Screening Methods of Anti-epileptic drugs

Screening Methods of
Anti-epileptic drugs
Presented by
A. Gowtham Sashtha |
st
M.Pharm
Department of pharmacology
K.M College of pharmacy

➜Epilepsy is very common disorder.It characterised
by abnormal or excessive in neuronal activity in brain,with or
without characteristic body movements.
➜Epilepsy was described by JH jackson.
Epilepsy ?
Classiﬁcation of seizure
➜Partial seizure
➜Generalized seizure
➜Unclassified seizure
➜Status epilepticus

→The usual approach to anti-convulsant drug testing in
animal is to observe the effect of prior drug administration on
seizures.
It produced by :
→Electrical stimulation of brain
→systemic administration of the convulsant drugs.
→Animal strains with spontaneous or sensory-evoked
convulsant
→Isolation of brain cells.
Preclinical Evaluation

➜The drug effects chosen for the study can be
assessed following parameters :
→Change in the threshold.
→A quantitative changes in pattern of the motor seizure.
→Changes in the EEG pattern.
→Changes in incident of seizure.
Measurement of drug e fects :

invivo models :
1) Electroshock induced seizure
2) Chemical induced seizure :
a) Pentylenetetrazole (Laptazol) induced seizures.
b) Strychnine induced convulsions.
c) Picrotoxin induced convulsions.
d) Isoniazid induced convulsions.
e) Bicuculine induced convulsions.
f) 4-aminopyridine induced seizure in mice.
Methods to induce Experimental epilepsy

3) Kindled rat seizure model.
4) Genetic rat model of epilepsy.
Invitro models :
1) Hippocampal slices.
2) Electrical recording from isolated brain cells.
3) Inhibitory amino acid binding assays.
a)GABAA
receptor binding assays
b)GABAB
Receptors binding assays.
4) Excitatory amino acid binding assay.
a) Glutamate receptors binding assays

Purpose :
→ Protection against electroshock induced seizures in
mice & rats in used as an indication for compounds which may
prove effective in grand mal epilepsy.
Two methods
: 1)corneal electrodes
: 2)Ear clip (or) pinna electrodes.
1) Electroshock induced seizures

☞The animals are observed closely for 2 mins.
☞Appearance / Disappearance of five different
phases of convulsions.
☞Drugs effectives against generalized tonic clonic
seizure such as phenytoin, carbamazepine, Phenobarbitone,
primidone and ethosuximide in effective in this test.
Evaluation :

In MES mothod in high intensity shock applied to the
head through corneal or ear electrodes. voltage as induced
50-60/sec current delivered via mice & rats.50mA in mice &
150mA in rats,
The electrodes are moistened with saline solution before
application for better conductance.
The MES convulsions produced in five phase such as tonic limb
flexion(about 1.5 sec),tonic limb extension(about 13 sec),clonic
convulsant,stupor and recovery or death.
Principle

Divided overnight fasted rats in to five groups comprising of six each
animals (five group)
administration of drug doses in three different groups.
1) Administer the drug / extract at administration of Group lll,lV,V in
orally (2ml / 100g body weight, extract should be suspended in 0.6%
sodium carboxy methyl cellulose) 60 mins before for induction of MES.
2) Phenytoin at administration of group ll in i.p (25mg /kg body weight)
30 mins before induction of MES.
3)Only administer vehicle(2ml / 100g body weight, extract should be
suspended in 0.6% sodium carboxy methyl cellulose) for group l.
Procedure

Apply a supramaximal electrical stimulus of 150 mA to the
animals for 0.2 seconds through ear clip electrodes.
Observe the rats for various phases like - tonic limb flexion,toni
limb extension and tonic clonic phase.
Abolition or decrease in the duration of extensor phases should be
taken as an index of anti-epileptic activity.

Purpose :
→To evaluate the Anti-epileptic drugs.
→Pentylenetetrazole (PTZ) is a CNS stimulant.
→It produce the jerky type of clonic convulsions in rat
& mice similar to petit mal type of convulsion in man.
→It causes direct depolarization of central neurons.
→Also interfere with GABAergic inhibition.
a)Pentylenetetrazole induced convulsions

Procedures :
Two group of 10 albino Swiss mice (20-25 gm)
First group is injected with Diazepam 4mg/kg(i.p)
Second group is control with normal vehicle saline 10ml/kg
30mins after Diazepam treatment inject with 75gm/kg of PTZ by
s.c to the both groups.
Each animals is observed for 1hr in plastic cage.
Seizure & myoclonic sezure are recorded.
at least 80% of animal 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.

Purpose :
Isoniazid (INH) regards as GABA synthesis Inhibitors.
It is known to produce convulsion in humans and animals.
b) Isoniazid induced convulsions

Procedure :
Two group of 10 albino mice (18-22gm)
First group is injected with Diazepam 5mg/kg.
Second group is control with normal vehicle saline 10ml/kg
15 mins after s.c injection/30 mins after i.p/60mins after oral
route,300mg/kg of INH is injected by s.c to the both groups.
During next 120mins clonic seizure , tonic seizure and death is
recorded.at least 80% of animal in control have to show convulsion.

Evaluation :
→The percentage of seizure or death occuring in
the control group is taken as 100% .
→The suppression of these effects in the treated
groups is calculated as percentage of control.ED 50
values are calculated.

Purpose :
Picrotoxin is a GABAa antagonist modifying the function of
chloride ion channel GABAa receptor complex.
Blocked by picrotoxin :
Activation of GABA receptor
Increased flow of cl ions into cells
hyperpolarization of neuron
C) Picrotoxin induced convulsions

Procedure :
Two group of 10 albino mice (18-20mg)
First group is treated with Diazepam 5mg/kg in i.p
Second group is treated with normal vehicle saline,as control group.
15mins after i.p diazepam & 30mins after saline,inject 3.5 mg/kg of
picrotoxin through i.p route.
Observe the symptoms during next 30mins.
(Tonic seizure, Clonic seizure,Death)
Time of onset of seizure and time to death are recorded.

Evaluation :
→ED 50 values are calculated by taking percentage of
Seizure in control group as 100%.

Purpose :
→The convulsive action of Strychnine is due to
interference with Post synaptic inhibition mediated by
glycine.
→Glycine is inhibitory neurotransmitters.
→Strychnine is act as selective and comparative
antagonist to block inhibitory effect of glycine to all Glycine
Receptors.
D) Strychnine induced convulsions

Procedure :
Two group of 10 albino mice (18-20gm)
First group is treated with Diazepam 5mg/kg in i.p
Second group is treated with normal vehicle saline,as control group.
one hour later ,mice are injected with 2mg/kg Strychnine in i.p
Time until occurance of tonic extensor convulsions and death is
noted during one hour.
At least 80% of animals in control have to show convulsion.

Evaluation :
→ED 50 values are calculated.
→For the time response curve interval between
treatment and Strychnine injection varries from 30-120
mins.

Purpose :
Bicuculline acts as a competitive antagonist at the
GABA-binding site of the GABAa receptor.
Block the inhibitory potential in most CNS synapes.
E) Bicuculline induced convulsions

Procedure :
Female Sprague dawley rats
The test components are administered in orally
after 1 (or) 2 hours injected i.v with 1mg/kg bicuculline.
at this dose ,a tonic convulsion appears in all rats within 30
sec after injection.
Dose response curve can be obtained.

Evaluation :
→percentage of protected animals in evaluated.
→ED 50 values and 95% confidence limits are
calculated by probir analysis.
Drug Evaluated :
→phenytoin
→phenobabital
→carbamazepine
→valproic acid

Purpose :
→The K+ channel antagonist 4-aminopyridine is a
powerful conversation in animal and human.
→The both excitatory and inhibitory transmission is
facilitated by 4-aminopyridine
→In mice parenterally administered 4-aminopyridine
induces clonic-tonic convulsions and lethality.
F) 4-aminopyridine induced seizure in mice.

Procedure :
Male Swiss mice (25-30 gm)
The test samples are administered by I.v.
15mins prior of injection of 4-aminopyridine at a dose of
13.3mg/kg (s.c)
Only produce exhibit characteristic behavioral sings,such as
hyper reactivity, trembling, clonic tonic convulsion and death
are recorded.

Evaluation :
The percentage of protected animals at each dose is
used to calculate as ED50 values.

Purpose :
→Kinding results from repetitive subconvulsive electrical
stimulation of certain areas of the brain.
→Initially ,local after discharge is associated with mild
behavioural signs; however, with continued electrical
stimulation activity presumably spreads, and generalized
convulsions occurs.
3) Kindled rat seizure model

Procedure :
Adult female Sprague dawley rats (270-400g)
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 rats , Duration,
amplitude, behavioral seizure duration and seizure stages are
recorded.

The rats are considered as kindled on the first stimulation causing
stage 5 seizure.
The animals are tested on the day before and after treatment with
the test components (i.v or orally)
Amygdala stimulation is applied at various time intervals.
Evaluation :
The occurrence and the degree of Seizures are compared
between control results and the those after administration of the
test comments.

Stages of kindling :
➜Stage 1
High frequency stimulation in the normal brain sites
produces elevation of the GABA and Glutamate release from
presynaptic terminals.
➜Stage 2
Activation NMDA receptors triggers intracellular
cascades that lead to function change in both glutamate and
GABA system.

➜Stage 3
Remodeling of excitatory and inhibitory and
production of abnormal pattern of neurotransmission in brain.
➜Stage 4
Hyper excitability develops in CA1 & CA3 neurons
and less frequent in the hippocampus.
➜Stage 5
This stage characterised by an imbalance between
interacting both glutmate and GABA.

➜ Extensively a strain of genetically epilepsy prone rats (GEPR) ,
susceptible to seizure induced by sounds , Hyperthermia, and various
electrical methods.
➜ With increasing seizure tendencies appearing over the period
2-4 weeks after birth.
➜ Animals from both colonies run about wildly and fall from
clonic jerk when stimulated by sounds.
➜ These seizure prone characters of these rats related in some
ways abnormalities in the auditory pathways including impairment of
cochlear function.
➜ Injection amino acid antagonist In to the inferior colliculus in
highly effective in blocking audiogenic seizure.
4) Genetic rat model of epilepsy

➜ The spontaneously epileptic rats (SER) exhibit 5-7/s
Spike wave like complexs in cortex and hippocampus area.
➜ Ethosuximide,trimethadione, Valproate &
Phenobarbital inhibit Absence seizure and phenytoin,
Valproate & Phenobarbital inhibit tonic seizure in SER rats.

Purpose :
→Especially useful due to the involvement of hippocampus
in generation of complex partial seizure.
Procedure :
→Hippocampus is dissected & slices about 0.5mm thickness
are made.
→Preserve the neurons synaptic circuit and associated
recurrent activities
→The neurons in the slice are done by passing micropipette
(<0.5mm) in to the under microscopic control.
Hippocampal slices

Evaluation :
→Adding drug to the slice medium and recording the
spontaneous firing of neuron.
Advantages :
→Mechanical stability and absence of anesthetics and
study of neurophysiological mechanisms.

→This methods used testing action of drugs on ion channel
in excitable cell membrane.
→The cells are either obtained from hippocampus or from
hypothalamus and then grown in tissue culture.
→Glass pipettes are directly connected to membrane in
response to voltage , ionic or chemical change.
→The isolation of cells are put in to path solution & drugs are
added to it & recording of capacity currents.
Electrical recording from isolation brain cells

GABAa & GABAb binding assay :
purpose :
The GABAA receptor directly gates a Cl– ionophore
and has modulatory binding sites for benzodiazepines,
barbiturates, neurosteroids and ethanol.
The GABAB receptors couple to Ca2+ and K+ channels
via G proteins and second messenger systems; they are
activated by baclofen.
Assay of GABAergic compounds

Procedure :
Male rats weighed about 100-150 g are decapitated & their
brains removed and placed into ice cold solution.
The assay tubes are prepared by serial homogenisation and
different centrifugation process of brain tissue of rat along with
adding some radio ligand.
All this done to increase the chemicals specific binding of the
receptors.
The radioactivity that can be displaced by a high
concentration of 3H GABA is calculated.

GABAa Receptors :
A synthetic derivative of GABA 3H-SR 95531
[2-(3-carboxy-2-propyl)-3-amino-6-methoxyphenylpyridazini
um bromide],
GABAb Receptors :
Baclofen, specific activity Tyrosine
Radio ligand
Centrifugation process :
1) 1 000 g at 4 °C for 10 min
2) 20,000 g for 20 min.
3) 48,000 g for 20 min
4) 50,000 g for 10 min...

The following parameters are calculated:
total binding
non-specific binding
specific binding=total binding– non-specific binding.
Evaluation :

Purpose :
Glutamate and possibly aspartate, function as the fast
excitatory neurotransmitter in brain.
Excessive excitatory neurotransmitter can produce
neuropathogenesis of epilepsy, stock, schizophrenia.
Antagonist at these receptors have been shown to
anti-convulsand property.
Glutamate receptor binding assay

Procedure :
same procedure of GABA binding assays.
Radio ligand or reagent :
l- glutamic acid
Test components.
Chlorpyriphos (CPP)
Triton - X

→Does not give any Proper idea about
pharmacokinetics and pharmacodynamics interaction of drug
in living animals.
→Not possible to study compensatory changes that
occurs in body when drug in given.
Disadvantage of invitro study

→The ideal model of epilepsy should show the
following characteristics :
→Development of spontaneously occuring seizure.
→Types of seizure similar to that seen in human
epilepsy.
→EEG correlates of epileptic - like activity.
→At present no models follows all criteria.
→Only genetic model come close.
Conclusion :

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Screening Methods of Anti-epileptic drugs

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