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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