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Analgesic screening methods

The document provides a comprehensive overview of pharmacological screening methods for analgesic agents, including the classification of pain and analgesics, mechanisms of action, and various in vivo and in vitro screening techniques. It describes acute and chronic pain, distinguishes between narcotic and non-narcotic analgesics, and presents specific tests like the hot plate and tail flick models for evaluating pain relief efficacy. The document emphasizes the importance of these models in assessing both central and peripheral analgesic properties of drugs.

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PHARMACOLOGICAL SCREENING OF ANALGESIC AGENTS PRESENTED BY: SHUBHA SHARMA M.SC. PHARMACOLOGY RUHS-CMS, JAIPUR
CONTENTS  Introduction.  Classification.  Mechanism of Action.  Screening techniques. In vivo. In vitro.
Introduction  Pain is an unpleasant sensory and emotional experience associated with actual and potential tissue damage.  Nociception : Perception of noxious stimuli.  An analgesic or painkiller is any member of the group of drugs used to achieve analgesia — relief from pain.  Analgesics is defined as the agents which selectively relieve pain by acting in the CNS or by peripheral pain mechanisms without significantly altering consciousness
Classification of Pain  Acute pain  Source is soft tissue damage, infection or inflammation  Short in duration  Serves to protect one after an injury  “Symptom of pain”  Chronic pain  Lasts 6 months or longer  Defined as ‘Disease of pain’  e.g. cancer pain, neuropathic pain, arthritic pain.
Classification of Analgesics  NARCOTIC ANALGESICS Act centrally Cause addiction Produce CNS depression Do not produce gastric irritation Show no anti inflammatory effect Eg. Morphine, Tramadol, Pethidine etc.  NON NARCOTIC ANALGESICS: Act peripherally Do not cause addiction Do not produce CNS depression Produce gastric irritation Show anti inflammatory effect Eg. Diclofenac, Ibuprofen, Aspirin etc.
Mechanism of Action  Analgesic drugs act in various ways on the peripheral and central nervous systems.  Opioids produce analgesia by binding to specific G – protein coupled receptors in brain and spinal cord.  NSAIDs inhibit the activity of both cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) and thereby the synthesis of prostaglandins and thromboxanes.  Inhibition of COX-2 leads to the anti-inflammatory, analgesic and antipyretic effects.
SCREENING MODELS
IN VIVO MODELS  1. Models using thermal stimuli: – Hot plate method – The tail flick model using radiant heat/immersion of the tail in hot water – Pain state models using cold stimuli •Cold tail flick test • Cold ethanol tail flick test (CET)  2. Models using electrical stimuli: – Stimulation of the tooth pulp – Electrical stimulation of the tail – Monkey shock titration test
 3. Models using chemical stimuli: – Formalin test –Writhing test – Stimulation of hollow organs • Rat sigmoid colon model • Inflammatory uterine pain model  4. Models using mechanical stimuli: – Haffner’s tail clip method – Randall Selitto Test
IN VITRO MODELS  3H-Naloxone binding assay.  𝜇 opiate receptor binding assay.  Receptor binding of cannabinoids.
IN VIVO MODELS
MODELS USING THERMAL STIMULUS
HOT- PLATE METHOD Purpose and rationale  The paws of mice and rats are sensitive to heat at temperatures which are not damaging to skin.  The responses are jumping, withdrawal of the paws and licking of the paws.  The responses are prolonged after administration of centrally acting analgesics, whereas peripheral analgesics of the acetylsalicylic acid or phenyl-acetic acid type do not generally affect these responses.
Procedure mice (18-22g) are used The temperature of the hot plate is maintained at 55° to 56°C. The animals are placed on the hot plate & time until either licking or jumping occurs is recorded. The latency is recorded before & after 20, 60 and 90 min after the administration of standard or test compound.
Evaluation  The prolongation of latency time between the test, standard and control animals are compared.  Using various doses ED50 values can be calculated.
TAIL FLICK MODEL Purpose and rationale  In tail flick test heat is used as the noxious stimulus.  Dependent variable is the time taken by the animal to flick its tail.  The morphine like drugs are capable of prolonging the reaction time.
The tail flick test using Radiant Heat
Procedure Mice (18-22g) are placed into small cage Leaving the tail exposed Appropriate temperature is maintained on the radiant source The tail of the mice is placed on the radiant source & time taken by the mice to withdraw its tail is recorded. Usually withdrawal time is within 2-10s The Tail-flick latency is recorded before & after the administration of standard or test compound.
Evaluation  The tail flick latency in the test, standard and control animals are compared.  Using various doses ED50 values can be calculated.
The tail flick test using Immersion of the tail Procedure  Wistar rat (170- 210gms) is used  Rat is placed into cage in such a way that their tail hangs freely.  Distal 5 cm of the tail of wistar rat is marked and immersed in a cup of warm water ( 55°Cto56°C) for 15 seconds.  The reaction time is determined periodically after administration of test drug (0.5, 1, 2, 3, 4, and 6 hours)
 Response and Evaluation: - Withdrawal of tail or attempt to escape or abrupt movement of tail and sometimes the recoiling of whole body is seen.  Centrally acting analgesics are capable of causing prolongation of tail withdrawal reflex( more than 6 sec.).  Modification: –Cold tail Flick test –Cold ethanol Tail flick test
MODELS USING ELECTRICAL STIMULUS
TOOTH PULP TEST Procedure  Rabbits 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 .
MONKEY SHOCK TITRATION TEST Procedure  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
MODELS USING CHEMICAL STIMULUS
WRITHING TEST Purpose and rationale  Pain is induced by injecting irritants like 0.6% acetic acid, Phenylquinone 0.02%, 4% NaCl into peritoneal cavity of mice.  The animals react with characteristic stereotyped behavior which is writhing. A writhe is indicated by stretching of the abdomen with simultaneous stretching of atleast 1 hind limb.  The test is suitable to detect analgesic activity of peripherally acting drugs.
Procedure Mice of either sex (20 and 25 g) is used Pain is induced by intraperitoneal injection of chemicals that irritate serous membranes The onset of writhing, abdominal contractions & trunk twist response are recorded for 10 min. The test and standard drug is administered 15 min prior to the acetic acid administration.
Evaluation  The writhing period is recorded and compared with the control group.  Writhing response in the drug treated must be less when compared to the control group.
FORMALIN TEST  Chronic pain model and sensitive to opioid like drugs.  Pain is induced in Male Wistar rats (180–300 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.
MODELS USING MECHANICAL STIMULUS
HAFFNER’S TAIL CLIP METHOD Purpose and rationale  Preferred sites for applying nociceptive mechanical stimuli are the hind paw and the tail  Highly sensitive for centrally acting drugs  Tests using constant pressure have been abandoned progressively for those applying gradually increasing pressures
Procedure An artery clip is placed at the root of tail of mice. A quick response is seen as biting the clip or tail, where clip has been placed. Then after 15, 30, and 60 minutes, the same procedure is repeated and the reaction time is measured.
RANDALL SELITTO TEST  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 Brewer’s 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.
ANIMAL MODELS OF CHRONIC PAIN  Neuropathic pain models  Vincristine induced neuropathy model  Diabetic neuropathy model  Persistent post-thoracotomy pain model  MODELS OF CANCER PAIN  Rat model of bone cancer pain
VINCRISTINE INDUCED NEUROPATHY MODEL:  Vincristine 100 μg/ 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.
DIABETIC NEUROPATHY MODEL:  Streptozotocin 75mg/kg i.p. in rats  diabetes development  Animals 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)
CANCER PAIN RAT MODEL OF BONE CANCER PAIN  Bone metastasis is one of the major causes of cancer related pain  Bone 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 3×103 cells.  Activity of animals is noted
IN VITRO MODELS
 3H-Naloxone binding assay  3H-Dihydromorphine binding to μ opiate receptors in rat brain  Inhibition of enkephalinase  Receptor binding of nociceptin  Receptor binding of cannabinoids  Vanilloid (capsaicin) receptor binding  Evaluation of vanilloid receptor antagonists  Vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP
DISCUSSION  Analgesic models are equally applicable for phasic or acute pain as well as tonic or chronic pain.  Different thermal, mechanical electrical and chemical stimuli are used.  In-vitro models are also used.  Centrally acting and peripherally acting analgesics. both can be evaluated by analgesic models.
 Haffner’s tail clip method, hot plate method, tail immersion method, formalin test in rats etc. are used mainly for central analgesic activity.  Writhing test, Randall Selitto test, mechanical/visceral pain models in the rat etc. Are used mainly for peripheral analgesic activity.
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