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2. EVALUATION SEMINAR ON
Preclinical
evaluation of
anxiolytics
Presented to : Dr. KVSRG PRASAD
Presented by:
P. Bindu,
M.Pharmacy 1st year
Department of pharmacology
2
3. Contents
o Introduction
o Types of anxiety
o Physiological Vs pathological anxiety
o Classification of anxiolytics
o Screening methods for anxiolytics
In vitro methods
In vivo methods
o Conclusion
o References
3
4. Introduction
o 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
4
5. Physiological and Pathological
Anxiety
jitter Panic attacks
Obsessions,
Stage-fright compulsions
Nervousness Flashbacks,
nightmares
Worrying Pathological fear
5
6. Types of anxiety disorders
a. Panic disorder
b. Generalised anxiety disorder
c. Social anxiety disorder.
d. Obsessive compulsive disorder.
e. Post traumatic stress disorder.
6
7. 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 theory
2. Serotonin theory
3. GABA receptor theory
7
8. 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.
8
9. GABA Receptor Theory
o 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.
9
10. 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.
10
13. 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)
13
14. In vivo methods
Methods based on unconditioned
(spontaneous) response:
o Exploratory activity
elevated plus-maze
light-dark model (two compartment
box)
o Social behaviour
social interaction
Isolation induced aggression
14
15. Methods based on conditioned (learned)
response:
o Conflict models
Vogel punished drinking/ Vogel’s lick
conflict model
Normal (adaptive) anxiety
o Elevated plus-maze test
o Social interaction
o Light-dark model
o Marble burying test
15
16. Stress-induced anxiety
o Vogel lick conflict test
Pathological anxiety
o Neurochemically - induced anxiety
mCPP induced anxiety in rats
16
17. 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. 17
18. 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.
18
19. Experimental Design
• Group I : control
• Group II : standard
• Group III : test treated with dose x
• Group IV : test treated with dose 2x ….
19
20. 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.
20
21. 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
21
22. 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.
22
23. 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.
23
24. 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.
24
25. 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 25
26. 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.
26
27. 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. 27
28. 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. 28
29. 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.
29
30. 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.
30
32. Methodology
Animals placed in groups of 5 each in a perspex open
topped box
1hr before test,2 rats from separate housing treated
with test compound orally
Placed in box with 60W bulb and behavior observed
for 10minutes
2types of activity – 1. social interactions like
sniffing, crawling over the partner
2. exploratory behavior 32
33. 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.
33
34. 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.
34
35. 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.
35
36. 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.
36
37. 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.
37
38. Vogel Lick-conflict (Vogel Punished
Drinking)
Source of anxiety:
thirsty, native rats
are administered
shocks while
licking water.
Animals used:
sprague dawley
rats.
38
39. 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..
39
40. Parameters measured:
o number of accepted punishments
(electric shock)
Anxiolytic effect :
o statistically significant increase in the
accepted shocks.
40
41. conclusion
• Anxiety disorderstress, tension, feardisorder and
associated with
is a psychological
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.
41
42. 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.
42