SCREENING METHODS OF ANALGESIC AGENTS. Introduction Classification of pain Analgesic drugs Mechanism of action Screening models In-vivo Models In-vitro models

1. Screening Methods of
Analgesic agent
Presented by
A. Gowtham Sashtha 1st M.pharm
Department of pharmacology
K.M College of pharmacy Madurai - 625107.

2. CONTENTS :
☞ Introduction
☞ Classification of pain
☞ Analgesic drugs
☞ Mechanism of action
☞ Screening models
☞ Invivo Models
☞ In-vitro models

3. Introduction
❯ Pain is the Unpleasant sensory and emotional experience
associated with actual and potential tissue damage.
❯ Nociception perception of noxious stimuli.
❯ Analgesics define as the agents wich is selectively relive
pain by acting in the CNS or peripheral pain mechanism
without significantly altering consciousness.

4. Classification of pain
1) Acute pain :
→Source is soft tissue damage,Infection or inflammation.
→Short in duration
→Serves to protect one after an injury.
→Define as "Symptoms of pain"
2) Chronic pain :
→last 6 months or longer
→Define as "disease of pain"
eg : cancer pain,Neuropathic pain,arthritic pain.

5. Analgesic drugs :
Norcotic analgesic. Non Norcotic analgesic
eg : eg :
Morphine,Tramadol. Diclofenac,ibuprofen,
pethidine. Asperin.

6. Mechanism of action :
❯ The analgesics drugs are acts as the various ways on
the peripheral nad central nervous system.
❯ Opioid produced analgesia by binding to the specific
G - protein couple receptor in brain and spinal cord.
❯ NSAID is inhibit the activity of both cyclooxygenase-1
(Cox-2) cyclooxygenase-2 (cox-2) and there by the
synthesis prostaglandins and thromboxanes.

7. Screening Methods
☞ In vivo
☞ In vitro

8. Invivo methods :
1) Methods using thermal stimuli
1)Eddy's hot plate methods
2)The tail flick model
→Radiant heat
→ Immersion of the tail in hot water.
3)pain state model using cold stimuli
→ Cold tail flick test
→ Cold ethanol tail flick test (CET)

9. 2)Methods using electrical stimuli :
1) Stimulation of the tooth pulp
2) Electrical stimulation of the tail
3) Monkey shock titration method
3) Methods using chemical stimuli.
1) Formalin test
2) Writhing test

10. 4) Methods using mechanical stimuli :
Haffner's tail clip method.
Randall selitto test.
1) 3H-Naloxone binding assay.
2) μ opiate receptor binding assay.
3) Cannabinoids receptors binding assay.
Invitro methods :

11. 1) Methods using thermal stimuli

12. Hot plate method
Purpose :
✓ The paws of mice and rats sensitive to the heats at
temperatures which are not damage to the skin.
✓ The responses are jumping, withdrawal of the paws
and licking of the paws.
✓ The analgesics drugs are response to this symptoms.

13. Procedure :
Mice are used (18-22g)
The temperature of the hot plate is maintained at 55o
C 56o
C
The animals placed on the hot plate & time until either
licking or jumping occurs is recorded.
The latency is recorded before & after the administration of
standard or test compounds.

16. Evaluation :
✓ The prologation of latency time between the test,
standard and control animals are compared.
✓ Using various doses ED50 values can be calculated.

17. Tail Flick Methods :
Purpose and rationale :
In tail flick test heat is used as the noxious stimulus.
Dependent variable is the time taken by the animal to flick
its tail.
The morphine like drugs are capable of prolonging the
reaction time.

18. The tail flick test using Radiant Heat

19. Procedure :
Mice (18-22g) are placed into small cage leaving the tail exposed
Appropriate temperature is maintained on the radiant source
The tail of the mice is placed on the radiant source & time taken
by the mice to withdraw its tail is recorded.
usually withdraw time is within 2-10s
The tail flick latency is recorded before & after the
administration of standard or test compound.

20. The tail flick test using immersion of the tail
procedure :
wistar rat (170-210 gms) is used Rat is placed in to cage in
such a way that their tail hangs freely.
Distal 5cm of the tail of wistar rat is marked and immersed
in a cup of warm water (55o
C to 56o
C) for 15 sec.
The reaction time is determined periodically after
administration of the test drug (0.5,1,2,3,4 and 6 hours)

21. Evaluation :
The tail flick latency in the test, standard and control animals
are compared.
Using various doses ED50 values can be calculated.
Modification methods :
→Cold tail Flick test.
→Cold ethanol tail flick test.

22. 2)Methods using electrical stimuli

23. Tooth pulp test :
Procedure :
✓ Rabbit 2-3 kg, anesthetized with thiopental sodium 15
mg/kg i.v
✓ Using dental drill,tooth pulp chambers are exposed close
to upper incision.
✓ Champing electrodes are placed into the drilled holes.
✓ After 30 min. electrically stimulus is applied by
rectangular current of frequency 50HZ up to 1sec
✓ Current is started 0.2 mA and increased until animal
starts licking and a threshold is determined.

24. Monkey shock Titration method
Procedure :
✓ Monkey are seated on restraining chairs.
✓ By coulbourn instrument programmable shocker electric
current is applied through electrodes to shaved portion of
tail.
✓ Current ranges from 0-4mA
✓ Monkey presses a bar to interrupt the shock.
✓ For each monkey stable base line shock level is recorded
day perior to drug administration.
✓ After 24hr of drug ,shock titration is measured.

26. 3) Methods using chemical stimuli

27. Writhing test :
Purpose :
✓ Pain is induced by injecting irritants like 0.6% acetic
acid,0.02% phenylquinone,4% Nacl into peritoneal
cavity of mice.
✓ Writhing is produced.writhing is indicated by
stretching of the abdomen with simultaneous
stretching of atleast 1 hind limb.
✓ The test is suitable to detect analgesics activity of the
peripherally acting drugs.

28. procedure :
Mice of either sex (20-25 gm) is used.
Pains is induced by intraperitoneal injection of chemicals that
irritate serous membranes.
The onset of writhing produced and recorded for 10 mins.
The test or standard drugs is administered 15 mins perior to
the acetic acid administration.

29. Evaluation :
✓ The writhing period is recorded and compared with
the control group.

30. Formalin Test :
Procedure :
✓ Pain is induced in male wistar rats (180-300 g) by a
subcutaneous injection of 0.05ml of 10 % formalin on the
dorsal surface of the right hind paw.
✓ Each individual rat is placed into a clear plastic cage for
observation.
✓ The response is the amount of time the animals spend in
elevation or favouring of the paw.

31. ✓ Two distinct periods of high licking activity can be
identified :
1) Early phase lasting first 3-5 mins
2) Late phase lasting from 15 to 30 min
✓ cenrally acting analgesics (morphine) are antinociceptive
in both phase
✓ Substance p and bradykinin act as the Early phase.
✓ NSAIDS (indomethacin and naproxen) inhibit by only late
phase.

32. 3) Methods using mechanical stimuli

33. HAFFNER'S Tail clip method :
Purpose :
✓ Preferred sites for applying nociceptive mechanical
stimuli are the hind paw and the tail.
✓ Highly sensitive for cenrally acting drugs.
✓ Tests using constant pressure have been abandoned
progressively for those applying gradually increasing
pressure.

34. Procedure :
An artery clip is placed at the
root of tail of mice
A quick response is seen as
biting clip or tail, where clip
has been placed.
Then after 15,30, and 60
minutes,the same procedure
is repeated and the reaction
time is measured.

35. RANDALL SELITTO Test :
Procedure :
✓ Male wistar rats (130-175 g) are used.
✓ 0.1ml of a 20% suspension of Brewer's yeast is injected in
s.c into plantar surface of hind paw and after 3hr, using a
special apparatus pressure is applied on the paw at a
constant rate until animal struggles and threshold is
determined.

37. Invitro methods

38. 3H Naloxone binding assays
Purpose :
✓ Naloxone is the opioid antagonist.
✓ Na+ enhances the binding of antagonists and reduces
the binding of agonist.
✓ antagonists by determining the IC50 values for
3H-Naloxone in the presence or absence of Na+
enhances.

39. Procedure :
✓ Male wister rats are used and their brain are
removed,the cerebella area weighted and homogenised
in50 volume of ice cold soln.
✓ The homogenate centrifuged at 40000 g in 15mins after
recentrifuged at 40000g.
✓ The tubes are incubated at 30mins at 370
C.
✓ The assay is stopped by vaccum filteration through
whatman filters washing 3 times.

40. ✓ The filters are counted in 10ml and add to the assay
soln between binding in the presence of dextrophan
and levorphanol agents are calculating this formula.
✓ specific binding = total binding- non specific binding

41. Assay reagent :
310 µl H2O
20 µl 5 µM dextrorphan (total binding) or
5 µM levorphanol (non-specific binding)
50 µl 2 M NaCl or H2O
50 µl 0.5 M Tris buffer, pH 7.7
20 µl drug or vehicle
50 µl 3H-naloxone
500 µl tissue suspension.

42. μ Opiate receptor binding assay
Purpose :
✓ Opioid drugs exert their analgesic action mainly
through μ opioid receptors only.
✓ The compounds that inhibit binding of 3H
dihydromorphine in a synaptic membrane preparation
from rat brain can be identified by this assay.

43. Procedure :
same procedure of Naloxone binding assays.
Assay reagent :
20nM stock soln of 3H Dihydromorphine
0.1 mM stock soln of levorphanol tartrate
1mM of test stock solution
Ice cold 0.05M Tris buffer

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