Ir aq i J.Pha rm.Sc i., Vol.15 (2 ) ,2006 Ph th alyl – N – tyros yl – glycin as a New Analgesic 46 Evaluation of Analgesic Activity of Newly Synthesized Phthalyl- tyrosyl-glycin Sodium Muthanna S. AL-Taee* , Kawkab Y. Saour*, Haider M. Mohammed**1 * Departmen t o f Pharmaceu tica l Chemistry, Co llege o f Ph armacy , Un ive rsity o f Bagh dad – Iraq. ** Depa rtme nt of Pharm aco logy and Toxicolog y , C olle ge of Pharm acy , Unive rsity of Baghdad –Iraq . Abstract Alteration in the ba ckbone structure of the endogenously re le ase d opioid peptides Leu 5/Met5 enkephalins may res ult in compounds having comparable profile of pha rma cologic al ac tivity but with different physicoche mic al prope rties and side effects. Phtha lyl amino ac id a nd phthalyl es te rs a re a mong the deriva tive s that have been s ynthe size d and e valuated for their a ntiba cteria l and a ntifungal a ctivitie s.This study was c onducted to eva luate the possible ana lgesic activity of phthalyl-tyrosyl-glycin sodium that ha s bee n re cently synthes ized by our team.The s tudy was c arried out on 24 albino mice us ing hot plate method. The anima ls we re alloca te d in to three groups; the firs t group rece ived saline and re prese nt a c ontrol group; the se cond group re ceive d morphine HCl as a s ta nda rd drug; and the third group rece ived phthalyl-tyrosyl-glycin sodium. The onse t with whic h the a nimal lift his fore arm and the number of jumps per 25 se conds were re corde d for eac h group. The results of this study showed that phthalyl-tyros yl-glycin sodium resulted in signific ant improvement (P<0.05) in a nalge sia sc ore a s well as s ignifica nt delay in the onse t of induce d hyperalges ia in comparis on to s aline-treated group, a nd in c ompa rison to morphine HCl, no signific ant differenc e (P>0.05) wa s obs erved in ana lgesia sc ore but with signific ant delay in induc ed hyperalges ia .The re sults obtained in this s tudy provide e xperimental evidences for the effe ctive nes s of the prepa re d compound as a nalge sic with comparable effe ct to that of morphine . K ey words: Phthalyl-tyros yl-glyc in s odium, phtha lyl group, analges ia . الخالصة ض م ان بع ى الببتيدات الحياتية ال ميائية عل كي عديالت ال ج )ليوسين ومثيونين انكفالين(الداخلية رفينيةوالت ها قد تنت مركبات ل ربة وائية مقا وية فعاليات د سكنات الم ق ي كم ع اخـتالف ـف ـم ة ة مختلـف وفيزياوـي ة وـي ص كيميا وا ـخ كن ب ض ول األعـراض بـع عن استخدامه ل . االجانبية الناتجة ى الفثالـي مركبات الحاوية عـل ض تعتبر ال رات كحـام ـت ي واس ميـن ـات أ ركب ن الم ل ـم الفثالـي طرية جرثومية والف مة فعاليتها ضد االلتهابات ال ـد وعليه المصنعة والمقي ة لببتي ة الدوائـي ميم هذه الدراسة لتقييم الفعالـي تم تص مسكن كب مر موعة الفثاليل ك كب ان. لأللميحتوي على مج Phthalyl-tyros(وهو هذا المر yl glycin s odium ( هو عة بغداد مياء الصيدالنية في كلية الصيدلة جام كي عه من قبل فرع ال 24الدراسـة باسـتخدام لقد تمت.مركب جديد قد تم تصني رة ذكرًا فأرًا ـا ة الصـفيحة الح تخدام طريـق ـس ن لأللم با مسك مركب الجديد ك وائية لل ـم (hot plate)أبيضا لتقييم الفعالية الد وت مالح ألولى بالماء ال موعة ا مجاميع٬ حقنت المج زيع الحيوانات على ثالثة sa)تو line موعة السيطرة والمجموعة ( مثل مج وت عقار رفين ك يالثانية بالمو كب قياس Phthalyl-tyros(والثالثة بمر yl glyc in s odium( ـم موضوع البحث وت و والذي ه حب الحيوان غرق ليس زات خالل احتساب الوقت المست وعدد القف عن الصفيحة الحارة هرت النتائج أن هناك .ثانية 25قدمه أظ زات خالل وكذلك في عدد القف ن الصفيحة حب الحيوان قدميه ع غرق ليس ست معنوي في الوقت الم واضح و ن ثانية في 25تحس طرة وعة السي مجم عن كب الجديد عالجة بالمر معد. الحيوانات الم معنوي قد لوحظ في رق وانات المعالجـة ال ف ل قفز الحي مورفين موعة المعالجة بال جيد بالمقارنة مع المج مركب ال رة كان بال حا سحب الحيوان لقدميه عن الصفيحة ال إال أن سرعة مركب .أبطأ عالية الدوائية ل ى الف عل سة أدلة تجريبية را ن هذه الد ج المستخلصة م ي النتائ Phthalyl-tyros(تعط yl- glyc in( ن مسك كب مورفينكمر عالية مقاربة لل .لأللم قوي وبف 1corresp ond in g author ema il r7 4th @yahoo.com Rec eived 24 -7-2006 Acce pted 20 -1 2-2 006 47 In troduction Opioid peptides , defined as pep tides with opia te -like pha rmacolo gical effe cts , are the oldest pha rma colo gical subs ta nces kno wn fo r th e i r a na lg e s ic , e up h o ri c a n d a dd ic ti v e effe cts(1,2).Morp hine is the firs t na rc otic analgesic alka lo id isolated fro m p la nt sourc e in 1806 (3) In 1 975 , Hughes and Kos terlitz succeeded in isolating two pentap ep tides, Leucine - and Methionine-enkephalin (Leu/Met enkep ha lin) fro m pig brain, whic h c ompe te strongly with morphine -like drugs fo r bind ing to recep to rs in the brain with p ha rma cological ac tions rese mbling those of mo rp hine itself [2]. Howeve r, to date, morp hine -like compounds re main the only class known to ac t b y mimic king thes e pe ptides (4 ). S eve ra l families of pe ptides were discove red with multiple ca te gories o f op io id recep to rs . Bind ing s tudies by Synd er and colleagues (1 973 ), demo ns trated tha t op io ids are reco gnized by sp ecific receptors (5). Various pharmaco logica l observa tions like analgesia, se da tion, a ntitussiv e, antidiarrhea l, pupilary co nstric tion, and bradyca rd ia prod uced b y different drugs implie d the mediation of more than o ne typ e of rec ep to rs . Recep to r cloning stud ies revealed the exis te nc e of s ix different opiate re cep tors na med as  (mu),  (de lta ),  (kappa),  (s igma),  (epsilon) and NFQ (nociceptin FQ)(6) The se re ceptors me diate sev eral pharmaco lo gica l and s ide e ffec ts o f opiates includ ing: a nalgesia (supraspinal, spinal, pe ripheral), res pira tory depress io n , p apillary co nstric tion, reduce d GI mo tility, e up horia , dy sphoria , sedatio n and phy sical d epend ence(3) . Conce rning their molecular p ha rma cology, thes e opioid receptors be long to the fa mily of inhibitory G-protein c oup led re ceptor, med ia te the re ductio n of intra ce llular cAMP leve l as a main ev ent beyond recep to r bind ing (7). Other mec hanisms invo lve the o pening of K + and bloc k of Ca2+-c hanne ls the reby red ucing both, ne urona l e xcitability a nd tra ns mitter release (8). Understa nding the p owe rful mo le cula r, biologic al a nd physio logical te rms o f opioids was used to dev elop a nalges ic c ompo unds with significant advanta ge s o ve r morphine (9). In this respect, inco rporation of a ne w gro up in the ma in ba ckbone of naturally oc curring enkep ha lin was a imed in s ynthesizing co mpounds with po ss ib le ana lges ic effe cts. On the other hand, one o f the earliest typ es of structural modific atio ns applied to the enkep ha lin was sho rtening of the peptid e chain by re moval of residues from the ess ential se quence or by remo val of residues fro m C- te rminal. These expe riments showed that significant potency re maine d in prod uc ing ana lges ia in v iv o as in Tyr-D-ala-phe-met amide ( 10,11). In pre vious wo rk, Mutha nna , et a l. (20 05) were suc ce ed in d esigning and s ynthesis of enkep halin a nalogue s having e ven shorte st cha in by re mov ing the C-te rmina l res id ue gly- phe -leu/met; ke ep ing N-tyros ine re sidue protecte d b y phthalyl gro up. Acco rd ingly, the newly s ynthe size d phthalyl-tyros yl-glycin and its sod ium s alt (Fig. 1 and 2) a re no vel comp ounds p ro duc ed in our la boratories with pos sible ana lges ic ac tiv ity [12]. The prese nt stud y was conduc ted to inve stigate the ana lges ic effec t of the sod ium sa lt of this comp ound on e xp erime ntal animals . Figure (1 ) Phtha ly l-ty rosyl-glyc in (12) Figure (2 ) Phtha lyl – tyrosyl – g ly cin so dium sa lt Material a nd Methods Animals: Twenty-four adult male albino mic e weighing 2 5.23 ± 3.1 gm we re used in this stud y. They w ere obtaine d from Ira qi S era and Vac cine Institute and were housed under standard co nditio ns in the animal hous e of the Colle ge of Pha rma cy-Univ ersity o f Baghda d. Animals were fe d commercial p elle t and ta p water in fre e ac ce ss ad libitum. Mate rials: Mo rp hine HCl was s upp lied by May and Baker Ltd , England. P htha lyl-tyro syl- glycin. has b ee n o btaine d from re fe re nce (13) and its c orre spo nd ing s od ium s alt has b een synthes ized ac cording to re fere nc e (14) to increas e its wa ter s olubility to b e suitab le for intrap eritonea l ad minis tration. All co mpounds N O O CH N H O H2 C OH OH O N O O CH N H O H2C OH O O Na 48 were disso lv ed in normal sa line a nd were ad minis te re d intra perito nially. Method s: Ho t plate metho d a s de scribed b y Woolfe a nd Ma cDo nald (13) was us ed for ev aluation of the analges ic effe ct o f the tes te d co mpound comp ared with morp hine as a re fe re nc e. Anima ls we re a lloc ated into thre e groups: firs t group re ce iv ed normal s aline, se cond group rece iv ed morphine HCl (Ma y and Ba ke r Ltd , Engla nd), and third group re ce iv ed phthalyl-N-tyros yl-glycin so dium. The p la te was he ated to 55 oC and the a nimal was p ut o n the plate . The ons et with which the anima l lift his forea rm and the number of jumps p er 2 5 se conds we re rec orded for e ac h group. The re sults were expres se d a s mea n ± standard e rror and were a na lyzed us ing ANOVA and unpaired Stud ent t-tes t. Results and Dis cuss ion The d ata pres ented in tab le (1) clea rly showe d that anima ls in c ontrol group lift the ir fo rea rms in abo ut (1.2 ± 0.66 ) s eco nds which rep re sents the no rma l onse t of he at-ind uce d hyperalge sia. The animal jumps 2 4.8 ± 2.4 time s/25 s eco nd s which rep re se nts the a nalge sia s core. Tab le (1) also s ho wed tha t mo rp hine s ignifica ntly (P <0.05 ) delayed the ons et o f hea t-induce d hyperalge sia (2 .6 ± 0.74) in comp aris on to co ntro l group and significa ntly (P<0.05) impro ve d the a na lges ia s core (1 1.0 ± 2 .4) in co mparison to c ontrol gro up (24.8 ± 2.4) (Fig. 3 and 4 ). Inte re stingly, phtha lyl-tyros yl-glycin so dium re sulted in s ignifica nt (P<0.05) impro ve ment in ana lges ia s core (10.8 ± 1.43 ), an e ffec t s eems comparab le to tha t of morphine (1 1.0 ± 2.4, P>0 .0 5). Howev er, p htha lyl- tyro syl-glyc in s odium showed slightly more significant de la y in the o ns et of hea t-induce d hyperalge sia ov er tha t of morphine (F ig. 3 and 4). The pha rma cologic re sults may ind ic ate that tyro sine is the ess ential a mino acid re sidue within the pe ptid e chain of leu / met enkep ha lin (15) ; and the enzymatic res is tanc e is doubtless an important fac to r in the high pote ncy of this a nalge sic [16] b ec aus e, chemica lly, the pres enc e of phtha lyl gro up ma y enhance the a va ilab ility a nd he nc e the bind ing of the compound to opioid rec eptors a nd this in turn may po te ntiate the pharmaco lo gica l effect of the te sted c ompo und , this may e xp la in why the co mpo und s ho wed a c ompa ra ble analge sic effe ct to that o f morphine . Furthe rmo re , the bulky phthalyl gro up ma y increa se the lipo philic ity and thus e nha nc e a suffic ient bioa vailability and tis sue penetra tion, a n effect which ma y e xplain why the co mpound ha s slightly fa ster o nse t of a ction than mo rphine. On the o ther ha nd, the pres ence of bulky phtha lyl gro up is sus pe cted to re duc e enzyme degra da tion of p hthalyl-tyro syl-glyc in ins ide the b od y b y a dding a sterric hind ra nce [17]; howe ve r, this e xpe ctation req uire s further pha rma co kinetic stud ie s. In conclus io n, phtha lyl-tyros yl-glycin sod ium showed promising ana lges ic activity app ro ximately eq ual to tha t of mo rp hine with re liab le onse t of ac tion, making it a goo d candida te for furthe r dev elopment in the field of mo rp hine -repla ceme nt therapy b as ed on the id ea tha t enke pha lins are co mpound s dev oid of add ic tive liab ility. Table (1) Effec t of Phtha ly l-tyro sy l-glyc in o dium and mo rph ine on the a nalge sia score and he a t-induce d hype ra lges ia in mice Groups Jumps/25 seconds Onset of hype ralgesia (se c.) Control group (n=6) 24 .8 ± 2 .4 a 1.2 ± 0.66 a Morphine- trea te d group (n=6) 11 .0 ± 2 .4 b 2.6 ± 0.74 b Phtha lyl- tyro syl-glyc in s odium-tre ated group (n=6) 1 0.8 ± 1.43 b 4.2 ± 1.04 c Fig ure (3) :The effect o f phtha ly l-tyro sy l - g ly cin s odiu m an d morphine o n a nalge sia sco re in mice re co rde d 25 second.Re sults are e xpresse d as the me an (jumps) ± SEM; n=8 ; non - ide ntica l supe rscripts (a , b) re prese nt s ig nific ant diffe re nce s among g ro ups by ANOVA (P<0.0 5) Value s are pre se nte d as me an ± SEM.Value s with non-ide ntica l s upe rs cripts (a , b, c ) amo ng diffe re nt groups are cons ide re d significa ntly diffe re nt (P<0 .0 5). 0 5 10 15 20 25 30 35 Control morphine phthalyl - tyrosyl glycin sodium A n a lg e s ia s c o re ( j u m b s /2 5 s e c ) a b c 49 Refere nces 1. Wille tte , R.E.: Ana lges ic a ge nts. In: Wilso n and Gis vo ld ’s Te xtbook of Organic Med ic inal and Pharmace utical Chemistry (1 2th ed.), Delgado, J.N. a nd Reme rs , W.A. (Eds .), J.B. Lippincott, Willia ms and Wilkins , P hila delphia, 2004, p. 6 87-709. 2. Rang, H.P.; Dale, M.M. and Ritte r, J .M. (Eds): Analgesic Drugs . In: P harmac ology (5 th ed .), Churchill Livingstone, Lond on, 20 03, pp. 589-595 . 3. Hardma n, J .G; Limbird, L.E. a nd Molinoff, P.B. (Eds .): Opioid analge sics . In: Goodma n and Gilman’s The P harmacological Basis of The rape utic s (10 th ed.), Mc Graw-Hill, New York, 2001, pp. 569 -6 05. 4. Cooper, J.R.; Bloo m, F.E. a nd Roth, R.H.: The biochemica l basis of neurop ha rma colo gy. Oxford Unive rsity Press, New York, 19 96; pp. 121-125 . 5. Synder, S .H.; P aste rnak, G.W. and P ert, C.: In: Handbook of ps yc hop ha rma cology, Inve rsen, L.L.; Inve rson, S.D. a nd Synder, S.H. (Eds.). Plenum, Ne w York, v ol.5 19 84 ; pp. 329-360 . 6. Dhawan, B.N.; Cesselin, F.; Raghubir, R. et al: Classificatio n o f opioid rece ptors. Pha rmaco l. Rev . 1996 ; 48 : 567-586 . 7. Wa y, W.L.; Fields , H.L. and S chumac he r, M.A.: Op io id a nalge sics a nd anta go nists. In: Bas ic a nd c linic al pharmacology (8 th ed.), Katzung, B.G. (Ed.), McGraw-Hill, New York, 2001, pp. 516 -517. 8. North, R.A: Op io id actions on me mbrane ion cha nnels. In: Ha ndb ook of e xp erime ntal pha rma co lo gy, Herz (Ed .). Springe rt- Verla g, Berlin, vo l 10 4, 1 99 3; p p. 9 8-101. 9. Americ an pain soc iety: P rinc iples of ana lges ic us e in the trea tme nt of a cute pain and ca nce r pain. Ame rican Pa in S oc ie ty, Glenview, I.L., 1 999 , pp .64. 10. S himohigashi, Y.; Matsuura, S.; Che n, H.C.; et al: Dimeric enkephalins display enhance d a ffini ty and selec tivity fo rt the - opiate receptors. Mo l. P harmacol. 1982; 2 1: 558 -563. 11. Mo rley, J.S .: Chemis try of opioid pe ptides. Br. Med. Bull. 1983 ; 39 : 5-10 . 12. AL-Taee , M.S.: Syn thes is o f ne w op ioid pep tide analogues with e xpected biologic al ac tivity. M.S c. thes is , 2005. Univ ersity of Baghdad, Baghda d. 13. Wo olfe , G. and Mc Dona ld , A.D.: J. Pha rma co l. Exp . Ther. 19 44; 80 : 300-307 . 14. F urmis s, B.S.; Hanna fo rd , A.J. and Roge rs, V. (Ed s.): Carbo xylic acid: cha ra cteristic che mical. In: Vo gel's te xt bo ok of p ra ctic al organic che mis try (4th ed.). Longman, Lo ndo n, 197 9; p p. 106 4-1065. 15. S asaki, Y.; Hirab uki, M.; Amb o, A.; e t a l: Enkephalin analogues with 2,6- dimethylp henylalanin rep la cing phenyla lanine in p ositio n 4. Bio org. Me d. Chem. Lett. 200 1; 11(3): 327-32 9. 16. Klob , V.M. and Scheine r, S .: J . Pharma. Sci. 1 98 4; 3 3: 3 . 17. Michea l, M.: Pep tide a nd p ro te in drugs . In: Foye’s Principle of medical c he mis try (5th e d.). David , A.W. a nd Thoma s, L.L. (Eds .), Lippincotte Williams and Wilkines, Philadelphia, 2002, pp. 120-123 . 0 1 2 3 4 5 6 Control morphine phthalyl - tyrosyl glycin sodium O n e st o f h yp er al g e si a (s e c ) a b c Figure (4) :Compariso n o f the e ffe ct of phthalyl-tyrosy l-glyc in so dium and mor phine on the onset of he at-induce d hy pe ralge sia in mice . Res ults a re ex presse d as the me an ± SEM; n=8; no n-ide ntic al supe rsc ripts (a , b , c ) re pre se nt significant diffe re nce s amo ng groups by ANOVA (P<0 .05)