INTRODUCTION:

Epilepsy is the fourth most common neurologic disorder and comprises roughly 1% of the world’s population¹. Epilepsy is known to cause brief impairment of consciousness, increasing an individual’s risk of bodily harm and often interfering with education and employment. Currently, the incidence of epilepsy is 0.3–0.5% in diverse populations throughout the world whereas the prevalence of epilepsy is approximated at 5–10 persons per 1000². Bromide was the first drug used for the treatment of epilepsy way back in 1857³, followed by phenobarbital in 1912.

Over the years we have seen the discovery of a number of drugs. Antiepileptic drugs (AEDs) not only have drawbacks like long duration of therapy but also tend to produce some serious side effects which require therapeutic drug monitoring. Hencethe need for an ideal antiepileptic agent that has a rapid onset of action with broad-spectrum activity, favorable adverse effect profile, and is cost-effective. Simvastatin competitively inhibits an enzyme called HMG-CoA reductasethatcatalyzesan important step in cholesterol synthesis which is also rate limiting and is hence used in the treatment of dyslipidaemia. Nonetheless, there are scanty reports about simvastatin having antiepileptic, neuro-protective and memory enhancement effects⁴. Statins are generally drugs with a high margin of safety and are well tolerated. So if they are proven to have an added anticonvulsant property they might be helpful in the prevention and/or treatment of epilepsy, epileptic seizures and convulsions. Hence this study was undertaken to evaluate the anticonvulsant effect of simvastatin in mice which might pave way for newer therapeutic approaches in the management of epilepsy.

MATERIALS AND METHODS:

The study was conducted post approval by the “Institutional Animal Ethics Committee”. Animals were acquired from the Institutional Central Animal Facilityin accordance with the guidelines of the “committee for the Control and Supervision of Experiments on Animals (CCSEA)”. 64 albino mice of either sex weighing 25-35g of normal behavior and activity, aged around 8-10 weeks were used for the study. “The animals were housed under standard conditions, 12:12 light-dark cycle, 50% humidity, and 28°C temperature, and given standard food granules and water ad libitum”. Drugs likePhenytoin sodium, Diazepam,and Simvastatin were procured from Microlabs Ltd whereas Pentylenetetrazole from Sigma Aldrich Ltd. Individual solutions of Phenytoin, Diazepam, Simvastatin, and Pentylentetrazolewere prepared by dissolving them in anappropriate solvent at room temperature to obtain a concentration of 1mg/ml, 0.1mg/ml, 1mg/ml and 10mg/ml respectively. In order to maintain the accuracy of preparation, all the drug solutions were sealed in vials, labelled and made airtight.

Albino mice were divided into 8 groups (n=8). The MES and PTZ model consisted of 4 groups each - Control group (polyethylene glycol 1ml/Kgi.p.), Test group1 (simvastatin 10mg/Kgi.p.)⁵, Test group2 (simvastatin 20mg/Kgi.p.); Standard group (phenytoin sodium 20mg/Kgi.p.)⁶anddiazepam 2mg/Kg i.p.⁷for MES and PTZ model respectively. 30 minutes post-treatment with the drugs, mice in all groups of MES model were exposed to maximal electroshock stimulus through transauricular electrodeswith a current strength of 80mA in 0.2 sec. They were under observation for a period of 60 minutes and subsequently studied for the following parameters–“Hind Limb Flexion, Hind Limb Extension, Clonus, Postictal Depression”. Tonic hind limb extensionoccurance was considered a positive response for MES seizures and reduction in duration or abolition of response was considered protective for the same. 30 minutes post-treatment with the drugs, mice in all groups of PTZ model were treated with PTZ 80mg/Kg s.c.⁸. Upon observation for a period of 60 minutes, parameters like seizure latency and duration of seizures were studied.Seizureoccurance for more than 5 seconds was considered as positive response whereas increase in latency, decrease in duration of seizures or total abolition was taken as protective for the same. Descriptive data was found for each group and used for analysis. “For simultaneous multiple group comparison,One way analysis of variance (ANOVA) was used followed by Tukey’s post hoc test for group wise analysis.p value<0.05 is significant and when p < 0.001it is considered highly significant.”

RESULTS:

Mean duration of tonic hind limb extension has decreased in standard group S (Phenytoin 20mg/Kg), test group T1 (Simvastatin 10mg/Kg) and test group T2 (Simvastatin 20mg/Kg) in comparison to that of control group C. There is complete abolition of the extensor phase which is statistically highly significant (p< 0.001) in standard group S. When compared with the control group, the test drug has shown statistically significant protection in both the test groups (p value < 0.001 for T1 and p value < 0.05 for T2). In terms of mean duration of tonic hind limb extension, there is statistically highly significant difference between standard group S and the test groups T1 and T2 (p value < 0.001). (Table I and figure 1).

Table-I: Statistical analysis showing a comparison of tonic hind limb extension in MES model

Groups	Mean±SD (Seconds)	Difference Between Groups
Groups	Mean±SD (Seconds)	Groups Compared	Mean Difference	Significance
Group I Control (C)	14.8 ± 2.1	I – II	+14.8	HS
		I – III	+5.5	HS
		I – IV	+2.4	S
Group II Standard (S)	0.0 ± 0.0	II – III	-9.3	HS
Group III Test group (T1)	9.3 ± 1.0	II – IV	-12.4	HS
Group IV Test group (T2)	12.4 ± 2.6	III – IV	-3.1	NS

SD – Standard deviation, HS – Highly significant (p<0.001), S – Significant (p<0.05), NS – Not significant (p>0.05)

Fig. 1: Mean duration of tonic hind limb extension in mes model

In comparison to the control group, the latency of seizures is increased in the standard group (diazepam) and is statistically significant (p value <0.001). It is reduced in the test groups when compared to control group. On comparing the results between standard and test groups, there is decrease in the latency of seizures in the test group and it is statistically highly significant (p value < 0.001). (Table II, figure 2).

Table-II: Statistical analysis showing a comparison of seizure latency in PTZ model

Groups	Mean±SD (Second)	Difference Between Groups
Groups	Mean±SD (Second)	Groups Compared	Mean Difference	Significance
Group V Control (C)	900.0 ± 531.8	V – VI	-900.0	HS
		V – VII	+360.0	NS
		V – VIII	+412.5	S
Group VI Standard (S)	1800.0 ± 0.0	VI – VII	+1260.0	HS
Group VII Test group (T1)	540.0 ± 120.0	VI – VIII	+1312.5	HS
Group VIII Test group (T2)	487.5 ± 174.0	VII – VIII	+52.5	NS

SD – Standard deviation, HS – Highly significant (p<0.001), S – Significant (p<0.05), NS – Not significant (p>0.05)

Fig. 2: Mean seizure latency in PTZ model

In comparison to the control group, the duration of seizures is decreased in all the groups and is statistically significant (p value < 0.001). On comparing the results between standard and test groups, it is seen that there is an increase in duration of seizures but it lacks statistical significance (p value > 0.05). (Table III, Figure 3).

Table-III: Statistical analysis showing a comparison of seizure duration in PTZ model

Groups	Mean ± SD (Second)	Difference Between Groups
Groups	Mean ± SD (Second)	Groups Compared	Mean Difference	Significance
Group V Control (C)	440.4 ± 182.2	V – VI	+411.9	HS
		V – VII	+349.6	HS
		V – VIII	+367.6	HS
Group VI Standard (S)	28.5 ± 68.6	VI – VII	-62.3	NS
Group VII Test group (T1)	90.8 ± 56.0	VI – VIII	-44.3	NS
Group VIII Test group (T2)	72.8 ± 47.3	VII – VIII	+18.0	NS

SD – Standard deviation, HS – Highly significant (p<0.001), S – Significant (p<0.05), NS – Not significant (p>0.05)

Fig. 3: Mean seizure duration in PTZ model

DISCUSSION:

Anumber of models can be used to screen for anticonvulsant activity, yet the MES and PTZ models remain the mainstay of any antiepileptic drug screening protocol⁹. In the present study, these two models were used to evaluate the anticonvulsant effect of simvastatin and compare with the respective standard drugs.

In generalized tonic-clonic seizures,MES model is the utmost validated experimental method for evaluation of antiepileptic drug. MES causes several changes at the cellular level with respect to calcium and sodium ions that too in large amounts thereby disrupting the signal transduction in the neurons^10,11. Currently available anticonvulsant medications like valproate and phenytoin act by modulating these ion channels. Hence for this study, phenytoin was chosen as the standard drug. Reduction in duration of tonic hind limb extension is consideredprotective ofMES induced convulsions¹⁰, simvastatin has anticonvulsant effect against MES convulsions at both 10mg/Kg and 20mg/Kg but this effect is not equal to that of phenytoin in the present study. In terms of duration of tonic hind limb flexion and post ictal depression, simvastatin 20mg/Kg dose has comparable results to that of phenytoin.

GABA is a major inhibitory neurotransmitter whose enhancement leads to diminution of convulsions. PTZ considered to be an antagonist at GABA_A receptor complex, inhibits the GABA pathway in the CNS leading to an imbalance between the ionic concentrations of the membrane causing seizures¹². Apart from drugs that reduce T type Ca²⁺ currents like ethosuximide, PTZ induced seizures can also be prevented by benzodiazepines and phenobarbital, drugs that enhance inhibitory neurotransmission (GABA_A receptor mediated)¹³. Hence in the present study, diazepam was used as a standard in PTZ model. For PTZ convulsions, an increase in latency, decrease in duration of seizures or total abolition of seizures is considered to be protective. In the present study, simvastatin has shown results comparable to that of diazepam in terms of seizure duration only and not seizure latency i.e., simvastatin decreased the seizure duration but did not delay the seizure onset.

The results of the present study agree with other studies that have reported an effective neuro-protective action of statins in various neuropathological conditions including epilepsy^14,15. A study was conducted byCitraro et al to investigate “the effects of statins on the development of absence seizures in a genetic animal model of absence epilepsy” and found that “early long-term statin treatment possesses antiepileptogenic properties, but when acutely administered they are not useful against established absence seizures”¹⁶.

Statins are touted to have plenty of pleiotropic effects like “improvement of endothelial dysfunction, inhibition of inflammatory responses, antioxidant properties, increased nitric oxide bioavailability,and stabilization of atherosclerotic plaques”. A lot has been speculated regarding the possible role of statins in treatment of epilepsy and it may be related or unrelated to their lipid lowering effect.These pleiotropic effects and the potent low-density lipoprotein cholesterol-lowering effect of simvastatin could act in concert and be responsible for its anticonvulsant effect.Smaller and lipophilic statins (simvastatin) easily penetrate the blood brain barrier(BBB)than larger and hydrophilic ones (atorvastatin)¹⁷. This is consistent with the findings of Sierra et al. who have shown in their study thatorder of predictive ability of statins to cross BBB in vivo is relatable to their anti-epileptic effects; highest being for simvastatin and least for pravastatin¹⁸. Reactive oxygen species (ROS) tend to cause tissue injury ranging from lipid peroxidation to enzyme inactivation and DNA damage if produced in excess. ROS along with excitotoxic syndrome are common denominators for neuropathological disorders such as hypoxia, ischemia, and epilepsy^19,20. In the pilocarpine model studied by Rangel et al, ROS producedmay have implications in the mechanisms involved with glutamatergic excitotoxicity both in vitro and in vivo²⁰. Zacco et al have shown that significant protection from NMDA excitotoxicity was obtained when cortical primary neuron cultures were pre-treated with simvastatin¹⁵. Different in vitro studies have revealed that statins particularlysimvastatin, exerts an antioxidant effect²¹ thus making it potentially useful in epilepsy.

It has been suggested that excitatoryamino acids like NMDA and kainite behave like proconvulsive endogenous substances. The activation of NMDA receptors may lead to the formation of nitric oxide (NO) simultaneously. Some studies have exposed that statins obstruct the production of NO in brain parenchyma, signifyingprobable role as an anticonvulsant agent. Statins are also anticipated to exert their anti-seizure and anti-excitotoxic activities through isoprenoidsynthesis inhibition which serve as lipid attachment for intracellular signalling molecules²². “Dysfunction of astrocytes has shown to play a key role in the pathogenesis of epilepsy substantiated by identification of astrogliosis as a hallmark in the brain of epileptics”²³. In transient global brain-ischemia model with hippocampal CA1 pyramidal neurons degenerated by excitotoxic damage, statin treatment ameliorated astrocyte activation²⁴. Statins may also decrease the pro-inflammatory cytokines and nitrergic system action which may lead to reduction in neuroinflammation mediated by them²⁵. Cholesterol homeostasis plays an important role for prevention of neurodegeneration. Cholesterol level reduction causes either a decrease in the availability of receptors in the membrane or reduced ability to associate with lipid raftsknown as DRM/DIM (Detergent Resistant/Insoluble Membrane domains)^14,22. Studies have shown that simvastatin is the most effective statin to decrease cholesterol levels in neuron cell lines independent of inhibition of the HMG-CoA reductase, and thus render a neuroprotective effect. Simvastatin has also shown to protect mice from excitotoxicity induced neuron damage and its neurological consequences like episodic memory impairment and seizures⁴.

Systematic reviews conducted to study the effect of statins have been shown to have reduced the risk of post stroke epilepsy^26,27,28.Existing molecules with favourable side effect profiles can be repurposed for antiepileptogenesis and statins are an excellent candidate for same²⁹.

CONCLUSION:

To conclude in our study, Simvastatin has shown in vivo anticonvulsant activity in both MES and PTZ models and maybe useful in the treatment of tonic clonic and absence seizures. Simvastatin has shown anticonvulsant property but was not comparable to the standard drug phenytoin in MES model. Simvastatin has anticonvulsant property in PTZ model; it produced reduction in seizure duration and was comparable to standard diazepam but did not prolong the seizure latency.Hence statins especially lipophilic ones like simvastatin show considerable anticonvulsant effect and thus might be useful in the management of epileptic patients who have pre-existing dyslipidemia.

LIMITATIONS OF THE STUDY:

The study has been carried out only in single species of animals namely mice and needs to be extended to other species as well. As only intraperitoneal route is used for administration of test compounds, testing needs to be done using other routes as well (oral route) for comparisons.

CONFLICT OF INTEREST:

Nil.

ACKNOWLEDGEMENTS:

I would like to thank the Department of Pharmacology, SSIMS & RC, Davangere for providing the necessary resources to conduct the study.

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Received on 03.08.2022 Modified on 29.03.2023

Research J. Pharm. and Tech 2023; 16(11):5193-5197.

DOI: 10.52711/0974-360X.2023.00842