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PRECLINICAL EVALUATION of Anti-Epileptics S K KANTHLAL Dept of Pharmacology Amrita School of Pharmacy Kochi

Screening of antiepileptic drugs

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Page 1: Screening of antiepileptic drugs

PRECLINICAL EVALUATION of Anti-Epileptics

S K KANTHLALDept of Pharmacology

Amrita School of PharmacyKochi

Page 2: Screening of antiepileptic drugs

Animal Models of SeizureAnimal Models of Seizure The usual approach to anticonvulsant drug testing in

animals is to observe the effect of prior drug

administration on seizures produced by

Electrical stimulation of brain,

Systemic administration of a convulsant drug,

Animal strains with spontaneous or sensory-evoked

convulsions.

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Measurement of drug effects:The drug effect chosen for the study can be assessed on

following parameters:i. Change in the threshold,ii. A qualitative change in pattern of the motor

seizure,iii. Changes in the EEG pattern,iv. Change in incidence of seizures.

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Models for Epilepsy

Induction of Seizure in normal animal

Genetic animal Model

Electrically induced Seizure

Chemically induced Seizure

Acute induced Seizure

MES PTZ

Chronic induced Seizure

Electrical or Chemical Kindling

Post Epilepticus model with spontaneous recurrent seizures

Electrical SE Induction

(Perforanth path)

Chemical SEInduction

( Pilocarpine)

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Methods (in vivo methods) 1. Electroshock seizures

i. Thresholod modelii. MES (Maximal Electro Shock)

2. Chemical induced seizures :i. Pentylenetetrazol ( Leptazol/PTZ) induced seizures.ii. Strychnine-induced convulsions.iii. Picrotoxin-induced convulsions.iv. Isoniazid-induced convulsions.v. Bicuculline tests in rats.vi. 4-Aminopyridine induced seizures in mice.

3. Epilepsy induced by Focal Seizures.

4. Kindled rat seizure model.5. Genetic rat model of epilepsy.

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Electroshock Seizures In Mice & Rats

The PHASES

Phase of tonic limb flexion (1.5 sec)

Full extension of hind limbs (10 sec)

Clonic interval (Variable)

Death/Recovery (in some animals)

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Principle:

• To determine the ability of the drug to alter the threshold for tonic

limb extension

•Effective drugs increases the threshold

•Good test for generalized tonic-clonic seizures (grandmal)

Animals: Mice

An electro-convulsiometer with Corneal or Ear electrodes

CV50/CC50 : Voltage/Current for inducing hind limb extension 50% of animal

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Maximal Electroshock Seizure Test

Protection against electroshock induced seizures in

mice and rats is used as an indication for compounds

which may prove effective in Generalized tonic clonic

seizures

Electric stimuli evoke tonic hind limb extensions, which

are suppressed by anti-epileptic drugs.

A model for screening drugs for generalized (tonic-

clonic) seizure

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ANIMALS: Groups of 6-10 male Swiss mice (20-32g) or Wistar rats (100-150g) are used.

ROUTE OF DRUG ADMINISTRATION: i. Intraperitoneal ii. Oral

30 min after i.p. injection and 60 min after oral administration the animals are subjected to electroshock.

An electro-convulsiometer with Corneal or Ear electrodes is used to deliver the shock.

Current used: o Rat: 150mA / 750 V Freq- 50-60/seco Mice : 50 mA/ 250 0.2 second duration

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The PHASES of maximal seizure shown by normal mice typically consists of : ◦ Phase of tonic limb flexion ( about 1.5 sec)◦ Full extension of limbs (about 10 sec )◦ Clonic interval ( variable )◦ Death (in some animals).

END POINT : Disappearance/reduction of hind limb extensor tonic convulsions is taken as criteria of protection.

The effect of drugs will be observed in terms of prolongation of latent phase, decrease in the duration of tonic phase.

◦ Ethosuccimide is ineffective in this model◦ No clue for probing MCM

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Principle : GABA antagonist causes direct depolarization of central neurons.It Produces jerky type of clonic convulsion in rats and mice similar

to petit mal type of convulsion in man.

Procedure: Groups of 10 Albino Swiss mice of either sex (20-25 gm) First group is injected with test & Diazepam 4mg/kg(i.p.) for std

groups 30min after treatment inject with 75 mg/kg of PTZ by s.c. Each animal is observed for 1 hr. in plastic cage

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Seizure & myoclonic convulsions are recordedObservations: seizures occurs in the following order

One/more jerks (twists)Clonic seizureLoss of righting reflexesMaximal tonic clonic seizureClonic seizure with loss of righting reflex end point

At least 80% of animals in control have to show convulsion.Evaluation: The number of protected animal in treated group is calculated

as percentage of affected animal in control group.ED 50 values calculated & time interval between PTZ injection

and occurance of seizure can be measured.

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Strychnine Induced SeizuresThe convulsant action of strychnine is due to

interference with post-synaptic inhibition that is mediated by Glycine.

It acts as a selective competitive antagonist to block the inhibitory effect of glycine at all glycine receptors.

The convulsions has a characteristic Motor pattern.

Dose : 2 mg/kg.Route : i.p.Time for onset of tonic extensor convulsions

and death of animals is noted.Strychnine abolishes the flexor latency

completely, leading to almost instantaneous onset of the extensor seizure.

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Picrotoxin-induced Picrotoxin-induced ConvulsionsConvulsions

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Bicuculine Tests In Rats

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4-Aminopyridine induced 4-Aminopyridine induced seizures in miceseizures in mice

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Kindled Rat Seizure ModelThe kindled seizure model in rats offer a method to study

the anticonvulsant activity on the basis of pathophysiological model.pathophysiological model.

PrinciplePrinciple::Repetitive admn of subconvulsive electrical stimulation of

certain areas of brain progressive stimulus induced seizure . On continued stimulation electrical activity spreads and

generalized convulsions occur.The animals are given stimulation through an electrode

implanted with in right amygdala.Kindling model “Epilepsy induced Epilepsy”

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Other brain areas like Neocortex, hippocampus in

rats

Electrical stimulus: 400-500 μA, 1 msec. wave pulse : 1 sec with 50-60/sec frequency

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Duration and amplitude, behavioural seizure duration and seizure stage are recorded

Seizure severity is graded into 5 stages.1: immobility, eye closure, twitching of vibrissae, sterotyping

sniffing2: facial clonus and head nodding3: facial clonus , head nodding and forelimb clonus4: rearing , often accompanied by bilateral forlimb clonus5: Rearing with loss of balance and falling accompanied by

generalized clonic seizuresRats are considered to be kindled causing all stages of

seizure

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EvaluationTest animals are tested on the day before and after the test

compound is given orally or i.p.Test and control are compared with four different

measures of efficacySeizure latency – time from stimulation to the first sign of

seizure activity Seizure severitySeizure durationAfter discharge duration

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Rat: 3o mg/kg of PTZ i.p. 3 dose/week for 9 weeksScoring :

0 - no response1 – ear and facial twitching2 – one to 20 myoclonic jerck3 – more than 20 body jerck4 – clonic forelimb convulsion5 – generalized convulsions with rearing and falling down

episodes6 – generelized convulsions with tonic extension episodes

and status epilepticus At the end of the 9th week 90% animals are kindledTest drug should be administered before each PTZEfficacy preventing seizure development

Page 24: Screening of antiepileptic drugs

EPILEPSY INDUCED BY FOCAL SEIZURESEPILEPSY INDUCED BY FOCAL SEIZURES Topical or intracerebral application of metal and chemical can

lead to simple partial seizures Cortical imlanted metals:

Alumina cream, cobalt, tungstic acid Appliead onto or into the cerebral cortex Injection of iron in brain cortex

Aluminium Hydroxide gel model 4% aluminium hydroxide is injected into surgically exposed monkey

neocortex One or two month after injection spontaneous and recurrent seizures

begins Model for focal epilepsy

Chemical Intrahippocampal – kainic acid, tetanus toxin Topical application – penicillin, picrotoxin, bicuculline