|Year : 2011 | Volume
| Issue : 2 | Page : 253-258
Experimental re-evaluation of flunarizine as add-on antiepileptic therapy
Anamika Thakur1, AK Sahai1, JS Thakur2
1 Department of Pharmacology, IG Medical College, Shimla, Himachal Pradesh - 171 001, India
2 Department of Otolaryngology - Head and Neck Surgery, IG Medical College, Shimla, Himachal Pradesh - 171 001, India
|Date of Submission||02-Oct-2010|
|Date of Decision||10-Nov-2010|
|Date of Acceptance||24-Nov-2010|
|Date of Web Publication||12-May-2011|
Department of Pharmacology, IG Medical College, Shimla, Himachal Pradesh - 171 001
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Experimental studies have found several calcium channel blockers with anticonvulsant property. Flunarizine is one of the most potent calcium channel blockers, which has shown anticonvulsant effect against pentylenetetrazole (PTZ) and maximal electroshock (MES)-induced seizures. However, further experimental and clinical trials have shown varied results. We conducted a PTZ model experimental study to re-evaluate the potential of flunarizine for add-on therapy in the management of refractory epilepsy. Materials and Methods: Experiments were conducted in PTZ model involving Swiss strain mice. Doses producing seizures in 50% and 99% mice, i.e. CD 50 and CD 99 values of PTZ were obtained from the dose-response study. Animals received graded, single dose of sodium valproate (100-300 mg/kg), lamotrigine (3-12 mg/kg) and flunarizine (5-20 mg/kg), and then each group of mice was injected with CD 99 dose of PTZ (65mg/kg i.p.). Another group of mice received single ED 50 dose (dose producing seizure protection in 50% mice) of sodium valproate and flunarizine separately in left and right side of abdomen. Results were analysed by Kruskal-Wallis ANOVA on Ranks test. Results: As compared to control, sodium valproate at 250 mg/kg and 300 mg/kg produced statistical significant seizure protection. At none of the pre-treatment dose levels of lamotrigine, the seizure score with PTZ differed significantly from that observed in the vehicle-treated group. Pre-treatment with flunarizine demonstrated dose-dependent decrease in the seizure score to PTZ administration. As compared to control group, flunarizine at 20 mg/kg produced statistical significant seizure protection. Conclusion: As combined use of sodium valproate and flunarizine has shown significant seizure protection in PTZ model, flunarizine has a potential for add-on therapy in refractory cases of partial seizures. It is therefore, we conclude that further experimental studies and multicenter clinical trials involving large sample size are needed to establish flunarizine as add-on therapy in refractory epilepsy.
Keywords: Flunarizine, lamotrigine, PTZ, seizure, sodium valproate
|How to cite this article:|
Thakur A, Sahai A K, Thakur J S. Experimental re-evaluation of flunarizine as add-on antiepileptic therapy. J Pharm Bioall Sci 2011;3:253-8
Epilepsy is a challenge to medicine as complete seizure control is still a dream because of its varied etiopathogenesis. Calcium has been implicated in the pathophysiology of seizure. ,, The bursting activity is an important stage in the seizure activity and caused by influx of extracellular calcium, which leads to a relatively long-lasting depolarization of the neuronal membrane, opening the voltage-dependent sodium channels and generation of repetitive action potentials. Calcium channel also mediate multiple cellular effects including fusion of neurotransmitter-containing vesicles with the presynaptic terminal membrane, and allowing release of neurotransmitter.  This role of calcium in seizure activity leads to hypothesis that calcium channel blockers may prevent seizure spread.
Experimental animal studies have found several calcium channel blockers with anticonvulsant activity. ,,,,,,,, Flunarizine is most potent calcium channel blocker,  which has shown anticonvulsant effect against pentylenetetrazole (PTZ) and maximal electroshock (MES)-induced seizures. ,,, However, Fischer et al. found that flunarizine exhibit anticonvulsant effect against tonic seizures induced by electroshock or other chemoconvulsants in mice but did not protect against PTZ-induced clonic seizures.  Gasior et al. found that flunarizine (up to 80 mg/kg) neither protect PTZ-induced seizure individually nor enhance (up to 20 mg/kg) antiepileptic effects of valproate or ethosuximide.
Similarly, flunarizine has shown varied efficacy in various clinical trials. ,,,,, Few double-blind clinical trials have found significant seizure reduction with flunarizine ,,, while study by Alvin et al. found no statistical significant difference between flunarizine and placebo in their double-blind study. Pharmacologically, addition of calcium antagonist with conventional antiepileptic drugs should provide better seizure control. Animal studies allow assessment of a much larger range of doses and dose combinations than do clinical studies.  The primary goal of rational polytherapy is to match drugs with differing mechanisms of action, and basic evidence for these mechanisms of action of antiepileptic drugs (AEDs) is provided by the animal studies.  It is therefore, we conducted an experimental study to re-evaluate the potential of flunarizine as an add-on therapy in treatment of refractory epilepsy.
| Materials and Methods|| |
All experiments were carried out on adult, male Swiss strain mice weighing between 20 and 30 g. Animals were kept in polystyrene cages under controlled environmental conditions i.e. at a temperature of 25 ± 1 o C and a relative humidity of 60 ± 2% and exposed to 12-h photoperiod. They had free access to food (Hindustan Lever Chow) and water. In the present study, a group size of six mice was used for each experiment. All drugs were administrated intraperitoneal (i.p.) in a volume not exceeding 10 ml/kg.
The research protocol was approved by the institutional animal ethics committee and utmost care was taken to ensure proper handling of animals.
Drugs and chemicals
PTZ was obtained from Sigma Chemicals Company (USA). Sodium valproate was sourced from Torrent Pharmaceuticals Ltd., India, while lamotrigine and flunarizine were obtained from Cipla Pharmaceuticals Ltd., India. All other chemicals and reagents used in the study were of analytical grade.
Preparation of the drug solutions
PTZ solution was prepared by dissolving 112.5 mg of PTZ in 10 ml of 0.9% w/v normal saline. Sodium valproate was dissolved in normal saline in a strength of 60 mg/ml. Lamotrigine was suspended in 1% aqueous solution of Tween 80. Flunarizine was weighed under subdued lighting and dissolved in 50% polyethylene glycol and 50% distilled water. All drugs solutions were prepared afresh and appropriately diluted prior to administration to mice.
PTZ-induced seizures in mice
PTZ solution (1.125% in 0.9% w/v normal saline) was injected i.p. in graded, single doses (35-65 mg/kg) to seven groups of mice. Each group consisted of six mice. After injection, each mouse was kept in a 30 × 18 cm Plexiglas cage, at ambient temperature of 25 ± 1 o C in a quiet room, and observed for 1 h. The seizure score was recorded according to the following scale: 0: No response; 1: Ear and facial twitching; 2: 1-20 myoclonic body jerks in 10 min; 3: >20 body jerks in 10 min; 4: Forelimb convulsions; 5: Generalized clonic convulsions with episodes of rearing and falling down and; 6: Generalized epilepticus. The response of PTZ was considered positive when the seizure score was ≥3.  CD 50 (Convulsive dose; dose producing seizures in 50% mice) and CD 99 (dose producing seizures in 99% mice) values of PTZ were obtained from the dose-response study.
PTZ-induced seizures in sodium valproate, lamotrigine and flunarizine pre-treated mice
The study was undertaken in four groups of mice. Each group or subgroup consisted of six mice. The first group of mice comprised of four subgroups and received graded, single dose of sodium valproate (100, 200, 250 and 300 mg/kg i.p.) with pre-treatment time of 30 min. Then, each subgroup of mice was injected with CD 99 dose of PTZ (65 mg/kg i.p.). Second group of mice comprised of four subgroups and received graded, single dose of lamotrigine (3, 6, 9 and 12 mg/kg i.p.) with pre-treatment time of 30 min. Then, each subgroup of mice was injected with 65 mg/kg i.p. dose of PTZ. Third group of mice comprised of four subgroups and received graded, single dose of flunarizine (5, 10, 15 and 20 mg/kg i.p.) with pre-treatment time of 30 min. Then, each subgroup of mice was injected i.p. with 65 mg/kg of PTZ. Approximately 0.2 ml of vehicle was injected i.p. in respective control groups and then 65 mg/kg i.p. dose of PTZ was injected. After injection with PTZ, each mouse was placed individually in 30×18 cm Plexiglas cage and observed for one hour for recording of seizure score (0-6 scale as mentioned earlier). The experiment provided the seizure score for PTZ, and that of PTZ in groups of mice pre-treated with sodium valproate, lamotrigine and flunarizine. Further, it provided ED 50 (effective dose; dose producing seizure protection in 50% mice) values of sodium valproate and flunarizine against PTZ-induced seizures. The fourth group of mice received single ED 50 dose of sodium valproate and flunarizine i.p. separately in left and right side of abdomen. After pre-treatment time of 30 min, PTZ (65 mg/kg i.p.) was injected. This combination was given after a wash out period of 18 days because of long half-life of flunarizine. Percentage of seizure protection was determined by failure to observe even a single episode of clonic spasm of at least five seconds duration for a period of one hour.
CD 50 and ED 50 values were calculated by least square regression analysis of log dose versus probit values for PTZ, sodium valproate and flunarizine. Raw data on seizure score was collected in Microsoft Excel sheet, and descriptive statistical analysis was done. The seizure score was expressed as mean ± standard error of mean (Mean ± SEM) for the different groups of mice and analysed by Kruskal-Wallis ANOVA on Ranks test. Statistical results with P<0.05 were considered significant.
| Results|| |
Sodium valproate at doses of ≥182 mg/kg produced ataxia, mild sedation and hypotonia, while there was mild motor in-coordination with lamotrigine at ≥6 mg/kg. Animals had no behavioral changes with flunarizine at a dose of 10 mg/kg but a dose of 20 mg/kg caused moderate motor in-coordination as compared with control. On co-administration of sodium valproate and flunarizine, restriction of motor activity, ataxia and hypotonia were slightly more apparent as compared to individual drugs. There was no mortality in 24 h of observation time.
[Table 1] shows dose-response study with PTZ in seven groups of mice. Data is expressed in terms of seizure score (Mean ± SEM) against graded doses of PTZ ranging from 35 to 65 mg/kg. Seizure score was 0.00 with 35 mg/kg i.p. dose of PTZ, while it increased to 5.3 ± 0.21 at 65 mg/kg i.p. dose. This seizure score data showed graded response at the intervening dose levels.
|Table 1: Dose-response study with PTZ in mice (n=6; seizure score=0-6 scale; observation time one hour)|
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[Figure 1] describes the graded log dose-probit curve derived from the percentage of positive responders against seven graded doses of PTZ ranging from 35-65 mg/kg administered i.p. to seven groups of mice. PTZ at 35 mg/kg i.p. dose had zero percent positive responders, intervening doses had graded response, and dose of 65 mg/kg produced 100% positive responders. CD 50 and CD 99 doses of PTZ were found as 48.9 mg/kg and 65 mg/kg, respectively.
|Figure 1: Log dose-probit curve of PTZ drawn from Table 1. Each probit value corresponds to 'percentage of positive responders' showing seizure score ≥3 to PTZ|
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Effect of graded doses of sodium valproate, lamotrigine and flunarizine on PTZ (65 mg/kg i.p.) induced seizures in mice [Table 2]
PTZ at a dose of 65 mg/kg i.p. resulted in a seizure score of 4.83 ± 0.17 in the vehicle-treated group. Pre-treatment with sodium valproate (100-300 mg/kg) demonstrated dose-dependent decrease in the seizure score to PTZ administration. As compared to control (seizure score 4.83 ± 0.17), sodium valproate at 250 mg/kg and 300 mg/kg produced statistical significant (P<0.05) seizure protection with seizure score of 2.50 ± 0.50 and 1.83 ± 0.31, respectively. All the used doses (3-12 mg/kg i.p.) of lamotrigine showed insignificant seizure score with PTZ in comparison to the vehicle-treated group. Pre-treatment with flunarizine (5-20 mg/kg i.p.) demonstrated dose-dependent decrease in the seizure score to PTZ administration. As compared to control group (seizure score 4.83 ± 0.17), flunarizine at 20 mg/kg produced statistical significant (P<0.05) seizure protection (seizure score: 2.17 ± 0.40). ED 50 values of sodium valproate and flunarizine were 182 mg/kg and 10 mg/kg, respectively [Figure 2] and [Figure 3].
|Figure 2: Log dose-probit curve of sodium valproate against CD99 dose (65 mg/kg i.p.) of PTZ in mice drawn from Table 2. Each probit value corresponds to 'percentage of positive responders' showing seizure score ≥3 to PTZ. ED50 value of sodium valproate was obtained as 181.97 mg/kg i.p.|
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|Figure 3: Log dose-probit curve of flunarizine against CD99 dose (65 mg/kg i.p.) of PTZ in mice drawn from Table 2. Each probit value corresponds to 'percentage of positive responders' showing seizure score ≥3 to PTZ. ED50 value of flunarizine was obtained as 10 mg/kg i.p.|
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|Table 2: Effect of graded doses of sodium valproate, lamotrigine and flunarizine on PTZ (65 mg/kg, i.p.) induced seizures in mice, n=6, Seizure scoring scale=0-6, Pretreatment time =30 min, Observation time=1 h|
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Effect of ED 50 doses of sodium valproate, flunarizine and co-administration on PTZ [Table 3]
Individual administration of ED 50 doses of sodium valproate (182 mg/kg) and flunarizine (10 mg kg) showed insignificant (P>0.05) seizure score reduction while co-administration of ED 50 doses of these two drugs produced statistical significant (P<0.05) reduction in seizure score.
|Table 3: Effect of combining ED50 dose of sodium valproate with ED50 dose of flunarizine seizure score in PTZ-induced seizures|
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| Discussion|| |
Epilepsy is one of the most common chronic neurological disorder affecting more than 50 million people worldwide. About one-third of these patients continue to have seizures despite adequate AEDs and this condition is termed as refractory or drug-resistant epilepsy. 
Valproate is a well-established AED prescribed worldwide. It controls the seizure activity by prolonging recovery of voltage-activated Na + channel from inactivation, reducing low threshold calcium current, stimulating GABA synthetic enzyme and inhibiting GABA degradative enzymes. Valproate inhibits tonic hind limb extension in MES seizure and kindled seizures at non-toxic doses. Valproic acid at sub-toxic doses inhibits clonic motor seizure induced by PTZ. Its efficacy in diverse models parallels its efficacy against absence as well as generalized tonic clonic seizures in human,  and this study corroborates these findings as sodium valproate at 250 mg/kg and 300 mg/kg doses produced statistical significant seizure reduction.
We observed that lamotrigine had no significant effect in PTZ-induced seizures at 3-12 mg/kg. These finding are comparable to the established fact that although lamotrigine suppress seizures in MES and kindled models but it does not suppress clonic motor seizures in PTZ model.  Lamotrigine blocks sustained repetitive firing of mouse spinal neurons and delays the recovery from inactivation of recombinant sodium channels. It inhibits the synaptic release of glutamate by acting at sodium channels themselves.  PTZ acts as convulsant by increasing glutamate levels,  whereas lamotrigine decreases it therefore, no significant anticonvulsant effect of lamotrigine was observed in PTZ model.
In this study, we used flunarizine in four doses (5, 10, 15 and 20 mg/kg) but statistical significant seizure reduction was seen with dose of 20 mg/kg only. These results were comparative to various studies showing anticonvulsant properties of flunarizine in experimental models of epilepsy. ,,
Flunarizine is a di-fluorinated piperazine derivative, and act as selective calcium entry blocker.  It has analogous mechanism of action like valproic acid on sodium channel but is ineffective against GABAergic inhibition.  It provides a direct neuro-protective effect against the damaging influx of calcium and prevents neuronal damage because of PTZ-induced seizures.  It reduces the trans-membrane fluxes of calcium in situation where calcium is stimulated to enter the cell in excess, thus preventing the deleterious consequences of calcium overload within the cell.  It readily crosses the blood-brain barrier and inhibits entry of calcium into neurons primarily under pathophysiological conditions, such as ischemia or seizure activity  without any effect on normal calcium hemostasis.
We observed statistical significant seizure reduction by sodium valproate at 250 and 300 mg/kg doses while flunarizine produced similar effect at 20 mg/kg. In this study, ED 50 doses of sodium valproate and flunarizine were 182 mg/kg and 10 mg/kg. On co-administration of ED 50 doses of sodium valproate and flunarizine, we found statistical significant (P<0.05) seizure reduction. This can be explained by the common target of action of these two drugs resulting in synergistic effect at calcium/sodium channels. These results are comparable to the experimental study done by Shantilal et al, which provided evidence for a synergistic effect of sodium valproate and flunarizine in experimental absence seizures and possible potential benefit in pharmaco-resistant seizures. In another study,  interaction of flunarizine with sodium valproate or ethosuximide in gamma-hydroxybutyrate induced absence seizures in rats showed an additive effect of sodium valproate and ethosuximide with flunarizine.
Clinical trials on flunarizine as add-on therapy has shown varied results. Overweg et al. performed a double-blind placebo-controlled crossover trial. Flunarizine was given as add-on therapy in 30 therapy-resistant adult patients. A lower seizure incidence was found with flunarizine as compared to placebo in 20 patients. Eleven patients showed improvement by 25% or more with flunarizine. Seven patients had a seizure reduction of over 50%. Moglia et al. and Froscher et al. also found significant seizure frequency reduction in patients with flunarizine as add-on therapy in double-blind placebo-controlled trials. Starreveld et al.  conducted an eight-month double-blind cross-over add-on trial with a daily dose of 15 mg flunarizine in 25 patients with long-standing therapy resistant epilepsy. They found 35% mean seizure reduction in patients on flunarizine. In another clinical trial of 47 adult patients with intractable epilepsy, flunarizine reduced seizure in patients with secondarily generalized epilepsy and neurological deficit. Flunarizine at 15-20 mg daily produced maximum seizure reduction. They concluded that increased seizure protection warrant further clinical trials.  Alvin et al. found no statistically significant difference between flunarizine 15 mg daily and placebo as adjunct therapy in total seizure frequency in a double-blind placebo-controlled study in 22 patients of complex partial seizures. Chaisewikul et al. conducted a systematic review of 11 studies reporting randomized placebo-controlled add-on trials of calcium antagonists in patients with drug-resistant epilepsy between 1966 and 2009. They concluded that flunarizine might have a weak effect on seizure frequency but should not be recommended for use as an add-on treatment due to significant withdrawal effect.
Although, clinical trials have shown varied results but these trial had few limitations. It is becoming clear that normal and epileptic patients respond differently as these epileptic patients have developed resistance due to prolonged and numerous AEDs use. ,, Small sample size and single institutional setting are the second drawback in majority of these clinical trials.
Rationale of polytherapy is to combine drugs to provide additive efficacy and antagonistic toxicity  as it is becoming increasingly apparent that several AEDs act in multiple ways that are not yet understood fully.  In order to support this assumption experimentally, flunarizine was combined with established antiepileptics (valproate and lamotrigine) and was found effective in PTZ model.
| Conclusion|| |
In this study, we observed that sodium valproate at 250 mg/kg and 300 mg/kg produced statistical significant (p<0.05) seizure protection with seizure score of 2.50 ± 0.50 and 1.83 ± 0.31, respectively, while flunarizine produced statistical significant seizure protection at 20 mg/kg. Individual administration of ED 50 doses of sodium valproate (182 mg/kg) and flunarizine (10 mg kg) showed non-significant seizure score reduction (2.90 ± 0.54 and 3.07 ± 0.61, respectively) while co-administration of ED 50 doses of these two drugs produced statistical significant (p<0.05) reduction in seizure score (1.17 ± 0.17).
As combined use of sodium valproate and flunarizine has shown significant seizure protection in PTZ model, flunarizine has a potential for add-on therapy in refractory cases of partial seizures. Exploration of different mechanisms of antiepileptic drugs will allow better understanding of the pathophysiology of seizure propagation and spread, and should lead ultimately to improved management of people with refractory epilepsy.  It is therefore, we conclude that further multicenter clinical trials involving large sample size are needed to establish flunarizine in epilepsy as add-on therapy.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3]
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