Evaluation of Antiepileptic activity of Mosapride in Albino wistar rats

 

Shubhika Jain1*, Bharti Chogtu2, Vybhava Krishna3, Isha Khadke3

1MBBS Undergraduate, Kasturba Medical College, Manipal Academy of Higher Education - 576104, India.

2Associate Professor, Department of Pharmacology, Kasturba Medical College, Manipal,

Manipal Academy of Higher Education, Manipal, Karnataka, India. 576104.

3Post Graduate Student, Department of Pharmacology, Kasturba Medical College, Manipal,

Manipal Academy of Higher Education, Manipal, Karnataka, India. 576104.

*Corresponding Author E-mail: shubhikajn24@gmail.com

 

ABSTRACT:

Serotonin causes a significant shift in the excitability of neurons and endogenous serotonin and drugs acting on serotonergic receptors play a role in pathogenesis of epilepsy.  This study was done to study the effect of Mosapride, a serotonin receptor 5HT4 agonist, in animal models of epilepsy. Albino Wistar rats were divided into 5 groups with six animals in each group. Group 1 was control group, group 2 was standard group and group 3, 4 and 5 received test drug mosapride in low dose (3mg/kg), high dose (6mg/kg) and mosapride plus standard antiepileptic drug respectively. The antiepileptic efficacy was evaluated using Maximal Electroshock Seizure model (MES) and Pentylenetetrazole (PTZ) induced convulsions. Data was analysed using ANOVA followed by post hoc Tukeys test. Mosapride treated animals showed statistically significant decrease (p<0.001) in the duration of flexion, hind limb extension and post ictal depression in MES model which was comparable to phenytoin group. In PTZ model, mosapride alone did not show any significant difference as compared to control group in terms of latency and duration of seizures (p>0.05). The antiepileptic efficacy of mosapride is similar to phenytoin in MES model. However, in PTZ model mosapride did not show any beneficial antiepileptic effect

 

KEYWORDS: Antiepileptic drugs, Mosapride, 5HT4 receptors, Serotonin, Epilepsy.

 

 


INTRODUCTION:

Epilepsy, a chronic neurologic disorder, classically presents as repeated episodes of seizure which are secondary to paroxysmal rampant discharges of neurons in the central nervous system.1 It is the second most common neurological condition that burdens individuals, families, and the health care system.2 Not only medical, epilepsy also poses social burden on different communities due to fear, unpredictability and associated social stigma leading to discrimination.3

 

 

Even patients with epilepsy have poor quality of life and this can further lead to mild to moderate depression.4 Imbalance between neurotransmitter Glutamine (excitatory) and GABA (inhibitoty)  is known to be a cause of seizure genesis. Changes in volatage, release and uptake of neurotransmitters have also been reported as mechanism of seizure development along with neurodegenrative and congenital developmental etiologies.5 Antiepileptic drugs serve as a mainstay of treatment in epilepsy by reversing the seizure genesis. The majority of the epileptic seizures are controlled by available antiepileptic drugs but about one-third of patients remain uncontrolled despite the drug therapy.6 Seizure frequency is also a significant predictor of psychological distress.7 Adverse effects with currently available antiepileptics lead to poor patient compliance. Thus a need for a new class of drugs with different mechanisms is always felt.

 

Seizure threshold can be modulated using serotonin neurotransmitters in both focal and generalized seizures.  Many studies have been conducted to test the efficiency of selective serotonin reuptake inhibitors (SSRI’s) on seizure threshold and have shown significant results.8-10 Evidence shows that the pathogenesis of epilepsy is significantly affected by endogenous serotonin and drugs acting as agonists and antagonists on serotonin receptors.11 5HT4 receptors have a facilitatory control over the release of serotonin.12 Audiogenic seizures in animal models of epilepsy have known to be controlled by 5-HT7 receptors antagonists.13

 

Mosapride, a prokinetic drug, is a 5HT4 agonist and is used for gastroesophageal reflux disorders14, functional dyspepsia, and irritable bowel syndrome. This study was designed to carry out the evaluation of the effect of mosapride in maximal electroshock seizures and pentylenetetrazole induced convulsion in animal models of epilepsy on the basis of the hypothesized linkage between serotonin and epilepsy.

 

MATERIAL AND METHODS:

Albino Wistar rats aged 6 months weighing between 150-200g were used in this study. The animals were kept in a standard environmental conditions (temperature of 22-240C with a 12 hour light/dark cycle) in an animal house with access to food and water. Institutional animal ethics committee approval was taken before conducting any experiments and CDSCO guidelines were followed.

 

The test drug used was mosapride in low and high doses of 3mg/kg and 6mg/kg respectively. Human therapeutic dose was converted into animal dose using Paget and Barnes’s table.15 Sodium valproate 108mg/kg16 and phenytoin 100mg/kg17 were used as the standard drugs for epilepsy. The animals were dosed for three days as previous studies report serotonin release and effect to be maximum on the third day of dosing.18 Carboxymethyl cellulose (CMC) 0.5% was administered to control group and Pentylenetetrazole 60mg/kg19 was used to induce seizures in the pentylenetetrazole model of epilepsy.

 

Experimental Design:

a) Maximal Electroshock Seizure (MES) Models:

In this model, 30 animals were used and divided into 5 groups as shown in table 1.

 

Table 1: Groups of MES model.

Group

Drug

Dose

Group 1 ( control)

CMC 0.5%

2ml

Group 2

Phenytoin

100mg/kg

Group 3

Mosapride

3mg/kg

Group 4

Mosapride

6mg/kg

Group 5

Mosapride+Phenytoin

3mg/kg+ 100mg/kg

 

Group 1 received CMC 0.5% as a vehicle for 3 days. Group 2, 3, 4, 5 received respective drugs intraperitoneally for 3 days.  On the third day after 30 minutes of receiving the drugs, electroshock at the intensity of 150mA, 50Hz for 0.2 sec was given. Later on, each animal was observed for the onset of seizures, duration of hind limb extension, the total duration of seizures, recovery/death in individual polypropylene cages. The scoring of the seizures was done according to the following scale: 0- No seizure, 1- Forelimb extension without hindlimb extension, 2- Complete forelimb extension with partial hindlimb extension, 3- Complete tonic hind limb extension (hind limb parallel to tail), 4- Postictal depression.

 

Percentage protection calculation = Number of animals with Total hind limb extension absent ×100

 

Total number of animals:

The endpoint of the experiment was considered as the absence/presence of tonic hind limb extension following drug treatment. This is a position during the generalized tonic-clonic seizures in rodents when the tail and both hind limbs are parallel to each other.

b) Pentylenetetrazole (PTZ) induced convulsions:

 

In this model 30 rats were taken and divided into 5 groups as shown in table 2.

 

Table 2: Groups of PTZ model.

Group

Drug

Dose

Group 1

(control)

Distilled water + Pentylenetetrazole

2ml

Group 2

Valproate + Pentylenetetrazole

108mg/kg + 60mg/kg

Group 3

Mosapride + Pentylenetetrazole

3mg/kg  + 60mg/kg

Group 4

Mosapride + Pentylenetetrazole

6mg/kg  + 60mg/kg

Group

Mosapride+valproate+ Pentylenetetrazole

3mg/kg + 208mg/kg + 60mg/kg

 

Group I received distilled water and Group 2, 3, 4, and 5 received drugs as shown respectively intraperitoneally. On the third day, after 30 minutes of receiving the drugs, 60mg/kg pentylenetetrazole was injected intraperitoneally to induce seizures. Afterward each animal was observed for a period of one hour in individual polypropylene cages. Seizures and tonic-clonic convulsions were recorded. The interval between administration of pentylenetetrazole and the occurrence of seizures was observed.

 

STATISTICAL ANALYSIS:

The analysis was done using SPSS 23.0. Intergroup analysis was done using one-way analysis of variance followed by post hoc Tukey’s test. Standard and control groups were compared with the test groups. P value of <0.05 was considered statistically significant and results were expressed in mean ± standard deviation.

The results are reported in (Table 3 and 4).

 

a) Maximal Electroshock Seizure Models:

The duration of flexion, hind limb extension and postictal depression is shown in Table 3.

 

Table 3. Duration of flexion, extension and postictal depression in MES model

Groups

Duration of flexion

(sec)

Duration of hind limb extension (sec)

Postictal depression (sec)

GROUP 1

13.33 ± 1.20

11.33 ± 0.49

162.67 ± 9.04

GROUP 2

1.83 ± 0.31a

1.67 ± 0.33a

1.67 ± 0.33a

GROUP 3

4.17 ± 0.31a

6.83  ± 0.40a e

53.50 ± 1.23a

GROUP 4

4.17 ± 0.17a

4.00 ± 0.37a b d

60.33 ± 1.48a

GROUP 5

3.50 ± 0.4a

3.67 ± 0.67a d c

54.67 ± 4.74a

(Values =  Mean ± SD)

SD = Stanadrd Deviation

a p value < 0.001 as compared to control           

bp value < 0.05 as compared to mosapride low dose

cp value <0.001 as compared to mosapride low dose

dp value < 0.05 as compared to phenytoin

ep value< 0.001 as compared to phenytoin

 

PERCENTAGE PROTECTION:

Figure 1 shows the percentage protection in different groups. Percentage protection is calculated by dividing the no. of animals without hind limb extension by total no. of animals in a group.  Mosapride low dose produced 83.3 % protection whereas standard and other test groups gave 100% protection.

 

Figure 1. Graph showing percentage protection in different MES groups.

 

b) Pentylenetetrazole induced convulsions:

The latency and duration of seizures in PTZ model in different groups is shown in Table 4. Only phenytoin and combination group showed a statistically significant increase in latency (p<0.001) as compared to control.

 

Table 4. Latency and duration of seizures in PTZ model

Groups

LATENCY (sec)

DURATION (sec)

GROUP 1

1.25 ± 0.25b d

3.67 ± 0.33c

GROUP 2

6.33 ± 0.67a

1.25 ± 0.25a

GROUP 3

1.62 ± 0.36b d

4.17  ± 0.48b

GROUP 4

2.50 ± 0.56 b d

4.83 ± 0.48b

GROUP 5

5.50 ± 0.43a

2.83 ± 0.40

Values (Mean ± SD)

SD = Standard Deviation

 a p value<0.001 as compared to control

bp value <0.001 as compared to valproate

c p value<0.01 as compared to valproate

dp value < 0.01 as compared to mosapride valproate combination

 

DISCUSSION:

Serotonin plays a significant role in the mechanism of action of psychiatric drugs like antidepressents, mood stabilisers, and sleep disorders and also as antiemetic.20) which works by modulating the levels of serotonin in the central nervous system.  The increased serotonin levels has even been found in metabolic disorders like diabetes.21  The role of serotonin in the generation of seizures has been documented. Studies have shown that chronic administration of phenytoin modifies the levels of serotonin and produces antiepileptic action by modulating the proportions of monoamine neurotransmitters in various areas in the brain in animal studies.22

 

Maximum Electric Shock (MES) model is a gold standard in the screening of antiepileptic drugs used in Grand Mal seizures because of its simple setup and high predictive value for efficacy of the drug used.23 This test has been used as a major initial screen for new drugs with a mechanism of action involving prolonged inactivation of the voltage-gated Na+ channels like phenytoin, carbamazepine, etc. In the present study, mosapride treated animals in the MES model showed a decrease in the duration of flexion, duration of hind limb extension and duration of postictal depression (p<0.001) in comparison to control which was statistically significant. (Table 1) The protective effect of mosapride treatment was comparable to the standard drug (phenytoin). No difference of statistical significance was shown between the two doses of mosapride and mosapride + phenytoin on different parameters in the MES model. However 100% protection was seen in phenytoin, Mosapride 6mg/kg and mosapride + phenytoin as compared to the lower dose of mosapride in which only 83% protection was seen (Figure 1.) In this study mosapride, a 5HT4 agonist has shown a protective effect in MES induced seizure model. Peripheral serum levels of serotonin play a role in seizures and post-seizure recovery.24 An animal study by Buchchan et al put forth that serotonin regulates seizure threshold and regulates the severity of seizures and augmentation of serotonin can help in the prevention of seizures. Serotonin related neurons raise seizure threshold and decrease mortality related to seizures.25 Phenytoin and carbamazepine increase extracellular serotonin and dopamine, using microdialysis in the hippocampus of freely moving rats.26 In comparison with the control rats, serotonin depleted rats showed spontaneous seizures with kainic acid administration, and the authors concluded that low serotonin is an important risk factor in epilepsy.27 On the contrary, stimulation of 5HT1A, 5HT2A, C, and 5HT3 receptors by fluoxetine did not alter the threshold and severity of seizures in male Wistar rats that was induced by electrical stimulation.28 In the present study, 5HT4 receptor agonist, mosapride has shown a favorable effect comparable to that of phenytoin on the duration of flexion and postictal depression in the MES model. This can be hypothesized due to increasing activity at 5HT4 receptors mediated by Mosapride. 

 

Drugs acting selectively on absence seizures can be identified by PTZ induced seizure models.23). In this model latency and duration of the seizure-induced after giving the drug are noted. Latency is the time taken to induce seizures in an animal. In the sodium Valproate group, significant increase in latency in comparison to control group was noted (Table 2). Mosapride low dose (p= 0.98) and mosapride high dose (p = 0.37) did not show any significant difference as compared to the control group. The effect of Mosapride and sodium valproate was similar to valproate alone showing that the addition of mosapride has no benefit in PTZ induced seizures. Similarly, the duration of convulsions also did not show any improvement in the mosapride group. On recording the duration of convulsions, Valproate and mosapride combination was comparable to valproate alone. Overall mosapride did not show any protective effect on PTZ induced seizures, the effect being comparable to that of control. Thalamus and cortex play a role in the generation of absence seizures in experimental models.29 There are monosynaptic connections between the thalamus and the hippocampus.30 Studies show a protective effect of serotonin underactivity in absence seizures. Serotonin depletion produced by administering parachlorophenylalanine in rats blocked generation and propagation of absence seizures in the hippocampus and thus protected against experimental absence seizures.31 However, in other studies, serotonin has shown a protective effect in absence seizures. In Groggy rats, serotonergic agonists acting on 5HT1A and 5HT2receptors decreased the incidence of absence like seizures. Also, SSRIs reduced the generation of seizures.32 5HT4 receptor knock out mice are more susceptible to PTZ related seizures. Authors further suggest that inhibitory influences on the excitability of neurons are mediated by 5HT4 receptors.33

 

To conclude, Mosapride has shown antiepileptic efficacy similar to phenytoin in the MES model, largely independent of dose. Owing to the burden of the disease and prevalence of current treatment failure, further experiments can be carried out to evaluate its efficacy in generalized tonic-clonic seizures. On the other hand, in the PTZ model of epilepsy, Mosapride did not show any promising effect.

 

ACKNOWLEDGEMENT:

Authors are much obliged to the Dean and the administration of Kasturba Medical College, Manipal, Manipal Academy of Higher Education for supporting this project.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

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Received on 13.10.2020            Modified on 10.12.2020

Accepted on 19.01.2021           © RJPT All right reserved

Research J. Pharm.and Tech 2021; 14(12):6364-6368.

DOI: 10.52711/0974-360X.2021.01100