Preclinical studies involve testing drugs or treatments in animals before human trials. This document discusses various types of preclinical studies including in vitro, in vivo, in situ, and in silico experiments. It also describes methods to study immunomodulators including inhibition of histamine release from mast cells and mitogen-induced lymphocyte proliferation assays. Various in vivo models are outlined such as acute systemic anaphylaxis in rats and delayed type hypersensitivity tests.

1. PRECLINICAL STUDIES
Preclinical studies refer to the testing
of a drug, procedure or other medical
treatment in animals before trials may
be carried out in humans. During
preclinical drug development, the
drug’s toxic and pharmacological
effects need to be evaluated through
in vitro and in vivo laboratory animal
testing.

2. SCREENING
It means thorough investigations
•measure the pharmacological activity of new or chemically
undefined substances
• investigate the function of endogenous mediators
• measure drug toxicity and unwanted effects
The main purposes of screening are to determine whether the new
substance are worthy for further attention and to indicate which
among them have the most interesting pharmacological
properties.

3. Types of Screening:
A. Simple screening
B. Blind screening
C. Programmed screening
A- Simple Screening:
It involves the use of one or two simple tests to find
substances having a particular property.
For example, a single test for conc. of glucose in blood can be
used to screen compound for hypoglycemic activity.

4. B- Blind Screening:
It is used to detect the pharmacological activities of new
drugs whose pharmacological activity is unknown. The chief
purposes is to demonstrate whether these new drugs are
worthy of further attention or not.
C- Programmed Screening:
It is used when a new drug of specific type is to be screened
for some pharmacological effects.
Examples are screening of certain drugs on the CVS, CNS,
kidney, blood etc. It includes the use of quantitative assay of
the compounds and their comparison with standard drugs
that are quite active representative members of their
pharmacological class.
It also provides indications of potential side effects.

5. TYPES OF EXPERIMENTS
INVIVO INVITRO INSITU INSILICO

6. DIFFERENCE BETWEEN
INVIVO AND INVITRO METHOD

7. IMMUNOMODULATORS
Immunomodulators are the agents that modulate the
immune system by suppress or stimulate the immune
response. So, these are divided into two parts that
are:
Immunomodulators
Immunosupr
-essants
Immunosti-
mulants

8. Immunosupressants
Immunosupressants drugs are used to dampen the
immune response in organ transplantation and
autoimmune disease.
CLASSIFICATION
SPECIFIC T –CELL INHIBITORS : 1-cyclosporin
2- tacrolimus
CYTOTOXIC DRUGS : 1- cyclophosphamide 2-
chlorambucil

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GLUCOCORTICOIDS- prednisolone
ANTIBIOTICS- antithymocyte
Immunostimulants
These drugs are used to stimulate the
immune response in case of
immunodeficiency:-
 Levamisole
Thalidomide
Interferons

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IMMUNOMODULATORS
INVITRO METHODS
I. Inhibition of histamine release from mast cells
II. Mitogen induced lymphocyte proliferation
III. Inhibition of T cell proliferation
 INHIBITION OF HISTAMINE RELEASE
FROM MAST CELLS:-
 Purpose and rationale:
• Hypersensitivity reaction can be elicited .
• The histamine concentration can be determined with
the o-pthalaldehyde reaction.

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Procedure
Preparation of mast cell suspension:-
Wister rats are decapitated.50ml of hanks balanced salt
solution (HBSS) are injected into the peritoneal cavity and
following massage of the body , the abdominal wall is opened.
The fluid containing peritoneal cells is collected in a
centrifuge tube and centrifuged at 2000 rpm. The cells are
suspended in HBSS.

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Then the cells suspension is brought to a final concentration.
Test compound administration and induction of
histamine release :-
1ml test drug is added to the mast cell suspension and the
mixture is incubated at 37˚C for 15 min.
The cells are made up to the volume of 3ml with HBBS.
An equal volume of calcium –ionophore is added.

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The suspension is incubated at 37˚C for 30 min followed by
centrifugation at 2500rpm.
EXTRACTION OF HISTAMINE :-
1ml of the top layer is transferred to a tube containing 300mg
Nacl and 1.25ml butanol.
The sample is alkalized to extract the histamine into butanol
by adding 1ml 3N NaOH following chemical shaking , the
sample is centrifuged for 5 min.
1ml of the top layer (butanol) is pipetted into a 5ml tube
containing 2ml of n-heptane and 0.4ml of 0.12N HCL.

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The tube is mixed by inverting it several times. Following
separation into aqueous and organic phases.
0.5ml of the aqueous phaseis transferred to another tube.
INDUCTION OF o-PTHALALDEHYDE COMPLEXING
REACTION :-
To each sample 100micro litre 0.2% o-pthaladehyde solution
after 2 min, the o-pthaladehyde complexing reaction is stopped
by addition of 50micro litre 3N HCL.
DETERMINATION OF HISTAMINE RELEASE :-
The total sample is transferred to an auto sample vial and the
histamine conc. is determined by a fluorescence detector

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At 350 and 450nm respectively.
EVALUATION
% histamine release can be expressed by the following
formula :-
Sample hist. release - spontaneous hist. release x 100
100% hist.release - spontaneous hist. release
•Spontaneous histamine release- contains only mast cells.
MODIFICATION OF THIS METHOD
Johnston et.al (1978) studied the inc. superoxide anion
production by immunologically activated and chemically
elicited macrophages.

16. MITOGEN INDUCED LYMPHPOCYTE
PROLIFERATION
PURPOSE AND RATIONALE :-
•Cultured lymphocytes can be stimulated to a proliferative response
and to DNA synthesis by various mitogens.
•Immunomodulating properties can be detected either by
pretreatment of the animals invivo or by adding the test drug to the
cultured lymphocytes.
MATERIAL :-
Sheep red blood cell (SRBC) specific antigen and the following
mitogens:-
Lipopolysaccharide 10-0.1microgram/ml
Dextran sulfate 30-7.5microgram/ml
Phytohaemogglutinin 0.5-0.12 %stock solution
Concanavallin A 0.5-0.12microgram/ml

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As standards levamisole , cyclosporineA , prednisolone are
used.
PROCEDURE
EX-VIVO
Animals receive the test compound once a day for 5
days. Thereafter, they are sacrified, spleens are
removed and a single cell suspension of 5*106 cells
/ml is prepared.
mitogens are titrated in 0.1/well and o.1ml of cell suspension
is added.

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Plates are incubtaed at 37˚c in 5% carbon dioxide in air for 48-
60 hr and for another 8 hr after addition of 0.25micro 3H-
thymidine per well.
Cells are harvested on glass fibre filters and after drying the
degree of radioactivity is determined.
IN-VITRO
Animals are sacrified and their spleens removed. A single cell
suspension of 10 7 cells /ml is prepared and 0.05ml placed in
each microliter well (4 replicates/groups).
Then the test compounds (4 times conc.) is added in 0.05ml.

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At last 0.1 ml of the double conc. Mitogen are added.
Plates are incubated and processed as describe above.
EVALUATION
Stimulation index = proliferation ratio according to
positive control , either with or without mean spleen
weight.
Statistical evaluation is carried out using the student
test ( comparison of positive and negative control to
experimental groups).

20. IN VIVO METHODS
Acute systemic anaphylaxis in rats
Anti- anaphylactic activity(Schuktz-Dale reaction)
Passive cutaneous anaphylaxis
Arthus type immediate hypersenstivity
Delayed type hypersenstivity
Reversed Passive Arthus reaction
Adjuvant arthritis in rats

21. ACUTE SYSTEMIC ANAPHYLAXIS IN
RATS
PURPOSE AND RATIONALE:-
Rats are immunized with ovalbumin and Bordetella
pertussis suspension as adjuvant.After 11 days the animals
are challenged by intervenous injection of ovalbumin. The
shock symptom can be inhibited by corticoids and
intravenous disodium cromoglycate.
PROCEDURE:-
Female Sprague-Dawley rats weighing 120g are
immunized by i.m. injection of 10mg/kg highly purified
ovalbumin

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Simultaneously 1ml of Bordetella pertusis
suspension (2⁎1010 organisms) is injected
intraperitoneally
IgE antibodies are induced and attached to the
surface of mast cells and basophilic granulocytes
Eleven days later the animals are challenged by
intravenous injection of 25mg/kg highly purified
ovalbumin

23. This results in formation of antigen- antibody complexes
on the surface of mast cells and basophilic granulocytes in
blood and in all organs with immediate release of various
mediators of anaphylaxis, such as histamine, serotonin,
prostaglandins; in shock symptoms.
Corticosteroids ,eg.- dexamethasone 1-10mg/kg
subcutaneous are given, 18hr prior to challenge, or
30mg/kg disodium cromoglycate i.v. before injection of
ovalbumin
EVALUATION :-
The shock symptoms are scored and mortality counted.
Results after treatment are compared with untreated
controls

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Pretreatment with corticosteroids or disodium
cromoglycate can inhibit death and ameliorate shock
symptoms
Stastical calculation is performed using the X2 test
MODIFICATION :-
•Desensitization by repeated ‘microshocks’ of
constant strength in given pigs has been reported by
Herxheimer(1952)
•Acute systemic anaphylaxis experiments have also
been performed in given pigs & mice. In guinea pigs
anaphlactic bronchospasm can be measured with
Konzett and Rossler method.

25. DELAYED TYPE HYPERSENSITIVITY
PURPOSE AND RATIONALE:-
Delayed type hypersensitivity is a reaction of cell mediated
immunity and becomes visible only after 16-24 hrs. The
same methods as for testing immediate type
hypersensitivity can be used.
PROCEDURE:-
Rats are sensitized in the same way by i.m. administration
of 0.5ml ovalbumin suspension 7 days prior to start the
experiment as described for testing immediate type of
hypersensitivity

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They are challenged by injection of 0.1ml of 0.04%
solution of highly purified ovalbumin in the left hind paw
Footpad thickness is measured immediately and 24hr
after ovalbumin administration
MODIFICATION:-
Mizukoshi et.al. (1994) injected female CDFI mice
intradermally with a suspension of 2⁎108 sheep red blood
cells/ 50μl into the left foot pad
A second booster of the same dose was given to the right
foot pad on day 4

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Thickness of the foot pads was measured on the
following day and the difference in the thickness
between the right & left pads was taken as the degree
of swelling.