Indian Journal of Experimental Biology Vol. 48, August 2010, pp. 858-860

Effect of combined treatment of thioperamide (THP), a selective H 3 receptor antagonist, is reported with some antiepileptic drugs on methionine- to have antiepileptic effects against a wide range of experimental models including the maximal electro- sulfoximine induced convulsions in mice 4 shock (MES) test , pentylenetetrazole (PTZ)-induced 5 4 1* 1 2 seizures , amygdaloid kindling etc. Such effects of Divya Vohora , Razia Khanam , Shanthi N Pal & K K Pillai 1 THP are known to be mediated via enhancement 1 of neuronal histamine release through an action on Department of Pharmacology, Faculty of Pharmacy, Jamia Ham- 5 dard, New Delhi 110062, India H3 receptors . Methionine-sulfoximine (MSO), a 2Health Technology & Pharmaceuticals, World Health Organiza- convulsant, increases the activity of histamine 6 tion, Geneva-27, Switzerland N-methyl transferase , the enzyme primarily responsible for the catabolism of histamine 7. There Received 5 October 2009; revised 29 March 2010 are various reports of agents inhibiting histamine N-methyl transferase to have anticonvulsant effects by Methionine-sulfoximine (MSO), a convulsant is known to increasing the brain histamine levels 2,8 . An example increase the activity of histamine N-methyl transferase. The effect 8 of a selective H3 receptor agonist R- ( α) methylhistamine of such an agent is metoprine . Thus, it is possible (RAMH) and antagonist (thioperamide, THP) and some that MSO, by increasing the activity of histamine antiepileptic drugs (gabapentin and sodium valproate) have been N-methyl transferase and hence decreasing brain evaluated on MSO-induced convulsions in mice. The effect of histamine, is responsible for its convulsant effects. THP was also evaluated in combination with these antiepileptic drugs. Sodium valproate (300 mg/kg, po) and gabapentin To test this hypothesis, the effects of an H 3 receptor (400 mg/kg, po) offered protection against MSO-induced convul- antagonist, THP, which acts as an anticonvulsant sions as evidenced by a significant prolongation of latency to via histaminergic mechanism, have been evaluated abnormal dorsoflexion and complete protection against mortality on MSO-induced convulsions. In addition, the effect within 6 h of administration. THP (15 mg/kg, ip) alone and in of R- ( α) methylhistamine (RAMH), a selective combination with sub-effective doses of gabapentin (75 mg/kg, po) and sodium valproate (75 mg/kg, po) revealed no significant H3 receptor agonist, was also investigated in the differences from the control group or either drug alone. Hence, MSO model. the convulsant action of MSO does not appear to be mediated Several studies indicate that GABA plays a role via histaminergic mechanisms. in the convulsive mechanisms of MSO 9,10 . Hence, the Keywords : Gabapentin, Methionine-sulfoximine, Sodium valproate, present study also evaluated the effects of combination Thioperamide of THP with antiepileptic drugs (AEDs) primarily acting via GABAergic mechanisms [sodium valproate Histamine is considered to be an anticonvulsant (SVP) and gabapentin (GBP)] on MSO-induced inhibitory neurotransmitter 1. Drugs that deplete brain convulsions in mice. The latter has not been evaluated histamine (for example, histidine decarboxylase for its effects on MSO-induced convulsions. inhibitors etc) are considered to have proconvulsant Eleven groups of Swiss strain male albino mice effects in experimental animals. On the other hand, (20-25 g) were used for the study. The guidelines of agents that enhances brain histamine levels (histamine the University Animal Ethics Committee were itself or histamine precursor l-histidine etc) show followed during the study. All animals were housed in potent anticonvulsant effects in experimental models 1,2 . cages kept at 23°-30°C with a natural light-dark cycle. Presynaptic control via histamine H 3 receptors They had free access to standard pellet diet (Amrut (autoreceptors) is an important mechanism of Laboratory Rat and Mice feed, Navmaharashtra histamine-mediated neurotransmission 3. Thioperamide Chakan Oils Mills Ltd. Pune, India) and tap water. —————— Convulsions were induced by MSO following the *Correspondent author method of Blizard and Balkoski 11 . The convulsant Telephone: 91-11-26059688, Ext 5657 dose of MSO employed by other workers ranged Fax: 91-11-26059663 9,11 E-mail: [email protected] from 100-300 mg/kg, ip . In the present study, a [email protected] median dose (200 mg/kg, ip) was used. This dose NOTE 859

produced mortality in 100% of animals within 6 h of affected MSO-induced convulsions. The latencies to administration. As the pilot experiments revealed a head twitches/ clonic jerks, tonic HLE and percent latency of 2-3 h, observations were begun 2 h after mortality were not significantly different from the MSO injection and continued for up to 4 h. The control group. SVP (300 mg/kg, ip) and GBP parameters included: (i) latency to abnormal (400 mg/kg, ip) offered protection against MSO- dorsoflexion (abrupt head twitches with clonic induced convulsions as evidenced by a significant jerking), (ii) latency to tonic hind limb extension prolongation of the latency to abnormal dorsoflexion (HLE), and (iii) mortality (%). MSO, THP and [F (6, 29) = 25.66, P<0.01, ANOVA; P<0.001, RAMH were procured from Sigma (USA), SVP from Dunnett's t test] and complete protection against Torrent, India and GBP from Intas, India. All drug mortality within 6 h of administration [ P<0.001, solutions were prepared fresh in distilled water except Chi-square test]. The latency to tonic HLE was also for RAMH, which was dissolved using pyrogen-free prolonged but it was found to be statistically sterile water for injection. All treatments were insignificant. The sub-effective dose (that offered no administered by intraperitoneal route (ip) in a volume protection) was found to be 75 mg/kg, ip for both not exceeding 10 ml/kg except RAMH, which was GBP and SVP. The combined treatment of THP given intracerebroventricularly (icv) following the (15 mg/kg, ip) with 75 mg/kg, ip of GBP and SVP method of Haley and McCormick 12 . The drug was revealed no significant differences from the control administered in a volume of 5 µl/mouse using a group or either drug alone. Hamilton's microlitre syringe in conscious animal. Both experimental seizures (MES, PTZ) and AEDs The site of injection was 2 mm from either side of the modulate brain histamine levels 13 . Consistent with midline on a line drawn through the anterior base of these findings, a decrease in histamine content the ears. of amygdala and hypothalamus has been reported The protective and sub-effective doses of SVP, following kindling 14 . A similar decrease in histamine GBP, THP and RAMH were as per earlier study 5. levels has also been reported in genetically epilepsy The pre-treatment timings were determined on the prone rats 15 . All these reports points toward an inverse basis of reported time of peak action and pilot relationship between brain histamine levels and experiments: 1 h for SVP, GBP and THP and 15 min epileptogenic activity. MSO is reported to deplete for RAMH 5. The data were analyzed by one-way brain histamine levels by enhancement of its ANOVA followed by Dunnett’s t-test except in case catabolism through a stimulatory action on histamine- of % mortality where Chi square test with Yate’s N-methyl transferase activity 6,7 . Since histaminergic correction was applied. agents that deplete brain histamine produces The results are summarized in Table 1. Neither proconvulsant effects in experimental animals, THP (15 mg/kg, ip) nor RAMH (5 µg/mouse, icv) whether this depletion of brain histamine by MSO Table 1 ―Interaction of sub-effective doses of thioperamide and antiepileptic drugs on methionine-sulfoximine-induced convulsions in mice

Treatments Dose (per kg) and route n Latency (mean ± SE, sec) Mortality with in 6 h of administration Head twitches/clonic jerking Tonic HLE (%) Distilled water 10 ml, po 6 123.33 ± 5.61 180.00 ± 6.23 100 THP 15 mg, ip 5 126.00 ±7.26 170.00 ± 8.00 100 RAMH 10 mg, icv 5 114.00 ± 4.56 184.00 ± 11.52 100 SVP 300 mg, po 5 299.00 ± 23.21* 364.00 ± 3.57 0** 75 mg, po 5 132.00 ± 5.01 195.00 ± 20.19 80 400 mg, po 5 274.00 ± 24.26* 337.00 ± 20.76 0** GBP 75 mg, po 5 136.00 ± 6.06 206.00 ± 18.24 100 15 mg, ip THP + 75 mg, ip 5 133.60 ± 6.95 200.00 ± 2.44 100 SVP 15 mg, ip THP + GBP 75 mg, ip 5 126.00 ± 8.17 196.00 ± 17.85 100 n = number of animals; HLE: Hind limb extension; SVP: Sodium valproate; GBP: Gabapentin; THP: Thioperamide; RAMH: R( α)-methylhistamine. P values: * <0.01; ** <0.001 860 INDIAN J EXP BIOL, AUGUST 2010

contributes to its convulsant effect is not known. 2 Vohora D & Pillai K. K. Epilepsy, in The third histamine Conversely, agents that inhibit histamine-N-methyl receptor selective ligands as potential therapeutic agents in CNS disorders , edited by D Vohora (CRC Press, Boca transferase (e.g. metoprine etc) thereby enhancing Raton) 2009, 305. brain histamine levels produce anticonvulsant effects 3 Arrang J M, Garbarg M & Schwartz J C, Autoinhibition of 8 in various animal models . In the present study, brain histamine release mediated by a novel class (H 3) of his- no modulation of MSO-induced convulsions by tamine receptor, Nature 302 (1983) 1. histaminergic modulators THP and RAMH alone or by 4 Harada C, Hirai T, Fujii Y, Harusawa S, Kurihara T & the combination of THP with AEDs was found. Kamei C, Intracerebroventricular administration of histamine H3 receptor antagonists decreases seizures in rat models of Therefore the convulsant action of MSO, which is epilepsia, Meth Find Exp Clin Pharmacol, 4 (2004) 263. 9 attributed to its modulatory effect on GABA synthesis , 5 Vohora D, Pal S N & Pillai K K, Thioperamide, a does not appear to be mediated via histaminergic selective histamine H3-receptor antagonist, protects against mechanisms. MSO is also known to increase ACh PTZ–induced seizures in mice, Life Sci, 66 (2000) 297. levels in rat brain 16 . However, even the latter effect 6 Schatz R A & Sellinger O Z, The elevation of cerebral 16 histamine-N-and catechol-O-methyltransferaze activitied by doesn’t contribute to its convulsant effects . L-methionine-dl-sulfoximine, J Neurochem, 25 (1975) 73. In the present study, both SVP and GBP 7 Schayer R W & Railly M A, In vivo formation and catabo- significantly protected mice against MSO-induced lism of histamine in mouse brain, J Neurochem, 17 (1970) convulsions. While SVP is known to have such 1649. effects, protective action of GBP in the MSO model 8 Tuomisto L, Tacke U & William A, Inhibition of sound in- is reported for the first time. Elevation of cerebral duced convulsions by metoprine in the audiogenic seizure susceptible rat, Agents and Actions, 20 (1987) 252. GABA levels is known to protect against a range of 9 Stone W E & Javid M J, Effects of prior administration of experimentally induced seizures. Bilateral injections methionine-sulfoximine on the thresholds of seizures induced of gamma-vinyl GABA in various brain regions in mice by 3-mercaptopropionic acid or pentylene tetrazole, were reported to protect against MSO-induced Biochem Pharmacol , 27 (1978) 2972. convulsions 10 . Several studies indicate that GABA 10 Toussi H R, Schatz R A & Waszczak B L, Suppression of plays a role in the convulsive mechanisms of methionine sulfoximine seizures by intra-nigral gamma-vinyl 9,10 injection, Eur JPharmacol, 137 (1987) 261. MSO . Results of the present study for effects of 11 Blizard D A & Balkoski V, Tryptophan availability, central SVP and GBP alone provide additional evidence serotonergic function and methionine-sulfoximine-induced for attributing the convulsant effects of MSO to convulsions, Neuropharmacol , 21 (1982) 27. GABAergic mechanisms. 12 Haley T J & McCormic W G, Pharmacological effects pro- To conclude, SVP and GBP exhibited a duced by intracerebral injections of drugs in the conscious Br J Pharmacol protective action against MSO-induced convulsions. mice, , 12 (1957) 12. 13 Vohora D, Pal S N & Pillai K K, Histamine and selective H 3- The latter were not modulated by THP and RAMH receptor ligands: A possible role in the mechanism and alone or by the combination of THP with SVP or management of epilepsy, Pharmacol Biochem Behav, 68 GBP. There is no evidence for the mediation of MSO- (2001) 735. induced convulsions via histaminergic mechanisms. 14 Kamei C, Ishizawa K, Kakinoki H & Fakunaga M, Financial support by All India Council for Histaminergic mechanisms in amygdaloid kindled seizures rats, Epilepsy Res, 30 (1998) 187. Technical Education (AICTE), New Delhi is 15 Onodera K, Tumisto L, Tacke U & Airaksinen M, Strain gratefully acknowledged. differences in regional brain histamine levels between genetically epilepsy-prone and resistant rats, Meth Find Exp References Clin Pharmacol, 14(1992) 13. 1 Vohora D, Pal S N & Pillai K K, Histamine as an Anticon- 16 Olivier R & Tobias H, Methionine sulfoximine increases vulsant Inhibitory Neurotransmitter, Current Neuropharma- acetylcholine level in the rat brain: No relation with col , 2 (2004) 419. epileptogenesis, Neuroreport, 6 (1995) 2027.