Screening of Anxiolytic Agents in Drug Discovery

For MD Pharmacology

Dr. Advaitha M V

Anxiety Disorders

• Definition : 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.

Preclinical anxiety models

• The goal of anxiety models is to produce a form of abnormally elevated anxiety.

• It should more closely resemble, by definition, the pathological nature of human anxiety disorders.

• Anxiety models are Models that generate lasting or permanently heightened anxiety.

This can be achieved by acutely or chronically subjecting animals to stressors before testing.

OR identifying genetic populations (inbred and selectively bred strains) or engineering mutant mice with innate anxiety-like phenotypes.

• This approach has proven to be valuable for screening novel anxiolytics and testing the pharmaco selectivity of putative anxiolytics .

• Emerging genetic technology is the optogenetics. It will be integral to future basic anxiety research.

Optogenetics

• It is the combination of genetic and optical methods to control specific events in targeted cells of living tissue in the millisecond timescale.

• Optogenetic approaches have been used to map neural circuits in the amygdala that contribute to fear conditioning.

• However , in general , it is only simple tests or assays which transiently evoke an anxiety-like behaviour in rhodents is commonly employed.

The five main anxiety disorders with preclinical tests which resembles human anxiety

• Generalized anxiety disorder (GAD)----MOST OF THE TESTS

• Post-traumatic stress disorder (PTSD)……. FEAR CONDITIONED TESTS

• Panic disorder………… ELEVATED T MAZE MODEL and MOUSE DEFENSE TEST BATTERY

• Obsessive compulsive disorder (OCD)…… ???????

• Social anxiety disorder (SAD)…… SOCIAL INTERACTION MODEL

PRECLINICAL EVALUATION OF ANXIOLYTICS

• In-vitro Methods

• In-vivo Methods

• Our main focus here would be In-vivo.

In- vitro methods

• GABAA receptor binding

• GABAB receptor binding

• Benzodiazepine receptor: [3H]-flunitrazepambinding 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 following parameters are calculated:

• Total binding

• Non-specific binding

• Specific binding= total binding– non-specific binding.

In Vivo Methods

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 .

Elevated plus –maze (EPM) test This has widely validated to measure anxiety to rodents

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.

Evaluation :

• The number of entries into the open arms.

(Entry an arm is defined as animal placing all four paws onto the arm. All tests are taped by using a video camera)

• 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

Methodology :

• The rats (200–250 g body weight) are housed in pairs for 10 days prior to testing in the apparatus.

• During this time the rats are handled by the investigator on alternate days to reduce stress.

• 6 rats are taken for each group.

• Thirty min after i.p. administration of the test drug or the standard, the rat is placed in the center of the maze facing one of the enclosed arms.

• Duration of test is 5 min where all data is collected.

• Test should be conducted in sound proof room.

• After each test, the maze is carefully cleaned up with wet tissue paper (10% ethanol solution)

• Anxiolytic treatments do not by themselves increase explorationin the tests but they decrease the stress-induced inhibition of exploration behaviour

light-dark Exploration model

• 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 locomotoractivity after treatment with anxiolytics.

Methodology

• Test apparatus- light and dark chamber divided by photocell equipped zone.

• Polypropylene animal cage 44x21x21 cm, darkened with black spray over on-third of surface.

• Partition containing 13cm long x 5cm high opening separates the dark one third from bright two thirds of the cage

• Cage rests on an animex activity monitor, which counts the locomotor activity.

• Four sets of photocells across the partition, automatically counts movement through the partition and clocks time spent in light and dark

• Naïve male mice or rats are placed in cage

• Animals are treated 30 min before the experiment with test or vehicle intraperitoneally and are observed for 10 min.

• Groups of 6-8 animals are used for each dose.

• Evaluation: number of crossings through the partition between the light and dark chamber are compared with total activity counts during 10 min

• Critical assessment of the method: anxiolyticslike diazepam, pentobarbital, meprobamate, produce dose dependent increase in crossings

• Test is relatively simple with no painful stimuli

Social interaction in rats

• Purpose and rationale : In an unfamiliar and brightly lit environment, the normal social interaction of rats ( sniffing, nipping, grooming) is suppressed.

• Anxiolytics counteract this suppression.

Procedure:

• Male sprague-dawley rats (225-275g) are housed in groups of 5 animals

• Apparatus: perspex open-topped box (51x51x 20cm) with 17x17cm marked areas on the floor.

• One hour prior to the test, 2 naïve rats from separate housing cages are treated with test compound orally

• They are placed into the box (with 60W bright illumination 17cm above) and their behaviouris observed over a 10 min period.

• 2 types of behaviour can be noted:

• Social interaction between the animals is determined by timing the sniffing of partner, crawling under or climbing over partner and following partner.

• Exploratory motion is measured as the number of crossings of the lines marked on the floor of the test box.

• Six pairs are used for each dose

• Evaluation: values of treated partners are compared with the data from 6 pairs of untreated animals using single factor analysis of variance followed by Dunnett’s t-test

• Open-field Test

• This test, originally designed by Hall on rats.

• It consists of placing an animal in an unknown environment with surrounding walls, so as to observe a number of behaviour patterns, including

The tendency to stay on the periphery of the field without entering the centre (called thigmotaxis and often interpreted as anxious behaviour)

level of defecation and urination.

• The open field floor is divided into squares

• Animals are tested individually, always being placed in the same position.

• Duration of test is usually 5 min.

• Higher levels of anxiety should mainly lead to decreases in the ratio ‘number of squares visited in centre/number of squares visited on periphery.

• Anxiolytic agents should lead to increase in this ratio

Vogel water-lick conflict test

• This test is a well-known method used in rats, designed by Vogel et al.

• Recently, this test has been reported to successfully detect anxiolytic-like action of diazepam .

• In this test, thirsty animals gain water reward through a water spout, but at the expense of receiving a mild electric shock delivered to the tongue.

• Licking in untreated/controls is suppressed due to the conflict.

• Anxiolytics release this suppressed behaviourand decrease the conflict.

• Diazepam and pentobarbital produces a significant anti-conflict effect, which means that -

These drugs increases the number of electric shocks the mice received during the test session.

• But drugs like baclofen, buspirone, chlorpromazine and also haloperidol did not produce anti-conflict effects.

Resident-intruder aggression test

Purpose and rational: To study the effects of drugs in a test for offensive aggression, the isolation-induced resident intruder aggression model in the rat.

• Procedure: Sprague-Dawley rats weighing 250 to 450 g are housed in a light-dark (12L:12D)-, temperature (ca. 22°C)- and humidity (ca. 55%)-controlled room.

Resident male rats (about 450 g) are tested in their home cages for aggression against a smaller (250 g) male intruder

• They are treated by intraperitoneal injection of test drug or saline 15 min before the test.

• The resident female is removed from the cage 30 min prior to the start of the test period.

• After placing the intruder rat in the territorial cage, the behavior of the resident male is observed.

• The time until the first attack (in seconds), number of attacks, and duration of each attack (in seconds) are recorded for the next 15 min by a blind observer.

• Different behavioral elements are scored and grouped into 7 behavioral categories: offensive, exploration, social interest, inactivity, avoidance, body care, defense.

• Evaluation: Paired and unpaired t-tests are used for comparisons of means of absolute values

Other Anti- Aggressive tests

• FOOT- SHOCK INDUCED AGGRESSION IN MICE

• ISOLATION INDUCED AGGRESSION

• MATERNAL AGGRESSION IN RATS

Anticipatory anxiety in mice

• Purpose and rationale: when group-housed mice are removed one by one from their home cage, the last mice removed have always higher rectal temperatures than those removed first.

• This phenomenon is caused by anticipatory fear for an aversive event .

• Increase in temperature prevented by prior treatment with diazepam and buspirone

• Procedure: groups of 18 male albino swissmice ( 25-30g) are housed in constant room temperature and relative humidity, for atleast7days.

• Test drugs or standard (diazepam) are administered orally in various doses to groups of 18 mice prior to the test.

• Thirty min later, first 3 mice are removed from the cage, rectal temperature recorded by inserting a silicone lubricated thermistorprobe (2mm diam)

• Average temperature of these 3 mice is taken as basal value

• Mice number 4 to 15 are simply removed and again returned to the cage.

• Thereafter body temperature is determined in the remaining three animals.

• The difference of the mean value of these mice and the basal values is calculated as increase.

• Vehicle treated test groups display increases of 1.1 to 1.3 °C.

• The mean increase values of treated groups are compared by ANOVA statistics with the controls .

mCPP induced anxiety in rats• Purpose and rational: The metabolite of the

antidepressant drug trazodone is

1- (3-chlorphenyl)piperazine (= mCPP).

• It is 5- HT1C/1B/2C agonist.

• It has been shown to be anxiogenic both in man and in rats.

• The compound induces hypophagia and hypolocomotion, inhibits social interaction in rats, diminishes exploratory activity of rats in the open field test

• In the light-dark box test induces hyperthermia

• Antagonism against these symptoms has been proposed as a screening model for anxiolyticdrugs.

Novelty-suppressed feeding • Purpose and rational: Placing a hungry rat

into an unfamiliar environment with access to food results in a suppression of feeding behavior relative to the condition when the test environment is familiar.

• This effect has been termed hyponeophagiaand occurs because of the novelty of the test environment.

• The avoidance of novel foods is termed food neophobia

• Both hyponeophagia and food neophobiahave been assumed to measure emotionality or anxiety by eliciting a conflict situation arising from a fear of the novel setting and foods, and the drive to eat.

Video downloads• http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2

762911/bin/jove-22-1088.flv

• http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3731199/bin/jove-77-4367.mov

• http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3727206/bin/jove-76-50324.mov

Thank you