Best protocol compitition: Third prize

A PROTOCOL FOR THE PROJECT WORK ENTITLED



“EVALUATION OF ANTICONVULSANT PROPERTIES OF NOVEL NOOTROPIC AGENTS IN SEIZURE MODELS OF MICE”

BY,
SUSHANT KAMATH
M Pharm
Department of Pharmacology
N G S M Institute of Pharmaceutical Sciences
Paneer, Deralakatte,
Mangalore – 574160.


1.
BRIEF RESUME OF THE INTENDED WORK:

INTRODUCTION:

Nootropics or Cognition enhancing agents are drugs used for the treatment of impaired brain function resulting from cerebral deficiencies like dementia or cerebral vascular disorders. Drug candidates with nootropic properties have been reported to enhance learning ability and improve experimentally induced amnesia in various animal models1.
Some standard nootropic drugs like piracetam have been reported to possess anticonvulsant properties or show synergistic effect in combination with antiepileptic drugs1.
In the present study, the anticonvulsant properties of novel nootropic drugs will be compared with standard antiepileptic drugs.



1.1 NEED FOR THE STUDY:
Epilepsy is a common chronic neurological disorder characterized by recurrent unprovoked seizures. These seizures are transient signs or symptoms of abnormal, excessive or synchronous neuronal activity in the brain. About 50 million people worldwide have epilepsy, with almost 90% of these people being in developing countries. Epilepsy is more likely to occur in young children or people over the age of 65 years, however it can occur at any time. Epilepsy is usually controlled, but not cured, with medication, although surgery may be considered in difficult cases. However, over 30% of people with epilepsy do not have seizure control even with the best available medications. Not all epilepsy syndromes are lifelong – some forms are confined to particular stages of childhood. Epilepsy should not be

understood as a single disorder, but rather as a group of syndromes with vastly divergent symptoms but all involving episodic abnormal electrical activity in the brain.
Epilepsy is one of the most common of the serious neurological disorders. Genetic, congenital and developmental conditions are mostly associated with it among younger patients; tumors are more likely over age 40; head trauma and CNS infections may occur at any age. The prevalence of active epilepsy is roughly in the range 5–10 per 1000 people. Up to 5% of people experience non febrile seizures at some point in life; epilepsy's lifetime prevalence is relatively high because most patients either stop having seizures or (less commonly) die of it. Epilepsy's approximate annual incidence rate is 40–70 per 100,000 in industrialized countries and 100–190 per 100,000 in resource-poor countries; socioeconomically deprived people are at higher risk. In industrialized countries the incidence rate decreased in children but increased among the elderly during the three decades prior to 2003, for reasons not fully understood. Hence efforts are needed to find newer antiepileptics for better therapy and to restore stability in lives of patients.
Early laboratory and clinical studies were conducted on potential nootropic drugs only to assess cognitive function. However as time passed by nootropic drugs showed various pharmacological effects and now their focus is on anticonvulsant and neuroprotective properties. Hence the present work will be undertaken to evaluate novel nootropic agents for their anticonvulsant activity.




1.2 REVIEW OF LITERATURE:

1. Shih TS et al1., reported testing of standard anticonvulsants for soman induced seizure activity. The report describes studies of anticonvulsants for the organophosphorous nerve agent soman: a basic research effort to understand how different pharmacological classes of compounds influence the expression of seizure produced by soman in rats, and a drug screening effort to determine whether clinically useful antiepileptics can modulate soman induced seizure in rats.

2. Semlitsch HV et al3., reported testing of drugs for impaired brain function in old age and on the effects of a single dose of bifemelane using topographic mapping of EEG and event related potentials and psychometric measurements in healthy elderly subjects.

3. Kitano Y et al4., reported effect of nefiracetam on amygdala kindled seizures in rats. Nefiracetam had no effect on amygdala kindled seizures but it inhibited electroencephalographic and behavioral seizures.

4. Faden IA et al5., reported the neuroprotective and nootropic properties of novel small peptides.

5. Smith MD et al6., demonstrated the effect of antiepileptic drugs on induced epileptiform in rat model of dysplasia. Rats were exposed to methylazoxymethanol (MAM) in utero, an animal model featuring nodular heterotopia to investigate the effect of antiepileptics in dysplastic brain.


1.3 OBJECTIVES OF THE STUDY:

The objective of the present study is:
1. To carryout the screening of agents using Maximal electroshock seizures and subcutaneous chemoconvulsants.

2.
MATERIALS AND METHODS:

2.1 Source of Data:

1. Laboratory based studies.
2. Journals and publications.
3. Internet.

2.2 METHOD OF COLLECTION OF DATA:

By animal experiments and laboratory investigations data will be collected from different in vivo and in vitro pharmacological experiments. The following experimental protocol is made so as to fulfill the maximum bio-statistical requirements. Animal experiments will be carried out as according to the OECD and CPCSEA guidelines.

2.3 OPERATIONAL PROCEDURES:

A. Maximal Electroshock seizures (MES):

Swiss albino mice of Mus musculus species belonging to the age group 8-10 weeks with average body weight 30 g will be used. The animals will be divided into nine groups. One group for control, four groups for the novel drug candidate and four for standard antiepileptic drug phenytoin treatment. Each group will contain n=6 animals. Electrical stimulation will be applied via corneal or ear electrodes with a stimulator that either derives constant current or constant voltage at a frequency of 50-60/sec. The electrodes and the ear of the mice will be moistened with saline solution before application. All animals will be stimulated with the same supramaximal current strength that is usually 2-5 times the threshold current strength. Stimulation parameter of 50 mA will be delivered via corneal electrodes for 0.2 sec. With constant voltage stimulators 250 V will be used for mice. The drug candidate and the standard drugs will be dissolved in 0.9% physiologic saline and will be administered intravenously, 30, 60, 90 and 120 min prior to electroshock stimulation. The resultant seizure will pass through various phases: phases of tonic limb flexion of about 1.5 sec duration followed by phase of tonic limb extension lasting about 10 sec and finally followed by a variable short clonic interval. Suppression of tonic limb extension will be taken as a measure of efficacy in this test2.

Statistical analysis:

ANOVA followed by student ‘t’ test will be used to analyze the significance of the results.

B. Pentylenetetrazol test:

Swiss albino mice of Mus musculus species belonging to the age group 8-10 weeks with average body weight 30 g will be used. The animals will be divided into nine groups. One group for control, four groups for the novel drug candidate and four groups for standard antiepileptic drug valproic acid treatment. Each group will contain n=6 animals. Prior to drug efficacy test, subcutaneous CD97( convulsive dose in 97% of animals) of PTZ will be determined. CD97¬ is usually about 80-100 mg/kg in mice. Mice will be given 1% solution of PTZ, 80-100 mg/kg subcutaneously. The drug candidate, valproic acid and piracetam will be administered 30, 60, 90 and 120 min prior to PTZ administration. Efficacy of the test drug will be measured by determining its ED50 for suppression of clonic seizure2.

Statistical analysis:

ANOVA followed by student ‘t’ test will be used to analyze the significance of the results.

Animal Ethical clearance will be obtained from the committee prior to experimentation.



3.
REFERENCES:

1. Shih TS, McDonough JH and Koplovitz I. Anticonvulsants for soman induced seizure activity. J Biomed Sci 1999;6:86-96.

2. Gupta SK. Drug Screening methods;1st edition, Jaypee Brothers, New Delhi. 2004. 84.

3. Semlitsch HV, Saletu B, Anderer P, Greunberger J and Linzmeyer L. Testing drugs for impaired brain function in old age: On the effects of a singe dose of Bifemelane using topographic mapping of EEG and event related potentials (P300) and psychometric measurements in healthy elderly subjects. Human Psychopharmacology 1996 ;11:379-390.

4. Kitano Y, Komiyama C, Makino M, Kasai Y, Takasuna K, Kinoshita M et al. Effects of Nefiracetam , a novel pyrrolidone type nootropic agent, on the amygdale kindled seizure in rats. Epilepsia 2005;46(10): 1561-1568.

5. Faden AI, Knoblach SM, Movsesyan VA and Cernak I. Novel small peptides with neuroprotective and nootropic properties. Journal of Alzheimer’s Disease 2004;6:S93-S97.

6. Smyth MD, Barbaro NM and Baraban SC. Effects of antiepileptic drugs on induced epileptiform activity in rat model of dysplasia. Epilepsy Res. 2002;50:251-64.