Screening of analgesics

1. Dr.Irfan Ahmad Khan
Deptt. Of Pharmacology,
JNMC, AMU.
Screening of Analgesics

2. Introduction
 Pain is an unpleasant sensory and emotional
experience associated with actual and potential
tissue damage.
 Nociception : Perception of noxious stimuli.
 Pain is a subjective experience and includes strong
affective component whereas nociception lacks
affective component.

3. Classification
 Acute pain
 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.

4. In vivo
ANIMAL MODELS OF ACUTE
PAIN

5. Models using Thermal stimulus
 Hot Plate Method
 Tail -Flick Test
 Tail-Flick Test using Radiant Heat
 Tail-Flick Test using Immersion of the Tail

6. Hot plate method
 Woolfe and MacDonald(1944)
 Paws of rats and mice are
sensitive to heat at temp. not
damaging skin.
 Animals are placed on the hot
plate which consists of
electrically heated surface
maintained at a temperature of
55± 1 ºC.
 Responses such as jumping,
withdrawal of paws and licking
of the paws are seen.
 The time period(latency period)
when animals are placed and
until responses occur is

7. Tail-Flick Test using Radiant Heat
 Ther et al(1963)
 Mice(18-22g) are place in
holder leaving tail exposed.
 Mouse tail is held by the
observer gently and a light
beam is exposed to proximal
one third of the tail.
 Tries to pull the tail away and
turns the head and is called
escape reaction .
 The time latency for escape
reaction is measured by stop
watch.

8. Tail immersion method
 Selective for morphine like analgesics.
 Rats are place in rat holder so that tail hangs freely.
 Distal 5 cm portion of the tail is immersed(max.upto
15 sec) in a cup filled with warm water maintained at
55 ±1 ºC
 The time taken by the rat to withdraw the tail from hot
water is noted as reaction time.

9. Modification
Cold tail flick test
 Distal 1-2cm tail is immersed in a cold mixture of
1:1 water and ethylene glycol at -10 ºC.

10. Models using Electrical stimulus
 Electrical stimulation of tail
 Grid shock test
 Tooth Pulp Test
 Monkey Shock Titration Test

11. Electrical stimulation of tail
 Kakunaga et al(1966)
 Here electrical stimuli are delivered by electrodes placed s.c.
in mice tail.
 Positive electrode placed at proximal end of tail.
 Rectangular wave pulses from a constant voltage stimulator
at an intensity of 40–50 V are applied.
 The frequency of the stimulation is 1 shock/s, and the pulse
duration 2.5 ms.
 When electrical stimuli is applied, a reflex movement of the
tail can be observed.
 The normal response time range of the stimuli is 3–4 s.
 Following administration of the drug, the response time is
registered at 15 min intervals until the reaction time returns to
control levels.

12. Grid shock test
 Blake et al. (1963)
 Approach to measure the analgesic properties by the “Flinch-
jump” procedure in rats
Male mice (18 and 20 g) are individually placed into clear plastic
chambers.
The floor of the box is wired with tightly strung stainless steel wire,
spaced about
1 mm apart.
The stimulus is given in the form of square wave pulses, 30 cycles
per second with a duration of 2 ms per pulse.
The output of the stimulator has to be connected to alternate wires
of the grid.
A fixed resistance is placed in series with the grid and in parallel to
an oscilloscope to allow calibration in milliamperes.
With increasing shock intensities the mice flinch, exhibit a startling
reaction, increase locomotion or attempt to jump.

13. Pain thresholds are determined in each individual mouse
twice before administration of the test drug and 15, 30, 60,
90 and 120 min after dosing.
The current as measured in milliamperes is recorded for
each animal before and after administration of the drug.

14. Tooth pulp test
 Kohl and Reffert (1938) for testing central analgesic activity
in rabbits
 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 .
 Animals serves as its own control and threshold is
determined before and after drug administration.

15. Monkey shock titration test
 Weiss and Laties(1958)
 This test is used for final
evaluation of new analgesic
agents.
 Monkeys are seated on restraining
chairs.
 By Coulbourn Instrument
Programmable Shocker electric
current is applied through
electrodes to shaved portion of tail.
 Current ranges from 0-4 mA
through 29 progressive steps.
 Monkey presses a bar to interrupt
the shock.
 For each monkey stable base line
shock level is recorded day prior to
drug administration.
 After 24hr of drug, shock titration is

16. Models using Chemical stimulus
 Formalin Test
 Writhing Test
 Distension of Hollow Organs Using Chemical
Stimulus
 Rat sigmoid colon model
 Inflammatory uterine pain model

17. Formalin test
 Dubuisson and Dennis (1977)
 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.

18.  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

19. Writhing test
 Collier et al(1968)
 Visceral or peritoneal pain model.
 Drugs having central and peripheral analgesic activity.
 Mice of either sex (20 and 25 g)
 Pain is induced by intraperitoneal injection of chemicals
that irritate serous membranes and provoke stereotype
behavior in mice known as writhing.
 A writhe is indicated by streching of the abdomen with
simultaneous streching of atleast 1 hind limb.
 The writhing response is considered to be a visceral
inflammatory pain model caused by the release of pain
mediators such as bradykinin, prostaglandins, histamine
and serotonin in the peritoneal fluid of mice.

20.  Chemicals: 0.6% acetic acid, Phenylquinone 0.02%,
4% NaCl etc. given i.p.
 The number of writhes per animal is counted for the
next 20 minutes.
 Percentage protection against abdominal constriction
is taken as an index of analgesia. It is calculated by–
writhes in (control group-test group) × 100
writhes in control group

21. Rat sigmoid colon model
 Miampamba et al (1994)
 Formalin (50μl,5%) administered into the rat sigmoid colon
produces a complex biphasic pain behavior .
 Initial phase  body stretching and contraction of either
the flanks or whole body.
 Second phase  abdomen licking and nibbling.
 A pain score can be calculated after analysis of the
formalin-induced behaviors.

22. Inflammatory uterine pain model
 Wesselmann et al(1998)
 Mustard oil is injected into one uterine horn in the rat to
produce chemical inflammation.
 Rat behavior is observed using non-stop videotape
recording for 7 days
 Rats with uterine inflammation show abnormal behavior
during the first 4 days such as licking of the lower
abdomen, repeated waves of contraction of the ipsilateral
oblique musculature with inward turning of the ipsilateral
hindlimb, squashing of the lower abdomen against the
floor.
 This is suggestive of visceral pain.
 Flank muscle hyperalgesia over 7 days indicative of
referred visceral pain.

23. Models using Mechanical stimulus
 Haffner’s tail clip method
 Randal Selitto Test

24. Haffner’s tail clip method
 Haffner(1929)
 Centrally acting analgesic drugs
 Male mice(18-25g) are used
 Artery clip is applied to root of the tail of mice (1 cm
from body)
 Response is seen as biting the clip or tail , where clip
has been placed.
 The reaction time between application of the clip and
response is noted by a stop watch.
 Cut-off time= Average reaction time+3 times the SD of
the
combined latencies of the control
mice.

25. Randall Selitto test
(Pain in inflamed tissue)
 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.

26. Antagonism against local effects of
bradykinin
 Deffenu et al.(1966)
 Paravascular sensory nerves which accompany blood
vessels throughout the body to end in unmyelinated free-
branching terminals close to the capillaries and venules
most likely carry the receptors of pain.
 Both central and peripheral analgesics.

27. Male Wistar rats (280–320 g) are anesthetized with ether.
A polyethylene catheter with an internal diameter of 0.5 mm is
inserted centripetally into the right carotid artery.
The catheter is passed through the subcutaneous tissues to
protrude from the back of the animal.
One hour after recovery from anesthesia, the first dose of
bradykinin is injected into the catheter producing dextro-rotation of
the head, flexing of the forelimb.
.The test compounds are applied subcutaneously or
intraperitoneally 15 min prior to injection of the threshold dose of
bradykinin.
The bradykinin injections are repeated in 5 min intervals until the
bradykinin effect reappears.
The criterion for protection is the disappearance of the bradykinin
effect after at least 2 consecutive doses of bradykinin.

28. 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

29. Neuropathic pain models
 Bennet and Xie (1988)
 Partial somatosensory nerve injury is the cause of
causalgiform pain disorders in man.
 Causalgia = spontaneous burning pain + hyperalgesia +
allodynia
 ProcedureMale Sparague-Dawley rats anesthetized with 4% halothane
Sciatic nerves of both legs are exposed at the level of mid thigh.
Four(4-0) chromic gut sutures are tied loosely with a square
knot around the Rt. sciatic nerve.
Lt. sciatic nerve is just mobilized. Incisions closed layer to layer.
During the next days, the animals show a mild eversion of the
affected paw and a mild-to-moderate degree of foot drop.

30. The time interval between the application of the light beam and the
brisk hind paw withdrawal response is measured.
The thermal nociceptive threshold is measured according to the
method of Hargreaves et al. (1988). The rats are placed beneath a
clear plastic
cage (10 × 20 × 24 cm) upon an elevated floor of clear glass.
A radiant heat source (halogen projector lamp) is placed beneath the
glass floor on a movable holder and positioned such that it focuses at
the plantar area of one hind paw.
After 7-8 days, test drug and vehicle injected intrathecally.
Paw withdrawal latency (PWL) of hind paws is recorded before and
after 5, 15, 30, 60 and 90 min after injection. To analyze the
magnitude of hyperesthesia, the difference score (DS) is calculated
by subtraction the maximum PWL of the control side (left side) from
the maximum PWL of the affected side (right side). Maximum PWL is
defined as the PWL which was the maximum during the first 30 min
after injection.

31. Vincristine induced neuropathy model:
 K. O. ALEY et al(1996)
 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.(the method of
Hargreaves et al. (1988).

32. Diabetic neuropathy model:
 Courteix et al(1993)
 Streptozotocin 75mg/kg i.p. in rats diabetes
development
 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)

33. Persistent post-thoracotomy pain
model
 Buvanendran et al(2004)
 Chronic post-thoracotomy pain recurs or persists after a
thoracotomy incision at least 2 months following the surgical
procedure.
 Procedure
 Male Sparague-Dawley rats anesthetized and Rt. 4th and 5th ribs
surgically exposed.
 Pleura opened b/w the ribs and a retractor placed under both ribs
and opened 8mm.
 Retractor is maintained for 60 min.
 Control animals given pleural incision only.(don’t develop
allodynia)
 2 days later animals are tested for mechanical allodynia using
calibrated von Frey filaments and cold allodynia using acetone
applied to incision site
 2 weeks after surgery, animals are tested for reduction of
allodynia with test drugs administration.

34. Cancer pain
Rat model of bone cancer pain:
 Medhurst et al(2002)
 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

35. In vitro methods

36.  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)

37. Thank you