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Screening methods for analgesics

This document summarizes screening methods for central and peripheral analgesics. For central analgesics, it describes in vivo methods like Haffner's tail clip, hot plate, tail immersion, and formalin tests that assess response to painful stimuli in mice and rats. For peripheral analgesics, it discusses writhing tests using acetic acid or phenylquinone in mice, Randall-Selitto testing in inflamed rat paws, and duodenum distension in rats to measure visceral pain responses. The document provides classifications and examples of different classes of central and peripheral analgesic agents and their mechanisms of action.

Health & Medicine◦
SCREENING METHODS FOR ANALGESICS DR. AMIT D. SHARMA Third year resident, Department of pharmacology SBKS MI & RC. Date : 09/05/2015
Contents  Definitions  Central analgesics : agents, mechanism, in vivo methods  Peripheral analgesics : agents, mechanism, in vivo methods
Definitions  Pain- an unpleasant sensory or emotional experience associated with actual or potential tissue damage, or described in terms of such damage (WHO).  Features of inflammation Pain (dolor) rubor (redness) tumor (swelling) calor (heat) functio laesa (loss of function).
 Analgesics – the drugs which possess significant pain- relieving properties by acting in the central nervous system or on peripheral pain receptors without significantly affecting consciousness.  Two groups : I. Narcotic/opioid /morphine like analgesics(central) II. Non-narcotic/ non steroidal anti-inflammatory- analgesic-antipyretic agents(peripheral)
CENTRALANALGESIC ACTIVITY
Classification  Morphine analogues: I. Agonists – morphine(prototype), diamorphine(heroin), codeine, pholcodeine, levorphanol II. Partial agonists – nalorphine, levallorphan  Synthetic derivatives unrelated to morphine : I. Agonists – Pethidine (meperidine), fentanyl, sufentanil, alfentanil, remifentanil, methadone, oxycodone II. Partial agonists – pentazocine, cyclazocine, buprenorphine, nalbuphine, butorphanol III. Mu receptor agonist with other mode of action – tramadol
IN VIVO METHODS
Haffner’s tail clip method, 1929  The test compounds are administered subcutaneously to fed mice or orally to fasted animals.  Then an artery clip is applied to the root of the tail (approximately 1 cm from the body) of mice.  The reaction time is noted.  Response : biting the clip or the tail near the location of the clip.
Hot plate method  Woolfe and Mac Donald (1944)  Mice are put on hot plate(temp 55° to 56 °C) and then latency is noted following administration of test drug.  Response – jumping or paw licking
Tail immersion method  Restraining the young female wistar rat, lower 5 cm portion of the tail is immersed in a cup of freshly filled water of exactly 55°C.  The reaction time is determined before and periodically after either oral or subcutaneous administration of the test substance.  The cut off time of the immersion is 15 seconds.  Response – withdrawal of tail or attempt to escape.
Grid shock test  Blake et al(1963)  Central and peripheral analgesics  Stimulus is given in the form of square wave pulses to male mice. The output of the stimulator is connected to the wires of grid with fixed resistance , which is kept parallel to oscilloscope.  Pain thresholds are determined in each individual mouse twice before and after administration of the test drug.  Response – on oscilloscope, a startling reaction, increase locomotion or attempt to jump.
Electrical stimulation of the tail  Burn et al, 1950  Central and peripheral analgesics  First test compound is given and then rectangular wave pulses from a constant voltage stimulator are applied to the alligator clips attached to the tail of the mice.  Response – spinal reflexes, complete vocalization or behavior changes(escape, aggression). The normal response time range of the stimuli is 3–4 sec.
Tooth pulp stimulation  Kohl and Reffert (1938)  Opioid agonists, pyrazolone derivatives  After anaesthetizing the rabbit with thiopental, current is applied through the electrodes passed in the dental drill holes exposed to pulp chambers.  Pain thresholds are determined in each individual rabbit twice before and after administration of the test drug.  Response - licking, biting, chewing or head flick.
Formalin test in rats  Dubuisson and Dennis,1977  Male wistar rats are administered 0.05 ml of 10% formalin in dorsum of the front paw along with test drug subcutaneously or orally.  Response - elevation or favoring of the paw or excessive licking and biting of the paw.
PERIPHERAL ANALGESIC ACTIVITY
Classification  Nonselective COX inhibitors (traditional NSAIDs) I. Salicylates – aspirin II. Propionic acid derivatives – ibuprofen, naproxen III. Fenamide – mephenamic acid IV. Enolic acid derivatives – piroxicam V. Acetic acid derivatives – ketorolac, indomethacin VI. Pyrazolone derivatives – phenylbutazone
 Preferential COX-2 inhibitors - nimesulide, diclofenac, aceclofenac, etodolac.  Selective COX-2 inhibitors – celecoxib, etoricoxib.  Analgesics-antipyretics with poor antiinflammatory action- I. Paraaminophenol derivative – paracetamol(acetaminophen). II. Pyrazolone derivatives – metamizol(dipyrone), prophenazone. III. Benzoxazocine derivative – nefopam.
IN VIVO METHODS
Writhing tests  Mice are administered the test drug before intraperitoneal injection of irritating agent like phenylquinone or acetic acid.  Pain sensation in acetic acid induced writhing paradigm is elicited by producing localized inflammatory response due to release of free arachidonic acid from tissue phospholipids via cyclo-oxygenase (COX), and producing prostaglandin specifically PGE2 and PGF2α, the level of lipoxygenase products may also increases in peritoneal fluids.
 Response – writhe (stretching of the abdomen with simultaneous stretching of at least one hind limb)  Aconitine can also be used instead of above drugs.  Rats – 4% sodium chloride.
Pain in inflamed tissue (RANDALL-SELITTO TEST)  Principle - inflammation increases the sensitivity to pain and that this sensitivity is susceptible to modification by analgesics.  Male wistar rats are given the test drug orally, subcutaneously or intraperitoneally.  Inflammation is induced by subcutaneous injection of Brewer’s yeast into the plantar surface of the left hind paw of the rat .Then pressure is applied through a tip to the plantar surface of the rat’s foot at a constant rate by a special apparatus to the point at which the animal struggles, squeals or attempts to bite.
Mechanical visceral pain model in the rat  Coburn et al 1989,deLeo et al 1989.  First test drug is administered into male Sprague Dawley rats and anaesthetized with nitrous oxide and halothane.  One piece balloon catheter is passed through gastrostomy tube into first part of duodenum.  Inflation of balloon distension of duodenum writhing.
Behavioural responses score 0 : Normal behaviour defined as exploration, escape attempts and resting 1 : Slightly modified behaviour defined as cessation of exploration, focusing, wet-dog shake, excessive facial grooming, teeth chattering and deep breathing 2 : Mildly to moderately modified behaviour defined as retching-like activity, hunching, abdominal grooming or nipping and immobility of the hind limbs (disappears with removal of the stimulus).
3 : Severely modified behaviour defined as stretching of the hind limbs, arching and dorsoflection of the hind paws. 4 : Intensive visceromotor activity defined as repetitive stretching of the body, extension of the hind limbs, and pelvis, frequent rotating sideward , i.e., writhing.
References  H. Gerhard Vogel et al. Drug discovery and evaluation : Pharmacological assays, third edition.  K D Tripathi. Essentials of medical pharmacology, seventh edition.  H L Sharma, K K Sharma. Principles of pharmacology, second edition.  Harsh Mohan. Textbook of pathology, sixth edition.
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