1. Anxiety is a normal human response to stress but can become pathological. It involves neurotransmitter and brain region abnormalities. 2. Common anxiolytics include benzodiazepines, azapirones, beta blockers, and carbamates. They work by enhancing GABA, blocking serotonin and histamine receptors, or other CNS mechanisms. 3. Animal models are used to screen for anxiolytics using tests like elevated plus maze and light/dark exploration to measure anxiety-like behaviors. In vitro assays measure drug binding to relevant receptors like GABA and histamine H3.

1. Submitted to
Prof. Harish R
By Maheshwari Hireholi
Mpharm first year
Government college of pharmacy-Bangaluru

2. Introduction
• Anxiety is an emotional state
caused by the perception of
real or perceived danger that
threatens the security of an individual.
• It is a normal human adaptive response to
stressful events.
• Physiological anxiety – transient in nature
• Pathological anxiety – needs treatment

3. Physiological and Pathological Anxiety
Normal Pathological
•Jitter •Panic attacks
•Stage-fright • Obsessions,
compulsions
•Nervousness •Flashbacks,
nightmares
•Worrying • Pathological fear

4. Pathophysiology of anxiety
• Neurotransmitters like GABA, nor adrenaline,
serotonin abnormalities – anxiety
• Amygdala, temporal lobe, hippocampus and
hypothalamus - involved in anxiety

5. Classification
1. Benzodiazepines- alprazolam,
chlordiazepoxide , diazepam
2. Azapirones -Buspirone, Ispapirone, gepirone
3. Beta blockers - Propranalol
4. Sedative antihistaminic - Hydroxyzine
5. Carbamates - Meprobamates

6. Mechanism of action
• Benzodiazepine
Enhances the response to GABA by
facilitating the opening of GABA activated
chlorine channels and increases affinity of GABA
for the receptor.
• Buspirone
Buspirone is a partial agonist at 5-HT 1A
receptor. 5-HT1A receptors are auto receptor that
reduce the release of 5-HT and other mediators.

7. Contd….
• Beta blockers
Beta blocker help anxious patient by cutting
vicious cycle and provide symptomatic relief.
• Carbamate It acts on several levels of CNS,
including limbic system, thalamus and spinal
cord. It has mild to moderate tranquilizing,
anticonvulsant and skeletal muscle relaxing
activity. It does not interact with GABA, but
inhibits multineuronal spinal reflexes

8. • Hydroxyzine
Functions principally as an inverse agonist
of the H1 histamine receptor and inhibhits
serotonin 5HT2A receptor sites which
contribute to anxiolytic activity

9. ANIMAL MODEL FOR ANXIETY
• Animal model is a living, a non-human species
that is extensively studied to understand
particular biological phenomena, with the
expectation that discoveries made in the
model organism will provide insight into the
workings of other organisms and without the
added risk of harming an actual human being
during the process.

10. Screening methods for Anxiolytics

11. In vivo methods
Conditioned response Unconditioned response
Geller-Seifter conflict Elevated plus maze (zero/T maze)
Vogel conflict Light/dark exploration (L/D)
Four-plate test Social interaction
Conditioned emotional response Open field
Conditioned taste aversion Ultrasonic vocalization (pain or
separation)
Fear-potentiated startle Fear/anxiety-defense test batteries
Defensive burying Staircase test
Active/passive avoidance Holeboard
Electrical brain stimulation Predator

12. IN-VITRO METHODS
Invitro assay for GABAergic compounds
• 3H GABA receptor binding
• GABA-A receptor binding
• GABA-B receptor binding
Benzodiazepine receptor
• H-Flunitrazepam binding assay
Serotonin receptor binding
• Binding of 3H 5-OH tryptamine
• Histamine H3 receptor binding in brain

13. ELEVATED PLUS MAZE TEST
o Most widely used method; male mice used
o Anxiolytics –decrease anxiety
o Increase open arm exploration time

14. •2 open arms and 2 closed
arms of 50 ˣ 10 ˣ 40cm
dimensions
•Open roof arrangement
•Two open arms are opposite
to each other.
•Maze elevated at 50cm
height

15. The rats weighing around 200-
300g selected; 6 animals 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 arm

16. Parameters Measured During Next 5
minutes:
• Time spent in the open arms
• Entries into the open arms
• Time spent in the closed arms
• Entries into the closed arms
• Total arm entries

17. Anxiolytic effect indicated by
• Increase in the proportion of time spent in
open arms
• Increase in the proportion of entries into
open arms

18. Anti-anxiety test (Light-dark model)
• Purpose and rational: A simple behavior
model in mice to detect compounds with
anxiolytic effects.
• Requirement
Animal: Male mice
Chemical: Drug.

19. Testing apparatus

20. Procedure
• The testing apparatus consists of a light and a
dark chamber divided by a photocell-
equipped zone.
• A polypropylene animal cage, 44 × 21 × 21
cm, is darkened with black spray over one-
third of its surface.
• A partition containing a 13 cm long × 5 cm
high opening separates the dark one third
from the bright two thirds of the cage.

21. • The cage rests on an activity monitor which
counts total locomotor activity.
• An electronic system using four sets of
photocells across the partition automatically
counts movements through the partition and
clocks the time spent in the light and dark
compartments.
• Naive male mice or rats are placed into the
cage.

22. Male mice with the weight
between 18-24g are used
Test drug is administered 30min
prior to experimentation by i.p.
and are then observed for 10 min.
The animal is placed in the cage.
Groups of 6–8 animals are used for
each dose

23. Evaluation
• Dose-response curves are obtained and the
number of crossings through the partition
between the light and the dark chamber are
compared with total activity counts during the
10 min.
• Loco motor activity also monitored.
• Anxiolytics increase locomotor activity and no.
of crossings

24. IN-VITRO METHODS
GABA RECEPTOR BINDING
Principle:
• To estimate the test drug’s binding
characteristic to the GABAA Receptor using
radioligand 3H-SR 95531 (3-carboxpropyl)-3-
amino,6-(4-methoxyphenyl)-pyridazinium
bromide

25. Procedure
MEMBRANE PREPARATION

26. Rats are killed by decapitation, brains are quickly
removed and separated from cerebellum
Placed into ice cold sucrose solution
homogenized and centrifuged at 1000 at 40C
10min
Pellets are discarded, supernatant is centrifuged at
20,000g for 20min.
Supernatant is discarded; pellets are lysed by
hypo-osmotic shock (addition of 20volume of ice
cold bi-distilled water) homogenisation
suspension

27. Suspension stirred under cooling for
20mincentrifuge at 48,000g for 20 min→
pellets resuspended in ice-cold bi-distilled
water suspension stirred and recentrifuged
Final pellets are resuspended in the
incubation buffer(1g brain wet weight/1ml
buffer)membrane suspension stored in
aliquots of 1ml at -20 0C.
Protein content determined according to
method of Lowry with bovine serum
albumin as standard.
On the day of the experiment, the required volume of
the membrane suspension is slowly thaweddiluted
to 1: 20 with bi-distilled water stirred for 10 min
centrifuged at 50,000 rpm for 10 min pellets are
re-suspended in ice-cold incubation buffer, yielding a
membrane suspension with a protein content of 1
mg/ml.

28. Assay
Saturation experiments
Estimation of total binding:
• 50ml 3H-SR 95531
• 50ml incubation buffer.
Estimation of non-specific binding:
• 50ml 3H-SR 95531
• 50ml (+) bicucculine (10-4M)
Competition Experiments
• 50ml 3H-SR 95531
• 50ml incubation buffer without or with non-labeled test
drug

29. • The binding reaction is started by adding
membrane suspension per incubation sample.
• The sample is incubated for 30min at 4oC.
• The reaction is stopped and subjected to rapid
vacuum filtration over glass fiber filters
thereby, the membrane bound radioactivity is
separated from free radioactivity.
• Filters are washed with approximately 20ml
ice cold rinse buffer (tris-Hcl, pH 7.4)/sample.
• The membrane bound radioactivity is
measured after addition of 2ml of liquid
scintillation cocktail per sample in packard
liquid scintillation counter.

30. Evaluation
• The following parameters are calculated :
Total binding
Non-specific binding
• Specific binding = total binding-non specific
binding

31. • Ki = KD3H × IC50/KD3H + 3H
• IC50 = concentration of the test drug, which
inhibits 50% of specifically bound 3H-SR 95531
in the competition experiment.
The Ki-value of the test drug is that
concentration, at which 50% of the receptors
are occupied by the test drug.

32. HISTAMINE H3 RECEPTOR BINDING IN
BRAIN
PURPOSE AND RATIONALE
• Histamine modulates its own synthesis and release
from depolarized brain slices or synaptosomes by
interacting with H3 auto-receptors with a
pharmacology distinct from that of H1 and H2
receptors. The R-isomer of α- methyl histamine (α-
MeHA) was identified as a highly selective H3-receptor
agonist active at nano molar concentrations.
Furthermore, this compound in 3H-labelled form is a
suitable probe for the H3-receptor.

33. PROCEDURE
• The cerebral cortex from guinea pigs is dissected
and homogenized in 50 volumes ice-cold 50 mM
KH2PO4/Na2HPO4 buffer, pH 7.5, in a Potter
homogenizer.
• The homogenate is centrifuged for 15 min at 750
rpm.
• The pellet is discarded and the supernatant
centrifuged at 42 000 rpm for 15 min.
• The supernatant is discarded and the pellet
washed superficially with and then re-suspended
in fresh buffer.

34. • The protein concentration of the membrane
suspension as determined.
• Aliquots of the membrane preparation are incubated
for 60 min at 25 °C with 3H(R) α-MeHA and
unlabeled substances in a final volume of 1 ml.
• The assay is stopped by dilution with 2 × 3 ml ice-
cold medium, followed by rapid filtration under
vacuum over Millipore AAWP filters which are then
rinsed twice with 5 ml of ice-cold medium.
• Radioactivity retained on the filters is measured by
liquid scintillation spectroscopy.

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

36. Reference
• H. Gerhard Vogel, ‘Drug Discovery and Evaluation:
Pharmacological Assays’, 3rd edition,585-613.
• P.Mahamuni Sagar, S.S.Nipate, ‘Preclinical
evaluation of anxiolytic agents: an overview’
“JOURNAL OF PHARMACEUTICAL RESEARCH AND
OPININION”
• Bourin Michel, ‘Animal models for screening
anxiolytic-like drugs: a perspective’, NCBI, 2015.