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Screening methods for analgesics
- 1. SCREENING METHODS FOR ANALGESICS SCREENING METHODS FOR ANALGESICS PRESENTED BY: AMEENA MEHABOOB
- 2. Pain is a symptom of many diseases requiring treatment with analgesics. Analgesics are drugs that selectively inhibit the perception (sensation) of the pain. INTRODUCTION
- 4. SCREENING METHODS IN VITRO • 3H-Naloxone binding assay • 3H-Bremazocine binding to κ opiate receptors in guinea pig cerebellum IN VIVO • HAFFNER’s tail clip method • Radiant heat method • Hot plate method • Tail immersion test • Electrical stimulation of the tail • Grid shock test • Tooth pulp stimulation • Monkey shock titration test • Formalin test in rats
- 5. IN VITRO TESTS 1) 3H-Naloxone binding assay PURPOSE AND RATIONALE- There is a correlation between the in vivo pharmacological potency of opiate agonists and antagonists with their ability to displace radiolabeled naloxone.
- 6. TISSUE PREPARATION Male wistar rats decapitated and brains removed Whole brain minus cerebella- weighed, homogenised in 50 vol of ice cold 0.05M Tris buffer Homogenate is centrifuged at 40000g, 15min Supernatent decanted, pellet resuspended in fresh 0.05M Tris buffer Yields tissue concentration of 10mg/ml
- 7. ASSAY 1 • Tubes containing H2O dextrorphan (total binding) or 5 μM levorphanol (non-specific binding), 2 M NaCl or H2O, 0.5 M Tris buffer, pH 7.7, drug or vehicle, 3H-naloxone tissue suspension are incubated for 30 min at 37 °C. 2 • The assay is stopped by vacuum filtration through Whatman GF/B filters. • Washed 3 times with icecold 0.05 M Tris buffer, pH 7 3 • Specific binding is roughly 1% of the total added ligand and 50% of the total bound in the absence of Na+ and 2% of the total added ligand and 65% of the total bound ligand in the presence of Na+ (100 mM). • The increase in binding is due to an increase in specific binding.
- 8. EVALUATION Data are converted into % stereospecific 3H-naloxone binding displaced by the test drug. IC50 values are determined from computer-derived analysis. The sodium shift is calculated from IC50 values with and without NaCl. High sodium shifts are found with agonists, low values with antagonists and medium values with mixed agonists-antagonists.
- 9. IN VIVO TESTS 1) HAFFNER’s tail clip method PURPOSE AND RATIONALE The raised tail (Straub phenomenon) in mice treated with morphine or similar opioid drugs and found the tail after drug treatment to be less sensitive to noxious stimuli.
- 10. PROCEDURE The animals were divided into four groups of six mice. An artery clip is applied to the root of the tail of mice and the reaction time is noted. The test compounds are administered orally to fasted animals. The drug is administered 15, 30 or 60 min prior testing. An artery clip is applied to the root of the tail (approximately 1 cm from the body) to induce pain. The animal quickly responds to this noxious stimuli by biting the clip or the tail near the location of the clip. The time between stimulation onset and response is measured by a stopwatch.
- 11. 2) EDDY’S HOT PLATE METHOD PROCEDURE The animals were divided into four groups of six mice. They are placed on Eddy’s hot plate heated to 55°C. The animal responds by either jumping or licking the paw. The response time prior to and after 30, 60, 120 minutes are taken. The cut off time is 15 seconds.
- 12. 3) TAIL IMMERSION METHOD PROCEDURE The animals are divided into four groups of six mice. The tail is immersed into hot water of 55°C. Within a few seconds, the mouse responds by withdrawing its tail. The reaction time is noted prior to and after 30, 60, 120, 180 minutes of drug administration
- 13. REFERENCES H. Gerhard Vogel, Drug Discovery and Evaluation, Pharmacological Assays, Second edition, Pages 385-693.