This document summarizes pain physiology and the evaluation of analgesics. It discusses the definition of pain, pain pathways, endogenous opioid peptides, and types of analgesics. Methods for evaluating analgesics include in vitro tests like receptor binding assays and in vivo tests using various pain models in animals. Acute pain models include thermal, electrical, chemical and mechanical stimuli. Chronic pain models examine neuropathic pain and cancer pain. While no single model perfectly mimics human pain, animal models remain important for assessing analgesic drug activity.

1. Dr. Sourav Chakrabarty
Post-graduate trainee
Department of Pharmacology
B.S. Medical College

2. “But pain is a perfect misery
The worst of evils
Excessive overturns
All patience”
John Milton - Paradise Lost

3. Overview
 Introduction
 Pain physiology
 Analgesics- Narcotic & Non-narcotic.
 Evaluation of analgesics.
 In vitro methods
 In vivo methods
 Discussion
 Conclusion.

4. Introduction
What is Pain?
Sherrington- “the physical adjunct of an imperative protective reflex.”
 Medical Definition
“Pain is an unpleasant sensory and emotional experience associated
with actual or potential tissue damage or described in terms of such
damage”- IASP
 Operative Definition
“Pain is whatever the experiencing person says it is, existing whenever
he/she says it does.”
 5th vital sign.

5. Contd……
 Types-
1. Acute pain/physiologic pain/good pain
2. Chronic pain/pathologic pain/bad pain
 But from pharmacological point of view
1. Nociceptive pain
2. Neuropathic pain
 Two components- sensory & affective.

6. Some basic terminologies
 Nociception: neural processes of encoding and processing noxious
stimuli.
 Hyperalgesia: an exaggerated response to a noxious stimulus
 Allodynia : a sensation of pain in response to a normally innocuous
stimulus.
 Nociceptive pain: pain arising from activation of nocieptors.
 Neuropathic pain: pain arising as a direct consequence of a lesion in the
neuron or disease affecting the somatosensory system.
 Pain threshold: the minimal intensity of stimulus that is perceived as
painful.

7. Pain pathway

8. Chemical signalling in the
nociceptive pathway

9. Modulation of pain

10. Nociceptive pain vs Neuropathic pain

11. Analgesics
 Analgesic: Agents which selectively relieve pain by acting in the CNS
or peripheral pain mechanisms without significantly altering
consciousness.
 Narcotic or Non-narcotics.
Narcotic analgesics Non-narcotic analgesics
Act centrally Act peripherally
Cause addiction Do not cause addiction
Produce CNS depression Do not produce CNS depression
No gastric irritation Produce gastric irritation
No anti-inflammatory effect Show anti-inflammatory effect
e.g. morphine,tramadol,pethidine. e.g. Diclofenac,Ibuprofen, aspirin etc

12. Level of drug action

13. Contd……

14. Endogenous Opioid Peptides
OPIOID
RECEPTOR
CLASS
EFFECTS
ASSOCIATED ENDOGENOUS
ENDORPHIN
Mu 1
Euphoria, supraspinal analgesia,
confusion, dizziness, nausea, low
addiction potential
Endormorphin 1,2=Beta-endorphin
>Enkephalin=Dynorphin
Mu 2
Respiratory depression, CVS and GI
effects, miosis, urinary retention
Beta-endorphin=Endormorphin
1,2>Enkephalin=Dynorphin
Delta
Spinal analgesia, Opioid
reinforcement CVS depression,
decreased brain and myocardial
oxygen Demand
Enkephalin=Beta-
endorphin>Dynorphin
Kappa
Supraspinal,Spinal ,Peripheral
analgesia, dysphoria, psychomimetic
effects, feedback inhibition of
endorphin system
Dynorphin A=beta-
endorphin>Enkephalin

15. Site of opioid action

16. Contd………..
reduce transmitter release
from presynaptic terminals of nociceptive
primary afferents
hyperpolarize
second-order pain transmission neurons by
increasing K+ conductance, evoking an inhibitory
postsynaptic potential

17. Newer analgesics
1. Nefopam- an inhibitor of amine uptake with some sodium channel
blocking properties.
 used in pain unresponsive to non-opioid drugs.
2. Ketamine- NMDA antagonist.
3. Ziconotide-a synthetic analogue of the N-type calcium-channel blocking
peptide ω-conotoxin MVIIA.
4. Sativex -an extract of the cannabis plant containing Δ9-
tetrahydrocannabinol (THC) .
 Used in multiple sclerosis

18. 5. Botulinum toxin
6. Ropinirole, pramipexole and rotigotine-used to treat restless leg
syndrome
7. cGRP antagonist- used in migraine.
8. Flupirtine

19. Future aspects
 monoclonal antibodies to NGF or its receptor TrkA .
 Trp channel ligands.
 Retigabine-Kv7 (M-current) opener (see Ch. 45) inhibits C-fibre-and
Aδ-fibre-mediated nociceptive responses in dorsal horn neurons in
both naive and neuropathic rats.
 Agonists at nicotinic acetylcholine receptors, based on epibatidine
 Antagonists of metabotropic glutamate receptors, mGluR1 and
mGluR5,

20. Evaluation of analgesics
 In vitro and In vivo screening
 In vitro tests can only partially substitute for animal experiments
involving pain
 In vivo tests should detect central & peripheral analgesic specifically
 What to measure?
 Change in pain threshold
 Change in the latency period of reaction

21. In vitro tests
1. 3H-Naloxone binding assay
2. 3H-Dihydromorphine binding to μ opiate receptors
3. 3H-Bremazocine binding to κ opiate receptors
4. Inhibition of enkephalinase
5. Receptor binding of nociceptin
6. Vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-
activating peptide(PACAP) binding assay.
7. Cannabinoid receptor binding assay.

22. In-vivo tests
 Models for acute pain
1. Thermal stimulus- Hot plate method/Tail-flick method/Cold tail flick
test
2. Electric stimulus- Tooth pulp test/ Tail stimulation test/Money shock
titration test
3. Chemical stimulus- Formalin test/ Writhing test/Rat sigmoid colon
model/ Inflammatory uterine pain model
4. Mechanical stimulus- Tail clip method/Randal Selitto test

23. Contd…...
 Models for chronic pain
1. Neuropathic pain model- mechanical/vincristine induced/diabetic
neuropathy
2. Chronic post thoracotomy pain model
3. Incisional pain model
4. Cancer pain model

24. Hot plate method
 Purpose- to evaluate central analgesics.
 Methods-
1. Mice weighing 18-22g are used.
2. Standard or test drug given orally/ sc
3. Animals placed on hot plate(55-56° C.)
4. Response- jumping/paw withdrawal/ licking of paws.
5. Latency period is measured after 20, 60 & 90 min.
6. Those increasing the latency period at least 50% are taken positive.
7. ED-50 values are calculated.
 Drawback- not for peripheral analgesics.
 False positive results with sedative/ muscle relaxants/ psychotomimetic.

25. Tail flick test
 Use of light beam exerting radiant heat, focused to proximal 1/3rd of
the tail.
 Nociceptive spinal reflex response- flicking tail away from heat source.
 Escape reaction- turning the head away.(more reliable)
 Latency period is compared.
 Minimal inter animal variation.

26. Tail immersion test
 Young female Wister rats(170-210 g).
 Placed in cages with tail hanging out freely.
 Distal 5 cm tail is immersed (max 15 sec)
in a cup of warm water(55°C)
 Tail withdrawal reflex is seen
 Recording done after ½ , 1,2,3,4 and 6 hours.
 latency period ˃6s is taken as positive.
 Modifications- cold mixture of water & ethylene glycol at -10°C./ cold
ethanol at -20°C.

27. Electric stimulus models
 Tooth pulp test-
1. Rabbits(2-3kg) are anaesthetized with thiopental at 15 mg/kg i.v.
2. Clamping electrodes are placed into tooth pulp chamber through drilled
holes.
3. After 30 min, stimulus given by rectangular current(50 Hz) upto 1 sec
4. Animals starts licking- current threshold is measured.
 Modification: stimulus given via subcutaneous electrodes at the tail
 Monkey shock titration test: shock given via Coulbourn Instrument
Programmable shocker at tail

28. Grid - shock test
 Drugs like Morphine, Acetylsalicylic acid can be measured by the Flinch –
jump response.
 Male mice (18-20g)
 The floor of the box is wired with stainless steel wire
 The stimulus in the form of
square wave pulses ( 30 cps).
 The output of stimulator is connected
to alternate wires of grid.
 The fixed resistance is placed with
the grid & parallel to an oscilloscope
to allow calibration in mill amperes.

29. Contd…..
 With increase in shock intensities the mice flinch, exhibit startling
reaction & increase locomotion or attempt to jump.
 The behavior is accurately reflected on the oscilloscope by marked
fluctuations of the displayed pulse.
 Pain thresholds are determined in each individual mouse twice before &
after the administration of the test drug.
 The average pain threshold values for each group at each time interval are
calculated and statistically compared with the control values.

30. Chemical stimulus models
 An irritant/ algogenic chemical agent given as nociceptive stimulus
 Slower mode of stimulation.- progressive & persisting for longer duration
 Both for central & peripheral analgesics
 Formalin test-
o 10% formalin injected at paw.
o Biphasic response
 Early: immediately due to chemical stimulation of nociceptors causing
C-fibre activation
 Late: after 10-15 min due to combination of an inflammatory reaction and
functional changes at dorsal horn of spinal cord.
 Opioid effective in both phase, but NSAIDs are effective in only second
phase.

31. Writhing test
 Model for visceral/peritoneal pain
 Used to detect peripheral analgesic activity of a compound.
 Mice(20-25g) are given i.p injection an aliquot of 0.25 ml of
phenylquinone(0.02%) suspended in 1% suspension of carboxy
methylcellulose
 A characteristic stereotyped behavior known as writhing is seen.

32.  Series of contraction along abdominal wall, turning movements of the
body and extension of hind limbs.
 Number of writhes ( stretching of the abdomen with at least one hind
limb) are recorded for 10 min
 Other chemicals- acetic acid, acetylcholine, bradykinin, PGE1, 4%
NaCl, ethacrynic acid.

34. Mechanical stimulus models
 Tail-clip method:
1. Noxious stimulus by using artery clip placed at the root of tail.
2. Response- biting the clip/tail.
3. Reaction time is noted.
4. Cut off time- average reaction time plus 3 times the sd of the
combined latencies of the control mice at all time period.

35.  Randal Selitto test: induction of hyperalgesia by producing
inflammation via sc injection of Brewar’s yeast at hind paw, followed by
application of pressure.
 Mechanical visceral pain: chronic intestinal distension by a
indwelling intraduodenal balloon catheter.

36. Chronic pain models
 Neuropathic pain- mechanical
1. Allodynia induced by partial somatosensory sciatic nerve injury.
2. Male Sprague-Dawley rat anesthetized with 4% halothane.
3. Sciatic nerves of both sides are exposed at the level of mid-thigh.
4. Right sciatic nerve is tied loosely with square knot using 4-0 chromic
gut sutures.
5. Layer by layer closure done.
6. Mild aversion of right paw & foot drop.

37. 1. Thermal nociceptive threshold is measured separately in each paws.
2. Drugs & vehicle given intrathecally.
3. PWL(paw withdrawal latency) is recorded.
4. Difference score(DS)=maximum PWL(LT)-maximum PWL(RT).
5. Dose is plotted against the change in DS.
 Vincristine induced neuropathy- By vincristine(100µg/kg) daily for
two weeks.

38. Diabetic neuropathy
 Streptozotocin(STZ) 75mg/kg ip for developing Diabetes.(BG≥14mM)
 4 weeks follow up with record of pain threshold, hyperglycemia &
clinical symptoms.
 Threshold to noxious heat stimuli/ non-painful thermal/
mechanical stimuli is observed.
 After 4 weeks, formalin injection is given to ascertain hyperalgesia.
 Ox-carbazepine can be evaluated in this model.

39. Models for cancer pain
 Induction of bone cancer by the syngeneic MRMT-1 mammary tumour
cell line.
 Sprague-Dawley rats are given intra-tibial injections of tumor cells.
 Control rats receive heat-killed cells or vehicle.
 Development of bone tumor and structural damage to the bone are
monitored.
 Mechanical allodynia & reduced weight bearing on affected limb,
beginning on day 12-14.
 Drugs are evaluated on the basis of their efficacy to reduce allodynia.

40. Discussion
 In humans, pain has a polymorphic nature.
 In animals, we focus on the latency period of response to noxious stimuli
or the change in the threshold of response.
 Limitations:
1. In most tests, responses are monitored around a nociceptive threshold,
whereas clinical pain is more severe.
2. Different scenario, when responses are evoked from healthy and
inflamed tissue.
3. Threshold to stimuli continue to increase in intensity

41. Conclusion
 No model is ideal, except chemical stimuli model, most closely mimicking
acute clinical pain.
 Neuropathic pain, very difficult to design the ideal model both for the
technical and ethical reasons.
 But still, animal models are very important to assess the analgesic activity
of drugs.
 Continued search should be maintained to find out novel drug targets & to
develop ideal animal model for their assessment.

42. THANK YOU