Evaluation of Anti-inflammatory drugs SEMINAR ONPRESENTER : 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 insultPfizer 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 INFLAMMATION12345

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

MEDIATORVASODILATATIONVASCULAR PERMEABILTYCHEMOTAXISPAINHistamine++ _ +Serotonin+/- _ +Bradykinin+++ _ +++Prostaglandins+++ +++ +Leukotrienes- +++ _

Animals used: Albino Guinea pigsTest drug is administered -30 min before exposureDuration of exposure to UV B rays : 20 secArea of exposure : Depilated skinDegree of erythema estimated after 2 hours visually on a scale 0-4

ADVANTAGE : Pure measure of the vasodilatory phaseDrawback: Accurate measurement of reaction not obtained, variability of responseDRUGS 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 IPSite : Subplantar region of left hind paw

Experimental injury cascade of events

Biphasic1 release of histamine,5 HT and kinins2- 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= %

Carrageenan COX MustardDextranEgg-white Phlogistic agentsCompound 48/80Yeast, Zymosan-LOXSerotonin, histamineKaolinSubstance p, nystatin, glass powder, cobra venom

an instrument for measuring changes in volume within anorgan 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 ratsIntra articular talc injections in pigeons

Locomotor activity with inflammed paws is impairedDrugs effective: NSAIDS, steroids ,DMARDSAnti-histamines

Induction of Pleurisy: Carrageenan , turpentine, Evans blue, Arabic gum, glycogen, dextran , antigens , microbes, mast cell degranulators

Advantage: Quantitation of anti-inflammatory activity and M.O.A of new test drugDrugs effective: Steroid, NSAIDS & GoldIndex of activity of drug: The volume of exudate is decreased

Croton oil induced ear edema ( Croton tiglium)TPA-induced ear edema - Protein kinase C , NF-B , phospholipase A2 PGArachidonic acid induced ear edema Xylol induced ear edema

Predictable model for topical anti-inflammatory drugsInflammatory 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 (55C) 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 edemaDrugs effective :Cortisone , Salicylates

To study repair phase characterized by fibroblast proliferation and blood vessel formationSuppression 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 ,groinDuration : 1-14 daysPellet along with granuloma removed and weighed

Weight of granulomatous tissue = Dry weight of granuloma cotton -initial weight of cotton pelletDrugs effective: corticosteroids

Immunologically mediated model of chronic inflammationIt a delayed HS response to mycobacterial Ag

Induction of Arthritis : Subplantar injection of Freunds complete adjuvant or mycobacteria suspended in oilDay 1 Day 4-5 Day 7 Local reaction Peak Systemic disease

Parameters : Foot thickness, wt, grip strength , deformity measuredEvaluation of chronically administered drug against inflammationDrugs effective: Steroids ,Immunosuppressants, gold ,d-penicillamine

Chronic exposure to foreign bodies like contact lenses leads to GPCLevels of IL3,IL 4,IL5 ,Ig G, Ig E and Ig M is increased in TearsProcedure : A sterile needle is inserted directly into conjunctiva of unsensitized animalPricking results in high number of neutrophil infiltration

VCAM-1,ICAM-1 and E-selectin are derived from HUVEC ,HDMEC cell linesCell based ELISA assay

HUVEC,HMECTNF- as reagentTest drug20 minBCECF labeled WBC

20 minNo drugAdherent 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 countedTest drug : Blocks AdhesionDrugs effective : NSAID ,chloroquine

Dianzani etalthe PMNs are challenged by FMLP to generate 02- for screening anti-inflammatory compoundsSuperoxide production was measured spectrophotometrically by measuring the superoxide dismutase inhibitable reductionDrugs effective: Anti-oxidant drugs

Stimulates A and C fibers producing painLowers BP by vasodilatationTest : 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 cytokineCirculating leptin levels are increased upon infectious and inflammatory stimuli such as LPS, turpentine, and cytokinesLeptin increased statin inhibitible free radical and cholestrol production invitroCRP increased in obesity and reduced by statinJUPITER TRIAL, Leptin and Insulin resistanceFructose fed rat is a model of metabolic syndrome.

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

A HAPPY TEACHERS DAY TO ALL TEACHERS ON BEHALF OF US

*The plan of my seminar is as follows*It can be defined as a protective response or vascular connective tissue response intended to eliminate the cause and the result of an original insult.It took Pfizer approximately 18 years to develop the AI drug Piroxicam which was launched during the golden age of rational dd in 1980The cardinal signs of inflammation are rubor (redness), calor (heat), tumor (swelling), dolor (pain), and loss of function. Seen here is skin with erythema, compared to the more normal skin at the far right.Can be broadly classified as acute char by haemodynamic changes such as vas ADC*5 rs but usually the regulation of response is not under control*Mast cells ,basophils and platelets release histamine in response to injury*Serotonin: vasodilatory effects similar to those of histamine; platelet dense-body granules; release triggered by platelet aggregation pain and itch of serotonin is triggered by 5 HT3 receptors and bronchial smooth muscle contraction

*Inflammation contributes or is a part whereas obesity and diabets are itself considered as inflammatory states*Inflammation is terminated when the offending agent is eliminated and the secreted mediators are broken down or dissipated.

There are active anti-inflammatory mechanisms that serve to control the response and prevent it from causing excessive damage to the host.

*Erythema is the earliest sign of inflammation and is yet not accompanied by plasma exudation and edema.>In this model Guinea pigs are the frequently used animals and the drug is given at a fixed time before and fixed duration to the depilated skin and degree of redness is estimated on visual scale.UV LAMP 180-200nm is used*This model is intended to study the acute and subacute phases of inflammation in rodents.Cascade of events leading to formation of exudates*Sub p tachykininfamily common carboxy terminal,smooth muscle contractor and pain neurotr*Auranofin orally aurthiomalate alter structure and fuction of macrophagesreduction in rheumatiod factor halh life 7 days 30 mg weekly*From expressed seeds tetra decanyoyl phrbol acetate ingredient o fcroton oil*Local imune complex disease *7 d(7 days : collagen and proliferative response well established*Formyl methionyllucylphenylalanineFormation of superoxide ion2O2 + NADPH 2O2-rad + NADP+ + H+ (NADPH oxidase)O2 + 2H+ H2O2 (dismutase)*