Screening of anti anxiety drugs
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Transcript of Screening of anti anxiety drugs
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Preclinical evaluation of
anxiolytics
Presented by:P. Bindu,
M.Pharmacy 1st yearDepartment of pharmacology
EVALUATION SEMINAR ON
Presented to : Dr. KVSRG PRASAD
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o Introductiono Types of anxietyo Physiological Vs pathological
anxietyo Classification of anxiolyticso Screening methods for anxiolytics
In vitro methodsIn vivo methods
o Conclusiono References
Contents
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Introductiono Anxiety is an emotional state caused
by the perception of real or perceived danger that threatens the security of an individual.
o It is normal human adaptive response to stressful events.
o Physiological anxiety – transient in nature
o Pathological anxiety – needs treatment
Physiological and Pathological Anxiety
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jitter
Stage-fright
Nervousness
Worrying
Panic attacks
Obsessions, compulsions
Flashbacks, nightmares
Pathological fear
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Types of anxiety disorders
a. Panic disorder
b. Generalised anxiety disorder
c. Social anxiety disorder.
d. Obsessive compulsive disorder.
e. Post traumatic stress disorder.
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Pathophysiology of anxiety
o Neurotransmitters like GABA, noradrenaline, serotonin abnormalities – anxiety.
o Amygdala, temporal lobe, hippocampus and hypothalamus - involved in anxeity
o Neurochemical theories :
1. Noradrenaline theory2. Serotonin theory3. GABA receptor theory
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Noradrenaline theory
o ANS of anxious patients- hypersensitive to stimuli.
o Locus cerulus – activates epinephrine release
o Anxiogenics – stimulate locus cerulus firing
o Anxiolytics- inhibits locus cerulus firing and decrease noradrenaline activity.
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GABA Receptor Theoryo GABA – inhibitory neurotransmitter in
brain.
o Has inhibitory and regulatory effects on serotonin, noradrenaline and dopamine.
o GABAA receptor involved in anxiety; decreases neuronal excitability
o Patients suffering from anxiety disorders have less level of GABA in cortex.
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Serotonin Theory
• Abnormalities in serotonin function i.e., release and uptake plays role in anxiety.
• Greater serotonin activity – reduces norepinephrine activity in locus cerulus.
• SSRIs – increases serotonin levels post synaptically – blocks symptoms of anxiety.
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Classification of anxiolytics
Benzodiazepines alprazolam, clonazepam, diazepam
Azapirones Buspirone, tandospirone, gapirone
Sedative antihistaminics Hydroxyzine
Beta blockers Propranalol
carbamates meprobamate
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Screening
methods for
anxiolytics
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In vitro methods
GABAA receptor binding
GABAB
receptor binding Benzodiazepine receptor: [3H]-flunitrazepam
binding assay Serotonin (5-HTIA) receptor: binding of [3H]-8-
hydroxy-2-(di-n-propylamino)-tetralin ([3H]-DPAT)
Serotonin (5-HTIB) receptors in brain: binding of [3H]5-hydroxytryptamine ([3H]5-HT)
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In vivo methods Methods based on unconditioned
(spontaneous) response:
o Exploratory activityelevated plus-maze light-dark model (two compartment box)
o Social behavioursocial interactionIsolation induced aggression
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Methods based on conditioned (learned) response:
o Conflict modelsVogel punished drinking/ Vogel’s lick conflict model
Normal (adaptive) anxietyo Elevated plus-maze test o Social interactiono Light-dark modelo Marble burying test
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Stress-induced anxietyo Vogel lick conflict test
Pathological anxiety o Neurochemically - induced
anxiety mCPP induced anxiety in rats
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Elevated Plus Maze Test
o Most widely used method; male mice used.
o For selective identification of anxiolytic and anxiogenic drugs
o Anxiolytics –decrease anxiety – increase open arm exploration time
o Anxiogenics – decrease open arm exploration time.
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Elevated plus maze
o 2 open arms and 2 closed arms of 50 ˣ 10 ˣ 40cm dimensions
o Open roof arrangement
o Two open arms are opposite to each other.
o Maze elevated at 50cm height.
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Experimental Design
• Group I : control• Group II : standard • Group III : test treated with dose
x • Group IV : test treated with dose
2x ….
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The rats weighing around 200g - housed in pairs for 10 days prior to testing;
6animals selected for each group
Test drug administered 30min prior to
experimentation by i.p route.
The rat is then placed in the centre of the maze
facing one of the enclosed arms.
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Parameters Measured During Next 5 minutes:
o time spent in the open arms
o entries into the open arms
o time spent in the closed arms
o entries into the closed arms
o total arm entries
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Anxiolytic effect indicated by:
o increase in the proportion of time spent in open arms i.e.,
time in open arms/total time in open or closed arms
o increase in the proportion of entries into open arms i.e.,
entries into open arms/total entries into open or closed arms.
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Evaluation of results:
o Motor activity and open arm exploratory activity determined.
o Values of treated groups expressed as % of control values.
o Benzodiazepines and valproate – decrease motor activity and increase exploratory time.
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Isolation induced aggression
o Male mice subjected to isolation develop aggressive behavior towards other animals of same sex.
o Compounds tested for their ability to suppress this isolation induced aggression.
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Animal used: Male NMRI strain mice (12g wt)
Mice kept isolated for 6weeks & aggressive behavior tested.
Male mice accustomed to live together placed in cage of isolated
mice for 5minutes
Isolated mice attacks intruder- aggressiveness observed
Drugs given to isolated mice s.c or orally; aggressive behavior tested
at 60, 120,240 minutes (oral route)
If drug active- decrease in aggressiveness
Attenuation of fighting reaction
PROCEDURE
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Evaluation of results:
o No. of animals with complete suppression of aggressiveness.
o Reaction time noted.
o Graduated scale of inhibition of aggressiveness is established.
o Results of test group animals is compared with the control group results.
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Anti – anxiety test(light – dark model)
o Rodents – have exploratory activity
o Animals placed in 2 chambered systems, where they can freely move between a brightly –lit open field and a dark corner.
o After treatment with anxiolytic - show more crossings between the two chambers and more locomotor activity.
o Number of crossings between the light and dark sites is recorded.
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Methodology o Apparatus - a dark and a
light chamber divided by a photocell equipped zone.
o Polypropylene animal cage (44 ˣ 21ˣ 21 cm) is darkened with black spray over 1/3rd of its surface.
o A partition containing 13cm(l) ,5 cm (h) opening is used for separating the dark one-third of the cage.
o This case rests on an activity monitor which counts total locomotor activity.
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o An electronic system consisting of 4 sets of photocells across the partition.
o It automatically counts movements
through the partition and records the time spent in the light and dark compartments.
o Animals- treated 30 min before the test with drugs or vehicle given i.p. placed in the cage and observed for 10 min.
o Groups of 6-8 animals used for each dose.
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o No. of crossings through the partition between the light and dark chambers compared with total activity counts during the 10 min.
o Loco motor activity also monitored.
o anxiolytics like diazepam & meprobamate increase locomotor activity and no. of crossings.
o non anxiolytics - not effective in this model.
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Social Interaction In Rats
o In unfamiliar and brightly lit environment, social interactions are suppressed.
o Anxiolytics –counteract this suppression.
o Animal used : male sprague dawley rats.
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2types of activity – 1. social interactions like sniffing, crawling over the partner
2. exploratory behavior
Placed in box with 60W bulb and behavior observed for 10minutes
1hr before test,2 rats from separate housing treated with test compound orally
Animals placed in groups of 5 each in a perspex open topped box
Methodology
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Parameters measured : o exploration, sniffing, rearing, social
contacts, sexual behaviour, attack, fighting, biting ,defensive posture, immobility and climbing over the partner.
Evaluation :o Values of treated partners compared
with data from control animals – ANOVA and t - test used.
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mCPP Induced Anxiety
o mCPP - [ 1-(3-chlorphenyl) piperazine] o Metabolite of antidepressant trazodone.
o mCPP induces hypophagia and hypolocomotion , inhibits social
interaction, diminishes exploratory activity in light-
dark box test.
o Antagonism of these symptoms is used for screening of anxiolytic drugs.
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Male Sprague Dawley rats (200- 250g) are housed in groups of 6; exposed to 12 hour light/dark cycle with free access to food and water.
mCPP induced anxiety in dark – light box
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Parameters measured : o time spent in both sides
(horizontal, vertical activity) o frequency of motion o number of transition
Anxiolytic effect : o increase in parameters measured
in the light/dark box or in number of transitions if test is active.
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mCPP Induced Anxiety -Locomotion Study
• Test compound or vehicle are administered orally 1h or i.p 30 min before the locomotion test.
• mCPP is injected i.p. in a dose of 7 mg/kg 20 min before the test.
• The animals are placed individually in an automated locomotor activity cages and locomotion is recorded for 10 min.
• Anxiolytic effect : disinhibition of locomotion.
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Vogel Lick-conflict (Vogel Punished Drinking)
Source of anxiety: thirsty, native
rats are administered shocks while licking water.
Animals used: sprague dawley
rats.
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Methodology
a water bottle with metal drinking tube is fitted to the animal housing
Electric circuit is connected between drinking tube and floor of
cage.
i.p injection of drugs are given; 30min later rats placed in cage and allowed to drink water and shock
given after 20 licks
For 3minutes next shocks are given for every 12th lick
No. of shocks delivered in 3min noted for each animal, no. of shocks received after treatment compared
with control..
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Parameters measured:o number of accepted punishments
(electric shock)
Anxiolytic effect :o statistically significant increase
in the accepted shocks.
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conclusion
• Anxiety disorder is a psychological disorder and
associated with stress, tension, fear and threat about
future.
• The pathophysiology of anxiety disorder is not fully
understood and hence improper diagnosis leads to
increase morbidity and mortality rates.
• Development of the screening methods resulted in introduction of many new anxiolytic agents.
• In future aspects more reliable and easy models for screening are to be developed.
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References • Joseph T. Dipiro, Robert L.Talbert, Gary C.Yee;
Pharmacotherapy-And-Pathophysiologic Approach; Seventh edition; 1161-1181.
• Parmar N.S, Shiv prakash; Screening Methods in Pharmacology; 98-107.
• Gerhard Vogel; Drug discovery and Evaluation – Pharmacological Assays; Second Edition; 401-458.
• Shenoy et.al/ Preclinical evaluation of anxiolytic agents: an overview; Journal of Pharmaceutical Research and Opinion; june 2011/volume 1/ issue 2/ 7-22 pgs.
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THANK YOU