1. Screening models of
anti-Psychotic
Drugs
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2. contents
❖Introduction
❖Clinical features of schizophrenia
❖Classification of antipsychotics
❖Pathophysiology of schizophrenia
❖Screening models for antipsychotics
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3. Introduction
➢Antipsychotic are the drugs having therapeutic
effect in psychoses.
Psychoses
Functional
disorder
Cognitive
disorder
Ex: Schizophrenia Ex: Delirium, Dementia
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4. Schizophrenia: It is a particular type of psychosis (that is
mental disorder caused by inherent dysfunction of the
brain).
➢ Symptoms are wide ranging and are found across
multiple cultural and ethnic group.
➢ It is characterized by delusions, hallucinations, and
thinking or speech disturbance.
➢Affects 1% of population.
➢Affects young and old.
➢Affects men and women equally.
➢Chronic and disabling disease.
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5. ❖ETIOLOGY
➢Genetic predisposition (10%)
➢ Prenatal (viral infections)
➢Prenatal (oxygen deprivation)
➢Postnatal (CNS infections)
➢Stress in adolescence
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6. CLINICAL FEATURES
Positive
Symptoms
Negative
symptoms
Cognitive
Symptoms
•Delusions
•Hallucination
•Thought disorders
•Introvert behavior
•Poor socialization
•Emotional blunting
•Lack of attention
•Loss of memory
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7. Pharmacological classification
➢ First-generation antipsychotic (low potency)
✓ Chlorpromazine
✓ Prochlorperazine
✓ Thioridazine
First generation antipsychotics(high potency)
✓ Fluphenazine
✓ Haloperidol
✓ Pimozide
✓ Thiothixene
➢Second generation antipsychotics
✓ Aripiprazole Olanzapine
✓ Asenapine Paliperidone
✓ Clozapine Risperidone
✓ Iloloperidol Ziprasidone
✓ Lurasidone
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8. ❖PATHOPHYSIOLOGY OF SCHIZOPHRENIA
❖Neurophysiological theory
➢ Dopamine theory
➢ Serotonin theory
➢ Glutamate theory
❖Dopamine theory
➢ Abnormal increase in dopamine
❖Hypo frontal hypothesis:
➢ Increase DA in mesolimbic site of brain
➢ Decrease DA in frontal, pre-frontal & the temporal
❖Cervices → Decrease metabolic activity
❖Other reason are increase serotonin (5HT, glutamate)
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9. ➢The psychosis behaviour mainly occurs due to excessive
release of Dopamine at D2 receptors.
No consistent bio-chemical evidence for excessive
dopamine synthesis or release.
➢The mesolimbic system is a dopaminergic tract that
originates in the ventral tegmental area (VTA) and projects
to the nucleus accumbens, ventral striatum, parts of the
hippocampus, and other components of the limbic system.
➢It involve the development of emotions & memory, and
some hypothesize that mesolimbic hyperactivity is
responsible for the positive symptoms of schizophrenia.
DOPAMINE THEORY
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12. IN VITRO MODELS IN VIVO MODELS
1) D1 receptor assay ³H –SCH 23 390
binding to rat striatal homogenates.
2) D2 receptor assay ³H spiroperidol
binding
3) D2 receptor auto radiography
³H spiperone binding
4) Binding to the D3 and D4 receptor
5) Determination of dopamine auto
receptor activity.
6) α1 adrenergic receptor binding in
brain
7) Serotonin 5HT receptor
autoradiography.
8) Binding to the sigma receptor.
9) Measurement of neurotransmitters
by intracranial micro-dialysis.
10)Simultaneous determination of
nor-epineprine, dopamine, DOPAC,
HVA, 5HIAA.
1) Golden hamster test
2) Influence on behavior of the
cotton rat
3) Open field test
4) Brain self stimulation.
5) Rota rod method
6) Amphetamine group toxicity
7) Inhibition of mouse jumping
8) Pole climb avoidance in rats.
9) Artificial hibernation in rats.
10) Grip strength
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13. IN VITRO MODELS
➢ D1 Receptor Assay
(³H- SCH 23 390 binding to rat stratial homogenates)
➢D2 Receptor assay
(³H spiroperidol binding)
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14. PURPOSE AND RATIONAL
➢Dopamine receptor are the primary targets in the development of drugs for the
treatment of schizophrenia, Parkinson’s diseases.
➢Multiple dopamine receptors are know. Designated as D1 and D2.
D1A
D1
DOPAMINE D2B
D2 D2S
D3 D2L
D5 D4
D1 Receptor assay
³H-SCH23390 Binding rat striatal homogenates
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15. ➢For typical neuroleptic agents, Haloperidol, good correlation
was found between D2 receptor binding and clinically
effective doses.
➢A typical neuroleptics, like clozapin, were found to be potent
inhibitors of D1 and D4 receptor binding, renewing interest
in these receptor types.
➢The compound SCH 23390 was found to be selective for
the D1 receptor.
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16. ❖ PROCEDURE
➢REAGENTS:
➢d-Butaclamol a 1 mM stock solution is made and diluted
1:20.
➢ 20 µl are added to 3 tubes for the determination of
nonspecific binding.
➢ For the test compounds a 1 mM stock solution is made
up in a suitable solvents and serially diluted, such that the
final concentration in the assays ranges from 10ˉ⁵ to 10ˉ⁸M.
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17. ❖Tissue preparation
Male Wister rates
decapitated, striate dissected.
Brains rapidly removed and weighed
The striata are homogenized in 100 volumes of 0.05 M tris buffer,
Mantained pH 7.7 using a Tekmar homogenizer.
centrifused at 40,000g for 20 min
The final pellet is re-suspended in the original volume of 0.05M Tris
buffer, pH 7.7, containing physiological ions
(NaCl 120mM, KCl 5mM, MgCI2 1mM and CaCl2 2mM).
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18. ❖ASSAY
➢50µl 0.5 M Tris buffer, pH 7.7, containing physiological
ions. 380µl H₂O, 20µl vehicle or d-butaclamol or
appropriate concentration of test compound.
➢5oµl 3H- SCH23390
➢500µl tissue suspension.
➢ The tubes are incubated at 37ºC for 30 min.
➢The assay is stopped by rapid filtration through
whatman’s GF/B filters using a brand-ell cell harvester.
➢The filter strips are then washed 3 times with ice-cold
0.05M this buffer, pH7.7, and counted in 10ml Liquiscint
scintillation cocktail.
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19. ❖EVALUATION
➢Specific binding is defined as the difference between total
binding in the presence of 1µM d-butaclamol.
➢ IC50 calculations are performed using log-Probit
analysis.
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20. ❖D2 RECEPTOR ASSAY: (³H) spiroperidol binding
❖PURPOSE AND 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,
dopamine enriched area of the brain, have been shown to be
predictive of in vivo dopamine receptor antagonism and
antipsychotic activity.
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21. ❖TISSUE PREPARATION
Male rats decapitated
striata removed and weighed
Homogenized in 50 volumes of ice cold 0.05M tris buffer ,
Ph 7.7
Then centrifuged at 40,000g for15 min
Again that pellets were re-centrifuse at 40,000g
Then the final pellets were re-suspended in Tris buffer
(physiological salt) resulting in a concentration of 10mg/ml.
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22. ❖ASSAY
The membrane preparation took
Incubated with ³H spiroperidol (0.25nm) and various concentration of
test drug at 37ºc for 20 min k/Na phosphate buffer.
Cooling in an ice bath for 45min
Bound ligand is separated by rapid filtration
Filters are washed 3 times with ice cold water
Dry it and shaken through with 3.5ml scintillation fluid
From the liquid scintillation counter radio activity is determined.
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23. ❖EVALUATION
From this assay
The following parameters are determined
✓Total binding of ³H spiroperidol
✓Non specific binding: binding of samples containing
2mM butaclamol
✓Specific binding = total binding – non specific binding
✓% inhibition :100- specific binding as % of control value.
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24. IN VIVO MODELS
➢Open field test
➢Rota road method
➢ Grip strength
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25. ❖Purpose and Rationale:
➢ Interruption of light beams as a measure of movements
of rats and mice in a cage (“open field”).
❖Procedure:
➢ Rats are observed in a square open field equipped with 2
rows of 8 photo cells, sensitive to IR.
➢ The photo cells are placed apart from each other and the
last photo cell in a row is spaced 25mm from the wall.
➢ Measurements are made in a ventilated, sound-
attenuating box.
➢ Interruptions of photocell beams can be collected by
micro-computer as follows
❖OPEN FIELD TEST
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26. ➢Motor activity: All interruptions of photo beams in the
lower rows.
➢Peripheral motor activity: Activation of photo beams in
the lower rows, provided that the photo beams from the wall
were collected.
➢Rearing: All the interruption of the photo beams in the
upper rows.
➢Locomotion: Successive interruptions of photo cells in the
lower rows, when the animal is moving in the same
direction.
➢Speed: The time between successive photo beam
interruptions during locomotion collected in 0.1s categories.
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28. ❖Evaluation:
➢Dose response curves can be obtained for sedative &
stimulant drugs, whereas the various parameters show
different results.
➢The effects of various drugs are compared statistically
with the values of controls & among themselves.
❖Modifications:
➢Besides interruption of light beams, devices based on
capacitance systems have been developed.
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29. ❖Purpose and Rationale:
➢ The test is used to evaluate the activity of the drugs
interfering with motor co-ordination which suggests that
the skeletal muscle relaxation induced by an compound.
❖Procedure:
➢ Its an horizontal wood or metal rod coat with rubber.
➢ The rod is divided into 6 sections by plastic discs, there by
allowing simultaneous testing of 6 mice.
➢ Rod is placed at an certain height above the table in order to
discourage the animals from jumping off the roller.
➢ Cages, below placed serves to place the animals, whenever
they fell down.
➢ Trained animals are selected. The test compounds are
administered either by oral or i.p.
❖Rota rod Method
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30. ROTARODAPPARATUS
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31. ➢ Mice are placed on the rod for 1min after administration
of drug.
➢ The number of animals falling down are noted along
with the time.
➢ ED50 is calculated by using different doses.
❖Calculation:
➢ % animals falling from the rota rod with the test period
is calculated for every drug concentration tested.
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32. ❖Grip strength:
❖PURPOSE AND RATIONALE:
➢ The test is being used to assess muscular strength or
Neuromuscular function in rodents.
❖PROCEDURE:
➢ Male or female mice are used.
➢ In a preliminary experiment the animals are tested for
their normal reactivity.
➢ The animals are exposed to a horizontal thin threat or
metallic wire suspended about 30 cm into the air which
they immediately grasp with the forepaws.
➢ The mouse is released to hang on with its forelimbs.
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33. ➢ Normal animals are able to catch the threat with the hind limbs and
to climb up within 5s.
➢ Only animals who fulfil this criterion are included into the
experiment.
➢ Ten mice are used in the control group and in the experimental
groups.
➢ Animals are tested every15 min.
➢ Animals which are not able to touch the threat with the hind limbs
within 5s or fall off from the threat are considered to be impaired.
➢ The test is continued for 2 hrs.
➢ The animals are observed for their behaviour in the cages.
➢ Only if their behaviour and their motility in the cage seem to be
normal the disturbance of the grasping reflex can be considered as
caused by central relaxation.
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35. ❖References
➢ Drug Discovery & Evaluation Pharmacological assay
– H. G. Vogel
➢ Rang and Dale Pharmacology sixth edition H. P. Rang,
M. M. Dale J. M. Ritter R. J. Flower
➢ Goodman and Gilman`s Manual of Pharmacology and
Therapeutics
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36. THANK YOU
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