SEMINAR ON

Evaluation of Anti-inflammatory
drugs

PRESENTER : Dr. Jill Mathew Dated :04/09/2013

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

1
2

CARDINAL SIGNS
OF
INFLAMMATION

Acute: vasodilatation & increased

capillary
permeability

Delayed: infiltration of leukocytes and

phagocyte cells

Chronic proliferative:
tissue degeneration and
fibrosis

(1)
(2)
(3)
(4 )
(5)

Recognition of the injurious agent

Recruitment of leukocytes
Removal of the agent
Regulation of the response
Resolution

ICAM-1
and
VCAM-1

MEDIATOR

VASO
DILATATION

Histamine

++

Serotonin

+/-

Bradykinin

+++

+++

Prostaglandin +++
s

+++

Leukotrienes

VASCULAR
PERMEABILT
Y

CHEMOTAXIS

+++

PAIN

+
_

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

Area 1: used as a control area and

was exposed to -UVB and treated
with cream base only twice daily.

Area 2: treated with topical H 1

antagonist (1.5% chlorphenoxamine
hydrochloride in the same cream
base, Allergex, Epico) twice daily.
Area 3: treated with topical H 2
antagonist (2% famotidine in the
same cream base) twice daily

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

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

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

% 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=
%

1.
2.
3.
4.
5.
6.
7.
8.
9.

Carrageenan COX
Mustard
Dextran
Egg-white
Phlogistic
agents
Compound 48/80
Yeast, Zymosan-LOX
Serotonin, histamine
Kaolin
Substance p, nystatin, glass powder,
cobra venom

an instrument for measuring changes in

volume within anorgan 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

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

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

Induction of Pleurisy:
Carrageenan , turpentine, Evans 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

3.

Croton oil induced ear edema ( Croton

tiglium)
TPA-induced ear edema Protein kinase C , NF-B ,
phospholipase A2
PG
Arachidonic acid induced ear edema

4.

Xylol induced ear edema

1.

2.

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

Right
-Control

Std duration (27 sec) and Std intensity

(55C)
thermostatically
controlled water

to the shaved abdomen of rat

Resultant edema can be measured by
excising skin

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

To study repair phase characterized by

fibroblast proliferation and blood vessel
formation
Suppression of granulation tissue
parallels with anti-inflammatory activity

For measurement of activity of antiinflammatory drugs on proliferative

component of subacute & chronic
inflammatory processes

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

Immunologically mediated model of

chronic inflammation
It a delayed HS response to
mycobacterial Ag

Induction of Arthritis : Subplantar injection of

Freunds complete adjuvant or mycobacteria
suspended in oil
Day 1
Day 4-5
Day 7

Local reaction

Parameters :
Foot thickness, wt, grip strength , deformity
measured
Evaluation of chronically administered drug against
inflammation
Drugs effective: Steroids ,Immunosuppressants,
gold ,d-penicillamine

Peak

Systemic disease

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

VCAM-1,ICAM-1 and E-selectin are

derived from HUVEC ,HDMEC cell lines
Cell based ELISA assay

BCECF
labeled WBC
20 min
TNF-
as
reagent

HUVEC,
HMEC

20 min

Test
drug

No
drug

Adherent cells are lysed and fluorescence measured at 485 nm

in ELISA reader

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

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

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 bindingnonspecific binding
% inhibition:100- specific binding

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.

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

Psoriasis