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PRECLINICAL EVALUATION of Anti-Epileptics
S K KANTHLALDept of Pharmacology
Amrita School of PharmacyKochi
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.
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.
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)
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.
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)
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
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
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
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
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
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.
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.
Picrotoxin-induced Picrotoxin-induced ConvulsionsConvulsions
Bicuculine Tests In Rats
4-Aminopyridine induced 4-Aminopyridine induced seizures in miceseizures in mice
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”
Other brain areas like Neocortex, hippocampus in
rats
Electrical stimulus: 400-500 μA, 1 msec. wave pulse : 1 sec with 50-60/sec frequency
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
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
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
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
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