Screening models of cns stimulant & anti depressant drugs-converted

Seminar on,
Screening models of CNS
Stimulant &Anti-depressant
drugs
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Department of Pharmacology BVVS
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➢ CNS is made of BRAIN and SPINAL CORD.
➢ The brain is divided into three parts – Forebrain,
Midbrain, and Hindbrain.
➢ Spinal cord begins from the brain stems and extends till
the lowest end of backbone.
➢ Both the brain and spinal cord contain fluid filled
spaces or cavities. The fluid in these spaces is called
cerebrospinal fluid (CSF), and contains nutrients,
hormones, white blood cells to maintain the CNS.
➢ Brain and Spinal cord mainly responsible for
information processing, imagination, memory and
communication.
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➢ Central Nervous System Stimulants may be used to reduce
tirednes and increase alertness, competitiveness and aggression.
➢ CNS stimulants may be defined as “Drug substance that most
specifically afford an enhancement in excitability either very
much within the different portions of the brain or the spinal cord
which may lead to convulsion.”
➢ CNS stimulants may be classified as below:
❖ Analeptics (convulsant)
1. Doxapram (respiratory stimulant)
2. Nikethamide (respiratory stimulant)
3. Strychnine
4. Picrotoxin
5. Bicuculline
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❖ Psychomotor stimulants
1. Amphetamine & Methamphetamine
2. Methylphenidate
3. Cocaine
4. Phentemine
5. Fenfluramine
❖ General cellular stimulants
1. Methylxanthines derivatives
2. Caffeine (Caffee)
3. Theophylline (Tea)
4. Theobromine (Chocolate)
❖ Clinical antidepressants
1. MAO inhibitors
2. Catecholamine reuptake inhibitors
❖ Psychotomimetic (Hallucinogenic)
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1. Sand displacement method
2. Runway test
3. Registration of motar activity test
4. Ptosis test
5. Open field test
6. Hole board test
7. Combined open filed test
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❖ Purpose & Rationale
➢ This method is useful for detecting stimulant of all types.
Amount displace sand in graduated cylinder is measuring
parameter of this method.
❖ Procedure:
➢ A cylinder diameter 10 cm, height 12cm. The cylinder cages
have a rubber torus around its lowest part to prevent motion
of cages. The rubber torus touches the funnel.
➢ The cage is loaded with 50 ml dry sand contains 10 ml blue
gel for absorption for moisture. Beneath the glass funnel,
graduated glass cylinder having volume (10 ml), capable of
being read to 0.1 ml. Quantity of sand is recorded every
15 minute.
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Evaluation: Amount of sand displaced by control
group is compare to the test group.
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➢ To study the effect of a drug on spontaneous
activity and motor coordination.
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➢ Age of 115-140 days wistar rats are used for the this
experiment. 8-19 rats are used for each dose the
apparatus is symmetrical Y shaped runway made of
wood and 13 inches high.
➢ Each arm is 15 inches long and 5 inches wide. A trial
consists of placing a rat in the centre of the Y and
leaving it in the apparatus for 5 minutes.
➢ The number of times it enter the arms of the apparatus,
so that all of its feet are arm, is recorded as a measure
of activity
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➢ In order to estimates the degree of ataxia, the rat is then
placed on a runway covered with paper, so that
footprint record of control rat shows that the regularity
of spacing, is a measure of ataxia.
➢ The group of control rats had 14.7 as the mean of
spontaneous activity. Amphetamine at a dose of
0.19mg/kg caused this number to increase to 20.
Amylobarbitone sodium at a dose of 15 mg/kg caused
it to increase to 22. But at high doses decreases in the
number of entries were found.
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❖ Evaluation
➢ There are increase the mean value of various CNS
stimulants at specific dose than the control animal
group.
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➢ This method may be used to detect increase motor
activity.
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❖ Procedure
➢ The rectangular cage is constructed with floor and ends of
wood, and with plastic sides.
➢ A beam of light is passed through a plastic side to a photo
electric cell, so adjusted that, when a mouse breaks the
beam of light, the cell activates a digital counter.
➢ The drug are dissolved in a 0.9% sodium chloride and
injected intraperitoneal the number of counts or interruption
of the light beam from the time of injection until 15minutes
later is noted.
❖ Evaluation
➢ The ratio of this count to the count for control mice is
measure of activity. For screening, a drug is tested initially
at dose level of 50% and 10% of its LD 50 for comparison,
amphetamine, 5mg/kg or more is used.
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➢ It belongs to heterogeneous group of mental disorder or mood
altering illness affecting energy, sleep, appetite, the ability to
function and psychomotor functions.
➢ The Biogenic amine theory propose that depression is due to
deficiency of monoamines such as nor-epinephrine, serotonin.
➢ Depression is major affective disorder.
➢ The physical and social dysfunction associated with depression-
outweighs many other chronic medical conditions including
hypertension, diabetes and arthritis.
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➢Unipolar depression (about 80% of the cases) in which
the mood swings in 1 direction only (either depression with
a feeling of worthlessness or depression with groundless
irritability).
➢ The other is Bipolar depression (or manic depression)
characterized by cyclic manifestations of depression
followed by mania (20% of cases).
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❖Monoamine theory
➢It states that depression is caused by a functional deficit of
monoamine transmitters at certain sites in the brain, while mania
results from a functional excess.
➢The major metabolites of noradrenaline and 5-HT, respectively, are
3-methoxy-4-hydroxyphenylglycol (MHPG) and
5-hydroxyindoleacetic acid (5-HIAA). These appear in the
cerebrospinal fluid, blood and urine.
➢ There are two fundamental problems in relating changes in the
concentration of these metabolites in body fluids to changes in
transmitter function in the brain.
• One is that many secondary factors can affect their concentration,
such as diet; transport between cerebrospinal fluid, blood and urine;
or release of monoamines from non-cerebral sites.
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➢ Second is that many patient receive
drug treatment, which affects the
metabolite conc. markedly.
➢Hypothalamic neurons controlling
pituitary function receive noradrenergic
and 5-HT inputs, which control the
discharge of these cells. Hypothalamic
cells release corticotrophin-releasing
hormone (CRH), which stimulates
pituitary cells to secrete
adrenocorticotrophic hormone (ACTH),
leading in turn to cortisol secretion. The
plasma cortisol concentration is usually
high in depressed patients.
Gene transcription
response
Neural apoptosis
Depressive Symptoms
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❖Neurotrophic Hypothesis
➢ BDNF are critical in regulation of neural plasticity and
neurogenesis.
➢ The evidence suggest that depression is associated with the
loss of neurotrophic support and that antidepressant
therapies increase neurogenesis and synaptic connectivity
in cortical areas such as the hippocampus.
➢ BDNF is thought to exert its influence on neuronal survival
and growth effects by activating
the tyrosine kinase receptor in
both ganglia and neurons.
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➢Hypothesis studies indicate that stress and pain are
associated with a drop in BDNF levels and this
contributes to structural changes in the hippocampus and
other areas such as medial frontal cortex & anterior
cingulated.
➢ Over 30 structural imaging studies suggest that major
depression is associated with a 5 to 10 % loss of volume
in the hippocampus.
➢ Depressant appears to be associated with a drop in BDNF
levels in the cerebrospinal fluid and serum as well as with
a decrease in tyrosine kinase receptor B activity.
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➢EMOTIONAL SYMPTOMS
➢A loss of pleasure and interest in usual activities, hobbies
or work.
➢Patients appear sad or depressed and they are often
pessimistic and believe that nothing will help them feel
better.
➢The presence of intense hopelessness may identify
patients at risk of suicide.
➢Anxiety symptoms are present in almost 90% patients.
➢Low self esteem indecisiveness and loss of motivation.
➢Depression with psychotic features requires
hospitalization, especially when patient becomes danger
to self and others.
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❖PHYSICAL SYMPTOMS
➢Chronic fatigue is the common complaint with decreased
ability to perform normal daily tasks. Fatigue usually
appears worst in the morning and does not improve with
rest.
➢Complaints of headache often accompany fatigue.
➢Sleep disturbances: terminal insomnia, with difficulty to
returning to sleep.
➢ They may co-exist with difficulty to falling a sleep
(initial insomnia) and frequent night time awakening.
➢Less frequently, depressed patients complain of increased
sleep (hypersomnia).
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❖ BIOLOGICAL SYMPTOMS
➢ Retardation of thought and action .
➢ Sleep disturbances and loss of appetite.
❖ TREATMENT
➢ Tricyclic/polycyclic antidepressant.
1. Amitriptylline
2. Imipramine
➢ Selective serotonin reuptake inhibitor.
1. Fluoxetine
2. Fluvoxomine
➢ Monoamine oxidase inhibitor.
1. Phenelzine
2. Tranylcypromine
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❖ Tricyclic antidepressant
➢The main immediate effect of TCAs is to block the
uptake of amines by nerve terminals, by competition for the
binding site of the amine transporter .
➢ Synthesis of amines, storage in synaptic vesicles, and
release are not directly affected, although some TCAs
appear to increase transmitter release indirectly by blocking
presynaptic α2-adrenoceptors.
➢Most TCAs inhibit nor-adrenaline and 5-HT uptake by
brain synaptosomes to a similar degree but have much less
effect on dopamine uptake.
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➢Currently these are the most commonly prescribed anti-depressants
and are used for some other psychiatric conditions as well.
➢They selectively increase the levels of serotonin in the synaptic cleft
by blocking its uptake by serotoninergic neurons.
➢In addition they have little ability to block dopamine transporter
also blocking activity at muscarinic, alpha Adrenergic and
histaminergic.
❖Selective Serotonin Reuptake inhibitors
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➢ Monoamino oxidase is a mitochondrial enzyme found in the nerve
and other tissues, such as the gut and liver.
➢ In the neuron, MAO functions as the safety valve to oxidatively
deaminate and inactivate any excess neurotransmitter molecules
(NE, DA and 5-HT) that may leak out of synaptic vesicles when
the neuron is at rest.
➢ Reversible or irreversible inactivate the enzyme, permitting NT
molecules to escape degradation and therefore, to both accumulate
within the presynaptic neuron and leak into the synaptic space.
➢ This causes activation of NE and serotonin receptors and it may be
responsible for the antidepressant action of these drugs.
➢ The MAO inhibitors increase brain amine levels by inhibiting their
metabolism in the nerve endings, resulting in a increase in the
vesicular stores of NA and 5-HT.
❖MAO Inhibitors
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➢ In vitro models
1. Inhibition of [3H]-norepinephrine uptake in rat brain
synaptosomes.
2. Inhibition of [3H]-serotonin uptake in synaptosomes.
3. Inhibition of 3H dopamine uptake in rat striatal
synaptosome.
➢ In vivo models
1. Catalepsy antagonism in chicken
2. Despair swim test
3. Tail suspension test in mice
4. Learned helplessness in rats
5. Muricide behavior in rat
6. Potentiation of norepinephrine toxicity
7. Compulsive gnawing in mice
8. Reserpine induced hypothermia
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1.Inhibition of [3H]-dopamine uptake in rat sterital
synaptosomes.
❖PURPOSE AND RATIONALE
➢High effinity, Saturabale, Temperature and Sodium
dependent of 3H dopamine has been observed in various
tissues preparations from different brain regions.
➢The area striata has a high content of dopamine & is
suitabale for uptake experiments.
➢The 3H dopamine uptake is inhibited by cocaine, certain
phenyl ethylamine & antidpressnts like nomifensine &
bupropion, but not by tricyclic antidepressants.
➢The test can be used to characterize the mode of action of
antidepressant action of antidepressant drugs.
IN-VITRO
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Tissue preparation
Male Wistar rats are decapitated
Brains rapidly removed.
Corpora striata are prepared
(weighed homogenized in 9 volumes
of ice-cold 0.32 M sucrose solution
( using a Potter-Elvejhem homogenizer)
Centrifuged at 1000 g at 0–4 °C
10 min.
❖ Procedure
Supernatent is
decanted
Used for uptake exp.
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100μl of tissue suspension
+ 900 μl 55.5 nM 3H-
dopamine sol in Krebs-
Henseleit bicarbonate buffer
20 ml of the appropriate
drug concentration at 37 °C
under a 95% O2.
(Incubation)
Each assay, 3 tubes are incubated
with 20 μl of vehicle at 0 °C in an
ice bath.
All tubes are immediately
centrifuged at 4000 g for 10 min.
Supernatant fluid is aspirated
Pellets dissolved adding 1 ml of
solubilizer (Triton X-100 + 50%
ethanol, 1 : 4).
Tubes are vigorously shaken, decanted
into scintillation vials, and counted in 10
ml of liquid scintillation cocktail
Active uptake is the difference
between cpm at 37 °C and 0 °C.
❖ ASSAY
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➢The percent inhibition at each drug concentration is the
mean of 3 determinations.
➢IC50 values are derived from log-Probit analysis.
➢IC50 values for nomifensine are 460nm but › 20000nm
for tricyclic antidepressant.
❖EVALUATION
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2.Inhibition of [3H]-serotonin uptake in synaptosomes
➢Serotonergic hypofunction leads to depression and
altering the serotonergic function leads to mood changes
associated with depression.
➢Antidepressants block the reuptake of 5-HT.
➢Therefore, the test can be used to detect compounds that
inhibit serotonin uptake into rat brain synaptosomes and
may be potential antidepressants.
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❖Procedure
❖Tissue preparation
➢Male Wistar rats are decapitated and the brains rapidly
removed.
➢Either the whole brain minus cerebellum or the
hypothalamus is weighed and homogenized in 9 volumes of
ice-cold 0.32 M sucrose solution using a Potter-Elvejhem
homogenizer.
➢The homogenate is centrifuged at 1000 g at 0–4°C for
10 min. The supernatant is decanted and used for further
uptake experiments
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200 μl of tissue suspension
+ 800 μl 62.5 nM 3H-5 HT in
Krebs-Henseleit bicarbonate
buffer
20 μl of the appropriate
drug concentration at 37 °C
under a 95% O2.
(Incubation)
Each assay, 3 tubes are incubated
with 20 μl of vehicle at 0 °C in an
ice bath.
All tubes are immediately
centrifuged at 4000 g for 10 min.
Supernatant fluid is aspirated
Pellets dissolved adding 1 ml of
solubilizer (Triton X-100 + 50%
ethanol, 1 : 4).
Tubes are vigorously shaken, decanted
into scintillation vials, and counted in 10
ml of liquid scintillation cocktail
Active uptake is the difference
between cpm at 37 °C and 0 °C.
❖ ASSAY
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❖EVALUATION
➢The percent inhibition of each drug concentration is the mean of
3 determinations.
➢IC50 values are calculated from log-probit analyses
❖MODIFICATION
➢Hallstrom et al. (1976) studied the platelet uptake of
5-hydroxytryptamine and dopamine in patients with depression.
➢Cloning of functional serotonin transporters has been described by
Blakely et al. (1991) and by Hoffman et al. (1991).
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IN-VIVO
❖Despair swim test
➢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.
➢This behavior reflects a state of despair which can reduced by
several agents which are therapeutically effective in human
depression.
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❖REQUIREMENTS
✓Animal- Male Sprague-Dawley rats
✓Drugs-Imipramine
✓Equipment- Plexiglas cylinder
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❖ PROCEDURE
➢Male Sprague-Dawley rats weighing 160–180gm are used.
➢They are brought to the laboratory at least one day before the
experiment and are housed separately in cages with free access to
food and water.
➢Rats are individually forced to swim inside a vertical Plexiglas
cylinder (height: 40 cm; diameter: 18 cm, containing 15 cm of water
maintained at 25 °C).
➢Rats placed in the cylinders for the first time are initially highly
active, vigorously swimming in circles, trying to climb the wall or
diving to the bottom.
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DESPAIR SWIM TEST
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➢After 2–3 min activity begins to subside and to be
depressed with phases of immobility or floating of
increasing length.
➢ After 5–6 min immobility reaches a plateau where the
rats remain immobile for approximately 80% of the time.
➢After 15 min in the water the rats are removed and
allowed to dry in a heated enclosure (32°C) before being
returned to their home cages.
➢They are again placed in the cylinder 24 hrs later and the
total duration of immobility is measured during a 5 min test.
➢Floating behavior during this 5 min period has been found
to be reproducible in different groups of rats.
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➢An animal is judged to be immobile whenever it remains
floating passively in the water in a slightly hunched but
upright position, its nose just above the surface.
➢Test drugs or standard are administered one hour prior to
testing.
➢Since experiments with the standard drug (imipramine)
showed that injections 1, 5 and 24 h prior the test gave the
most stable results in reducing floating these times are
chosen for the experiment.
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❖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.
➢ Dose-responses can be evaluated.
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❖ Modification
➢Wallach and Hedley (1979) reported that positive results
with antihistamines and Giardina and Ebert (1989) with
an ACE-inhibitor. Differentiation is achieved by the
simultaneous evaluation of motor activity.
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❖ Observation table Duration of immobility(min)
Sl no Wt of rats(gms) Control Std Test
1
2
3
4
5
6
Drug treated before 1 hr
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Duration of immobility(min)
1
2
3
4
5
6
1
2
3
4
5
6
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Final mean reading
Sl no Groups 1 hr 5 hr 24 hr
1 Control
2 Std
3 Test
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❖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
behavioral despair which in turn may reflect depressive disorders in
humans.
➢Clinically effective antidepressants reduce the immobility that mice
display after active and unsuccessful attempts to escape when
suspended by the tail
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❖ PROCEDURE
➢Male mice weighing 20–25 gm are used preferentially.
➢They are housed in plastic cages for at least 10 days prior
to testing in a 12 hrs light cycle with food and water freely
available.
➢Animals are transported from the housing room to the
testing area in their own cages and allowed to adapt to the
new environment for 1 hrs before testing.
➢Groups of 6 animals are treated with the test compounds
or the vehicle by intraperitoneal injection 30 min prior to
testing.
➢For the test the mice are suspended on the edge of a shelf
58 cm above a table top by adhesive tape placed
approximately 1 cm from the tip of the tail.
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Tail suspension test in mice
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➢The duration of immobility is recorded for a period of
5 min.
➢Mice are considered immobile when they hang passively
and completely motionless for at least 1 min.
❖Evaluation
➢The percentage of animals showing the passive behavior
is counted and compared with vehicle treated controls.
➢Using various doses, ED50 values can be calculated.
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❖ Modification
➢ Porsolt et al. (1987), Stéru et al. (1987) recommended
the use of the automated tail suspension test for the
primary screening of psychotropic agents.
➢ A specially developed computerized device
automatically measures the duration of immobility of 6
mice at one time and at the same time provides a
measure of the energy expended by each animal, the
power of the movements.
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❖ Observation table
Sl no Groups Wt of rats(gms) Dose
mg ml
Duration of
immobility(min
)
Normal Saline
1
2
3
4
5
6
Control
1
2
3
4
5
6
Test
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53
➢ Drug discovery and evaluation by H.Gerhard Vogel
➢ Goodman and Gilman’s. The pharmacological basis of
THERAPEUTICS. 11th ed
➢ Pharmacology by H.P.Rang and M.M.Dale
➢ www.google.com
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THANK U
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