This document describes various pharmacological screening techniques for analgesic agents. It discusses in vivo and in vitro models. In vivo models use thermal, electrical, mechanical, or chemical stimuli to induce pain in animals. Responses are measured before and after drug administration to test analgesic effects. Common in vivo models include the hot plate test, tail flick test, formalin test, and writhing test. In vitro models like receptor binding assays are also used to study mechanisms of analgesia. A variety of animal pain models exist to test treatments for both acute and chronic pain.
3. Introduction
Pain is an unpleasant sensory and emotional experience associated
with actual and potential tissue damage.
An analgesic or painkiller is any member of the group of drugs used to
achieve analgesia — relief from pain.
Analgesics is defined as the agents which selectively relieve pain by
acting in the CNS or by peripheral pain mechanisms without
significantly altering consciousness
4. Classification of Pain
Acute pain
Source is soft tissue damage, infection or inflammation
Short in duration
Serves to protect one after an injury
Chronic pain
Lasts 6 months or longer
Defined as ‘Disease of pain’
e.g. cancer pain, neuropathic pain, arthritic pain.
5. Classification of Analgesics
NARCOTIC ANALGESICS
Act centrally
Cause addiction
Produce CNS depression
Do not produce gastric
irritation
Show no anti inflammatory
effect
Eg. Morphine, Tramadol,
Pethidine etc.
NON NARCOTICANALGESICS:
Act peripherally
Do not cause addiction
Do not produce CNS depression
Produce gastric irritation
Show anti inflammatory effect
Eg. Diclofenac, Ibuprofen,
Aspirin etc.
6. Mechanism of Action
Analgesic drugs act in various ways on the peripheral and central
nervous systems.
Opioids produce analgesia by binding to specific G – protein coupled
receptors in brain and spinal cord.
NSAIDs inhibit the activity of both cyclooxygenase-1 (COX-1) and
cyclooxygenase-2 (COX-2) and thereby the synthesis
of prostaglandins and thromboxanes.
Inhibition of COX-2 leads to the anti-inflammatory, analgesic and
antipyretic effects.
8. IN VIVO MODELS
1. Models using thermal stimuli:
– Hot plate method
–The tail flick model using radiant heat/immersion of the tail
in hot water
– Pain state models using cold stimuli
•Cold tail flick test
• Cold ethanol tail flick test (CET)
2. Models using electrical stimuli:
– Stimulation of the tooth pulp
– Electrical stimulation of the tail
9. 3. Models using chemical stimuli:
– Formalin test
–Writhing test
– Stimulation of hollow organs
• Rat sigmoid colon model
• Inflammatory uterine pain model
4. Models using mechanical stimuli:
– Haffner’s tail clip method
– Randall Selitto Test
10. IN VITRO MODELS
3H-Naloxone binding assay.
𝜇opiate receptor binding assay.
Receptor binding of cannabinoids.
13. HOT- PLATE METHOD
Purpose and rationale
The paws of mice and rats are sensitive to heat at
temperatures which are not damaging to skin.
The responses are jumping, withdrawal of the paws and licking
of the paws.
The responses are prolonged after administration of centrally
acting analgesics, whereas peripheral analgesics of the
acetylsalicylic acid or phenyl-acetic acid type do not generally
affect these responses.
14. Procedure
mice (18-22g) are used
The temperature of the hot plate is maintained at 55° to 56°C.
The animals are placed on the hot plate & time until either
licking or jumping occurs is recorded.
The latency is recorded before & after 20, 60 and 90 min after
the administration of standard or test compound.
15.
16. Evaluation
The prolongation of latency time between the test,
standard and control animals are compared.
Using various doses ED50 values can be calculated.
17. TAIL FLICK MODEL
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.
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 withdrawal time is within 2-10s
The Tail-flick latency is recorded before & after the administration of standard
or test compound.
20.
21. Evaluation
The tail flick latency in the test, standard and control
animals are compared.
Using various doses ED50 values can be calculated.
22. The tail flick test using Immersion of the tail
Procedure
Wistar rat (170- 210gms) is used
Rat is placed into cage in such a way that their tail hangs
freely.
Distal 5 cm of the tail of wistar rat is marked and
immersed in a cup of warm water ( 55°Cto56°C) for 15
seconds.
The reaction time is determined periodically after
administration of test drug (0.5, 1, 2, 3, 4, and 6 hours)
23. Response and Evaluation: - Withdrawal of tail or attempt to
escape or abrupt movement of tail and sometimes the recoiling
of whole body is seen.
Centrally acting analgesics are capable of causing prolongation
of tail withdrawal reflex( more than 6 sec.).
Modification:
–Cold tail Flick test
–Cold ethanol Tail flick test
25. TOOTH PULP TEST
Procedure
Rabbits 2-3 kg, anesthetized with thiopental
sodium15mg/kg iv.
Using Dental drill, tooth pulp chambers are exposed close
to upper incisors .
Clamping electrodes are placed into the drilled holes.
After 30 min. electrical stimulus is applied by rectangular
current of frequency 50 Hz upto 1sec.
Current is started 0.2 mA and increased until animal starts
licking and a threshold is determined .
27. WRITHING TEST
Purpose and rationale
Pain is induced by injecting irritants like 0.6% acetic
acid, Phenylquinone 0.02%, 4% NaCl into peritoneal
cavity of mice.
The animals react with characteristic stereotyped
behavior which is writhing. A writhe is indicated by
stretching of the abdomen with simultaneous
stretching of atleast 1 hind limb.
The test is suitable to detect analgesic activity of
peripherally acting drugs.
28. Procedure
Mice of either sex (20 and 25 g) is used
Pain is induced by intraperitoneal injection of chemicals that
irritate serous membranes
The onset of writhing, abdominal contractions & trunk twist
response are recorded for 10 min.
The test and standard drug is administered 15 min prior to
the acetic acid administration.
29.
30. Evaluation
The writhing period is recorded and compared
with the control group.
Writhing response in the drug treated must be less
when compared to the control group.
31. FORMALIN TEST
Chronic pain model and sensitive to opioid like drugs.
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 favoring of the paw, licking and biting of the
injected paw.
32. Two distinct periods of high licking activity can be identified:
1.Early phase lasting the first 3-5 min (direct effect on nociceptors )
2.Late phase lasting from 15 to 30 min after the injection of formalin. (an
inflammatory reaction in the peripheral tissue and functional changes in
the dorsal horn of the spinal cord)
Centrally acting analgesics(Morphine) are antinociceptive in both phases.
NSAIDS(indomethacin and naproxen) inhibit only the late phase.
Substance P and bradykinin act as mediators in the first phase, while
histamine, serotonin, prostaglandin and bradykinin are involved in the
nociceptive response of the second stage.
34. HAFFNER’S TAIL CLIP METHOD
Purpose and rationale
Preferred sites for applying nociceptive mechanical
stimuli are the hind paw and the tail
Highly sensitive for centrally acting drugs
Tests using constant pressure have been abandoned
progressively for those applying gradually increasing
pressures
35. Procedure
An artery clip is placed at the root of tail of mice.
A quick response is seen as biting the 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.
36. RANDALL SELITTO TEST
Principal: Inflammation increases the pain
sensitivity and decreases pain threshold.
Male Wistar rats (130 to 175 g) are used.
0.1 ml of a 20% suspension of Brewer’s yeast is
injected 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. ANIMAL MODELS OF CHRONIC PAIN
Neuropathic pain models
Vincristine induced neuropathy model
Diabetic neuropathy model
Persistent post-thoracotomy pain model
MODELS OF CANCER PAIN
Rat model of bone cancer pain
38. VINCRISTINE INDUCED NEUROPATHY MODEL:
Vincristine 100 μg/ kg i.v. daily for 2 weeks in Male
Sprague-Dawley rats.
A decrease in mechanical(Randall Selitto paw- withdrawal
test) nociceptive threshold and hyperalgesia occurs after
2nd day of administration and chronic lowered threshold
occurs after 2 weeks.
Thermal hyperalgesia is also produced.
39. DIABETIC NEUROPATHY MODEL:
Streptozotocin 75mg/kg i.p. in rats
development
diabetes
Animals subjected to various painful stimuli- mechanical,
thermal and chemical.
A decrease in reaction threshold to noxious heat stimuli
and to non- painful thermal and mechanical stimulation
serves as evidence for hyperalgesia and allodynia.(2
weeks of establishment of diabetes)
40. CANCER PAIN
RAT MODEL OF BONE CANCER PAIN
Bone metastasis is one of the major causes of cancer related
pain
Bone cancer induced by intra-tibial injections of syngeneic
MRMT-1 mammary tumour cell line in Sparague-Dawley rats .
Leads to mechanical allodynia and hyperalgesia and decreased
weight bearing on the affected limb beginning on day12-14
following injection of 3×103 cells.
Activity of animals is noted
42. 3H-Naloxone binding assay
3H-Dihydromorphine binding to μ opiate receptors in rat brain
Inhibition of enkephalinase
Receptor binding of nociceptin
Receptor binding of cannabinoids
Vanilloid (capsaicin) receptor binding
Evaluation of vanilloid receptor antagonists
Vasoactive intestinal polypeptide (VIP) and pituitary adenylate
cyclase-activating peptide (PACAP
43. DISCUSSION
Analgesic models are equally applicable for phasic or
acute pain as well as tonic or chronic pain.
Different thermal, mechanical electrical and
chemical stimuli are used.
In-vitro models are also used.
Centrally acting and peripherally acting analgesics.
both can be evaluated by analgesic models.
44. Haffner’s tail clip method, hot plate method,
tail immersion method, formalin test in rats etc.
are used mainly for central analgesic activity.
Writhing test, Randall Selitto test,
mechanical/visceral pain models in the rat etc.
Are used mainly for peripheral analgesic activity.
