This document summarizes screening methods for evaluating potential anti-inflammatory drugs. It discusses the inflammatory response and various animal models used to test drug candidates, including carrageenan-induced paw edema, cotton pellet-induced granuloma, and UVB-induced erythema in guinea pigs. Several in vitro assays are also described, such as measuring COX inhibition and evaluating the ability of drugs to block mast cell degranulation and platelet-neutrophil adhesion. The goal of these screening methods is to effectively identify drug candidates that can target different phases and components of the inflammatory process.

1. SeminarOn
Screening Methods For Inflammatory Drugs
By: underguidance of:
SHAIKH ZOHRAMEENA MRs.ANURADHA
M.PHARMA (Pharmacology)
1st year

2.  Inflammation is a universal host defensive
process involving a complex network of cell-
cell, cell-mediator and tissue interaction.
 Inflammation is response to variety of harmful
stimuli physical, chemical, traumatic antigen
challenge, infectious agents and ionizing
radiations.

3. Factors
Exogenous: Physical, chemical, mechanical, nutritional and biological
etc.
Endogenous: immunological reactions, neurological and genetical
disorders.
Types of inflammation
Acute inflammation- short lasting.
Chronic inflammation- may persists for week, months and year.
Principle components of inflammatory response
Increased blood flow.
Increase capillary permeability.
Increased migration of leucocytes into effected area.

5.  Inflammation disease cover a broad spectrum of
conditions including auto immune diseases.
Examples- Rheumatoid Arthritis, Osteoarthritis, Inflammatory
bowel disease, Multiple sclerosis, Asthma, Chronic obstructive
pulmonary disease, Allergic Rhinitis, Infectious disease, various
types of Cancers and Cardiovascular diseases.
 Inflammation is regulated by a large number of pro and anti
inflammatory Medication such as histamine, PG (PGT2 and
Prostacyclin), leukotriene (LTB4), serotonin, bradykinin,
cytokines (IL-1, IL-6, IL-8, IL-n, TNF-a), Reactive oxygen
species, growth factors, lysosomes, contents of neutrophils,
Adipokines (leptin, adiponectins, resistin).

6.  COX assay.
 Mast cell degranulation.
 Inhibition of NO production induced by TNF-
a in mouse macrophages.
 Measurement of NO production in mouse
macrophages.
 Adhesion assays.
 Platelet- neutrophils adhesion.
 Cell based reporter gene assay.
 FMLP- induced O2 generation by
polymorphonuclear cells (PMNs).
 FMLP- induced adhesion of PMN of HUVEC.

7.  UV- B induced erythema in guinea pigs.
 Carrageen induced paw edema model.
 Plural exudation method.
 Cotton pellet induced granuloma.
 Adjuvant arthritis.
 Papaya latex induced arthritis.
 Candida albicans induced arthritis in mice.
 Air pouch model.
 Croton oil induced ear edema in mice.
 Arachidonic acid induced ear edema in mice.
 Dextran sulphate sodium induced colitis in
mice.

9.  COX-1
10ml of sample solution
added to 19ml of 0.1M
of L-adrenaline,
dihydrogen tartrate and
10mM of hematin.
After adding 0-2 units
of COX-1 it is pre
incubated for 5 mins by
adding 10 mL of 10%
formic acid.
The PGE2
concentration is
measured with a PGE2
enzyme immune assay.
 COX-2
Assay mixture consists of
100mm rod phosphate, 1mm
of hematin gelation, 2.5mL of
compound in DMSO.
It is pre incubated for 15 mins
at 22 C and the 20 mL of
solution of 1mM arachidonic
acid and 1mM TPMD in assay
buffer is added.
The absorbance at 400nm is
measured over the fast 36 sec
and % inhibition calculated.
The enzyme inhibition of
TPMD in absence of COX-2 is
also observe and subtracted
from activity in presence of
COX-2

10.  Heparinized Tyrode’s solution is injected into the
peritoneal cavity of exsanguinated rat (Sprague dawley
after abdominal massage, the cells in peritoneal fluid
are harvested and separated through 38% Bovine
Serum Albumin. Cells are washed and suspended in
Tyrode’s solution with 0.1% BSA.
 The cell suspension is pre incubated with test drugs at
37 C for 3 min. fifteen mins after addition of compound
48/80 (standard compound for mast cell degranulation),
glucuronidase (1mM phenolphthalein-D-glucuronide in
0.1 M acetic acid buffer, pH 4.5 is used as a substrate,
absorbance monitored at 550nm after alkalization) and
histamine (0.2% o-phthalaldehyde condensation in pH
12.5 fluorescence is monitored at 350/450 nm after
acidification) in the supernatant are determined.

11. o The total content is measured after treatment of the
cell suspension with Triton X-100.
o The percentage release determined is the index of anti-
inflammatory activity.

12.  Thrombin activated human platelets are
incubated with drug at 20 C for 10 mins,
and mixed with neutrophils at a ratio of
10:1.
 Neutrophil with two or more (number
positives) and one or no adherent
platelets (number negatives) are counted
as index of activity.
 The test drug block the adhesion with
respect to controls.

14.  Animal used- Albino guinea pigs
 Test drug is administered 30 mins
before exposure.
 Duration of exposure to UV- B: 20 sec.
 Area of exposure: Depilated skin.
 Degree of erythema estimated after 2
hours visually on scale of 0-4.
 Model can be use as a pure measure of
the vasodilatory phase in the
inflammation reaction.

15.  Rat paw edema (acute and subacute phases)
 Drugs: carageenan (1%, 0.1 ml SC),
indomethacin 2 mg/kg IP.
 Site: subplantar region of left hind paw.
 Biphasic
o 1- release of histamines, 5 HT and kinins.
o 2- release of prostaglandins.
Parameter : paw volume upto the ankle joint is
measured in treated and untreated groups before
and after carrageenan.

17.  For measurements of activity of anti-
inflammatory drugs on proliferative
components of subacute and chronic
inflammatory processes
 Procedure : Sterile cotton pellets (wt-
5mg-50mg).
• SC sites of back, axilla and groin.
duration: 1-14 days
• Pellet along with granuloma removed
and weighed.

18. Weight of granulomatous tissue =
Dry weight of granuloma cotton – initial
weight of cotton pellet.
Drugs effective: corticosteroids.