This includes various methods for analysis of analgesic activity of some compounds in animal models

1. SCREENING METHODS OF
ANALGESIC AGENTS
SUBMITTED BY:
BISWASH SAPKOTA
FIRST M.PHARM
PHARMACOLOGY
SUBMITTED TO:
RAJESH KOWTI
ASSISTANT PROFESSOR
DEPT of PHARMACOLOGY

2. Introduction
 Pain is an unpleasant sensory and emotional experience
associated with actual and potential tissue damage.
 Various types of pain are seen in humans for example somatic
pain, visceral pain, referred pain, neuropathic pain, cancer pain
etc.
 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.
 from pain.

3. Classification
Analgesic can be classified as
1. Narcotics : Morphine, Pethadine,
fentanyl
2. Non-Narcotics: NSAIDs

4. 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 ( µ, ƙ, δ) in brain and spinal cord which
decrease the release of neurotransmitter.
 NSAIDs inhibit the activity of both cyclooxygenase-1 (COX-
1) and cyclooxygenase-2 (COX-2) and thereby the synthesis
of prostaglandins and thromboxane.
 Inhibition of COX-2 leads to the anti-inflammatory, analgesic
and antipyretic effects.

5. Screening models
A. Invivo methods
 Pain - state models using Thermal stimuli
* Tail - flick model using radiant heat.
* Hot - plate test.
* Paw - withdrawal test.
 Pain - state models using Electrical stimuli
* Electrical stimulation of the tail.
* Grid - shock test.
* Tooth pulp test
 Pain - state models using Chemical stimuli
* Formalin test.
* Acetic acid induced writhing test.

6. Contd..
B. Invitro method
3H-Naloxone binding assay.
3H-Dihydromorphine binding to 𝜇 opiate receptors in rat brain.
 Receptor binding of nociceptin.
Bioassays for nociceptin.
 Receptor binding of cannabinoids.
 Vanilloid receptor binding.

7. 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 is 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.

8. Procedure
Groups of 10 mice (18-22g) are selected and
divided into standard,
test & control group respectively
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

9. Evaluation
 The latency time of the test, standard and control animals are
recorded and compared with values before administration
 Using various doses ED50 values can be calculated.

10. Tail flick method
Purpose and rationale
 The tail flick test with radiant heat is an simplified method.
 The application of thermal radiation to the tail of an animal
provokes the withdrawal of tail.
 The morphine like drugs are capable of prolonging the reaction
time.

11. Procedure
Wistar rats (170-210g) are selected and divided into
standard, test & control group respectively
Appropriate temperature is maintained on
the radiant source
The tail of the rat is placed on the radiant source & time
taken for the rat 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.

12. Evaluation
 The tail flick latency in the test, standard and control animals
are compared.
 Using various doses ED50 values can be calculated

13. Writhing test
Purpose and rationale
 Pain is induced by injecting irritants like acetic acid into
peritoneal cavity of mice.
 The animals react with characteristic stretching behavior
which is writhing.
 The test is suitable to detect analgesic activity of peripherally
acting drugs.

14. Procedure
Mice (20-25g) are selected and divided into
standard, test & control group respectively
re
Appropriate volume of acetic acid solution is
administered to the mice (control group) and
placed individually in the glass jar.
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.
n.

15. 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 acetic acid treated control

16. Tooth Pulp Test
Procedure
Rabbit weighing 2-3 kg are take and thiopental sodium of 15mg/kg I.V
body weight is given.
Using dental drill , tooth pulp chambers are exposed close to upper
incisors.
Clamping electrode are placed into drilled holes
After 30min electrical stimulus is applied by rectangular current of
frequency 50Hz upto 1sec.
Current is started 0.2mA and increased until animal starts licking and a
threshold is determined

17. Animal serves as its own control.
Test compound is administered orally or intravenously, After 15, 30, 60
and 120 min threshold current is measured and compared with the
threshold current prior to drug administration.

18. Formalin test
Purpose and rationale
The formalin test uses a 10% formalin solution as a
chemical noxious stimulus.
By injecting the formalin solution into the paw of a rat or
a mouse, a model of persistent (chronic) pain caused by
peripheral tissue injuries and inflammation is created.
This model is highly sensitive for opioids like drugs.

19. Procedure
Male Wistar rats weighing between 180-300 g are used
In the dorsum of front paw of the animal 0.05 ml of 10%
formalin is injected subcutaneously.
Each animal is placed into separate cage for observation of pain
responses in early and late phases.
These responses are elevation or favoring of the paw or excessive
licking and biting of the paw
Scoring of these pain responses is done according to a pain scale.

20. After administration of the test drug again scoring is done after
30, 60 min for comparison.
If both paws of the animal are allowed to rest on the floor with no
obvious favoring of the injected paw, this is taken as a positive
analgesic response.

21. Bioassay for Nociceptin
Purpose and Rationale
 Nociceptin receptors in the periphery can be characterized
by studies in isolated organs (Guerrini et al. 1998; Bigoni
et al. 1999): the guinea pig ileum according to
Paton(1957), the mouse vas deferens according to Hughes
et al. (1975), the rabbit vas deferens according to Oka et
al. (1980), the guinea pig renal pelvis (Giuliani and Maggi
1996).

22. Procedure
Tissues are taken from male Swiss mice, guinea pigs, Sprague
Dawley rats &New Zealand albino rabbit.
Suspended in 10 ml organ baths containing Krebs solution
oxygenated with 95% O2 & 5% CO2.
Temperature is set around 33°-35°C & a resting tension of 0.3-1g
is applied.
The tissues are stimulated through two platinum ring electrodes
The electrically evoked contractions are measured isotonically
with a strain gauge transducer and recorded on a multichannel
chart recorder.

23. After equilibration period of about 60 min the contractions
induced by electrical field stimulation are stable; at this time,
cumulative concentration response curves to nociception or
opioid peptides are performed.
Four electrical field stimulation are performed with each tissue at
30 min intervals.
Agonists & Antagonists are added to the bath.
Contractile responses to electrical field stimulation are expressed
as % increment to the spontaneous activity of the tissue.
The biological effects of the application of agonists or antagonists
are expressed as % inhibition of electrical filed stimulation-
induced contraction.

24. Evaluation
The agonists and antagonists potencies are recorded and
expressed as means of ±SEM.

25. References
 Patel PK, Sahu J, Chandel SS. A detailed review on
nociceptive models for the screening of analgesic
activity in experimental animals. Int J Neurol Phys
Ther. 2016;2:44-50.
 Milind P, Monu Y. Laboratory models for screening
analgesics. Int Res J Pharm. 2013;4(1):15-9.