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A. Gowtham Sashtha, profile picture
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Screening Methods of Analgesics Agents...

The document discusses various screening methods for analgesic agents, classifying pain into acute and chronic categories, and detailing the mechanisms of analgesics. It provides an overview of in vivo and in vitro models used to evaluate analgesic efficacy, including thermal, electrical, chemical, and mechanical stimuli tests. Additionally, it outlines the procedures for specific screening tests such as the tail flick test, writhing test, and formalin test.

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Screening Methods of Analgesic agent Presented by A. Gowtham Sashtha 1st M.pharm Department of pharmacology K.M College of pharmacy Madurai - 625107.
CONTENTS : ☞ Introduction ☞ Classification of pain ☞ Analgesic drugs ☞ Mechanism of action ☞ Screening models ☞ Invivo Models ☞ In-vitro models
Introduction ❯ Pain is the Unpleasant sensory and emotional experience associated with actual and potential tissue damage. ❯ Nociception perception of noxious stimuli. ❯ Analgesics define as the agents wich is selectively relive pain by acting in the CNS or peripheral pain mechanism without significantly altering consciousness.
Classification of pain 1) Acute pain : →Source is soft tissue damage,Infection or inflammation. →Short in duration →Serves to protect one after an injury. →Define as "Symptoms of pain" 2) Chronic pain : →last 6 months or longer →Define as "disease of pain" eg : cancer pain,Neuropathic pain,arthritic pain.
Analgesic drugs : Norcotic analgesic. Non Norcotic analgesic eg : eg : Morphine,Tramadol. Diclofenac,ibuprofen, pethidine. Asperin.
Mechanism of action : ❯ The analgesics drugs are acts as the various ways on the peripheral nad central nervous system. ❯ Opioid produced analgesia by binding to the specific G - protein couple receptor in brain and spinal cord. ❯ NSAID is inhibit the activity of both cyclooxygenase-1 (Cox-2) cyclooxygenase-2 (cox-2) and there by the synthesis prostaglandins and thromboxanes.
Screening Methods ☞ In vivo ☞ In vitro
Invivo methods : 1) Methods using thermal stimuli 1)Eddy's hot plate methods 2)The tail flick model →Radiant heat → Immersion of the tail in hot water. 3)pain state model using cold stimuli → Cold tail flick test → Cold ethanol tail flick test (CET)
2)Methods using electrical stimuli : 1) Stimulation of the tooth pulp 2) Electrical stimulation of the tail 3) Monkey shock titration method 3) Methods using chemical stimuli. 1) Formalin test 2) Writhing test
4) Methods using mechanical stimuli : Haffner's tail clip method. Randall selitto test. 1) 3H-Naloxone binding assay. 2) μ opiate receptor binding assay. 3) Cannabinoids receptors binding assay. Invitro methods :
1) Methods using thermal stimuli
Hot plate method Purpose : ✓ The paws of mice and rats sensitive to the heats at temperatures which are not damage to the skin. ✓ The responses are jumping, withdrawal of the paws and licking of the paws. ✓ The analgesics drugs are response to this symptoms.
Procedure : Mice are used (18-22g) The temperature of the hot plate is maintained at 55o C 56o C The animals placed on the hot plate & time until either licking or jumping occurs is recorded. The latency is recorded before & after the administration of standard or test compounds.
Evaluation : ✓ The prologation of latency time between the test, standard and control animals are compared. ✓ Using various doses ED50 values can be calculated.
Tail Flick Methods : 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 withdraw time is within 2-10s The tail flick latency is recorded before & after the administration of standard or test compound.
The tail flick test using immersion of the tail procedure : wistar rat (170-210 gms) is used Rat is placed in to cage in such a way that their tail hangs freely. Distal 5cm of the tail of wistar rat is marked and immersed in a cup of warm water (55o C to 56o C) for 15 sec. The reaction time is determined periodically after administration of the test drug (0.5,1,2,3,4 and 6 hours)
Evaluation : The tail flick latency in the test, standard and control animals are compared. Using various doses ED50 values can be calculated. Modification methods : →Cold tail Flick test. →Cold ethanol tail flick test.
2)Methods using electrical stimuli
Tooth pulp test : Procedure : ✓ Rabbit 2-3 kg, anesthetized with thiopental sodium 15 mg/kg i.v ✓ Using dental drill,tooth pulp chambers are exposed close to upper incision. ✓ Champing electrodes are placed into the drilled holes. ✓ After 30 min. electrically stimulus is applied by rectangular current of frequency 50HZ up to 1sec ✓ Current is started 0.2 mA and increased until animal starts licking and a threshold is determined.
Monkey shock Titration method Procedure : ✓ Monkey are seated on restraining chairs. ✓ By coulbourn instrument programmable shocker electric current is applied through electrodes to shaved portion of tail. ✓ Current ranges from 0-4mA ✓ Monkey presses a bar to interrupt the shock. ✓ For each monkey stable base line shock level is recorded day perior to drug administration. ✓ After 24hr of drug ,shock titration is measured.
3) Methods using chemical stimuli
Writhing test : Purpose : ✓ Pain is induced by injecting irritants like 0.6% acetic acid,0.02% phenylquinone,4% Nacl into peritoneal cavity of mice. ✓ Writhing is produced.writhing is indicated by stretching of the abdomen with simultaneous stretching of atleast 1 hind limb. ✓ The test is suitable to detect analgesics activity of the peripherally acting drugs.
procedure : Mice of either sex (20-25 gm) is used. Pains is induced by intraperitoneal injection of chemicals that irritate serous membranes. The onset of writhing produced and recorded for 10 mins. The test or standard drugs is administered 15 mins perior to the acetic acid administration.
Evaluation : ✓ The writhing period is recorded and compared with the control group.
Formalin Test : Procedure : ✓ 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 favouring of the paw.
✓ Two distinct periods of high licking activity can be identified : 1) Early phase lasting first 3-5 mins 2) Late phase lasting from 15 to 30 min ✓ cenrally acting analgesics (morphine) are antinociceptive in both phase ✓ Substance p and bradykinin act as the Early phase. ✓ NSAIDS (indomethacin and naproxen) inhibit by only late phase.
3) Methods using mechanical stimuli
HAFFNER'S Tail clip method : Purpose : ✓ Preferred sites for applying nociceptive mechanical stimuli are the hind paw and the tail. ✓ Highly sensitive for cenrally acting drugs. ✓ Tests using constant pressure have been abandoned progressively for those applying gradually increasing pressure.
Procedure : An artery clip is placed at the root of tail of mice A quick response is seen as biting 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 : Procedure : ✓ Male wistar rats (130-175 g) are used. ✓ 0.1ml of a 20% suspension of Brewer's yeast is injected in 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.
Invitro methods
3H Naloxone binding assays Purpose : ✓ Naloxone is the opioid antagonist. ✓ Na+ enhances the binding of antagonists and reduces the binding of agonist. ✓ antagonists by determining the IC50 values for 3H-Naloxone in the presence or absence of Na+ enhances.
Procedure : ✓ Male wister rats are used and their brain are removed,the cerebella area weighted and homogenised in50 volume of ice cold soln. ✓ The homogenate centrifuged at 40000 g in 15mins after recentrifuged at 40000g. ✓ The tubes are incubated at 30mins at 370 C. ✓ The assay is stopped by vaccum filteration through whatman filters washing 3 times.
✓ The filters are counted in 10ml and add to the assay soln between binding in the presence of dextrophan and levorphanol agents are calculating this formula. ✓ specific binding = total binding- non specific binding
Assay reagent : 310 µl H2O 20 µl 5 µM dextrorphan (total binding) or 5 µM levorphanol (non-specific binding) 50 µl 2 M NaCl or H2O 50 µl 0.5 M Tris buffer, pH 7.7 20 µl drug or vehicle 50 µl 3H-naloxone 500 µl tissue suspension.
μ Opiate receptor binding assay Purpose : ✓ Opioid drugs exert their analgesic action mainly through μ opioid receptors only. ✓ The compounds that inhibit binding of 3H dihydromorphine in a synaptic membrane preparation from rat brain can be identified by this assay.
Procedure : same procedure of Naloxone binding assays. Assay reagent : 20nM stock soln of 3H Dihydromorphine 0.1 mM stock soln of levorphanol tartrate 1mM of test stock solution Ice cold 0.05M Tris buffer
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