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Screening of antidepressant
1. Screening of antidepressants
DR MANU KUMAR
POST GRADUATE
DEPT OF PHARMACOLOGY
VMMC & SAFDARJUNG HOSPITAL
NEW DELHI
2. What is depression
Depression is a common mental disorder that presents
with low mood, loss of interest or pleasure(anhedonia),
feelings of guilt or low self-worth, disturbed sleep or
appetite, low energy, and poor concentration.
Suicide – 8,50,000 lives every year
4th leading contributor to the global burden of disease
(DALYs) in 2000
Prevalence
3-5% (point prevalence)
20% (lifetime prevalence)
3. For accurate diagnosis of MDD
Five of the following nine DSM-IV symptoms must be
present continuously for a minimum 2-week period:
“Depressed mood”
“Loss of interest or pleasure”
Significant weight or appetite alteration
Insomnia or hyposomnia
Psychomotor agitation or retardation
Fatigue or loss of energy
Feelings of worthlessness
Diminished ability to think or concentrate or
indecisiveness; and
Suicidal ideation.
4. Ethiopathogenesis
Multifactorial
History of antidepressants Henri Laborit
1950s- no treatment for psychiatric disorder.
Promethazine-promazine-Cl-imipramine
Ipraniazide- antitubercular drug
Reserpine – antihypertensive
1980s -SSRIs
6. Monoamine hypothesis
functional deficit of NE and/or
5-HT in certain sites of brain.
Antidepressants drugs acts –inhibiting uptake and
facilitate the NE/5-HT neurotransmission.
Reserpine inhibit the storage of 5-HT and NE –
depression
Tryptophan increase the 5-HT synthesis –elevate the
mood
7. Limitation of this hypothesis
Antidepressant action produce within hour but,
clinical benefit takes several weeks,
Amphetamine and cocaine not used as
antidepressant despite their ability to facilitate NE
transmission.
Atypical action antidepressants –tianeptine,
bupropion , mianserine
8.
9. Neuroendocrine hypothesis
Increase in CRF,- administration produce behaviour
changes similar to stress, (CRF antagonist CP-
154,526, R-121919)
Increase in ACTH
Weak response of plasma cortisol to exogenous
steroids (dexamethasone suppression test)
Dampened negative feedback mechanism ,
GR-II receptor- regulate HPA by negative feedback
10. Need for screening
Severe side effects of the existing drugs. So need for
more safe drugs
Need to find more efficacious drugs
Problems associated with screening of anti depressants
Lack of animal models that resemble depressive illness
in humans
Most of the existing models concentrate on monoamine
theory of depression only
11.
12. In vitro assays
Assays based on inhibition of amine uptake
Assays based on binding to the receptors
Though complicated but these are precise and accurate
13. Inhibition of [3H] norepinephrine uptake in rat brain
synaptosomes
Inhibition of [3H] dopamine uptake in rat striatal
synaptosomes
Inhibition of [3H] serotonin uptake in synaptosomes
Binding to monoamine transporters
Measurement of β adrenoceptors stimulated adenylate
cyclase
[3H] yohimbine binding to α2 adrenoceptors in rat
cerebral cortex
14. Assays based on inhibition of amine uptake
(Norepinephrine/Dopamine/Serotonin)
Isolate hypothalamus/ corpora striata / hypothalamus or whole brain minus
cerebellum. Homogenize with 0.32 M sucrose solution.
The homogenate is centrifuged
The supernatant is incubated with 3H-norepinephrine /Dopamine / serotonin
in Krebs-Henseleit bicarbonate buffer and appropriate drug concentration (or
the vehicle) at 37 °C and then centrifuge.
The supernatant fluid is aspirated and the pellets dissolved adding 1 ml of
solubilizer (Triton X-100 + 50% ethanol, 1 : 4).
15. Cont.
Take the count for radioactivity by liquid scintillography
IC50 are derived & compared with standards.
16. Basic procedure of receptor binding Assay
Isolate the cells with receptors
Add radioactive ligand for that receptor in presence and
absence of test drugs
Count the receptor ligand binding by liquid scintillography.
17. In vivo model
1. Gross behavior test.
2. Test based on inhibition of amine uptake.
3. Test Based on anticholinergic activity.
4. Test based on depletion of biogenic amine.
5. Hypermotility in olfactory-bulbectomized rats
18. Behavioural tests
Forced swim test
Tail suspension test in mice
Learned helpnessness in rats
Muricide behaviour in rats
Behavioural changes after neonatal clomipramine
treatment
Catalepsy antagonism in chicken
Open space swimming test
19. Forced swim test
Purpose and Rationale :
It was proposed as a model to test for
anti depressant activity by Porsolt et al.
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.
Procedure:
Naïve rats are individually forced to swim inside a vertical Plexiglas
cylinder (height: 40cm;diameter:18cm containing 15cm of water
maintained at 25deg cel)
Initially rats are hyperactive. After 2-3 min activity begins to subside
and is interspersed with phases of immobility or floating of increasing
length.
After 5-6 min immobility reaches a plateau where the rat remains
immobile for approx 80% of the time.
20. Forced swim test
After 15min in water rats are removed and allowed to dry in a heated
enclosuren (32deg cel) before being returned to their home cages.
They are again placed in the cylinder 24h later and the total duration of
immobility is measured during a 5min test.
Test drugs or standard are administered 1h prior to testing.
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 .
Differentiation is done by measurement of locomotor Activity by open
field test
21. Cont.
Open field apparatus - an arena 70 cm in diameter divided
into 9 or 18 approximately equal areas.
Each rat is individually placed in the center of the arena 15
h after the last treatment and its behavioural parameters
are recorded for 5 min.
Score is calculated
Locomotion (number of line crossings within 5 min)
Rearing frequencies (number of times an animal stood on
its hind legs).
22. 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 behavioural
despair which reflects depressive disorders in humans
Antidepressants reduce the immobility that rats display after active and
unsuccessful attempts to escape when suspended by the tail
23. Procedure
20 Male Balb/cJ mice (20-25 gm) divided in 2 groups
Housed in plastic cages with food and water ad libitum
Treated i.p with test drug or vehicle
30 min later, mice are suspended on the shelf 58 cm above the table top
by adhesive tape placed at 1 cm from the tip of the tail
Duration of immobility recorded for 6 min
Mice at considered immobile when they hang passively and completely
motionless for at least 1 min
24. Evaluation
Total immobility is compared with control , decrease in immobility
time after test drug indicate antidepressant action.
Using various doses, ED50 values can be calculated
Critical assessment
Easy method
SSRI are sensitive to this model .
25. Learned helplessness in rats
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 (300 g)
Apparatus - Box with a grid floor having a platform which can be
inserted through one side wall to allow a jump-up escape response.
Training - Exposure to electric shock (0.7 mA) for 1 h on a schedule of
10 s of shock/min.
The platform is not available
during training.
This training resulted in 80%
acquiring learned helplessness behavior.
26. Cont.
Trial
The platform is pushed into the box and a 0.4 mA shock initiated.
Shock is terminated in 10 s if the animal has not escaped onto the
platform by this time.
Ten such trials with an intertrial interval of 20 s are given.
EVALUATION
A drug is considered to be effective, if the learned helplessness is
reduced and the number of failures to escape is decreased.
27. Muricide behavior in rats
Male Sprague-Dawley rats (300–350 g)
Only rats consistently killing mice within 5 min after presentation are used for
the test.
Drugs are injected i.p. to the rats before the test. Mice are presented 30, 60 and
120 min after drug administration.
EVALUATION
Failure to kill a mouse within 5 min is considered inhibition of muricidal
behavior.
ED50 is calculated, the ED50 is defined as the dose which inhibits mouse
killing in 50% of the rats.
The mouse-killing behavior is also inhibited d-amphetamine some
antihistamines and some cholinergic drugs.
28. Some other models are…….
Behavioral changes after neonatal clomipramine
treatment.
Antidepressant-like activity in differential
reinforcement of low rate 72-second schedule.
Catalepsy antagonism in chicken.
30. Test based on inhibition of amine uptake
Potentiation of norepinephrine toxicity in mice
Male NMRI mice (22–25 g)
The test drug, the standard or the vehicle are given orally 1 h prior to the s.c.
injection of the sublethal dose of 3 mg/kg noradrenaline.
The mortality rate is assessed 48 h post-dosing. ED50 is calculated.
5-Hydroxytryptophan potentiation in mice/rats
DL-5-Hydroxytryptophan is used as the precursor of serotonin.
Enzymatic breakdown is inhibited by the MAO-inhibitor pargyline.
In mice the characteristic symptom of head-twitches is observed.
31. CONT.
0 min 30 min 120 min
Test Drug /vehicle
Pargyline Hcl s.c. 10 mg/kg DL -5 hydroxytryptophan
i.v.
A animal is considered to be positive if it shows head twitches 15 min
after 5- HTP injection. Enhancement is observed after treatment with
serotonin uptake blockers relative to animals pretreated with
pargyline only.
The head-twitch in mice can also be elicited without a MAO-inhibitor
by using higher doses (200 mg/kg) of DL-5-hydroxytryptophan.
32. .
3. Test Based on anticholinergic activity.
a) Compulsive gnawing in mice
Treatment of rodents with apomorphine causes compulsive gnawing
instead of vomiting due to dopaminergic stimulation.
Anticholinergics shift the balance between Ach & dopamine resulting
in an enhancement of apomorphine effect.
33. Cont.
NMRI mice(18–20 g) are injected s.c. with 10 mg/kg
apomorphine + test drug/vehicle at the same time.
Immediately , mice are placed in a cage with corrugated
paper the corrugation facing upwards for 1 hour.
The mice start to bite into the paper causing fine holes or
tear the paper.
Percentage of damaged paper in calculated.
This behavior is enhanced by antidepressants.
Not only antidepressants, but also centrally acting
anticholinergics and antihistaminics are active in this test.
34. Test based on depletion of biogenic amine
a) Tetrabenazine antagonism in mice
Tetrabenazine (TBZ) induces a depletion of biogenic
amines (e.g. noradrenaline, dopamine, serotonin)
without affecting their de novo synthesis.
Antidepressants antagonize the effect of TBZ
male NMRI mice (20–22 g)
Catalepsy and ptosis are used as criteria.
degree of ptosis and catalapsy is scored
The scores of the TBZ controls are taken as 100% and the
percentage is calculated for the treated animals.
35. Reserpine induced hypothermia
Depletion of biogenic amines (noradrenaline, 5-
hydroxytryptamine, dopamine) in the brain also induces
hypothermia in rodents.
The decrease of body temperature induced by reserpine is
antagonized by antidepressants.
male NMRI mice (19–21 g)
Rectal temperature is recorded every hour.
The difference in temperature from vehicle controls is
calculated for each time and the maximal difference is
scored.
The reversal of hypothermia is not specific for
antidepressants. The fall in body temperature can also be
antagonized by amphetamines, and some antipsychotic
agents (chlorpromazine).
36. Hypermotility in olfactory-
bulbectomized rats
Bilateral olfactory bulbectomy in the rat is associated with changes in
exploratory behavior that are reversed by chronic, but not acute
treatment with antidepressant drugs.
Male Sprague Dawley rats
The animals are allowed to recover for 14 days after surgery.
the animals are treated s.c. with the test drug / standard / vehicle once
daily for 14 days.
The behavior of the animals is tested from the 12th day onwards.
The rats are placed singly in the center of an open field apparatus.
37. Yohimbine toxicity enhancement
Purpose and rationale
Yohimbine occupies central α2 receptors and prevents NE
from binding to these receptors
Antidepressants block the reuptake of NE into the nerve
terminals
Combination of both can thus produce death in animals
due to NE toxicity
38. Procedure
20 male NMRI mice (25-28 gm) divided in 2 groups
Treated with test drug or the vehicle by oral or i.p route
After 30 min, 25 mg/kg yohimbine (sublethal dose) given
s/c
Evaluation
Mortality assessed at 1, 2, 3, 4, 5 and 24 hr after dosing
Mortality rates compared between 2 groups
Using various doses, ED50 values can be calculated.
Core clinical feature –anhedonia, Chronicstae in 5-10yr 12-17%, relapse of depression, mortality is due to suicides, disability-adjusted life year (DALY) is a measure of overall disease burden, expressed as the number of years lost due to ill-health, disability or early death, 2nd place in 2020. average 15% eventually die by suicide, and 79% of suicide is due to depression in US. 75% of pts develop major depression episode in 10yr, female to male 5.2%,,,
Diagnostic and statistical manual of mental health, al –IV, by american psychiatric assocoation provides common knowledge and std criteria for classification of metntaldisordder, DSM-V is expected may 2013, most of the day nearly every day, subjective or observational in child, change of more 5% body wt in month,
Multifactorial – genetic, neurotrasmitter dysfunction, psychosocial stress , chronic illness,1950 tretaament for pshiatric – insuline coma, ECT, henri – neurosurgeon, for op used to give antihistamicspromethazine, he asked ciba-geigy company( presently novartis) for more potent antihistamic, this leads to Imipremine, CSF concentartion of these metabolite- 5-HIAA, 3MHPG decraese in depression. Henri laborit
It has been suggested that this delayed onset of clinical effect may be due, at least in part, to the need for desensitization of somatodendritic 5-HT1A autoreceptors in the dorsal raphe (Le Poul et al, 1995). Hence, it has been hypothesized that concomitant therapy with an SSRI increasing 5-HT in the forebrain and a 5-HT1A receptor antagonist preventing feedback via blockade of the autoreceptors may result in a reduction of the time to onset of efficacy in the clinic. SB-649,915(by safonipharma) in phase II is a serotonin reuptake inhibitor and 5-HT1A and 5-HT1B receptor antagonist which is being investigated for its antidepressant effects……..elsonanaElzasonan (CP-448,187) is a selective 5-HT1B and 5-HT1D receptor antagonist that was under development by Pfizer for the treatment of depression, , Nd1251-Phosphoesterase 4 inhibitors- for depression is under clinical trial by sartorious companycAMP response element binding protein, regulate set of gene resposible for antidepressant action.BDNP, long term anti-d causes upregulation of cAMP and CREB system,
Tianeptin- enhance serotonine reuptake, bupropion- weak inhibitor of DNES reuptake, mianserine block presynaptic alpha receptor
R-121919 halted in phase IIa due to hepatotaxicity, it hs promised antidepressnteffcicay, NBI pharma, CRF –regulate tyrosine hydroxylase link of monoamine hypothesis
Imipramine – novartice, arvindcarlssonzemilitide first SSRi, then by fluoxetine Bryan Molloy and Robert Rathbun. Lilly,
The immobility is thought to reflect either a failure of persistence in escape-directed behaviour (i.e. behavioral despair total loss of hope, ) or the development of passive behaviour that disengages the animal from active forms of coping with stressful stimuli.
Rats wewre isolated for 6 week, then mice were introduced to rat cage,
Median effective dose ,(ED50) a dose that produces the desired effect in 50 per cent of a population, median curative dose (CD50) a dose that abolishes symptoms in 50 per cent of test subjects
Third, central administration of substance P produces behavioural and cardiovascular responses that resemble those seen following stressful stimuli. Fourth, there is significant overlap between 5-HT and noradrenaline pathways and substance P in limbic brain areas, and repeated administration of traditional antidepressants leads to decreased synthesis of substance P in certain brain regions. Fifth, mice in whom the NK1receptor has been knocked out are less anxious; neukinie receptor