Depression and methods of screening of antidepressants.

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

5. CP-448,187, antagonist, SR-46349 II
SB-649,915, NAD-299 III
ND-1251, phosphodiestarse 4 inhibitor
Sartorious I

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

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

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.

29. Tests based on mechanism of action

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.

39. Thank you