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1
UNDER GUIDANCE OF:

PRESENTED BY:

MRS. MEENU SINGH
ASST. PROFESSOR
DEPT OF PHARMACOLOGY
PESCP, BANGALORE

MISS SAYANTI SAU
I M. PHARM
DEPT OF
PHARMACOLOGY
PESCP, BANGALORE
OBJECTIVE
To understand the concepts behind Anxiolytics and to
reproduce the concepts of screening and clinical

evaluation of anxiolytics practically as well as
theoretically.
CONTENTS
o

o
o
o

o
o
o

o
o

Introduction
Physiological Vs pathological anxiety
Clinical presentation
Types of anxiety
Classification of anxiolytics
Pathophysiology of anxiety
Screening methods for anxiolytics
 In vitro methods
 In vivo methods
Conclusion
References

4
INTRODUCTION
•

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
nature

•

Pathological anxiety – needs
treatment
5
PHYSIOLOGICAL AND
PATHOLOGICAL ANXIETY

jitter
Stage-fright

Nervousness
Worrying

Panic attacks
Obsessions
compulsions

Flashbacks
nightmares
Pathological fear

6
TYPES OF ANXIETY DISORDER


GAD (Generalized Anxiety Disorder)



PD (Panic Disorder)



Agoraphobia



SAD (Social Anxiety Disorder)



Specific Phobia



Obsessive Compulsive Disorder



Post Traumatic Stress



Acute Stress
7
DIAGRAMMATIC VIEW OF ANXIETY
DISORDER

8
9
10
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.



COGNITIVE SYMPTOMS
Excessive Anxiety
Worries that are difficult to control
Poor concentration or mind going blank



PHYSICAL SYMPTOMS
Sleep disturbance
Irritability
Restlessness
10
PANIC DISORDER















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
occur.
Symptoms include Attacks usually last no more than about 10
minutes.
Symptoms
Intensive fear
Phobias
Dizziness
Racing heart
Perspiring
Shortness of breath
Tingling hands
Fear of dying or going crazy

11
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
anxious feelings.
Blushing
Profuse sweating
Trembling
Nausea
Shortness of breath
Racing heart
Difficulty talking
12
SPECIFIC DISORDER
 Is an intense fear of something that
posses little or no actual danger of a
object or situation.
 Some of the more common specific
phobias are

Centered around closed-in places

Heights

Escalators

Tunnels

Water

Flying dogs

Injuries involving blood

Injection

Insects

14
OBSESSIVE-COMPULSIVE DISORDER (OCD)
Obsessions (recurrent, intrusive and generally
distressing thoughts, images or feelings).
Compulsions(repetitive, ritualistic behaviors
aimed to alleviate obsessions).

14
POST TRAUMATIC STRESS DISORDER( PTSD)

•

Develops subsequent to experiencing or witnessing a traumatic
event

•

Symptoms (lasting at least 4-6 weeks )



Flash-backs



Disturbing memories



Nightmares



Intrusive images
15
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

Brain structures dealing with fear and
stress

16
Gaba receptor theory
GABA – inhibitory
neurotransmitter
• Inhibitory and
regulatory effects on
serotonin,
noradrenaline and
dopamine.
• GABAA receptor
involved in anxiety;
decreases neuronal
excitability.
• Patients suffering
from anxiety
disorders have less
level of GABA in
cortex.

Mechanism
• When GABA binds to the
GABAA receptor, the Clchannel opens, influx of
Cl• Causing hyper
polarization of
membrane & decreases
nerve cell excitability.
• The number of GABAA
receptors can change
with alterations in the
environment (e.g.,
chronic stress).

• The subunit expression
can be altered by
hormonal changes.

• In patients with GAD,
benzodiazepine binding
in left temporal lobe is
reduced.
17
18
Serotonin theory
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.,
cortex, amygdala,
hippocampus, and limbic
system).

Stimulation of the
postsynaptic 5-HT2A
receptors in the limbic
system results in anxiety and
avoidance behavior.

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
disorders.

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
release.

SSRIs – increases serotonin
levels post synaptically –
blocks symptoms of anxiety.

18
19
In response to threat or fearful
situations, the Locus cerulus
serves as an alarm center,
activating NE release and
stimulating the sympathetic and
parasympathetic nervous
systems.

Noradrenaline
theory

Chronic central noradrenergic
overactivity down- regulates
α2-adrenoreceptors in
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.
19
20
Classification of anxiolytic


BENZODIAZEPINES
• Diazepam
• Chlordiazepoxide
• Oxazepam
• Lorazepam
• Alprazolam



AZAPIRONES (NON-SEDATIVE)
• Buspirone
• Gepirone
• Ipsapirone



SEDATIVE ANTIHISTAMINICS
• Hyroxyzine



ANTIDEPRESSANTS
(useful in chronic anxiety and panic attacks)
• Fluoxetine



β-BLOCKERS
• Propranolol
21
SCREENING METHODS

22
2.SEROTONIN
RECEPTOR BINDING

1.GABAERGIC
COMPOUNDS
a.[3H]-GABA receptor
binding
b.GABAA receptor binding
c.GABAB receptor binding
d.Benzodiazepine receptor:
[3H]-flunitrazepam binding
assay

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
[3H]GR 65630

3. HISTAMINE H3
RECEPTOR
BINDING IN
BRAIN

In-vitro
methods
22
IN-VIVO METHODS
ANTICONVULSANT
ACTIVITY

ANTI-AGGRESSIVE
ACTIVITY

• Pentylenetetrazole
(Metrazol) induced
convulsions
• Strychnine-induced
convulsions
• Picrotoxin-induced
convulsions
• Isoniazid-induced
convulsions
• Yohimbine-induced
convulsions

• Foot-shock induced
aggression
• Isolation-induced
aggression
• Resident-intruder
aggression test
• Water competition
test
• Maternal aggression
in rats
• Rage reaction in cats

EFFECTS ON
BEHAVIOR
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•

Anti-anxiety test (light-dark model)
Anticipatory anxiety in mice
Social interaction in rats
Elevated plus maze test
Water maze test
Staircase 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
rats
Experimental anxiety in mice
mCPP induced anxiety in rats
Acoustic startle response in rats
Unconditioned conflict procedure
(Vogel test)
Novelty-suppressed feeding
Shock probe conflict procedure
Ultrasound induced defensive behavior
in rats
Anxiety/defense test battery in rats
Marmoset human threat test
Aversive brain stimulation

23
CONDITIONED
BEHAVIORAL RESPONSES

• Sidman avoidance
paradigm
• Geller conflict paradigm
• Conditioned defensive
burying in rats
• Taste aversion paradigm

EFFECTS ON THE
ENDOCRINE SYSTEM

• Plasma catecholamine
levels during and after
stress
• Plasma corticosterone
levels influenced by
psychotropic drugs

BENZODIAZEPINE
DEPENDENCE

• Benzodiazepine
tolerance and
dependence in rats

24
Elevated plus maze test

PRINCIPLE

The elevated plus maze
test has been extensively
used for the selective
evaluation of anxiolytic
drugs.
When the animals enter in
to open arm they show
fear like movement,
freeze and become
immobile.

Rodents have aversion
for high and open
space and prefer
enclosed arm and
therefore spend greater
amount of time in
enclosed arm.

ANXIOLYTIC

Advantages of this test
are :
1.simple fast and less
time consuming.
2.No prior training is
required.
For selective
identification of
anxiolytic and anxiogenic
drugs
Anxiolytics – decrease
anxiety – increase open
arm exploration time
Anxiogenics – decrease
open arm exploration
time.
25
REQUIREMENTS




Animal :
Rat(200-250 g)
Drugs:
Diazepam (1 mg/ kg ; i.p.)
Equipment : Elevated plus maze apparatus and a stop clock

26
Elevated plus maze apparatus
ELEVATED
PLUSMAZE

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.

28
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
route.

P
R
O
C
E
D
U
R
E

The mice is then placed in the
centre of the maze facing one of the
enclosed arms.
28
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
29
30
Anxiolytic effect indicated
parameter


Increase in the proportion of time spent in
open arms i.e.,
time in open arms/total time in open or
closed arms



Increase in the proportion of entries into open
arms i.e.,
entries into open arms/total entries
into open or closed arms.
31
EVALUATION

CRITICAL
ASESSMENT
OF THE
METHOD

• 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
exploratory time.

• 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
these difficulties.
32
LIGHT-DARK MODEL IN MICE AND RATS
Rodents have exploratory
activity
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
chambers
and
more
locomotor activity.
The number of crossings
between the light and dark
sites is recorded.
33
METHODS
The apparatus consists of
a dark and a light
chamber
which
are
divided by a photocell
equipped zone.
A polypropylene animal
cage (44 ˣ 21ˣ 21 cm) is
darkened with black
spray over one-third of
its surface.

Animal: Rats

A partition containing a
13cm(l) ,5 cm (h) opening
is used for separating the
dark one-third of the cage.

(200-250g)
Drugs: Diazepam

(1 mg/ kg , i.p.)

This case rests on an
activity monitor which
counts total locomotor
activity.
34
34
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
compartments.

M
E
T

They are treated 30 min before
the test drugs or vehicle given
i.p. placed in the cage and
observed for 10 min.

H

O
D

Experiments are conducted on
mice or rats.

Groups of 6-8 animals should
be used for each dose.
35
35
evaluation
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
monitored.

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

36
36
advantage
i.

Using black and white test box studied the effects of
anxiolytic agents and reported an anxiolytic effect of
dopamine receptor antagonists.

i.

Study the interaction of optical isomers modifying
rodent aversive behavior.

i.

Study anxiogenic and anxiolytic activity.

i.

Study animal models of anxiety and their relation to
serotonin interacting drugs.
37
[3H]-GABA receptor binding
PRINCIPLE


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
brain.



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.
38
PROCEDURE

REAGENTS

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)
TEST DRUGS-1mM
stock solutions are
initially prepared.These
are serially diluted to the
required concentrations
prior to the addition to the
incubation mixture. Final
concentrations are usually
from 2 10–8 to
1 10–5 M.

3H-GABA

(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.

39
TISSUE PREPARATION
Rats (100–150 g) are decapitated, brain removed.
Homogenized in 15 vol. of ice-cold 0.32
M sucrose.

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
40
stored frozen at –70 C.
40
ASSAY PROCEDURE
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
muscimol

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
buffer.
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.
41

41
EVALUATION
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
analysis.
42
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.
43
PROCEDURE


REAGENTS
1. Tris buffers, pH 7.7
2. 5-Hydroxy[G-3H] tryptamine creatinine sulfate
3. Serotonin creatinine sulfate
4. Spiroperidol
5. Test compounds

44
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.

45
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.
46
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
The KD value for [3H] 5-HT binding was found to
be 16.5 nM by Scatchard analysis of a receptor
saturation experiment.
47
conclusion
 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
required.

 Nevertheless, the knowledge gathered from animal studies
undoubtedly valuable therapeutically in the future studies.
48
REFFERENCES
Drug Discovery and Evaluation by H.
Gerhard Vogel
Pharmacological Screening &
Evaluation by S.K.Gupta
Internet source

Drugs The Straight Facts Anti –anxiety
Drugs
49
50
51

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Anxiolytics Screening Methods

  • 1. 1
  • 2. UNDER GUIDANCE OF: PRESENTED BY: MRS. MEENU SINGH ASST. PROFESSOR DEPT OF PHARMACOLOGY PESCP, BANGALORE MISS SAYANTI SAU I M. PHARM DEPT OF PHARMACOLOGY PESCP, BANGALORE
  • 3. OBJECTIVE To understand the concepts behind Anxiolytics and to reproduce the concepts of screening and clinical evaluation of anxiolytics practically as well as theoretically.
  • 4. CONTENTS o o o o o o o o o Introduction Physiological Vs pathological anxiety Clinical presentation Types of anxiety Classification of anxiolytics Pathophysiology of anxiety Screening methods for anxiolytics  In vitro methods  In vivo methods Conclusion References 4
  • 5. INTRODUCTION • 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 nature • Pathological anxiety – needs treatment 5
  • 6. PHYSIOLOGICAL AND PATHOLOGICAL ANXIETY jitter Stage-fright Nervousness Worrying Panic attacks Obsessions compulsions Flashbacks nightmares Pathological fear 6
  • 7. TYPES OF ANXIETY DISORDER  GAD (Generalized Anxiety Disorder)  PD (Panic Disorder)  Agoraphobia  SAD (Social Anxiety Disorder)  Specific Phobia  Obsessive Compulsive Disorder  Post Traumatic Stress  Acute Stress 7
  • 8. DIAGRAMMATIC VIEW OF ANXIETY DISORDER 8
  • 10. 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.  COGNITIVE SYMPTOMS Excessive Anxiety Worries that are difficult to control Poor concentration or mind going blank  PHYSICAL SYMPTOMS Sleep disturbance Irritability Restlessness 10
  • 11. PANIC DISORDER             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 occur. Symptoms include Attacks usually last no more than about 10 minutes. Symptoms Intensive fear Phobias Dizziness Racing heart Perspiring Shortness of breath Tingling hands Fear of dying or going crazy 11
  • 12. 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 anxious feelings. Blushing Profuse sweating Trembling Nausea Shortness of breath Racing heart Difficulty talking 12
  • 13. SPECIFIC DISORDER  Is an intense fear of something that posses little or no actual danger of a object or situation.  Some of the more common specific phobias are  Centered around closed-in places  Heights  Escalators  Tunnels  Water  Flying dogs  Injuries involving blood  Injection  Insects 14
  • 14. OBSESSIVE-COMPULSIVE DISORDER (OCD) Obsessions (recurrent, intrusive and generally distressing thoughts, images or feelings). Compulsions(repetitive, ritualistic behaviors aimed to alleviate obsessions). 14
  • 15. POST TRAUMATIC STRESS DISORDER( PTSD) • Develops subsequent to experiencing or witnessing a traumatic event • Symptoms (lasting at least 4-6 weeks )  Flash-backs  Disturbing memories  Nightmares  Intrusive images 15
  • 16. 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 Brain structures dealing with fear and stress 16
  • 17. Gaba receptor theory GABA – inhibitory neurotransmitter • Inhibitory and regulatory effects on serotonin, noradrenaline and dopamine. • GABAA receptor involved in anxiety; decreases neuronal excitability. • Patients suffering from anxiety disorders have less level of GABA in cortex. Mechanism • When GABA binds to the GABAA receptor, the Clchannel opens, influx of Cl• Causing hyper polarization of membrane & decreases nerve cell excitability. • The number of GABAA receptors can change with alterations in the environment (e.g., chronic stress). • The subunit expression can be altered by hormonal changes. • In patients with GAD, benzodiazepine binding in left temporal lobe is reduced. 17 18
  • 18. Serotonin theory 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., cortex, amygdala, hippocampus, and limbic system). Stimulation of the postsynaptic 5-HT2A receptors in the limbic system results in anxiety and avoidance behavior. 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 disorders. 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 release. SSRIs – increases serotonin levels post synaptically – blocks symptoms of anxiety. 18 19
  • 19. In response to threat or fearful situations, the Locus cerulus serves as an alarm center, activating NE release and stimulating the sympathetic and parasympathetic nervous systems. Noradrenaline theory Chronic central noradrenergic overactivity down- regulates α2-adrenoreceptors in 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. 19 20
  • 20. Classification of anxiolytic  BENZODIAZEPINES • Diazepam • Chlordiazepoxide • Oxazepam • Lorazepam • Alprazolam  AZAPIRONES (NON-SEDATIVE) • Buspirone • Gepirone • Ipsapirone  SEDATIVE ANTIHISTAMINICS • Hyroxyzine  ANTIDEPRESSANTS (useful in chronic anxiety and panic attacks) • Fluoxetine  β-BLOCKERS • Propranolol 21
  • 22. 2.SEROTONIN RECEPTOR BINDING 1.GABAERGIC COMPOUNDS a.[3H]-GABA receptor binding b.GABAA receptor binding c.GABAB receptor binding d.Benzodiazepine receptor: [3H]-flunitrazepam binding assay 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 [3H]GR 65630 3. HISTAMINE H3 RECEPTOR BINDING IN BRAIN In-vitro methods 22
  • 23. IN-VIVO METHODS ANTICONVULSANT ACTIVITY ANTI-AGGRESSIVE ACTIVITY • Pentylenetetrazole (Metrazol) induced convulsions • Strychnine-induced convulsions • Picrotoxin-induced convulsions • Isoniazid-induced convulsions • Yohimbine-induced convulsions • Foot-shock induced aggression • Isolation-induced aggression • Resident-intruder aggression test • Water competition test • Maternal aggression in rats • Rage reaction in cats EFFECTS ON BEHAVIOR • • • • • • • • • • • • • • • • • • • • • Anti-anxiety test (light-dark model) Anticipatory anxiety in mice Social interaction in rats Elevated plus maze test Water maze test Staircase 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 rats Experimental anxiety in mice mCPP induced anxiety in rats Acoustic startle response in rats Unconditioned conflict procedure (Vogel test) Novelty-suppressed feeding Shock probe conflict procedure Ultrasound induced defensive behavior in rats Anxiety/defense test battery in rats Marmoset human threat test Aversive brain stimulation 23
  • 24. CONDITIONED BEHAVIORAL RESPONSES • Sidman avoidance paradigm • Geller conflict paradigm • Conditioned defensive burying in rats • Taste aversion paradigm EFFECTS ON THE ENDOCRINE SYSTEM • Plasma catecholamine levels during and after stress • Plasma corticosterone levels influenced by psychotropic drugs BENZODIAZEPINE DEPENDENCE • Benzodiazepine tolerance and dependence in rats 24
  • 25. Elevated plus maze test PRINCIPLE The elevated plus maze test has been extensively used for the selective evaluation of anxiolytic drugs. When the animals enter in to open arm they show fear like movement, freeze and become immobile. Rodents have aversion for high and open space and prefer enclosed arm and therefore spend greater amount of time in enclosed arm. ANXIOLYTIC Advantages of this test are : 1.simple fast and less time consuming. 2.No prior training is required. For selective identification of anxiolytic and anxiogenic drugs Anxiolytics – decrease anxiety – increase open arm exploration time Anxiogenics – decrease open arm exploration time. 25
  • 26. REQUIREMENTS    Animal : Rat(200-250 g) Drugs: Diazepam (1 mg/ kg ; i.p.) Equipment : Elevated plus maze apparatus and a stop clock 26
  • 27. Elevated plus maze apparatus ELEVATED PLUSMAZE 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. 28
  • 28. 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 route. P R O C E D U R E The mice is then placed in the centre of the maze facing one of the enclosed arms. 28
  • 29. 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 29
  • 30. 30
  • 31. Anxiolytic effect indicated parameter  Increase in the proportion of time spent in open arms i.e., time in open arms/total time in open or closed arms  Increase in the proportion of entries into open arms i.e., entries into open arms/total entries into open or closed arms. 31
  • 32. EVALUATION CRITICAL ASESSMENT OF THE METHOD • 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 exploratory time. • 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 these difficulties. 32
  • 33. LIGHT-DARK MODEL IN MICE AND RATS Rodents have exploratory activity 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 chambers and more locomotor activity. The number of crossings between the light and dark sites is recorded. 33
  • 34. METHODS The apparatus consists of a dark and a light chamber which are divided by a photocell equipped zone. A polypropylene animal cage (44 ˣ 21ˣ 21 cm) is darkened with black spray over one-third of its surface. Animal: Rats A partition containing a 13cm(l) ,5 cm (h) opening is used for separating the dark one-third of the cage. (200-250g) Drugs: Diazepam (1 mg/ kg , i.p.) This case rests on an activity monitor which counts total locomotor activity. 34 34
  • 35. 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 compartments. M E T They are treated 30 min before the test drugs or vehicle given i.p. placed in the cage and observed for 10 min. H O D Experiments are conducted on mice or rats. Groups of 6-8 animals should be used for each dose. 35 35
  • 36. evaluation 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 monitored. 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 36 36
  • 37. advantage i. Using black and white test box studied the effects of anxiolytic agents and reported an anxiolytic effect of dopamine receptor antagonists. i. Study the interaction of optical isomers modifying rodent aversive behavior. i. Study anxiogenic and anxiolytic activity. i. Study animal models of anxiety and their relation to serotonin interacting drugs. 37
  • 38. [3H]-GABA receptor binding PRINCIPLE  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 brain.  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. 38
  • 39. PROCEDURE REAGENTS 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) TEST DRUGS-1mM stock solutions are initially prepared.These are serially diluted to the required concentrations prior to the addition to the incubation mixture. Final concentrations are usually from 2 10–8 to 1 10–5 M. 3H-GABA (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. 39
  • 40. TISSUE PREPARATION Rats (100–150 g) are decapitated, brain removed. Homogenized in 15 vol. of ice-cold 0.32 M sucrose. 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 40 stored frozen at –70 C. 40
  • 41. ASSAY PROCEDURE 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 muscimol 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 buffer. 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. 41 41
  • 42. EVALUATION 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 analysis. 42
  • 43. 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. 43
  • 44. PROCEDURE  REAGENTS 1. Tris buffers, pH 7.7 2. 5-Hydroxy[G-3H] tryptamine creatinine sulfate 3. Serotonin creatinine sulfate 4. Spiroperidol 5. Test compounds 44
  • 45. 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. 45
  • 46. 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. 46
  • 47. 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 The KD value for [3H] 5-HT binding was found to be 16.5 nM by Scatchard analysis of a receptor saturation experiment. 47
  • 48. conclusion  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 required.  Nevertheless, the knowledge gathered from animal studies undoubtedly valuable therapeutically in the future studies. 48
  • 49. REFFERENCES Drug Discovery and Evaluation by H. Gerhard Vogel Pharmacological Screening & Evaluation by S.K.Gupta Internet source Drugs The Straight Facts Anti –anxiety Drugs 49
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