INTRODUCTION:

Epilepsy is a disorder affecting brain and its parts there are recurrent, periodic and unpredictable episodes of seizures, with or without loss of unconsciousness and memory. it may affect whole brain or parts of brain characterized by abnormal excessive and synchronous neuronal activity in the brain, It is one of the serious disorder affecting the brain, it is having approximately 1% of diseases burden and it can be ranked with depression, Alzheimer’s disease and dementias which are among primary disorders of the brain. This condition is having occurrence with lung cancer in men and breast cancer in women.

Most of the patients with epilepsy can be found in developing nations like India affecting 0.5-1.0% of population and may be 5-10 million people developing nations took 80% of the burden of epilepsy.^[1]

Also worldwide mortality rate is higher among people with epilepsy up to 2-3 times higher than in normal population. Epidemiological data suggests this condition is affecting 0.4–0.8% of the population (Jones O.T.) and up to 50 million people worldwide. Whereas ccounting for almost one fifth of the global figure. Etiology of seizures in most of cases is Idiopathic: has no identifiable cause. Other physiologic factors, such as an alteration in blood gases, pH (extreme acidosis or alkalosis), electrolytes (hyponatrmia, hypocalcemia), and blood glucose (hypglycemia) changes in environmental factors, such as sleep deprivation, alcohol intake (rapid withdrawal from depressants), and stress. Epilepsy can be due to an underlying Genetic (heredity), Structural; CNS infection i) meningeal infection ii) tumors or metabolic defects or sometimes High Fever and individuals who have had only febrile seizures or only neonatal seizures (seizures in the first 30 days of life), and people with acute symptomatic seizures (seizures associated with acute systemic illness, intoxication, substance abuse or withdrawal, or acute neurological insults), and individuals with a single unprovoked seizure, are excluded from this category.^[3-4] the symptoms of epilepsy may consists of sudden and temporary abnormal conditions ranging from alterations of consciousness, changes in motor activities, changes in sensory, autonomic, or in some conditions there are also psychic events can be observed by patient or an observer. Seizures are classified by Commission on Classification and Terminology of Epilepsy of the International League Against Epilepsy according to their origins.^[5] these are Partial (focal) seizures; they start locally in a certain site of brain, and Generalized Seizures; they begin locally but spreads rapidly and, affects the whole brain,and both hemispheres may be convulsive or non convulsive, and there is immediate loss of consciousnessin the patient. (focal) seizures are divided further into: Simple partial: may occur at any age, without loss of consciousness, and could be of motor or sensory origin another type is Complex partial seizures also known as psychomotor or temporal lobe epilepsy; in which there is loss of consciousness ranging from 30 seconds to 2 minutes, also there are disturbances of cognitive, affective, and psychomotor (for eg. Patient may show chewing movement, diarrhoea, urination) or sensory hallucinations (non exciting smell or taste). Generalized Seizures are further classified intoTonic-clonic (grand-mal), Absence (petit-mal), Myoclonic, Febrile seizures, and Status epilepticus (Emergency condition)^[6].

Drugs:

Gabapentin:

Gabapentin is a centrally active GABA agonist, having high lipid solubility which allows it to easily cross blood brain barrier and can produce its GABA like action, which makes it a effective drug to prevent seizures and act as a antiepileptic drug, the chemical structure of gabapentin consists of a GABA molecule covalently bound to a lipophilic cyclohexane ring. The structure of gabapentin is:

Fig. 1 Cyclohexane acetic acid, 1-(aminomethyl)

Gabapentin is when tested in different models of epilepsy shows inhibition in seizures, in electroshock induced seizure model in mice it shows inhibition in tonic hind limb extension. And in clonic seizures induced by pentylenetetrazol italso shows inhibition. Its efficacy to inhibit seizures is almost as same as that of valproic acid, though gabapentin is a agonist for GABA it does not mimic GABA action when applied in the primary culture of neurons by iontophoretically, but it may promote vesicular release of GABA, which is also not completely understood so the exact mechanism of anticonvulsant action of gabapentin is unknown.

Gabapentin binds a protein in cortical membranes with an amino acid sequence identical to that of the α₂δ subunit of the L type of voltage-sensitive Ca²⁺ channel, yet gabapentin does not affect Ca²⁺ currents of the T, N, or L types of Ca²⁺ channels in dorsal root ganglion cells. It has not been found consistently to reduce sustained repetitive firing of action potentials, talking about pharmacokinetics of Gabapentin, it has good absorption when taken orally,and not completely metabolised in humans. Binding with plasma proteins is limited, excreted in unchanged form, through urine.it has halflife of 4 to 6 hours. With other antiseizure drugs it shows no known interactions. So to summarise the anticonvulsant gabapentin, it is well tolerated in the patients having some side effects like dizziness, ataxia, fatigue and somnolence. The tolerance for above stated side effects usually developed within 2 weeks of onset in continued treatment.^[7]

Nimodipine:

it is a dihydropyridine calcium channel blocker originally developed for treating hypertensive patients, and it is also having preferential cerebrovascular activity. It has Ca²⁺ channels blocking effect on specifically Voltage-sensitive Ca²⁺ channels (L-type or slow channels) and by this mechanism it shows marked lowering of blood pressure by dialating blood vessels including cerebrovascular dilating effects. As it is Ca²⁺ channel antagonists or also called Ca ²⁺ entry blockers, inhibit Ca²⁺ channel function. And due to this mechanism of action it also causes relaxation in vascular smooth muscle, leads to relaxation, its solubility properties are Practically insoluble in Water; sparingly soluble in absolute alcohol and freely soluble in ethyl acetate.

Fig. 2 Isopropyl 2-methoxyethyl 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl) pyridine-3,5-dicarboxylate

Cytosolic Ca²⁺ concentrations may be increased by various contractile stimuli. Thus many hormones and neurohormones increase Ca²⁺ influx through so-called receptor-operated channels, whereas high external concentrations of K⁺ and depolarizing electrical stimuli increase Ca²⁺ influx through voltage-sensitive, or "potential operated," channels. The Ca²⁺ channel antagonists produce their effects by binding to the α₁ subunit of the L-type Ca²⁺ channels and reducing Ca²⁺ flux through the channel. Voltage-sensitive channels contain domains of homologous sequence that are arranged in tandem within a single large subunit. In addition to the major channel-forming subunit (termed α₁), Ca²⁺ channels contain several other associated subunits (termed α₂, β, γ, and δ). Voltage-sensitive Ca²⁺ channels have been divided into at least three subtypes based on their conductance’s and sensitivities to voltage. The channels best characterized to date are the L, N, and T subtypes; P/Q and R channels also have been identified. Only the L-type channel is sensitive to the dihydropyridine Ca²⁺ channel blockers. Large divalent cations such as Cd²⁺ and Mn²⁺ block a wider range of Ca²⁺ channels. All approved Ca²⁺ channel blockers bind to the α₁ subunit of the L-type Ca²⁺ channel, which is the main pore-forming unit of the channel. This 200,000- to 250,000-dalton subunit is associated with a disulfide-linked α₂δ subunit of approximately 140,000 daltons and an intracellular β subunit of 55,000 to 72,000 daltons. The α₁ subunits share a common topology of four homologous domains (I, II, III, and IV), each of which is composed of six putative transmembrane segments (S1-S6). The α₂δ and β subunits modulate the α₁ subunit. The dihydropyridine Ca²⁺ channel blockers bind to transmembrane segment of both domain III (IIIS6) and domain IV (IVS6).

Pharmacokinetics of Nimodipine shows rapid absorption on oral administration from GIT and undergoes extensive first-pass metabolism in the liver, so oral bioavailability reduces upto 13% only. It has about 95% of plasma protein binding; Distributed all over body rapidly and also crosses blood-brain barrier. It is metabolized in liver and excreated through faeces and some parts through urine. The elimination half-life is about 9 hours.^[8]

MATERIAL AND METHODS:

Animals:

Swiss Albino mice of body weight 20-30 g were procured from Anuradha College of Pharmacy, Chikhli. With the exception of the short time that the animals were removed from their cages for testing, all animals were maintained on an adequate normal laboratory pellet diet and allowed free access to food and water. Each mouse was used for one seizure test only. Six animals were used for each group of study hence housed six mice per cage, and maintained on a 12-hour-on/12-hour off light schedule. All experiments were performed in between 11 am to 5 pm to avoid circadian influences. The experimental design approved by Institutional Animal Ethical Committee and the study was performed according to the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) guidelines for the use and care of animals

Induction of seizures:

Convulsions were induced by a method similar to that described by Kulkarni S. K. The convulsive dose of PTZ that causes minimum mortality in mice was determined in laboratory. PTZ was dissolved in water for injection to make 6.5 mg/ml PTZ solution. Mice were injected with PTZ in a dose of 65 mg/kg, i.p., following the injection, mice were put individually into open polypropylene cages and observed for 30 min for the occurrence seizures^[9]. In test group i.e. Group III to group V, PTZ was administered after suitable latency (2.5 and 1 hour after Gabapentin and nimodipine respectively) corresponding to the time peak effect after oral administration of the test drug.

Recording of the convulsive response:

Immediately after the administration of PTZ (65mg/kg), the animal was placed in the observational area. Severity of convulsions was carefully recorded onset of clonus, duration of clonus, tonic extension.^[10]

Experimental procedure for anticonvulsant activity:

Mice were divided into five groups. Each group contained six animals.

Group I (Normal control) received vehicle,

GroupII (Convulsive control) received pentylenetetrazole (65 mg/kg. ip)

Group III received pentylenetetrazole (65mg/kg ip.) and nimodipine (10mg/kg p.o.).

Group IV received pentylenetetrazole (65mg/kg ip.) and gabapentin (200mg/kg),

Group V received pentyleneterazole (65 mg/kg ip.), and combination of gabapentin 200mg/kg po.) And nimodipine (10mg/kg p.o.).

After the observation period blood was collected immediately by retro orbital method in eppendrops tubes and centrifuged at 3000 rpm for 15 min and stored at 0-4 ºC for the determination of lipidperoxidation.

Lipid per oxidation assay:

The lipid peroxidation assay was carried out by using the Modified Slater’s method. The MDA level was determined by a method based on the reaction with thiobarbituric acid (TBA) at 90–100 ºC. The TBA test reaction gives pink colouration on reacting with MDA or MDA-like substances which can be then tested at absorption maximum at 530 nm. The reaction was need to performed for 30 minutes at 90ºC. Concentration of TBARS was determined by standard graphic, which was prepared with serial dilutions of standard sulfuric acid to prepare a 10 μM solution. In this method, 2.5 ml of 20% trichloroacetic acid and 1.0 ml of 0.67% TBA were added to 0.5 ml of serum, and then the mixture was heated in a boiling water bath for 30 min. The resulting chromogen was extracted with 4.0 ml of n-butyl alcohol and the absorbance of the organic phase was determined at the wavelength of 530 nm. The determined values were expressed in terms of malondialdehyde (nmol/ml) used as reference standard ^[11].

Chimney test:

The influence of gabapentin alone or in combination with nimodipine on motor impairment was evaluated with the chimney test of Boissier et al, Briefly, mice had to climb up backwards in a plastic tube (3 cm inner diameter, 25 cm length). Animals unable to perform the task within 60 s were considered to display motor impairment. Motor impairment was quantified as the percentage of animals that failed to complete the test.

Statistical analysis:

Statistical analysis of data was carried out by applying oneway ANOVA followed by Tukey’s multiple comparison tests. P value < 0.05 was considered significant. P value of chimney test was calculated by applying Fisher’s exact test (two tailed) with 95% confidence interval. Results are expressed as mean ± SEM.

RESULT:

Effect of nimodipine alone and in combination with gabapentin against pentylenetetrazole induced seizure in mice:

Optimum dose of PTZ to induce clonic convulsion while avoiding mortality was found to be 65 mg/kg, (i.p.). All animals tested in vehical treated group exhibited seizures at the dose of PTZ (65 mg/kg, i.p.) used in study. The onset of seizure in vehical treated group was found to be 1.98 min, and the mean seizure duration was 71.5 sec. Table 1. Shows that,

1. Nimodipine pretreatment in group II animals significantly delayed the onset and decreased the duration, of PTZ induced seizure when compared with untreated animals (group I).

2. PTZ induced seizures in mice was significantly prevented by pretreatment of gabapentin alone in group III, when compared with untreated animals (group I).

3. Administration of combination of nimodipine and gabapentin significantly prevented PTZ induced seizures. Thus, nimodipine enhanced protective effect of gabapentin against PTZ induced seizures in mice.

Table- 1. Effect of nimodipine and its combination with gabapentin against PTZ induced seizure

Groups	Treatment (mg/kg, p.o.)	Onset of jerk (Min)	Durtation of clonus (Sec)
I (Toxic Control)	Saline	1.55	79
		2.7	65
		1.83	82
		2.0	81
		1.8	54
		2.01	68
Mean		1.98±0.15	71.5±4.46
II (Test 1)	NMD (10)	8.5	42
		9.91	58
		6.18	39
		7.68	47
		7.36	59
		5.8	31
Mean		7.57±0.61	46±4.48
III(Test 2)	GBP (200)	11.85	28
		14.28	30
		16.38	34
		10.46	42
		17.71	23
		9.0	45
Mean		13.28±1.39	33.66±3.45
IV(Test 3)	NMD (10) + GBP (200)	30	0
		19.45	10
		23.76	8
		26.13	5
		30	0
		22.9	7
Mean		25.37±1.70	5±1.71

Values are mean ± SEM (n=6). P<0.05 was considered to be statistically significant

Fig. 3 Effect of nimodipine alone and in combination with gabapentin on seizure latency against PTZ induced seizure in mice

Sr.No.	Treatment	Concentration of MDA nMol/ml
1	Normal saline	1.5
2		1.3
3		1.1
4		1.7
5		1.4
6		1.5
Mean		1.41±0.08
1	Pentylenetetrazole (65mg/kg i.p.)	5.4
2		5.8
3		6.1
4		5.9
5		6.0
6		5.6
Mean		5.8±0.10
1	Nimodipine (10mg/kg p.o.) + PTZ (65mg/kg i.p.)	4.4
2		3.9
3		4.0
4		3.9
5		4.2
6		4.2
Mean		4.1±0.08
1	Gabapentin (200mg/kg p.o.) + PTZ (65mg/kg i.p.)	4.0
2		3.8
3		4.2
4		3.6
5		4.2
6		3.6
Mean		3.9±.011
1	Nimodipine (200mg/kg p.o.) + Gabapentine (200mg/kg p.o.). + PTZ (65mg/kg i.p.)	2.5
2		2.6
3		2.0
4		2.2
5		2.3
6		2.5
Mean		2.35±0.09

Fig. 4 Effect of Nimodipine alone and in combination with gabapentine on seizure duration in PTZ induced seizure in mice.

Effect of Nimodipine alone and its combination with Gabapentin on serum lipid peroxidation level in PTZ induced oxidative stress in mice:

TBARS level, the indicator of lipid peroxidation, were significantly increased in serum of PTZ- induced epileptic mice when compared with controls. NMD and GBP significantly prevented these elevations; where as combination of NMD with GBP significantly potentiated this effect of GBP in mice.Values are mean ± SEM (n=6). P<0.05 was considered to be statistically significant.

Effect of Nimodipine alone and its combination with Gabapentin on serum lipidperoxidation level in PTZ induced oxidative stress in mice

Fig. 5 Effect of nimodipine alone and in combination with gabapentin on serum lipid peroxidation level in PTZ induced oxidative stress in mice.

Acute adverse effect of Nimodipine alone and in combination with Gabapentin in chimney test in mice:

NMD at the dose of (10 mg/kg, p.o.) and GBP (200 mg/kg, p.o.) had no significant effect on the motor performance of mice in chimney test. Combined treatment with GBP and NMD resulted in motor impairment, but was not significant as revealed by chimney test.

Acute adverse effect of Nimodipine alone and in combination with Gabapentin in chimney test in mice.

Sr.No.	Treatment (mg/kg p.o.)	% of animals failed
1	Normal saline	0
2	Nimodipine (10)	10
3	Gabapentin (200)	10
4	Nimodipine (10) + Gabapentin (200)	30

CONCLUSION:

In conclusion this study has demonstrated that NMD alone has mild anticonvulsant action against PTZ induce seizure, while in combination with gabapentin produced a synergistic and potent action as compared to gabapentin alone. The broder implication of this report suggests a role for calcium channel blockers for adjunctive therapy for epilepsy. As their degree of anticonvulsant activity not render them useful as anticonvulsants per se, NMD may be used as an add on therapy with GBP and combimation of both drugs may provide a greater effectiveness against epilepsy^[17]

PTZ administration produced an increased lipid peroxidation in serum of the mice treated with PTZ, and therefore, demonstrated and confirmed the possible involvement of free radical oxygen in the PTZ induced seizures. Treatment with both NMD, GBP individually and their combined treatment decreased serum MDA activity, increased by administration of PTZ, thereby suggesting that these drug acts positively on lipid peroxidation.

Combination of nimopdipine, with gabapentin may be promising for the treatment of epilepsy. Further studies of experimental epilepsy should determine whether the beneficial effects of such combinations persist over longer periods.

ACKNOWLEDGEMENT:

The authors are grateful to the authorities of Anuradha College College of Pharmacy, chikhali for the facilities.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 12.05.2018 Accepted on 06.09.2018

Asian J. Pharm. Res. 2018; 8(4): 215-220.

DOI: 10.5958/2231-5691.2018.00036.9