animal models for screening of antiepileptic drugs &

SCREENING OF ANTIEPILEPTIC DRUGS & THEIR RECENT ADVANCES

DR KAMAL OJAH

DEPT. OF PHARMACOLOGY

Outlines :

Brief overview on epilepsy

Antiepileptics drugs

Methods of screening of antiepileptics

Epilepsy : disorder of brain function characterized by a periodic and unpredictable occurrence of seizures.

Seizure : abnormal increase electrical activity in the brain.

Convulsion : major motor manifestations of a seizure(rhythmic jerking of the limbs).

Pathophysiology of epilepsy:

High frequency discharge of impulse by interconnected cerebral neurons.

Starts locally then spread.Enhancement of excitatory

transmission.Reduction of inhibitory transmission.

Causes of epilepsy :

Neurological diseasesMedical trauma as stroke InfectionHead injuryNeoplasm of brain hereditary

Types of epilepsy: Partial: involves small area brain , no loss of

consciousness.e.g. simple, complex. Generalised : spread over larger area of

brain,e.g. tonic-clonic, absence seizure,status epilepticus.

Secondarily generalised : generalised epilepsy that originates from partial event.

Screening methods for antiepileptics

Principle :

The ability of antiepileptics drug to partially or completely antagonize seizure induced either electrically or chemically.

Pharmacological screening:

involves sequential testing of drugs in

isolated organs followed by tests in

whole animals, mostly rats & mice but

also in higher animals if indicated.

Animal model:

Is a living animal used during the

research & investigation of human

disease for the purpose of better

understanding the disease without the

added risk of causing harm to an actual

human being.

In vivo methods: a) Electrically induced seizures

Threshold models Maximal electroshock seizure(MES) test Psychomotor seizures model Focal electrical stimulation such as

kindling

Threshold models: Determine ability of a drug to alter the seizure

threshold for tonic limb extension.

Procedure : Mice are used. Corneal or ear electrode are used . Electrical stimulation at frequency of 50-60/sec

& 50mA for 0.2 sec.

Evaluation : Elevation of threshold by the test drug is taken as a measure of its efficacy.

Maximal electroshock seizure(MES) test:This model is useful for screening of drugs effective against primary & secondary generalised tonic clonic seizure.

Procedure:

Step1: Weight the animal & mark properly. Divide animals into 3 groups. Stimulus given with ear electrode. Phases of convulsions are recorded in each

mouse. The drugs are injected.

Step 2:

Group1: Isotonic saline

Group2 : Phenytoin sodium, 30 mg/kg i.p.

Group 3: Phenobarbitone sodium,15 mg/kg ip

Step 3: Supramaximal shock given after 1 hrRecord whether ‘THLE’ present/absent.

The resultant seizure pass through the phases

Phase of tonic limb flexion for 1.5 sec

Phase of tonic limb extention for 10 sec

Clonic interval (variable)

Asphyxial death (sometimes)

The animals are observed closely for 2min

Disappearence of THLE used as +ve criterion.

Calculate percentage protection

Percentage protection = no. of animals with

THLE absent / total no. animal × 100

Evaluation :

Supression of tonic hind limb extension taken as a measure of efficacy.

Antiepiletic potency is determined by calculation of ED50 for THLE.

Drugs like phenytoin,CBZ, phenobarbitone are effective in this test.

Psychomotor seizures model: Mice are subjected to unidirectional current

Animal becomes stunned and shows automatism lasting for 15-20 sec.

drugs inhibiting the effect of current in this test are potentially useful in psychomotor seizures

Kindled rat seizure model:

Kindling is a phenomenon whereby repeated administration of an initially subconvulsive electrical or chemical stimulus results in progressive intensification of seizure activity culminating in a generalised seizure.

• The animals are given stimulation through an electrode implanted in right amygdala

• Duration ,amplitude & seizure stage are recorded.

Seizure severity is graded into 5 stages Facial clonus & head nodding Immobility ,eye closure, sterotyping

sniffing. Facial clonus, head nodding& forelimb

clonus

Rearing accompanied by B/L forelimb clonus

Rearing with loss of balance & falling accompanied by GTC.

Rats are considered to be kindled on the

1st stimulation causing a stage 5 seizure

which is followed by atleast 2

consecutive stage5 seizure.

Evaluation:

Animals are tested on the day before and after the test compound is given.

Test & control group are compared with 4 different measures.

a) Seizure latency

b) Seizure severity

c) Seizure duration

d) After discharge duration

Drug efficacy can be measured by determining separate ED 50 value for total suppression of

• GTC

• Focal seizure

• Amygdala after discharge

Kindling is used as a model for human temporal lobe epilepsy.

Other methods of kindling

• Corneal electroshock kindling

• Chemically induced kindling by PTZ

Chemical induced seizures :

i. Pentylenetetrazol(PTZ) induced seizures

ii. Picrotoxin induced seizures

iii. Bicuculine induced seizures

iv. Strychnine induced seizures

v. Isoniazide induced seizures

vi. 4-aminopyridine induced seizure.

PTZ induced seizure:

PTZ is a CNS stimulant. It produces jerky

type of clonic convulsion in mice

superceded by tonic convulsions.

Act by antagonising the inhibitory

GABAergic transmission.

Methods :

Animals : groups of 6-10 mice of either sex

Route of administration:

• Determine s.c. CD97

• 1% solution of PTZ , 80-100mg/kg in scruff of neck.

3 distinct phases constitutes PTZ seizure sequence i,e,

• Myoclonic jerk

• Clonic jerk

• THLE

• Death

Evaluation :

• First episode of clonic jerking last for 5 sec followed by loss of righting reflex.

• Efficacy measured by ED50 for suppression of clonic seizure.

• Ethosuximide, valproate are effective.

Picrotoxin induced convulsion:

• Picrotoxin is a GABA antagonist and modifies chloride ion channel of GABA receptor complex.

• Dose : 3.5 mg /kg

• Route : s.c.

Bicuculine tests :

• Bicuculine is a GABA antagonist

• Dose : 1mg/kg

• Route : iv

• Tonic cinvulsions appear in all treated rats within 30 secs of inj.

4-Aminopyridine induced seizures:

• 4-aminopyridine is K+ channel antagonist

• Epileptiform activity is mediated by non NMDA type receptors.

• Dose : 13.3 mg /kg

• Route: s.c

Genetic animal model for epilepsyTotterer Mice:• Homozygous (tg/tg) strain totterer mice are prone to spontaneous seizure.

• Develop both partial & absent seizure

• Two seizure type in one model

Lethergic mice:• Homozygous(lh/lh)

• Model for absent seizure

DBA/2J Mice:• Audiogenic seizure susceptible mice

GEPRs:

Seizures can be induced by various stimuli

• Sound

• Hyperthermia

• Chemical

• Electrical

Model for tonic-clonic convulsion

Photosensitive baboons:

• Intermittent light stimulation leads to seizure.

• Model for tonic clonic seizure.

Mongolian gebrils:

Seizure can be provoked by• Placing animal in new environment

• Bright light

• Vigorous shaking of cage

• Model for petitmal/myoclonous

In Vitro methods:

• Hippocampal slices model

• Electric recording from isolated brain cells

• GABA receptor binding assays

• Excitatory aminoacid receptor binding assays.

Disadvantages of in vitro study:

• Does not give any proper idea about PK-PD interaction of drugs in living animals.

• Not possible to study compensatory changes that occur in body when drug is given.

Conclusion :Ideal model of epilepsy should show following characteristics

• Development of spontaneously occurring seizures

• Type 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 to call ideal& resemble epilepsy in humans more closely than any other experimental model.

Recent advances in antiepileptic drugs

classical newer

• Phenytoin Lamotrigine

• Phenobarbital Felbamate

• Primidone Topiramate

• Carbamazipine Gabapentin

• Ethosuximide Tiagabine

• Valproate Vigabatrin

Oxycarbazepine

Levetiracetam

Fosphenytoin

Lacosamide

Classification of AEDs:

Schematics of mechanism of action

LAMOTRIGINE:

• Presently use as add-on therapy with valproic acid

• Supress rapid firing of neurons by inhibiting Na channels ,effective in partial seizures.

• Almost completely absorbed orally.

• T1/2 = 24 hrs

• Best safety record in pregnancy.

Adverse effect:

• Dizziness

• Headache

• Diplopia

• Nausea

• Somnolence

• High rate of dematological reaction(SJD)

TOPIRAMATE :

• Rapidly absorbed orally.

• T1/2= 20-30hrs, bioav. is ≥ 80%

• Block voltage dependant sod. Channels, also potentiates inhibitory effect of GABA, depresses excitatory action kainite on AMPA receptor.

Adverse effects :

• Somnolence

• Fatigue

• Dizziness

• Cognitive slowing

• confusion

FELBAMATE :

• Blocks glutamate NMDA receptors

• T1/2 is 20 hrs

• Effective in partial seizure

• Causes aplastic anaemia , hepatotoxicity.

ZONISAMIDE :

• Sulfonamide derivative

• T1/2= 1-3 days

• Inactivation of Na channels

• Add on therapy in partial & GTCS

Adverse effects:

• Drowsiness

• Cognitive impairment

• High incidence of renal stone

VIGABATRIN :

• Absorption is rapid

• Bioav. is 60% , T1/2 = 4-7 hrs

• Increases inhibitory effects of GABA by

GABA transaminase.

Adverse effect :

• Visual field defect

• Psychosis

• Depression

(limits its use.)

TIAGABINE :

• 100% bioav., highly protein bound

• T1/2 = 5-8 hrs

• GABA uptake inhibitor

• Add on therapy in partial and GTCS.

Adverse effect

• Dizziness

• Tremors

• Cognitive impairment

• Asthenia

• Skin rash

GABAPENTIN :

• Structural analogue of GABA

• Increases activity of GABA or inhibits its reuptake.

• Not metabolized and excreted unchanged in urine.

• T1/2 = 5-7 hrs

Adverse effect :

• Somnolence

• Dizziness

• Ataxia

• nystagmus

LACOSAMIDE :

• Bio av. 100%

• Inhibit Na channels, CRMP-2.

Adverse effect :

Headache

Nausea

dizziness

LEVETIRACETAM:

• Piracetam derivative

• Binds selectively to SV2A protein of synaptic vesicles & glutamate & GABA release.

Adverse effect :

• Dizziness

• Somnolence

• asthenia

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