antiinflammatory drugs ppt..............

Non-steroidal anti-inflammatory drugs, also known as NSAIDs are medicines that are used to relieve pain, and reduce swelling (inflammation). Examples include aspirin, naproxen, ibuprofen, diclofenac, and COX-2 inhibitors such as celecoxib and meloxicam.

1. EVALUATION OF ANALGESIC AND ANTIINFLAMMATORY DRUGS ms.Sapna rani mmcp,mm (du)

2. EVALUATION OF ANALGESIC Methods for evaluation In vivo methods In vitro methods Thermal methods Electrical methods Mechanical method Chemical method 3 H-Naloxone binding assay 3 H-Dihydromorphine binding to µ  opiate receptors in rat brain 3 H-Bremazocine binding to κ opiate receptors in guinea pig cerebellum Isolated tissue preparation

3. Introduction What is Analgesic ? A drug which diminishes the perception of pain by selectively depressing nociceptive mechanism. Pain is very difficult to define and measure Pain is differ in animal than in man by two ways Absence of report of pain felt  response are observed Placebo effect is not be expected

4. In vivo techniques Thermal methods Hot plate method Tail immersion method Radiant heat method Electrical methods Pododolorimeter Rectodolorimeter Tooth pulp stimulation Mechanical method Tail Clip Method Chemical method Writhing method

5. In vivo methods Criteria for selection of methods: Methods must permits quantitative determination of threshold value of nociceptive stimulus. It should yield quantitative information on difference in intensity of stimuli. Applicable to both animal and man .

6. In vivo methods Problems with analgesic agents: False positive  to avoid this discoordination test is performed. In this test animals are placed on slowly rotating drum covered with wire mesh, the animals that fall off are considered as discoordinated  Excluded from experiment

7. Thermal methods Hot plate method Purpose and rationale Evaluation of opioid analgesic Do not stimulate mechanoreceptor Stimulus is easy to control & applied to moving subject To determine Analgesic potency ,Analgesic peak time and Duration of analgesic drug. Response observed - licking of fore paw and/or hind paws ,jumping. Procedure :-

8. Hot plate method Hot plate analgesy meter

9. Hot plate method Analysis of data {post drug latency -pre drug latency } MPE = { cutoff time(60s)-pre drug latency } ED 50 : Plot graph of %MPE Vs Dose Peak analgesic time: Plot graph of mean group latency (s) Vs time (min) Duration of analgesic action Plot graph of mean group latency (s) Vs time (min) and find out AUC .

10. Tail immersion technique Purpose and rationale : Evaluation of centrally acting analgesic drug To determine Analgesic potency ,Analgesic peak time and Duration of analgesic drug. In contrast to hot plate, this test is useful in repeated nociceptive evaluation using the same animal. Response observed – Violent jerk of tail.

11. Tail immersion technique

12. Tail immersion technique Analysis of data {post drug latency -pre drug latency } MPE = { cutoff time(15s)-pre drug latency } ED 50 ; Plot graph of %MPE Vs Dose Peak analgesic time: Plot graph of mean group latency (s) Vs time (min) Duration of analgesic action Plot graph of mean group latency (s) Vs time (min) and find out AUC .

13. Radiant heat method Purpose and rationale The test is very useful centrally acting morphine-like analgesics. To determine Analgesic potency ,Analgesic peak time and Duration of analgesic drug. Response observed  Flicking of tail .

14. Radiant heat method Procedure : Mice or rats Normal reaction time is determined. The animal is put into a small cage with an opening for the tail at the rear wall. The tail is held gently by the investigator. By opening of a shutter, a light beam exerting radiant heat is directed to the proximal third of the tail. The test compounds and the standard are administered either orally or subcutaneously. The animals are exposed to the same testing procedure after 30, 60 and eventually 120 min

15. Radiant heat method Analysis of data {post drug latency - pre drug latency } MPE = { cut off time (60)- pre drug latency } ED 50 : Plot graph of Dose Vs %MPE Peak analgesic time Plot graph of time (min) Vs mean group latency (s) Duration of analgesic action Plot graph of time (min) Vs mean group latency (s) and find out AUC .

16. Radiant heat method Critical assessment of the test The radiant heat test on the tail of mice is very effective to estimate the efficacy and potency of central acting analgesic drugs.

17. Electrical methods Pododolorimeter It consist of cage capable of accommodating mouse or rat and having floor made of metallic rod which can be electrified and voltage is applied. Response observed  crying and struggling This method are used for evaluation of centrally and peripherally acting analgesic Rectodolorimeter In this floor of cage consist of single copper plate which is connected to pole of current supply and other pole is connected to small electrode inserted to animal rectum. In this method voltage require to produce the response is less than Pododolorimeter This method are used for evaluation of centrally and peripherally acting analgesic.

18. Tooth pulp stimulation Purpose Evaluation of centrally and peripherally acting analgesics Procedure Rabbits Pulp chambers are exposed close to the gingival line in the lateral margins of the two front upper incisors with a high-speed dental drill. On the day of the experiment, clamping electrodes are placed into the drilled holes. After an accommodation period of 30 min stimulation is started to determine the threshold value. The stimulus  frequency of 50 Hz with duration is 1 s. The electrical current is started with 0.2 mA Response observed- -licking, biting, chewing and head flick

19. Tooth pulp stimulation Evaluation Increase of threshold, expressed in mV, is calculated and find out ED 50 Critical assessment of the method Central analgesics found to be very active in this test. High sensitivity than hot plate method. In addition, non-opiate analgesics like ketamine and peripheral analgesics like pyrazolone derivatives give a positive response.

20. Mechanical method Tail clip method Procedure Male mice with a weight between 18 and 25 g are used. The test compounds are administered subcutaneously to fed mice or 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. Response observed  biting the clip or the tail near the location of the clip.

21. Tail clip method Analysis of data {post drug latency - pre drug latency } MPE = { cut off time (15s)- pre drug latency } ED 50 : Plot graph of Dose Vs %MPE Peak analgesic time Plot graph of time (min) Vs mean group latency (s) Duration of analgesic action Plot graph of time (min) Vs mean group latency (s) and find out AUC .

22. Chemical method Writhing test Purpose and rationale Pain is induced by injection of irritants into the peritoneal cavity of mice. The animals react with a characteristic stretching behavior which is called writhing. An irritating agent such as phenylquinone or acetic acid are used . Evaluation of peripheral analgesic Response observed  writhing

23. Writhing test Procedure Mice Phenylquinone in a concentration of 0.02% is suspended in a 1% suspension of carboxymethylcellulose. 0.25 ml suspension is injected intraperitoneally. Groups of 6 animals are used for controls and treated mice. The mice are placed individually into glass beakers and five min are allowed to elapse. The mice are then observed for a period of ten min and the number of writhes is recorded for each animal. For scoring purposes, a writhes is indicated by stretching of the abdomen with simultaneous stretching of at least one hindlimb.

24. Writhing test Evaluation % Inhibition is calculated avg writhes (control)-avg writhes (test) % Inhibition = 100 x avg writhes (control) ED 50 : Plot graph of Dose Vs % inhibition

25. Other methods Monkey shock titration test Formalin test in rats Pain in inflamed tissue (RANDALL-SELITTO-test

26. In vitro methods 3 H-Dihydromorphine binding to µ opiate receptors in rat brain 3 H-Naloxone binding assay 3 H-Bremazocine binding to κ o piate receptors in guinea pig cerebellum Isolated tissue preparation

27. 3 H-Dihydromorphine binding to µ receptors Purpose and rationale µ Receptors are considered to mediate the supraspinal activity of opioids. 3 H-Dihydromorphine ( 3 H-DHM)exhibits some selectivity for the µ receptor, a high affinity opiate binding site. Tissue preparation Male Wistar rats are sacrificed by decapitation. whole brains homogenated .

28. 3 H-Dihydromorphine binding to µ receptors Assay 1 850 µl tissue suspension 80 µl distilled water 20 µl vehicle, or levallorphan , or appropriate concentration of drug 50 µl [3H]DHM. Evaluation Specific binding is defined as the difference between total binding and binding in the presence of 0.1 mM levallorphan . IC 50 values are calculated from the percent specific binding at each drug concentration.

29. Isolated tissue preparations Electrically evoked contractions of isolated tissue preparations from different species : The contractions of the mouse vas deferens are inhibited by µ-, δ-, and κ-agonists . The guinea-pig myenteric plexus-longitudinal muscle preparation by µ- and κ-agonists Rabbit vas deferens by κ-agonists, and Hamster vas deferens by δ-agonists. The contractions of the rat vas deferens are inhibited mainly, but not exclusively, by δ-agonists.

30. EVALUATION OF ANTI-INFLAMMATORY AGENTS In vivo methods In vitro methods UV-erythema in guinea pigs Vascular permeability Oxazolone-induced ear edema in mice Paw edema Pleurisy test Granuloma pouch technique Cotton wool granuloma Glass rod granuloma PVC sponge granuloma . 3 H-Bradykinin receptor binding 3 H-Substance P receptor binding Methods for evaluation

31. Introduction What is inflammation ? Inflammation is a process which begins sublethal injury to tissues. The classical signs of inflammation are “ rubor ” (redness), “tumor” (swelling), “ calor ”(heat), “dolor”(pain), “ functia laesa ”(loss of function). The inflammatory process events elicited by infectious agents, ischemia, antigen-antibody interactions, chemical, thermal or mechanical injury.

32. Introduction Inflammatory responses : An acute, transient phase,  local vasodilatation and increased capillary permeability, A subacute phase,  infiltration of leukocytes and phagocytic cells, Chronic proliferative phase,  tissue degeneration and fibrosis occur.

33. In vivo methods Methods for testing acute and subacute inflammation are: UV-erythema in guinea pigs Vascular permeability Oxazolone-induced ear edema in mice Paw edema in rats (various modifications and various irritants) Pleurisy tests Granuloma pouch technique (various modifications and various irritants)

34. Methods for testing acute and subacute inflammation Ultraviolet erythema in guinea pigs Purpose Evaluation of anti –inflammatory agents Procedure Albino guinea pigs (average weight of 350 g ). Eighteen h prior testing, the animals are shaved on both flanks and on the back. On the next day, the test compound is dissolved (or suspended) in the vehicle and half the dose of the test compound is administered by orally (at 10 ml/kg) 30 min before ultraviolet exposure. Control animals are treated with the vehicle alone. The guinea pigs are placed in a leather cuff with a hole of 1.5 . 2.5 cm size punched in it, allowing the ultraviolet radiation to reach only this area.

35. Ultraviolet erythema in guinea pigs Evaluation The degree of erythema is evaluated visually by 2 different investigators in a double blinded manner. Scores are given: 0 = no erythema, 1 = weak erythema, 2 = strong erythema, 4 = very strong erythema. ED 50 values is calculated Critical assessment The test has the advantage of simplicity but needs training of the investigators. Corticosteroids after systemic application are ineffective in this test, however, can be evaluated after topical administration.

36. Vascular permeability method Purpose The test is used to evaluate the inhibitory activity of drugs against increased vascular permeability which is induced by a phlogistic substance. Procedure Rat. The ventral sides of the animal are shaved. Five ml/kg of an 1% solution of Evan’s blue are injected intravenously . 1 hr later the animals are dosed with the test compound orally or intraperitoneally or with the vehicle. contd..

37. Vascular permeability method 30min later, 0.05 ml of an 0.01% solution of compound 48/80 are injected intracutaneously at 3 sites both at the left and ventral side. Ninety minutes after the injection of compound 48/80 the animals are sacrificed by ether anesthesia. The abdominal skin is removed and the dye-infiltrated areas of the skin are measured . Evaluation The diameter of the dye-infiltrated areas is measured in millimeters in two perpendicular directions and the mean values of all injection sites in one animal are calculated.

38. Vascular permeability method . The percent inhibition in the treated animals as compared to the control group is calculated. ED 50 values is calculated Critical assessment Characterization of a new a nti-inflammatory compound. Sympathomimetic activity have a pronounced effect  cannot be regarded as a primary screening test for anti- inflammatory products. Together with an observation of writhing of mice  able to distinguish between narcotic and non narcotic analgesics

39. Oxazolone-induced ear edema in mice Purpose Evaluation of the topical and systemic anti-inflammatory activity of a compound following topical administration . Procedure The mice are sensitized by application of 0.1 ml 2% oxazolone on the inside of both ears under halothane anesthesia. The mice are challenged 8 days later again under anesthesia by applying 0.01 ml 2% oxazolone solution to the inside of the right ear (control). The left ear remains untreated. After 24 h the animals are sacrificed under anesthesia and a disc of 8 mm diameter is punched from both sides. The discs are immediately weighed on a balance .

40. Oxazolone-induced ear edema in mice Evaluation Average values of the change in weight are calculated for each treated group and compared statistically with the control group. Critical assessment The method is suitable for both steroidal and non-steroidal compounds as well as for the evaluation of various topical formulations.

41. Paw edema Purpose Evaluation of anti –inflammatory agents Procedure The rats  subcutaneous injection of 0.05 ml of 1% solution of carrageenan into the plantar side of the left hind paw. The paw is marked with ink at the level of the lateral malleolus and immersed in mercury up to this mark. The paw volume is measured plethysmographically immediately after injection, again 3 and 6 h, and eventually 24 h after challenge.

42. Paw edema Evaluation The increase of paw volume after 3 or 6 h is calculated as percentage compared with the volume measured immediately after injection of the irritant for each animal . A dose- response curve is run for active drugs and ED 50 values can be determined.

43. Paw edema Modifications 0.05 ml undiluted fresh egg white 0.1 ml of 1% ovalbumin solution 0.1 ml of 1% formalin 0.1 ml of 0.2% carrageenan solution 0.1 ml of 1% carrageenan solution plus 100 ng PGE2 or PGI2 0.1 ml of 1 to 3% dextran solution 0.1 ml of 2.5% brewer’s yeast powder suspension

44. Methods for testing proliferative phase Cotton wool granuloma Purpose granulomas were provoked in rats by subcutaneous implantation of pellets of compressed cotton. After several days, histologically giant cells and undifferentiated connective tissue can be observed besides the fluid infiltration. The method has been useful for evaluation of steroidal and nonsteroidal anti-inflammatory drugs .

45. Cotton wool granuloma Procedure Rats  anaesthetized  The back skin is shaved. An incision is made in the lumbar region. By a blunted forceps subcutaneous tunnels are formed and a sterilized cotton pellet is placed on both sides in the scapular region. The animals are treated for 7 days subcutaneously or orally. Than, the animals are sacrificed, the pellets are prepared and dried until the weight remains constant.

46. Cotton wool granuloma Evaluation The average weight of the pellets of the control group as well as of the test group is calculated. The percent change of granuloma weight relative to vehicle control group is determined .

47. Others methods Sponge implantation technique Glass rod granuloma

48. In vitro techniques 3 H-Bradykinin receptor binding 3 H-Substance P receptor binding

49. 3 H-bradykinin receptor binding Purpose The 3 H-bradykinin receptor binding is used to detect compounds that inhibit binding of 3 H-bradykinin in membrane preparations obtained from guinea-pig ileum. Procedure Ileum  homogenate centrifuge final pellets resuspended 150 l ileum suspension 50 l 3 H-bradykinin ( 0.005 x 10 -9 ) 50 l test compound (10 -5 to 10 -10 ) and total binding and non specific binding is determined.

50. 3 H-bradykinin receptor binding Evaluation Total binding of 3 H-bradykinin Non-specific binding in the presence of 10 µM bradykinin Specific binding = total binding – non-specific binding % inhibition: 100 – specific binding as percentage of control value and RBA is calculated

51. 3 H-Substance P receptor binding Purpose Evaluation of substance P antagonists as anti-inflammatory and analgesic drugs. Procedure porcine brains homogenate centrifuge final pellets resuspended 150 l suspension 50 l 3 H-substanceP ( 0.003 x 10 -9 M ) 50 l test compound (10 -5 to 10 -10 M ) total binding and non specific binding is determined.

52. 3 H-Substance P receptor binding Evaluation Total binding of 3 H substance P Non-specific binding in the presence of 25 µM substance P Specific binding = total binding – non-specific binding % inhibition: 100 – specific binding as percentage of control value and RBA is calculated

53. THANK YOU