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Transcript of 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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