2. Flow of Seminar
2
• Introduction
• Pathophysiology of seizure
• Seizure classification, Etiology
• Current treatments and limitations
• Methods of evaluation
• In vitro models
• In vivo models
• Clinical Evaluation
• Conclusion
3. Introduction
3
• Seizure : Paroxysmal event due to abnormal, excessive, high
frequency, hypersynchronous discharges from an aggregate of
neurons in central nervous system (CNS)
• Epilepsy : Recurrent episodes (two or more unprovoked
seizures) of such seizures due to chronic, underlying process
4. Continued…
4
• Epilepsy is second most common and frequently encountered neurological condition
• The word "epilepsy" being derived from the Greek word "epilambanein" which means
"to seize or attack
• 70 million persons with epilepsy worldwide, i.e. approx. 1% of the world population
• 12 million People With Epilepsy (PWE) expected to reside in India; contributes to
nearly one-sixth of global burden
• Prevalence 3.0-11.9 per 1,000 population
• Incidence 0.2-0.6 per 1,000 population per year
5. • Prevalent among other disability groups such as autism (25.5%), cerebral palsy (13%),
Down's syndrome (13.6%), and mental retardation (25.5%)
• For people with both cerebral palsy and mental retardation the prevalence is 40%
• More than one of every three persons with epilepsy are also affected by the mood
disorder
• People with a history of depression have a 3 to 7 times higher risk of developing epilepsy
• The mortality rate among people with epilepsy is two to three times higher than the
general population and the risk of sudden death is 24 times greater
6. Mechanism of seizure initiation
6
1. Initiation Phase
High-frequency bursts of action potentials
• Long-lasting depolarization of neuronal membrane due
to influx of extracellular calcium (Ca2+)
• Opening of voltage-dependent sodium (Na+) channels
• Influx of Na+ & generation of repetitive action potentials
7. 7
Hyper synchronization
• Increase in extracellular K+, which blunts hyperpolarization
and depolarizes neighbouring neurons
• Accumulation of Ca2+ in presynaptic terminals leading to
enhanced neurotransmitter release
8. 8
2. Propagation Phase
• Recruitment of sufficient number of neurons leads to loss of
surrounding inhibition
• Propagation of seizure activity into contiguous areas via local
cortical connections
• To distant areas via long commissural pathways such as the
corpus callosum
10. Epileptogenesis
10
• Process of brain acquiring an initial insult and secondarily undergoing series of events
until first observable seizure occurs
• Transformation of normal neuronal network into one which is chronically
hyperexcitable
• CNS injury like trauma, stroke, infection or first seizure initiates the process which
lowers seizure threshold in the affected region
• In idiopathic & genetic causes, developmental events are determinants
• Structural changes in neuronal networks, long term alterations in intrinsic, biochemical
properties of cells within neuronal network
11. Classification of Seizures
Seizures
Generalized Partial Unclassified
a. Absence (petit mal)
b. Tonic-clonic (grand mal)
c. Tonic
d. Clonic
e. Akinetic or Atonic
f. Myoclonic
a. Simple partial seizures
(with motor, sensory,
autonomic, or psychic
signs)
b. Complex partial seizures
c. Partial seizures with
secondary generalization
a. Febrile seizures
b. Infantile spasms
12. Current Classification [2016]
2017 revised classification of seizures. Available at: http://www.epilepsy.com/article/2016/12/2017-revised-classification-seizures [accessed 12/04/2018]. 12
15. 15Löscher W. Animal Models of Seizures and Epilepsy: Past, Present, and Future Role for the Discovery of Antiseizure Drugs. Neurochemical Research. 2017;42(7):1873-1888.
16. Mechanism of Action
1. Generalized seizures:-
a. Inhibition of Use-Dependant Na+ channels (Phenytoin, Carbamazepine, Valproate,
lamotrigine)
b. Enhancement of GABAergic Action (BZD, Phenobarbital, Vigabatrin, Tiagabine,
Valproic Acid)
c. Blockade of NMDA or AMPA receptors ( Felbamate, Rufinamide, Topiramate)
d. Blockage of Voltage-gated N-Type Ca2+ Channels (Lamotrigine, Gabapentin)
e. Selective Binding to Synaptic Vescicular Protein Sv2A (Levetiracetam)
f. Blocking Effects of Neurotrophic factor like BDNFs ( Lacosamide)
2. Partial Seizures:-
a. Inhibition of T-type Ca+2 channels (Ethosuximide)
17. NEED for new anti-epileptic
• Not controlled with the current options- approx. 1/3 of
patient
• AED will have adverse effects severe enough to require the
drug’s withdrawal- Approx. 1/4 of the patients
• Several epilepsy syndromes remain resistant to standard
therapies
• Additional indications for other CNS disorders (e.g., migraine
prophylaxis, neuropathic pain, anxiety, and bipolar disorder)
that amplify the rewards of this line of research
17
18. Limitations of current treatments
18
• Provide relief in only up to 75% patients with absence seizures
and in 85% patients with generalized tonic-clonic seizures
• 65% of patients with new-onset epilepsy respond
• Seizure recurrence in 5%, and 35% have uncontrolled epilepsy
• Possible risk of drug interactions who are enzyme inducers
• Drug resistant epilepsy
21. In vitro Methods
• Hippocampal slices
• Electrical recording from Isolated Brain cells
• In Vitro assays for GABAergic compounds
• Excitatory Amino Acid Receptor-binding Assays
22. Hippocampal slices
• Especially useful due to the involvement of hippocampus in generation of complex partial
seizures.
• Procedure:
a. Hippocampus is dissected out & slices of about 0.5 mm thickness are made
b. Preserve the three-neuron synaptic circuit and associated recurrent circuitry
c. 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: Adding drug to the slice medium and recording the spontaneous or shock evoked
repetitive firing of neurons
• Advantages:- Mechanical stability, absence of a bloodbrain barrier and absence of anesthetics
• Useful model for studying the neurophysiological mechanisms of convulsant and antiepileptic
drugs
23. Electrical recording from Isolated Brain cells
• Used for testing action of drugs on ion channels in excitable cell membranes
• The Cells are either obtained from hippocampus or from hypothalamus and then grown in
tissue culture
• Glass pipettes are directly opposed to membranes in order to record currents through
membrane in response to voltage, ionic or chemical change
• The isolated neurons are put in a bath solution and drugs are added to it & recording of
capacitative currents is done by Patch pipettes
• Used to explore voltage sensitive calcium and potassium channels, membrane response
to neurotransmitters and basic mechanisms of antiepileptic drugs.
24. Assays for GABAergic compounds
24
• Gamma aminobutyric acid (GABA) is the principal inhibitory neurotransmitter in
the central nervous system.
• Enhancing GABA-mediated synaptic inhibition reduces neuronal excitability and
raise the seizure threshold.
• Assay Methods:-
1. 3H-GABA receptor binding
2. GABAA receptor binding
3. GABAB receptor binding
4. 3H-GABA uptake in rat cerebral cortex
5. Others:- TBPS binding assay.
25. 3H-GABA Receptor-binding Assay
• Simple and sensitive method to evaluate compounds with GABAergic properties
• Purpose and Rationale:-
• Radiolabeled GABA is bound to synaptic membrane preparations of mammalian brains
and nonspecifically to plasma membranes
• Sodium-independent binding of 3H-GABA has characteristics consistent with the labeling
of GABA receptors
• The relative potencies of several amino acids in competing for these binding sites parallel
their abilities to mimic GABA neurophysiologically
• Therefore, the sodium-independent binding of 3H-GABA provides a simple and sensitive
method to evaluate compounds for GABA-mimetic properties
26. • Procedure:
• Male rats weighing about 100-150 g are decapitated and their brains removed
• The assay tubes are prepared by serial homogenization and centrifugation of brain
tissue of rat along with adding chemicals like Triton X, isoguvacaine or muscimol or
test drug. All this is done to increase the specific binding of the GABA receptors
• Evaluation: Specific 3H-GABA binding, i.e. the radioactivity that can be displaced by a
high concentration of unlabelled GABA is calculated
• Specific binding = total bound radioactivity – nonspecific bound radioactivity
• Percentage of specifically bound 3H-GABA displaced by a given concentration of the test
compound is calculated
27. GABAA receptor binding
• GABAA receptor mediates the bulk of postsynaptic inhibitory actions
of GABA. It is a ligand gated Cl- channel
• It exists as pentamer, composed of 3 diferent subunits (α, β, γ)
• Muscimol is a powerful agonist, whereas bicuculline, picrotoxin and
SR 95531 are antagonists
• Various centrally acting drugs like benzodiazepines, barbiturates and
neurosteroids also modulate GABAA receptor function. To examine
the GABAA binding sites, [3H] muscimol(agonist) and [3H] SR 95531
(antagonist) are used as radioligands
28. GABAB receptor binding
• GABAB is a metabotropic receptor, which acts by inhibiting
adenylyl cyclase, K+ channel opening or Ca2+ channel
blockade
• It mediates both presynaptic and postsynaptic inhibition in
the central nervous system
• Baclofen is an agonist at the GABAB receptor
• GABAB receptor-binding assay, using [3H] baclofen, allows the
screening of drugs with affinity for GABAB receptors
29. 3H-GABA uptake in rat cerebral cortex
• GABA action is terminated by uptake of GABA into neurons and glia via the GAT-1 transporter
• Increasing the concentration of GABA by blocking the transporter offers a useful mechanism for
anticonvulsant drugs
• Tiagabine, a recently introduced antiepileptic drug, acts by inhibition of GAT-1
• Various other uptake inhibitors such as nipecotic acid, guvacine and THPO also exhibit
anticonvulsant effects
• This assay is useful in screening of potential anticonvulsants that act by GABA uptake inhibition.
30. Excitatory Amino Acid Receptor binding
Assays
• Glutamate, and possibly aspartate, function as the principal fast excitatory
neurotransmitters in the brain
• Excessive excitatory amino acid neurotransmission has been implicated in the
neuropathogenesis of epilepsy, stroke, schizophrenia
• Antagonists at these receptors have been shown to act as anticonvulsants and
neuroprotective agents
• Eg:-
1. Glutamate receptors: [3H] CPP binding
2. NMDA receptor complex: [3H] TCP binding
3. Glycine binding
31. 31
• Advantages:
A large number of compounds can be evaluated in a short period of
time
Provide insight to mechanism of action of drugs
Less number of animals required
• Disadvantages:
Complicated procedures, take long time
Do not give any indication of PKPD interactions Require technical
expertise
Costly
32. Why We need animal model?
32
Discovery of new AEDs
Characterization of spectrum of anticonvulsant activity
Specific models for pharmaco-resistant seizures
Evaluation of change in efficacy of new AEDs during chronic
treatment
Comparison of adverse effects of new AEDs in epileptic vs. non-
epileptic animals
Estimation of effective plasma concentrations of new AEDs for
first clinical trials
33. Characteristics of ideal model of seizures
33
• Development of spontaneously occuring recurrent seizures
• Seizure type similar in clinical phenomenology to those in human epilepsy
• Clinical seizures should be accompained by epileptiform activity in EEG
• Pharmacokinetics of antiepileptic drugs should be similar to those in humans
• Effective plasma concentration of anti epileptic drugs similar to those required for
controlling particular seizure type in humans
• The animal model should display similar pathologies if the human condition is
characterized by specific pathological changes
• The condition and condition being modeled should respond to AEDs with similar
mechanisms of action
34. 34
Löscher W. Animal Models of Seizures and Epilepsy: Past, Present, and Future Role for the Discovery of Antiseizure Drugs. Neurochemical Research. 2017;42(7):1873-1888.
35. In vivo Methods
Animal Models
Models for GTCS
Models for
Absence
Seizures
Models for
Status
Epilepticus
Genetic Animal
Models
36. Models for GTC Seizures
• Electrically Induced seizures
• There are three major types of electrically induced seizure models:-
1. Maximal electroshock seizure (MES) test
2. Threshold models
3. Focal electrical stimulation such as kindling
37. Maximal Electroshock Seizure (MES)
test
• Anticonvulsant activity of Phenytoin was discovered using this test
• The purpose : to induce most intense physiologically possible seizure by
method analogous to human electroshock therapy.
• Useful for screening of drugs useful in GTC seizures
• -contd…..
37
38. Methodology
38
Animals:
Swiss mice (20-32g) or Wistar rats (100-150g) are used.
Electro-convuIsiometer used with Corneal or Ear electrodes.
Current used:
Rat : 150 mA, for 2s
Mice : 50 mA, for 2s second duration
Route of drug administration:
i. Intraperitoneal
ii. Oral
30 min after i.p. injection and 60 min after oral
administration the animals are subjected to electroshock.
39. Animals divided
into groups of 8-10
for single dose
All animals
stimulated with
same
supramaximal
current strength
• 2.5 times of
threshold
level
Animals pass
through various
phases of seizure
activity
• Tonic limb flexion -
1.5 sec
• Tonic limb
extension -10 sec
• Variable short
clonic intervals/
Death
Suppression of
tonic hind limb
extension is
efficacy
40. • Animals are observed for 2 min after shock
• End point
Disappearance of Tonic hind limb extension (THLE)
• Anticonvulsant potency - Calculation of ED50 for suppression of tonic hind
limb extension
• Percentage inhibition of seizures as compared to controls is calculated
• Drugs effective against GTCS such as phenytoin, carbamazepine,
phenobarbitone and primidone are effective while anti-absence seizure
drugs like ethosuximide are inefective in this test.
• Disadvantages
Does not give any clue regarding the mechanism of action of the
compound. 40
41. Threshold for Maximal (Tonic
Extension) Electro-convulsions
• Determines the ability of a drug to alter the seizure
threshold for tonic limb extension
• Good test for screening of drugs effective against GTC.
• Animals: Male Swiss mice (20-32g).
• Electro-convuIsiometer is used with Corneal or Ear
electrodes.
41
42. • Procedure
• For each stimulus intensity, mice (n=8-10) are used.
• Threshold = Current or voltage inducing hind limb
extension in 50% of the animals, i.e. CC50 and CV50
respectively.
• Control thresholds: 6-9mA (CC50) or 90-140 V (CV50)
depending on strain, age and method of stimulation.
• Evaluation:
I. Elevation of threshold by the test drug: measure
of efficacy
ii. Test drug should elevate the threshold by 20%
iii. Compare between the control group and test drug
group
42
Control and Test
drug Groups
identified
Give electrical
stimulation and
identify the threshold
Give the test drug
and see for the rise in
the threshold after
giving further
electrical stimuation
43. Kindled Rat Seizure Model
43
• Method to study anticonvulsant activity on the basis of pathophysiological
model.
• The kindling phenomenon is a manifestation of the fact that ‘epilepsy
induces epilepsy’
• Repeated administration of an initially subconvulsive electrical stimulus-
leads to progressive intensification of stimulus induced seizure activity,
culminating in a generalized seizure
44. • Methodology:-
• Animals used:-
• Adult female Sprague Dawley rats weighing 270 to 400 g are used
• Stimulation through electrode implanted with in right amygdala
45. Electrodes placed in animal
Animal is allowed to recover from
surgery for a minimum of 1-2
weeks
Daily electrical stimulus of A fixed
current strength (400-500 μA)
applied via the electrode
During the daily electrical
stimulation of amygdala, seizures
develop
Once Class 5 seizures have
developed- Rats are said to be fully
kindled
Test compound given (Orally or IP)
a day before and after the
stimulation
Comparison made between
animals with test drug and controls
Drug Efficacy measured
Class – 1: Immobility, eye closure, twitching of
vibrissae, stereotypic sniing
Class – 2: Facial clonus and head nodding
Class – 3: Facial clonus, head nodding and forelimb
clonus (contralateral to focus)
Class – 4: Rearing, often accompanied by bilateral
forelimb clonus
Class – 5: Rearing with loss of balance and falling
accompanied by generalized clonic seizures.
46. • Evaluation:-
The different measures for drug efficacy recorded in a kindled animal:
1. Seizure latency, i.e. time from stimulation to the irst sign of seizure activity.
2. Seizure severity
3. Seizure duration
4. After discharge duration.
Alternatively, drug efficacy can be measured by determining separate ED50 values for total
suppression of:
1. Generalized seizures (class 4 and 5)
2. Focal seizures (class 1-3)
3. Amygdaloid after discharges.
47. 47
Löscher W. Fit for purpose application of currently existing animal models in the discovery of novel epilepsy therapies. Epilepsy Research. 2016;126:157-184
48. Advantages:-
Efficacy of a drug against the process of epileptogenesis as well as against the fully kindled state
can be measured
Efficacy against generalized seizures - valid model for drugs effective in secondary generalized
seizures of partial epilepsy
Efficacy against the focal components of kindled seizures- valid model for drugs effective in
complex partial seizures
Phenobarbitone, diazepam and valproic acid block kindled seizures & kindling process
Phenytoin and carbamazepine block seizures once kindling has occurred, but not the
establishment of kindled seizures
49. Other methods of Kindling:-
1. Corneal electroshock kindling: Kindling done by giving electroshocks via corneal
electrodes.
2. Kindling by stimulation of other brain areas: Kindling done by stimulation of other
brain areas like neocortex or hippocampus in rats.
Eg:- Development of rapidly recurring hippocampal seizure (RRHS) model of
kindling in rats described by Lothman et al. (1985)
3. Chemical induced kindling: Eg:- Pentylenetetrazol (PTZ) can lead to long lasting
kindling in rats when given repeatedly in subconvulsive doses.
50. Models for absence seizure
PTZ (Pentylenetetrazol) in mice and rats
Strychnine in mice
Picrotoxin in mice
Isoniazid in mice
Bicuculline in Rats
4- Aminopyridine in mice
Systemic Penicillin Test in cats and rats
Seizures induced by focal lesions
General Principle:-
• In all the chemical induced seizures, give the test and standard drug
• then after fixed time, administer the convulsive chemical and then see for the time taken for the onset of seizures
• The test drug should be increasing the time required for the onset of seizures as compared to control group
50
Chemical induced Convulsions
51. PTZ (Metrozol) Induced Seizures
• Excitatory effects of PTZ are because acts by antagonizing the
inhibitory GABAergic neurotransmission and/ or decrease in neuronal
recovery time in the post synaptic pathway of spinal cord
• Produces Generalised Asynchronus clonic movements superceded by
tonic convulsion having flexion of limbs followed by extension
• Animal: Swiss albino mice (20-32 gms) or Wistar rats of either sex
(120-150 gms)
• Dose - S.C. 60 mg/kg of PTZ dissolved in 0.9 % normal saline, 30 min
after test drug
51
52. Select and divide the animals into test and control group of 6-10 each
Administer the test and standard drug either oral or by SC route
Administer 60 mg/kg of PTZ (metrozol) SC
Observe each animal for one hour
Record the seizures, Tonic-clonic convulsions & time taken for onset of Seizures
Atleast 80-90% of the animals in the control group must show convulsions
30 mins after SC or 60 mins after oral
53. Evaluation:-
1. The number of protected animals in the treated group is calculated as percentage of
animals showing seizures in the control group
2. ED50 values for suppression of clonic seizure for the test/ reference drugs are
calculated for comparison
3. The delay in onset of seizures caused by test drug and reference drug is also
calculated as compared to the control group
Eg:-
BZDs show anticonvulsant activity by this test
This test has been recommended for evaluation of Centrally Acting muscle Relaxants
54. Strychnine Induced Seizures
• Selective competitive antagonist: blocks inhibitory effect of glycine,
so blocks post synaptic inhibition by glycine
• Animal : Swiss mice of either sex weighing 20-25 gms
• Dose & Route : 2 mg/kg I.P, 60 min after oral test drug
• Evaluation: Time for onset of tonic extensor convulsions and death is
recorded till 1 hour after strychnine administration
• ED50 values are calculated using 3-4 doses of test/standard drugs
taking percentage of convulsing control mice as 100 %
54
55. Picrotoxin-induced Convulsions
• Picrotoxin is a GABA antagonist, modifies Cl ion channel
• Animal: Swiss mice of either sex weighing 20-25 gms
• Dose : 3.5 mg/kg, 30 min (IP) or 60 mins (oral) after test drug
• Route : S.C, animal observed for 30 min
• Endpoint: Time taken for the onset of seizure and causing death
• ED50 values are calculated using 3-4 doses of test/standard drugs
taking percentage of seizures in control mice as 100 %
55
56. Isoniazid-induced Convulsions
• INH is a inhibitor of GABA synthesis.
• The typical pattern is of tonic-clonic seizures
• Animal: Swiss mice of either sex weighing 20-25 gms
• Dose : 300 mg/kg, 30 min or 60 mins after test drug
• Route : S.C, animal observed for 2 hrs
• Endpoint: Occurrence of tonic-clonic seizure, ED50 values calculated
• Protection against death is calculated as percentage of controls
56
57. Bicuculine Tests In Rats
• Bicuculine is a competitive GABA antagonist
• Animal: Female Sprague Dawley rats of either sex weighing 100-150
gms
• Dose : 1 mg/kg, 1-2 hrs after test drug
• Route : Intravenous
• The tonic convulsions appear in all treated rats within 30 seconds of
injection.
• Endpoint: Occurrence tonic-clonic seizure
• Percentage of protected animals is calculated
57
58. 4-Aminopyridine induced seizures
• K+ channel antagonist, crosses BBB
• The epileptiform activity is predominantly mediated by non-NMDA type excitatory amino
acid receptors
• Produces Tonic-Clonic convulsions in mice and death
• Animal : Male NIH Swiss mice, observed for 10 mins after injection
• Dose & route: 13.3mg/kg, S.C, 15 mins after test drug
• Endpoint: Disappearance of hind limb extension
• Percentage of protected animals is used for calculation of ED50
58
59. Seizures Induced by Focal Lesions
• Intrahippocampal injections of noxious agents induce focal seizures in animals
• Kainic acid in a dose of 0.2 ml over 30 mins injected surgically in the hippocampus of
adult Rats after anaesthesizing them with Chloral hydrate
• Seizure activity is recorded using electrodes placed on the skull on an EEG graph
• Other Methods:-
• Cortically implanted Metals
• Aluminium Hydroxide Gel Model
• Miscellaneous chemicals:- Tetanus toxin, Topical application
of Penicillin, Atropine, cobalt powder, zinc, etc
• Now obsolete
59
60. Models for Status Epilepticus
These are animal models that can be used to screen drugs effective in pharmacotherapy of
status epilepticus.
1. Pilocarpine-induced status epilepticus: Behavioral and electroencephalographic seizures
suggestive of motor limbic seizures and status epilepticus in rats when given in a dose of
380-400 mg/kg i.p.
2. Lithium-pilocarpine induced status epilepticus: Status epilepticus can be induced in rats
by giving pilocarpine (30-40 mg/kg, i.p.) 24 h after pretreating with lithium (3 meq/kg i.p.)
3. Lithium-methomyl induced seizures in rats: Methomyl, a carbamate anticholinesterase,
in a dose of 5.2 mg/kg s.c., can induce long lasting status epilepticus in lithium pretreated
rats
61. Genetic Animal Models of Epilepsy
• Closely approximate human epilepsy.
• Opportunity to study genetic and biochemical basis of epilepsy
1. Photosensitive Baboons (Papiopapio)
• Intermittent light stimulation at frequencies close to 25Hz
• Seizures characterized to eyelid, then face
and body clonus and subsequently tonic spasms or
full tonic clonic convulsions
• Drugs like Valproic Acid, BZDs, Phenobarbital are effective here
61
62. Seizure-prone Mice Strains
i. Audiogenic Seizure Susceptible Mice:
a. DBA/2J mice exhibit sound induced seizures between the ages of 2-4 weeks
b. Audiogenic seizures can be prevented by phenytoin or phenobarbital or valproic
acid.
ii. Totterer Mice:
a. The homozygous (tg/tg) strain totterer mice are prone to spontaneous epileptic
seizures
b. By 3 weeks age- frequent partial and absence which can be suppressed by
diazepam
c. Also spontaneous petit mal seizures which are blocked by ethosuximide,
diazepam and phenobarbital while phenytoin is not effective
63. iii. E1 Mice:
a. Exhibit seizures in response to vestibular stimulation like tossing
or spinning.
b. These mice can serve as model for complex partial epilepsy with
secondary generalization.
c. Phenytoin and phenobarbitone are effective in this model.
iv. Quaking Mice:
a. These are C57BL/6J mutants
b. Spontaneous or stimulus-induced myoclonic and generalized
tonic-clonic seizures.
c. Seizures are blocked by phenytoin, phenobarbitone
carbamazepine and valproic acid
64. Seizure-prone Rat Strains
1. Genetically Epilepsy-prone Rats (GEPRs):
a. Seizures can be induced in these animals by various stimuli like sound,
hyperthermia, chemical and electrical
b. Drugs effective in MES test are effective in this model
2. Rats with Spontaneously Occurring Petit Mal Epilepsy:
a. 15-30% of Sprague Dawley and Wistar rats, both sexes, 14 to 18 weeks age
and above, exhibit spontaneous spike-wave discharges (7-11/sec) with
associated behavioral components
b. Drugs effective in absence seizures in humans suppress these seizures
65. • Mongolian Gerbils:
• Seizures precipitated by various stimuli like placing the animal in a new environment, onset
of bright light, audiogenic stimuli, vigorous shaking of cage and different handling
techniques.
• Young gerbils with minor seizures – Petit mal epilepsy Model
• Older gerbil- GTC seizure model
• Miscellaneous Genetically Seizure-prone Animals- Syrian golden hamsters, photosensitive
epileptic chickens and dogs
66. In vivo model
• Advantages:
• Clearly defined endpoints
• Require less technical expertise
• Permit a direct comparison of the anticonvulsant profile of a new
drug to that of the 'clinically effective therapeutic agents’
• Can be used for routine screening of a large number of potential
anticonvulsants
• Disadvantages:
• Provides little information regarding an active compound's
mechanism of action
66
67. 70Löscher 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.
69. Phase I
72
• A Double-blind/ open label, Randomised, Placebo-controlled, single dose/
multiple dose
• Objective To Investigate Safety, Tolerability, Steady State Pharmacokinetic
Profile [ AUC, T1/2, Cmax ]
Inclusion criteria:
• Normal healthy volunteers
• Adult male/female aged 18-45 years
Exclusion criteria:
• history of alcoholism
• history of drug abuse
• Pregnant females
• Psychiatric disorders
71. Phase II / III
74
• Multicenter, double-blind, placebo-controlled randomized
• Objective : Therapeutic efficacy and safety
• Serum level determinations of both the investigational and concomitant
antiepileptic drugs are highly recommended at least twice weekly
• Careful clinical observations should be made, with particular regard to
disturbances of thought processes, gait, speech, coordination,
nystagmus and lethargy.
72. 75
Specific inclusion criteria:-
a. Adults (ages 16 to 65) with seizures as per ILAE classification
b. Patients should have non-controlled seizures despite a stable regimen with 1-3
established appropriate AEDs (The vagal nerve stimulator is considered as a drug)
c. A defined minimum no. of seizures (e.g. > 6 observable seizures in 8 weeks)
Specific exclusion criteria:-
a) H/o status epilepticus in the past year
b) Non-epileptic attacks (syncope, pseudo seizures)
c) Significant psychiatric disorder.
d) Progressive CNS disorders (vascular malformations, high grade tumors, etc.)
e) Drug or alcohol abuse
f) Previous poor compliance with therapy
g) Pregnant or breastfeeding women
73. 76
• Primary endpoints :-
% change in seizure frequency at the end of 6 month
Responder rate (% of patients with a greater than 50% reduction in seizures
compared to baseline) at the end of 6 month
• Secondary endpoints:-
% of patients with seizure worsening (increase in seizures by 25% or more)
% of seizure-free patients
Change in seizure frequency and responder rate per dosage group
Incidence of adverse events
Changes in EEG pattern
74. Phase IV
77
• Long term safety
• Detect unusual effects
• Long -term adverse reactions
• Alterations in the therapeutic effect over a long period
• Possible exacerbation of seizures
• Teratogenic effect
• Patient adherence and provider compliance
• Cost effectiveness studies
75. Considerations for the Clinical Evaluation of
“Drugs in Infants and Children”
No inclusion in clinical trials until late-Phase II /III unless the seizure type under study is restricted
to the young-age period
Even in other forms of epilepsy if considered for inclusion prior to late-Phase II or Phase III,
selection on the basis of poor control on current medication or control obtained only at the cost
of unsatisfactory levels of side effects
In cases where children are to be included in Phase I and early Phase II studies hospitalization or
institutionalization with close and expert supervision is mandatory
Studies should involve children and infants of varying ages and seizure types
In addition to safety and efficacy studies, pharmacokinetic studies should be performed
Studies designed to test rates of learning and performance should also be included
76. Conclusion
A large number of in vitro and in vivo models for screening of antiepileptic drugs are
available
An ideal model of epilepsy should show the following characteristics:
• Development of spontaneously occurring seizures
• Type of seizure similar to that seen in human epilepsy
• EEG correlates of epileptic-like activity
• Age-dependency in the onset of epilepsy as is seen in many epileptic syndromes
At present, there are no models that satisfy all these criteria
77. Only the genetic animal models of epilepsy come closest to being called ideal, as they
resemble idiopathic epilepsy in humans more closely than any other experimental model
It must be emphasized that use of a single method for screening of antiepileptic drugs
cannot predict the full pharmacological profile of the drug
For successful development of a potential antiepileptic drug, effect of drug in various in
vitro and in vivo models must be studied together
Thus, there is need for combining scientific innovation with expertise in the drug
discovery and development process to develop new, affordable and effective
antiepileptic drugs
78. References
• Vogel H. Drug discovery and evaluation. 4th Edithion. Berlin: Springer; 2002
• Gupta SK. Drug Screening Methods. 3rd Edition. New Delhi: Jaypee Brothers; 2016
• Parmar NS. Screening Methods in Pharmacology. 2nd Edition. New Delhi: Narosa Publishing House; 2010
• Fisher R, Boas W, Blume W, Elger C, Genton P, Lee P et al. Epileptic Seizures and Epilepsy: Definitions
Proposed by the International League Against Epilepsy (ILAE) and the International Bureau for Epilepsy (IBE).
Epilepsia. 2005;46(4):470-472
• Amudhan S, Gururaj G, Satishchandra P. Epilepsy in India I: Epidemiology and public health. Ann Indian Acad
Neurol 2015;18:263-77
• Neuron-specific mechanisms for epilepsy self-termination. Molecular & Cellular Epilepsy. 2015
• Noe K, Williams K. Etiologies of Seizures. Adult Epilepsy. 2011;:83-97
• Schmidt D. Drug treatment of epilepsy: Options and limitations. Epilepsy & Behavior. 2009;15(1):56-65
• 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
• White H. Preclinical Development of Antiepileptic Drugs: Past, Present, and Future Directions. Epilepsia.
2003;44:2-8
• De Deyn P, D'Hooge R, Marescau B, Pei Y. Chemical models of epilepsy with some reference to their
applicability in the development of anticonvulsants. Epilepsy Research. 1992;12(2):87-110
Epilepsy can have profound socio-economic ,physical and psychological consequences.
ILAE- International League Against Epilepsy
Despite the relatively “crowded” market, pharmaceutical companies should be interested in further development of newer antiepileptic compounds.
Not only is epilepsy frequent and will increase further in frequency as the elderly population enlarges
Despite n number of drugs available for treatment of Epilepsy, still they provide limited relief
Keeping these problems in antiepileptic pharmacotherapy, it has been suggested that the current drug discovery process for antiepileptic drugs should be re-evaluated
The slices are preincubated for 2 h in a holding chamber in which they are kept moist in 28°C warm saline equilibrated with 95% O2 and 5% CO2.
Slices are then either kept in 3 mm thick layer of 32°C warm saline or submerged in liquid artificial cerebrospinal fluid.
containing 140 mMNaCl, 5 mMKCl, 0.5 mM CaCl2, 1 mM MgCl2, 5 mM HEPES, at pH 7.3.
Drugs are added to bath
Evaluation: Effect of drugs on capacitative component of current Ic is seen.
Ic = C dv/dt
where C = specific membrane capacitance dv/dt = rate of change of membrane potential
A number of antiepileptic drugs have been shown to act by enhancing the GABAergic inhibition, e.g. benzodiazepines, barbiturates, vigabatrin and tiagabine.
Abnormalities in the function of GABA system have been implicated in many diseases of the CNS including epilepsy.
The most prominent of them is the sodium-dependent binding of GABA to brain membranes, a process which appears to be associated with the transport (uptake) sites of GABA
and various neurodegenerative diseases
All these assays are used to assess the affinity of the test compounds for binding at the glutamate recpetors and thus antagonize them for their anti-epileptic activity
The procedure is same for all these assays, i.e. dissect the animal and take the brain tissue and prepare it with serial homogenization and centrifugation and then adding the chemicals like triton-X to increase the specificity of the receptors. And then calculating the Specific binding of the test compound or drug by assessing the diffence between total binding and non specific binding. Or In presence and absence
The purpose of this test is to induce the most intense physiologically possible seizure by a method analogous to human electroshock therapy.
The stimulation used in this method is considerably above threshold ( 5-10 times).
it is a good test for screening of drugs effective against generalized tonic—clonic (grand mal) seizures along with MES.
Corneal or ear electrodes are used to provide electrical stimulation of frequency of 50-60 Hz for 0.2 second duration.
(thresholds determined via ear electrodes are lower than via corneal electrodes).
Control threshold determination should be undertaken on each day parallel to threshold determinations in drug treated animals. Use of an animal more than once a day is not recommended as post-ictal rise in seizure threshold has been noted.
Recovery is imp otherwise the sensitivity of the animals to kindling is lowered.
If the stimulation is continued for a few weeks, rats develop ‘spontaneous’ epileptic seizures that persist for as long as 7 months following termination of the stimulation.
(PTZ), a proconvulsant chemical acting via antagonism of GABA function,
Bicuculine competitive antagonist
Picrotoxin non competitve antagonist
Crosses Blood Brain Barrier, Enhances neurotransmitter release, Facilitates excitatory and inhibitory synaptic transmissions both and thus Seizures precipitated
Used for differentiating the mode of action of Antiepileptic drugs
1.Cortically implanted Metals- A state of spontaneously recurrent simple partial seizures can be induced by topical application of certain metals such as alumina cream (aluminium hydroxide), cobalt and tungstic acid onto (or into) the cerebral cortex‘° or by injection of iron into the brain cortex.‘*
Aluminium hydroxide gel model is most commonly used- Four percent aluminium hydroxide is injected into surgically exposed monkey neocortex at few adjacent sites. One to two months after the injection, spontaneous and recurrent seizures begin. These seizures persist for several years. seizures consist of rhythmic jerking of an extremity or face contralateral to the lesion with occasional progression to secondarily generalized tonic—clonic seizures. Response to standard anticonvulsants parallels that of patients with focal epilepsy.
The seizures can be scored according to the classiication of Golarai et al. as follows:
Stage I - hypoactivity
Stage II - monoclonic jerks of the head, head bobbing and facial automatism
Stage III - whole body bilateral activity resembling wet dog shakes
Stage IV - rearing of forelimbs
Stage V - generalized clonic-tonic activity and loss of posture
Other methods are:-
1. Electrical stimulation of hippocampal perforant pathway
2. D, L-homocysteine induced status epilepticus
3. Generalized myoclonic seizures in baboons
Most of the animal models for screening of antiepileptic drugs are basically models of seizures rather than of epilepsy, which is a condition of chronically recurrent spontaneous seizures.
Photosensitive Baboons
25 flashes per second leads to seizures
Drugs useful against clinical tonic-clonic and myoclonic epilepsy like Valproic acid, benzodiazepines and phenobarbital are effective as anticonvulsants in them,
while phenytoin, carbamazepine and trimethadione provide less favourable therapeutic effects.
after which susceptibility gradually declines by 8 weeks of age and they are totally free of audiogenic seizures.
Handling induce seizures in quaking mice.
Others:-
v. Lethargic (lh/lh) mice: This is genetic model of absence seizures.
vi. Other mice strains showing spontaneous seizures (AE mice) or induced seizures (SJL/J) strain mice with noise-induced seizures)
The Antiepileptic Drug Development Program of the National Institute of Neurological and Communicative Disorders and Stroke (NINCDS) of the National Institute of Health (NIH), USA is primarily based on two seizure models, the MES test and the s.c. PTZ test, which predict drug efficacy against generalized tonic-clonic and absence seizures, respectively.
In vitro models are also very important for gaining insight into the pathophysiology of epilepsies and understanding the mechanism of action of drugs. They are useful in screening of drugs having specific mechanism of action, e.g. the use of [3H] GABA uptake assay for screening of drugs that act by inhibiting GABA uptake.