Advanced Pharmacology-I(PHR5001)

Lecture 6:ANTIDEPRESSANT

DRUGS

Dr. M G AzamAsstt. Professor

Dept. of Pharmacy, NSU1

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Depression• Thought to result from a deficiency of norepinephrine or

serotonin• Antidepressants increase the amounts of one of both of these

neurotransmitters in the CNS synapse by inhibiting their reuptake in the pre-synaptic neuron

• Most common mental illness characterized by depressed mood, feeling of sadness or emotional upset.

• Mild depression occurs in everyone as a normal response to left stressors and losses.

• No need for treatment

Symptoms of depression vary from person to person.2 key signs are loss of interest in things you like to do and sadness or irritability

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Major Depression• As defined by the American Psychiatric Associations

– Loss of energy, fatigue– Difficulty, thinking and concentrating– Loss of interest in appearance, work and leisure and sexual

activities– Inappropriate feelings of guild and worthlessness– Loss or appetite or excess eating– Sleep disorders (hypersomnia or insomnia)– Obsession with death, thoughts of suicide

• Major Depression causes symptoms that may:– Begin suddenly, possibly triggered by a loss, crisis or change– Interfere with normal functioning– Continue for months or years.

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Bipolar affective disorder• Depression with periods of mania

• People with this type of illness change back and forth

between periods of depression and periods of mania (an

extreme high).

• Symptoms of mania may include:– Less need for sleep– Overconfidence– Racing thoughts– Reckless behavior– Increased energy– Mood changes are usually gradual, but can be sudden

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What causes Depression?

Family History

– Having a family members who has depression may increase a person’s risk

– Imbalances of certain chemicals in the brain may lead to depression

Major Life Changes– Positive or negative events can trigger depression.

Examples include the death of a loved one or a promotion.

– Major Illnesses such as heart attack, stroke or cancer may trigger depression.

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PATHOPHYSIOLOGY

Biogenic Amine Hypothesis: ↓ NEPermissive Hypothesis: ↓ 5-HT ↓ DATheories of post synaptic changes in receptor

sensitivity

Not been fully understood yet. However, the development of antidepressant drugs associated with Norepinephrine, Serotonin & Dopamine systems

Following are the receptor important in depression5HT 1A5HT 2A5HT 6, 7D2

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Hypothalamus and Pituitary

also may play important role in

depression as they release hormone

Serotonin5HT and NorepinephrineNE in the brain

Limbic System

Locus Ceruleus

(NE Source)Raphe Nuclei (5-HT

source)

Prefrontal Cortex

PATHOPHYSIOLOGY

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Serotonin and Norepinephrine modulatory systems

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Serotonin (5-HT) Norepinephrine (NE)

Sex

Appetite

Aggression

Concentration

Interest

Motivation

Depressed Mood

Anxiety

Irritability

Thought process

• Dysregulation of Serotonin (5HT) and Norepinephrine (NE) in the brain are strongly associated with depression

• Dysregulation of 5HT and NE in the spinal cord may explain an increased pain perception among depressed patients

• Imbalances of 5HT and NE may explain the presence of both emotional and physical symptoms of depression

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Amine theoryAfter introduction of reserpine in 1950 it is evident that the drug those used for treatment of hypertension or schizophrenia may induce depression.Rserpine inhibit (deplete) storage of amine neurotransmitter such as serotonin and nor-epinephrine in the vesicles of presynaptic nerve ending

Monoamine Hypothesis: Depression results from a deficit in one or both of the serotonin and norepinephrine diffuse modulatory systems.

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Treatment approach1.Block the reuptake pump, such action help in temporary stay of neurotransmitter (serotonin and norepinephrine) at the postsynaptic ending and facilitate neurotransmission through corresponding receptors (by serotonin inhibition)

2. MAO inhibitor blocks major degradation pathway of neurotransmitter which permit more amount of neurotransmitter to accumulate and store at the presynaptic side.

Tricyclic antidepressantAmitryptalineNortryptalineProtryptalineImepramineDesipramineClopramineTrimipramineDoxepine

2nd and 3rd generationAmoxipineBupripionMeprotilineTrazodon3.Mirtazapine 3. Nefazodone3.Venlafexine

SSRIS:Selective 5HT

reuptake InhibitorCitalopramEsitalopram

Fluoxetine (Prozac)Fluvoxamine

Paroxetine (Paxil)Sertraline (Zoloft)

MAO inhibitorPhenelzineTranylcypra-mine

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Antidepressant Drugs• Monoamine oxidase inhibitors (MAOIs)

– Prevent breakdown of monoamines– Must avoid foods high in tyramine – ‘cheese effect’

• Tricyclic antidepressants– Block reuptake of serotonin and norepinephrine– Safer than MAOIs

• Selective serotonin-reuptake inhibitors (SSRIs)– Prozac, Paxil, Zoloft– No more effective than tricyclics, but side effects are few.

• Results are comparable with MAOIs, tricyclics, and SSRIs– About 50% improve, compared to 25% of controls

• Lithium – mood stabilizer– Not a drug – treats bipolar

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Tricyclic antidepressants (TCAs)

3rd generation

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Preclinical Evaluation of antidepressant drugs

Invivo Test1. Force swim test2. Rotarod performance

test3. Locomotors activity Open field Close field4. Elevated Plus maze Test

Invitro test1. Radioligand binding

Assay for GABAa receptor

2. Enzymatic fluromatric assessment of cAMP/Adenyle cyclase activity

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A new behavioral method for inducing a depressed state in mice. One hour after I. P administration of anti-depressant drugs mice

are dropped into the cylinder and left for 6 minutes. The immobility of last 4 minutes is counted.

Cylinder : Height- 25, diameter-10cm, depth of water- 6 cm , temp-21-23 ° C.

Antidepressant drug will reduce the immobility

This is a test of behavioral “despair” or learned helplesssness. Immobility is interpreted as a passive stress-coping strategy or depression-like behavior .

1. Forced Swimming Test

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2. Rotarod Performance Test

Rotarod performance test is based on a rotating rod with forced motor activity being applied, usually by a rodent.In the test , a rodent is placed on a horizontally oriented rotating cylinder (rod) suspend above a cage floor which is low enough not to injure the animals but high enough to induce avoidance of fall. Rodent naturally try to stay on the rotating cylinder and avoid falling to the ground.

The length of the time that a given

animal stay on this rotating rod is a measure of their

balance, coordination,

physical condition and motor planning

Scientific uses:Used to asses the motor function.

Drugs such asAnti-depressant

Anxiolytic ( Benz)Serotonin agonist

and antagonist

Specification:Animal diameter width Mice 30mm 60mmRats 60mm 85mm

Constant mode- 1-6 0rpm

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3.a. Open Field Locomotors Activityserve as good preliminary test to determine motor deficit and anxiety

Parameters are measured a. amount of distance traveled b. Observation of various horizontal, vertical and stereotype behavior. c. grooming (removal of dirt) d. Rearing

b. Closed field Locomotors activity measurementsUsing Opto 3 Multichannel activity monitor- Swiss albino mice are individually placed in plastic cages and then the crossing of each individual channel (ambulation) are counted from 2 to 6 minutes.

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Dark-Light Test

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4.Elevated Plus Maze Test (EPM):

Handling of animals before testing:Experience with handling , stress or injections can alter behavioral response of rodents in the elevated plus maze test. It is important to ensure that in the experiments using the elevated plus maze, handling of rodents and any experience with prior stress, particularly before testing is constant across animals and treatment groups.

Specification:Automated device produced by Campden Instrument LTD.Two open arms 50X10cmTwo enclosed arms- 50X 10X (30h) cmHeight from the floor grid- 50cm

Experimental requirements:The plus maze place in the dark room and center of the apparatus required to illuminate with 25 W electric bulb hanging above 100cm. This apparatus may connected to PC to facilitate the counting

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Open field test, elevator plus maze test, and dark/light box

These test can work as an antidepressant screen by measuring anxiety-related behavior as an accompanying depression. It is known that some antidepressants administration will cause a decrease behavior in these tests just like anxiolytics. However, the response to some antidepressants couldn’t be detected. Besides, these tests has their own problem. It is difficult to discriminate decreased anxiety-related avoidance from increased novelty-seeking in these tests.

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In Vitro Test

Receptors name Radioligands Site of the brain5HT1A (3H)-8 OH-DPAT Rat hippocampus5HT2A (3H)-Ketanserin Rat cortex

5HT6 (3H)-LSD HEK293

5HT7 (3H)-LSD HEK 293D2 (3H)-Spiperon Rat striatum

1. Radioligand Binding Assay

Total binding=specific binding + non-specific binding

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Sample are required to collect from the respective site in the isotonic phosphate buffer (50mM Na2/K phosphate buffer containing 37.8mM Na2HPO4 and 12.2 mM KH2PO4), PH -7.4 and homogenized using polytron homogenizer.The homogenate centrifuges at suitable (18000rmp) for 30 minutes at 4 0C. The pellets are resuspended in ice cold buffer (50 mM Na2/K- phosphate) and served as receptor sample for radioligand binding assay. In case of HEK 293, cells are lysed using hypertonic buffer and samples are used for radioligand binding assay.

Full length of 5HT6, 7 receptor gene can be obtained from PCR from human brain cDNA library, sub-cloned into a mammalian

vector, PcDNA (Invitrogent) and stably expressed in HEK 293 cells.

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2. Measurement of cAMP production/ Adenyl Cyclase activity

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Steps for reaction1. Conversion of cAMP & enzymatic destruction of non cyclic

adenosine nucleotide and phosphorylation2. Conversion of cAMP into ATP3. Enzymatic amplification of ATP4. Conversion of F6P to NADPH

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