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Evaluation of Anti-Epileptic Drugs

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Screening of anti-epileptic drugs, Evaluation of Anti-epileptic drugs, animal models for anti-epileptic drugs

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Evaluation of Anti-Epileptic Drugs

  1. 1. Evaluation of Anti- Epileptic drugs PG Guide- Dr. Shyamal. Sinha Presenter- Dr. Shivesh. Gupta
  2. 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. 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. 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. 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. 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. 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. 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
  9. 9. Cellular & Synaptic Mechanisms of Seizures 9 (From Brody et al., 1997)
  10. 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. 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. 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
  13. 13. Etiology of seizures 13 • Idiopathic • CNS Infection • Febrile seizures • Genetic disorders • Birth trauma • Perinatal hypoxia • Developmental disorders • Alcohol withdrawal • Primary or secondary CNS neoplasm • Metabolic disorders • Cerebrovascular diseases • Drugs of abuse • Alzheimer’s & other degenerative CNS disorders • Trauma
  14. 14. Drugs causing seizures 14 Drug class Examples Antimicrobials/Antivirals β-lactam, Quinolones Acyclovir, Ganciclovir, Isoniazid Anesthetics & Analgesics Meperidine, Tramadol, local anaesthetics Immunomodulatory drugs Cyclosporine, OKT3, Tacrolimus, Interferon Psychotropic Antidepressants, anti-psychotics, Lithium Sedative-hypnotic drug withdrawl Alcohol, barbiturates , Benzodiazepines Drugs of abuse Amphetamine, Cocaine, Phencyclidine, Methylphenidate Others Theophylline, Flumazenil, Radiographic contrast agents
  15. 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. 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. 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. 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
  19. 19. Evaluation
  20. 20. Evaluation of AEDs Experimental In vivo In vitro Clinical Phase l, ll, lll, lV 20
  21. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 35. In vivo Methods Animal Models Models for GTCS Models for Absence Seizures Models for Status Epilepticus Genetic Animal Models
  36. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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.
  68. 68. 71 CLINICAL EVALUATION
  69. 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
  70. 70. 73 • Evaluation:-  Laboratory parameters [ Hb, CBC, LFT, RFT ]  Pharmacokinetic parameters [AUC, T1/2, Cmax ]  Neuropsychological parameters  Side effects profile
  71. 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. 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. 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. 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. 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. 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. 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
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