DR MANU KUMARPOST GRADUATE

DEPT OF PHARMACOLOGYVMMC & SAFDARJUNG HOSPITAL

NEW DELHI

Screening of antidepressants

What is depression

Depression is a common mental disorder that presents with low mood, loss of interest or pleasure(anhedonia), feelings of guilt or low self-worth, disturbed sleep or appetite, low energy, and poor concentration.

Suicide – 8,50,000 lives every year4th  leading contributor to the global burden of disease

(DALYs) in 2000 Prevalence

3-5% (point prevalence)

20% (lifetime prevalence)

For accurate diagnosis of MDD

Five of the following nine DSM-IV symptoms must be present continuously for a minimum 2-week period:

“Depressed mood” “Loss of interest or pleasure” Significant weight or appetite alteration Insomnia or hyposomnia Psychomotor agitation or retardation Fatigue or loss of energy Feelings of worthlessness Diminished ability to think or concentrate or

indecisiveness; and Suicidal ideation.

Ethiopathogenesis

Multifactorial History of antidepressants1950s- no treatment for psychiatric disorder.Promethazine-promazine-Cl-imipramineIpraniazide- antitubercular drugReserpine – antihypertensive 1980s -SSRIs

Henri Laborit

ND-1251, phosphodiestarse 4 inhibitorsSartorious I

CP-448,187, antagonist, SR-46349 II

SB-649,915, NAD-299 III

Monoamine hypothesis

functional deficit of NE and/or 5-HT in certain sites of brain.Antidepressants drugs acts –inhibiting uptake

and facilitate the NE/5-HT neurotransmission.

Reserpine inhibit the storage of 5-HT and NE –depression

Tryptophan increase the 5-HT synthesis –elevate the mood

Limitation of this hypothesisAntidepressant action produce within hour

but, clinical benefit takes several weeks,Amphetamine and cocaine not used as

antidepressant despite their ability to facilitate NE transmission.

Atypical action antidepressants –tianeptine, bupropion , mianserine

Neuroendocrine hypothesis

Increase in CRF,- administration produce behaviour changes similar to stress, (CRF antagonist CP-154,526, R-121919)

Increase in ACTHWeak response of plasma cortisol to

exogenous steroids (dexamethasone suppression test)

Dampened negative feedback mechanism , GR-II receptor- regulate HPA by negative

feedback

Need for screening Severe side effects of the existing drugs. So

need for more safe drugs Need to find more efficacious drugs

Problems associated with screening of anti depressants Lack of animal models that resemble depressive

illness in humans Most of the existing models concentrate on

monoamine theory of depression only

In vitro assays

Assays based on inhibition of amine uptakeAssays based on binding to the receptors

Though complicated but these are precise and accurate

Inhibition of [3H] norepinephrine uptake in rat brain synaptosomes

Inhibition of [3H] dopamine uptake in rat striatal synaptosomes

Inhibition of [3H] serotonin uptake in synaptosomes

Binding to monoamine transportersMeasurement of β adrenoceptors stimulated

adenylate cyclase[3H] yohimbine binding to α2 adrenoceptors in

rat cerebral cortex

Assays based on inhibition of amine uptake (Norepinephrine/Dopamine/Serotonin)

Isolate hypothalamus/ corpora striata / hypothalamus or whole brain minus cerebellum. Homogenize with 0.32 M sucrose solution.

The homogenate is centrifuged

The supernatant is incubated with 3H-norepinephrine /Dopamine / serotonin in Krebs-Henseleit bicarbonate buffer and appropriate drug concentration (or the vehicle) at 37 °C and then centrifuge.

The supernatant fluid is aspirated and the pellets dissolved adding 1 ml of solubilizer (Triton X-100 + 50% ethanol, 1 : 4).

Cont.

Take the count for radioactivity by liquid scintillography

IC50 are derived & compared with standards.

Basic procedure of receptor binding Assay

Isolate the cells with receptors

Add radioactive ligand for that receptor in presence and absence of test drugs

Count the receptor ligand binding by liquid scintillography.

In vivo model

1. Gross behavior test.2. Test based on inhibition of amine uptake.3. Test Based on anticholinergic activity.4. Test based on depletion of biogenic amine.5. Hypermotility in olfactory-bulbectomized

rats

Behavioural tests

Forced swim testTail suspension test in miceLearned helpnessness in ratsMuricide behaviour in ratsBehavioural changes after neonatal

clomipramine treatmentCatalepsy antagonism in chickenOpen space swimming test

Forced swim test

Purpose and Rationale : It was proposed as a model to test for anti depressant activity by Porsolt et al. It was suggested that mice or rats forced to swim in a

restricted space from which they cannot escape are induced to a characteristic behavior of immobility.

Procedure: Naïve rats are individually forced to swim inside a vertical

Plexiglas cylinder (height: 40cm;diameter:18cm containing 15cm of water maintained at 25deg cel)

Initially rats are hyperactive. After 2-3 min activity begins to subside and is interspersed with phases of immobility or floating of increasing length.

After 5-6 min immobility reaches a plateau where the rat remains immobile for approx 80% of the time.

Forced swim test

After 15min in water rats are removed and allowed to dry in a heated enclosuren (32deg cel) before being returned to their home cages.

They are again placed in the cylinder 24h later and the total duration of immobility is measured during a 5min test.

Test drugs or standard are administered 1h prior to testing.

Evaluation: Duration of immobility is measured in controls and animals

treated with various doses of a test drug or standard. Antidepressant drugs but also stimulants like amphetamine

and caffeine reduce duration of immobility . Differentiation is done by measurement of locomotor Activity

by open field test

Cont.

Open field apparatus - an arena 70 cm in diameter divided into 9 or 18 approximately equal areas.

Each rat is individually placed in the center of the arena 15 h after the last treatment and its behavioural parameters are recorded for 5 min.

Score is calculatedLocomotion (number of line crossings within 5 min)

Rearing frequencies (number of times an animal

stood on its hind legs).

Tail suspension test in mice

Purpose and rationale The immobility displayed by rodents when subjected to an

unavoidable and inescapable stress has been hypothesized to reflect behavioural despair which reflects depressive disorders in humans

Antidepressants reduce the immobility that rats display after active and unsuccessful attempts to escape when suspended by the tail

Procedure 20 Male Balb/cJ mice (20-25 gm) divided in 2 groups Housed in plastic cages with food and water ad libitum Treated i.p with test drug or vehicle 30 min later, mice are suspended on the shelf 58 cm above the

table top by adhesive tape placed at 1 cm from the tip of the tail

Duration of immobility recorded for 6 min Mice at considered immobile when they hang passively and

completely motionless for at least 1 min

Evaluation Total immobility is compared with control , decrease in

immobility time after test drug indicate antidepressant action.

Using various doses, ED50 values can be calculated

Critical assessment Easy method SSRI are sensitive to this model .

Learned helplessness in rats

Animals exposed to inescapable and unavoidable electric shocks in one situation later fail to escape shock in a different situation when escape is possible.

Male SD rats (300 g) Apparatus - Box with a grid floor having a platform which can

be inserted through one side wall to allow a jump-up escape response.

Training - Exposure to electric shock (0.7 mA) for 1 h on a schedule of 10 s of shock/min.

The platform is not available during training. This training resulted in 80% acquiring learned helplessness behavior.

Cont.

Trial The platform is pushed into the box and a 0.4 mA shock

initiated. Shock is terminated in 10 s if the animal has not escaped onto

the platform by this time. Ten such trials with an intertrial interval of 20 s are given. EVALUATION A drug is considered to be effective, if the learned

helplessness is reduced and the number of failures to escape is decreased.

Muricide behavior in rats

Male Sprague-Dawley rats (300–350 g) Only rats consistently killing mice within 5 min after presentation

are used for the test. Drugs are injected i.p. to the rats before the test. Mice are presented

30, 60 and 120 min after drug administration. EVALUATION Failure to kill a mouse within 5 min is considered inhibition of

muricidal behavior. ED50 is calculated, the ED50 is defined as the dose which inhibits

mouse killing in 50% of the rats. The mouse-killing behavior is also inhibited d-amphetamine some

antihistamines and some cholinergic drugs.

Some other models are…….

Behavioral changes after neonatal clomipramine treatment.

Antidepressant-like activity in differential reinforcement of low rate 72-second schedule.

Catalepsy antagonism in chicken.

Tests based on mechanism of action

Test based on inhibition of amine uptake

Potentiation of norepinephrine toxicity in mice Male NMRI mice (22–25 g) The test drug, the standard or the vehicle are given orally 1 h

prior to the s.c. injection of the sublethal dose of 3 mg/kg noradrenaline.

The mortality rate is assessed 48 h post-dosing. ED50 is calculated.

5-Hydroxytryptophan potentiation in mice/rats DL-5-Hydroxytryptophan is used as the precursor of serotonin. Enzymatic breakdown is inhibited by the MAO-inhibitor pargyline. In mice the characteristic symptom of head-twitches is observed.

CONT.

A animal is considered to be positive if it shows head twitches 15 min after 5- HTP injection. Enhancement is observed after treatment with serotonin uptake blockers relative to animals pretreated with pargyline only.

The head-twitch in mice can also be elicited without a MAO-inhibitor by using higher doses (200 mg/kg) of DL-5-hydroxytryptophan.

Test Drug /vehiclei.v.

0 min

Pargyline Hcl s.c.10 mg/kg DL -5 hydroxytryptophan

30 min 120 min

.

3. Test Based on anticholinergic activity.a) Compulsive gnawing in mice Treatment of rodents with apomorphine causes compulsive

gnawing instead of vomiting due to dopaminergic stimulation.

Anticholinergics shift the balance between Ach & dopamine resulting in an enhancement of apomorphine effect.

Cont.

NMRI mice(18–20 g) are injected s.c. with 10 mg/kg apomorphine + test drug/vehicle at the same time.

Immediately , mice are placed in a cage with corrugated paper the corrugation facing upwards for 1 hour.

The mice start to bite into the paper causing fine holes or tear the paper.

Percentage of damaged paper in calculated.This behavior is enhanced by antidepressants.Not only antidepressants, but also centrally acting

anticholinergics and antihistaminics are active in this test.

Test based on depletion of biogenic amine

a) Tetrabenazine antagonism in mice

Tetrabenazine (TBZ) induces a depletion of biogenic amines (e.g. noradrenaline, dopamine, serotonin) without affecting their de novo synthesis.

Antidepressants antagonize the effect of TBZ male NMRI mice (20–22 g) Catalepsy and ptosis are used as criteria. degree of ptosis and catalapsy is scored The scores of the TBZ controls are taken as 100%

and the percentage is calculated for the treated animals.

Reserpine induced hypothermia

Depletion of biogenic amines (noradrenaline, 5-hydroxytryptamine, dopamine) in the brain also induces hypothermia in rodents.

The decrease of body temperature induced by reserpine is antagonized by antidepressants.

male NMRI mice (19–21 g)Rectal temperature is recorded every hour. The difference in temperature from vehicle controls

is calculated for each time and the maximal difference is scored.

The reversal of hypothermia is not specific for antidepressants. The fall in body temperature can also be antagonized by amphetamines, and some antipsychotic agents (chlorpromazine).

Hypermotility in olfactory-bulbectomized rats

Bilateral olfactory bulbectomy in the rat is associated with changes in exploratory behavior that are reversed by chronic, but not acute treatment with antidepressant drugs.

Male Sprague Dawley rats The animals are allowed to recover for 14 days after surgery. the animals are treated s.c. with the test drug / standard /

vehicle once daily for 14 days. The behavior of the animals is tested from the 12th day

onwards. The rats are placed singly in the center of an open field

apparatus.

Yohimbine toxicity enhancement

Purpose and rationale Yohimbine occupies central α2 receptors and

prevents NE from binding to these receptors Antidepressants block the reuptake of NE into

the nerve terminals Combination of both can thus produce death in

animals due to NE toxicity

Procedure 20 male NMRI mice (25-28 gm) divided in 2 groups Treated with test drug or the vehicle by oral or i.p

route After 30 min, 25 mg/kg yohimbine (sublethal dose)

given s/cEvaluation

Mortality assessed at 1, 2, 3, 4, 5 and 24 hr after dosing

Mortality rates compared between 2 groups Using various doses, ED50 values can be calculated.

Thank you

Substance p antagonist

CP-96,345.PF, pre

phosphod

iesta

rse 4

inhib

itors

Sarto

rious I

ND-1251,

5-HT2 receptor blocker

SR-46349 II

Antalarmine CRF-1

antagonist