Evaluation of anti-inflammatory

agents
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Inflammation
The immune response to the stress or tissue damage is called inflammation.
The 5 cardinal signs of inflammation:
1) Redness (Rubor)
2) Heat (Calor)
3) Pain (Dolor)
4) Swelling (Tumor)
5) Loss of functions
Factors:
a) Cut & Burns
b) Pathogens & Toxins
c) Foreign bodies dirt , pollen grains
d) Immune reaction due to hyper-sensitivity.
e) Chemical Irritants.
f ) Radiations
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Stages of inflammation:
1) Vasodilatation & increased permeability.
2) Phagocytic migration.
3) Tissue repair.

Mechanism of Inflammation:
The process of inflammation is initiated by cells present in tissue.
On activation cells release mediators.
a) Cells: b) Cell derived c) Plasma derived

1. Mast cells 1. Prostaglandins 1. Bradykinin

2. Histocytes 2. Cytokines 2. Thrombin
3. Macrophages 3. Leukotrienes 3. Plasmin
4. Dendritic cells 4. TNF- 4. Fibrin
5. Kupffer cells
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Significance & Disorders of Inflammation:

Significance:
1.Disposal of microbes, toxins or foreign materials.
2.Preparation of site for tissue repair.
3.Isolation of affected area of body.
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Anti-Inflammatory drugs
Mechanism of action:
Membrane Phospholipids

Arachidonic Acid

COX LOX

Prostaglandins Leukotrienes

Anti-Inflammatory Agents
 Anti-inflammatory Drugs 7

1. Non-steroidal anti-inflammatory agents:

e.g. Aspirin, Phenylbutazone, Ibuprofen, mefenamic acid, celecoxib,
diclofenac, Piroxicam, etc.

2. Corticosteroids:
e.g. Prednisolone, Betamethasone, Triamcinolone,etc.

3. Miscellaneous agents:
e.g. Auranofin, Bromelain
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In-vitro methods
1. 3H-Bradykinin receptor binding
2. H-Substance P receptor binding
3. Neurokinin receptor binding
4. Characterization of neurokinin agonists and antagonists by biological assays
5. Assay of polymorphonuclear leukocyte chemotaxis in vitro
6. Polymorphonuclear leukocytes aggregation induced by FMLP
7. Constitutive and inducible cellular arachidonic acid metabolism in vitro
a. Formation of leukotriene B4 in human white blood cells in vitro
b. Formation of lipoxygenase products from 14C-arachidonic acid in human
polymorphonuclear neutrophils (PMN) in vitro
c. Formation of eicosanoids from 14C-arachidonic acid in human platelets in vitro
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8. Stimulation of inducible prostaglandin pathway in human PMNL

9. COX-1 and COX-2 inhibition
10. Induced release of cytokines(Interleukin-1, IL-1, IL-6, IL-8 and TNF)
from human white blood cells in vitro
11. Flow cytometric analysis of intracellular cytokines
12. TNF-alpha antagonism
13. Binding to interferon receptors
14. Screening for interleukin-1 antagonists
15. Inhibition of interleukin-1- converting enzyme (ICE) .
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1) Formation of eicosanoids from 14C-arachidonic acid in

human platelets in vitro:

Purpose and rationale:

To test the inhibitory activity of the test substance on enzyme cyclo-oxygenase.

Procedure:
1) The platelet rich plasma obtained from blood of healthy volunteers mixed with
citrate/glucose solution and centrifuged.
2) The precipitate suspended in Dulbecco's solution and incubated with the test
compound.
3) Eicosanoid formation is started by the addition of 1-14C-arachidonic acid solution.
4) After stopping the reaction, the formed eicosanoids separated and analyzed.

Evaluation:
The radioactivity of the separated TXB2 and PGE2 is measured as a function of
drug concentration.
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2) Induced release of cytokines from Human white

blood cells: (IL-1alpha,IL-1beta, IL-6, IL-8, TNF- alpha)

Purpose and Rationale:

Cytokines are highly potent peptides involved in inflammation and
immunological responses.

Blocking their release has been successful in treating conditions like

rheumatoid arthritis, inflammatory bowel disease and graft Vs host disease.

The activity of the investigational drug , whether it possesses the inhibitory

effect on the release of cytokines can be found out.
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Procedure: Isolation of white blood cells

Synthesis and release of cytokines using Salmonella aborti equii

Incubation with the investigational drug

Cytokine levels determined using ELISA kit

Evaluation:
The cytokine-concentration is measured as a function of drug concentration,
and related to a control experiment without drug.
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3) 3H-Substance P receptor binding:

Purpose and rationale:

It functions as a neurotransmitter/ neuromodulator in a variety of

physiological processes.

Substance P is released in response to stress and noxious stimuli.

In causes vasodilatation and plasma extravasation, suggesting a role in

neurogenic inflammation.

Selective antagonists to substance P can be candidates for anti-inflammatory

and analgesic drugs.
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Procedure:
Fresh porcine brains homogenized in Tris-HCl buffer

The homogenates centrifuged and washed with Tris-HCl buffer

The pellets are resuspended in incubation buffer

Saturation Competition
binding binding
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In-vivo methods
Methods for testing acute and sub acute inflammation are:
UV-erythema in guinea pigs
Vascular permeability
Oxazolone-induced ear edema in mice
Croton-oil ear edema in rats and mice
Paw edema in rats (various modifications and various irritants)
Pleurisy tests
Granuloma pouch technique (various modifications and various irritants)

The proliferative phase is measured by methods for testing

granuloma formation, such as:
Cotton wool granuloma
Glass rod granuloma
PVC sponge granuloma.
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Methods for testing immunological factors

Adjuvant arthritis in rats (various modifications)

Experimental allergic encephalomyelitis
Schultz-Dale-reaction
Passive cutaneous anaphylaxis
Arthus type immediate hypersensitivity
Delayed type hypersensitivity
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Ultraviolet erythema in guinea pigs

Purpose and rationale:
To delay the development of ultraviolet erythema on albino guinea pig skin by
systemic pretreatment with clinically equivalent doses of phenylbutazone and
other NSAIDS.

Procedure:
Animals used: albino guinea pigs, both sexes, avg weight -350g
Number: 4 each for treatment and control
Pretreatment:
Animals are shaved on both the flanks and on the back.
They are chemically depilated (barium sulphate suspension)
The depilation paste and fur are rinsed off in running warm water.
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Treatment:
Test compound is dissolved or suspended in vehicle.
Half dose is administered by gavage 30 min before uv exposure.
Control animals are treated with vehicle alone.

Exposure:
Guinea pigs are placed in leather cuffs with a hole punched in it, allowing the
ultra violet radiation to reach only this area.

Burner used:
Original Hanau burner placed at affixed distance above the animal.

Post exposure:
Administration of remaining half of the dose.
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Evaluation:
Erythema is scored 2 to 4 hrs after exposure.
Method: visually detected by 2 different investigators in double blinded
manner.
Scores:
0 = no erythema,
1 = weak erythema,
2 = strong erythema,
4 = very strong erythema.
Doses of indomethacin and phenylbutazone were found to be effective.
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Vascular permeability
Purpose and rationale:
To evaluate the inhibitory activity of drugs against increased vascular permeability
which is induced by a phlogistic substance.
The drugs which can be used are H1- antihistaminics, inhibitors of arachidonic acid
metabolism, leucotriene receptor antagonists and membrane-stabilizing drugs.

Procedure:
Animals used: Male Sprague-Dawley rats
Number: 10 animals used each for test and control.
Pretreatment:
The ventral sides of the animal are shaved.
1% solution of Evans blue is injected intravenously.
Animals are dosed with the test compound orally or intraperitoneally or with the
vehicle.
Then the animals are briefly anaesthetized.
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Treatment:
Solution of compound 48/80 is injected intracutaneously at 3 sites both at the
left and ventral side.
Later animals are sacrificed by ether anesthesia.
The abdominal skin is removed and the dye infiltrated areas of the skin are
measured.

Evaluation:
The diameter of the dye-infiltrated areas is measured in millimeters and the
mean values of all injection sites in one animal are calculated.
The percent inhibition in the treated animals as compared to the control group
is calculated.
Phenylephrine was been found to be effective.
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Cotton Wool Granuloma

Purpose and rationale:
Foreign body granulomas are provoked in rats by subcutaneous implantation
of pellets of compressed cotton.

After several days, histologically giant cells and undifferentiated connective

tissue can be observed besides the fluid infiltration.

The amount of newly formed connective tissue can be measured by weighing

the dried pellets after removal.

More intensive granuloma formation has been observed if the cotton pellets
have been impregnated with carrageenin.
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Procedure:
Male Wistar rats with an average weight of 200 g are anaesthetized with
ether. The back skin is shaved and disinfected with 70% ethanol.
An incision is made in the lumbar region.
By a blunted forceps subcutaneous tunnels are formed and a sterilized
cotton pellet is placed on both sides in the scapular region.
The animals are treated for 7 days subcutaneously or orally.
The animals are sacrificed, the pellets prepared and dried until the weight
remains constant.
The net dry weight, i.e. after subtracting the weight of the cotton pellet is
determined.
Evaluation:
The average weight of the pellets of the control group as well as of the test
group is calculated.
The percent change of granuloma weight relative to vehicle control group
is determined.
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Thank You