SCREENING METHODS OF ANTI
ANXIETY DRUGS
Dr. Jitendra Agrawal
Second year resident
Introduction
o Anxiety is an emotional state caused by the
perception of real or perceived danger that
threatens the secur...
Physiological and Pathological
Anxiety
Normal

Pathological

• Jitter

• Panic attacks

• Stage-fright

• Obsessions, comp...
Pathophysiology of anxiety
o Neurotransmitters like GABA, noradrenaline,
serotonin abnormalities – anxiety
o Amygdala, tem...
Noradrenaline theory
o ANS of anxious patients- hypersensitive to
stimuli.
o Locus coeruleus – activates epinephrine relea...
GABA Receptor Theory
o GABA – inhibitory neurotransmitter in brain.
o Has inhibitory and regulatory effects on serotonin,
...
Serotonin Theory
• Abnormalities in serotonin function i.e., release
and uptake plays role in anxiety.
• Greater serotonin...
Classification of anxiolytics











Benzodiazepines
alprazolam, chlordiazepoxide , diazepam
Azapirones
Busp...
ANIMAL MODEL FOR ANXIETY
• Must add new insight or new dimension to the
process of human anxiety
• Should reproduce behavi...
• All animal model of anxiety are common in
– Induced “fear” as an analogy to human
anxiety and to give them a basic const...
BEHAVIOURAL MODEL
• Exteroceptive stimuli model
– Novel environment
•
•
•
•
•

Open field test
Elevated plus maze
Staircas...
• Interoceptive stimuli model
– Electrical stimulation of brain
– Pharmacological manipulation (drug discrimination
test)
...
Open field test
• This behavioral model is base on the induction of anxiety
state – ambulation or freezing by exposing the...
Elevated Plus Maze Test
o Most widely used method; male mice used
o Anxiolytics –decrease anxiety
o increase open arm expl...
o 2 open arms and 2 closed
arms of 50 ˣ 10 ˣ 40cm
dimensions

o Open roof arrangement
o Two open arms are opposite
to each...
The rats weighing around 200g housed in pairs for 10 days prior
to testing; 6animals selected
for each group

Test drug ad...
 Parameters Measured During Next 5
minutes:
o time spent in the open arms
o entries into the open arms

o time spent in t...
 Anxiolytic effect indicated by:
o increase in the proportion of time spent in
open arms
o increase in the proportion of ...
Staircase exploration
• When introduced into a novel environment, rodents
experience a conflict between anxiety and explor...
male mice (Charles River strain)
with a weight between
18 and 24 g are used

Test drug administered 30min
prior to experim...
Light – dark model
o Animals placed in 2 chambered systems, where
they can freely move between a bright and dark
compartme...
o Apparatus - a dark and a
light chamber divided by a
photocell equipped zone.
o This case rests on an activity
monitor wh...
male mice with a weight
between
18 and 24 g are used

Test drug administered 30min
prior to experimentation by i.p.
route....
o No. of crossings through the partition
between the light and dark chambers compared

with total activity counts during t...
Mirrored chamber
• Novel stimulation evokes both exploration and
anxiety and therefore generates approach
avoidance confli...
Geller seifter’s conflict test
• Conflict is produced by availability of
reinforcement with punishment
• Experimentally in...
Geller seifter’s conflict test
• Male albino rats with a body weight of 300–400
g are housed individually.
• They are food...
Geller seifter’s conflict test
• Anxiety or “conflict” segment (3 min)
• a dipper of milk is delivered in response to each...
Geller seifter’s conflict test
• “nonconflict” segment (15 min)
• lever presses produce a drop of milk only at
variable in...
Geller seifter’s conflict test
• Four cycles of 15 min nonshock variable
interval segments followed by a three minute
CRF-...
Vogel Lick-conflict (Vogel Punished
Drinking)
• Rats are deprived of
water for 48 hrs
• Then placed in
chamber with water
...
a water bottle with metal drinking tube is
fitted to the animal housing
Electric circuit is connected between
drinking tub...
o number of accepted punishments (electric
shock) are measured during conflict period

 Anxiolytic effect :
o increase in...
Social interaction test
• In unfamiliar and
brightly lit
environment, social
interactions are
suppressed.
• Anxiolytics –
...
Animals placed in groups of 5 each in a perspex open
topped box
1hr before test,2 rats from separate housing treated
with ...
Electric stimulation of brain
• Electrical stimulation of brain aversive areas, in
particular the midbrain central gray, i...
Foot shock induced aggression
• GABA is involved in control and agrgressive
behaviour of animals.
• Benzodiazepines are th...
Two mice are placed in a box with a grid floor consisting of
steel rods
A constant current of 0.6 or 0.8mA is supplied to ...
Isolation induced aggression
o Male mice
subjected to
isolation develop
aggressive
behavior towards
other animals of
same ...
Male Mice kept isolated for 6weeks &
aggressive behavior tested.
Male mice accustomed to live together
placed in cage of i...
In vitro methods
 GABAA receptor binding
 GABAB receptor binding
 Benzodiazepine receptor: [3H]-flunitrazepam
binding a...
conclusion
disorder and
• Anxiety disorder is a psychological and threat about
associated with stress, tension, fear

•
•
...
References
• Hand book of experimental pharmacology.
S.K. kulkarni. 3 rd edition
• Drug Discovery an Evaluation:Pharmacolo...
Screening methods of anxiolytics
Upcoming SlideShare
Loading in...5
×

Screening methods of anxiolytics

1,709

Published on

Screening Methods for Anxiolytic drugs. A very useful description about screening methods and usefool tool to understand.

1 Comment
3 Likes
Statistics
Notes
  • good very innovative
       Reply 
    Are you sure you want to  Yes  No
    Your message goes here
No Downloads
Views
Total Views
1,709
On Slideshare
0
From Embeds
0
Number of Embeds
0
Actions
Shares
0
Downloads
181
Comments
1
Likes
3
Embeds 0
No embeds

No notes for slide

Screening methods of anxiolytics

  1. 1. SCREENING METHODS OF ANTI ANXIETY DRUGS Dr. Jitendra Agrawal Second year resident
  2. 2. Introduction o Anxiety is an emotional state caused by the perception of real or perceived danger that threatens the security of an individual. • It is a normal human adaptive response to stressful events. • Physiological anxiety – transient in nature • Pathological anxiety – needs treatment
  3. 3. Physiological and Pathological Anxiety Normal Pathological • Jitter • Panic attacks • Stage-fright • Obsessions, compulsions • Nervousness • Flashbacks, nightmares • Worrying • Pathological fear
  4. 4. Pathophysiology of anxiety o Neurotransmitters like GABA, noradrenaline, serotonin abnormalities – anxiety o Amygdala, temporal lobe, hippocampus and hypothalamus - involved in anxeity o Neurochemical theories : 1. Noradrenaline theory 2. Serotonin theory 3. GABA receptor theory 4
  5. 5. Noradrenaline theory o ANS of anxious patients- hypersensitive to stimuli. o Locus coeruleus – activates epinephrine release o Anxiogenics – stimulate locus coeruleus firing o Anxiolytics- inhibits locus coeruleus firing and decrease noradrenaline activity.
  6. 6. GABA Receptor Theory o GABA – inhibitory neurotransmitter in brain. o Has inhibitory and regulatory effects on serotonin, noradrenaline and dopamine. o GABAA receptor involved in anxiety; decreases neuronal excitability o Patients suffering from anxiety disorders have less level of GABA in cortex.
  7. 7. Serotonin Theory • Abnormalities in serotonin function i.e., release and uptake plays role in anxiety. • Greater serotonin activity – reduces norepinephrine activity in locus cerulus. • SSRIs – increases serotonin levels post synaptically – blocks symptoms of anxiety.
  8. 8. Classification of anxiolytics           Benzodiazepines alprazolam, chlordiazepoxide , diazepam Azapirones Buspirone, Ispapirone, gepirone SSRI Citalopram ,Escitalopram ,Fluoxetine Beta blockers Propranalol Sedative antihistaminic Hydroxyzine
  9. 9. ANIMAL MODEL FOR ANXIETY • Must add new insight or new dimension to the process of human anxiety • Should reproduce behavioural and pathological features of anxiety • Should allow investigation for neurobiological mechanism that are not easily amenable to study in man • Permit reliable evaluation of anxiolytic drugs as well as anxiogenic drugs
  10. 10. • All animal model of anxiety are common in – Induced “fear” as an analogy to human anxiety and to give them a basic construct validity – The degree to which they can be taken as model of human anxiety is based upon their response to BZD’s
  11. 11. BEHAVIOURAL MODEL • Exteroceptive stimuli model – Novel environment • • • • • Open field test Elevated plus maze Staircase exploration Black and white test box Mirrored chamber – Conditioned aversive procedures – Conflict Procedures • Geller seifter’s conflict test • Vogel’s conflict test – Escape avoidance procedure – Social interaction test
  12. 12. • Interoceptive stimuli model – Electrical stimulation of brain – Pharmacological manipulation (drug discrimination test) • • • • • • Caffine induced anxiety Yohimbine induced anxiety Flumazenil induced anxiety Pentylenetetrazole induced anxiety Amphetamine induced anxiety Cacaine induced anxiety – Foot shock induced agression – Isolation induced agression
  13. 13. Open field test • This behavioral model is base on the induction of anxiety state – ambulation or freezing by exposing the animal to a highly novel field environment of a high sound and light • As a result their exploratory behavior is inhibited • Open field apparatus consist of circular arena with high sound and light sources • Simple sterotypy: when animal move from one segment to another – one ambulation score • Complex sterotypy: when animal stands on its hind limbs- one rearing score • Urination • defecation
  14. 14. Elevated Plus Maze Test o Most widely used method; male mice used o Anxiolytics –decrease anxiety o increase open arm exploration time
  15. 15. o 2 open arms and 2 closed arms of 50 ˣ 10 ˣ 40cm dimensions o Open roof arrangement o Two open arms are opposite to each other. o Maze elevated at 50cm height.
  16. 16. The rats weighing around 200g housed in pairs for 10 days prior to testing; 6animals selected for each group Test drug administered 30min prior to experimentation by i.p route. The rat is then placed in the centre of the maze facing one of the enclosed arms.
  17. 17.  Parameters Measured During Next 5 minutes: o time spent in the open arms o entries into the open arms o time spent in the closed arms o entries into the closed arms o total arm entries
  18. 18.  Anxiolytic effect indicated by: o increase in the proportion of time spent in open arms o increase in the proportion of entries into open arms
  19. 19. Staircase exploration • When introduced into a novel environment, rodents experience a conflict between anxiety and exploratory behavior manifested by increased vigilance and behavioral activity • Staircase climibing reflects exploratory or locomotor activity • Rearing behaviour is an index of anxiety state • The no. of rearing and steps climbed to be recorded for period of 5 minutes • Decrease in rearing behaviour and increase in steps climbed is characterisation of anxiolytic effect
  20. 20. male mice (Charles River strain) with a weight between 18 and 24 g are used Test drug administered 30min prior to experimentation by s.c. route. The animal is placed on the floor of the box with its back to the staircase.(staircase is composed of five identical steps 2.5 cm high, 10 cm wide )
  21. 21. Light – dark model o Animals placed in 2 chambered systems, where they can freely move between a bright and dark compartment o Number of crossings between the light and dark sites is recorded.
  22. 22. o Apparatus - a dark and a light chamber divided by a photocell equipped zone. o This case rests on an activity monitor which counts total locomotor activity.
  23. 23. male mice with a weight between 18 and 24 g are used Test drug administered 30min prior to experimentation by i.p. route. The animal is placed in the cage
  24. 24. o No. of crossings through the partition between the light and dark chambers compared with total activity counts during the 10 min. o Loco motor activity also monitored. o Anxiolytics increase locomotor activity and no. of crossings.
  25. 25. Mirrored chamber • Novel stimulation evokes both exploration and anxiety and therefore generates approach avoidance conflict behaviour • It is hypothesised that distortion of readily traversed environment by a chamber of mirror might produce aversion to entry • Mice are exposed to the chamber of mirror • Extended latency to enter the chamber of mirror used as a parameter for anxiety analogy • Anxiolytics reduce this latency in dose dependent manner.
  26. 26. Geller seifter’s conflict test • Conflict is produced by availability of reinforcement with punishment • Experimentally induced conflict by punishing food rewarded behavior has been used to differentiate between various psychoactive drugs by Geller and Seifter
  27. 27. Geller seifter’s conflict test • Male albino rats with a body weight of 300–400 g are housed individually. • They are food deprived until the body weight is gradually reduced by approximately 20% of original and it is maintained at this level by restricted food diet. • Conditioning is carried out with a flash of light, a single lever, a liquid dripper, and a grid-floor connected to a shocker • The animals are trained to lever press for the milk reward in two distinct response-reward sections.
  28. 28. Geller seifter’s conflict test • Anxiety or “conflict” segment (3 min) • a dipper of milk is delivered in response to each lever press (continuos reinforcement schedule =CRF), • accompanied by aversive foot-shock through the grid floor • Creates a conflict between milk reward and the a painful foot shock
  29. 29. Geller seifter’s conflict test • “nonconflict” segment (15 min) • lever presses produce a drop of milk only at variable intervals of time from 60 to 210 s with an average reward of once per 2min • No shocks are administered
  30. 30. Geller seifter’s conflict test • Four cycles of 15 min nonshock variable interval segments followed by a three minute CRF-conflict period phase • The total number of lever presses during the conflict periods (CRF) and the non-conflict periods (VI) are counted. • Anxiolytic effect :An increase of lever presses in the conflict periods
  31. 31. Vogel Lick-conflict (Vogel Punished Drinking) • Rats are deprived of water for 48 hrs • Then placed in chamber with water source • sprague dawley rats are used in this model
  32. 32. a water bottle with metal drinking tube is fitted to the animal housing Electric circuit is connected between drinking tube and floor of cage. i.p injection of drugs are given; 30min later rats placed in cage and allowed to drink water and shock given after 20 licks For 3minutes next shocks are given for every 12th lick No. of shocks delivered in 3min noted for each animal, no. of shocks received after treatment noted
  33. 33. o number of accepted punishments (electric shock) are measured during conflict period  Anxiolytic effect : o increase in the accepted shocks.
  34. 34. Social interaction test • In unfamiliar and brightly lit environment, social interactions are suppressed. • Anxiolytics – counteract this suppression. • Animal used : male sprague dawley rats.
  35. 35. Animals placed in groups of 5 each in a perspex open topped box 1hr before test,2 rats from separate housing treated with test compound orally Placed in box with 60W bulb and behavior observed for 10minutes social interactions like sniffing, rearing,crawling over the partner are observed 40
  36. 36. Electric stimulation of brain • Electrical stimulation of brain aversive areas, in particular the midbrain central gray, induces defensive reaction and/or flight behavior in several species • Used as an animal model of anxiety or of panic attack. • Most studies used intracerebral microinjections of neurotransmitters, their agonists and antagonists to elucidate the mechanisms of aversive or antiaversive effects
  37. 37. Foot shock induced aggression • GABA is involved in control and agrgressive behaviour of animals. • Benzodiazepines are thought to produce anxiolytic effects by binding to a specific high affinity site on GABA-A receptor • So aggressive and fighting behavior has been extended for GABAergic anxiolytic drug screening.
  38. 38. Two mice are placed in a box with a grid floor consisting of steel rods A constant current of 0.6 or 0.8mA is supplied to the grid floor is delivered for 5 s followed by 5 s. intermission for 3 min. The total number of fights are recorded for each pair during the 3-min period. The fighting behaviour consists of vocalization, leaping, running, rearing and facing each other with some attempt to attack by hitting, biting or boxing 43
  39. 39. Isolation induced aggression o Male mice subjected to isolation develop aggressive behavior towards other animals of same sex.
  40. 40. Male Mice kept isolated for 6weeks & aggressive behavior tested. Male mice accustomed to live together placed in cage of isolated mice for 5minutes Isolated mice attacks intruderaggressiveness observed Drugs given to isolated mice s.c or orally; aggressive behavior tested at 60, 120,240 minutes (oral route) If drug active- decrease in aggressiveness Attenuation of fighting reaction
  41. 41. In vitro methods  GABAA receptor binding  GABAB receptor binding  Benzodiazepine receptor: [3H]-flunitrazepam binding assay  Serotonin (5-HTIA) receptor: binding of [3H]8-hydroxy-2-(di-n-propylamino)-tetralin ([3H]DPAT)  Serotonin (5-HTIB) receptors in brain: binding of [3H]5-hydroxytryptamine ([3H]5-HT)
  42. 42. conclusion disorder and • Anxiety disorder is a psychological and threat about associated with stress, tension, fear • • future. The pathophysiology of anxiety disorder is not fully understood and hence improper diagnosis leads to increase morbidity and mortality rates. Development of the screening methods resulted in introduction of many new anxiolytic agents. • In future aspects more reliable and easy models for screening are to be developed.
  43. 43. References • Hand book of experimental pharmacology. S.K. kulkarni. 3 rd edition • Drug Discovery an Evaluation:Pharmacological Assays. H. Gerhard Vogel. 3rd edition • Shenoy et al. Preclinical evaluation of anxiolytic agents: an overview. Journal of Pharmaceutical Research and Opinion.2011 ;1(2):7-22.
  1. A particular slide catching your eye?

    Clipping is a handy way to collect important slides you want to go back to later.

×