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““A REVIEW ON PRECLINICAL A REVIEW ON PRECLINICAL EVALUATION OF ANTIDEPRESSANTS”EVALUATION OF ANTIDEPRESSANTS”

PRESENTING BY,PRESENTING BY,Ms. SONALI B. DIWATEMs. SONALI B. DIWATE

GUIDED BY,GUIDED BY,PROF. R. CHANSHETTI PROF. R. CHANSHETTI

MODERN C.O.P.,MOSHI, PUNE-412105 MODERN C.O.P.,MOSHI, PUNE-412105

To Review and Study the Preclinical Evaluation of Antidepressants To Review and Study the Preclinical Evaluation of Antidepressants

OBJECTIVEOBJECTIVE

1. COLLECT THE INFORMATION ABOUT DEPRESSION & ANTIDEPRESSANTS. 1. COLLECT THE INFORMATION ABOUT DEPRESSION & ANTIDEPRESSANTS. (LITERATURE SURVEY).(LITERATURE SURVEY).

2. SURVEY THE STATUS AND STANDARD ANTIDEPRESSANT DRUGS AVAILABLE IN 2. SURVEY THE STATUS AND STANDARD ANTIDEPRESSANT DRUGS AVAILABLE IN MARKET.MARKET.

3. STUDY THE COMMON ADR & USES OF ANTIDEPRESSANTS.3. STUDY THE COMMON ADR & USES OF ANTIDEPRESSANTS.4. STUDY THE PHRMACOKINETICS,PHARMACODYNAMICS OF ANTIDEPRESSANTS.4. STUDY THE PHRMACOKINETICS,PHARMACODYNAMICS OF ANTIDEPRESSANTS.5. EXPLAIN INVIVO & INVTRO MODELS FOR PRECLINICAL EVALUATION OF 5. EXPLAIN INVIVO & INVTRO MODELS FOR PRECLINICAL EVALUATION OF

ANTIDEPRESSANTS.ANTIDEPRESSANTS.6. ANALYZE ALL THE PARAMETERS RELATED TO ANTIDEPRESSANTS6. ANALYZE ALL THE PARAMETERS RELATED TO ANTIDEPRESSANTS7.INVESTIGATE RESEARCH SCOPE IN THIS AREA.7.INVESTIGATE RESEARCH SCOPE IN THIS AREA.8. AWARE PEOPLE ABOUT DEPRESSION & ANTIDEPRESSANTS. 8. AWARE PEOPLE ABOUT DEPRESSION & ANTIDEPRESSANTS. 9.SUMMERIZATION OF THE INFORMATION.9.SUMMERIZATION OF THE INFORMATION. 2

CONTENTS…..CONTENTS…..

1. AIM AND OBJECTIVE1. AIM AND OBJECTIVE2.INTRODUCTION TO DEPRESSION & ANTIDEPRESSANTS 2.INTRODUCTION TO DEPRESSION & ANTIDEPRESSANTS 3.STANDARD ANTIDEPRESSANTS AVAILABLE IN MARKET3.STANDARD ANTIDEPRESSANTS AVAILABLE IN MARKET4. COMMON ADR & USES OF ANTIDEPRESSANTS4. COMMON ADR & USES OF ANTIDEPRESSANTS5. PHARMACOKINETICS & PHARMACODYNAMICS5. PHARMACOKINETICS & PHARMACODYNAMICS6. PRECLINICAL SCREENING METHODS6. PRECLINICAL SCREENING METHODS7. SUMMERY7. SUMMERY8. CONCLUSION8. CONCLUSION9. BIBLIOGRAPHY9. BIBLIOGRAPHY

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INTRODUCTION TO DEPRESSIONINTRODUCTION TO DEPRESSION[3]

DEPRESSION is a word used in many different contexts and can cover many different meanings. in a medical context the term depression is used to describe a syndrome, that is a cluster of symptoms which often occur together, but where the underlying causes may vary.

The Symptoms of Depression1. Decreased energy, fatigue, feeling “slowed down”2. Depressed mood 3. Difficulty in concentrating or making decisions 4 .Feeling restless.5. Feelings of worthlessness or inappropriate guilt 6. Insomnia, early-morning awakening, or oversleeping 7. Loss of interest or pleasure in hobbies, work 8. Recurrent thoughts of death or suicide; suicide attempts

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TYPES OF DEPRESSION UNIPOLAR (EMOTIONAL SYMPTOMS) BIPOLAR (BIOLOGICAL SYMPTOMS)

HISTORY OF ANTIDEPRESSANTSHISTORY OF ANTIDEPRESSANTSThe drugs or agents which are used in treatment of depression is called as

antidepressantsAntidepressants acts by balancing brain neurotransmitters level to ease

depression.

CLASSIFICATION OF ANTIDEPRESSANTS

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INTRODUCTION TO ANTIDEPRESSANTSINTRODUCTION TO ANTIDEPRESSANTS[1]

1. TCAs: Amitriptyline, Imipramine, Clomipramine, Nortriptyline, Desipramine. 2. Tetracyclic antidepressants: Mianserin, Maprotiline. 3. SSRIs: Fluoxetine, Paroxetine, Sertraline, Fluvoxamine, Citalopram, Escitalopram. 4. SNRIs: Venlafaxine/ Venlafaxine XR, Duloxetine. 5. MAOIs: Moclobemide, Phenelzine, Isocarboxazid, Tranylcypromine. 6. Lithium salts: Lithium carbonate.

STANDARD DRUGS AVAILABLE IN MARKETSTANDARD DRUGS AVAILABLE IN MARKET[5]

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COMMON ADVERSE DRUG REACTIONCOMMON ADVERSE DRUG REACTION[5]

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DRUG INTERACTIONS [2]

CONTRAINDICATIONS [2]

The mechanism of action of tricyclic antidepressants Tricyclic antidepressants (TCAs) are a group of drugs used to treat affective, or ‘mood’,

disorders. Mood disorders are associated with reduced levels of monoamines in the brain. TCAs binding to 5-HT and noradrenalin re-uptake transporters prevents the re-uptake of these monoamines from the synaptic cleft and their subsequent degradation. This re-uptake blockade leads to the accumulation of 5-HT and noradrenalin in the synaptic cleft and the concentration returns to within the normal range.

The mechanism of action of SSRI antidepressants • SSRIs ease depression by affecting naturally occurring chemical messengers

(neurotransmitters), which are used to communicate between brain cells. SSRIs block the reabsorption (reuptake) of the neurotransmitter serotonin in the brain. Changing the balance of serotonin seems to help brain cells send and receive chemical messages, which in turn boosts mood.

• Most antidepressants work by changing the levels of one or more of these neurotransmitters. SSRIs are called selective because they seem to primarily affect serotonin, not other neurotransmitters.

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PHARMACOKINETICS & PHARMACODYNAMICS OF ANTIDEPRESSANTSPHARMACOKINETICS & PHARMACODYNAMICS OF ANTIDEPRESSANTS[ 3]

1) GIVEN BY ORAL ROUTE2) METABOLISM BY LIVER3) EXCREATION THROUGH URINE

PHARMACODYNAMICS OF ANTIDEPRESSANTSPHARMACODYNAMICS OF ANTIDEPRESSANTS

The mechanism of action of SNRI antidepressants• Serotonin (ser-o-TOE-nin) and norepinephrine (nor-ep-ih-NEF-rin) reuptake

inhibitors ease depression by affecting chemical messengers (neurotransmitters) used to communicate between brain cells. Like most antidepressants, SNRIs work by changing the levels of one or more of these naturally occurring brain chemicals.

• SNRIs block the absorption (reuptake) of the neurotransmitters serotonin and norepinephrine in the brain. They also affect certain other neurotransmitters. Changing the balance of these chemicals seems to help brain cells send and receive messages, which in turn boosts mood. Medications in this group of antidepressants are sometimes called dual-action antidepressants.

The mechanism of action of Lithium Salt• Lithium may also increase the release of serotonin by neurons in the brain.• The excitatory neurotransmitter glutamate could be involved in the effect of

lithium.• The other mechanisms by which lithium might help to regulate mood include the

alteration of gene expression.

SCREENING METHODS SCREENING METHODS [11][11]

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IN VIVO METHODS IN VITRO METHODS

BEHAVIORAL TESTS

ACTIVITY DEPENDS UPON MOA

CLASSIFICATION OF SCREENING METHODSCLASSIFICATION OF SCREENING METHODSIN VIVO IN VIVO METHODSMETHODS

BEHAVIORAL TESTS

1) Catalepsy antagonism in chicken

2) Despair swim test3) Tail suspension in mice4) Learned helplessness in rats5) Muricide behavior in rats6) Behavioral changes after

neonatal Clomipramine treatment

ACTIVITY DEPENDS ON MOA

1) Potenciation of NE toxicity in rats2) Compulsive gnawing in mice3) Reserpine induced hypothermia4) Tetrabenazine antagonism5) Yohimbin toxicity enhancement6) Apo morphine induced hypothermia7) 5HTpotentiation in rats8) Tryptamine potentiation in rats9) Syrotonine syndrome in rats10) Sexual behavior in rats

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CLASSIFICATION OF SCREENING METHODSCLASSIFICATION OF SCREENING METHODSIN VITRO IN VITRO METHODSMETHODS

1) Inhibition of NE uptake in rat brain2) Inhibition of Dopamine uptake in rat striatal3) Inhibition of Serotonin uptake in synapsome4) Binding to monoamine transporters5) Antagonism of p-chloramphetamine toxicity by inhibition of

seronine uptake6) Measurement of B-adrenoreceptor stimulated adenyl

cyclase7) Tests for anticholinergic properties in rat brain8) Moa inhibitors in rat synaptosomes Type-a Type-b 14

CATALEPSY ANTAGONISM IN CHICKENCATALEPSY ANTAGONISM IN CHICKEN

Adult white leghorn chickens are used

Grasped the animal & turn it on its back & hold it for 1 min

Cataleptic numbness occurs

Remove the hand carefully

It remains in cataleptic state

Clapping of hands above the head arouses the chicken which jumps up & runs away

Pretesting is done to ensure about cataleptic behavior

Control studies shows that, in untreated animals this phenomenon could be elicited 6 times (every 30 min for 5 days)

Inject test compound I.P.

The test is performed for 4 times (every 30 min during 2 hrs)

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PURPOSE This method is usedto screen the antidepressantActivity in chickens

EVALUATION 1. If cataleptic rigor does not occur

after treat.=Test +ve OR it is interrupted within 1 Min at

least twice during 2 hrs Period.

2. Arousal after hand clapping Or pulling on the wings is recorded to register central Sedative effect.

3. To obtained DRC 12 Animals/GRP are treated.

4. ED50 is calculated.

DESPAIR SWIM TESTDESPAIR SWIM TEST ADULT MALE SPRAGUE-DAWLEY RATS (WT=160-180gm)ARE USED.

NAÏVE RATS ARE INDIVIDUALLY FORCED TO SWIM INSIDE A VERTICAL PLEXIGLAS CYLINDER (HIGHT=40 cm , DIA.=18cm)

RATS ARE PLACED IN CYLINDER VIGOROUSLY SWIMMING IN CIRCLE (TEMP.=25’C)

(water level=15 cm) TRYING TO CLIMB ON WALLS OF CYLINDER & AFTER 2-3

MIN,ACTIVITY BEGINS TO SUBSIDE & FLOTING OF INCREASING LENGTH

AFTER 5-6 MIN IMMOBILITY REACHES A PLATEAU (RATS REMAINS IMMOBILE)

AFTER 15 MIN ,REMOVE RATS.ALLOW TO DRY THEM (32’C)

RATS ARE PLACED IN CYLINDER & IMMOBILITY IS CHECKED DURING 5 MIN IT HAS BEEN FOUND TO BE REPRODUCIBLE IN DIFF.GRP

IMMOBILE ANIMAL=WHICH REMAINS FLOATING PASSIVELY IN WATER IN A SLIGHTLY HUNCHED BUT UPRIGHT POSITION

INJECT TEST TEST/STD DRUG BEFORE 1 hr TESTING i.p.

PURPOSE

1. It was suggested that mice/rats Forced to swim in restricted space From which they can not escape are Induced to characteristic behavior Of immobility.

2. This behavior reflects a state of Despair which can reduced by several Agents which are therapeutically Effective in human depression.

EVALUATION

1. Duration of immobility is measured in Controls & treated animals with Various Doses of test drug & standard2. Antidepressants acts like stimulants Like amphetamine which reduces Duration of immobility3. Dose response can be evaluated.

TAIL SUSPENSION IN MICETAIL SUSPENSION IN MICE MALE BALB/CJ MICE(WT=20-25gm)ARE USED.

ANIMALS ARE TRANSFER INTO TESTING AREA TO ADAPT NEW ENVIRONMENT 1 hr BEFORE TESTING

GROUP OF 10 ANIMALS TREATED WITH TEST COMP./VEHICLE BEFORE 30 MIN OF TESTING(i.p)

FOR THE TEST MICE ARE SUSPENDED ON THE EDGE

OF SHELF 58cm ABOVE A TABLE TOP BY ADHESIVE TAPE PLACED 1cm FROM TIP OF THE TAIL.

THE DURATION OF IMMOBILITY IS RECORDED FOR PERIOD OF 5 MIN.

MICE ARE CONSIDERED AS IMMOBILE WHEN THEY HANG PASSIVELY & COMPLETELY MOTIONLESS FOR ATLEAST 1 MIN.

PURPOSE :

1. The immobility displayed by rodents , when Subjected to unavoidable & inescapable Stress has been hypothesized to reflect Behavioral distress which in turn may Depressive disorder in humans.2. Clinically effective antidepressants reduces The immobility that mice display after active & Unsuccessful attempts to escape when Suspended by the tail.

EVALUATION :1. % of animals showing passive Behavior is

counted & compared with Vehicle treated control.

2. Using various doses ed50 value can Be calculated.

Learned Helplessness in RatsLearned Helplessness in Rats MALE SPRAGUE RATS (WT=300gm)ARE USED.

LEARNED HELPLESSNESS IS PRODUCED BY EXPOSURE TO ELECTRIC SHOCK FOR 1 hr (0.7mA) ON SCHEDULE OF 10 S OF SHOCK/MIN

APPARATUS IS A 30*45*30 cm BOX WITH A GRID FLOOR (FLOOR=7.5*7.5 cm) (HIGHT=20 cm)

THE PLATFORM CAN BE INSERTED THROUGH ONE SIDE WALL TO ALLOW A JUMP-UP ESCAPE RESPONSE.

AFTER APPROPRIATE TREATMENT,THE ANIMALS ARE TESTED FOR ACQUISITION OF JUMP-UP ESCAPE IN THE SAME APPARATUS.

AT THE BEGINNING OF TRIAL ,PLATFORM IS PUSHED INTO THE BOX

0.4mA SHOCK IS INITIATED & TERMINTED IN 10 S.IF ANIMAL HAS NOT ESCAPED ONTO THE PLATFORM BY THIS TIME.

ESCAPE RESPONSE OCCURED.ANIMAL ALLOWED TO REMAIN ON PLATFORM(10SEC).RETURN IT TO GRID FLOOR.

10 TRIAL WITH TIME INTERVAL OF 20SEC ARE GIVEN

IN NAÏVE CONTROL GRP OF RATS,THIS TRAINING RESULTED IN 80% OF AQUIRING LEARNES HELPLESSNESS BEHAVIOUR. & DRUGS ARE GIVEN BEFORE TRAINING & THE TEST PERIOD.

PURPOSE 1. Animals are exposed to inescapable & Unavoidable electronic shocks in One situation later fail to escape Shock in a different situations when Escape is possible.2. This phenomenon was evaluated as A potential animal model of Depression.

Evaluation 1. Drug is considered to be effective if Learned helplessness is reduced & the no. of failures to escape is decreased.

2. Imipramine is found to be active only After repeated application.

3. Benzodiazepines=effective Chlorpromazine HCl= ineffective

MURICIDE BEHAVIOUR IN RATS MALE SPRAGUE-DAWLEY RATS (WT=300-350gm)ARE USED.

ONE MOUSE IS PLACED INTO THE RAT CAGE ABOUT 10-30% OF RATS KILL THE MOUSE BY BITING THE ANIMAL

THROUGH THE CERVICAL CORD

ONLY RATS CONSISTANTLY KILLING MICE WITHIN 5 MIN .

THE MICE ARE REMOVED AFTER 15-45 SEC TO AVOID EATING BY RATS

DRUGS ARE INJECTED (i.p.) TO THE RATS BEFORE TEST

MICE ARE PRESENTED FOR 30,60,120 MIN AFTER DRUG ADMINISTRATION.

ED50 VALUE IS CALCULATED.

PURPOSE :1.THIS METHOD CAN BE USED FOREVALUATION OF ANTIDEPRESSANTSLIKE TRICYCLIC & MAO INHIBITORS.

EVALUATION :

1. Failure to kill mouse within 5 Min is considered as inhibition of Muricide behaviour.

2. Performing dose response Experiment ed50 is defined as

the Dose which inhibits mouse Killing in 50% of the rats.

IN VITRO IN VITRO METHODMETHODINHIBITION OF [3H]- NOREPINEPHRINE UPTAKE IN RAT BRAIN INHIBITION OF [3H]- NOREPINEPHRINE UPTAKE IN RAT BRAIN

SYNAPTOSOMESSYNAPTOSOMES

TISSUE PREPARATION :

MALE WISTAR RATS ARE DECAPITATED

THE BRAINS RAPIDLY REMOVED

THE HYPOTHALAMIC REGION IS PREPARED, 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 oC

THE SUPERNATENT IS DECANTED &USED FOR EXPERIMENT

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PURPOSE :1. THE NEURONAL REUPTAKE OF NE IS

MOST IMPORTANT PHYSIOLOGICAL PROCESS FOR REMOVING & INACTIVATING NE IN SYNAPTIC CLEFT.

2. THIS UPTAKE IS INHIBITED BY ANTIDEPRESSANTS.

3. IN THE BRAIN THE HYPOTHALAMUS SHOWS GREATEST UPTAKE OF NA.THEREFORE,THIS REGION IS USED FOR TESTING POTENTIAL ANTIDEPRESSANT DRUGS.

EVALUATION :1.THE PERCENTAGE INHIBITION AT EACH DRUG CONCENTRATION IS THE MEAN OF 3 DETERMINATIONS.2.IC50 VALUES ARE DERIVED FROM LOG-PROBIT ANALYSIS.3.IC50 VALUES FOR THE STANDARD DRUGS DESIPRAMINE & NORTRYPTYLINE ARE AROUND 20nM.

CONTI…… CONTI…… IN VITRO IN VITRO METHODMETHODINHIBITION OF [3H]- NOREPINEPHRINE UPTAKE IN RAT BRAIN INHIBITION OF [3H]- NOREPINEPHRINE UPTAKE IN RAT BRAIN SYNAPTOSOMESSYNAPTOSOMES

ASSAY PROCEDURE

200µl OF TISSUE SUSPENSION ARE INCUBATED WITH 800µ l 62.5 Nm NE IN KREBS-HENSELEIT BICARBONATE BUFFER & 20 µl OF THE APPROPRIATE DRUG CONCENTRATION AT 37’C UNDER 95%O2 /5% CO2.

FOR EACH ASSAY TUBES ARE INCUBATED WITH 20µl OF VEHICLE AT 0’C IN ICE BATH.

AFTER THAT CENTRIFUGATION IS DONE 4000g FOR 10 MIN.

SUPERNATENT LIQUID IS ASPIRATED & PELLETS ARE DISSOLVED IN SOLUBILIZER.

SHAKING IS DONE & DECANTED INTO SCIENTILLATION VIALS & COUNTED IN 10 ML OF LIQUID SCINTILLATION COCKTAIL.

ACTIVE UPTAKE IS THE DIFFERENCE BETWEEN cmp at 37’C & 0 ‘C.

CONCLUSIONCONCLUSION Depression is an incapacitating disease which needs appropriate treatment.

This presentation reviews the pharmacology of antidepressant drugs and the future perspectives of

treating mood disorders such as depression.

The foremost theory for explaining the biological basis of depression has been the monoamine

hypothesis.

Depression is due to a deficiency in one or other biogenic monoamines (serotonin, 5-HT;

noradrenalin, NA; dopamine, DA). Antidepressant drugs are therefore classified according to their

ability to improve monoaminergic transmission.

Since this first theory, other explanations based on abnormal function of monoamine receptors or

associated with impaired signaling pathways have been suggested.

Notable progress has been accomplished in the treatment of major depressive disorders with new

compounds recently discovered (selective serotonin reuptake inhibitors: SSRI; serotonin noradrenalin

reuptake inhibitors: SNRI).

Behavioral, electrophysiological and micro dialysis studies have shown that serotonin (5-HT)

receptors, mainly 5-HT1A, 5-HT1B and 5-HT2C sub-types, exert a key role in modulating

antidepressant activity. 22

BIBLIOGRAPHYBIBLIOGRAPHY1. Lead Medical Assessor Anders Wessling ,Lead Health Economist Joakim Ramsberg, “The review of

Antidepressants”

2. “Antidepressants The Old and The New” ,October, 1998 iii

3. Review on Pharmacology of Antidepressants ,ELLIOTTRICHELSON, MD, Mayo Clin Proc. 2001;76:511-

527.

4. Ayflegül Y›ld›z, M.D., “Mechanism of Actions of Antidepressants: Beyond the Receptors”, 2002;12:194-

200.

5. http://ajp.psychiatryonline.org/cgi/reprint/157/11/1901

6. http://www.webmd.com/depression/

7. http://pn.psychiatryonline.org/content/41/24/21.full

8. http://www.mayoclinic.com/health/maois/MH00072

9. http://www.springerlink.com/content/b9b8668ff59f89d7/fulltext.pdf

10. http://www.emsam.com/pi_emsam.pdf

11. H.GERHARD VOGEL, “DRUG DISCOVERY & EVALUATION” PHARMACOLOGICAL ASSAY,SECOND

EDITION2002.23

Thank You…..

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Thank You…..

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