Screening of Analgesics

Dr.Irfan Ahmad KhanDeptt. Of Pharmacology, JNMC, AMU.

Screening of Analgesics

IntroductionPain is an unpleasant sensory and emotional experience associated with actual and potential tissue damage.

Nociception : Perception of noxious stimuli.

Pain is a subjective experience and includes strong affective component whereas nociception lacks affective component.

ClassificationAcute painShort in durationServes to protect one after an injury

Chronic painLasts 6 months or longerDefined as Disease of paine.g. cancer pain, neuropathic pain, arthritic pain.

In vivo ANIMAL MODELS OF ACUTE PAIN

Models using Thermal stimulusHot Plate Method

Tail -Flick TestTail-Flick Test using Radiant HeatTail-Flick Test using Immersion of the Tail

Hot plate methodWoolfe and MacDonald(1944)Paws of rats and mice are sensitive to heat at temp. not damaging skin.Animals are placed on the hot plate which consists of electrically heated surface maintained at a temperature of 55 1 C.Responses such as jumping, withdrawal of paws and licking of the paws are seen.The time period(latency period) when animals are placed and until responses occur is recorded by stop watch.Cut off time is 15 secs.

Tail-Flick Test using Radiant HeatTher et al(1963)Mice(18-22g) are place in holder leaving tail exposed.Mouse tail is held by the observer gently and a light beam is exposed to proximal one third of the tail.Tries to pull the tail away and turns the head and is called escape reaction .The time latency for escape reaction is measured by stop watch.

Tail immersion method

Selective for morphine like analgesics.Rats are place in rat holder so that tail hangs freely.Distal 5 cm portion of the tail is immersed(max.upto 15 sec) in a cup filled with warm water maintained at 55 1 CThe time taken by the rat to withdraw the tail from hot water is noted as reaction time.

ModificationCold tail flick testDistal 1-2cm tail is immersed in a cold mixture of 1:1 water and ethylene glycol at -10 C.

Models using Electrical stimulusElectrical stimulation of tail

Grid shock test

Tooth Pulp Test

Monkey Shock Titration Test

Electrical stimulation of tailKakunaga et al(1966)Here electrical stimuli are delivered by electrodes placed s.c. in mice tail.Positive electrode placed at proximal end of tail.Rectangular wave pulses from a constant voltage stimulator at an intensity of 4050 V are applied. The frequency of the stimulation is 1 shock/s, and the pulse duration 2.5 ms. When electrical stimuli is applied, a reflex movement of the tail can be observed.The normal response time range of the stimuli is 34 s. Following administration of the drug, the response time is registered at 15 min intervals until the reaction time returns to control levels.

Grid shock testBlake et al. (1963) Approach to measure the analgesic properties by the Flinch-jump procedure in ratsMale mice (18 and 20 g) are individually placed into clear plastic chambers. The floor of the box is wired with tightly strung stainless steel wire, spaced about 1 mm apart. The stimulus is given in the form of square wave pulses, 30 cycles per second with a duration of 2 ms per pulse. The output of the stimulator has to be connected to alternate wires of the grid. A fixed resistance is placed in series with the grid and in parallel to an oscilloscope to allow calibration in milliamperes.With increasing shock intensities the mice flinch, exhibit a startling reaction, increase locomotion or attempt to jump.

Pain thresholds are determined in each individual mouse twice before administration of the test drug and 15, 30, 60, 90 and 120 min after dosing.The current as measured in milliamperes is recorded for each animal before and after administration of the drug.

Tooth pulp testKohl and Reffert (1938) for testing central analgesic activity in rabbitsRabbits 2-3 kg, anesthetized with thiopental sodium15mg/kg iv.Using Dental drill, tooth pulp chambers are exposed close to upper incisors .Clamping electrodes are placed into the drilled holes.After 30 min. electrical stimulus is applied by rectangular current of frequency 50 Hz upto 1sec.Current is started 0.2 mA and increased until animal starts licking and a threshold is determined .Animals serves as its own control and threshold is determined before and after drug administration.

Monkey shock titration test

Weiss and Laties(1958)This test is used for final evaluation of new analgesic agents.Monkeys are seated on restraining chairs.By Coulbourn Instrument Programmable Shocker electric current is applied through electrodes to shaved portion of tail.Current ranges from 0-4 mA through 29 progressive steps.Monkey presses a bar to interrupt the shock.For each monkey stable base line shock level is recorded day prior to drug administration.After 24hr of drug, shock titration is measured according to a change in max. level of median shock intensity for drugs as compared to control levels.

Models using Chemical stimulusFormalin Test

Writhing Test

Distension of Hollow Organs Using Chemical StimulusRat sigmoid colon modelInflammatory uterine pain model

Formalin testDubuisson and Dennis (1977)Chronic pain model and sensitive to opioid like drugs.Pain is induced in Male Wistar rats (180300 g) by a subcutaneous injection of 0.05ml of 10% formalin on the dorsal surface of the right hind paw.Each individual rat is placed into a clear plastic cage for observation.The response is the amount of time the animals spend in elevation or favoring of the paw, licking and biting of the injected paw.

Two distinct periods of high licking activity can be identified: 1.Early phase lasting the first 3-5 min (direct effect on nociceptors ) 2.Late phase lasting from 15 to 30 min after the injection of formalin. (an inflammatory reaction in the peripheral tissue and functional changes in the dorsal horn of the spinal cord)Centrally acting analgesics(Morphine) are antinociceptive in both phases. NSAIDS(indomethacin and naproxen) inhibit only the late phase.

Substance P and bradykinin act as mediators in the first phase, while histamine, serotonin, prostaglandin and bradykinin are involved in the nociceptive response of the second stage.

Writhing testCollier et al(1968)Visceral or peritoneal pain model.Drugs having central and peripheral analgesic activity.Mice of either sex (20 and 25 g)Pain is induced by intraperitoneal injection of chemicals that irritate serous membranes and provoke stereotype behavior in mice known as writhing.A writhe is indicated by streching of the abdomen with simultaneous streching of atleast 1 hind limb.The writhing response is considered to be a visceral inflammatory pain model caused by the release of pain mediators such as bradykinin, prostaglandins, histamine and serotonin in the peritoneal fluid of mice.

Chemicals: 0.6% acetic acid, Phenylquinone 0.02%, 4% NaCl etc. given i.p.The number of writhes per animal is counted for the next 20 minutes.Percentage protection against abdominal constriction is taken as an index of analgesia. It is calculated by writhes in (control group-test group) 100 writhes in control group

Rat sigmoid colon modelMiampamba et al (1994)Formalin (50l,5%) administered into the rat sigmoid colon produces a complex biphasic pain behavior .Initial phase body stretching and contraction of either the flanks or whole body.Second phase abdomen licking and nibbling.A pain score can be calculated after analysis of the formalin-induced behaviors.

Inflammatory uterine pain model

Wesselmann et al(1998)Mustard oil is injected into one uterine horn in the rat to produce chemical inflammation.Rat behavior is observed using non-stop videotape recording for 7 daysRats with uterine inflammation show abnormal behavior during the first 4 days such as licking of the lower abdomen, repeated waves of contraction of the ipsilateral oblique musculature with inward turning of the ipsilateral hindlimb, squashing of the lower abdomen against the floor.This is suggestive of visceral pain.Flank muscle hyperalgesia over 7 days indicative of referred visceral pain.

Models using Mechanical stimulusHaffners tail clip method

Randal Selitto Test

Haffners tail clip methodHaffner(1929)Centrally acting analgesic drugsMale mice(18-25g) are usedArtery clip is applied to root of the tail of mice (1 cm from body)Response is seen as biting the clip or tail , where clip has been placed.The reaction time between application of the clip and response is noted by a stop watch.Cut-off time= Average reaction time+3 times the SD of the combined latencies of the control mice.

Randall Selitto test(Pain in inflamed tissue)Principal: Inflammation increases the pain sensitivity and decreases pain threshold.Male Wistar rats (130 to 175 g) are used.0.1 ml of a 20% suspension of Brewers yeast is injected s.c. into plantar surface of hind paw and after 3hr, using a special apparatus pressure is applied on the paw at a constant rate until animal struggles and threshold is determined.

Antagonism against local effects of bradykininDeffenu et al.(1966)Paravascular sensory nerves which accompany blood vessels throughout the body to end in unmyelinated free-branching terminals close to the capillaries and venules most likely carry the receptors of pain.Both central and peripheral analgesics.

Male Wistar rats (280320 g) are anesthetized with ether. A polyethylene catheter with an internal diameter of 0.5 mm is inserted centripetally into the right carotid artery. The catheter is passed through the subcutaneous tissues to protrude from the back of the animal.

One hour after recovery from anesthesia, the first dose of bradykinin is injected into the catheter producing dextro-rotation of the head, flexing of the forelimb.. The test compounds are applied subcutaneously or intraperitoneally 15 min prior to injection of the threshold dose of bradykinin.The bradykinin injections are repeated in 5 min intervals until the bradykinin effect reappears.

The criterion for protection is the disappearance of the bradykinin effect after at least 2 consecutive doses of bradykinin.

ANIMAL MODELS OF CHRONIC PAINNeuropathic pain modelsVincristine induced neuropathy modelDiabetic neuropathy modelPersistent post-thoracotomy pain modelMODELS OF CANCER PAINRat model of bone cancer pain

Neuropathic pain modelsBennet and Xie (1988)Partial somatosensory nerve injury is the cause of causalgiform pain disorders in man.Causalgia = spontaneous burning pain + hyperalgesia + allodyniaProcedureMale Sparague-Dawley rats anesthetized with 4% halothane

Sciatic nerves of both legs are exposed at the level of mid thigh. Four(4-0) chromic gut sutures are tied loosely with a square knot around the Rt. sciatic nerve. Lt. sciatic nerve is just mobilized. Incisions closed layer to layer.

During the next days, the animals show a mild eversion of the affected paw and a mild-to-moderate degree of foot drop.

The time interval between the application of the light beam and the brisk hind paw withdrawal response is measured. The thermal nociceptive threshold is measured according to the method of Hargreaves et al. (1988). The rats are placed beneath a clear plastic cage (10 20 24 cm) upon an elevated floor of clear glass. A radiant heat source (halogen projector lamp) is placed beneath the glass floor on a movable holder and positioned such that it focuses at the plantar area of one hind paw.

After 7-8 days, test drug and vehicle injected intrathecally. Paw withdrawal latency (PWL) of hind paws is recorded before and after 5, 15, 30, 60 and 90 min after injection. To analyze the magnitude of hyperesthesia, the difference score (DS) is calculated by subtraction the maximum PWL of the control side (left side) from the maximum PWL of the affected side (right side). Maximum PWL is defined as the PWL which was the maximum during the first 30 min after injection.

Vincristine induced neuropathy model:K. O. ALEY et al(1996)Vincristine 100 g/ kg i.v. daily for 2 weeks in Male Sprague-Dawley rats.A decrease in mechanical(Randall Selitto paw-withdrawal test) nociceptive threshold and hyperalgesia occurs after 2nd day of administration and chronic lowered threshold occurs after 2 weeks.Thermal hyperalgesia is also produced.(the method of Hargreaves et al. (1988).

Diabetic neuropathy model:Courteix et al(1993)Streptozotocin 75mg/kg i.p. in rats diabetes developmentAnimals subjected to various painful stimuli-mechanical, thermal and chemical. A decrease in reaction threshold to noxious heat stimuli and to non- painful thermal and mechanical stimulation serves as evidence for hyperalgesia and allodynia.(2 weeks of establishment of diabetes)

Persistent post-thoracotomy pain model

Buvanendran et al(2004)Chronic post-thoracotomy pain recurs or persists after a thoracotomy incision at least 2 months following the surgical procedure.ProcedureMale Sparague-Dawley rats anesthetized and Rt. 4th and 5th ribs surgically exposed.Pleura opened b/w the ribs and a retractor placed under both ribs and opened 8mm.Retractor is maintained for 60 min.Control animals given pleural incision only.(dont develop allodynia)2 days later animals are tested for mechanical allodynia using calibrated von Frey filaments and cold allodynia using acetone applied to incision site2 weeks after surgery, animals are tested for reduction of allodynia with test drugs administration.Allodynic animals show axon loss in the intercostal nerves of the retracted ribs.

Cancer painRat model of bone cancer pain:Medhurst et al(2002)Bone metastasis is one of the major causes of cancer related painBone cancer induced by intra-tibial injections of syngeneic MRMT-1 mammary tumour cell line in Sparague-Dawley rats .Leads to mechanical allodynia and hyperalgesia and decreased weight bearing on the affected limb beginning on day12-14 following injection of 3103 cells.Activity of animals is noted

In vitro methods

3H-Naloxone binding assay3H-Dihydromorphine binding to opiate receptors in rat brainInhibition of enkephalinaseReceptor binding of nociceptinReceptor binding of cannabinoidsVanilloid (capsaicin) receptor bindingEvaluation of vanilloid receptor antagonistsVasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP)

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