P r e s e n t e d b y : S h i v a m
M. Pharm I year Pharmacology
Under the Guideline of: Dr. Phoolchandra
D e p a r t m e n t o f P h a r m a c o l o g y
SCREENING OF
IMMUNOMODULATORS
School of Pharmaceutical Science
I F T M U n i v e r s i t y
M o r a d a b a d

IMMUNOMODULATORS
• Immunomodulators are the agents that
modulates the immune system by suppress
or depress the immune system. So, these are
also divide into two parts
• 1- Immunosuppressant
• 2- Immunostimulants

IMMUNESUPRESSENTS
• Immunosuppressive drugs are used to
dampen the immune response in the organ
transplantation and autoimmune disease.
a) Specific T-cell inhibitors: Cyclosporine, Tacrolimus
b) Cyclotoxic drugs: Azathioprine, Cyclophospamide,
Methottraxate, Chlorambusil, Mycophenolate
c) Glucocorticoids: Prednisolone
d) Antibodies: Muromonab CD3, Antithymocyte
globulin(ATG), Rho(D), Immunoglobulin

IMMUNOSTIMULANTS
• These drugs are used to stimulate the immune
response in case of immunodeficiency.
• a) Levamisole (ergamisole)
• b)Thalidomide (Thalomid)
• c) Bacillus Calmette- Guerin (BCG)
• d) Interferons- gamma 1b & 1a
• e) Interleukin-2

Screening Methods
• Acute systemic anaphylaxix in rats
• Anti-anaphylactic activity (schuitz-dale reaction)
• Passive cutaneus anaphylaxis
• Arthus type immediate hypersensitivity
• Delayed type hyper sensitivity
• Reversed passive arthus reaction
• Adjuvant arthritis in rats

1. Acute systemic anaphylaxis in rats
• Strain 10-20 female Sprague-Dawley rat (120g)
• Firstly immunized by i.m. injection of 10 mg/kg
highly purified ovalbumin.
• Simultaneously 1 ml of Bordetella pertusis
suspension injected intraperitoneally.
• After 11 days animals injected with i.v. injection
of 25 mg/kg highly purified obalbumin
• 18hr prior to challenge, test dexamethasone 1-
10mg/kg s.c. control vehicle.

• Evalution:
after treatment compared treated and
control group for their shock symptoms and
mortality counted.
Statistical calculation is performed using
the chi-square test.

Modification
• By Devis and Evans(1973)- this experiment have also
been performed in guniea pigs and in mice. Anaphylactic
bronchospasm can be measured in isolated guniea pig
lungs.
• By Ufkes and Ottenhoff (1984)- study on brown norway
rats.
Given a suspension of trinitrophenyl heptenized ovalbulin
together with aipo4.
Bronchial and cardiiovascular function were studied after
treatment with antiallergic agents and antigen challenge.

2. Anti anaphylactic Activity
• Animal guinea pig of both sex
• Body weight 300-350g
• Sensitized with alum precipitated egg
albumin
• Give injection of 0.125 ml of egg albumin
in each foot pad and 0.5 ml
subcutaneously.

• After four weeks animal are killed and the
ileum is dissected out.
• About 2-3 cm long are mounted in an organ
bath containing Tyrode solution at
37cetigrate.
• Contractility of the ileum strips is tested by
adding standard in one organ bath and test
compound in another organ bath.

Biochemical estimation
• Carbon clearance test- for phagocytic
response
phagocytic Index = K(sample)/K(control)
• Hemagglutinating antibody titer.

Evalution-
• The concentration are recorded by a
polygraph
Modification-
• This method modified by testing histamine
release lung after challenging with egg
albumin.
• Koppel et al.(1981) developed a method to
induce concentration of mouse trachea by
antigen.
• Omote et al. (1994) modified method by using
sensitized guinea pigs.

3. Passive coetaneous anaphylaxis
• Animal- male rats
• Body weight- 100 g
principle:
Formation of antigen antibody complex induce
the release of mediator from mast cells. This
results increase in permeability of the vessel
walls and leakage of plasma.

Procedure
• Antiserum are injected intradermally in to shaved dorsal
skin of rats.
• After 24hr each animal is challenged with the
intravenous administration of 0.1ml of 2.5% Evans blue
dye containing 25mg/ml of egg albumin.
• Test compound is also administer along with the antigen.
• After 30 min animals are scarified.
• Amount of Evans blue dye that leaked at the site of
reaction is extracted and determined calorimetrically at
620 milimicron wavelength.

Evaluation
• Amount of Evans blue that extracted from
passive coetaneous anaphylactic reaction of
control group is compared group.
Modification
• Gose and Blair (1969)- used Bordetella pert sis
as antigen in passive coetaneous anaphylaxis
experi

4. Arthus type immediate hypersensitivity
• Animal-rats of both sex
• Strain-Wistar or Spargue- Dawley
• Body weight 220-280g
Principle-
• Antigen antibody induced reaction to an
inflammatory factors that characterized by
edeme, hemorrhage and vasculitis.

Procedure
• Seven days prior to experiment rats are sensitized
by im administration of 0.5 ml of the ovalabulin
suspension.
• 1st group (treated group) 1hr prior test compound
are administered and challenged with 0.5ml of
ovalabulin in left hind paw
• 2nd group(positive group)-sensitized animal
treated with solvent alone.
• 3rd group (negative group) non sensitized animals
treated with solvent.

Evaluation
• The change in footpad thickness is expressed
as percent from the vehicle control group.
Thickness can be measured by calipers.
Modification
• Omote et al.(1994)-sheep red blood cell
suspension used for immunization.

5. Delayed type hypersensitivity
Principle
• Delayed type hypersensitivity is a reaction of
cell mediated immunity and becomes visible
after 16-18hr.
Procedure
• 7 days prior, rats are sensitized by im
administration of 0.5 ml ovalbulin.
• After seven days again 0.1 of 0.04% of
ovalbulin injected in the left hind paw.

• Footpad thickness is measured immediately
and 24hr after of administration.
Modification
• Mizukoshi et al.(1994)- they use sheep red
blood cells for immunization.