Advanced Pharmacology-I
(PHR5001)
Lecture 6:
Dr. M G Azam
Asstt. Professor
Dept. of Pharmacy, NSU
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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
Not been fully understood yet.
However, the development of
antidepressant drugs associated with
Norepinephrine, Serotonin &
Dopamine systems
Following
are
the
receptor important in
depression
5HT 1A
5HT 2A
5HT 6, 7
D2
Biogenic Amine Hypothesis: ↓ NE
Permissive Hypothesis: ↓ 5-HT
 ↓ DA
Theories of post synaptic changes in receptor
sensitivity
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PATHOPHYSIOLOGY
Serotonin5HT and NorepinephrineNE in the brain
Hypothalamus and
Pituitary
also may play
important role in
depression as they
release hormone
Prefrontal
Cortex
Limbic
System
Locus Ceruleus
Raphe
Nuclei (5-HT
source)
(NE Source)
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Serotonin and Norepinephrine modulatory systems
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Norepinephrine (NE)
Serotonin (5-HT)
Depressed
Mood
Concentration
Sex
Anxiety
Interest
Appetite
Irritability
Aggression
Thought
process
Motivation
• 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
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emotional and physical symptoms of depression
Amine theory
After 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 norepinephrine in the
vesicles of presynaptic
nerve ending
Monoamine Hypothesis: Depression results from a deficit in one or
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both of the serotonin and norepinephrine diffuse modulatory systems.
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Treatment approach
1. 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
antidepressant
Amitryptaline
Nortryptaline
Protryptaline
Imepramine
Desipramine
Clopramine
Trimipramine
Doxepine
2nd and 3rd generation
Amoxipine
Bupripion
Meprotiline MAO
Trazodon
inhibitor
3.Mirtazapine Phenelzine
3. Nefazodone Tranylcypra3.Venlafexine mine
SSRIS:
Selective 5HT
reuptake Inhibitor
Citalopram
Esitalopram
Fluoxetine (Prozac)
Fluvoxamine
Paroxetine (Paxil)
Sertraline (Zoloft)12
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 Test
1. Force swim test
2. Rotarod performance
test
3. Locomotors activity
Open field
Close field
4. Elevated Plus maze Test
Invitro test
1. Radioligand
binding
Assay
for
GABAa
receptor
2. Enzymatic fluromatric
assessment
of
cAMP/Adenyle
cyclase
activity
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1. Forced Swimming Test
This is a test of
behavioral “despair” or
learned helplesssness.
Immobility is
interpreted as a passive
stress-coping strategy
or depression-like
behavior .
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.
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Antidepressant drug will reduce the immobility
2. Rotarod Performance Test
Scientific uses:
Used to asses the
motor function.
Drugs such as
Anti-depressant
Anxiolytic ( Benz)
Serotonin agonist
and antagonist
Specification:
Animal diameter width
Mice 30mm
60mm
Rats
60mm
85mm
Constant mode- 1-6 0rpm
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
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and motor planning
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3.a.
Open
Field
Locomotors Activity
serve 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 measurements
Using 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 50X10cm
Two enclosed arms- 50X 10X (30h) cm
Height 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
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facilitate the counting
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
1. Radioligand Binding Assay
Receptors name
5HT1A
Radioligands
(3H)-8 OH-DPAT
Site of the brain
Rat hippocampus
5HT2A
(3H)-Ketanserin
Rat cortex
5HT6
(3H)-LSD
HEK293
5HT7
D2
(3H)-LSD
(3H)-Spiperon
HEK 293
Rat striatum
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 Na /K2
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
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vector, PcDNA (Invitrogent) and stably expressed in HEK 293 cells
.
2. Measurement of cAMP production/
Adenyl Cyclase activity
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Steps for reaction
1. Conversion of cAMP & enzymatic destruction of non cyclic
adenosine nucleotide and phosphorylation
2. Conversion of cAMP into ATP
3. Enzymatic amplification of ATP
4. Conversion of F6P to NADPH
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