Anti Inflammatory

PRECLINICAL SCREENING
OF ANTI-INFLAMMATORY
AGENTS
INTRODUCTION
 Inflammation is the body’s immune system’s response to

an irritant.
 Inflammation is universal host defensive process

involving a complex network of cell, cell mediators and
tissue interaction.
 Inflammation is response to variety of harmful stimuli

physical, chemical, traumatic antigen challenge,
infectious agents and ionizing radiation
Inflammation has different phases:
 The first phase is caused by an increase of vascular

permeability resulting in exudation of fluid from the
blood into the interstitial space.
 The second one by infiltration of leukocytes from the

blood into the tissues and the third one by granuloma
formation.
 Accordingly, anti-inflammatory tests have to be divided

into those measuring acute inflammation, subacute
inflammation and chronic repair processes.
 Predominantly, however, these studies are aimed to find

new drugs against polyarthritis and other rheumatic
diseases.
1|Page
PRE-CLINICAL SCREENING OF
ANTIINFLAMMATORY AGENTS
IN VITRO METHODS:
1. 3H Bradykinin receptor binding
2. 3H substance p receptor binding
IN VIVO METHODS:
1. UV Erythema in guinea pig
2. Oxazolone induced ear edema in mice
3. Croton oil ear edema in mice
4. Paw edema
5. Granuloma pouch technique
2|Page
IN VITRO METHODS
1. 3H BRADYKININ RECEPTOR BINDING
PURPOSE AND RATIONALE
The 3H -bradykinin receptor binding is used to detect

3
compounds that inhibit binding of H -bradykinin in
membrane preparations obtained from guinea-pig ileum.
PROCEDURE
Ileum from guinea pigs is cleaned from its content and

cut into pieces of 2cm length.
They are homogenized for 30 s in ice-cold TES buffer,

pH 6.8, containing 1mM 1,10-phenanthroline in a
homogenizer.
The homogenates are filtered through 3 layers of gauze

and centrifuged twice at 50000 g for 10 min with an
intermediate re-homogenization in buffer.
3|Page
For routine studies the final pellets are resuspended in 40 vol
of incubation buffer.
In the competition experiment, 50 µl 3H -bradykinin, 50 µl test

compound and 150 µl membrane suspension from guinea pig
ileum per sample are incubated in a shaker bath at 25 °C for
90 min.
Saturation experiments are performed with 12 concentrations

of 3H-bradykinin.
Total binding is determined in the presence of incubation

buffer, non-specific binding is determined in the presence of
non-labeled bradykinin.
The reaction is stopped by rapid vacuum filtration through

glass fibre filters.
Thereby the membrane bound radioactivity is separated from

the free one.
The retained membrane-bound radioactivity on the filter is

measured after addition of 3 ml liquid scintillation cocktail per
sample in a liquid scintillation counter.
4|Page
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 as percentage of control
value
2. 3H SUBSTANCE P RECEPTOR BINDING

Selective antagonists to substance P found in receptor binding
studies may elucidate the physiological role of substance P
and may be candidates for anti-inflammatory and analgesic
drugs.
PROCEDURE
Fresh porcine brains are obtained from the slaughterhouse.
Striata are dissected and homogenized in 50 mM ice-cold

Tris-HCl buffer, pH 7.4
5|Page
These homogenates are then incubated for 30 min at 4 °C
before being centrifuged at 30000 g for 20 min at 4 °C and
washed twice with 50 mM Tris-HCl (pH 7.4) buffer.
Pellets are resuspended in 0.32 M sucrose
60min incubations are carried out at room temperature in 50

mM Tris-HCl buffer, pH 7.4
Total binding and nonspecific binding are determined in

triplicate in the absence or presence of 1 mM unlabeled
substance P.
Incubations are terminated by adding 4 ml of ice-cold Tris-

HCl buffer (pH 7.4) and membranes are filtered on Whatman
glass fiber filters.
Filters are then washed three times using ice-cold Tris-HCl

buffer (pH 7.4).
Bound radio activities are determined using a liquid

scintillation counter
EVALUATION
Saturation and competition data are analyzed using a
computer program
6|Page
IN VIVO METHODS
1. UV ERYTHEMA IN GUINEA PIG

Method able to delay the development of ultraviolet erythema
on albino guinea pig skin by systemic pre-treatment with
clinically equivalent doses of phenylbutazone and other
nonsteroidal anti-inflammatory agents.
PROCEDURE
Albino guinea pigs of both sexes with an average weight of
350 g are used.
18h prior testing, the animals are shaved on both flanks and on
the back.
Then they are chemically depilated by a commercial

depilation product or by a suspension of barium sulfide.
20 min later, the depilation paste and the fur are rinsed off in
running warm water.
7|Page
On the next day, the test compound is dissolved in the vehicle
and half the dose of the test compound is administered by
gavage (at 10 ml/kg) 30 min before ultraviolet exposure.
Control animals are treated with the vehicle alone.
Four animals are used for each treatment group and control.
The guinea pigs are placed in a leather cuff with a hole of 1.5
× 2.5 cm size punched in it, allowing the ultraviolet radiation
to reach only this area.
An original ultraviolet burner is warmed up for about 30 min

prior to use and placed at a constant distance (20 cm) above
the animal.
Following a 2 min ultraviolet exposure, the remaining half of

the test compound is administered.
The investigator has to protect himself/herself by gloves and

ultraviolet glasses.
The erythema is scored 2 and 4 h after exposure.
8|Page
EVALUATION
The degree of erythema is evaluated visually by 2 different
investigators in a double-blinded manner.
The followings scores are given:
• 0 = no erythema,
• 1 = weak erythema,
• 2 = strong erythema,
• 4 = very strong erythema.
9|Page
2. OXAZOLONE INDUCED EAR EDEMA IN MICE

The oxazolone-induced ear odema model as first described in
mice is a model of delayed contact hypersensitivity that
permits the quantitative evaluation of the topical and systemic
anti inflammatory activity of a compound following topical
administration.
PROCEDURE
Mice of either sex with a weight of 25 g are used.
Before each use a fresh 2% solution of oxazolone in acetone is

prepared.
The mice are sensitized by application of 0.1 ml on the shaved

abdominal skin or 0.01 ml on the inside of both ears under
halothane anesthesia.
The mice are challenged 8 days later again under anesthesia

by applying 0.01 ml 2% oxazolone solution to the inside of
the right ear (control) or 0.01 ml of oxazolone solution, in
which the test compound or the standard is solved.
10 | P a g e
Special pipettes of 0.1 ml or 0.01 ml are used.
Groups of 10 to 15 animals are treated with the irritant alone

or with the solution of the test compound.
The left ear remains untreated.
The maximum of inflammation occurs 24 h later.
At this time the animals are sacrificed under anesthesia and a

disc of 8 mm diameter is punched from both sides.
The discs are immediately weighed on a balance.
The weight difference is an indicator of the inflammatory

edema.
11 | P a g e
EVALUATION
Average values of the increase of weight are calculated for
each treated group and compared statistically with the control
group.
3. CROTON-OIL EAR EDEMA IN RATS AND MICE

The method has been developed primarily as a bioassay for
the concomitant assessment of the antiphlogistic and
thymolytic activities of topically applied steroids.
12 | P a g e
PROCEDURE
For tests in mice the irritant is composed as follows (v/v):
1part Croton oil, 10 parts ethanol, 20 parts pyridine, 69 parts
ethyl ether.
For tests in rats the following mixture is prepared (v/v): 4

parts Croton oil, 10 parts ethanol, 20 parts pyridine, 66 parts
ethyl ether.
The standards and the test compounds are dissolved in this

solution.
For tests in mice male NMRI-mice with a weight of 22 g &

for tests in rats male Sprague-Dawley rats with a weight of 70
g are used.
10 animals are used for controls and each test group.
The test compounds are dissolved in a concentration of 0.03

mg/ml-1 mg/ml for mice and in a 3-10 times higher
concentration for rats in the irritant solution.
On both sides of the right ear 0.01 ml in mice or 0.02 ml in

rats are applied.
13 | P a g e
Controls receive only the irritant solvent.
The left ear remains untreated.
The irritant is applied under ether anesthesia.
4h after application the animals are sacrificed under

anesthesia.
Both ears are removed and discs of 8 mm diameter are

punched.
The discs are weighed immediately and the weight difference

between the treated and untreated ear is recorded indicating
the degree of inflammatory edema.
The animals were sacrificed 48 h after topical administration

and the thymus glands were removed, weighed and expressed
as mg thymus/100 g body weight.
EVALUATION
Antiphlogistic effect (%) = weight of the treated

ear-weight of control ear × 100
weight of control ear

14 | P a g e
4. PAW EDEMA

Among the many methods used for screening of
antiinflammatory drugs, one of the most commonly employed
techniques is based upon the ability of such agents to inhibit
the edema produced in the hind paw of the rat after injection
of a phlogistic agent.
PROCEDURE
Male or female Sprague-Dawley rats with a body weight
between 100 and 150 g are used.
The animals are starved overnight.
To ensure uniform hydration, the rats receive 5 ml of water by

stomach tube (controls) or the test drug dissolved or
suspended in the same volume
30min later, the rats are challenged by a subcutaneous

injection of 0.05 ml of 1% solution of carrageenan into the
plantar side of the left hind paw.
15 | P a g e
The paw is marked with ink at the level of the lateral
malleolus and immersed in mercury up to this mark.
The paw volume is measured plethysmographically

immediately after injection, again 3 and 6 h, and eventually 24
h after challenge.
Plethysmograph
EVALUATION
The increase of paw volume after 3 or 6 h is calculated as
percentage compared with the volume measured
immediately after injection of the irritant for each animal.
16 | P a g e
Percentage inhibition = Mean test × 100
Mean control
5. GRANULOMA POUCH TECHNIQUE

 The method has been developed for screening by using
croton oil as irritant.
 An aseptic inflammation resulting in large volumes of
haemorrhage exudate is elicited which resembles the
subacute type of inflammation.
 Instead of croton oil carrageenan can be used as irritant.
PROCEDURE
Male or female Sprague-Dawley rats with a body weight
between 150 and 200 g are used.
Ten animals are taken for controls and for test groups.
The back of the animals is shaved and disinfected.
17 | P a g e
With a very thin needle a pneumoderma is made in the
middle of the dorsal skin by injection of 20 ml of air
under ether anesthesia.
Into the resulting oval air pouch 0.5 ml of a 1% solution

of Croton oil in sesame oil is injected avoiding any
leakage of air.
48 hours later the air is withdrawn from the pouch and 72

h later any resulting adhesions are broken.
Instead of croton oil 1 ml of a 20% suspension of

carrageenan in sesame oil can be used as irritant.
Starting with the formation of the pouch, the animals are

treated every day either orally or subcutaneously with the
test compound or the standard.
For testing local activity, the test compound is injected

directly into the air sac at the same time as the irritant.
On the 4th or the 5th day the animals are sacrificed under
anesthesia.
18 | P a g e
The pouch is opened and the exudate is collected in glass
cylinders.
Controls have an exudate volume between 6 and 12 ml,

which is reduced dose dependent in the treated animals.
EVALUATION
 The average value of the exudate of the controls and the
test groups is calculated.
 Comparison is made by statistical means
19 | P a g e

Anti Inflammatory

Uploaded by

Copyright:

Available Formats

Anti Inflammatory

Uploaded by

Document Information

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Anti Inflammatory

Uploaded by

Copyright:

Available Formats

PRECLINICAL SCREENING

 Inflammation is the body’s immune system’s response to

 Inflammation is universal host defensive process

 Inflammation is response to variety of harmful stimuli

Inflammation has different phases:

 The first phase is caused by an increase of vascular

 The second one by infiltration of leukocytes from the

 Accordingly, anti-inflammatory tests have to be divided

 Predominantly, however, these studies are aimed to find

1. 3H Bradykinin receptor binding

2. 3H substance p receptor binding

1. UV Erythema in guinea pig

2. Oxazolone induced ear edema in mice

3. Croton oil ear edema in mice

5. Granuloma pouch technique

1. 3H BRADYKININ RECEPTOR BINDING

PURPOSE AND RATIONALE

The 3H -bradykinin receptor binding is used to detect

Ileum from guinea pigs is cleaned from its content and

They are homogenized for 30 s in ice-cold TES buffer,

The homogenates are filtered through 3 layers of gauze

In the competition experiment, 50 µl 3H -bradykinin, 50 µl test

Saturation experiments are performed with 12 concentrations

Total binding is determined in the presence of incubation

The reaction is stopped by rapid vacuum filtration through

Thereby the membrane bound radioactivity is separated from

The retained membrane-bound radioactivity on the filter is

2. 3H SUBSTANCE P RECEPTOR BINDING

PURPOSE AND RATIONALE

Striata are dissected and homogenized in 50 mM ice-cold

Pellets are resuspended in 0.32 M sucrose

60min incubations are carried out at room temperature in 50

Total binding and nonspecific binding are determined in

Incubations are terminated by adding 4 ml of ice-cold Tris-

Filters are then washed three times using ice-cold Tris-HCl

Bound radio activities are determined using a liquid

1. UV ERYTHEMA IN GUINEA PIG

PURPOSE AND RATIONALE

Then they are chemically depilated by a commercial

Control animals are treated with the vehicle alone.

An original ultraviolet burner is warmed up for about 30 min

Following a 2 min ultraviolet exposure, the remaining half of

The investigator has to protect himself/herself by gloves and

The erythema is scored 2 and 4 h after exposure.

PURPOSE AND RATIONALE

Before each use a fresh 2% solution of oxazolone in acetone is

The mice are sensitized by application of 0.1 ml on the shaved

The mice are challenged 8 days later again under anesthesia

Groups of 10 to 15 animals are treated with the irritant alone

The left ear remains untreated.

The maximum of inflammation occurs 24 h later.

At this time the animals are sacrificed under anesthesia and a

The discs are immediately weighed on a balance.

The weight difference is an indicator of the inflammatory

3. CROTON-OIL EAR EDEMA IN RATS AND MICE

PURPOSE AND RATIONALE

For tests in rats the following mixture is prepared (v/v): 4

The standards and the test compounds are dissolved in this

For tests in mice male NMRI-mice with a weight of 22 g &

10 animals are used for controls and each test group.