SCREENING METHODS OF ANTI ANXIETY DRUGS

Dr. Jitendra AgrawalSecond year resident

Introduction

o 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

Physiological and Pathological Anxiety

Normal Pathological

• Panic attacks

• Obsessions,

compulsions

• Flashbacks, nightmares

• Pathological fear

• Jitter

• Stage-fright

• Nervousness

• Worrying

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 4

Noradrenaline theory

o ANS of anxious patients- hypersensitive to stimuli.

o Locus coeruleus – activates epinephrine release

o Anxiogenics – stimulate locus coeruleus firing

o Anxiolytics- inhibits locus coeruleus firing and decrease noradrenaline activity.

GABA Receptor Theory

o GABA – inhibitory neurotransmitter in brain.

o Has inhibitory and regulatory effects on serotonin, noradrenaline and dopamine.

o GABAA receptor involved in anxiety; decreases neuronal excitability

o Patients suffering from anxiety disorders have less level of GABA in cortex.

Serotonin Theory

• Abnormalities in serotonin function i.e., release and uptake plays role in anxiety.

• Greater serotonin activity – reduces norepinephrine activity in locus cerulus.

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

Classification of anxiolytics

Benzodiazepines alprazolam, chlordiazepoxide , diazepam Azapirones Buspirone, Ispapirone, gepirone SSRI Citalopram ,Escitalopram ,Fluoxetine Beta blockers Propranalol Sedative antihistaminic Hydroxyzine

Screening methods of anxiolytics

ANIMAL MODEL FOR ANXIETY

• Must add new insight or new dimension to the process of human anxiety

• Should reproduce behavioural and pathological features of anxiety

• Should allow investigation for neurobiological mechanism that are not easily amenable to study in man

• Permit reliable evaluation of anxiolytic drugs as well as anxiogenic drugs

• All animal model of anxiety are common in

– Induced “fear” as an analogy to human anxiety and to give them a basic construct validity–The degree to which they can be taken as

model of human anxiety is based upon their response to BZD’s

BEHAVIOURAL MODEL• Exteroceptive stimuli model– Novel environment

• Open field test• Elevated plus maze• Staircase exploration• Black and white test box• Mirrored chamber

– Conditioned aversive procedures– Conflict Procedures

• Geller seifter’s conflict test• Vogel’s conflict test

– Escape avoidance procedure– Social interaction test

• Interoceptive stimuli model– Electrical stimulation of brain– Pharmacological manipulation (drug discrimination

test)• Caffine induced anxiety• Yohimbine induced anxiety• Flumazenil induced anxiety• Pentylenetetrazole induced anxiety• Amphetamine induced anxiety• Cacaine induced anxiety

– Foot shock induced agression– Isolation induced agression

Open field test• This behavioral model is base on the induction of anxiety

state – ambulation or freezing by exposing the animal to a highly novel field environment of a high sound and light

• As a result their exploratory behavior is inhibited• Open field apparatus consist of circular arena with high

sound and light sources• Simple sterotypy: when animal move from one segment

to another – one ambulation score• Complex sterotypy: when animal stands on its hind

limbs- one rearing score• Urination• defecation

Elevated Plus Maze Test

o Most widely used method; male mice used

o Anxiolytics –decrease anxiety

o increase open arm exploration time

Elevated plus maze

o 2 open arms and 2 closed arms of 50 ˣ 10 ˣ 40cm dimensions

o Open roof arrangement

o Two open arms are opposite to each other.

o Maze elevated at 50cm height.

The rats weighing around 200g - 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.

The rat is then placed in the centre of the maze facing one of the enclosed arms.

Parameters Measured During Next 5 minutes:

o time spent in the open arms

o entries into the open arms

o time spent in the closed arms

o entries into the closed arms

o total arm entries

Anxiolytic effect indicated by:

o increase in the proportion of time spent in open arms

o increase in the proportion of entries into open arms

Staircase exploration

• When introduced into a novel environment, rodents experience a conflict between anxiety and exploratory behavior manifested by increased vigilance and behavioral activity

• Staircase climibing reflects exploratory or locomotor activity

• Rearing behaviour is an index of anxiety state• The no. of rearing and steps climbed to be recorded

for period of 5 minutes• Decrease in rearing behaviour and increase in steps

climbed is characterisation of anxiolytic effect

male mice (Charles River strain) with a weight between18 and 24 g are used

Test drug administered 30min prior to experimentation by s.c. route.

The animal is placed on the floor of the box with its back to the staircase.(staircase is composed of five identical steps 2.5 cm high, 10 cm wide )

Light – dark model

o Animals placed in 2 chambered systems, where they can freely move between a bright and dark compartment

o Number of crossings between the light and dark sites is recorded.

o Apparatus - a dark and a light chamber divided by a photocell equipped zone.

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

male mice with a weight between18 and 24 g are used

Test drug administered 30min prior to experimentation by i.p. route.

The animal is placed in the cage

o No. of crossings through the partition

between the light and dark chambers

compared with total activity counts during the

10 min.

o Loco motor activity also monitored.

o Anxiolytics increase locomotor activity and

no. of crossings.

Mirrored chamber

• Novel stimulation evokes both exploration and anxiety and therefore generates approach avoidance conflict behaviour

• It is hypothesised that distortion of readily traversed environment by a chamber of mirror might produce aversion to entry

• Mice are exposed to the chamber of mirror • Extended latency to enter the chamber of mirror used as a

parameter for anxiety analogy• Anxiolytics reduce this latency in dose dependent manner.

Geller seifter’s conflict test

• Conflict is produced by availability of reinforcement with punishment

• Experimentally induced conflict by punishing food rewarded behavior has been used to differentiate between various psychoactive drugs by Geller and Seifter

Geller seifter’s conflict test

• Male albino rats with a body weight of 300–400 g are housed individually.

• They are food deprived until the body weight is gradually reduced by approximately 20% of original and it is maintained at this level by restricted food diet.

• Conditioning is carried out with a flash of light, a single lever, a liquid dripper, and a grid-floor connected to a shocker

• The animals are trained to lever press for the milk reward in two distinct response-reward sections.

Geller seifter’s conflict test

• Anxiety or “conflict” segment (3 min)• a dipper of milk is delivered in response to each lever

press (continuos reinforcement schedule =CRF),

• accompanied by aversive foot-shock through the grid floor

• Creates a conflict between milk reward and the a

painful foot shock

Geller seifter’s conflict test

• “nonconflict” segment (15 min)• lever presses produce a drop of milk only at

variable intervals of time from 60 to 210 s with an average reward of once per 2min

• No shocks are administered

Geller seifter’s conflict test

• Four cycles of 15 min nonshock variable interval segments followed by a three minute CRF-conflict period phase

• The total number of lever presses during the conflict periods (CRF) and the non-conflict periods (VI) are counted.

• Anxiolytic effect :An increase of lever presses in the conflict periods

Vogel Lick-conflict (Vogel Punished Drinking)

• Rats are deprived of water for 48 hrs

• Then placed in chamber with water source

• sprague dawley rats are used in this model

a water bottle with metal drinking tube is fitted to the animal housing

Electric circuit is connected between drinking tube and floor of cage.

i.p injection of drugs are given; 30min later rats placed in cage and allowed to drink water and shock given after 20 licks

For 3minutes next shocks are given for every 12t

h

lick

No. of shocks delivered in 3min noted for each animal, no. of shocks received after treatment noted

o number of accepted punishments (electric shock) are measured during conflict period

Anxiolytic effect :o increase in the accepted shocks.

Social interaction test• In unfamiliar and

brightly lit environment, social interactions are suppressed.

• Anxiolytics –counteract this suppression.

• Animal used : male sprague dawley rats.

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Electric stimulation of brain

• Electrical stimulation of brain aversive areas, in particular the midbrain central gray, induces defensive reaction and/or flight behavior in several species

• Used as an animal model of anxiety or of panic attack.

• Most studies used intracerebral microinjections of neurotransmitters, their agonists and antagonists to elucidate the mechanisms of aversive or antiaversive effects

Foot shock induced aggression

• GABA is involved in control and agrgressive behaviour of animals.

• Benzodiazepines are thought to produce anxiolytic effects by binding to a specific high affinity site on GABA-A receptor

• So aggressive and fighting behavior has been extended for GABAergic anxiolytic drug screening.

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Isolation induced aggressiono Male mice

subjected to isolation develop aggressive behavior towards other animals of same sex.

Male Mice kept isolated for 6weeks & aggressive behavior tested.

Male mice accustomed to live together placed in cage of isolated mice for 5minutes

Isolated mice attacks intruder- aggressiveness observed

Drugs given to isolated mice s.c or orally; aggressive behavior tested at 60, 120,240 minutes (oral route)

If drug active- decrease in aggressivenessAttenuation of fighting reaction

In vitro methods GABAA receptor binding

GABAB

receptor binding

Benzodiazepine receptor: [3H]-flunitrazepam binding assay

Serotonin (5-HTIA) receptor: binding of [3H]-8-hydroxy-2-(di-n-propylamino)-tetralin ([3H]-DPAT)

Serotonin (5-HTIB) receptors in brain: binding of [3H]5-hydroxytryptamine ([3H]5-HT)

conclusion

• Anxiety disorder is a psychological disorder and associated with

stress, tension, fear and threat about future.

• The pathophysiology of anxiety disorder is not fully understood

and hence improper diagnosis leads to increase morbidity and

mortality rates.

• Development of the screening methods resulted in introduction of many new anxiolytic agents.

• In future aspects more reliable and easy models for screening are to be developed.

References

• Hand book of experimental pharmacology. S.K. kulkarni. 3 rd edition

• Drug Discovery an Evaluation:Pharmacological Assays. H. Gerhard Vogel. 3rd edition

• Shenoy et al. Preclinical evaluation of anxiolytic agents: an overview. Journal of Pharmaceutical Research and Opinion.2011 ;1(2):7-22.