Utkarsh AlokFirst year ,M.pharm

Department of PharmacologyRIPS,BERHAMPUR

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SCREENING OF ANXIOLYTIC ACTIVITY

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Anxiety is an emotional state commonly caused by the perception of real or perceived danger that threatens the security of an individual.

Episodes of mild anxiety are common life experiences and do not warrant treatment. However, the symptoms of severe, chronic, debilitating anxiety may be treated with antianxiety drugs (sometimes called anxiolytic or minor tranquilizers) and/or some form of behavioral or psychotherapy.

Introduction

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Physiological and Pathological Anxiety

NORMAL•Jitter•Stage fright•Nervousness•Worrying

Pathological•Panic attack •Obsession,compulsion•Flashbacks,nightmares•Pathological fears

CLINICAL CLINICAL PRESENTATIONPRESENTATION

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ANXIETY DISORDER I

GAD (Generalized Anxiety Disorder)

PD (Panic Disorder)

Agoraphobia (fear of open or public places)

SAD (Social Anxiety Disorder)

Specific Phobia ANXIETY DISORDER IIObsessive Compulsive DisorderPost Traumatic StressAcute Stress

GENERALISED ANXIETY DISORDERGENERALISED ANXIETY DISORDER

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PHYSIOLOGIC & COGNITIVE SYMPTOMS Excessive AnxietyWorries that are difficult to control,poor concentration or

mind going blank PHYSICAL SYMPTOMS Sleep disturbance IrritabilityRestlessness

PANIC DISORDERPANIC DISORDER

•Unexpected panic attacks associated with physiological symptoms of autonomic nervous system.•Intensive fear•Phobias•Avoidance of places and situations where panic attacks occurred.

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. SOCIAL ANXIETY DISORDERSOCIAL ANXIETY DISORDER

•Intense, irrational and persistent fear of being negatively evaluated in at least one social or performance situation.•Blushing is the principal physical indicator SPECIFIC DISORDERSPECIFIC DISORDER

Marked and persistent fear of a circumscribed object or situation (e.g., insects, heights, or injections).

POST TRAUMATICSTRESSPOST TRAUMATICSTRESS DISORDER( PTSD)DISORDER( PTSD)

•Develops subsequent to experiencing or witnessing a traumatic event.•Symptoms (lasting at least 4-6 weeks ) : flash-backs, memories, nightmares

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OBSESSIVE-COMPULSIVE DISORDER (OCD)OBSESSIVE-COMPULSIVE DISORDER (OCD)

• Obsessions (recurrent, intrusive and generally distressing thoughts, images or feelings).

• Compulsions(repetitive,ritualistic behaviors aimed to alleviate obsessions

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Modulation of normal and pathologic anxiety states is associated with multiple regions of the brain and abnormal function in several neurotransmitter systems,which includes:-norepinephrine (NE) γ –aminobutyric acid (GABA) serotonin (5-HT)

Neuroanatomic models of fear (i.e., the response to danger) and anxiety (i.e., the feeling of fear that is disproportionate to the actual threat) include some key brain areas.Amygdala,(a temporal lobe structure,) role in the assessment of fear stimuli and learned response to fear.The locus ceruleus (LC), located in the brain stem, is the primary NE-containing site in the brain, with widespread projections to areas responsible for implementing fear responses .The hippocampus is integral in the consolidation of traumatic memory.The hypothalamus is the principal area for integrating neuroendocrine and autonomic responses to a threat.

PATHOPHYSIOLOGY

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CLASSIFICATION OF ANXIOLYTICS AND HYPNOTICS DRUGS

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SCREENING METHODS SCREENING METHODS

SCREENING METHODS OF ANXIETY I

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Methods based on unconditioned (spontaneous) response:

Exploratory activity elevated plus-maze light-dark (two compartment box)

Social behavior social interaction

Methods based on conditioned (learned) responseConflict models

Vogel punished drinking

SCREENING METHODS OF ANXIETY 2

Normal (adaptive) anxietyElevated plus-maze Social interactionLight-darkMarble burying

SCREENING METHODS OF ANXIETY 2 contd..

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Stress-induced anxietyVogel lick conflict

Pathological anxiety Neurochemically-induced anxiety.mcpp induced anxiety in rats.

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ANTI-ANXIETY TEST

LIGHT-DARK MODEL IN MICE AND RATS

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Purpose and Rationale 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.

METHODOLOGY: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. A partition containing a 13cm(l) ,5 cm (h) opening is used for

separating the dark one-third of the cage. This case rests on an activity monitor which counts total

locomotor activity.

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Another 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.

Experiments are conducted on mice or rats.

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

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

ELEVATED PLUS MAZE TESTELEVATED PLUS MAZE TEST

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PRINCIPLE: proposed for selective identification of anxiolytic and anxiogenic drugs. Anxiolytic compounds,by decreasing anxiety, increase the open arm exploration time; anxiogenic compounds have the oppositeeffect.• Dose response curves are plotted & number of crossings through the partition between the open and the closed arm are compared with total activity counts during the 10 min.• It has been reported that anxiolytics like diazepam and meprobamate produce a dose dependent facilitatory effect whereas the non anxiolytics are not effective in this model.

•Two open and two enclosed arms (50 ˣ 20 ˣ 40cm dimensions).•Open roof arrangement •Two open arms are opposite to each other

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The rats weighing around 200g are housed in pairs for 10 days prior to testing.

Six rats are taken for each test group. The test drug is administered 30 min prior to experimentation by i.p route. The rat is then placed in the center of the maze facing one of the enclosed

arms.

PROCEDURE:

Parameters Measured During Next 5minutes:•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 by:•increase in the proportion of time spent in the open arms (time in open arms/total time in open or closed arms)•increase in the proportion of entries into the open arms (entries into open arms/total entries into open or closed arms).

SOCIAL INTERACTIONSOCIAL INTERACTION

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Procedure :Male Sprague-Dawley rats (225–275 g

body weight) are housed in groups of 5 animals.

Apparatus used is Perspex open-topped

box (51 × 51 cm and 20 cm high) with 17 × 17 cm marked areas on the floor.

Two naive rats from separate housing cages are treated with the test compound orally 1 hr before the test.

They are placed into the box (with 60 W bright illumination 17 cm above) and their behavior is observed over a 10-min period by remote video recording

Source of anxiety: Presence of an unfamiliar social partner in a brightly lit environment.

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Parameters measured : exploration, sniffing, rearing, social contacts, sexual behavior, attack, fighting, biting ,defensive posture, immobility and climbing over the partner.

Anxiolytic effect:In an unfamiliar and brightly lit environment, the normal social interaction of rats is suppressed. Anxiolytics counteracts this suppression.

EVALUATION:The 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.

MARBLE BURYING MARBLE BURYING METHOD METHOD

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Source of anxiety : Presence of an unfamiliar objects (potential source of danger)Parameters measured:duration of burying of marbles.Purpose and rationale:The rats develops a peculiar behavior when shocked through a stationary prod by burying the shock source.

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PROCEDURE Male adult rats (250-400g) are used.Test performed in a chamber, covered with 5 cm of a commercial bedding material.In the centre a shock-prod is inserted through a small hole and is buried in the bedding material. Electric current is provided through the wires wrapped around the prod-the behavior of each rat is monitored for 15 minutes.

The rat is injected with the test drug i.p and placed into the test chamber.

When the rat first touches the prod with its forepaw, it receive a brief electric shock (1mA) which typically elicits a flinch away from the prod.

Afterwards, the rat moves towards the prod pushing the bedding material with its forepaws-the duration of burying is recorded.

Anxiolytic effect:Anxiolytics shortens the burying behavior dose-dependently at low shock intensity (1 mA).

VOGEL LICK-CONFLICT (VOGEL PUNISHED DRINKING)

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Source of anxiety: stressful situation (48h water deprivation) conflict between thirst and punishment after drinking Parameters measured: number of accepted punishment (electric shock).Anxiolytic effect: statistically significant increase in the accepted shocks.PROCEDURE :•The apparatus is a clear Plexiglas box (38 × 38 cm) with a black Plexiglas compartment (10 × 10.5 cm) attached to one wall and an opening from the large box to the small compartment, with a stainless-steel grid floor.

•A water bottle with a metal drinking tube is fitted to the outside of the small compartment, so that the tube extends into the box at a height 3 cm above the grid.

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•A water bottle with a metal drinking tube is fitted to the outside of the small compartment, so that the tube extended into the box at a height 3 cm above the grid.•A drinkometer circuit is connected between the drinking tube and the grid floor of the apparatus, so that the rat completes the circuit whenever it licks the tube. •Shock is administered to the feet of the animal by switching the connections to the drinking tube and grids from the drinkometer to a shocker.• Thirty min after intraperitoneal injection, the naive adult rat is placed in the apparatus and allowed to find the drinking tube and to complete 20 licks before shock (available at the tube for 2 s)is administered. •The rat controls shock duration by withdrawing from the tube. •A 3-min timer is automatically started after the termination of the first shock. During the 3-min period, shocks are delivered following each twentieth lick.• The number of shocks delivered during the 3-min session is recorded for each animal. EVALUATION:The number of shocks received after treatment is compared with untreated animals. Benzodiazepines increase dose-dependent the number of shocks.

mCPP INDUCED ANXIETY IN mCPP INDUCED ANXIETY IN RATSRATS

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mCPP - metabolite [ 1-(3-chlorphenyl) piperazine] of antidepressant drug trazodone.

mCPP induces hypophagia and hypolocomotion , inhibits social interaction in rats, diminishes exploratory activity of rats in the open field test and in the light-dark box test etc.

Antagonism of these symptoms has been used for the screening of anxiolytic drugs.

Parameters measured : time spent in both side (horizontal, vertical activity) frequency of motion, number of transition

Anxiolytic effect : statistically significant increase in parameters measured in the light/dark box or in number of transition

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PROCEDURE:Male Sprague Dawley rats

(200-250g) are housed in groups of six exposed to 12 h light/dark cycle with free access to food and water.

Test compound or vehicle are administered orally 1h or i.p 30 min before the locomotion test.

mCPP is injected i.p. in a dose of 7 mg/kg 20 min before the test.

The animals are placed individually in an automated locomotor activity cages and locomotion is recorded for 10 min.

Anxiolytic effect : Disinhibition of locomotion.

IN-VITRO IN-VITRO METHODMETHOD

Benzodiazepine receptor: [3H]-flunitrazepam binding assay

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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 antianxiety drugs

PROCEDURE:Reagents

[Methyl-3H]-Flunitrazepam. Clonazepam HCl

Tissue preparationMale 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.

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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 μl vehicle 0.1 mM Clonazepam 300 μl tissue suspension

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.

EVALUATION The percent inhibition at each drug concentration is the mean of

triplicate determinations. IC50 calculations are performed using log-probit analyses.

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SCREENING OF ANTISTRESS ACTIVITY

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STRESSStress represents reaction of a body to stimuli that tends to disturb its normal physiological equilibrium or homeostasis.

Common symptoms of stress include problems with sleep, depression, anxiety, fatigue, and lethargy.

BEHAVIORAL DEPRESSIONGeneralized and chronic stress induces behavioral depression.Methods used to assess depressive behavior are:•SWIM STRESS INDUCED ‘BEHAVIORAL DESPAIR’•LEARNED “HELPLESSNESS” TEST

SWIM STRESS INDUCED "BEHAVIORAL DESPAIR"

Purpose and Rationale: Rats are forced to swim in a

restricted space from which they cannot escape induce a characteristic behavior of immobility

This behavior is a state of despair which can be reduced by antistress agents.

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METHODMale Sprague- Dawley rats(160-180g)are individually forced to swim inside a vertical cylinderRats are initially highly activeAfter 2-3 min activity begins to subside immobility or floating After 5-6 min, immobility reaches a plateau

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After 15 min animals are removed, dried& returned to cages.

Again after 24 hour the test is repeated and immobility is measured during a 5 minute test .

Animal is judged to immobile when animal remains floating passively in water with head in upright position and nose just above surface.

The test drug is administered 1hr prior to the test Evaluation:Duration of immobility is measured in control and test.A decrease in immobility time in comparison to the control is measured.

Antistress effect: Statistically significant decrease in immobility.

LEARNED “HELPLESSNESS” TEST

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PURPOSE AND RATIONALE Animal is exposed to inescapable & unavoidable electric

shock in one situation later fail to escape shock in a different situation when escape is possible

METHOD Male Sprague –Drawly rats(300g) is used. Learned helplessness is produced by exposure to electric

shock 0.7mA; 10sof shock/min repeatedly given for 1 hr. A box with grid floor is taken.At 20cm height above the floor ,a platform can be inserted through one side wall to allow jump up escape.The platform is not available during training At the beginning of trial, after the appropriate treatment with the drug, the platform is pushed in the box and a 0.4mA shock initiated. If an escape response occur, animal is allowed to be on the platform for 10 sec and then returned to the grid floor. 10 trials with an interval for 20 sec are given.EVALUATION:A drug is considered to be effective if the animal helplessness is reduced and the number of failures to escape is decreased.

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