This document summarizes various in vitro and in vivo models for screening anti-inflammatory drugs. It describes the COX assay method to test inhibition of COX-1 and COX-2 enzymes. The mast cell degranulation assay measures compound 48/80-induced release of histamine and glucuronidase from rat peritoneal mast cells. The platelet-neutrophil adhesion assay examines inhibition of thrombin-activated platelet adhesion to neutrophils. In vivo models discussed are UV-B erythema in guinea pigs, carrageenan-induced rat paw edema, and cotton pellet-induced granuloma formation in rats over 14 days.

1. SCREENING MODELS OF
ANTI-INFLAMMATORY DRUGS
Noida Institute of Engineering and
Technology
(Pharmacy Institute)
Presented By:
Anas Siddiqui
M. Pharm 1st Sem
(Pharmocology)
Guided By:
Dr. Saumya Das
Professor H.O.D (Pharmacology)

2. Introduction
 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. Reasons
 Exogenous: Physical, chemical, mechanical,
nutritional and biological etc.
 Endogenous: immunological reactions,
neurological and genetical disorders.

4. Phases of Inflammation
 Acute: Vasodilation and increase capillary
permeability
 Delayed: Infiltration of leukocytes and phagocytes
cells
 Chronic proliferative: Tissue degeneration and
fibrosis

5. In Vitro Models
 COX Assay
 Mast cell Degranulation
 Platelet – neutrophils adhesion

6. COX Assay
COX-1
1. 10ml of sample solution added to 19ml of 0.1M of
L- adrenaline, dihydrogen tartrate and 10mM of
hematin.
2. After adding 0-2 units of COX-1 it is pre incubated
for 5 mins by adding 10 mL of 10% formic acid.
3. The PGE2 concentration is measured with a PGE2
enzyme immune assay.

7. COX-2
1. Assay mixture consists of 100mm rod phosphate,
1mm of hematin gelation, 2.5mL of compound in
DMSO.
2. It is pre incubated for 15 mins at 22 C and the 20
mL of solution of 1mM arachidonic acid and 1mM
TMPD in assay buffer is added.
3. The absorbance at 400nm is measured over the
fast 36 sec and % inhibition calculated.
4. The enzyme inhibition of TMPD in absence of COX-
2 is also observe and subtracted from activity in
presence of COX-2

8. MAST CELL DEGRANULATION
 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.

9.  pH 4.5 is used as a substrate, absorbance
monitored at 550nm after alkalization) and
histamine (0.2% opthlaldehyde condensation
in pH 12.5 fluorescence is monitored at
350/450 nm after acidification) in the
supernatant are determined.
 The total content is measured after treatment
of the cell suspension with Trito n X-100.
 The percentage release determined is the
index of anti- inflammatory activity.

10. PLATELET- NEUTROPHILS ADHESION
 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.

11. IN VIVO MODELS
 UV- B induced erythema in guinea pigs.
 Carrageen induced paw edema model.
 Cotton pellet induced granuloma.

12. UV- B INDUCED ERYTHEMA IN
GUINEA PIGS
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.

13. CARRAGEEN INDUCED PAW EDEMA
MODEL
 Rat paw edema (acute and sub-acute phases)
 Drugs: carrageenan (1%, 0.1 ml SC), indomethacin 2
mg/kg IP.
 Site: sub plantar region of left hind paw.
 Biphasic
1- release of histamines, 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.

14. Calculations

15. COTTON PELLET INDUCED
GRANULOMA
For measurements of activity of anti-inflammatory drugs
on proliferative components of sub acute 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.
Weight of granulomatous tissue =
Dry weight of granuloma cotton – initial weight of cotton pellet.
Drugs effective: corticosteroids.

16. REFERENCE
 Tripathi,K.D., ”Essentials of medical pharmacology”, Jaypee
publications, 2008(8):235-236
 Gupta,S.K., “Drug Screening Methods (Preclinical Evaluation
of Drugs)”, Jaypee Brothers Medical Publishers Pvt Ltd,2009
480-490.
 https://www.jpgmonline.com