Seizure: European Journal of Epilepsy
Volume 19, Issue 5 , Pages 300-302, June 2010

Trimetazidine exerts protection against increasing current electroshock seizure test in mice

Department of Pharmacology, University College of Medical Sciences & G.T.B. Hospital, University of Delhi, Delhi 110095, India

Received 1 September 2009; received in revised form 27 January 2010; accepted 1 April 2010. published online 05 May 2010.

Article Outline

Abstract 

Trimetazidine, a novel anti-ischemic agent, is used in the therapy of angina, vertigo and chorioretinal diseases. It has also been examined for its effect on nociception, inflammation and neuroprotection in various animal models. The present study was designed to investigate the effect of trimetazidine on electrically induced seizures in mice. Trimetazidine was administered orally in doses of 5, 10 and 20mg/kg (single dose) to observe its effect on the increasing current electroshock seizure (ICES) test in mice. Trimetazidine in 10 and 20mg/kg doses significantly raised the seizure-threshold current in the ICES test. Further, co-administration of per se ineffective dose of trimetazidine (5mg/kg, p.o.) with sub-anticonvulsant dose of nimodipine (10mg/kg, p.o.) and phenytoin (12.5mg/kg, p.o.) offered significant protection in the ICES test. These results indicate that trimetazidine possesses significant anticonvulsant activity against electro-convulsions in the mice.

Keywords: Trimetazidine, Anticonvulsant activity, Electroshock, Calcium

 

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1. Introduction 

Epilepsy is a complex neurological disorder that affects approximately 1% of the population worldwide. Currently available conventional antiepileptic drugs (AEDs) provide control in only 75–80% of epileptic patients.1 These agents mainly affect transport of ions across the cell membrane and/or modulate inhibitory or excitatory neurotransmission.2 A wide array of adverse effects associated with these agents are a major limitation for their long term use.3 Therefore, the development of a novel antiepileptic agent with better efficacy and minimal adverse effects would be an interesting prospect in the field of medical research.

Calcium ions (Ca2+) play an important role during epileptogenesis. It has been noticed that an increase in Ca2+ influx in the cell triggers seizurogenic activity in the neurons.4 Recently, Ca2+ channel blockers, particularly dihydropyridines per se and with AEDs have been shown to decrease the incidence of seizures in various experimental models of epilepsy.5, 6

Trimetazidine is a lipophilic piperazine derivative, a drug of interest in ischemic diseases. It has been found to improve symptoms and frequency of angina by maintaining Ca2+ homeostasis in the cells.7, 8, 9 In ischemic cardiomyopathy, trimetazidine has been found to prevent accumulation of calcium in cells by regulating exchange of ions across the membrane.10 It has demonstrated anti-oxidant, anti-inflammatory, antinociceptive and gastroprotective properties in various experimental animal models.11 Several studies have demonstrated that it protects not only heart8, 12 from ischemic injury but also brain,13, 14 lung,15 kidney,16 intestine17 and liver.18 A potent neuroprotective activity of trimetazidine has been observed against stroke in the gerbil model of transient global ischemia.14 In another study, trimetazidine was found to exhibit anti-excitotoxic activity in vestibular ganglionic neurons of rat. This anti-excitotoxic activity of trimetazidine was shown to be mediated via modulation of AMPA/kainate receptors.19 Most of these studies have focused on anti-ischemic profile of trimetazidine while data on its possible neurological actions especially on epilepsy are essentially lacking. Thus, the objective of the present study was to explore the possible effects of trimetazidine on electroshock-induced seizure in mice.

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2. Materials and methods 

2.1. Animals 

Swiss albino male mice weighing 24–35g were used in the study. Animals were procured from the Central Animal House, University College of Medical Sciences, University of Delhi, Delhi. Animals were housed in groups of six mice per cage (43cm×28.6cm×15.5cm) with a natural light/dark cycle and provided with free access to pellet diet and water. All the experiments were conducted between 9:30 a.m. and 6:30 p.m. in the neuropharmacology laboratory of the department. Procedures adopted during experiments on animals and their care were conducted in accordance with the guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India and was approved by Institutional Animal Ethics Committee, University College of Medical Sciences, University of Delhi, Delhi.

2.2. Drugs and dosing schedules 

The following drugs were used: trimetazidine (Trivedon-20mg tabs, Cipla Ltd., Mumbai, India), phenytoin (Sigma St Louis, MO, USA), and nimodipine (Nimodip 30mg tabs, USV Ltd., Mumbai, India). Trimetazidine, phenytoin and nimodipine were suspended with 1% carboxy methyl cellulose (CMC) in double-distilled water and administered once orally. Trimetazidine was administered in doses of 5, 10 and 20mg/kg while phenytoin was given in doses of 12.5 and 25mg/kg. Nimodipine was administered in doses of 10, 20 and 40mg/kg. Control groups were administered with 1% carboxy methyl cellulose. All drugs were administered in a volume of 10ml/kg body weight and the animals were tested 1h after drug administration.

2.3. Methodology 

2.3.1. Increasing current electroshock seizure test 

ICES test, as proposed by Kitano et al.20 and modified by Marwah et al.21 was used to determine seizure-threshold current (STC) for each animal. It is a widely used test to screen the anti- and pro-convulsant activity of a novel compound. The potential of an agent to increase seizure-threshold current can be assessed in this test. It was used in the present study to ascertain the efficacy of trimetazidine alone and in combination with phenytoin, a standard antiepileptic drug, and nimodipine, a calcium channel blocker, against electro-convulsions in mice.

Starting with a current of 2mA, electroshock was delivered to each mouse via ear electrodes as a single train of pulses (20Hz for 0.2s) with linearly increasing intensity of 2mA/2s using an electro-convulsiometer (Techno, India). The current at which tonic hind limb extension (HLE) occurred was recorded as the seizure-threshold current. If no tonic HLE was observed by the current of 30mA, electroshock was terminated and this cut-off current was used in the analysis.

2.4. Statistical analysis 

The results were expressed as mean±Standard Error of Mean (S.E.M.). Statistical analysis of the data was done using one-way analysis of variance (ANOVA) followed by post hoc Tukey's test. The p-values less than 0.05 were considered significant.

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3. Results 

3.1. Effect of trimetazidine on increasing current electroshock seizures test in mice 

Single dose administration of trimetazidine (10 and 20mg/kg), phenytoin (25mg/kg) and nimodipine (20 and 40mg/kg) significantly raised the seizure-threshold current as compared to control group. However, no significant elevation in seizure-threshold current was observed with administration of lower doses of trimetazidine (5mg/kg), nimodipine (10mg/kg) and phenytoin (12.5mg/kg) when compared with control.

The combined administration of trimetazidine and phenytoin in their sub-anticonvulsant doses, i.e. 5 and 12.5mg/kg, respectively caused significant increase in seizure-threshold current as compared to control, trimetazidine 5mg/kg and phenytoin 12.5mg/kg groups. Similarly, co-administration of trimetazidine (5mg/kg) and nimodipine (10mg/kg) in their sub-anticonvulsant doses significantly raised the seizure-threshold current when compared to control, trimetazidine (5mg/kg) and nimodipine (12.5mg/kg) alone treated groups (Table 1).

Table 1. Effect of trimetazidine, phenytoin, nimodipine and combinations on ICES test in mice.
Group (n=8)TreatmentDose (mg/kg, p.o.)STC (mA)
ICMC 1%10ml/kg14.2±0.45
IITrimetazidine515.5±0.50
IIITrimetazidine1018.7±1.46*
IVTrimetazidine2021.7±0.95#
VPhenytoin12.515.2±0.75
VIPhenytoin2524.7±0.36#
VIINimodipine1014.7±0.64
VIIINimodipine2019.0±1.30*
IXNimodipine4022.0±1.25#
XPhenytoin+trimetazidine12.5+525.7±0.59#,†,‡
XINimodipine+trimetazidine10+524.5±0.73#,†,¥

STC: seizure-threshold current; values are mean±S.E.M.

*P<0.01 vs. control (ANOVA followed by Tukey's test).

#P<0.001 vs. control (ANOVA followed by Tukey's test).

†P<0.001 vs. II group (ANOVA followed by Tukey's test).

‡P<0.001 vs. V group (ANOVA followed by Tukey's test).

¥P<0.001 vs. VII group (ANOVA followed by Tukey's test).

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4. Discussion 

Trimetazidine is a well-tolerated drug, indicated for clinical use in several ischemia related diseases.7, 8 Being lipophillic in nature it crosses the blood brain barrier.8 Trimetazidine inhibits the initiation of glutathione production and improves thiol component level in neuronal cells therefore, has shown beneficial effects on anti-oxidant enzymes in brain.22 Recently, it has demonstrated neuroprotective effect on crush injury of sciatic nerve in rats.23 Additionally, it has shown protection against animal models of inflammation, nociception, gastric injury11 and iron-induced epilepsy.24

In epilepsy it has been noticed that calcium plays an important role in generating seizures. Extensive literature has highlighted that Ca2+ is a primary mediator during epileptogenesis and status epilepticus-induced neurotoxicity.4, 25 It is well documented that epileptic depolarization of neurons is effectively suppressed by Ca2+ channel blocking agents. The Ca2+ channel inhibition by dihydropyridines exhibited protection in experimental models of seizures.6, 26 Several animal studies have reported that dihydropyridines by virtue of their brain penetrating property potentiate the protective efficacy of some antiepileptic drugs like phenytoin, carbamazepine, valproic acid etc.5, 25, 27 Various clinical reports have revealed beneficial effects of certain calcium channel blocking agents, i.e. nimodipine, cinnarizine and flunarizine as add-on treatment in epileptic seizures.28, 29

In the present study trimetazidine was tested in doses 5, 10 and 20mg/kg against electrically induced seizure in the mice. The results demonstrated that in ICES test trimetazidine in doses of 10 and 20mg/kg dose significantly increased the seizure-threshold current as compared to control group. Trimetazidine in a dose of 5mg/kg did not elicit protection when given alone against electrically induced seizure. However, co-administration of trimetazidine in 5mg/kg dose with sub-anticonvulsant dose of nimodipine 10mg/kg, a Ca2+ channel blocker significantly increased the seizure-threshold current as compared to control group. The protection demonstrated by trimetazidine in the present study is consistent with previous experimental reports where Ca2+ channel blockers have shown protection against various convulsive models of epilepsy.6, 27, 30 The additive effect obtained in the present study might be due to possible similar mechanism of action or due to interaction at blood brain barrier. Transport of various drugs at the blood brain barrier is mediated by many transporters. These drug efflux transporters thought to limit the drug distribution in brain parenchyma. Recently, it has been shown that adjuvant treatment with nimodipine restored the normal hippocampal concentration of phenytoin in phenytoin refractory epilepsy model in rats which is considered due to its P-gp inhibiting activity.31 In the present study the additive effect obtained might be due to increased concentration of these drugs at target site due to decrease efflux. Furthermore, the additive effect obtained may be due to possible similar mechanism of action. Nimodipine by inhibiting calcium channel has shown antiepileptic activity in various experimental models. Trimetazidine has also shown calcium antagonistic action. In a voltage clamp study using ventricular myocytes of guinea pig trimetazidine blocked the calcium channel and decreased the peak amplitude of calcium current.32 Furthermore, it decreased the calcium ion content in the cytoplasm of undifferentiated HL-60 cell, this inhibitory effect was considered due to blockade of Ca2+ channel.33

Many studies have demonstrated that conventional AEDs like phenytoin exert its anticonvulsant action partially due to interaction with voltage dependent calcium channels.34, 35 Schumacher et al.36 have observed that phenytoin altered the low voltage activated Ca2+ channels in cultured hippocampus neurons, rodent thalamic neurons and neuroblastoma cells. It partially antagonized the L-type Ca2+ current. In the present study the sub-anticonvulsant dose of trimetazidine was also co-administered with a sub-anticonvulsant dose of phenytoin. The combination has significantly increased the seizure-threshold current as compared to control group. These results are comparable with the effect obtained with trimetazidine when given alone in higher doses of 10 and 20mg/kg.

The findings of the present study suggest that trimetazidine possesses anticonvulsant activity against electroshock-induced seizure test in mice, possibly due to modulation of Ca2+ channels. However, our findings are preliminary and further chronic, biochemical and clinical studies are required to explore the exact mechanism of action of trimetazidine in epilepsy.

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Conflict of interest 

None.

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References 

  1. Kulak W, Sobaniec W, Wojtal K, Czuczwar SJ. Calcium modulation in epilepsy. Pol J Pharmacol. 2004;56:29–41
  2. Librowski T, Kubacka M, Meusel M, Scolari S, Muller CE, Gutschow M. Evaluation of anticonvulsant and analgesic effects of benzyl- and benzyhydryl ureides. Eur J Pharmacol. 2007;559:138–149
  3. Gupta YK, Malhotra J. Antiepileptic drug therapy in the twenty first century. Indian J Physiol Pharmacol. 2000;4:8–23
  4. Bading N, Hilmar M. Nuclear calcium-activated gene expression: possible roles in neuronal plasticity and epileptogenesis. Epilepsy Res. 1999;36:225–231
  5. Gupta MC, Garg SK, Das BP, Bhargava VK. Effect of nimodipine, a dihydropyridine calcium channel antagonist on the pharmacokinetics of carbamazepine in rhesus monkey. Indian J Physiol Pharmacol. 2003;47:347–351
  6. Kulak W, Sobaniec W. Anticonvulsant effects of calcium antagonists in the experimental seizures in the rat. Neurosci Jpn. 1993;19:107–112
  7. Albengres E, Tillement JP, Louet HL, Morin D. Trimetazidine: experimental and clinical update review. Cardiovasc Drug Rev. 1998;16:359–390
  8. Harpey C, Clauser P, Labrid C, Freyria JL, Poirier JP. Trimetazidine, a cellular anti-ischemic agent. Cardiovasc Drug Rev. 1989;6:292–312
  9. Iskesen I, Saribulbul O, Cerrahoglu M, Var A, Nazil Y, Sirin H. Trimetazidine reduces oxidative stress in cardiac surgery. Circ J. 2006;70:1169–1173
  10. Belardinelli R. Trimetazidine and the contractile response of dysfunctional myocardium in ischaemic cardiomyopathy. Rev Port Cardiol. 2002;19:35–39
  11. Abdel-Salam OME, El-Batran S. Pharmacological investigation of trimetazidine in models of inflammation, pain and gastric injury in rodents. Pharmacology. 2005;75:122–132
  12. Sambandam N, Lopaschuk GD, Brownsey RW, Allard MF. Energy metabolism in the hypertrophied heart. Heart Fail Rev. 2002;7:161–173
  13. Dhote V, Balaraman R. Anti-oxidant activity mediated neuroprotective potential of trimetazidine on focal cerebral ischaemia–reperfusion injury in rats. Clin Exp Pharmacol Physiol. 2008;35:630–637
  14. Iqbal S, Baziany A, Hussain M, James S, Wright S, Hemmings S, et al. Trimetazidine as a potential neuroprotection in transient global ischemia in gerbils: a behavioral and histological study. Brain Res. 2002;928:1–7
  15. Inci I, Dutly A, Rousson V, Bochler A, Weder W. Trimetazidine protects the energy status after ischemia and reduces reperfusion injury in a rat single lung transplant model. J Thorac Cardiovasc Surg. 2001;122:1155–1161
  16. Chander V, Singh D, Chopra K. Attenuation of glycerol-induced acute renal failure in rats by trimetazidine and deferoxamine. Pharmacology. 2003;67:41–48
  17. Tetik C, Ozden A, Calli N, Bilgihan A, Bostanci B, Yis O, et al. Cytoprotective effect of trimetazidine on 60min of intestinal ischemia-reperfusion injury in rats. Transpl Int. 1999;12:108–112
  18. Settaf A, Morin D, Lamchouri F, Elimadi A, Cherrah Y, Tillement JP. Trimetazidine ameliorates the hepatic injury associated with ischemia–reperfusion in rats. Pharmacol Res. 1999;39:211–216
  19. Dayanithi G, Gilles D, Travo C, Chabbert C, Sans A. Trimetazidine modulates AMPA/kainate receptors in rat vestibular ganglion neurons. Eur J Pharmacol. 2007;574:8–14
  20. Kitano Y, Usui C, Takasuna K, Hirohashi M, Nomura M. Increasing current electroshock seizure test: a new method for assessment of anti-and proconvulsant activities of drugs in mice. J Phamacol Toxicol Methods. 1996;35:25–29
  21. Marwah R, Pal SN, Pillai KK. Effect of fluoxetine on increasing current electro-shock seizures (ICES) in mice. Indian J Pharmacol. 1999;31:350–353
  22. Mironovan OP, Zarubina IV, Krivoruchko BJ, Smirnov AV. Antioxidant effects of amtizol and trimetazidine in brain ischemia. Patol Fiziol Eksp Ter. 2001;7:13–16
  23. Serarslan Y, Bal R, Altug ME, Kontas T, Keles ON, Unal D, et al. Effects of trimetazidine on crush injury of the sciatic nerve in rats: a biochemical and stereological study. Brain Res. 2009;1247:11–20
  24. Suzer T, Coskun E, Demir S, Tahta K. Lipid peroxidation and glutathione levels after cortical injection of ferric chloride in rats: effect of trimetazidine and deferoxamine. Res Exp Med. 2000;199:223–229
  25. Khosla P, Pandhi P. Anticonvulsant effect of nimodipine alone and in combination with diazepam and phenytoin in a mouse model of status epilepticus. Methods Find Exp Clin Pharmacol. 2000;22:731–736
  26. Shitak R, Sahai AK, Hota D, Chakrabarti A. Anti-seizure efficacy of nimodipine in pentylenetetrazole and kainic acid combined seizure models in mice. Indian J Physiol Pharmacol. 2006;50:265–272
  27. Luszczki JJ, Trojnar MK, Trojnar MP, Trojnar ZK, Szostakiewicz B, Zadrozniak A, et al. Effects of three calcium channel antagonists (amlodipine, diltiazem and verapamil) on the protective action of lamotrigine in the mouse maximal electroshock-induced seizure model. Pharmacol Rep. 2007;59:672–682
  28. Czuczwar SJ, Gasior M, Janusz W, Kleinrok Z. Influence of flunarizine, nicardipine and nimodipine on the anticonvulsant activity of different antiepileptic drugs in mice. Neuropharmacology. 1992;31:1179–1183
  29. Kulak W, Sobaniec W. Effects of cinnarizine, a calcium channel blocker on valproic acid concentration in epileptic children with epilepsy. Neurosci Jpn. 1994;20:187–190
  30. Dolin SJ, Hunter AB, Halsey MJ, Little HJ. Anticonvulsnat profile of the dihydropyridine calcium channel antagonists, nitrendipine and nimodipine. Eur J Pharmacol. 1988;152:19–27
  31. Hocht C, Lazarowski A, Gonzalez NN, Auzmendi J, Opezzo JAW, Bramgulia GF, et al. Nimodipine restores the altered hippocampal phenytoin pharmacokinetics in a refractory epileptic model. Neuroscience Letters. 2007;413:168–172
  32. Kiyosue T, Nakamura S, Arita M. Effects of trimetazidine on action potentials and membrane currents of guinea pig ventricular myocytes. J Mol Cell Cardiol. 1986;18:1301–1311
  33. Astashkin EI, Glezer MG, Grachev SV. Trimetazidine selectively inhibits SOC channels in plasma membranes of human HL-60 cells and does not affect the intracellular Ca2+ stores. Dokl Biol Sci. 2009;424:86–89
  34. Ali A, Pillai KK, Ahmad FJ, Dua Y, Vohora D. Anticonvulsant effect of amiloride in pentetrazole -induced status epilepticus in mice. Pharmacol Rep. 2006;58:242–245
  35. Stefani A, Spadoni F, Bernardi G. Voltage-activated calcium channels, targets of antiepileptic drug therapy?. Epilepsia. 1997;38:959–965
  36. Schumacher TB, Beck H, Steinhaiser C, Schramm J, Elger CE. Effects of phenytoin, carbamazepine and gabapentin on calcium channels in hippocampal granule cells from patients with temporal lobe epilepsy. Epilepsia. 1998;39:355–363

PII: S1059-1311(10)00072-5

doi:10.1016/j.seizure.2010.04.001

Seizure: European Journal of Epilepsy
Volume 19, Issue 5 , Pages 300-302, June 2010

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