UNDER GUIDANCE OF:
MRS. MEENU SINGH
DEPT OF PHARMACOLOGY
MISS SAYANTI SAU
I M. PHARM
To understand the concepts behind Anxiolytics and to
reproduce the concepts of screening and clinical
evaluation of anxiolytics practically as well as
Physiological Vs pathological anxiety
Types of anxiety
Classification of anxiolytics
Pathophysiology of anxiety
Screening methods for anxiolytics
In vitro methods
In vivo methods
Anxiety is an emotional state
commonly caused by the perception
of real or perceived danger that
threatens the security of an individual.
It is normal human adaptive response
to stressful events.
Physiological anxiety – transient in
Pathological anxiety – needs
TYPES OF ANXIETY DISORDER
GAD (Generalized Anxiety Disorder)
PD (Panic Disorder)
SAD (Social Anxiety Disorder)
Obsessive Compulsive Disorder
Post Traumatic Stress
GENERALISED ANXIETY DISORDER
It is chronic and fills a person’s day with exaggerated
worry and tension, even though there is little or nothing to
provoke it, associated with worrying excessively about
health, money, family, school or work. Concentration and
sleep problems are also common.
Worries that are difficult to control
Poor concentration or mind going blank
Unexpected panic attacks associated with physiological symptoms of
autonomic nervous system without any warning or apparent reason.
They can’t predict when an attack will occur, and many develop intense
anxiety between episodes, worrying when and where the next one will
Symptoms include Attacks usually last no more than about 10
Shortness of breath
Fear of dying or going crazy
SOCIAL ANXIETY DISORDER
Characterized by an intense, irrational,
persistant fear of situations, usually
social or performance situations, where
risk of embarrassment is present. It can
disrupt normal life, interfering with school,
work or social relationships.
Physical symptoms often accompany the
Shortness of breath
Is an intense fear of something that
posses little or no actual danger of a
object or situation.
Some of the more common specific
Centered around closed-in places
Injuries involving blood
OBSESSIVE-COMPULSIVE DISORDER (OCD)
Obsessions (recurrent, intrusive and generally
distressing thoughts, images or feelings).
Compulsions(repetitive, ritualistic behaviors
aimed to alleviate obsessions).
POST TRAUMATIC STRESS DISORDER( PTSD)
Develops subsequent to experiencing or witnessing a traumatic
Symptoms (lasting at least 4-6 weeks )
Pathophysiology of anxiety
Neurotransmitters like GABA, noradrenaline,
serotonin abnormalities – anxiety.
Amygdala, temporal lobe, hippocampus and
hypothalamus - involved in anxeity.
Neurochemical theories :
1. Noradrenaline theory
2. Serotonin theory
3. GABA receptor theory
Brain structures dealing with fear and
Gaba receptor theory
GABA – inhibitory
• Inhibitory and
regulatory effects on
• GABAA receptor
involved in anxiety;
• Patients suffering
disorders have less
level of GABA in
• When GABA binds to the
GABAA receptor, the Clchannel opens, influx of
Cl• Causing hyper
membrane & decreases
nerve cell excitability.
• The number of GABAA
receptors can change
with alterations in the
• The subunit expression
can be altered by
• In patients with GAD,
in left temporal lobe is
5-HT is an inhibitory
neurotransmitter, used by
neurons originating in the
raphe nuclei of the brain
stem and projecting diffusely
throughout the brain (e.g.,
hippocampus, and limbic
Stimulation of the
receptors in the limbic
system results in anxiety and
Abnormalities in serotonergic
functioning through release and
uptake at the presynaptic auto
receptors (5-HT1A/1D), the
serotonin reuptake transporter
site (SERT), or effect of 5-HT at
the postsynaptic receptors (e.g.,
5-HT1A, 5-HT2A, and 5-HT2C)
may play a role in anxiety
Low 5-HT activity may lead
to a dysregulation of other
neurotransmitters. NE may
act at presynaptic 5-HT
terminals to decrease 5-HT
release, and its activity at
postsynaptic receptors can
cause increased 5-HT
SSRIs – increases serotonin
levels post synaptically –
blocks symptoms of anxiety.
In response to threat or fearful
situations, the Locus cerulus
serves as an alarm center,
activating NE release and
stimulating the sympathetic and
Chronic central noradrenergic
overactivity down- regulates
patients with GAD.
Drugs with anxiogenic effects
(e.g., yohimbine) stimulate
LC firing & increase
noradrenergic activity. NE in
turn increases glutamate
release. This produces
subjective feelings of anxiety.
b.GABAA receptor binding
c.GABAB receptor binding
a.Serotonin (5-HT1A) receptor:
binding of [3H]-8-hydroxy-2-(din-propylamino) tetralin([3H]DPAT)
b. Serotonin (5-HT1B) receptors
in brain: binding of [3H]5hydroxytryptamine ([3H]5-HT)
c. 5-HT3 receptor in rat entorhinal
cortex membranes: binding of
3. HISTAMINE H3
• Foot-shock induced
• Water competition
• Maternal aggression
• Rage reaction in cats
Anti-anxiety test (light-dark model)
Anticipatory anxiety in mice
Social interaction in rats
Elevated plus maze test
Water maze test
Cork gnawing test in the rat
Distress vocalization in rat pups
Schedule induced polydipsia in rats
Four plate test in mice
Footshock induced freezing behavior in
Experimental anxiety in mice
mCPP induced anxiety in rats
Acoustic startle response in rats
Unconditioned conflict procedure
Shock probe conflict procedure
Ultrasound induced defensive behavior
Anxiety/defense test battery in rats
Marmoset human threat test
Aversive brain stimulation
• Sidman avoidance
• Geller conflict paradigm
• Conditioned defensive
burying in rats
• Taste aversion paradigm
EFFECTS ON THE
• Plasma catecholamine
levels during and after
• Plasma corticosterone
levels influenced by
dependence in rats
Elevated plus maze test
The elevated plus maze
test has been extensively
used for the selective
evaluation of anxiolytic
When the animals enter in
to open arm they show
fear like movement,
freeze and become
Rodents have aversion
for high and open
space and prefer
enclosed arm and
therefore spend greater
amount of time in
Advantages of this test
1.simple fast and less
2.No prior training is
anxiolytic and anxiogenic
Anxiolytics – decrease
anxiety – increase open
arm exploration time
Anxiogenics – decrease
open arm exploration
Diazepam (1 mg/ kg ; i.p.)
Equipment : Elevated plus maze apparatus and a stop clock
Elevated plus maze apparatus
2 open arms (16 x 5
cm for mice & 50 x
10 cm for rats)
2 closed arms (16 x 5 x
12 cm for mice and 50
x 10 x 40 cm for rats)
An open roof with entire maze elevated (25 cm
for mice and 50 cm for rats) from the floor.
Two open arms are opposite to each other.
The mice housed in pairs for 10
days prior to testing; 6animals
selected for each group
Test drug administered 30min
prior to experimentation by i.p
The mice is then placed in the
centre of the maze facing one of the
Parameters Measured During Next 5
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
Anxiolytic effect indicated
Increase in the proportion of time spent in
open arms i.e.,
time in open arms/total time in open or
Increase in the proportion of entries into open
entries into open arms/total entries
into open or closed arms.
• Motor activity and open arm
exploratory time are registered. The
values of treated groups are
expressed as percentage of controls.
Benzodiazepines decrease motor
activity and increase open arm
• The method is rather time
consuming, but can be regarded as a
reliable measure of anxiolytic
activity. Computerized automatic
systems are available for elevated
plus maze, radial maze, Y-maze, and
T-maze and may help to overcome
LIGHT-DARK MODEL IN MICE AND RATS
Rodents have exploratory
Animals are placed in a
two chambered systems,
where they can freely move
between a brightly –lit open
field and a dark corner.
After the treatment with
an anxiolytic they show more
crossings between the two
The number of crossings
between the light and dark
sites is recorded.
The apparatus consists of
a dark and a light
divided by a photocell
A polypropylene animal
cage (44 ˣ 21ˣ 21 cm) is
darkened with black
spray over one-third of
A partition containing a
13cm(l) ,5 cm (h) opening
is used for separating the
dark one-third of the cage.
(1 mg/ kg , i.p.)
This case rests on an
activity monitor which
counts total locomotor
A electronic system consisting of four sets of
photocells across the partition.
It automatically counts
movements through the
partition and records the time
spent in the light and dark
They are treated 30 min before
the test drugs or vehicle given
i.p. placed in the cage and
observed for 10 min.
Experiments are conducted on
mice or rats.
Groups of 6-8 animals should
be used for each dose.
No. of crossings through the
partition between the light
and dark chambers compared
with total activity counts
during the 10 min.
Anxiolytics like Diazepam
increase locomotor activity
and no. of crossings.
Loco motor activity also
Dose response curves are plotted
& number of crossings through
the partition between the light
and the dark chamber are
compared with total activity
counts during the 10 min.
It has been reported that
anxiolytics like Diazepam and
produce a dose dependent
facilitatory effect whereas the
non anxiolytics are not effective
in this model
Using black and white test box studied the effects of
anxiolytic agents and reported an anxiolytic effect of
dopamine receptor antagonists.
Study the interaction of optical isomers modifying
rodent aversive behavior.
Study anxiogenic and anxiolytic activity.
Study animal models of anxiety and their relation to
serotonin interacting drugs.
[3H]-GABA receptor binding
Abnormalities in GABA system have been found in neurological and psychiatric
diseases like anxiety, epilepsy etc.
Radio labeled GABA is bound to synaptic membrane preparations of mammalian
Labeling of the synaptic receptor with 3H-GABA requires careful attention to
possible interference from non synaptic binding since 3H-GABA can also bind nonspecifically to plasma membranes. The most prominent of which is Na dependent
binding of GABA to brain membranes.
Sodium-independent binding of 3H-GABA has characteristics consistent with
the labeling of GABA receptors.
Therefore, the sodium-independent binding of 3H-GABA provides a simple and
sensitive method to evaluate compounds for GABA-mimetic properties.
0.32 M Sucrose-109.5 g of sucrose are dissolved in
distilled water and filled up to 1000 ml. The solution
is stored at 4 C
0.05 M Tris-maleate buffer (pH 7.1)
stock solutions are
are serially diluted to the
prior to the addition to the
incubation mixture. Final
concentrations are usually
from 2 10–8 to
1 10–5 M.
(specific activity approximately 40 Ci/mmol) is made up to a concentration
of 780 nmol in distilled water and 20 μl is added to each test tube (yielding a final
concentration of 15 nmol in the assay). Isoguvacine or muscimol is prepared by
dissolving 8.35 mg of isoguvacine or 6.40 mg of muscimol in 10 ml water. 20 μl of
these solutions when added to 1 ml of incubation medium give a final concentration of
0.1 mM isoguvacine or muscimol.
Rats (100–150 g) are decapitated, brain removed.
Homogenized in 15 vol. of ice-cold 0.32
Centrifuged at 1000 g for 10 min.
Discard pellet & recentrifuged supernatant, 20000 g, 20 min.
Discard supernatant & pellet is
resuspended in 15 vol. disttiled water
using a Tekmar homogenizer.
The suspension is centrifuged at 8000 g
for 20 min.
Collect Supernatant and resuspend the pellet’s soft, upper, buffy layer.
Centrifuged at 48000 g for 20 min. The final pellets
are resuspended (without homogenization) in 15 vol.
disttiled H20 and centrifuged at 48000 g for 20min.
Supernatant is discarded, and the centrifuge tubes
containing pellet are capped with parafilm and
stored frozen at –70 C.
The standard Na-independent 3H-GABA binding assay procedure, aliquots of the previously
frozen, Triton treated crude synaptic membranes are incubated in triplicate at 4 C for 5 min
in 0.05 M Tris-maleate buffer (pH 7.1) containing 15 nM 3H-GABA alone or in the presence
of 0.1 mM isoguvacine or muscimol, or the test drug. The procedure is as follows
1 ml of the 0.05 M Trismaleate homogenate
20 μl of 3H-GABA
20 μl of test drug or 20 ml of
0.1 mM isoguvacine or
Incubate at 4 C for 5 min, the reaction is terminated by centrifugation for
15 min at 5000 rpm.
Supernatant fluid is aspirated & pellet washed twice with 1ml of the Tris-maleate
2ml of liquiscint are added to each tube which is then vigorously vortexed.
The contents of tubes are transferred to scintillation vials, tubes rinsed with
an additional 2ml of cocktail.
An additional 6 ml of liquiscint are added to each scintillation vial.
The radioactivity is measured by liquid scintillation photometry.
Specific 3H-GABA binding is defined as the radioactivity
which can be displaced by a high concentration of unlabeled
GABA and is obtained by subtracting from the total bound
radioactivity the amount of radioactivity bound in the
presence of 0.1 mM isoguvacine. Results are converted to
percent of specifically bound 3H-GABA displaced by a given
concentration of test drug. IC50 values with 95% confidence
limits are then obtained by computer derived linear regression
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 5HT1B 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 5HT1A 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.
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
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.
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.
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
The KD value for [3H] 5-HT binding was found to
be 16.5 nM by Scatchard analysis of a receptor
Anxiety is a leading disease now a days. To understand the
treatment of various types of anxiety, it is necessary to have a
detailed knowledge about anxiolytics. All anxiolytics do not act
similar way to understand the pharmacology and to invent more
safe and potent drugs different screening models are very
important . So further research on various sreening models are
Nevertheless, the knowledge gathered from animal studies
undoubtedly valuable therapeutically in the future studies.
Drug Discovery and Evaluation by H.
Pharmacological Screening &
Evaluation by S.K.Gupta
Drugs The Straight Facts Anti –anxiety