Raj Biotech (India) Pvt. Ltd.

D isco very S ervices D isco very S ervices

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Background
? The majority of our knowledge about the transduction,
transmission and modulation of nociceptive signals and
pain has been gained by studies in animals.
? Early studies used primarily acute pain models and
measured withdrawal responses to noxious heat (hot
plate, tail flick) or mechanical pressure either in nave
animals or shortly after inducing inflammation.
? These models have been used extensively and
successfully to discover and develop various opiate and
non-steroidal anti-inflammatory drugs, including the most
recent development of cyclo-oxygenase 2 (COX-2)
specific inhibitors.
Pharmacology Efficacy Models Pharmacology Efficacy Models
? Raj Biotech conducts pharmacology proof-
of-concept studies used in early stage
preclinical development to ascertain the
efficacy of test compounds.
? The various pain models of interest for
screening test compounds are as follows :
? Inflammation Pain
? Neuropathic Pain
? Visceral Pain
? Nociceptive Pain models
? Arthritis related pain
? Post operative pain
?Carrageenan Induced Inflammatory Pain
(rat model)
Male or female SD rat model. The paw edema method has been used by
many investigators and has been proven to be suitable for screening purposes
as well as for more in depth evaluations. Dependent on the irritant steroidal
and nonsteroidal anti-inflammatory drugs, antihistaminics and also, to a lesser
degree, serotonin antagonists are active in the paw edema tests.
Phenylbutazone is used as reference compound.
Inflammation Pain
Inflammation Pain
CFA Induced Acute Inflammatory
Pain (rat model)
Adjuvant arthritis in the rat is associated with chronic pain
Injections of complete Freunds adjuvant into the rat paw induces
inflammation as primary lesion with a maximum after 3 to 5 days.
Secondary lesions occur after a delay of approximately 11 to 12
days which are characterized by inflammation of non-injected sites
(hindleg, forepaws, ears, nose and tail), a decrease of weight and
immune responses.
Indomethacin and phenylbutazone are effective on the primary
lesions when dosage is started at the day of injection of the irritant.
They are not effective on the secondary lesions. In contrast,
immunosuppressants like cyclophosphamide inhibits the secondary
lesions even when started at day 9 or later.
Inflammation Pain
Pain in Inflammed tissue
(Randall Sellito Test)
Male wistar rat model. The tests are done at 15 min intervals after
subcutaneous administration and at 30 min intervals after oral
administration for any change in pain threshold. The interval of time which
indicates the greatest increase in pain threshold is regarded as the peak
time.
Na salicylate, Amidopyrine, Morphine, Codeine or Pethidine are used as
reference compounds.
Peripherally acting analgesics such as the NSAIDS drugs increase only the
threshold of the inflamed paw, whereas opiate analgesics increase also the
threshold of the intact paw
Neuropathic Pain
? Taxol Induced Neuropathic Pain (rat model)
Paclitaxel (Taxol) is injected once a day for 10 doses (i.p.) to create
neuropathic pain in male SD rats and thermal paw withdrawal is
measured using Hargreaves plantar compound. Mechanical
nociceptive threshold is evaluated by the RandallSellito paw
withdrawal test. Gabapentin is used as reference compound.
? STZ-Diabetic Neuropathic Pain (rat model)
The animal model of streptozotocin-induced diabetes and diabetic pain
proves to be more predictive of clinical outcomes in diabetic
neuropathy patients than the nerve injury models. STZ is injected in
male SD rats and screened to pass the inclusion criteria of fasting
blood sugar more than 250 mg/dl.
Neuropathic Pain
Spinal Nerve Ligation (Chung) Neuropathic Pain
(rat model)
Spinal nerve is ligated in male SD rats. 5-7 days after
ligation, rats showing extensive motor deficiency (paw
dragging) and failure to exhibit tactile allodynia are rejected
from study. Behavioural test is done after 1 week of
surgery using PWT (paw withdrawal threshold) of injured
hind paw to mechanical stimulation (Von Frey Filaments).
Ketamine is used as reference compound.
Sciatic Nerve Ligation (Bennet & Xie) (rat
model)
Bennet and Xie (1988) described a peripheral
neuropathy due to nerve constriction in the rat that
produces disorders of pain sensation like those seen
in man.
Experimental neuropathy is created by surgery in
male SD rats & thermal nociceptive threshold is
measured according to the method of Hargreaves.
Evaluation is done from paw withdrawal latency
(PWL)
Neuropathic Pain
Visceral Pain
? Visceral (acetic acid writhing) Nociceptive
Pain (Mouse model)
Visceral pain is induced by injection of irritants into the peritoneal cavity of mice. The
animals react with a characteristic stretching behavior which is called writhing.
Indomethacin, Acetylsalicylic acid, Amidopyrine and Phenacetin are used as reference
compounds.
? CRD (Colo Rectal Distension) Visceral Pain (Rat model)
CRD model is a surgery model where the pain threshold is measured as the minimal
pressure (kPa) inside the balloon when the rat showed flatting of abdomen during the
colorectal distension (CRD). The pain thresholds are compared with reference compound,
morphine 10, 15, 20 min after administration and evaluated.
? Mechanical visceral pain model in the rat
Male SD rat model using balloon catheter surgery is done for creating mechanical visceral
pain model. Behavioural responses are scored and evaluation are done. Morphine and
indomethacin are used as reference compounds
The majority of nociception measured in animal models
is evoked by mechanical or thermal stimuli.
Thermal sensitivities (heat or cold) are usually
measured from the latency to withdrawal of the
stimulated limb. Mechanical hypersensitivities can be
measured by paw pressure thresholds (mechanical
hyperalgesia), von Frey hair thresholds (static
mechanical allodynia) and latencies for removal from a
brush stimulus (dynamic mechanical allodynia). Other types of mechanical stimuli include
withdrawal pressure thresholds to joint compression and withdrawal or vocalization when
the joint is extended.
Mechanical hypersensitivity in an affected hind limb can also be estimated by
measuring the distribution of weight borne between the two hind limbs or by
analysis of gait in ambulatory animals. (Randall Sellito)
Tail Flick Nociceptive Pain
(rat model, mouse model) (described in upcoming slide)
Caspaicin-Induced Nociceptive Pain
(mouse model) (For NK2 receptors)
Inhibition of motor responses induced by intravesical admi nistration of capsaicin in rats
in vivo (Lecci et al. 1997) Capsazepine is used as reference compound.
Nociceptive Pain models
Arthritis related Pain
CFA-Induced MonoArthritic Pain (rat model)
MIA induced OA pain
ACLT OA pain
RA pain
Pain has been best studied in the MIA and menisectomy models. Both the MIA
and menisectomy models develop a mechanical allodynia (von Frey thresholds)
in the adjacent paw but mechanical hyperalgesia (paw pressure threshold) is only
seen in the MIA model. Both paracetamol and an NSAID (diclofenac) reverse the
early referred hyperalgesia suggesting an initial inflammatory phase, Gabapentin
reduces mechanical allodynia and morphine reverses both the weight bearing
difference and referred mechanical allodynia.
Post Operative Pain / Incident Pain
? Post-incisional (Brennan) Post-operative Pain (rat model)
Model is that it closely mimics the peripheral and central components of the human postoperative pain experience.
With this model, the analgesic effectiveness of some intrathecally administered analgesics such as morphine, glutamate
receptor antagonists, NK-1 receptor antagonist, and opioid receptor-like 1 receptor agonist have been studied (24).
The model might be also useful in assessing the ability of peripherally acting substances to alter pain behavior.
Withdrawal responses to punctuate mechanical stimulation were determined by using calibrated von Frey filaments
(0.0045 447 g bending force) applied from underneath the cage through openings (12 3 12 mm) in the wire mesh
floor to the area adjacent to the wound and to the same area on the non injured foot. The test was repeated three
times at each time point. A withdrawal response was considered to be complete lifting of the hind paw off the surface
of the cage or to be flinching. The least force producing a response was considered the withdrawal threshold
To measure responses to a non punctuate mechanical stimulus, a circular plastic disk (5 mm in diameter) attached to a
von Fey filament (447 g) was applied from underneath the cage through openings in the wire mesh floor directly to
the intended incision site. A response to the nonpunctate stimulus was defined as a withdrawal response or lifting of
the foot by touching the plastic disk without bending the filament. This test was repeated three times at each time
point; from these three trials, the response frequency was calculated.
? [Peripheral EP1 receptors in mechanical hyperalgesia produced by an incision]
? we examined whether the peripheral administration of the novel selective EP1 antagonist ONO-8711 would be
effective for controlling experimental postoperative pain (incident pain).
Von Frey
Acute pain models
Acute or nociceptive pain is part of a rapid warning relay instructing the
motor neurons of the central nervous system to minimize detected
physical harm.
? Tail flick test
The test is useful to differentiate central opioid like analgesics from
peripheral analgesics. Done using female wistar rats. Morphine and
Methadone are used as reference.
? Thermal paw stimulation (Hargraves test)
? Mechanical Allodynia (Von Frey filaments)
? Mechanical hyperalgesia (Randal Sellito)
Persistent central pain models
? Formalin paw test
(Male Wistar rat model, Pain responses are indicated by elevation or favoring of the
paw or excessive licking and biting of the paw. Analgesic response or protection is
indicated if both paws are resting on the floor with no obvious favoring of the injected
paw. Morphine, Pethidine are used as reference compounds)
The formalin test identifies mainly centrally active drugs, whereas peripherally acting
analgesics are almost ineffective. Therefore, the formalin test may allow a
dissociation between inflammatory and non-inflammatory pain, a rough classification
of analgesics.
The formalin test in rats is a model of chronic pain which is sensitive to centrally
active analgesic agents
? Abdominal writhing test
Already described in earlier slides
? Carrageenan injection
Already described in earlier slides
Chronic Pain models
Chronic pain is classified as a disease. Chronic pain encompasses
neuropathic pain, pain produced by damage to the neurons in the
peripheral and central nervous system, and inflammatory pain.
Chronic pain my involve a mix of both inflammatory and neuropathic
components, whereas inflammation may cause damage to the
neurons and produce neuropathic pain or neuronal injury may cause
an inflammatory reaction that contributes to the inflammatory pain
? Neuropathic pain models (already discussed)
? Inflammatory pain models (both OA & RA)
? Cancer pain model (Bone cancer pain)
? Post operative pain models (Brennan model of post incisional pain)
Behavioural testing
?Gross & fine motor functions
?Motor co ordination
?Cognition
?Anxiety & depression
?Gait analysis
?Irwin screen (FOB)
?Sensory system testing
Quality Assurance
? Independent Quality Assurance Unit
reporting only to the management.
? Well trained QA personnel's
? Audits as per guidelines
?
? Internal QA report directly send to
Sponsor on request
? Standard Operating Procedures
scrutinized by QA Dept. before
study.
? Audits in crucial phases of all
studies.
Thank you.
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