Anxiolytics screening methods.....it contains mechanism of action , screening methods and invivo and in vitro methods.

1. Presented by
K V S SAIKRISHNA
18T21S0104
M.Pharm 1st yr 2nd sem
CMR COLLEGE OF PHARMACY

2. INTRODUCTION
CLASSIFICATION
MECHANISM OF ACTION
ANIMAL MODEL FOR ANXIETY
SCREENING METHODS FOR ANXIOLYTICS
Pre clinical evaluation of anxiolytics
IN-VIVO METHODS
Elevated plus maze test
Light dark exploration model
IN-VITRO METHODS
GABA receptor binding
Histamine H3 receptor binding

3. DEFINITION: An anxiolytic (also antipanic or antianxiety
agent) is a medication or other intervention that inhibits
anxiety. This effect is in contrast to anxiogenic agents,
which increase anxiety. Together these categories of
psychoactive compounds or interventions may be referred
to as anxiotropic compounds or agents.
Anxiety Disorders: Anxiety is defined as a subjective
sense of unease, dread, or foreboding.
It is the most prevalent psychiatric illnesses in the
general community, are present in 15–20% of medical clinic
patients.
Physiological anxiety – transient in nature.
Pathological anxiety – needs treatment.

4. NORMAL PATHOLOGICAL
Jitter Panic attacks
Stage fright Obsessions,Compulsions
Nervousness Flashbacks,Nightmares
Worrying Pathological fear

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

6. 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.
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.
Hydroxyzine: Functions principally as an inverse agonist of the H1
histamine receptor and inhibhits serotonin 5HT2A receptor sites
which contribute to anxiolytic activity.

7. 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.

9. IN-VITRO METHODS
GABA-A receptor binding
GABA-B receptor binding
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)
The total following parameters are calculated:
Total binding,
Non-specific binding,
Specific binding: Total binding – non specific binding

10. IN-VIVO METHODS

11. Validity:
Face validity : A feature that is assessed (for a
test or model of anxiety) by determining how
closely the model or test resembles human anxiety
symptoms .
Predictive validity : whether the model or test
reliably responds to clinically efficacious anti-
anxiety medications .
Construct validity : Whether the degree to which
the model or test recruits the same underlying
neurobiology as implicated in human anxiety .

12. This has widely validated to measure anxiety to rodents.
Anxiolytics –decrease anxiety.
Increase open arm exploration time.

13. 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.

14. 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

15. Procedure:

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. Crawley and goodwin (1980)
 Mice and rats 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.

18. The testing apparatus consists of a light and a dark chamber
divided by a photo cell-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.
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.

20. • 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

21. Critical assessment of the method:
Anxiolytics like diazepam, pentobarbital,
meprobamate, produce dose dependent
increase in crossings
• Test is relatively simple with no painful
stimuli

22. 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. 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.

26. • 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.

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

28. 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.

29. 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.
 The protein concentration of the membrane
suspension as determined.

30. 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 icecold
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.

31. 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