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PHARMACOLOGICAL SCREENING OF ANTI-PSYCHOTIC AGENTS

Facilitated by

Mrs.MEENU SINGH

Asst.Professor

Dept.of pharmacology

PESCP,Bangalore

Presented by

ABIN JOY

1st year M-Pharm

Dept.of pharmacology

PESCP,Bangalore

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INTRODUCTION

DEFINITION

CLASSIFICATION

MOA OF ANTI-PSYCHOTIC AGENTS

SCREENING MODELS

IN VIVO

IN VITRO

REFERENCES

CONTENTS

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INTRODUCTION

The word psychosis was first

used by Ernst in 1845 and stems from the Greek words

psyche (mind) and osis (abnormal condition).

It has been used as an alternative to mania. The etiopathology of

psychosis is complex and genetic basis has been understood to play a

definitive role in its genesis.

Antipsychotic or Neuroleptic drugs are those agents which are used

to treat severe psychiatric illness, psychosis and mania.

The prominent symptom's of psychosis include hallucinations,

delusions and thought disorder. 4

PSYCHOSIS

Psychosis is a thought disorder characterized

by disturbances of reality and perception

(delusions and hallucinations), impaired

cognitive functioning and inappropriate or

diminished affect (mood).

Psychosis denotes many mental disorders.

Schizophrenia is a particular kind of psychosis

characterized mainly by a clear sensorium but

a marked thinking disturbance.

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Psychosis can be broadly Categorized in to four groups

1.Acute and chronic organic brain syndromes (cognitive disorders) such as, delirium and dementia, prominent features of confusion, disorientation, defective memory and disorganized behavior.

2.Functional disorders such as, memory and orientation mostly retained by emotion, thought, reasoning and behavior are altered.

3.Schizophrenia (split mind) i.e.splitting of perception and interpretation from reality- hallucination, inability to think coherently. Schizophrenia is often described in terms of positive or negative symptoms..

4.Paranoid state i.e. fixed delusions (false beliefs) and loss of insight in to abnormality.

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A thought disorder.

The disorder is characterized by a divorcement from reality in the

mind of the person (psychosis).

It may involve visual and auditory hallucinations, delusions, intense

suspicion, feelings of persecution or control by external forces

(paranoia), depersonalization and there is attachment of excessive

personal significance to daily events, called “ideas of reference”.

Schizophrenia

Positive symptoms Negative symptoms

Delusions Cognitive deficits

Hallucinations Emotional blunting

Paranoia Avolition

Ideas of reference Apathy, social withdrawal7

News paper cutting - DECCAN HERALD, September 18,2014

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Antipsychotic drugs are also

known as Neuroleptics, Ataractic,

Major Tranquilizer and Anti-

Schizophrenic drugs. A first

generation of antipsychotics,

known as Typical antipsychotics,

was discovered in the 1950s. Most

of the drugs in the second

generation, known as Atypical

antipsychotics, have been developed

more recently.

ANTI-PSYCHOTIC DRUGS

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Typical neuroleptics :

1.Phenothiazines:

a. Aliphatic side chain: Chlorpromazine,

Triflupromazine

b. Piperidine side chain: Thioridazine

c. Piperazine side chain: Trifluoperazine, Fluphenazine

2.Butyrophenones: Haloperidol, Trifluperidol, Penfluridol

3.Thioxanthenes: Flupenthixol

4. Other heterocyclics: Pimozide, Loxapine

Atypical neuroleptics:

Clozapine, Risperidone,Olanzapine, Quetiapine, Aripiprazole,

Ziprasidone.

CLASSIFICATION

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AII antipsychotics (except clozapine like atypical) have potent dopamine D₂ receptor blocking action; antipsychotic potency has shown good correlation with their capacity to bind to D₂receptor.

Blockade of dopaminergic projections to the temporal and prefrontal areas constituting the 'limbic system' and in mesocortical areas is probably responsible for the anti-psychotic action.

This along with the observation that drugs which increase DA

activity induce or exacerbate schizophrenia.

As an adaptive change to blockade of D₂ receptors, the firing

of DA neurones and DA turnover increases initially.

MECHANISM OF ACTION

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Antipsychotic blocks D₂receptors in the brain'sDopaminergic pathway.

Some also block or partiallyblock serotonin receptors(particularly 5HT2A, C and5HT1A receptors)

But antipsychotic drugs canalso block wide range ofreceptor targets.

Contd…..

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Dopaminergic pathway in Brain:

Mesolimbic- Mesocortical:

Control behavior, cognitive

funtion regulated by D₂

Receptor.

Nigrostriatal: Control

Voluntary Movement

regulated by D₁ and D₂

receptors.

Tuberoinfundibular:

Control prolactin secretion

regulated by D₂ receptor.

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In the Tuberoinfundibular pathway

Antipsychotics block:

Dopamine released at this

site regulates the secretion

of Prolactin from anterior

pituitary gland.

Antipsychotics blocks

receptor at this site.

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In the Mesolimbic - Mesocortical and Nigrostriatal

pathway Antipsychotic blocks:

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SCREENINGMODELS

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IN VIVO MODELS

Behavioral tests

1. Catalepsy in rodents.

2. Golden hamster test.

3. Pole climb avoidance in rats.

4. Conditioned avoidance reflex in rats.

Tests based on pharmacologic antagonism

1. Inhibition of Amphetamine-induced stereotypy in rats.

2. Inhibition of Apomorphine-induced stereotypy in mice.

3. Phencyclidine-induced bizarre pattern locomotor

activity and stereotypy.

4. Neuro developmental model. 17

1. D₁ - receptor assay

2. D₂ - receptor assay

3. Binding to D₃ - receptor

4. Binding to D₄ - receptor

5. ⍺₁ - adrenergic receptor binding in brain

6. Measurement of neurotransmitters by intracranial micro

dialysis.

7. Binding to sigma receptors

IN VITRO MODELS

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IN VIVO MODELS

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Purpose and rationale

Catalepsy in rats is defined as a failure to correct an externally

imposed, unusual posture over a prolonged period of time.

This is a typical effect of all agents which inhibits dopaminergic

system in nigrostratum.

CATALEPSY IN RODENTS

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PROCEDURE

Groupings→ Group I - treated with test drug

Group II - treated with standard drug (Haloperidol 0.2 mg/kg I.P)

Animals are placed inside the translucent plastic boxes.

After some time each animal is grasped gently around the shoulders and under

the forepaws and Placed carefully on the dowel.

The amount of time spent atleast with one fore paw on the bar is determined.

When the animal removes its paws, the time is recorded and the rat is

repositioned on the bar.

Four trials are conducted for each animal at 30, 60, 120 & 360 min. An animal

is considered cataleptic if it remains on the bar for 60 sec.

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EVALUATION

The percentage of cataleptic animal is calculated for each

group. ED₅₀ values are calculated for comparison &

elucidation of potency.

Haloperidol dose dependently increases catalepsy time.

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INHIBITION OF AMPHETAMINE INDUCED

STEREOTYPY IN RATS

Purpose & rationale

Amphetamine is an indirect acting sympathomimetic agent

which releases catacholamines from its neuronal storage pools.

In rats the drug induces characteristic stereotypic behavior and

this can be prevented by neuroleptics.

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PROCEDURE

Groupings;

Group I (control) - d-Amphetamine 10 mg/kg S.C.

Group II (treated) - Chlorpromazine 10 mg/kg S.C.+

d- Amphetamine 10 mg/kg S.C.

The rats with a body weight between 120 - 200 g are selected and grouped into 6

each in control and treated.

They are injected simultaneously with d-Amphetamine and test compound and

placed in individual cages.

After 60 min of drug administration the rats are observed.

In the group I animals which have been administered d-Amphetamine shows

stereotypic behaviour which is characterized by continuous sniffing, licking or

chewing and compulsive gnawing.

The stereotypic behaviour is reduced or abolished in drug treated group.24

EVALUATION

The % effectiveness of a drug is determined by the number of

animals protected in each group.

If the group of animals which treated with test drug is protected

by stereotypic behaviour, the efficacy of drug is confirmed as

neuroleptic agent.

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INHIBITION OF APOMORPHINE

INDUCED STEREOTYPY IN RATS

Purpose & rationale

Apomorphine (stimulate dopamine receptor in CTZ) induces a stereotyped

behavior in rats, characterized by licking, sniffing and gnawing in a

repetitive,compulsive manner, which is an indication of striatal dopaminergic

stimulation.

Compounds which prevent apomorphine-induced stereotypy antagonize

dopamine receptors in the nigrostriatal system.

Furthermore, antagonism of this behavior is predictive of propensity

(tendency to behave in certain way) for the development of extrapyramidal

side effects.26

For screening, group of 6 male Wistar rats with a body weight between

120 and 200 g are used.

The test drug or the standard are administered I.P. 60 min prior to

Apomorphine dosage.

Apomorphine HCl is injected S.C. at a dose of 1.5 mg/kg.

The animals are placed in individual plastic cages.

A 10 sec observation period is used to measure the presence of stereotypic

activity such as sniffing, licking and chewing 10 min after apomorphine

administration.

An animal is considered protected if this behavior is reduced or abolished.

PROCEDURE

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EVALUATION

The percent effectiveness of a drug is determined by the number of

animals protected in each group.

With a group size of 10 animals dose response curves are obtained

and ED₅₀ values calculated.

ED₅₀ values were found to be 0.2 mg/kg S.C. for Haloperidol and 5.0

mg/kg for Chlorpromazine, whereas Clozapine was ineffective even

at high doses.

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Purpose & RationaleThis test uses the innate behavior of the species ( Mesocricetus auratus)

for differentiation between neuroleptics & sedative-hypnotic activity. The

aggressive behaviour of male Golden hamster is suppressed by neuroleptic in

doses which do not impair motor function.

GOLDEN HAMSTER

TEST

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PROCEDURE

Control - vehicle treated (no.6)

Treated - standard drug (no.6)

The above grouped animals are placed together

in crowded conditions in specially designed

cages for at least 14 days.

The animals develop a characteristic fighting

behavior during this period.

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For selection of a test group

From the above, single animals are placed into a glass jars of 2 lit.

Animals assume a squatting resting position during day.

If animals are touched with a stick or forceps, they wake up from their day

time sleep & arouse immediately from resting position ( a characteristic

behavior is seen ).

Tries to hold the hamster with a blunted forceps.

The hamster throws himself onto his back & tries to bite & to push the

forceps away with his legs & utters angry shrieks. 31

Touching the animal is repeated up to 6 times followed by punching with

forceps.

Only the animals responding to stimulus with all defensive reactions

(turning, vocalizing, biting) are included for the test group.

The test drug is given by S.C. route. Six animals are used for each dose.

Observe the suppression of aggressive behavior of Golden hamsters in

doses.

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EVALUATION

The stimuli are applied every 20 min for 3 hr.

The suppression of defense reaction is evaluated.

If all defense reactions are suppressed even after

punching with forceps at least once during test period,

an animal is regarded to be completely ‘tamed’.

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IN VITROMODELS

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D₂ RECEPTOR ASSAY:

[³H]-SPIROPERIDOL BINDING

Purpose & Rationale The neuroleptic compound Haloperidol has been used as binding ligand to

study the activity of other neuroleptics.

The use of Haloperidol has been superseded by Spiroperidol.

Dopamine receptor binding assays employing dopaminergic antagonists in

mammalian striatal tissue, a dopamine enriched area of the brain, have been

shown to be predictive of in vivo dopamine receptor antagonism and

antipsychotic activity.

Spiroperidol is considered to be an antagonist specific for D₂ receptors.

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PROCEDURE

Tissue preparation

Male Wistar rats are decapitated, their corpora striate removed,

weighed and homogenized in 50 ml of ice-cold 0.05 M Tris buffer

( pH 7.7 ).

The homogenate is centrifuged at 40 000 rpm for 15 min.

The pellet is rehomogenized in fresh buffer and recentrifuged at 40 000

rpm.

The final pellet is then resuspended in Tris buffer.

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Assay

The membrane preparations are incubated with ³H-Spiroperidol.

Various concentrations of the test drug at 37 °C for 20 min in a K/Na

phosphate buffer (50 mM, pH 7.2), followed by cooling in an ice bath

for 45 min.

To determine non-specific binding, samples containing 2mM (+)-

Butaclamol are incubated under identical conditions without the test

compound.

Bound ligand is separated by rapid filtration through Whatman GF/B

glass fiber filters.

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The filters are washed three times with ice-cold buffer, dried, and

shaken thoroughly with 3.5 ml scintillation fluid.

Radioactivity is determined in a liquid scintillation counter.

Specific binding is defined as the difference between total binding

and the binding in the presence of 2.0 mM (+)-Butaclamol.

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SCINTILLATION COUNTER

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SCINTILLATION FLUID

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EVALUATION

The following parameters are determined:

• Total binding of ³H-spiroperidol

• Non-specific binding: binding of samples containing

2 mM Butaclamol

• Specific binding: total binding – non-specific

binding

• % inhibition: 100 – specific binding as percentage

of the control value.

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REFERENCES

1. Drug Discovery and Evaluation-H. Gerhard Vogel.

2. Drug screening methods – SK Gupta.

3. Essentials of Medical Pharmacology-KD Tripathi

4. Internet source

The Search for Novel Antipsychotic Drugs- Paul Kenyon

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