This document summarizes various methods used to evaluate anti-inflammatory drugs. It describes animal models used to induce and measure inflammation, including UV erythema, rat paw edema, and cotton pellet assays. Key mediators of inflammation like histamine, bradykinin, and prostaglandins are discussed. Tests to measure drug effects on leukocyte adhesion and superoxide production are also presented. The document provides an overview of the development process for the anti-inflammatory drug piroxicam and signs of both acute and chronic inflammation.

1. Evaluation of Anti-inflammatory
drugs
SEMINAR ON
PRESENTER : Dr. Jill Mathew Dated :04/09/2013

3.  Inflammation is a protective response intended
to eliminate the initial cause of cell injury as
well as the necrotic cells and tissues resulting
from the original insult
 Pfizer took about 18 years to develop the anti-
inflammatory drug piroxicam, which was
launched in 1980 during the “golden age of
rational drug discovery”

4. CARDINAL SIGNS OF
INFLAMMATION
1
2
34
5

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

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

8. ICAM-1
and
VCAM-1

9. MEDIATOR VASO
DILATATION
VASCULAR
PERMEABILTY
CHEMOTAXIS PAIN
Histamine ++ _ +
Serotonin +/- _ +
Bradykinin +++ _ +++
Prostaglandins +++ +++ +
Leukotrienes - +++ _

12.  Animals used: Albino Guinea pigs
 Test drug is administered -30 min before exposure
 Duration of exposure to UV –B rays : 20 sec
 Area of exposure : Depilated skin
 Degree of erythema estimated after 2 hours visually on
a scale 0-4
 ADVANTAGE : Pure measure of the vasodilatory phase
 Drawback: Accurate measurement of reaction not obtained,
variability of response
 DRUGS EFFECTIVE : NSAIDS like Phenyl butazone,
Aminopyrene, Aspirin and Sodium salicylate

13. Area 2: treated with topical H 1 antagonist
(1.5% chlorphenoxamine hydrochloride in the
same cream base, Allergex, Epico) twice daily.
Area 1: used as a control area and was exposed
to -UVB and treated with cream base only
twice daily.
Area 3: treated with topical H 2 antagonist (2%
famotidine in the same cream base) twice
daily

14.  Rat-paw edema (Acute and Subacute phases)
Drugs: Carrageenan (1%, 0.1 ml SC.), indomethacin
2 mg/kg IP
 Site : Subplantar region of left hind paw
Experimental injury cascade of events

15.  Biphasic
 1 – release of histamine,5 HT and kinins
 2- release of prostaglandins
 Parameter : Paw volume up to the ankle joint is
measured in treated and untreated groups
before and after carrageenan

16.  Advantage:
Simple ,easy, short lasting and reproducible
 Disadvantage :
Non –specific and difficult to quantify and
examine cells & their modification by anti-
inflammatory drugs
Avoid in both the hind paws because of
unalleviated pain and deformity

17.  % of edema produced by carrageenan alone
= A – B X 100 = X %
B
 % of edema produced by carrageenan after treatment
with drug
= C – D X 100 = Y %
D
 % reduction of edema by drug
= X –Y= %

18. 1. Carrageenan – COX
2. Mustard
3. Dextran
4. Egg-white Phlogistic agents
5. Compound 48/80
6. Yeast, Zymosan-LOX
7. Serotonin, histamine
8. Kaolin
9. Substance –p, nystatin, glass powder, cobra
venom

19.  an instrument for measuring changes in volume
within an organ whole body or limb
 The mercury displacement due to dipping of the
paw can be directly read from the scale attached
to the mercury column

20. TUBE A
TUBE B
RAT PAW IS
IMMERSED INTO
THE TUBE ‘A’ &
THE READING
IS CHECKED
THROUGH THE
TUBE ‘B ’ SOME
SET UPS HAVE A
DIGITAL DISPLAY
ATTACHED TO IT

21.  Inducer of arthritis:
 Silver nitrate in the tibiotarsal joint of rats
 Intra articular talc injections in pigeons
 Locomotor activity with inflammed paws
is impaired
 Drugs effective: NSAIDS, steroids ,DMARDS
 Anti-histamines

23.  Induction of Pleurisy:
 Carrageenan , turpentine, Evan’s blue, Arabic gum,
glycogen, dextran , antigens , microbes, mast cell
degranulators
 Advantage: Quantitation of anti-inflammatory
activity and M.O.A of new test drug
 Drugs effective: Steroid, NSAIDS & Gold
 Index of activity of drug:
The volume of exudate is decreased

24. 1. Croton oil induced ear edema ( Croton tiglium)
2. TPA-induced ear edema -
Protein kinase C , NF-кB , phospholipase A2
PG
3. Arachidonic acid induced ear edema
4. Xylol induced ear edema

25.  Predictable model for topical anti-inflammatory drugs
 Inflammatory response measured by increase in ear
plug weight & thickness at single time interval( at 6
hours)
 Drugs tested : Steroids and NSAIDS

26. Right
-Control

27.  Std duration (27 sec) and Std intensity (55◦C)
thermostatically controlled water
to the shaved abdomen of rat
Resultant edema can be measured by excising
skin

28. Egg albumin or horse serum
Passively sensitized by antibodies
Intra-dermal injection of Ag or Ab is made
Inflammatory reaction
ASSESSMENT : by dye leakage or edema
 Drugs effective :Cortisone , Salicylates

29.  To study repair phase characterized by fibroblast
proliferation and blood vessel formation
 Suppression of granulation tissue parallels with
anti-inflammatory activity

30.  For measurement of activity of anti-
inflammatory drugs on proliferative
component of ‘subacute & chronic
inflammatory processes’


31.  Sterile Cotton pellets (wt :5mg -50mg )
 sc sites of axilla , back ,groin
 Duration : 1-14 days
 Pellet along with granuloma removed and
weighed
 Weight of granulomatous tissue =
 Dry weight of granuloma cotton -initial weight
of cotton pellet
 Drugs effective: corticosteroids

32.  Immunologically mediated model of chronic
inflammation
 It a delayed HS response to mycobacterial Ag

33.  Induction of Arthritis : Subplantar injection of Freund’s
complete adjuvant or mycobacteria suspended in oil
 Day 1 Day 4-5 Day 7
 Local reaction Peak Systemic disease
 Parameters :
Foot thickness, wt, grip strength , deformity measured
 Evaluation of chronically administered drug against
inflammation
 Drugs effective: Steroids ,Immunosuppressants, gold
,d-penicillamine

34.  Chronic exposure to foreign bodies like contact
lenses leads to GPC
 Levels of IL3,IL 4,IL5 ,Ig G, Ig E and Ig M is
increased in Tears
 Procedure : A sterile needle is inserted directly
into conjunctiva of unsensitized animal
 Pricking results in high number of neutrophil
infiltration

36.  VCAM-1,ICAM-1 and E-selectin are derived from
HUVEC ,HDMEC cell lines
 Cell based ELISA –assay

37. HUVEC,
HMEC
TNF-ἀ
as
reagent
Test
drug
20 min
BCECF
labeled WBC
20 min
No
drug
Adherent cells are lysed and fluorescence measured at 485 nm
in ELISA reader

38.  Thrombin activated human platelets
Incubated with drug at 20 0
C for 10 min &
mixed with Neutrophils at a ratio 10:1
Index of activity : Neutrophils with 2 or more and
one or no adherent platelets are counted
Test drug : Blocks Adhesion
Drugs effective : NSAID ,chloroquine

39.  Dianzani etal
 the PMNs are challenged by FMLP to generate 02-
for screening anti-inflammatory compounds
 Superoxide production was measured
spectrophotometrically by measuring the
superoxide dismutase inhibitable reduction
 Drugs effective: Anti-oxidant drugs

40.  Stimulates A and C fibers producing pain
 Lowers BP by vasodilatation
 Test : detects compounds that inhibit binding of
3
H- Bradykinin in membrane preparation of guinea pig ileum
Evaluation:
specific binding= total binding- nonspecific binding
% inhibition:100- specific binding

41.  Leptin is a proinflammatory cytokine
 Circulating leptin levels are increased upon infectious and
inflammatory stimuli such as LPS, turpentine, and
cytokines
 Leptin increased statin inhibitible free radical and
cholestrol production invitro
 CRP increased in obesity and reduced by statin
 JUPITER TRIAL, Leptin and Insulin resistance
 Fructose fed rat is a model of metabolic syndrome.

42. The compounds were found to be useful in OA,RA and Psoriasis
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