screening of immunosuppressant and immunostimulant

1. PRE-CLINICAL SCREENING
OF IMMUNOMODULATORY
DRUGS
Presented by :- Akshay Joshi
M.Pharm (Sem- I) Department Of Pharmacology
Dr. D.Y Patil IPSR Pimpri, Pune

2. Contents :-
• Introduction
• Mechanism of action of immunosuppressant drug
• In-vitro methods
• In-vivo methods
• References

3. • IMMUNITY
The ability of an organism to resist
a particular infection or toxin.
WHAT IS ANTIGEN AND ANTIBODY ?
• ANTIGEN
An antigen is any foreign substance.
• ANTIBODY
It is an immunoglobulin produced by B-cells.

4. Introduction:-
• Immunomodulators are the agents that modulate immune system by suppressing
or by stimulating immune response
• Immunomodulation as part of immunotherapy, in which immune responses are
induced, amplified, attenuated, or prevented according to therapeutic goals
• Immunomodulators divided into two parts :-
- Immunosuppressant
- Immunostimulant

5. Immunostimulant drugs Immunosuppressant drugs
Stimulate response in case of
immunodeficiency
Inhibitory response in case of organ
transplantation
Levamisole Specific T-cell inhibitors- cyclosporin,
tacrolimus
Thalidomide Cytotoxic drugs- Azathiopurines
methotrexate,
Bacillus Calmette Guerin Glucocorticoids –Prednisolone
Interferons Antibodies –Muromonab CD3,
antithymocyte globulin
Interleukin-2

6. Mechanism of action of immunosuppressant drugs:-
1.Glucocorticoides
2.Cytotoxic drugs
3.T-cell inhibitor
4.Antibodies

7. • 1. Glucocorticoids inhibit MHC expression and IL-1, IL-2, IL-
6 production so that helper T-cells are not activated.
• 2. Cytotoxic drugs block proliferation and differentiation of
T and B cells.
• 3. Cyclosporine, tacrolimus and sirolimus inhibit antigen
stimulated activation and proliferation of helper T cells as
well as expression of IL-2 and other cytokines by them.
• 4. Antibodies like muromonab CD3, antithymocyte globulin
specifically bind to helper T cells, prevent their response
and deplete them.

8. Application
Organ
transplantation
Autoimmune
diseases
Asthma
Anti-
inflammatory
Uveitis

9. IN-VITRO METHODS
• Inhibition of histamine from mast cells
• Mitogen induced lymphocyte proliferation
• Inhibition of T cell proliferation
• Chemiluminescence in macrophages
• PFC (plaque forming colony) test in vitro
• Inhibition of dihydro-orotate dehydrogenase

10. IN-VIVO METHODS :-
• Acute systemic anaphylaxis in rats
• Antianaphylactic activity (Schultz-Dale reaction)
• Passive cutaneous anaphylaxis
• Arthus type immediate hypersensitivity
• Delayed type hypersensitivity
• Reversed passive arthus reaction
• Acute graft versus host disease in rats
• Collagen type-Ⅱ induced arthritis in rats
• Adjuvant arthritis in rats
• Porcine cardiac myosin-induced autoimmune myocarditis
in rats

11. Inhibition of histamine release from mast cells :-
• Purpose and rationale :-
Histamine release induce from mast cell by calcium ionophore 48/80.
Histamine concentration can be determined with o-phthalaldehyde reaction
• Procedure :-
1. Preparation Of mast cell suspension :-
Wistar rat decapitated and exsanguinated
50ml HBSS inj. Into peritoneal cavity
Massage body abdominal wall is open
Fluid containing peritoneal cell is collected
Centrifuge at 2000 rpm
Cells resuspended in HBSS
brought final concentration of 105 mast cells/100𝜇l

12. • Test compound administration and induction of histamine
release :-
 1ml test drug + mast cell suspension
 Incubate 37°c for 15 min
 Make 3ml vol with HBSS
 Equal vol of calcium ionophore with allergen
 Suspension incubate 37°c 30min
 Centrifuge at 2500 Rpm
Control solutions :-
• Spontaneous histamine release – only mast cells and solution
determine baseline
• Histamine release – mast cell + solution +calcium ionophore 10-6g/ml
• Test compound control – solutions +test compound

13. 1ml top layer transfer to
Tube contain 300mg Nacl+1.25 butanol
Sample alkalized by adding 1ml 3N NaoH
Centrifuge for 5min
1ml top layer (butanol) is pipetted out
In 5ml tube containing 2ml n-heptane+0.4ml of 0.12N HCl
Mixing by inverting tube
Separation of aq. and org. phase
0.5ml aqueous phase transfer to tube
Extraction of Histamine

14. • Induction of o-phthaladehyde complexing reaction :-
Sample + 100𝜇𝑙 1N NaOH 100𝜇𝑙 0.2%phthaladehyde solution After 2min, add 50 𝜇𝑙 3N
HCL
• Determination Of histamine release :-
Determine on fluorescence detector (350nm & 450nm)
• Evaluation :-
 Sample histamine release – spontaneous histamine release
100% histamine release – spontaneous histamine release
× 100

15. • Critical assessment of the method :-
Disodium cromoglycate reported to inhibit release of histamine and
degranulation of rat mast cells
• Modification of method :- sensitive colorimetric assay for release of
tryptase from human lung mast cells in vitro has been described by
Lavens et al. (1993)

16. Mitogen Induced Lymphocyte Proliferation :-
• Purpose and rationale :-
Immunomodulatory properties can detected either by pretreatment of
animal in vivo or by adding test drug to cultured lymphocyte
• Procedure :-
Mice of weight 18-20g or Rat 180-200g are used
Materials :-
Antigen and or following mitogens :
Lipopolysaccharide 10-0.1 micro/ml
Dextran sulfate 30-7.5 micro/ml
Phytohaemaglutinin 0.5-0.12 %stock solution
Concanavallin A 0.5-0.12 micro/ml
Standards – levamisole, cyclosporine A, prednisolone

17. After drying degree of radioactivity is determined
Cells are harvested on glass fibre filter
Another 8h after addition of 0.25𝜇𝐶 H-thymidine per well
Plates incubated at 37℃ in 5% CO2 in air for 48-60 h
Mitogens are titrated in 0.1ml/well and add 0.1ml suspension
Single cell suspension of 5× 106 cells /ml
Sacrifice and spleen is removed
Animal treated with test compound once a day for 5days
Ex-Vivo:-

18. • In-vitro :-
Animals sacrifice spleen removed
Single cell suspension of 107 cells /ml prepared
0.05ml place in each microtiter well
Test compound is added in 0.05ml
At last 0.1ml mitogen added
Plates incubated and processed as above
Evaluation:- stimulation index=proliferation ratio with or without spleen mean weight

19. IN-VIVO:- 1. Acute Systemic Anaphylaxis in rats :-
• Purpose and rationale :-
Detection of shock symptoms and it can be inhibited by corticosteroid and i.v
disodium cromoglycates
• Procedure :-
Female Sprague dawley rat 120g
Immunized by i.m inj. 10mg/kg high purified ovalbumin with 1ml Bordetella
pertussis suspension
IgE Ab are induce and attach to surface of mast cells, After 11 days
Animals challenged by i.v. inj. Of 25mg/kg ovalbumin

20. Formation of Ag-Ab complex in blood/organs
Then immediate anaphylaxis mediators release
Corticosteroid e.g. dexamethasone 1-10mg/kg s.c. Or 30mg/kg disodium cromoglycate
(before 18h challenged )

21. • Evaluation :- Shock symptoms are scored and mortality counted
• Modification of the method :-
elwood et al. (1992) studied the effect of dexamethasone and
cyclosporin A on allergen-induced airway hyperresponsiveness and
inflammatory cell responses in sensitizes brown –Norway rats.

22. Anti-anaphylactic activity (Schultz-dale reaction)
• Purpose and rationale :- guinea pig are sensitized against egg albumin.
Challenge after 3weeks cause isolated organs release mediators e.g. histamine
• Procedure :-
Guinea pig of either sex sensitized with alum precipitated egg albumin.
Alum egg albumin is prepared by dissolving egg albumin (1mg/ml)in 6% aluminum
hydroxide gel, suspended in saline.
Mixture is stirred and kept at room temperature.
Each animals receives at same time injection of 0.125ml of this mixture in each foot pad
and 0.5ml s.c
After 4weeks animals are killed and ileum is dissected out.

23. Cleaned pieces, about 2-3cm long, are mounted in organ bath containing Tyrode solution at
37℃.
Strips allowed to equilibrate for 15min.
Contractility of ileum strip is tested by adding 10-4 g/ml BaCl2 solution.
To one organ bath standard and to other test compound added.
After 3min ovalbumin in final concentration of 2× 10 -6g/ml is added
Contraction recorded with strain gauges by polygraph.

24. • Evaluation :-
If anti-anaphylactic activity is observed, ED50 value using different doses
are calculated.
• Modification of method :-
Trachea of sensitized guinea pigs was used by omote et al.
(1994)

25. References :-
• Hans Gerhard Vogel. Drug discovery and evaluation: Pharmacological assays, vol 2, 3rd edition. Pg
no.792-799
• KD Tripathi, Essentials of Medical Pharmacology, Jaypee publications, Seventh edition, Pg no.778-
788