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Background

? The majority of our knowledge about the transduction,transmission and modulation of nociceptive signals andpain has been gained by studies in animals.

? Early studies used primarily acute pain models and

measured withdrawal responses to noxious heat (hotplate, tail flick) or mechanical pressure either in naveanimals or shortly after inducing inflammation.

? These models have been used extensively andsuccessfully to discover and develop various opiate andnon-steroidal anti-inflammatory drugs, including the most

recent development of cyclo-oxygenase 2 (COX-2)specific inhibitors.

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Pharmacology Efficacy ModelsPharmacology 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

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?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 steroidaland 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

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Inflammation Pain

CFA Induced Acute InflammatoryPain (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 12days which are characterized by inflammation of non-injected sites(hindleg, forepaws, ears, nose and tail), a decrease of weight andimmune responses.

Indomethacin and phenylbutazone are effective on the primarylesions 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 secondarylesions even when started at day 9 or later.

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Inflammation Pain

Pain in Inflammed tissue

(Randall Sellito Test)

Male wistar rat model. The tests are done at 15 min intervals aftersubcutaneous administration and at 30 min intervals after oral

administration for any change in pain threshold. The interval of time whichindicates the greatest increase in pain threshold is regarded as the peaktime.

Na salicylate, Amidopyrine, Morphine, Codeine or Pethidine are used asreference compounds.

Peripherally acting analgesics such as the NSAIDS drugs increase only thethreshold of the inflamed paw, whereas opiate analgesics increase also thethreshold of the intact paw

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Neuropathic Pain

? Taxol Induced Neuropathic Pain (rat model)

Paclitaxel (Taxol) is injected once a day for 10 doses (i.p.) to createneuropathic pain in male SD rats and thermal paw withdrawal ismeasured using Hargreaves plantar compound. Mechanicalnociceptive 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 painproves to be more predictive of clinical outcomes in diabeticneuropathy patients than the nerve injury models. STZ is injected inmale SD rats and screened to pass the inclusion criteria of fastingblood sugar more than 250 mg/dl.

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Neuropathic Pain

Spinal Nerve Ligation (Chung) Neuropathic Pain

(rat model)

Spinal nerve is ligated in male SD rats. 5-7 days afterligation, rats showing extensive motor deficiency (pawdragging) and failure to exhibit tactile allodynia are rejectedfrom study. Behavioural test is done after 1 week ofsurgery using PWT (paw withdrawal threshold) of injuredhind paw to mechanical stimulation (Von Frey Filaments).Ketamine is used as reference compound.

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Sciatic Nerve Ligation (Bennet & Xie) (ratmodel)

Bennet and Xie (1988) described a peripheralneuropathy due to nerve constriction in the rat thatproduces disorders of pain sensation like those seenin man.

Experimental neuropathy is created by surgery in

male SD rats & thermal nociceptive threshold ismeasured according to the method of Hargreaves.Evaluation is done from paw withdrawal latency(PWL)

Neuropathic Pain

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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 referencecompounds.

? CRD (Colo Rectal Distension) Visceral Pain (Rat model)CRD model is a surgery model where the pain threshold is measured as the minimalpressure (kPa) inside the balloon when the rat showed flatting of abdomen during thecolorectal 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 ratMale SD rat model using balloon catheter surgery is done for creating mechanical visceralpain model. Behavioural responses are scored and evaluation are done. Morphine andindomethacin are used as reference compounds

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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 whenthe 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 administration of capsaicin in ratsin vivo (Lecci et al. 1997) Capsazepine is used as reference compound.

Nociceptive Pain models

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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, glutamatereceptor 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 threetimes at each time point. A withdrawal response was considered to be complete lifting of the hind paw off the surfaceof 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 avon Fey filament (447 g) was applied from underneath the cage through openings in the wire mesh floor directly tothe intended incision site. A response to the nonpunctate stimulus was defined as a withdrawal response or lifting ofthe 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 beeffective for controlling experimental postoperative pain (incident pain).

Von Frey

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Acute pain modelsAcute or nociceptive pain is part of a rapid warning relay instructing themotor neurons of the central nervous system to minimize detectedphysical harm.

? Tail flick test

The test is useful to differentiate central opioid like analgesics from

peripheral analgesics. Done using female wistar rats. Morphine andMethadone are used as reference.

? Thermal paw stimulation (Hargraves test)

? Mechanical Allodynia (Von Frey filaments)

? Mechanical hyperalgesia (Randal Sellito)

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Persistent central pain models? Formalin paw test

(Male Wistar rat model, Pain responses are indicated by elevation or favoring of thepaw or excessive licking and biting of the paw. Analgesic response or protection isindicated if both paws are resting on the floor with no obvious favoring of the injectedpaw. Morphine, Pethidine are used as reference compounds)

The formalin test identifies mainly centrally active drugs, whereas peripherally actinganalgesics are almost ineffective. Therefore, the formalin test may allow adissociation between inflammatory and non-inflammatory pain, a rough classificationof analgesics.

The formalin test in rats is a model of chronic pain which is sensitive to centrallyactive analgesic agents

? Abdominal writhing test

Already described in earlier slides

? Carrageenan injection

Already described in earlier slides

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Chronic Pain models

Chronic pain is classified as a disease. Chronic pain encompassesneuropathic pain, pain produced by damage to the neurons in theperipheral and central nervous system, and inflammatory pain.Chronic pain my involve a mix of both inflammatory and neuropathiccomponents, whereas inflammation may cause damage to the

neurons and produce neuropathic pain or neuronal injury may causean 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)

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Behavioural testing

?Gross & fine motor functions

?Motor co ordination

?

Cognition?Anxiety & depression

?Gait analysis

? Irwin screen (FOB)

?Sensory system testing

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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 toSponsor on request

? Standard Operating Proceduresscrutinized by QA Dept. beforestudy.

? Audits in crucial phases of allstudies.

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Thank you.

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