Determination of anticonvulsant activity of drugs using animal models

Neuro Pharmacology Practical overview

1. DETERMINATION OF ANTICONVULSANT ACTIVITY OF DRUGS USING ANIMAL MODELS Presenter : Dr. Nipa Mendapara Junior Resident Det. Of Pharmacology AIIMS, New Delhi

2. According to International League Against Epilepsy (ILAE) 2014 Seizure : An epileptic seizure is defined conceptually as “a transient occurrence of signs and/or symptoms due to abnormal excessive or synchronous neuronal activity in the brain.”  Epilepsy E pilepsy is diagnosed when an individual has: At least two unprovoked or reflex seizures >24 h apart, O ne unprovoked or reflex seizure and a probability of having another seizure similar to the general recurrence risk after two unprovoked seizures (at least 60%) over the next 10 years, or An epilepsy syndrome 

3. ILAE 2017 Classification Of Seizure  

4. Goodman’s and gilman’s 13th edition KNOWN TARGETS OF ANTI-EPILEPTIC DRUGS

5. A THOUGHT TO BE GIVEN AT…. Mechanisms of seizure initiation and propagation Mechanisms of epileptogenesis Genetic causes of epilepsy Mechanisms of action of antiepileptic drugs

6. WHAT IS THE NEED FOR SCREENING ? Identification of newer targets To find new drug therapy Refractory epilepsy (DRE) Several epilepsy syndromes remain resistant to standard therapies like Autosomal dominant nocturnal frontal lobe epilepsy(ADNFLE), Lennox- Gastaut syndrome(LGS), Dravet syndrome and West syndrome.

7. Screening methods for antiepileptics Principle: The ability of antiepileptic drug to partially or completely antagonize seizure induced either electrically or chemically. MODELS: I n vivo I n vitro

8. Screening methods (In Vivo): Electro shock seizure model Chemical induced seizure model Genetic model Kindling model Status epilepticus model

9. ELECTROSHOCK THRESHOLD MODEL: Initially Merritt and Putnam (1937) devised a simple and reliable method, the so-called electroshock threshold (EST) test in cats to assess drugs for antiseizure effects. Tonic seizures were induced in cats by applying rectangular current to the animal’s head through mouth and scalp electrodes attached to a 45-V battery. They demonstrated the antiseizure efficacy of phenytoin was a keystone event. “Threshold is usually determined as the current or voltage inducing hind limb extension in 50% of the animals.

10. 2 . MAXIMAL ELECTROSHOCK SEIZURE (MES): Determines the ability of the drug to alter the seizure threshold for tonic hind limb extension High correlation between ability of a drug to inhibit MES and its effectiveness in GTCS in humans Drugs effective against generalized tonic- clonic seizures such as phenytoin, carbamazepine, phenobarbitone and primidone are effective while anti-absence seizure drugs like ethosuximide are ineffective in this test. Electro- convulsiometer is used with corneal or ear electrodes. Rodents receive an electrical stimulus of sufficient intensity to induce maximal seizure of hind limbs A stimulus about 5-10 times higher than the seizure threshold of the rodent is applied.

11. Continue.. The conventional MES test has standardized parameters such as a 50-mA (mice) or 150-mA (rats) fixed current, a 50-60–Hz pulse frequency, a 0.6-ms pulse width and a 0.2-s stimulus duration. If bipolar corneal electrodes are used, a drop of an electrolyte(NS)/local anesthetic should be applied into the eyes before placement of the electrodes (not only to ensure adequate electrode contact and anesthesia, but, in mice, also to reduce the incidence of fatalities.

12. Ugo Basile Electro-Convulsive Device

13. Digital Electroconvulsiometer (Rodent Shocker) Digital Electroconvulsiometer is supplied with Corneal electrodes of three different sizes. Instrument is also supplied with Ear electrode with tension adjusted to such a level to not cause any damage to rodent ear and also has insulation arrangement to protect the user. Corneal electrode with tip size of 4mm, 5mm and 6mm - 1 each. Ear electrode-1 set.

14. MAXIMAL ELECTROSHOCK SEIZURE PHASES TIME PERIOD SIGNIFICANCE

15. Anticonvulsant activity using MES animal model

16. MES:SEIZURE SCORE

17. OUTCOMES MEASURED Positive test: Animals exhibit Tonic extensor seizure with hind limb extension (HLE) at > 90° from the body sustained for more > 3s following 10 sec after stimulation Measure efficacy of AED: Determined by its ability to abolish THLE Prolongation of latent phase and Decrease in the duration of tonic phase. MES is used to screen for AEDs active against GTCS Castel-Branco MM, Alves GL, Figueiredo IV, Falcão AC, Caramona MM. The maximal electroshock seizure (MES) model in the preclinical assessment of potential new antiepileptic drugs. Methods Find Exp Clin Pharmacol . 2009 Mar;31(2):101-6.

18. Chemical induced seizures: 1. Chemoconvulsants inducing generalized seizures after systemic administration e.g. Pentylenetetrazol(PTZ), bicuculline, picrotoxin, penicillin, isoniazid,, strychnine, pilocarpine, kainic acid 2. Chemoconvulsants inducing focal seizure after central administration (t he drug is applied locally to a specific site) e.g. penicillin, kainic acid, quinolinic acid, and pentylenetetrazol Experimental absence seizures have been studied mainly in animal models induced by chemical means.

19. Chemically Induced Generalised Seizures Bicuculline Strychnine Picrotoxin Isoniazid Competitive antagonist of GABA Competitive antagonist of glycine at all glycine receptors Non-competitive channel blocker for GABA A receptor chloride channels INH inhibits pyridoxal phosphate Tonic seizures within 30 secs of injection Convulsions with motor pattern Generalised convulsive seizure Tonic- clonic seizure Dose : 1 mg/kg Dose : 2 mg/kg Dose : 3.5 mg/kg Dose : 300 mg/kg Route : Intravenous Route: Intraperitoneal Route : subcutaneous Route : subcutaneous OTHERS : Kainic acid, 4-Aminopyridine, 4-nitropropionic acid

20. ABSENCE SEIZURES- Pentylenetetrazole (PTZ) model MECHANISM OF ACTION : Non-competitive antagonism of the gamma-aminobutyric acid (GABA A ) receptor complex Animals – Rats, mice Control mice develop a sequence of excitement, myoclonic jerks, clonic seizures, one or more maximal tonic seizures and death within 30 mins Endpoint First episode of clonic jerking lasting for 5 sec OR First clonic seizure with loss of righting reflex Dose: Rats : 60 mg/kg, Mice : 90 mg/kg Low dose induces nonmotor seizure, high dose produces motor seizures. Drugs effective in petit mal epilepsy like ethosuximide, valproic acid are effective while phenytoin, carbamazepine are not effective in this models using PTZ.

21. PTZ seizure severity score Score 0- No response Score1- Ear and facial twitching Score 2- Head nodding and myoclonic body jerks Score 3- Forelimb clonus Score 4- Generalized tonic- clonic seizures with loss of righting reflex or hind limb tonic extension https://youtu.be/PEgmyaOXCxM

22. Kindling refers to a seizure-induced plasticity phenomenon that occurs when repeated electrical stimulation in a specific brain region evokes a progressive enhancement of seizure susceptibility. Electrical stimulation for kindling: 1. Amygdala: 15 days 2. Hippocampus: 53 days • Chemical stimulation for kindling using PTZ: 1. Acute: 60-90 mg/kg X single dose 2. Chronic: 20-40 mg/kg X 14 days Electrical or chemical kindling are epileptogenic models used for understanding the epileptogenic process and for studying molecules that are preventing this process. KINDLING MODEL

23. Kindling: A model for temporal lobe epilepsy (TLE) Kindling Electrical Chemo convulsant Repeated dose ( Subconvulsive dose) SITES: amygdala, hippocampus, piriform cortex Pentylenetetrazol (PTZ) , Picrotoxin

24. Models of Status Epilepticus : 1. Pilocarpine-induced status epilepticus Pilocarpine, a cholinomimetic agent, can produce 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( LiP ) induced status epilepticus Status epilepticus can be induced in rats by giving pilocarpine (30-40 mg/kg, i.p. ) 24 hour after pre treating with lithium chloride 3 meq /kg ( 127 mg/kg) i.p. Glycopyrolate mg/kg s.c administered 1 hr before pilocarpine to limit peripheral side effects of pilocarpine (e.g. Salivation, diarrhea , and lacrimation) Status epilepticus induced by pilocarpine is terminated after 30-45 min with diazepam (10 g/kg, i.p. )

25. Genetic model: Genetic animal models of epilepsy comprise genetically predisposed animal species in which seizures either occur spontaneously or in response to sensory stimulation.  E pileptic dogs: human grand mal epilepsy. In tottering mice: spike-wave absence seizures and focal motor seizures. 

26. P hotosensitive baboons ( Papio papio ) : useful model of photomyoclonic seizures Audiogenic seizure (6Hz) susceptible mice and rats G erbils with seizures in response to different sensory stimuli : Gerbils can be subdivided into animals with minor (myoclonic) and major (mostly generalized tonic- clonic ) seizures .   Models with reflex seizures : G erbils

27. GENETIC MODELS MUTATIONS OUTCOME OBSERVATIONS 1. Lethargic ( lh / lh ) mice Hosford et al. 1992 Mutation of beta subunit Cchb4 on mouse Chromosome 2 Behavioural, EEG and anticonvulsant drug profiles similar to those in absence seizures in humans Ataxic gait by 3 weeks of age 2. GAERS(Genetic Absence Epilepsy Rat from Strasbourg) Vergnes et al . 1992 Single nucleotide mutation in T type Ca2+ channels , Cacna1h gene in Chr 10q12 Recurrent generalized non-convulsive seizures- bilateral and synchronous SWD Behavioural arrest, staring and sometimes twitching of vibrissae GENETIC MODELS FOR ABSENCE SEIZURES Hosford DA, Clark S, Cao Z, Wilson WA Jr, Lin FH, Morrisett RA, Huin A. The role of GABAB receptor activation in absence seizures of lethargic ( lh / lh ) mice. Science. 1992 Jul 17;257(5068):398-401. Marescaux C, Vergnes M, Depaulis A. Genetic absence epilepsy in rats from Strasbourg--a review. J Neural Transm Suppl. 1992;35:37-69.

28. In vivo models: 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

29. In vitro methods : I mportant for understanding mechanisms of epilepsy (seizure propagation, seizure initiation) and epileptic seizures P ropagation zones can be manipulated independently These preparations can be obtained from almost any species, the most widely used preparation in studies of epileptiform activity is the mammalian brain slice ( guinea pig, rodent, mice etc..) These thin brain slices can either be used acutely (the acute brain slice preparation ) or after preservation in culture in an incubator over a period of days or weeks ( organotypic brain slice preparation). H ippocampal slice W hole brain models.

30. The hippocampal slice Principle: The slice respond to administration of a convulsant agent with epileptiform discharge and Epileptiform discharge can be influenced by the subsequent administration of antiepileptic drugs. It has led to a better understanding of abnormal cellular and circuit properties which may prove important in human epilepsy.

31. Bathing medium (in mmol/L): NaCl 124, KCI 6, CaCI 2.4, MgSO 1.3, KH,PO 1.24, glucose 10, NaHCO % 26 Perfusion chamber for brain slices The best results were obtained from thinner slices (approximately 325 pm) taken from the smaller (200-300 g) animals.

32. Methodological parameters for in vitro model of seizure and epilepsy

33. Oliver AP, Hoffer BJ, Wyatt RJ. The hippocampal slice: a system for studying the pharmacology of seizures and for screening anticonvulsant drugs. Epilepsia . 1977 Dec;18(4):543-8.

34. In vitro method : Advantages : Indirect actions of drugs, such as local changes in blood supply or afferent input from other areas of the central nervous system can be eliminated. Effects of the seizure, such as apnea or hypoxia are minimized. To quantify the drug concentration in the perfusing medium and thus can construct dose-response curves at the level of the effector tissue. P owerful tool to explore the mechanisms and processes underlying epileptogenesis and ictogenesis. 

35. NEED FOR BETTER MODELS Development of spontaneously occurring seizures Type of seizure similar to that seen in human epilepsy Age dependency in the onset of epilepsy

36. Conclusion : 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.

37. References: Marescaux C, Vergnes M, Depaulis A. Genetic absence epilepsy in rats from Strasbourg--a review. J Neural Transm Suppl. 1992;35:37-69. Hosford DA, Clark S, Cao Z, Wilson WA Jr, Lin FH, Morrisett RA, Huin A. The role of GABAB receptor activation in absence seizures of lethargic ( lh / lh ) mice. Science. 1992 Jul 17;257(5068):398-401. Löscher W. Animal Models of Seizures and Epilepsy: Past, Present, and Future Role for the Discovery of Antiseizure Drugs. Neurochem Res. 2017 Jul;42(7):1873-1888. Kupferberg H. Animal models used in the screening of antiepileptic drugs. Epilepsia . 2001;42 Suppl 4:7-12. Raimondo JV, Heinemann U, de Curtis M, Goodkin HP, Dulla CG, Janigro D, Ikeda A, Lin CK, Jiruska P, Galanopoulou AS, Bernard C. Methodological standards for in vitro models of epilepsy and epileptic seizures. A TASK1-WG4 report of the AES/ILAE Translational Task Force of the ILAE. Epilepsia . 2017 Nov;58 Suppl 4( Suppl 4):40-52. Van Erum J, Van Dam D, De Deyn PP. PTZ-induced seizures in mice require a revised Racine scale. Epilepsy Behav . 2019 Jun;95:51-55. Dhir A. Pentylenetetrazol (PTZ) kindling model of epilepsy. Curr Protoc Neurosci . 2012;Chapter 9:Unit9.37. Sarangi SC, Pattnaik SS, Katyal J, Kaleekal T, Dinda AK. An interaction study of Ocimum sanctum L. and levetiracetam in pentylenetetrazole kindling model of epilepsy. J Ethnopharmacol . 2020 Mar 1;249:112389. Oliver AP, Hoffer BJ, Wyatt RJ. The hippocampal slice: a system for studying the pharmacology of seizures and for screening anticonvulsant drugs. Epilepsia . 1977 Dec;18(4):543-8. doi : 10.1111/j.1528-1157.1977.tb05002.x. PMID: 579341. Castel-Branco MM, Alves GL, Figueiredo IV, Falcão AC, Caramona MM. The maximal electroshock seizure (MES) model in the preclinical assessment of potential new antiepileptic drugs. Methods Find Exp Clin Pharmacol . 2009 Mar;31(2):101-6. doi : 10.1358/mf.2009.31.2.1338414. PMID: 19455265 Sk Gupta.

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