Preclinical screening of antidepressants involves testing drugs in vitro using assays that measure inhibition of neurotransmitter reuptake and in vivo using behavioral tests in animal models. Common animal models induce depression-like behaviors through methods like neonatal clomipramine treatment or tetrabenazine-induced depletion of monoamines. Tests evaluate parameters like immobility time in forced swim tests. Leading classes of antidepressants work by inhibiting the reuptake of monoamines like serotonin and norepinephrine.

1. Pre clinical screening of Anti
depressants
Submitted by Aquib Naseer
M.Pharm 1st sem (Pharmacology)
School of Pharmaceutical Education & research (SPER)
Jamia Hamdard New Delhi

2. Contents :
1. Introduction .
2. Monoamines synthesis & uptake in neurons.
3. Pathophysiology & associated hypothesis.
4. Classes of drugs used to treat Depression.
5. Animals used and Screening methods
6. Method of induction of disorder
7. Grouping and treatment of animals
8. Duration of Study
9. Parameters to be evaluated
10. Blank Observation table
11. References

3. Depression :
• Depression is the most common of the affective disorders (defined as disorders of mood rather than
disturbances of thought or cognition); it may range from a very mild condition, bordering on normality, to
severe (psychotic) depression accompanied by hallucinations and delusions.
• Depression is not a homogeneous disorder, but a complex phenomenon , which has many subtypes and
probably more than one etiology.
• Depression symptoms can vary from mild to severe and can include :-
1. Feeling sad or having a depressed mood
2. Loss of interest or pleasure in activities once enjoyed
3. Changes in appetite — weight loss or gain unrelated to dieting
4. Trouble sleeping or sleeping too much
5. Loss of energy or increased fatigue
6. Increase in purposeless physical activity (e.g., hand-wringing or pacing) or slowed movements and speech
(actions observable by others)
7. Feeling worthless or guilty
8. Difficulty thinking, concentrating or making decisions
9. Thoughts of death or suicide

4. Risk Factors for Depression :
• Depression can affect anyone—even a person who appears to live in relatively ideal
circumstances.
• Several factors can play a role in depression:
1. Biochemistry: Differences in certain chemicals in the brain may contribute to symptoms of
depression.
2. Genetics: Depression can run in families. For example, if one identical twin has depression, the
other has a 70 percent chance of having the illness sometime in life.
3. Personality: People with low self-esteem, who are easily overwhelmed by stress, or who are
generally pessimistic appear to be more likely to experience depression.
4. Environmental factors: Continuous exposure to violence, neglect, abuse or poverty may make
some people more vulnerable to depression.

5. • According to the criteria of the Diagnostic and Statistical Manual of Mental Health,Fourth Edition
(DSM-IV).
• For an appropriate diagnosis , five of the following nine DSM-IV symptoms must be present
continuously for a minimum 2-week period :
(I) Depressed mood .
(ii) loss of interest or pleasure .
(iii) Significant weight or appetite alteration .
(iv) Insomnia or hyposomnia .
(v) psychomotor agitation or retardation .
(vi) fatigue or loss of energy; (vii) feelings of worthlessness .
(viii) Diminished ability to think or concentrate or indecisiveness and
(ix) suicidal ideation.
Criteria of declaring a person as depressed :

6. Types :
• DSM-IV divides depression into two basic categories :
1)Bipolar depression. 2) Unipolar depressive disorders.
• Bipolar affective disorder depression alternates with mania whereas in
• unipolar depression mood changes are always in the same direction .

7. Physiology of Neurotransmitters (Biogenic monoamines) :

8. Biosynthesis of Monoamines :

9. Pathophysiology :

10. Mechanisms believed to be involved in the pathophysiology of depression :

11. Pathophysiologic hypothesis :
1) Monoamine hypothesis : (proposed by Schildkraut in 1965)
• This first major hypothesis proposed that the main symptoms of depression are due to a functional
deficiency of the brain monoaminergic transmitters :-
1. Norepinephrine (NE),
2. 5-HT, and/or dopamine (DA),
whereas mania is caused by functional excess of monoamines at critical synapses in the brain.
• Evidence for this hypothesis came from clinical observations and animal experiments, which showed that
the antihypertensive drug reserpine, which causes a depletion of presynaptic stores of NE, 5-HT, and DA,
induced a syndrome resembling depression.
• In contrast to the effects obtained with reserpine , euphoria and hyperactive behavior were observed in
some patients being treated with iproniazid , a compound synthesized for the treatment of tuberculosis,
which increased brain concentrations of NE and 5-HT by inhibiting the metabolic enzyme MAO.

12. • Repeated data showing decreased levels of the NE metabolite α-methoxy-4-hydroxyphenylglycol (MHPG) in
blood/urine of depressive patients , supporting the hypothesis of a deficient noradrenergic system .
• Similarly, the data on determinations of 5-HT and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) in
blood/urine depressed patients prove the hypothesis of exclusively reduced serotonergic transmission in
depressed patients.
Evaluation of neurotransmitter concentration :

13. 2) Endocrine processes in depression :
• A variety of hormonal abnormalities, such as altered levels of cortisol, growth hormone (GH), or
thyroid hormones, indicate the existence of endocrine disturbances, especially dysfunctions in
the hypothalamuspituitary- adrenal (HPA) axis.
• The observations include hypersecretion of hypothalamic corticotropin-releasing hormone (CRH)
and inadequate glucocorticoid feedback inhibition , increased cortisol levels .
• Alterations in thyroid function have been repeatedly linked to depression and the administration
of triiodothyronine (T3) seems to be an effective adjunctive treatment for many patients

14. Drugs used to treat depression :

15. TYPES OF ANTIDEPRESSANT DRUG :
Antidepressant drugs fall into the following categories :
1) Inhibitors of monoamine uptake .
2) Monoamine receptor antagonists.
3) Monoamine oxidase inhibitors (MAOIs).

16. Inhibitors of monoamine uptake :
• Selective serotonin (5-HT) reuptake inhibitors (SSRIs) (e.g. fluoxetine, fluvoxamine, paroxetine, sertraline,
citalopram, escitalopram).
• Classical tricyclic antidepressants (TCAs) (e.g. imipramine, desipramine, amitriptyline, nortriptyline,
clomipramine). These vary in their ability to inhibit noradrenaline and 5-HT reuptake.
• Newer, mixed 5-HT and noradrenaline reuptake inhibitors (e.g. venlafaxine [somewhat selective for 5-HT],
desvenlafaxine, duloxetine, milnacipran).
• Noradrenaline reuptake inhibitors (e.g. bupropion, reboxetine, atomoxetine).

17. Monoamine receptor antagonists :
• Drugs such as mirtazapine, trazodone, mianserin are non-selective and inhibit a range of amine
receptors including presynaptic α2 adrenoceptors (autoreceptors) and 5-HT2 receptors. They may
also have weak effects on monoamine uptake.
Monoamine oxidase inhibitors (MAOIs) :
Irreversible, non-competitive inhibitors (e.g. phenelzine, tranylcypromine), which are non-selective with respect
to the MAO-A and -B subtypes.
Reversible, MAO-A-selective inhibitors (e.g. moclobemide).

18. Site of action of 2 main classes of anti
depressants :-
1) TCAs
2) SSRIs

19. Drug screening methods :
Invitro methods Invivo methods (Behavioural test)
1) Inhibition of [3H]-norepinephrine uptake in rat
brain synaptosomes
1) Catalepsy antagonism in chicken
2) Inhibition of [3H]-dopamine uptake in rat striatal
synaptosomes
2) Despair swim test
3) Inhibition of [3H]-serotonin uptake
in synaptosomes
3) Tail suspension test in mice
4) Binding to monoamine transporters. 5)Muricide behavior in rats
5) Radioligand binding assays 7) Potentiation of norepinephrine toxicity

20. Animals used in in-vivo testing of anti depressants :
1. Male Sprague Dawley rats.
2. Albino –swiss rats.
3. Male wistar rats
4. Macca fasicularis or female cynomologus monkeys for post partum
depression.

21. Methods of induction of disorder :
• Induction of depression by any pharmacologically active compound in
animals is not yet available as such , yet changes in behaviour in animals
have been taken as a substitute , some of them are :
1. Clomipramine : neonatally administered clomipramine induces
endogenous like depression in adult animals leading to behavioural
changes during electroshock stimulus .
2. Tetrabenazine induced depletion of biogenic amines(DA,NA,5-
HT).Tetrabenazine antagonism in mice is evaluated by cataleptic and ptosis
criteria.
3. Reserpine induced hypothermia alongwith depletion of biogenic amines in
rodents is a criteria harvested for evaluation of anti depressants.

22. Drugs used as Standard :
• Desipramine & nor-tryptyline are used in in-vivo tests e.g Inhibition of [3H]-
norepinephrine uptake in rat brain synaptosomes
• Chlorimipramine in Inhibition of [3H]-serotonin uptake in synaptosomes.
• Imipramine in Learned helplessness in rats

23. Grouping and treatment of animals in
clomipramine treated rat neonates :
Group number Group name Volume (ml)
1 Control group N.S twice daily postnatal days (day 8 – 21
postnatal)
2 Inducing agent e.g clompiramine 15mg/kg clomipramine hydrochloride
3 Standard group _ml Inducing agent + _ml
Standard drug
4 Test drug (dose 1) group
TD1
_ml Inducing agent + _ml Test
drug (low dose)
5 Test drug (dose 2) group
TD2
_ml Inducing agent + _ml test
drug (high dose)
6 Standard drug per se _ml Std. drug (low dose)
7 Test drug per se _ml Test drug (high dose)

24. Duration of study :
• In-Vitro studies:
Usually 1-3 hour is utilized for the in-vitro preclinical studies. The time
may extend because of the preparation of the tissue or depends on the
type of bioassay employed for the study.
• In-Vivo studies:
The duration of study may vary from 24 hour to about 15 days as per the
method employed.
1. Catalepsy antagonism in chicken – 5 days.
2. Despair swim test – 24hrs .
3. Tail suspension test -5-6 mins
4. Learned helplesseness in rats – 5 mins maximum

25. 1. Inhibition of [3H]-norepinephrine uptake in rat brain synaptosomes ; 60-90 mins
minimum.
2. Inhibition of [3H]-dopamine uptake in rat striatal synaptosomes ; 60-90 mins minimum.
3. Inhibition of [3H]-serotonin uptake in synaptosomes ; 60-90 mins minimum.

26. Parameters to be evaluated :
• Invitro methods :
• IC50 values from log – probit analysis .
• Invivo methods (behavioural test parameters) :
• Catalepsy antagonism in chicken :-
1. Positive if cataleptic rigor does not occur after treatment or is interrupted
spontaneously within one min at least twice during the 2 hr test period.
2. ED50 values are calculated.
• Despair swim test :
1. Duration of immobility is measured in controls and animals treated with various doses
of a test drug or standard.
2. Dose responses can be evaluated.

27. • Tail suspension test in mice :
1. ED50 values are calculated .
2. Percentage of animals showing the passive behavior is counted and compared.
• Muricide behavior in rats :
1. Failure to kill a mouse within 5 min is considered inhibition of muricidal behavior.
2. ED50 is calculated.
• Behavioural changes after neonatal clomipramine treatment :
1. Individual behaviors of treatment and control groups are listed for offensive and defensive
behaviours on day 1 , 2 , 3 and total of scores are calculated for each group.
• Apomorphine induced hypothermia in mice :
1. Percent inhibition of apomorphine induced hypothermia in control group.
2. ED50 is calculated.

28. Blank observation table :
Group Treatment Tail suspension test /forced swim test.
Immobility time in seconds
Dose Response
1 Control ___ ±_____ ___ ±_____
2 Standard drug ___ ±_____ ___ ±_____
3 Test drug ___ ±_____ ___ ±_____
4
In-vitroIn-vivo
Values represented in mean (___ ±__ S.E.M)

29. References :
• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3181770/
• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3498773/
• https://www.webmd.com/depression/guide/depression-types#1
• https://www.medicinenet.com/depression/article.htm
• Goodman & Gilman’s the pharmacological basis of pharmacotherapeutics 11th edition/ chapter
17/DRUG THERAPY OF DEPRESSION AND Anxiety disorders / Ross J baldessarini.
• Rang and Dale’s Pharmacology/chapter 4 / unit 46/Antidepressant drugs.
• Drug discovery and evaluation pharmacological assays second edition 2nd edition /Wolfgang
H.Vogel Bernward A. Schölkens, Jürgen Sandow, Günter Müller ,Wolfgang F. Vogel.
• Drug screening and methods / SK Gupta / Jaypee publishers/chapter 27/Anti depressant Agents.