evaluation of antiepileptic drugs

8/8/2019 evaluation of antiepileptic drugs

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EVALUATION OF ANTI-

EPILEPTIC DRUGS

Presented ByPresented By

Rajpal SinghRajpal Singh

Dep't. Of PharmacologyDep't. Of PharmacologyISF collegeISF college of Pharmacyof Pharmacy


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EPILEPSY

Epilepsy is a group of disorders all of which are caused by

abnormal excessive electrical discharge in a part or whole of

brain and spread of electrical discharge in brain may be due to

decrease of diencephalon neurons inhibitory influence on

cortical neurons.

Pathophysiology

Reduction of inhibitory influence of GABA.

Abnormality in ion conduction (k+, Ca+).

Changes in nutrition (glucose and oxygen).

Enhancement of excitatory neurotransmitter activity including

Glutamate and Aspartate.

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Generalized seizures

Absence seizure (petitmal)

Involves both hemisphere along with thalamus, prevalent in children

lasting for 30 sec

Momentary loss of consciousness.

Generalized tonic clonic (Grandmal)

Unconsciousness

Aura - cry unconsciousness.

Tonic spasm of all body muscle and clonic jerking, prolonged sleep, CNS

depression. Myoclonic seizure

Lasts for seconds.

Brief shock like contraction of muscles which may be restricted to one

part of body or generalized 4


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IN VITRO MODELS

Radio ligand binding assaysa) 3H-GABA receptor binding

b) 3H-GABA uptake in rat cerebral cortex synaptosomes

c) GABA uptake and release in rat hippocampal slices

d) Glutamate receptors: [3H] CPP binding

e) NMDA receptor complex: [3H] TCP binding

f) Metabotropic glutamate receptors

g) Excitatory amino acid transporters

h) [35S]TBPS binding in rat cortical homogenates and sections

i) [3H]glycine binding in rat cerebral cortex

j)[3H]strychnine sensitive glycine receptor

Transverse hippocampal slice preparation

Electrical recordings isolated brain cells

Isolated neonatal rat spinal cord

Cell cultured neurons 5


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TRANSVERSE HIPPOCAMPAL SLICE

PREPARATION

Rationale

The transverse hippocampal slice can be easily maintained in-vitro.

The hippocampus slice has the advantage that each slice maycontain a hippocampal structures: The chain of neurons goes

from the perforant path to granule cells of the dentate gyrus,through mossy fibres to CA-3 pyramidal cells and then throughSchaffer collaterals to CA-1 cells with their axons leaving thehippocampus through the alveus.

Procedure

Male guinea pigs weighing 300400 g are anesthetized withether and the brain is removed, Transverse slices of thehippocampus (300400 m thick) are cut in parallel to thealvear fibers.

the slices are submerged in 28 C warm saline which isequilibrated with 95% O2 and 5% CO2.

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After a preincubation period of 2 h, slices are transferred in a

Perspex chamber and attached of hook.

The chamber is mounted on an inverted microscope.

Intracellular recordings are achieved by means of micropipettes

with tip diameters of less than 0.5 m which are filled with

3 mol/l potassium chloride and placed within the striatum

pyramidale.

The intracellular injections of drugs, e.g., pentylenetetrazol, are

made via the recording microelectrode, a passive bridge is used.

Alternatively, drugs are added to the Incubation bath.

Ev

aluation The resting membrane potential and paroxysmal depolarizations

are recorded before and after application of drugs.

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Application

Hippocampal slice is one of the most useful model for the study

of basic mechanism underlying the epilepsies.

It also recommended for screening of putative anti-convulsant

drugs.

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ELECTRICAL RECORDINGS IN ISOLATED

BRAIN CELLS (MES)

Rationale

The use of the cell-attached patch clamp configuration to recordaction potential currents has shown to have utility in the testingfor drug actions on ion channels in excitable cell membranes.

Procedure

Preparation of cultured cells

The cultured cells are obtained from the hippocampus or thehypothalamus of rat brain.

The hippocampal and hypothalamic neurons that are selectedfor electrophysiological recording are bipolar in shape with thelong axis dimension between 1015 m.

Electrophysiology

The cell-attached patch clamp configuration is used to recordspontaneous action potentials in the cultured neurons.

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The bath solution contains 140 mM NaCl, 5 mM KCl, 0.5 mM

CaCl2, 1 mM MgCl2, 5 mM HEPES, pH 7.3. The drugs used in the experiments are added to the bath

solution.

The spontaneous action and activity recorded at a sampling

frequency of 5 kHz, and the data analysed.

Evaluation

The capacitative component of current recorded by the patch

pipettes is proportional to the rate of change of membrane

potential and can be expressed as

IC= C dV/dt.

Where,

IC corresponding to the after-hyperpolarization component of

the action potential

C is the specific membrane capacitance10


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ISOLATED NEONATAL RAT SPINAL CORD

Rationale

In this preparation, ventral root potentials of ten seconds of

duration can be recorded after supramaximal electrical

stimulation of the lumbar dorsal root.

Procedure

Preparation of spinal cord

Male Wistar rats aged 69 days are used. Anaethesized with

ether and the spinal cord of the mid-thoracic to mid-sacral level

is then carefully removed.

After removal of the dura mater, the hemisected cord is

completely submerged in the recording chamber which is

perfused with physiological solution.

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Evaluation

Recording of monosynaptic reflexes

Test stimulations, composed of square wave pulses of 0.050.2

ms duration and 530 V, are applied to the dorsal root every 10s.

The discharges of the corresponding ventral root are recorded.

The mean values for the waveform of the monosynaptic reflex(amplitude, area and latency) are obtained from 618 successive

responses in each experiment before and during application of

drugs.

Statistical significance of the data is determined by repeated

measures analysis of variance (ANOVA) and, when appropriate,Students t-test.

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IN VIVO MODELS

Electroshock in mice

Pentylenetetrazol test in mice and rats

Strychnine-induced convulsions in mice

Picrotoxin-induced convulsions in mice

Isoniazid-induced convulsions in mice

Bicuculline test in rats

4-aminopyridine-induced seizures in mice

Epilepsy induced by focal lesions

Kindled rat seizure model

Post hypoxic myoclonus in rats

Genetic animal models of epilepsy

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IDEAL CHARACTERISTICS OF IN-VIVO

MODEL OF SEIZURES OR EPILEPSY Development of spontaneously occurring recurrent seizures.

Seizure type should similar in clinical phenomenology to those

in human epilepsy.

Age- dependent onset of epilepsy as in generalized epileptic

syndromes in human.

Clinical seizures should be accompanied by epileptiform activity

in the EEG.

Pharmacokinetics of antiepileptic drugs should be similar to

those in humans.

Effective plasma concentration of antiepileptic drugs similar to

those required for controlling the particular seizure type in

human.

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ELECTROSHOCK IN MICE

Rationale

Tonic hindlimb extension are evoked by electrical stimuli whichare suppressed by anti-epileptic drugs.

The electroshock assay in mice is used primarily as an indication

for compounds which are effective in grand mal epilepsy.

Procedure

Groups of 610 male swiss mice (1830 g) are used.

The test is started 30 min after i.p. injection or 60 min after oral

treatment with the test compound. An apparatus with corneal or ear electrodes is used to deliver

the stimuli.

The intensity of stimulus is dependent on the apparatus, e.g. 12mA, 50 Hz for 0.2 s have been used. 15


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Evaluation

Disappearance of the hind leg extensor tonic convulsion is usedas positive criterion.

Percent of inhibition of seizures relative to controls is calculated.

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PENTYLENETETRAZOL (METRAZOL)

INDUCED CONVULSIONS

Rationale

Pentylenetetrazole acts as a selective, competitive antagonist to blockthe inhibitory effects of glycine at all glycine receptors.

The convulsing action of pentylenetetrazole is due to interference with

postsynaptic inhibition mediated by glycine which is an importantinhibitory transmitter to motor neurons and interneurons in the spinalcord.

Procedure

Mice of either sex with a body weight between 18 -22 g are used.

The test compound or the reference drug is injected sc. or i.p. or given orally

to groups of 10 mice. Another group of 10 mice serves as control.

Fifteen min after sc.-injection, 30 min after i.p.-injection, or 60 min after oraladministration 60 mg/kg pentylenetetrazole are injected subcutaneously.

Seizures and tonic-clonic convulsions are recorded.

At least 80% of the animals in the control group have to show convulsions.17


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Evaluation

PTZ-injection and occurrence of seizures can be measured. The

delay of onset is calculated in comparison with the control

group.

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STRYCHNINE-INDUCED CONVULSIONS

Rationale

Strychnine acts as a selective, competitive antagonist to block the

inhibitory effects of glycine at all glycine receptors.

The convulsing action of strychnine is due to interference with

postsynaptic inhibition mediated by glycine which is an importantinhibitory transmitter to motor neurons and interneurons in the spinal

cord.

Procedure

Groups of 10 mice of either sex with a weight between 18 - 22 g are

used.

They are treated orally with the test compound or the standard (e.g.

diazepam 5 mg/kg).

One hour later the mice are injected with 2 mg/kg strychnine nitrate i.p.

The time until occurrence of tonic extensor convulsions and death is

noted during a 1 h period.19


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Evaluation

ED50-values are calculated using various doses taking thepercentage of the controls as 100%.

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PICROTOXIN-INDUCED CONVULSIONS

Rationale

Picrotoxin is a GABAA-antagonist modifying the function of the

chloride ion channel of the GABAA receptor complex.

Procedure

Groups of 10 mice of either sex with a weight between 18 - 22 g

are treated either orally or i.p. with the test compound or the

standard.

30 min after i.p. treatment or 60 min after oral administration

the animals are injected with 3.5 mg/kg s.c. Picrotoxin.

For the next 30 min. clonic seizures, tonic seizures, death are

observed.

Time of onset of seizures and time to death are recorded. 21


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Evaluation

Protection is expressed as percent inhibition relative to vehiclecontrol.

ED50- values are calculated taking the percentage of seizures in

the control group as 100%.

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ISONIAZID-INDUCED CONVULSIONS

Rationale

Isoniazide causes GABA synthesis inhibition.

Isoniazid can precipitate convulsions in patients with seizure

disorders.

Procedure

10 mice of either sex with a weight of 18 to 22 g are treated with

the test compound or the standard by oral or intraperitoneal

administration.

30 min after i.p. or 60 min after oral treatment the animals are

injected with a subcutaneous dose of 300 mg/kg isoniazid.

During the next 120 min the occurrence of clonic seizures, tonic

seizures and death is recorded. (Continued..)

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Evaluation

The suppression of seizures or death in the treated groups iscalculated as percentage of controls.

ED50-values are calculated.

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BICUCULLINE TEST IN RATS

Rationale

Seizures can be induced by the GABAA-antagonist bicuculline

and are antagonized by known anti-epileptics.

Procedure

Female Sprague-Dawley rats are injected i.v. with 1 mg/kg

bicuculline.

Tonic convulsion appears in all treated rats within 30 s after

injection.

Test compounds are administered orally 1 or 2 h before

bicuculline injection.

Evaluation

Percentage of protected animals is evaluated.25


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4-AMINOPYRIDINE-INDUCED SEIZURES

IN MICE

Rationale

4-aminopyridine is a k+ channel antagonist which producesconvulsions both in animal and men.

It readily penetrates BBB and induce seizure activity by enhancingspontaneous and evoked neurotransmitter release and consequent

excessive activation of non-NMD

A type exitatory amino acidreceptor.

In mice parentrally administered 4-aminopyridine induces clonic-tonic convulsions and lethality.

Procedure

Male swiss mice weighing 25-30 g are administered 4-aminopyridine (13.3 mg/kg, s.c.).

The test or standard drugs are administered in various doses i.p. 15min. prior to 4-aminopyridine injection.

The behavioral signs such as hyper-reactivity, trembling,intermittent forelimb hind limb clonus followed by hindlimbextension, tonic seizures and death will be noted.

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KINDLED RAT SEIZURE MODEL

Rationale

It was first described by Goddard et. al. (1969) kindling results fromrepetitive subconvulsive electrical stimulation of certain areas of thebrain.

Initially, local after discharge is associated with mild behavioralsigns; however, with continued stimulation electrical activitypresumably spreads, and generalized convulsions occur.

Procedure

Adult female Sprague-Dawley rats (270400 g) are used.

The rats are implanted with an electrode in the right amygdala. At least 1 week has to elapse before electrical stimulation of the

brain is started.

After discharge threshold is determined for each rat. Duration andamplitude, behavioral seizure duration and seizure stage arerecorded with increased stimuli afterdischarges.

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Seizure severity is classified into 5 stages

1. Immobility, eye closure, twitching of vibrissae

2. Facial clonus and head nodding

3. Unilateral forelimb clonus

4. Rearing often accompanied by bilateral fore limb clonus5. Rearing and failing accompanied by generalized clonic

seizure.

Rats are considered to be kindled on the first stimulationcausing a stage 5 seizure which is followed by at least 2consecutive stage 5 seizures.

Evaluation

The occurrence and the degree of seizures are comparedbetween control results and the those after administration of thetest compound.

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GENETIC ANIMAL MODELS OF EPILEPSY

Rationale.

Several animal species exhibit epilepsy with spontaneousrecurrent seizures such as dogs, rats, and mice (Lscher1984).

Serikawa and Yamada (1986) described spontaneous epilepticrats which are double mutants and exhibit both tonic and

absence-like seizures.

Procedure

Spontaneous epileptic rats are obtained by mating the tremorheterozygous rat (tm/+) with the zitter homozygous rat (zi/zi)

found in a Sprague-Dawley colony.

The frequency of tonic convulsions and wild jumping occurringin the absence of external stimuli are recorded.

An electrode is placed on the frontal cranium.

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The frequency of absence-like seizures and tonic convulsions, as

well as the duration of each seizure, are measured on the EEG. A mild tactile stimulus is given on the back of the animal every

2.5 min to induce consistent tonic convulsions.

Evaluation

The number of seizures and the duration of each seizure are

obtained and the total duration of the seizures (number

duration) is calculated every 5 min before and after injection of

the drug.

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Thankyou

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evaluation of antiepileptic drugs