Preclinical evaluation of

drugs for seizure disorders

Dr. Sayan Chatterjee

MD PGT 3RD YEAR(incoming!)
DEPT OF PHARMACOLOGY
IPGMER, KOLKATA
 01INTRODUCTION
02IN VIVO AND IN VITRO MODELS
OV 03 MODELS FOR STATUS EPILEPTICUS
ER
1 VI 04 GENETIC ANIMAL MODELS
EW 05 MODEL FOR INFANTILE SPASMS
06 IN SILICO MODEL
07 CONCLUSIONS
INTRODUCTION
Source: Löscher W. Animal Models of Seizures and Epilepsy: Past, Present, and Future Role for the
Discovery of Antiseizure Drugs. Neurochem Res. 2017;42(7):1873-1888. doi:10.1007/s11064-017-2222-z
Source: Löscher W. Animal Models of Seizures and Epilepsy: Past, Present, and Future Role for the
Discovery of Antiseizure Drugs. Neurochem Res. 2017;42(7):1873-1888. doi:10.1007/s11064-017-2222-z
WHY ANIMAL MODELS DEAR?

• Discovery of new ASDs

• Characterization of spectrum of anticonvulsant activity of new ASDs
• Evaluating whether efficacy of new ASD changes during chronic treatment of
epilepsy
• Estimation of effective plasma concentrations of new ASDs for first clinical
trials
• Comparison of adverse effects of new ASDs in epileptic vs non-epileptic
animals
• Specific models for pharmaco-resistant seizures
• Discovery of antiepileptogenic or disease modifying agents
 IN VIVO
MODELS

• Electrically induced seizures

• Chemically induced seizures

• Seizure induced by focal lesion

• Electrically induced seizures
A. Threshold for maximal (tonic extension) electroconvulsion

Ability of a drug to alter the seizure threshold for tonic hindlimb

Principle extension (THLE); in GTCS threshold↑

• Male mice[18-30g]; 8-10/group

• Corneal / ear electrode
Procedure • Constant current/ constant voltage at frequency 50-60/sec for 0.2sec
• Threshold for THLE determined in 50% animals ( CV50 and CC50 )

• Elevation in threshold by test drug taken as a measure of efficacy

• Comparison of drug effect done by calculating the dose which
Evaluation elevates the threshold by 20%
• Control threshold determined each day
• Electrically induced seizures
B. Maximal electroshock seizure(MES) model
Merritt and Putnami discovered anticonvulsant effect of diphenylhydantoin using MES
(1938)

Principle Screening of drugs for primary and secondary GTCS

• Rat/mice, 8-10/group
• Corneal/ear electrode
• Constant current/voltage at 50-60/sec for 0.2 sec
• Rat: 750V / 150mA ; mice: 250V / 50mA
Procedure • Resultant seizure passes through various phases:
a. Tonic limb flexion (1.5sec)
b. Tonic limb extension (10sec)
c. Variable short clonic interval
d. Asphyxial death

• Suppression of THLE taken as measure of efficacy

• Anticonvulsant activity calculated by ED50 for suppression
Evaluation • Drugs effective against GTCS like phenytoin, carbamazepine can
be tested
• Electrically induced seizures
C. Kindling rat seizure model

Kindling : Repeated administration of an initially subconvulsive

electrical or chemical stimulus results in progressive
intensification of seizure activity culminating in a generalised
seizure.

Principle
• Repetitive administration of subconvulsive electrical stimulation
of certain areas of brain  progressive stimulus induced seizure
• On continued stimulation electrical activity spreads and
generalized convulsions occur.
• The animals are given stimulation through an electrode
implanted with in right amygdala.
• Kindling model : “Epilepsy induced Epilepsy”

Contd.
 Procedure
Electrode implanted in right amygdala
Allowed to recover for 1-2 weeks
after surgery
Electrical stimulus: 400-500 μA, 1
Adult female SD msec. wave pulse : 1 sec with 50-
rats[270-400g] 60/sec frequency given

During daily electrical stimulation seizures develop in the following

stages
CLASSIFICATION

CLASS I immobility, eye closure, twitching of vibrissae, stereotypical

RACINE

sniffing
CLASS II facial clonus and head nodding
CLASS III facial clonus, head nodding and U/L forelimb clonus
CLASS IV rearing, B/L forelimb clonus
CLASS V rearing, loss of balance and GTCS
Contd.
Evaluation

Testing drug efficacy [ test drug vs. control ]

• Seizure latency [ time from stimulation to the first sign of
seizure activity ]
• Seizure severity
• Seizure duration
• After discharge duration

Alternatively, drug efficacy determined by calculating separate

ED50 for total suppression of
• Generalized seizures [ class IV, V ]
• Focal seizures [ class I-III ]
• Amygdaloid after discharges

Contd.
Other methods of kindling
• Corneal electroshock kindling
• Stimulation of other brain areas like hippocampus and neo-cortex
• Chemical induced kindling through Pentylenetetrazol [PTZ]

Adult SD rats  Seizures recorded and

 30mg/kg PTZ ip scoring done after every
 Thrice weekly injection
 9 weeks
 90% animals
kindled[ seizure score > 3]
Source: Van Erum J, Van Dam D, De Deyn PP. PTZ-induced seizures in mice require a revised
Racine scale. Epilepsy Behav. 2019;95:51-55
• Chemically induced seizures
Generalized seizures Focal seizures
[systemic administration] [central administration]

PTZ, Bicuculline, Picrotoxin, Penicillin

Penicillin, Isoniazid,
Thiosemicarbazide

Allylglycine, DMCM, β- CCM, Kainic acid

Strychnine

Pilocarpine, NMDA, Kainic Quinolinic acid

acid

γ- hydroxybutyric acid, DDT PTZ

IN VITRO MODELS
• Hippocampal slices

• Electrical recording from isolated brain cells

• In vitro assays for GABAergic compounds

 GABAA receptor binding assay

 GABAB receptor binding assay

• Excitatory amino acid receptor binding assays

• Hippocampal slices
Evaluation of drugs for complex partial seizures
Rodent decapitated , brain removed, hippocampus dissected out
PROCEDURE

Hippocampal slices of about 0.5mm made by vibrotome

3-neuron synaptic circuit and associated recurrent circuitry preserved

Slices pre-incubated for 2h in 28oC warm saline with 95% O2 , 5%CO2

Intracellular recordings by passing micropipettes into the stratum

pyramidale under microscopic control

E VA L U AT I O N
Readings taken by adding drug to the slice medium and recording
the spontaneous or shock evoked repetitive firing of neurons
• Electrical recordings from isolated brain cells
• Action of different drugs on ion channels are studied using
Patch clamp technique

• Neurons from hippocampus or hypothalamus are put in bath

• Glass pipette used to record the current through membrane in

response to voltage, ionic or chemical changes
Effect of drugs on capacitative component of current Ic seen
Ic = C dv/dt
C= specific membrane capacitance; dv/dt= rate of change of
membrane potential
Neurophysiologists have explored voltage sensitive calcium and
potassium channels , membrane response to neurotransmitters
and basic mechanisms of anti epileptic drugs using this
technique
Patch clamp technique
• In vitro assays for GABAergic compounds
 GABA – Principal inhibitory neurotransmitter
 ↓ Neuronal excitability; ↑Seizure threshold
 Through GABAA and GABAB receptors
 GABAA : ligand gated Cl- channel
 GABAB : GPCR family
[3H] GABA receptor binding assay

GABAA receptor binding assay

• [3H] Muscimol binding assay
• [3H] SR 95531 Binding assay

GABAB receptor binding assay

[3H] GABA Uptake in rat cerebral cortex

[35S] TBPS binding assay

[3H] GABA receptor binding assay

• Evaluation of GABAergic properties

• Assay tubes prepared in triplicate

[3H]GABA (15nM) in tris maleate buffer (0.05M)

• With test drug 40C, 5min,

Alone O • With isoguvacine centrifuged for
R • With muscimol 15min at 5000rpm

Radioactivity quantified with liquiscint using liquid scintillation photometry

Specific [3H] GABA binding calculated

Percentage of specifically bound [3H] GABA displaced by test compound

IC50 with 95% CI using computer derived linear regression analysis

• Excitatory amino acid receptor binding assay
 Glutamate and possibly aspartate are principal fast excitatory
neurotransmitters
Ligand gated ion channel receptors GPCRs
• AMPA
• NMDA
• Kinate

 Excessive excitatory NTs are involved in pathogenesis of

epilepsy, stroke, schizophrenia and other neurodegenerative
disorders
 NMDA receptor antagonists have shown anticonvulsant and
neuroprotective role
CPP binding assay TCP binding assay
Affinity of test compound for Affinity for phencyclidine binding
glutamate binding site on NMDA site of NMDA receptor
receptor complex
 MODELS FOR ABSENCE SEIZURES
• Subcutaneous PTZ induced seizure model
 Indicative of anticonvulsant activity of drugs against absence seizures
 S.C. CD97 determined
 Efficacy measured by ED50 for suppression of clonic seizure
 Effective for Ethosuximide,Valproate
 Non-effective against Phenytoin, Carbamazepine

• Genetic animal models

Totterer mice
• Homozygous(tg/tg) prone to
spontaneous epileptic seizure
• Also exhibit absence seizure with
synchronous 6-7/2nd spike wave
discharges in EEG
Lethargic mice#
• Homozygous(lh/lh)
• expresses spontaneous absence
seizures that share behavioral,
electrographic, and anticonvulsant
profiles with absence seizures in
patients.
• ↑GABAB receptors in thalamocortical
populations among lh/lh mice

#Source: Hosford DA, Lin FH, Wang Y, et al. Studies of the lethargic (lh/lh) mouse model of absence
seizures: regulatory mechanisms and identification of the lh gene. Adv Neurol. 1999;79:239-252.
 MODELS OF STATUS EPILEPTICUS

• Pilocarpine induced status epilepticus

• Lithium-pilocarpine induced status epilepticus

• Lithium-methonyl induced status epilepticus

• Electric stimulation of Hippocampal perforant

pathway

• D,L- Homocysteine induced status epilepticus

• Generalized myoclonic seizures in baboons

GENETIC ANIMAL MODELS OF EPILEPSY
• Photosensitive baboons

• Seizure prone mice strains

 Audiogenic seizure susceptible mice
 Totterer mice
 E1 mice
 Quaking mice
 Lethargic mice

• Seizure prone rat strains

 Genetically epilepsy prone rats[GEPRs]
 Rats with spontaneously occurring petit-mal epilepsy
 MODEL FOR INFANTILE SPASMS
Velisek et al. 2007

• Pregnant SD rats injected with 2 doses of

betamethasone[0.4mg/kg i.p.] at 8 AM and 6 PM on
gestational day 15

• Seizures produced in the pups on post-natal day 15 by

injecting NMDA 15mg/kg i.p.

• ACTH can delay the onset of NMDA induced seizures in these

rats

• Betamethasone proposed to sensitize the brain to onset of

NMDA induced seizures

• The model needs further evaluation and validation though

Source: Löscher W. Critical review of current animal models of seizures and epilepsy used in the discovery
and development of new antiepileptic drugs. Seizure. 2011;20(5):359-368. doi:10.1016/j.seizure.2011.01.003
In silico Screening and Evaluation of the Anticonvulsant Activity of
Docosahexaenoic Acid-Like Molecules in Experimental Models of Seizures#

3D spatial structures of the effective compounds 4-Phenylbutyric acid, 4-

Biphenylacetic acid, phenylacetic acid, and 2-Phenylbutyric acid suggest the
involvement of the anticonvulsant mechanisms similar to the parent compound
DHA.

Anticonvulsant activity was tested against clonic

seizures induced by pentylenetetrazole (PTZ, 60
mg/kg, i.p.) and tonic seizures induced by maximal
electroshock (MES, 50 mA, 50 Hz, 1 ms duration)
by intracerebroventricular (i.c.v.) injection of the
screened compounds to mice.

Gharibi Loron A, Sardari S, Narenjkar J, Sayyah M. In silico Screening and Evaluation of the
#

Anticonvulsant Activity of Docosahexaenoic Acid-Like Molecules in Experimental Models of

Seizures. Iran Biomed J. 2017;21(1):32-39.
 CONCLUSIONS
• Criteria for an ideal model for epilepsy:
 Development of spontaneously occurring seizures
 Type of seizures similar to human
 EEG correlation
 Age dependency in the onset of epilepsy

• Currently no single model qualifies..genetic models closest

• Genetic models very useful for drug resistant epilepsy

• Antiepileptic drug development prog. (NIH, USA) based on 2 test

models: MES test [GTCS] & PTZ test [petit mal epilepsy]

• In vitro models help to understand epileptogenesis and MOA of

drugs

• With the advent of AI and in silico modelling the existing models are
becoming outdated day by day.