EVALUATION OF ANTIEPILEPTIC DRUGS

Dr. Jitendra AgrawalSecond year residentANIMAL MODELS FOR ANTIEPILEPTIC DRUGS1Introduction A Seizure (from the Latin sacire, To take possession of) is a paroxysmal event due to abnormal, excessive, hyper synchronous discharges from an aggregate of central nervous system (CNS) neurons.

Epilepsy describes a condition in which a person has reccurent seizures due to a chronic, underlying process.2Classification Of SeizuresFocal seizure

With dyscongnitive featureWithout dyscognitive featureGeneralized seizure

AbsenceTonic clonicClonicTonicAtonicMyoclonic

Unclassified

Epileptic SpasmsWhy We need animal model?Discovery of new AEDCharacterization of spectrum of anticonvulsant activity of new AEDEvaluation whether efficacy of new AED changes during chronic treatment of epilepsyDiscovery of antiepileptogenic or disease modifying agent4Models for EpilepsyInduction of Seizure in normal animalGenetic animal ModelElectrically induced SeizureChemically induced SeizureAnimals with spontaneous Recurrent seizuresAcute induced SeizureMESPTZChronic induced SeizureElectrical or Chemical KindlingPost Epilepticus model with spontaneous recurrent seizuresElectrical SE Induction(Perforanth path)Chemical SEInduction( Pilocarpine)e.g. Rats or Mice with Spike wave discharge(lethargic mice,tottering mice)e.g. DBA/2 MiceGEPRs, Photosensetive baboons,Gebrils

Animal with reflex seizures5Electroshock Seizures In Mice & RatsProtection against electroshock induced seizures in mice and rats is used as an indication for compounds which may prove effective in Generalized tonic clonic seizuresElectric stimuli evoke tonic hind limb extensions, which are suppressed by anti-epileptic drugs.CC50 : current for inducing hind limb extension 50% of animal

6Maximal Electroshock SeizureMerritt and Putnam (1938) Animals are stimulated 2-5 times the threshold current strength The purpose of this test is to induce the most intense physiologically possible seizure by a method analogous to human electroshock therapy.

7MethodologyANIMALS: Groups of 6-10 male Swiss mice (20-32g) or Wistar rats (100-150g) are used.

ROUTE OF DRUG ADMINISTRATION: Intraperitoneal Oral30 min after i.p. injection and 60 min after oral administration the animals are subjected to electroshock.

8An electro-convulsiometer with Corneal or Ear electrodes is used to deliver the shock.

Current used: Rat: 150mAMice : 50 mA 0.2 second duration Methodology

9The PHASES of maximal seizure shown by normal mice typically consists of : Phase of tonic limb flexion Full extension of limbs Clonic interval ( variable )Death (in some animals)

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10Evaluation Supression of hind limb extenson - measure of eficcacyCalculation of ED50 for supression of tonic hind limb extension anticonvulsant potencyPhenytoin, carbmazepine, phenobarbitone effectiveEthosuximide - ineffective

11DisadvantageDo not give clue about mechanism of action of drug12Pentylenetetrazol Induced SeizuresPentylenetetrazol (PTZ) produces generalized asynchronized clonic movements which are superceded by tonic convulsions characterized by flexion of limbs followed by extension. Act by antagonizing the inhibitory GABAergic transmissionThe test is considered as indicative of anticonvulsant activity of drugs against Absence seizure13Methodology ANIMALS: Groups of 6-10 mice (18-22g) of either sex

ROUTE OF DRUG ADMINISTRATION: Determine S.C. CD97 (convulsive dose in 97% animals)1% solution of PTZ , 80-100mg/kg S.c. in scruff of neckThere are 3 distinct phases constituted the PTZ seizure sequence i.e.

Myoclonic jerkClonic seizuresTonic-clonic hind limb extension.Death

14EvaluationEnd pointFirst episode of clonic jerking last for 5 secFirst clonic seizure with loss of righting reflexEvaluationEfficacy: measured by ED50 for suppression of clonic seizureEthosuximide, valproate effectivePhenytoin, Carbamazepine not effective15Strychnine 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.16Picrotoxin-induced ConvulsionsPicrotoxin is a GABA-antagonist and it modifies the function of chloride ion channel of the GABA receptor complex.

Dose : 3.5 mg/kgRoute : subcutaneous

17Isoniazid-induced ConvulsionsINH is a inhibitor of GABA synthesis.It is known to produce convulsions in patients with a history of seizure disorders.The typical pattern is of tonic-clonic seizuresDose : 300 mg/kgRoute : subcutaneous18Bicuculine Tests In RatsBicuculine is a GABA-antagonist.Dose : 1 mg/kgRoute :Intravenous.The tonic convulsions appear in all treated rats within 30 seconds of injection.

194-Aminopyridine induced seizures in mice4-Aminopyridine, K+ channel antagonist is a powerful convulsant.The epileptiform activity is predominantly mediated by non-NMDA type excitatory amino acid receptors. Dose :13.3 mg/kgRoute : Subcutaneous

20Epilepsy Induced By Focal SeizuresTopical or intracerebral application of metal and chemical can lead to simple partial seizuresCortical imlanted metals:Alumina cream, cobalt, tungstic acidAppliead onto or into the cerebral cortexInjection of iron in brain cortexAluminium Hydroxide gel model4% aluminium hydroxide is injected into surgically exposed monkey neocortexOne or two month after injection spontaneous and recurrent seizures beginsModel for focal epilepsyChemicalIntrahippocampal kainic acid, tetanus toxinTopical application penicillin, picrotoxin, bicuculline

21Kindled Rat Seizure ModelThe kindled seizure model in rats offer a method to study the anticonvulsant activity on the basis of pathophysiological model.Kindling results from repetitive sub convulsive electrical stimulation of certain areas of brain . On continued stimulation electrical activity spreads and generalized convulsions occur.The animals are given stimulation through an electrode implanted with in right amygdala.

22Other brain areas likeNeocortex, hippocampus in rats23Duration and amplitude, behavioral seizure duration and seizure stage are recordedSeizure 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 on the 1st stimulation causing a stage 5 seizure which is followed by at least two consecutive stage 5 seizures24EvaluationTest 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

25Drug efficacy can be measured by determining separate ED50 value for total supression ofGeneralized seizure (stage 4,5)Focal seizure (Stage 1-3)Amygdala after discharges

26Advantage:Efficacy of drug :Process of epileptogenesis Fully kindled stateEfficacy against generalized seizures provides model for effective in secondary generalized seizures of partial epilepsyEfficacy against the focal component of kindled seizures provides a valid model for drugs effective in complex partial seizures27Other methods of kindlingCorneal Electroshock kindling Mice: once daily application of 3 mA current 60 Hz for 2 secRat : once daily application of 8 mA current 60 Hz for 4 secStage 5 seizure is considered as animal is kindled

28Chemical induced kindling

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 kindeledSeizure score more than or equal to 329Models for status epilepticusElectrical Stimulation of hippocampal perforant pathway:Implantation of bipolar stimulating eletrodeIn right angular bundle

Unipolar reccording electrode In right hippocampal dentate granule

Pathway is stimulated by2mA monopolar pulse for 50mcs, 20 Hz, for 2 h

Development of self sustained limbic status epilepticus30Chemical induced status epilepticusPilocarpineCholinomimeticCan produce status epilepticus in ratsDose : 380-400 mg/kgRoute : ipLithium- Pilocarpine;Pretreatment with lithium 3meq/kg ipFollowed by pilocarpine 30-40 mg/kg ipLithium methomyl Pretreatment with lithiumMethomyl 5.2mg / kg s.c.31Model for infantile spasmsEarly childhoodInsensitive to most of the available antiepilepticsVelisek (2007) developed model Pregnant sprague-dawley ratsBetamethasone 0.4mg/kg i.p. two doses at 8:oo am and 6:00 pm on gestational day15

Postnatal day 15Pups NMDA 15mg/kg ip

Twisting movements of tail, arching for several secondsFinally loss of righting reflexFlexion spasms with multiple recurrences.

32Genetic Animal Model for epilepsyTotterer Mice:Homozygous (tg/tg) strain totterer mice are prone to spontaneous epileptic seizureBroad based ataxic gateBy 3 to 4 weeks of age develop frequent partial seizureSpontaneous focal motor seizure occur a few times a day unilateral clonic jerk of limbs with secondary generalizationAlso exhibit absence seizure with synchronous 6-7 per second spike wave discharges in EEGTwo seizure type in one modelMutation in myelin protien ZeroA new spontaneous recessive mutation has been discovered and characterized at The Jackson Laboratory. Mice affected by the new totterer mutation have a neuromuscular phenotype causing a shaky gait. PCR and sequence analysisdetermined that this is a mutation in the myelin protein zero (Mpz) gene.

33Lethargic miceHomozygous (lh/lh)Model for absence seizureRecognized by ataxic gate at the age of 3 weeksBehavioural , EEG, and anticonvulsant profile is similar to those in absence seizure in humanLethargic (lh) mice are spontaneous mutant mice presenting a loss of 4 subunit ofvoltage-gated calcium channels (VGCC) due to a mutation in the gene encoding for 4,Cacnb4. These lh mice are characterized by a complex phenotype with notably severeneurobehavioral defects34Dba/2j miceInbred strain of house mouse (mus musculus)Audiogenic seizure susceptible miceBetween age 2-4 weeks these mice exhibit sound induced seizuresSusceptibility gradually declines at the 8 week totally free of audiogenic seizuresExposed to loud sound (12-16 kHz) Seizure pattern wild running phase clonic convulsion tonic extension respiratory arrest/ full recoverySensitive gross screening model for anticonvulsant drug35GEPRsGenetically epilepsy prone Rats:Seizures can be induced by various stimuliSoundHyperthermiaChemcalElectricalSeizure pattern wild running phase clonic jercks tonic extension respiratory arrest/ full recoveryModel for tonic-clonic convulsion

36Photosensitive BaboonsIntermittent light stimulation at frequencies close to 25 flashes/second leads to seizureEyelid, face, and body clonus and subsequently tonic spasms or full tonic clonic convulsionsModel for tonic clonic seizure, myoclonic seizure

37Mongolian gebrilsSeizure can be provoked byPlacing animal in new envioronmentOnset of bright lightAudiogenic stimulusVigorous shaking of cageSeizure can be myoclonic seizures (7 to 10 weeks)Model for petit mal epilepsyGenerelized tonic clonic in older animalsModel for tonic clonic epilepsy

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39ConclusionIdeal model of epilepsy should show the following characteristicsDevelopment of spontaneously occurring seizuresType of seizure similar to that seen in human epilepsyEEG correlates of epileptic like activityAge dependency in the onset of epilepsy as seen in many epileptic syndromesAt present no model follows all criteriaOnly genetic model come close to call idealResemble idiopathic epilepsy in humans more closely than any other experimental model

40The antiepileptic drug development program primarily based on two seizure model, the MES and the s.c. PTZSingle method of screening of antiepileptic drugs can not predict the full pharmacological profile of the drug.

41References Hans GV. Drug Discovery and Evaluation:Pharmacological Assays. Springer. 3rd edition. New York :Springer-Verlag Berlin Heidelberg ; 2008.Gupta SK. Drug Screening Methods (Preclinical Evaluation of New Drugs). 2nd edition.New Delhi:Jaypee Brothers Medical Publishers; 2009.Wolfgang L. Critical review of current animal models of seizures and epilepsy used in the discovery and development of new antiepileptic drugs. Seizure. 2011(20):359368.

42Thank you !