Brief description of screening methods for anti inflammatory drugs

1. SCREENING METHODS FOR
ANTI-INFLAMMATORY DRUGS
Presented by: Under Guidance of:
MOPURI ANKITHA Mrs. V.RAJANI (Ph.D)
M.Pharm 1st year (1st Sem) ASSOCIATE PROFESSOR
DEPARTMENT OF PHARMACOLOGY
SKU College of Pharmaceutical sciences,S.K.U,ATP

2. CONTENTS
Definition
Agents causing inflammation
Phase of inflammation
Symptoms of inflammation
Steps of the inflammatory response
Classification of inflammation
Mechanism action of Anti-inflammation
In-vivo Methods
In-vitro Methods

3. Definition
Inflammation is defined as local response of living
mammalian tissues to injury due to any agent. It is a defence
reaction in order to eliminate or limit the spread of injurious
agent, followed by removal of the necroses cells and tissues.
It consist in vascular, metabolic, cellular changes, triggered by
the entering of pathogenic agents in healthy tissues of the body

4. Agents causing inflammation
1. Infective agents: bacteria, virus, and other toxins, fungi
2. Immunological agents: cell mediated and antigeanti body reaction
3. Physical agents: heat, cold, radiation, mechanical trauma
4. Chemical agents: organic and inorganic poisons
5. Inert materials: foreign bodies

5. Phases of inflammation
 Acute : vasodilatation & increased capillary permeability
 Delayed: infiltration of leukocytes and phagocyte cells
 Chronic proliferative : tissue degeneration and fibrosis

6. Steps of the inflammatory response
(1) Recognition of the injurious agent
(2) Recruitment of leukocytes
(3) Removal of the agent
(4) Regulation of the response
(5) Resolution

7. symptoms of inflammation
1. Chronic inflammations (Cardinal sign of
inflammation)
2. redness (rubor)
3. pain (dolor)
4. hot (color)
5. swollen (tumour)
6. Loss of function (function losis)

8. Classification of Anti-inflammatory drugs
1.Salicylates : Aspirin (acetylsalicylic acid), Salicylic acid and
other salicylates
2.Propioninic derivatives: Ibuprofen , Naproxen
Dexibuprofen
3.Acetic acid derivatives: Indomethacin ,Tolmetin ,Diclofenac
4.Sulfonanilides : Nimesulide
5.Enolic acid (Oxicam) derivatives: Piroxicam, Meloxicam
Tenoxicam, Phenylbutazone
6.SelectiveCOX-2 inhibitors: Celecoxib,Rofecoxib, Etoricoxib
7.Anthranilic acid derivatives: Mefenamic acid, Meclofenamic
acidFlufenamic acid, Tolfenamic acid

10. In-vivo Method
ACUTE PHASE
1.Carrageenan induced paw edema in rats
2.Croton-oil induced ear edema
3.Oxazolone induced ear edema
4. UV erythema in guinea pigs
5. Pleurisy in ratsGranuloma air pouch technique
6.Vascular permeability
CHRONIC PHASE
The methods that include this chronic phase are
1.Cotton wool granuloma
2.Glass rod granuloma
3.Sponge implantation technique

11. 1.Carrageenan induced paw oedema in rats
Purpose and Rationale:
The carrageenan-induced paw edema is a well-defined model of acute
inflammation that a variety of inflammatory mediators involves in its
development. useful in detecting orally active anti-inflammatory
agents
Edema was induced by injecting 0.1 mL of 1% solution of carrageenan
into subplantar surface of right-hind paw.
Procedure:
a body weight between 150 and 170g are used. the animals are starved
for overnight.
Thirty minutes later, the rats are challenged by a subcutaneous injection
of 0.05ml of 1% solution of carrageenan

12. The paw is marked with ink at the level of lateral malleolus and
immersed in the mercury up to this mark.
EVALUATION:
 The paw volume was measured using plenthysmometer at 3 and 6 hr
The increase in paw volume at 3 and 6 hr is calculated
 The percentage increase in paw volume was measured by comparins
the difference of the average values between treated group animals
and control group
 A dose-response curve is run for the active drugs and ED50 values can
be determined.

13. 2.Croton-oil induced ear edema
Purpose and Rationale:
Croton Oil-Induced Ear Edema.Mice were randomly assigned into 6
per group. Melatonin, BBM or EBM solutions were prepared as 1%
(w/v) in acetone. Ten μL of melatonin or its derivatives (100 μg/ear)
was applied to the left outer ears of the mice 30 min prior to applying
5% croton oil to the left inner ears.
Procedure:
Both rats and mice are used
In this methodology 75microg of croton oil is applied to the inner
surface of the right ear of each mouse.

14. The animals are previously, anaesthetised with diethyl ether
Varying dose levels of test drug are applied to inner surface of the right
ear of each mouse inducing inflammation.
The animals are sacrificed by cervical dislocation, and a plug of 8mm
diameter is removed from each of the ear.
The difference in weight between the two plugs is taken as the measure
of edematous response

15. Evaluation:
This methodology is mainly used to detect antiinflammatory activity
of steroids.
The % antiinflammatory activity is calculated by
% anti inflammatory activity = (wt of treated ear-wt of untreated
ear/wt of control ear)*100

16. 3.Oxazolone induced ear edema
Croton oil , Tetradecanoyl , phorbol acetate, Xylol , Oxazolone
Topical application
Whole ear, punched discs, caliper, other ear

17. 4. UV erythema in guinea pigs
 Erythema earliest sign of inflammation.
 Depilated skin exposed to UV-B rays for 20-30 secs.
 Degree of erythema estimated after 2 hr visually
0 = no erythema
1 = mild erythema
2 = moderate erythema
3 strong erythema
4 = very strong erythema
 Pure measure of the vasodilatory phase
 No accompanying exudation or edema
 Drawbacks : Variable response
Accurate measurement of response
 Other sites/animals: Guinea pig ear
Sprague Dawley rats
Hairless mice (skin thickness)

18. 5.Pleurisy in ratsGranuloma air pouch technique
 Exudative Inflammation: Histamine, Bradykinin, Prostaglandins,
Dextran, Antigens, Microbes, irritants like turpentine and
carrageenan etc.
 Fluid extravasation: leukocyte migration, lysosomal enzyme
activities etc.

19. 6.Vascular permeability
 Inhibitory activity of drugs against increased vascular
permeability
 Extravasation of dye into surrounding tissue

20. CHRONIC PHASE
1.Cotton wool granuloma
Purpose and Rationale:
The cotton wool granuloma is widely used to evaluate the transudativand
proliferative components of chronic inflammation widely used to
assess the transudative, exudative and a proliferative phase of subacute
inflammation.
Procedure:
Foreign body granuloma Sterile Cotton pellets embedded
subcutaneously Back, Axilla, Groin After 7 days - Pellet + Granuloma
removed. in which granulomatous tissue formation is identified using
the dry weight of a cotton pellet
Evaluation:
Weight of granulomatous tissue =Dry weight of granuloma cotton -
intial weight of cotton pellet

21. 2.Glass Rod granuloma
Purpose and Rationale:
Granulomas seem to be a defensive mechanism that triggers the body
to "wall off" foreign invaders such as bacteria or fungi to keep them
from spreading. Common causes include an inflammatory condition
called sarcoidosis and infections such as histoplasmosis or
tuberculosis.
Procedure:
Foreign body granulomaGlass peices embedded subcutaneouslyBack,
Axilla, Groin. After 20-40 days - Granuloma + Connective removed
entirely. Glass rod can be removed
Evaluation:
Tensile strength and wet weight of granulomatous tissue can be
measured

22. In-vitro Methods
1) Induced human red blood cell stabilization
2) 3H-Bradykinin receptor binding
3) Isolated Tissue Preparations for NK receptors
4) Polymorphonuclear Leukocyte Chemotaxis
5) PMN leukocytes aggregation induced by FMLP
6) Constitutive and inducible cellular arachidonic acid metabolism in
vitro
7) COX-1 and COX-2 inhibition
8) Induced release of cytokines from human white blood cells
9) Flow cytometric analysis of intracellular cytokines

23. 1.Induced human red blood cell stabilization
Purpose and Rationale:
A method in estimating the anti- inflammatory property.
stabilization of human red blood cell membrane by hypo tonicity
induced membrane lysis. the integrity of the membranes is stabilized
by using anti- inflammatory drugs.
Procedure:
Animal selection (Each group has 6 animals.)
Group 1 Group 2 Group 3 Group 4 Group 5
(100ug/ml) (50ug/ml) (25ug/ml) (15.5ug/ml) (6.3ug/ml)

24. Bloods are collected from the healthy rats and sterilized Alsever solution
The blood was centrifuged at 3000 rpm and packed cells were washed
with iso-saline and a 10% (v/v) suspension was made with iso-saline
The two extracts taken separately, each extract added 1 ml phosphate
buffer, 2 ml of hypo-saline and 0.5 ml HRBC suspension.
Diclofenac sodium was used as the reference drug and distilled water
was used in the control group.

25. The assay mixtures were incubated at 37°C for 30 minutes and
centrifuged
The hemoglobin content in the supernatant solution was estimated using
UV analysis at 560 nm
Evaluation:
The percentage of HRBC membrane stabilization or protection was
calculated using the formula:
% inhibition of haemolysis= OD of test solution -OD of product control x 100
OD of test control

26. 2. 3H-Bradykinin receptor binding
Purpose and Rationale:
Bradykinin is one of the most potent inflammatory mediators in
humans. Bradykinin increases inflammation by widening the blood
vessels and allowing fluid and cells to leak from the vessels into
surrounding tissues. Bradykinin causes the production of histamine
and nitric oxide to increase.
Procedure:
Tissue homogenates in incubation bufferIncubated with 3H-
Bradykinin alone - Total Binding3H-Bradykinin + unlabelled
Bradykinin (saturated) - Non-specific Binding.
3H-Bradykinin + Test drug (subtract the non-specific binding)

27. Two types -Competition studies - increasing concentrations of Test drug
Saturation studies - increasing concentrations of 3H-BradykininTissues
G. pig ileum, rabbit carotid artery, canine cultured tracheal smooth
muscle cells, bovine aortic endothelial cells, human fibroblasts.
Evaluation:
 The following parameters are calculated:
 total binding of 3H-bradykinin.
 non-specific binding in the presence of 10 µM bradykinin
 specific binding = total binding - non-specific binding
 % inhibition: 100-specific binding aspercentage of control value
RBA =IC 50 standard compound /IC 50 compound X 100%

28. 3.Isolated Tissue Preparations for NK receptors
Purpose and Rationale:
these cells good therapeutic targets for controlling infection and
preventing the development and NK cells have the potential to act
both in driving inflammation and in restricting adaptive immune
responses of chronic inflammatory diseases
Procedure:
Isolated Tissue Preparations for NK receptors
Inhibition of Substance P-induced endothelium- dependent relaxation
of rabbit pulmonary artery, previously contracted with 0.1 μM
Noradrenaline

29. Inhibition of Substance P or Substance P sulfone- induced contractions
of guinea pig ileum in the presence of 3 µM Atropine and 3 µM
Mepyramine and Indomethac
inInhibition of Substance P-induced plasma extravasation in guinea-pig
bronchi

30. 4.Polymorphonuclear Leukocyte Chemotaxis
 Measures the chemotactic effects on the PMN Leukocytes
Migration rate- number of PMNs in the lower well number of PMNs
in the upper well x 100Dependent on the concentration of the
chemoattractant (e.g. zymosan -activated serum)
5.PMN leukocytes aggregation induced by FMLP
 FMLP (formyl-L-methionyl-Lleucyl- L-phenylalanine)
 PMNs cell suspensions + test compounds or standard
(pentoxiphylline) are dissolved in GBSS
 Incubated for 10 minSerial dilutions of FMLP added
 Change in transmittance

31. 6.Constitutive and inducible cellular arachidonic acid
metabolism in vitro
 Formation of leukotriene B4 in human white blood cells
 Formation of lipoxygenase products from 14C- arachidonic acid in
human polymorphonuclear neutrophils (PMN)
 Formation of eicosanoids from 14C-arachidonic acid in human
platelets
 Stimulation of inducible prostaglandin pathway in human PMNL
7.COX-1 and COX-2 inhibition
• Inhibition studies with recombinant human COX-1 and COX-2
• HPLC assay for oxygenation of radiolabelled arachidonic acid by
COX-1
• Determination of the stoichiometry of inhibitor binding
• Spectrophotometric assay of recombinant human COX-2Whole-cell
assays with transfected Chinese hamster (CHO) cells expressing COX-
1 and COX-2

32. 8.Induced release of cytokines from human white
blood cells
 Interleukin-1alpha, IL-1beta, IL-6, IL-8 and TNF-alphaCell fraction
from whole blood separatedExposed to LPS (Salmonella abortus equii)
with or without test drug
 Incubation
 Analysis of Cytokines
9. Flow cytometric analysis of intracellular cytokines
 Very powerful tool in which individual cells can be simultaneously
analyzed for several parameters
 Size, granularityExpression of surface and intracellular markers
 Defined by fluorescent antibodies
 Fluorescent anti-cytokine and anti-chemokine monoclonal antibodies