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SCREENING OF ANTI-EPILEPTIC DRGUS A REPORT SUBMITTED FOR CLASS SEMINAR SUBMITTED BY: Sunil Patel ( M.Pharm Sem - II ) GUIDED BY: Dr.Pankaj Tripathi ( M.pharm , Ph.D ) DEPARTMENT OF PHARMACOLOGY Shree S.K.Patel College Of Pharmaceutical Education & Research, Ganpat vidyanagar , Kherva

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Epilepsy is chronic neurological disorder that is characterized by recurrent unprovoked seizures. Seizures are transient signs and/or symptoms due to abnormal, excessive or synchronous neuronal activity in the brain. Mechanism Involved Seizures Ion Channels Neurotransmitters Hippocampal Sclerosis. Genetic Factors

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TYPES OF EPILEPSY 1) Partial Seizures ( Focal Seizures) a. Simple Partial Seizures. b. Complex Partial Seizures. 2) Generalized Seizures a. Tonic− Clonic (Grand Mal) Seizures. b. Absence (Petit Mal) Seizures.

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Methods to screen antiepileptic activity 1. IN VIVO METHODS 1.1 Electroshock in mice 1.2 Pentylenetetrazole Induced Convulsions in Mice & Rat 1.3 Strychnine-induced convulsions 1.4 Picrotoxin -induced convulsions. 1.5 Isoniazid -induced convulsions. 1.6 Yohimbine -induced convulsions 1.7 Bicuculline test in rats 1.8 4-Aminopyridine-induced seizures in mice 1.9 Epilepsy induced by focal lesions 1.10 Kindled rat seizure model

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2.In vitro method 2.1 [ 3 H] GABA Receptor-binding Assay 2.2 GABA A Receptor-binding Assay 2.2.A [ 3 H] Muscimol -binding Assay 2.2.B [ 3 H] SR 95531 Binding Assay 2.3 GABA B Receptor-binding Assay 2.4 [3H] GABA Uptake in Rat Cerebral Cortex 2.5 [ 35 S] TBPS Binding Assay 2.6 [3H] CPP-binding Assay 2.7 [ 3 H] TCP-binding Assay 2.8 [H] Glycine -binding Assay 2.9 Hippocampal Slices 2.10 Electrical Recording from Isolated Brain Cells

Electroshock in mice:

Purpose and rationale Effective in grandmal epilepsy. Tonic hind limb extensions are evoked by electric stimuli which are suppressed by anti-epileptics but also by other centrally active drugs. Requirements Animal: mice (n=6 to 10) Sex: male Weight: 18 to 30g Instrument: Electroconvulsiometer Procedure: animal trated with test or control i.p or orally deliver the stimuli using apparatus with corneal or ear electrode.( 12 mA , 50 Hz for 0.2 s) Electroshock in mice

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Evaluation Observed closely for 2 min. Disappearance of the hind leg extensor tonic convulsion I used as positive criterion. Percent of inhibition of seizures . ED 50-values PHASES: Flexon ( bendidg , 3-4 sec) Tonic (extension, up to 8 sec) Clonic (jerking, 4-5 sec) Stuper ( unconcious ) Recovery or death Modifications Increasing-current electro shock seizure test, a new method for assessment of anti- and pro- convulsant activities of drugs in mice. A single train of pulses of linearly increasing intensity from 5 to 30 mA was applied via ear electrodes. The current at which tonic hind limb extension occurred was recorded as the seizure threshold.

Strychnine-induced convulsions:

Strychnine-induced convulsions Purpose and rationale The convulsing action is due to interference with postsynaptic inhibition mediated by glycine . Strychnine acts as a selective, competitive antagonist to block the inhibitory effects of glycine at all glycine receptors . Requirement Animal: Swiss-Webster mice (M/F) (n= 10) Weight: 18 - 22 g Procedure treated orally with the test or the standard (e.g. diazepam 5 mg/kg). 1 hour later the mice are injected with 2 mg/kg strychnine nitrate i.p . time until occurrence of tonic extensor convulsions and death is noted during a 1 h period.

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Evaluation ED 50 -values are calculated using various doses taking the percentage of the controls as 100%. Critical assessment Useful to characterize CNS-active drugs. Modifications of the method McAllister (1992) induced spinal seizures in mice by rotating them along the body axis clockwise and anticlockwise alternatively three times following pretreatment with a sub convulsive dose of strychnine. Lambert (1994) tested the antagonism of a glycine derivative against seizures induced by 3-mercaptopropionic acid (3-MPA).

Picrotoxin-induced convulsions. :

Picrotoxin -induced convulsions. Purpose and rationale Used to further evaluate CNS-active compounds. Picrotoxin is regarded as a GABA A -antagonist modifying the function of the chloride ion channel of the GABA A receptor complex. Requirement Animal : mice (M/F) (n= 10) weight : 18 - 22 g

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Procedure Animal treated orally or i.p . with test or the standard (e.g. 10 mg/kg diazepam i.p .). 3o min ( i.p ) or 6o min (orally ) after inject with 3.5 mg/kg s.c . picrotoxin observe during the next 30 min for the following symptoms : clonic seizures, tonic seizures, death. Times of onset of seizures and time to death are recorded. Evaluation Time-response curves Percent inhibition relative to vehicle control. ED 50 -value

Bicuculline test in rats :

Bicuculline test in rats PURPOSE AND RATIONALE:- Induced by the GAGA A -antagonistic effect. Requirement Animal : Sprague- Dawley rats Sex : female Procedure. Test or standard compounds are administered orally after 1 to 2 hr inject Bicuculine i.v . (1 mg/kg) observe for 30 min

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Evaluation Percentage of protected animals is evaluated. ED 50 -values and 95% confidence limits are calculated by probit analysis. Critical assessment of the method Relatively specific for anti-epileptic activity. Modifications Czuczwar et al. (1985) studied the antagonism of N methyl-n D,L-aspartic acid-induced convulsions by antiepileptic drugs and other agents.

4-Aminopyridine-induced seizures in mice:

4-Aminopyridine-induced seizures in mice Purpose and rationale The K + channel antagonist The drug readily penetrates the blood-brain barrier and induce seizure activity by enhancing spontaneous and evoked neurotransmitter release. Requirement Animal : Swiss albino mice Sex : male Weight : 25 to 30 g Procedure Allowed to acclimatize with free access to food and water for 24-h before testing. Test drugs are administered in various doses intra peritoneally after 15 min administered with s.c .injection of 4-aminopyridine at a dose of 13.3 mg/kg

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Controls treated with 4-aminopyridine onlyexhibit characteristic behavioral signs, such as hyper reactivity, trembling, intermitted forelimb/hind limb clonus followed by hind limb extension, tonic seizures, opisthotonus and death. Evaluation The percentage of protected animals at each dose is used to calculate ED 50 values. Phenytoin -like anti- convulsants such as carbamazepine and broad spectrum anticonvulsants such as phenobarbital and valproate are effective whereas GABA-enhancers such as diazepam, several NMDA antagonists, and CA2+ channel antagonists such as nimodipine are not. Critical assessment of the method The test is useful to differentiate the mode of action of anticonvulsant drugs.

Epilepsy induced by focal lesions:

Epilepsy induced by focal lesions Purpose and rationale Intra hippocampal injections of noxious agents or certain cerebral lesions can induce seizures in animals. Cavalheiro et al. (1982) studied the long-term effects of intra hippocampal kainic acid injections in rats. Requirement Animal : Adult Wistar rats Sex : male Procedure Anesthetized with a chloral hydrate/Nembutal® mixture and placed in a stereotactic apparatus. insert 0.3 mm cannula is inserted through a burr hole in the calvarium .

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Kainic acid is dissolved in artificial serum and infused in various doses (0.1 to 3.0µg) in a volume of 0.2 µL over a period of 3 min. recorded EEG polygraph, using bipolar twisted electrodes (100µm) positioned stereotaxically Evaluation EEG recordings and observations of convulsive seizures are performed during the acute phase and during the chronic phase (up to 2 months) with and without drug treatment.

Kindled rat seizure model :

Kindled rat seizure model Purpose and rationale Kindling results from repetitive subconvulsive electrical stimulation of certain areas of the brain. Initially, local after discharge is associated with mild behavioral signs; however, with continued stimulation electrical activity presumably spreads, and generalized convulsions occur Requirement Animal : Adult Sprague- Dawley rats Sex : female Weight : 270–400 g Pracedure The rats are implanted with an electrode in the right amygdala : frontal, lateral, horizontal.

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after 1 week electrical stimulation of the brain is started. Duration and amplitude, behavioral seizure duration and seizure stage are recorded with increased stimuli after discharges. Seizure severity is classified into 5 stages. Rats are considered to be kindled on the first stimulation causing a stage 5 seizure which is followed by at least 2 consecutive stage 5 seizures. The animals are tested on the day before and after treatment with the test compound ( i.p . or orally). Evaluation occurrence and the degree of seizures are compared between control . Critical assessment of the method give more relevant results than the simpler methods using maximal electroshock or chemically induced convulsions.

[35S] TBPS Binding Assay :

[ 35 S] TBPS Binding Assay Purpose and rationale TBPS (t-butyl bicyclo phosphorothionate ) acts by blocking the GABAergic neurotransmission by interacting with picrotoxin sensitive site of GABA-benzodiazepine Cl - channel complex. Inhibition of binding of [ 35 S] TBPS to the rat cortical membranes by test compounds is used for screening of anticonvulsant drugs. Procedure: The tissue homogenate is incubated in buffer (0.05 M Tris maleate with 2 M KCI, pH 7.4) and distilled water along with [ 35 S] TBPS (2 nM ) and picrotoxin (10 M) or distilled water or the test compound for 150 minutes at 25°C, with agitation.

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binding is determined by rapid filtration over Whatman GF/B filters, which are presoaked in buffer. Radioactivity is quantified by counting with 10 ml liquiscint . Evaluation : Difference between binding in the absence or presence of picrotoxin gives the specific binding which is typically 85-90% of total binding. Percentage inhibition of [ 35 S] TBPS at each drug concentration is measured and IC 50 value calculated using log- probit analysis.

[3H] Glycine-binding Assay :

[3H] Glycine -binding Assay Purpose and rationale Glycine is a modulator of the NMDA receptor function Compounds with affinity for glycine -binding site provide novel substances for modulation of NMDA receptor function. Such compounds are assayed using the [ 3 H] glycine -binding assay. Procedure : The assay tubes are prepared in quadruplicate. The tissue homogenate is incubated with [ 3 H] glycine (10 nM ) in buffer (0.5 M Tris maleate , pH 7.4) along with glycine (10 M) or distilled water or the test drug for 20 minutes in an ice bath at 0-4°C.

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The binding is terminated by centrifugation for 20 minutes at 7000 rpm at 4°C. The pellet obtained is rinsed with ice-cold buffer and the radioactivity is quantified by counting with 10 ml of liquiscint . Evaluation : Difference of binding in the absence or presence of unlabeled glycine gives the specific binding, which is typically 60-70% of the total binding. Inhibition of binding of [ 3 H] glycine by the test compound is measured and IC value calculated using log- probit analysis.

Hippocampal Slices :

Hippocampal Slices Used for study of neurophysiological mechanisms of epilepsies. complex partial seizures. Procedure : Animal : rodent (rat, mouse, guinea pig) Decapitate animal, remove brain and dessect hippocampus. Using a vibrotome , slices of about 0.5 mm thickness are made. preincubated for 2 hours in a holding chamber in which they are kept moist and oxygenated in 28°C warm saline equilibrated with 95% 02 and 5% CO2. transferre the slices to a Perspex chamber & attached to its bottom.

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Intracellular recordings from the pyramidal neurons in the slice are done by passing micropipettes (tip diameter <0.5 mm) into the stratum pyramidale under microscopic control. Evaluation: Readings are taken by adding drug to the slice medium and recording the spontaneous or shock evoked repetitive firing of neurons. A dvantage of mechanical stability, absence of a blood- brain barrier for applied drugs and absence of anesthetics. Very useful model for studying the neurophysiological mechanisms of convulsant and anticonvulsant drugs.

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