Comparative Evaluation of Using Intranasal Desmopressin, Parenteral Diclofenac or their Combination in the Management of Acute Iraqi J.Pharm.Sci., Vol.16 (2) ,2007 Ergotamine, vitamin E, melatonin and total antioxidants 72 Effect of Ergotamine and its Combination with Vitamin E or Melatonin on Total Antioxidant Status in Migraine Patients Shahlaa H. Ali 1 , Salim A. Hamadi ** , Ashwaq N. Al- Jaff** ** Department of Clinical Pharmacy ,College of Pharmacy , University of Baghdad, Baghdad , Iraq Abstract Free radicals and oxidative damage caused by them have being suggested to be involved in the pathogenesis of migraine. These may result from distorted equilibrium of pro-oxidant/anti-oxidant system that continuously generates and detoxifies oxidants during normal aerobic metabolism. Escape of such system from equilibrium leads to damage of cellular elements with the depletion of cellular stores of anti- oxidants material such as glutathione and vitamin E. Therefore, free radical scavengers (vitamin E or melatonin) seems to be of potential benefit as prophylactic anti-migraine therapy by neutralizing free radicals overproduction and possibly preventing formation of highly toxic intermediates (such as nitric oxide). In addition of being powerful antioxidant, melatonin was shown to possess promising effects in modulating severity, frequency and duration of migraine attacks. For this reason the present study was conducted to investigate the involvement of changed anti-oxidant defense (measured as total antioxidant status “TAS”) during migraine attack and the possible modulation of such status by classical anti-migraine therapy (ergotamine), antioxidants (vitamin E and melatonin) and their combination. 23 normal subjects and 21 migraine patients with age range of (17-45) years were enrolled in the study. Patients were diagnosed according to neurologist decision to have migraine with and without aura. Migraine patients were divided into three treatment groups; first group treated with ergotamine alone, second group with ergotamine /vitamin E and third group with ergotamine /melatonin. All groups were advised to take their treatments during attacks. Blood samples were drawn from migraine patients and normal subjects before initiation of therapy and after pain has been relived (from migraine patients only) for the investigation of TAS . The results of the study showed that TAS was significantly lower in migraine patients in comparison to control healthy subjects (P<0.05) with a percent reduction ranged from 35.46% to 43.97%. However, there is no significant difference in the level of TAS among migraine patients (P>0.05). Treatment with ergotamine raised significantly the level of TAS by 157%. The addition of vitamin E or melatonin greatly raised TAS by 179% and 176% respectively. The addition of vitamin E to ergotamine showed superior effect to that when melatonin was added. The greater reduction in TAS seen in this study among migraine patients in comparison to control healthy subjects suggests the presence of generalized decrease in antioxidant defense elements. Elevation of TAS by all treatments was very clear. In conclusion the decrease in TAS can be implicated in the pathophysiology of migraine and enhancement of antioxidant system can add a beneficial effect for the management of migraine headache with the use of antioxidants (vitamin E or melatonin) with classical anti-migraine drug. الخالصـــــــــــة لذ سبب١ت داء اٌشم١مت ٚ٘زا اإلخٙاد اٌخأوسذٞحؼخبش اٌدزٚس اٌحشة ٚاإلخٙاد اٌخأوسذٞ إٌاخُ ػٕٙا ِٓ إٌظش٠اث اٌّفخشظت حذ٠ثاً فٟ ٠ٕشأ ػٓ ػذَ اٌخٛاصْ فٟ أٔظّت األوسذة ٚاألٔظّت اٌّعادة ٌألوسذة ٚاٌخٟ حٕخح خزٚساً حشة ٚحؼاٌح س١ّخٙا باسخّشاس خالي ػ١ٍّاث األ٠ط فت إٌٝ ٔفار اٌّحخٜٛ اٌخٍٛٞ ِٓ ِعاداث اٌخٍٛٞ. إْ أؼذاَ اٌس١طشة أٚ اٌخًٍ فٟ ػًّ ٘زٖ األٔظّت ٠ؤدٞ إٌٝ حٍف اٌّٛاد اٌخ٠ٍٛت باإلظا اد اٌمأصت ٌٍدزٚس اٌحشة ِثً ف١خا١ِٓ ٘ـ أٚ ا١ٌّالح١ٔٛٓ حبذٚ راث فائذة ٚلائ١ت ظذ ,األوسذة اٌطب١ؼ١ت ِثً اٌغٍٛحاث١ْٛ ٚف١خا١ِٓ ٘ـ. ٌزا فأْ اٌُ ٚس حشة خط١شة ِثً )أٚوس١ذ إٌخش٠ه(. إْ داء اٌشم١مت ِٓ خالي ِؼاٌدت اإلفشاغ فٟ إٔخاج اٌدزٚس اٌحشة ٚسبّا باٌخم١ًٍ ِٓ حىْٛ خز اٌشم١مت. ا١ٌّالح١ٔٛٓ باإلظافت إٌٝ وٛٔٗ ِعاداً فؼاالً ظذ األوسذة فأٗ لذ اظٙش فؼا١ٌت ِٛػٛدة فٟ اٌخم١ًٍ ِٓ حذة ٚحشدد ٚفخشاث اإلصابت بأٌُ ة )ِماساً بشىً "اٌحاٌت اٌذفاػ١ت اٌى١ٍت ظذ ٚػٍٝ ٘زا األساس أخش٠ج اٌذساست اٌحا١ٌت السخىشاف حذخً حغ١ش اٌؼٛاًِ اٌذفاػ١ت ظذ األوسذ )ف١خا١ِٓ ٘ـ ةاألوسذة"( خالي ٔٛباث صذاع اٌشم١مت ِٚذٜ إِىا١ٔت حغ١١ش ٘زٖ اٌحاٌت باٌؼالج اٌخم١ٍذٞ )االسغٛحا١ِٓ(، ِعاداث األوسذ ( سٕت. ُشخص 54-22ػّشٞ )ِش٠ط بذاء اٌشم١مت ٚبّؼً 72شخص س١ٍُ ٚ 72أُخش٠ج اٌذساست ػٍٝ ٚا١ٌّالح١ٔٛٓ( أٚ باالث١ٕٓ ِؼاً. اٌّشظٝ وُٛٔٙ ِصابْٛ بذاء اٌشم١مت ٚفك لشاس أخصائٟ اٌدٍّت اٌؼصب١ت. لسُ اٌّشظٝ إٌٝ ثالد ِدا١ِغ ػالخ١ت، ػٌٛدج اٌّدّٛػت الج خالي حذٚد األٌٚٝ باالسغٛحا١ِٓ ٚاٌثا١ٔت باالسغٛحا١ِٓ ِغ بف١خا١ِٓ ٘ـ ٚاٌثاٌثت باالسغٛحا١ِٓ ِغ ا١ٌّالح١ٔٛٓ. اسشذ اٌّشظٝ إٌٝ اخز اٌؼ اس ٔٛباث اٌُ اٌشم١مت. أُخزث ػ١ٕاث اٌذَ ِٓ ِشظٝ اٌشم١مت ٚاألصحاء لبً بذء اٌذساست ٚبؼذ حسى١ٓ اٌُ اٌشم١مت )ِٓ ِشظٝ اٌشم١مت فمػ( ٌم١ اٌحاٌت اٌذفاػ١ت اٌى١ٍت ظذ األوسذة. shahlaa_65@yahoo.com. mail : -E: ing authorCorrespond 1 Received : 26/7/2007 Accepted : 29/12/2007 mailto:shahlaa_65@yahoo.com mailto:shahlaa_65@yahoo.com Iraqi J.Pharm.Sci., Vol.16 (2) ,2007 Ergotamine, vitamin E, melatonin and total antioxidants 72 أظٙشث إٌخائح أْ اٌحاٌت اٌذفاػ١ت اٌى١ٍت ظذ األوسذة ِٕخفعت ٌذٜ ِشظٝ اٌشم١مت باٌّماسٔت ِغ األصحاء ٚوأج ٔسبت االٔخفاض بّسخٜٛ اٌحاٌت اٌذفاػ١ت اٌى١ٍت ظذ األوسذة ب١ٓ ِشظٝ %. ٚػٍٝ أ٠ت حاي ٌُ ٠ىٓ ٕ٘ان فشق ِٕطم52.32ٟ% إٌٝ 24.53حخشاٚذ ب١ٓ %. وّا ٚاْ إظافت 242اٌشم١مت أٔفسُٙ. إْ اٌؼالج باالسغٛحا١ِٓ لذ أدٜ إٌٝ اسحفاع ِٕطمٟ بّسخٜٛ اٌحاٌت اٌذفاػ١ت اٌى١ٍت ظذ األوسذة بٕسبت % ػٍٝ اٌخٛاٌٟ. ٚػٍٝ 223% ٚ 223ظذ األوسذة ٚبٕسبت ف١خا١ِٓ ٘ـ أٚ ا١ٌّالح١ٔٛٓ لذ أدٜ إٌٝ اسحفاع أوبش فٟ اٌحاٌت اٌذفاػ١ت اٌى١ٍت ١ش فٟ اٌؼَّٛ فمذ اظٙش ف١خا١ِٓ ٘ـ حفٛلاً فٟ سفغ اٌحاٌت اٌذفاػ١ت اٌى١ٍت ظذ األوسذة ػٓ حٍه اٌخٟ أظٙش٘ا ا١ٌّالح١ٔٛٓ. ٠ظٙش االٔخفاض اٌىب ٔمص ػاَ فٟ اٌؼٕاصش اٌذفاػ١ت ظذ األوسذة وّا ٚاْ اٌحاٌت اٌذفاػ١ت اٌى١ٍت ظذ األوسذة ب١ٓ ِشظٝ اٌشم١مت باٌّماسٔت ِغ األصحاء حذٚد حسخٕخح اٌذساست بأْ أخفاض اٌحاٌت اٌذفاػ١ت اٌى١ٍت ظذ خ١ّغ اٌّدا١ِغ اٌؼالخ١ت. ذسفغ اٌحاٌت اٌذفاػ١ت اٌى١ٍت ظذ األوسذة واْ ٚاظحاً ػٕ ٟ ػالج صذاع اٌشم١مت باسخخذاَ ِعاداث األوسذة ِثً األوسذة ٠ّىٓ أْ ٠ىْٛ ػاِالً فٟ سبب١ت داء اٌشم١مت ٚأْ ححس١ٕٙا لذ ٠ىْٛ را فائذة ف ف١خا١ِٓ ٘ـ أٚ ا١ٌّالح١ٔٛٓ ِعافتً إٌٝ ػالخاث داء اٌشم١مت اٌخم١ٍذ٠ت. Introduction The pathophysiological mechanisms of migraine have been discussed for years to be involving the humoral-vascular and neurogenic theories (1) . The constriction of cortical vessels may explain the aura, while dilation of extracortical (meningeal) vessels may underlay the throbbing headache (2) . Cerebral hypoxia secondary to vasospasm or platelet aggregation may explain impaired vision predisposing migraine headache (3) .Causative factors for cerebral vasospasm could be the alteration in ion concentration (low intracellular Mg +2 ) (4, 5) , abnormally released serotonin secondary to platelets aggregation (6) , endothelin receptor gene polymorphism (7) , impaired mitochondrial oxidative metabolism and altered nitric oxide synthesis and release (8,9,10) . The neurogenic theory is strongly postulated without excluding aspects of the humoral theory (2) . The familial nature of migraine is greatly linked to the hereditary abnormality of monoaminergic transmission (11) . This transmission is vulnerable to sudden changes in internal or external environment to emotional stress, or to overload of afferent systems by excessive glare, smell or other stimuli. Triggering factors, thus could induce a phase of excessive discharge followed by a state of monoamine depletion, hence pain gates would be opened, giving rise to spontaneous pain in the head and neck (12) . Many evidences suggest the involvement of serotonin and norepinephrine to be the monoamines of great interest in the pathophysiology of migraine; the effectiveness of anti-migraine drugs like methysergide , pizotifen (serotonin receptors antagonists) and β-blockers (block the action of norepinephrine) solidify such suggestion (13,14) . Beside that, the level of serotonin metabolities (5-hydroxyindolacetic acid) is highly elevated in plasma from patients during acute migraine (2) . Change in serotonin level can provoke a neurovascular reaction which involve not only constriction or dilation of cerebral and extracerebral blood vessels but also activate nociceptive trigeminovascular system, an effect enforced by releasing vasoactive neuropeptides (substance P, neurokinin A or calcitonin gen- related polypeptide) ended with sterile or neurogenic inflammation (15) . These events were shown to be blocked by sumatriptan or dihydroergotamine (16,17) . Calcium channel blockers on the other hand may diminish vasoconstriction whether produced by humoral agents or by intrinsic monoamine pathways; while non-steroidal anti-inflammatory drugs (NSAIDs) presumably suppress the sterile inflammatory response in vessel walls (18, 19) . Many authors believe that sterile inflammation within the trigeminovascular system is of great importance in the pathophysiology of migraine headache. This postulate the release of the neurotransmitter substance P, vasodilation, increased vessel permeability, edema of cranial blood vessels and sensitization of sensory nerve endings (20) . Biochemical mediators, like nitric oxide and prostaglandins (PGs) may participate in such scenario; where PGE2 and TXA2 levels are shown to be elevated in saliva from patients with migraine headache during acute attacks (21) . Furthermore, nitric oxide has the potential to induce oxidative stress by acting as a free radical through the peroxidation pathway (22) . The concept of oxidative stress has been accepted in the increasing association of diseases with advanced age. The plausible explanation for such association is based on the implication of free radicals in the pathogenesis of several disorders like cancer and atherosclerosis (23,24,25) .Oxidative stress, however, results from distorted equilibrium of pro-oxidant/anti-oxidant system in intact cell. Such system continuously generates and detoxifies oxidants during normal aerobic metabolism (26) . Outbalancing such equilibrium leads to damage of lipids, proteins, carbohydrates and nucleic acids; these events might deplete cellular stores of anti-oxidants material such as glutathione and vitamin E (27) . Treatment of acute attacks of migraine is achieved with the use of ergotamine. The effectiveness of ergotamine in treatment of migraine attacks was thought to be related to its α-antagonistic activity; in addition the drug is Iraqi J.Pharm.Sci., Vol.16 (2) ,2007 Ergotamine, vitamin E, melatonin and total antioxidants 73 known to stimulate 5-HTD1 receptors of the cerebrovascular system. However; several limitations may restrict the use of ergotamine for migraine patients, these include: pregnancy; sepsis; hypertension; cerebral, coronary and peripheral vascular diseases; hepatitis; and renal insufficiency. In addition, the drug has many side effects such as abdominal cramps, parasthesia, nausea and tightness of the chest (2 ) . Melatonin, an endogenous neurohormon, shows a promising effect in relieving migraine headache by means of reducing the severity, frequency and duration of migraine attacks (28) . The mechanism by which melatonin exert such effect has yet been proven, but this agent possessing many pharmacological properties like scavenging free radicals (29) , inhibiting nitric oxide synthase (30) , regulating neurovascular system (31) and modulating serotonin actions (32) . In the view of the association of various vascular disorders with oxidative stress (33) and because one of the theories of migraine postulate the change in cerebrovascular milieu, the present study was conducted to investigate the involvement of changed anti-oxidant defense (measured as total antioxidant status) during migraine attack and the possible modulation of such status by classical anti-migraine therapy (ergotamine), antioxidants (vitamin E and melatonin) and their combination. Materials and Methods Materials: Ergotamine tartrate (as Cafergot ® 1mg tablets, Novartis, Switzerland), Vitamin E (as 400 capsules, Cipla, India), Melatonin (as 3mg tablets, American Nutri-ceutical, USA), Total antioxidant status (TAS) Kit, (Randox Laboratories Ltd, UK). Patients Twenty-three normal subjects and 21 migraine patients with age range of (17-45) years were enrolled in this study for three months. For inclusion, patients had to have a long-term history of migraine with and without aura diagnosed according to neurologist decision at specialized neurological centers and were managed under neurologist supervision. Patients who are smokers, alcoholics or those with other apparent disease were excluded. No changes in patients’ medications were made during the study and patients were instructed to keep taking their medications. Migraine patients were divided into three treatment groups; first group treated with ergotamine (1mg) alone, second group with ergotamine (1mg)/vitamin E (400mg) and third group with ergotamine (1mg)/melatonin (3mg). All groups were advised to take their treatments during attacks. Blood samples were drawn from migraine patients and normal subjects before initiation of therapy and after pain has been relived (from migraine patients only) for the investigation of TAS using Randox TAS kit. Method of measurement was followed according to the instructions mentioned in Randox TAS kit. Data were expressed as mean ± standard deviation and differences between means were analyzed by Student’s t- test. P values less than 0.05 were considered significantly different. Results Table (1) and figure (1) showed that TAS was significantly lower in migraine patients (P<0.05) in comparison to control (normal subjects) with a percent reduction in TAS was ranged from 35.46% to 43.97%. However, there is no significant difference in the level of TAS among migraine patients (P>0.05). Table (1) clearly showed that ergotamine rise significantly the level of TAS by 157%. Interestingly, the addition of vitamin E and melatonin greatly raised TAS by 179% and 176% respectively. This accompanied by shortening the time required for pain to alleviate. Ergotamine treated group required (6.28 ± 3.77 hr) for the pain to alleviate; while ergotamine/vitamin E and ergotamine/melatonin required (4.84 ± 3.6 hr) and (2.77 ± 1.4 hr) respectively. However; only melatonin showed significant difference (P<0.05) in the time required for the pain to alleviate among other therapies. Although the percent improvement in TAS in patients treated with ergotamine/vitamin E and ergotamine/melatonin appears to be similar, there is a significant difference toward the superiority of vitamin E over melatonin when added to ergotamine. In this regard, it seems that the addition of melatonin did not significantly improve TAS over that produced by ergotamine alone (figure 1). Iraqi J.Pharm.Sci., Vol.16 (2) ,2007 Ergotamine, vitamin E, melatonin and total antioxidants 23 Table (1): Effect of treatments of migraine patients with ergotamine and its combination with vitamin E and melatonin on serum TAS. Serum Total Antioxidant Status (mmol/L) Control (n=23) Ergotamine (n=7) Ergotamine/ Vit E (n=8) Ergotamine/ Melatonin (n=6) Pre- treatment 1.41 ± 0.22 a 0.86 ± 0.15 b 0.91 ± 0.15 b 0.79 ± 0.12 b Post- treatment --- 1.35 ± 0.25 * a 1.63 ± 0.09 * b 1.39 ± 0.80 * a Data are resented as mean ± SD.n= number of patients.*P<0.05 with respect to pre-treatment value. Non-identical superscripts (a,b) among different groups considered significantly different, P<0.05. Fig. (1): Serum total antioxidant status in control subjects and migraine patients treated with ergotamine (n=7), Ergotamine/Vitamin E (n=8) and Ergotamine/Melatonin (n=6). Data are presented as mean ± SD. *P<0.05 with respect to pre-treatment value (by paired Student’s t-test). Non-identical superscripts (a,b) considered significantly different, P<0.05 (by unpaired Student’s t-test). Discussion: Free radicals in the brain and the implication of oxidative damage caused by them have recently being implicated to playing possible role in the pathogenesis of migraine headache. The greater reduction in TAS seen in this study among migraine patients in comparison to control healthy subjects suggests the presence of generalized decrease in antioxidant defense elements. TAS enables assessment of integrated antioxidant system which encompasses all biological components with antioxidant activity. Reduction in TAS has been implicated in several disease stats such as cancer, ischemic heart diseases and poor nutritional stats (34, 35) . The most convincing evidence for free radical activity comes from nitric oxide, which is a potent vasodilator and is an important biochemical in the trigeminal-vascular peripheral mechanism of migraine headache (36,37) . Infusion of NO donor (glyceryl trinitrate and sodium nitroprusside), immediately induce headache both in healthy subjects and patients suffering from primary headaches with higher intensity in maigiane patients; while slow injection of the nonspecific inhibitor of NO synthase (L- NAM) reduced the spontaneous activity in all neurons (8,38,39) . Koulchitsky and coworkers, suggest that NO can induce activation of central trigeminal neurons and that endogenous release of NO may contribute to the ongoing activity of these neurons. The delayed changes in neuronal activity may include gene expression of pro-nociceptive mediators (46) . In addition, migraine can be induced via a cGMP-dependent mechanism. Kruuse and coworkers showed that sildenafil significantly induce migraine symptoms and propose that triggering mechanisms may reside within the perivascular sensory nerve terminals or the brainstem (41) . NO may interact with other mechanisms for the precipitation of migraine. Strecker and coworkers showed that NO increases meningeal blood flow, an action depends partly on the release and vasodilatory action of calcitonin gene-related peptide (CGRP) from dural afferents; while, prostaglandins show only minimal interaction with NO in this respect (15) . Furthermore, platelet levels of nitric oxide, as well as nitric oxide metabolites such as nitrate/nitrite, are increased in migraine patients and rise further during attacks (9,10) . Therefore, free radical scavengers may provide a potential molecular basis for prophylactic antimigraine therapy by neutralizing nitric oxide overproduction and possibly preventing formation of highly toxic peroxynitrite. Interesting results were observed in this study by observing greater 0 0.4 0.8 1.2 1.6 2 2.4 T A S ( m m o l / L ) Pre-treatment Post-treatment Control Ergotamine Ergotamine/ Vit E Ergotamine/ Melatonin * * * ba a Iraqi J.Pharm.Sci., Vol.16 (2) ,2007 Ergotamine, vitamin E, melatonin and total antioxidants 22 elevation of TAS by anti-migraine treatments. In deed, the addition of antioxidants (vitamin E or melatonin) to the traditional treatment (ergotamine) greatly potentiate the effect . Although the present study did not concerned with the mechanism of such an elevation in TAS, it seems that the vasoconstriction effect of ergotamine may involved these events. However, the link between effect of ergotamine and the elevation in TAS is the spotlight for further investigation. Vitamin E is powerful antioxidant that scavenges free radicals within the lipid phase of the cell (42) . In addition, it have a structural role in stabilizing membranes (43) , thus nowadays, vitamin E is implicated in the therapy of many diseases (44, 45) . Melatonin, beside its known antioxidant activity, it was shown to possess the ability to minimize the intensity, frequency and duration of acute migraine attacks (28) . In addition, melatonin has many pharmacological effects, of them is the inhibition of nitric oxide synthase (30,46) , whether this feature is involved in the relief of migraine headache or in the elevation of TAS this is require further investigation. In our study, melatonin showed no superiority to vitamin E when added to ergotamine with high standard deviation. This may be explained by administration of single rather than maintenance dose of melatonin together with greater fluctuation in plasma melatonin level due to diurnal variation (melatonin plasma level is 10 times higher during night that during the daytime) (47) . The age is also an important factor; as we got older the plasma concentration of melatonin decline sharply (48) . The present study concluded that the decrease in TAS can be implicated in the pathophysiology of migraine. In addition, enhancement of antioxidant system can add a beneficial effect for the management of migraine headache with the use of antioxidants with classical anti-migraine drug. Acknowledgments The authors grateful Dr. Zuhair Abdul Gafoor (neurologist) and Dr. Ibrahem Adham, Dr. May AL-Sabag, and Dr. Haider Fakhry (Clinical Pharmacist) for their kind help in performing this study. This research is abstracted from High Diploma dissertation submitted to clinical pharmacy department . References 1. Crowell, G.F. and Carlin, L.I.: Neurologic complications of migraine. Am. Fam. Physicn. 1982; 26: 139-148. 2. Rang, H.P.; Dale, M.M. and Ritter, J.M.: Clinical conditions in which 5-HT plays a role. 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