Screening methods of anti anxiety agents Guided By - Dr. Saumya Das,Associate Professor,NIET(Pharmacy Institute),Greater Noida Presented By - Anupam Dubey,M.Pharm(Pharmacology) NIET(Pharmacy Institute) 2020-2021

1. Screening Methods Of Anti
Anxiety Agents
Presented By: Under Guidance:
Anupam Dubey Dr. Saumya Das
M.Pharm(Pharmacology) Associate Professor
PTSM-I NIET(Pharmacy Institute)
Greater Noida
NOIDA INSTITUTE OF ENGINEERING AND TECHNOLOGY
(PHARMACY INSTITUTE)
GREATER NOIDA

2. Introduction
• A mental health disorder characterized by feelings of worry, anxiety or fear
that are strong enough to interfere with one's daily activities
• Anxiety is a normal emotion. It’s your brain’s way of reacting to stress and
alerting you of potential danger ahead.
• Physiological Anxiety-transient in nature
• Pathological anxiety – needs treatment

3. Types Of Anxiety
The five major types of anxiety disorders are:
• Generalized Anxiety Disorder
Generalized Anxiety Disorder, GAD, is an anxiety disorder characterized by chronic anxiety, exaggerated worry and tension,
even when there is little or nothing to provoke it.
• Obsessive-Compulsive Disorder (OCD)
Obsessive-Compulsive Disorder, OCD, is an anxiety disorder and is characterized by recurrent, unwanted thoughts
(obsessions) and/or repetitive behaviors (compulsions). Repetitive behaviors such as hand washing, counting, checking, or
cleaning are often performed with the hope of preventing obsessive thoughts or making them go away. Performing these so-
called "rituals," however, provides only temporary relief, and not performing them markedly increases anxiety.
• Panic Disorder
Panic disorder is an anxiety disorder and is characterized by unexpected and repeated episodes of intense fear accompanied by
physical symptoms that may include chest pain, heart palpitations, shortness of breath, dizziness, or abdominal distress.

4. Cont….
• Post-Traumatic Stress Disorder (PTSD)
Post-Traumatic Stress Disorder, PTSD, is an anxiety disorder that can develop after exposure to a terrifying
event or ordeal in which grave physical harm occurred or was threatened. Traumatic events that may trigger
PTSD include violent personal assaults, natural or human-caused disasters, accidents, or military combat.
• Social Phobia (or Social Anxiety Disorder)
Social Phobia, or Social Anxiety Disorder, is an anxiety disorder characterized by overwhelming anxiety
and excessive self-consciousness in everyday social situations. Social phobia can be limited to only one type
of situation - such as a fear of speaking in formal or informal situations, or eating or drinking in front of
others - or, in its most severe form, may be so broad that a person experiences symptoms almost anytime
they are around other people.

5. PRECLINICAL EVALUATION OF ANXIOLYTICS
• In-vitro Methods
• In-vivo Methods
• Our main focus here would be In-vivo.

6. In- vitro methods
• GABAA receptorbinding
• GABAB receptorbinding
• Benzodiazepine receptor: [3H]-flunitrazepam binding assay
• Serotonin (5-HT1A) receptor: binding of [3H]-8- hydroxy-2-(di-n-
propylamino)-tetralin ([3H]- DPAT)
• Serotonin (5-HT1B) receptors in brain:binding of [3H]5-hydroxytryptamine
([3H]5-HT)

7. In Vivo
Methods

8. Elevated plus –maze (EPM) test
• This has widely validated to
measure anxiety to rodents

9. Principle
The test is based on the natural aversion of mice for open and elevated areas,
as well as natural aversion on their natural spontaneous exploratory behavior
in novel environments.
The apparatus consists of open arms and closed arms, crossed in the middle
perpendicularly to each other, and a center area.
Mice are given access to all of the arms and are allowed to move freely
between them.

10. Procedure
• The rats (200–250 g body weight) are housed in pairs for
10 days prior to testing in the apparatus.
• During this time the rats are handled by the investigator on
alternate days to reduce stress.
• 6 rats are taken for each group.
• Thirty min after i.p. administration of the test drug or the
standard, the rat is placed in the center of the maze facing
one of the enclosed arms.
• Duration of test is 5 min where all data is collected.
• Testshould be conducted in sound proof room.
• After each test,the maze is carefully cleaned up with wet
tissue paper (10% ethanol solution)

11. Evaluation
• The number of entries into the open arms.
• The time spent in the open arms are used as indices
of open space-induced anxiety
• Anxiolytic drugs specifically increase the
number of entries into the open arms and
the time spent there
• Anxiogenic drugs specifically decrease the
entry
• The total entries score and total distance are
considered a
Useful index of general activity.
The percentages of entries and time spent in
each arm constitute the index of primary anxiety

12. Light-dark Exploration model

13. • Crawley and Goodwin (1980)
• Mice tend to explore novel environment but retreat from brightly lit open
field.
• In a two chambered system, where animal can freely move between
brightly lit open field and dark corner, they show more crossings
between two chambers and more locomotor activity after treatment with
anxiolytics.

14. Procedure
• Test apparatus- light and dark chamber
divided by photocell equipped zone.
• Polypropylene animal cage 44x21x21 cm,
darkened with black spray over on-third of
surface.
• Partition containing 13cm long x 5cm high
opening separates the dark one third from bright
two thirds of the cage
• Cage rests on an animex activity monitor, which
counts the locomotor activity.
• Four sets of photocells across the partition,
automatically counts movement through the partition
and clocks time spent in light and dark
• male mice are placed in cage
• Animals are treated 30 min before the experiment with
test or vehicle intraperitoneally and are observed for 10
min.
• Groups of 6-8 animals are used for each dose.

15. Evaluation
• Number of crossings through the partition
between the light and dark chamber are
compared with total activity counts during 10
minute.
• Critical assessment of the method: Anxiolytics
like diazepam, pentobarbital, meprobamate,
produce dose dependent increase in crossings
• Test is relatively simple with no painful stimuli.

16. Vogel water-lick
conflict test

17. • This test is a well-known method used in rats, designed by Vogel et al.
• Recently, this test has been reported to successfully detect anxiolytic-like action of diazepam .
• In this test, thirsty animals gain water reward through a water spout, but at the expense of receiving a
mild electric shock delivered to the tongue.
•
Licking in untreated/controls is suppressed due to the conflict.
• Anxiolytics release this suppressedbehavior and decrease the conflict.
• Diazepam and pentobarbital produces a significant anti-conflict effect, which means that –
 These drugs increases the number of electric shocks the mice received during
the test session.
 But drugs like baclofen, buspirone, chlorpromazine and also haloperidol did not produce anti-
conflict effects.

18. Social interaction test in Rats

19. Purpose and rationale
• In an unfamiliar and brightly lit environment, the normal social interaction
of rats ( sniffing, nipping, grooming) is suppressed.
• Anxiolytics counteract this suppression.

20. Procedure
• Male sprague-dawley rats (225-275g) are housed in groups of 5 animals
• Apparatus: perspex open-topped box (51x51x 20cm) with 17x17cm marked areas on the floor.
• One hour prior to the test, 2 naïve rats from separate housing cages are treated with test compound orally
•
They are placed into the box (with 60W bright illumination 17cm above) and their behaviour is observed
over a 10 min period.
• 2 types of behaviour can be noted:
• Social interaction between the animals is determined by timing the sniffing of partner, crawling under or
climbing over partner and following partner.
•
Exploratory motion is measured as the number of crossings of the lines marked on the floor of the test
box.
• Six pairs are used for each dose

21. Evaluation
• Values of treated partners are compared with the data from 6 pairs of
untreated animals using single factor analysis of variance followed by
Dunnett’st-test.

22. IN-VITRO METHOD

23. 1-Benzodiazepine receptor: [3H]- flunitrazepam
binding
• PURPOSE AND RATIONALE
[3H]-flunitrazepam have specific binding sites in CNS membrane
preparations that satisfy the criteria for pharmacological receptors, e.g.
saturability, reversibility, stereo selectivity and significant correlation
with in vivo activities of the antianxietydrugs

24. PROCEDURE
• Reagents
• [Methyl-3H]-Flunitrazepam.
• Clonazepam HCl
• Tissue preparation
• Male Wistar rats are decapitated and the brains rapidly removed.
• The cerebral cortices are removed, weighed and homogenized with a Potter-Elvejhem homogenizer in 20 volumes of ice-cold 0.32 M
sucrose.
• This homogenate is centrifuged at 1000 g for 10 min. The pellet is discarded and the supernatant is centrifuged at 30 000 g for 20 min.
• The resulting membrane pellet is resuspended in 40 volumes of 0.05 M Tris buffer.

25. Assay
 1 ml 0.05 M Tris buffer, pH 6.9
 560 μl H2O
 70 μl 0.5 M Tris buffer, pH 6.9
 50 μl 3H-Flunitrazepam 20
 0.1 mM Clonazepam
 300μl tissue suspension

26. Cont…
• The tubes containing [3H] –flunitrazepam , buffer, drugs and water are then
incubated at 0–4 °C in an ice bath
• A 300µl aliquot of the tissue suspension is added to the tubes at 10 sec
intervals.
• The tubes are then incubated at 0-4ºC for 20 min and the assay stopped by
vacuum filtration through Whatman filters. This step is performed at 10 sec
intervals.
• Each filter is immediately rinsed with three 5ml washes of ice-cold buffer.
• The filters are counted in 10 ml of liquid scintillation counting cocktail.

27. EVALUATION
• The percent inhibition at each drug concentration is the mean of triplicate
determinations.
• IC50 calculations are performed using log-probit analyses

28. SEROTONIN (5-HT1B ) RECEPTORS IN BRAIN : BINDING
OF [3 H] 5-HYDROXYTRYPTAMINE ([3 H ]5-HT)
• PURPOSE AND RATIONALE
To determine the affinity of test compounds for the serotonin (5-HT1B ) receptor in brain.
The existence of two populations of 5-HT1 receptors in rat brain was shown by differential
sensitivity to Spiroperidol. The Spiroperidol-sensitive receptors were designated as the 5-
HT1A subtype and the insensitive receptors were referred to as the 5- HT1B subtype. The 5-
HT1B subtype has been identified in the brain of rats and mice and can be selectively labeled
by 5-HT in rat striatum when Spiroperidol is included to mask the 5- HT1A and 5-HT2
receptors. The distribution of 5-HT1B sites in rat brain is similar to that of 5-HT1D sites in
human brain. By comparing the results in the 5-HT1B assay with those in the 5-HT1A , 5-
HT2 and the 5-HT3 receptor binding assays the relative affinity of a test compound for the
major subclasses of 5-HT receptors in the rat brain can be determined.

29. PROCEDURE
• Reagents
1. Tris buffers, pH 7.7
2. 5-Hydroxy[G3H] tryptamine creatinine sulfate
3. Serotonin creatinine sulfate
4. Spiroperidol
5. Test compound

30. TISSUE PREPARATION
Rats are sacrificed by decapitation.
Striata are removed, weighed and homogenized in 20 vol. of 0.05 M Tris buffer, pH 7.7 The homogenate is
centrifuged at 48000 g for 10 min and discard supernatant.
The pellet is resuspended in an equal volume of 0.05 M Tris buffer, incubated at 37 C for 10 min and
recentrifuged at 48000 g for 10 min.
The final membrane pellet is resuspended in 0.05 M Tris buffer containing 4 mM CaCl2 , 0.1% Ascorbic acid
and 10 mM Pargyline

31. ASSAY
• 800 μl tissue
• 80 μl 0.05 M Tris+CaCl2+Pargyline+Ascorbic acid
• 20 μl vehicle/ 5-HT/ drug
• 50 μl [3H]5-HT
• 50 μl Spiroperidol
Tubes are incubated for 15 min at 25 C. The assay is stopped by vacuum filtration through
Whatman filters which are then washed 2 times with 5 ml of ice-cold 0.05 M Tris buffer.
The filters are then placed into scintillation vials with 10 ml of Liquiscint scintillation
cocktail and counted.

32. EVALUATION
• Specific binding is defined as the difference between total binding and
binding in the presence of 10 μM 5-HT. IC50 values are calculated from the
percent specific binding at each drug concentration. The Ki value may then
be calculated by the ChengPrusoff equation
Ki = IC50 / 1 + L /KD

33. References
• https://www.hhs.gov/answers/mental-health-and-substance-abuse/what-are-the-five-major-
types-of-anxiety-disorders/index.html
• https://www.slideshare.net/AdvaithaMv/screening-of-anxiolytics-44529278
• https://www.e-phy-science.com/services/behavioral-tests/elevated-plus-maze/
• https://en.m.wikipedia.org/wiki/Vogel_conflict_test
• https://www.slideshare.net/utkarshalok/anxiolytic-screening-
modelsutkarsh?from_action=save
• https://www.slideshare.net/bindupulugurtha/screening-of-anti-anxiety-drugs

34. THANK YOU