Evaluation of Anti-inflammatory drugs

SEMINAR ON

PRESENTER : Dr. Jill Mathew Dated :04/09/2013

Inflammation is a protective response intended to eliminate the initial cause of cell injury as well as the necrotic cells and tissues resulting from the original insult

Pfizer took about 18 years to develop the anti-inflammatory drug piroxicam, which was launched in 1980 during the “golden age of rational drug discovery”

CARDINAL SIGNS OF

INFLAMMATION

12

34

5

Acute: vasodilatation & increased capillary permeability

Delayed: infiltration of leukocytes and phagocyte cells

Chronic proliferative: tissue degeneration and

fibrosis

(1) Recognition of the injurious agent(2) Recruitment of leukocytes(3) Removal of the agent(4 ) Regulation of the response(5) Resolution

ICAM-1 and VCAM-1

MEDIATOR VASODILATATION

VASCULAR PERMEABILTY

CHEMOTAXIS PAIN

Histamine ++ _ +

Serotonin +/- _ +

Bradykinin +++ _ +++

Prostaglandins

+++ +++ +

Leukotrienes - +++ _

Animals used: Albino Guinea pigs Test drug is administered -30 min before exposure Duration of exposure to UV –B rays : 20 sec Area of exposure : Depilated skin Degree of erythema estimated after 2 hours

visually on a scale 0-4

ADVANTAGE : Pure measure of the vasodilatory phase

Drawback: Accurate measurement of reaction not obtained, variability of response

DRUGS EFFECTIVE : NSAIDS like Phenyl butazone, Aminopyrene, Aspirin and Sodium salicylate

Area 2: treated with topical H 1 antagonist (1.5% chlorphenoxamine hydrochloride in the same cream base, Allergex, Epico) twice daily.

Area 1: used as a control area and was exposed to -UVB and treated with cream base only twice daily.

Area 3: treated with topical H 2 antagonist (2% famotidine in the same cream base) twice daily

Rat-paw edema (Acute and Subacute phases) Drugs: Carrageenan (1%, 0.1 ml SC.),

indomethacin 2 mg/kg IP Site : Subplantar region of left hind paw

Experimental injury cascade of events

Biphasic 1 – release of histamine,5 HT and kinins 2- release of prostaglandins

Parameter : Paw volume up to the ankle joint is measured in treated and untreated groups before and after carrageenan

Advantage: Simple ,easy, short lasting and

reproducible

Disadvantage : Non –specific and difficult to quantify

and examine cells & their modification by anti-inflammatory drugs

Avoid in both the hind paws because of unalleviated pain and deformity

% of edema produced by carrageenan alone = A – B X 100 = X % B % of edema produced by carrageenan after

treatment with drug = C – D X 100 = Y % D % reduction of edema by drug = X –Y= %

1. Carrageenan – COX 2. Mustard3. Dextran4. Egg-white

Phlogistic agents5. Compound 48/806. Yeast, Zymosan-LOX7. Serotonin, histamine8. Kaolin9. Substance –p, nystatin, glass powder,

cobra venom

an instrument for measuring changes in volume within an organ whole body or limb

The mercury displacement due to dipping of the paw can be directly read from the scale attached to the mercury column

TUBE A

TUBE B

RAT PAW IS IMMERSED INTOTHE TUBE ‘A’ & THE READING IS CHECKED THROUGH THE TUBE ‘B ’ SOME SET UPS HAVE A DIGITAL DISPLAY ATTACHED TO IT

Inducer of arthritis: Silver nitrate in the tibiotarsal joint of rats Intra articular talc injections in pigeons

Locomotor activity with inflammed paws

is impaired Drugs effective: NSAIDS,

steroids ,DMARDS Anti-histamines

Induction of Pleurisy: Carrageenan , turpentine, Evan’s blue, Arabic

gum, glycogen, dextran , antigens , microbes, mast cell degranulators

Advantage: Quantitation of anti-inflammatory activity and M.O.A of new test drug

Drugs effective: Steroid, NSAIDS & Gold Index of activity of drug: The volume of exudate is decreased

1. Croton oil induced ear edema ( Croton tiglium)

2. TPA-induced ear edema - Protein kinase C , NF-кB ,

phospholipase A2 PG

3. Arachidonic acid induced ear edema

4. Xylol induced ear edema

Predictable model for topical anti-inflammatory drugs

Inflammatory response measured by increase in ear plug weight & thickness at single time interval( at 6 hours)

Drugs tested : Steroids and NSAIDS

Right -Control

Std duration (27 sec) and Std intensity (55◦C)

thermostatically

controlled water

to the shaved abdomen of rat

Resultant edema can be measured by excising skin

Egg albumin or horse serum Passively sensitized by

antibodies

Intra-dermal injection of Ag or Ab is made

Inflammatory reaction ASSESSMENT : by dye leakage or edema Drugs effective :Cortisone , Salicylates

To study repair phase characterized by fibroblast proliferation and blood vessel formation

Suppression of granulation tissue parallels with anti-inflammatory activity

For measurement of activity of anti-inflammatory drugs on proliferative component of ‘subacute & chronic inflammatory processes’

Sterile Cotton pellets (wt :5mg -50mg ) sc sites of axilla , back ,groin Duration : 1-14 days Pellet along with granuloma removed

and weighed

Weight of granulomatous tissue = Dry weight of granuloma cotton -initial

weight of cotton pellet Drugs effective: corticosteroids

Immunologically mediated model of chronic inflammation

It a delayed HS response to mycobacterial Ag

Induction of Arthritis : Subplantar injection of Freund’s complete adjuvant or mycobacteria suspended in oil

Day 1 Day 4-5 Day 7 Local reaction Peak Systemic disease

Parameters : Foot thickness, wt, grip strength , deformity

measured Evaluation of chronically administered drug against

inflammation Drugs effective: Steroids ,Immunosuppressants, gold

,d-penicillamine

Chronic exposure to foreign bodies like contact lenses leads to GPC

Levels of IL3,IL 4,IL5 ,Ig G, Ig E and Ig M is increased in Tears

Procedure : A sterile needle is inserted directly into conjunctiva of unsensitized animal

Pricking results in high number of neutrophil infiltration

VCAM-1,ICAM-1 and E-selectin are derived from HUVEC ,HDMEC cell lines

Cell based ELISA –assay

HUVEC,HMEC

TNF-ἀ as reagent

Test drug

20 min

BCECF labeled WBC

20 min

No drug

Adherent cells are lysed and fluorescence measured at 485 nmin ELISA reader

Thrombin activated human platelets

Incubated with drug at 20 0 C for 10 min & mixed with Neutrophils at a ratio 10:1

Index of activity : Neutrophils with 2 or more and one or no adherent platelets are counted

Test drug : Blocks Adhesion

Drugs effective : NSAID ,chloroquine

Dianzani etal the PMNs are challenged by FMLP to

generate 02- for screening anti-inflammatory compounds

Superoxide production was measured spectrophotometrically by measuring the superoxide dismutase inhibitable reduction

Drugs effective: Anti-oxidant drugs

Stimulates A and C fibers producing pain Lowers BP by vasodilatation Test : detects compounds that inhibit binding of 3 H- Bradykinin in membrane preparation of

guinea pig ileum Evaluation: specific binding= total binding-

nonspecific binding % inhibition:100- specific binding

Leptin is a proinflammatory cytokine Circulating leptin levels are increased upon

infectious and inflammatory stimuli such as LPS, turpentine, and cytokines

Leptin increased statin inhibitible free radical and cholestrol production invitro

CRP increased in obesity and reduced by statin JUPITER TRIAL, Leptin and Insulin resistance Fructose fed rat is a model of metabolic

syndrome.

The compounds were found to be useful in OA,RA and Psoriasis

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