circ arrhythm electrophysiol. 2009;2:652–659. 25. pratt cm, roy d, torp-pedersen c, et al; atrial arrhythmia conversion trial investigators. usefulness of vernakalant hydrochloride injection for rapid conversion of atrial fibrillation. am j cardiol. 2010;106:1277–1283. 26. stiell ig, roos js, kavanagh km, dickinson g. a multicenter, open-label study of vernakalant for the conversion of atrial fibrillation to sinus rhythm. am heart j. 2010;159:1095–1101. 27. dalyanoglu h, mehdiani a, minol jp, et al. conversion of atrial fibrillation after cardiosurgical procedures by vernakalant as an atrial repolarization delaying agent (arda). heart surg forum. 2018;21:e201–e208. 28. camm aj, capucci a, hohnloser sh, et al. a randomized active-controlled study comparing the efficacy and safety of vernakalant to amiodarone in recentonset atrial fibrillation. j am coll cardiol. 2011;57:313–321. 29. conde d, costabel jp, aragon m, et al. propafenone versus vernakalant for conversion of recent-onset atrial fibrillation. cardiovasc ther. 2013;31:377–380. 30. simon a, niederdoeckl j, skyllouriotis e, et al. vernakalant is superior to ibutilide for achieving sinus rhythm in patients with recent-onset atrial fibrillation: a randomized controlled trial at the emergency department. europace. 2017;19: 233–240. 31. carbajosa dalmau j, cosin-sales j, perez-dura mj, et al. vernakalant in hospital emergency practice: safety and effectiveness. emergencias. 2017;29: 397–402. 32. akel t, lafferty j. efficacy and safety of intravenous vernakalant for the rapid conversion of recent-onset atrial fibrillation: a meta-analysis. ann noninvasive electrocardiol. 2018;23:e12508. 33. pohjantähti-maaroos h, hyppola h, lekkala m, sinisalo e, heikkola a, hartikainen j. intravenous vernakalant in comparison with intravenous flecainide in the cardioversion of recent-onset atrial fibrillation. eur heart j acute cardiovasc care. 2019;8:114–120. 34. camm aj, lip gy, de caterina r, et al. 2012 focused update of the esc guidelines for the management of atrial fibrillation: an update of the 2010 esc guidelines for the management of atrial fibrillation. developed with the special contribution of the european heart rhythm association. eur heart j. 2012;33:2719–2747. 35. vogiatzis i, papavasiliou e, dapcevitch i, pittas s, koulouris e. vernakalant versus ibutilide for immediate conversion of recent-onset atrial fibrillation. hippokratia. 2017;21:67–73. 36. bash ld, buono jl, davies gm, et al. systematic review and meta-analysis of the efficacy of cardioversion by vernakalant and comparators in patients with atrial fibrillation. cardiovasc drugs ther. 2012;26:167–179. 37. conde d, costabel jp, caro m, et al. flecainide versus vernakalant for conversion of recent-onset atrial fibrillation. int j cardiol. 2013;168:2423–2425. https://orcid.org/0000-0002-7104-1323 kossaify 7 38. guerra f, matassini mv, scappini l, urbinati a, capucci a. intravenous vernakalant for the rapid conversion of recent onset atrial fibrillation: systematic review and meta-analysis. expert rev cardiovasc ther. 2014;12:1067–1075. 39. de riva-silva m, montero-cabezas jm, salgado-aranda r, lopez-gil m, fontenla-cerezuela a, arribas-ynsaurriaga f. 1:1 atrial flutter after vernakalant administration for atrial fibrillation cardioversion. rev esp cardiol (engl ed). 2012;65:1062–1064. 40. camm aj, toft e, torp-pedersen c, et al. efficacy and safety of vernakalant in patients with atrial flutter: a randomized, double-blind, placebo-controlled trial. europace. 2012;14:804–809. 41. torp-pedersen c, raev dh, dickinson g, butterfield nn, mangal b, beatch gn. a randomized, placebo-controlled study of vernakalant (oral) for the prevention of atrial fibrillation recurrence after cardioversion. circ arrhythm electrophysiol. 2011;4:637–643. 42. yan h, aung tt, guoqiang z, zhengnan z, lan j, zhiyu z. meta-analysis of effect of vernakalant on conversion of atrial fibrillation. bmc res notes. 2013;6:94. 43. costabel jp, lambardi f, aragon m, et al. predictors of conversion of recentonset atrial fibrillation treated with vernakalant. pacing clin electrophysiol. 2015;38:196–200. 44. manolis as, bethanis s, metaxa s, polytarchou k, sakellaris n, pyrros i. use of intravenous vernakalant for atrial fibrillation conversion in the regular ward under only bedside monitoring. hellenic j cardiol. epub ahead of print 15 february 2018. doi:10.1016/j.hjc.2018.02.003. 45. arrigo m, bettex d, rudiger a. treatment of atrial fibrillation in intensive care units and emergency departments. med klin intensivmed notfmed. 2015;110: 614–620. 46. airaksinen ke, gronberg t, nuotio i, et al. thromboembolic complications after cardioversion of acute atrial fibrillation: the fincv (finnish cardioversion) study. j am coll cardiol. 2013;62:1187–1192. 47. gronberg t, hartikainen je, nuotio i, biancari f, ylitalo a, airaksinen ke. anticoagulation, cha2ds2vasc score, and thromboembolic risk of cardioversion of acute atrial fibrillation (from the fincv study). am j cardiol. 2016;117:1294–1298. 48. garg a, khunger m, seicean s, chung mk, tchou pj. incidence of thromboembolic complications within 30 days of electrical cardioversion performed within 48 hours of atrial fibrillation onset. jacc clin electrophysiol. 2016;2: 487–494. 49. klein al, grimm ra, murray rd, et al; assessment of cardioversion using transesophageal echocardiography investigators. use of transesophageal echocardiography to guide cardioversion in patients with atrial fibrillation. n engl j med. 2001;344:1411–1420. 50. mussigbrodt a, john s, kosiuk j, richter s, hindricks g, bollmann a. vernakalant-facilitated electrical cardioversion: comparison of intravenous vernakalant and amiodarone for drug-enhanced electrical cardioversion of atrial fibrillation after failed electrical cardioversion. europace. 2016;18:51–56. 51. chen s, liu s, purerfellner h. vernakalant as a novel anti-arrhythmic agent for converting of atrial fibrillation, molecular mechanism, updated clinical efficacy, and future development. curr pharm des. 2015;21:4400–4408. dti drug target insights 2023; 17: 78-89issn 1177-3928 | doi: 10.33393/dti.2023.2596review drug target insights issn 1177-3928 www.aboutscience.eu/dti © 2023 the authors. this article is published by aboutscience and licensed under creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). commercial use is not permitted and is subject to publisher’s permissions. full information is available at www.aboutscience.eu efficacy of lamp assay for mycobacterial spp. detection to prevent treatment delays and onset of drug resistance: a systematic review and meta-analysis gurvinder singh bumbrah1, sarika jain2, zeeshan fatima3,4 and saif hameed3 1department of forensic sciences, amity school of applied sciences, amity university haryana, gurugram, manesar india 2department of mathematics, amity school of applied sciences, amity university haryana, gurugram, manesar india 3amity institute of biotechnology, amity university haryana, gurugram, manesar india 4department of medical laboratory sciences, college of applied medical sciences, university of bisha, bisha saudi arabia abstract background: tuberculosis (tb) remains a deadly disease affecting one-third population globally. long turnaround time and poor sensitivity of the conventional diagnostics are the major impediments for faster diagnosis of mycobacterial spp to prevent drug resistance. to overcome these issues, molecular diagnostics have been developed. they offer enhanced sensitivity but require sophisticated infrastructure, skilled manpower and remain expensive. methods: in that context, loop-mediated isothermal amplification (lamp) assay, recommended by the who in 2016 for tb diagnosis, sounds as a promising alternative that facilitates visual read outs. therefore, the aim of the present study is to conduct a meta-analysis to assess the diagnostic efficiency of lamp for the detection of a panel of mycobacterium spp. following prisma guidelines using scientific databases. from 1600 studies reported on the diagnosis of mycobacterium spp., a selection of 30 articles were identified as eligible to meet the criteria of lamp based diagnosis. results: it was found that most of the studies were conducted in high disease burden nations such as india, thailand, and japan with sputum as the most common specimen to be used for lamp assay. furthermore, is6110 gene and fluorescence-based detections ranked as the most used target and method respectively. the accuracy and precision rates mostly varied between 79.2% to 99.3% and 73.9% to 100%, respectively. lastly, a quality assessment based on quadas-2 of bias and applicability was conducted. conclusion: lamp technology could be considered as a feasible alternative to current diagnostics considering high burden for rapid testing in low resource regions. keywords: diagnosis, lamp, meta-analysis, mycobacteria, therapeutics, tuberculosis received: may 1, 2023 accepted: may 12, 2023 published online: june 7, 2023 corresponding authors: drs. saif hameed and zeeshan fatima amity institute of biotechnology amity university haryana gurugram, manesar-122413 india saifhameed@yahoo.co.in; drzeeshanfatima@gmail.com spine, or brain (1). worldwide, tb is the 13th leading cause of death and the second raging infectious killer after human immunodeficiency virus (hiv)/acquired immunodeficiency syndrome (aids) (2). in 2020, an estimated 10 million people got ill with tb worldwide, the infection being divided as 5.6 million men, 3.3 million women, and 1.1 million children. tb affects most of the countries among all age groups and can be fatal if not treated properly. moreover, the emergence of drug-resistant strains has further complicated the problem and has become a rising obstacle against efficient therapeutics (3). therapeutics are available but the effective control of the disease is impeded due to the lack of rapid and accurate diagnostics. under such significant circumstances, there is an urgent need for rapid, accurate, and cost-effective diagnostic test for tb to identify new cases and reduce the time-totreatment and prevent its further transmission. the current available methods are primarily based on smear microscopy (acid-fast staining), culture, and nucleic introduction tuberculosis (tb) caused by mycobacterium tuberculosis (mtb) remains a deadly disease affecting millions of people worldwide. it is estimated to affect approximately one-third of the global population and is becoming one of the most fatal infectious diseases. mtb usually attacks the lungs, but tb bacteria can infect any part of the body such as kidney, https://doi.org/10.33393/dti.2023.2596 https://creativecommons.org/licenses/by-nc/4.0/legalcode mailto:saifhameed@yahoo.co.in mailto:drzeeshanfatima@gmail.com bumbrah et al drug target insights 2023; 17: 79 © 2023 the authors. published by aboutscience www.aboutscience.eu acid amplification. although methods based on acid-fast staining are sensitive, they pose problems in low-resource places and are time-consuming (4). the solid culture method requires around 4-8 weeks, while liquid-based culture methods also require around 10-14 days (4). nucleic acid amplification techniques are based on polymerase chain reaction (pcr) or loop-mediated isothermal amplification (lamp). although hemi-nested pcr based on genexpert for mtb detection is rapid, sensitive, and specific, it also poses challenges of high cost and high end equipment dependency, which limits its implementation in low-resource regions (5). lamp is an isothermal dna amplification method that relies on four or six pairs of primers to amplify minute quantities of dna within a shorter period with simple operation, making it more suitable for low-resource regions (6). thus, research in tb diagnostics aims to find an efficient, reproducible, cost-effective tool with minimal infrastructure requirements. lamp is a popularly adopted new age technology for rapid nucleic acid amplification which is widely used for pathogen (virus, bacteria, and malaria) detection including severe acute respiratory syndrome coronavirus 2 (sars cov-2) (7-9). lamp-based detection methods have been proved to be more sensitive than genexpert assay. in fact, the world health organization (who) has endorsed lamp for tb as a replacement for smear microscopy for peripheral settings (10). in pursuit of developing better diagnostics, which are crucial for achieving global elimination of tb, we performed a systematic review and meta-analysis to access the diagnostic accuracy of lamp to detect mycobacteria. even if couple of studies have depicted the efficacy of lamp during the last decade, an updated version is missing. moreover, most of these studies were specific to either pulmonary or extrapulmonary tb. therefore, the present study not only offers an up-to-date diagnostic performance of lamp for tb detection but also covers other mycobacterium spp. the pooled sensitivity and specificity of lamp were analyzed against different references. further, diagnostic efficiency was determined based on reference methods, target genes, and detection methods of lamp. taken together, we aimed to evaluate the diagnostic potency of lamp as a tool for detection of mycobacteria to address the current tb diagnosis burden in lowresource places. methods the preferred reporting items for systematic review and meta-analyses (prisma) guidelines (11) were followed for identification of eligible studies in the present systematic review and meta-analysis. search strategy diverse scientific databases, for example, pubmed, google scholar, science direct, scopus, biorxiv, and medrxiv, were searched to screen for studies performed using lamp for tb diagnostics from the year 2000 till march 2022. the terms such as lamp, tuberculosis, mycobacterium and mycobacteria were used in various combinations during our research without any limitations: “lamp + tuberculosis” or “lamp + mycobacterium” or “lamp + mycobacteria” or “lamp + tb” or “lamp + tuberculosis + mycobacterium” or “lamp + tuberculosis + mycobacteria” for pubmed, science direct, and google scholar without using any language restriction. the retrieved results were screened for duplication and conformity with the prespecified eligibility criteria. study eligibility criteria inclusion criteria this systematic review and meta-analysis included: (1) both peer-reviewed and preprint original articles on lamp technology used for detection of any mycobacterial species such as mtb, m. bovis, and m. africanum; (2) only full-text articles written in english language; and (3) articles that contain data on true-positive (tp), false-positive (fp), falsenegative (fn), and true-negative (tn) values for the assay or have sufficient data so that the number of tp, fp, fn, and tn (performed on clinical samples) could be determined. exclusion criteria exclusion was made for: (1) studies based on non-isothermal amplification; (2) studies where data are irretrievable; (3) review articles, editorials, commentaries, proceedings, etc.; (4) foreign language articles (other than english) based on lamp-mediated detection of mycobacteria. data extraction potential articles after reviewing titles and abstracts followed by full text for inclusion were extracted by two authors (g.s.b. and z.h.). consultation from two independent authors (s.j. and s.h.) was made to eliminate the doubt about any discrepancy. the extracted information from included studies had authors, year of publication, location of study, sample size, types of specimens, target genes, detection method, and standard reference method. the data extracted for evaluation of diagnostic accuracy for lamp were performed by using either respiratory or non-respiratory specimens with any of the reference methods such as smear microscopy, culture, and genexpert. the important parameters in this meta-analysis such as tp, tn, fp, and fn of all studies were either extracted or calculated to provide their sensitivity and specificity values. the included studies (n = 30) were then assessed for their methodological quality to reduce systematic biases and inferential errors from the collected data. statistical analysis the quantitative analysis of the included studies (n = 30) from the data extracted such as the values of tp, fp, tn, fn and sample size was performed. furthermore, the values of sensitivity and sensitivity were mined or calculated from the available data. moreover, pooled sensitivity and specificity of lamp associated with 95% confidence interval (ci) were estimated. to maintain the accuracy and precision, the formulas: accuracy = [tp + tn/tp + tn + fp + fn]* 100 and precision = [tp/tp+fp]* 100 (12,13) were used. accuracy and precision are important characteristics of any measurement. mycobacterial diagnostic efficiency of lamp assay80 © 2023 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti accuracy is the degree of closeness of measured value to a standard value. however, precision provides the information regarding the closeness of multiple measured values to each other. accuracy and precision are independent of each other. forest plot for sensitivity and specificity were plotted using r-software along with summary receiver operating characteristic (sroc) for the given study. quality assessment quality assessment of diagnostic accuracy studies (quadas-2) tool was used to assess the methodological quality of the eligible studies. the risk of bias in the included studies (n = 30) was assessed from four areas of bias, for example, patient selection, index test, reference standards, and flow timing (14,15). for each quadas-2 domain specific yes/no questions were tailored. following these criteria, the eligible studies were then refereed for low, unclear, or high risks of bias. furthermore, we also judged to generate low, unclear, or high-risk applicability. results literature survey we followed the prisma guidelines (11) to search the literature for the present study (fig. 1). the major scientific databases viz. pubmed, science direct, scopus, biorxiv, and medrxiv have been extensively searched applying the above inclusion criteria and around 1,600 articles were extracted. from the 1,600 articles, we included the ones that were published after the year 2000 since the inception of lamp technology (6) and thus excluded 22 articles. further, only articles written in english language were considered and thus excluded 44 articles. reading the titles and abstracts of these studies allowed to exclude further 1,029 articles comprising the review articles, editorials, proceedings etc. following this exclusion, we removed the duplicated articles and further excluded 390 articles. additional 73 articles were irrelevant as they didn’t use lamp technology for the diagnosis of any mycobacterial species and were excluded, leaving a panel composed of 42 eligible studies. lastly, from the 42 included articles, further 12 articles were also eliminated because their tp, fp, tn, and tn values were either not specified in these articles or the sensitivity and specificity values could not be calculated. altogether, we observed that only 30 articles were eligible for detailed meta-analysis (fig. 1) considering all the exclusion criteria. study characteristics and meta-analysis table i shows the data extracted from the eligible studies mentioning the details of authors, year of publication, country of study, types of specimens, target genes, detection method, and reference methods. figure 2 shows the country-wise distribution of 30 identified articles included in the present study. most of the studies (43.3%; n = 13) were conducted in the high tb burden nations such as india followed fig. 1 preferred reporting items for systematic review and meta-analyses (prisma) flowchart depicts search of the literature and screening strategy for meta-analysis. bumbrah et al drug target insights 2023; 17: 81 © 2023 the authors. published by aboutscience www.aboutscience.eu ta bl e i c ha ra ct er is tic s an d ou tc om es o f t he in cl ud ed s tu di es (n  =  3 0) s. n o. a ut ho r jo ur na l co un tr y re fe re nc e m et ho d sp ec im en ta rg et g en e d et ec ti on m et ho d tp tn fp fn si ze se ns iti vi ty sp ec ifi ci ty 1 bo eh m e et a l ( 20 07 ) j c lin m ic ro bi ol sw itz er la nd cu ltu re , s m ea r m ic ro sc op y sp ut um gy rb fl uo re sc en ce , tu rb id it y 17 3 50 0  4  5  6 82 97 .7 0% 99 % 2 pa nd ey et a l ( 20 08 ) j m ed m ic ro bi ol ja pa n a ci dfa st s ta in in g, ba ct er ia l c ul tu re , ra di ol og y sp ut um 16 s rr n a fl uo re sc en ce  9 0  9 8  6  6  2 00 94 % 94 .2 0% 3 po ud el et a l ( 20 09 ) ka th m an du u ni v m ed j (k u m j) n ep al sm ea r m ic ro sc op y, cu ltu re , r ad io lo gy sp ut um 16 s rr n a fl uo re sc en ce  9 7  9 6  6  3  2 02 97 % 94 .1 2% 4 g eo jit h et a l ( 20 11 ) j m ic ro bi ol m et ho ds in di a cu ltu re , p cr r ev er se hy br id iz ati on li ne p ro be as sa y, g en ot yp e m tb e as sa y sp ut um ri m m co lo ri m et ry , g el el ec tr op ho re si s  1 7  1 7  1 21    56 44 .7 0% 94 .4 0% 5 g eo rg e et a l ( 20 11 ) pl os o ne in di a fl uo re sc en ce s m ea r m ic ro sc op y, c ul tu re sp ut um ri m m co lo ri m et ry , g el el ec tr op ho re si s  3 1  3 6  2  2    71 93 .9 0% 94 .7 0% 6 m it ar ai et a l ( 20 11 ) in t j t ub er c lu ng d is ja pa n cu ltu re , s m ea r m ic ro sc op y, n uc le ic a ci d am pl ifi ca tio n (n a a) sp ut um gy rb fl uo re sc en ce , tu rb id it y 19 6  8 8  9 27  3 20 87 .9 0% 90 .7 0% 7 n ag de v et a l ( 20 11 ) j c lin m ic ro bi ol in di a pc r ce re br os pi na l flu id is 61 10 tu rb id it y  1 5    8  2  2    27 88 .2 3% 80 % 8 se th i et a l ( 20 13 ) j c lin l ab a na l in di a sm ea r m ic ro sc op y, cu ltu re , p cr sp ut um 16 s rr n a , is 61 10 co lo ri m et ry , g el el ec tr op ho re si s  8 7  3 0  0 16  1 33 84 .5 0% 10 0% 9 ca o et a l ( 20 15 ) j m ic ro bi ol m et ho ds ch in a sm ea r m ic ro sc op y, cu ltu re , p cr sp ut um is 61 10 fl uo re sc en ce  9 8  1 8  5  2  1 23 98 .0 0% 78 .3 0% 10 jo on et a l ( 20 15 ) in t j t ub er c lu ng d is in di a sm ea r m ic ro sc op y, cu ltu re , p cr en do m et ria l fl ui d, ur in e, b lo od , s em en , ce re br os pi na l fl ui d, pl eu ra l fl ui d, p us , pe ric ar di al fl ui d, pe rit on ea l fl ui d, in te sti na l a nd ly m ph no de b io ps y tis su e is 61 10 , m pb 64 , sd aa co lo ri m et ry  2 8 26 2 23  2  3 15 93 .3 0% 91 .9 0% 11 m oo n et a l ( 20 15 ) j m ed m ic ro bi ol ko re a cu ltu re , s m ea r m ic ro sc op y sp ut um hs px co lo ri m et ry , tu rb id it y, g el el ec tr op ho re si s  3 2 25 5  3 13  3 03 71 .1 0% 98 .8 0% 12 bo ja ng et a l ( 20 16 ) j i nf ec t g am bi a sm ea r m ic ro sc op y, cu ltu re , g en ex pe rt m tb /r if sp ut um 16 s rr n a fl uo re sc en ce  9 8 15 7 10  1  2 66 99 .0 0% 94 .0 0% 13 g ra y et a l ( 20 16 ) j c lin m ic ro bi ol sw itz er la nd cu ltu re , s m ea r m ic ro sc op y sp ut um gy rb fl uo re sc en ce 33 1 ## # 52 61 17 77 84 .4 0% 96 .6 0% 14 ka ku et a l ( 20 16 ) jp n j i nf ec t d is ja pa n sm ea r m ic ro sc op y, cu ltu re sp ut um gy rb , is 61 10 fl uo re sc en ce 13 4 31 2  5  2 1 47 2 86 .5 0% 98 .4 0% 15 m od i et a l ( 20 16 ) in t j t ub er c lu ng d is in di a cu ltu re , r ad io lo gy , st ai ni ng , p cr ce re br os pi na l flu id is 61 10 , m pb 64 fl uo re sc en ce , g el el ec tr op ho re si s, tu rb id it y 14 4 10 0  0    6 25 0 96 .0 0% 10 0. 00 % (c on tin ue d) mycobacterial diagnostic efficiency of lamp assay82 © 2023 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti 16 sh ar m a et a l ( 20 16 ) tu be rc ul os is (e di nb ) in di a pc r, c ul tu re , s m ea r m ic ro sc op y n ee dl e as pi ra te is 61 10 , m pb 64 fl uo re sc en ce , g el el ec tr op ho re si s, tu rb id it y 10 8  5 0  0  1 2 17 0 90 .0 0% 10 0. 00 % 17 sh ar m a et al (2 01 6) j o rt ho p re s in di a cu ltu re , s ta in in g, p cr sy no vi al fl ui d, pu s is 61 10 , m pb 64 fl uo re sc en ce , g el el ec tr op ho re si s, tu rb id ity  8 1  5 0  0    9 14 0 90 .0 0% 10 0. 00 % 18 jo on e t a l (2 01 7) j m ic ro bi ol m et ho ds in di a pc r, c ul tu re , s m ea r m ic ro sc op y sp ut um is 61 10 , m pb 64 co lo ri m et ry , g el el ec tr op ho re si s  1 7 21 2  6    1 23 6 94 .4 0% 97 .2 0% 19 re dd y et a l (2 01 7) in t j t ub er c lu ng d is so ut h af ri ca cu ltu re , s m ea r m ic ro sc op y, x pe rt sp ut um gy rb fl uo re sc en ce 11 9 51 4 17  4 5 69 5 72 .6 0% 96 .8 0% 20 ya da v et a l (2 01 7) in t j t ub er c lu ng d is in di a cu ltu re , s m ea r m ic ro sc op y, g en ex pe rt sp ut um gy rb , is 61 10 fl uo re sc en ce  8 2 36 8  3    0 45 3 10 0. 00 % 99 .2 0% 21 ki m e t a l (2 01 8) a nn l ab m ed ko re a cu ltu re , s m ea r m ic ro sc op y, p cr sp ut um gy rb , is 61 10 fl uo re sc en ce , tu rb id it y  8 7 18 6  0  1 7 29 0 83 .6 0% 10 0. 00 % 22 n gu ye n et al (2 01 8) d ia gn m ic ro bi ol in fe ct d is v ie tn am sm ea r m ic ro sc op y, cu ltu re , x pe rt m tb /r if sp ut um gy rb , is 61 10 co lo ri m et ry , flu or es ce nc e  1 5 44 5 23  1 8 50 1 45 .5 0% 95 .1 0% 23 pe re ra e t a l (2 01 8) ce yl on m ed j sr i l an ka sm ea r m ic ro sc op y, cu ltu re cu ltu re is ol at es ri m m co lo ri m et ry  3 1  1 0  5    0  4 6 10 0. 00 % 66 .6 7% 24 jo on e t a l (2 01 9) j m ic ro bi ol m et ho ds in di a cu ltu re , s m ea r m ic ro sc op y, g en ex pe rt m tb /r if a ss ay , p cr , la m plf d a ss ay sp ut um s da a co lo ri m et ry  1 3  9 2  2    0 10 7 10 0. 00 % 97 .8 7% 25 ph et su ks ir i et a l ( 20 19 ) jp n j i nf ec t d is th ai la nd cu ltu re , i m m un och ro m at og ra ph ic te st sp ut um m pt 64 co lo ri m et ry 14 4    5  1    1 15 1 99 .3 1% 83 .3 3% 26 pu na ti et a l (2 01 9) br az j m ic ro bi ol in di a cu ltu re , p cr fe ca l s am pl es is 90 0 tu rb id it y, g el el ec tr op ho re si s, co lo ri m et ry , la te ra l fl ow de vi ce  8 6 29 4  9    0 38 9 10 0. 00 % 97 .0 2% 27 ra jp ut e t a l (2 01 9) j m ic ro bi ol m et ho ds in di a cu ltu re , s m ea r m ic ro sc op y, p cr fl ui ds , u ri ne , pu s is 61 10 , pa b, m pb 64 co lo ri m et ry , flu or es ce nc e  9 0  3 2  9  2 3 15 4 79 .6 5% 78 .0 5% 28 h an e t a l (2 02 0) bm c in fe ct d is ch in a xp er t m tb /r if , s at -t b as sa y pl eu ra l fl ui ds gy rb , is 61 10 fl uo re sc en ce  5 9  4 1  1 16 4 26 5 26 .5 0% 97 .6 0% 29 ph et su ks ir i et a l ( 20 20 ) jp n j i nf ec t d is th ai la nd m ic ro sc op y, c ul tu re , pc r, r ad io lo gy sp ut um 16 s rr n a co lo ri m et ry , flu or es ce nc e, g el el ec tr op ho re si s, im m un och ro m at og ra ph y 11 9 10 2 24 7 25 2 94 .4 4% 80 .9 5% 30 ph et su ks ir i et a l ( 20 20 ) re v in st m ed t ro p sa o pa ul o th ai la nd xp er t m tb /r if , c ul tu re , sm ea r m ic ro sc op y sp ut um 16 s rr n a co lo ri m et ry , flu or es ce nc e, g el el ec tr op ho re si s 12 6  7 1  0 7 20 4 94 .7 4% 10 0. 00 % la m p = lo op -m ed ia te d is ot he rm al a m pl ifi ca tio n; l fd = la te ra l fl ow d ip sti ck ; p cr = p ol ym er as e ch ai n re ac tio n. ta bl e i (c on tin ue d) bumbrah et al drug target insights 2023; 17: 83 © 2023 the authors. published by aboutscience www.aboutscience.eu fig. 2 country-wise distribution of included studies (n = 30) reported in the present investigation. table ii accuracy and precision of the included studies (n = 30) s. no. study accuracy precision 1 boehme et al (2007) 98.68 97.74 2 pandey et al (2008) 94.00 93.75 3 poudel et al (2009) 95.54 94.17 4 geojith et al (2011) 60.71 94.44 5 george et al (2011) 94.36 93.93 6 mitarai et al (2011) 88.75 95.60 7 nagdev et al (2011) 85.18 88.23 8 sethi et al (2013) 87.96 100.00 9 cao et al (2015) 94.30 95.14 10 joon et al (2015) 92.06 54.90 11 moon et al (2015) 94.71 91.42 12 bojang et al (2016) 95.86 90.74 13 gray et al (2016) 93.64 86.42 14 kaku et al (2016) 94.49 96.40 15 modi et al (2016) 97.60 100.00 16 sharma et al (2016) 92.94 100.00 17 joon et al (2017) 97.03 73.91 18 reddy et al (2017) 91.07 87.50 19 sharma et al (2016) 93.57 100.00 20 yadav et al (2017) 99.33 96.47 21 kim et al (2018) 94.13 100.00 22 nguyen et al (2018) 91.81 39.47 23 perera et al (2018) 89.13 86.11 24 joon et al (2019) 98.13 86.66 25 phetsuksiri et al (2019) 98.67 99.31 26 punati et al (2019) 97.68 90.52 27 rajput et al (2019) 79.22 90.90 28 han et al (2020) 37.73 98.33 29 phetsuksiri et al (2020) 87.69 83.21 30 phetsuksiri et al (2020) 96.56 100.00 values ranged from 0.67 to 1.00 (fig. 6). a total of 27 out of the 30 included studies showed pooled sensitivity greater than 70%. only three studies reported sensitivity values of 26% and 45% each (19-21). in terms of fp rate (1-specificity), 27 included studies showed a pooled fp rate higher than 80% (fig. 7). additionally, the accuracy and precision rates of included studies were calculated and varied between 37.73% and 99.33%. the analysis proved that 22 studies displayed more than 90% accuracy with only 4 studies depicting less than 80% accuracy (tab. ii). likewise, the precision rates varied between 39.47% and 100%. the analysis showed that 21 studies exhibited more than 90% precision rate with only 3 studies depicting less than 80%. of note, we observed that six studies displayed 100% precision rate. by thailand and japan (each 10%; n = 3). two studies each were also conducted in countries such as china, korea, and switzerland (6.3%; n = 2). apart from this, one study each, that is, 3.3%, was from countries included such as gambia, nepal, south africa, sri lanka, and vietnam. although most of the included articles do not mention about the patient details, the type of specimen (fig. 3) used in most of the studies was sputum (42.8%; n = 21). in addition, some studies have been tested on other specimens such as cerebrospinal fluid (n = 4), fecal samples (n = 1), urine (n = 3), blood (n = 2), and pleural fluid (n = 3) for the detection of mycobacteria by using lamp. furthermore, the standard smear microscopy, culture assay, and pcr-based methods were used as references either alone or in combination (n = 30). of note, radiology was also used (n = 4) to validate lamp results as a reference standard (tab. i), with one study using immunochromatography (16). next, we examined the various target genes used for the eligible studies. ten different types of target genes including hspx, is900, mpt64, pab, sdaa, rimm, 16srrna, mpb64, gyrb, and is6110 were used in the included studies (n = 30). is6110 gene was most frequently used in the included studies (n = 14; 31.18%) followed by gyrb (n = 9, 20.45%), 16srrna (n = 6, 13.63%), and mpb64 (n = 6, 13.63%) genes (fig. 4). furthermore, while analyzing detection methods used for these 30 studies, fluorescent method (n = 19, 32.39%) was the most frequently performed followed by colorimetry (n = 14, 25.35%), gel electrophoresis (n = 11, 20.00%) and turbidity (n = 9, 16.36%) methods (fig. 5). in 53.33% (n = 16) of studies, more than one detection method was used. in 16.66% (n = 5) of studies, combination of three methods was used while in only two studies (6.66%), combination of four different methods was reported (17,18). among all the eligible studies, 4 studies showed 100% sensitivity, while for 16 studies this parameter was higher than 90%. similarly, 6 studies exhibited 100% specificity while 90% or more specificity was observed in 24 studies (tab. i). furthermore, upon analysis of sensitivity and specificity using forest plot at 95% ci, we found that the sensitivity values varied between 0.26 and 1.00 and the specificity quality assessment of the study almost two-thirds of the included studies (22 out of 30 studies) have a high risk of patient selection bias due to mycobacterial diagnostic efficiency of lamp assay84 © 2023 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti fig. 3 distribution of type of specimen for detection of mycobacteria in the included studies (n = 30). fig. 4 distribution of target genes reported in the included studies (n = 30). non-random patient selection and case-control study design (fig. 8, tab. i). around 26% (8 out of 30) of the included studies have low risk of patient selection bias because these studies provided sufficient details about patient inclusion/ exclusion criteria; 86% of included articles (26 out of 30 studies) present low risk of index test bias because these tests clearly stated the quantitative detection read-outs with reported thresholds. moreover, these studies explicitly declared that their index and reference tests were done simultaneously in parallel to each other or that testing was blinded from each other. two studies (19,22) were reported without defined detection thresholds. one study (23) had unclear risk of index test bias as the quantitative detection thresholds were not explained. it was either unclear whether index test results were interpreted with knowledge of reference test results or if only qualitative read-out was used for bumbrah et al drug target insights 2023; 17: 85 © 2023 the authors. published by aboutscience www.aboutscience.eu fig. 5 distribution of type of detection method for mycobacteria in the included studies (n = 30). fig. 6 the forest plot of sensitivity and specificity of included studies (n = 30) on the diagnostic performance of loop-mediated isothermal amplification (lamp) technique. mycobacterial diagnostic efficiency of lamp assay86 © 2023 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti at high risk as it did not provide any information on whether the samples for a reference test and the index test were taken at the same time. our review question did not focus on any patient demographics. none of the included studies attempted to exclude patients based on demographics and thus had no “concern of patient selection applicability” (fig. 8, tab. i). index tests of all studies have generally been used for point-of-care test (pocts) and thus have low concern of index test applicability. reference standard tests of nearly all studies were culture, smear microscopy, xpert test, pcr, or combinations of them. thus, we graded these studies as having low concern of standard test applicability. discussion early and correct diagnosis of all the tb forms is pertinent for effective treatment of the disease and prevention of the spread of infection, particularly in nations which have high burden. the currently available diagnostics rely mostly on smear microscopy, culture, and pcr-based methods which are not only time-consuming and low sensitive but cumbersome and costly (25,26). lamp assay provides a faster and innovative point-of-care diagnostic alternative as it is costeffective, sensitive, and gives results in less than 1 hour due to amplification under isothermal condition by strand displacement activity of bst dna polymerase and visual readouts (27-30). in fact, the efficiency of lamp in diagnosis of pulmonary tb is evident from wide ranges of studies (31-35). additionally, lamp has been successfully deployed for diagnosis of other forms of tb such as tuberculous meningitis (36,37), osteoarticular tb (38), and tubercular lymphadenitis (39). although a few studies have evaluated the diagnostic validity of lamp by meta-analysis for diagnosis of mtb (40), fig. 7 summary receiver operating characteristic (sroc) depicts loop-mediated isothermal amplification (lamp) diagnostic performance in mycobacteria diagnosis. fig. 8 quality assessment of diagnostic accuracy studies 2 (quadas-2) summary of items for risk of bias and applicability in included studies (n = 30). green color depicts the low risk of biasedness, yellow color depicts the unclear risk of biasedness, and red color depicts the high risk of biasedness. reading the results. hence, index test bias of these studies was unclear. for the rest of the included studies, almost all (n = 30) have low risk of reference standard bias because they provided enough information about the standard reference test used in the study. half of the studies (15 out of 30) have an unclear risk of flow and timing bias as there is not enough information, whether reference standard results were interpreted with the knowledge of the results of the index test. one study (24) was bumbrah et al drug target insights 2023; 17: 87 © 2023 the authors. published by aboutscience www.aboutscience.eu pulmonary tb (41), and extrapulmonary tb (42), an updated meta-analysis covering all forms of mycobacteria was still missing. hence, the aim of the present study was to systematically review and perform the meta-analysis to assess the diagnostic accuracy of the lamp assay for detection of all forms of mycobacteria. this meta-analysis revealed that most of the studies were conducted in high tb burden countries such as india, thailand, and japan (fig. 2). we observed that for the detection of mycobacteria sputum could be considered as the most chosen sample (fig. 3). when considering the target genes, we found a variety of genes that were used in the included studies. however, is6110 ranked first among all evaluated genes in the included studies (fig. 4). this occurrence could be due to the presence of multiple copies of is6110 present in the mtb genome (43). however, other target genes such as 16s rrna and gyrb were also prominent. next, we considered the detection method that was used for assessing the lamp results. most of the studies used fluorescence-based methods followed by colorimetry, gel electrophoresis, and turbidity, with no justification of their choices (fig. 5). the prominence of fluorescence methods could be due to their increased sensitivity for the detection. exceptionally, only one study mentioned lateral flow-based detection method despite market applicability. forest plot was used to calculate the sensitivity and specificity. the pooled sensitivity values of meta-analysis ranged between 0.26 and 1.0 (fig. 6) and forest plot and sroc curve revealed a pooled specificity value between 0.67 and 1.0 (fig. 7) with 95% ci. the accuracy and precision were calculated for the included studies and for 16 studies we found that the accuracy rate was higher than their corresponding precision rates and vice versa for 14 articles upon intra-comparison of accuracy with precision (tab. ii). the current study also exhibited few limitations. firstly, we observed high risk of patient selection bias or index test bias in almost two-thirds of the eligible studies (fig. 8). therefore, the use of unbiased patient cohorts and double-blinded index test may be recommended for future studies. secondly, few studies showed the highest performance with 100% sensitivity and specificity, respectively, hence displaying the lowest quadas risk and concerns in all the domains. furthermore, lack of subgroup analysis and the use of solely peer-reviewed english language articles were also additional limitations. hence, although the current meta-analysis should be interpreted with caution, however, we believe that it will not impact the robustness of the analysis leading to further improved studies and reviews. particularly considering the growing significance of lamp-based detection for tb comparable to other methods, such studies may be encouraged (43-45). conclusion despite suffering from few disadvantages, like false positivity due to heavy reliance on indirect detection methods such as turbidity and nonspecific dyes and not providing any additional benefits like information on mutations, drug resistance etc., the lamp technique could be a promising molecular test to enhance case detection before conventional time-consuming culture. its simplicity, less turnaround time, and cost-effectiveness are major attractions for clinical laboratories. also, it will be unjust to rely on single point-of-care test for tb successfully in various kinds of populations and resource availability. although the unit cost is higher than smear microscopy and culture-based methods, it is likely to offer good value for money relative to conventional methods. in a nutshell, the present study endorses the use of lamp assay as a promising alternative for detection of mycobacteria, particularly in regions which are financially compromised, where drug-resistant strains are not prevalent and pcr-based tests cannot be done so frequently. the faster diagnosis through lamp could provide an alternative solution for failed medications to current therapeutics due to delayed diagnosis and subsequent development of drug resistance, thereby providing an opportunity to employ this new information in improving treatment strategies. however, the lamp assay still must be improved to turn to a strong and competitive alternative to other molecular diagnostic methods. acknowledgments we are grateful to akansha bhatt and reva gautam for their assistance in reviewing the literature and to muriel billamboz for assistance in english language editing. author contributions g.s.b.: search, data extraction, validation. g.s.b., s.j.: data analysis. z.f. and s.h.: supervision. g.s.b. and s.h.: writing, original draft. z.f. and s.h. contributed to the conception and design of the study and review and editing of the manuscript. disclosures conflict of interest: the authors declare no conflicts of interest, financial or otherwise. financial support: financial assistance to s.h. from the indian council of medical research (icmr), new delhi (5/8/5/9/itrc/diag/2022/ ecd-1), is sincerely acknowledged. references 1. norbis l, alagna r, tortoli e, codecasa lr, migliori gb, cirillo dm. challenges and perspectives in the diagnosis of extrapulmonary tuberculosis. expert rev anti infect ther. 2014;12(5):633-647. crossref pubmed 2. pai m, nicol mp, boehme cc. tuberculosis diagnostics: state of the art and future directions. microbiol spectr. 2016;4(5):1-15. crossref pubmed 3. world health organization. global tuberculosis report 2015. who 2015. 4. american thoracic society; centers for disease control and prevention; infectious diseases society of america. american thoracic society/centers for disease control and prevention/ infectious diseases society of america: controlling tuberculosis in the united states. am j respir crit care med. 2005;172(9): 1169-1227. crossref pubmed 5. park ks, kim jy, lee jw, et al. comparison of the xpert mtb/rif and cobas taqman mtb assays for detection of mycobacterium https://doi.org/10.1586/14787210.2014.899900 https://www.ncbi.nlm.nih.gov/pubmed/24717112 https://doi.org/10.1128/microbiolspec.tbtb2-0019-2016 https://www.ncbi.nlm.nih.gov/pubmed/27763258 https://doi.org/10.1164/rccm.2508001 https://www.ncbi.nlm.nih.gov/pubmed/16249321 mycobacterial diagnostic efficiency of lamp assay88 © 2023 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti tuberculosis in respiratory specimens. j clin microbiol. 2013; 51(10):3225-3227. crossref pubmed 6. notomi t, okayama h, masubuchi h, et al. loop-mediated isothermal amplification of dna. nucleic acids res. 2000;28(12): e63. crossref pubmed 7. bhatt a, fatima z, ruwali m, et al. clever assay: a visual and rapid rna extraction-free detection of sars-cov-2 based on crispr-cas integrated rt-lamp technology. j appl microbiol. 2022;133(2):410-421. crossref pubmed 8. huang we, lim b, hsu cc, et al. rt-lamp for rapid diagnosis of coronavirus sars-cov-2. microb biotechnol. 2020;13(4):950961. crossref pubmed 9. broughton jp, deng x, yu g, et al. crispr-cas12-based detection of sars-cov-2. nat biotechnol. 2020;38(7):870-874. crossref pubmed 10. world health organization. the use of loop-mediated isothermal amplification (tblamp) for the diagnosis of pulmonary tuberculosis: policy guidance. world health organization 2016;1-52. 11. moher d, liberati a, tetzlaff j, altman dg; prisma group. preferred reporting items for systematic reviews and meta analyses: the prisma statement. j clin epidemiol. 2009;62(10): 1006-1012. crossref pubmed 12. morillas av, gooch j, frascione n. feasibility of a handheld near infrared device for the qualitative analysis of bloodstains. talanta. 2018;184:1-6. crossref pubmed 13. gregório i, zapata f, torre m, garcía-ruiz c. statistical approach for atr-ftir screening of semen in sexual evidence. talanta. 2017;174:853-857. crossref pubmed 14. subali ad, wiyono l. reverse transcriptase loop mediated isothermal amplification (rt-lamp) for covid-19 diagnosis: a systematic review and meta-analysis. pathog glob health. 2021; 115(5):281-291. crossref pubmed 15. subsoontorn p, lohitnavy m, kongkaew c. the diagnostic accuracy of isothermal nucleic acid point-of-care tests for human coronaviruses: a systematic review and meta-analysis. sci rep. 2020;10(1):22349. crossref pubmed 16. phetsuksiri b, klayut w, rudeeaneksin j, et al. the performance of an in-house loop-mediated isothermal amplification for the rapid detection of mycobacterium tuberculosis in sputum samples in comparison with xpert mtb/rif, microscopy and culture. rev inst med trop são paulo. 2020;62:e36. crossref pubmed 17. phetsuksiri b, rudeeaneksin j, srisungngam s, et al. comparison of loop-mediated isothermal amplification, microscopy, culture, and pcr for diagnosis of pulmonary tuberculosis. jpn j infect dis. 2020;73(4):272-277. crossref pubmed 18. punati rd, mallepaddi pc, poonati r, et al. development and evaluation of lamp-coupled lateral flow device for the detection of map in livestock at point of care resource-limited areas. braz j microbiol. 2019;50(4):1105-1114. crossref pubmed 19. geojith g, dhanasekaran s, chandran sp, kenneth j. efficacy of loop mediated isothermal amplification (lamp) assay for the laboratory identification of mycobacterium tuberculosis isolates in a resource limited setting. j microbiol methods. 2011;84(1):71-73. crossref pubmed 20. nguyen vat, nguyen hv, dinh tv, et al. evaluation of loopamp™ mtbc detection kit for diagnosis of pulmonary tuberculosis at a peripheral laboratory in a high burden setting. diagn microbiol infect dis. 2018;90(3):190-195. crossref pubmed 21. han m, xiao h, yan l. diagnostic performance of nucleic acid tests in tuberculous pleurisy. bmc infect dis. 2020;20(1):242. crossref pubmed 22. perera su, navaratne v, nagahawatte a, et al. validating the loop mediated isothermal amplification (lamp) technique to detect tuberculosis in a sri lankan laboratory setting. ceylon med j. 2018 31;63(1):40-42. crossref pubmed 23. joon d, nimesh m, gupta s, kumar c, varma-basil m, saluja d. development and evaluation of rapid and specific sdaa lamplfd assay with xpert mtb/rif assay for diagnosis of tuberculosis. j microbiol methods. 2019;159:161-166. crossref pubmed 24. cao d, hu l, lin m, et al. real-time fluorescence loop-mediated isothermal amplification (lamp) for rapid and reliable diagnosis of pulmonary tuberculosis. j microbiol methods. 2015;109:74-78. crossref pubmed 25. bojang al, mendy fs, tientcheu ld, et al. comparison of tb-lamp, genexpert mtb/rif and culture for diagnosis of pulmonary tuberculosis in the gambia. j infect. 2016;72(3):332337. crossref pubmed 26. joon d, nimesh m, saluja d. loop-mediated isothermal amplification as alternative to pcr for the diagnosis of extra-pulmonary tuberculosis. int j tuberc lung dis. 2015;19(8):986-991. crossref pubmed 27. gray cm, katamba a, narang p, et al. feasibility and operational performance of tuberculosis detection by loop-mediated isothermal amplification platform in decentralized settings: results from a multicenter study. j clin microbiol. 2016;54(8):19841991. crossref pubmed 28. kaku t, minamoto f, d’meza r, et al. accuracy of lamp-tb method for diagnosing tuberculosis in haiti. jpn j infect dis. 2016;69(6):488-492. crossref pubmed 29. kim ck, cho ea, shin dm, choi sw, shin sy. comparative evaluation of the loop-mediated isothermal amplification assay for detecting pulmonary tuberculosis. ann lab med. 2018;38(2):119-124. crossref pubmed 30. mitarai s, okumura m, toyota e, et al. evaluation of a simple loop-mediated isothermal amplification test kit for the diagnosis of tuberculosis. int j tuberc lung dis. 2011;15(9):12111217, i. crossref pubmed 31. moon sh, kim ej, tomono j, et al. detection of mycobacterium tuberculosis complex in sputum specimens using a loop-mediated isothermal amplification assay in korea. j med microbiol. 2015;64(11):1335-1340. crossref pubmed 32. pandey bd, poudel a, yoda t, et al. development of an inhouse loop-mediated isothermal amplification (lamp) assay for detection of mycobacterium tuberculosis and evaluation in sputum samples of nepalese patients. j med microbiol. 2008;57(pt 4):439-443. crossref pubmed 33. phetsuksiri b, rudeeaneksin j, srisungngam s, et al. loopmediated isothermal amplification for rapid identification of mycobacterium tuberculosis in comparison with immunochromatographic sd bioline mpt64 rapid® in a high burden setting. jpn j infect dis. 2019;72(2):112-114. crossref pubmed 34. sethi s, singh s, dhatwalia sk, et al. evaluation of in-house loop-mediated isothermal amplification (lamp) assay for rapid diagnosis of m. tuberculosis in pulmonary specimens. j clin lab anal. 2013;27(4):272-276. crossref pubmed 35. yadav r, sharma n, khaneja r, et al. evaluation of the tb-lamp assay for the rapid diagnosis of pulmonary tuberculosis in northern india. int j tuberc lung dis. 2017;21(10):1150-1153. crossref pubmed 36. modi m, sharma k, sharma m, et al. multitargeted loop-mediated isothermal amplification for rapid diagnosis of tuberculous meningitis. int j tuberc lung dis. 2016;20(5):625-630. crossref pubmed 37. nagdev kj, kashyap rs, parida mm, et al. loop-mediated isothermal amplification for rapid and reliable diagnosis of tuberculous meningitis. j clin microbiol. 2011;49(5):1861-1865. crossref pubmed https://doi.org/10.1128/jcm.01335-13 https://www.ncbi.nlm.nih.gov/pubmed/23863563 https://doi.org/10.1093/nar/28.12.e63 https://www.ncbi.nlm.nih.gov/pubmed/10871386 https://doi.org/10.1111/jam.15571 https://www.ncbi.nlm.nih.gov/pubmed/35396760 https://doi.org/10.1111/1751-7915.13586 https://www.ncbi.nlm.nih.gov/pubmed/32333644 https://doi.org/10.1038/s41587-020-0513-4 https://www.ncbi.nlm.nih.gov/pubmed/32300245 https://doi.org/10.1016/j.jclinepi.2009.06.005 https://www.ncbi.nlm.nih.gov/pubmed/19631508 https://doi.org/10.1016/j.talanta.2018.02.110 https://www.ncbi.nlm.nih.gov/pubmed/29674018 https://doi.org/10.1016/j.talanta.2017.07.016 https://www.ncbi.nlm.nih.gov/pubmed/28738663 https://doi.org/10.1080/20477724.2021.1933335 https://www.ncbi.nlm.nih.gov/pubmed/34086539 https://doi.org/10.1038/s41598-020-79237-7 https://www.ncbi.nlm.nih.gov/pubmed/33339871 https://doi.org/10.1590/s1678-9946202062036 https://www.ncbi.nlm.nih.gov/pubmed/32520211 https://doi.org/10.7883/yoken.jjid.2019.335 https://www.ncbi.nlm.nih.gov/pubmed/32115540 https://doi.org/10.1007/s42770-019-00116-z https://www.ncbi.nlm.nih.gov/pubmed/31541428 https://doi.org/10.1016/j.mimet.2010.10.015 https://www.ncbi.nlm.nih.gov/pubmed/21047534 https://doi.org/10.1016/j.diagmicrobio.2017.11.009 https://www.ncbi.nlm.nih.gov/pubmed/29246776 https://doi.org/10.1186/s12879-020-04974-z https://www.ncbi.nlm.nih.gov/pubmed/32209054 https://doi.org/10.4038/cmj.v63i1.8625 https://www.ncbi.nlm.nih.gov/pubmed/29764096 https://doi.org/10.1016/j.mimet.2019.03.002 https://www.ncbi.nlm.nih.gov/pubmed/30858005 https://doi.org/10.1016/j.mimet.2014.12.013 https://www.ncbi.nlm.nih.gov/pubmed/25533219 https://doi.org/10.1016/j.jinf.2015.11.011 https://www.ncbi.nlm.nih.gov/pubmed/26724771 https://doi.org/10.5588/ijtld.14.0793 https://www.ncbi.nlm.nih.gov/pubmed/26162366 https://doi.org/10.1128/jcm.03036-15 https://www.ncbi.nlm.nih.gov/pubmed/27194691 https://doi.org/10.7883/yoken.jjid.2015.519 https://www.ncbi.nlm.nih.gov/pubmed/27000457 https://doi.org/10.3343/alm.2018.38.2.119 https://www.ncbi.nlm.nih.gov/pubmed/29214755 https://doi.org/10.5588/ijtld.10.0629 https://www.ncbi.nlm.nih.gov/pubmed/21943848 https://doi.org/10.1099/jmm.0.000164 https://www.ncbi.nlm.nih.gov/pubmed/26338293 https://doi.org/10.1099/jmm.0.47499-0 https://www.ncbi.nlm.nih.gov/pubmed/18349362 https://doi.org/10.7883/yoken.jjid.2018.128 https://www.ncbi.nlm.nih.gov/pubmed/30381677 https://doi.org/10.1002/jcla.21596 https://www.ncbi.nlm.nih.gov/pubmed/23852783 https://doi.org/10.5588/ijtld.17.0035 https://www.ncbi.nlm.nih.gov/pubmed/28911360 https://doi.org/10.5588/ijtld.15.0741 https://www.ncbi.nlm.nih.gov/pubmed/27084816 https://doi.org/10.1128/jcm.00824-10 https://www.ncbi.nlm.nih.gov/pubmed/21411583 bumbrah et al drug target insights 2023; 17: 89 © 2023 the authors. published by aboutscience www.aboutscience.eu 38. sharma k, sharma m, batra n, sharma a, dhillon ms. diagnostic potential of multi-targeted lamp (loop-mediated isothermal amplification) for osteoarticular tuberculosis. j orthop res. 2017;35(2):361-365. crossref pubmed 39. sharma m, sharma k, sharma a, gupta n, rajwanshi a. loopmediated isothermal amplification (lamp) assay for speedy diagnosis of tubercular lymphadenitis: the multi-targeted 60-minute approach. tuberculosis (edinb). 2016;100:114-117. crossref pubmed 40. nagai k, horita n, yamamoto m, et al. diagnostic test accuracy of loop-mediated isothermal amplification assay for mycobacterium tuberculosis: systematic review and metaanalysis. sci rep. 2016;6(1):39090. crossref pubmed 41. shete pb, farr k, strnad l, gray cm, cattamanchi a. diagnostic accuracy of tb-lamp for pulmonary tuberculosis: a systematic review and meta-analysis. bmc infect dis. 2019;19(1):268. crossref pubmed 42. yu g, shen y, zhong f, ye b, yang j, chen g. diagnostic accuracy of the loop-mediated isothermal amplification assay for extrapulmonary tuberculosis: a meta-analysis. plos one. 2018;13(6):e0199290. crossref pubmed 43. das s, mangold ka, shah ns, peterson lr, thomson rb jr, kaul kl. performance and utilization of a laboratory-developed nucleic acid amplification test (naat) for the diagnosis of pulmonary and extrapulmonary tuberculosis in a low-prevalence area. am j clin pathol. 2020;154(1):115-123. crossref pubmed 44. tayal d, sethi p, jain p. point-of-care test for tuberculosis – a boon in diagnosis. monaldi arch chest dis. 2023. crossref pubmed 45. ludi z, sule aa, samy rp, et al. diagnosis and biomarkers for ocular tuberculosis: from the present into the future. theranostics. 2023;13(7):2088-2113. crossref pubmed https://doi.org/10.1002/jor.23293 https://www.ncbi.nlm.nih.gov/pubmed/27175946 https://doi.org/10.1016/j.tube.2016.07.015 https://www.ncbi.nlm.nih.gov/pubmed/27553418 https://doi.org/10.1038/srep39090 https://www.ncbi.nlm.nih.gov/pubmed/27958360 https://doi.org/10.1186/s12879-019-3881-y https://www.ncbi.nlm.nih.gov/pubmed/30890135 https://doi.org/10.1371/journal.pone.0199290 https://www.ncbi.nlm.nih.gov/pubmed/29944682 https://doi.org/10.1093/ajcp/aqaa031 https://www.ncbi.nlm.nih.gov/pubmed/32249294 https://doi.org/10.4081/monaldi.2023.2528 https://www.ncbi.nlm.nih.gov/pubmed/37114932 https://doi.org/10.7150/thno.81488 https://www.ncbi.nlm.nih.gov/pubmed/37153734 creative commons cc by: this article is distributed under the terms of the creative commons attribution 4.0 license (http://www.creativecommons.org/licenses/by/4.0/) which permits any use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the sage and open access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage). https://doi.org/10.1177/1177392819886875 drug target insights volume 13: 1–7 © the author(s) 2019 doi: 10.1177/1177392819886875 we are at a pivotal moment in medical science and pharmacy practice where specific variations in the genetic code are being associated with differences in drug response, an individual’s propensity for developing certain drug side effects, and variation in the rate and extent of drug metabolism. termed pharmacogenomics (pgx), this new discipline is the study of the interplay between the human genome and the science of pharmacology.1 the curriculums of most pharmacy schools have only recently included coursework in genetics and pgx. pharmacists educated in the past may have had little exposure to the genetic principles underlying pgx. the intent of this review is to present basic concepts in genetics and genetic variation to provide a foundation for understanding important and highly evidenced gene-drug associations. whenever possible, the examples used will include medications where genetic testing is recommended by the us food and drug administration (fda) labeling or expert clinical practice guidelines. several reliable, online pgx references will also be presented. as pharmacists and scientists, the time is now to embrace these ensuing advancements in drug therapy and prepare ourselves to translate gene-drug associations into clinical practice. pharmacogenomics was born out of the findings f rom the human genome project (hgp; www.genome.gov).2 launched in 1990, the hgp was an international effort to identify and understand the structure of every gene human beings possess. the hgp will no doubt be viewed as the greatest scientific advancement of our lifetime and will have a profound impact on medical science and the practice of pharmacy. the main mission of the project was to crack the human genetic code by combining the power of scientists from universities and research centers in the united states, the united kingdom, france, germany, japan, and china. this worldwide endeavor stands as a testimony to the greater good that can be achieved when researchers work together to accomplish a common goal. the sequencing or mapping of the 2.91 billion base pairs that spell out the genetic blueprint within the molecule called deoxyribonucleic acid (dna) was completed years ahead of schedule in april of 2003. the entire code is known as the human genome and is housed in the nucleus of nearly every human cell. research to discover how variation and expression of this code influence human health is just beginning.3 the hgp has given rise to truly personalized medicine, which relates markers or patterns in an individual’s dna sequence to the causation and treatment of disease. pharmacists are the ideal practitioners to implement pgxguided drug therapy and to translate gene-drug associations into clinical practice. pharmacists will be needed to personalize the selection and dosing of medications and explain pgx risk information to both patients and providers. the intent of this review is to open a window into the future of drug therapy by presenting the basic terminology and concepts of pgx and provide educational resources that will help you remain up to date with these emerging changes in medical science and medication management. dna, chromosomes, and genes a foundational concept to keep in mind is that genes, which are stretches of dna, determine the composition, size, and shape of every protein a living organism builds. one gene may hold the “recipe” for one or hundreds of different proteins. an effort to identify every protein human beings produce called the human proteome project is also underway and once completed humans are estimated to make approximately 250 000 to 1 million different proteins.4 as enzymes, transporters, drug receptor, binding sites, cell structural components, and peptide hormones, protein molecules are central to nearly every biochemical and pharmacological reaction. basic concepts in genetics and pharmacogenomics for pharmacists kathleen b orrico1,2 1school of pharmacy, university of california, san francisco, san francisco, ca, usa. 2center for clinical research, stanford university school of medicine, stanford, ca, usa. abstract: this basic review of genetic principles will aid pharmacists in preparing for their eventual role of translating gene-drug associations into clinical practice. genes, which are stretches of deoxyribonucleic acid (dna) contained on the 23 pairs of human chromosomes, determine the size and shape of every protein a living organism builds. variation in pharmacogenes which encode for proteins central to drug action and toxicity serves as the basis of pharmacogenomics (pgx). important online resources such as pharmgkb.org, cpicpgx.org, and pharmvar.org provide the clinician with curated and summarized pgx associations and clinical guidelines. as genetic testing becomes increasingly affordable and accessible, the time is now for pharmacists to embrace pgx-guided medication selection and dosing to personalize and improve the safety and efficacy of drug therapy. keywords: pharmacogenomics, pharmacogenetics, personalized medicine, pharmacogenomics knowledge base received: september 25, 2019. accepted: october 7, 2019. type: review funding: the author(s) received no financial support for the research, authorship, and/or publication of this article. declaration of conflicting interests: the author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. corresponding author: kathleen b orrico, university of california san francisco, school of pharmacy, 1430 rosemary street, menlo park, ca 94025, usa. emails: kathleen.orrico@ucsf.edu; orricok@comcast.net 886875dti0010.1177/1177392819886875drug target insightsorrico review-article2019 www.genome.gov mailto:kathleen.orrico@ucsf.edu mailto:orricok@comcast.net 2 drug target insights proteins are unique molecules in that their function is greatly affected by their conformational shape. variation in the amino acid composition of a protein, which is translated from a gene(s), can influence molecular folding, and therefore the biological activity of the polypeptide molecule produced. for example, the cftr gene “encodes” for a protein structure called the cystic fibrosis transmembrane conductance regulator (cftr), which serves as a channel for chloride ions across exocrine cell membranes mainly in lung and pancreatic duct epithelium. cystic fibrosis is caused by variations or mutations in the cftr gene, which result in cftr proteins that differ in structure and possess limited to no ability to transport chloride and other ions. this changes the electrolyte composition and viscosity of exocrine secretions leading to cystic fibrosis disease sequelae. the most common cftr gene variation is caused by the deletion of 1 phenylalanine amino acid in the cftr protein produced. this singular change in cftr structure allows it to be sequestered and destroyed by regulatory factors before it can be placed within the cell membrane. the genome or complete set of genetic instructions for all cellular organisms such as plants, animals, and human beings is spelled out in the structure of dna and serves as the molecular basis of inheritance.5 the dna molecule is a large doublestranded polymer of nucleotide base units that resembles a twisted ladder and is often referred to as a double helix (figure 1). the side rails or backbone of the ladder are composed of repeating units of the 5-carbon sugar deoxyribose linked by an acidic phosphate group. the rungs of the ladder are formed from the hydrogen bonding of 2 of 4 different nitrogenous bases and serve as the connections between the 2 nucleotide strands. much as binary code is to computer language where every instruction is written as a series of 0s and 1s, it is the sequence or pattern of these 4 nitrogenous bases that ultimately spells out the directions to make every protein. the 4 bases in dna’s alphabet include the double-ring purines adenine (a) and guanine (g) and the single-ring pyrimidines cytosine (c) and thymine (t); 1 purine and 1 pyrimidine form the connecting or complementary base pairs in a very specific manner. adenine always pairs with thymine (at) using 2 hydrogen bonds, and guanine always pairs with cytosine (gc) using 3 hydrogen bonds. therefore, it is only necessary to determine the order of bases in 1 strand of the dna molecule to deduce the sequence of the complementary strand. within the nucleus of every human cell except red blood cells and platelets, dna is arranged into structures known as chromosomes. a small amount of dna, inherited through the maternal line only, exists in the mitochondria of cells and is known as mitochondrial dna (mtdna). if stretched end to end as a continuous strand, the dna contained in 1 cell nucleus called nuclear dna (ndna) measures over 6 feet in length and contains all of the approximately 3 billion base pairs that make up the human genome. before mitosis or cell division begins, ndna tightly coils around histone proteins to form structures called chromosomes. much more than inert spools, the function of histone proteins has only begun to be appreciated. through winding and unwinding of the dna strand in response to intracellular signaling, histones orchestrate gene exposure to the cellular environment and play a role in gene expression. chromosomes are not static structures but rather active arrangements of specific sections of the ndna strand that comprise the whole genome. the size of human chromosomes ranges from 50 000 000 to 300 000 000 base pairs and can contain hundreds to thousands of genes.6 chromosome 1, for example, which is the largest, contains approximately 2100 protein-coding genes, while the y chromosome contains the least number or 60 functional genes. chromosomes are typically depicted as in figure 2, when they are most visible. this arrangement occurs immediately before cell division and after figure 1. double helix structure of deoxyribonucleic acid (dna). source: genetics home reference.5 figure 2. the structure of a human chromosome. dna indicates deoxyribonucleic acid. source: genetics home reference.7 orrico 3 the dna has been replicated forming 2 sister chromatid strands held in contact by a centromere. within the nucleus of every cell, except sperm and egg cells, humans have a total of 46 chromosomes existing as 23 pairs composed of 1 chromosome contributed from the mother and 1 from the father; 22 of these pairs are called autosomes and are identified by number from the largest in size (chromosome 1) to the smallest (chromosomes 22). the 23rd or last pair are the non-identical sex chromosomes. named the x and y chromosomes, they determine the sex of the offspring. at the ends or tips of the dna strands that form chromosomes are repeating sequences of base pairs called telomeres.8 rather like caps or aglets at the end of shoelaces, telomeres protect the chromosome from breakdown by providing spare parts for the ongoing process of dna repair that occurs during replication. an association exists between the length of telomeres and the life span of a cell, and thus the ultimate age of a living organism. genes a gene is a specific section of the dna base pair sequence located on the chromosome, which acts as a recipe or code that is transcribed and then translated into the amino acid structure of a protein. of the estimated 20 000 protein-coding genes in the human genome, each makes an average of 3 proteins.7 the gene’s dna sequence is divided into regions called exons and introns. during transcription, messenger ribonucleic acid (mrna) copies or transcribes the code, and delivers it to the ribosome for translation to a protein. it is the exons or protein-coding portions of the sequence that ultimately determine which amino acids comprise a given protein. the intron sections perform regulatory functions and are spliced out. the size of a gene is expressed by the number of nitrogenous base pairs it contains and can range from a few hundred to over 2 million. many genes are named for the proteins they encode and are given a symbol. for example, the cytochrome p450 (cyp) 2d6 enzyme and gene share the same name, and the gene is assigned the symbol cyp2d6. the gene symbol ace designates the angiotensin i converting enzyme gene. the human genome organisation (hugo) gene nomenclature committee (genenames.org) establishes an official name and symbol for each gene; however, be aware that genes may be referred to by multiple names in the literature.9 genes are the basic units for the inheritance of traits. genes that encode for proteins involved in drug action, toxicity, or metabolism are often referred to as pharmacogenes. the term phenotype is used to designate the type or classification of a visible trait or characteristic resulting from gene expression such as eye color, height, or the rate of cytochrome p450 (cyp p450) metabolism. at the time of this writing, 66 “very important pharmacogenes” (vips) are listed by the premier curators of pgx information the pharmacogenomic knowledge base (pharmgkb).10 chief among these are genes for specific cyp p450 enzymes with known associations between variation in their genetic recipe and the phenotype or degree of enzyme activity produced by the resulting protein. human beings are diploid organisms, meaning we have 2 versions of every gene, 1 inherited from each parent. the process of meiosis determines which 1 of the 2 genes each parent possesses gets inherited by the offspring and is the basis for the mendelian law of segregation. when egg and sperm combine, the now-fertilized egg contains the entire genome of the offspring which in turn gets passed to future cells at every cell division. the term genotype refers to the specific combination of the 2 genes (alleles) an individual inherits.11 alleles are forms or variants of the same gene with small differences in their dna base sequence. differences in the dna base sequence can ultimately lead to variation in the structure of the encoded protein or the amount produced. some genes are more likely than others to be subject to variation, and the term polymorphism (multiple forms) is generally used to discuss genes with multiple alleles. most of the genes located on autosomal chromosomes have biallelic expression, meaning that both copies express the encoded protein. other factors involved in gene expression can influence when or whether a protein will be made and in what amount. understanding the implications of variation in pharmacogenes is the heart and soul of pgx and allows us to individualize drug therapy. as a species, human beings are nearly genetically homogeneous and share over 99.9% of our entire genome with each other. it is this less than 0.1% variation that makes each of us unique. a set or group of gene alleles typically located on the same chromosome and inherited together is referred to as a haplotype and may indicate a common line of descent in individuals who share one. the allele or version of a gene that produces a trait shared by a sizable group of individuals in a population is often referred to as the non-mutated or wild-type allele.11 the wild-type variant(s) is often noted using star (*) nomenclature and written as the gene symbol followed by an asterisk and number 1 (cyp2d6*1). it is possible to have more than 1 wild-type allele typically noted by a letter following *1. a person’s pharmacogenes can differ in multiple ways. genetic variation that results from differences in the dna structure of a gene or regulatory portions of the genome can be caused by deletions, insertions, duplications, inversions, or substitutions of nitrogenous bases within the usual sequence, thus changing the genetic code and possibly the proteins produced. a single-nucleotide polymorphism (snp, “snip”) occurs when 1 nitrogenous base, along with its paired complement, is substituted within the nucleotide sequence. single-nucleotide polymorphisms can be conceptualized as misspellings or “typos” in the usual order of bases. a snp can become consequential if it occurs within a gene or regulatory region, and alters the function of the protein produced or disrupts the usual recipe so that no protein is produced at all. for example, an allele of the 4 drug target insights cyp2c19 gene named cyp2c19*2a (“star 2a”) results from a substitution of an adenine instead of a guanine at exon position 681 (noted as 681g>a) and results in a non-functioning enzyme.12 several drugs, including the anti-platelet agent clopidogrel, require cyp2c19 for bioactivation to an active metabolite to be fully effective. people receiving clopidogrel, whose genotype includes 1 or 2 no function alleles such as cyp2c19*2a, are classified as poor metabolizers (pms) of the drug. the fda-approved label for clopidogrel recommends that people who are pms consider use of an alternative platelet p2y12 inhibitor to avoid poor outcomes.13 copy number variation (cnv ) occurs when sections of the usual dna sequence either repeat multiple times or are deleted and do not occur at all.14 copy number variations become significant when they encompass a regulatory or coding section of a gene. when cnv leads to greater than 2 copies of a gene, the encoded protein may be produced in greater amounts and enzymatic activity increased. a cnv, that results in multiple copies of the gene is a multiple, is typically noted as the gene symbol followed by the allele star number × n (*2 × n). if the protein is an enzyme involved in drug metabolism such as one of the cyp p450 enzymes, increased enzyme activity can result in accelerated metabolism and altered pharmacokinetics. conversely, cnv can also lead to the deletion or skipping of a gene leaving an individual with less than 2 copies. lacking one or both copies of a cyp p450 gene can result in less to no enzyme being produced leading to poor drug metabolism. variation can also exist in the factors that regulate the expression of genes but do not alter the nucleotide base sequence. epigenetics, meaning above or upon the gene, is the study of factors and processes within the cell or the environment that influence the expression or suppression of the genes mapped in the dna sequence. in other words, epigenetic factors such as methylation and transcription factor proteins signal the static dna blueprint to turn on and off. an organization called the pharmacogene variation consortium (pharmvar.org) has been formed to catalog and consistently name pharmacogene variants, especially alleles of the cyp p450 enzymes.15 as clinical genetic testing becomes widespread, it is important for researchers and providers to communicate pgx results in an accurate and standardized manner. as in the above example, variants of the cyp p450 enzymes are customarily named using star-allele nomenclature where the gene symbol is followed by an asterisk and a variant number. pharmvar will expand upon this convention and also serve as a repository for phenotypic information on pharmacogenes. variant tables for the polymorphic cyp2c9, cyp2c19, and cyp2d6 genes are posted in the pharmvar database and include the corresponding phenotypic enzyme activity level.16 clinically actionable pharmacogene variation generally, a gene-drug association is considered clinically actionable if information about genetic testing is included in the fda-approved labeling. currently, multiple gene-drug associations that are clinically actionable for pharmacists involve the cyp p450 enzymes. variation in the dna nitrogenous base sequence can produce different versions (alleles) of the cyp2c9, cyp2c19, and cyp2d6 genes.17 several known gene variants in turn produce cyp p450 enzymes that differ in the extent of enzyme activity or amount produced, and therefore in their ability to metabolize drugs. knowing the genotype or the 2 gene alleles a person inherits gives the pharmacist additional information to consider when selecting or dosing drugs. to illustrate several of these concepts, let us examine how cyp2d6 gene variation relates to both the effectiveness and safety of the opioid analgesic drug codeine. cyp2d6 polymorphism the cyp2d6 enzyme participates in the metabolism of approximately 25% of all drugs, including the bioactivation of codeine, tramadol, and several other pro-drugs. the cyp2d6 gene located on chromosome 22 is highly polymorphic and over 90 known variants or alleles have been identified which encode for multiple forms of the enzyme.18 for many of these alleles, a relationship has been established between the version of the gene and the enzymatic activity of the cyp2d6 protein produced. for example, the cyp2d6*4 allele results from a snp caused by the substitution of an adenine instead of a guanine (g>a), which shifts the code and produces a non-functioning enzyme. the cyp2d6 gene is also subject to inheritable cnv, which can include both multiple copies of the gene or deletion of one or both copies. one study found that 12.6% of 30 000 patient samples tested contained 0, 1, or 3 or more copies of the cyp2d6 gene.19 codeine is essentially a pro-drug that needs the cyp2d6 enzyme to convert 5% to 10% of each dose to morphine, which has a 200 times greater affinity for the mu opioid receptor than does the parent compound.20 the balance between adequate analgesic effect and over-sedation is dependent upon an individual’s cyp2d6 activity. based on genotype (the combination of inherited alleles), people can be placed into 1 of 4 phenotypic categories that predict their expected level of cyp2d6 enzyme activity. variation in the cyp2d6 gene leads to a spectrum of enzymatic activity that ranges from rapid conversion of codeine to morphine to an inability to metabolize codeine through this pathway. table 1 shows some common cyp2d6 alleles accompanied with the phenotypic enzyme activity level.21 when presented with a person’s genotype, the phenotype category is assigned based on the highest functioning cyp2d6 allele. people categorized as ultrarapid metabolizers (ums) possess multiple copies (more than 2) of active alleles and express greater amounts of the cyp2d6 enzyme and therefore rapidly convert codeine to morphine. an example genotype of an um might consist of a *1 allele (wild-type) and the cnv *2 × n (*1/*2 × n) resulting in the production of greater than normal amounts of the cyp2d6 enzyme activity. orrico 5 ultrarapid metabolizers can experience high blood levels of morphine and if breastfeeding can pass excessive amounts to a nursing infant. at the opposite end of the spectrum, pms possess 2 no function alleles that encode for cyp2d6 enzyme that is inactive or does not get produced at all. when given usual doses of codeine, pms experience little analgesic effect because they do not convert codeine to morphine to an appreciable degree. extensive metabolizer (em) and intermediate metabolizer (im) phenotypes are often grouped together and are typically considered to have enzyme activity within the normal range and classified as normal metabolizers (nms). an individual with a genotype of *1/*17, for example, would be classified as an nm because they possess at least 1 normal function allele. intermediate metabolizers possess a genotype consisting of 2 decreased function alleles or 1 decreased and 1 no function allele such as *9/*41 or *9/*3. once the cyp2d6 genotype and metabolizer class is determined, the pharmacist must integrate this information into the drug treatment plan. consulting the pharmgkb is a vital first step. pharmgkb.org provides a comprehensive and easily accessible online pgx resource developed and generously shared by experts at stanford university. the mission of pharmgkb is to collect, curate, and disseminate knowledge about the impact of human genetic variation on drug response.22 it is an invaluable repository of evidence-based pgx knowledge that is organized and annotated for clinicians and researchers alike. in addition to the aforementioned list of vips, pharmgkb houses clinical practice guidelines and drug labeling information from the united states, canada, japan, and the netherlands. navigating pharmgkb can be as simple as entering the name of the drug or gene into the search field and arriving at a monograph-type synopsis with a side bar section tool. another efficient way to navigate is to select the category link titled dosing guidelines displayed on the home page and scrolling to the drug name such as codeine, for example. displayed here is a side-by-side selection of the available vetted pgx clinical practice guidelines for codeine.23 accessing the link in the first column displays an annotation of the clinical pharmacogenetics implementation consortium (cpic) guidelines concerning codeine and cyp2d6. the cpic guidelines are composed by an international expert panel with a mission to help clinicians use genetic testing to inform safe prescribing (https://cpicpgx. org/). the cpic recommends using alternate analgesics for people categorized as ums and pms. for those categorized as nms (ims and ems), cpic advises following the fda labeling recommendations which contraindicate the use of codeine in children less than 18 years old and in breastfeeding women. next, guidelines from the royal dutch association for the advancement of pharmacy—pharmacogenetics working group (dpwg) are posted for comparison. notice that dpwg recommendations differ from cpic by advising that ims avoid codeine use as well as ums and pms. the last link leads to the canadian pharmacogenomics network for drug safety (cpnds) guidelines that match cpic recommendations on codeine use and cyp2d6 genotype. predicting the risk for adverse drug reactions one of the most promising applications of pgx information is the ability to predict an individual patient’s risk for developing certain drug side effects. approximately 15% of adverse drug reactions (adrs) are type b or idiosyncratic reactions, meaning they are not related to the expected pharmacology, pharmacokinetics, or systemic concentration of a medication.24 often these are immune-mediated, hypersensitivity reactions that in some cases have been associated with genetic risk factors. several strong associations between variants of the human leukocyte antigen (hla) gene complex and the occurrence of severe cutaneous reactions have been established for a range of drugs.25 for this reason, pharmgkb designates the human leukocyte antigen b (hla-b) gene as a vip and provides links to cpic guidelines on its website, which address hla gene testing and safe use recommendations for abacavir, allopurinol, phenytoin, oxcarbazepine, and carbamazepine.26 the hla-b gene is part of a complex of genes located on chromosome 6 that encode for cell surface proteins involved in presenting antigens to the immune system. specific allelic variants of hla-b have been associated with the development of table 1. cytochrome p450 cyp2d6 allelic variants and enzyme activity. phenotype: cyp2d6 enzyme activity level cyp2d6 gene variant (cyp2d6*x) no function (null) alleles *3 *4 *5 *6 *7 *8 *11 *12 *13 *15 *18 *19 *20 *21 *31 *36 *38 *40 *42 *44 *47 *51 *56 *57 *60 *62 *68 *69 *92 *96 *99 *100 *101 *114 decreased function alleles *9 *10 *14 *17 *29 *41*49 *50 *54 *55 *59 *72 *84 normal function alleles *1 (wild type) *2 *27 *33 *34 *35 *39 *45 *46 *48 *53 increased function alleles copy number variants *1 × n *2 × n *35 × 2 uncertain/unknown function alleles *22 *23 *24 *25 *26 *28 *30 *37 *43 *52 *58 *61 *63 *64 *65 *70 *71 *73 *74 *75 *81 *82 *83 *85 *86 *87 *88 *89 *90 *91 *93 *94 *95 *97 *98 *102 *103 *104 *105 *106 *107 *108 *109 *110 *111 *112 *113 source: adapted from pharmacogene variation consortium (pharmvar).21 https://cpicpgx.org/ https://cpicpgx.org/ 6 drug target insights severe cutaneous adrs following exposure to carbamazepine typically occurring early in therapy. known as stevens-johnson syndrome (sjs) and toxic epidermal necrolysis (ten), these maladies can result in blistering and sloughing of the skin and mucous membranes that can proceed to liver and other organ failure.27 stevens-johnson syndrome and ten are really a continuum of severity of the same adverse reaction. stevensjohnson syndrome is fatal in 10% of patients, whereas 50% of those who progress to ten die. possessing 1 copy of the hla-b*1502 allele places a person receiving carbamazepine at a greater risk for experiencing these rare but sometimes fatal delayed hypersensitivity reactions. for this reason, the fda mandates as a boxed warning targeted genetic testing for the hla-b*1502 allele before initiation of carbamazepine in ethnic groups more likely to possess this allelic variant and to avoid use in carriers. these groups include people of han chinese, thai, malaysian, indonesian, filipino, and south indian ethnicity who have as much as 10 times the risk for experiencing hypersensitivity reactions than do mainly caucasian populations. a review of the fda table of pharmacogenomic biomarkers in drug labeling identifies the location within the drug labeling of any recommendation regarding genetic testing.28 notice for carbamazepine that the fda also suggests but does not require testing for another hla complex gene variant, the hla-a*3101 allele, which is associated with an increased risk of drug reaction with eosinophilia and systemic symptoms (dress) and maculopapular exanthema (mpe) as well as sjs/ten. this differs from cpic guidelines for carbamazepine and hla-a and hla-b gene variants, which recommend that testing for both hlab*1502 and hla-a*1301 be conducted for all carbamazepinenaïve patients in high-risk ethnic groups.29 for clearly actionable and fda required genetic testing such as for newstarts on carbamazepine in specific ethnic populations, pharmacists can play an important role within their practice institutions by inquiring and conducting reviews to determine whether appropriate genetic testing is being done. genetic sequencing the increasing accessibility and affordability of genetic testing is driving the translation of genomic medicine into clinical practice. improvements in dna sequencing technologies have dramatically reduced both the cost and the time it takes to accomplish whole genome sequencing (wgs) and whole exome sequencing (wes), which selectively maps the proteincoding or exon regions of the genes. the hgp spent in excess of $500 million and took over 10 years to sequence the first single reference human genome.30 by comparison, in 2018, wgs conducted in standard clinical practice can deliver results in 2 to 8 weeks at a cost of less than $1000. testing for targeted gene variants can be accomplished in a matter of days at a cost of a few hundred dollars. in 1977, frederick sanger developed the original base sequencing technique that involved cleaving 1 strand of the dna molecule into millions of pieces, copying or amplifying the fragments, and painstakingly identifying the terminal nitrogenous base in each fragment by tagging it with a radioactive or fluorescent complementary base. the hgp used an updated version of the sanger method that automated the process and allowed multiple fragments to be tested at one time. today’s technologies called next-generation or high-throughput sequencing can sequence millions of dna fragments simultaneously and process dna from multiple individuals at the same time.31 an entire genetic testing industry has arisen devoted to exploring new technologies and marketing services not only to health care providers but directly to consumers.32 perhaps because of the success and public receptivity of ancestry genetic testing, personal medical testing is being marketed directly to the public with at-home dna sample collection. many companies offer clinical-grade genetic testing after delivery of a saliva sample and some but not all require a physician’s order. typically, a package of targeted genes intended to screen for an individual’s risk for developing specific diseases are characterized. several companies offer to test for gene variants predictive of cancers or inherited cardiovascular diseases such as cardiomyopathy. a few companies specifically offer pgx testing with 1 testing for 50 pharmacogenes. the directto-consumer marketing of genetic testing places the results into the consumer’s hands, and while most companies offer access to genetic counselors or help lines, people are often directed to discuss testing results with their health care providers. pharmacists may be called upon to evaluate a person’s drug regimen and tailor it based on pgx test results. reports usually include a clinical interpretation to help the recipient understand the implications of the test. if presented with a report from a patient, accessing the company’s website to obtain more information about which genes are tested and access clinical decision support tools may be helpful. certainly, consulting pharmgkb and cpic guidelines will be useful. conclusions pharmacists are the ideal providers to orchestrate the translation of pgx gene-drug associations into clinical practice and improve patient care. knowing the genetic information of our patients adds complexity to treatment options and gives us new attributes to consider when personalizing the selection, dosing, and monitoring of drug therapy. discovering and getting involved with committees focused on genomic medicine at your institution is important for establishing our eventual role early-on. consider adding pgx subcommittees to established pharmacy committees such as pharmacy and therapeutics and medication safety. hopefully, this review has inspired you to embrace this emerging body of knowledge and continue to learn about new discoveries in genomic medicine. as pgx driven drug therapy advances, in medical science lead to a paradigm shift in drug discovery and treatment, it is both necessary and professionally fulfilling to keep current and practice ready. orrico 7 orcid id kathleen b orrico https://orcid.org/0000-0001-9450-9040 supplemental material supplemental material for this article is available online. r efer ences 1. genetics home reference. what is pharmacogenomics? bethesda, md: us national library of medicine. https://ghr.nlm.nih.gov/primer/genomicresearch/pharmacogenomics. updated july 16, 2019. accessed august 6, 2019. 2. all about the human genome project (hgp). washington, dc: national institutes of health, national human genome research institute. https://www. genome.gov/human-genome-project. updated january 9, 2019. accessed september 1, 2019. 3. venter jc, adams md, myers ew, et al. the sequence of the human genome. science. 2001;291:1304-1351. 4. the human proteome project. vancouver, bc, canada: human proteome organization. https://hupo.org/human-proteome-project/. updated 2016. accessed september 1, 2019. 5. genetics home reference. what is dna? bethesda, md: us national library of medicine. https://ghr.nlm.nih.gov/primer/basics/dna. updated august 20, 2019. accessed september 2, 2019. 6. national human genome research institute. the human genome project completion: frequently asked questions. what is dna sequencing? https:// w w w.genome.gov/11006943/human-genome-project-completion-frequentlyasked-questions/. updated november 12, 2018. accessed september 2, 2019. 7. genetics home reference. what is a chromosome? bethesda, md: us national library of medicine. https://ghr.nlm.nih.gov/primer/basics/chromosome. update august 20, 2019. accessed september 2, 2019. 8. shammas ma. telomeres, lifestyle, cancer, and aging. curr opin clin nutr metab care. 2011;14:28-34. doi:10.1097/mco.0b013e32834121b1. 9. genetics home reference. how are genetic conditions and genes named? bethesda, md: us national library of medicine. https://ghr.nlm.nih.gov/ primer/mutationsanddisorders/naming. updated august 20, 2019. accessed september 2, 2019. 10. whirl-carrillo m, mcdonagh em, hebert jm, et al. pharmacogenomics knowledge for personalized medicine. clin pharmacol ther. 2012;92(4):414-417. doi:10.1038/clpt.2012.96. 11. talking glossary of genetic terms. washington, dc: national institutes of health, national human genome research institute. https://www.genome. gov/glossary/. updated march 26, 2019. accessed september 2, 2019. 12. johnson ja, cavallari lh, touyz rm. pharmacogenetics and cardiovascular disease—implications for personalized medicine. pharmacol rev. 2013;65(3):987-1009. 13. us food and drug administration. table of pharmacogenomic biomarkers in drug labeling: clopidogrel. https://www.fda.gov/drugs/science-research-drugs/ table-pharmacogenomic-biomarkers-drug-labeling. updated september 3, 2019. accessed september 5, 2019. 14. he y, hoskins jm, mcleod hl. copy number variants in pharmacogenetic genes. trends mol med. 2011;17:244-251. doi:10.1016/j.molmed.2011.01.007. 15. gaedigk a, ingelman-sundberg m, miller na, leeder js, whirl-carrillo m, klein te. the pharmacogene variation (pharmvar) consortium: incorporation of the human cytochrome p450 (cyp) allele nomenclature database. clin pharmacol ther. 2018;103(3):399-401. 16. pharmacogene variation consortium (pharmvar). genes. https://www.pharmvar.org/genes. updated june 21, 2019. accessed july 3, 2019. 17. zhou sf. polymorphism of human cytochrome p450 2d6 and its clinical significance: part i. clin pharmacokinet. 2009;48:689-723. 18. dean l. codeine therapy and cyp2d6 genotype. in: medical genetics summaries. https://www.ncbi.nlm.nih.gov/books/nbk100662/. updated march 16, 2017. accessed september 4, 2019. 19. beoris m, amos wilson j, garces ja, lukowiak aa. cyp2d6 copy number distribution in the us population. pharmacogenet genomics. 2016;26(2):96-99. 20. the medical letter. fda warns against use of codeine and tramadol in children and breastfeeding women. med lett drugs ther. 2017;59(1521):86-88. 21. pharmacogene variation consortium (pharmvar). cyp2d6. https://www. pharmvar.org/gene/cyp2d6. updated august 28, 2019. accessed september 4, 2019. 22. the pharmacogenomics knowledge base (pharmgkb). about us. https://www. pharmgkb.org/about. accessed september 4, 2019. 23. the pharmacogenomics knowledge base (pharmgkb). dosing guidelines. https://www.pharmgkb.org/guidelines. accessed september 4, 2019. 24. lee a. adverse drug reactions. 2nd ed. london, england: pharmaceutical press; 2006:7. 25. negrini s, becquemont l. hla-associated drug hypersensitivity and the prediction of adverse drug reactions. pharmacogenomics. 2017;18:1441-1457. doi:10.2217/pgs-2017-0090. 26. clinical pharmacogenetics implementation consortium (cpic). guidelines. https://cpicpgx.org/guidelines/. updated july 11, 2019. accessed september 5, 2019. 27. negrini s, becquemont l. pharmacogenetics of hypersensitivity drug reactions. therapie. 2017;72:231-243. 28. us food and drug administration. table of pharmacogenomic biomarkers in drug labeling: carbamazepine. https://www.fda.gov/drugs/science-researchdrugs/table-pharmacogenomic-biomarkers-drug-labeling. updated september 3, 2019. accessed september 5, 2019. 29. the pharmacogenomics knowledge base (pharmgkb). annotation of cpic guideline for carbamazepine and hla-a, hla-b. https://w w w.pharmgkb. org/guideline/pa166105008. updated march 20, 2019. accessed september 4, 2019. 30. national human genome research institute. the cost of sequencing a human genome. https://w w w.genome.gov/about-genomics/fact-sheets/sequencing -human-genome-cost. updated july 10, 2019. accessed september 5, 2019. 31. reuter ja, spacek d, snyder mp. high-throughput sequencing technologies. mol cell. 2015;58:586-597. 32. genetics home reference. what is direct-to-consumer genetic testing? bethesda, md: us national library of medicine. https://ghr.nlm.nih.gov/ primer/dtcgenetictesting/directtoconsumer. updated september 3, 2019. accessed september 4, 2019. https://orcid.org/0000-0001-9450-9040 https://ghr.nlm.nih.gov/primer/genomicresearch/pharmacogenomics https://ghr.nlm.nih.gov/primer/genomicresearch/pharmacogenomics https://www.genome.gov/human-genome-project https://www.genome.gov/human-genome-project https://hupo.org/human-proteome-project/ https://ghr.nlm.nih.gov/primer/basics/dna https://www.genome.gov/11006943/human-genome-project-completion-frequently-asked-questions/ https://www.genome.gov/11006943/human-genome-project-completion-frequently-asked-questions/ https://www.genome.gov/11006943/human-genome-project-completion-frequently-asked-questions/ https://ghr.nlm.nih.gov/primer/basics/chromosome https://ghr.nlm.nih.gov/primer/mutationsanddisorders/naming https://ghr.nlm.nih.gov/primer/mutationsanddisorders/naming https://www.genome.gov/glossary/ https://www.genome.gov/glossary/ https://www.fda.gov/drugs/science-research-drugs/table-pharmacogenomic-biomarkers-drug-labeling https://www.fda.gov/drugs/science-research-drugs/table-pharmacogenomic-biomarkers-drug-labeling https://www.pharmvar.org/genes https://www.pharmvar.org/genes https://www.ncbi.nlm.nih.gov/books/nbk100662/ https://www.pharmvar.org/gene/cyp2d6 https://www.pharmvar.org/gene/cyp2d6 https://www.pharmgkb.org/about https://www.pharmgkb.org/about https://www.pharmgkb.org/guidelines https://cpicpgx.org/guidelines/ https://www.fda.gov/drugs/science-research-drugs/table-pharmacogenomic-biomarkers-drug-labeling https://www.fda.gov/drugs/science-research-drugs/table-pharmacogenomic-biomarkers-drug-labeling https://www.pharmgkb.org/guideline/pa166105008 https://www.pharmgkb.org/guideline/pa166105008 https://www.genome.gov/about-genomics/fact-sheets/sequencing-human-genome-cost https://www.genome.gov/about-genomics/fact-sheets/sequencing-human-genome-cost https://ghr.nlm.nih.gov/primer/dtcgenetictesting/directtoconsumer https://ghr.nlm.nih.gov/primer/dtcgenetictesting/directtoconsumer 21drug target insights 2015:9 basal plasma levels of copeptin are elevated in inactive inflammatory bowel disease after bowel resection bodil ohlsson and olle melander department of clinical sciences, section of internal medicine, skåne university hospital, malmö, and lund university, lund, sweden. a bstr act: evidence of interactions between the enteric nervous system, neuropeptides, and the immune system is growing. the aim of this study was to examine basal plasma levels of a variety of peptide precursors in patients with inflammatory bowel disease (ibd). in two middle-aged cohorts, malmö preventive medicine (n = 5,415) and malmö diet and cost study (n = 6,103), individuals with the diagnosis of ibd were identified. medical records were scrutinized. three controls were matched for each patient. copeptin, midregional fragments of adrenomedullin, pro-atrial natriuretic peptide, and proenkephalin a, as well as n-terminal protachykinin a and proneurotensin were analyzed in the plasma. sixty-two ibd patients were identified. the only difference between patients and controls was higher copeptin levels in the patients compared with controls (p = 0.006), with higher copeptin levels in resected than unresected patients (p = 0.020). there was no difference in any precursor levels between crohn’s disease and ulcerative colitis, between different distributions of disease lesions, or between different treatments. k e y wor ds: copeptin, inflammatory bowel disease, irritable bowel syndrome-like symptoms, neuropeptides, precursors citation: ohlsson and melander. basal plasma levels of copeptin are elevated in inactive inflammatory bowel disease after bowel resection. drug target insights 2015:9 21–27 doi:10.4137/dti.s26589. received: march 24, 2015. resubmitted: june 1, 2015. accepted for publication: june 3, 2015. academic editor: anuj chauhan, editor in chief type: original research funding: this work was supported by the bengt ihre foundation, dir. albert påhlsson foundation, foundation of skåne university hospital, and the development foundation of region skåne. the authors confirm that the funder had no influence over the study design, content of the article, or selection of this journal. competing interests: authors disclose no potential conflicts of interest. copyright: © the authors, publisher and licensee libertas academica limited. this is an open-access article distributed under the terms of the creative commons cc-by-nc 3.0 license. correspondence: bodil.ohlsson@med.lu.se paper subject to independent expert blind peer review by minimum of two reviewers. all editorial decisions made by independent academic editor. upon submission manuscript was subject to anti-plagiarism scanning. prior to publication all authors have given signed confirmation of agreement to article publication and compliance with all applicable ethical and legal requirements, including the accuracy of author and contributor information, disclosure of competing interests and funding sources, compliance with ethical requirements relating to human and animal study participants, and compliance with any copyright requirements of third parties. this journal is a member of the committee on publication ethics (cope). published by libertas academica. learn more about this journal. introduction interactions between the enteric nervous system (ens) and the immune system have been the subject of much new evidence in recent years. inflammatory cells in the intestine express receptors for neuropeptides, and the enteric neurons are responsive to cytokines secreted from inflammatory cells.1,2 nevertheless, the focus in inflammatory bowel disease (ibd) has been the immune system, and few studies have examined the role of neuropeptides in the development and maintenance of the disease, especially in inactive disease. even when the disease is under control and no signs of inflammation are present, patients with ibd often suffer from abdominal pain and functional abdominal complaints, so-called irritable bowel syndrome (ibs)-like symptoms.3 the reason for these complaints is unknown, but hypersensitivity, lowgrade inflammation, or impaired epithelial barrier has been suggested.3 imbalance in neurotransmitters/neuropeptides has only been sparsely examined in relation to hypersensitivity and ibs-like symptoms. structural changes occur in the ens during ibd,4 and the precursor mrnas encoding substance p and related receptors have been found to be upregulated in crohn’s disease.5,6 recently, lower plasma levels of enkephalins were described in active ibd patients compared with controls.7 adrenomedullin, enkephalin a, neurotensin, and substance p, as well as their receptors, have been demonstrated in the gastrointestinal tract2,8–11 and found to be involved in inflammation and nociception.10–14 adrenomedullin has been shown to ameliorate the induction of colitis in animal models and to induce wound healing.10 enkephalins exert both antiinflammatory and anti-nociceptive effects in induced colitis in animal models.13 endogenous neurotensin facilitates visceral pain responses and is necessary for the development of irritantinduced hyperalgesia.12 substance p is involved in the regulation of mast cell activation,14 and the upregulation of substance p observed in the gastrointestinal tract in ibd patients seems to be associated with ongoing local inflammation, which can be clinically quiet.11 copeptin, the precursor of arginine vasopressin (avp), is secreted from the posterior pituitary gland, and all three vasopressin receptors have been found in the gastrointestinal tract.15 the main roles of vasopressin are water reabsorption through v2 receptors,16 regulation of vascular tone by v1a receptor activation,17 and physiological adaptations to stress through adrenocorticotropic hormone (acth) release by the posterior pituitary involving v1b receptors.18 a few studies have examined the pharmacological, but not the physiological, effect of vasopressin on water and sodium absorption and secretion in human intestines.19,20 in rodents, vasopressin has been reported to exert proinflammatory effects through mast cell activation and enhanced epithelial permeability in the colon.21 atrial natriuretic peptide (anp) is secreted from the heart and contributes to substantial fluid loss via the gastrointestinal tract.22 peptides have a short half-time in plasma, so it is advantageous to measure the highly stable precursors instead journal name: drug target insights journal type: original research year: 2015 volume: 9 running head verso: ohlsson and melander running head recto: basal plasma levels of copeptin in ibd http://www.la-press.com/drug-target-insights-journal-j23 http://www.la-press.com http://dx.doi.org/10.4137/dti.s26589 http://creativecommons.org/licenses/by-nc/3.0/ http://creativecommons.org/licenses/by-nc/3.0/ mailto:bodil.ohlsson@med.lu.se http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 ohlsson and melander 22 drug target insights 2015:9 of peptides, which are synthesized in stoichiometric amounts relative to the mature peptides.23,24 the aim of the present pilot study was to analyze the basal plasma levels of a variety of peptide precursors involved in the physiology of the gastrointestinal tract, ie, copeptin, midregional fragment of pro-adrenomedullin (mr-proadm), midregional fragment of pro-atrial natriuretic peptide (mr-proanp), midregional fragment of proenkephalin a (mr-penk a), n-terminal protachykinin a (nt-pta), and proneurotensin, in two well-defined populations of patients with ibd, as these precursors have been discussed in the development and activity of gastrointestinal diseases. material and methods the research protocol was in accordance with the declaration of helsinki, and the study was approved by the ethics committee of lund university (2013/609). study population. the malmö preventive medicine (mpm) study is a population-based, prospective, epidemiologic cohort of 22,444 men (born between 1921 and 1949) and 10,902 women (born between 1926 and 1949) from malmö, sweden, who underwent baseline examinations between 1974 and 1992.25 from this cohort, 18,240 were re-examined from 2002 to 2006, and new blood samples were collected. out of these, 5,415 were randomly chosen for precursor analyses. the malmö diet and cancer study (mdcs) is a population-based, prospective, epidemiologic cohort of 28,449 men (born between 1923 and 1945) and women (born between 1923 and 1950) from malmö, sweden, who underwent baseline examinations between 1991 and 1996. from this cohort, 6,103 individuals were randomly selected to participate in the mdcs cardiovascular cohort (mdc-cc), which was designed to investigate the epidemiology of carotid artery disease between 1991 and 1994.26 the baseline examination procedure has been described in detail previously.27 briefly, the mpm and mdc-cc baseline examinations included a dietary assessment; a self-administered questionnaire about marital status, education, employment, smoking habits, wine consumption, physical activity, medical conditions and medication; anthropometric measurements; and collection of blood samples. at baseline, no questions concerning bowel diseases were asked. thus, we used local registers to retrieve prevalent cases of ibd according to the international classification of diseases (icd) version 8, 9, and 10. we initially identified 35  patients with ibd in the mpm re-examination cohort with available precursors analyses, and 70 patients with ibd in the mdc-cc cohort. the diagnoses were validated by scrutinizing all the medical records, and all patients with a clinical diagnosis made after careful clinical examination including endoscopy and histopathological examination were included in the study. from the medical records, the activity and extent of disease, concomitant diseases, drug treatment, and bowel resections were recognized. disease involvement in more than one gastrointestinal segment was defined as extensive disease. six patients in the mpm cohort and nine patients in the mdccc were excluded due to wrong diagnosis, the coexistence of cardiovascular disease, diabetes mellitus, or malignancy, or the medical records not being found in the archive and hence validation according to prevalent or incident ibd, disease activity, and concomitant diseases could not be performed. thus, after validation of the information from the register using the medical records, 22 prevalent and 7 incident cases of ibd remained in the mpm cohort and 40 prevalent and 21 incident cases in the mdc-cc, at the time point when plasma samples were collected. analyses. at the mpm reexamination and at the mdccc baseline examination, blood was collected in the morning after 12 hours of fasting, and plasma was separated and immediately frozen at −80°c. in the mdc-cc, plasma samples were available for analyses of mr-penk, nt-pta, and proneurotensin in 4,632 participants, and for analyses of copeptin, mr-proadm, and mr-proanp in 4,742 participants. the excluded participants (due to lack of plasma samples) were slightly younger, but did not otherwise differ in terms of sex, smoking habits, diabetes, hypertension status, body mass index (bmi), or plasma lipids.27,28 copeptin, the stable precursor peptide of avp, was measured by a murine monoclonal antibody directed to amino acids 137–144 of proavp using a commercially available assay in the chemiluminescence/coated tube format (lumitest ct-proavp, brahms gmbh) as described previously.29 mr-proadm and mr-proanp were analyzed using sandwich immunoluminometric assays targeted against amino acids in the mid-regions of the respective peptide (brahms sevadil lia® and brahms seristra®, respectively, brahms gmbh).30,31 briefly, two polyclonal antibodies targeted to amino acids 45–92 of proadm was used for the measurement of mr-proadm,30 and polyclonal sheep antibodies specific for amino acids 73–90 were used for the measurement of mr-proanp.31 mr-penk a is the stable fragment of the peptide precursor of enkephalins, and nt-pta is the stable fragment of the peptide precursor of substance p. mr-penk a was measured by a sensitive chemiluminescence immunoassay against amino acids 119−159 of the mr-penk a precursor fragment (brahms gmbh).23 a similar sensitive chemiluminescence immunoassay was developed to detect amino acids 1−37 of nt-pta (brahms gmbh).32 proneurotensin was measured by a recent developed chemiluminometric sandwich immunoassay to detect a proneurotensin precursor fragment (pro nt/nmn 1–117) (brahms gmbh).33 statistical analyses. patients with valid, prevalent ibd in the mpm (n = 22) and mdc-cc (n = 40) cohorts were matched with three controls from their own cohort. those subjects with prevalent cardiovascular disease, diabetes mellitus, or malignancy prior to the blood sampling were excluded from both patients and controls, as these diseases http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 basal plasma levels of copeptin in ibd 23drug target insights 2015:9 may interact with plasma precursor levels.28 matching was performed considering age at baseline, date of inclusion, sex, smoking habits, and bmi. plasma analyses were not available in some of the subjects in the mdc-cc, so analyses were measured in 28 or 34 prevalent patients with ibd and in 106 or 115 controls, respectively. as plasma analyses were not available in several of the few incidental cases, these were omitted from statistical calculations. all calculations were performed with the spss version 22 (statistical package for the social sciences, ibm corporation). calculations were performed separately for the two cohorts compared with controls. furthermore, the two cohorts were added in an attempt to reach a greater cohort. as age differed between mpm and mdc-cc, and several peptide analyses correlated with age, the values were age-standardized using a linear regression model into which age was added as a covariate and the variables were expressed as z-scores. those precursors that did not differ between the mpm and mdc-cc cohorts were copeptin (p = 0.623), mr-proadm (p = 0.058), and mr-proanp (p = 0.500), which were thus calculated together. values are given as median [interquartile range (iqr)] or mean ± standard deviation (sd). the mann– whitney u-test was used to compare the differences in precursor levels between patients and controls and for subgroup analyses within the ibd group. fisher’s exact test was used for dichotomous variables. the spearman rank correlation test was used for correlations between precursor and disease duration. p  0.05 was considered statistically significant. results patient characteristics. mpm. the basal characteristics are described in table 1. the 22 included patients were in an inactive phase during their enrollment in the study. disease duration was 25.4 ± 13.4 years. none of the patients was receiving acute therapy, but six (27%) were on continuous treatment with anti-inflammatory or immune-modulating therapy. twelve patients (54%) had undergone intestinal resections due to ibd, and 10 of the patients (46%) had extensive disease. apart from ibd, six patients also suffered from hypertension and four from nephrolithiasis. sporadic cases of asthma bronchialis, bile stones, chronic obstructive pulmonary disease (copd), dyspepsia, hyperlipidemia, hypothyroidism, migraine, primary sjögren’s syndrome, reflux, and renal insufficiency were found. mdc-cc. all 40 included patients with prevalent ibd were in an inactive state during enrollment in the study. disease duration was 13.1 ± 10.0  years (table 1). none was undergoing acute therapy, and only three patients (8%) were on continuous treatment with anti-inflammatory or immunemodulating therapy. thirteen patients (32%) had undergone intestinal resections due to ibd, whereas 18 patients (45%) had an extensive distribution of the disease. sporadic concomitant diseases were present in the form of asthma bronchialis, copd, duodenal ulceration, dyspepsia, hiatal hernia, hypertension, hypothyroidism, inguinal hernia, ankylosing spondylitis, and venous thrombosis. precursor analyses. when precursor analyses were done together, the higher plasma level of copeptin was the only significant difference in plasma measurements between patients and controls (table 2). patients who had undergone bowel resection had higher plasma levels of copeptin compared with unresected patients [10.25 (7.94–13.75) and 6.33 (4.92–9.52) pmol/l, respectively, p = 0.020]. when the patients with a history of bowel resection were excluded (n = 23) together with their matched controls, there was no difference in copeptin levels between patients and controls [6.33 (4.92–9.52) and 5.81 (3.52–9.64) pmol/l, respectively, p = 0.501]. there was no diftable 1. patient characteristics of the two cohorts. prevalent ibd mpm n = 22 prevalent ibd mdc-cc n = 40 p-value age (years) 69.7 ± 7.2 56.0 ± 6.5 0.0001 sex (male/female) 12/10 19/21 0.791 smoking (n, %) 0.182 missing values (n) 2 smokers 11 (50) 12 (30) nonsmokers 11 (50) 26 (65) bmi (kg/m2) 23.8 ± 3.8 24.8 ± 3.5 0.237 duration of ibd (years) 25.4 ± 13.4 13.1 ± 10.0 0.0001 missing values (n) 2 crohn’s disease/ulcerative colitis 8/14 19/21 0.435 notes: n (%) = number and percentage of cases. values are given as mean ± standard deviation (sd). mann–whitney u-test or fisher’s exact test. p  0.05 was considered statistically significant. abbreviations: bmi, body mass index; ibd, inflammatory bowel syndrome; mdc-cc, malmö diet and cancer study cardiovascular cohort; mpm, malmö preventive medicine. http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 ohlsson and melander 24 drug target insights 2015:9 ference in copeptin levels whether or not the patients suffered from crohn’s disease or ulcerative colitis [7.19 (4.15–11.34) and 8.13 (5.03–11.68) pmol/l, respectively, p = 0.461], whether they were treated with anti-inflammatory drugs or not [4.69 (3.06–11.77) and 8.09 (5.60–11.43) pmol/l, respectively, p = 0.254], or whether they had a limited or extensive disease [8.03 (5.53–12.60) and 8.13 (5.24–11.05) pmol/l, respectively, p = 0.493]. since age and disease duration were strongly correlated (rs = 0.374, p = 0.003), the correlation between disease duration and plasma levels of mr-proadm (rs = 0.380, p = 0.005), nt-pta (rs = 0.483, p = 0.001), and mr-penk a (rs = 0.302, p = 0.037) was reflected by concomitant correlation with age (rs = 0.668, p  0.0001; rs = 0.483, p  0.0001; and rs = 0.315, p = 0.026, respectively). the expression of crohn’s disease or ulcerative colitis, treatment with anti-inflammatory drugs or not, limited or extensive disease, or a past bowel resection did not affect the plasma levels of any of the other precursors (data not shown). when calculated separately, there was a difference between mpm and controls regarding plasma levels of copeptin and mr-proadm but not regarding those of the other precursors (table 3). the level of copeptin was higher in patients who had undergone a bowel resection (n = 12) compared with the nonresected (n = 6, missing value = 4), although it did not reach statistical significance in the small cohort [11.46 (8.33–19.08) and 7.42 (5.68–14.44), respectively, p = 0.291]. neither was there any difference in plasma copeptin levels depending on crohn’s disease or ulcerative colitis [7.92 (5.68–11.01) and 10.41 (5.92–16.68), respectively, p = 0.365], were treated with anti-inflammatory drugs or not [9.71 (3.90–12.81) and 8.66 [6.09–18.16] pmol/l, respectively, p = 0.693) or a limited or extensive disease [11.75 (7.08–17.86) and 9.01 (5.90–13.82), respectively, p = 0.604]. regarding mr-proadm, there was no difference in plasma levels depending on bowel resection or not [0.83 (0.65–1.22) and 0.84 (0.73–1.05), respectively, p = 0.892], crohn’s disease or ulcerative colitis [0.75 (0.69–0.87) and 0.86 (0.66–1.11), respectively, p = 0525], were treated with anti-inflammatory drugs or not [0.84 (0.67–1.11) and 0.79 (0.68–1.01) pmol/l, respectively, p = 0.858], or a limited or extensive disease [0.89 (0.75–1.37) and 0.77 (0.64–1.04), respectively, p = 0.182]. in the mdc-cc cohort, no differences were seen between controls and patients in any plasma precursor analyses (table 4). neither were any differences in plasma levels of any of the precursors between the expression of crohn’s disease or ulcerative colitis, treatment with anti-inflammatory drugs or not, limited or extensive disease, or a past bowel resection (data not shown). discussion the main conclusion of this study is that basal plasma copeptin levels are elevated after complete bowel resection, but other peptide precursors are unaffected in middle-aged subjects in inactive ibd compared with matched controls. however, the elevated copeptin levels did not reach statistical significance when precursor levels were calculated separately in the mdccc cohort, or when the mpm cohort was separated into table 2. plasma peptide levels from all patients with inflammatory bowel disease and controls. ibd (n = 62) controls (n = 186) p-value copeptin (pmol/l) 8.0 (4.8–11.5) (56) 5.8 (3.4–8.5) (181) 0.006 mr-proadm (nmol/l) 0.6 (0.4–0.8) (56) 0.5 (0.4–0.7) (182) 0.127 mr-proanp (pmol/l) 78.0 (59.2–111.8) (56) 72.7 (54.1–101.3) (182) 0.367 notes: n = number of analyses. values are given as median [interquartile range (iqr)]. mann–whitney u-test. p  0.05 was considered statistically significant. abbreviations: ibd, inflammatory bowel disease; mr-proadm, midregional fragment of pro-adrenomedullin; mr-proanp, midregional fragment of pro-atrial natriuretic peptide. table 3. plasma peptide levels from patients in malmö preventive medicine (mpm) compared with controls. mpm (n = 22) controls (n = 66) p-value copeptin (pmol/l) 8.66 (5.90–12.81) 6.23 (3.79–9.19) 0.007 mr-proadm (nmol/l) 0.80 (0.68–1.04) 0.70 (0.57–0.84) 0.010 mr-proanp (pmol/l) 104.27 (77.10–149.13) 98.73 (68.14–145.69) 0.649 mr-penk (pmol/l) 59.48 (54.41–74.41) 60.89 (50.39–74.63) 0.785 nt-pta (pmol/l) 78.98 (64.11–98.66) 80.64 (67.12–97.40) 0.852 proneurotensin (pmol/l) 79.15 (63.15–130.45) 84.45 (62.18–119.65) 0.972 notes: n = number of analyses. values are given as median [interquartile range (iqr)]. mann–whitney u-test. p  0.05 was considered statistically significant. abbreviations: ibd, inflammatory bowel disease; mr-proadm, midregional fragment of pro-adrenomedullin; mr-proanp, midregional fragment of pro-atrial natriuretic peptide; mr-penk, midregional fragment of proenkephalin a; nt-pta, n-terminal polytachykinin a. http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 basal plasma levels of copeptin in ibd 25drug target insights 2015:9 resected and unresected groups. the elevated plasma level of mr-proadm observed in the mpm cohort was diluted when the two cohorts were calculated together. the etiology of ibd is unknown, but a complex interplay of genetic, microbial, immunologic, and environmental factors are discussed. the inflammation in ibd is mainly situated in the mucosa, but transmural inflammation has been documented in both crohn’s disease and ulcerative colitis.4 the majority of the neuropeptides are found both in the central nervous system (cns) and in the ens, and we do not know whether the plasma peptides are of gastrointestinal or cephalic origin. gastrointestinal peptides may be secreted from both enterochromaffin mucosal cells and enteric neurons.9,11,34 furthermore, ibd affects the brain–gut axis with possible subsequent effects on peptide release from the brain.35 the modest effect on plasma levels of precursors in the present study suggests that, despite the chronic nature of ibd, the disease does not have a major impact on peptide secretion. nevertheless, as a wide range of neuropeptides and inflammatory factors are involved in physiological and pathophysiological processes, the balance between peptides, rather than the actual concentration of one specific peptide, may be important. the levels of copeptin and vasopressin in plasma are correlated with each other in both healthy volunteers and sick patients.24 vasopressin has been shown to increase epithelial permeability,21 but its effect on water reabsorption in the gastrointestinal tract has only been studied in pharmacological doses of vasopressin, and the results are not conclusive.19,20 as patients with ibd and a history of bowel resection had higher plasma levels of copeptin, the question remains whether the altered precursor levels measured in plasma are a primary etiological factor or a secondary effect of the disease.7,36 the difference could hypothetically be explained by a compensatory mechanism due to a shorter bowel, with ensuing reduced amount of vasopressin receptors and/or reduced water reabsorption capacity of the bowel.15,24 elevated plasma levels of vasopressin have been found in diseases characterized by chronic inflammation.37 in mice, injections of vasopressin into the periaqueductal gray raised the plasma levels of all enkephalins,38 and stimulation of peripheral vasopressin receptors had proinflammatory effects in experimental colitis, whereas inhibition of the receptors abolished the vasopressin effect.21 the effect of vasopressin on encephalin secretion has not been studied in humans. proenkephalin is the precursor of met-enkephalin and leu-enkephalin, and in contrast to the findings in our study with unaffected mr-penk a, plasma levels of met-enkephalin have previously been shown to be lowered in ibd patients.7,36 the study by owczarek et al7 was performed in younger patients with active disease, which could exhaust the peptides in the blood circulation and explain the different results. the actual encephalin concentration in the gastrointestinal tissue is not measured, but agonists to the peripheral opioid receptors lead to anti-inflammatory and anti-nociceptive effects.13 these studies suggest a role of the enkephalins and their receptors in disease activity. the higher levels of copeptin in ibd patients with a bowel resection may thus be of interest, as vasopressin may modulate gastrointestinal inflammation, either directly38 or indirectly through modulation of proenkephalins and their stimulation of the opioid receptors.13,21 the role of copeptin to keep the ibd patient in remission deserves to be further examined. ibs-like symptoms are present in 35%–40% of ibd patients during remission. this well-described phenomenon has for several years been believed to depend on low-grade inflammation.3 very few human studies have examined the effect of neuropeptides on visceral pain, but a connection between spinal afferents, enteric mast cells, and the ens has been confirmed, suggesting how elevation of intestinal hypersensitivity might occur involving neural peptides, eg, neurotensin and substance p.12,14 substance p has also been shown to exert proinflammatory effects, especially on neurogenic inflammation.11,14 noninflamed small bowel samples from patients with crohn’s disease have shown increased expression of substance p mrna in one study,5 with more binding sites in the inflamed areas, although unchanged mrna levels were found in another study.6 plasma levels of substance p have previously been found to be increased in ibs patients but not in ibd patients with ibs-like symptoms.39 presence of ibs-like symptoms was not measured in this retrospective study, but the unaltered plasma levels of precursors in the current study do table 4. plasma peptide levels from patients in malmö diet and cancer study cardiovacsular cohort (mdc-cc) compared with controls. mdc-cc (n = 40) controls (n = 120) p-value copeptin (pmol/l) 6.70 (3.86–10.55) (34) 5.65 (3.23–8.42) (115) 0.182 mr-proadm (nmol/l) 0.45 (0.38–0.54) (34) 0.45 (0.38–0.51) (116) 0.489 mr-proanp (pmol/l) 66.60 (52.40–82.62) (34) 65.85 (47.38–85.65) (116) 0.499 mr-penk (pmol/l) 42.65 (37.45–47.52) (28) 46.15 (39.60–53.62) (106) 0.086 nt-pta (pmol/l) 51.86 (44.18–60.38) (28) 51.01 (41.00–61.67) (102) 0.854 proneurotensin (pmol/l) 96.28 (62.09–142.69) (28) 93.39 (67.37–138.27) (106) 0.860 notes: n = number of analyses. values are given as median [interquartile range (iqr)]. mann–whitney u-test. p  0.05 was considered statistically significant. abbreviations: ibd, inflammatory bowel disease; mr-proadm, midregional fragment of pro-adrenomedullin; mr-proanp, midregional fragment of pro-atrial natriuretic peptide; mr-penk, midregional fragment of proenkephalin a; nt-pta, n-terminal polytachykinin a. http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 ohlsson and melander 26 drug target insights 2015:9 not suggest involvement of peptides for development of gastrointestinal symptoms. the limitation of the present study is the small study population. however, previous studies in the field have been of smaller or similar sizes.7,36 the advantage of measuring precursors instead of peptides depends on the high stability of these precursors, which are synthesized in stoichiometric amounts relative to the mature peptides.23,24 the strength is that we have analyzed several precursors in well-characterized cohorts. higher plasma levels of precursors may be related to a reduced degradation/excretion in an elder cohort, rather than an altered expression/secretion.40 this means that multiple factors influence the precursor levels in plasma. we have matched for age, gender, smoking habits, and bmi, and excluded patients with cardiovascular diseases, diabetes mellitus, and malignancy, but factors such as dietary habits, alcohol intake, stress, physical activity, sleeping habits, and drug therapy may also influence the secretion and degradation of precursors. the difference between the two cohorts regarding plasma values of penk, pnt, and pta may be reflected by the different factors mentioned above. although not statistically significant, there were more cases of crohn’s disease in the mdc-cc cohort than in the mpm cohort, and also a different smoking prevalence between the two cohorts. as data on the mpm cohort were collected several years before those of the mdc-cc cohort, treatment regimens in the daily clinic also differed, which may have influenced the precursor secretion. thus, the two cohorts were not matched or identical in composition. in conclusion, higher basal plasma levels of copeptin were found in inactive ibd patients, whereas the basal plasma levels of mr-proadm, mr-proanp, mr-penk a, ntpta, and proneurotensin were not altered in inactive ibd compared with controls in middle-aged subjects. elevated copeptin levels were found in ibd patients who had undergone bowel resection. the role of copeptin to keep the ibd patient in remission deserves to be further examined. list of abbreviations avp, arginine vasopressin; bmi, body mass index; ibd, inflammatory bowel disease; mdc-cc, malmö diet and cancer study cardiovascular cohort; mpm, malmö preventive medicine; mr-proadm, midregional fragment of pro-adrenomedullin; mr-proanp, midregional fragment of pro-atrial natriuretic peptide; mr-penk, midregional fragment of proenkephalin a; nt-pta, n-terminal polytachykinin a. author contributions made substantial contributions to the conception and design of the study, participated in the interpretation of the statistical analysis, and financed the study: bo, om. validated the medical records and wrote the manuscript: bo. both authors were involved in revising the manuscript critically for important intellectual content, and approved the final manuscript. r efer ences 1. levite m, chowers y. nerve-driven immunity: neuropeptides regulate cytokine secretion of t cells and intestinal epithelial cells in a direct, powerful and contextual manner. ann oncol. 2001;12:s19–s25. 2. margolis kg, gershon md. neuropeptides and inflammatory bowel disease. curr opin gastroenterol. 2009;25:503–511. 3. vivinus-nébot m, frin-mathy g, bzioueche h, et al. functional bowel symptoms in quiescent inflammatory bowel diseases: role of epithelial barrier disruption and low-grade inflammation. gut. 2014;63:744–752. 4. ohlsson b, veress b, lindgren s, sundkvist g. enteric ganglioneuritis and abnormal interstitial cells of cajal; features of inflammatory bowel disease. inflamm bowel dis. 2007;132:45–51. 5. michalski cw, autschbach f, selvaggi f, et al. increase in substance p precursor mrna in noninflamed small-bowel sections in patients with crohn’s disease. am j surg. 2007;193:476–481. 6. ter beek wp, biemond i, muller es, van den berg m, lamers cb. substance p receptor expression in patients with inflammatory bowel disease. determination by three different techniques, i.e., storage phosphor autoradiography, rt-pcr and immunohistochemistry. neuropeptides. 2007;41:301–306. 7. owczarek d, cibor d, mach t, et al. met-enkephalins in patients with inflammatory bowel diseases. adv med sci. 2011;56:158–164. 8. monstein hj, grahn n, ohlsson b. proenkephalin-a mrna is widely expressed in tissues of the human gastrointestinal tract. eur surg res. 2006;38:464–468. 9. egerod kl, engelstoft ms, grunddal kv, et al. a major lineage of enteroendocrine cells coexpress cck, secretin, gip, glp-1, py y, and neurotensin but not somatostatin. endocrinology. 2012;153:5782–5795. 10. ashizuka s, inatsu h, inagaki-ohara k, kita t, kitamura k. adrenomedullin as a potential therapeutic agent for inflammatory bowel disease. curr protein pept sci. 2013;14:246–255. 11. demir ie, schäfer kh, tieftrunk e, friess h, ceyhan go. neural plasticity in the gastrointestinal tract: chronic inflammation, neurotrophic signals, and hypersensitivity. acta neuropathol. 2013;125:491–509. 12. gui x, carraway re, dobner pr. endogenous neurotensin facilitates visceral nociception and is required for stress-induced antinociception in mice and rats. neuroscience. 2004;126:1023–1032. 13. sobczak m, pilarczyk a, jonakowski m, et al. anti-inflammatory and antinociceptive action of the dimeric enkephalin peptide biphalin in the mouse model of colitis: new potential treatment of abdominal pain associated with inflammatory bowel diseases. peptides. 2014;60:102–106. 14. wang gd, wang xy, liu s, et al. innervation of enteric mast cells by primary spinal afferents in guinea pig and human small intestine. am j physiol gastrointest liver physiol. 2014;307:g719–g731. 15. monstein hj, truedsson m, ryberg a, ohlsson b. vasopressin receptor mrna expression in the human gastrointestinal tract. eur surg res. 2008;40:34–40. 16. deen pm, verdijk ma, knoers nv, et al. requirement of human renal water channel aquaporin-2 for vasopressin-dependent concentration of urine. science. 1994;264:92–95. 17. byron kl. vasoprressin stimulates ca2+ spiking activity in a7r5 vascular smooth muscle cells via activation of phospholipase a2. circ res. 1996;78:813–820. 18. decaux g, soupart a, vassart g. non-peptide arginine-vasopressin antagonists: the vptans. lancet. 2008;371:1624–1632. 19. levitan r, mauer i. effect of intravenous antidiuretic hormone administration on salt and water absorption from the human colon. j lab clin med. 1968;72: 739–746. 20. soergel kh, whalen ge, harris ja, geenen je. effect of antidiuretic hormone on human small intestinal water and solute transport. j clin invest. 1968;47: 1071–1082. 21. ferrier l, serradeil-le gal c, schulte am, et al. proinflammatory role of vasopressin through v1b receptors in hapten-induced experimental colitis in rodents: implication in ibd. am j physiol gastrointest liver physiol. 2010;299: g1298–g1307. 22. aardal s, helle kb. comparative aspects of the endocrine myocardium. acta physiol scand suppl. 1991;599:31–46. 23. ernst a, kohrle j, bergmann a. proenkephalin a 119–159, a stable proenkephalin a precursor fragment identified in human circulation. peptides. 2006;27: 1835–1840. 24. jochberger s, morgenthaler ng, mayr vd, et al. copeptin and arginine vasopressin concentrations in critically ill patients. j clin endocrinol metab. 2006;91: 4381–4386. 25. berglund g, eriksson kf, israelsson b, et al. cardiovascular risk groups and mortality in an urban swedish male population: the malmö preventive project. j intern med. 1996;239:489–497. 26. persson m, berglund g, nelson jj, hedblad b. lp-pla2 activity and mass are associated with increased incidence of ischemic stroke: a population-based cohort study from malmö, sweden. atherosclerosis. 2008;200:191–198. 27. enhörning s, wang tj, nilsson pm, et al. plasma copeptin and the risk of diabetes mellitus. circulation. 2010;121:2102–2108. http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 basal plasma levels of copeptin in ibd 27drug target insights 2015:9 28. melander o, maisel as, almgren p, et al. plasma proneurotensin and incidence of diabetes, cardiovascular disease, breast cancer, and mortality. jama. 2012;308:1469–1475. 29. fenske w, stork s, blechschmidt a, maier sg, morgenthaler ng, allolio b. copeptin in the differential diagnosis of hyponatremia. j clin endocrinol metab. 2009;94:123–129. 30. morgenthaler ng, struck j, thomas b, bergmann a. immunoluminometric assay for the midregion of pro-atrial natriuretic peptide in human plasma. clin chem. 2004;50:234–236. 31. morgenthaler ng, struck j, alonso c, bergmann a. measurement of midregional proadrenomedullin in plasma with an immunoluminometric assay. clin chem. 2005;51:1823–1829. 32. ernst a, suhr j, kohrle j, bergmann a. detection of stable n-terminal protachykinin a immunoreactivity in human plasma and cerebrospinal fluid. peptides. 2008; 29:1201–1206. 33. ernst a, hellmich s, bergmann a. proneurotensin 1–117, a stable neurotensin precursor fragment identified in human circulation. peptides. 2006;27:1787–1793. 34. khan wi, ghia je. gut hormones: emerging role in immune activation and inflammation. clin exp immunol. 2010;161:19–27. 35. bonaz bl, bernstein cn. brain-gut interactions in inflammatory bowel disease. gastroenterology. 2013;144:36–49. 36. wiedermann cj, sacerdote p, propst a, et al. decreased beta-endorphin content in peripheral blood mononuclear leukocytes from patients with crohn’s disease. brain behav immun. 1994;8:261–269. 37. han dm, zhang yq , bai qx, chen xq. assay of avp, crp, and lps in leukemia. int j lab hematol. 2007;29:185–189. 38. yang j, yang y, xu ht, chen jm, liu w y, lin bc. arginine vasopressin induces periaqueductal grey release of encephalin and endorphin relating to pain modulation in the rat. regul pept. 2007;142:29–36. 39. keszthelyi d, troost fj, jonkers dm, et al. alterations in mucosal neuropeptides in patients with irritable bowel syndrome and ulcerative colitis in remission: a role in pain symptom generation? eur j pain. 2013;17:1299–1306. 40. bhandari ss, loke i, davies je, squire ib, struck j, ng ll. gender and renal function influence plasma levels of copeptin in healthy individuals. clin sci. 2009; 116:257–263. http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 51drug target insights 2014:8 open access: full open access to this and thousands of other papers at http://www.la-press.com. drug target insights genome-wide analysis of mycoplasma hominis for the identification of putative therapeutic targets md. masud parvege, monzilur rahman and mohammad shahnoor hossain department of genetic engineering & biotechnology, university of dhaka, dhaka, bangladesh. a bstr act: ever increasing propensity of antibiotic resistance among pathogenic bacteria raises the demand for the development of novel therapeutic agents to control this grave problem. advances in the field of bioinformatics, genomics, and proteomics have greatly facilitated the discovery of alternative drugs by swift identification of new drug targets. in the present study, we employed comparative genomics and metabolic pathway analysis with an aim of identifying therapeutic targets in mycoplasma hominis. our study has revealed 40 annotated metabolic pathways, including five unique pathways of m. hominis. our study also identified 179 essential proteins, including 59 proteins having no similarity with human proteins. further filtering by molecular weight, subcellular localization, functional analysis, and protein network interaction, we identified 57 putative candidates for which new drugs can be developed. druggability analysis for each of the identified targets has prioritized 16 proteins as suitable for potential drug development. k e y wor ds: mycoplasma hominis, drug targets, bioinformatics, genomics, proteomics citation: parvege et al. genome-wide analysis of mycoplasma hominis for the identification of putative therapeutic targets. drug target insights 2014:8 51–62 doi:10.4137/dti.s19728. received: august 31, 2014. resubmitted: november 6, 2014. accepted for publication: november 10, 2014. academic editor: anuj chauhan, editor in chief type: original research funding: this work was supported by the research grant provided to dr. mohammad shahnoor hossain from biotechnology research centre, university of dhaka. the authors confirm that the funder had no influence over the study design, content of the article, or selection of this journal. competing interests: authors disclose no potential conflicts of interest. copyright: © the authors, publisher and licensee libertas academica limited. this is an open-access article distributed under the terms of the creative commons cc-by-nc 3.0 license. correspondence: mshahnoor@du.ac.bd paper subject to independent expert blind peer review by minimum of two reviewers. all editorial decisions made by independent academic editor. upon submission manuscript was subject to anti-plagiarism scanning. prior to publication all authors have given signed confirmation of agreement to article publication and compliance with all applicable ethical and legal requirements, including the accuracy of author and contributor information, disclosure of competing interests and funding sources, compliance with ethical requirements relating to human and animal study participants, and compliance with any copyright requirements of third parties. this journal is a member of the committee on publication ethics (cope). introduction mycoplasmas are cell wall-deficient, smallest, free-living organisms, which are resistant to many commonly used antimicrobial agents.1 although most of the mycoplasma are naturally found as commensals in the genitourinary tract, three species are wellknown pathogens: mycoplasma pneumoniae, m. hominis, and m. genitalium.2 to lead a parasitic lifestyle, m. hominis resides in the urogenital tract of women and sexually active men where it gains necessary nutrients.3 sometimes, the bacterium is found in a symbiotic relationship with protozoa, trichomonas vaginalis, an event that allows for more successful transfer of m. hominis from one person to another, and increases its resistance to antibiotics.3,4 this bacterium is the causative agent of numerous health problems, such as pelvic inflammatory disease, bacterial vaginosis, post-partum fever, and infertility in females.2,5–7 in addition, m. hominis is capable of causing diseases of the central nervous system in newborn babies8 and is associated with prostate cancer.9 currently, antibiotics are the only available treatment option against m. hominis infection. however, this species is inherently resistant to beta-lactam group antibiotics and macrolides because of their lack of cell wall and a mutation in 23s rrna, respectively.10 moreover, m. hominis has been found to carry resistance trait against ciprofloxacin and ofloxacin.11 the increasing trend of antibiotic resistance demands the discovery of alternative therapeutic agents for the treatment of infection caused by this bacterium. these issues require the need for exploring new drug targets in this bacterium, which will create the avenue for new drug discovery or will augment the sensitivity of the currently existing antimicrobial agents. increasing availability of related genomics, proteomics, metabolomics, and many other omics data of the infectious agents and information about molecules that can alter the capability of survival of the infectious organism have facilitated the search for new drugs or drug targets. as the genome journal name: drug target insights journal type: original research year: 2014 volume: 8 running head verso: parvege et al running head recto: identification of therapeutic targets in mycoplasma hominis http://www.la-press.com/drug-target-insights-journal-j23 http://www.la-press.com http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 http://dx.doi.org/10.4137/dti.s19728 http://creativecommons.org/licenses/by-nc/3.0/ http://creativecommons.org/licenses/by-nc/3.0/ mailto:mshahnoor@du.ac.bd parvege et al 52 drug target insights 2014:8 of m. hominis, the second smallest genome among the freeliving organisms, has already been sequenced, similar kind of study can also be possible for this bacterium.12 numerous in silico methods have already been developed for the prediction of potential drug targets in many bacteria and fungi.13–16 these approaches predict drug targets by comparing the proteins present in an infectious agent to those present or absent in the host. proteins that are unique to the infectious agent and essential for its survival can be potential therapeutic targets. in principle, any essential protein present in the organism can be targeted to control the growth of that organism.17 in the present study, we performed genomics, proteomics, and metabolic pathway analysis of m. hominis with an aim of identifying new drug targets. this study identified several drug targets and available inhibitors for those targets. we expect that present findings will not only extend our understanding of the molecular pathogenesis of m. hominis, but also open a new window to develop novel therapeutic agents against this deadly pathogen. methods identification of pathogen and host metabolic pathways. consideration of safety is the prime issue for the development of any therapeutic agent. if the therapeutic agent is not safe for the host, it will never get regulatory approval. although the overall similarity between eukaryotic and prokaryotic cell is very limited, the similarity in the coding region of a particular gene or functional domain of any protein may result in crossreactivity of a therapeutic agent against the host. in addition, if a drug inhibits any essential protein of the host, it might produce severe side effects to the host species. therefore, the starting point of this study was to compare the metabolic pathways of the pathogen and the host species. information of metabolic pathways was extracted from the kyoto encyclopedia of genes and genomes (kegg) pathway database.18,19 metabolic pathways and respective identification numbers of m. hominis and h. sapiens were retrieved from the ncbi database. subsequently, a manual comparison was made and pathways that appeared only in m. hominis but not in humans were selected as unique, and the remaining pathways of the pathogen were categorized as common pathways. proteins were identified from the unique and common pathways and the corresponding amino acid sequences were downloaded from the ncbi database. screening of essential genes. genes that are indispensable to supporting cellular life are called essential genes. database of essential genes (deg) includes all of the essential genes that are currently available. to identify essential genes of m. hominis, proteins of unique and common pathways were subjected to blastp against deg with an e-value cut-off of 10-10 and with a minimum bit score of 100.20 identification of non-host essential proteins. after the identification of essential proteins, they were compared with the human non-redundant protein sequence database (nr) to identify non-host proteins of m. hominis. a blastp analysis was performed and proteins without hits below the threshold e-value of 0.005 and 35% identity were picked out as nonhost essential proteins.21 molecular weight determination and protein 3-d structure identification. molecular weight (mw ) of each of the potential targets was determined using online tools followed by confirmation with the available literature. previous literature suggested that smaller proteins are suitable targets for drug development because they are more soluble and easier to purify in comparison with large proteins.22 proteins having mw larger than 110  kda were excluded, and the resultant list of proteins was enlisted as sigma (∑) and was used for qualitative analyses. the 3-d structure was scanned by searching the protein data bank (pdb)23,24 and modbase25,26 databases. pdb is the worldwide repository where experimentally determined structures of proteins, nucleic acids, and complex biomolecular assemblies are deposited. these structures are curated and annotated following the standards set by pdb. on the other hand, modbase is a database of protein structures that have been developed by computational approaches and validated by statistically significant sequence alignment and model assessment. qualitative characterization of the ∑ list. different structural and molecular criteria that facilitate prioritizing therapeutic targets in pathogens were assessed for the ∑ list.27 this involved the prediction of subcellular location, functional analysis, protein network analysis, broad spectrum analysis, and druggability analysis of the ∑ list. subcellular localization analysis. subcellular localization analysis of a protein reveals whether that protein is suitable as a drug or a vaccine target. cytoplasmic proteins can work better as drug targets while surface membrane proteins can be used as targets for vaccine development.28 psortb v3.0 server was used to predict the subcellular localization of the proteins,29 and the results were further crosschecked with predictions obtained from cello v.2.5,30,31 topcons,32 and tmhmm.33 psortb uses 11692 proteins of known localization from bacteria and archaea as a training set to sort out the location of a given protein. cello contains 9033 protein sequences as benchmark datasets for the prediction of protein localization using a support vector machine (svm) method. tmhmm is based on hidden markov model, and experimental evidence shows that it correctly predicts 97–98% of the transmembrane helices. functional analysis. the functions of the hypothetical proteins in the ∑ list were predicted using interpro online tool.34 interpro classifies hypothetical proteins into families and predicts domains and important sites by integrating various protein signature recognition methods and databases. broad spectrum analysis. a blastp search against a wide range of pathogenic bacteria with an expected threshold value of 0.005 was used to analyze proteins in the ∑ list for the identification of broad spectrum targets. a total of 240 diseasecausing bacteria from different genus were used in the broad spectrum analysis.35 from the homology analysis against each of the pathogen, it is speculated that close homologs present in http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 identification of therapeutic targets in mycoplasma hominis 53drug target insights 2014:8 maximum number of pathogens are better likely to be promising broad spectrum targets. interactome analysis. a network of protein–protein interaction was constructed for each of the ∑ listed proteins using string 9.1.36,37 string constructs protein–protein interaction networks based on experimental data, gene-based analysis (neighborhood, gene fusion, co-occurrence, and co-expression), curated pathways database, and various protein interactions databases. high confidence interactors with score greater than or equal to 0.700 alone were included in the protein network. to minimize false positives and false negatives, all interactors with low as well as medium confidence scores were removed from the network. druggability analysis. to be druggable, a target must have the potency to interact with drug or drug-like molecules with high affinity. in this study, the druggability potential of each protein of the ∑ list was assessed by drugbank.38 currently, drugbank is a huge and comprehensive collection of drugs with the target information. this database comprises 6816 experimental and fda-approved drugs, 4326 drug targets, and 169 drug enzymes/carriers. in drugbank, each of the ∑ list targets was explored for similar therapeutic targets with the same biological function. degree of homology was evaluated using the blastp program with an expected value of 10-05. presence of targets from the ∑ list in drugbank with the same biological function acts as evidence for their druggable property.39 in contrast, their absence suggests the novelty of the targets, and therefore they are classified as “novel targets.”40 ranking the druggable therapeutic targets based on quantitative characteristics. the effectiveness of a putative target may depend on its degree of essentiality for the survival of the pathogenic organism under diverse environmental conditions. some targets may prove essential for a limited number of physiological conditions, whereas others may prove essential irrespective of environmental conditions. the number of homologs found in deg and the number of interactors of the particular target are the two cardinal factors that determine the degree of essentiality. moreover, the number of available drugs present in drugbank also determines the druggability of a particular therapeutic target. therefore, we ranked the therapeutic targets by calculating the product of the number of homologs found in deg, the number of interactors of the target protein, and the number of drugs available in drugbank. the drug target score was calculated by dividing the product by 100, and the putative therapeutic targets were ranked according to the scores. results in the present study, we identified potential therapeutic targets in m. hominis employing comparative and subtractive genomic analyses of metabolic pathways. we used a systematic hierarchical approach that involved various computational tools utilization, databases search, and drug target prioritization analysis (fig. 1). figure 1. schematic representation of workflow for the identification of therapeutic targets. http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 parvege et al 54 drug target insights 2014:8 table 1. unique and common metabolic pathways of m. hominis with reference to h. sapiens. no unique pathways pathway id total proteins 1 polycyclic aromatic hydrocarbon degradation 00624 1 2 methane metabolism 00680 5 3 biosynthesis of secondary metabolites 01110 22 4 microbial metabolism in diverse environments 01120 20 5 bacterial secretion system 03070 8 no common pathways pathway id total proteins 1 glycolysis/gluconeogenesis 00010 10 2 pentose phosphate pathway 00030 9 3 fructose and mannose metabolism 00051 4 4 oxidative phosphorylation 00190 11 5 purine metabolism 00230 19 6 pyrimidine metabolism 00240 20 7 glycine, serine and threonine metabolism 00260 2 8 cysteine and methionine metabolism 00270 3 9 arginine and proline metabolism 00330 4 10 selenocompound metabolism 00450 3 11 cyanoamino acid metabolism 00460 2 12 glutathione metabolism 00480 2 13 starch and sucrose metabolism 00500 3 14 glycerolipid metabolism 00561 3 15 glycerophospholipid metabolism 00564 6 16 pyruvate metabolism 00620 3 17 propanoate metabolism 00640 2 18 one carbon pool by folate 00670 3 19 thiamine metabolism 00730 2 20 riboflavin metabolism 00740 1 21 nicotinate and nicotinamide metabolism 00760 4 22 aminoacyl-trna biosynthesis 00970 57 23 carbon metabolism 01200 16 24 biosynthesis of amino acids 01230 16 25 abc transporters 02010 16 26 ribosome 03010 58 27 rna degradation 03018 5 28 rna polymerase 03020 3 29 dna replication 03030 12 30 protein export 03060 9 31 base excision repair 03410 5 32 nucleotide excision repair 03420 6 33 mismatch repair 03430 9 34 homologous recombination 03440 14 35 sulfur relay system 04122 2 comparison bet ween m. hominis and h. sapiens metabolic pathways identified five unique pathways and 35 common pathways. primary information about the metabolic pathways of m. hominis and humans was retrieved from the kegg database. currently, the kegg database contains information about 40 metabolic pathways for m. hominis (table 1). comparison with human pathways revealed five pathways containing 36 proteins as unique to m. hominis while the remaining 35 pathways containing 197 proteins as common to m. hominis and humans. thirty-five http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 identification of therapeutic targets in mycoplasma hominis 55drug target insights 2014:8 out of 36 proteins of unique pathways are also present in common pathways. after removing redundant proteins, a total of 198 protein sequences were obtained from the ncbi database. homology search revealed 179 putative essential proteins for m. hominis. proteins that are required by pathogenic microorganisms to survive are called essential proteins. essential proteins are prime targets for drugs and vaccine development. to identify the essential proteins, 198 proteins of common and unique pathways were compared against the deg database. out of 198 input proteins, 179 proteins were found to be essential for the pathogen. the distribution of the 179 identified essential proteins in each of the 22 bacteria of deg is presented in figure 2. comparative analysis of the essential proteins revealed 59 of them as non-host. the aim of the non-homology analysis was to identify pathogen-specific proteins that are nonhomologous to the host. this step is important to avoid undesirable cross-reactivity of the drug arising from its binding to the active sites of the homologous proteins in the host. out of 179 proteins, blast search of essential proteins against nonredundant database of h. sapiens identified only 59 proteins as non-host but essential proteins. exclusion of high mw proteins generated a list of 57 putative therapeutic targets. proteins of low mw are preferable as drug targets because of their solubility and ease of purification. by mw analysis through online tools and literature study, 57 out of 59 proteins having mw below 110 kda were short listed (∑ list) for the qualitative characterization. although no experimentally solved 3-d structure was found for the ∑ listed proteins, computationally annotated 3-d models were available for 16 proteins of the ∑ list in the modbase database. cellular localization analysis mapped the proteins of ∑ list to different cellular locations. target proteins found in the cytoplasm can be used as potential drug targets, whereas extracellular and membrane-bound proteins can work as probable vaccine targets.28 various online tools were used for the prediction of subcellular localization and membrane topology. based on the localization score, psortb predicted the location of 32 targets in the cytoplasm and 13 in the membrane. however, 12 proteins could not be mapped to any cellular location with this software. cello predicted 43 targets in the cytoplasm and 14 in the membrane (table 2). this tool was able to predict the location of 12 targets previously uncharacterized by psortb, and the result of cello was in agreement with topcons prediction as well. psortb prediction varied with cello and topcons in the case of only one protein, mho_3910. 0 20 40 60 80 100 120 140 160 180 a ci ne to ba ct er b ay ly i a d p 1 b ac ill us s ub til is 1 68 b ac te ro id es fr ag ili s 63 8r b ac te ro id es th et ai ot ao m ic ro n v p i54 82 b ur kh ol de ria p se ud om al le i k 96 24 3 b ur kh ol de ria th ai la nd en si s e 26 4 c am py lo ba ct er je ju ni s ub sp . j ej un i… c au lo ba ct er c re sc en tu s e sc he ric hi a co li m g 16 55 i e sc he ric hi a co li m g 16 55 ii f ra nc is el la n ov ic id a u 11 2 h ae m op hi lu s in flu en za e r d k w 20 h el ic ob ac te r py lo ri 26 69 5 m yc ob ac te riu m tu be rc ul os is h 37 r v m yc ob ac te riu m tu be rc ul os is h 37 r v ii m yc ob ac te riu m tu be rc ul os is h 37 r v iii m yc op la sm a ge ni ta liu m g 37 m yc op la sm a pu lm on is u a b c t ip p or ph yr om on as g in gi va lis a t c c 3 32 77 p se ud om on as a er ug in os a p a o 1 p se ud om on as a er ug in os a u c b p p -p a 14 s al m on el la e nt er ic a se ro va r t yp hi s al m on el la e nt er ic a se ro va r t yp hi t y2 s al m on el la e nt er ic a s l1 34 4 s al m on el la e nt er ic a su bs p. e nt er ic a st r. … s al m on el la ty ph im ur iu m l t 2 s he w an el la o ne id en si s m r -1 s ph in go m on as w itt ic hi i r w 1 s ta ph yl oc oc cu s au re us n 31 5 s ta ph yl oc oc cu s au re us n c t c 8 32 5 s tr ep to co cc us p ne um on ia e s tr ep to co cc us s an gu in is v ib rio c ho le ra e n 16 96 1 n um be r of h its figure 2. comparison of m. hominis genome against deg. the height of the bars indicates the number of hits on other genomes. http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 parvege et al 56 drug target insights 2014:8 table 2. qualitative characterization of ∑ list targets. the subcellular localizations are based on the consensus results from prediction by psortb, cello, and topcons. tmh is transmembrane helix predicted by tmhmm. no kegg id gene product definition subcellular localization tmh broad spectrum property interactors 3d model druggability 1 mho_0670 fructose-bisphosphate aldolase cytoplasm 0 no (91) 13 yes druggable 2 mho_0100 putative nucleoside phosphorylase cytoplasm 0 no (71) 4 yes druggable 3 mho_0980 thymidylate kinase cytoplasm 0 yes (102) 10 no druggable 4 mho_0990 dna polymerase iii subunit cytoplasm 0 no (84) 10 no novel 5 mho_1260 putative nicotinate-nucleotide adenylyl transferase cytoplasm 0 no (98) 13 yes druggable 6 mho_1670 cytidylate kinase cytoplasm 0 yes (102) 8 yes druggable 7 mho_2380 riboflavin biosynthesis protein membrane 5 no (44) 12 no novel 8 mho_2730 dna-directed rna polymerase subunit alpha cytoplasm 0 no (91) 2 yes druggable 9 mho_0200 atp synthase subunit a cytoplasm 0 no (89) 50 no novel 10 mho_0210 atp synthase subunit c membrane 2 no (42) 13 no druggable 11 mho_3350 purine-nucleoside phosphorylase cytoplasm 0 no (74) 9 yes druggable 12 mho_0220 atp synthase subunit b membrane 1 no (34) 11 yes novel 13 mho_3650 aspartate—ammonia ligase cytoplasm 0 no (30) 2 no novel 14 mho_3810 putative 2,3-bisphosphoglycerate-independent phosphoglycerate mutase cytoplasm 0 no (59) 9 yes druggable 15 mho_3840 acetate kinase cytoplasm 0 no (96) 7 yes druggable 16 mho_3880 ribose-5-phosphate isomerase cytoplasm 0 no (76) 9 yes druggable 17 mho_4030 thiamine biosynthesis protein cytoplasm 0 no (80) 2 no novel 18 mho_4130 dna polymerase i cytoplasm 0 yes (127) 13 no druggable 19 mho_4370 putative glycerol-3-phosphate acyltransferase membrane 6 no (76) 3 no novel 20 mho_4600 uridylate kinase cytoplasm 0 yes (111) 6 yes novel 21 mho_4680 fatty acid/phospholipid synthesis protein cytoplasm 0 no (82) 9 yes novel 22 mho_2040 putative dna polymerase iii, delta subunit cytoplasm 0 no (41) 6 no novel 23 mho_0850 uvrabc system protein c cytoplasm 0 yes (102) 7 no novel 24 mho_0030 50s ribosomal protein l34 cytoplasm 0 no (64) 50 no novel 25 mho_0880 50s ribosomal protein l1 cytoplasm 0 no (97) 50 no novel 26 mho_1010 30s ribosomal protein s20 cytoplasm 0 no (72) 0 no novel 27 mho_1120 30s ribosomal protein s6 cytoplasm 0 no (59) 32 no novel 28 mho_1280 50s ribosomal protein l35 cytoplasm 0 no (72) 31 no novel 29 mho_1180 phenylalanyl-trna synthetase subunit beta cytoplasm 0 yes (116) 32 no druggable 30 mho_0050 dna polymerase iii subunit beta cytoplasm 0 yes (101) 24 yes druggable 31 mho_1520 oligopeptide transport system permease protein membrane 6 yes (119) 5 no novel 32 mho_1990 cobalt abc transporter permease protein membrane 5 no (57) 3 no novel 33 mho_0970 recombination protein recr cytoplasm 0 yes (104) 10 no novel 34 mho_1840 holliday junction dna helicase ruva cytoplasm 0 no (93) 7 no novel 35 mho_1130 single-strand binding protein cytoplasm 0 no (87) 4 no novel http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 identification of therapeutic targets in mycoplasma hominis 57drug target insights 2014:8 table 2. (continued) no kegg id gene product definition subcellular localization tmh broad spectrum property interactors 3d model druggability 36 mho_2160 dna primase cytoplasm 0 yes (129) 8 no novel 37 mho_3910 replicative dna helicase cytoplasm 0 yes (115) 14 yes novel 38 mho_3690 putative ribonuclease j cytoplasm 0 no (75) 1 no novel 39 mho_4170 phosphopentomutase cytoplasm 0 no (49) 6 yes novel 40 mho_0010 putative membrane insertase oxa1/alb3/yidc membrane 6 no (96) 4 no novel 41 mho_1860 protein-export membrane protein membrane 12 no (07) 6 no novel 42 mho_2790 preprotein translocase secy subunit membrane 10 yes (104) 49 no novel 43 mho_4440 not predicted membrane 1 no (06) 6 no novel 44 mho_4460 spermidine/putrescine abc transporter permease membrane 6 no (97) 6 no novel 45 mho_4470 spermidine/putrescine abc transporter permease membrane 6 no (68) 7 no novel 46 mho_4480 not predicted membrane 1 no (0) 4 no novel 47 mho_2610 50s ribosomal protein l19 cytoplasm 0 no (77) 50 no novel 48 mho_2660 50s ribosomal protein l21 cytoplasm 0 no (72) 50 no novel 49 mho_2700 50s ribosomal protein l10 cytoplasm 0 no (70) 43 no druggable 50 mho_2710 50s ribosomal protein l32 cytoplasm 0 no (44) 34 no druggable 51 mho_2820 50s ribosomal protein l18 membrane 0 no (85) 50 no novel 52 mho_2830 50s ribosomal protein l6 cytoplasm 0 no (90) 50 no novel 53 mho_2870 50s ribosomal protein l24 cytoplasm 0 no (76) 50 no novel 54 mho_2900 50s ribosomal protein l29 cytoplasm 0 no (79) 50 no novel 55 mho_3020 50s ribosomal protein l31 cytoplasm 0 no (78) 50 no novel 56 mho_3920 50s ribosomal protein l9 cytoplasm 0 no (71) 50 no novel 57 mho_0630 carbamate kinase cytoplasm 0 no (48) 3 yes novel tmhmm predicted the number of transmembrane helixes (tmh) in membrane proteins. biological functions were assigned to seven hypothetical proteins. nine proteins of the ∑ list with kegg id mho_0100, mho_1260, mho_3810, mho_4370, mho_2040, mho_3690, mho_0010, mho_4440, and mho_4480 were hypothetical. seven of them could be annotated using the interpro online server (table 2). mho_0100 was predicted to have a nucleoside phosphorylase domain with a catalytic role in the nucleoside metabolic process. mho_1260 is a probable member of the nicotinate-nucleotide adenylyltransferase protein family having a role in the nad biosynthetic pathway. mho_3810 probably plays a role as phosphoglycerate mutase 2,3-bisphosphoglycerate-independent enzyme in glucose metabolism. mho_4370 and mho_2040 were recognized as glycerol-3-phosphate acyltransferase (plsy ) and dna polymerase iii (delta subunit) type proteins, respectively. mho_3690 protein was predicted to be beta-lactamase-like protein having rnaand metal ion-binding potential. the mho_0010 was recognized as membrane insertase yidc/oxa1 (c-terminal) having a pivotal role in protein insertion into the membrane. interpro could not predict any function for mho_4440 and mho_4480 as no homolog was found in the database. comparison of proteomes of 240 pathogens identified 12 broad spectrum targets. blastp homology search for proteins in the ∑ list against the whole proteome of each of the 240 bacterial pathogens identified ideal broad spectrum targets. this comparative sequence analysis revealed 12 proteins having homologs in more than 100 pathogens, whereas homologs were found in more than 50 pathogens for 33 proteins present in the ∑ list. ∑ listed proteins exhibit maximum homology to the proteome of mycoplasma pathogens like m. capricolum, m. gallisepticum, m. genitallium, m. penetrans, and m. pneumoniae. proteins having homologs in more than 100 pathogens were considered as broad spectrum target candidates (table 2). interactome analysis identified 23 targets having maximum interactors. protein–protein interactions are important determinants of protein function. to evaluate the functional importance of ∑ listed targets in the metabolic network, analysis on protein interaction network was performed using the string database. target protein interacting with http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 parvege et al 58 drug target insights 2014:8 table 3. non-host essential proteins similar to fda-approved/experimental drug targets and the list of fda-approved drugs for the targets. no kegg id associated pathways drugbank id drug name drug group 1 mho_0670 glycolysis/gluconeogenesis, pentose phosphate pathway, fructose and mannose metabolism db03026 phosphoglycolohydroxamic acid experimental 2 mho_0100 cysteine and methionine metabolism, biosynthesis of amino acids db07463, db00173 adenine approved 3 mho_0980 pyrimidine metabolism db03280 p1-(5′-adenosyl)p5-(5′-thymidyl) pentaphosphate experimental 4 mho_1260 nicotinate and nicotinamide metabolism db04099, db04272 deamido-nad+, citric acid experimental, nutraceutical 5 mho_1670 pyrimidine metabolism db02456, db02883, db03403 db04555 cytosine arabinose-5′-phosphate, 2′,3′-dideoxycytidine-5′-monophosphate, cytidine-5′-monophosphate, cytidine-5′-diphosphate experimental 6 mho_2730 purine metabolism, pyrimidine metabolism, rna polymerase db00615, db08226, db08266 rifabutin, myxopyronin b, methyl carbamate approved, experimental 7 mho_0210 oxidative phosphorylation db04464, db03143 n-formylmethionine, nonan-1-ol experimental 8 mho_3350 purine metabolism, pyrimidine metabolism, nicotinate and nicotinamide metabolism, biosynthesis of secondary metabolites db02857, db04627 guanosine, cyclouridine experimental 9 mho_3810 glycolysis/gluconeogenesis, glycine, serine and threonine metabolism, methane metabolism db01709, db04510 2-phosphoglyceric acid, 3-phosphoglyceric acid experimental 10 mho_3840 pyruvate metabolism, propanoate metabolism, methane metabolism db01942 formic acid experimental 11 mho_3880 pentose phosphate pathway, fructose and mannose metabolism, biosynthesis of secondary metabolites db03661, db03108 cysteinesulfonic acid, 4-phospho-d-erythronate experimental 12 mho_4130 purine metabolism, pyrimidine metabolism, nucleotide excision repair, homologous recombination db00548, db03152 azelaic acid, b-2-octylglucoside approved, experimental 13 mho_1180 aminoacyl-trna biosynthesis db07817 1-{3-[(4-pyridin-2-ylpiperazin-1-14yl) sulfonyl]phenyl}-3-(1,3-thiazol-2-yl)urea experimental 14 mho_0050 purine metabolism, pyrimidine metabolism, dna replication, mismatch repair, homologous recombination db06998 [(5r)-5-(2,3-dibromo-5-ethoxy-4hydroxybenzyl)-4-oxo-2-thioxo-1,3thiazolidin-3-yl]acetic acid experimental 15 mho_2700 ribosome db00778, db01190, db01211, db01369, db01627 roxithromycin, clindamycin, clarithromycin, quinupristin, lincomycin approved 16 mho_2710 ribosome db01361 troleandomycin approved more proteins is considered as metabolically important active protein, which can act as an appropriate drug target.41,42 out of 57 input proteins, 12 were found to have less than five interactors and 23 to have more than 10 interactors (table 2). drugbank database search identified 16 druggable targets. the probability of being druggable of the potential targets can be evaluated by sequence similarity search against the targets from drugbank.38,39 blastp search against drugbank targets with fda-approved drugs, nutraceuticals, and experimental drugs revealed that 16 targets in the ∑ list are homologous to drugbank targets (table 3). five targets were found to have homologies to fda-approved drug targets. among them, mho_2700  is homologous to a known target (50s ribosomal protein l10) and has five approved drugs against it, named roxithromycin (db00778), clindamycin (db01190), clarithromycin (db01190), quinupristin (db01211), and lincomycin (db01369), which are used to treat shigella infection. other four fda-approved drug targets have one approved drug against each. proteins in the ∑ list that did not hit with drugbank database are novel therapeutic targets for which new drug and vaccines can be developed. ranking of the drug targets suggests that 50s ribosomal protein l10 has the highest potential to be an effective drug target. it is useful to rank the putative therapeutic targets based http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 identification of therapeutic targets in mycoplasma hominis 59drug target insights 2014:8 table 4. rankings of the druggable targets based on drug target score. kegg id gene product definition homologs in deg interactors drugbank hits drug target score rank mho_2700 50s ribosomal protein l10 7 43 5 15.05 1 mho_1180 phenylalanyl-trna synthetase subunit beta 26 32 1 8.32 2 mho_1670 cytidylate kinase 24 8 4 7.68 3 mho_2710 50s ribosomal protein l32 15 34 1 5.1 4 mho_1260 putative nicotinate-nucleotide adenylyl transferase 19 13 2 4.94 5 mho_0050 dna polymerase iii subunit beta 19 24 1 4.56 6 mho_4130 dna polymerase i 14 13 2 3.64 7 mho_0980 thymidylate kinase 21 10 1 2.1 8 mho_2730 dna-directed rna polymerase subunit alpha 27 2 3 1.62 9 mho_0670 fructose-bisphosphate aldolase 12 13 1 1.56 10 mho_3810 putative 2, 3-bisphosphoglycerate-independent phosphoglycerate mutase 8 9 2 1.44 11 mho_3880 ribose-5-phosphate isomerase 5 9 2 0.9 12 mho_0210 atp synthase subunit c 2 13 2 0.52 13 mho_3840 acetate kinase 6 7 1 0.42 14 mho_3350 purine-nucleoside phosphorylase 2 9 2 0.36 15 mho_0100 putative nucleoside phosphorylase 2 4 2 0.16 16 note: drug target score = homologs in deg × interactors × drugbank hits/100. on their quantitative values in order to decide which target has a higher probability of being effective in laboratory experiments. we ranked the putative therapeutic targets based on the number of homologs found in deg, the number of interactors of the target protein, and the number of drugs available. the ranking of the putative drug targets is presented in table 4 and showed that 50s ribosomal protein l10 has the highest potential to be an effective therapeutic target. cytidylate kinase, 50s ribosomal protein l32, and putative nicotinate-nucleotide adenylyl transferase also fall in the group of top five putative therapeutic targets. discussion the increasing trend of bacterial resistance to antibiotics is posing an imminent public health concern. researchers around the world are giving attention to find new drug targets so that bacterial infections can be prevented. a vast array of omics data and the availability of various computational tools have speeded up the therapeutic target identification process. the potential of a protein to be a therapeutic target depends on two factors, essentiality and absence in the host. essential proteins are required for bacterial survival and blocking them inhibits bacterial growth. non-homologous proteins are preferred because they reduce the probability of side effects. in this study, we extracted metabolic data from kegg database and used different bioinformatics and computational databases and tools for the identification of appropriate therapeutic targets of m. hominis. systematic computational analyses revealed 57 possible drug targets in m. hominis; among them 16 are druggable targets, which have homologs in drugbank. we further utilized different drug prioritization parameters to make a short list of potential drug and vaccine targets. predicted drug targets belong to a diverse range of cellular activity and fall mostly into ribosome synthesis, pyrimidine metabolism, homologous recombination, dna replication, and abc transporter pathways (fig. 3). the assembly of bacterial ribosomes has been considered prominently as a potential target for antibacterial drugs.43 we identified 16 potential drug targets from the ribosome synthesis and assembly pathway. among them, two are druggable targets that include 50s ribosomal protein l10 and l32. 50s ribosomal protein l10 interacting with acidic l7/l12 proteins constitutes the ribosomal stalk. this stalk is involved in the binding of elongation factors ef-tu and ef-g and plays a crucial role in activating the gtpase center. this target is highly similar to an already available drug target with five fda-approved drugs used to control shigella infection. our http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 parvege et al 60 drug target insights 2014:8 0 5 10 15 20 25 30 others (24 pathways) pentose phosphate pathway protein export microbial metabolism in diverse… biosynthesis of amino acids biosynthesis of secondary metabolites abc transporters homologous recombination ribosome number of proteins figure 3. distribution of putative therapeutic targets in their associated pathways. the percentage distribution of other pathways ranges from 0 to 4.5. study revealed that 50s ribosomal protein l10 holds the highest promise to be an effective drug target (table 4). hence, the potential of it as a drug target remains open for experimental validation. 50s ribosomal protein l32 is a structural constituent of ribosomes. this target was found to have a homologous target in drugbank for an fda-approved drug called troleandomycin. however, m. hominis is intrinsically resistant to this antibiotic because of mutations in 23s rrna, which has stimulated the search for other drug targets.10 de novo nucleotide synthesis is crucial for the successful growth of bacteria in human blood.44 nucleotides synthesized in purine and pyrimidine metabolic pathways are important substrates not only for dna synthesis but also for dna repair. we have found five and eight drug targets for purine and pyrimidine metabolism, respectively. dna polymerase i (pol i) and dna-directed rna polymerase subunit alpha (rpoa), which is involved in nucleotide metabolism as well as other important metabolic pathways, have homologs in drugbank. azelaic acid, an approved fda drug, can be used to treat m. hominis infection by inhibiting the activity of pol i. this drug is currently being used as an inhibitor of pol i in escherichia coli. rpoa activity can be blocked by an approved drug, rifabutin, which is highly specific to bacterial rna polymerase. in the last few years, aminoacyl-trna synthetases (aars) have drawn much attention as therapeutic targets because of their crucial roles in protein synthesis and conservation across different pathogens.45 here, we highlighted phenylalanyl-trna synthetase subunit beta (phet) as a therapeutic target, which showed considerable homology with a target in drugbank database. phenyl-thiazolylurea-sulfonamide, a successful inhibitor of phenylalanyl-trna synthetase (phe-rs),  can be tested in the laboratory to determine its efficacy against m. hominis.46 glycolysis/gluconeogenesis is perceived as a promising target for new drugs against bacterial pathogens because many of the proteins involved in this pathway are significantly different from human proteins. here, we identified two proteins of the glycolytic pathway as potential therapeutic targets—one is fructose-bisphosphate aldolase (fba) and the other is a hypothetical protein, mho_3810, which is found to have 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (ipgm) activity. fba catalyses an important reversible reaction required for both glycolysis and gluconeogenesis.47 previously, this enzyme has been reported as a target of antifungal and antiprotozoal drugs. downregulation of  ipgm using rnai resulted in embryonic and larval lethality in caenorhabditis elegans.48 in addition to druggable targets, we have also identified several other targets that are involved in crucial bacterial metabolic pathways. bacterial protein secretion system pathway modulates biotic association as well as pathogenicity. therefore, proteins from the bacterial secretion system were identified as drug targets in many bacteria.49,50 this secretion system of m. hominis includes eight proteins. among them three have been proposed as potential therapeutic targets in our study. the proposed three targets are secd, secy, and yidc/oxa1 family membrane proteins, and this result is consistent with two previous studies.51,52 extensive research is going on in the development of protein secretion inhibitors like salicylidene acylhydrazides53 and 2-imino-5-arylidene thiazolidinone.54 earlier studies reported that abc transporters play an important role in bacterial physiological processes such as the import of important nutrients required for bacterial growth55 and export of toxic substances outside of the cell.56 here, we have reported six abc transporters as novel therapeutic targets. these transporters can be used for the development of antibacterial vaccines.57 conclusion this study has identified several proteins that can be targeted for effective drug development. since some of the identified http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 identification of therapeutic targets in mycoplasma hominis 61drug target insights 2014:8 drug targets play important roles in metabolism, a synchronized approach to develop new drugs would be promising to control m. hominis infection. in addition, results of this study can bring a substantial advancement to test the effectiveness of the currently available drugs. further wet lab experiments are essential to validate the obtained findings. author contributions conceived and designed the experiments: msh and mmp. analyzed the data: mmp and mr. wrote the first draft of the manuscript: mmp. contributed to the writing of the manuscript: mr. agree with manuscript results and conclusions: mmp, mr, and msh. jointly developed the structure and arguments for the paper: mmp and mr. made critical revisions and approved final version: msh. all authors reviewed and approved of the final manuscript. r efer ences 1. razin s, yogev d, naot y. molecular biology and pathogenicity of mycoplasmas. microbiol mol biol rev. 1998;62(4):1094–1156. 2. waites k, talkington d. new developments in human diseases due to mycoplasmas. in: blanchard a, browning g, eds. mycoplasmas molecular biology pathogenicity and strategies for control. norfolk: horizon bioscience; 2005:289–354. 3. xiao jc, xie lf, fang sl, et al. symbiosis of mycoplasma hominis in trichomonas vaginalis may link metronidazole resistance in vitro. parasitol res. 2006;100(1): 123–130. 4. mammen-tobin a, wilson jd. management of metronidazole-resistant trichomonas vaginalis—a new approach. int j std aids. 2005;16(7):488–490. 5. kataoka s, yamada t, chou k, et al. association between preterm birth and vaginal colonization by mycoplasmas in early pregnancy. j clin microbiol. 2006;44(1): 51–55. 6. stellrecht ka, woron am, mishrik ng, venezia ra. comparison of multiplex pcr assay with culture for detection of genital mycoplasmas. j clin microbiol. 2004; 42(4):1528–1533. 7. ladefoged sa. molecular dissection of mycoplasma hominis. apmis suppl. 2000; 97:1–45. 8. mcnaughton rd, robertson ja, ratzlaff vj, molberg cr. mycoplasma hominis infection of the central nervous system in a neonate. can med assoc. 1983;129(4):353–354. 9. barykova ya, logunov dy, shmarov mm, et al. association of mycoplasma hominis infection with prostate cancer. oncotarget. 2011;2(4):289–297. 10. pereyre s, gonzalez p, de barbeyrac b, et al. mutations in 23s rrna account for intrinsic resistance to macrolides in mycoplasma hominis and mycoplasma fermentans and for acquired resistance to macrolides in m. hominis. antimicrob agents chemother. 2002;46(10):3142–3150. 11. mihai m, valentin n, bogdan d, carmen cm, coralia b, demetra s. antibiotic susceptibility profiles of mycoplasma hominis and ureaplasma urealyticum isolated during a population-based study concerning women infertility in northeast romania. braz j microbiol. 2011;42(1):256–260. 12. pereyre s, sirand-pugnet p, beven l, et al. life on arginine for mycoplasma hominis: clues from its minimal genome and comparison with other human urogenital mycoplasmas. plos genet. 2009;5(10):e1000677. 13. sakharkar kr, sakharkar mk, chow vt. a novel genomics approach for the identification of drug targets in pathogens, with special reference to pseudomonas aeruginosa. in silico biol. 2004;4(3):355–360. 14. perumal d, lim cs, sakharkar kr, sakharkar mk. differential genome analyses of metabolic enzymes in pseudomonas aeruginosa for drug target identification. in silico biol. 2007;7(4–5):453–465. 15. amineni u, pradhan d, marisetty h. in silico identification of common putative drug targets in leptospira interrogans. j chem biol. 2010;3(4):165–173. 16. abadio ak, kioshima es, teixeira mm, martins nf, maigret b, felipe ms. comparative genomics allowed the identification of drug targets against human fungal pathogens. bmc genomics. 2011;12:75. 17. galperin my, koonin ev. searching for drug targets in microbial genomes. curr opin biotechnol. 1999;10(6):571–578. 18. kanehisa m, goto s, hattori m, et al. from genomics to chemical genomics: new developments in kegg. nucleic acids res. 2006;34(database issue):d354–d357. 19. kanehisa m, goto s, furumichi m, tanabe m, hirakawa m. kegg for representation and analysis of molecular networks involving diseases and drugs. nucleic acids res. 2010;38(database issue):d355–d360. 20. zhang r, ou hy, zhang ct. deg: a database of essential genes. nucleic acids res. 2004;32(database issue):d271–d272. 21. altschul sf, madden tl, schäffer aa, et al. gapped blast and psi-blast: a new generation of protein database search programs. nucleic acids res. 1997;25(17): 3389–3402. 22. duffield m, cooper i, mcalister e, bayliss m, ford d, oyston p. predicting conserved essential genes in bacteria: in silico identification of putative drug targets. mol biosyst. 2010;6(12):2482–2489. 23. berman h, henrick k, nakamura h, markley jl. the worldwide protein data bank (wwpdb): ensuring a single, uniform archive of pdb data. nucleic acids res. 2007;35(database issue):d301–d303. 24. kouranov a, xie l, de lacruz j, et al. the rcsb pdb information portal for structural genomics. nucleic acids res. 2006;34(database issue):d302–d305. 25. pieper u, webb bm, barkan dt, et al. modbase, a database of annotated comparative protein structure models, and associated resources. nucleic acids res. 2011;39(database issue):d465–d474. 26. pieper u, webb bm, dong gq , et al. modbase, a database of annotated comparative protein structure models and associated resources. nucleic acids res. 2014;42(database issue):d336–d346. 27. agüero f, al-lazikani b, aslett m, et al. genomic-scale prioritization of drug targets: the tdr targets database. nat rev. 2008;7(11):900–907. 28. barh d, tiwari s, jain n, et al. in silico subtractive genomics for target identification in human bacterial pathogens. drug dev res. 2011;72(2):162–177. 29. yu ny, wagner jr, laird mr, et al. psortb 3.0: improved protein subcellular localization prediction with refined localization subcategories and predictive capabilities for all prokaryotes. bioinformatics (oxford, england). 2010;26(13): 1608–1615. 30. yu c-s, lin c-j, hwang j-k. predicting subcellular localization of proteins for gram-negative bacteria by support vector machines based on n-peptide compositions. protein sci. 2004;13(5):1402–1406. 31. yu c-s, chen y-c, lu c-h, hwang j-k. prediction of protein subcellular localization. proteins. 2006;64(3):643–651. 32. bernsel a, viklund h, hennerdal a, elofsson a. topcons: consensus prediction of membrane protein topology. nucleic acids res. 2009;37(web server issue):w465–w468. 33. krogh a, larsson b, von heijne g, sonnhammer el. predicting transmembrane protein topology with a hidden markov model: application to complete genomes. j mol biol. 2001;305(3):567–580. 34. hunter s, jones p, mitchell a, et al. interpro in 2011: new developments in the family and domain prediction database. nucleic acids res. 2012;40(database issue): d306–d312. 35. raman k, yeturu k, chandra n. targettb: a target identification pipeline for mycobacterium tuberculosis through an interactome, reactome and genome-scale structural analysis. bmc syst biol. 2008;2:109. 36. franceschini a, szklarczyk d, frankild s, et al. string v9.1: protein-protein interaction networks, with increased coverage and integration. nucleic acids res. 2013;41(database issue):d808–d815. 37. von mering c, huynen m, jaeggi d, schmidt s, bork p, snel b. string: a database of predicted functional associations between proteins. nucleic acids res. 2003;31(1):258–261. 38. knox c, law v, jewison t, et al. drugbank 3.0: a comprehensive resource for ‘omics’ research on drugs. nucleic acids res. 2011;39(database issue):d1035–d1041. 39. hasan s, daugelat s, rao ps, schreiber m. prioritizing genomic drug targets in pathogens: application to mycobacterium tuberculosis. plos comput biol. 2006; 2(6):e61. 40. crowther gj, shanmugam d, carmona sj, et al. identification of attractive drug targets in neglected-disease pathogens using an in silico approach. plos negl trop dis. 2010;4(8):e804. 41. kushwaha sk, shakya m. protein interaction network analysis—approach for potential drug target identification in mycobacterium tuberculosis. j theor biol. 2010; 262(2):284–294. 42. cui t, zhang l, wang x, he zg. uncovering new signaling proteins and potential drug targets through the interactome analysis of mycobacterium tuberculosis. bmc genomics. 2009;10:118. 43. maguire ba. inhibition of bacterial ribosome assembly: a suitable drug target? microbiol mol biol rev. 2009;73(1):22–35. 44. samant s, lee h, ghassemi m, et al. nucleotide biosynthesis is critical for growth of bacteria in human blood. plos pathog. 2008;4(2):e37. 45. hurdle jg, o’neill aj, chopra i. prospects for aminoacyl-trna synthetase inhibitors as new antimicrobial agents. antimicrob agents chemother. 2005;49(12):4821–4833. 46. beyer d, kroll hp, endermann r, et al. new class of bacterial phenylalanyltrna synthetase inhibitors with high potency and broad-spectrum activity. antimicrob agents chemother. 2004;48(2):525–532. 47. rodaki a, young t, brown aj. effects of depleting the essential central metabolic enzyme fructose-1,6-bisphosphate aldolase on the growth and viability of candida albicans: implications for antifungal drug target discovery. eukaryot cell. 2006;5(8): 1371–1377. http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 parvege et al 62 drug target insights 2014:8 48. zhang y, foster jm, kumar s, fougere m, carlow ck. cofactor-independent phosphoglycerate mutase has an essential role in caenorhabditis elegans and is conserved in parasitic nematodes. j biol chem. 2004;279(35):37185–37190. 49. chhabra g, sharma p, anant a, et al. identification and modeling of a drug target for clostridium perfringens sm101. bioinformation. 2010;4(7):278–289. 50. gotoh y, eguchi y, watanabe t, okamoto s, doi a, utsumi r. two-component signal transduction as potential drug targets in pathogenic bacteria. curr opin microbiol. 2010;13(2):232–239. 51. barh d, kumar a. in silico identification of candidate drug and vaccine targets from various pathways in neisseria gonorrhoeae. in silico biol. 2009;9(4):225–231. 52. chong ce, lim bs, nathan s, mohamed r. in silico analysis of burkholderia pseudomallei genome sequence for potential drug targets. in silico biol. 2006;6(4): 341–346. 53. duncan mc, linington rg, auerbuch v. chemical inhibitors of the type three secretion system: disarming bacterial pathogens. antimicrob agents chemother. 2012; 56(11):5433–5441. 54. felise hb, nguyen hv, pfuetzner ra, et al. an inhibitor of gram-negative bacterial virulence protein secretion. cell host microbe. 2008;4(4):325–336. 55. koster w. abc transporter-mediated uptake of iron, siderophores, heme and vitamin b12. res microbiol. 2001;152(3–4):291–301. 56. van veen hw, higgins cf, konings wn. multidrug transport by atp binding cassette transporters: a proposed two-cylinder engine mechanism. res microbiol. 2001;152(3–4):365–374. 57. garmory hs, titball rw. atp-binding cassette transporters are targets for the development of antibacterial vaccines and therapies. infect immun. 2004;72(12): 6757–6763. http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 1drug target insights 2015:9 7-ni and odq disturbs memory in the elevated plus maze, morris water maze, and radial arm maze tests in mice oguz mutlu1, furuzan akar1, ipek komsuoglu celikyurt1, pelin tanyeri2, guner ulak1 and faruk erden1 1department of pharmacology, kocaeli university medical faculty, kocaeli, turkey. 2department of pharmacology, faculty of medicine, sakarya university, sakarya, turkey. a bstr ac t: nitric oxide (no) is an atypical neurotransmitter that causes changes in cognition. nitric oxide synthase (nos) and guanylate cyclase (gc) inhibitors have been shown to exert some effects on cognition in previous studies; however, the findings have been controversial. this study was aimed at understanding the effects of an nos inhibitor, 7-nitroindazole (7-ni), and a guanylate cyclase inhibitor, 1h-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (odq ), on spatial memory in modified elevated plus maze (mepm), morris water maze (mwm), and radial arm maze (r am) tests. male balb-c mice were treated via intraperitoneal injections with 7-ni (15 mg/kg), odq (3, 10 mg/kg), l-arginine (100 mg/kg) + 7-ni (15 mg/kg), or physiological saline. odq (3 mg/kg) and 7-ni (15 mg/kg) significantly increased the second-day latency in the mepm test. 7-ni (15 mg/kg) and odq (10  mg/kg) significantly increased the escape latency in second, third, and fourth sessions, decreased the time spent in the escape platform’s quadrant, and increased the mean distance to the platform in the probe trial of the mwm test. odq (3, 10 mg/kg) and 7-ni (15 mg/kg) significantly increased the number of errors, whereas only 7-ni increased the latency in the r am test. the administration of l-arginine (100  mg/kg) prior to 7-ni inverted the effects of 7-ni, which supports the role of no on cognition. our study shows that the no/cgmp/gs pathway can regulate spatial memory in mice. k e y wor ds: 7-ni, odq , memory, mice citation: mutlu et al. 7-ni and odq disturbs memory in the elevated plus maze, morris water maze, and radial arm maze tests in mice. drug target insights 2015:9 1–8 doi:10.4137/dti.s23378. received: january 9, 2015. resubmitted: february 2, 2015. accepted for publication: february 10, 2015. academic editor: anuj chauhan, editor in chief type: original research funding: authors disclose no funding sources. competing interests: authors disclose no potential conflicts of interest. copyright: © the authors, publisher and licensee libertas academica limited. this is an open-access article distributed under the terms of the creative commons cc-by-nc 3.0 license. correspondence: oguzmutlu80@hotmail.com paper subject to independent expert blind peer review by minimum of two reviewers. all editorial decisions made by independent academic editor. upon submission manuscript was subject to anti-plagiarism scanning. prior to publication all authors have given signed confirmation of agreement to article publication and compliance with all applicable ethical and legal requirements, including the accuracy of author and contributor information, disclosure of competing interests and funding sources, compliance with ethical requirements relating to human and animal study participants, and compliance with any copyright requirements of third parties. this journal is a member of the committee on publication ethics (cope). published by libertas academica. learn more about this journal. introduction nitric oxide (no) is synthesized from l-arginine by no synthase (nos) as a result of ca2+ influx, which is activated by induction of n-methyl-d-aspartate (nmda) receptors by excitatory amino acids. no acts as an atypical neuromediator inside brain cells because it reacts with heme moieties of guanyl cyclase in the synaptic junction and induces cyclic guanosine monophosphate (cgmp)-mediated presynaptic glutamate release. no is produced both presynaptically and postsynaptically in the brain as a result of the increase in cytosolic ca2+ concentration; it subsequently diffuses outside and affects the neighboring neuronal structures and glial cells.1 no has several effects on behavior, cognition, and emotion, and has been shown to play role in depression, anxiety, locomotion, aggression, tolerance, addiction, and learning.2–4 no has effects on the modulation of cognition;5 however, role of no in learning is not completely understood. multiple mechanisms modulate synaptic efficacy and its actions, including the regulation of synaptic plasticity and the modulation of cgmp.6 there is evidence of soluble guanylyl cyclase (sgc) activation in memory formation.7–9 the activation of sgc may represent a major pathway that regulates no messenger function in the brain10,11 because it has been reported that the induction of long-term potentiation (ltp) in hippocampal slices can be blocked with sgc inhibitors.12,13 in different rodent models, many studies have investigated drugs that affect no levels to examine the role on cognition; however, the results have been controversial. in some studies, it was shown that compounds that block nos inhibited learning,14,15 whereas some others did not support these findings.16,17 in the morris water maze (mwm) test, systemic inhibition of no had disturbing effects in some studies,18,19 whereas others showed different results.17,20 besides, ltp playing a role in no-mediated cognition was completely revered after the administration of nos inhibitors in some studies,21,22 whereas others studies have demonstrated a partial inhibition13,23 or no effect at all.24,25 several studies regarding nos inhibitors demonstrated that 7-nitroindazole (7-ni) exerted some impairing effects on cognition in rodents. injections of 7-ni disturbed spatial memory and object recognition in rats and also had impairing effects in passive avoidance test in animal models.26–28 journal name: drug target insights journal type: original research year: 2015 volume: 9 running head verso: mutlu et al running head recto: 7-ni and odq disturbs memory in the elevated plus maze http://www.la-press.com/drug-target-insights-journal-j23 http://www.la-press.com http://dx.doi.org/10.4137/dti.s23378 http://creativecommons.org/licenses/by-nc/3.0/ http://creativecommons.org/licenses/by-nc/3.0/ mailto:oguzmutlu80@hotmail.com http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 mutlu et al 2 drug target insights 2015:9 the goal of this study was to further evaluate the effects of 7-ni (a nonselective inhibitor of nos), l-arginine (an no precursor combined with 7-ni), and 1h-[1,2,4]oxadiazole[4,3a]-quinoxaline-1-one (odq , a highly selective, irreversible inhibitor of sgc) on spatial memory in the modified elevated plus maze (mepm), mwm, and radial arm maze (ram) tests. furthermore, these studies were aimed at further understanding the effects of no on cognition because of the controversy in the literature. methods animals. ninety-six male inbred balb/c byj mice (uludağ university, bursa, turkey) aged 8 weeks were used in this study. the animals (4–5 per cage) were kept in the laboratory for 2  weeks prior to experimentation. the animal room had a temperature of 21  ±  1.5°c with 60% relative humidity, and a 12-hour light/dark cycle (light on at 8.00 p.m.). all procedures that involved animals were in compliance with the european community council directive of november 24, 1986, and ethical approval was granted by the kocaeli university ethics committee (number: aek 9/4-2010, kocaeli, turkey). modified elevated plus-maze test. cognitive behavior was determined using the mepm, which measures spatial long-term memory.29 the maze was composed of wood; it comprised two open arms (29 × 5 cm) surrounded by a short (1  cm) plexiglass edge to avoid falls and two enclosed arms (29 × 5 × 15 cm) arranged such that the two open arms were opposite to each other. the arms were connected by a central platform (5 × 5 cm). the maze was kept 40 cm above the floor. the principle of this experiment is based on aversion of rodents to open spaces and heights. the animals prefer the enclosed, protected areas of the maze.29 the procedure was as previously described.29–32 in the acquisition session (day 1), each mouse was placed at the distal end of an open arm facing away from central platform. the time required for mice to move from the open arm to either of the enclosed arms (transfer latency) was recorded. training (repeated exposure of the animals to the open arms) shortened this parameter, most likely as a result of learning acquisition and retention. after entering the enclosed arm, the mice were allowed to move freely in the maze regardless of open and enclosed arms for 10 seconds. the retention session followed 24 hours after the acquisition session. the mice were placed in the open arm, and the transfer latency was recorded again. the experiments were conducted between 10:00 and 14:00 hours in a dimly lit, semi-soundproof room under natural light.29–32 morris water maze test. the mwm comprised a circular pool (90 cm diameter and 30 cm height) filled with water (22°c) to a depth of 14 cm and rendered opaque by addition of small black balls. the pool was located in a dimly lit, soundproof test room with various visual cues, including a white/ black colored poster on the wall, a halogen lamp, a camera, and the experimenter. the maze was divided into four quadrants, and three equally spaced points served as starting positions around the edge of the pool. the order of the release positions was varied systematically throughout the experiment. a circular escape platform (6  cm diameter and 12  cm high) was located in one quadrant 1 cm above the water surface during the familiarization session and 1 cm below the water surface during the other sessions.17,20 video tracking was conducted with a video camera focused on the full diameter of the pool. navigation parameters were analyzed using the ethovision 3.1 video analysis system (noldus). mice were trained in mwm five times per day (familiarization session, s1, s2, s3, and s4). one familiarization and four acquisition sessions were carried out using the mwm. during the familiarization session and acquisition phase of experiment, each mouse underwent three trials. the delay between trials was 60 seconds, and a 1-day interval was used between each session. for each trial, the mouse was removed from the home cage and placed in the water maze at one of three randomly determined locations with its head facing the center of the water maze. after the mouse had found and climbed onto the platform, the trial was terminated and the escape latency was recorded. if the mouse did not climb onto the platform in 60 seconds, the trial was terminated, and experimenter guided the mouse to the platform; an escape latency of 60 seconds was recorded.17,20 twenty-four hours after the final acquisition session, a “probe trial” was used to assess the spatial memory retention of the location of the hidden platform. during this trial, the platform was removed from the maze and the mouse was allowed to search the pool for 60 seconds. the percent of time spent in each quadrant was recorded.17,20 radial arm maze test. the experimental device comprised an elevated maze with eight open arms (32 cm long and 5 cm wide) that led to an 8-cm square platform, which radiated from a central circular platform 44  cm in diameter with 1-cm high sides surrounding each arm.33 a small cup, 1 cm in diameter, was embedded in each distal platform, and it contained a hidden 10-mg noodle used as reinforcement. the maze was oriented in a small room, which had four large black, white, or black and white striped patterns hung on walls; these patterns provided particularly salient visual extramaze cues. for further details on the apparatus, see beuzen et al.33 twenty-four hours prior to training, the mice were deprived of food but not water; their weight loss reached 15%–20% of the initial body weight by the start of the testing. the ram procedure was applied according to belzung et al.34 the mice were first subjected to two pretraining sessions at 24-hour intervals. groups of four mice were placed on the maze at the same time and for 20 minutes per session; the mice could freely explore the eight arms, which contained abundant food. following pretraining, the mice were subjected to five training sessions at 90-minute intervals. after baiting the eight arms with 10-mg noodles, a mouse was placed on the central platform. sessions were terminated http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 7-ni and odq disturbs memory in the elevated plus maze 3drug target insights 2015:9 when the animal had visited all eight arms and consumed the rewards, after 16 arms were visited (regardless of which arms), or after a maximum of 15  minutes. an error was recorded when the mouse entered an arm previously visited during the retention session. the total number of errors and the latency of the retention session (time taken to complete the task) were recorded.34 drug administration. 7-ni, odq, and l-arginine were procured from sigma chemical company. odq and l-arginine were dissolved in saline, whereas 7-ni was dissolved in saline supplemented with 10% dimethylsulfoxide (dmso). all drugs were freshly prepared and administered in a volume of 0.1  ml per 10  g body weight. the control groups received the same volume of vehicle. 7-ni (15 mg/kg), odq (3 and 10  mg/kg), l-arginine, or the vehicle was administered via an intraperitoneal (i.p.) injection 30, 30, and 60 minutes, respectively, prior to the first session (acquisition session, day 1) of mepm test, prior to the retention trial of ram test, and for 6 days prior to the acquisition trials and the probe trial of the mwm test. the number of animals per group ranged from 6 to 7. effective dose of each drug was selected according to previous behavioral and neurochemical studies.3,35–37 statistics. a two-way analysis of variance (anova) and post hoc tukey test were used to analyze the mepm, mwm, and ram tests. the data are expressed as the mean ± sem. p , 0.05 was considered statistically significant. results effects of 7-ni, odq , and 7-ni ± l-arginine on learning and memory in the mepm test. when 7-ni (15  mg/kg), odq (3 and 10  mg/kg), or 7-ni (15  mg/kg) + l-arg (100  mg/kg) was administered prior to the acquisition session (training; day 1), there was no significant effect of the drugs [f(3,29)  =  1.81; fig. 1a] or their combination [f(1,29)  =  1.71; fig. 1a] in the mepm test. there was a significant effect of the drugs [f(3,29)  =  7.07, p  =  0.001; fig. 1b] and their combination [f(1,29) = 14.17, p , 0.001; fig. 1b] on the second-day latency in the mepm test. odq (3  mg/kg) and 7-ni (15  mg/kg) significantly prolonged the latency (tl2) on the second day compared with the control group when the drugs were administered prior to the acquisition session (p  ,  0.05 and p  ,  0.01, respectively; fig. 1b). l-arginine (100 mg/kg) combined with 7-ni (15 mg/kg) significantly shortened the tl2 compared with 7-ni (15 mg/kg) alone (p , 0.001; fig. 1b). effects of 7-ni, odq, and 7-ni ± l-arginine on learning and memory in the mwm test. there was a significant difference in the escape latency in the first, second, third, and fourth sessions during the evaluation of the drug groups [f(3,35)  =  6.58, p  =  0.001; f(3,35)  =  11.71, p  ,  0.001; f(3,35)  =  7.41, p  ,  0.001; and f(3,35)  =  10.24, p  ,  0.001; respectively; fig. 2a]. 7-ni (15 mg/kg) significantly increased the escape latency during the first, second, third, and fourth sessions (p  =  0.01; p  ,  0.01; p  ,  0.01; and p  ,  0.001, figure 1. drug effects on (a) transfer latency 1 (tl-1) and (b) transfer latency 2 (tl-2) (n = 6) in the mepm test in mice. odq (3 and 10 mg/kg), 7-ni (15 mg/kg), or l-arginine (100 mg/kg) was administered 30, 30, and 60 minutes, respectively, prior to the acquisition trial of the mepm test. the data are expressed as the mean ± sem values of the animals. *p , 0.05, **p , 0.001 compared with the control group. #p , 0.001 compared with the 7-ni group. http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 mutlu et al 4 drug target insights 2015:9 morris water maze test 0 5 10 15 20 25 30 35 drugs (mg/kg) ti m e sp en t i n es ca pe pl at fo rm ’s q ua dr an t ( s) e sc ap e la te nc y (s ) 180 160 140 120 100 80 60 40 20 0 fam ses 1st ses 2nd ses 3rd ses 4th ses morris water maze testa b c d control odq 3 odq 10 7-ni (30) 7-ni (30) + l-arg (100) sessions morris water maze test morris water maze test 0 5 10 15 20 25 30 35 drugs (mg/kg) drugs (mg/kg) m ea n di st an ce to pl at fo rm (c m ) control odq 3 odq 10 7-ni (15) 7-ni (15) + l-arg (100) # 14.5 15 15.5 16 16.5 17 17.5 18 18.5 19 19.5 s pe ed (c m /s ) ** ** *** # figure 2. drug effects on (a) the escape latency in five acquisition sessions of the mwm test, (b) the time spent in the escape platform’s quadrant in the probe trial (60 seconds) in the mwm test, (c) the mean distance to the platform in the probe trial (60 seconds) of the mwm test, and (d) the swimming speed in the probe trial (60 seconds) of the mwm test. odq (3 and 10 mg/kg), 7ni (15 mg/kg), or l-arg (100 mg/kg) was administered daily 30, 30, and 60 minutes, respectively, prior to the first trial of the day for 6 days. results are expressed as the mean ± sem. n = 7 per group. *p , 0.05, **p , 0.01, ***p , 0.001 compared with the control group; #p , 0.001 compared with the 7-ni group. http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 7-ni and odq disturbs memory in the elevated plus maze 5drug target insights 2015:9 respectively), whereas odq 10  mg/kg significantly increased the escape latency during the second, third, and fourth sessions (p , 0.01; p , 0.05; and p , 0.05, respectively) compared with the control in naive mice. l-arginine (100 mg/kg) significantly reversed the effects of 7-ni (15  mg/kg) on the escape latency in the first, second, third, and fourth sessions [f(1,35)  =  13.49; p  ,  0.001; f(1,35)  =  23.29; p  ,  0.001; f(1,35) = 15.23; p , 0.001; and f(1,35) = 29.19; p , 0.001, respectively; fig. 2a]. a significant difference was noted between all drug groups in the time spent in the target quadrant [f(3,35) = 6.04; p = 0.002; fig. 2b]. 7-ni (15 mg/kg) and odq (10 mg/kg) significantly decreased the time spent in the escape platform’s quadrant (p , 0.01 and p , 0.05, respectively). l-arg (100  mg/kg) combined with 7-ni significantly increased the decrease in the time spent in the escape platform’s quadrant in the 7-ni only group [f(1,35) = 12.06; p = 0.002]. the mean distance traveled by the mice to the platform in the probe trial of the mwm test was significantly different between the drug groups [f(3,35) = 6.94; p = 0.001; fig. 2c]. 7-ni 15 mg/kg and odq (10 mg/kg) significantly increased the mean distance traveled to the platform (p , 0.01). l-arg combined with 7-ni significantly reversed the effects of 7-ni 15 mg/kg [f(1,35) = 12.06; p = 0.002; fig. 2c]. the treatment groups were not significantly different in swimming speed [f(3,35) = 0.06; p = 0.97; fig. 2d], and the combination had no effect on the speed of the animals in the probe trial of the mwm test [f(1, 35) = 0.02; p = 0.86; fig. 2d]. effects of 7-ni, odq , and 7-ni ± l-arginine on learning and memory in the r am test. in the evaluation of the effects of acute treatment with 7-ni (15  mg/kg) and odq (3 and 10  mg/kg) administered 30  minutes prior to the retention trial on the number of errors in the ram test, a significant difference between the groups was identified [f(3,29) = 19.08; p , 0.001]. 7-ni (15 mg/kg) and odq (3 and 10 mg/kg) significantly enhanced the number of working memory errors in the ram test in mice (p , 0.001). l-arg (100  mg/kg) significantly decreased the number of working memory errors in the 7-ni-treated mice [f(1,29)  =  33.86; p , 0.001] in the ram test (fig. 3a). when the effects of 7-ni (15 mg/kg) and odq (3 and 10  mg/kg) on the latency (time taken to complete the task) of the animals in the ram test were evaluated, there was a significant difference between the groups [f(3,29)  =  15.11; p , 0.001]. 7-ni (15 mg/kg) significantly enhanced the latency of the animals (p , 0.001), whereas odq (3 and 10 mg/kg) had no significant effect (p . 0.05). l-arg (100 mg/kg) combined with 7-ni (15 mg/kg) significantly decreased the latency figure 3. (a) drug effects on working memory errors in the ram test. (b) drug effects on the latency in the ram test. odq (3 and 10 mg/kg), 7-ni (15 mg/kg), or l-arginine (100 mg/kg) was administered 30, 30, and 60 minutes, respectively, prior to the retention trial of the ram test. results are expressed as the mean ± sem. n = 6 per group. *p , 0.001 compared with the control group; #p , 0.001 compared with the 7-ni group. http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 mutlu et al 6 drug target insights 2015:9 of the ram test compared with 7-ni alone [f(1,29) = 19,01; p , 0.001; fig. 3b]. discussion in our study, both the gc inhibitor odq (3 mg/kg) and the nos inhibitor 7-ni (15 mg/kg) increased the retention latency in the mepm test. 7-ni (15  mg/kg) and odq (10  mg/kg) significantly increased the escape latency in the second, third, and fourth sessions, diminished the time spent in the escape platform’s quadrant, and enhanced the mean distance to the platform in the probe trial of the mwm test. odq (3, 10 mg/kg) and 7-ni (15 mg/kg) significantly increased the number of errors, whereas only 7-ni increased the latency in the ram test. the 7-ni (15  mg/kg)-induced effects in the mepm, mwm, and ram tests were reversed by the no precursor, l-arginine (100 mg/kg). mwm is a spatial, long-term memory evaluation test.38 the daily decrease in escape latencies reflects learning, and it is related to long-term reference memory. this study revealed that odq (10  mg/kg) and 7-ni (15  mg/kg) increased the escape latency during the acquisition sessions, diminished the time spent in the escape platform quadrant, and enhanced the mean distance traveled to the platform during the probe test. therefore, both odq and 7-ni disturbed the spatial memory. the drug treatment did not alter the swimming speed of the mice, which suggests odq and 7-ni did not alter their motor activity. because the position of the platform did not change throughout the experiment, these results indicate that odq and 7-ni disturbed the reference spatial memory. it has been shown that the ability to make a series of correct choices in the ram test depends on the spatial information from extramaze cues.39 in our protocol, the effects of the drugs on spatial memory were evaluated because performance in the maze requires sufficient memory of the spatial environment. each arm was baited with food, and reentry to a previously visited arm was categorized as an error; thus, spatial working memory was thought to be examined.39 both odq (3 and 10 mg/kg) and 7-ni (15 mg/kg) disturbed the spatial working memory in the ram test in our study. the mepm test is a simple method that evaluates spatial memory. a shortened transfer latency in the second trial is used as a parameter to measure the retention or consolidation of memory, and drug treatment prior to the first day may be utilized to determine the effects on memory acquisition.40 the evaluation of drug effects in the first trial may be confounded by nonspecific effects, such as effects on anxiety, locomotion, and motility.41 since there was no significant difference between groups for the first-day latency in the mepm test, we can exclude these nonspecific effects. both odq (3  mg/kg) and 7-ni (15 mg/kg) treatment led to memory deterioration in the epm test in our study, which is in accordance with previous studies.28,37,42 the effects of odq did not follow a typical dose–response curve of a drug. the experimental condition can cause some differences between the results. we studied with standard experimental conditions and standard equipment and obtained these results. recent studies suggest that both neuronal (nnos) and endothelial (enos) nitric oxide isoforms have a role in memory formation. additionally, in a passive avoidance test, nnos cannot substitute for the role of enos on ltp.43 in research using “knock-out” mice, it has been demonstrated that both neuronal and endothelial nos isoforms were expressed in hippocampal ca1 pyramidal cells, and both endothelial and neuronal nos deletion resulted in the loss of ltp.44 in contrast, when only one isoform mutant was used, the mice had preserved their normal ltp capacity.44 taken together, these results support the claim that both isoforms have a role in ltp and can substitute for each other; however, the mechanisms that underlie their actions and the degree to which enos can compensate nnos remain unknown. these findings can explain why the nonselective nnos and enos inhibitor 7-ni impaired learning and memory in our study, as well as the discrepancies in the studies with nos inhibitors on memory. the differences between the animal strains, types, and protocols of the test, drug type, pharmacokinetics, specificity, dose, and administration route of the drugs can explain, in part, the discrepancies between different studies. no inhibitors or donors are known to exert effects on blood pressure.45 no donors can cause hypotension, whereas nos inhibitors can lead to hypertension. the effects of these drug groups on cognitive functions can be the result of their effects on cardiovascular functions. there is controversy regarding the effects of 7-ni on blood pressure, although prickaerts et al14 reported that the disruption of 7-ni effects was not correlated with its effects on blood pressure. hölscher et al26 observed that 30 mg/kg 7-ni impaired spatial learning without changing blood pressure in both the water maze and eight-arm radial maze tests. in our study, we administered the drugs systematically; thus, we could not avoid nonspecific effects, such as hypertension. however, in recent studies, it was shown that 7-ni did not have a significant hypertensive effect at the doses used in our study.26 no affects the release and reuptake of various neurotransmitters,46 which results in the ltp of synaptic transmission and the neuronal basis of memory formation. some studies have reported that nos inhibitors do not change the learning performance or memory,47,48 whereas other studies have reported that these drugs inhibited the retention trial of the passive avoidance test,49,50 spatial learning in the water maze test,51,52 or object recognition14 in the radial arm maze.53 7-ni has been shown to impair learning and memory in most tests,26,28,37 and these findings are similar to the results observed in our study. our study is in accordance with the studies that demonstrated the disturbing effect of nos inhibitors on learning and memory. the reversal of the 7-ni effects by l-arginine supports the theory that these effects are specific to nos. no is known http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 7-ni and odq disturbs memory in the elevated plus maze 7drug target insights 2015:9 to play a role in some forms of memory processing;14,52,53 however, the exact function is not known. the activation of the gc/cgmp/protein kinase g (pkg) pathway causes most no-mediated physiological processes. edwards et al54 reported that the inhibition of gc and pkg impairs retention for the passive avoidance task. kleppsich et al55 reported that pkgs are not involved in ltp in mice, but no induces ltp through an alternative cgmp-independent pathway, possibly adenosine diphosphate (adp) ribosylation. our study supports the role of cgmp on cognition and the disruptive effects of odq on learning and memory. in conclusion, the present study demonstrates that both the gs inhibitor odq and the nos inhibitor 7-ni disturbed spatial memory in the mepm, mwm, and ram tests. l-arginine, the no precursor, reversed 7-ni-induced changes, which confirms that the effects of 7-ni were no dependent. our findings suggest that the nnos/sgc/cgmp pathway is involved in the pathophysiology of memory functions. author contributions conceived and designed the experiments: om, fa, ikc, pt. analyzed the data: om, fa, ikc. wrote the first draft of the manuscript: om, fa, gu. contributed to the writing of the manuscript: om, gu, fe. agree with manuscript results and conclusions: om, fa, ikc, pt, gu, fe. jointly developed the structure and arguments for the paper: fe, gu, pt. made critical revisions and approved final version: fa, ikc, pt. all authors reviewed and approved of the final manuscript. r efer ences 1. prast h, philippu a. nitric oxide as a modulator of neuronal function. prog neurobiol. 2001;64:51–56. 2. yildiz f, erden f, ulak g, utkan t, gacar n. antidepressant-like effect of 7-nitroindazole in the forced swimming test in rats. psychopharmacology. 2000; 149:41–44. 3. yildiz f, ulak g, erden f, gacar n. anxiolytic-like effects of 7-nitroindazole in the rat plus-maze test. pharmacol biochem behav. 2000;65:199–202. 4. trainor bc, workman jl, jessen r, nelson rj. impaired nitric oxide synthase signaling dissociates social investigation and aggression. behav neurosci. 2007; 121:362–369. 5. susswein aj, katzoff a, miller n, hurwitz i. nitric oxide and memory. neuroscientist. 2004;10:153–162. 6. barnstable cj, wei jy, han mh. modulation of synaptic function by cgmp and gmp-gated cation channels. neurochem int. 2004;45:875–884. 7. chien wl, liang kc, teng cm, kuo sc, lee fy, fu wm. enhancement of learning behaviour by a potent nitric oxide-guanylate cyclase activator yc-1. eur j neurosci. 2005;21:1679–1688. 8. chien wl, liang kc, fu wm. enhancement of active shuttle avoidance response by the no-cgmp-pkg activator yc-1. eur j pharmacol. 2008;590: 233–240. 9. zhuo m, hu y, schultz c, kandel er, hawkins rd. role of guanylyl cyclase and cgmpdependent protein kinases in long-term potentiation. nature. 1994; 368:635–639. 10. garthwaite j. glutamate, nitric oxide and cell–cell signalling in the nervous system. trends neurosci. 1991;14:60–67. 11. southam e, garthwaite j. the nitric oxide-cyclic gmp signalling pathway in rat brain. neuropharmacology. 1993;32:1267–1277. 12. arancio o, kandel er, hawkins rd. activity-dependent long-term enhancement of transmitter release by presynaptic 3x, 5xcyclic-gmp in cultured hippocampal neurons. nature. 1995;376:74–80. 13. boulton cl, southam e, garthwaite j. nitric oxide-dependent long-term potentiation is blocked by a specific inhibitor of soluble guanylyl cyclase. neuroscience. 1995;69:699–703. 14. prickaerts j, steinbusch hw, smits jf, vente j. possible role of nitric oxidecyclic gmp pathway in object recognition memory: effects of 7-nitroindazole and zaprinast. eur j pharmacol. 1997;33:125–136. 15. meyer cr, spangler el, patel n, london ed, ingram dk. impaired learning in rats in a 14-unit t-maze by 7-nitroindazole, a neuronal nitric oxide synthase inhibitor, is attenuated by the nitric oxide donor, molsidomine. eur j pharmacol. 1998;341:17–22. 16. knepper br, kurylo dd. effects of nitric oxide synthase inhibitor ng-nitro-larginine methyl ester on spatial and cued learning. neuroscience. 1998;83:837–841. 17. blokland a, de vente j, prickaerts j, honig w, markerink-van ittersum m, steinsbuch h. local inhibition of hippocampal nitric oxide synthase does not impair place learning in the morris water escape task in the rat. eur j neurosci. 1999; 11:223–232. 18. estall lb, grant sj, cicala ga. inhibition of nitric oxide (no) production selectively impairs learning and memory in the rat. pharmacol biochem behav. 1993; 46:959–962. 19. yamada k, noda y, nakayama s, et al. role of nitric oxide in learning and memory and in monoamine metabolism in the rat brain. br j pharmacol. 1995;115: 852–858. 20. bannerman dm, chapman pf, kelly pa, butcher sp, morris rg. inhibition of nitric oxide synthase does not impair spatial learning. j neurosci. 1994;14: 7404–7414. 21. bon c, böhme ga, doble a, stutzmann j-m, blanchard j-c. a role for nitric oxide in long-term potentiation. eur j pharmacol. 1992;4:420–424. 22. doyle c, hölscher c, rowan mj, anwyl r. the selective neuronal no synthase inhibitor 7-nitro-indazole blocks both long-term potentiation and depotentiation of field epsps in rat hippocampal ca1 in vivo. j neurosci. 1996;16:418–426. 23. southam e, charles sl, garthwaite j. the nitric oxide-cyclic gmp pathway and synaptic plasticity in the rat superior cervical ganglion. br j pharmacol. 1996; 119:527–532. 24. cummings ja, nicola sm, malenka rc. induction in the rat hippocampus of long-term potentiation (ltp) and long-term depression (ltd) in the presence of a nitric oxide synthase inhibitor. neurosci lett. 1994;176:110–114. 25. murphy kp, williams jh, bettache n, bliss tv. photolytic release of nitric oxide modulates nmda receptor-mediated transmission but does not induce long-term potentiation at hippocampal synapses. neuropharmacology. 1994;33: 1375–1385. 26. hölscher c, mcglinchey l, anwyl r, rowan mj. 7-nitroindazole, a selective neuronal nitric oxide synthase inhibitor in vivo, impairs spatial learning in the rat. learn mem. 1996;2:267–278. 27. mizuno m, yamada k, olariu a, nawa h, nabeshima t. involvement of brainderived neurotrophic factor in spatial memory formation and maintenance in a radial arm maze test in rats. j neurosci. 2000;20:7116–7121. 28. yildiz-akar f, ulak g, tanyeri p, erden f, utkan t, gacar n. 7-nitroindazole, a neuronal nitric oxide synthase inhibitor, impairs passive-avoidance and elevated plus-maze memory performance in rats. pharmacol biochem behav. 2007;87:434–443. 29. reddy ds, kulkarni sk. possible role of nitric oxide in the nootropic and antiamnesic effects of neurosteroids on agingand dizocilpine-induced learning impairment. brain res. 1998;799:215–229. 30. hlinak z, krejci i. concurrent administration of subeffective doses of scopolamine and mk-801 produces a short-term amnesia for the elevated plus-maze in mice. behav brain res. 1998;91:83–89. 31. hlinak z, krejci i. mk-801 induced amnesia for the elevated plus-maze in mice. behav brain res. 2002;131:221–225. 32. hlinak z, krejci i. oxiracetam prevents the mk-801 induced amnesia for the elevated plus-maze in mice. behav brain res. 2000;117:147–151. 33. beuzen a, belzung c, roullet p. drug effects in a radial maze designed for dissociation of cues used by mice. pharmacol biochem behav. 1994;48:23–29. 34. belzung c, le guisquet am, griebel g. beta-cct, a selective bz-v receptor antagonist, blocks the anti-anxiety but not the amnesic action of chlordiazepoxide in mice. behav pharmacol. 2000;11:125–131. 35. ergün y, ergün ug. prevention of pro-depressant effect of l-arginine in the forced swim test by ng-nitro-l-arginine and [1h-[1,2,4]oxadiazole[4,3-a] quinoxalin-1-one]. eur j pharmacol. 2007;554:150–154. 36. yıldız akar f, komsuoğlu fi, ulak g, mutlu o. effects of l-arginine on 7-nitroindazole-induced reference and working memory performance of rats. pharmacology. 2009;84:211–218. 37. mutlu o, ulak g, belzung c. effects of nitric oxide synthase inhibitors 1-(2 trifluoromethylphenyl)—imidazole (trim) and 7-nitroindazole (7-ni) on learning and memory in mice. fundam clin pharmacol. 2011;25:368–377. 38. cain dp. testing the nmda, long-term potentiation, and cholinergic hypotheses of spatial learning. neurosci biobehav rev. 1998;22:181–193. 39. nagai t, kamei h, dohniwa m, et al. involvement of hippocampal extracellular signal-regulated kinase 1/2 in spatial working memory in rats. neuroreport. 2006; 17:1453–1457. 40. sharma ac, kulkarni sk. evaluation of learning and memory mechanisms employing elevated plus-maze in rats and mice. prog neuropsychopharmacol biol psychiatry. 1992;16:117–125. http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 mutlu et al 8 drug target insights 2015:9 41. hunter b, zornetzer sf, jarvik me, mcgaugh jl. modulation of learning and memory: effects of drugs influencing neurotransmitters. in: iversen ll, iversen sd, snyder sh, eds. handbook of psychopharmacology. vol 19. new york: plenum press; 1988:531–577. 42. komsuoglu-celikyurt i, gocmez ss, mutlu o, gacar n, aricioglu f, utkan t. evidence for the involvement of neuronal nitric oxide synthase and soluble guanylate cyclase on cognitive functions in rats. life sci. 2011;89(23–24):905–910. 43. rickard ns, gibbs me, ng kt. inhibition of the endothelial isoform of nitric oxide synthase impairs long-term memory formation in the chick. learn mem. 1999;6(5):6458–6466. 44. son h, hawkins rd, martin k, kiebler m, huang pl, fishman mc. longterm potentiation is reduced in mice that are doubly mutant in endothelial and neuronal nitric oxide synthase. cell. 1996;87:1015–1023. 45. pitsikas n, rigamonti ae, cella sg, locatelli v, sala m, muller ee. effects of molsidomine on scopolamine-induced amnesia and hypermotility in the rat. eur j pharmacol. 2001;426:193–200. 46. guevara-guzman r, emson pc, kendrick km. modulation of in vivo striatal transmitter release by nitric oxide and cyclic gmp. j neurochem. 1994;62: 807–810. 47. böhme ga, bon c, lemaire m, et al. altered synaptic plasticity and memory formation in nitric oxide synthase inhibitor-treated rats. proc natl acad sci u s a. 1993;90:9191–9194. 48. telegdy g, kokavszky r. the role of nitric oxide in passive avoidance learning. neuropharmacology. 1997;36:1583–1587. 49. finn c, da cunha c, bromberg e, et al. experiments suggesting a role for nitric oxide in the hippocampus in memory processes. neurobiol learn mem. 1995;63: 113–115. 50. kopf sr, benton sr, kanfin r, giovannini mg, pepeu g. no synthesis inhibition decreases cortical ach release and impairs retention of a conditioned response. brain res. 2001;894:141–144. 51. toyoda m, saito h, matsuki n. nitric oxide but not carbonmonoxide is involved in spatial learning of mice. jpn j pharmacol. 1996;71:205–211. 52. prendergast ma, buccafusco jj, terry av jr. nitric oxide inhibition impairs spatial navigation learning and induces contitioned taste aversion. pharmacol biochem behav. 1998;57:347–352. 53. zou lb, yamada k, tanaka t, kameyama t, nabeshima t. nitric oxide synthase inhibitors impair reference memory formation in a radial arm maze. neuropharmacology. 1998;37:323–330. 54. edwards tm, rickard ns, ng kt. inhibition of guanylate cyclase and protein kinase g impairs retention for the passive avoidance in the day old chick. neurobiol learn mem. 2002;77:313–326. 55. kleppisch t, pfeifer a, klatt p, et al. long-term potentiation in the hippocampal ca1 region ofmice lacking cgmp-dependent kinases is normal and susceptible to inhibition of nitric oxide synthase. j neurosci. 1999;19:48–55. http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 drug target insights 2013:7 19–25 doi: 10.4137/dti.s12109 this article is available from http://www.la-press.com. © the author(s), publisher and licensee libertas academica ltd. this is an open access article published under the creative commons cc-by-nc 3.0 license. open access full open access to this and thousands of other papers at http://www.la-press.com. drug target insights r a p i d c o m m u n i c a t i o n drug target insights 2013:7 19 microscopic colitis is associated with several concomitant diseases bodil roth1, jonas manjer2 and bodil ohlsson1 1department of clinical sciences, section of internal medicine. 2department of clinical sciences, plastic surgery, skåne university hospital, lund university, sweden. corresponding author email: bodil.ohlsson@med.lu.se abstract: microscopic colitis (mc) is a disease with intestinal mucosal inflammation causing diarrhea, affecting predominantly middle-aged women. the etiology is unknown, but increased prevalence of autoimmune diseases in these patients has been described, although not compared with controls or adjusted for confounding factors. the aim of this study was to examine the prevalence of common diseases in patients with mc and controls from the general population. hypertension, rheumatoid arthritis, asthma or bronchitis, ischemia, and diabetes mellitus were more prevalent in patients than in controls. the prevalence of gastric ulcer and cancer did not differ between the groups. besides corticosteroids, many patients were also being treated with proton pump inhibitors, antidepressant drugs, angiotensin-converting enzyme inhibitors or angiotensin ii receptor antagonists, statins, thyroid hormones, and beta-blockers. more patients than controls were former or current smokers (72.5% versus 57.7%). thus, mc patients have an increased prevalence of several diseases, not only of autoimmune origin. keywords: concomitant diseases, drug treatments, microscopic colitis, women http://dx.doi.org/10.4137/dti.s12109 http://www.la-press.com http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 http://www.la-press.com mailto:bodil.ohlsson@med.lu.se roth et al 20 drug target insights 2013:7 introduction primary microscopic colitis (mc) is a clinical and histopathological disease of unknown etiology, characterized by chronic gastrointestinal symptoms, and a macroscopically normal or near normal colonic mucosa. the entity includes 2 basic forms: collagenous colitis (cc) and lymphocytic colitis (lc).1 some studies have shown a female predominance in both cc and lc,2 mainly affecting middle-aged women, whereas others have not been able to confirm this in lc.3,4 besides primary mc, a wide array of conditions may lead to secondary lymphocytic inflammation in the intestinal mucosa, which should be distinguished from real mc.1. an increased prevalence of autoimmune diseases and use of anti-inflammatory drugs has been described in retrospective studies of patients with mc. on this basis autoimmunity has been suggested as an etiology.3,5–7 asthma has been associated with lc, but not with cc.8 however, these studies have not compared the prevalence of diseases in patients with mc with controls from the general population, and no adjustment for confounding factors has been performed. furthermore, the coexistence of autoimmune diseases and mc may be due to a high consumption of anti-inflammatory drugs, rather than a common causality.3,5–7 smoking and advanced age are risk factors for developing mc, and individuals over 65 years of age are at least 5 times more likely to be diagnosed as having mc than younger individuals.2,9 in these women of upper middle age, it may be difficult to determine whether the mc is a primary disease, or a secondary effect due to other concomitant diseases and drug treatments influencing the colonic mucosa.1 the aim of this cross-sectional study was to compare the prevalence of concomitant diseases in patients with mc and controls from the same geographic area, after adjustment for confounding factors. materials and methods the ethics committee of lund university approved the study protocol for both patients (dnr 2009/565 and 2011/209) and controls (dnr 51-90). all participants gave their written informed consent to take part in the study. subjects women who had been treated for mc at any outpatient clinic of the departments of gastroenterology, skåne, between 2002 and 2010, were identified by a search for the icd-10 classification for the 2 forms cc and lc (k52.8) in outpatient records, as well as in the local register at the department of pathology, skåne university hospital, malmö. about 1/3 of the total number of identified patients were excluded because they were over 73 years of age, since they had many other concomitant diseases and drug therapies, obscuring the picture as to whether they were suffering from primary or secondary mc.1 of the patients recognized, only the 240 patients (median 63 years, range 22–73 years) who had the diagnoses verified by examination of colonic biopsies by a pathologist specialized in gastrointestinal pathology were invited to participate in the present study. altogether, 159 (median 63 years, range 22–73 years) of the 240 patients invited accepted and were enrolled in the study. one patient was excluded due to another ibd diagnosis a few weeks after inclusion, leaving 158 patients (66%), and of these, 133 also agreed to provide blood samples. these patients represent the majority of female cases of diagnosed mc in the southernmost districts of sweden under the age of 73 years. microscopic colitis is more frequent in women than in men, the small male cohort in our region being unsuitable for statistical calculations. furthermore, as the quality of life and experience of symptoms differ between the genders,2 we chose to include only women in the study. controls the malmö diet and cancer study (mdcs) the mdcs, a population-based prospective cohort study, invited all women in malmö born 1923–1950. recruitment was carried out between 1991 and 1996, and 41% of eligible subjects participated. in all, 17,035 women completed the baseline examination.10 the mdcs baseline examination included a dietary assessment, a self-administered questionnaire about marital status, education, employment, smoking habits, wine consumption, physical activity, medical conditions and medication, anthropometric measurements, and the collection of blood samples.11 menopausal status was defined using information on previous surgery and menstrual status. the classification of pre-, periand postmenopausal women has been described in detail elsewhere.12 http://www.la-press.com mc and concomitant diseases drug target insights 2013:7 21 women selected as controls in a previous study on breast cancer were used in the present study as controls. in all, 737 subjects (median age 56 years, range 45–73 years) were available and the only exclusion criterion was that they should not have had a previous breast cancer at baseline.13 patient recruitment and study design between march and june 2011, invitations including study information and the same self-administered questionnaire as was sent to the controls were sent by mail to all 240 women with mc. in addition, questionnaires about gastrointestinal symptoms, psychological well-being and rome iii criteria were sent. the patients were also invited to visit the outpatient clinics of the departments of gastroenterology, skåne university hospital, malmö or the central hospital in kristianstad, to provide blood samples. a reminding letter was sent a month after the invitation letter to those who had not answered. questionnaires were completed 1–3 weeks before blood samples were collected. medical records were scrutinized, and age, gastrointestinal symptoms, examinations, and treatments were recorded, as well as whether the patients had had a single attack of mc, or had persistent disease. patients were compared with controls from the mdsc study. questionnaire a self-administered questionnaire about marital status, education, employment, smoking habits, wine consumption, medical conditions and medication was completed by both controls and patients. one of the questions was: “have you ever been treated for any of the following diseases, namely, hypertension, rheumatoid arthritis, asthma or chronic bronchitis, gastric ulcer, ischemia including myocardial infarction, intermittent claudication and stroke, cancer, or diabetes mellitus?”. statistical analyses the data were analyzed using the statistical software package spss for windows© (release 20.0; ibm, ny, usa). the patients were significantly older, with a wider age range than controls. therefore, the 12 patients younger and the two patients older than the controls were excluded, as were patients with celiac disease and gastroenteritis (13 patients), leaving 131 of the original 158 patients for statistical analysis. thus, both controls and patients were within the age range 45–73 years. first, the distribution of continuous variables (age, disease duration, days of drinking wine/month) was tested using a onesample kolmogorov-smirnov test. all these distributions differed significantly (p , 0.05) from a normal distribution. therefore, the factors studied were categorized and values were given as median (interquartile range). there was no difference between cc and lc for any characteristics in this mc cohort14 and therefore all calculations were performed independent of the category cc or lc. the number of patients in the study cohort (131 patients) who were under treatment with a drug was given as the percentage of drug users. differences between groups were calculated by the 2-tailed mann–whitney u test. fisher’s exact test was used for categorical variables. p-values , 0.05 were considered statistically significant. age was divided into 5-year intervals. the cohort was divided into quartiles of the number of days wine was taken per month. smoking was divided into 3 categories: subjects who had never smoked, subjects who had stopped smoking, and current smokers, including both regular and occasional smokers. employment was divided into 3 categories: employed, retired, or others, where others included housewives, students, and unemployed. education was divided into patients with or without a university education. some answers concerning days of drinking wine/month and level of education were lacking. these were labeled as separate categories. factors intended to be studied (independent variables) were initially examined using univariate analyses to calculate odds ratios with 95% confidence intervals (or with 95% ci). analyses were then adjusted for age at baseline, smoking, the number of days of drinking wine/month, level of education, and employment, as these characteristics differed by .5 percentage points between controls and patients. results patient characteristics in total, 131 women (median age 63 (59–67) years) with mc were included in the statistical calculations (table 1). collagenous colitis was diagnosed in 82 patients (62.6%) and lc in 49 patients (37.4%). http://www.la-press.com roth et al 22 drug target insights 2013:7 table 1. patient and control characteristics. controls n = 737 microscopic colitis n = 131 age at study (years) 56.16 (50.47–62.36) 63.00 (58.94–67.15) age groups (%) 45–49 17.1 4.6 50–54 22.3 6.9 55–59 22.3 13.7 60–64 19.5 32.1 65–69 11.7 26.0 70–74 7.2 16.8 smoking habits (%) never smoked 42.3 27.5 stopped smoking 29.9 36.6 current smokers 27.8 35.9 days of drinking wine/month (%) missing data 6.8 5.3 0–2 49.5 42.7 3–4 15.2 12.2 5–7 12.3 8.4 .7 16.1 31.3 married women (%) 61.9 58.0 level of education (%) missing 0 2.3 #12 years at school 76.1 67.9 .12 years at school 23.9 29.8 employment (%) employed 65.7 44.3 retired 26.5 49.6 others* 7.9 6.1 notes: *includes housewives, students and unemployed. values are given as median (interquartile range). the duration of the disease was 7 (3–14) years. measurements of hemoglobin (hb) in blood and creactive protein (crp) in plasma were in the majority of patients within reference values, showing that the patients were in an overall inactive phase (data not shown). of the patients, 91.7% were born in sweden compared with 90.2% of the controls. more patients than controls had completed a university degree (p = 0.001). as the patients were older than the controls, more patients were retired (p , 0.001) (table 1). smoking and drinking habits there were more controls than patients who had never smoked, and the prevalence of both current and former smokers was higher among the patients (table 1). more patients than controls drank wine .7 days a month (table 1). concomitant diseases the presence of any concomitant disease was more prevalent in patients with mc (58.8%) than in controls (35.5%) (adjusted or = 1.81, 95% ci = 1.18–2.81). hypertension was present in more than 1/3 of the patients. rheumatoid arthritis was 6 times more common and asthma and bronchitis 3 times as common in patients as in controls (table 2). the type of diabetes mellitus is not known in controls, but 2 of the patients with mc had type 1 diabetes and 7 had type 2 diabetes. there was no difference between those who had had a single attack of mc or a persistent mc in those with concomitant diseases and those without (p = 0.930). there was no difference in duration of mc, or age at inclusion, between those with concomitant diseases and those without in addition to the mc (p = 0.564 and p = 0.146, respectively). the patients were currently being treated with several drugs at the time of inclusion. the most common drug treatments as a percentage of the study cohort were corticosteroids (32.1%), proton pump inhibitors (26.0%), antidepressant drugs, specifically selective serotonin reuptake inhibitors (21.4%), angiotensinconverting enzyme inhibitors or angiotensin ii receptor antagonists (18.3%), statins (17.6%), thyroid hormones (17.6%), and beta-blockers (16.0%). patients on any of these drug treatments were older at inclusion (64.92 (60.00–68.34) years and 62.07 (55.55–64.24) years, respectively, p = 0.012). those who had persistent mc had a higher prevalence of current drug treatment (p = 0.024). 8 of the 31 patients with rheumatoid arthritis used non-steroidal anti inflammatory drugs as well as many other drugs. there was no difference in the prevalence of cc and lc in patients who were on any of these drugs or had any of the concomitant diseases (p = 1.000 and p = 0.931, respectively). discussion in spite of excluding all those over 73 years of age, to get a fairly healthy group with real mc, several concomitant diseases and drugs were still present. all chronic diseases measured were over-represented in patients, in contrast to a history of gastric ulcer or cancer. previous studies have been retrospective, collecting patient cohorts seen at tertiary centers.5–7 in our present study, we used a cross-sectional design, http://www.la-press.com mc and concomitant diseases drug target insights 2013:7 23 collecting patients from the whole region at primary, secondary and tertiary centers. this approach reflects the patient group in a better way, as patients handled at tertiary centers are often selected cases.15 as patients with mc are women of upper middle age with former or current smoking in the anamnesis, it is to be expected that asthma, bronchitis, and cardiovascular diseases will be frequently seen in such a cohort, apart from diseases of autoimmune origin. in the present study, hypertension was the most common concomitant disease, and recent research confirms that smokers have a higher prevalence of hypertension than non-smokers.16 a high prevalence of cardiovascular diseases in patients with mc has been described previously, but this was not compared with a control population.17 the medication of the controls is not reported here because drug recommendations have been table 2. the prevalence of different diseases in microscopic colitis (mc) and controls. controls n = 737 mc n = 131 crude or 95% ci or 95% ci hypertension (%) missing 8.4 1.5 – – no hypertension 77.7 62.6 1.00 1.00 hypertension 13.8 35.9 3.22 (2.12–4.88) 2.73 (1.49–3.78) rheumatoid arthritis (%) missing 8.4 1.5 – – no rheumatoid arthritis 87.9 74.0 1.00 1.00 rheumatoid arthritis 3.7 23.7 7.67 (4.39–13.40) 7.21 (3.81–13.64) asthma and bronchitis (%) missing 8.4 1.5 – – no asthma 85.5 80.9 1.00 1.00 asthma 6.0 16.8 2.97 (1.71–5.16) 3.18 (1.68–6.00) cancer (%) missing 8.4 1.5 – – no cancer 85.9 89.3 1.00 1.00 cancer 5.7 8.4 1.42 (0.71–2.83) 1.14 (0.53–2.47) diabetes mellitus (%) missing 8.4 1.5 – – no diabetes mellitus 90.5 90.8 1.00 1.00 diabetes mellitus 1.1 6.9 6.31 (2.38–16.67) 4.91 (1.62–14.87) gastric ulcer (%) missing 8.4 1.5 – – no gastric ulcer 84.3 80.9 1.00 1.00 gastric ulcer 7.3 16.8 2.39 (1.40–4.08) 1.77 (0.95–3.30) ischemia* (%) missing 8.4 1.5 – – no ischemia 88.7 88.5 1.00 1.00 ischemia 2.8 9.9 3.49 (1.70–7.16) 2.96 (1.30–6.76) notes: *including myocardial infarction, intermittent claudication and stroke. analyses were performed adjusted for age at baseline, smoking, days of drinking wine/month, level of education, and employment, as these characteristics differed by .5 percentage between controls and patients. abbreviations: or, odds ratio; ci, confidence interval. changed since the control cohort was recruited. however, medication in controls should be less than of the patients as they were healthier. in accordance with previous reports,18 the present patients who were taking drugs were older than un-treated ones. it has been suggested in previous studies that the drugs being consumed extensively by the patient group are associated with mc and could explain the persistent character of the disease.6,18–20 the consensus is that drugs suspected to induce mc should be withdrawn prior to diagnosis, and that the introduction of treatment against mc may not be followed in the daily clinic.2 this could contribute to the high prevalence figures of mc in the growing elderly population, with more efficient treatment regimens for cardiovascular diseases.2 prospective studies are needed to determine whether the introduction of a new drug precedes the development of the disease, and whether the disease http://www.la-press.com roth et al 24 drug target insights 2013:7 remains resolved after drug withdrawal, in order to estimate appropriate prevalence figures of mc. ischemic colitis is another frequently diagnosed condition in elderly patients with a history of smoking, cardiovascular diseases and diabetes mellitus, and in those who are on vasoactive drugs.21 these characteristics are similar to those described for the mc population.2,3,5 corticosteroids are used in the treatment of mc, with a better response than antiinflammatory drugs, and corticosteroids can also be useful in the treatment of ischemic, radiatic, and toxic colitis.2,21–23 there are several limitations in this study. one is the use of an external control group, and that recommendations for drug prescription have been changed since our recruitment of controls. it is very difficult to recruit healthy volunteers to clinical studies. the response rate of our control group was 41%, and it is possible that these subjects are healthier than those who did not agree to participate. however, it is a strength of the study to, for the first time, compare patients with mc to such a well-defined control group.13 furthermore, the data concerning smoking, overweight status, and level of education were similar in this control group to a study with 80% participation from the same population.10 we could not find from the medical records whether mc was developed prior to or after the introduction of new drugs, and therefore it is impossible to determine whether the disease is primary or secondary. an additional strength of the study was that the control group is derived from the same geographic area as the patient group, and that calculations are adjusted for age differences, life style factors, and socioeconomic factors. in conclusion, patients with a diagnosis of mc are a selection of middle-aged women, former or current smokers, with several concomitant diseases and cardiovascular ageing, and therefore are under treatment with a wide range of diverse drugs. it is not surprising that these patients exhibit gastrointestinal symptoms and microscopic, intestinal, mucosal inflammation. these changes must be interpreted with caution, before considering them as a separate entity of autoimmune origin, instead of secondary reactions to ischemia and toxic stimulants. efforts must be made to better classify and diagnose patients with real, primary mc, to avoid overprescription of corticosteroids. author contributions conceived and designed the experiments: br, jm, bo. analyzed the data: bo. wrote the first draft of the manuscript: bo. contributed to the writing of the manuscript: br, jm, bo. agree with manuscript results and conclusions: br, jm, bo. jointly developed the structure and arguments for the paper: br, jm, bo. made critical revisions and approved final version: br, jm, bo. all authors reviewed and approved of the final manuscript. funding this study was sponsored by grants from the bengt ihre foundation and the development foundation of region skåne. competing interests authors disclose no potential conflicts of interest. disclosures and ethics as a requirement of publication the authors have provided signed confirmation of their compliance with ethical and legal obligations including but not limited to compliance with icmje authorship and competing interests guidelines, that the article is neither under consideration for publication nor published elsewhere, of their compliance with legal and ethical guidelines concerning human and animal research participants (if applicable), and that permission has been obtained for reproduction of any copyrighted material. this article was subject to blind, independent, expert peer review. the reviewers reported no competing interests. references 1. carmack sw, lash rh, gulizia jm, genta rm. lymphocytic disorders of the gastrointestinal tract: a review for the practicing pathologist. adv anat pathol. 2009;16(5):290–306. 2. pardi ds, kelly cp. microscopic colitis. gastroenterology. 2011;140(4): 1155–65. 3. olesen m, eriksson s, bohr j, järnerot g, tysk c. lymphocytic colitis: a retrospective clinical study of 199 swedish patients. gut. 2004;53(4):536–41. 4. fernández-bañares f, salas a, forné m, esteve m, espinós j, viver jm. incidence of collagenous and lymphocytic colitis: a 5-year population-based study. am j gastroenterol. 1999;94(2):418–23. 5. bohr j, tysk c, eriksson s, abrahamsson h, järnerot g. collagenous colitis: a retrospective study of clinical presentation and treatment in 163 patients. gut. 1996;39(6):846–51. 6. pardi ds, ramnath vr, loftus ev, tremaine wj, sandborn wj. lymphocytic colitis: clinical features, treatment, and outcomes. am j gastroenterol. 2002;97:2829–33. 7. chande n, driman dk, reynolds rp. collagenous colitis and lymphocytic colitis: patient characteristics and clinical presentation. scand j gastroenterol. 2005;40(3):343–7. http://www.la-press.com mc and concomitant diseases drug target insights 2013:7 25 8. koskela rm, niemelä se, karttunen tj, lehtola jk. clinical characteristics of collagenous and lymphocytic colitis. scand j gastroenterol. 2004;39(9):837–45. 9. yen ef, pokhrel b, du h, et al. current and past cigarette smoking significantly increase risk for microscopic colitis. inflamm bowel dis. 2012;18(10):1835–41. 10. manjer j, carlsson s, elmståhl s, et al. the malmö diet and cancer study: representativity, cancer incidence and mortality in participants and nonparticipants. eur j cancer prev. 2001;10(6):489–99. 11. manjer j, elmståhl s, janzon l, berglund g. invitation to a populationbased cohort study: differences between subjects recruited using various strategies. scand j public health. 2002;30(2):103–12. 12. manjer j, johansson r, berglund g, et al. postmenopausal breast cancer risk in relation to sex steroid hormones, prolactin and shbg (sweden). cancer causes control. 2003;14(7):599–607. 13. almquist m, bondeson ag, bondeson l, malm j, manjer j. serum levels of vitamin d, pth and calcium and breast cancer risk-a prospective nested case-control study. int j cancer. 2010;127(9):2159–68. 14. roth b, gustafsson rj, ohlsson b. auto-antibodies and their association with clinical findings in women diagnosed with microscopic colitis. plos one. 2013;8(6):e66088. 15. zankel e, rogler g, andus t, reng cm, schölmerich j, timmer a. crohn’s disease patient characteristics in a tertiary referral center: comparison with patients from a population-based cohort. eur j gastroenterol hepatol. 2005;17(4):395–401. 16. d’elia l, de palma d, rossi g, et al. not smoking is associated with lower risk of hypertension: results of the olivetti heart study. eur j public health. 2013. 17. bjørnbak c, engel pj, nielsen pl, munck lk. microscopic colitis: clinical findings, topography and persistence of histopathological subgroups. aliment pharmacol ther. 2011;34(10):1225–34. 18. rasmussen ma, munck lk. systematic review: are lymphocytic colitis and collagenous colitis two subtypes of the same disease—microscopic colitis? aliment pharmacol ther. 2012;36(2):79–90. 19. cindoruk m, tuncer c, dursun a, et al. increased colonic intraepithelial lymphocytes in patients with hashimoto’s thyroiditis. j clin gastroenterol. 2002;34(3):237–9. 20. beaugerie l, pardi ds. review article: drug-induced microscopic colitis—proposal for a scoring system and review of the literature. aliment pharmacol ther. 2005;22(4):277–84. 21. o’neill s, yalamarthi s. systematic review of the management of ischaemic colitis. colorectal dis. 2012;14(11):e751–63. 22. kochhar r, patel f, dhar a, et al. radiation-induced proctosigmoiditis. prospective, randomized, double-blind controlled trial of oral sulfasalazine plus rectal steroids versus rectal sucralfate. dig dis sci. 1991;36(1):103–7. 23. onishi h, oosegi t, machida y. efficacy and toxicity of eudragit-coated chitosan-succinyl-prednisolone conjugate microspheres using rats with 2,4,6-trinitrobenzenesulfonic acid-induced colitis. int j pharm. 2008; 358(1–2):296–302. http://www.la-press.com dti drug target insights 2023; 17: 5-30issn 1177-3928 | doi: 10.33393/dti.2023.2477review drug target insights issn 1177-3928 www.aboutscience.eu/dti © 2023 the authors. this article is published by aboutscience and licensed under creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). commercial use is not permitted and is subject to publisher’s permissions. full information is available at www.aboutscience.eu a systematic review of mucoadhesive vaginal tablet testing ismin zainol abidin1, emma j. murphy 1–3, gustavo waltzer fehrenbach4, emanuele rezoagli5,6, noel gately1, ian major1 1prism research institute, technological university of the shannon: midlands midwest, athlone campus, westmeath ireland 2 shannon applied biotechnology centre, department of applied science, faculty of applied sciences and technology, technological university of the shannon: midlands midwest, moylish campus, limerick ireland 3life—health and biosciences research institute, technological university of the shannon, midwest campus, limerick ireland 4bioscience research institute, technological university of the shannon: midlands midwest, athlone campus, westmeath ireland 5department of emergency and intensive care, san gerardo university hospital, monza italy 6school of medicine and surgery, university of milano-bicocca, monza italy abstract drug administration through the vaginal tract is one of the oldest modalities of pharmacotherapy, and it is also one of the most explored. since the vaginal cavity has a wide surface area, a plentiful blood supply, and a complex network of blood arteries, it can evade hepatic first-pass metabolism and obtain high local drug concentrations. vaginal pills look to be a good dose form since they are simple to use, portable, and can easily deliver the required amount of medicine. vaginal formulations, on the other hand, are vulnerable to rapid expulsion due to the vaginal tract’s self-cleaning action, which reduces the formulation’s efficiency. currently, there is an increasing amount of focus on mucoadhesive vaginal formulation research and development to fix the formulation at the place where the medicine can be released and/or absorbed. this article examines all of the strategies used by researchers to develop a mucoadhesive vaginal tablet that is safe, effective, and comfortable for the user. keywords: dissolution, mucoadhesion, physicochemical, swelling received: july 27, 2022 accepted: december 7, 2022 published online: january 16, 2023 corresponding author: ian major prism research institute tus athlone campus dublin road n37 hd68 athlone ireland ian.major@tus.ie surface area, rich blood supply, and the presence of a dense network of blood vessels, the vagina serves as a promising site for systemic drug delivery (3). its relatively high permeability to many drug compounds (including several with high molecular weight) also allows for drug transport across the vaginal mucosa and access into the blood circulation, presenting in many cases higher flux levels than those observed via intestinal tissues (2,4). the vaginal route may prove to be of particular importance in the case of drugs undergoing extensive hepatic metabolism, since it avoids the hepatic first-pass effect (1). furthermore, it permits the elimination of possible degradation in the gastrointestinal tract and the effect of the drug directly at the site of application (5). traditionally, the vaginal route has been used for delivery of locally acting drugs such as antibacterial, antifungal, antiprotozoal, antiviral, labor-inducing, and spermicidal agents, prostaglandins, and steroids (6). recently, there has been increased interest and effort in the development of vaginal formulations such as microbicides that provide effective contraception and protection against transmission of various sexually transmitted diseases (stds), including acquired immunodeficiency syndrome (aids) (3). there are many different vaginal products in the market to treat different vaginal conditions, for example, canesten® for antifungal and nuvaring® as contraceptive (7,8). one of the leading vaginal introduction over the past three decades, the vaginal route has gained relevance in modern medicine as a route for drug delivery and is now considered an option for several therapeutic strategies, specifically for female-related conditions. several advantages have been claimed for vaginal drug delivery in managing local conditions and achieving systemic effects. in the case of managing local conditions, vaginal administration means that lower doses can be effective (compared to the oral route), which frequently leads to reduced systemic exposure and can prevent side effects (1,2). owing to its large https://doi.org/10.33393/dti.2023.2477 https://creativecommons.org/licenses/by-nc/4.0/legalcode mailto:ian.major@tus.ie mucoadhesive vaginal tablet testing6 © 2023 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti tablets available in the market is vagifem® by novo nordisk, which was first introduced in 1988. it is an option for local estrogen therapy that continuously releases steroid in the vagina for a consistent dose of hormone (9). there are many other vaginal tablets that are currently in the process of patent application and are listed in table i. these applications are in various stages of the patenting process, some has been granted and anticipating expiry, some are still pending, and some have just published a research article under the patent id. vaginal products do not need to be sterile; they are usually cheap and relatively easy to manufacture (2). it also allows easy and comfortable self-administration and rarely requires the intervention of a health-care provider (1). marketed vaginal dosage forms include solutions (douches), semisolids (creams, ointments, and gels), and solid formulations (tampons, capsules, pessaries, suppositories, films, sponges, powders, and special controlled release devices like the intravaginal ring) as well as other types of formulations such as aerosols and particulate systems integrated in adequate drug delivery systems (3,6,10). however, the use of vaginal formulations can be limited due to poor drug retention in the vaginal tract, as they are removed in a short time by the tract’s self-cleansing action (3). the low residence time often leads to disappointing experiences such as leakages and messiness, which cause loss of formulation from the application site, giving rise to inadequate formulation and hence lack of effectiveness (11). therefore, frequent daily doses are often required to maintain an effective drug concentration, which further complicates application and contributes to low patient acceptability and, thus, poor compliance (3,4,6,12,13). extensive research and innovative attempts have been made to develop vaginal formulations to meet clinical and user requirements. to overcome these limitations, researchers have focused their attention on the development of new delivery systems that can prolong the drug residence time in the vaginal cavity, basically by using mucoadhesive formulations (3,12,14,15). the general principles of the mucoadhesive vaginal drug delivery system will be discussed further in a later section of this review. mucoadhesive vaginal drug delivery systems are superior to conventional ones due to their ability to prolong drug residence time at the application site, leading to improved bioavailability and efficacy (12). the number of products based on new vaginal drug delivery systems has significantly increased, and this growth is expected to continue in the near future (3). with various types of formulations available, the popularity of vaginal products can be different among women from different backgrounds and countries (6). nonetheless, tablets and gels (films) are among the most popular vaginal formulations (3,6,12). even with major advancement in the gel (hydrogel) and film formulations, moderate vaginal leakage was still observed and daily administration was required (16). therefore, vaginal tablets still often represent the typically acceptable dosage form with stability-related advantages and an economical choice for both manufacturers and users (17,18), thus advocating its role and relevance in the vaginal drug delivery system. so, it won’t be obsolete just yet. this review aims to assemble and discuss the key parameters and unique methodologies that should be considered when evaluating vaginal tablet formulations. this review will not go into any specific medicinal substances or polymers; instead, it will concentrate on the tactics and evaluation table i list of some vaginal tablet patent applications for different treatments patent id author/inventor title treatment/ condition date of application current status cn1307986c chen z. vaginal effervescent tablets for inflammation and their preparation gynecological inflammation 2004-06-08 granted (2007-04-04) anticipated expiration (2024-06-08) au2012210296b2 mogna g., mogna l., strozzi g.p. effervescent composition in solid form for use in vaginal application for the treatment of vaginal infections vaginal infections 2011-01-28 granted (2017-01-05) anticipated expiration (2032-01-24) cn106420726a jiang dingyu, zhang yongwei, zhu ming clotrimazole vaginal tablets vulvovaginal candidiasis 2016-06-30 pending wo2020201515a1 ellervik u., manner s., sterner o., strevens h., lindberg n., säfholm a. vaginal tablet formulation vaginal microbial infections 2019-04-05 publication (2020-10-08) us20200155621a1 thoral c., tchoreloff p., mazel v., busignies v., nivoliez a. mucoadhesive sustainedrelease vaginal tablet probiotic strain: lactobacillus 2014-03-10 pending w02022218487a1 crouzier t., schimpf u. a vaginal contraceptive composition for reinforcement of the cervical mucus barrier properties contraceptive 2021-04-12 publication (2022-10-20) these patents have been sourced from google patent and accessed on november 28, 2022. abidin et al drug target insights 2023; 17: 7 © 2023 the authors. published by aboutscience www.aboutscience.eu methods that can aid in the development of an effective vaginal tablet formulation. to the author’s knowledge, this is the first systematic review that compiles numerous assessment methodologies required to develop a new mucoadhesive vaginal tablet formulation. methods eligibility criteria inclusion criteria i. all original studies designing and formulating mucoadhesive vaginal tablets, regardless of its therapeutic treatment ii. publication years between 2000 and november 2021 iii. articles in english language iv. articles published in scholarly peer-reviewed journals exclusion criteria i. articles of studies on other mucoadhesive vaginal dosage forms (e.g., semisolids and liquids) ii. articles of studies on vaginal pessaries, suppositories, and pellets because it has been identified that they are different from tablet dosage forms iii. articles of studies of mucoadhesive vaginal tablet for veterinary use iv. articles of studies that compare between two different dosage forms (e.g., tablets and films) information sources a search of relevant papers between years 2000 and 2021 was made via chosen electronic databases available in the technology university of shannon: midlands midwest (tus) library online search engine. the databases included pubmed, science direct, multidisciplinary digital publishing institute (mdpi) and academic search complete (ebsco). the search was done between august 19 and november 11, 2021, for any new relevant publications. search strategy the search in the databases was carried out using the keyword “mucoadhesive vaginal tablets” or “vaginal tablets + mucoadhesive” or “vaginal tablets + evaluations” or “vaginal tablet testing + mucoadhesion.” study selection and data collection process the initial stage involves screening the titles. titles that specify a dosage form other than vaginal tablets (e.g., films, gels) and for veterinary purposes are essentially excluded. in the case of vague titles, a quick scan through the abstract is conducted to identify the words, vaginal tablet or tablet/s. titles that do not use the term “vaginal tablet” are considered as vague titles. following that, the abstracts were screened to ensure that they meet the inclusion criteria. the full text of eligible abstracts is then accessed and reviewed. two reviewers were involved in the selection process. articles were collected individually; the other reviewer replicated the search strategy and listed the articles deemed eligible for the review. excluded articles from each reviewer were confirmed with each other and eligible articles are compiled. studies that were deemed eligible were compiled in an excel spreadsheet (microsoft 365 application, version 2201). data were collected systematically and analyzed using excel spreadsheet. data items data extracted from each study include: (i) information of the tablets (e.g., use, physical appearance, etc.), (ii) all the methods used to measure the physicochemical properties of the tablets, (iii) all the methods used to assess the mucoadhesion property of the tablet, (iv) all the methods used to evaluate drug release profile of the tablet, and (v) other relevant methods used to evaluate the technical working ability of the tablet. for each outcome, effect measures were determined via difference in means and standard deviation. all graphs to presenting the results were prepared using excel software (microsoft 365 application, version 2201). study risk of bias assessment each article was assessed for risk of bias using the cochrane collaborations tool (19). the following biases were examined: (i) bias in selection/sampling, (ii) performance bias associated with the allocation of interventions during the study, (iii) attrition bias associated with the handling of incomplete outcome data, (iv) reporting bias associated with selective outcome reporting, (v) measurement bias associated with the use of non-validated data collection criteria, and (vi) analysis bias associated with the omission of necessary statistical coefficients associated with the study. synthesis methods the full text of each article was scrutinized in sequence and grouped according to the data items mentioned in section 2.5. data were collected systematically and tabulated and analyzed (means and standard deviation) using excel software (microsoft 365 application, version 2201). all graphs prepared to present the results were prepared using the same software. results study selection there were 772 research articles that were identified as a result of the keyword search of mucoadhesive vaginal tablets across five electronic databases. the breakdown is as follows: (i) pubmed, 65 articles; (ii) science direct, 624 articles; (iii) mdpi, 5 articles; (iv) scopus, 77 articles, and (v) ebsco, 1 article. from the initial screening of the titles, a total of 63 research articles were deemed eligible to proceed for abstract screening. at this stage, the research articles mucoadhesive vaginal tablet testing8 © 2023 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti were sub-divided into two categories according to the study characteristic: (i) research articles with a single formulation of vaginal tablet (study of inter-batches variation) and (ii) vaginal tablets formulation with polymer intervention to improving the mucoadhesive properties of the formulation (comparison study between modified and non-modified formulations). the subcategories were to facilitate the reviewers in understanding the study design and characteristic of the articles. following the abstract screening, 43 research articles were deemed eligible to proceed for full-text review and data collection. for most excluded research articles, the study focuses more on a specify drug compound or polymer rather than the technology of tablet manufacturing and performance. in addition, four other research articles were then excluded during the full-text review, due to one being outdated (20) and three research articles having non-vaginal tablet-related outcomes (21-23). in these research articles, most formulations were simply made into kbr disks to test the mucoadhesive property exclusively, no further tablet manufacturing parameters were considered. thus, it is considered non-vaginal tablet-related outcomes. to avoid further limitations, unaccessible research articles were agreed to not be included in this review. figure 1 shows the flow diagram of the selection process and that at the end of this process, 39 research articles were included in this review. study characteristics table ii summarizes the study characteristics of the research articles reviewed. all of the vaginal tablets were prepared by direct compression, with most having flat-faced and round or cylindrical shape. more of the study characteristics are discussed in section 4.2. the studies were divided into two subcategories depending on the study design: either (a) inter-batch comparison or (b) polymer intervention. the study background of the articles reviewed is summarized in table iii. inter-batch comparison studies involved vaginal tablet formulations that were prepared in different batches varying in different types and/or ratios, and/or combinations of mucoadhesive polymers in each batch. some are natural polymers, some in combination fig. 1 prisma flow diagram of the study selection process. abidin et al drug target insights 2023; 17: 9 © 2023 the authors. published by aboutscience www.aboutscience.eu table ii demographic of the research articles reviewed in alphabetical order research article author study design vaginal tablet design treatment application study title 1 abidin et al (2020) inter-batch comparison (n* = 6) bilayer tablet, flat faced, round shaped anticancer a bilayer vaginal tablet for the localized delivery of disulfiram and 5-fluorouracil to the cervix 2 abu el-enin et al (2020) inter-batch comparison (n* = 6) core-in-cup tablet flat faced, round shaped preterm labor formulation, development, in vivo pharmacokinetics and pharmacological efficacy evaluation of novel vaginal bioadhesive sustained core-in-cup salbutamol sulphate tablets for preterm labor 3 baki et al (2009) inter-batch comparison (n* = 6) flat faced, round shaped vaginal health management formulation of a solid intravaginal matrix system to prolong the ph-decreasing effect of lactic acid 4 baloglu et al (2011) polymer intervention flat faced, round shaped antifungal in vitro evaluation of mucoadhesive vaginal tablets of antifungal drugs prepared with thiolated polymer and development of a new dissolution technique for vaginal formulations 5 bartkowiak et al (2018) inter-batch comparison (n* = 6) flat faced, round shaped antifungal surface and swelling properties of mucoadhesive blends and their ability to release fluconazole in a mucin environment 6 bhat et al (2010) inter-batch comparison (n* = 6) flat faced, round shaped vaginal infection bioadhesive controlled release clotrimazole vaginal tablets 7 cazorla-luna et al (2019) inter-batch comparison (n* = 15) flat faced, round shaped hiv prevention chitosan-based mucoadhesive vaginal tablets for controlled release of the anti-hiv drug tenofovir 8 cevher et al (2014) inter-batch comparison (n* = 4) flat faced, round shaped antifungal bioadhesive tablets containing cyclodextrin complex of itraconazole for the treatment of vaginal candidiasis 9 cevher et al (2008) inter-batch comparison (n* = 3) flat faced, round shaped antifungal preparation and characterisation of natamycin: g-cyclodextrin inclusion complex and its evaluation in vaginal mucoadhesive formulations 10 el-kamel et al (2002) inter-batch comparison (n* = 4) flat faced, round shaped antibacterial chitosan and sodium alginate–based bioadhesive vaginal tablets 11 fitaihi et al (2017) inter-batch comparison (n* = 17) flat faced, round shaped antifungal role of chitosan on controlling the characteristics and antifungal activity of bioadhesive fluconazole vaginal tablets 12 gupta et al (2013) inter-batch comparison (n* = 6) flat faced, round shaped antimycotic bioadhesive vaginal tablets containing spray dried microspheres loaded with clotrimazole for treatment of vaginal candidiasis 13 gök et al (2017) polymer intervention flat faced, round shaped did not specify the effects of the thiolation with thioglycolic acid and l-cysteine on the mucoadhesion properties of the starch-graft-poly(acrylic acid) 14 hani et al (2016) inter-batch comparison (n* = 10) flat faced, round shaped antifungal development of a curcumin bioadhesive monolithic tablet for treatment of vaginal candidiasis 15 hassan et al (2017) inter-batch comparison (n* = 12) flat faced, round shaped hormone therapy mucoadhesive tablets for the vaginal delivery of progesterone: in vitro evaluation and pharmacokinetics/ pharmacodynamics in female rabbits 16 hombach et al (2009) polymer intervention flat faced, round shaped vaginal infection development and in vitro evaluation of a mucoadhesive vaginal delivery system for nystatin 17 kailasam et al (2010) inter-batch comparison (n* = 5) flat faced, round shaped antibacterial formulation and evaluation of once daily mucoadhesive vaginal tablet of metronidazole (continued) mucoadhesive vaginal tablet testing10 © 2023 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti research article author study design vaginal tablet design treatment application study title 18 kast et al (2002) polymer intervention flat faced, round shaped vaginal infection design and in vitro evaluation of a novel bioadhesive vaginal drug delivery system for clotrimazole 19 khan et al (2017) inter-batch comparison (n* = 8) did not specify hiv prevention formulation and evaluation of once daily mucoahdesive vaginal tablet of metronidazole 20 khan et al (2014) inter-batch comparison (n* = 8) did not specify hiv prevention formulation and evaluation of mucoadhesive vaginal tablets of tenofovir disoproxil fumarate 21 lupo et al (2017) polymer intervention flat faced, round shaped antibacterial entirely s-protected chitosan: a promising mucoadhesive excipient for metronidazole vaginal tablets 22 notario-pérez et al (2019) inter-batch comparison (n* = 5) flat faced, round shaped hiv prevention tenofovir hot-melt granulation using gelucire® to develop sustained-release vaginal systems for weekly protection against sexual transmission of hiv 23 anotario-pérez et al (2017) inter-batch comparison (n* = 12) flat faced, round shaped hiv prevention optimization of tenofovir release from mucoadhesive vaginal tablets by polymer combination to prevent sexual transmission of hiv 24 bnotario-pérez et al (2017) inter-batch comparison (n* = 12) flat faced, round shaped hiv prevention influence of chitosan swelling behaviour on controlled release of tenofovir from mucoadhesive vaginal systems for prevention of sexual transmission of hiv 25 nowak et al (2015) polymer intervention flat faced, round shaped did not specify preactivated hyaluronic acid: a potential mucoadhesive polymer for vaginal delivery 26 pacheco-quito et al (2020) inter-batch comparison (n* = 8) flat faced, round shaped vaginal infection carrageenan-based acyclovir mucoadhesive vaginal tablets for prevention of genital herpes 27 paczkowska et al (2020) inter-batch comparison (n* = 6) did not specify vaginal infection mucoadhesive chitosan delivery system with chelidonii herba lyophilized extract as a promising strategy for vaginitis treatment 28 palade et al (2013) inter-batch comparison (n* = 12) did not specify did not specify in vitro evaluation of 5-fluorouracil dissolution profiles from vaginal bioadhesive tablets 29 patel a. et al (2012) inter-batch comparison (n* = 9) did not specify antifungal design, development and in vitro evaluation of sertaconazole mucoadhesive vaginal tablet 30 patel a. et al (2011) inter-batch comparison (n* = 10) did not specify antifungal development and evaluation of mucoadhesive vaginal tablet of sertaconazole for vaginal candidiasis 31 patel g.m. et al (2010) inter-batch comparison (n* = 9) did not specify antifungal a novel effervescent bioadhesive vaginal tablet of ketoconazole: formulation and invitro evaluation 32 pendekal et al (2013) inter-batch comparison (n* = 9) flat faced, round shaped anticancer hybrid drug delivery system for oropharyngeal, cervical and colorectal cancer—in vitro and in vivo evaluation 33 pendekal et al (2012) inter-batch comparison (n* = 9) flat faced, round shaped anticancer development and characterization of chitosanpolycarbophil interpolyelectrolyte complex-based 5-fluorouracil formulations for buccal, vaginal and rectal application 34 perioli et al (2009) inter-batch comparison (n* = 6) flat faced, round shaped antibacterial fg90 chitosan as a new polymer for metronidazole mucoadhesive tablets for vaginal administration 35 perioli et al (2011) inter-batch comparison (n* = 5) flat faced, round shaped vaginal infection new solid mucoadhesive systems for benzydamine vaginal administration table ii (continued) table ii (continued) abidin et al drug target insights 2023; 17: 11 © 2023 the authors. published by aboutscience www.aboutscience.eu research article author study design vaginal tablet design treatment application study title 36 sánchez et al (2017) inter-batch comparison (n* = 4) double layer, flat faced, round shaped vaginal infection a novel double-layer mucoadhesive tablet containing probiotic strain for vaginal administration: design, development and technological evaluation 37 szymańska et al (2014) inter-batch comparison (n* = 4) flat faced, round shaped vaginal infection vaginal chitosan tablets with clotrimazole—design and evaluation of mucoadhesive properties using porcine vaginal mucosa, mucin and gelatine 38 tunpanich et al (2019) inter-batch comparison (n* = 5) capsule shaped hormone therapy mucoadhesive sustained-release tablets for vaginal delivery of curcuma comosa extracts: preparation and characterization 39 valenta et al (2001) polymer intervention flat faced, round shaped hormone therapy development and in vitro evaluation of a mucoadhesive vaginal delivery system for progesterone hiv = human immunodeficiency virus. *n = number of batches formulated and compared to. table iii background characteristic of research articles reviewed author study background polymers used abidin et al (2020) designed a bilayer vaginal tablet. a comparison study between batches formulated using different ratios of polymers in individual layers. chitosan, poly(acrylic acid) abu el-enin et al (2020) designed a core-in-cup vaginal tablet. a comparison study between batches formulated using different combinations of mucoadhesive polymers. carbopol, hydroxypropyl methylcellulose, hydroxyethylcellulose, polyethylene glycol baki et al (2009) studied the applicability of solid matrix system for tablet formulations. a comparison study between batches formulated with different concentrations of api. hydroxypropyl methylcellulose, microcrystalline cellulose baloglu et al (2011) a comparison study between batches formulated using thiolated and nonthiolated mucoadhesive polymers. article includes polymer modification steps and structure characterization methods. poly(acrylic acid), poly(acrylic acid)cysteine (thiolated) bartkowiak et al (2018) a comparison study between batches formulated using different blends of mucoadhesive polymers. carbopol, polycarbophil, chitosan, hydroxyethylcellulose, hydroxypropyl methylcellulose bhat et al (2010) a comparison study between batches formulated using mixtures of natural mucoadhesive polymer with hpmc in different ratios. hydroxypropyl methylcellulose, sodium carboxymethylcellulose, guar gum cazorla-luna et al (2019) a comparison study between batches formulated using chn alone and in combination with natural mucoadhesive polymers. the different blends of polymers were used to generate spontaneous polyelectrolyte complexes. article includes structure characterization methods of the complexes formed. chitosan, pectin, locust bean gum cevher et al (2014) this study focuses on forming inclusion complex of cyclodextrin with the chosen api to improve their aqueous solubility. the complex was then formulated into sustained-release vaginal tablet. hydroxypropyl methylcellulose, carbopol, xanthan gum cevher et al (2008) a comparison study between batches formulated using different polymer ratios. this study focuses on forming inclusion complex of cyclodextrin with the chosen api to improve their aqueous solubility. the complex was then formulated with a polymer at different ratios until high mucoadhesion to the vaginal mucosa is achieved. hydroxypropyl methylcellulose, carbopol, xanthan gum el-kamel et al (2002) a comparison study between batches formulated using mixtures of anionic polymers with chn. the polymer combinations were used to generate spontaneous polyelectrolyte complexes. chitosan, microcrystalline cellulose, sodium carboxymethylcellulose, sodium alginate fitaihi et al (2017) a comparison study between batches formulated using different types of polymers physically blended with chn at different ratios. chitosan, hydroxypropyl methylcellulose, guar gum, sodium carboxymethylcellulose, polyvinyl pyrrolidone (continued) mucoadhesive vaginal tablet testing12 © 2023 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti author study background polymers used gupta et al (2013) incorporated microspheres of the api into a vaginal tablet formulation. a comparison study between batches formulated using the api in the pure form and the api microspheres. eudragit rs®, eudragit rl, carbopol, hydroxypropyl methylcellulose, sodium carboxymethylcellulose gök et al (2017) this study investigated the effect of thiolation reagents on the thiolation process. article includes polymer modification and confirmation methods. a comparison study between batches formulated using thiolated and nonthiolated mucoadhesive polymers. poly(acrylic acid), poly(acrylic acid)cysteine (thiolated), poly(acrylic acid)thioglycolic acid (thiolated) hani et al (2016) a comparison study between batches formulated using different types of polymers and at different ratios. hydroxypropyl methylcellulose, xantham gum, guar gum hassan et al (2017) a comparison study between batches formulated using different types of polymers. carbopol, hydroxypropyl methylcellulose, chitosan, sodium alginate hombach et al (2009) a comparison study between batches formulated using two different thiolated polymers (thiomers); the conjugates of poly(acrylic acid). poly(acrylic acid), poly(acrylic acid)cysteine, poly(acrylic acid)-cysteamine kailasam et al (2010) a comparison study between batches formulated using wo different types of polymer in different ratios. carbopol, hydroxypropyl methylcellulose kast et al (2002) investigated the effect of thiolation by using various amounts of thiolating agent. a comparison study between batches formulated using thiolated and non-thiolated polymers. chitosan, chitosan-thioglycolic acid (thiolated) khan et al (2017) investigated the application of pcp as the matrix forming polymer with other types of polymers. a comparison study between batches formulated with different polymer combinations. carbopol, chitosan, sodium carboxymethylcellulose khan et al (2014) formulation and evaluation of mucoadhesive vaginal tablets of tenofovir disoproxil fumarate. hydroxypropyl methylcellulose, carbopol, chitosan, sodium carboxymethylcellulose lupo et al (2017) investigated the effect of enhanced modification by protecting the thiol groups on thiolated polymers to avoid oxidation and increase the stability of the polymer. a comparison study between batches formulated using s-protected thiomer and non-thiolated polymer. chitosan, s-protected chitosan notario-pérez et al (2019) evaluated and compared batches of vaginal tablets formulated using granules prepared by hot-melt granulation method. chitosan, hydroxypropyl methylcellulose, polyvinyl pyrrolidone notario-pérez et al (a) (2017) a comparison study between batches formulated using different polymer combinations. hydroxypropyl methylcellulose, chitosan, eudragit rs®, guar gum notario-pérez et al (b) (2017) a comparison study between batches formulated using natural, semisynthetic, and synthetic polymers. chitosan, hydroxypropyl methylcellulose, eudragit rs®, guar gum nowak et al (2015) investigated the effect of enhanced modification by using preactivated thiomer on polymer stability. hyaluronic acid-cysteine-6mercaptonicotinamide pacheco-quito et al (2020) a comparison study between batches formulated using different types of polymers and at different amount. iota-carrageenan, hydroxypropyl methylcellulose paczkowska et al (2020) developed vaginal drug delivery system using lyophilized extract with chn carrier. chitosan, hydroxypropyl methylcellulose, microcrystalline cellulose palade et al (2013) a comparison study between batches formulated using acrylic acid derivatives and cellulose derivatives. carbopol, metolose patel a. et al (2012) a comparison study between batches formulated with different combination of polymers. in addition, this study included an effervescent feature in the formulation to enhance the swelling of the tablets. carbopol, chitosan, sodium carboxymethylcellulose, methyl cellulose, hydroxypropyl methylcellulose, hydroxyethylcellulose, sodium alginate patel a. et al (2011) a comparison study between batches formulated with different combination of polymers. in addition, this study included an effervescent feature in the formulation to enhance the swelling of the tablets. carbopol, chitosan, sodium carboxymethylcellulose, methyl cellulose, hydroxypropyl methylcellulose, hydroxyethylcellulose, sodium alginate patel g.m. et al (2010) a comparison study between batches formulated with different combination of polymers. in addition, this study included an effervescent feature in the formulation to enhance the swelling of the tablets. hydroxypropyl methylcellulose, carbopol, sodium carboxymethylcellulose, chitosan, methyl cellulose, sodium alginate table iii (continued) abidin et al drug target insights 2023; 17: 13 © 2023 the authors. published by aboutscience www.aboutscience.eu author study background polymers used pendekal et al (2013) this study investigated spontaneous interpolymer complexes between chn and alginate. it is thought to be a newer and efficient form of polymeric carriers and will make the tablet more versatile in its application. chitosan, alginate pendekal et al (2012) this study investigated spontaneous interpolymer complexes between chn and cp. it is thought to be a newer and efficient form of polymeric carriers and will make the tablet more versatile in its application. chitosan, carbopol perioli et al (2009) a comparison study between batches that includes different type of polymers blended in different ratios. chitosan, polyvinyl pyrrolidone, polycarbophil perioli et al (2011) a comparison study between batches using mucoadhesive polymers alone and in combinations. hydroxypropyl methylcellulose, carbopol sánchez et al (2017) designed a two-layered vaginal tablet. a comparison study between batches formulated using mixtures with different polymeric ratios. sodium carboxymethylcellulose, carbopol, chitosan szymańska et al (2014) this study formulated vaginal tablets using chn as a matrix and investigated two different mucosa surrogate as simple adhesive models. chitosan, silicified microcrystalline cellulose, sodium stearyl fumarate tunpanich et al (2019) a comparison study between batches formulated using different types of polymers and their blends in different ratios. polycarbophil, hydroxypropyl methylcellulose valenta et al (2001) a comparison study between batches formulated using non-thiolated and thiolated chn. the effect of varied amount of tga on the thiomer was investigated. the study included modification steps and structure characterization methods. chitosan, chitosan-thioglycolic acid (thiolated) with synthetics ones. in addition, there were some articles that compared different batches that were made using solutions with different ph (13), some with different modification of the drug compound itself instead of the polymer (24). on the other hand, studies with polymer intervention involved modification to a selected mucoadhesive polymer into thiomers. thiomers (thiolated polymers) are currently thought to be a new generation of mucoadhesive polymers, as they have thiol group side chains that can form interand/or intrachain disulfide bonds and improve the cohesive property of a formulation (25). therefore, these studies include tablet evaluations comparing between batches formulated using non-thiolated and thiolated polymers. risk of bias in studies the risk of bias from the reviewed articles is shown in table iv. the risk of bias is indicated by symbols; positive “+” indicates that there was risk of bias and negative “–” indicates that there were no/less risk of bias. there was no risk of bias in selection and sampling as this was not applicable to the study design of all the reviewed articles. performance bias refers to the improved results when a control goes through intervention. this was seen in articles with polymer intervention as formulation using manipulated polymers performed better compared to formulations with non-manipulated polymer. therefore, there was some risk of bias mostly in articles that included polymer interventions. all reviewed articles were thought to have low risk of both attrition and reporting bias as there were no incomplete outcome data and there were reports on both good and bad batches in the articles, respectively. some performance assessments were done using self-constructed or modified apparatus setup, thus increasing the risk of measurement bias, as compared to if they were to use a standardized and calibrated apparatus/equipment. results in all the articles were expressed as mean values ± standard deviation and are indicated in the text. however, some studies conducted further statistical analysis to establish significant differences between groups (formulated batches) compared. these studies have reduced their risk of analysis bias. discussion mucoadhesive drug delivery systems there has been extensive research done to circumvent the limitations of discomfort (i.e., messiness, leakage), short stay, and frequent dosing due to insufficient therapeutic dosage of vaginal formulations (3,14,15). innovative and novel attempts have been made to develop vaginal formulations that consider both the clinical as well as the user’s requirements. mucoadhesive drug delivery systems exploit the useful property of mucoadhesion of certain biopolymers on interaction with mucus that is present at the targeted physiological sites, for example, the vaginal mucosa (24), though in the absence of goblet cells and the lack of direct release of mucin, the vaginal epithelium is still considered as a mucosal surface (24). mucin is a glycoprotein that makes up the most part of mucus on mucosal surfaces. mucin exhibits electrostatic, hydrophobic, and coupling effect, which makes it possible for good adherence of a number of substances to the vaginal mucosa (5). mucoadhesive polymers that bind to mucin or epithelial surfaces increase the residence time of the dosage form at the action or absorption site, and thus could be useful in solving bioavailability problems resulting from a too short stay of the pharmaceutical dosage form at the absorption site (26). mucoadhesive vaginal tablet testing14 © 2023 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti table iv bias assessment for each research article reviewed author sampling bias performance bias attrition bias reporting bias measurement bias bias in analysis abidin et al (2020) – – – – – – abu el-enin et al (2020) – – – – – – baki et al (2009) – – – – – + baloglu et al (2011) – + – – + – bartkowiak et al (2018) – – – – – + bhat et al (2010) – – – – + – cazorla-luna et al (2019) – – – – – – cevher et al (2014) – – – – – – cevher et al (2008) – – – – – – el-kamel et al (2002) – – – – + + fitaihi et al (2017) – – – – – – gupta et al (2013) – – – – + – gök et al (2017) – + – – – + hani et al (2016) – – – – + + hassan et al (2017) – – – – + – hombach et al (2009) – + – – – – kailasam et al (2010) – – – – + + kast et al (2002) – + – – – – khan et al (2017) – – – – + + khan et al (2014) – – – – + + lupo et al (2017) – + – – – – notario-pérez et al (2019) – – – – – – anotario-pérez et al (2017) – – – – – – bnotario-pérez et al (2017) – – – – – + nowak et al (2015) – + – – + + pacheco-quito et al (2020) – – – – – – paczkowska et al (2020) – – – – – + palade et al (2013) – – – – – – patel a. et al (2012) – + – – + – patel a. et al (2011) – + – – + + patel g.m. et al (2010) – + – – + – pendekal et al (2013) – – – – + – pendekal et al (2012) – – – – + + perioli et al (2009) – – – – + + perioli et al (2011) – – – – + + sánchez et al (2017) – – – – + + szymańska et al (2014) – – – – – – tunpanich et al (2019) – – – – – – valenta et al (2001) – + – – + + abidin et al drug target insights 2023; 17: 15 © 2023 the authors. published by aboutscience www.aboutscience.eu the inclusion of mucoadhesive polymers into vaginal formulations intensifies the contact between formulation and the vaginal mucosa (27,28). mucoadhesion happens in two stages. suitable parameters such as wettability, swelling, and hydration of the polymer can ensure close contact between polymer and mucosal layer. this establishment of contact is the first stage of mucoadhesion. any material can adhere to the mucosa thanks to its viscous nature, but there can be no real adhesion without an interrelation between some specific chemical groups in the polymers and biological tissues, or without establishing an interpenetration of chains (29). therefore, the second stage of mucoadhesion involves the activation of the polymer in the presence of moisture (hydration), including wetting and swelling of the formulation. the moisture plasticizes the system, which allows the release of mucoadhesive particles and their connection with the mucin by forming van der waals or hydrogen bonds (5). this then facilitates intimate contact between the formulation and the underlying absorptive surface (14). the main purpose of mucoadhesive drug delivery system is to remain fixed (localization) at the point where the drug’s release and/or absorption can occur (29). this is the great advantage as it prolongs the residence time at the targeted site of application (3,27,30). hence, the drug’s uptake and bioavailability may be increased, frequency of dosing reduced, and patient compliance improved (24,31). apart from prolongation of drug release at the site of absorption, drug targeting to the affected site can also be realized (24). therefore, the polymers used in these mucoadhesive formulations must be able to adhere to the vaginal mucosa and modulate the drug release from the dosage form. mucoadhesive polymers should ideally be biocompatible, biodegradable, and non-toxic (11). commonly employed mucoadhesive polymers can be derived either from synthetic or natural sources (24). additionally, after hydration, these polymers form hydrophilic matrices that can often be used to produce controlled/sustained-release formulations through their hydration, swelling, and/or erosion (28). properly designed mucoadhesive vaginal tablets should be able to hydrate and gel very slowly, leading to a prolonged release of the drug to provide a long-term therapeutic effect with improved efficacy, reduced frequency of administration, and minimized drug side effect (32,33). vaginal tablets as mentioned, vaginal dosage forms have been developed and used clinically for many years to deliver gynecological drugs and/or for local therapy of female-related conditions. as shown in figure 2, the vaginal tablets formulated in all the research articles reviewed is a form of local therapy for female-related conditions. it has been reported that vaginal infections affect nearly 75% of adult women at least once in their lifetime (13). therefore, it reflects on the research (tab. ii) that has been done with 56% formulated vaginal tablets to treat vaginal infections, including formulations that are antibacterial, antifungal, and antimycotic. there are increasing attention and more recent developments to tackle other female-related conditions such as hiv preventions (15%), vaginal health and management such as hormone therapy (10%), cervical cancer (8%), and preterm labor (3%). about 8% of the research articles reviewed did not specify the application for the vaginal tablet formulated because their research focuses on developing the roles of the carrier in their mucoadhesive vaginal tablets regardless of its therapeutic purpose. compared to semi-solid systems, solid formulations have the advantage of high dose accuracy and long-term stability (29). tablets are one of the best means of drug delivery because they are uncomplicated to formulate, and have the feasibility for mass industrial production at a low cost (11,34). tablets are usually prepared by direct compression, which is an easy, rapid, and cheap method (35). similar to tablets intended for other administrative routes, vaginal tablets also have advantages including portability, avoidance or antimicrobial agents for preservation, and ease of storage and handling (11,18). furthermore, the application is convenient, with no applicators or supervision needed, giving the user an added advantage of discretion (18,34). tablets are a recurring trend because they permit controlling effects on the drug dissolution. recently, considerable attention has been paid on novel and controlled release systems to provide a long-term therapeutic concentration of drug following a single administration (36). there has been major advancement in the development of vaginal tablets that increase vaginal residence time and are capable of delivering the active agent for an extended period at a predictable rate using mucoadhesive polymers (3,27,32). it can be an appropriate therapeutic strategy as the required quantity of the drug can be readily administered; with the prolonged residence time, high drug levels at the target site are achievable, simultaneously minimizing unnecessary drug exposure and side effect in other parts of the body (26,32). mucoadhesive vaginal tablets are relatively easy to insert and do not cause leakages (37). a controlled drug release can be achieved over several hours, if the delivery system does not disintegrate too early (38). in general, the tablet softens and adheres to the vaginal mucosa and is retained in position until dissolution fig. 2 overview of the application for the mucoadhesive vaginal tablets formulated in the 39 research articles reviewed. mucoadhesive vaginal tablet testing16 © 2023 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti and/or release is complete. they are designed to melt in the vaginal cavity and release the drug for several hours (3). a controlled drug release can be achieved over several hours if the formulation does not disintegrate too early (38). after a short time, the presence of tablet is reported to be no longer noticeable to the patient (3). referring to the tablet shapes and designs from the reviewed studies, 80% (31 research articles) are flat-faced cylindrical-shaped tablets, compared to a few (3 research articles) that used an ovoid, concave, and capsule shape and 5 research articles that did not specify the shape of the tablet (tab. ii). however, in these studies the research articles did mention using a disc when testing for the mucoadhesive property of the formulations. the diameters of the tablets range from 15 to 5 mm; 13 mm is the most popular diameter (11 research articles). however, recent research has started using smaller sized tablets. tablets kept at a smaller size offer a more efficient drug release ability, reduce the risk of an inhomogeneous blend or non-uniform drug content in finished tablets, and can also improve patient comfort and thus compliance (18). strategies in formulating mucoadhesive vaginal tablets the strategies in designing a mucoadhesive vaginal tablet adopted by the reviewed articles are divided into six main categories (fig. 3). the strategies used are listed as follows: (i) polymer blends (46%), (ii) thiomers (18%), (iii) intermolecular complexes (18%), (iv) physical design (10%), (v) micro-particulate technology (5%), and (vi) formulation technique (3%). the percentages are the number of research articles that used the design strategy respectively. the categories were decided by the reviewers based on the theoretical application/theme used in designing the tablets. as the advancement of mucoadhesive drug system continues, there is a vast number of novel strategies that are being invented and it can get quite competitive. therefore, this review focuses greatly on the assessment methods that are conducted by the research articles reviewed, to evaluate the degree of mucoadhesion for vaginal tablet formulation. physical evaluations of tablets/physicochemical properties tablet formulations are evaluated to comply to specific pharmacopoeia requirements, including precompression (e.g. tabletability, flowability), tablet uniformity, friability, disintegration time, tablet ejection force, and dissolution performance and any other relevant evaluations (18). if any of these requirements are not met, the formulation should be excluded and new formulation is designed and re-evaluated to address identified deficiencies (18). precompression evaluation investigates the powdered mixtures’ compressibility profile or matrix granules before compression. the flowability determines the mechanical behavior of the powders, which can affect the tablets’ weight, hardness, and content uniformity (39). for example, powder flowability can determine the ease with which the powder can be fed into the die and non-flowing powders can also cause the non-uniformity incorporation of drug compounds in the powders (40). therefore, flow properties of pharmaceutical powders are critical to manufacturing. there are various methods that can be used as an indication of powder flowability, including measurement of angle of repose, bulk density, tapped density, and carr’s compressibility index (ci) or hausner ratio (hr) (39). angle of repose (°) is the steepest slope of the unconfined material, measured from a horizontal plane (41). repose angles are reported within the range of 30°–55°. it is described that less than 30° are powders that has high flowability and more than 55° are non-flowing powders (41). bulk and tapped densities are commonly reported together, as it measures the volume occupied by the powder (of known mass), g/ml, in a graduated cylinder mounted onto a tapping platform. the volume occupied before tapping is reported as the bulk density and after a range of 250–1,000 taps, the volume obtained is reported as the tapped density (18). both these densities are used to calculate ci and hr to characterize flowability. ci uses a percentage scale of 0–100, where 0–10% indicates excellent flowability. hr has a range of 1.00–1.50, where 1.00–1.11 indicates excellent flowability (18,40). poor flowability of powders are indicated with >31 ci and >1.60 hr. any value in between can describe the powder’s flowability as good, fair, and passable. these evaluations are to some extent interrelated to one another and help to describe the particles’ flow and how readily the material undergoes a change in volume when compressed (18,32). good compressibility, on the other hand, describes a material capable of achieving desired tablet hardness at low compression pressure. it is important to remember that any unnecessary increase in compression pressure can induce physical changes upon the compressed material (37). therefore, careful selection of optimal compression pressure to be employed in manufacturing the tablets is important. a pressure will not physically damage the material and can produce tablets with good tensile strength. figure 4 shows the list of the types of tablet evaluations that have been conducted by the research articles reviewed (tab. v). about 54% and 44% of the research articles conducted the hardness and friability test, respectively. these are considered fundamental evaluations as it ensures that the formulations have satisfactory tablet strength to withstand fig. 3 the main categories of the strategies adopted in the research articles reviewed in designing their mucoadhesive vaginal tablets. abidin et al drug target insights 2023; 17: 17 © 2023 the authors. published by aboutscience www.aboutscience.eu fig. 4 this bar chart shows the type and frequency of the tablet evaluations conducted within the research articles reviewed. table v physicochemical evaluations conducted by each research article reviewed author precompression uniformity measurement durability measurement weight height/ thickness diameter drug content hardness friability abidin et al (2020) √ √ √ √ √ √ √ abu el-enin et al (2020) – √ – – √ √ √ baki et al (2009) √ – √ √ – – √ baloglu et al (2011) – √ √ √ – √ √ bartkowiak et al (2018) – – – – – – – bhat et al (2010) – √ – – √ √ √ cazorla-luna et al (2019) – √ √ √ – √ – cevher et al (2014) – √ – – – √ √ cevher et al (2008) – – – – – – – el-kamel et al (2002) – – – – √ – – fitaihi et al (2017) √ √ √ – √ √ √ gupta et al (2013) – √ √ – √ √ √ gök et al (2017) – – – – – – – hani et al (2016) – √ √ – √ √ √ hassan et al (2017) – √ √ – √ √ √ hombach et al (2009) – – – – – – – kailasam et al (2010) – – – – – – – kast et al (2002) – – – – – – – khan et al (2017) √ √ √ √ – √ √ khan et al (2014) √ √ √ √ – √ √ lupo et al (2017) – – – – – – – notario-pérez et al (2019) – – – – – – – anotario-pérez et al (2017) – – – – – – – bnotario-pérez et al (2017) – – – – – – – (continued) mucoadhesive vaginal tablet testing18 © 2023 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti abrasion or chipping during packaging, handling, and shipping (18,26). evaluations of tablet’s hardness typically involve crushing actions of individual tablets and the crushing force used can be reported in newtons (n) and/or kilograms (kg). the crushing force reported serves as an information to avoid crushing the tablets during manufacturing and packaging processes. friability evaluation involves subjecting tablets in circular motions and the result is reported as the weight difference of the tablets before and after the test, in percentage. weight loss of below 1% is ideal. the friability test can help indicate the brittleness of the tablets, which helps to prevent chipping and abrasion of the tablets (18). weight, thickness/height, diameter, and drug content measurements are evaluations of uniformity in the tablets manufactured. the evaluations of tablet uniformity are typically reported as an average and standard deviation. weight, thickness/height, and diameter of any individual tablets should not deviate more than 5% from the average measurement. drug content within 95–105% from the expected content is considered acceptable (18). the tablet uniformity is important as it is an indication of a consistent tablet formulation and manufacturing process (26). furthermore, uniformed tablets can also contribute to consistent tablet performance. swelling assessment mucoadhesive drug delivery systems require mucoadhesive polymers that can adhere to the vaginal mucosa and swell rapidly in aqueous environmental conditions (36). some studies have suggested that tablet swelling is an important parameter to be studied before considering mucoadhesion (35). the swelling characteristic of a polymer contributes to their adhesive capacity and in order to manifest maximum adhesive strength, an optimum water uptake (hydration) is needed for the polymer particles (26). however, if the hydration is too high, the adhesion property is expected to be reduced due to the competition between water molecules and the active groups in the mucin chains of the vaginal mucosa to bind to the functional groups of the polymer (18). therefore, the swelling property (degree of hydration) should be investigated and considered when designing the mucoadhesive vaginal tablet formulations. about 90% of the vaginal tablets in the research articles reviewed were subjected to swelling tests and the results were used to consider the tablet size and improve drug retention and drug release kinetics. the swelling characteristic is typically done by subjecting the tablet to hydration in an aqueous solvent used in dissolution or disintegration studies. the degree of hydration may be reported using units, including swelling index (si), swelling ratio (sr/q), water uptake ratio (wur), mass swelling factor (msf), swelling degree (q e ), water uptake (wu), swelling percentage, hydration percentage, matrix erosion (me), and matric dissolution (ds). although there are various units to report the numerical data of swelling, all of these measure the degree of hydration by the change in weight that occurs in the tablet formulations after hydration. therefore, it is essential to record the weight of individual tablets prior to hydration. positive values indicate that the percentage of swelling or weight gain is greater than the initial weight of the dry formulation, while negative values indicate the weight is less than the dry formulations and this can be due to erosion or dissolution of the system in the solvent (11). from the research articles reviewed, the swelling test was done using three different methods: (i) immersion (49%), (ii) gravimetric (31%), and (iii) water absorbing method (10%) (tab. vi). the different method describes the different setting author precompression uniformity measurement durability measurement weight height/ thickness diameter drug content hardness friability nowak et al (2015) – – – – – – – pacheco-quito et al (2020) – √ √ √ – – – paczkowska et al (2020) – √ √ √ – √ – palade et al (2013) – – – – – – – patel a. et al (2012) – √ √ – – √ – patel a. et al (2011) – – – – – √ √ patel g.m. et al (2010) – √ √ – – √ – pendekal et al (2013) – – – – – – – pendekal et al (2012) – – – – – – – perioli et al (2009) – – √ – – √ √ perioli et al (2011) – – √ – – √ √ sánchez et al (2017) – √ √ √ – √ – szymańska et al (2014) – √ √ √ √ √ √ tunpanich et al (2019) √ √ √ – √ √ √ valenta et al (2001) – – – – – – – table v (continued) abidin et al drug target insights 2023; 17: 19 © 2023 the authors. published by aboutscience www.aboutscience.eu table vi swelling assessment conducted by each research article reviewed author method shaking motion incubation temperature (°c) reporting unit swelling witness testimmersion gravimetric water absorbing abidin et al (2020) √ – 37 ± 1 swelling index (si) – abu el-enin et al (2020) √ – ambient swelling index (si) – baki et al (2009) – – – – 37 ± 1 – – baloglu et al (2011) √ – 37 ± 1 water uptake ratio (wur) – bartkowiak et al (2018) √ – 37 ± 1 mass swelling factor (msf) – bhat et al (2010) √ – 37 ± 1 swelling index (si) – cazorla-luna et al (2019) √ √ 37 ± 1 swelling ratio (sr/q) √ cevher et al (2014) √ – 37 ± 1 swelling index (si) – cevher et al (2008) √ – 37 ± 1 swelling index and matrix erosion (me) – el-kamel et al (2002) √ – 37 ± 1 swelling index (si) – fitaihi et al (2017) √ – 37 ± 1 swelling percentage (%s) – gupta et al (2013) √ – 37 ± 1 reweighed – gök et al (2017) √ – 37 ± 1 swelling degree (qe) – hani et al (2016) √ – 37 ± 1 percentage of hydration – hassan et al (2017) √ – 37 ± 1 swelling index (si) – hombach et al (2009) √ – 37 ± 1 reweighed – kailasam et al (2010) √ – 37 ± 1 swelling index (si) – kast et al (2002) √ – 37 ± 1 reweighed – khan et al (2017) √ – ambient swelling index (si) – khan et al (2014) √ – 37 ± 1 swelling index (si) – lupo et al (2017) √ – 37 ± 1 water uptake (wu) – notario-pérez et al (2019) √ √ 37 ± 1 swelling ratio (sr) – anotario-pérez et al (2017) √ √ 37 ± 1 swelling ratio (sr) √ bnotario-pérez et al (2017) √ √ 37 ± 1 swelling ratio (sr) √ nowak et al (2015) √ – 37 ± 1 water uptake percentage – pacheco-quito et al (2020) √ √ 37 ± 1 swelling ratio (sr) √ paczkowska et al (2020) – – – – 37 ± 1 – – palade et al (2013) – – – – 37 ± 1 – – patel a. et al (2012) √ – 37 ± 1 swelling percentage (%s) – patel a. et al (2011) √ – 37 ± 1 swelling percentage (%s) – patel g.m. et al (2010) √ – 37 ± 1 swelling percentage (%s) – pendekal et al (2013) √ – 37 ± 1 swelling index (si) – pendekal et al (2012) √ – 37 ± 1 swelling index (si) – perioli et al (2009) √ – 37 ± 1 hydration percentage and matrix erosion (me) – perioli et al (2011) √ √ 37 ± 1 hydration percentage and matrix erosion (me) – sánchez et al (2017) √ – 37 ± 1 hydration percentage and matrix dissolution (ds) – szymańska et al (2014) √ – 37 ± 1 swelling index (si) – tunpanich et al (2019) √ – 37 ± 1 swelling index (si) and matrix erosion (me) – valenta et al (2001) – – – – 37 ± 1 – – mucoadhesive vaginal tablet testing20 © 2023 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti in hydrating the tablets. the immersion method involves total submergence of the tablet into a solvent. gravimetric method involves fixing the tablet to a needle and suspending it into a solvent. this method is also called the tea bag method in some research articles. there was no specific volume; from the research articles reviewed there was a range of volume from 5 ml up to 1000 ml of solvent used for these methods. in the third method, individual vaginal tablet is placed on 2% agar gel plates to assess the water absorbing capacity of each tablet. most of these swelling methods were conducted in incubators or water bath, kept at 37 ± 1°c, and some remained stationary or was put in a shaking motion. two research articles reported to conduct the swelling test in ambient temperature (14,42). individual tablets are taken out at scheduled time intervals and excess surface water was carefully removed. the swollen tablets were then reweighed for the swelling assessment. proper swelling characteristic will contribute to effective mucoadhesion and controlled release of the drug (14). the polymer will start to swell upon hydration, and a viscous gel layer should start to form around the tablet core (26). as it reaches the maximum swelling capacity, erosion occurs until complete dissolving or erosion of tablets (14). the formation of this gel layer has been regarded as an essential first step that would govern the drug release from the vaginal tablet formulation (26). swelling witness test in the past few years, researchers have started to include a complementary technique using prepared swelling witnesses to better understand the swelling behavior of the mucoadhesive polymers used and structural characterization of the tablets (16,43). it is a method that evaluates the tablet’s capacity to absorb water or determine the entry pattern of the solvent into the tablet during the hydration process (16). the mobility (movement) of the solvent within the tablet formulation controls the swelling and erosion of the tablets, thus it plays a role in the drug release as well (11). tablet formulations were left to hydrate and swell in a chosen solvent in accordance with predetermined time and/or until it reaches the maximum swelling capacity. the swollen tablets were then immediately lyophilized (freezedried). water is removed during freeze-drying, and the space that was originally occupied by the solvent is transformed into pores (hollow gaps), obtaining porous structures similar to a sponge, that are now called swelling witnesses (16,43). the swelling witnesses can then be observed and analyzed by field emission scanning electron microscopy (sem) (11,16). corresponding sem micrographs shows the witness’ microstructures which vary depending on the type of polymer and different formulations. mercury porosimetry can be used to determine the pore size distributions (psds) of the witnesses, which reflects the pores (gaps/hollows) that were occupied by the solvent before the freeze-drying process (16). the gaps inside the polymers are related to the swelling ratio, as the gaps get larger the higher its ability to capture water, and the tablet formulation swells more (43). polymers with good swelling capacity will produce a narrow psd (the pores are closer together) with high pore sizes. in contrast, polymers with minimal swelling properties produce a wider psd (the pores are further apart) and smaller pore sizes. the wider psds, however, do have an appealing advantage, where it will help the tablet to maintain the shape while the drug diffuses slowly between them (43). furthermore, formulations capturing less water are expected to be more comfortable for the user. there were four research articles that included the swelling witness in their research, and they have reported varying microstructures in their witnesses depending on the nature of their polymers and the changes in the swelling witness in different formulations (11,16,29,43). when a solvent enters the polymer during swelling, it creates different microstructures depending on the nature of the polymer. figure 5 is a compilation of sem micrographs from the research articles that depict the most distinctive microstructures corresponding to the type of drug release that it can achieve. figure 5a shows a channeled microstructure (elongated channels) allowing gradual uptake of the surrounding solvent which translates into a moderate swelling behavior (29). the moderate swelling can help maintain the shape of the tablet while the drug diffuses slowly between them (43). figure 5b shows a spongelike microstructure with numerous pores, which the solvent can circulate with some difficulty, which would also result in moderate swelling capacity of the formulation (11). figure 5c shows a microstructure arranged in parallel sheets with the absorbed solvent between them (29). formulations using this type of polymer will swell the most and can remain swollen the longest, as there is a high capacity for very effectively retaining water between the sheets. however, although the water cannot escape, the drug is able to diffuse through the polymer sheets, thus reducing their ability to retain the drug longer (43). figure 5d is included to show one of the examples when a formulation was unable to swell (29). this formulation fig. 5 electron microscopy micrographs of distinct swollen witness in different polymers compiled from four research articles reviewed. the different panels show the different type of microstructures observed: a) channeled microstructure (250 μm) (29); b) sponge-like microstructure (1 mm) (11); c) parallel sheets microstructure (250 μm) (29); and d) a material that is unable to swell (250 μm) (29). (pictures are reproduced from references (11) and (29) under the creative common attributions license.) abidin et al drug target insights 2023; 17: 21 © 2023 the authors. published by aboutscience www.aboutscience.eu showed a grainy microstructure with different-sized particles and was reported to have failed in controlling drug delivery (43). these images are compiled from research articles reviewed and reproduced from references (11) and (29) under the creative common attributions license. this assessment helps to identify the effects or modification in the microstructure arrangement, wu process, and swelling capability, when different polymers are mixed and/or drugs are incorporated into the formulation. the presence of other materials that do not swell can make it difficult for the polymer to swell as usual. in some cases, materials that do not swell can clog the pores, thus reducing the sizes of the pores or hindering the drug diffusion through the polymer (43). therefore, wu rates are reduced and can be the cause for slower drug release from the tablet formulations (16). although the key criteria for selecting the optimum formulation are control over the drug release and mucoadhesion to the vaginal mucosa, the amount of swelling must be taken into account in formulations that can achieve these criteria but capturing less water, as it will be more comfortable (11). drug release assessment the terms “drug dissolution” and “drug release” are not synonyms, although they are often not appropriately distinguished. drug dissolution refers to the process of diffusion of the drug into the solvent. unless the drug dissolution process is the factor that can manipulate the release of the drug, the drug dissolution and drug release can be considered synonyms. in all other cases, the drug release is the more appropriate term. upon contact with the dissolution solvent, water penetrates the tablet formulation and can dissolve the drug content. the dissolved drug substance subsequently diffuses from the tablet due to the concentration gradient. additionally, the tablet formulation might also undergo several changes including swelling and consequent dissolution in the solvent, all contributing to the overall drug release process (44). assessing drug dissolution/release from the tablet formulation is extremely important within the absorption process to be effective and it is indicative of the tablet’s potential in vivo performance and clinical applications (44,45). ideally, 100% of drug dissolution is targeted in all formulations as it can increase bioavailability and the efficacy of the formulation. however, it depends on the objective of the therapy to have an immediate or controlled/extended drug release. a constant drug release rate over the targeted time also contributes to the formulation performance (18). the in vitro dissolution/release test represents an important tool for this purpose as it can be used to assess the dissolution and release profile of the formulation in an artificial vaginal environment (45). drug releases are primarily recorded at predetermined time intervals to observe the difference in drug concentration over time. it has become an important tool in the drug product development phase and its quality control and the regulatory approval process (44). for many types of mucosal formulations including vaginal tablets, drug testing is performed using the apparatus developed for oral formulations (44). today four apparatuses for dissolution testing of solid dosage forms are described in pharmacopoeias: paddle apparatus, basket apparatus, reciprocating cylinder, and flow through cell. from the research articles reviewed (tab. vii), 46% have used the paddle table vii drug release assessment conducted by each research article reviewed author novel setup/ other dissolution medium ph volume (ml) sink conditions rpm usp paddle usp basket shaking water bath still water bath disintegration test abidin et al (2020) 2% sodium dodecyl sulfate aqueous solution 4.2 900 √ 100 √ x abu el-enin et al (2020) simulated vaginal fluid 4.5 100 √ 50 √ x baki et al (2009) phosphate buffer 4.6 4 x x √ x baloglu et al (2011) √ simulated vaginal fluid x 6 x √ bartkowiak et al (2018) simulated vaginal fluid 5.8 500 √ 50 √ x bhat et al (2010) 100 mm acetate buffer 6 500 √ 25 √ x cazorla-luna et al (2019) simulated vaginal fluid x 80 √ 15 √ x cevher et al (2014) lactate buffer 5 250 x 75 √ x cevher et al (2008) lactate buffer 5 500 √ 75 √ x el-kamel et al (2002) citrate buffer 5.5 650 √ 25 √ x fitaihi et al (2017) mcilvaine’s citrate buffer 4.8 250 √ 50 √ x (continued) mucoadhesive vaginal tablet testing22 © 2023 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti author novel setup/ other dissolution medium ph volume (ml) sink conditions rpm usp paddle usp basket shaking water bath still water bath disintegration test gupta et al (2013) citrate phosphate buffer 4.5 600 √ 100 √ x gök et al (2017) lactate buffer x 500 x 75 √ x hani et al (2016) simulated vaginal fluid x 900 √ 100 √ x hassan et al (2017) citrate buffer 4.5 900 √ 50 √ x hombach et al (2009) simulated vaginal fluid 4.2 500 √ 20 √ x kailasam et al (2010) 1 m phosphate buffer 4.0 x x 50 √ x kast et al (2002) 100 mm acetate buffer 6.0 4 √ 100 √ √ khan et al (2017) simulated vaginal fluid 4.2 100 √ 50 √ x khan et al (2014) simulated vaginal fluid 4.2 100 √ 50 √ x lupo et al (2017) √ simulated vaginal fluid 4.2 6 √ 630 √ notario-pérez et al (2019) simulated vaginal fluid x 80 √ 15 √ x anotario-pérez et al (2017) simulated vaginal fluid x 80 x 15 √ x bnotario-pérez et al (2017) simulated vaginal fluid x 80 √ 15 √ x nowak et al (2015 – – – – – – – – – √ pacheco-quito et al (2020) simulated vaginal fluid x 80 √ 15 √ x paczkowska et al (2020) phosphate buffer 4.5 150 √ 50 √ x palade et al (2013) acetate buffer 4.2 900 √ 60 √ x patel a. et al (2012) phosphate buffer 4.0 500 √ 30 √ x patel a. et al (2011) phosphate buffer 4.0 500 √ 30 √ x patel g.m. et al (2010) citrate buffer 4.0 500 √ 50 √ x pendekal et al (2013) simulated vaginal fluid 4.2 500 √ 50 √ x pendekal et al (2012) simulated vaginal fluid 4.2 500 √ 50 √ x perioli et al (2009) simulated vaginal fluid x 900 √ 100 √ x perioli et al (2011) simulated vaginal fluid x x √ 100 √ x sánchez et al (2017) simulated vaginal fluid 5.5 600 √ 100 √ √ szymańska et al (2014) 0.08 m acetic buffer 4.5 900 √ 75 √ x tunpanich et al (2019) 1% sodium dodecyl sulfate aqueous solution 5.5 900 √ 75 √ x valenta et al (2001) 100 mm phosphate buffer 6.0 20 √ 100 √ x *nb: x = did not specify; “–“ = was not conducted. table vii (continued) abidin et al drug target insights 2023; 17: 23 © 2023 the authors. published by aboutscience www.aboutscience.eu apparatus and 26% used the basket method, and others have used alternative dissolution apparatus. most methods used are designed to mimic the general conditions encountered in the physiological environment of the vagina, including the ph of the dissolution solvent and maintaining a temperature of 37 ± 1°c during testing. a total of 72% from the reviewed studies have specified the ph of the dissolution medium used and the mean ph was 4.7, which is within the range of the ph of the vaginal fluid (ph 4–5) (46,47). although it is acceptable for research articles to use the apparatus mentioned, it should be pointed out that these methods utilize a large volume of dissolution solvent and the rotational movement (36,44). these are generally far from the real in vivo conditions at the vaginal mucosa, thus the method used would not have given an accurate information. some articles have taken the initiative to use smaller-volume apparatus, and some have designed a novel drug release technique that considers the correct amount of vaginal fluid and its turnover in the vaginal lumen. as reviewed (tab. vii), 59% of the studies used larger dissolution solvent volumes ranging from 150 to 900 ml; 23% have tried reducing the volume, using 20–100 ml of dissolution solvent; however, even these volumes are significantly higher than those available to the mucoadhesive vaginal tablet on the vaginal mucosa (44). the daily production of vaginal fluid is approximately 6 ml and 0.5–0.75 ml is continually present in the vagina (36). only 10% of the studies have considered using the correct estimated volumes ranging from 4 to 7 ml to closely mimic the in vivo conditions for vaginal administration. as in the case of the rotational movement, there is no specification. most studies have chosen a setting of revolutions per minute (rpm) that is appropriate only to their tablet formulations. however, 100 and 50 rpm are commonly used. the rpm used in the research articles reviewed ranges from 15 to 100 rpm and one study (48) used an astounding 630 rpm as its dissolution technique employs a thermomixer. even though some methods do not represent the correct vaginal environment, the results of these evaluations can still contribute to preliminary findings of the formulation. an increasing number of research have modified and designed drug release techniques that can better simulate the specific conditions of the vaginal environment for mucoadhesive vaginal tablets. various parameters must be considered when designing a dissolution apparatus, including selection of apparatus, volume and composition of the dissolution medium, environmental conditions of the absorption site (e.g., agitation), and surface exposure of the tablet (36,44). however, as new technique emerges, there is a need now to further validate and standardize the methods developed (44). for further advancement of drug release techniques, more physiological vaginal conditions should be considered. for instance, different conditions occur during menstrual cycle or at different ages, along with the enzymatic activity of the vaginal microflora. in particular, the ph and composition of vaginal fluid change from low ph values (3.5–4.5) during the ovulation phase to a higher ph during menstruation (49). novel release study method by baloglu et al (36) in this study, a new simple technique mimicking the vaginal environment was developed to investigate the release behavior of the vaginal tablet formulation. the apparatus mainly consists of a perfusor and syringe which are connected with a thin latex connector and a sample collection vessel as illustrated in figure 6. (pictures are reproduced from (36) and have received copyrights permission by the publisher on april 28, 2022.) the syringe used (without needle) has an internal diameter of 20 mm and total length of 75 mm to simulate the vaginal physiology. tablets were placed at the bottom of the syringe and the assembly was dipped into a water at 37 ± 0.5°c. a perfusor was connected to the top of the syringe to supply a total of 6 ml of vaginal fluid to the tablets in 24 hours. the same amount of sample was collected concurrently from the bottom (36). kinetic analysis to understand the mechanism of drug release from mucoadhesive tablets, some research articles have plotted the in vitro drug release data in kinetic equation models. many model-dependent approaches can be used to fig. 6 schematic drawing of the in vitro release studies developed by baloglu et al. (36). (picture was taken from reference (36) and has received copyrights permission by the publisher on april 28, 2022.) mucoadhesive vaginal tablet testing24 © 2023 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti investigate the best-fit parameters (32). the common equation used in most kinetic studies of the research articles reviewed is the korsmeyer-peppas semi-empirical model (eq. [1]), m m k tt kp n ∞ = equation 1 where m t /m ∞ is the fractional amount of drug release at time t, k kp is the release rate constant, and n is the diffusional exponent that characterizes the type of release mechanism used during the dissolution process (26). the values n and k kp were estimated using a linear regression of log (m t /m ∞ ) compared with log t. for the case of cylindrical tablets, n = 0.45 corresponds to a fickian diffusion release (case i diffusional), 0.45 < n < 0.89 to a non-fickian or anomalous diffusion, n = 0.89 to a case-ii transport or typical zero-order release and n > 0.89 to a super case-ii transport (13). most research articles have obtained diffusional exponent that indicates a non-fickian or anomalous diffusion, which involves a combination of both diffusion and erosion mechanism (13,26). there are other types of kinetic release models including higuchi (32), weibull (32), hopfenberg (11), hixson and crowell (11), and moore and flanner (37) that can be used; however, as mentioned the korsmeyer-peppas kinetic model is the most relevant and commonly used. mucoadhesion assessment defining the mucoadhesive characteristic of the vaginal tablet formulation is of great importance to prevent a decrease in the detachment of the tablet from the vaginal mucosa, prolonging the residence time of the formulation at the site of administration (36). as drug diffuses from the gel layer of the mucoadhesive polymer to the mucus and absorbed by the tissue lining the vaginal cavity, it is essential to quantify the interaction between the mucoadhesive formulation and the vaginal mucosal surface (25,36). this is reflected in figure 7, showing only 8% of the research articles did not conduct any mucoadhesion assessment. the main feature of mucoadhesion is the tablet formulation’s attachment strength (tensile strength) to the vaginal mucosa. there are numerous tests proposed and adopted in the research articles reviewed to determine the attachment strength. the test methods reviewed can be divided into two major categories: (i) in vitro/ex vivo methods and (ii) in vivo methods. it can be summarized from the research articles reviewed (fig. 7 and tab. viii) that 33% are exclusive to only one type of in vitro/ex vivo assessment method, 41% have used multiple in vitro/ex vivo methods and 18% have assessed the mucoadhesion strength in combination of both in vitro/ex vivo and in vivo methods. the in vitro/ex vivo method is by far the most common as in vivo mucoadhesive studies are costly, time consuming, and ethically sensitive (3). however, when a study does include in vivo mucoadhesive studies, it would normally only involve assessment of the most optimal vaginal tablet formulation. in vitro/ex vivo mucoadhesive assessment the in vitro/ex vivo methods can be further divided into forced detachment and residence time methods. in conducting these tests, it is recommended to also test blank formulations and compare it to the drug formulations. it can be useful in confirming and establishing the mucoadhesive properties of the chosen polymers or polymer blends (16). it can also be a control to evaluate if the mucoadhesive properties are altered after the addition of the drug substance with the polymers. forced detachment method this in vitro/ex vivo method is based on measuring the force required for destructing the adhesive bond between the vaginal tablet and the vaginal tissue. it involves a physical act of pulling apart the vaginal tablet from the tissue, fig. 7 summary of the type of methods in the research articles reviewed in assessing the mucoadhesive property of their vaginal tablet formulations. abidin et al drug target insights 2023; 17: 25 © 2023 the authors. published by aboutscience www.aboutscience.eu table viii mucoadhesion assessments conducted by each research article reviewed author tensile method to measure force methods to measure over time in vivo ta-xt novel/modified dissolution apparatus wash-off/rinsing method immersion/ submerged abidin et al (2020) √ abu el-enin et al (2020) √ √ √ baki et al (2009) – – – – – – baloglu et al (2011) √ √ bartkowiak et al (2018) – – – – – – bhat et al (2010) √ cazorla-luna et al (2019) √ √ cevher et al (2014) √ cevher et al (2008) √ el-kamel et al (2002) √ fitaihi et al (2017) √ √ gupta et al (2013) √ √ gök et al (2017) √ √ hani et al (2016) √ √ √ hassan et al (2017) √ √ √ hombach et al (2009) √ √ kailasam et al (2010) √ kast et al (2002) √ khan et al (2017) √ √ khan et al (2014) √ √ lupo et al (2017) √ notario-pérez et al (2019) √ anotario-pérez et al (2017) √ bnotario-pérez et al (2017) √ nowak et al (2015) √ √ pacheco-quito et al (2020) √ √ paczkowska et al (2020) √ √ palade et al (2013) – – – – – – patel a. et al (2012) √ √ patel a. et al (2011) √ √ patel g.m. et al (2010) √ √ pendekal et al (2013) √ √ pendekal et al (2012) √ √ perioli et al (2009) √ √ perioli et al (2011) √ √ sánchez et al (2017) √ √ szymańska et al (2014) √ √ tunpanich et al (2019) √ valenta et al (2001) √ mucoadhesive vaginal tablet testing26 © 2023 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti representing complete detachment (32). this can be done using a tensile tester (e.g., ta-xtplus), where the vaginal tissue is fixed onto the lower clamp/plate and the vaginal tablet can be fixed onto the upper clamp/probe of the tensile tester (16). the vaginal tablet should be wetted with the dissolution medium, then both the tablet and tissue are brought into contact with a slight contact force for a minimum of 30 seconds to allow the formation of the adhesive (13). next the tablet and the mucosa were pulled apart at a constant speed until complete detachment and the force applied to pull them apart is recorded (14,23,32). there are a total of 31 research articles that have conducted this type of assessment method; 13 research articles have used a tensile tester and the remaining 18 research articles have devised a simple apparatus or have used other alternatives, however, still in keeping with the basic principle of this assessment method. a simple apparatus called the dynamometer was used to perform the forced detachment test in several studies (30,33,50). some research articles have devised a simple apparatus by modifying an analytical balance to measure the force required to remove the vaginal tablet from the vaginal tissue. an example of this apparatus setup is shown in figure 8. any modifications can be done according to what is available to the researchers. therefore, only a general apparatus setup will be discussed. one arm of the balance is used to hold the vaginal tablet and contact the vaginal tissue fixed on a plank/platform. a pre-load force is used to ensure adhesion bond formation when contact is made. the other arm is used to place the weights that will be added until the tablet is pulled apart from the vaginal tissue by the weights’ gravity (26). in some cases, researchers used addition of water at a constant rate, instead of weights (12,38). the addition of water is stopped when the tablet detaches from the vaginal tissue. the weight required to detach the tablet formulation from the mucosa was noted (26). time measurement test an optimal vaginal tablet formulation must not only be able to have a good drug release control but must also remain adhered to the vaginal mucosa for a similar period of time as the drugs are released to be therapeutically effective (16). a total of 15 research articles in this review have conducted an in vitro/ex vivo test to measure how long the vaginal tablets remain attached to the vaginal mucosa. this assessment was conducted in a few different ways, by (i) total immersion, (ii) modified disintegration apparatus, and (iii) wash-off method. the mucoadhesion time was assessed over time by observation of the samples, including erosion and complete detachment of the tablet from the vaginal mucosa (16). although there are a few different methods the general principle of a detachment test involves first attaching the vaginal tablet to the vaginal tissue, then immersing them in a dissolution solvent. this then is kept at body temperature of 37 ± 1°c in an incubator or a water bath with or without agitation depending on the study. visually, the time taken for erosion and completed detachment of the vaginal tablet is recorded as the mucoadhesion retention time (34). the method by total immersion involves mounting the vaginal tissue onto a glass slide or stainless steel plate using cyanoacrylate glue. a vaginal tablet was wetted and allowed to attach to the vaginal tissue with a slight force (14). the glass slide can then be inserted in a beaker containing the dissolution solvent, at an angle (fig. 9) or vertically with an aid of a clamp. figure 9 shows a schematic diagram of the total immersion method at an angle to measure the mucoadhesion time of a mucoadhesive vaginal tablet formulation. illustration includes the swelling, formation of gel layer, and detachment of the tablet. (picture was taken from pacheco-quito et al (11), figure 8, page 12 of 19 and reproduced under the creative common attribution license.) a study by hani et al (38) glued the vaginal tissue directly on the inner side of the beaker, attached a vaginal tablet on to the tissue, and filled the beaker with dissolution solvent. this assemble was then left in a shaking incubator (38). in vivo mucoadhesive assessment the in vivo mucoadhesive studies include administering the vaginal tablet intravaginally to live healthy animal models, fig. 9 a schematic diagram showing the total immersion method at an angle to measure the mucoadhesion time of a mucoadhesive vaginal tablet formulation. illustration includes the swelling, formation of a gel layer, and detachment of the tablet. (picture was taken from pacheco-quito et al (11), figure 8, page 12 and 19 and reproduced under the creative common attribution license.) fig. 8 a schematic diagram of the modified analytical balance devise to measure the weight required to detach the mucoadhesive vaginal tablet from the vaginal tissue. abidin et al drug target insights 2023; 17: 27 © 2023 the authors. published by aboutscience www.aboutscience.eu typically rats or rabbits. it is primarily applied to evaluate the behavior of vaginal tablets (appearance, physical changes, swelling behavior, and residence time in vagina) (25). the physical status of the tablets can be observed at certain time intervals using various methods including vaginal speculum (25), pictures taken using laparoscopic probe with a camera (27), and x-ray (51), as shown in figure 10. the images illustrate that the vaginal tablet was able to swell, remains intact, and adhered to the vaginal mucosa (51). (pictures are taken from (51), figure 8, page 184 and reproduced under the creative commons license.) from this assessment, the retention time of the tablet formulation can be established in vivo and evidently show that the tablet can remain intact and adhered to the vaginal mucosa for an expected amount of time (27). some research articles expanded the in vivo assessment by pharmacokinetic evaluation of the drug substance present in the blood and plasma of the animal taken at different time intervals (12,14) and histological examinations (12). this can then be correlated with the drug release profile of the tablet formulation. simulated vaginal fluid to optimize the formulations destined for the vaginal site, a reliable in vitro method must be put in place that may better mimic the real biological environment in the vagina, in particular in the presence of vaginal fluids. this can be essential as to better understand the behavior of the formulated tablets in the target site (47). the vaginal fluid itself has become an essential tool to evaluate the mucoadhesive strength of the formulation. it demonstrates the interaction between the vaginal fluid to the formulation. furthermore, it helps to better understand the behavior of the formulated tablets at the target site. therefore, simulated vaginal fluid (svf) is used in many methods (e.g., swelling studies, dissolution studies). vaginal fluid originates from a number of different sources. the fluid is mostly transudate from vaginal and cervical cells and also contains vulvar secretions from sebaceous, sweat, bartholin, and skene glands, cervical mucus, endometrial and oviductal fluids, microorganisms, and their metabolic products (47). because of the limited quantity of human vaginal fluid and its rapid degradation once collected from its source, researchers have developed a svf in order to model the fluid properties originating in the vagina (47). many research articles had referred and modified the svf proposed by owen and katz’s original research (52). a liter of svf is prepared using nacl (3.51 g), koh (1.40 g), ca(oh) 2 (0.222 g), albumin (0.18 g), acetic acid (1.00 g), lactic acid (2.0 g), glycerol (0.16 g), urea (0.4 g), glucose (5.0 g) mixed in 1000 ml water and stirred well until complete dissolution (14). ph of the svf can be adjusted to 4–5 with either hcl or acetic acid according to different research articles. other relevant assessments there are many other assessment methods that were conducted in the research articles reviewed, as reported in table ix. we believe they contribute additional information to strengthen the sense of the formulated tablets in terms of stability and performance. physicochemical interaction studies in this type of study, the compatibility of the drug substance and the excipients are assessed. investigations can be performed using fourier transform infrared spectrometry (ftir) and/or differential scanning calorimetry (dsc) on drugs and excipients separately and in a mixed state. the results from these techniques will provide the degree of compatibility between the drug substance–excipient as well as excipient–excipient (38). it can be concluded that there are no chemical interactions in the mixes tested, if there are no changes in the characteristic peaks obtained in the ir spectra and dsc thermograms of individual substances (18,38). ex vivo permeation study a research article by pendekal et al (51) conducted an ex vivo permeation study that was carried out for the optimized formulation using franz diffusion cell. the tablet was placed in the donor compartment on the sheep mucosa. the mucosal layer is on donor compartment. the receptor fig. 10 x-ray radiographic images of a rabbit’s vaginal cavity after 1 and 8 hours of vaginal tablet administration. the images demonstrate that this vaginal tablet formulation was able to swell, remains intact, and adhered to the vaginal mucosa over the time allowed (51). (pictures are taken from (51), figure 8, page 184 and reproduced under the creative commons license.) mucoadhesive vaginal tablet testing28 © 2023 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti table ix other relevant assessment included in each research article reviewed author dsc ftir nmr size analysis rheology sem abidin et al (2020) √ √ abu el-enin et al (2020) – – – – – – baki et al (2009) – – – – – – baloglu et al (2011) √ bartkowiak et al (2018) – – – – – – bhat et al (2010) √ √ cazorla-luna et al (2019) – – – – – – cevher et al (2014) √ √ cevher et al (2008) √ √ √ √ el-kamel et al (2002) – – – – – – fitaihi et al (2017) – – – – – – gupta et al (2013) √ √ √ √ gök et al (2017) – – – – – – hani et al (2016) √ √ hassan et al (2017) – – – – – – hombach et al (2009) √ √ kailasam et al (2010) – – – – – – kast et al (2002) – – – – – – khan et al (2017) √ khan et al (2014) √ lupo et al (2017) √ √ notario-pérez et al (2019) – – – – – – anotario-pérez et al (2017) – – – – – – bnotario-pérez et al (2017) – – – – – – nowak et al (2015) – – – – – – pacheco-quito et al (2020) √ √ paczkowska et al (2020) – – – – – – palade et al (2013) – – – – – – patel a. et al (2012) – – – – – – patel a. et al (2011) – – – – – – patel g.m. et al (2010) – – – – – – pendekal et al (2013) √ √ pendekal et al (2012) √ √ perioli et al (2009) √ perioli et al (2011) √ sánchez et al (2017) – – – – – – szymańska et al (2014) √ √ tunpanich et al (2019) √ √ valenta et al (2001) √ dsc = differential scanning calorimetry; ftir = fourier transform infrared spectrometry; nmr = nuclear magnetic resonance; sem = scanning electron microscopy. abidin et al drug target insights 2023; 17: 29 © 2023 the authors. published by aboutscience www.aboutscience.eu compartment was filled with a dissolution solvent and the temperature was maintained at 37 ± 0.5°c and 50 rpm. the amount of drug substances permeated through the sheep mucosa was determined by taking sample aliquots from the receptor compartment using a syringe and immediately replacing the same volume of solvent (51). limitations the initial strategy was to exclude any unaccessible articles to reduce the limitation in conducting this review. there were no research articles that were unaccessible. the authors also acknowledge the large number of articles selected for this review. however, it was thought that it helps to map out the tests available in evaluating a mucoadhesive vaginal tablet formulation. conclusions although drug-controlled release profiles, mucoadhesion force, and mucoadhesion residence periods are utilized to determine the optimal formulation, vaginal formulations must be created for women’s convenience, which can enhance patient compliance. because the system must have two unique properties: (i) immobilization and (ii) controlled release characteristics, mucoadhesive drug delivery is quite complex. as a result, the approaches discussed in this study can be used to assess the balance between the two features without sacrificing one. understanding the goals and concepts of each assessment approach can aid researchers in evaluating experimental formulations and obtaining an optimal formulation more rapidly. disclosures funding: funding for this work came from the ait president’s seed fund (psf2017-im). conflict of interest: the authors declare no conflict of interest. author contributions: i.m. and e.j.m. provided relevant references and resources. i.z.a. and g.w.f. compiled, interpreted, and analyzed the data. i.z.a., e.j.m., and i.m. wrote the article. all authors revised the article and gave final approval of the version to be submitted. all authors have read and agreed to the published version of the manuscript. references 1. das neves j, palmeira-de-oliveira r, palmeira-de-oliveira a, rodrigues f, sarmento b. vaginal mucosa and drug delivery. in: khutoryanskiy vv, ed. mucoadhesive materials and drug delivery systems. chichester: john wiley & sons 2014; 99-132. crossref 2. das neves j, notario-pérez f, sarmento b. women-specific routes of administration for drugs: a critical overview. adv drug deliv rev. 2021;176:113865. crossref pubmed 3. de araújo pereira rr, bruschi ml. vaginal mucoadhesive drug delivery systems. drug dev ind pharm. 2012;38(6):643-652. crossref pubmed 4. acartürk f. mucoadhesive vaginal drug delivery systems. recent pat drug deliv formul. 2009;3(3):193-205. crossref pubmed 5. bartkowiak a, rojewska m, hyla k, zembrzuska j, prochaska k. surface and swelling properties of mucoadhesive blends and their ability to release fluconazole in a mucin environment. colloids surf b biointerfaces. 2018;172:586-593. crossref pubmed 6. vermani k, garg s. the scope and potential of vaginal drug delivery. pharm sci technol today. 2000;3(10):359-364. crossref pubmed 7. zhang l, de salvo r, ehret a, young k, trapp s. vulvovaginal candidiasis: a real-world evidence study of the perceived benefits of canesten®. sage open med. 2022;10:20503121221085437. crossref pubmed 8. mccoy cf, spence p, dallal bashi yh, et al. use of simulated vaginal and menstrual fluids to model in vivo discolouration of silicone elastomer vaginal rings. int j pharm x. 2021;3:100081. crossref pubmed 9. chollet ja. efficacy and safety of ultra-low-dose vagifem (10 mcg). patient prefer adherence. 2011;5:571-574. crossref pubmed 10. machado rm, palmeira-de-oliveira a, gaspar c, martinez-deoliveira j, palmeira-de-oliveira r. studies and methodologies on vaginal drug permeation. adv drug deliv rev. 2015;92:1426. crossref pubmed 11. pacheco-quito e-m, ruiz-caro r, rubio j, tamayo a, veiga md. carrageenan-based acyclovir mucoadhesive vaginal tablets for prevention of genital herpes. mar drugs. 2020;18(5):249. crossref pubmed 12. hassan as, soliman gm, ali mf, el-mahdy mm, el-gindy gea. mucoadhesive tablets for the vaginal delivery of progesterone: in vitro evaluation and pharmacokinetics/pharmacodynamics in female rabbits. drug dev ind pharm. 2018;44(2):224-232. crossref pubmed 13. cevher e, açma a, sinani g, aksu b, zloh m, mülazımoğlu l. bioadhesive tablets containing cyclodextrin complex of itraconazole for the treatment of vaginal candidiasis. int j biol macromol. 2014;69:124-136. crossref pubmed 14. abu el-enin asm, elbakry am, el hosary r, fouad lotfy ma, yahia r. formulation, development, in vivo pharmacokinetics and pharmacological efficacy evaluation of novel vaginal bioadhesive sustained core-in-cup salbutamol sulphate tablets for preterm labor. j drug deliv sci technol. 2020;60:102076. crossref 15. garg s, goldman d, krumme m, rohan lc, smoot s, friend dr. advances in development, scale-up and manufacturing of microbicide gels, films, and tablets. antiviral res. 2010;88(suppl 1): s19-s29. crossref pubmed 16. cazorla-luna r, notario-pérez f, martín-illana a, et al. chitosanbased mucoadhesive vaginal tablets for controlled release of the anti-hiv drug tenofovir. pharmaceutics. 2019;11(1):20. crossref pubmed 17. clark mr, peet mm, davis s, doncel gf, friend dr. evaluation of rapidly disintegrating vaginal tablets of tenofovir, emtricitabine and their combination for hiv-1 prevention. pharmaceutics. 2014;6(4):616-631. crossref pubmed 18. abidin iz, rezoagli e, simonassi-paiva b, et al. a bilayer vaginal tablet for the localized delivery of disulfiram and 5-fluorouracil to the cervix. pharmaceutics. 2020;12(12):1185. crossref pubmed 19. higgins jp, green s. cochrane handbook for systematic reviews of interventions. chichester: john wiley & sons; 2019. crossref 20. lee c-h, chien yw. development and evaluation of a mucoadhesive drug delivery system for dual-controlled delivery of nonoxynol-9. j control release. 1996;39(1):93-103. crossref 21. perrone m, lopalco a, lopedota a, et al. s-preactivated thiolated glycol chitosan useful to combine mucoadhesion and https://doi.org/10.1002/9781118794203.ch05 https://doi.org/10.1016/j.addr.2021.113865 https://www.ncbi.nlm.nih.gov/pubmed/34280514 https://doi.org/10.3109/03639045.2011.623355 https://www.ncbi.nlm.nih.gov/pubmed/21999572 https://doi.org/10.2174/187221109789105658 https://www.ncbi.nlm.nih.gov/pubmed/19925443 https://doi.org/10.1016/j.colsurfb.2018.09.014 https://www.ncbi.nlm.nih.gov/pubmed/30218984 https://doi.org/10.1016/s1461-5347(00)00296-0 https://www.ncbi.nlm.nih.gov/pubmed/11050460 https://doi.org/10.1177/20503121221085437 https://www.ncbi.nlm.nih.gov/pubmed/35371482 https://doi.org/10.1016/j.ijpx.2021.100081 https://www.ncbi.nlm.nih.gov/pubmed/34027386 https://doi.org/10.2147/ppa.s22940 https://www.ncbi.nlm.nih.gov/pubmed/22163155 https://doi.org/10.1016/j.addr.2015.02.003 https://www.ncbi.nlm.nih.gov/pubmed/25689736 https://doi.org/10.3390/md18050249 https://www.ncbi.nlm.nih.gov/pubmed/32403219 https://doi.org/10.1080/03639045.2017.1386203 https://www.ncbi.nlm.nih.gov/pubmed/28956650 https://doi.org/10.1016/j.ijbiomac.2014.05.033 https://www.ncbi.nlm.nih.gov/pubmed/24857873 https://doi.org/10.1016/j.jddst.2020.102076 https://doi.org/10.1016/j.antiviral.2010.09.010 https://www.ncbi.nlm.nih.gov/pubmed/21109064 https://doi.org/10.3390/pharmaceutics11010020 https://www.ncbi.nlm.nih.gov/pubmed/30621307 https://doi.org/10.3390/pharmaceutics6040616 https://www.ncbi.nlm.nih.gov/pubmed/25494201 https://doi.org/10.3390/pharmaceutics12121185 https://www.ncbi.nlm.nih.gov/pubmed/33291349 https://doi.org/10.1002/9781119536604 https://doi.org/10.1016/0168-3659(95)00142-5 mucoadhesive vaginal tablet testing30 © 2023 the authors. drug target insights issn 1177-3928 www.aboutscience.eu/dti drug delivery. eur j pharm biopharm. 2018;132:103-111. crossref pubmed 22. rojewska m, bartkowiak a, milanowski b, prochaska k, lulek j. physicochemical and release studies of new mucoadhesive fluconazole delivery systems. colloids surf a physicochem eng asp. 2019;566:11-20. crossref 23. szymańska e, winnicka k, amelian a, cwalina u. vaginal chitosan tablets with clotrimazole-design and evaluation of mucoadhesive properties using porcine vaginal mucosa, mucin and gelatine. chem pharm bull (tokyo). 2014;62(2):160-167. crossref pubmed 24. cevher e, sensoy d, zloh m, mülazimoğlu l. preparation and characterisation of natamycin: γ-cyclodextrin inclusion complex and its evaluation in vaginal mucoadhesive formulations. j pharm sci. 2008;97(10):4319-4335. crossref pubmed 25. gök mk, demir k, cevher e, et al. the effects of the thiolation with thioglycolic acid and l-cysteine on the mucoadhesion properties of the starch-graft-poly(acrylic acid). carbohydr polym. 2017;163:129-136. crossref pubmed 26. bhat s, shivakumar h. bioadhesive controlled release clotrimazole vaginal tablets. trop j pharm res. 2010;9:339-346. crossref 27. gupta nv, natasha s, getyala a, bhat rs. bioadhesive vaginal tablets containing spray dried microspheres loaded with clotrimazole for treatment of vaginal candidiasis. acta pharm. 2013;63(3):359-372. crossref pubmed 28. tunpanich p, limpongsa e, pongjanyakul t, sripanidkulchai b, jaipakdee n. mucoadhesive sustained-release tablets for vaginal delivery of curcuma comosa extracts: preparation and characterization. j drug deliv sci technol. 2019;51:559-568. crossref 29. notario-pérez f, martín-illana a, cazorla-luna r, et al. influence of chitosan swelling behaviour on controlled release of tenofovir from mucoadhesive vaginal systems for prevention of sexual transmission of hiv. mar drugs. 2017;15(2):50. crossref pubmed 30. perioli l, ambrogi v, pagano c, scuota s, rossi c. fg90 chitosan as a new polymer for metronidazole mucoadhesive tablets for vaginal administration. int j pharm. 2009;377(1-2):120-127. crossref pubmed 31. kailasam p, jamunadhevi v, kaur g. formulation and evaluation of once daily mucoadhesive vaginal tablet of metronidazole. int j res pharm sci. 2010;1:308-312. online 32. fitaihi ra, aleanizy fs, elsamaligy s, mahmoud ha, bayomi ma. role of chitosan on controlling the characteristics and antifungal activity of bioadhesive fluconazole vaginal tablets. saudi pharm j. 2018;26(2):151-161. crossref pubmed 33. sánchez mt, ruiz ma, castán h, morales me. a novel doublelayer mucoadhesive tablet containing probiotic strain for vaginal administration: design, development and technological evaluation. eur j pharm sci. 2018;112:63-70. crossref pubmed 34. khan ab, thakur rs. design and evaluation of mucoadhesive vaginal tablets of tenofovir disoproxil fumarate for pre-exposure prophylaxis of hiv. drug dev ind pharm. 2018;44(3):472483. crossref pubmed 35. el-kamel a, sokar m, naggar v, al gamal s. chitosan and sodium alginate-based bioadhesive vaginal tablets. aaps pharmsci. 2002;4(4):e44. crossref pubmed 36. baloglu e, ay senyıgıt z, karavana sy, et al. in vitro evaluation of mucoadhesive vaginal tablets of antifungal drugs prepared with thiolated polymer and development of a new dissolution technique for vaginal formulations. chem pharm bull (tokyo). 2011;59(8):952-958. crossref pubmed 37. paczkowska m, chanaj-kaczmarek j, romaniuk-drapała a, et al. mucoadhesive chitosan delivery system with chelidonii herba lyophilized extract as a promising strategy for vaginitis treatment. j clin med. 2020;9(4):1208. crossref pubmed 38. hani u, shivakumar hg, osmani ra, srivastava a, kumar varma ns. development of a curcumin bioadhesive monolithic tablet for treatment of vaginal candidiasis. iran j pharm res. 2016;15(1):23-34. pubmed 39. shah rb, tawakkul ma, khan ma. comparative evaluation of flow for pharmaceutical powders and granules. aaps pharmscitech. 2008;9(1):250-258. crossref pubmed 40. jallo lj, ghoroi c, gurumurthy l, patel u, davé rn. improvement of flow and bulk density of pharmaceutical powders using surface modification. int j pharm. 2012;423(2):213-225. crossref pubmed 41. beakawi al-hashemi hm, baghabra al-amoudi os. a review on the angle of repose of granular materials. powder technol. 2018;330:397-417. crossref 42. cazorla-luna r, martín-illana a, notario-pérez f, et al. vaginal polyelectrolyte layer-by-layer films based on chitosan derivatives and eudragit® s100 for ph responsive release of tenofovir. mar drugs. 2020;18(1):44. crossref pubmed 43. notario-pérez f, cazorla-luna r, martín-illana a, et al. optimization of tenofovir release from mucoadhesive vaginal tablets by polymer combination to prevent sexual transmission of hiv. carbohydr polym. 2018;179:305-316. crossref pubmed 44. jug m, hafner a, lovrić j, et al. an overview of in vitro dissolution/release methods for novel mucosal drug delivery systems. j pharm biomed anal. 2018;147:350-366. crossref pubmed 45. palade l, popovici i, cojocaru i. in vitro evaluation of 5-fluorouracil dissolution profiles from vaginal bioadhesive tablets. farmacia. 2013;61(4):640-647. 46. baki g, bajdik j, kelemen a, pintye-hódi k. formulation of a solid intravaginal matrix system to prolong the ph-decreasing effect of lactic acid. j drug deliv sci technol. 2009;19(2):133137. crossref 47. fernandes l, costa r, henriques m, rodrigues me. simulated vaginal fluid: candida resistant strains’ biofilm characterization and vapor phase of essential oil effect. j mycol med. 2022;33(1):101329. crossref pubmed 48. lupo n, fodor b, muhammad i, yaqoob m, matuszczak b, bernkop-schnürch a. entirely s-protected chitosan: a promising mucoadhesive excipient for metronidazole vaginal tablets. acta biomater. 2017;64:106-115. crossref pubmed 49. colombo n, carinelli s, colombo a, et al. esmo guidelines working group. cervical cancer: esmo clinical practice guidelines for diagnosis, treatment and follow-up. ann oncol. 2012; suppl 7:vii27-32. crossref pubmed 50. perioli l, ambrogi v, pagano c, massetti e, rossi c. new solid mucoadhesive systems for benzydamine vaginal administration. colloids surf b biointerfaces. 2011;84(2):413-420. crossref pubmed 51. pendekal ms, tegginamat pk. hybrid drug delivery system for oropharyngeal, cervical and colorectal cancer—in vitro and in vivo evaluation. saudi pharm j. 2013;21(2):177-186. crossref pubmed 52. owen dh, katz df. a vaginal fluid simulant. contraception. 1999;59(2):91-95. crossref pubmed https://doi.org/10.1016/j.ejpb.2018.09.015 https://www.ncbi.nlm.nih.gov/pubmed/30253185 https://doi.org/10.1016/j.colsurfa.2018.12.058 https://doi.org/10.1248/cpb.c13-00689 https://www.ncbi.nlm.nih.gov/pubmed/24492586 https://doi.org/10.1002/jps.21312 https://www.ncbi.nlm.nih.gov/pubmed/18288724 https://doi.org/10.1016/j.carbpol.2017.01.065 https://www.ncbi.nlm.nih.gov/pubmed/28267489 https://doi.org/10.4314/tjpr.v9i4.58924 https://doi.org/10.2478/acph-2013-0027 https://www.ncbi.nlm.nih.gov/pubmed/24152896 https://doi.org/10.1016/j.jddst.2019.03.030 https://doi.org/10.3390/md15020050 https://www.ncbi.nlm.nih.gov/pubmed/28230790 https://doi.org/10.1016/j.ijpharm.2009.05.016 https://www.ncbi.nlm.nih.gov/pubmed/19454304 https://ijrps.com/index.php/home/article/view/2922 https://doi.org/10.1016/j.jsps.2017.12.016 https://www.ncbi.nlm.nih.gov/pubmed/30166911 https://doi.org/10.1016/j.ejps.2017.11.006 https://www.ncbi.nlm.nih.gov/pubmed/29129498 https://doi.org/10.1080/03639045.2017.1399272 https://www.ncbi.nlm.nih.gov/pubmed/29082767 https://doi.org/10.1208/ps040444 https://www.ncbi.nlm.nih.gov/pubmed/12646014 https://doi.org/10.1248/cpb.59.952 https://www.ncbi.nlm.nih.gov/pubmed/21804238 https://doi.org/10.3390/jcm9041208 https://www.ncbi.nlm.nih.gov/pubmed/32331437 https://www.ncbi.nlm.nih.gov/pubmed/27610145 https://doi.org/10.1208/s12249-008-9046-8 https://www.ncbi.nlm.nih.gov/pubmed/18446489 https://doi.org/10.1016/j.ijpharm.2011.12.012 https://www.ncbi.nlm.nih.gov/pubmed/22197769 https://doi.org/10.1016/j.powtec.2018.02.003 https://doi.org/10.3390/md18010044 https://www.ncbi.nlm.nih.gov/pubmed/31936439 https://doi.org/10.1016/j.carbpol.2017.10.001 https://www.ncbi.nlm.nih.gov/pubmed/29111056 https://doi.org/10.1016/j.jpba.2017.06.072 https://www.ncbi.nlm.nih.gov/pubmed/28720350 https://doi.org/10.1016/s1773-2247(09)50021-4 https://doi.org/10.1016/j.mycmed.2022.101329 https://www.ncbi.nlm.nih.gov/pubmed/36270212 https://doi.org/10.1016/j.actbio.2017.10.014 https://www.ncbi.nlm.nih.gov/pubmed/29030305 https://doi.org/10.1093/annonc/mds268 https://www.ncbi.nlm.nih.gov/pubmed/22997451 https://doi.org/10.1016/j.colsurfb.2011.01.035 https://www.ncbi.nlm.nih.gov/pubmed/21354770 https://doi.org/10.1016/j.jsps.2012.07.002 https://www.ncbi.nlm.nih.gov/pubmed/23960833 https://doi.org/10.1016/s0010-7824(99)00010-4 https://www.ncbi.nlm.nih.gov/pubmed/10361623 9drug target insights 2015:9 medicinal plants: a potential source of compounds for targeting cell division ihsan n. zulkipli, sheba r. david, rajan rajabalaya and adi idris pap rashidah sa’adatul bolkiah institute of health sciences, universiti brunei darussalam, gadong, brunei darussalam. a bstr act: modern medicinal plant drug discovery has provided pharmacologically active compounds targeted against a multitude of conditions and diseases, such as infection, inflammation, and cancer. to date, natural products from medicinal plants remain a solid niche as a source from which cancer therapies can be derived. among other properties, one favorable characteristic of an anticancer drug is its ability to block the uncontrollable process of cell division, as cancer cells are notorious for their abnormal cell division. there are numerous other documented works on the potential anticancer activity of drugs derived from medicinal plants, and their effects on cell division are an attractive and growing therapeutic target. despite this, there remains a vast number of unidentified natural products that are potentially promising sources for medical applications. this mini review aims to revise the current knowledge of the effects of natural plant products on cell division. k e y wor ds: cell division, cancer, medicinal plants, microtubule, natural products citation: zulkipli et al. medicinal plants: a potential source of compounds for targeting cell division. drug target insights 2015:9 9–19 doi:10.4137/dti.s24946. received: february 13, 2015. resubmitted: april 7, 2015. accepted for publication: april 20, 2015. academic editor: anuj chauhan, editor in chief type: short review funding: we would like to thank the universiti brunei darussalam (ubd) research grant (ubcd/pnc2/2/rg/1(322)) for funding this research. the authors confirm that the funder had no influence over the study design, content of the article, or selection of this journal. competing interests: authors disclose no potential conflicts of interest. copyright: © the authors, publisher and licensee libertas academica limited. this is an open-access article distributed under the terms of the creative commons cc-by-nc 3.0 license. correspondence: yusri.idris@ubd.edu.bn paper subject to independent expert blind peer review by minimum of two reviewers. all editorial decisions made by independent academic editor. upon submission manuscript was subject to anti-plagiarism scanning. prior to publication all authors have given signed confirmation of agreement to article publication and compliance with all applicable ethical and legal requirements, including the accuracy of author and contributor information, disclosure of competing interests and funding sources, compliance with ethical requirements relating to human and animal study participants, and compliance with any copyright requirements of third parties. this journal is a member of the committee on publication ethics (cope). published by libertas academica. learn more about this journal. introduction human beings have long used plants as a medicinal source. their use has grown more sophisticated with modern chemists using compounds isolated from plants as a basis for generating novel compounds with additional benefits, such as its lower toxicity and potential for combating drug-resistant diseases. between 1981 and 2010, naturally derived products and their mimics composed an estimated 70% of new chemical compounds reported.1 naturally derived compounds with anticancer activity have also been used as the basis for original synthetic analogs, forming their own novel class of chemical compounds.2 mammalian microtubules appear to be a common target for naturally occurring toxic molecules produced by a large number of flora, presumably with the original intent of self-defense. microtubules are a component of the cytoskeleton, found throughout the cell cytoplasm, which is important in the process of mitosis (ie, cell division). most microtubule-targeting compounds have been discovered in large-scale screens of natural products3,4 (table 1). approximately 75% of the available anticancer drugs between 1940 and 2010 were naturally derived products or their mimics. additionally, of the seven anticancer drugs approved in 2010, almost half of them exert their effects by binding onto microtubules.1 one of the biggest success stories of microtubule-targeted compounds from a naturally derived source is paclitaxel (commercially known as taxol), a member of the taxane family. paclitaxel is extracted from the bark of the pacific yew tree (taxus brevifolia) and acts as an antimitotic drug, by binding to microtubules, thus stabilizing them and arresting cells in mitosis.5–9 taxol and its derivatives have successfully been used clinically to treat ovarian cancer, breast cancer, and nonsmall cell lung cancer for almost 40 years, making taxol the best-selling anticancer drug currently manufactured. its success has sparked the search for similar microtubule-stabilizing compounds. another class of microtubule-targeted compounds from a naturally derived source is the vinca alkaloids, vincristine and vinblastine, which were initially isolated from the madagascar periwinkle plant (catharanthus roseus).10 the vinca alkaloids are microtubule destabilizers and have proven to be particularly effective against hematological malignancies,11 and their success has generated several semisynthetic derivatives. semisynthetic and synthetic derivatives may offer advantages over a fully natural source, as the bioactive natural compound may be present only in trace amounts. natural compounds may instead act as lead compounds, where analogs with higher potencies and lower toxicities may be developed12,13 (table 2). natural products are ideal as lead compounds as their chemical structures are complex and diverse (table 3). the biggest study looking into the isolation of compounds with clinical bioactivity, specifically anticancer activity, from natural sources was done by the national cancer institute (nci) of the national journal name: drug target insights journal type: short review year: 2015 volume: 9 running head verso: zulkipli et al running head recto: medicinal plants: a potential source of compounds for targeting cell division http://www.la-press.com/drug-target-insights-journal-j23 http://www.la-press.com http://dx.doi.org/10.4137/dti.s24946 http://creativecommons.org/licenses/by-nc/3.0/ http://creativecommons.org/licenses/by-nc/3.0/ mailto:yusri.idris@ubd.edu.bn http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 zulkipli et al 10 drug target insights 2015:9 ta b le 1 . s el ec te d co m po un ds o rig in al ly is ol at ed fr om n at ur al s ou rc es th at a ct o n m ic ro tu bu le s. b io lo g ic a ll y a c t iv e c o m p o u n d (s ) a n d t h e ir s t r u c t u r e s c ie n t if ic n a m e (s ) o f n a t u r a l s o u r c e m e c h a n is m o f a c t io n o n m ic r o tu b u le s s ta t u s a s a n t ic a n c e r d r u g t e s t e d c a n c e r t y p e s r e fe r e n c e s p ac lit ax el ta xu s br ev ifo lia s ta bi liz es m ic ro tu bu le s in c lin ic al u se o va ria n ca nc er , b re as t c an ce r, no nsm al l c el l l un g ca nc er , a dv an ce d k ap os i sa rc om a 47 ,4 8 v in bl as tin e v in cr is tin e c at ha ra nt hu s ro se us d es ta bi liz es m ic ro tu bu le s in c lin ic al u se a cu te ly m ph ob la st ic le uk ae m ia , b re as t ca nc er , c ho rio ca rc in om a, h od gk in ly m ph om a, k ap os i s ar co m a, m yc os is fu ng oi de s, h od gk in a nd n on -h od gk in ly m ph om a, te st ic ul ar c an ce r, ne ur obl as to m a, r ha dd om yc os ar co m a, w ilm s tu m ou r, bl ad de r ca nc er , t es tic ul ar c an ce r, br ea st c an ce r, ch or io ca rc in om a, lu ng c an ce r, m ul tip le m ye lo m a, s of t t is su e sa rc om a, b ra in tu m ou rs . l eu ka em ia , he ad a nd n ec k ca nc er s 10 ,1 1, 49 ,5 0 c ol ch ic in e c ol ch ic um a ut um na le d es ta bi liz es m ic ro tu bu le s fa ile d an tica nc er tr ia ls du e to to xi ci ty ; i n cl in ic al us e fo r go ut th er ap y h ep at oc el lu la r ca rc in om a, m ul tip le m ye lo m a, h od gk in ’s ly m ph om a, c hr on ic ly m ph at ic le uk ae m ia , b re as t c an ce r, lu ng ca nc er , c hr on ic ly m ph oc yt ic le uk ae m ia 51 – 53 p od op hy llo to xi n p od op hy llu m s pp . d es ta bi liz es m ic ro tu bu le s in u se fo r th e to pi ca l t re at m en t o f e xt er na l g en ita l w ar ts n on e 43 ,5 4, 55 c om br et as ta tin s c om br et um c af fr um d es ta bi liz es m ic ro tu bu le s p ha se i, ii c lin ic al tr ia ls ; s em isy nt he tic d er iv at iv e in p ha se ii i c lin ic al tr ia ls a cu te m ye lo id le uk ae m ia , m ye lo dy sp la stic s yn dr om e, th yr oi d ca nc er , n on -s m al l ce ll lu ng c an ce r, va rio us s ol id tu m ou rs 56 n os ca pi ne p ap av er ac ea e sp p. s up pr es se s m ic ro tu bu le dy na m ic s p ha se ii c lin ic al tr ia ls m ul tip le m ye lo m a 57 n o te : d at a in th is ta bl e w er e ob ta in ed fr om a c om bi na tio n of n c i d ru g d ic tio na ry ( ht tp :// w w w .c an ce r.g ov /d ru gd ic tio na ry ), p ub lis he d lit er at ur e, a nd c om pa ny w eb s ite s. http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 11drug target insights 2015:9 medicinal plants: a potential source of compounds for targeting cell division table 2. selected synthetic and semisynthetic compounds originally isolated from natural sources that act on microtubules. compound and structure original compound mechanism of action on microtubules status as anticancer drug tested cancer types references vindesine vinca alkaloids destabilizes microtubules in clinical use various lung cancers, various haematological malignancies, melanoma, renal cancer, colorectal cancer and breast cancer. currently in clinical trials for other cancer types 58 vinorelbine vinca alkaloids destabilizes microtubules in clinical use non-small cell lung cancer, metastatic breast cancer, renal cancer 42,59,60 vinflunine vinca alkaloids destabilizes microtubules in clinical use bladder cancer, urethral cancer, ureteral cancer, cancer of the renal pelvis 42 docetaxel paclitaxel stabilizes microtubules in clinical use breast cancer, gastric cancer, nonsmall cell lung cancer, prostrate cancer, squamous cell carcinoma of the head and neck, stomach cancer 61 cabazitaxel paclitaxel stabilizes microtubules in clinical use metastatic prostrate cancer 62,63 larotaxel paclitaxel stabilizes microtubules phase iii clinical trials breast cancer, pancreatic cancer, urothelial tract cancer, bladder cancer, various solid tumours 63 tesetaxel paclitaxel stabilizes microtubules phase ii clinical trials gastric cancer, melanoma, bladder cancer, breast cancer, prostate cancer, various solid tumours 63 ombrabulin combrestatin destabilizes microtubules discontinued, due to insufficient clinical benefit soft tissue sarcoma, non-small cell lung cancer, ovarian cancer, various solid tumours 55,64 fosbretabulin combrestatin destabilizes microtubules phase i and phase ii clinical trials ovarian cancer, gastrointestinal neuroendocrine tumours, ovarian epithelial, fallopian tube, and primary peritoneal cancers, gliomas, thyroid cancer 65 crolibulin combrestatin destabilizes microtubules phase i and phase ii clinical trials thyroid cancer 66 verubulin combrestatin destabilizes microtubules phase i and phase ii clinical trials glioblastoma 66–68 note: data in this table were obtained from a combination of nci drug dictionary (http://www.cancer.gov/drugdictionary), published literature, and company web sites. institute of health in usa from 1960 to 1980.14 however, it is estimated that .90% of plant species worldwide remain understudied. discovery of drug molecules has been limited because of genomic instability and drug resistance characteristics in certain cancer cells.15 therefore, modern drug discovery has shifted to personalized treatment of patients, where drugs are selected for specific molecular targets, taking advantage of the vulnerabilities of cancer in a particular patient, leading to increased interest in studying traditional herbs as an alternative source of anticancer drugs because of its multitargeted characteristic.16 the role of the microtubule in cell division microtubules are a class of the cytoskeletal proteins present in all eukaryotic cells. they form long, filamentous, polymeric structures within the cell, composed of αand β-tubulin heterodimers, of which there are several isotypes.17 the different isotypes of tubulin in human beings are summarized in table 4. microtubules play many roles in eukaryotic cells, including development and maintenance of cell shape,18 intracellular transport,19 cell motility,20,21 cell signaling,22 and cell division.23 cell division, or mitosis, is a crucial event in the cell cycle that results in the division of a single cell into two identical daughter cells with the equal distribution of genetic materials (fig. 1). during mitosis, the cytoskeleton forms a superstructure called the mitotic spindle, which facilitates many of the cell division processes. mitosis involves a series of stages. the initial prophase and prometaphase stages are where there is condensation of chromosomes, which then attaches to the mitotic spindle. the chromosomes then align at the equator of the mitotic spindle (metaphase) before the sister chromosomes segregate into daughter cells (anaphase). the final stage is where the chromosomes decondense and the cells divide fully into two daughter cells (telophase). all the stages of mitosis must be regulated for the proper development and function of a multicellular organism. central to the function of microtubules is the regulation of microtubule dynamics. microtubule filaments are able to polymerize and depolymerize stochastically within a cell, in what is termed http://www.la-press.com/drug-target-insights-journal-j23 http://www.la-press.com zulkipli et al 12 drug target insights 2015:9 ta b le 3 . c he m ic al s tr uc tu re s of n at ur al m ic ro tu bu le -t ar ge tin g co m po un ds a nd th ei r sy nt he tic a nd s em is yn th et ic d er iv at iv es . v in b la st in e v in cr is ti ne v in d es in e v in fl u n in e v in o re lb in e http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 13drug target insights 2015:9 medicinal plants: a potential source of compounds for targeting cell division p ac lit ax el d o ce ta xe l c ab az it ax el l ar o ta xe l te se ta xe l c om br es ta tin o m br ab ul in fi sb re ta b u lin (c on tin ue d) http://www.la-press.com/drug-target-insights-journal-j23 http://www.la-press.com zulkipli et al 14 drug target insights 2015:9 c ro lib u lin v er u b u lin c ol ch ic in e p o d o p hy llo to xi n n o sc ap in e ta b le 3 . ( c on tin ue d ) http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 15drug target insights 2015:9 medicinal plants: a potential source of compounds for targeting cell division table 4. subtypes and isoforms of microtubules. tubulin subtype isotype gene length (amino acids) tissue distribution putative function (if any) altered expression in cancers? α 1a tuba1a 451 ubiquitous no isoform-specific function identified no 1b tuba1b 451 ubiquitous no 1c tuba1c 449 ubiquitous no 3c tuba3c 450 enriched expression in testis, fallopian tube, soft tissues, central nervous system and other selected tissues variable expression 3d tuba3d 450 enriched expression in testis, fallopian tube, soft tissues, central nervous system and other selected tissues decreased 3e tuba3e 448 enriched expression in testis, fallopian tube, soft tissues, central nervous system and other selected tissues decreased 4a tuba4a 446 ubiquitous no 8 tuba8 449 ubiquitous, but enriched in heart muscle, skeletal muscle and testis decreased β 1 tubb1 451 enriched in haematopoietic cells may play a role in microtubule stability, as well as interaction with actin increased on exposure to microtubuletargeting drugs 2a tubb2a 445 ubiquitous, enriched in brain may play a role in neuronal differentation increased in microtubule-targeting drug-resistant cancers 2b tubb2b 445 ubiquitous, enriched in brain may play a role in neuronal differentation no 3 tubb3 450 mostly expressed in central and peripheral nervous system may play a role in neuronal differentiation. may help cells cope with oxidative stress overexpressed in aggressive tumours 4a tubb4a 444 highly expressed in brain, moderate levels in testis, very low levels in other tissues occurs in axonemes, may be required for determination of axonemal microtubule structure increased on exposure to microtubuletargeting drugs 4b tubb4b 445 ubiquitous occurs in axonemes, may be required for determination of axonemal microtubule structure no 5 tubb 444 ubiquitously expressed with highest levels in spleen, thymus and immature brain unknown unknown 6 tubb6 446 ubiquitous, with highest expression in the breast and lung unknown largely decreased 8 tubb8 444 ubiquitous, enriched in cliliated cells and lymphoid tissue unknown unknown γ 1 tubg1 451 ubiquitous important for nucleation and polarity of microtubules, mostly found in microtubuleorganising centres unknown 2 tubg2 451 ubiquitous important for nucleation and polarity of microtubules, mostly found in microtubuleorganising centres unknown δ – tubd1 453 ubiquitous sperm differentiation decreased ε – tube1 475 majority of tissues centrosome cycle decreased notes: data in this table were obtained from uniprot (http://www.uniprot.org) and proteinatlas (http://www.proteinatlas.org). http://www.la-press.com/drug-target-insights-journal-j23 http://www.la-press.com zulkipli et al 16 drug target insights 2015:9 figure 1. the process of cell division in mammalian cells. this figure illustrates the different microtubule structures present during different stages of the cell cycle. in the interphase stage of the cell cycle, microtubules (green) emanate out from the microtubule-organizing center, the centrosome (dark blue circle), forming an array that extends toward the cell periphery. during the mitotic stage of the cell cycle, the centrosomes are duplicated and separated to form spindle poles, while the microtubule cytoskeleton is reorganized to form a superstructure called the mitotic spindle. the mitotic spindle is responsible for mitotic events such as chromosome congression and chromosome segregation. two stages of the mitotic stage of the cell cycle are illustrated—metaphase and anaphase. at metaphase, the mitotic spindle holds sister chromatids (blue) together at the cell equator. at anaphase, the cell elongates the spindle poles move further apart and the sister chromatids move toward the opposite poles. black arrows indicate the path normally followed by a cell in a cell cycle. when the cell cycle is disrupted at mitosis by tubulin-binding agents, the cell is unable to complete mitosis and follows an alternative pathway (red arrows) where it undergoes mitotic arrest and eventually cell death. all stages of mitosis must be regulated for proper development and function of a multicellular organism. unregulated mitosis may lead to an overgrowth of cells, as in cancer. the ability to carry out an infinite number of cell divisions is one of the hallmarks of cancer. blockage of any stage of mitosis may not allow the cells to complete mitosis, resulting in cell cycle arrest and ultimately, cell death. as microtubule dynamicity.24 microtubule dynamics are tightly regulated within cells, through the binding of various regulatory proteins, expression of different tubulin isotypes, and posttranslational modifications of tubulin subunits.25,26 dynamic microtubules have a very short half-life of a few minutes, or even seconds, whereas stable microtubules have half-lives of minutes to hours.27 during mitosis, microtubules are the main components of the mitotic spindle, where microtubule dynamics are increased significantly.27,28 dynamic microtubules are required for all stages of mitosis: from capturing and congressing chromosomes to the metaphase plate,29 pulling chromosomes toward opposite poles and initiating anaphase,30 and finally cytokinesis to complete mitosis.31,32 microtubule-binding compounds may either stabilize microtubules (promoting growth and not supporting shrinkage of the microtubule filament) or destabilize microtubules (promoting shrinkage and not supporting growth of the microtubule filament). any alterations in microtubule dynamics will affect the different events in mitosis. for example, if microtubule dynamics are suppressed, chromosomes may not be able to congress to the metaphase plate.33,34 the presence of a single uncongressed chromosome is enough to induce mitotic arrest.1 accordingly, altered microtubule dynamics is among the major causes of mitotic arrest. a cell that is arrested in mitosis for a prolonged time may eventually undergo apoptosis, or programed cell death.35 at present, most of the drugs used to treat cancer target microtubule dynamics in order to arrest cancerous cells in mitosis. microtubules—a potential target for cancer therapy unregulated cell division may lead to an overgrowth of cells, as in cancer. the ability to carry out an infinite number of cell divisions is one of the hallmarks of cancer.36 blockage of any stage of mitosis may not allow the cells to complete mitosis, resulting in cell cycle arrest and ultimately, cell death. microtubules represent the best and most successful target thus far http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 17drug target insights 2015:9 medicinal plants: a potential source of compounds for targeting cell division identified in cancer treatment.37–39 cancer cells are sensitive to microtubule poisons that arrest cells in mitosis because they undergo mitosis more frequently than normal cells. at high concentrations, anticancer drugs that target microtubules may act in one of the two ways. each approach has different effects, including affecting the polymerized microtubule mass, destabilization of microtubules (decreases microtubule mass), and stabilization of microtubules (increases microtubule mass), dependent on the site of binding on the microtubule lattice.40 the effects of each compound on microtubules are indicated in both tables 1 and 2. currently, there are two main classes of microtubulebinding anticancer drugs. these are the microtubule destabilizers, such as the vinca alkaloids,41–44 and microtubule stabilizers that prevent microtubule disassembly without affecting their polymerization, such as the taxanes.6 however, studies have shown that various microtubule-targeting drugs, irrespective of their effects on polymerized microtubule mass at high concentrations, all suppress microtubule dynamics at lower concentrations, ie, prevent the growth or shrinkage of microtubules without changing the microtubule polymer mass6,8,33,34,42–45 (fig. 2). in this way, changes in microtubule dynamics can be used as an indicator of the efficacy of the anticancer activities of a naturally derived compound. conversely, tumors can acquire resistance to microtubuletargeting drugs. although a discussion on the resistance mechanisms to these drugs is beyond the scope of this review, the possible methods of resistance include multidrug resistance pumps, altered drug binding, altered microtubule assembly, altered tubulin synthesis, and alterations in microtubuleinteracting proteins (refer to fojo and menefee46 for a more extensive review). as with all drugs, the toxic side effects of microtubule-targeting agents must be taken into account. owing to the physiological functions of microtubules, treatment with microtubule-targeting agents often exhibits myelosuppression and peripheral neuropathy. the specif icity of each compound must therefore be tested. the cancers identif ied to be susceptible to each drug are illustrated in tables 1 and 2. conclusion mitosis is an important stage of the cell cycle, which is deregulated in cancer, leading to uncontrolled cancer growth. an important facilitator of mitosis is the microtubule cytoskeleton. hence, many anticancer drugs target the microtubule skeleton in order to arrest cancer cells in mitosis, which eventually leads to cell death. most of these microtubuletargeting drugs act by suppressing microtubule dynamics, which is particularly important for the microtubule function in mitosis. interestingly, many of the microtubule-binding anticancer drugs are derived from natural sources, including taxol and the vinca alkaloids, two very successful classes of anticancer drugs. therefore, there is great potential for the isolation of compounds with similar microtubule-targeting figure 2. microtubule dynamic instability. the figure illustrates the growth and shrinkage of a single microtubule, with each row representative of a single time point. microtubules are composed of stable αβ-tubulin heterodimers that are arranged in a head-to-tail fashion, forming a polar structure. each heterodimer is illustrated as a single circle. microtubules therefore consist of two distinct ends: the plus (+) end and the minus (-) end. in vivo, the—ends are anchored at the microtubule-organizing centers. the + ends are more dynamic than the—ends, with the microtubule end constantly switching between growth and shrinkage in what is termed dynamic instability. microtubules are normally very dynamic (top), with tubulin subunits randomly added or lost from both ends. in vivo, microtubule elongation usually occurs in the plus end. when microtubule dynamics are suppressed (for example, through the action of tubulin-binding agents) (bottom), tubulin subunits are rarely added or lost from the microtubule ends. activities from medicinal plants. future aims for the development of novel microtubule-binding agents are the development of compounds specific to cancer cells, thereby reducing potential toxic side effects, as well as the development of compounds that are able to overcome current drug-resistant cancers. author contributions prepared the first draft of the manuscript: inz. contributed to the writing of the manuscript: srd, rr, and ai. jointly developed the structure and arguments for the paper: inz, srd, rr, and ai. made critical revisions and approved the final version: ai. all the authors reviewed and approved the final manuscript. http://www.la-press.com/drug-target-insights-journal-j23 http://www.la-press.com zulkipli et al 18 drug target insights 2015:9 r efer ences 1. newman dj, cragg gm. natural products as sources of new drugs over the 30 years from 1981 to 2010. j nat prod. 2012;75(3):311–335. 2. loong hh, yeo w. microtubule-targeting agents in oncology and therapeutic potential in hepatocellular carcinoma. onco targets ther. 2014;7:575–585. 3. balunas mj, kinghorn ad. drug discovery from medicinal plants. life sci. 2005;78(5):431–441. 4. gurib-fakim a. medicinal plants: traditions of yesterday and drugs of tomorrow. mol aspects med. 2006;27(1):1–93. 5. arnal i, wade rh. how does taxol stabilize microtubules? curr biol. 1995;5(8): 900–908. 6. jordan ma, toso rj, thrower d, wilson l. mechanism of mitotic block and inhibition of cell proliferation by taxol at low concentrations. proc natl acad sci u s a. 1993;90(20):9552–9556. 7. xiao h, wang h, zhang x, et al. structural evidence for cooperative microtubule stabilization by taxol and the endogenous dynamics regulator map4. acs chem biol. 2012;7(4):744–752. 8. yvon am, wadsworth p, jordan ma. taxol suppresses dynamics of individual microtubules in living human tumor cells. mol biol cell. 1999;10(4):947–959. 9. wani mc, taylor hl, wall me, coggon p, mcphail at. plant antitumor agents. vi. the isolation and structure of taxol, a novel antileukemic and antitumor agent from taxus brevifolia. j am chem soc. 1971;93(9):2325–2327. 10. noble rl. the discovery of the vinca alkaloids—chemotherapeutic agents against cancer. biochem cell biol. 1990;68(12):1344–1351. 11. gidding ce, kellie sj, kamps wa, de graaf ss. vincristine revisited. crit rev oncol hematol. 1999;29(3):267–287. 12. chin y-w, balunas mj, chai hb, kinghorn ad. drug discovery from natural sources. a aps j. 2006;8(2):e239–e253. 13. lahlou m. the success of natural products in drug discovery. pharmacol pharm. 2013;4(3a):17–31. 14. cragg gm, boyd mr, cardellina jh ii, newman dj, snader km, mccloud tg. ethnobotany and drug discovery: the experience of the us national cancer institute. ciba found symp. 1994;185:178–190. [discussion 176–190]. 15. tyagi a, prasad s. drug discovery inspired by mother nature for cancer therapy. biochem physiol. 2015;4:e128. 16. hoelder s, clarke pa, workman p. discovery of small molecule cancer drugs: successes, challenges and opportunities. mol oncol. 2012;6(2):155–176. 17. downing kh, nogales e. tubulin structure: insights into microtubule properties and functions. curr opin struct biol. 1998;8(6):785–791. 18. mogilner a, keren k. the shape of motile cells. curr biol. 2009;19(17):r762–r771. 19. vale rd. intracellular transport using microtubule-based motors. annu rev cell biol. 1987;3:347–378. 20. ridley aj, schwartz ma, burridge k, et al. cell migration: integrating signals from front to back. science. 2003;302(5651):1704–1709. 21. stehbens s, wittmann t. targeting and transport: how microtubules control focal adhesion dynamics. j cell biol. 2012;198(4):481–489. 22. gundersen gg, cook ta. microtubules and signal transduction. curr opin cell biol. 1999;11(1):81–94. 23. khodjakov a, rieder cl. mitosis: too much of a good thing (can be bad). curr biol. 2009;19(22):r1032–r1034. 24. mitchison t, kirschner m. dynamic instability of microtubule growth. nature. 1984;312(5991):237–242. 25. etienne-manneville s. from signaling pathways to microtubule dynamics: the key players. curr opin cell biol. 2010;22(1):104–111. 26. van der vaart b, akhmanova a, straube a. regulation of microtubule dynamic instability. biochem soc trans. 2009;37(pt 5):1007–1013. 27. saxton wm, stemple dl, leslie rj, salmon ed, zavortink m, mcintosh jr. tubulin dynamics in cultured mammalian cells. j cell biol. 1984;99(6):2175–2186. 28. salmon ed, leslie rj, saxton wm, karow ml, mcintosh jr. spindle microtubule dynamics in sea urchin embryos: analysis using a fluorescein-labeled tubulin and measurements of fluorescence redistribution after laser photobleaching. j cell biol. 1984;99(6):2165–2174. 29. kapoor tm, compton da. searching for the middle ground: mechanisms of chromosome alignment during mitosis. j cell biol. 2002;157(4):551–556. 30. gorbsky gj, sammak pj, borisy gg. chromosomes move poleward in anaphase along stationary microtubules that coordinately disassemble from their kinetochore ends. j cell biol. 1987;104(1):9–18. 31. strickland li, wen y, gundersen gg, burgess dr. interaction between eb1 and p150glued is required for anaphase astral microtubule elongation and stimulation of cytokinesis. curr biol. 2005;15(24):2249–2255. 32. ferreira jg, pereira aj, akhmanova a, maiato h. aurora b spatially regulates eb3 phosphorylation to coordinate daughter cell adhesion with cytokinesis. j cell biol. 2013;201(5):709–724. 33. honore s, kamath k, braguer d, wilson l, briand c, jordan ma. suppression of microtubule dynamics by discodermolide by a novel mechanism is associated with mitotic arrest and inhibition of tumor cell proliferation. mol cancer ther. 2003; 2(12):1303–1311. 34. kamath k, okouneva t, larson g, panda d, wilson l, jordan ma. 2-methoxyestradiol suppresses microtubule dynamics and arrests mitosis without depolymerizing microtubules. mol cancer ther. 2006;5(9):2225–2233. 35. gascoigne ke, taylor ss. how do anti-mitotic drugs kill cancer cells? j cell sci. 2009;122(pt 15):2579–2585. 36. hanahan d, weinberg ra. hallmarks of cancer: the next generation. cell. 2011;144(5):646–674. 37. jordan ma, wilson l. microtubules as a target for anticancer drugs. nat rev cancer. 2004;4(4):253–265. 38. pasquier e, kavallaris m. microtubules: a dynamic target in cancer therapy. iubmb life. 2008;60(3):165–170. 39. dumontet c, jordan ma. microtubule-binding agents: a dynamic field of cancer therapeutics. nat rev drug discov. 2010;9(10):790–803. 40. yue q-x, liu x, guo d-a. microtubule-binding natural products for cancer therapy. planta med. 2010;76(11):1037. 41. jordan ma, thrower d, wilson l. mechanism of inhibition of cell proliferation by vinca alkaloids. cancer res. 1991;51(8):2212–2222. 42. ngan vk, bellman k, hill bt, wilson l, jordan ma. mechanism of mitotic block and inhibition of cell proliferation by the semisynthetic vinca alkaloids vinorelbine and its newer derivative vinflunine. mol pharmacol. 2001;60(1):225–232. 43. jordan ma, thrower d, wilson l. effects of vinblastine, podophyllotoxin and nocodazole on mitotic spindles. implications for the role of microtubule dynamics in mitosis. j cell sci. 1992;102(pt 3):401–416. 44. dhamodharan r, jordan ma, thrower d, wilson l, wadsworth p. vinblastine suppresses dynamics of individual microtubules in living interphase cells. mol biol cell. 1995;6(9):1215–1229. 45. gan pp, mccarroll ja, po’uha st, kamath k, jordan ma, kavallaris m. microtubule dynamics, mitotic arrest, and apoptosis: drug-induced differential effects of betaiii-tubulin. mol cancer ther. 2010;9(5):1339–1348. 46. fojo t, menefee m. mechanisms of multidrug resistance: the potential role of microtubule-stabilizing agents. ann oncol. 2007;18(suppl 5):v3–v8. 47. schiff pb, horwitz sb. taxol stabilizes microtubules in mouse fibroblast cells. proc natl acad sci u s a. 1980;77(3):1561–1565. 48. manfredi jj, parness j, horwitz sb. taxol binds to cellular microtubules. j cell biol. 1982;94(3):688–696. 49. panda d, jordan ma, chu kc, wilson l. differential effects of vinblastine on polymerization and dynamics at opposite microtubule ends. j biol chem. 1996; 271(47):29807–29812. 50. prakash v, timasheff sn. the interaction of vincristine with calf brain tubulin. j biol chem. 1983;258(3):1689–1697. 51. vandecandelaere a, martin sr, engelborghs y. response of microtubules to the addition of colchicine and tubulin-colchicine: evaluation of models for the interaction of drugs with microtubules. biochem j. 1997;323(pt 1):189–196. 52. taylor ew. the mechanism of colchicine inhibition of mitosis. i. kinetics of inhibition and the binding of h3-colchicine. j cell biol. 1965;25(suppl):145–160. 53. tangutoori s, ohta a, gatley s, campbell r. repurposing an erstwhile cancer drug: a quantitative and therapeutic evaluation of alternative nanosystems for the delivery of colchicine to solid tumors. j cancer sci ther. 2014;6:236–246. 54. schilstra mj, martin sr, bayley pm. the effect of podophyllotoxin on microtubule dynamics. j biol chem. 1989;264(15):8827–8834. 55. bruschi m, rindone b, tollpa el, et al. podophyllotoxin and antitumor synthetic aryltetralines. toward a biomimetic preparation. croatia: intech open access publisher; 2010. 56. tozer gm, kanthou c, parkins cs, hill sa. the biology of the combretastatins as tumour vascular targeting agents. int j exp pathol. 2002;83(1):21–38. 57. landen jw, lang r, mcmahon sj, et al. noscapine alters microtubule dynamics in living cells and inhibits the progression of melanoma. cancer res. 2002;62(14): 4109–4114. 58. jordan ma, himes rh, wilson l. comparison of the effects of vinblastine, vincristine, vindesine, and vinepidine on microtubule dynamics and cell proliferation in vitro. cancer res. 1985;45(6):2741–2747. 59. klotz dm, nelson sa, kroboth k, et al. the microtubule poison vinorelbine kills cells independently of mitotic arrest and targets cells lacking the apc tumour suppressor more effectively. j cell sci. 2012;125(pt 4):887–895. 60. zhang j, qi hw, zheng h, et al. etoposide-cisplatin alternating with vinorelbinecisplatin versus etoposide-cisplatin alone in patients with extensive disease combined with small cell lung cancer. asian pac j cancer prev. 2014;15(10):4159. 61. garcia p, braguer d, carles g, et al. comparative effects of taxol and taxotere on two different human carcinoma cell lines. cancer chemother pharmacol. 1994; 34(4):335–343. 62. kunos ca, stefan t, jacobberger jw. cabazitaxel-induced stabilization of microtubules enhances radiosensitivity in ovarian cancer cells. front oncol. 2013;3:226. 63. yared ja, tkaczuk kh. update on taxane development: new analogs and new formulations. drug des devel ther. 2012;6:371–384. 64. nihei y, suzuki m, okano a, et al. evaluation of antivascular and antimitotic effects of tubulin binding agents in solid tumor therapy. jpn j cancer res. 1999; 90(12):1387–1395. http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 19drug target insights 2015:9 medicinal plants: a potential source of compounds for targeting cell division 65. mooney cj, nagaiah g, fu p, et al. a phase ii trial of fosbretabulin in advanced anaplastic thyroid carcinoma and correlation of baseline serum-soluble intracellular adhesion molecule-1 with outcome. thyroid. 2009;19(3):233–240. 66. subbiah im, lenihan dj, tsimberidou am. cardiovascular toxicity profiles of vascular-disrupting agents. oncologist. 2011;16(8):1120–1130. 67. grossmann kf, colman h, akerley wa, et al. phase i trial of verubulin (mpc6827) plus carboplatin in patients with relapsed glioblastoma multiforme. j neurooncol. 2012;110(2):257–264. 68. mahal k, resch m, ficner r, schobert r, biersack b, mueller t. effects of the tumor-vasculature-disrupting agent verubulin and two heteroaryl analogues on cancer cells, endothelial cells, and blood vessels. chem med chem. 2014;9(4):847–854. http://www.la-press.com/drug-target-insights-journal-j23 http://www.la-press.com drug target insights 2013:7 27–34 doi: 10.4137/dti.s12519 this article is available from http://www.la-press.com. © the author(s), publisher and licensee libertas academica ltd. this is an open access article published under the creative commons cc-by-nc 3.0 license. open access full open access to this and thousands of other papers at http://www.la-press.com. drug target insights s h o r t r e v i e w drug target insights 2013:7 27 p-glycoprotein inhibition for optimal drug delivery md. lutful amin department of pharmacy, stamford university bangladesh, dhaka, bangladesh. corresponding author email: lutful_amin@yahoo.com abstract: p-glycoprotein (p-gp), an efflux membrane transporter, is widely distributed throughout the body and is responsible for limiting cellular uptake and the distribution of xenobiotics and toxic substances. hundreds of structurally diverse therapeutic agents are substrates to it and it impedes the absorption, permeability, and retention of the drugs, extruding them out of the cells. it is overexpressed in cancer cells and accountable for obstructing cell internalization of chemotherapeutic agents and for developing transporter mediated resistance by cancer cells during anti-tumor treatments. as it jeopardizes the success of drug delivery and cancer targeting, strategies are being developed to overcome p-gp mediated drug transport. this concise review represents a brief discussion on p-gp mediated drug transport and how it hinders the success of various therapies. its main focus is on various strategies used to tackle this curb in the field of drug delivery and targeting. keywords: p-glycoprotein, drug efflux, bioavailability, drug delivery, drug resistance, p-glycoprotein inhibitor http://dx.doi.org/10.4137/dti.s12519 http://www.la-press.com http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 http://www.la-press.com mailto:lutful_amin@yahoo.com amin 28 drug target insights 2013:7 introduction p-glycoprotein (p-gp) is one of the first members of the atp-binding cassette (abc) transporter which acts as a physiological barrier by extruding toxins and xenobiotics out of cells.1,2 it is being extensively studied and experimented upon recently and is gaining much importance in numerous researches. p-gp is primarily found in epithelial cells which have the excretory roles including apical surface of epithelial cells lining the colon, small intestine, pancreatic ductules, bile ductules, kidney proximal tubules, and the adrenal gland.3–5 it is also located in the endothelial cells of the blood brain barrier (bbb).6 the transporter is overexpressed on the surface of many neoplastic cells and restricts cell entry. the role of p-gp is likely to protect these susceptible organs from toxic compounds, preventing them to enter the cytosol and extrude them to the exterior.7 thus it also enhances the secretion of metabolites and xenobiotics into bile, urine, and the lumen of gastrointestinal tract.1 p-gp in human forms a small gene family with two isoforms. the class i isoform (mdr1/abcb1) is a drug transporter while the class ii isoform (mdr2/3/ abcb4) carries out export of phosphatidylcholine into the bile.2,8 a single p-gp molecule can recognize and transport numerous drugs with a wide range of chemical structures, ranging from a molecular weight of 250 g/mol (cimetidine) to 1202 g/mol (cyclosporin).9 p-gp can extrude a wide range of structurally diverse compounds out of the cells. hundreds of substrates (usually hydrophobic) interact with this atp dependent transporter including anticancer agents, immunosuppressants, steroid hormones, calcium channel blockers, beta-adrenoreceptor blockers, cardiac glycosides, among others.2,10,11 less permeable drugs (weak substrates) may also undergo a substantial extrusion. thus it contributes greatly in the extrusion of many drugs from the blood into the intestinal lumen. p-gp is also responsible for enhancing the excretion of drugs out of hepatocytes and renal tubules into the adjacent luminal space. therefore, p-gp can potentially reduce the absorption and oral bioavailability and decrease the retention time of a number of drugs.9,10 additionally, it has a role in limiting cellular uptake of drugs from blood circulation into the brain while being present in the bbb.6 p-gp is overexpressed in cancer cells and is responsible for drug efflux in tumors. it prevents cell internalization of chemotherapeutic agents and makes the chemotherapy almost ineffective in many cases (fig. 1). hence, this protein is one of the main barriers in cancer treatment by chemotherapy.12,13 a variety of strategies are being developed to overcome the difficulties associated with p-gp in optimum drug delivery. those include not only inhibition of p-gp, but also various techniques to bypass it.1,4,15,16 these promising approaches for optimizing drug delivery and targeting will be the focus of discussions in this review. inhibition of p-gp the inhibition of efflux pump is mainly done in order to improve the delivery of therapeutic agents. in general, p-gp can be inhibited by three mechanisms: (i) blocking drug binding site either competitively, non-competitively (fig. 2) or allosterically; (ii) interfering with atp hydrolysis; and (iii) altering integrity of cell membrane lipids.1,10,17–19 the goal is to achieve improved drug bioavailability, uptake of drug in the targeted organ, and more efficacious cancer chemotherapy through the ability to selectively block the action of p-gp. inhibitors are as structurally diverse as substrates.19 many inhibitors figure 1. drug efflux by p-glycoprotein. http://www.la-press.com p-glycoprotein inhibition for optimal drug delivery drug target insights 2013:7 29 (verapamil, cyclosporin a, trans-flupenthixol, etc.) are themselves transported by p-gp.2 p-gp inhibitors are classified into three generations based on their specificity, affinity, and toxicity. first generation inhibitors are pharmacologically active substances which are clinically used for specific treatments but have the ability to inhibit p-gp. the usage of first generation inhibitors is limited due to their high serum concentrations (at the doses that are required to inhibit p-gp) and potential toxicity.20,21 they are substrate to other transporters and enzyme systems, resulting in unpredictable pharmacokinetic interactions. second generation inhibitors lack the pharmacological activities and possess a greater p-gp affinity which include non-immunosuppressive analogues of cyclosporin a (psc833) and d-isomer of verapamil (dexverapamil). however, second generation inhibitors inhibit the cypa4 enzyme and other abc transporters. hence, metabolizing rate decreases and inhibition of two or more abc transporters lead to complicated pharmacokinetic alterations. third generation p-gp inhibitors are under clinical development, aiming to inhibit p-gp with higher specificity and lower toxicity (eg, tariquidar). they are developed by using structure activity relationships and many were found to be very specific and effective against p-gp, having minimal toxicity.22–24 table 1 illustrates the detailed classification of p-gp inhibitors. figure 2. inhibition of p-glycoprotein to prevent drug efflux. table 1. classification of p-gp inhibitors.1,20,23,25–27 generations examples specificity limitations first generation verapamil, cyclosporin a, reserpine, quinidine, yohimbine, tamoxifen and toremifena non-selective and low binding affinities. they are substrates to other transporters and enzyme systems. they are pharmacologically active. they themselves are transported by p-gp. second generation dexverapamil, dexniguldipine, valspodar (psc 833), and dofequidar fumarate (ms-209) higher specificity then first generation inhibitors but interact with other systems. they are substrates to cyp 3a4 enzyme and other abc transporters. third generation cyclopropyldibenzosuberane zosuquidar (ly335979), laniquidar (r101933), mitotane (nsc-38721), biricodar (vx-710), elacridar (gf120918/gg918), ont-093, tariquidar (xr9576), and hm30181 highest specificity that specifically and potently inhibit p-gp function. no limitations like the first and the second generation inhibitors. http://www.la-press.com amin 30 drug target insights 2013:7 monoclonal antibodies can be effectively used against p-gp in multidrug resistance (mdr) tumor cells. many anti-p-gp monoclonal antibodies, such as mrk16 and mrk17, have been developed to overcome multidrug resistance. conjugated monoclonal antibodies, such as bispecific antibody, immunotoxin, and radioisotope conjugates, have also been constructed to enhance the anti-tumor activity of monoclonal antibodies.28 the conformation-sensitive uic2 monoclonal antibody can inhibit p-gp mediated substrate transport (calcein-am, daunorubicin, and 99 mtc-hexakis-2-methoxybutylisonitrile). however, inhibition by uic2 alone is usually partial because uic2 binds only 10% to 40% of p-gp present in the cell membrane. this antibody recognizes and inhibits the rest of the p-gp molecules only in the presence of certain inhibitors, including vinblastine, cyclosporine a, and psc 833 (valspodar). therefore, simultaneous application of any of these inhibitors and uic2 can enhance accumulation of certain substrates of p-gp by total inhibition of p-gp pump activity.29 enhancement of bioavailability and transport even though p-gp decreases drug absorption of all of its substrates, it quantitatively does not have an equal significant impact on overall drug absorption for all of them. in the case of fast absorbing drugs having larger doses, efflux by p-gp poses less impact on drug absorption and therefore is not important in terms of bioavailability or pharmacokinetic properties. this is because the transport activity of p-gp becomes saturated by high concentrations of drug in the intestinal lumen. in the case of drugs requiring a very small dose for their pharmacological actions or the drugs that have very slow dissolution and diffusion rates, p-gp mediated drug efflux greatly interferes with their delivery. as it decreases drug absorption, those small amounts of drugs cannot reach the blood circulation in sufficient quantity and, at times, can be life threatening. moreover, it can make the sustained release dosage forms of the substrates completely ineffective by limiting their absorptions.9 usually, an inhibitor of p-gp is coadministered with the drug to enhance drug absorption.30,31 methods are being developed in preparing clinically useful oral formulations of drugs having poor oral absorption, which are administered only by parenteral routes. when a drug and an inhibitor are coadministered, p-gp mediated transport mechanism remains the same for both the drug and the inhibitor, if the inhibitor is a substrate of it. in this process, drug and inhibitor must be discriminable by p-gp at molecular level as drug molecules should not be extruded. this is why a large difference between the rate of efflux of the substrate and the inhibitor is created. when the inhibitor molecules are effluxed, they rapidly bind to the p-gp binding sites again and thus the inhibition of p-gp by the modulators is cycled repeatedly, preventing efflux of drugs. this process depends on the hydrophobicity of the compounds.32,33 hm30181, a newly developed third generation p-gp inhibitor, showed promising results for increasing oral absorption of some drugs in recent studies. kwak et al34 examined its pharmacologic characteristics and it showed the highest potency among several p-gp inhibitors, including cyclosporin a, xr9576, and gf120918. the inhibitory activity of hm30181 was highly selective to p-gp and it did not inhibit other abc transporters. its coadministration (10 mg/kg) greatly increased oral bioavailability of paclitaxel from 3.4% to 41.3% in rats. importantly, oral coadministration of paclitaxel and hm30181showed a tumor inhibitory strength equal or superior to that of intravenous paclitaxel in the xenograft model in nude mice.34 p-gp inhibitors may have a great impact on altering pharmacokinetics of a drug.10 since p-gp molecules are present in many organs like bbb, kidney proximal tubule, and bile ductule, their inhibition can potentially improve not only the absorption, but also the distribution, metabolism, and elimination of their substrates.10,35 asperen et al36 observed a 10-fold increased oral bioavailability of paclitaxel in mice administered along with a p-gp blocker (valspodar). sugie et al37 articulated that coadministration of azithromycin and cyclosporine resulted in decreased billiary excretion of azithromycin. bbb is considered as the main barrier to prevent drugs entering the central nervous system (cns). presence of p-gp in the bbb makes it additionally difficult, effluxing the drug molecules from the cns.2,38 p-gp inhibition can prevent p-gp mediated drug efflux and assist the substrate molecules to enter the cns. kemper et al38 reported a 5-fold increase in brain uptake of paclitaxel by gf120918 (elacridar), a third http://www.la-press.com p-glycoprotein inhibition for optimal drug delivery drug target insights 2013:7 31 generation p-gp inhibitor. a reduced clearance of paclitaxel was noticed, assuming the inhibition of p-gp in elimination pathway.38 p-gp inhibition can also increase the half-lives of the substrates as the inhibition may reduce billiary excretion and the clearance of the substrates in kidney proximal tubule, increasing renal reuptake. orally coadministered doxorubicin and verapamil have shown to increase peak plasma level, prolong elimination of half-life, and increase volume of distribution of doxorubicin after oral administration.39 both natural and synthetic inhibitors can be coadministered with drugs in oral dosage forms including polyethylene glycol (peg) based surfactants, anionic gums, sodium alginate, poloxamers, thiomers, dendrimers, etc. among these inhibitors, surfactants are considered to be the better choice as they were already approved for routine use in pharmaceutical formulations. these surfactants act by altering integrity of membrane lipids by changing the secondary and tertiary structure. thus the function of p-gp is hampered due to disturbance in hydrophobic environment.10,40 antimicrobial therapy drug efflux is a common mechanism of resistance in microorganisms, along the same lines as target modification or production of antibiotic inactivating enzymes. efflux pumps are now recognized as one of the most important factors in accumulation of antimicrobials in all cell types, from prokaryotes to superior eukaryotes. mdr, mediated through efflux pumps, has been described for various organisms, including bacteria, fungi, and protozoa. all transporters, but the abc family, function as secondary transporters. p-gp is one of the main abc transporters that is greatly responsible for mdr in microorganisms.41–43 p-gp molecules prevent antimicrobial agents from entering the microorganisms. they reduce intracellular drug concentrations and thereby impede accessibility of drugs to their sites of action, ultimately leading to reduced susceptibility. this transport mechanism may result in treatment failure in many infectious diseases.44,45 mdr is a major problem in bacterial infections because it limits the option of antimicrobial agents. the treatment becomes troublesome in the case of the infections where p-gp substrates are the only choice. it poses several complications including increased costs, prolonged duration of hospital stay, and higher morbidity and mortality rates.46–48 the use of inhibitors may be a good strategy to overcome transporter mediated bacterial multidrug resistance. they can be used in conjunction with antibiotics and can extend the lifetime of the antibiotics, improving therapeutic efficacy. they can also suppress the emergence of resistant variants that may arise during the treatment. these inhibitors increase bacterial susceptibility towards antimicrobial agents.41,49–51 seral et al52 examined the influence of inhibitors of p-gp (verapamil, cyclosporine, and gf120918) on the antimicrobial activity of macrolides (erythromycin, clarithromycin, roxithromycin, azithromycin, and telithromycin) in j774 murine macrophages. the result showed that p-gp enhanced the extrusion of azithromycin, erythromycin, telithromycin, and roxithromycin, resulting in suboptimal drug accumulation. hence, inhibitors can enhance their accumulations inside the cells and increase antimicrobial actions.52 leitner et al49 investigated the potency of tariquidar, a third-generation p-gp inhibitor, for overcoming bacterial resistance towards ciprofloxacin. activity of tariquidar was evaluated by staphylococcus aureus strains. the addition of tariquidar resulted in a 10-fold reduction in the minimum inhibitory concentration (mic) of ciprofloxacin. the result suggested that tariquidar significantly increased susceptibility of s. aureus towards ciprofloxacin. their high activity can be very promising for applications in infectious diseases. their study also showed the improved susceptibility of stenotrophomonas maltophilia towards elacridar.48 cancer chemotherapy p-gp is overexpressed on the surface of cancer cells and prevents drug accumulation inside the tumor, acting as the efflux pump. it extrudes anticancer drugs before they can reach the intended target. further, it often mediates the development of resistance of the cells to anticancer drugs. therefore, the administered drugs remain ineffective or cannot bring the desired output. several approaches have been taken to overcome p-gp mediated drug resistance.53,54 p-gp locates drugs which are localized in the plasma membrane only. concurrent administration of cytotoxic drugs and inhibiting agents, like verapamil or cyclosporine, can restrain p-gp mediated extrusion and facilitate the http://www.la-press.com amin 32 drug target insights 2013:7 drug in reaching the targeted area. thus both chemotherapeutic agent and inhibiting agent are incorporated into the carrier system to overcome the difficulty associated with p-gp.55,56 another strategy could be the involvement of anti-p-gp monoclonal antibody in refraining p-gp from extruding drugs. in this process, the antibody is conjugated to the drug loaded carrier system, which can sufficiently inhibit drug efflux. vincristine-loaded lipid nanoparticles conjugated to an anti-p-gp monoclonal antibody (mrk-16), showed greater cytotoxicity in resistant human myelogenous leukemia cell lines than non-targeted particles.14 goda et al29 reported that combination therapy with uic2 monoclonal antibody and cyclosporine a, a first generation p-gp inhibitor administered at the dose ineffective when applied alone, dramatically increased daunorubicin accumulation in xenotransplanted pgp+ tumors. it established that combined application of uic2 antibody and a class of modulators, used at low concentrations, can be a specific and effective way of blocking p-gp function in vivo.29 danson et al57 developed sp1049c, a non-ionic block copolymer composed of a hydrophobic core and hydrophilic tail, which contained doxorubicin and was able to circumvent p-gp mediated drug resistance in a mouse model of leukaemia.57,58 tidefelt et al59 found that by interacting with p-gp, valspodar can increase the cellular uptake of daunorubicin in leukemic blasts in vivo. in another study, folic acid, attached to peg derivatized distearoyl-phosphatidylethanolamine in doxorubicin loaded liposomes, was used to target folate receptor overexpressing tumor cells. folate receptor mediated cell uptake of targeted liposomal doxorubicin into a multidrug resistant subline of m109-hifr cells (m109r-hifr) was clearly unaffected by p-gp mediated drug efflux, in sharp contrast to uptake of free doxorubicin.15 conclusion p-gp is one of the main barriers for delivering drugs properly. a variety of approaches are being tested to develop p-gp inhibitors or mechanisms to bypass it. proper inhibition will allow not only an increase in cellular uptake, transport, and half-lives of drugs, but also to predict their pharmacokinetics accurately and fine tune them for targeting specific region. these advances will result in cost effective therapy by saving the additional amount of drugs that was previously wasted by p-gp transport. furthermore, it will shorten the treatment time with optimal drug delivery. thus it can bring a great revolution in the field of drug delivery. author contributions conceived and designed the experiments: mla. analyzed the data: mla. wrote the first draft of the manuscript: mla. contributed to the writing of the manuscript: mla. agree with manuscript results and conclusions: mla. jointly developed the structure and arguments for the paper: mla. made critical revisions and approved final version: mla. author reviewed and approved of the final manuscript. funding author(s) disclose no funding sources. competing interests author(s) disclose no potential conflicts of interest. disclosures and ethics as a requirement of publication the author has provided signed confirmation of compliance with ethical and legal obligations including but not limited to compliance with icmje authorship and competing interests guidelines, that the article is neither under consideration for publication nor published elsewhere, of their compliance with legal and ethical guidelines concerning human and animal research participants (if applicable), and that permission has been obtained for reproduction of any copyrighted material. this article was subject to blind, independent, expert peer review. the reviewers reported no competing interests. references 1. srivalli kmr, lakshmi pk. overview of p-glycoprotein inhibitors: a rational outlook. braz j pharm sci. 2012;48(3):353–67. 2. sharom fj. the p-glycoprotein multidrug transporter. essays biochem. 2011;50(1):161–78. 3. edwards je, alcorn j, savolainen j, anderson bd, mcnamara pj. role of p-glycoprotein in distribution of nelfinavir across the blood-mammary tissue barrier and blood-brain barrier. antimicrob agents chemother. 2005;49(4): 1626–8. 4. melaine n, liénard mo, dorval i, le goascogne c, lejeune h, jégou b. multidrug resistance genes and p-glycoprotein in the testis of the rat, mouse, guinea pig, and human. biol reprod. 2002;67(6):1699–707. 5. beaulieu e, demeule m, ghitescu l, beliveau r. p-glycoprotein is strongly expressed in the luminal membranes of the endothelium of blood vessels in the brain. biochem j. 1997;326(pt 2):539–44. 6. ma jd, tsunoda sm, bertino js jr, trivedi m, beale kk, nafziger an. evaluation of in vivo p-glycoprotein phenotyping probes: a need for validation. clin pharmacokinet. 2010;49(4):223–37. http://www.la-press.com p-glycoprotein inhibition for optimal drug delivery drug target insights 2013:7 33 7. fortuna a, alves g, falcao a. in vitro and in vivo relevance of the p-glycoprotein probe substrates in drug discovery and development: focus on rhodamine 123, digoxin and talinolol. j bioequiv availab. 2011:s2. 8. ruetz s, gros p. phosphatidylcholine translocase: a physiological role for the mdr2 gene. cell. 1994;77(7):1071–81. 9. lin jh, yamazaki m. role of p-glycoprotein in parmacokinetics: clinical implications. clin pharmacokinet. 2003;42(1):59–98. 10. varma mvs, ashokraj y, dey cs, panchagnula r. p-glycoprotein inhibitors and their screening: a perspective from bioavailability enhancement. pharmacol res. 2003;48(4):347–59. 11. sharom fj, liu r, qu q, romsicki y. exploring the structure and function of the pglycoprotein multidrug transporter using fluorescence spectroscopic tools. seminars cell dev biol. 2001;12(3):257–65. 12. martins a, vasas a, schelz z, et al. constituents of carpobrotus edulis inhibit p-glycoprotein of mdr1-transfected mouse lymphoma cells. anticancer res. 2010;30(3):829–35. 13. bansal t, jaggi m, khar rk, talegaonkar s. emerging significance of flavonoids as p-glycoprotein inhibitors in cancer chemotherapy. j pharm pharm sci. 2009;12(1):46–78. 14. matsuo h, wakasugi m, takanaga h, et al. possibility of the reversal of multidrug resistance and the avoidance of side effects by liposomes modified with mrk-16, a monoclonal antibody to p-glycoprotein. j control release. 2001;77(1–2):77–86. 15. goren d, horowitz at, tzemach d, tarshish m, zalipsky s, gabizon a. nuclear delivery of doxorubicin via folate-targeted liposomes with bypass of multidrug-resistance efflux pump. clin cancer res. 2000;6(5): 1949–57. 16. mazel m, clair p, rousselle c, et al. doxorubicin—peptide conjugates overcome multidrug resistance. anticancer drugs. 2001;12(2):107–16. 17. shapiro ab, ling v. effect of quercetin on hoechst 33342 transport by purified and reconstituted p-glycoprotein. biochem pharmacol. 1997;53(4): 587–96. 18. drori s, eytan gd, assaraf yg. potentiation of anticancer drug cytotoxicity by multidrug-resistance chemosensitizers involves alterations in membrane fluidity leading to increased membrane permeability. eur j biochem. 1995;228(3):1020–9. 19. robert j, jarry c. multidrug resistance reversal agents. j med chem. 2003;46(23):4805–17. 20. lomovskaya o, bostian ka. practical applications and feasibility of efflux pump inhibitors in the clinic—a vision for applied use. biochem pharmacol. 2006;71(7):910–8. 21. lomovskaya o, zgurskaya hi, totrov m, watkins wj. waltzing transporters and ‘the dance macabre’ between humans and bacteria. nat rev drug discov. 2007;6(1):56–65. 22. kuppens ie, witteveen eo, jewell rc, et al. a phase i, randomized, open-label, parallel-cohort, dose-finding study of elacridar (gf120918) and oral topotecan in cancer patients. clin cancer res. 2007;13(11): 3276–85. 23. pusztai l, wagner p, ibrahim n, et al. phase ii study of tariquidar, a selective p-glycoprotein inhibitor, in patients with chemotherapy-resistant, advanced breast carcinoma. cancer. 2005;104(4):682–91. 24. malingré mm, beijnen jh, rosing h, et al. co-administration of gf120918 significantly increases the systemic exposure to oral paclitaxel in cancer patients. br j cancer. 2001;84(1):42–7. 25. thomas h, coley hm. overcoming multidrug resistance in cancer: an update on the clinical strategy of inhibiting p-glycoprotein. cancer control. 2003;10(2):159–65. 26. krishna r, mayer ld. multidrug resistance (mdr) in cancer. mechanisms, reversal using modulators of mdr and the role of mdr modulators in influencing the pharmacokinetics of anticancer drugs. eur j pharm sci. 2000;11(4):265–83. 27. newman mj, rodarte jc, benbatoul kd, et al. discovery and characterization of oc144–093, a novel inhibitor of p-glycoprotein-mediated multidrug resistance. cancer res. 2000;60(11):2964–72. 28. nokihara h, yano s, sone s. overcoming of multidrug resistance by antip-glycoprotein monoclonal antibody. nippon rinsho. 1997;55(5):1128–34. japanese. 29. goda k, fenyvesi f, bacsó z, et al. complete inhibition of p-glycoprotein by simultaneous treatment with a distinct class of modulators and the uic2 monoclonal antibody. j pharmacol exp ther. 2007;320(1):81–8. 30. woo js, lee ch, shim ck, hwang sj. enhanced oral bioavailability of paclitaxel by coadministration of the p-glycoprotein inhibitor kr30031. pharm res. 2003;20(1):24–30. 31. banerjee sk, jagannath c, hunter rl, dasgupta a. bioavailability of tobramycin after oral delivery in fvb mice using crl-1605 copolymer, an inhibitor of p-glycoprotein. life sci. 2000;67(16):2011–16. 32. eytan gd, regev r, oren g, assaraf yg. the role of passive transbilayer drug movement in multidrug resistance and its modulation. j biol chem. 1996;271(22):12897–902. 33. döppenschmitt s, spahn-langguth h, regårdh cg, langguth p. role of p-glycoprotein-mediated secretion in absorptive drug permeability: an approach using passive membrane permeability and affinity to p-glycoprotein. j pharm sci. 1999;88(10):1067–72. 34. kwak jo, lee sh, lee gs, et al. selective inhibition of mdr1 (abcb1) by hm30181 increases oral bioavailability and therapeutic efficacy of paclitaxel. eur j pharmacol. 2010;627(1–3):92–8. 35. batrakova ev, miller dw, li s, alakhov vy, kabanov av, elmquist wf. pluronic p85 enhances the delivery of digoxin to the brain: in vitro and in vivo studies. j pharmacol exp ther. 2001;296(2):551–7. 36. van asperen j, van tellingen o, sparreboom a, et al. enhanced oral bioavailability of paclitaxel in mice treated with the p-glycoprotein blocker sdz psc 833. br j cancer. 1997;76(9):1181–3. 37. sugie m, asakura e, zhao yl, et al. possible involvement of the drug transporters p glycoprotein and multidrug resistance-associated protein mrp2 in disposition of azithromycin. antimicrob agents chemother. 2004;48(3):809–14. 38. kemper em, van zandbergen ae, cleypool c, et al. increased penetration of paclitaxel into the brain by inhibition of p-glycoprotein. clin cancer res. 2003;9(7):2849–55. 39. kerr dj, graham j, cummings j, et al. the effect of verapamil on the pharmacokinetics of adriamycin. cancer chemother pharmacol. 1986;18(3):239–42. 40. werle m. natural and synthetic polymers as inhibitors of drug efflux pumps. pharm res. 2008;25(3):500–11. 41. gibbons s, oluwatuyi m, kaatz gw. a novel inhibitor of multidrug efflux pumps in staphylococcus aureus. j antimicrob chemother. 2003;51(1): 13–7. 42. poole k. efflux-mediated antimicrobial resistance. j antimicrob chemother. 2005;56(1):20–51. 43. normark bh, normark s. evolution and spread of antibiotic resistance. j intern med. 2002;252(2):91–106. 44. poole k. mechanisms of bacterial biocide and antibiotic resistance. j appl microbiol. 2002;92:55s–64. 45. wright gd. mechanisms of resistance to antibiotics. curr opin chem biol. 2003;7(5):563–9. 46. huet aa, raygada jl, mendiratta k, seo sm, kaatz gw. multidrug efflux pump overexpression in staphylococcus aureus after single and multiple in vitro exposures to biocides and dyes. microbiology (reading, engl). 2008;154(pt 10):3144–53. 47. escribano i, rodríguez jc, llorca b, garcía-pachon e, ruiz m, royo g. importance of the efflux pump systems in the resistance of mycobacterium tuberculosis to fluoroquinolones and linezolid. chemotherapy. 2007;53(6): 397–401. 48. levy sb, marshall b. antibacterial resistance worldwide: causes, challenges and responses. nat med. 2004;10(suppl 12):s122–9. 49. leitner i, nemeth j, feurstein t, et al. the third-generation p-glycoprotein inhibitor tariquidar may overcome bacterial multidrug resistance by increasing intracellular drug concentration. j antimicrob chemother. 2011;66(4): 834–9. 50. van bambeke f, pagès jm, lee vj. inhibitors of bacterial efflux pumps as adjuvants in antibiotic treatments and diagnostic tools for detection of resistance by efflux. recent pat antiinfect drug discov. 2006;1(2): 157–75. 51. renau te, léger r, flamme em, et al. inhibitors of efflux pumps in pseudomonas aeruginosa potentiate the activity of the fluoroquinolone antibacterial levofloxacin. j med chem. 1999;42(24):4928–31. http://www.la-press.com amin 34 drug target insights 2013:7 52. seral c, michot jm, chanteux h, mingeot-leclercq mp, tulkens pm, van bambeke f. influence of p-glycoprotein inhibitors on accumulation of macrolides in j774 murine macrophages. antimicrob agents chemother. 2003;47(3):1047–51. 53. mazel m, clair p, rousselle c, et al. doxorubicin-peptide conjugates overcome multidrug resistance. anticancer drugs. 2001;12(2):107–16. 54. raub tj. p-glycoprotein recognition of substrates and circumvention through rational drug design. mol pharm. 2005;3(1):3–25. 55. brigger i, dubernet c, couvreur p. nanoparticles in cancer therapy and diagnosis. adv drug deliv rev. 2002;54(5):631–51. 56. soma ce, dubernet c, bentolila d, benita s, couvreur p. reversion of multidrug resistance by co-encapsulation of doxorubicin and cyclosporin a in polyalkylcyanoacrylate nanoparticles. biomaterials. 2000;21(1):1–7. 57. danson s, ferry d, alakhov v, et al. phase i dose escalation and pharmacokinetic study of pluronic polymer-bound doxorubicin (sp1049c) in patients with advanced cancer. br j cancer. 2004;90(11): 2085–91. 58. batrakova ev, dorodnych ty, klinskii ey, et al. anthracycline antibiotics non-covalently incorporated into the block copolymer micelles: in vivo evaluation of anti-cancer activity. br j cancer. 1996;74(10):1545–52. 59. tidefelt u, liliemark j, gruber a, et al. p-glycoprotein inhibitor valspodar (psc 833) increases the intracellular concentrations of daunorubicin in vivo in patients with p-glycoprotein-positive acute myeloid leukemia. j clin oncol. 2000;18(9):1837–44. http://www.la-press.com drug target insights 2013:7 9–17 doi: 10.4137/dti.s11802 this article is available from http://www.la-press.com. © the author(s), publisher and licensee libertas academica ltd. this is an open access article published under the creative commons cc-by-nc 3.0 license. open access full open access to this and thousands of other papers at http://www.la-press.com. drug target insights o r i g i n a l r e s e a r c h drug target insights 2013:7 9 impact of hepatocyte growth factor on skeletal myoblast transplantation late after myocardial infarction stacy b. o’blenes1–3, audrey w. li3, chris bowen4, drew debay4, mohammed althobaiti5 and james clarke5 1iwk health centre, halifax, nova scotia, canada. 2dalhousie university department of surgery, halifax, nova scotia, canada. 3dalhousie university department of physiology and biophysics. 4nrc biomedical mri research lab, halifax, nova scotia, canada. 5dalhousie university department of radiology, nova scotia, canada. corresponding author email: stacy.oblenes@iwk.nshealth.ca abstract: in clinical studies, skeletal myoblast (skmb) transplantation late after myocardial infarction (mi) has minimal impact on left ventricular (lv) function. this may be related to our previous observation that the extent of skmb engraftment is minimal in chronic mi when compared to acute mi, which correlates with decreased hepatocyte growth factor (hgf) expression, an important regulator of skmb function. here, we investigated delivery of exogenous hgf as a strategy for augmenting skmb engraftment late after mi. rats underwent skmb transplantation 4 weeks after coronary ligation. hgf or vehicle control was delivered intravenously during the subsequent 2 weeks. lv function was assessed by mri before and 2 weeks after skmb transplantation. we evaluated hgf delivery, skmb engraftment, and expression of genes associated with post-mi remodeling. serum hgf was 6.2 ± 2.4 ng/ml after 2 weeks of hgf infusion (n = 7), but undetectable in controls (n = 7). lv end-diastolic volume and ejection fraction did not improve with hgf treatment (321 ± 27 mm3, 42% ± 2% vs. 285 ± 33 mm3, 43% ± 2%, hgf vs. control). mis were larger in hgf-treated animals (50 ± 7 vs. 30 ± 6 mm3, p = 0.046), but the volume of engrafted skmbs or percentage of mis occupied by skmbs did not increase with hgf (1.7 ± 0.3 mm3, 4.7% ± 1.9% vs. 1.4 ± 0.4 mm3, 5.3% ± 1.6%, hgf vs. control). expression of genes associated with post-infarction remodeling was not altered by hgf. delivery of exogenous hgf failed to augment skmb engraftment and functional recovery in chronic mi. expression of genes associated with lv remodeling was not altered by hgf. alternative strategies to enhance engraftment of skmb must be explored to optimize the clinical efficacy of skmb transplantation. keywords: cell transplantation, heart failure, myocardial infarction http://dx.doi.org/10.4137/dti.s11802 http://www.la-press.com http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 http://www.la-press.com mailto:stacy.oblenes@iwk.nshealth.ca o’blenes et al 10 drug target insights 2013:7 introduction skeletal myoblast (skmb) transplantation has been extensively investigated as a strategy to positively influence ventricular remodeling following myocardial infarction (mi). while skmbs transplanted by direct injection improved ventricular function in animal models of myocardial injury,1,2 a randomized trial in patients with chronic mi did not show improved ventricular function compared to controls.3 we previously reported that the timing of skmb transplantation relative to the mi influences the extent of engraftment.4 if skmb transplantation is delayed until a chronic mi scar has developed, there is a 95% reduction in the volume of engrafted skmbs compared to transplantation immediately after mi.4 this may be why in the clinical setting, skmb transplantation into a chronic mi scar does not have a major impact on ventricular remodeling.3,5 strategies to augment engraftment of skmbs transplanted late after mi may enhance the utility of this therapy in the clinical situation, where the time required for isolation and expansion of autologous skmbs in culture precludes immediate delivery after an mi. we observed a correlation between the degree of skmb engraftment and the amount of hepatocyte growth factor (hgf) present in the mi, which declined over time.4 hgf is the primary regulator of skeletal muscle repair by skmbs.6–9 we showed in vitro that recombinant human hgf can augment skmb proliferation and protect against hypoxia and oxidative stress,4 effects that may assist skmb engraftment following transplantation into an mi. since decreased hgf in the mi scar over time correlates with degree of skmb engraftment,4 the aim of this study was to determine whether delivery of exogenous hgf could enhance engraftment of skmbs transplanted into a chronic mi scar. methods animals inbred dark agouti transgenic rats expressing β-galactosidase under the cag promoter (datg(cag-lacz)30 jmsk)10 were obtained from the rat resource and research center (columbia, mo, usa) and a breeding colony established at our center. these animals have widespread β-galactosidase activity that is particularly strong in skeletal muscle (fig. 1a). we maintained inbred transgene positive and wild-type figure 1. characterization of β-galactosidase staining. representative images from x-gal stained tissues and cultured cells. (a) skeletal muscle from transgene positive rats used as skmb donors showing widespread x-gal staining. (b) myocardium from transgene negative rats identical to those which are subjected to coronary ligation and skmb transplantation showing no staining for x-gal. (c) only a small percentage of skmbs used for transplantation stained positive with x-gal. notes: scale bars = 10 mm (a and b) and 70 µm (c). lines, which were used as skmb donors and recipients, respectively. all animals were genotyped prior to inclusion in the study using the redextract-n-amp tissue pcr kit (sigma chemical co., st. louis, mo, usa) according to manufacturer’s instructions and the primers gaatctctatcgtgcggtggttga (forward) and gccgtgggtttcaatattggcttc (reverse). animals were cared for in accordance with the canadian council on animal care guidelines. the dalhousie university committee on laboratory animals approved the experimental protocol. experimental protocol the experimental protocol was 6 weeks long (fig. 2). male transgene negative rats (skmb recipients, approximately 20 weeks old) underwent coronary ligation. during week 4, cardiac magnetic resonance imaging (mri) was performed and skmbs were harvested from male transgene-positive rats (skmb donors, approximately 24 weeks old). implantation of http://www.la-press.com impact of hgf on myoblast transplant drug target insights 2013:7 11 end-diastole and end-systole were determined based on visual inspection of short axis images (fig. 3). manual planimetry of endocardial contours from 500-µm thick and short continuous axis slices through the entire left ventricle (18 to 20 per heart) was used to calculate lv volumes. skeletal myoblast isolation and culture skmbs were isolated as previously described.4 briefly, donor rats were anesthetized and the soleus muscles were removed and enzymatically dissociated. cells isolated by centrifugation were plated in laminin-coated dishes (1 µg/cm2, sigma) and cultured in ham’s f-12 media (invitrogen, carlsbad, ca, usa) containing 20% fetal bovine serum (fbs, invitrogen). after 7 days in culture, the cells were harvested by trypsinization and re-suspended in minimum essential medium (sigma) at a final concentration of 1 × 106 cells/85 µl. this technique yields cells with approximately 95% viability according to trypan blue exclusion and approximately 90% positive labeling with antibodies against desmin, a cytoskeletal protein expressed in myoblasts and myocytes. only 5% ± 1% of cultured cells showed β-galactosidase activity (fig. 1c). implantation of osmotic pump and skeletal myoblast transplantation implantable osmotic intravenous infusion pumps (200 µl, 0.5 µl/h, alzet, cupertino, ca, usa) were loaded with human recombinant hgf (2.5 mg/ml, genentech, san francisco, ca, usa) in buffer (500 mm nacl, 20 mm tris-hcl, dextran sulfate 25 mg/ml) or vehicle control (buffer only) and primed overnight (37 °c) in 0.9% nacl before implantation. the activity of hgf used in this experiment for promoting skmb proliferation had been confirmed in a cell culture assay (data not shown). lad ligation 1st mri pump implantation skmb transplantation 2nd mri control hgf week 0 2 4 6 figure 2. experimental protocol. notes: skmb transplantation occurred 4 weeks after coronary ligation. hgf or vehicle control was delivered over a two-week period following skmb transplantation by implanted minipump. cardiac mri was performed before skmb transplantation and prior to the end of the experiment. figure 3. cardiac mri. representative images from mri data showing examples of short axis lv chamber contours determined at end diastole (a) and end systole (b). notes: contours collected at 500 µm intervals throughout the lv were used to generate lv volumes. scale bar = 5 mm. an osmotic pump carrying hgf or vehicle control, and transplantation of skmbs into mi scars of recipients occurred at the end of week 4. a second cardiac mri was performed at the end of week 6. coronary ligation rats were anesthetized with ketamine hydrochloride (26 mg/kg intraperitoneally), xylazine (4.8 mg/kg intraperitoneally), and isoflurane (approximately 2%), intubated, and ventilated. the mid-left anterior descending coronary artery was ligated through a left thoracotomy. animals were extubated when breathing spontaneously. ketoprofen (5 mg/kg subcutaneously) and buprenorphine (0.03 mg/kg subcutaneously, every 8 to 12 h × 4 doses) were used for analgesia. cardiac mri mri scans were performed using a magnex scientific scanner (oxford, uk) interfaced with a direct drive spectrometer (varian inc., palo alto, ca, usa). a custom quadrature radio frequency (rf) transmit/ receive volume coil was used for imaging. rats were anesthetized with isoflurane (2%–3%) and were spontaneously breathing. a 3d balanced steadystate, free precession, self-gated sequence adapted from a previously described technique11 was used to obtain 500 µm isotropic resolution volumetric images (approximately 26 per cardiac cycle). raw 3d image sets were imported into rview (colin studholme, university of california), de-identified, and provided to a cardiac radiologist for assessment of left ventricular (lv) volumes and ejection fraction (ef). http://www.la-press.com o’blenes et al 12 drug target insights 2013:7 rats were anesthetized as described above, the left jugular vein was cannulated, and a bolus injection of hgf (30 µg in 500 µl buffer) or vehicle control (buffer only) was administered. the pump cannula was secured in the jugular vein and the pump was implanted subcutaneously. previous thoracotomy was reopened and skmb transplantation was performed as previously described.4 four injections of 1 × 106 skmbs were performed in a diamond pattern within each infarct (total of 4 × 106 cells per animal). animals were recovered as described above. sample collection rats were euthanized 2 weeks after skmb transplantation. serum was collected for assessment of hgf concentration. the left ventricle was opened longitudinally opposite to the mi, pinned to a dish with the endocardium exposed, and fixed in 2% paraformaldehyde followed by 30% sucrose. sections (40-µm thick) were cut parallel to the endocardial surface using a freezing microtome. samples of left ventricular myocardium remote from the infarct were snap-frozen in liquid nitrogen for later mrna isolation. remaining fluid in the osmotic pump was collected and quantified. hepatocyte growth factor elisa serum hgf levels were quantified using the quantikine human hgf immunoassay kit (r&d systems, minneapolis, mn, usa) according to the manufacturer’s instructions. a standard curve was generated using recombinant human hgf (genentech). the assay has a detection limit of 40 pg/ml. immunofluorescence and β-galactosidase staining, quantification of infarct size, and skeletal myoblast engraftment every fifth section through the ventricular wall was stained for β-galactosidase activity (novaultra special stain kit, ihc world, woodstock, md, usa) according to the manufacturer’s instructions. the same sections were then immunofluorescence double-labeled as described previously4 with antibodies against skeletal myosin to identify engrafted skmbs and connexin 43 to identify surviving myocardium. immunofluorescence staining was quantified using a computerized image analysis system as previously described.4 briefly, total infarct area (absence of connexin 43 labeling) and area occupied by engrafted skmb’s (positive for skeletal myosin) was quantified on each section and the corresponding volumes calculated from the entire stack of serial sections. mrna isolation and quantitative pcr left ventricular myocardium remote from the infarct was collected and immediately frozen in liquid nitrogen. rna was extracted using the aurum total rna fatty and fibrous tissue kit (bio-rad, hercules, ca, usa), quantified using the qubit rna assay kit (invitrogen), and reverse-transcribed using the iscriptcdna synthesis kit (bio-rad). quantitative gene expression was assessed using a cfx96 realtime pcr detection system (bio-rad) and sybr green technology (bio-rad). fibronectin (fn), collagen-i (c-i), collagen iii (c-iii), matrix metalloproteinases (mmp)-2 and -9, membrane type 1-mmp (mt1-mmp), and monocyte chemoattractant peptide-1 (mcp-1) gene expression was assessed using the primers shown in table 1. pcr conditions were optimized for each set of primers. melting curve analysis showed a single pcr product for each gene amplified. the pcr cycling profile consisted of an initial denaturation at 95 °c for 30 s and 35 cycles of 95 °c (5 s) and 58 or 60 °c (5 s), depending on the primer set (table 1), for annealing with extension. the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (gapdh) was used for normalization. relative gene expression was calculated using the 2-∆∆ct method.12 results coronary artery ligation and skeletal myoblast transplantation thirty-one rats underwent lad ligation. nine (30%) died, either from arrhythmia immediately after ligation or from euthanasia due to respiratory distress in the post-operative period. twenty-two rats returned to the operating room 4 weeks later for implantation of the osmotic pump and skmb transplantation. three animals died intraoperatively due to bleeding during re-thoracotomy and three died during the postoperative period (1 control animal and 2 hgf-treated animals). two animals showed no evidence of mi and were excluded. a total of 14 rats (7 control and 7 hgf) successfully completed the protocol. initial weights at the start of the experiment were similar (263 ± 8 g for control vs. 270 ± 8 g for hgf), and http://www.la-press.com impact of hgf on myoblast transplant drug target insights 2013:7 13 weight changes during the experiment did not differ (-8 ± 2 g for control vs. -3 ± 4 g for hgf, p = 0.35). hepatocyte growth factor delivery based on the amount of residual fluid in the osmotic pumps at the end of the experiment, 192 ± 11 µl of buffer containing hfg was delivered over the 2-week period. including the initial 30 µg bolus of hgf delivered at the time of pump implantation, the amount of hgf delivered over the 2-week period was 509 ± 28 µg. serum hgf levels measured by enzyme-linked immunosorbent assay (elisa) were 6.2 ± 2.4 ng/ml in the hgf group and were undetectable in the plasma of all animals in the control group. ventricular dimensions lv volumes and ef were assessed by mri before and 2 weeks after skmb transplantation (fig 2). lv volumes and ef did not differ between control and hgf groups prior to skmb transplantation (table 2). there was no progression in lv dilation during table 1. primer sequences for real-time pcr analysis of gene expression. gene primer annealing temp. (°c) accession number gapdh 60 af 106860 forward 5′-atgactctacccacggcaag-3′ reverse 5′-ctggaagatggtgatgggtt-3′ fibronectin 60 nm_019143 forward 5′-gcgactctgactggccttac-3′ reverse 5′-ccgtgtaagggtcaaagcat-3′ collagen i 60 xm_213440 forward 5′-tgctgccttttctgttcctt-3′ reverse 5′-aaggtgctgggtagggaagt-3′ collagen iii 58 bc_087039 forward 5′-gtccacgaggtgacaaaggt-3′ reverse 5′-catcttttccaggaggtcca-3′ mmp-2 60 nm_031054 forward 5′-accgtcgcccatcatcaa-3′ reverse 5′-ttgcactgccaactctttgtct-3′ mmp-9 60 nm_031055 forward 5′-tcgaaggcg acctcaagtg-3′ reverse 5′-ttcggtgtagctt tggatcca-3′ mt1-mmp 60 – forward 5′-ggatacccaatgcccattggcca-3′ reverse 5′-ccattgggcatccagaagagagc-3′ mcp-1 60 – forward 5′-cacctgctgctactcattcact-3′ reverse 5′-gttctctgtcatactggtcacttct-3′ abbreviations: gapdh, glyceraldehyde-3-phosphate dehydrogenase; mmp, matrix metalloproteinase; mt1, membrane type 1; mcp, monocyte chemoattractant peptide. the 2 weeks after skmb transplantation and slight improvement in lv volumes and ef were generally observed at the second mri, although the differences were not statistically significant. there were no differences between animals that received hgf and those that did not (table 2). infarct size and skeletal myoblast engraftment two weeks after skmb transplantation (6 weeks after mi), the volume of the mi scar and skeletal myoblast engraftment were quantified from immunofluorescence labeled histological sections (fig. 4). the volume of the mi scar was larger in hgf-treated animals compared with in controls (fig. 5a, 50 ± 7 vs. 30 ± 6 mm3, p = 0.046). mis occupied a larger area in the hgf-treated group; however, the difference was not statistically significant (fig. 4b, 54 ± 8 vs. 40 ± 8 mm2, p = 0.24). the volume of engrafted skmbs detected was low and did not increase following administration of hgf (fig. 5a, 1.7 ± 0.3 vs. http://www.la-press.com o’blenes et al 14 drug target insights 2013:7 figure 4. identification of mi and engrafted skmbs. (a) representative merged image from a typical section cut parallel to the endocardial surface. the area shown demonstrates surviving myocardium (arrows) identified by immunofluorescence labeling using antibodies against connexin 43 (green) and engrafted skmbs (arrow heads) identified by immunofluorescence labeling using antibodies against fast skeletal myosin (red). (b) the same sections were also stained for β-galactosidase activity (blue). β-galactosidase-positive cells (arrows) were found exclusively in areas containing engrafted skmbs.identified by fast skeletal myosin staining. note: scale bar = 100 µm. table 2. cardiac mri results. pre skmbtx post skmbtx δ (%) control lvedv (mm3) 312 ± 35 285 ± 33a -27 ± 40 (-2 ± 17) lvesv (mm3) 195 ± 21 162 ± 17b -33 ± 30 (-7 ± 19) lvef (%) 37 ± 3 43 ± 2c +5 ± 2 (+19 ± 9) hgf lvedv (mm3) 383 ± 55 321 ± 27d -62 ± 56 (-14 ± 16)g lvesv (mm3) 249 ± 41 189 ± 29e -60 ± 37 (-17 ± 9)h lvef (%) 34 ± 5 42 ± 2f +8 ± 4 (+28 ± 13)i notes: ap = 0.51 vs. pre-skmbtx; bp = 0.25 vs. pre-skmbtx; cp = 0.06 vs. pre-skmbtx; dp = 0.31 vs. pre-skmbtx; ep = 0.26 vs. pre-skmbtx; fp = 0.07 vs. pre-skmbtx; gp = 0.92 vs. control; hp = 0.69 vs. control; ip = 0.60 vs. control. abbreviations: skmb tx, skeletal myoblast transplantation; lvedv, left ventricular end diastolic volume; lvesv, left ventricular end systolic volume; lvef, left ventricular ejection fraction. 1.4 ± 0.4 mm3, hgf vs. control, p = 0.58). similarly, the percentage of the infarct scar occupied by skmbs did not increase following hgf treatment (4.7 ± 1.9 vs. 5.3% ± 1.6%, hgf vs. control, p = 0.81). expression of genes associated with post-mi ventricular remodeling expression of genes associated with post-mi remodeling19,20 was assessed in lv myocardium remote from the mi (fig. 6). expression (2-∆∆ct) of fn (5.3 ± 0.3 vs. 5.8 ± 0.2, hgf vs. control), c-i (15.4 ± 0.4 vs. 14.7 ± 0.4, hgf vs. control), c-iii (3.3 ± 0.2 vs. 3.7 ± 0.4, hgf vs. control), mmp-2 (4.7 ± 0.1 vs. 5.1 ± 0.2, hgf vs. control), mmp-9 (13.7 ± 0.1 vs. 12.7 ± 0.6, hgf vs. control), mt1-mmp (7.3 ± 0.1 vs. 7.7 ± 0.2, hgf vs. control), and mcp-1 (16.6 ± 0.5 vs. 16.3 ± 0.2, hgf vs. control) was not altered by hgf administration. comment in the rat model of mi, infarcts develop into a chronic scar by 4 weeks after coronary ligation. in the present study, we found that skmbs occupy only about 5% of the mi scar when transplantated 4 weeks after coronary ligation. this result is consistent with our previous report that showed 2.5% of the mi occupied by skmbs transplanted 5 weeks after mi, which is a 95% reduction in engraftment compared to when the same number of skmbs were transplanted in the setting of an acute mi.4 mccue et al also reported that only a small percentage of skmbs transplanted 1 month after mi in rabbits can be identified in the heart.13 in humans, only about 1% of skmbs transplanted into chronic mis can be identified through histologic examination.14 the limited engraftment observed when skmbs are transplanted into a chronic infarct scar may explain the modest benefit observed in clinical trials.3,5 because of the time required to expand autologous skmbs in culture before transplantation, it is not practical to perform transplantation very early after mi. therefore, strategies for augmenting engraftment in the setting of chronic mi are necessary for enhancing the clinical impact of this therapy. in our previous study, we found that the extent of myoblast engraftment correlated with hgf immunoreactivity in the mi scar, which declined over time. hgf plays an important role in mediating repair of skeletal muscle by skmbs.6–9 hgf promotes skmb proliferation in vitro4,15 and protects against hypoxia and oxidative stress,4,16 conditions that are present in mi and heart failure.16–18 we therefore hypothesized that delivery of exogenous hgf would enhance engraftment and positively influence lv remodeling. however, we found that delivery of exogenous hgf did not enhance the extent of skmb engraftment when skmbs were transplanted late after mi. http://www.la-press.com impact of hgf on myoblast transplant drug target insights 2013:7 15 there was also no impact on ventricular function, and the expression of genes associated with post-mi ventricular remodeling19,20 in the remote un-infarcted myocardium was not altered. in fact, mi volume was somewhat larger in the hgf group, but this is likely related to variability in the size of the initial infarct rather than an adverse impact on remodeling by hgf. ventricular dimensions were stable during the two week period following skmb transplantation and were not affected by hgf. our results are in contrast with those of tambara et al in which hgf was delivered locally by sustained release matrix placed at the time of transplantation of 5 × 106 skeletal myoblasts 4 weeks after mi (21). in control animals, the volume of skmb engraftment was similar to that observed in our study (5 ± 0.7 mm3). however, with the addition of local hgf delivery, tambara et al observed an approximately 7-fold increase in the volume of engrafted skmbs, whereas we found no impact of hgf delivered systemically. while it is possible that neonatal skmbs used in their study behaved differently than the more clinically relevant adult skmbs used in our model, it is also possible that the route of hgf delivery is important. in the setting of mi, systemic delivery may not provide adequate tissue concentrations of hgf within the infarct scar. poppe et al examined the effect of transfected neonatal skmbs overexpressing hgf on lv function and infarct size following transplantation 2 weeks after coronary ligation.22 consistent with our results, they found that transfecting skmbs overexpressing hgf had no impact on ventricular function after transplantation. however, skmb engraftment was not quantified in the study. it is possible that had we chosen a local delivery method to target the transplanted skmbs with hgf, we would have observed an impact on engraftment rate. we used a dose of hgf based on a previously reported protocol in which human recombinant hgf was delivered in rats at a dose of 100 µg/kg/day for 14 days by osmotic intravenous pump, which was 60 infarct myoblasts 50 40 30 20 v o lu m e ( m m 3 ) 10 0 control hgf * a 80 60 40 20 a re a ( m m 2 ) 0 control hgf b 1.5 1.0 0.5 t h ic k n e s s ( m m ) 0 control hgf c figure 5. delivering exogenous hgf did not enhance engraftment of skmbs. (a) graph of total infarct volume and volume of engrafted skmbs. the total infarct volume was somewhat larger in the hgf group. however, the volume of engrafted skmbs (black bars) was not different in hgf treated animals when compared to controls. (b) graph showing mi area. average area was not significantly different in animals treated with hgf when compared to controls. (c) graph showing average thickness of mi. mi thickness was not significantly different in either group. note: *p , 0.05. 20 control 2 − ∆ ∆ c t hgf 15 10 5 0 f n c -i c -i ii m m p -2 m m p -9 m t 1 -m m p m c p -1 figure 6. expression of genes associated with lv remodeling in noninfracted myocardium was not altered by delivery of exogenous hgf. notes: graph showing gene expression levels of several genes reported to be associated with post-mi remodeling. expression levels were similar in animals receiving hgf (black bars) and controls (white bars). abbreviations: fn, fibronectin; c-i and c-iii, collagen-1 and 3; mmp-2 and mmp-9, matrix metalloproteinase-2 and -9; mt1-mmp, membrane type 1-mmp; mcp-1, monocyte chemoattractant peptide-1. http://www.la-press.com o’blenes et al 16 drug target insights 2013:7 associated with a biological effect in a model of kidney disease.23 while it is possible that a higher dose of hgf would alter our results, high concentrations of hgf are known to have a negative impact on myoblast proliferation by inducing myostatin expression.24 yamada et al demonstrated that hgf concentrations of 2.5 and 10 ng/ml have a positive influence on skmb proliferation; however, beyond this dose range, skmb proliferation is reduced.24 the hgf concentrations used in our experiment (6.2 ± 2.4 ng/ml) appear to be in the optimal range. conclusions in summary, delivery of exogenous hgf by intravenous infusion did not enhance engraftment of adult skmbs transplanted in the setting of a chronic mi scar or positively impact lv remodeling. this finding is in contrast with a previous report demonstrating that local delivery of hgf does enhance engraftment of fetal skmbs transplanted into mis of a similar age. it appears that alternative strategies for enhancing engraftment of adult skmbs in the setting of chronic mi will be required to optimize the potential clinical impact of this therapy. author contributions conceived and designed the experiments: so, al, cb, dd. analysed the data: so, al, cb, dd, ma, jc. wrote the first draft of the manuscript: so. contributed to the writing of the manuscript: so, al, cb, dd, ma, jc. agree with manuscript results and conclusions: so, al, cb, dd, ma, jc. jointly developed the structure and arguments for the paper: so, al, cb, dd, ma, jc. made critical revisions and approved final version: so, al, cb, dd, ma, jc. all authors reviewed and approved of the final manuscript. funding this work was funded in part by the heart and stroke foundation of new brunswick. competing interests author(s) disclose no potential conflicts of interest. disclosures and ethics as a requirement of publication the authors have provided signed confirmation of their compliance with ethical and legal obligations including but not limited to compliance with icmje authorship and competing interests guidelines, that the article is neither under consideration for publication nor published elsewhere, of their compliance with legal and ethical guidelines concerning human and animal research participants (if applicable), and that permission has been obtained for reproduction of any copyrighted material. this article was subject to blind, independent, expert peer review. the reviewers reported no competing interests. references 1. ghostine s, carrion c, souza lc, et al. long-term efficacy of myoblast transplantation on regional structure and function after myocardial infarction. circulation. 2002;106(12 suppl 1):i131–6. 2. al attar n, carrion c, ghostine s, et al. long-term (1 year) functional and histological results of autologous skeletal muscle cells transplantation in rat. cardiovasc res. 2003;58(1):142–8. 3. menasche p, alfieri o, janssens s, et al. the myoblast autologous grafting in ischemic cardiomyopathy (magic) trial: first randomized placebocontrolled study of myoblast transplantation. circulation. 2008;117(9): 1189–200. 4. o’blenes sb, li aw, chen r, arora rc, horackova m. engraftment is optimal when myoblasts are transplanted early: the role of hepatocyte growth factor. ann thorac surg. 2010;89(3):829–35. 5. duckers hj, houtgraaf j, hehrlein c, et al. final results of a phase iia, randomised, open-label trial to evaluate the percutaneous intramyocardial transplantation of autologous skeletal myoblasts in congestive heart failure patients: the seismic trial. eurointervention. 2011;6(7):805–12. 6. morgan je, partridge ta. muscle satellite cells. int j biochem cell biol. 2003;35(8):1151–6. 7. hawke tj, garry dj. myogenic satellite cells: physiology to molecular biology. j appl physiol. 2001;91(2):534–51. 8. husmann i, soulet l, gautron j, martelly i, barritault d. growth factors in skeletal muscle regeneration. cytokine growth factor rev. 1996;7(3): 249–58. 9. wagers aj, conboy im. cellular and molecular signatures of muscle regeneration: current concepts and controversies in adult myogenesis. cell. 2005;122(5):659–67. 10. inoue h, ohsawa i, murakami t, et al. development of new inbred transgenic strains of rats with lacz or gfp. biochem biophys res commun. 2005;329(1):288–95. 11. nieman bj, szulc ku, turnbull dh. three-dimensional, in vivo mri with self-gating and image coregistration in the mouse. magn reson med. 2009;61(5):1148–57. 12. livak kj, schmittgen td. analysis of relative gene expression data using real-time quantitative pcr and the 2(-delta delta c(t)) method. methods. 2001;25(4):402–8. 13. mccue jd, swingen c, feldberg t, et al. the real estate of myoblast cardiac transplantation: negative remodeling is associated with location. j heart lung transplant. 2008;27(1):116–23. 14. pagani fd, dersimonian h, zawadzka a, et al. autologous skeletal myoblasts transplanted to ischemia-damaged myocardium in humans. histological analysis of cell survival and differentiation. j am coll cardiol. 2003;41(5):879–88. 15. sheehan sm, tatsumi r, temm-grove cj, allen re. hgf is an autocrine growth factor for skeletal muscle satellite cells in vitro. muscle nerve. 2000;23(2):239–45. 16. sun y. myocardial repair/remodelling following infarction: roles of local factors. cardiovasc res. 2009;81(3):482–90. http://www.la-press.com impact of hgf on myoblast transplant drug target insights 2013:7 17 17. inoue t, ide t, yamato m, et al. time-dependent changes of myocardial and systemic oxidative stress are dissociated after myocardial infarction. free radic res. 2009;43(1):37–46. 18. hill mf, singal pk. antioxidant and oxidative stress changes during heart failure subsequent to myocardial infarction in rats. am j pathol. 1996; 148(1):291–300. 19. purdham dm, rajapurohitam v, zeidan a, huang c, gross gj, karmazyn m. a neutralizing leptin receptor antibody mitigates hypertrophy and hemodynamic dysfunction in the postinfarcted rat heart. am j physiol heart circ physiol. 2008;295(1):h441–6. 20. gajarsa jj, kloner ra. left ventricular remodeling in the post-infarction heart: a review of cellular, molecular mechanisms, and therapeutic modalities. heart fail rev. 2011;16(1):13–21. 21. tambara k, premaratne gu, sakaguchi g, et al. administration of controlreleased hepatocyte growth factor enhances the efficacy of skeletal myoblast transplantation in rat infarcted hearts by greatly increasing both quantity and quality of the graft. circulation. 2005;112(suppl 9):i129–34. 22. poppe a, golsong p, blumenthal b, et al. hepatocyte growth factor transfected skeletal myoblasts to limit the development of postinfarction heart failure. artif organs. 2012;36(3):238–46. 23. gong r, rifai a, dworkin ld. anti-inflammatory effect of hepatocyte growth factor in chronic kidney disease: targeting the inflamed vascular endothelium. j am soc nephrol. 2006;17(9):2464–73. 24. yamada m, tatsumi r, yamanouchi k, et al. high concentrations of hgf inhibit skeletal muscle satellite cell proliferation in vitro by inducing expression of myostatin: a possible mechanism for reestablishing satellite cell quiescence in vivo. am j physiol cell physiol. 2010;298(3):c465–76. http://www.la-press.com begleiter et al.indd drug target insights 2009:4 1–8 1 original research correspondence: asher begleiter, manitoba institute of cell biology, cancercare manitoba, departments of internal medicine and pharmacology and therapeutics, university of manitoba, 675 mcdermot avenue, winnipeg, manitoba r3e 0v9 canada. tel: +1-204-787-2155; fax: +1-204-787-2190; email: begleit@cc.umanitoba.ca copyright in this article, its metadata, and any supplementary data is held by its author or authors. it is published under the creative commons attribution by licence. for further information go to: http://creativecommons.org/licenses/by/3.0/. a model for nad(p)h:quinoneoxidoreductase 1 (nqo1) targeted individualized cancer chemotherapy asher begleiter1, nadia el-gabalawy2, laurie lange2, marsha k. leith2, lynn j. guziec3 and frank s. guziec jr3 1manitoba institute of cell biology, cancercare manitoba, departments of internal medicine and pharmacology and therapeutics, university of manitoba, 675 mcdermot avenue, winnipeg, manitoba r3e 0v9 canada. 2manitoba institute of cell biology, cancercare manitoba, 675 mcdermot avenue, winnipeg, manitoba r3e 0v9 canada. 3department of chemistry and biochemistry, southwestern university, georgetown, texas 78628 u.s.a. abstract: nqo1 (nad(p)h:quinoneoxidoreductase 1) is a reductive enzyme that is an important activator of bioreductive antitumor agents. nqo1 activity varies in individual tumors but is generally higher in tumor cells than in normal cells. nqo1 has been used as a target for tumor specifi c drug development. we investigated a series of bioreductive benzoquinone mustard analogs as a model for nqo1 targeted individualized cancer chemotherapy. we compared the tumor cell growth inhibitory activity of benzoquinone mustard analogs with sterically bulky groups of different size and placed at different positions on the benzoquinone ring, using tumor cell lines with different levels of nqo1. we demonstrated that functional groups of different steric size could be used to produce a series of bioreductive antitumor agents that were activated by different levels of nqo1 in tumor cells. this series of drugs could then be used to target cells with specifi c levels of nqo1 for growth inhibition and to avoid damage to normal cells, like bone marrow cells, that have low levels of nqo1. this approach could be used to develop new bioreductive antitumor agents for nqo1 targeted individualized cancer chemotherapy. keywords: individualized cancer chemotherapy, tumor targeting, bioreductive agents, nqo1, benzoquinone mustard introduction current anticancer agents are generally potent enough to kill most cancer cells, but their use is limited by toxic side effects. similarities between cancer and normal cells result in a narrow therapeutic index. the use of chemotherapy could be greatly enhanced by new drugs that specifi cally target cancer cells. an important strategy to improve selectivity of anticancer drugs for cancer cells is enzyme-directed tumor targeting.1,2 in this strategy drugs activated by a specifi c enzyme are used to treat tumors with high levels of that enzyme. bioreductive agents are a class of anticancer drugs that must be activated in cells by reductive enzymes. these agents are preferentially active in solid tumors, which represent the majority of cancers and produce the highest mortality. the clinical potential3,4 and mode of action5,6 of these agents have been actively studied, and the use of the prototype drug mitomycin c (mmc) in the clinic has been reviewed.7 other agents like porfi romycin, diaziquone (azq), 3-hydroxymethyl-5-aziridinyl-1-methyl2-(1h-indole-4,7-dione)prop-β-en-α-ol (eo9), tirapazamine (tpz) and 2,5-diaziridinyl-3(hydroxymethyl)-6-methyl-1,4-benzoquinone (rh1) have been tested clinically,7–15 and there is also ongoing work to develop new agents.16,17 these drugs are potent antitumor agents, but produce a variety of side effects including lung,7,18 hearing, muscle and renal toxicity.10,12 however, marrow toxicity is often the dose limiting toxicity for these agents.7,9,10,19 bioreductive agents can be activated by two electron reducing enzymes like nad(p)h:quinoneoxidoreductase 1 (nqo1; dt-diaphorase)1,20 or one electron reducing enzymes like nadph cytochrome p450 reductase (p450 red).5,21 nqo1 is a fl avoprotein that catalyzes 2-electron reduction of quinones and n-oxides.1 several human diaphorases are known,1,22,23 but nqo1 appears to be most important for activating bioreductive agents.1,22,24 nqo1 is a homodimer that uses nad(p)h as an electron donor.1 the enzyme is mainly cytosolic, but a signifi cant proportion of the enzyme is present in the nucleus of cancer cells.25 it is ubiquitous in eukaryotes http://creativecommons.org/licenses/by/3.0/ http://creativecommons.org/licenses/by/3.0/ 2 begleiter et al drug target insights 2009:4 but levels vary in different tissues,1,24,26 with low levels in hematopoetic cells.26 activity is usually higher in tumor than normal cells.24,26,26 nqo1 has been shown to play a role in the activation of cancer chemotherapeutic agents,1,20,28 detoxifi cation of xenobiotics29 and cancer prevention.20,28 activation of bioreductive agents by nqo1 has been extensively studied.1,20 cells with elevated nqo1 levels are more sensitive to mmc and drug activity is decreased by the nqo1 inhibitor, dicoumarol.30–32 thus, nqo1 is a major activating enzyme for mmc. nqo1 is also the major activating enzyme for eo9, 2,5-diaziridinyl3,6-dimethyl-1,4-benzoquinone (medzq) and rh1.1,4,14,33,34 bioreductive agents are ideally suited for enzymedirected tumor targeting because they must be activated in cells by reductive enzymes.1,2 nqo1 has been extensively used as a target for this strategy because it is found in all tissues,24,26 and is preferentially expressed in tumor cells.24,26,27 in addition, nqo1 levels are generally low in hematopoetic cells.26 thus, agents, like rh1, that are selectively activated by nqo1,14 have been developed to target tumors with high nqo1 levels and to avoid bone marrow toxicity. current bioreductive agents have demonstrated the potential of this strategy for improved cancer chemotherapy; however, a number of problems have hindered the development of new targeted anticancer agents. to use nqo1 as a target for enzyme-directed tumor targeting requires an agent specifi cally activated by nqo1. if the drug is also activated by p450 red, which is found at appreciable levels in hematopoetic cells and other organs, this may produce toxic side effects as is seen with mmc.7 however, nqo1 levels vary in individual human tumors. thus, antitumor agents that have a high affi nity for nqo1 may be activated by the low levels of this enzyme in marrow cells resulting in marrow toxicity, while agents that have a low affi nity for nqo1 may not be fully activated in tumors with lower levels of this enzyme resulting in a poor tumor response. the former situation is illustrated by the observation that rh1, which is highly specific for activation by nqo114 but has very high affi nity for this enzyme,35 produces signifi cant marrow toxicity.19 an example of the latter situation is the resistance to mmc observed in tumor cells with low levels of nqo1.30,36,37 bioreductive agents need a bioreductive element that is reduced and a cytotoxic element that is activated by this reduction and produces the cytotoxic effects. current bioreductive agents generally use quinones or nitrogen-oxides as bioreductive elements and alkylating groups like aziridines or nitrogen mustards as cytotoxic elements. alkylating groups bind to dna to produce dna crosslinks1,38,39 leading to cell death by apoptosis.40 using a series of benzoquinone mustards (bm) analogs, we found that sterically bulky groups on the benzoquinone ring signifi cantly decreased the rate of reduction of the bm analogs by nqo1 and reduced their cytotoxic and dna crosslinking activities.41,42 this was likely due to interference by the bulky groups with the ability of the benzoquinone to fi t into the active site of nqo1. the effect of these bulky groups was also infl uenced by the position of the group on the benzoquinone ring moiety. thus, adding an appropriate sterically bulky group to the benzoquinone structure could decrease the affi nity for nqo1. this would make it more diffi cult to activate the new agent in cells with very low levels of nqo1, like bone marrow cells, but could still allow activation in tumor cells with higher levels of this enzyme. since the level of nqo1 in tumors varies in different individuals, an anticancer agent with the minimum affi nity for nqo1 to be fully activated by the level of enzyme in the tumor of a patient would provide the maximum antitumor effect, while minimizing activation in the marrow and the resulting marrow toxicity for that individual. this strategy could provide an optimized therapeutic index for each patient. in this study, we investigated a series of bm analogs as a model for nqo1 targeted individualized cancer chemotherapy. we compared the tumor cell growth inhibitory activity of bm analogs with sterically bulky groups of different size and placed at different positions on the benzoquinone ring, using tumor cell lines with different levels of nqo1. we demonstrated that it was possible to design bioreductive agents with the appropriate nqo1 affi nity to target tumors with specifi c levels of nqo1. materials and methods all media, fetal bovine serum and hank’s balanced salt solutions were obtained from invitrogen (burlington on). all reagents for the nqo1 activity and mtt assays were obtained from sigmaaldrich (st. louis, mo). the fadu, hct116 and 3 a model for nqo1 targeted individualized chemotherapy drug target insights 2009:4 t47d cell lines were obtained from the american type culture collection (rockville, md). fadu, human pharynx squamous carcinoma cells, hct116, human colon carcinoma cells and t47d, human breast ductal carcinoma cells were grown in dulbecco’s modifi ed eagle’s medium: hams f12 (1:1) media with 10% fetal bovine serum. the syntheses of the bm analogs p-mebm, m-mebm, p-pbm and m-pbm (fig. 1) have been previously reported.41,42 m-nprbm was synthesized as described below. for the preparation of the m-nprbm precursor 2-n-propylbenzoquinone, benzoquinone (5.184 g, 48.0 mmol), butyric acid (3.524 g, 40.0 mmol), and silver nitrate (1.02 g, 6.0 mmol) in a acetonitrile-water mixture (1:1) (200 ml) were heated to refl ux while a solution of 1.0 m ammonium persulfate (48 ml) was added dropwise over 1 h. after an additional 1.25 h refl ux, the mixture was cooled to room temperature and solid sodium bicarbonate (3.36 g, 40 mmol) was added in portions to neutralize excess butyric acid. the mixture was extracted with ethyl ether (3x100 ml) and the ether phase washed with 0.5 m sodium bicarbonate, brine and dried over anhydrous sodium sulfate. the crude mixture was concentrated and dissolved in hexanes-ethyl acetate (7:1) (30 ml), and fi ltered through a short column of (50 g) silica gel. concentration afforded the crude product as a yellow oil, 3.51 g. a portion of crude mixture (665 mg) was chromatographed on silica using hexanes-ethyl acetate (7:1), affording pure 2-n-propylbenzoquinone (229 mg) as a yellow semi-solid in 24% overall yield. nmr (cdcl3): δ 6.69–6.78 (complex, 2h), 6.57 (m, 1h), 2.40 (t, 2h), 1.55 (sextet, 2h), 0.98 (t, 3h). for preparation of m-nprbm 6-n-propyl-2[bis(2-chloroethylamino)]-1,4-benzoquinone (fig. 1), potassium fl uoride (0.266 g, 4.58 mmol) was added at room temperature to a stirred suspension of 2-n-propylbenzoquinone (0.229 g, 1.53 mmol), bis(2-chloroethylamine) hydrochloride (0.817 g, 4.58 mmol) and cupric acetate (0.187 g, 2.29 mmol) in 95% ethanol (6.0 ml). the mixture was stirred open to the air but protected from light for 69 h. the mixture was fi ltered with suction through a celite pad to remove copper salts and the precipitate washed with ethyl acetate (2 × 10 ml). the fi ltrates were concentrated, and the residue was taken up in ethyl acetate (30 ml). the organic bm analog p-mebm p-pbm m-pbm m-mebm m-nprbm r1 r1 r2 r2 ch3 ch3 ch3ch2ch2 ch2ch2cl ch2ch2cl h h h h h phenyl phenyl n o o 1 2 3 4 5 6 figure 1. structures of bm analogs. 4 begleiter et al drug target insights 2009:4 phase was washed with 0.15 m hcl (15 ml), brine, and then dried over anhydrous sodium sulfate. concentration, followed by silica chromatography using ethyl acetate-hexanes (1:2) afforded the 6-npropyl-2-[bis(2-chloroethylamino)]-1,4-benzoquinone as a red semi-solid, 0.440 g, 10% yield. 1h nmr (cdcl3): δ 6.42 (m, 1h), 5.63 (d, 1h, j = 2.4 hz), 3.82–3.78 (m, 4h), 3.74–3.69 (m, 4h), 2.37 (t, 2h), 1.54 (sextet, 2h), 0.97 (t, 3h); 13c nmr (cdcl3): δ 186.0, 185.6, 149.0, 147.5, 133.0, 106.0, 54.8, 40.4, 31.2, 21.1, 13.8. nqo1 activity in the cell lines was determined as we have previously described43 using 2, 6-dichlorophenolindophenol (dcpip) as the electron acceptor. the statistical signifi cance of differences in the mean values of the nqo1 activities in the three cell lines was evaluated using a oneway analysis of variance. for cell growth inhibition studies, bm analogs were prepared at various concentrations in dimethylformamide and were added to cells in media. the fi nal concentration of dimethylformamide in the cells was 1%. the cells were incubated with the bm analog at 37 ºc for 1 h. the cells were washed and incubated for 4 cell doublings at 37 °c. cell growth inhibition was determined by 3-(4, 5-dimethylthiazo2-yl)-2,5-diphenyltetrazolium bromide (mtt) assay as we have previously described.44,45 the results are presented as relative absorbance compared with control vs drug concentration curves, and as ic50 values (concentration of drug reducing the relative absorbance to 0.5) obtained from the linear regression lines of the relative absorbance vs drug concentration curves for each bm analog. the ic50 values for the 5 bm analogs in each cell line were compared by one way analysis of variance with student-newman-kuels pairwise multiple comparison. results description of the bm analogs and cell lines the synthesis of the benzoquinone mustards, p-mebm, m-mebm, p-pbm and m-pbm was previously reported.41,42 here we report the synthesis of a new bm analog, m-nprbm (fig. 1). p-mebm and p-pbm have functional group substitutions at the c5 position of the benzoquinone ring, while m-mebm, m-nprbm and m-pbm have functional group substitution at the c6 position of the ring. the relative steric size of the functional groups is ch3 � ch3ch2ch2 � phenyl. the tumor cell growth inhibitory effects of the bm analogs in human cancer cells with different levels of nqo1 were studied to determine if the size and position of the functional groups could determine the level of nqo1 required to activate the analogs. tumor cell growth inhibition studies were carried out in fadu human pharynx squamous carcinoma cells, hct116 human colon carcinoma cells or t47d human breast carcinoma cells. the mean ± standard error of the mean (sem) nqo1 activity in these cells was 557.6 ± 120.3, 230.4 ± 31.3 and 39.7 ± 12.1 nmol.min−1.mg protein−1, respectively, and these activities were statistically different (p � 0.02) (fig. 2). tumor cell growth inhibition by bm analogs fadu, hct116 or t47d cells were treated with various concentrations of each bm analog for 1 h and tumor cell growth inhibition was determined by mtt assay.44,45 figure 3 shows the growth inhibition curves for each of the 5 bm analogs in the 3 tumor cell lines. in all three cell lines, p-mebm had the greatest inhibitory effect while m-pbm cells fadu hct116 t47d n q o 1 a ct iv ity (n m ol .m in –1 .m g pr ot ei n– 1 ) 600 400 200 0 figure 2. nqo1 activity in tumor cell lines. nqo1 activities in fadu, hct116 and t47d cells were determined as described.43 bars represent the mean ± sem of 3 or 4 determinations. 5 a model for nqo1 targeted individualized chemotherapy drug target insights 2009:4 had the smallest effect. in fadu cells, the other 3 bm analogs produced intermediate effects. in hct116 cells, m-mebm and p-pbm continued to produce an intermediate inhibitory effect; however, tumor cell growth inhibition by mnprbm was lower and was similar to that produced by m-pbm. in t47d cells, m-mebm again produced an intermediate tumor cell growth inhibitory effect, while p-pbm, m-nprbm and m-pbm all produced lower but similar inhibitory effects. table 1 shows the ic50 values for the tumor cell growth inhibitory effects of the bm analogs in fadu, hct116 and t47d cells. there were differences in the overall sensitivity of the cell lines to the bm analogs; however, there were also specifi c differences in the sensitivities of the cell lines to individual drugs. in fadu cells, the relative tumor cell growth inhibitory effects of the bm analogs were: p-mebm � m-mebm = p-pbm � m-nprbm � m-pbm. in hct116 cells, the relative inhibitory effects were: p-mebm � m-mebm = p-pbm � m-nprbm = m-pbm. while in t47d cells, the relative inhibitory effects were: pmebm � m-mebm � p-pbm = m-nprbm = mpbm. the differences in activities of the different analogs in each cell line were statistically signifi cant by one way analysis of variance (p � 0.001). discussion we have previously demonstrated that functional groups can signifi cantly infl uence the reduction and activation of bm bioreductive agents by nqo1.41,42 activation of these agents was decreased as the steric size of the functional group increased, and this affect was greater when the functional group was at the c6 position on the benzoquinone ring compared with the c5 position of the ring. we made use of these fi ndings to design a series of bm analogs with different affi nities for activation by nqo1, as a model for developing t47dfadu hct116 r el at iv e a bs or ba nc e p-mebm m-nprbm p-pbm m-pbm m-mebm bm analog concentration (μm) 0 0.1 0.25 0.5 0.75 1 1 2 3 4 5 0 2 4 6 8 10 0.0 0.5 1.0 1.5 2.0 figure 3. cell growth inhibition produced by bm analogs in fadu, hct116 and t47d cells. cells were incubated at 37 °c for 1 h with various concentrations of each bm analog and cell growth inhibition was determined by mtt assay as we have previously described.44,45 the results are presented as relative absorbance compared with control vs. drug concentration. points represent the mean ± sem of 3 or 4 determinations. the lines are linear regression lines. 6 begleiter et al drug target insights 2009:4 nqo1 targeted individualized cancer chemotherapy. the analogs p-mebm and m-mebm have small methyl functional groups at c5 and c6 of the benzoquinone ring, respectively. we expected these analogs to be most easily activated by nqo1 with p-mebm being activated by a lower level of nqo1 activity than m-mebm. the p-pbm and m-pbm analogs have larger phenyl functional groups at c5 and c6 of the ring, respectively. we expected these analogs to be less easily activated by nqo1 than the methyl analogs, with m-pbm requiring the highest level of nqo1 activity. the m-nprbm analog has an n-propyl functional group at c6 of the benzoquinone ring. this group is smaller than the phenyl group and should produce a bm analog that is activated by intermediate levels of nqo1. human tumor cells have varied levels of nqo1 activity ranging from � 10 nmol. min−1.mg protein−1 to � 1000 nmol. min−1.mg protein−1. in contrast, human bone marrow cells generally have a level of nqo1 activity � 30 nmol. min−1.mg protein−1. since bone marrow toxicity is often the dose limiting toxicity for bioreductive agents, an agent that is fully activated by the level of nqo1 in the tumor but is not activated by the level of nqo1 in bone marrow cells would produce little bone marrow toxicity and would have the highest therapeutic index. if a series of bioreductive agents that were activated by different levels of nqo1 were available, a patient could be treated with an agent that was maximally activated by the level of nqo1 in their tumor but minimally activated by the level of nqo1 in their bone marrow. to test this hypothesis, we measured tumor cell growth inhibition produced by the series of bm analogs having different affi nities for activation by nqo1, in human tumor cell lines with a wide range of nqo1 activity. fadu human pharynx squamous carcinoma cells had a high level of nqo1 activity, 557.6 ± 120.3 nmol.min−1.mg protein−1, while hct116 human colon carcinoma cells had a level of nqo1 activity that was close to the average for human tumors, 230.4 ± 31 nmol.min−1.mg protein−1 (fig. 2). in contrast, t47d human breast carcinoma cells had a low level of nqo1 activity similar to that found in human bone marrow cells, 39.7 ± 12.1 nmol.min−1.mg protein−1. when we compared the inhibition of tumor cell growth produced by the bm analogs in the three tumor cell lines we observed a number of signifi cant differences. looking at the overall activities of the bm analogs, the fadu and hct116 cells appeared to have similar sensitivities to the bm analogs as a group, with ic50 values ranging from approximately 0.25 to 6.00 μm in both cell lines. in contrast, the t47d cells appeared to be overall approximately 5-fold more sensitive to these agents as a group (fig. 3, table 1). these differences likely refl ect general tissue type and/or cell line variations in sensitivity to these agents due to differences in drug uptake, drug detoxifi cation, dna repair, induction of apoptosis and activation by other enzymes, and are probably not related to activation of the analogs by nqo1. this suggestion is supported by the fi nding that in relative terms, p-mebm had the largest growth inhibitory effect; m-pbm had the smallest inhibitory effect, and the other bm analogs had intermediate inhibitory effects in all three cell lines. when we examined the growth inhibitory activities of the fi ve bm analogs within each cell line, we observed signifi cant differences in the relative activity of the analogs in the different cell lines. in the fadu cells with the highest nqo1 activity, the relative tumor cell growth inhibitory table 1. tumor cell growth inhibition by bm analogs in cell lines with different nqo1 activities. ic50 (μm) bm analog fadu (n)1 hct116 (n)1 t47d (n)1 p-mebm 0.30 ± 0.08 (4) 0.22 ± 0.01 (3) 0.08 ± 0.01 (4) m-mebm 1.27 ± 0.13 (4) 2.69 ± 0.25 (4) 0.39 ± 0.04 (4) p-pbm 1.39 ± 0.31 (4) 1.85 ± 0.13 (3) 0.63 ± 0.08 (4) m-nprbm 3.10 ± 0.95 (4) 6.10 ± 1.18 (4) 0.80 ± 0.08 (4) m-pbm 5.77 ± 1.31 (4) 6.68 ± 1.53 (3) 0.76 ± 0.02 (3) 1mean ± sem. n = number of determinations. 7 a model for nqo1 targeted individualized chemotherapy drug target insights 2009:4 activity was p-mebm � m-mebm = p-pbm � m-nprbm � m-pbm. this suggests that p-mebm was most activated; m-mebm and p-pbm were substantially activated; m-nprbm was somewhat activated, and m-pbm was poorly activated by the level of nqo1 in these cells. this is consistent with our hypothesis based on the steric size and location of the functional groups. in the hct116 cells with an intermediate level of nqo1 activity, the relative inhibitory activity was p-mebm � m-mebm = p-pbm � m-nprbm = m-pbm, suggesting that p-mebm was most activated; m-mebm and ppbm were substantially activated, and m-nprbm and m-pbm were poorly activated by the level of nqo1 in these cells. this represents a decrease in the relative growth inhibitory effect of m-nprbm in the hct116 cells compared with the fadu cells. this suggests that the n-propyl functional group at the c6 position produces a greater steric effect than the methyl group at the c6 position and the phenyl group at the c5 position. in addition, this result suggests that m-nprbm would not be fully activated in tumors with intermediate levels of nqo1 and would only be maximally effective in tumors with high levels of nqo1 activity. in t47d cells with the lowest nqo1 activity, the relative growth inhibitory activity was p-mebm � m-mebm � p-pbm = m-nprbm = m-pbm, suggesting that p-mebm was most activated; m-mebm was substantially activated, and p-pbm, m-nprbm and m-pbm were poorly activated by the level of nqo1 in these cells. this represents a decrease in the relative growth inhibitory effect of p-pbm in t47d cells compared with hct116 cells. this result suggests that p-pbm would not be fully activated in tumors with low levels of nqo1 and would only be effective in tumors with average or higher nqo1 activity. furthermore, these fi ndings indicate that m-mebm and p-mebm are at least partially activated in cells with low levels of nqo1 activity like bone marrow cells, and would likely produce some bone marrow toxicity. since m-nprbm and p-pbm showed good tumor cell growth inhibition in the fadu cells but poor activity in the t47d cells, these bm analogs would likely have good antitumor activity in tumors with high nqo1 activity but little activity in bone marrow cells. because p-pbm also showed good growth inhibition in hct116 cells, it would likely also have good antitumor activity in tumors with average levels of nqo1, again with little bone marrow toxicity. thus, m-nprbm or p-pbm might be suitable agents for use in patients having tumors with high nqo1; p-pbm might be suitable for use in patients having tumors with average nqo1, and m-mebm might be used in patients having tumors with low nqo1. however, the use of m-mebm would likely result in at least some bone marrow toxicity. the use of p-mebm would likely produce signifi cant bone marrow toxicity in most patients. these results demonstrate the potential of using a series of bioreductive agents with different affi nities for activation by nqo1 for targeted individualized cancer chemotherapy. they also suggest that using functional groups of varying steric size with quinone based bioreductive agents represents a feasible approach to designing new agents with a range of affi nities for activation by nqo1. in summary this study has demonstrated that functional groups of different steric size can be used to produce a series of bioreductive antitumor agents that are activated by different levels of nqo1 in tumor cells. a series of drugs of this type might be used to target cells with specifi c levels of nqo1 for growth inhibition and to avoid damage to normal cells, like bone marrow cells, that have low levels of nqo1. this approach could be used to develop new bioreductive antitumor agents for nqo1 targeted individualized cancer chemotherapy that produce the optimal therapeutic index for each patient. acknowledgments this work was supported by grants from the cancercare manitoba foundation (ab) and from the herbert and kate dishman endowment at southwestern university (fsg) and the robert a. welch foundation (grant af-0005) (fsg). the authors thank lindsay jones for technical assistance. disclosure the authors report no confl icts of interest. references 1. riley rj., workman p. dt-diaphorase and cancer chemotherapy. biochem pharmacol. 1992;43:1657–69. 2. workman p. enzyme-directed bioreductive drug development revisited: a commentary on recent progress and future prospects with emphasis on quinone anticancer agents and quinone metabolizing enzymes, particularly dt-diaphorase. oncol res. 1994;6:461–75. 3. sartorelli ac. therapeutic attack of hypoxic cells of solid tumors: presidential address. cancer res. 1988;48:775–8. 4. workman p, stratford ij. the experimental development of bioreductive drugs and their role in cancer therapy. cancer metastasis rev. 1993;12:73–82. 8 begleiter et al drug target insights 2009:4 5. rockwell s, sartorelli ac, tomasz m, kennedy ka. cellular pharmacology of quinone bioreductive alkylating agents. cancer metastasis rev. 1993;12:165–76. 6. rauth am, marshall rs, kuehl bl. cellular approaches to bioreductive drug mechanisms. cancer metastasis rev. 1993;12:153–64. 7. begleiter a. clinical applications of quinone alkylating agents. frontiers bioscience. 2000;5:e153–71. 8. rockwell s, keyes sr, sartorelli ac. preclinical studies of porfi romycin as an adjunct to radiotherapy. radiat. res. 1988;116:100–13. 9. chamberlain mc, prados md, silver p, levin va. a phase i/ii study of 24 hour intravenous azq. in recurrent primary brain tumors. j neurooncol. 1988;6:319–23. 10. schellens jhm, planting ast, van acker bac, et al. phase i and pharmacologic study of the novel indoloquinone bioreductive alkylating cytotoxic drug e09. j natl cancer inst. 1994;86:906–12. 11. dirix ly, tonnesen f, cassidy j, et al. eo9 phase ii study in advanced breast, gastric, pancreatic and colorectal carcinoma by the eortc early clinical studies group. eur j cancer [a]. 1996;32a:2019–22. 12. senan s, rampling r, graham ma, et al. phase i and pharmacokinetic study of tirapazamine (sr. 4233) administered every three weeks. clin cancer res. 1997;3:31–8. 13. lee dj, trotti a, spencer s, et al. concurrent tirapazamine and radiotherapy for advanced head and neck carcinomas: a phase ii study. int j radiat oncol biol phys. 1998;42:811–5. 14. winski sl, hargreaves rh, butler j, ross d. a new screening system for nad(p)h:quinone oxidoreductase (nqo1)-directed antitumor quinones: identifi cation of a new aziridinylbenzoquinone, rh1, as a nqo1-directed antitumor agent. clin. cancer res. 1998;4:3083–8. 15. loadman pm, phillips rm, lim le, bibby mc. pharmacological properties of a new aziridinylbenzoquinone, rh1 (2,5-diaziridinyl-3(hydroxymethyl)-6-methyl-1,4-benzoquinone), in mice. biochem pharmacol. 2000;59:831–7. 16. loadman pm, bibby mc, phillips rm. pharmacological approach towards the development of indolequinone bioreductive drugs based on the clinically inactive agent eo9. br j pharmacol. 2002;137:701–9. 17. jaffar m, phillips rm, williams kj, et al. 3-substituted-5-aziridinyl1-methylindole-4,7-diones as nqo1-directed antitumour agents: mechanism of activation and cytotoxicity in vitro. biochem pharmacol. 2003;66:1199–206. 18. hortobagyi gn. mitomycin: its evolving role in the treatment of breast cancer. oncology (basel). 1993;50 suppl 1:1–8. 19. digby t, leith mk, thliveris ja, begleiter a. effect of nqo1 induction on the antitumor activity of rh1 in human tumors in vitro and in vivo. cancer chemother pharmacol. 2005;56:307–16. 20. ross d, siegel d, beall h, prakash as, mulcahy rt, gibson nw. dt-diaphorase in activation and detoxifi cation of quinones. cancer metastasis rev. 1993;12:83–101. 21. pan ss, andrews pa, glover cj, bachur nr. reductive activation of mitomycin c and mitomycin c metabolites catalyzed by nadphcytochrome p-450 reductase and xanthine oxidase. j biol chem. 1984;259:959–66. 22. jaiswal ak. human nad(p)h: quinone oxidoreductase (nqo-1) gene structure and induction by dioxin. biochemistry. 1991;30:10647–53. 23. jaiswal ak, burnett p, adesnick m, mcbride ow. nucleotide and deduced amino acid sequence of a human cdna (nqo2) corresponding to a second member of the nad(p)h: quinone oxidoreductase gene family. extensive polymorphism at the nqo2 gene locus on chromosome 6. biochemistry. 1990;29:1899–906. 24. belinsky m, jaiswal ak. nad(p)h:quinone oxidoreductase1 (dt-diaphorase) expression in normal and tumor tissues. cancer metastasis rev. 1993;12:103–17. 25. winski sl, koutalos y., bentley dl, ross d. subcellular localization of nad(p)h:quinone oxidoreductase 1 in human cancer cells. cancer res. 2002;62:1420–4. 26. schlager jj. and powis g. cytosolic nad(p)h:(quinoneacceptor)oxidoreductase in human normal and tumor tissue: effects of cigarette smoking and alcohol. int j cancer. 1990;45:403–9. 27. smitskamp-wilms eg, giaccone g, pinedo hm, van der laan bfam, peters gj. dt-diaphorase activity in normal and neoplastic human tissues; an indicator for sensitivity to bioreductive agents? br j cancer. 1995;72:917–21. 28. begleiter a, leith mk, curphey tj., doherty gp. induction of dt-diaphorase in cancer chemoprevention and chemotherapy. oncol res. 1997;9:371–82. 29. twerdok le, rembish sj, trush ma. induction of quinone reductase and glutathione in bone marrow cells by 1,2-dithiole-3-thione: effect on hydroquinone-induced cytotoxicity. toxicol applied pharmacol. 1992;112:273–81. 30. begleiter a, robotham e, lacey g, leith mk. increased sensitivity of quinone resistant cells to mitomycin c. cancer lett. 1989;45:173–6. 31. siegel d, gibson nw, preusch pc, ross d. metabolism of mitomycin c by dt-diaphorase: role in mitomycin c-induced dna damage and cytotoxicity in human colon carcinoma cells. cancer res. 1990; 50:7483–9. 32. pan s, akman sa, forrest gl, hipsher c, johnson r. the role of nad(p)h:quinone oxidoreductase in mitomycin cand porfi romycinresistant hct 116 human colon-cancer cells. cancer chemother pharmacol. 1992;31:23–31. 33. beall hd, murphy am, siegel d, hargreaves rhj, butler j, ross d. nicotinamide adenine dinucleotide (phosphate):quinone oxidoreductase (dt-diaphorase) as a target for bioreductive antitumour quinones: quinone cytotoxicity and selectivity in human lung and breast cancer cell lines. mol pharmacol. 1995;48:499–504. 34. plumb ja, gerritsen m, milroy r, thomson p, workman p. relative importance of dt-diaphorase and hypoxia in the bioactivation of eo9 by human lung tumor cell lines. int j radiat oncol biol phys. 1994;29:295–9. 35. winski sl, swann e, hargreaves rhj, et al. relationship between nad(p)h:quinone oxidoreductase 1 (nqo1) levels in a series of stably transfected cell lines and susceptibility to antitumor quinones. biochem pharmacol.. 2001;61:1509–16. 36. malkinson am, siegel d, forrest gl, et al. elevated dt-diaphorase activity and messenger rna content in human non-small lung carcinoma: relationship to the response of lung xenografts to mitomycin c. cancer res. 1992;52:4752–7. 37. marshall rs, patterson mc, rauth am. dt-diaphorase activity and mitomycin c sensitivity in non-transformed cell strains derived from members of a cancer-prone family. carcinogenesis. 1991;12:1175–80. 38. dusre l, covey jm, collins c, sinha bk. dna damage, cytotoxicity and free radical formation by mitomycin c in human cells. chem biol interac. 1989;71:63–78. 39. begleiter a and leith mk. activity of quinone alkylating agents in quinone-resistant cells. cancer res. 1990;50:2872–6. 40. begleiter a, lee k, israels lg, mowat mra, johnston jb. chlorambucil induced apoptosis in chronic lymphocytic leukemia (cll) and its relationship to clinical effi cacy. leukemia. 1994;8(suppl 1):s103–6. 41. fourie j, oleschuk c, guziec f jr, et al. the effect of functional groups on reduction and activation of quinone bioreductive agents by dt-diaphorase. cancer chemother pharmacol. 2002;49:101–10. 42. fourie j, guziec f jr, guziec l, fiterman dj, monterrosa c, begleiter a. structure-activity study of benzoquinone bioreductive alkylating agents: effects on dt-diaphorase mediated dna cross-link and strand break formation and relation to mechanisms of cytotoxicity. cancer chemother pharmacol. 2004;53:191–203. 43. doherty gp, leith mk, wang x, curphey tj, begleiter a. induction of dt-diaphorase by 1,2-dithiole-3-thiones in human tumour and normal cells and effect on antitumour activity of bioreductive agents. br j cancer. 1998;77:1241–52. 44. johnston jb, verburg l, shore t, williams m, israels lg, begleiter a. combination therapy with nucleoside analogs and alkylating agents. leukemia. 1994;8(suppl 1):s140–3. 45. kirkpatrick dl, duke m, goh ts. chemosensitivity testing of fresh human leukemia cells using both a dye exclusion assay and a tetrazolium dye (mtt) assay. leuk res. 1990;14:459–66. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true 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drug target insights 2008:3 37–44 37 review correspondence: dr. n. wilczak, university medical center groningen, department neurology, postbus 30001, hanzeplein 1, 9700 rb groningen, the netherlands. tel: 0031-50-3637719; fax: 0031-50-3611707. copyright in this article, its metadata, and any supplementary data is held by its author or authors. it is published under the creative commons attribution by licence. for further information go to: http://creativecommons.org/licenses/by/3.0/. targeting insulin-like growth factor-1 signaling into the central nervous system for promoting myelin repair nadine wilczak, jacques de keyser and daniel chesik department of neurology, university medical center groningen, the netherlands. abstract: multiple sclerosis (ms) is the most common demyelinating disease of the central nervous system (cns). without myelin, nerve impulses in the cns are slowed or stopped, leading to a constellation of neurological symptoms. demyelination also provides a permitting condition for irreversible axonal damage. remyelination of ms lesions largely fails, although oligodendrocyte precursors and premyelinating oligodendrocytes (myelin forming cells) are present in many demyelinated plaques. insulin-like growth factor (igf)-1 is a growth factor that should provide the appropriate signals to promote repair of ms lesions, because it acts as a survival factor for cells of the oligodendrocyte lineage and stimulates myelin synthesis. in a pilot study on ms patients, no detectable remyelinating effects in the cns were observed following subcutaneous administration of igf-1. a number of reasons might explain a lack of benefi cial effects: a) it is unlikely that subcutaneous administration of igf-1 provides suffi cient passage across the blood-brain-barrier and into the cns, b) the biological actions of igf-1 are tightly regulated by several insulin-like growth factor binding proteins (igfbps), which become upregulated in the demyelinated lesions and may prevent access of igf-1 to its receptor, c) igf-1 not only acts on oligodendrocytes, but also stimulates the proliferation of astrocytes, which form the glial scar that impedes repair processes. in this review, we will discuss strategies to enhance igf-1 signaling in the cns utilizing a) alternative routes of administration, b) igf analogues that displace igf-1 from regulatory igfbps and c) strategies to selectively target igf-1 to oligodendrocytes. keywords: insulin-like growth factor (igf)-1, igf-1 analogues, igf-binding proteins, igfbp ligand inhibitors, micrornas, multiple sclerosis introduction multiple sclerosis (ms) is a chronic multifocal demyelinating disease of the central nervous system (cns) of unknown etiology. one in thousand persons in europe are affl icted with this disease which generally occurs between the ages of 30–40 (noseworthy et al. 2000). pathological hallmarks of ms are infl ammation, demyelination, degeneration and loss of oligodendrocytes, proliferation of astrocytes, and axonal damage in the cns (wolswijk, 2000). figure 1 shows a chronic ms lesion, characterized by demyelination and astrogliosis. the underlying cause of myelin destruction and death of oligodendrocytes in ms is not completely understood, however data suggest that ms may be a disease of autoimmune nature (steinman, 1996). disruption of the myelin sheaths, axonal injury and glial scar formation is responsible for the clinical symptoms that accumulate in the course of disease. for a disease with such a high impact, the present therapeutic options are disappointing with restricted clinical benefi ts. in order to prevent further damage from occurring in the cns, current therapies target the immune system and are aimed at reducing relapses, which are caused by the formation of new demyelinating lesions. current immunomodulatory therapies, including interferon-γ (fillipini et al. 2003) and glatiramer acetate, show only a modest protective effect against relapses. therapies directed at repairing damage, e.g. based on remyelination strategies, have not yet been developed. following demyelination, some remyelination of ms lesions can be observed during the early stages of the disease (prineas et al. 1993), although this is often limited in its extent, and largely fails as the disease progresses. most chronic lesions of ms demonstrate no remyelination, although oligodendrocyte precursor cells and premyelinating oligodendrocytes are present in many demyelinated plaques (chang et al. 2002; scolding et al. 1998; wolswijk, 2002). this suggests that the microenvironment in chronic ms lesions may possess the potential for remyelination, yet failure of this event is likely due to a lack of appropriate signals to stimulate remyelination. reduced (neuro) trophic support to http://creativecommons.org/licenses/by/3.0/ http://creativecommons.org/licenses/by/3.0/ 38 wilczak et al drug target insights 2008:3 oligodendrocytes might be implicated in oligodendrocyte apoptosis and the failure of remyelination in ms. insulin-like growth factor (igf)-1 is a (neuro)trophic growth factor with insulin-like metabolic activities which possesses potential clinical applications, particularly in (neuro) degenerative disorders of the cns. short overview of the igf system the components of the igf system include igf-1 and -2 (igfs), type-1 and -2 igf receptors and six insulin-like growth factor binding proteins (igfbps) . in the cns, both igf-1 and -2 are produced as paracrine and autocrine hormones. igf-2 is genetically related to igf-1 and both hormones display approximately 62 percent sequence homology. whereas igf-1 expression in the cns is high in neuronal rich regions of the brain (bondy et al. 1992), igf-2 is highly expressed in mesenchymal support structures of the cns, including the choroid plexus (logan et al. 1994). biological actions of these growth factors on target cells are mediated by cell-surface receptors. two types of igf receptors have been identifi ed in human brain, the type-1 and type-2 igf receptor. the type-1 igf receptor is a membrane glycoprotein consisting of two α-subunits and two β-subunits (yamasaki et al. 1993). the α-subunit is entirely extracellular and contains the ligandbinding site. the β-subunit contains a transmembrane domain with a short extracellular region, and a tyrosine kinase domain in its cytoplasmic portion. the type-2 igf receptor is structurally and functionally quite different from the type-1 igf receptor. the type-2 igf receptor consists of a single glycosylated polypeptide. the type-2 igf receptor lies primarily extracellular with a short cytoplasmic tail and consists of 15 repeated mannose6-phosphate (m6p)-binding units (kornfeld et al. 1992). the major functions of the type-2 igf receptor binding appear to be lysosomal enzyme traffi cking (kornfeld et al. 1992) and igf-2 degradation via receptor mediated-internalization (morgan et al. 1987). type-2 igf receptors are not thought to be involved in cell signaling. there is ample consensus today that the biological actions of both igf-1 and -2 are mediated through type-1 igf receptors. in vitro and in vivo, igf-1 stimulates dna synthesis and cell growth. a cellular action of igf1 that is complementary to its stimulation of cell proliferation is its capacity in certain cells to inhibit apoptosis. igf-1 also induces differentiation of neurons and oligodendrocytes (feldman et al. 1997). two major pathways induce the actions of igf-1 and igf-2 through type-1 igf receptors, the fi rst is the mitogen-activated protein (map) kinase pathway and the second is the serine-threonine protein kinase akt pathway. the map kinase pathway is often associated in proliferation and differentiation (kim et al. 1997), whereas the akt pathway is induced in igf-1 mediated cell survival (brunet et al. 1999) and protection from apoptosis (kermer et al. 2000). biological actions of igf-1 and igf-2 are modulated through six igfbps. these proteins contain high affi nity binding sites for igf-1 and igf-2, and they possess an 80% sequence homology with each other (rajaram et al. 1997). in the circulation, the major igfbp form is igfbp-3, which binds igf-1 and igf-2 with similar affi nities (baxter, 1994). circulating igfbp-3 inhibits insulin-like activity, regulates the rate of transport figure 1. luxol-fast blue staining (myelin staining) of the white matter from ms (a). the pale area is a demyelinated plaque (black arrows) and the surrounding non-affected area is myelinated white matter (white arrows). gfap-staining of astrocytes in a ms plaque (b) showing the presence of large reactive astrocytes in ms (black arrows) and normal astrocytes surrounding the ms plaque (white arrows). astrocytes in the ms plaques are stained using an antibody against glial fi brillary acidic protein (gfap) a specifi c marker for astrocytes. 39 targeting insulin-like growth factor-1 drug target insights 2008:3 and prolongs half-lives of plasma igf-1 and igf-2. as carrier proteins of igf-1 and igf-2, a major function of igfbps is to transport and target igfs to specifi c tissues and cell types. therefore, these proteins have a central position in igf ligand-receptor interactions by infl uencing bioavailability and extracellular distribution. igfbps are able to enhance or inhibit igf effects by regulating binding to type-1 igf receptors, a mechanism that is thought to be dependent on specifi c cell and tissue properties (firth and baxter, 2002). binding of igf/igfbp complexes to components of the extracellular matrix (ecm) and the cell-surface can facilitate the release of igfs leading to enhanced delivery to type-1 and type-2 igf receptors. for example, the binding of igfbp-2 to chondroitin6-sulphate, an ecm component, decreases the binding affi nity of igfbp-2 to igf-1 by 3-fold in rat brain, leading to increased levels of biologically active igf-1 (russo et al. 1997). another mechanism involved in igf regulation are specifi c proteases, which are secreted by cells and act as growth stimulators by increasing local igf availability (conover et al. 1993). circulating and tissue specifi c igfbps bind igf-1 and igf-2 by forming biologically inactive igf/igfbp complexes. igf-1 and -2 can be released from these complexes, for example by proteolysis of igfbps. igfbp fragments generated by the action of cellular proteases show a marked loss of igf binding affi nity. in conclusion, igfbps can influence igf actions in different ways. the multitude of these effects depends on the cell type in which they are expressed, how they interact with cell surfaces and ecm, the presence of specifi c proteases and the formation of high affi nity binding proteins complexed with igfs. the role of insulin-like growth factor-1 in multiple sclerosis during nervous system development, igf-1 plays a crucial role in cell proliferation, differentiation, and survival (bondy and cheng, 2004; russo et al. 2005). during that period igf-1 and type-1 igf receptors are highly expressed in neuronal rich regions, such as the spinal cord, midbrain, cerebral cortex, hippocampus, and olfactory bulb (anlar et al. 1999; beck et al. 1988). we have shown that cerebral white matter in human neonates, undergoing active myelination, contains a 3-fold higher density of type-1 igf receptors than in adults, indicating that igf-1 also plays a important role in the myelination of the human cns (de keyser et al. 1994b). in vitro experiments have demonstrated that igf-1 greatly enhances oligodendrocyte survival (mcmorris et al. 1986), myelin production (roth et al. 1995) and proliferation of oligodendrocyte precursors (mozell and mcmorris, 1991). the effects of igf-1 are not only restricted to oligodendrocytes of the cns, but also apply to schwann cells of the pheripheral nervous system, which demonstrate enhanced differentiation, myelin production, and increased survival in response to igf-1 stimulation (cheng et al. 1999; ogata et al. 2004; syroid et al. 1999). the importance of igf-1 in myelin production has also been demonstrated in several animal models. in the cuprizone model, demyelination occurs in the corpus callossum and superior cerebellar peduncles, and remyelination in these areas ensues when treatment with cuprizone is terminated. cuprizone-induced demyelination in mice defi cient of type-1 igf receptors has demonstrated inadequate remyelination and lack of oligodendrocyte progenitor cells (opc) accummulation in the site of injury (mason et al. 2003). transgenic mice that overexpress igf-1 show increased brain growth and myelination (carson et al. 1993). myelin content in these animals was increased by 130%. by comparison, igf-1 knock-out mice displayed reduced brain size, hypomyelination, reduced density of oligodendrocytes, loss of neuron populations, as well as reduced glucose uptake (beck et al. 1995; cheng et al. 2000; liu et al. 1993). transgenic mice overexpressing igfbp-1, an inhibitory binding protein for igf-1, showed reduced myelinated axons and thickness of the myelin sheets, this feature was induced by reduction in myelin expression (ye et al. 1995). other studies have shown that systemic application of igf-1 in acute demyelinating experimental autoimmune encephalomyelitis (eae), an animal model for ms, signifi cantly reduced the number and area of the demyelinated lesions in the spinal cord (liu and yao, 1995; yao et al. 1995). igf-1 increased the number of remyelinated axons, reduced the permeability of the blood-spinal cord barrier, and enhanced myelin expression. however, in chronic-relapsing eae, systemic administration of igf-1 together with igfbp-3 after disease onset resulted in increased severity and or relapses (lovett-racke et al. 1998). in line 40 wilczak et al drug target insights 2008:3 with these results, long-term application of igf-1 in chronicrelapsing eae from the standpoint of myelin gene expression and repair, showed no positive effects of igf-1 (canella et al. 2000). in a clinical trial with systemically administered recombinant igf-1 in ms patients, no effects on either new lesion formation or remyelination of existing lesions could be demonstrated (frank et al. 2002). data from this clinical trial, as well as from the above mentioned study on chronic relapsing eae raise doubts about the effectiveness of systemic administered igf-1 in chronic forms of demyelination. in the following sections, we dicuss several obstacles that might explain a lack of benefi cial effects of systemic administered igf-1. enhancing the level of free igf-1 and targeting igf-1 into the cns one possible obstacle in an igf-1 based therapy is the limited penetration across the blood brain barrier (bbb) that prevents free passage of large proteins into the cns. however, there is some evidence that igf-1 from the circulation might be transported across these barriers, albeit to a limited extent. one possible mechanism to transport igf-1 from the circulation into the cns is throughreceptor mediated transcytosis (reinhardt and bondy, 1994). however, it is still not clear which receptor is involved in such transport system. it has been suggested that the presence of type-1 igf receptors on the endothelium might play a role in such transport. the transport of peripheral igf-1 into the cns may also be infl uenced by igfbps which become saturable at the bbb (weihong and kastin, 2000). other studies have shown that the uptake of circulating igfs into cerebrospinal fl uid (csf) and probably into the cns appears to be independent of type-1 igf receptors as well as igf-binding proteins. there is evidence that choroid plexus megalin is involved in neuroprotection by serum igf-1 in alzheimer disease by its dual effects on transporting igf-1 in across the bbb, and by enhancing the clearance of brain amyloidβ (carro et al. 2005). however, it remains questionable whether systemic levels of igf-1 would indeed raise ligand levels in the deep white matter of the cns in which demyelination occurs. the amount of igf-1 eventually reaching the deep white matter will depend on the concentration of circulating igf-1 and how avidly the bbb transport system uptakes it. approaches that bypass the bbb and show that igf-1 has neuroprotective effects in the cns of animals use surgically invasive procedures, such as the intracerebrovascular or intraparenchymal administration. these methods are not applicable to ms patients but do demonstrate a need for targeting. intranasal administration of igf-1 offers an alternative strategy to bypass the bbb and is a method that provides several advantages; it is a non-invasive way of drug application, there is a direct delivery of the drug into the cns, levels of the drugs are more concentrated by avoiding the diluting effects of the circulation, and degradation and destruction of drugs are minimized by avoiding the gastrointestinal tracts. thorne and co-workers have demonstrated that intranasal application of igf-1 results in a rapid delivery of igf-1 into multiple areas of the cns, including the deep white matter in which demyelination can occur (thorne et al. 2004). in this study, igf-1 was delivered from the nasal cavity along olfactory and trigeminal pathways and was accompanied by activation of signaling pathways in several areas that express high levels of type-1 igf receptors. thorne and coworkers suggested an extracellular route of transportation from the nasal passages into the cns associated with components of the trigeminal nerve. this mode of transport provides evidence for a rapid and direct pathway for protein transport into the cns following intranasal administration. a second obstacle in an igf-1 based therapy lies in the complex regulation of igf-actions. the biological effects of igf-1 on target cells are mediated by interactions with the type-1 igf receptor (leroith et al. 1995). however, six igfbps govern this interaction (clemmons, 1997; spagnoli and rosenfeld, 1997). all six igfbps may inhibit igf-1 actions by sequestering igf-1, thereby preventing the interaction of igf-1 to its receptor. we have found that igfbp-1 and igfbp-6 were upregulated on oligodendrocytes in the periplaque white matter in ms lesions (unpublished results). furthermore, it has been demonstrated that these igfbps bind igf-1 with high affi nity, and inhibit igf-1 induced survival and myelin production of primary oligodendrocytes (kühl et al. 2002, 2003). these results suggest that the up-regulation of igfbps lead to a shortage of biologically active (free) igf-1 in the demyelinated ms plaques, contributing to the lack of (re)-myelination. however, the exact function of igfbp-1 and igfbp-6 41 targeting insulin-like growth factor-1 drug target insights 2008:3 in physiological and pathological situations is yet unknown. igf-1 analogues that display low affi nity for igfbps, and igf-1 analogues that display high affi nity for igfbps, which are able to displace endogenous igf-1 and or igf-2 from igfbps, may be suitable candidates for stimulating (re) myelination in ms (fig. 2). des(1-3) igf-1 is a truncated form of igf-1 lacking the tripeptide glycine-proline-glutamate (gpe) at the aminoterminus of the full-length form (sara et al. 1986). the biological potency of this analogue is signifi cantly higher than that of the full-length form and is explained by its reduced affi nity for igfbps. igf-1 analogues that display high affi nity for igfbps, which can therefore displace endogenous figure 2. a schematic overview: astrocytes regulate their own cellular action through igf-1, and astrocytic igf-1 has no effect on (pre)myelinating oligodendrocytes in ms. astrocytes become astrogliotic forming the tissue scar in ms lesions and preventing remyelination by oligodendrocytes (a). through intranasal application of igf-1 analogues and or igfbp ligand inhibitors, igf-1 is released from igfbps which are present on oligodendrocytes and igf-1 become available to oligodendrocytes, resulting in remyelination (b). 42 wilczak et al drug target insights 2008:3 igf-1 from igfbps might also be considered. this approach has already been studied in rats with focal cerebral ischemia. in this model, the intracerebroventricular administration of the igf-1 analogue [(leu24,59,60, ala31) higf-1] with high affi nity to igfbps and no affi nity to type-1 igf receptors, increased levels of biologically active igf-1 and provided potent neuroprotection (loddick et al. 1998). a third obstacle lies in the targeting of igf-1 signaling to the myelin producing cells: the oligodendrocyte. such a targeting is complicated by the high expression of type-1 igf receptors on other cells in the cns. we have demonstrated that type-1 igf receptors were present on neurons and other glial cells, including microglia and astrocytes (de keyser et al. 1994a; wilczak and de keyser, 1997). this may have important implications for the clinical use of igf-1 in ms. chronic plaques of ms contain a dense network of astrocytes, which are responsible for the characteristic astrogliotic plaque. in vitro studies have shown that igf-1 enhances the proliferation of astrocytes (chesik et al. 2004; tranque et al. 1992). as acute ms lesions are rapidly invaded by reactive astrocytes, enhancing the levels of igf-1 in ms lesions may not only protect oligodendrocytes and stimulate remyelination but also enhance the astrogliosis that creates a glial scar, limiting remyelinating processes. for these reasons, targeting igf-1 effects to oligodendrocytes is crucial. a feasible approach for targeting oligodendrocytes consists of intervening in igf-1 regulation by igfbps. in ms lesions, we have shown a unique distribution of igfbps expression on oligodendrocytes and astrocytes. oligodendrocytes in demyelinated ms plaques display an upregulation of igfbp-1 and igfbp-6, whereas astrocytes show increased expression of igfbp-2 and igfbp-4 (chesik et al. 2006). an interesting prospect is the use of non-peptide small molecules that act as specifi c igfbp ligand inhibitors and prevent binding of igf-1 to specifi c igfbps (chen et al. 2001; zhu et al. 2003). this approach would result in an elevation of biological active igf-1 that is available in the vicinity of olgodendrocytes. nbi-31772 is a recently developed non-peptide small molecule that binds to all six known igfbps and displaces biologically active igf-1 from all six binding proteins (liu et al. 2001).the neuroprotective effects of nbi31772 has been studied in experimental models of cerebral ischemia. this study has demonstrated that intracerebroventricular administration of nbi31772 signifi cantly reduced ischemic brain damage and infarct size. (mackay et al. 2003). new potent ligand-inhibitors could be designed which selectively bind individual igfbps in order to enhance and target bioactive igf-1. another approach to elevate igf-1 levels as well as target igf-1 into oligodendrocytes is altering the expression level of igfbps on oligodendrocytes and astrocytes in ms. igfbp-2 is of particular interest because hypertrophic astrocytes in ms lesions express high levels of igfbp-2 (chesik et al. 2006). astrocytes are the primary source of igf-1 in damaged cns, and it has been suggested that this growth factor assists in neuronal protection as well as in facilitation of myelin production. we have shown that reactive astrocytes in vitro and in situ upregulate igfbp-2 and that combined treatment of igfbp-2 and igf-1 does not inhibit igf-1 stimulated astrocyte proliferation, whereas it inhibits igf-1 stimulated survival of oligodendrocytes (chesik et al. 2004). we propose that an upregulation of igfbp-2 in ms facilitates the process of astrogliosis by targeting igf-1 to these cells. inhibition of endogenous expression of igfbp-2 in astrocytes by means of micrornas (mirnas) might have implications on cell proliferation and maturation of these cells. in ms, astrocytes become reactive and form the glial scar, which is thought to impede remyelination processes. mirnas are a growing family of singlestranded forms of rna, which regulate the expression and production of proteins. such an approach was already investigated in neuroblastoma (nb) cells (tanno et al. 2005). neuroblastoma is a malignant childhood tumor, in which igfbp-5 is frequently expressed. by suppressing the expression level of igfbp-5 in nb cells through mirnas, nb cells become more prone to apoptosis (tanno et al. 2005). the use of these molecules as therapeutics to infl uence the expression of igfbps during demyelination has a long way to go. however, regulating the expressing of igfbps through mirnas opens a new window to selectively study the role of igfbps in several diseases such as ms. conclusion the use of igf-1 analogues, igfbp ligand inhibitors and micrornas to modify actions of igf-1 signaling and induce remyelination in the cns 43 targeting insulin-like growth factor-1 drug target insights 2008:3 would be a tremendous breakthrough in the treatment of human demyelinating diseases, such as ms. the unique distribution of igfbps in ms lesions as well as a unique functional characteristic of the igf-1 receptor on oligodendrocytes offers a means to specifi cally target this cell type. acknowledgments this work was supported by the school of behavior and cognitative neurosciences (bcn) groningen and “msanders”, the netherlands. references anlar, b., sullivan, k.a. and feldman, e.l. 1999. insulin-like growth factor-i and central nervous system development. horm. metab. res., 31:120–5. baxter, r.c. 1994. insulin-like growth factor binding proteins in the human circulation: a review. horm. res., 42:140–4. beck, f., samani, n.j., byrne, s. et al. 1988. histochemical localization of igf-1 and igf-ii mrna in the rat between birth and adulthood. development, 104:29–39. beck, k.d., powell-braxton, l., widmer, h.r. et al. 1995. igf1 gene disruption results in reduced brain size, cns hypomyelination, and loss of granule and striatal parvalbumin-containing neurons. neuron, 14:717–30. bondy, c.a. and cheng, c.m. 2004. signaling by insulin-like growth factor i in brain. eur. j. pharmacol., 490:25–31. bondy, c., werner, h., roberts, ct., jr. et al. 1992. cellular pattern of type-i insulin-like growth factor receptor gene expression during maturation of the rat brain: comparison with insulin-like growth factors i and ii. neuroscience, 46:909–23. brunet, a., bonni, a., zigmond, m.j. et al. 1999. akt promotes cell survival by phosphorylating and inhibiting a forkhead transcription factor. cell, 96:857–68. canella, b., pitt, d., capello, e. et al. 2000. insulin-like growth factor-1 fails to enhance central nervous system myelin repair during autoimmune demyelination. am. j. pathol., 157:933–43. carson, m.j., behringer, r.r., brinster, r.l. et al. 1993. insulin-like growth factor-i increases brain growth and central nervous system myelination in transgenic mice. neuron, 10:729–40. caro, e., spuch, c., trejo, j.l. et al. 2005. choroid plexus megalin is involved in neuroprotection by serum insulin-like growth factor-i. j. neurosci., 25:10884–93. chang, a., tourtellotte, w.w., rudick, r. et al. 2002. premyelinating oligodendrocytes in chronic lesions of multiple sclerosis. n. engl. j. med., 346:165–73. chen, c., zhu, y.f., liu, x.j. et al. 2001. discovery of a series of non-peptide small molecules that inhibit the binding of insulin-like growth factor (igf) to igf-binding proteins. j. med. chem., 44:4001–10. cheng, c.m., reinhardt, r.r., lee, w.h. et al. 2000. insulin-like growth factor-i regulates developing brain glucose metabolism. proc. natl. acad. sci., u.s.a., 97:10236–41. cheng, h.l., russel, j.w., feldman, e.l. et al. 1999. igf-i promotes peripheral nervous system myelination. ann. n.y. acad. sci., 883:124–30. chesik, d., de keyser, j., glazenburg, l. et al. 2006. insulin-like growth factor binding proteins: regulation in chronic active plaques in multiple sclerosis and functional analysis on glial cells. eur. j. neurosci., 24:1645–52. chesik, d., kühl, n.m., wilczak, n. et al. 2004. enhanced production and proteolytic degradation of insulin-like growth factor binding protein2 in proliferating astrocytes. j. neurosci. res., 77:354–62. clemmons, d.r. 1997. insulin-like growth factor binding proteins and their role in controlling igf actions. cytokine growth factor rev., 8:45–62. conover, c.a., kiefer, m.c. and zapf, j. 1993. posttransitional regulation of insulin-like growth factor binding protein-4 in normal and transformed human fi broblasts. insulin-like growth factor dependence and biological study. j. clin. endocrinol. metab., 91:1129–37. de keyser, j., wilczak, n., de backer, j.p. et al. 1994a. insulin-like growth factor-i receptors in human brain and pituitary gland: an autoradiographic study. synapse, 17:196–202. de keyser, j., wilczak, n. and goossens, a. 1994b. insulin-like growth factor-i receptor densities in human frontal cortex and white matter during aging, in alzheimer’s de disease, and in huntington's disease. neurosci. lett., 172:93–6. feldman, e.l., sullivan, k.a., kim, b. et al. 1997. insulin-like growth factors regulate neuronal differentiation and survival. neurobiol. dis., 4:201–14. fillipini, g., munari, l., incorvaia, b. et al. 2003. interferon’s in relapsing remitting multiple sclerose: a systematic review. lancet, 361:545–52. firth, s.m. and baxter, r.c. 2002. cellular actions of the insulin-like growth factor binding proteins. endocr. rev., 23:824–54. frank, j.a., richert, n., lewis, b. et al. 2002. a pilot study of recombinant insulin-like growth factor-1 in seven multiple sclerosis patients. mult. scler., 8:24–9. kermer, p., klocker, n., labes, m. et al. 2000. insulin-like growth factor-i protects axotomised rat retinal ganglion cells from secondary death via pi3-k-dependent akt phosphorylation and inhibition of caspase3 in vivo. j. neurosci., 20:2–8. kim, b., leventhal, p.s., saltiel, a.r. et al. 1997. insulin-like growth factor-i mediated neurite outgrowth in vitro requires mitogen-activated protein kinase activation. j. biol. chem., 272:21268–73. kornfeld, s. 1992. structure and function of the mannose-6-phosphate/ insulin-like growth factor-ii receptors. ann. rev. biochem., 61:307–30. kühl, n.m., de keyser, j., de vries, h. et al. 2002. insulin-like growth factor binding protein-1 and -2 differentially inhibit rat oligodendrocyte precursor survival and differentiation in vitro. j. neurosci., 69:207–16. kühl, n.m., hoekstra, d., de vries, h. et al. 2003. insulin-like growth factor binding protein-6 inhibits survival and differentiation of rat oilgodendrocyte precursor cells. glia, 44:91–101. leroith, d., werner, h., beitner-johnson, d. et al. 1995. molecular and cellular aspects of the insulin-like growth factor i receptor. endocr. rev., 16:143–63. liu, j.p., baker, j., perkins, a.s. et al. 1993. mice carrying null mutations of the genes encoding insulin-like growth factor i (igf-1) and type 1 igf receptor (igf-1r). cell, 75:59–72. liu, x. and yao, d.l. 1995. insulin-like growth factor-i treatment reduces clinical defi cits and plaques severity in acute demyelinating experimental autoimmune encephalitis. mult. scler., 1:2–9. liu, x.j., xie, q., zhu, y.f. et al. 2001. identifi cation of a nonpeptide ligand that release bioactive insulin-like growth factor-i from its binding protein complex. j. biol. chem., 276:32419–22. loddick, s.a., liu, x.j., lu, z.x. et al. 1998. displacement of insulin-like growth factors from their binding proteins as a potential treatment for stroke. proc. natl. acad. sci., u.s.a., 95:1894–8. logan, a., gonzalez, a.m., hill, d. et al. 1994. coordinated pattern of expression and localization of insulin-like growth factor-ii (igf-ii) and igf-binding protein-2 in the adult brain. endocrinology, 135:2255–64. lovett-racke, a.e., bittner, p. and cross, a.h. 1998. regulation of experimental autoimmune enceplamyelitis with insulin-like growth factor (igf-i) and igf-i/igf-binding protein-3 complex (igf-i/ igfbp-3). j. clin. invest., 101:1797–804. mackay, k.b., loddick, s.a., naeve, g.s. et al. 2003. neuroprotective effects of insulin-like growth factor-binding protein ligand inhibitors in vitro and in vivo. j. cereb. blood flow metab., 23:1160–7. 44 wilczak et al drug target insights 2008:3 mason, j.l., xuan, s. and dragatsis, i. 2003. insulin-like growth factor (igf) signaling through type 1 igf receptor plays an important role in remyelination. j. neurosci., 23:7710–8. mcmorris, f.a., smith, t.m., desalvo, s. et al. 1986. insulin-like growth factor i/somatomedin c: a potent inducer of oligodendrocyte development. proc. natl. acad. sci., u.s.a., 83:822–6. morgan, d.o., edman, j.c., standring, d.n. et al. 1987. insulin-like growth factor ii receptor as a multifunctional binding protein. nature, 329:301–7. mozell, r.l. and mcmorris, f.a. 1991. insulin-like growth factor i stimulates oligodendrocyte development and myelination in rat brain aggregate cultures. j. neurosci. res., 30:382–90. noseworthy, j.h., luchinetti, c., rodriguez, m. et al. 2000. multiple sclerose. n. engl. j. med., 343:938–52. ogata, t., iijima, s., hoshikawa, s. et al. 2004. opposing extracellular signal-regulated kinase and akt pathways controll schwann cell myelination. j. neurosci., 24:6724–32. prineas, j.w., barnard, r.o., kwon, e.e. et al. 1993. multiple sclerosis: remyelination of nascent plaques. ann. neurol., 33:137–51. rajaram, s., baylink, d.j. and mohan, s. 1997. insulin-like growth factorbinding proteins in serum and other biological fl uids: regulation and function. endocr. rev., 18:801–31. reinhardt, r.r. and bondy, c.a. 1994. insulin-like growth factors cross the blood-brain barrier. endocrinology, 135:1753–61. roth, g.a., spada, v., hamill, k. et al. 1995. insulin-like growth factor i increases myelination and inhibit demyelination in cultured organotypic nerve tissue. brain res. dev. brain res., 88:102–8. russo, v.c., bach, l.a., fosang, a.j. et al. 1997. insulin-like growth factor binding protein-2 binds to cell surface proteoglycans in the rat brain olfactory bulb. endocrinology, 138:4858–67. russo, v.c., gluckman, p.d., feldman, e.l. et al. 2005. the insulin-like growth factor system and its pleiotrophic functions in brain. endocr. rev., 26:916–43. sara, v.r., carlsson-skwirut, c., anderson, c. et al. 1986. characterization of somatomedins from human fetal brain: identifi cation of a variant form of insulin-like growth factor-i. proc. natl. acad. sci., u.s.a., 83:4904–7. scolding, n., franklin, r., stevens, s. et al. 1998. oligodendrocyte progenitors are present in the normal adult human cns and in the lesions of multiple sclerosis. brain, 121:2221–8. spagnoli, a. and rosenfeld, r.g. 1997. insulin-like growth factor binding proteins. curr. opinion endocrinol., 4:1–9. steinman, l. 1996. multiple sclerosis: a coordinated immunological attack against myelin in the central nervous system. cell, 85:299–302. syroid, d.e., zorick, t.s., arbet-engels, c. et al. 1999. a role for insulinlike growth factor-i in the regulation of schwann cell survival. ageing res. rev., 4:195–212. tanno, b., cesi, v., vitali, r. et al. 2005. silencing of endogenous igfbp-5 by micro rna interference affects proliferation, apoptosis and differentiation of neuroblastoma cells. cell. death diff., 12:213–23. thorne, r.g., pronk, g.j., padmanabhan, v. et al. 2004. delivery of insulinlike growth factor-i to the rat brain and spinal cord along olfactory and trigeminal pathways following intranasal administration. neuroscience, 127:481–96. tranque, p.a., calle, r., naftolin, f. et al. 1992. involvement of protein kinase-c in the mitogenic effects of insulin-like growth factor i on rat astrocytes. endocrinology, 131:1948–54. weihong, p. and kastin, a.j. 2000. interactions of igf-1 with the bloodbrain barrier in vivo and in situ. neuroendocrinology, 72:171–8. wilczak, n. and de keyser, j. 1997. insulin-like growth factor-i receptors in normal appearing white matter and chronic plaques in multiple sclerosis. brain res., 772:243–6. wolswijk, g. 2000. oligodendrocyte survival, loss and birth in plaques of chronic-stage multiple sclerosis. brain, 123:105–15. wolswijk, g. 2002. oligodendrocyte precursor cells in demyelinated multiple sclerosis spinal cord. brain, 125:338–49. yamasaki, h., prager, d. and melmed, s. 1993. structure-function of the human insulin-like growth factor receptor-i: a discordance of somatotroph internalization and signaling. mol. endocrinol., 7:681–5. yao, d.l., liu, x., hudson, l.d. et al. 1995. insulin-like growth factor i treatment reduces demyelination and up-regulates gene expression of myelin-related prpteins in experimental autoimmune encephalomyelitis. proc. natl. acad. sci., u.s.a., 92:6190–4. ye, p., carson, j. and d' ercole, a.j. 1995. in vivo actions of insulin-like growth factor-i (igf-i) on brain myeliation: studies of igf-i and igf binding protein-1 (igfbp-1) transgenic mice. j. neurosci., 15:7344–56. zhu, y.f., wang, x.c., connors, p. et al. 2003. quinoline-carboxylic acids are potent inhibitors that inhibit the binding insulin-like growth factor (igf) to igf-binding proteins. bioorganic. med. chem. lett., 13:1931–4. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true 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drug target insights 2008:3 77–85 77 review correspondence: rita moretti, m.d., department of neurology, university of trieste, cattinara hospital 34149 trieste, italy. tel: +39-40-3994321; fax: +39-40-910861; email: moretti@univ.trieste.it copyright in this article, its metadata, and any supplementary data is held by its author or authors. it is published under the creative commons attribution by licence. for further information go to: http://creativecommons.org/licenses/by/3.0/. memantine: reality and potentiality rita moretti, paola torre, cristina vilotti, davide manganaro, luca zanet and rodolfo m. antonello department of medicine and neurology, university of trieste, trieste, italy. abstract: memantine protects cultured neurons from excitotoxin-induced cell-death; it attenuated loss of cholinergic neurons in the cns induced by injection of nmda into the basal forebrain of rats. it has been shown that memantine induced production of brain-derived neurotrophic factor (bdnf), a substance shown to promote survival and differentiation of cns neuron. due to the preclinical effects of memantine owing to its anti-ischemic and anti-excitotoxic properties, recent clinical effi cacy has been demonstrated in patients with advanced dementia of vascular origins. therefore, it has been employed in different trials, in vascular dementia, showing a potential benefi t and no unbearable side effects. different studies underline the possible role of memantine in parkinson disease. keywords: memantine, nmda, glutamate, degeneration glutamate is the principal excitatory neurotransmitter in the brain and is active in about one-third of all the synapses in the central nervous system. although glutamate is a crucial mediator of physiological communication between neuronal cells, under certain conditions activation of glutamate receptors kills neurones—a term called “excitotoxicity” (rothman et al. 1987). it has been implied that excitotoxicity is involved in many types of acute and chronic cns neurodegenerative disorders and is connected with ca2+ overload (choi et al. 1995). disturbance of glutamate homeostasis probably plays a pivotal role in neuropathology triggered by other factors such as: energy defi cits, free radicals formation, etc. that facilitate the neurotoxic potential of endogenous glutamate (danysz et al. 2000). glutamate activates three major types of ionotropic receptors, namely a-amino-3hydroxy-5-methyl4-isoxazolepropionic acid (ampa), kainate and n-methyl-d-aspartate (nmda) and several types of metabotropic receptors. ampa receptors are probably involved in all forms of fast glutamatergic neurotransmission (danysz et al. 2000). there are four known subunits glur1 to glur4 which form functional receptors as tetrameric subunit assemblies. all ampa receptors are permeable to na+ and k+ while complexes lacking glur2 subunits are also permeable to ca2+. nmda-sensitive ionotropic glutamate receptors are coupled to high conductance cationic channels permeable to na+, k+, and ca2+ (danysz et al. 2000). the nmda channel is blocked in a useand voltage dependent manner by mg2+ and many exogenous agents. nmda receptors are only activated following depolarisation of the postsynaptic membrane which physiologically follows ampa receptor stimulation which relieves blockade by mg2+. this unique feature and the high ca2+ permeability renders nmda receptors inherently suitable as mediators of synaptic plasticity (e.g. learning and memory). similar to mg2+, uncompetitive nmda receptor antagonists such as ketamine, dextromethorphan, memantine, phencyclidine and (+)-5-methyl-10,1l-dihydro-5hdibenzocyclohepten-5,10-imine meleate (+)mk-801 block the nmda channel in the open state, although the blocking kinetics and voltage dependence of this effect vary considerably (danysz et al. 2000). glutamate and nmda receptors are involved in long-term potentiation (ltp), a fundamental process for memory consolidation, whereby brief high-frequency stimulation leads to an increased response after subsequent activation. nmda receptors are involved in mediating the postsynaptic components of ltp in the hippocampus e.g. in the schaffer collateral projection from ca3 to ca1 (danysz et al. 1995; parsons et al. 1998; fischer et al. 1977). under normal physiological resting conditions, the ion channels of the nmda receptors are blocked by magnesium ions in a voltage-dependent manner. the small amounts of glutamate that are released http://creativecommons.org/licenses/by/3.0/. http://creativecommons.org/licenses/by/3.0/. 78 moretti et al drug target insights 2008:3 are not suffi cient to displace the magnesium ions and remove the blockade of the nmda receptor channels; there is a so-called “low background noise”. during normal synaptic activity, larger concentrations of glutamate are released, the postsynaptic membrane is depolarised, the magnesium blockade is transiently inactivated, calcium ions enter the cell and a signal is generated. under conditions of impaired metabolism, there is a sustained release of glutamate, and excessive glutamate activity is associated with excitotoxicity. the magnesium ions are lost from the nmda receptors—allowing a continuous infl ux of calcium ions into the cell-creating a high level of background noise and impairing the recognition of signals, resulting from physiological activation of the receptor. high intracellular concentrations of calcium eventually lead to neuronal degeneration and cell death. memantine blocks nmda receptor channels in cultured neurons in a voltage-dependent manner as measured by patch clamp technique (chen et al. 1997; blanpied et al. 1997; borman et al. 1989). the effects of memantine were not reversed by exposure to glycine �100 micromol/l (borman et al. 1989). memantine induces open-channel blockade of nmda receptors (ambrozi et al. 1988) and is ‘partially trapped’ in nmda receptor channels (chen et al. 1999; frankiewicz et al. 1999). memantine could be washed from approximately one-sixth of channels during in vitro experiments (frankiewicz et al. 1999). memantine protects cultured neurons from excitotoxin-induced cell death (parsons et al. 1993; krieglstein et al. 1996; erdo et al. 1991; weller et al. 1993; pellegrini et al. 1993). memantine exerts neuroprotective effects in several models of brain injury. the drug attenuated loss of cholinergic neurons in the cns induced by injection of nmda into the basal forebrain of rats (wenk et al. 1997). memantine also attenuated neuronal injury in various rat models including traumatic brain injury (rao et al. 2001), ischaemic stroke induced by occlusion of cerebral (gorgiilii et al. 2000; dogan et al. 1999) or carotid arteries (seif ej nasi’ et al. 1990; heim et al. 1995), a photo-induced thrombotic model of cerebral focal ischaemia (stieg et al. 1999) and quinolinic acid-induced hippocampal damage (keilhoff et al. 1992). at a dosage of 5–50 mg/kg in rats, memantine induced production of brain-derived neurotrophic factor (bdnf), a substance shown to promote survival and differentiation of cns neurons, and trkb, a tyrosine kinase receptor for bdnf (keilhoff et al. 1992). mrna for bdnf and trkb was detected in limbic cortex slices by in situ hybridisation (lundbeck ais, 2002). clinical therapy due to its preclinical effects, above described, memantine, late in 2003, was approved by the fda as a drug for ad treatment. memantine is indicated for the treatment of moderate to severe alzheimer’s disease, refl ecting a license extension granted by the european commission in october 2005 to include the moderate ad patient population. post-mortem orepidemiological studies suggest a strong association between glutamate dysfunction and alzheimer ’s disease. some authors observed co-localisation of glutamatergic neurones and pathological alterations (neurofi brillary tangles and senile plaques) in post-mortem analysis of the brains of alzheimer’s patients (braak et al. 1993; francis et al. 1993). in alzheimer's disease there is an increase of glutamate, caused by a decrease of uptake and/or increase of release. there is a decrease in astroglial glutamate carrier eaa2 in the frontal cortex of post-mortem samples from brains of alzheimer’s patients (li et al. 1997). in vitro constituents of senile plaques stimulate microglia to produce an unknown neurotoxin having agonistic properties at nmda receptors (giulian et al. 1995; klegeris et al. 1997). beta-amyloid peptide either activates nmda receptors or enhances their sensitivity (goodwin et al. 1995); in fact, beta-amyloid (1–40) stimulates nitric oxid (no) production by microglia (goodwin et al. 1995)—no is known to enhance glutamate release and to inhibit uptake (brorson et al. 1995). beta-amyloid peptide enhances the toxicity of glutamate in in vitro test (mattson et al. 1992; wu et al. 1995) and augments nmda receptor mediated transmission (cullen et al. 1996). in vivo injection of beta-amyloid produces long lasting depression of epsps in the hippocampus, which is an expression of ongoing mild excitotoxicity. it is prevented by the nmda receptor antagonist 3-(2-carboxypiperazin4-yl)propyl-1-phosphonic acid (cpp) (cullen et al. 1996). in multi centre placebo-controlled trials, memantine has demonstrated effi cacy and safety 79 memantine: reality and potentiality drug target insights 2008:3 in ad patients. the n-methyl-d-aspartate (nmda) antagonist memantine has been shown to be effective in moderately-severe to severe ad. 252 patients were enrolled, with equal numbers (n = 126) receiving either memantine 20 mg/day or placebo for 28 weeks. effi cacy was assessed using the clinician’s interview-based impression of change (nyu cibicplus), the modified alzheimer’s disease cooperative study activities of daily living (adcs-adlsev) inventory, the severe impairment battery (sib), and functional assessment staging (fast) (reisberg et al. 2003; reisberg et al. 2000). in study two, 166 patients were enrolled to the study and randomised to receive either memantine 10 mg/day or placebo for 12 weeks. the primary effi cacy endpoints were measured using the clinical global impression of change (cgi-c) and the behavioural rating scale for geriatric patients (bgp). the modifi ed d-scale (arnold/ferm) was used to assess basic activities of daily living as a secondary endpoint. both studies demonstrated that patients treated with memantine show improvements in the three main ad domains: function, cognition and global response. memantine was well tolerated in both studies, being the incidence of adverse events (aes) and serious aes was similar in both groups (winblad et al. 1999). in conclusion, memantine treatment offered functional improvements and reduced care dependence for patients with moderately-severe to severe ad. another well designed study, involving patients with moderate-to-severe alzheimer’s disease, who were randomly assigned to receive placebo or 20 mg of memantine daily for 28 weeks has been conducted (bleich et al. 2003). 252 patients were enrolled. of these, 181 (72%) completed the study and were evaluated at week 28. 71 patients discontinued treatment prematurely (42 taking placebo and 29 taking memantine). patients receiving memantine had a better outcome than those receiving placebo, according to the results of the clinical global impression (p = 0.06 with the last observation carried forward, p = 0.03 for observed cases), of the activities of daily living (p = 0.02 with the last observation carried forward, p = 0.003 for observed cases), and the severe impairment battery (p � 0.001 with the last observation carried forward, p = 0.002 for observed cases). memantine was not associated with a signifi cant frequency of adverse events. the conclusions of the authors are well stigmatized by the following sentence: “antiglutamatergic treatment reduced clinical deterioration in moderate-to-severe alzheimer’s disease, a phase associated with distress for patients and burden on caregivers, for which other treatments are not available” (bleich et al. 2003). it has been demonstrated a favourable effect of memantine, even in the association with cholinesterase inhibitors. a 24-week, randomised, doubleblind, parallel-arm, placebo controlled trial was performed in 37 us centres to study the safety and effi cacy of memantine in patients with moderate to severe ad treated with donepezil (hartmann et al. 2003). inclusion criteria of the study were: a diagnosis of probable ad by nincds-adrda, mmse (5–14), mri or ct scan consistent with probable ad, and 6-month daily achei (donepezil) therapy (stable dose for the past 3 months). primary outcome assessments were: cognition and function in daily living. a pharmacokinetic study in 24 healthy volunteers showed no pharmacokinetic or pharmacodynamic interactions and the combination was well tolerated. the global effect was in general favourable to the population who received both the drugs. a postmarketing surveillance study was performed in germany to assess the tolerability of memantine in combination with an achei (84% donepezil and 15% rivastigmine) based on 200 questionnaires. the results demonstrate that combining memantine with a commonly used achei is safe and superior to the achei alone in moderate to severe ad (tariot et al. 2003; tariot et al. 2004; knopman, 2005; pomara et al. 2004; sudhir, 2004). to investigate, the cognitive effect of the memantine, a group of scientists (schmitt et al. 2006) executed a post-hoc exploratory reanalysis of a 24 week randomized, double-blind, placebocontrolled, parallel group clinical trial comparing memantine to placebo in patients with moderate to severe ad receiving treatment with cholinesterase inhibitor, donepezil. these post-hoc analyses support the benefi cial effects of memantine on cognition observed in a previously reported clinical trial. the results suggested an effect of memantine on memory, language, and praxis in patients with moderate to severe ad and support the effi cacy of memantine for the treatment of cognitive defects in ad (schmitt et al. 2006). a very recent work (jones et al. 2007) confi rmed the statistically signifi cant benefi ts of memantine given twice-daily to treat moderate to severe ad have already been 80 moretti et al drug target insights 2008:3 shown by multiple studies in pivotal ad domains (function, cognition and global performance) (winblad et al. 2006). no clinically relevant differences in adverse effects or vital signs were observed between the different dosing schedules (jones et al. 2007). memantine and vascular dementia in course of a cerebrovascular event, there is a run-down of energy in neurons. there are number of microdialysis studies indicating that there is also a consistent increase in extracellular glutamate concentration. in humans, there is also an increase in cerebrospinal fl uid (csf) and plasma content of glutamate in patients with progressive, but not stable stroke. thus, other factors may increase neuronal vulnerability to physiological levels of glutamate by, for example, a decrease of resting membrane potential or intracellular buffering of ca2+ ions. apart from glutamate, oxidative stress, inflammatory reactions and breakdown of the blood-brain barrier may also play a pivotal role. the toxic effects of glutamate are mediated largely through n-methyld-aspartate (nmda) receptors. activation of nmda-associated channels leads to the passage of sodium and chloride into the cell followed by the obligatory movement of water resulting in cytotoxic oedema. if calcium is present early, it tends to pass through the channel into the cell where it recruits second messengers, with concomitant activation of kinases and proteases that eventually lead to irreversible injury. this is believed to be the underlying mechanism behind delayed glutamate toxicity. glutamate excitotoxicity can lead to a state of self-amplifi cation, followed by an increase of intracellular calcium. these events lead to a second messenger activation, giving rise to changes that make the cell more permeable to additional calcium entry and further glutamate release. glutamate release also gives rise to oxygen free radical production, which maintains the further release of glutamate. controversial are the results from animal studies: often short delays (1–3 days) for analysis of infarct size are used. there are data showing that some treatments delay, but do not really prevent neuronal death. therefore, the protective effects are seen when analyzed at three days but not 7–28 days after insult. infarct volume or cell damage is not always predictive of functional outcome. there is signifi cant strain and vendor variability of infarct size and neuroprotective efficacy of nmda receptor antagonists, adding to the already large methodological diversity (mortimer et al. 1991; orgogozo et al. 2002). physiological nmda receptor activity, however, is also essential for normal neuronal function; potential neuroprotective agents that block virtually all nmda receptor activity will very likely have unacceptable clinical side effects. for this reason many nmda receptor antagonists have disappointingly failed advanced clinical trials for a number of diseases including stroke and neurodegenerative disorders such as huntington’s disease. in contrast, studies by lipton (2004) were the fi rst to show that memantine preferentially blocks excessive nmda receptor activity without disrupting normal activity (orgogozo et al. 2002). based on the hypothesis of glutamate-induced neurotoxicity (excitotoxicity) in cerebral ischemia, different studies examined the effi cacy and tolerability of memantine, an uncompetitive n-methyld-aspartate antagonist, in the treatment of mild to moderate vascular dementia. in a multicenter, 28-week trial carried out in france, 321 patients received 10 mg/d memantine or placebo twice a day; 288 patients were valid for intent-to-treat analysis. patients had to meet the criteria for probable vascular dementia and have a mini-mental state (mmse) score between 12 and 20 at inclusion. the 2 primary end points were the cognitive subscale of the alzheimers disease assessment scale (adas-cog) and the global clinician’s interview based impression of change (cibic-plus) (wilkock et al. 2002). after 28 weeks, the mean adas-cog scores were signifi cantly improved relative to placebo. in the intention-to-treat population, the memantine group mean score had gained an average of 0.4 points, whereas the placebo group mean score had declined by 1.6 points (95% confi dence interval, 0.49 to 3.60). the response rate for cibic-plus, defi ned as improved or stable, was 60% with memantine compared with 52% with placebo (p = 0.227, intention to treat). among the secondary effi cacy parameters, which were analyzed in the per-protocol subset, mmse was signifi cantly improved with memantine compared with deterioration with placebo (p = 0.003) (wilkock et al. 2002). another work has been conducted on the topic. the aim of the reported trial was to investigate the safety and effi cacy of memantine in mild to moderate vascular dementia (vad) (jarvis et al. 2002). 81 memantine: reality and potentiality drug target insights 2008:3 it was a 28-week, double-blind, parallel, randomized controlled trial of memantine 20 mg daily versus placebo, in probable vad which was conducted in 54 centres in the u.k. primary effi cacy parameters were cognition and the clinical global impression. a total of 579 patients were randomized and 548 patients with at least one post baseline effi cacy assessment qualifi ed for the intent-to-treat analysis. at endpoint, memantine was shown to improve cognition relative to placebo in vad: the change of cognition scores from baseline differed by a mean of −1.75 points (95% confi dence intervals −3.023 to −0.49) and a median of 2 points between the two groups, while clinical general impression on the global status rating scores showed no signifi cant differences between treatment groups. demaerschalk and wingerchuk (2007) examined in a complete meta-analysis eight relevant systematic reviews and randomized controlled trials were identifi ed and served as the principal sources of information. the best evidence to date revealed that donepezil 5 mg/d [number needed to treat (nnt) = 10] was the most effective and best tolerated [number needed to harm (nnh) = 50] of the available agents. galantamine 24 mg/d (nnt = 7) was also effective but less well tolerated (nnh = 7). due to insuffi cient evidence, rivastigmine could not yet be recommended for the treatment of vascular dementia. memantine appeared to be safe and well tolerated but did not demonstrate effectiveness across all cognitive outcomes and clinical global measures. acetylcholinesterase inhibitors and nmda receptor antagonists, in general, displayed promise as treatments for patients with vascular dementia and vascular cognitive impairment. the most effective, evidence-based treatments were donepezil and galantamine memantine and parkinson’s disease because of its peculiar pharmacological properties, memantine is proposed to be benefi cial, as it blocks excessive nmda receptors activation, without interfering with their physiological activity. an anti-parkinsonian activity has been described for memantine, in animal models of parkinson’s disease and in parkinsonian patients (merello et al. 1999). in addition, memantine prevents cell death induced by 1-methyl-4-phenyl1,2,3,6tetrahydropyridine (kucheryanu and kryzhanovskii, 2000). therefore, an italian group (giustizieri et al. 2007) focused our attention onto the action of this drug on the dopamine neurons of the substantia nigra pars compacta (snc), whose progressive degeneration is a hallmark of parkinson’s disease. indeed, several lines of evidence indicate an overstimulation of glutamate receptors, especially of the nmda subtype, as the main cause of the progressive loss of this neuronal population (gardoni and di luca, 2006). memantine has been showed not only to increase the fi ring rate of the dopamine neurons, but also changed occasionally their fi ring mode, from tonic to bursting behavior. this change in the fi ring pattern may have important functional implications, as burst fi ring of the dopamine neurons has been associated to increased release of dopamine in the areas of nigral projection (floresco et al. 2003; phillips et al. 2003). according to the observations of giustizieri et al. (2007), memantine does not affect the basal fi ring activity of the dopamine neurons in physiological conditions, while, in conditions of metabolic stress, a signifi cant effect of memantine emerges, resulting in recovery of fi ring activity of previously silenced dopamine neurons. this property may be particularly relevant in terms of fi ring dependent dopamine release and in relation to prevention of neuronal loss in parkinson’s disease. the activity of complex i of the mitochondrial respiratory chain is reduced in dopamine neurons of parkinsonian patients (schapira, 2001), and drugs acting as inhibitors of complex i, like rotenone or 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine induce dopamine neurons degeneration (przedborski and vila, 2003). a recent report by liss and colleagues (2005) proposed that the selective vulnerability of the snc dopamine neurons in parkinson’s disease is casually correlated with the opening of katp conductances in these neurons; thus, the presence of functional katp channels promotes the selective loss of snc dopamine neurons in both a genetic model of parkinson’s disease and in response to mitochondrial complex i inhibition. at present, the mechanism through which katp channel opening contributes to dopamine neurons degeneration is still unclear. there is evidence that increasing neuronal excitability protects dopamine neurons from degeneration (salthunlassalle et al. 2004), for this reason liss and colleagues (2005) proposed that drugs acting at katp channels of snc dopamine neurons, should cause 82 moretti et al drug target insights 2008:3 a recovery from their functional silencing, thus providing a clinical benefi t in the treatment of parkinson’s disease. indeed, memantine has been shown to prevent cell death associated to parkinson’s disease (merello et al. 1999; kucheryanu and kryzhanovskii, 2000), although prevention of excitotoxic neuronal damage through an uncompetitive inhibition of nmda receptors has been proposed as its underlying mechanism of action. giustizieri et al. results (2007) results show that memantine does inhibit nmda responses in the snc, however, memantine may also result benefi cial in parkinson’s disease patients because it reduces dopamine neurons silencing through closure of katp conductances. quite balancing the preclinical studies, there are not so consistent data in parkinson patients in a clinical perspective. an open-fashion study, involving 14 parkinsonian patients with motor fl uctuations taking l-dopa, has been conducted (rabey et al. 1992); these patients were given a supplement of memantine 30 mg/day. after one month, 10 patients completed the treatment (4 discontinued it due to abdominal pain, psychomotor agitation, confusion and dizziness). in 5 patients, the main parkinsonian features improved signifi cantly (1 point or more on the webster scale). in 6 patients, “off ” episodes improved (from daily mean of 273 minutes, to 172 minutes). in summary, the authors postulated that memantine addition to parkinsonian features, could form a basis for novel therapeutic strategies directed to neutralize the effects of glutamate at striatal and subthalamic levels. another work has been conducted quite later (merello et al. 1999); the aim was to evaluate the effect of memantine on cardinal symptoms of parkinson’s disease and on the latency, duration, and magnitude of the response to a single dose of l-dopa and on drug-induced dyskinesias. twelve hoehn-yahr iii-iv patients with idiopathic parkinson’s disease with motor fl uctuations and drug-induced dyskinesias were randomized to the nmda antagonist memantine or placebo in a cross-over design. a single-dose l-dopa challenge was performed after each medication arm. a signifi cant drug effect on the unifi ed parkinson’s disease rating scale motor score was observed in “off ” and “on” states (f(1,11) = 13.5; p � 0.003). no signifi cant effect on drug-induced dyskinesias was seen. the results suggest that memantine may improve parkinsonian symptoms independently of dopaminergic drugs and, in contrast to recent fi ndings with amantadine, it has no effect on druginduced dyskinesias. very recently, a new study has been conducted to determine the effect of memantine on parkinson disease (seeman et al. 2007), considering that memantine is reported to improve symptoms in moderate cases of alzheimer’s disease and parkinson’s disease, but is also known to trigger psychosis in some parkinson patients. because these clinical features suggested a possible dopamine component of memantine action, we measured the potency of memantine on the functional high-affi nity state of dopamine d2 receptors, or d2(high). using [(3)h]domperidone to label d2 receptors, the memantine dissociation constant at d2(high) was 917 +/− 23 nm for rat striatal d2 receptors and 137 +/− 19 nm for human cloned d2long receptors. the memantine dissociation constant for striatal n-methyl-d-aspartate (nmda) receptors labeled by [(3)h] mk 801 was 2200 +/− 400 nm. memantine stimulated the incorporation of [(35)s]gtpgamma-s into d2-expressing chinese hamster ovary cells with a dissociation constant of 1200 +/− 400 nm. memantine, between 200 and 2000 nm, directly acted on d2(high) to inhibit the release of prolactin from isolated anterior pituitary cells in culture. because the memantine potencies at nmda receptors and dopamine d2(high) receptors are of a similar order of magnitude, it is likely that the clinical features of memantine can be attributed to its action at both types of receptors. conclusions memantine protects cultured neurons from excitotoxin-induced cell-death. memantine exerts neuroprotective effects in several models of brain injury. the drug attenuated loss of cholinergic neurons in the cns induced by injection of nmda into the basal forebrain of rats. it has been shown that memantine induced production of brain-derived neurotrophic factor (bdnf), a substance shown to promote survival and differentiation of cns neuron. due to the preclinical effects of memantine owing to its anti-ischemic and anti-excitotoxic properties, recent clinical effi cacy has been demonstrated in patients with advanced dementia of vascular origins. therefore, it has been employed in different trials, in vascular dementia, showing a potential benefi t and no unbearable side effects. memantine has a small benefi cial, clinically detectable effect on cognitive function and 83 memantine: reality and potentiality drug target insights 2008:3 functional decline measured at 6 months in patients with moderate to severe alzheimer’s disease (ad). in patients with mild to moderate dementia, the small benefi cial effect on cognition was not clinically detectable in those with vascular dementia and barely detectable in those with ad. it is well tolerated (mcshane et al. 2007). main results can be summarized as follows: 1. moderate to severe ad. two out of three six month studies show a small benefi cial effect of memantine. pooled data indicate a benefi cial effect at six months on cognition (2.97 points on the 100 point sib, 95% ci 1.68 to 4.26, p � 0.00001), activities of daily living (1.27 points on the 54 point adcs-adlsev, 95% ci 0.44 to 2.09, p = 0.003) and behaviour (2.76 points on the 144 point npi, 95% ci 0.88 to 4.63, p = 0.004), supported by clinical impression of change (0.28 points on the 7 point cibic+, 95% ci 0.15 to 0.41, p � 0.0001). 2. mild to moderate ad. pooled data from three unpublished studies indicate a marginal benefical effect at six months on itt cognition (0.99 points on the 70 point adas-cog, 95% ci 0.21 to 1.78, p = 0.01) which was barely detectable clinically (0.13 cibic+ points, 95% ci 0.01 to 0.25, p = 0.03) but no effect on behaviour, activities of daily living or oc analysis of cognition. 3. mild to moderate vascular dementia. pooled data from two six month studies indicated a small benefi cial effect of memantine on cognition (1.85 adas-cog points, 95% ci 0.88 to 2.83, p = 0.0002), and behaviour (0.84 95% ci 0.06 to 0.91, p = 0.03) but this was not supported by clinical global measures. 4. patients taking memantine were slightly less likely to develop agitation (134/1739, 7.7% versus 175/1873, 9.3% or 0.78, 95% ci 0.61 to 0.99, p = 0.04). this effect was slightly larger, but still small, in moderate to severe ad (58/506 [12%] vs 88/499 [18%]; or = 0.6, 95% ci 0.42 to 0.86, p = 0.005). there is no evidence either way about whether it has an effect on agitation which is already present. importantly, however, it does appear that the dementia caused by brain vascular disease may share similar anatomic substrates with ad, supporting the notion of a common substrate to dementia. different studies suggest that memantine should be studied in a wider and broader population, even in parkinson disease, but results are quite experimental. it might be postulated that many studies should be designed to define the real clinical relevance of the laboratory and pre-clinical observations. note the authors express their gratitude to ms mara conte for her precious help for the corrections of the text. references ambrozi, l. and danieczyk, w. 1988, may. treatment of impaired cerebral function in psychogeriatric patients with memantine—results of a phase ii double-blind study. pharmacopsychiatry, 21(3):144–6. blanpied, t.a., boeckman, f.a., aizenman, e. et al. 1997, jan. trapping channel block of nmda-activated responses by amantadine and memantine. j. neurophysiol., 77(i):309–23. bleich, s., wiltfang, j., kornhuber, j., reisberg, b., doody, r. and möbius, h.j. 2003. memantine in moderate-to-severe alzheimer’s disease. n. engl. j. med., 349:609–10. bormann, j. 1989, aug 3. memantine is a potent blocker of n.-methyldaspartate (nmda) receptor channels. eur. j. pharmacol., 166(3):591–2. braak, h., braak, e. and bohl, j. 1993. staging of alzheimer related cortical destruction. eur. neurol., 33:403–8. brorson, j.r., bindokas, y.p., iwama, i., marcuccilli, c.j., chisholm, j.c. and miller, r.j. 1995. the ca2+ infl ux induced by beta-amyloid peptide 25–35 in cultured hippocampal neurons results from network excitation. j. neurobiol., 26:325–38. chapira, a.h. 2001. causes of neuronal death in parkinson’s disease. adv. neurol., 86:155–62. chen, h-s. and lipton, s.a. 1997, feb 15. mechanism of memantine block of nmda-activated channels in rat retinal ganglion cells: uncompetitive antagonism. j. physiol., 499(pt i):27–46. choi, d.w. 1995. calcium: still center-stage in hypoxicischemic neuronal death. trends neurosci., 18:58–60. cullen, w.k., wu, j.q., anwyl, r. and rowan, m.j. 1996. beta amyloid produces a delayed nmda receptor-dependent reduction in synaptic transmission in rat hippocampus. neuroreport., 8:87–92. danysz, w., zajaczkowski, w. and parsons, c.g. 1995. modulation of learning processes by ionotropic glutamate receptor ligands. behav. pharmacal., 6:455–74. danysz, w., parsons, c.g., mobius, h.j., stoffl er, a. and quack, g. 2000. neuroprotective and symptomatological action of memantine relevant for alzheimer’s disease—a unifi ed glutamatergic hypothesis on the mechanism of action. neurotoxicity res., 2:85–97. demaerschalk, b.m. and wingerchuk, d.m. 2007, jan. treatment of vascular dementia and vascular cognitive impairment. neurologist, 13(1):37–41. dogan, a., eras, m.a., rao, vlr. et al. 1999. protective effects of memantine against ischemia-reperfusion injury in spontaneously hypertensive rats. acta. neurochir (wien)., 141(10):1107–13. erdo, s.l. and schafer, m. 1991, jun 6. memantine is highly potent in protecting cortical cultures against excitotoxic cell death evoked by glutamate and n.-methyj.-d-aspartate. eur. j. pharmacol., 198(2–3):215–7. fischer, p-a., jacobi, p., schneider, e. et al. 1977. effects of intravenous administration of memantine in parkinsonian patients (in ger-23 man]. arzneimittelforschung, 27(ii)(7):1487–9. floresco, s.b., west, a.r., ash, b., moore, h. and grace, a.a. 2003. afferent modulation of dopamine neuron fi ring differentially regulates tonic and phasic dopamine transmission. nat. neurosci., 6:968–73. 84 moretti et al drug target insights 2008:3 francis, p.i., sims, n.r., procter, a.w. and bowen, d.m. 1993. cortical pyramidal neurone loss may cause glutamatergic hypoactivity and cognitive impairment in alzheimer’s disease—investigative and therapeutic perspectives. j. neurochem., 60:1589–604. frankiewicz, t. and parsons, c.g. 1999, sep. memantine restores long term potentiation impaired by tonic n-methyl-d-aspartate (nmda) receptor activation following reduction of mg2+ in hippocampal slices. neuropharmacology, 38(9):1253–9. gardoni, f. and di luca, m. 2006. new targets for pharmacological intervention in the glutamatergic synapse. eur. j. pharmacol., 545:2–10. giulian, d., haverkamp, l.j., li, l., karshin, w.l., yu, l., tom, d., li, x. and kirkpatrick, j.b. 1995. senile plaques stimulate microglia to release a neurotoxin found in alzheimer brain. neurochem. int., 27:119–37. giustizieri, m., cucchiaroni, m.l., guatteo, e., bernardi, g., mercuri, n.b. and berretta, n. 2007, may 11. memantine inhibits atp-dependent k+ conductances in dopamine neurons of the rat substantia nigra pars compacta jpet fast forward. published on, as doi:10.1124/ jpet.107.122036. goodwin, j.l., demura, e. and cunnick, j.e. 1995. microglial release of nitric oxide by the synergistic action of beta-amyloid and ifn-gamma. brain res., 692:207–14. gorgiilii, a., kins, t., cobanoglu, s. et al. 2000. reduction of edema and infarction by memantine and mk-80l after focal cerebral ischaemia and reperfusion in rat. acta. neurochir (wien), 142(11):1287–92. hartmann, s. and mobius, h. 2003. tolerability of memantine in combination with cholinesterase inhibitors in dementia therapy. int. clin. psychopharmacol., 18:81–5. heim, c. and sontag, k.h. 1995. memantine prevents progressive functional neurodegeneration in rats. j. neura.l transm, 46(supp1):17–30. jarvis, b. and figgitt, d.p. 2003. memantine. drugs and aging, 20(6):465–76. jones, r.w., bayer, a., inglis, f. et al. 2007, january. safety and tolerability of once-daily versus twice-daily memantine: a randomised, doubleblind study in moderate to severe alzheimer’s disease. int. j. geriatr. p s y c h i a t r. , h t t p : / / w w w 3 . i n t e r s c i e n c e . w i l e y. c o m / c g i – b i n / jhome/4294. keilhoff, g. and wolf, g. 1992, sep 4. memantine prevents quinolinic a c i d i n d u c e d h i p p o c a m p a l d a m a g e . e u r. j . p h a r m a c o l . , 219(3):451–4. klegeris, a. and mcgeer, p.l. 1997. beta-amyloid protein enhances macrophage production of oxygen free radicals and glutamate. j. neurosci. res., 49:229–35. knopman, d. 2005. san francisco. where we stand with symptomatic treatments for alzheimer disease. american academy of neurology, 1kp003:1–34. krieglstein, j., lippert, k. and poch, g. 1996. apparent independent action of nimodipine and glutamate antagonists to protect cultured neurons against glutamate-induced damage. neuropharmacology, 35(no. j2):1737–42. kucheryanu, v.g. and kryzhanovskii, g.n. 2000. effect of glutamate and antagonists of nmethyld-aspartate receptors on experimental parkinsonian syndrome in rats. bull exp. biol. med., 130:629–32. li, s., mallory, m., alford, m., tanaka, s. and masliah, e. 1997. glutamate transporter alterations in alzheimer disease are possibly associated with abnormal app expression. j. neuropathal. exp. neurol., 56:901–11. liss, b., haeckel, o., wildmann, j., miki, t., seino, s. and roeper, j. 2005. k-atp channels promote the differential degeneration of dopaminergic midbrain neurons. nat. neurosci., 8:1742–51. lundbeck als, h. 2002. summary of product characteristics. ebixa (memantine hydrochloride). h. lundbeck ais, valby, denmark l–13. mattson, m.p., cheng, b., davis, d., bryant, k., lieberburg, 1 and rydel, r.e. 1992. beta-amyloid peptides destabilize calcium homeostasis and render human cortical neurons vulnerable to excitotoxicity. j. neurosci., 12:376–89. mcshane, r., areosa sastre, a. and minakaran, n. memantine for dementiathe cochrane library the cochrane database of systematic reviews 2007 issue 1. merello, m., nouzeilles, m.i., cammarota, a. and leiguarda, r. 1999. effect of memantine (nmda antagonist) on parkinson’s disease: a doubleblind crossover randomized study. clin. neuropharmacol., 22:273–6. mortimer, j.a., vanduijn, c.m., chandra, v., fratiglioni, l., graves, a.b., heyman, a., jorm, a.e., kokmen, e., kondo, k., rocca, w.a., shalat, s.l., soinien, s.l. and hofman, a. 1991. head trauma as a risk factor for alzheimer’s disease: a collaborative re-analysis of case control studies. int. j. epidemiol., 20(suppl 2):s28–s35. orgogozo, j.m., rigaud, a.s., stoffl er, a., mobius, h.j. and forette, f. 2002. effi cacy and safety of memantine in patients with mild to moderate vascular dementia. a randomized, placebo-controlled trial (mmm 300) stroke 33:1834–9. parsons, c.g., gruner, r., rozental, j. et al. 1993, dec. patch clamp studies on the kinetics and selectivity of n.-methyl-d aspartate receptor antagonism by memantine (l-amino-3,5-dimethyjadamantan). neuropharmacology, 32(12):1337–50. parsons, c.g., danysz, w. and quack, g. 1998. glutamate in cns disorders as a target for drug development. an update. drug news perspect, 11:523–69. pellegrini, j.w. and lipton, s.a. l993. delayed administration of memantine prevents n-methyl-d-aspartate receptor-mediated neurotoxicity. ann. neurol., 33(4):403–7. phillips, p.e., stuber, g.d., heien, m.l., wightman, r.m. and carelli, r.m. 2003. subsecond dopamine release promotes cocaine seeking. nature, 422:614–8. pomara, n., peskind, e.r., potkin, s.g. et al. 2004. memantine monotherapy is effective and safe for the treatment of mild to moderate alzheimer’s disease: a randomized controlled trial. neurobiol. aging, 25:1. p r z e d b o r s k i , s . a n d vi l a , m . 2 0 0 3 . t h e 1 m e t h y l 4 p h e n y l 1,2,3,6-tetrahydropyridine mouse model: a tool to explore the pathogenesis of parkinson’s disease. ann. n.y. acad. sci., 991:189–98. rabey, j.m., nissipeanu, p. and korczyn, a. 1992. d effi cacy of memantine, an nmda receptor antagonist, in the treatment of parkinson’s disease. journal of neural transmission, 4(4):277–82. rao, vlr., dogan, a., todd, k.g. et al. 2001, aug 17. neuroprotection by memantine, a non-competitive nmda receptor antagonist after traumatic brain injury in rats. brain res., 911(1):96–100. reisberg, b., mobius, h.j., stomer, a. et al. 2000, july 20–25. long-term treatment with the nmda antagonist memantine; results of a 24-week, open-label extension study in moderately severe to severe alzheimer’s disease [abstract]. 8th international conference on alzheimer’s disease and related disorders, stockholm. reisberg, b., doody, r., stamer, a. et al. 2003. a randomized, placebo controlled study of memantine, an uncompetitive nmda antagonist, in patients with moderate to severe alzheimer’s disease. n. engl. j. med., 348:1333–41. rothman, s.m. and olney, j.w. 1987. excitotoxicity and the nmda receptor. trends neurosci., 10:299–302. salthun-lassalle, b., hirsch, e.c., wolfart, j., ruberg, m. and michel, p.p. 2004. rescue of mesencephalic dopaminergic neurons in culture by low-level stimulation of voltage-gated sodium channels. j. neurosi, 24:5922–30. schmitt, f., van dyck, c., wichems, c., olin, j. and for the mem md 02 study group, 2006. cognitive response to memantine in moderate to severe alzheimer disease patients already receiving donepezil: an exploratory reanalysis. alzh. dis. ass. dis., 20(4):255–62. seeman, p., caruso, c. and lasaga, m. 2007, nov 13. memantine agonist action at dopamine d2(high) receptors. synapse, 62(2):149–53. seif ej. nasi’, m., peruche, b., rossberg, c. et al. 1990, aug 21. neuroprotective effect of memantine demonstrated in vivo and in vitro. eur. j. pharmacol., j. 85(1):19–24. stieg, p.e., sathi, s., warach, s. et al. 1999, jun 30. neuroprotection by the nmda receptor-associated open-channel blocker memantine in a photothrombotic model of cerebral focal ischemia in neonatal rat. eur. j. pharmacol., 375(1–3):115–20. 85 memantine: reality and potentiality drug target insights 2008:3 sudhir, k. 2004. memantine: pharmacological properties and clinical uses. neurology india, 52(3):307–9. tariot, p., farlow, m., grossberg, g. et al. 2003, may. memantine/donepezil dual therapy is superior to placebo/donepezil therapy for treatment of moderate to severe alzheimer’s disease. poster presented at the american psychiatric association annual meeting, san francisco, u.s.a. tariot, p.n., farlow, m.r., grossberg, g.t., graham, s.m., mcdonald, s. and gergel, i. 2004. memantine treatment in patients with moderate to severe alzheimer disease already receiving donepezil: a randomized controlled trial. jama, 291:317–24. weller, m., finiels-marlier, f. and paul, s.m. 1993. nmda receptormediated glutamate toxicity of cultured cerebellar, cortical and mesencephalic neurons: neuroprotective properties of amantadine and memantine. brain res., 613:143–8. wenk, g.l., zajaczkowski, w. and danysz, w. 1997, feb. neuroprotection of acetylcholinergic basal forebrain neurons by memantine and neurokinin b. behav. brain res., 83 (1–2):129–33. wilcock, g., mobius, h.j. and stoffler, a. 2002. on behalf of the mmm 500 group a double-blind, placebo-controlled multi centre study of memantine in mild to moderate vascular dementia (mmm500). international clinical psychopharmacology, 17:297–305. winblad, b., mobius, h.j. and stoffl er, a. 2002. glutamate receptors as a target for alzheimer’s disease—are clinical results supporting the hope? j. neural. transm., 62(suppl):217–25. winblad, b., jones, r.w., wirth, y. et al. 2006, september. meta-analysis of clinical trial data with memantine in moderate to severe alzheimer’s disease. poster presented at efns. wu, j.q., anwyl, r. and rowan, m.j. 1995. 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drug target insights 2008:3 137–151 137 review correspondence: rikio yoshimura, m.d., ph.d., department of urology, osaka city university hospital, 1-4-3 asahi-machi, abeno-ku, osaka 545-8585, japan. tel: 81-6-6645-3857; fax: 81-6-6647-4426; email: jasmin@med.osaka-cu.ac.jp copyright in this article, its metadata, and any supplementary data is held by its author or authors. it is published under the creative commons attribution by licence. for further information go to: http://creativecommons.org/licenses/by/3.0/. the target of 5-lipoxygenase is a novel strategy over human urological tumors than the target of cyclooxygenase-2 masahide matsuyama and rikio yoshimura department of urology, osaka city university graduate school of medicine, add: 1-4-3 asahi-machi, abeno-ku, osaka, 545-8585, japan. abstract: the metabolism of arachidonic acid by either the cyclooxygenase (cox) or lipoxygenase (lox) pathway generates eicosanoids, which have been implicated in the pathogenesis of a variety of human diseases, including cancer. it is now considered that they play important roles in tumor promotion, progression, and metastasis, also, the involvement of cox and lox expression and function in tumor growth and metastasis has been reported in human tumor cell lines. in this study, we examined the expression of cox and lox in human urological tumors (renal cell carcinoma, bladder tumor, prostate cancer, testicular cancer) by immunohistochemistry and rt-pcr, and we also examined the effects of cox and lox (5and 12-lox) inhibitors in those cells by mtt assay, hoechest staining, and fl ow cytometry. cox-2, 5-lox and 12-lox expressions were signifi cantly more extensive and intense in malignant tissues than in normal tissues. furthermore, 5-lox inhibitor induced the reduction of malignant cell viability through early apoptosis. these results demonstrated cox-2 and lox were induced in urological tumors, and 5-lox inhibitor may mediate potent antiproliferative effects against urological tumors cells. thus, 5-lox may become a new target in the treatment of urological tumors. keywords: cyclooxygenase-2, 5-lipoxygenase, 12-lipoxygenase, renal cell carcinoma, bladder tumor, prostate cancer, testicular cancer introduction angiogenetic factors play important roles in urological tumors as well as in other cancers. in recent years, the expression of angiogenic factors in solid human tumors has been widely reported [1]. growth factors secreted by tumor cells such as fi broblast growth factor, and transforming growth factor, have increased neovascularization in vivo and in vitro [2]. the metabolism of arachidonic acid (aa) by either the cyclooxygenase (cox) pathway or the lipoxygenase (lox) pathway generates eicosanoids, have been implicated in the pathogenesis of a variety of human diseases, including cancer, and are considered important in tumor promotion, progression, and metastasis. cox is the fi rst enzyme in the pathway for producing prostaglandin (pg) and thromboxane (tx) from arachidonic acid, and can occur as three isoforms, cox-1, cox-2 and cox-3. the enzymes of both cox-1 and cox-2 are transformed from the cell membrane phospholipid to arachidonic acid by the phospholipasea2, and then transform arachidonic acid to pgh2 through pgg2 (fig. 1). cox-1 occurs in tissues and cells and works to protect the cell. cox-2 express momentarily and strongly in response to growth factors and some endotoxins. it is involved with infl ammation, cell proliferation and differentiation [3]. cox-2 has also been shown to play an important role in carcinogenesis. although the existence of cox-3 has recently been reported, it continues to be argued [4]. lox is the fi rst enzyme in the pathway for producing leukotrienes (lt) from arachidonic acid. isoenzymes of lox include 5-lox, 12-lox and two 15-lox isoforms (15-lox-1, 15-lox-2). these catalyze the biosynthesis of biologically active compounds such as lts and hydroxyeicosatetraenoic acids (hetes) [5, 6]. 5-lox catalyzes the fi rst step in oxygenation of arachidonic acid to produce 5-hydroperoxyeicosatetraenoic acid (5-hpete), and the subsequent metabolism of 5-hpete to 5-hete and lts (fig. 1). lts belong to an important group of pro-infl ammatory mediators that are synthesized from arachidonic acid via the 5-lox pathway. the activity of 5-lox leads to the formation of unstable lta4, which can be converted into either ltb4, or cysteinyl lts (ltc4, ltd4 and lte4) [7]. http://creativecommons.org/licenses/by/3.0/ http://creativecommons.org/licenses/by/3.0/ 138 matsuyama and yoshimura drug target insights 2008:3 the 12-lox, includes platelet 12-lox, and leukocyte 12-lox that oxygenate arachidonic acid at position c-12 to produce 12-hydroperoxyeicosatetraenoic acid and then 12-hete [8]. whereas 5-lox, 12-lox and 15-lox-1, have procarcinogenic roles, 15-lox-2 appears to have an anti-carcinogenic roles. our research focuses on the relationship between cox-2 and lox (5and 12-lox) and urological tumors (renal cell carcinoma, bladder tumor, prostate cancer and testicular cancer) and on the antitumor effects of cox and lox inhibitors. methods tumor specimens all tissue specimens were obtained from osaka city university hospital. tumor tissues, nontumor tissues, vascular endothelium, and interstitial tissues from the subjects were preserved in 10% formalin and embedded in paraffi n, serially sectioned onto microscope slides at a thickness of 4 μm. a) cox renal cell carcinoma (rcc) specimens specimens were obtained from 108 patients with rcc and 20 patients with normal kidney (nk) tissues who underwent total nephroureterectomy due to ureteral cancer. bladder tumor (bt) specimens specimens were obtained from 118 patients with bt (including 68 who underwent total cystectomy and 50 who underwent transureteral resection of arachidonic acid 5-lox 15-lox-1 15-lox-2 12-lox 12-hete 5-hete 15-hete lta4 ltc4 ltb4 pgg2 pgh2 pgf2 pge2pgd2 pgj2 txa2 pgi2 ltd4 lte4 membrane phospholipida2 phospholipasea2 cox-2 cox-1 figure 1. map of arachidonic acid (aa) cascade. cyclooxygenase (cox) is the fi rst enzyme in the pathway for producing prostaglandin (pg) and thromboxane (tx) from aa, and there are two isoforms, cox-1 and cox-2. the enzymes of both cox-1 and cox-2 are transformed from the cell membrane phospholipid to aa by the phospholipase a2, and then transform aa to pgh2 through pgg2. lipoxygenase (lox) is an initial enzyme in the pathway for producing leukotrien (lt) from aa and there are 5-, 12-lox and two isoenzymes of 15-lox (15-lox-1, 15-lox-2) as isoforms catalyzing the biosynthesis of biologically active compounds such as lts and hydroxyeicosatetraenoic acids (hetes). 139 5-lipoxygenase in urological tumor drug target insights 2008:3 bladder tumor) and 10 patients with chronic cystitis (cc) and 8 with normal bladder tissues (nb) who underwent total prostatectomy because of prostate cancer. prostate cancer (pc) specimens specimens were obtained from 28 patients with pc, 8 patients with benign prostatic hyperplasia (bph), 1 patient with prostatic intraepithelial neoplasia (pin) who underwent total prostatectomy or subcapsular prostatectomy, and 8 patients with normal prostate (np) tissues who underwent total cystectomy because of bladder tumor. testicular cancer (tc) specimens specimens were obtained from 72 patients with tc, 20 patients with normal testis (nt) tissues who underwent orchiectomy for prostate cancer. b) lox rcc specimens specimens were obtained from 50 patients with rcc and 10 patients with nk tissues. bt specimens specimens were obtained from 170 patients with bt (87 total cystectomy and 83 transureteral resection) and 20 patients with cc and 20 patients with nb tissues. pc specimens specimens were obtained from 174 patients with pc, 20 patients with bph, 20 patients with pin, and 8 patients with np tissues. tc specimens specimens were obtained from 72 patients with tc, 20 patients with nt tissues. immunohistochemical staining immunohistochemical staining was performed with a vectastain (vector laboratories, burlingame, california) avidin-biotin preoxidase complex kit, as previously described [9]. primary antibodies against rabbit cox-1 (dhhilhvavdv) (1:100 dilution in pbs), rabbit cox-2 (lddinptvllker) (1:100 dilution in pbs), rabbit 5-lox (cayman chemical, ann arbor, michigan) (1:100 dilution in pbs), rabbit 12-lox (oxford biomedical research, oxford, michigan) (1:100 dilution in pbs) and control pbs were used. immunohistochemical analysis staining was classifi ed into 5 grades from 0 to 4 according to staining intensity and number of positive cells by two blind observers on two separate occasions using coded slides. an average score was calculated. a 4 grade indicated that all staining was maximally intense throughout the specimen, while 0 indicated that staining was absent throughout the specimen. micro-anatomical staining sites were also recorded. this method was perfomed as previously described [9]. all results are presented as the mean ± sd. data analysis were performed using anova [10]. pt-pcr concerning cox, total rna was extracted from rcc, bt, pc and tc tissues. concerning lox, total rna was extracted from rcc tissues, bt and pc cell lines by the acid guanidium thiocyanatephenol-chloroform method [11]. a) cox for polymerase chain reaction (pcr) analysis of rna, complementary dna (cdna) was made by reverse-transcription of 2 μg of each rna sample using super script preamplifi cation system for fi rst-strand cdna synthesis (gibco brl, md, u.s.a.). pcr reactions were performed with 3 μl of each cdna, 3 μl of each sense and antisense primers (20 μm), and 1 unit of taq polymerase (nippon gene, toyama, japan). 35 cycles of denaturation, annealing, and extension (94ºc for 45 sec, 54 ºc for 45 sec, and 72 ºc for 2 min) were performed on automatic heat-block (model pj2000 dna thermal cycler, perkin elmer, nj, u.s.a.). the primers used were: human cox-1 sense (5’-tgccc agctcctggcccgccgctt-3’) and antisense (5’-gtgcatcaacacaggcgcctctt c-3’); human cox-2 sense (5’-ttcaaatgagattgtgggaaaattgct-3’) and antisense (5’-agatcatctctgcctgagtatctt-3’); 140 matsuyama and yoshimura drug target insights 2008:3 h u m a n g l y c e r a l d e h y d e 3 p h o s p h a t e dehydrogenase (g3pdh) sense (5’-ccacccatggcaaattccatggca-3’) and antisense (5’-tctagagggcaggtcaggtccacc-3’). b) lox after the rt reaction, nested pcr was used to examine 5and 12-lox mrna expression. for 5and 12-lox, the fi rst run pcr profi le was 94 ºc, 15s to denature; 61 ºc, 30s for annealing and extension for 30 cycles with upstream (5’cttcccgtgctaccgctg-3’) and downstream (5’-tggggttggcaccattgag-3’) primers. 5 μl of fi rst run pcr product was used for the nested pcr with the profi le of 94 ºc, 15s to denature; 61 ºc, 30s for annealing and extension for 25 cycles with nested primers (upstream 5’-ccaggagacaatgctttggaca-3’; downstream 5’-gaacaactcatcatcctgccag-3’). reagents and materials rpmi 1640 was purchased from nissui pharmaceutical company (tokyo, japan), fetal bovine serum (fbs) and penicillin-streptomycin mixture from biowhitteker (walkerville, md), and trypsin/ edta from gibco brl (rockville, md). ibupurofen, piroxicam, meloxicam, and nimesulide were obtained from biomol research laboratories inc (u.s.a.), naproxen was obtained from cayman chemical company (st. louis, u.s.a.), indomethacin and ns 398 were obtained from wako pure chemical industries ltd (osaka, japan), and etodolac was obtained from nippon shinyaku co. ltd (kyoto, japan). ibupurofen, piroxicam, meloxicam, nimesulide naproxen, indomethacin, ns 398, and etodolac were all cox inhibitors. 5-lox inhibitor (caffeic acid) and 12-lox inhibitor (baicalein) were obtained from biomol. research laboratories inc, u.s.a. nonspecifi c lox inhibitor (ndga) was obtained from cayman chemical, u.s.a. all data characteristics of these inhibitors were published in their product information. cell cultures the human rcc cell line (caki-1) and normal prostate stromal cell were provided by dr. shinichi ikemoto (dept. of urology, osaka city university school of medicine, osaka, japan). the human bt cell line (t24), pc cell lines (pc3, du-145), tc cell line (nec-8) and normal proximal tubular endothelial cell (prtec) were obtained from health science research resources bank (osaka, japan). normal bladder cell line was obtained from the patients with normal bladder tissues who underwent total prostatectomy due to prostate cancer. cells were grown in culture flask (nunc, roskilde, denmark) in rpmi 1640 supplemented with 10% fbs, 100 u/ml of penicillin and 100 μg/ ml of streptomycin in a humidifi ed 5% co2 atmosphere at 37 ºc. the media were changed every 3 days and the cells were separated via trypsinization, using trypsin/edta when they reached subconfl uence. cell proliferative studies approximately 1.0 × 104 cells placed onto 8 × 8 mm diameter multichamber slides (nunc, copenhagen, denmark) were treated with cox and lox inhibitors (10–80 μm) dissolved in ethanol. the fi nal concentration of ethanol was �0.05%. cell viability was measured after 48 hours by a microplate reader using a modifi ed 3-[4, 5-dimethylthiazol-2-thiazolyl]-2, 5diphenyltetrazolium bromide (mtt) assay (wst-1 assay; dojindo, kumamoto, japan) and presented as the percentage of control-culture conditions. flow cytometry (annexin v and propidium iodide staining) the effects of lox inhibitors on human urological tumors cell lines were determined by dual staining with annexin v-fitc and propidium iodide using annexin v-fitc apoptosis detection kit i (biosiences pharmingen). annexin v-fitc and propidium iodide (pi) were added to the cellular suspension as in the manufacturer’s instruction, and sample fl uorescence of 1.0 × 104 cells was analyzed fl ow cytometry. flow cytometry was with facscan (becton dickinson, germany). cell which were annexin v-fitc positive and pi negative were identifi ed as early apoptosis. cell which were annexin v-fitc positive and pi positive were identified as late apoptosis or necrosis. flow cytometry (identifi cation of dna fragmentation) the assay was performed by tdt-mediated dutp nick end labelling (tunel) method using 141 5-lipoxygenase in urological tumor drug target insights 2008:3 apo-directtm kit (becton dickinson, germany). following the experiments, human urological tumors cell lines in suspension (1 × 106/ml) were fi xed with 1% pbs, washed in pbs, and suspended in 70%(v/v) ice-cold ethanol. the cells were stored in ethanol at –20 ºc until use. the positive and negative controls and the sample were stained with fitc-dutp by incubation in terminal deoxynucleotidyl transferase buffer as in the manufacturer’s instruction, and sample fl uorescence of 1 × 104 cells was analyzed by fl ow cytometry (becton dickinson, germany). results are given as % of tunel-positive cells. detection of apoptosis by hoechst staining dna chromatin morphology was assessed using hoechst staining. human urological tumors cell (5 × 105 cells) were incubated with 50 μm lox inhibitor for 24 hour. cells were washed by rpmi1640 and labeled with 8 mg/ml of hoechest 33342 (sigma-aldrich japan k.k. tokyo, japan) for 10 min; pi (sigma-aldrich japan k.k. tokyo, japan) was added (10mg/ml fi nal concentration), and the cells were examined by fl uorescence microscopy. results expression of cox and lox 1) immunohistochemistry a) cox rcc tissue sample cox-2 expression was observed in proximal and distal tubules of nk tissues. however, in epithelial cells, blood vessels and stromal tissues, while cox-2 was not expressed in nk tissues, cox-2 was strongly expressed in all rcc tissues. bt tissue sample cox-1 was weakly expressed in cc tissues but no expression was found in any bt tissues. on the other hand, cox-2 was strongly expressed in all bt tissues with an intense expression in high-grade bt group. neither cox-1 nor cox-2 were expressed in nb tissues. pc tissue sample the expression of cox-1 was very weak in pc, pin, bph and np tissues. however, cox-2 was strongly expressed in all pc tissues, although very weak expression of cox-2 was found in pin, bph and np tissues. tc tissue sample cox-1 and cox-2 were strongly expressed in all tc group tissues, although very weak expression of cox-1 and cox-2 were found in nt tissues. b) lox rcc tissue sample 5and 12-lox were strongly expressed in all grades rcc tissues (a: rcc -g1, b: rcc -g2, c: rcc -g3) and other types of rcc tissues (d: rcc papillary cell type, e: rcc chromphobe cell type, f: rcc collecting duct type) although very weak expressions of 5and 12-lox were found in nk tissues (g). immunostaining with pbs was negative (h) (fig. 2). bt tissue sample 5and 12-lox were strongly expressed in all grades of bt tissues, although very weak expressions of 5and 12-lox were found in cc and nb tissues. pc tissue sample 5and 12-lox were strongly expressed in all grades of pc and pin tissues, although very weak expressions of 5and 12-lox were found in bph and np tissues. tc tissue sample 5and 12-lox were strongly expressed in all tc group tissues, although very weak expressions of 5and 12-lox were found in nt tissues. statistical analysis of immunohistochemistry a) cox rcc tissue sample we classified 3 categories (epithelium, blood vessel, a small quantity of stromal tissue) in 142 matsuyama and yoshimura drug target insights 2008:3 a b dc cce f g h figure 2. 5-lipoxygenase (lox) immunostaining in renal cell carcinoma (rcc) and normal kidney (nk) tissues. 5-lox was strongly expressed in all slides from cancer specimens, clear cell rcc -g1, -g2 and g3 (a, b and c) and other types of rcc tissues (d: rcc papillary cell type, e: rcc chromphobe cell type, f: rcc collecting duct type). in nk tissues, expression of 5-lox was observed only in tubules and was not expressed in tissues from nk in epithelial cells, blood vessels or stromal tissues (g). immunostaining with pbs was negative (h). 143 5-lipoxygenase in urological tumor drug target insights 2008:3 rcc tissues, and examined them for intensity of cox-2 immunostaining. cox-2 expression score was signifi cantly more extensive and intense in all categories of rcc tissues than nk tissues. cox-2 expression score was higher in g1 cancer than in g3 cancer. however, no difference was seen in all categories among grades. another comparison between stages, the expression score was higher in early stage cancer pt1 than in advanced cancer (pt2 or above). however, this comparison among stages also shows no signifi cant difference among the categories. bt tissue sample we classified 3 categories (epithelium, blood vessel, stromal tissue) in bt tissues, and examined them for intensity of cox-1 and cox-2 immunostaining. cox-2 expression score was signifi cantly more extensive and intense in epithelial cells of bt and cc than in epithelial cells of nb. cox-2 expression score was higher in g3 cancer than in g1 cancer. moreover, the expression score was higher in advanced cancer (pt2 or above) than in early stage cancer (pt1 or below). on the other hand, no difference was seen in blood vessels a n d s t r o m a l t i s s u e s b e t w e e n n b a n d b t tissues. pc tissue sample we classified 3 categories (epithelium, blood vessel, stromal tissue) in pc tissues, and examined them for intensity of cox-1 and cox-2 immunostaining. cox-2 expression score was signifi cantly more extensive and intense in epithelial cells of all grades pc tissues than in bph and pin tissues. on the other hand, cox-2 expression score was high in the blood vessels, and the stromal tissues of pc in the study groups with no signifi cant difference between grades. however, cox-2 expression score in the blood vessels and stromal tissues from bph, pin and np were at basic level. tc tissue sample we classified 2 categories (epithelium, blood vessel) in tc tissues, and examined them for intensity of cox-1 and cox-2 immunostaining. cox-1 expression score was signifi cantly more extensive and intense in all categories of tc tissues than in nt tissues. cox-2 expression score was also signifi cantly more extensive and intense in all categories of tc tissues in the studied groups than in nt tissues. however, there were no signifi cant differences among the five histopathological groups in all categories. b) lox rcc tissue sample we classifi ed 3 categories (epithelium, blood vessel, a small quantity of stromal tissue) in rcc tissues, and examined them for intensity of 5and 12-lox immunostaining. 5and 12-lox expression scores were signifi cantly more extensive and intense in all categories of rcc tissues than nk tissues. only in epithelium, 5and 12-lox expression scores were higher in g1 cancer than in g3 cancer. however, no differences were seen in blood vessels and stromal tissues among the three grades. bt tissue sample we classifi ed 3 categories (epithelium, blood vessel, stromal tissue) in bt tissues, and examined them for intensity of 5and 12-lox immunostaining. 5and 12-lox expression scores were signifi cantly more extensive and intense in bt tissues than in cc and nb. a signifi cant difference was seen only in epithelium, showing that staining was intensifi ed as the grade increased. no difference was seen in blood vessels and stromal tissues between grades. comparison of early and advanced stages shows a signifi cant difference only in epithelium. no difference was seen in blood vessels and stromal tissues between early stage (pt1 or below) and advanced cancer (pt2 or above). pc tissue sample we classifi ed 3 categories (epithelium, blood vessel, stromal tissue) in pc tissues, and examined them for intensity of 5and 12-lox immunostaining. 5and 12-lox expression scores were signifi cantly more extensive and intense in pc and pin tissues than bph and np tissues in all categories. there was no significant difference between grades. 144 matsuyama and yoshimura drug target insights 2008:3 tc tissue sample we classified 2 categories (epithelium, blood vessel) in tc tissues, and examined them for intensity of 5and 12-lox immunostaining. 5and 12-lox expression scores were signifi cantly more extensive and intense in all tc groups than nt tissues in all categories. however, there were no signifi cant differences among the fi ve histopathological groups in all categories (table 1). rt-pcr a) cox rcc tissue we detected a specifi c band of cox-2 mrna band in rcc, whereas sample of from nk displayed no band of cox-2 mrna. bt tissue we detected specifi c band of cox-1 mrna in all samples (bt, cc and nb). however, we detected specifi c band of cox-2 in bt, while a weak band was displayed in cc and no clear band was displayed in nb. pc tissue we detected a specifi c band of cox-1 mrna in all samples (pc, bph and np). however, we detected a specifi c band of cox-2 was detected in pc, while a weak band was displayed in bph and no clear band was displayed in np. tc tissue we detected a specifi c band of cox-1 and cox2 mrna all tc groups. b) lox rcc tissue we detected a specifi c band of 5and 12-lox mrna in rcc, whereas the sample of from nk displayed no band of—and 12-lox mrna. bt cell line we detected a specifi c band of 5and 12-lox mrna (a: 5-lox, b: 12-lox, lane 2) in bt cell line, whereas the sample of from nb cells displayed no band of—and 12-lox mrna (a: 5-lox, b: 12-lox, lane 1) (fig. 3). pc cell line we detected a specifi c band of 5and 12-lox mrna in pc cell line, whereas a sample of from np cells displayed no band of—and 12-lox mrna. 2) effect of cox and lox inhibitors mtt assay a) cox rcc cell line all cox inhibitors were unable to induce a reduction of cell viability with the half-maximal concentration of growth inhibition of rcc cells in the range of 10–80 μm and were unable to stop the growth of rcc cells. all cox inhibitors had no effect on normal proximal tubular endothelial cells (prtec) proliferation. bt cell line similar to rcc cells, cox inhibitors could not induce a reduction of cell viability with the halfmaximal concentration of growth inhibition of bt cells in the range of 10–80 μm and could not stop the growth of bt cells. cox inhibitors had no effect on normal bladder cells proliferation. pc cell line similar to rcc and bt cells, none of the cox inhibitors could induce a reduction of cell viability with the half-maximal concentration of growth inhibition of pc cells in the range of 10–80 μm, neither could they stop the growth of pc cells. cox inhibitors had no effect on normal prostate stromal cells proliferation (table 2). tc cell line regarding rcc, bt and pc cells, some forms of cox inhibitors induced a slight reduction of tc cells growth in 80 μm, but we were unable to detect the induction of tc cells apoptosis in 80 μm cox inhibitors. 145 5-lipoxygenase in urological tumor drug target insights 2008:3 a) lox rcc cell line lox inhibitors induced a reduction of cell viability with the half-maximal concentration of growth inhibition of rcc cells in the range of 10–80 μm. although the effect of non-specifi c lox inhibitor was strongest, the effect of 5-lox inhibitor was stronger than that of 12-lox inhibitor. no lox inhibitors had any effect on normal proximal tubular endothelial cells (prtec) proliferation. bt cell line similar to rcc cells, lox inhibitors induced a reduction of cell viability with the half-maximal concentration of growth inhibition of bt cells in table 1. effects of cox and lox inhibitors in viabity of human prostate cancer and normal prostate stromal cells. % of control culture 10 μm 20 μm 40 μm 80 μm du-145 cox inhibitors ibupurofen 96.4% 107.5% 94.8% 96.7% piroxicam 127.5% 126.9% 93.0% 94.7% meloxicam 114.6% 110.2% 108.3% 97.6% nimesulide 110.1% 99.1% 99.8% 105.9% naproxen 121.6% 114.2% 106.5% 85.4% indomethacin 120.6% 117.8% 114.5% 97.9% ns398 91.5% 80.7% 81.9% 61.3% etodolac 105.1.% 106.7% 104.4% 95.1% lox inhibitors baicalein 102.4% 99.1% 85.3% 63.5% caffeic acid 80.7% 69.2% 22.2% 8.1% ndga 67.7% 42.3% 9.9% 5.2% pc3 cox inhibitors ibupurofen 90.0% 86.2% 75.3% 62.9% piroxicam 93.6% 87.4% 78.2% 64.3% meloxicam 98.1% 97.9% 89.3% 67.4% nimesulide 83.1% 91.5% 78.1% 94.1% naproxen 87.4% 90.7% 94.7% 105.8% indomethacin 95.1% 96.8% 86.6% 65.9% ns398 88.9% 77.5% 67.1% 58.1% etodolac 93.6% 88.3% 89.1% 77.5% lox inhibitors baicalein 117.8% 100.2% 103.8% 76.5% caffeic acid 112.5% 96.7% 78.8% 45.3% ndga 113.0% 101.7% 51.1% 18.5% normal prostate stromal cell cox inhibitors ibupurofen 97.6% 95.6% 92.3% 81.7% piroxicam 99.5% 105.1% 98.3% 108.1% meloxicam 115.3% 97.0% 103.6% 108.7% nimesulide 96.1% 95.1% 99.6% 103.3% naproxen 95.2% 94.9% 94.6% 107.7% indomethacin 98.4% 116.5% 118.1% 113.4% ns398 106.0% 92.1% 90.5% 90.3% etodolac 101.0% 104.1% 104.8% 98.6% lox inhibitors baicalein 97.2% 84.8% 87.0% 81.6% caffeic acid 89.7% 80.1% 81.8% 84.1% ndga 107.3% 86.9% 88.6% 80.7% the dose-response analysis of viability in human prostate cancer and normal prostate stromal cells treated with cox and lox inhibitors (10-80 μm, 48 hr) was measured by the mtt assay and expressed as% of control culture conditions. 146 matsuyama and yoshimura drug target insights 2008:3 the range of 10–80 μm. although the effect of non-specifi c lox inhibitor was strongest, the effect of 5-lox inhibitor was stronger than that of 12-lox inhibitor. no lox inhibitors had any effect on normal bladder cells proliferation. pc cell line similar to rcc and bt cells, lox inhibitors induced a reduction of cell viability with the half-maximal concentration of growth inhibition of pc cells in the range of 10–80 μm. although the effect of nonspecifi c lox inhibitor was strongest, the effect of 5-lox inhibitor was stronger than that of 12-lox inhibitor. no lox inhibitors had any effect on normal prostate stromal cells proliferation (table 2). tc cell line similar to rcc, bt and pc cells, lox inhibitors induced a reduction of cell viability with the halfmaximal concentration of growth inhibition of tc cells in the range of 10–80 μm. although the effect of non-specifi c lox inhibitor was strongest, the effect of 5-lox inhibitor was stronger than that of 12-lox inhibitor. 3) apoptosis effect of lox inhibitor a) flow cytometry rcc cell line rcc cells treated with 100 μm 5-lox inhibitor could induce early apoptosis, not late apoptosis or necrosis and dna fragmentation. however, treated with 100 μm 5-lox inhibitor did not induce apoptosis in normal proximal tubular endothelial cellss (prtec). diagram of fitcannexin v/pi fl ow cytometry (fig. 4) and typical fl ow cytometry analysis histogram are presented (fig. 5). bt cell line similar to rcc cells, bt cells treated with 100 μm 5-lox inhibitor could induce early apoptosis, not late apoptosis or necrosis and dna fragmentation. figure 3. rt-pcr analysis of 5and 12-lipoxygenase (lox) in bladder tumor (bt) cell line and normal bladder (nb) cell line. using specifi c primers for 5and 12-lox, the amplifi cation predicted fragments of 337 bp for 5-lox and 345 bp for 12-lox in length. a; 5-lox, b; 12-lox. lane 1; nb cells, lane 2; bt cells. bt cells expressed signifi cant 5and 12-lox mrna bands while nb cells expressed no 5and 12-lox mrna bands. table 2. statistical analysis of 5and 12-lox immunostaining. av. ± sd tumor type epithelium blood vessel 5-lox immunostaining seminoma 2.3 ± 0.8* 1.9 ± 0.7* embryonal carcinoma 2.8 ± 0.8* 2.3 ± 0.7* yolk sac tumor 1.6 ± 0.6* 1.4 ± 0.6* choriocarcinoma 2.4 ± 0.6* 1.9 ± 0.5* teratoma 2.5 ± 0.7* 2.0 ± 0.6* normal testis 0.9 ± 0.5 0.7 ± 0.5 12-lox immunostaining seminoma 2.0 ± 0.8* 1.6 ± 0.8* embryonal carcinoma 2.4 ± 0.6* 1.9 ± 0.9* yolk sac tumor 1.3 ± 0.5* 1.2 ± 0.6* choriocarcinoma 2.1 ± 0.7* 1.7 ± 0.5* teratoma 2.3 ± 0.8* 1.9 ± 0.7* normal testis 0.9 ± 0.5 0.6 ± 0.4 note: graded 0 to 4 on the coded sections by two observers in a blinded manner. 0, no staining; 4, maximum intensity. statistical analysis was performed using the analysis of variance (p value; anova). 5-and 12-lox immunostaining were more intense and diffuse in testicular tumor tissues than in the normal testicular tissues. p � 0.001. a b 147 5-lipoxygenase in urological tumor drug target insights 2008:3 pc cell line similar to rcc and bt cells, pc cells treated with 100 μm 5-lox inhibitor could induce early apoptosis, not late apoptosis or necrosis and dna fragmentation. tc cell line similar to rcc, bt and pc cells, tc cells treated with 100 μm 5-lox inhibitor could induce early apoptosis, not late apoptosis or necrosis and dna fragmentation. 2) hoechest staining rcc cell line rcc cells treated with 50 μm 5-lox inhibitors caffeic acid, and non-specifi c lox inhibitor ndga showed chromatin condensation, cellular shrinkage, small membrane-bound bodies (apoptotic bodies), and cytoplasmic condensation. cells with 12-lox inhibitor baicalein showed the same apoptotic changes slightly. in contrast, untreated cells maintained normal chromatin patterns and cell size. caki-1 prtec a nn ex in -v -f it c a nn ex in -v -f it c a nn ex in -v -f it c a nn ex in -v -f it c 99.5 % 74.2 % 25.8 % control control 100μm 5-lox inhibitor 100μm 5-lox inhibitor 99.6 % 95.9 %pi pi pi pi 4.1 %0.4 % 0.5 % figure 4. effects of 5-lipoxygenase (lox) inhibitor on early and late apoptosis as shown by fl ow cytometry on human renal cell carcinoma (rcc) cells. treatment with 100 μm 5-lox inhibitor induced early apoptosis in most of the total percentage of rcc cells. however, treatment with 100μm 5-lox inhibitor did not induce apoptosis in normal proximal tubular endothelial cells (prtec). the top left quadrants represent early apoptosis (annexin v-fitc-positive cells and pi-negative cells). the top right quadrants represent late necrosis and necrosis (annexin v-fitc-positive cells and pi-positive cells). diagram of fitc-annexin v/pi fl ow cytometry in a representative experiment are presented. 148 matsuyama and yoshimura drug target insights 2008:3 bt cell line similar to rcc cells, bt cells treated with 50 μm 5-lox inhibitors caffeic acid (b), and non-specifi c lox inhibitor ndga (d) showed chromatin condensation, cellular shrinkage, apoptotic bodies, and cytoplasmic condensation. cells with 12-lox inhibitor baicalein (c) showed the same apoptotic changes slightly. in contrast, untreated cells maintained normal chromatin patterns and cell size (a) (fig. 6). pc cell line similar to rcc and bt cells, pc cells treated with 50 μm 5-lox inhibitors caffeic acid, and nonspecifi c lox inhibitor ndga showed chromatin condensation, cellular shrinkage, apoptotic bodies, and cytoplasmic condensation. cells with 12-lox inhibitor baicalein showed the same apoptotic changes slightly. in contrast, untreated cells maintained normal chromatin patterns and cell size. tc cell line similar to rcc, bt and pc cells, tc cells treated with 50 μm 5-lox inhibitors caffeic acid, and non-specific lox inhibitor ndga showed chromatin condensation, cellular shrinkage, apoptotic bodies, and cytoplasmic condensation. cells with 12-lox inhibitor baicalein showed the same apoptotic changes slightly. in contrast, untreated cells maintained normal chromatin patterns and cell size. discussion with recent increases in routine medical check-ups and progress in diagnostic imaging techniques, the discoveries of rcc have risen. the cause of rcc is unknown. rcc generally does not respond well to radiotherapy and chemotherapy compared to many other types of cancers, and anticancer drugs as interleukin-2 is used with relative success. surgery is currently the only therapeutic option. hence, new molecular targets are needed for the treatment and prevention of rcc. the natural history of bt is not well understood, but exposure to carcinogens, including aromatic amines, is considered a major risk factors for the development of bt. workers exposed to aromatic caki-1 1.2 % 75.9 % dutp fitc dutp fitc control 100μm 5-lox inhibitor dutp fitc dutp fitc control 100μm 5-lox inhibitor prtec 1.6 % 11.5 % figure 5. 5-lipoxygenase induced dna fragmentation in human renal cell carcinoma (rcc) cells. treatment with 100 μm 5-lox inhibitor induced dna fragmentation in rcc cells. however, treatment with 100μm 5-lox inhibitor did not induce dna fragmentation in normal proximal tubular endothelial cells (prtec). typical fl ow cytometry analysis histogram in representative experiment are presented. 149 5-lipoxygenase in urological tumor drug target insights 2008:3 amines frequently have a mutated p53 gene, a tumor suppressor gene involved in the carcinogenesis of many tumors. pc comprises 32% of all cancers in american men and is on the rise worldwide. because of increased screening, pc is frequently diagnosed at a clinically localized stage, making it amenable to the therapy. nevertheless, it remains the second most common cause of cancer death in men. these patients generally respond to androgen deprivation therapy, but the vast majority eventually experience disease progression and become refractory to sustained hormonal manipulation. typically, such patients progress with a rise in their serum prostatespecific antigen level. unfortunately, standard therapeutic options at this stage of disease are limited, and while there has been some success with chemotherapy for hormone-refractory pc patients, the response is generally short-lived [12]. tc is very rare with over 90% of all tc being germ cell tumors (seminoma and non-seminoma), and the remaining percentage non-germinal tumors. the survival rate of tc has improved in recent years, refl ecting the development and refi nement of effective combination chemotherapy. however, it is still necessary to improve the treatment of tc. n o n s t e r o i d a l a n t i i n f l a m m a t o r y d r u g s (nsaids) have anti-tumor effects on human urological tumors (rcc, bt, pc and tc) and have attracted a great deal of attention. the typical target of nsaids is cox. in recent reports, a number of patients have had significantly low-risk of colorectal cancer while they continued using nsaids typifi ed by aspirin. consequently, the suppression of carcinogenesis by administering nsaids has come into focus. it was also reported that the size and number of adenoma were markedly reduced when sulindac which is a type of nsaids was given to patients with the familial adenomatous polyposis, a high risk group for colorectal cancer [13]. a b c d figure 6. effects of lipoxygenase (lox) inhibitor in induction of apoptosis on human bladder tumor (bt) cells. bt cells treated with 5-lox inhibitors caffeic acid (b), and non-specifi c lox inhibitor ndga (d) showed chromatin condensation, cellular shrinkage, small membrane-bound bodies (apoptotic bodies), and cytoplasmic condensation. cells with 12-lox inhibitor baicalein showed the same apoptotic changes slightly (c). in contrast, untreated cells (a) maintained normal chromatin patterns and cell size. 150 matsuyama and yoshimura drug target insights 2008:3 regarding cox-2 in rcc, bt and pc, cox-2 expression in malignant tissues was stronger than that in normal tissues using immunohistochemical staining and rt-pcr [9, 14, 15]. both cox-1 and cox-2 expressions in all tissue types of tc were stronger than those in normal tissues using immunohistochemical staining and rt-pcr [16]. both cox-1 and cox-2 expressions appeared stronger in all tissue types of tc possibly due to the amount of pg increased in tc. evidence revealed the pg quantity increased in breast cancer tissue compared to normal breast tissue and the expression of both cox-1 and cox-2 increased [17]. many publications have reported cox-2 expression in malignant tissue was stronger than in normal tissue, and cox-2 expression in malignant tissue was stronger than cox-1 expression in malignant tissue. however, the correlation between the grade or stage, and cox-2 expression can be argued. although many papers have reported nsaids produce anti-tumor effects, our studies confi rmed that eight kinds of cox inhibitors were unable to induce reduction of the viability in human urological tumors cells in the range of 10–80 μm by mtt assay [18]. our results suggest cox-2 expression is strong in treating urological tumors, but the antitumor effect of cox inhibitor (including cox-2 inhibitor) is very weak in urological tumor patients in a single administration at a clinical dose. cox-2 inhibitor is suitable for chemopreventive therapy. regarding 5and 12-lox in urological tumors, 5and 12-lox expressions in malignant tissues were stronger than those in normal tissues using immunohistochemical staining and rt-pcr [8, 19–21]. similar to cox-2, the correlation between the grade or stage, and lox expression can be argued. furthermore, lox inhibitor (particularly 5-lox inhibitor) could induce reduction of the viability in human urological tumors cells in the range of 10–80 μm by mtt assay. the effect of 5-lox inhibitor was stronger than that of 12-lox inhibitor [22]. furthermore, urological tumors cells treated with 5-lox inhibitor could induce early apoptosis and dna fragmantation in urological tumors cells using fl ow cytometry and hoechest staining. several papers have reported lox inhibitors to be targets for development of new chemopreventive or chemotherapeutic strategies for pc. ghosh j et al. reported that inhibition of arachidonate 5-lox triggers massive apoptosis in both androgen-sensitive (lncap) and androgenrefractory (pc3) human pc cells [23]. furthermore, ghosh j also reported the metabolites of arachidonate 5-lox promoted survival of pc cells involving down-regulation of stress-activated protein kinase [24]. pommery n et al. reported dual cox-2/5-lox inhibitors induced agents potentially useful in pc chemotherapy through apoptosis [25]. ghosh j also reported the combination of selenium and 5-lox inhibitors may be a more effective regimen for pc control [26]. research strongly suggests lox expression is strong in urological tumors, but the anti-tumor effect of 5-lox inhibitor is weak in urological tumor patients receiving a single administration at a clinical dose. 5-lox inhibitor is suitable as a chemo-preventive therapy. in conclusion, it is clear that cox-2 and lox (particularly 5-lox) are involved in the initiation and promotion of urological tumor tissues. it may be possible to use cox-2 and lox inhibitors as anti-tumor drugs from the viewpoint of preventing cancer. however, it may be diffi cult to use the cox-2 inhibitor or 5-lox inhibitor at the clinical dose with expectation of a suppressive effect on the cancer. although the clinical application of 5-lox inhibitor requires further research and consideration, the target of 5-lox is a novel strategy in the treatment of human urological tumors. acknowledements this manuscript was edited by hilah edney, bs, ms. references [1] weidner, n., folkman, j., pozza, f., bevilaqua, p., allred, e.n. and moore, d.h. 1992. tumor angiogenesis: a new signifi cant and independent prognostic indicator in early stage brest carcinoma. j. nalt. cancer inst., 84:1875–87. [2] lafyatis, r., thompson, n.l., remmers, e.f., flanders, k.c., roche, n.s. and kim, s.j. 1989. transforming growth factor-beta production by synovial tissues from rheumatoid patients and streptococcal cell wall arthritic rats. studies on secretion by synovial fi broblast-like cells and immunohistologic localization. j. immunol., 143:1142–8. [3] xie, w., chipman, j.g., robertson, d.l., erikson, r.l. and simmons, d.l. 1991. expression of a mitogen-responsive gene encoding prostaglandin synthase is regulated by mrna splicing. proc. natl. acad. sci. u.s.a., 88:2692–6. [4] matsuyama, m. and yoshimura, r. 2004. relationship between cyclooxygenase (cox)-2 and malignant tumors. recent res. devel. cancer., 6:243–51. 151 5-lipoxygenase in urological tumor drug target insights 2008:3 [5] sigal, e. 1991. the molecular biology of mammalian arachidonic acid metabolism. am. j. physiol., 260:13–28. [6] funk, c.d. 1996. the molecular biology of mammalian lipoxygenases and the quest for eicosanoid functions using lipoxygenase-defi cient mice. biochim. biophys. acta., 11:65–84. [7] matsuyama, m., hayama, t., funao, k., kawahito, y., sano, h., takemoto, y., nakatani, t. and yoshimura, r. 2007. overexpression of cysteinyllt1 receptor in prostate cancer and cyslt1r antagonist inhibits prostate cancer cell growth through apoptosis. oncol. rep., 18:99–104. [8] yoshimura, r., matsuyama, m., tsuchida, k., kawahito, y., sano, h. and nakatani, t. 2003. expression of lipoxygenase in human bladder carcinoma and growth inhibition by its inhibitors. j. urol., 170:1994–9. [9] yoshimura, r., sano, h., masuda, c., kawamura, m., tsubouchi, y., chargui, j., yoshimura, n., hla, t. and wada, s. 2000. expression of cyclooxygenase-2 in prostate carcinoma. cancer, 89:589–96. [10] fitzgerald, s.m. and flinn, s. 2000. evaluating research studies using the analysis of variance (anova): issues and interpretations. j. hand. ther., 13:56–60. [11] kawahito, y., sano, h., mukai, s., asai, k., kimura, s., yamamura, s., kato, h., chrousos, g.p., wilder, r.l. and kondo, m. 1995. corticotoropin-releasing hormone in colonic mucosa in patients with ulcerative colitis. gut, 37:544–51. [12] oh, w.k. and kantoff, p.w. 1998. management of hormone refractory prostate cancer: current standards and future prospects. j. urol, 160:1220–9. [13] sano, h., kawahito, y., wilder, r.l., hashiramoto, a., mukai, s., asai, k., kimura, s., kato, h., kondo, m. and hla, t. 1995. expression of cyclooxygenase-1 and -2 in human colorectal cancer. cancer res., 55:3785–9. [14] yoshimura, r., matsuyama, m., kawahito, y., tsuchida, k., kuratsukuri, k., takemoto, y., mitsuhashi, m., sano, y. and nakatani, t. 2004. stydy of cyclooxygenase-2 in renal cell carcinoma. int. j. mol. med., 13:229–33. [15] yoshimura, r., sano, h., mitsuhashi, m., kohno, m., chargui, j. and wada, s. 2001. expression of cyclooxygenase-2 in patients with bladder carcinoma. j. urol., 165:1468–72. [16] hase, t., yoshimura, r., matsuyama, m., kawahito, y., wada, s., tsuchida, k., sano, h. and nakatani, t. 2003. cyclooxygenase-1 and -2 in human testicular tumours. eur. j. cancer, 39:2043–9. [17] hwang, d., scollard, d., byrne, j. and levine, e. 1998. expression of cyclooxygenase-1 and cyclooxygenase-2 in human breast cancer. j. natl. cancer inst., 90:455–60. [18] yoshimura, r., matsuyama, m., kawahito, y., takemoto, y., tsuchida, k., kuratsukuri, k., segawa, y., sinnka, t., sano, y. and nakatani, t. 2004. the effect of cyclooxygenase-2 inhibitors on urological cancer cells. int. j. mol. med., 13:789–93. [19] matsuyama, m., yoshimura, r., mitsuhashi, m., tsuchida, k., takemoto, y., kawahito, y., sano, h. and nakatani, t. 2005. 5-lipoxygenase inhibitors attenuate growth of human renal cell carcinoma and induce apoptosis through arachidonic acid pathway. oncol. rep., 14:73–9. [20] matsuyama, m., yoshimura, r., mitsuhashi, m., hase, t., tsuchida, k., takemoto, y., kawahito, y., sano, h. and nakatani, t. 2004. expression of lipoxygenase in human prostate cancer and growth reduction by its inhibitors. int. j. oncol., 24:821–7. [21] yoshimura, r., matsuyama, m., mitsuhashi, m., takemoto, y., tsuchida, k., kawahito, y., sano, h. and nakatani, t. 2004. relationship between lipoxygenase and human testicular cancer. int. j. mol. med., 13:389–93. [22] matsuyama, m., yoshimuira, r., tsuchida, k., takemoto, y., segawa, y., shinnka, t., kawahito, y., sano, h. and nakatani, t. 2004. lipoxygenase inhibitors prevent urological cancer cell growth. int. j. mol. med., 13:665–8. [23] ghosh, j. and myers, c.e. 1998. inhibition of arachidonate 5-lipoxygenase triggers massive apoptosis in human prostate cancer cells. proc. natl. acad. sci. u.s.a., 95:13182–7. [24] ghosh, j. 2003. inhibition of arachidonate 5-lipoxygenase triggers prostate cancer cell death through rapid activation of c-jun n-terminal kinase. biochem. biophys. res. commun., 307:342–9. [25] pommery, n., taverne, t., telliez, a., goossens, l., charlier, c., pommery, j., goossens, j.f., houssin, r., durant, f. and henichart, j.p. 2004. new cox-2/5-lox inhibitors: apoptosis-inducing agents potentially useful in prostate cancer chemotherapy. j. med. chem., 47:6195–206. [26] ghosh, j. 2004. rapid induction of apoptosis in prostate cancer cells by selenium: reversal by metabolites of arachidonate 5-lipoxygenase. biochem. biophys. res. commun., 315:624–35. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.1000 /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 1048576 /lockdistillerparams false 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drug target insights 2008:3 1–11 1 original research correspondence: dr. j.a. garrote. research unit. hospital clinico universitario. c/ ramon y cajal 3. 47011 valladolid. (spain). tel: +34983420000 (ext. 20422); email: jgarrote@hcuv.sacyl.es copyright in this article, its metadata, and any supplementary data is held by its author or authors. it is published under the creative commons attribution by licence. for further information go to: http://creativecommons.org/licenses/by/3.0/. cytokine, chemokine and immune activation pathway profi les in celiac disease: an immune system activity screening by expression macroarrays josé a. garrote1,2, emma gómez1, alberto j. león1, david bernardo1, carmen calvo3, luis fernández-salazar4, alfredo blanco-quirós1 and eduardo arranz1 1group of mucosal immunology. pediatrics and immunology areasinstituto de biologia y genética molecular (ibgm). university of valladolid. (spain); and 2research unit, 3pediatrics gastroenterology and 4adults digestive diseases services. hospital clinico universitario of valladolid. (spain). abstract: the aims of the study were to assess the usefulness of expression macroarrays to determine the pattern of expression of cytokines, chemokines and molecules related to immune system activation pathways, in non-stimulated intact intestinal tissue specimens from patients with active cd (acd) and on a gluten-free diet (gfd), to compare it with two groups of controls with either normal or altered mucosal architecture, and to establish putative targets for diagnostic markers or therapeutic intervention. we have experienced the lack of sensitivity to detect signal of genes with low level of expression. in spite of that, active cd seems to show a th1 cytokine pattern, but with signs of th2 activity. cytokines such as il-9, il-11, il-21 or mif might be involved in mucosal infl ammation in cd. in gfd, some memory cells and dc’s activity remains, and factors that maintain this remnant activation might be responsible of the fast mucosal response on gluten challenge. stat3 and stat5 pathways, and their regulatory molecules socs’s may result keys for understanding mucosal infl ammation in gut and putative targets for further research. keywords: th2, il-21, mif, cx3cr1, stat3, stat5, celiac disease introduction celiac disease (cd) is an immune-mediated enteropathy caused by the ingestion of gluten, a group of store proteins of certain cereals (wheat, rye, barley and probably oats) in genetically predisposed individuals (maki and collin, 1997). the typical celiac intestinal mucosa shows fl attened villi, crypt hyperplasia, and intraepithelial infi ltration of lymphocytes (iel). the current treatment is a life-long strict gluten-free diet (gfd), after which a complete remission of the symptoms and mucosal recovery are found. t cells have a central role in the immunopathogenesis of cd, and cytokines released during a t cellmediated hypersensitivity may trigger the development of the enteropathy (macdonald and spencer, 1988; sollid, 2000). specifi c lamina propria t helper cells recognize gluten peptides modifi ed by the enzyme tissue transglutaminase in the context of hla-dq2 or dq8 molecules (godkin and jewell, 1998; schuppan, 2000; sollid, 2002). it has been reported that the immune response to gluten may follow two complementary (and sometimes parallel) pathways, mediated by the adaptive and innate immunity (maiuri, ciacci et al. 2003). previous reports on cytokine expression in cd have studied biopsies from untreated patients (nilsen, jahnsen et al. 1998; monteleone, pender et al. 2001; forsberg, hernell et al. 2002), after ex vivo stimulation with gluten. gluten-specifi c t helper cell clones, hla-dq restricted, isolated from the intestine of cd patients show a th1 cytokine pattern following gluten challenge (lundin, scott et al. 1993; nilsen, lundin et al. 1995), and a similar profi le, characterized by high expression of ifnγ, but no il12, has been found by mrna expression in biopsy homogenates (nilsen, jahnsen et al. 1998; troncone, gianfrani et al. 1998; monteleone, pender et al. 2001), or isolated t cell populations (forsberg, hernell et al. 2002), from patients with active cd. gluten challenge has been reported to induce also the expression of il-2, il4, il5, il6, and tnfα (nilsen, jahnsen et al. 1998), as well as il-18, il-15 (maiuri, ciacci et al. 2000; monteleone, pender et al. 2001; salvati, macdonald et al. 2002), and tgfβ, but not http://creativecommons.org/licenses/by/3.0/ http://creativecommons.org/licenses/by/3.0/ 2 garrote et al drug target insights 2008:3 of il10 (nilsen, jahnsen et al. 1998; lionetti, pazzaglia et al. 1999; forsberg, hernell et al. 2002; hansson, ulfgren et al. 2002). these fi ndings may be secondary to the acute response triggered by gluten on tissue specimens or isolated t cell populations, but not refl ect the situation in vivo of a long-standing infl ammatory reaction occurring in cd patients at diagnosis. the use of intact non-stimulated intestinal tissue specimens may help to identify the cytokine profi le in the intestine with a well-established on-going chronic infl ammation. the aims of the study were to assess the usefulness of low density expression arrays (or macroarrays) to determine the pattern of expression of cytokines, chemokines and molecules and transcription factors related to immune system activity pathways, in non-stimulated intact intestinal tissue specimens from adults and children with active cd (acd) and on a gluten-free diet (gfd), and to compare it with two groups of controls with either normal or altered mucosal architecture, and to establish putative targets for diagnostic markers or therapeutic intervention. patients and methods patients intestinal biopsy specimens where collected from 8 cd patients, (mean age 20 years, range 3–52 years). from these, 4 cases were untreated (active celiac disease group acd) and 4 cases were on a gluten-free diet for at least 3 months (gfd group). all patients attended the adult and pediatric gastroenterology clinics, hospital clínico universitario of valladolid, as part of the routine diagnostic procedures for suspicion or follow up of cd. adult intestinal small bowel biopsies were obtained during upper gastrointestinal endoscopy using a fybergastroscope with forceps (olympus, tokyo, japan), and jejunal biopsies from children were obtained using a pediatric crosby capsule. patients with active cd showed altered biopsy histology, positive anti-transglutaminase antibodies and the hla-dq2 genotype, and the diagnosis was later confi rmed by the remission of symptoms and recovery of the histological and serological markers after treatment. mucosal abnormalities in biopsies were described following the modifi ed marsh classification (marsh, 1992; united european gastroenterology, 2001). cd patients on gfd presented normal mucosal histology and negative anti-transglutaminase antibodies. two other groups of patients were studied: a) a group of controls with normal biopsy histology, which includes 4 patients, (mean age 27.7 years, range 8–53), who underwent diagnostic investigations due to clinical suspicion of a gastrointestinal disorder which was later ruled out due to the fi nding of a normal biopsy histology, and no signs of intestinal infection, infl ammation or allergy (healthy control group, hc). b) 2 adult patients (mean age 35 years, range 14–56) with signs of non-specifi c intestinal infl ammation, altered intestinal biopsy histology, and negative both serological markers of cd and the hla-dq2 genotype, but presenting small bowel symptoms (diseased or pathologic control group, pc). informed consent was obtained from patients and/or their parents following the institutional protocols and recommendations, and the study protocol was approved by the ethics committee of the university hospital. sample preparation (rna extraction and cdna preparation) after collection, samples were immediately submerged in 1ml of rna-later® solution (ambion inc, tx, usa) and stored at −20 °c to preserve rna integrity until processing. total rna was purifi ed from intact biopsy specimens using the trizol® reagent (invitrogen, life technologies, usa). samples were placed in sterile tubes, submerged in 1ml of trizol, and homogenised using a diax 900 tissue homogeniser (heidolph, germany), followed by the steps detailed in the protocol provided by the manufacturer. following steps, transcription with annealing by random primers, linear polymerase reaction (lpr) and labelling by means of biotin-16-dudp, were carried out using the superscript® first-stand synthesis system for rt-pcr kit (invitrogen, life technologies, usa) and superarray ampolabelling (lpr) kit, according to the instructions of the manufacturers to produce biotin-labeled probes. gene expression testing gearray tm q series (superarray bioscence cop. usa) membranes were used to test human infl ammatory cytokines/chemokines and receptors (hs015.2, gearray® q series human infl ammatory cytokines and receptors gene array), and jak/ stat pathways and transcription factor molecules 3 macroarray immune screening of celiac disease drug target insights 2008:3 (hs-039, gearray® q series human jak/stat signaling pathway gene array). the membranes were hybridated with the biotin-labeled probes following the manufacturer’s protocol. previously, a label test was performed by successive dilution of the probes to assess the suitable working dilution. after hybridation step, the membranes were revealed with superarray gearray tm chemiluminiscent detection kit and autoradiographied. each kind of q series presented 114 clovershaped dots formed by 4 spots of 60 mer oligonucleotids corresponding to 96 specifi c genes, 5 housekeeping genes and blank dots. infl ammatory cytokines/chemokines and receptors membranes allow testing the following specifi c genes expression: blr1, ccr1, ccr2, ccr3, ccr4, ccr5, ccr6, ccr7, ccr8, ccr9, xcr1, cx3cr1, chemiokine (cxc) motif receptor (cxcr) 4, ifnγ, il-10, il-10rα, il-10rβ, il-11, il-11rα, il-12p35, il-12p40, il-12rβ1, il-12rβ2, il-13, il-13rα1, il-13rα2, il-15, il-15rα, il-16, il-17, il-17r, il-18, il-18r1, il-1α, il1β, il1r1, il-1r2, il-2, il-20, il-21, c19orf10, il2rα, il-2rβ, il-2rγ, il-4, il-5, il-5rα, il-6, il-6r, il-6st, il-9, il-9r, leptin (lep), linfotoxin (lt)α, ltβ, ltβr, mif, ccl1, ccl11, ccl13, il-3, ccl15, ccl16, ccl17, ccl18, ccl19, ccl2, ccl20, ccl21, ccl22, ccl23, ccl24, ccl25, ccl3, ccl4, ccl5, ccl7, ccl8, cxcl10, cxcl11, cxcl13, cxcl5, cxcl6, xcl1, xcl2, cx3cl1, small inducible cytokine subfamily e (scye) 1, cxcl12, stromal cell-derived factor (sdf2), tgfα, tgfβ1, tgfβ2, tgfβ3, tnf, tnfrsf1α y tnfrsf1β. and jak/stat pathways and transcription factors membranes, the following: alpha 2 macroglobulin (a2m), bcl2-related gene (bcl-x), cyclin d1, cyclin-dependent kinase inhibitor 1 a (cip1 o cdkn1a) 1a, ccaat/enhacer binding protein beta (cebpβ), creb binding protein (crebbp), v-crk sarcoma virus ct10 oncogene holomolog (avian)-like (crkl), c-reactive protein pentraxin-related (crp), csf1r, csf2rb, casein beta (csn2), cxcl9, egfr, epor, fc fragment of ige high affi nity i receptor for alpha subunit (fcer1α), fc fragment of ige low affi nity ii receptor (fcer2), fc fragment of igg high affi nity i receptor for alpha subunit (fcgr1α), interferon alpha inducible protein clone ifi-6-16 (g1p3), gata3, guanylate binding protein 1 (gbp1), interferon induced protein ifi-15k (g1p2), high mobility group at-hook 1 (hmga1), ifnαr1, ifnαr2, ifnγ, ifnγr1, ifnγr2, immunoglobulin heavy constant delta (ighd), il-10rα, il-10rβ, il-10rα, il-22rα1, il-2rα, il-2rγ, il-4, il-4r, gp130 o il-6st, indoleamine-pyrrole 2,3 dioxygenase (indo), irf1, p48/irf9, jak1, jak2, jak3, o v-jun avian sarcoma virus 17 oncogene homolog (jun), junb, smad1, smad2, smad3, smad4, smad5, smad6, smad7, smad9, v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog (maf), cell division cycle 46 (cdc46 o mcm5), mhc class ii transactivator (mhciita), pias2, mmp3, myeloproliferative leucemia virus oncogene (mpl), v-myc avian myelocytomatosis viral oncogene homolog (myc), nuclear receptor coactivator 1 (ncoa1), nf-κb1, nmyc interactor (nmi), nitric oxide synthase 2a (nos2a), oligoadenylate synthetase 1 (oas1), osm, pias1, pias3, pias4, oncogen pim1, protein tyrosin phosphatase non receptor type 1 (ptptn1), ptpns1, cd45 o ptprc, adaptor protein (sh2b), socs1, socs2, socs3, socs4, socs5, socs6, specificity protein 1 (sp1), hematopoietic transcription factor pu-1, oncogene c-src, signal transducing adaptor molecule (stam), stat1, stat2, stat3, stat4, stat5a, stat5b, stat6, stip1 homologous u box containing protein 1 (stub1), fas antigen (fas), tyk2, upstream transcription factor 1 (usf1) and transcription factor yy1. as housekeeping genes: puc18 (3 dots), glyceraldehyde 3 phosphate dehydrogenase (gapdh, 2 dots), cyclophilin a (ppia, 4 dots), ribosomal protein l 13a (rpl13a, 2 dots) and β-actin (2 dots). densitometric measurements and statistic analysis films were scanned and processed with gearray expression analysis suite version 1.0 of superarray bioscience corporation, usa (http://geasuite. superarray.com/) for densitometric analysis. the average density of each spot was measured with “clover” mode on. background was corrected with the “local background” mode. the results were normalized with the housekeeping genes expression. the application considers “absent” gene (non-expressed) when the spot density is lower than the 75 percentile of the average local 4 garrote et al drug target insights 2008:3 background of the spots non-classified as “bleeding”. the remainders are considered “present” (expressed). bleeding spots are those ones with an average density higher than the average value of all the spots and that it differs from its local background less than 30%. all genes classifi ed as “absent” or “bleeding” were visually assessed and 0 values was assigned to “absent” ones and the value of the normalized measure of density with the general background correction to “bleeding” ones. differential gene expression between two groups was carried out by calculating the ratio of the median values of expression for each gene. genes with a ratio equal or higher than 2 were considered over-expressed, and equal or lower than 0.5 sub-expressed. the statistical signifi cance of these differences was checked by non-parametric tests: krustall-wallis and mann-whitney u tests. this simple statistical treatment was previously use for a similar approach with microarrays in ibd (costello, mah et al. 2005). results infl ammatory cytokines and chemokines. (tables 1 and 2) il-11rα (r:e/p = 0.005), il-1r2 (r:12.026/ p = 0.019) and il-2rβ (r:e/p = 0.025) genes r e s u l t e d o v e re x p r e s s e d a n d n o n e u n d e rexpressed in active celiac disease (acd) as compared with healthy controls (hc). however, xcr1, cx3cr1, il-11, il-11rα and mif are over-expressed in gfd cd small intestine vs hc (r:3.278/p = 0.026, r:e/p = 0.01, r:2.474/p = 0.007, r:e/p = 0.001 and r:2.402/p = 0.01 respectively) and the gene of the chemokine cxcl11 underexpressed (r:0/p = 0.01). il-1r2 and il9r resulted over-expressed in acd when compared with gfd (r:4.974/p = 0.029 and r:e/p = 0.048). when compared with diseased (pathologic) controls (pc), ccr9 (r:e/p = 0.027), ifnγ (r:e/p = 0.034), il-21 (r:13,31/p = 0.005) and il-5 (r:21,319/p = 0.005) were over-expressed in acd group. however, only cxcl5 (r:e/p = 0.037) resulted over-expressed in pc as compared with hc, and ifnγ (r:0/p = 0.006), il-21 (r:0.107/ p = 0.006), il-5 (r:0.077/p = 0.006) and chemokine ccl17 (r:0/p = 0.017) were under-expressed. jak/stat pathway. (tables 3 and 4) egfr (r:e, p = 0.045), gata3(r:e/p = 0.028), hmga (r:e/p = 0.045), jak3 (r:e/p = 0.045), junb (r:e/p = 0.014), smad3 (r:e/p = 0.045), smad5 (r:e/p = 0.045), maf (r:411.205/ p = 0.049), pim1 (r:17.769/p = 0.014), ptprc (r:e, p = 0.014), socs1 (r:2.642/p = 0.025), src (r:e/p = 0.05), stam (r:e/p = 0.045), tyk2 (r:e/p = 0.045) and yyf (r:e/p = 0.045) were over-expressed in acd intestinal mucosa as compared with hc group, while ccnd1 (r:0.174/ p = 0.045) y stat3 (r:0/p = 0.014) resulted underexpressed. in gfd group, csf2rb (r:4.154/p = 0.01),csn2 (r:e/p = 0.008), fcre2 (r:23.827/p = 0.01), g1p3 (r:29.956/p = 0.01), smad2 (r:3.834/p = 0.01), pias2 (r:51.937/p = 0.032) and socs2 (r:5.405/ p = 0.01) were over-expressed and ptpns1 (r:0/ p = 0.008), stat3 (r:0/p = 0.023) and stat5a (r:0/p = 0.023) under-expressed when compared with hc group. when compared acd and dsg groups, we found gata3 (r:e/p = 0.038), il6st (r:e/ p = 0.038), junb (r:e/p = 0.038), mcm5 (r:e/ p = 0.038), nf-κb1 (r:e/p = 0.013), pias4 (r:e/ p = 0.038), pim1 (r:e/p = 0.013), ptpns1 (r:e/ p = 0.023), ptprc (r:e/p = 0.038), src (r:e/ p = 0.038) and fas (r:e/p = 0.038) over-expressed in acd group and ccnd1 (r:0.185/p = 0.046), epor (r:0.355/p = 0.046), fcer2 (r:0.044 p = 0.003) and il-10rα (r:0/p = 0.038) underexpressed. smad2, maf and ptprc are also overexpressed in acd group vs pc group (r:e/ p = 0.038, r:e/p = 0.003 y r:e/p = 0.038) respectively, and ccnd1 (r:0.156/p = 0.013), stat2 (r:0.346/p = 0.052) under-expressed. and pc group had gata3 (r:0/p = 0.006) and osm (r:2.162/p = 0.011) over-expressed, and stat3 and stat5a (r:0/p = 0.023 and r:0/p = 0.023, respectively) under-expressed when compared with hc group. discussion expression arrays techniques have resulted important tools in genomic studies, such as in positional genetics as in the study of tissues physiology and in the changes induced by disease in them. however, these techniques are susceptible of methodologic and interpretative variability, resulting in an obstacle for fi ndings reproducibility 5 macroarray immune screening of celiac disease drug target insights 2008:3 table 1. medians fold ratios (marn) and p values of comparations amongst groups of expression of cytokines and chemokines genes. gen p(total)*= acd/hc p*= gfd/ hc p*= pc/hc p*= acd/ gfd p*= acd/pc p*= ccr9 0.069 4.660 0.109 3.369 0.192 0 0.113 1.382 e 0.027 xcr1 0.106 0.297 0.569 3.278 0.026 0.729 0.090 0.271 0.408 cx3cr1 0.039 ne e 0.01 ne 0 0.339 ne cxcr4 0.194 ne e 0.153 ne 0 0.213 ne ifng 0.086 0.898 1.405 0 0.006 0.639 e 0.034 il10ra 0.382 0.705 1.611 0 0.256 0.437 0.373 e 0.174 il11 0.252 1.697 2.474 0.007 2.136 0.865 0.685 0.794 il11ra 0.02 e 0.005 e 0.01 ne 4.191 0.322 e 0.162 il12b 0.133 ne e 0.071 ne 0 0.908 ne il16 0.231 ne e 0.198 ne 0 0.817 ne il17r 0.121 1.021 1.413 0.406 0.174 0.722 2.509 0.103 il1r1 0.34 0 0.403 1.033 0 0.053 0 0.087 ne il1r2 0.101 12.026 0.019 2.417 1 10.193 0.468 4.974 0.029 1.179 il21 0.016 1.424 1.403 0.107 0.006 1.015 13.310 0.005 il2rb 0.079 e 0.025 ne ne e 0.811 e 0.099 il4 0.2 e 0.097 e 0.198 ne 10.576 0.436 e 0.131 il5 0.041 1.659 1.293 0.077 0.006 1.283 21.319 0.005 il5ra 0.22 1.973 1.389 0 0.124 1.420 e 0.083 il9r 0.163 e 0.153 ne ne e 0.048 e 0.481 lep 0.384 ne e 0.391 ne 0 1 ne lta 0.836 0.897 2.347 3.425 0.61 0.382 0.514 0.262 1 ltb 0.457 1.415 1.021 0.315 0.126 1.385 4.486 0.247 ltbr 0.269 ne e 0.153 ne 0 0.081 ne mif 0.178 0.714 2.402 0.01 1.653 0.297 0.126 0.432 1 ccl1 0.2 2.376 0.091 1.558 0.914 1.524 2.598 0.12 ccl15 0.242 1.132 1.144 0.152 0.126 0.989 7.418 0.065 ccl16 0.665 ne e 0.382 e 0.449 0 0.271 0 0.51 ccl17 0.123 0.629 0.571 0 0.017 1.101 e 0.24 ccl18 0.698 0 0.922 0.366 0.893 0 0.256 0 0.728 ne ccl21 1.247 1.301 1.639 0.958 0.760 ccl22 0.486 0.357 0.619 1.521 0.541 0.234 0.322 0.660 ccl23 1.142 1.129 1.059 1.011 1.078 ccl24 0.736 0.273 0.39 1.419 2.400 0.864 0.192 0.271 0.114 0.651 ccl25 0.275 ne e 0.215 ne 0 0.643 ne ccl4 0.088 1.388 0.566 1.332 2.450 0.051 1.041 cxcl11 0.071 0 0.306 0 0.01 0.792 ne 0 0.546 cxcl13 0.271 1.873 2.476 0.396 2.301 0.396 0.756 0.814 cxcl5 0.174 ne ne e 0.037 ne 0 0.168 cxcl6 0.545 3.345 0.18 0.315 0.231 6.646 0.231 10.589 0.444 0.503 xcl1 0.82 e 0.363 ne ne e 0.633 e 0.695 scye1 0.258 0 0.063 0.218 0.235 0.343 0.3 0 0.473 0 0.539 cxcl12 0.544 0 0.204 6.617 0.687 2.597 0.856 0 0.213 0 0.479 sdf2 0.325 0.413 1 1.449 0 570 0.285 0.155 e 0.365 tgfa ne ne e 0.686 ne 0 0.288 tnf 0.228 0 0.41 0.777 2.873 0.73 0 0.356 0 0.156 tnfrsf1b 0.549 ne ne e 0.705 ne 0 0.156 p*(total): krustal-wallis test (p � 0.05); p: mann-whitney non parametric test (p � 0.05); e: overexpression with divisor = 0; ne: non expressed. red case, over-expression. white case, 2�ratio�0.5. green case: under-expression. acd: active celiac disease; gfd: celiac patients on gluten free diet; pc: pathological contols; hc: healthy contols. 6 garrote et al drug target insights 2008:3 and comparison amongst different laboratories (li, gu et al. 2002). low density arrays or macroarrays are more user-friendly variants than microarrays, designed specifi cally for one system or pathway, to explore some tens or a few hundreds of genes. however we have not to forget that both versions are screening tools, and that the fi ndings should be considered as a fi rst approach, and susceptible of been validated by other techniques. this technique has been used by numerous research groups and its limitations are well known. we have experienced the lack of sensitivity to detect signal of genes with low level of expression. perhaps, increasing the number of cycles of the sample amplifi cation, it would be possible to achieve the number of copies and to enhance the performance of the hybridation. another pitfall for sensitivity is the use of intact whole tissue (intestinal mucosa in our case). in this case the coexistence of multiple cell lineages would tend to mask some changes of expression in one of the lineages. this technique shows better performances with homogeneous samples (of one cell lineage)(torres-munoz, stockton et al. 2001). we found an inter-assay variability for the same sample similar to the intra-assay one (determined for housekeeping genes -data not shown-) in an acceptable range. however, we found a wide biological variability for each one of the analytes amongst the several samples of the same group, what claims for a higher casuistic for more robust results. we should not forget that arrays techniques were designed with the aim of detect wide variations in the expression of multiple genes, to determine pathways or patterns of gene activation/repression with gross differences (being used inicially in oncology). this means, wide changes in multiple related genes behaviour, in a parallel manner, and not to observe individual differences in one gene separately. we have tried to extrapolate from this use to a group of diseases with subtler changes, and we have found that the sensitivity of the technique result a limiting factor. complex tissues tend to buffer the changes in one of its several components versus to very homogeneous tissues, as tumors are. on the other hand, the quantity of the difference of gene expression used to be lower in diseases non so extrem as neoplastic tissues. as a consequence of all the previously exposed, we have not pretended to draw any conclusion about isolated molecules, but to get an overall view of the immunologic activity in each group of patients, regarding to the cytokine/chemokine pattern and the activation pathways activated. the quantitative expression analysis of cytokine and chemokines in intestinal mucosa mucosa shows that, although th1 pattern factors are expressed, th2 pattern related molecules have also a role in active cd intestinal mucosa. cytokine receptor il-11rα, il-1r2 and il-2r gene expression is increased in active ec as compared with healthy controls and il9r as compared with cd table 2. resume of molecules with differential expression between groups, by functional families in cytokines/ chemokines array. r: fold ratio. p: statistical signifi cance by mann-whitney test. gene acd vs hc gfd vs hc acd vs gfd pc vs hc acd vs pc ifnγ r:0/p = 0.006 r:e/p=0.034 il-5 r:0.077/p = 0.006 r:21.319/p = 0.005 il-11 r:2.474/p = 0.007 il-21 r:0.107/p = 0.006 r:13.31/p = 0.005 mif r:2.402/p = 0.01 il-1r2 r:12.026/p = 0.019 r:4.974/p = 0.029 il-2rβ r:e/p = 0.025 il-9r r:e/p = 0.048 il-11rα r:e/p = 0.005 r:e/p = 0.001 ccl17 r:0/p = 0.017 cxcl11 r:0/p = 0.01 cxcl5 r:e/p = 0.037 ccr9 r:e/p = 0.027 xcr1 r:3.278/p = 0.026 cx3cr1 r:e/p = 0.01 7 macroarray immune screening of celiac disease drug target insights 2008:3 table 3. medians fold ratios (marn) and p values of comparations amongst groups of expression of jak/stat pathway genes. gen p(total)*= acd/hc p*= gfd/hc p*= pc/hc p*= acd/gfd p*= acd/pc p*= ccnd1 0.028 0.174 0.045 0.938z 1.114 0.185 0.046 0.156 0.013 cebpb 0.332 0 0.286 0 0.116 3427.909 0.82 ne 0 0.339 crebbp 0.17 ne ne e 0.068 ne 0 0.577 csf2rb 1.922 4.154 0.01 2.533 0.392 0.462 0.051 0.758 csn2 0.145 e 0.217 e 0.008 e 337 0.256 0.135 0.323 0.796 cxcl9 0.161 e 0.11 ne e 0.19 e 0.094 0.656 egfr e 0.045 ne e 0.068 e 0.094 0.222 0.796 epor 0.546 1.537 0.998 0.355 0.046 0.547 fcer2 0.004 1.071 23.827 0.01 0 0.055 0.044 0.003 e 0.094 g1p3 0.219 22.143 0.252 29.956 0.01 24.927 0.087 0.739 0.888 gata3 0.005 e 0.028 ne e 0.006 e 0.038 0.291 0.143 gbp1 0.161 e 0.11 ne e 0.19 e 0.094 0.667 g1p2 0.239 e 0.217 ne e 0.337 e 0.094 0.412 0.796 hmga1 0.079 e 0.045 ne e 0.068 e 0.094 0.048 ifngr2 0.489 e 0.303 ne e 0.19 e 0.094 0.169 0.796 ighd 0.326 ne ne e 0.19 ne 0 0.577 il10ra 0.189 ne e 0.078 e 1 0 0.038 0 1 il4r 0.369 169.793 1 271.786 0.392 162.841 0.66 0.624 0.051 1.042 il6st 0.219 e 0.433 ne e 0.337 e 0.038 0.775 jak3 0.083 e 0.045 ne e 0.063 e 0.094 0.080 0.796 junb 0.014 e 0.014 ne e 0.068 e 0.038 3.027 0.135 smad2 0.032 1.918 3.864 0.01 0 0.055 0.496 0.392 e 0.038 smad3 0.036 e 0.045 ne ne e 0.094 e 0.094 smad5 0.036 e 0.045 ne ne e 0.094 e 0.094 smad9 0.234 e 0.695 ne e 1 e 0.038 0.793 maf 0.008 411.205 0.049 221.186 0.392 0 0.055 1.859 e 0.003 mcm5 0.164 27.868 1 0 0.055 14.565 0.66 e 0.038 1.913 mhc2ta 0.12 0 0.764 0 0.055 0 0.055 ne ne pias2 0.21 12.883 0.845 51.937 0.032 11.865 0.66 0.248 0.319 1.085 nfkb1 0.118 21.944 0.572 0 0.055 19.626 0.66 e 0.013 1.118 nmi 0.445 e 1 ne e 1 e 0.094 0.258 0.796 nos2a 0.084 0 0.217 0 0.055 0 0.055 ne ne oas1 0.171 e 0.681 ne ne e 0.094 e 0.094 osm 0.071 1.037 1.587 2.162 0.011 0.653 0.479 0.052 pias4 0.041 4.337 1 0 0.055 7.143 0.392 e 0.038 0.607 0.051 pim1 0.014 17.769 0.014 0 0.055 6.144 0.66 e 0.013 2.891 0.222 ptpns1 0.044 3.327 1 0 0.008 0.681 e 0.023 4.882 0.11 ptprc 0.006 e 0.014 ne ne e 0.038 e 0.038 socs1 0.121 2.642 0.025 1.701 2.514 0.087 1.552 1.050 socs2 0.041 3.996 0.09 5.405 0.01 2.824 0.087 0.739 1.414 socs4 0.114 e 0.537 e 0.187 ne 0.269 0.262 e 0.094 sp1 0.709 2.669 0.349 2.100 0.394 2.115 0.394 1.271 1.262 src 0.07 e 0.05 ne e 0.337 e 0.038 0.338 0.618 stam 0.036 e 0.045 ne ne e 0.094 e 0.094 stat2 0.790 1.228 2.279 0.088 0.643 0.346 0.052 stat3 0.008 0 0.014 0 0.023 0 0.023 ne ne stat4 0.077 1.176 0 0.055 0 0.055 e 0.094 e 0.094 stat5a 0.018 0.171 0.101 0 0.023 0 0.023 e 0.094 e 0.094 stat5b 0.228 0 0.123 0 0.116 3.612 0.82 ne 0 0.339 stub1 ne ne e 0.068 ne 0 0.658 fas 0.166 e 0.695 ne e 0.379 e 0.038 1.399 tyk2 0.036 e 0.045 ne ne e 0.094 e 0.094 yy1 0.036 e 0.045 ne ne e 0.094 e 0.094 p*(total): krustal-wallis test (p � 0.05); p: mann-whitney non parametric test (p � 0.05); e: overexpression with divisor = 0; ne: non expressed. red case, over-expression. white case, 2�ratio�0.5. green case, under-expression. acd: active celiac disease; gfd: celiac patients on gluten free diet; pc: pathological contols; hc: healthy contols. 8 garrote et al drug target insights 2008:3 table 4. resume of molecules with differential expression between groups, by functional families of jak/stat pathway array. r: fold ratio. p: statistical signifi cance by mann-whitney test. gene acd vs hc gfd vs hc acd vs gfd pc vs hc acd vs pc stat2 r:0.346/p = 0.052 stat3 r:0/p = 0.014 r:0/p = 0.023 r:0/p = 0.023 stat5a r:0/p = 0.023 r:0/p = 0.023 gata3 r:e/p = 0.028 r:e/p = 0.038 r:0/p = 0.006 junb r:e/p = 0.014 r:e/p = 0.038 maf r:411.205/p = 0.049 r:e/p = 0.003 nfkb1 r:e/p = 0.013 smad2 r:3.834/p = 0.01 r:e/p = 0.038 smad3 r:e/p = 0.045 smad5 r:e/p = 0.045 pias2 r:51.937/p = 0.032 pias4 r:e/p=0.038 socs1 r:2.642/p = 0.025 socs2 r:5.405/p = 0.01 jak3 r:e/p = 0.045 tyk2 r:e/p = 0.045 pim1 r:17.769/p = 0.014 r:e/p = 0.013 src r:e/p = 0.05 r:e/p = 0.038 fas r:e/p = 0.038 egfr r:e. p = 0.045 csf2rb r:4.154/p = 0.01 fcre2 r:23.827/p = 0.01 r:0.044/p = 0.003 epor r:0.355/p = 0.046 il-10ra r:0/p = 0.038 il-6st r:e/p = 0.038 cd45/ptprc r:e. p = 0.014 r:e/p = 0.038 r:e/p = 0.038 ptpns1 r:0/p = 0.008 r:e/p = 0.023 g1p3 r:29.956/p = 0.01 osm r:2.162/p = 0.011 stam r:e/p = 0.045 yyf r:e/p = 0.045 hmga r:e/p = 0.045 ccdn1 r:0.174/p = 0.045 r:0.185/p = 0.046 r:0.156/p = 0.013 csn2 r:e/p = 0.008 mcm5 r:e/p = 0.038 9 macroarray immune screening of celiac disease drug target insights 2008:3 patients in remission. both il-11rα and il-9r are receptors related to th2 response regulation. il-11 is regulated by the antiinfl ammatory cytokine il10. il-9 is growth factor whose receptor is expressed in eosinophils, very abundant in cd intestinal mucosa, and it may contribute synergically with il-13 to mucosal infl ammation. this double faced immune pattern may be also found in ulcerative colitis, whose immune pattern is simplistically described as th2, but it shows increased presence of typically th1 cytokines in injured colonic mucosa (gordon, di sabatino et al. 2005). cytokines ifnγ, il-21 and il-5 genes seem specifi cally expressed in active cd mucosa, but not in diseased controls. il-5 also is a cytokine related to the regulation of eosinophil activation, as il-9, and it is a paradigmatic component of th2 pattern (broide, hoffman et al. 1999). the presence of il-5 in cd is controversial. it has not been found in cd intestinal mucosa by some authors (nilsen, johansen et al. 1998), but described by others (desreumaux, delaporte et al. 1998). il-21 is a cytokine that modules as th1 immune response as th2 type, and it has been related to innate immune response and nk cells activity (mehta, wurster et al. 2005). il-21 gene maps in a genome region that recently has been described as linked to cd susceptibility (van heel, franke et al. 2007), and its expression has been found increased in cd. ifnγ has been yet previous described as the main cytokine responsible of mucosal damage in cd (wapenaar, van belzen et al. 2004; leon, sanchez et al. 2005; leon, garrote et al. 2006), but not specifi c of this disease. there are also other target genes involved in the in active phase of this enteropathy, as il-1r2 or il-2rβ. il-1r2 is a receptor induced by il-4, and it might have a role in negative regulation of il-1 expression (colotta, re et al. 1993). il-2rβ is the signal transducer of il-2 and il-15, and it is an important receptor as in innate as in adaptive response, controlling t lymphs expansion and autoimmunity (suzuki, kundig et al. 1995). surprisingly, in the group of cd patients in gfd, we found overexpression in a group of chemokine receptor genes related to dendritic cell (dc) functionality: cx3cr1 y xcr1. the fi rst one is expressed in monocytes, nk and memory cells (sozzani, allavena et al. 1999) and it is involved in the emission of transepithelial dendrites by cds, with the possible function of sampling antigens in gut lumen, with the consequent impact in infl ammation or tolerance triggering (niess, brand et al. 2005; rescigno and chieppa, 2007). xcr1 is the receptor for lymphotactin, highly expressed by myeloid cells and chemoattractant of nk cells and t lymphocytes, amongst other non well known actions (luttichau, johnsen et al. 2007). these expressions might indicate an increased basal activity of dc’s in cd, even in remission. there are other proinfl ammatory factors overexpressed in gfd cd patients as the macrophage migration inhibitory factor (mif). this molecule is involved in infl ammatory and autoimmune processes. a polymorphism in the gene promoter of mif has been related to cd genetic susceptibility (nunez, rueda et al. 2007). in contrast with our results, o’keeffe et al. found overexpression of mif in intestinal epithelial cells of active cd patients (o’keeffe, lynch et al. 2001). cytokines, chemokines and growing factors regulate large aspects of hematopoiesis and immune response through interaction with their specific receptors. these ones trigger their responses through signalling pathways activity. in our results, we fi nd overexpressed molecules of pathways related to th2 pattern in active cd intestinal mucosa. there is an increase in the expression of gata3 and junb genes in the group of active cd patients as compared with healthy control group or gfd group. gata3 mediate in the th1 pattern inhibition and th2 induction. junb and maf (also overexpressed in acd group) are also related with nf-kb activity. maf seems specifi c of cd in activity, with and increased expression above healthy or diseased controls. it is an activating factor of cell differentiation (blank and andrews, 1997), and also promoter of th2 immune response, through il4 gene upregulation (valanciute, le gouvello et al. 2004). maf expression is controlled by nf-kb (nenci, becker et al. 2007). junb is an oncogene, member of ap-1 family of transcription factors. when t lymphs are stimulated the degradation process of junb is increased, controlling the cytokine production of effector t cells (gao, labuda et al. 2004). junb is also capable of inducing proinfl ammatoy response through nf-kb activation (mathas, hinz et al. 2002). gata3 and stat6 compose a tandem that induces the polarization of th0 lymphocytes towards th2. in previous studies no differences have been found between cd patients and controls in gata3 expression (monteleone, monteleone et al. 2004). 10 garrote et al drug target insights 2008:3 the oncogene scr codifi es for a kinase that has been related with several signalling pathways of some importance in cell growth, migration or cell survival. it is activated by several kinds of receptors: integrins, cytokines or growing factors and it is a factor in charge of regulating the cell communication for cell growth (azarnia, reddy et al. 1988), in relation with trance, a family member of tnf (wong, besser et al. 1999). experiments of in vitro inhibition of scr in intestinal epithelial cell lines have resulted in a parallel decrease of stat3, an important mediator of the antiapoptotic response. so, scr expression prevents cell apoptosis, increasing the mucosal infl ammation (bhattacharya, ray et al. 2006). we fi nd increased expression of scr gene in the group of active cd compared with diseased controls. in cd patients in remission (gfd), we fi nd a decrease in stat3 and stat5 expression, with an increase in socs2, smad2 and pias2 as compared to healthy controls. stat3 is related to signal transmission of cytokine receptors sharing gp130 (il-6, il-11, il-12 or il-23 receptors), while stat5 is related to some members of γc receptor family (il-2, il-9 or il-21 receptors) or to the single chain receptor family (epo, gh or prolactine receptors). stat3 pathway activity and its fi nal actions on gene expression is modulated by socs3 and il-10 (kinjyo, inoue et al. 2006; qasimi, ming-lum et al. 2006). stat3 pathway activation had been previously described in infl ammatory bowel disease and in ec (mazzarella, macdonald et al. 2003; musso, dentelli et al. 2005) and it may mediate in il-17 production. socs2 increased expression might explain the decrease of stat5 expression, as socs2 downregulates gene products that activate stat5 (il-6, il-9, epo or gh), and it may result the key for intestinal inflammation controlling in these patients. pias2 product is a nuclear level inhibitor of stat4 (chen, daines et al. 2004), and downregulator of proinfl ammatory genes expression. smad2 is a positive transcription factor in tgfβ signalling pathway (becker, fantini et al. 2006), the main regulatory cytokine in intestinal mucosa. diseased controls present an expression pattern of the molecules of the activation pathways between the patients with active cd and patients in gfd, with an increase of gata3 expression, but a low expression of stat3 or stat5. in conclusion, active cd seems to show a th1 cytokine pattern, but with signs of th2 activity. cytokines such as il-9, il-11, il-21 or mif might be involved in mucosal infl ammation in cd. in spite of the mucosal recovery following gfd, some memory cells and dc’s activity remains, and factors that maintain this remnant activation might be responsible of the fast mucosal response on gluten challenge, and they should be inquired. stat3 and stat5 pathways, and their regulatory molecules socs's may result keys for understanding mucosal infl ammation in gut and putative targets for research. competeting interests no exist funding this work has been partially funded by the spanish ministry of education (fpu, ap2002-2696), the spanish ministry of health (fis, pi020895; 02/3068) and the junta de castilla y leon (san1052-va02/05 va057/04). references azarnia, r., reddy, s. et al. 1988. the cellular src gene product regulates junctional cell-to-cell communication. science, 239(4838):398– 401. becker, c., fantini, m.c. et al. 2006. tgf-beta as a t cell regulator in colitis and colon cancer. cytokine growth factor rev., 17(1–2):97–106. bhattacharya, s., ray, r.m. et al. 2006. integrin beta3-mediated src activation regulates apoptosis in iec-6 cells via akt and stat3. biochem. j., 397(3):437–47. blank, v. and andrews n.c. 1997. the maf transcription factors: regulators of differentiation. trends biochem. sci., 22(11):437–41. broide, d.h., hoffman, h. et al. 1999. genes that regulate eosinophilic infl ammation. am. j. hum. genet., 65(2):302–7. colotta, f., re, f. et al. 1993. interleukin-1 type ii receptor: a decoy target for il-1 that is regulated by il-4. science, 261(5120):472–5. costello, c.m., mah, n. et al. 2005. dissection of the infl ammatory bowel disease transcriptome using genome-wide cdna microarrays. plos med., 2(8):e199. chen, w., daines, m.o. et al. 2004. turning off signal transducer and activator of transcription (stat): the negative regulation of stat signaling. j. allergy. clin. immunol., 114(3):476–89; quiz 490. desreumaux, p., delaporte, e. et al. 1998. similar il-5, il-3, and gm-csf syntheses by eosinophils in the jejunal mucosa of patients with celiac disease and dermatitis herpetiformis. clin. immunol. immunopathol., 88(1):14–21. forsberg, g., hernell, o. et al. 2002. paradoxical coexpression of proinfl ammatory and down-regulatory cytokines in intestinal t cells in childhood celiac disease. gastroenterology, 123(3):667–78. gao, m., labuda, t. et al. 2004. jun turnover is controlled through jnkdependent phosphorylation of the e3 ligase itch. science, 306(5694):271–5. godkin, a. and jewell, d. 1998. the pathogenesis of celiac disease. gastroenterology 115(1):206–10. 11 macroarray immune screening of celiac disease drug target insights 2008:3 gordon, j.n., di sabatino, a. et al. 2005. the pathophysiologic rationale for biological therapies in infl ammatory bowel disease. curr. opin. gastroenterol., 21(4):431–7. hansson, t., ulfgren, a.k. et al. 2002. transforming growth factor-beta (tgfbeta) and tissue transglutaminase expression in the small intestine in children with coeliac disease. scand. j. immunol., 56(5):530–7. kinjyo, i., inoue, h. et al. 2006. loss of socs3 in t helper cells resulted in reduced immune responses and hyperproduction of interleukin 10 and transforming growth factor-beta 1. j. exp. med., 203(4):1021–31. leon, a.j., garrote, j.a. et al. 2006. interleukin 18 maintains a long-standing infl ammation in coeliac disease patients. clin. exp. immunol., 146(3):479–85. leon, f., sanchez, l. et al. 2005. cytokine production by intestinal intraepithelial lymphocyte subsets in celiac disease. dig. dis. sci., 50(3):593–600. li, x., gu, w. et al. 2002. dna microarrays: their use and misuse. microcirculation, 9(1):13–22. lionetti, p., pazzaglia, a. et al. 1999. differing patterns of transforming growth factor-beta expression in normal intestinal mucosa and in active celiac disease. j. pediatr. gastroenterol. nutr., 29(3):308–13. lundin, k.e., scott, h. et al. 1993. gliadin-specific, hla-dq(alpha 1*0501,beta 1*0201) restricted t cells isolated from the small intestinal mucosa of celiac disease patients. j. exp. med., 178(1):187–96. luttichau, h.r., johnsen, a.h. et al. 2007. kaposi sarcoma-associated herpes virus targets the lymphotactin receptor with both a broad spectrum antagonist vccl2 and a highly selective and potent agonist vccl3. j. biol. chem., 282(24):17794–805. macdonald, t.t. and spencer j. 1988. evidence that activated mucosal t cells play a role in the pathogenesis of enteropathy in human small intestine. j. exp. med., 167(4):1341–9. maiuri, l., ciacci, c. et al. 2000. interleukin 15 mediates epithelial changes in celiac disease. gastroenterology, 119(4):996–1006. maiuri, l., ciacci, c. et al. 2003. association between innate response to gliadin and activation of pathogenic t cells in coeliac disease. lancet, 362(9377):30–7. maki, m. and collin p. 1997. coeliac disease. lancet, 349(9067):1755–9. marsh, m.n. 1992. gluten, major histocompatibility complex, and the small intestine. a molecular and immunobiologic approach to the spectrum of gluten sensitivity (‘celiac sprue’). gastroenterology, 102(1):330–54. mathas, s., hinz, m. et al. 2002. aberrantly expressed c-jun and junb are a hallmark of hodgkin lymphoma cells, stimulate proliferation and synergize with nf-kappa b. embo. j., 21(15):4104–13. mazzarella, g., macdonald, t.t. et al. 2003. constitutive activation of the signal transducer and activator of transcription pathway in celiac disease lesions. am. j. pathol., 162(6):1845–55. mehta, d.s., wurster, a.l. et al. 2005. nfatc2 and t-bet contribute to t-helper-cell-subset-specifi c regulation of il-21 expression. proc. natl. acad. sci. u.s.a., 102(6):2016–21. monteleone, g., pender, s.l. et al. 2001. role of interferon alpha in promoting t helper cell type 1 responses in the small intestine in coeliac disease. gut., 48(3):425–9. monteleone, i., monteleone, g. et al. 2004. regulation of the t helper cell type 1 transcription factor t-bet in coeliac disease mucosa. gut., 53(8):1090–5. musso, a., dentelli, p. et al. 2005. signal transducers and activators of transcription 3 signaling pathway: an essential mediator of infl ammatory bowel disease and other forms of intestinal infl ammation. infl amm. bowel. dis., 11(2):91–8. nenci, a., becker, c. et al. 2007. epithelial nemo links innate immunity to chronic intestinal infl ammation. nature, 446(7135):557–61. niess, j.h., brand, s. et al. 2005. cx3cr1-mediated dendritic cell access to the intestinal lumen and bacterial clearance. science, 307(5707):254–8. nilsen, e.m., jahnsen, f.l. et al. 1998. gluten induces an intestinal cytokine response strongly dominated by interferon gamma in patients with celiac disease. gastroenterology, 115(3):551–63. nilsen, e.m., jahnsen, f.l. et al. 1998. gluten induces an intestinal cytokine response strongly dominated by interferon gamma in patients with celiac disease. gastroenterology, 115(3):551–63. nilsen, e.m., johansen, f.e. et al. 1998. cytokine profi les of cultured microvascular endothelial cells from the human intestine. gut., 42(5):635–42. nilsen, e.m., lundin, k.e. et al. 1995. gluten specifi c, hla-dq restricted t cells from coeliac mucosa produce cytokines with th1 or th0 profi le dominated by interferon gamma. gut., 37(6):766–76. nunez, c., rueda, b. et al. 2007. involvement of macrophage migration inhibitory factor gene in celiac disease susceptibility. genes immun., 8(2):168–70. o’keeffe, j., lynch, s. et al. 2001. flow cytometric measurement of intracellular migration inhibition factor and tumour necrosis factor alpha in the mucosa of patients with coeliac disease. clin. exp. immunol., 125(3):376–82. qasimi, p., ming-lum, a. et al. 2006. divergent mechanisms utilized by socs3 to mediate interleukin-10 inhibition of tumor necrosis factor alpha and nitric oxide production by macrophages. j. biol. chem., 281(10):6316–24. rescigno, m. and chieppa m. 2007. gut-level decisions in peace and war. nat. med., 11(3):254–255. salvati, v.m., macdonald, t.t. et al. 2002. interleukin 18 and associated markers of t helper cell type 1 activity in coeliac disease. gut., 50(2):186–90. schuppan, d. 2000. current concepts of celiac disease pathogenesis. gastroenterology, 119(1):234–42. sollid, l.m. 2000. molecular basis of celiac disease. annu. rev. immunol., 18:53–81. sollid, l.m. 2002. coeliac disease: dissecting a complex infl ammatory disorder. nat. rev. immunol., 2(9):647–55. sozzani, s., allavena, p. et al. 1999. the role of chemokies in the regulation of dendritic cell traffi king. j. leukoc. biol., 66:1–9. suzuki, h., kundig, t.m. et al. 1995. deregulated t cell activation and autoimmunity in mice lacking interleukin-2 receptor beta. science, 268(5216):1472–6. torres-munoz, j., stockton, p. et al. 2001. detection of hiv-1 gene sequences in hippocampal neurons isolated from postmortem aids brains by laser capture microdissection. j. neuropathol. exp. neurol., 60(9):885–92. troncone, r., gianfrani, c. et al. 1998. majority of gliadin-specifi c t-cell clones from celiac small intestinal mucosa produce interferon-gamma and interleukin-4. dig. dis. sci., 43(1):156–61. united european gastroenterology. 2001. when is a coeliac a coeliac? report of a working group of the united european gastroenterology week in amsterdam, 2001. eur. j. gastroenterol. hepatol., 13(9):1123–8. valanciute, a., le gouvello, s. et al. 2004. nf-kappa b p65 antagonizes il-4 induction by c-maf in minimal change nephrotic syndrome. j. immunol., 172(1):688–98. van heel, d.a., franke, l. et al. 2007. a genome-wide association study for celiac disease identifi es risk variants in the region harboring il2 and il21. nat genet 39(7):827–9. wapenaar, m.c., van belzen, m.j et al. 2004. the interferon gamma gene in celiac disease: augmented expression correlates with tissue damage but no evidence for genetic susceptibility. j. autoimmun., 23(2):183–90. wong, b.r., besser, d. et al. 1999. trance, a tnf family member, activates akt/pkb through a signaling complex involving traf6 and c-src. mol. cell, 4(6):1041–9. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) 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<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> >> >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice drug target insights 2013:7 53–62 doi: 10.4137/dti.s12889 this article is available from http://www.la-press.com. © the author(s), publisher and licensee libertas academica ltd. this is an open access article published under the creative commons cc-by-nc 3.0 license. open access full open access to this and thousands of other papers at http://www.la-press.com. drug target insights o r i g i n a l r e s e a r c h drug target insights 2013:7 53 microscopic colitis and reproductive factors related to exposure to estrogens and progesterone bodil roth1, jonas manjer2 and bodil ohlsson1 1department of clinical sciences, division of internal medicine, skåne university hospital, malmö, lund university, lund, sweden. 2department of clinical sciences, division of plastic surgery, skåne university hospital, malmö, lund university, lund, sweden. corresponding author email: bodil.ohlsson@med.lu.se abstract: microscopic colitis (mc) often debuts around or after menopause and is divided into lymphocyticand collagenous colitis. the aim of this study was to examine whether factors influencing sex hormone levels differed between subgroups of mc as well as between patients and controls. a self-administered questionnaire about parity was completed which included questions surrounding age at first childbirth, menarche and menopause, the use of oral contraceptives, and hormonal replacement therapy. patients with lymphocytic colitis had children less often compared to those with collagenous colitis (or = 0.20, 95% ci = 0.05–0.86), however no differences were observed between patients with persistent or transient disease. patients were less often older than 15 years of age at menarche (or = 0.48, 95% ci = 0.26–0.91) and were younger at menopause (or = 0.30, 95% ci = 0.16–0.56) compared with controls. thus, no obvious association between factors influencing sex hormone levels and presence of mc could be found. keywords: microscopic colitis, reproductive factors, estrogen, progesterone http://dx.doi.org/10.4137/dti.s12889 http://www.la-press.com http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 http://www.la-press.com mailto:bodil.ohlsson@med.lu.se roth et al 54 drug target insights 2013:7 introduction microscopic colitis (mc) is a chronic disease of unknown etiology with mucosal, colonic inflammation. the disease is divided into collagenous colitis (cc) and lymphocytic colitis (lc), depending on the histopathological picture.1 interestingly, mc often debuts in women of midor upper middle age, at the time when endogenous sex hormone levels are diminished.2 thus, the involvement of sex hormones as well as autoimmunity has been discussed in the pathophysiology of mc.2 sex hormones may influence the function of the gastrointestinal tract. both estrogens and progesterone have been shown to reduce the inflammation in experimentally induced colitis in rats.3,4 estrogens exhibit anti-inflammatory and epithelial barrier-enhancing properties in colitic rats, and the protective effect of a fermented soy germ extract are based on protease inhibition and is partly mediated by activity of an estrogen receptor ligand.5 the assumption among health care professionals is, therefore, that the fall of estrogenand progesterone levels at menopause could predispose to the debut of a colonic inflammation, triggered by luminal factors. however, associations between factors influencing sex hormone levels and mc have never been examined. the aim of the present study was to compare parity age at first childbirth, menarche and menopause, as well as the use of oral contraceptives (oc) and hormonal replacement therapy (hrt)—factors that may affect the level of sex hormones during life time —between subgroups of patients suffering from mc and between patients and a population-based control group. material and methods patients women who had been treated for mc at any outpatient clinic of the departments of gastroenterology, skåne, between 2002 and 2010, were identified through a search for the icd-10 classification for the two forms cc and lc (k52.8) in outpatient records, as well as in the local register at the department of pathology, skåne university hospital, malmö. about one-third of the total number of patients identified were excluded as they were over 73 years of age, since they had many other concomitant diseases and drug therapies, representing secondary mc.6 of the patients recognized, only the 240 patients (median age 63 years, range 22–73 years) who had the diagnoses verified by histopathological examination of colonic biopsies were invited to participate in the present study. altogether, 159 (median age 63 years, range 22–73 years) of the 240 patients invited accepted to participate and were enrolled in the study. one patient was excluded due to another inflammatory bowel disease (ibd) diagnosis a few weeks after inclusion, leaving 158 patients (66%), and of these, 133 also agreed to provide blood samples. these patients represent the majority of female cases of diagnosed mc in the southernmost districts of sweden under the age of 73 years. controls the malmö diet and cancer study (mdcs) the malmö diet and cancer study (mdcs), a population-based prospective cohort study, invited all women in malmö born between 1923 and 1950 to participate. recruitment was carried out between 1991 and 1996, and 41% of eligible subjects participated. in all, 17,035 women completed the baseline examination.7 the mdcs baseline examination included a dietary assessment, a self administered questionnaire about marital status, education, employment, smokingand alcohol habits, parity, age at first childbirth, menarche, and menopause, exposure to oc (ever/never), current use of hrt (yes/no), and medical conditions and medication, as well as anthropometric measurements and the collection of blood samples.8 menopausal status was defined using information on previous surgery and menstrual status. the classification of pre-, periand postmenopausal women has been described in detail elsewhere.9 women selected as controls in a previous study on breast cancer were used in the present study as controls. in all, 737 subjects (median age 56 years, range 45–73 years) were available, the only exclusion criterion was that they should not have had a previous breast cancer at baseline.10 patient recruitment and study design between march and june 2011, invitations including information about the study and the questionnaire described above were sent by mail to all 240 women identified with mc. they were also invited to visit the outpatient clinics of the departments of gastroenterology, skåne university hospital, malmö or the http://www.la-press.com mc and hormonal factors drug target insights 2013:7 55 central hospital in kristianstad, to provide blood samples. a reminder letter was sent out a month after the invitation letter to those who had not answered. questionnaires were completed 1–3 weeks before blood samples were collected. medical records were scrutinized, and age, gastrointestinal symptoms, examinations, and treatments were recorded. the diagnosis of either cc or lc was registered and patients were divided into two groups based on their clinical presentation of mc. one group included patients with at least two episodes of watery diarrhea, and/or their dependence on long-term treatment of corticosteroids to maintain remission and/or two pathological intestinal mucosa biopsies (mc1, n = 78, in the final population for statistical calculation). these criteria are in line with the consensus for diagnosing ibd.11 the other group included patients who had had only one episode of severe diarrhea or had had a normal biopsy after the initial pathological intestinal biopsy, in combination with clinical remission (mc2, n = 53, in the final population for statistical calculation). patients with concomitant celiac disease (9 patients) or an acute gastroenteritis briefly prior to the diagnostic colonoscopy (4 patients) were excluded as they had an obvious organic explanation for the intestinal inflammation, and were considered to be suffering from secondary mc.6 concomitant diseases and drug treatment in the controls and the patient population have been described previously.12 in addition to mc, the patient group also suffered from many other diseases, where hypertension (36%), rheumatoid arthritis (24%), and asthma (17%) were the most prevalent. these diseases were more prevalent in the mc group than in the controls.12 patients were compared to controls from the mdsc study. statistical analyses the data were analyzed using the statistical software package spss for windows© (release 20.0; ibm, ny, usa). the patients were significantly older, with a wider age range than the controls. therefore, the 12 patients younger and the two patients older than the controls were excluded, as were patients with celiac disease and gastroenteritis (13 patients), leaving 131 of the original 158 patients for statistical analysis of patients compared to controls. thus, both controls and patients were within the age range 45–73 years. first, the distribution of continuous variables (age, disease duration, and body mass index (bmi)) was tested using a one-sample kolmogorov-smirnov test. all these distributions differed significantly (p , 0.05) from a normal distribution. therefore, the factors studied were categorized and the values were given as median (interquartile range). there were missing values in some variables, which were given a category of their own. differences between groups were calculated by the 2-tailed mann-whitney u-test. fisher’s exact test was used for categorical variables. a p-value , 0.05 was considered statistically significant. age was divided into 5-year intervals. smoking was divided into three categories: subjects who had never smoked, subjects who had stopped smoking, and current smokers, including both regular and occasional smokers. subjects who denied intake of beer, wine, and alcoholic liquids during the previous year were defined as having no alcohol intake. subjects were divided into three groups: subjects consuming no alcohol, subjects who had drunk some alcohol in the previous year, but not in the past month, and subjects who had drunk some alcohol in the previous month. employment was divided into three categories: employed, retired, or other, where other included housewives, students, and the unemployed. education was divided into having a university education or not. parity was dichotomized as nulliparous and parous in order to yield larger groups. age at first childbirth was also dichotomized as #25 years of age and .25 years of age. hrt was defined as non-use, and use of either estrogen or progesterone replacement therapy, or combined estrogen and progesterone therapy. age at menopause was categorized as #45 years, .45 and ,53 years, and $53 years. these classifications were in accordance with previous classifications.13 the first category was used as reference. factors intended to be studied (independent variables), namely, age at menarche, first childbirth, and menopause; parity; exposure to oc (ever/ never); exposure to hrt (current/none); or bilateral oophorectomy (yes/no), were initially examined using an unconditional logistic regression to calculate odds ratios with 95% confidence intervals (or with 95% ci). analyses were adjusted in a second model for age at baseline, smokingand alcohol habits, level of education, and employment, as these characteristics http://www.la-press.com roth et al 56 drug target insights 2013:7 differed by .5 percentage between controls and patients, and between mc1 and mc2. analyses were also adjusted for age at baseline, smokingand alcohol habits, employment, and civil status in the calculations between cc and lc. calculations were first performed on the whole patient group compared with controls, and then separately for patients with cc compared to patients with lc, and patients with mc1 compared to patients with mc2 (dependent variables). ethical considerations the ethics committee of lund university approved the study protocol for patients (dnr 2009/565 and 2011/209) and controls (dnr 51-90). all participants gave their written, informed consent to take part in the study. results patient characteristics in total, 131 women (median age 63 (59–67) years) with mc were included in the statistical calculations; cc was diagnosed in 82 patients (62.6%) and lc in 49 patients (37.4%) (table 1). although identical in age range, the median age was higher in the patient group (p , 0.001). the duration of the disease was 7 (3–14) years. measurements of hemoglobin (hb) in blood and c-reactive protein (crp) in plasma were in the majority of patients within reference values, showing that the patients were in an overall inactive phase (data not shown). of the patients, 91.7% were born in sweden compared with 90.2% of the controls. the number of married patients did not differ between cc and lc (p = 0.362). as patients with table 1. patient and control characteristics. controls n = 737 microscopic colitis n = 131 p-value cc n = 82 lc n = 49 p-value age at study (years) 56.16 (50.47–62.36) 63.00 (58.94–67.15) 0.000 62.77 (58.94–67.30) 63.78 (58.74–66.85) 0.761 age groups (%) 0.000 0.763 45–49 17.1 4.6 4.9 4.1 50–54 22.3 6.9 4.9 10.2 55–59 22.3 13.7 15.9 10.2 60–64 19.5 32.1 34.1 28.6 65–69 11.7 26.0 24.4 28.6 70–74 7.2 16.8 15.9 18.4 smoking habits (%) 0.076 0.380 never smoked 42.3 27.5 26.8 28.6 former smokers 29.9 36.6 32.9 42.9 current smokers 27.8 35.9 40.2 28.6 alcohol habits (%) missing value 0.3 3.8 0.206 6.1 0 0.573 nothing last year 11.0 16.0 13.4 20.4 something last year (not last month) 12.3 13.0 13.4 12.2 something last month 76.4 67.2 67.1 67.3 bmi (kg/m2) 24.84 (22.55–27.79) 24.88 (22.62–29.15) 0.451 24.70 (21.85–29.49) 24.90 (23.10–28.33) 0.977 missing value (%) 0 44.3 40.2 51.0 married women (%) 61.9 58.0 0.542 61.0 53.1 0.362 level of education (%) missing value 0 2.3 0.002 3.7 0 0.578 #12 years at school 76.1 67.9 67.1 69.4 .12 years at school 23.9 29.8 29.3 30.6 employment (%) 0.001 0.567 employed 65.7 44.3 46.3 40.8 retired 26.5 49.6 46.3 55.1 others* 7.9 6.1 7.3 4.1 notes: *includes housewives, students, and unemployed. values are given as median (interquartile range). mann-whitney u-test or fischer’s exact test were used for statistical calculations. p , 0.05 was considered statistically significant. http://www.la-press.com mc and hormonal factors drug target insights 2013:7 57 lc increased in age, the number of retired persons was greater, but these changes did not reach statistical significance (p = 0.763 and p = 0.567, respectively). there was no difference between cc and lc whether mc was persistent or transient (p = 0.273). smoking and drinking habits more patients than controls were former or current smokers (table 1). there was no statistically significant difference between cc and lc, or between mc1 and mc2, concerning smoking habits (p = 0.380 and p = 0.128, respectively). there were only a few patients and controls who consumed beer and stronger alcoholic beverages. thus, the alcohol intake consisted mainly of wine. the majority of subjects who drank imbibed 1–2 glasses a day. only a minority of the subjects were non-users of alcohol (table 1). reproductive factors the only difference between cc and lc was that among patients with lc, where there was a higher percentage of nulliparity (table 2). furthermore, patients with lc had fewer children than patients with cc (2 (1–2) and 2 (1–3), respectively), although this did not reach statistical significance (p = 0.057). there was no difference in reproductive factors influencing sex hormone levels between those patients who had a transient mc and those with persistent mc (table 3). table 2. differences between collagenous colitis (cc) and lymphocytic colitis (lc). cc n = 82 % lc n = 49 % cc/lc crude or, 95% ci or, 95% ci age at menarche (year) missing value 9.8 6.1 “–” “–” #12 (reference) 28.0 18.4 1.00 1.00 .12 to ,15 47.6 51.0 1.64 (0.65–4.11) 1.68 (0.62–4.54) $ 15 14.6 24.5 2.56 (0.84–7.76) 3.42 (0.96–12.14) parity missing value 3.7 2.0 “–” “–” nullipara (reference) 4.9 16.3 1.00 1.00 parous 91.5 81.6 0.27 (0.08–0.94) 0.20 (0.05–0.86) age at first childbirth (year) missing value 4.9 2.0 “–” “–” nullipara (reference) 4.9 16.3 1.00 1.00 #25 62.2 61.2 0.29 (0.08–1.06) 0.22 (0.05–0.98) .25 28.0 20.4 0.22 (0.05–0.89) 0.16 (0.03–0.79) age at menopause (year) missing value* 25.6 16.3 “–” “–” #45 (reference) 22.0 18.4 1.00 1.00 .45 to ,53 28.0 40.8 1.74 (0.64–4.73) 1.92 (0.64–5.81) $ 53 24.4 24.5 1.20 (0.41–3.51) 1.09 (0.33–3.64) exposure to oc missing value 2.4 0 “–” “–” never (reference) 23.2 24.5 1.00 1.00 ever 74.4 75.5 0.96 (0.42–2.20) 0.97 (0.40–2.37) exposure to hrt missing value 0 0 “–” “–” non (reference) 91.5 91.8 1.00 1.00 current 8.5 8.2 0.95 (0.26–3.44) 0.91 (0.23–3.59) bilateral oophorectomy missing value 7.3 8.2 “–” “–” no (reference) 79.3 71.4 1.00 1.00 yes 13.4 20.4 1.69 (0.65–4.36) 1.68 (0.58–4.88) notes: *missing values includes missing values and premenopausal women. calculations were adjusted for age, smoking habits, alcohol habits, civil status, and employment. abbreviations: hrt, hormonal replacement therapy; ci, confidence interval; oc, oral contraceptives; or, odds ratio. http://www.la-press.com roth et al 58 drug target insights 2013:7 more of the patients had their menarche before the age of 15 years. many controls reached menopause between 45–53 years of age, whereas more patients with mc were younger than 45 years when they reached menopause (table 4). there was no difference in parity or age at first childbirth between the groups (table 4). the majority of mc patients had used oc at some period of their life. in contrast, fewer patients than controls were on current treatment with hrt (table 4). discussion the present study showed no differences in the influence of sex hormones between the subgroups, except that patients with lc were more often nulliparous than patients with cc. this is interesting as some studies have shown a female predominance in both cc and lc,2 whereas others have not been able to confirm this in lc.14,15 patients with mc differed from the external control group as they had reached menarche and menopause earlier, but there was no difference in parity or age at first childbirth. more of the patients than controls had been exposed to oc at any time during their lives, and fewer were exposed to current hrt. although the debut of mc often occurs in predominantly middle-aged women, the role of sex hormones has never been examined in this entity. mc is table 3. differences between persistent (mc1) and transient (mc2) microscopic colitis. mc1 n = 78 % mc2 n = 53 % mc1/mc2 crude or 95% ci or 95% ci age at menarche (year) missing value 10.3 5.7 “–” “–” #12 (reference) 24.4 24.5 1.00 1.00 .12 to ,15 47.4 50.9 0.94 (0.40–2.22) 0.94 (0.37–2.37) $15 17.9 18.9 0.96 (0.33–2.81) 0.94 (0.29–3.04) age at first childbirth (year) missing value 3.8 3.8 “–” “–” nullipara (reference) 10.3 7.5 1.00 1.00 #25 64.1 58.6 0.81 (0.22–2.90) 1.40 (0.33–5.97) .25 21.8 30.2 0.53 (0.13–2.11) 0.67 (0.15–3.05) parity missing value 3.8 1.9 “–” “–” nullipara (reference) 10.3 7.5 1.00 1.00 parous 85.9 90.6 0.70 (0.20–2.45) 1.03 (0.26–4.14) age at menopause (year) missing value* 23.1 20.8 “–” “–” #45 (reference) 19.2 22.6 1.00 1.00 .45 to ,53 34.6 30.2 1.35 (0.51–3.59) 1.50 (0.52–4.30) $53 23.1 26.4 1.03 (0.37–2.88) 1.42 (0.45–4.42) exposure to oc missing value 2.6 0 “–” “–” never (reference) 19.2 30.2 1.00 1.00 ever 78.2 69.8 1.76 (0.78–3.97) 2.24 (0.91–5.55) exposure to hrt missing value 0 0 “–” “–” non (reference) 88.5 96.2 1.00 1.00 current 11.5 3.8 3.33 (0.69–16.06) 3.48 (0.68–17.73) bilateral oophorectomy missing value 7.7 7.5 “–” “–” no (reference) 17.9 13.2 1.00 1.00 yes 74.4 79.2 1.45 (0.54–3.90) 1.43 (0.49–4.15) notes: *missing values includes missing values and premenopausal women. calculations were adjusted for age, smoking habits, alcohol habits, level of education, and employment. abbreviations: hrt, hormonal replacement therapy; ci, confidence interval; oc, oral contraceptives; or, odds ratio. http://www.la-press.com mc and hormonal factors drug target insights 2013:7 59 table 4. differences between controls and patients with microscopic colitis (mc). controls n = 737 % mc n = 131 % controls/microscopic colitis crude or, 95% ci or, 95% ci age at menarche (year) missing value 0 8.4 “–” “–” #12 (reference) 19.1 24.4 1.00 1.00 .12 to ,15 54.3 48.9 0.70 (0.44–1.12) 0.65 (0.38–1.09) $15 26.1 18.3 0.55 (0.31–0.98) 0.48 (0.26–0.91) parity missing value 2.6 3.1 “–” “–” nullipara (reference) 11.4 9.2 1.00 1.00 parous 86.0 87.8 1.27 (0.67–2.40) 1.21 (0.61–2.40) age at first childbirth (year) missing value 2.6 3.8 “–” “–” nullipara (reference) 11.1 9.2 1.00 1.00 #25 53.6 61.8 1.40 (0.73–2.69) 1.46 (0.72–2.96) .25 32.4 25.2 0.94 (0.46–1.91) 0.79 (0.37–1.69) age at menopause (year) missing value* 32.8 22.1 “–” “–” #45 (reference) 11.0 20.6 1.00 1.00 .45 to ,53 39.9 32.8 0.44 (0.26–0.75) 0.30 (0.16–0.56) $53 16.0 24.4 0.81 (0.45–1.46) 0.53 (0.28–1.03) exposure to oc missing value 0 1.5 “–” “–” never (reference) 50.2 23.7 1.00 1.00 ever 49.8 74.8 3.19 (2.08–4.89) 7.49 (4.46–12.43) exposure to hrt missing value 0.4 0 “–” “–” non (reference) 79.5 91.6 1.00 1.00 current 20.1 8.4 0.36 (0.19–0.69) 0.42 (0.22–00.83) bilateral oophorectomy missing value 66.5 7.6 “–” “–” no (reference) 29.0 76.3 1.00 1.00 yes 4.2 16.0 1.45 (0.79–2.65) 1.19 (0.60–2.35) notes: *missing values includes missing values and premenopausal women. calculations were adjusted for age, smoking habits, alcohol habits, level of education, and employment. abbreviations: hrt, hormonal replacement therapy; ci, confidence interval; oc, oral contraceptives; or, odds ratio. sometimes characterized as a subgroup of ibd,16 and autoimmunity is assumed to be involved in the pathogenesis of ibd.2 autoimmune diseases are often in remission during pregnancy, since the elevated estrogen levels during pregnancy influence the cytokine profile in general, not only the cytokine profile in the gut.17 prior studies on hormonal influences have been performed in patients with ibd, and studies have shown an association between oc and a risk to develop ibd, especially crohn’s disease, among younger, premenopausal women. this increased risk of ibd reverts to that of the non-exposed population when the women stop the use of oc.18 in a large prospective study, postmenopausal hormone therapy was associated with an increased risk of ulcerative colitis (uc), but not crohn’s disease.19 when scrutinizing all papers written about colonic toxicity of administered drugs and chemicals, a hypercoagulable state with ischemic colitis due to mesenteric vein thrombosis was the only association found between oc and the gastrointestinal tract.20 both estrogenand progesterone receptors are expressed in the gastrointestinal tract under normal conditions,21,22 with predominance of estrogen receptor β in the colon, mainly located in epithelial cells.21 sex steroids have been shown to influence colonic transit time,23 chloride ion secretion,24 and epithelium formation.25 one important function of the intestinal epithelium is to provide a http://www.la-press.com roth et al 60 drug target insights 2013:7 protective barrier for the internal milieu against luminal factors. the physical barrier is dependent on intercellular tight junctions sealing the intercellular spaces between the epithelial cells.26 increased intercellular permeability has been implicated in the pathogenesis of chronic, mucosal inflammation.27,28 there is a physiological link between circulating estrogens and estrogen receptor β-mediated increase in tight junction proteins, with pivotal functions in the maintenance of intercellular spaces in female rats.29 the protective role of estradiol in decreasing paracellular permeability enhances its beneficial effects on intestinal barrier function.4 in recent years, progesterone has been reported to suppress inflammatory responses to reduce lipid peroxidation and cell membrane damage due to free oxygen radicals in clinicaland experimental studies.3 although sex hormones strenghten the epithelial barrier in experimental trials in rats,4,29 pharmacological levels of estrogens and progesterone in oc and hrt taken over a long time span seem to increase the risk of ibd.18,19 severe mc has to be treated by a derivation of fecals from the colonic mucosa, which heals after diversion.30 this has raised the hypothesis that luminal factors trigger the mucosal inflammation. the fall in levels of estrogens and progesterone at menopause could theoretically impair the epithelial barrier function, and the mucosa could be influenced to a greater extent by luminal factors, e.g. drugs. at the same time, the older the person, the more drugs are used, and the combination of different drugs may have a synergetic effect on the mucosa. as there was no difference in sex hormone influences between mc1 and mc2, it can be suggested that other factors are further involved in the pathophysiology and maintenance of inflammation. however, one limitation in this study is that we have not measured the sex hormone levels directly, only registered factors that indirectly affect sex hormone levels. another limitation is the small cohort in the study. nevertheless, in light of the current knowledge on mc, a colonic, epithelial dysfunction seems more pertinent in the pathophysiology of mc than autoimmunity.31 intestinal ischemia, drugs, and environmental factors, such as smoking, may trigger mucosal changes which could represent an intestinal reaction to diverse irritants rather than being a specific entity.12,32 there are several differences which affect sex hormone levels between patients in our study and controls. these differences must be interpreted with caution as the controls are an external group. it is very difficult to recruit healthy volunteers to clinical studies. the response rate of our control group was 41%, and therefore it can be assumed that these subjects are healthier than those who did not agree to participate. however, it is a strength for the first time to compare patients with mc with such a well-defined control group.10 furthermore, the data concerning smoking, overweight, and level of education were similar to a study with 80% participation of the same population.7 the difference in oc consumption may be explained by the fact that the external control group was recruited two decades previously, and therefore may have not been exposed to oc to the same extent as the women born later. in the same way, the use of hrt was highest during the nineties, at the time when the control group was recruited. later on, when the side effects were better known, the consumption of hrt had diminished.33 a higher use of oc earlier in life should not be important as previous reports have shown that the increased risk of crohn’s disease during oc treatment is reversed after cessation.18 in addition, it would have been useful to know the past use of hrt, not only the current use. however, as the response rate to this question was very low, we chose not to calculate with this parameter. due to severe side effects of hrt, prevention of mc by prescribing hrt is not an option,33 and thus, of no clinical interest. the study is cross-sectional, and a prospective study is necessary to determine the time of initiation of mc in relation to hormonal and environmental changes. furthermore, few controls may also suffer from mc. however, as the prevalence of mc in the population is around 1–12 per 1000002 this could not affect the results. in conclusion, there were no differences in exposure to factors influencing sex hormones between cc and lc, or between mc1 and mc2, excepting that more patients with lc were nulliparous. patients with mc reach menarche and menopause earlier and have been exposed to oc to a greater extent, and to a lesser extent to hrt, compared with the previously recruited controls. since we found no differences in exposure to hormonal treatments between patients with transient and persistent mc, factors other than hormonal levels may affect the susceptibility to develop colonic, mucosal http://www.la-press.com mc and hormonal factors drug target insights 2013:7 61 inflammation to a greater extent, and luminal factors should be further examined for causality in prospective studies. author contributions conceived and designed the experiments: br, jm, bo. analyzed the data: bo. wrote the first draft of the manuscript: bo. contributed to the writing of the manuscript: br, jm. agree with manuscript results and conclusions: br, jm, bo. jointly developed the structure and arguments for the paper: br, jm, bo. made critical revisions and approved final version: br, jm. all authors reviewed and approved of the final manuscript. funding this study was sponsored by grants from the bengt ihre foundation, ruth and richard julin foundation, and the development foundation of region skåne. competing interests author(s) disclose no potential conflicts of interest. disclosures and ethics as a requirement of publication the authors have provided signed confirmation of their compliance with ethical and legal obligations including but not limited to compliance with icmje authorship and competing interests guidelines, that the article is neither under consideration for publication nor published elsewhere, of their compliance with legal and ethical guidelines concerning human and animal research participants (if applicable), and that permission has been obtained for reproduction of any copyrighted material. this article was subject to blind, independent, expert peer review. the reviewers reported no competing interests. references 1. rasmussen ma, munck lk. systematic review: are lymphocytic colitis and collagenous colitis two subtypes of the same disease—microscopic colitis? aliment pharmacol ther. 2012;36(2):79–90. 2. pardi ds, kelly cp. microscopic colitis. gastroenterology. 2011;140(4): 1155–65. 3. karatepe o, altiok m, battal m, et al. the effect of progesterone in the prevention of the chemically induced experimental colitis in rats. acta cir bras. 2012;27(1):23–9. 4. günal o, oktar bk, ozçinar e, sungur m, arbak s, yeğen b. estradiol treatment ameliorates acetic acid-induced gastric and colonic injuries in rats. inflammation. 2003;27(6):351–9. 5. moussa l, bézirard v, salvador-cartier c, et al. a low dose of fermented soy germ alleviates gut barrier injury, hyperalgesia and faecal protease activity in a rat model of inflammatory bowel disease. plos one. 2012;7(11): e49547. 6. carmack sw, lash rh, gulizia jm, genta rm. lymphocytic disorders of the gastrointestinal tract: a review for the practicing pathologist. adv anat pathol. 2009;16:290–306. 7. manjer j, carlsson s, elmståhl s, et al. the malmö diet and cancer study: representativity, cancer incidence and mortality in participants and nonparticipants. eur j cancer prev. 2001;10(6):489–99. 8. manjer j, elmståhl s, janzon l, berglund g. invitation to a populationbased cohort study: differences between subjects recruited using various strategies. scand j public health. 2002;30(2):103–12. 9. manjer j, johansson r, berglund g, et al. postmenopausal breast cancer risk in relation to sex steroid hormones, prolactin and shbg (sweden). cancer causes control. 2003;14(7):599–607. 10. almquist m, bondeson ag, bondeson l, malm j, manjer j. serum levels of vitamin d, pth and calcium and breast cancer risk-a prospective nested case-control study. int j cancer. 2010;127(9):2159–68. 11. henriksen m, jahnsen j, lygren i, et al. ibsen study group. change of diagnosis during the first five years after onset of inflammatory bowel disease: results of a prospective follow-up study (the ibsen study). scand j gastroenterol. 2006;41(9):1037–43. 12. roth b, manjer j, ohlsson b. microscopic colitis is associated with several concomitant diseases. drug target insights. 2013;7:19–25. 13. butt s, harlid s, borgquist s, et al. genetic predisposition, parity, age at first childbirth and risk for breast cancer. bmc res notes. 2012;5:414. 14. olesen m, eriksson s, bohr j, järnerot g, tysk c. lymphocytic colitis: a retrospective clinical study of 199 swedish patients. gut. 2004;53(4):536–41. 15. fernández-bañares f, salas a, forné m, esteve m, espinós j, viver jm. incidence of collagenous and lymphocytic colitis: a 5-year populationbased study. am j gastroenterol. 1999;94(2):418–23. 16. münch a, aust d, bohr j, et al; european microscopic colitis group (emcg). microscopic colitis: current status, present and future challenges: statements of the european microscopic colitis group. j crohns colitis. 2012;6(9):932–45. 17. gonzález da, díaz bb, rodríguez pérez mdel c, hernández ag, chico bn, de león ac. sex hormones and autoimmunity. immunol lett. 2010;133(1): 6–13. 18. cornish ja, tan e, simillis c, clark sk, teare j, tekkis pp. the risk of oral contraceptives in the etiology of inflammatory bowel disease: a metaanalysis. am j gastroenterol. 2008;103(9):2394–400. 19. khalili h, higuchi lm, ananthakrishnan an, et al. hormone therapy increases risk of ulcerative colitis but not crohn’s disease. gastroenterology. 2012;143(5):1199–206. 20. cappell ms. colonic toxicity of administered drugs and chemicals. am j gastroenterol. 2004;99(6):1175–90. 21. konstantinopoulos pa, kominea a, vandoros g, et al. oestrogen receptor beta (erbeta) is abundantly expressed in normal colonic mucosa, but declines in colon adenocarcinoma paralleling the tumour’s dedifferentiation. eur j cancer. 2003;39(9):1251–8. 22. xiao zl, pricolo v, biancani p, behar j. role of progesterone signaling in the regulation of g-protein levels in female chronic constipation. gastroenterology. 2005;128(3):667–75. 23. cong p, pricolo v, biancani p, behar j. abnormalities of prostaglandins and cyclooxygenase enzymes in female patients with slow-transit constipation. gastroenterology. 2007;133(2):445–53. 24. o’mahony f, alzamora r, betts v, et al. female gender-specific inhibition of kcnq1 channels and chloride secretion by 17beta-estradiol in rat distal colonic crypts. j biol chem. 2007;282(34):24563–73. 25. wada-hiraike o, imamov o, hiraike h, et al. role of estrogen receptor beta in colonic epithelium. proc natl acad sci u s a. 2006;103(8):2959–64. 26. turner jr. molecular basis of epithelial barrier regulation: from basic mechanisms to clinical application. am j pathol. 2006;169(6):1901–9. 27. meddings jb. review article: intestinal permeability in crohn’s disease. aliment pharmacol ther. 1997;11 suppl 3:47–53. 28. teshima cw, dieleman la, meddings jb. abnormal intestinal permeability in crohn’s disease pathogenesis. ann n y acad sci. 2012;1258:159–65. http://www.la-press.com roth et al 62 drug target insights 2013:7 29. braniste v, leveque m, buisson-brenac c, bueno l, fioramonti j, houdeau e. oestradiol decreases colonic permeability through oestrogen receptor beta-mediated up-regulation of occludin and junctional adhesion molecule-a in epithelial cells. j physiol (lond). 2009;587(pt 13):3317–28. 30. stroehlein jr. microscopic colitis. curr treat options gastroenterol. 2007;10(3):231–6. 31. roth b, gustafsson rj, ohlsson b. auto-antibodies and their association with clinical findings in women diagnosed with microscopic colitis. plos one. 2013;8(6):e66088. 32. roth b, bengtsson m, ohlsson b. diarrhoea is not the only symptom that needs to be treated in patients with microscopic colitis. eur j intern med. 2013;24:573–8. 33. królik m, milnerowicz h. the effect of using estrogens in the light of scientific research. adv clin exp med. 2012;21(4):535–43. http://www.la-press.com satoh et al.indd review correspondence: dr. hiroyasu satoh, department of pharmacology, nara medical university, kashihara, nara 634-8521, japan. tel: +81-744-29-8831; fax: +81-744-29-0510; email: hysat@naramed-u.ac.jp please note that this article may not be used for commercial purposes. for further information please refer to the copyright statement at http://www.la-press.com/copyright.htm cardiovascular pharmacology of sinomenine: the mechanical and electropharmacological actions seiichiro nishida and hiroyasu satoh department of pharmacology, division of traditional medicine, nara medical university, kashihara, nara 634-8521, japan abstract: sinomenine is one of the alkaloids extracted from chinese medical plant, sinomenium acutum rehder et wilson. sinomenine has been used for rheumatoid arthritis as an anti-infl ammatory and immunomodulative drugs. we have so far been investigated the cardiovascular pharmacological actions of sinomenine. sinomenine dilated ne (5 µm)-, kcl (60 mm)and pdb (300 nm)-induced vasoconstrictions. the pretreatment with nicardipine (0.1 µm), staurosporine (30 nm), l-nmma (100 µm), indomethacin (10 µm) or propranolol signifi cantly attenuated the sinomenine-induced vasorelaxation. therefore, these results indicate that sinomenine causes the vasorelaxation by the involvement with the inhibitions of ca2+ current (ica) and pk-c, β-adrenoceptor stimulation, and the activation of no and pgi2 syntheses in endothelium. on the other hand, in the ventricular cardiomyocytes of guinea pig, sinomenine inhibits ica and simultaneously decreases the delayed rectifi er k+ current (ik), resulting in the prolongation of action potential duration. sinomenine also suppresses the dysrhysmias induced by triggered activities under the ca2+ overload condition. therefore, sinomenine may be expected as one of effective therapeutic drugs for heart failure and dysrhythmias, and may maintain the cardiovascular functions due to modulation of cardiac ionic channels and blood vessels. keywords: sinomenine, vasodilation, cardioprotective action, ca2+ channel, aorta, cardiomyocytes introduction sinomenine (7, 8-didehydro-4-hydroxy-3, 7-dimethoxy-17-methyl-9α,13α,14α-morphinan-6-one) is one of the alkaloids extracted from chinese herbs, sinomenium acutum rehder et wilson (li et al. 2004) or sinomenium acutum var. cinereum (zhao et al. 2005). in chinese traditional medicine, sinomenine acutum, a vine plant, has been used for rheumatic diseases for thousands years (yamasaki, 1976; liu et al. 1996). its main constituent of sinomenine acutum is sinomenine, which has also used for clinical treatment of rheumatoid arthritis (ra), due to the anti-infl ammatory and immunomodulative actions (yamasaki, 1976; liu et al. 1996). we have already investigated the cardiovascular pharmacological actions of sinomenine. mokuboito (mu-fang-yi-tang), a kind of kampo formulation containing sinomenium acutum, has been used for heart failure (inaki et al. 2005), which improves heart failure symptom and reduces new york heart association (nyha) class and plasma brain natriuretic peptide (bnp) concentration (yakubo et al. 2002). mokuboito consists of sinomeni caulis et rhizoma (rhizome of sinomenium acutum rehder et wilson), cinnamomi cortex (bark of cinnamomum cassia blume), ginseng radix (roots of panax ginseng c. a. meyer) and gypsum fibrosum. in our recent reports (satoh, 2005; nishida and satoh, 2006; 2007), mokuboito, sinomenium acutum and sinomenine might improve chronic heart failure, resulting from the modulation of cardiac and vascular systems. mokuboito can exert the vasodilating and cardioprotective actions (satoh, 2005), as discussed in the following parts. in general, the basic treatments of heart failure consist of (1) reducing workload of heart, (2) protection of cardiomyocytes and (3) restriction and control of waters and sodium. in order to reduce both preand after-loads, the dilations of arterioles and veins are strongly required in the case of elevated fi lling pressures and reduced cardiac output. as a cardiovascular protective drug, sinomenine might contribute to clinical treatments via modulation of the ionic currents of cardiac cells, and control of the tension of blood vessels. satoh et al.indd 1 5/14/2007 8:58:41 pm 97drug target insights 2007: 2 97–104 nishida and satoh drug target insights 2007: 2 in this review, the cardiovascular pharmacological actions of sinomenine are mainly shown and discussed. source of sinomenine sinomenine is derived from natural plant such as sinomenium acutum (li et al. 2004) and sinomenium acutum var. cinereum (zhao et al. 2005). sinomenium acutum and sinomenium acutum var. cinereum are widely in a lot of regions of china. caulis sinomeni is the dried plant stems of sinomenium acutum and sinomenium acutum var. cinereum. there is less or no difference in sinomenine content of caulis sinomeni between the species and the varieties of growing regions. the variation is found among the samples collected from different parts of plant. the content of sinomenine is dependent on the size (diameter) of stem. the sinomenine content in sinomenine acutum is 1.63 ± 0.64 (%, w/w) in large (>3 cm) stem, 0.96 ± 0.45 (%, w/w) in 1–3 cm stem, and 0.49 ± 0.16 (%, w/w) in <1 cm stem (zhao et al. 2005). pharmacokinetics of sinomenine the pharmacokinetics and tissue distribution of sinomenine have been studied in rats (liu et al. 2005). sinomenine achieves high bioavilability (about 80%) by oral administration of 90 mg/kg. at 45min later, sinomenine is found widely in internal organs such as kidney, liver, lung, spleen, heart, brain and testis. sinomenine is metabolized and eliminated by kidney and liver. tmax is 39.5 ± 8.49 min, cmax is 13.89 ± 4.29 µg/ml, t1/2a phase is 61.28 ± 53.62 min, auc0-t is 2331.53 ± 1172.77 µg-min/ml, and cl is 42.95 ± 14.4 ml/min per kg. in clinical studies, oral administration of 80 mg sinomenine is performed for healthy volunteers (yan et al. 1997). in the pharmacokinetic parameters, t1/2α is 1.04 ± 0.491h, t1/2β is 9.397 ± 2.425h, tmax is 1.04 ± 0.274h, cmax is 246.6 ± 71.165 ng/ml. furthermore, auc is 2651.158 ± 1039.050 ng.h/ml, and cl is 0.033 ± 0.010 ng/ml. vascular pharmacology male wistar rats (5–10 week-old) were anesthetized with ether, and euthanized by exsanguination. the thoracic aorta was quickly removed, and then the isolated aorta was cut into rings of 3-mm in length. all rings were stretched to generate a resting tension of 1.2 g in krebs solution. after 40 min of resting, norepinephrine (ne) (5 µm) was added to the tissue bath. after the contractile response became steady, the drugs were cumulatively administrated into the bath solution. the similar methods are described in our previous reports (nishida and satoh, 2003; 2006). the aorta ring strip of rat had a strong contraction after an initial application of 5 µm ne. sinomenine (0.1 to 100 µm) applications potently relaxed the contraction induced by ne in a concentration-dependent manner (nishida and satoh, 2006). the relaxation was produced at the concentrations of over 0.3 µm sinomenine, and at 100 µm decreased the contractions by 68.8 ± 5.1% (n = 6, p < 0.001). pdb (300 nm) and kcl (60 mm) also caused strong contractions. sinomenine dilated both pdband kcl-induced contractions in a concentration-dependent manner; at 100 µm by 49.9 ± 9.8% (n = 6, p < 0.001) and 86.9 ± 8.5% (n = 6, p < 0.001), respectively (fig. 1). furthermore, the relaxation of sinomenine (0.1 to 100 µm) was attenuated signifi cantly by the pretreatment with nicardipine (fig. 2). at 100 µm the relaxation decreased from 68.8 ± 5.1% (n = 6) to 35.5 ± 6.9% (n = 5, p < 0.001). since pk-c inhibition may be related to sinomenine-induced vasorelaxation, the pretreatment with staurosporine (30 nm) for 30 min was carried out (satoh, 1996; nishida and satoh, 2004). staurosporine attenuated the sinomenine-induced vasorelaxation; at 100 µm from 68.8 ± 5.1% (n = 6) to 49.5 ± 7.7% (n = 5, p < 0.001) (fig. 2). in addition, propranolol (0.3 µm) also signifi cantly attenuated the sinomenineinduced relaxation. the vasorelaxation at 100 µm sinomenine was attenuated to 45.2 ± 4.2% (n = 5, p < 0.01). therefore, the modulation of both ca2+ channel and pk-c is largely contributed to sinomenineinduced actions, and sinomenine also vasodilates mediated through β-adrenoceptor stimulation of aortic smooth muscle cells. for involvement with endothelium-dependent relaxation via no activation, a pretreatment with 100 µm l-nmma (a non-selective no synthesis inhibitor) was carried out (nishida and satoh, 2003; 2007). under the conditions, the vasorelaxation induced by sinomenine was attenuated from 68.8 ± 5.1% (n = 6) to 25.3 ± 2.3% (n = 5, p < 0.01) (fig. 2). the attenuation was supported by the results using the aorta with removal of endothelium; at 100 µm sinomenine by 53.7 ± 1.8% (n = 5, p < 0.01). indomethacin (10 µm), as an satoh et al.indd 2 5/14/2007 8:59:09 pm 98 cardiovascular pharmacology of sinomenine drug target insights 2007: 2 inhibitor of prostanoid production, also strongly reduced the vasorelaxation induced by 100 µm sinomenine to 37.1 ± 9.3% (n = 5, p < 0.001). thus, both l-nmma and indomethacin affected the sinomenine-induced relaxation significantly. therefore, sinomenine possesses the pharmacological characteristics for modulation of no synthesis and pg production, as well as the inhibitions of ca2+ channel and pk-c and the stimulation of β-adrenoceptor. the possible mechanisms for the vasodilating actions of sinomenine are summarized in fig. 3. cardiac electropharmacology cardiac cells were taken from the ventricle muscles of guinea pig hearts, using methods similar to those described previously (satoh, 2003, 2005). currentclamp and whole-cell voltage-clamp were performed using an axopatch patch-clamp amplifi er (axon instruments, burlingame, c.a, u.s.a.) and standard techniques. current-clamp experiments were carried out to examine the modulation of the action potential confi guration in guinea pig ventricular muscles (fig. 4a) (satoh, 2005). sinomenine at 300 µm and 1 mm increased the 75% repolarization of action potential duration (apd75) by 24.9 ± 3.5% (n = 6, p < 0.05) and by 43.7 ± 3.3% (n = 6, p < 0.001), respectively. sinomenine at 1 mm decreased the amplitude (apa), but not signifi cantly (0.9 ± 2.1%, n = 5). the resting potential was not affected. the modulation of l-type ca2+ current (ica) was investigated. test pulses were applied to 0 mv from a holding potential of –30 mv. the average capacitance was 84.1 ± 2.4 pf (n = 23). at 1 mm sinomenine inhibited the ica at 0 mv by 18.2 ± 2.1% (n = 6, p < 0.05). at 1 mm, the delayed rectifi er k+ currents (ik) at 60 mv was inhibited by 16.2 ± 2.6% (n = 6, p < 0.05) (fig. 4b), and the inwardly rectifying k+ current (ik1) at -120 mv by 47.2 ± 3.8% (n = 6, p < 0.01). s inome nine 0 10 20 30 40 50 60 70 80 90 100 0.1 0.3 1 3 10 30 100 r el ax at io nn (% ) ne p db kc μ m figure 1. concentration-dependent relaxation of sinomenine on ne-, pdband kcl-induced vasorelaxations. symbols used are sinomenine in pretreatments with ne (open circles, n = 6), pdb (triangles, n = 6) and kcl (squares, n = 6). values (%) represent mean ± s.e.m. *: p < 0.05, **: p < 0.01, ***: p < 0.001, with respect to control value. satoh et al.indd 3 5/14/2007 8:59:10 pm 99 nishida and satoh drug target insights 2007: 2 in multicellular preparations, the modulation of action potential confi guration by sinomenine was also examined (satoh, 2005). the preparations were stimulated at 1 hz. sinomenine (100 µm to 1 mm) had inhibitory effects on the action potentials, and tended to increase the action potential durations (apd); at 1 mm, by 4.1± 1.6% (n = 6) in apd50 and by 4.5 ± 1.4% (n = 6) in apd90 (but not signifi cantly). the maximum rate of depolarization (vmax) was also inhibited by 20.0 ± 2.4% (n = 6, p < 0.05) at 300 µm and by 32.1 ± 3.3% (n = 6, p < 0.05) at 1 mm of sinomenine. in high extracellular ca2+ ([ca2+]o) solution (5.4 mm) to cause cellular ca2+ overload, abnormal action potentials occurred irregularly, in spite of constant stimulation (1 hz) (satoh, 2005). application of 300 µm sinomenine suppressed and abolished the abnormal action potentials (dysrhythmias) (fig. 5). the changes in the action potential confi gurations and the modulation of the ionic channel currents on the membrane of cardiomyocyte by sinomenine are summarized in fig. 6. immunomodulative action sinomenium acutum has been used for treatment for various rheumatic diseases as a chinese traditional medicine (yamasaki, 1976; liu et al. 1996). in basic pharmacological studies of sinomenium acutum, the inhibitory actions on some enzymes relating to infl ammation have been shown (li et al. 2003). one of most famous pharmacological actions of sinomenine is an immunomodulative effect. sinomenine is also clinically used for ra treatment (yamasaki, 1976; liu et al. 1996). a lot of reports concerning about not only an anti-rheumatic effect, but also anti-inflammatory and immunomodulative effects have already been shown. as anti-rheumatic direct effects, it has been reported that sinomenine reduces infl ammatory parameters and attenuates proliferation of figure 2. modulation by several inhibitors of the relaxation induced by sinomenine. symbols used are in the absence (open circles, n = 6), in the presence of 100 µm l-nmma (open triangles, n = 5), 10 µm indomethacin (open squares, n = 5), 0.1 µm nicardipine (closed circles, n = 5), 0.3 µm propranolol (closed triangles, n = 5), and 30 nm staurosporine (closed squares, n = 5). values (%) represent mean ± s.e.m. *: p < 0.05, **: p < 0.01, ***: p < 0.001, with respect to control value. satoh et al.indd 4 5/14/2007 8:59:10 pm 100 cardiovascular pharmacology of sinomenine drug target insights 2007: 2 synovial fibroblasts in rat adjuvant arthritis models (liu et al. 1996). the anti-infl ammatory and immunomodulative actions of sinomenine are responsible for various mechanisms via complex modulation of leukocytes and cytokine. sinomenine reduces the production of prostagrandin (pg) e2 and no from macrophage (liu et al. 1994a). also, sinomenine possesses anti-proliferative effects on lymphocytes (liu et al. 1994b), contributing to anti-infl ammatory and anti-rheumatic effects. in addition, sinomenine depressed mrna expression of tumor necrosis factor (tnf)-α and interleukin (il)-β of peritoneal macrophages (wang et al. 2005). therefore, sinomenine may act as an anti-rheumatic drug through the anti-infl ammatory effects on lymphocytes and cytokine. sinomenine inhibited bfgf-induced angiogenesis in vitro and in vivo (kok et al. 2005). sinomenine also attenuates transmigration of granulocyte. the inhibition of leukocytes migration across the vessel wall and anti-angiogeneic effect of sinomenine may also contribute to therapeutic effects for ra. immunomodulative actions have been studied as the other aspect of sinomenine concerning about the cardiac transplantation model. it has been reported that acute and chronic cardiac allograft ejections are blocked by the immunomodulatory effects of sinomenine (mark et al. 2003). conclusion endothelium-dependent and -independent relaxations we have been demonstrated that sinomenine possesses strong vasodilating actions by multiple mechanisms (nishida and satoh, 2006). the summarized mechanisms of sinomenine-induced vasorelaxation are shown in fig. 3. sinomenine possesses endothelium-dependent vasorelaxation via no and pgi2 releasing from endothelium. nos activation and pgi2 release are elicited by an increase in the intracellular ca2+ concentration ([ca2+]i) in endothelium cells (busse et al. 1998; quignard et al. 1999). the mechanisms for the endothelium-dependent relaxations are not yet unclear. however, sinomenine might increase [ca2+]i in endothelium cells and then, activates figure 3. summary of the multiple mechanisms induced by sinomenine. sinomenine produces the vasorelaxation via no and pgi2 releasings from endothelium. also, sinomenine causes the vasorelaxation via modulation of ca2+ channels and pk-c activity in smooth muscle cells. in addition, β-adrenoceptor stimulation is caused. nos: nitric oxide synthetase, pk-c: protein kinase c, pgi2 : prostaglandin i2. satoh et al.indd 5 5/14/2007 8:59:10 pm 101 nishida and satoh drug target insights 2007: 2 nos activity and pgi2 releasing, as reported previously (nishida and satoh, 2003). sinomenine causes the vasorelaxation via modulation of ca2+ channels and pk-c activity in vascular smooth muscle cells. in vascular muscle cells, the contraction systems and the ion channels are regulated through the intracellular signal conditions (satoh and sperelakis, 1991, 1995; satoh, 1996). therefore, sinomenine might modulate the contraction systems, ca2+ and na+ channels, delayed rectifi er k+ channels, and ca2+-activated k+ (kca) channel, accompanied with the activation of pk-c (nishida and satoh, 2007). also, sinomenine possesses β-adrenoceptor stimulating action to inhibit the aortic constriction. clinically possibility of cardiovascular pharmacological effects sinomenine is included in sinomenine acutum of mokuboito. mokuboito is traditionally used for dyspnea and edema (shuji et al. 2002). therefore, sinomenine may be expected as one of the therapeutic agents for heart failure. most recently, satoh (2005) has demonstrated that sinomenine effectively modulates cardiac ionic channels. sinomenine inhibits ica , and simultaneously produces the ik decerease in cardiomyocytes which results in the apd prolongation. modulation of ca2+ channel induced by sinomenine is similarly exerted in vascular smooth muscle cells. in addition, sinomenine possesses the regulatory actions for dysrhythmias under ca2+ overload conditions. it has been well known that under the ischemia and heart failure, the cellular ca2+ overload of heart muscles elicits some arrhythmias and dysfunctions (satoh, 2001; 2003). the regulation of ca2+ infl ux may modulate ca2+ overload in cardiomyocytes and produces protective actions for ca2+-overloaded myocardial cells (satoh and spererakis, 1998). therefore, sinomenine might restrain the cell damages of heart muscles via modulation of [ca2+]i, and as a result, exert a cardioprotective action. cardiopotective action of sinomenine on rat acute myocardial ischemia has also been demonstrated. reperfusion injury is induced by ligating the rat left coronary artery for 15 min and reopening. sinomenine can inhibit the incidence of b a figure 4. modulation by sinomenine on the action potentials in guinea pig ventricular cardiomyocytes. a: concentration-dependent changes in the action potential confi guration by sinomenine. short lines at the left of action potential recordings represent zero mv level. b: the percentage inhibitions by sinomenine of cardiac ionic currents. vertical bars represent mean ± sem. figure 5. antiarrhythmic actions of sinomenine in guinea pig papillary muscles. sinomenine abolishes the abnormal action potentials in high ca2+ concentration (5.4 mm). dots above the action potential recordings are represented the regular rhythms induced by 1 hz stimulation. the horizontal line indicates zero mv. satoh et al.indd 6 5/14/2007 8:59:10 pm 102 cardiovascular pharmacology of sinomenine drug target insights 2007: 2 arrhythmias and reduce intracellular ca2+ concentration (xie et al. 1993), well consistent with our results. sinomenine has multiple vasodilating mechanisms. the vasodilating agent is one of the great useful tools for heart failure and regulates preand afterloads of cardiovascular systems. therefore, sinomenine-induced vasodilating actions may improve cardiac functions via the regulation of both preand after-loads under heart failure. in summary, sinomenine caused a concentrationdependent vasorelaxation on ne-, kcland pdb-induced contractions, and sinomenineinduced vasorelaxation is attenuated by the pretreatments with l-nmma, indomethacin, staurosporine, nicardipine and propranolol. in electropharmacological mechanisms, sinomenine inhibits the ica and the ik in cardiomyocytes which results in the apd prolongation. in addition, sinomenine depressed the dysrhysmias induced by triggered activities under the ca2+ overload. finally, sinomenine also possesses the antiinfl ammatory and immunomodulative actions. in future, therefore, sinomenine as a cardioprotective drug may be expected to the respectable effectiveness for heart failure, mediated through the modulation of cardiac ion channels (including the regulation for dysrhythmias) and blood vessels. further experiments need to elucidate more in detail mechanisms of sinomenine. acknowledgements the authors wish to express thanks for the supply of mokuboito and sinomenium acutum extracts (tsumura co.). this work was in part supported by the research fund of japan kampo medicine manufactures association. references busse, r., fichter, h., luckhoff, a. et al. 1998. hyperpolarizaion and increased free calcium in acethylcholine-stimulated endothelium cells. am. j. physiol., 255:h965–9. inaki, k. 2005. diseases and kampo. in sato, y., hanawa, t., arai, m., cyong, j., fukuzawa, m., mitani, k., ogihara, y., sakiyama, t., shimada, y., toriizuka, k., ymamada, t. ed. introduction to kampo. elsevier japan. p125. kok, t.w., yue, p.y., mak, n.k. et al. 2005. the anti-angiogenic effect of sinomenine. angiogenesis, 8(1):3–12. kubota, y., tanaka, t. and umegaki, k. 2001. ginkgo biloba extract-induced relaxation of rat aorta is associated with increase in endothelial intracellular calcium level. life sci., 69:2327–36. li, r.w., david, lin, g., myers, s.p. et al. 2003. anti-inflammatory activity of chinese medical vine plants. j. ethnopharmacol. 85(1):61–7. li, y., cui, s., cheng, y. et al. 2004. application of nonaqueous capillary electrophoresis for quantitative analysis of quinolizidine alkaloids in chinese herbs. anal. chim. acta., 508:17–22. liu, l., riese, j., resch, k. et al. 1994 (a). impairment of macropharge eicosanoids and nitric oxide production by alkaloid from sinomenium acutum. arzneimittelforschung, 44:1223–26. liu, l., resch, k., and kaever, v. 1994 (b). inhibition of lymphocyte proliferation by the anti-arthritc drug of sinomenine. int. j. immunopharmacol., 16(8):685–91. liu, l., buchner, e., beitze, d. et al. 1996. amelioration of rat experimental arthritides by treatment with the alkaloid sinomenine. int. j. immunopharmacol., 18(10):529–43. liu, z.q., chan, k., zhou, h. et al. 2005. the pharmacokinetics and tissues distribution of sinomenine in rats and its protein binding ability in vitro. life sci., 77(25):3197–209. lopez-jaramillo, p., gonzalez, m.c., palmer, r.m. et al. 1990. the crucial role of physiological ca2+ concentrations in the production of endothelial nitric oxide and the control of vascular tone. br. j. pharmacol., 101(2):489–93. mark, w., schneeberger, s., seiler, r. et al. 2003. sinomenine blocks tissue remodeling in a rat model of chronic cardiac allograft rejection. transplantation, 15:940–5. nishida, s., and satoh, h. 2003. mechanisms for the vasodilations induced by ginkgo biloba extract and its main constituent, bilobalide, in rat aorta. life sci., 72:2659–67. nishida, s., and satoh, h. 2004. comparative vasodilating actions among terpenoids and fl avonoids contained in ginkgo biloba extract. clinica chimica acta., 339:129–33. nishida, s., and satoh, h. 2006. in vitro pharmacological actions of sinomenine on the smooth muscle and endothelial cell activity in rat aorta. life sci., 79:1203–06. nishida, s., and satoh, h. 2007. vascular pharmacology of mokuboito (mu-fang-yi-tang) and its constituents on the smooth muscle and the endothelium in rat aorta. ecam (in press). quignard, j.f., feleton, m., thollon, c. et al. 1999. pottasium ions and endothelium-derived hyperpolarizing factor in guinea-pig carotid and porcine coronary arteries. br. j. pharmacol., 127:27–37. figure 6. shame for the changes in cardiac ionic channels induced by sinomenine. superimposed action potentials are in control (solid line) and in sinomenine (dash line). ina: the na + channel, ica: the ca 2+ channel, ik: the delayed rectifi er k +channel, ik1: the inwardly rectifying k+channel. satoh et al.indd 7 5/14/2007 8:59:10 pm 103 nishida and satoh drug target insights 2007: 2 satoh, h. 1996. modulation of ca2+-activated k+ current by isoprenaline, carbachol, and phorbol ester in cultured (and fresh) rat aortic vascular smooth muscle cells. gen. pharmacol., 27:319–24. satoh, h. 2001. [ca2+]idependent actions of taurine in spontaneously beating rabbit sino-atrial nodal cells. eur. j. pharmacol., 424:19–25. satoh, h. 2003. effects of ginkgo biloba extract and bilobalide, a main constituent, on the ionic currents in guinea pig ventricular cardiomyocytes. arzneimittelforschung, 53:407–13. satoh, h. 2005. electropharmacology of sinomeni caulis et rhizome and its constituents in cardiomyocytes. am. j. chin. med., 33:967–79. satoh, h., and sperelakis, n. 1991. calcium and potassium currents in cultured rat aortic vascular smooth muscle cell lines. in sperelakis n ed. ion channels of vascular smooth muscle cells and endothelial cells. academic press, new york: p55–63. satoh, h. and sperelakis, n. 1995. modulation of l-type ca2+ current by isoprenaline, carbachol and phorbol ester in cultured rat aortic vascular smooth muscle (a7r5) cells. gen. pharmacol., 26: 369–79. satoh, h. and sperelakis, n. 1998. review of some actions of taurine on ion channels of cardiac muscle cells and others. gen. pharmacol., 30:451–63. shuji, y., kinoshita, y., arakawa, y. et al. 2002. clinical evaluation of moku-boi-to(mu-fang-yi-tang): a japanese and chinese traditional medicine for heart failure. j. trad. med., 19:159–63. wang, y., fang, y., huang, w. et al. 2005. effect of sinomenine on cytokine expression of macrophages and synoviocytes in adjuvant arthritis rats. j. ethopharmacol., 98:37–43. yamasaki, h. 1976. pharmacology of sinomenine, an anti-rheumatic alkaloids from sinomenine acutum. acta med okayama, 30:1–20. xie, s.x., and jin, q.q. 1993. prevention of sinomenine on isolated rat myocardial reperfusion injury. zhongguo yao li xue bao, suppl: s12–5. yan, x.h., li, h.d., peng, w.x. et al. 1997. determination of sinomenine hcl in serum and urine by hplc and its pharmacokinetics in normal volunteers. yao xue xue bao, 32(8):620–4. zhao, z.z., liang, z.t., zhou, h. et al. 2005. quantifi cation of sinomenine in caulis sinomenii collected from different growing regions and wholesale herbal markets by a modifi ed hplc method. biol. pharm. bull., 28(1):105–9. satoh et al.indd 8 5/14/2007 8:59:11 pm 104 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 1048576 /lockdistillerparams false /maxsubsetpct 100 /optimize true /opm 1 /parsedsccomments true /parsedsccommentsfordocinfo true /preservecopypage true 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setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice untitled drug target insights 2007:2 221–228 221 original research correspondence: copyright in this article, its metadata, and any supplementary data is held by its author or authors. it is published under the creative commons attribution by licence. for further information go to: http://creativecommons.org/licenses/by/3.0/. effect of live salmonella ty21a in dextran sulfate sodium-induced colitis gunnar nysœter1, kari erichsen2, anne marita milde3, eva colás4, einar kristoffersen5 and arnold berstad6 1department of medicine, section for gastroenterology, 2children’s clinic, 3department of biological and medical psychology, 4innovest,5section for microbiology and immunology, haukeland university hospital, bergen, norway, 6institute of medicine, university of bergen, norway. abstract background: intestinal microbiota seems to play an essential role in the development of infl ammatory bowel diseases (ibd). we hypothesised that an oral vaccine based on live salmonella typhi would be well tolerated and could even attenuate dextran sulfate sodium (dss) induced colitis in rats, an animal model of ibd. methods: nine male wistar rats was used for an initial tolerance study, in which we used 3 dose-levels of salmonella ty21a, 0.5 × 109, 1 × 109, and 2 × 109cfu, each dose being tested in 3 rats. four treatment groups consisting of 8 male wistar rats per group: 1) control group given standard food and water, 2) control group given four daily administrations of salmonella ty21a 1 × 109 cfu, 3) water with 5% dss the last 7 days, 4) four daily administrations of salmonella ty21a before water with 5% dss the last 7 days. the salmonella ty21a was administered by gastric gavage on day 1, 3, 5 and 16, while dss was given with the drinking water from day 15 to 22. the animals were sacrifi ced and colonic tissue removed for analysis 22 days after gavage of the fi rst vaccine dose. results: the animals in the tolerance study got no signs of disease. in the treatment study, all animals receiving dss had histologic indications of colitis, particularly in the distal part of the colon. administration of salmonella ty21a had no signifi cant effect on crypt and infl ammation scores (p � 0.05). conclusion: gastric administration of live vaccine strain salmonella ty21a was well tolerated, but did not provide any signifi cant protection against development of dss induced colitis in rats. keywords: salmonella ty21a, colitis, rats, infl ammatory bowel disease introduction about 0.2% of the population in scandinavia suffer from ulcerative colitis or crohn’s disease, collectively called chronic infl ammatory bowel disease (ibd) (fonager, sorensen and olsen, 1997; lapidus 2006; moum et al. 1996). but while the prevalence and cost of the disease is increasing, to fi nd the aetiology and causal treatment remain a huge challenge. traditionally, ibd is considered an autoimmune disease, and treatment with immunosuppressive drugs has had a prominent position. more recently, ibd has been ascribed to an inadequate mucosal immune response to the intestinal microbiota in genetically susceptible individuals. consequently it might be better to stimulate the immune defence of the intestine rather than suppressing a secondary infl ammatory response. there is in fact mounting evidence that immune stimulation of the intestinal epithelium is one way to treat infl ammatory conditions in the gut. thus, a study with granulocyte—macrophage colony-stimulating factor (gm-csf), a myeloid growth factor, has shown promising results in crohn’s disease (korzenik et al. 2005). several animal studies on experimental colitis have shown a protective or therapeutic effect from probiotics or vaccines (boirivant et al. 2001; fujiwara et al. 2003; madsen et al. 2001; ohman 2005; osman et al. 2004). a similar effect on humans with ibd has presently not been shown, except possibly for probiotics (bibiloni et al. 2005; kanauchi et al. 2003). the vaccine strain salmonella ty21a is of interest in this connection. containing live bacilli it may theoretically combine the effects of probiotics and vaccines used in other studies. extensive clinical use has proved that the vaccine is http://creativecommons.org/licenses/by/3.0/ http://creativecommons.org/licenses/by/3.0/ 222 nysœter et al drug target insights 2007:2 remarkably well tolerated, even by ibd-patients with moderate disease activity (engels et al. 1998; sands et al. 2004). one may speculate whether salmonella infections have some relation to ibd, considering the inverse prevalence of the two diseases. we even have reports from a few of our ibd-patients that the course of their ibd has become milder after they have been vaccinated against typhoid fever with this live vaccine. this has been a surprising observation as they were vaccinated before travelling to endemic areas and did not anticipate any effect on their chronic disease. results from animal studies have limited value for the understanding of ibd. but as a fi rst step we wanted to see if experimental colitis could be infl uenced by live salmonella vaccine in a similar way that was previously found with probiotics and some other vaccines. we therefore studied whether administration of the live vaccine strain, salmonella ty21a would influence the induction of colitis by dextran sulphate sodium (dss) in rats, a well-known model of ulcerative colitis (kim and berstad, 1992). materials and methods animals and husbandry forty-one male wistar rats (taconics europe, skensved, denmark), 6 weeks old with mean weight of 147 g (95% ci 143.5–149.9), were housed individually in makrolon iii cages in an open system. they were kept under standard laboratory conditions with a temperature of 21 ± 1 °c, dark/light cycles of 12/12 hours, relative humidity of 55% ± 5%, and 20 air changes per hour. access to food, sds rm1 (e) (scanbur bk as, nittedal, norway) was ad libitum. tap water was given ad libitum if not otherwise stated. the norwegian animal research authority approved the protocol. induction of colitis acute colitis was induced by 50 g/l of dss (mw 44000; tdb consultancy ab, uppsala, sweden) given in distilled drinking water for 7 days. test substance salmonella serovar ty21a (vivotif®; berna, como italy), 2 × 109 colony forming units (cfu) freeze dried live bacilli per capsule. the content of one capsule was dissolved in 2 ml nacl 0.9% immediately before use. antibody response serum salmonella antibodies were measured semiquantitatively by bacteria agglutination using the widal reaction with o-antigens (sifin gmbh, berlin, germany). experimental protocol after 7 days of acclimatisation, 9 animals were divided at random into 3 groups of 3 animals for the dose-tolerance study, while the remaining 32 animals waited for the colitis study starting one week later. dose-tolerance study the 9 animals were given ty21a; one group with 0.5 × 109, one with 1 × 109, and one with 2 × 109 cfu. each dose was given three times at two days interval. six days after the fi rst dose the animals were sacrifi ced and blood culture was taken. we registered general condition, consistency and blood content of the stool daily. treatment study the animals were divided into the following four groups of 8 rats in each group: (1) control. (2) ty21a alone. (3) dss alone. (4) dss + ty21a. rats in the groups 2 and 4 got 1 × 109 cfu salmonella ty21a in 1 ml saline 0.9% through a metallic oral tube on day 1, 3, 5 and 16. in group 3 and 4 drinking water was replaced by dss 5% in distilled water from day 15 to 21. group 1 remained on normal food and drinking water during the whole test period. the rats were weighed at start of treatment with ty21a (day 1), after 7 and 14 days, and then daily until sacrifi ce (day 22). on day 22 the animals were anaesthetised by subcutaneous injection of a combination of phenantyl citrate + fl uanisone (hypnorm; jansen pharmaceutica, beerse, belgium) and midazolam (dormicum; roche, oslo, norway). (phenantyl citrate 0.079 mg/ml, fl uanisone 2.5 mg/ml, midazolam 1.25 mg/ml given at dose 0.2 ml/100g.) thoracotomy, cardiac puncture and exsanguination were performed. the colon was taken out. 223 effect of salmonella ty21a on experimental colitis drug target insights 2007:2 sample collection and analyses stool was observed daily from day 15 to 21 for the presence of diarrhoea or blood. we used a guyac method (haemofec. med-kjemi a/s, asker, norway). granulocyte marker protein (gmp), the rat equivalent to human calprotectin, was measured in stool (kristinson et al. 2002; milde et al. 2003). samples for analysis were collected on day 1, 7 and 21. they were stored at –20 °c until analysis for gmp. on exsanguination blood was drawn for blood culture and for analysis of salmonella antibodies. tissue preparation and histologic examination colon from the colocecal junction to the anal verge was removed. the length of the colon was recorded. the colon was rinsed with phosphate-buffered saline, opened longitudinally, and divided into one proximal and one distal segment, which were fi xed in 10% formalin and embedded in paraffi n. eight pieces per segment were stained with hematoxylin and eosin and specimens coded and randomised before microscopic examination. crypt and infl ammatory scores were determined according to a validated scoring system (carrier et al. 2001). crypt injury was scored as follows: grade 0, intact crypts; grade 1, loss of the bottom third of crypts; grade 2, loss of the bottom two thirds of crypts; grade 3, loss of entire crypt with the surface epithelium remaining intact; grade 4, loss of entire crypt and surface epithelium. the severity of infl ammation was scored as follows: grade 0, normal; grade 1, focal infl ammatory cell infi ltration; grade 2, infl ammatory cell infi ltration, gland drop out, and crypt abscess. both scores include a measure of involvement as follows: grade1, 1% to 25%; grade 2, 26% to 50%; grade 3, 51% to 75%; grade 4, 76% to 100%. the score was the product of either the crypt or infl ammation grade by the involvement grade. statistical analysis data were analysed using the graphpad prism version 4 (graphpad software, san diego, calif.) statistical software package. results are presented as mean ± sem. differences between means were evaluated with 1-way anova and bonferroni posttest for selected pairs of columns. differences between means and 95% confi dence intervals (cis) are given if not otherwise stated. p values less than 0.05 were considered statistically signifi cant. results tolerance study all the animals were well during the test period. none got diarrhoea or bloody stools. blood cultures were all negative. treatment study all the animals appeared to be at good condition until sacrifi ce. dss intake total intake of dss-containing water was similar in the dss alone (161.0 ± 8.1ml) and the dss + ty21a groups (168 ± 8.4 ml; p = 0.45). there was no signifi cant difference of drinking volume in the four groups. weight change all rats gained weight without signifi cant differences between groups until dss treatment was started on day 15. during the following period until day 22 both groups on dss had signifi cantly less weight gain than the animals in the control or ty21a-groups. (fig. 1) there was no signifi cant difference in weight change between the dss group (4.75 ± 2.15) and the dss + ty21a group (−0.75 ± 2.82; p = 0.25). stool changes none of the animals got diarrhoea. blood in stools was found in six animals in both the dss group and those on dss + ty21a, four with occult blood and two with traces of visible blood in each group. colon length colon length did not differ signifi cantly between the groups, though there was a tendency to shorter colon in the groups that got dss (14.13 ± 0.42 cm) or dss + ty21a (13.69 ± 0.49 cm; p = 0.35). macroscopic lesions of the gut intramucosal bleeding or erosions was observed in the colon of a few animals both in the dss group (4 animals) and the dss + ty21a group (5 animals), 224 nysœter et al drug target insights 2007:2 while in the other groups, the colons were macroscopically normal. granulocyte marker protein compared with controls, dss-induced colitis signifi cantly increased faecal gmp by 100 mg/l (95% ci 118-81; p � 0.0001). there was no signifi cant difference of gmp-levels in animals on dss (105 ± 8) or dss + ty21a (108 ± 8; p = 0.5). (fig. 2). salmonella antibodies salmonella antibodies were found in 7 of the 8 animals that got ty21a and in all of the eight animals that got dss + ty21a. the other animals had no salmonella antibodies. there was no correlation between antibody levels and tissue damage. blood culture there was growth of gram positive cocci, not salmonella, in one sample from an animal in the control group, no growth in culture from the others. as no rats showed signs of septicaemia, the positive blood culture most likely refl ects contamination upon blood sampling. histology histologic fi ndings were normal or close to normal in all animals in the control group and the ty21a group, while all animals on dss, with or without the addition of ty21a developed inflammatory changes in the colon (fig. 3). the mean value of crypt score was slightly less for the dss + ty21a group compared to the dss group (fig. 4), but the difference was not statistically signifi cant (95% ci of diff. −0.63 to 1.55; p � 0.05). analysis of infl ammatory score showed no signifi cant differences between the two groups (95% ci of diff. –0.63 to 0.73; p � 0.05), (fig. 4). there was a signifi cant correlation between gmp values and histologic fi ndings (pearson’s r = 0.77; p � 0.0001). discussion we failed to fi nd a protective effect of salmonella ty21a in dss-induced colitis. both histological signs of colitis and gmp levels in blood indicated similar degree of infl ammation in dss and dss + ty21a treated groups. the two methods support each other as they both depend on the amount of granulocytes infi ltrating the intestinal mucosa. mucosal damage as measured by crypt score was numerically less pronounced in vaccinated animals, but the difference was not statistically signifi cant. however, neither the initial tolerance study nor the controlled study indicated any adverse effects of the treatment. although the results should be carefully extrapolated, the fi nding of no deterioration of the colitis, suggests that control ty21a dss dss + ty21a-15 -10 -5 0 5 10 15 20 25 ns w ei g h t (g ra m ) p = 0.045 figure 1. weight gain from day 14–22 (dss-period). 225 effect of salmonella ty21a on experimental colitis drug target insights 2007:2 figure 3.a b c d figure 2. salmonella ty21a does not affect the level of granulocyte marker protein (gmp) in rats. figure 3. h&e × 20. histologic normal mucosa in healthy control rat (a) and healthy ty21a rat (b). mucosa from dss-group (c) and dssty21a-group (d) with severe infl ammatory changes with crypt loss, crypt abscess and infi ltration with infl ammatory cells. 0 50 100 150 control g m p m g /l ty21a dss dss + ty21a 226 nysœter et al drug target insights 2007:2 figure 4. histologic fi ndings (crypt score and infl ammatory score) in dextran sulphate induced colitis in rats is not signifi cantly infl uenced by treatment with salmonella ty21a. control ty21a dss dss − ty21a 0 1 2 3 4 control ty21a dss dss + ty21a 0 1 2 3 p > 0.05 p > 0.05 c ry p t sc o re in fl a m m a to ry s co re 227 effect of salmonella ty21a on experimental colitis drug target insights 2007:2 vaccination with salmonella ty21a in humans with ibd is safe. this corresponds well with extensive experience in humans. current guidelines for vaccination of patients with ibd allow oral administration of salmonella ty21a provided the patient is not signifi cantly malnourished or immune compromised (sands, cuffari, katz, kugathasan, onken, vitek and orenstein, 2004). any information as to whether the vaccine infl uences the ibd itself is not available. for human use salmonella ty21a is taken in the encapsulated form to avoid destruction of the microbes by pepsin and acid in the stomach. our rats got unprotected vaccine by gavage through a metallic sonde, assuming that a suffi cient proportion of the high dose of live microbes (half that given to an adult person) would reach the gut. the three fi rst doses of vaccine were given on day 1, 3 and 5 as is the recommended schedule for human use. the following two weeks interval enabled the animals to face the dss-challenge from day 15 with an immune system triggered by ty21a. all the vaccinated rats, except one, produced salmonella antibodies, thus proving the vaccine’s contact with the immune system. to achieve a possible probiotic effect, a higher vaccine dose could be necessary. but instead we chose to give a fourth dose the day after dss-treatment was started (day 16), in order to have live salmonella present in the gut during the colitis period. the choice of timing for vaccination as well as duration of the colitis period may be essential for the effect (di giacinto et al. 2005; jun et al. 2005). to have a better parallel to a clinical situation, we might have induced a chronic colitis fi rst, and then introduced the vaccine to study its therapeutic effect. all animals receiving dss got colitis, particularly in the distal part of the colon, but the infl ammation was remarkably weak compared with what we observed in a recent study performed by us (erichsen et al. 2005; milde and murison, 2002). the relevance of this fact, is not known. a vaccine containing live bacilli could theoretically have the advantages of both probiotic and vaccine components. accordingly we gave the vaccine both two weeks before and during the induction of colitis. as assumed, the adaptive immune system had started production of salmonella antibodies after two weeks. others have found that in humans, oral vaccination with live salmonella ty21a stimulated, during the fi rst few weeks, large amounts of ifn-γ but no il-10 or tgf-β (lundin, johansson, and svennerholm, 2002), which theoretically could be an unfavourable effect of salmonella ty21a in ibd. however, the activity of such pro-infl ammatory th1 cytokines may be subdued by subsequent induction of cd4+cd25+ regulatory t lymphocytes producing both il-10 and tgfβ with a healing effect on colitis (holmen et al. 2006; uhlig et al. 2006). in case of the latter, a study with a longer observation period would be of interest. a live vaccine against enterotoxigenic eschericia coli (etec) suppressed the proinfl ammatory response (jun, gilmore, callis, rynda, haddad and pascual, 2005) and reduced the production of tnf-α, il-1 and il-6, while it increased the production of il-4, il-10, and il-13. interestingly, toxins associated with another tropic diarrhoeal disease, cholera, has an anti-infl ammatory effect in experimental tnbs-colitis. oral administration of recombinant cholera toxin subunit b has been shown to inhibit murine tnbsinduced experimental colitis (boirivant, fuss, ferroni, de pascale and strober, 2001) and promote a th2 response with tr1 cells, enhanced il-10 production and inhibition of il-12 and tnfα secretion (lavelle et al. 2004). even vaccines unrelated to intestinal disease may alleviate experimental colitis. thus, a three-component bordetella pertussis vaccine attenuated colitis in gαi2defi cient mice (ohman, 2005). contrary to the adaptive immunity, the innate immune system would respond immediately (fellermann et al. 2003). the innate immune system is supposed to play a key role in the protection against ibd through production of defensins and other protective mechanisms (wehkamp et al. 2003; wehkamp, 2005; wehkamp, schauber and stange, 2007). however, an important pathogenetic trait of salmonella is to down-regulate the production of defensins, enabling bacterial invasion of the gut mucosa (salzman et al. 2003). experiments on pigs have shown that salmonella enterica serovar typhimurium infection might up-regulate cathelicidine genes and stimulate the production of another family of antimicrobial peptides prominent in the host defence mechanisms of several mammalian species (wu et al. 2000). if immunisation with the non-pathogenous salmonella ty21a has the same effect, it would contribute to protection of the host. treating chronic colitis with a vaccine that is well known, very well tolerated and cheap is an attractive idea. several animal experiments support 228 nysœter et al drug target insights 2007:2 the idea. unfortunately our results do not. but further studies are warranted before we exclude a positive effect of ty21a in this context. references bibiloni, r., fedorak, r.n., tannock, g.w., madsen, k.l., gionchetti, p., campieri, m., de simone, c. and sartor, r.b. 2005. vsl#3 probiotic-mixture induces remission in patients with active ulcerative colitis, am. j. gastroenterol., 100(7):1539–46. boirivant, m., fuss, i.j., ferroni, l., de pascale, m. and strober, w. 2001. oral administration of recombinant cholera toxin subunit b inhibits il-12-mediated murine experimental (trinitrobenzene sulfonic acid) colitis, j. immunol., 166(5):3522–32. carrier, j., aghdassi, e., platt, i., cullen, j. and allard, j.p. 2001. effect of oral iron supplementation on oxidative stress and colonic infl ammation in rats with induced colitis, aliment. pharmacol. ther., 15(12):1989–99. di giacinto, c., marinaro, m., sanchez, m., strober, w. and boirivant, m. 2005. probiotics ameliorate recurrent th1-mediated murine colitis by inducing il-10 and il-10-dependent tgf-beta-bearing regulatory cells, j. immunol., 174(6):3237–46. engels, e.a., falagas, m.e., lau, j. and bennish, m.l. 1998. typhoid fever vaccines: a meta-analysis of studies on effi cacy and toxicity, bmj, 316(7125):110–6. erichsen, k., milde, a.m., arslan, g., helgeland, l., gudbrandsen, o.a., ulvik, r.j., berge, r.k., hausken, t. and berstad, a. 2005. low-dose oral ferrous fumarate aggravated intestinal infl ammation in rats with dss-induced colitis, infl amm. bowel. dis., 11(8):744–8. fellermann, k., wehkamp, j., herrlinger, k.r. and stange, e.f. 2003. crohn’s disease: a defensin defi ciency syndrome?, eur. j. gastroenterol. hepatol., 15(6):627–34. fonager, k., sorensen, h.t. and olsen, j. 1997. change in incidence of crohn’s disease and ulcerative colitis in denmark. a study based on the national registry of patients, 1981–1992, int. j. epidemiol., 26(5):1003–8. fujiwara, m., kaneko, t., iwana, h., taketomo, n., tsunoo, h., kanno, j., ohkusa, t. and okayasu, i. 2003. inhibitory effects of bifi dobacterium longum on experimental ulcerative colitis induced in mice by synthetic dextran sulfate sodium, digestion, 67(1–2):90–5. holmen, n., lundgren, a., lundin, s., bergin, a.m., rudin, a., sjovall, h. and ohman, l. 2006. functional cd4+cd25high regulatory t cells are enriched in the colonic mucosa of patients with active ulcerative colitis and increase with disease activity, infl amm. bowel. dis., 12(6):447–56. jun, s., gilmore, w., callis, g., rynda, a., haddad, a. and pascual, d.w. 2005. a live diarrheal vaccine imprints a th2 cell bias and acts as an anti-infl ammatory vaccine, j. immunol., 175(10):6733–40. kanauchi, o., mitsuyama, k., araki, y. and andoh, a. 2003. modifi cation of intestinal fl ora in the treatment of infl ammatory bowel disease, curr. pharm. des, 9(4):333–46. kim, h.s. and berstad, a. 1992, experimental colitis in animal models, scand. j. gastroenterol., 27(7):529–37. korzenik, j.r., dieckgraefe, b.k., valentine, j.f., hausman, d.f. and gilbert, m.j. 2005. sargramostim for active crohn’s disease, n.engl. j. med., 352(21):2193–201. kristinson, j., nygaard, k., sundseth, a., aadland, e. and fagerhol, m.k. 2002, comparison of faecal and intestinal concentrations of granulocyte marker protein and localization of gastrointestinal tumours in rats, scandinavian journal of gastroenterology, 37(9):1029–33. lapidus, a. 2006, crohn’s disease in stockholm county during 1990–2001: an epidemiological update, world j. gastroenterol., 12(1):75–81. lavelle, e.c., jarnicki, a., mcneela, e., armstrong, m.e., higgins, s.c., leavy, o. and mills, k.h. 2004, “effects of cholera toxin on innate and adaptive immunity and its application as an immunomodulatory agent”, j. leukoc. biol., 75(5):756–63. lundin, b.s., johansson, c. and svennerholm, a.m. 2002. oral immunization with a salmonella enterica serovar typhi vaccine induces specifi c circulating mucosa-homing cd4(+) and cd8(+) t cells in humans, infect. immun., 70(10):5622–7. madsen, k., cornish, a., soper, p., mckaigney, c., jijon, h., yachimec, c., doyle, j., jewell, l. and de simone, c. 2001. probiotic bacteria enhance murine and human intestinal epithelial barrier function, gastroenterology, 121(3):580–91. milde, a.m., arslan, g., roseth, a., berstad, a., overmier, j.b. and murison, r. 2003. intestinal permeability and faecal granulocyte marker protein in dextran sulphate sodium—induced colitis in rats, scandinavian journal of laboratory animal science, 30(4):169–75. milde, a.m. and murison, r. 2002. a study of the effects of restraint stress on colitis induced by dextran sulphate sodium in singly housed rats, integr. physiol behav. sci., 37(2):140–50. moum, b., vatn, m.h., ekbom, a., aadland, e., fausa, o., lygren, i., stray, n., sauar, j. and schulz, t. 1996. incidence of crohn’s disease in four counties in southeastern norway, 1990–93. a prospective population-based study. the infl ammatory bowel south-eastern norway (ibsen) study group of gastroenterologists, scand. j. gastroenterol., 31(4):355–61. ohman, l. 2005. acellular bordetella pertussis vaccine enhances mucosal interleukin-10 production, induces apoptosis of activated th1 cells and attenuates colitis in galphai2-deficient mice, clinical and experimental immunology, 141(1):37–46. osman, n., adawi, d., ahrne, s., jeppsson, b. and molin, g. 2004. modulation of the effect of dextran sulfate sodium-induced acute colitis by the administration of different probiotic strains of lactobacillus and bifi dobacterium, dig. dis. sci., 49(2):320–7. salzman, n.h., ghosh, d., huttner, k.m., paterson, y. and bevins, c.l. 2003. protection against enteric salmonellosis in transgenic mice expressing a human intestinal defensin, nature, 422(6931):522–6. sands, b.e., cuffari, c., katz, j., kugathasan, s., onken, j., vitek, c. and orenstein, w. 2004. guidelines for immunizations in patients with infl ammatory bowel disease, infl amm. bowel dis., 10(5):677–92. uhlig, h.h., coombes, j., mottet, c., izcue, a., thompson, c., fanger, a., tannapfel, a., fontenot, j.d., ramsdell, f. and powrie, f. 2006. characterization of foxp3+cd4+cd25+ and il-10secreting cd4+cd25+ t cells during cure of colitis, j. immunol., 177( 9):5852–60. wehkamp, j. 2005. reduced paneth cell alpha-defensins in ileal crohn’s disease, proceedings of the national academy of sciences of the united states of america, 102(50):18129–34. wehkamp, j., harder, j., weichenthal, m., mueller, o., herrlinger, k.r., fellermann, k., schroeder, j.m. and stange, e.f. 2003. inducible and constitutive beta-defensins are differentially expressed in crohn’s disease and ulcerative colitis, inflamm. bowel. dis., 9(4):215–23. wehkamp, j., schauber, j. and stange, e.f. 2007. defensins and cathelicidins in gastrointestinal infections, curr. opin. gastroenterol., 23(1):32–8. wu, h., zhang, g., minton, j.e., ross, c.r. and blecha, f. 2000. regulation of cathelicidin gene expression: induction by lipopolysaccharide, interleukin-6, retinoic acid, and salmonella enterica serovar typhimurium infection, infect. immun., 68(10):5552–8. zangari et al.indd drug target insights 2008:3 87–97 87 review correspondence: maurizio zangari, m.d., 30 north 1900 east, som room 5c402, salt lake city, utah, 84132. tel: 801-585-3229; fax: 801-585-3432; email: maurizio.zangari@hsc.utah.edu copyright in this article, its metadata, and any supplementary data is held by its author or authors. it is published under the creative commons attribution by licence. for further information go to: http://creativecommons.org/licenses/by/3.0/. thrombophilia maurizio zangari1, francesca elice2, guido tricot1 and louis fink3 1university of utah school of medicine, department of hematology, blood/marrow transplant and myeloma, salt lake city, ut, u.s.a. 2department of hematology, san bortolo hospital, vicenza, italy. 3nevada cancer institute, las vegas, nevada, u.s.a. thrombophilia or hypercoagulable state is a clinical condition characterized by a tendency to develop venous and (less frequently) arterial thrombosis. thrombosis is defi ned as the obstructive clot formation within a vessel. since the fi rst observation of virchow, three major pathogenic causes of thrombosis have been identifi ed: changes in the vessel wall, in the blood fl ow and in the blood composition. although all these mechanisms may contribute to thrombosis, arterial events are mainly determined by changes in the vessel wall, in particular atherosclerosis, while stasis and prothrombotic blood abnormalities play a major role in venous thrombosis. venous thrombosis is a sudden event that occurs during a shortor long-lasting period of increased risk, but clinical symptoms may sometimes be mild, leading to diagnostic diffi culties. the development of a venous thromboembolic episode (vte) is often the result of multiple risk factors, including both congenital procoagulant defects and enviromental factors such as age, male sex, obesity, exposure to “risk periods” of immobilization, trauma, cancer, pregnancy, use of exogenous hormones or chemotherapy. hereditary thrombophilia is a genetically determined increased risk of thrombosis; acquired or secondary thrombophilia is a physiologic or pathologic condition that predispose affected persons to thromboembolic diseases. hereditary thrombophilia should be suspected in persons with a family history of thrombosis, especially if the thrombotic events occurred in young patients or when trigger factors are absent or minimal. a congenital or acquired hypercoagulable state should also be suspected in the case of idiopathic recurrent vte or in thrombosis involving atypical locations, like upper extremities, visceral veins (hepatic, portal, mesenteric) or cerebral veins.1 table 1 summarizes the most frequently inherited and acquired thrombophilic conditions in a population of patients with a fi rst episode of vte. in patients with venous thrombosis before the early nineteen-ninties a biologic cause of thrombophilia was detectable in only 5% to 15% of cases and was confi ned to defi ciencies of antithrombin, protein c, and protein s. the discovery of two prothrombotic mutations prevalent in white populations, the factor v-arg506gln mutation (factor v leiden) and the prothrombin g20210a mutation has signifi cantly increased the number of patients with recognizable hereditary risk factor. factor v leiden mutationis apparently not present in african blacks, japanese or native american populations and less than 1% in chinese.24 the incidence of vte is higher in africans and lower in asian populations, however, the prevalence of hereditary or acquired thrombophilic factors in these ethnic groups is less known. hereditary thrombophilia the most common inherited defects include activated protein c resistance caused by the factor v leiden mutation, the prothrombin gene g20210a mutation and hyperhomocysteinemia. less common disorders include defi ciencies of antithrombin, protein c, protein s, plasminogen and dysfi brinogenemias. these thrombophilic defects either enhancing procoagulant reactions or inhibiting natural anticoagulant mechanisms, promote hypercoagulability. deep vein thrombosis (dvt) or pulmonary embolism are the most common manifestations of these disorders, although arterial thromboembolism can also manifest in a minority of patients. the fi rst identifi ed coagulation defects were rare but strong prothrombotic factors whereas the more recently described abnormalities usually cause thrombosis only in the presence of additional risk factors. http://creativecommons.org/licenses/by/3.0/ http://creativecommons.org/licenses/by/3.0/ 88 zangari et al drug target insights 2008:3 the family history of the patient is itself an independent thrombotic risk factor because, even when a specifi c defect has been identifi ed, carriers of thrombophilic defects that belong to a thrombophilic family have a worse clinical course. these patients are younger at onset and have a more severe phenotype compared to carriers of the same defects with a silent family history.2,3 in fact, thrombophilic families harbor synergistic genetic defects (both characterized and uncharacterized) that contribute to the thrombotic risk. defi ciencies of natural anticoagulant proteins are frequently identifi ed in patients with thrombosis, while they are observed in less than 1% of the general population. figure 1 illustrates the inhibitory activity of the natural anticoagulants (antithrombin, the protein c system) on the coagulation cascade. defi ciency of protein c, s or antithrombin increases the risk of thrombosis approximately 10-fold in heterozygotes, while homozygotes may develop purpura fulminans (with laboratory evidence of dic) shortly after birth.4 levels of natural coagulation inhibitors should be measured if indicated before beginning anticoagulant therapy or after its discontinuation, because treatment affects the tests results. antithrombin defi ciency this a rare defect inherited in an autosomal dominant fashion which prevalence is estimated to be one in 1/2000 to 1/5000 persons.5 the prevalence of antithrombin defi ciency in patients presenting with a fi rst thrombotic episode is 1%.6 antithrombin table 1. hereditary and acquired thrombophilia. for the most frequent conditions, the prevalence in patients presenting with a fi rst episode of venous thromboembolism is reported.1 hereditary thrombophilia prevalence antithrombin defi ciency 1,1% 6 protein c defi ciency 0,5–4% 78 protein s defi ciency 1,3% 80 factor v leiden mutation 12–40% 5,6 prothrombin gene g20210a mutation 6–18% 6 mthfr mutation 1,4–15% 79 factor xii defi ciency 2,3% 81,§ dysfi brinogenemias, plasminogen defi ciency unknown acquired thrombophilia elderly trauma, surgery, especially orthopedic immobilization, long distance travel obesity pregnancy and puerperium oral contraceptives and hormone replacement therapy disseminated intravascular coagulopathy (dic) malignancy chemotherapy, tamoxifen, central venous catheter heparin-induced thrombocytopenia nephrotic syndrome, congestive heart failure antiphospholipid antibody syndrome myeloproliferative disorders (polcythemia vera; essential thrombocythemia) hyperviscosity (waldenstrom’s macroglobulinemia, multiple myeloma) paroxysmal nocturnal hemoglobinuria, sickle cell anemia unknown or mixed etiology hyperhomocystinemia acquired apc (activated protein c) resistance high levels of factor viii, factor xi, factor ix high levels of tafi (thrombin-activatable fi brinolysis inhibitor) low levels of free tfpi (tissue factor pathway inhibitor) §prevalenve in the general population. 89 thrombophilia drug target insights 2008:3 (or antithrombin iii) is a plasma anti protease that belong to the serpin group inhibits thrombin by irreversibly binding in a 1:1 complex. type i defi ciency is characterized by low antithrombin antigen levels, whereas in type ii is caused by a mutation in the thrombin binding site producing a dysfunctional molecule, with normal antigen level and reduced antithrombin activity.7 there are no clinical differences between the two types. the best single screening test for this disorder is the antithrombin-heparin cofactor assay that measures factor xa inhibition. heterozygotes have 8.1 times higher probability of developing thrombosis.8 recurrent thrombotic episodes occur in 60% of patients9 and 40% exhibit pulmonary embolism.10 patients with an acute thrombotic episode should be treated with heparin, although in some patients antithrombin iii replacement may be useful.11 f va f viiia f xf xa f xa tissue factor factor viia t prothrombin fibrinogen fibrin thrombomodulin t pc s c4bp apc tm t s apc f ixa thrombin protein s protein c antithrombin intrinsic pathway he pa ran su lfa te f va f viiia t prothrombin fibrinogen fibrin t pc s apc t s apc thrombin antithrombin f vaf va f viiiaf viiia tt prothrombinprothrombin tt pcpcpc ss apcapc tt s apc ss apcapc antithrombin figure 1. inhibitory effect of natural anticoagulants on the coagulation cascade. thrombin (t), beside its procoagulant enzymatic activity of fi brinogen activation, is able to bind thrombomodulin (tm) and activate protein c to activated protein c (apc). apc binds free protein s (s), which acts as a cofactor by enhancing the activity of apc. the complex can inhibit the coagulation cascade by the inactivation of factor va and factor viiia. antithrombin associated with heparan sulfate molecules on the surface of vascular endothelium inactivates thrombin and factor xa. abbreviations: c4bp: c4b binding protein; pc: protein c; apc: activated protein c; tm: thrombomodulin; s: protein s; t: thrombin. 90 zangari et al drug target insights 2008:3 protein c and protein s defi ciencies protein c defi ciency is a common defect in the general caucasian population, where the prevalence of heterozygous protein c defi ciency in non symptomatic subjects ranges from 1/200 to 1/500 and 1/3000–1/5000 in symptomatic patients. heterozygous protein c defi ciency can be inherited in an autosomal dominant fashion or in a more severe autosomal recessive manner.5 type i (reduced enzymatic and immunological activity) and type ii (dysfunctional protein c) defi ciencies have been described.12 heterozygous protein c defi ciency produces a 7.3 fold increase risk of thrombosis.8 warfarin-induced skin necrosis has been associated with this disorder, but it is not specifi c for this condition. this syndrome develops in the fi rst few days of warfarin treatment, when protein c levels can decrease to 50% of normal level, causing an active prothrombotic state.13 rare cases of purpura fulminans have been described in newborns with less than 1% protein c activity, which resulted from homozygous or double heterozygous mutations for protein c defi ciency.14 in order to avoid skin necrosis in patients with known protein c defi ciency warfarin treatment should start at a low dose and only after full heparinization. in patients with a previous episode of skin necrosis, administration of protein c concentrates (or alternatively fresh frozen plasma) can be protective at the beginning of oral anticoagulant therapy. protein c defi ciency can be acquired in conditions like liver diseases, dic, sepsis,and in malignancies treated with l-asparaginase, methotrexate, fl uorouracil, cyclophosphamide.15 protein s defi ciency is a common thrombophilic abnormality that can originate both from a congenital genetic defect or, more often, from acquired plasma perturbations. inherited heterozygotes prevalence at fi rst thrombotic episode is between 1% and 7%. patients with protein s defi ciency have an increased risk of vte but a clear association with arterial thrombosis has not been demonstrated. heterozygotes have an 8.5 times higher risk of developing thrombosis.8 neonatal purpura fulminans or recurrent vte at a young age have been described in homozygotes or double heterozygotes.18 under normal conditions, about 60% of protein s is bound in plasma to c4b binding protein (c4bp). it is generally accepted that only the free protein s (about 40% of the total) is functionally active, consequently an increase of c4bp levels produces a reduced protein s activity, despite normal antigen levels.19 recent report never less has shown that protein s-c4b couples retain apc cofactor activity (20). total protein s levels are 15% to 30% of normal in healthy newborns, but c4bp is also reduced (20% of normal levels), with only slightly reduced functional level compared to adults. acquired defi ciency of protein s is observed during pregnancy, oral contraceptive use, during an acute thromboembolic disease, anticoagulant therapy, dic and liver diseases.20 free protein s antigen and functional activity can decrease during infl ammatory disorders, possibly due to higher levels of c4bp.21 symptomatic cases should be treated with full anticoagulation; like for protein c defi ciency, warfarin treatment should start at a low dose and only after full heparinization. factor v leiden mutation the most common inherited prothrombotic condition is due to a mutation of factor v, called factor v leiden. the mutation is at the cleavage site, where apc inactivates factor va. this single point mutation leads factor v leiden to be relatively resistant to proteolytic inactivation by apc. the slower inactivation of factor va results in its persistent presence in the blood, producing a prothrombotic state. this defect was identifi ed in 1993, when dahlbäck observed that plasma from a patient with a personal and family history of vte showed a reduced response to the addition of apc in an aptt-based test. this phenomenon called apc resistance has been associated in most cases to a single amino acid substitution (arg residue at position 506 is replaced by gln).22,23 the prevalence of this mutation in the general caucasian population is between 1% and 7%, while it is very rare in other ethnic groups.24 patients with heterozygous factor v leiden mutation have a relative risk 7-fold increased in overall risk of vte (relative risk corrected for sex and family status is 2.2),8 relative risk increases to 50–80 fold in homozygous patients.25,26 compared with defi ciency of natural anticoagulants, factor v leiden is a weaker risk factor for vte, but it is far more common, as it can be found in about 20% of patients with venous thrombosis. homozygotes are not so rare, with a prevalence of 1/500 in the general population.26 the main clinical manifestation of this defect is the increased vte development. of particular interest is the observation of a high incidence of vte in women with factor v leiden mutation taking oral contraceptives; in these cases the risk of thrombosis 91 thrombophilia drug target insights 2008:3 is 35 fold higher.27 discordant results have been obtained from different studies testing the correlation between factor v leiden and the risk of myocardial infarct or arterial thrombosis: it seems that factor v leiden can increase the risk of arterial events only in patients with an already present cardiovascular risk factor like cigarette smoking.28 the presence of factor v leiden mutation, as well as other hereditary thrombophilic factors, has been associated with a high risk of fetal loss.29 resistance to apc is caused in most cases by the factor v leiden mutation; however, an acquired state without any genetic mutation (acquired apc resistance) has been associated to pregnancy, use of estroprogestone therapy and cancer (see below). prothrombin g20210a mutation this is a quite common mutation observed almost exclusively in caucasian people. it is present in about 2% of healthy individuals and in 6% of patients with vte.30 as a consequence of this mutation, levels of prothrombin in the blood are increased and the risk of vte is 3 times higher in patients with this mutation. this defect is not a risk factor for arterial thrombosis or fetal loss.31 mthfr mutation the gene for methylenetetrahydrofl ate reductase (mthfr) plays a role in homocysteine metabolism; in particular it is essential for the methylation of homocysteine and formation of methionine. the c677t mutation is quite common and has been shown to be associated with mildly elevated homocysteine levels. as later explained, elevated homocysterine levels are associated with an increased risk of thrombosis. although this variant is common (about 10% of the general population are homozygous carriers), it produces a slight elevation of homocysteine levels and only a small number of patients with the homozygous defect shows premature vascular disease and thrombosis.32,33 a less common genetic defect in the homocysteine metabolism is the defi ciency of cystathionine-βsynthase, which causes elevated homocysteine levels in the blood and result in early death due to cv disease. factor xii defi ciency patients with factor xii defi ciency show a prolonged activated partial thromboplastin time (aptt), but they do not have a bleeding diathesis. an increased rate of vte has been observed in subjects carrying this abnormality. the thrombophilic tendency associated with severe factor xii defi ciency (�1% factor xii activity) has been attributed to reduced plasma fi brinolytic activity.34 however, different rates of vte have been reported in different studies including patients with factor xii defi ciency and a clear role of this defect for the risk of vte has not been established.35,36 dysfi brinogenemias this group of disorders is characterized by qualitative abnormalities of fi brinogen, usually inherited in an autosomal dominant fashion. some variants are associated with an increased risk of thrombosis and can be detected by a prolonged thrombin time (tt) and reptilase time and by the discrepancy between the functional and the antigenic levels of fi brinogen.1 treatment treatment of acute episodes of vte begins with heparinization to obtain a full coagulation, that can be switched to oral anticoagulant therapy with inr in range 2–3. the decision to extend therapy beyond 6–12 months after a thrombotic event must be made on an individual basis, depending on the presence of concomitant transient risk factors, location and severity of the thrombosis. the risk of vte associated with the inherited thrombophilic defect should be weighted against the hemorrhagic risk associated with a long-term anticoagulant therapy. current guidelines suggest continuing anticoagulation for individuals with antithrombin defi ciency and a previous thrombotic event, with homozygous thrombophilic defects or with two or more prothrombotic abnormalities (see also below).82 acquired thrombophilia classic risk factors for vte include cancer, surgery, prolonged immobilization, fractures, puerperium, paralysis, use of oral contraceptives, and antiphospolipid antibody; these may trigger thrombosis in people with inherited thrombophilic abnormalities. combined genetic defects as well as the combination of a genetic defect with one or more acquired risk factors and the combination of 92 zangari et al drug target insights 2008:3 two acquired risk factors result in a risk of vte that exceeds the sum of single factors effect. pregnancy, puerperium, oral contraceptives and hormone replacement therapy the risk of vte is approximately 10-fold increased during pregnancy and puerperium, leading to an overall rate of vte of about 1%.37,38 it has been estimated that 12% of the fatalities during pregnancy are attributable to pulmonary embolism;37,39 the presence of a hereditary thrombophilia represents a major risk factor in this setting. thrombosis during pregnancy and puerperium is attributable both to venous stasis (caused by the compression from the gravid uterus), to estrogen-dependent alterations of the hemostatic mechanisms like elevation of procoagulant factors (thrombin, tissue factor, fi brinogen, factor vii, ix, x, xii, xiii vwf), to the decline of the natural anticoagulant protein s and antithrombin40,41,42 and to impaired fi brinolysis.43,44 the prothrombotic effect of the estrogens produces also a 2-to 5-fold increased risk of venous and arterial thrombosis in women taking oral contraceptives.45 it is noteworthy that incidence of arterial thrombosis is signifi cant in this setting. this risk decreased slightly after the reduction of the estrogens dose content in contraceptives (from fi rst to second generation pills), but further dose reduction in the latest contraceptives preparation did not produce any additional benefi t on the thrombotic risk. many studies have observed that third-generation contraceptives containing the progestogens desogestrel or gestodene carry a higher thrombotic risk compared to second generation pills containing levonorgestrel. this difference is not due to different estrogens content but presumably by the less compensated effect of desogestrel compared to levonorgestrel. hormone replacement therapy, which often consists in a combination of conjugated estrogens with medroxyprogesterone, is associated with a 2to 4-fold higher risk of venous and arterial thrombosis.46,47 for either oral contraceptives or hormone replacement therapy, the risk of thrombosis is highest shortly after the beginning of therapy. acquired factors like obesity, age, and the coexistence of hereditary thrombophilic disorder further increase the thrombotic risk. in particular, antithrombin, protein s or c defi ciency and factor v leiden greatly enhance this risk: women with factor v leiden have a 15to 30-fold thrombotic risk while taking oral contraceptives.48,27 cancer after the fi rst report of an association between malignacies and thrombosis, many large studies have confi rmed the higher risk of thromboembolic events in the cancer population. the rates of vte in cancer patients have a wide variability in different trials. in women with breast cancer, the vte rate ranges from 0.1% in untreated stage i patients to 17% in chemotherapy treated women for advanced stages. the mega study accrued 3220 unselected patients with vte and 2131 controls; the presence of a malignancy increased the thrombotic risk 4.3 fold.49 in patients with cancer vte represents an important case of morbidity and mortality. it has been estimated that mortality in one of every 7 hospitalized cancer patients is associated to pulmonary embolism. according to “medicare provider analysis and review record”, the rate of initial or recurrent thromboembolism in patients with cancer greatly exceeds the cardiovascular complications recorded in those without malignancy, and occurs with similar frequency among cancers of virtually all body systems. the most common co-morbidities which produce a higher risk of vte in cancer patients include immobilization, surgery, chemotherapy with or without adjuvant hormone therapy, and the insertion of central venous catheters. the relationship between cancer and venous thromboembolism is further emphasized by the high rate of cancer development in patients with unprovoked venous thrombosis.50 multiple studies have consistently shown 4–5 times higher risk in patients with idiopathic rather than in subjects with secondary thrombosis. three largescale prevention studies involving over 5500 medically ill patients have shown that 11%–15% will have vte and 4%–5% will have proximal-vein thrombosis as identifi ed by screening studies in the absence of prophylaxis. carriers of the factor v leiden mutation who developed cancer had a 12fold higher dvt risk compared to individuals without malignancy and factor v leiden mutation; similar results were observed in carriers of prothrombin gene 20210a variant. serine proteases such as thrombin and tf/viia operate not only in promoting clot formation but 93 thrombophilia drug target insights 2008:3 function as signaling factors modulating cellular behavior.51,52 serine proteases communicate with cells through a family of protease activated receptors (par1, par2, par3, par4). thrombin can activate par 1, 3 and 4 while either the tf/fviia or the more effective tf/viia/xa complex activates par 2. par is expressed primarily by cells in the vasculature, but also by tumor cells with high metastatic potential. thrombin and the tf/ fviia or tf/fviia/xa complex also initiate signal transduction activating a number of pathways that shapes the microenvironment of the tumor. tf has been found a wide variety of tumor cells.53,54 increasing expression of tf correlates with advanced stages of disease and poorer survival rate.55,56 the fi brinolytic system functions either within the vascular space or in the tissue compartment. plasminogen plays a critical role in the extravascular space serving as the key mediator of extracellular proteolysis a process that is essential for cell migration. plasmin-mediated degradation of extracellular matrix enables malignant cells to invade surrounding tissue and also facilitates a tumor’s ability to metastasize. angiogenesis is also dependent on the tissue plasminogen system.57 different model systems have now provide evidence that oncogene activation or tumor suppressor gene inactivation upregulate clotting pathways in vivo. targeting activated human met oncogene to mouse liver with a lentivrial vector and liver-specifi c promoter has recently been described as a model for human liver carcinoma.58 progressive hepatocarcinogenesis was preceded and accompanied by a thrombohemorrhagic state, which was indistinguishable from trousseau’s syndrome with disseminated intravascular coagulation (dic).57 the contribution of platelet activation to tumor dissemination has been recently elucidated; palumbo and colleagues have been studied mice lacking gαq, a g protein critical for platelet activation. loss of platelet activation resulted in a profound decrease in both experimental and spontaneous metastases after injection of either lewis lung carcinoma cells or b16 melanoma cells. radiolabeled tumor cells distribution demonstrated that diminished platelet function and decreased fi brinogen, signifi cantly improved the survival of circulation tumor cells in the pulmonary vasculature. the prometastatic effect conferred by either platelets or fi brinogen was linked to a reduction in natural killer cell function.59 medically ill patients the frequency of dvt in medically ill patients, in the absence of prophylaxis, varies from 10% to 26%. about 10% of deaths that occur in hospitals are associated to pulmonary embolism and 75% of fatal pulmonary emboli develop in medical population. numerous risk factors for vte have been identifi ed. these clinical risk factors include increasing age, heart and respiratory failure, prolonged immobility, stroke or paralysis, previous vte, cancer chemotherapy and, acute infection, dehydration, hormonal treatment, varicose veins; incidence of dvt has been also noted to rise in association with acute infl ammatory bowel disease, rheumatologic disease, and nephrotic syndrome. patient carriers of proximalvein thrombosis have an unexpectedly high risk of in-hospital death. risks factors for vte in medically ill patients have a cumulative effect; hospitalized subjects with thrombophilia or a history of thrombosis are at increased risk of vte, as well as patients with lower limb paralysis from acute ischaemic stroke. acquired activated protein c (apc) resistance abnormally increased resistance to apc has been observed in patients not carrying factor v leiden mutation; this phenomenon has been defi ned as acquired apc resistance. in a large cohort of 15,109 unselected subjects, 2.3% showed an apc resistance in the absence of factor v mutation.60 the presence of an apc resistance increases the thrombotic risk, independently from presence of a genetic defect.61,62 resistance to apc has been also described with cerebrovascular diseases and preeclampsia.63,64 many physiologic and pathologic conditions have been associated with the presence of acquired apc resistance:65 pregnancy, oral contraceptives and hormone replacement therapy, lupus anticoagulant syndrome, and neoplasia. several authors have recently described the presence of apc resistance in patients with malignancies; in addition, multiple reports indicated an association between low apc levels and increased thrombotic risk in cancer.66,67,68 testing cancer patients for baseline apc resistance seems to be an appealing screening method to identify hypercoagulable subjects with impaired natural anticoagulant system. however, the initiation of an anticoagulant therapy or 94 zangari et al drug target insights 2008:3 prophylaxis based only on the presence of apc resistance is not fully justifi ed by current data. antiphospholipid antibodies antiphospholipid antibodies are a heterogenous group of autoantibodies directed against anionic phospholipids. there are two classes of antiphospholipid antibodies: anticardiolipin antibodies and lupus anticoagulants. anticardiolipin antibodies, which can be igg or, less often, igm, may be directed against β2-glycoprotein 1 and quantifi ed by elisa that uses cardiolipin as the antigen.69 high-titer igg anticardiolipin antibodies are most strongly associated with clinical manifestations. lupus anticoagulants are very common in normal children and are frequently identifi ed prior to scheduled tonsillectomy/adenoidectomy or the basis of a prolonged aptt. in this setting, they are not a signifi cant risk factor for thrombosis. lupus anticoagulants induce a dose dependent prolongation in phospholipiddependent clotting assays such as the aptt using a sensitive reagent, the dilute pt, russell viper venom time or kaolin clotting time. the presence of such antibodies can be indicated by the failure of aptt to correct when normal plasma is added in mixing studies. the mechanism by which antiphospholipid antibodies cause thrombosis is unclear. there is evidence that the antibodies interfere with the protein c pathway by impairing both protein c activation and the function of apc. endothelial cell dysfunction with reduced prostacyclin synthesis and antibody-induce platelet activation have also been described. the antiphospholipid antibody syndrome is defi ned by thrombosis or pregnancy morbidity in association with a persistent elevation (�12 weeks) of lupus anticoagulant, anticardiolipin, or antiβ2 glycoprotein i antibodies.69 clinical manifestations are venous or arterial thrombosis, recurrent fetal loss, and livedo reticularis. the clinical signifi cance of transient antiphospholipid antibodies is unclear and testing should be repeated at 6–12 weeks. although antiphospholipid antibody syndrome can be idiopathic, it is frequently associated to systemic lupus erythematosus, or cancer (such as lymphoma) or infections (pneumocystis carinii pneumonia, in hiv patients), and in association with drugs such as hydralazine or procainamide. all patients �65 years of age who present with transient ischemic attacks or ischemic stroke should be screened for antiphospholipid antibodies. management of thrombotic defects asymptomatic patients with hereditary thrombophilia identifi ed through family studies should not receive long-term oral anticoagulation. they should, however, receive counselling regarding their diagnosis and need for prophylaxis during high-risk periods.70 in patients who have a fi rst venous thrombotic even in the setting of a transient triggering factor, anticoagulation can be discontinued after 3 to 6 months after removal of the triggering factor. patients with idiopathic thromboembolism without triggering factors are generally treated for 6 months. extended anticoagulation should be considered for single unprovoked venous thrombotic events in the presence of more than one allelic abnormality (for example, homozygous factor v leiden and combined heterozygosity for factor v leiden and prothrombin g20210a mutation), and initial life-threatening thrombosis (such as massive pulmonary embolism or cerebral, mesenteric, portal, or hepatic venous thrombosis), after second unprovoked thrombotic episode. in the setting of acute thrombosis, the presence of factor v leiden of prothrombin g20210a does not alter the initial anticoagulation regimen. patients with a diagnosis of one of the less common thrombophilias (defi ciencies of antithrombin, protein c, or protein s) are generally initially treated as patients without one of these defects. treatment therapy for patients with deep venous thrombosis and pulmonary embolism typically includes administration of unfractionated or low-molecular-weigh heparin in therapeutic doses, followed by anticoagulation with warfarin at an inr between 2 and 3 for 3 to 6 months. after cessation of anticoagulant therapy for patients with a fi rst episode of symptomatic venous thromboembolism the cumulative incidence of recurrent venous thrombosis is 5% to 15% at 1 year and approximately 25% at 5 years. recurrences are much less frequent when the initial event was associated with surgery or trauma. it is unclear whether risk of recurrence is higher among patients with a fi rst episode of venous thromboembolism associated with the factor v leiden or prothrombin g20210a mutations than in those without a prothrombotic mutation.71 a statistically signifi cant higher incidence of recurrence has been reported 95 thrombophilia drug target insights 2008:3 in a subset of patients who are heterozygous for both mutations.72 in patients with unprovoked thromboembolism, the risk of recurrent thrombosis in the presence of antithrombin, protein c, and protein s defi ciencies is not known. it is common practice that patients with heterozygous antithrombin deficiency receive anticoagulation for an indefi nite period of time because they appear more prone to thrombosis than patients with other single heritable abnormalities. in the setting of arterial thrombosis, most studies indicate that the presence of hereditary thrombophilias does not constitute a risk factor. it is not recommended to investigate for the hereditary thrombophilias in patients who have isolated arterial thrombosis, in the presence of other independent cardiovascular risk factors (hypertension or diabetes mellitus or if they smoke or have hyperlipidemia). most patients with an antiphospholipid antibody are adequately treated with warfarin administered to achieve an inr of 2.0–3.0. the addition of aspirin to warfarin for those patients with arterial thrombosis is reasonable. patients with recurrent thrombosis despite “usual intensity warfarin” therapy can be treated with heparin or low molecular weight heparin (lmwh) administered subcutaneously in therapeutic doses. higher doses of warfarin (target inr of 3.0–4.0 in combination with aspirin) might also be considered in such patients. because of the high risk of recurrent thrombosis off anticoagulation, retrospective studies have suggested that patients with antiphospholipid antibody syndrome require indefi nite treatment. even if a signifi cant body of evidence73,74 suggests a survival advantage in cancer patients treated with lmwh, routine administration of anticoagulant is not recommended. primary prophylaxis for thromboembolism is recommended unfractionated heparin (ufh), or lmwh in cancer patients who are to undergo surgery; such patients have a post operative thromboembolic risk 3 times higher of non cancer individuals. all hospitalized acutely ill individuals with active cancer should receive anticoagulant prophylaxis with low dose ufh or lmwh.75,76 with the current practice in oncology being dominated by outpatient care with the more frequent use of active anticancer drugs with prothrombogenic activity, the physician should watch for signs or symptoms of vte and patients seeking immediate medical attention for symptoms such as chest pain, shortness of breath or lower extremities swelling. in patients treated with a combination of immunomodulatory drugs such as thalidomide, with chemotherapy or steroids, has now become common practice with the use of a prophylactic dose of lmwh or coumadin, especially during the early courses of cancer chemotherapy.77 acknowledgments we wish to thank ashlie finlayson for excellent technical assistance. references [1] bauer, k.a. new york, 2005. hypercoagulable states. in hoffman r. (ed): hematology-basic and principle and practice, 4th ed. churchill livingstone, 2197–224. [2] lensen, r.p., bertina, r.m., de ronde, h., vandenbroucke, j.p. and rosendaal, f.r. 2000a. venous thrombotic risk in family members of unselected individuals with factor v leiden. thromb. haemost., 83:817–21. [3] lensen, r., rosendaal, f., vandenbroucke, j. and bertina, r. 2000b. factor v leiden: the venous thrombotic risk in thrombophilic families. br. j. haematol., 110:939–45. [4] koster, t., rosendaal, f.r., briët, e. et al. 1995. protein c defi ciency in a controlled series of unselected outpatients: an infrequent but clear risk factor for venous thrombosis (leiden thrombophilia study). blood, 85:2756–61. [5] tait, r.c., walker, i.d., perry, d.j. et al. 1994. prevalence of antithrombin defi ciency in the healthy population. br. j. haematol., 87:106–12. [6] van der meer, f.j., koster, t., vandenbroucke, j.p., briet, e. and rosendaal, f.r. 1997 jul. the leiden thrombophilia study (lets). thromb. haemost., 78(1):631–5. [7] lane, d.a., bayston, t., olds, r.j. et al. 1997. antithrombin mutation database: 2nd (1997) update. for the plasma coagulation inhibitors subcommittee of the scientifi c and standardization committee of the international society on thrombosis and haemostasis. throm. haemost., 77:197. [8] martinelli, i., mannucci, p.m., de stefano, v., taioli, e., rossi, v., crosti, f., paciaroni, k., leone, g. and faioni, e.m. 1998. different risks of thrombosis in four coagulation defects associated with inherited thrombophilia: a study of 150 families. blood, 92(7):2353–8. [9] thaler, e. and lechner, k. 1981. antithrombin iii defi ciency and thromboembolism. clin. haematol., 10:369. [10] broekmans, a.w. and bertina, r.m. 1985. in poller l (ed): recent advances in blood coagulation. new york, churchikk livingstone117. [11] lechner, k. and kyrle, p.a. 1995 mar. antithrombin iii concentrates are they clinically useful? thromb. haemost., 73(3):340–8. [12] reitsma, p.h., bernardi, f., doig, r.g., gandrille, s., greengard, j.s., ireland, h., krawczak, m., lind, b., long, g.l., poort, s.r. et al. 1995 may. protein c defi ciency: a database of mutations, 1995 update. on behalf of the subcommittee on plasma coagulation inhibitors of the scientifi c and standardization committee of the isth. thromb. haemost, 73(5):876–89. [13] mcgehee, w.g., klotz, t.a., epstein, d.j. and rapaport, s.i. 1984 jul. coumarin necrosis associated with hereditary protein c defi ciency. ann. intern. med., 101(1):59–60. [14] seligsohn, u., berger, a., abend, m., rubin, l., attias, d., zivelin, a. and rapaport, s.i. 1984 mar 1. homozygous protein c defi ciency manifested by massive venous thrombosis in the newborn. n. engl. j. med., 310(9):559–62. 96 zangari et al drug target insights 2008:3 [15] buchanan, g.r. and holtkamp, c.a. 1980. reduced antithrombin iii levels during l-asparaginase therapy. med. pediatr. oncol., 8:7. [16] wall, g., weiss, r.b., norton, l. et al. 1989. arterial thrombosis associated with adjuvant chemotherapy for breast carcinoma: a cancer and leukaemia group b. study. am. j. med., 87:501. [17] feffer, s.e., carmosino, l.s. and fox, r.l. 1989. acquired protein c defi ciency in patients with breast cancer receiving cyclophosphamide, methotrexate, and 5-fl uorouracil. cancer, 63:1303. [18] comp, p.c., nixon, r.r., cooper, m.r. and esmon, c.t. 1984. familial protein s defi ciency is associated with recurrent thrombosis. j. clin. invest., 74:2082–8. [19] esmon, c.t., esmon, n.l. and protein, c. 1984 apr. activation. semin. thromb. hemost., 10(2):122–30. [20] maurissen lf, thomassen mc, nicolases ga, dahlback b, rosing j, hackeng tm. 2008 mar 15. re-evaluation of the role of the protein s-c4b binding protein complex in activated protein c-catalyzed vainactivation. blood, 111(6):3043–41. [21] d’angelo, a., vigano-d’angelo, s., esmon, c.t. and comp, p.c. 1988 may. acquired defi ciencies of protein s. protein s activity during oral anticoagulation, in liver disease, and in disseminated intravascular coagulation. j. clin. invest., 81(5):1445–54. [22] rezende, s.m., simmonds, r.e. and lane, d.a. 2004 feb 15. coagulation, infl ammation, and apoptosis: different roles for protein s and the protein s-c4b binding protein complex. blood, 103(4):1192–201 allaartaronsonruys1990. [23] dahlback, b., carlsson, m. and svensson, p.j. 1993. familial thrombophilia due to a previously unrecognized mechanism characterized by poor anticoagulant response to activated protein c: prediction of a cofactor to activated protein c. proc. natl. acad. sci. u.s.a., 90(3):1004–8. [24] bertina, r.m., koeleman, b.p., koster, t., rosendaal, f.r., dirven, r.j., de ronde, h., van der velden, p.a. and reitsma, p.h. 1994. mutation in blood coagulation factor v associated with resistance to activated protein c. nature, 369(6475):64–7. [25] rees, d.c., cox, m. and clegg, j.b. 1995. world distribution of factor v leiden. lancet, 346(8983):1133–4. [26] koster, t., rosendaal, f.r., de ronde, h., briet, e., vandenbroucke, j.p. and bertina, r.m. 1993. venous thrombosis due to poor anticoagulant response to activated protein c: leiden thrombophilia study. lancet, 342(8886–8887):1503–6. [27] rosendaal, f.r., koster, t., vandenbroucke, j.p. and reitsma, p.h. 1995. high risk of thrombosis in patients homozygous for factor v leiden (activated protein c resistance). blood, 85(6):1504–8. [28] vandenbroucke, j.p., koster, t., briët, e., reitsma, p.h., bertina, r.m. and rosendaal, f.r. 1994. increased risk of venous thrombosis in oral-contraceptive users who are carriers of factor v leiden mutation. lancet, 344:1453–7. [29] rosendaal, f.r., siscovick, d.s., schwartz, s.m., beverly, r.k., psaty, b.m., longstreth, w.t. jr, raghunathan, t.e., koepsell, t.d. and reitsma, p.h. 1997 apr 15. factor v leiden (resistance to activated protein c) increases the risk of myocardial infarction in young women. blood, 89(8):2817–21. [30] preston, f.e., rosendaal, f.r., walker, i.d., briet, e., berntorp, e., conard, j., fontcuberta, j., makris, m., mariani, g., noteboom, w., pabinger, i., legnani, c., scharrer, i., schulman, s. and van der meer f.j. 1996 oct 5. increased fetal loss in women with heritable thrombophilia. lancet, 348(9032):913–6. [31] poort, s.r., rosendaal, f.r., reitsma, p.h. and bertina, r.m. 1996 nov 15. a common genetic variation in the 3’-untranslated region of the prothrombin gene is associated with elevated plasma prothrombin levels and an increase in venous thrombosis. blood, 88(10):3698–703. [32] bank, i., libourel, e.j., middeldorp, s. et al. 2004. prothrombin 20210a mutation: a mild risk factor for venous thromboembolism but not for arterial thrombotic disease and pregnancy-related complications in a family study. arch. intern. med., 164:1932–7. [33] frosst, p., blom, h.j., milos, r., goyette, p., sheppard, c.a., matthews, r.g., boers, g.j., den heijer, m., kluijtmans, l.a., van den heuvel, l.p. et al. 1995 may. a candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase. nat. genet., 10(1):111–3. [34] den heijer, m., lewington, s. and clarke, r. 2005. homocysteine, mthfr. and risk of venous thrombosis: a meta-analysis of published epidemiological studies. j. thromb. haemost, 3:292–9. [35] lodi, s., isa, l., pollini, e., bravo, a.f. and scalvini, a. 1984 jul. defective intrinsic fi brinolytic activity in a patient with severe factor xii-defi ciency and myocardial infarction. scand j. haematol, 33(1):80–2. lammle et al.,1991; factorxiirodeghiero et al., 1992) factorxii. [36] lammle, b., wuillemin, w.a., huer, i., krauskopf, m., zurcher, c., pfl ugshaupt, r. and furlan, m. 1991. thromboembolism and bleeding tendency in congenital factor xii defi ciency—a study on 74 subjects from 14 swiss families. thromb. haemost, 65:117–21. [37] rodeghiero, f., castaman, g., ruggeri, m. and tosetto, a. 1992. thrombosis in subjects with homozygous and heterozygous factor xii defi ciency. thromb. haemost, 67:590–1. [38] kierkegaard, a. 1983. incidence and diagnosis of deep vein thrombosis associated with pregnancy. acta. obstet. gynecol. scand, 62:239–43. [39] mccoll, m.d., ramsay, j.e., tait, r.c. et al. 1997. risk factors for pregnancy associated venous thromboembolism. thromb. haemost, 78:1183–8. [40] sachs, b.p., brown, d.a., driscoll, s.g., schulman, e., acker, d., ransil, b.j. and jewett, j.f. 1987 mar 12. maternal mortality in massachusetts. trends and prevention. n. engl. j. med., 316(11):667–72. [41] comp, p.c., thurnau, g.r., welsh, j. and esmon, c.t. oct 1986. functional and immunologic protein s levels are decreased during pregnancy. blood, 68:881–5. [42] rosing, j., middeldorp, s., curvers, j. et al. 1999. low-dose oral contraceptives and acquired resistance to activated protein c: a randomised cross-over study. lancet, 354:2036–40. [43] middeldorp, s., meijers, j.c.m., van den ende, a.e. et al. 2000. effects on coagulation of levonorgestreland desogestrelcontaining low dose oral contraceptives: a cross-over study. thromb. haemost, 84:4–8. [44] booth, n.a., reith, a. and bennett, b. 1988 feb 25. a plasminogen activator inhibitor (pai-2) circulates in two molecular forms during pregnancy. thromb. haemost, 59(1):77–9. [45] meijers, j.c.m., middeldorp, s., tekelenburg, w. et al. 2000. increased fi brinolytic activity during use of oral contraceptives is counteracted by an enhanced factor xi-independent down regulation of fi brinolysis: a randomized crossover study of two low-dose oral contraceptives. thromb. haemost, 84:9–14. [46] world health organization, 1996. ischaemic stroke and combined oral contraceptives: results of an international, multicentre, casecontrol study. who collaborative study of cardiovascular disease and steroid hormone contraception. lancet, 348:498–505. [47] daly, e., vessey, m.p., hawkins, m.m., carson, j.l., gough, p. and marsh, s. 1996. risk of venous thromboembolism in users of hormone replacement therapy. lancet, 348:977–80. [48] grady, d. and furberg, c. 1997. venous thromboembolic events associated with hormone replacement therapy. jama, 278:477. [49] bloemenkamp, k.w.m., rosendaal, f.r., helmerhorst, f.m., b.üller, h.r. and vandenbroucke, j.p. 1995. enhancement by factor v leiden mutation of risk of deep-vein thrombosis associated with oral contraceptives containing a third-generation progestagen. lancet, 346:1593–6. [50] blom, j.w., doggen, c.j., osanto, s. and rosendaal, f.r. 2005. malignancies, prothrombotic mutations, and the risk of venous thrombosis. jama, 293:715–22. [51] prandoni, p., lensing, a.w., buller, h.r., cogo, a., prins, m.h., cattelan, a.m., cuppini, s., noventa, f. and ten cate, j.w. 1992. deep-vein thrombosis and incidence of subsequent symptomatic cancer. n. eng j. med., 327(16):1128–33. [52] rickles, f.r., patierno, s. and fernandez, p.m. 2003. tissue factor, thrombin and cancer. chest (supplement), 124:58s–68s. 97 thrombophilia drug target insights 2008:3 [53] koizume, s., jin, m., miyagi, e. et al. 2006. activation of cancer cells migration and invasion by ectopic synthesis of coagulation factor vii. cancer res., 66:9453–60. [54] callander, n.s., varki, n. and rao, l.v.m. 1992. immunohistochemical identifi cation of tissue factor in solid tumors. cancer, 70:1194–201. [55] rong, y., post, d.e., pieper, r.o. et al. 2005. pten. and hypoxia regulate tissue factor expression and plasma coagulation by glioblastoma. cancer res., 65:1406–13. [56] fernandez, p.m., patierno, s.r. and rickles, f.r. 2004. tissue factor and fi brin in tumor angiogenesis. semin. thromb. hemost., 30:31–44. [57] fernandez, p.m. and rickles, f.r. 2002. tissue factor and angiogenesis in cancer. curr. opin. hematol., 9:401–6. [58] rak, j., yu, j.l., luyendyk, j. et al. 2006. oncogenes, trousseau syndrome, and cancer-related changes in the coagulome of mice and humans. cancer res., 66:10643–6. [59] boccaccio, c., sabatino, g., medico, e. et al. 2005. the met oncogene drives a genetic programme linking cancer to haemostasis. nature, 434:396–400. [60] palumbo, j.s., talmage, k.e., massari, j.v. et al. 2005. platelets and fi brin(ogen) increase metastatic potential by pmpeding natural killer cell-mediated elimination of tumor cells. blood, 178–85. [61] tosetto, a., missiaglia, e., gatto, e. and rodeghiero, f. 1997. the vita project: phenotypic resistance to activated protein c and fv leiden mutation in the general population. vicenza thrombophilia and atherosclerosis. thromb. haemost, 78(2):859–63. [62] de visser, m.c., rosendaal, f.r. and bertina, r.m. 1999. a reduced sensitivity for activated protein c in the absence of factor v leiden increases the risk of venous thrombosis. blood, 93:1271–6. [63] rodeghiero, f. and tosetto, a. 1999. activated protein c resistance and factor v leiden mutation are independent risk factors for venous thromboembolism. annals. intern. med., 130:643–50. [64] van der bom, j.g., bots, m.l., haverkate, f., slagboom, p.e., meijer, p., de jong, p.t., hofman, a., grobbee, d.e. et al. 1996. reduced reponse to activated protein c is associated with increased risk for cerebrovascular disease. ann. intern. med., 125:265–9. [65] clark, p., sattar, n., walker, i.d. and greer, i.a. 2001a. the glasgow outcome, apcr. and lipid (goal) pregnancy study: signifi cance of pregnancy associated activated protein c resistance. thromb. haemost, 85(1):30–5. [66] clark, p. and walker, i.d. 2001b. the phenomenon known as acquired activated protein c resistance. br. j. haematol., 115(4):767–73. [67] green, d., maliekel, k., sushko, e., akhtar, r. and soff, g.a. 1997. activated-protein-c resistance in cancer patients. haemostasis, 27(3):112–8. [68] haim, n., lanir, n., hoffman, r., haim, a., tsalik, m. and brenner, b. 2001. acquired activated protein c resistance is common in cancer patients and is associated with venous thromboembolism. am. j. med., 110(2):91–6. [69] zangari, m., saghafi far, f., anaissie, e., badros, a., desikan, r., fassas, a., mehta, p., morris, c., toor, a., whitfi eld, d., siegel, e., barlogie, b., fink, l. and tricot, g. 2002. activated protein c resistance in the absence of factor v leiden mutation is a common fi nding in multiple myeloma and is associated with an increased risk of thrombotic complications. blood coagul. fibrinolysis, 13(3):187–92. [70] miyakis, s., lockshin, m.d., atsumi, t., branch, d.w., brey, r.l., cervera, r., derksen, r.h.w.m., de groot, p.g., koike, t., meroni, p.l., reber, g., shoenfeld, y., tincani, a., vlachoyiannopoulos, p.g. and krilis, s.a. 2006. international consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (aps). j. thromb. haem., 4:295–306. [71] middeldorp, s., meinardi, j.r., koopman, m.m., van pampus, e.c., hamulyak, k., van der meer, j. et al. 2001. a prospective study of asymptomatic carriers of the faxtor v leiden mutation to determine the incidence of venous thromboembolism. ann. intern. med., 135:322–7. [72] lensing, a.w. and prins, m.h. 1999. recurrent deep vein thrombosis and two coagulation factor gene mutations: quo vadis? thromb. haemost., 82:1564–6. [73] de stefano, v., martinelli, i., mannucci, p.m., paciaroni, k., chiusolo, p., casorelli, i. et al. 1999. the risk of recurrent deep venous thromobisis among heterozygous carriers of both factor v leiden and the g20210a prothrombin mutation. n. engl. j. med., 341:801–6. [74] lee, ayy., rickles, f.r., julian, j.a. et al. 2005. randomized comparison of low molecular weight heparin and coumarin derivatives on the survival of patients with cancer and venous thromboembolism. j. clin. oncol., 23:2123–9. [75] klerk, c.p.w., smorenburg, s.m., otten, h.m. et al. 2005. the effect of low molecular weight heparin on survival in patients with advanced malignancy. j. clin. oncol., 23:2130–5. [76] geerts, wh., pineo, g.f., heit, j.a. et al. 2004. prevention of venous thromboembolism. the seventh accp consensus conference on antithrombotic and thrombolytic therapy. chest, 136:338s–400s. [77] büller, h.r., agnelli, g., hull, r.d. et al. 2004. antithrombotic therapy of r venous thromboembolic disease. the seventh accp consensus confernce on antithrombotic and thrombolytic therapy. chest, 126:401s–28s. [78] zangari, m., barlogie, b., anaissie, e., saghafi far, f., eddlemon, p., jacobson, j. et al. 2004. deep vein thrombosis in patients treated with thalidomide and chemotherapy: effects of prophylactic and therapeutic anticoagulation. br. j. haematol., 126:715–21. [79] heijboer, h., brandjes, d.p.m., b.üller, h.r. et al. 1990. defi ciencies of coagulation-inhibiting and fi brinolytic proteins in outpatients with deep venous thrombosis. n. engl. j. md., 323:1512. [80] d’angelo, a. and selhub, j. 1997. homocysteine and thrombotic disease. blood, 90:1. [81] pabinger, i., brucker, s., kyrle, p.a., schneider, b., korninger, h.c., niessner, h. and lechner, k. 1992. hereditary defi ciency of antithrombin iii, protein c and protein s: prevalence in patients with a history of venous thrombosis and criteria for rational patient screening. blood coagul. fibrinolysis, 3:547–53. [82] halbmeyer, w.m., haushofer, a., schon, r., mannhalter, c., strohmer, e., baumgarten, k. and fischer, m. 1994. the prevalence of moderate and severe fxii (hageman factor) defi ciency among the normal population: evaluation of the incidence of fxii defi ciency among 300 healthy blood donors. thromb. haemost, 71:68–72. [83] bockenstedt, p.l. 2006. management of hereditary hypercoagulable disorders. hematology am. soc. hematol. educ. program, 444–9. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 1048576 /lockdistillerparams false /maxsubsetpct 100 /optimize true /opm 1 /parsedsccomments true /parsedsccommentsfordocinfo true /preservecopypage true /preserveepsinfo 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drug target insights 2007: 2 111–117 111 review correspondence: petros perimenis, university hospital of patras, department of urology, 26500 rio patras, greece. email: petperim@upatras.gr please note that this article may not be used for commercial purposes. for further information please refer to the copyright statement at http://www.la-press.com/copyright.htm endothelial dysfunction, erectile dysfunction and phosphodiesterase 5 inhibitors. an update of the current data and future perspectives angelis konstantinopoulos, konstantinos giannitsas, spiros raptis and petros perimenis department of urology, university hospital of patras, greece. abstract: endothelial dysfunction is a pathological entity that multiply affects the health status. erectile dysfunction is being recognized as a condition that is strongly interrelated with endothelial dysfunction, being a vascular event itself. oral pharmacotherapy for erectile dysfunction has provided us with a new armamentarium on this condition. phosphodiesterase 5 inhibitors have been investigated and proved useful in clinical practice for erectile dysfunction but in addition to this, the results seem promising of a benefi cial effect on endothelial dysfunction, as well. keywords: endothelial dysfunction, erectile dysfunction, phosphodiesterase 5 inhibitors introduction the vascular endothelium consists of a monolayer of specialised, fl attened, orthogonal cells lining the inner surface of the blood vessels of any diameter, as well as spaces like the surface of the sinusoids of tissues like the corpus cavernosum of the penis. its role is regulatory of the vascular tone, coagulation, metabolism and permeability of the vessels. endothelial dysfunction results in abnormal regulation of blood pressure, response to infl ammation, impairment of the sensitive balance between the vasoconstricting and vasodilating agents and stimuli and coagulation disorders. endothelial dysfunction is strongly related to hypertension, diabetes melittus (kirby, 2005), ischaemic heart disease, congestive heart failure (chong et al. 2003), pulmonary hypertension (budhiraja et al. 2004) and atheromatosis, but also with diseases like erectile dysfunction and pathological states like lower urinary tract symptoms, benign prostate hyperplasia and bladder outlet obstruction (rosen, 2006). erectile dysfunction is largely a vascular problem, both in the macroscopic and the microscopic level. excluding hormonal disorders, vascular or neural anatomical defects, it is a process that is directly related to the functional status of the endothelium of the small resistance arteries of the penis and the penile corpus cavernosum. pathologic conditions like heart disease, high blood pressure, diabetes, atheromatosis, hypercholesterolemia, are strongly interrelated as well as related to erectile dysfunction (feldman et al. 1994). from 1998 onwards, a new class of drugs has entered the daily practice, fi rst and mainly in andrology but increasingly also in other specialties like cardiology and pulmonology. these drugs are the phosphodiesterase type 5 inhibitors (pde5is). they interfere with the availability of cyclic guanosin monophosphate (cgmp) in the vascular smooth muscle cells, a second messenger of nitric oxide (no) release from neurons and endothelial cells. erectile dysfunction the national consensus development panel of the nih has defi ned erectile dysfunction as the inability to achieve or maintain an erection suffi cient for satisfactory sexual performance (nih consensus development panel on impotence, 1993). it has been estimated to affect about 30 million men in the united states, according to epidemiological data from the past decade (feldman et al. 1994). while in 1995 it was estimated that over 152 million men were having some degree of erectile dysfunction, 112 perimenis et al drug target insights 2007: 2 epidemiological projections predict that in 2025 the impotent men will reach 322 million (ayta et al. 1999). physiology of erectile function vascular, neurologic, hormonal and psychological factors interact to result in normal erectile function. erectile function is largely a vasculogenic process, both from the macroand the micro-anatomical point of view. tumescence and rigidity of the erect penis is the result of an increased arterial infl ow, relative to the venous outfl ow of blood to and from the sinousoidal structures of the corpora cavernosa. anatomical and functional integrity of the vascular mechanism of erection (a mechanism that incorporates internal iliac artery, internal pudendal artery, penile artery, bulbourethral artery, cavernous deep penile arteries, helicine arteries within the corpora cavernosa, lateral circumflex arterial branches of the dorsal artery of the penis, corporal sinusoids, deep dorsal, superfi cial and circumfl ex veins, cavernous, corporal, emissary veins, periprostatic venous plexous and finaly internal pudendal vein) is of primary importance in the process. sympathetic and parasympathetic innervation reaches the corpus cavernosum via the cavernous nerves, a branch of the pelvic plexous. parasympathetic innervation arises from s2–s4 sacral levels, while sympathetic innervation comes from the thoracolumbar (t10–l2) region of the spinal cord. somatic innervation, bringing proprioceptive and sensory information to the central nervous system is incorporated by the pudendal nerves, which have a different anatomical route from the autonomic innervation (cavernous nerves). a baseline sympathetic tone derived by the interomediolateral gray matter thoracolumbar portion of the spinal cord keeps the penis in the fl accid state when there is no sexual stimulus. penile arterioles, sinousoidal smooth muscle and endothelial cells receive norepinephrinergic stimulation from the penile adrenergic nerve endings, which result in vasoconstriction and significant resistance in arterial blood inflow. sexual stimulation induces a parasympathetic (acetylcholine mediated) activity that reverses the vasomotor balance in favor of the vasodilatation. increased intracellular concentrations of cgmp are responsible for sinousoidal and small arteries smooth muscle relaxation, the molecular mechanism of which is structured over potassium channel-mediated decrease of intracellular calcium concentrations. the increased levels of intracellular cgmp are a consequence of the release of no both from endothelial cells and nonadrenergic, noncholinergic neurons that end in the presynaptic areas on the vascular smooth muscle. detumescence is the result of the return of the sympathetic tone on withdrawal of the sexual stimulus and the degradation of cgmp by phosphodiesterase (mainly isoenzyme 5) within the erectile tissue (figure 1). causes and risk factors of erectile dysfunction erectile dysfunction may be the result of functional or anatomical impairment of the structures that are involved in the process, in all of the previously, briefl y presented levels (from the central nervous system down to the fi nal synapses and from the arteries of the lesser pelvis down to the endothelial and smooth muscle cells of the corpus cavernosum). arteriogenic erectile dysfunction may result from trauma that disrupts arterial tree integrity at any pre-corporal level. atherosclerosis is a condition that results similar to trauma, as blood fl ow in the arterial tree that brings blood into the penis is obstructed. montorsi et al. in an editorial for european urology consider erectile dysfunction as the “tip of the iceberg” of a systemic vascular disorder although they pinpoint exceptions to the no larginine citrulline nos guanylyl cyclase relaxation cgmp tumescence and erection ca ++ & k+ channels gtp contraction detumescence and flaccidity pde5 cgmp breakdown pde5 inhibitors smooth muscle sexual stimulation figure 1. molecular mechanisms involved in erectile function. 113 endothelial and erectile dysfunction and pde5is. present and future drug target insights 2007: 2 rule like patients with myocardial infarction as the fi rst clinical presentation of coronary artery disease (montorsi et al. 2003). they consider erectile dysfunction as the clinical manifestation of a disorder involving penile circulation in the same way as angina pectoris is the clinical manifestation of a disorder involving coronary circulation. according to the “artery size hypothesis” that they introduced, at the time that signifi cant vascular obstruction is evident in the smaller size penile arteries, the same plaque burden will not have any interference with blood fl ow in bigger caliber arteries like coronal arteries, internal carotid or femoral arteries. in an animal model, it has been observed that in a population of hypercholesterolemic mice with more than 50% occlusion of the iliohypogastric arteries, almost all had erectile dysfunction. but they also noted that in mice with minimal occlusive lesions, 33% had developed erectile dysfunction. that lead to the hypothesis that there may be other factors associated with atherosclerosis and impotence, such as the possible concomitant hypercholesterolemic and atherosclerotic induced alterations in the local reactivity of corpus cavernosum smooth muscle and lacunar space endothelial cells (azadzoi and goldstein, 1992). in this way, erectile dysfunction should represent a marker of sub-clinical vascular disease early in the atherosclerotic process. according to montorsi et al. in a patient with erectile dysfunction, the chance of detecting concomitant coronary artery disease is low, whereas in a patient with clinically evident coronary artery disease, the chance of having erectile dysfunction is high. also, symptoms of erectile dysfunction should come before symptoms of coronary artery disease (montorsi et al. 2004). shared risk factors for erectile dysfunction and coronary artery disease include diabetes mellitus, hyperlipidemia, hypertension and cigarette smoking. the latter may be held responsible for venous leakage (in addition to diseases that affect the macroanatomy of the draining system of the corpora cavernosa, like peyronie’s disease), as it negatively affects the elasticity of the venous wall. in a follow-up study of 9457 patients, a strong association between erectile dysfunction and subsequent cardiovascular disease was found. this association was in the range of risk associated with current smoking or a family history of myocardial infarction (thompson et al. 2005). in a milestone study on erectile dysfunction and its physiological associations, supported by numerous references, it is noted that men on treatment for diabetes have a 3-fold probability of erectile dysfunction than non diabetic age-matched controls. also, a signifi cant correlation was found between erectile dysfunction, heart disease, hypertension and low serum hdl (feldman et al. 1994). it has also been found that hyperlipidemia, especially high hdl concertrations and total cholesterol/hdl ratio are predictors of erectile dysfunction. considering that these patients are at increased risk of developing coronary heart disease in the future, they concluded that ed is a sentinel event for coronary heart disease (roumeguere et al. 2003). neurogenic causes of erectile dysfunction may constitute neurological disorders affecting the integrity of the nervous pathways (surgery, diabetic neuropathy, spinal trauma). diabetic neuropathy is also considered to play a role in the pathophysiology of erectile dysfunction, yet another negative fi nal outcome of diabetes mellitus apart of the vascular implications. diabetics also show a high prevalence of hypogonadism, probably related to their higher body mass index, further leading to erectile dysfunction. they suffer from more severe erectile dysfunction than non diabetic individuals (corona et al. 2004), and they do not respond well to pde5i therapy (vickers and satyanarayana, 2002). vascular endothelium physiology and pathophysiology the vascular endothelium can be considered an endocrine organ in its own right (chong et al. 2003) as it plays an active role in functions like hemostasis, fi brinolysis, regulation of vascular tone and permeability and synthesis of growth factors (lip and blann, 1997). factors secreted by the endothelial cells under the infl uence of a variety of stimuli include no, endothelins, tissue factor, tissue plasminogen activator and von willebrand factor. thrombomodulin, ecto-enzymes, cell adhesion molecules (vcam, icam, selectins), binding sites for factors ix and x, human leukocyte antigen (hla) are molecules and structures on the cell membrane of the endothelial cells that play several roles in the processes of blood coagulation and anticoagulation, infi ltration and oedema and leukocyte adherence. a function largely regulated by the endothelium is vasoconstriction and vasodilatation for the regulation of the vascular tone. substances which mainly are involved in the process of 114 perimenis et al drug target insights 2007: 2 vascular smooth muscle tone regulation are no, endothelin (et) and, of specifi c interest to tumescence and erection, cgmp. many years ago (furchgott and zawadzki, 1980) the role of the endothelium in vasodilatation was demonstrated. the regulation of the vascular tone is largely a function carried out by no, a gas that some years ago was merely considered to be an atmospheric pollutant. in the endothelial cells, it is produced by two isoforms of the enzyme nitric oxide synthase (nos), endothelial nos (enos) and inducible nos (inos). no synthase, acting constitutively or in response to specifi c signals, catalyzes the formation of nitric oxide from arginine and o2. once formed, nitric oxide diffuses only locally through tissues and is highly labile with a half-life of from 2 to 30 seconds. it plays an important role in mediating many local cellular interactions. release of acetylcholine from adjacent tissues promotes infl ux of ca2+ into endothelial cells lining blood vessels. after ca2+ binds to calmodulin, the resulting complex stimulates the activity of no synthase. the nitric oxide that is formed diffuses from the endothelial cell and into neighboring smooth muscle cells where it binds to and activates soluble guanylate cyclase. the subsequent increase in cgmp then leads to muscle relaxation and dilation of the vessel. no’s pivotal role in the maintenance of vascular tone and reactivity is recognized, as it is the main determinant of basal vascular smooth muscle tone, it negates the actions of vasoconstrictors like angiotensin ii and endothelin i, inhibits platelet and white cell activation and maintains the vascular smooth muscle in a nonproliferative state (verma et al. 2003). physical activation of the endothelial cells by shear stress and pulsatile fl ow as well as no release by non adrenergic non-cholinergic neural terminals in the smooth muscle vascular bed (rand, 1992) is the basis of the vasodilatory action of no. on the other hand, a substance also produced by the same endothelial cells that produce no, endothelin, is a most potent vasoconstrictor. it is 21 aminoacid peptide produced also by other cell types like adrenal cortex cells, smooth muscle cells, renal tubular epithelial cells, glomerular mesangial cells, glial cells, macrophages, mast cells and pituitary cells. of the 4 isoforms that have been identifi ed (et 1-4), et 1 is primarily produced by endothelial cells and acts on the underlying smooth muscle cells (chong et al. 2003). et 1 is produced and directly acts on its target receptors, after stimulation by hypoxia, shear stress and ischemia (cines et al. 1998). vascular smooth muscle cells, along with other types of cells, express two subtypes of et 1 receptors, eta and et b. eta mediated actions on the vascular muscle cells include vasoconstriction and smooth muscle proliferation (newby and webb, 1996). increasing vascular smooth muscle cell tone is an action mediated by increase in the intracellular calcium ions (ca++) concentration. interestingly, this action persists long after endothelin has dissociated from the receptor, but no accelerates the restoration of intracellular calcium and shortens the duration of the vasoconstricting effect of et 1 (goligorsky et al. 1994). et 1 and catecholamines potentiate each other’s vasoconstricting actions (cines et al. 1998). etb receptors mediate vasodilatation as a consequence of no and prostacycline release by endothelial cells, but etb receptors on the vascular smooth muscle cause vasoconstriction (verhaar et al. 1998). in endothelial dysfunction, there is an imbalance between the actions of no and endothelin, due to the decrease in the bioactive concentrations of no. the vasoconstricting and smooth muscle proliferative actions of endothelin are left unopposed (lopez et al. 1990). cyclic guanosin monophosphate (cgmp) is a cyclic mononucleotide that acts as a second messenger for numerous molecular messages in the cell. synthesis of cgmp is induced by both peptide hormones and no. although cgmp was discovered more than thirty years ago, its role in as a second messenger has long been overshadowed by that of camp. synthesis of cgmp is catalyzed by two types of guanylyl cyclase: a soluble cytosolic form and a transmembrane form. soluble guanylyl cyclases are activated by no. these enzymes are heterodimers and contain a bound heme molecule that interacts with both subunits. binding of nitric oxide to the heme leads to a conformational change in the enzyme and stimulates its catalytic activity. table 1. main features and roles of the vascular endothelium. vasoconstriction vasodilatation hemostasis fibrinolysis regulation of vascular permeability smooth muscle proliferation antiproliferation synthesis of growth factors 115 endothelial and erectile dysfunction and pde5is. present and future drug target insights 2007: 2 the common mechanism that underlies endothelial dysfunction is oxidative stress. reactive oxygen species can be derived by enzymatic processes of different types. final products of superoxide anion (o2 )̄ interactions within the endothelium are hydroxyl radicals (ho) and peroxynirite (onoo )̄. they either cause cell damage through peroxidation of lipids and sulfydryl groups or regulate several classes of genes, including those controlling the formation of adhesion molecules, chemotactic substances and antioxidant enzymes. they can inhibit the endothelium-dependent vasodilator pathways of no, prostacyclin and endothelium-derived hyperpolarizing factor (edhf) and they directly inhibit soluble guanylyl cyclase. they also decrease the activity of calcium-activated potassium channels involved in vasodilatory responses. they have a direct contractile effect on vascular smooth muscle by helping mobilization of calcium and increasing the sensibility of contractile proteins, but they are involved in a number of other mechanisms resulting also in contracted vascular smooth muscle bed (feletu 2006). assesment of endothelial dysfunction assessment of endothelial function/dysfunction is an issue of great diversity. the methods that are currently used vary from ultrasound doppler measurement of the brachial artery diameter and blood fl ow to quantifi cation of soluble substances produced by the endothelium in plasma or serum. the gold standard is still uncertain, as different approaches to the matter estimate or measure different aspects of endothelial function, dysfunction, activation or damage. flow mediated dilatation (fmd) induced by reactive hyperemia has been shown to be endothelium dependent and can be assessed by highresolution ultrasound in superficial arteries following a well defined and standardized methodology (corretti et al. 2002; sorensen et al. 1995) for the non invasive assessment of endothelial function in vivo. several molecules have been measured as markers of different aspects of endothelial function. their evaluation in relation with disease states or outcomes of treatments have been thus far only for research. their clinical usefulness has not yet been proven or ever more, put in practice. they can offer not only useful diagnostic tests for diseases, but also give a helping hand in assessing the prognosis of pathological states affected by or originating from the vascular endothelium, an organ that is not to be underestimated in its powerfulness to affect almost all functions and anatomical formations of the human body. the major advantage of biochemical measures of endothelial function is that they are inexpensive and offer excellent reproducibility (verma et al. 2003), as well as a potential role in future mass screening for vascular pathology. pde5is effects on the endothelium under conditions of sexual stimulation, nonadrenergic-noncholinergic neurons and vascular endothelial cells release no, which is responsible for increasing levels of cgmp in the smooth muscle cells of the corpora cavernosa of the penis. this is mediated by activation of the enzyme guanylate cyclase. cgmp levels are lowered by a cgmp specifi c hydrolyzing enzyme, phosphodiesterase (pde), the isoenzyme 5 of which (pde5) is found in high concentrations in penile corporal tissue. pde5 inhibitors maintain high cgmp levels by preventing its degradation, thus promoting tumescence and erection. three pde5is have been marketed since 1998, sildenafi l, tadalafi l and vardenafi l. all three molecules have proven their effi cacy in erectile dysfunction treatment (cirino et al. 2006). they have similarities but also signifi cant differences in pharmacokinetics and pde isoenzyme selectivity and specifi city. these differences are refl ected in differences in effi cacy and also in safety profi les (gupta, 2005). recently, a new mechanism of sildenafi l’s effect on ed treatment was proposed, that is by inhibition of superoxide formation (by inhibiting nadph oxidase expression and reducing oxygen free radicals (o2 )̄ formation, the amount of bioavailable no would be enhanced. adding the inhibitory effect of no itself on nadph oxidase, this could constitute of a positive feedback mechanism of reducing the superoxide burden on the endothelium, that is, oxidative stress. they think that their fi ndings could become the basis of repeated dosing of sildenafi l, which may reduce intrapenile oxidative stress both in the short and the long term (jeremy et al. 2005). acute and also chronic sildenafi l treatment has favorable effects on brachial artery fl owmediated dilatation up to 24 h post-dose in men with and without erectile dysfunction. sildenafi l 116 perimenis et al drug target insights 2007: 2 has been demonstrated to improve the vasomotor aspect of endothelial dysfunction in patients with heart failure (katz et al. 2000): there was a change in fl ow mediated dilatation after administration of single doses of 12.5, 25 and 50 mg of sildenafi l and the authors concluded that sildenafi l improves endothelium dependent vasodilatation in patients with endothelial dysfunction due to chronic heart failure. another group of investigators evaluated brachial artery diameter as a measure of fl ow mediated dilatation one hour after a single oral dose of sildenafi l 25 mg and also after 2 weeks of daily dosing of 25 mg of sildenafi l, 24 hours after the last dose, in type 2 diabetic patients with erectile dysfunction without overt clinical heart disease, and found it signifi cantly improved, in contrast to placebo (desouza et al. 2002). other researchers treated men with increased cardiovascular risk with tadalafi l 20 mg on alternate days and they found that after 4 weeks they had a signifi cantly improved brachial artery fl ow mediated dilatation, as well as increased levels of nitrite/nitrate levels and decreased levels of et 1 at the same time intervals, changes signifi cantly different from their placebo counterparts, even after 2 weeks of tadalafi l discontinuation (rosano et al. 2005), providing optimistic messages of a more sustained effect of chronic pde5i use on endothelial function in general, not limited to penile tissue. opinions and fi ndings opposing to those previously described also exist (robinson et al. 2006). in a study evaluating the effect of sildenafi l on altitude-induced hypoxemia and pulmonary hypertension, treatment with sildenafi l induced an increase in plasma levels of cgmp (richalet et al. 2005). recently, the effects of da-8159 (udenafi l), a novel pde5i, on endothelial cell, smooth muscle and tgf-beta expression on streptozotocin induced diabetic rats corpous cavernosum was evaluated. subchronic treatment with da-8159 prevented the structural degradation of the corpus cavernosum, in terms of reduction of smooth muscle, endothelial cell content, immunoreactivity of tgf-beta 1expression and intracorporeal fi brosis (ahn et al. 2005). increased fl ow mediated dilatation after acute sildenafi l therapy was observed in patients with heart failure, a condition characterized by endothelial dysfunction. the same effect was also produced by acute administration of an angiotensin converting enzyme inhibitor, ramipril (hryniewicz et al. 2005). novel molecules and therapies of endothelial dysfunction recently, a method of treating endothelial dysfunction, and oxidative stress was claimed with the administration of d-chiroinositol, d-pinitol and 3,4-di-o-butyryl-d-chiroinositol. the d-chiroinositol and the d-pinitol are claimed to be antioxidants, glucose scavengers, pro-oxidant scavengers, peroxide radical scavengers and superoxide radical scavengers. the d-chiroinositol acts as a glucose uptake promoter and a metabolic normalizer. administration of 3,4-di-o-butyryld-chiroinositol (20 mg/kg) in hyperglycemic and control rats prevented endothelial dysfunction and had a positive effect on microvascular endothelial dysfunction (larner, 2006). conclusions erectile dysfunction is a global male population health and quality of life problem. since 1998, the clinical value of pde5i’s in on demand use has been proved in clinical trials and in everyday clinical practice. ed also is more and more recognized as a problem that is refl ective of a more complicate problem: endothelial dysfunction. therapies that could improve endothelial function may be benefi cial to erectile function, as well. phosphodiesterase inhibition has been shown to positively affect endothelial function. these fi ndings could serve as a fi rst indication of the benefi cial results of regular pde5is administration to patients with erectile dysfunction, as a means of “endothelial rehabilitation” (sommer and schulze, 2005) in our effort to improve or even reverse endothelial dysfunction and restore spontaneous sexual behaviour in patients with erectile dysfunction. molecular mechanisms of the endothelium, biological markers of endothelial function, genes of interest to the endothelial function are being investigated and the future lies in the effort for a more consistent intervention on endothelial dysfunction. references ahn, g.j., sohn, y.s., et al. 2005. the effect of pde5 inhibition on the erectile function in streptozotocin-induced diabetic rats. int. j. impot. res., 17(2):134–41. ayta, i.a., mckinlay, j.b., krane, r.j. 1999. the likely worldwide increase in erectile dysfunction between 1995 and 2025 and some possible policy consequences. b. j. u. int., 84:50–6. azadzoi, k.m. and goldstein, i. 1992. erectile dysfunction due to atherosclerotic vascular disease: the development of an animal model. j. urol., 147:1675–81. 117 endothelial and erectile dysfunction and pde5is. present and future drug target insights 2007: 2 budhiraja, r., tuder, r.m., hassoun, p.m. 2004. endothelial dysfunction in pulmonary hypertension. circulation, 109:159–65. chong, a.y., blann, a.d., and lip, g.y.h. 2003. assessment of endothelial damage and dysfunction: observations in relation to heart failure. q. j. med., 96:253. cines, d.b., pollak, e.s., buck, c.a. et al. 1998. endothelial cells in physiology and in the pathophysiology of vascular disorders. blood, 91:3527–61. cirino, g., fusco, f., imbimbo, c. et al. 2006. pharmacology of erectile dysfunction in man. pharmacol. ther., 111:400–23. corona, g., mannucci, e., mansani, r. et al. 2004. organic, relational and psychological factors in erectile dysfunction in men with diabetes mellitus. eur. urol., 46:222–8. corretti, m.c., anderson, t.j., benjamin, e.j. et al. 2002. guidelines for the ultrasound assessment of endothelial-dependent fl ow-mediated vasodilation of the brachial artery. a report of the international brachial artery reactivity task force. j. am. coll. cardiol., 39:257–65. desouza, c., parulkar, a., lumpkin, d. et al. 2002. acute and prolonged effects of sildenafi l on brachial artery fl ow-mediated dilatation in type 2 diabetes. diabetes care, 25(8):1336–9. feldman, h.a., goldstein, i., hatzichristou d.g. et al. 1994. impotence and its medical and psychosocial correlates: results of the massachusetts male aging study. j. urol., 151:54–61. feletu, m., vanhoutte, p.m. 2006. endothelial dysfunction: a multifaced disorder (the wiggers award lecture). am. j. physiol. heart. circ. physiol., 291:985–1002. furchgott, r.f., zawadzki, j.v. 1980. the obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. nature, 288:373–6. goligorsky, m.s., tsukahara, h., magazine, h. et al. 1994. termination of endothelin signaling: role of nitric oxide. j. cell physiol., 158:485–94. gupta, m., kovar, a., meibohm, b. 2005. the clinical pharmacokinetics of phosphodiesterase-5 inhibitors for erectile dysfunction. j. clin. pharmacol., 45:987–1003. hryniewicz, k., dimayuga, c., hudaihed, a. et al. 2005. inhibition of angiotensin-converting enzyme and phosphodiesterase type 5 improves endothelial function in heart failure. clin. sci. (lond.)., 108(4):331–8. jeremy, j., koupparis, a., muzaffar, s. et al. 2005. is the therapeutic action of sildenafi l mediated partly through the inhibition of superoxide formation? b. j. u. int., 95:930–1. katz, s.d., balidemaj, k., homma, s. et al. 2000. acute type 5-phosphodiesterase inhibition with sildenafi l enhances fl ow-mediated vasodilation in patients with chronic heart failure. j. am. coll. cardiol., 36:845–51. kirby, m., jackson, g., simonsen, u. 2005. endothelial dysfunction links erectile dysfunction to heart disease. int. j. clin. pract., 59:225–9. larner, j. 2006. (us): us20060004096-a1. lip, g.y.h., blann, a. 1997. von willebrand factor: a marker of endothelial dysfunction in vascular disorders? cardiovasc. res., 34:255–65. lopez, j.a., armstrong, m.l., piegors, d.j. et al. 1990. vascular responses to endothelin-1 in atherosclerotic primates. arteriosclerosis, 10:1113–8. montorsi, p., montorsi, f., schulman, c.c. 2003. is erectile dysfunction the “tip of the iceberg” of a systemic vascular disorder? eur. urol., 44:352. montorsi, p., ravagnani, p.m., galli, s. et al. 2004. common grounds for erectile dysfunction and coronary artery disease. curr. opin. urol., 14:361. newby, d.e., webb, d.j. 1996. advances in clinical pharmacology and therapeutics: endothelin. br. j. hosp. med., 56:360–4. nih consensus conference. impotence. nih consensus development panel on impotence. 1993. jama, 270: 83–90. rand, m.j. 1992. nitrergic transmission: nitric oxide as a mediator of nonadrenergic, non-cholinergic neuro-effector transmission. clin. exp. pharmacol. physiol., 19:147–69. rosen, r.c., riley, a., wagner, g. et al. 1997. the international index of erectile function (iief): a multidimensional scale for assesment of erectile dysfunction. urology, 49:822–30. rosen, r.c. 2006. update on the relationship between sexual dysfunction and lower urinary tract symptoms/benign prostatic hyperplasia. curr. opin. urol., 16:11–9. roumeguere, t., wespes, e., carpentier, y. et al. 2003. erectile dysfunction is associated with a high prevalence of hyperlipidemia and coronary artery disease. eur. urol., 44:355–9. rosano, g., aversa, a., vitale, c. et al. 2005. chronic treatment with tadalafi l improves endothelial function in men with increased cardiovascular risk. eur. urol., 47:214–22. robinson, s.d., ludlam, c.a., boon, n.a. et al. 2006. phosphodiesterase type 5 inhibition does not reverse endothelial dysfunction in patients with coronary heart disease. heart, 92(2):170–6. richalet, j.p., gratadour, p., robach, p. et al. 2005. sildenafi l inhibits altitudeinduced hypoxemia and pulmonary hypertension. am. j. respir. crit. care med., 171:275–81. sorensen, e., celermajer, s., spiegelhalter, d. et al. 1995. non-invasive measurement of human endothelium dependent arterial responses: accuracy and reproducibility. br. heart. j., 74:247–53. sommer, f., schulze, w. 2005. treating erectile dysfunction by endothelial rehabilitation with phosphodiesterase 5 inhibitors. world. j. urol., 23:385–92. thompson, i., tangen, c., goodman, p. et al. 2005. erectile dysfunction and subsequent cardiovascular disease. jama, 294:2996–3002. verma, s., buchanan, m.r., anderson, t.j. 2003. endothelial function testing as a biomarker of vascular disease. circulation, 108:2054–9. verhaar, m.c., strachan, f.e., newby, d.e. et al. 1998. endothelin-a receptor antagonist-mediated vasodilatation is attenuated by inhibition of nitric oxide synthesis and by endothelin-b receptor blockade. circulation, 97:752–6. vickers, m.a., satyanarayana, r. 2002. 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drug target insights 2007: 2 119–128 119 review the relationship of arginine deprivation, argininosuccinate synthetase and cell death in melanoma niramol savaraj1, chunjing wu2, marcus tien kuo3, min you2, medhi wangpaichitr2, carlos robles1, seth spector1 and lynn feun2 1va medical center, hematology-oncology, miami, florida, u.s.a. 2university of miami, hematology-oncology, miami, florida, u.s.a. 3m.d. anderson cancer center, molecular pathology, houston, texas, u.s.a. abstract: it has been shown that melanoma cells do not express argininosuccinate synthetase (ass) and therefore are unable to synthesize arginine from citrulline. depleting arginine using pegylated arginine deiminase (adi-peg20) results in cell death in melanoma but not normal cells. this concept was translated into clinical trial and responses were seen. however, induction of ass expression does occur which results in resistance to adi -peg20. we have used 4 melanoma cell lines to study factors which may govern ass expression. although these 4 melanoma cell lines do not express ass protein or mrna as detected by both immunoblot and northernblot analysis, ass protein can be induced after these cells are grown in the presence of adi-peg20, but again repressed after replenishing arginine in the media. the levels of induction are different and one cell line could not be induced. interestingly, a melanoma cell line with the highest level of induction could also be made resistant to adi-peg20. this resistant line possesses high levels of ass mrna and protein expression which cannot be repressed with arginine. our study indicates that ass expression in melanoma cells is complex and governed by biochemical parameters which are different among melanoma cells. keywords: melanoma, arginine deiminase, argininosuccinate synthetase. introduction treatment of advanced malignant melanoma has not signifi cantly improved in the past 20 years. most chemotherapeutic agents have response rates of 15–20% or less (devita et al. 2001). immunotherapeutic agents including interleukin-2 , adoptive immunotherapy, interferon alpha and vaccine therapy may produce signifi cant responses in a very small subset of patients, but their overall response rate is not better than chemotherapy (brinckerhoff et al. 2000; riker et al. 2007). combinations of chemotherapy and immunotherapy also have not produced signifi cant response rates in randomized trials (keilholz and gore, 2002; punt et al. 2006; sasse et al. 2007). recently, blocking of raf signaling has been explored for the treatment of melanoma (alsina et al. 2003; hoefl ich et al. 2006; scott et al. 2000; sridhar et al. 2005), however, the fi rst generation of raf inhibitors such as sorafenib has not shown signifi cant antitumor activity in malignant melanoma. thus, new effective agents are needed for the treatment of this disease. we and others have shown that melanoma cell lines do not express argininosuccinate synthetase (ass) (ensor et al. 2002; feun and savaraj, 2006; scott et al. 2000; shen et al. 2006; wheatley, 2005; wheatley and campbell, 2002; wheatley and campbell, 2003) a key enzyme which converts citrulline to argininosuccinate. argininosuccinate is then converted to arginine by argininosuccinate lyase. thus, melanoma cells are particularly vulnerable to arginine depletion while normal cells are able to survive. in fact, exposure of melanoma cells to arginine deiminase(adi), an enzyme which catalyzes the hydrolysis of arginine to citrulline results in cell death (ensor et al. 2002; shen and shen, 2006; sugimura et al. 1992; takaku et al. 1995; takaku et al. 1993). this laboratory fi nding has been developed into clinical trial, and antitumor activity has been documented in melanoma patients (ascierto et al. 2005; feun and savaraj, 2006). however, not all patients responded to treatment and the duration of response varied. in this communication we explore the role of ass gene expression in adi response/resistance using 4 melanoma cell lines as models. correspondence: nirmal savaraj, va medical center, hematology-oncology, 1201 nw 16th street, miami, florida 33125. tel: 305-575-3143; email: nsavaraj@med.miami.edu please note that this article may not be used for commercial purposes. for further information please refer to the copyright statement at http://www.la-press.com/copyright.htm 120 savaraj et al drug target insights 2007: 2 material and methods cell lines four human melanoma cell lines were used: a375, sk-mel-2, a2058 and mel-1220. the fi rst three cell lines (a375, sk-mel-2 and a2058) were from atcc, and mel-1220 was established in our laboratory from a subcutaneous biopsy of a melanoma patient. this cell line produces melanin pigment and is positive for s-100. a telomerase immortalized normal foreskin fi broblast cell line (bj-1) was purchased from clonetech to be used as normal control. nsclcs was derived from metastatic adenocarcinoma to the brain from primary lung cancer. this cell line grows in monlayer and was positive for keratin and epithelial membrane antigen. all cells were cultured in mem with 10% fcs and penicillin/streptomycin. cells were checked for mycoplasma every month using mycoalert mycoplasma detection kit purchased from cambrex. reagent adi-peg20 was supplied by polaris inc. larg-free medium was purchased from invitrogen. ass antibody was purchased from bd bioscience. ass antibody was purchased from bd-biosciences. parp antibody was purchased from cell signaling and actin antibody was purchased from sigma. growth inhibitory effect 0.6 × 105 cells were seeded onto 24-well plates and allowed 6 hr. for attachment. various concentrations of adi were added to each well. each concentration was performed in duplicate. after 72 hr. exposure, cells were counted in the presence of 0.2% trypan blue. the growth inhibitory effect (id50) was determined by plotting the number of viable cells as a percentage of control against the compound concentration. detection of apoptosis cells (1 × 104) were seeded onto lab-tek chamber slides (nalge nunc international) and then exposed to adi-peg20 at 0.1 ug/ml for 72 hrs. the apoptotic cells were detected by an in situ endlabeling assay using a kit from oncor. briefl y, cells were fi xed in 4% neutral buffered formalin, treated with 2% hydrogen peroxide, and incubated with terminal deoxynucleotide transferase enzyme and digoxigenin-11-dutp under a plastic coverslip for 1 hr. anti-digoxigenin peroxidase was applied to the slide, followed by the chromogenic substrate diaminobenzidine and counterstained with hematoxylin. cells that underwent apoptosis showed dark brown staining in the nuclei. we have also used poly adp-ribose polymerase cleavage assay to detect apoptosis. during apoptosis ice family such as caspase-3 and caspase-7 cleave parp to yield 85 and 25 kda fragments. briefl y, cells were treated with adi-peg20 for 72 hrs, nuclear protein was obtained and immunoblot was performed using parp rabbit polyclonal antibody purchased from cell signaling. analysis of arginine and citrulline by hplc arginine and citrulline analysis was performed by cation exchange hewlett packard 1100 series hplc with a post-column derivitization(11). this instrument utilizes a cation exchange column and a guard column. the temperature was set at 34 °c and the reactor temperature was set at 39 °c. amino acid standards arginine and citrulline were prepared in li220 diluent (all mobile phase reagents were obtained as pre-mixed preparations from pickering laboratories). the mobile phase reagents were pump a li280, pump b li750, pump c rg003. the initial conditions were 100% a for 12 min., followed by a linear gradient of 015% b over the next 16 min. the mobile phase was then switched to 92% b and 8% c and an isocratic gradient was run for an additional 37 min. a constant fl ow rate of 0.3 ml/minute was used. ass expression detected by reverse transcriptase polymerase chain reaction total rna was extracted using the kit from invitrogen. first-strand cdna was generated from 0.2ug of total rna using mlv reverse-transcription. the amplifi cation was carried out using the following primers: forward primer: ggccaaaaaggtgttcattg (nt: 240–259); reverse primer: attccaatgaagcggttctc (nt: 883–902) and set as follows: denature: 45 sec. at 94 °c, annealing 45 second at 60 °c and extension 1 min. at 72 °c for a total of 30 cycles. gapdh was used as control. the primers for gapdh were as follows: 121 argininosuccinate synthetase in melanoma drug target insights 2007: 2 forward primer: gaaggtgaaggtcggagtc; reverse primer: caaagttgtcatggatgacc. southernblot analysis of ass gene 10 ug of nuclear dna were digested with ecori restriction enzyme and electrophoresed using 0.8% agarose gels at 3 cm/v for 10–16 hrs in tae buffer (40 mm tris-acetate, 1 mm edta, ph8.0). the nuclear dna was transferred onto a nylon fi lter (hybond-n; amersham) and hybridized with 32p-dctp ass probe overnight, washed, and autoradiography was performed. ass probe was generated from bj-1 cell line. briefl y, the ass pcr product was obtained using the primers mentioned above, then eluted from the agorose gel using the kit from qiagen and labeled by 32p-dctp using random oligolabeling technique. northernblot analysis of ass gene total rna was extracted using the kit from invitrogen. 10 ug total rna was separated in 1% agarose gel in mops -formaldehyde buffer, transferred to a nylon membrane (hybond-n; amersham) and hybridized with 32p-dctp labeled ass probe. autoradiography was carried out. westernblot analysis of ass cells were lysed with ripa buffer (10 mmtris ph7.4 100 mm nacl, 1 mm edta, 20 mm na4p2o7, 2 mm na3vo4, 1% np-40, 0.5% deoxycholate, 1 mm pmsf) and protease inhibitor cocktail from sigma, passed several times through a 23g needle, and centrifuged. the total protein was separated on 12% sds-page, transferred onto membrane and immunoblot with a specifi c antibody and detected by chemiluminescence. construction of human argininosuccinate synthetase expression vector to construct pcdna3-ass, we amplified the human argininosuccinate synthetase mrna sequence from 79 to 1314 nt (using genbank accession number x01630 as the reference) by pcr with primer pairs containing sequences 5’gcggccgc tccagcaaaggc tccgtgg (sense; underscore sequence contains the not i site) and 5’-gcggccgctatttggcagtgaccttgc (antisense; underscore sequence contains not i site) using human pancrease cdna (clonetech, mountain view, ca) as the template. the pcr product was cloned into pcr®ii-topo vector (invitrogen). the noti fragment containing the respective cdna was then transferred into the noti site of cin-ha-pcdna3 vector, which contains a hemagglutinin tag, enhancer cin sequence for expression, and a neomycin resistance marker for transfection selection. transfection of ass gene. ass cdna was introduced into a375 and a2058 cell line using lipofectamine from invitrogen and selected with g-418. these transfectants were then tested for their sensitivity to adi-peg20. results growth inhibitory effect of adi-peg20 the growth inhibitory effect as well as the arginine and citrulline concentrations in the media are shown in table i. the id50 ranged from 0.05– 0.08 ug/ml. however, there are no viable cells in all three cell lines (a375, sk-mel-2 and mel-1220) after exposure to adi-peg20 for seven days, whereas in a2058 there is 2–3% viable cell left. from the id50 results, it appears that in sk-mel-2 and a2058 cell lines require more adi-peg20 to deplete arginine in the media. at 72 hr. there is still arginine remaining in the media (12.8 um for skmel-2 and 23.5 um for a2058) whereas in a375 and mel-1220 there is no detectable arginine level in the media. these fi ndings may be related to the capability of the intracellular machinery to maintain ass actin a 37 5 s km el -2 a 20 58 m e l12 20 b j1 n s c lc s figure 1. immunoblot of ass protein using commercially available monoclonal antibody (bd-bioscience). all four melanoma cell lines (a375, sk-mel2, a2058 and mel-1220) do not express ass protein whereas nsclcs and bj-1 possess high levels of ass protein. actin was used as control. 122 savaraj et al drug target insights 2007: 2 arginine either by degradation of certain unessential proteins or turning on ass or other unknown mechanisms. all these 4 melanoma cell lines do not express ass protein (as detected by westernblot, fig.1) and neither at the transcriptional levels as detected by northernblot analysis. this data also correspond to what has been reported in the literature (dillon et al. 2004; ensor et al. 2002). however, the ass dna can be detected readily by southernblot analysis (fig. 2). thus, lack of ass expression is not from gene deletion. in contrast, degradation of arginine by adi-peg20 did not have growth inhibitory effect on normal fi broblast bj-1 and nsclcs cell line (table 1). these two cell lines possess high levels of ass and hence are able to synthesize arginine from citrulline. these two cell lines also have arginine in the media at 0.05 ug/ml of adi-peg20. at this concentration there is no arginine in the media when emem media was incubated with adi-peg20 alone with no cells for 3 days. adi induces apoptosis in melanoma cell line we have exposed melanoma cells to arginine free emem media with citrulline and nh4cl supplement or adi at 0.05 ug/ml for 3 days and assay for apoptosis. the results are shown in figure 3a & 3b. all melanoma cell lines undergo apoptosis by both parp assay and in situ end labeling assay whereas bj-1 and nsclcs cells do not undergo apoptosis (data not shown). arginine deprivation induces melanoma ass gene expression it has been shown that arginine levels can regulate ass expression in lymphoblastoma cells and epithelial cells (jackson et al. 1996; philip et al. 2003). however, whether this phenomenon occurs in melanoma cells which lack ass expression is not known. we have studied this possibility by exposing four melanoma cell lines (a375, sk-mel-2, a2058 and mel-1220) to adi-peg20 for 3 days and assay for ass expression . the results are shown in figure 4. it is of interest that despite the similar levels of arginine deprivation, the levels of ass expression as detected by westernblot are different. a2058 has more ass expression (2.76 fold) followed by sk-mel-2 (2.03) and a375(1.52) while mel-1220 is unable to turn on ass gene. to further confi rm this fi nding we have cultured all four cell lines in the arginine free media with or without citrulline (0.4 um) and nh4cl (0.4 um) for 72 hr. as well as using mem media which has been incubated with 0.05 ug of adi-peg20 for 72 hr. to deplete arginine. at 24, 48 and 72 hr., cells were harvested and assayed for ass expression. after 72 hrs cells were placed on normal emem media. at 24, 48 and 72 hrs after being in normal media cells were harvested table i. growth inhibitory effect of adi-peg20. cell lines id50(ug/ml) arginine um citrulline um (72 h) (72 h) a2058 0.088 ± 0.008 23.5 367.5 a375 0.055 ± 0.001 nd 317.5 sk-mel-2 0.07 ± 0.008 12.8 329.2 mel-1220 0.09 ± 0.005 0 3 bj-1 >1 1.6 404 nsclcs* >1 20.27 603 *rpmi media (base line arginine: 971um) emem media (base line arginine: 400um) a 37 5 s km el -2 a 20 58 m e l12 20 b j1 ass actin figure 2. southernblot analysis of ass in 4 melanoma cell lines and bj-1 cell lines. all fi ve cell lines show similar levels of ass dna. 123 argininosuccinate synthetase in melanoma drug target insights 2007: 2 and assayed for ass expression. the results are shown in figure 5. the three type of media yield similar results. similar to data in figure 4, a2058 appears to have highest ass levels in arginine free media followed by sk-mel-2 and a375, while mel 1220 has no effect (data not shown). thus, our results indicate that the levels of arginine in the media regulate ass protein levels, and that the amount of citrulline did not have any effect. interestingly, ass gene expression cannot be detected by northernblot analysis (fig. 8), but may be slightly increased by rt-pcr (data not shown). thus, it appears that increased translation of ass proceeds transcription. arginine deprivation does not induce ass expression in nsclc we further investigated whether arginine deprivation also induced ass expression in tumor cell lines which constitutively express ass. we have chosen nsclcs which was established from metastatic adenocarcinoma of the lung to the brain. this cell line expresses ass and is not sensitive to adi-peg20. cells were seeded in arginine free media with citrulline supplement or adi-peg20 treated media to degrade all arginine for 3 day and assay for ass expression by westernblot. the results are shown in figure 6. ass expression is similar in normal media or arginine depleted media. thus, it appears that arginine deprivation does not have effect on ass expression in tumor cell lines which constitutively express high levels of ass. ass gene transfection results in resistance to adi we have transfected ass cdna into a375, and a2058, and then assay for growth inhibitory effect of adi-peg20. we are able to obtain only 2 fold increase in ass expression as shown by rt-pcr and westernblot (fig. 7a & 7b). the id50 of these a b figure 3a. in situ end labeling apoptosis assay in a375 cell line. a: control. b: after exposure to 0.08 ug/ml for 72 hr. treated cells undergo apopotosis shown brown staining in the nuclei. 116kd 89kd actin + + + +adi a375 sk-mel-2 a2058 mel-1220 parp figure 3b. apoptosis as detected by parp cleavage in 4 melanoma cell lines (a375, sk-mel-2, a2058 and mel-1220). untreated cell showed uncleaved parp at 116 kd whereas treated cells showed cleaved parp seen at 89 kd. 124 savaraj et al drug target insights 2007: 2 generation of adi-peg20 resistant cell line since we were unable to generate a stable ass transfected cell line with high levels of ass expression, we have exposed a2058 cell lines to adi-peg20 at 0.05 ug/ml for 5 days with 2 days off × 6 weeks. we were able to establish a2058r with high levels of ass expression by northernblot (fig. 8a) and by westernblot analysis (fig. 8b). this cell line is resistant to adi-peg20 with id50 >1 ug/ml. interestingly, the levels of ass protein also are not affected by arginine deprivation (data not shown). discussion it has been reported that melanoma cell lines as well as tumor samples do not express ass and thus are auxotrophic for arginine. arginine deprivation using arginine deiminase or arginase has ass actin ass actin ass actin a375 sk-mel-2 a2058 1 2 3 4 5 6 7 figure 5. immunoblot of ass protein in 3 melanoma cell lines (a375, sk-mel-2, a2058) before and after exposure arginine free media supplemented with citrulline and nh4cl for 24, 48 and 72 hrs. afterward, cells were washed, and replenished with normal emem media for 24, 48 and 72hrs. lane 1: control. lane 2: 24 hrs on arginine free media lane 3: 48hrs on arginine free media lane 4: 72 hrs in arginine free media lane 5: removal of arginine free media and changed to normal emem media for 24 hrs. lane 6: 48 hrs in normal media. lane 7: 72 hrs in normal media. similar results were obtained with arginine free media with no citrulline supplement and adi-peg20 treated media. ass actin 1 2 3 nsclcs figure 6. immunoblot of ass in nsclcs: lane 1: control (normal media ). lane 2: adi-peg20 treated media for 72 hr. lane 3 : after exposure to arginine free media with citrulline and nh4cl supplement for 72 hrs. transfectants were 0.1 ± 0.05 ug/ml for a375, and 0.2 ± 0.05 ug/ml for a2058, respectively. although we are unable to yield high levels of ass expression in these transfectants, this modest increase in ass expression does affect slightly the id50 of adi-peg20 with 2 fold increase. a375 sk-mel-2 a2058 mel-1220 ass actin + + + +adi ratio of ass 1.52 2.03 2.76 1 figure 4. immunoblot of ass protein in 4 melanoma cell lines (a375, sk-mel-2, a2058 and mel-1220) before and after exposure to adi-peg20 for 3 days. the ratio of ass depicts the intensity of ass expression by densitometer after exposure to adi versus control background. a2058 exhibited the highest level of expression whereas mel-1220 did not express ass after exposure to adi-peg20. 125 argininosuccinate synthetase in melanoma drug target insights 2007: 2 been shown by several investigators to have antitumor activity in melanoma cell lines as well as in other tumor cell lines which lack ass in vitro (dillon et al. 2004; ensor et al. 2002; gong et al. 2000; miyazaki et al. 1990; noh et al. 2004; shen et al. 2006; sugimura et al. 1992; szlosarek et al. 2006; takaku et al. 1995; takaku et al. 1993; yoon et al. 2007). correlation of antiproliferative effect of adi-peg20 with endogenous ass levels also has been reported (ensor et al. 2002; shen et al. 2003). however, these enzymes have a short half life and high antigenicity which makes them not suitable for in vivo use. to overcome this problem, a pegylated form of adi (adi-peg20) was developed by polaris inc. and has been shown to increase the half life as well as decrease the antigenicity (izzo et al. 2004). this compound has been shown to have antitumor activity in vivo (ensor et al. 2002). clinical trials also look promising with antitumor responses seen in a number of melanoma patients (ascierto et al. 2005; feun and savaraj, 2006). however, not all patients respond to adi-peg20 and resistance to this compound does occur after treatment. our limited clinical data suggest that ass expression occurs in patients who develop resistance to adi-peg20 (feun and savaraj, 2006). thus, it appears that exposure to adi-peg20 which results in continuous arginine depletion in the serum results in ass expression and hence drug resistance. in this communication, we have shown that exposure of melanoma cells to adi-peg20 or arginine free media can result in ass protein production which again become negligible when cells were exposed to normal media. interestingly, the levels of ass protein being induced while cells are deprived with arginine vary among the 4 melanoma cell lines. a2058 has the highest of ass protein followed by sk-mel-2 and a375. one melanoma cell line (mel -1220 ) cannot produce ass protein despite being cultured in the arginine depleted media. interestingly, a2058 can be selected to become resistant to adi-peg20 rather rapidly in 6 weeks. the primary mechanism of resistance involves increase in ass protein production followed by ass transcription as detected by northernblot analysis. the resistance also is irreversible despite replacement with normal emem media. on the other hand, mel-1220 cannot be made resistant to adipeg20. these fi ndings may mirror what we have found in melanoma patients. this is the fi rst report which demonstrated that extracellular arginine does control ass protein in certain melanoma cells. the underlying mechanism(s) on how arginine controls ass translation/transcription in melanoma are not known. it is possible that when the arginine levels is low, the cells will attempt to increase the translation of ass in order to synthesize arginine from citrulline. we are m ar ke r a 37 52 a 37 53 a 37 54 a 37 5 a 20 58 -5 a 20 58 -6 a 20 58 ass gapdh ass actin 1 2 3 4 figure 7b. immunoblot of ass in control and transfected cells. lane 1: a-375. lane 2: a375-3. lane 3: a2058. lane 4: a2058-6 (a375-3 and a2058-6 transfected with ass). figure 7a. rt-pcr of ass in a375 and a2058 and their ass transfected clones a375-2, a375-3, a375-4. and a2058-5,a2058-6, respectively. a375-3, and a2058-6 possess high levels of expression and were selected to study the growth inhibitory effect of adi-peg20. 126 savaraj et al drug target insights 2007: 2 currently investigating both transcription and translation control of ass in both normal and arginine free media. interestingly, the levels of induction are different among the melanoma cell lines tested. it appears that the cell line which has high levels of induction is capable of becoming resistant to adi -eg20 in a short period of time (within 6 weeks). on the other hand, the cell line which has no induction of ass (mel-1220) is not capable of turning on ass gene transcription and becoming resistant to adi-peg20. whether these fi ndings occur in patients’ tumor samples is not known. however, we have observed a melanoma patient who was ass(-) prior to treatment and become ass positive at the time of treatment failure which suggest that arginine deprivation can result in ass expression. as mention earlier, the mechanism by which arginine in the media control ass expression is not known, we are currently investigating how ass gene transcription is regulated in the presence and absence of arginine. ass cdna has been cloned in 1981 (su et al. 1981). the kinetic properties of ass enzyme has been extensively studied and the crystal structure in bacteria has been identifi ed (husson et al. 2003; ratner, 1973). the transcriptional and translational control of ass gene are not well understood and appear to be tissue specifi c (husson et al. 2003). several hormones such as glucocorticoids, glucagons, insulin and fatty acid have been shown to positively or negatively infl uence ass expression (husson et al. 1975; husson et al. 1986; husson and vaillant, 1979; husson and vaillant, 1982; lin et al. 1982). our preliminary data does not indicate that these hormones infl uence ass expression in melanoma cells. however, arginine in the media can mediate ass expression in certain melanoma cells. in contrast, arginine in the media has no effect in a2058r (selected by adi-peg20) or nsclcs, both have high levels of ass expression. this fi nding is similar to the previous report (boyce et al. 1986) which showed that arginine mediated ass repression does not occur in canavanine (arginine analog) resistant variant of rpmi 2650 cells which has high levels of ass expression. ass actin a 37 5 a 37 5+ a d i s km el -2 s km el -2 +a d i m e l12 20 m e l12 20 +a d i a 20 58 a 20 58 +a d i a 20 58 r b j1 b j1+ a d i figure 8a. northernblot analysis of ass in a panel of melanoma cell line and bj-1 cells. lane 1: a375. lane 2: a375 after exposure to adi-peg20 for 72hrs. lane 3: sk-mel-2 lane 4: sk-mel-2 after exposure to adi-peg20 for 72 hrs. lane 5: mel-1220. lane 6: mel-1220 after exposure to adi-peg20 for 72 hrs. lane 7: a2058. lane 8: a2058 after exposure to adi-peg20 for 72 hrs. lane 9: a2058r. lane 10: bj-1. lane 11: bj-1 after exposure to adi-peg20 for 72 hrs. note: only a2058r and bj-1 possess 1.9 kb ass mrna and there is no differences in ass mrna in bj-1 cells after exposure to adi-peg20. 1 2 3 4 5 ass actin figure 8b. immunoblot of ass. lane 1: a2058. lane 2: a2058 in arginine free media with out citrulline and nh4cl2. lane 3: a2058 in arginine free media supplemented with citrulline and nh4cl. lane 4: a2058r. lane 5: bj-1 cells. ass protein in a2058r is only slightly less than bj-1. 127 argininosuccinate synthetase in melanoma drug target insights 2007: 2 nevertheless, the key question remains what are the factors which govern ass expression in melanoma cells. if these factors can be identifi ed, one can attempt to repress the ass expression and hence evade adi-peg20 resistance. it is not yet clear why transfection with ass cdna in melanoma cells did not yield high levels of ass expression and we are unable to generate stable transfected cell line(s). it is conceivable that certain factors in melanoma cells prevent this cdna to express at high levels and/or arginine in the media may also play a role in repressing the expression of transfected ass. we are currently investigating these possibilities. acknowledgment this work was supported by a grant (r01ca 109578) from nih and a research grant from va. we would like to thank polaris inc. for providing adi-peg20 and analysis of arginine and citrulline. references alsina, j., gorsk, d.h., germino, f.j. et al. 2003. detection of mutations in the mitogen-activated protein kinase pathway in human melanoma. clin. cancer res., 9:6419–25. ascierto, p.a., scala, s., castello, g. et al. 2005. pegylated arginine deiminase treatment of patients with metastatic melanoma: results from phase i and ii studies. j. clin. oncol., 23:7660–8. boyce, f.m., anderson, g.m., rusk, c.d. et al. 1986. human argininosuccinate synthetase minigenes are subject to arginine-mediated repression but not to trans induction. mol. cell. biol., 6:1244–52. brinckerhoff, l.h., thompson, l.w. and slingluff, c.l. jr. 2000. melanoma vaccines. curr. opin. oncol., 12:163–73. devita, v.t., hellman, s. and rosenberg, s.a. 2001. cancer, principles and practice of oncology. philadelphia: lippincott, williams & wilkins. dillon, b.j., prieto, v.g., curley, s.a. et al. 2004. incidence and distribution of argininosuccinate synthetase defi ciency in human cancers: a method for identifying cancers sensitive to arginine deprivation. cancer, 100:826–33 ensor, c.m., holtsberg, f.w., bomalaski, j.s. et al. 2002. pegylated arginine deiminase (adi ss peg20,000 mw) inhibits human melanomas and hepatocellular carcinomas in vitro and in vivo. cancer res., 62:5443–50. feun, l. and savaraj, n. 2006. pegylated arginine deiminase: a novel anticancer enzyme agent. expert. opin. investig. drugs, 15:815–22. gong, h., zolzer, f., von, recklinghausen, g. et al. 2000. arginine deiminase inhibits proliferation of human leukemia cells more potently than asparaginase by inducing cell cycle arrest and apoptosis. leukemia, 14:826–9. hoefl ich, k.p., gray, d.c., eby, m.t. et al. 2006. oncogenic braf is required for tumor growth and maintenance in melanoma models. cancer res., 66:999–1006. husson, a., gautier, c. and vaillant r. 1975. [control activities and regulation by the glucocorticoids of three urea-cycle enzymes in rat foetal liver: argininosuccinate synthetase, argininosuccinase and arginase (author’s transl)]. experientia, 31:1403–5. husson, a. and vaillant, r. 1979. hormonal regulation of three urea cycle enzymes in rat fetal liver. biol. neonate., 35:74–81. husson, a. and vaillant, r. 1982. effects of glucocorticosteroids and glucagon on argininosuccinate synthetase, argninosuccinase, and arginase in fetal rat liver. endocrinology, 110:227–32. husson, a., guechairi, m., fairand, a. et al. 1986. effects of pancreatic hormones and glucocorticosteroids on argininosuccinate synthetase and argininosuccinase activities of rat liver during the perinatal period: in vivo and in vitro studies. endocrinology, 119:1171–7. husson, a., brasse-lagnel, c., fairand, a. et al. 2003. argininosuccinate synthetase from the urea cycle to the citrulline-no cycle. eur. j. biochem., 270:1887–99. izzo, f., marra, p., beneduce, g. et al. 2004. pegylated arginine deiminase treatment of patients with unresectable hepatocellular carcinoma: results from phase i/ii studies. j. clin. oncol., 22:1815–22. jackson, m.j., zielke, h.r. and zielke, c.l. 1996. induction of astrocyte argininosuccinate synthetase and argininosuccinate lyase by dibutyryl cyclic amp and dexamethasone. neurochem. res., 21:1161–5. keilholz, u. and gore, m.e. 2002. biochemotherapy for advanced melanoma. semin. oncol., 29:456–61. lin, r.c., snodgrass, p.j. and rabier, d. 1982. induction of urea cycle enzymes by glucagon and dexamethasone in monolayer cultures of adult rat hepatocytes. j. biol. chem., 257:5061–7. miyazaki, k., takaku, h., umeda, m. et al. 1990. potent growth inhibition of human tumor cells in culture by arginine deiminase purifi ed from a culture medium of a mycoplasma-infected cell line. cancer res., 50:4522–7. noh, e.j., kang, s.w., shin, y.j. et al. 2004. arginine deiminase enhances dexamethasone-induced cytotoxicity in human t-lymphoblastic leukemia ccrf-cem cells. int. j. cancer, 112:502–8. philip, r., campbell, e. and wheatley, d.n. 2003. arginine deprivation, growth inhibition and tumour cell death: 2. enzymatic degradation of arginine in normal and malignant cell cultures. br. j. cancer, 88:613–23. punt, c.j., suciu, s., gore, m.a. et al. 2006. chemoimmunotherapy with dacarbazine, cisplatin, interferon-alpha2b and interleukin-2 versus two cycles of dacarbazine followed by chemoimmunotherapy in patients with metastatic melanoma: a randomised phase ii study of the european organization for research and treatment of cancer melanoma group. eur. j. cancer, 42:2991–5. ratner, s. 1973. enzymes of arginine and urea synthesis. adv enzymol relat areas mol. biol., 39:1–90. riker, a.i., radfar, s., liu, s. et al. 2007. immunotherapy of melanoma: a critical review of current concepts and future strategies. expert. opin. biol. ther., 7:345–58. sasse, a., sasse, e., clark, l. et al. 2007. chemoimmunotherapy versus chemotherapy for metastatic malignant melanoma. cochrane database syst. rev., cd005413. scott, l., lamb, j., smith, s. et al. 2000. single amino acid (arginine) deprivation: rapid and selective death of cultured transformed and malignant cells. br. j. cancer, 83:800–10. shen, l.j., lin, w.c., beloussow, k. et al. 2003. resistance to the antiproliferative activity of recombinant arginine deiminase in cell culture correlates with the endogenous enzyme, argininosuccinate synthetase. cancer lett., 191:165–70. shen, l.j., beloussow, k. and shen, w.c. 2006. modulation of arginine metabolic pathways as the potential anti-tumor mechanism of recombinant arginine deiminase. cancer lett., 231:30–5. shen, l.j. and shen, w.c. 2006. drug evaluation: adi-peg-20—a pegylated arginine deiminase for arginine-auxotrophic cancers. curr opin mol ther, 8:240–8 sridhar ss, hedley d and siu ll. 2005. raf kinase as a target for anticancer therapeutics. mol. cancer ther., 4:677–85. su, t.s., bock, h.g., o’brien, w.e. et al. 1981. cloning of cdna for argininosuccinate synthetase mrna and study of enzyme over production in a human cell line. j. biol. chem., 256:11826–31. sugimura, k., ohno, t., kusuyama, t. et al. 1992. high sensitivity of human melanoma cell lines to the growth inhibitory activity of mycoplasmal arginine deiminase in vitro. melanoma. res., 2:191–6 szlosarek, p.w., klabatsa, a., pallaska, a. et al. 2006. in vivo loss of expression of argininosuccinate synthetase in malignant pleural mesothelioma is a biomarker for susceptibility to arginine depletion. clin. cancer res., 12:7126–31. 128 savaraj et al drug target insights 2007: 2 takaku, h., misawa, s., hayashi, h. et al. 1993. chemical modifi cation by polyethylene glycol of the anti-tumor enzyme arginine deiminase from mycoplasma arginini. jpn. j. cancer res., 84:1195–200. takaku, h., matsumoto, m., misawa, s. et al. 1995. anti-tumor activity of arginine deiminase from mycoplasma argini and its growth-inhibitory mechanism. jpn. j. cancer res., 86:840–6. wheatley, d.n. and campbell, e. 2002. arginine catabolism, liver extracts and cancer. pathol. oncol. res., 8:18–25. wheatley, d.n. and campbell, e. 2003. arginine deprivation, growth inhibition and tumour cell death: 3. defi cient utilization of citrulline by malignant cells. br. j. cancer, 89:573–6. wheatley, d.n. 2005. arginine deprivation and metabolomics: important aspects of intermediary metabolism in relation to the differential sensitivity of normal and tumour cells. semin. cancer biol., 15:247–53. yoon, c.y., shim, y.j., kim, e.h. et al. 2007. renal cell carcinoma does not express argininosuccinate synthetase and is highly sensitive to arginine deprivation via arginine deiminase. int. j. cancer, 120:897–905. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile 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drug target insights 2008:3 55–66 55 review correspondence: john j. nemunaitis, m.d., 1700 pacifi c avenue, suite 1100, dallas, tx 75201. tel: 214-658-1965; fax: 214-658-1992; email: jnemunaitis@marycrowley.org copyright in this article, its metadata, and any supplementary data is held by its author or authors. it is published under the creative commons attribution by licence. for further information go to: http://creativecommons.org/licenses/by/3.0/. use of proteomics analysis for molecular precision approaches in cancer therapy yuqiao shen1, neil n. senzer1,2 and john j. nemunaitis2 1lead therapeutics, inc., san bruno, ca. 2mary crowley cancer research centers, dallas, tx. abstract: the rapidly expanding data sets derived from genomic and transcriptomic analyses have allowed greater understanding of structural and functional network patterns within the genome resulting in a realignment of thinking within a systems biologic framework of cancer. however, insofar as spatially and temporally dynamic differential gene expression at the protein level is the mediate effector of cellular behavior and, in view of extensive post translational modifi cation (ptm), the need for sensitive, quantitative, and high throughput proteomic analytic techniques has emerged. to circumvent the problems of tissue sample heterogeneity, laser capture microdissection (lcm) allows for the acquisition of homogeneous cell populations. using different fl uorescent dyes to label protein samples prior to gel electrophoresis, 2-d dige (twodimensional differential in-gel electrophoresis) can, with reasonable sensitivity, process three protein samples on the same gel allowing for intragel relative quantifi cation. mudpit (multidimensional protein identifi cation technology) is a non-gel approach exploiting the unique physical properties of charge and hydrophobicity which allows the separation of peptide mixtures as well as direct ms (mass spectrometry) and database searching. the introduction of itraq (isobaric tags for relative and absolute quantifi cation) achieves labeling of all peptides by employing an 8-plex set of amine reactive tags to derivatize peptides at the n-terminus and lysine side chains allowing for absolute quantifi cation and assessment of ptm. these and other new laboratory technologies, along with improved bioinformatics tools, have started to make signifi cant contributions in cancer diagnostics and treatments. keywords: proteomics, molecular profi ling, targeted cancer therapy introduction for 70 years the oncology community has been exploring therapeutic opportunities involving cytotoxic based therapy for cancer control. unfortunately, principles derived from the application of cytotoxic based therapies now provide diminishing return with respect to patient benefi t. recent developments in genetics, molecular biology, and molecular pharmacology promise to dramatically alter strategies of cancer management. our ability to differentially characterize the neoplastic process in malignant cells on the bases of receptor overexpression, signaling process modulation, and genetic and epigenetic aberrations is now enabling us to realistically entertain the concept of specifi cally matching the “right patient with the right therapeutic”. molecular characterization of relatively sensitive and differentially specifi c cancer structural and process components has resulted in a new wave of “targeted” therapies. in a scale-free, hierarchical, modular system such as cancer, redundancy enables most neoplastic cells to bypass or buffer the effect of any single gene/target modifi cation, thereby minimizing targeted therapeutic effectiveness and the capacity for long term durable response (carlson and doyle, 2002; ciliberti et al. 2007). however, systems biology analysis suggests that coordinated and integrated targeted (rather than ‘random’) network disruptions can expose an “attack vulnerability” (carlson and doyle, 2002; hartwell et al. 1997). as such, the disordered system circuitry can become, almost paradoxically, more highly dependent on a specifi c rewired pathway (i.e. pathway addiction). in other words, the disruption of pathways that produce robustness to certain insults are often associated with enhanced fragility to other perturbations thereby exposing an “achilles’ heel” of cancer. in such an approach, the most intriguing targets derived from a patient’s differential genomic-proteomic profi le, which we are studying, would involve highly interconnected “hub” genes which control cancer cell competitive survival, metastagenicity and/or cancer stem-cell renewal (albert et al. 2000; jeong et al. 2001). we and others have previously demonstrated that semi-quantitative proteomic (feldman et al. 2004; nemunaitis et al. 2007; petricoin et al. 2004; zhou et al. 2002) profi ling derived by comparing malignant http://creativecommons.org/licenses/by/3.0/ http://creativecommons.org/licenses/by/3.0/ 56 shen et al drug target insights 2008:3 and non-malignant tissue from patients with progressive cancer can be analyzed in the context of global protein interaction networks in order to generate a prioritized list of potential protein and gene targets. compared to cdna microarray studies, analysis of differential gene expression at the protein level presents unique advantages as proteins are the mediate, if not direct, effectors of cellular behavior. furthermore, the products of posttranslational modifi cations and differential rna splicing, as well as samples with limited nucleic acids (e.g. serum) are most effectively analyzed using proteomics approaches. thus far, proteomics analyses have been used to probe the cellular and molecular mechanisms of transformation (young et al. 2005), search for determination of potential tumor biomarkers (zhou et al. 2002), screen and allow early detection of cancer (feldman et al. 2004; petricoin et al. 2004), prognosticate, predict response and support therapeutic management of cancer (nagata et al. 2004; volpi et al. 2003). it is hoped that one day the knowledge of proteomic signals within individual cancer specimens prior to treatment will provide a more optimal match between the patient and a molecular based targeted therapy opportunity, thereby enhancing clinical response predictability for the clinical treatment team and the patient. proteomics technologies while many proteomics strategies have been designed and tested, only a handful have been widely used. these technologies along with their advantages and disadvantages are discussed below. polyacrylamide gel electrophoresis (page) polyacrylamide gel electrophoresis (page) is one of the most widely used techniques for separation of complex protein mixtures (somiari et al. 2005). upon completion of page, the proteins of interest are excised from the gel and their identities are determined by mass spectrometry. there are mainly two versions of page: one-dimensional gel electrophoresis (1-d page or sds-page) and twodimensional gel electrophoresis (2-d page). in 1-d page, the protein samples are dissolved in a loading buffer that usually contains a reducing agent (for example, dithiothreitol) and a denaturing agent [for example, sodium dodecyl sulphate (sds)]. separation is primarily based on the molecular weight of the proteins in the samples. after electrophoresis, proteins are visualized either by using antibody-detection techniques or by staining the gel with coomassie brilliant blue dye, silver stain or a fl uorescent dye. the degree of protein separation (resolution) is rather low. a single protein band may contain several hundred proteins. thus, 1-d page is of little utility in proteomic analysis of complex protein samples. for many years, 2-d page has been the “benchmark” for large-scale separation of complex protein mixtures. it separates proteins by the sequential orthogonal use of two different electrophoretic techniques based on two different properties of the proteins. proteins are fi rst separated by iso-electric focusing (ief) based on their isoelectric point. subsequently, the proteins are separated in the presence of sds according to their molecular weights. protein detection after 2d-page utilizes either antibody-detection techniques or gel staining techniques as described for 1-d page. the proteins on the gel are detected as distinct spots which can be identifi ed by mass spectrometry. a few hundred to a few thousand spots can be detected on a 2-d gel based on the specifi cs of the gel size and electrophoresis conditions. 2d-page was fi rst developed by o’farrell (1975) but has subsequently undergone several modifications designed to improve its resolution, reliability and sensitivity [for example, see yan et al. (yan et al. 1999)]. integrated imaging and bioinformatics tools are now available for proteomic characterization of biological specimens. the recently introduced modifi cation of 2-d page, termed two-dimensional differential in-gel electrophoresis (2-d dige), has significantly improved the speed, reproducibility and sensitivity of 2-d page based proteomics (friedman, 2007; friedman et al. 2004; somiari et al. 2003; wu et al. 2006; wu, 2006). using different fl uorescent dyes to label protein samples prior to gel electrophoresis, the dige technique allows multiple samples to be co-separated and visualized on one 2-d gel (unlu et al. 1997). up to three protein extracts, for example one control and two treated, are labeled covalently with different fl uorescent dyes (cy2, cy3 and/or cy5), then combined and separated by 2-d page. up to three coincident images of the gel are captured—using the cy2, 57 proteomics in personalized cancer therapy drug target insights 2008:3 cy3 and/or cy5 excitation wavelengths. the images are then merged, and differences between them can be determined using image analysis software. 2-d dige effectively eliminates gel-togel variability that is associated with standard 2-d page and improves the accuracy of quantitative protein profi ling (friedman, 2007). this technology has been used for protein profi ling in numerous organisms including bacteria (rathsam et al. 2005), yeast (hu et al. 2003), plants (bohler et al. 2007; keeler et al. 2007), animals (henkel et al. 2006; kisby et al. 2006), and human (jin et al. 2007; reynolds et al. 2007). it is also used for analysis of phosphoproteins (stasyk et al. 2005) and protein-protein interaction (lyakhovich et al. 2007). the dyes are purported to have a linear response to variation in protein concentration over five orders of magnitude, offer sub-nanogram sensitivity, and are compatible with ms analysis. main disadvantages to this technology include: 1) the high cost involved in acquiring equipment as well as expendable supplies, such as the fl uorescent dyes; 2) the labor-intensive nature of the method, which limits its usefulness as a highthroughput analytical method; 3) proteins that are very acidic (ph � 4) or very basic (ph � 9), have high (�150 kda) or low molecular weight (�15 kda) and proteins that are very hydrophobic are diffi cult to analyze using 2-d dige; and 4) its detection sensitivity dictates that many low abundance proteins (such as transcription factors, many serum biomarkers) are hard to detect with this method. moreover, both microheterogeneity due to post translational modifi cations (i.e. one protein producing multiple spots) and comigration (one spot containing two or more proteins) have been observed. two approaches of dige, termed “minimal” and “saturation” labeling procedures, have been described. the minimal labeling procedure (lilley and friedman, 2004; tannu and hemby, 2006b) is more widely used. in this procedure, the cydyes are covalently attached to the lysine residue side chain. the protein-to-dye ratio is deliberately kept high: only 2%–5% of the protein molecules are labeled (thus “minimal labeling”), so that only the proteins containing a single dye molecule are visualized on the gel. saturation labeling procedure (kondo and hirohashi, 2006; sitek et al. 2006) attaches dye molecules to cysteine residues, and all available cysteine residues are labeled (“saturation labeling”), thereby increasing the fl uorescent signal and protein detection sensitivity. the saturation labeling procedure is used specifi cally for the analyses of scarce protein samples, for example, samples obtained by laser capture microdissection (kondo and hirohashi, 2006; sitek et al. 2006). however, saturation labelling has also several drawbacks: (i) the 2-d spot pattern is signifi cantly altered compared to that of unlabelled or minimal-labeled proteins; (ii) the labeling reaction must be optimized for each sample to produce a uniform labeling of proteins, which is a timeconsuming and laborious procedure; (iii) proteins may precipitate during the labeling reaction due to the introduction of the hydrophobic dye molecule; and (iv) currently only two different saturation labeling dyes are available, which excludes the use of an internal standard (shaw et al. 2003). multidimensional protein identifi cation technology (mudpit) mudpit (multidimensional protein identifi cation technology) is a non-gel approach to the identifi cation of proteins from complex mixtures (kislinger et al. 2005). it offsets many of the disadvantages associated with two-dimensional gel electrophoresis. mudpit uses two liquid chromatography steps interfaced back-to-back in a fused silica capillary to permit two-dimensional high performance liquid chromatography (breci and haynes, 2007). typically, the fi rst column containing a strong cation exchange (scx) material is coupled to the second column containing reversed phase (rp) materials, which is in turn connected to a tandem mass spectrometer (ms) (fig. 1). by exploiting a peptide’s unique physical properties of charge and hydrophobicity, complex mixtures can be separated prior to sequencing by mass spectrometry. a complex peptide mixture generated from protein lysates is loaded onto the biphasic columns (florens and washburn, 2006). sample preparation is relatively straightforward, the samples are denatured, the cysteines reduced and alkylated and the proteins digested with a protease such as trypsin. charged peptides bind to the scx column, whereas any uncharged peptides pass through and bind to a reverse phase trap column. chromatography proceeds in steps with increasing salt concentration to release proteins from the scx resin in steps unto the rp resin. a reversed phase gradient with increasing hydrophobicity is then slowly introduced 58 shen et al drug target insights 2008:3 to progressively elute peptides from the rp resin into the mass spectrometer. peptide fragmentation data is then obtained to identify the peptides and hence the proteins from which they are derived. in the next step, a buffer with increased salt concentration is injected onto the scx column, displacing further peptides from it onto the rp trap column. salt is removed by washing the column and again an analytical rp separation is performed and the eluting peptides analyzed by mass spectrometry. incremental increases of salt are used (salt step gradient from around 0–200 mm). the end result is multiple protein identifi cations from each salt step (wu and maccoss, 2002). the biphasic column which is placed in-line with the hplc system acts as an ion source for tandem ms. this design reduces dead volumes and band broadening, thereby maximizing resolution and sensitivity. isotope coded affi nity tags (icat) chemical tagging (usually stable isotope labeling) of proteins/peptides allows for relative quantifi cation of protein samples by liquid chromatography/mass spectrometry (lc-ms) analyses. the prototypical stable-isotope labeling for quantitative proteomics was isotope-coded affi nity tags (icat) technology. developed in the laboratory of dr. reudi aebersold, this technology simultaneously quantifi es and identifi es protein differences in paired samples (gygi et al. 1999). essentially, proteins from the two states to be compared are labeled at cysteine residues with light and heavy tags, respectively. like the cyanine dyes used in 2-d dige, the isotopic tags are similar in structure and chemical properties, but are different in mass. the icat reagents consist of three functional elements: a cleavable biotin group, an isotopic tag that occurs in a “heavy” or “light” state, and a cysteine-reactive group. two proteins samples are separately labeled using the heavy (deuterated or 13c) icat reagent for one protein sample and the light (non-deuterated or 12c) icat reagent for the other (han et al. 2001). the two labeled mixtures are then combined, proteolytically digested and run on an avidin column to pull out only the labeled peptides via the biotin tag. this reduces the complexity of the sample prior to analysis by nano-scale lc-ms/ms (shiio and aebersold, 2006). peaks corresponding to the same peptide are identifi ed as doublets in mass spectra due to the mass difference between light and heavy isotopes. the peak intensities of the peptides correlate directly with the relative abundance of the proteins in the two states. selected peptides (usually those that show a signifi cant difference in abundance between the two samples) will be subjected to ms/ms analysis to reveal the amino acid sequence and thus the protein identity (tannu and hemby, 2006a; turecek, 2002). icat is a powerful protein profi ling technology t h a t a l l o w s s i m u l t a n e o u s d e t e c t i o n a n d figure 1. summary of steps utilized by multidimensional protein identifi cation technology (mudpit). scx = strong cation exchange; rp = reversed phase. 59 proteomics in personalized cancer therapy drug target insights 2008:3 quantification of protein differences between biological samples as well as identifi cation of the proteins. icat has the power to quantitatively identify proteins including acidic and basic proteins, membrane proteins, low copy number proteins, and high molecular weight proteins (griffi n et al. 2003; smolka et al. 2001). the weaknesses of icat include (ciordia et al. 2006; tannu and hemby, 2006a): 1) proteins that do not contain cysteine, or proteins that do not contain cysteine residues on tractable peptides upon proteolytic digestion, will not be detected due to the nature of the labeling procedure (moseley, 2001). 2) high sample complexity and the data-acquisition rate of the mass spectrometer used may limit coverage of differentially expressed proteins (moseley, 2001). 3) the number of proteins that can be identifi ed in an icat experiment (resolution) is far smaller than what is typically achieved with 2-d page technology (somiari et al. 2005), although this has been partially improved with technological developments and introduction of more powerful softwares (bouyssie et al. 2007). 4) the cysteine-based icat tags will generally not yield information on changes in the proteome based on post-translational modifi cations. 5) avidin columns used to concentrate labeled peptides may further complicate the analysis due to non-specifi c binding and/or irreversible binding of the peptides (moseley, 2001). stable isotope labeling with amino acids in cell culture (silac) is a somewhat similar approach which is used for incorporation of a label into proteins for mass spectrometry (ms)-based quantitative proteomics (liang et al. 2006). silac relies on metabolic incorporation of a given ‘light’ or ‘heavy’ form of the amino acid into the proteins. thus in an experiment, two cell populations are grown in culture media that are identical except that one of them contains a ‘light’ and the other a ‘heavy’ form of a particular amino acid (e.g. 12c and 13c labeled l-lysine, respectively). it is becoming one of the highly effective techniques in cell biology. however, because metabolic labeling is required, this technique is unlikely to be used for proteomic analyses in cancer patients. isobaric tags for relative and absolute quantitation (itraq) recently a new quantitative method, isobaric tags for relative and absolute quantitation (itraq), was developed (chen et al. 2007; overall and dean, 2006; ross et al. 2004). this technology employs amine-reactive isobaric tags to label peptides at the n-terminus and the lysine side chains, thereby labeling all peptides in a digest mixture. currently, eight isobaric tags are available, allowing for the labeling and simultaneous comparison of 8 protein samples by mass spectrometry. the itraq-based protocol contains four steps (aggarwal et al. 2006; zieske, 2006), as shown in figure 1. first, protein extracts are prepared from each sample to be analyzed. second, proteins are separately digested into polypeptides. third, each set of the polypeptides are labeled with itraq reagent individually. finally, labeled peptide samples are mixed and the mixture is analyzed by tandem mass spectrometry (ms/ms) to obtain an ms/ms spectrum. due to the large number of peptides produced, chromatographic methods are often used individually or in combination to fractionate the peptide mixture prior to lc-maldi tof/tof or lc-esi tof/tof. each of the four isobaric reagents has a mass of 145 daltons, and consists of three groups: reporter, balance, and reactive groups (ross et al. 2004). the reporter groups have molecular weights of 114, 115, 116, and 117 daltons, respectively. the carbonyl balance groups ensure that all the itraqlabeled peptides have the same mass. the reactive groups are attached to the n-terminal and the lysine residues of sample proteins. in single ms, peptides labeled with any of the isotopic tags are indistinguishable (isobaric). upon fragmentation in ms/ ms, however, the reporter groups of the itraq reagents will split from the peptide and form small fragments with mass/charges (m/z) of either 114, 115, 116, and 117 (4-plex) or 113, 114, 115, 116, 117, 118, 119, and 121 (8-plex), respectively. intensity of each of these peaks represents quantity of small reporter group fragment and thus represents the quantity of a peptide. ms/ms analysis will also generate peaks from polypeptides which allow the identifi cation of peptide sequences and therefore protein sequences. the advantages of itraq include: 1) all tryptic peptides are labeled resulting in increased confi dence and higher quality data; 2) up to 8 labels can be used for multiplexing experiments; 3) improved ms/ms fragmentation results in more confi dent peptide or protein identifi cations; and 4) post translational modifi cations, such as phosphorylation, can be analyzed (gafken and lampe, 2006). the key 60 shen et al drug target insights 2008:3 disadvantages of itraq include: 1) more mass spectrometry time is required because of the increased number of peptides; and 2) samples must be prepared according to very strict guidelines (aggarwal et al. 2006). surface enhanced laser desorption ionization-time of fl ight (seldi-tof) surface enhanced laser desorption ionization-time of fl ight (seldi-tof) is a relatively novel and straightforward proteomic technology that can be used for quantitative analysis of protein mixtures after selectively capturing proteins with unique attributes on activated surfaces (maurya et al. 2007; poon, 2007). this technique utilizes stainless steel or aluminum-based supports, or chips, engineered with binding features that have either chemical (hydrophilic, hydrophobic, pre-activated, normal-phase, immobilized metal affi nity, and cationic or anionic) or biological (antibody, antigen binding fragments (e.g. scfv), dna, enzyme, or receptor) bait surfaces (roelofsen et al. 2007; szalowska et al. 2007). solubilized tissue protein samples or body fl uids are directly applied to the chips, where proteins can bind to different chromatographic surfaces to retain proteins with specifi c features. after removing unbound proteins through serial washes, the bound proteins are subjected to analysis by laser desorption ionizationtime of flight mass spectrometry. masses of proteins ranging from small peptides of less than 1 kda up to proteins of greater than 300 kda are calculated based on time-of-fl ight. as mixtures of proteins will be analyzed within different samples, a unique sample fi ngerprint or signature will result for each sample tested. consequently, patterns of masses rather than actual protein identifi cations are produced by seldi analysis. this allows for comparison of spectra of a large number of samples within an abbreviated time frame and acquisition of differentially expressed proteomic patterns. seldi-tof is a potentially powerful clinical proteomics tool for identifi cation of patients at risk for development of cancer based on the direct analysis of body fl uids like serum, plasma, ductal lavage, cerebro spinal fl uid and urine (maurya et al. 2007; poon, 2007; zhang et al. 2006). a high profile and well publicized ovarian cancer study utilized seldi-tof to identify protein peaks in serum that distinguished patients with ovarian cancer from those without ovarian cancer (petricoin et al. 2002). while the study design, sensitivity and specifi city reported have generated counter comments, the seldi-tof is an emerging and potentially powerful proteomic tool that has attributes, e.g. cost and ease of use, that are lacking in other proteomics technologies. protein array protein arrays are solid-phase ligand binding assay systems comprising of immobilized biological molecules on solid surfaces which include glass, membranes, microtiter wells, mass spectrometer plates, and beads or other particles (clarke and chan, 2005; lueking et al. 2005). the assays are highly parallel (multiplexed) and often miniaturized (microarrays, protein chips). biological molecules such as antibodies, proteins, protein fragments, peptides, aptamers or carbohydrate are affi xed in a grid-like pattern on small surfaces thus forming a microscopic array (collett et al. 2005; reid et al. 2007). thus, protein arrays represent a proteomic tool that closely emulates the dna microarray technology. protein arrays are used to perform protein expression profi ling, to analyze protein-protein interactions, to determine the substrates of protein kinases, to identify the targets of biologically active small molecules, or to detect new disease biomarkers (uttamchandani et al. 2006). one of the chief formats of protein array is the capture array, in which ligand-binding reagents, which are usually antibodies but may also be alternative protein scaffolds, peptides or nucleic acid aptamers, are used to capture target molecules in mixtures such as plasma or tissue extracts. protein arrays using antibodies as the capture moieties are called antibody arrays (kopf and zharhary, 2007), which fall into one of two subtypes: those using matched antibody pairs for sandwich-type assays (forward phase arrays) and those utilizing single antibodies and a sample labeling methodology (reverse phase arrays). the former platform requires the use of a “detector” antibody with is either modifi ed with a directly detectable label (enzyme, fluorescent molecule, isotope, etc.), or it is biotinylated for detection after subsequent probing with labeled streptavidin. this platform essentially resembles elisa. the latter requires that protein samples be labeled beforehand (e.g. with fl uorescent molecule, isotope, or biotin), thus obviating use of a detector antibody. antibody array technology is attracting 61 proteomics in personalized cancer therapy drug target insights 2008:3 a lot of attention because of the potential of analyzing the levels of hundreds of proteins within a pathway of interest (borrebaeck, 2006). when other ligand-binding molecules are used, the capture arrays can be used to study functional proteomics such as protein-protein, protein-dna, protein-drug, receptor-ligand, enzyme-substrate interactions, etc. (cho and cheng, 2007). they may also be used to correlate the polymorphic changes resulting from snps with protein function. the capture reagents themselves will need to be selected and screened against many proteins, which can also be done in a multiplex array format against multiple protein targets. another protein array platform, known as profusion™ (phylos, inc., lexington, ma) arrays, utilizes surface-bound dna probes that hybridize to a protein via its encoding mrna. taken together, these protein array technologies offer advantages include being rapid and automatable, capable of high sensitivity, economical on reagents, and giving an abundance of data for a single experiment (becker et al. 2006; hall et al. 2007). however, the potential of antibody arrays is currently limited by the high cost of producing antibodies and the availability of antibodies that have both high specifi city (to eliminate cross reactions with non-specifi c proteins within the sample) and high affi nity for the target of interest (to allow detection of small quantities within a sample). additionally, the diffi culty associated with preserving proteins in their biologically active conformation before analysis with protein arrays further limits the application of this technology as a routine proteomic strategy. given the amount of information generated from one experiment, significant input of bioinformatics support is important; the data handling demands sophisticated software and data comparison analysis. fortunately some of the software can be adapted from that used for dna arrays, as can much of the hardware and detection systems. laser capture microdissection (lcm) the power of proteomic analytical techniques relies heavily on the preparation of homogeneous cell populations. tumors are heterogeneous microenvironments comprised of stroma, normal cells, and cancer cells at various stages of differentiation. identifi cation of protein level changes specifi c to cancer cells is hampered by the composite interspersed cell subpopulations in acquired tissue specimens. in order to overcome this diffi culty, michael emmert-buck and colleagues (emmert-buck et al. 1996; emmertbuck et al. 1994) at the national institutes of health (nih) developed lcm and demonstrated its usefulness for studying various tissue types at the dna, mrna, and protein levels. lcm technology allows researchers to isolate distinct cell subpopulations from stained tumor sections. tissue sections are mounted on standard glass slides. a transparent, 100-mm-thick, ethylene—vinyl acetate fi lm is then placed over the dry section (bonner et al. 1997). during lcm, cells are isolated using an inverted microscope fi tted with a low-power near-infrared laser. the laser provides enough energy to transiently melt this thermoplastic fi lm (momentarily heats to 90 oc) in a precise location, binding it to the underlying cells in that location. to improve the convenience of the technique, the transfer fi lm is usually mounted on a transparent cap. after the appropriate cells have been selected, the film and adherent cells are removed, and the unselected tissue remains in contact with the glass slide. the laser diameter can be adjusted from 7.5 to 30 µm so that individual cells or a cluster of cells can be selected. the fi lm holding the captured cells is then transferred to a tube, where an extraction buffer is used to remove the cells for further molecular analysis. up to 3000 transfers can be performed on one fi lm. therefore, up to 3,000–5,000 cells can be isolated from a single slide in this fashion (simone et al. 1998). because the plastic fi lm absorbs most of the thermal energy and the pulse lasts for only a fraction of a second, biological macromolecules are not damaged (bonner et al. 1997; emmert-buck et al. 1996). lcm is compatible with many common methods for the preparation of tissue sections. tissues are typically fi xed by alcohol-based precipitation techniques. although aldehyde-based fi xation may also be used, covalent cross-linking of macromolecules can potentially interfere with subsequent analysis of proteins. tissue sections are stained prior to lcm procedures by standard methods such as hematoxylin and eosin, methylene green nuclear stain, fl uorescence in situ hybridization, or immunohistochemistry for identifi cation of tissue morphology and cell populations of interest. capture of approximately 50,000 cells is suffi cient for 2-d page separation and visualization of several hundred distinct spots using silver or fl uorescent stains. image analysis can then be used to identify differentially expressed proteins. 62 shen et al drug target insights 2008:3 the major challenge in using lcm for proteomic studies is the limited amounts of material collected. for most proteomic applications, it is necessary to procure dozens of slides to get enough material for a single experiment. although this technique is faster compthan the previously usedmicrodissection methods, isolation of large numbers of cells from many sections can require considerable time (vogel et al. 2007; von eggeling et al. 2007). this shortcoming also signifi cantly limits one’s ability to detect and quantify proteins that are present at low levels, such as transcription factors, signal transduction molecules, hormones, etc., even when highly sensitive protein analysis systems are used. thus far, lcm has been used more successfully with nucleic acids (dna or rna), for which amplifi cation methods are available. another disadvantage is that cover slips and mounting solutions are not compatible with lcm and, as with other microdissection techniques, visualization of samples can be diffi cult (maitra et al. 2001). the newer versions of the lcm have a built-in optical system allowing the operator to confi rm the histology of the area to be microdissected without transferring the slide. for an experienced histopathologist, this suboptimal visualization should not be a problem. another limitation of lcm is that it is not compatible with live-cell analysis, but for these applications fl ow cytometry have been used routinely. bioinformatic analysis of proteomics data technological advances highlighted above have created a bottleneck at the level of data analysis and resulted in an accumulation of experimental data at a rate far exceeding the current ability to assimilate that data. transforming the rapidly proliferating quantities of experimental data into a usable form in order to facilitate data analysis is a challenging task (meunier et al. 2007). although interpretation of experimental datasets in an interactive and intuitive way will remain a big challenge in the foreseeable future, numerous specialized databases and graphical tools are beginning to make signifi cant contributions toward dissecting protein complex structures, generating functional data organization and revealing hierarchical relationships (ashburner et al. 2000; bader et al. 2001; bohannon, 2002; demir et al. 2002; duan et al. 2002; karp, 1998; karp, 2001; ruths et al. 2000; salamonsen et al. 1999; sirava et al. 2002). a detailed discussion of these bioinformatics tools are outside the scope of this review, but a number of excellent reviews are available to interested readers (cannataro, 2008; deutsch et al. 2008; ferre, 2005; palagi et al. 2006). discussion proteins are the direct effectors of cellular behavior rather than their dna and mrna templates. characterization of protein expression provides the most immediate assessment of cellular functional capacity. the use of laser capture microdissection (lcm) to maximize the concentration of cell populations of interest within the context of their microenvironment so as to optimize protein signal detection is critical for quantitative and comparative analysis to non malignant expression patterns. use of isobaric tags for relative and absolute quantifi cation and liquid chromatography may provide the broadest quantitative protein analysis achievable for unknown protein comparison with today’s technology. proteins assemble themselves into networks through a variety of protein-protein interactions (jensen, 2006). the resolution of these coupling events and in silico prediction of outcome from the disruption of these events are likely to provide the functional basis for defi ning novel and, potentially, effective targets for drug therapy (aksenov et al. 2005; christopher et al. 2004; pearl, 2000; senzer et al. 2005). pathogenic gene mutations, gene loss, and gene duplication or amplifi cation as well as epigenetic modifi cations can result in defective, absent or overexpressed proteins. these proteins realign within the cellular protein network in a “degenerative” pattern resulting in an oncopathologic hostile takeover (ajani and allgood, 2005). the culling of these unique tumor proteomic patterns provides the basis for a systematic approach to the development of personalized cancer therapeutics, especially when coupled with functional analysis of individual candidate gene/protein couplets. correlation of gene expression patterns (protein mediated activity) with disease outcome (survival) has been demonstrated in a variety of cancers (bhattacharjee et al. 2001; garber et al. 2001; perou et al. 2000; rosenwald et al. 2002; rosenwald et al. 2003; shipp et al. 2002). however, gene transcript levels often show poor correlation with protein levels and they cannot predict post translational modifi cations. 63 proteomics in personalized cancer therapy drug target insights 2008:3 by using the following criteria: (1) differential expression, (2) linkage to essential oncogenic processes, (3) high connectivity, and (4) high bottleneck centrality, it is feasible to reduce a fi nite number of overexpressed proteins in malignant tissue into a smaller subset of candidate targets, against which potentially therapeutic sirna or shrna agents can be constructed (nemunaitis et al. 2007). these products can then be used to enable a systematic loss-of-function analysis, in order to validate an integrated and coordinated complex of biologically relevant “gene targets” for trial investigation. it is envisioned that future rnai based gene therapy (one possible approach) for cancer can be prescribed based on the integrated mrna-proteomic co-expression profi le of each individual’s tumor. there are available data allowing preliminary assessment of the potential comparative therapeutic benefi ts of sirna versus shrna as rnai effectors. synthetic sirna can be delivered to the cytosol for direct incorporation into the rnainduced silencing complex (risc), whereas shrna requires dicer processing or, if delivered in a plasmid, nuclear penetration and translational processing to produce the hairpin shrna for dicer. conversely, dicer processing may result in a more potent rnai effector (siolas et al. 2005). furthermore, by using mir-30 based shrna, pol ii promoters (rather than pol iii) can be used to drive shrna-mir expression, allow for greater regulation and, perhaps, minimize the potential risk of exportin-5 saturation (dickins et al. 2005). obversely, sirna cannot be amplifi ed intracellularly as can plasmid-expressed shrna. preliminary comparisons between sirna and shrna (two methods of controlling rna expression and target protein expression) indicate that shrna induced knockdown is more durable and effi cient than sirna (lage, 2005; mcanuff et al. 2007; stein, 2006; vlassov et al. 2007) and, furthermore, amenable to second and third layers of tumor specifi city via tumor-targeted vector delivery and tumor specifi c promoter control thereby minimizing non malignant cell uptake and limiting potential toxic effects to non target agents (dickins et al. 2005; gossen and bujard, 1992; grimm and kay, 2006; gu and fang, 2003; gupta et al. 2004; yu et al. 2002). comparing patient-specifi c molecular profi les with known and developing human tissue-based pathways and with networks derived from protein interaction databases provides a means for identifying biologically relevent oncodependent target clusters—in other words, “matching up the right patient with the right therapeutic”. references aggarwal, k., choe, l.h. and lee, k.h. 2006. shotgun proteomics using the itraq. isobaric tags. brief funct genomic proteomic, 5:112–20. ajani, j. and allgood, v. 2005. molecular mechanisms in cancer: what should clinicians know? semin. oncol., 32:2–4. aksenov, s.v., church, b., dhiman, a., georgieva, a., sarangapani, r., helmlinger, g. and khalil, i.g. 2005. an integrated approach for inference and mechanistic modeling for advancing drug development. febs lett, 579:1878–83. albert, r., jeong, h. and barabasi, a.l. 2000. error and attack tolerance of complex networks. nature, 406:378–82. ashburner, m., ball, c.a., blake, j.a., botstein, d., butler, h., cherry, j.m., davis, a.p., dolinski, k., dwight, s.s., eppig, j.t., harris, m.a., hill, d.p., issel-tarver, l., kasarskis, a., lewis, s., matese, j.c., richardson, j.e., ringwald, m., rubin, g.m. and sherlock, g. 2000. gene ontology: tool for the unifi cation of biology. the gene ontology consortium. nat. genet., 25:25–9. bader, g.d., donaldson, i., wolting, c., ouellette, b.f., pawson, t. and hogue, c.w. 2001. bind—the biomolecular interaction network database. nucleic acids res., 29:242–5. becker, k.f., metzger, v., hipp, s. and hofl er, h. 2006. clinical proteomics: new trends for protein microarrays. curr. med. chem., 13:1831–7. bhattacharjee, a., richards, w.g., staunton, j., li, c., monti, s., vasa, p., ladd, c., beheshti, j., bueno, r., gillette, m., loda, m., weber, g., mark, e.j., lander, e.s., wong, w., johnson, b.e., golub, t.r., sugarbaker, d.j. and meyerson, m. 2001. classifi cation of human lung carcinomas by mrna expression profi ling reveals distinct adenocarcinoma subclasses. proc. natl. acad. sci. u.s.a., 98:13790–5. bohannon, j. 2002. bioinformatics. the human genome in 3d, at your fi ngertips. science, 298:737. bohler, s., bagard, m., oufi r, m., planchon, s., hoffmann, l., jolivet, y., hausman, j.f., dizengremel, p. and renaut, j. 2007. a dige analysis of developing poplar leaves subjected to ozone reveals major changes in carbon metabolism. proteomics, 7:1584–99. bonner, r.f., emmert-buck, m., cole, k., pohida, t., chuaqui, r., goldstein, s. and liotta, l.a. 1997. laser capture microdissection: molecular analysis of tissue. science, 278:1481,1483. borrebaeck, c.a. 2006. antibody microarray-based oncoproteomics. expert opin. biol. ther., 6:833–8. bouyssie, d., gonzalez de peredo, a., mouton, e., albigot, r., roussel, l., ortega, n., cayrol, c., burlet-schiltz, o., girard, j.p. and monsarrat, b. 2007. mfpaq, a new software to parse, validate, and quantify proteomic data generated by icat and silac mass spectrometric analyses: application to the proteomic study of membrane proteins from primary human endothelial cells. mol. cell. proteomics. breci, l. and haynes, p.a. 2007. two-dimensional nanofl ow liquid chromatography-tandem mass spectrometry of proteins extracted from rice leaves and roots. methods mol. biol., 355:249–66. cannataro, m. 2008. computational proteomics: management and analysis of proteomics data. brief bioinform. carlson, j.m. and doyle, j. 2002. complexity and robustness. proc. natl. acad. sci. u.s.a., 99(suppl 1):2538–45. chen, x., sun, l., yu, y., xue, y. and yang, p. 2007. amino acid-coded tagging approaches in quantitative proteomics. expert rev. proteomics, 4:25–37. cho, w.c. and cheng, c.h. 2007. oncoproteomics: current trends and future perspectives. expert rev. proteomics, 4:401–10. christopher, r., dhiman, a., fox, j., gendelman, r., haberitcher, t., kagle, d., spizz, g., khalil, i.g. and hill, c. 2004. data-driven computer simulation of human cancer cell. ann. n. y. acad. sci., 1020:132–53. 64 shen et al drug target insights 2008:3 ciordia, s., de los rios, v. and albar, j.p. 2006. contributions of advanced proteomics technologies to cancer diagnosis. clin. transl. oncol., 8:566–80. clarke, w. and chan, d.w. 2005. proteinchips: the essential tools for proteomic biomarker discovery and future clinical diagnostics. clin. chem. lab med., 43:1279–80. collett, j.r., cho, e.j. and ellington, a.d. 2005. production and processing of aptamer microarrays. methods, 37:4–15. demir, e., babur, o., dogrusoz, u., gursoy, a., nisanci, g., cetin-atalay, r. and ozturk, m. 2002. patika: an integrated visual environment for collaborative construction and analysis of cellular pathways. bioinformatics, 18:996–1003. deutsch, e.w., lam, h. and aebersold, r. 2008. data analysis and bioinformatics tools for tandem mass spectrometry in proteomics. physiol. genomics. dickins, r.a., hemann, m.t., zilfou, j.t., simpson, d.r., ibarra, i., hannon, g.j. and lowe, s.w. 2005. probing tumor phenotypes using stable and regulated synthetic microrna precursors. nat. genet., 37:1289–95. duan, x.j., xenarios, i. and eisenberg, d. 2002. describing biological protein interactions in terms of protein states and state transitions: the livedip database. mol. cell. proteomics, 1:104–16. emmert-buck, m.r., bonner, r.f., smith, p.d., chuaqui, r.f., zhuang, z., goldstein, s.r., weiss, r.a. and liotta, l.a. 1996. laser capture microdissection. science, 274:998–1001. emmert-buck, m.r., roth, m.j., zhuang, z., campo, e., rozhin, j., sloane, b.f., liotta, l.a. and stetler-stevenson, w.g. 1994. increased gelatinase a (mmp-2) and cathepsin b. activity in invasive tumor regions of human colon cancer samples. am. j. pathol., 145:1285–90. feldman, a.l., espina, v., petricoin, e.f., liotta, l.a. and rosenblatt, k.p. 2004. use of proteomic patterns to screen for gastrointestinal malignancies. surgery, 135:243–7. ferre, f. 2005. integrated bioinformatics software at ncbi, encyclopedia of genetics, genomics, proteomics and bioinformatics. part 4— bioinformatics. john wiley and sons, ltd. florens, l. and washburn, m.p. 2006. proteomic analysis by multidimensional protein identifi cation technology. methods mol. biol., 328:159–75. friedman, d.b. 2007. quantitative proteomics for two-dimensional gels using difference gel electrophoresis. methods mol. biol., 367:219–39. friedman, d.b., hill, s., keller, j.w., merchant, n.b., levy, s.e., coffey, r.j. and caprioli, r.m. 2004. proteome analysis of human colon cancer by two-dimensional difference gel electrophoresis and mass spectrometry. proteomics, 4:793–811. gafken, p.r. and lampe, p.d. 2006. methodologies for characterizing phosphoproteins by mass spectrometry. cell. commun. adhes, 13:249–62. garber, m.e., troyanskaya, o.g., schluens, k., petersen, s., thaesler, z., pacyna-gengelbach, m., van de rijn, m., rosen, g.d., perou, c.m., whyte, r.i., altman, r.b., brown, p.o., botstein, d. and petersen, i. 2001. diversity of gene expression in adenocarcinoma of the lung. proc. natl. acad. sci. u.s.a., 98:13784–9. gossen, m. and bujard, h. 1992. tight control of gene expression in mammalian cells by tetracycline-responsive promoters. proc. natl. acad. sci. u.s.a., 89:5547–51. griffi n, t.j., lock, c.m., li, x.j., patel, a., chervetsova, i., lee, h., wright, m.e., ranish, j.a., chen, s.s. and aebersold, r. 2003. abundance ratio-dependent proteomic analysis by mass spectrometry. anal. chem., 75:867–74. grimm, d. and kay, m.a. 2006. therapeutic short hairpin rna expression in the liver: viral targets and vectors. gene ther., 13:563–75. gu, j. and fang, b. 2003. telomerase promoter-driven cancer gene therapy. cancer biol. ther., 2:s64–70. gupta, s., schoer, r.a., egan, j.e., hannon, g.j. and mittal, v. 2004. inducible, reversible, and stable rna interference in mammalian cells. proc. natl. acad. sci. u.s.a., 101:1927–32. gygi, s.p., rist, b., gerber, s.a., turecek, f., gelb, m.h. and aebersold, r. 1999. quantitative analysis of complex protein mixtures using isotope-coded affi nity tags. nat. biotechnol., 17:994–9. hall, d.a., ptacek, j. and snyder, m. 2007. protein microarray technology. mech. ageing dev., 128:161–7. han, d.k., eng, j., zhou, h. and aebersold, r. 2001. quantitative profi ling of differentiation-induced microsomal proteins using isotope-coded affi nity tags and mass spectrometry. nat. biotechnol., 19:946–51. hartwell, l.h., szankasi, p., roberts, c.j., murray, a.w. and friend, s.h. 1997. integrating genetic approaches into the discovery of anticancer drugs. science, 278:1064–8. henkel, c., roderfeld, m., weiskirchen, r., berres, m.l., hillebrandt, s., lammert, f., meyer, h.e., stuhler, k., graf, j. and roeb, e. 2006. changes of the hepatic proteome in murine models for toxically induced fi brogenesis and sclerosing cholangitis. proteomics, 6:6538–48. hu, y., wang, g., chen, g.y., fu, x. and yao, s.q. 2003. proteome analysis of saccharomyces cerevisiae under metal stress by two-dimensional differential gel electrophoresis. electrophoresis, 24:1458–70. jensen, o.n. 2006. interpreting the protein language using proteomics. nat. rev. mol. cell. biol., 7:391–403. jeong, h., mason, s.p., barabasi, a.l. and oltvai, z.n. 2001. lethality and centrality in protein networks. nature, 411:41–2. jin, t., hu, l.s., chang, m., wu, j., winblad, b. and zhu, j. 2007. proteomic identifi cation of potential protein markers in cerebrospinal fl uid of gbs patients. eur. j. neurol., 14:563–8. karp, p.d. 1998. metabolic databases. trends biochem. sci., 23:114–6. karp, p.d. 2001. pathway databases: a case study in computational symbolic theories. science, 293:2040–4. keeler, m., letarte, j., hattrup, e., hickman, f. and haynes, p.a. 2007. twodimensional differential in-gel electrophoresis (dige) of leaf and roots of lycopersicon esculentum. methods mol. biol., 355:157–74. kisby, g.e., standley, m., park, t., olivas, a., fei, s., jacob, t., reddy, a., lu, x., pattee, p. and nagalla, s.r. 2006. proteomic analysis of the genotoxicant methylazoxymethanol (mam)-induced changes in the developing cerebellum. j. proteome res., 5:2656–65. kislinger, t., gramolini, a.o., maclennan, d.h. and emili, a. 2005. multidimensional protein identifi cation technology (mudpit): technical overview of a profi ling method optimized for the comprehensive proteomic investigation of normal and diseased heart tissue. j. am. soc. mass spectrom, 16:1207–20. kondo, t. and hirohashi, s. 2006. application of highly sensitive fl uorescent dyes (cydye dige fluor saturation dyes) to laser microdissection and two-dimensional difference gel electrophoresis (2d-dige) for cancer proteomics. nat. protoc., 1:2940–56. kopf, e. and zharhary, d. 2007. antibody arrays-an emerging tool in cancer proteomics. int. j. biochem. cell. biol., 39:1305–17. lage, h. 2005. potential applications of rna interference technology in the treatment of cancer. future oncol., 1:103–13. liang, x., hajivandi, m., veach, d., wisniewski, d., clarkson, b., resh, m.d. and pope, r.m. 2006. quantifi cation of change in phosphorylation of bcr-abl kinase and its substrates in response to imatinib treatment in human chronic myelogenous leukemia cells. proteomics, 6:4554–64. lilley, k.s. and friedman, d.b. 2004. all about dige: quantifi cation technology for differential-display 2d-gel proteomics. expert rev. proteomics, 1:401–9. lueking, a., cahill, d.j. and mullner, s. 2005. protein biochips: a new and versatile platform technology for molecular medicine. drug discov. today, 10:789–94. lyakhovich, a., canals, f., nosov, m. and surralles, j. 2007. a dige-based approach to study interacting proteins. j. biochem. biophys. methods, 70:693–5. maitra, a., wistuba, i.i. and gazdar, a.f. 2001. microdissection and the study of cancer pathways. curr. mol. med., 1:153–62. maurya, p., meleady, p., dowling, p. and clynes, m. 2007. proteomic approaches for serum biomarker discovery in cancer. anticancer res., 27:1247–55. mcanuff, m.a., rettig, g.r. and rice, k.g. 2007. potency of sirna versus shrna mediated knockdown in vivo. j. pharm sci., 96:2922–30. meunier, b., dumas, e., piec, i., bechet, d., hebraud, m. and hocquette, j.f. 2007. assessment of hierarchical clustering methodologies for proteomic data mining. j. proteome res., 6:358–66. 65 proteomics in personalized cancer therapy drug target insights 2008:3 moseley, m.a. 2001. current trends in differential expression proteomics: isotopically coded tags. trends biotechnol., 19:s10–6. nagata, y., lan, k.h., zhou, x., tan, m., esteva, f.j., sahin, a.a., klos, k.s., li, p., monia, b.p., nguyen, n.t., hortobagyi, g.n., hung, m.c. and yu, d. 2004. pten. activation contributes to tumor inhibition by trastuzumab, and loss of pten. predicts trastuzumab resistance in patients. cancer cell., 6:117–27. nemunaitis, j., senzer, n., khalil, i., shen, y., kumar, p., tong, a., kuhn, j., lamont, j., nemunaitis, m., rao, d., zhang, y.a., zhou, y., vorhies, j., maples, p., hill, c. and shanahan, d. 2007. proof concept for clinical justifi cation of network mapping for personalized cancer therapeutics. cancer gene ther., 14:686–95. overall, c.m. and dean, r.a. 2006. degradomics: systems biology of the protease web. pleiotropic roles of mmps in cancer. cancer metastasis rev., 25:69–75. palagi, p.m., hernandez, p., walther, d. and appel, r.d. 2006. proteome informatics i: bioinformatics tools for processing experimental data. proteomics, 6:5435–44. pearl, j. 2000. causality: models, reasoning, and inference. new york, cambridge university press: cambridge, u.k. perou, c.m., sorlie, t., eisen, m.b., van de rijn, m., jeffrey, s.s., rees, c.a., pollack, j.r., ross, d.t., johnsen, h., akslen, l.a., fluge, o., pergamenschikov, a., williams, c., zhu, s.x., lonning, p.e., borresen-dale, a.l., brown, p.o. and botstein, d. 2000. molecular portraits of human breast tumours. nature, 406:747–52. petricoin, e., wulfkuhle, j., espina, v. and liotta, l.a. 2004. clinical proteomics: revolutionizing disease detection and patient tailoring therapy. j. proteome res., 3:209–17. poon, t.c. 2007. opportunities and limitations of seldi-tof-ms in biomedical research: practical advices. expert rev. proteomics, 4:51–65. rathsam, c., eaton, r.e., simpson, c.l., browne, g.v., valova, v.a., harty, d.w. and jacques, n.a. 2005. two-dimensional fl uorescence difference gel electrophoretic analysis of streptococcus mutans biofi lms. j. proteome res., 4:2161–73. reid, j.d., parker, c.e. and borchers, c.h. 2007. protein arrays for biomarker discovery. curr. opin. mol. ther., 9:216–21. reynolds, j.l., mahajan, s.d., sykes, d.e., schwartz, s.a. and nair, m.p. 2007. proteomic analyses of methamphetamine (meth)-induced differential protein expression by immature dendritic cells (idc). biochim. biophys. acta., 1774:433–42. roelofsen, h., alvarez-llamas, g., schepers, m., landman, k. and vonk, r.j. 2007. proteomics profi ling of urine with surface enhanced laser desorption/ionization time of fl ight mass spectrometry. proteome sci., 5:2. rosenwald, a., wright, g., chan, w.c., connors, j.m., campo, e., fisher, r.i., gascoyne, r.d., muller-hermelink, h.k., smeland, e.b., giltnane, j.m., hurt, e.m., zhao, h., averett, l., yang, l., wilson, w.h., jaffe, e.s., simon, r., klausner, r.d., powell, j., duffey, p.l., longo, d.l., greiner, t.c., weisenburger, d.d., sanger, w.g., dave, b.j., lynch, j.c., vose, j., armitage, j.o., montserrat, e., lopez-guillermo, a., grogan, t.m., miller, t.p., leblanc, m., ott, g., kvaloy, s., delabie, j., holte, h., krajci, p., stokke, t. and staudt, l.m. 2002. the use of molecular profi ling to predict survival after chemotherapy for diffuse large-b-cell lymphoma. n. engl. j. med., 346:1937–47. rosenwald, a., wright, g., wiestner, a., chan, w.c., connors, j.m., campo, e., gascoyne, r.d., grogan, t.m., muller-hermelink, h.k., smeland, e.b., chiorazzi, m., giltnane, j.m., hurt, e.m., zhao, h., averett, l., henrickson, s., yang, l., powell, j., wilson, w.h., jaffe, e.s., simon, r., klausner, r.d., montserrat, e., bosch, f., greiner, t.c., weisenburger, d.d., sanger, w.g., dave, b.j., lynch, j.c., vose, j., armitage, j.o., fisher, r.i., miller, t.p., leblanc, m., ott, g., kvaloy, s., holte, h., delabie, j. and staudt, l.m. 2003. the proliferation gene expression signature is a quantitative integrator of oncogenic events that predicts survival in mantle cell lymphoma. cancer cell., 3:185–97. ross, p.l., huang, y.n., marchese, j.n., williamson, b., parker, k., hattan, s., khainovski, n., pillai, s., dey, s., daniels, s., purkayastha, s., juhasz, p., martin, s., bartlet-jones, m., he, f., jacobson, a. and pappin, d.j. 2004. multiplexed protein quantitation in saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. mol. cell. proteomics, 3:1154–69. ruths, d.a., chen, e.s. and ellis, l. 2000. arbor 3d: an interactive environment for examining phylogenetic and taxonomic trees in multiple dimensions. bioinformatics, 16:1003–9. salamonsen, w., mok, k.y., kolatkar, p. and subbiah, s. 1999. biojake: a tool for the creation, visualization and manipulation of metabolic pathways. pac. symp. biocomput., 392–400. senzer, n., shen, y., hill, c. and nemunaitis, j. 2005. individualised cancer therapeutics: dream or reality? expert opin. ther. targets, 9:1189–201. shaw, j., rowlinson, r., nickson, j., stone, t., sweet, a., williams, k. and tonge, r. 2003. evaluation of saturation labelling two-dimensional difference gel electrophoresis fl uorescent dyes. proteomics, 3:1181–95. shiio, y. and aebersold, r. 2006. quantitative proteome analysis using isotope-coded affi nity tags and mass spectrometry. nat. protoc., 1:139–45. shipp, m.a., ross, k.n., tamayo, p., weng, a.p., kutok, j.l., aguiar, r.c., gaasenbeek, m., angelo, m., reich, m., pinkus, g.s., ray, t.s., koval, m.a., last, k.w., norton, a., lister, t.a., mesirov, j., neuberg, d.s., lander, e.s., aster, j.c. and golub, t.r. 2002. diffuse large b-cell lymphoma outcome prediction by gene-expression profi ling and supervised machine learning. nat. med., 8:68–74. simone, n.l., bonner, r.f., gillespie, j.w., emmert-buck, m.r. and liotta, l.a. 1998. laser-capture microdissection: opening the microscopic frontier to molecular analysis. trends genet., 14:272–6. siolas, d., lerner, c., burchard, j., ge, w., linsley, p.s., paddison, p.j., hannon, g.j. and cleary, m.a. 2005. synthetic shrnas as potent rnai triggers. nat. biotechnol., 23:227–31. sirava, m., schafer, t., eiglsperger, m., kaufmann, m., kohlbacher, o., bornberg-bauer, e. and lenhof, h.p. 2002. biominer—modeling, analyzing, and visualizing biochemical pathways and networks. bioinformatics, 18(suppl 2):s219–30. sitek, b., potthoff, s., schulenborg, t., stegbauer, j., vinke, t., rump, l.c., meyer, h.e., vonend, o. and stuhler, k. 2006. novel approaches to analyse glomerular proteins from smallest scale murine and human samples using dige saturation labelling. proteomics, 6:4337–45. smolka, m.b., zhou, h., purkayastha, s. and aebersold, r. 2001. optimization of the isotope-coded affi nity tag-labeling procedure for quantitative proteome analysis. anal. biochem., 297:25–31. somiari, r.i., somiari, s., russell, s. and shriver, c.d. 2005. proteomics of breast carcinoma. j. chromatogr b. analyt technol biomed life sci., 815:215–25. somiari, r.i., sullivan, a., russell, s., somiari, s., hu, h., jordan, r., george, a., katenhusen, r., buchowiecka, a., arciero, c., brzeski, h., hooke, j. and shriver, c. 2003. high-throughput proteomic analysis of human infi ltrating ductal carcinoma of the breast. proteomics, 3:1863–73. stasyk, t., morandell, s., bakry, r., feuerstein, i., huck, c.w., stecher, g., bonn, g.k. and huber, l.a. 2005. quantitative detection of phosphoproteins by combination of two-dimensional difference gel electrophoresis and phosphospecifi c fl uorescent staining. electrophoresis, 26:2850–4. stein, u., stege, alexandra., walther, wolfgang., lage, hermann. 2006. complete in vivo reversal of the multidrug resistance (mdr) phenotype in a breast cancer model by jet-injection of anti-mdr1 short hairpin rna-encoding plasmid dna. molecular therapy, in press. szalowska, e., hijum, s.a., roelofsen, h., hoek, a., vonk, r.j. and meerman, g.j. 2007. fractional factorial design for optimization of the seldi protocol for human adipose tissue culture media. biotechnol. prog., 23:217–24. tannu, n.s. and hemby, s.e. 2006a. methods for proteomics in neuroscience. prog. brain res., 158:41–82. 66 shen et al drug target insights 2008:3 tannu, n.s. and hemby, s.e. 2006b. two-dimensional fluorescence difference gel electrophoresis for comparative proteomics profi ling. nat. protoc., 1:1732–42. turecek, f. 2002. mass spectrometry in coupling with affi nity capture-release and isotope-coded affi nity tags for quantitative protein analysis. j. mass spectrom., 37:1–14. unlu, m., morgan, m.e. and minden, j.s. 1997. difference gel electrophoresis: a single gel method for detecting changes in protein extracts. electrophoresis, 18:2071–7. uttamchandani, m., wang, j. and yao, s.q. 2006. protein and small molecule microarrays: powerful tools for high-throughput proteomics. mol. biosyst., 2:58–68. vlassov, a.v., korba, b., farrar, k., mukerjee, s., seyhan, a.a., ilves, h., kaspar, r.l., leake, d., kazakov, s.a. and johnston, b.h. 2007. shrnas targeting hepatitis c: effects of sequence and structural features, and comparision with sirna. oligonucleotides, 17:223–36. vogel, a., horneffer, v., lorenz, k., linz, n., huttmann, g. and gebert, a. 2007. principles of laser microdissection and catapulting of histologic specimens and live cells. methods cell. biol., 82:153–205. volpi, a., nanni, o., de paola, f., granato, a.m., mangia, a., monti, f., schittulli, f., de lena, m., scarpi, e., rosetti, p., monti, m., gianni, l., amadori, d. and paradiso, a. 2003. j. clin. oncol., 21:2708–12. von eggeling, f., melle, c. and ernst, g. 2007. microdissecting the proteome. proteomics, 7:2729–37. wu, c.c. and maccoss, m.j. 2002. shotgun proteomics: tools for the analysis of complex biological systems. curr. opin. mol. ther., 4:242–50. wu, h., pan, c.l., yao, y.c., chang, s.s., li, s.l. and wu, t.f. 2006. proteomic analysis of the effect of antrodia camphorata extract on human lung cancer a549 cell. proteomics, 6:826–35. wu, t.l. 2006. two-dimensional difference gel electrophoresis. methods mol. biol., 328:71–95. yan, j.x., sanchez, j.c., rouge, v., williams, k.l. and hochstrasser, d.f. 1999. modifi ed immobilized ph gradient gel strip equilibration procedure in swiss-2dpage protocols. electrophoresis, 20:723–6. young, t., mei, f., liu, j., bast, r.c., kurosky, a. and cheng, x. 2005. proteomics analysis of h-ras-mediated oncogenic transformation in a genetically defi ned human ovarian cancer model. oncogene, 24:6174–84. yu, j.y., deruiter, s.l. and turner, d.l. 2002. rna interference by expression of short-interfering rnas and hairpin rnas in mammalian cells. proc. natl. acad. sci. u.s.a., 99:6047–52. zhang, h., kong, b., qu, x., jia, l., deng, b. and yang, q. 2006. biomarker discovery for ovarian cancer using seldi-tof-ms. gynecol. oncol., 102:61–6. zhou, g., li, h., decamp, d., chen, s., shu, h., gong, y., flaig, m., gillespie, j.w., hu, n., taylor, p.r., emmert-buck, m.r., liotta, l.a., petricoin, e.f. and zhao, y. 2002. 2d differential in-gel electrophoresis for the identifi cation of esophageal scans cell cancer-specifi c protein markers. mol. cell. proteomics, 1:117–24. zieske, l.r. 2006. a perspective on the use of itraq. reagent technology for protein complex and profi ling studies. j. exp. bot., 57:1501–8. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left 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drug target insights 2008:3 27–29 27 original research correspondence: hubert h. fernandez, m.d., department of neurology/mcknight brain institute. university of florida, po box 100236, gainesville, florida 32610. fax: (352) 273 5575; email: fernandez@neurology.ufl .edu copyright in this article, its metadata, and any supplementary data is held by its author or authors. it is published under the creative commons attribution by licence. for further information go to: http://creativecommons.org/licenses/by/3.0/. new-onset diabetes mellitus among parkinsonian patients treated with long-term quetiapine hubert h. fernandez1, katie m. mccown1, janet romrell1, martha e. trieschmann2, joseph h. friedman2, charles e. jacobson iv1 and michael s. okun1 1dept. of neurology, university of florida, gainesville, fl. 2dept. of clinical neurosciences, brown university, providence, ri. abstract: atypical antipsychotics (aa) are commonly used to manage drug-induced psychosis (dip) in parkinsonian patients. in the treatment of schizophrenia, aa’s have been associated with increasing reports of new-onset diabetes mellitus (dm). this study examined the risk of developing new-onset dm among parkinsonian patients on long-term, low dose quetiapine. fifty-three parkinsonian subjects (mean age: 71.3 years) taking an average quetiapine dose of 70.5 mg/day (range: 12.5–350 mg/day) for a mean duration of 21.3 months (range 3–61 months) were reviewed. eight out of 53 subjects carried a diagnosis of dm prior to quetiapine treatment. four out of 45 patients (8.9%) met criteria for new diagnosis of dm, giving a total prevalence rate of 22.6% (12 out of 53). this prevalence rate of 22.6% was slightly higher than that reported in the aged-matched general population (year 2003 dm prevalence = 17.3% for 65–74 years) but methodological differences could explain the difference. larger epidemiologic studies will be needed to confi rm these results as they could potentially impact a signifi cant number of patients. keywords: quetiapine, diabetes, atypical antipsychotic, parkinson’s disease introduction atypical antipsychotics (aas) have become fi rst line treatment in the management of drug-induced psychosis (dip) in patients with parkinsonian diseases. they are considered fi rst line because of their lower risk of extrapyramidal side effects (eps) when compared to conventional neuroleptics [1]. currently, clozapine is considered the most effi cacious and perhaps best aa for parkinsonism [1–3]. however, its use is limited by the rare associated risk of agranulocytosis, and consequent weekly blood draws as well as intensive monitoring. quetiapine is the closest aa to clozapine in terms of effi cacy in parkinsonism, and is currently the most employed alternative to clozapine by movement disorder specialists [4]. given its low eps profi le and lack of hematologic risk, many authors consider quetiapine to now be the fi rst line treatment of dip in parkinsonism [4]. several of the aas, most notably olanzapine and clozapine, have been associated with the development of diabetes mellitus (dm) when used to treat schizophrenia [5,6]. though rare, there are also case reports of new-onset dm associated with quetiapine use in schizophrenia [5–7]. in the setting of parkinsonism, the association with dm is unclear and has not been carefully investigated. previously we described a lower incidence of dm with clozapine in the parkinsonian population when compared to age-matched controls [8]. although published reports indicate no dose relationship between clozapine and glucose tolerance, most studies involve schizophrenia patients who are typically on 200–800 mg/day of clozapine. since the average dose of clozapine was lower in parkinsonism than in schizophrenia, we hypothesized that perhaps the risk of dm could be dose-related. since quetiapine is the ‘practical fi rst line treatment’ for dip in parkinsonism, and because the published mean daily dose of quetiapine in the parkinsonian population is slightly higher compared to that reported for clozapine [1,4], we sought to study the prevalence of newly-diagnosed dm among parkinsonian patients on long-term quetiapine. method we reviewed the medical records of all quetiapine-treated parkinsonian patients actively followed at two movement disorder centers, brown university and the university of florida. patients were excluded http://creativecommons.org/licenses/by/3.0/ http://creativecommons.org/licenses/by/3.0/ 28 fernandez et al drug target insights 2008:3 if they had been on quetiapine for less than three months or were no longer taking the agent. to determine true prevalence, patients with previous and recent diagnoses of dm, and/or use of hypoglycemic agents prior to quetiapine initiation were included. the dose and duration of quetiapine use were recorded, as well as the patient’s age and sex. a fasting blood glucose (fbg) was obtained on all non-diabetic patients taking quetiapine. the diagnosis of new-onset dm while on quetiapine was determined by (1) a diagnosis made by the patient’s primary care physician, (2) the initiation of oral hypoglycemic agents or insulin by their primary care physician after initiation of quetiapine, (3) or a fbs greater than or equal to 126 mg/dl which is the ‘gold standard’ level warranting a diagnosis of dm [9]. the prevalence of dm in this cohort was then compared to the latest reported prevalence (year 2000) of dm in the (age-matched) general population [10]. the prevalence of dm in the general population was taken from the national health and nutrition examination survey (nhanes) based on a house to house survey. results fifty-three parkinsonian subjects (36 males, 17 females) with a mean: age of 71.3 years (range: 51–91 years), quetiapine dose of 70.5 mg/day (range: 12.5–350 mg/day), and treatment duration of 21.3 months (range 3–61 months) were identifi ed. nine out of the 53 subjects resided in longterm care facilities. because quetiapine was the fi rst line treatment for psychosis in both participating institutions, none of the subjects were previously exposed to other antipsychotic agents. eight out of 53 patients carried a prior diagnosis of dm prior to quetiapine initiation. among those without a prior diagnosis of dm, three undiagnosed patients had fbg �126 mg/dl, and a fourth patient was started on a hypoglycemic agent (after obtaining a fbg �126 mg/dl by the primary care physician) while taking quetiapine (total: 4/45; 8.9%). therefore, the prevalence of dm in our cohort was 22.6% (12/53). this rate was slightly higher than that reported in the age-matched general population (year 2003 dm prevalence = 17.3% for ages 65–74 years [10]). however, if 3 of the newly diagnosed dm cases based on “active diagnostic intervention” were excluded, and only known/recorded cases of dm (before and after quetiapine treatment) were included, then our cohort would have a “natural” prevalence rate of 16.9% (9/53). discussion this is, to our knowledge, the fi rst study to evaluate the prevalence of dm among parkinsonian patients on quetiapine. how quetiapine and other aas are related to dm remains unclear. quetiapine has been associated with weight gain [12], and this increase in body weight may at least in part be responsible for the slightly higher increased risk of new-onset dm. the aa drugs have also been linked to hypertriglyceridemia [13]. increased appetite, insulin resistance, and other endocrine changes may also play a role [12,14]. although this study found a slightly higher rate of dm in parkinsonian patients taking quetiapine than in the age-matched general population, there were several weaknesses in this study. the sample size was small making it diffi cult to draw fi rm conclusions from the data. ideal body weights versus actual body weights were not calculated, and therefore we cannot carefully account for who was at risk for type ii dm. additionally, in this study there was no screening for dm with fbg prior to quetiapine initiation, and no careful screening for endocrinopathies was performed. a repeat fbg was not drawn in the three patients who were high (�126 mg/dl). in addition, fbg may be a less stringent criteria for diagnosing dm than is postprandial blood glucose which was not measured in this study. post prandial blood glucose is an independent risk factor for mortality in patients with new-onset dm, while fbg is not [15]. these study weaknesses could have led to an overestimation of the prevalence of dm in this cohort. also of note was that this study utilized agematched historical controls. the prevalence rate of dm at the nhanes study was determined from a “naturalistic” survey (i.e. based on know/ recorded diagnosis of dm) without active diagnostic intervention; whereas the overall prevalence rate of dm in our cohort was calculated from a combination of data recording/inquiry and active diagnostic intervention. thus, employing the same method used at the nhanes study would yield a prevalence rate of 16.9%, but including newly diagnosed dm cases from mandatory blood testing of patients without a prior diagnosis would yield a higher prevalence rate. 29 new-onset diabetes mellitus among parkinsonian patients treated drug target insights 2008:3 we included only patients who were taking quetiapine for 3 months or more. most studies on the prevalence of diabetes in the schizophrenia population required 2–3 months of antipsychotic exposure prior to inclusion in their cohort. however, in schizophrenia, there are confl icting reports as to whether duration of disease is a signifi cant risk factor for dm development. two reports found no association [16,17], while one found a signifi cant association with olanzapine, clozapine and conventional antipsychotic agents [18]. no association with duration of disease was found in our cohort. larger epidemiologic studies are needed to better assess, and verify, these preliminary fi ndings for the risk of new-onset dm associated with quetiapine. neurologists, psychiatrists and primary care physicians should be aware of this potential slight increased risk of dm related to quetiapine and be cautious. references [1] fernandez, h.h. and friedman, j.h. 1999. the role of atypical antipsychotics in the treatment of movement disorders. cns drugs, 11(6):467–83. [2] the french clozapine parkinson study group, clozapine in drug induced psychosis in parkinson’s disease. 1999. lancet, 353(9169):2041–2. [3] the parkinson study group, low-dose clozapine for the treatment of drug-induced psychosis in parkinson’s disease. 1999. n. engl. j. med., 340(10):757–63. [4] friedman, j. and fernandez, h. 2002. atypical antipsychotics in parkinson-sensitive populations. journal of geriatric psychiatry and neurology, 15:156–70. [5] jin, h., meyer, j. and jeste, d. 2002. phenomenology of and risk factors for new-onset diabetes mellitus and diabetic ketoacidosis associated with atypical antipsychotics: an analysis of 45 published cases. annals of clinical psychiatry, 14(1):59–64. [6] sernyak, m., leslie, d., alarcon, r., losonczy, m. and rosenheck, r. 2002. association of diabetes mellitus with use of atypical neuroleptics in the treatment of schizophrenia. am. j. psychiatry, 159(4):561–6. [7] sobel, m., jaggers, e.d. and franz, m.a. 1999. new-onset diabetes mellitus associated with the initiation of quetiapine treatment. j. clin. psychiatry, 60(8):556–7. [8] fernandez, h., friedman, j., lansang, m., factor, s., molho, e. and coskun, d. 2004. diabetes mellitus among parkinsonian patients treated chronically with clozapine. parkinsonism and related disorders, 10:439–41. [9] powers, a.c. 2005. diabetes mellitus. in: kasper dl, braunwald e, fauci as, et al., eds. harrison's principles of internal medicine. 16th ed. new york: mcgraw-hill, 2152–54. [10] cdc. national health and nutrition examination survey 1999–2000 data fi les. available at http://www.cdc.gov/nchs/data/hhis/earlyrelease/2004 [11] expert committee on the diagnosis and classifi cation of diabetes mellitus, report of the expert committee on the diagnosis and classifi cation of diabetes mellitus. 2003. diabetes care, 26(suppl 1): s5–s20. [12] baptista, t., kin, n., beaulieu, s. and debaptista, e. 2002. obesity and related metabolic abnormalities during antipsychotic drug administration: mechanisms, management, and research perspectives. pharmacopsychiatry, 35(6):205–19. [13] meyer, j. 2001. novel antipsychotics and severe hyperlipidemia. journal of clinical psychopharmacology, 21(4):369–74. [14] lindenmayer, j., nathan, a. and smith, r. 2001. hyperglycemia associated with the use of atypical antipsychotics. journal of clinical psychiatry, 62(suppl 23):30–8. [15] bastyr, e., stuart, c., brodows, r., schwartz, s., graf, c., zagar, a. and robertson, k. 2000. therapy focused on lowering postprandial glucose, not fasting glucose, may be superior for lowering hba1c. diabetes care, 23(9):1236–41. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 1048576 /lockdistillerparams false /maxsubsetpct 100 /optimize true 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insights 2006: 1 1–4 1 review correspondence: 11 jalan tan tock seng, singapore 308433. tel: (65) 6357-7533; fax: (65) 6256-9178; email: obgpjt@nus.edu.sg please note that this article may not be used for commercial purposes. for further information please refer to the copyright statement at http://www.la-press.com/copyright.htm neurogenesis and alzheimer’s disease philippe taupin1,2,3 1national neuroscience institute, singapore. 2national university of singapore. 3nanyang technological university, singapore. abstract: alzheimer’s disease (ad) is a neurodegenerative disease, characterized in the brain by amyloid plaque deposits and neurofi brillary tangles. it is the most common form of dementia among older people. there is at present no cure for ad, and current treatments consist mainly in drug therapy. potential therapies for ad involve gene and cellular therapy. the recent confi rmation that neurogenesis occurs in the adult brain and neural stem cells (nscs) reside in the adult central nervous system (cns) provide new opportunities for cellular therapy in the cns, particularly for ad, and to better understand brain physiopathology. hence, researchers have aimed at characterizing neurogenesis in patients with ad. studies show that neurogenesis is increased in these patients, and in animal models of ad. the effect of drugs used to treat ad on neurogenesis is currently being investigated, to identify whether neurogenesis contributes to their therapeutic activities. keywords: neural stem cells, hippocampus, learning and memory, acetylcholinesterase inhibitors, n-methyl-d-aspartateglutamate receptor antagonist, cellular therapy. introduction the recent confi rmation that neurogenesis occurs in the adult brain and nscs reside in the adult cns in various species including humans, is as important for cellular therapy in the cns, particularly for neurodegenerative diseases like ad, as for our understanding of developmental biology (gage, 2000; taupin and gage, 2002). environmental enrichment, drugs, trophic factors, neurotransmitters, and a broad range of physiopathological conditions, including ad, modulate adult neurogenesis (taupin, 2005). recently, using a combination of mouse models and x-irradiation, to inhibit neurogenesis, it was reported that antidepressants, like fl uoxetine, increase hippocampal neurogenesis, which contribute to their behavioral effects (santarelli et al. 2003). researchers have aimed to investigate whether neurogenesis may contribute to the therapeutic effects of drugs used to treat other neurological diseases and disorders, particularly ad (jin et al. 2006). alzheimer’s disease is associated with the loss of nerve cells in areas of the brain that are vital to memory and other mental abilities, like the hippocampus (hardy and selkoe, 2002; st george-hyslop and petit, 2005). hence, cognitive impairments that worsen overtime are major disabilities of ad. two classes of drugs are currently used to treat patients with ad: acetylcholinesterase (ache) inhibitors, like tacrine, donepezil, galantamine and rivastigmine, and an n-methyl-d-aspartate (nmda)-glutamate receptor antagonist, like memantine (arrieta et al. 1998; scarpini et al. 2003; wilkinson et al. 2004; mcshane et al. 2006). these drugs produce improvements in cognitive and behavioral symptoms, but their role in the pathogenesis of ad is unknown. ache inhibitors are thought to improve cognitive functions by enhancing cholinergic neurotransmission in affected brain regions of ad. neurogenesis in alzheimer’s disease jin et al. (2004) studied neurogenesis from autopsies of ad brain patients. the expression of markers for immature neuronal cells, doublecortin, polysialylated nerve cell adhesion molecule and neurogenic differentiation factor, increase in the subgranular zone (sgz), granular layer of the dentate gyrus (dg), and ca1 region of hippocampal ammon’s horn (jin et al. 2004a). the sgz is a layer beneath the granular layer. newly generated neuronal cells in the sgz migrate to the granular layer where they differentiate into neuronal cells of the dg (taupin and gage, 2002). studies also show that neurogenesis is modulated in animal models of ad. animal models have been devised to study genes involved in ad, like presenilin 1 (psen1) and amyloid-beta protein precursor (app) (german and eisch, 2004). drug target insights 2006: 12 philippe taupin psen1 and app are associated with most cases of early-onset ad, a rare hereditary form of dementia (st george-hyslop and petit, 2005). neurogenesis is positively regulated in the dg of transgenic mice that express the swedish and indiana app mutations, a mutant form of human app (jin et al. 2004b), and negatively regulated in the dg and subventricular zone (svz) of knock-out mice for psen1 and app (feng et al. 2001; wen et al. 2002). the dg and svz are the two main regions of the cns where neurogenesis occurs in the adult (taupin and gage, 2002). these animal studies were performed using bromodeoxyurine (brdu) labeling, a thymidine analog that incorporates into the dna of dividing cells during the s-phase of the cell cycle, and is used for birthdating cells and monitoring cell proliferation (miller and nowakowski, 1988; taupin and gage, 2002). the discrepancies between the studies could be explained by the limitation of the transgenic animal models as representative of complex diseases, and to study adult phenotypes, like adult neurogenesis (dodart et al. 2002). particularly, mutant or defi cient mice for single genes, like psen1 and app, may not fully reproduce the features of ad, associated with loss of multiple cell types. four to 10% of nerve cells in regions in which degeneration occurs in ad, like the hippocampus, are tetraploids (yang et al. 2001). nerve cells may have entered the cell cycle and underwent dna replication, but did not complete the cell cycle. it is proposed that cell cycle re-entry and dna duplication precedes neuronal death in degenerating regions of the cns (herrup et al. 2004). as brdu incorporates dna of dividing cells during the sphase of the cell cycle, brdu labeling will not allow to discriminate cell proliferation versus cell cycle re-entry and dna duplication without cell division. the existence of aneuploid cells may account for some of the newly generated neuronal cells observed using brdu-labeling in experimental models of ad. therefore, though reports suggest that neurogenesis is enhanced in ad. this remains to be further confi rmed in the light of recent data showing the existence of tetraploid cells in regions in which degeneration occurs in ad. effect of drugs used to treat ad on neurogenesis researchers have aimed to identify the effect of drugs used to treat ad on neurogenesis. the effects of tacrine, galantamine and memantine on neurogenesis were assessed in adult mice, using the brdu-labeling paradigm (jin et al. 2006). the three drugs increase neurogenesis in the dg and svz by 26–45%, except tacrine that does not alter brdu labeling in the dg. these results show that drugs used to treat ad increase neurogenesis in the adult brain, which may contribute to their therapeutic effects (jin et al. 2006). neurogenesis is enhanced in ad (jin et al. 2004), and drugs used to treat ad also increase neurogenesis. the function of increased neurogenesis in ad brain, and by drugs used to treat ad remain to be elucidated. some speculations can be raised. the increase neurogenesis in ad may represent a regenerative attempt by the cns, to compensate for the loss of nerve cells. it may also represent a compensatory process to increase cns plasticity in the diseased brain (taupin, 2006). one can speculate that drugs used to treat ad would then attempt to amplify such processes. the mechanism of action of these drugs on neurogenesis remains to be elucidated, as well. lesion of the cholinergic forebrain impairs hippocampal neurogenesis in adult rats and muscarinic receptors have been identifi ed on newly generated neuronal cells in the sgz and svz. this suggests that the cholinergic pathway promotes neurogenesis (cooper-kuhn et al. 2004; mohapel et al. 2005). since ache inhibitors are thought to improve cognitive function by enhancing cholinergic neurotransmission in affected brain regions of ad and, tacrine, galantamine may promote neurogenesis through a similar mechanism. the reason why tacrine that do not alter brdu labeling in the dg remains to be further investigated. nmda receptor antagonists on the one hand promote neurodegeneration (ikonomidou et al. 1999). on the other hand, they promote neurogenesis in the adult brain (cameron et al. 1995, 1998; gould et al. 1997; nacher et al. 2001, 2003). therefore, the therapeutic effect of memantine, an nmda-glutamate receptor antagonist, in ad may also be mediated through stimulation of neurogenesis. these hypotheses and the mechanisms of action of drugs used for the treatment of ad remain to be confi rmed, and investigated. stem cell therapy for the treatment of ad because ad is associated with the loss of nerve cells, cellular therapy is considered for the drug target insights 2006: 1 3 neurogenesis and ad treatment of this disease. with the recent evidence that neurogenesis occurs in the adult brain and nscs reside in the adult cns, new strategies for the treatment of neurodegenerative diseases, and particularly ad, are being considered and are promising: the transplantation of adult-derived neural progenitor and stem cells, and the stimulation of endogenous neural progenitor cells. experimental studies reveal that adult derived-neural progenitor and stem cells engraft the host tissues (gage et al. 1995; shihabuddin et al. 2000), and promote functional recovery in animal models of neurodegenerative diseases, like multiple sclerosis (pluchino et al. 2003). in ad, like multiple sclerosis, the degeneration is widespread, therefore direct transplantation of neural progenitor and stem cells in the brain may not offer an optimum strategy for treating these diseases. neural progenitor and stem cell migrate to diseased and injured sites in the brain, when administered by systemic injection (macklis et al. 1993; pluchino et al. 2003; fujiwara et al. 2004). such ways of delivering neural progenitor and stem cells, that are also noninvasive, may prove to be valuable for the treatment of ad. neurogenesis is enhanced in the diseased brain, particularly in ad (jin et al. 2004a). this suggests that the brain has the potential to self-repair, and that endogenous progenitor cells may be recruited to replace degenerated nerve cells and promote functional recovery. future studies will aim at identifying factors that promote neurogeneis in ad, as candidates for cellular therapy. conclusion neurogenesis is enhanced in ad, and drugs used to treat ad, though acting through different mechanisms of action, increase neurogenesis. this may contribute to their therapeutic effects. santarelli et al. (2003) reported using a combination of mouse models and x-irradiation -to inhibit neurogenesis-, that antidepressants, like fluoxetine, increase hippocampal neurogenesis, which contribute to their behavioral activities. meshi et al. (2006), using the same approaches, reported that neurogenesis does not mediate the behavioral effects of environmental enrichment (meshi et al. 2006). inhibition of neurogenesis is therefore key to determine causal relationship between neurogenesis and a physiological or pathological function. therefore, it remains to evaluate the consequence of inhibiting neurogenesis on the effect of drugs used for the treatment of ad, to establish a causal relationship between their therapeutic effects and the stimulation of neurogenesis. nonetheless, the evidence that drugs used to treat ad positively regulate neurogenesis may lead to new drugs design, and new strategies to treat ad. to this aim, unraveling the cellular and molecular mechanisms of action of drugs to treat ad, on neurogenesis, will be a key factor. acknowledgments p.t. is supported by grants from the nmrc, bmrc, and the juvenile diabetes research foundation. references arrieta, j.l. and artalejo, f.r. 1998. methodology, results and quality of clinical trials of tacrine in the treatment of alzheimer’s disease: a systematic review of the literature. age ageing, 27:161–79. cameron, h.a., mcewen, b.s. and gould, e. 1995. regulation of adult neurogenesis by excitatory input and nmda receptor activation in the dentate gyrus. j. neurosci., 15:4687–92. cameron. h.a., tanapat, p. and gould, e. 1998. adrenal steroids and nmethyl-d-aspartate receptor activation regulate neurogenesis in the dentate gyrus of adult rats through a common pathway. neurosci., 82:349–54. cooper-kuhn, c.m., winkler, j. and kuhn, h.g. 2004. decreased neurogenesis after cholinergic forebrain lesion in the adult rat. j. neurosci. res., 77:155–65. dodart, j.c., mathis, c. and bales, k.r. et al. 2002. does my mouse have alzheimer’s disease? genes brain behav., 1:142–55. feng, r., rampon, c. and tang, y.p. et al. 2001. defi cient neurogenesis in forebrain-specifi c presenilin-1 knockout mice is associated with reduced clearance of hippocampal memory traces. neuron, 32:911–26. erratum in: 2002. neuron, 33:313. fujiwara, y., tanaka, n. and ishida, o. et al. 2004. intravenously injected neural progenitor cells of transgenic rats can migrate to the injured spinal cord and differentiate into neurons, astrocytes and oligodendrocytes. neurosci. lett., 366:287–91. gage, f.h., coates, p.w. and palmer, t.d. et al. 1995. survival and differentiation of adult neuronal progenitor cells transplanted to the adult brain. proc. natl. acad. sci., u.s.a., 92:11879–83. gage, f.h. 2000. mammalian neural stem cells. science, 287:1433–8. german, d.c. and eisch, a.j. 2004. mouse models of alzheimer’s disease: insight into treatment. rev. neurosci., 15:353–69. gould, e., mcewen, b.s. and tanapat, p. et al. 1997. neurogenesis in the dentate gyrus of the adult tree shrew is regulated by psychosocial stress and nmda receptor activation. j. neurosci., 17:2492–8. hardy, j. and selkoe, d.j. 2002. the amyloid hypothesis of alzheimer’s disease: progress and problems on the road to therapeutics. science, 297:353–6. erratum in: 2002. science, 297:2209. herrup, k., neve, r. and ackerman, s.l. et al. 2004. divide and die: cell cycle events as triggers of nerve cell death. j. neurosci., 24:9232–9. ikonomidou, c., bosch, f. and miksa, m. et al. 1999. blockade of nmda receptors and apoptotic neurodegeneration in the developing brain. science, 283:70–4. jin, k., peel, a.l. and mao, x.o. et al. 2004a. increased hippocampal neurogenesis in alzheimer’s disease. proc. natl. acad. sci., u.s.a., 101:343–7. jin, k., galvan, v. and xie, l. et al. 2004b. enhanced neurogenesis in alzheimer’s disease transgenic (pdgf-appsw,ind) mice. proc. natl. acad. sci., u.s.a., 101:13363–7. drug target insights 2006: 14 philippe taupin jin, k., xie, l. and mao, x.o. et al. 2006. alzheimer’s disease drugs promote neurogenesis. brain res., 1085:183–8. macklis, j.d. 1993. transplanted neocortical neurons migrate selectively into regions of neuronal degeneration produced by chromophoretargeted laser photolysis. j. neurosci., 13:3848–63. meshi, d., drew, m.r. and saxe, m. et al. 2006. hippocampal neurogenesis is not required for behavioral effects of environmental enrichment. nat. neurosci., 9:729–31. mcshane, r., areosa sastre, a. and minakaran, n. 2006. memantine for dementia. cochrane database syst. rev., 2:cd003154. miller, m.w., nowakowski, r.s. 1988. use of bromodeoxyuridine-immunohistochemistry to examine the proliferation, migration and time of origin of cells in the central nervous system. brain res., 457:44–52. mohapel, p., leanza, g. and kokaia, m. et al. 2005. forebrain acetylcholine regulates adult hippocampal neurogenesis and learning. neurobiol. aging, 26:939–46. nacher, j., rosell, d.r. and alonso-llosa, g. et al. 2001. nmda receptor antagonist treatment induces a long-lasting increase in the number of proliferating cells, psa-ncam-immunoreactive granule neurons and radial glia in the adult rat dentate gyrus. eur. j. neurosci., 13:512–20. nacher, j., alonso-llosa, g. and rosell, d.r. et al. 2003. nmda receptor antagonist treatment increases the production of new neurons in the aged rat hippocampus. neurobiol. aging, 24:273–84. pluchino, s., quattrini, a. and brambilla, e. et al. 2003. injection of adult neurospheres induces recovery in a chronic model of multiple sclerosis. nature, 422:688–94. santarelli, l., saxe, m. and gross, c. et al. 2003. requirement of hippocampal neurogenesis for the behavioral effects of antidepressants. science, 301:805–9. scarpini, e., scheltens, p. and feldman, h. 2003. treatment of alzheimer’s disease: current status and new perspectives. lancet. neurol., 2:539–47. shihabuddin, l.s., horner, p.j. and ray, j. et al. 2000. adult spinal cord stem cells generate neurons after transplantation in the adult dentate gyrus. j. neurosci., 20:8727–35. st. george-hyslop, p.h. and petit, a. 2005. molecular biology and genetics of alzheimer’s disease. c.r. biol., 328:119–30. taupin, p. and gage, f.h. 2002. adult neurogenesis and neural stem cells of the central nervous system in mammals. j. neurosci. res., 69:745–9. taupin, p. 2005. consideration of adult neurogenesis from basic science to therapy. med. sci. monit., 11:le16–17. taupin, p. 2006. adult neurogenesis and neuroplasticity. restor. neurol. neurosci., 24:9–15. wen, p.h., shao, x. and shao, z. et al. 2002. overexpression of wild type but not an fad mutant presenilin-1 promotes neurogenesis in the hippocampus of adult mice. neurobiol. dis., 10:8–19. wilkinson, d.g., francis, p.t. and schwam, e. et al. 2004. cholinesterase inhibitors used in the treatment of alzheimer’s disease: the relationship between pharmacological effects and clinical effi cacy. drugs aging, 21:453–78. yang, y., geldmacher, d.s. and herrup, k. 2001. dna replication precedes neuronal cell death in alzheimer’s disease. j. neurosci., 21:2661–8. 11drug target insights 2014:8 open access: full open access to this and thousands of other papers at http://www.la-press.com. drug target insights introduction several lines of evidence have suggested that impaired cognition is an element of depression and that antidepressant therapy may improve cognitive function.1 classic antidepressants with a central monoaminergic function are associated with a delayed therapeutic response and side effects, thereby limiting their usage. although the pathology of depression and the mechanism of action of antidepressant drugs are well known, the adequacy of current antidepressant targets is questionable. stress is an important risk factor in the development of depression,2 and stress-induced memory impairments are commonly reported.3 therefore, the use of an antidepressant to ameliorate stress-induced cognitive deficits is of therapeutic relevance. drugs that elevate the mood of depressed patients are associated with synaptic effects, that is, the ability to the antidepressant agomelatine improves memory deterioration and upregulates creb and bdnf gene expression levels in unpredictable chronic mild stress (ucms)-exposed mice esen gumuslu1, oguz mutlu2, deniz sunnetci1, guner ulak2, ipek k. celikyurt2, naci cine1, furuzan akar2, hakan savlı1 and faruk erden2 1department of medical genetics, kocaeli university medical faculty, kocaeli, turkey. 2pharmacology, kocaeli university medical faculty, kocaeli, turkey. abstr act: agomelatine, a novel antidepressant with established clinical efficacy, acts as an agonist of melatonergic mt1 and mt2 receptors and as an antagonist of 5-ht2c receptors. the present study was undertaken to investigate whether chronic treatment with agomelatine would block unpredictable chronic mild stress (ucms)-induced cognitive deterioration in mice in passive avoidance (pa), modified elevated plus maze (mepm), novel object recognition (nor), and morris water maze (mwm) tests. moreover, the effects of stress and agomelatine on brain-derived neurotrophic factor (bdnf) and cyclic adenosine monophosphate (camp) response element binding protein (creb) messenger ribonucleic acid (mrna) levels in the hippocampus was also determined using quantitative real-time polymerase chain reaction (rt-pcr). male inbred balb/c mice were treated with agomelatine (10 mg/kg, i.p.), melatonin (10 mg/kg), or vehicle daily for five weeks. the results of this study revealed that ucms-exposed animals exhibited memory deterioration in the pa, mepm, nor, and mwm tests. the chronic administration of melatonin had a positive effect in the pa and +mepm tests, whereas agomelatine had a partial effect. both agomelatine and melatonin blocked stress-induced impairment in visual memory in the nor test and reversed spatial learning and memory impairment in the stressed group in the mwm test. quantitative rt-pcr revealed that creb and bdnf gene expression levels were downregulated in ucms-exposed mice, and these alterations were reversed by chronic agomelatine or melatonin treatment. thus, agomelatine plays an important role in blocking stress-induced hippocampal memory deterioration and activates molecular mechanisms of memory storage in response to a learning experience. key words: agomelatine, melatonin, depression, memory, bdnf, creb citation: gumuslu et al. the antidepressant agomelatine improves memory deterioration and upregulates creb and bdnf gene expression levels in unpredictable chronic mild stress (ucms)-exposed mice. drug target insights 2014:8 11–21 doi: 10.4137/dti.s13870. received: december 11, 2013. resubmitted: january 19, 2014. accepted for publication: february 6, 2014. academic editor: prithviraj bose, editor in chief type: original research funding: author(s) disclose no funding sources. competing interests: author(s) disclose no potential conflicts of interest. copyright: © the authors, publisher and licensee libertas academica limited. this is an open-access article distributed under the terms of the creative commons cc-by-nc 3.0 license. correspondence: oguzmutlu80@hotmail.com http://www.la-press.com http://www.la-press.com http://dx.doi.org/10.4137/dti.s13870 mailto:oguzmutlu80@hotmail.com gumuslu et al 12 drug target insights 2014:8 increase the extracellular levels of serotonin, noradrenaline, and dopamine in the brain.4 the monoamine hypothesis of depression fails to explain all the effects of antidepressants5 and receptor activation as a consequence of the elevation in synaptic monoamines, which represent the primary molecular results of antidepressants. one of the more recent novel antidepressant mechanisms identified is the control of circadian rhythms. melatonin, the major hormone of the pineal gland, is an endogenous agonist of mt1/mt2 receptors and is involved in the regulation of the sleep–wake cycle. the recognition that circadian rhythm desynchronization also plays a key role in mood disorders has led to the development of agomelatine. agomelatine, the first melatonergic antidepressant, is an agonist of the melatonergic mt1 and mt2 receptors 6 and an antagonist of the serotonergic 5-ht2c receptors, 7 and it mimics the actions of melatonin in the synchronization of circadian rhythm patterns in rodents.8 the antidepressant-like activity of agomelatine is attributed to the synergy between these sets of receptors, which are important components of the circadian timing system. agomelatine has demonstrated antidepressant-like activity in several animal models of depression9 and in a transgenic mouse model of depression.10 clinically, it has demonstrated efficacy in major depressive disorders in several trials.11 agomelatine is an effective treatment for depression because it resynchronizes circadian rhythms12 that are disturbed in depression. indeed, it has been speculated for a considerable amount of time that the disorganization of internal circadian rhythms plays a critical role in the development of major depression.13 agomelatine has a favorable clinical profile of antidepressant properties and fewer side effects than traditional antidepressants.11 unpredictable chronic mild stress (ucms) is an important behavioral model that resembles human depression.14 the hippocampus and its connections within limbic–cortical networks may play a crucial role in the pathogenesis of major depression. acute stress and chronic stress disturb hippocampal-dependent memory and prevent the formation of longterm potentialization, which plays a role in the formation of synaptic plasticity and memory. the expression of genes implicated in neuronal plasticity, such as brain-derived neurotrophic factor (bdnf) and cyclic adenosine monophosphate (camp) response element binding protein (creb), have been shown to be downregulated in stressed mice.15 bdnf is a neurotrophin that modulates neuronal plasticity, which is frequently associated with antidepressant treatment.16 neurotrophin expression is activitydependent17 and may be regulated by the light and dark cycle in rats18 and in humans.19 the regulation of the neurotrophin bdnf, whose gene and protein expression and function may be defective in mood disorders,20 has been extensively investigated in recent years as one of the mechanisms of antidepressants. the modulation of bdnf represents a key element in long-term adaptive changes induced by antidepressant drugs. moreover, such molecular analyses were preceded by behavioral assays to investigate the antidepressant activity of agomelatine via the forced swimming test21 and to evaluate the effect of agomelatine on recognition memory in the novel object recognition (nor) task.22 chronic administration of agomelatine leads to the upregulation of bdnf-ltp (longterm potentialization)-related genes and reverses depressionlike symptoms. creb is a core component of the molecular switch that converts short-term memory to long-term memory. recent studies have established the role of creb in learning and memory in mammals in addition to providing insight into the molecular mechanisms of creb regulation and function. the involvement of creb and the upstream signaling pathways leading to its activation in learningassociated plasticity makes them attractive targets for drugs aimed at improving memory function in both diseased and healthy individuals.23 as stress plays an important role in the development of depression, we aimed to investigate whether chronic treatment with agomelatine, a novel antidepressant that has a unique receptor profile as a mt1/mt2 melatonergic agonist 6 and 5-ht2c receptor antagonist, 7 would block ucmsinduced cognitive deterioration in mice in the passive avoidance (pa), modified elevated plus maze (mepm), nor, and morris water maze (mwm) tests. as the genes involved in neurite remodeling are among the primary targets of regulation by chronic stress, the effects of stress and the chronic administration of agomelatine on bdnf and creb messenger ribonucleic acid (mrna) expression in the hippocampus of stressed mice were also determined using quantitative realtime polymerase chain reaction (rt-pcr). methods animals. male, inbred balb/cbyj mice (mam tubi̇tak, gebze, kocaeli, turkey), seven to eight weeks old at their arrival to the laboratory, were used in this study. the animals (four to five per cage) were kept in the laboratory at 21 ± 1.5 °c with 60% relative humidity under a 12 hour light/dark cycle (lights on at 8:00 p.m.) for two weeks before experimentation. the animals were assigned to one of two treatment groups, the non-stressed group (controls) and mice subjected to the ucms procedure. non-stressed mice were housed in groups (eight mice per cage) during the experiment, whereas mice in the stressed group were housed individually in cages (length: 268 mm, width: 135 mm, height: 81 mm) from the start of the chronic stress until the end of the study. all animals received food and water ad libitum. a grouphoused control group was preferred to an individual-housing condition because social isolation is highly stressful for mice and should thus per se contribute to the effects of chronic stress.24,25 all procedures described in this paper were conducted in accordance with the european community council directive for the ethical treatment of animals (86/609/eec) and with the ethical approval of the kocaeli university ethics committee (number: aek 9/3 2010, kocaeli, turkey). http://www.la-press.com agomelatin improves memory in stress exposed mice 13drug target insights 2014:8 all animals were naive to the experimental apparatus, and different animals were used for each test. experimental groups and drug administration. melatonin was purchased from merck chemical company (merck, hohenbrunn, germany), and agomelatine was purchased from wuhan sunrise technology development company limited (wuhan, china). both were dissolved in saline supplemented with 10% dmso. all drugs were freshly prepared and administered in a volume of 0.1 ml per 10 g body weight. the control groups received the same volume of vehicle. at the end of two weeks of drug-free ucms, the mice were assigned to six experimental groups (n = 15 per group) in a semi-randomized manner such that the initial coat state and body weights were equivalent in all of the groups. melatonin (10 mg/kg), agomelatine (10 mg/kg), or vehicle was administered intraperitoneally (i.p.) each day at 17:00 hours (two hours before the lights were turned off ) for five weeks to both stressed and non-stressed animals. on the final day of injections (day 35), agomelatine-, melatonin-, or vehicle-treated mice (n = 7 per group) were sacrificed without behavioral testing to examine the effects of the drugs on the gene expression levels of bdnf and creb. the remaining animals (n = 8 per group) underwent training in the elevated plus maze, pa, and mwm tests. all behavioral testing and tissue and blood sampling were conducted two to five hours following the final injection, between 19:00 and 22:00 hours. ucms procedure. the ucms regimen used in this study was based on the procedure originally designed by willner et al.26 and adapted to mice.27 this stress model consists of repeated mild physical and psychological stressors. mice were subjected to different types of stressors in a chronic, inevitable, and unpredictable way several times a day for seven weeks. stressors were administered in a pseudo-random manner and could occur at any time of night or day. in this respect, the stressor sequence was changed every week to make the stress procedure unpredictable. in all of the experiments, the first two drug-free weeks of ucms were followed by five weeks of ucms application during which the mice were treated with drug or vehicle. for further details on the procedure, see yalcin et al.28 pa test. animals were trained in a one-trial, stepthrough pa apparatus to evaluate memory based on contextual fear conditioning and instrumental learning.29 a decrease in retention latency indicates an impairment in memory in the pa task. the apparatus consisted of a box with an illuminated part (l 7 × 12.5 × h 14 cm) and a dark part (l 24 × 12.5 × h 14 cm), both equipped with a grid floor composed of steel bars (0.3 cm diameter) spaced 0.9 cm apart. the inhibitory avoidance task consisted of two trials. on the first day of training, the mice were individually placed into the light compartment and allowed to explore the boxes. the intercompartment door was opened after a 60 second acclimation period. in the acquisition trial, each mouse was placed in the illuminated compartment, which was lit by a bright bulb (2000 lux). the animals received drugs prior to acquisition training. if the mouse stepped into the dark compartment (2/3 of the tail in the dark compartment), the door was closed by the experimenter, and an inescapable foot shock (0.25 ma/1 second) was delivered through the grid floor of the dark compartment. a cutoff time of five minutes was selected. the time taken to enter the dark compartment (training latency) was recorded. immediately after the shock, the mouse was returned to the home cage. the retention trial started 24 hours after the end of the acquisition trial. each mouse was placed in the illuminated compartment as in the training trial. the door was opened after a 30 second acclimation period. the step-through latency in the retention trial (with a maximum 300 seconds cutoff time) was used as the index of retention of the learned experience. a shock was not applied during the retention trial. mepm test. cognitive behavior was evaluated using the mepm learning task, which measures spatial long-term memory.30 the maze was made of wood and consisted of two open arms (29 × 5 cm) surrounded by a short (1 cm) plexiglas edge to avoid falls and two enclosed arms (29 × 5 × 15 cm) arranged such that the two open arms were opposite to each other. the arms were connected by a central platform (5 × 5 cm). the maze was elevated 40 cm above the floor. the principle of this experiment is based upon the aversion of rodents to open spaces and heights. the animals prefer the enclosed, protected areas of the maze. the procedure was performed as described previously.30,31 during the acquisition session (day 1), each mouse was gently placed at the distal end of an open arm facing away from the central platform. the time required for the mice to move from the open arm to either of the enclosed arms (transfer latency) was recorded. training (repeated exposure of animals to the open arms) shortened this parameter, possibly as a consequence of learning acquisition and retention. if the mouse did not enter the enclosed arm within 90 seconds, it was excluded from further experimentation. animal entry into the enclosed arm required the animal to cross an imaginary line separating the enclosed arm from the central space with all four legs. after entering the enclosed arm, mice were allowed to move freely in the maze in both the open and enclosed arms for 10 seconds. mice were then returned to their home cage. the retention session occurred 24 hours after the acquisition session (on day 2). mice were placed in the open arm, and the transfer latency was recorded again. experiments were conducted between 10:00 and 14:00 hours in a dimly lit, semisoundproof room under natural light. nor. we used a nor test protocol based on that of ennaceur and delacour22 with slight modifications. the apparatus consisted of a circular open field (40 cm diameter and 30 cm height) made of pvc with a black-and-white striped cardboard pattern (30 × 20 cm) nailed to one of the walls and a plexiglas floor. a light bulb above the central section provided constant illumination of approximately 100 lux. the nor http://www.la-press.com gumuslu et al 14 drug target insights 2014:8 task procedure consisted of the following three components: habituation, training, and retention. each mouse was individually habituated to the apparatus for five minutes in the absence of objects (habituation trial). the mouse was placed in the apparatus for the training trial and two identical objects (moon or butterfly) were placed in a symmetrical position 10 cm above the side wall 30 minutes after the habituation trial. the order of objects used for each subject per trial was determined randomly. the total time spent exploring the two objects was recorded by the experimenter over five minutes. exploration of an object was defined as directing the nose toward the object and/or touching it with the nose. after a predetermined retention interval of one hour, the mouse was placed back into the apparatus for the retention trial; however, during this trial, two dissimilar objects were presented, a familiar one and a new one. the object not used in the training trial was used as the novel object in the retention trial. the animals were allowed to explore freely for five minutes and the time spent exploring each object was recorded. if recognition memory was intact, the mouse would be expected to spend more time exploring the novel object.30 the ratio index (ri) was calculated as the time spent exploring the new object (n) divided by the total time exploring both objects (n + r) multiplied by 100. a higher ri was considered to reflect greater memory retention. mwm. the mwm was a circular pool (90 cm diameter and 30 cm height) filled with water (22ºc) to a depth of 14 cm and rendered opaque by the addition of small black balls. the pool was located in a dimly lit, soundproof test room with a various visual cues, including a white and black poster on the wall, a halogen lamp, a camera, and the experimenter. the maze was divided into four quadrants, and three equally spaced points served as starting positions around the edge of the pool. the order of the release positions varied systematically throughout the experiment. a circular escape platform (6 cm diameter and 12 cm high) was located in one quadrant 1 cm above the water surface during the familiarization session and 1 cm below the water surface during the other sessions. video tracking was conducted with a video camera focused on the full diameter of the pool. the navigation parameters were analyzed using the ethovision 3.1 video analysis system (noldus, the netherlands). the mice were trained in the mwm five times daily (familiarization session, s1, s2, s3, s4). one familiarization and four acquisition sessions were performed using the mwm. during the familiarization session and acquisition phase of the experiment, each mouse was subjected to three trials. the delay between the trials was 60 seconds, and a one-day interval was used between each session. for each trial, the mouse was taken from the home cage and placed into the water maze at one of three randomly determined locations with its head facing the center of the water maze. after the mouse found and climbed onto the platform, the trial was stopped, and the escape latency was recorded. if the mouse did not climb onto the platform in 60 seconds, the trial was stopped, and the experimenter guided the mouse to the platform; an escape latency of 60 seconds was recorded. a “probe trial” was used to assess the spatial memory retention of the location of the hidden platform 24 hours after the last acquisition session. during this trial, the platform was removed from the maze, and the mouse was allowed to search the pool for 60 seconds. the percent of time spent in each quadrant was recorded. tissue sampling, rna isolation, and quantitative rt-pcr. one day after the final stress session, the mice were decapitated by cervical dislocation. the left and right hippocampi were surgically removed and stored in liquid nitrogen. total rna was isolated with the rneasy mini kit extraction procedure (qiagen, valencia, ca, usa). briefly, tissues were homogenized in rlt lysis buffer containing β-mercaptoethanol using a thermo savant fastprep fp120 homogenizer. sample homogenates were applied to rneasy mini spin columns (qiagen) and processed according to the manufacturer’s instructions. an on-column dnase digestion was performed to remove any residual genomic dna contamination. rna samples were eluted in rnase-free water, and the concentration was measured spectrophotometrically using the nanodrop nd-1000 spectrophotometer (nanodrop nd-1000; nanodrop technologies, wilmington, de). subsequently, cdna was synthesized using a revertaid first strand cdna synthesis kit (fermentas inc., maryland, usa). quantitative rt-pcr was performed according to the methods described in previous studies.32,33 standard curves were obtained via serial dilutions of the beta-globulin gene. primers specific to the genes under investigation (table 1) were obtained from integrated dna technologies (iowa, usa) and iontek inc. (merter, istanbul, turkey). the gene expression values obtained were normalized using the bact housekeeping gene. gene expression levels were calculated with the rest (relative expression software tool) program. changes in the creb and bdnf gene expression levels were calculated in the stress-exposed and non-stressed (n = 7/each group) animal groups. the effects of agomelatine and melatonin (n = 7/each group) on creb and bdnf expression levels were also evaluated in the stress-exposed group. statistics one-way analysis of variance (anova) and the post-hoc tukey’s test were used to analyze the mepm, mwm, and nor tests. to evaluate the differences among drug treatment groups during the first and second transfer latencies in the pa test, the kruskal–wallis non-parametric test was used followed by dunn’s post-hoc test. data are expressed as the mean values ± sem. p , 0.05 was accepted as statistically significant. statistical evaluation of bdnf and creb gene expressions was performed with the rest (relative expression software tool) program. http://www.la-press.com agomelatin improves memory in stress exposed mice 15drug target insights 2014:8 results effects of drugs on learning and memor y in the pa test. there was no significant difference in first day latency among the groups (h = 6.98, p . 0.05, figure 1a). the second day latency (retention latency) significantly differed between the groups (h = 17.82, p = 0.003). stress significantly shortened the retention latency compared to the nonstressed control group (p , 0.05). melatonin prolonged the retention latency in stressed animals (p , 0.05), while agomelatine had a partial but statistically insignificant effect (fig. 1b). effects of drugs on learning and memory in the mepm test. after chronic injection of melatonin (10 mg/kg) or agomelatine (10 mg/kg) for five weeks, there was no significant difference in first day latency (tl1) among the groups [f(5,35) = 2.22; p = 0.07, figure 2a]. tl2 (latency on the second day) was significantly different when all groups were compared [f(5,35) = 3.38; p = 0.01, figure 2b]. tl2 significantly increased in the stressed control group compared to the non-stressed control group (p , 0.05), and this effect was significantly reversed by melatonin (p , 0.05), while agomelatine had a partial effect but failed to reach to a statistically significant value (fig. 2b). effects of drugs on visual memory in the nor test. a significant difference was observed between the groups [f(5,41) = 9.80; p , 0.001] when the effects of melatonin or agomelatine were evaluated during the retention trial of the nor test. the ri between the stressed control and nonstressed control mice were significantly different (p , 0.001). both melatonin and agomelatine significantly increased the table 1. primary sequences of genetic studies. gene primary sequence beta2 microglobulin (f) 5’ tga ctt tgt cac agc cca aga ta 3’ (r) 5’ aat cca aat gcg gca tct tc 3’ bact (f) 5’ agc cat gta cgt agc cat cca 3’ (r) 5’ tct ccg gag tcc atc aca atg3’ creb (f) 5’ agc tgg cct gtc cca ctg ct 3’ (r) 5’ acc att ctg aac aca aag cag cca3’ bdnf (f) 5’ gcc caa cga aga aaa cca taa 3’ (r) 5’ gga ggc tcc aaa ggc act t 3’ pa test 0 10 20 30 40 50 60 70 drugs f ir s t d a y l a te n c y nc nm na sc sm sa pa test 0 10 20 30 40 50 60 70 80 90 drugs r e te n ti o n l a te n c y nc nm na sc sm sa a b figure 1. effects of melatonin (10 mg/kg) or agomelatine (10 mg/kg) on (a) first day latency and (b) retention latency in the passive avoidance test in mice. notes: the data are expressed as the mean ± sem values. *p , 0.05 vs. non-stressed control group. #p , 0.05 vs. stressed control group. http://www.la-press.com gumuslu et al 16 drug target insights 2014:8 mepm test 0 10 20 30 40 50 60 70 drugs t l -1 nc nm na sc sm sa mepm test 0 2 4 6 8 10 12 14 drugs t l -2 nc nm na sc sm sa a b figure 2. effects of melatonin (10 mg/kg) or agomelatine (10 mg/kg) on (a) transfer latency on the first day (b) transfer latency on the second day. notes: the data are expressed as mean ± sem values. *p , 0.05 vs. non-stressed control group. #p , 0.05 vs. stressed control group. nor test 0 10 20 30 40 50 60 70 80 90 drugs r a ti o i n d e x nc nm na sc sm sa figure 3. effect of melatonin (10 mg/kg) or agomelatine (10 mg/kg) on the ri in the novel object recognition test. notes: the data are expressed as mean ± sem values. *p , 0.001 vs. non-stressed control group. #p , 0.001 vs. stressed control group. ri compared to stress-exposed control mice (p , 0.001) (fig. 3). effects of drugs on learning and memory in the mwm test. there was a significant difference in escape latency in all sessions during the evaluation of drug groups [f(5,41) = 8.22, p , 0.001; f(5,41) = 10.77, p , 0.001; f(5,41) = 8.93, p , 0.001; f(5,41) = 12.40, p , 0.001; f(5,41) = 10.32, p , 0.001, respectively; figure 4a]. stress significantly increased the escape latency during all sessions (p , 0.001) in control animals, whereas both melatonin and agomelatine http://www.la-press.com agomelatin improves memory in stress exposed mice 17drug target insights 2014:8 mwm test 0 20 40 60 80 100 120 140 160 sessions nc nm na sc sm sa mwm test 0 5 10 15 20 25 30 35 40 drugs nc nm na sc sm sa nc nm na sc sm sa nc nm na sc sm sa mwm test 0 5 10 15 20 25 30 35 40 drugs mwm test 0 5 10 15 20 25 drugs e s c a p e l a te n c y ( s ) t im e s p e n t in t a rg e t q u a d ra n t in p ro b e t ri a l (s ) m e a n d is ta n c e t o p la tf o rm i n p ro b e t ri a l (c m ) s p e e d ( c m /s ) a b c d fam ses 1st ses 2nd ses 3rd ses 4th ses figure 4. effects of melatonin (10 mg/kg) or agomelatine (10 mg/kg) on (a) escape latency, (b) the time spent in escape platform quadrant, (c) mean distance to platform, and (d) swim speed in the probe trial (60 seconds) of the mwm test. the data are expressed as the mean ± sem values. *p , 0.001 vs. non-stressed control group. #p , 0.05, ##p , 0.01, ###p , 0.001 vs. stressed control group. significantly shortened the escape latency in the familiarization session (p , 0.05 and p , 0.01; respectively) and in the other sessions (p , 0.001) in stressed animals (fig. 4a). a significant difference was observed among all drug groups in the time spent in the target quadrant in probe trial of mwm test [f(5,41) = 6.27; p = 0.0003; figure 4b]. stressed control animals significantly decreased the time spent in the escape platform quadrant (p , 0.001) compared to non-stressed animals, and both melatonin (p , 0.05) and agomelatine reversed this effect; agomelatine had a higher impact than melatonin (p , 0.01; figure 4b). the mean distance traveled by the mice to the platform in the probe trial of the mwm test was significantly different between the drug groups [f(5,41) = 3.98; p = 0.005; http://www.la-press.com gumuslu et al 18 drug target insights 2014:8 figure 4c]. stress significantly increased the mean distance traveled to the platform (p , 0.01) compared to the nonstressed control group. melatonin (p , 0.05) and agomelatine significantly reversed this effect; agomelatine had a higher impact (p , 0.01; figure 4c). each treatment group did not significantly differ in swimming speed [f(5,41) = 1.32; p = 0.27; figure 4d] in the probe trial of the mwm test. effects of drugs on creb and bdnf gene expression. in evaluating plasticity-related genes, we measured mrna levels in the hippocampus of mice subjected to ucms/drug treatment using quantitative rt-pcr. decreased bdnf and creb expression might indicate both stress and cognitive impairment. to determine whether downregulation of gene expression can be prevented by antidepressant treatment, melatonin or agomelatine was administered for 35 days to mice subjected to ucms. our results demonstrated that mrna levels of the neurotrophin family member bdnf, which has been studied in relation to the stress response, were reduced in the hippocampus of the mice subjected to ucms. we also measured hippocampal mrna expression levels for creb, a transcription factor that regulates bdnf in response to drug treatment. ucms caused a reduction in creb mrna levels in stressed animals. melatonin or agomelatine treatment significantly reversed ucms-induced downregulation of creb and bdnf gene expression. our results are in agreement with data from similar studies.34,35 the gene expression observed in each group is shown in table 2. discussion stress is a known risk factor in the development of many neuropsychiatric disorders, including depression.2 moreover, because stress-induced memory impairment is commonly reported in stress-related psychopathologies,3 there is therapeutic relevance of the use of antidepressant treatments to prevent stress-induced cognitive deficits.1 both chronic mild stress and learned helplessness significantly diminish the cognitive performance of mice in the mwm test, and animals treated with antidepressants exhibit significantly enhanced cognitive performance.35 the chronic administration of agomelatine produced antidepressant-like effects in the chronic mild stress model of depression36 and improved learned helplessness in a model of depression-induced avoidance learning deficits. in our study, stress-induced a significant deterioration of memory in the pa, mepm, nor, and mwm tests, and agomelatine ameliorated these effects in nor and mwm tests while it had a partial effect in the pa and mepm tests. agomelatine, a novel antidepressant with established clinical efficacy,6 is an agonist of the melatonin mt1 and mt2 receptors 37 and has a potent antagonistic activity on serotonergic 5-ht2c receptors. 7 the affinity of agomelatine for melatonin receptors is comparable with that of melatonin.38 melatonin produced in the pineal gland during periods of darkness plays a key role in the regulation of circadian rhythms.8 it has a short half-life and is extensively metabolized, leading to poor bioavailability. moreover, the antidepressant-like activity of agomelatine in the rat cms model of depression is independent of the time of drug administration, while melatonin has no antidepressant-like activity after administration in the morning.9 the search for metabolically stable analogs with new and innovative properties resulted in the discovery of agomelatine.39 agomelatine-induced molecular changes may play a role in its antidepressant and pro-cognitive effects and may be attributed to synergy between the 5-ht2c antagonist and melatonergic agonist properties of the drug.6 agomelatine increased the release of noradrenaline and dopamine in accordance with its 5-ht2c antagonist properties. 7 these may in turn be associated with the functional output of β-adrenergic d1 and d2 receptors and activation or inhibition of the camppka pathway, which is postulated to modulate microtubule dynamics40 and synaptic plasticity.41 numerous findings indicate that reduced function of 5-ht2c receptors may be involved in the mechanism by which antidepressants alleviate depression.42 the 5-ht2c receptor is involved in circadian rhythm resynchronization,43 and 5-ht2c receptor antagonists prevent the inhibitory effects of light on melatonin synthesis. in mammals, specific m1 and m2 receptors are located mainly in suprachiasmatic nuclei in the central nervous system and in some peripheral sites. moreover, the mt1 and mt2 agonistic properties of agomelatine might also play a role because both receptors modulate several signaling pathways such as pka and protein kinase c (pkc), which have been implicated in synaptic plasticity regulation.40 circadian rhythms are disturbed in depressed patients,44 and the ucms procedure causes a generalized disorganization of circadian rhythms, which is suggested to play an important role in the pathophysiology of depression, among other biochemical, physiological, and behavioral impairments.45 agomelatine can resynchronize experimentally disturbed circadian rhythms,36 an effect that is independent of the time of administration.36 table 2. gene expression levels in ucmsexposed mice and effects of drugs on gene expressions in ucmsexposed mice. melatonin (10 mg/kg) or agomelatine (10 mg/kg) was given intraperitoneally for 35 days to mice subjected to unpredictable chronic mild stress (ucms) (n = 7/each group). all of the treatments begun after 2 weeks of stress regimen and were administered during 5 weeks. groups creb bdnf ucms + vehicle 2,408 ↓ 1,208 ↓ ucms + melatonin 6,409 ↑ 1,164 ↑ ucms + agomelatine 2,088 ↑ 1,060 ↑ notes: ↓, decrease in expression. ↑, increase in expression. abbreviations: creb, cyclic adenosine monophosphate (camp) response element binding protein; bdnf, brain-derived neurotrophic factor. http://www.la-press.com agomelatin improves memory in stress exposed mice 19drug target insights 2014:8 basic and clinical studies provide evidence for the neurotrophic hypothesis of depression and antidepressant activity.20 the neurotrophin family member bdnf is involved in neuronal differentiation and survival as well as the synaptic plasticity associated with learning and memory.46 the camp signaling pathway (in particular, the downstream effector creb) has also been shown to play an important role in neuronal and synaptic plasticity.47 therefore, decreased expression of bdnf or creb could contribute to the atrophy of the hippocampus in response to stress, and the upregulation of bdnf and creb could contribute to the action of antidepressant therapy.48 it has been postulated that chronic stress caused a downregulation of hippocampal bdnf or creb levels and that this reduction could be upregulated through antidepressant therapy.48 moreover, molteni et al.49 demonstrated that acute agomelatine treatment modulates the expression of bdnf through a functional interaction between melatonergic mt1/ mt2 and serotonergic 5-ht2c receptors, supporting the concept that intracellular events can be regulated via the synergistic activity of different neuromodulatory systems. our results are consistent with this hypothesis and demonstrate that agomelatine treatment improves ucms-induced memory deterioration and upregulates hippocampal creb and bdnf gene expression levels. bdnf belongs to the neurotrophic factor family and plays a crucial role in the development, regeneration, survival, and maintenance of neuronal function in the central nervous system.16 these neurotrophins are abundantly expressed in the hippocampus,50 where they are important modulators of spatial learning51 and activity-dependent synaptic plasticity, such as long-term potentiation.52 moreover, bdnf plays an important role in the formation, retention, and recall of spatial memory, and decreases in bdnf expression result in the impairment of spatial learning and memory.53 the results of clinical studies have shown that depressive patients exhibit diminished plasma bdnf levels and that antidepressant treatment increases plasma bdnf levels.54 interestingly, the expression of bdnf is also influenced by light and dark cycles in rats55 as well as in humans.19 it is speculated that acute agomelatine treatment can upregulate the expression of bdnf mrna levels in the prefrontal cortex through the functional interaction between melatonergic mt1/mt2 and serotonergic 5-ht2c receptors,56 thus preventing the circadian downregulation of the neurotrophin. soumier et al.57 postulated that agomelatine produced major transcriptional changes in the hippocampus, where significant upregulation of bdnf was observed. moreover, the levels of bdnf protein were elevated by agomelatine in both the hippocampus and the prefrontal cortex.58 these findings support the hypothesis that alteration of hippocampal bdnf expression is correlated with antidepressant response in the hippocampus.16 chronic agomelatine treatment decreased bdnf expression in the amygdale and this effect might be related to a negative feedback mechanism in response to the high magnitude of neuronal remodeling or to a distinct and yet unknown neurochemical event.57 chronic agomelatine has neurogenic effects in the hippocampus in rats57 and a reversed depressioninduced decrease in neurogenesis.59 agomelatine exposure increases neurite outgrowth of granule cells in hippocampal primary cell culture and accelerates the maturation of newly formed granule cells in rats.57 the results of our study revealed that chronic agomelatine increased bdnf expression in the hippocampus, and our findings are consistent with those of recent studies.57,60 these findings provide new information regarding the molecular mechanisms that contribute to the chronic effects of the new antidepressant agomelatine on brain function. the ability of agomelatine to modulate the expression of these neuroplastic molecules, which follow a circadian rhythm, may contribute to its antidepressant action. bdnf is involved in the etiology of mood disorders, and it is thought to participate in the structural remodeling associated with antidepressant therapy.58 as neurotrophin expression is activity-dependent17 and may be regulated by the light and dark cycle in rats18 and humans,19 it may be inferred that its transcription can be modulated by acute agomelatine administration and may represent a downstream target of its synaptic effects. on this basis, we investigated bdnf mrna levels in the rat hippocampus. the camp-creb signal transduction cascade is known to be responsible for the sustained alterations that occur in cellular and behavioral models of learning and memory.61 chronic but not acute antidepressant administration, including norepinephrine and selective serotonin reuptake inhibitors, increases creb expression, phosphorylation, and function in limbic brain structures including the hippocampus and cerebral cortex.62 agomelatine has a favorable clinical safety and tolerability profile of antidepressant properties with few side effects and no withdrawal syndrome.11 in contrast to ssris,63 it has few sexual side effects and there are no reports of impotence, ejaculation difficulties, or decreased libido. the novel mode of action of agomelatine (selective binding profile, not inducing serotonin release or increasing extracellular serotonin levels and no effect on 5-ht1 a receptors)7 might be responsible for its favorable safety profile. overall, the present study suggests that chronic administration of the novel antidepressant agomelatine, with its distinct mechanism of action based on synergy between the melatonergic and 5-ht2c pathways and the advantages of a favorable clinical safety/tolerability profile, improves memory deterioration and upregulates creb and bdnf gene expression levels in ucms-exposed mice. thus, agomelatine appears to play an important role in blocking stress-induced hippocampal memory deterioration and activates the molecular mechanisms of memory storage in response to a learning experience. http://www.la-press.com gumuslu et al 20 drug target insights 2014:8 author contributions eg, om, ds, and gu conceived and designed the experiments. eg, om, ds, ikc, and nc analyzed the data. eg, om, gu, and ikc wrote the first draft of the manuscript. nc, fa, hs, and fe contributed to the writing of the manuscript. eg, om, ds, gu, ikc, nc, fa, hs, and fe agree with manuscript results and conclusions. eg, om, gu, fa, hs, and fe jointly developed the structure and arguments for the paper. fa, hs, and fe made critical revisions and approved final version. all authors reviewed and approved the final manuscript. disclosures and ethics as a requirement of publication the authors have provided signed confirmation of their compliance with ethical and legal obligations including but not limited to compliance with icmje authorship and competing interests guidelines, that the article is neither under consideration for publication nor published elsewhere, of their compliance with legal and ethical guidelines concerning human and animal research participants (if applicable), and that permission has been obtained for reproduction of any copyrighted material. this article was subject to blind, independent, expert peer review. the reviewers reported no competing interests. references 1. yau jl, noble j, hibberd c, et al. chronic treatment with the antidepressant amitriptyline prevents impairments in water maze learning in aging rats. j neurosci. 2002;22:1436–42. 2. bremner jd, vermetten e. stress and development: behavioral and biological consequences. dev psychopathol. 2001;13:473–89. 3. de quervain dj, roozendaal b, nitsch rm, mcgaugh jl, hock c. acute cortisone administration impairs retrieval of long-term declarative memory in humans. nat neurosci. 2000;3:313–4. 4. hirschfeld rm. history and evolution of the monoamine hypothesis of depression. j clin psychiatry. 2000;61(6):4–6. 5. castren e. is mood chemistry? nat rev neurosci. 2005;6:241–6. 6. audinot v, mailliet f, lahaye-brasseur c, et al. new selective ligands of human cloned melatonin mt1 and mt2 receptors. naunyn schmiedebergs arch pharmacol. 2003;367:553–61. 7. millan mj, gobert a, lejeune f, et al. the novel melatonin agonist agomelatine (s20098) is an antagonist at 5-hydroxytryptamine2c receptors, blockade of which enhances the activity of frontocortical dopaminergic and adrenergic pathways. j pharmacol exp ther. 2003;306:954–64. 8. pitrosky b, kirsch r, malan a, mocaer e, pevet p. organization of rat circadian rhythms during daily infusion of melatonin or s20098, a melatonin agonist. am j phys. 1999;277:812–28. 9. bertaina-anglade v, la rochelle cd, boyer pa, mocaer e. antidepressant-like effects of agomelatine (s 20098) in the learned helplessness model. behav pharmacol. 2006;17:703–13. 10. barden n, shink e, labbe m, vacher r, rochford j, mocaer e. antidepressant action of agomelatine (s 20098) in a transgenic mouse model. prog neuropsychopharmacol biol psychiatry. 2005;29:908–16. 11. kennedy sh, emsley r. placebo-controlled trial of agomelatine in the treatment of major depressive disorder. eur neuropsychopharm. 2006;16:93–100. 12. quera salva ma, vanier b, laredo j, et al. major depressive disorder, sleep eeg and agomelatine: an open label study. int j neuropsychopharmacol. 2007;10:691–6. 13. mcclung ca. circadian genes, rhythms and the biology of mood disorders. pharmacol ther. 2007;114:222–32. 14. willner p. validity, reliability and utility of the chronic mild stress model of depression: a 10 years review and evaluation. psychopharmacology. 1997;134:319–29. 15. gumuslu e, mutlu o, sunnetci d, et al. the effects of tianeptine, olanzapine and fluoxetine on the cognitive behaviors of unpredictable chronic mild stressexposed mice. drug res. 2013;63:532–9. 16. tardito d, perez j, tiraboschi e, musazzi l, racagni g, popoli m. signaling pathways regulating gene expression, neuroplasticity, and neurotrophic mechanisms in the action of antidepressants: a critical overview. pharmacol rev. 2006;58:115–34. 17. bramham cr, messaoudi e. bdnf function in adult synaptic plasticity: the synaptic consolidation hypothesis. prog neurobiol. 2005;76:99–125. 18. cirelli c, tononi g. differential expression of plasticity related genes in waking and sleep and their regulation by the noradrenergic system. j neurosci. 2000;20:9187–94. 19. begliuomini s, lenzi e, ninni f, et al. plasma brain-derived neurotrophic factor daily variations in men: correlation with cortisol circadian rhythm. j endocrinol. 2008;197:429–35. 20. calabrese f, molteni r, racagni g, riva ma. neuronal plasticity: a link between stress and mood disorders. psychoneuroendocrinology. 2009;34(1):208–16. 21. porsolt rd, le pichon m, jalfre m. depression: a new animal model sensitive to antidepressant treatments. nature. 1977;266:730–2. 22. ennaceur a, delacour j. a new one-trial test for neurobiological studies of memory in rats. 1 behavioral data. behav brain res. 1988;31:47–59. 23. barco a, pittenger c, kandel er. creb, memory enhancement and the treatment of memory disorders: promises, pitfalls and prospects. expert opin ther targets. 2013;7(1):101–14. 24. muscat r, willner p. suppression of sucrose drinking by chronic mild unpredictable stress: a methodological analysis. neurosci biobehav rev. 1992;16:507–17. 25. arbe md, einstein r, lavidis na. stressful animal housing conditions and their potential effect on sympathetic neurotransmission in mice. am j physiol regul integr comp physiol. 2002;282:1422–8. 26. willner p, muscat r, papp m. chronic mild stress-induced anhedonia: a realistic animal model of depression. neurosci biobehav rev. 1992;16:525–34. 27. ducottet c, belzung c. behaviour in the elevated plus-maze predicts coping after subchronic mild stress in mice. physiol behav. 2004;81:417–26. 28. yalcin i, aksu f, belzung c. effects of desipramine and tramadol in a chronic mild stress model in mice are altered by yohimbine but not by pindolol. eur j pharmacol. 2005;514:165–74. 29. ogren so, johansson c, magnusson o. forebrain serotonergic involvement in avoidance learning, neurosci lett. 1985;58:305–9. 30. reddy ds, kulkarni sk. possible role of nitric oxide in the nootropic and antiamnesic effects of neurosteroids on agingand dizocilpine-induced learning impairment. brain res. 1998;799:215–29. 31. hlinak z, krejci i. concurrent administration of subeffective doses of scopolamine and mk-801 produces a short-term amnesia for the elevated plus-maze in mice. behav brain res. 1998;91:83–9. 32. savlı h, aalto y, nagy b, knuutila s, pakkala s. gene expression analysis of 1,25(oh)2d3 dependent differentiation of hl-60 cells—a cdna array study. br j haematol. 2002;118(4):1065–72. 33. savlı h, karadenizli a, kolaylı f, gundes s, ozbek u, vahaboglu h. expression stability of six housekeeping genes: a proposal for resistance gene quantification studies of pseudomonas aeruginosa by real-time quantitative rt-pcr. j med microbiol. 2003;52:403–8. 34. alfonso j, frasch ac, flugge g. chronic stress, depression and antidepressants: effects on gene transcription in the hippocampus. rev neurosci. 2005;16:43–56. 35. song l, che w, min-wei w, murakami y, matsumoto k. impairment of the spatial learning and memory induced by learned helplessness and chronic mild stress. pharmacol biochem behav. 2006;83:186–93. 36. papp m, gruca p, boyer pa, mocaer e. effect of agomelatine in the chronic mild stress model of depression in the rat. neuropsychopharmacol. 2003;28:694–703. 37. ying sw, rusak b, delagrange p, mocaer e, renard p, guardiolalemaitre b. melatonin analogues as agonists and antagonists in the circadian system and other brain areas. eur j pharmacol. 1996;296:33–42. 38. bonnefond c, martinet l, lesieur d, adam g, guardiola-lemaître b. characterization of s-20098, a new melatonin analogue. in: touitou y, arendt j, pevet p, eds. melatonin and the pineal gland. amsterdam: elsevier science publishers; 1993:123–6. 39. guardiola-lemaitre b. melatoninergic receptor agonists and antagonists: pharmacological aspects and therapeutic perspective. ann pharm fr. 2005;63:385–400. 40. bianchi m, hagan jj, heidbreder ca. neuronal plasticity, stress and depression: involvement of the cytoskeletal microtubular system? curr drug targets cns neurol disord. 2005;4:597–611. 41. warner-schmidt jl, duman rs. hippocampal neurogenesis: opposing effects of stress and antidepressant treatment. hippocampus. 2006;16:239–49. 42. sanchez c, hyttel j. comparison of the effects of antidepressants and their metabolites on reuptake of biogenic amines and on receptor binding. cell mol neurobiol. 1999;19:467–89. 43. kennaway dj, moyer rw. serotonin 5-ht2c agonists mimic the effect of light pulses on circadian rhythms. brain res. 1998;806:257–70. 44. wirz-justice a. biological rhythms in mood disorders. in: bloom fe, kupfer dj, eds. psychopharmacology: the fourth generation of progress. new york: raven pres; 1995:999–1017. 45. wehr ta, wirz-justice a. circadian rhythm mechanisms in affective illness and in antidepressant drug action. pharmacopsychiatry.1982;15:31–9. 46. branchi i, francia n, alleva e. epigenetic control of neurobehavioural plasticity: the role of neurotrophins. behav pharmacol. 2004;15:353–62. 47. schulman h. protein phosphorylation in neuronal plasticity and gene expression. curr opin neurobiol. 1995;5:375–81. http://www.la-press.com agomelatin improves memory in stress exposed mice 21drug target insights 2014:8 48. duman rs, malberg j, nakagawa s, d’sa c. neuronal plasticity and survival in mood disorders. biol psychiatry. 2000;48:732–9. 49. molteni r, calabrese f, cattaneo a, et al. acute stress responsiveness of the neurotrophin bdnf in the rat hippocampus is modulated by chronic treatment with the antidepressant duloxetine. neuropsychopharmacology. 2009;34:1523–32. 50. ernfors p, ibáñez cf, ebendal t, olson l, persson h. molecular cloning and neurotrophic activities of a protein with structural similarities to nerve growth factor: developmental and topographical expression in the brain. proc natl acad sci u s a. 1990;87:5454–8. 51. scaccianoce s, del bianco p, caricasole a, nicoletti f, catalani a. relationship between learning, stress and hippocampal brain-derived neurotrophic factor. neuroscience. 2003;121:825–8. 52. lu b. bdnf and activity-dependent synaptic modulation. learn mem. 2003;10:86–98. 53. mu js, li wp, yao zb, zhou xf. deprivation of endogenous brain-derived neurotrophic factor results in impairment of spatial learning and memory in adult rats. brain res. 1999;835:259–65. 54. aydemir c, yalcin es, aksaray s, et al. brain-derived neurotrophic factor (bdnf) changes in the serum of depressed women. prog neuropsychopharmacol biol psychiatry. 2006;30:1256–60. 55. bova r, micheli mr, qualadrucci p, zucconi gg. bdnf and trkb mrnas oscillate in rat brain during the light-dark cycle. brain res mol brain res. 1998;57:321–4. 56. molteni r, calabrese f, pisoni s, et al. synergistic mechanisms in the modulation of the neurotrophin bdnf in the rat prefrontal cortex following acute agomelatine administration. world j biol psychiatry. 2010;11:148–53. 57. soumier a, banasr m, lortet s, et al. mechanisms contributing to the phasedependent regulation of neurogenesis by the novel antidepressant, agomelatine, in the adult rat hippocampus. neuropsychopharmacol. 2009;34:2390–403. 58. calabrese f, molteni r, maj pf, et al. chronic duloxetine treatment induces specific changes in the expression of bdnf transcripts and in the subcellular localization of the neurotrophin protein. neuropsychopharmacology. 2007;32:2351–9. 59. morley-fletcher s, mairesse j, soumier a, et al. chronic agomelatine treatment corrects behavioral, cellular, and biochemical abnormalities induced by prenatal stress in rats. psychopharmacology (berl). 2011;217(3):301–13. 60. ladurelle n, gabriel c, viggiano a, mocaër e, baulieu ee, bianchi m. agomelatine (s20098) modulates the expression of cytoskeletal microtubular proteins, synaptic markers and bdnf in the rat hippocampus, amygdala and pfc. psychopharmacology. 2012;221:493–509. 61. barad m, bourtchouladze r, winder dg, golan h, kandel e. rolipram, a type iv specific phosphodiesterase inhibitor, facilitates the establishment of long-lasting long-term potentiation and improves memory. proc natl acad sci usa. 1998;95:15020–5. 62. thome j, sakai n, shin k, et al. camp response element-mediated gene transcription is upregulated by chronic antidepressant treatment. j neurosci. 2000;20:4030–6. 63. ferguson jm, shrisvastava rk, stahl sm, et al. re-emergence of sexual dysfunction in patients with major depressive disorder: doubleblind comparison of nefadozone and sertraline. j clin psychiatry. 2001;62:24–9. http://www.la-press.com untitled drug target insights 2007: 2 1–7 1 original research long-term l-carnitine administration reduces erythropoietin resistance in chronic hemodialysis patients with thalassemia minor biagio r. di iorio1, pasquale guastaferro2, nicola cillo1, emanuele cucciniello1 and vincenzo bellizzi1 1unità operativa di nefrologia e dialisi, ospedale “a landolfi”, solofra (av), italy. 2unità operativa di nefrologia e dialisi, ospedale, san angelo dei lombardi (av), italy. abstract background and aim: both thalassemia and carnitine defi ciency represent independent causes of erythropoietin resistance, and thus anemia, in uremic patients. we evaluated the unknown long-term effects of l-carnitine administration in β-thalassemic on chronic hemodialysis. methods: we studied twelve subjects (m = 8; f = 4) affected by β-thalassemia minor (β-thal; hba2 level = 6.6 ± 0.6%) and forty non-thalassemic subjects (m = 24; f = 16) as controls (c), on chronic hemodialysis treatment. patients and controls were at target hemoglobin levels (11–12g/dl) prior to the study and underwent to i.v. l-carnitine administration for a one year period-time. results: groups were comparable for age, gender, serum levels of hemoglobin (hb), iron, ferritine, pth and aluminum, transferrin saturation, and dialysis modalities. during the study both groups showed signifi cant hb increase and erythropoietin (epo) decrease; as a difference, such changes emerged at the 3rd month in c but at the 8th month in β-thal. at start, during the dialysis session the erythrocyte mcv reduced in c but not in β-thal (65.3 ± 3.2 to 65.5 ± 3.2 fl ; ns); along carnitine administration period, however, mcv during dialysis decreased also in β-thal, starting since the 9th month of treatment. conclusion: this study provides evidence of the lowering of epo resistance in β-thalassemia patients on hemodialysis due to long-term carnitine administration. thus, prolonged carnitine supplementation should be suggested to patients on dialysis affected by β-thalassemia with poorly responsive anemia, or requiring large doses of erythropoietin. keywords: l-carnitine, thalassemia minor, hemodialysis. introduction anemia is very common in chronic dialysis patients and it is related to relative erythropoietin defi ciency, to blood losses from both gastrointestinal tract and dialysis fi lter or lines, and to blood drawings for laboratory tests (1–4). also, reduced levels of hemopoiesis compounds (iron, folic acid, vitamin b12) and reduced erythrocytes life are major contributors to anemia in dialysis (1–4). in addition, carnitine defi ciency represents an independent cause of erythropoietin resistance in uremic patients. indeed, the lack of an effi cient carnitine pathway in uremia further reduces the erythrocyte half-life and the erythropoietin effectiveness (5–8); likely, the impairment of carnitine metabolism in uremia induces the worsening of erythrocyte membrane functioning (9–10). it has been demonstrated that carnitine administration in dialysis patients improves the management of anemia and reduces the erythropoietin resistance (11–12). β-thalassemia minor (β-thal) represents a not rare cause of erythropoietin resistance in uremia in several regions, such as mediterranean areas, but worldwide due to migrations. indeed, in patients with β-thal on chronic dialysis, higher erythropoietin doses are needed to correct anemia, even if all the major known factors that contribute to erythropoietin resistance are corrected (13–15). the impact of carnitine defi ciency on erythropoietin resistance in β-thalassemic patients on hemodialysis, however, has never been explored. thus, the aim of this study was to evaluate the long-term effect of l-carnitine administration on the erythropoietin resistance in β-thalassemic patients on chronic hemodialysis. correspondence: biagio r. di iorio m.d., c.da san tommaso, 286 83100 avellino, italy. fax: ++39 0825 530363; email: br.diiorio@libero.it please note that this article may not be used for commercial purposes. for further information please refer to the copyright statement at http://www.la-press.com/copyright.htm drug target insights 2007: 22 iorio et al methods patients patients were included into the study on the basis of the following criteria: adult age; chronic hemodialysis treatment at a trice weekly schedule; βthalassemia minor; stable hemoglobin (hb) levels within the target levels (11–12g/dl) obtained by high erythropoietin (epo) dosages. we studied β-thalassemia minor hemodialysis patients (β-thal) which were previously treated during a one-year period by progressively higher epo doses to reach the hb target; the control of anemia was then prolonged for a further additional one-year time (16). diagnosis of β-thal was made on the basis of the hba2 level (>3.5%). in order to evaluate the response of thalassemic patients to carnitine, we also studied as controls a group of hemodialyzed patients without thalassemia (c). all subjects gave their informed consent to the study. study design prior to study, we performed a six-month run-in period in order to verify the stability of hb levels (coeffi cient of variation <3%). after the run-in, l-carnitine was administered at the end of each of the three hd session of the week, at the dose of 2 g for each hemodialysis session, along a 1 year period-time both in c and β-thal-m groups. thereafter, a six months wash-out period was performed. during the study, iron gluconate was administered i.v. once a week, at the end of the third hd session of the week, to patients with iron defi ciency (serum ferritin <100 mg/dl or transferrin saturation [tsat] <20%), to keep both tsat above 20% and serum ferritine between 100 and 500 mg/dl. also folate and vitamin b12 were regularly administered to all patients. anemia was evaluated monthly, prior to the second dialysis of the week (16), by drowning blood samples from the arterial line prior to the infusion of saline or heparin and before starting the blood pump. blood samples were also obtained at three month intervals at the end of dialysis, after 5 minutes stop-fl ow (blood pump rate 50 ml/min and no dialysate fl ow), for estimation of dialysis dose. at the end of each dialysis session, dry body weight (bw) was measured and the effective epo dose administered was reported into the chart. hematocrit (ht), hemoglobin, mean corpuscular volume (mcv) and red blood cells count were detected monthly. serum levels of urea nitrogen (sun), parathyroid hormone (pth), aluminum, iron, ferritine, transferrine and tsat%, were evaluated every three months. analytical determinations urea nitrogen was measured using an autoanalyzer (olympus au 400, olympus italia, segratemilano, italy). hemoglobin, red blood cells, erythrocyte volume and hematocrit were measured by a blood cell counter (cell-dyn 3500 r, abbot diagnostics, germany). pth, plasma iron, transferrine and ferritine were evaluated with standard technique. aluminum was determined by atomic absorbiment (17, 18). statistical analysis values are reported as mean ±sd. anova for repeated measures and newman-keuls post-hoc test was applied for intra-group repeated measures comparison. unpaired t-test was used for intergroup comparison. a p value <0.05 was considered statistically signifi cant. results we studied twelve subjects (m = 8; f = 4) affected by β-thalassemia minor (β-thal; hba2 level = 6.6 ± 0.6 %) and forty non-thalassemic subjects (m = 24; f = 16) as control (c). in the β-thal and c groups the renal disease respectively was: primary glomerulonephritis in 6 and 12 patients, hypertensive nephrosclerosis in 3 and 11 subjects, diabetic nephropathy in 3 and 9 subjects, and unknown in 8 controls. no patient suffered of hepatic dysfunction. patients were on chronic thrice weekly bicarbonate hemodialysis; the vascular access was always native arterovenous fi stula. a total of 5/12 β-thal and 22/40 c subjects used ace-inhibitors. all patients received β-recombinant epo at a thrice weekly schedule; the drug was administered intravenously through the infl ow needle the end of each hd session. patients and controls were comparable for gender, age, dialysis age and dose, nutrition, hemoglobin and iron status (table 1). also serum levels of pth (187 ± 98 and 205 ± 78 pg/ml; ns) and aluminum (38 ± 16 and 45 ± 12 mcg/ml; ns) did drug target insights 2007: 2 3 l-carnitine in dialysis patients with thalassemia minor not differ between thalassemic and controls. during the study period, body weight, and dialysis both dose and uf rate, were stable in the two groups (table 2). figure 1 shows the behavior of both hemoglobin and epo dosage in β-thal (1a) and c (1b) during l-carnitine administration. c showed signifi cant both hb increase and epo dose decline since the third month during the study; β-thal group evidenced similar changes in hb and epo, but they were evident after 8 months of carnitine administration. at start, epo dose was 280 and 100 ui/kg/ week in β-thal and c groups, respectively; at the maximum response to epo during the study (12th month) the epo dose was 170 and 60 ui/kg/week in β-thal and c (stable since the 3rd–6th month) groups, respectively; thus, during carnitine administration, epo dose decreased by the maximum of 39% in β-thal and 40% in control patients. of interest, because of the intra to extracellular water shift due to the hemoconcentration determined by the ultrafi ltration during the dialysis procedure, the erythrocyte mcv reduced at the end of dialysis as compared to pre-dialysis values in c, but not in β-thal; in this latter, at the start of the study, the post-dialysis mcv resulted slightly increased (65.3 ± 3.2 to 65.5 ± 3.2 fl ; ns) (figure 2). during the carnitine administration period, however, the intradialysis mcv reduced also in β-thal since the 9th month of treatment; conversely, since the fi rst month of the recovery period after carnitine stop, the mcv in β-thal resumed to increase during dialysis. discussion there are compelling indicators of carnitine defi ciency in chronic dialysis patients as a result of low dietary intakes and increased losses during the table 1. baseline characteristics of thalassemic and control subjects. controls β-thal p number 40 12 females (%) 40 33 ns age (years) 52 ± 18 59 ± 8 ns dialysis age (months) 66 ± 23 58 ± 18 ns kt/v 1.33 ± 0.5 1.31 ± 0.3 ns ultrafi ltration (ml/min) 10.7 ± 3.1 11.9 ± 2.6 ns body weight (kg) 60.9 ± 12.4 57.9 ± 14.2 ns albumin (g/dl) 3.96 ± 0.08 3.95 ± 0.07 ns pna (g/kg/day) 1.21 ± 0.04 1.19 ± 0.06 ns hb (g/dl) 11.6 ± 1.2 11.1 ± 1.3 ns serum iron (mg/l) 78 ± 30 89 ± 28 ns tsat (%) 30 ± 4 29 ± 5 ns ferritine (mcg/l) 245 ± 56 256 ± 60 ns pna = protein nitrogen appearance; hb = hemoglobin; tsat = transferrine saturation. table 2. body weight (bw), dialysis ultrafi ltration (uf) and kt/v during the study period in controls (c) and β-thal (t) groups. 0 mo 6 mo 12 mo p bw (kg) c 58.6 ± 6.1 58.6 ± 6.4 58.4 ± 6.0 ns t 55.3 ± 4.1 55.0 ± 5.0 55.5 ± 6.2 ns uf (ml/min) c 10.7 ± 3.1 10.6 ± 3.3 10.9 ± 3.4 ns t 11.9 ± 2.6 11.0 ± 3.4 11.3 ± 3.5 ns kt/v c 1.33 ± 0.5 1.30 ± 0.6 1.31 ± 0.4 ns t 1.31 ± 0.3 1.19 ± 0.4 1.30 ± 0.5 ns drug target insights 2007: 24 iorio et al dialysis procedure. koistra fi rst described a relationship between carnitine and anemia in uremia; indeed, he reported that esrd patients with severe anemia requiring erythropoietin had lower carnitine levels than patients who had less severe or no anemia (19). matsamura showed a significant inverse correlation between the epo dose required to maintain target hematocrit in dialysis and both total and free carnitine serum levels (20). all together, these data suggest that carnitine defi ciency may contribute to the need for greater epo doses in anemic, uremic patients (12, 21–25). this is the fi rst study investigating the effect of long-term carnitine supplementation on epo resistance in patients on chronic hemodialysis affected by β-thalassemia. the study evidences the lowering of epo need in β-thal by mean of 39% after 1 year carnitine administration, as compared to a 40% reduction that occurred in non thalassemic patients after 3–6 months; such epo reduction was associated figure 1. serum hemoglobin levels and amount of epo dose in β-thal (1a) and controls (1b) subjects, prior, during and after carnitine administration. (1a) epo: * = p < 0.05 vs –6, –3, 0, 3, 6, 15 and 18 months; (1b) epo: * = p < 0.05 vs –6, –3, 0, 15 and 18 months; hb: ° p < 0.05 vs –6, –3, 0 and 18 months. a b drug target insights 2007: 2 5 l-carnitine in dialysis patients with thalassemia minor with stable hemoglobin levels within the target and was reversible after carnitine discontinuation. a large body of evidences show that mcv results reduced during the hemodialysis due to the intravascular, and consequently intra-cellular, water removal (26–29); such erythrocyte capability is strictly dependent on the cell membrane elasticity (27–28). our data evidenced a reduction of mcv during the hemodialysis session in the control group, but not in β-thal subjects, possibly due to the rigidity of red blood cell membrane. in β-thal patients the erythrocyte recovered such intra-dialysis capability to reduce cell volume after 8 months of carnitine treatment and again losted such a capacity 4 weeks after stopping the drug administration. such an erythrocyte behavior is possibly related to the improvement of cell-membrane elasticity during carnitine treatment. indeed, several authors suggested that in hemodialysis patients without β-thal the effectiveness of carnitine is related to the increasing of erythrocyte stability (30). furthermore, it was showed in uremic rats that in vivo administration of carnitine increases the osmotic erythrocyte resistance, due to the increase of erythrocyte membrane na+ – k+ pump activity (13–14, 31–32). thus, our data may suggest that carnitine administration possibly induces the improvement of erythrocyte deformability in β-thal uremic patients, likely as in non thalassemic uremics; however, such effect is obtained only after a longtime treatment. it can be hypothesized that in β-thal on dialysis only the long-term carnitine administration improves the strictness of rbc, and conversely enhances the cell half-life. indeed, other authors showed that abnormalities in rbc deformability improves after carnitine administration, which is also responsible for hematocrit increase and epo dose reduction (33). in this study, the epo dose reduced from 280 to 170 ui/kg/day (–39%) at the 12th month of treatment in thal, and all patients were responders (epo reduction range: –29 to –45%). in the control group the epo dose decreases from 100 to 60 ui/kg/day (–40%) at the 3rd month of carnitine treatment. two groups of expert evaluated the effi cacy of carnitine in the treatment of anemia in dialysis patients. the american association of kidney patients consensus group concluded there is a defi nite role for carnitine in dialysis patients, particularly for certain conditions that do not adequately respond to the standard therapy (34). a trial supplementation period may be indicated to exclude carnitine defi ciency as a cause of epo resistance in selected patients (34). more recently, figure 2. intra dialysis mcv changes (pre minus post dialysis values) in β-thal and controls at start, during and after carnitine administration. drug target insights 2007: 26 iorio et al the nkf/doqi clinical practice guidelines for nutrition in chronic renal failure reported that the most promising of the proposed applications for carnitine in dialysis patients is in the treatment of epo resistant anemia (35). it has been suggested that a 4-month trial of carnitine was reasonable in selected patients with anemia and/ or very large epo requirements and should be of adequate duration to reliably assess the response to carnitine. recently, handelman based on the analysis of a database of the registered carnitine trials, concluded that carnitine benefi ts are not real and, consequently, the dialysis administration of carnitine is not justifi ed (36). however, several authors, either before handelman observation (11–13, 19–25), or even more recently (38), showed the positive effects of carnitine in patients on chronic hemodialysis. our study adds new inside in this fi eld, evidencing for the fi rst time the effects of long carnitine administration also in β-thalassemic patients in chronic dialysis. in summary, carnitine administration has been shown to be effective in many patients for the adjunctive treatment of anemia associated with chronic kidney disease. this study suggests that a very long-term carnitine administration should be useful also in uremic patients on chronic dialysis affected by β-thalassemia on target hemoglobin, by reducing the doses of erythropoietin needed to manage the anemia. references [1] nfk-doqi clinical practice guidelines for the treatment of anemia in crf. 2001. am. j. kidney dis., 37:s182–s238. [2] canadian clinical practice guidelines for the management of anemia coexistent with crf. 1999. jasn, 10:s292–6. [3] european best practice guidelines for the management of anemia in patient with crf. 2000. nephrol. dial. trasplant., 15:s1–s63. [4] horl, w.h., jacobs, c., macdougall, valderrabano, f., parrondo, i., thompson, k. and carveth, b.g. 2000. european best practice guidelines 14–16: inadequate response to epoietin. nephrol. dial. transplant., 15(s4):43–50. [5] rocco, m.v., bedinger, m.r. and milam, r. et al. 2001. duration of dialysis and its relationship to dialysis adequacy, anemia management and serum albumin level. am. j. kidney dis., 38:813–823. [6] battistella, p.a., angelini, c., vergani, l., bertoli, m. and lorenzi, s. 1978. carnitine defi ciency induced during haemodialysis. lancet, 2:939. [7] bohmer, t., bergrem, h. and eiklid, k. 1978. carnitine defi ciency induced during intermittent haemodialysis for renal failure. lancet, 1:126–128. [8] bommer, j. 1999. saving erythropoietin by administering l-carnitine? nephrol. dial. transplant., 14:2819–21. [9] arduini, a., rossi, m., mancinelli, g., belfiglio, m., scurti, r., radotti, g. and shohet, s.b. 1990. effect of l-carnitine and acetyll-carnitine on the human erythrocite membrane stability and deformability. life sci., 47:2395–400. [10] bayon, j.e., alvarez, a.i. and barrio, i.p. et al. 1993. effects of stanazolol and l-carnitine on erythrocyte osmotic fragility during aerobic exercise in rats. comp. haematol. int., 3:196–200. [11] hurot, j.m., cucherat, m., haugh, m. and fouque, d. 2002. effect of l-carnitine supplementation in maintenance hemodialysis patients: a systematic review. j. am. soc. nephrol., 13:708–714. [12] golper, t.a., goral, s., becker, b.n. and langman, c.b. 2003. lcarnitine treatment of anemia. am. j. kidney. dis., 41(s4):27–34. [13] tarng, d.c., huang, t.p., chen,t.w., fan, c.y. and chang, j.g. 1996. resistance to recombinant human erythropoietin treatment in thalassaemic patients on chronic hemodialysis: a real clinical entity. nephrol. dial. transplant., 11:1893–5. [14] di iorio, b., aucella, f., stallone, c., aversano, a., rubino, r. and bellizzi, v. 2001. the use of recombinat erythropoietin in hemoglobinopathy and uremia. dial. and transplant., 30:268–272. [15] di iorio, b., guastaferro, p. and bellizzi, v. 2003. relationship between resistance to epo and high anomalous hb levels in hemodialysis patients with beta-thalassemia minor. blood purif., 21:376–380. [16] di iorio, b., de nicola, l., bellizzi, v., minutolo, r., zamboli, p., rubino, r., fuiano, g. and conte, g. 2004. effi cacy of erythropoietin on dialysis in patients with beta thalassemia minor. blood purif., 22:453–460. [17] andress, d.l., kopp, j.b. and maloney, n.a. et al. 1987. early deposition of aluminium in bone in diabetic patients on hemodialysis. n. engl. j. med., 316:292–296. [18] consensus conference: diagnosis and treatment of aluminium overload in end-stage renal disease. 1993. nephrol. dial. transplant., 8(s1):1–4. [19] koistra, m.p., struyvenberg, a. and van es, a. 1991. the response to recombinant huma erythropoietin in patients with anemia of esrd is correlated with serum carnitine levels. nephron, 57:127–128. [20] matsumura, m., hatakeyama, s. and koni, i. et al. 1996. correlation between serum carnitine levels and erythrocyte osmotic fragility in hemodialysis patients. nephron, 72:574–578. [21] kletzmayr, j., mayer, g. and legenstein, e. et al. 1999. anemia and carnitine supplementation in hemodialyzed patients. kidney int., 69: s93–s106. [22] caruso, u., leone, l., cravotto, e. and nava, d. 1998. effects of carnitine on anemia in aged hemodialysis patients treated with recombinant human erythropoietin: a pilot study. dial. transplant., 28:499–505. [23] labonia, w.d. 1995. carnitine effects on anemia in hemodialyzed patients treated with erythropoietin. am. j. kidney. dis., 26:757–764. [24] trovato, g.m., ginardi, v. and di marco, v. et al. 1982. long term carnitine treatment of chronic anemia of patients with esrd. curr. ther. res., 31:1042–1049. [25] matsumoto, y., amano, i. and hirose, s. et al. 2001. effects of carnitine supplementation on renal anemia in poor responders to erythropoietin. blood purif., 55:24–32. [26] de nicola, l., bellizzi, v., minutolo, r., cioffi , m., giannattasio, p., terracciano, v., iodice, c., uccello, f., memoli, b., di iorio, b.r. and conte, g. 2000. effect of dialysate sodium concentration on interdialytic increase of potassium. jasn, 11:2337–2343. [27] fleming, s.j., wilkinson, j.s., greenwood, r.n., aldridge, c., baker, l.r. and cattell, w.r. 1987. effect of dialysate composition on intercompartmental shift. kidney int., 32:267–273. [28] fleming, s.j., wilkinson, j.s., aldridge, c., greenwood, r.n., muggleston, s.d. and cattell, w.r. 1988. dialysis-induced change in erythocyte volume: effect on change in volume calculated from packed cell volume. clin. nephrol., 29:63–68. [29] de vries, p.m., kouw, p.m., meijer, j.h., oe, l.p., schneider, h. and donker, a.j. 1988. changes in blood parameters during hemodialysis as determine conductivity measurements. asaio trans., 34:623–626. [30] vlassopoulos, d.a., hadjiyannakos, d.k. and anogiatis, a.g. et al. 2002. carnitine action on red blood cell osmotic resistance in hemodialysis patients. j. of nephrology, 15:68–73. [31] labonia, w.d., morelli, j.r., jimenez, m.i., freuler, p.v. and morelli, d.h. 1987. effect of the l-carnitine on sodium transport in erythrocytes from dialyzed uremic patients. kidney int., 32:754–9. drug target insights 2007: 2 7 l-carnitine in dialysis patients with thalassemia minor [32] donatelli, a., terrizzi, c., zumma, g., russo, v., bucalo, m.l. and scarpinato, a. 1987. effects of the l-carnitine on chronic anaemia and erythrocyte adenosine triphosphate concentration in hemodialysi patients. curr. ther. res., 41:620–4. [33] sotirakopoulos, n., athanasiou, g., tsitsios, t., stambolidou, m., missirlis, y. and mavromatidis, k. 2000. effect of l-carnitine supplementation on red blood cells deformability in hemodialysis patients. renal failure, 22:73–80. [34] consensus group statement. role of l-carnitine in treating renal dialysis patients. 1994. dial. transplant., 23:177–81. [35] nfk-doqi practice recommandation for the use of l-carnitine in dialysis-related carnitine disorders. 2006. am. j. kidney dis., 41: s1–s91. [36] handelman, g.j. 2006. debate forum: carnitine supplements have not been demonstrated as effective patients on long-term dialysis therapy. blood purif., 24:140–142. [37] kazmi, w.h., obrador, g.t. and sternberg, m. et al. 2005. carnitine therapy is associated with decreased hospital utilization among hemodialysis patients. am. j. nephrol., 25:106–115. nysaeter et al.indd original research correspondence: dr. gunnar nysæter, department of medicine, haukeland university hospital, 5021 bergen, norway. tel: +47 55976739; fax: +47 55974973; email: gunnar.nysaeter@helse-bergen.no copyright in this article, its metadata, and any supplementary data is held by its author or authors. it is published under the creative commons attribution by licence. for further information go to: http://creativecommons.org/licenses/by/3.0/. live typhoid vaccine for ibd-patients—well tolerated and with possible therapeutic effect gunnar nysæter1 and arnold berstad2 1department of medicine, section for gastroenterology, haukeland university hospital, bergen, norway. 2institute of medicine, university of bergen, norway. abstract background: our incidental observation of a remarkable improvement of disease activity following vaccination against typhoid in a patient with infl ammatory bowel disease (ibd) was the incentive of this pilot study. methods: ten ibd-patients (7 with ulcerative colitis and 3 with crohn’s disease) with disease activity grade 2–10 on simple colitis index were included in the study. the use of 5-asa and prednisolone �12.5 mg/day, but no other immunosuppressive drugs, were allowed during the trial. live typhoid vaccine containing salmonella serovar ty21a (vivotif ®, berna) was given in standard doses on day 1, 3 and 5. symptoms and endoscopic fi ndings were followed up for a 3-months-period. results: improvement of abdominal symptoms was recorded in 8 patients after 90 days, one patient was unchanged and one slightly worse. endoscopic fi ndings improved in 4 patients and were unchanged in 5 patients after 90 days. no side effects were observed. conclusion: our results indicate that a live typhoid vaccine is well tolerated by patients with ibd of moderate activity. the symptomatic and endoscopic improvements were not dramatic, but encouraging enough to warrant further studies on the potential therapeutic effect of live typhoid vaccine on patients with ibd. keywords: salmonella ty21a, colitis, infl ammatory bowel disease introduction vaccination has been one of the most rewarding efforts in modern medicine, reducing or even abolishing the threat of several serious infectious diseases. a limited number of vaccines are necessary when living in the western world. but with increasing international travel, the need for a wider spectrum of vaccines is rising. current vaccination guidelines for chronic infl ammatory bowel disease (ibd) patients allow extensive use of vaccines, even live vaccines when the patient is not immunosuppressed (1). still ibd-patients tend to underutilize immunisation against vaccine-preventable diseases (2). vaccines like live typhoid vaccine or cholera vaccine may temporally cause slight abdominal symptoms. information about this may deter some ibd-patients from taking the vaccine and thus increase their risk for attracting dangerous infections. some animal studies show a protective or therapeutic effect of vaccines on experimental colitis (3–5). but whether human ibd is infl uenced by vaccines seems to be unknown. the experience of one of our ulcerative colitis patients evoked our interest on this question. she is a 46 years old female, with a 20 years’ history of left-sided ulcerative colitis documented by colonoscopy and biopsies. for the fi rst 10 years she had almost continuous symptoms, had three colonocopies, and was treated with oral 5-asa and intermittent cures with oral prednisone and steroid enemas. then she prepared for travel to india and therefore had an oral vaccination with a live typhoid vaccine (vivotif®, berna). the fi rst two weeks thereafter she had slight worsening of the diarrhea, but then followed a 3 months’ period with practically no symptoms. she thought this must be an effect of the vaccination as she found no better explanation. so when the colitis symptoms started to recur, she repeated the vaccination with the same drug target insights 2008:3 119–123 119 http://creativecommons.org/licenses/by/3.0/ http://creativecommons.org/licenses/by/3.0/ nysæter and berstad drug target insights 2008:3 vaccine. again improvement ensued, and she stayed symptom free for 9 months. after this she has continued to take oral typhoid vaccine about once a year for the last 9 years, being convinced of the vaccine’s positive effect on her ulcerative colitis. but she also continued to take oral 5-asa during these years. a recent sigmoideoscopy showed a slight proctitis. with this story in mind we discovered two other ulcerative colitis patients who could report something similar after oral vaccination, though their improvement was less convincing. these observations inspired us to perform a pilot study in order to disclose any modifying effects, positive or negative of live typhoid vaccine on the course of ibd. material and methods since few data exist on the effect of live oral typhoid vaccine’s effect on ibd, we did an open pilot study, well aware of its limitations when the numbers are so small. ten ibd-patients were included, 7 with ulcerative colitis or proctitis, 3 with crohn’s disease. median age was 43 years, range 21–52 years. male/female ratio 4/6. duration of disease: median 7,5 years, range 2 months–23 years. inclusion criteria: 1. patients with verifi ed ibd by endoscopy, pathologic anatomic diagnosis of chronic inflammation or ibd, negative stool examinations for enteropathogenic microbes and giardia lamblia. 2. good general condition, and ibd activity index �10 for the last two weeks. 3. age between 18 and 70 years. exclusion criteria: 1. simple colitis index �10 with affected general condition. 2. fever �38 °c. 3. altered immunocompetence due to diseases or drugs like tnfα-inhibitors, azathioprin or corticosteroids in doses � the equivalent of prednisolone 12,5 mg pr. day. 4. ongoing use of antibiotics. 5. known allergy to any of the ingredients in the vaccine. 6. pregnancy and lactation. written informed consent was obtained from each patient, and the protocol was approved by regional commitee for medical research ethics, norwegian medicines agency and norwegian social science data service. the patients were treated with a currently available oral vaccine containing the salmonella ty21a strain (vivotif®, berna) using the standard dosage for such vaccination against typhoid fever. one enterocapsule was taken with drinking water on day one, three and fi ve. the patients took the capsules at home and noted on a report form when they were taken. current medication was not changed unless medically indicated during the test period. thus, seven ulcerative colitis patients were on oral or rectal medication with 5-asa, two of these were steroid dependant, one using oral prednisolon 10 mg daily, the other hydrocortisone enema daily. bowel symptoms before treatment, on day 5, 15, and 90 from treatment start were scored. we used special diaries allowing scoring of disease activity index, the harvey-bradshaw simple index of crohn’s disease activity for patients with crohn’s disease, or the simple clinical colitis activity index (walmsley) for patients with ulcerative colitis (6–8). the patients were examined with fl exible sigmoidoscopy by one of the investigators. to confi rm the diagnosis, biopsies for histopathologic examination were taken from the recto sigmoid mucosa. macroscopic changes in the mucosa were monitored by fl exible sigmoideoscopy, at treatment start and at the following three consultations. the endoscopic changes were graded according to a simple scale: normal = 0, redness and edema = 1, erosions = 2, ulcers = 3. (9) blood tests were not included in the study because typically such tests give very little information as long as ibd activity is low to moderate. statistics data were analysed using the graphpad prism 5.0 (graphpad software inc., u.s.a.) statistical software package. differences between means were evaluated with nonparametric two-tailed wilcoxon matched pairs test. p values less than 0.05 were considered statistically signifi cant. results at fi rst follow-up, 5 days after last vaccination day, there was a signifi cant improvement of clinical symptoms, as compared with baseline, and, on average, this improvement remained for the following three months (fig. 1). none of the patients became worse during the fi rst fi ve days. but one patient with a 17 years history of ulcerative colitis, who was on prednisolone 10 mg pr. day at the start, had a fl are-up of symptoms after 15 days and had to increase the dose to 30 mg prednisolone per day. disease activity 120 live typhoid vaccine for ibd-patients drug target insights 2008:3 was only slightly changed for the patients with crohn’s disease. a seemingly endoscopic improvement did not reach statistical signifi cance (fig. 2). no adverse effects appeared in any of the patients. discussion we used a vaccine that has been in extensive use for decades in the combat of typhoid and paratyphoid fever. its effi cacy and safety is well documented through mass studies and millions of vaccinations world wide (10). but the vaccine contains live microbes that are supposed to alert the host’s immune system through contact with intestinal mucosal immune receptors. it was unknown whether this contact would worsen the already infl amed mucosa of ibd-patients. our previous patient contacts suggested there might be a period of slight exacerbation of symptoms after vaccination. we therefore followed up our patients closely, both endoscopically and with respect to symptoms, but no such early exacerbation was observed. because the patients had symptoms at baseline, we could not withdraw their current treatment with 5-asa or prednisolone, as this could have worsened their condition. already at day fi ve of treatment, a signifi cant improvement without any initial exacerbation was recorded. this fi nding encourages an active attitude to protect ibd-patients with vaccination when typhoid is a relevant threat. whether the vaccine has a real therapeutic effect in ibd, cannot be properly answered by this small pilot study. both placebo effect and simultaneous medication infl uence our results. still there was an improvement that we had not otherwise expected. an actual therapeutic effect of live typhoid vaccine in ibd can therefore not be excluded. also, the suggestion is in fact supported by several prior experimental studies. in animal models, the gut fl ora is essential for the development of colitis, and colitis may be infl uenced by antibiotics and certain strains of live bacilli able to alter or suppressing the fl ora. thus, oral administration of lactobacillus and bifi dobacterium attenuated dss-colitis in mice (11; 12), while administration of the probiotic mixture vsl#3 (a mixture of bifi dobacteria, lactobacilli, and streptococcus salivarius) to mice with il-10 defi ciency also reduced microscopic infl ammation along with a reduction in mucosal secretion of tnf-α and ifn-γ (13). a vaccine stimulates the immune system’s targeted defence against defi ned pathogens, without having their mutilating effect. in addition to protection against specifi c microbes, vaccines may also strengthen the general defence mechanisms of the intestinal mucosa. an apparently irrelevant vaccine for the gastrointestinal tract proved to have an effect on the mouse colon, as a three-component bordetella pertussis vaccine attenuated colitis in gαi2-defi cient mice (4). and administration of recombinant cholera toxin subunit b has been shown to inhibit murine tnbs experimental colitis (3). cholera toxin may promote the induction of th2 and tr1 cells, enhance il-10 production and inhibit secretion figure 1. as compared with baseline, symptoms decreased signifi cantly fi ve days after oral treatment with live typhoid vaccine. improvement persisted three months later. * = p < 0.05. ● = ulcerative colitis. ○ = crohn’s disease. figure 2. endoscopic fi ndings after oral treatment with live typhoid vaccine. improvement appeared in some individuals but did not reach statistical signifi cance for the whole group. error bars = sd. 121 nysæter and berstad drug target insights 2008:3 of il-12 and tnf-α (14). whether salmonella ty21a has similar effects, has not been shown. but experiments by neish et al. showed that cells with previous contact with apathogenic salmonella got decreased infl ammatory response when subsequently exposed to pathogenic strains (15). a vaccine against enterotoxigenic eschericia coli (etec) composed of a live, attenuated salmonella vector-expressing enterotoxigenic e. coli fimbriae, stimulated a biphasic th cell response when given orally and suppressed the normally produced proinfl ammatory response. the vaccine reduced the production of tnf-α, il-1 and il-6, while it increased production of il-4, il-10, and il-13 (5). these results indicate that selected strains of microbes or vaccines may have a benefi cial effect on a chronically infl amed mucosa in animal models of ibd. also in human ibd the interplay between intestinal microfl ora and the gut seems to have a key role (16). the mucosa senses the luminal microbes through its complex system of receptors. production of anti-microbial factors like defensins is one of the results, thereby supporting the intestinal surface with a protective layer that stops microbial invasion of the intestinal wall. some microbes evade these protective mechanisms, by tolerating the defending peptides or down-regulating the sensing mechanisms of the mucosa, and thereby manage to invade the host. pathogenic salmonella is able to down-regulate the production of defensins, (17), and shigella seems to suppress the production of cathelicidin, another important defence molecule of the colonic mucosa (18). in the case of nod2/card 15 mutations, as seen in about one third of patients with crohn’s disease, the host has a defect sensing of microbes. parallel to this a reduced amount of defensin (hd5) in terminal ileum of crohn’s disease patients has been reported (19). in these patients the amount of microbes inside the mucus covering the terminal ileal mucosa is also severely increased (20). these genetic and protective factors have not been examined in our pilot study as it contains very few crohn’s patients. but a therapeutic effect of vaccination might well be related to some change in these parameters. in ulcerative colitis, the findings are more obscure, but here too the intestinal mucus layer is abnormally infested with microbes, suggesting a defective mucosal protection. recent clinical studies have given hope that altering or up-regulating the mucosal immune system may improve ibd-patients’ health. stimulation of the mucosa by means of apatogenous paracites (21) or by granulocyte macrophage colony-stimulating factor (gm-csf) both seem to have a therapeutic effect (22). this may signal a new policy in the treatment of ibd; to stimulate the mucosal immune system in a well designed way instead of applying a regimen for general immunosuppresion. the positive results of the present study support the idea of applying immunostimulation in the treatment of ibd, and further studies along these lines are warranted. statement of interests declaration of personal interests: none. declaration of funding interests: this study was fully funded by helse vest rhf, project number 911305. references [1] sands, b.e., cuffari, c., katz, j., kugathasan, s., onken, j., vitek, c. and orenstein, w. 2004. guidelines for immunizations in patients with infl ammatory bowel disease. infl amm. bowel dis., 10:677–92. [2] melmed, g.y. 2006. patients with infl ammatory bowel disease are at risk for vaccine-preventable illnesses. the american journal of gastroenterology, 101:1834–40. [3] boirivant, m., fuss, i.j., ferroni, l., de pascale, m. and strober, w. 2001. oral administration of recombinant cholera toxin subunit b. inhibits il-12-mediated murine experimental (trinitrobenzene sulfonic acid) colitis. j. immunol., 166:3522–32. [4] ohman, l. 2005. acellular bordetella pertussis vaccine enhances mucosal interleukin-10 production, induces apoptosis of activated th1 cells and attenuates colitis in galphai2-defi cient mice. clinical and experimental immunology, 141:37–46. [5] jun, s., gilmore, w., callis, g., rynda, a., haddad, a. and pascual, d.w. 2005. a live diarrheal vaccine imprints a th2 cell bias and acts as an anti-infl ammatory vaccine. j. immunol., 175:6733–40. [6] harvey, r.f. and bradshaw, j.m. 1980. a simple index of crohn’sdisease activity. lancet, 1:514. [7] higgins, p.d., schwartz, m., mapili, j., krokos, i., leung, j. and zimmermann, e.m. 2005. patient defi ned dichotomous end points for remission and clinical improvement in ulcerative colitis. gut., 54:782–8. [8] walmsley, r.s., ayres, r.c., pounder, r.e. and allan, r.n. 1998. a simple clinical colitis activity index. gut., 43:29–32. [9] azzolini, f., pagnini, c., camellini, l., scarcelli, a., merighi, a., primerano, a.m., bertani, a., antonioli, a., manenti, f. and rigo, g.p. 2005. proposal of a new clinical index predictive of endoscopic severity in ulcerative colitis. dig. dis. sci., 50:246–51. [10] engels, e.a., falagas, m.e., lau, j. and bennish, m.l. 1998. typhoid fever vaccines: a meta-analysis of studies on effi cacy and toxicity. bmj., 316:110–6. [11] fujiwara, m., kaneko, t., iwana, h., taketomo, n., tsunoo, h., kanno, j., ohkusa, t. and okayasu, i. 2003. inhibitory effects of bifi dobacterium longum on experimental ulcerative colitis induced in mice by synthetic dextran sulfate sodium. digestion, 67:90–5. 122 live typhoid vaccine for ibd-patients drug target insights 2008:3 [12] osman, n., adawi, d., ahrne, s., jeppsson, b. and molin, g. 2004. modulation of the effect of dextran sulfate sodium-induced acute colitis by the administration of different probiotic strains of lactobacillus and bifi dobacterium. dig. dis. sci., 49:320–7. [13] madsen, k., cornish, a., soper, p., mckaigney, c., jijon, h., yachimec, c., doyle, j., jewell, l. and de simone, c. 2001. probiotic bacteria enhance murine and human intestinal epithelial barrier function. gastroenterology, 121:580–91. [14] lavelle, e.c., jarnicki, a., mcneela, e., armstrong, m.e., higgins, s.c., leavy, o. and mills, k.h. 2004. effects of cholera toxin on innate and adaptive immunity and its application as an immunomodulatory agent. j. leukoc. biol., 75:756–63. [15] neish, a.s., gewirtz, a.t., zeng, h., young, a.n., hobert, m.e., karmali, v., rao, a.s. and madara, j.l. 2000. prokaryotic regulation of epithelial responses by inhibition of ikappab.-alpha ubiquitination. science, 289:1560–3. [16] neish, a.s. 2002. the gut microfl ora and intestinal epithelial cells: a continuing dialogue. microbes infect., 4:309–17. [17] salzman, n.h., ghosh, d., huttner, k.m., paterson, y. and bevins, c.l. 2003. protection against enteric salmonellosis in transgenic mice expressing a human intestinal defensin. nature, 422:522–6. [18] islam, d., bandholtz, l., nilsson, j., wigzell, h., christensson, b., agerberth, b. and gudmundsson, g. 2001. downregulation of bactericidal peptides in enteric infections: a novel immune escape mechanism with bacterial dna as a potential regulator. nat. med., 7:180–5. [19] wehkamp, j. 2005. reduced paneth cell alpha-defensins in ileal crohn’s disease. proceedings of the national academy of sciences of the united states of america, 102:18129–34. [20] swidsinski, a., weber, j., loening-baucke, v., hale, l.p. and lochs, h. 2005. spatial organization and composition of the mucosal fl ora in patients with inflammatory bowel disease. j. clin. microbiol., 43:3380–9. [21] weinstock, j.v., summers, r.w. and elliott, d.e. 2005. role of helminths in regulating mucosal infl ammation. springer semin. immunopathol., 27:249–71. [22] korzenik, j.r., dieckgraefe, b.k., valentine, j.f., hausman, d.f. and gilbert, m.j. 2005. sargramostim for active crohn’s disease. n. engl. j. med., 352:2193–201. 123 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 1048576 /lockdistillerparams false /maxsubsetpct 100 /optimize true /opm 1 /parsedsccomments true /parsedsccommentsfordocinfo true 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<feff005500730065002000740068006500730065002000730065007400740069006e0067007300200074006f0020006300720065006100740065002000500044004600200064006f00630075006d0065006e0074007300200077006900740068002000680069006700680065007200200069006d0061006700650020007200650073006f006c007500740069006f006e00200066006f0072002000680069006700680020007100750061006c0069007400790020007000720065002d007000720065007300730020007000720069006e00740069006e0067002e0020005400680065002000500044004600200064006f00630075006d0065006e00740073002000630061006e0020006200650020006f00700065006e00650064002000770069007400680020004100630072006f00620061007400200061006e0064002000520065006100640065007200200035002e003000200061006e00640020006c0061007400650072002e002000540068006500730065002000730065007400740069006e006700730020007200650071007500690072006500200066006f006e007400200065006d00620065006400640069006e0067002e> >> >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice untitled 19 review correspondence: omar m.e. abdel salam, (m.d., ph.d.), department of pharmacology, national research centre, tahrir st., dokki, cairo, egypt. email: omasalam@hotmail.com please note that this article may not be used for commercial purposes. for further information please refer to the copyright statement at http://www.la-press.com/copyright.htm effect of ribavirin alone or combined with silymarin on carbon tetrachloride induced hepatic damage in rats omar m.e. abdel salam1, amany a. sleem1, enayat a. omara2 and nabila s. hassan2 1department of pharmacology, national research centre, tahrir st., dokki, cairo, egypt. 2department of pathology, national research centre, tahrir st., dokki, cairo, egypt. abstract: the effect of the antiviral agent ribavirin given alone or in combination with silymarin on the development of liver injury induced in rats with carbon tetrachloride (ccl4; 2.8 ml/kg followed by 1.4 ml/kg after one week) was studied. ribavirin at three dose levels (30, 60 or 90 mg/kg), silymarin (25 mg/kg) or combination of ribavirin (60 mg/kg) and silymarin (25 mg/kg) was administered once daily orally for 14 days, starting at time of administration of ccl4. the administration of ribavirin decreased the elevations in serum alanine aminotransferase (alt) by 78.5, 82.1, 75.1%, aspartate aminotransferase (ast) 47.5, 37.4, 38.8%, and alkaline phosphatase (alp) by 23.4, 16, 21.6%, respectively and also prevented the development of hepatic necrosis caused by ccl4. in comparison, the elevated serum alt, ast and alp levels decreased to 43.3%, 46%, and 37.5% of controls, respectively by silymarin. when silymarin was combined with ribavirin, the serum activities of ast and alp were further decreased, indicating a benefi cial additive effect. morphometric analysis indicated signifi cant reduction in the area of necrosis and fi brosis on ribavirin treatment and this was further reduced after the addition of silymarin. metabolic pertuberations caused by ccl4 as refl ected in a decrease in intracellular protein content in hepatocytes were improved by ribavirin monotherapy and to higher extent by combined silymarin and ribavirin therapy. proliferating cell nuclear antigen was reduced in nuclei of hepatocytes by ribavirin montherapy or the combination of ribavirin and silymarin compared with ccl4-control group. the study demonstrates that ribavirin treatment in the model of ccl4induced liver injury results in less liver damage. results also indicate that the combined application of ribavirin and silymarin is likely to be a useful additive in reducing liver injury. keywords: ribavirin, silymarin, carbon tetrachloride, liver injury, rat. introduction ribavirin (1-β-d -ribofuranosyl-1,2,4, triazole-3 carboxamide) is an orally active synthetic guanosine analogue with antiviral and immunomodulatory actions. ribavirin is a broad-spectrum antiviral drug, preventing the replication of a large number of rna and dna viruses by inhibiting the enzyme inosine monophosphate dehydrogenase, which is required for the synthesis of guanosine triphosphate. the fi nal step in this chain of events is lethal mutagenesis of the rna genome (cameron and castro, 2001). when used alone in the treatment of chronic hepatitis c virus infection, the drug normalizes serum aminotransferases, an effect that is not sustained and relapse was reported after discontinuing treatment. in patients with chronic hepatitis c, ribavirin is used more often in regimens employing interferon-alpha (inf-α) (wartelle-bladou et al. 2006). the addition of ribavirin to interferon alpha is superior to interferon alpha in terms of virologic, biochemical, and histologic end points, resulting in improved end-of-treatment and sustained response rates, with an overall 41% sustained virological response rate in patients treated for 48 weeks (pianko and mchutchison, 2000; mukherjee and lyden, 2006). this combined therapy has also resulted in an increased toxicity profi le, which made therapy more diffi cult for both the patient and managing physician and prompted its discontinuation or a dosage reduction in a signifi cant proportion of patients (pianko and mchutchison, 2000; chutaputti, 2000; bonaccorsoa et al. 2000; collier and chapman, 2001; fried et al. 2002; burra et al. 2006). in addition, response is not obtained in up to 50% of cases and even in those where a response occurs, there is a 30% chance of relapse (pianko and mchutchison, 2000; hoofnagle et al. 2003). in most studies, ribavirin monotherapy, improved liver enzyme levels, but without signifi cant effects on hcv viraemia (gane et al. 1995, 1996; di_bisceglie et al. 1995; dusheiko et al. 1996; cattral drug target insights 2007: 2 19–27 20 salam et al et al. 1999; kamar, 2003; hoofnagle et al. 2003). nevertheless, histological improvement with reduction in hepatic necro-infl ammation has been reported (gane et al. 1995, 1998; di bisceglie et al. 1995; hoofnagle et al. 2003) and ribavirin has been shown to possess anti-infl ammatory properties and to decrease the synthesis of proinfl ammatory cytokines (e.g. ifn-gamma) (meier et al. 2003; barnes et al. 2004). in the present study, it was aimed to examine whether ribavirin alone could exert protective effects in the ccl4 model of liver toxicity and if there is any benefi t from combining ribavirin and silymarin. the latter, a standardized plant extract, derived from the milk thistle plant is widely used as a hepatoprotective agent, because of its antioxidant and membrane stabilizing properties (flora et al. 1998; muriel and mourelle, 1990; farghali et al. 2000; wellington et al. 2001). the effect of ribavirin was evaluated on biochemical markers, histologically as well as by histochemical techniques. the area of damage or necrosis was calculated by image analysis system and immune staining by avidin biotin-peroxidae method for detection of proliferating cell nuclear antigen (pcna), an endogenous cell replication marker (shiina et al. 1996) was used. materials and methods animals sprague-dawley rats of both sex, weighing 150–160 g were used throughout the experiments and fed with standard laboratory chow and water ad libitum. drugs and chemicals carbon tertrachloride (bdh chemicals, england), ribavirin (virazole, october pharma, cairo) and silymarin (sedico pharmaceutical co. cairo) were used in the experiments. the carbon tetrachloride induced hepatic damage hepatic injury was induced by treating rats by gavage with ccl4-olive oil (1:1, 2.8 ml/kg followed by 1.4 ml/kg after one week) . starting on the time of the fi rst dose of ccl4 administration, rats also orally received either saline, silymarin (25 mg/kg), ribavirin (at three dose levels of 30, 60 and 90 mg/kg) alone or combined with silymarin 25 mg/kg. control rats were treated with olive oil (2.8 ml/kg followed by 1.4 ml/kg after one week). the animals were killed on day 15 after the fi rst dose of ccl4 or olive oil administration. rats had free access to food and drinking water during the study. biochemical assessment at the end of the experiments, blood samples were obtained from the retro-orbital vein plexuses, under ether anaesthesia. alt and ast activities in serum were measured according to reitmanfrankel colorimetric transaminase procedure (crowley, 1967), whereas colorimetric determination of alp activity was done according to the method of belfi eld and goldberg (1971), using commercially available kits (biomérieux, france).total protein in serum was measured spectrophotometrically (bradford, 1976). glucose concentrations in serum were measured enzymatically (bauer, 1982). histopathological and histochemical studies after the end of the treatment period, rats were killed, livers were excised and fixed in 10% formalin saline, bouin’s and carnoy’s fluids. sections were prepared and stained with hematoxylin and eosin (h & e) for the histological investigations. bromophenol blue stain for intracellular proteins and avidin biotin-peroxidae method for detection of proliferating cell nuclear antigen (pcna), an endogenous cell replication marker were used. further histopathgological evaluation was done with morphometry. the percentage of liver tissue affected by necrosis and fi brosis (damaged area) was determined using a computer-assisted automated image analyzer. qwin leica image processing and analysis system (cambridge, england) was used for interactive automatic measurement of the percentage of damaged areas on slides stained by h & e by analyzing 15 random fi elds per slide. statistical analysis all results are expressed as means ± se. multiple group comparisons were performed by anova followed by duncan test. p < 0.05 was considered statistically signifi cant. drug target insights 2007: 2 21 effect of ribavirin and silymain on liver injury results biochemical changes results are presented in table 1. serum alanine aminotransferase (alt), aspartate aminotransferase (ast), and alkaline phosphatase (alp) levels were signifi cantly higher in ccl4-treated rats compared with the vehicle-treated control group. ribavirin administered to ccl4-treated rats at 30, 60 or 90 mg/kg resulted in a signifi cant reduction in the levels of the serum enzymes. this effect of ribavirin was not dose-dependent. thus, compared with ccl4 control group, serum alt levels was reduced by the above doses of ribavirin by 78.5, 82.1 and 75.1%, respectively. when ribavirin and silymarin were given in combination, no further decrease in alt values was noted (82.7, 80.2 and 82.1% vs ccl4 control group, respectively). also a signifi cant reduction in ast values by 47.5, 37.4 and 38.8% was noted after 30, 60 or 90 mg/kg of ribavirin, respectively. ribavirin at these doses combined with silymarin caused 67.5, 66.2 and 68.6% decrease in serum ast levels, respectively. serum alp levels were reduced after ribavirin treatment by 23.4, 16 and 21.6%, respectively. however, 40 and 59.4% decrease in serum alp was observed when ribavirin at 60 or 90 mg/kg was combined with silymarin. in comparison, silymarin given alone at 25 mg/kg to ccl4-treated rats decreased the elevated serum alt, ast and alp levels to 43.3%, 46%, and 37.5% of controls, respectively. serum proteins increased by 19.2% after ribavirin monotherapy at 90 mg/kg, while the combined treatment with ribavirin (30, 60 or 90 mg/kg) plus silymarin resulted in 22.9, 20.5 and 35% increase in serum proteins compared with the ccl4 control group. serum glucose level was reduced by 46.9% in ccl4-treated compared to vehicle-treated control. signifi cant increases in serum glucose by 56.9, 45.7, and 51.1% were observed after ribavirin monotherapy at 30, 60 or 90 mg/kg compared with the ccl4 control group. when ribavirin (30, 60 or 90 mg/kg) was combined with silymarin, 49.2, 93.5 and 120.2% increase in serum glucose was observed as compared to the ccl4 control group. histopathological changes examination of h & e stained sections of control liver showed the characteristic hepatic architecture (fig. 1a). in rats treated with ccl4, patchy areas of necrosis, damaged bile ducts and focal infl ammatory cell infi ltrate were observed (fig. 1b). the hepatocytes from rats treated with silymarin (fig. 2a), the high dose of ribavirin (fig. 2c) or ribavirin (60 mg/kg) plus silymarin (fig. 2b) showed more or less normal appearance. quantitative analysis of the area of damage signifi cant increase in the percentage of damaged areas was observed in ccl4-treated rats when compared to normal animals; 21.6 ± 1.4% vs 0.2 ± 0.1%. morphometric analysis of liver sections showed that ribavirin administration to a b figure 1. (a) a photomicrograph from a section of control rat liver showing normal hepatic architecture: central vein with radiating cords of liver cells, the hepatocytes had vesicular nuclei and granular cytopolasm and blood sinusoids were evident between the cords of hepatocytes (h× & ε× 300). (b) a photomicrograph from a section of rat liver treated with ccl4 showing loss of normal architecture, patchy areas of necrosis, damaged bile ducts, vascular odema, infl ammatory cellular infi ltrate (h× & e × 150). drug target insights 2007: 2 22 salam et al ccl4-treated rats resulted in a signifi cant and dose-dependent decrease in damaged areas; 9.6 ± 0.8, 6.2 ± 0.5, 4.3 ± 0.5% vs control value of 21.6 ± 1.4% and vs 9.0 ± 1.1% for silymarin at 25 mg/kg. when ribavirin and silymarin were given in combination, further reduction in the damaged area by 16.1% (p < 0.05) was noted in rats given 60 mg/kg ribavirin plus silymarin (5.2 ± 0.4 vs 6.2 ± 0.5) (table 1). a b a b c figure 2. (a) a photomicrograph from a section of rat liver treated ccl4 and silymarin showing normal hepatic architecture, while the sinusoids were dilated (h× & e × 500). (b) a photomicrograph from a section of control rat liver treated with ccl4 and a combination of ribavirin (60 mg/kg) and silymarin, showing that hepatocytes have almost regained their normal pattern (h× & e × 150). (c) a photomicrograph from a section of rat liver treated with ccl4 and ribavirin at 90 mg/kg, showing more or less normal hepatocytes and blood sinusoids (h× & e × 150). ta bl e 1. e ffe ct o f rib av iri n, s ily m ar in o r rib av iri n co m bi ne d w ith s ily m ar in o n se ru m a la ni ne a m in ot ra ns fe ra se ( a lt ), a sp ar ta te m in ot ra ns fe ra se ( a s t ), al ka lin e ph os ph at as e (a lp ), to ta l p ro te in s an d gl uc os e in c c l 4tr ea te d ra ts . a lt (u /l) a s t (u /l) a lp to ta l p ro te in g lu co se a re a of g lu co se (iu /l) (g /d l) (m g/ dl ) da m ag e (% ) (m g/ dl ) tr ea tm en t s al in e co nt ro l 67 .4 ± 1 .7 77 .8 ± 3 .9 12 2. 6 ± 11 .1 9. 1 ± 0. 6 96 .0 ± 7 .6 0. 2 ± 0. 1 96 .0 ± 7 .6 c c l 4 co nt ro l 12 5. 5 ± 6. 3 15 6. 2 ± 4. 5 19 8. 4 ± 5. 8 8. 3 ± 0. 2 51 .0 ± 4 .5 21 .6 ± 1 .4 51 .0 ± 4 .5 + s ily m ar in 2 5 m g/ kg 71 .2 ± 6 .6 * 84 .8 ± 7 .4 * 12 4. 0 ± 11 .6 * 8. 7 ± 0. 5 59 .0 ± 7 .8 9. 0 ± 1. 1* 59 .0 ± 7 .8 + r ib av iri n 30 m g/ kg 27 .0 ± 3 .1 * 82 .0 ± 5 .7 * 15 2. 0 ± 12 .4 * 8. 8 ± 0. 4 80 .0 ± 4 .9 * 9. 6 ± 0. 8* 80 .0 ± 4 .9 + r ib av iri n 60 m g/ kg 22 .5 ± 2 .8 * 97 .8 ± 6 .7 16 6. 6 ± 14 .6 8. 9 ± 1. 0 74 .3 ± 4 .1 * 6. 2 ± 0. 5* 74 .3 ± 4 .1 + r ib av iri n 90 m g/ kg 31 .2 ± 2 .6 * 95 .6 ± 8 .4 * 15 5. 6 ± 1 3. 5 9. 9 ± 0. 6 77 .1 ± 6 .9 * 4. 3 ± 0. 5* 77 .1 ± 6 .9 + r ib av iri n 30 m g/ kg 21 .7 ± 3 .4 * 50 .7 ± 6 .0 *+ 15 2. 6 ± 7. 8 10 .2 ± 0 .6 76 .1 ± 6 .1 * 8. 9 ± 0. 6* 76 .1 ± 6 .1 + si ly m ar in 2 5 m g/ kg + r ib av iri n 60 m g/ kg 24 .9 ± 2 .3 * 52 .8 ± 5 .7 *+ 11 9. 0 ± 12 .8 *+ 10 .0 ± 0 .7 98 .7 ± 8 .3 *+ 5. 2 ± 0. 4* 98 .7 ± 8 .3 + si ly m ar in 2 5 m g/ kg + r ib av iri n 90 m g/ kg 22 .5 ± 1 .8 * 49 .0 ± 4 .1 *+ 80 .6 ± 7 .0 *+ 11 .2 ± 0 .7 * 11 2. 3 ± 9. 6* + 4. 8 ± 0. 5* 11 2. 3 ± 9. 6 + si ly m ar in 2 5 m g/ kg r es ul ts a re m ea ns ± s .e . d at a w er e an al yz ed b y on e w ay a n o va a nd m ea ns o f d iff er en t g ro up s w er e co m pa re d by d un ca n’ s m ul tip le r an ge te st . t w ota ile d pr ob ab ili tie s of le ss th an 0 .0 5 w er e co ns id er ed s ig ni fi c an t. *p < 0 .0 5 vs c c l 4 co nt ro l. +p < 0 .0 5 vs c or re sp on di ng r ib av iri n al on etr ea te d gr ou p. drug target insights 2007: 2 23 effect of ribavirin and silymain on liver injury histochemical observations as regards bromophenol blue reactivity, the hepatocytes of control rats showed +ve reaction and moderate protein content (fig. 3a). in ccl4-treated rats, the hepatocytes in pericentral and periportal regions showed faint reaction, resulting from a decrease in protein contents (fig. 3b ). after silymarin and ribavirin (60 mg/kg) treatment, marked increase in protein content was observed compared to the ccl4 control group (fig. 3c). treatment with ribavirin at 90 mg/kg, resulted in moderate improvement in protein content in liver cells (fig. 3d). immunohistochemistry for pcna proliferating cell nuclear antigen (pcna) known as cyclin, is a non-histone nuclear protein whose level of synthesis correlates directly with rates of cellular proliferation and dna synthesis (shiina et al. 1996). cells were considered pcna positive if there was brown nuclear staining of the cells and negative nuclei not stained and appear blue. in the control group there were few nuclei that showed positive reaction (brown nuclei) (fig. 4a). increase in the number of pcna staining of hepatocyte nuclei was evident in ccl4-treated rats (fig. 4b). in rat liver given ccl4 and a combination of ribavirin (60 mg/kg) and silymarin there was a marked reduction in the number of pcna positive nuclei especially in peripheral zones compared with sections from rats treated with ccl4-olive oil (fig. 4c). the hepatocyte nuclei of rats treated with ccl4 + ribavirin showed a reduction in pcna +ve reaction compared to ccl4 control group (fig. 4d). discussion the present study provides evidence that in the ccl4 model of hepatic toxicity, ribavirin, an antiviral drug, exerts hepatic protective effects. leakage of hepatocellular enzymes alt and ast into plasma was signifi cantly reduced and the histological degree of hepatocyte necrosis was attenuated. improved liver function tests are likely a consequence of the lower degree of organ damage a c d b figure 3. (a) a photomicrograph from a section of control rat liver showing normal protein distribution in hepatocytes cytoplasm (bromophenol blue reaction × 300). (b) a photomicrograph from a section of rat liver given ccl4 showing marked reduction in protein content especially in damaged areas (bromophenol blue reaction × 300). (c) a photomicrograph from a section of rat liver given ccl4 and a combination of ribavirin (60 mg/kg) and silymarin. marked improvement of protein content in cytoplasm of hepatic cells is seen. (bromophenol blue reaction × 300). (d) a photomicrograph from a section of rat liver given ccl4 with ribavirin at 90 mg/kg, showing moderate improvement in protein content in liver cells (bromophenol blue reaction × 300). drug target insights 2007: 2 24 salam et al and fi brosis. the hepatocytes from rats treated with the high dose of ribavirin showed more or less normal appearance. morphometric analysis of liver sections showed that ribavirin administration to ccl4-treated rats resulted in a signifi cant and dosedependent decrease in damaged areas. metabolic pertuberations caused by the hepatotoxin ccl4 as refl ected in a decrease in intracellular protein content in hepatocytes were improved by ribavirin monotherapy and also by combined silymarin and ribavirin therapy. these results point to a hepatic protective effect of ribavirin distinct from its antiviral activity. one of the principal functions of the liver is the regulation of carbohydrate metabolism and blood glucose homeostasis. in the present study, serum glucose was reduced by 50% in ccl4-treated rats which was prevented by ribavirin montherapy and also by ribavirin plus silymarin. studies have demonstrated a decreased hepatic glycogen content after treatment with ccl4, refl ecting decreased gluconeogenesis by the liver (muriel et al. 2001). without sufficient glycogen levels to provide glucose to drive glycolysis, cellular atp levels may drop below critical levels when secondary stress such as hypoxia is present, precipitating cell death (ulrich et al. 2001). glucose also has a role in protecting cells from oxidative injury (tian et al. 1999). low o2 tensions which are found in the centrilobular areas of the liver favor conversion of ccl4 to free radical products which cannot be detoxifi ed by the glutathione-dependent mechanism (burk et al. 1984). abnormalities in glucose metabolism are also present in patients with liver disease and type 2 diabetes mellitus seems to be more common in patients with chronic hepatitis c infection (allison et al. 1994; caronia et al. 1999). insulin resistance, hepatocyte dysfunction, or an hcv-related autoimmune process might be implicated (alexander, 2000). insulin resistance in chronic hepatitis c is relevant because it promotes steatosis and fi brosis. insulin resistance together c d ba figure 4. (a) a photomicrograph from a section of control rat liver showing normal hepatocytes with few pcna positive nuclei (arrows) (pcna immunostaining × 300). (b) a photomicrograph from a section of rat liver given ccl4 showing prominent increase in the number of pcna positive nuclei (proliferating cells) in regenerated cells in necrotic areas around central veins (pcna immunoperoxidae × 300). (c) a photomicrograph from a section of rat liver given ccl4 and a combination of ribavirin (60 mg/kg)and silymarin showing marked reduction in the number of pcna positive nuclei especially in peripheral zones compared with sections from rats treated with ccl4-olive oil (pcna immunostaining × 300). (d) a photomicrograph from a section of rat liver given ccl4 and ribavirin at 90 mg/kg showing few number of pcna positive nuclei in hepatocytes adjacent to the central vein (pcna immunostaining × 300). drug target insights 2007: 2 25 effect of ribavirin and silymain on liver injury with fi brosis and genotype has been found to be independently associated with impaired response rate to peginterferon plus ribavirin (romerogomez, 2006). alternatively, diabetic status is one of the more important variables determining the severity of hcv recurrence after liver transplantation (foxton et al. 2006). in decompensated cirrhosis, there may be, however, acute post absorptive hypoglycaemia, primarily due to a reduction in hepatic glycogen capacity (mccullough and tavill, 1991; krahenbuhl et al. 1991) and there is evidence for altered hepatic gluconeogenesis (changani et al. 2001). the present study also provides evidence of additional benefi cial effect of combining silymarin with ribavirin. silymarin (milk thistle, silybum marianum) is a commonly used herbal therapy, particularly by patients who have liver disease including those with chronic hepatitis c infection. there are clinical data to support its use in chronic alcoholic liver disease (feher et al. 1989; pares et al. 1998) where it resulted in reducing serum bilirubin, and transaminases. others, however, failed to demonstrate such benefi t from silymarin (trinchet et al. 1989; buzzelli et al. 1993). in patients with chronic hepatitis c, silymarin has been shown not to affect serum hcv rna, alt levels, quality of life or psychological well-being in subjects with this condition (gordon et al. 2006). in animal models of hepatotoxicity, silymarin showed marked protective properties. this hepatoprotective effect of silymarin is due to membranestabilizing action, free radicals scavenging properties, inhibition of lipid peroxidation and modulation of hepatocyte ca++ (muriel and mourelle, 1990; flora et al. 1998; farghali et al. 2000). in rats with secondary biliary cirrhosis, silymarin reduced hepatic collagen accumulation by 35% (boigk et al. 1997). silymarin thus might be of benefi t in reducing liver injury in patients on ribavirin therapy. cell proliferation is of interest since abnormal cell proliferation is a precursor of tumorigensis. identifi cation of proliferating cells was studied by the avidin-biotin-peroxidase technique. proliferating cell nuclear antigen (pcna) known as cyclin, is a non-histone nuclear protein whose level of synthesis correlates directly with rates of cellular proliferation and dna synthesis (shiina et al. 1996). the elevated levels of pcna expression appear in the nucleus during the late g1 phase with maximum expression during s-phase and decline during g2 and m phase. therefore, the accumulation of pcna gene products in cycling cells can be an index of the degree of cellular proliferation and dna synthesis (bravo et al. 1987). carbon tetrachloride administration in rats concomitantly induces both processes in acute injury and liver regeneration. hepatocyte growth factor and pcna were induced in the early stage (6 h) and 36 h, respectively (taniguchi et al. 2004). liver cirrhosis induced by ccl4 is associated with alterations in cell cycle-related proteins, and that the expression of these proteins is responsible for hepatocyte regeneration in the damaged liver and may be involved in liver carcinogenesis (jeong et al. 2001). in the present study hepatocyte proliferation in ccl4-treated rats, as evidenced by the increase in the number of pcna staining of hepatocyte nuclei was reduced by ribavirin administration. ribavirin monotherapy has been employed in hepatitis c virus infection such as in patients with hepatitis c recurring after liver transplant (quadri et al. 2002) and renal transplant patients (kamar et al. 2003), often with discrepancy in results. in most studies, ribavirin monotherapy, improved liver enzyme levels, but without signifi cant effects on hcv viraemia (gane et al. 1995, 1998; di_bisceglie et al. 1995; dusheiko et al. 1996; cattral et al. 1999; stanimirovic et al. 2002; hoofnagle et al. 2003; kamar et al. 2003). others noted slight, albeit not signifi cant, decrease of serum hcv rna level and intrahepatic hcv antigen staining score (quadri et al. 2002). signifi cant histological improvement with reduction in hepatic infl ammation and necrosis (gane et al. 1995, 1996; di_bisceglie et al. 1995; stanimirovic et al. 2002; hoofnagle et al. 2003), no change in necroinfl ammation (cattral et al. 1999; izopet and rostaing, 2003) or even worsening of fi brosis (quadri et al. 2002; kamar et al. 2003) has been reported. the exact mechanism of action of ribavirin is unknown and direct antiviral properties are unclear (lee et al. 1998; querenghi et al. 2001). it was suggested that the benefi cial effects of ribavirin are mediated by inhibition of induction of macrophage proinfl ammatory cytokines and th2 cytokines while preserving th1 cytokines (ning et al. 1998). ribavirin could decrease the synthesis of proinfl ammatory cytokines (e.g. ifn-gamma) by an inhibition of total dna-, rna-, and proteinsynthesis and by induction of apoptosis in the cells of the infl ammatory infi ltrate (meier et al. 2003). ribavirin at physiological doses markedly suppressed the production of tnf-alpha, il-10, and il-12 (p70) drug target insights 2007: 2 26 salam et al which may explain the reduction in hepatic infl ammation observed during ribavirin monotherapy (barnes et al. 2004). in liver injury induced by ccl4, secondary hepatic injury occurs from infl ammatory processes originating from products released by activated kupffer cells, which play a central role in hepatic infl ammation. kupffer cells isolated from rats with ccl4-induced steatonecrosis produced more reactive oxygen intermediates than cells isolated from normal rats. these oxidants could activate nf-kappa b and lead to an overexpression of tnfalpha, observed in liver tissue sections. this cytokine expressed in the ccl4-induced infl ammatory process is associated with the development of fi brosis and may contribute to disease severity (orfi la et al. 1999, 2000). in ccl4-induced liver injury il-6, il-1beta, tnf-alpha and ifn-gamma upregulation was found at the maximum 12 hours after administration of the toxin (sheikh et al. 2006). studies also linked protection from ccl4-induced hepatic injury with suppression of tnf-alpha level (yang et al. 2005, 2006). based on the above data, it is suggested that the inhibition of proinfl ammatory cytokines by ribavirin might be involved in the protection observed in the present study. in conclusion, protective effects for the antiviral agent ribavirin were observed against liver damage induced by ccl4 treatment. additional benefi t from combining ribavirin and silymarin was observed. these results suggest that ribavirin lessens hepatic necro-infl ammation through mechanisms distinct from antiviral activity. furthermore, these effects of ribavirin were associated with a reduction in pcna staining of hepatocyte nuclei, a fi nding that could have relevance for humans in view of the risk of developing hepatocellular carcinoma in patients with chronic hepatitis c infection. the results suggest that a therapeutic approach using both ribavirin and silymarin could be effective in chronic liver disease. this regimen is likely to benefi t those patients who can not tolerate, failed to respond to, or relapsed after combined interferon and ribavirin therapy. references alexander, g., j.m. 2000. an association between hepatitis c virus infection and type 2 diabetes mellitus: what is the connection? 2000. ann. intern. med., 33:650–652. allison, m.e., wreghitt, t., palmer, c.r. and alexander g.j. 1994. evidence for a link between hepatitis c virus infection and diabetes mellitus in a cirrhotic population. j. hepatol., 21:1135–9. bauer, j.d. 1982. clinical laboratory methods, 9th ed. c.v. mosby company, st.louis, toronto, london. belfi eld, a. and goldberg, d.m. 1971. revised assay for serum phenyl phosphatase activity using 4-amino-antipyrine. enzyme, 12:561–73. boigk, g., stroedter, l., herbst, h., waldschmidt, j., riecken, e.o. and schuppan d. 1997. silymarin retards collagen accumulation in early and advanced biliary fi brosis secondary to complete bile duct obliteration in rats. hepatology, 26:643–9. bonaccorsoa, s., meltzerc, h. and maes, m. 2000. psychological and behavioural effects of interferons. curr. opin. psychiat., 13:673–677. bradford, m.m. 1976. a rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. anal. biochem., 72:248–254. bravo, r., frank, r., blundell, p.a. and macdonald-bravo, h. 1997. cyclin/pcna is the auxiliary protein of dna polymerase-delta. nature, 326:515–517. burk, r.f., lane, j.m. and patel, k. 1984. relationship of oxygen and glutathione in protection against carbon tetrachloride-induced hepatic microsomal lipid peroxidation and covalent binding in the rat. rationale for the use of hyperbaric oxygen to treat carbon tetrachloride ingestion. j. clin. invest.,74:1996–2001. burra, p., targhetta, s., pevere, s., boninsegna, s., guido, m., canova, d., brolese, a., masier, a., d’aloiso, c., germani, g., tomat, s. and fagiuoli s. 2006. antiviral therapy for hepatitis c virus recurrence following liver transplantation: long-term results from a single center experience. transplant. proc., 38:1127–1130. buzzelli, g., moscarella, s., giusti, a., duchini, a., marena, c. and lampertico, m. 1993. a pilot study on the liver protective effect of silybin-phosphatidylcholine complex (idb1016) in chronic active hepatitis. int. j. clin. pharmacol. ther. toxicol., 31:456–60. cameron, c.e. and castro, c. 2001. the mechanism of action of ribavirin: lethal mutagenesis of rna virus genomes mediated by the viral rna-dependent rna polymerase. curr. opin. infect. dis., 14:757–764. caronia, s., taylor, k., pagliaro, l., carr, c., palazzo, u., petrik, j., o’rahilly, s., shore, s., tom, b.d. and alexander, g.j. 1999. further evidence for an association between non-insulin-dependent diabetes mellitus and chronic hepatitis c virus infection. hepatology, 30:1059–63. cattral, m.s., hemming, a.w., wanless, i.r., al, ashgar, h., krajden, m., lilly, l., greig, p.d. and levy, g.a. 1999. outcome of long-term ribavirin therapy for recurrent hepatitis c after liver transplantation. transplantation, 67:1277–1280. changani, k.k., jalan, r., cox, i.j., ala-korpela, m., bhakoo, k., taylorrobinson, s.d. and bell, j.d. 2001. evidence for altered hepatic gluconeogenesis in patients with cirrhosis using in vivo 31-phosphorus magnetic resonance spectroscopy. gut., 49:557–564. chutaputti, a. 2000. management of hepatitis c. adverse effects and other safety aspects of the hepatitis c antivirals. j. gastroenterol. hepatol., 15 (suppl):e156–163. collier, j. and chapman, r. 2001. combination therapy with interferon-alpha and ribavirin for hepatitis c: practical treatment issues. biodrugs, 15: 225–238. crowley, l.v. 1967. the reitman-frankel colorimetric transaminase procedure in suspected myocardial infarction. clin. chem., 13:482–487. di-bisceglie, a.m., conjeevaram, h.s., fried, m.w., sallie, r.f., park, y., yurdaydin, c., swain, m., kleiner, d.e., mahaney, k. and hoofnagle j.h. 1995. ribavirin as therapy for chronic hepatitis c. a randomized, double-blind, placebo-controlled trial. ann. intern. med., 123:897–903. dusheiko, g., main, j., thomas, h., reichard, o., lee, c., dhillon, a., rassam, s., fryden, a., reesink, h., bassendine, m., norkrans, g., cuypers, t., lelie, n., telfer, p., watson, j., weegink, c. and sillikens, p., weiland, o. 1996. ribavirin treatment for patients with chronic hepatitis c: results of a placebo-controlled study. j. hepatol., 25:591–598. gane, e.j., tibbs, c.j., ramage, j.k., portmann, b.c. and williams, r. 1995. ribavirin therapy for hepatitis c infection following liver transplantation. transpl. int., 8:61–64. gane, e.j., lo, s.k., riordan, s.m., portmann, b.c., lau, j.y., naoumov, n.v. and williams, r. 1998. a randomized study comparing ribavirin and interferon alfa monotherapy for hepatitis c recurrence after liver transplantation. hepatology, 27:1403–1407. drug target insights 2007: 2 27 effect of ribavirin and silymain on liver injury gordon, a., hobbs, d.a., bowden, d.s., bailey, m.j., mitchell, j., francis, a. j.p. and roberts, s.k. 2006. effects of silybum marianum on serum hepatitis c virus rna, alanine aminotransferase levels and well-being in patients with chronic hepatitis c. j. gastroenterol. hepatol., 21: 275–280. farghali, h., kamenikova, l., hynie, s. and kmonickova, e. 2000. silymarin effects on intracellular calcuim and cytotoxicity: a study in perfused rat hepatocytes after oxidative stress injury. pharmacol. res., 41: 231–237. feher, j., deak, g., muzes, g., lang, i., niederland, v., nekam, k. and karteszi, m. 1989. liver protective action of silymarin therapy in chronic alcoholic liver diseases. orv. hetil., 130:2723–7. flora, k., hahn, m., rosen, h. and benner, k. 1998. milk thistle (silybum marianum) for the therapy of liver disease. am. j. gastroenterol., 93: 139–143. foxton, m.r., quaglia, a., muiesan, p., heneghan, m.a., portmann, b., norris, s., heaton, n.d. and o’grady, j.g. 2006. the impact of diabetes mellitus on fi brosis progression in patients transplanted for hepatitis c. am. j. transplant, 6:1922–9. fried, m.w., shiffman, m.l, reddy, k.r., smith, c., marinos, g., goncales, f.l., haussinger, d., diago, m., carosi, g., dhumeaux, d., craxi, a., lin, a., hoffman, j. and yu, j. 2002. peginterferon alfa-2a plus ribavirin for chronic hepatitis c virus infection. n. engl. j. med., 347:975–982. hoofnagle, j.h., ghany, m.g., kleiner, d.e., doo, e., heller, t., promrat, k., ong, j., khokhar, f., soza, a., herion, d., park, y., everhart, j.e. and liang, t.j. 2003. maintenance therapy with ribavirin in patients with chronic hepatitis c who fail to respond to combination therapy with interferon alfa and ribavirin. hepatology, 38:66–74. jeong, d.h., jang, j.j., lee, s.j., lee, j.h., lim, i.k., lee, m.j. and lee y.s. 2001. expression patterns of cell cycle-related proteins in a rat cirrhotic model induced by ccl4 or thioacetamide. j. gastroenterol., 36:24–32. kamar, n., sandres-saune, k., selves, j., ribes, d., cointault, o., durand, d., izopet, j. and rostaing, l. 2003. long-term ribavirin therapy in hepatitis c virus-positive renal transplant patients: effects on renal function and liver histology. am. j. kidney. dis., 42:184–192. krahenbuhl, s., weber, f.l., jr. and brass, e.p. 1991. decreased hepatic glycogen content and accelerated response to starvation in rats with carbon tetrachloride-induced cirrhosis. hepatology, 14:1189–95. lee, j.h., von_wagner, m., roth, w.k., teuber, g., sarrazin, c. and zeuzem, s. 1998. effect of ribavirin on virus load and quasispecies distribution in patients infected with hepatitis c virus. j. hepatol., 29:29–35. mccullough, a.j. and tavill, a.s. 1991.disordered energy and protein metabolism in liver disease. semin. liver. dis., 11:265–277. mchutchison, j.g. and poynard, t. 1999. combination therapy with interferon plus ribavirin for the initial treatment of chronic hepatitis c. semin liver. dis.,19 suppl 1:57–65. meier, v., burger, e., mihm, s., saile, b. and ramadori g. 2003. ribavirin inhibits dna, rna, and protein synthesis in pha-stimulated human peripheral blood mononuclear cells: possible explanation for therapeutic efficacy in patients with chronic hcv infection. j. med. virol., 69:50–58. mukherjee, s. and lyden, e. 2006. impact of pegylated interferon alpha-2b and ribavirin on hepatic fi brosis in liver transplant patients with recurrent hepatitis c: an open-label series. 1: liver. int., 26:529–535. muriel, p. and mourelle, m. 1990. prevention by silymarin of membrane alterations in acute ccl4 liver damage. j. appl. toxicol., 10:275–279. muriel, p., alba, n., perez-alvarez, v.m., shibayama, m. and tsutsumi, v.k. 2001. kupffer cells inhibition prevents hepatic lipid peroxidation and damage induced by carbon tetrachloride. comp. biochem. physiol. c. toxicol. pharmacol., 130:219–26. ning, q., brown, d., parodo, j., cattral, m., gorczynski, r., cole, e., fung, l., ding, j.w., liu, m.f., rotstein, o., phillips, m.j. and levy, g. 1998. ribavirin inhibits viral-induced macrophage production of tnf, il-1, the procoagulant fgl2 prothrombinase and preserves th1 cytokine production but inhibits th2 cytokine response. j. immunol., 160: 3487–93. orfi la, c., lepert, j.c., alric, l., carrera, g., beraud, m., vinel, j.p. and pipy, b. 1999. expression of tnf-alpha and immunohistochemical distribution of hepatic macrophage surface markers in carbon tetrachloride-induced chronic liver injury in rats. histochem. j., 31: 677–685. orfi la, c., lepert, j.c., alric, l., carrera, g., beraud, m. and pipy, b. 2005. immunohistochemical distribution of activated nuclear factor kappab and peroxisome proliferator-activated receptors in carbon tetrachlorideinduced chronic liver injury in rats. histochem. cell biol., 123: 585–593. pares, a., planas, r., torres, m., caballeria, j., viver, j.m., acero, d., panes, j., rigau, j., santos, j. and rodes, j.1998. effects of silymarin in alcoholic patients with cirrhosis of the liver: results of a controlled, double-blind, randomized and multicenter trial. j. hepatol., 28:615–21. pianko, s. and mchutchison, j.g. 2000. treatment of hepatitis c with interferon and ribavirin. j. gastroenterol. hepatol., 15:581–586. quadri, r., giostra, e., roskams, t., pawlotsky, j.m., mentha, g., rubbia-brandt, l., perrin, l., hadengue, a. and negro, f. 2002. immunological and virological effects of ribavirin in hepatitis c after liver transplantation. transplantation, 73:373–378. querenghi, f., yu, q., billaud, g., maertens, g., trepo, c. and zoulim, f. 2001. evolution of hepatitis c virus genome in chronically infected patients receiving ribavirin monotherapy. j. viral. hepat., 8:120–131. sheikh, n., batusic, d.s., dudas, j., tron, k., neubauer, k., saile, b. and ramadori, g. 2006. hepcidin and hemojuvelin gene-expression in rat liver damage: in vivo and in vitro studies. am. j. physiol., [in press]. shiina, h., igawa, m., nagami, h., yagi, h., urakami, s., yoneda, t., shirakawa, h., ishibe, t. and kawanishi, m. 1996. immunohistochemical analysis of proliferating cell nuclear antigen, p53 protein and nm23 protein, and nuclear dna content in transitional cell carcinoma of the bladder. cancer, 78:1762–1774. stanimirovic, v., nikolic, d., stanimirovic, b., nikolic, a. and cucak, s. galenika. 2002. r&d institute, belgrade. evaluation of ribavirin effi cacy and tolerance in subjects with chronic hepatitis c virus infection. vojnosanit pregl., 59:479–484. taniguchi, m., takeuchi, t., nakatsuka, r., watanabe, t. and sato, k. 2004. molecular process in acute liver injury and regeneration induced by carbon tetrachloride. life sci., 75:1539–1549. trinchet, j.c., coste, t, levy, v.g., vivet, f., duchatelle, v., legendre, c., gotheil, c. and beaugrand, m. 1989. treatment of alcoholic hepatitis with silymarin. a double-blind comparative study in 116 patients. gastroenterol. clin. biol., 13:120–124. ulrich, r.g., bacon, j.a., brass, e.p., cramer, c.t., petrella, d.k. and sun, e.l. 2001. metabolic, idiosyncratic toxicity of drugs: overview of the hepatic toxicity induced by the anxiolytic, panadiplon. chemico-biological interactions, 134:251–270. wang-ni, tian, leigh, d., braunstein pang, j., stuhlmeier, k.m., xi qc, tian, x. and stanton, r.c. 1999. importance of glucose-6-phosphate dehydrogenase activity in cell death. am. j. physiol., 276:c1121–31. wang, h., wei, w., wang, n.p., gui, s.y., wu, l., sun, w.y. and xu, s.y. 2005. melatonin ameliorates carbon tetrachloride-induced hepatic fi brogenesis in rats via inhibition of oxidative stress. life sci., 77:1902–1915. wartelle-bladou, c., arpurt, j.p., renou, c., pariente, a., pillon, d., nalet, b., picon, m., glibert, a., chousterman, m., grasset, d., morin, t., bernard, p., fischer, d., ramdani, m., lagier, e. and rotily, m. 2006. viral hepatitis group of the angh. high dose daily interferon-alpha induction and secondary adjunction of ribavirin in treatment-naive patients with chronic hepatitis c. a multicentric, randomised, controlled trial. gastroenterol. clin. biol., 30:525–532. wellington, k. and jarvis, b. 2001. silymarin: a review of its clinical properties in the management of hepatic disorders. biodrugs, 15:465–489. yang, x.j., liu, j., ye, l.b., yang, f., ye, l., gao, j.r. and wu, z.h. 2006. in vitro and in vivo protective effects of proteoglycan isolated from mycelia of ganoderma lucidum on carbon tetrachloride-induced liver injury. world. j. gastroenterol., 12:1379–1385. drug target insights 2007: 2 untitled drug target insights 2007: 2 29–38 29 review correspondence: omar m.e. abdel-salam, email: omasalam@hotmail.com please note that this article may not be used for commercial purposes. for further information please refer to the copyright statement at http://www.la-press.com/copyright.htm modulation of visceral nociception, infl ammation and gastric mucosal injury by cinnarizine omar m.e. abdel-salam department of pharmacology, national research centre, tahrir st., dokki, cairo, egypt. abstract: the effect of cinnarizine, a drug used for the treatment of vertigo was assessed in animal models of visceral nociception, infl ammation and gastric mucosal injury. cinnarizine (1.25–20 mg/kg, s.c.) caused dose-dependent inhibition of the abdominal constrictions evoked by i.p. injection of acetic acid by 38.7–99.4%. this effect of cinnarizine (2.5 mg/kg) was unaffected by co-administration of the centrally acting dopamine d2 receptor antagonists, sulpiride, haloperidol or metoclopramide, the peripherally acting d2 receptor antagonist domperidone, but increased by the d2 receptor agonist bromocryptine and by the non-selective dopamine receptor antagonist chlorpromazine. the antinociception caused by cinnarizine was naloxone insenstive, but enhanced by propranolol, atropine and by yohimbine. the antinociceptive effect of cinnarizine was prevented by co-treatment with the adenosine receptor blocker theophylline or by the atp-sensitive potassium channel (katp) blocker glibenclamide. cinnarizine at 2.5 mg/kg reversed the baclofen-induced antinociception. cinnarizine at 2.5 mg/kg reduced immobility time in the porsolt’s forced-swimming test by 24%. cinnarizine inhibited the paw oedema response to carrageenan and reduced gastric mucosal lesions caused by indomethacin in rats. it is suggested that cinnarizine exerts anti-infl ammatory, antinociceptive and gastric protective properties. the mechanism by which cinnarizine modulates pain transmission is likely to involve adenosine receptors and katp channels. keywords: cinnarizine, visceral pain, infl ammation, gastric mucosa, rat, mice. introduction cinnarizine (stugeron r) is a calcium-channel blocker used in treatment of vertiginous disorders (pianese et al. 2002) and migraine (mansooreh et al. 2006). among its most rare adverse effects are extrapyramidal symptoms and depression; these effects can persist during weeks, months or years after the withdrawal of the drug (negrotti and calzetti, 1997; fabiani et al. 2004; teive et al. 2004; hirose, 2006) and can be explained by the inhibition of the passage of calcium in striatal neurons and a direct anti-dopaminergic features (dopamine d1 and d2 receptor blockade) because of the similar chemical structure with neuroleptic drugs (reiriz et al. 1994; brucke et al. 1995) with more than 80% d2-receptor occupancy being required for drug-induced parkinsonism to appear (hirose, 2006). cinnarizine has been reported to possess anti-infl ammatory and pain alleviating properties. cinnarizine inhibited the ear oedema induced by croton oil or capsaicin in mice and reduced oedema induced in the rat hind paw by subplantar injection of carrageenan (blazso et al. 1999). intraperitoneal or intrathecal cinnarizine caused a dose-dependent antinociception in the rat tail-fl ick test (rego et al. 1990). cinnarizine administered via subcutaneous (del pozo et al. 1987) or intracerebroventricular route (miranda et al. 1993) produced a dose-dependent antinociception in acetic acid writhing test in mice. this effect is naloxone insensitive (miranda et al. 1993). cinnarizine (20 mg/kg), inhibited 100% ethanol-induced lesion formation by 71% (lozeva et al. 1994). the present study aimed to investigate the effects of cinnarizine on visceral pain caused by intraperitoneal injection of acetic acid in mice, a model of visceral infl ammatory pain (chemonociception) and to pharmacologically characterize and investigate the possible neural pathways involved in its analgesic effect. in addition, the behavioral effect of cinnarizine on locomotor activity and on immobility time in porsolt’s forced-swimming test, its effect on acute infl ammation caused by subplantar carrageenan and the effects of the drug on gastric mucosal damage caused by indomethacin was studied. drug target insights 2007: 230 omar m.e. abdel-salam materials and methods animals sprague-dawley strain rats weighing 120–130 g of body weight or swiss male albino mice 20–22 g of body weight were used (national research centre, cairo). standard laboratory food and water were provided ad libitum. animal procedures were performed in accordance with the ethics committee of the national research centre and followed the recommendations of the national institutes of health guide for care and use of laboratory animals (publication no. 85–23, revised 1985). equal groups of 6 mice each were used in all experiments. the doses of cinnarizine used in the study were based upon the human dose after conversion to that of rat according to paget and barnes (1964). drugs cinnarizine (arab drug co., cairo), guanethidine, propranolol hydrochloride, yohimbine hydrochloride, naloxone hydrochloride (sigma, st. louis, u.s.a.), bromocryptine (novartis pharma, cairo), haloperidol, indomethacin (kahira pharm & chem. ind co., cairo), glibenclamide (hoechst orient, cairo), atropine sulphate, baclofen (misr pharm co., cairo), domperidone (janssen-cilag, switz) were used. analytical-grade glacial acetic acid (sigma, st. louis, u.s.a.) was diluted with pyrogen-free saline to provide a 0.6% solution for i.p. injection. all drugs were dissolved in isotonic (0.9% nacl) saline solution immediately before use. indomethacin was dissolved in a 5% solution of sodium bicarbonate. acetic acid-induced writhing separate groups of 6 mice each were administered vehicle or drug (1.5, 2.5, 5, 10 or 20 mg/kg, s.c.). after 30-min pretreatment interval, 0.6% acetic acid (0.2 ml/mice) was intraperitoneally (i.p.) administered (koster et al. 1959). each mouse was then placed in an individual clear plastic observational chamber, and the total number of writhes made by each mouse was counted for 30 min after acetic acid administration. further experiments were designed in an attempt to elucidate the mechanisms by which cinnarizine exerts its anti-nociceptive effect. the dose of 2.5 mg/kg of cinnarizine was selected to be used in the subsequent experiments. thus, the effect of co-administration of the alpha-2 adrenoreceptor antagonist yohimbine (5 mg/kg, i.p.), the beta adrenoreceptor antagonist, propranolol (2 mg/kg, i.p.), the muscarinic acetylcholine receptor antagonist atropine (2 mg/kg, i.p.), the non-selective opioid receptor antagonist naloxone (5 mg/kg, i.p.), the non-selective adenosine receptor antagonist theophylline (20 mg/kg, i.p.), the gaba agonist baclofen (5 mg/kg, i.p.), and the potassium channel blocker glibenclamide (5 mg/ kg, i.p.), indomethacin (5 mg/kg, i.p.) were examined on antinociception caused by cinnarizine. furthermore, the effect of the centrally acting dopamine d2 receptor antagonists, sulpiride (10 mg/kg, i.p.) and haloperidol (1.5 mg/kg, i.p.), the peripherally acting d2 receptor antagonist domperidone (10 mg/kg, i.p.) or d2 receptor agonist bromocryptine (3 mg/kg, i.p.), the d2 receptor antagonist metoclopramide (10 mg/kg) and the non-selective dopamine receptor antagonist chlorpromazine (3 mg/kg, i.p.) was examined. all drugs were administered 30 min prior to the abdominal constriction assay. rotarod testing motor performance was measured as the latency to fall from an accelerating rotarod located over plates connected to an automatic counter (ugo basile, varese, italy). mice were trained to remain on a rotating rod for 2 min as the rod rotated toward the animal. after the 2-min training period, the mice were administered vehicle (saline) or drug and 30 min later placed on the rotating rod as it accelerated from 4 to 40 rpm over 5 min and the time that they could remain on the accelerating rod was noted (millan et al. 1994). the cutoff time was 600 sec. the time was measured from the start of the acceleration period. the test was repeated 2 h after vehicle or drug injection. six animals were used per dose and for the controls. porsolt’s forced-swimming test each mouse was placed individually in a glass cylinder (diameter 12 cm, height 24 cm) fi lled with water at a height of 12 cm. water temperature was maintained at 22–23°c. the animal was forced to swim for 6 min and the duration of immobility was measured. the mouse was considered as immobile when it stopped struggling and moved only to remain fl oating in the water, keeping its head above water. the fl oating time, which is used as the measure of despair (porsolt et al. 1977), was recorded after treatment after treatment with saline, cinnarizine (2.5, 5, 10 or 20 mg/kg, s.c.) or imipramine (15 mg/kg, s.c.). drug target insights 2007: 2 31 modulation of visceral nociception, infl ammation and gastric mucosal injury carageenan-induced paw oedema paw swelling was elicited by sub-plantar injection of 100 μl of 1% sterile lambda carrageenan suspension in saline into the right hind paw (winter et al. 1962). contralateral paw received an equal volume of saline. the oedema component of infl ammation was quantifi ed by measuring the increase in paw volume (ml) with a plethysmometer (ugo basile, milan, italy) before carrageenan injection and at selected times thereafter. oedema was expressed as a percentage of change from control (pre-drug) values. the effect cinnarizine (1.25, 2.5, 5, 10 or 20 mg/kg, s.c., 0.2 ml/rat, n = 6/ group) was studied. cinnarizine was administered 30 min before the injection of the carrageenan suspension. the control groups received saline (0.2 ml/rat, n = 6 per group; s.c.). gastric ulcerogenic studies gastric mucosal damage was evoked by indomethacin (20 mg/kg, s.c.). rats received either saline (0.2 ml/rat, s.c., n = 6) (control) or cinnarizine (2.5, 5 or 10 mg/kg, 0.2 ml/rat, s.c., n = 6 per group). rats were killed 48 h later. gastric mucosal lesions were scaled as described earlier (mózsik et al. 1982). statistical analyses data are expressed as mean ± s.e. the effects of different drugs used in the abdominal constriction assay are also expressed as percent inhibition (%) compared to the control value. differences between vehicle (control) and treatment groups were determined by using one and two-way anova followed by multiple comparison by the tukey’s honestly signifi cant difference. a probability value less than 0.05 was considered statistically signifi cant. results effect of cinnarizine on abdominal constrictions induced by acetic acid cinnarizine (1.25, 2.5, 5, 10 or 20 mg/kg, s.c.) caused dose-dependent inhibition of the abdominal constrictions evoked by i.p. injection of acetic acid by 38.7–99.4% (fig. 1). this effect of cinnarizine (2.5 mg/kg) was unaffected by co-administration of the centrally acting dopamine d2 receptor antagonists, sulpiride, haloperidol or metoclopramide, the peripherally acting d2 receptor antagonist domperidone, but increased by the d2 receptor agonist bromocryptine and by the non-selective dopamine receptor antagonist chlorpromazine (fig. 2). the antinociception caused by cinnarizine was unaffected by the opioid receptor antagonist naloxone, but enhanced by the beta-adrenergic antagonist propranolol, the muscarinic receptor antagonist atropine and by the alpha2-adrenergic antagonist yohimbine (fig. 3). the antinociceptive effect of cinnarizine was prevented by co-treatment with the adenosine receptor blocker theophylline (fig. 4) and by k-channel blocker glibenclamide (fig. 5). cinnarizine at 2.5 mg/kg reversed the baclofen-induced antinociception (fig. 5). cinnarizine enhanced the antinociceptive effect of piracetam or vinpocetine (fig. 6). when indomethacin (5 mg/kg, i.p.) was administered in combination with cinnarizine (2.5 mg/kg, s.c), an additive effect was noted (fig. 7). rotarod testing cinnarizine (1.5–20 mg/kg) did not produce any signifi cant changes on the rotarod performances of the mice. there was no signifi cant difference between the control group and cinnarizine-treated groups in the latency to fall (table 1). effect of cinnaizine on immobility time in porsolt’s forced-swimming test cinnarizine administered at 2.5 mg/kg reduced immobility time in the porsolt’s forced-swimming figure 1. effect of different doses of cinnarizine (1.25, 2.5, 5, 10 and 20 mg/kg) on abdominal constrictions caused by i.p. injection of dilute acetic acid in mice. saline (control) or cinnarizine was s.c. administered 30 min prior to testing. data are expressed as mean ± s.e. percent inhibition (%) compared to the control animals are also shown. *p < 0.05 vs. control. six mice were used per each group. co ntr ol ci nn ar izi ne 1. 25 m g/k g ci nn ar izi ne 2. 5 m g/k g ci nn ar izi ne 5 mg /kg ci nn ar izi ne 10 m g/k g ci nn ar izi ne 20 m g/k g 0 10 20 30 40 50 60 70 * * * * * -38.7% -53.1% -94.5% -99.4% -58.6% n um be r of a bd om in al c on st ri ct io ns drug target insights 2007: 232 omar m.e. abdel-salam test by 24%, although higher doses of the drug failed to alter immobility time (fig. 8). effect of cinnarizine on the carrageenan-induced paw oedema carrageenan injected into the rat hind paw elicited an infl ammation (swelling and erythema) and a time-dependent increase in paw volume. in the control group, paw volume increased by 128.5 ± 10.6 % at 4 h after injection of carrageenan. cinnarizine at 1.25 or 2.5 administered s.c., 30 min prior to carrageenan had no significant effect on the paw oedema. cinnarizine at 5, 10 and 20 mg/kg induced a dose-dependent inhibition of paw oedema response to carrageenan (100 ml/paw) which was apparent within 1 h of carrageenan injection and with a maximal inhibitory effect of –22.7, –29.9 and – 43.4%, respectively (fig. 9). the percentages of inhibition of the oedema response were –27.3, –22.7, –17.8, –19.6% by 5 mg/kg cinnarizine; –23.6, –28.8, –29.9, –21.2% by 10 mg/kg cinnarizine and –43.4, –38.7, –37, –26.4% by 20 mg/kg cinnarizine at 1, 2, 3 and 4 h post-carrageenan, respectively. two-way anova revealed a signifi cant main effect for treatment (f3, 84 = 6.9; p < 0.001) and time (f3, 85 = 64.2; p < 0.001). post-hoc analysis showed signifi cant inhibition of oedema formation by 5, 10 or 20 mg/kg of cinnarizine at all time points in the test. rats treated with cinnarizine at 20 mg/kg showed signifi cantly less oedema than those given 2.5 or 5 mg/kg cinnarizine at 1, 2 and 3 h time points and than those treated with 1.25 mg/kg cinnarizine at all time points in the test. figure 2. effect of haloperidol (1.5 mg/kg, i.p.), sulpiride (10 mg/kg, i.p.), domperidone (10 mg/kg, i.p.), metoclopramide (10 mg/kg, i.p.), bromocryptine (3 mg/kg, i.p.) and chlorpromazine (3 mg/kg, i.p.) on antinociception caused by cinnarizine (2.5 mg/kg, s.c.) in the abdominal constriction assay in mice. drugs or saline (control) were administered 30 min prior to testing. data expressed as mean ± s.e. percent inhibition (%) compared to the control animals is shown. *p < 0.05 compared to control and between different groups as shown in the fi gure. the plus sign (+) indicates signifi cant change from the cinnarizine alone (2.5 mg/kg)-treated group. the (#) sign indicates signifi cant difference from the cinnarizine + chlorpromazine-treated group. six mice were used per each group. figure 3. effect of naloxone (5 mg/kg, i.p.), yohimbine (5 mg/kg, i.p.), atropine (2 mg/kg, i.p.) and propranolol (2 mg/kg, i.p.) on antinociception caused by cinnarizine (2.5 mg/kg, s.c.) in the abdominal constriction assay. drugs or saline (control) were administered 30 min prior to testing. data expressed as mean ± s.e. percent inhibition (%) compared to the control animals is shown. *p < 0.05 vs. control and between different groups as shown in the fi gure. the plus sign (+) indicates signifi cant difference from the cinnarizine + naloxonetreated group. six mice were used per each group. table 1. assessment of motor coordination in cinnarizine-treated mice in the rotarod test. treatment rotarod latency (sec) saline 446.4 ± 54.1 cinnarizine 1.25 mg/kg 465.4 ± 43.5 cinnarizine 2.5 mg/kg 512.8 ± 35.7 cinnarizine 5.0 mg/kg 498.0 ± 42.2 cinnarizine 10.0 mg/kg 392.8 ± 20.3 cinnarizine 20.0 mg/kg 375.4 ± 46.0 data represent mean ± s.e. co ntr ol ci nn ar izi ne 2. 5 m g/k g ha lop er ido l + c inn ar izi ne su lpi rid e + c inn ar izi ne do mp er ido ne ci nn ar izi ne me tac lop ra mi de + c inn ar izi ne br om oc ryp tin e + c inn ar izi ne ch lor pr om az ine + c inn ar izi ne 0 10 20 30 40 50 60 70 80 90 * * * * * -60.5% -70.1% -66.4% -53.8% -48.1% -74.2% -96.1% * *+ + # # # # # # n um be r of a bd om in al c on st ri ct io ns * * + co ntr ol ci nn ar izi ne 2. 5 m g/k g na lox on e + c inn ar izi ne yo him bin e + c inn ar izi ne at ro pin e + c inn ar izi ne pr op ra no lol + c inn ar izi ne 0 10 20 30 40 50 60 70 * * * * * -48.3% -45.3% -80.4% -80.4% -89.2% + + + n um be r of a bd om in al c on st ri ct io ns drug target insights 2007: 2 33 modulation of visceral nociception, infl ammation and gastric mucosal injury ruling out the confounding infl uence of a possible sedative effect. in man induction of extrapyramidal signs by cinnarizine has been reported, due to its antagonistic properties at dopamine d1 and d2 receptors (fabiani et al. 2004; teive et al. 2004). it is likely that higher doses are required in mice for cinnarizine to impair motor coordination signifi cantly. in other studies, cinnarizine (75 and 200 mg/kg) antagonized the ethanol-induced impairment of locomotor activity on rota-rod test in mice (czarnecka and kubik-bogucka, 1993). cinnarizine induced no catalepsy in mice at the dose of 20 mg/kg, inducing only mild catalepsy at the doses of 60 and 180 mg/kg (dall’igna, 2005). cinnarizine administered via subcutaneous (del pozo et al. 1987) or intracerebroventricular route (miranda et al. 1993) produced a dose-dependent antinociception in acetic acid writhing test in mice. the writhing response to acetic acid is brought about by the release of prostacyclin synthesized by cyclo-oxygenase in the abdominal cavity of the mice (berkenkopf and weichman, 1988). it is reduced by cyclo-oxygenase inhibitors such as meloxicam or diclofenac (santos et al. 1998), by figure 4. effect of theophylline (20 mg/kg, i.p.) on antinociception caused by cinnarizine (2.5 mg/kg, s.c.) in the abdominal constriction assay. drugs or saline (control) were administered 30 min prior to testing. data expressed as mean ± s.e. *p < 0.05 compared to control group. the plus sign (+) indicates signifi cant difference from the cinnarizine alone-treated group. six mice were used per each group. figure 5. effect of glibenclamide (5 mg/kg, i.p.) or baclofen (5 or 10 mg/kg, i.p.) on antinociception caused by cinnarizine (2.5 mg/kg, s.c.) in the abdominal constriction assay. drugs or saline (control) were administered 30 min prior to testing. data expressed as mean ± s.e. percent inhibition (%) compared to the control animals is shown. *p < 0.05 compared to control group and between different groups as shown in the fi gure. six mice were used per each group. effect of cinnarizine on gastric mucosal lesions induced by indomethacin in the indomethacin control group, the number and severity of gastric mucosal lesions were 5 ± 0.68 and 7 ± 1.0, respectively. this was signifi cantly reduced by co-administration of cinnarizine at 2.5, 5 or 10 mg/kg. it was noted however that the lower doses of the drug i.e. 2.5 or 5 mg/kg were more effective in inhibiting the development of gastric lesions than the higher dose of 10 mg/kg. thus cinnarizine at doses of 2.5 or 5 mg/kg, reduced the number and severity of gastric mucosal lesions caused by indomethacin by 67 & 76% and by 68.6 & 74.4%. cinnarizine at 10 mg/kg, reduced the number and severity of gastric lesions by 32 & 14.3% (fig. 10). discussion the present study provides evidence that cinnarizine exerts different effects on visceral pain, infl ammation and on the development of gastric mucosal damage in mice and rat. cinnarizine (1.25–20 mg/kg, s.c.) inhibited visceral pain evoked by i.p. acetic acid injection in mice. cinnarizine at the doses used in the present study did not impair motor performance in the rota-rod test, thus co ntr ol ci nn ar izi ne 2. 5 m g/k g th eo ph yll ine 20 m g/k g ci nn ar izi ne + t he op hy llin e 0 10 20 30 40 50 60 70 * ++ -49.7% n um be r of a bd om in al c on st ri ct io ns co ntr ol ci nn ar izi ne 2. 5 m g/k g gl ibe nc lam ide 5 mg /kg + ci nn ar izi ne 2. 5 m g/k g ba clo fen 5 mg /kg + ci nn ar izi ne 2. 5 m g/k g ba clo fen 10 m g/k g + ci nn ar izi ne 2. 5 m g/k g 0 25 50 75 * * * -61.2% -68.7% -48.6% * -97.5% -23.6% -35.9% * * * * * * n um be r of a bd om in al c on st ri ct io ns drug target insights 2007: 234 omar m.e. abdel-salam morphine (baamonde et al. 1989) and by antidepressant drugs (singh et al. 2001). in the present study, an attempt was made to pharmacologically characterize and investigate the possible neural pathways involved in the analgesic effect of cinnarizine. the possible involvement of neurotransmitter systems, such as dopaminergic, opioid, purinergic, cholinergic, catecholaminergic, gabaergic, systems as well as atp-gated potassium channels was evaluated. cinnarizine possesses direct anti-dopaminergic features (dopamine d1 and d2 receptor blockade) that are likely to contribute to the ability of this drug to cause extrapyramdial symptoms (reiriz et al. 1994; brucke et al. 1995). dopamine d2 receptors are involved in modulation of nociceptive responses and dopamine d2-receptor antagonists e.g. sulpiride caused antinociception in different pain models (ben-sreti et al. 1983; rooney and sewell, 1989; frussa-filho et al.1996). there is also an evidence of dopaminemediated descending nociceptive inhibition of spinal neurons (burkey et al. 1999). therefore, the involvement of the dopamine receptors in antinociception induced by cinnarizine was investigated. the effect of cinnarizine was unaffected by coadministration of the centrally acting dopamine d2 receptor antagonists, sulpiride, haloperidol or metoclopramide, the peripherally acting d2 receptor antagonist domperidone, but increased by the d2 receptor agonist bromocryptine and by the non-selective dopamine receptor antagonist chlorpromazine. these data do not suggest the involvement of dopamine d2 receptors in the visceral analgesic properties of cinnarizine. the antinociception caused by cinnarizine was in also unaffected by the opioid receptor antagonist naloxone, which is in agreement with earlier reports (miranda et al. 1993). the inhibition of adrenergic and cholinergic systems appears to facilitate cinnarizine-induced antinociception, since the co-administration of the beta-adrenergic antagonist propranolol, the muscarinic receptor antagonist atropine and the alpha2adrenergic antagonist yohimbine rather enhanced the effect of cinnarizine observed in the present study. most forms of pain arising from the gastrointestinal tract are mediated by activity in visceral afferent fibres running in sympathetic nerves (cervero, 1988). coeliac plexus block relieves visceral pain that is caused by carcinoma of the pancreas, stomach, gall bladder or liver (brown et al. 1987; eisenberg et al. 1995). chemical sympathectomy attenuated visceral nociceptive responses figure 6. effect of piracetam (300 mg/kg, i.p.) or vinpocetine (1.8 mg/kg, i.p.) on antinociception caused by cinnarizine (2.5 mg/kg, s.c.) in the abdominal constriction assay. drugs or saline (control) were administered 30 min prior to testing. data expressed as mean ± s.e. percent inhibition (%) compared to the control animals is shown. *p < 0.05 compared to control and between different groups as shown in the fi gure. six mice were used per each group. figure 7. effect of indomethacin (ind; 5 mg/kg, i.p.) or indomethacin (5 mg/kg, i.p.) + cinnarizine (2.5 mg/kg, s.c.) visceral pain in the abdominal constriction assay. drugs or saline (control) were administered 30 min prior to testing. data expressed as mean ± s.e. percent inhibition (%) compared to the control animals is shown. *p < 0.05 compared to control and between indomethacin or indomethacin + cinnarizine as shown in the fi gure. six mice were used per each group. co ntr ol ci nn ar izi ne 2. 5 m g/k g pi ra ce tam 30 0 m g/k g + ci nn ar izi ne 2. 5 m g/ kg vi np oc eti ne 1. 8 m g/k g + ci nn ar izi ne 2. 5 m g/k g 0 10 20 30 40 50 60 70 * * * * * * -51% -67.2% -54.5% -74.2% -56.1% * n um be r of a bd om in al c on st ri ct io ns co ntr ol in d in d + ci nn ar izi ne 2. 5 m g/k g 0 25 50 75 100 125 * * -61% -78.8% * n um be r of a bd om in al c on st ri ct io ns drug target insights 2007: 2 35 modulation of visceral nociception, infl ammation and gastric mucosal injury (kalmari et al. 2001), while the adrenergic neurone blocker guanethidine reduced the number of abdominal constrictions induced by acetic acid in mice (duarte et al. 1988). beta adrenoreceptor antagonists e.g. propranolol and metoprolol reduced visceral pain caused by i.p. injection of acetic acid in rat (korzeniewska-rybicka and plaznik, 2001). the spinal cholinergic system and muscarinic receptors are also important for regulation of nociception. spinally administered muscarinic receptor agonists can produce effective analgesia (iwamoto and marion, 1993). in the mouse acetic acid writhing test, m1-muscarinic agonists increased the pain threshold (bartolini et al. 1992), while atropine, a cholinergic muscarinic antagonist caused hyperalgesia only when administered at high doses of 5 mg/kg (ghelardini et al. 1990). in contrast, atropine administered at low doses of 1–100 μg/kg, resulted in analgesia, which might have been due to amplification of cholinergic transmission by a selective blockade of presynaptic muscarinic autoreceptors (ghelardini et al. 1990). in the present study, atropine administered ip at 2 mg/kg increased the analgesic effect of cinnarizine, thereby, suggesting an interaction at the muscarinic receptors. adenosine is an endogenous purine nucleoside that functions as an extracellular signalling molecule. it is released locally at sites of cellular trauma, and interacts with specifi c cell-surface purinergic receptors near its site of release to exert its effects. adenosine acts as an inhibitory neuromodulator in the central and peripheral nervous system (kowaluk, 1998; sawynok, 1999). blockade of adenosine receptors by theophylline, a nonselective adenosine receptor antagonist at a1 and a2 receptors, was shown to induce hyperalgesia (paalzow, 1994). adenosine a1 receptor agonists are effective antinociceptive agents in neuropathic and infl ammatory pain (curros-criado and herrero, 2005) and mice lacking the adenosine a1 receptor are hyperalgesic (wu et al. 2005). in the present study the antinociceptive effect of cinnarizine was prevented by co-treatment with the adenosine receptor blocker theophylline, suggesting that cinnarizine antinociception involves adenosine receptors. adenosine triphosphate (atp)-sensitive k+ channels (katp) play an important role in the mechanisms of pain modulation (asano et al. 2000; han et al. 2004; rodrigues et al. 2004). intrathecal administration of katp channel openers produces antinociception (asano et al. 2000). they can also contribute to the sensitization of primary afferents observed in gastrointestinal pain states (cervero and laird, 2003). in the present study, antinociception induced by cinnarizine was prevented by the administration of glibenclamide, a blocker of katp channel. this may suggests that this antinociceptive effect of cinnarizine may also rely on atpgated potassium channels. in the present study also the administration of cinnarizine reversed the baclofen—induced antinociception. baclofen, a prototypical agonist for gabab receptors, alters nociception at the level of the spinal cord by acting on gabab receptors located on primary afferent terminals and is known to produce analgesia in man and animals (dirig and yaksh, 1995; hara et al. 2004). cinnarizine inhibits the reuptake of gaba by sections of the rat brain cortex (mirzoian et al. 1998), which is likely to account for the observed effect of cinnarizine on the baclofen-antinociception. in the present study, the effect of cinnaizine on immobility time in porsolt’s forced-swimming test, a commonly used tool for screening of potential antidepressants (porsolt et al. 1977) was examined. only at the dose of 2.5 mg/kg, did cinnarizine reduced immobility time by 24%, although higher doses of the drug were without effect. other researches reported a decrease of immobility time by 5 mg/kg of cinnarizine (sushma et al. 2004). figure 8. effect of different doses of cinnarizine (1.25, 2.5, 5, 10 and 20 mg/kg) on the fl oating time in porsolt’s forced-swimming test in mice. data expressed as mean ± s.e. percent inhibition (%) compared to the control animals is shown. *p < 0.05 compared to saline control. six mice were used per each group. co ntr ol ci nn ar izi ne 1. 25 m g/k g ci nn ar izi ne 2. 5 m g/k g ci nn ar izi ne 5 mg /kg ci nn ar izi ne 10 m g/k g ci nn ar izi ne 20 m g/k g 0 100 200 300 * -24.7% se co nd s "i m m ob il e" ti m e ou t o f 6 m in ut es drug target insights 2007: 236 omar m.e. abdel-salam dopamine is implicated in the symptoms of depression (willner, 1995; brunswick et al. 2003; remy et al. 2005) and dopamine re-uptake inhibitors, bupropion and nomifensine reduce immobility in the forced swimming test by activation of d1 and d2 receptors (yamada et al. 2004). cinnarizine, however, exerts direct anti-dopaminergic effects (dopamine d1 and d2 receptor blockade) (reiriz et al. 1994; brucke et al. 1995). it is worthy to mention that cinnarizine also displays inhibitory activity on catecholamine uptake in storage vesicles (terland and flatmark, 1999) which could be involved at least in part in the observed decrease in immobility time by the drug. in paw oedema caused by carrageenan, cinnarizine at doses of 5–20 mg/kg, produced a dosedependent and marked inhibition of paw oedema response to carrageenan. cinnarizine in low doses failed to reduce the infl ammatory response. this result is in accordance to what has been reported previously of the anti-infl ammatory property of cinnarizine (blazso et al. 1999). time (h) 0 1 2 3 4 5 % in cr ea se in p aw v ol um e (o ed em a) 0 20 40 60 80 100 120 140 160 carrageenan control cinnarizine 1.25 mg/kg cinnarizine 2.5 mg/kg cinnarizine 5 mg/kg cinnarizine 10 mg/kg cinnarizine 20 mg/kg * * * * * * * * * * * figure 9. effect of different doses of cinnarizine (1.25, 2.5, 5, 10 and 20 mg/kg) on the paw odema caused by sub-plantar injection of carrageenan in rats. data expressed as mean ± s.e. percent inhibition (%) compared to the control animals is shown. *p < 0.05 compared to saline control. six rats were used per each group. figure 10. effect of cinnarizine (2.5, 5 and 10 mg/kg) on the number and severity of gastric lesions caused by s.c. indomethacin (20 mg/kg) in rats. data expressed as mean ± s.e. * p < 0.05 compared to saline control. the plus sign (+) indicates signifi cant difference from the 2.5 mg/kg cinnarizine group. six rats were used per each group. in d co ntr ol + ci nn ar izi ne 2. 5 m g/k g + ci nn ar izi ne 5 mg /kg + ci nn ar izi ne 10 m g/k g in d co ntr ol + ci nn ar izi ne 2. 5 m g/k g + ci nn ar izi ne 5 mg /kg + ci nn ar izi ne 10 m g/k g 0 1 2 3 4 5 6 7 8 * * * * * number of lesions -64% -76% -32% -68.6% -74.3% -14.3% severity of lesions + + g as tr ic m uc os al le si on s drug target insights 2007: 2 37 modulation of visceral nociception, infl ammation and gastric mucosal injury the effect of cinnarizine on gastric mucosa was also examined in the present study. gastric lesions induced by indomethacin were reduced dose-dependently by co-administration of cinnarizine, although it was noted that this effect was more evident with lower doses of 2.5, 5 mg/kg. studies indicated that cold/restraint stressand ethanol-induced lesions was decreased by the administration of cinnarizine, possibly due to decrease in the elevated histamine content by the drug (marazova et al. 1993; lozeva et al. 1994). cinnarizine has a complex the complex mechanism of action . in addition to a calcium channel blocking activity and antihistaminic properties, binding to both h1 and h2 receptors (nagai et al. 1986; nguyen et al. 2001), the drug displayed dopamine d1 and d2 receptor blocking effects as well as inhibitory effects on the reuptake of gaba (mirzoian et al. 1998) and on catecholamine uptake (terland and flatmark, 1999). the antihistaminic or catecholamine reuptake blocking properties might be involved in the antioedema effect observed in the present study. the benefi cial effect of cinnarizine on gastric lesions can be attributed to inhibition of gastric acid secretion (bouclier and spedding, 1985), to its vasodilator properties (izumo et al. 1999), leading to an increase in gastric mucosal blood fl ow or to its antihistaminic properties (nagai et al. 1986; nguyen et al. 2001). in summary the present study confi rms and extends previous studies suggesting anti-infl ammatory, antinociceptive and gastric protective properties for cinnarizine. the study indicates that mechanism by which cinnarizine modulates pain transmission is likely to involve adenosine receptors and atpgated potassium channels. the study in addition shows that cinnarizine inhibits gaba-mediated antinociception. references asano, t., dohi, s. and iida h. 2000. antinociceptive action of epidural k1 atp channel openers via interaction with morphine and an 2-adrenergic agonist in rats. anesth. analg., 90:1146–51. baamonde, a., hidalgo, a. and andres-trelles, f. 1989. sex-related differences in the effects of morphine and stress on visceral pain. neuropharmacology, 28:967–970. bartolini, a., ghelardini, c., fantetti, l., malcangio, m., malmberg-aiello, p. and giotti, a. 1992. role of muscarinic receptor subtypes in central antinociception. br. j. pharmacol., 105:77–82. ben-sreti, m.m., gonzalez, j.p. and sewell, r.d. 1983. differential effects of skf 38393 and ly 141865 on nociception and morphine analgesia. life sci., 33 (suppl 1):665–658. berkenkopf, j.w. and weichman, b.m. 1988. production of prostacyclin in mice following intraperitoneal injection of acetic acid, phenylbenzoquinone and zymosan: its role in the writhing response. prostaglandins, 36:693–709. blazso, g., razga, z. and gabor, m. 1999. effects of cinnarizine on different experimentally induced oedemas. fundam. clin. pharmacol., 13:91–95. bouclier, m. and spedding, m. 1985. differential effects of calcium channel antagonists on histamine and pentagastrin-stimulated gastric acid secretion in the rat. agents actions,16:491–495. brown, d.l., bulley, c.k. and quiel, e.l. 1987. neurolytic celiac plexus block for pancreatic cancer pain. anesth. analg., 66:869–873. brucke, t., wober, c., podreka, i., wober-bingol, c., asenbaum, s., aull, s., wenger, s., ilieva, d., harasko-van der meer, c. and wessely p. et al. 1995. d2 receptor blockade by fl unarizine and cinnarizine explains extrapyramidal side effects. a spect study. j. cereb. blood. flow. metab., 15:513–518. brunswick, d.j., amsterdam, j.d., mozley, p.d. and newberg, a. 2003. greater availability of brain dopamine transporters in major depression shown by [99mtc]trodat-1 spect imaging. am. j. psychiatry., 160:1836–1841. burkey, a.r., carstens, e. and jasmin, l. 1999. dopamine reuptake inhibition in the rostral agranular insular cortex produces antinociception. j. neurosci., 19:4169–4179. cervero, f. 1988. neurophysiology of gastrointestinal pain. baillieres. clin. gastroenterol., 2:183–99. cervero, f. and laird, j.m. 2003. role of ion channels in mechanisms controlling gastrointestinal pain pathways. curr. opin. pharmacol., 3:608–612. curros-criado, m.m. and herrero, j.f. 2005. the antinociceptive effects of the systemic adenosine a1 receptor agonist cpa in the absence and in the presence of spinal cord sensitization. pharmacol. biochem. behav., 82:721–726. czarnecka, e. and kubik-bogucka, e. 1993. effects of calcium antagonists on central actions of ethanol: comparative studies with nifedipine, verapamil and cinnarizine. alcohol. alcohol., 28:649–655. dall’igna, o.p. 2005. cinnarizine has an atypical antipsychotic profi le in animal models of psychosis. j. psychopharmacol., 19:342–346. del pozo, e., caro, g. and baeyens, j.m. 1987. analgesic effects of several calcium channel blockers in mice. eur. j. pharmacol., 137:155–160. dirig, d.m. and yaksh, t.l. 1995. intrathecal baclofen and muscimol, but not midazolam, are antinociceptive using the rat-formalin model. j. pharmacol. exp. ther., 275:219–227. duarte, i.d., nakamura, m. and ferreira, s.h. 1988. participation of the sympathetic system in acetic acid-induced writhing in mice. braz. j. med. biol. res., 21:341–343. eisenberg, e., carr, d.b. and chalmers, t.c. 1995. neurolytic celiac plexus block for treatment of cancer pain: a meta-analysis. anesth. analg., 80:290–295. fabiani, g., pastro, p.c. and froehner, c. 2004. parkinsonism and other movement disorders in outpatients in chronic use of cinnarizine and fl unarizine. arq. neuropsiquiatr., 62:784–788. frussa-filho, r., rocha, j.b. and conceicao, i.m. 1996. effects of dopaminergic agents on visceral pain measured by the mouse writhing test. arch. int. pharmacodyn. ther., 331:74–93. ghelardini, c., malmberg-aiello, p., giotti, a., malcangio, m. and bartolini a. 1990. investigation into atropine-induced antinociception. br. j. pharmacol., 101:49–54. han, b.f., zhang, c., reyes-vazquez, c., qiao, j.t. and dafny, n. 2004. atp-sensitive potassium channels and endogenous adenosine are involved in spinal antinociception produced by locus coeruleus stimulation. int. j. neurosci., 114:961–974 hara, k., saito, y., kirihara, y. and sakura, s. 2004. the interaction between gamma-aminobutyric acid agonists and diltiazem in visceral antinociception in rats. anesth. analg, 98:1380–1384. hirose, g. 2006. drug induced parkinsonism: a review. j. neurol., 253 suppl 3:iii22–iii24. iwamoto, e.t. and marion, l. 1993. characterization of the antinociception produced by intrathecally administered muscarinic agonists in rats. j. pharmacol. exp. ther., 266:329–38. drug target insights 2007: 238 omar m.e. abdel-salam izumo, t., suzuki, g., chen, z., fujii, y. and kamei, c. 1999. effects of certain cerebral circulation activating drugs on regional cerebral blood fl ow in rats. methods find. exp. clin. pharmacol., 21:279–83. kalmari, j., niissalo, s., konttinen, y.t. and pertovaara a. 2001. modulation of visceral nociceptive responses of rat spinal dorsal horn neurons by sympathectomy. neuroreport., 12:797–801. korzeniewska-rybicka, i. and plaznik a. 2001. role of serotonergic and noradrenergic systems in a model of visceral pain. pol. j. pharmacol., 53:475–480. koster, r., anderson, m. and de beer, e.j. 1959. acetic acid for analgesic screening. fed. proc., 18:412. kowaluk, e.a. 1998. adenosine modulation: a novel approach to analgesia and infl ammation. expert opin. investig. drugs, 7:535–543. lozeva, v., marazova, k. and belcheva, a. 1994. gastric histamine content and ulcer formation in rats with ethanol-induced injury. effects of cinnarizine and fl unarizine. agents actions, 41 spec no:c91–92. mansooreh, t., hossein, a. and parvin, t. 2006. open-label trial of cinnarizine in migraine prophylaxis. headache, 46:498–502. marazova, k., lozeva, v. and belcheva, a. 1993. gastric histamine content and gastric ulcer formation in cold/restraint stressed rats: effects of cinnarizine and fl unarizine. infl ammation research, 38:c301–c303. miranda, h.f., pelissier, t. and sierralta, f. 1993. analgesic effects of intracerebroventricular administration of calcium channel blockers in mice. gen. pharmacol., 24:201–204. mirzoian, n.r., akopian, v.p., kocharian, a.z., knarian, v.a., arakelian, l. and gevorkian, v.a. 1998. gaba reuptake by rat brain slices in hypokinesia and under the infl uence of cinnarizine and fl unarizine. eksp. klin. farmakol., 61:15–17. mózsik, gy., móron, f. and jávor, t. 1982. cellular mechanisms of the development of gastric mucosal damage and of gastric cytoprotection induced by prostacyclin in rats a pharmacological study. prostaglandins leukot. med., 9:71–84. nagai, h., yakuo, i., yamada, h., inagaki, n., goto, s. and koda, a. 1986. effect of cinnarizine on ige antibody mediated allergic reaction in mice and rats. j. pharmacobiodyn., 9:923–927. negrotti, a. and calzetti, s. 1997. a long-term follow-up study of cinnarizine and fl unarizine-induced parkinsonism. mov. disord., 12:107–110. nguyen, t., shapiro, d.a., george, s.r., setola, v., lee, d.k., cheng, r., rauser, l., lee, s.p., lynch, k.r., roth b.l. and o’dowd, b.f. 2001. discovery of a novel member of the histamine receptor family. mol. pharmacol., 59:427−433. paget, g.e. and barnes, j.m. toxicity tests. 1964. in evaluation of drug activities pharmacometics. eds. laurence dr, bacharach al. london and newyork: academic press, pp. 1–135. paalzow, g.h. 1994. noradrenaline but not dopamine involved in nmda receptor-mediated hyperalgesiainduced by theophylline in awake rats. eur. j. pharmacol., 252:87–97. porsolt, r.d., le pichon, m. and jalfre, m. 1977. depression: a new animal model sensitive to antidepressant treatments. nature, 266:730–732. rego, e.m., corrado, a.p. and prado, w.a. 1990. antinociceptive effects of calcium channel blockers in the rat. braz. j. med. biol. res., 23:297–305. reiriz, j., ambrosio, s., cobos, a., ballarin, m., tolosa, e. and mahy, n. 1994. dopaminergic function in rat brain after oral administration of calcium-channel blockers or haloperidol. a microdialysis study. j. neural. transm. gen. sect., 95:195–207. remy, p., doder, m., lees, a., turjanski, n. and brooks, d. 2005. depression in parkinson’s disease: loss of dopamine and noradrenaline innervation in the limbic system. brain, 128:1314–1322. rodrigues, a.r.a., castro, m.s.a., francischi, j.n., perez, a.c. and duarte, i.d.g. 2004. participation of atp-sensitive k+ channels in the peripheral antinociceptive effect of fentanyl in rats. braz. j. med. biol. res., 38:91–97. rooney, k.f. and sewell, r.d. 1989. evaluation of selective actions of dopamine d-1 and d-2 receptor agonists and antagonists on opioid antinociception. eur. j. pharmacol., 168:329–336. santos, a.r., vedana, e.m. and de-freitas, g.a. 1998. antinociceptive effect of meloxicam, in neurogenic and infl ammatory nociceptive models in mice. infl amm., res., 47:302–307. sawynok, j. 1998. adenosine receptor activation and nociception. eur. j. pharmacol., 347:1–11. singh, v.p., jain, n.k. and kulkarni, s.k. 2001. on the antinociceptive effect of fl uoxetine, a selective serotonin reuptake inhibitor. brain. res., 915:218–226. sushma, m., sudha, s. and guido, s. 2004. effect of calcium channel blockers on antidepressant activity following electroconvulsive shock in mice. indian j. pharmacol., 36:317–319 teive, h.a., troiano, a.r., germiniani, f.m. and werneck, l.c. 2004. flunarizine and cinnarizine-induced parkinsonism: a historical and clinical analysis. parkinsonism relat. disord., 10:243–245. terland, o. and flatmark, t. 1999. drug-induced parkinsonism: cinnarizine and fl unarizine are potent uncouplers of the vacuolar h+-atpase in catecholamine storage vesicles. neuropharmacology, 38:879–882. willner, p. 1995. dopaminergic mechanisms in depression and mania, in psychopharmacology: the fourth generation of progress. edited by bloom, f.e. and kupfer, d.j. new york, raven press, pp. 921–931. winter, c.a., risley, e.a. and nuss, g.w. 1962. carrageenan-induced edema in hind paw of the rat as an assay for antiinfl ammatory drugs. proc. soc. exp. biol. med., 111:544–52. wu, w.p., hao, j.x., halldner, l., lovdahl, c., delander, g.e., wiesenfeldhallin, z., fredholm, b.b. and xu, x.j. 2005. increased nociceptive response in mice lacking the adenosine a1 receptor. pain, 113:395– 404. yamadaa, j., sugimotoa, y. and yamadab s. 2004. involvement of dopamine receptors in the anti-immobility effects of dopamine re-uptake inhibitors in the forced swimming test. eur. j. pharmacol., 504:207–211. untitled 9 correspondence: kunitada shimotohno, ph. d., laboratory of human tumor viruses, institute for virus research, kyoto university, 53 kawaharacho, shogoin, sakyo-ku, kyoto, 606-8507, japan. tel: +81-75-751-4000; fax: +81-75-751-3998; email: kshimoto@virus.kyoto-u.ac.jp please note that this article may not be used for commercial purposes. for further information please refer to the copyright statement at http://www.la-press.com/copyright.htm review cyclophilin and viruses: cyclophilin as a cofactor for viral infection and possible anti-viral target koichi watashi and kunitada shimotohno department of viral oncology, institute for virus research, kyoto university, kyoto, japan. abstract: cyclophilin (cyp) is a peptidyl prolyl cis/trans isomerase, catalyzing the cis-trans isomerization of proline residues in proteins. cyp plays key roles in several different aspects of cellular physiology including the immune response, transcription, mitochondrial function, cell death, and chemotaxis. in addition to these cellular events, a number of reports demonstrated that cyp plays a critical role in the life cycle of viruses, especially human immunodefi ciency virus (hiv) and hepatitis c virus (hcv). these two viruses are signifi cant causes of morbidity and mortality worldwide, but current therapies are often insuffi cient. cyp may provide a novel therapeutic target for the management and/or cure of these diseases, in particular hcv. keywords: cyclosporin, hiv, hcv, virus, replication, mptp. immunophilins and immunosuppressants cyclophilin (cyp) and fk506 binding protein (fkbp) are peptidyl-prolyl cis-trans isomerases (ppiases), enzymes that catalyze the cis-trans interconversion of peptide bonds amino terminal to proline residues (fischer et al. 1989; harding et al. 1989; takahashi, 1999; takahashi et al. 1989). cyp and fkbp are originally identifi ed as cellular factors that bind csa and fk506, respectively, both of which are immunosuppressants used clinically for the prevention of graft rejection following organ transplantation (handschumacher et al. 1984; harding et al. 1989). therefore, these ppiases are also called immunophilin. the action of ppiases leads to changes in protein conformation (takahashi, 1999), but the binding of csa and fk506 to cyp and fkbp, respectively, inhibits the activity of these enzymes (fischer et al. 1989; rosen et al. 1990; takahashi et al. 1989). however, the inhibition of ppiase activity by csa and fk506 is an insuffi cient requirement for their immunosuppressive function (bierer et al. 1990; schreiber, 1991). the csa/cyp or fk506/fkbp complex, subsequently interacts with and inhibits calcineurin (cn), a phosphatase involved in the activation of the transcription factor nf-at. proper nf-at function is essential for the generation of a productive t cell response (clipstone and crabtree, 1992; fruman et al. 1992; liu et al. 1991). in the absence of immunosuppressants, cn dephosphorylates cytoplasmic nf-at, leading to nf-at nuclear translocation and transactivation of downstream genes participating in the immune response (liu et al. 1992; mccaffrey et al. 1993). csa and fk506 prevent the dephosphorylation and subsequent nuclear translocation of nf-at leading to immunosuppression. role of cyp family members in cellular events more than 10 cyp subtypes are found in mammals (table 1). the subcellular localization of cyps varies. cypa is primarily found in the cytoplasm, while cypb, cypd, cype, and ranbp2 are distributed in the endoplasmic reticulum (er), mitochondria, nucleus, and nuclear pore, respectively. members of the cyp family play roles in a variety of cellular processes including the immune response, transcription, mitochondrial function, cell death, and chemotaxis, as described below. while a number of cyp family members have been ideitifi ed, intensive functional analysis has been performed on only a few including cypa, cypb, cypd, and cyp40. cypa is the most abundant cyp subtype found in the cells (waldmeier et al. 2003), and it is the primary factor mediating the immunosuppressive effects of csa (colgan et al. 2005). however, even in the absence of csa, cypa plays an important role in regulating the immune responses as seen in drug target insights 2007: 2 9–18 10 watashi and shimotohno cypa-defi cient mice. cypa-knockout mice have an “allergic” phenotype with increased serum igg1 and ige levels and tissue infi ltration by mononuclear cells, eosinophils, and mast cells (colgan et al. 2004), related to increased and dysregulated activity of th2 cd4+ t cells. in cypa-knockout cells, interleukin-2 tyrosine kinase (itk), a signaling molecule crucial for the development of a th2 response, is constitutively activated. itk is a member of the tec family of sh2/sh3-containing tyrosine kinases, and it participates in the signal transduction cascade leading to t cell activation. cypa can bind itk, and this negatively regulates itk activity (brazin et al. 2002). thus, cypa plays a suppressive role in the development of cd4+ t cell responses through its interaction with itk. o t h e r s t u d i e s h a v e r e p o r t e d s e v e r a l non-immune system roles for cypa. cypa interacts with apoptosis-inducing factor (aif) and promotes aif-mediated chromatinolysis during apoptosis (cande et al. 2004). additionally, cypa interacts with membrane-bound guanylate cyclase-a (gc-a), a receptor for atrial natriuretic factor (anf) (chen et al. 2004). gc-a and anf are involved in cardiovascular homeostasis, and cypa appears to function as an endogenous inhibitor of gc-a activation by competing for anf binding. further interactions of cypa with prolactin receptor (syed et al. 2003) and transcription factor yy1 (yang et al. 1995) have been observed, but the exact role of cypa in these processes remains unclear. cypa was also observed to bind dna in a zinc-dependent manner in a mouse macrophage cell line (krummrei et al. 1995). however, the best-characterized role identified for cypa is not in normal cellular physiology, but rather as co-factor during the human immunodefi ciency virus-1 (hiv-1) viral life cycle (see below). cypb was originally identifi ed as a cyp family member bearing a signal sequence leading to the er lumen or the secretory pathway (price et al. 1991), but the specifi c function of cypb is poorly understood. a yeast two-hybrid screening using cypb as a bait identified an interaction with calcium-signal modulating cyclophilin ligand (caml) (bram and crabtree, 1994). caml is located on the cytoplasmic face of the er membrane (holloway and bram, 1998). caml participates in calcium signal transduction pathway and it is essential for peripheral t cell development (tran et al. 2005). however, the importance of cypb binding to caml function remains unknown. cypb also enhances prolactin-driven cell proliferation (rycyzyn et al. 2000) and promotes the nuclear retrotranslocation of prolactin through a direct interaction. additionally, cypb potentiates prolactin-induced stat5 transactivation by promoting the dissociation of pias3, a stat5 repressor (rycyzyn and clevenger, 2002). cypb can also associate with interferon regulatory factor (irf)-3 (obata et al. 2005). extracellular cypb can bind platelets (allain et al. 1999) and table 1. human cyclophilin subtypes. protein name length genbank accession no. reference cypa 165 aa nm_021130 liu et al. 1990 cypb 216 aa nm_000942 price et al. 1991 cypc 212 aa nm_000943 friedman et al. 1991 cyp40 370 aa nm_005038 kieffer et al. 1992 cype, cyp33 301 aa nm_006112 mi et al. 1996 cypd, cypf, cyp3 207 aa nm_005729 bergsma et al. 1991 cypg, cars-cyp,srcyp 754 aa nm_004792 nestel et al. 1996 cyph, usa-cyp, snucyp-20 177 aa nm_006347 horowitz et al. 1997 ppi-l1 166 aa nm_016059 ozaki et al. 1996 ppi-l2, cyp60 520 aa nm_014337 wang et al. 1996 ppil3 165 aa nm_032472 zhou et al. 2001 ppi-l4 492 aa nm_139126 zeng et al. 2001 ppi-l5, lrr-1 414 aa nm_152329 jang et al. 2001 ranbp2 3224 aa nm_006267 yokoyama et al. 1995 drug target insights 2007: 2 11 cyclophilin and viruses this initiates a transmembranous infl ux of calcium ion, kinase activation, and platelet adhesion to collagen. accumulating evidence suggests that cyps, in particular cypa and cypb, can mediate intercellular communication similar to cytokines. cyps are secreted from cells in response to infl ammatory stimuli or oxidative stress (jin et al. 2000; seko et al. 2004; sherry et al. 1992; xu et al. 1992) and they can act as potent chemoattractants for neutrophils (sherry et al. 1992), eosinophils (xu et al. 1992), and t cells (allain et al. 2002). cypa and cypb are recognized by the cell surface receptor cd147, and cyp binding leads to erk activation and chemotaxis (pushkarsky et al. 2001; yurchenko et al. 2001; yurchenko et al. 2002). cypd plays a critical role in mitochondrial function and cell death (tanveer et al. 1996). during ischemia-induced necrosis, e.g. following a heart attack or stroke, the accumulation of calcium and increase of reactive oxygen species (ros) trigger the opening of a pore in the inner mitochondrial membrane, known as the membrane permeability transition pore (mptp) (halestrap, 1999). calcium overload and ros induce a conformational change in adenine nucleotide translocase (ant), a key component regulating the opening of mptp at the inner mitochondrial membrane. the opening of mptp leads to mitochondrial swelling, rupture of the outer membrane, and the release of small molecules (waldmeier et al. 2003). cypd is located within the matrix of the mitochondria and it binds ant facilitating its conformational change (crompton et al. 1998; woodfi eld et al. 1998). in cypd-knockout cells, necrosis induced by calcium and ros was decreased, but apoptotic cell death induced by cytokines or anticancer agents was unaffected (baines et al. 2005; nakagawa et al. 2005). cypd-knockout mice also experienced reduced cardiac injury following reperfusion after ischemia. thus, cypd is a key molecule involved in the cell death process. cyp40 regulates the activity of steroid receptors (srs) (duina et al. 1996; owens-grillo et al. 1995; ratajczak et al. 1993). srs including the glucocorticoid receptor, estrogen receptor, androgen receptor and progesterone receptor are nuclear hormone receptors that exert transcriptional activity following steroid ligand binding and nuclear translocation. in the absence of steroid ligands, srs form complexes with heat shock protein 90 (hsp90) together with the immunophilins cyp40, fkbp51, or fkbp52 in the cytoplasm. these immunophilins control sr activity by increasing receptor avidity for hormone ligands through ppiase-dependent conformational changes. upon hormone binding, this sr/hsp90/ immunophilin complex dissociates, leaving homodimeric sr, which then translocates into the nucleus to transactivate downstream genes. although there are some reports on other cyp subtypes (table 1), the precise functions and signifi cances of them are largely unknown. viruses requiring cyps as described above, cyps play essential roles in diverse cellular processes. interestingly, several viruses have evolved to use cyps during their life cycles. in particular, cyps are demonstrated to be involved in the proliferation of hiv-1 and hepatitis c virus (hcv). other viruses using cyps during their life cycle include vaccinia virus (vv), vesicular stomatitis virus (vsv), and sars-coronavirus. vaccinia virus a signifi cant role of cyp in vv replication was fi rst identifi ed through the analysis of several csa analogs. the ability of cyclosporins to suppress vv replication correlated with the inhibition of cyp function (damaso and moussatche, 1998). vv infection stabilizes cypa, leading to the accumulation of cypa (castro et al. 2003). in vv infected cells, cypa relocalizes to the peripheral region of the nucleus, colocalizing with sites of virus production. cypa is incorporated into viral particles and is located in the viral core. vesicular stomatitis virus cypa interacted with the nucleocapsid protein of vsv (bose et al. 2003), and, like vv, cypa is incorporated into vsv viral particles. although the binding and incorporation of cypa occurred beyond the virus serotypes, the functional role of cypa in the viral life cycle appears to be straindependent. inhibition of cyp activity by csa reduced primary transcription of vsv-new jersey (vsv-nj) but not vsv-indiana (vsv-ind) serotype, and cypa activity was required for the replication of vsv-nj to a greater extent than vsv-ind. the authors suggest that differential requirements of cypa are likely the results of evolutionary pressure during lineage development. drug target insights 2007: 2 12 watashi and shimotohno sars coronavirus the nucleocapsid protein (np) of severe acute respiratory syndrome coronavirus (sars-cov) binds cypa (luo et al. 2004), and another group reported that cypa is incorporated into sars-cov particles (chen et al. 2005). extracellular cypa binds cd147 on the cell surface, and treatment with a peptide that blocks cd147 binding inhibits viral infection. thus, cypa may be involved in sars-cov invasion into host cells through interaction with np and cd147, respectively. cypa and hiv-1 cypa plays an important role in the viral life cycle of hiv-1. in 1993, cypa was found to interact with hiv-1 gag (luban et al. 1993), and in 1994, cypa was reportedly incorporated into viral particles (franke et al. 1994; thali et al. 1994). a gene targeting study demonstrated that only cypa among cyp subtypes was essential for hiv-1 proliferation (braaten and luban, 2001). within the hiv-1 life cycle, cypa plays multiple roles through different interaction partners, including an early step prior to reverse transcription (braaten et al. 1996; mlynar et al. 1997; steinkasserer et al.1995). although cypa is incorporated into virions through binding to the ca domain of the gag polyprotein (franke et al. 1994; ott et al. 1995; thali et al. 1994), this incorporation is not required for viral infection. instead, target cell expressed cypa is important for productive infection and viral replication (hatziioannou et al. 2005; sokolskaja et al. 2004). it has been known for several decades that host cells express different restriction factors to prevent infection by certain retroviruses (cullen, 2003), and several recent studies have suggested that cypa modulates sensitivity to such a restriction factor early in the hiv-1 life cycle prior to reverse transcription. trim5α is a host restriction factor originally identifi ed using expression cloning that recognizes ca limiting retrovirus proliferation (stremlau et al. 2004). towers et al. showed that cypa regulates the activity of a host restriction factor (towers et al. 2003). disruption of cypa-ca binding by introducing of point mutation into ca or treating human cells with csa decreases hiv-1 infectivity. conversely, the loss of cypa-ca binding greatly enhanced hiv-1 infectivity in simian cells (berthoux et al. 2005; kootstra et al. 2003; sayah et al. 2004). from the results, the hypothesis was proposed by luban et al. that ca binding by cypa prevented normal antiviral effects mediated by trim5α during hiv-1 infection of human cells, but this same interaction mediated hiv-1 restriction in nonhuman primate cells (sokolskaja et al. 2006; luban, in press). both the mechanism of trim5α restriction of hiv-1 and the modulation of ca recognition by cypa remain unclear, and further studies are clearly needed to resolve these important issues in the hiv-1 life cycle and the host response to hiv-1 infection. cypa may be important for other aspects of hiv-1 infection. cypa interacts with cd147 (pushkarsky et al. 2001), heparans (saphire et al. 1999), vpr (zander et al. 2003), and envelope glycoprotein gp120 (endrich and gehring, 1998), although their relevances of the interactions should be further verifi ed. cypb and hcv current therapy against hcv hcv is a major causative agent of chronic liver diseases such as chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (liang et al. 1993). hcv infection is a serious health problem affecting approximately 170 million individuals worldwide (poynard et al. 2003). the current standard therapy for hcv is restricted to interferon (ifn) or pegylated-ifn either alone or in combination with ribavirin. because treatment with these agents, however, fails to produce sustained virus elimination in about one half of the patients (di bisceglie et al. 2002), alternative and effective strategies to combat against hcv are greatly needed. hcv encodes a single polypeptide that is cleaved by host and hcv-encoded protease including ns3 to generate a set of functional proteins. its genome is replicated by the hcv-encoded rna-dependent rna polymerase (rdrp) ns5b. both of these proteins, ns3 and ns5b, are essential for hcv genome replication and are possible targets for the development of anti-hcv therapeutics (di bisceglie et al. 2002). small molecule compounds targeting ns3 and ns5b have been developed, and their effi cacy has been examined in clinical trials (di bisceglie et al. 2002). in addition to these viral enzymes, host cell factors are required for viral replication, and these may provide other options for the development of novel anti-viral agents. disrupting the function of drug target insights 2007: 2 13 cyclophilin and viruses host cell derived factors is particularly appealing as the mutation rate of host proteins is much less than that of viral encoded proteins and should less give rise to drug-resistant viruses. however, while some host factors required for viral genome replication have been identifi ed, other host proteins need to be identifi ed to develop optimal anti-viral therapies with few side effects. at this time, only a limited number of host proteins have been found to be involved in hcv genome replication with biological relevance. hvap-33 is one of snare family proteins that regulate vesicle biogenesis, protein sorting, and membrane fusion. hvap-33 binds hcv ns5a and ns5b, and this interaction regulates the presence of hcv proteins in the subcellular compartment performing viral genome replication (evans et al. 2004; gao et al. 2004; tu et al. 1999). fbl2 is a member of the f-box protein family, involved in the ubiquitination pathway. fbl2 is geranylgeranylated in cells (wang et al. 2005), and associates with ns5a in a geranylgeranylation-dependent manner to regulate hcv genome replication. however, the mechanism of action of fbl2 in hcv genome replication is not known. additionally, we recently found that cypb is a cofactor for hcv replication in host cells, and this may represent a new target for anti-hcv therapeutics (watashi et al. 2005). we will fi rst discuss the role of cypb in hcv genome replication followed by the therapeutic implications of this discovery in hcv treatment. anti-hcv activity of cyclosporin we identifi ed csa as an anti-hcv agent using a hcv replicon system, a cell culture system supporting hcv genome replication (lohmann et al. 1999), and csa inhibits hcv genome replication as potently as ifnα (watashi et al. 2003). since that time, several groups have made similar observations (firpi et al. 2006; nakagawa et al. 2004; paeshuyse et al. 2006). as shown in fig.1a, cellular treatment with 1 µg/ml csa decreases hcv rna levels by approximately 1/500 (watashi et al. 2003). csa also reduces the expression of hcv-encoded proteins to undetectable levels (fig. 1b). in contrast, fk506 has no effect on the production of hcv rna or proteins (fig. 1a and b). the differences between csa and fk506 suggest that csa prevents viral genome replication independently of cn, an effector ( c o p y /p g t o ta l r n a ) a m o u n t o f h c v r n a figure. 1 csa suppresses hcv genome replication. (a) hcv rna was quantifi ed in total rna isolated from hcv replicon-bearing cells treated with various concentrations of csa, fk506, or ifnα for 7 days. the amount of hcv rna per 1 pg total rna was plotted against the concentration of csa (µg/ml), fk506 (µg/ml), or ifnα (× 100 iu/ml). (b) the expression of hcv ns5a and protein disulfi de isomerase (pdi) as a cellular protein was examined in the hcv replicon-bearing cells treated without (control) or with 100 iu/ml ifnα, 1�µg/ml csa, or 1�µg/ml fk506 for 7 days. drug target insights 2007: 2 14 watashi and shimotohno common to both csa and fk506 mediated immunosuppression. and the anti-hcv effects of csa are mediated by pathway(s) distinct from those of ifnα (watashi et al. 2003). cypb as a cellular cofactor of hcv genome replication the ability of csa to inhibit hcv genome replication correlates with the inhibition of cyp activity (watashi et al. 2005). moreover, an alternative cyp inhibitor, sanglifehrin, also decreases the levels of hcv rna. thus, the inhibition of cyp activity is essential for the anti-hcv effect of csa, and this strongly suggests that cyp plays a direct, important role in hcv genome replication. moreover, the specifi c knockdown of cypb by rnai reduced hcv rna titer, but knockdown of cypa, cypc, cype, or cyph had no effect on hcv replication activity. these data indicate that cypb plays a critical role in hcv genome replication. regulation of ns5b by cypb the effects of cypb on hcv genome replication are mediated through a direct interaction with ns5b as demonstrated in both in vitro and in cells (watashi et al. 2005) (fig. 2a). cypb do not bind any other hcv proteins involved in viral replication. ns5b binds hcv genome rna in order to function as a rdrp. cypb but not cypa promotes the rna binding activity of ns5b and stimulates hcv genome replication in cells (fig. 2a). this functional support by cypb to ns5b is essential for the effi cient replication of the hcv genome, and csa blocks the interaction of cypb with ns5b, leading to reduced rna binding (fig. 2b). figure. 2 cypb regulates the activity of ns5b. (a) in the absence of csa (normal conditions), ns5b associates with cellular cypb to effi ciently bind to the hcv genome rna and drive genome replication. (b) in the presence of csa, cypb does not interact with ns5b. free ns5b less functions, and viral genome replication is impaired in the absence of functional cypb. drug target insights 2007: 2 15 cyclophilin and viruses thus, cypb serves as a cellular cofactor for hcv genome replication. therapeutic implications of cyp inhibition for the treatment for hcv the anti-hcv activity of csa analogs correlates with their ability to inhibit cyp function (watashi et al. 2005). the dissociation of cypb and ns5b greatly reduces the extent of hcv genome replication. these observations suggest that the inhibition of cypb may represent a novel therapeutic strategy against hcv. this possibility has been examined by two reports using stronger cyp inhibitors than csa. paeshuyse et al. used the csa analog debio-025 to inhibit cyp activity (paeshuyse et al. 2006), and this compound inhibited hcv replication 10-fold more potently than csa. the authors speculated that debio-025 might be an attractive drug candidate for the treatment of individuals with hcv/hiv coinfection because csa derivatives should also inhibit hiv-1 replication. we used the non-immunosuppressive csa derivative nim811 to target cyp (goto et al. 2006; ishii et al. 2006). nim811 inhibits cyp enzymatic activity two-fold more than csa (rosenwirth et al. 1994), and this increased inhibition correlates with greater suppression of hcv genome replication than csa, especially at lower doses. cotreatment of cells with nim811 and ifnα led to a synergistic anti-hcv effect at higher doses of nim811. treatment of nim811 for three weeks eliminated hcv rna from host cells to under detectable level. because the immunosuppression in patients during a viral infection is undesirable, these non-immunosuppressive variants of csa that inhibit cyp activity are likely to offer great promise for the treatment of patients with chronic hcv infection. conclusion cyps are cellular ppiases that catalyze conformational changes in proteins, but the role(s) and substrates for this protein family in cells are not well-characterized. however, discoveries of the signifi cance of cyp in life cycles for several viruses make this protein virologically notable and present novel therapeutic anti-viral targets. further analyses of the role of cyps in viral life cycles should reveal novel functions for these proteins as well as provide mechanistic insight into possible therapeutic targets. references allain, f., durieux, s., denys, a., carpentier, m. and spik, g. 1999. cyclophilin b binding to platelets supports calcium-dependent adhesion to collagen. blood, 94:976–983. allain, f., vanpouille, c., carpentier, m., slomianny, m. c., durieux, s. and spik, g. 2002. interaction with glycosaminoglycans is required for cyclophilin b to trigger integrin-mediated adhesion of peripheral blood t lymphocytes to extracellular matrix. proc. natl. acad. sci. u.s.a., 99:2714–2719. baines, c.p., kaiser, r.a., purcell, n.h., blair, n.s., osinska, h., hambleton, m.a., brunskill, e.w., sayen, m.r., gottlieb, r.a. and dorn, g. w. et al. 2005. loss of cyclophilin d reveals a critical role for mitochondrial permeability transition in cell death. nature, 434:658–662. bergsma, d.j., eder, c., gross, m., kersten, h., sylvester, d., appelbaum, e., cusimano, d., livi, g.p., mclaughlin, m.m. and kasyan, k. et al. 1991. the cyclophilin multigene family of peptidyl-prolyl isomerases. characterization of three separate human isoforms. j. biol. chem., 266:23204–23214. berthoux, l., sebastian, s., sokolskaja, e. and luban, j. 2005. cyclophilin a is required for trim5{alpha}-mediated resistance to hiv-1 in old world monkey cells. proc. natl. acad. sci. u.s.a., 102:14849– 14853. bierer, b.e., somers, p.k., wandless, t.j., burakoff, s.j. and schreiber, s.l. 1990. probing immunosuppressant action with a nonnatural immunophilin ligand. science, 250:556–559. bose, s., mathur, m., bates, p., joshi, n. and banerjee, a.k. 2003. requirement for cyclophilin a for the replication of vesicular stomatitis virus new jersey serotype. j. gen. virol., 84:1687–1699. braaten, d., franke, e.k. and luban, j. 1996. cyclophilin a is required for an early step in the life cycle of human immunodefi ciency virus type 1 before the initiation of reverse transcription. j. virol., 70:3551–3560. braaten, d. and luban, j. 2001. cyclophilin a regulates hiv-1 infectivity, as demonstrated by gene targeting in human t cells. embo. j., 20: 1300–1309. bram, r.j. and crabtree, g.r. 1994. calcium signalling in t cells stimulated by a cyclophilin b-binding protein. nature, 371:355–358. brazin, k.n., mallis, r.j., fulton, d.b. and andreotti, a.h. 2002. regulation of the tyrosine kinase itk by the peptidyl-prolyl isomerase cyclophilin a. proc. natl. acad. sci. u.s.a., 99:1899–1904. cande, c., vahsen, n., kouranti, i., schmitt, e., daugas, e., spahr, c., luban, j., kroemer, r.t., giordanetto, f., garrido, c. et al. 2004. aif and cyclophilin a cooperate in apoptosis-associated chromatinolysis. oncogene, 23:1514–1521. castro, a.p., carvalho, t.m., moussatche, n. and damaso, c.r. 2003. redistribution of cyclophilin a to viral factories during vaccinia virus infection and its incorporation into mature particles. j .virol. 77:9052– 9068. chen, z., mi, l., xu, j., yu, j., wang, x., jiang, j., xing, j., shang, p., qian, a. and li, y. et al. 2005. function of hab18g/cd147 in invasion of host cells by severe acute respiratory syndrome coronavirus. j. infect. dis., 191:755–760. chen, z.j., vetter, m., chang, g.d., liu, s., che, d., ding, y., kim, s.s. and chang, c.h. 2004. cyclophilin a functions as an endogenous inhibitor for membrane-bound guanylate cyclase-a. hypertension, 44:963–968. clipstone, n.a. and crabtree, g.r. 1992. identifi cation of calcineurin as a key signalling enzyme in t-lymphocyte activation. nature, 357: 695–697. colgan, j., asmal, m., neagu, m., yu, b., schneidkraut, j., lee, y., sokolskaja, e., andreotti, a. and luban, j. 2004. cyclophilin a regulates tcr signal strength in cd4+ t cells via a proline-directed conformational switch in itk. immunity, 21:189–201. colgan, j., asmal, m., yu, b. and luban, j. 2005. cyclophilin a-defi cient mice are resistant to immunosuppression by cyclosporine. j. immunol., 174:6030–6038. drug target insights 2007: 2 16 watashi and shimotohno crompton, m., virji, s. and ward, j.m. 1998. cyclophilin-d binds strongly to complexes of the voltage-dependent anion channel and the adenine nucleotide translocase to form the permeability transition pore. eur. j. biochem, 258:729–735. cullen, b.r. 2003. hiv-1 infection: fooling the gatekeeper. nat. med., 9: 1112–1113. damaso, c.r. and moussatche, n. 1998. inhibition of vaccinia virus replication by cyclosporin a analogues correlates with their affi nity for cellular cyclophilins. j. gen. virol., 79 (pt 2):339–346. di bisceglie, a.m., mchutchison, j. and rice, c.m. 2002. new therapeutic strategies for hepatitis c. hepatology 35:224–231. duina, a.a., chang, h.c., marsh, j.a., lindquist, s. and gaber, r. f. 1996. a cyclophilin function in hsp90-dependent signal transduction. science, 274:1713–1715. endrich, m.m. and gehring, h. 1998. the v3 loop of human immunodefi ciency virus type-1 envelope protein is a high-affi nity ligand for immunophilins present in human blood. eur. j. biochem., 252: 441–446. evans, m.j., rice, c.m. and goff, s.p. 2004. phosphorylation of hepatitis c virus nonstructural protein 5a modulates its protein interactions and viral rna replication. proc. natl. acad. sci. u.s.a., 101: 13038–13043. firpi, r.j., zhu, h., morelli, g., abdelmalek, m.f., soldevila-pico, c., machicao, v.i., cabrera, r., reed, a.i., liu, c. and nelson, d.r. 2006. cyclosporine suppresses hepatitis c virus in vitro and increases the chance of a sustained virological response after liver transplantation. liver transpl., 12:51–57. fischer, g., wittmann-liebold, b., lang, k., kiefhaber, t. and schmid, f. x. 1989. cyclophilin and peptidyl-prolyl cis-trans isomerase are probably identical proteins. nature, 337:476–478. franke, e. k., yuan, h. e. and luban, j. 1994. specifi c incorporation of cyclophilin a into hiv-1 virions. nature, 372:359–362. friedman, j. and weissman, i. 1991. two cytoplasmic candidates for immunophilin action are revealed by affi nity for a new cyclophilin: one in the presence and one in the absence of csa. cell, 66:799–806. fruman, d.a., klee, c.b., bierer, b.e. and burakoff, s.j. 1992. calcineurin phosphatase activity in t lymphocytes is inhibited by fk 506 and cyclosporin a. proc. natl. acad. sci. u.s.a., 89:3686–3690. gao, l., aizaki, h., he, j. w. and lai, m.m. 2004. interactions between viral nonstructural proteins and host protein hvap-33 mediate the formation of hepatitis c virus rna replication complex on lipid raft. j. virol., 78:3480–3488. goto, k., watashi, k., murata, t., hishiki, t., hijikata, m. and shimotohno, k. 2006. evaluation of the anti-hepatitis c virus effects of cyclophilin inhibitors, cyclosporin a and nim811. biochem. biophys. res. commun., 343:879–884. halestrap, a.p. 1999. the mitochondrial permeability transition: its molecular mechanism and role in reperfusion injury. biochem. soc. symp., 66:181–203. handschumacher, r.e., harding, m.w., rice, j., drugge, r.j. and speicher, d.w. 1984. cyclophilin: a specifi c cytosolic binding protein for cyclosporin a. science, 226:544–547. harding, m.w., galat, a., uehling, d.e. and schreiber, s.l. 1989. a receptor for the immunosuppressant fk506 is a cis-trans peptidyl-prolyl isomerase. nature, 341:758–760. hatziioannou, t., perez-caballero, d., cowan, s. and bieniasz, p.d. 2005. cyclophilin interactions with incoming human immunodefi ciency virus type 1 capsids with opposing effects on infectivity in human cells. j. virol, 79:176–183. holloway, m.p. and bram, r.j. 1998. co-localization of calcium-modulating cyclophilin ligand with intracellular calcium pools. j. biol. chem, 273:16346–16350. horowitz, d.s., kobayashi, r. and krainer, a.r. 1997. a new cyclophilin and the human homologues of yeast prp3 and prp4 form a complex associated with u4/u6 snrnps. rna, 3:1374–1387. ishii, n., watashi, k., hishiki, t., goto, k., inoue, d., hijikata, m., wakita, t., kato, n., and shimotohno, k. 2006. diverse effects of cyclosporine on hepatitis c virus strain replication. j. virol., 80:4510–4520. jang, l.k., lee, z. h., kim, h.h., hill, j.m., kim, j.d. and kwon, b.s. 2001. a novel leucine-rich repeat protein (lrr-1): potential involvement in 4-1bb-mediated signal transduction. mol. cells., 12:304– 312. jin, z.g., melaragno, m.g., liao, d.f., yan, c., haendeler, j., suh, y.a., lambeth, j.d. and berk, b.c. 2000. cyclophilin a is a secreted growth factor induced by oxidative stress. circ. res., 87:789–796. kieffer, l.j., thalhammer, t. and handschumacher, r.e. 1992. isolation and characterization of a 40-kda cyclophilin-related protein. j. biol. chem., 267:5503–5507. kootstra, n.a., munk, c., tonnu, n., landau, n.r. and verma, i.m. 2003. abrogation of postentry restriction of hiv-1-based lentiviral vector transduction in simian cells. proc. natl. acad. sci. u.s.a., 100: 1298–1303. krummrei, u., bang, r., schmidtchen, r., brune, k. and bang, h. 1995. cyclophilin-a is a zinc-dependent dna binding protein in macrophages. febs. lett., 371:47–51. liang, t.j., jeffers, l.j., reddy, k.r., de medina, m., parker, i.t., cheinquer, h., idrovo, v., rabassa, a. and schiff, e.r. 1993. viral pathogenesis of hepatocellular carcinoma in the united states. hepatology, 18:1326–1333. liu, j., albers, m.w., chen, c.m., schreiber, s.l. and walsh, c.t. 1990. cloning, expression, and purifi cation of human cyclophilin in escherichia coli and assessment of the catalytic role of cysteines by sitedirected mutagenesis. proc. natl. acad. sci. u.s.a., 87:2304–2308. liu, j., albers, m.w., wandless, t.j., luan, s., alberg, d.g., belshaw, p. j., cohen, p., mackintosh, c., klee, c.b. and schreiber, s.l. 1992. inhibition of t cell signaling by immunophilin-ligand complexes correlates with loss of calcineurin phosphatase activity. biochemistry., 31:3896–3901. liu, j., farmer, j.d. jr., lane, w.s., friedman, j., weissman, i. and schreiber, s.l. 1991. calcineurin is a common target of cyclophilin-cyclosporin a and fkbp-fk506 complexes. cell, 66:807–815. lohmann, v., korner, f., koch, j., herian, u., theilmann, l. and bartenschlager, r. 1999. replication of subgenomic hepatitis c virus rnas in a hepatoma cell line. science, 285:110–113. luban, j., bossolt, k.l., franke, e.k., kalpana, g.v. and goff, s.p. 1993. human immunodefi ciency virus type 1 gag protein binds to cyclophilins a and b. cell., 73:1067–1078. luban, j. (in press). cyclophilin a, trim5 and resistance to hiv-1 infection. j. virol., [epub ahead of print]. luo, c., luo, h., zheng, s., gui, c., yue, l., yu, c., sun, t., he, p., chen, j., shen, j. et al. 2004. nucleocapsid protein of sars coronavirus tightly binds to human cyclophilin a. biochem. biophys. res. commun., 321:557–565. mccaffrey, p.g., perrino, b.a., soderling, t.r. and rao, a. 1993. nf-atp, a t lymphocyte dna-binding protein that is a target for calcineurin and immunosuppressive drugs. j. biol. chem., 268:3747–3752. mi, h., kops, o., zimmermann, e., jaschke, a. and tropschug, m. 1996. a nuclear rna-binding cyclophilin in human t cells. febs. lett., 398:201–205. mlynar, e., bevec, d., billich, a., rosenwirth, b. and steinkasserer, a. 1997. the non-immunosuppressive cyclosporin a analogue sdz nim 811 inhibits cyclophilin a incorporation into virions and virus replication in human immunodefi ciency virus type 1-infected primary and growth-arrested t cells. j. gen. virol., 78 (pt 4):825– 835. nakagawa, m., sakamoto, n., enomoto, n., tanabe, y., kanazawa, n., koyama, t., kurosaki, m., maekawa, s., yamashiro, t., chen, c. h. et al. 2004. specifi c inhibition of hepatitis c virus replication by cyclosporin a. biochem. biophys. res. commun., 313:42–47. drug target insights 2007: 2 17 cyclophilin and viruses nakagawa, t., shimizu, s., watanabe, t., yamaguchi, o., otsu, k., yamagata, h., inohara, h., kubo, t. and tsujimoto, y. 2005. cyclophilin d-dependent mitochondrial permeability transition regulates some necrotic but not apoptotic cell death. nature, 434:652–658. nestel, f. p., colwill, k., harper, s., pawson, t. and anderson, s. k. 1996. rs cyclophilins: identifi cation of an nk-tr1-related cyclophilin. gene., 180:151–155. obata, y., yamamoto, k., miyazaki, m., shimotohno, k., kohno, s. and matsuyama, t. 2005. role of cyclophilin b in activation of interferon regulatory factor-3. j. biol. chem., 280:18355–18360. ott, d.e., coren, l.v., johnson, d.g., sowder, r.c., 2nd, arthur, l.o. and henderson, l.e. 1995. analysis and localization of cyclophilin a found in the virions of human immunodefi ciency virus type 1 mn strain. aids. res. hum. retroviruses 11:1003–1006. owens-grillo, j.k., hoffmann, k., hutchison, k.a., yem, a.w., deibel, m.r. jr., handschumacher, r. e. and pratt, w.b. 1995. the cyclosporin a-binding immunophilin cyp-40 and the fk506-binding immunophilin hsp56 bind to a common site on hsp90 and exist in independent cytosolic heterocomplexes with the untransformed glucocorticoid receptor. j. biol. chem., 270:20479–20484. ozaki, k., fujiwara, t., kawai, a., shimizu, f., takami, s., okuno, s., takeda, s., shimada, y., nagata, m. and watanabe, t. et al. 1996. cloning, expression and chromosomal mapping of a novel cyclophilin-related gene (ppil1) from human fetal brain. cytogenet. cell. genet., 72:242–245. paeshuyse, j., kaul, a., de clercq, e., rosenwirth, b., dumont, j.m., scalfaro, p., bartenschlager, r. and neyts, j. 2006. the non-immunosuppressive cyclosporin debio-025 is a potent inhibitor of hepatitis c virus replication in vitro. hepatology, 43:761–770. poynard, t., yuen, m.f., ratziu, v. and lai, c.l. 2003. viral hepatitis c. lancet., 362:2095–2100. price, e.r., zydowsky, l.d., jin, m.j., baker, c.h., mckeon, f.d. and walsh, c.t. 1991. human cyclophilin b: a second cyclophilin gene encodes a peptidyl-prolyl isomerase with a signal sequence. proc. natl. acad. sci. u.s.a., 88:1903–1907. pushkarsky, t., zybarth, g., dubrovsky, l., yurchenko, v., tang, h., guo, h., toole, b., sherry, b. and bukrinsky, m. 2001. cd147 facilitates hiv-1 infection by interacting with virus-associated cyclophilin a. proc. natl. acad. sci. u.s.a., 98:6360–6365. ratajczak, t., carrello, a., mark, p.j., warner, b.j., simpson, r.j., moritz, r.l. and house, a.k. 1993. the cyclophilin component of the unactivated estrogen receptor contains a tetratricopeptide repeat domain and shares identity with p59 (fkbp59). j. biol. chem., 268:13187–13192. rosen, m.k., standaert, r.f., galat, a., nakatsuka, m. and schreiber, s. l. 1990. inhibition of fkbp rotamase activity by immunosuppressant fk506: twisted amide surrogate. science, 248:863–866. rosenwirth, b., billich, a., datema, r., donatsch, p., hammerschmid, f., harrison, r., hiestand, p., jaksche, h., mayer, p. and peichl, p. et al. 1994. inhibition of human immunodefi ciency virus type 1 replication by sdz nim 811, a nonimmunosuppressive cyclosporine analog. antimicrob. agents. chemother., 38:1763–1772. rycyzyn, m.a. and clevenger, c.v. 2002. the intranuclear prolactin/cyclophilin b complex as a transcriptional inducer. proc. natl. acad. sci. u.s.a., 99:6790–6795. rycyzyn, m.a., reilly, s.c., o’malley, k. and clevenger, c.v. 2000. role of cyclophilin b in prolactin signal transduction and nuclear retrotranslocation. mol. endocrinol. 14:1175–1186. saphire, a.c., bobardt, m.d. and gallay, p.a. 1999. host cyclophilin a mediates hiv-1 attachment to target cells via heparans. embo. j. 18: 6771–6785. sayah, d.m., sokolskaja, e., berthoux, l. and luban, j. 2004. cyclophilin a retrotransposition into trim5 explains owl monkey resistance to hiv-1. nature, 430:569–573. schreiber, s.l. 1991. chemistry and biology of the immunophilins and their immunosuppressive ligands. science, 251:283–287. seko, y., fujimura, t., taka, h., mineki, r., murayama, k. and nagai, r. 2004. hypoxia followed by reoxygenation induces secretion of cyclophilin a from cultured rat cardiac myocytes. biochem. biophys res. commun., 317:162–168. sherry, b., yarlett, n., strupp, a. and cerami, a. 1992. identifi cation of cyclophilin as a proinfl ammatory secretory product of lipopolysaccharide-activated macrophages. proc. natl. acad. sci. u.s.a., 89: 3511–3515. sokolskaja, e., sayah, d.m. and luban, j. 2004. target cell cyclophilin a modulates human immunodefi ciency virus type 1 infectivity. j. virol., 78:12800–12808. sokolskaja, e. and luban, j. 2006. cyclophilin a, trim5, and innate immunity to hiv-1. curr. opin. microbiol., 9:404–408. steinkasserer, a., harrison, r., billich, a., hammerschmid, f., werner, g., wolff, b., peichl, p., palfi , g., schnitzel, w. and mlynar, e. et al. 1995. mode of action of sdz nim 811, a nonimmunosuppressive cyclosporin a analog with activity against human immunodefi ciency virus type 1 (hiv-1): interference with early and late events in hiv-1 replication. j. virol., 69:814–824. stremlau, m., owens, c.m., perron, m.j., kiessling, m., autissier, p. and sodroski, j. 2004. the cytoplasmic body component trim5alpha restricts hiv-1 infection in old world monkeys. nature, 427:848–853. syed, f., rycyzyn, m.a., westgate, l. and clevenger, c.v. 2003. a novel and functional interaction between cyclophilin a and prolactin receptor. endocrine, 20:83–90. takahashi, n. 1999. pharmacodynamics of ciclosporin a (cyclosporine). clin. exp. nephrol., 3:s16–s26. takahashi, n., hayano, t. and suzuki, m. 1989. peptidyl-prolyl cis-trans isomerase is the cyclosporin a-binding protein cyclophilin. nature, 337:473–475. tanveer, a., virji, s., andreeva, l., totty, n.f., hsuan, j.j., ward, j.m. and crompton, m. 1996. involvement of cyclophilin d in the activation of a mitochondrial pore by ca2+ and oxidant stress. eur. j. biochem., 238:166–172. thali, m., bukovsky, a., kondo, e., rosenwirth, b., walsh, c.t., sodroski, j. and gottlinger, h.g. 1994. functional association of cyclophilin a with hiv-1 virions. nature, 372:363–365. towers, g.j., hatziioannou, t., cowan, s., goff, s.p., luban, j. and bieniasz, p.d. 2003. cyclophilin a modulates the sensitivity of hiv-1 to host restriction factors. nat. med., 9:1138–1143. tran, d.d., edgar, c.e., heckman, k.l., sutor, s.l., huntoon, c.j., van deursen, j., mckean, d.l. and bram, r.j. 2005. caml is a p56lck-interacting protein that is required for thymocyte development. immunity, 23:139–152. tu, h., gao, l., shi, s.t., taylor, d.r., yang, t., mircheff, a.k., wen, y., gorbalenya, a.e., hwang, s.b. and lai, m.m. 1999. hepatitis c virus rna polymerase and ns5a complex with a snare-like protein. virology, 263:30–41. waldmeier, p.c., zimmermann, k., qian, t., tintelnot-blomley, m. and lemasters, j.j. 2003. cyclophilin d as a drug target. curr. med. chem., 10:1485–1506. wang, b.b., hayenga, k.j., payan, d.g. and fisher, j.m. 1996. identifi cation of a nuclear-specifi c cyclophilin which interacts with the proteinase inhibitor eglin c. biochem. j., 314(pt 1):313–319. wang, c., gale, m., jr., keller, b. c., huang, h., brown, m.s., goldstein, j.l. and ye, j. 2005. identifi cation of fbl2 as a geranylgeranylated cellular protein required for hepatitis c virus rna replication. mol. cell., 18:425–434. watashi, k., hijikata, m., hosaka, m., yamaji, m. and shimotohno, k. 2003. cyclosporin a suppresses replication of hepatitis c virus genome in cultured hepatocytes. hepatology, 38:1282–1288. watashi, k., ishii, n., hijikata, m., inoue, d., murata, t., miyanari, y. and shimotohno, k. 2005. cyclophilin b is a functional regulator of hepatitis c virus rna polymerase. mol. cell., 19:111–122. drug target insights 2007: 2 18 watashi and shimotohno woodfi eld, k., ruck, a., brdiczka, d. and halestrap, a.p. 1998. direct demonstration of a specifi c interaction between cyclophilin-d and the adenine nucleotide translocase confi rms their role in the mitochondrial permeability transition. biochem. j., 336(pt 2):287–290. xu, q., leiva, m.c., fischkoff, s.a., handschumacher, r.e. and lyttle, c. r. 1992. leukocyte chemotactic activity of cyclophilin. j. biol. chem., 267:11968–11971. yang, w.m., inouye, c.j. and seto, e. 1995. cyclophilin a and fkbp12 interact with yy1 and alter its transcriptional activity. j. biol. chem., 270:15187–15193. yokoyama, n., hayashi, n., seki, t., pante, n., ohba, t., nishii, k., kuma, k., hayashida, t., miyata, t., aebi, u. and et al. 1995. a giant nucleopore protein that binds ran/tc4. nature, 376:184–188. yurchenko, v., o’connor, m., dai, w. w., guo, h., toole, b., sherry, b. and bukrinsky, m. 2001. cd147 is a signaling receptor for cyclophilin b. biochem. biophys. res. commun., 288:786–788. yurchenko, v., zybarth, g., o’connor, m., dai, w. w., franchin, g., hao, t., guo, h., hung, h. c., toole, b. and gallay, p. et al. 2002. active site residues of cyclophilin a are crucial for its signaling activity via cd147. j. biol. chem. 277:22959–22965. zander, k., sherman, m.p., tessmer, u., bruns, k., wray, v., prechtel, a. t., schubert, e., henklein, p., luban, j. and neidleman, j. et al. 2003. cyclophilin a interacts with hiv-1 vpr and is required for its functional expression. j. biol. chem., 278:43202–43213. zeng, l., zhou, z., xu, j., zhao, w., wang, w., huang, y., cheng, c., xu, m., xie, y. and mao, y. 2001. molecular cloning, structure and expression of a novel nuclear rna-binding cyclophilin-like gene (ppil4) from human fetal brain. cytogenet. cell. genet., 95:43–47. zhou, z., ying, k., dai, j., tang, r., wang, w., huang, y., zhao, w., xie, y. and mao, y. 2001. molecular cloning and characterization of a novel peptidylprolyl isomerase (cyclophilin)-like gene (ppil3) from human fetal brain. cytogenet. cell. genet., 92:231–236. drug target insights 2007: 2 chiang et al.indd durg target insights 2008:3 153–159 153 original research correspondence: thomas m. chiang, memphis va med, ctr., research service (151), 1030 jefferson ave., memphis, tn 38104. tel: 901-523-8990; ext: 7608; fax: 901-577-7273; email: tchiang@utmem.edu copyright in this article, its metadata, and any supplementary data is held by its author or authors. it is published under the creative commons attribution by licence. for further information go to: http://creativecommons.org/licenses/by/3.0/. the toxicity of a chemically synthesized peptide derived from non-integrin platelet collagen receptors thomas m. chiang and v. woo-rasberry from the department of veterans affairs and the departments of medicine and molecular sciences, memphis, tn 38104. abstract: a chemically synthesized peptide derived from platelet non-integrin collagen receptor has been shown to be an effective agent for inhibiting collagen-induced platelet aggregation and adhesion of washed radiolabeled platelets onto natural matrices and collagen coated microtiter plates. in order to be a therapeutic agent, we have used a cell culturing system and an animal model to test its cytotoxicities. in cell culture experiments, the peptide is not toxic to meg-01, a megakaryoblastic cell line. prior to performing experiments in rats, the existence of both platelet type i and type iii collagen receptors and its functional roles in rat platelets had to be established. in this investigation, we report that rat platelets contain both receptors and the chyb peptide inhibits both type i and type iii collagen-induced rat platelet aggregation. in addition, analysis of the rat sera collected at various time intervals following an injection of chyb into the rat-tail vein, did not show an increase in the activity of key enzymes which indicate tissue and/or organ damage. these results suggest that the chyb peptide is safe and its development into a potential therapeutic agent for inhibiting thrombi formation is possible. keywords: collagen, platelet, platelet aggregation inhibitor, thrombosis introduction ligand-receptor interactions with the subsequent transduction of signals have long been recognized as a major mechanism for regulating cellular activities in eukaryotic cells. this mechanism is also operative when platelets interact with collagen exposed by damage to the endothelial surfaces of blood vessels. in blood vessels, type i and type iii collagens are the major collagen components. the vessels also contain small amounts of types iv and v collagen and minor amounts of several other types of collagen. after platelets adhere to the exposed connective tissue, they aggregate, secreting biologically active substances, and effect hemostasis. although many studies have centered on the role of the integrin and non-integrin collagen receptors in regulating platelet function, others have shown that platelets possess additional distinct reactive sites for type i collagen (fitzsimmons et al. 1986) and type iii collagen (morton et al. 1987). defi nitions of the platelet collagen receptors must be fully established before logical interventions can be identifi ed and used to alter the course of abnormal hemostasis when platelets encounter denuded vascular surfaces. development of an inhibitor(s) from platelet collagen receptors to inhibit the collageninduced platelet activation and aggregation will prevent the risk of thrombosis. we have characterized a non-integrin platelet receptor for type i collagen (chiang et al. 1997) and defi ned its active peptide (chiang and kang, 1997; chiang, 2000). in addition, we have characterized the platelet receptor for type iii collagen and defi ned two active peptides in its sequences (chiang et al. 2002). we have chemically synthesized a hybrid peptide (chyb), which contains one of each active peptide of platelet types i and iii collagen receptors with a linker of 12 amino acid residues. the chyb can inhibit types i and iii collagen-induced platelet aggregation and other functions in vitro (du et al. 2007). in the present investigation, we have studied the cytotoxicity of the chyb in a human cell line and in rats, to determine its feasibility as a therapeutic agent. materials and methods reagents we have purchased cytotoxicity detection kit (roche diagnostic corp., indianapolis, il) to assay the activity of ldh). assay kits for alt and ast were from diagnostic chemical ltd (oxford, ct). http://creativecommons.org/licenses/by/3.0/ http://creativecommons.org/licenses/by/3.0/ 154 chiang and rasberry drug target insights 2008:3 reagents for the gs assay method were purchased from sigma chemical inc. (st. louis, mo). all other chemical reagents were purchased from sigma chemical co. (st. louis, mo), bio-rad (hercules, ca), fisher scientifi c (st louis, mo), and pierce (rockford, il). [3h]-thymidine and meg-01 cells were purchased from perkin elmer (waltham, ma) and american type culture collection (manassas, va), respectively. preparation of platelet-rich plasma (prp) human blood (9 parts) from normal volunteers were collected in polypropylene tubes containing 1 part 3.8% sodium citrate following an overnight fast. prp were prepared by centrifuging the citrated blood at room temperature for 10 min at 226 × g (chiang et al. 1975). whole blood and prp were exposed to plastic surfaces or siliconized vessels only. platelet counts of the prp ranged from 200,000 to 300,000 per mm3. the method to obtain prp from rats was the same method as from humans for aggregation studies. platelet aggregation: platelet aggregations were performed according to the method established by born (1962). preparation of types i and iii collagen type i and type iii collagens were prepared from a human placenta of a normal delivery according the method developed by seyer et al. (1976, 1980). collagen concentrations were determined according the method described by bergman and loxley (1963). western blot analysis rat platelets were obtained from heparinized blood by centrifugation (225 × g) for 5 minutes and washed with 20 mm tris-130 mm nacl-1 mm edta, ph 7.3 (tris-edta), sonicated, and protein concentration determined. human and rat platelet lysates (100 µg) were separated on 7.5% sdspage and then transferred onto a nitrocellulose membrane. following transfer, the membrane was incubated with 3% difco skim milk in 20 mm tris/500 mm nacl/0.05% tween 20 (tbst) for 1 hr at room temperature, then washed with tbst three times and probed with either anti65-kda antibodies (1:3000) or anti-47-kda antibodies (1:5000) diluted in tbst/1% difco skim milk at 4° overnight. the membrane was washed with tbst three times and probed with appropriate secondary antibodies (cappel, solon, oh) at 1:20000 dilution in tbst for 1 hr at room temperature, washed with tbs-t three times and visualized with chemiluminescent substrate (pierce, rockford, il). culturing meg-01 cells meg-01 cells were plated at a density of 1 × 105 cells/ml and cultured in rpmi 1640 medium with 2 mm l-glutamine, 10 mm hepes, 1 mm sodium pyruvate, 4.5 g/l glucose, adjusted to contain 1.5 g/l sodium bicarbonate, 10 mm hepes, and 10% fetal bovine serum and grown at 37 °c and 5% co2. the sub cultivation ratio was 1:2 to 1:3 as recommended by the manufacturer. the toxicity of the chyb was examined in a cultured cell line to ensure the chemical synthesis of the chyb did not contain cytotoxic component(s) (chiang and postlethwaite, 2007). first, the effect of chyb on cultured meg-01 cells was tested. we have used the cell line (5 × 105), cultured in the presence of various concentrations of chyb and added [3h]-thymidine for incorporation at different culturing times. table1shows that the chyb does affect the cell growth at 48 hr compared to 24 hr of different concentrations, which inhibit the adhesion of washed labeled platelets on rabbit aortic segments. these results are an acute effect. we will perform experiments with longer time exposures (1, 2, and 3 weeks), multiple additions, and higher doses to ensure the chyb is safe to use. cytotoxicity studies in rats the rats (two per group) were injected with either 0.25 ml of controls (0.5% dmso in pbs or pbs alone) and 120 µg/kg chyb as listed in the table. the initial venipuncture drawn immediately following injection counted as time zero. all withdrawn blood, time zero and each time point following injection (1, 3, 6, 24, and 36 hr), were placed in a microfuge tube containing 1/10 of heparin, centrifuged, plasma collected, and frozen until assay for activities of lactic acid dehydrogenase (ldh), glutamine synthase (gs), alanine aminotransferase (alt), and aspartate aminotransferase (ast) could be performed. the methods for the determining ldh, alt, and ast were according to the manufacturer’s protocol. the gs assay method was adapted from kingdon et al. (1968). 155 platelet-collagen interaction drug target insights 2008:3 results rat platelets possess 65-kda and 47-kda proteins rat platelets possess immunoreactive proteins to anti-65-kda and anti-47-kda antibodies. we have performed experiments showing that rat platelets possess the 65-kda (panel a) and 47-kda (panel b) receptors by western blots. figure 1 shows rat platelets possess anti-65-kda active peptide antibody (panel a) and anti47-kda antibody (panel b) reactive bands. this result demonstrates that rat platelets contain immunoreactive proteins to human platelets. the chyb inhibits collagen-induced rat platelet aggregation. the chyb inhibits both type i and type iii collagen-induced platelet aggregation similar to that of human platelets. figure 2 shows the type i collagen-induced rat platelet aggregations are inhibiting dose-dependently by chyb. initially, we performed aggregation experiments with prp from rats, which had not fasted overnight and observed that platelets required more collagen and longer delay time to induce platelet aggregation. we then performed experiments with the prp from rats, which were fasted overnight and we did observe the inhibitory effect of chyb on type iii collagen-induced platelet aggregation (fig. 3). the differences in lag-time of type i (prp was prepared from rats’ blood which had not fasted overnight) and type iii collagen (prp was prepared from rats who were fasted overnight). toxicity of the chyb was examined in cultured the meg-01 cell line the cytotoxicity of the chyb was examined using a cultured cell line to ensure that the chemical synthesis of the chyb does not contain cytotoxic component(s). first, we tested the effect of chyb on cultured meg-01 cells. cells were cultured (5 × 105) in the presence of various concentrations of chyb (concentrations which inhibited the adhesion of washed labeled platelets on rabbit aortic segments) for various time points, then [3h]-thymidine is added for incorporation by the cells. table 1 shows that the chyb does affect the cell growth at 24 hr nor 48 hr with differing concentrations of peptide. these results are an acute effect. next, we tested the toxicity of chyb in multiple doses and table 1. cytotoxicity of chyb on short-term cultured meg-01 cells. treatments 24 hr culture 48 hr culture vehicle control 110950 + 8269 185000 + 8544 chyb 1 µm 160500 + 3500 174000 +1527 chyb 2 µm 155500 + 9500 163500 + 5500 chyb 4 µm 158000 + 3500 178000 + 1000 chyb 8 µm 170000 + 3852 169666 + 6807 chyb 16 µm 162000 + 4795 176000 + 5031 meg-01 cells were harvested, washed with pbs × 2, resuspended in growth media and aliquoted 5 × 105 cells/100ul/ well. the peptide, chyb, was dissolved in dmso, diluted with media (0.7% dmso-fi nal concentration), fi lter-sterilized and added to individual wells in appropriate concentrations. various concentrations of hybrid peptide and vehicle control (0.7% dmso) were added to appropriate wells for 24 and 48 hr, in triplicate. following incubation, an aliquot of 1 µci/10 µl/well of [3h]-thymidine was added to each sample, cultured for an additional 24 hr, harvested using the packard harvester filtermate 196 cell harvester and quantifi ed with the packard matri × 96 direct beta counter. data were mean + s.d. of triplicate determinations. there is no signifi cant effect by comparing the vehicle control and each individual concentration of chyb (student t test). a b 1 2 1 2 figure 1. platelets in rats possess immunoreactive bands with anti-65-kda and 47-kda antibodies. human platelet membranes (100 µg, lane 1) and rat platelet membranes (100 µg, lane 2) western blotted with anti-65-kda (panel a) and anti-47 kda (panel b) antibodies. the dilution of fi rst antibody was 1/1000 and the second antibody was 1/10000. bands were visualized with enhanced chemiluminescence solutions. 156 chiang and rasberry drug target insights 2008:3 long-term cultures and these results are in table 2 and table 3. these data demonstrated that there are no toxic effects by chyb on meg-01 cells. we intend to perform longer-term cultures (1, 2, and 3 weeks), additional multiple dosing, and extension the dose response curve to ensure that use of chyb is safe. toxicity of the chyb was examined with rats by i. v. injection we have also used the ldh cytotoxicity detection kit (lactate dehydrogenase—ldh) to examine the plasma damage effect by chyb in rats. we measured the release of ldh at different time intervals following injection of the tail veins. results of the study (table 4) show that the release/ activity of ldh does not increase with the time, at the concentration of chyb used. these results are also an acute effect. in order to determine whether chyb has a damaging effect on various tissues (heart, muscle, and lung), we assayed the activity of three enzymes i.e. glutamine synthtetase (gs), aspartate amino transferase (ast), and alanine amino transferase (alt) of chyb injected rat sera. results show that these enzyme activities were undetectable in these samples (data not shown). figure 2. chyb inhibits type i collagen-induced platelet aggregation. panel a shows the platelet aggregation-induced by pbs (trace 1) and various amounts of type i collagen (traces 2 and 3 are 1 µg, and 2 µg, respectively). panel b shows the effect of chyb on type i collagen (1 µg)-induced platelet aggregation (traces 1, 2, and 3 are 60 µg, 30 µg, and 15 µg, respectively). panel c shows the inhibitory effect of chyb (30 µg) on type i collagen-induced platelet aggregation can be reversed by adding higher amounts of type i collagen (traces 1, 2, and 3 are 3 µg, 2 µg and 1 µg, respectively). following pre-incubations of various amounts of chyb with collagen, an aliquot of 0.45 ml prp was added to the cuvette and platelet aggregation was immediately (at time 1, x-axis) monitored with a chronolog lumi-aggregometer. figure 3. chyb inhibits type iii collagen-induced platelet aggregation. panel a shows the platelet aggregation-induced by pbs (trace 1) and various amounts of type iii collagen (traces 2, 3, and 4 are 0.25 µg, 0.5 µg, and 1 µg, respectively). panel b shows the effect of chyb on type iii collagen (0.5 µg)-induced platelet aggregation (traces 1, 2, and 3 are 60 µg, 30 µg, and 15 µg, respectively). panel c shows the inhibitory effect of chyb (30 µg) on type iii collageninduced platelet aggregation can be reversed by adding higher amounts of type i collagen (traces 1, 2, and 3 are 0.25 µg, 0.5 µg and 1 µg, respectively). following pre-incubations of various amounts with collagen, an aliquot of 0.45 ml prp was added to a cuvette and platelet aggregation was immediately (at time 1, x-axis) monitored with a chronolog lumi-aggregometer. 157 platelet-collagen interaction drug target insights 2008:3 discussion the use of an animal model is essential for the discovery of new drugs effective in the prophylaxis and treatment of arterial thrombosis. a modern approach used in attempts to unravel the physiological role of the various integrin and non-integrin receptors has been the use of specifi c “knock out” mice. elimination of several integrin subunits (β1, β5, β6, and αv) has been performed and has lead to embryonic or prenatal lethal phenotype, however, ablation of ß3 or β1 does not affect development or viability. conversely, mice lacking α2 or αiib have still not been breed. this area has been reviewed (chen and sheppard, 2007). a knockout mouse of the non-integrin collagen receptor, gp iv (cd36) has been successful but its role in platelet function has not been defi ned (goudriaan, 2002). many other animal models have been used to test the effectiveness of an inhibitor on the thrombi formation (philip et al. 1978; hynes and bader, 1997; kuez et al. 1990; gaber et al. 2004). leger et al. (15) developed a guinea pig arterial thrombosis model to study a protease-activated receptor 1–4 heterodimer in platelet-mediated thrombosis. hladovec (1971) has described an experimental model of arterial thrombosis in rat, which was induced in the carotid artery by electric current. kurt et al. (1990) modifi ed this model by using 30% fecl3 instead of an electric current to injure vessel walls thus preventing swift corrosion of the artery. gaber et al. (2004) used the rat cremaster muscle to study the leukocyte-endothelial cell interactions in microvessels. this rat model appeared to be a good candidate for our purpose. the rat model was selected due to the manageable size of the rats and their blood vessels for our studies. others, including table 2. the effect of multiple doses of various concentrations of chyb on long-term meg-01 cultured cells. treatments mean + s.d. med. control 48541 + 1999 med. + vehicle control (dmso) 51634 + 4851 chyb 1 µm 46797 + 1509 chyb 2 µm 46779 + 3721 chyb 4 µm 59068 + 3801 chyb 8 µm 53304 + 4743 meg-01 cells were harvested, washed with pbs, resuspended in growth media and aliquoted as in table 1. the peptide was prepared (as in table 1) and appropriately added to individual wells with fi nal concentrations of 1, 2, 4, and 8 µm, respectively, in triplicate. media and media with dmso are controls. the cells were maintained at 37 °c with 5% co2. following 24 and 48 hr of incubation, corresponding wells received a second and third dose of the peptide, respectively. after 72 hr of incubation, each well received 1 µci/10 µl of [h3]-thymidine and allowed to incubate an additional 24 hr. the cells were harvested and quantifi ed as described in table 1. there is no signifi cant effect by comparing the vehicle control and each individual concentration of chyb (student t test). table 3. the effect of various concentrations of chyb on meg-01 cell growth. treatments mean + s.d. med. control 45439 + 1211 med. + vehicle control (dmso) 46182 + 8045 chyb 1 µm 51155 + 6777 chyb 2 µm 45613 + 3288 chyb 4 µm 43519 + 6705 chyb 8 µm 48658 + 1904 meg-01 cells were harvested, washed with pbs × 2, and aliquoted as described in table 1. also, the dissolution and initial distribution of the peptide, controls and culturing conditions were the same as described in table 1. following 48 hr of incubation, the cells received a second dose of peptide, incubated for an additional 24 hr before the addition of 1 µci/10 µl of [h3]-thymidine. again, the cells were cultured for thymidine uptake, harvested, and quantifi ed as described in table 1. there is no signifi cant effect by comparing the vehicle control and each individual concentration of chyb (student t test). 158 chiang and rasberry drug target insights 2008:3 chen et al. (2000) have applied this model to study the effects of fi sh oil on arterial thrombogenesis, platelet aggregation, and superoxide dismutase activity. hynes et al. (1997) have used targeted mutations in integrins and their ligands to study the receptor and ligand interaction. although, we do not have animal model for the role of the 65-kda and 47-kda proteins, we have used rat thrombosis model to test the effective of chyb on thrombi formation (du et al. 2007). in that report, we have established that chyb can inhibit types i and iii collagen-induced platelet aggregation, adhesion of platelets onto types i and iii collagen-coated microtiter wells, and rabbit aortic segments in a dose-dependent manner. to advance the peptide as a useful therapeutic agent, we tested its cytotoxicity in cells and rats. results from the present study suggest that the chyb is not toxic to cultured cells by using [3h]-thymidine incorporation at different time intervals and multiple additions of the chyb. the chyb does not have a toxic effect on the meg-01 cells suggesting it is safe for further studies in an animal model. results from our earlier studies have shown that the chyb is a specifi c and functional inhibitor in vitro experiments (du et al. 2007; zhu et al. 2007). for the therapeutic usefulness, we have injected chyb into tail vein of rats, taken blood samples at different time intervals, and measured the activity of ldh, ast, alt, and gs in the duration of 36 hr. the activity of these enzymes did not increase in these samples suggesting that the chyb did not damage organs (muscle, heart, and lung). we will perform experiments with multiple doses and longer time frames (1, 2, and 3 weeks) to study the effect of chyb on tissue damage. additional studies to fully characterize the cytotoxicity of selected organs (liver, heart, kidney, and brain) are needed to justify its usefulness. acknowledgements the authors wish to thank mr. a. wright for his assistance with the rat experiments. the office of biomedical laboratory research, department of veterans affairs supported the present investigation. abbreviations used were prp, platelet-rich plasma; chyb, chemical synthesized peptide containing one of active peptide derived from both platelet type i and type iii collagen receptor with 12 amino acid residues as a linker; ldh, lactic acid dehydrogenase, gs, glutamine synthase, alt, alanine aminotransferase, and ast, asparate aminotransf e r a s e ; tr i s e d ta , 2 0 m m tr i s 1 3 0 m m nacl-1 mm edta, ph 7.3; tbs, 20 mm tris500 mm nacl; tbst, 20 mm tris-500 mm nacl-0.05% tween 20. disclosure the authors report no confl icts of interest. references bergman, i. and loxley, r. 1963. two improved and simplifi ed methods for the spectrophotometric determination of hydroxyproline. anal. chem., 35:1961–65. born, g.v.r. 1962. aggregation of platelets by adenosine diphosphate and its reversal. nature, 194:927–9. chen, c. and sheppard, d. 2007. identifi cation and molecular characterization of multiple phenotypes in integrin knockout mice. methods in enzymol., 426:291–305. chen, l.y., jokela, r., bowry, a., sandler, h., sjoquist, m., saldeen, t. and mehta, j.l. 2000. effect of stable fi sh oil on arterial thrombogenesis, platelet aggregation, and superoxide dismutase activity. j. cardiovasc. pharmacol., 35:502–5. chiang, t.m. 2000. a nonapeptide derived from the cloned platelet type i collagen receptor inhibits type i collagen-platelet interaction. am. j. med. sci., 320:362–7. table 4. the activity of ldh in chyb injected rat sera. treatments 0 hr 1.5 hr 3 hr 6 hr 24 hr 36 hr pbs control 0.076 0.131 0.045 0.072 0.023 0.022 vehicle control 0.054 0.105 0.128 0.051 0.130 0.041 120 µg chyb/kg 0.063 0.109 0.062 0.079 0.082 0.050 rats (two per group) were injected with 0.25 ml of either: control (pbs) or vehicle control (pbs/0.7% dmso), or chyb as listed in the table, following venipuncture as zero time. at each time point, blood was withdrawn, placed in a microfuge tube with 1/10 heparin, centrifuged, the plasma collected separately, and frozen at −80 ºc until ldh activity could be measured. determination of ldh activity for each time point was according to manufacturer’s suggestions, using 100 µg/well, in triplicate. a positive control of 0.055u ldh yielded o.d 490 nm = 1.788). 159 platelet-collagen interaction drug target insights 2008:3 chiang, t.m. and kang, a.h. 1997. a synthetic peptide derived from the sequence of type i collagen receptor inhibits type i collagen-mediated platelet aggregation. j. clin. invest., 100:2079–84. chiang, t.m. and postlethwaite, a.e. 2007. a cell model to study the regulation of phosphoitdylinositide 3-kinase and protein kinase b activity. biochem. biophys. acta., 1770:1181–6. chiang, t.m., beachey, e.h. and kang, a.h. 1975. interaction of chick skin collagen fragment (α1-cb5) with human platelets. biochemical studies during the aggregation and release reaction. j. biol. chem., 250:6916–22. chiang, t.m., cole, f. and woo-rasberry, v. 2002. cloning, characterization, and functional studies of a 47-kda platelet receptor for type iii collagen. j. biol. chem., 277:34896–901. chiang, t.m., rinaldy, a. and kang, a.h. 1997. cloning, characterization, and functional studies of a platelet receptor for type i collagen. j. clin. invest., 100:514–21. du, h., zawaski, j.a., gaber, w. and chiang, t.m. 2007. a recombinant hybrid peptide and chemically synthesized peptide inhibit thrombi formation in fecl3-induced thrombosis model in rat. thromb. res., 121:419–26. fitzsimmons, c.m., cawston, t.e. and barnes, m.j. 1986. the reactivity of collagen type i: evidence for multiple platelet-reactive sites in type i collagen molecule. thromb. and haemost., 56:95–9. gaber, m.w., yuan, h., killmar, j.t., naimark, m.d., kiani, m.f. and merchant, t.e. 2004. an intravital microscopy study of radiationinduced changes in permeability and leukocyte-endothelial cell interactions in the microvessels of the rat pia mater and cremaster muscle. brain res. protocols., 13:1–10. goudriaan, j.r., dahlmans, v.e.h., febbraio, m., teusink, b., romijn, j. a., havekes, l.m. and voshol, p.j. 2002. intestinal lipid absorption is not affected in cd 36 defi cient mice. molecular and cellular biochem., 239:199–202. hladovec, j. 1971. experimental arterial thrombosis in rats with continuous registration. thromb. diath. haemorrh., 26:407–10. hynes, r.o. and bader, b.l. 1997. targeted mutations in integrins and their ligands: their implications for vascular biology. thromb. haemost., 78:83–7. kingdon, h.s., hubbard, j.s. and stadtman, e.r. 1968. regulation of glutamine synthetase. xi. the nature and implications of a lag phase in the escherichia coli glutamine synthetase reaction. biochemistry, 7:2136–42. kurz, k.d., main, b.w. and sandusky, g.e. 1990. rat model of arterial thrombosis induced by ferric chloride. thromb. res., 60:269–80. leger, a.j., jacques, s.l., badar, j., kaneider, n.c., derian, c.k., andradegordon, p., covic, l. and kuliopulos, a. 2006. blocking the proteaseactivated receptor 1–4 heterodimer in platelet-mediated thrombosis. circulation, 113:1244–54. morton, l.f., fitzsimmons, c.m., rauterberg, j. and barnes, m.j. 1987. platelet-reactive sites in collagen: collagens i and iii possess different aggregatory sites. biochem. j., 248:483–7. philp, r.b., francey, i. and warren, b.a. 1978. comparison of antithrombotic activity of heparin, asa, sulfi npyazone and vk744 in a rat model of arterial thrombosis. haemostasis, 7:282–93. seyer, j.m. and kang, a.h. 1980. covalent structure of collagen: amino acid sequence of (1(iii)-cb5 from type iii collagen of human liver. biochemistry, 20:2621–27. seyer, j.m., hutchesion, e.t. and kang, a.h. 1976. collagen polymorphism in idiopathic chronic pulmonary fi brosis. j. clin. invest., 57:1498–507. zhu, j., cole, f., woo-rasberry, v., fang, x.r. and chiang, t.m. 2007. platelet receptors for type i and type iii collagens: evidences support type specifi c active peptides. thromb. res., 119:111–9. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true 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249 correspondence: bilgehan erkut, assist prof, atatürk bulvari, eda apartmani palandoken polikliniği üstü, kat: 3, no: 3, 25080 yenişehir, erzurum, türkiye. tel: (+90 533) 745 10 06; fax: (+90 442) 316 63 40; email: bilgehanerkut@yahoo.com copyright in this article, its metadata, and any supplementary data is held by its author or authors. it is published under the creative commons attribution by licence. for further information go to: http://creativecommons.org/licenses/by/3.0/. effects of ascorbic acid, alpha-tocopherol and allopurinol on ischemia-reperfusion injury in rabbit skeletal muscle: an experimental study bilgehan erkut1, ahmet özyazıcıoğlu2, bekir sami karapolat1, cevdet uğur koçoğulları3, sait keles4, azman ateş1, cemal gundogdu5, hikmet kocak1 1department of cardiovascular surgery, atatürk university medical faculty, erzurum, turkey. 2department of cardiovascular surgery, yüksek i̇htisas hospital, bursa, turkey. 3department of cardiovascular surgery, afyon kocatepe university medical faculty, afyon, turkey. 4department of biochemistry, atatürk university medical faculty, erzurum, turkey. 5department of pathology, medical faculty of atatürk university, erzurum, turkey. abstract purpose: ischemia reperfusion injury to skeletal muscle, following an acute arterial occlusion is important cause of morbidity and mortality. the aim of the present study was to determine and evaluate the effects of ascorbic acide, alpha-tocopherol and allopurinol on ischemia reperfusion injury in rabbit skeletal muscle. methods: forty-eight new zealand white rabbits, all male, weighing between 2.5 to 3.0 (mean 2.8) kg, were used in the study. they were separated into four groups. group i was the control group without any drugs. the other groups were treatment groups (groups ii, iii, and iv). group ii rabbits administrated 50 mg/kg ascorbic acide and 100 mg/kg alpha-tocopherol 3 days prior to ischemia, group iii rabbits received 50 mg/kg allopurinol 2 days prior to ischemia, and group iv rabbits were administrated both 50 mg/kg ascorbic acide, 100 mg/kg alpha-tocopherol 3 days prior to ischemia and 50 mg/kg allopurinol 2 days prior to ischemia. two hours ischemia and 2 hours reperfusion were underwent to the treatment groups. at the end of the reperfusion periods, muscle samples were taken from rectus femoris muscle for determination of superoxide dismutase, catalase and glutathione peroxidase activities as antioxidant enzymes, and malondialdehyde as an indicator of lipid peroxidation and xanthine oxidase levels as source hydroxyl radical. besides, histopathological changes (edema, infl ammation, ring formation and splitting formation) were evaluated in the muscle specimens. results: in the treatment groups; superoxide dismutase (u/mgprotein), catalase (u/mgprotein), and glutathione peroxidase (u/mgprotein) levels increased, malondialdehyde (nmol/mgprotein) and xanthine oksidase (mu/mgprotein) levels decreased compared to control i ( p � 0.05). increase of superoxide dismutase, catalase, and glutathione peroxidase levels were the highest and decrease of malondialdehyde and xanthine oxidase levels were the highest in group iv compared to groups ii and iii, but no signifi cant as statistically. also amount of cellular injury in group ii, iii, and iv were lower than group i. conclusions: antioxidant medication may help lowering ischemia reperfusion injury. in our study, all drug medications are shown to be able to have an effective role for preventing ischemia reperfusion injury. moreover, ascorbic acide + alphatocopherol + allopurinol group (group iv) may have a benefi cial effect to decrease the local and systemic damage due to ischemia-reperfusion injury. keywords: ischemia-reperfusion injury, antioxidant agents, ascorbic acid, alpha-tocopherol, allopurinol introduction ischemia-reperfusion (i/r) injury is an important adverse clinical outcome in a wide range of vascular conditions and surgical interventions including stroke, transplantation, cardiopulmonary bypass, trauma, and abdominal aortic aneurysm repair. it is a complex and serious condition and may life-threatening (1). experimental studies have shown that the tissue-destructive effects of i/r injury are mediated by free oxygen radicals (h2o2, o2 −, oh−), which damage cellular components and they cause lipid peroxidation of cellular membranes and generate more free radicals (frs) in a self-propagating cycle, leading to cell death by necrosis (1–3). drug target insights 2007:2 249–258 original research http://creativecommons.org/licenses/by/3.0/ http://creativecommons.org/licenses/by/3.0/ 250 erkut et al drug target insights 2007:2 the physiopathology of i/r injury is a complex cascade of events starting from the point of release of frs followed by lipid peroxidation that ends by procuding substances such as malonyldialdehyde (mda) (4,5). the determination of mda can be used as a marker of fr formation (6). during ischemia, atp production is diminished related to the limited oxygen availability. secondly, the changes in membrane ion gradients cause an infl ux of calcium in damaged membranes. this leads to an elevation of the cytosolic calcium concentration that activates proteases capable of transforming xanthine dehydrogenase to xanthine oxidase (xo). during reperfusion, the provided molecular oxygen is converted to superoxide radicals by xo (6). there is evidence that xo levels are elevated during ischemia (6,7). the fi rst line defence mechanism includes antioxidant enzymes such as superoxide dismutase (sod), catalase (cat) and glutathione peroxidase (gpx) which they are the enzymatic part represented by fr scavenger enzymes (8). these enzymes catalyse the conversion of frs into less reactive species. another mechanism is the nonenzymatic part including a large number of natural or synthetic antioxidant compounds, which have the ability to inhibit the oxidative damage by scavenging the highly destructive fr species (9). ascorbic acide is a well-known antioxidant agent and can protect the endothelium from direct injury by oxidants, including h2o2, and prevent microvascular dysfunction (10,11). we and others have also shown that ischaemia-reperfusion injury is reduced by the administration of ascorbic acide (10–12). alpha-tocopherol is a potent antioxidant and shown that it prevents reperfusion injury in miscellaneous tissue (13). allopurinol is a xo inhibitor that prevents the generation of frs and may play a role in the protection of the cells during cerebral ischemia. previous studies have indicated that allopurinol can improve the tissue energy metabolism during reperfusion after ischemia (14,15). a number of authors have examined the role of frs species in ischemic damage to skeletal muscle and experimentally, and the effect of fr scavengers have been evaluated in skeletal muscle (16,17). besides, in many studies, the antioxidant activity was shown the roles of ascorbic acide, alphatocopherol and allopurinol in i/r injury through biochemical enzyme studies in addition to histopathological studies (18–21). but, according to our knowledge, there was no report to show the effects of ascorbic acide, alpha-tocopherol and allopurinol on reducing reperfusion injury in muscle of rabbit. the aim of this study was to determine the protective effect of the ascorbic acide, alphatocopherol and allopurinol as antioxidant agents against frs in extremity ischemia and reperfusion. for this purpose, we measured sod, cat, gpx and mda, xo levels, and evaluated as histopathologically in rabbit muscle in ischemia reperfusion model. our fi ndings suggest that all drug groups, especially group iv, reduces i/r injury. materials and methods the experiment was performed in compliance with the principles of laboratory animal care formulated by the national institutes of health. the experiment and animal care protocol, and all procedures were approved by the local ethics committee in animal experiments. animals fourty-eight rabbits were used as subjects in our study. male adult new zealand type rabbits weighing 2,500–3,000 g (2,610 ± 1,122 g) were kept in a light-controlled room with a 12:12-h light–dark cycle; temperature (22 ± 0.5 °c) and relative humidity (65%–70%) were kept constant. animals received a standard rabbit diet and water and libitum. they had not been used in priory another study and they had not been given a drug regularly, in addition they had not a disease, previously. the rats were deprived of food for 12 h before the experiment but had free access to water. the subjects were inserted 22 no branul through ear veins. experiments were carried out under sterile conditions and antibiotic prophylaxis with cefazolin sodium (30 mg/kg intramuscularly, single preoperative dose) was given. isotonic nacl solution was given intravenously at the rate of 3 ml/kg/h. during all experimental manipulations, to prevent the effects of hypothermia and to provide the stability of hemodynamic parameters, the body temperature was maintained at 37.2 °c with a rectal probe. for this, animals were placed on an operating table with thermoregulatory, and was used heat pad. artifi cial respiration the rabbits were shaved from abdomen to leg. the surgical area was painted with batticon. surgical 251 ischemia reperfusion injury drug target insights 2007:2 area was cleaned and draped. the rabbits were intubated via endotracheal cannulation (16-g vasofi x, b. braun melsungen, ag, and germany). tidal volume and respiratory rate were adjusted to 10 ml/kg (3 ml for an average of 250–300 g subject) and 60 times per minute, respectively. ventilator (ugo basile, biological research apparatus, comerio, and varesee, italy) was used for artifi cial respiration. experimental groups before starting the experimental protocols, rabbits were divided into four groups of twelve animals. the fi rst group was the control group (group i) without any treatment, the treatment groups were the group ii, which was medicated intravenous with 50 mg/kg ascorbic acide (redoxan 500 mg ampule, roche, germany) and intramuscular with 100 mg/kg alpha-tocopherol (α-t; evigen 300 mg ampule, aksu farma, turkey) for 3 days prior experiment, the group iii, which was medicated with 50 mg/kg allopurinol (urikoliz 300 mg, ilsan, turkey) for 2 days via per oral prior experiment, and the group iv, which was medicated with both ascorbic acide + alpha-tocopherol + allopurinol. the dosage and timing of each antioxidant were based on our preliminary data on the metabolism of the drugs, and on published data, which determined the peak serum concentrations of the drugs at the time reperfusion was initiated (22–25). the protective effects against increasing of lipid peroxidation caused by i/r were shown in rats treated with different dosages (range, 30–100 mg/kg) of ascorbic acide (22). in light of these past studies, we used 50 mg/kg of ascorbic acide as the dosage in our study. dosage of alpha–tocopherol and allopurinol were chosed on on the basis of earlier studies (24,26,27). in experiment day, all animals were performed ischemia for 2 hours, and then performed reperfusion for 2 hours. hoballah’s i/r model was used with direct occlusion of femoral artery and vein occlusions in the medial part of rectus femoris muscle (28). surgical method the rabbits were anesthetized with intramuscular injection of 15 mg/kg ketamine hydrochloride (ketalar; pfi zer, istanbul, turkey) and 2 mg/kg xylazine hydrochloride (rompun, bayer, turkey) before the surgical procedure. longitudinal incision was performed in venteromedial of right thigh. after the femoral area exploration, we reached to the rectus femoris muscle. heparin of 400 u/kg (liquemine, roche, brazil) was given via ear vein. the medial part of rectus femoris muscle was cut and separated from surrounding tissues. the i/r model was constituted by using the direct occlusion procedure as the study of hoballah (fig.1) (28). we exposed the muscle to ischemia for 2 hours. during the ischemic waiting period, incision was closed and the rabbits were freed. at the end of the ischemic period, was applied intraperitoneally ketamine hydrochloride and incision was opened, again. microvascular clamps were removed and the wounds were closed. it was exposed to reperfusion for 2 hours. at the end of the reperfusion, muscle tissue samples were collected for biochemical and histopathological examination after the incision was opened. the muscle pieces from proximal and distal were anastomozed with one another. the femoral area was closed with silk suture. during these surgical interventions intervals, they were given same sort analgesics. biochemical assay sod, cat, gpx, xo, mda were detected in muscle tissue cuts. each tissue was stocked in a separate bowl at −80 °c till analysis. tris tampon of 10 ml was added into each one gram of frozen tissues. homogenates are to be centrifuged at 10.000 × g for 10 minutes after homogenization. supernatants were kept in stock at −80 °c till analysis. analysis of tissue samples was carried out spectrophotometrically as below. results were expressed as units per miligram protein for sod, cat, gpx, and nanomoles per milligram for mda and miliunits per milligram for xo. tissue sod assay: the method is based on the inhibition of nitroblue tetrazolium (nbt) reduction by the xanthine-xo system as a superoxide generator by using yi-sun method (29). study solution was prepared by mixing xanthine (0.3 mmol/l), ethylenediaminetetraacetate (edta) (0.6 mmol/l), nbt (0.15 mmol/l), sodium carbonate (na2co3) (400 mmol/l), bovine serum albumin (1 g/l). study solution of 2850 ul, 100 ul supernatan, 100 ul distilled water and 50 ul xo (5 u/l) were incubated for 25 minutes at 20 °c. following 30 seconds, absorbance was recorded. one unit is the amount of sod that inhibits the rate by 50%. tissue cat assay: catalase activity was assayed according to the methods of cohen et al. (30) 252 erkut et al drug target insights 2007:2 to a 100 µl aliquot of tissue extract, ethanol was added to a concentration of 0.17 mol/l (10 µl ethanol/ml) and samples were incubated in an ice bath for 30 min. after 30 min, 10% triton x–100 was added to a fi nal concentration of 1% and samples were kept at room temperature. reactions were performed at room temperature. the enzyme-catalysed decomposition of h2o2 was measured. in a tube containing 200 µl phosphate buffer and 50 µl tissue extract, 1 ml of 6.0 mmol/l h2o2 (in phosphate buffer) was added and mixed thoroughly. the reaction was stopped after exactly 3 min by the addition of 100 µl of 6 mol/l h2so4. the excess h2o2 was measured by reacting it with a standard excess of kmno4 and then measuring the residual kmno4 spectrophotometrically at 480 nm within 30–60 s using 1.0 absorbance unit for standard kmno4. tissue gpx assay: gpx catalyzes oxidation of glutathione (gsh) by using hydrogen peroxide. the activity of gpx was determined by the beutler method (31). briefly, solutions of tris-hcl (1000 mmol/l), edta (5 mmol/l) (ph = 8.0), gsh (100 mmol/l), gsh reductase (10 u/ml), nicotinamide adenine dinucleotide phosphate (nadph) (2 mmol/l), and t-butil hydroperoxide (7 mmol/l) were incubated with 10 ul hemolysate for figure 1. occlusion appearance of two vascular systems of rectus femoris muscle (direct occlusion) as described by hoballah. 253 ischemia reperfusion injury drug target insights 2007:2 10 minutes at 37 °c. decrease in nadph was foolewed spectrophotometically at 340 nm. tissue mda assay: thiobarbituric acid (tba) reactive substances, mda and product of fatty acid peroxidation, reacts with tba to form a colored complex that has maximum absorbance at 532 nm by ohkawa method (32). 200 ul sodium dodecyl sulfate (8.1%), 1.5 ml acetic acid (20%), 1.5 ml tba (0.8%), 0.6 ml distilled water were mixed by 200 ul tissue homogenate (10%) and heated in boiling water for 60 minutes. after cooling, 1 ml distilled water and 5 ml butanol/pyrimidine (15:1) were added. after centrifuging at 4000 rpm for 10 minutes, absornance of the supernatant was read at 532 nm. tissue xo assay: xo activity was determined spectrophotometrically by the method of hashimoto (33), based on the formation of uric acid from xanthine at 293 nm. histopathological examination the muscle biopsy samples of the subjects were fi xed in neutral formalin solution (3%) for 24 hours. paraffi n blocks were prepared after biopsy samples were exposed to routine tissue examination. later 1 to 2 micro hematoxylin-eosin (he) cuts of paraffin blocks were prepared. light microscopy fi ndings were scored as 0 to + 3 which corresponds to no change, mild, moderate and severe changes, respectively. assessments were made for interstitial edema, infl ammation, splitting formation and ring formation in tissues through the use of nikon optiphot-2 microscope. statistical analysis all the results were obtained as mean ± sem for each study group. all statistical analyses were carried out using spss 10.0 statistical software (spss inc, chicago, il). the signifi cance of differences between the groups was assessed using the nonparametric analyses with mann-whitney u-test, and the kruskal-wallis test was used to compare group medians for histopathological. statistical signifi cance was set up p � 0.05. results biochemical evaluation sod, cat, and gpx levels were measured in skeletal muscle after 2 hours reperfusion, and the levels increased in the treatment groups compared to control group (from 0,17 ± 0,02 to 0,23 ± 0,02, 0,23 ± 0,03 and 0,25 ± 0,04, respectively for sod; from 6,74 ± 1,00 to 9,02 ± 0,60, 8,22 ± 1,35 and 9,09 ± 1,16, respectively for cat; from 18,52 ± 2,26 to 22,48 ± 2,38, 23,06 ± 2,34 and 25,67 ± 3,72, respectively for gpx) (fig. 2), and it was signifi cant as statistically (p � 0.05). however, there was no a statistically signifi cant among group ii, iii, and iv in terms of the increasing in enzymes levels. xo levels in the skeletal muscle were found to be higher in the control group. however, in treatment groups lowered (from 2,86 ± 0,35 to 2,19 ± 0,34, 1,94 ± 0,41 and 1,02 ± 0,32, respectively) the levels of xo during ischemia and reperfusion (fig. 3). tissue mda levels were decreased (from 0,38 ± 0,13 to 0,12 ± 0,04, 0,13 ± 0,07 and 0,10 ± 0,02, respectively) in the treatment group compared to the control group. the results of mda assays are shown in figure 3. histopathological evaluation cell infi ltration, edema, splitting and ring formations were evaluated for 4 groups. there was a signifi cant difference in terms of pathological parameters between treatment groups and control group as histopathological scores ( p � 0.05). figure 4 shows markedly ring formation, splitting formation, interstitial edema, and neutrophil cell infi ltration in control group after 2 hour reperfusion. in treatment groups ring formation, splitting formation, interstitial edema, and neutrophil cell infi ltration decreased markedly compared to control group. although ring formation was rare in treatment groups, there was not a difference among 3 treatment groups ( p � 0.05). splitting formation, interstitial edema and neutrophil cell infi ltration were less determined in treatment groups ( p � 0.05). when treatment groups were compared to each other, it was found that splitting formation, interstitial edema and neutrophil cell infi ltration was less in group iv ( p � 0.05). discussion because skeletal muscle is more resistant to the ischemia, we applied total skeletal muscle ischemia model in this study. the transformation of xanthine dehydrogenase into xo during frs constitution is slower in skeletal muscle, which explains why skeletal muscle is more resistant to ischemia (28,34,35). recently advances in the understanding of reperfusion injury and the pharmacology of 254 erkut et al drug target insights 2007:2 antioxidants have made the interruption of reperfusion injury clinically promising and fr mediated tissue injury can be limited by the use of antioxidant therapy and several studies have suggested a positive role for antioxidant therapy in skeletal muscle reperfusion injury (36,37). it is well recognized that ischaemia followed by reperfusion in skeletal muscle represents an important clinical problem in many vascular diseases and musculoskeletal trauma. the signifi cant mortality and morbidity can be due to compartment syndrome, rhabdomyolysis, renal failure, limb loss, systemic infl ammatory syndrome and respiratory and mesenteric injuries. it has been emphasized the importance of ischaemia duration as a progressive increase of ultrastructural lesions takes place between 2and 7-h ischaemia insult in skeletal muscular tissues (38). two hours after ischaemia, it is already possible to identify, with histochemical analysis, small muscular lesions which become severe according to the ischaemia duration (39). regarding reperfusion injury, several reports have figure 2. between groups sod, cat, and gpx enzyme levels. increased sod, cat and gpx levels in treatment groups compared to control group. figure 3. between groups mda and xo enzyme levels. decreased mda and xo levels in treatment groups compared to control group. sod; superoxide dismutase, cat; catalase, gpx; glutathione peroxidase, mda; malonyldialdehyde, xo; xanthine oxidase, ap: allopurinol. 255 ischemia reperfusion injury drug target insights 2007:2 showed that a period over 2 h of reperfusion is enough to establish the muscular lesion (40–42). most study was made relation to administration time of drugs in order to decrease i/r damage (17–19). these studies were carried out in which antioxidants were given before ischemia. however, antioxidant agents were injected before reperfusion in other study (43). in a study, feller showed that sod and dimethylsulfoxide (oh _ radical scavenger) given before reperfusion and decreased reperfusion injury, and in late term, the muscle functions were excellent (44). we administrated antioxidant agents to the drug groups before ischemia in our study. frs are normal by-products of cellular metabolic processes. the human body has a complex antioxidant defense system that includes the antioxidant enzymes (sod, gpx and cat) and nonenzymatic antioxidant components such as glutathione, a-tocopherol, ascorbic acide, and b-carotene. these prevent the initiation or propogantation of free radical chain reactions. post-ischemic reperfusion injury is associated with the generation of frs which damage cellular components and initiate the lipid peroxidation process. in many studies, antioxidant activity was tried to be shown through biochemical enzyme studies in addition to histopathological studies. we examined that the vitamin combinations given 3 days before the ischemia and allopurinol given 2 days before the ischemia for decreased i/r damage, both histopathological and biochemical. we could not fi nd any report showing the effect of ascorbic acide, alpha-tocopherol, and allopurinol on recuding i/r injury in lower extremity muscle of rabbit as enzymatic and biochemically. sod is an enzyme which catalyses the transformation of the o2 − into h2o2, and it is one of the primary and signifi cant defensive systems against oxidative damage. the physiological function of this enzyme is to protect the cells of oxygen against frs harmful effects (45,46). when oxidant stress increases in organism, sod enzyme levels increase (45). the function of cat is to divide h2o2 into o2 − and h2o by participating into the reaction with h2o2. through this, it prevents the formation of oh− radicals, which are more toxic. criado found that cat enzyme levels increased in 30 minutes after i/r damage (47). for gpx catalyses h2o2 and lipid hydroperoxidase, it protects to cellular membranes from damage, prevents the start and development of lipid peroxidation. lin showed in a myocardial ischemia reperfusion injury model found that gpx levels increased with the antioxidant figure 4. the fi gure shows (a) ring formation, (b) splitting formation, (c) interstitial edema, (d) neutrofi l cell infi ltration in control groups (the arrows were depicted pathological changes) × 400 he. 256 erkut et al drug target insights 2007:2 agent (48). in our study, sod, cat and gpx levels were increased in the treatment groups compared to control group ( p � 0.05). although there was no signifi cant different between treatment groups, the increase of the enzymes levels was higher in group iv than group ii and iii. the end production of lipid peroxidation includes aldehydes, hydrocarbon gases, and mda. it is good markers for increased systemic oxidative stress (48). mda levels indicate the amount of cellular damage secondary to lipid peroxidation and has been widely adopted as a measure of free radical formation. lipid peroxidation can cause changer leading to the deaths of cells. feng found that mda increase depended on fr appearance (49). a signifi cant elevation of mda level after the 30 min of ischemia and 45 min of reperfusion was observed in tissues (50). in our study, the mda levels had signifi cantly increased after ischemia reperfusion in the control group, because of the high level of hydroxyl radicals. in the antioxidant treatment groups, the levels were found have decreased, which may show the effects of antioxidant medication on limiting ischemia reperfusion injury. xo plays an important role in i/r injury (6,51). there is evidence that xo levels are elevated during ischemia (52). xo is the fi rst-known o2 − radical source (16). during ischemia, adenosine triphosphate is degraded to hypoxanthine and xanthine dehydrogenase is converted to xo. during reperfusion, xo catalyzes the conversion of hypoxanthine to uric acid with release of the o2 − radical. hammerman showed that lipid peroxidation was prevented in the group together with the decrease in xo activity (53). allopurinol is considered to be an xo inhibitor. smith found that i/r injury were decreased with tungsten and allopurinol (54). in our study, there were signifi cant differences in the levels of xo between treatment groups and control group ( p � 0.05). moreover, the decrease was highest in group iii and iv administrated allopurinol. ascorbic acide is a powerful antioxidant agent. it is a critical component of the oxidant shield in skeletal muscle, being actively accumulated by muscle endothelium (55) according to niki et al. (20), ascorbic acide has an antioxidant effect of the superoxide and hydrophilic radicals. it also acts on limiting lipidic peroxidation and scavenges reactive oxidants produced immediately after reperfusion (21). in our study, in group ii and iv, which was used ascorbic acide, decreased level of xo and mda enzyme and increased antioxidant enzymes (sod, cat and gpx) levels, and these results were supported with histopathological fi ndings. αlpha-tocopherol is an antioxidant, and is protector against frs. the preventive effects of alpha-tocopherol has been demonstrated in different experimental models (56,57). it protects cell membrane from oxidative damage and lipid peroxidation (57). allopurinol, a specifi c inhibitor of the enzyme xo, blocks the synthesis of xanthine from hypoxanthine and therefore avoids the formation of the free radical superoxide. the studies showed that it is decrease the level of frs production and reduce the tissue injury associated with i/r injury (23, 58). it is not only a potent inhibitor of xo but may also be an agent that improves ischemia-induced mitochondrial dysfunction (14,58). our data show that fr overproduction induced by i/r causes lipid peroxidation of rabbit skeletal muscle. administration of ascorbic acide, alphatocopherol (before 3 days) and allopurinol (before 2 days) skeletal i/r decreased mda and xo levels and increased in sod, cat and gpx enzyme activities in the skeletal muscle and this result was suggested with hystopathological results. results of this study show that prophylactic administration of ascorbic acide, alpha-tocopherol and allopurinol ischemia condition prevents reperfusion injuries by eliminating oxygen radicals and inhibiting lipid peroxidation. besides, the combination of ascorbic acide, alpha-tocopherol and allopurinol may be suffi cient to more effi ciently prevent subsequent oxidative stress in the tissues and improve their function after i/r. references [1] nanobashvili, j., neumayer, c., fuegl, a. et al. 2003. development of ‘no-refl ow’ phenomenon in ischemia/reperfusion injury: failure of active vasomotility and not simply passive vasoconstriction. eur. surg. res., 35:417–4. [2] chander, v. and chopra, k. 2006. protective effect of nitric oxide pathway in resveratrol renal ischemia-reperfusion injury in rats. arch. med. res., 37:19–26. [3] waters, s., fae, a., gondalia, j. et al. 2004. effects of pretreatment with a xanthine oxidase inhibitor on free radical levels during carotid endarterectomy. free radic. res., 38:283–93. [4] draper, h.h. and hadley, m. 1990. malondialdehyde determination as index of lipid peroxidation. methods enzymol., 186:421. [5] ozcan, a.v., sacar, m., aybek, h. et al. 2007. the effects of iloprost and vitamin c on kidney as a remote organ after ischemia/reperfusion of lower extremities. j. surg. res., 140:20–6. 257 ischemia reperfusion injury drug target insights 2007:2 [6] inal, m., altinisik, m. and bilgin, m.d. 2002. the effect of quercetin on renal ischemia and reperfusion injury in the rat. cell. biochem. funct., 20:291–6. [7] engerson, t.d., mckelvey, t.g., rhyne, d.b., boggio, e.b., snyder, s.j. and jones, h.p. 1987. conversion of xanthine dehydrogenase to oxidase in ischemic rat tissues. j. clin. invest, 79:1564–70. [8] mccord, j.m. 2000. the evolution of free radicals and oxidative stress. am. j. med., 108:652–9. [9] concannon, m.j., kester, c.g., welsh, c.f. et al. 1992. patterns of freeradical production after tourniquet ischemia: implications for the hand surgeon. plast reconstr. surg., 89:846–52. [10] kearns, s.r., daly, a.f., sheehan, k., murray, p., kelly, c. and bouchier-hayes, d. 2004. oral vitamin c reduces the injury to skeletal muscle caused by compartment syndrome. j. bone joint surg. br., 86:906–11. [11] armour, j., tyml, k., lidlington, d. and wilson, j.x. 2001. ascorbate prevents microvascular dysfunction in the skeletal muscle of the septic rat. j. appl. physiol., 90:795–803. [12] kearns, s.r., kelly, c.j., barry, m. et al. 1999. vitamin c reduces ischaemia-reperfusioninduced acute lung injury. eur. j. vasc endovasc surg., 17:533–6. [13] yagmurdur, m.c., ozdemir, a., ozenc, a. and kilinc, k. 2003. the effects of alpha-tocopherol and verapamil on mucosal functions after gut ischemia/reperfusion. turk j. gastroenterol., 14:26–32. [14] isik, n., berkman, m.z., pamir, m.n., kalelioglu, m. and sav, a. 2005. effect of allopurinol in focal cerebral ischemia in rats: an experimental study. surg. neurol., 64:suppl 2:s5–10. [15] jeon, b.r., yeom, d.h. and lee, s.m. 2001. protective effect of allopurinol on hepatic energy metabolism in ischemic and reperfused rat liver. shock, 15:112–7. [16] lindsay, t., romaschin, a. and walker, p.m. 1989. free radical mediated damage in skeletal muscle. microcirc endothelium lymphatic, 5:57–70. [17] punz, a., nanobashvili, j., neumayer, c. et al. 1999. multivitamin administration before ischemia reduces ischemia-reperfusion injury in rabbit skeletal muscle. clin. nutr., 18:219–26. [18] akgun, s., tekeli, a., isbir, s.c., civelek, a., ak, k. and sirvanci, s. 2004. fk506 to prevent lung injury after hindlimb ischemia and reperfusion in a rat model: an electron microscopic study. surg. today, 34:678–84. [19] tanaka, m., terry, r.d., mokhtari, g.k., inagaki, k., koyanagi, t. and kofi dis, t. 2004. suppression of graft coronary artery disease by a brief treatment with a selective epsilon pkc activator and a deltapkc inhibitor in murine cardiac allografts. circulation, 14:110 (11 suppl 1): ii194–99. [20] niki, e. 1991. action of ascorbic acid as a scavenger of active and stable oxygen radicals. am. j. clin. nutr., 54:1119s–1124s. [21] higa, o.h., parra, e.r., ab’saber, a.m., farhat, c., higa, r. and capelozzi, v.l. 2007. protective effects of ascorbic acid pretreatment in a rat model of intestinal ischemia-reperfusion injury: a histomorphometric study. clinics, 62:315–20. [22] seo, m.y. and lee, s.m. 2002. protective effect of low dose of ascorbic acid on hepatobiliary function in hepatic ischemia/reperfusion in rats. j. hepatol., 36:72. [23] lee, w.y. and lee, s.m. 2006. synergistic protective effect of ischemic preconditioning and allopurinol on ischemia/reperfusion injury in rat liver. biochem. biophys. res. commun., 349:1087–93. [24] akgur, f.m., klinic, k., aktug, t. and olguner, m. 1994. the effect of allopurinol pretreatment before detorting testicular torsion. j. urol., 151:1715–7. [25] nakamura, m., ozaki, m., fuchinoue, s., teraoka, s. and ota, k. 1997. ascorbic acid prevents ischemic-reperfusion injury in the rat small intestine. transplant int., 10:89–95. [26] gupta, y.k. and sharma, m. 2001. reversal of pyrogallol-induced delay in gastric emptying in rats by ginger (zingiber offi cinale). methods find exp. clin. pharmacol., 23:501–3. [27] cadenas, s., lertsiri, s., otsuka, m., barja, g. and miyazawa, t. 1996. phospholipid hydroperoxides and lipid peroxidation in liver and plasma of ods rats supplemented with alpha-tocopherol and ascorbic acid. free radic. res., 24:485–93. [28] hoballah, j.j., mohan, c.r., schipper, p.h., chalmers, r.t., corry, d.c. and corson, j.d. 1996. rabbit rectus femoris muscle for ischemiareperfusion studies: an improved model. j. surg. res., 66:21–4. [29] sun, y., oberley, l.w. and li, y. 1988. a simple method for clinical assay of superoxide dismutase. clin. chem., 34:497–500. [30] cohen, g., dembiec, d. and marcus, j. 1970. measuement of catalase activity in tissue extracts. anal. biochem., 34:30–8. [31] beutler, e. 1973. red cell metabolism. a manuel of biocemical methods. new york: in grune and stratton.74. [32] ohkawa, h., ohishi, n. and yagi, k. 1979. assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. anal. biochem., 95:351–8. [33] hashimato, s.a. 1974. new spectrophotometric assay method of xanthine oxidase in crude tissue homogenate. anal. biochem., 62:425–35. [34] hickey, m.j., hurley, j.v., angel, m.f. and o’brien, b.m. 1992. the response of the rabbit rectus femoris muscle to ischemia and reperfusion. j. surg.. res., 53:369–377. [35] belkin, m., lamorte, w.i., wright, j.g. and hobson, r.w. 1989. 2nd. the role of leucocytes in the pathophysiology of skeletal muscle ischemic injury. j. vasc. surg., 10:14–9. [36] mohler, l.r., pedowitz, r.a., ohara, w.m. et al. 1996. effects of an antioxidant in a rabbit model of tourniquet induced skeletal muscle ischemia-reperfusion injury. j. surg. res., 60:23–8. [37] bushell, a., klenerman, l., davies, h. et al. 1996. ischemia reperfusion-induced muscle damage: protective effect of corticosteroids and antioxidants in rabbits. acta. orthop. scand, 67:393–8. [38] harris, k., walker, p.m., mickle, d.a.g. et al. 1986. metabolic response of skeletal muscle to ischemia. am. j. physiol., 250:213–20. [39] makitie, j. and teravainen, h. 1977. histochemical studies of striated muscle after temporary ischemia in the rat. acta. neuropathol., 37:247–53. [40] appell, h.j., duarte, j.a., gloser, s. et al. 1997. administration of tourniquet. ii. prevention of postischemic oxidative stres can reduce muscle edema. arch. orthop. trauma surg., 116:101–5. [41] santavirta, s., luoma, a. and arstila, a.u. 1979. morphological and biochemical changes in rabbit striated muscle of after experimental tourniquet ischemia. res. exp. med., 174:245–51. [42] strock, p.e. and majno, g. 1969. vascular responses to experimental tourniquet ischemia. surg. gynecol. obstet., 129:309–18. [43] bozkurt, a.k. 2002. alpha-tocopherol (vitamin e) and iloprost attenuate reperfusion injury in skeletal muscle ischemia/reperfusion injury. j. cardiovasc surg. (torino), 43:693–6. [44] feller, a.m., roth, a.c., russell, r.c., eagleton, b., suchy, h. and debs, n. 1989. experimental evaluation of oxygen free radical scavengers in the prevention of reperfusion injury to skeletal muscle. ann. plast surg., 22:321–31. [45] seven, a. and candan, g. 1996. antioxidant defences systems. cerrahpaşa j. med., 27. [46] kanko, m., maral, h., akbas, m.h. et al. 2005. protective effects of clopidogrel on oxidant damage in a rat model of acute ischemia. tohoku j. exp. med., 205:133–9. [47] garcia-criado, f.j., palma-vargas, j.m., valdunciel-garcia, j.j., gomez-alonso, a., srivastava, o. and ezrin, a. 1997. sulfo-lewis(x) diminishes neutrophil infi ltration and free radicals with minimal effect on serum cytokines after liver ischemia and reperfusion. j. surg. res., 70:187–94. erratum in: j. surg. res., 73:95. [48] lin, l.n., wang, w.t. and xu, z.j. 1997. clinical study on ligustrazine in treating myocardial ischemia and reperfusion injury. zhongguo zhong xi yi jie he za zhi, 17:261–3. [49] feng, f. 1990. biochemical metabolism and oxygen free radical changes following ischemic and reperfused injured limbs. an experimental study. zhonghua wai ke za zhi, 28:693–6. 258 erkut et al drug target insights 2007:2 [50] campos, r., maureira, f., garrido, a. and valenzuela, a. 1993. different glutathione redox status and lipid peroxidation in the cortex and the medulla of the rat kidney subjected to ischemia–reperfusion stress. comp. biochem. physiol. b., 105:157–63. [51] joannidis, m., gstraunthaler, g. and pfaller, w. 1990. xanthine oxidase: evidence against a causative role in renal reperfusion injury. am. j. physiol., 258:f232–f236. [52] engerson, t.d., mckelvey, t.g., rhyne, d.b., boggio, e.b., snyder, s.j. and jones, h.p. 1987. conversion of xanthine dehydrogenase to oxidase in ischemic rat tissues. j. clin. invest, 79:1564–70. [53] hammerman, c., goldschmidt, d., caplan, m.s., kaplan, m., schimmel, m.s. and eidelman, a.i. 1999. amelioration of ischemiareperfusion injury in rat intestine by pentoxifylline-mediated inhibition of xanthine oxidase. j. pediatr gastroenterol. nutr., 29:69–74. [54] smith, j.k., carden, d.i. and korthuis, r.j. 1987. role of xanthine oxidase in post ischemic microvascular injury in skeletal muscle. am. j. physiol., 257:1782–9. [55] wilson, j.x., dixon, s.j., yu, j., nees, s. and tyml, k. 1996. ascorbate uptake by microvascular endothelial cells of rate skeletal muscle. microcirculation, 3:211–21. [56] demirbas, a., bozoklu, s., ozdemir, a. and haberal, m. 1993. effect of alpha tocopherol on the prevention of reperfusion injury caused by free oxygen radicals in the canine kidney autotransplantation model. transplant proc., 25:2274. [57] hsiao, g., lee, j.j., chen, y.c. et al. 2007. neuroprotective effects of pmc, a potent alpha-tocopherol derivative, in brain ischemiareperfusion: reduced neutrophil activation and anti-oxidant actions. biochem. pharmacol., 73:682–93. [58] canbaz, s., duran, e., ege, t., sunar, h., cikirikcioglu, m. and acipayam, m. 2003. the effects of intracoronary administration of vitamin e on myocardial ischemia-reperfusion injury during coronary artery surgery. thorac cardiovasc surg., 51:57–61. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 1048576 /lockdistillerparams false /maxsubsetpct 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drug target insights 2006: 1 13–17 13 correspondence: 11 jalan tan tock seng, singapore 308433. tel: (65) 6357-7533; fax: (65) 6256-9178; email: obgpjt@nus.edu.sg please note that this article may not be used for commercial purposes. for further information please refer to the copyright statement at http://www.la-press.com/copyright.htm review neurogenesis and the effect of antidepressants philippe taupin1,2,3 1national neuroscience institute, singapore. 2national university of singapore. 3nanyang technological university, singapore. abstract: the recent evidence that neurogenesis occurs throughout adulthood and neural stem cells (nscs) reside in the adult central nervous system (cns) suggests that the cns has the potential for self-repair. beside this potential, the function of newly generated neuronal cells in the adult brain remains the focus of intense research. the hippocampus of patients with depression show signs of atrophy and neuronal loss. this suggests that adult neurogenesis may contribute to the biology of depression. the observations that antidepressants, like fl uoxetine, increase neurogenesis in the dentate gyrus (dg) and neurogenesis is required for the behavioral effect of antidepressants, lead to a new theory for depression and the design of new strategies and drugs for the treatment of depression. however, the role of adult neurogenesis in the etiology of depression remains the source of controversies and debates. keywords: neural stem cells, hippocampus, depression, fl uoxetine, cellular therapy. introduction neurogenesis, the generation of new neuronal cells, occurs in the adult brain of mammals (gage, 2000; gross, 2000), including human (eriksson et al. 1998). neurogenesis occurs primarily in two regions of the adult brain: the dg and the subventricular zone (svz) (taupin and gage, 2001). newly generated neuronal cells establish synaptic and functional connections with nerve cells of the pre-existing network (stanfi eld and trice, 1988; markakis and gage, 1999; van praag et al. 2002). it is hypothesized that newborn neuronal cells arise from stem cells in the adult brain. nscs are the self-renewing multipotent cells that generate the main phenotypes of the nervous system, neurons, astrocytes and oligodendrocytes; as such they hold the potential to treat a broad range of cns diseases and injuries (mckay, 1997). neural progenitor and stem cells have been isolated and characterized in vitro from the adult brain, further supporting the existence of nscs in the adult cns (reynolds and weiss, 1992; gage et al. 1995). the existence of nscs in the adult brain has tremendous consequences for cellular therapy in the cns, but also for our understanding of developmental biology (taupin, 2006a). depression is a major public health problem that affects 12–17% of the population (kessler et al. 1994). various classes of drugs are currently prescribed for the treatment of depression (wong and licinio, 2001; brunello et al. 2002). among them selective serotonin reuptake inhibitors (ssris), like fl uoxetine, monoamine oxidase inhibitors (maois), like tranylcypromine, specifi c norepinephrine reuptake inhibitors (snris), like reboxetine and phosphodiesterase-iv inhibitors, like rolipram, alleviate symptoms of depression. it is hypothesized that an imbalance in serotonin (5-hydroxytryptamine or 5-ht) and noradrenaline (na) pathways may underlie the pathogenesis of depressive disorders (hindmarch, 2001; owens, 2004). ssris, like fl uoxetine, may produce their therapeutic effects by increasing brain levels of 5-ht, a neurotransmitter implicated in the modulation of mood and anxiety-related disorders (whittington et al. 2004; ryan, 2005). among the 5-ht receptor subtypes, the 5-ht1a receptor has been prominently implicated in the modulation of mood and anxiety-related disorders (gross et al. 2002). there are increasing evidences that the hippocampus, a structure classically involved in leaning and memory, is involved in the modulation of emotional responses, particularly depression. clinical magnetic resonance imaging and post-mortem studies in depression patients, as well as in animal studies, reveal that chronic stress and depression result in loss of nerve cells and atrophy in the hippocampus, and that these effects can be reversed by antidepressants (watanabe et al. 1992a; sheline et al. 1996; czeh et al. 2001; campbell et al. 2004; videbech and ravnkilde, 2004; colla et al. 2006). drug target insights 2006: 114 philippe taupin this suggests that neurogenesis may be an underlying factor in the contribution of the hippocampus to depression. in support of this contention, glucocorticoids, stress-related hormones, induce brain atrophy (sapolsky, 2000; mcewen, 2001) and decrease neurogenesis (gould et al. 1991; cameron and gould, 1994), whereas antidepressants, like fl uoxetine, promote neurogenesis (malberg et al. 2000; malberg and duman, 2003). investigators have aimed at confirming and unraveling the mechanism underlying the involvement of adult neurogenesis to the etiology of depression. neurogenesis contributes to the therapeutic effects of antidepressants the effect of antidepressants, like fl uoxetine, tranylcypromine, reboxetine and rolipram, on adult neurogenesis was assessed by means of bromodeoxyuridine (brdu) labeling, immunohistochemistry for neuronal specifi c markers and confocal microscopy (malberg et al. 2000, 2004). brdu is a thymidine analog that incorporates into the dna of dividing cells and is used for birthdating cells and monitoring cell proliferation (miller and nowakowski, 1988; kuhn et al. 1996; taupin, 2006b). chronic administration of these antidepressants increases neurogenesis in the dg, but not the svz in adult rats, suggesting that hippocampal neurogenesis contributes to the therapeutic effects of antidepressants (malberg et al. 2000, 2004). to study the functional implication of such observations, santarelli et al. (2003) aimed at characterizing whether an increase in neurogenesis is required for the effect of antidepressants. x-ray irradiation of the hippocampal area in adult rats causes long-term reductions in cell proliferation in the dg (tada et al. 2000). hippocampal x-ray irradiation, but not irradiation of other brain areas, like the svz or the cerebellar region, prevented the neurogenic effect of antidepressants, like fl uoxetine, in adult mice (santarelli et al. 2003). the behavioral effect of the antidepressants on the novelty-suppressed feeding (nsf) test was also abolished after hippocampal irradiation. the nsf test, in which animals are food deprived, then placed into a novel environment containing food, and assessed for the latency to begin eating, is devised to assess chronic antidepressant effi cacy in rodents (bodnoff et al. 1988). further, 5-ht1a receptor null mice were insensitive to the neurogenic and behavioral effects of fl uoxetine. in all, these data show that ssris, like fl uoxetine, increase hippocampal neurogenesis, which contributes to their behavioral effects (santarelli et al. 2003). a neurogenic theory of depression stress, an environmental factor, is an important causal factor in precipitating episodes of depression in human, and potently suppresses hippocampal neurogenesis in adult monkey (gould et al. 1998; malberg and duman, 2003), probably due to increased glucocorticoid release (gould et al. 1991; cameron and gould, 1994). neurogenesis plays an important role in biology of depression; particularly the stimulation of neurogenesis by antidepressants contributes to their behavioral effects (malberg et al. 2000; santarelli et al. 2003). it is proposed that stress-induced decrease of neurogenesis in the dg is an important causal factor in precipitating episodes of depression. the waning and waxing of neurogenesis in the hippocampal formation are therefore important causal factors, respectively, in the precipitation of, and recovery from, episodes of clinical depression, probably mediated through the increase in brain serotonin levels (jacobs et al. 2000). the mechanism underlying the increased neurogenesis mediated by antidepressants remains to be identifi ed. studies reveal that the 5-ht, particularly 5-ht1a, receptor subtypes mediate the involvement of adult neurogenesis in depression (banasr et al. 2004), and that fl uoxetine targets a population of early progenitor cells in the dg, rather than stem-like cells in the dg (encinas et al. 2006). the effect of antidepressants on neurogenesis may be mediated by trophic factors, like brain-derived neurotrophic factor (bdnf). on the one hand, antidepressant treatments increase the level of expression of bdnf in the patents’ brain, and bdnf has an antidepressant effect (siuciak et al. 1997; chen et al. 2001; saarelainen et al. 2003). on the other hand, administration of bdnf increases adult neurogenesis in the hippocampus (scharfman et al. 2005). this suggests that the effect of antidepressants on neurogenesis may be mediated by bdnf, through its signaling pathway, particularly the mitogen-activated protein (map) kinase pathway (duman et al. 2006). the map kinase pathway is a bdnf signaling cascade mediated drug target insights 2006: 1 15 neurogenesis and depression by the activation of map kinase (mapk) that phosphorylates and activates the extracellular signal-regulated kinase (erk) pathway (huang and reichardt, 2003). a hypothesis supported by recent fi ndings showing that exercise promotes hippocampal neurogenesis, bdnf expression, and has an antidepressant effect (van praag et al. 1999; eadie et al. 2005; russo-neustadt and chen, 2005; bjornebekk et al. 2005; ernst et al. 2006). though these studies provide compelling evidences of the role of bdnf in depression and neurogenesis, it remains to link the activity of bdnf on the increase of neurogenesis mediated by antidepressants. there are however controversies and debates over the involvement of the hippocampus and adult neurogenesis in the etiology of depression. among them, i) a link between neurogenesis, loss of nerve cells, atrophy and decrease of hippocampal volume in depression subjects is yet to be demonstrated, ii) studies show that hippocampal volume remains unchanged in depressive patients (axelson et al. 1991; inagaki et al. 2004; bielau et al. 2005), iii) the hippocampal formation may not be primarily involved in depressive episodes, as other areas of the brain may play a critical role in depression (nestler et al. 2002; ebmeier et al. 2006), iv) there are questions over validity of animal models of depression as representative of the human disorder, and v) neurogenesis may be more a contributing factor of cns plasticity, rather than to specific physiological or pathological processes (taupin, 2006c). the involvement of adult neurogenesis in depression remains therefore speculative (feldmann et al. 2006). in all these data involved the hippocampus, a structure classically involved in leaning and memory, and adult neurogenesis in depression and anxiety disorders (thomas and peterson, 2003). antidepressant treatments may increase neural plasticity and adult neurogenesis, especially in the hippocampus. however, the neurogenic theory of depression remains the source of debates and controversies, and to be further confi rmed (feldmann et al. 2006). more data and evidences are needed to confi rm the involvement of adult neurogenesis in depression. conclusion these studies show that antidepressants increase hippocampal neurogenesis, and establish a causal relation between the stimulation of neurogenesis and the effect of antidepressants. new neuronal cells that survived and integrate the pre-existing network survive for long period, over two years in human (eriksson et al. 1998). therefore, antidepressants may have long-term consequence on the architecture, and functioning of the cns. the function of newly generated neuronal cells in the adult brain remains the source of intense research and debates. though the hippocampus and neurogenesis play an important role in depression, these data remain the source of controversies and debates, and the involvement of adult neurogenesis in the etiology of depression to be further characterized. nonetheless, the evidence that stimulation of neurogenesis contributes to the effects of antidepressants may hold the key for the understanding of the long-term consequences of the effects of antidepressants of the physiopathology of the cns, and lead to new drugs design, and new strategies to treat depressive disorders. to this aim, determining the cellular and molecular mechanisms of action of antidepressants on neurogenesis will be a determining factor. acknowledgments p.t. is supported by grants from the nmrc, bmrc, and the juvenile diabetes research foundation. references axelson, d.a., doraisw amy, p.m., mcdonald, w.m., boyko, o.b., tupler, l.a., patterson, l.j., nemeroff, c.b., ellinwood, e.h. jr. and krishnan, k.r. 1993. hypercortisolemia and hippocampal changes in depression. psychiatry res., 47:163–73. banasr, m., hery, m. and printemps, r., et al. 2004. serotonin-induced increases in adult cell proliferation and neurogenesis are mediated through different and common 5-ht receptor subtypes in the dentate gyrus and the subventricular zone. neuropsychopharmacology, 29:450–60. bbielau, h., trubner, k., krell, d., agelink, m.w., bernstein, h.g., stauch, ielau, h., trubner, k., krell, d., agelink, m.w., bernstein, h.g., stauch, r., mawrin, c., danos, p., gerhard, l., bogerts, b. and baumann, br., mawrin, c., danos, p., gerhard, l., bogerts, b. and baumann, b.. 2005. volume defi cits of subcortical nuclei in mood disorders a postmortem study. eur. arch. psychiatry clin. neurosci., 255:401–12. bjornebekk, a., mathe, a.a. and brene, s. 2005. the antidepressant effect of running is associated with increased hippocampal cell proliferation. int. j. neuropsychopharmacol., 8:357–68. bodnoff, s.r., suranyi-cadotte, b. and aitken, d.h. et al. 1988. the effects of chronic antidepressant treatment in an animal model of anxiety. psychopharmacology (berl.), 95:298–302. brezun, j.m. and daszuta, a. 1999. depletion in serotonin decreases neurogenesis in the dentate gyrus and the subventricular zone of adult rats. neurosci., 89:999–1002. brunello, n., mendlewicz, j. and kasper, s. et al. 2002. the role of noradrenaline and selective noradrenaline reuptake inhibition in depression. eur. neuropsychopharmacol., 12:461–75. cameron, h.a. and gould, e. 1994. adult neurogenesis is regulated by adrenal steroids in the dentate gyrus. neurosci., 61:203–9. drug target insights 2006: 116 philippe taupin campbell, s., marriott, m., nahmias, c. and macqueen, g.m. 2004. lower hippocampal volume in patients suffering from depression: a meta-analysis. am. j. psychiatry., 161:598–607. chen, b., dowlatshahi, d., macqueen, g.m., wang, j.f. and young, l.t. 2001. increased hippocampal bdnf immunoreactivity in subjects treated with antidepressant medication. biol. psychiatry., 50:260–5. colla, m., kronenberg, g., deuschle, m., meichel, k., hagen, t., bohrer, m. and heuser, i. 2006. hippocampal volume reduction and hpasystem activity in major depression. j. psychiatr. res., in press. czeh, b., michaelis, t. and watanabe, t. et al. 2001. stress-induced changes in cerebral metabolites, hippocampal volume, and cell proliferation are prevented by antidepressant treatment with tianeptine. proc. natl. acad. sci. u.s.a., 98:12796–801. duman, c.h., schlesinger, l., kodama, m., russell, d.s. and duman, r.s. 2006. a role for map kinase signaling in behavioral models of depression and antidepressant treatment. biol. psychiatry, in press. eadie, b.d., redila, v.a. and christie, b.r. 2005. voluntary exercise alters the cytoarchitecture of the adult dentate gyrus by increasing cellular proliferation, dendritic complexity, and spine density. j. comp. neurol., 486:39–47. ebmeier, k.p., donaghey, c., and steele, j.d. 2006. recent developments and current controversies in depression. lancet., 367:153–67. encinas, j.m., vaahtokari, a. and enikolopov, g. 2006. fluoxetine targets early progenitor cells in the adult brain. proc. natl. acad. sci. u.s.a., 103:8233–8. eriksson, p.s., perfi lieva, e. and bjork-eriksson, t. et al. 1998. neurogenesis in the adult human hippocampus. nat. med., 4:1313–7. ernst, c., olson, a.k., pinel, j.p., lam, r.w. and christie, b.r. 2006. antidepressant effects of exercise: evidence for an adult-neurogenesis hypothesis? j. psychiatry neurosci., 31:84–92. feldmann, r.e. jr., sawa, a. and seidler, g.h. 2006. causality of stem cell based neurogenesis and depression to be or not to be, is that the question? j. psychiatr. res., in press. gage, f.h. 2000. mammalian neural stem cells. science, 287:1433–8. gage, f.h., coates, p.w. and palmer, t.d. et al. 1995. survival and differentiation of adult neuronal progenitor cells transplanted to the adult brain. proc. natl. acad. sci. u.s.a., 92:11879–83. gould, e., woolley, c.s. and cameron, h.a. et al. 1991. adrenal steroids regulate postnatal development of the rat dentate gyrus: ii. effects of glucocorticoids and mineralocorticoids on cell birth. j. comp. neurol., 313:486–93. gould, e., tanapat, p. and mcewen, b.s. et al. 1998. proliferation of granule cell precursors in the dentate gyrus of adult monkeys is diminished by stress. proc. natl. acad. sci. u.s.a., 95:3168–71. gross, c.g. 2000. neurogenesis in the adult brain: death of a dogma. nat. rev. neurosci., 1:67–73. gross, c., zhuang, x. and stark, k. et al. 2002. serotonin1a receptor acts during development to establish normal anxiety-like behaviour in the adult. nature, 416:396–400. hindmarch, i. 2001. expanding the horizons of depression: beyond the monoamine hypothesis. hum. psychopharmacol., 16:203–18. huang, e.j. and reichardt, l.f. 2003. trk receptors: roles in neuronal signal transduction. annu. rev. biochem., 72:609–42. inagaki, m., matsuoka, y., sugahara, y., nakano, t., akechi, t., fujimori, m., imoto, s., murakami, k. and uchitomi, y. 2004. hippocampal volume and fi rst major depressive episode after cancer diagnosis in breast cancer survivors. am. j. psychiatry, 161:2263–70. jacobs, b.l., praag, h. and gage, f.h. 2000. adult brain neurogenesis and psychiatry: a novel theory of depression. mol. psychiatry, 5:262–9. kessler, r.c., mcgonagle, k.a. and zhao, s. et al. 1994. lifetime and 12-month prevalence of dsm-iii-r psychiatric disorders in the united states. results from the national comorbidity survey. arch. gen. psychiatry, 51:8–19. kuhn, h.g., winkler and j. kempermann, g. et al. 1997. epidermal growth factor and fi broblast growth factor-2 have different effects on neural progenitors in the adult rat brain. j. neurosci., 17:5820–9. malberg, j.e., eisch, a.j. and nestler, e.j. et al. 2000. chronic antidepressant treatment increases neurogenesis in adult rat hippocampus. j. neurosci., 20:9104–10. malberg, j.e. and duman, r.s. 2003. cell proliferation in adult hippocampus is decreased by inescapable stress: reversal by fl uoxetine treatment. neuropsychopharmacology, 28:1562–71. malberg, j.e. 2004. implications of adult hippocampal neurogenesis in antidepressant action. j. psychiatry neurosci., 29:196–205. markakis, e.a. and gage, f.h. 1999. adult-generated neurons in the dentate gyrus send axonal projections to fi eld ca3 and are surrounded by synaptic vesicles. j. comp. neurol., 406:449–60. mcewen, b.s. 2001. plasticity of the hippocampus: adaptation to chronic stress and allostatic load. ann. n.y. acad. sci., 933:265–77. mckay, r. 1997. stem cells in the central nervous system. science, 276:66–71. miller, m.w. and nowakowski, r.s. 1988. use of bromodeoxyuridine-immunohistochemistry to examine the proliferation, migration and time of origin of cells in the central nervous system. brain res., 457:44–52. nestler, e.j., barrot, m., dileone, r.j., eisch, a.j., gold, s.j. and monteggia, l.m. 2002. neurobiology of depression. neuron, 34:13–25. owens, m.j. 2004. selectivity of antidepressants: from the monoamine hypothesis of depression to the ssri revolution and beyond. j. clin. psychiatry, 65 suppl 4:5–10. reynolds, b.a. and weiss, s. 1992. generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. science, 255:1707–10. ryan, n.d. 2005. treatment of depression in children and adolescents. lancet., 366:933–40. russo-neustadt, a.a., chen, m.j. 2005. brain-derived neurotrophic factor and antidepressant activity. curr. pharm. des., 11:1495–510. saarelainen, t., hendolin, p., lucas, g., koponen, e., sairanen, m., macdonald, e., agerman, k., haapasalo, a., nawa, h., aloyz, r., ernfors, p. and castren, e. 2003. activation of the trkb neurotrophin receptor is induced by antidepressant drugs and is required for antidepressantinduced behavioral effects. j. neurosci., 23:349–57. santarelli, l., saxe, m. and gross, c. et al. 2003. requirement of hippocampal neurogenesis for the behavioral effects of antidepressants. science, 301:805–9. sapolsky, r.m.2000. glucocorticoids and hippocampal atrophy in neuropsychiatric disorders. arch. gen. psychiatry, 57:925–35. scharfman, h., goodman, j., macleod, a., phani, s., antonelli, c. and croll, s. 2005. increased neurogenesis and the ectopic granule cells after intrahippocampal bdnf infusion in adult rats. exp. neurol., 192:348–56. sheline, y.i., wang, p.w. and gado, m.h. et al. 1996. hippocampal atrophy in recurrent major depression. proc. natl. acad. sci., u.s.a., 93:3908–13. siuciak, j.a., lewis, d.r., wiegand, s.j. and lindsay, r.m. 1997. antidepressant-like effect of brain-derived neurotrophic factor (bdnf). pharmacol. biochem. behav., 56:131–7. stanfi eld, b.b. and trice, j.e. 1988. evidence that granule cells generated in the dentate gyrus of adult rats extend axonal projections. exp. brain. res., 72:399–406. tada, e., parent, j.m. and lowenstein, d.h. et al. 2000. x-irradiation causes a prolonged reduction in cell proliferation in the dentate gyrus of adult rats. neurosci., 99:33–41. taupin, p. and gage, f.h. 2002. adult neurogenesis and neural stem cells of the central nervous system in mammals. j. neurosci. res., 69:745–9. taupin, p. 2006a. neurogenesis in the adult central nervous system. c.r. biol., 329:465–75. taupin, p. 2006b. brdu immunohistochemistry for studying adult neurogenesis: paradigms, pitfalls, limitations, and validation. brain res. rev., in press. taupin, p. 2006c. adult neurogenesis and neuroplasticity. restor. neurol. neurosci., 24:9–15. thomas, r.m. and peterson, d.a. 2003. a neurogenic theory of depression gains momentum. mol. interv., 3:441–4. drug target insights 2006: 1 17 neurogenesis and depression van praag, h., kempermann, g. and gage, f.h. 1999. running increases cell proliferation and neurogenesis in the adult mouse dentate gyrus. nat. neurosci., 2:266–70. van praag, h., schinder, a.f. and christie, b.r. et al. 2002. functional neurogenesis in the adult hippocampus. nature, 415:1030–4. videbech, p. and ravnkilde, b.2004. hippocampal volume and depression: a meta-analysis of mri studies. am. j. psychiatry, 161:1957–66. watanabe, y., gould, e. and mcewen, b.s. 1992a. stress induces atrophy of apical dendrites of hippocampal ca3 pyramidal neurons. brain res., 588:341–5. watanabe, y., gould, e. and daniels, d.c. et al. 1992b. tianeptine attenuates stress-induced morphological changes in the hippocampus. eur. j. pharmacol., 222:157–62. whittington, c.j., kendall, t. and fonagy, p. et al. 2004. selective serotonin reuptake inhibitors in childhood depression: systematic review of published versus unpublished data. lancet., 363:1341–5. wong, m.l. and licinio, j. 2001. research and treatment approaches to depression. nat. rev. neurosci., 2:343–51. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile 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rapid communication correspondence: yasunori okubo, department of oral and maxillofacial surgery,graduate school of medicine, kyoto university, 54 kawahara-cho, shogoin, sakyo-ku, kyoto, 606-8507, japan. tel: +81-75-751-3405; fax: +81-75-761-9732; email: okubo@kuhp.kyoto-u.ac.jp please note that this article may not be used for commercial purposes. for further information please refer to the copyright statement at http://www.la-press.com/copyright.htm accelerators of osteogenesis by recombinant human bone morphogenetic protein-2 yasunori okubo1, kenji kusumoto2 and kazuhisa bessho1 1department of oral and maxillofacial surgery, graduate school of medicine, kyoto university, 54 kawahara-cho, shogoin, sakyo-ku, kyoto, 606-8507, japan. 2department of plastic and reconstructive surgery, kansai medical university, 10-15 fumizono-cho, moriguchi, osaka, 570-8507, japan. abstract: bone morphogenetic protein (bmp) appears to be one of the most promising cytokine and for clinical use in reconstructive surgery for bony defects and augmentation. to evaluate the effect of basic fi broblast growth factor (bfgf), fk506, elcatonin, and hyperbaric oxygenation (hbo) on osteoinduction by recombinant human bone morphogenetic protein-2 (rhbmp-2), 2 or 5 µg of rhbmp-2 was implanted into intramuscular sites of rats. at 21 days after implantation, the osteoinductive activity in the treatment group and control group was compared radiographically, biochemically, and histologically. the amount of new bone in the treatment group was signifi cantly greater than that in the control group. the alkaline phosphatase activity and calcium content in the treatment group were signifi cantly higher than those in the control group. these results suggest that bfgf, fk506, elcatonin, and hbo accelerated the activity and rate of osteoinduction by rhbmp2. these results may be useful when bmp is applied clinically in near future. keywords: fk506, basic fi broblast growth factor (bfgf), elcatonin, hyperbaric oxygenation (hbo), recombinant human bone morphogenetic protein. introduction bone morphogenetic protein (bmp) appears to be one of the most promising biomaterial and for clinical use in reconstructive surgery for bony defects and augmentation. therefore, bmp is noted in the fi eld of bone reconstructive surgery. since recombinant human bone morphogenetic protein-2 (rhbmp-2) has become available, many animal studies on osteoinduction by rhbmp-2 have been performed (fujimura et al.1995; okubo et al. 1999; okubo et al. 2000). however for clinical application of rhbmp-2 to tissue with low blood supply tissue, e.g. scarred tissue or irradiated tissue, it is necessary to evaluate the factors that enhance osteoinduction by rhbmp-2. in the present study, the basic mechanism of osteoinduction by rhbmp-2 and preclinical studies are discussed and reviewed mainly referring to our previous research regarding accelerators of osteogenesis and related studies. effect of basic fibroblast growth factor (bfgf) fgf has various effects on cellular proliferation and it has a strong proliferative affected on endothelial cells, osteocytes and chondrocytes (connolly et al. 1987; gospodarowicz et al. 1987; globus et al. 1988). in addition, fgf and transforming growth factor ß (tgfß) are co-active on proliferating chondrocytes and osteoblasts (iwamoto et al. 1989; inoue et al. 1989; nakamura et al. 1995). we evaluated the effect of fgf on the osteoinductive activity by rhbmp-2. bmp-2 (genetics institute, ma) was provided by yamanouchi pharmaceutical co. ltd. (tokyo, japan). it was dissolved in a buffer (ph 4.5) containing 5 mm glutamic acid, 2.5% glycine, 0.5% sucrose and 0.01% tween 80, and stored at −80°c. fgf-was provided by kaken pharmaceutical co. ltd. (tokyo, japan). type i collagen solution (3 mg/ml, ph 3.0) (cellmatrix la®; nitta gelatin inc., osaka, japan) was used as the carrier for bmp-2 and fgf-2. this collagen was purifi ed from fresh porcine skin, and the telopeptide was removed by proteolytic digestion. bmp-2 (2 µg) and 0, 16, 80 and 400 ng, and 2, 10 and 50 µg of fgf-2 (n = 10 drug target insights 2007: 2 55–60 56 okubo et al in each group) were mixed with 3 mg of type i collagen. the mixtures were lyophilized (0.04 torr) (eyela® type fdu-830; tokyo rika inc., tokyo, japan) and shaped into discs (4 mm diameter; 1.5 mm thickness). seventy male 10-week-old wistar rats weighing 240–260 g were used. they were divided into seven groups. all the rats were anaesthetized with an intraperitoneal injection of sodium pentobarbital (4.0 mg solidus 100 g body weight). after disinfecting the operative region and incising the skin, a disc containing bmp-2 (2 µg), bfgf (0, 16, 80 and 400 ng, and 2, 10 and 50 µg in each) and type i collagen (3 mg) were implanted into the right calf muscles of the rats. these seven groups (n = 10 in each group) consisted of group 1 (control), bmp-2 + fgf (0 ng) + type i collagen; group 2, bmp-2 + fgf (16 ng) + type i collagen; group 3, bmp-2 + fgf (80 ng) + type i collagen; group 4, bmp-2 + fgf (400 ng) + type i collagen; group 5, bmp-2 + fgf (2 µg) + type i collagen; group 6, bmp-2 + fgf (10 µg) + type i collagen; and group 7, bmp-2 + fgf (50 µg) + type i collagen. they were fed rodent chow (certifi ed diet mf; oriental koubo inc., tokyo, japan) for the period of the study. three weeks after the operation, all the animals were killed with an intraperitoneal injection of excess sodium pentobarbital. the specimens with peripheral tissues were fi xed in 10% formalin neutral buffer solution (ph 7.4), demineralized in edta, and embedded in paraffi n. they were cut into 4 µm-thick sections and stained with hematoxylin and eosin. the samples for quantitative analysis were weighed and then homogenized in 0.25 m sucrose in a polytron homogenizer (bio-mixer; type abm, nissei inc., osaka, japan). the sediment was demineralized in 0.5 n hcl, and the calcium (ca) content of the soluble fraction was determined by the orthocresolphthalein complexone method. the alkaline phosphatase (alp) activity and total protein in the resultant supernatant were determined by the 4-nitrophenylphosphate method. the ca content (µg/mg of tissue) and the alp activity (iu/mg of protein) were used as indices of bone formation. the treatment of each animal was conducted according to the 1988 guidelines for animal experiments at kyoto university. three weeks after implantation, alp was increased in the 16, 80 and 400 ng fgf-2-treated groups but decreased in the 50 µg fgf-2-treated group. histological examination revealed increased bone formation in the 16, 80 and 400 ng fgf-2-treated groups (table 1). these results show that combined treatment with fgf-2 and bmp-2 has a biphasic effect on osteoinductive activity, i.e. it increases with low doses of fgf-2 and decreases with high doses of fgf-2 (fujimura et al. 2002). effect of fk506 fk506 has generally been used as an immunosuppressant for organ transplantation. we evaluated the effect of fk506 on osteoinduction by rhbmp-2. one hundred and twenty male wistar rats (10 weeks old and weighting 230–250 g) were randomly divided into the following four groups of 30 rats each: 1) the short-term fk506 group (sfg) received a daily intramuscular (i.m.) injection of 0.1 ml of fk506 (1 mg/kg) from 2 days before the implantation of lyophilized specimens until the day of implantation. then the animals received a daily injection of 0.1 ml of saline i.m. from the day of implantation until sacrifi ce. 2) the medium-term fk506 group (mfg) received a table 1. dose of fgf histological fi ndings alp activity radiological fi ndings percentage of bone area (%) iu/mg protein rediopacity area (mm2) 0 15.2 (0.15) 0.88 (0.27) 2.8 (0.6) 16 ng 30.4 (4.6)* 6.1 (2.6)* 5.4 (1.6)* 80 ng 28.2 (4.3)* 4.0 (1.1)* 7.7 (1.8)* 400 ng 32.5 (9.0)* 17.1 (4.3)* 9.0 (2.3)* 2 µg 19.6 (5.9) 0.69 (0.43) 3.5 (1.9) 10 µg 7.6 (6.5) 0.62 (0.34) 1.7 (0.8) 50 µg 0 0.17 (0.1) 0 data are means (sd). *signifi cant difference at p < 0.05, compared with fgf on g group. drug target insights 2007: 2 57 accelerators of osteogenesis by rhbmp-2 the sfg than in the other groups. twenty-one days after implantation, the trabecular bone area was increased in the cg, but not in the mfg. in the sfg and lfg, it was decreased at the border of the implant, and fatty marrow occupied most of the marrow tissue. twenty-one days after implantation, the alp activity was 6.37 ± 0.37 in the cg, 3.34 ± 0.19 in the sfg, 5.40 ± 0.46 in the mfg, and 1.30 ± 0.24 in the lfg. the values in the sfg, and lfg were signifi cantly lower than in the cg and mfg. twenty-one days after implantation, the ca content was 33.81 ± 3.44 in the cg, 18.43 ± 1.94 in the sfg, 24.11 ± 2.61 in the mfg, and 15.24 ± 1.96 in the lfg. values in the sfg and lfg were signifi cantly lower than in the cg (table 2). these fi ndings demonstrate that short-term administration of fk506 promotes early osteoinduction. however, long-term administration accelerates both bone formation and bone resorption, and insuffi cient oxygen supply leads to failure of bone matrix maturation, resulting in poor bone formation (kaihara et al. 2002). effect of elcatonin elcatonin is a derivative of eel calcitonin synthesized by substituting an ethylene bond for the disulfi de bond (morikawa et al. 1976; otani et al. 1978; orimo et al. 1990). it has also been reported from in vivo and in vitro studies that elcatonin suppresses bone resorption (orimo et al. 1990; yamamoto i et al. 1981). in the clinical fi eld, elcatonin is used currently for the treatment of paget’s disease, and osteoporosis. however, the role of this hormone in producing an anabolic effect on osteoblasts is not yet fully understood. we evaluated the effect of elcatonin on osteoinduction by rhbmp-2, especially the anabolic effect on osteoblasts. daily injection of 0.1 ml of fk506 (1 mg/kg i.m.) from 2 days before implantation until 7 days after implantation. then a daily injection of 0.1 ml of saline i.m. was given for the next 7 days until sacrifi ce. 3) the long-term fk506 group (lfg) received a daily injection of 0.1 ml of fk506 (1 mg/kg i.m.) from 2 days before implantation until sacrifi ce. 4) the control group (cg) received a daily injection of 0.1 ml of saline i.m. from 2 days before implantation until sacrifi ce. rhbmp-2 was obtained from w. sebald (würzburg university, germany, ruppert et al. 1996). atelopeptide type-i collagen (cl) (ph 3.0 was used as the carrier). rhbmp-2 (5 µg) was mixed with 3 mg of cl and was lyophilized (eyela fdu-830; tokyo rikakikai inc., tokyo, japan). then the material was compressed in a syringe to form discs (4 mm in diameter and 1.5 mm thick). rats were anesthetized with intraperitoneal sodium pentobarbital (5.0 mg per 100 g of body weight) and lyophilized disc specimens were implanted into the right calf muscle. after implantation, the fascia and skin were sutured. fk506 (tacrolimus; fujisawa pharmaceutical co., ltd., osaka, japan) was suspended in saline and injected into the left calf muscle of each rat (1 mg/kg/day). this dose has already been given intramuscularly in rat organ transplantation models (akahane et al. 1999). in the radiographic fi ndings, the area of the shadows at 21days after implantation was in the order of cg > mfg > sfg > lfg. in the histological fi ndings, fourteen days after implantation, new bone surrounded by immature mesenchymaltype cells was present at border of the implant around almost the entire circumference in every group. however, cartilage was still observed in the lfg. the immature new bone area was larger in table 2. fk506 ca content alp activity µg/mg tissue iu/mg protein day 7 day 14 day 21 day 7 day 14 day 21 sfg 0.15 (0) 33.5 (1.1)* 18.4 (1.9)* 2.4 (0.4)* 1.9 (0.2) 3.3 (0.2)* mfg 0.14 (0) 24.4 (3.1) 24.1 (2.6) 2.3 (0.2)* 2.4 (0.3) 5.4 (0.3) lfg 0.13 (0) 13.3 (1.0)* 15.4 (1.9)* 2.4 (0.3)* 3.1 (0.3) 1.3 (0.4)* cg 0.1 (0) 23.4 (2.9) 33.8 (3.4) 1.8 (0.4) 2.7 (0.4) 6.3 (0.4) data are means (sd). *signifi cant difference at p < 0.05, compared with cg. drug target insights 2007: 2 58 okubo et al twenty wistar rats (male; 10 weeks old; weight 240–260 g) were used. four groups, consisting of a high elcatonin group (heg), medium elcatonin group (meg), low elcatonin group (leg) and control group (cg), were established with 5 rats in each group. rhbmp-2 derived from e. coli was obtained from w. sebald (würzburg university, germany, rupport et al. 1996). cl (ph 3.0) was used as a carrier. five µg of rhbmp-2 mixed with 3 mg of cl was lyophilized (eyela fdu-830; tokyo rikakikai inc., tokyo, japan). the material was compressed in the injection syringe to discal form (4 mm in diameter, 1.5 mm in thickness). as the pharmacological agent, 14-day doses of elcatonin (elcitonin®; asahi chemical industry co., ltd., tokyo, japan) were prepared, 80 u for heg, 8 u for meg, and 0.8 u for leg. the total volume of the elcatonin agent in physiological saline solution was 0.2 ml for each rhbmp-2 implanted group. the elcatonin solution was placed into a mini-osmotic pump (alzet® model 2002; alza co., ca), that would pump out the solution continuously at a constant rate of 0.5 µl/hour for 14 days. for cg, only 0.2 ml of physiological saline was placed into the mini-osmotic pump. all rats were anesthetized with intraperitoneal administration of sodium pentobarbital. the lyophilized discal specimens were implanted into a right calf muscle. after the implantation, the fascia and skin were sutured. a one-cm-long incision was made in the paramedian abdominal wall, including the skin, muscle, and the peritoneum and the mini-osmotic pump, which had been previously prepared for each group, was inserted into the peritoneal space. the abdominal wall was then closed by suturing layer by layer. twenty-one days after the implantation, all rats were sacrifi ced with an overdose of sodium pentobarbital. the implanted region was excised with the surrounding tissue and a radiograph was taken. each excised specimen was removed and then cut into 2 halves, one for histological analysis and the other for biochemical analysis. the soft tissue radiographs revealed opaque shadows morphologically identical to the implanted specimens. these opaque shadows were observed in each of the specimens of all groups. in heg, there was a relatively vigorous trabecular bone on the outermost edge of the implanted material. lining osteoblasts were observed around the trabecular bone. bone marrow, including angioid tissue, was rich at the central side of the trabecular bone. fatty marrow occupied a major part of the marrow tissue. in meg, there was trabecular bone on the outermost edge of the implanted material. the trabecular bone was thinner than that in heg. bone marrow included fatty tissue. collagen fi bers remained at the center of the implanted material. in leg, less trabecular bone and marrow were observed compared to the respective amounts in meg and heg. at the central side of the newly formed trabeculae, a small area of bone marrow was observed. in cg, especially, the amount of trabecular bone was clearly less than in the other groups and few osteoblasts were observed. the values of alp activity on day 21 were 5.87 ± 0.43 (mean ± sd iu/mg protein) in cg, 6.41 ± 0.37 in leg, 7.10 ± 0.37 in meg, and 7.37 ± 0.50 in heg (table 3). the value was highest in heg and lowest in cg. the values of ca content on day 21 were 25.0 ± 1.61 (mean ± sd µg/mg tissue) in cg, 26.6 ± 0.96 in leg, 29.0 ± 0.60 in meg, and 31.3 ± 1.56 in heg. the alp activity and ca content in heg were highest and lowest in cg. in heg and meg, the values of alp activity and ca content were signifi cantly lower than in cg and leg (p < 0.01). these results suggested that elcatonin is effective in enhancing osteoinduction by rhbmp-2, and that elcatonin has an anabolic effect on osteoblasts in addition to an anti-resorptive effect (okubo et al. 2000). effect of hyperbaric oxygenation (hbo) hyperbaric oxygen (hbo) therapy is an oxygenation method use to treat anoxia by increasing dissolved oxygen. hbo therapy has been shown to increase collagen synthesis, capillary ingrowth (hunt et al. 1972), neovascularization, and osteogenesis (nilson et al. 1988). recently, the use of table 3. elcatonin ca content alp activity µg/mg tissue iu/mg protein leg 26.6 (1.0) 6.4 (0.4) meg 29.0 (0.6)* 7.1 (0.4)* heg 31.3 (1.6)* 7.3 (0.5)* cg 25.0 (1.6) 5.9 (0.4) data are means (sd). *signifi cant difference at p < 0.01, compared with cg. drug target insights 2007: 2 59 accelerators of osteogenesis by rhbmp-2 hbo therapy to improve the rate of bone healing in conjunction with surgery for dental implant, osteomyelitis and osteonecrosis has increased. we compared osteoinduction by rhbmp-2 with and without hbo therapy. thirty wistar rats were randomly assigned to an hbo group and a control group of 15 rats each. cl was used as a carrier. five µg of rhbmp-2 mixed with 3 mg of cl was lyophilized (eyela fdu-830; tokyo rikakikai inc., tokyo, japan). the material was compressed in an injection syringe to discal form (4 mm in diameter, 1.5 mm in thickness). all rats were anaesthetized by intraperitoneal administration of sodium pentobarbital (5.0 mg per 100 g of body weight). following disinfection of the operative region, the lyophilized discal specimens were implanted into a right calf muscle pouch. the fascia and skin were sutured. the rats in the hbo group were placed in a pressure chamber (kho-100; kawasaki engineering inc., hyogo, japan) and exposed to a pressure of 2.0 ata 100% inspired fl ow oxygen for 60 minutes everyday for 3, 7, and 21 days. during the fi rst 15 minutes of hbo therapy, the pressure was increased to 2.0 ata, and decompression proceeded for 15 minutes after the treatment. three, 7, and 21 days after the implantation, the rats were sacrifi ced by an overdose of sodium pentobarbital. then the implanted region was excised together with the surrounding tissue and soft x-rayed. each excised specimen was removed and cut into 2 halves, one for histological analysis and the other for biochemical analysis. on day 21, soft x-ray revealed opaque shadows morphologically identical to the implanted specimens in both groups. the oval shadows in the hbo group were larger with slighter high radio-opacity than those in the control group. on day 7 after the implantation, in the hbo group, cartilage tissue was induced at the outer edge of implanted material. in the control group, no cartilage or chondrocytes were detected in these fi ndings. on day 21 after the implantation, new bone formation was found in both groups. around the trabecular bone, lining osteoblasts and a few osteoclasts were observed in both groups. in the control group, trabecular bone tissue was observed at the outer edge of the implanted material. in the hbo group, the trabecular area was greater than that in the control group. the bone marrow area in the hbo group, including fatty marrow in part, was wider than that in the control group. the trabecular area bone in the hbo group was wider than that in the control group. the results of the micrograph analysis of the trabecular area and the percentage of the trabeculum occupying the overall area are summarized in table 4. the alp activity and ca content of the hbo group and the control group are shown in table 3. the alp activity and the ca content in the hbo group were signifi cantly higher than those in the control group on days 7 and 21. the present results suggest that hbo therapy accelerates the activity and rate of osteoinduction by rhbmp-2, since hyperbaric oxygenation may enhance the effects of rhbmp-2 on the differentiation from immature mesenchymal cells to osteoblasts (okubo et al. 2001). conclusions in skeletal reconstruction using bmps, lower amount of bmps had better induce more bony tissue. therefore, some materials have been studied in vivo for the promotion of osteoinduction. to date, fgf, fk506, elcatonin, hbo, and prostaglandin e1 and other materials have been studied as the accelerators in our group. these results may be useful when bmp is applied clinically in near future. acknowledgements this work was supported in part by grant-in-aid for young scientists b(no. 17791453) the japanese ministry of education, science, sports and culture. table 4. histological fi ndings ca content alp activity percentage of bone area (%) µg/mg tissue iu/mg protein hbo group 30.1 (2.2)* 41.0 (3.6)* 7.5 (0.8)* control group 16.9 (1.2) 24.0 (2.9) 3.8 (1.2) data are means (sd). *signifi cant difference at p < 0.05, compared with fgf on g group. drug target insights 2007: 2 60 okubo et al references akahane, m., ohgushi, h. and yoshikawa, t. et al. 1999. osteogeneic phenotype expression of allogeneic rat marrow cells in porous hydroxyapatite ceramics. j. bone miner. res., 14:561–8. connetry, h.v. and briggs, a.r. 1966. determination of serum calcium by means of orthocresolphthalein complexone. am. j. clin. pathol., 45:290–6. fujimura, k., bessho, k. and kusumoto, k. et al. 1995. experimental studies on bone inducing activity of composites of atelopeptide type i collagen as a carrier for ectopic osteoinduction by rhbmp-2. biochem. biophys. res. commun., 208:316–22. fujimura, k., bessho, k. and okubo, y. et al. 2002. the effect of fi broblast growth factor-2 on the osteoinductive activity of recombinant human bone morphogenetic protein-2 in rat muscle. arch. oral biol., 47:577–84. globus, r.k., patterson-buckendahl, p. and gospodarowicz, d. 1988. regulation of bovine bone cell proliferation by fi broblast growth factor and transforming growth factor β. endocrinology, 123:98–105. gospodarowicz, d., ferrara, n. and schweigerer, l. et al. 1987. structural characterization and biological functions of fi broblast growth factor. endocr. rev., 8:95–114. hunt, t.k. and pai, m.p. 1972. the effect of varying ambient oxygen tensions on the wound metabolism and collagen synthesis. surg. gynecol. obstet., 135:561–3. inoue, h., kato, y. and iwamoto, m. et al. 1989. stimulation of cartilagematrix proteoglycan synthesis by morphologically transformed chondrocytes grown in the presence of fi broblast growth factor and transforming growth factor-beta. j. cell. physiol., 138:329–37. iwamoto, m., sato, k. and nakashima, k. et al. 1989. regulation of colony formation differentiated chondrocytes in soft agar by transforming growth factor-beta. biochem. biophys. res. commun., 159:1006–11. kaihara, s., bessho, k. and okubo, y. et al. 2002. effect of fk506 on osteoinduction by recombinant human bone morphogenetic protein-2. life. sci., 72:247–56. morikawa, t., munekata, e. and sakakibara, s. et al. 1976. synthesis of eel-calcitonin and (asu1, 7)-eel-calcitonin: contribution of the disulfi de bond to the hormonal activity. experientia., 32:1104–1106. nilsson, p., albrektson, t. and granström, g. et al. 1988. the effect of hyperbaric oxygen treatment on bone generation: an experimental study using the bone harvest chamber in the rabbit. int. j. oral maxillofac. implants, 3:43–48. nakamura, t., hanada, k. and tamura, t. et al. 1995. stimulation of endosteal bone formation by systemic injections of recombinant basic fi broblast growth factor in rats. endocrinology, 136:1276–84. okubo, y., bessho, k. and fujimura, k. et al. 1999. comparative study of intramuscular and intraskeletal osteogenesis by recombinant human bone morphogenetic protein-2. oral surg. oral med. oral pathol. oral radiol. endod., 87:34–8. okubo, y., bessho, k. and fujimura, k. et al. 2000. osteoriduction by recombinant human bone morphogenetic protein-2 at intramuscular, intermusucular, subcutaneous and intrafatty sites. int. j. oral maxillofac. surg., 29:62–6 okubo, y., bessho, k. and fujimura, k. et al. 2000. effect of elcatonin on osteoinduction by recombinant human bone morphogenetic protein-2. biochem. biophys. res. commun., 269:317–21. okubo, y., bessho, k. and fujimura, k. 2001. effect of hyperbaric oxygenation on bone induced by recombinant human bone morphogenetic protein-2. br. j. oral maxillofac. surg., 39:91–5. orimo, h. 1990. the development of novel therapy for bone disease, the discovery of elcatonin. in christiansen, c. and overgard, k., eds. osteoporosis 1st ed. copenhagen: publisher. p. 5–9. otani, m., kitazawa, s. and yamaguchi, h. et al. 1978. stability and the biological activity of eel calcitonin in rats. horm. metab. res., 10:252–6. ruppert, r., hoffmann, e. and sebald, w. 1996. human bone morphogenetic protein-2 contains a heparin-binding site which modifi es its biological activity. eur. j. biochem., 237:295–302. yamamoto, i., morita, r. and fukunaga, m. et al. 1981. adenylate cyclase activation and 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target insights 2008:3 13–25 13 original research correspondence: robert p. doyle, department of chemistry, syracuse university, syracuse, ny 13244-4100, u.s.a. tel: +1 315 443 3584; email: rpdoyle@syr.edu copyright in this article, its metadata, and any supplementary data is held by its author or authors. it is published under the creative commons attribution by licence. for further information go to: http://creativecommons.org/licenses/by/3.0/. targeting gallium to cancer cells through the folate receptor nerissa viola-villegas, anthony vortherms and robert p. doyle department of chemistry, syracuse university, syracuse, ny 13244-4100, u.s.a. abstract: the development of gallium(iii) compounds as anti-cancer agents for both treatment and diagnosis is a rapidly developing fi eld of research. problems remain in exploring the full potential of gallium(iii) as a safe and successful therapeutic agent or as an imaging agent. one of the major issues is that gallium(iii) compounds have little tropism for cancer cells. we have combined the targeting properties of folic acid (fa) with long chain liquid polymer poly(ethylene glycol) (peg) ‘spacers’. this fa-peg unit has been coupled to the gallium coordination complex of 1,4,7,10-tetraazacyclo-dodecane-n,n′,n′′,n′′′-tetraacetic acid (dota) through amide linkages for delivery into target cells overexpressing the folate receptor (fr). in vitro cytotoxicity assays were conducted against a multi-drug resistant ovarian cell line (a2780/ad) that overexpresses the fr and contrasted against a fr free chinese hamster ovary (cho) cell line. results are rationalized taking into account stability studies conducted in rpmi 1640 media and hepes buffer at ph 7.4. keywords: folate receptor, gallium, dota, targeting, cytotoxicity introduction the anti-cancer properties of gallium(iii) have been extensively investigated since 1971 (hart et al. 1971). gallium has numerous ways to induce cell death, including dna binding and modifi cation (hedley et al. 1988), enzyme inhibition (especially ribonucleotide reductase) (chitambar et al. 1988), and ion transport disruption (such as calcium effl ux from mitochondria), a known trigger of cellular apoptosis (collery et al. 1996). in general, the poor pharmacokinetic properties of gallium salts investigated have prevented their widespread use in chemotherapy. efforts to develop gallium complexes to improve its profi le, by addressing the problems of hydrolysis, poor absorption, poor solubility, rapid renal excretion and little tropism for cancer cells are currently underway (keppler and jakupec, 2004; desoize, 2004). complexes such as those produced by the groups of keppler (keppler et al. 2006), sharma (sharma et al. 2007), low and green (low and green et al. 1996) have been successful in increasing plasma concentrations of gallium, providing better antiproliferative effects or improved imaging of cancer cells (when using gallium radioactive isotopes (67ga γ, 68ga β+)) (greenwood and earnshaw, 2005). problems still remain however especially in regards to renal retention times and cancer cell targeting. folic acid (fa) (see fig. 1) is a vitamin potentially capable of delivering agents specifi cally to folic acid-receptor (fr) positive tumors (lee and sudimack, 2000). frs are membrane glyco-proteins (anderson et al. 1990) overexpressed by a number of tumor cell types such as ovarian, breast, cervical, colorectal, renal and nasopharyngeal cancers (antony, 1996). cells overexpressing the fr bind fa-drug conjugates tightly (kd ∼ 0.42 × 10 −9 m) (shen et al. 1995) and endocytose them inside (anderson et al. 1988), provided that chemical modifi cation of the fa upon conjugation does not disrupt recognition by the fr (liu et al. 2005). targeting the fr is attractive because in addition to being overexpressed in tumor lines, it is downregulated (and inaccessible to blood circulation) in healthy adult cells (anderson et al. 1988). we are primarily focused on ovarian tumors since they have been shown to greatly overexpress the fr (see table 1). current treatments for ovarian cancer have a number of serious side effects associated with their use including kidney damage, hearing loss and even secondary cancers (sun et al. 2002). in addition, over 75% of patients are diagnosed when the disease has already progressed to stage iii or iv, with only a 10%–20% 5 year survival rates, respectively (ries, 1993). new ways to diagnose and/or treat this illness are therefore urgently needed. http://creativecommons.org/licenses/by/3.0/ http://creativecommons.org/licenses/by/3.0/ 14 viola-villegas et al drug target insights 2008:3 we set out to synthesize, purify and evaluate in vitro a new fa bioconjugate of gallium and compare its activity to the unconjugated gallium analog. we began by initially complexing gallium (iii) to the 1,4,7,10-tetraazacyclo-dodecanen,n′,n′′,n′′′-tetraacetic acid (dota) ligand (doyle et al. 2006). we have previously described the synthesis and solid state structure of this system (see fig. 2). preceding literature reports have demonstrated good kinetic and thermodynamic stability provided by the dota ligand in its coordination chemistry and we wished to exploit this coupled with folate receptor (fr) targeting conjugates. in addition to coupling the gallium-dota complex to folic acid we wished to include poly(ethylene glycol) (peg) polymer linkers between the fa and gallium complex since ‘pegylated’ fa conjugates have been shown to have greater affi nity for the fr than free fa (low and lee, 1994). pathways that can break down or effl ux certain fa conjugates are inhibited by the polymer-fa conjugate and renal retention times of certain pharmaceuticals have been improved by conjugation to peg units (anderson et al. 2005). hence, the use of fa-peg conjugates yields synergistic traits that are of particular interest. conjugation of peg to fa through the glutamate moiety produces two regioisomer products at the αand γ-carboxylic acid functional groups that need to be separated. fa modifi ed at the α-carboxylic acid loses its affi nity for the fr, making it unsuitable as a targeting agent (yan and ratnam, 1995). this separation is diffi cult when using polydisperse peg units. such peg units are typically all that are commercially available but are approved for use by the fda (qui and bae, 2006). a facile route to separation was previously reported by us and this route was used here to allow access to pure γ-fa-peg-nh2 for subsequent conjugation to the gallium-(hdota) complex (doyle et al. 2008). in vitro cytotoxicity assays were conducted against adriamycin resistant ovarian cancer cell line (a2780/ad), which overexpresses the fr, and contrasted against a non-fr expressing chinese hamster ovary (cho) control line. experimental chemicals the following reagents were purchased and used without further purifi cation: folic acid (fa) (98%, sigma), n,n’-dicyclohexylcarbodiimide (dcc, �99%, fluka), polyethylene glycol bis(amine) (peg, mw: 2000) (fluka), n-hydroxysuccinimide (nhs, �97%, fluka), and 1,4,7,10-tetraazacyclododecane-n,n’,n’’,n’’’-tetraacetic acid (dota, 98%, strem chemicals), n-hydroxysulfosuccinimide sodium salt (sulfo-nhs, 98.5%, fluka), 1(3-dimethylaminopropyl)-3-ethylcarbodiimide (edc, 98%, alfa aesar) and trifl uoroacetic acid (99%, aldrich). gacl3 (99.9%) was purchased from alfa aesar and dissolved in 100 mm ammonium acetate (ph 4.8) to make a stock concentration of 1.325 m. dimethylsulfoxide (dmso) (min. 99.9%, sigma) was dried by running the solvent through a column of 4 å molecular sieves (mallincrodt) dried previously overnight at 120 °c. solvents used for hplc and growth media are fi ltered with 0.45 µm fi lter (fisher). pyridine (99.9%) was obtained from fisher. triethylamine (99.5%) was purchased from sigma aldrich. 3′-azido-3′deoxythymidine (azt; used as internal control in cytotoxicity assays) was purchased from sigma aldrich. all other reagents and buffers used were of reagent grade or higher. ultra pure water (18.6 mω) was used through out the investigation. all syntheses except for 1 were performed in a figure 1. fa with its three major structural components including the αand γ-carboxylic acid group of the glutamate moiety indicated. table 1. comparison of fr overexpression investigated in different cancer tissues via immunohistochemistry (ihc) and reverse transcriptase—polymerase chain techniques (rt-pcr) (low and leamon, 2005). tissue ihc (%) rt-pcr (%) ovarian 93 100 endometrial 91 100 breast 21 80 lung 33 33 colorectal 22 20 kidney 50 100 15 targeting gallium to cancer cells through the folate receptor drug target insights 2008:3 dark-room under a 15 w red light. all reactions were conducted under nitrogen gas at ambient conditions unless otherwise stated with sample transfer conducted by cannula (24 inch, 16 gauge). physical measurements and instrumentation an agilent 1100 reverse phase high pressure liquid chromatography (hplc) with manual injection and automated fraction collector was fi tted with a zorbax c18 analytical column (42 × 10 mm) for analytical trace analysis with a flow rate of 0.7 ml/min. purifi cation was achieved using a c18 (9.4 × 250 mm) semi-preparative column using a fl ow rate of 2 ml/min. detection was by ultra violet monitoring at 280 nm. the linear gradient used was: (1) 90% 5 mm na2hpo4 (ph 7.0) and 10% acetonitrile over 10 minutes; (2) 40% 5 mm na2hpo4 and 60% acetonitrile over 20 minutes. ion exchange chromatography (iec) was conducted on an akta prime plus with primeview 5.0 software. the anx (1 ml) and the pd10 sephadex g-25m desalting (10 ml) columns were purchased from ge healthsciences. 1h nuclear magnetic resonance (1h nmr) was performed on bruker avance dpx 500 mhz. a shimadzu lcms-2010 a mass spectrometer and an applied biosystems voyager-de linear matrix assisted laser desorption ionization—time of flight mass spectrometer (maldi-tof) were used for mass spectrometry analysis. infrared (ir) analyses were performed as kbr pellets on a nicolet magna-ir 850 series ii spectrophotometer. a perkin elmer elan 6100 was used to conduct inductively coupled plasma analysis (icp). centrifugation was performed using a sorvall legend rt centrifuge typically as 10 minute runs at 4000 rpm at 4 ºc. optical densities were measured with a thermo multiskan ex 96-well plate reader equipped with ascent software version 2.6 with 450 nm fi lter. chemical synthesis synthesis of gahdota (1) 1 was synthesized as reported previously by doyle et al. (doyle et al. 2006). synthesis of γ-fa-peg-nh2 (γ-2) fa (0.0441 g, 0.100 mmol) was dissolved in 3 ml of dry dmso. to this solution, 0.0127 g of nhs (0.110 mmol) was added. the mixture was stirred for 5 minutes after which 0.023 g (0.110 mmol) of dcc was added. the solution was then stirred overnight. the activated fa was fi ltered through a 0.45 µm fi lter to remove the dicyclohexylurea side product. the fa-nhs solution was then added dropwise to peg2000 (0.200 g, 0.100 mmol) previously dissolved in 3 ml dmso. 100 µl of pyridine was then added and the reaction stirred overnight. approximately 25 ml of chilled isopropanol (−78 °c) was added forming a light yellow precipitate. the precipitate was obtained via centrifugation. the γand α-isomers of 2 were separated via iec using the following method [doyle et al. 2008). 2 was redissolved in water to give a [20 mg/ml] concentration. this solution was desalted using a 10 ml sephadex pd10 desalting column eluting the product in water. 500 µl of this solution was injected into a 1 ml anx weak anion exchange column. the fl ow rate was set at 0.1 ml/min. the column was then washed with figure 2. crystal structure of ga(hdota) [20]. 16 viola-villegas et al drug target insights 2008:3 5 column volumes of water. after the fi rst peak was eluted, the column was then washed following a gradient (solvent a, water; solvent b, 100 mm ammonium acetate, ph 10) of 10% b for 17 column volumes, 50% b for 17 column volumes, 80% b for 15 column volumes. a column volume of 5 ml of 0.5 m nacl was used to fl ush the column. 1h nmr (d2o): δ 8.62 (s, 1h), δ 7.83 (t, 2h), δ 6.63 (d, 2h), δ 3.50–3.80 (m, peg). yield: 60% based on peg. only the isolated γ -isomer (γ -2) was used for subsequent coupling. synthesis of γ-fa-peg-h3dota ( γ-3) the sulfo-succinamide ester of dota was prepared by activating 0.121 g (0.300 mmol) of the ligand with 26.5 µl (0.150 mmol) edc and 0.0260 g (0.120 mmol) sulfo-nhs in 2 ml water. γ-2 (0.0726 g, 0.03 mmol) was dissolved in 2 ml water and cooled to 4 °c. to the dota solution, γ-2 was added dropwise and the ph was adjusted to 8.5. the reaction was left to stand overnight. γ-3 was obtained via hplc with a retention time, of tr = 20.4 minutes. 1h nmr (d2o): δ 8.76 (s, 1h), δ 7.62 (d, 2h), δ 6.73 (d, 2h), δ 4.61 (s, 2h), δ 4.48 (m, 2h), δ 3.90 – 3.29 (m, peg), δ 2.87 (d, 2h). yield: 64.4% based on γ-2. synthesis of γ-fa-peg-ga(hdota) ( γ-4) 1 (3.42 mg, 0.00726 mmol) was dissolved in 1 ml of 20:80 water:dmso solution. a volume of 1 ml containing dissolved nhs (0.800 mg, 0.00695 mmol) and dcc (1.5 mg, 0.00727 mmol) was added to the solution of 1. this mixture was stirred for 30 minutes. γ-2 (17.6 mg, 0.00726 mmol) was subsequently dissolved in 1 ml of dmso. a volume of 100 µl of triethylamine was added to this solution and was also stirred for 30 minutes. the solution of γ-2 was then added dropwise to the solution of 1. the mixture was left to react overnight. the resulting solution was filtered with a 0.45 µm filter and the crude product precipitated with 25 ml of chilled isopropanol (−78 °c). a yellow solid was isolated by centrifugation and redissolved with 1 ml water and purifi ed by hplc. 1h nmr (d2o): δ 8.64 (s, 1h), δ 7.68 (d, 2h), δ 6.85 (d, 2h), δ 4.61 (s, 2h), δ 3.83 – 3.20 (m, peg), δ 2.32 (d, 12h), δ 1.18 – 1.13 (t, 11h). maldi-tof: 2715.00 m/z (m+h+) calculated 2876.25 for γ-4. yield: 82.9% based on γ-2. cell lines and culture conditions adriamycin resistant ovarian cancer cell line (a2780/ad) and chinese hamster ovary (cho) cell line were cultured as adherent monolayers in rpmi 1640 (invitrogen) growth media containing l-glutamine and fa supplemented with 10,000 units penicillin and 10 mg/ml streptomycin (sigma), 10% (v/v) fetal bovine serum (sigma). cho cells were obtained from the atcc. the a2780/ad cell line used for testing was provided by the fox chase cancer centre, philadelphia. cells were incubated and grown in a vwr mammalian incubator at 5% co2 and 95% humidity. the presence of the fr in the a2780/ad line (and indeed absence in cho cells) was followed by rt-pcr and confocal microscopy (doyle et al, unpublished results). all preparations for cell culture and assays were conducted in a sterile environment under a labconco purifi er i laminar fl ow hood. cells were cultured in millipore 250 ml culture bottles with vented lids. drug cytotoxicity the proliferation of the exponential phase cultures of a2780/ad and cho cells was assessed by colorimetric assay. wsk-8 (dojindo) was performed according to manufacturer’s instructions. adherent cell cultures were harvested by stripping of culture fl asks by a non-enzymatic cell stripper (mediatech) after a 30 minute incubation period. the cells were then collected. the cell densities were adjusted using fa-free rpmi 1640 media to 3.0 × 104 cells/ml to guarantee exponential growth for the period of drug exposure. to each well, aliquots of 100 µl were inoculated. after a 24 hour incubation time to facilitate adherence, the fa free rpmi media was removed and replaced with 200 µl of fresh media containing different concentrations of 1, γ-2, γ-3, γ-4, dota and a control of azt. the cells were then incubated for 72 hours. optical densities were measured at 450 nm using a plate reader. the percentage of cell viability was determined relative to untreated control microcultures. stability studies 2 mm solutions of γ-4 were prepared from 25 mm hepes (ph 7.4) and rpmi 1640 fa-free media. these solutions were incubated over 72 hours at 37 °c. solutions made from the media were fi ltered by centrifugation using a centrifugal fi lter (pall life sciences, mw: 1000 g/mol) at 4,000 rpm over 17 targeting gallium to cancer cells through the folate receptor drug target insights 2008:3 15 minutes. analytical c18 reverse phase hplc analysis was conducted at 0, 1, 24, 48 and 72 hours. at 72 hours, fractions were analyzed for the presence of gallium via inductively coupled plasma (icp). results and discussion chemical synthesis the synthesis of 1 (scheme 1) was prepared by direct addition of stoichiometric equivalents of gallium to dota under acidic (ph 4.8) conditions. crystals were grown after concentrating the solution to its saturation point. five volume equivalents of acetone was then added and the suspension fi ltered. the clear, colorless solution was then placed at 4 °c. colorless needle-shaped crystals formed after 24 hours. fa was activated by a reaction with dcc/nhs to couple it to peg. α/γ-2 was subsequently separated via iec using a weak anion exchange column as shown in scheme 2. the fi rst and second peak eluted both isomers. upon increasing the conductivity to 3 ms/cm, a third peak eluted to give γ-2. analytical hplc runs of fractions collected from iec confi rmed the identity of the isomers. previous work separating both isomers established the α-2 eluting at a later time than the γ-isomer with reverse phase hplc (doyle et al. 2008). the identities of the peaks from fractions collected from the iec were confi rmed with γ-2 eluting at tr = 23.64 minutes and α-2 subsequently eluting at tr = 26.34 minutes. γ-3 was made with sulfo-nhs/edc water soluble cross linkers to give the metal-free compound. edc formed an o-acylisourea intermediate with dota. this converts to the sulfo-hydroxysuccinimide dota ester in the presence of the amine reactive sulfo-nhs. amidation proceeds upon addition of the amine group of γ-2 (see scheme 3). coupling of γ-2 and 1 proceeded using dcc/ nhs coupling agents (see scheme 4). tea was added to γ-2 improving the nucleophilicity of the amine end of the conjugate. since 1 is only soluble in water, it was dissolved in a minimal amount of water followed by addition of dmso. the water to dmso ratio was 20:80. mass spectra a maldi-tof mass spectrum of the commercial peg displayed a mass range of 1900–2100 m/z due to the polydispersity of the polymer. the observed mass of γ-2 is centered at 2422 m/z in agreement with the calculated theoretical mass of ~ 2400 m/z for the polydisperse peg containing system. the calculated theoretical mass of γ-4 is ~ 2900 m/z. a central range at 2846 m/z was observed from maldi-tof mass spectrometry analysis. the 44 m/z spacing is indicative of one unit of ethylene glycol (mw: 44 g/mol). figure 3a–b displays the mass spectra obtained for γ-2 and γ-4 respectively. in vitro biological activity ic50 concentrations were calculated using an exponential fi t. table 2 shows the potency of 1, γ-2, γ-3, scheme 1. synthesis of 1 involving chelation of ga(iii) to dota in ammonium acetate buffer (ph 4.8). 18 viola-villegas et al drug target insights 2008:3 scheme 2. synthesis and separation, via iec of the regioisomers of the αand γ-isomers of 2. scheme 3. synthesis of γ-3 using edc/sulfo-nhs as coupling agents. 19 targeting gallium to cancer cells through the folate receptor drug target insights 2008:3 γ-4 and dota against both the a2780/ad and cho cell lines. azt was used as an internal control (data not shown). in all cases toxicity was greater in the a2780/ad line over the cho line. 1, γ-3, γ-4 and dota displayed between [0.18 and 1.85 mm] activity against a2780/ad cells and between [0.8 and 2.93 mm] in cho cells. γ-2 provided no ic50 concentration over 72 hours at concentrations up to [100 mm]. the fact that the fa-peg moiety is not toxic indicates that the activity of the completed conjugates stems from the gallium metal or dota ligand itself. interestingly gallium compounds were noted as less toxic than free dota containing controls. this was the case in both cell lines. the ic50 concentrations for both compounds containing gallium (namely 1 and γ-4) indicate that gallium is in fact reducing the toxicity of the dota moiety, presumably by chelation, hence preventing the scavenging by dota of other essential metals. toxicity of the fa-peg containing dota compound (γ-3) against cho cells can then also be explained as metal scavenging outside the cell, with uptake not necessary (and not possible in cho without the fr). dota and γ-3 displayed ic50 concentrations of 800 µm and 1.35 mm against cho and 580 µm and 180 µm against a2780/ad cell lines respectively. the presence of the fr receptor in a2780/ad and more rapid division in a2780/ad over cho helps explain the greater toxicity. toxicity of γ-4 in cho cells was noted as 2.93 mm and 1.85 mm in a2780/ad cells. the reduced toxicity of the dota compounds previously complexed with gallium is consistent with the observed toxicity of free dota and supports the idea that the toxicity lies with metal scavenging by dota and supercedes any gallium related toxicity. the toxicity in cho cells can only be explained then if gallium is leaching from the dota macrocyle, since this would produce a gallium salt or complex and would leave dota now uncomplexed and free to chelate other metals. gallium decomplexation from a ligand such as dota that renders such thermodynamic and kinetic stability is possible even with a reported stability constant of log k ~ 21.33 (clark and martell, 1991) (compared to say to open-chain multidentate ligands like ethylenediamine (log k ∼ 17.2) (harris and martell, 1976)). a key scheme 4. synthesis of γ-4 illustrates the coupling of γ-2 and 1 using dcc and nhs as coupling agents in dry dmso. 20 viola-villegas et al drug target insights 2008:3 factor in this release is an increase in dota’s electron density due to inductive effects contributed by the ethylene bridges (hancock and martell, 1995). a plausible explanation for the possibility of gallium release concerns the formation of the fa conjugate. the stability of γ-4 may be affected by the conjugation of one of the pendant carboxylate arms of dota. this phenomenon has been observed by several investigations that involve modifi cation of the dota side arms. sherry et al. in their work involving gadolinium-dota conjugated to a propylamide group via one carboxylate arm has reported a stability constant that is considerably lower (105 fold) than the dota complex owing to the decrease in basicity of the amine macrocycles (sherry et al. 1989). a recent investigation reported that substitution with a p-no2-benzyl group at either one of the dota arms resulted in a reduction in the cooperative binding of the ligand and a lower thermodynamic stability constant compared to unmodifi ed metaldota complex (sherry et al. 2004). of course thermodynamic stability does not necessarily translate into in vivo stability with kinetic inertness often being of greater importance. this may also have a role to play in gallum’s release. structural studies by csajbok et al. via 1h nmr reveal the occurrence of ring inversion and fl uxionality in dota with an increase in temperature (a) (b) figure 3. maldi-tof mass spectrometry analysis of a) γ-2 and b) γ-4 showing a central peak at ca. 2400 m/z and 2846 m/z respectively. icp also confi rmed the presence of gallium in γ-4. 21 targeting gallium to cancer cells through the folate receptor drug target insights 2008:3 (csajbok et al. 2004). similarly, proton exchange can occur between ring amine groups and the carboxylate pendant arms, which may trigger decomplexation (goddard et al. 2001). with the ligand’s dynamic exchange process occurring in solution decomplexation can occur. a whole series of gallium compounds have been screened for in vitro cytotoxicity. a series of gallium compounds with signifi cant toxicity are shown for comparision in table 3. to prove that gallium has indeed been “freed” from its macrocyclic cage, stability studies in hepes buffer and rpmi 1640 media over 72 hours coupled with hplc and icp techniques were conducted. new peaks were observed between 48 and 72 hours indicative of gallium release (see supplemental material). these peaks were analyzed via icp and gallium was noted. in addition, a slight precipitate could be removed by fi ltration (0.22 µm fi lters) and icp confi rmed the presence of gallium in the collected solid fraction. attempts to identify the new species were unsuccessful by electrospray mass spectrometry and 1h nmr and attempts to obtain crystals for x-ray structural analysis also proved unsuccessful. it is likely that both soluble and insoluble gallium salts (such as gallium hydroxides) are forming and/or gallium is complexing with compounds found in the rpmi media. the comparable observed cytotoxicities on both cell lines can then be ascribed to gallium leaching from dota over 72 hours. table 2. ic50 concentrations for 1, γ-2, γ-3, γ-4 and dota against a2780/ad ovarian cells and chinese hamster ovary (cho) cells. (−) indicates no ic50 was recorded. azt was used as a control and returned an ic50 concentration of ∼ 6–8 mm consistent with literature values (doyle et al. 2008). compound ic50 (mm) (72hrs) cho a2780/ad 1 1.61 0.77 γ-2 (−) (−) γ-3 1.35 0.18 γ-4 2.93 1.85 dota 0.80 0.58 table 3. ga compounds of various ligands (l) tested on cell lines showing signifi cant antiproliferative activity. ic50 concentrations were obtained at 72 hours unless otherwise noted. pih is pyridoxal isonicotinoyl hydrazone. ligand (l) ic50 cell line references 2-acetylpyridine 4n dimethylthiosemicarbazone 1.33 +/− 0.43 nm – 96 hr 2.10 +/− 0.90 nm – 96 hr 0.18 +/− 0.02 nm – 96 hr ovarian: 41m mammary: sk-br3 colon: sw480 26 kenpaullone �1 µm – 48 hr lung: ccrf-cem; 27 k-562; mlt-4 �5 µm – 48 hr colon: hct-116; hct-15; ht29; sw-620 �1 µm – 48 hr melanoma: sk-mel-28; sk-mel-5 �10 µm – 48 hr ovarian: ovcar-3 �10 µm -48 hr breast: mcf7 pih 50 µm lung: ccrf-cem 28 chloride 175 µm – 48 hr leukemia: l1210 29 16 µm – 96 hr transferrin 1.1 +/0.2 µm leukemia: hl60 30 nitrate 120 µm lung: ccrf-cem 2 80 µm s-phase arrest lung 1 1.61 mm ovarian: cho this work 0.77 mm ovarian: a2789/ad γ-3 1.35 mm ovarian: cho this work 180 µm ovarian: a2789/ad γ-4 2.93 mm ovarian: cho this work 1.85 mm ovarian: a2789/ad 22 viola-villegas et al drug target insights 2008:3 conclusion we have successfully synthesized, characterized, and investigated the in vitro cytotoxicity studies of dota based gallium complexes and conducted controls to track the source of the toxicity. these results demonstrate that while a ligand of extraordinary kinetic and thermodynamic stability gallium can ‘leach’ from dota over a 72-hour period. what is also clear is that dota itself has between [500–800 µm] toxicity, an interesting note in and of itself. toxicity in both lines could be explained by the uptake, by diffusion, of free dota or gallium-dota, or the presence of uncomplexed dota and/or the release of gallium from the conjugate as applicable in the fa-peg containing systems. clearly the fact that free dota has greater toxicity than the gallium complexed forms described herein, make them unsuitable as anticancer agents themselves. there is however a signifi cant difference on fr containing cells over non-fr containing cells in terms of selectivity, as well as suffi cient stability, to suggest that coupling the γ-emitting 67ga isotope or the β-emitting 68ga isotope to the fa-peg conjugate unit may provide a suitable route to targeting radioisotopes of gallium to cell lines for use as diagnostic agents. this work is currently being investigated in the group. acknowledgments the authors wish to thank syracuse university and the ilearn program for funding. we also thank karen l. howard (state university of new york, esf) and chris incarvito (yale university) for assistance obtaining maldi-tof mass spectra and colin fuss (cese, su) for conducting icp. supporting material hplc stability traces and ic50 graphs showing exponential plots. references anderson, r.g., kamen, b.a., wang, m.t/ et al. 1988. delivery of folates to the cytoplasm of ma104 cells is mediated by a surface membrane receptor that recycles. j. biol. chem., 263:13602–9. anderson, r.g., rothberg, k.g., ying, y.s. et al. 1990. the glycophospholipidlinked folate receptor internalizes folate without entering the clathrincoated pit endocytic pathway. j. cell. biol., 110:637–49. antony, a.c. 1996. folate receptors. annu. rev. nutr., 16:501–21. chitambar, c.r., matthaeus, w.g., antholine, w.e. et al. 1988. inhibition of leukemic hl60 cell growth by transferrin-gallium: effects on ribonucleotide reductase and demonstration of drug synergy with hydroxyurea. blood, 72:1930–6. clarke, e.t. and martell, a.e. 1991. stabilities of trivalent metal ion complexes of the tetraacetate derivatives of 12-, 13and 14-membered tetraazamacrocycles. inorg. chimica. acta., 190:37–46. csajbok, e., banyai, i. and brucher, e. 2004. dynamic nmr properties of dota ligand: variable ph and temperature 1h nmr study on [k(hxdota)](3-x)species. dalton trans.,14:2152–6. delgado, r., sun, y., martell, a.e. et al. 1993. stabilities of divalent and trivalent metal ion complexes of macrocyclic triazatriacetic acids. inorg. chem., 32:3320–6. desoize, b. 2004. metals and metal compounds in cancer treatment. anticancer res., 24:1529–44. doyle, r.p., viola, n.a, ouellette, w. et al. 2006. synthesis, structure and thermal analysis of the gallium complex of 1,4,7,10-tetraazacyclododecane-n,n’,n’’,n’’’-tetraacetic acid (dota). polyhedron, 25:3457–62. doyle, r.p., vortherms, a.r., sinko, d.g. et al. 2008. synthesis, characterization and in vitro assay of folic acid conjugates of 3′(-azido3′(-deoxythymidine (azt): towards targeted azt based anti-cancer therapeutics. nucleosides, nucleotides and nucleic acids, 27:173–85. gogvadze, v., khassanova, z., collery, p. et al. 1996. the effect of gallium on the calcium retention capacity of rat liver mitochondria. in: collery, p., ed., metal ions in biology and medicine, proceedings of the international symposium on metal ions in biology and medicine, 4th, barcelona. p. 249–52. greenwood, n.n., earnshaw, a. 1997. aluminum, gallium, indium and thallium. chemistry of the elements 2nd ed. elsevier science ltd., burlington, ma: publisher. p. 216–67. hancock, r.d., martell, a.e. 1995. lewis acid-base behavior in aqueous solution: some implications for metal ions in biology. adv. inorg. chem., 42:89–146. harpstrite, s.e., prior, j.l., sharma, v. et al. 2007. metalloprobes: synthesis, characterization, and potency of a novel gallium(iii) complex in human epidermal carcinoma cells. j. inorg. biochem., 101:1347–53. harris, w.r., martell, a.e. 1976. aqueous complexes of gallium(iii). inorg. chem., 15:713–20. hart, m.m., yancey, s.t., adamson, r.h. et al. 1971. toxicity and antitumor activity of gallium nitrate and periodically related metal salts. j. natl. cancer inst., 47:1121–7. hedley, d.w., tripp, e.h., mann, g.j. et al. 1988. effect of gallium on dna synthesis by human t-cell lymphoblasts. cancer res., 48:3014–18. jakupec, m., keppler, b. 2004. gallium and other main group metal compounds as antitumor agents. in siegel, a. and siegel, j., eds. metal ions in biological systems, vol. 42. dekker, new york: publisher. p. 425–62. keire, d.a., jang, y.h., shively, j.e. et al. 2001. chelators for radioimmunotherapy: i. nmr and ab initio calculation studies on 1,4,7,10-tetra(carboxyethyl)-1,4,7,10-tetraazacyclododecane (do4pr) and 1,4,7-tris(carboxymethyl)-10-(carboxyethyl)-1,4,7,10tetraazacyclododecane (do3a1pr). inorg. chem., 40:4310–18. liu, m., xu, w., ling-jie, x., gao-ren, z. et al. 2005. synthesis and biological evaluation of diethylenetriamine pentaacetic acidpolyethylene glycol-folate: a new folate-derived, 99mtc-based radiopharmaceutical. j. am. chem., soc., 6–8. low, p.s., lee, r.j. 1994. delivery of liposomes into cultured kb. cells via folate receptor-mediated endocytosis. j. biol. chem., 269:3198–204. low, p.s., mathias, c.j., green, m.a. et al. 1996. tumor-selective radiopharmaceutical targeting via receptor-mediated endocytosis of gallium-67-deferoxamine-folate. j. nucl. med., 37:1003–8. low, p.s., leamon, c.p. 2005. receptor mediated drug delivery. in wang, b., siahaan, t., soltero, r.a., eds. drug delivery: principles and applications. john wiley and sons inc., new jersey: publisher. p. 167–88. qiu, l.y., bae, y.h. 2006. polymer architecture and drug delivery. pharm. res., 23:1–30. 23 targeting gallium to cancer cells through the folate receptor drug target insights 2008:3 ries, l. a. 1993. ovarian cancer. survival and treatment differences by age. cancer , 71:524–9. rudnev, a.v., foteeva, l.s., keppler, b.k. et al. 2006. preclinical characterization of anticancer gallium(iii) complexes: solubility, stability, lipophilicity and binding to serum proteins. j. inorg. biochem., 100:1819–26. shen, f., wu, m.d., ratnam, m. et al. 1995. folate receptor type gamma is primarily a secretory protein due to lack of an effi cient signal for glycosylphosphatidylinositol modifi cation: protein characterization and cell type specifi city. biochem., 34:5660–5. sherry, a.d., brown, r.d., spiller, m. et al. 1989. synthesis and characterization of the gadolinium(3+) complex of dota-propylamide: a model dota-protein conjugate. inorg. chem., 28:620–22. sherry, a.d., woods, m., kovacs, z. et al. 2004. solution dynamics and stability of lanthanide(iii) (s)-2-(p-nitrobenzyl)dota complexes. inorg. chem., 43:2845–51. sun, c.c., bodurka, d.c., gershenson, d.m. et al. 2002. patient preferences regarding side effects of chemotherapy for ovarian cancer: do they change over time? gynecologic oncology, 87:118–28. veronese, f.m., schiavon, o., duncan, r. et al. 2005. peg-doxorubicin conjugates: infl uence of polymer structure on drug release, in vitro cytotoxicity, biodistribution, and antitumor activity. bioconj. chem., 16:775–84. yan, w., ratnam, m. 1995. preferred sites of glycosylphosphatidylinositol modifi cation in folate receptors and constraints in the primary structure of the hydrophobic portion of the signal. biochem., 34:14594–600. 24 viola-villegas et al drug target insights 2008:3 targeting gallium to cancer cells through the folate receptor nerissa viola-villegas, anthony vortherms and robert p. doyle department of chemistry, syracuse university, syracuse, ny 13244-4100, u.s.a. supplementary data (a) (b) (c) (d) figure s1. hplc traces of γ-4 displaying peaks after incubation at 37 °c in 25 mm hepes buffer (ph 7.4) at a) 0 hr b) 24 hrs c) 48 hrs d) 72 hrs. icp confi rmed presence of gallium at the new peaks growing after 72 hrs at a retention time of tr = 15.6–16.3 min. 25 targeting gallium to cancer cells through the folate receptor drug target insights 2008:3 figure s2. cytotoxic effects of 1 (♦), γ-3 (▲), γ-4 ( ) and dota ( ) against cho cancer cells. error bars represent the standard deviation of the mean of three experiments (where n = 3 for each experiment) calculated for each concentration. lines are exponential fi ts with r2 values of 0.8333, 0.9982, 0.9942 and 0.9652 for 1, γ-3, γ-4 and dota respectively. figure s3. cytotoxic effects of 1 (♦), γ-3 (▲), γ-4 ( ) and dota ( ) against a2780/ad cells. error bars represent the standard deviation of the mean of three experiments (where n = 3 for each experiment) calculated for each concentration. lines are exponential fi ts with r2 values of 0.9905, 0.7274, 0.9444, and 0.9645 for 1, γ-3, γ-4 and dota. 0 20 40 60 80 100 120 140 % c el l v ia bi lit y 0 20 40 60 80 100 120 0 0.5 1 1.5 2 2.5 % c el l v ia b i lit y 3 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages 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insights 2006: 1 29–34 29 correspondence: xing qing pan please note that this article may not be used for commercial purposes. for further information please refer to the copyright statement at http://www.la-press.com/copyright.htm original research the way that pegyl-dspc liposomal doxorubicin particles penetrate into solid tumor tissue xing qing pan1, susie jones2 and karen cox2 1college of pharmacy, 2department of pathology, medical center. the ohio state university, columbus, oh 43210. abstract background: for enhancement of drug effectiveness and reduction of drug toxicity, liposomal drugs have been studied in laboratories and clinics for decades. although the results obtained from in vitro are encouraging, but the results from in vivo tests were not satisfactory. the main reasons for this situation were that we do not have enough information about the way how liposomal particles penetrating into solid tumor tissue, and what happening to the liposome particles after they got into the tumor tissue. in this paper, we are going to report the results from our observations on the way folic acid targeted and non-targeted pegyl-dspc liposomal doxorubicin particles penetrate into solid tumor tissue. methods: subcutaneous transplanted murine l1210jf solid tumors in mice were used as a model. pegyl liposomal doxorubicins were injected through tail venue, and tumor tissue samples were collected at special time points. cryosections were cut and dried by a fl owing of air after mounted on the slides right away. then the dried cryosections were stained in water systems; the blood vessel cells were stained with green fl uorescent fitc labeled antibody against cd31 antigen; the nuclei of the living cells were stained with a blue fl uorescent dye dapi. since the whole procedure was carried out in aquatic system, the red color fl uorescent liposomal doxorubicin particles remain visible under fl uorescence microscope. results: both folate conjugated and non-conjugated pegyl-dspc liposomal doxorubicin particles were only leaking out from the broken holes of blood vessels with a special direction and spread out for a limited distance, which was similar to the results showed before, in that observation a latex microsphere sample was used as a model. abbreviations: dspc:1, 2-distearoyl-sn-glycero-3-phosphatylcholine; peg2000-dspc:1, 2-distearoyl-sn-glycero 3-phosphatidylethanolamine-n-[methoxy(polyethylene glycol)-2000]; folate-peg3400-dspe:1, 2-distearoyl-sn-glycero3-phosphatidylethanolamine-n-[polyethylene glycol-3400]-folate. keywords: solid tumor, liposome particles, blood vessel penetration. introduction the toxicity of all the available anticancer drugs are too high, commonly the side effect occurs at their functional dosage. liposomal drugs have been proven capable of changing the drug distribution in vivo, increasing the drug effectiveness, and decreasing the drug toxicity (vail et al. 2004). after targeting liposomal drug technique was developed, we can see in culture systems the liposome particles binding onto tumor cell surface and taken up by the cells quickly. however, at the time the liposomes being used in animals, the results were showed not as good as what we expected (jain, 2001; nagayasu et al. 1999; patel, 1996). people were too busy in making and testing different types of liposomes, number of papers even only reported their results without animal tests, did not pay enough attention to the study of knowing further about the real processing of the liposomal particles in the solid tumor. actually, since we do not know the exact way from which liposomal particles getting into solid tumor tissue, and also the fortune of those liposomal particles after they got inside the solid tumor tissue. clearly, it is not really clear what is the right direction to further improve the effectiveness of liposomal drugs in animal or clinic yet, especially in the targeted liposomes and the solid tumor cases. a number of researchers have done a lot of studies in this area with various types of equipment and experimental designs (lichtenbeld et al. 1996; hashizume et al. 2000; davorak et al. 1999; kohn et al. 1992; uster et al. 1998). from the results of those studies we agreed that the liposomal particles may pass through drug target insights 2006: 130 pan et al the broken hole of the tumor blood vessels and spread out in the tumor tissue, but so far a strong or direct evidence about this hypothesis is still not available. the main obstacle in this part of study is that the liposomal particles are composed of lipid membranes, which will be destroyed immediately when the solid tumor tissue were treated in organic solvents during the procedures of histological stains. because of this reason we have tried to use rhodamine labeled latex microsphere particles with the same size (100 nm in diameter) and negative electro charge to mimic the behaving of the liposomal particles (pan et al. 2004). the results were very good, from the fl uorescent color, we can see the red color particles leaking out from green color blood vessels through the broken areas, but not from everywhere or passing through the blood vessel wall. however, the methods of using latex microspheres has limitations, fi rst they are rigid particles, possibly still not totally the same to the real liposomal particles, second, they will not be up taken up by the cell and kill the cancer cells as the liposomal particles probably will do. these week points become critical, when we want to compare the different situations between the nontargeted and the targeted liposomal particles. it is not impossible to label the latex microspheres, make them become targeted or carry drugs, but it is kind of diffi cult to control, anyway, it will be another research, no longer the problem of liposomal drugs we want to see at present time. nevertheless, based on the experience with the use of fl uorescent latex microspheres, we realized if we omitted the acetone fi xing step, instead, dried the thin cancer tissue under an air fl ow immediately after the cryosections have been mounted on the glass slices, then stained the tumor sections with antibody or chemical reagent in water solutions, we may be able to avoided the damage of the liposomal particles, and see the liposome particles, if the liposome particles carried a red color fl uorescence. thus, in this study, we are using the real pegyl-dspc liposomal doxorubicin as a tool to run the observation. the advantage of this liposome is fi rst we can locate the liposome by the red color fl uorescence of drug doxorubicine with the same method of fl uorescent stains as we have used in the fl uorescent latex microspheres; and the second, these folic acid targeted and non-targeted liposomes have been used in set of liposome studies in our study in the past. in this case, with the limited changes, more results can be used in comparasion, with that we may be able to obtain some better ideas about it. meanwhile, in another experiment, we also observed the fortune of these liposomal particles after they got into the solid tumor tissue, and compared the anticancer effectiveness of these liposomal doxorubicin in mice. the results were surprisely different to what we used to believe, the better anticancer effectiveness was not because of more folic acid targeted particles getting into tumor tissue, the better anticancer effectiveness came from the non-targeted liposomal particles kept in the tumor tissue for long time with no change, kept the drug doxorubicin inside the lipid membrane and no chance to meet the cancer cells. those results will be reported in another paper. the abstract of these results have been present in the meeting os aacs (pan et al. 2005). we hope based on these new information we may be able to fi nd out what should we do to improve the effectiveness of the liposomal drug treatment in vivo. materials and methods reagents folic acid free rpmi 1640 and fetal calf serum were purchased from gibco (grand island, ny, u.s.a.). dspc and peg2000-dspc were purchased from avanti polar lipids, inc. folate-ped3400dspc was obtained from the nih. the other reagents such as cholesterol, doxorubicin and sepharose cl-4b chromatography resin were obtained from sigma chemical co (st. louis, missouri). rat anti-mouse cd31 antibody was obtained from bd pharmingen (san diego, ca, u.s.a., catalog number 550274). the biotinylated rabbit anti-rat, mouse absorbed antibody was purchased from vector, inc. (burlingame, ca, u.s.a., catalog number ba-4001), and the fitcstreptavidin was obtained from dako inc. (carpinteria, ca, u.s.a., catalog number f0422). the nuclei stain fl uorescent dye dapi (4’, 6diamidino-2-phenylindole) containing mount medium was a gift from dako inc. the tissue-tek o.c.t. compound was obtained from sakura finetek usa inc. (torrance, ca, u.s.a.). pegyl-dspc liposomal doxorubicin preparation dspc/cholesterol/peg-dspe (65:31:4, mole/ mole) non-targeting liposome and dspc/cholesdrug target insights 2006: 1 31 observation of liposomal drug particles penetrating into solid tumor tissue. terol/peg2000-dspe/folate-peg3400-dspe (60:31:3.5:0.5 mole/mole) folate targeted liposomes were prepared by the procedure we used in previous studies (pan et al. 2003; pan et al. 2002). briefl y, mixed the lipid composition in chloroform solution in a round bottom fl ask, totally weight of the lipid mixture was 30 mg. then this mixture solution was dried under nitrogen. the residue was desiccated under vacuum for two hours, and re-hydrated in 5 ml ph 4.0, 400 mm sodium citrate solution, treated with ultrasonic and vortex, freezing and thawing alternately. the production was then passed through a 100-nm pore-sized polycarbonate membrane under nitrogen at 60o c in a lipid extruder (lipextm, northern lipids, inc., vancouver, canada). the mean size of the liposome particles was controlled at 100 nm in diameter (ishida et al. 1999), which was measured by photon-correlation spectroscopy on a nicomp 370 submicron particle analyzer. after purifi ed from sepharose cl-4b column, the unilamellar liposome particles were remote-loaded with doxorubicin based on transmembrane ph gradient of 4.0–7.4. the drug-tolipid weight ratio was at 1:10 (wt/wt). the doxorubicin containing liposome sample was purifi ed by passing through sepharose cl-4b column again to separate off small amount of free doxorubicin. then the liposomal doxorubicin preparations were diluted in ph 7.4 pbs to yield a doxorubicin concentration of 1 mg/ml suspension. the fi nal production of liposomal doxorubicin samples were stored under 4o c, covered with aluminum foil. in our observations the liposome sample were all fresh made and used within one month, although they have been proven stable for much longer time. mice model of l1210jf solid tumor murine leukemia l1210jf cells were cultured in a folic acid free rpmi 1640 medium, supplemented with 10% fetal calf serum, 100 u/ml penicillin and 100 mg/ml streptomycin, in a humidifi ed incubator, atmosphere containing 5% co2, and the temperature was 37o c. before injection, the cells were spun down and washed with ph 7.4 pbs solution three times. dba2 mice weighting 18–22 g were purchased from charles river laboratories (wilmington, massachusetts, u.s.a.). the mice were fed a folate free special rodent diet (catalogue no. 117772, dyets inc.) for one week before use (pan et al. 2003). they were then inoculated subcutaneously with 0.1 ml suspension of 1 × 106 l1210jf cells in pbs at the left fl ank. the tumor was allowed to grow until it reached a size of about 0.5 cm3. in our animal experiments, all the procedures strictly followed the regulations of the ohio state university institutional animal care and use committee. cryosections of solid tumor tissue sample preparation. each mouse was injected through the tail vein with pegyl-dspc-liposome sample containing 0.2 mg doxorubicin. at each of the selected time points, three mice were sacrifi ced with co2 gas. then the tumor tissues were collected immediately and embedded into tissue-tek o.c.t. under dry ice right away. all the frozen samples kept under minus 80oc before use. six μm thick cryosections were cut and mounted on positive-charged glass slides, and dried right away under an electric fan. the dried sections were kept in a minus 20o c freezer before staining. the staining procedure was processed on a dako autostainer. briefl y, fi rst the sections were rehydrated in ph 7.4 pbs, and blocked with 10 % normal rabbit serum. a 1:50 dilution of rat antimouse cd31 primary antibody was applied and then the slides were incubated for 30 minute at room temperature (rubin et al. 1999; rogatsch et al. 1997; pan et al. 2004). after wash, a 1:200 dilution of biotinylated rabbit anti-rat, mouse absorbed antibody was used as the second antibody. after 30 minutes incubation, the sections were washed and then a 1:40 dilution of fitcstreptavidin solution was applied. lastly, the slides were counterstained with fl uorescent dye dapi contained mounting medium to stain the alive cell nuclei and covered with a regular cover glass. observation on the penetration and distribution of the liposomal doxorubicin stained cryosections were viewed immediately under a zeiss fl uorescence microscope. a 480 nm ultraviolet exciting, 535 nm emission system was used for green fl uorescent labeled blood vessel observation, and 360 nm/420 nm system was applied for observation of color fl uorescent labeled cell nuclei and a 545 nm/610 nm system was used for red fl uorescent doxorubicin containing liposome particle observation. drug target insights 2006: 132 pan et al results and discussion the results of the observation showed that the methods used in cryosection cutting, the tissue stainings and the selection of the liposomes were suitable. the omission of the acetone fi xing step did not interfere the staining of the blood vessel cells and the nuclei of the tumor cells. clearly, the red color doxorubicin containing liposomal particles were not penetrating out from the blood vessel wall, since the red color liposomal particles did not distributed along the blood vessels like a sleeve or as a cycle surround the cross section of the blood vessel. the liposomal particles were only passing though the holes of the broken blood vessels with a direction and spreading into the cancer tissue in the space between the cells in the tumor tissue. compare to the total number of blood vessels in the sections, the number of leaking blood vessels were limited in our l1210jf experimental cancer tissues. those broken blood vessels commonly located near the necrosis areas, or those areas where the necrosis is starting, since in those necrosis areas, the number of the cells along the blood vessel were high, but in a short distance showed no many alive cells there. it could be nicer if we could have a chance to quantitatively determine the ratio or the number of leaking holes in different tumor tissue samples, however, from our observations in different experiments, the frequency of the necrosis happening in the tumor tissues might different from different species of the tumors, and determined by the location on back or at the fl ank of the mouse where the tumor was growing. the level of necrosis also related to the size of the tumor and time how long the solid cancer had been growing. as early as six hours after liposome injection, the red color liposome particles already showing in the tumor tissue. at 24 hours after liposome injection the liposomal particles spread out for a longer distances and then the liposomal particles stopped leaking out from the blood vessel at about 48 hours after liposome injection. in this paper, for showing the clearer picture of the way by which non-targeted and folic acid targeted liposomal particles penetrating out from the blood vessel, we showed two photos for each liposome and all of tumor tissue samples were taken at the time 12 hours after liposome injection (see fig. 1). in figure 1, a and b showing the non-targeting liposomal doxorubicin particles penetrating in l1210jf solid tumor tissue; c and d are showing the folic acid targeted liposomal doxorubicin particles penetrating in l1210jf solid tumors. no obvious differences could be pointed out in figure between the non-targeted liposome and folic acid targeted liposome, only the diffusion of the folic acid targeted liposomal particles looked faster. we have been wary about that the low temperature frozen cryosection cutting step might break the liposomal particles or changed particle size. but, from the results we obtained, we believed that was not happened, or only happened at very limited level. anyway, even it happened, at the time the crysections were cutting, the liposome particles already leaked out from the blood vessel, the size change at that time will not interfere our observation. also under the experimental condition the limited amount of free doxorubicin which leaked out from the broken liposomal particles would be wash off from the thin section tissue during the staining procedure, it would not be seeable. as showed quite often, that there are not many living cells near the leaking blood vessels, we believe those broken blood vessels are located closely to the necrotic areas or those areas where the solid tumor tissue was starting to form necrotic area. lately, kirpotin et al. observed the distribution and up taken of anti-her2 antibody targeted long circulating liposomal doxorubicin particles and its correlated non-targeted liposome in her2 expressed bt-474 cell solid tumor and non-her2 antigen expressed mcf-7 cell solid tumor in nude mice (kirpotin et al. 2006). in their report, the amount of liposomal particles distributed into the solid tumor tissues were the same, no difference in both antigen expressed bt-474 solid tumor or her2 antigen not expressed mcf-7 solid tumor. and no difference between the antibody targeted or non-targeted liposome. it is agreeable, the amount of liposomal particles can penetrate into solid tumor tissue is determined by the hole size and number of the blood vessels, but not determined by the targeting group which located on the liposomal particle surface. however, after got into the tumor tissue the functional folic acid ligands bound on to folic acid receptors. besides, as it was already reported that a number activated macrophages were presenting in the solid tumor tissue (murdoch et al. 2004) they also actively expressing folic acid receptors on their surface. so, in our folic acid targeted liposomal particle case, the macrophage cells also up taken the folic acid targeted liposomal particles, then free drug doxorubicin drug target insights 2006: 1 33 observation of liposomal drug particles penetrating into solid tumor tissue. released out after the lipid membrane broken down, which killed the tumor cells in neighborhood as well. after the cancer cells were killed, the blood vessel further damaged, we saw quite often the red blood cells presented in the damage tumor tissue. in this situation, more space and bigger holes allowed the targeted liposomal particles leaked into cancer tissue. no doubt the folic acid targeted liposomal doxorubicin showed better anticancer effectiveness compared to the non-targeted same liposomal doxorubicin. we will discuss about this in another paper. here, we need to mention that since the liposomal particles penetrated through directly into solid tumor the blood-brain barrier will no longer show ( vail et al. 2004), which will be good for the treatment of brain tumors. but the problem is since the number of liposomal particles, in another words the amount of liposomal drug which can leak into the solid tumor is determined by the number of the leaking blood vessels, or say the nacrosis level of the solid cancer tissue. so, in the case even the drug sensitivity of the cancer cells are the same, the effi ciency of the cancer treatment will be still different at least at the beginning will be determined by situation of the blood vessels say by the necrotic level of the solid tumor tissue. the same problem will be more important for the study on early diagnosis of cancer, people nowadays are trying hard to use liposomal particles or other nanoparticles to carry special reagents or radio active labeled materials to fi nd the location of the earliest stage tumor. here we can see that we need to re-consider that strategy, since with that idea we figure 1. the pegyl-dspc liposomal doxorubicin particles are leaking out from broken blood vessels in l1210jf solid tumor tissue growing subcutaneously in dba2 mice. photo a & b samples were collected at 12 hours after non-targeting liposomal doxorubicin was injected from the tail vein; c & d samples were taken at 12 hours after folic acid targeting liposomal doxorubicin was injected from the tail vein. drug target insights 2006: 134 pan et al may not be able to show the tumor location before the tumor grow into special size, and the break blood vessels showed up. furthermore, some cancer even naturally do not have blood vessels, such as some of the non-small-cell lung carcinoma (pezzella et al. 1997). at the end of this paper, we would like to make a suggestion that it might be interesting, if we mix the rhodamine labeled latex microspheres with an anticancer drug sample, or a liposomal form drog suspension, we may be able to trace the drug penetration process in a solid tumor tissue, not like doxorubicin its fl uorescence will be diminished after doxorubicin reacted to the cells, the fl uorescence of rhodamine can last much longer. acknowledgements the authors are grateful to dr. robert j lee and dr. julius kreier for their creative discussion and encouragements. the authors would also like to express our thanks to mr. brian kemmenoe of ohio state university campus microscopy & imaging core for his technical help. references dvorak, h.f., nagy, j.a. and feng, d. et al. 1999. vascular permeability factor/vascular endothelial growth factor and the signifi cance of microvascular hyperpermeability in angiogenesis. curr. top. microbiol. immunol., 237:97–132. forssn, e.a., male-brune, r. and adler-moore, j.p. et al. 1996. fluorescence imaging studies for the disposition of daunorubicin liposomes (daunoxome) within tumor tissue. cancer res., 56:2066–2075. hashizume, h., baluk, p. and morikawa, s. et al. 2000. openings between defective endothelial cells explain tumor vessel leakiness. am. soc. invest. pathol., 156:1363–1380. ishida, o., maruyama, k. and sasaki, k. et al. 1999. size-dependent extravasation and interstitial location of polyethyleneglycol liposomes in solid tumor-bearing mice. international j. pharmaceutics, 100:49–56. jain, r.k. 2001. delivery of molecular medicine to solid tumors: lessons from in vivo imaging of gene expression and function. j. control release, 74:7–25. kirpotin, d.b., drummond, d.c. and shao, y. et al. 2006. antibody targeting of long-circulating lipidic nanoparticles does not increase tumor localization but does increase internalization in animal models. cancer res., 66:6732–6740. kohn, s., nagy, j.a. and dvorak, h.f. 1992. pathways of macromolecular tracer transport across venules and small veins: structural basis for the hyperpermeability of tumor blood vessels. lab invest., 67:596– 607. lichtenbeld, h.c., yuan, f. and michel, c.c. 1996. perfusion of single tumor microvessels: application to vascular permeability measurement. microcirculation, 3:349–357. murdoch, c., giannoudis, a. and lewis, c.e. 2004. mechanisms regulating the recruitment of macrophages into hypoxic areas of tumors and other ischemic tissues. blood, 104:2224–2234. nagayasu, a., uchiyama, k. and kiwada, h. 1999. the size of liposomes: a factor which affects their targeting effi ciency to tumors and therapeutic activity of liposomal antitumor drugs. adv. drug delivery rev., 40:75–87. pan, x.q., zheng, x. and shi, g.f. 2002. strategy for the treatment of acute myelogenous leukemia based on folate receptor-targeted liposomal doxorubicin combined with receptor induction using all-trans retinoic acid. blood, 100:594–602. pan, x.q., wang, h. and lee, r.j. 2003. antitumor activity of folate receptor-targeted liposomal doxorubicin in a kb oral carcinoma murine xenograft model. pharmaceutical res., 20:417–422. pan, x.q., lee, r.j. and ratnam, m. 2004a. penetration into solid tumor tissue of fl uorescent latex microspheres: a mimic of liposome particles. anticancer res., 24:3005–3008. patel, j. 1996. liposomal doxorubicin: doxil. j. oncol. practice, 2:201–210. pezzella, f., pastorino, u. and tagliabue, e. et al. 1997. non-small-cell lung carcinoma growth without morphological evidence of neoangiogenesis. american j. pathol., 151:1417–1423. rogatsch, h., hittmair, a. and reissigl, a. et al. 1997. microvessel density in core biopsies of prostatic adenocarcinoma: a stage predictor. j. pathol., 182:205–210. rubin, m.a. buyyounouski, m. and bagilla, e. et al. 1999. microvessel density in prostate cancer: lack of correlation with tumor grad, pathologic stage, and clinical outcome. urology, 53:542–547. uster, p.s., working, p.k. and vaage, j. 1998. pegylated liposomal doxorubicin (doxil, caelyx) distribution in tumour models observed with confocal laser scanning microscopy. international j. pharmaceutics, 162:77–86. vaage, j., donovan, d. and wipff, e. 1999. therapy of a xenografted human colonic carcinoma using cisplatin or doxorubicin encapsulated in long-circulating pegylated stealth liposomes. int. j. cancer, 80: 134–137. vail, d.m., amantea, m.a. and colbern, g.t. et al. 2004. pegylated liposomeal doxorybicin: proof of principle using preclinical animal models and pharmacokinetic studies. semin. oncol., 31 (6 suppl) 13:16–35. chiang et al.indd drug target insights 2008:3 67–76 67 original research correspondence: thomas m. chiang, research service (151), veterans affairs medical center, 1030 jefferson avenue, memphis, tn 38104, u.s.a. tel: 901-523-8990 × 7608; fax: 901-577-7273; email:tchiang@utmem.edu copyright in this article, its metadata, and any supplementary data is held by its author or authors. it is published under the creative commons attribution by licence. for further information go to: http://creativecommons.org/licenses/by/3.0/. the beta3 499–513 peptide region is required for alphaiib/ beta3 active complex formation and fibrinogen binding virginia woo-rasberry1 and thomas m. chiang1,2,3 1veterans affairs medical center and 2departments of medicine and 3molecular sciences, university of tennessee health science center, memphis, tn 38163, u.s.a. abstract background: alphaiib/beta3 (αiib/β3) complex is an important integrin that is involved in the fi nal step of platelet aggregation. peptides derived from either αiib or β3 have demonstrated to have an effect on the activation of the complex and its ability to bind fi brinogen. we have previously defi ned a peptide from β3, which inhibits agonists-induced platelet aggregation. methods: we used standard methodologies for construction of clones and expression of cdnas, establishment of stable cell lines that contained these cdnas. expression of proteins was detected with immunoblots. flow cytometric analyses were used to verify the presence of the active and inactive complexes with different antibodies. in addition, a fi brinogenbinding assay was used to determine the inhibition of the active complex by the peptide. results and discussion: a stable cell line of the co-transfected cdnas of αiib, β3 wild type and mutants of β3 (scrambled sequence of the peptide region, replacement of c499a and c512a), expressing the inactive complex on cho cells, has allowed us to examine the important role of the peptide sequence and the cysteine residues within the peptide. the peptide inhibits the active complex formation and thereby inhibits the binding of fitc-pac-1 in a dose dependent manner by fl ow cytometric analyses, as well as binding of [3h]-fi brinogen. in addition, creation of a second stable cell line containing wild type αiib and the mutated region of β3 (residues 499–513) shows that the binding of fitc-pac-1 and [3h]-fi brinogen on the mutant activated complex was much lower than the wild type activated complex. our results indicate that the region 499–513 in β3 is one of the important sites for αiib/β3 active complex formation and the cysteines play an important role in the process. keywords: α2ß3 integrin, fl ow cytometry, disulphide bond, fi brinogen, integrin activation, mutagenesis introduction alphaiibbeta3 (αiib/β3) complex plays an important role in platelet aggregation. the αiib/β3 complex exists in the inactive and active conformational states. the activated complex serves as ligand-binding sites (luo et al. 2004; schwartz et al. 1995) for four macromolecules (fi brinogen, von willebrand factor, fi bronectin and vitronectin). the binding of fi brinogen to the activated αiib/β3 complex mediates the fi nal step of platelet aggregation. in damaged vessel walls, αiib/β3 undergoes a conformational change from an inactive to an active state that is able to initiate thrombi formation. many studies have proposed that the interaction between αiib and β3 reside in several regions (filizola et al. 2004; feuston, 2003 and d’souza et al. 1994). there are two discrete peptides defi ned from β3 with amino acid residues of 211–222 and 217–230, which have been reported to inhibit fi brinogen binding and affect platelet aggregation (charo et al. 1991 and steiner et al. 1993). in addition, two complex forming sequences of αiib and β3 (αiib: 94–314 and ß3: 118–131, and αiib: e117 and β3:r214), which correlate with both ligand and cation binding within the αiib/β3 complex, have been identifi ed (feuston, 2003; d’souza et al. 1994; d’souza et al. 1991 and charo et al. 1991). other investigators (kashiwagi et al. 1999; sun et al. 2002; beglova et al. 2002 and butta et al. 2003) have addressed the important function of cysteine residues in β3. the consensus is that cysteine residues play an important role in the formation of the active complex. wang et al. (1997) have found that disruption of the disulfi de bond between cys 406 and cys 665 of β3 did not affect αiib/β3 ligand binding. however, in some cases, disruption of certain disulfi de bonds resulted in the formation of an http://creativecommons.org/licenses/by/3.0/ http://creativecommons.org/licenses/by/3.0/ 68 woo-rasberry and chiang drug target insights 2008:3 active complex. kashiwagi et al. (1999) created a single amino acid mutation in the extracellular cysteine-rich repeat region of the β3 subunit (t562n), and activated αiib/β3. although, many laboratories have studied the extracellular, transmembrane, cytoplasmic regions and carboxylterminal of β3 in relation to the formation of the functional complex, the exact site remains to be determined. previously, we have reported that a platelet 90-kda glycoprotein (gp 90) is involved in collagen-, epinephrine-, and thrombin-platelet interaction (chiang et al. 1989). we have identified the protein as β3 by using twodimensional gel (2d gel) electrophoresis, immunblotted with anti-90-kda and anti-β3 antibodies, and matrix assisted laser desorption/ ionization-time of flight (maldi-tof). searches of the swiss-prot database for the identity with the mass spectra of the trypsinized fragments, isoelectric point, and molecular weight resulted in a match with human platelet β3. recently, we have defi ned a region of β3 (residues 499–513, named p4), which inhibits antagonists-induced platelet activation by inhibiting fitc-pac-1 binding. a chemically synthesized peptide also inhibits adp-, type i collagenand type iii collagen-induced platelet aggregation in a dose-dependent manner through the inhibition of αiib/β3 complex formation (chiang et al. 2005). in the present study, we have constructed three mutant cdnas of β3 by scrambling the amino acid residues and the other two by substituting the cysteine residues with alanine (c499a and c512a) of the p4 peptide region and expressing them with wild type αiib, as well as expressing αiib/β3-wild type in chinese hamster ovary cells (cho). we examined the binding ability of the mutant compared with wild type to fitc-pac-1 and [3h]-fi brinogen. results show that co-transfection with the mutated cdnas expresses an inactive complex and binds less fitc-pac-1 as compared to wild type. in addition, a chemically synthesized peptide (of the defi ned region) inhibited the binding of fitc-pac-1 as well as inhibiting the binding of [ 3 h]-fibrinogen onto mn2+ -activated wild type αiib/β3 expressed in cho cells. these results suggest that the p4 peptide of β3 and its cysteine residues play an important role in the formation of the active complex of αiib/β3. material and methods reagents monoclonal anti-β3 was purchased from r&d systems, inc. (minneapolis, mn) and monoclonal anti-αiib (clone sz22) was purchased from beckman coulter (fullerton, ca). the monoclonal antibody, which recognizes the inactive complex of αiib/β3, ap2, was purchased from gti diagnostics (waukesha, wi). the chinese hamster ovary (chok1) cell line was purchased from america type culture collection (atcc) (manassas, va). the transfection reagent, lipofectamine 2000 and fluorescent dye alexa fluor 647 were from invitrogen (carlsbad, ca). all other chemicals were from sigma chemical co. (st. louis, mo), fisher scientifi c (st. louis, mo), bio-rad (hercules, ca), and pierce (rockford, il). gp iib cdna construct is a gift from dr. peter newman (the blood center of southeastern wisconsin, inc. milwaukee, wi). preparation of platelet-rich plasma (prp) human blood (9 parts) from normal volunteers, were collected following an overnight fast, in polypropylene tubes containing 1 part 3.8% sodium citrate. prp was prepared by centrifuging the citrated blood at room temperature for 10 minutes at 226 × g (chiang et al. 1976). whole blood and prp were exposed to plastic surfaces or siliconized vessels only. platelet counts of the prp ranged from 200,000 to 300,000 per mm3. the washed platelets for binding assays were prepared by gently mixing equal volumes of prp and 20 mm tris-hcl-130 mm nacl-1 mm edta, ph 7.4 (tris-edta), centrifuged at 1,000 × g for 5 min, washed once with tris-edta and then resuspended in the tyrode’s buffer. binding of [3h]-fi brinogen to washed human platelets fibrinogen was labeled with [3h]-formaldehyde as described by others (whitnack et al. 1985; rice et al. 1971 and grinnell, 1980). briefl y, human fibrinogen was dissolved in pbs at a concentration of 3.4 mg of clottable protein per ml (10 µm), dialyzed against pbs to remove salts, and stored at –20 °c. fibrinogen was dissolved in 0.2 m sodium borate (ph 7.4) at a concentration of 10 µm and dialyzed against the 69 the beta3 499–513 peptide region is required for alphaiib/beta3 active complex drug target insights 2008:3 same buffer. the fi brinogen was then subjected to reductive n-methylation at 4 °c with 0.1 volume of a 1% aqueous solution of [3h]-formaldehyde, followed by 0.3 volume of sodium borohydride. the labeled fibrinogen was dialyzed against pbs, assessed for amount of bound radiolabel, assayed for binding capacity and fi nally, stored at −20 °c. the binding mixture consisted of washed human platelets (108) in tyrode’s buffer (5 mm hepes, 2 mm mgcl2, 0.3 mm nah2po4, 3 mm kcl, 12 mm nahco3, 0.1% glucose, 0.1% bsa, and 1 mm cacl2, ph 7.0). various amounts of p4 and scrambled p4s peptides (0–80 µm) were used to determine the dose response of inhibition of binding by fi brinogen for 30 minutes at room temperature. initiation of the assay began with the addition of adp (final concentration of 0.5 µm), incubated at room temperature for 5 minutes, followed by adding [3h]-fi brinogen with further incubation at room temperature for an hour. then unbound [3h]-fi brinogen was removed by washing with tyrode’s buffer three times, the pellet was then suspended in the same buffer and transferred to scintillation vials containing 7.5 ml of scintillation solution (scintiverse bd, fisher scientifi c, pittsburgh, pa). radioactivity was assessed with a packard liquid scintillation analyzer (perkin elmer life and analytical sci., inc., boston, ma). construction and expression of wild type and mutant β3 cdnas construction of a plasmid encoding for the fulllength cdna of platelet β3 were from several cdna fragments deposited at atcc and subcloned into the vectors pet-24a and pcdna3.1. the β3-mut cdna is a scrambling of the peptide sequence from 499–513 (473–487 according to numbering by kamata et al. 2001) of β3 by pcr. the two sets of primers used were, forward primer: 5’-acagatcttgcgagtattccgagcagtgcccccgggagggtcagcc-3 and reverse primer: 5’caagatctgtcgctaggctgctcgtcctcacactgggatcccagc-3’. the following primers were used to construct the cdna with c499a using forward primer: 5’-ggatcccagtgtgaggcctcagag-3’ and reverse primer: 5’-ctctgaggcctcacactgggatcc-3’ and the second cdna with c512a using forward primer: 5’-gcaggacgaggccagcccccgg-3’ and reverse primer: 5’-cgggggctggcctcgtcctgc-3’. the template used for all pcr reactions was β3-wild type in pcdna3.1. all of the fi nal pcr products carrying the desired mutations in the full-length cdna were treated with dpn i, purifi ed, transformed into dh5α, selectively screened, and sequence verified before proceeding with expression. peptides for inhibition studies the first chemically synthesized peptide is c(acm)seedyrp sqqdec(acm)s, which we referred to as p4. the second chemically synthesized peptide is the scrambled version of p4, sdc (acm)eypeseqrsqdc(acm), which served as a control and is referred to as p4s. establishment of stable cell lines for eukaryotic expression in cho cells and immunoblot analyses wild type β3 or mutant β3 (β3-mut) (4 µg of each construct) and wild type αiib (4 µg) were cotransfected into cho cell using lipofectamine 2000 according to the manufacturer’s specifi cations. cells were incubated 37 °c for 48 hours following transfection, allowing for transgene expression in selective media containing 700 µg/ml of geneticin for two weeks. selected single colonies were expanded to 24-well plates and the selected integrant was screened by fl ow cytometry in a bd facs calibur using the ap2 antibody conjugated to the fl uorescent dye (alexa fluor 647). following selection, the cells are maintained in selective medium with 700 µg/ml of geneticin. immunoblot analysis with both, αiib and β3 antibodies also verifi ed the successful co-transfections. flow cytometry analysis of activated of αiib/β3 complex stable cells (wild type αiib/β3 and αiib/β3-mut) were harvested with 0.5 mm edta/pbs ph 7.4, washed twice with 50 mm hepes/1% glucose/2 mm ca2+/2 mm mg2+ ph 7.4 (hepes buffer i) and resuspended in 50 mm hepes/1% glucose/ 1 mm ca2+/1 mm mg2+ ph 7.4. (hepes buffer ii). activation of the washed cells (1 × 106/ml) were initiated by the addition of 2 mm mn2+ for 45 minutes (litvinov et al. 2004), then incubated with fitc-pac-1 for an additional 60 minutes at 70 woo-rasberry and chiang drug target insights 2008:3 room temperature. following three washes with hepes buffer ii, the cells were resuspended in the same buffer and the amount of fitc-pac-1 binding was analyzed by fl ow cytometry. inhibition of αiib/β3 active complex by peptides the chemically synthesized peptides, p4 and p4s, were used to determine whether they could inhibit the activation of the wild type αiib/β3 complex. again, cells were prepared as previously stated, incubated with varying amounts of peptide, (either p4 or p4s, 0–80µm) and 2 mm mn2+ for 45 minutes, followed by incubation with fitc-pac-1 for an additional 60 minutes at room temperature. following immunostaining, all cells were washed three times in hepes buffer ii, resuspended in the same buffer and binding was analyzed by fl ow cytometry. binding of [3h]-fi brinogen to stable co-transfected cho cells binding mixture consisted of co-transfected cho cells (either wild type or mutant, 5 × 106) in tyrode’s buffer. the assay was initiated by adding mn2+ to fi nal a concentration of 2 mm, incubated at room temperature for 45 minutes, then added of p4 or p4s, followed by the addition of [3h]fi brinogen and incubated at room temperature for an additional hour. following incubation, the bound and unbound [3h]-fi brinogen, were separated by washing with tyrode’s buffer three times, resuspended in the same buffer and transferred to scintillation vials containing 7.5 ml of scintillation solution. radioactivity was determined with a packard tri-carb 2000ca liquid scintillation analyzer (perkin elmer life and analytical sci., inc., boston, ma). the effect of peptides, p4 and p4s, on [3h]-fi brinogen binding followed the same binding assays as previously stated, with the exception that varying amounts of each peptide are incubated with the mn2+. results binding of [3h]-fi brinogen on washed human platelets we have established the optimum binding conditions [(time (one hour), room temperature, number of platelets (108) or co-transfected cho cells (5 × 106), and concentration of fi brinogen (0.5 µm)] for the binding assay (data not shown). using the established conditions, we tested the effects of p4 and p4s peptide on the binding of [3h]-fi brinogen on washed human platelets. the amount of [3h]fi brinogen binding to the platelets was reduced by p4 (fig. 1, line with diamonds). the percent inhibition by p4 is dose-dependent. treatment with p4s did not signifi cantly reduce fi brinogen binding (fig. 1, line with fi lled squares). in addition, the p4 peptide could not inhibit the binding of fi brinogen to platelets without the addition of suboptimal concentrations of adp (data not shown). these results suggest that the peptide probably binds on gp iib to inhibit the αiib/β3 active complex formation rather than directly affecting the fi brinogen binding to the activated complex. in order to study the effects of p4 (wild type β3) on the formation of active αiib/β3, we have engineered a construct containing the scrambled sequence of the active peptide (p4s) in β3 cdna. it served as a control (named as β-mut) in cotransfection with wild type αiib cdna into cho cells and proved that the p4 peptide is an inhibitor of αiib/β3 active complex formation. recombinant proteins of αiib, β3, and β3-mut expressed in cho cells expressions of co-transfected proteins, αiib with wild type-β3 or β3-mut were examined by immunoblot analyses. figure 2 shows that both wild type αiib and wild type β3 proteins and wild type αiib and β3-mut were detected in co-transfected cho cell lysates. panels a and b shows a set of immunoblots of co-transfected cho cells (lane 1: mock, lane 2: αiib/β3 wild type, and lane 3: αiib/β3-mut). the αiib antibody recognized αiib expressed in cho cells expressing wild type αiib/β3 (lane 2) and αiib/β3-mut (lane 3) as shown in panel a (arrow). in addition, the β3 antibody recognized β3 proteins expressed in the wild type αiib/β3 (lane 2) and less in αiib/β3-mut (lane 3) as shown in panel b (arrow). we have then used fl ow cytometry to study the αiib/β3 expression in co-transfected cho cells. figure 3, panel a showed the presence of an inactive complex, identifi ed by the ap2 antibody in both αiib/β3 (line 2) and αiib/β3-mut (line 3). the binding capacity of ap2 (calculated as percentage gated subset) was similar between 71 the beta3 499–513 peptide region is required for alphaiib/beta3 active complex drug target insights 2008:3 wild type β3 and β3-mut. the mutation made in β3 did not interrupt the ability for the protein to be expressed, as well as being transported to the cell surface and forming the complex. panel b showed that in the absence of mn2+, the fitcpac-1 binding level (calculated as percentage gated subset) was also similar in both wild type (line 2) and αiib/β3-mut expressed complexes (line 3). however, in panel c, following incubation with 2 mm mn2+, the ability to bind fitcpac-1 increased in the αiib/β3 wild type expressed complex (line 2) but not the αiib/β3mut expressed complex (line 3). in addition to the wild type β3 and β3-mut, we have constructed two other mutants to examine the role of the cysteine residues within the peptide. we have performed replacement of cystine residues (499 and 512) one at a time with alanine. table 1 shows following mn2+ treatment, the wild type αiib/β3 had a higher percentage (92%) of pac-1 binding, compared to a lower percentage with the αiib/β3-mut (61%). replacement of either 499 (89%) or 512 (91%) with ala, the binding of pac-1 did not change signifi cantly compared to wild type. the binding of pac-1 by gp iib/iiia-c499a (89%) or gp iib/iiia-c512a (91%) did not change signifi cantly compared to wild type. however, these results do indicate that mn2+ could not fully activate αiib/β3-mutants resulting in fewer numbers of the formation of the active complex and thus less binding of fitc-pac-1. we have assayed and analyzed the effect of both the p4 and p4s peptides to the mn2+-activated αiib/β3 complex by measuring the binding of fitc-pac-1. figure 4 shows the effects of the addition of the p4 peptide. panel a shows the control cho cells while panel b represents the fitcpac-1 bound to mn2+-activated αiib/β3 complex. when cells are pre-incubated with p4 and mn2+, the binding of fitc-pac-1 is inhibited in a dosedependent manner. the percentage of inhibition is figure 1. inhibition of [3h]-fibrinogen binding to human platelets by p4 and scrambled p4 (p4s) peptides. binding assay of [3h]-fi brinogen to human platelets in the presence of various amounts of p4 (line with fi lled diamonds) and scrambled p4 peptides (line with closed squares) 0–80 µm for 30 minutes at room temperature and assessed for radioactivity. the data expressed is an average of duplicates per experiment and repeated three times with similar results. a representative study is shown. 72 woo-rasberry and chiang drug target insights 2008:3 28% (panel c, 60 µm) and 60% (panel d, 80 µm), respectively. the concentration of peptide required for inhibition was similar to that used in washed human platelets (schwartz et al. 1995). the p4s did not show any signifi cant inhibition on the binding to the mn2+-activated αiib/β3 complex (data not shown). we have established the optimal conditions for [3h]-fi brinogen binding assays [fi brinogen fi nal concentration (80 nm, cell number (5 × 106), temperature, and incubation time (60 minutes)] to determine the effects of the p4 and p4s peptides. we investigated the ability of the p4 peptide to inhibit fibrinogen binding to αiib/β3 in the presence of 2 mm mn2+. the p4s peptide was used as the control in the [3h]-fi brinogen binding assays. the addition of p4 peptide decreased the amount of fibrinogen binding to the mn2+activated αiib/β3 complex in a dose-dependent fashion (fig. 5, line with open circles), as the peptide dose increased, the amount of fi brinogen binding to the cell surface decreased. the scrambled peptide, p4s, did not inhibit the fi brinogen binding signifi cantly (fig. 5, line with fi lled squares), indicating that the order of amino acid residues in the p4 peptide sequence is important for inhibiting αiib/β3 complex formation, thus inhibiting fi brinogen binding. although, this experiment may not represent the cellular physiology and biochemistry of human platelets, it may be another useful tool to study the interaction processes. figure 2. immunoblots of wild type αiib, β3 and β3-mut proteins. the wild type cdnas of αiib and β3 as well as αiib/β3-mut were cotransfected into cho cells allowing for transgene expression 48 hours following transfection. the cells are harvested, washed, lysed, and protein concentration determined. equal amounts of protein were separated by sds-page and electroblotted for immunoblot analysis. panel a and b are 10% and 7.5% reduced sds-page, respectively. monoclonal antibodies (anti-αiib-panel a and anti β3-panel b, preabsorbed with cho cells) are used to detect the presence of each integrin. lane 1, mock control; lane 2, co-transfected cells of wild type αiib/β3 cdnas; lane 3, co-transfected cells of wild αiib and β3-mut cdnas. arrows indicates αiib protein in panel a and β3 protein in panel b. figure 3. analysis of expressed αiib/β3 wild type and αiib/β3-mut by fl ow cytometry. stable lines expressing wild type or mutant β3 and wild type αiib were incubated with different monoclonal reagents. panel a: monoclonal antibody ap2 labeled with fl uorescent dye alexa fluor 647 binds to the αiib/β3 inactive complex. panel b: pac-1-fitc binds to non-activated αiib/β3 complex. panel c: pac-1-fitc bound to 2 mm mn2+ stimulated αiib/β3 complex. line 1, mock control (cells without cdna insert); line 2, cells of wild type αiib with β3-mut cdnas lane 3, cells of wild type αiib cdna and β3 cdnas. 73 the beta3 499–513 peptide region is required for alphaiib/beta3 active complex drug target insights 2008:3 figure 4. effect of the defi ned β3 peptide (p4) on the binding of fitc-pac-1 to co-transfected cho cells. the cho cells expressing αiib/β3 inactive complex were activated with 2 mm mn2+ in the presence of various amounts of p4 for 45 minutes at room temperature. following incubation, an aliquot of pac-1-fitc was added and incubated at room temperature for an additional 60 minutes. following three washings, the fl uorescent stringency was detected by fl ow cytometry. panel a: control, (cells without cdna insert). panel b: cells expressing αiib/β3 activated with 2 mm mn2+. panel c: wild type cells activated with 2 mm mn2+ and 60 µm of peptide. panel d: cells activated with 2 mm mn2+ and 80 µm of peptide. figure 5. binding of [3h]-fi brinogen on cho cells expressing the αiib/β3 complex and the effect of the p4 peptide (open-circle line) and the p4s (square-fi lled line) from β3. the cells were incubated with 2 mm mn2+ and different amounts of peptide (0–80 µm). the y-axis indicates the percentage of [3h]-fi brinogen binding. the peptide concentrations were 10 µm, 20 µm, 40 µm and 80 µm for various time points beginning with time 0, respectively (p4, line with open circles; p4s, line with fi lled squares). the data expressed is an average of duplicates per experiment and repeated three times with similar results. a representative study is shown. 0 20 40 60 80 100 120 0 10 20 40 80 peptide concentration (µm) f ib ri n o g e n b in d in g ( % ) discussion we have previously defi ned a peptide (p4), amino acid residues from 499–513 of β3 as one of several important sites of the active conformational state of the complex αiib/ß3 (chiang and zhu, 2005). in this investigation, we have performed experiments to study its function on the formation of the αiib/β3 active complex. we have established a stable cell line that was co-transfected with wild type αiib and wild type β3 cdnas in cho cells. results revealed that the p4 peptide inhibited both fitc-pac-1 and [3h]-fi brinogen binding to the 74 woo-rasberry and chiang drug target insights 2008:3 cell surface expressed mn2+-activated αiib/β3 complex. in addition, three stable cell lines of cotransfected wild type αiib and β3-mut cdnas also demonstrated a decrease in fitc-pac-1 binding and [3h]-fi brinogen binding. these results are consistence with our earlier report that the p4 peptide inhibits binding of fitc-pac-1 on human platelets (chiang and zhu, 2005). collagen-induced platelet aggregation is mediated by the released of adp from the collagen-activated platelets. in the present studies, we have used suboptimal concentrations of adp to trigger partial platelet activation for the binding of [3h]-fi brinogen. however, under these same conditions, the addition of p4s does not inhibit the binding of [3h]-fi brinogen. in contrast, the addition of p4 after platelets become fully activated, does not inhibit fi brinogen binding. the 2,3,5,6-tetrafl uorophenyl ester (purchased from molecular probes)-labeled p4 and p4s could not bind on the fi brinogen-coated wells. these fi ndings suggest that the peptide does not bind to fi brinogen per se to lower [3h]-fi brinogen concentration in turn to decrease the binding. parise et al. (1987) and du et al. (1991) have reported that the rgd peptide (a recognition sequence for fibrinogen binding) or rgdderived peptides inhibit(s) the binding site exposed by conformational changes in platelets. our results showed that p4 peptide inhibits the binding of both fitc-pac-1 and [3h]-fi brinogen on the stably co-transfected mn2+-activated αiib/β3 complex. the dose-dependent inhibitory effect of p4 peptide indirectly leads us to postulate that the peptide binds to αiib and prevents the formation of the active αiib/β3 complex. alternatively, p4 modifi es the αiib/β3 complex conformation in some fashion to keep it in the inactive state. in our results, the peptide inhibitory effects are partial. a plausible explanation would be that there is more than one region involved in the activation of the complex formation. our results would support takagi’s fi nding, in which he suggests a “2-site docking model” from his study of ligand recognition by rgddependent integrins (takagi, 2004). blystone et al. (1995) has reported that exposure to mn2+ shifts the β3 integrin to their high affi nity state and possibly activation of the receptor or the subsequent function of β3 following activation. in addition, takagi et al. (2002) have reported that integrins, αvβ3 and αiibβ3, have a highly bent conformation and had low affinity for biological ligands under physiological condition. the addition of mn2+ resulted in a switchblade-like opening to an extended structure that has high affinity for biological ligands. our results are consistence with theirs in that upon exposure to mn2+, the αiib/β3 complex shifts from its inactive to the active state as shown by the increase in fitcpac-1 binding. cysteine residues in β3 play important roles in αiib/β3 complex activation. sun et al. (2002) reported that disruption of the long-range β3 cys5cys435 disulfi de bond resulted in the production of constitutively active αiib/β3 integrin complex. another report proposed that the cysteine residues located from 616–690 of the carboxyl-terminal region was important in enhancing ligand binding (butta et al. 2003). kashiwagi et al. (1999) found that disrupting only a single disulfi de bond in the cysteine-rich repeat region of β3 was enough to activate αiib/β3. the p4 peptide, which we have identifi ed is present within the cysteine-rich region of β3 and contains two cysteine residues, which may form a disulfi de bond with other cysteines of αiib. we used the dipro 2.0 software to predict the location, which the disulfi de bond formation would reoccur (cheng et al. 2006). according to the software results of proposed β3-mut, the predicted disulfi de bridge reforms at 513–527. altering the positions of these two cysteine residues may affect the ability of mn2+ to activate αiib/β3. the β3-mut cdna we created and expressed in cho cells was not clearly defi ned by coomassie brilliant blue stained sds-page or by immunoblot analysis with an anti-β3 monoclonal antibody (fig. 1, panel b, lane3). however, with flow cytometry, we observed the presence of the inactive complex of αiib/β3-mut with ap2 (an antibody, which recognizes the αiib/β3 inactive complex). the percentage-gated subset of ap2 binding was similar between the two different stable lines αiib/β3 and αiib/β3-mut. these results indicate that expression and transport of the αiib/β3-mut proteins to the cell surface does occur. the poor immunoblot result of β3-mut may be a consequence of the inability of the antibody to recognize the mutated sequence of amino acid residues. the αiibβ3-mut cdna expressed in cho cells did not show strong activation of the inactive complex with the addition of mn2+. our results of substitution of c499a and c512a 75 the beta3 499–513 peptide region is required for alphaiib/beta3 active complex drug target insights 2008:3 resulted in less pac-1 binding suggesting these two cysteines are also important. integrins have become attractive therapeutic targets. many drugs, which inhibit integrin αiib/ β3 binding can effectively block agonists induced platelet aggregation in vitro. however, intravenous infusion or oral ingestion of these drugs failed to effectively block pathological thrombosis in certain patients. this raises a question of “how effective is the ligand-mimetic integrin blockade?” our results demonstrates that the p4 peptide is important for interfering with αiib/β3 activation, thus inhibiting binding of fi brinogen on activated platelets and platelet aggregation in vitro, may be another candidate as a therapeutic agent. however, its functional signifi cance in vivo is not clear and requires further investigation. acknowledgements the authors wish to thank mrs. x. r. fang for her expert technical assistance, dr zhu for discussion, and dr. peter newman, the blood center of southeastern wisconsin, inc. milwaukee, wi for a αiib construct. the offi ce of biomedical laboratory research, department of veterans affairs supported the present investigation. abbreviations prp, platelet-rich plasma; tris-edta, 20 mm tris-hcl-130 mm nacl-1 mm edta, ph 7.4; ty r o d e ’s b u f f e r, 5 m m h e p e s , 2 m m mgcl2, 0.3 mm nah2po4, 3 mm kcl, 12 mm nahco3, 0.1% glucose, 0.1% bsa, and 1 mm cacl2, ph 7.0; (hepes buffer, 50 mm hepes/1% glucose/2 mm ca2+/2 mm mg2+ ph 7.4; fitc-pac-1, fluroscence isothiocyanate conjugated anti-gp iib/iiia; and cho, chinese hamster ovary cell. references beglova, n., blacklow, s.c., takagi, j. and springer, t.a. 2002. cysteinerich module structure reveals a fulcrum for integrin rearrangement upon activation. nat. struct. biol., 9:282–7. blystone, s.d., lindberg, f.p., laflamme, s.e. and brown, e.j. 1995. integrin beta 3 cytoplasmic tail is necessary and suffi cient for regulation of alpha 5 beta 1 phagocytosis by alpha v beta 3 and integrinassociated protein. j. cell biol., 130:745–54. butta, n., arias-salgado, e.g., gonzalez-manchon, c., ferrer, m., larrucea, s., ayuso, m.s. and parrilla, r. 2003. disruption of the beta3 663–687 disulfi de bridge confers constitutive activity to beta3 integrins. blood, 102:2491–7. charo, i.f., nannizzi, l., phillips, d.r., hsu, m.a. and scarborough, r.m. 1991. inhibition of fi brinogen binding to gp iib-iiia by a gp iiia peptide. j. biol. chem., 266:1415–21. chiang, t.m., beachey, e.h. and kang, a.h. 1975. interaction of a chick skin collagen fragment (α1-cb5) with human platelets. biochemical studies during the aggregation and release reaction. j. biol. chem., 250:6916–24. chiang, t.m., jin, a., hasty, k. and kang, a.h. 1989. collagen-platelet interaction: inhibition by a monoclonal antibody, which binds a 90000 dalton platelet glycoprotein. thromb. res., 53:129–43. chiang, t.m. and zhu, j. 2005. a defi ned peptide that inhibits the formation of the glycoprotein iib and iiia complex. thromb. res., 115:503–8. cheng, j., saigo, h. and baldi, p. 2006. large-scale prediction of disulphide bridges using kernel methods, two-dimensional recursive neural networks, and weighted graph matching. proteins, 62:617–29. d’souza, s.e., ginsberg, m.e., matsueda, g.r. and plow, e.f. 1991. a discrete sequence in a platelet integrin is involved in ligand recognition. nature, 350:66–8. d’souza, s.e., haas, t.a., piotrowicz, r.s., byers-ward, v., mcgrath, d.e., soule, r.d., cierniewski, c., plow, e.f. and smith, j.w. 1994. ligand and cation binding are dual functions of a discrete segment of the integrin beta 3 subunit: cation displacement is involved in ligand binding. cell, 79:659–67. du, x.p., plow, e.f., frelinger, a.l., o’toole, t.e., loftus, j.c. and ginsberg, m.h. 1991. ligands “activate” integrin alpha iib beta 3 (platelet gpiib-iiia). cell, 65:409–16. feuston, b.p. 2003. molecular model of the alpha(iib)beta(3) integrin. j. med. chem., 46:5316–25. filizola, m., hassan, s.a., artoni, a., coller, b.a. and weinstein, s. 2004. mechanistic insights from a refi ned three-dimensional model of integrin alphaiibbeta3. j. biol. chem., 279:24624–30. grinnell, f. 1980. fibroblast receptor for cell-substratum adhesion: studies on the interaction of baby hamster kidney cells with latex beads coated by cold insoluble globulin (plasma fibronectin). j. cell biol., 86:104–12. kamata, t., tieu, k.k., irie, a. and springer, t.a. 2001. amino acid residues in the alpha iib subunit that are critical for ligand binding to integrin alphaiibbeta3 are clustered in the beta-propeller model. j. biol. chem., 276:44275–83. kashiwagi, h., tomiyama, y., tadokoro, s., honda, s., shiraga, m., mizutani, h., handa, m., kurata, y., matsuzaw, y. and shattil, s.j. 1999. a mutation in the extracellular cysteine-rich repeats region of the beta3 subunit activates integrins alphaiibbeta3 and alphavbeta3. blood, 93:2559–68. litvinov, r.i., nagaswami, c., vilaire, g., shuman, h., bennett, j.s. and weisel, j.w. 2004. functional and structural correlations of individual alphaiibbeta3 molecules. blood, 104:3979–85. luo, b.h., takagi, j. and springer, t.a. 2004. locking the beta3 integrin i-like domain into high and low affi nity conformations with disulfi des. j. biol. chem., 279:10215–21. parise, l.v., helgerson, s.l., steiner, b. and phillips, d.r. 1987. synthetic peptides derived from fi brinogen and fi bronectin change the conformation of purifi ed platelet glycoprotein iib-iiia. j. biol. chem., 262:12597–602. rice, r.h. and means, g.e. 1971. radioactive labeling of proteins in vitro. j. biol. chem., 246:831–2. schwartz, m.a., schaller, m.d. and ginsberg, m.h. 1995. integrins: emerging paradigms of signal transduction. annu rev. cell dev. biol., 11:549–99. steiner, b., trzeciak, a., pfenninger, g. and kouns, w.c. 1993. peptides derived from a sequence within beta 3 integrin bind to platelet al.phaiib beta3 (gpiib-iiia) and inhibit ligand binding. j. biol. chem., 268:6870–3. sun, q.h., liu, c.y., wang, r., paddock, c. and newman, p.j. 2002. disruption of the long-range gpiiia cys(5)-cys(435) disulfi de bond results in the production of constitutively active gpiib-iiia (alpha(iib)beta(3)) integrin complexes. blood, 100:2094–101. takagi, j., petre, b.m., walz, t. and springer, t.a. 2002. global conformational rearrangements in integrin extracellular domains in outsidein and inside-out signaling. cell, 110:599–611. 76 woo-rasberry and chiang drug target insights 2008:3 takagi, j. 2004. structural basis for ligand recognition by rgd (arg-gly-asp)dependent integrins. biochem. soc. trans., 32:403–6. wang, r., peterson, j., aster, r.h. and newman, p.j. 1997. disruption of a long-range disulfi de bond between residues cys406 and cys655 in glycoprotein iiia does not affect the function of platelet glycoprotein iib-iiia. blood, 90:1718–9. whitnack, e. and beachey, e.h. 1985. biochemical and biological properties of the binding of human fi brinogen to m protein in group a streptococci. j. bacteriology, 164:350–8. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) 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inhibition of intrinsic thrombin generation thomas w. stief department of clinical chemistry, university hospital giessen & marburg, germany. abstract background: the contact phase of coagulation is of physiologic/pathophysiologic importance, whenever unphysiologic polynegative substances such as cell fragments (microparticles) get in contact with blood. there are several clinically used inhibitors of intrinsic thrombin generation. here the inhibitory concentrations 50% (ic50) of these anticoagulants are measured by the highly specifi c thrombin generation assay inca. methods: unfrozen pooled normal citrated plasma in polystyrole tubes was supplemented at 23°c in duplicate with 0–2 iu/ml low molecular weight heparin (dalteparin), 0–2 iu/ml unfractionated heparin, 0–500 kiu/ml aprotinin, or 0–40 mm arginine. 50 µl plasma or 1 iu/ml thrombin standard were pipetted into a polystyrole microtiter plate with fl at bottom. 5 µl sio2/ cacl2 reagent (inca activator) were added and after 0–30 min incubation at 37°c 100 µl 2.5 m arginine, ph 8.6, were added; arginine inhibits hemostasis activation and depolymerizes generated fi brin within 20 min at 23°c. the in the physiologic 37°c incubation phase generated thrombin was then chromogenically detected. the intra-assay cv values were < 5%. results and discussion: the approximate ic50 were 0.01 iu/ml dalteparin, 0.02 iu/ml heparin, 25 kiu/ml aprotinin, and 12 mm arginine. the effi ciency of any anticoagulant on intrinsic thrombin generation should be measured for each individual patient. abbreviations: iia, thrombin; ∆a, increase in absorbance; aptt, activated partial thromboplastin time; crt, coagulation reaction time (at 37°c in water-bath); f-wells, polystyrole microtiter plates with fl at bottom; ic50, inhibitory concentration 50%; inca, intrinsic coagulation activity assay; iu, international units; kiu, kallikrein inhibiting unis; lmwh, low molecular weight heparin; ma, milli-absorbance units; psl, pathromtin sl®; rt, room temperature (23°c); u-wells, polystyrole microtiter plates with round bottom. keywords: inca, thrombin, lmw-heparin, dalteparin, heparin, aprotinin, arginine introduction low molecular weight heparins (lmwh) are essential drugs for patients inside and outside the hospital; unfortunately, the global hemostasis assay aptt is not sensitive to lmwh [fareed et al. 2004]. there is clinical need for a simple physiologic global hemostasis test that monitors the anticoagulant power of lmwh. the only lmwh routine assay currently available is the anti-xa assay, that is available in a clotting [denson and bonnar, 1973] and in a chromogenic version [teien et al. 1976]. however, the target therapeutic dose range in the anti-xa assay is 0.4–0.7 iu/ml heparin in the anti-xa assay but only 0.2–0.4 iu/ml heparin in the aptt [kitchen, 2000]. this discrepancy indicates that the xa activity added in the anti-xa assays might be supra-physiological and that the other pharmacologic actions of the heparins than just the inhibition of activated factor x are not refl ected by the anti-xa assay. recently, a new test for thrombin activity in plasma was developed [stief, 2006; stief et al. 2006]. this assay uses (i) a chromogenic thrombin substrate at fi nal concentrations < 0.6 mm and (ii) arginine at fi nal concentrations > 1 m, resulting in highly specifi c thrombin determination. of diagnostic importance are the following chromogenic thrombin tests that all base on this new technique: 1. basal thrombin activity (iia) 2. recalcifi ed coagulation activity assay (reca) 3. intrinsic coagulation activity assay (inca) 4. extrinsic coagulation activity assay (exca). as equipment only a normal microtiterplate reader is required; these only 1 or 2 measuring point based tests are easy to handle with high reproducibility (all have intra-assay cv values less than 5%), correspondence: t.w. stief, md, priv.-doz. department of clinical chemistry, university hospital of giessen & marburg, d-35033 marburg, germany. fax: +49-6421-286 5594; email: thstief@med.uni-marburg.de please note that this article may not be used for commercial purposes. for further information please refer to the copyright statement at http://www.la-press.com/copyright.htm perspectives drug target insights 2006:1 5-10 5 inhibition of intrinsic thrombin generation they are fast and economical, and can thus be used in routine diagnostic. material and methods inca coagulation reaction time the inca is a new simple global chromogenic hemostasis test that requires only a two-point determination of thrombin generation in the important ascending part of the thrombin activity curve, i.e. the ratio between thrombin at a second time point divided by thrombin at a fi rst time point should be > 1. 50 µl unfrozen plasma (1 part 106 mm citrate + 9 parts of venous blood; centrifuged at 2800 g (4000 rotations per minute at 23°c) are pipetted into flat bottom polystyrole microtiter plate wells (f-wells, polysorp®, nunc, wiesbaden, germany; article nr. 446140). the inca is started by addition of 5 µl sio2/cacl2 reagent (freshly with 278 mm cacl2 1:10 diluted pathromtin sl®, dadebehring, marburg, germany). always h2o-rinsed completely emptied new disposable polypropylene tips for the eppendorf-multipette® are used: if this disposable tip of the multipette for addition of the inca-activator to the reaction well is not rinsed, the intrinsic thrombin generation is strongly inhibited; some releasable plastic material in the new tip seems to act like a heparinoid; if the untreated polystyrole f-well plate (polysorp®) is replaced by an irradiated one (maxisorp®) or a u-well plate (for sample volumes < 25 µl u-wells result in higher precision than f-wells), then this plastic material inhibits the intrinsic coagulation to some extent. inca inhibition by heparins unfrozen pooled normal citrated plasma in 5 ml polystyrole tubes was supplemented with 0–2 iu/ ml dalteparin (a lmwh; fragmin p®, pharmacia, erlangen, germany) or with 0–2 iu/ml unfractionated heparin (roche, basel, switzerland) and kept at 23°c. 50 µl samples were pipetted into f-wells, and the inca was started by addition of 5 µl sio2/ cacl2 reagent. after 3 min coagulation reaction time (a crt of 3 min is the initial time point of thrombin generation) at 37°c in the water-bath, 100 µl 2.5 m arginine, ph 8.6 (sigma, deisenhofen, germany) were added. the plate was withdrawn from the water-bath. after 20 min (23°c = rt) 10 µl 3.85 mm chromogenic thrombin substrate hd-chg-ala-arg-pna (pentapharm, basel, switzerland) in h2o were added and ∆a/t was determined by a microtiterplate reader with a 1 ma resolution (milenia-dpc, los angeles, usa). the result was standardized against 1 iu/ml iia in 6.7 % human albumin (kabi, stockholm, sweden) replacing the plasma sample. inca kinetic in plasma supplemented with heparins 20 µl unfrozen pooled normal plasma, supplemented with 0 iu/ml, 0.063 iu/ml, 0.13 iu/ml, 0.25 iu/ml dalteparin or 0 iu/ml, 0.063 iu/ml, 0.13 iu/ml, 0.25 iu/ml, or 0.5 iu/ml unfractionated heparin were incubated in u-wells (nunc) with 2 µl sio2/cacl2 reagent. after 0–30 min (37°c), 50 µl 2.5 m arginine, ph 8.6, were added. the plate was withdrawn from the water-bath. after 20 min (23°c) 20 µl 0.77 mm chromogenic thrombin substrate in 2 m arginine was added and the linear ∆a/t (rt) was determined. inca inhibition by aprotinin unfrozen pooled normal plasma in polystyrole tubes was supplemented with 0–400 kiu/ml aprotinin (bayer, leverkusen, germany). the inca was performed with 20 µl plasma samples in u-wells and 2 µl inca-activator. after 0–30 min (37°c), 50 µl 2.5 m arginine, ph 8.6, were added. after 20 min (rt) 20 µl 0.77 mm chromogenic substrate in 2 m arginine was added and the linear ∆a/t (rt) was determined. additionly, the aptt of the aprotinin-supplemented plasma samples was measured by a behring coagulation timer®. inca inhibition by arginine unfrozen pooled normal plasma in polystyrole tubes was supplemented with 0–40 mm arginine (braun, melsungen, germany) or alkaline arginine (ph = 8.7; sigma). 50 µl samples were tested in the inca (f-wells). infl uence of fi brin on anticoagulants in inca unfrozen pooled normal plasma with 2.8 g/l fi brinogen was unsupplemented and supplemented with purifi ed human fi brinogen (haemocomplettan, aventis, frankfurt, germany; the preparation drug target insights 2006:16 thomas w. stief contained about 1% soluble fi brin [stief, 2000] that might act as antithrombin i [mosesson, 2005]) to a fi nal active fi brinogen concentration of 4.0 g/l. the plasmas were then unsupplemented or supplemented with 0.01 iu/ml dalteparin, 0.02 iu/ml heparin, 25 kiu/ml aprotinin, or 12 mm arginine. the inca was performed as described above. addition of 0.1% triton x 100® (sigma) fi nal test concentration accelerates the decrease of the fi brin-related turbidity about 7fold, i.e. instead of 20 min arginine reaction time 3 min is suffi cient before starting the iia detection phase of the assay. a turbidity increase in plasma occurs within minutes, if the fi nal thrombin activity exceeds 0.02 iu/ml [stief, 2007 in press]. results the action of heparins on inca is shown in figure 1. the 50% inhibitory concentrations (ic50) were about 0.01 iu/ml for lmwh and 0.02 iu/ml for unfractionated heparin (fig. 1a). in therapeutical heparin concentrations (about 0.2 iu/ml), 0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0 0.05 0.1 0.1 5 0.2 0.25 0.3 heparin conc. [iu/ml] ii a a ct iv it y 3 m in c r t [ iu /m l] figure 1. inhibition of intrinsic thrombin generation by heparins. figure 1a. ic50 determination. unfrozen pooled normal plasma, supplemented with dalteparin (●) or unfractionated heparin (■) was analyzed in the inca (50 µl sample into f-wells). the inca was performed with a thrombin generation time = coagulation reaction time of 3 min (initial phase of thrombin generation). 10 µl 3.85 mm chg-ala-arg-pna in h 2o were added and ∆a/t was measured. 1 iu/ml iia had 12.2 ma/min rt. 0 1 2 3 4 5 6 7 0 5 10 5 crt [min] iia a ct iv ity [i u /m l] 1 20 25 30 35 figure 1b. reaction kinetic of dalteparin. 20 µl unfrozen pooled normal plasma, supplemented with 0 iu/ml (o), 0.063 iu/ml (■), 0.13 iu/ml (♦), or 0.25 iu/ml (▲) dalteparin was incubated with 2 µl sio2/cacl2 reagent. after 0–30 min crt at 37°c in the water-bath 50 µl 2.5 m arginine, ph 8.6, was added. after 20 min (rt) 20 µl 0.77 mm chromogenic thrombin substrate in 2 m arginine was added and the linear ∆a/t (rt) was determined. drug target insights 2006:1 7 inhibition of intrinsic thrombin generation 0 1 2 3 4 5 6 7 0 5 10 15 20 25 30 35 iia a ct iv ity [i u /m l] crt [min] the thrombin generation in the inca at crt < 20 min does not exceed 0.5 iu thrombin/ml sample (fig. 1b,c). the inca curves are evaluated in their important pre-maximum phase. the ic50 of aprotinin on the inca is about 25 kiu/ml (fig. 2). this reflects the extreme sensitivity of the inca: in the usual aptt the aprotinin concentration that prolongs the normal aptt 1.5 fold is 300 kiu/ml. arginine dose-dependently inhibits inca (fig. 3). the ic50 values were about 12 mm for commercially available arginine for i.v. infusion and about 8 mm for alkaline arginine (at ph 8.7). normal plasma supplemented with 1.2 g/l purifi ed fi brinogen containing 1% soluble fi brin resulted in a 50% decrease of iia–generation: plasmatic fi brin entraps generated thrombin, that is why fi brin can be considered as antithrombin i (8). figure 1c. reaction kinetic of unfractionated heparin. 20 µl unfrozen pooled normal plasma, supplemented with 0 iu/ml (o), 0.063 iu/ml (■), 0.13 iu/ml (♦), 0.25 iu/ml (▲), or 0.5 iu/ml (●) heparin was incubated with 2 µl sio2/cacl2 reagent. after 0–30 min crt at 37°c in the water-bath 50 µl 2.5 m arginine, ph 8.6, was added. after 20 min (rt) 20 µl 0.77 mm chromogenic thrombin substrate in 2 m arginine was added and the linear ∆a/t (rt) was determined. 0 0.5 1 1.5 2 2.5 3 0 5 10 15 20 25 30 35 iia a ct iv it y [i u /m l] crt [min] figure 2. inhibition of intrinsic thrombin generation by aprotinin. figure 2a. 20 µl unfrozen pooled normal plasma, supplemented with 0 kiu/ml (o), 25 kiu/ml (∆), 50 kiu/ml (□), 100 kiu/ml (■), 200 kiu/ml (▲), or 400 kiu/ml (●) aprotinin, were tested in the inca (u-wells). drug target insights 2006:18 thomas w. stief this decrease in iia-generation was independent of added anticoagulant. discussion the aptt does not refl ect the effi ciency of some important clinically used anticoagulants, such as e.g. the lmwh [fareed et al. 2004]. the sio2 amount used to trigger the inca is about 100fold below the contact activator amount used for the aptt, and in contrast to the usual global coagulation test aptt the plasma matrix in the inca is not signifi cantly changed (only 1 part of reagent to 10 parts of citrate plasma). the inca monitors the anticoagulant potency of anti-factor xa or anti-thrombin drugs [tobu et al. 2004]: lmw-heparins are powerful inhibitors of iia generation, especially via inhibition of factor xa. in an inca test version with an assay incubation at 37°c prolonged to 12 min (inca-12) patient aptt values of about 40 s correspond to about 1 iu/ml iia generation, aptt values of 50–60 s to about 0.2 iu/ml iia (< 36 s = 100% of norm aptt; 4.3 ± 1.4 iu/ml iia = 100% of norm iia generation) [stief et al. 2006]. the usual plasmatic unfractionated heparin concentrations in therapeutic anticoagulation are about 10fold higher than the ic50 values observed here in the inca. the superior anticoagulant capacity of lmwh compared to unfractionated heparin might be due to some contact activating 0 0.5 1 1.5 2 2.5 3 0 50 100 150 200 250 300 350 400 450 iia a ct iv ity [i u /m l] aprotinin activity [klu/ml] figure 2b. crt = 10 min (●), crt = 15 min (■). 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 0 2 4 6 8 1 4 16 iia a ct iv ity [i u /m l] 0 112 crt [min] figure 3. inhibition of intrinsic thrombin generation by arginine. figure 3a. unfrozen pooled normal plasma was supplemented with 0–40 mm arginine. 50 µl samples in f-wells were tested in the inca: 0 mm (o), 5 mm (▲), 10 mm (●), 20 mm (♦), or 40 mm (■) arginine. drug target insights 2006:1 9 inhibition of intrinsic thrombin generation (pre-kallikrein to kallikrein folding) potential of unfractionated heparin, a polynegatively charged molecule of a molecular mass > 15000 dalton [kongsgaard et al. 1992]. in many clinical situations cells can fragment into phospholipidmicroparticles that activate the intrinsic pathway of hemostasis and are thus of great pathophysiologic importance [soriano et al. 2005; boulanger et al. 2006]. 10–20 mm arginine are common plasmatic concentrations in the so-called arginine-test in internal medicine [appleton et al. 2002; cylwik et al. 2005]. therefore, arginine might be a new therapeutic option in patients with severe disturbances of the coagulation system [lee and downey; 2000]. it is suggested to measure the effi ciency of any anticoagulant on intrinsic thrombin generation for each individual patient. references appleton, j., arginine, 2002. clinical potential of a semi-essential amino. alternative medicine revue, 7:512–522. boulanger, c.m., amabile, n. and tedgui, a. 2006. circulating microparticles: a potential prognostic marker for atherosclerotic vascular disease. hypertension, 48:180–186. cylwik, d., mogielnicki, a. and buczko, w. 2005. arginine and cardiovascular system. pharmacological reports, 57:14–22. denson, k.w.e. and bonnar, j. 1973. the measurement of heparin: a method based on the potentiation of anti factor xa. thromb. diath. haemorrh., 30:471–79. fareed, j., ma, q., florian, m., maddineni, j., iqbal, o., hoppensteadt, d. and bick, r.l. 2004. differentiation of low-molecular-weight heparins: impact on the future of the management of thrombosis. seminars in thrombosis and hemostasis, 30 suppl. 1:89–104. kitchen, s. 2000. problems in laboratory monitoring of heparin dosage. brit. j. haematol., 111:397–406. kongsgaard, u.e., aasen, a.o., smith-erichsen, n. and bjornskau, l. 1992. effects of heparin on proteolytic activities in human plasma. eur. surg. res., 24:119–28. lee, w.l., downey, g.p. 2000. coagulation inhibitors in sepsis and disseminated intravascular coagulation. intensive care medicine, 26:1701–1706. mosesson, m.w. 2005. fibrinogen and fi brin structure and functions. j. thromb. haemost., 3:1894–904. soriano, a.o., jy, w., chirinos, j.a., valdivia, m.a., velasquez, h.s., jimenez, j.j., horstman, l.l., kett, d.h., schein, r.m. and ahn, y.s. 2005. levels of endothelial and platelet microparticles and their interactions with leukocytes negatively correlate with organ dysfunction and predict mortality in severe sepsis. crit. care med. 33:2540–2546. stief , t.w. 2000. functional determination of soluble fi brin polymers (sfp) in plasma. thromb. haemost., 84:1120–1121. stief, t.w. 2006 specifi c determination of plasmatic thrombin activity. clin. appl. thromb. hemost., 12:324–329. stief, t.w., the fi brinogen function turbidimetric assay (fifta). clin. appl. thrombosis/hemostasis (in press). stief, t.w., otto, s. and renz, h. 2006. the intrinsic coagulation activity assay. blood coagul. fibrinol., 17:369–78. teien, a.n., lie, m. and abildgaard, u. 1976. assay of heparin in plasma using a chromogenic substrate for activated factor x. thromb. res., 8:413–16. tobu, m., iqbal, o., hoppensteadt, d., neville b., messmore h.l. and fareed, j. 2004. anti-xa and anti-iia drugs alter international normalized ratio measurements: potential problems in the monitoring of oral anticoagulants. clinical and applied thrombosis/ hemostasis 10:301–309. 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 0 5 10 15 20 25 30 35 40 45 arginine conc. [m m] iia a ct iv ity [i u /m l] figure 3b. 4 min crt (▲), 5 min crt (●), 6 min crt (■), 7 min crt (x), 8 min crt (*). drug target insights 2006:110 debouzy et al.indd original research correspondence: jean-claude debouzy, centre de recherche du service de santé des armées, 24, avenue des maquis du grésivaudan, bp 87, 38702 la tronche cedex france. tel: (33) 4 76 63 69 39; fax: (33) 4 76 63 69 22; email: jcdebouzy@crssa.net copyright in this article, its metadata, and any supplementary data is held by its author or authors. it is published under the creative commons attribution by licence. for further information go to: http://creativecommons.org/licenses/by/3.0/. study of alkylglycerol containing shark liver oil: a physico chemical support for biological effect? jean-claude debouzy1, david crouzier1, bertrand lefebvre2 and vincent dabouis1 1unité bcm centre de recherches du service santé des armées, 24, avenue des maquis du grésivaudan, bp 87-38 702 la tronche cedex, france. 2unité de biospectrométrie centre de recherches du service santé des armées, 24, avenue des maquis du grésivaudan, bp 87-38 702 la tronche cedex, france. abstract: shark liver oil (slo), is used in natural medicine as immunity stimulant, cardiovascular protector and anti ageing reagent. these properties were related with the high amounts of alkylglycerols (22%) obtained from greenland shark liver. after a control of the mean slo composition by nmr and ms, surface and membrane interactions and antioxidant properties were investigated using nmr, esr and st measurements and the in vitro consequences on erythrocytes and cells were studied. an estimation of the composition of this extract was performed. moreover, slo was found not haemolytic (a concentration inducing 50% haemolysis, hc50 could not be reached) and superfi cial tension measurements revealed slight tension active properties. the 31p and 2h-nmr and esr studies of phospholipid dispersions (dimyristoyl phosphatidyl cholin, dmpc) in the presence of slo showed a signifi cant increase in membrane fl uidity at low temperature (below phase transition temperature) predominantly observed at the surface level. the anti oxidant activity was also confi rmed, similar as that observed for vitamin e. keywords: alkylglycerol, membrane fl uidity, esr, nmr, anti oxidant properties 1. introduction greenland sharks, and especially somnosius microcephalus are very robust species well adapted to hard environmental conditions such as deep and cold surroundings. in empiric traditional scandinavian medicine, meat and oil from greenland shark have been extensively used for healing of wounds, physical stress tolerance, and also immune stimulation and antitumor properties. these uses continuously faded out during the 19th century until early 20th when specifi c lipids (up to 50% in shark livers [1]) were identifi ed as alkylglycerols [2] (1-o-alkyl-2,3-diacylglycerols and their metoxy derivatives [3]). this led to the fi rst trials of a.brohult [4] who evidenced increased production of granulocytes and thrombocytes and proposed the use of alkylglycerols to counterbalance the bone marrow depletion after radiotherapy in the therapy of carcinomas of the uterine cervix [5]. later, alkyglycerols have been found to inhibit the growth and spread of transplanted or chemically induced tumors [6]. immunological system stimulation was also identifi ed, leading to bacteriostatic properties. it is noteworthy that the same substance, shown to be effective per os, exhibited both immunoreactivity stimulating and antitumor activity. among the different mechanisms proposed, the ability of alkylglycerol to penetrate the cell membranes would stimulate the body’ s own defense system, mainly the macrophages. besides, the results found when alkylglycerol was given before radiotherapy would also support the hypothesis of a direct interaction with radio induced free radical production. however, no precision of the mechanism involved was clearly proposed at the molecular level. this led us to investigate the biophysical properties of shark liver oil (slo), the commercial form, by using both nmr and esr spectroscopies, and assignment biophysics methods both in synthetic systems (phospholipidic membranes) and in red blood cells. experimental materials alkyrol® alkyrol® oil extract from greenland shark liver was purchased by nutrilys® company (divonne les bains, france) and used without further purifi cation. as this extract is a natural mixture, the amounts drug target insights 2008:3 125-135 125 http://creativecommons.org/licenses/by/3.0/ http://creativecommons.org/licenses/by/3.0/ debouzy et al drug target insights 2008:3 of slo are better expressed in this paper in mg rather than in mm concentrations (even if the apparent density was estimated at 0.71). this product was characterized by nmr (see fig. 1 for peak assignment) and ms (es+) analysis. as major ms peaks were found at m/z = 701; and also 633; 517; 351(m/2z), and from 1h-nmr peak integration and 13c direct spectra and dept datas, an very coarse estimation of the mean apparent molecular weight m#650 was proposed slo. the corresponding molar ratios might be considered as only indicative and close to w/w ratios (molecular mass for dimyristoylphosphatidylcholine, dmpc, is 678). chemicals dimyristoylphosphatidylcholine (dmpc), egg yolk phosphatidylcholine (epc), and deuterated solvents were purchased from sigma (la verpillère, france) and were used as received. chain perdeuterated dmpc-d54 was from interchim, montluçon, france. multibilayers (mlv) dmpc liposomes for 31p experiments were prepared in pure deuterated water by successive freezing and thawing cycles [7] until an homogenous milky sample was obtained. [8] the suspensions were degassed under nitrogen gas then introduced into nmr tubes and sealed. the fi nal lipid concentration was 50 mm (in 500 μl samples), while slo/dmpc in mixed systems was ranged from 1/50 to 1/25, mg/m. the same procedure was used for multilayers for 2h-nmr experiments, except that 25% dmpc with perdeuterated chains were used (dmpc-d54) to build the liposomes. methods haemolytic activity all procedures were in accordance with the standards for animal care established by our institute and were approved by our animal use ethic committee (decree 87-848 19 october 1987). blood from male sprague-dawley rats was collected in heparinated tubes and washed twice using isotonic nacl solution; the hematocrit was then brought to 10%. 1 ml cuves were fi lled with the slo solutions to test (0 to 32 μl) in 50 μl of dmso and with 100 μl of the diluted blood in saline. the samples were stocked for 1 hour at 37 °c, then centrifuged at 2400 rpm, 4 °c for 10 minutes. absorption measurements were fi nally performed on a shimazu mcs-2000 absorption spectrometer at 540 nm, as described elsewhere. [1, 9] the haemolytic activities were expressed in terms of hc50, the concentration giving 50% haemolysis as referenced to i) the total haemolysis induced by triton x-100 addition or on sonicated samples ii) the absence of any haemolysis (0% haemolysis) evaluated on samples where only isotonic nacl (0.9% w/w) solution was added. nmr experiments all nmr experiments were recorded on a brüker am-400 spectrometer. 1h-nmr spectra in d2o were acquired at 298 k using a presaturation of the water resonance and a spectral width of 10 ppm. the chemical shifts were referenced by setting the water resonance at 4.75 ppm. 1h-nmr control spectra were recorded using classical 1d and 2d (cosy, tocsy (sanders, 1989) experiments at 300 k, 2 mg, in perdeuterated di methyl sulfoxide (dmso-d6). in 1h-nmr t1 and t2 measurements in water preparation (1 mg, d2o, 298 k) used the inversion recovery method [10] with a 10 ppm line width and a 5sec recycling delay to ensure relaxation. figure 1. top: proton nomenclature used for acyl chain labeling; middle: 1h-nmr spectrum of slo; 1 mg in cdcl3, 298 k: bottom: 1 mg in d2o, 298 k; glycerol proton labeling: g1, g3, methylenic groups, g2, methinic group. 126 study of alkylglycerol containing shark liver oil drug target insights 2008:3 partition coefficient (logp) calculation was realized by using an nmr method derived from the classical shake-fl ask method [9]: a fi rst 1h-nmr spectrum was recorded acquired as previously in water, while using. 1 mg slo in 1 ml d2o to ensure that the nmr observation area is fully included in the aqueous solution. then an equal volume (1 ml) of perdeuterated octanol was added, and the sample vortexed and centrifugated to allow phase separation. the octanol phase was then removed. another spectrum was the acquired and the intensity signal i (that of ch3 at 0.89 ppm) compared to the intensity of slo in pure water, (io). thus, if r = i/io, i.e. the remaining fraction of slo in the water, p = (1-r)/r gives the partition in the sample, and the partition coeffi cient logp is obtained by logp = log((1−r)/r) 31p-nmr experiments were performed at 162 mhz. phosphorus spectra were recorded using a dipolar echo sequence (π/2-t-π-t) [11] with a t value of 12 μsec and a broadband two levels proton decoupling π/2 pulse was 4.8 μs, recycling delay of 5sec. phosphoric acid (85%) was used as external reference. undecoupled spectra and partial continuous wave proton low level decoupling (24l) were also used to measure phosphorusproton coupling constants. 2h-nmr experiments were performed at 61 mhz. mlv were formed as for 31p experiments whereas in deuterium depleted water. deuterium spectra were recorded by using a quadrupolar echo sequence (π/2-t-π/2-t) with a t value of 20 μsec; π/2 pulse was 8 μs and recycling delay of 15sec. the free induction decay was shifted by fractions of the dwelling time to ensure that its effective time for the fourier transform started at the top of the echo. surface tension measurements measurements were done on a tensiometer csc-du nouy (csc n°70535) using the ring method of measurement in 20 ml of water. pure water from milliq (18.2 mω.cm) was used as reference (75.8 mn/m at 293 k). electron spin resonance (esr): spin trapping investigation anti radical activity was assessed by in vitro spin trapping experiment. reactive oxygen species were generated immediately before esr experiment by a fenton reaction (feso4, 0.1 mm and h2o2, 0.1 mm). the formation of short-lived radical species (.oh) was evidenced by addition of water soluble α-(4-pyridyl-1-oxide)-n-t-butylnitrone (4-pobn) (sigma, france) at 150 mm (in dmso/ h2o solution 5% v/v) spin trapping agent. reaction was performed in an eppendorf tube, 100 μl of feso4 were mixed with 100 μl of 4-pobn spin trap and with 2 μl of alkyrol®. the trigger of reaction was performed by adding 100 μl of h2o2. reference samples were prepared by replacing alkyrol® by distilled water, anti radical properties were also compared by replacing alkyrol® by 2 μl of vit e. the samples were transferred in 20 μl pyrex capillary tube, an placed in 3 mm diameter quartz holder. the spectra were acquired using the continuous wave mode with a esp 380 (brucker) esr spectrometer, operating at a microwaves frequency of 9.71 ghz. the instrumental parameter were: microwave power of 10 mw, modulation frequency at 100 khz with a modulation amplitude of 0.51 g, receiver gain was 6.30 × 104 and scan range was 70 g with magnetic fi eld centred at 3430 g. each sample was scanned 3 times at controlled temperature 295 k, with the following acquisition parameters: time constant 20.48 ms, conversion time 20.48 ms and 5 repetitions. figure 6 b shows typical esr spectra of the control groups with the 4-pobn spin trap. an estimation of free radical promotion was obtained by measuring the amplitude of the central doublet. esr spin label study the fl uidity of rat red cell membrane was investigated by esr spin label experiments. two spin labels (sigma france) were used: 5 nitroxide stearate (5 ns) and 16 nitroxide stearate (16 ns). this fatty acids self incorporate the membrane and the nitroxide groups provide information of motional freedom of the label in biological membrane. so the former probes the superfi cial part of the membrane layer, the latter in its hydrophobic core. [12] the experiments were performed on rat red cells. the erythrocytes were isolated from fresh b l o o d b y c e n t r i f u g a t i o n a t 4 ° c ( 1 0 m i n , 1000 × g), then rinsed using saline, recentrifuged, 127 debouzy et al drug target insights 2008:3 this procedure being repeated until a clear supernatant was obtained, then brought to 30% packed cell volume. 2 μl of alkyglycerol solution were added in each 1 ml sample and then labelled with 20 μl of spin label solution (5 ns 10−3m or 16 ns 10−3m). after 30 min incubation at room temperature, sample were transferred by capillarity in 20 μl pyrex capillary tube. this tube was placed in a 3 mm diameter quartz holder, and insert into the cavity of the esr spectrometer. the esr spectra were recorded at different controlled temperature (288, 293, 298, 303, 310 and 315 k) with the following conditions: microwave power 10.00 mw, modulation frequency 100 khz, modulation amplitude 2.05 g, receiver gain 6.105conversion time 40.96 ms, time constant 20.48 ms. sweep range was 160 g with a central fi eld value of 3435 g. the complete membrane incorporation of the spin labels was ascertained by the absence on the spectra of the extremely resolved esr lines corresponding to free rotating markers. 5 ns experimentations: the value of outer and inner hyperfine splitting were measured (2t// and 2t⊥ respectively), on esr specra (fig. 5b), and order parameter s was calculated following the equation: [13] s t t c t t c = × − ⊥ +( ) + ⊥ +( ) 1 723 2 . // // with c t t= − × − ⊥( )1 4 0 053. . // the increase of the order parameter value means a decrease of local membrane fl uidity. 16 ns experimentations: the changes in freedom motion of 16 ns were analyzed with the calculation of τc, the rotational correlation time. τc was calculated following the formula: [14] tc k w h h= × ( ) −( )−δ 0 0 1 1/ with k = 6.5 × 10−10 s.g−1 in this formula, δw0 is the peak-to-peak line width of the central line; h0 and h−1 are the peak high of the central and high-fi eld lines respectively (fig. 5c). the decrease of the rotational correlation time means a decrease of local membrane fl uidity. mass spectroscopy the es-control spectra were acquired in ch2cl2 as solvent with 1% formic acid, using a vg.quatro ii spectrometer from micromass/waters, and treated with the masslink 4.00 v software. the capillary tension was 3.88 kv, and the cone tension and ion energy 88 v and 1.8 v, resolution values were set to 15.2, and the multipliers 1 and 2 set to 650 v. investigations and results slo structure evaluation in solution and in water samples chloroformic solution as expected a true solution of slo was obtained in chloroform (see fig. 1 top trace) and the control of slo main composition could be easily obtained from standard 1d and 2d 1h-13c-31p nmr and es-ms experiments. especially, no other hydrophobic components such as phospholipids, sterols an squalene were detected and the resonances of glycerol moiety (labeled g1,2,3) and those of the chain (see the nomenclature on the fig. 1) were clearly identifi ed. as relaxation times were quite homogenous (relaxation times t1 and t2 close to 1sec) [15] an estimation of the average length and insaturation of the chains was obtained by building indexes from 1h-nmr peak integrals as follows: as shown on figure 1 the resonance labeled (4) at 5.2 ppm is representative of methynic group; however, since this resonance is completely overlapped by glycerol signal (g2), the unambiguous resonance of methylenic groups (3) nei ghbouring methynic (4) was used. the resonances (5) were representative of polyunsaturation, and terminal methyle peaks (7) of number of chains. for each group, the value of the integral was divided by the corresponding number of protons (3, for methyl, 2 for methylen) to allow a count of the number of groups. finally, an estimation of the chain length reference, a, was obtained by adding all the weighted resonances 1,2,3,4,5,6 and 7, with subtraction of half the contribution of g1 methylenic group of glycerol (at 4 ppm) to overcome the g2 (ch) contribution at 5.2 ppm. 128 study of alkylglycerol containing shark liver oil drug target insights 2008:3 within the different samples controlled, no variation exceeded 10% from the following values: number of groups per chain; a/(7) = 19 +/− 2 chain insaturation index: (3)/a = 10% +/−1% chain polyinsaturation index: (5)/a = 4.9% +/− 0.5%, thus indicating a good homogeneity within the different samples used. ms spectra confi rmed this homogeneity by giving exclusively a dominant line at m/z = 351.9, another of half the intensity at m/z = 517 and four minor components at m/z = 301-417-467-633. aqueous samples depending on chain length and insaturation found, partial apparent solubilization in the water was not excluded. [16] hence, 1h-nmr lines were detected on the nmr aqueous sample containing 1 mg slo (see fig. 1, bottom trace). however, the linewidthes measured (from 30 to 50 hz) suggested the that supramolecular assemblies had been formed, such as micelles or droplets. this led to measure t1 and t2 relaxation times. these parameters are closely related to the correlation time τc and the volume of the system as classically described following the relations: [17] 1/t1 = r1 = a . τc . [(1/(1 + ω 2τc 2) + 4/(1 + 4ω2τc 2)] (1) 1/t2 = r2 = 6a . τc . [4 + 9/(1 + ω 2τc 2) + 6/(1 + 4ω2τc 2)] (2) with ω = 400 mhz; a = γ4.(h/2π)2/r6; γ the gyromagnétic factor, (h/2π) planck’s constant and r the inter spins distance. similar relaxation values were found within the molecule (t2 = 30+/− 10 ms, t1 = 420+/− 20 ms) except for terminal methyl groups (resonance 7, t2 = 50 ms, t1 = 310 ms). this allowed to calculate the range of correlation time by using the ratio t1/t2 as follows: r1/r2 = [(1/(1 + ω2τc 2) + 4/(1 + 4ω2τc 2)]/ 6[4 + 9/(1 + ω2τc 2) + 6/(1 + 4ω2τc 2)] (3) relation in ω2τc 2 simplifi ed in a second degree equation giving ωτc.limits (from 1 to 3). then, assuming a spherical approximation for the molecular assembly, the stockes-einstein relation allows an evaluation of the average apparent volume: τc = ηv/kt, soit v = kt. τc/η (4) where η = 0.9 × 10−3p, (n.s/m2 at 298 k), k = 1.38 × 10−23j/kg, t = 297 and v the volume (m3). finally, 6 � v � 12 nm3 corresponding to a 50å diameter. such assemblies are signifi cantly smaller than small unilameller vesicles of phospholipids (typically of 10–20 nm radius, with t1 in the 450–900 ms range and linewidths of 40 to 120 hz) [18]. from these features, collective properties of slo could be studied by using these aqueous dispersion in biological medium that organic solutions precluded. by considering the great importance of interfacial systems in biology, such as cell surfaces, complementary physico chemical tests were then performed. surface properties the partition coeffi cient (logp) was calculated as described in the method section. the value logp = 1.3 well confi rmed that, even if the solubility in organic solvent—octanolis more than tenfold that in water, the presence of slo at the interfacial area is highly probable. as a consequence, possible tensioactive properties had to be tested. the result is shown on figure 2: starting from 75.8 mn/m (pure water at 293 k) successive additions of slo resulted in progressive diminution figure 2. superfi cial tension (dg-g°, mn/m), 298 k as a function of slo concentration (mg/ml) (•), and percentage of haemolysis following the concentration of slo (• ). 129 debouzy et al drug target insights 2008:3 figure 3. 31p-nmr of dmpc: column: a) typical spectrum of (top) dmpc bilayers (50 mm) close to transition temperature (296 k) (bottom) and in the presence of slo (slo/dmpc = 1/25 w/w) ; column b) spectra of ghosts prepared from rat erythrocytes (top), and in the presence of 3 mg slo (bottom). bottom traces: temperature dependence of the chemical shift anisotropy for pure dmpc (•), and slo/dmpc systems, 1/25 w/w ( ) and 2/25 w/w (∆). the arrow indicates the point of the curve corresponding to the top traces. of surface tension down to st = 53 mn/m around 5 mg/ml. higher amounts of slo induced no further decrease of st. such a limited evolution runs counter any detergent effect or soap-like interactions of slo, as found for instance for sds or amphiphilic [19] molecules like cyclodextrins. [20] however, these negative tensioactive properties suggest interactions with membranes. this point is the topic of the next section. membrane structure and dynamics study by 2h and 31p-nmr 31p and 2h-nmr spectroscopies of phospholipid dispersions (mlv) were used to observe the structural and dynamics consequences of the presence of slo at the polar head (31p) and chain (2h) levels of the membrane. the polar head group level as shown figure 3a (bottom of column), the spectrum of pure dmpc dispersion (mlv) is typical of an axially symetric powder pattern, with a chemical shift anisotropy of 69 ppm, classical of dmpc bilayers in their liquid crystallin phase around (296 k) phase transition7 the chemical shift difference between the lowfi eld and the highfi eld edges of the 31p-nmr spectrum is called chemical shift anisotropy (csa, ppm) and is directly related to the fl uidity-reorientationat the polar head level where the phosphorus nuclei are located. on such spectra a mobile phosphorus group gives a single narrow resonance (several hz) as detected in true solution or for small structures (micelles), while solide state phosphorus gives extremely broad contributions (more than 100 ppm). note that membrane fl uidity increases (and csa decreases) with temperature, with a special jump at the transition temperature between gel phase and liquid crystal structure (around 297 k for dmpc),). thus the plot of csa as a function of temperature provides a good overview of membrane dynamics at the polar head level where phosphorus nuclei are located, while the lineshape allows to identify the overall membrane organisation (bilayer, hexagonal, isotropic phases). such plots are presented on the bottom traces of the figure 3: for pure dmpc dispersions and for slo containing mlv (slo/ dmpc weigh ratios of 1/25 and 2/25 mg/mg) as expected a csa decrease (around 18–20 ppm) was observed on pure dmpc systems with the transition-related jump around 297 k. such was also the case for the spectra recorded under the same conditions on slo containing systems at various temperatures. especially, no isotropic contribution typical of detergent effect was observed. however, a signifi cant reduction in csa value were measured at low temperature (under transition temperature, see figure 3a, bottom trace); this increase in local fl uidity was not detected at higher temperatures while transition temperature was found unaffected by the presence of slo (297 k). the presence of structural rearrangements was also supported by this decrease in csa at low temperature, with a normal transition temperature (297 k) and csa values close to those of dmpc at higher temperatures. 130 study of alkylglycerol containing shark liver oil drug target insights 2008:3 the acyl chain level 2h-nmr lineshape figure 4a (top) shows the spectrum of dmpc-d54 (dimyristoyl phosphatidyl choline with perdeuterated chains) dispersions. this spectrum is typical of phospholipid bilayers in the liquid crystal phase close to transition temperature (296 k) [21]. such a spectrum appears as a superimposition of symetrical doublets, each doublet corresponding to a methylenic cd2 group of the acyl chain. for a given doublet, the splitting (δνq) is directly related to the local order following the relation: δνq = [a*(3*cos2θ–1)]/2, where a is 170 khz (for the cd2 bound in dmpc) and θ the averaged value of the solid angle of reorientation. this splitting can be used in a fi rst approximation as an order parameter. as the acyl chain fl uidity decreases from the terminal methyl group (cd3, as shown on the expanded part of the spectrum m on fig. 4b) to the methylenic groups close to the polar head of the lipids (the so called “plateau region”, from c-2 to c-8 of the chain), the resulting spectrum consists of i) an inner doublet with a quadrupolar splitting of 3800 hz attributed to the cd3 methyl group, a is found, ii) doublets with increasing quadrupolar splittings assigned to successive cd2 groups from c14 to c9; iii) the external edge doublet, attributed to the deuterium of the c2-c8 plateau region where a 29 khz quadrupolar splitting is measured. figure 4. 2h-nmr spectrum of a) pure dmpc-d54 dispersions at 296 k (the spectrum is expanded in b to show the splitting of cd3 groups of pure dmpc –topand in the presence of 1 mg slo—bottom-), bottom traces temperature dependence of the half quadrupolar splitting (khz) for pure dmpc (•), and slo/dmpc systems, 1/25 w/w ( ) and 2/25 w/w (∆), for plateau resonances (c) and terminal cd3 group resonances (d). 131 debouzy et al drug target insights 2008:3 the main spectrum recorded under the same conditions (296 k) in the presence of slo (r = 1/25 and 2/25 w/w) also shows a dramatic reduction in quadrupolar splittings both at the superficial level (the plateau region) and in the deep part of the membrane (right traces and curves fig. 4). besides, no other contribution i n d i c a t i v e o f i s o t r o p i c r a p i d m o t i o n w a s found. from this part, one can conclude that slo induces an overall fl uifi zation of synthetic membrane, exclusively present below transition temperature, without inducing any detergent effect and membrane structure and dynamics modifi cation over phase transition. the following step was to test the relevance of these observations in biological systems, i.e. red blood cells, by using macroscopic haemolysis tests and esr biophysical measurements. biological relevance of biophysical results haemolytic activity the haemolysis curve is shown on the figure 2. by comparison with well identifi ed haemytic molecules (for instance natural β-cyclodextrin has a 50% haemolytic concentration of 13 mm,) [22] slo haemolytic activity is found very low, since the maximum haemolysis obtained was 6.6% (21 mg/ml slo) and 50% haemolysis could not be obtained. 31p-nmr of erythrocyte ghosts the figure 3b (top) shows a typical spectrum of red blood cell membranes (100 mg ghosts in d2o for a total sample volume of 500 μl) recorded at 296 k with the same parameters as dmpc dispersions. due to cellular organization (cytoskeleton, proteins) the overall membrane structure is signifi cantly more rigid than synthetic systems, according with a csa (chemical shift anisotropy) of 110 ppm. the addition of 3 mg slo results in a signifi cant reduction of this value (83 ppm), revealing an increase in collective fl uidity without local membrane damages that should have been evidenced by the presence of isotropic line at 0 ppm. however, this effect required at least 2 mg slo and was not observed for lower amounts. esr spin labeling experiments spin label experiments were then realised to investigate the red cells membrane fl uidity in different temperature conditions. two probes were separately used, 5 ns gives information about superfi cial membrane fl uidity, while 16 ns concerned the inner membrane region. the results are shown on figure 5. an increase of the mobility of the two probes contribution could be observed in slo groups, related to a global enhancement of the membrane fl uidity. furthermore, at low temperature (288 k and 293 k), a drop in the order parameter was measured in the slo group compared to control, that disappeared at the physiological and at the above temperature (up to 315 k). the same observation was done for the rotational correlation time of the 16 ns probe with a decrease of τ in slo group. this feature indicated an overall increase of the membrane fl uidity of the erythrocytes induced by slo at the lowest temperature. this effect was not noticeable at physiological temperature. esr spin trapping experiments the fenton reaction in presence of 4-pobn yields characteristic six-line spectra (fig. 6b showing 4-pobn results). the spin adduct hyperfi ne splitting constants were an = 15.73 g and ah = 2.57 g. according to finkelstein et al. [23] value: 4-pobn spin trap: an = 15.60 g and ah = 2.55 g and to augusto et al. [24] who found an = 15.50 g and ah = 2.50 g; these hyperfi ne splitting constants correspond to a α-hydroxyethyl adduct. this is stable adduct results from a reaction between hydroxyl radicals initially generated and the spin trap. the histogram presented on figure 6a, shows the free radicals promotion for 3 different concentrations of slo (8 mg/ml, 0,8 mg/ml and 0,08 mg/ ml) versus control and vitamine e (8 mg/ml). statistical comparisons were achieved using nonparametric tests (kruskal-wallis). vitamine e is a reference antioxidant molecule able to recombine with free radical. in the presence of vitamine e and alkylglycerol in the same concentration, a signifi cant strong decrease in the trace amplitude compared to control was observed (−26% slo and −28% vit e). for lower concentrations of slo (0.8 and 0.08 mg/ml) no signifi cant decrease in spin adduct detection could be shown. 132 study of alkylglycerol containing shark liver oil drug target insights 2008:3 figure 5. esr spin labeling experiment. (a) 5 ns and 16 ns results. left y axis: temperature dependance of the order parameter (5 ns) for control red cells (black diamond) and red cell in presence of slo (grey square). right y axis: temperature dependence of the rotational correlation time (16 ns) for control red cells (black triangle) and red cell in presence of slo (grey circle). (b) typical 5 ns spectrum, parameter used for order parameter estimation are inner (2t┴) and outer hyperfi ne (2t //) splitting. (c) typical 16 ns spectrum, parameter used for rotational correlation time was central peak intensity h0, high fi eld peak intensity and the with of the mid-fi eld line w0. 0 5000 10000 15000 20000 25000 control alkyrol 8 mg/ml alkyrol 0,8 mg/ml alkyrol 0,08 mg/ml vit e 8 mg/ml a rb it ra ry u n it s a * * b figure 6. esr spin trapping experiment. (a) mean free radical production after exposure using spin trap n-tert-butyl-α-(4-pyridyl)nitrone n’oxide (4-pobn). for each group the value was the average of 3 measurements ± sd. (b) typical esr of n-tert-butyl-α-(4-pyridyl)nitrone n’oxide (4-pobn) radical adducts following fenton reaction. (*) represents p � 0.05. 133 debouzy et al drug target insights 2008:3 discussion beside the well established effects of alkyglycerols and polyunsaturated fatty acids on platelet aggregation and infl ammatory reactions, the aim of the present work was to investigate physico chemical properties of slo, especially membrane interactions. this work could be undertaken due to two initial conditions fulfi lled: the average composition was found homogenous between numerous samples tested; this was also in agreement with previous analysis showing a composition exclusively made of alkylglycerols and polyinsatured fatty acids [25] (pufa); due to relatively amphiphilic properties, slo exhibits a signifi cant solubility in the water, by the way of self-organisation in supramolecular assemblies with an average diameter of 50å. this point allows the use of slo in aqueous preparation without requiring to other organic cosolvents (dmso…) or special preparations (encapsulation…). the fundamental part of the study, performed on phospholipidic synthetic membranes allowed to identify the fl uidifi zation of the membrane, as evoqued elsewhere. [26] an intercalation of the oil into the hydrophobic core of the membrane have been observed, affecting the order, packing and overall mobility of the lipid acyl chains. this effect was only observed when the chains are in the gel phase and ordered. this intercalation in the membrane, coupled with possible antioxidant effect should constitute the basis for a reasonably model for its action. however, our results clearly show that a dramatic fl uidifi zation is obtained at low temperature, while this effect completely vanishes at temperature (over 296 k). this feature observed both in synthetic systems and in erythrocytes would be of interest in cold environmental conditions if related with the clinical effects expected (a better resistance to intense training…). however, it is worth to note that temperature regulation in sharks is very limited, even at very low temperature. under this point of view, increased fl uidity at low temperature would contribute to maintain cell and tissue functions in extreme environments where sharks live in. by the way of contrast, homeotherms such as humans generally maintain their internal temperature around 37 °c by active metabolic mechanisms such as increasing blood pressure, vasoconstriction and active shivering (from 37 to 34 °c) [27]. lower temperatures lead to collapses and severe hypothermia to death by ventricular fi brillation. here the observed effect of slo at low temperature could appear of limited practical interest. however, in cold environments (e.g. high mountain training, outside work in winter…) cutaneous and subcutaneous temperature are often dramatically lower and slo properties would play here a physiological role. this is particularly true in some pathologies such as raynaud’s syndrom or microcirculation abnormalities (malan’s syndrome) where both blood cell viscosity and capillary membrane fl uidity are involved [28]. also, cardiac surgery frequently uses extra corporal circulation systems: during operating time, central temperature is set down to 15 °c to protect the brain from the consequences of long lasting hypoxia (20–30min or more). possible benefi ts in these circumstances remain to study. slo antioxidant properties are also of interest: hence, vitamin e is routinely used as adjuvant agent in radiotherapy, used for its antiradical properties in the prevention of radio induced fi brosis [29]. another advantage is the apparent extremely low toxicity as tested by insignifi cant hemolytic activity and complete absence of detergent effect. another point of interest is that the amphiphilic properties of slo are very favorable to overcome biological barrier (cellular membranes, intestinal wall…) by allowing both surface binding and spontaneous cell integration as shown by paramagnetic broadening experiments in cells (not shown). [30] also, the anti oxidant properties were found similar as those of vitamin e and were related mainly to the general properties of polyinsatured fatty acids. [31] finally, the presence of non specifi c membrane properties of slo, associated with its low toxicity is consistent with the great diversity of biological effects evoqued in the past, [32] such as anticancer, antioxidant and anti-infl ammatory properties. a promising way for future research would be to evaluate the specific applications for work or physical effort in cold environments. the extreme environmental conditions (pressure and cold) met in all day life of greenland sharks would also been related with an adaptative evolutional process leading to optimize biochemical composition of this species. from this point of view, the properties of slo under high pressure conditions (diving) should also be evaluated. 134 study of alkylglycerol containing shark liver oil drug target insights 2008:3 aknowledgments thanks to j.morin and t.lerond for stimulating discussions, and prof. j.hàn-peuh-pluu for manuscript relecture. abbreviations 4-pobn: α-(4-pyridyl-1-oxide)-n-t-butylnitrone; 5 ns: 5 nitroxide stearate; 16 ns: 16 nitroxide stearate; csa: chemical shift anisotropy; epc: egg yolk phosphatidylcholine; esr: electron spin resonance; dmpc: dimyristoyl phosphatidyl cholin; hc50: hemolytic constant 50%; mlv: multibilayer vesicle; es-ms: electron spray-mass spectroscopy; nmr: nuclear magnetic resonance; paf: platelet activating factor; slo: shark liver oil; st: superfi cial tension; vit e: vitamin e. references [1] hallgren, b. and larsson, s. 1962. the glycerol ethers in elasmobranch fi sh. lipid res., 3:31–8. [2] tsujimoto, m. and toyama, s. 1922. studies on unsaponifi able fractions from some fish liver oil. tokyo inst. technol., 16:471–510. [3] hallgren, b. and stallberg, g. 1967. methoxy-substituted glycerol ethers isolated from greenland shark liver oil. acta. chem. scand. b., 21:1519–29. [4] brohult, a., brohult, j. and brohult, s. 1972. effects of alkylglycerols on the frequency of injuries following radiation therapy. experientia, 57:79–85. [5] brohult, a., brohult, j. and brohult, s. 1978. regression of tumour growth after administration of alkoxyglycerols. acta. obstet. gynecol. scand., 57(1):79–83. [6] hallgren, b., niklasson, a., stallberg, g. and thorin, h. 1974. on the occurrence of 1-o-(2-methoxyalkyl)glycerols and l-o-phytanylglycerol in marine animals. acta. chem. scand. b., 28(9):1035–40. [7] dufourc, e.j., mayer, c., stohrer, j., althoff, g. and kothe, g. 1992. dynamics of phosphate head group in biomembranes. biophys. j., 61:42–57. [8] girault, l., lemaire, p., boudou, a., debouzy, j.c. and dufourc, e.j. 1996. interaction of inorganic mercury with phospholipid micelles and model membranes. a 31p-nmr study. eur. biophys. j., 24:413–21. [9] rappel, c., galanski, m., yasemi, a., habala, l. and keppler, b.k. 2005. analysis of anticancer platinium(ii) complexes by microemulsion electrokinetic chromatography: separation of diastezreoisomers and estimation of octanol-water partition coeffi cient. electrophoresis, 26(4–5):878–84. [10] sanders, j.k.m., constable, e.c. and hunter, b.k. modern nmr spectroscopy, ed. o.u. press. 1989, oxford. [11] mavromoustakos, t., daliani, i. and matsoukas, j. 1999. the application of biophysical methods to study drug: membranes interactions, bioactive peptids in drug discovery and design: medical aspects 13–24. [12] debouzy, j.c., crouzier, d. and gadelle, a. 2007. physico chemical properies and membranes interactions of per (6-desoxy-6-halogenated) cyclodextrins. annales pharmacologiques francaises, 65:331–41. [13] gaffney, b.j. pratical considerations for the calculation of order parameters for fatty acid or phospholipid spin labels in membranes., in spin labelling. theory and applications, r.j. berliner, editor. 1976, academic press: new york london. 567–71. [14] gornicki, a. and gutsze, a. 2000. in vitro effects of ozone on human erythrocyte membranes: an epr study. acta. biochim. pol., 47(4):963–71. [15] kaplan, j.i. and fraenkel, g. nmr of exchanging systems, ed. a. press. 1980, new york. [16] dennis, e.a. and plückthun, a. p31-nmr of phospholipids and micelles, in phosphorus 31p-nmr: principles and applications, gorenstein, editor. 1984, academic press: london 423–80. [17] canet, d., boubel, j.c. and soulas, e. la rmn, concepts, méthodes et applications, ed. dunod. 2002, paris. 235. [18] neumann, j.m., zachowski, a., tran-dinh, s. and devaux, p.f. 1985. high resolution proton magnetic resonance of sonicated phospholipids. eur. biophys. j., 11:219–23. [19] gremy, f. and leterrier, f. elementrs de biophysique, flammarion, editor. 1981: paris 295–304. [20] runhua, l., jincheng, h., hanqing, w. and luinhui, l. 1997. surface tension measurements and 1h-nmr studies of inclusion complex of beta-cyclodextrin with sodium alkyl sulfonate. j. of inclusion phenomena and molecular recognition in chemistry, 28:213–21. [21] douliez, j.p., léonard, a. and dufourc, e.j. 1996. conformational order of dmpc sn-1 versus sn-2 chains and membrane thickness: an approach to molecular protrusion by solid-state 2h-nmr and neutron diffraction. j. phys. chem., 100(18):400–57. [22] leray, e., leroy-lechat, f., parrot-lopez, h. and duchene, d. 1995. reduction of the haemolytic effect in biologically recognisable betacyclodextrin. supramol. chem., 5:149–51. [23] finkelstein, e., rosen, g.m. and rauckman, e.j. 1982. production of hydroxyl radical by decomposition of superoxide spin-trapped adducts. mol. pharmacol., 21(2):262–5. [24] augusto, o., weingrill, c.l., schreier, s. and amemiya, h. 1986. hydroxyl radical formation as a result of the interaction between primaquine and reduced pyridine nucleotides. catalysis by hemoglobin and microsomes. arch. biochem. biophys., 244:147–55. [25] centre regional de lutte contre le cancer and montpellier., c.v.-d.a., métabolisme des lipides pharmacologiques de l’huile de foie de requin. compte rendu du comité d’interface de recherché clinique, 1999. [26] singh, g. and chandran, r.k. 1988. biochemical and biological effects of fi sh and fi shoils. prog. food nutr. sci., 12(4):371–419. [27] meliet, j.l. 2000. eléments de médecine de la plongée. bull med. sub. hyp., 10:116. [28] malan, e. morphogenesis of pezripherical blood vessel-angiodysplasias, ed. c.e. fond. 1974. [29] delanian, s., porcher, r., balla-mekias, s. and lefaix, j.l. 2003. randomized, placebo-controlled trial of combined pentoxifi llin and tocopherol for regression of superfi cial radiation induced fi brosis. j. of clinical oncology, 21(13):2545–50. [30] debouzy, j.c., neumann, j.m., hervé, m., daveloose, d., viret, j.j. and apitz-castro, r. 1989. interaction of antiagregant molecule ajoene with membranes. eur. biophys. j., 17:211–6. [31] descher, e.e., lyttle, j.s., wong, g., ruperto, j.f. and newmark, h.l. 1990. the effect of dietary omega-3 fatty acids (fi sh oil) on azowymethanol induced focal areas of dysplasia and colon tumor incidence. cancer, 66(11):2350–6. 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target insights 2007:2 239–247 239 review correspondence: hideto kameda, m.d./ph.d., division of rheumatology/clinical immunology, department of internal medicine, saitama medical center, saitama medical university, 1981 kamoda, tsujido-machi, kawagoe, saitama 350-8550, japan. tel/fax: +81-49-228-3574; email: kamehide@saitama-med.ac.jp copyright in this article, its metadata, and any supplementary data is held by its author or authors. it is published under the creative commons attribution by licence. for further information go to: http://creativecommons.org/licenses/by/3.0/. platelet-derived growth factor as a therapeutic target for systemic autoimmune diseases hideto kameda, miyuki suzuki and tsutomu takeuchi division of rheumatology/clinical immunology, department of internal medicine, saitama medical center, saitama medical university, kawagoe, saitama, japan. abstract: some systemic rheumatic diseases and disorders, especially fi brotic and vascular disorders, are often refractory to corticosteroid therapy. recently, ever accumulating evidence suggests that platelet-derived growth factor (pdgf) is involved in those refractory diseases. imatinib mesylate inhibits the activation of pdgf receptor as well as c-abl, bcr-abl and c-kit tyrosine kinases. it has therefore been widely used for the treatment of chronic myeloid leukemia and gastrointestinal stromal tumors. imatinib effectively suppresses the activation and proliferation of fi broblasts, mesangial cells and smooth muscle cells both in vitro and in vivo. additionally, it has recently been reported that some patients with rheumatoid arthritis or idiopathic pulmonary arterial hypertension demonstrated a good clinical response to imatinib therapy. imatinib may therefore overcome the limitations of current therapeutic strategy with corticosteroids and immunosuppressive agents for refractory diseases, such as systemic sclerosis and interstitial lung diseases, without clinical intolerability. keywords: imatinib mesylate, rheumatoid arthritis, fi broblast, rheumatic diseases, interstitial lung disease introduction the developmental process of systemic autoimmune diseases has been an unsolved mystery for decades. despite the identifi cation of autoreactive t lymphocytes or autoantibodies, both of which may participate in the pathogenesis of the diseases, any precise insight into “immunodistortion” present in each patient is lacking. therefore, overall immunosuppression, instead of a specifi c correction of “immunodistortion,” has been a common choice in clinical practice. corticosteroids and immunosuppressive agents have been the mainstay of the therapeutic strategy of systemic rheumatic diseases. however, the presence of refractory diseases/patients to such approaches, and various adverse effects such as increased risk of severe infections, limits their effi cacy. recent progress in our general understanding of molecular pathophysiology of diseases possibly downstream of the “immunodistortion” has led us to develop molecular targeted therapies in which the function of the key molecule(s) of the diseases is manipulated in order to control disease activity. we have then an excellent safety profi le in terms of an eventual lack of lethal dosage enabled most of the biologic response modifi ers (biologics) that can be applied at the dosage with which they suffi ciently inhibit the function of the targeted molecule. for example, tumor necrosis factor α (tnfα) inhibitors dramatically improved the outcome of patients with rheumatic diseases such as rheumatoid arthritis (ra) and seronegative spondyloarthropathies [takeuchi, 2005]. those agents include a chimeric antitnfα monoclonal antibody (infl iximab), a humanized anti-tnfα monoclonal antibody (adalimumab), and a p75 tnf-receptor/igg-fc fusion protein (etanercept). in addition, a chimeric anti-cd20 monoclonal antibody (rituximab), anti-interleukin-6 receptor monoclonal antibody (tocilizumab), and cytotoxic t-lymphocyte antigen 4 (ctla-4; cd152)/igg-fc fusion protein (abatacept) have been also successful in ra. on the other hand, low molecular weight chemical agents developed as molecular targeted therapies do have some drawbacks compared to biologics in terms of selectivity and limitations in dosage. nonetheless, their intracellular activity which large-molecular agents such as biologics do not possess, although slightly less selective, is still indispensable in the treatment of diseases in which various cytoplasmic/nuclear molecules play important roles. in diseases refractory to glucocorticoids and conventional immunosuppressants such as cyclophosphamide—both of which are chiefl y targeted to leukocytes—, cells of mesenchymal origin http://creativecommons.org/licenses/by/3.0/ http://creativecommons.org/licenses/by/3.0/ 240 kameda et al drug target insights 2007:2 such as fi broblast and vascular cells play a pivotal role. novel molecular targeted therapies should therefore include growth factors participating in the remodeling of mesenchymal tissues. among various growth factors, platelet-derived growth factor (pdgf) is an excellent candidate due to its multipotent roles in many rheumatic diseases [östman and heldin, 2001; paniagua and robinson, 2007]. it can be interfered with monoclonal antibodies against pdgf or its receptor (pdgf-r). another approach in inhibiting pdgf signaling is the application of tyrosine kinase inhibitors of pdgf-r. in contrast to non-selective tyrosine kinase inhibitors such as genestein, imatinib mesylate (sti571; gleevec® or glivec®) has been widely used for the treatment of chronic myeloid leukemia (cml) [druker et al. 2001a; druker et al. 2001b] and gastrointestinal stromal tumors (gist) [demetri et al. 2002]. therefore, in order to speculate on the clinical importance of pdgf-targeted therapy in refractory diseases and patients, this review article summarizes the involvement of pdgf in systemic autoimmune diseases, as well as preclinical or clinical results of imatinib or other pdgf-targeted therapies. signaling from pdgf-r and its inhibition by imatinib pdgf is a family of homoor hetero-dimeric molecules of disulfi de-bonded polypeptide chains with a conserved sequence of approximately 100 amino acid residues containing a characteristic motif of 8 cystein residues [östman and heldin, 2001; heldin and westermark, 1999; pietras et al. 2003]. combinations of subunits aa, ab, bb, cc and dd have been identifi ed to date. pdgf is synthesized by many cell types such as platelets, endothelial cells, fi broblasts, macrophages, and vascular smooth muscle cells. the pdgf-rs occur as α (~170 kda) and β (~180 kda) homodimers or heterodimers. the receptor sequences have 5 immunoglobulin-like domains in their extracellular parts and tyrosine kinase domains intracellularly into which is inserted an interrupting sequence of about 100 amino acids. pdgf-aa binds to pdgf-rαα, ab and cc to αα and αβ, bb binds to all 3 combinations of receptors, and dd binds to αβ and ββ [pietras et al. 2003]. the dimerization and activation of receptor tyrosine kinases lead to cellular activation of targeted cells including fibroblasts, smooth muscle cells and mesangial cells. a large number of sh2 domain-containing protein kinases/phosphatases and adaptor proteins, such as phosphatidylinositol 3-kinase (pi3k), phospholipase cγ (plcγ), sh2containing protein tyrosine phosphatase shp-2, grb2, nck and shc, link pdgf-r with downstream signaling molecules and lead to upregulated gene expression and proliferation, or transformation (fig. 1) [östman and heldin, 2001; heldin and westermark, 1999]. originally, imatinib focused on attempts to inhibit pdgf-r kinase with an ic50 of 0.12– 0.15 µm. however, it was soon noted that imatinib effectively inhibited c-abl (ic50 0.1–0.3 µm), bcr-abl (ic50 0.25 µm) and c-kit (ic50 � 1 µm) [carroll et al. 1997; druker et al. 1996; capdeville et al. 2002]. imatinib was also reported to be well tolerated and have signifi cant antileukemic activity in patients with cml with whom treatment with interferon α had failed, as well as those with blast crisis of cml or bcr-abl-positive acute lymphoblastic leukemia [druker et al. 2001a; druker et al. 2001b]. imatinib was later revealed to induce a sustained objective response in more than half of patients with an advanced unresectable or metastatic gist [demetri et al. 2002]. imatinib has now been widely approved for the treatment of cml and unresectable and/or metastatic c-kit (cd117)-positive gist. systemic sclerosis systemic sclerosis (ssc; scleroderma) is characterized by vascular damage and excessive fi brotic response on the basis of immunologic abnormalities (“immunodistortion”). the progression of organ damage is usually insidious in ssc, with the exception of scleroderma renal crisis which can be successfully treated with angiotensinconverting enzyme inhibitors. therefore, overall disappointing response to corticosteroids has been attributed to the paucity of involvement of active infl ammation, characterized by massive leukocyte infi ltration. for this reason, disease-modifying drugs for ssc have not been available to date [wollheim, 2007]. as for vascular damage, inhibitors targeting endothelin receptors, such as bosentan, represent a breakthrough in the treatment of pulmonary arterial hypertension (pah), a complication with one of the worst prognosis in ssc, systemic lupus erythematosus (sle) or mixed connective tissue 241 pdgf as a target for systemic autoimmune diseases drug target insights 2007:2 disease (mctd) [denton et al. 2006; kondo, 2001]. bosentan may also reduce the fibrotic process via its inhibitory effect on fibroblasts [braun-moscovici et al. 2004], although these effects have not yet been proven in clinical trials. at the same time, a recent report describing a stimulatory autoantibody against pdgf-r is of great interest in view of the pathogenesis and management of fi brosis in ssc [baroni et al. 2006]. although the determination of autoantibodies recognizing nuclear components have been useful in defi ning clinical subgroup of ssc, antinuclear antibodies (anas) have not been shown to be directly pathogenic. in contrast to this, stimulatory anti-pdgf-r antibodies exclusively found in sera from ssc patients were demonstrated to activate pdgf-r and induce the upregulation of the ha-ras-erk1/2 (external signal regulated kinases 1 and 2) cascade. this stimulated the expression of type i collagen gene expression and α-smooth muscle actin (α-sma) [baroni et al. 2006] which resembles the case of anti-thyroid stimulating hormone antibodies developing hyperthyroidism. early in 2007, distler et al. reported that imatinib reduced basal and pdgfor tgf-β-stimulated synthesis of extracellular matrix (ecm) proteins such as collagen and fibronectin; and that a 50–150 mg/kg/day intraperitoneal administration of imatinib effectively inhibited dermal thickness, the number of myofi broblasts and synthesis of ecm proteins in bleomycin-induced dermal fi brosis model—all without evidence of toxic adverse effects [distler et al. 2007]. a signifi cant increase in activated pdgf-rβ expression in idiopathic pah lungs compared with healthy donor lungs, and successful reversal of pulmonary vascular remodeling, in both monocrotaline-induced rat model and hypoxia-induced mouse model of pah [schermuly et al. 2005], led to the compassionate treatment of a patient with idiopathic pah with a daily administration of 200 mg of oral imatinib [ghofrani et al. 2005]. the patient’s condition imatinib gene regulation figure 1. signaling through the pdgf-r. the binding of pdgf dimer (pdgf-bb, for example) to pdgf-r results in the tyrosine phosphorylation of its cytoplasmic domain, where many adapter proteins and kinases/phosphatases assemble and augment the activation signaling leading to upregulation of gene expressions. imatinib inhibits the activation of pdgf-r tyrosine kinase. 242 kameda et al drug target insights 2007:2 improved impressively over 3 months in pulmonary vascular resistance (from 1056 dyn.sec.cm−5 to 815 dyn.sec.cm−5), six-minute walk distance (from 260 m to 383 m), and new york heart association functional class (from iv to ii) [ghofrani et al. 2005]. these reports provide the possibility that imatinib may be a disease-modifying drug for ssc. on the other hand, recombinant human anti-tgfβ1 antibody (cat-192) showed no evidence of effi cacy in a multicenter, randomized, placebocontrolled phase i/ii trial for early-stage diffuse cutaneous ssc [denton et al. 2007]. the blockade of multiple isoforms of tgfβ may be required for an effective inhibition of fi brosis. in this context, the pan-isoform-specifi c anti-tgfβ antibody 1d11 could be potentially effective for fi brotic diseases. nonetheless, imatinib has the advantage of inhibiting both pdgf and tgfβ signaling via pdgf-r and c-abl, respectively. interstitial lung disease (ild) in ssc will be discussed in the following section. ild in systemic rheumatic diseases the lung is one of the vital organs involved most frequently in systemic rheumatic diseases, and the presence of ild is a signifi cant prognostic factor of polymyositis/dermatomyositis (pm/dm), rheumatoid arthritis (ra), systemic vasculitis syndromes and ssc. the clinical courses of patients with ild associated with those diseases can be categorized into 4 groups: 1) a/sip with rapid deterioration within a month (acute) or within 2–3 months (subacute); 2) chronic progression of pulmonary fibrosis causing non-productive coughing, breath-shortening upon exertion, and occasionally leading to respiratory failure after more than 6 months; 3) acute or subacute exacerbation of chronic ild that is recurrent in some cases; and 4) asymptomatic ild detected in a milder form by radiographic examination or pulmonary function tests in the absence of clinically apparent signs and symptoms throughout the observation period [kameda and takeuchi, 2006]. the american thoracic society/european respiratory society (ats/ers) international multidisciplinary consensus classifi cation is usually used for the classifi cation of idiopathic interstitial pneumonias (iip) [american thoracic society; european respiratory society 2002]. diffuse alveolar damage (dad) and organizing pneumonia (op) usually develop acutely or subacutely, while non-specifi c organizing pneumonia (nsip) shows a subacute course, and usual interstitial pneumonia (uip) represents a chronic form of ild called pulmonary fi brosis. however, whether ild associated with systemic rheumatic diseases resembles iip remains debatable: the histological fi ndings may consist of the overlapping features of two or more patterns, nsip and op, or nsip and uip. moreover, patients having idiopathic uip (clinically idiopathic pulmonary fi brosis) show poorer prognosis compared to patients with systemic rheumatic diseases, such as ssc demonstrating uip in lung histology [bouros et al. 2002]. nevertheless, patients with ild associated with systemic rheumatic diseases also tend to show a favorable response to corticosteroids when the lung histology reveals op or cellular nsip, while they demonstrate a relatively poor response when the lung histology shows dad or uip; fi brotic nsip stands for an intermediate response. the addition of a single immunosuppressive agent or the combination of 2 or more improved the mortality and morbidity of rapidly or slowly progressive ild associated with pm/dm or ssc [white et al. 2000; kameda et al. 2005]. despite the intensifi ed immunosuppressive strategies, a signifi cant number of ild patients resulted in fatal outcomes. because the pathological examination of ild with poor prognosis, dad and uip, shares an excessive fibrosis, another therapeutic strategy such as anti-fi brotic agents is necessary. pirfenidone, nacetylcysteine and interferon-γ have been shown to be potentially effective for the prevention of lung fi brosis [american thoracic society 2000]. at the end of the last century, it was reported that the inhibition of the autophosporylation of pdgf-r by ag1296 effectively prevented v2o5stimulated proliferation of alveolar epithelial/ mesenchymal cells as well as hydroxyproline accumulation [rice et al. 1999]. in that model, a specific inhibition of epidermal growth factor (egf) receptor activation by ag1478 also showed a preventive efficacy, although slightly less compared to ag1296. another approach using an expression plasmid of the extracellular domain of pdgf-rβ for bleomycin-treated c57bl/6 mice also ameliorated the increases in the wet weight, hydroxyproline content and the histologic changes in the lung [yoshida et al. 1999]. daniel et al. proved that imatinib dramatically reduced hydroxyproline content and prevented 243 pdgf as a target for systemic autoimmune diseases drug target insights 2007:2 histopathologic changes in bleomycin-treated lungs of 129tsvems mice [daniel et al. 2004]. they also suggested that the effi cacy of imatinib is mediated, in part, by the inhibition of serine/threonine tgf-β receptor kinase signaling through c-abl tyrosine kinase. aono et al. also showed that imatinib (50 mg/kg) signifi cantly attenuated bleomycin-induced pulmonary fi brosis in terms of histology and collagen deposition in c57bl/6 mice. interestingly, the early treatment with imatinib (from day 0 to 14), but not the late treatment (from day 15 to 28) significantly inhibited bleomycin-induced lung fi brosis in that model [aono et al. 2005]. however, another bleomycininduced lung fi brosis model using rats showed that imatinib (50 mg/kg) treatment commenced at day 10 was still effective, while prednisolone was effective exclusively in the preventive model started at day 1 [chaudhary et al. 2006]. in the radiation (20 gy)-induced lung fi brosis model using c57bl/6 mice, treatment with either of 3 pdgf-r inhibitors (su9518, su11657 and imatinib) markedly attenuated the development of pulmonary fi brosis in excellent correlation with clinical, histological, ct results, and life span. in addition, no decline in their effi cacy was observed when they were started after thoracic irradiation compared to a prevention schedule [abdollahi et al. 2005]. these results may provide a possibility that imatinib could be chosen for the treatment of corticosteroid(/immunosuppresants)-refractory ild. in contrast to the above, a recent report failed to demonstrate the effi cacy of imatinib (10 mg/kg, intraperitoneal or oral administration) in the protection against bleomycin-induced pulmonary fi brosis in c57bl/6 mice [vittal et al. 2007]. lupus nephritis systemic lupus erythematosus (sle) is a prototype of systemic autoimmune diseases and its renal involvement has been posing major challenges for physicians. renal accumulation of immune complexes followed by complement activation, and antibody-effector-cell interactions through the fc receptor have been implicated in the pathogenesis of lupus nephritis. high-dose corticosteroids combined with immunosuppressive agents such as cyclophosphamide or mycophenolate mofetil are the mainstay in current strategy for the induction of response in lupus nephritis. some patients however do not respond favorably to the above regimens or show repeated relapses, and ultimately progress to end-stage renal disease. renal expression of pdgf, which is synthesized by mesangial cells, endothelial cells, macrophages, and smooth muscle cells in the kidney, and of pdgf-r was signifi cantly increased in the glomeruli of patients with mesangial proliferative glomerulonephritis (iga nephropathy, henochschönlein purpura nephritis, and lupus nephritis) compared with normal glomeruli [matsuda et al. 1997]. thus, pdgf/pdgf-r axis is considered a novel therapeutic target of lupus nephritis and other forms of glomerulonephritis. indeed, administration of neutralizing anti-pdgf igg [johnson et al. 1992] or oligonucleotide aptamer [floege et al. 1999], which specifi cally binds to the pdgf-b chain and inhibits its activity to anti-thy-1 glomerulonephritis rats resulted in a signifi cant reduction in mesangial cell proliferation and largely prevented the increased deposition of ecm. imatinib (0.013–2.0 µm) inhibited pdgfstimulated, but not fgf-stimulated, mesangial cell proliferation in a dose-dependent fashion in vitro [gilbert et al. 2001]. moreover, the effi cacy of imatinib in vivo has been proven in 3 different models of (lupus) nephritis. first, pdgf-r tyrosine kinase blockade by imatinib (50 mg/kg/day) was associated with a signifi cant reduction in mesangial cell proliferation, the number of α-sma-positive mesangial cells, and glomerular type iv collagen deposition in male wistar rats with anti-thy-1.1 glomerulonephritis [gilbert et al. 2001]. second, 50 mg/kg imatinib inhibited proliferation of glomerular cells and crescent formation, and also prolonged the life span of mrl/lpr female mice [sadanaga et al. 2005]. intriguingly, attenuation of lymphadenopathy and salivary gland infl ammation, as well as reduction in serum anti-doublestranded dna antibodies, was also observed in the imatinib-treated mice. third, (nzb/w)f1 mice treated with imatinib (50 mg/kg b.i.d./day) showed ameliorated survival, delayed onset of proteinuria, and preserved renal function [zoja et al. 2006]. histologic examination provided evidence of reduced glomerular hypercellularity, deposits, tubulointerstitial damage, and accumulation of α-sma-positive myofi broblasts. rheumatoid arthritis the synovial membrane in patients with ra is characterized by hyperplasia, angiogenesis, and an 244 kameda et al drug target insights 2007:2 infi ltrate of infl ammatory cells including cd4+ t lymphocytes [choy and panayi, 2001]. synovial fi broblast-like (sfl) cells from ra patients show transformed characteristics [firestein and zvaifl er, 2002; müller-ladner et al. 2000; yamanishi and firestein, 2001]: altered morphology, anchorageindependent growth [lafyatis et al. 1989], loss of contact inhibition, oncogene activation [müllerladner et al. 1995], monoclonal or oligoclonal expansion [imamura et al. 1998], cartilage invasion in severe combined immunodefi cient mice [müllerladner et al. 1996], etc. pdgf-rs are abundantly expressed on the surface of ra-sfl cells, and stimulation with pdgf enhances both the anchorage-dependent and -independent growth of ra-sfl cells and thus implicate pdgf in the activation and transformation of ra-sfl cells [lafyatis et al. 1989; rubin et al. 1998; remmers et al. 1991; sano et al. 1993; watanabe et al.]. indeed, pdgf immunostaining of ra synovia is more extensive and intense than that of osteoarthritis (oa) or normal synovia. also, pdgf-r expression is elevated in ra synovia compared with oa and normal synovia [remmers et al. 1991]. moreover, pdgf, together with tnfα, were identified as the major growth factors of ra-sfl cells [thornton et al. 1991]. furthermore, thrombin activity in synovial fl uid is signifi cantly higher in the patients with ra than with oa, and the mitogenic activity of thrombin toward ra-sfl cells is associated with an increase in the expression of mrna of pdgf-rs [ohba et al. 1996]. we demonstrated that 1 µm of imatinib effectively inhibited the pdgf-stimulated tyrosinephosphorylation of pdgf-r in ra-sfl cells, as well as pdgf-enhanced anchorage-dependent and -independent ra-sfl cell proliferation in vitro [kameda et al. 2006]. inhibition of pdgf-r signaling by 1 µm of imatinib did not induce apoptosis in cultured ra-sfl cells [sandler et al. 2006]. the effi cacy of imatinib in vivo has been almost simultaneously reported. imatinib (33– 100 mg/kg) effectively prevented and treated collagen-induced arthritis model of dba/1 mice in terms of synovitis, pannus formation and joint erosion, although preventive administration was more effi cacious than therapeutic administration [paniagua et al. 2006]. notably, imatinib inhibited mast cell c-kit activation, proinflammatory cytokine production, immunoglobulin production from b cells, and t cell response. however, collagen-induced arthritis in lewis rats were resistant to imatinib treatment: only high-dose (150 mg/kg, not 50 mg/kg) imatinib showed a signifi cant inhibition of osteoclast formation and joint destruction, and failed to reduce paw swelling [ando et al. 2006]. although the downstream signaling pathway from pdgf-r in ra-sfl cells has not been clarifi ed, many adaptor proteins are likely to be involved. for example, gab1 and gab2 were expressed in ra-sfl cells, and both adaptor proteins were rapidly tyrosine-phosphorylated after the stimulation of ra-sfl cells with 10 ng/ml of pdgf [kameda et al. 2006]. the fact that the expression of gab1 lacking the pleckstrin homology domain is associated with the enhanced anchorage-independent growth of syrian hamster embryo fi broblasts under growth factor stimulation suggests that similar alteration in signaling proteins might be involved in the acquisition of the transformed phenotype of ra-sfl cells [kameda et al. 2001]. this is supported by the fact that pdgf stimulation enhanced anchorage-independent growth of ra-sfl cells [kameda et al. 2006]. recent reports on ra patients successfully treated with imatinib were more encouraging than above in vitro and in vivo results. miyachi et al. reported a case with ra and chronic myeloid leukemia, both of which were successfully treated using imatinib [miyachi et al. 2003]. moreover, eklund et al. described three patients with severe ra who were treated for 12 weeks with escalating daily doses (from 100 to 400 mg) of imatinib [eklund and joensuu, 2003]. all three patients had failed to respond to prior anti-rheumatic medications, including methotrexate and infl iximab. all reported less pain and disease activity, and their health assessment questionnaire scores improved subsequent to the imatinib treatment. therefore, the addition of, or the switching to, imatinib may be benefi cial to patients with ra who failed in conventional therapies. perspective and conclusion besides those described above, pdgf is a fascinating molecular target in various other systemic rheumatic diseases such as spondyloarthropathy and systemic vasculitis. the strategies of pdgf signaling inhibition include antibodies against pdgf or pdgf-r, soluble recombinant fusion proteins of pdgf-r and igg, oligonucleotide 245 pdgf as a target for systemic autoimmune diseases drug target insights 2007:2 figure 2. predicted molecular mechanisms and possible therapeutic targets of rheumatic infl ammation and fi brosis. various cell types shown here are likely to be involved in the development and progression of rheumatic infl ammation. autoimmune responses and the subsequent infi ltration of neutrophils and lymphocytes are known to occur. resident macrophage activation, increased apoptosis of epithelial cells and endothelial dysfuction may result in the formation of fi brin clots and a provisional matrix, initiating the proliferation of fi broblasts. any of those cells could serve as therapeutic targets in the treatment of systemic rheumatic diseases, and a simultaneous control of them seems to be essential in overwhelming the persistent infl ammation. table 1. summary of preclinical fi ndings positively supporting the application of imatinib for systemic rheumatic diseases. ssc: inhibition of the differentiation of fi broblast into myofi broblast, proliferation, and ecm production [distler et al. 2007, in vitro and in vivo] pah: inhibition of medical thickening of pulmonary arteries leading to the improvement in right ventricular pressure, cardiac output, and arterial pressure of oxygen [schermyuly et al. 2005, in vivo] ild: inhibition of the differentiation of fi broblast into myofi broblast, proliferation, and ecm production, leading to histologic effi cacy [daniels et al. 2004; aono et al. 2005; in vitro and in vivo; abdollahi et al. 2005; chaudhary et al. 2006, in vivo] lupus nephritis: inhibition of mesangial proliferation, ecm production, and crescentic formation leading to decrease in proteinuria and improvement in renal failure [gilbert et al. 2001; sadanaga et al. 2005; zoja et al. 2006, in vivo] ra: inhibition of transformation/proliferation of synovial fi broblasts [kameda et al. 2006; sandler et al. 2006, in vitro], delay in joint destruction [paniagua et al. 2006; ando et al. 2006, in vivo] endothelial cell # anti-thrombosis epithelial cell # antigen presentation # apoptosis # cytokine production alveolar macrophage # tnf α production # chemokine production neutrophil # superoxide production # proteinase secretion lymphocyte # lymphokine production # autoantibody production # complement activation fibroblast # myofibroblast differentiation # migration and proliferation # provisional ecm secretion # angiotensin ii secretion 246 kameda et al drug target insights 2007:2 aptamer interfering pdgf-r signaling, and i n h i b i t o r s o f p d g f r t y r o s i n e k i n a s e o r downstream signaling molecules. among many candidates, imatinib possesses some advantages in: 1) clinical use with well acceptable tolerability; 2) additional inhibition of abl and c-kit, which leads to the suppression of tgf-β and mast cell activity, respectively. thus, imatinib is likely to play a multipotent role in the treatment of various systemic rheumatic diseases (table 1). and indeed, several clinical trials examining the effi cacy of imatinib for skin sclerosis, ild and pah are ongoing. to date, the principal of the treatment for systemic rheumatic diseases has been chiefl y targeting leukocytes. thus, future therapeutic strategies should focus on other cell types, including fi broblasts, epithelial cells and endothelial cells (fig. 2). combined therapy of antiinfl ammatory and anti-fi brotic agents may shed light on the treatment of refractory diseases described in this review and further improve the long-term survival of patients with systemic rheumatic diseases. references abdollahi, a., li, m., ping, g. et al. 2005. inhibition of platelet-derived growth factor signaling attenuate pulmonary fi brosis. j. exp. med., 201:925–35. american thoracic society. 2000. idiopathic pulmonary fi brosis: diagnosis and treatment. international consensus statement. am. j. respir. crit. care med., 161:646–64. american thoracic society; european respiratory society. 2002. american thoracic society/european respiratory society interstitial multidisciplinary consensus classifi cation of the idiopathic interstitial pneumonias. am. j. respir. crit. care med., 165:277–304. ando, w., hashimoto, j., nampei, a. et al. 2006. imatinib mesylate inhibits osteoclastogenesis and joint destruction in rats with collageninduced arthritis (cia). j. bone miner. metab., 24:274–82. aono, y., nishioka, y., inamiya, m. et al. 2005. imatinib as a novel antifi brotic agent in bleomycin-induced pulmonary fi brosis in mice. am. j. respir. crit. care med., 171:1279–85. baroni, s.s., santillo, m., bevilacqua, f. et al. 2006. stimulatory autoantibodies to the pdgf receptor in systemic sclerosis. n. engl. j. med., 354:2667–76. bouros, d., wells, a.u., nicholson, a.g. et al. 2002. histopathologic subsets of fi brosing alveolitis in patients with systemic sclerosis and their relationship to outcome. am. j. respir. crit. care med., 165:1581–6. braun-moscovici, y., nahir, a.m. and balbir-gurman, a. 2004. endothelin and pulmonary arterial hypertension. semin. arthritis rheum., 34:442–53. capdeville, r., buchdunger, e., zimmermann, j. and matter, a. 2002. glivec (sti571, imatinib), a rationally developed, targeted anticancer drug. nat. rev. drug discov., 1:493–502. carroll, m., ohno-jones, s., tamura, s. et al. 1997. cgp57148, a tyrosine kinase inhibitor, inhibits the growth of cells expressing bcr-abl, tel-abl, and tel-pdgfr fusion proteins. blood, 90:4947–52. chaudhary, n.i., schnapp, a. and park, j.e. 2006. pharmacologic differentiation of infl ammation and fi brosis in the rat bleomycin model. am. j. respir. crit. care med., 173:769–76. choy, e.h. and panayi, g.s. 2001. cytokine pathways and joint infl ammation in rheumatoid arthritis. n. eng j. med., 344:907–16. daniels, c.e., wikes, m.c., edens, m. et al. 2004. imatinib mesylate inhibits the profi brogenic activity of tgf-β and prevents bleomycin-mediated lung fi brosis. j. clin. invest., 114:1308–16. demetri, g.d., von mehren, m., blanke, c.d. et al. 2002. effi cacy and safety of imatinib mesylate in advanced gastrointestinal stromal tumors. n. engl. j. med., 347:472–80. denton, c.p., humbert, m., rubin, l. and black, c.m. 2006. bosentan treatment for pulmonary arterial hypertension related to connective tissue disease: a subgroup analysis of the pivotal clinical trials and their open-label extensions. ann. rheum. dis., 65:1336–40. denton, c.r., merkel, p.a., furst, d.e. et al. 2007. recombinant human anti-transforming growth factor β1 antibody therapy in systemic sclerosis. a multicenter, randomized, placebo-controlled phase i/ii trial of cat-192. arthritis rheum., 56:323–33. distler, j.h.w., jüngel, a., huber, l.c. et al. 2007. imatinib mesylate reduces production of extracellular matrix and prevents development of experimental dermal fi brosis. arthritis rheum., 56:311–22. druker, b.j., tamura, s., buchdunger, e. et al. 1996. effects of a selective inhibitor of the abl tyrosine kinase on the growth of bcr-abl positive cells. nat. med., 2:561–6. druker, b.j., talpaz, m., resta, d.j. et al. 2001a. effi cacy and safety of a specifi c inhibitor of the bcr-abl tyrosine kinase in chronic myeloid leukemia. n. engl. j. med., 344:1031–7. druker, b.j., sawyers, c.l., kantarjian, h. et al. 2001b. activity of a specifi c inhibitor of the bcr-abl tyrosine kinase in the blast crisis of chronic myeloid leukemia and acute lymphoblastic leukemia with the philadelphia chromosome. n. engl. j. med., 344:1038–42. eklund, k.k. and joensuu, h. 2003. treatment of rheumatoid arthritis with imatinib mesylate: clinical improvement in three refractory cases. ann. med., 35:362–7. firestein, g.s. and zvaifl er, n.j. 2002. how important are t cells in chronic rheumatoid synovitis? ii. t cell-independent mechanisms from beginning to end. arthritis rheum., 46:298–308. floege, j., ostendorf, t., janssen, u. et al. 1999. novel approach to specifi c growth factor inhibition in vivo. antagonism of platelet-derived growth factor in glomerulonephritis by aptamers. am. j. pathol., 154:169–79. ghofrani, h.a., seeger, w. and grimminger, f. 2005. imatinib for the treatment of pulmonary arterial hypertension. n. engl. j. med., 353:1412–3. gilbert, r.e., kelly, d.j., mckay, t. et al. 2001. pdgf signal transduction inhibition ameliorates experimental mesangial proliferative glomerulonephritis. kidney int., 59:1324–32. heldin, c-h. and westermark, b. 1999. mechanism of action and in vivo role of platelet-derived growth factor. physiol. rev., 79:1283–316. imamura, f., aono, h., hasunuma, t. et al. 1998. monoclonal expansion of synoviocytes in rheumatoid arthritis. arthritis rheum., 41:1979–86. johnson, r.j., raines, e.w., floege, j. et al. 1992. inhibition of mesangial cell proliferation and matrix expression in glomerulonephritis in the rat by antibody to platelet-derived growth factor. j. exp. med., 175:1413–6. kameda, h., risinger, j.i., han, b-b. et al. 2001. expression of gab1 lacking the preckstrin homology domain is associated with neoplastic progression. mol. cell. biol., 21:6895–905. kameda, h., nagasawa, h., ogawa, h. et al. 2005. combination therapy with corticosteroids, cyclosporine a, and intravenous pulse cyclophosphamide for acute/subacute interstitial pneumonia in patients with dermatomyositis. j. rheumatol., 32:1719–26. kameda, h. and takeuchi, t. 2006. recent advances in the treatment of interstitial lung disease in patients with polymyositis/dermatomyositis. endocr. metab. immune. disord. drug targets, 6:409–15. 247 pdgf as a target for systemic autoimmune diseases drug target insights 2007:2 kameda, h., ishigami, h., suzuki, m. et al. 2006. imatinib mesylate inhibits proliferation of rheumatoid synovial fibroblast-like cells and phosphorylation of gab adapter proteins activated by platelet-derived growth factor. clin. exp. immunol., 144:335–41. kondo, h. 2001. vascular disease in mixed connective tissue disease (mctd). intern. med., 40:1176. lafyatis, r., remmers, e.f., roberts, a.b. et al. 1989. anchorageindependent growth of synoviocytes from arthritic and normal joints. stimulation by exogenous platelet-derived growth factor and inhibition by transforming growth factor-beta and retinoids. j. clin. invest., 83:1267–76. matsuda, m., shikata, k., makino, h. et al. 1997. gene expression of pdgf and pdgf receptor in various forms of glomerulonephritis. am. j. nephrol., 17:25–31. miyachi, k., ihara, a., hankins, r.w. et al. 2003. effi cacy of imatinib mesylate (sti571) treatment for a patient with rheumatoid arthritis developing chronic myelogenous leukemia. clin. rheumatol., 22:329–32. müller-ladner, u., kriegsmann, j., gay, r.e. et al. 1995. oncogenes in rheumatoid arthritis. rheum dis. clin. north am., 21:675–90. müller-ladner, u., kriegsmann, j., franklin, b.n. et al. 1996. synovial fi broblasts of patients with rheumatoid arthritis attach to and invade normal human cartilage when engrafted into scid mice. am. j. pathol., 149:1607–15. müller-ladner, u., gay, r.e. and gay, s. 2000. activation of synoviocytes. curr. opin. rheumatol., 12:186–94. ohba, t., takase, y., ohhara, m. et al. 1996. thrombin in the synovial fl uid of patients with rheumatoid arthritis mediates proliferation of synovial fi broblast-like cells by induction of platelet-derived growth factor. j. rheumatol., 23:1505–11. östman, a. and heldin, c-h. 2001. involvement of platelet-derived growth factor in disease: development of specifi c antagonists. adv cancer res., 80:1–38. paniagua, r.t., sharpe, o., ho, p.p. et al. 2006. selective tyrosine kinase inhibition by imatinib mesylate for the treatment of autoimmune arthritis. j. clin. invest., 116:2633–42. paniagua, r.t. and robinson, w.h. 2007. imatinib for the treatment of rheumatic diseases. nat. clin. pract. rheumatol., 3:190–1. pietras, k., sjöblom, t., rubin, k. et al. 2003. pdgf receptors as cancer drug targets. cancer cell., 3:439–43. remmers, e.f., sano, h. and wilder, r.l. 1991. platelet-derived growth factors and heparin-binding (fi broblast) growth factors in the synovial tissue pathology of rheumatoid arthritis. semin. arthritis rheum., 21:191–9. rice, a.b., moomaw, c.r., morgan, d.l. and bonner, j.c. 1999. specifi c inhibitors of platelet-derived growth factor or epidermal growth factor receptor tyrosine kinase reduce pulmonary fi brosis in rats. am. j. pathol., 155:213–21. rubin, k., terracio, l., ronnstrand, l. et al. 1988. expression of plateletderived growth factor receptors is induced on connective tissue cells during chronic synovial infl ammation. scand. j. immunol., 27:285–94. sadanaga, a., nakashima, h., matsutani, k. et al. 2005. amelioration of autoimmune nephritis by imatinib in mrl/lpr mice. arthritis rheum., 52:3987–96. sandler, c., joutsiniemi, s., lindstedt, k.a. et al. 2006. imatinib mesylate inhibits platelet derived growth factor stimulated proliferation of rheumatoid synovial fi broblasts. biochem. biophys. res. commun., 347:31–5. sano, h., engleka, k., mathern, p. et al. 1993. coexpression of phosphotyrosine-containing proteins, platelet-derived growth factor-b, and fi broblast growth factor-1 in situ in synovial tissues of patients with rheumatoid arthritis and lewis rats with adjuvant or streptococcal cell wall arthritis. j. clin. invest., 91:553–65. schermuly, r.t., dony, e., ghofrani, h.a. et al. 2005. reversal of experimental pulmonary hypertension by pdgf inhibition. j. clin. invest., 115:2811–21. takeuchi, t., amano, k., kameda, h. and abe, t. 2005. anti-tnf biological agents in rheumatoid arthritis and other infl ammatory diseases. allergology international, 54:191–202. thornton, s.c., por, s.b., penny, r., richter, m., shelley, l. and breit, s.n. 1991. identifi cation of the major fi broblast growth factors released spontaneously in infl ammatory arthritis as platelet-derived growth factor and tumor necrosis factor-alpha. clin. exp. imunol., 86:79–86. vittal, r., zhang, h., han, m.k., moore, b.b., horowitz, j.c. and thannickal, v.j. 2007. effects of the protein kinase inhibitor, imatinib mesylate, on epithelial/mesenchymal phenotypes: implications for treatment of fi brotic diseases. j. pharmacol. exp. ther., 321:35–44. watanabe, n., ando, k., yoshida, s. et al. 2002. gene expression profi le analysis of rheumatoid synovial fi broblast cultures revealing the overexpression of genes responsible for tumor-like growth of rheumatoid synovium. biochem. biophys. res. commun., 294:1121–9. white, b., moore, w.c., wigly, f.m., xiao, h.q. and wise, r.a. 2000. cyclophosphamide is associated with pulmonary function and survival benefi t in patients with scleroderma and alveolitis. ann. intern. med., 132:947–54. wollheim, f.a. 2007. treatment of pulmonary fi brosis in systemic sclerosis: light at the end of the tunnel? arthritis rheum., 56:9–12. yamanishi, y. and firestein, g.s. 2001. pathogenesis of rheumatoid arthritis: the role of synoviocytes. rheum dis. clin. north am., 27:355–71. yoshida, m., sakuma-mochizuki, j., abe, k. et al. 1999. in vivo gene transfer of an extracellular domain of platelet-derived growth factor β receptor by the hvj-liposome method ameliorates bleomycininduced pulmonary fi brosis. biochem. biophys. res. commun., 265:503–8. zoja, c., corna, d., rottoli, d., zanchi, c., abbate, m. and remuzzi, g. 2006. imatinib ameliorates renal disease and survival in murine lupus autoimmune disease. kidney int., 70:97–103. << /ascii85encodepages false /allowtransparency false 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insights 2007: 2 71–81 71 review correspondence: j.h. hamman, ph.d., school of pharmacy, tshwane university of technology, private bag x680, pretoria, 0001, south africa. tel: 27 12 382 6397; fax: 27 12 382 6243; email: hammanjh@tut.ac.za please note that this article may not be used for commercial purposes. for further information please refer to the copyright statement at http://www.la-press.com/copyright.htm targeting receptors, transporters and site of absorption to improve oral drug delivery j.h. hamman, p.h. demana and e.i. olivier school of pharmacy, tshwane university of technology, private bag x680, pretoria, 0001, south africa. abstract: although the oral route of drug administration is the most acceptable way of self-medication with a high degree of patient compliance, the intestinal absorption of many drugs is severely hampered by different biological barriers. these barriers comprise of biochemical and physical components. the biochemical barrier includes enzymatic degradation in the gastrointestinal lumen, brush border and in the cytoplasm of the epithelial cells as well as effl ux transporters that pump drug molecules from inside the epithelial cell back to the gastrointestinal lumen. the physical barrier consists of the epithelial cell membranes, tight junctions and mucus layer. different strategies have been applied to improve the absorption of drugs after oral administration, which range from chemical modifi cation of drug molecules and formulation technologies to the targeting of receptors, transporters and specialized cells such as the gut-associated lymphoid tissues. this review focuses specifi cally on the targeting of receptor-mediated endocytosis, transporters and the absorption-site as methods of optimizing intestinal drug absorption. intestinal epithelial cells express several nutrient transporters that can be targeted by modifying the drug molecule in such a way that it is recognized as a substrate. receptor-mediated endocytosis is a transport mechanism that can be targeted for instance by linking a receptor substrate to the drug molecule of interest. many formulation strategies exist for enhancing drug absorption of which one is to deliver drugs at a specifi c site in the gastrointestinal tract where optimum drug absorption takes place. keywords: oral drug delivery, absorption enhancement, receptor-mediated endocytosis, active transporters, site-specifi c drug delivery. introduction oral delivery remains the most favorable and preferred route for drug administration. currently more than 60% of drugs are marketed as oral products (masaoka et al. 2006). however, many drugs cannot be effectively delivered by the oral route of administration in their original form due to reasons of instability, low membrane permeability, poor solubility and effl ux transport mechanisms. overcoming these barriers is currently one of the most challenging goals in oral drug delivery (majumdar and mitra, 2006; leonard et al. 2006; hamman et al. 2005; ghilzai, 2004). the main function of the gastrointestinal tract is to digest and absorb nutrients and fl uids. in addition, it also has to prevent the invasion of toxins, antigens and pathogens. the barriers that exist to fulfi ll this protective task are also responsible for hampering the absorption of drug molecules after oral administration. the physical barrier of the gastrointestinal tract can be attributed to the cell membranes, the tight junctions between adjacent epithelial cells and the mucus layer, while the biochemical barrier comprises of the catabolic enzymes and effl ux systems that pump molecules back into the gastrointestinal lumen (hunter and hirst, 1997; lennernäs, 1998; gabor et al. 2004). the barrier function of the gastrointestinal tract is schematically illustrated in figure 1. the implications of a barrier against drug absorption from the gastrointestinal tract include low drug bioavailability after oral administration. when the bioavailability of a drug is low, it is most likely that insuffi cient drug will become available at the site of action and it will therefore also not produce its pharmacological effect (aungst, 1993; aungst, 2000). several strategies have been employed to improve the bioavailability of drugs after oral administration. some strategies aim at maximizing the intestinal uptake while others focus on protecting the drug molecules from degradation, but combinations there of have also been reported. these strategies include the formation of pro-drugs and/or drug conjugates, modifying the chemical structure of the drug and drug target insights 2007: 272 hamman et al formulation design approaches (gomez-orellana, 2005). although some of these approaches have been demonstrated to be successful in laboratory scale research, they still present challenges in terms of long-term safety and reproducibility in the clinical situation. morishita and peppas (2006) suggested that the development of an effective oral delivery system for a new generation of macromolecular drugs should consider the following three approaches: modifi cation of a physicochemical property of the drug molecule (e.g. lipophilicity and enzyme susceptibility) or addition of novel functionality (e.g. receptor recognition or cell permeability) or the use of a novel drug delivery carrier system. these strategies may be applied alone or in combination to provide a solution to the problem of poor bioavailability. since it is desirable that efforts do not compromise the integrity of the intestinal mucosa (both tight junctions and cell membranes), more safe and practical approaches seem to be targeting of receptors, transporters or absorption sites in the gastrointestinal tract in terms of enhancing the absorption of drugs with poor bioavailabilities. these appealing approaches for optimizing oral drug delivery will be the focus of the discussions in this review. receptor-mediated endocytosis the pharmacological effects of a large number of macromolecules such as proteins and oligonucleotides for the treatment of several human diseases are determined by many factors, including receptor binding, cellular internalization, intracellular sorting and targeting as well as transcellular transport. therefore, the therapeutic applications of most proteins and macromolecular drugs depend largely on their ability to be endocytosed (shen et al. 1992). the main mechanism important in this regard is known as receptor-mediated endocytosis. receptor-mediated endocytosis is a process of internalization of extracellular molecules during which binding occurs between these molecules and the receptors. the receptors are considered as membrane-associated proteins and the intracellular molecules which specifi cally bind to these receptors are known as ligands (shen et al. 1992). following binding to the receptor on the cell surface, the resultant ligand-receptor complex is internalized via a clathrin-dependent or a clathrinindependent endocytotic process. in the clathrin-dependent pathway, the formation of ligand-receptor complex is followed by concentration in clathrin-coated regions or coated pits of the plasma membrane. these coated pits invaginate from the plasma membranes and turn into coated vesicles by pinching inward from the membrane. after their formation, the coated vesicles lose the clathrin coats rapidly, and as a result, smooth membrane vesicles and tubules are formed (morris et al. 1989; vyas and sihorkar, 2000). these early endosomes, which carry receptors and ligands, subsequently participate in a sequence of intracellular processing and sorting events. the clathrin-independent pathway, on the other hand, involves vesicle formation derived from the invagination of non-clathrin-coated plasma membrane. in general, this type of receptor-mediated endocytosis occurs in receptors figure 1. schematical illustration of the barrier properties of the intestinal mucosa. a) the physical barrier includes the tight junctions that limit paracellular transport and the epithelial cell membrane that limits the transcellular transport and b) the biochemical barrier includes brush border and/or intracellular metabolism and apical polarized effl ux (with permission from pauletti et al. 1996). drug target insights 2007: 2 73 targeted oral drug delivery with a low population and a slow rate of internalization, when compared to that of the clathrindependent pathway. the exact internalization mechanism of this pathway is still largely unknown (shen et al. 1992). following receptor-mediated endocytosis process, the endocytosed material may be processed in one of four ways. most ligands are dissociated from their receptors by the low ph (i.e. <5.5) encountered within the endosomes or the receptosomes or the compartment for uncoupling of receptors or ligands (curl) (geuze et al. 1983). the receptors may either be recycled to the cell surface or degraded, while the ligand is routed to the lysosomes for degradation (mostov et al. 1985). alternatively, the binding between the receptor and ligand may be unaffected by acidifi cation and the receptor-ligand complexes are directly sorted to lysosomes for degradation e.g. insulin receptor (féger et al. 1994). the internalization of transferrin-bound iron represents a third process, in which the iron dissociates from the transferrin, which is then returned to the cell surface (russeljones, 2001). the fourth case is characteristic of epithelial and enterocytic cells, and results in the endocytosed material being transcytosed across the cell. in this process, the ligands such as thyroglobulin bind to their receptors on either the apical or basolateral membrane. the complex is then endocytosed and transported to endosomes via coated vesicles. the endosomal material is uncoupled from its receptor and then transported across the cell in an as yet to be identifi ed membrane vesicle (simons et al. 1985). the use of receptor-mediated endocytosis in the gut is very important for oral drug delivery because it can be used to delay intestinal transit of drugs (kilpatrick et al. 1985; king et al. 1986; woodley and naisbet, 1988; lehr et al. 1992), to target drugs to the intestinal epithelial cells (russel-jones, 2001) and for systemic drug delivery (de aizpurua and russel-jones, 1987; pusztai, 1989; lindner et al. 1994). signifi cant developments in the oral delivery of peptides and proteins have been conducted involving receptor-mediated endocytosis process in the vitamin b12 uptake system (de aizpurua et al. 1986; russel-jones and de aizpurua, 1988; habberfi eld et al. 1996; russeljones, 2001), folate absorption (ward et al. 2000; ni et al. 2002; lu, 2002) and also system in which transferrin-receptors are activated (qian et al. 2002; kovar et al. 2002; kursa et al. 2003). vitamin b12 (cyanocobalamine) vitamin b12 is a much larger molecule than other vitamins and therefore cannot enter the body through simple diffusion, facililated diffusion or active transport (russel-jones, 2001). during the absorption of vitamin b12, intrinsic factor (if) produced in the stomach binds to vitamin b12 forming a complex which passes down the small intestine until it reaches the ileum. here the complex binds to a specifi c if receptor (ifr) located on the apical membrane of the villous enterocyte depending on the concentration of calcium ions. the complex is then internalized by the enterocyte via receptor-mediated endocytosis. once inside the cell, the vitamin b12 is released from if following the action of cathepsin l on if (fyfe et al. 1991; schohn et al. 1991; guéant et al. 1992). research has demonstrated that it is possible to link chemically peptides such as luteinizing hormone-releasing hormone (lhrh), and protein such as erythropoietin (epo), granulocyte-colony stimulating factor (g-csf) or interferon-α to vitamin b12 in a way which is capable of shuttling these molecules across the intestinal epithelia without interfering with the ability of vitamin b12 to bind to if (de aizpurua et al. 1986; russel-jones and de aizpurua, 1988; russel-jones, 1995; habberfi eld et al. 1996). vitamin b12 conjugated to an analogue of lhrh was found to be active in stimulating ovulation signifi cantly better in experimental mice than in control mice following an oral dose (russel-jones, 1995). habberfi eld and coworkers linked vitamin b12 to epo and also to g-csf and used these complexes to examine the potential of the vitamin b12 uptake system to transport these systems from the small intestine to the circulation in rats (habberfi eld et al. 1996). it was shown that the vitamin b12 uptake system could deliver epo or g-csf to the circulation in rats at a level 4-fold higher than similar administration of epo or g-csf alone. the use of receptor-mediated endocytosis for oral delivery of nanoparticles linked to vitamin b12 for systemic circulation has also been demonstrated (russel-jones, 1995). nanoparticles containing a fluorochrome have been chemically linked to vitamin b12 and administered to rats orally. upon histological examination, the fl uorescent particles were initially found to be bound to the surface of the intestinal villous cells. some time later, the nanoparticles could be found to have crossed the drug target insights 2007: 274 hamman et al villous epithelial cells and were observed below the mucosal cell layer congregating in the central lacteal gland for systemic circulation (russeljones, 1995). folate in rapidly dividing cells such as cancer cells, receptors are up-regulated and can thus be differentially targeted in drug delivery strategies. the folate receptor is an ideal candidate for tumor-targeted drug delivery because it is upregulated in many human cancers. access to the folate receptor in normal tissues can be severely limited due to its location on the apical membrane of polarized epithelia, and the density of folate receptors appears to increase as the stage or grade of the cancer worsens (lu et al. 2002). the conjugation of folic acid via its γ-carboxylic group has resulted in drug binding to cells expressing the folate receptor and consequently endocytosis taking place (lu et al. 2002). folic acid has been linked to both drugs of low molecular weight and the macromolecular complexes as a means of targeting the attached molecules to malignant cells (lu et al. 2002). although this conjugation has been shown to enhance the delivery of macromolecules to folate receptor-expressing cancer cells in almost all in vitro situations tested, mixed effects have however, been observed when conducting similar studies in vivo conditions. despite these mixed effects, prominent examples do exist where folate targeting has signifi cantly improved the outcome of a macromolecule-based therapy, leading to complete remission of established tumors (ward, 2000; lu et al. 2002). for example, folate receptor-targeted delivery of liposomal daunorubicin to folate receptor expressing cells was found to have signifi cantly increased drug cellular uptake and cytotoxicity compared to other cells (ni et al. 2002). transferrin the use of the transferrin receptor for targeted drug delivery has also been receiving attention in literature in recent years (xu et al. 2001; qian et al. 2002; kovar et al. 2002; kursa et al. 2003). high levels of transferrin receptors are expressed on the surface of actively metabolizing cells (iacopetta et al. 1982; banerjee et al. 1986), certain tumors (faulk et al. 1980), and the brain capillary endothelium (jefferies et al. 1984). the expression of high levels of transferrin receptor in the brain capillary endothelium is particularly important because there is a possibility of delivering drugs across the blood-brain barrier. this was demonstrated using a conjugate of methotrexate with anti-transferrin receptor antibody which was shown to bind and traverse the blood-brain barrier (frieden et al. 1980). apart from delivering drugs across the blood-brain barrier, conjugates of transferrin have been successfully used to selectively kill cell lines expressing the transferrin receptor in certain cancers (cawley et al. 1981; raso and basala, 1984). furthermore, polylysine conjugates of transferrin in particular have been used to deliver dna sequences to cell lines in culture for the development of gene therapy (wagner et al. 1990). the potential problem with manipulation of transferrin uptake as a means of drug delivery across cells is the recyling pathway through which both transferrin and its receptor undergo (dautryvarsat, 1986). however, research fi ndings have now suggested possible means of modulating the recycling pathway to achieve greater transport across the cells. this was demonstrated with brefeldin a, a drug that causes the disruption of transport of secretory proteins from the endoplasmic reticulum to golgi cisternae (wan et al. 1991). this drug showed a capacity to cause a missorting of the transferrin receptor from the basal to the apical membrane and a consequent 30and 100-fold increase in the transcytosis of transferrin in the basal-to-apical and the apical-to-basal direction, respectively (wan et al. 1991). similarly, monensin which is a drug with reversible disrupting activity on the golgi apparatus (wan et al. 1990) has been shown to increase the transcotysis of transferrin and its conjugates in the basal-to-apical direction by up to 26-fold (wan et al. 1991). membrane transporters many organic solutes such as nutrients (i.e. amino acids, sugars, vitamins and bile acids) and neurotransmitters are transferred across cell membranes by means of specialized transporters. these carrier systems comprise integral membrane proteins that are capable of transferring substrates across cell membranes by means of a passive process (i.e. through channels or facilitated transporters) or an active process (i.e. with carriers). carrier-mediated active transport requires energy drug target insights 2007: 2 75 targeted oral drug delivery obtained by adenosine tri-phosphate (atp) hydrolysis or by coupling to the co-transport of a counterion down its electrochemical gradient (e.g. na+, h+, cl–). several drugs and pro-drugs share this transport pathway with nutrients and it has been shown that targeting drugs to these transporter carriers can infl uence their bioavailability as well as their distribution (zhang et al. 2002; steffansen, 2004). targeting drug delivery to intestinal nutrient transporters has emerged as an important strategy to improve oral bioavailability of poorly permeating therapeutic agents. this approach usually entails linking the drug molecule to a natural ligand in order to be recognized as a substrate by a specifi c nutrient transporter in the apical membrane of the epithelial cell. alternatively, the drug molecule can be designed or changed (e.g. formation of derivatives or pro-drugs) in such a way that it mimics the three-dimensional features of natural ligands. these pro-moieties are then either cleaved within the intracellular environment of the epithelial cells or elsewhere in the body to free the active drug (zhang et al. 2002; majumdar et al. 2004). in general, transporter proteins that can be targeted for this purpose are those that provide transport mechanisms for amino acids, dipeptides, monosaccharides, monocarboxylic acids, organic cations, phosphates, nucleosides and water-soluble vitamins (lee, 2000). peptide transporters exogenic peptides are rapidly metabolized in the gastrointestinal tract by proteolytic enzymes into smaller oligopeptides, tripeptides, dipeptides as well as amino acids. while the absorption of larger peptides across intestinal epithelial cells is restricted, large amounts of amino acids and di/ tripeptides cross the enterocytic membrane by means of transporter systems (steffansen et al. 2005). human intestinal membrane transporters involved in the uptake of di/tripeptides include the peptide transporter pept1, the peptide/histidin transporters pht1, pht2 and the peptide transporter pt1. the peptide transporter pept2 is found in other types of tissue than the small intestine and only limited information is available on pht1 and pt1, but pept1 is widely described in the literature (steffansen et al. 2004). pept1 is an h+-coupled, active transport system with a broad substrate specifi city, which may range from natural substrates in food such as diand tripeptides to peptide-like therapeutic agents such as β-lactam antibiotics and angiotensin-converting enzyme (ace) inhibitors (zhang et al. 2002). even substances without an obvious peptide bond such as δ-amino-levulinic acid and ω-amino fatty acids are substrates for this transporter (lee, 2000). due to the wide substrate specifi city of pept1, various approaches with pro-drugs that are aimed at targeting this transporter have been attempted. one approach is to form dipeptidyl based pro-drugs by linking dipeptides with intrinsic affi nity for pept1 to the drug molecule such as asp-sar and glu-sar. it was shown that a variety of derivatized dipeptides target pept1 to improve bioavailability, for example the dipeptidyl derivatives of α-methyldopa as well as p-glu-l-dopa-pro and l-dopa-phe showed enhanced permeability as compared to the parent drugs respectively. another approach to target pept1 is to form amino acid pro-drugs, for example the l-valyl ester pro-drug of acyclovir increased its oral bioavailability 3–5 times (steffansen et al. 2004). enalapril is an ester pro-drug of the ace-inhibitor, enalaprilat and is a substrate for pept1. formation of this pro-drug of enalaprilat resulted in an increase of the oral bioavailability from 3–12% to 60–70% (zhang et al. 2002). although pept1 targeted amino acid and dipeptidyl pro-drugs show potential for the effective delivery of di/tripeptidomimetics and small drug molecules, it seems to be limited for the delivery of larger peptides or macromolecules (steffansen et al. 2005). amino acid transporters amino acid transporters are widely expressed by almost all living cells and are responsible for the absorption of amino acids from the gastrointestinal tract into the systemic circulation and distribution into tissues. seven amino acid transport systems have been identifi ed in the brush border of the small intestine of which some exhibit overlapping substrate specifi cities (hidalgo and li, 1996). these amino acid transporters present a potential target for improving the absorption of drugs and numerous studies have investigated the possibility of targeting pro-drugs and derivatives to be absorbed via these carriers. the absorption of gabapentin from the small intestine, for example, drug target insights 2007: 276 hamman et al is mediated by the large neutral amino acid transporter (majumdar and mitra, 2006). nucleoside transporters although nucleosides and nucleotides are essential precursors for the synthesis of nucleic acids, they are not required to be taken up by most cells because these compounds are synthesized intracellularly. however, the synthesis of purines and pyrimidines in enterocytes is insufficient to support their rapid division. these building blocks of nucleic acids are therefore absorbed from the intestinal lumen through equilibrative (facilitated diffusion) transport systems and na +-dependent concentrative (energy-dependent active transport) mechanisms (hidalgo and li, 1996; lee, 2000). examples of drugs that are absorbed by equilibrative nucleoside transporters include s-adneosylmethionine, fludarabine, arabinosylcytosine and azidothymidine (majumdar et al. 2004). bile acid transporters bile acids are synthesized in the liver and secreted into the duodenum after ingestion of a meal, to facilitate the digestion and absorption of fats. approximately 90% of the bile acids that are secreted into the small intestinal lumen are recycled back to the liver to prevent the continuous re-synthesis of large amounts of bile acids. re-absorption of bile acids occurs by passive diffusion in the jejunum, but by active transport in the ileum. the na+ -dependent bile acid active transport carriers are located in the apical membrane of the epithelial cells of the ileum and the na+ gradient required is provided by na+ /k+ atpase located in the basolateral cell membrane. the strategy to enhance drug absorption via this active transporter system involves formation of bile acid-drug conjugates. it was shown that the size of the molecule conjugated to the bile acid plays an important role in its ability to be absorbed via the bile acid transporters. another challenge to be overcome, before this strategy can be used for drug absorption, is to avoid biliary secretion of the conjugates back into the gastrointestinal lumen. it seems that if the drug is not released from the conjugate before reaching the liver, it will most probably be secreted into the bile (hidalgo and li, 1996). monocarboxylic acid transporters the transport of lactic acid, which is produced during the metabolic reactions to generate atp, into and out of cells is mediated by h+ -dependent monocarboxylic acid transporter family. in addition, monocarboxylate drugs such as valproic acid, salicylic acid and pravastatin have been shown to be transported by monocarboxylic acid transporters in the intestine. however, because the retinal pigmented epithelium expresses a monocarboxylic acid transporter, pro-drugs targeted at these transporters may be useful in enhanced retinal drug permeation to achieve higher drug concentrations in the deeper layers of the cornea and aqueous humor (lee, 2000; majumdar et al. 2004). miscellaneous (glucose, fatty acid, vitamin, organic cation and phosphate) transporters two types of transporters exist for the transport of monosaccharides across biological membranes, these include the sodium-dependent na+/glucose co-transporters (sglt) and sodium-independent glucose transporters (glut). because sglt1 exhibits a high capacity and broad substrate specificity, targeting this receptor for drug delivery offers an exciting opportunity to improve the bioavailability of drugs (steffansen et al. 2004). three types of fatty acid transporter proteins have been identifi ed of which fatp4 is located in the apical membrane of the small intestine with long chain fatty acids as substrates (e.g. myristate, oleate and palmitate). not much information is currently available on these transporters and further investigation is needed to determine their usefulness in drug delivery (steffansen et al. 2004). although transporters for uptake of watersoluble vitamins are expressed in the intestine such as those for vitamin c (ascorbic acid) and biotin, their general low capacities make them poor transporter candidates to target for enhancement of drug absorption (steffansen et al. 2004). many drugs that carry a positive charge at physiological ph values (e.g. antihistamines) are transported by the organic cation transporters. phosphate transporters hold some potential for the delivery of drugs and fosfomycin as well as foscarnet have been shown to be substrates for these transport carrier systems (majumdar et al. 2004). drug target insights 2007: 2 77 targeted oral drug delivery p-glycoprotein effl ux transporters p-glycoprotein (p-gp), an mdr1 gene product, is the most extensively studied member of the superfamily of atp-binding cassette (abc) transporters. p-gp is associated with multi-drug resistance (mdr) in cancer cells, which is responsible for failure of chemotherapy with many drugs. although p-gp is over-expressed in tumors, it is also localized in several tissues, particularly in the columnar epithelial cells of the lower gastrointestinal tract, capillary endothelial cells of the brain and testis, canalicular surface of the hepatocytes and on the apical surface of the proximal tubules in the kidney. clinically, this efflux transporter plays an important role in the absorption, disposition, metabolism and excretion of a variety of drugs. it constitutes a formidable barrier against drug absorption by limiting drug uptake from the intestinal lumen into the systemic circulation. furthermore, it pumps drug molecules out from hepatocytes into the canalicular system, prevents distribution of drugs to the brain and restricts re-absorption of drug into the systemic circulation from renal tubules (katragadda et al. 2005; ambudkar et al. 2006; varma et al. 2006). the hypothesis that inhibition of p-gp improves the bioavailability of drugs that are substrates for this effl ux transporter is gaining widespread recognition. moreover, the pharmacokinetic advantages of p-gp inhibition includes improved effi cacy of chemotherapeutic agents, enhanced intestinal absorption and reduced clearance. oral co-administration of the p-gp inhibitor, verapamil, has demonstrated an increase in the peak plasma level and volume of distribution as well as a prolonged halflife of doxorubicin. however, these fi rst generation p-gp inhibitors pose a pharmacological effect themselves and therefore possible toxic and or other unwanted effects may occur. this has led to the design of second and third generation p-gp inhibitors with the potential to enhance the absorption of p-gp substrates without undesirable pharmacologic or toxic effects (varma et al. 2003). examples of pro-drugs that target an active transporter and simultaneously decrease the substrate’s interaction with p-gp are the dipeptides derivatives of saquinavir, namely l-valine-l-valinesaquinavir and l-glycine-l-valine-saquinavir. these dipeptides pro-drugs that target peptide transporters and diminish interaction with p-gp exhibited an overall increased transport from the apical to basolateral side in caco-2 cell monolayers. this example shows the potential of rational pro-drug design to decrease p-gp mediated efflux and thereby increase the absorption of drugs that are substrates for this effl ux pump (majumdar and mitra, 2006). site of absorption site-specific absorption occurs in the gastrointestinal tract because of differences in the composition and thickness of the mucus layer, ph, surface area and enzyme activity (hamman et al. 2005). furthermore, the physicochemical properties of the drug not only influence the site of absorption but also the mechanism of absorption. despite these differences the most important site for intestinal drug absorption is the small intestine (lacombe et al. 2004, masaoka et al. 2006). in general, drug permeability is accepted to be higher in the upper region of the gastrointestinal tract compared to the lower parts (masaoka et al. 2006). timing of drug delivery is therefore important for optimized absorption and in diseases that are related to the circadian rhythm such as asthma and rheumatoid arthritis (weidner, 2001). in a recent study by masaoke et al. (2006), various factors that may contribute to the regional absorption of drugs from the intestine were studied. they concluded that the epithelial surface area should not be a determining factor in drug absorption for highly permeable drugs in the different regions of the gastrointestinal tract. in contrast, the effects of the mucus layer and fl uidity of the cell membrane of the different regions were found to contribute to dissimilarities in intestinal drug permeability. regional membrane fluidity decreased from the upper to the lower parts of the gastrointestinal tract. atenolol, a drug with low permeability, was observed to be absorbed in the middle and lower portions of the jejunum, while highly permeable drugs such as antipyrine and metoprolol were generally absorbed in the upper part of the intestine and also possibly in the stomach. the drug permeability of griseofulvin and naproxen was higher in the colon compared to the jejunum. it was found that removal of the mucus layer of the jejunum signifi cantly enhances griseofulvin absorption to almost the same levels as those observed in the ileum and colon. they concluded that the main factors affecting drug drug target insights 2007: 278 hamman et al absorption are membrane permeability, luminal drug concentration and residence time in the different parts of the gastrointestinal tract, while regional ph differences are specifi cally important for poorly permeable drugs. upper gastrointestinal delivery the stomach is responsible for initial digestion, temporary food storage and controlled release of the resulting chime into the duodenum. the small surface area and short residence time in the stomach limits gastric absorption, however, gastric retentive systems can be used for local action in the stomach (e.g. antacids, misoprostol, antibiotics for helicobacter pylori), absorption of drugs in the stomach and upper small intestine (e.g. l-dopa, p-aminobenzoic acid, furosemide, ribofl avin and fl avin mononucleotide), drugs that are unstable in the intestine and colon (e.g. captopril and ranitidine) or for drugs that exhibit low solubility at high ph values (e.g. diazepam, chlordiazepoxide and verapamil). gastric retention is not desirable when drugs cause gastric irritation (e.g. non-steroidal anti-infl ammatory drugs), are unstable in the acid ph of the stomach or for drugs that exhibit significant fi rst-pass liver metabolism (e.g. nifedipine) (streubel et al. 2006). various formulation techniques have been used to achieve gastric retention, including bioadhesive systems, gastric swellable systems, density controlled systems that fl oat or sink in gastric fl uid and magnetic systems that require positioning of an external magnet. each of the above techniques face their own challenges such as the high turnover rate of gastric mucus for bioadhesive systems, the low-density fl oating systems are dependent on the fl uid volume in the stomach and magnetic systems require accurate external magnet positioning that patients may not be able to comply with (bardonnet et al. 2006; streubel et al. 2006). enteric coating enteric coating is employed to delay release of the active ingredient until it reaches the small intestine. this coating technique has been used to release drugs in the small intestine such as aspirin in order to reduce gastric irritation and erythromycin that exhibits acid degradation. various polymers have been used as enteric polymers that become “soluble” once the ph of the environment reaches the range between 5 and 7. polymers that degrade above a ph of 7 have been used in an attempt to target colonic drug delivery in diseases such as colitis (gibaldi, 1984). however, the use of enteric coating to obtain colonic delivery has been reported to be less successful (basit et al. 2004). magnesium chloride is an example of a compound that is prone to gastric irritation due to excessive formation of hydrochloric acid in the stomach when formulated into immediate release products. magnesium is actively absorbed from the small intestine (reynolds, 1993) and attempts have been made to target this area by means of enteric coating. the targeting of the proximal regions of the small intestine by enteric coating has, however, been criticized because release of the active ingredient may only occur 1–2 hours after expulsion from the stomach (basit et al. 2004). this suggests release of the drug in the distal parts of the small intestine. another potential drawback is that enteric coated tablets may be retained in the stomach for an extended period of time when taken with a heavy breakfast (friend, 2005). ranitidine was used as a model drug to investigate differences in the bioavailability when administered in the form of immediate release, enteric coated and colon targeted delivery systems. the absolute mean bioavailability of ranitidine was found to be statistically similar for the immediate and enteric coated formulations, while it was much lower for the colonic release formulation. this was despite the fact that effective colonic release was demonstrated which was achieved by using a mixture of amylose and ethylcellose. amylose is susceptible to degradation by amylase producing bacteria that reside in the colon (basit et al. 2004). in this case the poor colonic bioavailability of ranitidine was ascribed to colonic bacterial metabolism (friend, 2005). colonic delivery targeting of the colon as a site of absorption has recently received attention by various authors because of its favorable properties particularly for the absorption of peptide drugs, proteins and biotechnical molecules (weidner, 2001, gazzaniga et al. 2006). some of the advantages of colonic delivery for these types of drugs include the reduced concentration of enzymes such as peptidases that degrade peptide drugs, the colon is a site with significant absorption due to the long drug target insights 2007: 2 79 targeted oral drug delivery residence time in this part of the gastrointestinal tract, it exhibits enhanced sensitivity to absorption enhancers, demonstrates natural absorptive characteristics and the abundance of lymphoid tissue follicles may be responsible for macromolecule uptake (weidner, 2001, hamman et al. 2005, gazzaniga et al. 2006). the colon has been used as a target for treatment of conditions that affect this part of the gastrointestinal tract such as ulcerative colitis, crohn’s disease and adenocarcinoma (weidner, 2001, gazzaniga et al. 2006). colonic targeting has been studied using various formulation techniques such as reservoir systems with rupturable, erodible-, diffusive polymeric coats, release controlling polymeric plugs and osmotic systems. currently only micro fl ora-, ph dependent pressureand time controlled technologies are available on the market. potential problems associated with some of the above systems include intraand inter-subject intestinal ph variability, physiological fl uctuation and disease conditions. micro flora imbalances due to diet and habit changes are also a cause for concern when targeting the colon particularly when using systems based on metabolism of coatings for colonic delivery. most of these disadvantages can be circumvented by using time-controlled systems (gazzaniga et al. 2006), however in patients with irritable bowl syndrome intestinal transit times can vary from those observed in healthy subjects and patients with ulcerative colitis commonly experience diarrhea (friend, 2005). specialized delivery systems superporous hydrogels and composites thereof have been described for use in specialized systems designed for the delivery of peptide drug. they swell very quickly and mechanically interact with intestinal membranes at the specifi c site of absorption. the lag time provided by the system enables drug release from the core to achieve optimal absorption (dorkoosh et al. 2001). other specialized dosage forms include particulate systems that are designed to protect the drug against enzymatic degradation and to provide a high transfer rate of drug across the epithelial mucosa. some particulate systems are capable of being taken up through peyer’s patches without addition of absorption enhancers. these systems include nanoparticles, liposomes, microspheres and lipid based systems. despite increased oral peptide delivery with modifi ed liposomes, solid particles appear to be more effective for the delivery of hydrophilic macromolecules (morishita and peppas, 2006). nano-sized particles such as chitosan-coated nanoparticles have illustrated limited success as peptide delivery systems. these systems have also been linked to ligands to target specifi c absorption carriers, however, these types of systems still have serious problems with the manufacturing process and safety issues such as accumulation of the carrier in tissues (hamman et al. 2005, morishita and peppas, 2006). examples of marketed drug products and drugs under investigation are listed in table 1. references ambudkar, s.v., kim, i-w. and sauna, z.e. 2006. the power of the pump: mechanisms of action of p-glycoprotein (abcb1). eur. j. pharm. sci., 27:392–400. aungst, b.j. 1993. novel formulation strategies for improving oral bioavailability of drugs with poor membrane permeation or presystemic metabolism. j. pharm. sci., 82(10):979–87. aungst, b.j. 2000. intestinal permeation enhancers. j. pharm. sci., 89(4):429–42. banerjee, d., flanagan, p.r. and cluett, j. et al. 1986. transferrin receptors in the human gastrointestinal tract. gastroenterology, 91:861–9. bardonnet, p.l., faive, v. and pugh, w.j. et al. 2006. gastroretentive dosage forms: overview and special case of helicobacter pilori. j. control release, 111:1–18. drug formulation trade name mesalamine eudragit-s/-l coated tablets asacol®/salofac® budesonide eudragit-l coated beads entocort® acetazolamide sustained release capsules diamox sa® aminophyline matrix tablets pecram® aspirin enteric coated tablets caprin® carbamazepine modifi ed release tablet tegretol retard® diclofenac enteric coated tablets voltaren® table 1. some examples of drug products on the market associated with targeting the site of drug delivery (lund, 1994; friend, 2005). drug target insights 2007: 280 hamman et al basit, a.w., podczeck, f. and newton, j.m. et al. 2004. the use of formulation technology to assess regional gastrointestinal drug absorption in humans. eur. j. pharm. sci., 21:179–89. cawley, d.b., simpson, d.l. and hershman, h.r. 1981. asiologlycoprotein receptor mediates the toxic effects of an asiolofetuin-diptheria toxin fragment a conjugate on cultured rat hepatocytes. proc. natl. acad. sci. u.s.a., 78:3383. dautry-varsat, a. 1986. receptor-mediated endocytosis: the intracellular journey of transferrin and its receptor. biochimie. 68:375–81. de aizpurua, h.j., burge, h.j. and howe, p.a. et al. 1986. oral delivery system pct/au86/0299. de aizpurua, h.j. and russel-jones, g.j. 1987. oral vaccination: identifi cation of classes of proteins which provoke response upon oral feeding. j. exp. med., 167:440–51. dorkoosh, f.a., verhoef, j.c. and borchard, g. et al. 2001. development and characterization of a novel peroral peptide drug delivery system. j. control release, 17:307–18. faulk, w.p., his, b.l. and stevens, p.j. 1980. transferrin and transferrin receptors in carcinoma of the breast. lancet., ii:390–2. féger, j., gil-falcon, s., lamaze, c. 1994. cell receptors: defi nition, mechanisms and regulation of receptor-mediated endocytosis. cell. mol. biol., 40:1039–61. frieden, p.m., walus, l.r. and musso, g.f. et al. 1991. anti-transferrin receptor antibody and antibody-conjugates cross the blood brain barrier. proc. natl. acad. sci. u.s.a., 88:4771–5. friend, d.r. 2005. new oral delivery systems for the treatment of infl ammatory bowel disease. adv. drug deliv. rev., 57:247–65. fyfe, j.c., ramanujam, j.c. and ramasamy, k.s. et al. 1991. defective brush-border expression of intrinsic factor-cobalamin receptor in canine inherited intestinal cobalamin malabsorption. j. biol. chem., 266:4489–94. gabor, f., bogner, e., weissenboeck, a. et al. 2004. the lectin-cell interaction and its implications to intestinal lectin-mediated drug delivery. adv. drug deliv. rev., 56:459–80. gazzaniga, a., marconi, a. and sangalli, m.e. et al. 2006. time-controlled oral delivery systems for colon targeting. expert. opin. drug deliv., 3(5):583–97. geuze, j.j., slot, j.w. and strou, g.j. 1983. intracellular site of asiologlycoprotein receptor-ligand uncoupling: double-label immunoelectronmicroscopy during receptor-mediated endocytosis. cell. 35:277–287. ghilzai, m.k. 2004. advances in the delivery of large-size drug molecules. innovations in pharmaceutical technology, jun.,:103–8. gibaldi, m. 1984. gastrointestinal absorption—role of the dosage form. in: biopharmaceutics and clinical pharmacokinetics. 3rd ed. lea and febiger:philadelphia p. 71–3. gomez-orellana, i. 2005. strategies to improve oral drug bioavailability. expert opin. drug deliv., 2(3):419–33. guéant, j.l., masson, d. and schohn, h. et al. 1992. receptor-mediated endocytosis of intrinsic factor-cobalamin complex in ht 29, a human colon carcinoma cell line. febs lett., 297:229–32. habberfi eld, a., jensen-pippo, k. and ralph, l. et al. 1995. vitamin b12mediated uptake of recombinant therapeutic proteins from the gut. nature med., submitted. habberfi eld, a., jensen-pippo, k. and ralph, l. et al. 1996. vitamin b12mediated uptake of erythropoietin and granulocyte colony stimulating factor in vitro and in vivo. int. j. pharm., 145:1–8. hamman, j.h., enslin, g.m. and kotze, a.f. 2005. oral delivery of peptide drugs biodrugs, 19:165–77. hidalgo, i.j. and li, j. 1996. carrier-mediated transport and effl ux mechanisms in caco-2 cells. adv. drug deliv. rev., 22:53–66. hunter, j. and hirst, b.h. 1997. intestinal secretions of drugs: the role of p-glycoprotein and related drug effl ux systems in limiting oral drug absorption. adv. drug deliv. rev., 25:129–57. iacopetta, b.j., morgan, e.h. and yeoh, gct. 1982. transferrin receptors and iron uptake during erythroid cell development. biochim. biophys. acta., 687:204–10. jefferies, a.e., brandon, m.r. and hunt, s.v. et al. 1984. transferrin receptors on endothelium of brain capillaries. nature, 312:162–163. katragadda, s., budda, b. and anand, a. et al. 2005. role of effl ux pumps and metabolising enzymes in drug delivery. expert. opin. drug deliv., 2(4):683–705. kilpatrick, d.c., pusztai, a. and grant, g. et al. 1985. tomato lectin resists digestion in mammalian alimentary canal and binds to intestinal villi without deleterious effects. febs lett., 185:299–305. king, t.p., pusztai, a. and grant, g. et al. 1986. immunogold localization of ingested kidney bean (phaseolus vulgaries) lectin in epithelial cells of the rat small intestine. histochem. j., 18:413–20. kovar, m., strohalm, j. and ulbrich, k. et al. 2002. in vitro and in vivo effect of hpma copolymer-bound doxorubicin targeted transferrin receptor to b-cell lymphoma 38c13. drug target, 10:23–30. kursa, m., walker, g.f., roessler, v. and ogris, m. et al. 2003. novel shielded transferring-polyethylene glycol-polyethylenimine/dna complexes for systemic tumor-targeted gene transfer. bioconj.chem., 14:222–31. lacombe, o., woodley, j., solleux, c. and delbos, j-m. et al. 2004. localization of drug permeability along the rat small intestine, using markers of the paracellular, transcellular and some transporter routes. eur. j. pharm. sci., 23:385–91. lee, vhl. 2000. membrane transporters. eur. j. pharm. sci., 11:s41–50. lehr, c.m., bouwstra, j.a. and kok, w. et al. 1992. bioadhesion by means of specifi c binding of tomato lectin. pharmacol. res., 9:547–53. lennernäs, h. 1998. human intestinal permeability. j. pharm. sci., 87(4): 403–10. leonard, t.w., lynch, j. and mckenna, m.j. et al. 2006. promoting absorption of drugs in humans medium chain fatty acid-based solid dosage forms: gipet™. expert. opin. drug. deliv., 3(5):685–92. lindner, j., geczy, a.f. and russel-jones, g.j. 1994. identifi cation of the site of uptake of the e-coli heat labile enterotoxin, ltb. scand. j. immunol., 40:564–72. lu y. and low, p.s. 2002. folate-mediated delivery of macromolecular anticancer therapeutic agents. adv. drug deliv. rev., 54:675–93 lund, w., ed. 1994. the pharmaceutical codex. london: the pharmaceutical press. majumdar, s., duvuri, s. and mitra, a.k. 2004. membrane transporter/ receptor-targeted prodrug design: strategies for human and veterinary drug development. adv. drug deliv. rev., 56:1437–52. majumdar, s. and mitra, a. 2006. chemical modifi cation and formulation approaches to elevated drug transport across cell membranes. expert opin. drug deliv., 3(4):511–27. masaoka, y., tanaka, y. and kataoke, m. et al. 2006. site of absorption after oral administration: assessment of membrane permeability and luminal concentration of drugs in each segment of gastrointestinal tract. eur. j. pharm. sci., in press. morishita, m. and peppas, n.a. 2006. is the oral route possible for peptide and protein drug delivery? drug discov. today., 11:905–10. morris, s.a., ahle, s. and ungewickell, e. 1989. clathrin-coated vesicles. curr. opin. cell. biol., 1:684–90. mostov, k.e. and simister, n.e. 1985. transcytosis. cell., 43:389–90. ni, s., stephenson, s.m. and r.j. 2002. folate receptor targeted delivery of liposomal daunorubicin into tumor cells. anticancer res., 22:2131–2135. pauletti, g.m., gangwar, s. and knipp, g.t. et al. 1996. structural requirements for intestinal absorption of peptide drugs. j. control release, 41:3–17. pusztai, a. 1989. transport of proteins through the membranes of the adult gastro-intestinal tract — a potential for drug delivery? adv. drug deliv. rev., 3:215–28. qian, z.m., li, h. and sun, h. et al. 2002. targeted drug delivery via transferring receptor-mediated endocytosis pathway. pharmacol. rev., 54:561–587. raso, v. and basala, m. 1984. a highly cytotoxic human transferrin-ricin a chain conjugate used to select receptor-modifi ed cells. j. biol. chem., 259:1143–49. drug target insights 2007: 2 81 targeted oral drug delivery reynolds, j.e.f. 1993. magnesium. in: martindale the extra pharmacopoeia. 30th ed. the pharmaceutical press:london p. 857. russel-jones, g.j. and de aizpurua, h.j. 1988. vitamin b12: a novel carrier for orally presented antigens. proc. int. symp. control rel. bioact. mater, 15:142–3. russel-jones, g.j. 1995. oral delivery of therapeutic proteins and peptides by the vitamin b12 uptake system. in: taylor md, amidon g, eds. peptide-based drug design: controlling transport and metabolism, acs books, washington, dc, pp 181–98. russell-jones, g.j. 2001. the potential use of receptor-mediated endocytosis for oral drug delivery. adv. drug deliv. rev., 46:59–73. schohn, h., guéant, j.l. and girr, m. et al. 1991. synthesis and secretion of a cobalamin-binding protein by ht 29 cell line. biochem. j., 280:427–30. shen, w., wan, j. and ekrami, h. 1992. enhancement of polypeptide and protein absorption by macromolecular carriers via endocytosis and transcytosis. adv. drug deliv. rev., 8:93–113. simons, k. and fuller, s.d. 1985. cell surface polarity in epithelia. annu. rev. cell. biol., 1:243–48. steffansen, b., nielsen, c.u. and brodin, b. et al. 2004. intestinal solute carriers: an overview of trends and strategies for improving oral drug absorption. eur. j. pharm. biopharm., 21:3–16. steffansen, b., nielsen, c.u. and frokjaer, s. 2005. delivery aspects of small peptides and substrates for peptide transporters. eur. j. pharm. biopharm., 60:241–5. steubel, a., siepmann, j. and bodmeier, r. 2006. gastroretentive drug delivery systems. expert opin. drug deliv., 3(2):217–33. varma, m.v.s., perumal, o.p. and panchagnula, r. 2006. functional role of p-glycoprotein in limiting peroral drug absorption: optimizing drug delivery. curr. opin. chem. biol., 10:367–73. varma, m.v.s., ashokraj, y. and dey, c.s. et al. 2003. p-glycoprotein inhibitors and their screening: a perspective from bioavailability enhancement. pharmacol. res., 48:347–359. vyas, s.p. and sihorkar, v. 2000. endogenous carriers and ligands in nonimmunogenic site-specifi c drug delivery. adv. drug deliv. rev., 43:101–164. wagner, e., zenke, m. and cotten, m. et al. 1990. transferrin-polycation conjugates as carriers for dna uptake into cells. used to select receptor-modifi ed cells. proc. natl. acad. sci. u.s.a., 87:3410–14. wan, j. and shen, w.c. 1990. transcytosis of transferring is enhanced by monensin in fi lter grown mdck epithelial cells. j. cell biol., 111:83a. wan, j., shah, d. and shen, w.c. 1991. brefeldin a (bfa) induces missorting of transferrin receptors (tfr) and increases receptor-mediated transcytosis of transferrin (tf) in fi lter grown mdck cells. j. cell biol., 115:259a. ward, c.m. 2000. folate-targeted non-viral dna vectors for cancer gene therapy. curr. opin. mol. ther., 2:182–187. weidner, j. 2001. drug delivery drug discov. today, 6(19):1028–9. woodley, j.f. and naisbett, b. the potential for delaying the gastro-intestinal transit of drugs. proc. int. sym. control rel. bioact. mater., 15 (1988) 125–126. xu, l., tang, w.h. and huang, c.c. 2001. systemic p53 gene therapy of cancer with immunolipoplexes targeted by anti-transferrin receptor scfz. mol. med., 7:723–34. zhang, e.y., phelps, m.a. and cheng, c. et al. 2002. modeling of active transport systems. adv. drug deliv. rev., 54:329–54. https://doi.org/10.1177/1177392819866412 drug target insights volume 13: 1–13 © the author(s) 2019 article reuse guidelines: sagepub.com/journals-permissions doi: 10.1177/1177392819866412 creative commons non commercial cc by-nc: this article is distributed under the terms of the creative commons attribution-noncommercial 4.0 license (http://www.creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the sage and open access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage). alzheimer disease (ad) is a devastating, progressive, and irreversible neurodegenerative disorder, which is clinically characterized by the deterioration of memory, disorientation, increased confusion, and other psychological as well as physical manifestations (figure 1).1 the appearance of extracellular amyloid-beta (aβ) deposits in senile plaques and the development of intracellular neurofibrillary tangles, reactive microgliosis, and astrogliosis are the primary histopathological characteristics of ad.2 alzheimer disease primarily affects the elderly,3 is the most common and feared type of dementia, represents 70% of all dementia cases, and is a worldwide epidemic. bertram et al4 postulated that in addition to the sporadic form of ad, for which aging is the primary factor, mutations in the amyloid-beta precursor protein (aβpp), presenilin 1 (psen1), and presenilin 2 (psen2) cause autosomal dominant earlyonset familial ad. polidori et al5 found that genetic and environmental factors, vascular pathology, and other risk factors also play crucial roles in ad pathogenesis. due to the lack of effective disease-modifying treatments, findings on pharmacological or nonpharmacological strategies to slow disease progression are of significant importance. in addition, the failure of potential pharmaceuticals in human clinical trials has highlighted the need for research into early ad diagnosis. as synaptic and neuronal loss along with brain shrinkage has already occurred when ad’s clinical symptoms appear, current treatments that seek to slow disease progress are more likely to be effective before the onset of ad symptoms, ideally at the earliest preclinical stage. the lack of effective ad treatments and pharmaceuticals has led to the assessment of alternative therapeutics, such as nutraceuticals. for example, many antioxidants may enhance cognitive ability.6–8 nutraceuticals have an effect on various neurodegenerative diseases as they modulate signaling pathways.9 nutraceuticals are nutrients, herbals, and dietary supplements that can help in maintaining physical wellbeing, work against various diseases, and ensure a better quality of life. bacosides from bacopa monnieri (b monnieri) are examples of valuable therapeutic agent for ad due to their anti-inflammatory, antioxidant, and aβ aggregation inhibitor properties. this review presents current clinical studies and scientific evidences that document the therapeutic potential of b monnieri extracts (bme) such as bacosides in ad. traditional aspects of b monnieri according to world health organization, traditional medicine is defined as “the sum total of knowledge, skills and practices based on the theories, beliefs and experiences of different cultures that are used to maintain health, as well as to prevent, diagnose, improve or treat physical and mental illnesses.”10 many population in the developing countries have reverted to the use of traditional plants in maintaining their health and wellbeing.11 in this age where migration has taken a leap, immigrants tend to bring traditional plants from their country of origin to use as supplements. this has caused the promotion bacopa monnieri, a neuroprotective lead in alzheimer disease: a review on its properties, mechanisms of action, and preclinical and clinical studies aimi syamima abdul manap1, shantini vijayabalan2, priya madhavan3 , yoke yin chia1, aditya arya3, eng hwa wong3, farzana rizwan3, umesh bindal3 and shajan koshy3 1school of biosciences, faculty of health and medical sciences, taylor’s university, subang jaya, malaysia. 2school of pharmacy, faculty of health and medical sciences, taylor’s university, subang jaya, malaysia. 3school of medicine, faculty of health and medical sciences, taylor’s university, subang jaya, malaysia. abstract: alzheimer disease is a neurodegenerative disease that is signified by cognitive decline, memory loss, and erratic behavior. till date, no cure for alzheimer exists and the current alzheimer medications have limited effectiveness. however, herbal medicines may slow down the disease’s progression, which may hopefully reduce the number of cases in the years to come. numerous studies have been done on characterizing the neuroprotective properties from plants belonging to scrophulariaceae family, particularly bacopa monnieri and its polyphenolic compounds known as bacosides. this review presents the findings on bacosides in therapeutic plants and their impact on alzheimer disease pathology. these reports present data on the clinical, cellular activities, phytochemistry, and biological applications that may be used in new drug treatment for alzheimer disease. keywords: alzheimer, aging, therapeutic plant, bacopa monnieri, bacosides received: july 5, 2019. accepted: july 8, 2019. type: review funding: the author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: this study was supported by taylor’s university flagship research grant (tufr/2017/002/04). declaration of conflicting interests: the author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. corresponding author: priya madhavan, school of medicine, faculty of health and medical sciences, taylor’s university, lakeside campus, no. 1 jalan taylor’s, 47500 subang jaya, selangor darul ehsan, malaysia. email: priya.madhavan@taylors.edu.my 866412dti0010.1177/1177392819866412drug target insightsmanap et al research-article2019 https://uk.sagepub.com/en-gb/journals-permissions mailto:priya.madhavan@taylors.edu.my 2 drug target insights of such non-native plants in a foreign country, particularly the ones used in ayurvedic and chinese traditional medicine.12 these plants or plant compounds are known as complementary or alternative medicines in non-native countries. notably, b monnieri, otherwise known as brahmi and aindri (sanskrit) is classified into the scrophulariaceae family and found throughout the indian subcontinent in moist soil, humid, and muddy environments.13 the genus bacopa has 146 aquatic herbal species dispersed throughout the subtropical regions of the globe, including nepal, india, sri lanka, taiwan, china, and vietnam, as well as florida and other us southern regions. although it can be seen in the united states, these plants are perceived as weeds in rice fields and abundantly grown in wetlands and marshes of warmer districts.14 as shown in figure 2, brahmi is a succulent herb commonly grown in subtropical nations up to 1500 m altitude. brahmi, which is traditionally known as “medhya rasayana,” which means brain tonic or nootropic, or in sanskrit word, referring to intellectual, cognition, and rejuvenation because it enhances the brain’s cognitive properties, is popular among ayurveda practitioners, who use it to treat various ailments, (ie, memory loss, inflammation, epilepsy, fever, and asthma).15 structure and components of bacosides the chemical compound that has neuropharmacological properties and pseudo-jujubogenin moieties, known as aglycone units, is bacoside a (dammarane-type triterpenoid saponin; figure 3).16,17 this compound is composed of bacopaside iii, bacopaside x, bacoside a3, and bacopasaponin c.15 through structural similitude analysis, 12 analogues derived from the bacosides have been characterized and various saponin types have been identified as essential ingredients, known as bacopasides i-xii.18 bacopa’s additional components include apigenin, cucurbitacin, alkaloids brahmine, monnierin, hersaponin, monnierasides i-iii, plantain side b, d-mannitol, herpestine, and nicotine.19,20 table 1 shows molecular composition of bacoside a. figure 1. clinical symptoms of alzheimer disease. figure 2. bacopa monnieri plants. manap et al 3 the neuropharmacological activity of bacoside a numerous studies suggested that b monnieri’s bioactive components (ie, bacosides) protect the brain against oxidative damage and age-related cognitive deterioration with several mechanisms of action.22,23 in addition, bacosides prevent aß aggregation and formation of fibrils24 as well as protect neurons against aβ-induced toxicity.25 from high-performance liquid chromatography (hplc) analysis, the bioactive constituent, bacoside a, was present in the b monnieri extract (bme)treated rat serum and could directly or indirectly interact with the neurotransmitter systems to improve memory and learning ability.26 bacosides present in b monnieri are commonly nonpolar glycosides,27 which enable it to cross the blood-brain barrier (bbb) via simple lipid-mediated passive diffusion.28 similarly, the bioavailability in the brain has been affirmed by the radiopharmaceuticals biodistribution.29 de et  al, using an animal model, described bme as being capable of altering the uptake of radioactivity of 99mtc-labeled ethylene dicysteine diethyl ester (99mtc-ecd) and 99mtc-labeled cystine dimethyl ester (99mtc-cdm) in brain and other organs. the results revealed an increased and significant uptake (p < .05) of 99mtcecd and 99mtc-cdm in brain and other organs after treatment with bme. as bme is a good antioxidant and has cognitive function on human memory, these findings have evaluated pharmacokinetic interactions of bme and suggested that bme can act on the biodistribution of 99mtc-ecd and 99mtc-cdm in specific organs.29 likewise, clinical studies also showed that oral treatment with b monnieri was able to enhance memory in both adults and children. the effects of b monnieri administration on hepatic and intestinal p-glycoprotein (pgp) as well as cytochrome 3a (cyp3a) expression levels were examined in watkins’ studies.30 according to him, individually, pgpmediated efflux and cytochrome p450 (cyp45o)-mediated metabolism play a vital role in modulating the oral bioavailability of corresponding drug. however, b monnieri mediating effects on cyp3a4 and alterations in pgp were measured according to the mrna expression level and functional activity in the intestine and liver of male sprague dawley (sd) rats after a week of b. monnieri administration. the results showed that b. monnieri downregulated both intestinal pgp and cyp3a expression levels, depending on the testosterone hydroxylase catalytic activity in liver and intestine.31 further studies also showed that in vivo pharmacokinetic interaction between digoxin (pgp substrate) and carbamazepine (cyp 3a substrate) along with the administration of b monnieri extract in male sd rats could alter the pharmacokinetics of both pgp and cyp3a probe drugs. probe drug is known to lessen both biological and technical risk factors of tracking a particular target to be selective as well as potent to their target. the results showed that treatment with b monnieri and carbamazepine caused a change in the carbamazepine pharmacokinetic profile with a significant increase in cmax (maximum serum concentration of the drug achieved in the plasma) and auc (the area under the plasma drug concentration-time curve) (0-∞) as well reduction in cl/f (apparent total clearance of the drug from plasma after oral administration) opposing to the vehicle control rats. the role of other compounds in bme cdr1-08 also known as synapsa, or keenmind, a nootropic cdr1-08 is a well-characterized ethanolic extract of b monnieri. several lines of evidence demonstrated that cdri-08 significantly enhances the cognitive performance in the elderly and patients with impaired neurological functions32–34 as well as healthy human participants.35,36 moreover, bacosides present in the cdri-08 are nonpolar glycosides, and it can enter the brain by crossing the bbb through lipid-mediated passive diffusion.28 the biodistribution in brain also has been affirmed by radiopharmaceuticals.29 study by preethi et  al investigated whether treatment with the cdri-08 could change the methylation figure 3. chemical structure of bacoside a.1 4 drug target insights status of reelin and brain-derived neurotrophic factor (bdnf) to enhance the memory through the interaction of n-methyld-aspartate receptor (nmdar) with synaptic proteins. using rat pups as a model in the study, after treatment with cdr108/5-azacytidine (80 mg/kg/3.2 mg/kg), their results demonstrate a higher discrimination toward novel objects than with old objects during the testing. they also observed an elevated level of unmethylated dna in reelin and bdnf-promoted region, which suggested that this mechanism might contribute to the modulation of synaptic plasticity and thus can enhance learning and memory.37 however, study by rai et  al provides the evidence for the mechanism underlying the role of the cdri-08 in restoring spatial memory in amnesic mice. in their study, upon daily oral administration of cdri-08 (200 mg/kg body weight [bw ]) to scopolamine-treated amnesic mice for 7 days, the spatial memory was restored, which was found to be related with significant upregulation of the glun2b (ionotropic glutamate receptors) subunit expression and reduction in the acetylcholinesterase activity in prefrontal cortex as well as hippocampus.38 bacognize® the standardized extract of b monnieri (bacognize) has been shown to improve some aspects of cognitive functions in a 6-month trial in geriatric alzheimer patients.39 in this study, all patients who took 300 mg of bacognize orally twice a day showed a statistically significant improvement in various components of mini-mental state examination scale (mmses) including orientation of time, place and person, table 1. molecular composition of bacoside-a21. composition functional unit bacopaside 3-o-α-l-arabinofuranosyl-(1→2)-[6-o-sulfonyl-β-d-glucopyranosyl-(1→3)]-α-l-arabinopyranoside, 3-o-ß-dglucopyranosyl-(1→3)-α-l-arabinofuranosyl bacopaside v 3-o-α-l-arabinofuranosyl-(1→2)-[6-o-sulfonyl-β-d-glucopyranosyl-(1→3)]-α-l-arabinopyranoside, 3-o-ß-dglucopyranosyl-(1→3)-α-l-arabinofuranosyl bacopaside ii 3-o-α-l-arabinofuranosyl-(1→2)-[β-d-glucopyranosyl-(1→3)]-β-d-glucopyranoside bacopaside iii 3-o-(6-o-sulfonyl-β-d-glucopyranosyl-[1→3])-α-l-arabinopyranoside bacopaside iv 3-o-ß-d-glucopyranosyl-(1→3)-α-l-arabinopyranosyl 3-o-{ß-d-glucopyranosyl(1→4)(α-l-arabinofuranosyl-[1→2])-ß-d-glucopyranosyl}-20-o-α-l-arabinopyranosyl bacopaside ix 3-o-ß-d-glucopyranosyl-(1→3)-α-l-arabinopyranosyl 3-o-{ß-d-glucopyranosyl(1→4)(α-l-arabinofuranosyl-[1→2])-ß-d-glucopyranosyl}-20-o-α-l-arabinopyranosyl bacopaside xi 3-o-{ß-d-glucopyranosyl(1→4)(α-l-arabinofuranosyl -[1→2])-ß-d-glucopyranosyl}-20-o-α-l-arabinopyranosyl bacopaside xii-12 3-o-{ß-d-glucopyranosyl(1→3)[ß-d-arabinofuranosyl(1→2)]-ß-d-glucopyranosyl}-20-obacopasaponin a 3,20-di-o-α-l-arabinopyranoside bacoside a1 3-o-(α-l-arabinofuranosyl[1→3])-α-l-arabinopyranoside bacoside a2 3-o-α-l-arabinopyranosyl-(1→5)-[α-l-arabinofuranosyl-(1→6)]-α-d-glucofuranoside bacoside a3 3-o-α-l-arabinofuranosyl-(1→2)-[β-d-glucopyranosyl-(1→3)]-β-d-glucopyranoside bacopasaponin b 3-o-(α-l-arabinofuranosyl-[1→2])-α-l-arabinopyranoside bacopasaponin c 3-o-α-l-arabinofuranosyl-(1→2)-[β-d-glucopyranosyl-(1→3)]-α-l-arabinopyranoside bacopasaponin d 3-o-(α-l-arabinofuranosyl-[1→2])-β-d-glucopyranoside bacopasaponin e 3-o-α-l-arabinofuranosyl-(1→2)-[β-d-glucopyranosyl-(1→3)]-α-l-arabinopyranoside, 20o-α-l-arabinopyranoside bacopasaponin f 3-o-α-l-arabinofuranosyl-(1→2)-[β-d-glucopyranosyl-(1→3)]-β-d-glucopyranoside,20-oα-l-arabinopyranoside bacopasaponin g 3-o-(α-l-arabinofuranosyl-[1→2])-α-l-arabinopyranoside bacopasaponin h 3-o-[α-l-arabinopyranosyl] manap et al 5 attention, and their language ability in terms of reading, writing, and comprehension at the end of trial. another study refers this extract to be safe and have sustained cognitive effects when used for 12 weeks in healthy older adults.40 kumar et al had evaluated the effect of bacognize on memory of 60 medical students with 42 days of administration. this randomized placebo-controlled trial exhibited a significant improvement in the tests relating to the cognitive functions in the participants who had taken 150 mg of bacognize.41 the poor solubility of bacognize also has been improved by recent study of thakkar et al using the inclusion complex of bacognize (contained 16% bacosides) and β-cyclodextrin prepared in different molar ratios of b monnieri via co-precipitation method. the results revealed that the inclusion of complex at molar ratio of 1:4 can enhance threefold solubility and stability of b monnieri in inclusion complex.42 mechanisms as a neuroprotective agent the mechanisms that underlie the progression of neuronal degeneration are described in the following sections. figure 4 illustrates the neuroprotective effects of bacoside from b monnieri from various studies.43–45 bacosides and reactive oxygen species wide studies have reported the role of superoxide anion, hydroxyl radical, hydrogen peroxide, and nitric oxide in the oxidative stress-mediated neurodegeneration in ad.46,47 neuronal lesions can activate microglia activation, which further generates excessive superoxide radicals.48 thus, mitochondrial autophagy serves as a vital source of reactive oxygen species (ros) production.49 as mitochondria functions as both the source and target of toxic ros, mechanisms by which mitochondrial dysfunction leads to neuron degeneration in ad are believed to be associated with ros generation, activation of mitochondrial permeability transition, excitotoxicity, impaired production of adenosine triphosphate, and altered calcium homeostasis.50 various studies have shown an increased level of 4-hydroxynonenal, the byproduct of oxidative stress in the brain of ad patients.51,52 an increased level of lipid peroxidation (lpo) marker has been reported as well.53,54 besides that, iron-induced oxidative stress, as demonstrated by iron accumulation in the brain of ad, is responsible for neurodegeneration in patients diagnosed with ad.55 extensive studies have been performed on neuroprotection of b monnieri against ros. the administration of b monnieri inhibited lpo especially in the hippocampus, prefrontal cortex, and striatum areas of the rat cerebrum.56 as for the rat’s astrocytes, it significantly reduced the harm done by high concentrations of nitric oxide.57 likewise, different reports recommended that bioactive components from b monnieri can protect the brain against oxidative harm and improve cognitive capability via a few mechanisms.22,23 the enhanced cognitive capability was attributed to the free radical scavenging properties of the bacosides. superoxide dismutase (sod), heat shock protein 70 (hsp70), and cytochrome p450 (cytp450) in the rat’s cerebrum have critical role in both the production of ros and scavenging activity.58 the detoxification and binding of free radical scavenging metal ions or increasing the antioxidant properties are the mechanisms involved in the neuroprotection from bacosides25,34,58 (figure 5). it also reduces the formation of lipid peroxides, divalent metals, scavenging ros, and restraining lipoxygenase action. as results indicated that ros level had declined when neurons were treated with bme, we propose that it may control the intracellular oxidative stress.25 figure 4. neuroprotective effects of bacoside from bacopa monnieri. 6 drug target insights likewise, in neonatal hypoglycaemia, b monnieri has potent neuroprotective capability in reversing the modified dopamine d1 receptor function, bax (bcl2 associated x, apoptosis regulator), and gene expressions, respectively. thus, sod level is lowered, which in turn causes cortical cell death.59 furthermore, in rat models of neurotoxicity incitation by ibotenic corrosive and colchicine, b monnieri indicated a dosage-related intellectual deficiencies.60 acrolein is an exceptionally active compound shaped as a lpo byproduct and acts as an oxidative stress inducer by framing adducts of cell nucleophilic groups. it demonstrates a significant elevation of acrolein levels in the hippocampus; b monnieri extract is accounted to have neuroprotection in human neuroblastoma cell line sk-n-sh against hydrogen peroxide and acrolein-induced toxicity.3 it also protects through ros scavenging, maintains the mitochondrial membrane integrity, modulates the expression of several redox regulatory proteins, that is, sirt1 (sirtuin 1), nf-κb (nuclear factor kappa-light-chain-enhancer of activated b cells), p66shc (a member of the shc family of protein adapters), and erk1/2 (extracellular signal-regulated protein kinases 1 and 2), and protects the cells from oxidative stress. the counter pressure impact of bacosides of b monnieri was studied in adult male sd rats. the results portrayed that it is able to de-stress the modulation of sod, hsp70, and cytp450 under unfavourable conditions, for example, stress.58 bacoside a and beta amyloid toxicity a significant inhibitory effect of cytotoxicity, fibrillation, and membrane interactions of beta-amyloid (1-42) were observed by preincubation of bacoside a with aβ42 in sh-sy5y cell line model.43 aβ is a peptide that plays a prominent role in ad progression and toxicity. in ad, aβ assembles into insoluble amyloid fibrils that aggregate in extracellular neuritic or senile plaques61,62 and is accompanied by synaptic dysfunction, neuronal deterioration, dementia, and cognitive declination.63 therefore, it can suggest that aβ may be directly toxic to neuronal cells and synapses. the previous study showed that extracts containing soluble aβ aggregates can induce amyloidosis in an animal model that otherwise never develop amyloid plaque.64 inhibition of aβ aggregates and assembly is one of the primary therapeutic strategies in ad treatment and prevention. another study reported a substantial link between the toxic peptide of aβ with its membrane interaction.43 in their experiment, aβ42 monomer initially produces oligomeric species that were membrane-active and cytotoxic. it then aggregated into fibrils, which were promoted through interactions with the bilayer interface. however, aggregation of aβ42 was reduced and its membrane interaction was inhibited following incubation with bacoside a. figure 6 shows the mechanism of action of bacoside on aβ.43,61 synergistic action of b monnieri the synergistic effects of b monnieri have been investigated, providing information on its possible neuropharmacological effects between this herbal medicine and other plant extracts or synthetic drugs. the synergistic action of b monnieri (320 mg), l-theanine (100 mg), crocus sativus (30 mg), copper (2 mg), folate (400 µg) with vitamin b (450-9 µg) and vitamin d (25 µg) in a cohort of elderly subjects (1 capsule per day) for 8 weeks of treatment were investigated.66 the results showed a significant improvement of cognitive decline, perceived stress, and depression tested with mini-mental state examination (mmse), perceived stress questionnaire (psq) index, and self-rating depression scale (srds) scores. in another study done in an in vivo model, combination of b monnieri (100 mg/kg) with rivastigmine (5 mg/kg) showed significant protection against aluminum chloride (alcl3)-induced figure 5. the action mechanism of bacoside against ros induces mitochondrial damage. ros indicates reactive oxygen species; sod, superoxide dismutase. manap et al 7 memory impairment in rats compared to those treated with alcl3 per se.67 in their study, chronic administration of alcl3 caused functional deficits in learning and memory skills, which were tested using the morris water maze and elevated plus maze (epm) tasks. however, rats treated with combination of rivastigmine and b monnieri showed better acquisition and retention latencies compared to groups treated only with alcl3, indicating significant protection against alcl3-induced deterioration in learning and memory skills. they concluded that b monnieri and rivastigmine act through synergistic mechanisms to prevent neuronal damage and enhance cholinergic neurotransmission, thus showing better therapeutic effect compared to treatment alone. antidementia and anticholinesterase activities in adult male swiss mice also were studied using combined extracts of b monnieri and ginkgo biloba (gb).68 in this study, anti-dementia activity was tested against scopolamine (3 mg/kg bw )-induced impairment in passive avoidance (pa) test. their results indicate a significant increase in transfer latency time (tlt) and no transfer response (ntr) after treatment of combined extracts of b monnieri at 30 mg/kg and gb at 15, 30, and 60 mg/kg for 7 days of administration. all the extracts showed potent effects toward attenuating the effects of dementia. in vivo and in vitro study on neuroprotective effects of bme research on neuroprotective effects of bme has been widely studied before by in vivo and in vitro models. most of the study conducted by in vivo were performed on male wistar rats and male swiss albino mice and rats. however, for in vitro study, different cell line models were chosen to study the effect of bme such as pc12, sh-sy5y, as well as primary cortical neuron cells. uabundit et al demonstrated protective effects of bme in male wistar rat model that had been induced with 2 nmol/2 μl ethylcholine aziridinium ion (af64a). result showed that 20, 40, and 80 mg/kg bw of bme was able to mitigate the memory impairment and neurodegeneration in the rats by enhancing the escape latency time (p < .01) in the morris water maze test. they also observed that both cholinergic neuron and neuron density reduction were lessened.69 other than that, bme administered orally at 40 mg/kg/day for 5 weeks was able to prevent the neurotoxicity in the cerebral cortex of male wistar rat brain exposed with aluminum chloride (alcl3).70 research done by khan et al revealed that bme given orally at 30 mg/kg bw for 2 weeks significantly improved the memory and learning capability in intracerebroventricularstreptozotocin (icv-stz)-induced male wistar rats. their finding demonstrates the therapeutic efficacy of bme on cognitive impairment and oxidative damage, observed by significant reduction in lpo levels, increased gsh (glutathione) contents, and upregulated antioxidant enzymes activity such as sod, gst (glutathione s-transferases), cat (catalase), and gpx (glutathione peroxidase) in the hippocampus infused by icv-stz model.71 on a different study, bme at 100 mg/kg bw for 180 days lessened both the sodium nitrate (nano2) and d-galactose (d-gal) levels, which improved the bw, memory, and learning skills. b monnieri extracts also normalized the atpase system in ad-induced mice.72 dwivedi et al73 figure 6. mechanism of action of bacoside against beta-amyloid (adaptation and modifications from previous works2,3). however, bacoside a exhibited anti-amyloid toxicity properties upon membrane interactions and bilayer-induced fibrillation of pathogenic substance prion protein (prp). the experimental data revealed that preincubation of prp (106-126) with bacoside a before addition to vesicle bilayers might possibly enhance fibril formation and in parallel had inhibited membrane interactions of the peptide assemblies. the findings from this study revealed a significant interaction of the compound with the amyloidogenic determinant of prp and noticeable effects upon the structural and functional properties of the peptide even though bacoside a has not been explored yet in conjunction with the prion protein. in a more extensive context, the anti-amyloid properties of bacoside a might be discovered to its impact in ameliorating the amyloid protein toxicity via stimulating and enhancing fibrillation.65 8 drug target insights also demonstrate attenuation of okadoic acid (oka)-induced memory dysfunction in sd rats treated with bme at 40 and 80 mg/kg bw for 13 days. moreover, interesting extensive finding by rastogi et  al revealed the protective effect of bacosides, against the age-associated neurodegeneration and promotion of healthy brain aging in female wistar rats. in this study, bacosides were administered orally at 200 mg/kg bw for 3 months in middle aged and aged rats, and its impact on the prevention of senile dementia of alzheimer type (sdat) was evaluated. their findings demonstrated that bacosides was found to display significant anti-aging property by preventing the lipofuscin aggregation in the brain cortex of middle-aged and aged rat. other than that, cholinergic neurotransmission was observed in aged-rat brain cortex, and treatment with bacosides was able to mitigate this age-associated cholinergic degeneration. based on the potential findings on bacosides, they suggested that bacosides exerted multitargeted pharmacological action by preventing the lipofuscin accumulation, enhancing the synthesis of cholinergic neurotransmitter acetylcholine, modulating the metabolism of monoaminergic neurotransmitters, and inhibiting lpo in the aged rats.74 a previous study also investigated a new nanotechnology approach for the brain delivery of the bacoside a for the treatment of neurodegenerative disorders using poly-(d, l)-lactide-co-glycolide (plga) as surfactant. bacoside-a-loaded plga nanoparticles were prepared via oil-in-water (o/w) emulsion solvent evaporation technique. surface of nanoparticles were modified by coating with polysorbate 80 to enhance the crossing of bbb. the ability of nanoparticles in targeting the brain was evaluated by in vivo studies using wistar albino rats. their results suggested that plga nano bacoside a formulation with a size range of 70-200 nm and a relatively low polydispersity index of 0.391 ± 1.2 showed encapsulation efficiency at 57.11% ± 7.11%, with a drug loading capacity of 20.5% ± 1.98%. scanning electron microscopy (sem) and x-ray studies also revealed its spherical shape and low crystallinity. this verified that there were no chemical interactions between both polymer and drug molecules. the in vitro study showed a constant pattern with maximum release of 83.04% ± 2.55% in 48 hours, while in vivo study showed a higher brain concentration of bacoside a (23.94 ± 1.74 μg/g tissues) that implied a significant role of surface-coated nanoparticles on brain targeting. the overall results suggested the efficiency of surface-modified plga nanoparticles in delivery of bacoside a to the brain.75 in vitro study demonstrated that scopolamine induced pc12 cell death was significantly ameliorated by bme pretreatment, and the viability was restored at 85.75% of the control with 100 µg/ml of bme. b monnieri extracts pretreated cells also showed a decreased release of lactate dehydrogenase (ldh) up to 22.42% of total as compared with 30% of scopolamine-treated group. b monnieri extracts also found to ameliorate scopolamine effect by downregulating acetylcholinesterase (ache) and upregulating bdnf as well as muscarinic-1 receptor expression.76 while pretreatment of bme with different doses (2.5-100 µg/ml) for 3 hours in sk-n-sh cells prior to the addition of 200 µm of h2o2 or 15 µm of acrolein can significantly protect against acrolein-induced cytotoxicity. b monnieri extracts also showed to inhibit the generation of intracellular ros in addition to preserving the mitochondrial membrane potential. b monnieri extracts pretreatment also prevented the modifications caused by the activity of several redox regulated protein.3 furthermore, limpeanchob et  al revealed the neuroprotective effect of bme against aβ-induced cell death in primary cortical cultured neuron cells. they found that the cell viability of cultured cortical cells was increased when treated with 100 µg/ml of bme. from their study, they postulated that brahmi extract can diminish neuronal death induced by aβ peptide through the suppression of ache activity. brahmi extract also exhibited antioxidant properties in both in vitro and cell-based assays.25 by using sh-sy5y cell as a model, bme at 0.1 to 25 µm significantly reduced neurotoxicity of oxidized low-density lipoprotein (ldl) in a dosedependent manner as well as suppressed the elevation of cellular ache activity mediated by oxidized ldl.77 using the same sh-sy5y model, bacoside a at 50 μm exerted significant inhibitory effects upon cytotoxicity, fibrillation, and particularly membrane interactions of aβ (1-42) (aβ42).43 table 2 outlines the specific effects of bacosides on various study designs (in vivo and in vitro) of ad. clinical studies in humans using bme upon the promising neuroprotective effect of b monnieri in in vitro and in vivo studies, numerous clinical studies on human subjects have been performed using b monnieri for cognitive improvement. a clinical study of standardized extract of b monnieri (150 mg) on 60 medical students from government medical college, nagpur, india over a period of 15 days revealed significant improvement in biochemical analyses, that is, significant elevation in serum calcium levels and enhanced memory test.41 another group of researchers reported that individual doses of b monnieri and sideritis scardica extracts in 10 mild cognitive impairment subjects from germany (mean age: 61.88 ± 6.69 years) resulted in improvement in the d2-concentration test.79 however, treatment with b monnieri (2 × 150 mg) for 90 days in 107 participants (between ages 18 and 60 years) in swinburne university, australia led to an improved performance in a structural working remembrance task in healthy participants with no history of neurological diseases, gastrointestinal disorders, as well as chronic infections. above it all, none of the healthy participants took any cognitive-enhancing drugs.80 besides cognitive improvement, b monnieri can also enhance learning capability. consumption of b monnieri for 3 months in 76 human subjects between 40 and 65 years of age in university of wollongong, australia resulted in significant effects on retention of new information.81 the consistent consumption of manap et al 9 ta b le 2 . e ffe ct s o f b a c o p a m o n n ie ri e xt ra ct ( b m e ) o n v a ri o u s st u d y d e si g n s o f a d . t h e m o d e l u s e d a n d s t u d y d e s ig n d o s e o r f r e q u e n c y e f f e c t o f b m e t r e a t m e n t r e f e r e n c e s in v iv o m o d e l e th y lc h o lin e a zi ri d in iu m i o n ( a f 6 4 a ) (2 n m o l/ 2 μ l )— ic v -i n d u c e d m a le w is ta r ra ts 2 0 , 4 0 , a n d 8 0 m g /k g b w b m e e n h a n c e d t h e e s c a p e l a te n c y ti m e ( p < .0 1) in t h e m o rr is w a te r m a ze t e st . b o th c h o lin e rg ic n e u ro n a n d n e u ro n d e n s it ie s re d u c ti o n w e re le s s e n e d . u a b u n d it e t a l6 9 o ra l a d m in is tr a ti o n o f a lu m in u m c h lo ri d e (a lc l 3 ; 5 0 m g /k g , p . o .) — ip -i n d u c e d m a le w is ta r ra ts 4 0 a n d 5 0 m g /k g b w b m e t re a te d s ig n ifi c a n tl y p re ve n te d t h e r e d u c ti o n in s o d a c ti v it y a n d d e c re a s e d t h e li p id p e ro x id e s a n d p ro te in o x id a ti o n . b o th e le c tr o n a n d fl u o re s c e n c e m ic ro s c o p ic s tu d ie s u n c o ve re d s u b st a n ti a l i n h ib it io n o f n e c ro ti c c h a n g e a n d in tr a -n e u ro n a l l ip o fu s c in in t h e h ip p o c a m p u s c a 1 r e g io n . j yo ti e t a l7 0 s tr e p to zo to c in ( s t z ) (3 m g /k g , p . o .) — ic v in d u c e d m a le w is ta r ra ts 3 0 m g /k g b w b m t re a te d im p ro ve d t h e m e m o ry a n d l e a rn in g c a p a b ili ty in i c v -s t z r a ts . b m e t re a te d s ig n ifi c a n tl y re d u c e d in l p o l e ve ls , in c re a s e g s h c o n te n ts a n d in c re a s e t h e a c ti v it y o f a n ti o x id a n t e n z y m e s su c h a s s o d g s t , c a t a n d g p x in t h e h ip p o c a m p u s in fu s e d b y ic v -s t z m o d e l. k h a n e t a l7 1 d -g a la c to s e ( 1 2 0 m g /k g , p . o .) a n d s o d iu m n it ri te ( 9 0 m g /k g , p . o .) — ip -i n d u c e d m a le a lb in o m ic e 1 0 0 m g /k g b w b m e l e s s e n e d b o th t h e n a n o 2 a n d d -g a l l e ve ls , w h ic h im p ro ve d t h e b o d y w e ig h t, m e m o ry , a n d l e a rn in g s k ill s. b m e a ls o n o rm a liz e d t h e a t p a s e s ys te m in a d -i n d u c e d m ic e . k u n te a n d k u n a 7 2 o k a d a ic a c id ( 3 0 0 m g /k g , p . o .) — ic v in d u c e d m a le s p ra g u e d a w le y ra ts 4 0 a n d 8 0 m g /k g b w b m e t re a te d s ig n ifi c a n tl y e n h a n c e d t h e m e m o ry -e n h a n c e d m e m o ry d ys fu n c ti o n in a d r a ts a s a p p e a re d b y a r e d u c ti o n in p a th l e n g th a n d la te n c y ti m e . b m a ls o r e st o re d g c l c , h o 1, a n d n rf 2 a s w e ll re d u c e d t h e n e u ro n a l l o s s, o x id a ti ve s tr e s s a n d n e u ro in fl a m m a ti o n d w iv e d i e t a l7 3 f e m a le w is ta r ra ts : yo u n g ( 2 -3 m o n th s) , m id d le -a g e d ( 17 -1 8 m o n th s) , a g e d (> 2 4 m o n th s o ld ) 2 0 0 m g /k g b w b m e ( b a c o s id e s) s ig n ifi c a n tl y (p < .0 5 ) p re ve n ts t h e li p o fu s c in a g g re g a ti o n in t h e m id d le -a g e d a n d a g e d r a t b ra in c o rt e x. b a c o s id e s a ls o e n h a n c e s th e s y n th e s is o f c h o lin e rg ic n e u ro tr a n s m it te r a c e ty lc h o lin e , m o d u la te s th e m e ta b o lis m o f m o n o a m in e rg ic n e u ro tr a n s m it te rs a n d in h ib it s lip id p e ro x id a ti o n in t h e a g e d b ra in r a ts . r a st o g i e t a l7 4 a d u lt a lb in o w is ta r ra ts 2 0 m g /k g b w p l g a n a n o b m e f o rm u la ti o n w it h a s iz e r a n g e o f 7 0 -2 0 0 n m a n d a r e la ti ve ly l o w p o ly d is p e rs it y in d e x o f 0 .3 9 1 ± 1 .2 s h o w e d t h a t e n c a p su la ti o n e ffi c ie n c y w a s 5 7. 11 % ± 7 .1 1% , w it h a d ru g l o a d in g c a p a c it y o f 2 0 .5 % ± 1 .9 8 % . s e m r e ve a le d t h e p l g a n a n o p a rt ic le s’ s p h e ri c a l s h a p e , a s w e ll a s it a p p e a re d t o h a ve l o w c ry st a lli n it y b y x -r a y st u d ie s. t h is v e ri fi e d t h e re w e re n o c h e m ic a l i n te ra c ti o n s b e tw e e n b o th p o ly m e r a n d d ru g m o le c u le s. t h e in v it ro s tu d y s h o w e d a c o n st a n t p a tt e rn w it h a m a x im u m r e le a s e o f 8 3 .0 4 % ± 2 .5 5 % in 4 8 h o u rs a n d in v iv o s tu d y s h o w e d h ig h e r b ra in c o n c e n tr a ti o n o f b a c o s id e a ( 2 3 .9 4 ± 1 .7 4 μ g /g t is su e s) , w h ic h im p lie d a s ig n ifi c a n t ro le o f su rf a c e -c o a te d n a n o p a rt ic le s o n b ra in t a rg e ti n g . j o s e e t a l7 5 m a le s w is s a lb in o m ic e a n d w is ta r ra ts w e re u s e d t o e va lu a te n o o tr o p ic a c ti v it y a n d b io a va ila b ili ty s tu d ie s, r e s p e c ti ve ly . 4 0 m g /k g b w b a c o p a e p h o s p h o lip id c o m p le x (b p c ) p o rt ra ye d 2 e n d o th e rm a l p e a ks ( 8 0 .9 0 °c a n d 1 7 1 °c ) in d s c s tu d ie s. b p c t re a te d s ig n ifi c a n tl y im p ro ve d c o g n it iv e a b ili ty a n d a n ti -a m n e s ic a c ti v it y in a g e d m ic e in m o st m e m o ry -r e la te d m o d e ls s tu d ie d . b p c a ls o r e ta in e d e ff e c ti ve b a c o p a s id e s c o n c e n tr a ti o n f o r a l o n g e r p e ri o d in r a t s e ru m . h a b b u e t a l7 8 s c o p o la m in e — ip -i n d u c e d m a le s w is s a lb in o m ic e 1 2 0 m g /k g b w b m e t re a te d r e ve rs e d b o th r e tr o g ra d e a n d a n te ro g ra d e a m n e s ia . s a ra f e t a l2 2 p s a p p m ic e 4 0 a n d 1 6 0 m g /k g b w b m e r e ve rs e d b o th y -m a ze a n d o p e n -fi e ld h y p e rl o c o m o ti o n b e h a v io ra l c h a n g e s in p s a p p m ic e . t h u s, s u g g e st e d b m e r e d u c e d a β 1 -4 0 a n d 1 -4 2 l e ve ls in t h e c o rt e x b y 6 0 % . h o lc o m b e t a l2 4 in v it ro m o d e l p c 1 2 c e ll lin e 1 0 0 μ g /m l b m e a m e lio ra te d t h e m it o c h o n d ri a l a n d p la s m a m e m b ra n e d a m a g e in d u c e d b y 3 μ g /m l s c o p o la m in e t o 5 4 .8 3 % a n d 3 0 .3 0 % . p a n d a re e s h a n d a n a n d 7 6 s k -n -s h c e ll lin e 1 2 .5 , 2 5 , 5 0 , 7 5 , a n d 1 0 0 μ g /m l b m e p re tr e a tm e n t s ig n ifi c a n tl y p ro te c ts a g a in st h 2 o 2 a n d a c ro le in -i n d u c e d c y to to x ic it y a n d in h ib it e d t h e g e n e ra ti o n o f in tr a c e llu la r re a c ti ve o x yg e n s p e c ie s in a d d it io n t o p re s e rv in g t h e m it o c h o n d ri a l m e m b ra n e p o te n ti a l. s in g h e t a l3 p ri m a ry c o rt ic a l c u lt u re d n e u ro n s c e ll lin e 1 0 0 g /m l b m e p ro te c te d n e u ro n s fr o m b e ta -a m y lo id -i n d u c e d c e ll d e a th . b m e in h ib it e d t h e li p id p e ro x id a ti o n r e a c ti o n o f b ra in h o m o g e n a te in a d o s e -d e p e n d e n t m a n n e r. l im p e a n c h o b e t a l2 5 s h -s y 5 y c e ll lin e 0 .1 t o 2 5 μ m b m e d im in is h e d t h e n e u ro to x ic it y o f o x id iz e d l d l in a d o s e -d e p e n d e n t m a n n e r p o te n ti a lly b y su p p re s s io n o f c e llu la r o x id a ti ve s tr e s s. y a m c h u e n e t a l7 7 s h -s y 5 y c e ll lin e b a c o s id e -a (5 0 μ m ) b a c o s id e -a e xe rt e d s ig n ifi c a n t in h ib it o ry e ff e c ts u p o n c y to to x ic it y, fi b ri lla ti o n , a n d p a rt ic u la rl y m e m b ra n e in te ra c ti o n s o f a m y lo id -b e ta ( 1 -4 2 ) (a β 4 2 ). m a lis h e v e t a l4 3 a b b re vi a tio n s: a lc i3 , a lu m in u m c h lo ri d e ; a d , a lz h e im e r d is e a se ; b m e , b a c o p a m o n n ie ri e xt ra ct ; b p c , b a co p a e p h o sp h o lip id c o m p le x; b w , b o d y w e ig h t; c a t, c a ta la se ; d -g a l, d -g a la ct o se ; g c l c , g lu ta m a te -c ys te in e li g a se ca ta ly tic s u b u n it; g s h , g lu ta th io n e , g s t, g lu ta th io n e s -t ra n sf e ra se ; g p x, g lu ta th io n e p e ro xi d a se ; h o 1 , h e m e o xy g e n a se 1 ; h 2 o 2 ; h yd ro g e n p e ro xi d e ; i c v -s t z , in tr a ce re b ro ve n tr ic u la rst re p to zo to ci n ; i p, in tr a p e ri to n e a l; l d l , lo w -d e n si ty li p o p ro te in ; l p o , lip id p e ro xi d a tio n ; n a n o 2 , so d iu m n itr a te ; n rf 2 , n u cl e a r fa ct o r e ry th ro id 2 -r e la te d f a ct o r 2 ; o k a , o ka d o ic a ci d ; p l g a , p o ly -( d , l )la ct id e -c o -g ly co lid e ; p s a p p, p re se n ili n /a m yl o id p re cu rs o r p ro te in ; s e m , sc a n n in g e le ct ro n m ic ro sc o p y; s o d , su p e ro xi d e d is m u ta se ; d s c , d iff e re n tia l s ca n n in g c a lo ri m e tr y. 10 drug target insights ta b le 3 . s u m m a ry o f cl in ic a l s tu d ie s o f b a c o p a e xt ra ct in c o g n iti o n . p a r t ic ip a n t s /s t u d y d e s ig n /g e o g r a p h ic a l r e g io n in t e r v e n t io n c l in ic a l o u t c o m e r e f e r e n c e s h e a lt h y c h ild re n , 6 -8 y e a rs f ro m r u ra l i n d ia . d o u b le -b lin d , ra n d o m iz e d p la c e b o -c o n tr o lle d in d e p e n d e n t g ro u p s tu d y w a s e m p lo ye d o n e t e a s p o o n fu l o f b a c o p a s yr u p 3 t im e s d a ily fo r 3 m o n th s. ( e a c h t e a s p o o n fu l w a s e q u iv a le n t to 3 5 0 m g o f c ru d e b ra h m i.) s tr e n g th e n e d e x p lo ra to ry d ri ve ( a s m e a su re d b y m a ze l e a rn in g ), im p ro ve d p e rc e p tu a l i m a g e s o f p a tt e rn s, a n d in c re a s e d p e rc e p tu a l o rg a n iz a ti o n a n d r e a s o n in g a b ili ty ( a s m e a su re d b y re a c ti o n t im e ) s h a rm a e t a l8 3 h e a lt h y a d u lt s, b e tw e e n 1 8 a n d 6 0 y e a rs , in s w in b u rn e u n iv e rs it y, a u st ra lia . a d o u b le -b lin d , p la c e b o -c o n tr o lle d in d e p e n d e n t g ro u p d e s ig n in w h ic h s u b je c ts w e re r a n d o m ly a llo c a te d t o 1 o f 2 t re a tm e n t c o n d it io n s. b a c o p a e x tr a c t, 3 0 0 m g d a ily , fo r 1 2 w e e ks s ig n ifi c a n t im p ro ve m e n t in s p e e d o f v is u a l i n fo rm a ti o n p ro c e s s in g m e a su re d b y th e i t t a s k , le a rn in g r a te , a n d m e m o ry c o n s o lid a ti o n m e a su re d b y th e a v lt ( p < .0 5 ) a n d s ta te a n x ie ty ( p < .0 0 1) c o m p a re d t o p la c e b o , w it h m a x im a l e ff e c ts e v id e n t a ft e r 1 2 w e e ks . s to u g h e t a l8 4 h e a lt h y a d u lt s, b e tw e e n t h e a g e s o f 4 0 a n d 6 5 y e a rs in u n iv e rs it y o f w o llo n g o n g , a u st ra lia . d o u b le -b lin d , ra n d o m iz e d p la c e b o -c o n tr o lle d in d e p e n d e n t g ro u p s tu d y w a s e m p lo ye d b a c o p a e x tr a c t, 3 0 0 m g i f su b je c t < 9 0 k g a n d 4 5 0 m g i f > 9 0 k g , fo r 1 2 w e e ks s ig n ifi c a n t e ff e c t o n a t a s k re q u ir in g t h e r e te n ti o n o f n e w in fo rm a ti o n ( p < .0 5 ) w h e re t h e g ro u p w h o re c e iv e d t h e b ra h m i r e ta in e d m o re w o rd p a ir s o ve r th e d e la y th a n t h e p la c e b o g ro u p . r o o d e n ry s a t a l8 1 h e a lt h y a d u lt s (m e a n a g e 7 3 .5 y e a rs ) in u n iv e rs it y o f c a ta n ia , it a ly . d o u b le -b lin d , ra n d o m iz e d p la c e b o -c o n tr o lle d c lin ic a l tr ia l w it h a p la c e b o r u n -i n o f 6 w e e ks b a c o p a e x tr a c t, 3 0 0 m g d a ily , fo r 1 2 w e e ks e n h a n c e d a v lt d e la ye d w o rd r e c a ll m e m o ry s c o re s re la ti ve t o p la c e b o , s ig n ifi c a n t im p ro ve m e n t in s tr o o p re su lt s (p < .0 5 ) a n d a ls o d e c re a s e d in c e s d -1 0 d e p re s s io n s c o re s o ve r ti m e , a s w e ll a s d e c re a s e d in c o m b in e d s ta te p lu s tr a it a n x ie ty s c o re s a n d h e a rt a tt a c k . c a la b re s e e t a l3 3 h e a lt h y a d u lt s, b e tw e e n 1 8 a n d 6 0 y e a rs , in s w in b u rn e u n iv e rs it y, a u st ra lia . a d o u b le -b lin d , p la c e b o -c o n tr o lle d in d e p e n d e n t g ro u p d e s ig n w a s e m p lo ye d b a c o p a e x tr a c t, 3 0 0 m g d a ily , fo r 9 0 d a ys s ig n ifi c a n t im p ro ve m e n t in w o rk in g m e m o ry ( p = .0 3 5 ), s p a ti a l w o rk in g m e m o ry ( p = .0 5 1 0 ), a n d s ig n ifi c a n t re d u c ti o n ( p = .0 2 9 ) in t h e a m o u n t o f fa ls e a la rm s p ro d u c e d d u ri n g r v ip t a s k . s to u g h a t a l8 0 c h ild re n r e q u ir in g in d iv id u a l e d u c a ti o n a l s u p p o rt , 1 0 .5 y e a rs in c e n te r fo r r e s e a rc h in m e n ta l r e ta rd a ti o n ( c r e m e r e ), m u m b a i, in d ia . t h e s tu d y w a s c o n d u c te d a s o u tp a ti e n t p ro c e d u re in h o s p it a l s e tt in g s w it h c lo s e m o n it o ri n g . b a c o p a e x tr a c t, 2 2 5 m g d a ily , fo r 1 6 w e e ks s ig n ifi c a n t c h a n g e in t h e b a s e lin e v a lu e o f w o rk in g m e m o ry a n d s h o rt -t e rm v e rb a l m e m o ry f ro m 5 .2 1 ± 0 .3 2 t o 6 .3 8 ± 0 .2 5 ( p ⩽ .0 5 ) a n d 5 .3 3 ± 0 .4 4 t o 6 .5 4 ± 0 .3 5 ( p ⩽ .0 5 ). s ig n ifi c a n t im p ro ve m e n t (p ⩽ .0 5 ) w a s a ls o s e e n in l o g ic a l m e m o ry , m e m o ry r e la te d t o p e rs o n a l l if e a n d a ls o in v is u a l a s w e ll a s a u d it o ry m e m o ry . u s h a e t a l8 5 n in e ty e ig h t h e a lt h y su b je c ts , a g e ⩾ 5 5 y e a rs in l is m o re , n e w s o u th w a le s, a u st ra lia . d o u b le -b lin d , ra n d o m iz e d p la c e b o -c o n tr o lle d d e s ig n w a s e m p lo ye d b a c o p a e x tr a c t, 3 0 0 m g d a ily , fo r 1 2 w e e ks s ig n ifi c a n tl y e n h a n c e d t h e m e m o ry a c q u is it io n , ve rb a l l e a rn in g , a n d d e la ye d r e c a ll m e a su re b y r e y a u d it o ry v e rb a l l e a rn in g t e st ( a v lt ); t ri a l a 4 ( p = .0 0 0 ), t ri a l a 5 ( p = .0 1 6 ); t ri a l a 6 ( p = .0 0 0 ); t ri a l a 7 (d e la ye d r e c a ll, p = .0 0 1) ; t o ta l l e a rn in g ( p = .0 11 ) a s w e ll re tr o a c ti ve in te rf e re n c e ( p = .0 4 8 ). s c o re s in c lu d in g m a c -q , t m t , a n d c f t im p ro ve d t h e g ro u p d if fe re n c e s a n d n e ve rt h e le s s w e re n o t s ig n ifi c a n t 8 2 s ix ty h e a lt h y a d u lt s, m e a n a g e : 6 2 .6 2 ± 6 .4 6 y e a rs ( 3 7 fe m a le s a n d 2 3 m a le s) in t h a ila n d . d o u b le -b lin d , ra n d o m iz e d p la c e b o -c o n tr o lle d d e s ig n w a s e m p lo ye d b a c o p a e x tr a c t, 3 0 0 m g o r 6 0 0 m g d a ily , fo r 1 2 w e e ks t re a te d e x tr a c t g ro u p d is p la ye d a n e n h a n c e d w o rk in g m e m o ry a s w e ll a r e d u c ti o n in b o th p 3 0 0 a n d n 1 0 0 la te n c ie s. t h e p la s m a a c h e a c ti v it y su p p re s s io n w a s a ls o s e e n , w h ic h s u g g e st t h a t it c o u ld e n h a n c e t h e c o g n it iv e a b ili ty a n d w o rk in g m e m o ry a n d im p ro ve a tt e n ti o n p e th -n u i e t a l8 6 s e ve n te e n h e a lt h y vo lu n te e rs ( 1 3 f e m a le s a n d 4 m a le s) , m e a n a g e 2 5 .2 3 ± 5 .9 7 in m e lb o u rn e , a u st ra lia . d o u b le -b lin d , p la c e b o -c o n tr o lle d c ro s s -o ve r st u d y w a s e m p lo ye d b a c o p a e x tr a c t, 3 2 0 m g o r 6 4 0 m g d a ily , 1 h o u r a n d 2 h o u r b a c o p a c o n su m p ti o n s h o w e d a c h a n g e f ro m b a s e lin e s c o re in d ic a ti ve o f p o s it iv e c o g n it iv e e ff e c ts a t fir st a n d s e c o n d h o u r p o st c o n su m p ti o n o n t h e s tr o o p t a s ks a s w e ll l e tt e r s e a rc h . it p ro d u c e d s o m e n o o tr o p ic a n d a d a p to g e n ic e ff e c ts . p o s it iv e m o d e ff e c ts a n d r e d u c ti o n in c o rt is o l l e ve ls ( p h ys io lo g ic a l s tr e s s re s p o n s e ) w e re a s s o c ia te d w it h b a c o p a c o n su m p ti o n b y p a rt ic ip a n ts . b e n s o n e t a l3 5 s ix ty h e a lt h y a d u lt s b e tw e e n 1 9 a n d 2 2 y e a rs f ro m g o ve rn m e n t m e d ic a l c o lle g e , n a g p u r, i n d ia . d o u b le -b lin d , ra n d o m iz e d p la c e b o -c o n tr o lle d n o -c ro s s o ve r, p a ra lle l t ri a l w a s e m p lo ye d b a c o p a e x tr a c t, 1 5 0 m g , fo r 1 5 d a ys s ig n ifi c a n t im p ro ve m e n t in m e m o ry t e st , n e u ro p sy c h o lo g ic a l t e st s (d ig it s p a n m e m o ry t a s k , p a ir e d a s s o c ia te t a s k , lo g ic a l m e m o ry t e st [ st o ry r e c a ll] , m e m o ry s p a n f o r n o n s e n s e s y lla b le s) a n d c o m p u te ri ze d te st s (fi n g e r ta p p in g t e st , s im p le r e a c ti o n t e st , c h o ic e r e a c ti o n t e st , c h o ic e d is c ri m in a ti o n t e st , a n d d ig it p ic tu re s u b st it u ti o n t e st ( sy m b o l d ig it m o d a lit ie s te st ). b lo o d b io c h e m is tr y s h o w e d s ig n ifi c a n t e le va ti o n in s e ru m c a lc iu m l e ve ls ( st ill w it h in n o rm a l r a n g e ). k u m a r e t a l4 1 t e n s u b je c ts ( m e a n a g e : 6 1. 8 8 ± 6 .6 9 y e a rs ) fr o m g e rm a n y w it h m ild c o g n it iv e im p a ir m e n t. s id e ri ti s e x tr a c t, 5 0 0 m g c o m b in e d w it h b a c o p a e x tr a c t, 1 6 0 a n d 3 2 0 m g s id e ri ti s e x tr a c t c o m b in e d w it h b a c o p a e x tr a c t in d ic a te d b e tt e r p e rf o rm a n c e s in d 2 -t e st t e st o n ly c o n tr a st e d w it h m e m o ry t e st a n d a ri th m e ti c c a lc u la ti o n t e st ( c p t ). q u a n ti ta ti ve e e g a s s e s s m e n t re ve a le d th a t s id e ri ti s e x tr a c t c o m b in e d w it h b a c o p a e x tr a c t a t lo w e r d o s e ( 1 6 0 m g ) in c re a s e d t h e s p e c tr a l p o w e r w h ile c o m b in e d w it h b a c o p a e x tr a c t a t h ig h e r d o s e ( 3 2 0 m g ) fo rm e d a tt e n u a ti o n o f a ll w a ve s e xc e p t fo r d e lt a in f ro n ta lte m p o ra l b ra in a re a s, in d ic a ti n g m a s s iv e d if fe re n c e s b e tw e e n b o th e x tr a c ts . d im p fe l e t a l7 9 t h ir ty e ld e rl y su b je c ts m e a n a g e 6 6 ± 3 y e a rs in b o lo g n a , it a ly . d o u b le -b lin d , c ro s s -o ve r d e s ig n e d t ri a l v e rs u s p la c e b o g ro u p s tu d y w a s e m p lo ye d c o m b in e d n u tr a c e u ti c a ls c o n ta in in g b a c o p a d ry e x tr a c t (3 2 0 m g ), l -t h e a n in e ( 1 0 0 m g ), c ro c u s sa ti vu s (3 0 m g ), c o p p e r (2 m g ), f o la te ( 4 0 0 µ g ), a n d v it a m in s o f b ( 4 5 0 µ g -9 m g ) a n d d ( 2 5 µ g ) a ft e r 2 m o n th s o f n u tr a c e u ti c a l t h e ra p y, m m s e a n d p s q i n d e x s ig n ifi c a n tl y im p ro ve d in t h e a c ti ve tr e a tm e n t a rm , b o th v e rs u s b a s e lin e a n d v e rs u s th e p a ra lle l a rm . b o th g ro u p s e x p e ri e n c e d a s ig n ifi c a n t im p ro v in g in t h e s r d s s c o re s c ic e ro e t a l6 6 a b b re vi a tio n s: a v lt , r e y a u d ito ry v e rb a l l e a rn in g t e st ; c e s d , c e n te r fo r e p id e m io lo g ic s tu d ie s d e p re ss io n s ca le ; c f t, c o m p le x f ig u re t e st ; m a c -q , m e m o ry c o m p la in t q u e st io n n a ir e ; m m s e , m in im e n ta l s ta te e xa m in a tio n ; p s q i n d e x, p e rc e iv e d s tr e ss q u e st io n n a ir e ; r v ip , r a p id v is u a l i n fo rm a tio n p ro ce ss in g ; s r d s , s e lfr a tin g d e p re ss io n s ca le ; t m t, t ra il m a ki n g t e st . manap et al 11 bme (300 mg/day) for 84 days in participants without dementia aged 65 years and above in university of catania, italy also showed improvement in their performance in a restraint recall and stroop task, that is, evaluating the capability to bypass unnecessary input.33 moreover, in lismore, new south wales, australia, the administration with b monnieri (300 mg/day) in healthy volunteers over 55 years of age showed improvement in their oral learning, memory attainment, and suppressed recall.82 in another research done at swinburne university of technology, melbourne, australia using higher single dose in a double-blind, placebo-controlled trial among normal healthy subjects between the age of 18 and 44 years demonstrated an improved and preserved cognitive ability.36 significant enhancement in prompt memory and response performance was also observed when bacopa in the form of syrup (proportionate to 10 g dried bacopa daily) was administered in 40 school children aged between 6 and 8 years for 90 days from rural india.83 the overall clinical trials in humans using bme are summarized in table 3. conclusions many traditional plants especially b monnieri have intricate mixtures of chemical compounds, which exhibit various pharmacological and biological activities. they have been used as traditional medicines and for anti-aging. according to the long-established hypothesis, plant compounds are able to maintain the fundamental vitality in the body and have various neuroprotective mechanisms that empower them to be used as part of our well-being. this review reveals the effective use of b monnieri in cognition and neuroprotection and its phytoconstituents that can be used in novel drug discovery. author contributions asam, sv and pm designed the flow and wrote sub-sections of the manuscript. all other authors contributed to manuscript revision, proof reading and approval of the submitted version. orcid id priya madhavan https://orcid.org/0000-0001-6714-1279 r efer ences 1. jadoopat r. review of alzheimer’s disease treatment and potential future therapies. annu rev chang healthc. 2018;2. 2. heneka mt, o’banion mk. inflammatory processes in alzheimer’s disease. j neuroimmunol. 2007;184:69–91. 3. singh m, murthy v, ramassamy c. modulation of hydrogen peroxide and acrolein-induced oxidative stress, mitochondrial dysfunctions and redox regulated pathways by the bacopa monniera extract: potential implication in alzheimer’s disease. j alzheimers dis. 2010;21:229–247. 4. bertram l, lill cm, tanzi re. the genetics of alzheimer disease: back to the future. neuron. 2010;68:270–281. 5. polidori mc, pientka l, mecocci p. a review of the major vascular risk factors related to alzheimer’s disease. j alzheimers dis. 2012;32:521–530. 6. calabrese v, butterfield da, stella a. nutritional antioxidants and the heme oxygenase pathway of stress tolerance: novel targets for neuroprotection in alzheimer’s disease. ital j biochem. 2003;52:177–181. 7. emilien g, beyreuther k, masters cl, maloteaux j-m. prospects for pharmacological intervention in alzheimer disease. arch neurol. 2000;57:454–459. 8. kontush a, schekatolina s. vitamin e in neurodegenerative disorders: alzheimer’s disease. ann n y acad sci. 2004;1031:249–262. 9. maity s, nandy s, mukherjee a, dey a. recent trends in drug discovery against alzheimer’s disease: use of natural products and nutraceuticals from botanicals. in: ullah, mf, ahmad, a, eds. nutraceuticals and natural product derivatives: disease prevention & drug discovery. hoboken, nj: john wiley; 2019:237–278. 10. world health organization (who). traditional medicine: fact sheet no. 134. https://apps.who.int/gb/ebwha/pdf_files/eb134/b134_24-en.pdf. updated 2008. 11. robinson mm, zhang x. the world medicines situation 2011, traditional medicines: global situation, issues and challenges. geneva, switzerland: world health organization; 2011. 12. sandhu ds, heinrich m. the use of health foods, spices and other botanicals in the sikh community in london. phytother res. 2005;19:633–642. 13. satyavati g, raina m, sharma m. indian medicinal plants. new delhi, india: indian council of medical research; 1976:20–35. 14. barrett sc, strother jl. taxonomy and natural history of bacopa (scrophulariaceae) in california. syst bot. 1978;3:408–419. 15. vishnupriya p, padma vv. a review on the antioxidant and therapeutic potential of bacopa monnieri. react oxygen spec. 2017;3:111–120. 16. anand t, prakash kb, pandareesh m, khanum f. development of bacoside enriched date syrup juice and its evaluation for physical endurance. j food sci technol. 2014;51:4026–4032. 17. blazquez-sanchez mt, de matos am, rauter ap. exploring anti-prion glycobased and aromatic scaffolds: a chemical strategy for the quality of life. molecules. 2017;22:864. 18. rauf k, subhan f, al-othman a, khan i, zarrelli a, shah m. preclinical profile of bacopasides from bacopa monnieri (bm) as an emerging class of therapeutics for management of chronic pains. curr med chem. 2013;20:1028–1037. 19. deepak m, amit a. the need for establishing identities of “bacoside a and b,” the putative major bioactive saponins of indian medicinal plant bacopa monnieri. phytomedicine. 2004;11:264–268. 20. kapoor r, srivastava s, kakkar p. bacopa monnieri modulates antioxidant responses in brain and kidney of diabetic rats. environ toxicol pharmacol. 2009;27:62–69. 21. deepak m, sangli g, arun p, amit a. quantitative determination of the major saponin mixture bacoside a in bacopa monnieri by hplc. phytochem anal. 2005;16:24–29. 22. saraf mk, prabhakar s, khanduja kl, anand a. bacopa monniera attenuates scopolamine-induced impairment of spatial memory in mice. evid based complement alternat med. 2011;2011:236186. 23. mukherjee s, dugad s, bhandare r, et al. evaluation of comparative free-radical quenching potential of brahmi (bacopa monnieri) and mandookparni (centella asiatica). ayu. 2011;32:258–264. 24. holcomb la, dhanasekaran m, hitt ar, young ka, riggs m, manyam bv. bacopa monniera extract reduces amyloid levels in psapp mice. j alzheimers dis. 2006;9:243–251. 25. limpeanchob n, jaipan s, rattanakaruna s, phrompittayarat w, ingkaninan k. neuroprotective effect of bacopa monnieri on beta-amyloid-induced cell death in primary cortical culture. j ethnopharmacol. 2008;120:112–117. 26. rajan ke, preethi j, singh hk. molecular and functional characterization of bacopa monniera: a retrospective review. evid based complement alternat med. 2015;2015:945217. 27. chakravarty ak, sarkar t, masuda k, shiojima k, nakane t, kawahara n. bacopaside i and ii: two pseudojujubogenin glycosides from bacopa monniera. phytochemistry. 2001;58:553–556. 28. pardridge wm. blood-brain barrier biology and methodology. j neurovirol. 1999;5:556–569. 29. de k, chandra s, misra m. evaluation of the biological effect of brahmi (bacopa monnieri linn) extract on the biodistribution of technetium-99m radiopharmaceuticals. life sci j. 2008;5:45–49. 30. watkins pb. the barrier function of cyp3a4 and p-glycoprotein in the small bowel. adv drug deliv rev. 1997;27:161–170. 31. singh r, panduri j, kumar d, et al. evaluation of memory enhancing clinically available standardized extract of bacopa monniera on p-glycoprotein and cytochrome p450 3a in sprague-dawley rats. plos one. 2013;8:e72517. 32. barbhaiya h, desai r, saxena v, et al. efficacy and tolerability of bacomind on memory improvement in elderly participants—a double blind placebo controlled study. j pharmacol toxicol. 2008;3:425–434. 33. calabrese c, gregory wl, leo m, kraemer d, bone k, oken b. effects of a standardized bacopa monnieri extract on cognitive performance, anxiety, and depression in the elderly: a randomized, double-blind, placebo-controlled trial. j altern complement med. 2008;14:707–713. 34. stough c, scholey a, cropley v, et al. examining the cognitive effects of a special extract of bacopa monniera (cdri08: keenmnd): a review of ten years of research at swinburne university. j pharm pharm sci. 2013;16:254–258. 35. benson s, downey la, stough c, wetherell m, zangara a, scholey a. an acute, double-blind, placebo-controlled cross-over study of 320 mg and 640 mg https://orcid.org/0000-0001-6714-1279 https://apps.who.int/gb/ebwha/pdf_files/eb134/b134_24-en.pdf 12 drug target insights doses of bacopa monnieri (cdri 08) on multitasking stress reactivity and mood. phytother res. 2014;28:551–559. 36. downey la, kean j, nemeh f, et al. an acute, double-blind, placebo-controlled crossover study of 320 mg and 640 mg doses of a special extract of bacopa monnieri (cdri 08) on sustained cognitive performance. phytother res. 2013;27:1407–1413. 37. preethi j, singh hk, rajan ke. possible involvement of standardized bacopa monniera extract (cdri-08) in epigenetic regulation of reelin and brain-derived neurotrophic factor to enhance memory. front pharmacol. 2016;7:166. 38. rai r, singh hk, prasad s. a special extract of bacopa monnieri (cdri-08) restores learning and memory by upregulating expression of the nmda receptor subunit glun2b in the brain of scopolamine-induced amnesic mice. evid based complement alternat med. 2015;2015:254303. 39. goswami s, saoji a, kumar n, thawani v, tiwari m, thawani m. effect of bacopa monnieri on cognitive functions in alzheimer’s disease patients. int j collaborat res int med public health. 2011;3:285–293. 40. hingorani l, patel s, ebersole b. sustained cognitive effects and safety of hplc-standardized bacopa monnieri extract: a randomized, placebo controlled clinical trial. planta medica. 2012;78:ph22. 41. kumar n, abichandani l, thawani v, gharpure k, naidu m, venkat ramana g. efficacy of standardized extract of bacopa monnieri (bacognize®) on cognitive functions of medical students: a six-week, randomized placebo-controlled trial. evid based complement alternat med. 2016;2016:4103423. 42. thakkar vt, deshmukh a, hingorani l, et al. development and optimization of dispersible tablet of bacopa monnieri with improved functionality for memory enhancement. j pharm bioallied sci. 2017;9:208–215. 43. malishev r, shaham-niv s, nandi s, kolusheva s, gazit e, jelinek r. bacosidea, an indian traditional-medicine substance, inhibits β-amyloid cytotoxicity, fibrillation, and membrane interactions. acs chem neurosci. 2017;8:884–891. 44. farooqui aa, farooqui t. contribution of diabetes and metabolic syndrome in the pathogenesis of alzheimer’s disease. in: farooqui, t, farooqui, aa, eds. role of the mediterranean diet in the brain and neurodegenerative diseases. london, england: elsevier; 2018:301–316. 45. chaudhari ks, tiwari nr, tiwari rr, sharma rs. neurocognitive effect of nootropic drug brahmi (bacopa monnieri) in alzheimer’s disease. ann neurosci. 2017;24:111–122. 46. cheignon c, tomas m, bonnefont-rousselot d, faller p, hureau c, collin f. oxidative stress and the amyloid beta peptide in alzheimer’s disease. redox biology. 2018;14:450–464. 47. manoharan s, guillemin gj, abiramasundari rs, essa mm, akbar m, akbar md. the role of reactive oxygen species in the pathogenesis of alzheimer’s disease, parkinson’s disease, and huntington’s disease: a mini review. oxid med cell longev. 2016;2016:8590578. 48. magalingam kb, radhakrishnan a, ping ns, haleagrahara n. current concepts of neurodegenerative mechanisms in alzheimer’s disease. biomed res int. 2018;2018:3740461. 49. flannery pj, trushina e. mitochondrial dysfunction in alzheimer’s disease and progress in mitochondria-targeted therapeutics [published online ahead of print june 8, 2019]. curr behav neurosci rep. doi:10.1007/s40473-019-00179-0. 50. huang w, zhang x, chen w. role of oxidative stress in alzheimer’s disease. biomed rep. 2016;4:519–522. 51. mecocci p, boccardi v, cecchetti r, et al. a long journey into aging, brain aging, and alzheimer’s disease following the oxidative stress tracks. j alzheimers dis. 2018;62:1319–1335. 52. ramesh s, govindarajulu m, jones e, suppiramaniam v, moore t, dhanasekaran m. mitochondrial dysfunction and the role of mitophagy in alzheimer’s disease. https://pdfs.semanticscholar.org/548a/b1df84b2168d94bb851644d90e 695523443a.pdf. updated 2018. 53. bhatia s, kim ws, shepherd ce, halliday gm. apolipoprotein d upregulation in alzheimer’s disease but not frontotemporal dementia. j mol neurosci. 2019;67:125–132. 54. massaccesi l, galliera e, galimberti d, et al. lag-time in alzheimer’s disease patients: a potential plasmatic oxidative stress marker associated with apoe4 isoform. immun ageing. 2019;16:7. 55. nikseresht s, bush ai, ayton s. treating alzheimer’s disease by targeting iron [published online ahead of print february 2019]. br j pharmacol. doi:10.1111/ bph.14567. 56. russo a, borrelli f, campisi a, acquaviva r, raciti g, vanella a. nitric oxiderelated toxicity in cultured astrocytes: effect of bacopa monniera. life sci. 2003;73:1517–1526. 57. tripathi yb, chaurasia s, tripathi e, upadhyay a, dubey gp. bacopa monniera linn. as an antioxidant: mechanism of action. indian j exp biol. 1996;34:523–526. 58. chowdhuri dk, parmar d, kakkar p, shukla r, seth p, srimal r. antistress effects of bacosides of bacopa monnieri: modulation of hsp70 expression, superoxide dismutase and cytochrome p450 activity in rat brain. phytother res. 2002;16:639–645. 59. thomas rb, joy s, ajayan m, paulose c. neuroprotective potential of bacopa monnieri and bacoside a against dopamine receptor dysfunction in the cerebral cortex of neonatal hypoglycaemic rats. cell mol neurobiol. 2013;33:1065–1074. 60. singh h, dhawan b. drugs affecting learning and memory. lect neuro. 1992;1:189–207. 61. batarseh ys, duong q-v, mousa ym, al rihani sb, elfakhri k, kaddoumi a. amyloid-β and astrocytes interplay in amyloid-β related disorders. int j mol sci. 2016;17:338. 62. selkoe dj, hardy j. the amyloid hypothesis of alzheimer’s disease at 25 years. embo mol med. 2016;8:595–608. 63. khan ua, liu l, provenzano fa, et al. molecular drivers and cortical spread of lateral entorhinal cortex dysfunction in preclinical alzheimer’s disease. nat neurosci. 2014;17:304–311. 64. nussbaum jm, schilling s, cynis h, et al. prion-like behaviour and taudependent cytotoxicit y of pyroglutamylated amyloid-β. nature. 2012;485: 651–655. 65. malishev r, nandi s, kolusheva s, shaham-niv s, gazit e, jelinek r. bacoside-a, an anti-amyloid natural substance, inhibits membrane disruption by the amyloidogenic determinant of prion protein through accelerating fibril formation. biochim biophys acta. 2016;1858:2208–2214. 66. cicero a, bove m, colletti a, et al. short-term impact of a combined nutraceutical on cognitive function, perceived stress and depression in young elderly with cognitive impairment: a pilot, double-blind, randomized clinical trial. j prev alz dis. 2016;4:12–15. 67. thippeswamy ah, rafiq m, viswantha gl, kavya kj, anturlikar sd, patki ps. evaluation of bacopa monniera for its synergistic activity with rivastigmine in reversing aluminum-induced memory loss and learning deficit in rats. j acupunct meridian stud. 2013;6:208–213. 68. das a, shanker g, nath c, pal r, singh s, singh hk. a comparative study in rodents of standardized extracts of bacopa monniera and ginkgo biloba: anticholinesterase and cognitive enhancing activities. pharmacol biochem behav. 2002;73:893–900. 69. uabundit n, wattanathorn j, mucimapura s, ingkaninan k. cognitive enhancement and neuroprotective effects of bacopa monnieri in alzheimer’s disease model. j ethnopharmacol. 2010;127:26–31. 70. jyoti a, sethi p, sharma d. bacopa monniera prevents from aluminium neurotoxicity in the cerebral cortex of rat brain. j ethnopharmacol. 2007;111:56–62. 71. khan mb, ahmad m, ahmad s, et al. bacopa monniera ameliorates cognitive impairment and neurodegeneration induced by intracerebroventricular-streptozotocin in rat: behavioral, biochemical, immunohistochemical and histopathological evidences. metab brain dis. 2015;30:115–127. 72. kunte kb, kuna y. neuroprotective effect of bacopa monniera on memory deficits and atpase system in alzheimer’s disease (ad) induced mice. j sci innov res. 2013;2:719–735. 73. dwivedi s, nagarajan r, hanif k, siddiqui hh, nath c, shukla r. standardized extract of bacopa monniera attenuates okadaic acid induced memory dysfunction in rats: effect on nrf2 pathway. evid based complement alternat med. 2013;2013:294501. 74. rastogi m, ojha rp, prabu p, devi bp, agrawal a, dubey g. prevention of age-associated neurodegeneration and promotion of healthy brain ageing in female wistar rats by long term use of bacosides. biogerontology. 2012;13: 183–195. 75. jose s, sowmya s, cinu t, aleykutty n, thomas s, souto e. surface modified plga nanoparticles for brain targeting of bacoside-a. eur j pharm sci. 2014;63:29–35. 76. pandareesh m, anand t. neuromodulatory propensity of bacopa monniera against scopolamine-induced cytotoxicity in pc12 cells via down-regulation of ache and up-regulation of bdnf and muscarnic-1 receptor expression. cell mol neurobiol. 2013;33:875–884. 77. yamchuen p, chaiwiang n, lapphanichayakool p, ingkaninan k, limpeanchob n. neuroprotective effect of bacopa monnieri extract on oxidized low density lipoprotein-induced neurotoxicity in sh-sy5y neuroblastoma cells. thai j pharm. 2017;39:5–18. 78. habbu p, madagundi s, kulkarni r, jadav s, vanakudri r, kulkarni v. preparation and evaluation of bacopa–phospholipid complex for antiamnesic activity in rodents. drug invention today. 2013;5:13–21. 79. dimpfel w, schombert l, biller a. psychophysiological effects of sideritis and bacopa extract and three combinations thereof—a quantitative eeg study in subjects suffering from mild cognitive impairment (mci). advances in alzheimer’s disease. 2016;5:1–22. 80. stough c, downey la, lloyd j, et al. examining the nootropic effects of a special extract of bacopa monniera on human cognitive functioning: 90 day doubleblind placebo-controlled randomized trial. phytother res. 2008;22:1629–1634. 81. roodenrys s, booth d, bulzomi s, phipps a, micallef c, smoker j. chronic effects of brahmi (bacopa monnieri) on human memory. neuropsychopharmacology. 2002;27:279–281. https://pdfs.semanticscholar.org/548a/b1df84b2168d94bb851644d90e695523443a.pdf https://pdfs.semanticscholar.org/548a/b1df84b2168d94bb851644d90e695523443a.pdf manap et al 13 82. morgan a, stevens j. does bacopa monnieri improve memory performance in older persons? results of a randomized, placebo-controlled, double-blind trial. j altern complement med. 2010;16:753–759. 83. sharma r, chaturvedi c, tewari p. efficacy of bacopa monniera in revitalizing intellectual functions in children. j res edu ind med. 1987;1:12. 84. stough c, lloyd j, clarke j, et al. the chronic effects of an extract of bacopa monniera (brahmi) on cognitive function in healthy human subjects. psychopharmacology. 2001;156:481–484. 85. usha p, wasim p, joshua j, et al. bacomind®: a cognitive enhancer in children requiring individual education programme. j pharmacol toxicol. 2008;3: 302–310. 86. peth-nui t, wattanathorn j, muchimapura s, et al. effects of 12-week bacopa monnieri consumption on attention, cognitive processing, working memor y, and functions of both cholinergic and monoaminergic systems in healthy elderly volunteers. evid based complement alternat med. 2012;2012: 606424. guterres et al.indd drug target insights 2007: 2 147–157 147 review correspondence: prof. sílvia s. guterres or prof. a. r. pohlmann, faculdade de farmácia, universidade federal do rio grande do sul, av. ipiranga 2752, porto alegre, 90610-000, rs, brazil. tel: 55 51 33165500; fax: 55 51 33165437; email: nanoc@farmacia.ufrgs.br or pohlmann@iq.ufrgs.br please note that this article may not be used for commercial purposes. for further information please refer to the copyright statement at http://www.la-press.com/copyright.htm polymeric nanoparticles, nanospheres and nanocapsules, for cutaneous applications sílvia s. guterres1, marta p. alves1 and adriana r. pohlmann2 1programa de pós-graduação em ciências farmacêuticas, faculdade de farmácia, universidade federal do rio grande do sul, ufrgs, porto alegre, rs, brazil. 2departamento de química orgânica, instituto de química, ufrgs, porto alegre, rs, brazil. abstract: this review presents an overview about pharmaceutical and cosmetic topical products containing polymeric nanoparticles (nanospheres and nanocapsules), reporting the main preparation and characterization methods and the studies of penetration and transport of substances through the skin. the penetration and transport extent of those systems through the skin depends on the ingredients chemical composition, on the encapsulation mechanism infl uencing the drug release, on the size of nanoparticles and on the viscosity of the formulations. the polymeric nanoparticles are able to modify the activity of drugs, delay and control the drug release, and increase the drug adhesivity or its time of permanence in the skin. briefl y, the nanoparticles can be useful as reservoirs of lipophilic drugs to deliver them in the stratum corneum becoming an important strategy to control their permeation into the skin. keywords: polymeric nanoparticles, nanocapsules, semi-solid formulations, topical formulation, skin, sunscreen. introduction topical administration of drugs has advantages such as minimal systemic effects and targeting only the areas of disease (ting et al. 2004). nevertheless, the stratum corneum, which is the non-viable uppermost layer of the epidermis, is an obstacle for the delivery of many molecules at therapeutic levels. in this way, the transport of drugs across the stratum corneum is complex (kalia and guy, 2001). therefore, in order to enhance the transfer of a molecule across this layer, parameters such as partition, diffusion and solubility coeffi cients need to be manipulated and targeted. in addition, factors including the physicochemical properties of the drug, its interaction with the membrane and its pharmacokinetic properties also infl uence the penetration-absorption of a molecule (kalia and guy, 2001). in the last 30 years, different carrier systems have been extensively studied with the aim of controlling the drug release and improving the effi cacy and selectivity of formulations (barratt, 2000; couvreur et al. 2002; schaffazick et al. 2003). the controlled drug release systems are designed to provide appropriated response at the required site of action for prolonged time periods, improving the treatment (vauthier et al. 2003). those systems can be administered by different routes including intravenous, ocular, oral, intraperitoneal, intramuscular, subcutaneous and cutaneous (barratt, 2000). drug targeting is defi ned as a selective drug release at specifi c physiological sites, organs, tissues or cells, in which the pharmacological effect is required (yokoyama and okano, 1996; soppimath et al. 2001). besides, in the case of a local treatment, the drug targeting systems can increase the therapeutic index due to the reduction of the systemic absorption and/or side effects of drugs, which occur when the drug acts at non-specifi c sites (kreuter, 1994; yokoyama and okano, 1996). the technological development of new dosage forms have been a promising approach to increase and control the drug skin penetration (lboutounne et al. 2002; müller et al. 2002; alvarez-román et al. 2004a; alvarez-román et al. 2004b). in the past years, different strategies have been proposed to increase drug skin permeation and to circumvent the inadequate physico-chemical characteristics of several substances (bonina et al. 2001). nanometric systems have a great surface area, which renders them highly satisfactory for the application of lipophilic substances promoting a homogeneous drug release (bouchemal et al. 2004). among the different approaches, nanostructured systems, that are colloidal aqueous suspensions, have been developed. the types of those systems are nanospheres (shim et al. 148 guterres et al drug target insights 2007: 2 2004), nanocapsules (alvarez-román et al. 2001; milão et al. 2003; miyazaki et al. 2003; alvarezromán et al. 2004a; alvarez-román et al. 2004b; müller-goymann, 2004; shim et al. 2004), nanoemulsion (calvo et al. 1996; müller-goymann, 2004; sonneville-aubrun et al. 2004; yilmaz and borchert, 2005), solid lipid nanoparticles (jenning et al. 2000a; jenning et al. 2000b; lippacher et al. 2001; mehnert and mäder, 2001; müller et al. 2002; wissing and müller, 2002a; wissing and müller, 2002b; wissing and müller, 2002c), microemulsions (kreilgaard, 2002), liposomes (barratt, 2000; maghraby et al. 2000; verma et al. 2003; fang et al. 2006) and niosomes (shahiwala and misra, 2002). moreover, such structures have been investigated as alternatives to the classical formulations based on chemical skin permeation enhancers (asbill and michniak, 2000; foldvari, 2000; santoyo and ygartua, 2000). applied epicutaneously, those systems modulate the transdermic diffusion, modifying the molecule activity and/or its partition and diffusivity. in consequence, their use can alter the drug pharmacokinetic and biodistribution through the skin (cevc, 2004). additionally, the nanostructure systems have small size which facilitates their formulation in dermatological products and enable confortable application to the skin (perugini et al. 2002). liposomes were the fi rst carriers introduced for topical delivery of drugs and, since then, they have been extensively studied (barratt, 2000; maghraby et al. 2000; verma et al. 2003; fang et al. 2006). they present advantages for example not being toxic or invasive, as well as they are able to deliver hydrophilic and/or lipophilic substances. many drugs and cosmetic ingredients are already on the marked in liposomal formulations, presenting better dose/effect ratio and less adverse reactions compared to the free substances at the same concentration (redziniak, 2003). in this way, some reviews have been consecrated to describe and discuss the preparation, physico-chemical characterization and cutaneous applications of liposomes (redziniak, 2003; choi and maibach, 2005; fang et al. 2006). more recently, solid lipid nanoparticles (sln) have been developed as novel topical carrier systems for cosmetic and pharmaceutical drugs (mühlen et al. 1998; müller-goymann, 2004). sln are formed by a matrix of lipids which are biodegradable raw materials that are physiologically well tolerated (wissing and müller, 2001). the main advantages of these systems include protection of labile substances from chemical degradation, control of the release of substances due to the solid state of the lipid matrix, and formation of fi lms over the skin showing occlusive properties (müller et al. 2000; müller et al. 2002). additional features are the avoidance of organic solvents during the preparation and no problem concerning large scale production and sterilization. furthermore, the great ability of sln to facilitate the contact of active substances with the stratum corneum, because of the small size of the particles and consequently the high surface area, leads to the high permeation of the carried substances through the viable skin (jenning et al. 2000b; maia et al. 2000). however, according to mehnert and mäder (2001) “sln do not, as proposed, combine the advantages of other colloidal carriers and avoid the disadvantages of them”. even though sln are compounded by physiological ingredients and can be easily produced, they present some disadvantages such as low drug-loading capacities, presence of simultaneous alternative colloidal structures (micelles, liposomes, mixted micelles and drug nanocrystals), as well as physical instability during storage or administration due to the complexicity of the physical state of the lipid (mehnert and mäder, 2001). in the last decade, several articles and reviews have been published on this topic, a first review being published by müller and co-workers (1995). the use of polymeric materials for encapsulating drugs or other active substances is an important approach to mask the physico-chemical intrinsic properties of substances facilitating their skin penetration (alvarez-román et al. 2004a). polymeric nanoparticles are carrier systems presenting diameters lower than 1 µm that can be named nanocapsules or nanospheres depending on their composition. the presence of oil in the nanocapsules leads to a vesicular structure while its absence in nanospheres provide a matricial organization of the polymeric chains (soppimath et al. 2001; couvreur et al. 2002; schaffazick et al. 2003). considering the encapsulation mechanisms (lopes et al. 2001; schaffazick et al. 2003, cruz et al. 2006a; cruz et al. 2006b), the drug can be entrapped, dispersed, dissolved within or adsorbed on the nanoparticles (fig. 1). taking those considerations into account, the review focus is on the insights and data generated over the last few years based on the dermatological 149 nanoparticles for cutaneous applications drug target insights 2007: 2 applications of pharmaceutical and cosmetic formulations containing polymeric nanoparticles reporting the main preparation and characterization methods and the studies of penetration and transport of substances through the skin. to our knowledge, this is the fi rst review centered on the cutaneous applications of polymeric nanoparticles (nanospheres and nanocapsules). the skin as a topical route of administration the skin is composed of the epidermis, the dermis and the hypodermis (fig. 2) being a complex barrier as a consequence of its anatomical organization and special chemical composition. at the epidermis, the stratum corneum consists of 10 to 15 layers of corneocytes presenting a thickness of 10 to 20 µm (foldvari, 2000). the intercellular junctions have corneocyte lipid envelopes and desmosomes (cell structures specialized for cellto-cell adhesion). quality and crystallinity as well as quantity of lipids in the stratum corneum determine the perfection of the skin barrier (cevc, 2004). in this way, the stratum corneum and its compact structure are the main obstacle for the penetration of substances topically administrated on the skin (suhonen et al. 1999; hadgraft, 2001; kalia and guy, 2001; morganti et al. 2001; moser et al. 2001; essa et al. 2002; blanco et al. 2003; ting et al. 2004). many factors are able to govern the drug release into the skin after administration of topical formulations. those factors include molecular weight and lipophilicity of the substance, type of formulation, presence of chemical penetration enhancers and physical state of the stratum corneum (verma et al. 2003). furthermore, the degree of hydration of the stratum corneum is important to determine the rate of drug percutaneous absorption. the hydration level is a function of the water concentration gradient between the dermis and the surface of the skin. hence, an increase in skin water permeability corresponds to an augmentation in permeability to topical applied compounds (morganti et al. 2001). the skin metabolic activity should also be regarded although its biotransformation capacity is considerably lower than the metabolic activity in the gut or in the liver (tauber, 1989). the partition coeffi cient between the vehicle and the stratum corneum is one of the factors which control the drug permeability, establishing a high initial drug concentration on the external layers of the skin (morganti et al. 2001; ting et al. 2004). the effi cacy of a product for cutaneous application depends on the correlation between the permeability coeffi cients in the stratum corneum and the drug chemical characteristics. the absorption degree of a drug through the cutaneous structure is infl uenced by the physico-chemical characteristics of either the susbstance or the vehicle. then, besides the partition coefficient between the vehicle and the stratum corneum lipids, the drug absorption is a function of the drug diffusion in the stratum corneum, the drug partition between the stratum corneum and the viable epidermis, the drug diffusion in the epidermis and dermis, as well as the drug ability to reach the systemic circulation through the cutaneous microvascularization figure 1. encapsulation mechanism models: drug entrapped in, dissolved or dispersed within, and adsorbed on: a) nanocapsules and b) nanospheres. figure 2. scheme of the skin: a) epidermis, b) dermis and c) subcutaneous fat. 150 guterres et al drug target insights 2007: 2 (morganti et al. 2001). regarding the cosmetic and dermatological formulations, the systemic absorption must be avoided and only the skin permeation by the diffusion of the active substances in the epidermis is required. polymeric nanoparticles for cutaneous administration preparation and characterization different methods are described in the literature to obtain polymeric nanoparticles. from a general point of view, those methods are based on in situ polymerization or precipitation of pre-formed polymers (fessi et al. 1989; soppimath et al. 2001; couvreur et al. 2002, schaffazick et al. 2003; sinha et al. 2004). the polymerization of alkyl cyanoacrylates in emulsion leads to matricial nanoparticles called nanospheres (fig. 3a), while the addition of an organic solvent and oil in this medium gives vesicular nanostructures, the nanocapsules, by interfacial polymerization (fig. 3b). moreover, nanospheres and nanocapsules can be also prepared using pre-formed polymers by nanoprecipitation (omitting the oil in the formulation) or interfacial deposition of polymer (containing the oil), respectively (fig. 3c). the method of emulsifi cation-diffusion has been also introduced to obtain nanocapsules (moinard-checot et al. 2006) (fig. 3d), being able to produce nanospheres by omitting the oil in the formulations. nanoparticles can also be prepared using a solvent extraction method, in which the o/w emulsion is high-speed homogenized followed by addition of water and solvent evaporation (luengo et al. 2006). the techniques commonly used for the characterization of nanoparticles include size exclusion chromatography, liquid chromatography, ultrafi ltration-centrifugation and ultracentrifugation, dynamic and static light scattering, electrophoretic mobility, potentiometry, small angle x-ray scattering, differential scanning calorimetry, atomic force microscopy, and scanning and transmission electron microscopies (kumar et al. 2002; schaffazick et al. 2003; müller-goymann, 2004; astete and sabliov, 2006; pohlmann et al. 2007). the size exclusion chromatography provides polymer weight and weight distribution after the nanoparticle preparation furnishing information about the polymerization process or polymer degradation after storage. the liquid chromatography is employed to determine the drug loading (drug total content in the formulation), as well as it is used to quantify the drug in the supernatant or in the ultrafi ltrate informing the non-encapsulated concentration of drug in the formulation (free drug concentration). the drug encapsulation rate is calculated by the ratio between the subtraction of the total content and the free drug concentration and the drug total content, multiplied by 100. the ultracentrifugation or the ultrafi ltration-centrifugation of the native nanoparticle suspension is unable to distinguish either the mechanism of drug encapsulation (fig. 1) or the simultaneous presence of nanocrystals and nanostructures in suspension. dynamic light scattering measurements give the particle size and size distribution, and the static light scattering can provide the gyration radius of particles as well as the depolarization ratio, which is calculated by the correlation between the depolarized scattered light and the polarized scattered light, indicating the shape of the particles in suspension. regarding the electrophoretic mobility, the attraction from the colloidal particles in suspensions causes some of the counter-ions to form a fi rmly attached layer around the surface of the nanoparticle. this layer of counter-ions is known as the stern layer. the counter-ions have a high concentration near the surface which gradually decreases with the distance. the dynamic equilibrium of those counter-ions forms the diffuse layer, where they are repelled by the stern layer. the electrical potential at the junction between the stern layer and the diffuse layer is related to the mobility of the particles and is called zeta potential. the zeta potential is important to evaluate the physical stability of suspensions, to determine either the effectiveness of surface coating or the drug adsorption on the nanoparticles. furthermore, the chemical stability of suspensions can be evaluated by monitoring the ph because its decrease can indicate the degradation of the polymer or other ingredient. dsc and saxs analyses can provide information about the organization at a molecular level of the nanoparticle components. additionally, microscopy techniques (sem, tem and afm) characterize the surface morphology, shape and size of the particles as well as, in the case of tem, the polymeric wall of nanocapsules. 151 nanoparticles for cutaneous applications drug target insights 2007: 2 figure 3. preparation of polymeric nanoparticles by a) polymerization in emulsion, b) interfacial polymerization, c) nanoprecipitation or interfacial deposition of pre-formed polymers, and d) emulsifi cation-diffusion. 152 guterres et al drug target insights 2007: 2 formulating polymeric nanoparticles for cutaneous applications up to now, the nanoprecipitation method is the most commonly used to formulate polymeric nanoparticles intended for cutaneous applications (alverez-román et al. 2001; lboutounne et al. 2002; milão et al. 2003; alvarez-román et al. 2004b; jiménez et al. 2004a; jiménez et al. 2004b; lboutounne et al. 2004; shim et al. 2004; kim et al. 2006). the advantage of this method is the spontaneous, simple, effi cient and reproducible formation of small particles exhibiting a high drug loading capacity (jiménez et al. 2004a). other methods of nanoparticle preparation include in situ polymerization (miyazaki et al. 2003; simeonova et al. 2003; diaz-torrez et al. 2005), solvent extraction (luppi et al. 2004; luengo et al. 2006), emulsifi cation-diffusion (olvera-martinez et al. 2005) and salting out (perugini et al. 2002). concerning the polymers, the poly(ε-caprolactone) (alvarez-roman et al. 2001; alvarez-roman et al. 2004b; jiménez et al. 2004a) is the most employed due to its biocompatibility, biodegradability and mechanical properties (kim and rhee 2003). because poly(ε-caprolactone) is a semi-cristalline polymer its degradation is delayed compared to amorphous polyesters, like poly(lactide) and its copolymers with glycolide. other polymers have also been used to prepare nanoparticulated systems, such as poly (lactide-co-glycolide) (perugini et al. 2002), poly(ε-caprolactone)-block-poly(ethylene glycol) (shim et al. 2004), poly(butyl cyanoacrylate) (simeonova et al. 2003), poly(ethyl cyanoacrylate) (diaztorres et al. 2005), ethyl cellulose (perugini et al. 2002), cellulose acetate phtalate (calderilla-fajardo et al. 2006) and a fatty acid-conjugated poly(vinyl alcohol) (luppi et al. 2004). regarding the reported works, the nanoparticles presented diameters varying between 100 and 615 nm, excepting the particles prepared with poly(lactide-co-glycolide) by salting out, which sizes ranged from 0.68 to 5.71 µm. in this work (perugini et al. 2002) the presence of chloroform in the organic phase probably infl uenced the size of those particles because organic solvents of low water solubility in this phase result in slow precipitation, and microparticles are formed (choi et al. 2002). semi-solid formulations containing polymeric nanoparticles skin care formulations are often based on emulsions and gels because of the technological ability of controlling their viscosity, which can provide appropriated characteristics for cutaneous application by the patient/costumers. the choice of their ingredients defi nes the rheology of formulations that is refl ected by spreading properties on the skin. besides, rheological properties affect all stages of manufacture such as mixing, pumping and fi lling; in addition they are valuable tools in quality control (lippacher et al. 2001). nevertheless, only few studies have been carried out on semi-solid formulations containing polymeric nanoparticles (alvarez-román et al. 2001; milão et al. 2003; miyazaki et al. 2003; jiménez et al. 2004a; alves et al. 2005; luengo et al. 2006). the thickening agents usually used to prepare gel formulations containing polymeric nanoparticles are: 1) pluronic f127 [poly(oxyethylene)-bpoly(oxypropylene)] (miyazaki et al. 2003), 2) satiaxane cx 91 (purifi ed xanthan gum) (alvarezromán et al. 2001), 3) natrosol® 250 m (hydroxyethyl cellulose) (luengo et al. 2006) and 4) carbopol® 940 [cross-linked poly(acrylic acid)] (milão et al. 2003; alves et al. 2005). two emulsions containing nanoparticles, one oil-in-water (o/w) and another water-in-oil (w/o), have also been studied (jiménez et al. 2004a). our research group reported the only two works, as far as we know, concerning the rheological characterization of semi-solid formulations containing polymeric nanoparticles (milão et al. 2003; alves et al. 2005). the non-newtonian behavior and the pseudo-plastic character of the hydrogels have not been affected by the incorporation of nanocapsules or nanospheres. cutaneous applications in 1995, the polymeric nanocapsules were introduced in the cosmetic market by l’oreal. subsequently, scientifi c articles based on skin penetration and distribution of drugs or cosmetic ingredients, encapsulated in polymeric nanoparticles, were published. those works are commented in the following sections according to the type of encapsulating active ingredient: sunscreens or other drugs. sunscreen formulations formulations containing sunscreens are usually applied superfi cially to large skin areas. consequently, their effectiveness indicates that sunscreen molecules adhere to the skin like a protecting fi lm. the uv fi lters are designed to remain on the upper153 nanoparticles for cutaneous applications drug target insights 2007: 2 most layers of the skin (jiang et al. 1997). ideally sunscreen molecules should be bound to the outer section of the stratum corneum being immobilized near to the skin sufarce. defi nitely, penetration to the viable tissues and beyond characterizes a loss from the desired deposition sites being counterproductive (gupta et al. 1999). the main application of polymeric nanoparticles is focused on the new formulations of sunscreens as a result of the ability of those nanoparticles in carrying highly lipophilic substances and their potentialities in modifying and/or masking the physico-chemical properties of loaded drugs; in addition to the need of developing new sunscreen formulations showing an important remanence and a limited penetration in the skin. the works reported in the literature concerning the use of polymeric structures to the nanoencapsulation of sunscreens have been published in the last 5 years. table 1 summarizes the type of nanostructure, the preparation method, the particle size, the active ingredient, the vehicle used and the type of skin evaluation. alvarez-román and co-workers (2001) have conducted the fi rst study concerning the nanoencapsulation of a sunscreen, the octyl methoxycinnamate (omc). omc-loaded nanocapsules have been evaluated regarding the in vitro omc release, during the time of contact with the skin, and the in vivo ability to protect the skin against uvb radiation. omc-loaded nanocapsules have been prepared as aqueous suspension and incorporated in gel formulation. for these suspension and gel, the omc release profi les showed similar shape, but the release rate was faster from the nanocapsule suspension than from the gel. the higher viscosity of the gel compared to the nanocapsule suspension could explain the fi ndings. in addition, the gel containing omc nanocapsules significantly reduced uv-induced erythema, compared to the corresponding omc-free gel. the authors attributed those results to the nanocapsule fi lm formation on the skin surface. other subsequent studies have been performed to further define the passive skin penetration, permeation and distribution of omc encapsulated within nanoparticles (alvarez-román et al. 2004b). for comparison, omc-loaded formulations have been prepared by interfacial deposition (nanocapsules) and by spontaneous emulsifi cation (emulsion). the penetration of omc into the stratum corneum from the nanocapsules was 3.4-fold higher than that from the emulsion. furthermore, after 6 hours of experiment the sunscreen was not detected in the receptor compartment. the authors suggested that the thermodynamic activity of the nanoencapsulated sunscreen molecules, compared to solution formulations, could be higher facilitating their partitioning into the membrane. in addition, the high surface area of nanoparticulated systems may also play an important role in dermal penetration, and facilitates the contact of the encapsulated molecules with the stratum corneum. confocal laser scanning microscopy has been used to visualize the skin penetration of nile red, a fluorescent probe, from nanoparticles. the confocal images clearly demonstrated the enhanced distribution of nile red when delivered from nanoparticles. however, the study did not allow determining unequivocally whether the fl uorescence observed in the images have been originated from nile red associated with the nanoparticles or from free nile red. on the other hand, confocal microscopy studies performed using fl uorescein-conjugated polystyrene nanoparticles showed that they preferentially accumulate in the follicular openings in a time dependentmanner (alvarez-román et al. 2004a). moreover, the infl uence of the particle size (20 or 200 nm) on the skin deposition and/or permeation was also examined. the accumulation was higher as the smaller the particles were. the nanoparticles have been also detected in the furrows on the skin. the infl uence of omc nanoencapsulation on the in vitro transdermal permeation and skin accumulation has been evaluated applying four different formulations: oil-in-water (o/w) and water-in-oil (w/o) emulsions containing the free sunscreen, and similar emulsions containing omc-loaded nanocapsules (jiménez et al. 2004a). results have shown that the incorporation of omc in nanocapsules decreased the release compared to the free omc emulsions. the encapsulation of omc in nanocapsules decreased the penetration of the sunscreen in the skin compared to the free omc emulsions, as well as omc has shown a slower diffusion rate when nanoencapsulated. in consequence, the sunscreen remained longer on the surface of the skin where it was designed to act. stratum corneum penetration degree of omc formulated in nanocapsules has been compared to those obtained for omc-loaded nanoemulsion and for a conventional o/w emulsion containing omc (olvera-martínez et al. 2005). 154 guterres et al drug target insights 2007: 2 ta bl e 1. c ha ra ct er is tic s of fo rm ul at io ns c on ta in in g su ns cr ee nlo ad ed n an st ru ct ur es a nd ty pe s of c ut an eo us e va lu at io n. ty pe p ol ym er m et ho d s iz e (n m ) s un sc re en ve hi cl e c ut an eo us e va lu at io n r ef er en ce n c p c l na no pr ec ip ita tio n 25 5 ± 3 to 4 27 ± 4 o m c g el s ir ra di at io n of g ui ne a pi g sk in w ith u v 3 65 /3 12 n m a lv ar ez -r om an et a l. 20 01 n p p c l na no pr ec ip ita tio n 25 0 o m c s us pe ns io n d iff us io n ce lls a nd ta pe s tr ip pi ng us in g p or ci ne e ar s ki n a lv ar ez -r om an et a l. 20 04 b n c p c l na no pr ec ip ita tio n 37 4 o m c o /w a nd w /o em ul si on s s ta tic d iff us io n ce lls in a m od ifi ed f ra nz c el ls a nd ta pe s tr ip pi ng us in g fl a nk o f f em al e pi gs ji m én ez e t a l. 20 04 a n c a nd n e c a p – em ul si fi c at io ndi ffu si on fo r bo th 39 6 ± 41 to 6 15 ± 2 7 16 2 ± 19 o m c o m c s us pe ns io n s us pe ns io n s tr at um c or ne um p en et ra tio n af te r ta pe s tr ip pi ng in h ea lth y vo lu nt ee rs o lv er am ar tin ez et a l. 20 05 n c a nd n e c a p – em ul si fi c at io ndi ffu si on fo r bo th 36 3 ± 40 to 4 58 ± 2 6 12 4 ± 15 to 1 62 ± 2 4 o m c s us pe ns io n s us pe ns io n s tr at um c or ne um p en et ra tio n af te r ta pe s tr ip pi ng in h ea lth y vo lu nt ee rs c al de ril la f aj ar do e t a l. 20 06 n p p va -f a so lv en t e xt ra ct io n 31 2 ± 10 to 4 40 ± 2 5 b z p s us pe ns io n s ta tic d iff us io n ce ll ba se d on th e f ra nz d es ig n an d ta pe s tr ip pi ng us in g pi g ea r sk in lu pp i e t a l. 20 04 n c , n an oc ap su le s; n p, n an op ar tic le s, n e , n an oe m ul si on ; p c l, p ol y( εca pr ol ac to ne ); c a p, c el lu lo se a ce ta te p ht ha la te ; p va -f a , f at ty a ci d co nj ug at ed p ol y( vi ny l a lc oh ol ); o m c , o ct yl m et ho xy ci nn am at e; b z p, b en zo ph en on e3; o /w , o ilin -w at er ; w /o , w at er -in -o il. 155 nanoparticles for cutaneous applications drug target insights 2007: 2 i n c o r p o r a t i o n o f o m c i n n a n o e m u l s i o n increased the penetration rate compared to its incorporation in nanocapsules or in the conventional emulsion. the aptitude of nanoemulsion to enhance the penetration of omc have been attributed to the size and flexibility of the droplets compared to those of nanocapsules, which are larger and have a rigid structure, or those of conventional emulsion showing the largest droplet size. these authors have also studied the effect of sucrose laurate and sucrose oleate on the in vivo percutaneous penetration of omc from nanocapsules and nanoemulsion (calderilla-fajardo et al. 2006). the inclusion of sucrose laurate in the nanoemulsion increased the transport of omc into the stratum corneum. this enhancement was not the result of only one factor such as the size, the nature of the systems or the type of enhancer, but a combination of them. indeed the enhancement was a result of a synergic effect of the size, the interaction of sucrose laurate with intercellular lipids, and the deformability of the globules. in the case of nanocapsules, the interaction of sucrose esters with the lipid domain of stratum corneum did not enhance the penetration, probably because the rigidity of the particles due to the polymeric matrix of nanocapsule wall. the degree of sunscreen penetration through the skin depends mainly on the physico-chemical properties and nature of the carrier. varying the chemical nature of polymers used to formulate polymeric nanoparticles, different physicochemical and functional properties can be obtained. in this way, the effects of various fatty acid-conjugated pva (pva-fa) on the skin permeation of benzophenone-3 have been evaluated with the purpose of developing a new formulation that can limit the benzophenone-3 penetration into the skin and into the systemic circulation (luppi et al. 2004). nanoparticles, prepared using pva-fa at two different degrees of substitution (40% and 80%), have shown the prevention of the movement of benzophenone3 towards the skin, as a result of the limitation of its percutaneous absorption. precisely, nanoparticles prepared with a low degree of substitution have been the best formulations for enhancing sunscreen location in the epidermis, while nanoparticles prepared with a high degree of substitution have prevented benzophenone-3 percutaneous absorption. other drugs the topical antimicrobial effi cacy of chlorhexidineloaded nanocapsules has been compared to the effi cacy of a disinfectant-detergent solution of chlorhexidine digluconate (lboutounne et al. 2002). a sustained release of chlorhexidine from those nanocapsules enhanced the drug delivery by mediating a more direct and prolonged contact between the carrier and bacteria, skin surface and skin follicles. furthermore, the formulation presented a prolonged ex vivo topical antimicrobial activity against staphylococcus epidermidis. more recently, the transport of chlorexidine-loaded poly(ε-caprolactone) nanocapsules through fullthickness and the stripped hairless rat skin has been investigated in static diffusion cells (lboutounne et al. 2004). after modeling the permeation profi les using the fickian diffusion equation, data showed that the drug encapsulation decreased the percutaneous absorption through stripped skin. confocal laser microscopy confi rmed that nanocapsules transport has taken place by the skin conducts. the small wetting of nanocapsules on the stratum corneum surface, estimated by the contact angle and the surface tension, has maintained the mechanical integrity of the nanocapsules. that is, the fl exibility of the nanocapsules has guaranteed a bioadhesion to the skin while the rigidity of the nanoparticle restricted the molecular leakage into the skin, controlling the chlorexidine delivery. the penetration mechanism of minoxidil encapsulated in polymeric nanoparticles prepared with a diblock, the poly(ε-caprolactone)-b-poly(ethylene glycol), has been studied (shim et al. 2004). in addition, the effect of the nanoparticle diameters on the permeation on both hairy and hairless guinea pig skin using franz diffusion cells has been evaluated. in the hairy guinea pig skin, minoxidil permeated 1.5-fold higher in the epidermal layer and 1.7-fold higher in the receptor solution, when encapsulated in the smaller nanocapsules (40 nm) compared to the larger ones (130 nm). on the other hand, regarding the hairless guinea pig, the permeation of minoxidil has not been dependent upon the nanoparticle sizes. in this way, nanoparticles released minoxidil in the skin mainly by the hair follicles. distribution of poly(lactide-co-glycolide) (4.6 ± 0.8 µm) in porcine skin after its topical administration has been studied in vitro using rhodamine as a fl uorescent probe (jalón et al. 2001a). fluorescence photomicrographs revealed that microparticles 156 guterres et al drug target insights 2007: 2 penetrated through the stratum corneum and reached the epidermis. a subsequent study (jalón et al. 2001b) showed that acyclovir-loaded microparticles can increase drug retention in the porcine basal epidermis. at 6 and 24 h, the quantity of drug was similar to that obtained with the control suspension, while after 88 h the acylovir reservoir in the basal epidermis was higher with the microparticles in comparison with the control suspension. the infl uence of nanoencapsulation of fl ufenamic acid, used as lipophilic model of drug, on its transport into excised human skin has been investigated (luengo et al. 2006). in order to estimate drug penetration, the saarbrücken model has been employed. in this model the skin itself acts as a receptor compartment. a tape stripping technique of the deeper skin layers allowed quantifying penetrated drug concentration. additionally, drug release and permeation through the epidermis have been measured using static franz diffusion cells. in the case of the stratum corneum no differences have been found between the nanoencapsulated and the free drug. on the contrary, the drug accumulation in deeper layers of the skin has been slightly delayed for the nanoencapsulated drug compared to the free drug after shorter incubation times (t <12 h). after longer incubation times (t >12 h), the drug transport has been enhanced for the nanoencapsulated drug compared to the free drug. despite all the works mentioned in this section (cutaneous applications) have reported the use of polymeric nanoparticles as local delivery systems, miyazaki and co-workers (2003) showed the ability of poly(n-butyl cyanoacrylate) nanocapsules containing indomethacin to deliver the drug systemically after topical application. confocal laser microscopy (rhodamin 6g-loaded nanoparticles), in vitro release, in vitro permeation and in vivo percutaneous absorption have been used to compare the behavior of the indomethacin-loaded nanocapsule suspension with a gel containing the drug loadednanocapsules and a conventional gel containing free indomethacin. indomethacin-loaded nanocapsules improved the transdermal delivery of indomethacin compared to the conventional gel. furthermore, the authors suggested that the nanocapsules have been penetrated intact through the rat skin. concluding remarks polymeric nanoparticles intended for cutaneous delivery are prepared with biocompatible polymers generally presenting particle diameters arround 200 to 300 nm. the penetration and transport extent of those systems through the skin seem to be mainly dependent on the chemical composition of ingredients, on the encapsulation mechanism, which, by consequence, infl uences the drug release mechanism, on the size of nanoparticles and, as much as, on the viscosity of formulations. despite some of the reports have compared formulations in a qualitative way, the infl uence of the rheological properties of formulations have not been considered under a quantitative point of view in the case of the studies carried out in vivo. this is an important feature because the topical application of polymeric nanoparticles implies the use of semisolid formulations due to the low viscosity of the nanoparticle suspensions. taking all fi ndings toghether, it was clearly demonstrated that polymeric nanoparticles are able to modify the activity of drugs by altering the physico-chemical properties of formulations, to delay and control the drug release and increase the drug adhesivity or its time of permanence in the skin. briefl y, the polymeric nanoparticles can be useful as reservoirs of lipophilic drugs to deliver them in the stratum corneum being an important strategy to control their permeation into the skin. acknowledgments the authors thank cnpq/brasil, rede nanocosméticos cnpq/mct, capes/cofecub, rede brasil/frança cnpq/mct and fapergs. references alvarez-román. r., barré, g., guy, r.h. and fessi, h. 2001. eur. j. pharm. biopharm., 52:191–5. alvarez-román, r., naik, a., kalia, y.n., guy, r.h. and fessi, h. 2004a. j. control release, 99:53–62. alvarez-román, r,, naik, a., kalia, y.n., guy, r.h. and fessi, h. 2004b. pharm. res., 21:1818–24. alves, m.p., pohlmann, a.r. and guterres, ss. 2005. pharmazie, 60:900–4. asbill, c.s. and michniak, b.b. 2000. pharm. sci. & tech. today, 3:36–41. astete, c.e. and sabliov, c.m. 2006. j. biomaterials sci. polymer ed., 17:247–89. barratt, g.m. 2000. pstt, 3:163–71. blanco, m.d., bernardo, m.v., teijón, c., sastre, r.l. and teijón, m.j. 2003. int. j. pharm., 255:99–107. bonina, f.p., puglia, c., barbuzzi, t., caprariis, p., palagiano, f., rimoli, m.g. and saija, a. 2001. eur. j. pharm. sci., 14:123–34. bouchemal, k., briançon, s., perrier, e., fessi, h., bonnet, i. and zydowicz, n. 2004. int. j. pharm., 269:89–100. calderilla-fajardo, s.b., cazares-delgadillo, j., villalobos-garcía, r., quintanar-guerrero, d., ganem-quintanar, a. and robles, r. 2006. drug dev. ind. pharm., 32:107–13. calvo, p., alonso, m.j., vila-jato, j.l. and robinson, j.r. 1996. j. pharm. pharmacol., 48:1147–52. 157 nanoparticles for cutaneous applications drug target insights 2007: 2 cevc, g. 2004. adv. drug. delivery rev., 56:671–5. choi, m.j. and maibach, h.i. 2005. skin pharmacol. physiol., 18:209– 19. choi, s.w., kwon, h.i., kim, w.s. and kim, j.h. 2002. coll. surfaces a., 201:283–9. couvreur, p., barrat, g., fattal, e., legrand, p. and vauthier, c. 2002. crit. rev. ther. drug carrier syst., 19:99–134. cruz, l., schaffazick, s.r., dalla costa, t., soares, l.u., mezzalira, g., da silveira, n.p., shapoval, e.s., pohlmann, a.r. and guterres ss. 2006a. j. nanosci. nanotechnol., 6:3154–62. cruz, l., soares, l.u., dalla-costa, t., mezzalira, g., da silveira, n.p., guterres, s.s. and pohlmann, a.r. 2006b. int. j. pharm., 313:198–205. diaz-torres, r., castano, v.m., ganem-quintanar, a., quintanar-guerrero, d. and rodriguez-romo, s. 2005. nanotechnology, 16:2612–8. essa, e.a., boner, m.c. and barry, b.w. 2002. j. pharm. pharmacol., 54:1481–90. fang, j.y., hwang, t.l., huang, y.l. 2006. curr. nanoscience, 2:55–70. fessi, h., puisieux, f., devissaguet, j.p., amoury, n. and benita s. 1989. int. j. pharm., 113:r1–r4. foldvari, m. 2000. pharm. sci. & tech. today, 3:417–25. gupta, v.k., zatz, j.l. and rerek m. 1999. pharm. res., 16:1602–7. hadgraft, j. 2001. int. j. pharm., 224:1–18. jalón, e,g,, blanco-príeto, m.j., ygartua, p. and santoyo, s. 2001a. int. j. pharm., 226:181–4. jalón eg, blanco-príeto, m.j., ygartua, p. and santoyo, s. 2001b. j. control release, 75:191–7. jenning, v., gysler, a., schäfer-korting, m. and gohla, s. 2000a. eur. j. pharm. sci., 49:211–18. jenning, v., schäfer-korting, m. and gohla, s. 2000b. j. control release, 66:115–26. jiang, r., roberts, m.s., prankerd, r.j. and benson, h.a. 1997. j. pharm. sci., 86:791–6. jiménez, m.m., pelletier, j., bobin, m.f. and martini, m.c. 2004a. int. j. pharm., 272:45–55. jiménez, m.m., pelletier, j., bobin, m.f., martini, m.c. and fessi, h. 2004b. pharm. dev. tech., 9:329–39. kalia, n.y. and guy, r.h. 2001. adv. drug delivery rev., 48:159–72. kim, d.g., jeong, y.i., choi, c., roh, s.h., kang, s.k., jang, m.k. and nah, j.w. 2006. int. j. pharm., 319:330–8. kim, d.y., rhee, y.h. 2003. appl. microbiol. biotechnol., 61:300–8. kreilgaard, m. 2002. adv. drug delivery rev., 54:s77–s98. kreuter, j. 1994. nanoparticles. in kreuter, j., ed. colloidal drug delivery systems ed. new york: marcel dekker. p. 219–342. kumar, m.n.v.r., kumar, n., domb, a.j. and arora, m. filled elastomers drug del. systems adv. polymer sci., 160:45–117. lboutounne, h., chaulet, j., ploton, c., falson, f. and pirot, f. 2002. j. control release, 82:319–34. lboutounne, h., faivre, v., falson, f. and pirot, f. 2004. skin pharmacol. physiol., 17:176–82. lippacher, a., müller, r.h. and mäder, k. 2001. int. j. pharm., 214:9– 12. lopes, e., pohlmann, a.r., bassani, v. and guterres, s.s. 2000. pharmazie, 55:527–530. luengo, j., weiss, b., schneider, m., ehlers, a., stracke, f., könig, k., kostka, k.h., lehr, c.m. and schaefer, u.f. 2006. skin pharmacol. physiol., 19:190–7. luppi, b., cerchiara, t., bigucci, f., basile, r. and zecchi, v. 2004. j. pharm. pharmacol., 56:407–11. maghraby, g.m.m., williams, a.c. and barry, b.w. 2000. int. j. pharm., 196:63–74. maia, c.s., mehnert, w. and schäfer-korting, m. 2000. int. j. pharm., 196:165–67. mehnert, w. and mäder, k. 2001. adv. drug delivery rev., 47:165–96. milão, d., knorst, m.t. and guterres, s.s. 2003. pharmazie, 58:325–9. miyazaki, s., takahashi, a., kubo, w., bachynsky, j. and löbenberg, r. 2003. j. pharm. pharmaceutical sci., 6:238–45. moinard-checot, d., chevalier, y., briancon, s., fessi, h. and guinebretiere s. 2006. j. nanosci. nanotechnol., 6:2664–81. morganti, p., ruocco, e., wolf, r. and ruocco, v. 2001. clin. dermatol., 19:489–501. moser, k., kriwet, k., naik, a., kalia, y.n. and guy, r.h. 2001. eur. j. pharm. biopharm., 52:103–12. mühlen, a., schwarz, c. and mehnert w. 1998. eur. j. pharm. biopharm., 45:149–55. müller, r.h., mäder, k. and gohla, s. 2000. eur. j. pharm. biopharm., 50:161–77. müller, r.h., mehnert, w., lucks, j.s., schwarz, c., zurmuhlen, a., weyhers, h., freitas, c. and ruhl, d. 1995. eur. j. pharm. biopharm., 41:62–9. müller, r.h., radtke, m. and wissing, s.a. 2002. adv. drug delivery rev., 54:s131–s155. müller-goymann, c.c. 2004. eur. j. pharm. biopharm., 58:343–56. olvera-martínez, b.i., cázeres-delgadillo, j., calderilla-fajardo, s.b., villalobos-garcía, r., ganem-quintanar, a. and quintanar-guerrero, d. 2005. j. pharm. sci., 94:1552–9. perugini, p., simeoni, s., scalia, s., genta, i., modena, t., conti, b. and pavanetto, f. 2002. int. j. pharm., 246:37–45. pohlmann, a.r., cruz, l., mezzalira, g., soares, l.u., da silveira, n.p. and guterres, s.s. 2007. int. j. nanotechnol., 4:in press. redziniak, g. 2003. phathologie bio., 51:279–81. santoyo, s. and ygartua, p. 2000. eur. j. pharm. biopharm., 50:245–50. schaffazick, s.r., guterres, s.s., freitas, l.l. and pohlmann, a.r. 2003. química nova, 26:726–37. shahiwala, a. and misra, a. 2002. j. pharm. pharma. sci., 5:220–25. shim, j., kang, h.s., park, w., han, s., kim, j. and chang, i. 2004. j. control release, 97:477–84. simeonova, m., velichkova, r., ivanova, g., enchev, v. and abrahams, i. 2003. int. j. pharm., 263:133–40. sinha, v.r., bansal, k., kaushik, r., kumria, r. and trehan a. 2004. int. j. pharm., 278:1–23. sonneville-aubrun, o., simonnet, j.t. and l’alloret, f. 2004. adv. coll. interf. sci., 108–109:145–9. soppimath, k.s., aminabhavi, t.m., kulkarni, a.r. and rudzinski, w.e. 2001. j. control release, 70:1–20. suhonen, m.t. and bouwstra, j.a., urtti, a. 1999. j. control release, 59:149–61. tauber, u. 1989. drug metabolism in the skin: advantages and disadvantages. in hadgraft, j.r.h., ed. transdermal drug delivery: developmental issues and research initiatives ed. newyork: marcel dekker. p. 99–112. ting, w.w., vest, c.d. and sontheimer, r.d. 2004. int. j. dermatol., 43:538–47. vauthier, c., dubernet, c., fattal, e., pinto-alphandary, h. and couvreur, p. 2003. adv. drug delivery rev., 55:519–48. verma, d.d., verma, s., blume, g. and fahr, a. 2003. int. j. pharm., 258:141–51. wissing, s.a. and müller, r.h. 2001. int. j. pharm., 23:233–43. wissing, s.a. and müller, r.h. 2002a. int. j. pharm., 242:377–9. wissing, s.a. and 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target insights 2007:2 183–196 183 review correspondence: henning ulrich, departamento de bioquímica, instituto de química, universidade de são paulo, av. prof. lineu prestes 748, 05508-900, são paulo, sp, brazil. tel: + 55-11-3091-3810, ext. 223; email: henning@iq.usp.br please note that this article may not be used for commercial purposes. for further information please refer to the copyright statement at http://www.la-press.com/copyright.htm delivery systems for in vivo use of nucleic acid drugs resende r.r1,4, torres h.a.m2, yuahasi k.k3, majumder p1 and ulrich h1 1departamento de bioquímica, instituto de química, universidade de são paulo, são paulo 05508-900, sp, brazil. 2departamento de biofísica, universidade federal de são paulo, são paulo 04023-062, sp, brazil. 3departamento de neurologia e neurocirurgia, universidade federal de são paulo, são paulo, sp, brazil. present address 4departamento de fisiologia e biofísica, instituto de ciências biomédicas, universidade de são paulo, 05508-900 são paulo, sp, brazil. abstract: the notorious biotechnological advance of the last few decades has allowed the development of experimental methods for understanding molecular mechanisms of genes and new therapeutic approaches. gene therapy is maturing into a viable, practical method with the potential to cure a variety of human illnesses. some nucleic-acid-based drugs are now available for controlling the progression of genetic diseases by inhibiting gene expression or the activity of their gene products. new therapeutic strategies employ a wide range of molecular tools such as bacterial plasmids containing transgenic inserts, rna interference and aptamers. a nucleic-acid based constitution confers a lower immunogenic potential and as result of the high stringency selection of large molecular variety, these drugs have high affi nity and selectivity for their targets. however, nucleic acids have poor biostability thus requiring chemical modifi cations and delivery systems to maintain their activity and ease their cellular internalization. this review discusses some of the mechanisms of action and the application of therapies based on nucleic acids such as aptamers and rna interference as well as platforms for cellular uptake and intracellular delivery of therapeutic oligonucleotides and their trade-offs. keywords: aptamers, rna interference (rnai), drug delivery systems, nucleic-acid-based drugs. introduction the discovery of the dna molecule was one of the most important achievements in the understanding of the fundamental basis of life and now its untapped therapeutic potential is being revealed. therapies based on nucleic acids (nas) including plasmids containing transgenes used in gene therapy, antisense and antigene oligonucleotides, ribozymes, dnazymes, dna and rna aptamers and small interfering rnas, have been developed over the past couple of decades (crooke, 1998; stull and szoka, 1995; patil et al. 2005; ulrich et al. 2006). although most na-based drugs are in the early stages of clinical trials, this molecule class has emerged during recent years as promising drug candidates able to act in a large range of diseases including aids, cancer and neurological and cardiovascular disorders (stull and szoka, 1995; patil et al. 2005; ulrich et al. 2006). the sequencing of the human genome and the transcriptome and proteome projects are providing additional platforms for the advancement of nabased therapies by supplying new targets for the design, screening and selection of drugs. one of the most signifi cant advantages of na-based drugs over conventional pharmaceutical drugs is its high selectivity towards its molecular targets resulting in very specifi c physiological action. these drugs can be used to investigate the genetic disease condition or used in prophylactic measures, thereby preventing disease progression and/or complications in its early stages. for instance, gene therapy usually involves the correction of a malfunctioning gene by the introduction and expression of its correct copy, thus resulting in a corrected protein product. similarly, na-based drugs involved in gene ablation turn off only selected genes, guaranteeing specifi c control of the disease state. thus, at least theoretically, the use of na-based therapies can result in null or minimal collateral effects when compared to conventional, often less specifi c, pharmaceutical drugs. however, the effects of na drugs on human exposition must be completely understood, with emphasis to unforeseen long-term effects. there is yet little knowledge about pharmacokinetics of na-based drugs. the greatest challenge facing the therapeutic utility of nas is to overcome the low cellular absorption inherent to their highly polar molecular structure. the innate ability of these drugs to cross membranes is minimal in normal circumstances. in addition, their low biostability 184 ulrich et al drug target insights 2007:2 results in unpredictable pharmacokinetics. na molecules that happen to enter the cell are subsequently subjected to intracellular degradation by nucleases effectively narrowing the drug’s activity time-span. the fi rst na-drug approved in november 1998 by the fda was denominated vitravene®, an oligonucleotide discovered by isis® as antisense drug for the treatment of cytomegalovirus retinitis in aids patients (perry and balfour, 1999). vitravene® was marketed by novartis ophthalmics® and revealed to be a powerful aids medication. in preclinical studies, antisense inhibition of c-raf kinase was associated with a reduction in the formation of new blood vessels in the eye involved in both, age-related macular degeneration (amd) and diabetic retinopathy (danis et al. 2003). the approval of this fi rst nucleic-acid based drug serves as an encouragement for scientists to further use selex systematic evolution of ligands by exponential enrichment and rna interference (rnai) approaches for drug development (see fig.1a for a scheme of oligonucleotide action on gene expression and protein acivity). nucleic-acid based therapies mechanisms of action small interfering rnas artifi cial modulation of gene expression is mainly based on the inhibition of gene transcription or mrna degradation. the phenomenon of rnai was first observed in the nematode worm caenorhabditis elegans in response to a doublestranded rna (dsrna) treatment which resulted in sequence-specifi c gene silencing (fire et al. 1998). later, the same phenomenon was observed in a large variety of biological systems including several invertebrates and, more recently, also vertebrates such as xenopus and mice (nakano et al. 2000; wianny and zernicka-goetz, 2000). for induction of rnai, small double stranded rnas (termed small interfering rnas – sirnas) are produced by the cleavage of long dsrnas (tuschl et al. 1999; zamore et al. 2000; hamilton and baulcombe, 1999; hammond et al. 2000). the cytoplasmic, highly conserved dicer protein, member of the family of rnase iii–like enzymes, forms a characteristical 21–23-nucleotide long dsrna duplex with symmetric twoto threenucleotide 3' overhangs (bernstein et al. 2001; elbashir et al. 2001a). the duplex small-interfering (si)-rnas (products of the long dsrna cleavage) are integrated into the risc complex (rnainduced silencing complex). the complex becomes activated by the unwinding of the duplex upon the loss of one strand of the si-rna duplex by an rna helicase activity. depending on several target and si-rna properties that are not entirely understood, risc can either specifi cally cleave and degrade target mrna (yekta et al. 2004; meister et al. 2004; zamore et al. 2000; bagga et al. 2005; giraldez et al. 2005; wu et al. 2006) or inhibit its translation without initiating its sequence-specifi c mrna degradation process (olsen and ambros, 1999; reinhart et al. 2000; wightman et al. 1993). the target mrna is cleaved by the risc complex at the middle of the complementary region, ten nucleotides upstream of the nucleotide paired with the 5' end of the guide sirna (elbashir et al. 2001b). the cleavage reaction guided by risc does not require atp (nykänen et al. 2001; hutvágner and zamore, 2002). however, multiple rounds of mrna cleavage, which requires the release of cleaved mrna products, are more effi cient in the presence of atp (hutvágner and zamore, 2002). when the dsrna is of endogenous origin, the dicer cleavage products are named micro rna (mirna). the mirna-guided mechanism of translational regulation is not as well understood. studies of mutant or transgenic c. elegans, showed that mirnas inhibited target-protein synthesis without affecting mrna levels (bartel, 2004). the target mrna contains three-prime untranslated regions with several binding sites for the mirna, and both the target and the mirna were found to be associated with polyribosomes. this suggested that mirnas block translation elongation or termination rather than translational initiation (olsen and ambros, 1999; seggerson et al. 2002). by using protein mutants various genes involved in the rnai process were identifi ed, including some highly homologous helicases and other enzymes involved in the transposition of mobile elements, such as rnase d (bertrand et al. 2002; mcmanus and sharp, 2002; scherr et al. 2003). results like these corroborate the currently accepted hypothesis that the response to dsrnas plays a defensive physiologic role against deleterious rnas such as virus transcripts. therefore mirnas are essential for the maintenance of genome integrity in a large range of biological 185 delivery systems of nucleic acids drugs drug target insights 2007:2 figure 1. nucleic-acid based therapies. (a) actions of antisense oligonucleotides, small interfering rnas, aptamers and intramers (intracellularly acting aptamers) on target cells. the antisense technology is based on the introduction of a complementary oligonucleotide sequence to the target mrna resulting in rnase h activation and target rna degradation. for rna interference process 21–23 nucleotide-long sirnas induced into the cell activate the risc complex leading to degradation of target mrna. rna or dna aptamers target and inhibit the products of gene expression such as intracellular or extracellular protein. (b) conventional delivery of sirna to target cells by transfection. therapeutic applications are hindered due to poor cellular uptake and absence of a mechanism to deliver sirnas specifi cally to target cells. (c) aptamer-directed sirna delivery to target cells. the si-rna is coupled to an aptamer which specifically binds a surface epitope on target cells (i.e. prostate-specifi c membrane antigen), thereby possibiliting the specific down-regulation of gene expression in cancer cells. following binding of the sirna-aptamer chimera to its cell-surface receptor, the receptor-oligonucleotide complex is internalized followed by induction of rna interference. 186 ulrich et al drug target insights 2007:2 systems (hutvágner and zamore, 2005). the interference effect of dsrna is non-stoichiometric in relation to homologous mrna, since very low amounts of dsrna cause strong interference. this suggests that the rnai effect involves a catalytic stage and cannot be based on the titration of endogenous mrna (sverdlov, 2001). therapeutical applications of rnai currently, sirna drugs are being developed in order to inhibit cell infection by hiv (martinez et al. 2002) and infl uenza viruses (ge et al. 2003) and to treat autoimmune hepatitis (song et al. 2003) (fig. 1b). rnai has also been used as a tool to study signaling pathways involved in neurogenesis and neurodegeneration (miller et al. 2005). as an example, the function of vascular endothelial growth factor (vegf) in directing neurogenesis was verifi ed via rnai (cao et al. 2004). another approach uses adenoviral, lentiviral and aav (adeno-associated viral) delivery systems (chen et al. 2006) to treat spinocebellar ataxia (xia et al. 2004), amyotrophic lateral sclerosis (als) (ralph et al. 2005; raoul et al. 2005), huntington´s (harper et al. 2005; rodriguez-lebron et al. 2006) and alzheimer’s disease (singer et al. 2005). when compared to other gene ablation tools such as antinsense oligonucleotides, sirnas are notably superior due to their higher degree of mrna degradation and low potential of inducting immune responses (bertrand et al. 2002). since sirnas molecules are not integrated in the genome as plasmids potentially are, they cause collateral effects and thus are much more therapeutically advantageous. small-interfering rnas do not need to be transferred into the nucleus in order to present activity, requiring less sophisticated delivery systems. in addition, due to their relative small size, the delivery of a cocktail of sirnas targeting the expression of multiple disease causing genes at the same time should be feasible. vegf gene expression was the fi rst target for clinical trials using sirna in the treatment of age-related macular degeneration (amd). rnai is also being used to combat infection by the respiratory syncytial virus (rsv) genome. the vehicle used in both cases was a saline formulation. the success of these trials may be due to the direct administration of sirnas at the diseased organs. the advantage of the direct administration is the high concentrations of the sirna available at the target side. a sirna-based drug denominated cand6 which also suppresses vegf gene expression is currently being tested in a phase 2 trial with patients suffering from amd. moreover, early in 2006, cand5 was also tested in phase 2 clinical trials for the treatment of diabetic macular edema (http://www.acuitypharma.com/). the sirna aln-rsv01 developed by alnylam pharmaceuticals, inc., for treatment of rsv infection has completed two phase 1 trials and now appears to be appropriate for tests in humans. other sirnas targeting influenza and hepatitis c viruses (protiva biotherapeutics) are expected to be available for clinical use within the coming year (protiva biotherapeutics). stability of sirna in plasma sirna molecules are unstable in serum as a result of degradation by serum nucleases and thus have very short half-lives in vivo (soutschek et al. 2004). stability against nuclease degradation can be achieved by introducing a phosphorothioate (p = s) backbone linkage at the 3′ end for exonuclease resistance and 2′�modifi cations (2′-ome, 2′-f or related) for endonuclease resistance (vornlocher et al. 2005; li et al. 2005; choung et al. 2006). moreover, sirna molecules consisting entirely of 2′-o-methyl and 2′-fl uoro-modifi ed nucleotides demonstrated enhanced plasma stability and increased in vitro potency. duplexes containing the 4′-thioribose modifi cation present increased thermal stability and are 600 fold more resistant to degradation in plasma than natural rna duplexes are (hoshika et al. 2004). substantial improvements in sirna activity and plasma stability have also been achieved by judicious combination of 4′-thioribose with 2′-o-me and 2′-o-methoxyethyl modifi cations (dande et al. 2006). aptamers aptamers are oligonucleotides identifi ed by an in vitro selection process as high-affi nity binders to a given target molecule. for this purpose, a dna library is synthesized containing an inner randomized sequence of typically 20–100 nucleotides fl anked by two outer constant regions of 20–40 nucleotides. a t7 promoter site is incorporated in one of the constant sequences, if a rna aptamer is to be selected. the chemically synthesized dna pool is amplified by pcr in the presence of senseand anti-sense-primers. the dna template can now either be transcribed in vitro to the rna 187 delivery systems of nucleic acids drugs drug target insights 2007:2 pool or be denatured to originate a single-stranded (ss) dna pool to be used in the in vitro selection process. in many cases 2′-f-modifi ed pyrimidines are employed in the in vitro transcription reaction to improve nuclease-resistance of generated rna molecules (reviewed by ulrich et al. 2004). reiterative cycles of in vitro selection, also denominated as systematic evolution of ligands by exponential enrichment (selex), are carried out by incubating the target protein or another molecule of biological importance with the combinatorial dna or rna pool, followed by elution and amplifi cation of target binders by rt-pcr or pcr techniques. selection stringency is increased with the numbers of selex cycles. increased stringency can be achieved by augmenting the number of dna or rna molecules relative to possible target binding sites as well as by extensive washing for removal of low-affi nity binders. these procedures ensure that the original random pool containing 1013–1015 different sequences becomes narrowed down to a more homogenous population of high-affi nity target binders. when the binding affi nity of a selected rna or dna library to its target cannot be any longer improved by subsequent selex cycles, this fi nal pool is sequenced for identifi cation of aptamers. at this stage one expects that similar sequence motifs have been preserved in aptamers with binding affi nity to their targets and that in most cases these consensus sequences fall into conserved stem-loop motifs. these rna or dna molecules with unique binding characteristics, also denominated as aptamers (from latin aptus = to fi t) can be used for basic research, clinical and diagnostic purposes. basic research purposes include the characterization of aptamer-target protein interaction in its cellular context (ulrich et al. 1998, 2002), the study of the mechanism of protein activation and inactivation (hess et al. 2000), the mapping of binding sites (shi et al. 2007), the dissection of intracellular signaling pathways (famulok et al. 2001) as well as the inhibition of intracellular virus replication (toulmé et al. 2003). an example for an intracellular acting aptamer (intramer) is an rna molecule selected as highaffi nity ligand of the b52 protein. the protein b52 is expressed during drosophila development and acts there in the gene-splicing process. as the exact function of this protein was not fully understood, an anti-b52 intramer was developed and expressed as a pentameric structure in developing drosophila cells. the observed drastic reduction of drosophila survival in the presence of the aptamer indicated crucial functions of the b52 protein (shi et al. 1999). another approach in therapeutics makes use of an aptamer that binds the intracellular domain of the β−2 integrin lymphocyte function-associated antigen-1 (lfa-1). this portion mediates cell adhesion by binding to the intercellular adhesion molecule-1 (icam-1). the specifi c blockage of signaling pathways in vivo by intramers could potentially be applied to any signal-transduction cascade (blind et al. 1999). the intramer against the rev protein, which is involved in the cycle of replication of hiv resulted in inhibition of the virus replication in cell culture (good et al. 1997), and in human lymphocyte cells (chaloin et al. 2002). these results indicate that intramers may be an alternative to rnai by specifi cally suppressing the activity of gene products instead of inducing degradation of mrnas coding for these proteins. the ability to modulate intramer activity would drastically increase the effi ciency of regulation of intracellular signaling by oligonucleotides. the activity of these aptamers could be put under the allosteric control of a second molecule (tang and breaker, 1997) or their expression could be allosterically regulated. the sequence coding for the intramer can be introduced in the 5′-promoter position and work as an inductive promoter. some intramers were developed against kanamicin and tobramicin and expressed in the position of the 5′-gene promoter. the addition of those antibiotics to the cell system resulted in the shut down of the 5′-gene transcription (werstuck and green, 1998). the high specifi city of aptamers in acting just on an isotype or a splice-variant of a target protein makes them excellent drug candidates. in this regard, theis and colleagues identifi ed intramers which bind and switch off the cytohesin-2 guanine nucleotide exchanger but do not affect the homologous protein cytohesin-1 that has a different function. this effect was observed following six hours of hela cell transfection with the aptamer construct (theis et al. 2004). these properties of intramers make them promising tools for development of therapeutics with applications in vaccine development, blockage of intracellular transduction pathways, in viral infection control and timedependent gene knockdown of protein activity. the suppression of target protein activity at desired time points followed by aptamer inactivation may gain importance in the control of important 188 ulrich et al drug target insights 2007:2 physiological functions such as blood coagulation (rusconi et al. 2004). inhibition of coagulation is for instance desirable in a pathological condition of high blood pressure, but must be immediately reversed in case of hemorrhage. moreover, aptamers are very potent inhibitors of extracellular protein activity, for instance by blocking growth factor-receptor binding. an anti-vegf165 aptamer was developed that blocks pathological vegf165-receptor binding, thereby leaving other vital vegf isoform action unaffected. an antivegf165 aptamer formulation has been recently approved for therapeutic use by the fda and trade-named as macugen (reviewed by ulrich et al. 2006; vavvas and d’amico, 2006; ng et al. 2006). another promising aptamer tested in clinical trials has been denominated reg1 (regado biosciences). this therapeutic aptamer targets factor ixa (drug, rb006) with anti-coagulation activity. the aptamer and its complementary oligonucleotide antidote (rb007) were tested in healthy volunteers in a phase 1a pharmacodynamic evaluation (dyke et al. 2006). there was not any signifi cant bleeding occurrence associated with rb006 treatment, and both aptamer drug and antidote were well tolerated. the overall results of the pharmacokinetics of the two compounds in healthy volunteers indicated their safe use in humans encouraging further studies (dyke et al. 2006). in addition to their possible therapeutic relevance, aptamers may be used in diagnostic applications, such as differentiating between normal and tumoral vasculature (blank et al. 2001), the pathogenic form of the prion protein from its normal conformation (rhie et al. 2003), as well as identifying possible biohazards, such as anthrax spores (bruno and kiel, 1999). the possible pharmaceutical and therapeutic importance of aptamers is mainly related to the following characteristics: (i) their affi nity for their targets with dissociation constants in the nanoto picomolar range, similar to those found on monoclonal antibody-antigen complexes. high-affi nity aptamer-protein interactions result from specifi c hydrogen-bond formation between bases and amino acids of the target proteins in addition to interactions between oligonucleotide backbones and protein secondary and tertiary structures. (ii) aptamers can be chemically modified to acquire more stability for in vivo applicatons, resulting in an increase of their halflife time from a couple of seconds to days (ulrich et al. 2004). these modifi cations in oligonucleotide structure can be done prior or following the selex process. (iii) identifi ed aptamer sequences can be truncated to their minimal sequences, represented by a single loop structure which is suffi cient for binding and biological activity. (iv) aptamers can be easily enzymatically reproduced or produced in a large scale by chemical synthesis (reviewed by ulrich et al. 2006). vehicles for oligonucleotide delivery the combination of target-specifi c drug delivery and its controlled release (langer, 1998) is an important goal in the search for more effi cient and less hazardous treatment of tumoral diseases. it is desirable that the cytotoxic drug dosage is delivered to target cells for a long time span, thereby sparing the healthy cells of the surrounding tissue. in order to attain this objective, it is critical to develop specialized vessels which encapsulate the chemotherapeutical drugs for its controlled release and that such vessels are directed to cancer cells (i.e. by presenting appropriate ligands which recognize specifi c cancer-cell antigens). a wide variety of address-molecules have been investigated for their effi ciency to deliver oligonucleotides to cancer cells. humanized antibodies and single chain variable fragments generated by murine hybridomas or phage displayed, minibodies and peptides were among the tested delivery vehicles (reviewed by weiner and adams, 2000). for the development of vehicles, some pre-requisites must be satisfi ed in order to improve their chances of passing functional and clinical trials. the drug encapsulating particle-system must be composed of biocompatible, biodegradable polymers approved for clinical use by the drug regulating agencies. moreover, the vessel particles must effi ciently bind the negative charges of na chains while minimally adverse-effecting their tridimensional folding and thus their binding properties. the delivery vessels must yet effi ciently and selectively bind to target cells, as well as have a long half-life in circulation in order to reach the target before being degraded and releasing their contents. vehicles may be classifi ed as such (i) improving oligonucleotide pharmacokinetics by attaching a high-molecular weight lipophilic molecule to an aptamer in order to augment the half-life of a therapeutic oligonucleotide in the plasma, and as 189 delivery systems of nucleic acids drugs drug target insights 2007:2 such (ii) permitting the immobilized oligonucleotide to pass physiological barriers such as the brainblood barrier or plasma membranes to be delivered into cells. in addition to their ability to act as therapeutic drugs by themselves, aptamers can also be used as vehicles themselves to deliver another oligonucleotides to specifi c target cells (farokhzad et al. 2006). applications for oligonucleotidedirected drug delivery prostate-specifi c membrane antigen (psma) is a type-2 integral membrane glycoprotein, expressed at the prostate carcinoma surface and on new vessels formed by various other solid tumors. this antigen is highly expressed in every stage of prostate cancer development (rajasekaran et al. 2005) and, therefore, is a strong molecular target candidate for immunotherapy and prostate-cancer imaging. previous efforts to selectively destroy cancer cells generally have made use of antibodies to deliver cytotoxic packages (wu and senter, 2005). however, aptamers developed against psma as protein or another molecule of biological importance potent target binders can be used instead of antibodies to deliver cytotoxic agents to cancer cells (farokhzad et al. 2006). gelonin (gel) is a protein toxin with n-glycosidase activity promoting cell death by cleavage of a specific glycosidic bond of rrna thereby promoting inhibition of protein synthesis and resulting in elimination of target cells. however, gelonin does not contain a translocation domain such as those of many other ribosomal toxins do, and is not incorporated into cells at considerable quantities (rosenblum et al. 1999). in order to further improve its cytotoxicity, a recombinant gel (rgel) was developed. although this recombinant variant provoked some toxicity on target cells, membrane-permeability was not signifi cantly improved (rosenblum et al. 1999). this problem was solved by chemical conjugation of rgel or its genetic fusion with the delivery package’s recognition molecules and by addition of cysteine residues to form antibody immunoconjugates (rosenblum et al. 2003; better et al. 1994). for instance, the cytokine vegf was coupled to gelonin and the resulting conjugate specifi cally killed cancer cells overexpressing the vegf receptor flt-1 (veenendaal et al. 2002). a selective drug that can home in a specifi c target cell or tissue as therapeutical nanoparticle is the most desirable aim of any delivery system. previously selected rna aptamers specifi cally binding to psma were used to escort gel to tumoral cells expressing psma at their surface. the conjugated toxin destroyed prostate cancer cells with an ic50 value of 27 nm, presenting an increase in toxicity of more than 600 times in comparison to cells which do not express psma (chu et al. 2006). the extracellular domain of psma can now be recognized by a biocompatible and biodegradable polymeric nanoparticle encapsulated with a docetaxel (dtxl) surface functionalized with a stable, nuclease-resistant rna aptamer containing 2′-f-modified pyrimidine bases (farokhzad et al. 2006). these aptamernanoparticle bioconjugates (dtxl-np-apt) bound to psma proteins expressed at the surface of prostate epithelial cells lncap were easily incorporated by cancer cells with cytotoxic effects in vitro. encouragingly, dtxl-np-apt bioconjugates also presented remarkable effi cacy and reduced side effects in vivo. these observations strongly indicate a potential therapeutic application of aptamer-nanoparticle bioconjugates to specifi cally target and destroy cancer cells. another approach with possible therapeutic applications makes use of packaging rna (prna) as part of the dna-packaging machinery of the bacteriophage phi29. this prna was genetically engineered to originate chimeric rna that forms dimers via interlocking rightand left-handed loops (guo et al. 2005). fusing prna with either receptor-binding rna aptamers, folate, small interfering rna (sirna), ribozyme, or another chemical group did not disturb dimer formation or interfere with the function of the inserted moieties. incubation of cancer cells with the prna dimer with one subunit harboring the receptor-binding moiety and the other containining the gene expression-silencing molecule resulted in targetcell recognition, uptake into these cells and subsequent silencing of anti-apoptotic gene expression. the chimeric prna complex was found to be processed into functional double-stranded sirna by the rna-specifi c endonuclease dicer. animal trials confi rmed the suppression of tumorigenicity of cancer cells by ex vivo delivery (guo et al. 2005; khaled et al. 2005). these small-size rna nanoparticles will allow repeated long-term administration and avoid the problems of short retention time of small molecules and will also 190 ulrich et al drug target insights 2007:2 avoid the delivery problems of particles larger than 100 nm. farokhzad and collaborators (2004) synthesized a polylactic acid (pla)-block-polyethylene glycol (peg) copolymer with a carboxilic terminal functional group (pla-peg-cooh), and encapsulated rhodamine-labeled dextran inside pla-peg-cooh nanoparticles. these nanoparticles have negatively charged carboxilic groups on their surfaces, which minimize unspecifi c interactions with negatively charged nas and, for instance, can be conjugated with aminomodifi ed nas. clinical trials revealed that the presence of anti-psma aptamers at target cells is increased 77-fold when it is bioconjugated to a nanoparticle (farokhzad et al. 2004). the incubation of protein-free nanoscale particles containing a receptor-binding aptamer or other ligands may result in the binding and internalization of the trivalent therapeutic particles (dtxl-np-apt or pla-peg-cooh-apt) subsequently modulating prostate cancer cell apoptosis. these bioconjugates were based on materials which had been priorly approved for clinical use by the fda. since these molecules are small, relatively stable, non-immunogenic and easy to synthesize, the translation of these bioconjugates into clinical practice is facilitated. therefore, therapeutic and diagnostic nanoparticle-aptamer bioconjugates will be shortly developed for other important human diseases. in this regard, rna molecules might be used as building blocks in many associations in nanotechnology. the delivery of macromolecules (including globular proteins and aptamers) to the sclera as therapeutics in eye disease has been described in recent studies (ambati et al. 2000a; ambati et al. 2000b). the transport and potential diffusion of molecules to the sclera takes place through an extensive surface area containing a high percentage of water. water is the main constituent of the extracellular matrix containing few cells and unchanging permeability during aging (olsen et al. 1998; olsen et al. 1995; boubriak et al. 2000). the use of this route of administration could avoid problems and limitations of other delivery approaches in the treatment of viral and systemic diseases (kamei et al. 1999; dayle, 2001; lang, 1995). since some transscleral delivery systems may be destructive (i.e. iontophoresis) occasionally provoking retinal necrosis and gliosis (lam et al. 1991), the ideal approach would be to develop biodegradable polimeric particles with prolonged delivery capacity. lenghtened protein and na release time-spans would allow more effi cient addressing of drugs to specifi c target tissues (carrasquillo et al. 1999; carrasquillo et al. 2001a; carrasquillo et al. 2001b). frazza and schmitt (1971) developed a poly (lactic-co-glycolic) acid (plga) polymer, which since then has been widely used in clinical procedures as suture for tissue engineering (hasirci et al. 2001; ma and choi, 2001). plga may be locally applied, allowing intralesional administration of drugs while minimizing adverse systemic effects. the possibility of local application of plga constitutes an important pharmacological advantage (mallery et al. 2000; moritera et al. 1991). the employment of plga as a drug delivery system for the therapy of ocular diseases did not reveal any signs of ocular toxicity or infl ammatory processes even during long treatment periods (moritera et al. 1991; giordano et al. 1995). however, adverse side effects resulted from the sclerotomy as result of the delivery procedure of small encapsulated synthetic drugs. carrasquillo et al. (2003) developed a drug delivery system that continuously releases the eye001 anti-vegf aptamer (macugen) in a controlled fashion during signifi cant time spans when it is locally administered at the external area of the sclera. the use of the proposed delivery system for the release of macugen illustrates a promissing alternative for the tansscleral delivery of drugs for the treatment of ocular and choroidal illnesses. another way to successfully home aptamers at their subcellular targets is to express them in the cells of interest. in this case the expressed drugs would be useful for both the treatment of hereditary diseases as well as of viral diseases such as aids. the tar region of the rna genome of hiv-1 is an attractive target for inhibitory nabased drugs. tar is a 57 nucleotide regulatory element present at the 5′ end of every viral rna particle. it exerts a crucial role on viral transcription, as it is recognized by the ternary complex composed of the tat viral protein (trans-activator of transcription) and of two cellular proteins named cyclin t1 and cdk9 (herrmann and mancini, 2001; richter et al. 2002).cdk9 when associated to tat, hyperphosphorilates the carboxy-terminal of rna polymerase ii and subsequently activates the transcription machinery triggering the effi cient synthesis of the entire viral rna. a na-based drug strongly interacting with tar competes with the formation of the 191 delivery systems of nucleic acids drugs drug target insights 2007:2 transcription complex and consequently inhibits the trans-activation of the transcription apparatus. thus, hiv-1 replication is compromised. moreover, since the tar element is located in close proximity to the 5′ end of the mature mrna, a tar binder could interfere with the ribosomal machinery as well. nas, including antisense oligonuvleotides, sirna and aptamers were employed in many studies for targeting the tar element (turner et al. 2005; yoshinari et al. 2004; ducongé and toulmé, 1999). the anti-tar rna aptamer (ducongé and toulmé, 1999) was optimized by chemical modifi cations towards improved stability regarding nuclease resistance and decreased trans-activation of transcription in cell nuclei extract assays (darfeuille et al. 2002a; darfeuille et al. 2002b; darfeuille et al. 2004; kolb et al. 2005; toulmé et al. 2001). other aptamers were also developed interfering with hiv-1 gene expression, such as the anti-hiv rev-binding aptamer (rbeapt) (konopka et al. 1998). konopka and collaborators used cationic liposomes as delivery vessels carrying the association of rbeapt and a ribozyme that acts against the hiv-1 env gene inhibiting viral production (konopka et al. 1998). these data provide strong evidence for the therapeutical potential of na ligands as anti-hiv agents when their intracellular delivery is effi cient. delivery systems for sirna conjugation of na terminals with lipophilic molecules has been reported to improve or direct cellular uptake. for example, sirnas conjugated with cholesterol improved in vitro and in vivo permeation of liver cells (lorenz et al. 2004). a number of approaches—including lipid-based formulation, transmessenger (niu et al. 2006) and complexation with polyethylenimine (grzelinski et al. 2006), cholesterol-oligoarginine (kim et al. 2006), a protamine-fab fusion protein (song et al. 2005) and atelocollagen (takei et al. 2004; minakuchi et al. 2004)—have been shown to facilitate delivery into tumor cells. aptamer-sirna chimeric rnas have also been successfully used to facilitate sirna delivery in vivo (mcnamara et al. 2006; chu et al. 2006) (fig.1 c). in 2004, soutschek and coworkers demonstrated effective silencing of gene expression of apolipoprotein apob by intravenous administration of chemically modifi ed sirna which resulted in silencing of the apob mrna in liver and jejunum, decreased plasma levels of apob protein, and reduced total cholesterol concentration in mice (soutschek et al. 2004). judge et al (2005) made use of sirna duplexes formulated in stable na lipid particles (snalps) to attain gene silencing of apob in mice (zimmermann et al. 2006). moreover, the general applicability of snalp formulations for hepatic delivery of sirna has been demonstrated in animal models of hbv and ebola virus infection (morrissey et al. 2005; geisbert et al. 2006). in oncology, direct delivery of sirnas and viral delivery of small hairpin (sh)-rnas to tumors have been shown to successfully inhibit xenograft growth in mouse models. combination of sirna and aptamers in therapeutics technologies that mediate targeted delivery of sirnas are needed to improve their therapeutic effi cacy and safety for use in humans. lupold et al. (2002) identifi ed two aptamers that bind with low nanomolar affinity to the extracellular portion of psma. the two aptamers, denominated as xpsm-a9 and xpsm-a10, did not share any consensus sequences and bound to different sites of psma. distinct modes of inhibition suggested that each aptamer identifi es a unique extracellular epitope of recombinant psma (xpsm). these aptamers were the fi rst recognizing specifi c prostate cancer markers. chu et al. (2006) coupled sirnas interfering with laminin and gapdh gene expression by a modular streptavidin bridge to the aptamer a9 which binds to prostate-cancer cells. comparison of oligofectamine (invitrogen) and aptamer-mediated sirna transfection resulted in similar inhibition of target-protein expression as evaluated by real-time pcr experiments. psma endocytosis in lncap cells is thought to predominantly proceed via clathrin-coated pits. the rate of internalization has been previously measured using antibodies directed against psma and also in the presence of the anti-psma aptamer a10. internalization was shown to take place for both types of targeting agents within hours of binding (farokhzad et al. 2004). inhibition of target-gene expression was detected after 72 h of sirnaaptamer transfection. mcnamara et al. (2006) generated a chimera of a10 and a sirna construct targeting polo-like 192 ulrich et al drug target insights 2007:2 kinase 1 (plk1) and bcl2 (yano et al. 2004; reagan-shaw and ahmad, 2005) as two survival genes overexpressed in most human tumors (takai et al. 2004; eckerdt et al. 2005; cory and adams, 2005). since dicer also acts on chimeric rnas, aptamer-sirnas are directed into the rnai pathway and silence their cognate mrnas. aptamer-sirna chimera–mediated gene silencing is dependent on dicer activation and occurs via the rnai pathway. however, inhibition of dicer activation had no effect on transfected plk1 sirnamediated silencing, as 21to 23-nucleotide containing sirnas have been shown to bypass the dicer step (murchison et al. 2005). the chimera a10-sirna specifi cally bound to psma on the surface of lncap cells but did not interact with pc-3 prostate cancer cells which do not express psma (see scheme in fig.1c). the combination of shrna and aptamer for specifi c gene expression control in target cells was accomplished by an et al (2006). this research group constructed a vector containing the theophyllinebinding aptamer (jenison et al. 1994; zimmermann et al. 1997) and the loop region of the shrna targeting the enhanced green fl uorescent protein (egfp) under u6 promoter expression. when this construct was co-transfected with a construct coding for egfp expression (pegfp-n1) into hek cells, shrna-induced egfp gene expression silencing was dose-dependently inhibited by increasing concentrations of theophylline, as aptamer-bound theophylline interfered with the dicer-cleavage site. this study proved the feasibility of modulation of gene expression under the control of intracellular proteins or cell metabolites (an et al. 2006). recent clinical developments one of the fastest approvals ever obtained for a drug by the fda was for imatinib (imatinib mesylate [gleevec], from novartis) for the treatment of chronic myelogenous leukemia (cml). although the incidence of cml is low, the rate of cure with conventional treatment is poor. gleevec is a tyrosine kinase inhibitor and affects only the leukemic cells that are caused by fusion of two genes, bcr and abl, a chromosomal shuffling between chromosomes 9 and 22. although expensive, gleevec, an oral drug, led to remission in 90% to 95% of cml-relapsed patients. however, it has not worked well in blast crisis. current activities of the cytrx corporation are concentrated in the development of small molecule drugs, rnai drug discovery and dna vaccines and a delivery technology with multiple applications in the area of dna vaccine and gene therapy. its proprietary poloxamer compound, tranzfect, has revealed good results regarding its transfection ability, immunoadjuvant activity and toxicity profi le as prerequisites for dna-based vaccines. this company also participates in the development of gene-silencing technologies for treatment of amyotrophic lateral sclerosis, type ii diabetes, cmv retinitis, obesity, and cancer. targeting of the epidermal growth factor (egf) receptor by pegylated immunoliposomes carrying a plasmid coding for the shrna prolonged the survival of mice with intracranial human brain cancer (zhang et al. 2004). the observation that encapsulated oligonucleotides readily passed the blood-brain barrier, encourages the development of na-based therapies for neurodegenerative diseases (reviewed by sa, 2004). conclusions in the nanotechnology fi eld, aptamers have the potential to act as targeting molecules by directing the delivery of nanoparticles to antigens present on the surface of target cells. in general terms, therapeutic nanoparticles are components of specialized delivery vehicles of an encapsulated drug. drug release should occur in a regulated and defi ned manner. depending on the therapeutic demand, such devices are designed to ensure continuous or immediate drug release. the combination of targeted delivery and controlled release of drugs at affected tissue sites will lead to the development of “smart therapeutics”, which are more effective and will have less undesired side effects than drugs available today. several studies have shown that nanoparticles can be attached to nas in a way that na-binding properties to their targets are preserved, thus reducing their potentially associated deleterious side effects. these new approaches can supplement the conventional chemotherapy and radiotherapy in cancer treatment, prevent drug resistance and damage to normal tissues. in view of the impact of the genomic revolution on improving medicines and healthcare, state-of-theart na carriers together with highly specifi c oligonucleotide drugs may help in reducing the side effects of drugs during therapy. 193 delivery systems of nucleic acids drugs drug target insights 2007:2 acknowledgments h.u. is grateful for grant support from fapesp (fundação de amparo à pesquisa do estado de são paulo) and cnpq (conselho nacional de desenvolvimento científico e tecnologico), brazil. r.r.r. ph.d. thesis was supported by coordenação de aperfeiçoamento de pessoal de nível superior (capes), brazil, and his post-doctoral research is currently supported by fapesp. h.a.t. is supported by a fellowship from capes. k.k.y.’s and p.m.’s ph.d.theses are or were supported by fellowships from fapesp. references ambati, j., canakis, c.s., miller, j.w., et al. 2000a. diffusion of high molecular weight compounds through sclera. invest ophthalmol. vis. sci., 41:1181–5. ambati, j., gragoudas, e.s., miller, j.w., et al. 2000b. transscleral delivery of bioactive protein to the choroid and retina. invest ophthalmol vis. sci., 41:1186–91. an, c.i., trinh, v.b. and yokobayashi, y. 2006. artifi cial control of gene expression in mammalian cells by modulating rna interference through aptamer-small molecule interaction. rna, 12:710–6. bagga, s., bracht, j., hunter, s., et al. 2005. regulation by let-7 and lin-4 mirnas results in target mrna degradation. cell, 122:553–63. bartel, d.p. 2004. micrornas: genomics, biogenesis, mechanism, and function. cell, 116:281−97. bernstein, e., caudy, a.a., hammond, s.m., et al. 2001. role for a bidentate ribonuclease in the initiation step of rna interference. nature, 409:363–6. bertrand, j.r., pottier, m., vekris, a., et al. 2002. comparison of antisense oligonucleotides and sirnas in cell culture and in vivo. biochem. biophys. res. commun., 296:1000–4. better, m., bernhard, s.l., fishwild, d.m., et al. 1994. gelonin analogs with engineered cysteine residues form antibody immunoconjugates with unique properties. j. biol. chem., 269:9644–50. blank, m., weinschenk, t., priemer, m., et al. 2001. systematic evolution of a dna aptamer binding to rat brain tumor microvessels. selective targeting of endothelial regulatory protein pigpen. j. biol. chem., 276:16464–8. blind, m., kolanus, w. and famulok, m. 1999. cytoplasmic rna modulators of an inside-out signal-transduction cascade. proc. natl. acad. sci. u.s.a., 96:3606–10. boubriak, o.a., urban, j.p.g., akhtar, k.m., et al. 2000. the effect of hydration and matrix composition on solute diffusion in rabbit sclera. exp. eye res., 71:503–14. bruno, j.g. and kiel, j.l. 1999. in vitro selection of dna aptamers to anthrax spores with electrochemiluminescence detection. biosens. bioelectron., 14:457–64. cao, l., jiao, x., zuzga, d.s., et al. 2004. vegf links hippocampal activity with neurogenesis, learning and memory. nat. genet., 36:827–35. carrasquillo, k.g., carro, j.c., alejandro, a., et al. 2001a. reduction of structural perturbations in bovine serum albumin by non-aqueous microencapsulation. j pharm. pharmacol., 53:115–20. carrasquillo, k.g., costantino, h.r., cordero, r.a., et al. 1999. on the structural preservation of recombinant human growth hormone in a dried fi lm of a synthetic biodegradable polymer. j pharm. sci., 88:166–73. carrasquillo, k.g., ricker, j.a., rigas, i.k., et al. 2003. controlled delivery of the anti-vegf aptamer eye001 with poly(lactic-co-glycolic)acid microspheres. invest ophthalmol vis. sci., 44:290–9. carrasquillo, k.g., stanley, a.m., aponte, j.c., et al. 2001b. non-aqueous encapsulation of excipient-stabilized spray freeze-dried bsa into poly (lactide-co-glycolide) microspheres results in release of native protein. j. control release, 76:199–208. chaloin, l., lehmann, m.j., sczakiel, g., et al. 2002. endogenous expression of a high-affi nity pseudoknot rna aptamer suppresses replication of hiv-1. nucleic acids res., 30:4001–8. chen, y., chen, h., hoffmann, a., et al. 2006 adenovirus-mediated smallinterference rna for in vivo silencing of angiotensin at1a receptors in mouse brain. hypertension, 47, 230–37. choung, s., kim, y.j., kim, s., et al. 2006. chemical modifi cation of sirnas to improve serum stability without loss of effi cacy. biochem. biophys. res. commun., 342:919–27. chu, t.c., marks, j.w., 3rd, lavery, l.a., et al. 2006. aptamer:toxin conjugates that specifi cally target prostate tumor cells. cancer res., 66:5989–92. cory, s. and adams, j.m. 2005. killing cancer cells by fl ipping the bcl2/bax switch. cancer cell, 8:5–6. crooke, s.t. 1998. an overview of progress in antisense therapeutics. antisense nucleic acid. drug dev., 8:115–22. dande, p., prakash, t.p., sioufi , n., et al. 2006. improving rna interference in mammalian cells by 4’-thio-modifi ed small interfering rna (sirna): effect on sirna activity and nuclease stability when used in combination with 2’-o-alkyl modifications. j. med. chem., 49:1624–34. danis, m. 2003. therapeutic advances against malaria in 2003. med. trop., 63:267–70. darfeuille, f., arzumanov, a., gait, m.j., et al. 2002a. 2′-o-methyl-rna hairpins generate loop-loop complexes and selectively inhibit hiv-1 tat-mediated transcription. biochemistry, 41:12186–92. darfeuille, f., arzumanov, a., gryaznov, s., et al. 2002b. loop-loop interaction of hiv-1 tar rna with n3′-->p5′ deoxyphosphoramidate aptamers inhibits in vitro tat-mediated transcription. proc. natl. acad. sci. u.s.a., 99:9709–14. darfeuille, f., hansen, j.b., orum, h., et al. 2004. lna/dna chimeric oligomers mimic rna aptamers targeted to the tar rna element of hiv-1. nucleic acids res., 32:3101–7. dayle, h. 2001. transscleral drug delivery for posterior segment disease. adv. drug deliv. rev., 52:37–48. ducongé, f. and toulmé, j.j. 1999. in vitro selection identifi es key determinants for loop-loop interactions: rna aptamers selective for the tar rna element of hiv-1. rna, 5:1605–14. dyke, c.k., steinhubl, s.r., kleiman, n.s., et al. 2006. first-in-human experience of an antidote-controlled anticoagulant using rna aptamer technology: a phase 1a pharmacodynamic evaluation of a drug-antidote pair for the controlled regulation of factor ixa activity. circulation, 114:2490–7. eckerdt, f., yuan, j. and strebhardt, k. 2005. polo-like kinases and oncogenesis. oncogene, 24:267–76. elbashir, s.m., lendeckel, w. and tuschl, t. 2001a. rna interference is mediated by 21 and 22 nt rnas. genes. dev., 15:188−200. elbashir, s.m., martinez, j., patkaniowska, a., et al. 2001b. functional anatomy of sirnas for mediating effi cient rnai in drosophila melanogaster embryo lysate. embo j, 20:6877–88. famulok, m., blind, m. and mayer, g. 2001. intramers as promising new tools in functional proteomics. chem. biol., 8: 931–9. farokhzad, o.c., cheng, j., teply, b.a., et al. 2006. targeted nanoparticleaptamer bioconjugates for cancer chemotherapy in vivo. proc. natl. acad. sci. u.s.a., 103:6315–20. farokhzad, o.c., jon, s., khademhosseini, a., et al. 2004. nanoparticleaptamer bioconjugates: a new approach for targeting prostate cancer cells. cancer res., 64:7668–72. fire, a., xu, s., montgomery, m.k., et al. 1998. potent and specifi c genetic interference by double-stranded rna in caenorhabditis elegans. nature, 391:806–11. frazza, e.j. and schmitt, e.e. 1971. a new absorbable suture. j biomed. mater res. symp., 1:43–58. 194 ulrich et al drug target insights 2007:2 ge, q., mcmanus, m.t., nguyen, t., et al. 2003. rna interference of infl uenza virus production by directly targeting mrna for degradation and indirectly inhibiting all viral rna transcription. proc. natl. acad. sci. u.s.a., 100:2718–23. geisbert, t.w., hensley, l.e., kagan, e., et al. 2006. postexposure protection of guinea pigs against a lethal ebola virus challenge is conferred by rna interference. j. infect dis., 193:1650–7. giordano, g.g., chevez-barrios, p., refojo, m.f., et al. 1995. biodegradation and tissue reaction to intravitreous biodegradable poly(dl-lactic-coglycolic)acid microspheres. curr. eye res., 14:761–8. giraldez, a.j., cinalli, r.m., glasner, m.e., et al. 2005. micrornas regulate brain morphogenesis in zebrafi sh. science, 308:833–8. good, p.d., krikos, a.j., li, s.x., et al. 1997. expression of small, therapeutic rnas in human cell nuclei. gene. ther., 4:45–54. grzelinski, m., urban-klein, b., martens, t., et al. 2006. rna interferencemediated gene silencing of pleiotrophin through polyethyleniminecomplexed small interfering rnas in vivo exerts antitumoral effects in glioblastoma xenografts. hum. gene. ther., 17:751–66. guo, s., tschammer, n., mohammed, s., et al. 2005. specifi c delivery of therapeutic rnas to cancer cells via the dimerization mechanism of phi29 motor prna. hum. gene. ther., 16:1097–109. hamilton, a.j. and baulcombe, d.c. 1999. a species of small antisense rna in posttranscriptional gene silencing in plants. science, 286:950–2. hammond, s.m., bernstein, e., beach, d., et al. 2000. an rna-directed nuclease mediates post-transcriptional gene silencing in drosophila cells. nature, 404:293–6. harper, s.q., staber, p.d., he, x., et al. 2005. rna interference improves motor and neuropathological abnormalities in a huntington’s disease mouse model. proc. nat.l acad. sci. u.s.a., 102:5820–5. hasirci, v., berthiaume, f., bondre, s.p., et al. 2001. expression of liverspecifi c functions by rat hepatocytes seeded in treated poly(lactic-coglycolic) acid biodegradable foams. tissue eng., 7:385–94. herrmann, c.h. and mancini, m.a. 2001. the cdk9 and cyclin t subunits of tak/p-tefb localize to splicing factor-rich nuclear speckle regions. j. cell sci., 114:1491–503. hess, g.p., ulrich, h., breitinger, h.g., et al. 2000. mechanism-based discovery of ligands that counteract inhibition of the nicotinic acetylcholine receptor by cocaine and mk-801. proc. natl. acad. sci. u.s.a., 97:13895–900. hoshika, s., minakawa, n. and matsuda, a. 2004. synthesis and physical and physiologicalproperties of 4’-thiorna: application to postmodifi cation of rna aptamer toward nf-κb. nucleic. acids. res., 32:3815–3825. hutvágner, g. and zamore, p.d. 2002. a microrna in a multiple-turnover rnai enzyme complex. science, 297:2056−60. hutvágner, g. and zamore, p.d. 2005. rnai: nature abhors a double-strand. curr. opin. genet. dev., 12:225–32. jenison, r.d., gill, s.c., pardi, a., et al. 1994. high-resolution molecular discrimination by rna. science, 263:1425–9. judge, a.d., sood, v., shaw, j.r., et al. 2005. sequence-dependent stimulation of the mammalian innate immune response by synthetic sirna. nat. biotechnol., 23:457–62. kamei, m., misono, k. and lewis, h. 1999. a study of the ability of tissue plasminogen activator to diffuse into the subretinal space after intravitreal injection in rabbits. am. j. ophthalmol., 128:739–46. khaled, a., guo, s., li, f. and guo, p. 2005. controllable self-assembly of nanoparticles for specifi c delivery of multiple therapeutic molecules to cancer cells using rna nanotechnology. nano. lett., 5:1797–808. kim, w.j., christensen, l.v., jo, s., et al. 2006. cholesteryl oligoarginine delivering vascular endothelial growth factor sirna effectively inhibits tumor growth in colon adenocarcinoma. mol. ther., 14:343–50. kolb, g., reigadas, s., boiziau, c., et al. 2005. hexitol nucleic acidcontaining aptamers are effi cient ligands of hiv-1 tar rna. biochemistry, 44:2926–33. konopka, k., duzgunes, n., rossi, j., et al. 1998. receptor ligand-facilitated cationic liposome delivery of anti-hiv-1 rev-binding aptamer and ribozyme dnas. j. drug target, 5:247–59. lam, t.t., fu, j., tso, m.o., et al. 1991. a histopathologic study of retinal lesions infl icted by transscleral iontophoresis. graefes arch. clin. exp. ophthalmol, 229:389–94. lang, j.c. 1995. ocular drug delivery: conventional ocular formulations. adv. drug deliv. rev., 16:39–43. langer, r. 1998. drug delivery and targeting. nature, 392:5–10. li, b.j., tang, q., cheng, d., et al. 2005. using sirna in prophylactic and therapeutic regimens against sars coronavirus in rhesus macaque. nat. med., 11:944–51. lorenz, c., hadwiger, p., john, m., et al. 2004. steroid and lipid conjugates of sirnas to enhance cellular uptake and gene silencing in liver cells. bioorg. med. chem. lett., 14:4975–7. lupold, s.e., hicke, b.j., lin, y., et al. 2002. identifi cation and characterization of nuclease-stabilized rna molecules that bind human prostate cancer cells via the prostate-specifi c membrane antigen. cancer res., 62:4029–33. ma, p.x. and choi, j.w. 2001. biodegradable polymer scaffolds with welldefi ned interconnected spherical pore network. tissue eng., 7:23–33. mallery, s.r., kang, p.p., ness, g.m., et al. 2000. controlled-release of doxorubicin from poly(lactic-co-glycolic) microspheres signifi cantly enhances cytotoxicity against cultured aids-related kaposi’s sarcoma cells. anticancer res., 20:2817–25. martinez, m.a., gutierrez, a., armand-ugon, m., et al. 2002. suppression of chemokine receptor expression by rna interference allows for inhibition of hiv-1 replication. aids, 16:2385–90. mcmanus, m.t. and sharp, p.a. 2002. gene silencing in mammals by small interfering rnas. nat. rev. genet., 3:737–47. mcnamara, j.o., 2nd, andrechek, e.r., wang, y., et al. 2006. cell typespecifi c delivery of sirnas with aptamer-sirna chimeras. nat. biotechnol., 24:1005–15. meister, g., landthaler, m., patkaniowska, a., et al. 2004. human argonaute2 mediates rna cleavage tarfeted by mirnas and sirnas. mol. cell, 15:185–97. miller, v.m., paulson, h.l. and gonzalez-alegre, p. 2005. rna interference in neuroscience: progress and challenges. cell mol. neurobiol., 25:1195–207. minakuchi, y., takeshita, f., kosaka, n., et al. 2004. atelocollagen-mediated synthetic small interfering rna delivery for effective gene silencing in vitro and in vivo. nucleic acids res., 32:e109. moritera, t., ogura, y., honda, y., et al. 1991. microspheres of biodegradable polymers as a drug-delivery system in the vitreous. invest ophthalmol. vis. sci., 32:1785–90. morrissey, d.v., lockridge, j.a., shaw, l., et al. 2005. potent and persistent in vivo anti-hbv activity of chemically modifi ed sirnas. nat. biotechnol., 23:1002–7. murchison, e.p., partridge, j.f., tam, o.h., et al. 2005. characterization of dicer-defi cient murine embryonic stem cells. proc. natl. acad. sci. u.s.a., 102:12135–40. nakano, h., amemiya, s., shiokawa, k., et al. 2000. rna interference for the organizer-specifi c gene xlim-1 in xenopus embryos. biochem. biophys. res. commun., 274:434–9. ng, e.w., shima, d.t., calias, p., et al. 2006. pegaptanib, a targeted antivegf aptamer for ocular vascular disease. nat. rev. drug. discov., 5:123–32. niu, x.y., peng, z.l., duan, w.q., et al. 2006. inhibition of hpv 16 e6 oncogene expression by rna interference in vitro and in vivo. int. j. gynecol. cancer, 16, 743–51. nykänen, a., haley, b. and zamore, p.d. 2001. atp requirements and small interfering rna structure in the rna interference pathway. cell, 107:309–21. olsen, p.h. and ambros, v. 1999. the lin-4 regulatory rna controls developmental timing in caenorhabditis elegans by blocking lin-14 protein synthesis after the initiation of translation. dev. biol., 216:671–80. 195 delivery systems of nucleic acids drugs drug target insights 2007:2 olsen, t.w., edelhauser, h.f., lim, j.i., et al. 1995. human scleral permeability. invest ophthalmol. vis. sci., 36:1893–903. olsen, t.w., aaberg, s.y., geroski, d.h., et al. 1998. human sclera: thickness and surface area. am. j. ophthalmol., 125:237–41. patil, s.d., rhodes, d.g. and burgess, d.j. 2005. dna-based therapeutics and dna delivery systems: a comprehensive review. aaps j, 7: e61–77. perry, c.m., balfour, j.a., fomivirsen. 1999. drugs, 57:375–80. rajasekaran, a.k., anilkumar, g. and christiansen, j.j. 2005. is prostatespecifi c membrane antigen a multifunctional protein? am. j. physiol. cell physiol., 288:c975–81. ralph, g.s., radcliffe, p.a., day, d.m., et al. 2005. silencing mutant sod1 using rnai protects against neurodegeneration and extends survival in an als model. nat. med., 11:429–33. raoul, c., abbas-terki, t., bensadoun, j.c., et al. 2005. lentiviral-mediated silencing of sod1 through rna interference retards disease onset and progression in a mouse model of als. nat. med., 11:423–8. reagan-shaw, s. and ahmad, n. 2005. silencing of polo-like kinase (plk) 1 via sirna causes induction of apoptosis and impairment of mitosis machinery in human prostate cancer cells: implications for the treatment of prostate cancer. faseb j, 19:611–3. reinhart, b.j., slack, f.j., basson, m., et al. 2000. the 21-nucleotide let-7 rna regulates developmental timing in caenorhabditis elegans. nature, 403:901–6. rhie, a., kirby, l., sayer, n., et al. 2003. characterization of 2’-fl uororna aptamers that bind preferentially to disease-associated conformations of prion protein and inhibit conversion. j. biol. chem., 278:39697–705. richter, s., cao, h. and rana, t.m. 2002. specific hiv-1 tar rna loop sequence and functional groups are required for human cyclin t1-tat-tar ternary complex formation. biochemistry, 41:6391–7. rodriguez-lebron, e. and paulson, h.l. 2006. allele-specifi c rna interference for neurological disease. gene. ther., 13:576–81. rosenblum, m.g., cheung, l.h., liu, y., et al. 2003. design, expression, purifi cation, and characterization, in vitro and in vivo, of an antimelanoma single-chain fv antibody fused to the toxin gelonin. cancer res., 63:3995–4002. rosenblum, m.g., marks, j.w. and cheung, l.h. 1999. comparative cytotoxicity and pharmacokinetics of antimelanoma immunotoxins containing either natural or recombinant gelonin. cancer chemother. pharmacol., 44:343–8. rusconi, c.p., roberts, j.d., nimjee, s.m., et al. 2004. antidote-mediated control of an anticoagulant aptamer in vivo. nat. biotechnol., 22:1423–8. sa, d.w.y. 2004. therapeutic potential of rna interference for neurological disorders. life sci., 79:1773–80. scherr, m., morgan, m.a. and eder, m. 2003. gene silencing mediated by small interfering rnas in mammalian cells. curr. med. chem., 10:245–56. seggerson, k., tang, l. and moss, e.g. 2002. two genetic circuits repress the caenorhabditis elegans heterochronic gene lin-28 after translation initiation. dev. biol., 243:215−25. shi, h., fan, x., sevilimedu, a. and lis, j.t. 2007. rna aptamers directed to discrete functional sites on a single protein structural domain. proc. natl. acad. sci. u.s.a. 104:3742–6. shi, h., hoffman, b.e. and lis, j.t. 1999. rna aptamers as effective protein antagonists in a multicellular organism. proc. natl. acad. sci. u.s.a., 96:10033–8. singer, o., marr, r.a., rockenstein, e., et al. 2005. targeting bace1 with sirnas ameliorates alzheimer disease neuropathology in a transgenic model. nat. neurosci., 8:1343–9. song, e., lee, s.k., wang, j., et al. 2003. rna interference targeting fas protects mice from fulminant hepatitis. nat. med., 9:347–51. song, e., zhu, p., lee, s.k., et al. 2005. antibody mediated in vivo delivery of small interfering rnas via cell-surface receptors. nat. biotechnol., 3:709–17. soutschek, j., akinc, a., bramlage, b., et al. 2004. therapeutic silencing of an endogenous gene by systemic administration of modifi ed sirnas. nature, 11;432:173–8. stull, r.a., szoka fc jr. 1995. antigene, ribozyme and aptamer nucleic acid drugs: progress and prospects. pharm. res., 12:465–83. sverdlov, e.d. 2001. rna interferencea novel mechanism of regulation of gene expression and a novel method of study of their functions. russian journal of bioorganic chemistry, 27:209–12. takai, n., ueda, t., nishida, m., et al. 2004. the relationship between oncogene expression and clinical outcome in endometrial carcinoma. curr. cancer drug targets, 4:511–20. takei, y., kadomatsu, k., yuzawa, y., et al. 2004. a small interfering rna targeting vascular endothelial growth factor as cancer therapeutics. cancer res., 64:3365–70. tang, j., breaker, r.r. 1997. rational design of allosteric ribozymes. chem. biol., 4:453–9. theis, m.g., knorre, a., kellersch, b., et al. 2004. discriminatory aptamer reveals serum response element transcription regulated by cytohesin-2. proc. natl. acad. sci. u.s.a., 101:11221–6. toulmé jj, darfeuille, f., kolb, g., et al. 2003. modulating viral gene expression by aptamers to rna structures. biol. cell, 95: 229–38. toulmé jj, di primo, c., moreau, s. 2001. modulation of rna function by oligonucleotides recognizing rna structure. prog. nucleic acid res. mol. biol., 69:1–46. turner, j.j., ivanova, g.d., verbeure, b., et al. 2005. cell-penetrating peptide conjugates of peptide nucleic acids (pna) as inhibitors of hiv-1 tatdependent trans-activation in cells. nucleic acids res., 33:6837–49. tuschl, t., zamore, p.d., lehmann, r., et al. 1999. targeted mrna degradation by double-stranded rna in vitro. genes dev., 13:3191–7. ulrich, h., ippolito, j.e., pagan, o.r., et al. 1998. in vitro selection of rna molecules that displace cocaine from the membrane-bound nicotinic acetylcholine receptor. proc. natl. acad. sci. u.s.a., 95:14051–6. ulrich, h., magdesian, m.h., alves, m.j. and colli, w. 2002. in vitro selection of rna aptamers that bind to cell adhesion receptors of trypanosoma cruzi and inhibit cell invasion. j. biol. chem., 277:20756–62. ulrich, h., martins, a.h.b. and pesquero, j.b. 2004. rna and dna aptamers in cytomics analysis. cytometry, 59a:220–31. ulrich, h., trujillo, c.a., nery, a.a., et al. 2006. dna and rna aptamers: from tools for basic research towards therapeutic applications. comb. chem. high throughput screen, 9:619–32. vavvas, d. and d’amico, d.j. 2006. pegaptanib (macugen): treating neovascular age-related macular degeneration and current role in clinical practice. ophthalmol clin. north am., 19:353–60. veenendaal, l.m., jin, h., ran, s., et al. 2002. in vitro and in vivo studies of a vegf121/rgelonin chimeric fusion toxin targeting the neovasculature of solid tumors. proc. natl. acad. sci. u.s.a., 99:7866–71. vornlocher, h.p., zimmermann, t.s., manoharan, m., et al.2005 a. nuclease-resistant double-stranded rna for rna interference. patent cooperation treaty international application wo2005115481. weiner, l.m. and adams, g.p. 2000. new approaches to antibody therapy. oncogene, 19:6144–51. werstuck, g. and green, m.r. 1998. controlling gene expression in living cells through small molecule-rna interactions. science, 282:296–8. wianny, f. and zernicka-goetz, m. 2000. specifi c interference with gene function by double-stranded rna in early mouse development. nat. cell biol., 2:70–5. wightman, b., ha, i. and ruykun, g. 1993. posttranscriptional regulation of the heterochronic gene lin-14 mediates temporal pattern formation in c. elegans. cell, 75:855–62. wu, a.m. and senter, p.d. 2005. arming antibodies: prospects and challenges for immunoconjugates. nat. biotechnol., 23:1137–46. wu, l., fan, j. and belasco, j.g. 2006. micrornas direct rapid deadenylation of mrna. proc. natl. acad. sci. u.s.a., 103:4034–9. xia, h., mao, q., eliason, s.l., et al. 2004. rnai suppresses polyglutamineinduced neurodegeneration in a model of spinocerebellar ataxia. nat. med., 10:816–20. 196 ulrich et al drug target insights 2007:2 yano, j., hirabayashi, k., nakagawa, s., et al. 2004. antitumor activity of small interfering rna/cationic liposome complex in mouse models of cancer. clin. cancer res., 10:7721–6. yekta, s., shih, i.h. and bartel, d.p. 2004. microrna-directed cleavage of hoxb8 mrna. science, 304:594–6. yoshinari, k., miyagishi, m. and taira, k. 2004. effects on rnai of the tight structure, sequence and position of the targeted region. nucleic acids res., 32:691–9. zamore, p.d., tuschl, t., sharp, p.a., et al. 2000. rnai: double-stranded rna directs the atp-dependent cleavage of mrna at 21 to 23 nucleotide intervals. cell, 101:25–33. zhang, y., zhang, y.f., bryant j et al. 2004. intravenous rna interference gene therapy targeting the human epidermal growth factor receptor prolongs survival in intracranial brain cancer. clin. cancer. res., 10:3667–77. zimmermann, g.r., jenison, r.d., wick, c.l., et al. 1997. interlocking structural motifs mediate molecular discrimination by a theophyllinebinding rna. nat. struct. biol., 4:644–9. zimmermann, t.s., lee, a.c., akinc, a., et al. 2006. rnai-mediated 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drug target insights 2007:2 229–237 229 review correspondence: bodil ohlsson, entrance 35, 205 02 malmö, sweden. tel: +46 40 33 10 00; fax: +46 40 33 62 08; email: bodil.ohlsson@med.lu.se copyright in this article, its metadata, and any supplementary data is held by its author or authors. it is published under the creative commons attribution by licence. for further information go to: http://creativecommons.org/licenses/by/3.0/. new insights into the understanding of gastrointestinal dysmotility bodil ohlsson1 and sabina janciauskiene2 department of clinical sciences, gastroenterology division1, wallenberg laboratory2, entrance 46, 2nd floor, university hospital malmö, lund university, 20502 malmö, sweden. abstract: our understanding of the physiology of digestion, absorption, secretion, and motility in the gastrointestinal tract has improved immensely. today it is well established that the gross functions of the gastrointestinal tract depend on the coordination between the muscles, nerves and hormones. the enteric nervous system (ens) is involved in most of the physiological and pathophysiological processes in the gastrointestinal tract. therefore, clinical and experimental studies on the ens provide the basis for a better understanding of the mechanisms involved in gastrointestinal disorders and promote the development of therapeutic options. this review outlines some of the current views on the role of the ens and its related hormones in gastrointestinal motility. keywords: gonadotropin-releasing hormone (gnrh), oxytocin, chronic intestinal pseudo-obstruction (cipo), apoptosis introduction gastrointestinal motility is a term used to describe the contraction of the muscles in the gastrointestinal tract. the normal motility of the gastrointestinal tract is dependent on the function of the enteric nervous system (ens), the smooth muscle layers, and the interstitial cells of cajal (iccs) (fig. 1) (goyal and hirano, 1996; wood, 2000). diseases characterised by gastrointestinal dysmotility are highly prevalent conditions. for instance, the mild form of dysmotility, also called irritable bowel syndrome (ibs), is assumed to affect almost 15%–20% of the population (gwee, 2005). chronic intestinal pseudo-obstruction (cipo) is the most diffi cult of these clinical challenges, characterised by the presence of chronic dysmotility and intestinal dilatation in the absence of mechanical obstruction (de giorgio et al. 2004a). the pathogenesis of ibs and cipo remains unclear, although putative mechanisms, including infl ammation, altered calcium signalling, mitochondrial dysfunction, free radical production, and others, may contribute to the degeneration and loss of enteric neurons (hall and wiley, 1998; spiller, 2003). over recent years much effort has been made to try to improve the motility and decrease the abdominal pain occasioned by these conditions by targeting enteric peptides and their receptors. one example is motilin, an important peptide in digestive motility whose receptor is the site of action for erythromycin in the treatment of gastro paresis (galligan and vanner, 2005). the peptide ghrelin, related to motilin, which originates primarily in the stomach (möller et al. 2003), has recently also been shown to enhance gastric emptying in idiopathic gastro paresis (tack et al. 2005). serotonin and its receptor have been the goal for an intensive effort to develop different agonists and antagonists (johanson, 2004; cash and chey, 2005; wessinger et al. 2005). so far, these attempts have not been very successful. recent fi ndings support a new concept that alterations in intracellular mechanisms of neuronal survival might play a crucial role in the degeneration of the enteric nervous system (de giorgio et al. 2000; bassotti et al. 2006). furthermore, two newly discovered peptides localized in the human gastrointestinal tract, oxytocin and gonadotropin releasing hormone (gnrh) seem to play crucial roles in the regulation of gastrointestinal dysmotility (monstein et al. 2004; ohlsson et al. 2006a; ohlsson et al. 2007). oxytocin is known to enhance gastric emptying (hashmonai et al. 1979; petring, 1989). our own studies have shown that oxytocin is expressed in the myenteric and submucous ganglia and nerve fi bres along the entire human gastrointestinal tract (monstein et al. 2004; ohlsson et al. 2006b), and that it increases colonic peristalsis while the receptor antagonist delays the gastric emptying rate (ohlsson et al. 2004; ohlsson et al. 2006a). similarly, gnrh has also been shown to stimulate intestinal motor http://creativecommons.org/licenses/by/3.0/. 230 ohlsson and janciauskiene drug target insights 2007:2 activity in rat (khanna et al. 1992; ducker et al. 1996), and a loss of gnrh-containing neurons in the ens has been related to cipo development (ohlsson et al. 2007). the enteric nervous system and gastrointestinal dysmotility enteric neuron apoptosis the ens is a highly integrated neural system which consists of distinct subclasses of enteric neurons localized within the wall of the alimentary tract throughout its entire length. the ens closely resembles the central nervous system (gershon et al. 1994), and has a unique ability to control virtually all gut functions, including motility, independently of the central nervous system (cns) (furness et al. 2005). remarkably, in response to different types of stimuli/conditions, enteric neurons are able to change their structural, functional and chemical phenotype (lomax et al. 2005). these changes in the functional and/or structural integrity of the ens may occur as a consequence of normal aging or due to pathologies ranging from enteric neuropathies enteric nervous system neurones iccs glial cells hormones and neurotransmitters trh gnrh oxytocin glp 1 cck gip secreto neurin crh ghrelin motilin pp vip serotonin figure 1. a schematic overview over the enteric nervous system and its most important peptides according to motility. cck = cholecystokinin; crh = corticotrophin-releasing hormone; iccs = interstitiasl cells of cajal; gip = gastric inhibitory polypeptide; glp-1 = glucagon-like peptide 1; gnrh = gonadotropin-releasing hormone; pp = pancreatic polypeptide; trh = thyrotropin-releasing hormone; vip = vasoactive intestinal peptide. 231 gastroinestinal motility drug target insights 2007:2 (i.e. hirschsprung’s disease) to intestinal or extra intestinal diseases (i.e. ulcerative colitis and crohn`s disease, amyloidosis, scleroderma and etc) (di lorenzo, 1999; de giorgio and camilleri, 2004b; de giorgio et al. 2004c). it has been suggested that some motility disorders originate from developmental defects, i.e. hirschsprung’s disease (kim et al. 2006), whereas others are due to neurodegeneration. in fact, pathological changes of the ens are often accompanied by nerve process degeneration and necrosis (dvorak et al. 1993). ultra structural evaluation of tissue specimens from patients with crohn’s disease and ulcerative colitis have shown swollen, empty axons, fi lled with large vacuoles, swollen mitochondria and concentrated neurofi brils (vasina et al. 2006). the b-cell leukaemia/lymphoma-2 (bcl-2) protein has the functional role of blocking apoptosis, i.e. programmed cell death. this protein is widely expressed in the developing central and peripheral nervous systems. the expression of bcl-2 is also displayed in enteric neurons (wester et al. 1999). the reduced expression of bcl-2 has been demonstrated in degenerative disorders of both the central nervous system and ens (merry and korsmeyer, 1997). thus, the current state of knowledge allows speculations that alterations in the intracellular mechanisms involved in neuronal survival may play a critical role in various gastrointestinal motility disorders. to support of this postulation, the decreased expression of the bcl-2 protein in enteric neurons has been demonstrated in patients with severe forms of cipo (de giorgio et al. 2000; de giorgio et al. 2004a; de giorgio and camilleri, 2004b). we found further support when, examining full thickness biopsies, we noticed signifi cantly lower bcl-2 expression in a patient with cipo than in controls. in parallel, histological examination revealed the presence of swollen or shrunken neurons of the myenteric plexus with or without vacuolisation of the cytoplasm (ohlsson et al. 2007). the increased number of apoptotic enteric neurons and decreased expression of bcl-2 have also been found in patients with slow-transit constipation (bassotti et al. 2006) and in patients with hirschsprung’s disease (song et al. 2002). thus, the improved knowledge on the changes and regulation in proteins associated with apoptosis in the cells of ens may be crucial for regulating the apoptosis program. a number of animal models such as a rat hemispheric ischemia/reperfusion model (gabryel et al. 2006) and an apoptosisdependent emphysema mouse model (petrache et al. 2006), as well as age-related macular degeneration (glotin et al. 2006) have demonstrated that substances controlling intracellular pathways of cell apoptosis can reduce disease processes characterised by excessive cell apoptosis. therefore, we believe that clinical and experimental studies on the role of enteric neuronal apoptosis are of fundamental importance because they may improve our understanding regarding the pathophysiology of gastrointestinal dysmotility and may also provide the basis for new therapeutic approaches. enteric glial cells the ens is composed of both neurons and enteric glial cells, which play a central role in sustaining the structural and functional integrity of enteric neurons. enteric glial cells were fi rst described by dogiel in 1899 as nucleated satellite cells accompanying enteric neuronal cells. dogiel assumed that enteric glia represented a kind of connective tissue, and consequently very little research was conducted to reveal their functions (dogiel, 1889). today, there is evidence from transgenic animal models that enteric glial cells are essential for gastrointestinal integrity and function, but still little is known about the underlying mechanisms. for example, in genetically modifi ed animals, loss of enteric glia results in neuronal degeneration and changes in the neurochemical coding of enteric neurons (bush et al. 1998; aube et al. 2003). new data suggest that enteric glial cells have an important role in maintaining the integrity of the mucosal barrier of the gut, and may also serve as a link between the nervous and immune systems of the gut as indicated by their potential to synthesize cytokines, present antigens and respond to infl ammatory insults. the role of enteric glia in human disease has not yet been systematically studied, but, based on the evidence available it can be predicted that enteric glia are involved in the aetiopathogenesis of various pathological processes in the gut, particularly those with neuroinfl ammatory or neurodegenerative components (ruhl, 2005). the number of glia cells has been shown to increase in response to pro-infl ammatory cytokines, such as interleukin-1 (il-1) and tumour necrose factor alpha (tnfα), or lipopolysaccharide (von boyen et al. 2004). notably, several studies have described 232 ohlsson and janciauskiene drug target insights 2007:2 an association between increased glial cell proliferation and neuronal disintegration in patients with infl ammatory bowel diseases (cabarrocas et al. 2003; lomax et al. 2005). in another study, examination of non-involved intestinal tissue from patients with crohn’s disease, ulcerative colitis, or histological normal controls demonstrated that the enteric glia cell network was signifi cantly disrupted in crohn’s disease, but not in ulcerative colitis (cornet et al. 2001). in patients with slow transit constipation a remarkable decrease both in the number of enteric neurons and interstitial cells of cajal (iccs), and also in the number of glial cells has been found. these patients had signifi cantly more apoptotic enteric neurons than controls (bassotti et al. 2006). it is likely that a dynamic equilibrium between enteric neurons and glia plays an important role in vivo. therefore, the insufficient support of enteric neurons by glial cells may lead to enhanced neuronal apoptosis and neurodegeneration, characteristic features of gastrointestinal dysmotility disorders such as idiopathic chronic constipation, ibs and cipo (törnblom et al. 2002; de giorgio et al. 2004a; de giorgio and camilleri, 2004b; de giorgio et al. 2004c; bassotti et al. 2006). interstitial cells of cajal (iccs) interstitial cells of cajal (iccs) were originally described in the gut more than a century ago by ramóny cajal (he et al. 2001). iccs are a unique class of mesenchymal cells found in the gastrointestinal tract of mammals. in the region of the gastric corpus and antrum, multipolar iccs form two-dimensional networks, and have been mistaken for neurons, glial cells, smooth muscle cells, macrophages and fi broblasts. iccs are the pacemaker cells responsible both for initiating slow wave activity in gastrointestinal muscles and for the active propagation of the electrical slow waves (thomsen et al. 1998). iccs can be recognised either by their characteristic ultra structure by electron microscopy or by the immunohistochemical demonstration of their surface receptor tyrosine kinase kit. recent studies demonstrated that the c-kit receptor is essential for the development of iccs. mesenchymal icc precursors that carry the c-kit receptor require the kit ligand, which can be provided by neuronal cells or smooth muscle cells. accordingly the iccs develop as either myenteric or muscular iccs (wu et al. 2000). the evidence from experimental models and human diseases increasingly point to a central role of iccs in the aetiology of human gastrointestinal dysmotility. many gastrointestinal motor disorders like gastro paresis, abnormal small bowel motility, infl ammatory bowel disease, cipo, gastrointestinal stromal and multiple autonomic tumours, achalasia and hirschsprung’s disease show a changed number and/or structure of iccs (he et al. 2000; sanders et al. 1999; hagger et al. 2000). gastro paresis is associated with electrical abnormalities, and deviations from normal slowwave rhythm (dysrhythmias) have been reported to result in delayed gastric emptying. in a diabetic rat model it has been demonstrated that degeneration of iccs is responsible for these gastroelectrical dysrhythmias (ordög et al. 2000). therefore, the identifi cation of abnormalities in iccs which are linked to specifi c gastrointestinal motor disorders should be taken more into focus in the future. newly discovered peptides in the enteric nervous system (ens) a wide range of peptides are described as having a decreased expression in dysmotility. it is not known whether these down-regulated peptides are primary or secondary to development of the disease (krischnamurthy and schuffl er, 1993; de giorgio and camilleri, 2004b). gut peptides exert diverse effects, regulating gastrointestinal motility and acid secretion, epithelial integrity, and both nutrient absorption and disposal. these actions are initiated by the activation of specifi c g protein-coupled receptors and may be mediated by direct or indirect effects on target cells (kutchai, 2004). more recent evidence demonstrates that gut peptides, such as glucagon-like peptides-1 and 2, also directly regulate signalling pathways coupled to cell proliferation and apoptosis (drucker, 2003). a number of signalling pathways between mesenchymal and neural crest cells are required for the development of the ens (natarajan et al. 2002). these signalling pathways involve peptides secreted by intestinal mesenchymal cells such as endothelin-3, glial cell line-derived neurotrophic factor (gdnf), neuroturin, neurotrophin-3 (nt-3), and netrin-1 (chalazonitis et al. 1998; young et al. 2004; nagy and goldstein, 2006). the presence of both motilin and ghrelin in guinea-pig myenteric neurons is suggested to play a role in the 233 gastroinestinal motility drug target insights 2007:2 activation of the ens and hence in the regulation of gastrointestinal motility (xu et al. 2006), which is further supported by a close relationship between ghrelin and gastric motility in rats (masuda et al. 2000). the fi ndings that patients with functional dyspepsia (fd) have altered plasma profi le of ghrelin suggest a possible role for this peptide in the pathophysiology of fd (takamori, 2006). obestatin, a newly discovered ghrelin-associated peptide, was initially suggested to decrease gastric emptying (zhang, 2005). unfortunately, recent studies have not been able to confi rm these results, and existing reports do not support obestatin as a regulator of digestive motility (gourcerol and taché, 2007). serotonin is a biochemical neurotransmitter, found primarily in the cns, gastrointestinal tract, and blood platelets (vialli, 1966). the bowel exhibits refl exes in the absence of cns input. to do so, epithelial sensory transducers, such as enterochromaffi n (ec) cells, activate the mucosal processes of intrinsic and extrinsic primary afferent (sensory) neurons by secretion of serotonin (5-ht) in response to mucosal stimuli (gershon, 2005). the enteric serotonin reuptake transporter has been proposed to play a critical role in serotonergic neurotransmision and in the initiation of peristaltic and secretory refl exes (chen et al. 2001). the current knowledge suggests that serotonin initiates peristaltic and secretory refl exes because of its ability to stimulate secretion of acetylcholine (ach) and calcitonin gene related peptide (cgrp) (pan et al. 1994; sidhu et al. 1995; grider, 1994, 2003). these afferent refl ex pathways also lead to perceptions of nausea, and discomfort and pain from the gastrointestinal tract (grundy, 2002). serotonin is thus implicated in the pathology of irritable bowel syndrome (ibs), which is characterised by visceral hypersensitivity and altered motility (simrén et al. 2003; costedio et al. 2006). multiple receptor families explain the broad physiological actions and distribution of serotonin, therefore, many agonists and antagonists to the serotonin receptors have been developed and clinically used. so far, no one has given successful results in the treatment of ibs (mclean et al. 2006). the neuropeptide vasoactive intestinal peptide (vip) is the most important peptidergic transmitter in intestinal relaxation, which regulates smooth muscleand epithelial function. for the fi rst time, vip/pituitary adenylate cyclase activating peptide (pacap) receptors have been detected in the human gastrointestinal tract by the use of specifi c antibodies (rettenbacher and reubi, 2001). observed correlation between delayed gastrointestinal transit and an increase of vip neurons in a rat ischemia/reperfusion model suggests that changes in enteric transmitters might contribute to gastrointestinal dysmotility (calcina et al. 2005). secretoneurin is a functional neuropeptide derived from secretogranin ii (chromogranin c). both in the myenteric and submucous plexuses, nerve fi bres and the majority of ganglion cells were found to be secretoneurin-immunoreactive. thus, secretoneurin is a new major peptide within the human enteric neuroendocrine system. its abundant presence in myenteric ganglion cells may imply a role in the modulation of gastrointestinal motility. the chemotactic properties of secretoneurin and its possible localization in sensory fi bres suggest that this peptide may be involved in the genesis of intestinal infl ammation (schurmann et al. 1995). oxytocin oxytocin is a hormone with its most well-known effects on myoepithelial cells of the breast during lactation and the uterine contractions during parturition. oxytocin is detected not only in plasma but also in almost all segments of the gastrointestinal tract (monstein et al. 2004). the indirect immunofl uorescence approach has shown that oxytocin is expressed in myenteric and submucous ganglia, suggesting that it is important for both gastrointestinal sensitivity and motility (ohlsson et al. 2006b). oxytocin is released into plasma in response to a meal (ohlsson et al. 2002), and has been shown to stimulate gastric emptying (hashmoni et al. 1979; petring, 1989) and colonic peristalsis (ohlsson et al. 2004). in addition, inhibition of the binding of endogenous oxytocin by the receptor antagonist atosiban delayed gastric emptying (ohlsson et al. 2006a). the prokinetic effect of oxytocin on the gastrointestinal tract is speculated to be similar to the one in uterine myometrium and mammary myoepitheal cells; intracellular release of ca2+ which leads to muscle contraction via myosin light kinase activity (gimpl and fahrenholz, 2001). a woman with chronic gastro paresis demanding continuous treatment with prokinetic drugs, was completely out of symptoms during pregnancy and breast feeding, and could stop medicamentation 234 ohlsson and janciauskiene drug target insights 2007:2 every time she was pregnant (ohlsson, 2006c). although the mechanism behind this phenomenon is not proven, these states are characterised by elevated oxytocin levels in plasma (chiodera et al. 1991, silber et al. 1991), and together with other observations mentioned above, one may speculate whether oxytocin defi ciency may be the aetiology to the gastro paresis in this woman (ohlsson, 2006c). despite the stimulatory effect of oxytocin on peristalsis, treatment with nasally administered oxytocin did not improve the stool habits in women with refractory constipation (ohlsson et al. 2005). oxytocin is also known to have analgesic effects (petersson et al. 1996), and its plasma levels are found to be decreased in patients suffering from dyspepsia and ibs, conditions characterised by abdominal pain and discomfort (uvnäs-moberg et al. 1991). furthermore, children suffering from recurrent abdominal pain exhibit lower plasma levels of oxytocin than healthy controls (alfven, 2004). interestingly, both depression and fi bromylagia are associated with ibs (lydiard et al. 1993; sperber et al. 1999), and both of these conditions are also characterised by low plasma levels of oxytocin (frash et al. 1995; anderberg and uvnäsmoberg, 2000). accordingly, treatment of ibs patients with intravenously (louvel et al. 1996) or nasally (ohlsson et al. 2005) administered oxytocin resulted in the reduction of abdominal pain and reduced depression. the questions remain as to whether oxytocin could be used clinically to improve the suffering of patients with ibs and cipo by reducing their pain and their depressive mood rather than by attempting to improve motility. further randomised clinical trials are needed to answer these questions. gonadotropin releasing hormone (gnrh) the central core of the hypothalamic-pituitarygonadal axis, in all vertebrate species, is the group of neurons that produce and secrete gonadotropinreleasing hormone (gnrh). over the past 20 years, techniques have become available to identify the gnrh-producing neurons and measure both gnrh content and levels of gnrh mrna in brain tissue. indeed, several types of differentiated lymphocytes, including spleenocytes, thymocytes, peripheral tand b-lymphocytes, and mast cells have been demonstrated to produce gnrh or a gnrh-like peptide (marchetti et al. 1996). although it is not known whether different forms of gnrh might have different receptor types, gnrh receptors have been found throughout the human body (fekete et al. 1989; kakar and jennes, 1995), but have not been studied in the gastrointestinal tract. in rats, gnrh mrna has been found in parietal cells of gastric glands, the epithelium of the small and large intestine, and in parasympathetic ganglion cells of the myenteric plexus. in addition, the gnrh receptor has been found in the epithelium of gastric pits (huang et al. 2001) and gnrh receptor mrna in the myenteric neurons in the rat (ho et al. 1996). gnrh has also been detected in rat pancreas (wang et al. 2001). in the dog, gnrh has been shown to inhibit the release of gastric secretions and gastrin release (soldani et al. 1982), possibly due to diminished vagal activity. apart from its effects on reproduction, these fi ndings suggest a role for gnrh also in the regulation of the gastrointestinal tract. accordingly, the gnrh analogue leuprolide (pglu-histrp-ser-tyr-dleu-arg-pro-etnh2) has been shown to stimulate intestinal motor activity in rats (khanna et al. 1992; ducker et al. 1996). furthermore, symptom resolution and alleviation of intestinal motility abnormality after treatment with leuprolide have been reported in a patient with cipo (mathias et al. 1992). in a study of the effect of leuprolide in the treatment of ibs, the overall symptom score was improved, but the greatest therapeutic effect was seen on abdominal pain and nausea (mathias et al. 1994a; mathias et al. 1998). this effect persisted when the treatment was continued for up to 6–12 months (mathias et al. 1994b; palomba et al. 2005). other gnrh analogues, such as buserelin, are used in the treatment of in vitro fertilization (ivf), endometriosis, polycystic ovary syndrome, prostate cancer, uterine leiomyoma and precocious puberty. gastrointestinal side effects are considered infrequent (who) but nausea and abdominal pain have been reported in 7%–17% of women treated with buserelin for endometriosis and uterine leiomyoma (fass, micromedex). the aetiology to these side effects is not known. recently we demonstrated for the fi rst time gnrh positive neurons in the human gastrointestinal tract and have shown a decreased number of gnrh positive neurons in a cipo patient. the patient, who had been treated with buserelin, 235 gastroinestinal motility drug target insights 2007:2 developed cipo with pronounced abdominal pain and nausea/vomiting. remarkably, immunohistochemical analysis of intestinal resects revealed that in the patient only 3% of myenteric neurons are gnrh positive as compared to 53% in controls (ohlsson et al. 2007). the patient had high plasma titres of anti-gnrh antibodies that correlated with the occasions of the treatment with buserelin. the latter led us to the hypothesis that the patient developed cipo due to buserelin-induced formation of anti-gnrh antibodies which destroyed gnrhproducing neurons of the myenteric plexus. we believe that gnrh plays a pivotal role not only in the regulation of different hormones involved in reproduction, but also in the regulation of the motor activity of the gastrointestinal tract. degeneration of gnrh neurons might be of central importance for the pathophysiology of different forms of ibs and cipo. concluding remarks over recent decades, a number of peptides have been characterised, which led to an explosion in our understanding of their biological action and function in the central and enteric nervous system. gut hormones, including cholecystokinin, corticotrophin-releasing hormone, gastrin, gastric inhibitory polypeptide, ghrelin, glucagon-like peptide-1, motilin, neurotensin, pancreatic polypeptide, secretoneurin, serotonin, thyrotrophic-releasing hormone and vip have been shown to play a role in modulating gastrointestinal motility. new experimental and clinical data point to gnrh and oxytocin, two other peptide candidates, as being involved in controlling gastrointestinal motility. these fi ndings open new, fascinating perspectives for research and the therapeutic potential of the peptidal role in gastrointestinal diseases. furthermore, the role of neuronal apoptosis and agents which improve neuronal survival deserves further attention concerning their function in preventing neuronal degeneration which results in dysmotility. references alfven, g. 2004. plasma oxytocin in children with recurrent abdominal pain. j. pediatr. gastroenterol. nutr., 38:513–7. anderberg, u.m. and uvnäs-moberg, k. 2000. plasma oxytocin levels in female fi bromyalgia syndrome patients. z. rheumatol., 59:373–9. aube, a.c., cabarrocas, j., bauer, j., et al. 2006. changes in enteric neurone phenotype and intestinal functions in a transgenic mouse model of enteric glia disruption. gut., 55:630–7. bassotti, g., villanacci, v., maurer, c.a., et al. 2006. the role of glial cells and apoptosis of enteric neurons in the neuropathology of intractable slow transit constipation. gut., 55:41–6. bush, t.g., savidge, t.c., freeman, t.c., et al. 1998. fulminant jejunoileitis following ablation of enteric glia in adult transgenic mice. cell, 93:189–201. cabarrocas, j., savidge, t.c. and liblau, r.s. 2003. role of enteric glial cells in infl ammatory bowel disease. glia., 41:81–93. calcina, f., barocelli, e., bertoni, s., et al. 2005. effect of n-methyl-d-aspartate receptor blockade on neuronal plasticity and gastrointestinal transit delay induced by ischemia/reperfusion in rats. neuroscience, 134:39–49. cash, b.d., chey, w.d. 2005. review article: the role of serotonergic agents in the treatment of patients with primary chronic constipation. aliment. pharmacol. ther., 22:1047–60. chalazonitis, a., rothman, t.p., chen, j., et al. 1998. age-dependent differences in the effects of gdnf and nt-3 on the development of neurons and glia from neural crest-derived precursors immunoselected from the fetal rat gut: expression of gfralpha-1 in vitro and vivo. dev. biol., 204:385–406. chen, j.j., zhishan, l., pan, h., murphy, d.l., tamir, h., koepsell, h. and gershon, m.d. 2001. maintenance of serotonin in the intestinal mucosa and ganglia of mice that lack the high-affi nity serotonin transporter (sert): abnormal intestinal motility and the expression of cation transporters. j. neurosci., 21:6348–61. chiodera, p., salvarani, c., bacchi-modena, a., et al. 1991. relationship between plasma profi les of oxytocin and adrenocorticotropic hormone during suckling or breast stimulation in women. horm. res., 35:119–23. cornet, a., savidge, t.c., cabarrocas, j., et al. 2001. enterocolitis induced by autoimmune targeting of enteric glial cells: a possible mechanism in crohn’s disease? proc. natl. acad. sci. u.s.a., 98:13306–11. costedio, m.m., hyman, n. and mawe, g.m. 2006. serotonin and its role in colonic function and in gastrointestinal disorders. dis. colon. rectum, 50:376–88. de giorgio, r., barbara, g. and stanghellini, v. 2000. reduced bcl-2 expression in the enteric nervous system as a marker for neuronal degeneration in patients with gastrointestinal motor disorders. gastroenterology, 118:a867 (abstract). de giorgio, r., sarnelli, g., corinaldesi, r., et al. 2004a. advances in our understanding of the pathology of chronic intestinal pseudoobstruction. gut, 53:1549–52. de giorgio, r. and camilleri, m. 2004b. human enteric neuropathies: morphology and molecular pathology. neurogastroenterol. motil., 16:515–31. de giorgio, r., guerrini, s., barbara, g., et al. 2004c. new insights into human enteric neuropathies. neurogastroenterol motil., 16:143–7. di lorenzo, c. 1999. pseudo-obstruction: current approaches. gastroenterology, 116:980–7. dogiel, a.s. 1889. ueber den bau der ganglien in den gefl echten des darmes und der gallenblase des menschen und der säugethiere. z. naturforsch. b., 5:130–58. drucker, d.j. 2003. glucagon-like peptides: regulators of cell proliferation, differentiation, and apoptosis. mol. endocrinol., 17:161–71. ducker, t.e., boss, j.w., altug, s.a., et al. 1996. luteinizing hormone and human chorionic gonadotropin fragment the migrating myoelectrical complex in rat small intestine. neurogastroenterol motil., 8:95–100. dvorak, a.m., onderdonk, a.b., mcleod, r.s., et al. 1993. axonal necrosis of enteric autonomic nerves in continent ileal pouches. possible implications for pathogenesis of crohn’s disease. ann. surg., 217:260–71. fassr, the swedish register of drugs. fekete, m., redding, t.w. and comaru-schallu, a.m. 1989. receptors for luteinizing hormone-releasing hormone, somatostatin, prolactin, and epidermal growth factor in rat and human prostate cancers and in benign prostate hyperplasia. prostate, 14:191–208. 236 ohlsson and janciauskiene drug target insights 2007:2 frasch, a., zetzsche, t., steiger, a., et al. 1995. reduction of plasma oxytocin levels in patients suffering from major depression. adv. exp. med. biol., 395:257–8. furness 2005, goyal, r.k. and hirano, i. 1996. the enteric nervous system. n. engl. j. med., 334:1106–15. gabryel, b., chalimoniuk, m., stolecka, a., et al. 2006. inhibition of arachidonic acid release by cytosolic phospholipase a (2) is involved in the antiapoptotic effect of fk506 and cyclosporine a on astrocytes exposed to simulated ischemia in vitro. j. pharmacol. sci., 102:77–87. galligan, j.j. and vanner, s. 2005. basic and clinical pharmacology of new motility promoting agents. neurogastroenterol motil., 17:643–53. gershon, m.d., kirchgessner, a.l. and wade, p.r. 1994. functional anatomy of the enteric nervous system. in: johnson, l.r., ed. physiology of the gastrointestinal tract. 3rd ed. new york: raven press, p381–422. gershon, m.d. 2005. review article: nerves, refl exes, and the enteric nervous system. pathogenesis of the irritable bowel syndrome. j. clin. gastroenterol., 39:s184–s193. gimpl, g. and fahrenholz, f. 2001. the oxytocin receptor system: structure, function, and regulation. physiol. rev., 81:629–83. glotin, a.l., calipel, a., brossas, j.y., et al. 2006. sustained versus transient erk1/2 signaling underlines the antiand proapoptotic effects of oxidative stress in human rpe cells. invest. ophthalmol. vis. sci., 47:4614–23. gourcerol, g. and taché, y. 2007. obestatina ghrelin-associated peptide that does not hold its promise to suppress food intake and motility. neurogastroenterol motil., 19:161–5. goyal, r.k. and hirano, i. 1996. the enteric nervous system. n. engl. j. med., 334:1106–15. grider, j.r. 1994. cgrp as a transmitter in the sensory pathway mediating peristaltic refl ex. am. j. physiol., 26:g1139-g1145. grider, j.r. 2003. neurotransmitters mediating the intestinal peristaltic refl ex in the mouse. j. pharmacol. exp. ther., 307:460–7. grundy, d. 2002. towards a reduction of rectal pain? neurogastroenterol motil., 14:217–9. gwee, k.a. 2005. irritable bowel syndrome in developing countries-a disorder of civilization or colonization? neurogastroenterol motil., 17:317–24. haggers, r., finlayson, c., kahn, f., et al. 2000. a defi ciency of interstitial cells of cajal in chagasic megacolon. j. auton. nerv. syst., 80:108–11. hall, k.e. and wiley, j.w. 1998. neural injury, repair and adaptation in the gi tract. i. new insights into neuronal injury: a cautionary tale. am. j. physiol., 274:g978–83. hashmonai, m., torem, s., argov, s., et al. 1979. prolonged post-vagotomy gastric atony treated by oxytocin. br. j. surg., 66:550–51. he, c.l., burgart, l., wang, l., et al. 2000. decreased intestitial cell of cajal volume in patients with slow-transit constipation. gastroenterology, 118:14–21. he, c.l., soffer, e.e., ferris, c.d., et al. 2001. loss of interstitial cells of cajal and inhibitory innervation in insulin-dependent diabetes. gastroenterology, 121:427–34. ho, j.s., nagle, g.t., mathias, j.r., et al. 1996. presence of gonadotropinreleasing hormone (gnrh) receptor mrna in rat myenteric plexus cells. comp. biochem. physiol., 113:817–21. huang, w., yao, b., sun, l., et al. 2001. immunohistochemical and in situ hybridization studies of gonadotropin releasing hormone (gnrh) and its receptor in rat digestive tract. life. sci., 68:1727–34. johanson, j.f. 2004. options for patients with irritable bowel syndrome: contrasting traditional and novel serotonergic therapies. neurogastroenterol motil., 16:701–11. kakar, s.s. and jennes, l. 1995. expression of gonadotropin-releasing hormone and gonadotropin-releasing hormone receptor mrnas in various non-reproductive human tissues. cancer lett., 98:57–62. khanna, r., browne, r.m., heiner, a.d., et al. 1992. leuprolide acetate affects intestinal motility in female rats before and after ovariectomy. am. j. physiol., 262:g185–g190. kim, j.h., yoon, k.o., kim, j.k., et al. 2006. novel mutations of ret gene in korean patients with sporadic hirschsprung’s disease. j. pediatr. surg., 41:1250–54. krishnamurthy, s. and schuffl er, m.d. 1993. pathology of neuromuscular disorders of the small intestine and colon. gastroenterology, 104:1398–1408. kutchai, h.c. 2004. the gastrointestinal system. in: berne, r.m., levy, m.n., koeppen, b.m., stanton, b.a., edr. physology. fifth edition. mosby, st louis, missouri. lomax, a.e., fernandez, e. and sharkey, k.a. 2005. plasticity of the enteric nervous system during intestinal infl ammation. neurogastroenterol motil., 17:4–15. louvel, d., delvaux, m., felez, a., et al. 1996. oxytocin increases thresholds of colonic visceral perception in patients with irritable bowel syndrome. gut., 39:741–7. lydiard, r.b., fossey, m.d., marsh, w., et al. 1993. prevalence of psychiatric disorders in patients with irritable bowel syndrome. psychosomatics, 34:229–34. masuda, y., tanaka, t., inomata, n., et al. 2000. ghrelin stimulates gastric acid secretion and motility in rats. biochem. biophys. res. commun., 276:905–8. marchetti, b., gallo, f., farinella, z., et al. 1996. luteinizing hormonereleasing hormone (lhrh) receptors in the neuroendocrine-immune network. biochemical bases and implications for reproductive physiopathology. ann. n. y. acad. sci., 784:209–36. mathias, j.r., baskin, g.s., reeves-darby, v.g., et al. 1992. chronic intestinal pseudoobstruction in a patient with heart-lung transplant. therapeutic effect of leuprolide acetate. dig. dis. sci., 37:1761–8. mathias, j.r., clench, m.h., reeves-darby, v.g., et al. 1994a. effect of leuprolide acetate in patients with moderate to severe functional bowel disease. double-blind, placebo-controlled study. dig. dis. sci., 39:1155–62. mathias, j.r., clench, m.h., roberts, p.h., et al. 1994b. effect of leuprolide acetate in patients with functional bowel disease. long-term follow-up after double-blind, placebo-controlled study. dig. dis. sci., 39:1163–70. mathias, j.r., clench, m.h., abell, t.l., et al. 1998. effect of leuprolide acetate in treatment of abdominal pain and nausea in premenopausal women with functional bowel disease: a double-blind, placebocontrolled, randomized study. dig. dis. sci., 43:1347–55. mclean, p.g., borman, r.a. and lee, k. 2006. 5-ht in the enteric nervous system: gut function and neuropharmacology. trends in neurosciences, 30:9–13. merry, d.e., korsmeyer, s.j. 1997. bcl-2 gene family in the nervous system. annu. rev. neurosci., 20:245–67. micromedex, drugdex, drug evaluation. monstein, h-j., grahn, n., truedsson, m., et al. 2004. oxytocin and oxytocin receptor mrna expression in the human gastrointestinal tract: a polymerase chain reaction study. regul. pept., 119:39–44. möller, n., nygren, j., hansen, t.k., et al. 2003. splanchnic release of ghrelin in humans. j. clin. endocrinol. metabol., 88:850–52. nagy, n. and goldstein, a.m. 2006. endothelin-3 regulates neural crest cell proliferation and differentiation in the hindgut enteric nervous system. dev. biol., 293:203–17. natarajan, d., marcos-gutierrez, c., pachnis, v., et al. 2002. requirement of signalling by receptor tyrosine kinase ret for the directed migration of enteric nervous system progenitor cells during mammalian embryogenesis. development, 129:5151–60. ohlsson, b., forsling, m.l., rehfeld, j.f., et al. 2002. cholecystokinin leads to increased oxytocin secretion in healthy women. eur. j. surg., 168:114–18. ohlsson, b., ringström, g., abrahamsson, h., et al. 2004. oxytocin stimulates colonic motor activity in healthy women. neurogastroenterol mot., 16:33–40. ohlsson, b., björgell, o., ekberg, o., et al. 2006a. the oxytocin/vasopressin receptor antagonist atosiban delays the gastric emptying of a semisolid meal compared to saline in human. bmc. gastroenterology, 6:11 (16 mars 2006). 237 gastroinestinal motility drug target insights 2007:2 ohlsson, b., truedsson, m., djerf, p., et al. 2006b. oxytocin is present throughout the human gastrointestinal tract. reg. pept., 135:7–11. ohlsson, b. 2006c. a case report on a patient suffering from recurrent vomiting episodes, whose condition improved remarkedly during pregnancy and breast feeding. bmc. gastroenterology, 6:28. ohlsson, b., veréss, b., janciauskiene, s., et al. 2007. chronic intestinal pseudo-obstruction due to buserelin-induced formation of anti-gnrh antibodies. gastroenterology, 132:45–51. ordög, t., takayama, i., cheung, w.k.t., et al. 2000. remodeling of networks of interstitial cells of cajal in a murine model of diabetic gastroparesis. diabetes, 49:1731–9. palomba, s., orio, f., manguso, f., et al. 2005. leuprolide acetate treatment with and without coadministration of tibolone in premenopausal women with menstrual cycle-related irritable bowel syndrome. fertility and sterility, 83:1012–20. pan, h. and galligan, j.j. 1994. 5-ht1a and 5-ht4 receptors mediate inhibition and facilitation of fast synaptic transmission in enteric neurons. am. j. physiol., 266:g230–g238. petersson, m., alster, p., lundeberg, t., et al. 1996. oxytocin increases nociceptive thresholds in a long-term perspective in female and male rats. neurosci. lett., 212:87–90. petrache, i., fijalkowska, i., medler, t.r., et al. 2006. [alpha]-1 antitrypsin inhibits caspase-3 activitiy, preventing lung endothelial cell apoptosis. am. j. pathol., 169:1155–66. petring, o.u. 1989. the effect of oxytocin on basal and pethidine-induced delayed gastric emptying. br. j. clin. pharmacol., 28:329–32. rettenbacher, m. and reubi, j.c. 2001. localization and characterization of neuropeptide receptors in human colon. naunyn. schmiedebergs. arch. pharmacol., 364:291–304. ruhl, a. 2005. glial cells in the gut. neurogastroenterol motil., 17:777–90. sanders, k.m., ordög, t., koh, s.d., et al. 1999. development and plasticity of interstitial cells of cajal. neurogastroenterol motil, 11:311–38. schurmann, g., bishop, a.e., facer, p., et al. 1995. secretoneurin: a new peptide in the human enteric nervous system. histochem. cell biol., 104:11–9. sidhu, m. and cooke, h.j. 1995. role for 5-ht and ach in submucosal refl exes mediating colonic secretion. am. j. physiol. gatsrointest. liver. physiol., 269:g346–g351. silber, m., larsson, b. and uvnäs-moberg, k. 1991. oxytocin, somatostatin, insulin and gastrin concentrations vis-à-vis late pregnancy, breastfeeding and oral contraceptives. acta. obstet. gynecol. scand., 70:283–9. simren, m., simms, l., d’souza, d., abrahamsson, h., bjornsson, e.s. 2003. lipid-induced colonic hypersensitivity in irritable bowel syndrome: the role of 5-ht3 receptors. aliment. pharmacol. ther., 17:279–87. soldani, g., del tacca, m., bambini, g., et al. 1982. effects of gonadotropinreleasing hormone (gnrh) on gastric secretion and gastrin release in the dog. j. endocrinol. invest., 5:393–6. song, y., li, j.c., li, m.j. 2002. bcl-2 expression in enteric neurons of hirschsprung’s disease and its signifi cance. shi yan sheng wu xue bao, 35:155–8. sperber, a.d., atzmon, y., neumann, l., et al. 1999. fibromyalgia in the irritable bowel syndrome: studies of prevalence and clinical implications. am. j. gastroenterol, 94:3541–6. spiller, r.c. 2003. postinfectious irritable bowel syndrome. gastroenterology, 124:1662–71. tack, j., depoortere 1, bisschops, r., et al. 2005. infl uence of ghrelin on gastric emptying and meal-related symptoms in idiopathic gastroparesis. aliment. pharmacol. ther., 22:847–53. takamori, k.i., mizuta, y., takshima, f., et al. 2007. relation among plasma ghrelin level, gastric emptying and psychologic condition in patients with functional dyspepsia. j. clin. gastroenterol., 41:477–83. thomsen, l., robinson, t.l., lee, j.c.f., et al. 1998. interstitial cells of cajal generate a rhythmic pacemaker current. nat. med., 4:848–50. törnblom, h., lindberg, g., nyberg, b., et al. 2002. full-thickness biopsy of the jejunum reveals infl ammation and enteric neuropathy in irritable bowel syndrome. gastroenterology, 123:1972–79. uvnäs-moberg, k., arn, i., theorell, i., et al. 1991. gastrin, somatostatin and oxytocin levels in the patients with functional disorders of the gastrointestinal tract and their response to feeding and interaction. j. psychosom. res., 35:525–33. vasina, v., barbara, g., talamonti, l., et al. 2006. enteric neuroplasticity evoked by infl ammation. auton. neurosci., 126–127:264–72. vialli, m. 1966. histology of the enterochromaffin cell system. in: erspamer, v., ed. handbook of experimental pharmacology: 5-hydroxytrypramine and related indolealkylamines. vol 19. new york: springer-verlag, p 1–65. von boyen, g.b., steinkamp, m., reinshagen, m., et al. 2004. proinfl ammatory cytokines increase glial fi brillary acidic protein expression in enteric glia. gut., 53:222–8. wang, l., xie, l.p., huang, w.q., et al. 2001. presence of gonadotropinreleasing hormone (gnrh) and its mrna in rat pancreas. mol. cell. endocrinol., 14:172–85. wessinger, s., jones, m.p. and crowell, m.d. 2005. editorial overview: serotonergic agents in functional gi disorders: targeting the brain-gut axis. curr. opin. invest. drugs., 6:663–6. wester, t., olsson, y. and olsen, l. 1999. expression of bcl-2 in enteric neurons in normal human bowel and hirschsprung disease. arch. pathol. lab. med., 123:1264–8. wood, j.d. 2000. neuropathy in the brain-in-gut. eur. j. gastroenterol hepatol., 12:597–600. wu, j.j., rothman, t.p., gershon, m.d. 2000. development of the interstitial cell of cajal: origin, kit dependence and neuronal and nonneuronal sources of kit ligand. j. neurosci. res., 59:384–401. xu, l., depoortere, i., tomasetto, c., et al. 2005. evidence for the presence of motilin, ghrelin, and the motilin and ghrelin receptor in neurons of the myenteric plexus. regul. pept., 124:119–25. young, h.m., anderson, r.b. and anderson, c.r. 2004. guidance cues involved in the development of the peripheral autonomic nervous system. auton. neurosci., 112:1–14. zhang, j.v., ren, p.g., avsian-kretchmer, o., et al. 2005. obestatin, a peptide encoded by the ghrelin gene, opposes ghrelin’s effects on food intake. science, 310:996–9. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding 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drug target insights 2007: 2 197–207 197 review correspondence: ningaraj, n.s., department of pediatric neurooncology and molecular pharmacology, hoskins center, curtis and elizabeth anderson cancer institute, memorial health university medical center, mercer university medical school, 4700 waters avenue, savannah, ga 31404, u.s.a.tel: +1 9123500958; fax: +1 9123501269; email: ningana1@memorialhealth.com please note that this article may not be used for commercial purposes. for further information please refer to the copyright statement at http://www.la-press.com/copyright.htm targeted brain tumor treatment-current perspectives ningaraj n.s1, salimath b.p2, sankpal u.t1, perera r1 and vats t1 1department of pediatric neurooncology and molecular pharmacology, hoskins center, curtis and elizabeth anderson cancer institute, memorial health university medical center, mercer university medical school, 4700 waters avenue, savannah, ga 31404, u.s.a. 2department of biotechnology, university of mysore, mysore 570006, karnataka, india. abstract: brain tumor is associated with poor prognosis. the treatment option is severely limited for a patient with brain tumor, despite great advances in understanding the etiology and molecular biology of brain tumors that have lead to breakthroughs in developing pharmaceutical strategies, and ongoing nci/pharma-sponsored clinical trials. we reviewed the literature on molecular targeted agents in preclinical and clinical studies in brain tumor for the past decade, and observed that the molecular targeting in brain tumors is complex. this is because no single gene or protein can be affected by single molecular agent, requiring the use of combination molecular therapy with cytotoxic agents. in this review, we briefl y discuss the potential molecular targets, and the challenges of targeted brain tumor treatment. for example, glial tumors are associated with over-expression of calcium-dependent potassium (kca) channels, and high grade glioma express specifi c kca channel gene (gbk) splice variants, and mutant epidermal growth factor receptors (egfrviii). these specifi c genes are promising targets for molecular targeted treatment in brain tumors. in addition, drugs like avastin and gleevec target the molecular targets such as vascular endothelial cell growth factor receptor, platelet-derived growth factor receptors, and brc-abl/akt. recent discovery of non-coding rna, specifi cally micrornas could be used as potential targeted drugs. finally, we discuss the role of anti-cancer drug delivery to brain tumors by breaching the blood-brain tumor barrier. this non-invasive strategy is particularly useful as novel molecules and humanized monoclonal antibodies that target receptor tyrosine kinase receptors are rapidly being developed. abbreviations: bbb: blood-brain barrier; btb: blood-tumor barrier; kca: calcium-dependent potassium channels; ns-1619/ ns 004: 1,3-dihydro-1-5-(trifl uoromethyl)-2h benzimidazol-2-one; hbmvec: human brain microvascular endothelial cells; facs: fl uorescence activated cell sorting; pdgfr: platelet-derived growth factor receptor; rtkis: receptor tyrosine kinase inhibitors; egfr: epidermal growth factor receptor; egfrviii: variant iii of the human egfr; gbk channel: glioma specifi c spice variant of kca channel gene; katp: atp sensitive potassium channels; minoxidil sulfate (ms: katp channel agonist); trastuzumab (herceptin, her-2 inhibitor, genentech inc.). keywords: brain tumor, bbb, drug delivery, therapeutic targets in brain tumors introduction brain tumor nearly 20,000 new primary brain tumors and about 200,000 metastatic brain tumor cases are reported each year in the u.s.a. (levin, 2007). the overall survival of these patients is dismal and the majority of survivors suffer disabling toxicities from their treatments. standard treatment for brain tumors includes combination of surgery, radiation therapy, and chemotherapy. brain tumor poses unique challenges due to its distinct biology, genetics, treatment response, and survival. despite extensive characterization of the brain tumor pathways, molecularly targeted approach is not available to brain tumor patients. future research in brain tumors needs to focus on strategies for improving drug delivery, disruption of blood-brain-barrier (bbb), and molecular profi ling of tumors. in addition, careful studies are needed to delineate pathways that aid and abate brain tumor progression. identifi cation of potential markers (genes and proteins) for targeted therapy will defi nitely help the clinicians to design the treatment accordingly. usually, after surgical treatment, brain tumor recurs, severely shortening life expectancy (friedman, kerby and calvert, 2000). conventional treatments using radiation and intravenous chemotherapy are not sucessful 198 ningaraj et al drug target insights 2007: 2 because the cancer cells develop resistance to treatment. anti-cancer drugs fail to penetrate the bbb in suffi cient quantities (pardridge, 2001), allowing cancer cells to develop resistance to these agents. therefore, understanding the biochemical regulation of the bbb (fig. 1) in normal and tumor-invaded brain is of great importance to develop therapeutics that breach or circumvent bbb and directly target brain tumor cells (ningaraj, 2006). the focus is now on the targeted cancer therapies (butowski and chang, 2005) that complement conventional treatments and reduce the drug resistance in cancer cells and the toxicity in normal brain (newton, 2003). novel cancer therapies include anti-angiogenic agents, immunotherapy, bacterial agents, viral oncolysis, cyclin-dependent kinases and receptor tyrosine kinase inhibitors (rtkis), anti-sense agents, gene therapy, microrna (mirna), and combinations of various methods (butowski and chang, 2005). chemotherapy chemotherapy is a form of targeted therapy where cytotoxic drugs act on multiplying tumor cells. the drugs can also be used as sensitizers to augment the effects of radiation therapy. chemotherapeutic drugs can be delivered directly to brain tumors through a polymer wafer implant such as a biodegradable wafer soaked with bcnu (carmustine). besides bcnu, several other chemotherapy drugs are used to treat brain tumors, which are administered by various routes. the chemotherapeutic drugs taken orally include temozolomide (tmz, temodar), procarbazine (matulane), and lomustine (ccnu). the intravenously administered drugs include vincristine (oncovin or vincasar pfs), cisplatin (platinol), carmustine (bcnu, bicnu), carboplatin (paraplatin), while methotrexate (rheumatrex or trexall) may be taken orally, by injection, or intrathecally. treating brain tumors with chemotherapy can be diffi cult because the brain is protected by bbb, which keeps out harmful substances such as bacteria and chemotherapeutic drugs. among many cytotoxic agents in the clinician’s arsenal, temozolomide (tmz) has shown some promise in treatment of low grade gliomas (friedman, kerby and calvert, 2000), however, the effect on patient survival was modest (balana et al. 2004). the problem is that glioblastoma multiforme (gbm) exhibits varying responses to tmz (hirose, berger and pieper, 2001), and in some cases gliomas have increased o6-methyl guanine methyl transferase (mgmt) activity, which results in complete resistance to tmz (bocangel et al. 2002). the clinical utility of tmz against all types of brain tumors remains limited due to its btb penetration (some authors claim tmz metabolite (mtic) concentration in csf to be as high as 30%), which demand repeated high doses to achieve in vivo therapeutically effective concentrations in brain tumors (yung et al. 1999), and different phenotypes and genotypes that render some form of resistance against tmz (kanzawa et al. 2003). most importantly, an extensive literature search and preliminary work on bbb/btb penetration of tmz did not convince us that suffi cient amount of drug penetrates the bbb or btb to elicit anti-tumor effect. to circumvent the penetration problem, chemotherapy drugs can be delivered by figure 1. a schematic representation of normal and abnormal blood-brain barriers. we reason that genes in brain cancers/vasculature are distinct from normal brain/vasculature. they are attractive targets for the design of therapies that can penetrate the btb and selectively kill brain cancer cells. we are studying the genes that direct the formation of the normal and abnormal (cancer) human brain vasculature, and with this knowledge develop new treatment strategies for brain tumor patients. ec: endothelial cells; tj: tight junction proteins; n: nucleus. 199 targeted brain tumor treatment drug target insights 2007: 2 intratumoral route or by drug impregnated wafers to attain higher concentration of drugs in the tumor cells, but the procedures are highly invasive. targeted brain tumor treatment the human genome project has raised the expectation of the development of novel therapies for brain tumor because the conventional treatment strategies have not yielded any significant clinical outcome. brain tumor treatment differs according to the grade and location of the tumor. hence, combination of surgery, chemotherapy, and radiotherapy can be used in treating brain tumor patients (stupp et al. 2005). most promising anti-cancer drugs for pediatric and adult patients that are effective against cancers outside the brain have failed against brain tumors in clinical trails, in part, due to poor penetration across the bbb. for instance, aberrant expression of src family kinase (lck) (fabian et al. 2005) or mutation of c-kit are involved in the pathogenesis of many cancers. studies using imatinib mesylate (sti 571, gleevec, novartis, u.s.a.), an inhibitor of the tyrosine kinases brc-abl, c-kit, and pdgfr, have shown signifi cant response in patients with chronic myelogenous leukemia (cml) and gastrointestinal stromal tumor (gist). clinical trials were recently conducted to test the effi cacy of gleevec in brain tumors (reardon et al. 2005; wen et al. 2006; pollack et al. 2007). gleevec is an effective agent that targets specifi c gene/protein in cancer cells without harming normal cells and tissues. drugs like gleevec and temozolomide attack abnormal chemical signals or molecules inside the cells or on the surface of the cells that have enabled brain tumor cells to escape the normal growth controls. therefore, combating many forms of cancer will probably require a variety of targeted drugs used in combination, as cancer involves different types of dysfunctional genes and no single or two drugs will be sufficient. some cancers, particularly primary and metastatic brain tumors of the breast and lung are diffi cult to treat because they are caused by multiple signaling pathways that are running amok, rather than just one, as observed in cml and gist (butowski and chang, 2005). gleevec may potentially target the above mentioned oncogenes in brain tumor (holdhoff et al. 2005) provided it penetrates the bbb (leis et al. 2004). careful molecular studies would identify the stem cell factor/c-kit pathways in pediatric brain tumors, which might be the target of gleevec. characterizing the genetic and proteomic events that play a role in the biology of these tumors may allow molecular sub-typing which could lead to the development of novel therapeutic strategies, including treatment with gleevec or with potassium channel modulators targeting tumor and tumor blood vessel endothelial cells (ningaraj, 2006). targeting brain tumors targeting tumor and tumor blood vessel-specific marker(s) is a good strategy to control tumor growth (robinson et al. 2003). it is, however, critical to study whether tumor-specific drug delivery has the potential to minimize toxicity to normal tissues, and to improve the bioavailability of cytotoxic agents to neoplasms. existing site-specific drug delivery systems include delivery to endothelial receptor αvβ3, and tumor specific antigens. antibody conjugation to cytotoxic agents has shown promise in achieving the goal of tumor-targeted cytotoxicity. this approach may be limited by the small subsets of tumors that can be targeted by these antibodies and by poor biodistribution of these a n t i b o d i e s i n t o s o l i d t u m o r s . a l t e r n a t i v e approaches to target all neoplasms exploit differences in human tumor blood vessel characteristics when compared to normal brain blood vessels (black and ningaraj, 2004; ningaraj et al. 2002; ningaraj, rao and black, 2003a). epidermal growth factor receptor (egfr) is often amplified and mutated in human gliomas, but the expression is low or undetectable in normal brain. recently, egfr’s mutant isoform, variant iii of the human egfr (egfrviii), is under intensive investigation as potential molecular target for the specific delivery of the diagnostic and the therapeutic agents to brain tumors (yang et al. 2005). the therapeutic monoclonal antibodies (mab) targeting growth factor pathways are being developed. the purpose of antibody treatment of cancer is to induce the direct or indirect destruction of cancer cells, either by specifically targeting the tumor or the tumor vasculature (butowski and chang, 2005). examples of therapeutic antibodies which are effective in treating cancer includes the humanized igg antibody herceptin for the treatment of breast cancer, cetuximab, abx-egf, emd 720000 and h-r3 directed at extracellular receptor domain that inhibits the ligand-receptor interactions. other antibodies 200 ningaraj et al drug target insights 2007: 2 like y10 and mab806, which are directed towards the extracellular portion of egfrviii in gliomas have also shown some activity in clinical trials (rich and bigner, 2004). suramin (polysulfonated napthylurea), which acts by interfering with the binding of several growth factors-including egf, platelet derived growth factor (pdgf), and insulin growth factor (igf1) with their putative receptors, is being tested in clinical trails. these mabs, however, have poor penetration into brain tumors, which results in recurrence in brain tumor patients. kinase inhibitors kinase inhibitors show great promise as a new class of therapeutics to control gliomas. the specifi city of rtkis, including those that are in clinical use or in development widely varies, and is not strongly correlated with chemical structure of the identity of the intended target. many novel interactions were recently identifi ed (fabian et al. 2005). egfr and pdgfr are abnormal genes identifi ed in gliomas (rich and bigner, 2004), whose expression is linked to an increased rate of tumor cell proliferation, resistance to chemotherapy, invasion, and apoptosis, and hence decreased survival in patients with malignant gliomas. pdgf ligands bind to pdgfrs to induce phosphorylation and activation of downstream signaling pathways such as ras, mapk, and akt. therefore, therapies using gleevec, suramin, and mabs are directed at pdgfr to control glioma growth. pdgfr inhibitors may also provide additional benefit by blocking pericytes-assisted angiogenesis (bergers et al. 2003). clinical trials with egfr and pdgfr inhibitors have shown promise for glioma therapy, although their ability to penetrate bbb in suffi cient amounts is largely unknown. we transiently opened the btb with kca and atp-sensitive potassium (katp) channel agonists (black and ningaraj, 2004; ningaraj et al. 2002; ningaraj, rao and black, 2003a,b; rao and ningaraj, 2001) to increase the delivery of gleevec and herceptin to human glioma xenografts grown in murine brains. kca channels in gliomas membrane ion channels are essential for cell proliferation and appears to play a role in the development of cancer (ningaraj, 2006). the kca channels are highly expressed in gliomas (weaver, liu and sontheimer, 2004) supporting the hypothesis that these channels play an important role in brain tumor growth and possibly the progression of low grade anaplastic astrocytomas (grade ii) to a deadly high grade gbm (who grade iv). in addition, studies have shown that modulation of the biological function of kca channels with specific inhibitors attenuate glioma growth (rao and ningaraj, 2001). another study showed that the activation of intermediate kc a channels with its opener caused down-regulation of these channels and attenuated the non-excitable cell growth and its proliferation (kraft et al. 2003). we showed that chronic activation of kca channels with its specific openers ns-1619 and ns-004 elicited apoptosis in vitro and in vivo (rao and ningaraj, 2001). however, the role of kca channels in progression from a treatable low grade to an untreatable high grade glioma in pediatric as well as in adult patients is not fully understood. recently, glioma kca/bk channels (gbk) splice variant of the kcnma1 gene was characterized by enhanced sensitivity to intracellular calcium levels (weaver, bomben and sontheimer, 2006). the study also showed that the expression of functional gbk channels appears to be regulated in a growth-factor-dependent manner. it is well established that egf activates egfr. several molecular agents targeting egfr are undergoing clinical trails as potential therapies in neurooncology (rich and bigner, 2004). for example, zd1839 (iressa) an orally active, selective egfr-tyrosine kinase inhibitor has anti-tumor activity against malignant human cancer cell lines (31). glioma cells also show up-regulation and constitutive activation of her2 neu, and its expression which correlates positively with aggressive malignancy (mellinghoff et al. 2005). a correlation has been demonstrated for the expression of gbk/kca channels and her-2 neu, which implies gbk/kca channels as a downstream target for her-2 neu signaling (olsen et al. 2004). how kca channel modulates egfr tyrosine kinase or vice versa is poorly understood. it appears to occur via changes in intracellular calcium levels without change in channel expression or phosphorylation (weaver, bomben and sontheimer, 2006). in a transgenic glioma mouse model, a loss of egfr overexpression was observed by egfrviii introduction 201 targeted brain tumor treatment drug target insights 2007: 2 (gullick, 2001). this model of high grade glioma is useful in evaluating targeted molecular therapies in brain tumor. targeting angiogenesis in brain tumors angiogenesis plays a crucial role in malignant primary brain tumor growth. several preclinical and clinical studies have confirmed that the vascular endothelial cell growth factor (vegf) and the bfgf bind to their receptors to promote glioma growth. vegfr is expressed in human high grade glioma but not found in normal brain. increased concentration of angiogenic factors and their receptors is correlated with tumor vasculature and malignant human gliomas. furthermore, it is shown that the endogenous inhibitor of angiogenesis, thrombospondin-1 (tsp-1) is produced by normal brain and low grade gliomas, but is completely absent in high grade gliomas. the gbm is among the most “endothelial rich” brain tumors studied. in children with brain tumors, microvascular density correlates with tumor recurrence, and patient mortality. as tumor vascularity is highly correlated with disease outcome in neuroblastoma, novel therapeutic that targets the vascular endothelium is a suitable clinical trial target candidate. the molecules like vegf, bfgf, pdgf as well as endothelial integrins are linked to advanced malignancy, which provided the rationale for developing anti-angiogenic therapies in brain tumors. the potential of anti-angiogenic therapy in human brain tumors is demonstrated in experimental brain tumor models. a wide range of anti-angiogenic agents such as endogenous angiogenesis inhibitors, synthetic angiogenic inhibitors, antibodies, and anti-angiogenic gene therapy are investigated with radiation therapy. anti-angiogenic drugs have low potential for toxicity and resistance because they specifi cally target endothelial cells. the potential of antiangiogenic agents to augment the anti-tumor activity of standard cytotoxic chemotherapeutic agents is being investigated (bernsen and van der kogel, 1999; reijneveld, voest and taphoorn, 2000; takano et al. 2004). the evidence for glioma anti-angiogenesis therapy, with or without chemotherapy has been described in several preclinical animal models. the anti-angiogenic function of tsp-1 is known for a long time. the tsp-1 transfected glioma cells lacked vegf expression ability, which supports the rationale for using vegf and bfgf antibodies in clinical trails. anti-angiogenic d r u g , t h a l i d o m i d e e x h i b i t s s y n e r g i s t i c anti-glioma activity when combined with dna alkylating agent temozolomide, and increased median survival from 63 weeks to 103 weeks compared to thalidomide only group (baumann et al. 2004). while evaluating anti-angiogenic drugs for clinical development, it is important to analyze if such drugs penetrate the bbb, and survive p-glycoprotein-mediated drug efflux system. at present, there is a great deal of interest in combination therapy using conventional cytotoxic therapy with chemotherapeutics and radiotherapy. anti-angiogenic agents like tnp-470, angiostatin, dc 101, su5416, anti-vegf and vegf-r antibodies and vegf monoclonal antibody a4.6.1, tyrosine kinase inhibitors, cox-2 inhibitors, and anti-egfr inhibitors are used in combination with radiation. the synthetic fumagillin analogue, tnp-470 was shown to interfere with angiogenesis through inhibition of endothelial cell proliferation and migration in murine and human neuroblastoma xenograft model. now it is being evaluated in phase i/ii clinical trials. in brain tumor models, tnp470 and minocycline together increased 9l glioma sensitivity to bcnu and andriamycin (shusterman et al. 2001), while lund, bastholm and kristjansen, (2000) found that tnp-470 increased radiation sensitivity of human u87 glioblastoma xenografts. a phase ii study with anti-angiogenic monoclonal antibody bevacizumab (avastin) and anti-cytokine irinotecan in brain tumor patients is also being conducted (nct00381797). endogenous inhibitors of angiogenesis such as angiostatin, endostatin, pex, pigment epithelial-derived factor, and thrombospondin (tsp-1&2) are shown to be effi cacious. they exert their effects through multiple mechanisms, including induction of apoptosis of micro vascular endothelial cells, inhibition of proliferation of endothelial cells, inhibition of function, and regulation of proangiogenic molecules. these endogenous inhibitors offer a novel treatment option because they are unlikely to trigger a host immune response. angiostatin, a proteolytic fragment of plasminogen inhibits angiogenesis and attenuate the growth of primary and metastatic tumors. angiostatin was effectively used in combination with 202 ningaraj et al drug target insights 2007: 2 fractional radiation therapy in human glioma models (mauceri et al. 1998; rege, fears and gladson, 2005). recently, gene therapy has hit a snag, but offers a promising alternate treatment strategy. brain tumors are attractive for gene therapy because the brain is an immunologically privileged organ, and the bbb provides a natural immunological barrier. mice when treated with a retrovirus encoding a dominant negative mutant of the vegf receptor flk-1 resulted in reduced tumor growth and decreased blood vessel density. recombinant adeno-associated virus (aav) vector with the angiostatin gene was used to reduce tumor growth and angiogenesis in a c6 glioma model. antiangiogenic therapy using semliki forest virus (sfv) carrying endostatin gene significantly reduced the tumor growth in animals. therefore, gene therapy with endostatin delivered via sfv may be a viable treatment strategy for brain tumors (ma et al. 2002; yamanaka et al. 2003). although, the gene therapy in general is in its infancy, it provides an alternate strategy to treat hard-to treat brain tumors. epigenetic genes as brain tumor targets epigenetic events are genetic modifi cations (dna methylation and covalent histone modifi cations) that are heritable through cell division, which affect gene expression without causing changes to the dna coding sequence. cancer cells exhibit global hypomethylation of the genome accompanied by region-specifi c hypermethylation events. the hypomethylation mainly occurs in the repetitive sequences leading to genomic instability and tumor formation. aberrant hypermethylation occurs at cpg islands found in the promoter region of genes, which is usually associated with the transcriptional silencing of that gene (baylin et al. 2001). dna methylation changes (palanichamy, erkkinen and chakravarti, 2006), particularly cpg island hypermethylation is frequent, early, and common event (as common as mutations) in many types of cancers leading to the inactivation of tumor suppressor genes. several genetic changes have been identifi ed in aas and gbms involving heterozygous deletion of 19q13, inactivation/deletion of tumor suppressor genes namely p16ink4a (hegi et al. 1997), p14arf (ichimura et al. 2000), rb1 (ichimura et al. 1996), pten and p53 gene (mashiyama et al. 1991) and amplifi cation of egfr gene (libermann et al. 1985). epigenetic research in glioma pathogenesis revealed several epigenetic genes silenced by promoter cpg island hypermethylation, such as, cell cycle regulatory proteins rb1 (nakamura et al. 1996), p16ink4a (costello et al. 1996; fueyo et al. 1996), myelin related gene emp3 (alaminos et al. 2005), and matrix metalloproteinases inhibitor timp3 (bachman et al. 1999). comprehensive whole-genome microarray studies using inhibitors of epigenetic modifi cation have identified several genes including cst6 (putative metastatic suppressor), bik (apoptosis inducer), tspyl5 (unknown function), bex1, and bex2 (uncharacterized function) as putative tumor suppressors that are frequently methylated in primary gliomas (kim et al. 2006; foltz et al. 2006). another genome-wide study using restriction landmark genomic scanning has identifi ed as many as 1500 cpg islands to be aberrantly methylated in low grade gliomas (costello et al. 2000). these studies have highlighted a role for dna methylation in gliomagenesis. to date very few genetic assays are available to accurately provide information regarding patient prognosis or response to therapy. it has been hypothesized that aberrant dna methylation plays a key role in tumor initiation. therefore identifying such modifi cations helps in early detection of cancer, and might also provide information regarding the mechanisms that control glioma progression (costello, 2003). in addition to being a diagnostic marker, dna methylation can also serve as an useful prognostic marker as shown by the methylation of the dna repair gene, mgmt, in gliomas. epigenetic silencing of the gene (involved in the repair of dna damaged by alkylating agents) is associated with the increased survival in patients treated with alkylating drug temozolomide (esteller et al. 2000; komine et al. 2003). current laboratory studies are aimed at discovering novel methylation markers in tumor tissue as well as in the patient’s body fl uids (belinsky et al. 2006; cairns et al. 2001). since the primary dna sequence of epigenetically modifi ed genes remains intact, it is possible to reactivate genes using inhibitors of dna methylation or histone modifi cations (daskalakis et al. 2002; plumb et al. 2000). clinical trials using dna methylation and histone deacetylase inhibitors, which reactivate silenced genes in cancers, are in various development stages. the dna methyltransferase inhibitors, 203 targeted brain tumor treatment drug target insights 2007: 2 5-azacytidine (vidaza) and 5-aza-2′-deoxycytidine (decitabine), are used with reasonable success in the treatment of hematologic malignancies (lubbert, 2000), but have limited success in solid tumors. combination of hdac inhibitors with dna methyltransferase inhibitors appear to synergistically induce the expression of silenced genes (cameron et al. 1999). however, these drugs have drawbacks such as extreme instability, serious side effects, and sometimes these drugs at high doses may promote malignant transformation. alternative approaches include the use of sirna targeted against the dna methyltransferase enzyme (goffi n and eisenhauer, 2002) and developing stable small molecule inhibitors that can overcome the bbb. small interfering rna (sirna) to target brain tumor gene(s) the sirna directs the targeted destruction of mrna encoding a specific protein, in a process known as rna interference (rnai). this process stops translation of the targeted mrna into protein, effectively silencing the gene. rnai is a recent discovery, identified in mammalian cells in 2001, but it has rapidly advanced into practical technique, and is being used increasingly to investigate mammalian gene function. tools are available to induce rnai in cell lines, intact tissue preparations, and even in in vivo. depending on the method used, loss of gene expression may be transient or sustained, enabling a wide range of functions to be investigated. the rnai is a powerful technique that can be used to produce targeted knockout of genes in mammalian cells (gurney and hunter, 2005). its applications potentially include identification of protein function in health and disease, identification of novel genes, and drug target validation. effective rnai requires an appropriate sirna sequence to be designed and an efficient method for delivering the sirna to the cells of interest. since not all potential sirna sequences are effective, it is important to verify the loss of gene expression by measuring the level of protein remaining. limitations for delivering sirna are one of the main obstacles to produce efficient rnai, especially in intact tissue preparations. a successful in vitro method for targeted rnai against the task-1 potassium channel gene (gurney and h u n t e r, 2 0 0 5 ) w a s d e s c r i b e d . i n c r e a s i n g evidence show that microrna (mirna) represent a new class of genes involved in oncogenesis (ciafre et al. 2005). mirnas as druggable targets the mirnas are non-coding, double stranded rna molecules with an average size of 22 bp, and serve as posttranscriptional regulators of gene expression in higher eukaryotes. the mirnas play an important role in development and other cellular processes by hybridizing to complementary target mrna transcripts and destabilizing the latter by preventing their translation (ambros, 2003; bartel and bartel, 2003; bartel, 2004). although a few hundred mirnas have been discovered in a variety of organisms, little is known about their cellular functions. they have been implicated, among others, in regulation of developmental timing and pattern formation, restriction of differentiation potential, regulation of insulin secretion, resistance to viral infection, and in genomic rearrangements associated with carcinogenesis or other genetic disorders, such as the fragile x syndrome. recent evidence suggests that the number of unique mirna genes in human ranges from 1000 to 20,000. it is estimated that 20%–30% of all human mrna genes are mirna targets, and hence special attention has been given to mirnas as candidate drug targets in brain tumor. several recent reviews and research articles have illustrated the involvement of mirnas in cancer (calin and croce, 2006a, 2006b; jannot and simard, 2006; kent and mendell, 2006; jovanovic and hengartner, 2006; dalmay and edwards, 2006; hutvagner, 2006; osada and takahashi, 2006; zhang and coukos, 2006). therefore, we will restrict this section to the general concepts. in a recent study the mirna expression levels in gbm was investigated (ciafre et al. 2005; chan, krichevsky and kosik, 2005). the analysis of both glioblastoma tissues and glioblastoma cell lines showed a signifi cantly altered mirna expression. the most interesting mirna is mir-21, which is signifi cantly upregulated in glioblastoma. in another study, knockdown of mirna-21 in cultured glioblastoma cells triggers activation of caspases that leads to increased apoptotic cell death (chan, krichevsky and kosik, 2005). these data suggest that aberrantly expressed mir-21 may contribute to the malignant phenotype by blocking expression 204 ningaraj et al drug target insights 2007: 2 of critical apoptosis-related genes. a set of brain-enriched mirnas, mir-128, mir-181a, mir-181b, and mir-181c, are down-regulated in glioblastoma is also discovered (ciafre et al. 2005; o’driscoll, 2006). one of the early works that demonstrates mirnas as potential candidate drug target was performed by obstructing the adipocyte differentiation process in human primary adipocytes (esau et al. 2004). major hurdles are expected before a mirna-based drug is successfully developed against cancer. in fairness this is only the beginning of the impact of the discovery of mirna on understanding the brain tumor etiology, and developing cancer treatment strategies. anti-cancer drug delivery to brain tumor drug delivery in the treatment of brain tumors is a crucial consideration in the development of anticancer agent because the delivery of all substances into the brain is tightly regulated by bbb. brain tumor cells diffuse into the normal brain and are protected by intact bbb (rich and bigner, 2004), where anti-cancer drug delivery is very critical. we showed that improved drug delivery in human glioma xenograft models (ningaraj, 2006) has the potential to be extrapolated to patients with brain tumors for better control of the disease. in this direction, our laboratory is developing methods for high-throughput screening of rtkis for selective delivery to brain tumors, simultaneously monitor dosing, delivery, and pharmacological effi cacy of rtk inhibitors in animal brain tumor models. the challenges and opportunities of the biochemical modulation of bbb for selective drug delivery to brain tumor was reviewed recently (ningaraj, 2006). we showed that intravenously administered, potassium channel agonists increase tmz (fig. 2) and her-2 mab (herceptin) (ningaraj, rao and black, 2003b) delivery across the btb to elicit anti-tumor activity and increase survival in nude mice with intracranially implanted human glial tumor. our study suggested that the btb allows a small amount of tmz into brain tumors. potassium channel agonist-mediated biochemical modulation signifi cantly increased btb permeability allowing greater amounts of figure 2. quantitative increases in btb permeability. a signifi cant increase in the mean ki for [ 14c]-temozolomide (tmz) after i.v. infusion of 100 µg/kg/min for 15 min of ns-1619 and ms compared to a vehicle-treated group was observed. the increase in [14c]-tmz uptake in tumor center was signifi cant although a slight increase in uptake of the radiotracer was observed in the brain tissue-surrounding tumor. no [14c]-tmz uptake in contralateral normal brain, which served as internal control, was observed in all the groups. data are presented as mean ± s.d (n = 6), ***p < 0.001 versus vehicle-treated group. precaution was taken to avoid necrotic area during the ki measurement by comparing the qar brain section with a corresponding h&e stained serial brain tumor section. 205 targeted brain tumor treatment drug target insights 2007: 2 tmz, selectively to reach brain tumor and brain tissue surrounding tumor, which represents proliferating edges of tumor where the bbb may be intact (pardridge et al. 1992). furthermore, we showed that trastuzumab combined with tmz co-administered with potassium channel agonists signifi cantly increased survival rates in mice with intracranial gbm xenograft (unpublished data). these results are consistent with our earlier study, where we showed that potassium channel activator (minoxidil sulfate: ms) infusion selectively enhanced carboplatin delivery to tumor tissue without increasing delivery to normal brain (ningaraj, rao and black, 2003b). ms co-infusion with carboplatin in rats resulted in tumor regression, significantly increasing survival (black and ningaraj, 2004). the ability to deliver her-2 neu targeting drug herceptin (trastuzumab) by potassium channel-mediated btb modulation in human xenografts may be clinically useful because gbms frequently have altered receptor tyrosine kinase genes (fuller and bigner, 1992), including her-2 neu that is over expressed in about 17%–20% of gbm patients (forseen et al. 2002) resulting in poor prognosis and patient survival. a molecular target-based therapy using trastuzumab and pertuzumab (omnitarg, 2c4) is developed by genentech inc., for brain tumor, but their delivery across the btb remains a major concern. molecular medicine to conclude, the future of molecular targeted therapy is to achieve customized treatment strategy for brain tumor patients, where individual patient treatment will be based on the molecular profi le of the disease. the information based on the changing levels of active genes/proteins inside tumor cells in response to an anti-cancer drug, could help physicians to determine early in the treatment whether a drug works effectively or not. researchers have identifi ed gene/protein markers that are useful in individualizing treatment in prostate, breast, and ovarian cancer patients. although, brain tumor tissue is heterogeneous, the genetic profi ling of tumor tissue gives valuable molecular information. as a case in point, high-throughput gene profi ling of brain tumor biopsy samples by gene array technique can be compared with genomic data generated using tumor samples to predict whether patients would benefit from anti-cancer drug (like gleevec) treatment or by potassium channel modulation in tumor and tumor vascular endothelial cells. acknowledgments we thank robert bishop, ph.d., schering-plough research institute, kenilworth, new jersey for kindly providing radiolabeled and non-radiolabeled temozolomide. we also acknowledge american cancer society award to nsn. references ambros, v. 2003. microrna pathways in fl ies and worms: growth, death, fat, stress, and timing. cell, 113:673–6. alaminos, m. et al. 2005. emp3, a myelin-related gene located in the critical 19q13.3 region, is epigenetically silenced and exhibits features of a candidate tumor suppressor in glioma and neuroblastoma. cancer res., 65:2565–71. bachman, k.e. et al. 1999. methylation-associated silencing of the tissue inhibitor of metalloproteinase-3 gene suggest a suppressor role in kidney, brain, and other human cancers. cancer res., 59:798–802. balana, c.et al. 2004. phase ii study of temozolomide and cisplatin as primary treatment prior to radiotherapy in newly diagnosed glioblastoma multiforme patients with measurable disease. a study of the spanish medical neuro-oncology group (genom). j. neurooncol., 70:359–69. bartel, b. and bartel, d.p. 2003. micrornas: at the root of plant development? plant physiol., 132:709–17. bartel, d.p. 2004. micrornas: genomics, biogenesis, mechanism, and function. cell, 116:281–97. baumann, f. et al. 2004. combined thalidomide and temozolomide treatment in patients with glioblastoma multiforme. j. neurooncol., 67:191–200. baylin, s.b. et al. 2001. aberrant patterns of dna methylation, chromatin formation and gene expression in cancer. hum. mol. genet., 10:687–92. bernsen, h.j. and van der kogel, a.j. 1999. antiangiogenic therapy in brain tumor models. j. neurooncol., 45:247–55. bergers, g. et al. 2003. benefi ts of targeting both pericytes and endothelial cells in the tumor vasculature with kinase inhibitors. j. clin. invest., 111:1287–95. belinsky, s.a. et al. 2006. promoter hypermethylation of multiple genes in sputum precedes lung cancer incidence in a high-risk cohort. cancer res., 66:3338–44. black, k.l. and ningaraj, n.s. 2004. modulation of brain tumor capillaries for enhanced drug delivery selectively to brain tumor. cancer control, 11:165–73. bocangel, d.b. et al. 2002. multifaceted resistance of gliomas to temozolomide. clin. cancer res., 8:2725–34. butowski, n. and chang, s.m. 2005. small molecule and monoclonal antibody therapies in neurooncology. cancer control, 12:116–24. cameron, e.e. et al. 1999. synergy of demethylation and histone deacetylase inhibition in the re-expression of genes silenced in cancer. nat. genet., 21:103–7. cairns, p. et al. 2001. molecular detection of prostate cancer in urine by gstp1 hypermethylation. clin. cancer res., 7:2727–30. calin, g.a. and croce, c.m. 2006a. genomics of chronic lymphocytic leukemia micrornas as new players with clinical signifi cance. semin. oncol., 33:167–73. calin, g.a. and croce, c.m. 2006b. micrornas and chromosomal abnormalities in cancer cells. oncogene, 25:6202–10. chan, j.a., krichevsky, a.m. and kosik, k.s. 2005. microrna-21 is an antiapoptotic factor in human glioblastoma cells. cancer res., 65:6029–33. ciafre, s.a. et al. 2005. extensive modulation of a set of micrornas in primary glioblastoma. biochem. biophys. res. commun., 334:1351–8. costello, j.f. et al. 1996. silencing of p16/cdkn2 expression in human gliomas by methylation and chromatin condensation. cancer res., 56:2405–10. costello, j.f. et al. 2000. aberrant cpg-island methylation has non-random and tumour-type-specifi c patterns. nat. genet., 24:132–8. 206 ningaraj et al drug target insights 2007: 2 costello, j.f. 2003. dna methylation in brain development and gliomagenesis. front biosci., 8:s175–84. dalmay, t. and edwards, d.r. 2006. micrornas and the hallmarks of cancer. oncogene, 25:6170–5. daskalakis, m. et al. 2002. demethylation of a hypermethylated p15/ink4b gene in patients with myelodysplastic syndrome by 5-aza-2′deoxycytidine (decitabine) treatment. blood, 100:2957–64. fabian, m.a. et al. 2005. a small molecule-kinase interaction map for clinical kinase inhibitors. nat. biotechnol., 23:329–36. forseen, s.e. et al. 2002. identifi cation and relationship of her-2/neu overexpression to short-term mortality in primary malignant brain tumors. anticancer res., 22:1599–602. foltz, g. et al. 2006. genome-wide analysis of epigenetic silencing identifi es bex1 and bex2 as candidate tumor suppressor genes in malignant glioma. cancer res., 66:6665–74. friedman, h.s., kerby, t. and calvert, h. 2000. temozolomide and treatment of malignant glioma. clin. cancer res., 6:2585–97. fuller, g.n. and bigner, s.h. 1992. amplifi ed cellular oncogenes in neoplasms of the human central nervous system. mutat. res., 276:299–306. fueyo, j. et al. 1996. hypermethylation of the cpg island of p16/cdkn2 correlates with gene inactivation in gliomas. oncogene, 13:1615–9. gullick, w.j. 2001. update on her-2 as a target for cancer therapy: alternative strategies for targeting the epidermal growth factor system in cancer. breast cancer res., 3:390–4. goffi n, j. and eisenhauer, e. 2002. dna methyltransferase inhibitors-state of the art. ann. oncol., 13:1699–716. gurney, a.m. and hunter, e. 2005. the use of small interfering rna to elucidate the activity and function of ion channel genes in an intact tissue. j. pharmacol. toxicol. methods, 51:253–62. hegi, m.e. et al. 1997. hemizygous or homozygous deletion of the chromosomal region containing the p16ink4a gene is associated with amplifi cation of the egf receptor gene in glioblastomas. int. j. cancer., 73:57–63. hirose, y. berger, m.s. and pieper, r.o. 2001. p53 effects both the duration of g2/m arrest and the fate of temozolomide-treated human glioblastoma cells. cancer res., 61:1957–63. hutvagner, g. 2006. micrornas and cancer: issue summary. oncogene, 25:6154–5. ichimura, k. et al. 1996. human glioblastomas with no alterations of the cdkn2a (p16ink4a, mts1) and cdk4 genes have frequent mutations of the retinoblastoma gene. oncogene., 13:1065–72. ichimura, k. et al. 2000. deregulation of the p14arf/mdm2/p53 pathway is a prerequisite for human astrocytic gliomas with g1-s transition control gene abnormalities. cancer res., 60:417–24. jannot, g. and simard, m.j. 2006. tumour-related micrornas functions in caenorhabditis elegans. oncogene, 25:6197–201. jovanovic, m. and hengartner, m.o. 2006. mirnas and apoptosis: rnas to die for. oncogene, 25:6176–87. kanzawa, t. et al. 2003. inhibition of dna repair for sensitizing resistant glioma cells to temozolomide. j. neurosurg., 99:1047–52. kent, o.a. and mendell, j.t. 2006. a small piece in the cancer puzzle: micrornas as tumor suppressors and oncogenes. oncogene, 25:6188–96. kim, t.y. et al. 2006. epigenomic profi ling reveals novel and frequent targets of aberrant dna methylation-mediated silencing in malignant glioma. cancer res., 66:7490–501. komine, c. et al. 2003. promoter hypermethylation of the dna repair gene o6-methylguanine-dna methyltransferase is an independent predictor of shortened progression free survival in patients with low grade diffuse astrocytomas. brain pathol., 13:176–84. kraft, r. et al. 2003. bk channel openers inhibit migration of human glioma cells. pfl ugers arch., 446:248–55. leis, j.f. et al. 2004. central nervous system failure in patients with chronic myelogenous leukemia lymphoid blast crisis and philadelphia chromosome positive acute lymphoblastic leukemia treated with imatinib (sti-571). leuk lymphoma., 45:695–8. levin, v.a. 2007. are gliomas preventable? recent results cancer res., 174:205–5. libermann, t.a. et al. 1985. amplifi cation, enhanced expression and possible rearrangement of egf receptor gene in primary human brain tumours of glial origin. nature, 313:144–7. lund, e.l., bastholm, l. and kristjansen, p.e. 2000. therapeutic synergy of tnp-470 and ionizing radiation: effects on tumor growth, vessel morphology, and angiogenesis in human glioblastoma multiforme xenografts. clin. cancer res., 6:971–8. lubbert, m. 2000. dna methylation inhibitors in the treatment of leukemias, myelodysplastic syndromes and hemoglobinopathies: clinical results and possible mechanisms of action. curr. top microbiol. immunol., 249:135–64. mashiyama, s. et al. 1991. detection of p53 gene mutations in human brain tumors by single-strand conformation polymorphism analysis of polymerase chain reaction products. oncogene., 6:1313–8. mauceri, h.j. et al. 1998. combined effects of angiostatin and ionizing radiation in antitumour therapy. nature, 394:287–91. ma, h.i. et al. 2002. intratumoral gene therapy of malignant brain tumor in a rat model with angiostatin delivered by adeno-associated viral (aav) vector. gene ther., 9:2–11. mellinghoff, i.k. et al. 2005. molecular determinants of the response of glioblastomas to egfr kinase inhibitors. n. engl. j. med., 353:2012–24. nakamura, m. et al. 1996. promoter hypermethylation of the rb1 gene in glioblastomas. lab invest., 81:77–82. newton, h.b. 2003. molecular neuro-oncology and development of targeted therapeutic strategies for brain tumors. part 1: growth factor and ras signaling pathways. expert rev. anticancer ther., 3:595–614. ningaraj, n.s. et al. 2002. regulation of blood-brain tumor barrier permeability by calcium-activated potassium channels. j. pharmacol. exp. ther., 301:838–51. ningaraj, n.s., rao, m.k. and black, k.l. 2003a. adenosine 5′-triphosphate-sensitive potassium channel-mediated blood-brain tumor barrier permeability increase in a rat brain tumor model. cancer res., 63:8899–911. ningaraj, n.s. 2006. drug delivery to brain tumours: challenges and progress. expert opin. drug deliv., 3:499–509. o’driscoll l. 2006. the emerging world of micrornas. anticancer res., 26:4271–8. olsen, m.l., weaver, a.k., ritch, p.s., sontheimer, h. 2004. modulation of glioma bk channels via erbb2, 81:2179–89. osada, h. and takahashi, t. 2006. micrornas in biological processes and carcinogenesis. carcinogenesis, 28:2–12. pardridge, w.m. et al. 1992. blood-brain barrier and new approaches to brain drug delivery. west j. med., 156:281–6. pardridge, w.m. 2001. drug targeting, drug discovery, and brain drug development. in: brain drug targeting: the future of brain drug development. cambridge: cambridge university press, pp. 1–13. plumb, j.a. et al. 2000. reversal of drug resistance in human tumor xenografts by 2′-deoxy-5-azacytidine-induced demethylation of the hmlh1 gene promoter. cancer res., 60:6039–44. pollack, i.f. et al. 2007. phase i trial of imatinib in children with newly diagnosed brainstem and recurrent malignant gliomas: a pediatric brain tumor consortium report. neuro-oncol., 9:145–60. rao, m. and ningaraj, n. 2001. activation of calcium-dependent potassium channels elicits selective glioma cell death. in: aacr-ncieortc. rege, t.a., fears, c.y. and gladson, c.l. 2005. endogenous inhibitors of angiogenesis in malignant gliomas: nature’s antiangiogenic therapy. neuro-oncol., 7:106–21. reijneveld, j.c., voest, e.e. and taphoorn, m.j. 2000. angiogenesis in malignant primary and metastatic brain tumors. j. neurol., 247:597–608. reardon, d.a. et al. 2005. phase ii study of imatinib mesylate plus hydroxyurea in adults with recurrent glioblastoma multiforme. j. clin. oncol., 23:9359–68. rich, j.n. and bigner, d.d. 2004. development of novel targeted therapies in the treatment of malignant glioma. nat. rev. drug discov., 3:430–46. 207 targeted brain tumor treatment drug target insights 2007: 2 robinson, s.p. et al. 2003. tumour dose response to the antivascular agent zd6126 assessed by magnetic resonance imaging. br. j. cancer., 88:1592–7. saito, y. and jones, p.a. 2006. epigenetic activation of tumor suppressor micrornas in human cancer cells. cell cycle, 5:2220–22. shusterman, s. et al. 2001. the angiogenesis inhibitor tnp-470 effectively inhibits human neuroblastoma xenograft growth, especially in the setting of subclinical disease. clin. cancer res., 7:977–84. stupp, r. et al. 2005. radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. n. engl. j. med., 352:987–96. takano, s. et al. 2004. angiogenesis and antiangiogenic therapy for malignant gliomas. brain tumor pathol., 21:69–73. thomas, m. 2003. epidermal growth factor receptor tyrosine kinase inhibitors: application in non-small cell lung cancer. cancer nurs., 26: 21s–25s. weaver, a.k., bomben, v.c. and sontheimer, h. 2006. expression and function of calcium-activated potassium channels in human glioma cells. glia., 54:223–33. weaver, a.k., liu, x. and sontheimer, h. 2004. role for calcium-activated potassium channels (bk) in growth control of human malignant glioma cells. j. neurosci. res., 78:224–34. wen, p.y. et al. 2006. phase i/ii study of imatinib mesylate for recurrent malignant gliomas: north american brain tumor consortium study 99–08. clin. cancer res., 12:4899–907. yamanaka, r. et al. 2003. induction of an antitumor immunological response by an intratumoral injection of dendritic cells pulsed with genetically engineered semliki forest virus to produce interleukin-18 combined with the systemic administration of interleukin-12. j. neurosurg., 99:746–53. yang, w. et al. 2005. development of a syngeneic rat brain tumor model expressing egfrviii and its use for molecular targeting studies with monoclonal antibody l8a4. clin. cancer res., 11:341–50. yung, w.k. et al. 1999. multicenter phase ii trial of temozolomide in patients with anaplastic astrocytoma or anaplastic oligoastrocytoma at first relapse. temodal brain tumor group. j. clin. oncol., 17:2762–71. zhang, l. and coukos, g. 2006. micrornas: a new insight into cancer genome. cell cycle, 5:2216–19. << /ascii85encodepages false 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target insights 2007: 2 209–219 209 original research correspondence: dr. med. harald j. hamre, ifaemm e. v., böcklerstr. 5, d-79110 freiburg, germany, tel. +49 761 15 60 307, fax +49 761 15 60 306. email: harald.hamre@ifaemm.de please note that this article may not be used for commercial purposes. for further information please refer to the copyright statement at http://www.la-press.com/copyright.htm use and safety of anthroposophic medications for acute respiratory and ear infections: a prospective cohort study harald j. hamre1, anja glockmann1, michael fischer2, david s. riley3, erik baars4 and helmut kiene1 1institute for applied epistemology and medical methodology, freiburg, germany. 2clinresearch gmbh, cologne, germany. 3university of new mexico school of medicine, santa fe, new mexico, u.s.a. 4louis bolk instituut, driebergen, the netherlands. abstract objective: anthroposophic medications (amed) are widely used, but safety data on amed from large prospective studies are sparse. the objective of this analysis was to determine the frequency of adverse drug reactions (adr) to amed in outpatients using amed for acute respiratory and ear infections. methods: a prospective four-week observational cohort study was conducted in 21 primary care practices in europe and the u.s.a. the cohort comprised 715 consecutive outpatients aged �1 month, treated by anthroposophic physicians for acute otitis and respiratory infections. physicians’ prescription data and patient reports of adverse events were analyzed. main outcome measures were use of amed and adr to amed. results: two patients had confi rmed adr to amed: 1) swelling and redness at the injection site after subcutaneous injections of prunus spinosa 5%, 2) sleeplessness after intake of pneumodoron® 2 liquid. these adr lasted one and two days respectively; both subsided after dose reduction; none were unexpected; none were serious. the frequency of confi rmed adr to amed was 0.61% (2/327) of all different amed used, 0.28% (2/715) of patients, and 0.004% (3/73,443) of applications. conclusion: in this prospective study, anthroposophic medications used by primary care patients with acute respiratory or ear infections were well tolerated. abbreviations: a-: anthroposophy; adr: adverse drug reactions; ae: adverse events; am: anthroposophic medicine; amed: am medication; c-: conventional; ene-patients: eligible, not enrolled patients; iipcos: international primary care outcomes study keywords: adverse effects, complementary therapies, drug monitoring, otitis media, respiratory tract infections introduction anthroposophic medicine (am) is a system of medicine founded by rudolf steiner and ita wegman (steiner and wegman, 2000). am is provided by physicians in 56 countries worldwide. a cornerstone of am therapy is am medication (amed). amed includes more than 2,000 different products of mineral, botanical or zoological origin as well as chemically defi ned substances. amed are prepared in concentrated form or in homeopathic potencies (iaap 2005). all amed are manufactured according to good manufacturing practice and national drug regulations; quality standards of raw materials and manufacturing methods are described in the anthroposophic pharmaceutical codex (iaap 2005). almost all amed in current use have been on the market since the 1970s, some amed even since the 1920s. pre-clinical testing, pharmacovigilance reports, surveys, and 190 clinical studies suggest that adverse drug reactions (adr) to amed are infrequent and mostly mild to moderate (kienle et al. 2006). however, safety data from the clinical trials are often sparse, and in two-thirds of the trials, the number of patients using amed was less than 100. the study iipcos-anthroposophy (international integrative primary care outcomes study (hamre et al. 2005)) provided an opportunity to investigate the use and safety of amed in a large patient sample. 210 hamre et al drug target insights 2007: 2 iipcos-anthroposophy was a prospective, observational comparative study of patients with acute respiratory or ear infections seeing am (n = 715 a-patients) or conventional physicians (n = 301 cpatients). compared to c-patients, a-patients had more favorable clinical outcomes: adjusted odds ratios were 1.54 (95% confi dence interval 1.03– 2.31) for fi rst improvement within 24 hours, 1.61 (1.16–2.22) for fi rst improvement within 3 days, 1.50 (1.07–2.11) for response (major improvement or complete recovery) within 7 days, and 1.29 (0.82–2.00) for response within 14 days. a-patients were also more satisfi ed with therapy (odds ratio for “very satisfi ed”: 1.39 (0.98–1.95)). nineteen (2.7%) a-patients and 18 (6.0%) c-patients reported adverse events (aes) with a possible or probable relationship to medication taken during the study. in the primary analysis (hamre et al. 2005), these aes were not further investigated but were all classifi ed as adr. here we present a more detailed analysis of amed use and safety in a-patients from the iipcos-anthroposophy study. material and methods objective and design the primary objective was to investigate the use and safety of amed. in this study, amed was defi ned as any medication produced by the pharmaceutical companies weleda ag, arlesheim, switzerland or wala-heilmittel gmbh, eckwälden, germany. the secondary objective was to investigate the safety of all (amed + non-am) medications used in am settings. for this purpose we analyzed physicians’ prescription data and patient reports of aes in the am arm of a prospective observational comparative study of am vs. conventional treatment of respiratory and ear infections. setting, physicians, patients, and therapy the study was conducted 1999–2000 in primary care practices in austria, germany, the netherlands, u.k., and u.s.a. participating am physicians were recruited through national am physicians’ associations. all participating physicians had at least 5 years’ clinical experience and were regularly prescribing at least 75% amed in acute respiratory and ear infections. physicians were also required to have computers with internet access available, in order to collect data by remote data entry. the physicians enrolled consecutive outpatients fulfi lling the eligibility criteria. inclusion criteria were (1) age �1 month, (2) chief complaint of sore throat, ear pain, sinus pain, runny nose or cough, (3) onset of chief complaint within seven days. exclusion criteria were dementia, schizophrenia, psychosis, spinal cord injury, stroke, renal failure, severe hepatic disease, ongoing immunosuppressive treatment, chemotherapy or radiotherapy, alcohol or drug abuse. patients were treated according to the physician’s discretion. outcomes medication use medication use was assessed (for amed and for non-am medications, respectively) by the number of different medications used, the number of users and the number of applications. patient selfreporting of compliance with medication prescription was also recorded. medication safety for each ae the causal relationship of ae to all medication used by the patient between study entry and end of the ae was assessed (probable, possible, improbable, no relationship, unable to evaluate). in addition, the most probable cause of the ae (amed, non-am medication, primary illness, intercurrent illness, other) was noted. aes with probable or possible causal relationship to any medication, confi rmed by this analysis, were classified as confirmed adr and described as follows: • name and duration of the adr, intensity: mild/moderate/severe = no/some/complete impairment of normal daily activities. • necessary actions taken against the adr: none, dose reduction/withdrawal/change of medication, admit to hospital, therapeutic counter actions, others. • outcome of the adr: subsided, still being treated, uncertain—still under observation, patient lost to follow-up, permanent health damage, patient died. • adr serious: yes/no (yes: necessary action: “admit to hospital” or outcome: “permanent health damage” or “patient died”). • adr expected: yes/no (yes: adr previously 211 anthroposophic medications drug target insights 2007: 2 reported or may be expected because of a known mechanism of action of ingredients). the frequency of adr (to amed and to any medication) was assessed in relation to the number of different medications, the number of users, and the number of applications. data collection on day 0, the physicians documented primary and concomitant diseases, ongoing medication, and all medication or non-medication therapy prescribed: name, dose, medication form, dosing frequency, number of days prescribed. on days 7, 14, and 28, patients (for children: legal guardians) were interviewed by telephone. the interviews included the degree of compliance with medication prescription, change in medication, and aes. aes were defi ned as any disorders of health, subjective and objective symptoms of illness including changes in laboratory fi ndings, intercurrent medical problems, and accidents observed during the study, regardless of a possible causal relationship to any medication. for patients with complete recovery on days 7 or 14, study participation was terminated and no further followup interviews were performed. data collection, follow-up interviews, and queries were performed by the institute for numerical statistics (now: omnicare clinical research), cologne, germany. except for patients’ day 0 questionnaire, all items were documented by remote data entry. for patients with aes with a probable or possible causal relationship to any medication, according to patient response, the study physicians were contacted by telephone and the following items were checked: diagnosis of chief complaint; complaint-related symptoms and concomitant disease present at study entry; prescribed therapy (name, duration); any consultation between study entry and end of the ae; beginning, end, outcome, and necessary actions against the ae. information about previously reported or expected adr was obtained from the manufacturers. aes were coded according to the world health organization adverse reaction terminology. quality assurance, adherence to regulations the study was approved by local ethics committees and conducted according to the helsinki declaration, the international conference on harmonisation good clinical practice guidelines, and legal requirements. written informed consent was obtained from all patients before enrolment. data analysis medication use patients fulfi lling all eligibility criteria with at least one follow-up interview were included in the prescription analysis. the statistical analysis (spss® 13.0.1) was descriptive. for the prescription analysis, amed with identical ingredients and dosage form but different concentrations were grouped together. medication safety the safety analysis comprised all aes reported by the patient (or legal guardian) as having a possible or probable causal relationship to any medication. for each medication used between study entry and end of the ae, the causal relationship to the reported ae was classifi ed by the fi rst author (hjh) according to criteria formulated in the study protocol (table 1) as probable, possible, improbable, no relationship or “unable to evaluate”. results patient recruitment 26 am physicians (19 general practitioners, three internists and four pediatricians) from 21 different practices in 20 different municipalities participated. the physicians had an average of 18.0 (sd 8.8) years in practice. 853 patients were enrolled: 715 patients were evaluable for prescription analysis; 138 were not evaluable (protocol violations: n = 98, no followup interview: n = 40). the last follow-up interview was performed an average of 16.2 (sd 8.5) days after inclusion. a total of 878 patients were screened but not enrolled: 111 patients refused to participate; 306 did not fulfi ll all eligibility criteria; 461 (100%) screened patients fulfi lled all eligibility criteria (“eligible, not enrolled patients” = ene-patients). reasons for non-enrolment of ene-patients were: physician too busy (68.1%, 314/481 patients), 212 hamre et al drug target insights 2007: 2 practical/technical (12.1%), ongoing therapy for chief complaint (2.0%), special diagnoses, e.g. mental handicap or scarlet fever (5.6%), other or not specifi ed (12.1%). ene-patients (n = 461) did not differ from evaluable patients (n = 715) regarding gender or chief complaint severity; enepatients were median 1.13 years younger (95% confi dence interval: 0.38–1.95, p = 0.0036) and more ene-patients were prescribed antibiotics on day 0 (2.8% vs. 0.8%, p = 0.0153). a total of 83.1% (383/461) of screened, not enrolled patients were prescribed amed. patient characteristics the patients were recruited from germany (50.6%, 362/715 patients), the netherlands (21.3%), austria (14.1%), u.k. (7.3%), and the u.s.a. (6.7%). fifty-three percent (382/715 patients) were females; age groups were 0–17 years (68.1%, 487/715 patients), 18–64 years (30.2%), and � 65 years (1.5%). patients’ chief complaint was cough (39.9%, 285/715 patients), sore throat (26.3%), ear pain (20.0%), sinus pain (7.0%), and runny nose (6.9%). physicians’ diagnosis of chief complaint was pharyngitis/ tonsillitis (25.9%, 185/715 patients), bronchitis (19.3%), otitis media (17.2%), laryngitis/tracheitis (15.1%), rhinitis/common cold/upper respiratory infection unspecifi ed (14.4%), sinusitis (7.4%), and other (0.7%). medication use at study entry, 10.5% (75/715) of patients were using amed for concomitant diseases, and all patients were prescribed amed for their chief complaint. during follow-up (day 1–28), 18.2% (130/715) of patients had at least one further amed prescription. altogether 73,443 applications of 327 different amed were documented, thereof 265 different amed prescribed on day 0–28 (table 2). eight amed were prescribed to at least 50 patients each (table 3), 53 amed were prescribed to at least ten patients each. the most frequent administration forms were liquids (35.4%, n = 830 of 2,346 prescriptions day 0–28), pillules (11.2%), powders (11.0%), ointments (8.7%), ampoules (5.8%), tablets (4.7%), and eardrops (3.8%). the most common indications for amed were acute otitis (18.9%, 467 of 2,468 prescriptions day 0–28 + ongoing medication), bronchitis (15.7%), laryngotracheitis (14.4%), pharyngitis (9.6%), tonsillitis (9.2%), sinusitis (7.0%), and the common cold (5.4%). in addition to amed, patients used 12,130 applications of 218 different non-am medications table 1. criteria for classifi cation of causal relationship between adverse events and medication. probable • rational temporal relationship to the time of intake of the medication. • ae is already known to be a side effect of the medication or may be expected. • regression or disappearance of the ae after discontinuation of medication or dose reduction. • reappearance of the ae after repeated exposure. • ae cannot be explained in a reasonable manner by the clinical state of the patient. possible • rational temporal relationship to the time of intake of the medication. • ae is already known as a side effect of the medication or may be expected. • ae could be explained by numerous other factors. improbable • rational temporal relationship to the time of intake of the medication. • ae has not been reported so far as a side effect of the medication or cannot be expected. • ae persists after discontinuation of the medication or dose reduction. • repeated exposure does not lead to reappearance of the ae. • ae could be explained by numerous other factors. no relationship • no rational temporal relationship to the time of intake of the medication. • ae is evidently caused by other factors, e.g. symptom of a concomitant disease. unable to evaluate • amount and content of data do not permit a judgment of the relationship to the medication. 213 anthroposophic medications drug target insights 2007: 2 during the study, altogether 85,573 applications of 545 different (amed + non-am) medications. 89.7% (641/715) of patients reported taking their medication as prescribed at all evaluable follow-ups. safety of anthroposophic medications reported aes aes were reported by 136 patients. the relationship between the medication used and these aes was, according to patient responses: probable (n = 9 patients), possible (n = 10), improbable (n = 7), no relationship (n = 97), unable to evaluate (n =13). aes with possible or probable causal relationship to any medication, according to patient responses, (n = 19 patients aged 0–53 years, male/ female = 11/8, table 4) were included in the safety analysis. the intensity of these aes was mild (n = 17 patients), moderate (n = 1), and severe (n = 1). median ae duration was 4 days (interquartile range 1–7 days). no ae was serious. between study enrolment and end of the ae the 19 patients had used altogether 62 amed (thereof 57 different amed), 32 non-am medications, and 13 nonmedication therapies, with a median of 4 (range 0–8) medications/therapies per patient. for the 62 amed in question, the causal relationship to the ae was classifi ed as probable (n = 0), possible (n = 2, table 5), improbable (n = 28), and no relationship (n = 32). the most probable cause of the ae was primary or intercurrent illness (n = 13 patients), non-am medication (n = 3), amed (n = 2), other (n = 1). seven patients reported a total of 11 ae with improbable causal relationship to medication used, according to patient responses: asthma, coughing, diarrhea, dry skin, erythema, hay fever, mesenterial adenitis, rash, rhinorrhea, upper respiratory tract infection, and whooping cough. ninety-seven patients reported a total of 151 ae with no relationship to medication used; the most common of these ae were: coughing (n = 22 patients), rhinitis (n = 22), diarrhea (n = 11), gastroenteritis (n = 8), fever (n = 7), and viral infection (n = 7). patients reporting aes with no relationship to medication were asked about the suspected cause of their aes; in 93% (97/104) of interviews, the reported cause was a concomitant illness. frequency of confi rmed adr to amed throughout the study, the patients used 327 different amed, of which two (0.61%) amed were associated with confi rmed adr. a total of 715 patients used amed; in two (0.28%) patients, adr to amed occurred. overall, 73,443 amed applications were documented; three applications (0.004% or one in 24,481 applications) were associated with an adr. two (0.003%) applications were associated with an adr of severe intensity. safety of all medication description of confi rmed adr to any medication five adr were confi rmed (table 4). median adr duration was 2 (range 1–8) days; the fi ve adr were observed in conjunction with altogether 25 medication applications. the adr intensity was mild in four patients and severe in one patient. all fi ve adr subsided after dose reduction (n = 2) or withdrawal (n = 3) of the causative medication. none of the adr were serious, none were unexpected. no adverse reactions to non-medication therapies were found. table 2. overview of medication use. patients with medication different medications applications medication amed non-am all amed non-am all amed non-am all a) ongoing at study entry, 75 78 125 94 59 153 8,656 3,576 12,232 used day 0–28 b) prescribed at day 0 715 255 715 223 76 299 59,090 3,842 62,932 c) prescribed at day 1–28 131 179 211 130 119 249 5,697 4,712 10,409 b+c) prescribed at day 0–28 715 377 715 265 171 436 64,787 8,554 73,341 a+b+c) ongoing 715 429 715 327 218 545 73,443 12,130 85,573 + prescribed day 0–28 214 hamre et al drug target insights 2007: 2 ta bl e 3. m os t f re qu en tly p re sc rib ed a nt hr op os op hi c m ed ic at io ns o n d ay 0 –2 8. p at ie nt s w ith a pp lic at io ns pr es cr ip tio n* m ed ic at io n in gr ed ie nt s m an uf ac tu re r n % n % p la nt ag o b ro nc hi al b al m 10 0 g co nt ai ns : c am ph or a 2 g, c er a fl a va 1 5 g, d ro se ra r ot un di fo lia / w al a 11 7 16 .4 % 19 45 3. 0% in te rm ed ia /a ng lic a e pl an ta to ta fe rm . d 3 1. 0 g, e uc al yp ti ae th er ol eu m 0 .5 g , p et as ite s hy br id us e r ad ic e fe rm . d 1 1. 0 g, p la nt ag o la nc eo la ta e fo lii s fe rm . d 1 1. 0 g, t er eb in th in a la ric in a 5. 0 g, t hy m i a et he ro le um 0 .5 g e ry si do ro n® 1 l iq ui d 10 g c on ta in s: a pi s m el lifi c a d 2 1 g, b el la do nn a d 2 1 g w el ed a 98 13 .7 % 36 05 5. 6% c in na ba r co m p. p ow de r 10 g c on ta in s: a pi si nu m d 5 3. 3 g, b el la do nn a d 3 3. 3 g, w el ed a 96 13 .4 % 30 37 4. 7% c in na ba r d 5, 3 .3 g p ne um od or on ® 1 l iq ui d 10 g ( = 10 .5 m l) co nt ai ns : a co ni tu m n ap el lu s. d 2 0. 5 g, w el ed a 70 9. 8% 17 89 2. 8% b ry on ia d 2 1 g c in na ba r/ p yr it ta bl et s 1 ta bl et c on ta in s: c in na ba r d 20 1 76 m g, p yr it d 2 20 m g w el ed a 69 9. 7% 20 75 3. 2% b ol us e uc al yp ti co m p. 10 g c on ta in s: a pi s m el lifi c a ∅ ( = d 1) 0 .1 g , b el la do nn a ∅ 0 .0 02 g , w el ed a 59 8. 3% 22 23 3. 4% p ow de r e uc al yp tu s ∅ ( = d 1) 0 .1 g , w hi te c la y 9. 97 –1 0 g p in e r ev iv in g b at h m ilk c on ta in s: w at er ( a qu a) , p ot as si um o liv at e, a bi es a lb a w el ed a 53 7. 4% 45 9 0. 7% le af o il, a bi es s ib iri ca o il, l im on en e b er do ni a n os e s pr ay 1 g co nt ai ns : b er be ris v ul ga ris e fr uc tib us fe rm d 2 0. 1 g, c itr us w al a 50 7. 0% 15 99 2. 5% lim on e fr uc tib us fe rm d 1 0. 1 g, c yd on ia o bl on ga e fr uc tib us fe rm d 1 0. 1 g, q ua rz ( s ili ce a) d 19 0 .1 g e ch in ac ea c om p. 10 0 g co nt ai ns : a rg en tu m n itr ic um d 13 1 .0 g , c al en du la o ffi ci na lis , w al a 49 6. 9% 10 62 1. 6% m ou th sp ra y fl o s re c. 1 0. 0 g, e ch in ac ea p al lid a, h er ba r ec . 1 0. 0 g, e uc al yp tu s gl ob ul us e fo lii s fe rm . d 1 1. 0 g, g in gi va b ov is g l d 4 1. 0 g, g in gi va b ov is g l d il. d 8 1. 0 g, s al vi a of fi c in al is , f ol iu m r ec . 1 0. 0 g, to ns ill ae p al at in ae b ov is g l d 4 1. 0 g, t on si lla e pa la tin ae b ov is g l d 8 1. 0 g s tic ta l iq ui d s tic ta d 3/ d 6 w el ed a 48 6. 7% 21 44 3. 3% h ep ar s ul fu ris p ow de r h ep ar s ul fu ris d 3/ d 4/ d 6/ d 12 /d 30 w el ed a 47 6. 6% 19 70 3. 0% c ha m om ill a co m p. 1 su pp os ito ry ( 1 g) c on ta in s: b el la do nn a d 3 20 m g, c ha m om ill a w el ed a 43 6. 0% 55 5 0. 9% s up po si to ry re cu tit a, r ad ix , e th an ol . d ec oc tu m d 2 20 m g, e ch in ac ea p ur pu re a, pl an ta to ta ∅ 1 35 m g, p ap av er s om ni fe ru m , f ru ct us im m at . d 3 20 m g, a rg en tu m m et al lic um p ra ep ar at um d 19 2 0 m g a co ni tu m c om p. e ar dr op s 10 g co nt ai ns : a co ni tu m n ap el lu s e tu be re fe rm . d 9 1. 0 g, w al a 42 5. 9% 16 80 2. 6% c am ph or a 0. 1 g, l av an du la e ae th er ol eu m 0 .1 g , q ua rz ( s ili ce a) d 9 1. 0 g 215 anthroposophic medications drug target insights 2007: 2 frequency of confi rmed adr to any medication throughout the study, the patients used 545 different (amed + non-am) medications, of which fi ve (0.92%) medications were associated with confi rmed adr. a total of 715 patients used medication; in fi ve (0.70%) patients adr occurred. adr of severe intensity occurred in one (0.14%) patient. overall, 85,573 medication applications were documented; 25 applications (0.03% or one in 3,423 applications) were associated with an adr. two (0.002%) applications were associated with an adr of severe intensity. discussion overall study fi ndings this is one of the fi rst detailed analyses (hamre et al. 2006) of use and safety of amed within a large prospective cohort study. in outpatients with acute respiratory and ear infections we found a low frequency of confirmed adr to amed (0.28% of amed users and 0.004% of amed applications). strengths and limitations this study has several strengths: data collection was prospective with extensive quality assurance guaranteeing high data quality (100% source data verifi cation performed for all baseline prescription data, i.e. for 88% of amed applications). patients were recruited by experienced physicians (average 18 years in practice) in a range of healthcare settings (20 different municipalities in five countries). follow-up rates were high (only 5% of otherwise evaluable patients were lost to follow-up). aes were documented in all patients at all follow-ups (instead of relying on spontaneous reporting). in the safety analysis, aes were investigated with respect to a causal relationship to all ongoing medication according to predefi ned criteria, checking each case with physicians and patients. selection bias is unlikely for this study: screening data suggest that enrolled patients are representative for eligible patients. moreover, the percentage of eligible but not enrolled patients prescribed amed (83%) was lower—and not higher—than the percentage of evaluable patients prescribed amed (100%). therefore, if any signifi cant “selection-out” of patients from the in fl u do ® l iq ui d 10 g ( = 11 .1 m l) co nt ai ns : a co ni tu m n ap el lu s d 3 1 g, b ry on ia w el ed a 40 5. 6% 11 30 1. 7% d 2 0. 6 g, e uc al yp tu s d 2 0, 5g , e up at or iu m p er fo lia tu m d 2 0. 4 g, p ho sp ho ru s d 4 1 g, s ab ad ill a d 3 1 g le vi st ic um r h li qu id le vi st ic um d 2/ d 3/ d 4/ d 6/ d 10 w el ed a 40 5. 6% 12 69 2. 0% c ou gh e lix ir 10 0g ( = 76 m l) co nt ai ns : 5 g aq ue ou s ex tr ac t f ro m 0 .6 g w el ed a 34 4. 8% 91 6 1. 4% a lth ae ae r ad ix , 3 0 g aq ue ou s de co ct um fr om ( 0. 15 g s ol an um du lc am ar a, s tip ite s si cc .; 0. 35 g m ar ru bi um v ul ga re , h er ba s ic c. ; 0. 5 g a ni si fr uc tu s; 0 .3 5 g s er py lli h er ba ; 2 .8 5g t hy m i h er ba ), d ro se ra d 2 0. 1g , e xt ra ct um m al ti 5 g, ip ec ac ua nh a, e th an ol . de co ct um ∅ ( = d 1) 0 .1 g , p ul sa til la v ul ga ris d 3 0. 01 g k al iu m c ar bo ni cu m l iq ui d k al iu m c ar bo ni cu m d 3/ d 4/ d 6/ d 10 /d 12 /d 20 /d 30 w el ed a 34 4. 8% 15 41 2. 4% s ili ce a (q ua rz ) 1% e ar dr op s 10 g ( = 10 .9 m l) co nt ai ns : q ua rz ( s ili ce a) 0 .1 g w el ed a 34 4. 8% 67 6 1. 0% n os e b al m fo r c hi ld re n 10 g c on ta in s: b al sa m um p er uv ia nu m 0 .0 5 g, b er be ris v ul ga ris w al a 32 4. 5% 61 1 0. 9% e fr uc tib us fe rm . ∅ 1 .0 0 g, p ru nu s s pi no sa , f ru ct us r ec . 0 .5 0 g, s ili ce a co llo id al is 0 .0 5 g c ap si cu m a nn uu m l iq ui d c ap si cu m a nn uu m d 3/ d 4/ d 6/ d 10 w el ed a 32 4. 5% 41 8 0. 6% o th er m ed ic at io ns ( n = 24 5) 34 08 3 52 .6 % to ta l 71 5 10 0. 0% 6 47 87 10 0. 0% *m ul tip le r es po ns es p os si bl e, s um o f p er ce nt ag es � 10 0% . d : d ec im al p ot en ci es ( 1: 10 d ilu tio n; e .g . d 3 = 1: 10 00 ). ∅ : m ot he r tin ct ur e. g l: m ot he r tin ct ur e pr ep ar ed u si ng g ly ce ro l./ : m ed ic atio n ex is ts in d iff er en t c on ce nt ra tio ns g ro up ed to ge th er . 216 hamre et al drug target insights 2007: 2 (c on tin ue d) ta bl e 4. a dv er se e ve nt s (a e ) re po rt ed w ith p os si bl e or p ro ba bl e ca us al r el at io ns hi p to a ny m ed ic at io n, a cc or di ng to p at ie nt fo llo w -u p re sp on se . p at ie nt no . a ge ye ar s s ex d ia gn os is c on co m ita nt di se as e n th er ap ie s a dv er se e ve nt n am e in te nsi ty d ur at io n d ay s m os t p ro ba bl e ca us e* 1 7 f a cu te to ns ill iti s p ur ul en t r hi ni tis 4 n as al c on ge st io n m ild � 1 in te rc ur re nt il ln es s 2 0 m a cu te o tit is m ed ia n o 7 c on di tio n ag gr ava te d, fe ve r m ild 1 p rim ar y ill ne ss 3 23 f a cu te ph ar yn gi tis n o 2 s el fcr iti ci sm m ild 2 o th er 4 45 f a cu te u r i un sp ec ifi ed d us t m ite a lle rg y 6 n au se a m ild 4 p rim ar y or in te rc ur re nt il ln es s 5 8 m a cu te to ns ill iti s n o 4 c ra m p ab do m in al , v om itin g m ild � 1 p rim ar y ill ne ss ( m es en te ria l ad en iti s) a bd om in al p ai n m ild � 26 6 2 m a cu te p ha ry ngi tis n o 4 d ia rr he a m ild 1 c on co m ita nt m ed ic at io n (iv y le af e xt ra ct ) 7 6 m b ro nc hi tis a to pi c de rm at iti s 8 e ye lid e de m a m ild 3 c on co m ita nt m ed ic at io n (s od iu m c ro m og ly ca te a nd / or s al bu ta m ol ) 8 1 f a cu te la ry ng iti s an d tr ac he iti s n o 3 r es tle ss ne ss a t ni gh t m ild 8 p rim ar y ill ne ss 9 1 m a cu te o tit is m ed ia n o 6 f ac ia l r as h m ild � 7 in te rc ur re nt il ln es s 10 40 f a cu te s in us iti s n o 3 g as tr oin te st in al di so rd er n o s m ild 8 c on co m ita nt m ed ic at io n (m yr to l) 11 8 m b ro nc hi tis a to pi c de rm at iti s 2 r es tle ss ne ss m ild 8 p rim ar y ill ne ss 12 39 f a cu te to ns ill iti s n o 3 r as h m ild 7 p rim ar y ill ne ss 13 53 f a cu te la ry ng iti s an d tr ac he iti s n o 2 m ou th d ry m ild 4 p rim ar y or in te rc ur re nt il ln es s 14 32 f a cu te to ns ill iti s n o 0 a bd om in al p ai n m ild � 1 p rim ar y or in te rc ur re nt il ln es s 15 39 f b ro nc ho pn eu m on ia n o 3 s le ep d iffi c ul t s ev er e 2 p ne um od or on ® 2 l iq ui d, w el ed a* * 16 14 m a cu te to ns ill iti s n o 6 in je ct io n si te sw el lin g an d re dn es s m ild 1 p ru nu s s pi no sa 5 % in je ct io n, w el ed a* * 217 anthroposophic medications drug target insights 2007: 2 study took place, these would have been patients not prescribed amed, which would not affect the present analysis. a limitation of our safety analysis is its restriction to aes reported by patients as having a possible or probable causal relationship to any medication. unidentifi ed adr could be present among the other aes. however, inspection of these aes suggests that in the overwhelming majority of cases they were symptoms of primary disease or intercurrent illness. in the safety analysis, aes were classifi ed as “confi rmed adr” (probable/possible relationship to a medication) or “not confi rmed adr” (improbable/ no relationship/unable to evaluate). false-negative classifications (true adr is not confirmed) are unlikely, since for all medication for which an adr was not confirmed, there was either no rational temporal relationship to the ae, or another cause (primary or intercurrent illness or another medication) was much more likely. however, false-positive classifi cations cannot be ruled out; the three “confi rmed adr” to non-am medication might instead be symptoms of primary or intercurrent illness. implication for research classifi cation of causal relationship between medication and aes in this study most aes were disease symptoms and other subjective symptoms of 1–7 days’ duration. thus a major challenge of the safety analysis was to distinguish between true adr and symptoms of primary or intercurrent illness. for example, in 13 analyzed patients the ae started the same day as the medication was first t a k e n o r w i t h i n t h e f o l l o w i n g 1 – 2 d a y s , suggesting a “rational temporal relationship to the time of intake of the medication” (table 1). however, if the ae is a symptom of the primary disease for which treatment is sought, a temporal relationship between beginning of treatment and beginning of the ae does not in itself indicate a causal relationship between the treatment and the ae. the same applies to cases where the end of the ae coincided with end of treatment, since am treatment of respiratory and ear infections is usually applied as long as symptoms persist. these problems will have to be addressed in future safety research into amed. 17 35 f a cu te n as oph ar yn gi tis (c om m on c ol d) n o 5 d ry li ps m ild 4 p rim ar y ill ne ss 18 29 m a cu te to ns ill iti s n o 3 c on ce nt ra tio n im pa ire d, fe el in g ba d, u rin e ab no rm al m od era te 5 p rim ar y ill ne ss 19 5 f b ro nc hi tis a st hm a 5 in cr ea se d bo w el m ov em en ts m ild 11 p rim ar y or in te rc ur re nt il ln es s n th er ap ie s: n um be r of d iff er en t m ed ic at io ns o r no nm ed ic at io n th er ap ie s us ed b et w ee n st ud y en tr y an d en d of th e a e ( al l p at ie nt s ex ce pt n o. 1 4 us ed a nt hr op os op hi c m ed ic at io ns b et w ee n st ud y en tr y an d en d of a e ). c on fi r m ed a dv er se d ru g re ac tio ns in b ol d ty pe s. * m os t p ro ba bl e ca us e w as c la ss ifi ed b y th e au th or s; o th er it em s w er e do cu m en te d by p hy si ci an s an d pa tie nt s. ** s ee t ab le 5 . 218 hamre et al drug target insights 2007: 2 table 5. confi rmed adverse reactions to anthroposophic medications. patient no. 15: a woman aged 39 with bronchopneumonia (including severe cough, very severe hemoptysis, moderate shortness of breath, moderate expiratory wheezing, severe sputum expectoration, moderate pain with coughing or breathing, severe discomfort, and fever � 39.5 °c) was treated with three amed (pneumodoron® 1 liquid 10 drops hourly, pneumodoron® 2 liquid 10 drops hourly, tabulettae calcarea cum ferro three times daily) and quark compresses twice daily. the fi rst two subsequent nights she experienced severe sleeplessness which subsided after she stopped taking pneumodoron® 2 at night. another possible explanation for this ae is severe illness present at study entry. she has however taken pneumodoron® 2 once after the study upon which she experienced sleeplessness which again subsided after stopping taking pneumodoron® 2 at night. pneumodoron® 2 (10 g = 11.1 ml contains: phosphorus d4 1 g, tartarus stibiatus d2 1g) was classifi ed as the most probable cause of the ae but her bronchopneumonia may have contributed to the intensity of the ae. according to the manufacturer’s information, this ae can be expected from pneumodoron® 2 in sensitive individuals, but has not been previously reported. patient no. 16: a 14-year old boy with acute tonsillitis was treated with daily subcutaneous injections of prunus spinosa, summitates 5% and three further amed and developed mild swelling and redness at the injection site. the reaction was observed after the fi rst injection and subsided after subsequent dose reduction of prunus (the dose of the other three amed was not reduced). this adr has not been reported to the manufacturer previously, but has been observed repeatedly in other patients by the boy’s physician. prescription profi le of amed—implications for safety research a striking finding of this study is the broad prescription profi le of amed: 715 patients were prescribed altogether 265 different amed; 53 amed were prescribed to at least 10 patients each. safety analysis assessed amed as a single package: 2.7% (19/715) of patients reported aes suspected to be adr and 0.3% (2/715) of patients had confi rmed adr to amed. a conventional approach, assessing safety of individual amed would not have been possible: to detect adr from a single medication in a hypothetical frequency of 1% with suffi cient power, at least 500 patients per medication are needed. to detect adr from the 53 most common amed with a frequency of 0.3% (as in this analysis), a sample size of approximately 90,000 patients would be required. if 2.7% of patients report aes suspected to be adr (as here), a study of this size would necessitate examining more than 2,000 aes. this task would, if performed as in the present analysis, hardly be feasible. therefore, safety studies of amed for indications with broad prescription profi les will generally have to assess amed as a package rather than as single medications. implications for risk-benefi t assessment for most individual amed, scientifi c evidence of effectiveness is limited. this study evaluated the effectiveness and safety of am treatment (265 individual amed, adjunctive non-am medication, and adjunctive non-medication therapies) of respiratory and ear infections (hamre et al. 2005). using a conventional approach, focusing on each of the 265 individual amed, a benefi t-risk assessment would not have been possible: effectiveness was not proven for any individual amed (the study was not designed for this purpose) and adr from two amed were found, leading to a negative benefi t-risk profi le for two amed and inconclusive data for the remaining 263 amed. instead, all am treatment was analyzed as one therapy package, thus a benefi t-risk assessment in comparison to conventional treatment was possible. a broad amed prescription profi le is typical not only for respiratory and ear infections but for many other indications (husemann and wolff 1987; ritchie et al. 2001). therefore, therapy package evaluation will probably have an important role in future benefit-risk assessments of amed. implications for practice safety of am this study of acute respiratory and ear infections demonstrated an excellent safety profi le, both for comprehensive am treatment (adr in 0.7% of patients and 0.03% of applications with a median duration two days; severe intensity adr in 0.1% of patients and 0.003% of applications) and for amed (adr in 0.3% of patients and 0.004% of 219 anthroposophic medications drug target insights 2007: 2 applications; severe intensity adr in 0.1% of patients and 0.003% of applications). comparative risk-benefi t assessment in the primary analysis of this study (hamre et al. 2005), am treatment had a signifi cantly lower frequency of reported adr (2.7%, n = 19/715 patients) than conventional (c-) treatment (6.0%, n = 18/301) (fisher ’s exact test, 2-tailed, p = 0.0157). for practical reasons, the present more precise secondary safety analysis was restricted to a-patients, confi rming adr in 0.7% (5/715) of a-patients. in c-patients the frequency of confi rmable adr may be between 0/301 and 18/301. at both ends of this range, this frequency will not be lower but will either be comparable to (p = 0.3295) or signifi cantly higher (p � 0.00005) than the frequency in a-patients. in other words: am had comparable or lower risk than conventional treatment. since am had more favorable clinical outcomes, am had a more favorable benefi t-risk profi le than conventional treatment. conclusion in this prospective study of 715 outpatients with acute respiratory and ear infections, we found a low frequency of adr and no serious adr to amed. study results suggest that short-term amed therapy for acute respiratory and ear infections is well tolerated. acknowledgments this analysis was funded by grants from weleda and wala. the sponsors had no infl uence on study design or planning; on the collection, analysis, or interpretation of data; on the writing of the manuscript; or on the decision to submit the manuscript for publication. we thank peter vögele, wala and jasmin peschke, weleda for providing safety data on amed, and gunver s. kienle and wilfried tröger for valuable help and advice. our special thanks go to the study physicians and their patients for participating. confl ict of interest: all authors declare that they have no confl icts of interest. references hamre, h.j., fischer, m., heger, m. et al. 2005. anthroposophic vs. conventional therapy of acute respiratory and ear infections: a prospective outcomes study. wien klin wochenschr., 117:256–68. hamre, h.j., witt, c.m., glockmann, a. et al. 2006. use and safety of anthroposophic medications in chronic disease: a 2-year prospective analysis. drug saf., 29:1173–89. husemann, f. and wolff, o. 1987. the anthroposophical approach to medicine. volume 2. london: rudolf steiner press. iaap. 2005. anthroposophic pharmaceutical codex apc. dornach: the international association of anthroposophic pharmacists. kienle, g.s., kiene, h. and albonico, h.u. 2006. anthroposophic medicine: effectiveness, utility, costs, safety. stuttgart, new york: schattauer verlag. ritchie, j., wilkinson, j., gantley, m. et al. 2001. a model of integrated primary care: anthroposophic medicine. london: department of general practice and primary care, st bartholomew’s and the royal london school of medicine and dentistry, queen mary university of london. steiner, r. and wegman, i. 2000. extending practical medicine. fundamental principles based on the science of the spirit. ga 27. bristol: rudolf steiner press. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedjoboptions true /dscreportinglevel 0 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review correspondence: m. omar f. khan, tel: (1)580-774-3064; fax: (1)580-774-7020; email: faruk.khan@swoasu.edu please note that this article may not be used for commercial purposes. for further information please refer to the copyright statement at http://www.la-press.com/copyright.htm trypanothione reductase: a viable chemotherapeutic target for antitrypanosomal and antileishmanial drug design m. omar f. khan college of pharmacy, southwestern oklahoma state university, 100 campus drive, weatherford, ok 73096, u.s.a. abstract: trypanosomiasis and leishmaniasis are two debilitating disease groups caused by parasites of trypanosoma and leishmania spp. and affecting millions of people worldwide. a brief outline of the potential targets for rational drug design against these diseases are presented, with an emphasis placed on the enzyme trypanothione reductase. trypanothione reductase was identifi ed as unique to parasites and proposed to be an effective target against trypanosomiasis and leishmaniasis. the biochemical basis of selecting this enzyme as a target, with reference to the simile and contrast to human analogous enzyme glutathione reductase, and the structural aspects of its active site are presented. the process of designing selective inhibitors for the enzyme trypanothione reductase has been discussed. an overview of the different chemical classes of inhibitors of trypanothione reductase with their inhibitory activities against the parasites and their prospects as future chemotherapeutic agents are briefl y revealed. key words: trypanothione, glutathione, chagas disease, sleeping sickness, rational drug design introduction trypanosomiasis and leishmaniasis are among the major debilitating and devastating tropical diseases that are targets for the world health organization’s special program research and training in tropical diseases (tdr) (hyde, 1990) and most recently the drugs for neglected diseases initiative (dndi, www.dndi.org). these diseases are caused by the parasites of the genus trypanosoma and leishmania. the present review focuses on the major human diseases caused by trypanosomal and leishmanial infections and the potential targets for designing chemotherapeutic agents against these diseases with special emphasis on trypanothione reductase. table 1 gives an outline of the major human trypanosomiasis and leishmaniasis with their global annual disease burdens (as of 1999) in terms of disability adjusted life years (daly). table 1.the major trypanosomiasis and leishmaniasis, causative agents, their global burdens in terms of disability adjusted life years (daly) and current treatments. disease causative agents daly* current treatments (million/year) african trypanosoma brucei 1.2 suramine, pentamidine, trypanosomiasis gambiense and dfmo, tryparsamide or sleeping t. b. rhodesiense sickness american t. cruzi 0.6 benznidazole, nifurtimox trypanosomiasis or chagas disease visceral leishmania donovani 1.7 pentostam, glucantime, leishmaniasis or kalazar dermal l. major, l. tropica, aminosidine leishmaniasis l. braziliensis, l. mexicana or tropical sore *taken from world health report 1999, publ. world health organization geneva (1999). drug target insights 2007: 2 129–146 130 khan suramine (1) and pentamidine (2) are useful drugs for treating human african trypanosomiasis (hat) during early infection, but being highly charged, cannot cross the blood brain barrier and are of no use for late stage infection with involvement of central nervous system (cns) with either trypanosoma brucei gambiense or t. b. rhodesiense. melarsoprol (3), a trivalent arsenical, or tryparsamide (4) is then used. difl uoromethylornithine (dfmo, 5) is effective in treatment of hat, but is not very effective against rhodesiense sleeping sickness where large doses must be used, resulting in significant side-effects, including bone marrow suppression (meshnick, 1984; neva and brown, 1994). nifurtimox (6), a nitrofuran derivative, is the best drug currently available for treating chagas disease but is still considered investigational. it is thought to kill trypanosomes selectively through futile cycling by the formation of hydrogen peroxide and toxic oxygen species (docampo and stoppani, 1979; le trant et al. 1983; docampo and moreno, 1984a and b, 1986; neva and brown, 1994). benznidazole (7) is of equivalent effectiveness and is used in south america in the acute stage. both drugs must be given for several months and are associated with severe side effects (neva and brown, 1994). recently, the present and future prospects of chemotherapy of hat, in addition to the possible mode of action and the mechanism of resistance of the current chemotherapeutic agents have been reviewed (fairlamb, 2003a). pentavalent antimonials e.g. pentostam (8) is the recommended treatment for visceral leishmaniasis but are toxic and developed resistance (croft, 1988; grogl et al. 1992). relapse of disease or only partial response is more common in kenya, the sudan, and india than in mediterranean or latin american kala azar, where a second or longer course of treatment is often needed. overall, the demand for chemotherapeutic agents for the treatment of chagas disease, sleeping sickness and kala azar is desperate. those needing treatment are mainly in impoverished rural and urban communities with poor housing and limited access to medical attention, and in countries where basic healthcare infrastructures have yet to be developed. approved chemotherapies that are available were developed in the fi rst half of the last century (suramine, pentamidine, arsenicals and antimonials); some would fail today’s more stringent standards for drug safety. given the initial success of a largely empirical approach, progress in drug development in recent years has been poor and, undoubtedly there is great need for new less toxic treatments for human diseases by parasitic trypanosomes and leishmanias. in view of the economies of the third world countries suffering most from these diseases, such drugs will have to be cheap and simple to administer. so3 h n ch3 h n n h o-o3s so3 o o 2 1 +h2n h2n o(ch2)5o nh2 + nh2 2 n n n n h as s s ch2oh h2n nh2 3 as o o-na+ho nhch2conh24 h2n chf2 nh2 cooh 5 o n n o2n so2 ch3 6 n n ch2conhch2 no2 7 o sb o o sb o o ho oh ohoh ho oh cooh cooh o o 8 figure 1. structures of the currently available drugs for the treatment of trypanosomiasis and leishmaniasis. drug target insights 2007: 2 131 target for antitrypanosomal and antileishmanial drug design potential chemotherapeutic targets for trypanosomiasis and leishmaniasis in this genomic, proteomic, and bioinformatics era of target identifi cation, scores of potential targets for antitrypanosomal and antileishmanial chemotherapy will be emerging. in the pregenomic era, basic research in molecular biology and multidimensional research initiatives has identifi ed some biological features associated with the development of trypanosomiasis that have been well documented and studied extensively. ergosterol biosynthesis, parasite specifi c proteases and reductases, purine salvage and phospholipid biosynthesis (for review see urbina, 2003) could be turned into targets, provided that the following two requirements can be met: (a) the target must be essential for the survival of the parasites, and (b) the target must be such that a counterpart in the mammalian host either does not exist or is sufficiently different to allow selective inhibition (wang, 1995). the technological advancements related to post-target selection are also important criteria. because the target must be suitable for study at the molecular level that include most importantly the high throughput screening for which convenient, cheap and sensitive assay methods are highly desirable. glycolytic pathway as trypanosomal cells are completely energetically dependent on glycolysis it might be used to develop new trypanocidal drugs (michels, 1988). compertmentation of glycolytic enzymes is generally assumed to result in an enhancement of the rate of glycolysis in bloodstream trypanosomes, approximately 50-times higher than that in mammalian cells (brohn and clarkson, 1980). this appears to be necessary to compensate for poor yields of energy. trypanosomes need to replicate every 6 to 8 h in mammalian blood and varian surface glycoproteins (vsgs) must be replaced frequently to evade a host immune response (donelson and rice-ficht, 1985). thus it has been suggested that inhibition of any one of the glycolytic enzymes inside the glycosomes may block glycolytic activity and kill bloodstream trypanosomes (clarkson and brohn, 1976; michels, 1988). the three dimensional (3d) structure of t. brucei glycosomal triosephosphate isomerase (tim), determined at 2.4 å resolution, was found to be very similar to that of mammalian tim (wierenga et al. 1987). the 3d structure of glycosomal glyceraldehyde-3-phosphate dehydrogenase (gadph) (vellieux et al. 1993) could provide opportunities for designing selective inhibitors as it differs from the mammalian homolog (verlinde et al. 1994; wang, 1995). bloodstream t. b. brucei imports glucose by facilitated diffusion and the uptake of glucose apparently represents the rate-limiting step in glycolysis. the genes encoding trypanosomal glucose transporters are tandemly arranged in a multigene family consisting of two homologous groups, trypanosome hexos transporter (tht)1 and tht2. tht1-encoded glucose transporters, preferentially expressed in a bloodstream form, have a moderate sensitivity to cytochalasin b and recognize d-fructose as substrate, thereby distinguishing them from the human erythrocyte glucose transporter. they are potential targets for antitrypanosomal chemotherapy (for review, see wang, 1995). dna topoisomerases many of the established antiprotozoal agents are known to bind to dna. there are two potential sites for dna binding in members of the kinetoplastida: nuclear and kinetoplast dna. in general, dna binding agents would be expected to be active against protozoa, but toxicity is a major factor. it was assumed that binding to dna leads directly to inhibition of dna-dependent processes, but it is now generally accepted that intercalating agents induce topoisomerase ii – mediated strand breaks in dna (brown, 1987). trypanosomal topoisomerase ii inhibitors affect both nuclear and mitochondrial dna and may prove to be effective and safe antitrypanosomal drugs (shapiro, 1993) as they differ structurally from mammalian topoisomerase ii (shapiro and showalter, 1994). dna topoisomerase i could also serve as an intracellular target, as its inhibition can cause dna-cleavage and ultimate death of trypanosomes (bodley et al. 1995). ergosterol biosynthesis ergosterol biosynthesis is a novel metabolic pathway essential for parasitic survival lacking a counterpart in the host. several enzymes of this pathway, e.g. squalene synthase, fernesylpyrophosphate synthase are capable of depleting drug target insights 2007: 2 132 khan as shown in figure 2, ornithine decarboxylase (odc), s-adenosyl-l-methionine decarboxylase ( s a m d c ) a n d s p e r m i d i n e s y n t h e t a s e i n trypanosomes serve crucial functions (fairlamb and bowman, 1980) and may be potential targets for antitrypanosomal chemotherapy. little is known about trypanosomal samdc except that it did not cross-react with human samdc antiserum (tekwani et al. 1992). detailed comparison of mammalian and trypanosomal samdcs have not yet been done nor have crystal structure and amino acid sequence been determined, steps important for designing drugs active against this enzyme. trypanothione is a conjugate of glutathione and the polyamine spermidine. this polyamine component of the structure of trypanothione disulfi de (t[s]2) rationalized the actions of several antitrypanosomal and antileishmanial drugs. for example, dfmo (5), the fi rst new drug licensed to treat hat for over 50 years, inhibits odc, which catalyzes the initial step in polyamine biosynthesis (fig. 2), decreasing the trypanothione pool. the origin of the trypanocidal effect of dfmo has not been established (fairlamb and cerami, 1992). figure 2. metabolism and function of trypanothione, showing possible sites of action of trypanocidal compounds. the insert above illustrates the futile redox cycling by nitro compounds (rno2) to form hydrogen peroxide (h2o2) and hydroxyl radicals (oh ●). abbreviations: bso, buthionine sulfoximine; dfmo, difl uoromethylornithine; r-as=o, melarsen oxide; mel t, melarsen trypanothione adduct; put, putrescine; spd, spermidine; dsam, decarboxylated s-adenosylmethionine; mta, methylthioadenosine (modifi ed from krauth-siegel et al. 1987). nadph nadp+ rno2 rno o2 o2 o2 h2o2 oh + oh + o2 2h2o glu + cys bso -glu-cys gly gsh spd n1-gsh-spd n8-gsh-spd t[sh]2 gsh gsh t[s]2 gssg 2gsh nadphnadp+ ras=o mel t rno2 r as s s t (mel t) put odc co2 dfmo mta dsam co2 sam met + atppi, ppi endogenous sterols, and therefore represent viable chemotherapeutic targets (for review, see linares et al. 2006). purine salvage pathway some striking differences between parasites and their mammalian host are apparent in purine metabolism. unlike their mammalian host, most parasites lack the de novo purine biosynthetic mechanisms and rely on salvage pathways to meet their purine needs. there are suffi cient distinctions between enzymes of the purine salvage pathway in host and parasite that can be exploited to design specifi c inhibitors or “subversive substrates” for the parasitic enzymes. furthermore, the specifi cities of purine transport, the fi rst step in purine salvage, differ signifi cantly between parasites and their mammalian host to allow selective inhibitor design (for review see el kouni, 2003). polyamine biosynthesis the ability to synthesize polyamines (fig. 2) is vitally important for the proliferation of bloodstream hat in an environment defi cient in polyamines. drug target insights 2007: 2 133 target for antitrypanosomal and antileishmanial drug design trypanothione reductase (tr) and the synthetic enzyme of trypanothione metabolism found in trypanosomatids exemplify unique features of the organisms. as shown in figure 2, trypanothione synthesis proceeds from glutathione; the similar redox potentials allow for nonenzymatic thiol-disulfi de exchange reactions to occur (fairlamb and cerami, 1992). considerable advances have been made in characterization and inhibitor design against tr. the key enzyme of the two-step trypanothione biosynthesis, glutathionyl spermidine synthetase, was isolated, partially sequenced and kinetically analyzed (koenig et al. 1997). the utilization of trypanothione for the reduction of hydroperoxides has remained a matter of debate. a trypanothione peroxide activity, presumed to substitute for the glutathione peroxide activity typical of host metabolism (flohé, 1989), was observed in crude extracts of various trypanosomatids (henderson et al. 1987a). consistent failures to isolate putative trypanothione peroxidases, and substantial spontaneous reaction rates between trypanothione and hydrogen peroxide, led to the conclusion that trypanothione-dependent peroxide metabolism may represent a non-enzymatic event (carnieri et al. 1993). recent discoveries have demonstrated (nogoceke et al. 1997) that the reduction of peroxides by trypanothione is an enzymatic process, but in contrast to previous expectations, is catalyzed by two distinct proteins in concert (flohé, 1998). lipoamide dehydrogenase (lipdh) is another fl avoprotein dependent enzyme, which has been discussed as a target molecule for antitrypanosomal therapy. in t. cruzi, an organism highly susceptible to oxidative stress, lipdh participates in the redox cycling of nifurtimox, one of the most effective anti-chagas agents (krauth-siegel and schöneck, 1995). trypanothione reductase as a chemotherapeutic target in the pre-genomic era investigation of mode of action of arsenical drugs and glutathione biosynthesis inhibitor, buthionine sulfoximine (bso), gave rise to the discovery of trypanothione, unique to trypanosomes and absent from the mammalian cells (fairlamb et al. 1985). in mammals, potential redox damage meets the glutathione (gsh)-based system as a fi rst defense, during the course of which glutathione disulfi de (gssg) is formed (equation 1). regeneration of protective gsh from gssg is catalyzed by gr. in trypanosomes and leishmanias an analogous system has evolved (fairlamb and cerami, 1992) insofar as the disulfi de (t[sh]2) differs from gssg by the presence of a spermidine cross-link between the two glycyl carboxyl groups (compare gssg and t[s]2). the enzyme tr reduces t[s]2 to dithiol t[sh]2 in a manner analogous to gr (fig. 3). with this discovery of a fundamental metabolic difference, tr was proposed and amplifi ed further as a target for the rational design of antitrypanosomal and antileishmanial drugs (fairlamb et al. 1985; shames et al. 1986; benson et al. 1992; hunter et al. 1992; schirmer et al. 1995). unlike human hosts, trypanosomes contain tr instead of analogous enzyme gr to process their cognate substrates trypanothione and glutathione, nadph nadp+ gr gssg gsh nadph nadp+ tr t[s]2 t[sh]2 reactive oxygen species host parasite figure 3. outline of glutathione and trypanothione based redox defences. +h3n co2 conh conh s s co2 +h3n co2 conh conh co2 glutathion disulphide (gssg) gsh gssg (1) +h3n co2 conh conh s s conh +h3n co2 conh conh conh nh2 + trypanothion disulphide (t[s]2) scheme 1. glutathion and trypanothione. drug target insights 2007: 2 134 khan respectively. parasite tr does not process gssg and host gr does not reduce t[s]2 (shames et al. 1986; krauth-siegel et al. 1987). selective inhibitor design is probable, due to the mutually exclusive recognition and rejection of cognate substrates between host and parasite (shames et al. 1986; krauth-siegel et al. 1987; schirmer et al. 1995). efficient selective blockade of tr would be expected to compromise the redox defences of the parasites, increasing their sensitivity to redox-damage based drugs, e.g. nifurtimox. a tr inhibitor might be expected to be drug in its own right or for co-administration with a redoxactive drug e.g. nifurtimox. the later case may even provide synergy, allowing use of lowered doses of the redox drug (chan et al. 1998). tr is a member of the large well-characterized protein family of fad-dependent nadph oxidoreductases (reviewed in williams, 1992) and share close structural and mechanistic similarities with that of gr (summarized in table 2). it is a dimeric protein of monomer molecular mass 52kda, providing fad-binding, nadph-binding, central and interface domains. there are two identical active sites, formed by residues of the fad, nadph, and central domains of one monomer and the interface domain of the other (fig. 4). validating suitability of trypanothione reductase as a target to validate tr as a viable target for rational drug design, inhibition of tr in vitro should correlate with an observable effect in vivo. this is a diffi cult test to satisfy unambiguously; biochemical and molecular biological attempts had been made to probe tr as a reasonable drug target. in absolute terms, disruption or deletion of the tr genes in parasites should be lethal. over-expression of tr in transfected l. donovani promastigotes was not found to alter the sensitivity to hydrogen peroxide indicating that the regeneration of t[sh]2 from its disulfi de after oxidative challenge is not the ratedetermining feature of the defense system (kelly et al. 1993). over-expression of tr in t. cruzi led to gene rearrangements when antisense modulation of tr expression was attempted (tovar and fairlamb, 1996). disruption of two of the tr alleles of leishmania failed to produce a null mutant, and actually produced a third copy of tr gene by genomic rearrangement to a larger chromosome (dumas et al. 1997). down-regulation of tr using a trans-dominant mutation strategy demonstrated that even with only 15% residual tr activity, promastigote growth is still supported in culture (tovar et al. 1998a). it was proposed that any rationally designed inhibitor of tr must attain >85% inhibition for activity as an antileishmanial species, but it was pointed out that such cells are more sensitive to oxidative insult, such as the oxidative burst of macrophages, and that clinically usable tr inhibitors may need to be as effective in vivo (tovar et al. 1998a). later it had been demonstrated that tr absence is incompatible with parasite survival and validated tr as a bonafi de drug target. as it was not possible to obtain viable leishmania devoid of tr catalytic activity, specifi c inhibitors of this enzyme are likely to be useful antileishmanial agents for chemotherapeutic use (tovar et al. 1998b). sequence alignment of trs and human grs (hgrs) showed that relative to tr hgr has an nterminal extension, a c-terminal truncation and several deletion and insertions throughout the sequence. the most striking regions of homology are 14-residue sequence containing the redox-active cysteine residues and the 10-residue sequence containing the active site histidine. although the disulfi de-binding site in tr in general rather closely resembles that for gssg in gr, they have over 1000-fold selectivity for their cognate substrates, the molecular basis for which involves size, charge and hydrophobicity of their active sites. tr binding site is much wider in the outer region (~22 å × 20 å × 28 å) due to different orientations of two helices figure 4. structure of trypanothione reductase with fad, nadph and trypanothione bound (modifi ed from bond et al. 1999). fad trypanothione nadph interface drug target insights 2007: 2 135 target for antitrypanosomal and antileishmanial drug design ta bl e 2. p ro pe rt ie s of tr yp an ot hi on e re du ct as e an d gl ut at hi on e re du ct as e; ta ke n fr om li te ra tu re ( w ill ia m s et a l. 19 92 ). p ro pe rt y tr yp an ot hi on e re du ct as e g lu ta th io ne r ed uc ta se c . f as ci cu la ta l. d on ov an i t. c ru zi t. c on go le ns e e .c ol i h um an f la vi n fa d n. d. fa d fa d fa d fa d p yr id in e di nu cl eo tid e n a d p h n a d p h n a d p h n a d p h n a d p h n a d p h s ub un it m r ( d a) 54 00 0 52 94 0 53 90 0 53 40 0 48 70 0 52 50 0 a m in o ac id s/ su bu ni t 49 1 49 1 49 2 49 2 45 0 47 8 o lig om er ic s tr uc tu re di m er di m er di m er di m er di m er di m er e ox , λ m ax ( nm ) 46 4 46 3 46 1 46 4 46 2 46 0 ε 0 a t λ m ax ( m m –1 cm –1 ) 11 .3 11 .5 11 .2 10 .6 11 .3 ch ar ge tr an sf er in e h 2 ye s ye s ye s ye s ye s ye s ε 0 a t λ 53 0 (m m –1 cm –1 ) 3. 63 4. 2 4. 9 3. 7 4. 5 k m ( µm ) tr yp an ot hi on e 53 ( 51 , 5 8) 36 45 ( 55 , 5 0) 31 ( 18 ) 20 00 g lu ta th io ne 66 ( 61 , 7 0) 65 k c at ( m in –1 ) tr yp an ot hi on e 31 00 0 (2 86 00 0) 10 76 0 14 29 9 96 00 61 00 9. 6 gl ut at hi on e 3. 1 <2 44 00 0 12 60 0 k c at /k m ( m –1 se c– 1 ) 9. 8 x 10 6 5. 0 x 10 6 5. 3 x 10 6 5. 2 x 10 6 6. 2 x 10 6 3. 1 x 10 6 k m ap p (n a d p h ) 7 9 5 5 16 ( 25 ) 9 drug target insights 2007: 2 136 khan in the fad domains (kuriyan et al. 1991). the tr active site is more hydrophobic and has an overall negative charge to attract its positively charged and more hydrophobic polyamine containing cognate substrate trypanothione and repel the negatively charged smaller host substrate glutathione. conversely, the gr active site is smaller and positively charged, repelling the larger and positively charged trypanothione. the x-ray crystal structures of trs from crithedia fasciculata (kuriyan et al. 1991; hunter et al. 1992), and t. cruzi have been solved (lantwin et al. 1994; zhang et al. 1996). the crystal structure of tr in complex with trypanothione was solved (bond et al. 1999) as was the crystal structure of tr complexed with the alternative substrate glutathionyl spermidine (bailey et al. 1993) and with the weak, but selective, inhibitor mepacrine (jacoby et al. 1996). as already pointed out, a convenient screening technology is highly desirable for a suitable substrate to expedite the drug discovery process. to this end, a convenient colorimetric plate assay suitable for automated highthroughput screening has been developed by hamilton et al. (2003). one disadvantage of selecting tr as a drug target for the development of broad-spectrum antiparasitics is that intracellular trypanothione concentrations vary from 0.3 mm in african trypanosomes to ~3.0 mm in leishmania spp., >99% of which is in the reduced form. as soon as tr is inhibited, t[s]2 will start to accumulate due to continuing intracellular oxidant process. it is not currently known what t[s]2/t[sh]2 ratio is lethal to the parasite. it has been suggested that the design of competitive inhibitors with ki-values in nm range will be required, and the design of irreversible inhibitors may represent a better strategy for drug development (fairlamb, 2003b). the following section will focus on recent discoveries of different classes of tr inhibitors, which demonstrated antiparasitic activity and promise of development of newer chemotherapeutic agents. rational drug design based on trypanothione reductase as a target the homology modeling of tr (benson et al. 1992) with that of hgr shed light on the structural aspects of the enzyme also in addition to its substratebinding mode from a theoretical perspective. studies with various alternative peptide substrates demonstrated that the spermidine moiety of trypanothione could be conveniently replaced with n,n-dimethylaminopropylamide (dmapa) without loss of substrate activity. subsequently, a series of alternative γ-glutamyl-modifi ed trypanothione substrates were synthesized. the discovery that a benzyloxycarbonyl (z) group represented a suitable replacement resulted in (z.cys.gly.dmapa)2 as a convenient alternative assay substrate in which the z group occupies a hydrophobic pocket near phe396’ of tr (henderson et al. 1987b; el-waer et al. 1991, 1993a and b; marsh and bradley, 1997). this pocket was since named the z-site, approximately enclosed by phe396’, pro398’ and leu399’. this initial advancement in structural aspects of tr active site prompted the discovery of peptide inhibitors (garforth et al. 1994; mckie et al. 2001; chan et al. 2002). the fi rst rationally designed non-peptide inhibitors are represented by the tricyclic ring structures that are competitive versus trypanothione (benson et al. 1992). progress has been made in discovering large classes of inhibitors of tr over recent years, which are also found to be lethal to parasites in vitro. the tricyclic compounds and congeners molecular modeling approaches were useful in identifying tricyclic antidepressants including phenothiazines and related structures (e.g. 9, 10) and mepacrine (11) (fig. 5) as competitive inhibitors of tr but not gr (benson et al. 1992; jacoby et al. 1996; chan et al. 1998). the tricyclic moiety of these compounds were shown to lodge against the hydrophobic wall of tr active site formed by trp21 and met113, with the aminopropyl side chain pointing towards the glu466’ and glu467’ residues. the synthesis and biological evaluation of a series of dibenzazepine analogs (garforth et al. 1997), n-acylpromazines, 2-substituted phenothiazines, and trisubstituted promazines failed to show any improvements over the parent leads (chan et al. 1998). the antimalarial drug mepacrine (11), an acridine derivative, is also a competitive inhibitor of tr. the acridine ring also aligns to the hydrophobic wall of tr formed by trp21 and met113, but the alkylamino side chain point towards glu18, unlike the tricyclics described above (bonse et al. 1999). sulfonamides and urea derivatives of mepacrine with varying methylene spacer lengths (e.g. 12, 13) were found to be superior inhibitors of tr relative to mepacrine with the best inhibitors drug target insights 2007: 2 137 target for antitrypanosomal and antileishmanial drug design n s nh3c h3c cl chlorpromazine, 9 ki = 10 m (benson et al.1992) n nh3c h3c cl clomipramine, 10 ki = 6.6 m (benson et al. 1992 ) n hn ch3 ch2ch2ch2net2 mepacrine, 11 ki = 19 m (jacoby et al. 1996) cl och3 n hn cl och3 h n r r = o2s (chibale et al. 2001) r = ochn i50 = 3.3 m i50 = 13.1 m o h3c ch3 nho n ch3 ch3 nho n ch3 ch3 14, i50 = 35.7 m (chibale et al. 2003) 12, 13, figure 5. structures and activities of tricyclic inhibitors of trypanothione reductase. being 40-fold more potent (chibale et al. 2001). a series of synthetic 9,9-dimethylxanthene derivatives (e.g. 14) were shown to be competitive inhibitors of tr which are comparable to known tricyclic inhibitors of tr (chibale et al. 2003). it is important to note that as required in principle for an ideal drug target, inhibition of tr in vitro should also correlate with antiparasitic activity in vitro against t. brucei, t. cruzi and l. donovani. however, no strong correlation between tr-inhibitory activity and in vitro antiparasitic activity was observed indicative of unfavorable pharmacokinetic profi les. 2-aminodiphenylsulfi des and its congeners modifi cation of the central ring of the phenothiazines furnished the 2-aminodiphenylsulfides, termed ‘open chain chlorpromazines’. using a microplate assay to screen tr inhibitors, this group of compounds was discovered to be potent competitive inhibitors (fig. 6). compound 15 was the best inhibitor in the preliminary series (fernandezgomez et al. 1995). based on these results and molecular modeling studies, a series of bis (2-aminodiphenylsulfi des) were synthesized and compound 16 was shown to be the most potent in this series (girault et al. 1997). further improvement was evident with a newer generation of bis(2-aminodiphenylsulfides) (e.g. 17) corresponding to attachment of an additional hydrophobic side to an analog of compound 16. the large volume of the tr active site justifi ed the introduction of an additional hydrophobic group to the end of the side chain (girault et al. 2001). all the compounds demonstrated antiparasitic activity in vitro at low micromolar ranges. however, no correlations between tr inhibition and in vitro antiparasitic activity were apparent. quaternary alkylammonium compounds the quaternization of tertiary alkylamine ω-nitrogen atom of chlorpromazine by substituted benzyl and other aromatic or heterocyclic groups resulted the discovery of quaternary alkylammonium phenothiazines as a new class of linear competitive inhibitors of tr (fig. 7) (khan et al. 2000). the permanent positive charge on the distal nitrogen atom of the tricyclic’s side chain contributed to binding was estimated as ≥5.6 kcal.mol-1 by comparison with the analog with cationic nitrogen atom of the quaternary (18) replaced by an ether oxygen atom (19). the major contribution to improving ki value and inhibition strength was accomplished by incorporating the hydrophobic n-benzyl substituents. the best inhibitor identified was compound 19, containing a 3, drug target insights 2007: 2 138 khan n s n+h3c h3c cl cl clcl 18, ki = 0.12 m (khan et al. 2000) n s o cl cl cl 19, no inhibition at 50 m (khan et al. 2000) nh s n+h3c h3c cl cl clcl 20, ki = 1.7 m (parveen et al. 2005) figure 7. structures of quaternary alkylammonium compounds with their anti-tr activities. 4-dichlorobenzyl substituent (~2 orders of magnitude >chlorpromazine). detailed molecular modeling studies helped to establish docking orientations and energies by revealing involvement of: (i) the major hydrophobic pocket (trp21, met113, tyr110), (ii) the “z”-site (phe396’, pro398’, leu399’) and (iii) the ionic interactions possible for the quaternary cationic nitrogen with nearby side chains of glu466’ and glu467’ (fig. 8) (austin et al. 1999, khan et al. 2000). this “three point attachment”, a concept initially designed by ogston (1948) to explain chiral specifi city of enzymes, has allowed on average a 30-fold improvement of ki values against tr (khan et al. 2000). similarly, the quaternization of the side chain tertiary nitrogen atom of the ‘open chain chlorpromazines’ also showed improvements in inhibition up to nh s n cl n h3c 15, ki = 25 m (fernandez-gomez et al. 1995) nh s n br n h3c h n h n hn s n br n ch3 o o 16, i50 = 0.55 m (girault et al. 1997) nh s n br n h3c h n n h n hn s n br n ch3 o o nh n o o 17, i50 = 0.25 m (girault et al. 20 01) figure 6. structures of 2-aminodiphenylsulfi des with their anti-tr activities. drug target insights 2007: 2 139 target for antitrypanosomal and antileishmanial drug design 40-fold. the most potent compound synthesized from this series was the 3, 4-dichlorobenzyl analog (20) (parveen et al. 2005). all analogs demonstrated strong inhibition, some in lower nm ranges, against the blood stream form of t. brucei. antiparasitic activity was not solely determined by the inhibition strength against tr; a strong contribution from hydrophobicity was also observed. although active against l. donovani, none showed major improvement in this activity relative to their parent compounds. some of the analogs also showed improved inhibition against the amastigote stage of t. cruzi with ed50 values <1 µm (khan et al. 2000; parveen et al. 2005). polyamine derivatives as shown in scheme 1, the natural disulfide substrate of tr, t[s]2, differs from the analogous host’s gssg only by the presence of a spermidine (a polyamine) cross-link between the two glycyl carboxyl groups. this discrimination between parasite and host substrate served as a criterion for developing polyamine derivatives as selective inhibitors of tr (o’sullivan and zhau, 1995; o’sullivan et al. 1996, 1997; baillet et al. 1996; li et al. 2001; bi et al. 2006). this approach led to the discovery of series of potent selective competitive inhibitors of tr, several selected compounds are included in figure 9 (compounds 21, 22). solid phase synthesis approaches allowed for the expedient synthesis of polyamine-based focused libraries as potential tr inhibitors. several potent inhibitors were identifi ed with low nm activity (orain and bradley, 2001; de luca et al. 2003). most of these compounds also displayed potent in vitro antiparasitic activity against t. brucei with ed50 values <1 µm (o’sullivan et al. 1997; li et al. 2001; bi et al. 2006). these preliminary antiparasitic activity in vitro are suggestive of their tr involvement although well correlation between tr-inhibition and trypanocidal activity were not evident. bisbenzylisoquinoline alkaloids the bisbenzylisoquinoline alkaloids were also shown to be potent inhibitors of tr and trypanocidal agents (fournet et al. 1998, 2000). six of the alkaloids evaluated displayed potent trypanocidal activity in vitro against t. cruzi with ed50 values <100 µm. the best tr inhibitor, cepharanthine figure 8. compound 18 docked into active site of tr to show major interactions (taken from austin et al. 1999). figure 9. structures and trypanothione reductase inhibitory activities of polyamine derivatives. n n n nh ph ph ph ph ph 21, ki = 151 nm (li et al. 20 01) r n h n h n h n h n h n h n h n h r nh nh nh nh 22, r = diphenylpropyl; i50 = 950 nm (bi et al. 2006) 5 n o o o meo o n ch3h h h3c ome 23, i50 = 15 m (fournet et al. 1998 ) figure 10. bisbenzyleisoquinoline alkaloid. drug target insights 2007: 2 140 khan (23), had an i50 value of 15 µm which is the same order of magnitude as its ed50 against the parasites, suggesting that inhibition of tr could be the mechanism of their trypanocidal activities. natural products natural products may be considered as “nature’s medicine chest” and have served as a potential source of tr-inhibitors. the natural antihypertensive agent, kukoamine a (24), a bis (trihydrocinnamoyl) spermidine derivative, fi rst isolated from lycium chinense, was identifi ed as a mixed type of inhibitor of tr (ponasik et al. 1995). virtual screening of a large number of chemical databases, using the tr active site as the targeted template, identified the natural spermine-based macrocyclic alkaloid lunarin (25) (originally isolated from lunaria biennis) as tr-inhibitor, which was shown to inhibit the enzyme in a time-dependent manner (bond et al. 1999). further investigation with synthetic derivatives confi rmed the importance of the unique structure of the tricyclic core as a motif for inhibitor design and revealed that the non-natural enantiomer may be a more suitable scaffold upon which thiophilic groups may be presented (hamilton et al. 2006). ajoen (26) a garlic derived natural sulfur-containing compound was established as an irreversible inhibitor and subversive substrate of both tr and gr (gallwitz et al. 1999). most of these natural products also inhibited the parasites in vitro in one way or another. irreversible inhibitors the anticancer nitrosourea drug carmustin was the fi rst ligand to display irreversible inactivation of tr, but non-specifi cally as also inactivated hgr, by carbamoylating an active site cystinyl residue, which becomes accessible after being reduced by nadph (schirmer et al. 1995). ajoen (26), as previously mentioned as a natural product inhibitor, is an irreversible non-specifi c inhibitor of tr (gallwitz et al. 1999). the fi rst rationally designed and selective irreversible inhibitors of tr include the pt-complexes of terpyridine derivatives (e.g. 27), which inhibit the reduced tr, presumably through occupying the cys52 by replacing its fourth pyridine ligand (bonse et al. 2000). coupling the irreversible ligand (terpyridine)pt2+ complex with an 9-aminoacridine derivative (a competitive inhibitor) furnished mixed-type inhibitors (e.g. 28) (inhoff et al. 2002). an important advancement was made with the quinacrine mustard (29), which was shown to selectively and irreversibly inactivate tr in a time-dependent manner with a stoichiometry of two inhibitors bound per monomer. the rate of inactivation was dependent upon the oxidative states of tr, with nadph-reduced tr form being inactivated faster. the structure of tr-quinacrine mustard-adduct solved to 2.7å revealed that two molecules of ligand are bound in the trypanothione binding site of tr. each acridine moiety interacts through πstacking, while only one of the acridine groups interacts with a trypanothione residue in a similar fashion (saravanamuthu et al. 2004). recently, several mannic bases (e.g. 30) were shown by lee et al. (2005) to be irreversible inhibitors of tr. hplc, nmr and ms analyses were performed to delineate their mechanism of action and found that divinyl ketone are the key intermediates to irreversibly inhibit tr. esiand maldi-tof-ms of tr, modifi ed by mannic base or corresponding divinyl ketone, demonstrated n h n h n h n h oh ho oh oh o o 3 4 3 24, ki = 1.8 m, ki' = 13 m (ponasik et al. 1995) o o nh h n nhoo 25, ki = 144 m, kinac = 0.116 min-1 (bond et al. 1999) s s s o 26, modify cys52 (gallwitz et al. 19 99) figure 11. the natural product inhibitors of trypanothione reductase. drug target insights 2007: 2 141 target for antitrypanosomal and antileishmanial drug design specifi c alkylation of cys52 in a manner as shown in figure 13 (lee et al. 2005). sixteen novel pd-complexes of the bioactive nitrofuryl thiosemicarbazones were synthesized and tested for their in vitro activity (otero et al. 2006). most complexes showed higher in vitro trypanocidal activity against t. cruzi than the standard drug nifurtimox. overall, the activities of pd complexes ≥their parent compounds. it has been suggested that main trypanocidal mechanism was the production of oxidative stress as a result of their bioreduction with the reductive enzymes, although strong dna-adduct formation was also evident (otero et al. 2006). subversive substrates of trypanothione reductase nifurtimox (6) and related compounds exert their parasiticidal activity through acting as futile, superoxide ion producing substrates of tr . tr and gr reduce these futile, superoxide ion producing substrates in a single electron step (nadph + 2rno2 → 2rno2 •‾ + 2h+; 2rno2 •‾ + o2 → 2rno2 + o2 •‾), a process by which nadph and o2 are wasted and t[sh]2 is inhibited/oxidized by scores of newly formed superoxide ions causing a reduction in thiol/disulfi de titer and thus producing oxidative stress (henderson et al. 1988; schirmer et al. 1995). this group of compounds, known as ‘subversive substrates’ or ‘turncoat inhibitors’, are best represented by nitrofurans or quinones, chemically modified to take into account the substrate specifi city for tr over gr (cenas et al. 1994a and b). thus, chinifur (31), a nitrofuran derivative with a positively charged side chain, was discovered as a selective inhibitor and subversive substrate of tr (cenas et al. 1994a). recently, the anti-tr activities of a series of nitrofuran and nitroimidazole derivatives were studied to examine the mechanism of action of different types of n nn pt2+ n br 27 n nn pt2+ s s oh h n nh+ 2no3 meo cl 28 hn n meo cl ch3 n cl cl 29 figure 12. structures of irreversible inhibitors of trypanothione reductase. o n o cl 30 rsh , -unsaturated mannic base 1st michael addition o n o cl h sr on+ o cl sr h ocl sr hn o ocl rshdivinyl ketone intermediate rsh 2nd michael addition ocl sr figure 13. proposed reaction mechanisms for the modifi cation of protein thiol (rsh), e.g. cys52 in t. cruzi trypanothione reductase by an unsaturated mannic base such as 30 (modifi ed from lee et al. 2005). drug target insights 2007: 2 142 khan nitro-group containing compounds. the results indicated that the nitrofurans, e.g. nifurtimox act as futile-cyclers as discussed above, whereas 5-nitroimidazoles, e.g. megazole (32) act as thiol scavengers particularly for t[sh]2 thus reducing the thiol/disulfi de titer which is detrimental for the parasites’ survival (maya et al. 2003). vega-teijido et al (2006) explored three possible binding sites of tr and gr, i.e. the active site, the dimer interface and the nadph binding site to study the mechanism of action of nitrofuran and nitrothiophene analogs. it has been suggested that this class of compounds act as either uncompetitive or mixed inhibitors of tr. moreover, it has also been indicated that the presence of an α-helix connecting the active site of tr with the interface may be crucial for charge-transfer processes. 1,4-naphthaqiuinone derivatives (e.g. 33) are examples of quinine-based subversive substrates of tr, designed after plumbagin. compound 33 proved to be a potent subversive substrate and an effective uncompetitive inhibitor of tr versus t[sh]2 and nadph (salmon-chemin et al. 2001). most of these compounds were potent inhibitors o f t h e p a r a s i t e s i n v i t ro . f o r a s e r i e s o f naphthaquinones, a correlation between their potency as subversive substrates in vitro and trypanocidal activity in vivo has been demonstrated (salmon-chemin et al. 2001). this ‘alternative approach to chemotherapy of chagas disease’ has been considered one of the most promising advances. chemotherapeutic prospects of the trypanothione reductase inhibitors for an enzyme inhibitor to become a practical drug, several criteria must be met: (i) the biochemical pathway that is inhibited must be related to the disease state in such a way that inhibition of that pathway in a patient is therapeutic; (ii) the enzyme inhibitor must be specifi c so that unwanted inhibition of other pathways or receptors does not occur at therapeutic doses; (iii) the compounds must have the pharmacokinetic characteristics of a practical drug, i.e. must be absorbed, must penetrate to the site of action and must have a reasonably predictable doseresponse relationship and duration of action; (iv) the compound must have an acceptable toxicological profi le in animals, and the results of clinical studies in humans must demonstrate an appropriate balance between benefi ts and risks in therapeutic use; (v) the compound must survive a long and expensive clinical development process and ultimately be approved by regulatory agencies; (vi) the compound must be economically viable in the marketplace and compete successfully with other therapeutic alternatives (crout, 1989). the criteria (i) and (ii) have already been addressed while selecting the target, and thus, all of the above mentioned inhibitors have fulfi lled them. pharmacological and toxicological studies in animal models are needed to meet other criteria. although many potent inhibitors of tr have been discovered through enzyme screening, in vivo evaluation of these lead compounds are scant. several polyamine derivatives that were shown to be potent trypanocidals in vitro (with ed50 values in submicromolar ranges) failed to prolong the lives of experimental mice infected with trypanosomes or to cause a signifi cant decrease in bloodstream parasitemia of infected mice (o’sullivan et al. 1997). however, none of the compounds exerted overt toxicity in mice. it has been suggested that the lack of in vivo trypanocidal activity may be due to their rapid elimination and/or metabolism. since these compounds are reversible inhibitors of tr, concentrations of oo2n n nh n c2h5 c2h5o h3c 31, ki = 4.5 m (cenas et al. 1994a) n no2n ch3 n n s nh2 32, thiol scavenger (maya et al. 2003 ) o o ch3 n h n h n h o o h3c o o 4 4oh oh 33, i50 = 0.45 m, km 28 m, (salmon-chemin et al. 1994a) figure 14. structures and activity of subversive substrates of trypanothione reductase. drug target insights 2007: 2 143 target for antitrypanosomal and antileishmanial drug design these compounds may not be maintained, and as a result, tr activity will not be signifi cantly decreased (o’sullivan et al. 1997). it has also been suggested that converting reversible inhibitors into irreversible inhibitors through complexation with metal ions might be a reasonable strategy for identifying improved leads with enhanced in vivo profi les. clomipramine and thioridazine were shown to be effective in treatment of mice with experimental chagas disease (rivarola et al. 2001, 2002). the investigation of effects of clomipramine on t. cruzi infected mice demonstrated that clomipramine at 5 mg/kg daily doses for 30 days, or two doses of clomipramine 40 mg/kg given intraperitoneally at 1h and 7 days after infection, was not toxic for the host, but was effective against the parasite. parasitamiasis became negative and only mild heart structural and elecctrocardiographic alterations were detected in chronic phase in the group treated with clomipramine 5 mg/kg. in mice treated with 40 mg/kg, none of these alterations was detected (rivarola et al. 2001). overall, it has been shown that clomipramine and thioridazine signifi cantly modifi ed the natural evolution of the infection. cardiac function and survival of infected and treated animals were not different from noninfected animals. thioridazine or clomipramine are currently registered as drugs (antipsychotics) and meet all the above mentioned criteria. however, to consider them as antitrypanosomal drugs, dose becomes an important obstacle, since they need to be active at a dose which is known to be tolerated by psychiatric patients without causing adverse events. apart from their potent in vitro activity and a preliminary report of in vivo activity, more detail studies are needed to establish their effi cacy and safety and also to improve their antiparasitic potency. overall, it would not be overenthusiastic to consider phenothiazines and related tricyclic antidepressants and the other inhibitors of tr as important drug leads for development of future chemotherapy against trypanosomal and leishmanial infections. conclusion the demand for chemotherapeutic agents for the treatment of chagas disease, sleeping sickness and kala azar is desperate. an account of the different possible targets for rational drug design with special emphasis on trypanothione reductase has been focused. tr has been established as a potential target in several studies although it is not universally accepted. the literature, molecular graphics and other medicinal chemistry approaches have led to the development of a large class of compounds as inhibitors of tr and the parasites. considerable progress have been made in terms of identifi cation, validation and inhibitor design based on trypanothione reductase as a target, which will be milestones towards the goal of developing chemotherapy against these devastating diseases. this review has revealed several approaches based on tr towards the development of new drugs against the parasitic diseases. reasonable activity against parasites living in both culture and in mouse macrophases has been demonstrated by most classes of compounds, full activity against whole animal model to be achieved. it has been suggested that the alternative approaches like subversive substrates and metal bound inhibitors would be advantageous in achieving reasonable in vivo activity against the parasite infection. only the phenothiazines and related tricyclic antidepressants were shown to reduce parasite burden in infected mice. development of the quaternary alkylammonium chlorpromazines and congeners, with an additional hydrophobic moiety provided, on an average, up to about 30-fold more potent inhibitors of tr. the charge on n+ is needed for interaction at the glu466’ or glu467’ of the enzyme active site, the tricyclic or equivalent moiety interacts with the major hydrophobic cleft and the second hydrophobic moiety may interact with z-site. studies with other classes of inhibitors, in addition to the tr:inhibitor complex’s x-ray crystal structure, provided similar tr:inhibitor interactive motifs. as with any rational drug design case at the current stage of computational development, the problem of bioavailability, pharmacokinetic, metabolism and targeting have not been addressed. the prodrug approach might be a reasonable step in correcting some of the aforementioned problems in developing tr inhibitors with observable effects in vivo and might open a new path towards the development of drugs against these parasitic diseases. acknowledgements the author wish to gratefully acknowledge professors k. t. douglas of manchester, a. h. fairlamb of dundee and r. l. krauth-siegel of heidelberg and others who are pioneering the drug target insights 2007: 2 144 khan research in the endeavor of trypanothione reductase and many of their research have been cited in this review. references austin, s.e., khan, m.o.f. and douglas, k.t. 1999. rational drug design using trypanothione reductase as a target for antitrypanosomal and anti-leishmanial drug leads. drug des. discov., 16:5–23. bailey, s., smith, k., fairlamb, a.h. et al. 1993. substrate interactions between trypanothione reductase and n1-glutathionylspermidine disulphide at 0.28-nm resolution. eur. j. biochem. / febs, 213:67–75. baillet, s., buisine, e., horvath, d. et al. 1996. 2-amino diphenylsulfi des as inhibitors of trypanothione reductase: modifi cation of the side chain. bioorg. med. chem., 4:891–9. benson, t.j., mckie, j.h., garforth, j. et al. 1992. rationally designed selective inhibitors of trypanothione reductase. phenothiazines and related tricyclics as lead structures. biochem. j., 286:9–11. bi, x., lopez, c., bacchi, c.j. et al. 2006. novel alkylpolyaminoguanidines and alkylpolyaminobiguanides with potent antitrypanosomal activity. bioorg. med. chem. lett., 16:3229–32. bodley, a.l., wani, m.c., wall, m.e. et al. 1995. antitrypanosomal activity of camptothecin analogs. structure-activity correlations. biochem. pharmacol., 50:937–42. bond, c.s., zhang, y., berriman, m. et al. 1999. crystal structure of trypanosoma cruzi trypanothione reductase in complex with trypanothione, and the structure-based discovery of new natural product inhibitors. structure, 7:81–9. bonse, s., santelli-rouvier, c., barbe, j. et al. 1999. inhibition of trypanosoma cruzi trypanothione reductase by acridines: kinetic studies and structure-activity relationships. j. med. chem., 42:5448–54. bonse, s., richards, j.m., ross, s.a. et al. 2000. (2,2’:6’,2”terpyridine)platinum(ii) complexes are irreversible inhibitors of trypanosoma cruzi trypanothione reductase but not of human glutathione reductase. j. med. chem., 43:4812–21. brown, j.r. 1987. trypanosomiasis and leishmaniasis. in critical reports on applied chemistry, vol 21(chemother trop dis), pp72–101. brohn, f.h. and clarkson, a.b.jr., 1980. trypanosoma brucei brucei: patterns of glycolysis at 37°c in vitro. mol. biochem. parasitol., 15:291–305. carnieri, e.g., moreno, s.n. and docampo, r. 1993. trypanothione-dependent peroxide metabolism in trypanosoma cruz different stages. mol. biochem. parasitol., 61:79–86. cenas, n., bironaite, d., dickancaite, e. et al. 1994a. chinifur, a selective inhibitor and “subversive substrate” for trypanosoma congolense trypanothione reductase. biochem. biophys. res. comm., 204:224–9. cenas, n.k., arscott, d., williams, chjr. et al. 1994b. mechanism of reduction of quinones by trypanosoma congolense trypanothione reductase. biochemistry, 33:2509–15. chan, c., yin, h., garforth, j. et al. 1998. phenothiazine inhibitors of trypanothione reductase as potential antitrypanosomal and antileishmanial drugs. j. med. chem., 41:148–56. chan, c., yin, h., mckie, j.h. et al. 2002. peptoid inhibition of trypanothione reductase as a potential antitrypanosomal and antileishmanial drug lead. amino acids, 22:297–308. chibale, k., haupt, h., kendrick, h. et al. 2001. antiprotozoal and cytotoxicity evaluation of sulfonamide and urea analogues of quinacrine. bioorg. med. chem. lett, 11:2655–7. chibale, k., visser, m., van schalkwyk, d. et al. 2003. exploring the potential of xanthene derivatives as trypanothione reductase inhibitors and chloroquine potentiating agents. tetrahedron, 59:2289–96. clarkson, abjr, brohn., f.h., 1976. trypanosomiasis: an approach to chemotherapy by the inhibition of carbohydrate catabolism. science, 194(4261):204–6. croft, s.l., 1988. recent developments in the chemotherapy of leishmaniasis. trends. pharmacol. sci., 9:376–81. crout, j.r., 1989. enzymes as targets for drug design. in palfreyman, mccann lovenberg, et al. ed. london: academic press. de luca, s., ulhaq, s., dixon, m.j. et al. 2003. solid phase synthesis of focused library of trypanothione reductase inhibitors. tet lett, 44:3195–7. docampo, r. and stoppani, a.o.m., 1979. generation of superoxide anion and hydrogen peroxide induced by nifurtimox in trypanosoma cruzi. arch. biochem. biophys., 197:317–21. docampo, r. and moreno, s.n.j., 1984a. free-radical intermediates in the antiparasitic action of drugs and phagocytic cells. free radicals. biol., 6:243–88. docampo, r. and moreno, s.n.j., 1984b. free radical metabolites in the mode of action of chemotherapeutic agents and phagocytic cells on trypanosoma cruzi. rev. infect. dis., 6:223–38. docampo, r. and moreno, s.n.j., 1986. free radical metabolism of antiparasitic agents. fed. proc., 45:2471–6. donelson, j.e. and rice-ficht, a.c., 1985. molecular biology of trypanosome antigenic variation. microbiol. rev., 49:107–25. dumas, c., ouellette, m., tovar, j. et al. 1997. disruption of the trypanothione reductase gene of leishmania decreases its ability to survive oxidative stress in macrophages. embo, j., 16:2590–8. el kouni, m.h., 2003. potential chemotherapeutic targets in the purine metabolism of parasites. pharmacol. therapeut., 99:283–309. el-waer, a., douglas, k.t., smith, k. et al. 1991. synthesis of n-benzyloxycarbonyl-l-cysteinylglycine 3-dimethylaminopropylamide disulfide: a cheap and convenient new assay for trypanothione reductase. anal. biochem., 198:212–16. el-waer, a.f., smith, k., mckie, j.h. et al. 1993a. the glutamyl binding site of trypanothione reductase from crithidia fasciculata: enzyme kinetic properties of γ-glutamyl-modifi ed substrate analogs. biochim. biophys, acta, prot. struc. mol. enzymol., 1203:93–8. el-waer, a.f., benson, t., douglas. k.t., 1993b. synthesis of substrate analogs for trypanothione reductase. int. j. pept. prot. res., 41:141–6. fairlamb, a.h., bowman, i.b.r., 1980. trypanosoma brucei: maintenance of concentrated suspensions of bloodstream trypomastigotes in vitro using continuous dialysis for measurement of endocytosis. exp parasitol, 49:366–80. fairlamb, a.h., blackburn, p., ulrich, p. et al. 1985. trypanothione: a novel bis(glutathionyl)spermidine cofactor for glutathione reductase in trypanosomatids. science, 227(4693):1485–7. fairlamb, a.h., cerami, a., 1992. metabolism and functions of trypanothione in the kinetoplastida. annu. rev. microbiol., 46:695–729. fairlamb, a.h., 2003a. chemotherapy of human african trypanosomiasis: current and future prospects. trends in parasitology., 19(11):488–494. fairlamb, a.h., 2003b. target discovery and validation with special reference to trypanothione. in fairlamb, a.h., ridley, r.g., vial, h.j., ed. drugs against parasitic diseases: r&d methodologies and issues. ed. geneva: who tdr. pp107–18. fernandez-gomez, r., moutiez, m., aumercier, m. et al. 1995. 2-amino diphenylsulfi des as new inhibitors of trypanothione reductase. int. j. antimicrob. agents., 6:111–18. flohé l, 1989. glutathione—chemical, biochemical and medical aspects. in dolphine d, poulson r and avramovic o, ed. new york: wiley interscience. flohé l, 1998. the achilles’ heel of trypanosomatids: trypanothione-mediated hydroperoxide metabolism. biofactors, 8:87–91. fournet, a., inchausti, a., yaluff, g. et al. 1998. trypanocidal bisbenzylisoquinoline alkaloids are inhibitors of trypanothione reductase. j. enz inhibit, 13:1–9. fournet, a., de arias, r.a., ferreira, m.e. et al. 2000. effi cacy of bisbenzylisoquinoline alkaloids in acute and chronic trypanosoma cruzi murine model. int. j. antimicrob. agents., 13:189–95. gallwitz, h., bonse, s., martinez-cruz, a. et al. 1999. ajoene is an inhibitor and subversive substrate of human glutathione reductase and trypanosoma cruzi trypanothione reductase: crystallographic, kinetic, and spectroscopic studies. j. med. chem., 42:364–72. garforth, j., mckie, j.h., jaouhari, r. et al. 1994. rational design of peptide -based inhibitors of trypanothione reductase as potential antitrypanosomal drugs. amino acids, 6:295–9. drug target insights 2007: 2 145 target for antitrypanosomal and antileishmanial drug design garforth, j., yin, h., mckie, j.h. et al. 1997. rational design of selective ligands for trypanothione reductase from trypanosoma cruzi. structural effects on the inhibition by dibenzazepines based on imipramine. j. enz. inhibit., 12:161–73. girault, s., baillet, s., horvath, d. et al. 1997. new potent inhibitors of trypanothione reductase from trypanosoma cruzi in the 2-aminodiphenylsulfi de series. eur. j. med. chem., 32:39–52. girault, s., davioud-charvet, t.e., maes, l. et al. 2001. potent and specifi c inhibitors of trypanothione reductase from trypanosoma cruzi: bis(2-aminodiphenylsulfi des) for fl uorescent labeling studies. bioorg. med. chem., 9:837–46. grogl, m., thomason, t.n. and franke, e.d., 1992. drug resistance in leishmaniasis: its implication in systemic chemotherapy of cutaneous and mucocutaneous disease. am. j. trop. med. hyg., 47:117–26. hamilton, c.j., saravanamuthu, a., eggleston, i.m. et al. 2003. ellman’sreagent-mediated regeneration of trypanothione in situ: substrateeconomical microplate and time-dependent inhibition assays for trypanothione reductase. biochem. j., 369:529–537. hamilton, c.j., saravanamuthu, a., poupat, c. et al. 2006. time-dependent inhibitors of trypanothione reductase: analogues of the spermidine alkaloid lunarine and related natural products. bioorg. med. chem., 14:2266–78. henderson, g.b., fairlamb, a.h. and cerami, a. 1987a. trypanothione dependent peroxide metabolism in crithidia fasciculata and trypanosoma brucei. mol. biochem. parasitol., 24:39–45. henderson, g.b., fairlamb, a.h., ulrich, p. et al. 1987b. substrate specifi city of the fl avoprotein trypanothione disulfi de reductase from crithidia fasciculata. biochemistry, 26:3023–7. henderson, g.b., ulrich, p., fairlamb, a.h. et al. 1988. “subversive” substrates for the enzyme trypanothione disulfi de reductase: alternative approach to chemotherapy of chagas disease. proce. natl. acad. sci. u.s.a,, 85:5374–8. hyde, j.e., 1990. molecular parasitology. milton keynes: open university press. hunter, w.n., bailey, s., habash, j. et al. 1992. active site of trypanothione reductase. a target for rational drug design. j. mol. biol., 227:322–33. inhoff, o., richards, j.m., briet, j.w. et al. 2002. coupling of a competitive and an irreversible ligand generates mixed type inhibitors of trypanosoma cruzi trypanothione reductase. j. med. chem., 45:4524–30. jacoby, e.m., schlichting, i., lantwin, c.b. et al. 1996. crystal structure of the trypanosoma cruzi trypanothione reductase.mepacrine complex. proteins, 24:73–80. kelly, j.m., taylor, m.c., smith, k. et al. 1993. phenotype of recombinant leishmania donovani and trypanosoma cruzi which overexpress trypanothione reductase. sensitivity towards agents that are thought to induce oxidative stress. eur. j. biochem. / febs., 218:29–37. khan, m.o.f., austin, s.e., chan, c. et al. 2000. use of an additional hydrophobic binding site, the z site, in the rational drug design of a new class of stronger trypanothione reductase inhibitor, quaternary alkylammonium phenothiazines. j. med. chem., 43:3148–56. koenig, k., menge, u., kiess, m. et al. 1997. convenient isolation and kinetic mechanism of glutathionylspermidine synthetase from crithidia fasciculata. j. biol. chem., 272:11908–11915. krauth-siegel, r.l., enders, b., henderson, g.b. et al. 1987. trypanothione reductase from trypanosoma cruzi. purifi cation and characterization of the crystalline enzyme. eur. j. biochem., 164:123–8. krauth-siegel, r.l. and schoneck, r., 1995. flavoprotein structure and mechanism. 5. trypanothione reductase and lipoamide dehydrogenase as targets for a structure-based drug design. faseb. j., 9:1138–46. kuriyan, j., kong, x.p., krishna, t.s. et al. 1991. x-ray structure of trypanothione reductase from crithidia fasciculata at 2.4-å resolution. proc. natl. acad. sci., u.s.a., 88:8764–8. lantwin, c.b., schlichting, i., kabsch, w. et al. 1994. the structure of trypanosoma cruzi trypanothione reductase in the oxidized and nadph reduced state. proteins, 18:161–73. le trant, n., meshnick, s.r., kitchener, k., eaton, j.w. and cerami, a. 1983. iron-containing superoxide dismutase from crithidia fasciculata. purifi cation, characterization, and similarity to leishmanial and trypanosomal enzymes. j. biol. chem., 258:125–30. lee, b., bauer, h., melchers, j. et al. 2005. irreversible inactivation of trypanothione reductase by unsaturated mannich bases: a divinyl ketone as key intermediate. j. med. chem., 48:7400–10. li, z., fennie, m.w., ganem, b. et al. 2001. polyamines with n-(3-phenylpropyl) substituents are effective competitive inhibitors of trypanothione reductase and trypanocidal agents. bioorg. med. chem. lett, 11:251–4. linares, g.e.g., ravaschino, e.l. and rodriguez, j.b. 2006. progresses in the fi eld of drug design to combat tropical protozoan parasitic diseases. curr. med. chem., 13:335–60. marsh, i.r. and bradley, m. 1997. substrate specifi city of trypanothione reductase. eur. j. biochem., 243:690–4. maya, j.d., bollo, s., nunez-vergara, l.j. et al. 2003. trypanosoma cruzi: effect and mode of action of nitroimidazole and nitrofuran derivatives. biochem. pharmacol., 65:999–1006. mckie, j.h., garforth, j., jaouhari, r. et al. 2001. specifi c peptide inhibitors of trypanothione reductase with backbone structures unrelated to that of substrate: potential rational drug design lead frameworks. amino. acids, 20:145–53. meshnick, s.r., 1984. parasitic diseases vol 2. mansfi eld jm, ed. new york: marcel dekker. michels, p.m., 1988. compartmentation of glycolysis in trypanosomes: a potential target for new trypanocidal drugs. biol. cell., 64:157–64. neva, f.a. and brown, h.w., 1994. basic clinical parasitology. norwalk, connecticut: appleton & lange. nogoceke, e., gommel, d.u., kiess, m. et al. 1997. a unique cascade of oxidoreductases catalyses trypanothione-mediated peroxide metabolism in crithidia fasciculata. biol. chem., 378:827–36. ogston, a.g, 1948. interpretation of experiments on metabolic processes, using isotopic tracer elements. nature, 162:963. orain, d. and bradley, m. 2001. solid phase synthesis of trypanothione reductase inhibitors—towards single bead screening. tet lett, 42:515–8. o’sullivan, m.c. and zhou, q. 1995. novel polyamine derivatives as potent competitive inhibitors of trypanosoma cruzi trypanothione reductase. bioorg. med. chem. lett., 5:1957–60. o’sullivan, m.c., dalrymple, d.m. and zhou, q. 1996. inhibiting effects of spermidine derivatives on trypanosoma cruzi trypanothione reductase. j. enz. inhibition., 11:97–114. o’sullivan, m.c., zhou, q., li, z. et al. 1997. polyamine derivatives as inhibitors of trypanothione reductase and assessment of their trypanocidal activities. bioorg. med. chem., 5:2145–55. otero, l., vieites, m., boiani, l. et al. 2006. novel antitrypanosomal agents based on palladium nitrofurylthiosemicarbazone complexes: dna and redox metabolism as potential therapeutic targets. j. med. chem., 49:3322–31. parveen, s., khan, m.o.f., austin, s.e. et al. 2005. anti-trypanosomal, anti-leishmanial and anti-malarial activities of quaternary arylalkylammonium 2-amino-4-chlorophenyl phenyl sulfi des, a new class of trypanothione reductase inhibitor, and of n-acyl derivatives of 2amino-4-chlorophenyl phenyl sulfi de. j. med. chem., 48:8087–97. ponasik, j.a., strickland, c., faerman, c. et al. 1995. kukoamine a and other hydrophobic acylpolyamines: potent and selective inhibitors of crithidia fasciculata trypanothione reductase. biochem. j., 311:371–5. rivarola, h.w., fernandez, a.r., enders, j.e. et al. 2001. effects of clomipramine on trypanosoma cruzi infection in mice. trans. royal. soc. trop. med. hyg., 95:529–33. rivarola, h.w. and paglini-oliva, p.a. 2002. trypanosoma cruzi trypanothione reductase inhibitors: phenothiazines and related compounds modify experimental chagas’ disease evolution. curr drug targets: cardiovasc. haematol. disord., 2:43–52. salmon-chemin, l., buisine, e., yardley, v. et al. 2001. 2and 3-substituted 1,4-naphthoquinone derivatives as subversive substrates of trypanothione reductase and lipoamide dehydrogenase from trypanosoma cruzi: synthesis and correlation between redox cycling activities and in vitro cytotoxicity. j. med. chem., 44:548–65. drug target insights 2007: 2 146 khan saravanamuthu, a., vickers, t.j., bond, c.s. et al. 2004. two interacting binding sites for quinacrine derivatives in the active site of trypanothione reductase: a template for drug design. j. biol. chem., 279:29493–500. schirmer, r.h., mueller, j.g., krauth-siegel, r.l., 1995. disulfi de-reductase inhibitors as chemotherapeutic agents: the design of drugs for trypanosomiasis and malaria. angew. chem. int. ed, 34:141–54. shames, s.l., fairlamb, a.h., cerami, a. et al. 1986. purifi cation and characterization of trypanothione reductase from crithidia fasciculata, a newly discovered member of the family of disulfi de-containing fl avoprotein reductases. biochemistry, 25:3519–26. shapiro, t.a., 1993. inhibition of topoisomerases in african trypanosomes. acta tropica, 54:251–60. shapiro, t.a. and showalter, a.f. 1994. in vivo inhibition of trypanosome mitochondrial topoisomerase ii: effects on kinetoplast dna maxicircles. mol. cell. biol., 14:5891–7. tekwani, b.l., bacchi, c.j., pegg, a.e. 1992. putrescine activated s-adenosylmethionine decarboxylase from trypanosoma brucei brucei. mol. cell. biochem., 117:53–61. tovar, j. and fairlamb, a.h. 1996. extrachromosomal homologous expression of trypanothione reductase and its complementary mrna in trypanosoma cruzi. nucl. acids. res., 24:2942–9. tovar, j., cunningham, m.l., smith, a.c. et al. 1998a. down-regulation of leishmania donovani trypanothione reductase by heterologous expression of a trans-dominant mutant homolog: effect on parasite intracellular survival. proc. natl. acad. sci. u.s.a., 95:5311–5316. tovar, j., wilkinson, s., mottram, j.c. et al. 1998b. evidence that trypanothione reductase is an essential enzyme in leishmania by targeted replacement of the trya gene locus. mol. microbiol., 29:653–60. urbina, j.a., 2003. rational approach to specifi c chemotherapy of chagas disease. world class parasites, 7:127–35. vega-teijido, m., caracelli, i., zuckerman-schpector, j. et al. 2006. conformational analyses and docking studies of a series of 5-nitrofuran and 5-nitrothiophenesemicarbazone derivatives in three possible binding sites of trypanothione and glutathione reductases. j. mol. graph. model., 24:349–55. vellieux, f.m.d., hajdu, j., verlinde, c.l.m.j. et al. 1993. structure of glycosomal glyceraldehyde-3-phosphate dehydrogenase from trypanosoma brucei determined from laue data. proc. natl. acad. sci. u.s.a., 90:2355–9. verlinde, c.l.m.j., callens, m., van calenbergh, s. et al. 1994. selective inhibition of trypanosomal glyceraldehyde-3-phosphate dehydrogenase by protein structure-based design: toward new drugs for the treatment of sleeping sickness. j. med. chem., 37:3605–13. wang, c.c. 1995. molecular mechanisms and therapeutic approaches to the treatment of african trypanosomiasis. annu. rev. pharmacol. toxicol., 35:93–127. wierenga, r.k., kalk, k.h. and hol, w.g.j. 1987. structure determination of the glycosomal triosephosphate isomerase from trypanosoma brucei brucei at 2.4 å resolution. j. mol. biol., 198:109–21. williams, c.h.jr, 1992. lipoamide dehydrogenase, glutathione reductase, thioredoxin reductase, and mercuric ion reductase. a family of fl avoenzyme transhydrogenases. chem. biochem. flavoenzymes., 3:121–211. zhang, y., bond, c.s., bailey, s. et al. 1996. the crystal structure of trypanothione reductase from the human pathogen trypanosoma cruzi at 2.3 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drug target insights 2006: 1 19–28 19 review correspondence: junichi kitanaka, department of pharmacology, hyogo college of medicine, 1-1 mukogawa-cho, nishinomiya, hyogo 663-8501, japan. tel: +81 798 456333; fax: +81 798 456332; email: kitanaka-hyg@umin.net please note that this article may not be used for commercial purposes. for further information please refer to the copyright statement at http://www.la-press.com/copyright.htm modifi cation of monoaminergic activity by mao inhibitors infl uences methamphetamine actions junichi kitanaka, nobue kitanaka and motohiko takemura department of pharmacology, hyogo college of medicine, nishinomiya, hyogo 663-8501, japan. abstract: methamphetamine (meth) abuse is a serious health and social problem worldwide. at present, however, there are no effective medications for the treatment of meth abuse. of the intracellular meth target proteins, monoamine oxidase (mao) is involved in the regulation of monoaminergic tone in the brain, resulting in the modulation of methinduced behavioral abnormalities in mammals. the meth-induced expression of increased motor activity, stereotypy, and sensitization is closely associated with monoaminergic transmission in the brain. modifi cation of mao activity by mao inhibitors can infl uence meth action. of the mao inhibitors, the propargylamine derivative clorgyline, an irreversible mao-a inhibitor, effectively blocks meth-induced hyperlocomotion and behavioral sensitization in rodents. analysis of the associated monoaminergic activity indicates an involvement of altered striatal serotonergic transmission as well as an increased dopaminergic tone. some effects of mao inhibitors on meth action appear to be independent of mao, suggesting complex mechanisms of action of mao inhibitors in meth abuse. this review describes current research to fi nd effective treatment for meth abuse, using mao inhibitors. keywords: methamphetamine, hyperlocomotion, stereotypy, behavioral sensitization, clorgyline, selegiline, monoamine turnover, monoamine oxidase, 5-ht, striatum. introduction of amphetamines and their related compounds, d-methamphetamine (meth) is one of the most powerful drugs of abuse recognized worldwide on the basis of an increasing number of health and social problems, both acute and chronic (murray, 1998). in japan, the third epidemic of meth abuse among the population is in progress (ujike and sato, 2004), and about 18000 persons were arrested in 2004 for illegal drug abuse. the u.s. national survey on drug use and health estimated in 2004 that 1.4 million americans aged 12 or older had used meth in the past year (nsduh 2005). the direct cns effects of meth include increased wakefulness, increased respiration, enhanced body movements, hyperthermia, euphoria, and decreased appetite, as well as the following psychiatric sequelae: confusion, anxiety, paranoia, delirium, increased agitation, and aggressiveness (nida 2004). recently, evidence has accumulated that meth can cause neurodegeneration in the brain (davidson et al. 2001; kita et al. 2003; cadet et al. 2005). in addition, increased infectious diseases such as hiv and hepatitis b and c are likely to be consequences of increased meth abusers through sharing contaminated syringes (poshyachinda, 1993). at present, however, there are no effective medications for the treatment of meth abuse (kantak, 2003). the molecular basis of amphetamine action has been investigated. meth targets in subcellular components include the cocaine-sensitive dopamine transporter (dat) located in presynaptic plasma membranes, the vesicular monoamine transporter-2 (vmat-2) located in vesicular membranes, and the monoamine oxidase (mao) enzyme located in mitochondrial outer membranes (seiden et al. 1993). the direct action of meth on dat reverses dopamine transport, resulting in enhanced release of dopamine from presynaptic terminals into extracellular space (sulzer et al. 1995; jones et al. 1998; khoshbouei et al. 2003). meth also enters the presynaptic neurons via lipophilic diffusion and through dat as a structural analogue of dopamine (zaczek et al. 1991a, b), thereby allowing the drug to enter synaptic vesicles through vmat-2 and/or by diffusion. the resulting meth-induced disruption of vesicular ph produces enhanced release of dopamine from the vesicles through vmat-2 (sulzer et al. 1995; jones et al. 1998). once inside the presynaptic terminals, meth inhibits (i) vmat-2; resulting drug target insights 2006: 120 kitanaka et al in a decrease in vesicular dopamine uptake (brown et al. 2002; ugarte et al. 2003), and (ii) mao, which catalyzes the oxidative deamination of monoamines, resulting in increased levels of monoamines such as dopamine and serotonin (5-hydroxytryptamine, 5-ht) and a decrease in their metabolites (felner and waldmeier, 1979; egashira et al. 1987). overall, the actions of meth on these three target proteins result in the massive outfl ow of dopamine from the presynaptic terminal into the synaptic cleft (kuczenski et al. 1995). the activation of dopamine receptors by aberrant levels of released dopamine in mesolimbic and mesocortical areas is closely related to meth-induced abnormal behavior and the rewarding property of the drug in rodents and humans (robinson and becker, 1986; seiden et al. 1993; self and nestler, 1995; giros et al. 1996; everitt and robbins, 2005). pre-clinical investigations suggest that dat blockers such as vanoxerine (also known as gbr 12909) are effective for the prevention of cocaine effects such as cocaine-self administration and cocaine-induced increases in extracellular dopamine levels, because cocaine exerts its effects predominantly through an interaction with dat (gorelick et al. 2004). pre-clinical evaluation of vanoxerine as a potential medication for meth addiction is also in progress (baumann et al. 2002). alternatively, mechanism(s) other than dat have been proposed for meth action in transgenic studies (scearce-levie et al. 1999; budygin et al. 2004), because meth targets are multiple (seiden et al. 1993). in the recent literature, some attempts have been made to fi nd an effective treatment for meth abuse through one of the three targets listed above. this review describes current insights into the pharmacology of mao inhibitors from the behavior of rodents administered with various doses of single and repeated meth, since relatively moderate and high doses of single and repeated meth administration in rodents serve as an animal model for meth abuse in humans (described below). although amphetamines inhibit mao reversibly, a role for mao inhibition in amphetamine-induced behaviors has not brought much scientific interest since amphetamineinduced mao inhibition is weak (mantle et al. 1976; miller et al. 1980). however, recent evidence that meth (or d-amphetamine) in combination with mao inhibitors produces unique behavioral effects, and implicates a role for mao inhibitors in the protection against and improvement of meth abuse, especially, through the actions on the striatal serotonin system. meth-treated rodents as animal models of meth abuse the use of naïve rodents administered with single or repeated meth serves as an animal model for meth abuse in humans, since animal models show abnormal behavior such as increased motor activity (abekawa et al. 1995; kitanaka et al. 2003, 2005a), repetitive and compulsive behavior called stereotypy (nishikawa et al. 1983; kuczenski et al. 1995; abekawa et al. 1995; tatsuta et al. 2005, 2006), self-injurious behavior (halladay et al. 2003; mori et al. 2004), and rewarding properties (ranaldi and poeggel, 2002; justinova et al. 2003; kitanaka et al. 2006), which resemble human symptoms of amphetamine psychosis (randrup and munkvad, 1967; groves and rebec, 1976; winchel and stanley, 1991). in addition to the effects of meth listed above, repeated administration of meth also induces a progressive augmentation of locomotor activity in response to treatment, a phenomenon referred to as behavioral sensitization (post, 1980; shimosato and saito, 1993; ohno and watanabe, 1995; itzhak, 1997; ito et al. 2000; itzhak and ali 2002; kitanaka et al. 2003, 2005b; okabe and murphy, 2004). the progressive augmentation of locomotor activity in response to repeated meth in rodents resembles the meth-induced psychiatric symptoms which show progressive quantitative alteration in meth addicts (ellinwood and kilbey, 1977; robinson and becker, 1986; itzhak and ali, 2002; ujike and sato, 2004). mao, mao inhibitors, and amphetamines in mammals,the mao enzyme exists in two isoforms, termed mao-a and mao-b, which differ in substrate specifi city (johnston, 1968; murphy, 1978) and have been identifi ed as separate gene products (shih et al. 1999). selective and non-selective mao inhibitors have been developed for pre-clinical and clinical purposes, especially as treatments for major depression and parkinson’s disease (finberg and youdim, 1983; worms et al. 1987; pletscher 1991; aubin et al. 2004). the fi rst mao inhibitor to be discovered drug target insights 2006: 1 21 meth action and monoamine turnover was the hydrazine derivative iproniazid, which was originally developed for the treatment of tuberculosis. iproniazid and the related compounds are highly toxic to the liver when taken excess (sinha, 1987), resulting in the withdrawal of many hydrazine derivatives from the clinic. then, the propargyl compounds were developed as mao inhibitors with less undesirable side-effects (swett et al. 1963). among them, pargyline (n-methyl-n-2propynylbenzylamine) and clorgyline (n-methyln-propargyl-3-(2, 4-dichlorophenoxy) propylamine) were reported to be irreversible inhibitors of maoa/b (non-selective) and mao-a, respectively. selective irreversible mao-b inhibitors were also developed (knoll et al. 1965). one of them is selegiline (n, α-dimethyl-n-2-propynylphenethylamine; also know as l-deprenyl), which is a propargyl derivative of l-amphetamine. besides being potent monoamine releasing agents, amphetamines are also relatively potent, non-selective mao inhibitors (seiden et al. 1993), while the inhibition is weak compared with the actions of synthetic mao inhibitors (mantle et al. 1976; miller et al. 1980). while a valuable review highlights the advantages of mao inhibitors (youdim et al. 2006), these propargyl compounds have received little attention in the literature in recent years because of (1) the side effects associated with this drug class, including possible hypertensive crisis, (2) the development of new and more specifi c types of agents for the treatment of mental diseases and, (3) the complex mechanism of action of mao inhibitors, that are at least partially unresolved. as for the clinical use of psychostimulant-mao inhibitor combinations, amphetamine and mao inhibitor combination therapy has been used to augment antidepressant treatment (feinberg, 2004). other clinical trials of amphetamine and mao inhibitor combination appear to be highly restricted because of the lack of therapeutic benefi ts. in the animal experiments, however, the effects of clorgyline, selegiline, and pargyline on methinduced behavior have been well documented. modifi cation of meth action by clorgyline as shown in table 1, clorgyline has no effect on spontaneous locomotor activity in naïve mice and rats (table 1). however, when co-administered with meth, clorgyline exerts signifi cant inhibitory effects on meth (1 mg/kg, i.p.)-induced hyperlocomotion after single and repeated (5 days) clorgyline administration (kitanaka et al. 2005a). the effect of clorgyline on meth-induced behaviors lasts up to 40 min after meth challenge, suggesting that the development of meth-induced hyperlocomotion was effectively inhibited by clorgyline pretreatment (kitanaka et al. 2005a). it should be noted that no enhanced stereotypic behavior including “mouthing” behavior in parallel with the decrease in hyperlocomotion after meth in mice pretreated with clorgyline was observed in terms of the rearing index (kitanaka et al. 2005a). this effect was not observed with selegiline (kitanaka et al. 2005a). in this study, the association between the inhibitory action of clorgyline and mao-a activity in the brain was simultaneously investigated. for this purpose, the activity of monoaminergic transmission was evaluated in terms of apparent monoamine turnover. with respect to 5-ht metabolism, the ratio of 5-hydroxyindoleacetic acid (5-hiaa), a metabolite of 5-ht, to 5-ht is a good index of apparent 5-ht turnover (de vries and odink, 1991; torres et al. 2002). 5-ht turnover was signifi cantly increased in the regions of the striatum and nucleus accumbens after single meth administration due to a signifi cant decrease in 5-ht levels (kitanaka et al. 2003). clorgyline pretreatment, in turn, signifi cantly increased and decreased 5-ht and 5-hiaa contents, respectively, by inhibiting mao-a, resulting in a signifi cant decrease in the 5-ht turnover index in the striatum and nucleus accumbens (kitanaka et al. 2005a). this effect of clorgyline on 5-ht turnover is closely correlated with the drug’s inhibitory action on meth-induced hyperlocomotion, evaluated by anova analysis (kitanaka et al. 2005a). this fi nding suggests that the improvement of meth-induced abnormal 5-ht turnover in the striatal region by clorgyline treatment may play a role in the suppression of meth-induced increase in motor activity. since selegiline had no effect on 5-ht turnover in vivo, meth-induced hyperlocomotion and 5-ht turnover were not affected by selegiline pretreatment (kitanaka et al. 2005a; table 1). clorgyline action on meth-induced hyperlocomotion in mice (kitanaka et al. 2005a) appears paradoxical, because clorgyline and meth were assumed to inhibit mao-a activity additively, presumably resulting in enhanced meth toxicity (i.e. hypermotility) with clorgyline pretreatment. drug target insights 2006: 122 kitanaka et al ta bl e 1. e ffe ct s of m a o in hi bi to rs o n be ha vi or o f n aï ve a nd m e t h ( or d -a m ph et am in e) -c ha lle ng ed r od en ts . m ea su re m en t m a o i s pe ci es , d os e an d in je ct io n s ch ed ul e e ff ec ta r ef er en ce n aï ve lo co m ot io n c lo rg yl in e r at , 4 m g/ kg , i p x 1 d n c s eg al e t a l. 19 92 c lo rg yl in e m ou se , 1 m g/ kg , s c x 1 d or 5 d n c k ita na ka e t a l. 20 05 a s el eg ili ne r at , 0 .2 5 m g/ kg , s c x 42 d n c ti m ár e t a l. 19 86 s el eg ili ne r at , 1 –2 0 m g/ kg , s c x 1 d n c ti m ár e t a l. 19 96 s el eg ili ne r at , 1 0 m g/ kg , i p x 1 d in cr ea se o ku da e t a l. 19 92 s el eg ili ne r at , 1 0 m g/ kg , i p x 1 d in cr ea se t he m an n et a l. 20 02 s el eg ili ne m ou se , 0 .3 m g/ kg , s c x 1 d or 5 d n c k ita na ka e t a l. 20 05 a p ar gy lin e r at , 1 0 m g/ kg , s c x 24 d in cr ea se b ar be liv ie n et a l. 20 01 s te re ot yp y s el eg ili ne r at , 1 –1 0 m g/ kg , s c x 1 d n d ti m ár e t a l. 19 96 s el eg ili ne r at , 2 0 m g/ kg , s c x 1 d in cr ea se ti m ár e t a l. 19 96 b eh av io ra l s en si tiz at io n c lo rg yl in e m ou se , 1 m g/ kg , s c x 5 d n d k ita na ka e t a l. 20 05 b s el eg ili ne r at , 1 0 m g/ kg , i p x 8 d in cr ea se t he m an n et a l. 20 02 c p p in de x c lo rg yl in e m ou se , 0 .1 –1 0 m g/ kg , s c x 6 d in cr ea se k ita na ka e t a l. 20 06 s el eg ili ne r at , 1 –2 0 m g/ kg , s c x 4 d n d ti m ár e t a l. 19 96 s el eg ili ne m ou se , 1 0 an d 25 m g/ kg , i p x 5 d in cr ea se w u an d z hu 1 99 9 # of c a r s el eg ili ne r at , 1 –2 0 m g/ kg , s c x 5 d n d ti m ár e t a l. 19 96 d s e b s el eg ili ne r at , 1 0– 30 m g/ kg , i p x 1 in cr ea se ya sa r et a l. 19 93 a fte r m e t h ( or d -a m ph et am in e) c ha lle ng e h yp er lo co m ot io n c lo rg yl in e r at , 4 m g/ kg , i p x 1 d d ec re as e s eg al e t a l. 19 92 c lo rg yl in e m ou se , 1 m g/ kg , s c x 1 d or 5 d d ec re as e k ita na ka e t a l. 20 05 a, b s el eg ili ne m ou se , 0 .3 m g/ kg , s c x 1 d or 5 d n c k ita na ka e t a l. 20 05 a p ar gy lin e m ou se , 5 0 m g/ kg , i p x 1 d n c le w e t a l. 19 71 s te re ot yp y c lo rg yl in e r at , 4 m g/ kg , i p x 1 d in cr ea se s eg al e t a l. 19 92 c lo rg yl in e r at , 0 .1 –1 0 m g/ kg , s c x 1 d n c ta ts ut a et a l. 20 05 c lo rg yl in e m ou se , 0 .1 n ot 1 –1 0 m g/ kg , s c x 1 d d ec re as e ta ts ut a et a l. 20 05 s el eg ili ne r at , 0 .2 5– 5 m g/ kg , s c x 1 d d ec re as e ti m ár e t a l. 19 93 s el eg ili ne m ou se , 0 .1 , 1 a nd 1 0 m g/ kg , s c x 1 d n c ta ts ut a et a l. 20 05 b eh av io ra l s en si tiz at io n c lo rg yl in e m ou se , 1 m g/ kg , s c x 5 d d ec re as e k ita na ka e t a l. 20 05 b c p p in de x c lo rg yl in e m ou se , 0 .1 –1 0 m g/ kg , s c x 6 d n c k ita na ka e t a l. 20 06 d s e b s el eg ili ne r at , 3 a nd 5 .6 m g/ kg , i p x 1 in cr ea se ya sa r et a l. 19 93 m or ta lit y s el eg ili ne m ou se , 2 m g/ kg , s c, x 1 3 d n c g ra si ng e t a l. 20 01 s el eg ili ne m ou se , 0 .0 2 an d 0. 2 m g/ kg , s c, x 1 3 d d ec re as e g ra si ng e t a l. 20 01 t hi s ta bl e co m pa re s th e pu bl is he d ef fe ct s of m a o in hi bi to rs ( m a o i) o n na ïv e an d m e t h -t re at ed m ic e. c a r : c on di tio ne d av oi da nc e re sp on se s; c p p : c on di tio ne d pl ac e pr ef er en ce ; d s e : d is cr im in at iv e st im ul us e ffe ct ; n c : n o ch an ge ; n d : n ot d et ec te d; ip : i nt ra pe rit on ea l i nj ec tio n; s c: s ub cu ta ne ou s in je ct io n. “ s el eg ili ne ” m ea ns “ l-i so m er o f s el eg ili ne ” in th is ta bl e. a c om pa ris on b et w ee n m a o i a nd v eh ic le -p re tr ea te d su bj ec ts . b m ic e w er e us ed a fte r th ey w er e tr ai ne d un de r a 5re sp on se , fi x ed r at io s ch ed ul e of s tim ul us -s ho ck te rm in at io n or a 1 0re sp on se , fi x ed r at io s ch ed ul e of fo od p re se nt at io n to d is cr im in at e be tw ee n dam ph et am in e (1 m g/ kg ) an d sa lin e in a tw ole ve r, co nd iti on in g pr oc ed ur e. drug target insights 2006: 1 23 meth action and monoamine turnover indeed, for apparent dopamine turnover, an additive inhibition of the ratio of 3, 4-dihydroxyhenylacetic acid (dopac), a metabolite of dopamine, to dopamine by single meth administration after clorgyline pretreatment was observed in the regions of the striatum and nucleus accumbens in mice (kitanaka et al. 2005a). in light of these fi ndings, why did the 5-ht content selectively decrease in the regions of the striatum and nucleus accumbens in mice after a relatively moderate dose of meth (1 mg/kg)? a depletion effect similar to this is often interpreted as evidence that the neurotoxic dosage of meth induces neurodegeneration in the terminals of monoaminergic neurons. in the rat brain striatum, neurotoxic meth destroys the terminal arbors of fi ne 5-htergic axons that arise from the dorsal raphe nucleus, resulting in the acute depletion of 5-ht from the axons (axt and molliver, 1991; brown and molliver, 2000). similar neurodegeneration is observed in the dopaminergic axons of rats (ricaurte et al. 1982, 1984; xu et al. 2005), with a signifi cant decrease in the striatal dopamine content (broening et al. 2005). neurotoxic dosages of meth in mice (four injections of 15 mg/kg meth every 2 h or a single administration with 30 mg/kg) have been shown to produce a signifi cant decrease in the striatal contents of dopamine and 5-ht (fumagalli et al. 1998), similar to the observations in rats. unfortunately, the possible neurodegenerative effects of meth at a moderate dose (1 mg/kg) on the striatal neuronal system in mice has not yet been studied. provided that monoaminergic axons in the striatum express properties that determine which are relatively vulnerable to meth at even moderate doses, there exists an obvious difference between dopamine and 5-ht fi bers which result in the meth vulnerability. since there is evidence of 10-30-fold higher content of dopamine than 5-ht in the mouse striatum (kitanaka et al. 2003, 2005a, 2006), the density of dopamine-containing fi bers might be higher than that of 5-ht-containing fi bers, provided that the percentages of synapses occupied by dopamine and 5-ht present in the brain are identical (krieger, 1983). it is possible that the low density of striatal 5-ht fi bers can be damaged more seriously than the dopamine fi bers by meth at moderate doses. striatal 5-ht neurotransmission is suggested to play a crucial role in the regulation of meth-induced hyperlocomotion in mice (kitanaka et al. 2005a). therefore, it is possible that mao-a inhibition increases the content of 5-ht, resulting in a signifi cant improvement in 5-ht neurotransmission which protects against meth-induced hyperlocomotion via the activation of 5-ht turnover. several investigations have shown that the development of sensitization to the locomotor stimulating effect of meth in rodents can be blocked effectively by pharmacological agents such as a protein synthesis inhibitor cycloheximide, nitric oxide synthase inhibitors ng-nitro-l-arginine and 7-nitroindazole, and a benzodiazepine clonazepam administered prior to, or simultaneously with meth (1 mg/kg once per day for 5–10 consecutive days or 2 mg/kg once per day, 5 times at intervals of 3–4 days) (shimosato and saito, 1993; ohno and watanabe, 1995; itzhak, 1997; ito et al. 2000). unfortunately, a protocol involving agent pretreatment or co-treatment is not easily applied to human meth addictions. to overcome this, we applied a modifi ed treatment protocol to mice which have already acquired behavioral sensitization to meth (1 mg/kg, i.p. once per day for 5 consecutive days). during the drug-free period (4 days) after the repeated meth administration, the mice were treated with 1 mg/kg of clorgyline once per day. this treatment successfully blocked the expression of behavioral sensitization to meth (table 1; kitanaka et al. 2005b). it is suggested that behavioral sensitization may have some common properties with learning and memory (lidow et al. 1998); therefore, the cerebral cortex is one region of interest. after the meth challenge, signifi cantly enhanced dopamine metabolism (i.e. increased overall dopamine turnover, the ratio of homovanillic acid (hva) to dopamine) was observed in the cerebral cortex of meth-sensitized mice compared with non-sensitized animals (kitanaka et al. 2005b). in particular, cerebral cortical dopamine metabolism increased approximately 3-fold in sensitized mice treated with repeated clorgyline compared with mice without clorgyline treatment. it is likely that brain dopamine in mice, which is not metabolized by mao-a after repeated clorgyline treatment (kitanaka et al. 2005a) was, in turn, exposed to catechol-o-methyltransferase (comt), another dopamine metabolizing enzyme. in the cerebral cortex, dopamine metabolism is more sensitive to comt than that in the striatum or hypothalamus (gogos et al. 1998), or cerebral cortical mao activity is lower than in other regions. drug target insights 2006: 124 kitanaka et al therefore, dopamine released in the cerebral cortex by a single meth challenge was metabolized largely by comt after clorgyline treatment, resulting in inhibition of the expression of behavioral sensitization to meth. it is of interest to note the possible relationship between the activities of mao-a and comt, infl uenced by each other, in the brain; however, to our knowledge, there is no direct evidence of molecular interaction between the two enzymes. clorgyline exhibits no behavioral sensitization per se (table 1). in rats, segal et al. (1992) reported that clorgyline (4 mg/kg) pretreatment signifi cantly reduced locomotion (increased crossover plus rearing) during the fi rst 1-h interval after the amphetamine challenge (0.25 and 2.5 mg/kg) in parallel with a significant increase in the total period of the observed stereotypy (table 1). this effect is interpreted by experimental evidence that mao-a inhibition by clorgyline increases the extracellular dopamine concentration in the nucleus accumbens, assessed by in vivo microdialysis. in contrast, no change in the intensity of meth (10 mg/kg)induced stereotypy was observed in rats pretreated with clorgyline (0.1–10 mg) (table 1; tatsuta et al. 2005). in mice, the lowest dose of clorgyline tested (0.1 mg/kg) signifi cantly increased and decreased hyperlocomotion and stereotypy, respectively, during the fi rst 20-min interval at which the mice showed a submaximal intensity of stereotypy (tatsuta et al. 2005). however, clorgyline pretreatment (1 and 10 mg/kg) did not signifi cantly alter horizontal hyperlocomotion in mice during the fi rst 20-min interval after meth challenge (10 mg/kg) compared with the mice pretreated with vehicle (saline). the molecular action of the clorgyline is likely to be independent of mao-a because (1) change in the intensity of meth-induced stereotypy was not correlated with the change in the striatal monoamine turnover during the fi rst 20-min interval (tatsuta et al. 2006) and, (2) the clorgyline (0.1 mg/kg)-induced shift in the meth response was not correlated with the degree of mao-a inhibition estimated by apparent monoamine turnover (tatsuta et al. 2005). possible interactions of clorgyline with sigma receptors (itzhak and kassim, 1990; itzhak et al. 1991), imidazoline i2 receptors (alemany et al. 1995; macinnes and duty, 2004), and/or mao inhibitor-displaceable quinpirole binding sites (culver and szechtman, 2003) should not be neglected to understand the mode of action of clorgyline, since these binding sites are involved in psychiatric disorders (eglen et al. 1998; bermack and debonnel, 2005). clorgyline displays high affi nity for both mao-a and sigma receptors with relatively identical affi nities (ic50 value of 10 nm and 3 nm, respectively) (egashira et al. 1987; itzhak et al. 1991), and clorgyline-sensitive sigma receptors are suggested to coexist with a subcellular fraction with mao activity (itzhak et al. 1991). therefore, the doses of clorgyline used in the in vivo studies appear to fully activate the sigma receptors. for the meth-induced rewarding property, clorgyline pretreatment (0.1–10 mg/kg) failed to block the meth (0.5 mg/kg)-induced increase in the conditioned place preference (cpp) index in mice (table 1; kitanaka et al. 2006). the monoamine turnover index (ratios of dopac to dopamine, hva to dopamine, and 5-hiaa to 5-ht) in the striatum and nucleus accumbens was not different between mice conditioned with and without meth, indicating that the inhibitory effect of various doses of clorgyline on mao activity was independent of meth (0.5 mg/kg) action. it should be noted that the saline/saline pairing groups pretreated with clorgyline at a dose of 1 mg/kg showed an increased cpp index, similar to the result from meth/saline pairing group (kitanaka et al. 2006). this might mean that the mice in the saline/saline pairing group entered and stayed in each cpp compartment independent of the given visual and texture cues on the testing day after the pretreatment with 1 mg/kg clorgyline. modifi cation of meth action by selegiline selegiline in appropriate doses exhibits amphetamine-like properties per se, such as increased motor activity, rewarding effect, and behavioral sensitization (table 1), since selegiline is metabolized in part to l-meth and l-amphetamine (reynolds et al. 1978; elsworth et al. 1982; lajtha et al. 1996; gerlach et al. 1996; baker et al. 1999). because of the potential of the selegiline metabolites for abuse liability, it is likely that selegiline could enhance meth action when given in combination. indeed, selegiline (20 mg/kg) induced the stereotyped head movement in rats, while the stereotypy was not observed when the rats were administered with 1–10 mg/kg of selegiline (table 1; timár et al. 1996). selegiline in doses of drug target insights 2006: 1 25 meth action and monoamine turnover 1–20 mg/kg had no effect on locomotion, cpp index, nor the number of conditioned avoidance responses (timár et al. 1996). increased discriminative stimulus effects were reported using 3 and 5.6 mg/kg of selegiline in combination with d-amphetamine (1 mg/kg) compared with d-amphetamine alone in mice which have been trained under a 5-response, fi xed ratio schedule of stimulus-shock termination or a 10-response, fi xed ratio schedule of food presentation to discriminate between d-amphetamine (1 mg/kg) and saline in a two-lever, conditioning procedure (table 1; yasar et al. 1993). also, high doses of selegiline (17–30 mg/kg) produced full generalization to d-amphetamine (yasar et al. 1993). these observations as well as those of timár et al. (1996) suggest that the amphetamine-like properties can be observed when selegiline l-isomer) in high doses is treated. small doses of selegiline fail to induce any amphetamine-like hyperlocomotion (timár et al. 1986). timár et al. (1993) reported that selegiline at small doses can decrease amphetamine-induced stereotypy in rats, without change in the index of dopamine turnover in the olfactory tubercle, compared with saline-pretreated animals. they suggest that the uptake of amphetamine might be reduced by pretreatment with selegiline, resulting in the decrease of stereotyped behavior; however, the same selegiline treatment protocol as that reported by timár et al. (1993) enhances striatal dopamine turnover (zsilla et al. 1982). therefore, the relationship between selegiline action on amphetamine-induced stereotypy and mao-b activity needs to be clarifi ed by further studies. only one report shows the effect of selegiline on the lethal action of meth. repeated administration with lower doses of selegiline (0.02 and 0.2 mg/kg) to mice treated with toxic meth dosage (10 mg/kg # 4 within one day, two-hour intervals) blocked the meth-induced increase in mortality (table 1; grasing et al. 2001). this selegiline effect was not detected at 2 mg/kg, suggesting that the mode of action is mao-b independent. regarding this point, lamensdorf et al. (1999) reported that increased dat expression was observed after chronic (21 days) treatment with selegiline (0.25 mg/kg). this might explain the results of timár et al. (1993) and of grasing et al. (2001), since the increased dat expression induces increased extracellular dopamine clearance in the brain. molecular mechanism(s) of the enhanced dat expression by selegiline are unclear, although selegiline has been shown to alter the expression of mrna for a variety of proteins including tyrosine hydroxylase and laromatic amino acid decarboxylase (vrana et al. 1992; li et al. 1992). in mice, stereotypy can be induced by meth at doses of 10 mg/kg by a single injection (tatsuta et al. 2005). when administered of mice with 20 mg/kg meth, the mice show self-injurious behavior (mori et al. 2004). tatsuta et al. (2005) reported that a wide range of selegiline (0.1–10 mg/kg) showed no enhancement of meth-induced stereotyped behavior nor a shift of the abnormal behavior from stereotypy to self-injurious behavior in mice, suggesting that 10 mg/kg of selegiline could not have an amphetamine (10 mg/kg)-like property after the systemic injection and following metabolism. modifi cation of meth action by non-selective mao inhibitors lew et al. (1971) reported that pretreatment of mice with 50 mg/kg of pargyline, a non-selective mao inhibitor, had no effect on d-amphetamineinduced hyperlocomotion in mice (table 1). however, treatment of rats with 10 mg/kg of pargyline increases locomotor activity per se (table 1; barbelivien et al. 2001); this effect might be interpreted by evidence that mao-a inhibition by clorgyline (and probably by pargyline at high doses) increases extracellular dopamine concentration in the nucleus accumbens (segal et al. 1992). the possible effect of metabolites of pargyline (benzylamine, n-methylbenzylamine, and n-propargylbenzylamine) on spontaneous locomotion in rodents can not be ruled out, but no reports have not been published. aubin et al. (2004) reported the behavioral profi le of a newly developed, mixed-reversible mao-a/b inhibitor, sl25.1131, in mice. the agent can improve decreased dopaminergic tone in the striatum by inhibiting mao-a and –b and locomotion disrupted by treatment with mptp (1-methyl-4-pheny l–1,2,3,6-tetrahydropyridine). mixed mao inhibitors possess attractive potential properties for the treatment of meth abuse, since selective, irreversible mao inhibitors can block meth (or d-amphetamine)-induced abnormal behavior in rodents (table 1), although the mechanisms of action are complex. drug target insights 2006: 126 kitanaka et al conclusions meth-induced motor activity, stereotypy, and sensitization are closely associated with monoaminergic transmission. modifi cation of mao activity by mao inhibitors can infl uence meth action. although some pre-clinical studies cited in this review suggest the feasibility of mao inhibitors for the treatment of meth abuse, careful attention should be directed to the potential risk similar to that reported in the treatment of depressive disorder. based on the current research on the mechanisms of mao inhibitors, they exhibit a putative ‘novel’ mode of action which is independent of mao and might infl uence monoaminergicrelated behavior, as well as ‘classical’ mao inhibition. for pre-clinical studies of the exact mode of action of mao inhibitors and the effects of the mao inhibitors on meth-induced abnormal behavior, meth-induced abnormal behavior in rodents may serve as an appropriate animal model for meth abuse in humans. acknowledgments research by one of the authors (nk) described herein was supported in part by a grant-in-aid for researchers, hyogo college of medicine. we thank anonymous reviewers for their very helpful comments on an earlier version of the manuscript. references abekawa, t., ohmori, t. and koyama, t. 1995. effects of nitric oxide (no) synthesis inhibition on the development of supersensitivity to stereotypy and locomotion stimulating effects of methamphetamine. brain res., 679:200–4. alemany, r., olemos, g. and garcía-sevilla, j.a. 1995. the effects of phenelzine and other monoamine oxidase inhibitor antidepressants on brain and liver i2 imidazoline-preferring receptors. br. j. pharmacol., 114:837–45. aubin, n. et al. 2004. sl25.1131 [3(s), 3a(s)-3-methoxymethyl-7-[4,4,4t r i f l u o r o b u t o x y ] 3 , 3 a , 4 , 5 t e t r a h y d r o 1 , 3 o x a z o l o [ 3 , 4 a ] quinolin-1-one], a new, reversible, and mixed inhibitor of monoamine oxidase-a and monoamine oxidase-b: biochemical and behavioral profi le. j. pharmacol. exp. ther., 310:1171–82. axt, k.j. and molliver, m.e. 1991. immunocytochemical evidence for methamphetamine-induced serotonergic axon loss in the rat brain. synapse, 9:302–13. baker, g.b. et al. 1999. metabolism of monoamine oxidase inhibitors. cell mol. neurobiol., 19:411–26. barbelivien, a. et al. 2001. inhibition of mao-a activity enhances behavioural activity of rats assessed using water maze and open arena tasks. pharmacol. toxicol., 88:304–12. baumann, m.h. et al. 2002. preclinical evaluation of gbr 12909 decanoate as a long-acting medication for methamphetamine dependence. ann. ny acad. sci., 965:92–18. bermack, j.e. and debonnel, g. 2005. the role of sigma receptors in depression. j. pharmacol. sci., 97:317–36. broening, h.w., morford, l.l. and vorhees, c.v. 2005. interactions of dopamine d1 and d2 receptor antagonists with d-methamphetamineinduced hyperthermia and striatal dopamine and serotonin reductions. synapse, 56:84–93. brown, p. and molliver, m.e. 2002. dual serotonin (5-ht) projections to the nucleus accumbens core and shell: relation of the 5-ht transporter to amphetamine-induced neurotoxicity. j. neurosci., 20:1952–63. brown, j.m. et al. 2002. a single methamphetamine administration rapidly decreases vesicular dopamine uptake. j. pharmacol. exp. ther., 302:497–501. budygin, e.a. et al. 2004. dissociation of rewarding and dopamine transporter-mediated properties of amphetamine. proc. natl. acad. sci. u.s.a., 101:7781–6. cadet, j.l., jayanthi, s. and deng, x. 2005. methamphetamine-induced neuronal apoptosis involves the activation of multiple death pathways. review. neurotoxicity res., 8:199–206. culver, k.e. and szechtman, h. 2003. clorgyline-induced switch from locomotion to mouthing in sensitization to the dopamine d2/d3 agonist quinpirole in rats: role of sigma and imidazoline i2 receptors. psychopharmacology (berl.), 167:211–8. davidson, c. et al. 2001. methamphetamine neurotoxicity: necrotic and apoptotic mechanisms and relevance to human abuse and treatment. brain res. rev., 36:1–22. de vries, m. and odink, j. 1991. simultaneous measurement of serotonin and 5-hydroxyindoleacetic acid in rat brain using a liquid chromatographic method with electrochemical detection. j. chromatogr, 564:250–7. egashira, t., yamamoto, t. and yamanaka, y. 1987. effects of d-methamphetamine on monkey brain monoamine oxidase, in vivo and in vitro. jpn. j. pharmacol., 45:79–88. eglen, r.m. et al. 1998. ‘seeing through a glass darkly’: casting light on imidazoline ‘i’ sites. trends pharmacol. sci., 19:381–90. ellinwood, e.h., jr. and kilbey, m.m. 1977. chronic stimulant intoxication models of psychosis. in hanin i and usdin e, eds. animal models in psychiatry and neurology. new york: pergamon press ltd. p 61–74. elsworth, j.d. et al. 1982. the contribution of amphetamine metabolites of (-)-deprenyl to its antiparkinsonian properties. j. neural. transm., 54:105–10. everitt, b.j. and robbins, t.w. 2005. neural systems of reinforcement for drug addiction: from actions to habits to compulsion. nature neurosci., 8:1481–9. feinberg, s.s. 2004. combining stimulants with monoamine oxidase inhibitors: a review of use and one possible additional indication. j. clin. psychiatry, 65:1520–4. felner, a.e. and waldmeier, p.c. 1979. cumulative effects of irreversible mao inhibitors in vivo. biochem. pharmacol., 28:995–1002. finberg, j.p. and youdim, m.b. 1983. selective mao a and b inhibitors: their mechanism of action and pharmacology. neuropharmacology, 22:441–6. fumagalli, f. et al. 1998. role of dopamine transporter in methamphetamineinduced neurotoxicity: evidence from mice lacking the transporter. j. neurosci., 18:4861–9. gerlach, m., youdim, m.b.h. and riederer, p. 1996. pharmacology of selegiline. neurology, 47:s137–s145. giros, b. et al. 1996. hyperlocomotion and indifference to cocaine and amphetamine in mice lacking the dopamine transporter. nature, 379:606–12. gogos, j.a. et al. 1998. catechol-o-methyltransferase-defi cient mice exhibit sexually dimorphic changes in catecholamine levels and behavior. proc. natl. acad. sci. u.s.a., 95:9991–6. gorelick, d.a., gardner, e.l. and xi, z-x. 2004. agents in development for the management of cocaine abuse. drugs, 64:1547–73. grasing, k, et al. 2001. biphasic effects of selegiline on striatal dopamine: lack of effect on methamphetamine-induced dopamine depletion. neurochem. res., 26:65–74. groves, p.m. and rebec, g.v. 1976. biochemistry and behavior: some central actions of amphetamine and antipsychotic drugs. annu. rev. psychol., 27:91–127. drug target insights 2006: 1 27 meth action and monoamine turnover halladay, a.k. et al. 2003. relationship between methamphetamine-induced dopamine release, hyperthermia, self-injurious behaviour and long term dopamine depletion in balb/c and c57bl/6 mice. pharmacol. toxicol., 93:33–41. ito, k. et al. 2000. the role of benzodiazepine receptors in the acquisition and expression of behavioral sensitization to methamphetamine. pharmacol. biochem. behav., 65:705–10. itzhak, y. 1997. modulation of cocaineand methamphetamine-induced behavioral sensitization by inhibition of brain nitric oxide synthase. j. pharmacol. exp. ther., 282:521–7. itzhak, y. and ali, s.f. 2002. behavioral consequences of methamphetamineinduced neurotoxicity in mice: relevance to the psychopathology of methamphetamine addiction. ann. ny acad. sci., 965:127–35. itzhak, y. and kassim, c.o. 1990. clorgyline displays high affi nity for sigma binding sites in c57bl/6 mouse brain. eur. j. pharmacol., 176:107–8. itzhak, y. et al. 1991. binding of sigma-ligands to c57bl/6 mouse brain membranes: effects of monoamine oxidase inhibitors and subcellular distribution studies suggest the existence of sigma-receptor subtypes. j. pharmacol. exp. ther., 257:141–8. johnston, j.p. 1968. some observations upon a new inhibitor of monoamine oxidase in brain tissue. biochem. pharmacol., 17:1285–97. jones, s.r. et al. 1998. mechanisms of amphetamine action revealed in mice lacking the dopamine transporter. j. neurosci., 18:1979–86. justinova, z, et al. 2003. involvement of adenosine a1 and a2 receptors in the adenosinergic modulation of the discriminative-stimulus effects of cocaine and methamphetamine in rats. j. pharmacol. exp. ther., 307:977–86. kantak, k.m. 2003. vaccines against drugs of abuse: a viable treatment option? drugs, 63:341–52. khoshbouei, h. et al. 1993. amphetamine-induced dopamine effl ux: a voltage-sensitive and intracellular na+-dependent mechanism. j. biol. chem., 278:12070–7. kita, t., wagner, g.c. and nakashima, t. 2003. current research on methamphetamine-induced neurotoxicity: animal models of monoamine disruption. j. pharmacol. sci., 92:178–95. kitanaka, n., kitanaka, j. and takemura, m. 2003. behavioral sensitization and alteration in monoamine metabolism in mice after single versus repeated methamphetamine administration. eur. j. pharmacol., 474:63–70. kitanaka, n., kitanaka, j. and takemura, m. 2005a. inhibition of methamphetamine-induced hyperlocomotion in mice by clorgyline, a monoamine oxidase-a inhibitor, through alteration of the 5-hydroxytryptamine turnover. neuroscience, 130:295–308. kitanaka, n., kitanaka, j. and takemura, m. 2005b. repeated clorgyline treatment inhibits methamphetamine-induced behavioral sensitization in mice. neurochem. res., 30:445–51. kitanaka, n. et al. 2006. methamphetamine reward in mice as assessed by conditioned place preference test with supermex® sensors: effects of subchronic clorgyline pretreatment. neurochem. res., 31:805–813. knoll, j. et al. 1965. phenylisopropylmethylpropinylamine (e-250): a new spectrum psychic energizer. arch. int. pharmacodyn ther., 155:154–64. krieger, d.t. 1983. brain peptides: what, where, and why? science, 222:975–85. kuczenski, r. et al. 1995. hippocampus norepinephrine, caudate dopamine and serotonin, and behavioral responses to the stereoisomers of amphetamine and methamphetamine. j. neurosci., 15:1308–17. lajtha, a. et al. 1996. metabolism of (-)-deprenyl and pf-(-)-deprenyl in brain after central and peripheral administration. neurochem. res., 21:1155–60. lamensdorf, i. et al. 1999. effect of low-dose treatment with selegiline on dopamine transporter (dat) expression and amphetamine-induced dopamine release in vivo. br. j. pharmacol., 126:997–1002. lew, c., iversen, s.d. and iversen, l.l. 1971. effects of imipramine, desipramine and monoamine oxidase inhibitors on the metabolism and psychomotor stimulant actions of d-amphetamine in mice. eur. j. pharmacol., 14:351–9. li, x-m. et al. 1992. specifi c irreversible monoamine oxidase b inhibitors stimulate gene expression of aromatic amino acid decarboxylase in pc12 cells. j. neurochem., 59:2324–7. lidow, m.s., williams, g.v. and goldman-rakic, p.s. 1998: the cerebral cortex: a case for common site of action of antipsychotics. trends pharmacol. sci., 19:136–40. macinnes, n. and duty, s. 2004. locomotor effects of imidazoline i2-sitespecifi c ligands and monoamine oxidase inhibitors in rats with a unilateral 6-hydroxydopamine lesion of the nigrostriatal pathway. br. j. pharmacol., 143:952–9. mantle, t.j. tipton, k.f. and garrett, n.j. 1976. inhibition of monoamine oxidase by amphetamine and related compounds. biochem. pharmacol., 25:2073–7. miller, h.h., shore, p.a. and clarke, d.e. 1980. in vivo monoamine oxidase inhibition by d-amphetamine. biochem. pharmacol., 29:1347–54. mori, t. et al. 2004. effects of dopamineand serotonin-related compounds on methamphetamine-induced self-injurious behavior in mice. j. pharmacol. sci., 96:459–64. murray, j.b. 1998. psychophysiological aspects of amphetamine-methamphetamine abuse. j. physiol., 132:227–37. murphy, d.l. 1978. substrate-selective monoamine oxidases: inhibitor, tissue, species and functional differences. biochem. pharmacol., 27:1889–93. national institute on drug abuse. 2004. nida infofacts: methamphetamine. accessed 3 august 2006. url: http://www.nida.nih.gov/infofacts/ methamphetamine.html national survey on drug use and health. 2005. the nsduh reportmethamphetamine use, abuse, and dependence: 2002, 2003, and 2004. accessed 14 july 2006. url: http://oas.samhsa.gov/2k5/meth/ meth.pdf nishikawa, t. et al. 1983. behavioral sensitization and relative hyperresponsiveness of striatal and limbic dopaminergic neurons after repeated methamphetamine treatment. eur. j. pharmacol., 88:195–203. ohno, m. and watanabe, s. 1995. nitric oxide synthase inhibitors block b e h a v i o r a l s e n s i t i z a t i o n t o m e t h a m p h e t a m i n e i n m i c e . eur. j. pharmacol., 275:39–44. okabe, c. and murphy, n.p. 2004. short-term effects of the nociceptin receptor antagonist compound b on the development of methamphetamine sensitization in mice: a behavioral and c-fos expression mapping study. brain res., 1017:1–12. okuda, c., segal, d.s. and kuczenski, r. 1992. deprenyl alters behavior and caudate dopamine through an amphetamine-like action. pharmacol. biochem. behav., 43:1075–80. pletscher, a. 1991. the discovery of antidepressants: a winding path. experientia, 47:4–8. poshyachinda, v. 1993. drug injecting and hiv infection among the population of drug abusers in asia. bull. narc., 45:77–90. post, r.m. 1980. intermittent versus continuous stimulation: effect of time interval on the development of sensitization. life sci., 26:1275–82. ranaldi, r. and poeggel, k. 2002. baclofen decreases methamphetamine self-administration in rats. neuroreport, 13:1107–10. randrup, a. and munkvad, i. 1967. stereotyped activities produced by amphetamine in several animal species and man. psychopharmacologia (berl.), 11:300–10. reynolds, g.p. et al. 1978. deprenyl is metabolized to methamphetamine and amphetamine in man. br. j. clin. pharmacol., 6:542–4. ricaurte, g.a. et al. 1982. dopamine nerve terminal degeneration produced high doses of methylamphetamine in the rat brain. brain res., 235:93–103. ricaurte, g.a., seiden, l.s. and schuster, c.r. 1984. further evidence that amphetamines produce long-lasting dopamine neurochemical defi cits by destroying dopamine nerve terminals. brain res., 303:359–364. sinha, b.k. 1987. activation of hydrazine derivatives to free radicals in the perfused rat liver: a spin-trapping study. biochim. biophys. acta., 924:261–9. robinson, t.e. and becker, j.b. 1986. enduring changes in brain and behavior produced by chronic amphetamine administration: a review and evaluation of animal model of amphetamine psychosis. brain res. rev., 11:157–98. drug target insights 2006: 128 kitanaka et al scearce-levie, k. et al. 1999. 5-ht receptor knockout mice: pharmacological tools or models of psychiatric disorders. ann. ny acad. sci., 868:701–15. segal, d.s., kuczenski, r. and okuda, c. 1992. clorgyline-induced increases in presynaptic da: changes in the behavioral and neurochemical effects of amphetamine using in vivo microdialysis. pharmacol. biochem. behav., 42:421–9. seiden, l.s., sabol, k.e. and ricaurte, g.a. 1993. amphetamine: effects on catecholamine systems and behavior. annu. rev. pharmacol. toxicol., 32:639–77. self, d.w. and nestler, e.j. 1995. molecular mechanisms of drug reinforcement and addiction. annu. rev. neurosci., 18:463–95. shih, j.c., chen, k. and ridd, m.j. 1999. monoamine oxidase: from genes to behavior. annu. rev. neurosci., 22:197–217. shimosato, k. and saito, t. 1993. suppressive effect of cycloheximide on behavioral sensitization to methamphetamine in mice. eur. j. pharmacol., 234:67–75. sulzer, d. et al. 1995. amphetamine redistributes dopamine from synaptic vesicles to the sytosol and promotes reverse transport. j. neurosci., 15:4102–8. swett, l.r. et al. 1963. structure-activity relations in the pargyline series. ann. ny acad. sci., 107:891–8. tatsuta, t. et al. 2005. effects of monoamine oxidase inhibitors on methamphetamine-induced stereotypy in mice and rats. neurochem. res., 30:1377–85. tatsuta, t. et al. 2006. lobeline attenuates methamphetamine-induced stereotypy in adolescent mice. neurochem. res., in press. themann, c. et al. 2002. effect of repeated treatment with high doses of selegiline on behaviour, striatal dopaminergic transmission and tyrosine hydroxylase mrna levels. naunyn-schmiedeberg’s arch. pharmacol., 365:22–8. timár, j. knoll, b. and knoll, j. 1986. long-term administration of (-)deprenyl (selegiline), a compound which facilitates dopaminergic tone in the brain, leaves the sensitivity of dopamine receptors to apomorphine unchanged. arch. int. pharmacodyn, 284:255–66. timár, j. et al. 1993. further proof that (-)deprenyl fails to facilitate mesolimbic dopaminergic activity. pharmacol. biochem. behav., 46:709–14. timár, j. et al. 1996. differences in some behavioural effects of deprenyl and amphetamine enantiomers in rats. physiol. behav., 60:581–7. torres, i.l.s et al. 2002. effects of chronic restraint stress on feeding behavior and on monoamine levels in different brain structures in rats. neurochem. res., 27:519–25. ugarte, y.v. et al. 2003. methamphetamine rapidly decreases mouse vesicular dopamine uptake: role of hyperthermia and dopamine d2 receptors. eur. j. pharmacol., 472:165-71. ujike, h. and sato, m. 2004. clinical features of sensitization to methamphetamine observed in patients with methamphetamine dependence and psychosis. ann. ny acad. sci., 1025:279–87. vrana, s.l., azzaro, a.j. and vrana, k.e. 1992. chronic selegiline administration transiently decreases tyrosine hydroxylase activity and mrna in the rat nigrostriatal pathway. mol. pharmacol., 41:839–44. winchel, r.m. and stanley, m. 1991. self-injurious behavior: a review of the behavior and biology of self-mutilation. am. j. psychiatry., 148:306–17. worms, p. et al. 1987. sr 95191, a selective inhibitor of type a monoamine oxidase with dopaminergic properties. i. psychopharmacological profi le in rodents. j. pharmacol. exp. ther., 240:241–50. wu, w-r. and zhu, x-z. 1999. the amphetamine-like reinforcing effect and mechanism of l-deprenyl on conditioned place preference in mice. eur. j. pharmacol., 364:1–6. xu, w., zhu, j.p.q. and angulo, j.a. 2005. induction of striatal preand postsynaptic damage by methamphetamine requires the dopamine receptors. synapse, 58:110–21. yasar, s. et al. 1993. evaluation of the stereoisomers of deprenyl for amphetamine-like discriminative stimulus effects in rats. j. pharmacol. exp. ther., 265:1–6. youdim, m.b.h., edmondson, d. and tipton, k.f. 2006. the therapeutic potential of monoamine oxidase inhibitors. nature rev. neurosci., 7:295–309. zaczek, r., culp, s. and de souza, e.b. 1991a. interactions of [3h]amphetamine with rat brain synaptosomes. ii. active transport. j. pharmacol. exp. ther., 257:830–5. zaczek, r. et al. 1991b. interactions of [3h]amphetamine with rat brain synaptosomes. i. saturable sequestration. j. pharmacol. exp. ther., 257:820–9. zsilla, g. et al. 1982. the action of (-)deprenyl on monoamine turnover rate in rat brain. adv. biochem. psychopharmacol., 31:211–7. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true 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39 original research correspondence: a. panossian, swedish herbal institute research and development, spårvägen 2, åsklöster 43296, sweden. tel / fax: (46) 430-23723; email: alexander.panossian@shi.se please note that this article may not be used for commercial purposes. for further information please refer to the copyright statement at http://www.la-press.com/copyright.htm the adaptogens rhodiola and schizandra modify the response to immobilization stress in rabbits by suppressing the increase of phosphorylated stress-activated protein kinase, nitric oxide and cortisol alexander panossian1, marina hambardzumyan2, areg hovhanissyan2 and georg wikman1 1 swedish herbal institute research and development, spårvägen 2, åsklöster 43296, sweden. 2 “exlab” expert analytical laboratory of armenia drug agency, komitas ave. 49/4, 375051 yerevan, armenia. abstract: adaptogens possess anti-fatigue and anti-stress activities that can increase mental and physical working performance against a background of fatigue or stress. the aim of the present study was to ascertain which mediators of stress response are signifi cantly involved in the mechanisms of action of adaptogens, and to determine their relevance as biochemical markers for evaluating anti-stress effects in rabbits subjected to restraint stress. blood levels of stress-activated protein kinase (sapk/jnk), the phosphorylated kinase p-sapk/p-jnk, nitric oxide (no), cortisol, testosterone, prostaglandin e2, leukotriene b4 and thromboxane b2 were determined in groups of animals prior to daily oral administration of placebo, rhodioloside or extracts of eleutherococcus senticosus, schizandra chinensis, rhodiola rosea, bryonia alba and panax ginseng over a 7 day period. ten minutes after the fi nal treatment, animals were immobilized for 2 hours and blood levels of the markers re-determined. in the placebo group, only p-sapk/p-jnk, no and cortisol were increased signifi cantly (by 200–300% cf basal levels) following restraint stress, whilst in animals that had received multiple doses of adaptogens/stress-protectors, the levels of no and cortisol remained practically unchanged after acute stress. rhodioloside and extracts of s. chinensis and r. rosea were the most active inhibitors of stress-induced p-sapk/p-jnk. e. senticosus, b. alba and p. ginseng exerted little effect on p-sapk/p-jnk levels. it is suggested that the inhibitory effects of r. rosea and s. chinensis on p-sapk/p-jnk activation may be associated with their anti-depressant activity as well as their positive effects on mental performance under stress. keywords: adaptogens, stress, rhodiola rosea, schizandra chinensis, rhodioloside, p-sapk/p-jnk, nitric oxide, cortisol. introduction the term “adaptogen” was coined in the middle of the 20th century by the russian scientist lazarev (brekhman and dardymov, 1968) to describe medicinal plants that are able to enhance the so-called “state of non-specifi c resistance” of an organism to stress. it is now accepted that true adaptogens must: (i) possess stress-protective effects (i.e. reduction of stress-induced damage) such as anti-fatigue, antiinfection and restorative activities; (ii) present stimulating effects, following both single and multiple administration, that give rise to an increase in working capacity and mental performance against a background of fatigue and stress (such stimulating effects must be different from those of conventional cns stimulants and anabolics that deplete energy and plastic resources of the organism and are accompanied by negative side effects including drug withdrawal syndrome); and (iii) be innocuous and not disturb the normal level of body functions, but rather present a normalizing infl uence on the pathologic state, independent of the nature of that state (brekhman and dardymov, 1968). it should be noted that only schizandra chinensis, eleutherococcus senticosus and rhodiola rosea have been found to be fully compliant with this specifi c defi nition of adaptogen (panossian and wagner, 2005). whilst the concept of an adaptogen is readily understood from the physiological standpoint, it is not so easy to accept pharmacologically when it is necessary to defi ne the mechanism of action of a medicine and to formulate indications of its use. in particular, the stress-protective effect of an adaptogen results from the adaptation of the organism to repeated stimulating effects of the drug (brekhman and drug target insights 2007: 2 39–54 40 panossian et al dardymov, 1968; wagner et al. 1994; panossian et al. 1999a; panossian, 2003; panossian and wagner, 2005). since adaptation to stress is associated with the interactions of numerous mediators of the nervous, endocrine and immune systems, and is regulated at all levels of organization (cellular, regulating systems, whole organism) (fink, 2000), it is very unlikely that different stressprotectors have the same mechanism of action. the active components of stress-protective plants and adaptogens (table 1) can be formally d i v i d e d i n t o t h r e e m a i n g r o u p s , n a m e l y, tetra(penta)cyclic terpenoids, phenyland phenylethyl-propanoids and derivatives, and oxylipins. on the basis of the chemical nature of their active principles, some indication of the possible mechanism of action of these plants may be derived. thus, the extracts of panax ginseng, withania somnifera, bryonia alba and aralia mandshurica contain phytosterols, and tetracyclic and pentacyclic triterpenes, that likely exert their effect on the hypothalamus-pituitary-adrenal (hpa) axis in which cortisone plays a key role during stress. typically these adaptogens prevent or at least decrease certain hormonal changes, such as the increased level of cortisone, that are characteristic of a stress reaction (panossian et al. 1999b; kim et al. 2003a). plants such as r. rosea and s. chinensis accumulate phenolic secondary metabolites such as phenyland phenylethylpropanoids and their dimeric lignans (wagner et al. 1996; saratikov and krasnov 2004). such compounds can play an active role in stress response in respect of achieving a state of maximum work capacity as would be required in fi ght-orfl ight situations (lüllmann et al. 2005). interestingly, e. senticosus contains both types of biologically active, low molecular weight compound and exhibits a very wide range of pharmacological effects (world health organization, 2002). the pharmacological assessment of adaptogens typically includes evaluation of their stimulating, tonic and stress-protective activities in model systems in which animals are subjected to various stress conditions (panossian and wikman, 2005). despite considerable research effort, however, it still remains somewhat unclear which mediators of stress response are predominantly involved in table 1. classes of secondary metabolites identifi ed in panax ginseng, bryonia alba, withania somnifera, aralia mandshurica, rhodiola rosea, schizandra chinensis and eleutherococcus senticosus. group i: stress-protectors group ii: adaptogens panax ginseng rhodiola rosea tetracyclic triterpenes and their glycosides phenylethyl glycosides pentacyclic triterpenes and their glycosides phenylpropanoids polyacetylenes flavonoids bryonia alba phenolics tetracyclic triterpenes and their glycosides polyphenolics pentacyclic triterpenes lignans sterols and their glycosides flavolignans oxylipins and glycolipids withania somnifera schizandra chinensis tetracyclic triterpene lactones and their glycosides dibenzo[a,c]cyclooctadiene sterols and their glycosides sterols alkaloids organic and fatty acids, aralia mandshurica vitamins a,c and e pentacyclic triterpenes and their glycosides sterols eleutherococcus senticosus pentacyclic and tetracyclic triterpene glycosides phenylpropanoids sterols lignans polysaccharides (heteroglycans, eleutherans) coumarins drug target insights 2007: 2 41 effect of adaptogens in restraint stress the mechanisms of action of adaptogens, and which biochemical markers need to be assayed in the evaluation of drug effi cacy. in order to address this problem further, we have determined blood levels of the potential stress response markers (fink, 2000) stress-activated protein kinase/jun n-terminal protein kinase (sapk/jnk), the phosphorylated kinase p-sapk/ p-jnk, no, cortisol, testosterone, prostaglandin e2, leukotriene b4 and thromboxane b2 in laboratory rabbits, treated with stress-protectors and adaptogens, both before and after immobilization stress. the most extensively studied stressprotectors, b. alba and p. ginseng, and the adaptogens, e. senticosus, r. rosea, and s. chinensis (panossian et al. 1997; upton, 1999; world health organization, 1999, 2002; saratikov and krasnov, 2004), together with rhodioloside, an active ingredient of r. rosea (aksenova et al. 1968), were employed in this study. materials and methods details of the project were submitted to and approved by the ethics committee of the armenian drug and medical technology agency of the ministry of health of the republic of armenia. the principles of laboratory animal care, as delineated in eec directive 75/318 (1994), were followed throughout the study. study animals male chinchilla rabbits were obtained from the breeding unit of the institute of fine organic chemistry of the national academy of science, yerevan, armenia. all animals were clinically examined upon arrival and any that showed signs of abnormality or disease were excluded. the 39 animals employed in the study were maintained in the animal house under a 12 h light / dark cycle for 10–15 days prior to the commencement of the study and were offered standard rat chow ad libitum. any animals considered to be unsuitable were replaced before the start of the study, and no animals were replaced after the study had commenced. the weights of the study animals immediately prior to the commencement of the study were in the range 2.5–3.0 kg. during the study period, animals were kept separately in cages (150 × 100 × 100 cm) consisting of polystyrene cases and lattice framed steel lids: wood-sawdust was used as bedding. the target ranges for the temperature and the relative humidity of the animal house were 22 ± 4°c and 40 ± 5%, respectively, and the air was changed 1–2 times / h. throughout the study, a standardized diet (combi/ yerevan combi-corm plant) was provided, but feeding was discontinued prior to the administration of a test material. only drinking water was offered ad libitum. plant extracts extracts of e. senticosus roots (she-3, batch ex 20729 standardized for the content of eleutherosides e and b), r. rosea roots (shr-5, batch ex 20715 standardized for the content of rhodioloside, tyrosole, triandrin and rosavin) and s. chinensis berries (shs-2, batch ex 20646 standardized for the content of schizandrin and γ-schizandrin) were supplied by the swedish herbal institute (gothenburg, sweden). extracts of b. alba roots (batch 026 containing 4% w/w of total cucurbitacins expressed as cucurbitacin r equivalents) and of p. ginseng roots (containing 10.5% w/w of ginsenosides) were prepared by extraction of the herbal material with 70% ethanol and manufactured using commonly employed commercial processes. rhodoloside (syn. salidroside; batch s10402) was obtained from vilar (moscow, russia). dosage regimes and immobilization of study animals the doses employed for the six different treatments involved in this study were: e. senticosus root extract –6.5 mg/kg, r. rosea root extract –1 mg/kg, s. chinensis berry extract –22 mg/kg; b. alba root extract –15 mg/kg, p. ginseng root extract –6 mg/kg, and rhodioloside –0.5 mg/kg. in each case, a suspension of the test material was prepared freshly each day by shaking an appropriate amount in distilled water such that the a 10 ml volume of the final suspension contained the stated dosage amount per kg body weight. starting on day 2, study animals were treated each day (during the period 10.00–10.30) for 7 consecutive days with an appropriate volume of suspension (10 ml/kg), which was shaken gently immediately prior to administration and delivered by oral gavage. equivalent volumes of distilled water were supplied to placebo animals during the same period. the study animals were divided into three groups and treated as follows: those in group a (3 animals) were treated with distilled water for 7 drug target insights 2007: 2 42 panossian et al days and subjected to forced immobilization on days 2 and 8; those in group b (3 animals in each of 6 different treatment sub-groups) were treated with a study drug for 7 days but were not subjected to immobilization; and those in group c (3 animals in each of 6 different treatment sub-groups) were treated with a study drug for 7 days and subjected to forced immobilization on days 2 and 8. immobilization, which was conducted 10 min after the administration of drug or placebo, was carried out by fi xing the head and pads of the rabbit to a 1.1 × 0.4 m plate and maintaining the animal in this state for 2 h without food and water. blood sampling a 10 ml sample of blood was collected from each rabbit on day 1 of the study (i.e. the day prior to the commencement of drug administration). blood samples were collected from the heart cavity under aseptic conditions by inserting the needle of a 20 ml syringe into the 3rd intercostal space at a location 3–4 mm from the left hand end of the sternum. over the following 5 min period, a 20 ml volume of warm sterile saline solution was administered subcutaneously and the animal was transferred to a standard cage and given free access to food and water. for rabbits receiving adaptogens/stress-protectors, blood samples (10 ml) were similarly collected on day 2 either immediately after immobilization (group c) or at the same designated time (group b). on day 8 of the study, blood samples were collected in a similar manner from all animals, either immediately after immobilization (groups a and c) or at the same designated time (group b). plasma was obtained by transferring a portion of the freshly collected blood sample into a 4 ml sterile heparinised (lithium heparin) vacuette® tube (greiner bio-one gmbh, kremsmuenster, austria) and centrifuging at 600 g for 15 min. the remaining portion of the blood sample was allowed to clot at room temperature in the original plain collecting tube, and serum was separated by centrifugation in a micro-centrifuge at 3000 rpm for 10 min. plasma and serum samples were stored at –40oc until required for assay. biochemical assays p-jnk1/2 assays were performed using phospho jnk1 colorimetric (eia) titerzyme® kits (assay designs, ann arbor, mi, u.s.a.; product number 900–106) containing mouse monoclonal antibody specifi c to jnk immobilized on a microtitre plate. blood plasma (100 μl) was mixed with 0.9 ml of ripa cell lysis buffer [50 mm tris hcl (ph 7.4), 150 mm nacl, 1 mm edta, 1 mm egta, 1% triton x-100, 1% sodium deoxycholate and 0.1% sds] containing 0.5 ml/l protease inhibitor cocktail (pic; sigma, st. louis, mo, u.s.a.), 1 mm phenylmethylsulfonyl fl uoride (pmsf), 2 mm sodium orthovanadate and 20 mm sodium pyrophosphate, and mixed thoroughly for 5 min. the test solution was prepared by diluting 1 ml of this mixture with 9 ml of assay buffer solution (mopso buffered saline containing proteins, detergents, phosphatase inhibitor, 0.5 ml/l pic and 1 mm pmsf). aliquots (100 μl) of test solutions, or reference standards, were pipetted in duplicate into wells of the microtitre plate and incubated at room temperature for 1 h. wells were washed with tris buffered saline containing detergents, and 100 μl of biotinylated mouse monoclonal antibody to p-jnk was added to each well in order to bind the immobilized analyte. after a further incubation of 1 h, excess antibody was washed out and streptavidin conjugated to horseradish peroxidase was added (except to the blank) to bind to the biotinylated monoclonal p-jnk antibody. following a 30 min incubation, excess conjugate was washed out, 100 μl of substrate solution (3, 3’, 5, 5’tetramethylbenzidine and hydrogen peroxide) was added and the mixture incubated at room temperature for 30 min. the enzyme reaction was stopped by the addition of 100 μl of 1 m hcl, the optical density was measured at 450 nm (with correction between 570 and 590 nm) on a dynatech medicinal products (guernsey, channel island, u.k.) mx microplate reader, and the concentration of pjnk1/2 determined directly from a calibration curve generated using recombinant phosphorylated c-junn-terminal protein kinase. total jnk1/2 assays were performed using total jnk1 colorimetric (eia) titerzyme® kits (assay designs; product number 900–107) employing a protocol that was essentially the same as that for p-jnk1/2. nitric oxide assays were performed using total nitric oxide kits (assay designs; product number 917–020). blood serum (25 µl) was diluted with 25 µl of drug target insights 2007: 2 43 effect of adaptogens in restraint stress hepes buffer containing detergents and preservatives, and incubated with nitrate reductase in the presence of nadh for 30 min at 37oc. the total nitrite formed was determined by griess reaction w i t h a s o l u t i o n o f s u l f a n i l a m i d e a n d n (1-naphthyl)ethylenediamine in 2m hydrochloric acid in wells of a microtitre plate. after a 10 min incubation, the optical density of the colored azo-dye product was measured at 570 nm and the concentration of total no determined directly from a calibration curve generated using nitrate standard. cortisol assays were performed using cortisol colorimetric (eia) correlate® kits (assay designs; product number 900–0071) containing goat antibody specifi c to mouse igg immobilized on a microtitre plate. blood serum (0.5 ml) was extracted twice with equal volumes of diethyl ether, the organic phases were separated, combined, evaporated to dryness under nitrogen, and the resulting residue stored in a freezer at–20oc. frozen residues were dissolved in 0.5 ml of assay buffer containing sodium azide immediately prior to assay. test solutions, or reference standards, and mouse monoclonal antibody to cortisol were pipetted in duplicate into wells of the microtitre plate and incubated at room temperature for 1 h. alkaline phosphatase-cortisol eia conjugate was added and, following a further incubation of 1 h, excess reagents were washed out and the substrate solution (p-nitrophenyl phosphate) added. after a further incubation of 1 h, the enzyme reaction was stopped, the optical density measured at 405 nm, and the concentration of cortisol determined directly from a calibration curve generated using cortisol standard. testosterone assays were performed using testosterone colorimetric (eia) correlate® kits (assay designs; product number 900–065) employing a protocol that was essentially the same as that for cortisol. leukotriene b4 assays were performed using leukotriene b4 colorimetric (eia) correlate® kits (assay designs; product number 900–068) containing goat antibody specifi c to rabbit igg immobilized on a microtitre plate. blood serum (0.5 ml) was acidifi ed to ph 3.5 by the addition of 25 μl of 2м нс1, incubated at 4°c for 15 min and centrifuged at 2000 rpm in a micro-centrifuge for 2 min. the supernatant was separated and applied to a supelco (belefonte, pa, u.s.a.) supelclean lc-18 spe reverse phase cartridge that had previously been washed with 10 ml of ethanol and 10 ml of deionized water. the cartridge was eluted sequentially with 5 ml of water, 5 ml of 15% ethanol, 5 ml hexane and 5 ml ethyl acetate. the ethyl acetate fraction was evaporated to dryness on a rotary evaporator and dissolved in 500 μl of tris buffered saline containing proteins, detergents and sodium azide as preservative. test solutions, suitably diluted with assay buffer, or reference standards, rabbit polyclonal antibody to leukotriene b4 and alkaline phosphatase-leukotriene b4 eia conjugate were pipetted in duplicate into wells of the microtitre plate and incubated at room temperature for 2 h. excess reagents were washed out and the substrate solution (p-nitrophenyl phosphate) added. after a further incubation of 2 h, the enzyme reaction was stopped, the optical density measured at 405 nm, and the concentration of leukotriene b4 determined directly from a calibration curve generated using leukotriene b4 standard. prostaglandin e2 and thromboxane b2 assays were performed using assay design colorimetric (eia) correlate® kits for prostaglandin e2 and thromboxane b2 (product numbers 900– 001 and 900–002, respectively) employing protocols that were similar to that employed in the assay of leukotriene b4. statistical analysis data management and statistical analyses were performed using graphpad (san diego, ca, u.s.a.) prism software (version 3.03 for windows). the signifi cance of between-group differences (at 95% confi dence intervals) in the normalized mean values of analytes measured in blood plasma of rabbits were examined using two-tailed unpaired t-tests or mann-whitney tests; within-group comparisons were made using paired t-tests. the signifi cance of the between-group differences in the normalized mean values of analytes measured on days 1 and 8 of treatment, and of those measured before and after stress, were determined using oneway anova with tukey’s or dunnett’s multiplecomparison ad hoc tests. drug target insights 2007: 2 44 panossian et al results water (placebo group a) or rhodioloside or an extract of e. senticosus, r. rosea, s. chinensis, b. alba or p. ginseng (verum groups b and c) was administered orally to rabbits each day (commencing on day 2) for 7 days. ten minutes after the administration of drug or placebo, animals in groups a and c were subjected to 2 h of stress by immobilization on days 2 and 8; animals in group b were not subjected to stress. blood samples were taken from all rabbits on days 1 and 8, and also on day 2 for animals in groups b and c. sampling on days 2 and 8 took place immediately after the application of stress (groups a and c) or at the same designated time (group b). blood samples were analyzed for content of sapk/jnk, p-sapk/pjnk, no, cortisol, testosterone, prostaglandin e2, leukotriene b4, and thromboxane b2. the coeffi cients of variation in the levels of the assayed markers at the beginning of the study were found to vary from 2 to 100% (table 2) even though the animals had all been kept under identical conditions. hence all data were normalized separately for each rabbit with respect to the initial (day 1) level (taken as 100%) such that analyte concentrations could be expressed as a percentage of this basal value. figure 1 shows the levels of assayed markers measured in the blood of placebo rabbits (group a) sampled immediately after immobilization stress applied on day 8 and expressed as percentages of the basal levels determined on day 1 of the study. only the contents of p-sapk/p-jnk, no and cortisol were signifi cantly increased (by between 200–300% cf. to basal levels: table 3) following the application of stress. in fig. 2, the post-stress levels (expressed as percentages of basal levels) of p-sapk/p-jnk, no and cortisol measured on day 8 in the blood of verum group c animals, who had received multiple doses of study drugs, are compared with those of the placebo group. it is clear that, following the repeated administration of adaptogens/stress-protectors, the levels of the stress markers no and cortisol remained practically unchanged (p > 0.05) from the basal values after the period of acute stress (table 3). rhodioloside and extracts of s. chinensis and r. rosea were the most active adaptogens with respect to their capability to inhibit p-sapk/p-jnk formation during stress. the percentage changes in the blood levels of stress markers in verum group c animals after single (day 2) and multiple (day 8) administration of adaptogens, in comparison with those sa pk /jn k p-s ap k/ p-j nk n o co rti so l te sto ste ro ne pg e2 lt b4 tx b2 0 100 200 300 400 * * * p er ce nt ag e in cr ea se cf no rm al iz ed b as al le ve l pr io r to s tr es s ev en t (t ak en a s 10 0% ) * p < 0.05 vs basal level figure 1. stress-induced changes in the concentration of analytes in the blood of rabbits treated with placebo (group a). sapk/jnk: stress-activated protein kinase; p-sapk/jnk: phosphorylated-sapk/jnk; no: nitric oxide; pge2: prostaglandin e2; ltb4: leukotriene b4; txb2: thromboxane b2. drug target insights 2007: 2 45 effect of adaptogens in restraint stress table 2. mean basal levels of biochemical markersa determined in rabbits on 1 day of the study prior to treatment or stress conditions. study sapk/jnk p-sapk/p-jnk no cortisol testosterone pge2 ltb4 txb2 animal (ng/ml) (ng/ml) (nmol/ml) (pg/ml) (pg/ml) (pg/cl) (pg/cl) (pg/cl) 1 1952 45 145 1767 1391 3844 4580 3346 2 1832 59 133 2793 1117 3657 3568 5917 3 2272 35 66 1657 1437 3332 4817 2446 4 1517 45 106 1177 1362 3869 3278 8894 5 3112 66 40 7456 653 3426 3198 8129 6 1833 83 59 1642 1718 2276 3190 10728 7 5558 118 99 2969 1857 1938 4161 7963 8 1910 120 64 8017 1373 2276 2269 5098 9 1378 93 216 2500 1791 1886 1451 5377 10 1456 77 321 4549 1631 2731 3705 1673 11 1376 185 173 4817 1185 2053 3854 5460 12 2123 203 322 2597 1296 2676 4010 7971 13 2595 291 95 8491 1472 1085 1840 4163 14 1877 281 221 6708 1043 773 1783 7361 15 1326 251 258 1218 1070 1847 4014 9825 16 4164 48 48 1388 1552 3292 1700 3970 17 802 20 37 2708 718 1551 3906 4830 18 1758 53 45 6018 1726 1220 4588 2198 19 4914 26 31 8206 1461 4068 3840 1014 20 1832 10 30 8917 745 4054 3826 4340 21 1614 43 324 10000 2554 1118 3650 1018 22 1373 276 178 2169 428 2442 3362 9551 23 1997 165 96 4315 1016 2161 3122 10040 24 1407 175 56 9913 3132 1451 3049 10025 25 5200 275 179 10000 730 1512 2113 1776 26 1914 277 197 12700 1383 1481 3198 1604 27 2109 209 61 12500 1608 991 3100 1624 28 1717 36 56 1632 1002 3272 2949 3199 29 1493 42 66 3710 735 4248 2758 3710 30 1779 108 41 6020 1127 3878 2850 3121 31 2807 63 214 4269 1219 2442 2953 9081 32 1972 140 233 5267 1080 3033 2273 8597 33 2007 167 176 7241 1506 2383 2454 7960 34 2238 57 285 8974 1182 2677 3353 3303 35 1324 129 25 9606 1053 4320 2582 9790 36 1675 106 77 5469 1149 2730 1338 9382 37 3550 212 95 5733 2464 2248 2871 1669 38 2033 163 17 12600 1472 2690 2515 2839 39 1346 245 43 6237 2301 3102 2328 2780 mean 2183.1 128.1 126.3 5742.2 1378.0 2564.9 3087.1 5430.0 sd 1091 88 93 3448 547 1001 872 3152 cv% 50 68 74 60 40 39 28 58 minimum 802 10 17 1177 428 773 1338 1014 maximum 5558 291 324 12700 3132 4320 4817 10728 range 4757 280 308 11523 2704 3547 3479 9714 min/max 7 28 20 11 7 6 4 11 asapk/jnk: stress-activated protein kinase; p-sapk/jnk: phosphorylated-sapk/jnk; no: nitric oxide; pge2: prostaglandin e2; ltb4: leukotriene b4; txb2: thromboxane b2. drug target insights 2007: 2 46 panossian et al determined at the start of the study (day 1), are presented in table 4. there were few signifi cant changes in levels of the stress markers in resting animals after single or repeated administration of adaptogens. thus, total jnk increased after single (but not multiple) application of r. rosea extract and its active component rhodioloside. it would appear that repeated treatment with these drugs results in adaptation of the organism to the “stressors”. a single dose of rhodioloside or of an extract of s. chinensis or r. rosea decreased the thromboxane b2 level implying inhibition of platelet aggregation, blood clotting and an antistress effect. testosterone signifi cantly increased after repeated administration of an extract of e. senticosus, an effect that is consistent with previous observations (winterhoff et al. 1993) also indicating an anti-stress effect of this drug discussion the mechanisms of action of plant-derived stressprotectors and adaptogens, and the biochemical markers that need to be assayed in order to evaluate the effi cacy of such drugs, have yet to be fully elucidated. thus the aim of the present study was to develop a simple laboratory test for the evaluation of the effi cacy of adaptogens. typically, blood (serum or plasma) is the most common and convenient biological fl uid for routine tests since it is not necessary to kill the animal and no special skills or facilities for surgery are required in order to obtain samples. blood is the liquid that is in contact with all tissues, including the brain (where the most important regulatory processes associated with the effects of adaptogens take place), and provides a unique source in which all of the studied hormones, including cortisone, testosterone and thromboxane b2, can be measured. for these reasons, the experiments described in this paper were conducted using blood samples rather than brain tissues. the sources of jnk and p-jnk are blood plasma cells, presumably the pmnl and lymphocytes, which were subjected to lysis prior to assay. it is assumed that the nitric oxide originated both from blood cells and from many other tissues, including the brain. in the present study, the blood levels of potential stress response markers were determined in laboratory rats in both the resting state and after restraint stress. the basis for choosing the specifi c stress markers assayed is described below. sapk/jnk and p-sapk/p-jnk: these kinases belong to a family of enzymes (the mitogen-activated protein kinases, mapk) that act within the signaling systems by which cells transduce extracellular stimuli into intracellular responses. such signal transduction mediators are distributed extensively throughout the cns and regulate a diverse array of cellular functions. the most common mapks are the extracellular signalregulated kinases that primarily regulate cellular (a) pl ac eb o r. ro sea rh od iol os ide s.c hin en sis e. sen tic os us p. gin sen g b. alb a 0 100 200 300 * * * p er ce nt ag e in cr ea se cf no rm al iz ed b as al le ve l pr io r to t re at m en t an d st re ss e ve nt ( ta ke n as 10 0% ) * p < 0.05 vs placebo drug target insights 2007: 2 47 effect of adaptogens in restraint stress (b) pl ac eb o r. ro sea rh od iol os ide s. ch ine ns e. sen tic os us p. gin sen g b. alb a 0 100 200 300 * * * * p er ce nt ag e in cr ea se cf no rm al iz ed b as al le ve l pr io r to t re at m en t an d st re ss e ve nt ( ta ke n as 10 0% ) * p < 0.05 vs placebo (c) pl ac eb o r. ro sea rh od iol os ide s.c hin en sis e. sen tic os us p. gin sen g b. alb a 0 100 200 300 400 * p<0.05 vs placebo * * * * * * p er ce nt ag e in cr ea se cf no rm al iz ed b as al le ve l pr io r to t re at m en t an d st re ss e ve nt ( ta ke n as 10 0% ) figure 2. stress-induced changes in the concentration of: (a) phosphorylated stress-activated protein kinase (p-sapk/p-jnk), (b) nitric oxide and (c) cortisol in the blood of rabbits treated with a placebo or multiple doses of adaptogens/stress-protectors. growth, differentiation, apoptosis, survival, differentiation and adaptation to stress (schaeffer and weber, 1999; kyriakis and avruch, 2001). sapk/ jnk is activated by diverse stress and pro-infl ammatory stimuli including cytokines, growth factors, irradiation, hyperosmolality, cold, heat and shear stress, muscle contraction and exercise (williamson et al. 2003; shen et al. 2004). the activation of sapk/jnk occurs through phosphorylation at thr183 and tyr185, and the resulting phosphorylated kinase can translocate to the nucleus where it regulates transcription through its effects on c-jun, activating transcription factor-2 (atf-2) and activator protein 1 (ap-1) transcription factor. the prolonged activation of jnk and the subsequent phosphorylation of various transcription factors have been implicated in the initiation of the apoptosis cascade in some cell lines, and may represent the initiating factor in the pathogenesis of overuse injuries. moreover, sapk/jnk is believed to be important in neuronal development, cd4 t-helper-cell differentiation, t-cell activation, drug target insights 2007: 2 48 panossian et al ta bl e 3. p er ce nt ag e di ffe re nc es in c ha ng es in b lo od le ve ls o f s tr es s m ar ke rs b et w ee n gr ou p a ( pl ac eb o) a nd g ro up c (v er um ) an im al s fo llo w in g im m ob iliz at io n at th e en d of th e st ud y (d ay 8 ). m ar ke ra n or m al iz ed c on te nt s ta tis tic p ar am et er s r ho di ol a b ry on ia p an ax s ch iz an dr a e le ut he ro co cc us r ho di ol os id e in s tr es s cf . t o ba se ro se a al ba gi ns en g ch in en si s se nt ic os us le ve l ( 10 0% )b [in iti al le ve lb ; c v % ] s a p k /j n k 88 .3 02 ± 1 6. 3% d iff er en ce b et w ee n m ea ns –3 +2 5 +3 8 –2 9 +1 0 –2 gr ou ps a a nd c ( % , n = 3 ) [2 01 8 ± 22 7 ng /m l; 11 .2 ] p v al ue c >0 .0 5 >0 .0 5 >0 .0 5 >0 .0 5 >0 .0 5 >0 .0 5 ps a p k / 21 0. 9 ± 50 .7 % d iff er en ce b et w ee n m ea ns –1 62 –1 55 –3 2 –1 64 –5 3 –1 39 gr ou ps a a nd c ( % , n = 3 ) pjn k [4 6 ± 11 ng /m l; 24 .9 ] p v al ue 0. 01 * >0 .0 5 >0 .0 5 <0 .0 5 >0 .0 5 0. 01 1* n o 20 9. 6 ± 55 .6 % d iff er en ce b et w ee n m ea ns –1 17 –8 0 –1 19 –7 6 –1 43 –1 41 gr ou ps a a nd c ( % , n = 3 ) [1 14 ± 4 2 nm ol /m l; 37 .1 ] p v al ue 0. 03 * >0 .0 5 0. 04 7* * >0 .0 5 0. 04 6* 0. 02 * c or tis ol 29 1. 9 ± 18 3% d iff er en ce b et w ee n m ea ns –2 09 –6 3 –1 77 –1 92 –2 05 –2 23 gr ou ps a a nd c ( % , n = 3 ) [2 07 1 ± 62 6 pg /m l; 30 .2 ] p v al ue 0. 02 * >0 .0 5 0. 01 2* 0. 02 7* * 0. 02 5* 0. 02 * te st os te ro ne 8 6. 9 ± 7. 3% d iff er en ce b et w ee n m ea ns +1 5 –3 –1 1 +6 +4 1 +4 gr ou ps a a nd c ( % , n = 3 ) [1 31 5 ± 17 3 pg /m l; 13 .2 ] p v al ue >0 .0 5 >0 .0 5 0. 02 6* >0 .0 5 0. 02 1* >0 .0 5 p g e 2 85 .8 ± 2 9. 4% d iff er en ce b et w ee n m ea ns +4 3 +5 2 +3 3 +4 8 +4 +1 9 gr ou ps a a nd c ( % , n = 3 ) [3 61 ± 2 6 pg /m l; 7. 1] p v al ue >0 .0 5 >0 .0 5 0. 00 1* ** >0 .0 5 >0 .0 5 >0 .0 5 lt b 4 98 .6 ± 1 4. 7% d iff er en ce b et w ee n m ea ns +3 3 +5 7 +8 –7 +1 6 –5 gr ou ps a a nd c ( % , n = 3 ) [4 32 ± 6 6 pg /m l; 15 .3 ] p v al ue >0 .0 5 0. 03 6* >0 .0 5 >0 .0 5 >0 .0 5 >0 .0 5 tx b 2 10 5. 5 ± 51 .8 % d iff er en ce b et w ee n m ea ns +8 8 –1 9 -4 95 +1 34 -5 5 –3 gr ou ps a a nd c ( % , n = 3 ) [3 90 ± 1 80 p g/ m l; 46 .1 ] p v al ue >0 .0 5 >0 .0 5 >0 .0 5 >0 .0 5 >0 .0 5 >0 .0 5 a s a p k /j n k : s tr es sac tiv at ed p ro te in k in as e; p -s a p k /j n k : p ho sp ho ry la te ds a p k /j n k ; n o : n itr ic o xi de ; p g e 2: p ro st ag la nd in e 2; l t b 4: le uk ot rie ne b 4; t xb 2: th ro m bo xa ne b 2. b v al ue s sh ow n ar e m ea n ± st an da rd d ev ia tio n. c m ea ns s ig ni fi c an tly d iff er en t a t v al ue s of p < 0 .0 5 (in di ca te d by * ), p < 0 .0 1 (in di ca te d by * *) o r p < 0 .0 01 ( in di ca te d by * ** ). drug target insights 2007: 2 49 effect of adaptogens in restraint stress ta bl e 4. p er ce nt ag e ch an ge s in th e bl oo d le ve l o f s tr es s m ar ke rs in g ro up c ( ve ru m ) an im al s af te r a si ng le ( da y 2) a nd m ul tip le ( da y 8) a dm in is tr at io n of ad ap to ge ns in c om pa ris on w ith th os e de te rm in ed a t t he s ta rt o f t he s tu dy ( da y 1) . m ar ke r s ta tis tic p ar am et er s r ho di ol a b ry on ia p an ax s ch iz an dr a e le ut he ro co cc us r ho di ol os id e ro se a al ba gi ns en g ch in en si s se nt ic os us d ay 2 d ay 8 d ay 2 d ay 8 d ay 2 d ay 8 d ay 2 d ay 8 d ay 2 d ay 8 d ay 2 d ay 8 s a p k /j n k d iff er en ce b et w ee n +7 +1 8 +/ – +/ – +/ – +/ – +3 8 +/ – +3 –2 1 +7 +/ – m ea ns ( % ; n = 3 ) p v al ue a <0 .0 5* >0 .0 5 >0 .0 5 >0 .0 5 >0 .0 5 >0 .0 5 >0 .0 5 >0 .0 5 >0 .0 5 >0 .0 5 <0 .0 5* >0 .0 5 ps a p k /p -j n k d iff er en ce b et w ee n –3 5 –4 5 +/ – +/ – –4 6 –3 3 +/ – –6 3 +/ – +/ – –4 6 +3 6 m ea ns ( % ; n = 3 ) p v al ue >0 .0 5 >0 .0 5 >0 .0 5 >0 .0 5 >0 .0 5 >0 .0 5 >0 .0 5 <0 .0 5* >0 .0 5 >0 .0 5 >0 .0 5 >0 .0 5 n o d iff er en ce b et w ee n –4 6 –4 5 +/ – +/ – +/ – –3 5 +1 8 +3 1 +3 7 +7 1 -5 3 +1 15 m ea ns ( % ; n = 3 ) p v al ue >0 .0 5 >0 .0 5 >0 .0 5 >0 .0 5 >0 .0 5 >0 .0 5 >0 .0 5 >0 .0 5 >0 .0 5 >0 .0 5 >0 .0 5 >0 .0 5 c or tis ol d iff er en ce b et w ee n –5 3 –5 8 +/ – +/ – –9 +/ – +/ – +/ – +3 2 +/ – -5 4 –3 1 m ea ns ( % ; n = 3 ) p v al ue >0 .0 5 >0 .0 5 >0 .0 5 >0 .0 5 >0 .0 5 >0 .0 5 >0 .0 5 >0 .0 5 >0 .0 5 >0 .0 5 >0 .0 5 >0 .0 5 te st os te ro ne d iff er en ce b et w ee n +1 01 +4 5 –1 9 +/ – –9 +/ – +/ – +/ – +8 4 +9 9 +1 00 –2 3 m ea ns ( % ; n = 3 ) p v al ue >0 .0 5 >0 .0 5 >0 .0 5 >0 .0 5 >0 .0 5 >0 .0 5 >0 .0 5 >0 .0 5 >0 .0 5 <0 .0 5* >0 .0 5 >0 .0 5 p g e 2 d iff er en ce b et w ee n +/ – +2 4 +/ – +/ – +/ – +/ – +/ – +2 5 +/ – +/ – +/ – +1 0 m ea ns ( % ; n = 3 ) p v al ue >0 .0 5 >0 .0 5 >0 .0 5 >0 .0 5 >0 .0 5 >0 .0 5 >0 .0 5 >0 .0 5 >0 .0 5 >0 .0 5 >0 .0 5 >0 .0 5 lt b 4 d iff er en ce b et w ee n +/ – +/ – –9 –3 +/ – +/ – +/ – +/ – +/ – +3 +/ – +/ – m ea ns ( % ; n = 3 ) p v al ue >0 .0 5 >0 .0 5 >0 .0 5 >0 .0 5 >0 .0 5 >0 .0 5 >0 .0 5 >0 .0 5 >0 .0 5 >0 .0 5 >0 .0 5 >0 .0 5 t xb 2 d iff er en ce b et w ee n –5 8 –5 9 +/ – +/ – –2 5 +/ – –3 3 +/ – +/ – +6 0 –5 8 +/ – m ea ns ( % ; n = 3 ) p v al ue <0 .0 5* >0 .0 5 >0 .0 5 >0 .0 5 >0 .0 5 >0 .0 5 <0 .0 5* * >0 .0 5 >0 .0 5 >0 .0 5 <0 .0 5* >0 .0 5 a m ea ns s ig ni fi c an tly d iff er en t a t v al ue s of p < 0 .0 5 (in di ca te d by * ) or p < 0 .0 1 (in di ca te d by * *) . drug target insights 2007: 2 50 panossian et al and the pro-apoptotic response to genotoxins (kyriakis and avruch, 2001). nitric oxide no is a short-lived free radical that can be produced in mammalian cells by a family of no synthases (nos), including neuronal (nnos), endothelial (enos) and inducible (inos) enzymes, members of which have been shown to function as intracellular signaling regulators in a variety of cellular events (nathan and xie, 1994). while nnos and enos are constitutively expressed and their enzymatic activities regulated by changes in intracellular concentrations of free ca2+, inos is regulated at the transcriptional level. the expression of inos can be induced by cytokines and lipopolysaccharides in many cell types including macrophages. inos, which produces high levels of no, plays a role in cellular immune responses (nathan, 1997), exerting many of its functions, including signal transduction, dna repair, host defense, blood pressure control, and neurotransmission, through snitrosylation of proteins (stamler, 1994). changes in gene expression and enzymatic activity of nnos in the hypothalamus, pituitary and adrenal glands of rats subjected to immobilization stress have been reported (liu et al. 1996). furthermore, psychological and/or physiological stress causes no release in, and may modulate stress-induced activation of, the hpa axis and the sympatho-adrenal medullary system (kishimoto et al. 1996). endogenous no can suppress jnk/sapk through a thiol-redox mechanism (park et al. 2000). cortisol this corticosteroid hormone is produced by the adrenal cortex and is known to be involved in the response to stress suppression in the immune system. increased serum cortisol levels have been observed in connection with clinical depression and psychological stress involving stressors such as hypoglycemia, illness, fever, trauma, surgery, fear, pain, physical exertion or extremes of temperature. in normal release, cortisol has widespread actions that help restore homeostasis after stress. it acts as a physiological antagonist to insulin by promoting gluconeogenesis, breakdown of lipids and proteins, and mobilization of extrahepatic amino acids and ketone bodies. this leads to increased blood glucose concentrations, resulting in increased glycogen formation in the liver (freeman, 2005). in chronic stress, prolonged cortisol secretion causes muscle wastage, hyperglycemia, and suppresses immune/inflammatory responses. moreover, long-term exposure to cortisol results in damage to cells of the hippocampus that may cause impaired learning. however, short-term exposure of cortisol helps to create memory, and constitutes the proposed mechanism for the storage of fl ash bulb memories. testosterone the principal male sex hormone and an anabolic steroid that promotes cell growth and division resulting in the growth of several types of tissues, especially muscle and bone. administration of testosterone produces numerous physiological effects including increased protein synthesis, muscle mass, strength, appetite, bone growth, increased libido, etc. natural levels of testosterone decline gradually with age in men and also decrease during stress. prostaglandin e2, leukotriene b4 and thromboxane a2 these are eicosanoids that are known to play important roles in the early stages of stress response exerting both proand anti-infl ammatory actions. these mediators are involved in the initiation, transmission and modulation of stress, as well as in the expression of stress symptoms. the synthesis of prostaglandins has been found to be stimulated by most stressors including heat pain, trauma, exercise, bacteria, restraint, water immersion, cage switching, examination, surgery, immobilization etc. the prostaglandins are known to activate the hpa axis through direct stimulation of corticotrophin releasing factor (crf), vasopressin (avp) neurons and noradrenergic neurons in the paraventricular nucleus (fink, 2000). of the potential stress markers assayed in the present study, only p-sapk/p-jnk, no and cortisol were increased signifi cantly above resting levels following application of immobilization stress to laboratory rabbits. however, after repeated administration of the adaptogens/stress-protectors, e. senticosus, r. rosea, s. chinensis, b. alba, p. ginseng and rhodioloside, the levels of no and cortisol remained practically unchanged from normal during restraint stress. the inhibition of stress-induced no production demonstrated in this study is noteworthy since it may drug target insights 2007: 2 51 effect of adaptogens in restraint stress provide an explanation of the energy bursting effects of adaptogens in which the phase of endurance is prolonged and that of fatigue postponed. in this context, it is known that no formation can strongly inhibit the production of cellular energy through two mechanisms: (i) inhibition of mitochondrial respiration by reversible (from constitutive isoforms of nos) and irreversible (from inos) inhibition of cytochrome p450 (brown, 2001), and (ii) the inhibition of glycolysis through modifi cation of sh-groups of glyceraldehyde-3-phosphate dehydrogenase (hara et al. 2006). it has previously been reported that ginseng saponins act primarily on the hypothalamus and/or hypophysis producing a stimulation in the secretion of adrenocorticotrophic hormone (acth) within 30–90 min after single oral or intraperitoneal treatment, together with increased synthesis of corticosterone in the adrenal cortex and an increase in the concentration of corticosterone in the plasma (hiai et al. 1979a, b; filaretov et al. 1988). the mild stress-protective activity of the ginseng saponins is believed to be mediated through the blocking of acth action in the adrenal gland (kim et al. 2003a, b) and by inducing no production in the brain (kim et al. 1998). administration of a single dose of p. ginseng increased working capacity in rats by up to 132% (filaretov et al. 1988): interestingly, although the effect of repeated administration over a 7 day period was more pronounced (179%), it was not accompanied by further changes in blood cortisone level. additionally, ginseng saponins have been shown to affect brain monoamine levels in heat-stressed mice causing a reduction in the stress-induced increase of noradrenalin and serotonin. similar effects on the hpa axis under both normal and stress conditions were reported in experiments involving another tetracyclic triterpene glycoside, namely, cucurbitacin r diglucosides an active principle of b. alba, giving rise to a stress-protective and stimulating effect in animals (panossian et al. 1997, 1999b). the action of a steroid hormone is mainly attributed to its binding with a receptor, which results in the activation of the nuclear genome and subsequent alterations in protein synthesis. however, such a mechanism cannot account for the rapid effects of some adaptogens, particularly the single dose effects of s. chinensis and r. rosea (panossian and wagner, 2005). moreover it has been shown that these adaptogens are able to enhance resistance in simple organisms, such as developing snail (lymnaea stagnalis) embryos, silk worm (bombix mori) larvae, round worms (caenorhabditis elegans), and isolated cells (reuber h35 hepatoma and isolated cardiac cells), against various conditions including cold-induced viral infection, bacillus thuringiensis infection, stress induced by menadione, formalin, heat, and toxic metal ions (chernykh et al. 1985; boon-niermeijer et al. 2000). such results clearly cannot be explained in terms of regulation of endocrine, immune or cns systems, but rather confi rm that adaptogens are universal enhancers of non-specifi c resistance of living organisms at various levels of organization, and that they can adapt cells and organisms to stress by mechanisms of regulation of intracellular communications. whilst the effects of adaptogens on the hpa axis (filaretov et al. 1986; kimura and sumiyoshi, 2004), on no (park et al. 1996; panossian et al. 1999c) and eicosanoids (panossian et al. 1988; okhura et al. 1990; park et al. 2005) are well documented, nothing is known about the involvement of sapk/jpk, and its phosphorylated form, in the mechanism of action of plant adaptogens. the results of the present study show that immobilization stress increases the levels of p-jnk in the blood cells of rabbits signifi cantly (p < 0.05) up to 200–300% compared with the initial basal level. previously, a single 15 min session of forced swimming was found to increase p-jnk (p-jnk1 and/or p-jnk2/3) levels in all regions of the brain (hippocampus, neocortex, prefrontal cortex, amygdala and striatum) by ca 2–5-fold (shen et al. 2004). moreover, acute sessions of bicycle ergometry have been reported to produce increases in signaling intermediates from the sapk/jnk pathways (williamson et al. 2003). interestingly, it was found that older men exhibited signifi cantly (p < 0.05) higher resting levels of p-sapk/p-jnk compared with younger men, but lower comparative levels after a session of the resistance exercise. in the present study, the blood levels of p-sapk/p-jnk in rats that had been treated with multiple doses of the adaptogens r. rosea, s. chinensis and rhodioloside remained practically unchanged after the period of acute stress. these adaptogens contain mainly phenolic compounds and do not contain triterpenes. in contrast, repeated administration of stress-protectors that contained mainly triterpenes but no phenolics, produced little drug target insights 2007: 2 52 panossian et al effect on p-sapk/p-jnk during stress. under resting conditions, r. rosea and rhodioloside induced increases in total jnk after single (but not repeated) dose administration. it appears, therefore, that repeated administration of r. rosea and rhodioloside results in adaptation of the organism to these “stressors.” the curative effect of an extract of s. chinensis on patients with asthenia and depressive syndromes has been established in several studies (staritsina, 1946; zakharova, 1948; leman, 1952). furthermore, it has recently been demonstrated that the standardized extract shr-5 from r. rosea possesses a clear and signifi cant anti-depressive activity in patients suffering from mild to moderate depression (darbinyan et al. 2006). it can be hypothesized that the antidepressant effects of r. rosea and s. chinensis are associated with the inhibition of emotional stress induced by the over-activation of p-sapk/p-jnk. the sapk/jnk pathway is known to be involved in the pathogenesis of glucocorticoid resistance (gr) found in certain chronic immune/infl ammatory diseases and in subgroups of patients with major depression, and activation of sapk/jnk has been reported to inhibit gr function (wang et al. 2005). moreover, physiologic activation of sapk/jnk appears necessary for the induction of long term depression, and overactivation of these kinases by cytokines at pathophysiological concentrations is detrimental to long term potentiation. it has thus been suggested that sapk/jnk pathways may represent a therapeutic target for the normalization of gr function in these disorders (wang et al. 2005). since sapk/jnk is activated in alzheimer disease (lagalwar et al. 2006), the inhibition of such activation might provide some protection from stress-induced apoptotic cell death. in this context, adaptogens could induce a positive effect in neurodegenerative disorders characterized by the loss of neurons in brain regions involved in learning and memory. thus, it is suggested that the benefi cial effects of r. rosea and rhodioloside on mental performance in stress, as well as the protection against neurotoxicity offered by s. chinensis, might be associated with their inhibitory effect on the formation of p–sapk. related data may be considered to add further support to the hypothesis that adaptogens have a therapeutic effect in neurological and neurodegenerative disorders. thus, both r. rosea and rhodioloside inhibit propyl endoperoxidase, which is known to play a role in the degradation of neuropeptides involved in the process of learning and memory (fan et al. 2001). moreover, rhodioloside may protect pc12 cells against the excitotoxic action of glutamate by suppressing the excessive entry of ca2+ and the release of calcium stores (cao et al. 2006), whilst an extract of s. chinensis fruit, and the active components schizandrins a, b and c, signifi cantly reduce the neurotoxic action of glutamate (kim et al. 2004). conclusion it has been demonstrated that nitric acid and cortisol are appropriate stress markers that can be employed in the evaluation of the anti-stress effects of stress-protectors and adaptogens. it is noteworthy that only p-sapk/p-jnk appears to be a potential marker in bioassays of adaptogens and presumably of potential antidepressants. notice of confl ict of interest this study was funded with project grants from the research and development division of the swedish herbal institute (the sponsor): all materials were supplied by the sponsor. the funding sponsor, however, had no role in any practical aspect of the study including experiments, data collection, management, analysis and interpretation of the data the study was conceived by ap and the protocol of the study was formulated by ap. ah was responsible for experiments with animals and for the collection of samples for bioassays; ma performed the bioassays; ah and ma were involved in the data analysis, statistical evaluation and preparation of the draft report of the experimental part of the study. ap drafted the manuscript, and all authors (gw,ap, ah and mh) were involved in its critical appraisal and fi nal approval. ap is employed by the sponsor (shi) on a permanent basis. mh has no commercial associations or fi nancial interests with respect to this study: the work described forms part of her research project on adaptogens. ah receives an honorarium from the sponsor for contract research carried out on behalf of shi.gw is the director of research and development at shi and is an shi stockholder. references aksenova, r.a., zotova, m.i. and nekhoda, mf. et al. 1968. comparative characteristics of the stimulating and adaptogenic effects of rhodiola rosea preparations. in saratikov as, ed. stimulants of the central nervous system. vol. 2. tomsk:tomsk university press, p. 3–12. drug target insights 2007: 2 53 effect of adaptogens in restraint stress boon-niermeijer, e.k., van den, berg, a. and wikman, g. et al. 2000. phytoadaptogens protect against environmental stress-induced death of embryos from the freshwater snail lymnea stragnalis. phytomedicine, 7:389–400. brekhman, ii. and dardymov, iv. 1968. new substances of plant origin which increase non-specific resistance. ann. rev. pharmacol., 8:419–30. brown, g.c. 2001. regulation of mitochondrial respiration by nitric oxide inhibition of cytochrome c oxidase. biochim biophys acta bioenergetics, 1504:46–57. cao, l.l., du, g.h. and wang, m.w. 2006. the effect of salidroside on cell damage induced by glutamate and intracellular free calcium in pc12 cells. j. asian nat. prod. res., 8:159–65. chernykh, s.i., lukhtanov, v.a. and simonenko, n.p. 1985. adaptation to damage in the silkworm bombyx mori l. (lepidoptera bombycidae). iii. adaptogens and larval resistance to stress-induced activation of latent viral infection. entomol. obozrenye, 2:267–72. darbinyan, v., aslanyan, g. and amroyan, e. et al. 2006. clinical trial of rhodiola rosea l. extract shr-5 in the treatment of mild to moderate depression. nordic. j. psychiatry, accepted for publication on 21 july 2006. fan, w., tezuka, y. and ni, k.m. et al. 2001. propyl endopeptidase inhibitors from the underground part of rhodiola sachalinensis. chem pharm. bull., 49:396–401. filaretov, a.a., bogdanova, t.s. and mitiushov, m.i. et al. 1986. effect of adaptogens on the activity of the pituitary-adrenocortical system in rats. biull. eksp. biol. med., 101:573–4. filaretov, a.a., bogdanova, t.s. and podvigina t.t. et al. 1988. role of pituitary-adrenocortical system in body adaptation abilities. exp. clin. endocrinol., 92:129–36. fink, g. 2000. encyclopaedia of stress. san diego: academic press. 1–3. freeman, s. 2005 . biological science. 2nd ed. englewood cliffs: pearson prentice hall. hara, m.r., cascio, m.b. and sawa, a. 2006. gapdh as a sensor of no stress. biochim. biophys. acta., 1762:502–9. hiai, s., yokoyama, h. and oura, h. 1979a. features of ginseng saponininduced corticosterone secretion. endocrinol. jpn., 26:737–40. hiai, s., yokoyama, h. and oura, h. et al. 1979b. stimulation of pituitaryadrenocortical system by ginseng saponin. endocrinol. jpn., 26:661–5. kim, d.h., jung, j.s. and suh, h.w. et al. 1998. inhibition of stressinduced plasma corticosterone levels by ginsenosides in mice: involvement of nitric oxide. neuroreport, 9:2261–4. kim, d.h., moon, y.s. and jung, j.s. et al. 2003a. effects of ginseng saponin administered intraperitoneally on the hypothalamopituitary-adrenal axis in mice. neurosci. lett, 343:62–6. kim, d.h., jung, j.s. and moon, y.s. et al. 2003b. inhibition of intracerebroventricular injection stress-induced plasma corticosterone levels by intracerebroventricularly administered compound k, a ginseng saponin metabolite, in mice. biol. pharm. bull, 26:1035–8. kim, s.r., lee, m.k., koo and k.a. et al. 2004. dibenzocyclooctadiene lignans from schisandra chinensis protect primary cultures of rat cortical cells from glutamate-induced toxicity. j. neurosci. res., 76:397–405. kimura, y. and sumiyoshi, m. 2004. effects of various eleutherococcus senticosus cortex on swimming time, natural killer activity and corticosterone level in forced swimming stressed mice. j. ethnopharmacol, 95:447–53. kishimoto, j., tsuchiya, t. and emson, p.c. et al. 1996. immobilizationinduced stress activates neuronal nitric oxide synthase (nnos) mrna and protein in hypothalamic-pituitary-adrenal axis in rats. brain res., 720: 159–71. kyriakis, j.m. and avruch, j. 2001. mammalian mitogen-activated protein kinase pathways activated by stress and infl ammation. physiol. rev., 81:807–69. lagalwar, s., guillozet-bongaarts, a.l. and berry, r.w. et al. 2006. formation of phospho-sapk/jnk granules in the hippocampus is an early event in alzheimer disease. j. neuropathol. exp. neurol., 65:455–64. leman, m.f. 1952. treatment of reactive and asthenic states of exogenous etiology using the far east schizandra. j. nevropatol. psychiatr., 52: 67–70. liu, j., wang, x. and shigenaga, m.k. et al. 1996. immobilization stress causes oxidative damage to lipid, protein, and dna in the brain of rats. faseb j., 10:1532–8. lüllmann, h., mohr, k. and hein, l. et al. 2005. color atlas of pharmacology. 3rd ed. stuttgart: georg thieme verlag. nathan, c. 1997. inducible nitric oxide synthase: what difference does it make? j. clin. invest., 100:2417–23. nathan, c. and xie, q.w. 1994. nitric oxide synthases: roles, tolls, and controls. cell, 78:915–8. ohkura, y., mizoguchi, y. and morisawa, s. et al. 1990. effect of gomisin a (tjn-101) on arachidonic acid cascade in macrophages. jpn. j. pharmacol., 52:331–6. panossian, a. 2003. adaptogens: tonic herbs for fatigue and stress. alt comp. therap., 9:327–32. panossian, a. and wagner, h. 2005. stimulating effects of adaptogens: an overview of clinical trials of adaptogens with particular reference to their effi cacy on single dose administration. phytother. res., 19:819–38. panossian, a. and wikman, g. 2005. effect of adaptogens on the central nervous system. arq. bras. fitomed. cient., 2:108–30. panossian, a.g., sprygin, v.g., dardimov, iv. 1988. infl uence of eleutherococcus and eleutherosides a, b, c, d and e on the arachidonic acid release and metabolism. chem. pharm. j., 7:776–9. panossian, a., gabrielian, e. and wagner, h. 1997. plant adaptogens ii. bryonia as an adaptogen. phytomedicine, 4:83–97. panossian, a.,wikman, g. and wagner, h. 1999a. plant adaptogens iii. earlier and more recent aspects and concepts on their mode of action. phytomedicine, 6, 287–300. panossian, a., gabrielian, e. and wagner, h. 1999b. on the mechanism of action of plant adaptogens with particular references on cucurbitacin r diglucoside. phytomedicine, 6:147–55. panossian, a.g., oganessian, a.s. and ambartsumian, m. et al. 1999c. effects of heavy physical exercise and adaptogens on nitric oxide content in human saliva. phytomedicine, 6:17–26. park, y.c., lee, c.h. and kang, h.s. et al. 1996. ginsenoside-rh1 and rh2 inhibit the induction of nitric oxide synthesis in murine peritoneal macrophages. biochem. mol. biol. int., 40:751–7. park, h-s., huh, s-h. and kim, m-s. et al. 2000. nitric oxide negatively regulates c-jun n-terminal kinase/stress-activated protein kinase by means of s-nitrosylation. proc. natl. acad. sci. u.s.a., 97:14382–7. park, e.k., shin, y.w. and lee, h.u. et al. 2005. inhibitory effect of ginsenoside rb1 and compound k on no and prostaglandin e2 biosyntheses of raw264.7 cells induced by lipopolysaccharide. biol. pharm. bull, 28:652–6. saratikov, a.s. and krasnov, e.a. 2004. rhodiola rosea (golden root): a valuable medicinal plant. tomsk: tomsk university press. p 1– 205. schaeffer, h.j. and weber, m.j. 1999. mitogen-activated protein kinases: specifi c messages from ubiquitous messengers. mol. cell. biol., 19:2435–44. shen, c., tsimberg, y. and salvadore, c. et al. 2004. activation of erk and jnk mapk pathways by acute swim stress in rat brain regions. bmc neuroscience, 5:36. stamler, j.s. 1994. redox signaling: nitrosylation and related target interactions of nitric oxide. cell, 78:931–6. staritsina, on. 1946. the experience of using schizandra preparations against depressive states in psychiatry. zdravookhr kazakhstana, 6–7:42–4. drug target insights 2007: 2 54 panossian et al upton, r. 1999. schisandra berry. schisandra chinensis. analytical, quality control and therapeutic monograph. american herbal pharmacopoea and therapeutic compendium. 1–26. wagner, h., norr, h. and winterhoff, h. 1994. plant adaptogens. phytomedicine, 1:63–76. wagner, h., bauer, r. and peigen, x. et al. 1996. fructus schisandrae. chinese drug monographs and analysis: radix bupleuri (chaihu). verlag für ganzheit medizin koetzting/bayer, 4:1–8. wang, x., wu, h., lakdawala, v.s. and hu, f. et al. 2005. inhibition of jun n-terminal kinase (jnk) enhances glucocorticoid receptormediated function in mouse hippocampal ht22 cells. neuropsychopharmacology, 30:242–9. williamson, d., gallagher, p. and harber, m. et al. 2003. mitogenactivated protein kinase (mapk) pathway activation: effects of age and acute exercise on human skeletal muscle. j. physiol., 547:977– 87. winterhoff, h., gumbinger, h.g. and vahlensieck, u. et al. 1993. effects of eleutherococcus senticosus on the pituitary-adrenal system of rats. pharmacol lett., 3:99–102. world health organisation. 1999. who monographs on selected medicinal plants. vol 1. geneva: who. world health organisation. 2002. who monographs on selected medicinal plants. vol 2. geneva: who. zakharova pm. 1948. the experience of using schizandra chinensis in psychiatric practice. in krasnushkina ek, ed. problems of social and clinical psychiatry. vol. 9. moscow: moscow 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drug target insights 2007: 2 159–182 159 review correspondence: gilda tachedjian, ph.d., molecular interactions group, the macfarlane burnet institute for medical research and public health, gpo box 2284, melbourne, victoria, 3001, australia. tel: 61 3 9282 2256; fax: 61 3 9282 2100; email: gildat@burnet.edu.au please note that this article may not be used for commercial purposes. for further information please refer to the copyright statement at http://www.la-press.com/copyright.htm targeting human immunodefi ciency virus type 1 assembly, maturation and budding johanna wapling1,2, seema srivastava1, miranda shehu-xhilaga3,4 and gilda tachedjian1,2,3 1molecular interactions group, macfarlane burnet institute for medical research and public health, melbourne, victoria, 3004, australia. 2department of microbiology, monash university, clayton, victoria 3168, australia. 3department of medicine, monash university, prahran, victoria 3181, australia. 4infectious diseases unit, alfred hospital, prahran, victoria 3181, australia. abstract: the targets for licensed drugs used for the treatment of human immunodefi ciency virus type 1 (hiv-1) are confi ned to the viral reverse transcriptase (rt), protease (pr), and the gp41 transmembrane protein (tm). while currently approved drugs are effective in controlling hiv-1 infections, new drug targets and agents are needed due to the eventual emergence of drug resistant strains and drug toxicity. our increased understanding of the virus life-cycle and how the virus interacts with the host cell has unveiled novel mechanisms for blocking hiv-1 replication. this review focuses on inhibitors that target the late stages of virus replication including the synthesis and traffi cking of the viral polyproteins, viral assembly, maturation and budding. novel approaches to blocking the oligomerization of viral enzymes and the interactions between viral proteins and host cell factors, including their feasibility as drug targets, are discussed. keywords: hiv-1, antiretroviral drugs, drug targets, assembly, maturation, budding, protease dimerization, reverse transcriptase dimerization. introduction hiv-1 is a major public health problem affecting an estimated 40 million individuals worldwide (www. unaids.org). although it has been over 20 years since hiv-1 was identifi ed as the etiologic cause of acquired immune defi ciency syndrome (aids) an effective vaccine is not available. thus, apart from public health measures that aim at hiv-1 prevention, the only effective strategy for controlling hiv-1 infections and lowering hiv-1 transmission is the use of antiretroviral drugs either for the treatment or prevention of infections. current antiretroviral drugs belong to four classes, the nucleoside/nucleotide reverse transcriptase inhibitors (nrtis), nonnucleoside reverse transcriptase inhibitors (nnrtis), protease (pr) inhibitors (pi) and fusion inhibitors (vivet-boudou et al. 2006; de clercq, 1998; abdel-rahman et al. 2002; manfredi and sabbatani, 2006). nrtis and nnrtis are respectively, competitive and allosteric inhibitors of the hiv-1 reverse transcriptase (rt) and act early in the viral life-cycle by blocking the conversion of the viral rna genome into a double stranded proviral dna precursor (shehu-xhilaga et al. 2005). fuzeon (enfuvirtide or t20) is a peptide that also acts early in the virus life-cycle by preventing viral entry through interaction with the gp41 transmembrane protein (shehu-xhilaga et al. 2005). in contrast, pis inhibit the late stage of virus replication by blocking the specifi c cleavage of gag and gag-pol polyproteins to mature structural proteins and enzymes (shehu-xhilaga et al. 2005). early antiretroviral regimens consisted of one or two rtis, which were delivered as sequential monotherapy and led to treatment failure (piacenti, 2006). the advent of combination therapy, or highly active antiretroviral therapy (haart) since 1996 has been responsible for a dramatic decrease in aids mortality (palella et al. 1998). current haart regimens generally comprise three antiretroviral drugs, usually two nrtis and either a pi or an nnrti (yeni et al. 2002). while an armoury of agents is available for the treatment of hiv-1 patients, new drugs and drug targets need to be identifi ed due to drug toxicity and the eventual emergence of drug resistant strains to current antiretroviral inhibitors (clavel and hance, 2004). moreover, resistance to one drug normally results 160 wapling et al drug target insights 2007: 2 in cross-resistance to inhibitors of the same class, rendering a large number of agents to limited clinical use (clavel and hance, 2004). therefore, the development or availability of new drugs such as fuzeon, the hiv-1 integrase inhibitor raltegravir (mk-0518) (grinsztejn et al. 2007) and the ccr5 antagonist maraviroc (stephenson, 2007) that remain active against drug resistant virus is essential for the continuing success of haart (yeni, 2006). the increased understanding of how hiv-1 reproduces and interacts with the host cell machinery has resulted in the identifi cation of potential drug targets, which can be exploited for the development of new classes of inhibitors. here we describe strategies and agents that block the late stages of hiv-1 replication including the synthesis and traffi cking of viral polyproteins, viral assembly, maturation and budding. novel approaches to blocking the oligomerization of viral enzymes and the interactions between viral proteins and host cell factors are discussed including their feasibility as drug targets. while peptidomimetic pis act at the late stage of hiv-1 replication to block viral maturation, this review will deal with agents that inhibit hiv-1 pr by novel mechanisms that are distinct to these transition state mimetics that are competitive inhibitors of the hiv-1 pr. late stages of the hiv-1 life cycle following virus attachment, fusion and uncoating the single stranded positive sense rna genome of hiv-1 is reverse transcribed by the viral rt into a proviral dna precursor in a reverse transcription complex (rtc) containing viral and possibly host cell factors (fig. 1). the rtc matures into a preintegration complex (pic) and traffi cs to the nucleus where the viral cdna is inserted into the host cell chromosome by the hiv-1 integrase (in) (telesnitsky a. and goff, 1997). the processes from entry up to and including integration are defi ned as the early steps in the viral life cycle. the late stage of virus replication begins with transcription of the viral mrnas from the integrated provirus (fig.1). singly and multiply spliced mrnas encode the hiv-1 envelope proteins and regulatory/accessory proteins, respectively (rabson and graves, 1997). pr55gag (gag) and pr160gag-pol (gag-pol) polyproteins are translated from unspliced mrnas (swanstrom, 1997). formation of two types of polyproteins from the same unspliced mrna is mediated by a ribosomal frameshifting mechanism that brings the pol sequence in the same reading frame as gag. perturbation of ribosomal frameshifting leads to changes in the gag and gag-pol ratio that is detrimental to virus assembly, morphogenesis and release (swanstrom, 1997). gag encodes the viral structural proteins matrix (ma), capsid (ca), nucleocapsid (nc), p6 and two spacer peptides, p1 and p2. gag-pol also encodes ma, ca and nc in addition to the three viral enzymes, pr, rt and in. after translation, gag and gag-pol are targeted to the host cell plasma membrane, a process that is dependent on the myristoylation of the n-terminus of gag (fig. 1) (swanstrom, 1997). inhibition of myristoylation disrupts the proper targeting of gag and gag-pol to the plasma membrane (swanstrom, 1997). gag-gag, gag/gag-pol and gag-rna interactions are also essential for the proper assembly and maturation of infectious virions. gag and gag-pol assemble at the plasma membrane along with viral envelope glycoproteins gp120 and gp41 to form immature viral particles (fig. 1). gag is necessary and suffi cient for virus particle formation (freed, 1998; swanstrom, 1997). the viral genomic rna is also packaged into virions through interactions with the nc of gag and a psi packaging signal in the genome (swanstrom, 1997). as the newly assembled virions bud from the cell it is believed that gag-pol polyproteins oligomerize in order to activate the hiv-1 pr by forming an active pr homodimer. this results in the sequential cleavage of gag and gag-pol into the mature structural proteins and enzymes (kaplan et al. 1994; pettit et al. 1998). agents that bind to domains in gag or gag-pol and modulate their oligomerization are likely to have a negative effect on virus assembly, maturation and budding (fig. 1). agents that interfere with hiv-1 pr mediated cleavage of gag and gag-pol result in the production of immature viral particles that are non-infectious (kohl et al. 1988). virus particle budding and egress is mediated by interactions of viral proteins such as the p6 late domain with components of the endosomal sorting machinery. ion channels formed by viral protein u (vpu) also facilitate viral particle egress from the host cell. below we describe in more detail the 161 targeting the late stages of hiv-1 replication drug target insights 2007: 2 specifi c processes required for viral assembly, maturation and budding and agents that have been described that block these steps. inhibitors of gag and gag-pol expression: targeting ribosomal frameshifting hiv-1 gag and gag-pol polyproteins are encoded by overlapping open reading frames on the same unspliced mrna. during translation gag-pol is synthesized by a -1 ribosomal frameshifting mechanism that occurs at a frequency of 5 to 10% of gag translation events (jacks et al. 1988b). similar frameshifting mechanisms are also used by other retroviruses including rous sarcoma virus and mouse mammary tumor virus in order to regulate expression of gag-pol (jacks and varmus, 1985; jacks et al. 1987; jacks et al. 1988a). the hiv-1 frameshift site is a heptanucleotide au-rich sequence (uuuuuua) found at the 3′ end of the nc coding sequence and is conserved amongst hiv-1 isolates. this slippery sequence and a downstream rna stem loop structure stall the ribosome during the synthesis of gag, allowing the ribosome to slip figure 1. overview of the hiv-1 life-cycle. early events in virus replication include attachment, fusion and uncoating of the virus followed by reverse transcription in the cytoplasm of the cell, nuclear import of the preintegration complex and integration of the proviral dna precursor into the host cell chromosome. late events begin with transcription of unspliced and spliced rna from the provirus and export of the mrnas to the cytoplasm, resulting in the translation of gag, gag-pol, env and the accessory and regulatory proteins of hiv-1. regulation of gag-pol synthesis is mediated by a ribosomal frameshifting mechanism from unspliced mrna that also expresses gag. myristoylation of gag is necessary for traffi cking of gag and gag-pol to the site of viral assembly. assembly is driven by interactions between gag-gag, gag/gag-pol, gag-rna. viral budding and egress involves host cell factors. during or shortly after budding the hiv-1 pr cleaves the gag and gag-pol polyproteins resulting in a mature and infectious viral particle. late event targets frameshifting myristoylation & trafficking assembly budding & egress maturation early events late events recognition & attachment fusion uncoating & reverse transcription nuclear import mrna export translation myristoylation & trafficking maturation integration transcription & splicing assembly budding & egress rtcpic gag/gag-pol ratio myristoylation gag/gag interactions gag/gag-pol interactions gag/rna interactions vpu p6 ptat motif pr dimerization rt dimerization in dimerization gag cleavage 162 wapling et al drug target insights 2007: 2 back one nucleotide and enable synthesis of the gag-pol fusion protein (jacks et al. 1988b). this sequence, the stem-loop structure and its stability and adjacent interacting sequences are believed to be the key components of the frameshifting signal (jacks et al. 1988b; kollmus et al. 1994; hill et al. 2005). details of a recently reported nmr structure and an analysis of current hiv-1 frameshifting models have recently been reviewed (brierley and dos ramos, 2006). studies demonstrate that perturbation of the gag/gag-pol ratio result in major defects in virus replication, suggesting that interfering with ribosomal frameshifting represents a viable drug target. alteration of the gag/gag-pol ratio, by engineering vectors with gag and pol genes in the same open reading frame, results in major defects in assembly and budding (karacostas et al. 1993; park and morrow, 1991). the block in virus assembly is partially overcome by inhibition of the hiv-1 pr, suggesting that increased hiv-1 pr activity is responsible for the defect (karacostas et al. 1993). a later study, in which the impact of decreasing the ratio of gag/gag-pol on virion production was determined by co-transfection of plasmids expressing gag and gag-pol alone demonstrate that the maintenance of this ratio is not only important for hiv-1 replication but also for virion rna dimer formation and stability (shehu-xhilaga et al. 2001a). furthermore, a decrease in gag-pol translation results in major defects in virus maturation and hiv-1 infectivity (dulude et al. 2006). the small molecule, 1,4bis-[n-(3-n,n-dimethylpropyl)amidino]benzene tetrahydrochloride (rg501, table 1), is thought to enhance ribosomal frameshifting of hiv-1 by binding to the rna stem loop structure of the ribosomal frameshifting signal resulting in increased ribosomal pausing (hung et al. 1998). the imbalance in the resulting gag/gag-pol ratio is associated with inhibition of acute and chronic hiv-1 infection in ccrf-cem cells and peripheral blood mononuclear cells. targeting gag and gag-pol traffi cking during the late phase of the viral life cycle, gag polyproteins are targeted to the plasma membrane, where they are believed to colocalise to lipid raft microdomains for assembly into immature virions (morikawa et al. 1996; bryant and ratner, 1990; bouamr et al. 2003; ding et al. 2003; holm et al. 2003; tang et al. 2004). membrane targeting of gag is mediated by the n-terminal myristoyl group in concert with conserved basic amino acids at the n-terminus of the ma domain of gag (bryant and ratner, 1990; facke et al. 1993; ono and freed, 1999; ono et al. 2000). myristic acid is a saturated 14-carbon fatty acid, post transationally attached to the n-terminal glycine of both gag and gag-pol (veronese et al. 1988). myristoylation of gag but not gag-pol is critical for targeting these polyproteins to the plasma membrane (park and morrow, 1992; smith et al. 1993). mutations that interfere with gag myristoylation inhibit viral budding and misdirect virion assembly to the cytosolic fraction (gottlinger et al. 1989; bryant and ratner, 1990). however, complete inhibition of gag myristoylation is necessary to block hiv-1 budding (morikawa et al. 1996). myristoylation is a two-step process involving activation of myristate to myristoyl-coa by acylcoa synthetase and transfer of the myristoyl moiety from the myristoyl-coa substrate to the n-terminal glycine of gag by the enzyme n-myristoyltransferase (nmt) (morikawa et al. 1996; veronese et al. 1988). this pathway has been utilized to deliver alternate myristoylation substrates that perturb viral assembly. the myristic acid analogue 12-azidododecanoic acid is a potent inhibitor of hiv-1 production in acute and chronically infected t-cell lines, exhibiting a maximum inhibitory effect between 10–50 µm at noncytotoxic concentrations, however the mechanism of action is not defi ned (devadas et al. 1992). another analogue, 4-oxatetra-decanoic acid, reduces hiv-1 replication in a t-cell line at 18 µm (langner et al. 1 9 9 2 ) . heteroatom-substituted analogs of myristic acid such as 12-methoxydodecanoate (13-oxamyristate or 13-oxamyr), 5-octyloxypentanoate (6-oxamyristate or 6-oxamyr), 11-ethylthioundecanoic acid and 12-thioethyldodecanoic acid act as alternate substrates for gag myristoylation (bryant et al. 1989; bryant et al. 1991; parang et al. 1997) and can prevent membrane binding of the modifi ed gag proteins (bryant et al. 1989; bryant et al. 1991). of the heteroatom substituted analogs, 13-oxamyr is the most potent inhibitor. 13-oxamyr is added to gag with an effi cacy similar to that of myristate and alters viral polyprotein processing, which is suggested to be a consequence of inhibiting gag and 163 targeting the late stages of hiv-1 replication drug target insights 2007: 2 table 1. inhibitors of the late stages of hiv-1 replication. inhibitor description reference ribosomal frameshifting 1,4-bis-[n-(3-n,n small molecule (hung et al. 1998). dimethylpropyl)amidino]benzene tetrahydrochloride (rg501) myristoylation and traffi cking 12-azidododecanoic acid myristic acid analogue (devadas et al. 1992) 4-oxatetra-decanoic acid (langner et al. 1992) 12-methoxydodecanoate heteroatom-substituted (bryant et al. 1989) 5-ocytl-oxypentanoate myristic acid analogues (bryant et al. 1991) 11-ethylthioundecanoic acid (parang et al. 1997) 12-thioethyldodecanoic acid 5-cis-tetradecenoic acid unsaturated 14-carbon (lindwasser and resh, 2002). (physeteric acid) fatty acids 5-cis,8-cis-tetradecenoic acid (goshuyic acid) assembly—gag/gag interactions cap-1 small molecule (tang et al. 2003) paatleemmta ca derived peptide (niedrig et al. 1994). gpg-nh2 ca derived tripeptide amide (hoglund et al. 2002) cai peptide (sticht et al. 2005) (ternois et al. 2005) maturation—gag processing 3-0-(3′-3′-dimethylsuccinyl)-betulinic small molecule (li et al. 2003) acid (zhou et al. 2004) (pa-457/bevirimat) (sakalian et al. 2006) electrophilic disulfi de-substituted nc zn fi nger inhibitor (rice et al. 1995) benzamides (dibas) (turpin et al. 1996) 1,2-dithiane-4,5-diol,1,1-dioxide nc zn fi nger inhibitor (rice et al. 1997) (nsc 624151) s-acyl 2-mercaptobenzamide thioester nc zn fi nger inhibitor (schito et al. 2006). (samt) maturation—pr dimerisation ac-tlnf-oh pr c-terminal tetrapeptide (zhang et al. 1991) pal-ydl-oh modifi ed pr c-terminal (schramm et al. 1999) pal-yd-(biphenylalaine)-oh lipopeptides pal-ydt-oh apam(2)-yd-thyroxine-oh (dumond et al. 2003) pqitl(ggg)ctlnf glycine linked pr interface (babe et al. 1992) tetra-peptides ho-fnlts-nh-(ch2)n-n-pqitlw-oh alkyl linked pr interface (zutshi et al. 1997) peptides (ulysse and chmielewski, 1998) (zutshi and chmielewski, 2000) (continued) 164 wapling et al drug target insights 2007: 2 molecular tongs scaffold constrained pr (bouras et al. 1999) interface peptides (breccia et al. 2003) (merabet et al. 2004) (hwang and chmielewski, 2005) (bannwarth et al. 2006) β-sheet peptide/peptidomimetic pr interface derived (song et al. 2001) peptidomimetics pqitl-rkkrrqrrrppqv-sfnfpr c-terminal fusion (davis et al. 2006) c/atln (p27/p27a) peptide bocfψ[ch2nh]fef-nh-ch2-colinked pr c-terminal (uhlikova et al. 1996) tlnf-oh tetrapeptide—active site (skalova et al. 2003) inhibitor pentaester 13e didemnaketal a analogue (fan et al. 1998) ursolic acid triterpene (quere et al. 1996) nhgrnlltqi (s8) pr les peptide (broglia et al. 2005) (broglia et al. 2006) ivqvdaeg (p51) random peptide (park and raines, 2000). vpr-(spacer)-tlnf-oh vpr, pr c-terminal fusion peptide (cartas et al. 2001). maturation—rt dimerisation [2′,5′-bis-o-(tert-butyldimethylsilyl)-βsmall molecule (sluis-cremer et al. 2000) d-ribofuranosyl]-3′-spiro-5″-(4″-amino (rodriguez-barrios et al. 1′,2″-oxathiole-2″,2″-dioxide) 2001) thymine (tsao-t) n-(4-tert-butylbenzoyl)-2-hydroxy-1small molecule (arion et al. 2002) naphthaldehyde hydrazone) (bbnh) (sluis-cremer and tachedjian, 2002) (himmel et al.) ketwetwwte (pep-7) rt connection subdomain peptide (morris et al. 1999) (depollier et al. 2005) tlmalelkgklllaglapsaflplsfp designed peptide targeting (campbell et al. 2002) egl (tlma2993) rt connection subdomain (hosokawa et al. 2004) maturation—in dimerisation ini 1 host cell factor (yung et al. 2001) (sorin et al. 2006) (ariumi et al. 2006) (kalpana et al. 1994) budding and egress 5-(n,n-hexamethylene)amiloride (hma) amiloride analogue (ewart et al. 2002) 5-(n,n-dimethyl)amiloride (dma) (ewart et al. 2004) vpu binding protein (ubp) host cell factor (callahan et al. 1998) (handley et al. 2001) (harila et al. 2006) (neil et al. 2006) tsg101 host cell factor (garrus et al. 2001) (demirov et al. 2002) (goila-gaur et al. 2003) zlll/mg-132 lactocystin proteosome inhibitor (schubert et al. 2000) (ott et al. 2003) abbreviations: ac: acetylation; pam: palmitoyl; apam: 2-aminopalmitic acid table 1. (continued) inhibitor description reference 165 targeting the late stages of hiv-1 replication drug target insights 2007: 2 gag-pol traffi cking (bryant et al. 1991). in an acutely infected t-cell line 13-oxamyr reduces hiv-1 replication in the 40–80 µm range (bryant et al. 1989). 13-oxamyr also inhibits viral production in chronically infected h9/iiib cells, which is consistent for an inhibitor that targets the late stage of hiv-1 replication (bryant et al. 1991). 13-oxamyr exhibits a synergistic antihiv-1 effect with azt suggesting its potential for use in combination therapy (bryant et al. 1991). the therapeutic effi cacy of 13-oxamyr can be further enhanced by conjugation with glycerophospholipid l-∝-phosphatidylethanolamine (pidgeon et al. 1993). the selectivity of these heteroatom analogs for the target protein is dependent on the position of the substituted heteroatom, thus they can be exploited as a therapeutic antiretroviral strategy. nevertheless, heteroatom-substituted myristic acid analogs are still expected to adversely affect a substantial range of cellular processes that depend on protein n-myristoylation (lindwasser and resh, 2002). an alternative strategy for targeting gag myristoylation is the exogenous treatment of cells with unsaturated 14-carbon fatty acids including 5-cistetradecenoic acid (14:1n-9, physeteric acid) and 5-cis,8-cis-tetradecadienoic acid (14:2n-6, goshuyic acid) (lindwasser and resh, 2002). as lipid rafts have preference for saturated fatty acids, treatment with unsaturated analogs interferes with membrane targeting of gag and consequentially viral assembly and production (lindwasser and resh, 2002). these inhibitors also interfere with certain srckinase mediated cellular pathways, although they appear to have no effect on cell proliferation (campbell and vogt, 1995). it is suggested that direct dietary intake of physeteric acid and goshuyic acid could be a useful therapeutic strategy for the treatment of hiv-1 infections. however, the effect of long term intake of these unsaturated fatty acids and their effect on n-myristoylated signaling proteins such as src, g-proteins, arf and heterogeneously n-acylated retinal proteins needs to be assessed (lindwasser and resh, 2002). recent studies also indicate a role for phosphatidylinositide 4,5-bisphosphate [pi(4,5)p2] in regulating gag localization (ono et al. 2004). in hiv-1, binding of pi(4,5)p2 to the ma domain in gag activates the “myristyol switch” and also acts as the point of membrane attachment (saad et al. 2006). the binding site of pi(4,5)p2 on ma is highly conserved amongst hiv-1 strains and therefore represents an attractive antiviral target (shkriabai et al. 2006; saad et al. 2006). targeting hiv-1 ca ca plays an important role in the hiv-1 life-cycle by promoting gag-gag interactions during virion maturation. the nand c-terminal domains of this protein serve distinct functions. as shown by mutational analysis, the n terminal domain of ca (n-ca), otherwise known as the ntd, is responsible for maintaining the proper conformation of ca during the assembly process (worthylake et al. 1999; li et al. 2000). the c-terminal domain of ca (c-ca) or the ctd, is critical for gag-gag interactions during assembly and maturation (gamble et al. 1996; gamble et al. 1997) and described mutations in this region have major consequences on virion maturation and infectivity (von schwedler et al. 2003; ganser-pornillos et al. 2004). the nmr structure of ca has demonstrated that the protein consists mainly of seven α-helices, two β-hairpins and a loop structure (momany et al. 1996; gitti et al. 1996). five of the α-helices form a coiled-coiled structure while one of the β-hairpins is located on the surface of the n-terminal domain of the protein (momany et al. 1996). the second β-hairpin is predicted to be formed after cleavage by the hiv-1 pr (tang et al. 2002). cleavage of ca from its neighbouring proteins is necessary for core condensation and conical capsid shell formation (vogt, 1996; wiegers et al. 1998). compounds that bind to these regions would be expected to disrupt proper ca shell formation and virion infectivity making ca an important and attractive target for the development of antiretroviral agents. a proof of concept study, demonstrating the potential of inhibiting ca-ca interactions as an antiretroviral target has been published (tang et al. 2003). computational high throughput screening of a small molecule library and nmr analysis for binding specifi city resulted in the identifi cation of cap-1 and cap-2 which bind to an apical site on the ntd of both immature and mature ca (tang et al. 2003). while cap-2, is toxic to u1 cells, cap-1 reduces viral infectivity by 95% at 100 µm. the released virions lack cone shaped cores and resemble viral particles that have been observed in hiv-1 expressing mutations that disrupt ca-ca interactions (dorfman et al. 1994; reicin et al. 1996; von schwedler et al. 2003; 166 wapling et al drug target insights 2007: 2 lanman et al. 2003). despite aberrant viral morphology cap-1 does not affect viral particle release or proteolytic processing (tang et al. 2003). cap-1 and cap-2 bind to a common site within the ntd thus preventing ca-ca interactions and proper gag assembly. peptides derived from hiv-1 ca have also been described to affect viral morphogenesis by interfering with capsid formation (niedrig et al. 1994). the synthetic peptide, paatleemmta, inhibits hiv-1 replication in cell culture assays at 20–200 µg/ml and results in the production of immature and aberrant viral particles (niedrig et al. 1994). tripeptide amides derived from the carboxyl terminus of ca inhibit hiv-1 replication, with the three most potent peptides interacting with ca as demonstrated by capillary electrophoresis analysis (hoglund et al. 2002). glycyl-prolyl-glycine-amide (gpg-nh2) interferes with the formation of hiv-1 particles with a normal conical core structure (hoglund et al. 2002). g-nh2 is an active metabolite of gpg-nh2 indicating that the latter acts as a pro-drug (andersson et al. 2005). however, the development of hiv-1 resistance to either g-nh2 or gpg-nh2 has been elusive suggesting that the peptides mediate their effects through a host cell or other factor (andersson et al. 2004). cai, a small peptide selected by phage display screening, acts as an inhibitor of the assembly of immature gag in vitro (sticht et al. 2005; ternois et al. 2005). cai binds to the c-terminus of ca (kd ~ 800 µm), thus preventing the necessary conformational changes in ca that lead to the formation of mature cores (sticht et al. 2005). the structure of cai complexed with ca has revealed that the cai binding region is a highly conserved hydrophobic pocket within the c terminus of ca where the peptide forms an extra α-helix, which binds to the four α-helices of ca (ternois et al. 2005). the resulting proteinpeptide complex is therefore a fi ve α-helix bundle with reduced ca-ca dimerization contacts that destabilizes the dimer interface. binding of cai to the c-ca not only affects the assembly of the immature capsid particles but also reduces the amount of correctly assembled mature capsids in vitro, thus acting as a promising two-step inhibitor (sticht et al. 2005). the c-terminal domain of gag in the context of gag-pol is essential for its interaction with gag and its incorporation into the virion (srinivasakumar et al. 1995; chiu et al. 2002; chien et al. 2006). this sequence includes a highly conserved “major homology region” (mhr) in the ca domain of gag and the adjacent ca-sp1 (srinivasakumar et al. 1995; chien et al. 2006). these sequences are also critical for hiv-1 gag assembly as they drive gag oligomerization. however, the magnitude of the virion incorporation defect of gag-pol mhr deletion mutants varies between different studies making the value of targeting this region of gag-pol unclear with respect to inhibition of the late stages of hiv-1 replication (mammano et al. 1994; srinivasakumar et al. 1995; chiu et al. 2002; chien et al. 2006). sequences involved in gag and gag-pol interactions are assumed to be similar to those involved in gag-gag interactions. however, virions generated in the presence of cap-1 are unlikely to affect gag/gag-pol interactions as defects in proteolytic processing in the virus or virion associated rt activity were not observed (tang et al. 2003). the proline rich region of p6 has also been implicated in the packaging of cleaved pol proteins into virions, which is suggested to be mediated by host cell proteins (dettenhofer and yu, 1999; cen et al. 2004). identifying the host cell factor implicated in the virion incorporation of cleaved pol will be necessary for establishing this process as a viable drug target. targeting hiv-1 nc the hiv-1 nc (ncp7) contains two highly conserved zinc fi nger motifs c-x2-c-x4-h-x4-c (x, any amino acid). the zinc fi ngers of nc are critical in the early and late stages of hiv-1 replication with mutations in the zinc chelating amino acids resulting in formation of noninfectious virus (aldovini and young, 1990). the zinc fi ngers of ncp7 are required for initiation, elongation and effi cient template switching d u r i n g r e v e r s e t r a n s c r i p t i o n ( r o d r i g u e z rodriguez et al. 1995; tanchou et al. 1995). ncp7 is also involved in hiv-1 genomic rna dimerization, in cleavage activity and coats the viral rna genome protecting it from nucleases (lapadat-tapolsky et al. 1993). given the critical role of ncp7 zinc fi ngers in hiv-1 replication it is not surprising that agents that covalently modify the zinc chelating residues of ncp7 have been described as inhibitors of hiv-1 replication (rice et al. 1995). the electrophilic 167 targeting the late stages of hiv-1 replication drug target insights 2007: 2 disulfi de-substituted benzamides (dibas) inactivate cell free virus and inhibit the early and late stages of hiv-1 replication by interfering with reverse transcription and viral particle maturation (rice et al. 1995; turpin et al. 1996). in the u1 cell line treatment with dibas results in the inhibition of virus particle release, processing of gag, and the production of virions with reduced infectivity (turpin et al. 1996). the defect in viral particle release and maturation was attributed to the formation of intermolecular cross-linkages between the zinc fi ngers of adjacent gag molecules, thereby preventing effi cient cleavage by the hiv-1 pr (turpin et al. 1996). t h e n o n d i s s o c i a b l e t e t h e r e d d i t h i a n e compound 1,2-dithiane-4,5-diol,1,1-dioxide, (nsc 624151) also mediates similar defects in gag processing (rice et al. 1997). although cellular proteins also contain zinc fingers, these inhibitors appear to preferentially target retroviral zinc fingers. this may be explained by the inaccessibility of these inhibitors to the appropriate cellular compartments where zinc finger containing cellular proteins are located. the in vivo anti-hiv-1 activity of zinc finger inhibitors has been demonstrated in a transgenic murine model where infectious hiv-1 is induced from an integrated provirus (schito et al. 2003). a recent study in a nonhuman primate model demonstrated a reduction in the levels of siv/ deltab670 in peripheral blood mononuclear cells during therapy with the zinc finger inhibitor, s-acyl 2-mercaptobenzamide thioester (samt), although there was no effect on viral load (schito et al. 2006). further studies are in progress to optimise the bioavailability and pharmacokinetics of this promising inhibitor. targeting hiv-1 pr much of our understanding of how the pr domain in gag-pol is activated and the processing cascade of gag and gag-pol are due to the contributions of kaplan and colleagues (kaplan et al. 1994; pettit et al. 2005). strict regulation of pr function is critical for effi cient production of mature viral particles. premature activation, partial inhibition, or over-expression of hiv-1 pr leads to major defects in viral assembly and the production of non-infectious viral particles (krausslich, 1991; kaplan et al. 1993; karacostas et al. 1993). hence novel inhibitors designed to prevent or perturb pr dimerization could potentially inhibit the mature pr homodimer and the immature gag-pol embedded pr. targeting the pr dimer interface with interface peptides hiv-1 pr is a homodimeric aspartyl protease formed by the symmetrical association of two 99 amino acid subunits. the crystal structure reveals a compact, predominantly β-strand structure with a short α-helix region near the c terminus (wlodawer et al. 1989). dimerization of the pr monomers generates both the substrate-binding pocket and the catalytic centre and is essential for pr activity (cheng et al. 1990). the pr dimer has a dissociation constant of 50 nm and gibbs free energy of dimer stabilisation of 10 kcal/mol (25 oc, ph 3.4). nearly 75% of the binding energy is contributed by the four-stranded β-sheet formed by the nand c-termini (todd et al. 1998).the four-stranded β-sheet comprising the nand c-termini from each pr monomer represents an attractive drug target for the following reasons: 1. it is the major stabilising region of the active dimer, 2. the region is relatively free of known pr resistance mutations, 3. the sequence is highly conserved in most hiv-1 and hiv-2 isolates and 4. it provides a unique target minimising potential toxicity issues for eukaryotic aspartyl proteases (gustchina and weber, 1991). a standard methodology for analysing potential pr inhibitors that prevent pr dimerization (dissociative inhibition) or target and bind to the pr active site (competitive inhibition) has been described (zhang et al. 1991). an example of a dissociative inhibitor is the c-terminal tetrapeptide, ac-t-l-n-f, which exhibits activity in the micromolar range (ki 45 µm) (zhang et al. 1991). other studies have also shown the capacity of nand c-terminal peptides, or ‘interface’ peptides, to bind to pr monomers and thus prevent pr dimerization and activity (babe et al. 1991; franciskovich et al. 1993; schramm et al. 1991; schramm et al. 1996). the identifi cation of these lead peptides provides proof of concept that targeting the pr β-sheet region constitutes a viable strategy for the development of novel inhibitors of hiv-1 pr. the potency of c-terminal tetrapeptides are increased by truncation to a core tripeptide, amino acid modifi cation, and the addition of a linear hydrophobic moiety such as palmitoyl to the amino 168 wapling et al drug target insights 2007: 2 hiv-1 pr monmer n-terminal c-terminal peptide peptide scaffold ‘molecular tong’ terminus of the peptide. the lipid moiety is thought to increase the dissociative activity of the peptides by directing it to the hydrophobic pr interface (schramm et al. 1999). however, despite their capacity to inhibit pr activity at low nanomolar concentrations, these lipopeptides are poorly soluble and susceptible to protein degradation. further modifi cations have been made to the lipopeptides by making them less peptide-like (cafl isch et al. 2000) and by modifying the lipid moiety to increase their solubility while retaining potency (dumond et al. 2003). cross-linking interfacial peptides represent another strategy, with the aim to increase the affi nity of the peptides by presenting them in a conformation similar to a pr monomer. the fi rst interface tetrapeptides tethered with a glycine linker display greater potency (pf1, ic50= 40 µm) compared to free tetrapeptides (ic50 ≥ 150 µm) (babe et al. 1992). this approach has evolved to linking peptides with fl exible alkyl tethers (zutshi and chmielewski, 2000) and semirigid alkyl based tethers (ulysse and chmielewski, 1998), which increase the distance between the peptides to approximate that of the pr termini in the dimer (~10 å). the conformational freedom of these linked peptides was addressed by the use of pyridinediol and naphthalene based molecularly constrained scaffolds (bouras et al. 1999; song et al. 2001; merabet et al. 2004; bannwarth et al. 2006). known as ‘molecular tongs’, these compounds are designed to position the interface peptides to clamp the termini of a pr monomer (fig. 2). these studies have culminated in a set of optimised tongs with symmetrical peptidomimetic sequences based on an optimised pr c-terminal sequence. the tongs inhibit the activity of hiv-1 pr that are either sensitive or resistant to pis with ki values from 0.4–4.8 µm in cell free assays (bannwarth et al. 2006). other variations on the theme of interface peptides include combining the advantages of lipopeptides and molecular tongs. interface peptides have been linked to lipophilic groups by a rigid bicyclic guanidinium scaffold (breccia et al. 2003). the most potent compound demonstrates pr inhibitory activity similar to tethered peptides and molecular tongs. cross-linked interfacial peptides have been designed to irreversibly inhibit hiv-1 pr by formation of a disulfi de bond between the peptide and the conserved pr residues c-95 and c-67, and demonstrate a ki in the low micromolar range (zutshi and chmielewski, 2000). the c-terminal tetrapeptide has also been tethered to a peptidic pr active site inhibitor, combining both dissociative and competitive methods of inhibition in one molecule (uhlikova et al. 1996). random peptides that are dissociative inhibitors of hiv-1 pr have been described. the bacteriophage lambda repressor protein was utilised to develop a powerful two-hybrid pr dimerization assay. from a library of 5 × 108 random peptides, 300 were identifi ed as potential pr dimerization inhibitors. the most potent peptide identifi ed, p52, was a pure dissociative inhibitor with low ki of 780 nm (park and raines, 2000). ultimately, one of the major hurdles in developing peptidic inhibitors is to obtain a biologically stable compound that can be delivered inside the cell. one mechanism to achieve this is to fuse the peptide to amino acid sequences that promote either encapsidation into viral particles or entry into the host cell. virus protein r (vpr) is a hiv-1 accessory protein packaged into virions by its trans association with the gag p6 motif. both viral and cellular proteins have been successfully delivered into viral particles as vpr fusion proteins (wu et al. 1997). inhibition of hiv-1 replication has been reported by the fusion of vpr to viral pr recognition sequences (serio et al. 2000). expression of the pr c-terminal tetrapeptide as a vpr fusion [vpr-(spacer)-t-l-n-f-oh] attenuates hiv-1 replication in chronically infected cells and in single-round replication assays (cartas et al. 2001). most recently, inhibition of hiv-1 replication has been demonstrated by delivering pr interface peptides as a fusion peptide utilising the hiv-1 tat derived cell permeable protein transduction domain figure 2. a molecular tong bound to the c-terminus of the hiv-1 pr monomer. 169 targeting the late stages of hiv-1 replication drug target insights 2007: 2 (davis et al. 2006). peptides p27/a are pr dimerization inhibitors that inhibit the activity of wild-type and drug resistant pr in cell free assays with ic50 values in the 0.28–0.58 µm range. these peptides are successfully delivered into chronically hiv-1 infected cells and reduce viral particle production. this was observed by a reduction in p24, rather than inhibition of gag processing which suggests that the peptide may interact with the gag-pol embedded pr and disrupt the ordered processing of gag-pol leading a decrease in viral particle production (davis et al. 2006). pr folding inhibitors local elementary structures (les) are comprised of strongly interacting, highly conserved amino acids that are usually hydrophobic. these amino acids are suggested to direct the folding of a protein into its native conformation. short peptides corresponding to or mimicking the les are hypothesised to act as folding inhibitors, preventing the protein achieving its native conformation (broglia et al. 2005). a peptide has been identifi ed from a les in the hiv-1 pr (peptide s8, amino acids 83–93) that inhibits pr activity with a ki of 2.58 µm and results in disorganisation of the pr secondary structure by reducing the β-sheet content from 30% to 14% (broglia et al. 2005; broglia et al. 2006). current efforts are directed towards developing a shorter less hydrophobic peptide or mimetic based on the s8 lead peptide. catalytically inactive pr subunits as dominant negative inhibitors of pr activity catalytically inactive pr monomers act in a dominant negative fashion to inhibit wild-type hiv-1 pr by forming inactive heterodimers in recombinant protein assays (babe and craik, 1991). when virus expressing a pr active site mutation is co-transfected with wild-type hiv-1, both viral replication and virus infectivity are reduced (babe et al. 1995). computer modelling has been used to successfully design an optimised dominant-negative pr expressing d25k, g49w and i50w (kww) (mcphee et al. 1996), which also reduces viral replication and infectivity (junker et al. 1996). biochemical studies on recombinant dominant negative prs confi rm that the mechanism of action is by formation of inactive heterodimers (rozzelle et al. 2000). interestingly, the mutant prs cannot homodimerize and they fold only when expressed with wild-type pr. pr heterodimers are also more stable that the wild-type homodimer (rozzelle et al. 2000). hence inactive pr heterodimers form the dominant species. such a dominant negative strategy for the inhibition of hiv-1 pr would require in vivo delivery by a genetherapy system, the therapeutic use of which is unlikely in the near future. non-peptide inhibitors of hiv-1 pr a screen of a crude extract from the marine organism magenta ascidian didemnum identifi ed two didemnaketals, a (a bicyclic ketal) and b (a linear heptaprenoid), that inhibit hiv-1 pr activity with ic50 values of 2 µm and 10 µm, respectively (potts et al. 1991). these compounds are unsuitable drug candidates, but have given rise to a novel class of pentaesters, the most potent of which is a dissociative inhibitor of pr with a ki of 2.1 µm (fan et al. 1998). a novel class of pr dimerization inhibitors were identifi ed by searching the cambridge structural database for pharmacophores that mimic the action of previously identified inhibitory interface peptides (quere et al. 1996). several triterpene structures were identifi ed, of which ursolic acid acts as a dissociative inhibitor of pr with an ic50 of 2 µm. it has been suggested that triterpene could provide another basic scaffold for building more effective peptidomimetics. interestingly, another member of the triterpene family, pa-457, acts as a novel inhibitor of hiv-1 maturation which is discussed later in this review. a β-sheet mimetic was tested for its ability to inhibit pr homodimerization by perturbation of β-sheet formation (song et al. 2001). the β-sheet mimetic had a relatively high ic50 of 30 µm and the method of inhibition appears to be complex, however the structure provides a non-peptidic lead compound for pr inhibitors. targeting gag processing the rate and the specifi city of gag cleavage by the hiv-1 pr is dependent on the amino acid composition of the different cleavage sites recognized by the viral pr (swanstrom, 1997). based on the order of proteolysis by hiv-1 pr, these sites are classified as primary (p2/nc), secondary (ma/ca and p1/p6) or tertiary (ca/p2 170 wapling et al drug target insights 2007: 2 or ca/sp2) cleavage sites. the lack of processing of any of these sites by pr results in the formation of aberrant particles (swanstrom, 1997). in particular, inhibition of cleavage at the ca/p2 site has severe consequences for core formation, stability and virion infectivity (pettit et al. 1994; wiegers et al. 1998; pettit et al. 1998; shehuxhilaga et al. 2001b). the α-helical structure that stretches between the c-terminus of ca and the n-terminus of sp1 is critical for virion assembly and p2 function (accola et al. 1998). clearly, these pr cleavage sites are potential targets for antiretroviral drug design. in this regard, a compound that interferes with viral maturation by blocking ca/p2 cleavage has been identifi ed. 3-o-(3′,3′-dimethysuccinyl) betulinic acid (pa-457 or bevirimat) potently inhibits hiv-1 maturation and infectivity (li et al. 2003; zhou et al. 2004). pa-457 specifi cally blocks the cleavage of ca/p2 in cell based (li et al. 2003) and in cell free assays (zhou et al. 2005; sakalian et al. 2006), thus inhibiting core condensation and virion maturation. inhibition of gag processing at the ca/p2 junction results in the generation of the uncleaved p25 product in transfected cells at 0.1 µg/ml of pa-457 (li et al. 2003). consistent with the proposed mechanism, pa-457 resistant hiv-1 selected in long term cultures in the presence of betulinic acid contain mutations in the regions that fl ank the p-p’ scissile bond (adamson et al. 2006; zhou et al. 2006). these mutated sites in gag are recognized by the viral pr during proteolysis. in addition, other single amino acid substitutions have been identifi ed that confer resistance to pa-457 and are exclusively located either at the c terminus of ca or within the fi rst three amino acids of the p2 spacer peptide (adamson et al. 2006). interestingly, they all conferred resistance independently and were located within the boundaries of the ca/p2 proteins, a region well known to promote gag multimerization (adamson et al. 2006). these data suggest that there is more than one mechanism by which these mutants have acquired resistance to pa-457. pa-457 has successfully undergone phase 1 and 2 clinical trials and is currently in a phase 2b trial to test the effi cacy of different doses of pa-457 in combination with approved hiv-1 inhibitors as part of an optimised regimen in patients failing therapy due to the emergence of drug resistant virus. targeting the rt domain in gag-pol like other hiv-1 enzymes, rt subunits must oligomerize to form an active enzyme. the biologically relevant form that is present in the virion is an asymmetric heterodimer comprised of the p66 (66 kda) and the p51 (51 kda) subunits (jacobo-molina et al. 1993; kohlstaedt et al. 1992). the rt heterodimer is extremely stable and has an extensive protein surface area (4800 å2) that is buried upon subunit dimerization. thermodynamic measurements of the association between the p66 and p51 rt subunits have estimated gibbs free energy of dimer stabilization of approximately 10–12 kcal/mol–1, corresponding to a dissociation constant of approximately 3 × 10–7 m (venezia et al. 2006). for an extensive review on hiv-1 rt dimerization see srivastava et al. 2006. regions both upstream and downstream of the pr region in gag-pol have been investigated for effects on pr activation (bukovsky and gottlinger, 1996; partin et al. 1991; louis et al. 1999). large deletions within or c-terminal truncations of rt in the context of gag-pol result in an increase in virion associated gag processing intermediates, suggesting a defect in pr activity (cherry et al. 1998; liao and wang, 2004; quillent et al. 1996). these studies suggest that modulating rt dimerization in the context of gag-pol may have a negative impact on pr activation and hiv-1 maturation. the importance of the rt region in gag-pol for both rt maturation and viral particle production has been demonstrated by the study of rt point mutations that prevent rt heterodimerization and p66 homodimerization. mutations at w401, a component of the highly conserved tryptophan repeat motif in the connection subdomain, blocks rt dimerization in vitro (tachedjian et al. 2003; tachedjian et al. 2005b). when expressed in hiv-1 it manifests as defects in reverse transcription, aberrant processing of rt, and low levels of infectivity (wapling et al. 2005). the l234a mutation, located in the rt primer grip region, prevents rt dimerization and decreases gag-pol stability (tachedjian et al. 2000; yu et al. 1998). this mutation reduces pr incorporation into virions, increases the accumulation of gag processing intermediates, and results in the production of non-infectious virus particles (tachedjian et al. 2000; yu et al. 1998). these examples demonstrate the potential for 171 targeting the late stages of hiv-1 replication drug target insights 2007: 2 targeting the gag-pol embedded rt for blocking hiv-1 maturation. inhibitors that modulate hiv-1 rt subunit interaction apart from classical nnrtis, there exists a class of unconventional nnrtis that bind to hiv-1 rt and inhibit enzyme activity by decreasing the overall stability of the heterodimer without dissociating the complex (sluis-cremer et al. 2000; sluis-cremer and tachedjian, 2002; sluis-cremer et al. 2006; camarasa et al. 2006). the tsao-t derivatives ([2′,5′-bis-o(tert-butyldimethylsilyl)-β-d-ribofuranosyl]-3′spiro-5″-(4″-amino-1″,2″-oxathiole-2″,2″-dioxide) thymine) destabilise both rt heterodimers and p66 homodimers by inducing changes at the rt dimer interface (sluis-cremer et al. 2000; rodriguezbarrios et al. 2001). the putative binding site is at the rt dimer interface and overlaps in part with the nnrti binding pocket (rodriguez-barrios et al. 2001). resistance mutations to the tsao drugs are readily generated in cell culture indicating that these drugs are specific inhibitors of the hiv-1 rt (balzarini et al. 1993). while tsao represent the fi rst class of small molecules that destabilize the rt heterodimer, preclinical studies demonstrate that the pharmacological profile of tsao inhibitors is unfavourable for further clinical development (camarasa et al. 2006). the n-acylhydrazone derivative n-(4-tertbutylbenzoyl)-2-hydroxy-1-naphthaldehyde hydrazone (bbnh) binds to both the dna polymerase and rnase h domains of rt and inhibits both enzymatic activities of the rt (arion et al. 2002). similar to tsao, bbnh prevents rt activity through destabilizing, but not dissociating the subunits. bbnh derivatives that bind to the dna polymerase domain alone are suffi cient to induce dimer destabilization (sluis-cremer and tachedjian, 2002). the recently resolved structure of hiv-1 rt bound to a bbnh derivative has confi rmed that the binding site is in close proximity to, but distinct from both the polymerase active site and nnibp. it is thought that bbnh destabilizes the rt heterodimer by inducing changes in the primer-grip motif, which is an important region for rt dimer stability (himmel et al. 2006; srivastava et al. 2006). by targeting the rt dimerization interface, it is possible that unconventional nnrtis may have an affect on the late stage of virus replication. in particular, the tsao drugs that destabilize the p66 homodimer, may also perturb the process of rt maturation to the heterodimer, and arguably even target the rt domain in gag-pol and interfere with pr activation. however, these possible effects have not been described. further elucidation of the impact of nonclassical nnrtis on rt maturation, and the mechanism of destabilization would be advantageous for designing more potent inhibitors of both rt function and rt maturation. classical nnrtis as rt inhibitors acting at the late stage of viral replication interestingly, several classical nnrtis have been shown to confer a concentration dependant increase in rt heterodimer formation, corresponding with a loss of rt polymerase function (tachedjian et al. 2001; venezia et al. 2006). efavirenz (efv) is a strong enhancer of rt dimerization, and also enhances the formation of p66 and p51 homodimers (tachedjian et al. 2005a). the exact mechanism for increasing rt subunit interactions is unknown but it is suspected that the binding of efv to the rt mediates conformational changes in the p66 subunit that promotes interaction with p51 (tachedjian et al. 2001). efv has also demonstrated the capacity to enhance the homodimerization of p66 in vitro, and a 90kda model pol protein in an inducible bacterial expression system (sluis-cremer et al. 2004; tachedjian et al. 2005a). it has recently been demonstrated that nnrtis enhance gag-pol dimerization, resulting in premature pr activation and a decrease in viral particle release (figueiredo et al. 2006). in hiv-1 transfected cells, efv, tmc120 and tmc125 increased gag and gag-pol processing, and caused up to 45% decrease in viral particle production. similar effects were not observed for nnrtis that do not signifi cantly enhance p66 homodimerization and nrtis (figueiredo et al. 2006). hence, nnrtis that are potent enhancers of rt dimerization also affect the late stage of viral replication, which represents a novel inhibitory mechanism for these drugs. however, the concentrations required to mediate this effect are two to three orders of magnitude higher then concentrations that block rt function. this is likely due to reduced binding affi nity of the nnrtis to the proposed target which is the rt domain of gag-pol. strategies to identify drugs that are more potent 172 wapling et al drug target insights 2007: 2 inhibitors of this late stage in the viral life-cycle could be identifi ed by screening for molecules that enhance gag-pol dimerization. such a screen could be enhanced by incorporating mutations in the rt that are known to confer decreased susceptibility to current nnrtis in order to select for drugs that have the potential to block nnrti resistant strains of hiv-1. peptide based inhibitors of rt dimerization two strategies have been utilised to generate peptides designed to target the rt dimer interface in order to block hiv-1 rt function, including peptides corresponding to regions that are known to have an important role in rt dimerization (debyser and de clercq, 1996; depollier et al. 2005; divita et al. 1994; morris et al. 1999b; morris et al. 1999a). the most successful of these peptides, pep-7, corresponds to rt residues 395–404, derived from the highly conserved tryptophan repeat motif (w398–w414) (depollier et al. 2005). pep-7 interacts with p51, and destablizes both the rt heterodimer and the p66 homodimer. similar to unconventional nnrtis, pep-7 is unable to induce rt dissociation (depollier et al. 2005). pep-7 based peptides are potent suppressors of hiv-1 replication at noncytotoxic concentrations (morris et al. 1999b). the method of inhibition in hiv-1 infected cells has not been elucidated. however, given that pep-7 cannot induce rt subunit dissociation, it has been suggested that it acts at the late stage of virus replication by preventing the formation of an active rt heterodimer (morris et al. 1999b). rational strategies utilising the available rt structures to direct the design and manufacture of mimetic peptides targeting subunit interaction is a recent development (campbell et al. 2002; hosokawa et al. 2004). these studies have led to the synthesis of a peptide, tlma2993, which also targets the rt connection subdomain. tlma2993 inhibits rt activity at micromolar concentrations (campbell et al. 2002). cells stably transfected with this peptide are protected from hiv-1 infection in a concentration dependant manner due to inhibition of reverse transcription, as observed by a decrease in hiv-1 dna (hosokawa et al. 2004). targeting the in domain in gag-pol in catalyses the insertion of viral dna into the host chromosome and thus inhibits an early crucial step in the virus life cycle. in is also implicated in reverse transcription, nuclear import of the pre-integration complex, viral assembly and budding (engelman et al. 1995; hehl et al. 2004). despite the numerous roles of in in hiv-1 replication, new approaches for inhibiting viral replication have focused on targeting the catalytic activity of in that is required for proviral dna integration. two in inhibitors, mk-0518 and gs-9137 (jtk-303) have entered clinical trials (cotelle, 2006; makhija, 2006) and have shown effi cacy in phase iii clinical trials (stephenson, 2007). since in is expressed as part of gag-pol, agents that bind to the in domain in this polyprotein are likely to impact on the late stages of replication. consistent with this notion, mutations in in have been reported to effect virion formation (shin et al. 1994). truncations of in at the c-terminus of gag-pol result in aberrant virion core structures, with a reduction in the overall levels of cell-associated viral gag, suggesting a defect in gag-pol processing (engelman et al. 1995; bukovsky and gottlinger, 1996). in requires oligomerization for activity. therefore, inhibitors of in function that mediate their effects through negating in subunit interactions are also likely to interfere with viral assembly. this would be manifested by interfering with gag-pol/gag-pol interactions leading to subsequent effects on hiv-1 pr activation (muriaux et al. 2004). in this regard peptide inhibitors of in dimerization have been reported, however their effects on the late stages of the virus life-cycle remains to be determined (maroun et al. 2001; zhao et al. 2003). certain host cell factors are incorporated into the virion by interaction with the in domain of gag-pol. a cellular factor that has been implicated in affecting the late stage of the virus life cycle is integrase interactor 1 (ini1). ini1 was identifi ed in a yeast two-hybrid screen for host cell proteins interacting with hiv-1 in (yung et al. 2001;yung et al. 2004; kalpana et al. 1994). ini1 mutants that abrogate interaction with in or cells defi cient in ini1 exhibit a substantial reduction in viral production (yung et al. 2001). ini1 affects several steps during hiv-1 replication (ariumi et al. 2006; sorin et al. 2006; yung et al. 2001) and is also packaged into hiv-1 particles (kalpana et al. 1994). a 110amino-acid fragment of ini1 (s6) with a minimal in-interaction domain inhibits viral production (yung et al. 2001; yung et al. 2004). the inhibitory effect of s6 on hiv-1 production is mediated by 173 targeting the late stages of hiv-1 replication drug target insights 2007: 2 binding of the ectopically expressed s6 to the gag-pol embedded in. furthermore, stable expression of a transdominant s6 mutant inhibits infection in t-cells. s6 represents a potential lead for the development of inhibitors of the late stage of hiv-1 replication (yung et al. 2001). proteosome inhibitors intracellular degradation of misfolded, damaged or unwanted proteins is mediated by the proteosome, which is a multisubunit proteolytic complex of 26s (schubert et al. 2000). proteins are tagged for proteolytic destruction by the covalent attachment of a chain of ubiquitin polypeptides on lysine residues of the protein (schubert et al. 2000). proteosome inhibitors inhibit the late stages of the hiv-1 life-cycle by interfering with viral particle release and maturation (schubert et al. 2000). decreased budding has also been demonstrated for retroviruses expressing the pppyor ptap containing late domains but not those that use the ypdl type late domain (schubert et al. 2000; ott et al. 2003). the effect is not dependent on the viral particle assembly site (i.e. cytoplasm or plasma membrane) or on monoubiquitination of gag (ott et al. 2003). in addition to a decrease in viral particle release (4-fold), virions released from cells treated with proteosome inhibitors have approximately a 10-fold decrease in infectivity (schubert et al. 2000). the impact of proteosome inhibitors is dependent on an active hiv-1 pr and the presence of the p6 late domain but is independent of vpu function. inhibition of hiv-1 maturation and budding is observed with reversible (zlll also known as mg-132) and irreversible (lactocystin) proteosome inhibitors (schubert et al. 2000). proteosome inhibitors also interfere with the activity of the hiv-1 viral infectivity factor (vif) on the antiviral function of apolipoprotein b mrnaediting enzyme catalytic polypeptide-like 3g (apobec3g) in virus producing cells (stopak et al. 2003; sheehy et al. 2003; mehle et al. 2004; yu et al. 2003). wild-type viruses expressing vif are able to prevent incorporation of apobec3g into the virion by promoting its degradation in the cytoplasm of the producer cell. inhibition of apobec3g incorporation in the virus prevents hypermutation in newly synthesized viral dna following infection of target cells due to c to u modifi cations during minus stand dna synthesis mediated by apobec3g. proteosome inhibitors interfere with vif dependent degradation of apobec3g suggesting that these inhibitors can impede the mechanisms used by the virus to evade the innate defences of the host cell (stopak et al. 2003; sheehy et al. 2003; mehle et al. 2004; yu et al. 2003). targeting an essential cellular process like the proteosome is anticipated to be cytotoxic and not well tolerated in vivo. nevertheless, the highly specifi c proteosome inhibitor epoxomicin, which also inhibits hiv-1 maturation, is well tolerated in mice (meng et al. 1999). the proteosome inhibitor, ps-341, is approved as a last resort treatment of multiple myelomas and is associated with adverse effects (kane et al. 2006). the use of proteosome inhibitors in hiv-1 infected individuals needs to be considered in the context of the potential risk benefi t and the net effect on inhibition of hiv-1 replication as proteosome inhibitors also enhance the early step of the virus life-cycle by preventing degradation of the reverse transcription complex mediated by trim5α (schwartz et al. 1998; wu et al. 2006; wei et al. 2005). targeting hiv-1 egress mediated by vpu the hiv-1 accessory protein, vpu is a 16 kda type 1 integral membrane protein that is indispensable for viral pathogenesis (li et al. 2005). vpu plays two distinctive roles in the viral life-cycle that include down regulating host cell cd4 receptors (willey et al. 1992) and enhancing viral particle release from the cell surface, the latter associated with its ion channel forming properties (schubert et al. 1996a). vpu is unique to hiv-1/sivcpz viruses (binette and cohen, 2004). interestingly the two closely related retroviruses hiv-2 and siv, which lack vpu, are less pathogenic (bour and strebel, 2003). the role of vpu in the viral budding process is coupled to its ion channel forming properties, which is predicted to be a pentameric structure composed of fi ve transmembrane domains (grice et al. 1997). the vpu ion channel is thought to function by altering the electric potential at the plasma membrane or alternatively by overcoming host restriction factors for viral release (neil et al. 2006). the hiv-1 vpu is a member of viral ion channel proteins called viroporins and is structurally similar to the m2 ion channel protein of infl uenza (gonzalez and carrasco, 2003; hout et al. 2006). an interesting feature of vpu is its role in 174 wapling et al drug target insights 2007: 2 viral particle release from nondividing cells such as macrophages (deora and ratner, 2001). in this regard the rate of host cell proliferation is a determining factor for vpu mediated viral particle release (deora and ratner, 2001). analogues of amiloride (a sodium channel blocker) inhibit vpu ion channel activity. the amiloride analogues, 5-(n,n-hexamethylene) amiloride (hma) and 5-(n,n-dimethyl)amiloride (dma) inhibit vpu mediated virus budding and viral replication in macrophages (ewart et al. 2002; ewart et al. 2004). the inhibitory effects are observed in the absence of cytotoxicity. both analogs exhibit strong inhibition of hiv-1 replication as measured by viral p24 levels in culture supernatants (ewart et al. 2004). hma at 4 µm suppresses viral p24 in culture supernatants to undetectable levels for more than 10 days in culture (ewart et al. 2004). while amiloride analogues demonstrate activity in macrophages, they fail to inhibit hiv-1 replication in t-cells (ewart et al. 2002). nevertheless, vpu ion channel inhibitors have the potential for use in combination therapy, targeting viral reservoirs and drug-resistant variants. an amiloride derivative, bit225, is currently being pursued for drug development (biotron limited, sydney, nsw, australia). bit225 represents a promising antiretroviral therapeutic although the evaluation of its in vivo effi cacy will present a challenge since inhibition of hiv-1 replication appears to be restricted to nonproliferating cells. recent studies also suggest that rimantadine, an ion channel blocker of infl uenza a viruses, can be a useful lead compound for designing vpu inhibitors. rimantadine and amantadine belong to class of polycyclic amines that are active against the m2 ion channel of infl uenza a but not against hiv-1 vpu (hout et al. 2006). studies indicate that mutating histidine at residue 19 to alanine results in a rimantadine sensitive vpu ion channel demonstrating the potential of this class of inhibitor as hiv-1 ion channel blockers (hout et al. 2006). vpu is also implicated to interact with certain host cell restriction factors that interfere with viral particle egress. the host cell protein, vpu-binding protein (ubp), is suggested to be a negative factor for virus assembly (callahan et al. 1998; bour and strebel, 2003). ubp is a 34-kda protein that exhibits competitive binding with vpu and gag (callahan et al. 1998; handley et al. 2001). overexpression of ubp has been reported to significantly suppress viral particle release suggesting that ubp is a negative factor that requires displacement by gag or vpu (callahan et al. 1998). it is suggested that vpu mediates its effect on viral egress by facilitating membrane targeting of gag precursors (handley et al. 2001; harila et al. 2006; neil et al. 2006). supporting this notion, vpu-defective particles appear in internal membrane-bound compartments suggesting a gag targeting defect (klimkait et al. 1990). another host cell restriction factor implicated in viral release is an acid-sensitive potassium channel-forming protein, task-1, which is down regulated during viral infection (hsu et al. 2004). due to the structural homology of task-1 and hiv-1 vpu, task-1 has been suggested to form hetero-oligomers with vpu which interferes with both task-1 mediated conductance and vpu ion channel function (hsu et al. 2004). further delineation of the how these host cell factors interact with vpu is required in order to design small molecule inhibitors that inhibit viral particle release. targeting hiv-1 egress mediated by the p6 late domain in the recent years, major advances have been made in our understanding of how hiv-1 and other retroviruses are released from infected cells. in the early 1990s it was reported that mutations in the p6 late domain inhibit virion particle release (gottlinger et al. 1991). moreover, a ptap sequence, encompassing amino acids 7 to 10 of p6, is critical for virus particle production (huang et al. 1995). the ptap motif binds specifi cally to the host cell protein, tumor suppressor gene 101 (tsg101), resulting in the recruitment of components of the endosomal sorting complex required for transport-i (escrt-i) (verplank et al. 2001; martin-serrano et al. 2001; garrus et al. 2001; demirov et al. 2002). deletion of the ptap motif results in approximately 80% reduction in hiv-1 particle release. similarly, overexpression and silencing of tsg101 abolish viral egress (garrus et al. 2001; demirov et al. 2002; goila-gaur et al. 2003). nmr structure analysis of a 14 amino acid peptide derived from p6 encompassing the ptap motif complexed with the uev domain of tsg101 has revealed that the ptap motif binds to a groove in tsg101. this binding creates two main pockets: the “a-p” pocket through contact of amino acids 7–10 and the “p” pocket through the binding of amino acids 9–10 of the peptide (pornillos et al. 175 targeting the late stages of hiv-1 replication drug target insights 2007: 2 2002a; pornillos et al. 2002b). interruption of this viral host protein-protein interaction with compounds that bind to this pocket in tsg101 and compete with gag would potentially inhibit viral particle release and the rate of cell-cell hiv-1 transmission (bieniasz, 2006). so far there has been one report that describes the synthesis and selection of small molecules with up to fi ve fold better binding capacity than a peptide that contains the sequence of the wild type ptap motif in the l domain of hiv-1 (liu et al. 2006). in this study, the authors describe an approach previously employed to obtain peptoids in which a key proline residue is substituted with glycine at the n-terminus of the parent pro-rich sequence in order to improve binding specifi city to src homology 3 (sh3) domains (nguyen et al. 2000). similarly, the proline rich ptap domain in p6 was considered a good candidate for the n-substituted glycine residue approach. in this study, the tsg101 binding affi nity (kd) of the 9-mer wild-type ptap containing peptide was 50 µm (liu et al. 2006). binding constants for the highest affi nity peptoidhydrazones (designated 11q and 11p) were 17.5 and 9.8 µm, respectively. the highest affi nity peptoid hydrazone was found to be the n-butyl-containing 11p peptide, with a fi ve-fold increase in binding affi nity compared to the wild-type 9-mer ptap peptide. the capacity of these peptides to compete with hiv-1 gag for tsg101 binding and their effect on hiv-1 egress in vitro and in vivo remains to be determined. although disruption of viral-host cell interactions is a very attractive approach to abolish virion release and infectivity, interfering with the host cell machinery could have major consequences for the host (bieniasz, 2006). tsg101 is a multifunctional protein and plays a critical role in cell proliferation as shown by studies conducted in tsg101 defi cient mice (ruland et al. 2001). tsg101 is involved in cellular transcription and plays a central role in endosomal sorting of cargo protein that is destined for degradation by the proteasome. tsg101 is recruited to the endosomal sorting pathway by a specifi c interaction with the host cell protein, hepatocyte growth factor-regulated tyrosine kinase substrate (hrs), which binds to tsg101 through a psap motif (lu et al. 2003). disruption of this interaction inhibits delivery of epidermal growth factor receptor (egfr) to the late endosomes. in recruiting tsg101, it is believed that hiv-1 mimics hrs in order to enter the endosomal sorting pathway and negotiate its release from the infected cell (pornillos et al. 2003). thus, targeting tsg101 would result in the accumulation of proteins at the plasma membrane and the disruption of protein sorting within the infected cell. specifi c inhibition of hiv-1 budding by targeting the late domain binding site on tsg101 will require preferential inhibition of p6 binding compared to hrs. conclusion considerable progress has been made in understanding the late steps of the viral life-cycle leading to the production of infectious viral particles. many of these processes rely on protein:protein interactions either between viral proteins or viral proteins and host cell factors. the interactions between the host and viral proteins have either a role in facilitating virus replication, as is observed for the viral p6 and tsg101, or are necessary to overcome negative effects of the host on virus replication, as mediated by vif. hiv-1 also relies on posttranslational modifi cations mediated by the host cell machinery in order for viral polyproteins to be traffi cked to the appropriate compartment of the cell for viral assembly and budding. arguably, one of the most effective drugs used to treat hiv-1 infected individuals are the hiv-1 pr inhibitors that block viral maturation. this underscores the effectiveness of targeting the late stage of virus replication. nevertheless, while some of the strategies described in this review inhibit virus replication in cell culture assays, they require improvements in potency, specifi city and delivery before being viable for use in the clinic. acknowledgements j.w. is supported by an australian postgraduate award, s.s. and m. s-x are supported by funding from the australian centre for hiv and hepatitis virology research and g.t. is supported by funding from nhmrc r.d. wright biomedical career development grant 235102 and nhmrc project grant 381703. johanna wapling and seema srivastava made equal contribution to the review. references abdel-rahman, h.m., al-karamany, g.s., el-koussi, n.a., youssef, a.f. and kiso, y. 2002. hiv protease inhibitors: peptidomimetic drugs and future perspectives. curr. med. chem., 9:1905–1922. accola, m.a., hoglund, s. and gottlinger, h.g. 1998. a putative alphahelical structure which overlaps the capsid-p2 boundary in the human immunodefi ciency virus type 1 gag precursor is crucial for viral particle assembly. j. virol., 72:2072–2078. 176 wapling et al drug target insights 2007: 2 adamson, c.s., ablan, s.d., boeras, i., goila-gaur, r., soheilian, f., nagashima, k., li, f., salzwedel, k., sakalian, m., wild, c.t. and freed, e.o. 2006. in vitro resistance to the human immunodefi ciency virus type 1 maturation inhibitor pa-457 (bevirimat). j. virol., 80:10957–10971. aldovini, a. and young, r.a. 1990. mutations of rna and protein sequences involved in human immunodeficiency virus type 1 packaging result in production of noninfectious virus. j. virol., 64:1920–1926. andersson, e., horal, p., jejcic, a., hoglund, s., balzarini, j., vahlne, a. and svennerholm, b. 2005. glycine-amide is an active metabolite of the antiretroviral tripeptide glycyl-prolyl-glycine-amide. antimicrob. agents chemother., 49:40–44. andersson, e., horal, p., vahlne, a. and svennerholm, b. 2004. no crossresistance or selection of hiv-1 resistant mutants in vitro to the antiretroviral tripeptide glycyl-prolyl-glycine-amide. antiviral res., 61:119–124. arion, d., sluis-cremer, n., min, k.l., abram, m.e., fletcher, r.s. and parniak, m.a. 2002. mutational analysis of tyr-501 of hiv-1 reverse transcriptase. effects on ribonuclease h activity and inhibition of this activity by n-acylhydrazones. j. biol. chem., 277:1370–1374. ariumi, y., serhan, f., turelli, p., telenti, a. and trono, d. 2006. the integrase interactor 1 (ini1) proteins facilitate tat-mediated human immunodefi ciency virus type 1 transcription. retrovirology, 3:47. babe, l.m. and craik, c.s. 1991. time dependent heterodimer formation leads to inhibition of hiv protease activity. adv. exp. med. biol., 306:543–547. babe, l.m., pichuantes, s. and craik, c.s. 1991. inhibition of hiv protease activity by heterodimer formation. biochemistry, 30:106–111. babe, l.m., rose, j. and craik, c.s. 1992. synthetic “interface” peptides alter dimeric assembly of the hiv 1 and 2 proteases. protein sci., 1:1244–1253. babe, l.m., rose, j. and craik, c.s. 1995. trans-dominant inhibitory human immunodefi ciency virus type 1 protease monomers prevent protease activation and virion maturation. proc. natl. acad. sci. u.s.a., 92:10069–10073. balzarini, j., karlsson, a., vandamme, a.m., perez-perez, m.j., zhang, h., vrang, l., oberg, b., backbro, k., unge, t., san-felix, a. et al. 1993. human immunodefi ciency virus type 1 (hiv-1) strains selected for resistance against the hiv-1-specific [2′,5′-bis-o-(tertbutyldimethylsilyl)-3′-spiro-5″-(4″-amino-1″,2″-oxathiole2″,2″-dioxide)]-beta-d-pentofurano syl (tsao) nucleoside analogues retain sensitivity to hiv-1-specifi c nonnucleoside inhibitors. proc. natl. acad. sci. u.s.a., 90:6952–6956. bannwarth, l., kessler, a., pethe, s., collinet, b., merabet, n., boggetto, n., sicsic, s., reboud-ravaux, m. and ongeri, s. 2006. molecular tongs containing amino acid mimetic fragments: new inhibitors of wildtype and mutated hiv-1 protease dimerization. j. med. chem., 49:4657–4664. bieniasz, p.d. 2006. late budding domains and host proteins in enveloped virus release. virology, 344:55–63. binette, j. and cohen, e.a. 2004. recent advances in the understanding of hiv-1 vpu accessory protein functions. curr. drug. targets immune endocr. metabol. disord., 4:297–307. bouamr, f., scarlata, s. and carter, c. 2003. role of myristylation in hiv-1 gag assembly. biochemistry, 42:6408–6417. bour, s. and strebel, k. 2003. the hiv-1 vpu protein: a multifunctional enhancer of viral particle release. microbes infect., 5:1029–1039. bouras, a., boggetto, n., benatalah, z., de rosny, e., sicsic, s. and reboudravaux, m. 1999. design, synthesis, and evaluation of conformationally constrained tongs, new inhibitors of hiv-1 protease dimerization. j. med. chem., 42:957–962. breccia, p., boggetto, n., perez-fernandez, r., van gool, m., takahashi, m., rene, l., prados, p., badet, b., reboud-ravaux, m. and de mendoza, j. 2003. dimerization inhibitors of hiv-1 protease based on a bicyclic guanidinium subunit. j. med. chem., 46:5196–5207. brierley, i. and dos ramos, f.j. 2006. programmed ribosomal frameshifting in hiv-1 and the sars-cov. virus res., 119:29–42. broglia, r.a., provasi, d., vasile, f., ottolina, g., longhi, r. and tiana, g. 2006. a folding inhibitor of the hiv-1 protease. proteins, 62:928–933. broglia, r.a., tiana, g., sutto, l., provasi, d. and simona, f. 2005. design of hiv-1-pr inhibitors that do not create resistance: blocking the folding of single monomers. protein sci., 14:2668–2681. bryant, m. ratner, l. 1990. myristoylation-dependent replication and assembly of human immunodefi ciency virus 1. proc. natl. acad. sci. u.s.a, 87:523–527. bryant, m.l., heuckeroth, r.o., kimata, j.t., ratner, l. and gordon, j.i. 1989. replication of human immunodefi ciency virus 1 and moloney murine leukemia virus is inhibited by different heteroatom-containing analogs of myristic acid. proc. natl. acad sci. u.s.a., 86:8655– 8659. bryant, m.l., ratner, l., duronio, r.j., kishore, n.s., devadas, b., adams, s.p. and gordon, j.i. 1991. incorporation of 12-methoxydodecanoate into the human immunodefi ciency virus 1 gag polyprotein precursor inhibits its proteolytic processing and virus production in a chronically infected human lymphoid cell line. proc. natl. acad. sci. u.s.a., 88:2055–2059. bukovsky, a. and gottlinger, h. 1996. lack of integrase can markedly affect human immunodefi ciency virus type 1 particle production in the presence of an active viral protease. j. virol., 70:6820–6825. cafl isch, a., schramm, h.j. and karplus, m. 2000. design of dimerization inhibitors of hiv-1 aspartic proteinase: a computer-based combinatorial approach. j. comput. aided mol. des., 14:161–179. callahan, m.a., handley, m.a., lee, y.h., talbot, k.j., harper, j.w. and panganiban, a.t. 1998. functional interaction of human immunodefi ciency virus type 1 vpu and gag with a novel member of the tetratricopeptide repeat protein family. j. virol., 72:5189–5197. camarasa, m.j., velazquez, s., san-felix, a., perez-perez, m.j., bonache, m.c. and de castro, s. 2006. tsao derivatives, inhibitors of hiv-1 reverse transcriptase dimerization: recent progress. curr. pharm. des., 12:1895–1907. campbell, s. and vogt, v.m. 1995. self-assembly in vitro of purifi ed ca-nc proteins from rous sarcoma virus and human immunodefi ciency virus type 1. j. virol., 69:6487–6497. campbell, w., kleiman, l., barany, l., li, z., khorchid, a., fujita, e., okada, n. and okada, h. 2002. a novel genetic algorithm for designing mimetic peptides that interfere with the function of a target molecule. microbiol. immunol., 46:211–215. cartas, m., singh, s.p., serio, d., rizvi, t.a., kalyanaraman, v.s., goldsmith, c.s., zaki, s.r., weber, i.t. and srinivasan, a. 2001. intravirion display of a peptide corresponding to the dimer structure of protease attenuates hiv-1 replication. dna cell biol., 20:797–805. cen, s., niu, m., saadatmand, j., guo, f., huang, y., nabel, g.j. and kleiman, l. 2004. incorporation of pol into human immunodefi ciency virus type 1 gag virus-like particles occurs independently of the upstream gag domain in gag-pol. j. virol., 78:1042–1049. cheng, y.s., yin, f.h., foundling, s., blomstrom, d. and kettner, c.a. 1990. stability and activity of human immunodefi ciency virus protease: comparison of the natural dimer with a homologous, singlechain tethered dimer. proc. natl. acad. sci. u.s.a., 87:9660–9664. cherry, e., liang, c., rong, l., quan, y., inouye, p., li, x., morin, n., kotler, m. and wainberg, m.a. 1998. characterization of human immunodefi ciency virus type-1 (hiv-1) particles that express protease-reverse transcriptase fusion proteins. j. mol. biol., 284: 43–56. chien, a.i., liao, w.h., yang, d.m. and wang, c.t. 2006. a domain directly c-terminal to the major homology region of human immunodefi ciency t-ype 1 capsid protein plays a crucial role in directing both virus assembly and incorporation of gag-pol. virology, 348:84–95. chiu, h.c., yao, s.y. and wang, c.t. 2002. coding sequences upstream of the human immunodefi ciency virus type 1 reverse transcriptase domain in gag-pol are not essential for incorporation of the pr160(gag-pol) into virus particles. j. virol., 76:3221–3231. clavel, f. and hance, a.j. 2004. hiv drug resistance. n. engl. j. med., 350: 1023–1035. 177 targeting the late stages of hiv-1 replication drug target insights 2007: 2 cotelle, p. 2006. patented hiv-1 integrase inhibitors (1998–2005). recent patents on anti-infective drug discovery, 1:1–15. davis, d.a., brown, c.a., singer, k.e., wang, v., kaufman, j., stahl, s.j., wingfi eld, p., maeda, k., harada, s., yoshimura, k., et al. 2006. inhibition of hiv-1 replication by a peptide dimerization inhibitor of hiv-1 protease. antiviral res., 72:89–99. de clercq, e. 1998. the role of non-nucleoside reverse transcriptase inhibitors (nnrtis) in the therapy of hiv-1 infection. antiviral res., 38:153–179. debyser, z. and de clercq, e. 1996. chemical crosslinking of the subunits of hiv-1 reverse transcriptase. protein sci., 5:278–286. demirov, d.g., ono, a., orenstein, j.m. and freed, e.o. 2002. overexpression of the n-terminal domain of tsg101 inhibits hiv-1 budding by blocking late domain function. proc. natl. acad. sci. u.s.a., 99: 955–960. deora, a. and ratner, l. 2001. viral protein u (vpu)-mediated enhancement of human immunodefi ciency virus type 1 particle release depends on the rate of cellular proliferation. j. virol., 75:6714–6718. depollier, j., hourdou, m.l., aldrian-herrada, g., rothwell, p., restle, t. and divita, g. 2005. insight into the mechanism of a peptide inhibitor of hiv reverse transcriptase dimerization. biochemistry, 44: 1909–1918. dettenhofer, m. and yu, x.f. 1999. proline residues in human immunodefi ciency virus type 1 p6(gag) exert a cell type-dependent effect on viral replication and virion incorporation of pol proteins. j. virol., 73:4696–4704. devadas, b., lu, t., katoh, a., kishore, n.s., wade, a.c., mehta, p.p., rudnick, d.a., bryant, m.l., adams, s.p., li, q. et al. 1992. substrate specifi city of saccharomyces cerevisiae myristoyl-coa: protein n-myristoyltransferase. analysis of fatty acid analogs containing carbonyl groups, nitrogen heteroatoms, and nitrogen heterocycles in an in vitro enzyme assay and subsequent identifi cation of inhibitors of human immunodefi ciency virus i replication. j. biol. chem., 267: 7224–7239. ding, l., derdowski, a., wang, j.j. and spearman, p. 2003. independent segregation of human immunodefi ciency virus type 1 gag protein complexes and lipid rafts. j. virol., 77:1916–1926. divita, g., restle, t., goody, r.s., chermann, j.c. and baillon, j.g. 1994. inhibition of human immunodefi ciency virus type 1 reverse transcriptase dimerization using synthetic peptides derived from the connection domain. j. biol. chem., 269:13080–13083. dorfman, t., bukovsky, a., ohagen, a., hoglund, s. and gottlinger, h.g. 1994. functional domains of the capsid protein of human immunodefi ciency virus type 1. j. virol., 68:8180–8187. dulude, d., berchiche, y.a., gendron, k., brakier-gingras, l. and heveker, n. 2006. decreasing the frameshift effi ciency translates into an equivalent reduction of the replication of the human immunodefi ciency virus type 1. virology, 345:127–136. dumond, j., boggetto, n., schramm, h.j., schramm, w., takahashi, m. and reboud-ravaux, m. 2003. thyroxine-derivatives of lipopeptides: bifunctional dimerization inhibitors of human immunodefi ciency virus-1 protease. biochem. pharmacol., 65:1097–1102. engelman, a., englund, g., orenstein, j.m., martin, m.a. and craigie, r. 1995. multiple effects of mutations in human immunodefi ciency virus type 1 integrase on viral replication. j. virol., 69:2729–2736. ewart, g.d., mills, k., cox, g.b. and gage, p.w. 2002. amiloride derivatives block ion channel activity and enhancement of virus-like particle budding caused by hiv-1 protein vpu. eur. biophys. j., 31: 26–35. ewart, g.d., nasr, n., naif, h., cox, g.b., cunningham, a.l. and gage, p.w. 2004. potential new anti-human immunodefi ciency virus type 1 compounds depress virus replication in cultured human macrophages. antimicrob. agents chemother., 48:2325–2330. facke, m., janetzko, a., shoeman, r.l. and krausslich, h.g. 1993. a large deletion in the matrix domain of the human immunodefi ciency virus gag gene redirects virus particle assembly from the plasma membrane to the endoplasmic reticulum. j. virol., 67:4972–4980. fan, x., flentke, g.f. and rich, d.h. 1998. inhibition of hiv-1 protease by a subunit of didemnaketal a. journal of the american chemical society, 120:8893–8894. figueiredo, a., moore, k.l., mak, j., sluis-cremer, n., de bethune, m.p. and tachedjian, g. 2006. potent nonnucleoside reverse transcriptase inhibitors target hiv-1 gag-pol. plos pathog., 2: e119. franciskovich, j., houseman, k., meuller, r. and chmielewski, j. 1993. a systematic evaluation of the inhibition of hiv-1 protease by its cand n-terminal peptides. bioorg. med. chem. lett., 765–768. freed, e.o. 1998. hiv-1 gag proteins: diverse functions in the virus life cycle. virology, 251:1–15. gamble, t.r., vajdos, f.f., yoo, s., worthylake, d.k., houseweart, m., sundquist, w.i. and hill, c.p. 1996. crystal structure of human cyclophilin a bound to the amino-terminal domain of hiv-1 capsid. cell, 87:1285–1294. gamble, t.r., yoo, s., vajdos, f.f., von schwedler, u.k., worthylake, d.k., wang, h., mccutcheon, j.p., sundquist, w.i. and hill, c.p. 1997. structure of the carboxyl-terminal dimerization domain of the hiv1 capsid protein. science, 278:849–853. ganser-pornillos, b.k., von schwedler, u.k., stray, k.m., aiken, c. and sundquist, w.i. 2004. assembly properties of the human immunodefi ciency virus type 1 ca protein. j. virol., 78:2545–2552. garrus, j.e., von schwedler, u.k., pornillos, o.w., morham, s.g., zavitz, k.h., wang, h.e., wettstein, d.a., stray, k.m., cote, m., rich, r.l., myszka, d.g. and sundquist, w.i. 2001. tsg101 and the vacuolar protein sorting pathway are essential for hiv-1 budding. cell, 107: 55–65. gitti, r.k., lee, b.m., walker, j., summers, m.f., yoo, s. and sundquist, w.i. 1996. structure of the amino-terminal core domain of the hiv1 capsid protein. science, 273:231–235. goila-gaur, r., demirov, d.g., orenstein, j.m., ono, a. and freed, e.o. 2003. defects in human immunodefi ciency virus budding and endosomal sorting induced by tsg101 overexpression. j. virol., 77: 6507–6519. gonzalez, m.e. and carrasco, l. 2003. viroporins. febs lett., 552:28–34. gottlinger, h.g., dorfman, t., sodroski, j.g. and haseltine, w.a. 1991. effect of mutations affecting the p6 gag protein on human immunodefi ciency virus particle release. proc. natl. acad. sci. u.s.a., 88: 3195–3199. gottlinger, h.g., sodroski, j.g. and haseltine, w.a. 1989. role of capsid precursor processing and myristoylation in morphogenesis and infectivity of human immunodefi ciency virus type 1. proc. natl. acad. sci. u.s.a., 86:5781–5785. grice, a.l., kerr, i.d. and sansom, m.s. 1997. ion channels formed by hiv-1 vpu: a modelling and simulation study. febs lett., 405: 299–304. grinsztejn, b., nguyen, b.y., katlama, c., gatell, j.m., lazzarin, a., vittecoq, d., gonzalez, c.j., chen, j., harvey, c.m. and isaacs, r.d. 2007. safety and effi cacy of the hiv-1 integrase inhibitor raltegravir (mk-0518) in treatment-experienced patients with multidrug-resistant virus: a phase ii randomised controlled trial. lancet, 369:1261–1269. gustchina, a. and weber, i.t. 1991. comparative analysis of the sequences and structures of hiv-1 and hiv-2 proteases. proteins, 10:325– 339. handley, m.a., paddock, s., dall, a. and panganiban, a.t. 2001. association of vpu-binding protein with microtubules and vpu-dependent redistribution of hiv-1 gag protein. virology, 291:198–207. harila, k., prior, i., sjoberg, m., salminen, a., hinkula, j. and suomalainen, m. 2006. vpu and tsg101 regulate intracellular targeting of the human immunodefi ciency virus type 1 core protein precursor pr55gag. j. virol., 80:3765–3772. hehl, e.a., joshi, p., kalpana, g.v. and prasad, v.r. 2004. interaction between human immunodefi ciency virus type 1 reverse transcriptase and integrase proteins. j. virol., 78:5056–5067. hill, m., tachedjian, g. and mak, j. 2005. the packaging and maturation of the hiv-1 pol proteins. curr. hiv. res., 3:73–85. 178 wapling et al drug target insights 2007: 2 himmel, d.m., sarafi anos, s.g., dharmasena, s., hossain, m.m., mccoysimandle, k., ilina, t., clark, a.d., jr., knight, j.l., julias, j.g., clark, p.k. et al. 2006. hiv-1 reverse transcriptase structure with rnase h inhibitor dihydroxy benzoyl naphthyl hydrazone bound at a novel site. acs chem. biol., 1:702–712. hoglund, s., su, j., reneby, s.s., vegvari, a., hjerten, s., sintorn, i.m., foster, h., wu, y.p., nystrom, i. and vahlne, a. 2002. tripeptide interference with human immunodefi ciency virus type 1 morphogenesis. antimicrob agents chemother., 46:3597–3605. holm, k., weclewicz, k., hewson, r. and suomalainen, m. 2003. human immunodefi ciency virus type 1 assembly and lipid rafts: pr55(gag) associates with membrane domains that are largely resistant to brij98 but sensitive to triton x-100. j. virol., 77:4805–4817. hosokawa, m., imai, m., okada, h. and okada, n. 2004. inhibition of hiv-1 infection in cells expressing an artifi cial complementary peptide. biochem. biophys. res. commun., 324:236–240. hout, d.r., gomez, l.m., pacyniak, e., miller, j.m., hill, m.s. and stephens, e.b. 2006. a single amino acid substitution within the transmembrane domain of the human immunodefi ciency virus type 1 vpu protein renders simian-human immunodefi ciency virus (shiv(ku-1bmc33)) susceptible to rimantadine. virology, 348:449–461. hsu, k., seharaseyon, j., dong, p., bour, s. and marban, e. 2004. mutual functional destruction of hiv-1 vpu and host task-1 channel. mol. cell, 14:259–267. huang, m., orenstein, j.m., martin, m.a. and freed, e.o. 1995. p6gag is required for particle production from full-length human immunodefi ciency virus type 1 molecular clones expressing protease. j. virol., 69:6810–6818. hung, m., patel, p., davis, s. and green, s.r. 1998. importance of ribosomal frameshifting for human immunodefi ciency virus type 1 particle assembly and replication. j. virol., 72:4819–4824. jacks, t., madhani, h.d., masiarz, f.r. and varmus, h.e. 1988a. signals for ribosomal frameshifting in the rous sarcoma virus gag-pol region. cell, 55:447–458. jacks, t., power, m.d., masiarz, f.r., luciw, p.a., barr, p.j. and varmus, h.e. 1988b. characterization of ribosomal frameshifting in hiv-1 gag-pol expression. nature, 331:280–283. jacks, t., townsley, k., varmus, and h.e. majors, j. 1987. two effi cient ribosomal frameshifting events are required for synthesis of mouse mammary tumor virus gag-related polyproteins. proc. natl. acad. sci. u.s.a., 84:4298–4302. jacks, t. and varmus, h.e. 1985. expression of the rous sarcoma virus pol gene by ribosomal frameshifting. science, 230:1237–1242. jacobo-molina, a., ding, j., nanni, r.g., clark, a.d., jr., lu, x., tantillo, c., williams, r.l., kamer, g., ferris, a.l., clark, p. et al. 1993. crystal structure of human immunodefi ciency virus type 1 reverse transcriptase complexed with double-stranded dna at 3.0 a resolution shows bent dna. proc. natl. acad. sci. u.s.a., 90:6320–6324. junker, u., escaich, s., plavec, i., baker, j., mcphee, f., rose, j.r., craik, c.s. and bohnlein, e. 1996. intracellular expression of human immunodefi ciency virus type 1 (hiv-1) protease variants inhibits replication of wild-type and protease inhibitor-resistant hiv-1 strains in human t-cell lines. j. virol., 70:7765–7772. kalpana, g.v., marmon, s., wang, w., crabtree, g.r. and goff, s.p. 1994. binding and stimulation of hiv-1 integrase by a human homolog of yeast transcription factor snf5. science, 266:2002–2006. kane, r.c., farrell, a.t., sridhara, r. and pazdur, r. 2006. united states food and drug administration approval summary: bortezomib for the treatment of progressive multiple myeloma after one prior therapy. clin. cancer res., 12:2955–2960. kaplan, a.h., manchester, m. and swanstrom, r. 1994. the activity of the protease of human immunodefi ciency virus type 1 is initiated at the membrane of infected cells before the release of viral proteins and is required for release to occur with maximum effi ciency. j. virol., 68:6782–6786. kaplan, a.h., zack, j.a., knigge, m., paul, d.a., kempf, d.j., norbeck, d.w. and swanstrom, r. 1993. partial inhibition of the human immunodefi ciency virus type 1 protease results in aberrant virus assembly and the formation of noninfectious particles. j. virol., 67: 4050–4055. karacostas, v., wolffe, e.j., nagashima, k., gonda, m.a. and moss, b. 1993. overexpression of the hiv-1 gag-pol polyprotein results in intracellular activation of hiv-1 protease and inhibition of assembly and budding of virus-like particles. virology, 193:661–671. klimkait, t., strebel, k., hoggan, m.d., martin, m.a. and orenstein, j.m. 1990. the human immunodefi ciency virus type 1-specifi c protein vpu is required for effi cient virus maturation and release. j. virol., 64:621–629. kohl, n.e., emini, e.a., schleif, w.a., davis, l.j., heimbach, j.c., dixon, r.a., scolnick, e.m. and sigal, i.s. 1988. active human immunodefi ciency virus protease is required for viral infectivity. proc. natl. acad. sci. u.s.a., 85:4686–4690. kohlstaedt, l.a., wang, j., friedman, j.m., rice, p.a. and steitz, t.a. 1992. crystal structure at 3.5 a resolution of hiv-1 reverse transcriptase complexed with an inhibitor. science, 256:1783–1790. kollmus, h., honigman, a., panet, a. and hauser, h. 1994. the sequences of and distance between two cis-acting signals determine the effi ciency of ribosomal frameshifting in human immunodefi ciency virus type 1 and human t-cell leukemia virus type ii in vivo. j. virol., 68:6087–6091. krausslich, h.g. 1991. human immunodefi ciency virus proteinase dimer as component of the viral polyprotein prevents particle assembly and viral infectivity. proc. natl. acad. sci. u.s.a., 88:3213–3217. langner, c.a., lodge, j.k., travis, s.j., caldwell, j.e., lu, t., li, q., bryant, m.l., devadas, b., gokel, g.w., kobayashi, g.s. et al. 1992. 4-oxatetradecanoic acid is fungicidal for cryptococcus neoformans and inhibits replication of human immunodefi ciency virus i. j. biol. chem., 267:17159–17169. lanman, j., lam, t.t., barnes, s., sakalian, m., emmett, m.r., marshall, a.g. and prevelige, p.e., jr. 2003. identifi cation of novel interactions in hiv-1 capsid protein assembly by high-resolution mass spectrometry. j. mol. biol., 325:759–772. lapadat-tapolsky, m., de rocquigny, h., van gent, d., roques, b., plasterk, r. and darlix, j.l. 1993. interactions between hiv-1 nucleocapsid protein and viral dna may have important functions in the viral life cycle. nucleic acids res., 21:831–839. li, f., goila-gaur, r., salzwedel, k., kilgore, n.r., reddick, m., matallana, c., castillo, a., zoumplis, d., martin, d.e., orenstein, j.m., allaway, g.p., freed, e.o. and wild, c.t. 2003. pa-457: a potent hiv inhibitor that disrupts core condensation by targeting a late step in gag processing. proc. natl. acad. sci. u.s.a., 100:13555–13560. li, l., li, h.s., pauza, c.d., bukrinsky, m. and zhao, r.y. 2005. roles of hiv-1 auxiliary proteins in viral pathogenesis and host-pathogen interactions. cell res., 15:923–934. li, s., hill, c.p., sundquist, w.i. and finch, j.t. 2000. image reconstructions of helical assemblies of the hiv-1 ca protein. nature, 407:409–413. liao, w.h. and wang, c.t. 2004. characterization of human immunodefi ciency virus type 1 pr160 gag-pol mutants with truncations downstream of the protease domain. virology, 329:180–188. lindwasser, o.w. and resh, m.d. 2002. myristoylation as a target for inhibiting hiv assembly: unsaturated fatty acids block viral budding. proc. natl. acad. sci. u.s.a., 99:13037–13042. liu, f., stephen, a.g., adamson, c.s., gousset, k., aman, m.j., freed, e.o., fisher, r.j. and burke, t.r., jr. 2006. hydrazoneand hydrazidecontaining n-substituted glycines as peptoid surrogates for expedited library synthesis: application to the preparation of tsg101-directed hiv-1 budding antagonists. org. lett., 8:5165–5168. louis, j.m., clore, g.m. and gronenborn, a.m. 1999. autoprocessing of hiv-1 protease is tightly coupled to protein folding. nat. struct. biol., 6:868–875. lu, q., hope, l.w., brasch, m., reinhard, c. and cohen, s.n. 2003. tsg101 interaction with hrs mediates endosomal traffi cking and receptor down-regulation. proc. natl. acad. sci. u.s.a, 100:7626–7631. makhija, m.t. 2006. designing hiv integrase inhibitors--shooting the last arrow. curr. med. chem., 13:2429–2441. mammano, f., ohagen, a., hoglund, s. and gottlinger, h.g. 1994. role of the major homology region of human immunodefi ciency virus type 1 in virion morphogenesis. j. virol., 68:4927–4936. 179 targeting the late stages of hiv-1 replication drug target insights 2007: 2 manfredi, r. and sabbatani, s. 2006. a novel antiretroviral class (fusion inhibitors) in the management of hiv infection. present features and future perspectives of enfuvirtide (t-20). curr. med. chem., 13: 2369–2384. maroun, r.g., gayet, s., benleulmi, m.s., porumb, h., zargarian, l., merad, h., leh, h., mouscadet, j.f., troalen, f. and fermandjian, s. 2001. peptide inhibitors of hiv-1 integrase dissociate the enzyme oligomers. biochemistry, 40:13840–13848. martin-serrano, j., zang, t. and bieniasz, p.d. 2001. hiv-1 and ebola virus encode small peptide motifs that recruit tsg101 to sites of particle assembly to facilitate egress. nat. med., 7:1313–1319. mcphee, f., good, a.c., kuntz, i.d. and craik, c.s. 1996. engineering human immunodefi ciency virus 1 protease heterodimers as macromolecular inhibitors of viral maturation. proc. natl. acad. sci. u.s.a., 93:11477–11481. mehle, a., strack, b., ancuta, p., zhang, c., mcpike, m. and gabuzda, d. 2004. vif overcomes the innate antiviral activity of apobec3g by promoting its degradation in the ubiquitin-proteasome pathway. j. biol. chem., 279:7792–7798. meng, l., mohan, r., kwok, b.h., elofsson, m., sin, n. and crews, c.m. 1999. epoxomicin, a potent and selective proteasome inhibitor, exhibits in vivo antiinfl ammatory activity. proc. natl. acad. sci. u.s.a., 96:10403–10408. merabet, n., dumond, j., collinet, b., van baelinghem, l., boggetto, n., ongeri, s., ressad, f., reboud-ravaux, m. and sicsic, s. 2004. new constrained “molecular tongs” designed to dissociate hiv-1 protease dimer. j. med. chem., 47:6392–6400. momany, c., kovari, l.c., prongay, a.j., keller, w., gitti, r.k., lee, b.m., gorbalenya, a.e., tong, l., mcclure, j., ehrlich, l.s., summers, m.f., carter, c. and rossmann, m.g. 1996. crystal structure of dimeric hiv-1 capsid protein. nat. struct. biol., 3:763–770. morikawa, y., hinata, s., tomoda, h., goto, t., nakai, m., aizawa, c., tanaka, h. and omura, s. 1996. complete inhibition of human immunodefi ciency virus gag myristoylation is necessary for inhibition of particle budding. j. biol. chem., 271:2868–2873. morris, m.c., berducou, c., mery, j., heitz, f. and divita, g. 1999a. the thumb domain of the p51-subunit is essential for activation of hiv reverse transcriptase. biochemistry, 38:15097–15103. morris, m.c., robert-hebmann, v., chaloin, l., mery, j., heitz, f., devaux, c., goody, r.s. and divita, g. 1999b. a new potent hiv-1 reverse transcriptase inhibitor. a synthetic peptide derived from the interface subunit domains. j. biol. chem., 274:24941–24946. muriaux, d., darlix, j.l. and cimarelli, a. 2004. targeting the assembly of the human immunodefi ciency virus type i. curr. pharm. des., 10:3725–3739. neil, s.j., eastman, s.w., jouvenet, n. and bieniasz, p.d. 2006. hiv-1 vpu promotes release and prevents endocytosis of nascent retrovirus particles from the plasma membrane. plos pathog., 2:e39. nguyen, j.t., porter, m., amoui, m., miller, w.t., zuckermann, r.n. and lim, w.a. 2000. improving sh3 domain ligand selectivity using a non-natural scaffold. chem. biol., 7:463–473. niedrig, m., gelderblom, h.r., pauli, g., marz, j., bickhard, h., wolf, h. and modrow, s. 1994. inhibition of infectious human immunodefi ciency virus type 1 particle formation by gag protein-derived peptides. j. gen. virol., 75:1469–1474. ono, a., ablan, s.d., lockett, s.j., nagashima, k. and freed, e.o. 2004. phosphatidylinositol (4,5) bisphosphate regulates hiv-1 gag targeting to the plasma membrane. proc. natl. acad. sci. u.s.a., 101:14889–14894. ono, a. and freed, e.o. 1999. binding of human immunodefi ciency virus type 1 gag to membrane: role of the matrix amino terminus. j. virol., 73:4136–4144. ono, a., orenstein, j.m. and freed, e.o. 2000. role of the gag matrix domain in targeting human immunodefi ciency virus type 1 assembly. j. virol., 74:2855–2866. ott, d.e., coren, l.v., sowder, r.c., adams, j. and schubert, u. 2003. retroviruses have differing requirements for proteasome function in the budding process. j. virol., 77:3384–3393. palella, f.j., jr., delaney, k.m., moorman, a.c., loveless, m.o., fuhrer, j., satten, g.a., aschman, d.j. and holmberg, s.d. 1998. declining morbidity and mortality among patients with advanced human immunodefi ciency virus infection. hiv outpatient study investigators. n. engl. j. med., 338:853–860. parang, k., wiebe, l.i., knaus, e.e., huang, j.s., tyrrell, d.l. and csizmadia, f. 1997. in vitro antiviral activities of myristic acid analogs against human immunodefi ciency and hepatitis b viruses. antiviral res., 34:75–90. park, j. and morrow, c.d. 1991. overexpression of the gag-pol precursor from human immunodefi ciency virus type 1 proviral genomes results in effi cient proteolytic processing in the absence of virion production. j. virol., 65:5111–5117. park, j. and morrow, c.d. 1992. the nonmyristylated pr160gag-pol polyprotein of human immunodefi ciency virus type 1 interacts with pr55gag and is incorporated into virus like particles. j. virol., 66: 6304–6313. park, s.h. and raines, r.t. 2000. genetic selection for dissociative inhibitors of designated protein-protein interactions. nat. biotechnol., 18:847–851. partin, k., zybarth, g., ehrlich, l., decrombrugghe, m., wimmer, e. and carter, c. 1991. deletion of sequences upstream of the proteinase improves the proteolytic processing of human immunodefi ciency virus type 1. proc. natl. acad. sci. u.s.a., 88:4776–4780. pettit, s.c., lindquist, j.n., kaplan, a.h. and swanstrom, r. 2005. processing sites in the human immunodefi ciency virus type 1 (hiv-1) gag-pro-pol precursor are cleaved by the viral protease at different rates. retrovirology, 2:66. pettit, s.c., moody, m.d., wehbie, r.s., kaplan, a.h., nantermet, p.v., klein, c.a. and swanstrom, r. 1994. the p2 domain of human immunodefi ciency virus type 1 gag regulates sequential proteolytic processing and is required to produce fully infectious virions. j. virol., 68:8017–8027. pettit, s.c., sheng, n., tritch, r., erickson-viitanen, s. and swanstrom, r. 1998. the regulation of sequential processing of hiv-1 gag by the viral protease. adv. exp. med. biol., 436:15–25. piacenti, f.j. 2006. an update and review of antiretroviral therapy. pharmacotherapy, 26:1111–1133. pidgeon, c., markovich, r.j., liu, m.d., holzer, t.j., novak, r.m. and keyer, k.a. 1993. antiviral phospholipids. anti-hiv drugs conjugated to the glycerobackbone of phospholipids. j. biol. chem., 268: 7773–7778. pornillos, o., alam, s.l., davis, d.r. and sundquist, w.i. 2002a. structure of the tsg101 uev domain in complex with the ptap motif of the hiv-1 p6 protein. nat. struct. biol., 9:812–817. pornillos, o., alam, s.l., rich, r.l., myszka, d.g., davis, d.r. and sundquist, w.i. 2002b. structure and functional interactions of the tsg101 uev domain. embo j., 21:2397–2406. pornillos, o., higginson, d.s., stray, k.m., fisher, r.d., garrus, j.e., payne, m., he, g.p., wang, h.e., morham, s.g. and sundquist, w.i. 2003. hiv gag mimics the tsg101-recruiting activity of the human hrs protein. j. cell biol., 162:425–434. potts, b.c.m., faulkner, d.j., chan, j.a., simolike, g.c., offen, p., hemling, m.e. and francis, t.e. 1991. didemnaketal a and b, hiv-1 protease inhibitors from the accidian didemnum sp. journal of the american chemical society, 113:6321–6322. quere, l., wenger, t. and schramm, h.j. 1996. triterpenes as potential dimerization inhibitors of hiv-1 protease. biochem. biophys. res. commun., 227:484–488. quillent, c., borman, a.m., paulous, s., dauguet, c. and clavel, f. 1996. extensive regions of pol are required for effi cient human immunodefi ciency virus polyprotein processing and particle maturation. virology, 219:29–36. rabson, a.b. and graves, b.j. 1997. synthesis and processing of viral rna. in coffi n, j.m., hughes, s. h. & varmus, h.e. ed. retroviruses. new york, cold spring harbor laboratory press. reicin, a.s., ohagen, a., yin, l., hoglund, s. and goff, s.p. 1996. the role of gag in human immunodefi ciency virus type 1 virion morphogenesis and early steps of the viral life cycle. j. virol., 70:8645–8652. 180 wapling et al drug target insights 2007: 2 rice, w.g., baker, d.c., schaeffer, c.a., graham, l., bu, m., terpening, s., clanton, d., schultz, r., bader, j.p., buckheit, r.w., jr., field, l., singh, p.k. and turpin, j.a. 1997. inhibition of multiple phases of human immunodefi ciency virus type 1 replication by a dithiane compound that attacks the conserved zinc fingers of retroviral nucleocapsid proteins. antimicrob. agents chemother., 41:419–426. rice, w.g., supko, j.g., malspeis, l., buckheit, r.w., jr., clanton, d., bu, m., graham, l., schaeffer, c.a., turpin, j.a., domagala, j., gogliotti, r., bader, j.p., halliday, s.m., coren, l., sowder, r.c., arthur, l.o. and henderson, l.e. 1995. inhibitors of hiv nucleocapsid protein zinc fingers as candidates for the treatment of aids. science, 270:1194–1197. rodriguez-barrios, f., perez, c., lobaton, e., velazquez, s., chamorro, c., san-felix, a., perez-perez, m.j., camarasa, m.j., pelemans, h., balzarini, j. and gago, f. 2001. identifi cation of a putative binding site for [2′,5′-bis-o-(tert-butyldimethylsilyl)-beta-d-ribofuranosyl]3′-spiro-5″-(4″-amino-1″,2″-oxathiole-2″,2″-dioxide)thymine (tsao) derivatives at the p51-p66 interface of hiv-1 reverse transcriptase. j. med. chem., 44:1853–1865. rodriguez-rodriguez, l., tsuchihashi, z., fuentes, g.m., bambara, r.a. and fay, p.j. 1995. infl uence of human immunodefi ciency virus nucleocapsid protein on synthesis and strand transfer by the reverse transcriptase in vitro. j. biol. chem., 270:15005–15011. rozzelle, j.e., dauber, d.s., todd, s., kelley, r. and craik, c.s. 2000. macromolecular inhibitors of hiv-1 protease. characterization of designed heterodimers. j. biol. chem., 275:7080–7086. ruland, j., sirard, c., elia, a., macpherson, d., wakeham, a., li, l., de la pompa, j.l., cohen, s.n. and mak, t.w. 2001. p53 accumulation, defective cell proliferation, and early embryonic lethality in mice lacking tsg101. proc. natl. acad. sci. u.s.a., 98:1859–1864. saad, j.s., miller, j., tai, j., kim, a., ghanam, r.h. and summers, m.f. 2006. structural basis for targeting hiv-1 gag proteins to the plasma membrane for virus assembly. proc. natl. acad. sci. u.s.a., 103:11364–11369. sakalian, m., mcmurtrey, c.p., deeg, f.j., maloy, c.w., li, f., wild, c.t. and salzwedel, k. 2006. 3-o-(3′,3′-dimethysuccinyl) betulinic acid inhibits maturation of the human immunodefi ciency virus type 1 gag precursor assembled in vitro. j. virol., 80:5716–5722. schito, m.l., goel, a., song, y., inman, j.k., fattah, r.j., rice, w.g., turpin, j.a., sher, a. and appella, e. 2003. in vivo antiviral activity of novel human immunodefi ciency virus type 1 nucleocapsid p7 zinc fi nger inhibitors in a transgenic murine model. aids res. hum. retroviruses, 19:91–101. schito, m.l., soloff, a.c., slovitz, d., trichel, a., inman, j.k., appella, e., turpin, j.a. and barratt-boyes, s.m. 2006. preclinical evaluation of a zinc fi nger inhibitor targeting lentivirus nucleocapsid protein in siv-infected monkeys. curr. hiv res., 4:379–386. schramm, h.j., boetzel, j., buttner, j., fritsche, e., gohring, w., jaeger, e., konig, s., thumfart, o., wenger, t., nagel, n.e. and schramm, w. 1996. the inhibition of human immunodefi ciency virus proteases by ‘interface peptides’. antiviral res., 30: 155-170. schramm, h.j., de rosny, e., reboud-ravaux, m., buttner, j., dick, a. and schramm, w. 1999. lipopeptides as dimerization inhibitors of hiv-1 protease. biol. chem., 380:593–596. schramm, h.j., nakashima, h., schramm, w., wakayama, h. and yamamoto, n. 1991. hiv-1 reproduction is inhibited by peptides derived from the nand c-termini of hiv-1 protease. biochem. biophys. res. commun., 179:847–851. schubert, u., ferrer-montiel, a.v., oblatt-montal, m., henklein, p., strebel, k. and montal, m. 1996a. identifi cation of an ion channel activity of the vpu transmembrane domain and its involvement in the regulation of virus release from hiv-1-infected cells. febs lett, 398:12–18. schubert, u., ott, d.e., chertova, e.n., welker, r., tessmer, u., princiotta, m.f., bennink, j.r., krausslich, h.g. and yewdell, j.w. 2000. proteasome inhibition interferes with gag polyprotein processing, release, and maturation of hiv-1 and hiv-2. proc. natl. acad. sci. u.s.a., 97:13057–13062. schwartz, o., marechal, v., friguet, b., arenzana-seisdedos, f. and heard, j.m. 1998. antiviral activity of the proteasome on incoming human immunodefi ciency virus type 1. j. virol., 72: 3845–3850. serio, d., singh, s.p., cartas, m.a., weber, i.t., harrison, r.w., louis, j.m. and srinivasan, a. 2000. antiviral agent based on the non-structural protein targeting the maturation process of hiv-1: expression and susceptibility of chimeric vpr as a substrate for cleavage by hiv-1 protease. protein. eng., 13:431–436. sheehy, a.m., gaddis, n.c. and malim, m.h. 2003. the antiretroviral enzyme apobec3g is degraded by the proteasome in response to hiv-1 vif. nat. med., 9:1404–1407. shehu-xhilaga, m., crowe, s.m. and mak, j. 2001a. maintenance of the gag/gag-pol ratio is important for human immunodefi ciency virus type 1 rna dimerization and viral infectivity. j. virol., 75:1834–1841. shehu-xhilaga, m., kraeusslich, h.g., pettit, s., swanstrom, r., lee, j.y., marshall, j.a., crowe, s.m. and mak, j. 2001b. proteolytic processing of the p2/nucleocapsid cleavage site is critical for human immunodefi ciency virus type 1 rna dimer maturation. j. virol., 75: 9156–9164. shehu-xhilaga, m., tachedjian, g., crowe, s.m. and kedzierska, k. 2005. antiretroviral compounds: mechanisms underlying failure of haart to eradicate hiv-1. curr. med. chem., 12:1705–1719. shin, c.g., taddeo, b., haseltine, w.a. and farnet, c.m. 1994. genetic analysis of the human immunodefi ciency virus type 1 integrase protein. j. virol., 68:1633–1642. shkriabai, n., datta, s.a., zhao, z., hess, s., rein, and a. kvaratskhelia, m. 2006. interactions of hiv-1 gag with assembly cofactors. biochemistry, 45:4077–4083. sluis-cremer, n., arion, d., abram, m.e. and parniak, m.a. 2004. proteolytic processing of an hiv-1 pol polyprotein precursor: insights into the mechanism of reverse transcriptase p66/p51 heterodimer formation. int. j. biochem. cell. biol., 36:1836–1847. sluis-cremer, n., dmitrienko, g.i., balzarini, j., camarasa, m.j. and parniak, m.a. 2000. human immunodefi ciency virus type 1 reverse transcriptase dimer destabilization by 1-[spiro[4″-amino-2″,2″-dioxo-1″,2″ oxathiole-5″,3′-[2′, 5′-bis-o-(tert-butyldimethylsilyl)-beta-d-ribofuranosyl]]]-3-ethylthy mine. biochemistry, 39:1427–1433. -cremer, n., hamamouch, n., san felix, a., velazquez, s., balzarini, j. and camarasa, m.j. 2006. structure-activity relationships of [2′,5′bis-o-(tert-butyldimethylsilyl)-beta-d-ribofuranosyl]3′-spiro-5″(4″-amino-1″,2″-oxathiole-2″,2″-dioxide)thymine derivatives as inhibitors of hiv-1 reverse transcriptase dimerization. j. med. chem., 49:4834–4841. sluis-cremer, n. and tachedjian, g. 2002. modulation of the oligomeric structures of hiv-1 retroviral enzymes by synthetic peptides and small molecules. eur. j. biochem., 269:5103–5111. smith, a.j., srinivasakumar, n., hammarskjold, m.l. and rekosh, d. 1993. requirements for incorporation of pr160gag-pol from human immunodefi ciency virus type 1 into virus-like particles. j. virol., 67: 2266–2275. song, m., rajesh, s., hayashi, y. and kiso, y. 2001. design and synthesis of new inhibitors of hiv-1 protease dimerization with conformationally constrained templates. bioorg. med. chem. lett., 11:2465– 2468. sorin, m., yung, e., wu, x. and kalpana, g.v. 2006. hiv-1 replication in cell lines harboring ini1/hsnf5 mutations. retrovirology, 3:56. srinivasakumar, n., hammarskjold, m.l. and rekosh, d. 1995. characterization of deletion mutations in the capsid region of human immunodefi ciency virus type 1 that affect particle formation and gag-pol precursor incorporation. j. virol., 69:6106–6114. srivastava, s., sluis-cremer, n. and tachedjian, g. 2006. dimerization of human immunodefi ciency virus type 1 reverse transcriptase as an antiviral target. curr. pharm. des., 12:1879–1894. stephenson, j. 2007. researchers buoyed by novel hiv drugs: will expand drug arsenal against resistant virus. jama, 297:1535–1536. sticht, j., humbert, m., findlow, s., bodem, j., muller, b., dietrich, u., werner, j. and krausslich, h.g. 2005. a peptide inhibitor of hiv-1 assembly in vitro. nat. struct. mol. biol., 12:671–677. 181 targeting the late stages of hiv-1 replication drug target insights 2007: 2 stopak, k., de noronha, c., yonemoto, w. and greene, w.c. 2003. hiv-1 vif blocks the antiviral activity of apobec3g by impairing both its translation and intracellular stability. mol. cell., 12:591–601. swanstrom, r., and j.w. wills. 1997. synthesis, assembly, and processing of viral proteins. in coffi n, j. m., hughes, s. h. and varmus h. e. (ed.), retroviruses. cold spring harbor laboratory press, cold spring harbor, n.y., p. 263–334. tachedjian, g., aronson, h.e., de los santos, m., seehra, j., mccoy, j.m. and goff, s.p. 2003. role of residues in the tryptophan repeat motif for hiv-1 reverse transcriptase dimerization. j. mol. biol., 326:381– 396. tachedjian, g., aronson, h.e. and goff, s.p. 2000. analysis of mutations and suppressors affecting interactions between the subunits of the hiv type 1 reverse transcriptase. proc. natl. acad. sci. u.s.a., 97: 6334–6339. tachedjian, g., moore, k.l., goff, s.p. and sluis-cremer, n. 2005a. efavirenz enhances the proteolytic processing of an hiv-1 pol polyprotein precursor and reverse transcriptase homodimer formation. febs lett., 579:379–384. tachedjian, g., orlova, m., sarafi anos, s.g., arnold, e. and goff, s.p. 2001. nonnucleoside reverse transcriptase inhibitors are chemical enhancers of dimerization of the hiv type 1 reverse transcriptase. proc. natl. acad. sci. u.s.a., 98:7188–7193. tachedjian, g., radzio, j. and sluis-cremer, n. 2005b. relationship between enzyme activity and dimeric structure of recombinant hiv-1 reverse transcriptase. proteins, 60:5–13. tanchou, v., gabus, c., rogemond, v. and darlix, j.l. 1995. formation of stable and functional hiv-1 nucleoprotein complexes in vitro. j. mol. biol., 252:563–571. tang, c., loeliger, e., kinde, i., kyere, s., mayo, k., barklis, e., sun, y., huang, m. and summers, m.f. 2003. antiviral inhibition of the hiv-1 capsid protein. j. mol. biol., 327:1013–1020. tang, c., loeliger, e., luncsford, p., kinde, i., beckett, d. and summers, m.f. 2004. entropic switch regulates myristate exposure in the hiv-1 matrix protein. proc. natl. acad. sci. u.s.a., 101:517–522. tang, c., ndassa, y. and summers, m.f. 2002. structure of the n-terminal 283-residue fragment of the immature hiv-1 gag polyprotein. nat. struct. biol., 9:537–543. telesnitsky a. and goff, s. 1997. reverse transcriptase and the generation of retroviral dna. in coffi n, j.m., hughes, s. h. and varmus, h.e. (ed.), retroviruses. cold spring harbor laboratory press, new york. ternois, f., sticht, j., duquerroy, s., krausslich, h.g. and rey, f.a. 2005. the hiv-1 capsid protein c-terminal domain in complex with a virus assembly inhibitor. nat. struct. mol. biol., 12:678–682. todd, m.j., semo, n. and freire, e. 1998. the structural stability of the hiv-1 protease. j. mol. biol., 283:475–488. turpin, j.a., terpening, s.j., schaeffer, c.a., yu, g., glover, c.j., felsted, r.l., sausville, e.a. and rice, w.g. 1996. inhibitors of human immunodefi ciency virus type 1 zinc fi ngers prevent normal processing of gag precursors and result in the release of noninfectious virus particles. j. virol., 70:6180–6189. uhlikova, t., konvalinka, j., pichova, i., soucek, m., krausslich, h.g. and vondrasek, j. 1996. a modular approach to hiv-1 proteinase inhibitor design. biochem. biophys. res. commun., 222:38–43. ulysse, l.g. and chmielewski, j. 1998. restricting the fl exibility of crosslinked, interfacial peptide inhibitors of hiv-1 protease. bioorg. med. chem. lett., 8:3281–3286. venezia, c.f., howard, k.j., ignatov, m.e., holladay, l.a. and barkley, m.d. 2006. effects of efavirenz binding on the subunit equilibria of hiv-1 reverse transcriptase. biochemistry, 45:2779–2789. veronese, f.d., copeland, t.d., oroszlan, s., gallo, r.c. and sarngadharan, m.g. 1988. biochemical and immunological analysis of human immunodefi ciency virus gag gene products p17 and p24. j. virol., 62: 795–801. verplank, l., bouamr, f., lagrassa, t.j., agresta, b., kikonyogo, a., leis, j. and carter, c.a. 2001. tsg101, a homologue of ubiquitin-conjugating (e2) enzymes, binds the l domain in hiv type 1 pr55(gag). proc. natl. acad. sci., u.s.a., 98: 7724–7729. vivet-boudou, v., didierjean, j., isel, c. and marquet, r. 2006. nucleoside and nucleotide inhibitors of hiv-1 replication. cell. mol. life. sci., 63:163–186. vogt, v.m. 1996. proteolytic processing and particle maturation. curr. top. microbiol. immunol., 214:95–131. von schwedler, u.k., stray, k.m., garrus, j.e. and sundquist, w.i. 2003. functional surfaces of the human immunodefi ciency virus type 1 capsid protein. j. virol., 77:5439–5450. wapling, j., moore, k.l., sonza, s., mak, j. and tachedjian, g. 2005. mutations that abrogate human immunodefi ciency virus type 1 reverse transcriptase dimerization affect maturation of the reverse transcriptase heterodimer. j. virol., 79:10247–10257. wei, b.l., denton, p.w., o’neill, e., luo, t., foster, j.l. and garcia, j.v. 2005. inhibition of lysosome and proteasome function enhances human immunodeficiency virus type 1 infection. j. virol., 79: 5705–5712. wiegers, k., rutter, g., kottler, h., tessmer, u., hohenberg, h. and krausslich, h.g. 1998. sequential steps in human immunodefi ciency virus particle maturation revealed by alterations of individual gag polyprotein cleavage sites. j. virol., 72:2846–2854. willey, r.l., maldarelli, f., martin, m.a. and strebel, k. 1992. human immunodefi ciency virus type 1 vpu protein induces rapid degradation of cd4. j. virol, 66:7193–7200. wlodawer, a., miller, m., jaskolski, m., sathyanarayana, b.k., baldwin, e., weber, i.t., selk, l.m., clawson, l., schneider, j. and kent, s.b. 1989. conserved folding in retroviral proteases: crystal structure of a synthetic hiv-1 protease. science, 245:616–621. worthylake, d.k., wang, h., yoo, s., sundquist, w.i. and hill, c.p. 1999. structures of the hiv-1 capsid protein dimerization domain at 2.6 a resolution. acta. crystallogr. d. biol. crystallogr., 55:85–92. wu, x., anderson, j.l., campbell, e.m., joseph, a.m. and hope, t.j. 2006. proteasome inhibitors uncouple rhesus trim5alpha restriction of hiv-1 reverse transcription and infection. proc. natl. acad. sci. u.s.a., 103:7465–7470. wu, x., liu, h., xiao, h., conway, j.a., hunter, e. and kappes, j.c. 1997. functional rt and in incorporated into hiv-1 particles independently of the gag/pol precursor protein. embo j., 16:5113–5122. yeni, p. 2006. update on haart in hiv. j. hepatol., 44:s100–103. yeni, p.g., hammer, s.m., carpenter, c.c., cooper, d.a., fischl, m.a., gatell, j.m., gazzard, b.g., hirsch, m.s., jacobsen, d.m., katzenstein, d.a., et al. 2002. antiretroviral treatment for adult hiv infection in 2002: updated recommendations of the international aids society-usa panel. jama, 288:222–235. yu, q., ottmann, m., pechoux, c., le grice, s. and darlix, j.l. 1998. mutations in the primer grip of human immunodefi ciency virus type 1 reverse transcriptase impair proviral dna synthesis and virion maturation. j. virol., 72:7676–7680. yu, x., yu, y., liu, b., luo, k., kong, w., mao, p. and yu, x.f. 2003. induction of apobec3g ubiquitination and degradation by an hiv-1 vif-cul5-scf complex. science, 302:1056–1060. yung, e., sorin, m., pal, a., craig, e., morozov, a., delattre, o., kappes, j., ott, d. and kalpana, g.v. 2001. inhibition of hiv-1 virion production by a transdominant mutant of integrase interactor 1. nat. med., 7:920–926. yung, e., sorin, m., wang, e.j., perumal, s., ott, d. and kalpana, g.v. 2004. specifi city of interaction of ini1/hsnf5 with retroviral integrases and its functional signifi cance. j. virol., 78:2222–2231. zhang, z.y., poorman, r.a., maggiora, l.l., heinrikson, r.l. and kezdy, f.j. 1991. dissociative inhibition of dimeric enzymes. kinetic characterization of the inhibition of hiv-1 protease by its cooh-terminal tetrapeptide. j. biol. chem., 266:15591–15594. zhao, l., o’reilly, m.k., shultz, m.d. and chmielewski, j. 2003. interfacial peptide inhibitors of hiv-1 integrase activity and dimerization. bioorg. med. chem. lett., 13:1175-1177. zhou, j., chen, c.h. and aiken, c. 2004. the sequence of the ca-sp1 junction accounts for the differential sensitivity of hiv-1 and siv to the small molecule maturation inhibitor 3-o-{3′,3′-dimethylsuccinyl}-betulinic acid. retrovirology, 1:15. 182 wapling et al drug target insights 2007: 2 zhou, j., chen, c.h. and aiken, c. 2006. hiv-1 resistance to the small molecule maturation inhibitor 3-o-{3′,3′-dimethylsuccinyl}-betulinic acid is conferred by a variety of single amino acid substitutions at the ca-sp1 cleavage site in gag. j. virol., 80:12095–12101. zhou, j., huang, l., hachey, d.l., chen, c.h. and aiken, c. 2005. inhibition of hiv-1 maturation via drug association with the viral gag protein in immature hiv-1 particles. j. biol. chem., 280:42149– 42155. zutshi, r. and chmielewski, j. 2000. targeting the dimerization interface for irreversible inhibition of hiv-1 protease. bioorg. med. chem. lett., 10:1901–1903. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true 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