1.
SCREENING METHODS FOR
ANALGESICS
DR. AMIT D. SHARMA
Third year resident,
Department of pharmacology
SBKS MI & RC.
Date : 09/05/2015

2.
Contents
 Definitions
 Central analgesics : agents, mechanism, in vivo
methods
 Peripheral analgesics : agents, mechanism, in vivo
methods

3.
Definitions
 Pain- an unpleasant sensory or emotional experience
associated with actual or potential tissue damage, or described
in terms of such damage (WHO).
 Features of inflammation
Pain (dolor)
rubor (redness)
tumor (swelling)
calor (heat)
functio laesa (loss of function).

4.
 Analgesics – the drugs which possess significant pain-
relieving properties by acting in the central nervous
system or on peripheral pain receptors without
significantly affecting consciousness.
 Two groups :
I. Narcotic/opioid /morphine like analgesics(central)
II. Non-narcotic/ non steroidal anti-inflammatory-
analgesic-antipyretic agents(peripheral)

5.
CENTRALANALGESIC
ACTIVITY

6.
Classification
 Morphine analogues:
I. Agonists – morphine(prototype), diamorphine(heroin),
codeine, pholcodeine, levorphanol
II. Partial agonists – nalorphine, levallorphan
 Synthetic derivatives unrelated to morphine :
I. Agonists – Pethidine (meperidine), fentanyl, sufentanil,
alfentanil, remifentanil, methadone, oxycodone
II. Partial agonists – pentazocine, cyclazocine,
buprenorphine, nalbuphine, butorphanol
III. Mu receptor agonist with other mode of action –
tramadol

7.
IN VIVO METHODS

8.
Haffner’s tail clip method, 1929
 The test compounds are administered subcutaneously to
fed mice or orally to fasted animals.
 Then an artery clip is applied to the root of the tail
(approximately 1 cm from the body) of mice.
 The reaction time is noted.
 Response : biting the clip or the tail near the location of the
clip.

9.
Hot plate method
 Woolfe and Mac Donald (1944)
 Mice are put on hot plate(temp 55° to 56 °C) and then
latency is noted following administration of test drug.
 Response – jumping or paw licking

10.
Tail immersion method
 Restraining the young female wistar rat, lower 5 cm
portion of the tail is immersed in a cup of freshly filled
water of exactly 55°C.
 The reaction time is determined before and periodically
after either oral or subcutaneous administration of the
test substance.
 The cut off time of the immersion is 15 seconds.
 Response – withdrawal of tail or attempt to escape.

11.
Grid shock test
 Blake et al(1963)
 Central and peripheral analgesics
 Stimulus is given in the form of square wave pulses to male
mice. The output of the stimulator is connected to the wires of
grid with fixed resistance , which is kept parallel to
oscilloscope.
 Pain thresholds are determined in each individual mouse
twice before and after administration of the test drug.
 Response – on oscilloscope, a startling reaction, increase
locomotion or attempt to jump.

12.
Electrical stimulation of the tail
 Burn et al, 1950
 Central and peripheral analgesics
 First test compound is given and then rectangular wave
pulses from a constant voltage stimulator are applied to
the alligator clips attached to the tail of the mice.
 Response – spinal reflexes, complete vocalization or
behavior changes(escape, aggression). The normal
response time range of the stimuli is 3–4 sec.

13.
Tooth pulp stimulation
 Kohl and Reffert (1938)
 Opioid agonists, pyrazolone derivatives
 After anaesthetizing the rabbit with thiopental, current is
applied through the electrodes passed in the dental drill
holes exposed to pulp chambers.
 Pain thresholds are determined in each individual rabbit
twice before and after administration of the test drug.
 Response - licking, biting, chewing or head flick.

14.
Formalin test in rats
 Dubuisson and Dennis,1977
 Male wistar rats are administered 0.05 ml of 10%
formalin in dorsum of the front paw along with test drug
subcutaneously or orally.
 Response - elevation or favoring of the paw or
excessive licking and biting of the paw.

15.
PERIPHERAL
ANALGESIC ACTIVITY

16.
Classification
 Nonselective COX inhibitors (traditional NSAIDs)
I. Salicylates – aspirin
II. Propionic acid derivatives – ibuprofen, naproxen
III. Fenamide – mephenamic acid
IV. Enolic acid derivatives – piroxicam
V. Acetic acid derivatives – ketorolac, indomethacin
VI. Pyrazolone derivatives – phenylbutazone

17.
 Preferential COX-2 inhibitors - nimesulide, diclofenac,
aceclofenac, etodolac.
 Selective COX-2 inhibitors – celecoxib, etoricoxib.
 Analgesics-antipyretics with poor antiinflammatory
action-
I. Paraaminophenol derivative – paracetamol(acetaminophen).
II. Pyrazolone derivatives – metamizol(dipyrone),
prophenazone.
III. Benzoxazocine derivative – nefopam.

18.
IN VIVO METHODS

19.
Writhing tests
 Mice are administered the test drug before intraperitoneal
injection of irritating agent like phenylquinone or acetic
acid.
 Pain sensation in acetic acid induced writhing paradigm is
elicited by producing localized inflammatory response due
to release of free arachidonic acid from tissue phospholipids
via cyclo-oxygenase (COX), and producing prostaglandin
specifically PGE2 and PGF2α, the level of lipoxygenase
products may also increases in peritoneal fluids.

20.
 Response – writhe (stretching of the abdomen with
simultaneous stretching of at least one hind limb)
 Aconitine can also be used instead of above drugs.
 Rats – 4% sodium chloride.

21.
Pain in inflamed tissue
(RANDALL-SELITTO TEST)
 Principle - inflammation increases the sensitivity to pain and
that this sensitivity is susceptible to modification by
analgesics.
 Male wistar rats are given the test drug orally,
subcutaneously or intraperitoneally.
 Inflammation is induced by subcutaneous injection of
Brewer’s yeast into the plantar surface of the left hind paw of
the rat .Then pressure is applied through a tip to the plantar
surface of the rat’s foot at a constant rate by a special
apparatus to the point at which the animal struggles, squeals
or attempts to bite.

22.
Mechanical visceral pain model in the rat
 Coburn et al 1989,deLeo et al 1989.
 First test drug is administered into male Sprague Dawley
rats and anaesthetized with nitrous oxide and halothane.
 One piece balloon catheter is passed through
gastrostomy tube into first part of duodenum.
 Inflation of balloon distension of duodenum
writhing.

23.
Behavioural responses score
0 : Normal behaviour defined as exploration, escape attempts
and resting
1 : Slightly modified behaviour defined as cessation of
exploration, focusing, wet-dog shake, excessive facial
grooming, teeth chattering and deep breathing
2 : Mildly to moderately modified behaviour defined as
retching-like activity, hunching, abdominal grooming or
nipping and immobility of the hind limbs (disappears with
removal of the stimulus).

24.
3 : Severely modified behaviour defined as stretching of
the hind limbs, arching and dorsoflection of the hind
paws.
4 : Intensive visceromotor activity defined as repetitive
stretching of the body, extension of the hind limbs, and
pelvis, frequent rotating sideward , i.e., writhing.

25.
References
 H. Gerhard Vogel et al. Drug discovery and evaluation :
Pharmacological assays, third edition.
 K D Tripathi. Essentials of medical pharmacology,
seventh edition.
 H L Sharma, K K Sharma. Principles of pharmacology,
second edition.
 Harsh Mohan. Textbook of pathology, sixth edition.

26.
THANK YOU