Pharmacological screening of Anti-psychotic agents

PHARMACOLOGICAL SCREENINGOF 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
2

 INTRODUCTION
 DEFINITION
 CLASSIFICATION
 MOA OF ANTI-PSYCHOTIC AGENTS
 SCREENING MODELS
IN VIVO
IN VITRO
 REFERENCES
CONTENTS
3

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.
5

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.
6

 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 withdrawal
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News paper cutting - DECCANHERALD, September 18,2014
8

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
9

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
10

 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 D₂
receptor.
 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 D₂
receptors in the brain's
Dopaminergic pathway.
Some also block or partially
block serotonin receptors
(particularly 5HT2A, C and
5HT1A receptors)
But antipsychotic drugs can
also block wide range of
receptor targets.
Contd…..
12

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.
13

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.
14

In the Mesolimbic - Mesocortical and Nigrostriatal
pathway Antipsychotic blocks:
15

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
18

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.
21

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.
22

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.
23

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.
25

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.
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 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
27

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.
28

Purpose & Rationale
This 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
29

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.
30

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.
32

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’.
33

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.
35

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.
36

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.
37

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.
38

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.
41

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
42

Pharmacological screening of Anti-psychotic agents

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