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.
Thursday, September 3, 2009
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.
“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.
Best protocol competition: Second prize
Pharmacological investigation for prevention and /or treatment of Alzheimer's disease with investigational drug/s: A methodic study protocol
By:
Dr.Nishan Mathias
Post Graduate (MD Pharmacology)
Department of Pharmacology,
Father Muller Medical College,
Kankanady, Mangalore – 575002.
Introduction:
Alzheimer’s disease is an age-dependent, progressive, neurodegenerative disorder characterized by multiple cognitive deficits, gradual and an irreversible disturbance in calcium homeostasis, which is often, accompanied by behavioral disorders and mood changes (Qiu et al., 2009). Recent data indicates that in the year 2005, around 24 million people suffered from dementia. Further estimation are that the number may increase to 42 million by 2020 and 81 million by 2040; assuming no change in mortality, and any effective prevention strategies or curative treatment is invented (Ferri et al, 2005).
With enhance in longevity of most populations, the prevalence of Alzheimer's disease is likely to increase, imposing greater social and economic burdens on society and healthcare systems (Ferri et al, 2005). Alzheimer's and other dementias are projected to show a 66% increase of disability adjusted life years from 2005 to 2030 (WHO, 2009)
The cardinal features of Alzheimer’s disease (AD) include formation of extracellular protein deposits in the brain, consisting predominantly of aggregates of -amyloid protein (senile plaques of 39- 43 amino acid peptide), neurofibrilary tangles (hyperphosphorylated tau protein) in the intracellular compartments, disturbances in calcium homeostasis and degeneration/loss of synapses and neurons (Behl et al., 1994).
-Amyloid peptide has been shown to induce oxidative stress and inflammation in the brain, which are postulated to play important roles in the pathogenesis of Alzheimer's disease (Behl, 1999). -Amyloid plaque induces the production of hydrogen peroxide and lipid peroxide in neurons (Behl et al., 1994) and in turn the accumulation and precipitation of proteins may be aggravated by oxidative damage. This may in turn cause more oxidative damage by interfering with the function of the proteasome which then parallely increases the levels of oxidative damage not only to proteins but also to other biomolecules (Halliwell, 2001). A high incidence of RNA and DNA strand breaks are also reported in patients with Alzheimer's disease (Adamec et al., 1999; Nunomura et al., 2009).
Alzheimer's disease has tremendous impact on the affected individuals, caregivers, and society in both developed and developing nations. This has made it imperative to investigate the genetic and molecular basis for the causes and progression of Alzheimer's disease; and also on the search for pharmacological agent that can interfere with the pathological progress (Qiu et al., 2009). In clinics, the control of Alzheimer’s disease is ensued by enhancing the cholinergic activities of the brain (Brunto et al., 2006). Precursors of acetyl choline like choline chloride and phosphatidyl choline were used. However their clinical efficacies are limited. The use of anti-cholinesterase like physostigmine is also limited by its short duration of action and systemic cholinergic effects.
Recently, the use of lecithin, tacrine, donepazil, rivastigmine and galactamine is common. Donepazil has a long half life, hence once daily dosing is better in compliance than that of osfrivastigmine and galactamine which are to be given twice daily. Nephrotoxicity is not seen with rivastigmine, donepazil and galactamine. However, the use of tacrine is associated with adverse effects on the gastro intestinal tract like abdominal cramps, diarrhea, vomiting etc. Menantine causes dose dependent blockade of NMDA receptors with mild adverse effects (Brunto et al., 2006). In milieu of these observations it is realized that the present day therapies are far from the optimal justification and therefore studies are on to discover effective non toxic drug/s.
Objective of the study:
Dementia is a mental disorder characterized by loss of intellectual ability, sufficiently severe to interfere with one's occupational or social activities. Studies have shown that these disorders result form deterioration of neurons or their myelin sheath which over time lead to dysfunction and disabilities. Current estimates are that more than 25 million people in the world today are affected by dementia (Qiu et al., 2009).
Alzheimer's disease is the most common form of dementia, accounting for approximately 70% of the dementia cases in most industrialized countries (Qiu et al., 2009). The current treatment protocol and pharmacological agents are observed to be not uniformly effective and are associated with inherent toxicity. Therefore the call of time is to investigate and develop a drug that is uniformly active and is devoid of untoward effects. Here in the proposal the standard experimental models and the assay points needed to be evaluated would be enlisted with suitable justification.
In vitro models of Alzheimer's disease (Choi et al., 2007; Kumaria and Tolias, 2008; Apel et al., 2009): Organotypic explant (primary) cultures of hippocampal and ventral mesencephalic neurons from mice and rats have been used for studying the effect of investigational drugs. The cells are grown as per the standard tissue culture procedures and then treated with amyloid beta peptide.
The standard protective drug/s as well as the experimental investigational drug under inestigation is added either before (for preventive effective) or after the addition of amyloid beta peptide (for therapeutic effect). At the end of the experimental time schedule (1, 3, 5, 7 or 30 days) the assays need to be performed to understand both early and late effects. Assay end points like the cell viability, cytotoxicity, clonogenecity, microscopic observation, apoptosis and the biochemical and molecular markers/targets may be assayed as per the standard protocols (Apel et al., 2009).
Note: Immortalized neural cell lines and continuous primary cultures (like the neural stem cells; NSC-34, PC 12) may also be used instead of the primary cultures as it is experimentally less cumbersome and does not involve sacrifice of pregnant or new born mice/rats, which at times can be traumatic to the investigator/s (Choi et al., 2007; Kumaria and Tolias, 2008).
In vivo models of Alzheimer's disease (Manzano et al., 2009; Raghavendra et al 2009): Animal models aim to replicate the symptoms, the lesions or the cause(s) of Alzheimer's disease and also allow in designing new therapeutic approaches. After in vitro studies it is mandatory to observe similar results in animals and accordingly the study may be performed with lab mouse/rats. The most commonly used Alzheimer's disease animal model is by the intracerabral administration of -amyloid peptide in to mice. The pathogenesis and molecular cascade ensued by -amyloid peptide is akin to the clinico-pathological manifestations. Later, studies with transgenic animals will be more appropriate (explained later).
Experimental groups:
1. Normal animals (no treatment at all).
2. Animals + Saline only (vehicle of Alzheimer's-inducing agent -amyloid peptide)
3. Animals + -amyloid peptide dissolved in saline.
4. Animals + -amyloid peptide dissolved in saline + standard protective drug (Positive control, preferably a clinically used marketed drug).
5. Animals + -amyloid peptide dissolved in saline + Test drug/s (at least four concentrations, preferably 1/50th, 1/25th, 1/10th and 1/5th, of LD50)
6. Animals + Saline only + 1/5th, of LD50 (to check for whether experimental drug has any inherent toxic effects).
Note: Animal study/s is to be taken up after having the due permission from CPCSEA and the institutional ethical committee permission.
ASSAY END POINTS TO BE STUDIED:
Behavioral studies
1. Open-field test
2. Elevated plus-maze test
3. Porsolt's swim test
4. Learned helplessness test
(4a) Inescapable shock pretreatment
(4b) Conditioned avoidance training
5. Morris' water maze test
6. Y-maze test
7. Passive avoidance test
Biochemical analysis:
1. Assessment of antioxidant status: Total thiols, glutathione, protein thiol, vitamin
E levels, super oxide dismutase, catalase and glutathione peroxidase.
2. Assessment of Phase I enzymes: Cytochrome p450 and Cytochrome b5.
3. Assessment of Phase II enzymes: Glutathione S transferase, UDP-glucuronosyl
transferases, DT diaphorase and NAD(P)H-quinone oxidoreductase 1 (NQO1).
4. Assessment of levels of Nitric oxide and the levels of Nitric oxide synthase.
5. Assessment of macromolecular damage: Lipid peroxidation, Diene formation
and protein carbonyl contents.
6. Acetylcholinesterase activity.
7. Neuroamine studies by HPLC for GABA, serotonin, acetylcholine etc.
Cytological analysis:
1. Normal hemotoxylin and eosin histopathology studies
2. Immunohistochemistry for
The -Amyloid peptide (end point for quantitative reduction and
localization of plaques),
Apoptosis (end point for estimating the cell death)
Protein oxidation (DNPH reactive to understand protein oxidation)
Hydrogen peroxide (surrogate end point for quantitative reduction in
oxidative stress)
8-Oxo-7, 8-dihydroguanosine (Quantitative DNA damage).
3. Comet assay for quantative estimation of DNA damage.
Statistical analysis: The results of means ± S.D will be tabulated and data will be analyzed using Duncan’s multiple range tests. The statistical significance of differences among groups will be calculated by one-way ANOVA.
EXTENSION OF THE STUDIES:
If the experimental drug/s is observed to be effective in the chemical-induced model of Alzheimer's disease, the best dose may be proceeded with for detailed mechanistic studies in the transgenic animals adopting the earlier route and schedule. Literature studies show that there are genetic knockout mice based on the metabolism of the amyloid precursor protein; based on Presenilin (transgenic mice over expressing the wild-type or the mutant ones); the double mutants Presenilin / amyloid precursor protein that develop lesions earlier; based on tau protein (tau-JNPL3); the triple transgenic Presenilin / amyloid precursor protein/ tau-JNPL3; altered expression of neprilysin, the main degrading enzyme of Aß and models based on Apolipoprotein E (Manzano et al., 2009). The whole process of drug development is summarized in the preceding figure.
REFERENCES:
Adamec E, Vonsattel JP, Nixon RA (1999). DNA strand breaks in Alzheimer's disease. Brain Res. 849: 67-77.
Apel C, Forlenza OV, de Paula VJ, Talib LL, Denecke B, Eduardo CP, Gattaz WF (2009). The neuroprotective effect of dental pulp cells in models of Alzheimer's and Parkinson's disease. J Neural Transm. 116: 71-8.
Behl C (1999). Alzheimer's disease and oxidative stress: implications for novel therapeutic approaches. Prog. Neurobiol. 57: 301-323.
Behl C, Davis J, Lesley R and Schubert D (1994). Hydrogen peroxide mediates amyloid beta protein toxicity. Cell 77: 817-827.
Brunto LL, Lazo SJ, Parker KK (2006). The Pharmacological basis of therapeutics (Goodman and Gilman’s. Eleventh edition, McGraw Hill, Chicago USA.
Choi SJ, Kim MJ, Heo HJ, Hong B, Cho HY, Kim YJ, Kim HK, Lim ST, Jun WJ, Kim EK, Shin DH (2007). Ameliorating effect of Gardenia jasminoides extract on amyloid beta peptide-induced neuronal cell deficit. Mol Cells 24:113-8.
Ferri CP, Prince M, Brayne C, Brodaty M, Fratiglioni L, Ganguli M et al., (2005) Global prevelance of dementia: a Delphi concensus study. Lancet 366:2112-17
Halliwell B (2001). Role of free radicals in the neurodegenerative diseases: therapeutic implications for antioxidant treatment. Drugs Aging 18: 685-716.
Kumaria A and Tolias CM (2008). In vitro models of neurotrauma. Br J Neurosurg. 22: 200-6.
Manzano S, González J, Marcos A, Payno M, Villanueva C, Matías-Guiu J (2009). Experimental models in Alzheimer's disease. Neurologia. 24: 255-62.
WHO (2009), Neurological disorders: Public health challenges – Chapter 2, Global burden of neurological disorders, estimates and projections.
Nunomura A, Hofer T, Moreira PI, Castellani RJ, Smith MA, Perry G (2009). RNA oxidation in Alzheimer disease and related neurodegenerative disorders. Acta Neuropathol 118: 151-66.
Qiu C, Kivipelto M, von Strauss E (2009). Epidemiology of Alzheimer's disease: occurrence, determinants, and strategies toward intervention. Dialogues Clin Neurosci 11:111-28.
Raghavendra M, Maiti R, Kumar S, Acharya SB (2009). Role of Ocimum sanctum in the experimental model of Alzheimer's disease in rats. Int J Green Pharm 3:6-15.
By:
Dr.Nishan Mathias
Post Graduate (MD Pharmacology)
Department of Pharmacology,
Father Muller Medical College,
Kankanady, Mangalore – 575002.
Introduction:
Alzheimer’s disease is an age-dependent, progressive, neurodegenerative disorder characterized by multiple cognitive deficits, gradual and an irreversible disturbance in calcium homeostasis, which is often, accompanied by behavioral disorders and mood changes (Qiu et al., 2009). Recent data indicates that in the year 2005, around 24 million people suffered from dementia. Further estimation are that the number may increase to 42 million by 2020 and 81 million by 2040; assuming no change in mortality, and any effective prevention strategies or curative treatment is invented (Ferri et al, 2005).
With enhance in longevity of most populations, the prevalence of Alzheimer's disease is likely to increase, imposing greater social and economic burdens on society and healthcare systems (Ferri et al, 2005). Alzheimer's and other dementias are projected to show a 66% increase of disability adjusted life years from 2005 to 2030 (WHO, 2009)
The cardinal features of Alzheimer’s disease (AD) include formation of extracellular protein deposits in the brain, consisting predominantly of aggregates of -amyloid protein (senile plaques of 39- 43 amino acid peptide), neurofibrilary tangles (hyperphosphorylated tau protein) in the intracellular compartments, disturbances in calcium homeostasis and degeneration/loss of synapses and neurons (Behl et al., 1994).
-Amyloid peptide has been shown to induce oxidative stress and inflammation in the brain, which are postulated to play important roles in the pathogenesis of Alzheimer's disease (Behl, 1999). -Amyloid plaque induces the production of hydrogen peroxide and lipid peroxide in neurons (Behl et al., 1994) and in turn the accumulation and precipitation of proteins may be aggravated by oxidative damage. This may in turn cause more oxidative damage by interfering with the function of the proteasome which then parallely increases the levels of oxidative damage not only to proteins but also to other biomolecules (Halliwell, 2001). A high incidence of RNA and DNA strand breaks are also reported in patients with Alzheimer's disease (Adamec et al., 1999; Nunomura et al., 2009).
Alzheimer's disease has tremendous impact on the affected individuals, caregivers, and society in both developed and developing nations. This has made it imperative to investigate the genetic and molecular basis for the causes and progression of Alzheimer's disease; and also on the search for pharmacological agent that can interfere with the pathological progress (Qiu et al., 2009). In clinics, the control of Alzheimer’s disease is ensued by enhancing the cholinergic activities of the brain (Brunto et al., 2006). Precursors of acetyl choline like choline chloride and phosphatidyl choline were used. However their clinical efficacies are limited. The use of anti-cholinesterase like physostigmine is also limited by its short duration of action and systemic cholinergic effects.
Recently, the use of lecithin, tacrine, donepazil, rivastigmine and galactamine is common. Donepazil has a long half life, hence once daily dosing is better in compliance than that of osfrivastigmine and galactamine which are to be given twice daily. Nephrotoxicity is not seen with rivastigmine, donepazil and galactamine. However, the use of tacrine is associated with adverse effects on the gastro intestinal tract like abdominal cramps, diarrhea, vomiting etc. Menantine causes dose dependent blockade of NMDA receptors with mild adverse effects (Brunto et al., 2006). In milieu of these observations it is realized that the present day therapies are far from the optimal justification and therefore studies are on to discover effective non toxic drug/s.
Objective of the study:
Dementia is a mental disorder characterized by loss of intellectual ability, sufficiently severe to interfere with one's occupational or social activities. Studies have shown that these disorders result form deterioration of neurons or their myelin sheath which over time lead to dysfunction and disabilities. Current estimates are that more than 25 million people in the world today are affected by dementia (Qiu et al., 2009).
Alzheimer's disease is the most common form of dementia, accounting for approximately 70% of the dementia cases in most industrialized countries (Qiu et al., 2009). The current treatment protocol and pharmacological agents are observed to be not uniformly effective and are associated with inherent toxicity. Therefore the call of time is to investigate and develop a drug that is uniformly active and is devoid of untoward effects. Here in the proposal the standard experimental models and the assay points needed to be evaluated would be enlisted with suitable justification.
In vitro models of Alzheimer's disease (Choi et al., 2007; Kumaria and Tolias, 2008; Apel et al., 2009): Organotypic explant (primary) cultures of hippocampal and ventral mesencephalic neurons from mice and rats have been used for studying the effect of investigational drugs. The cells are grown as per the standard tissue culture procedures and then treated with amyloid beta peptide.
The standard protective drug/s as well as the experimental investigational drug under inestigation is added either before (for preventive effective) or after the addition of amyloid beta peptide (for therapeutic effect). At the end of the experimental time schedule (1, 3, 5, 7 or 30 days) the assays need to be performed to understand both early and late effects. Assay end points like the cell viability, cytotoxicity, clonogenecity, microscopic observation, apoptosis and the biochemical and molecular markers/targets may be assayed as per the standard protocols (Apel et al., 2009).
Note: Immortalized neural cell lines and continuous primary cultures (like the neural stem cells; NSC-34, PC 12) may also be used instead of the primary cultures as it is experimentally less cumbersome and does not involve sacrifice of pregnant or new born mice/rats, which at times can be traumatic to the investigator/s (Choi et al., 2007; Kumaria and Tolias, 2008).
In vivo models of Alzheimer's disease (Manzano et al., 2009; Raghavendra et al 2009): Animal models aim to replicate the symptoms, the lesions or the cause(s) of Alzheimer's disease and also allow in designing new therapeutic approaches. After in vitro studies it is mandatory to observe similar results in animals and accordingly the study may be performed with lab mouse/rats. The most commonly used Alzheimer's disease animal model is by the intracerabral administration of -amyloid peptide in to mice. The pathogenesis and molecular cascade ensued by -amyloid peptide is akin to the clinico-pathological manifestations. Later, studies with transgenic animals will be more appropriate (explained later).
Experimental groups:
1. Normal animals (no treatment at all).
2. Animals + Saline only (vehicle of Alzheimer's-inducing agent -amyloid peptide)
3. Animals + -amyloid peptide dissolved in saline.
4. Animals + -amyloid peptide dissolved in saline + standard protective drug (Positive control, preferably a clinically used marketed drug).
5. Animals + -amyloid peptide dissolved in saline + Test drug/s (at least four concentrations, preferably 1/50th, 1/25th, 1/10th and 1/5th, of LD50)
6. Animals + Saline only + 1/5th, of LD50 (to check for whether experimental drug has any inherent toxic effects).
Note: Animal study/s is to be taken up after having the due permission from CPCSEA and the institutional ethical committee permission.
ASSAY END POINTS TO BE STUDIED:
Behavioral studies
1. Open-field test
2. Elevated plus-maze test
3. Porsolt's swim test
4. Learned helplessness test
(4a) Inescapable shock pretreatment
(4b) Conditioned avoidance training
5. Morris' water maze test
6. Y-maze test
7. Passive avoidance test
Biochemical analysis:
1. Assessment of antioxidant status: Total thiols, glutathione, protein thiol, vitamin
E levels, super oxide dismutase, catalase and glutathione peroxidase.
2. Assessment of Phase I enzymes: Cytochrome p450 and Cytochrome b5.
3. Assessment of Phase II enzymes: Glutathione S transferase, UDP-glucuronosyl
transferases, DT diaphorase and NAD(P)H-quinone oxidoreductase 1 (NQO1).
4. Assessment of levels of Nitric oxide and the levels of Nitric oxide synthase.
5. Assessment of macromolecular damage: Lipid peroxidation, Diene formation
and protein carbonyl contents.
6. Acetylcholinesterase activity.
7. Neuroamine studies by HPLC for GABA, serotonin, acetylcholine etc.
Cytological analysis:
1. Normal hemotoxylin and eosin histopathology studies
2. Immunohistochemistry for
The -Amyloid peptide (end point for quantitative reduction and
localization of plaques),
Apoptosis (end point for estimating the cell death)
Protein oxidation (DNPH reactive to understand protein oxidation)
Hydrogen peroxide (surrogate end point for quantitative reduction in
oxidative stress)
8-Oxo-7, 8-dihydroguanosine (Quantitative DNA damage).
3. Comet assay for quantative estimation of DNA damage.
Statistical analysis: The results of means ± S.D will be tabulated and data will be analyzed using Duncan’s multiple range tests. The statistical significance of differences among groups will be calculated by one-way ANOVA.
EXTENSION OF THE STUDIES:
If the experimental drug/s is observed to be effective in the chemical-induced model of Alzheimer's disease, the best dose may be proceeded with for detailed mechanistic studies in the transgenic animals adopting the earlier route and schedule. Literature studies show that there are genetic knockout mice based on the metabolism of the amyloid precursor protein; based on Presenilin (transgenic mice over expressing the wild-type or the mutant ones); the double mutants Presenilin / amyloid precursor protein that develop lesions earlier; based on tau protein (tau-JNPL3); the triple transgenic Presenilin / amyloid precursor protein/ tau-JNPL3; altered expression of neprilysin, the main degrading enzyme of Aß and models based on Apolipoprotein E (Manzano et al., 2009). The whole process of drug development is summarized in the preceding figure.
REFERENCES:
Adamec E, Vonsattel JP, Nixon RA (1999). DNA strand breaks in Alzheimer's disease. Brain Res. 849: 67-77.
Apel C, Forlenza OV, de Paula VJ, Talib LL, Denecke B, Eduardo CP, Gattaz WF (2009). The neuroprotective effect of dental pulp cells in models of Alzheimer's and Parkinson's disease. J Neural Transm. 116: 71-8.
Behl C (1999). Alzheimer's disease and oxidative stress: implications for novel therapeutic approaches. Prog. Neurobiol. 57: 301-323.
Behl C, Davis J, Lesley R and Schubert D (1994). Hydrogen peroxide mediates amyloid beta protein toxicity. Cell 77: 817-827.
Brunto LL, Lazo SJ, Parker KK (2006). The Pharmacological basis of therapeutics (Goodman and Gilman’s. Eleventh edition, McGraw Hill, Chicago USA.
Choi SJ, Kim MJ, Heo HJ, Hong B, Cho HY, Kim YJ, Kim HK, Lim ST, Jun WJ, Kim EK, Shin DH (2007). Ameliorating effect of Gardenia jasminoides extract on amyloid beta peptide-induced neuronal cell deficit. Mol Cells 24:113-8.
Ferri CP, Prince M, Brayne C, Brodaty M, Fratiglioni L, Ganguli M et al., (2005) Global prevelance of dementia: a Delphi concensus study. Lancet 366:2112-17
Halliwell B (2001). Role of free radicals in the neurodegenerative diseases: therapeutic implications for antioxidant treatment. Drugs Aging 18: 685-716.
Kumaria A and Tolias CM (2008). In vitro models of neurotrauma. Br J Neurosurg. 22: 200-6.
Manzano S, González J, Marcos A, Payno M, Villanueva C, Matías-Guiu J (2009). Experimental models in Alzheimer's disease. Neurologia. 24: 255-62.
WHO (2009), Neurological disorders: Public health challenges – Chapter 2, Global burden of neurological disorders, estimates and projections.
Nunomura A, Hofer T, Moreira PI, Castellani RJ, Smith MA, Perry G (2009). RNA oxidation in Alzheimer disease and related neurodegenerative disorders. Acta Neuropathol 118: 151-66.
Qiu C, Kivipelto M, von Strauss E (2009). Epidemiology of Alzheimer's disease: occurrence, determinants, and strategies toward intervention. Dialogues Clin Neurosci 11:111-28.
Raghavendra M, Maiti R, Kumar S, Acharya SB (2009). Role of Ocimum sanctum in the experimental model of Alzheimer's disease in rats. Int J Green Pharm 3:6-15.
Tuesday, September 1, 2009
Best protocol competition-First prize
PROTOCOL FOR EVALUATION OF THE EFFECTS OF SJM111 IN COMPARISON WITH DIAZEPAM IN RATS AFTER ETHANOL WITHDRAWAL
CONTENTS
Page no.
1. INTRODUCTION 2
2. NEED FOR THE STUDY 2
3. REVIEW OF LITERATURE 3
4. METHODS 3-7
5. STATISTICAL ANALYSIS 8
6. TIMELINES 8
7. BUDGET 8
8. REFERENCES 9
1. INTRODUCTION
Anxiety, unlike other psychiatric illness can present both as a normal emotion as well as a psychiatric disorder. When the symptoms are disruptive & maladaptive interfering with normal functioning of the individual, anxiety should be regarded as pathological. Anxiety disorder in India is drastically becoming a common illness mainly due to lifestyle changes.
The prevalence of generalized anxiety disorder ranges between 3-5% 1. The rate is higher when associated with co morbidities such as Diabetes mellitus, chronic obstructive pulmonary disorder ranging 14-31%2,3.
The advent of benzodiazepine four decades ago led to intensive research in the field of anxiety. Ever since then, benzodiazepines have been the drug of choice for anxiety. However, due to tolerance, sedation and physical dependence of benzodiazepines, the search for newer, relatively safer alternatives have become the current topic of research in the field of anxiety.
2. NEED FOR THE STUDY
Drug preparations from nature’s bounty have been known to be effective and safer as compared to their chemical counterparts. Thus, the search for a newer, effective and safer alternative to benzodiazepines from the plant kingdom seems to be a promising option. In our study, we aim to evaluate anxiolytic effects of the herbal preparation SJM111 (a hypothetical plant) in comparison with diazepam. Invitro & toxicity studies to study the effect of this preparation on vital organs have shown a good safety profile.
3. REVIEW OF LITERATURE
In comparison to conditions such as pain and inflammation which can be induced in animals, psychiatric illness similar to that as in humans is difficult to be induced. Several models such as the elevated plus maze, open field chamber, conflict models, social interaction tests have been used in anxiety screening. Some of the drugs used as anxiogenics are Pentylene tetrazole, Yohimbine, Corticosteroids and Amphetamines 4. Recent studies have shown rats to exhibit anxiety like behavior post ethanol withdrawal5. Another study has shown that in chronic doses of 2g/kg IP of ethyl alcohol daily for 5days, an anxiety like state can be produced which can further be evaluated using the anxiety screening techniques 6. Based on these studies we propose to use ethanol as an anxiogenic drug, Elevated plus maze and open field chamber as our screening techniques to evaluate the effects of SJM111.
OBJECTIVE
To evaluate the effects of SJM111 in comparison with Diazepam in rats post ethanol withdrawal using elevated plus maze and open field chamber.
4. METHODS
4.1 PLANT MATERIALS:
• SJM111 will be used as a water extract.
• The extract will be prepared according to following procedure.
• The whole plant of SJM111 is to be coarsely powdered and refluxed with water (1:5) for 3 times for 1 hour each and filtered through cloth.
• The filtrate should then be concentrated to 10% w/w total soluble solids.
4.2 DRUGS:
• Diazepam- will be used as the standard anxiolytic agent. The dose to be used is 1mg/kg body weight IP at a concentration of 2.5mg /ml.
• Ethanol (15%w/v) at a specific gravity of 0.787 will be prepared using 95% absolute alcohol. Will be used in a dose of 2gm/kg IP on the basis of previous studies6. The animals will be injected with ethanol for 5 consecutive days at the same time each day between 9am-1pm.
• Previous studies5,6 show that animals exhibit withdrawal symptoms including anxiety like behavior 9hrs after the last dose. Thus we intend to do screening 9hrs after last dose with elevated plus maze.
• Vehicle- Distilled water at a dose of 3ml will be used in the control group for 5 days and then screened 9hrs after last dose.
• SJM111- the water extract of this preparation will be used in a dose of 300mg/kg body weight IP. The animals to be injected 30mins prior to screening. (All drugs used provided by SJMC gardens, SJNAHS, Bangalore.)
4.3 ANIMALS:
• Male Wistar rats, 6weeks old weighing 180-200gm to be used.
• A total of 24animals will be chosen. They will be caged in groups of 3-4.
• The animals will be provided with food & water ad- libitum and will be allowed to acclimatize to the environs of the screening room and to gentle human handling every day for a period of 1 week prior to the beginning of screening.
• The 12 hr day-night cycle will be maintained throughout the study period.
• They will then be randomized into 3 groups of 8 animals in each. Will be caged in individual cages.
• The 3 groups are as depicted in table 1
Table 1: Study groups, drug dose and duration of treatment.
Group(n=8) Treatment(I.P) & duration Screening
Control Distill water 3ml x 5days 9 hrs post last dose
Standard-Diazepam 15 %( w/v) Ethanol 2gm/kg x 5days. Watch for withdrawal symptoms 9 hrs post last dose. Inject diazepam 1mg/kg 30mins prior to screening 9 hrs post last ethanol dose
Test- SJM111 15% (w/v) Ethanol 2gm/kg x 5days . Watch for withdrawal symptoms 9 hrs post last dose. Inject SJM111 30mins prior to screening at a dose of 300mg/kg 9 hrs post last ethanol dose
4.4 ETHICS APPROVAL:
The study will be conducted after obtaining IERB approval and the CPCSEA guidelines will be followed throughout.
4.5 PROCEDURE:
Screening model (1) Elevated plus maze
This model has been extensively used for evaluation of novel anxiolytic agents and also to investigate the neurochemical & psychological basis of anxiety. A typical elevated maze consists of 2 opposite open arms 50x10cm crossed with 2 closed arms of the same dimension having walls 50cm in height. The arms are connected by a central square 10x10cm. The model is kept in a dimly lit room & elevated 50cm above the floor.
Principle: An approach avoidance conflict. i.e. fear of balancing on an elevated open arm maze.
The naive treated animal is to be tested individually. It should be placed in centre of the maze, facing an open arm. Thereafter the following parameters should be monitored for 5mins:
• Number of open arm entries and closed arm entries - all 4 paws should be within open/closed arm
• Time spent in open arm & closed arm
• Number of head dips
• Number of rearing
• Number of stretch attend postures
To avoid possible bias during screening, we intend to video record the entire screening procedure for all animals. Evaluation will then be done independently by each investigator of this study.
Screening model 2: Open field chamber
The apparatus comprises of a large green chamber 96x96cm with high walls 96cm. The floor is divided into 16squares by white lines and is placed in dimly lit room. Naïve treated rat is then placed at one corner of the apparatus and is observed thereafter for 5mins. We intend to video- record screening of each animal. The parameters to be evaluated are:
Ambulation (no of squares crossed)
Freeze
Rearing
Scratching & licking
Defecation/urination
An anxious animal is one which shows reduced ambulation with periodic freeze and reduced normal behavior like rearing/grooming and augmented autonomic activity such as defecation/urination.
5. STATISTICAL ANALYSIS
ANOVA- one way analysis of variance followed by post hoc Tukey test will be used. P value of 0.05 will be taken as statistically significant difference between the groups.
6. TIME LINES FOR THE STUDY
Events Aug.09 Sep.09 Oct.09 Nov.09 Dec.09 Jan.10
Protocol Design
Ethics approval
Application for Funding
Animal & Drug procurement
Study duration
Data Analysis
Publication
7. PROPOSED BUDGET FOR THE STUDY
Sl no Items Amount in Rs
1
Animals/Drug
a Procurement charges 3500
b Diet (2 bags ) 1000
c Maintenance charges 1500
d IERB approval 1000
2
Study Requirements
a Diazepam vials/ syrup (10/ 2) 100
b Syringes/gloves 600
3
Publishing charges
a Write up & printing 1000
TOTAL
8700/-
8. REFERENCES
1. Wittchen. Generalized anxiety disorder, prevalence, burden and cost to society. Depression and anxiety 2002; 161:162-171
2. Grigsby, Anderson et al. Prevalence of anxiety in adults with diabetes mellitus- Systematic review. Psychosomatic research 2002;53:1053-1060
3. COPD and anxiety. Chest 2005;127(4):1205-1211
4. S.K Bhattacharya, K.S Satyan. Experimental methods for evaluation of psychotropic agents in rodents: Anti-anxiety agents. IJEB 1997; 35:565-75
5. Rasmussen DD, Mitton DR, Green J, Puchalski. Chronic daily ethanol & withdrawal: 2 Behavioral changes during prolonged abstinence. Alc Cli Exp Res 2001; 25: 999-1005
6. Zhongqi Zhang, Andrew Morse, George Koob and Gerry. Dose and time dependent expression of anxiogenic like behavior in the elevated plus maze during withdrawal from acute and repeated intermittent ethanol intoxication in rats. Alcohol Clin Exp Res 2007; 31(1): 1811-1819
PROTOCOL WRTTEN & SUBMITTED BY
Dr.Leena. A
St. John’s Medical College, Bangalore
CONTENTS
Page no.
1. INTRODUCTION 2
2. NEED FOR THE STUDY 2
3. REVIEW OF LITERATURE 3
4. METHODS 3-7
5. STATISTICAL ANALYSIS 8
6. TIMELINES 8
7. BUDGET 8
8. REFERENCES 9
1. INTRODUCTION
Anxiety, unlike other psychiatric illness can present both as a normal emotion as well as a psychiatric disorder. When the symptoms are disruptive & maladaptive interfering with normal functioning of the individual, anxiety should be regarded as pathological. Anxiety disorder in India is drastically becoming a common illness mainly due to lifestyle changes.
The prevalence of generalized anxiety disorder ranges between 3-5% 1. The rate is higher when associated with co morbidities such as Diabetes mellitus, chronic obstructive pulmonary disorder ranging 14-31%2,3.
The advent of benzodiazepine four decades ago led to intensive research in the field of anxiety. Ever since then, benzodiazepines have been the drug of choice for anxiety. However, due to tolerance, sedation and physical dependence of benzodiazepines, the search for newer, relatively safer alternatives have become the current topic of research in the field of anxiety.
2. NEED FOR THE STUDY
Drug preparations from nature’s bounty have been known to be effective and safer as compared to their chemical counterparts. Thus, the search for a newer, effective and safer alternative to benzodiazepines from the plant kingdom seems to be a promising option. In our study, we aim to evaluate anxiolytic effects of the herbal preparation SJM111 (a hypothetical plant) in comparison with diazepam. Invitro & toxicity studies to study the effect of this preparation on vital organs have shown a good safety profile.
3. REVIEW OF LITERATURE
In comparison to conditions such as pain and inflammation which can be induced in animals, psychiatric illness similar to that as in humans is difficult to be induced. Several models such as the elevated plus maze, open field chamber, conflict models, social interaction tests have been used in anxiety screening. Some of the drugs used as anxiogenics are Pentylene tetrazole, Yohimbine, Corticosteroids and Amphetamines 4. Recent studies have shown rats to exhibit anxiety like behavior post ethanol withdrawal5. Another study has shown that in chronic doses of 2g/kg IP of ethyl alcohol daily for 5days, an anxiety like state can be produced which can further be evaluated using the anxiety screening techniques 6. Based on these studies we propose to use ethanol as an anxiogenic drug, Elevated plus maze and open field chamber as our screening techniques to evaluate the effects of SJM111.
OBJECTIVE
To evaluate the effects of SJM111 in comparison with Diazepam in rats post ethanol withdrawal using elevated plus maze and open field chamber.
4. METHODS
4.1 PLANT MATERIALS:
• SJM111 will be used as a water extract.
• The extract will be prepared according to following procedure.
• The whole plant of SJM111 is to be coarsely powdered and refluxed with water (1:5) for 3 times for 1 hour each and filtered through cloth.
• The filtrate should then be concentrated to 10% w/w total soluble solids.
4.2 DRUGS:
• Diazepam- will be used as the standard anxiolytic agent. The dose to be used is 1mg/kg body weight IP at a concentration of 2.5mg /ml.
• Ethanol (15%w/v) at a specific gravity of 0.787 will be prepared using 95% absolute alcohol. Will be used in a dose of 2gm/kg IP on the basis of previous studies6. The animals will be injected with ethanol for 5 consecutive days at the same time each day between 9am-1pm.
• Previous studies5,6 show that animals exhibit withdrawal symptoms including anxiety like behavior 9hrs after the last dose. Thus we intend to do screening 9hrs after last dose with elevated plus maze.
• Vehicle- Distilled water at a dose of 3ml will be used in the control group for 5 days and then screened 9hrs after last dose.
• SJM111- the water extract of this preparation will be used in a dose of 300mg/kg body weight IP. The animals to be injected 30mins prior to screening. (All drugs used provided by SJMC gardens, SJNAHS, Bangalore.)
4.3 ANIMALS:
• Male Wistar rats, 6weeks old weighing 180-200gm to be used.
• A total of 24animals will be chosen. They will be caged in groups of 3-4.
• The animals will be provided with food & water ad- libitum and will be allowed to acclimatize to the environs of the screening room and to gentle human handling every day for a period of 1 week prior to the beginning of screening.
• The 12 hr day-night cycle will be maintained throughout the study period.
• They will then be randomized into 3 groups of 8 animals in each. Will be caged in individual cages.
• The 3 groups are as depicted in table 1
Table 1: Study groups, drug dose and duration of treatment.
Group(n=8) Treatment(I.P) & duration Screening
Control Distill water 3ml x 5days 9 hrs post last dose
Standard-Diazepam 15 %( w/v) Ethanol 2gm/kg x 5days. Watch for withdrawal symptoms 9 hrs post last dose. Inject diazepam 1mg/kg 30mins prior to screening 9 hrs post last ethanol dose
Test- SJM111 15% (w/v) Ethanol 2gm/kg x 5days . Watch for withdrawal symptoms 9 hrs post last dose. Inject SJM111 30mins prior to screening at a dose of 300mg/kg 9 hrs post last ethanol dose
4.4 ETHICS APPROVAL:
The study will be conducted after obtaining IERB approval and the CPCSEA guidelines will be followed throughout.
4.5 PROCEDURE:
Screening model (1) Elevated plus maze
This model has been extensively used for evaluation of novel anxiolytic agents and also to investigate the neurochemical & psychological basis of anxiety. A typical elevated maze consists of 2 opposite open arms 50x10cm crossed with 2 closed arms of the same dimension having walls 50cm in height. The arms are connected by a central square 10x10cm. The model is kept in a dimly lit room & elevated 50cm above the floor.
Principle: An approach avoidance conflict. i.e. fear of balancing on an elevated open arm maze.
The naive treated animal is to be tested individually. It should be placed in centre of the maze, facing an open arm. Thereafter the following parameters should be monitored for 5mins:
• Number of open arm entries and closed arm entries - all 4 paws should be within open/closed arm
• Time spent in open arm & closed arm
• Number of head dips
• Number of rearing
• Number of stretch attend postures
To avoid possible bias during screening, we intend to video record the entire screening procedure for all animals. Evaluation will then be done independently by each investigator of this study.
Screening model 2: Open field chamber
The apparatus comprises of a large green chamber 96x96cm with high walls 96cm. The floor is divided into 16squares by white lines and is placed in dimly lit room. Naïve treated rat is then placed at one corner of the apparatus and is observed thereafter for 5mins. We intend to video- record screening of each animal. The parameters to be evaluated are:
Ambulation (no of squares crossed)
Freeze
Rearing
Scratching & licking
Defecation/urination
An anxious animal is one which shows reduced ambulation with periodic freeze and reduced normal behavior like rearing/grooming and augmented autonomic activity such as defecation/urination.
5. STATISTICAL ANALYSIS
ANOVA- one way analysis of variance followed by post hoc Tukey test will be used. P value of 0.05 will be taken as statistically significant difference between the groups.
6. TIME LINES FOR THE STUDY
Events Aug.09 Sep.09 Oct.09 Nov.09 Dec.09 Jan.10
Protocol Design
Ethics approval
Application for Funding
Animal & Drug procurement
Study duration
Data Analysis
Publication
7. PROPOSED BUDGET FOR THE STUDY
Sl no Items Amount in Rs
1
Animals/Drug
a Procurement charges 3500
b Diet (2 bags ) 1000
c Maintenance charges 1500
d IERB approval 1000
2
Study Requirements
a Diazepam vials/ syrup (10/ 2) 100
b Syringes/gloves 600
3
Publishing charges
a Write up & printing 1000
TOTAL
8700/-
8. REFERENCES
1. Wittchen. Generalized anxiety disorder, prevalence, burden and cost to society. Depression and anxiety 2002; 161:162-171
2. Grigsby, Anderson et al. Prevalence of anxiety in adults with diabetes mellitus- Systematic review. Psychosomatic research 2002;53:1053-1060
3. COPD and anxiety. Chest 2005;127(4):1205-1211
4. S.K Bhattacharya, K.S Satyan. Experimental methods for evaluation of psychotropic agents in rodents: Anti-anxiety agents. IJEB 1997; 35:565-75
5. Rasmussen DD, Mitton DR, Green J, Puchalski. Chronic daily ethanol & withdrawal: 2 Behavioral changes during prolonged abstinence. Alc Cli Exp Res 2001; 25: 999-1005
6. Zhongqi Zhang, Andrew Morse, George Koob and Gerry. Dose and time dependent expression of anxiogenic like behavior in the elevated plus maze during withdrawal from acute and repeated intermittent ethanol intoxication in rats. Alcohol Clin Exp Res 2007; 31(1): 1811-1819
PROTOCOL WRTTEN & SUBMITTED BY
Dr.Leena. A
St. John’s Medical College, Bangalore
A Big Thank You
All of us at department of pharmacology, K.M.C. Mangalore would like to thank all the delegates, dignitaries and resource persons who attended the workshop. When we started thinking about the workshop a few months back, we never imagined we would have 230 delegates from twenty institutions attending the event. 50 to 70 was the number expected by the most optimistic among us. And to think we had almost as many competing for the ‘Best protocol competition’! We know the no. of delegates were probably was on the higher side for a workshop. But then we did not know that entries which started as a trickle are going to end in a torrent, by the time last date was to be over. We did not have the heart to say ‘no’ to endless requests and pleadings we kept receiving from individuals who obviously were very very keen to attend the workshop. At the end of the day we are happy that we did our best to accommodate all the requests. It was a pleasure interacting with you guys! Thank you again for giving us an opportunity to meet you all. Hope you enjoyed as much as we did.
Subscribe to:
Posts (Atom)
