Screening methods of anti-epileptics

ByT. Sravanthi,

12DG1SO1O9,Pharmacology,

M.Pharmacy

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Epilepsy is a very common disorder, characterised by

seizures, which take various forms and results from

neurongal discharges, with or without characteristic

body movement

Epilepsy was described by JH Jackson.

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Primary Genralised Partial Seizure Unclassified Seizure

Tonic – Clonic seizure

Absence seizure

Atonic seizure

Myoclonic seizure

Simple Partial Seizure

Complex Partial Seizure

Partial Seizures

Secondarily generalized.

Infantile Spasms

Status

Epilepticus

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Generalised seizure

Whole brain is involved

Characterised by immediate loss of consciousness

Major Types

1. Tonic – Clonic Seizure (Grand Mal Epilepsy):-

Cry – Unconsciousness- Strong (Tonic) contraction of all body muscles –

Clonic Jerking – Prolonged sleep & Depression.

1-2 min

2. Absence Seizure (Petit Mal Epilepsy):-

Prevalant in Children.

Momemtary loss of consciousnees

Patient freezes & stares in one direction

No muscular movement & jerking.

½ mins

3. Atonic Seizure:-

Due to excessive inhabitory dischagre

Relaxation & Patient may fall.4

Partial seizure

Discharge begins locally, remains localised.

Symptoms depend on the brain region involved.

Also known as Psychomotor Epilepsy.

1. Simple partial seizures (Cortical Focal Epilepsy):-

Involvement of area of cortex causes convulsions.

No loss of consciousness.

½ to 1 min.

2. Complex partial Seizures

(Temporal lobe epilepsy):-

Attacks of bizarre, purposeless

movement.

Impairment of consciousness.

1 – 2 min.

3. Partial Seizures Secondarily

generalized.

Partial seizures occurs first & evolves into generalized tonic-clonic seizure. 5

Selection Of Anti-epileptic Drug

Primary GTC Partial Absence Atypical ,

Myoclonic, Atonic

Valpraote Carbamazepine Valpraote Valpraote

Lamotrigine Phenytoin Ethosuximide Lamotrigine

Topiramate Lamotrigine Topiramate

Valpraote

Oxcarbazepine

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PRECLINICAL EVALUATION

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Animal 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.

For industrial preliminary testing the acute method i.e. drugs tested 1

or 2 hr. after administration of drug, instead of chronically, is used for

economical reasons.

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Methods (in vivo methods)

1. Electroshock seizures

2. Chemical induced seizures :

1. Pentylenetetrazol ( Leptazol/PTZ) induced seizures.

2. Strychnine-induced convulsions.

3. Picrotoxin-induced convulsions.

4. Isoniazid-induced convulsions.

5. Bicuculine tests in rats.

6. 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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Hippocampal slices.

Electrial recording from isolated brain cells.

GABA uptake in rat cerebral cortex.

TBPS binding assay.

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Electroshock Seizures In Mice & Rats

Protection against electroshock induced seizures in mice and

rats is used as an indication for compounds which may prove

effective in

Electric stimuli evoke tonic hind limb extensions, which are

suppressed by anti-epileptic drugs.

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Supramaximal Electroshock.

Extensor Seizure Latency.

Psychomotor Electroshock.

Minimal Electroshock Threshold.

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The purpose of this test is to induce the most intense

physiologically possible seizure by a method analogous to

human electroshock therapy.

The stimulation used in this method is considerably above

threshold ( 5-10 times).

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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

o Mice : 50 mA 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 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.

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Purpose : To evaluate antiepileptic drugs. Pentylenetetrazole is aCNS Stimulant. It Produces jerky type of clonic convulsion inrats and mice similar to petit mal type of convulsion in man.

It causes direct depolarization of central neurons.Also interfere with GABAergic inhibition.

Procedure:

Two groups of 10 Albino Swiss mice of either sex (20-25 gm)

First group is injected with Diazepam 4mg/kg(i.p.)

Second group as control

30min after Diazepam treatment inject with 75 mg/kg of PTZ bys.c.

Each animal is observed for 1 hr. in plastic cage18

Seizure & myoclonic convulsions are recorded

At least 80% of animals in control have to show convulsion.

Evaluation:

The number of protected animal in treated group is calculatedas percentage of affected animal in control group.

ED 50 values calculated & time interval between PTZinjection and occurance of seizure can be measured.

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Purpose : Isoniazid is regarded as GABA synthesis inhibitor.It is known to produce convulsions in patients having history ofseizures.

Procedure:

o Two groups each of 10 albino mice (18-22gm)

o First group is injected with Diazepam 5mg/kg. (i.p.)

o Second group as control, receive vehicle saline 10ml/kg .

o 15 min after s.c. injection/30min after i.p./60 min after oral route,300 mg/kg of INH is injected by s.c. to the both groups.

o During next 120 minutes clonic seizure, tonic seizure and death isrecorded

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o At least 80% of animals in control have to show convulsion.

Evaluation:

o The percentage of seizures or death occuring in the control group

is taken as 100%

o The supression of these effects in the treated groups is calculated

as percentage of controls. ED 50 values are calculated.

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Purpose : Picrotoxin is GABAA - Antagonist modifying the function of

chloride ion channel of GABAA receptor complex.Activation of GABA receptor

Increased flow of cl ions into cell Blocked by picrotoxin

hyperpolarisation of neuron

Procedure:

Two groups of 10 Albino mice of either sex (18-20 gm)

First group is treated with 5mg/kg Diazepam i.p.

Second group is treated with normal saline, as control group

15mins after i.p. Diazepam and 30mins after saline, inject 3.5 mg/kg ofPicrotoxin through i.p. route

Observe the symptoms during next 30 min.

(Clonic seizure, Tonic seizure, Death)

Time of onset of seizure and time to death are recorded 22

Evaluation:

Protection is expressed as percent inhibition relative to vehicle

control . The time period with greatest inhibition is said to be

the peak time of drug activity.

ED 50 values are calculated taking percentage of seizures in

the control group as 100%.

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Purpose

The convulsive action of strychnine is due to interference with post synapticinhibition mediated by glycine.

Glycine is inhibitory neurotransmitter.

Stychnine act as selective and compitative antagonist to block inhibitoryeffect of glycine to all glycine receptors.

Procedure:

Two groups of 10 Albino mice of either sex (18-20 gm)

First group receive 5mg/kg Diazepam i.p.

Second group is control, receive normal saline as vehicle.

One hour later, mice are injected with 2mg/kg stychnine (i.p.)

Time until occurance of tonic extensor convulsions and death is noted duringone hour 24

At least 80% of animals in control have to show convulsion.

Evaluation:

ED 50 values calculated. For the time response curve the interval

between treatment and stychnine injection varries from 30-

120min.

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INH is a

It is known to produce convulsions in patients with a

history of seizure disorders.

The typical pattern is of tonic-clonic seizures.

Dose : 300 mg/kg

Route : subcutaneous

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Bicuculine is a

Dose : 1 mg/kg

Route : Intravenous.

The tonic convulsions appear in all treated rats within

30 seconds of injection.

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Different animal models used are:

pilocarpine induced status epilepticus.

Lithium pilocarpine induced status epilepticus.

Lithium methomyl induced seizure in rats.

Electrical stimulation of hippocampal perforant

pathways.

D,L-homocysteine induced status epilepticus.

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Electrical Stimulation of hippocampal perforant pathway:

Implantation of bipolar stimulating eletrode

In right angular bundle

Unipolar reccording electrode

In right hippocampal dentate granule

Pathway is stimulated by

2mA monopolar pulse for 50mcs, 20 Hz, for 2 h

Development of self sustained limbic status epilepticus

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Pilocarpine◦ Cholinomimetic

◦ Can produce status epilepticus in rats

◦ Dose : 380-400 mg/kg

◦ Route : ip

Lithium- Pilocarpine;◦ Pretreatment with lithium – 3meq/kg ip

◦ Followed by pilocarpine – 30-40 mg/kg ip

Lithium – methomyl◦ Pretreatment with lithium

◦ Methomyl – 5.2mg / kg s.c.

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Totterer Mice:

Homozygous strain totterer mice are prone to spontaneous

epileptic seizure

Broad based ataxic gate

By 3 to 4 weeks of age → develop frequent partial seizure

Spontaneous focal motor seizure occur a few times a day →

unilateral clonic jerk of limbs with secondary generalization

Also exhibit absence seizure with synchronous 6-7 per

second spike wave discharges in EEG

Two seizure type in one model

Ideal model of epilepsy should show the following

characteristics

◦ Development of spontaneously occurring seizures

◦ Type of seizure similar to that seen in human epilepsy

◦ EEG correlates of epileptic –like activity

◦ Age dependency in the onset of epilepsy as seen in many

epileptic syndromes

At present no model follows all criteria

Only genetic model come close

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The antiepileptic drug development program primarily based

on two seizure model, the MES and the PTZ.

Single method of screening of antiepileptic drugs can not

predict the full pharmacological profile of the drug.

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S. K. Kulkarni, Handbook of Experimental

Pharmacology.

S.K Gupta, text book of screening methods and toxicology,

page no:401-419

Tripathi KD, Essentials of medical pharmacology, 6th edition,

chapter-7 page no:401-413

N S Parmar, screening methods of pharmacology, chapter-5

page no:90-97

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Thank you