Screening of Antidepressants

8/11/2019 Screening of Antidepressants

1/29

By,

Dr. Manoj Upadhya,

SKBCOP, Kamptee


2/29

Extremely common psychiatric condition

oIntense feeling of sadness

oHopelessness

oDespair

oInability to experience pleasure in usual activities


3/29


4/29


5/29

Dopamine

Pleasure,

Reward,

Motivation/Drive

Norepinephrine

Alertness,

Concentration,

Energy.

Serotonin

Obsession &

Compulsions,

Memory.

Mood,

Cognitive

Function

Anxiety,

Impulse,

Irritability

Appetite,

Sex,

Aggression

Attention


6/29


7/29

oTricyclic /polycyclic antidepressants

Imipramine, Desipramine, Amitriptyline etc.

oSelective serotonin reuptake inhibitors (SSRI)

Fluoxetine, Venlafaxine, Sertraline etc.

oMonoamine Oxidase Inhibitors (MAO inhibitors)

Isocarboxazid, Phenelzine, Tranylcypromine etc.


8/29


9/29

Based on reuptake inhibition concept

Procedure:

Tissue Preparation

W. Rats 200-250 g

Brain is removed rapidly

Hypothalamic region is prepared, weighed and homogenized in 1:9 ice cold 0.32 M sucrosesolution

Supernatant decanted and used for assay

(Decapitation)

(Centrifuge at 1000g, 0-4OC for 10 min)

Assay procedure

200l tissue suspension + 800l 62.5nM [3H]-norepinephrine in Krebs-Hanseleit bicarbonate

buffer and 20l of appropriate drug concentration or vehicle

(incubate at 37OC under 95% O2:5%CO2for 5

min, parallel reaction at 0OC)

Immediately centrifuge at 4000g for 10min.


10/29

Supernatant is aspirated and the pallets are dissolved adding 1ml solubilizer

(Triton X-100 + 50 % ethanol, 1:4)

Decanted into scintillation vials, and counted in 10ml of liquid scintillation cocktail

Evaluation

Corpora striata

Hypothalamus or whole brain minus cerebellum


11/29

Procedure:

Expression of Human transporters

Human serotonin transporter cDNA ligated into the expression vector pRc/CMV

Or

Human dopamine transporter cDNA ligated into the expression vector pcDNA3

Transfected to HEK293(human embryonic kidney) cells by Ca2+method

Cell lines are grown and harvested

The growth medium is removed by aspiration

Cell lines are washed with modified Pucks D1 solution

Cell lines are incubated with D1 solution for 5 min

(Centrifuge at 110g for 5 min)

Pellets are suspended in respective binding assay buffers


12/29

Mixture is centrifuged at 35600g for 10 min at 4OC

Supernatant decanted and the final pellets are suspended in respective buffers and stored

at -80OC until assay

Radioligand binding assay

[3H]imipramine binding to human serotonin transporter

Total protein conc. is determined by the Lowry assay using bovine serum albumin as standard


13/29

[3H]nisoxetine binding to human norepinephrine transporter

[3H]WIN35428 binding to human dopamine transporter

Methods similar as above

Methods similar as above

o p-Chloramphetamine (p-CA) causes selective toxicity to serotonin neurons.

o At 10mg/kg i.p. p-CA causes long term decrease in 5-HT, [3H]-5-HT uptake and tryptophan

hydroxylase.

o antagonism of the long-term p-CA induced reduction of synaptosomal 3H-5-HT in-vitro

uptake is a highly useful index of compounds ability to inhibit 5-HT re-uptake in-vivo

Procedure:

Drug treatment

Group of 8 male W.Rats (150-200g)molar equivalent doses of test drugs are

injectedafter 30 min , 4rats from each group are injected with saline or with p-CA (10mg/kg;

i.p.). Three days after treatment the rats are sacrificed.


14/29

Tissue preperation

Rats are decapitated and brains are rapidly removed

Whole brain minus cerebellum is weighed and homoginized 1:9 vol of ice-cold 0.32M sucrose

The supernatant is decanted and used for uptake experiments

(Centrifuge at 1000g at 0-4OC for 10 min)

Assay procedure

200l tissue suspension + 800l Krebs-Hanseleit bicarbonate buffer + [3H]-5-HT

Incubate at 37OC under 95% O2:5%CO2for 5min

Supernatant is aspirated and pellets dissolved by adding 1ml of solubilizer (Triton X-100 and

50% ethanol, 1:4 v/v)

Counted in scintillation counting cocktail

(Centrifuge at 4000g at 0-4OC for 10 min)


15/29


16/29

Adult white leghorn chicken

The animal is grasped with both hands (left hand slightly pushes the chicken down and the righthand supports the animal from the ventral side)

Immediately the chicken is turned on its back and hold for 1min

The chicken remains in cataleptic state for several min up to 1hr

(it may be interrupted by noise or fast movements of the observer)

The animals are pretested in order to be sure about cataleptic activity

After control experiment the animals are treated with test compound or vehicle

The test is performed 4 times every 30 min during 2 h


17/29

Evaluation
http://localhost/var/www/apps/conversion/tmp/scratch_9/The%20Mouse%20Forced%20Swim%20Test%20_%20JoVE%20Video.mov


18/29

S.Mice (20-25g) brought to laboratory at least one day before the experiment

Forced to swim inside a vertical plexiglass cylinder(30cm height with 20cm diameter containing 15cm water at 25OC)

After initial 2-3 min activity the phase of immobility or floating increases

After 5-6 min the immobility reaches to plateau

After 15 min in water the animals are taken out and dried in a heated enclosure (32OC)

After 24h the total immobility time is recorded during 5 min test for each animal

(test drug or standard are administered 1 h prior to testing)

Evaluation
http://localhost/var/www/apps/conversion/tmp/scratch_9/The%20Mouse%20Forced%20Swim%20Test%20_%20JoVE%20Video.movhttp://localhost/var/www/apps/conversion/tmp/scratch_9/The%20Tail%20Suspension%20Test%20_%20JoVE%20Video.mov


19/29

S.Mice (20-25g) brought to test area at least 1 h before the experiment

Test animal is suspended on the edge of the shelf about 58cm above the table top with adhesivetape placed approximately 1cm from the tip of the tail.

The duration of immobility recording is for 5min

(considered immobile when they hand passively and completely motionless for at least 1 min)
http://localhost/var/www/apps/conversion/tmp/scratch_9/The%20Tail%20Suspension%20Test%20_%20JoVE%20Video.mov


20/29

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 (250 to 300g)

Exposed to electric shock (0.7mA) for 1 h on a

schedule of 10s shock/min.

(Platform not available during training)

During test the platform is attached

Grid floor Platform for escape

0.4mA shock is initiated for 10s if animal not escape

If escape occurs the animal is allowed to rest on platform for 10s and then returned to the grid

floor

10 trials in 20s interval


21/29

SD rats (250-300g) are isolated for 6 weeks in individual cages

(Free access to food and water)

After 6 weeks one mouse is introduced into the rats cage

About 10-30 % of rats kill the mouse by biting the animal through the cervical cord

Only rats consistently killing mice within 5 min after presentation are used for the test

Drugs are injected to the rats before the test

Mice are presented 30,60 and 120 min after drug administration


22/29


23/29

Antidepressants block the re-uptake of biogenic amines into nervous tissue. The toxic

effect of norepinephrine are potentiated.

Mice (20-25g)

Test drugs or standard are administered 1h prior to the administration of sub-lethal dose of

3mg/kg of noradrenaline

The mortality rate is assessed 48h post-dosing

ED50 or dose which causes death of 50% of treated animals is calculated


24/29

Apomorphine induces emesis in humans but in rodents it causes compulsive gnawing

instead of vomiting. This is due to dopaminergic stimulation.

Therefore many compounds with psychotropic activity are known to have an

apomorphin-synergistic effect. TCAs also enhance this behavior.

Mice (20-25g)

10mg/kg apomorphine at the same time treated with test drug or vehicle if i.p.or s.c.

(forp.o. activity the drug is administered 30min prior to apomorphine)

Immediately after apomorphine the mice are kept in cages having corrugated paper

The mice start biting the paper causing fine holes or tearing the paper

(Mice are kept for 1h in cage)

This behavior is enhanced by antidepressants


25/29

Evaluation


26/29

Apomorphine induces hypothermia in mice which can be prevented by

antidepressants

Mice (20-25g)

1 h after oral administration of test or vehicle a dose of 16mg/kg apomorphine in injected s.c.

The rectal temperature of each mouse is measured with electronic thermometer immediatelyprior to apomorphine administration and 10, 20 and 30 min later.

Evaluation


27/29

Olfactory bulbactomy in rats is associated with changes in exploratory behavior that

are reversed by chronic , but not acute treatment with antidepressants.

W.Rats (200-250g)

Skull is exposed and a burr hole is drilled at points 7mm anterior to bregma and 2mm either side

of midline

The olfactory bulbs are removed by suction and the holes are filled with sterile hemostatic

sponge. (Sham surgery is also carried out)

The wound is closed with due care and application of antibiotic and stitched

Animals are allowed to recover for 14days after surgery

Animals are treated with test drug or standard or vehicle at 09:00 daily for 14 days


28/29

The behavior is tested 12thday onwards in open-field apparatus

Ambulation (No. of squares crossed), Rearing (forepaws raised from the floor), grooming and

defecation scores are recorded for 3min period observation.

Evaluation


29/29

Screening of Antidepressants

Documents

Transcript of Screening of Antidepressants