Assignment and Presentation on

Pharmacological Screening of Anti-Epileptic Drugs

Subject: 4106- Advanced Pharmaceutical Analysis

Submitted to
Prof. Sanjay Singh Department of Pharmaceutics IT-BHU

Submitted by Gangineni Ravi Teja Roll no- 08421EN008 M.Pharm. IDD Part-IV Department of Pharmaceutics IT-BHU

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Contents:_________________________________________________________________________________________Page No. a) b) c) d) e) Seizure--------------------------------------------------------------------------------------------------------3 Types of seizures-------------------------------------------------------------------------------------------3 Mechanisms of treatment for Seizures---------------------------------------------------------------4 Classification of drugs------------------------------------------------------------------------------------5 Screening methods----------------------------------------------------------------------------------------6 (i) Invitro Methods-----------------------------------------------------------------------------------7 (ii) Invivo Methods------------------------------------------------------------------------------------9

References: 1) Drug Discovery and Evaluation: Pharmacological Assays by H.Gerhard Vogel(Ed.) 3rd Edition. 2) GOODMAN & GILMAN'S THE PHARMACOLOGICAL BASIS OF THERAPEUTICS - 11th Ed.
(2006)

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Pharmacological Screening of Anti-Epileptic Drugs a) Seizure: The term seizure refers to a transient alteration of behavior due to the disordered, synchronous, and rhythmic firing of populations of brain neurons. Epilepsy: The term epilepsy refers to a disorder of brain function characterized by the periodic and unpredictable occurrence of seizures. Seizures can be "nonepileptic" when evoked in a normal brain by treatments such as electroshock or chemical convulsants or "epileptic" when occurring without evident provocation. Pharmacological agents in current clinical use inhibit seizures, and thus are referred to as antiseizure drugs. Whether any of these prevent the development of epilepsy (epileptogenesis) is uncertain. Seizures are thought to arise from the cerebral cortex, and not from other central nervous system (CNS) structures such as the thalamus, brainstem, or cerebellum. b) Types of Seizures:

SEIZURE TYPE

FEATURES

CONVENTIONA L ANTISEIZURE DRUGS

RECENTLY DEVELOPED ANTISEIZURE DRUGS

Simple Partial

Complex partial

Diverse manifestations determined by the region of cortex activated by the seizure (e.g., if motor cortex representing left thumb, clonic jerking of left thumb results; if somatosensory cortex representing left thumb, paresthesia of left thumb results), lasting approximating 20 to 60 seconds. Key feature is preservation of consciousness Impaired consciousness lasting 30 seconds to 2 minutes, often associated with purposeless movements such as lip smacking or hand wringing.

Carbamazepine, phenytoin, valproate

Gabapentin, lamotrigine, levetiracetam, tiagabine, topiramate, zonisamide

Carbamazepine, phenytoin, valproate

Gabapentin, lamotrigine, levetiracetam, tiagabine, topiramate, zonisamide Gabapentin, lamotrigine, levetiracetam, tiagabine, topiramate, zonisamide

Partial with secondarily generalized tonicclonic seizure

Simple or complex partial seizure evolves into a tonic-clonic seizure with loss of consciousness and sustained contractions (tonic) of muscles throughout the body followed by periods of muscle

Carbamazepine, phenobarbital, phenytoin, primidone, valproate

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Absence seizure

Myoclonic seizure

Tonic-clonic seizure

contraction alternating with periods of relaxation (clonic), typically lasting 1 to 2 minutes. Abrupt onset of impaired consciousness associated with staring and cessation of ongoing activities typically lasting less than 30 seconds. A brief (perhaps a second), shocklike contraction of muscles which may be restricted to part of one extremity or may be generalized As described above for partial with secondarily generalized tonic-clonic seizures except that it is not preceded by a partial seizure.

Lamotrigine

Lamotrigine

Ethosuximide, valproate
Valproate

Lamotrigine, topiramate

Carbamazepine, phenobarbital, phenytoin, primidone, valproate

Lamotrigine, topiramate

Epileptic seizures have been classified into partial seizures, those beginning focally in a cortical site, and generalized seizures, those that involve both hemispheres widely from the outset Simple partial seizure is associated with preservation of consciousness. A complex partial seizure is associated with impairment of consciousness. The majority of complex partial seizures originate from the temporal lobe. Generalized-onset seizures arise from the reciprocal firing of the thalamus and cerebral cortex These reverberatory, low-frequency rhythms are made possible by a combination of factors, including reciprocal excitatory synaptic connections between the neocortex and thalamus as well as intrinsic properties of neurons in the thalamus C) Mechanisms of treatment for Seizures: The pivotal role of synapses in mediating communication among neurons in the mammalian brain suggested that defective synaptic function might lead to a seizure. That is, a reduction of inhibitory synaptic activity or enhancement of excitatory synaptic activity might be expected to trigger a seizure; pharmacological studies of seizures supported this notion. The neurotransmitters mediating the bulk of synaptic transmission in the mammalian brain are amino acids, with -aminobutyric acid (GABA) and glutamate being the principal inhibitory and excitatory neurotransmitters, respectively. Pharmacological studies disclosed that antagonists of the GABAA receptor or agonists of different glutamate-receptor subtypes (NMDA, AMPA, or kainic acid) trigger seizures in experimental animals in vivo. Conversely, pharmacological agents that enhance GABA-mediated synaptic inhibition suppress seizures in diverse models. Glutamate-receptor antagonists also inhibit seizures in diverse models, including seizures evoked by electroshock and chemical convulsants such as pentylenetetrazol.

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Inhibiting voltage-regulated ion channels is a common mechanism of action of antiseizure drugs, with anti-partial-seizure drugs inhibiting voltage-activated Na+ channels and anti-absenceseizure drugs inhibiting voltage-activated Ca2+ channels. Therapeutic Aspects. The ideal antiseizure drug would suppress all seizures without causing any unwanted effects. Unfortunately, the drugs used currently not only fail to control seizure activity in some patients, but frequently cause unwanted effects that range in severity from minimal impairment of the CNS to death from aplastic anemia or hepatic failure. The clinician who treats patients with epilepsy is thus faced with the task of selecting the appropriate drug or combination of drugs that best controls seizures in an individual patient at an acceptable level of untoward effects. As a general rule, complete control of seizures can be achieved in up to 50% of patients, while another 25% can be improved significantly. The degree of success varies as a function of seizure type, cause, and other factors. To minimize toxicity, treatment with a single drug is preferred. If seizures are not controlled with the initial agent at adequate plasma concentrations, substitution of a second drug is preferred to the concurrent administration of another agent. However, multiple-drug therapy may be required, especially when two or more types of seizure occur in the same patient. Measurement of drug concentrations in plasma facilitates optimizing antiseizure medication, especially when therapy is initiated, after dosage adjustments, in the event of therapeutic failure, when toxic effects appear, or when multiple-drug therapy is instituted. However, clinical effects of some drugs do not correlate well with their concentrations in plasma, and recommended concentrations are only guidelines for therapy. The ultimate therapeutic regimen must be determined by clinical assessment of effect and toxicity. d) Classification of drugs: 1.hydantoins Effect is mediated by a slowing of the rate of recovery of voltage-activated Na+ channels from inactivation, an action that is both voltage- (greater effect if membrane is depolarized) and use-dependent. 2. The mechanism by which phenobarbital inhibits seizures likely involves potentiation of synaptic inhibition through an action on the GABAA receptor, increased the GABAA receptormediated current by increasing the duration of bursts of GABAA receptor-mediated currents without changing the frequency of bursts. 3. Ethosuximide reduces low threshold Ca2+ currents (T currents) in thalamic neurons. The thalamus plays an important role in generation of 3-Hz spike-and-wave rhythms typical of absence seizures. Neurons in the thalamus exhibit a large-amplitude T-current spike that underlies bursts of action potentials and likely plays an important role in thalamic oscillatory activity such as 3-Hz spike-and-wave activity. Ethosuximide reduces this current without modifying the voltage dependence of steady-state inactivation or the time course of recovery from inactivation.

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4. Valproic Acid: voltage-activated Na+ channels from inactivation, small reductions of the lowthreshold (T) Ca2+ current, stimulate the activity of the GABA synthetic enzyme, glutamic acid decarboxylase, and inhibit GABA degradative enzymes, GABA transaminase and succinic semialdehyde dehydrogenase. 5. Benzodiazepines act at subsets of GABAA receptors and increase the frequency, but not duration, of openings at GABA-activated Cl- channels, reduce sustained high-frequency firing of neurons, 6. GABAPENTIN-Enhances GABA by non vesicular release of GABAby poorly understood mechanism, it also binds to a protein of L type of voltage-sensitive Ca2+ channel, yet gabapentin does not affect Ca2+ currents of the T, N, or L types of Ca2+ channels in dorsal root ganglion cells 7- A stereo-selective binding site has been identified in rat brain membranes and the synaptic vesicle protein SVZA has been shown to be a brain-binding target of levetiracetam 8- Tiagabine inhibits the GABA transporter, GAT-1, and thereby reduces GABA uptake into neurons and glia. 9. Topiramate activates a hyperpolarizing K+ current, enhances postsynaptic GABAA-receptor currents, and also limits activation of the AMPA-kainate-subtype(s) of glutamate receptor e) Screening methods: Epilepsy becomes drug resistant in 20%30% of patients. Out of the animal models, a) The amygdala-kindled rat seems to be a suitable approach (Lscher1997,1998,2002a, 2002b). b)The rat cortical dysplasia model is recommended (Smythetal.2002). Induction of epilepsy: Several biochemical hypotheses have been advanced, involving the inhibitory GABAergic system and the system of the excitatory amino acids glutamate and aspartate. Excitatory receptors have been divided into subtypes according to the actions of specic agonists or antagonists. Agents which reduce GABAA synaptic function provoke convulsions. A convulsive state is induced by the direct blockade of GABAA receptors (e.g.to the action of bicuculline)or a reduction in the GABA- mediated opening of the chloride ion channel (e.g.,by picrotoxin). One major factor in epileptogenesis seems to be a decreased function of GABAA synapses. Recently , research has focused on the therapeutic potential of blocking excitatory aminoacidsin particular glutamate. Of the three receptors of glutamate, the NMDA(N-methyl-D-aspartate)receptor is considered the one of most interest in epilepsy and competitive NMDA receptor antagonists are proposed as potential anti-epileptic drugs. Excitatory amino acid receptors have been classied into atleast three subtypes by electrophysiological criteria: NMDA, quisqualic acid(QA) and kainic acid (KA).

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(i)

Invitro Methods: 1) GABA Receptor Binding: Amino butyric acid(GABA) is known to be an important inhibitory neurotransmitter in the brain. GABA receptors have been divided into GABAA, GABAB and GABAC receptor sub types which by themselves form receptor families. GABAA receptor agonists are CNS depressants, muscle relaxants, and possess some nociceptive properties, whereas the receptor antagonists are convulsants. GABAB receptor: place where research is going on to develop new therapeutic compounds. GABAC receptors are ligand-gated chloride channels that are present in many parts of the brain including the superior colliculus, cerebellum, hippocampus, and most prominently, the retina

[3H]-GABA Receptor Binding: Radio labeled GABA is bound to synaptic membrane preparations of mammalian brain. The labeling of the synaptic receptor with 3H GABA requires careful attention to possible interference from non synaptic binding since 3H-GABA can also bind nonspecically to plasma membranes. The most prominent of which is the sodium dependent binding of GABA to brain membranes, a process which appears to be associated with the transport (uptake) sites of GABA. Sodium independent binding of 3H- GABA has characteristics consistent with the labeling of GABA receptors. In addition, 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. GABAA Receptor Binding: Two sub types of GABA receptors have been identied: 1. GABAA receptor for which muscimol is the typical agonist,where as bicucculine, picrotoxin, and SR95531 are antagonists, and 2. GABAB receptor, for which baclofen is the typical agonist. In rat brain membranes, only the GABAA receptor is labeled with the GABA A selective radio ligand 3H-SR95531 in the given concentration range. The assay allows specically the estimation of the test drugs binding characteristics to the GABAA receptor sub population.

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We Estimate Ki and IC50 values of test drug compared with standard ligand. GABAB Receptor Binding In rat cerebellar membranes ,only the GABAB receptor is labeled in the given concentration range of the GABAB selective agonist 3H-()baclofen. The assay allows specically the estimation of the test drugs binding characteristics at the GABAB subtype receptor population.
3H-GABA

Uptake in Rat Cerebral Cortex Synaptosomes

Inhibitory action of the amino acid amino butyric acid (GABA) is the ne tuning control for pacemaker neurons. Disruption of this interplay due to in adequacies of the GABA system result in various disorders, in particular convulsive seizures. Demonstration of the high afnity mechanism that best reects the invivo condition utilizes GABA depleted cerebral cortex synaptosomes. Fluspirilene was found to be equivalent to the most potent uptake inhibitors known. The assay is used as a biochemical screen for potential anti convulsants or GABA (-amino butyric acid) mimetic compounds that act by inhibiting GABA uptake. Glutamate Receptors:[3H] CPP Binding NMDA, AMPA and Kainate. The following assay is used to assess the afnity of compounds for the excitatory amino acid binding site of the NMDA receptor complex.[3H] CPP3-[()-2-carboxypiperazin-4-yl]-1phosphonic acid is a structurally rigid analog of the selective NMDA receptor antagonist 2AP7(2-amino-7-phosphono heptanoic acid). NMDA Receptor Complex:[3H] TCP Binding. The purpose of this assay is to determine the binding afnity of potential noncompetitive NMDA antagonists at the phencyclidine(PCP) binding site which is believed to be within or near the NMDA-regulated ionchannel.TCP,1-[1-(2-thienyl)cyclohexyl]-piperidine,is a thienyl derivative of PCP. Metabotropic Glutamate Receptors: Three groups of native receptors are distinguishable on the basis of similarities of agonist pharmacology, primary sequence, and G protein-effector coupling. GroupI (mglu1 and mglu5 and splicevariants) are coupled via Gq/11 to phosphoinositide hydrolysis. GroupII (mglu2 and mglu3) are negatively coupled via Gi /Go to adenylylcyclase and inhibit the formation of cAMP following exposure of cells to forskolin or activation of an intrinsic GsCoupled receptor

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The groupIII receptors (mglu4, mglu6, mglu7, and mglu8)also inhibit forskolin-stimulated adenylylcyclase. Dose-response curves for inhibition of forskolin stimulated cAMP formation and for percentage of phosphoinositide hydrolysis are established for each test compound. [35S] TBPS Binding in Rat Cortical Homogenates and Sections: To screen potential anticonvulsant agents which interact at the convulsants binding site of the benzodiazepine/GABA /chloride ionophore complex by measuring the inhibition of binding of [35S] TBPS to rat cortical membranes. (ii) In Vivo Methods: 1. Electroshock in Mice: The electroshock assay in mice is used primarily as an indication for compounds which are effective in grandma epilepsy. Tonic hind limb extensions are evoked by electric stimuli which are suppressed by anti-epileptics but also by other centrally active drugs. 2. Pentylenetetrazol(PTZ) Test in Mice and Rats: This assay has been used primarily to evaluate anti epileptic drugs. However, it has been shown that most anxiolytic agents are also able to prevent or antagonize Metrazol(PTZ)-induced convulsions. Pentylenetetrazol is considered a GABA antagonist.The mechanism of the epileptogenic action of pentylenetetrazol at the cellular neuronal level is still unclear. Electrophysiological studies have shown it acts at cell membrane level decreasing the recovery time between action potentials by increasing potassium permeability of the axon. Other studies have implicated an increase in membrane currents of several other ions, such as sodium and calcium, leading to an overall increase in excitability of the neuron membrane. 3. Strychnine-Induced Convulsions in Mice: The convulsing action of strychnine is due to interference with post synaptic inhibition mediated by glycine. Glycine is an important inhibitory transmitter to motor neurons and inter neurons in the spinalcord, and strychnine acts as a selective, competitive antagonist to block the inhibitory effects of glycine at all glycine receptors. Strychnine sensitive post synaptic inhibition in higher centers of the CNS is also mediated by glycine. 4. Picrotoxin-Induced Convulsions in Mice: Picrotoxin induced convulsions are used to further evaluate CNS-active compounds. Picrotoxin is regarded as a GABAA- antagonist modifying the function of the chloride ion channel of the GABAA receptor complex. 5. Isoniazid-Induced Convulsions in Mice: Isoniazid can precipitate convulsions in patients with seizure disorders. The compound is regarded as a GABA-synthesis inhibitor (Costaetal.1975).

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Clonic-tonic seizures are elicited in mice which are antagonized by anxiolytic drugs. 6. Bicuculline Test in Rats: Seizures can be induced by the GABAA- antagonist bicuculline and are antagonized by known anti-epileptics. 7. 4-Aminopyridine-Induced Seizures in Mice: The K channel antagonist 4-aminopyridine is powerful convulsants in animals and in man. The drug readily penetrates the blood-brain barrier and is believed to induce seizure activity by enhancing spontaneous and evoked neurotransmitter release. Although both excitatory and inhibitory synaptic transmission is facilitated by 4-aminopyridine, the epileptiform activity induced by the drug is predominantly mediated by non-NMDA type excitatory amino acid receptors. In mice, parenterally administered 4-aminopyridine induces clonic-tonic convulsions and lethality. 8. 3-Nitropropionic Acid-Induced Seizures in Mice 3-Nitropropionic acid is a naturally occurring toxin demonstrated to impair energy metabolism via irreversible inhibition of a mitochondrial complex II component, succinate dehydrogenase. 3-Nitropropionic acid evokes seizures in mice after i.p. injectionof100200mg/kg evaluated anticonvulsants for their protective effect against 3-nitropropionic-acid- induced seizures. 9. Epilepsy Induced by Focal Lesions: Intra Hippocampal injections of noxious agents or certain cerebral lesions can induce seizures in animals. Cavalheiroetal.(1982) studied the long-term effects of intra Hippocampal kainicacid injections in rats. 10. Kindled Rat Seizure Model: Kindling, results from repetitive sub convulsive electrical stimulation of certain areas of the brain. Initially, local after discharge is associated with mild behavioral signs; however, with continued stimulation electrical activity presumably spreads, and generalized convulsions occur. Although the pathogenesis of kindled seizures is not fully understood, it serves as a useful tool for investigating the efficacy of experimental anticonvulsant agents. 11. Rat Kainate Model of Epilepsy: The kainate- treated rat is one of several models used to study temporal lobe epilepsy. Examination of the hippo campus and dentate gyrus from kainite treated rats has revealed a similar pattern of neurodegeneration in the hippocampus and the presence of mossy ber sprouting in the inner molecular level of the dentate gyrus. 12. Pilocarpine Model of Epilepsy: When rats are pretreated with lithium chloride, status epilepticus can be produced with a substantially lower dose of pilocarpine, and rats display the same clinical and EEG features of status epilepticus as with pilocarpine alone 13. Self-Sustained Status Epilepticus:

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Status epilepticus causes neuronal damage that is associated with cognitive impairment. Self sustained status epilepticus (SSSE) can be induced in rats bye lectrical stimulation of the perforant pathway. 14. Rat Model of Cortical Dysplasia Cortical dysplasia is implicated as a major contributing factor of many types of epileptic disorders that are resistant to pharmacological intervention. Several animal models of cortical dysplasia withs pecic clinical pathologies have been described. 15. Genetic Animal Models of Epilepsy NER rat strain (Noda epileptic rat) as a genetic model in epilepsy research, Which was developed by inbreeding rats with spontaneous toni clonic seizures in a stock of Crj:Wistar.

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