EVALUATION OF A LOCAL ELECTROSHOCK CONVULSION MODEL FOR SCREENING POTENTIAL ANTI-ELEPTIC DRUGS

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EVALUATION OF A LOCAL ELECTROSHOCK CONVULSION MODEL FOR SCREENING POTENTIAL ANTI-ELEPTIC DRUGS

TABLE OF CONTENTS

TITLE PAGE

DECLARATION

DEDICATION

ACKNOWLEDGEMENT

TABLE OF CONTENTS

LIST OF TABLES

LIST OF FIGURES

ABSTRACT

CHAPTER ONE

INTRODUCTION

1.1 CLASSIFICATION OF SEIZURE TYPES

1.2 METHODS OF ASSESSING ANTIEPILEPTIC DRUGS

1.3 AIM OF STUDY

1.4 RELEVANT PHARMACOLOGY OF DRUGS USED IN THIS STUDY

1.4.1 GENERAL MODE OF ACTION OF ANTICONVULSANT DRUGS

1.4.2 PHENOBARBITONE

1.4.2.1 MECHANISM OF ACTION AND ANTICONVULSANT PROPERTIES

1.4.2.2 PHARMACOKINETICS

1.4.2.3 TOXICITY

1.4.2.4 THERAPEUTIC USES

1.4.3 DIAZEPAM

1.4.3.1 MECHANISM OF ACTION

1.4.3.2 ANTICONVULSANT PROPERTIES

1.4.3.3 PHARMACOKINETICS

1.4.3.4 TOXICITY

1.4.3.5 THERAPEUTIC USES

1.4.4 NATIVE PREPARATION

CHAPTER TWO

METHODOLOGY AND MATERIALS

2.1 ANIMALS AND DRUG ADMINISTRATION

2.2 ELECTROCONVULSIVE SHOCK ADMINISTRATION

2.3 DETERMINATION OF CONVULSION AND ANTICONVULSANT ACTIVITY

CHAPTER THREE

RESULTS

3.1 QUALITATIVE OBSERVATION OF ANIMALS AFTER DRUG TREATMENT

3.2 ANTICONVULSANT ACTIVITY

CHAPTER FOUR

DISCUSSION

4.1 TOXICITY AND BEHAVIOURAL CHANGES

4.2 GENERAL COMMENTS

4.3 CONCLUSION

REFERENCES

ABSTRACT

This project was designed to set up and validate an animal model of epilepsy using electroconvulsive shocks in rats. This was accomplished by evaluating the effects of two conventional anti-epileptic drugs, diazepam and phenobarbitone on the protection afforded rats against the tonic hind-limb extension of this study, the possible anticonvulsant effects of an indigenous native preparation was also assessed. Saline was used as control throughout this study.

Fifty-eight healthy rats were used and chosen in a randomized fashion for this study. The drugs were given on a single dose basis using the intraperitoneal route of administration.

The results indicates that the anticonvulsant effect of diazepam was not significantly different from control (0.05 < 0.08). phenobarbitone and the native preparation caused significant anticonvulsant effects of phenobarbitone and the native preparation were not significantly different from each other (p>0.1).

Considering the obvious constraints of apparatus and funds, the results in this study have been able to establish an animal model for screening antiepileptic drugs.

CHAPTER ONE

INTRODUCTION

An epileptic seizure may be defined as a brief disorder of cerebral function, usually associated with a disturbance of consciousness and accompanied by a sudden, excessive electrical discharge of cerebral neurones. The electrical activity recorded by the electrocepphalogram (EEG) is of high voltage relative to the background and results from an unphysiological hyper-synchronous discharge of an aggregation of neurones.

In many, perhaps the majority of cases epilepsy arises from causes which at present cannot be identified. This large category of cryptogenic or idiopathic epilepsy includes many cases in which generalized seizures first occur in children whose relative are similarly affected but also includes many other cases without a family history or with atypical seizure.

Any intracranial disease may give rise to epilepsy, either as a manifestation of an active pathological process or a sequel thereof. Important causes of symptomatic epilepsy include cerebral tumours, head injuries and cerebrovascular disease. Seizures may occur as a result of disease elsewhere than in the brain. Hypoglycaemia, Uraemia, heart block, ingestion or sudden withdrawal of alcohol or drugs are but a few of the conditions which may evoke seizures.

Before discovery of the antiepileptic drugs treatment of epilepsy consisted of trephining, cupping and the use of herbal medicines and animal extracts.

In 1857, Sir Charles Locock reported the successful use of potassium bromide in the treatment of what is now known as catamenial epilepsy. In 1912, phenobarbitone was first used for epilepsy. And in the next 25 years, 35 analogs of phenobarbitone were studied as anticonvulsants, in 1938, phenytoin was found to be effective against experimental seizures in cats.

Between 1935 and 1960, tremendous strides were made both in the development of experimental models and in methods for screening and tasting new antiepileptic drugs (Porter et al. 1984). During that period 13 new antiepileptic drugs were developed and marketed.

Following the enactment of requirements for proof of drug efficacy, anti-epileptic drug development slowed dramatically. In the last 25 years, only 5 new anti-epileptic drugs have reached the market place (Meldrum et al 1986).

For a long time it was assumed that single drug could be developed for treatment of all forms of epilepsy. It now appears unlikely that the wide variety of epileptic seizures can be managed successfully with just one drug. More than one mechanism may be responsible for the various seizures and drugs useful for one seizure type may occasionally aggravate other types.

CLASSIFICATION OF SEIZURE TYPES

For proper and clear management, epilepsies have been classified into 2 main types viz:

PARTIAL SEIZUREAS

Simple partial seizures

Complex partial seizures

Partial seizures seco0ndarily generalised

GENERALISED SEIZURES

Generalised tonic-clonic (grandma) seizures

Absence (petit-mal) seizures.

Tonic seizures

Atonic seizures

Clonic and myoclonic seizures.

METHODS OF ASSESSING ANTIEPILEPTIC DRUGS

The general heading of epilepsy gathers a variety of convulsive syndromes that differ in their sensitivity to anticonvulsant drugs (Eadie, 1985; Theodore 1985). Thus to elucidate the etiology of the human disease and to find new therapeutic approaches it is important to develop animal models that mimic the pathological features and the pharmacological sensitivity of the different kinds of epilepsy as closely as possible.

A number of experimental models have been used to investigate the mechanisms responsible for the genesis of epileptic seizures (Symposium 1972, 1981). Application of alumina cream to the motor cortex of the monkey produces chronically recurring spontaneous convulsive seizures of focal onset.

A potentially more informative model is that produced in a variety of animal species by a procedure termed ‘’kindling’’ (symposium 1981). Thus involves delivery of brief, localized trains of electrical stimuli to various areas of the brain at widely spaced intervals. With time, progressively longer and more intense periods of after discharges are produced at sites both near to and remote from the point of stimulation. In the advanced stages of kindling in rats, the seizure consists of a running fit sometimes including periods of tonus at the beginning and end of the seizure (pin, in symposium 1981).

Other experimental models utilize animals that are genetically ay appropriate sensory stimuli (symposium 1972). These include audiogenic seizures in certain strains of mice and seizures elicited by intermitted photic stimulation, prevalent in a specific group of baboons. More recently, two inbred strains of mice have been developed, one characterized by spontaneous tonic-clonic convulsions and the other by behavioural and electro-graphic evidence of spontaneous absence seizures (Heller et al, 1983; Maxson et al., 1983). In addition to providing opportunities for the evaluation of antiepileptic drugs, these models call attention to inherited factors that are suspected to play a role in the genesis of human epilepsy.

In addition, it is well known that groups of neurones display epileptic form discharges in the presence of a variety of drugs that interfere with the function of gamma-aminobutyric acid (GABA), the inhibitory neuro transmitter in the brain; these include bicucullline, picrotoxin, pentylenetetrazol, and certain B – lactam antibiotics. As recorded by intracellular electrodes these neuronal burst arise from sudden, large depolarization, termed paroxysmal depolarizing shift (PDS). The PDS displays the characteristics of an ‘’giant’’ excitatory postsynaptic potential and appears to results from synchronous activity within a neuronal network (Johnson and Brown 1984). It can occur spontaneously or be triggered by external stimuli. The use of drugs to induce seizure is referred to as chenoshock.

A popularly employed model is the electroshock techniques, whereby electroconvulsive shocks are administered through car-clip electrodes to the animal. It is proposed that a convulsion results in an inhibition of GABA synthesis (Green et al., 1987).

For the purposes of this study the electro convulsive shock techniques (ESC) was employed. This was necessitated by lack of funds to run the other types of models. However this model has been shown by a number of workers to be appropriate to screen potential antiepileptic drugs (Green et al., 1978)

AIM OF STUDY

There is need for the development of newer antiepileptic drugs. It was therefore the aim of this study to ascertain in the effect of two standard antiepileptic drugs on the conclusion elicited by the electroconvulsive technique.

And also to extend the researched into the screening of potentially new antiepileptic drugs such as the native preparation under study; as a preliminary study or guide for the screening of potential antiepileptic drugs.

RELEVANT PHARMACOLOGICAL OF DRUGS USED IN THIS STUDY

GENERAL MODE OF ACTION OF ANTICONCULSANT DRUGS:

Hughlins Jackson, some hundred years ago put forward the theory that epileptic seizures were caused by ‘’occasional sudden excessive local discharges of grey matter’’ and that generalized epileptic convulsions developed when normal brain tissue became invaded by the electrical currents generated in the abnormal focus. Hughlins Jackson’s theory has been substantially confirmed by electroenceplographic investigations.

Antiepileptic drugs could thus act (1) by inhibiting or damping the seizure focus itself or (2) by preventing the spread for the spread of the abnormal activity to normal brain tissue. Most experimental evidence suggests that antiepileptic drugs generally act by the second mechanism and this is supported by the finding that these drugs can prevent epileptic form convulsion artificially induced by electrical stimulation of the brain.

PHENOBARBITONE

Phenobartone was the first effective organic antiepileptic agent. It has relatively low toxicity, is inexpensive, and is still one of the more effective and widely used drugs for this purpose.

MECHANICAL OF ACTION AND ANTI-CONVULSANT PROPERTIES

The exact mechanism of action of phenobarbitone is unknown. It is thought to markedly prol;ong posttetanic potentiation and enhances presynaptic inhibition. Recent data indicate that phenobarbitone may act only onb abnormal neurons, inhibiting the spread and suppressing firing from the foci. Like phenytoin, phenobarbitone decreases sodium and potassium conductance, but only at high concentrations. Phenobarbitone may exert its effect by binding to the dihydropicrotoxinin sites on the GABA receptors, thereby enhancing chloride conductance. However, direct activation of GABA receptors appears to correlate better with the hypnotic effect than with the anticonvulsant action of phenobarbitone. At therapeutic concentrations, phenobarbitone antagonizes glutamate excitation while at the same time greatly enhancing GABA inhibition. Quite a host on barbiturates have anticonvulsant properties. Nevertheless, the capacity of some of these agents such as phenobarbitone, to exert maximal anticonvulsant properties ad doses below those required for hypnosis determines their clinical utility as antiepileptics.

PHARMACOKINETICS

Phenobarbitone is completely absorbed after oral administration. Peak plasma concentrations occur several hours after a single dose, suggestive of a slow absorption rate. It is 40 to 60% bound t plasma proteins and bound to as similar extent in tissues, including the brain.

The PH of phenobarbitone is 7.3 and up to 25% of a dose is eliminated by PH – dependent renal excretion of the unchanged drug: the remainder is in-activated by hepatic micronal enzyme,es. The plasma half-life of phenobarbitone is about 100 hours in adults, it is somewhat longer in neonates, while it is shorter and more variable in children.

TOXICITY

The adverse effects of phenobarbitone have been reviewed by Mattson and Gramer (Symposium 1982a). This include: sedation, Nystagmus, ataxia. Phenobarbityone sometimes produces irritability and hyperactivity in children and agitation and confusion in the elderly. Scarlatiniform or morbiliform rash may occur in a small population of patients. Hypoprothrombinemia with haemorrhage have been noticed in the new-born of mothers who have received phenobarbitone during pregnancy

1.4.2.4 THERAPEUTIC USES

Phenobarbitone is an effective agent for the following:

Gemeralized tonic-Clonic seizures

Partial seizures

1.4.3 DIAZEPAM

Diazepam is one of the five benzodiazepines (BZ) that play a prominent role in the therapy of epilepsy.

Intravenously administered diazepam is in fact the drugs of choice for stopping continuous seizure activity; especially generalized tonic-clonic seizure.

1.4.3.1 MECHANISM OF ACTION

The mechanism of action of diazepam like other benzordiazepines has raised a great interest over the last few years due to their pharmacological significance as anticonvulsants, antianxiety agents, muscle relaxants and hypnotics.

It has been experimental shown that the benzodiazepine (BZ) kind to specific receptors on the rat brain membranes (Square and Braistrup 1977). The displacement of BZ from the specific binding occurs only with pharmacologically active benzodiazepines. This is in support of considering these sites as receptors mediating the action these drug within the CNS. (Williamson et al., 1978; Duka et al., 1979). It was furthermore reported that there is an excellent correlation between BZ receptor occupancy by diazepam and protection against pentylenetetrazol induced seizures (pant et al., 1979). It has also been suggested that BZ might modify the affinity of the GABA receptor or the coupling between receptors and ionophore (Macdonald and Bawler, 1982).

Following this idea the binding site for the benzodiazepines might be part of the GABA/BZ/ionophore complex and the binding of BZ would modify the conformation (Tallman et al. 1978). It has been proposed that the BZs increase the action of GABA at its receptors sites by displacing an endogenous modulator protein (GABA modulin), which forms part of a marcromoleculer complex constituting the GABA-receptor Ionophore (Iversen, 1978). Coster et al. (1979) have suggested that the recognition site for BZ may be closely associated with a chloride ion channel which in turn is closely linked to the GABA-recognition site. Moreover, there are data about an apparent enhancement of the BZ affinity towards their receptors mediated by GABA and some GABA agonists like muscimol and also that the action of BZ is antagonized by bicuculline a well-known GABA antagonist (Tallman et al 1978).

1.4.3.2 ANTICONVULSANT PROPERTIES

In animals, prevention of pentylenetetazol – induced seizures by the benzodiazepines is much more prominent than their modification of the maximal electroshock seizure pattern. In various experimental models, however it has been shown that the benzodiazepines including diazepam, suppress the spread of seizure activity produced by epileptogenic foci in the cortex, thalamus and limbic structures but do not abolish the abnormal discharge of the focus. Further both diazepam and clonazepam supress stimulus-induced generalized convulsions in kindled rats, but they produce little or no reduction in stimulus induced after discharges (Albright and Burnnharm 1980).

1.4.3.3 PHARMACOKINETICS

Generally benzodiazepines (including diazepam) are well absorbed after oral administration and concentrations in plasma are usually maximal within 1 to 4 hours (symposium 1982a, Browne in symposium, 1983a). Central effects develop promptly but wane rapidly as the drugs move to other tissues. Diazepam is about 99% bound to plasma proteins.

The major metabolite of diazepam N – desmethyldiazepam is about as active as the parent drug. Both diazepam and N- desmethyldiazepam are slowly hydroxylated to other active metabolites such as oxazepam. The half-life of diazepam in plasma average is between 1 and 2 days.

1.4.3.4 toxicity

The acute toxicity of the benzodiazepines is low relative to usual clinical dosage. However, cardiovascular and respiratory depression may occur after the intravenous administration of diazepam, particularly if other anti-convulsants or central depressants have been administered previously (Symposium 1983d).

Other side effects include, hypotonia, dysathria and dizziness. Seizures are sometimes exacerbated (Browne, in symposium 1983a), and status epilepticus may be precipitated if the drug is discontinued abruptly.

1.4.3.5 THERAPEUTIC USES

Diazepam is currently the drug of choice for the treatment of status epilepticus, its relative short duration of action is a disadvantage. However, diazepam is effective in about 80-90% of cases for this type of epilepsy.

1.4.4 NATIVE PREPARATION

As mentioned above, before the advent of antiepileptic drugs there was a wide use of a host of herbal medicines and animal extracts by different peoples of the world. The use of herbal medicines for epilepsy and other ailment is not new to this part of the world. The use of herbal medicines for epilepsy and other ailment is not new to this part of the world. For till this day it forms the first line medication for a substantial part of the population (Onuaguluchi, 1986). This no doubt can be linked to the population believe amongst the illiterates that epilepsy can be linked to with craft and spirits.

Several studies have been undertaken into various tradomedical manuvoures. One of such in epilepsy is that reported by anuagulchi (1986) about the anticonvulsant activities of a cow’s urine galenical preparation used in the western part of Nigeria. It is also common knowledge that here in the east a lot of preparations or traditional medical therapies are available for epilepsy. Amongst the ‘’Ikwerres’’ (Rivers state) there is a common practise of the use of extracts of onion, a white thick liquid from an edible plant and also the use of the native gin as therapy. The onion is applied to the eyelids of the patient (mostly children) and this is thought to bring them ‘’back’’

Nevertheless the most impressive preparation so far is a powder made from four edible plant products. The native doctor make several small razor blade cuts in the skin along the dorsal aspect of both arms and rub in the powder. In a few cases the process is repeated a week later on the skin of the back. Patients so treated a week later on the skin of the back. Patients so treated a week later on the skin of the back. Patients so treated once are in claimed to remain free of seizures for several years and in many cases permanently; without any further orthodox or unorthodox treatment. Historical evaluation suggests that the powder has been used for treatment of various types of epilepsy but mostly generalised tonic-clonic and complex partial seizures. A few of the beneficiaries of this treatment have been examined medically and found to be in apparent good health.

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