Screening methods of Anti inflammatory drugs

Screening methods of Anti Inflammatory drugs including Acute Phases of inflammation, Sub Acute Phases of Inflammation and Chronic Phases of Inflammation

1. Screening of Anti-inflammatory Drugs Presenter: Dr. Nitesh Shukla (JR-2) Moderator: Dr. Shoebul Haque (SR) Peer Support: Dr. Snehashis Singha (JR-2) Department of Pharmacology & Therapeutics King George’s medical University , Lucknow, U.P, India Email id- [email protected]

2. Contents : Introduction Phases of inflammation Types of screening methods In-vitro methods of screening In-vivo methods of screening Summary References

3. Specific learning objectives : At the end of this teaching learning session, the audience will able to- Understand about the inflammation and its phases Enumerate in-vitro methods of screening of anti-inflammatory drugs Enumerate in-vivo methods of screening of anti-inflammatory drugs

4. Introduction : Inflammation : “ Inflammation is a response of vascularized tissue to infections and damaged tissue that brings cell and molecules of host defense from the circulation to the sites where they are needed, in order to eliminate the offending agents”.

5. Exogenous stimuli : Physical Chemical Mechanical Nutritional Biological Endogenous stimuli : Immunological Neurological Genetic origin

6. Phases of inflammation : Acute phase- An immediate response to any insult , lasts for few days Subacute phase- It is a transitional phase, lasts from 2 to 6 weeks Chronic phase- It can persist for months or even years

7. Acute Inflammation : The classic triad of acute inflammation is Pain, fever & swelling , which is caused by increased blood low Increased capillary permeability Subacute inflammation : Increased migration of leukocytes out of which neutrophils are first to ingress into the affected tissue area Chronic Inflammation : Defined by tissue proliferation, granuloma, and repair

8. Cardinal features of inflammation : Erythema - Initiated by vasodilation following release of Nitric oxide from endothelium Edema- Initiated by increased capillary permeability Hyperalgesia and Pain- Elicited because of increased viscosity (Stasis)

9. Cellular events of inflammation :

10. Types of screening methods

11. In-vivo methods : Animal m odel for acute p hase : Carrageenan induced paw oedema in rats Histamine induced paw oedema in rats Xylene induced ear oedema Arachidonic acid induced ear oedema

12. Animal m odel for acute p hase ( Cont.) : Croton oil induced ear oedema Oxazolone induced ear oedema UV erythema in guinea pigs Pleurisy in rats Vascular permeability

13. Animal model for subacute phase : Carrageenan induced granuloma pouch model Formalin induced paw oedema

14. Animal model for chronic phase : Cotton wool granuloma Glass rod granuloma Sponge implantation technique

15. Acute phase experimental model

16. Carrageenan-induced paw edema : It is based on principle of release of various inflammatory mediators by carrageenan which leads to oedema in rat paw Biphasic event Initial phase : Attributed to release of histamine and serotonin Second phase : Due to release of prostaglandins , protease and lysosome S/c injection of carrageenan into rat paw produced inflammation

17. Methodology : Test and standard drugs are administered 1 hour prior to procedure Animals are divided into groups Sprague-Dawley rats of either sex with body weight 150 to 170 gm Paw is marked at the level lateral malleolus & immersed in mercury up to this mark After 1 hour s/c injection of 0.05 ml of 1% solution of carrageenan into plantar side of hind limb

18. Paw volume measured using plethysmometer at 3 hr., 6 hr., and 24 hr. Increase in paw volume at 3 and 6 hours is calculated % increase in paw volume is measured by comparing the difference of average value between treated group and control group animals at each interval Vt and Vc are oedema volume in average in the drug treated and control group % oedema inhibition = [( V c -V t )/ V c ] X 100 Evaluation :

19. Study of anti-inflammatory activity using carrageenan induced paw oedema method YouTube. Available from: https://www.youtube.com/watch?v=xX41v7vUDhQ

20. Histamine-induced paw oedema in rats : Histamine injection of 0.1 ml at subplantar surface induces paw oedema Occurs due to vascular reaction (vasodilation and increase vascular permeability ) Evokes the release of neuropeptides as well as release of prostaglandins from endothelial cell leading to hyperalgesia and other inflammatory effects

21. Arachidonic acid-induced ear oedema : Metabolism of arachidonic acid by COX (cyclo-oxygenase) leads to generation of PGs and Thromboxane that mediate pain and oedema Inhibition of these mediators by test drug is evaluated Inflammation is induced by topical application of arachidonic acid, over right ear of each mouse whereas left ear is regarded as control Percentage of ear oedema is calculated based on left ear

22. Croton oil-induced ear oedema in mice : It induces the activation of phospholipase A2 ,which releases arachidonic acid metabolites leading to generation of PGs and leukotrienes Ability of a test drug to inhibit oedema by inhibition of COX and 5-lipoxygenase and are tested by this procedure Steps involved : Administer test drug and standard drug before application of croton oil Administration of test drug and standard drug are repeated, 2 hrs after croton oil application

23. Apply the croton oil with a micropipette (20 µl/ear) to inner surface of right ear Apply acetone to left ear which serves as control After croton oil application the mouse are sacrificed by cervical dislocation Left ear (acetone), right ear (croton oil in acetone) both are removed, and Degree of inflammation is measured and compared

24. Oxazolone-induced ear oedema in mice : It is the model of delayed contact hypersensitivity which permits quantitative evaluation of inflammatory mediators Animals divided into various groups(n=6) Mice are sensitized by application of 0.1 ml of 2% oxazolone solution in acetone on the shaved abdominal skin or 0.01 ml, inside both the ear under anesthesia

25. After 8 days, 0.01 ml of 2% oxazolone solution is given again inside right ear The untreated left ear are painted on both side with ethanol/acetone mixture Maximum inflammation occurs 24 hrs later Animals are sacrificed under anesthesia and disc of 7 mm is taken from both side The disc are immediately weighed, weight difference is indicator of inflammatory oedema

26. Xylene-induced ear oedema : Xylene induces neurogenous oedema associated with substance p In periphery release of substance P from sensory neurons causes vasodilation and plasma extravasation Produces oedema in ear of the mice

27. Xylene-induced ear oedema (Thickness parameter) : The mice are divided into groups (n=6) & fasted overnight, water ad-libitum Animals are administered test drug and standard drug After 1 hour each animal received 30 ml of xylene is applied over right ear and left ear is considered as control After 1 hr. thickness of the ear is determined using Digimatic Calliper Percentage of ear oedema is calculated based on left ear (without xylene)

28. Xylene-induced ear oedema (weight parameter ) : Animals are sacrificed by access ether anaesthesia and both ears are removed Circular sections are taken using cork borer with diameter of 7 mm and weighted Percentage of ear oedema is calculated based on left ear (without xylene) and weight difference is indicator of inflammatory oedema

29. UV-B-induced erythema in guinea Pigs : Guinea pigs of either sex weighing about 350 gm are the frequently used animals to study the anti-inflammatory activity of drugs in this model 18 hrs prior to experiment, the animal are shaved on both the sides, flanks and on back Guinea pigs are pretreated with the test drugs 30 min before UV-exposure In a lbino guinea pigs, the pure erythema reaction appears 2 h after exposure of the depilated skin to ultraviolet radiation

30. Model can be used as a pure measure of the vasodilatory phase of inflammation Erythema is scored after 2 and 4 hours of exposure Following scores are given : 0- No erythema 1- weak erythema 2- strong erythema 4- Very strong erythema Animal with score of 0 or 1 are said to be protected under the influence of pretreated test drug

31. Sub-acute phase experimental model

32. Carrageenan induced granuloma pouch model Carrageenan is indicated into s/c air pouch on dorsal surface of rat initiates inflammatory response Procedure : Rats are divided into respective groups(n=6) Fasted overnight with free access to water Animals are administered with vehicle, test and standard drug 1 hour later the back of animal is shaved and disinfected

33. S/c dorsal granuloma is made by injecting 6 mL of air followed by 4 ml of 2% carrageenan in normal saline Treatment is continued for seven consecutive days On 8 th day amount of exudate is collected by means of syringe under anesthesia Average volume of exudates, and weight of granuloma is determined Average value of exudate in controls and test groups is calculated and compared

34. Chronic phase experimental model

35. Cotton wool granuloma : Granuloma is induced in rat by subcutaneous implantation of pellets of compressed cotton Histologically , giant cells and undifferentiated connective tissue along with fluid infiltration can be observed Amount of newly formed connective tissue can be measured by weighing the dried pellet after removal Cotton pellet impregnated with carrageenan induces more extensive granuloma

36. Procedure : Rats are divided into respective groups (n=6) and fasted overnight allowed free access to water Animals are administered with vehicle , test and standard drug One hour after first dosing, animals are anesthetised and cotton pellet is implanted in each axilla and groin region by subcutaneous incision and sutured with catgut Animals are sacrificed by excess anaesthesia

37. On 8 th day cotton pellets are removed and seperated by extraneous tissue Dried at 60 ̊C until weight become constant Net dry weight after subtracting initial weight of cotton pellet is measured Average weight of control group and test group will be calculated and compared % Inhibition = {( W c -W d )/ W c } X 100 W d = difference in pellet weight of the drug treated group W c = difference in pellet weight of the control group

38. Cotton Wool Granuloma (Method for Anti-inflammatory study) Available from: https://youtu.be/Gjzxn53sTAA

39. In vitro methods Measurement of NO Production in LPS/ IFN-γ- co-stimulated and unstimulated murine macrophage RAW264.7 cell line Inhibition of oxidative stress by reducing reactive oxygen species (ROS) generation Blocking Inflammasome Activation Induction of autophagy

40. Measurement of cytokine expression in murine macrophages Measurement of Inflammatory Gene Expression Adhesion Assays Interleukin 1beta-stimulated human articular chondrocytes Cyclo-oxygenase (COX) assays Mast cell degranulation

41. Summary Inflammation is the body’s defense against infections and injuries. It has acute, subacute, and chronic phases. Anti-inflammatory drugs are tested using in vivo & in vitro methods . In-vivo models include carrageenan-induced paw edema , histamine-induced oedema , and cotton wool granuloma. In vitro methods assess NO production, oxidative stress, cytokine levels. Screening helps develop effective anti-inflammatory drugs for clinical use.

42. References Sarkar S, Srivastava V, Mohanty M. Postgraduate pharmacology . 1st ed. New Delhi: Paras Medical Publishers; 2020. p. 444-447 Medhi B, Prakash A. Anti-inflammatory . In: Practical Manual of Experimental and Clinical Pharmacology . New Delhi: Jaypee Publication ; p. 254-256 Robbins SL, Cotran RS. Robbins and Cotran Pathologic Basis of Disease. 10th ed. Philadelphia: Elsevier; 2020. p. 68-72

43. Questions What is Inflammation , define different phases of inflammation? Enumerate in-vivo methods of screening anti-inflammatory drugs? Enumerate in-vitro methods of screening anti-inflammatory drugs? Explain in brief about Carrageenan induced paw oedema in rats? What is the principle behind Plethysmometer ?

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