PC-610_ Pharmacological Screening and Assays_Pain

Pharmacological screening models

for Pain research
Pain: Types

•Pain:
• According to the International Association for the Study of Pain (IASP), pain is “an
unpleasant sensory and emotional experience associated with actual or
potential tissue damage, or described in terms of such damage.”
• Mental suffering or distress
 • Acute pain is a normal defensive function that is not associated with
a pathological condition but signals threat to the integrity of the
organism.

• Pain is a symptom of many diseases requiring treatment with

analgesics. Severe pain due to cancer metastases needs the use of strong
analgesics, that means opioid drugs. The addiction liability of opioids led
to intensive research for compounds without this side effect.

Pain • The role of endogenous peptides such as enkephalins and endorphins

gives more insight into brain processes and the action of central
analgesics.

• Lim and Guzman (1968) differentiated between antipyretic analgesics

causing analgesia by blocking impulse generation at pain receptors in the
periphery while the narcotic analgesics block synaptic transmission of
impulses signaling pain in the central nervous system.
 • Somatic pain comes from the skin. muscles, and soft tissues,
while visceral pain comes from the internal organs.

• Allodynia: Pain resulting from a stimulus that does not normally provoke
pain.

• Polymodal Receptors

• Substance P; Substance P belongs to the tachykinin family of Peptides.

Bioassay extracts from spinal dorsal roots implicated substance P as a
pain neurotransmitter.

Pain • Neurokinins belong like substance P to a group of neuropeptides named

tachykinins.

• Following the discovery of neurokinin A and neurokinin B, three distinct

G protein-coupled receptors, NK1, NK2 and NK3, were described

• Dorsal Root Ganglia (DRG)

• calcitonin-gene-related peptide (CGRP)
• von Frey fibres : Nylon monofilaments that, when pressed against tissue
until they bend, exert a calibrated amount of force. They are used to
measure mechanical sensibility.
 • The measurement of ‘pain sensation’ in animals is
largely indirect.

• Aversive Behavior (vocalization or

biting/licking/shaking the affected limb)
Measurement
of pain in • all measurements are based on the nocifensive
reflex.
animals
• latency of hindpaw withdrawal on exposure to
increasing heat or as the intensity of pressure that
produces withdrawal
 An over-reliance on measures of hypersensitivity?
• The behaviors typically measured are either spinal reflexes (such
as withdrawal from experimenter-applied stimuli), spino-bulbo
spinal reflexes (such as jumping or abdominal stretching), simple
innate behaviors (such as vocalization, scratching, biting, licking and
guarding)

Behavioural • Evoked withdrawal responses measure not pain itself but rather
measures of the hypersensitivity (allodynia and hyperalgesia) that often
accompanies pain.
pain • Conditioned place preference or aversion: A behavioral task
during which a subject learns to associate an experience with a
specific physical environment. A subject will choose to spend more
time in an environment in which it previously had a rewarding
experience (for example, an analgesic drug) and less time in an
environment in which it had an aversive experience (for example,
inflammatory pain).
Pain: Types

Nociceptive Inflammatory Pain Neuropathic Pain

Pain
Stimulus Noxious Inflammation Neural Damage and Ectopic
firing
Sensory Neurons Nociceptor Nociceptor & non- Nociceptor & non-
nociceptor nociceptor
Site PNS PNS & CNS PNS & CNS
Clinical settings Acute trauma Post-operative pain, PNS & CNS lesions, SCI,
Arthritis Diabetic Neuropathy
Function Protective Healing/Repair Pathological
Pain sensitivity High threshold Low threshold Low threshold
Intensity Decrease with Decrease with Increase with duration
duration duration
Tactile Allodynia Absent Absent Present
Nerve Fiber

Type of Nerve Information Carried Myelin Diameter Conduction

Fibre Sheath Speed
A-alpha [Aα] Proprioception Yes 13–20 μm 80–120 m/s
A-beta [Aβ] Touch, cutaneous Yes 6–12 μm 33–75 m/s
mechanoreceptor
A-delta [Aδ] Mechanical and Yes 1–5 μm 3–30 m/s
Thermal
C Polymodal NO 0.2–1.5 μm 0.5–2.0 m/s
Neuroanatomy, Spinothalamic tracts

o It is responsible for the transmission of pain,

temperature, and crude touch to the somatosensory
region of the thalamus

o Spinothalamic tract is also referred to as the ventrolateral

(anterolateral) system. It is composed of four tracts:
 Anterior spinothalamic tract
 Lateral spinothalamic tract
 Spinoreticular tract
 Spinotectal tract

DCML: fine touch (tactile sensation), vibration, two-

point discrimination and proprioception
We can contrast the spinothalamic tract with the
dorsal column/medial lemniscus pathway. The
former crosses/decussates at the level of the spinal
nerve, where as the latter crosses at the level of
the medulla. If a lesion occurs in the brainstem or
higher, the patient presents with loss of pain
perception, crude touch and temperature sensation
contralateral (other side) to the lesion.
However, with spinal cord hemisections, the loss of
fine touch and proprioception is ipsilateral, while
that of pain perception and temperature sensation
is contralateral. This specific condition is known as
the Brown-Sequard syndrome.
 Homunculus

A cortical
homunculus (from Latin homunculus 'littl
e man, miniature human') is a distorted
representation of the human body, based
on a neurological "map" of the areas and
proportions of the human brain dedicated
to processing motor functions, or sensory
functions, for different parts of the body
Peripheral analgesic activity
• Peripheral analgesics possess anti-inflammatory properties and in some cases also antipyretic
activity besides analgesia. Mode of action has been elucidated as an inhibition of cyclooxygenase in
the prostaglandin pathway.

• Writhing tests: Pain is induced by injection of irritants (phenylquinone or acetic acid) into the
peritoneal cavity of mice. The animals react with a characteristic stretching behavior which is called
writhing. Caution is required in interpreting the results, until other tests have been performed.
central and peripheral analgesics are detected.

• Pain in inflamed tissue; (RANDALL-SELITTO-test) The mean applied force is determined for each
time interval tested. The percentage increase in pain threshold is calculated by subtracting the
applied force of the vehicle control from the applied force of the drug group which is divided by the
applied force of the vehicle control in order to give the percentage of increase in pain threshold of
the drug group.

• Mechanical visceral pain model in the rat

• Antagonism against local effects of bradykinin
Acute assays
wide use at the time— involve applying a noxious stimulus (which may be thermal,
mechanical, electrical or chemical) to a convenient body part (usually the hindpaws, tail or
abdomen), leading to nocifensive withdrawal or to other simple behaviours that can be
easily scored.

Inflammatory assays.
inflammatory assays featuring behavioural responses that involve injecting formalin, which
is now known to act as a direct transient receptor potential cation channel, subfamily A,
member 1 (TRPA1), capsaicin, which directly activates TRPV1 receptors
carrageenan, complete Freund’s adjuvant, urate crystals, zymosan, AITC

Neuropathic assays
Neuroma model [complete nerve transection in an intact limb and results in autotomy
behaviour (self-mutilation of the digits)];
chronic constriction injury

Painful disease assays

surgical assays of neuropathic pain produce injuries that are similar to those that cause
complex regional pain syndrome (also known as causalgia)
painful diabetic neuropathy, post-herpetic neuralgia, persistent post-surgical pain,
trigeminal neuralgia, cancer-associated neuropathic pain and spinal cord injury-related
pain, burn-related pain,
Commonly used animal models of pain
Model
Acute pain
o Tail-flick/hot-plate Used to study nociceptive modalities and to develop analgesic drugs

Persistent/central pain
o Capsaicin
Used as a valuable tool to study selective activation of polymodal nociceptors and
‘central sensitization’ in the spinal cord

o Formalin An extensively used model as it features both peripheral and central components;
easy to use with a high throughput capacity; no relevance to chronic pain
conditions
Chronic/Inflammatory
pain
o FCA Only monoarthritic or localized form is used for pain research; long-lasting, reliable
model for inflammatory pain; time-course might differ between species
o Carrageenan and
Produce inflammation with a 3–7 day duration and substantial oedema formation
turpentine models

o UV-irradiation Various irradiation periods with UV-B produce skin inflammation with different
time courses
Commonly used animal models of pain contd….
Model
Chronic/neuropathic
pain
Bennett model (loose Chromic Frequently used model, particularly for behavioural studies; high frequency of
ligature of the sciatic nerve) autotomy is a disadvantage
Chronic Constriction Injury
(CCI)
Seltzer model (partial tight High reproducibility, easy to perform, excellent for behavioural studies; however,
ligation of the sciatic nerve) difficult to study changes in the DRG as damaged and undamaged primary
Partial sciatic nerve ligation afferents are mixed in the nerves
(pSNL)
Chung’s model (tight ligation Fairly extensive surgery with muscle damage might complicate the
of one of the two spinal nerves pathomechanism; the damaged and undamaged fibres of the peripheral nerve
of the sciatic nerve) originate from distinct DRGs
Spinal nerve ligation (SNL)
Diabetic neuropathy Produces severe distress to the animal with deterioration of general condition;
difficult to interpret data or obtain clear pain scores; insulin treatment abolishes
pain and hyperalgesia
THE DISCOVERY PROCESS – Pre-clinical testing Strategy (in vivo)
Animal model(s) of Neuropathic Pain
 • The in vivo pharmacological potency of opiate agonists and
antagonists parallels the in vitro displacement of 3H-naloxone, a
potent narcotic antagonist. [Radioligand] RIA

In vitro • According to different pharmacological profiles of opiates,

several receptor types have been identified designated as μ, κ, δ,

methods and σ receptor.

• Analgesia is thought to involve activation of μ receptors

for (largely at supraspinal sites) and κ receptors (principally within the
spinal cord); δ receptors may also be involved at the spinal and
supraspinal level.

central • Other consequences of μ activation include respiratory

depression, miosis, reduced gastrointestinal motility, and

analgesic euphoria. [endomorphins]

• Endogenous ligands for δ receptors are enkephalins.

activity Endogenous ligands for κ receptors are dynorphins.

• (OP for opioid) OP1 stands for δ, OP2 for κ, and OP3 for μ
receptor.
In vivo Several methods are available for testing central
methods analgesic activity, such as

for • HAFFNER’s tail clip method in mice,

• Tail flick or other radiant heat methods,
testing • Tail immersion tests,
• Hot plate methods in mice or rats,
central • Electrical stimulation (grid shock, stimulation
of tooth pulp or tail)
analgesic • Monkey shock titration,
• Formalin test in rats.
activity
 • Respiratory depression: [Three animals (rabbits) are used for the
test compound and the standard. Dose-response curves of the
effect on respiratory frequency and volume are compared]

• Decrease of body temperature [Not specific test since other drugs

Side can also cause)

effects of
• Tolerance [The increase of the ED50 values before and after
subchronic treatment indicates the phenomenon of tolerance]
radiant heat or the hot plate method

central • Physical dependence [Abstinence phenomena similar to those in

man after abrupt cessation of chronic treatment with opioid

analgesic analgesics have been reported in rats and monkeys] Withdrawal

symptoms are recorded during a 30 to 60 min period. A 10 point
score is used: writhing, 3; squealing, either spontaneous or
provoked by touch, 2; diarrhea, 2; teeth chattering, 1; eyelid ptosis,

drugs 1; wet-dog-type shaking 1.

• Abuse liability.

• Great effort has been made to synthesize compounds with a

better ratio of analgesic activity versus side effects.
 Mechanical Hyperalgesia
Effect of Compound X on CCI induced
mechanical hyperalgesia in male SD rats
82%
100 # 75%
** Vehicle
#
% reversal of mechanical

68% ***
mean  SEM (n = 5 - 6)

61% 66% Standard 10 mg/kg, p.o.

62%
75
*** ** *** * Compound X 0.3 g/kg, p.o.
hyperalgesia

45%
46%
42% Compound X 1 mg/kg, p.o.
50 *** 26%
Compound X 3 mg/kg, p.o.
27%
Compound X 10 mg/kg, p.o.
25 13% 16% 13%
6% 9% Compound X 30 mg/kg, p.o.
0

-25
Day 1 - 2 h Day 8 - 0 h Day 8 - 2 h Acute ED50 : 4.9 m g/kg (2 h)

Time (post - dose) Chronic ED50 : 12.4 m g/kg (day 8)

*** p < 0.001, ** p < 0.01 vs Veh, One-way ANOVA / Tukey's test
: 6.3 m g/kg (day 8 + 2 h)

FCA, pSNL, SNL,MIA,STZ, Paclitaxel can be used to cause

Mechanical hyperalgesia
 Thermal Hyperalgesia
Effect of Compound X on FCA induced
thermal hyperalgesia in male SD rats
100
mean ± SEM (n = 5 - 6)
%reversal of thermal

80 57% 50% Vehicle

hyperalgesia

60
* * Standard 15 mg/kg, p.o.
37%
Compound X 1 mg/kg, p.o.
25%
40 Compound X 3 mg/kg, p.o.
Compound X 10 mg/kg, p.o.
20 3%
Copound X 30 mg/kg, p.o.

0 * p < 0.05 vs Vehicle

Treatment

Capsaicin, Carrageenan, Post-operative pain can be used

to cause Thermal hyperalgesia