Slide 1

SCREENING METHODS OF ANTI ANXIETY DRUGSDr. Jitendra AgrawalSecond year residentIntroductionAnxiety 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 AnxietyNormalPathologicalPanic attacksObsessions, compulsionsFlashbacks, nightmaresPathological fear

JitterStage-frightNervousnessWorrying

Pathophysiology of anxietyNeurotransmitters like GABA, noradrenaline, serotonin abnormalities anxiety

Amygdala, temporal lobe, hippocampus and hypothalamus - involved in anxeity

Neurochemical theories :

Noradrenaline theorySerotonin theoryGABA receptor theory

4Noradrenaline theoryANS of anxious patients- hypersensitive to stimuli.

Locus coeruleus activates epinephrine release

Anxiogenics stimulate locus coeruleus firing

Anxiolytics- inhibits locus coeruleus firing and decrease noradrenaline activity.

GABA Receptor TheoryGABA inhibitory neurotransmitter in brain.

Has 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.Serotonin TheoryAbnormalities 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 anxiolyticsBenzodiazepines alprazolam, chlordiazepoxide , diazepamAzapironesBuspirone, Ispapirone, gepironeSSRICitalopram ,Escitalopram ,FluoxetineBeta blockersPropranalolSedative antihistaminicHydroxyzine Screening methods of anxiolyticsANIMAL MODEL FOR ANXIETYMust add new insight or new dimension to the process of human anxietyShould reproduce behavioural and pathological features of anxietyShould allow investigation for neurobiological mechanism that are not easily amenable to study in manPermit reliable evaluation of anxiolytic drugs as well as anxiogenic drugs

10All animal model of anxiety are common in

Induced fear as an analogy to human anxiety and to give them a basic construct validityThe degree to which they can be taken as model of human anxiety is based upon their response to BZDsBEHAVIOURAL MODELExteroceptive stimuli modelNovel environmentOpen field testElevated plus mazeStaircase explorationBlack and white test boxMirrored chamberConditioned aversive proceduresConflict ProceduresGeller seifters conflict testVogels conflict testEscape avoidance procedureSocial interaction testInteroceptive stimuli modelElectrical stimulation of brainPharmacological manipulation (drug discrimination test)Caffine induced anxietyYohimbine induced anxietyFlumazenil induced anxietyPentylenetetrazole induced anxietyAmphetamine induced anxietyCacaine induced anxietyFoot shock induced agressionIsolation induced agressionOpen 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 lightAs a result their exploratory behavior is inhibitedOpen field apparatus consist of circular arena with high sound and light sourcesSimple sterotypy: when animal move from one segment to another one ambulation scoreComplex sterotypy: when animal stands on its hind limbs- one rearing scoreUrinationdefecatioN

Elevated Plus Maze TestMost widely used method; male mice used

Anxiolytics decrease anxiety

increase open arm exploration time

Elevated plus maze2 open arms and 2 closed arms of 50 10 40cm dimensions

Open roof arrangement

Two open arms are opposite to each other.

Maze elevated at 50cm height.

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

Anxiolytic effect indicated by:

increase in the proportion of time spent in open arms

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 activityStaircase climibing reflects exploratory or locomotor activityRearing behaviour is an index of anxiety stateThe no. of rearing and steps climbed to be recorded for period of 5 minutesDecrease in rearing behaviour and increase in steps climbed is characterisation of anxiolytic effectBlack and white test box

Test box is divided into two compartments

Dark compartment

Rats tends to spend more time Brightly illumintaed compartmentLight dark modelAnimals placed in 2 chambered systems, where they can freely move between a bright and dark compartment

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

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

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

No. of crossings through the partition between the light and dark chambers compared with total activity counts during the 10 min.Loco motor activity also monitored.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 entryMice are exposed to the chamber of mirror Extended latency to enter the chamber of mirror used as a parameter for anxiety analogyAnxiolytics reduce this latency in dose dependent manner.Conditioned aversive proceduresA classicaly conditioned fear stimulus is introduced in to environment in which an ongoing response is occuringThe conditioned stimulus is created by mainly an originally neutral stimulus such as a tone or light, with an unconditioned aversive stimulus such as foot shock.After training , presentation of conditioned stimulus alone elicits defensive reaction from the subject, resulting in cessation of the ongoing response.

Geller seifters conflict test

Conflict is produced by availability of reinforcement with punishmentExperimentally induced conflict by punishing food rewarded behavior has been used to differentiate between various psychoactive drugs by Geller and Seifter

Geller seifters conflict testMale albino rats with a body weight of 300400 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 shockerThe animals are trained to lever press for the milk reward in two distinct response-reward sections.

Geller seifters conflict testAnxiety 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 seifters conflict testnonconflict 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 administeredGeller seifters conflict testFour 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 hrsThen placed in chamber with water sourcesprague dawley rats are used in this model

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

Anxiolytic effect :increase in the accepted shocks.Social interaction testIn unfamiliar and brightly lit environment, social interactions are suppressed.Anxiolytics counteract this suppression.Animal used : male sprague dawley rats.

Social interaction testSocial interactions : exploration, sniffing, rearing, social contacts, fighting, biting ,defensive posture climbing over the partner.

40

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 speciesUsed 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 effectsFoot 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 receptorSo aggressive and fighting behavior has been extended for GABAergic anxiolytic drug screening.43Isolation induced aggressionMale mice subjected to isolation develop aggressive behavior towards other animals of same sex.

In vitro methodsGABAA 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) conclusionAnxiety 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.ReferencesHand book of experimental pharmacology. S.K. kulkarni. 3 rd editionDrug 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.