3. Introduction
• Inflammation – self-defense mechanism
• Protective response intended to eliminate the
initial cause of cell injury as well as the
necrotic cells and tissues resulting from the
original insult
• Symptom of a wide variety of diseases
3Dr. Aditi M. Panditrao
7. Steps in inflammatory response
1. Recognition of the injurious agent
2. Recruitment of leukocytes
3. Removal of the agent
4. Regulation of the response
5. Resolution
7Dr. Aditi M. Panditrao
10. 10
Anti-inflammatory Analgesic Anti-pyretic
In vitro In vivo
• 3H-Bradykinin receptor binding
• 3H-Substance P receptor binding
• Assay of PMNLchemotaxis in vitro
• PMNL aggregation
• Constitutive and inducible cellular
arachidonic acid metabolism in
vitro
• Formation of leukotriene B4 in
human white blood cells in vitro
• Formation of lipoxygenase
products from 14C-arachidonic acid
in human PMN in vitro
• Formation of eicosanoids from
14C-arachidonic acid in human
platelets in vitro
• Methods for testing acute and
subacute inflammation
• Methods for testing the
proliferative phase (granuloma
formation)
• Side effects of anti-inflammatory
agents
Dr. Aditi M. Panditrao
13. Hageman
factor (XII)
Activation
Prekallikrein
HMW Kininogen
LMW Kininogen
Kallikrein
Kallikrein
Bradykinin
Kallidin
Des Arg
Bradykinin
Trauma,
Tissue injury
• Vasodilation
• ↑ Vascular Permeability
• Bronchoconstriction
• Pain
B1
• Pain
•Histamine release
• Recruit Neutrophils
• IL-1, TNF-α, etc.
INFLAMMATION
Role of Bradykinin in Inflammation
13Dr. Aditi M. Panditrao
14. • Tissue homogenates in incubation buffer
• Incubated with
• 3H-Bradykinin alone – Total Binding
• 3H-Bradykinin + unlabelled Bradykinin (saturated) – Non-specific
Binding
• 3H-Bradykinin + Test drug (subtract the non-specific binding)
• Two types –
• Competition studies – increasing concentrations of Test drug
• Saturation studies – increasing concentrations of 3H-Bradykinin
• Tissues – G. pig ileum, rabbit carotid artery, canine cultured
tracheal smooth muscle cells, bovine aortic endothelial
cells, human fibroblasts, etc.
14Dr. Aditi M. Panditrao
15. 3H-Substance P receptor binding
• Substance P – inflammatory mediator
• Bind to NK1 and NK2 receptor
• Direct and Indirect Actions
• ↑ Levels – Acute and Chronic Inflammatory
processes
15Dr. Aditi M. Panditrao
16. Trauma,
Tissue injury
Peripheral
Nerves
Substance P
Direct Actions
• Pain
• Vasodilation
• ↑ Vascular Permeability
• Neutrophil recruitment
• Activation of Macrophages
and Mast cells
• Release of ILs, Chemokines
NK1, NK2
Indirect Actions
• Angiogenesis
• Stimulation of Adhesion
molecules
• Induces Apoptosis?
16Dr. Aditi M. Panditrao
17. • Tissue homogenates in incubation buffer
• Incubated with
• 3H-Substance P alone – Total Binding
• 3H-Substance P + unlabelled Substance P (saturated) – Non-
specific Binding
• 3H-Substance P + Test drug (subtract the non-specific binding)
• Two types –
• Competition studies – increasing concentrations of Test drug
• Saturation studies – increasing concentrations of 3H-Substance P
• Tissues – Chinese Hamster Ovary (CHO), CHO cells
transfected with human ileum NK2 receptor, Guinea pig
cortical membranes, human lymphoblasts,
17Dr. Aditi M. Panditrao
18. Isolated Tissue Preparations for NK receptors
• Inhibition of Substance P-induced endothelium-
dependent relaxation of rabbit pulmonary artery,
previously contracted with 0.1 μM Noradrenaline
• Inhibition of Substance P or Substance P sulfone-
induced contractions of guinea pig ileum in the
presence of 3 μM Atropine and 3 μM Mepyramine
and Indomethacin
• Inhibition of Substance P-induced plasma
extravasation in guinea-pig bronchi
18Dr. Aditi M. Panditrao
22. Polymorphonuclear Leukocyte Chemotaxis
• Measures the chemotactic effects on the PMN
Leukocytes
• Migration rate –
number of PMNs in the lower well x 100
number of PMNs in the upper well
• Dependent on the concentration of the
chemoattractant (e.g., zymosan-activated
serum)
22Dr. Aditi M. Panditrao
23. PMN leukocytes aggregation induced by FMLP
• FMLP (formyl-L-methionyl-Lleucyl- L-phenylalanine)
• PMNs cell suspensions + test compounds or standard
(pentoxiphylline) are dissolved in GBSS
• Incubated for 10 min
• Serial dilutions of FMLP added
• Change in transmittance
23Dr. Aditi M. Panditrao
24. Constitutive and inducible cellular
arachidonic acid metabolism in vitro
• Formation of leukotriene B4 in human white blood
cells
• Formation of lipoxygenase products from 14C-
arachidonic acid in human polymorphonuclear
neutrophils (PMN)
• Formation of eicosanoids from 14C-arachidonic acid
in human platelets
• Stimulation of inducible prostaglandin pathway in
human PMNL
24Dr. Aditi M. Panditrao
25. COX-1 and COX-2 inhibition
• Inhibition studies with recombinant human COX-1
and COX-2
• HPLC assay for oxygenation of radiolabelled
arachidonic acid by COX-1
• Determination of the stoichiometry of inhibitor
binding
• Spectrophotometric assay of recombinant human
COX-2
• Whole-cell assays with transfected Chinese hamster
(CHO) cells expressing COX-1 and COX-2
25Dr. Aditi M. Panditrao
26. Induced release of cytokines
from human white blood cells
• Interleukin-1alpha, IL-1beta, IL-6, IL-8 and TNF-alpha
• Cell fraction from whole blood separated
• Exposed to LPS (Salmonella abortus equii) with or
without test drug
• Incubation
• Analysis of Cytokines
26Dr. Aditi M. Panditrao
27. Flow cytometric analysis of intracellular
cytokines
• Very powerful tool in which individual cells can be
simultaneously analyzed for several parameters:
• Size, granularity
• Expression of surface and intracellular markers
• Defined by fluorescent antibodies.
• Fluorescent anti-cytokine and anti-chemokine
monoclonal antibodies
27Dr. Aditi M. Panditrao
29. • Based on the symptoms observed during the
inflammation
• Acute/Transient phase: In this phase vasodilation
and increased capillary permeability are observed.
• Subacute/Delayed phase : Infiltration of
leucocytes and phagocytes in blood.
• Chronic inflammatory phase: granuloma
formation is observed in this phase
29Dr. Aditi M. Panditrao
30. • Acute and Subacute phase:
• Carrageenan induced paw oedema in rats
• Croton-oil induced ear edema
• Oxazolone induced ear edema
• UV-B erythema in guinea pigs
• Pleurisy in rats
• Vascular permeability
• Chronic phase:
• Cotton wool granuloma
• Glass rod granuloma
30Dr. Aditi M. Panditrao
31. Paw edema
• Most commonly employed method
• Various substances used:
• Carrageenin, Brewer’s yeast, Formaldehyde, Dextran, Egg
albumin, Kaolin, Zymosan
• Serotonin, Histamine, Substance P, Cobra Venom
• Subplantar region Left Hind Paw (0.05 ml-SC)
• Immediately after injection, again 3 and 6 h, and
eventually 24 h after challenge
31Dr. Aditi M. Panditrao
37. • Pure measure of the vasodilatory phase
• No accompanying exudation or edema
• Drawbacks:
• Variable response
• Accurate measurement of response
• Other sites/animals -
• Guinea pig ear
• Sprague Dawley rats
• Hairless mice (skin thickness)
37Dr. Aditi M. Panditrao
38. Pleurisy test
• Exudative Inflammation
• Histamine, Bradykinin, Prostaglandins,
Dextran, Antigens, Microbes, irritants like
turpentine and carrageenan etc.
• Fluid extravasation, leukocyte migration,
lysosomal enzyme activities etc.
38Dr. Aditi M. Panditrao
39. Vascular permeability
• Inhibitory activity of drugs against increased
vascular permeability
• Extravasation of dye into surrounding tissue
39Dr. Aditi M. Panditrao
40. Cotton wool granuloma
• Foreign body granuloma
• Sterile Cotton pellets embedded
subcutaneously
• Back, Axilla, Groin
• After 7 days – Pellet + Granuloma removed
• Weight of granulomatous tissue =
Dry weight of granuloma cotton – initial weight
of cotton pellet
40Dr. Aditi M. Panditrao
42. Glass Rod granuloma
• Foreign body granuloma
• Glass peices embedded subcutaneously
• Back, Axilla, Groin
• After 20-40 days – Granuloma + Connective
removed entirely
• Glass rod can be removed
• Tensile strength and wet weight of
granulomatous tissue can be measured
42Dr. Aditi M. Panditrao
44. Side Effects of Anti-
inflammatory Agents
44Dr. Aditi M. Panditrao
45. Ulcerogenic effect in rats
• Gastric irritation
• 48 hrs fasting
• 3, 5, 7 hrs after drug administration –
sacrificed
• Check gastric lining for hyperemia and ulcers
45Dr. Aditi M. Panditrao
46. Measurement of gastric mucosal damage
by intragastric inulin
• Inulin in the gastric wall – measure of gastric
damage
• Given intragastric
• 3H-inulin radioactivity is expressed as
disintegrations/min/ml of plasma or per gram
of tissue
46Dr. Aditi M. Panditrao
47. Determination of blood loss
• Microbleeding which is not detected by
observation of overt ulcers
• Feces, Stomach and intestinal contents
• Heme - spectrophotometry
47Dr. Aditi M. Panditrao
49. Adjuvant Arthritis in Rats
• Mimics human rheumatoid arthritis
• Freund’s Adjuvant – Mycobacterium
butyricum
• Delayed hypersensitivity response to
mycobacterial antigen
• Subplantar region of left hind paw
49Dr. Aditi M. Panditrao
50. • Day 1 Day 4 Day 7
Local Peak Systemic
reaction disease
• Parameters :
• Paw edema, weight, grip strength , deformity
measured
• Evaluation of chronically administered drug
against inflammation
• On Day 5 and 21: Plethysmometer
50Dr. Aditi M. Panditrao
52. • Day 5 : Primary Lesions
• Day 21 : Secondary Lesions
• Arthritic Index : Sum of scores of each animal
• Total % change =
percent inhibition of the injected paw on day 5 +
percent inhibition of the non-injected paw on day 21
+ percent change of the arthritic index
52Dr. Aditi M. Panditrao
53. Schultz-Dale Reaction
• Anti-anaphylactic activity
• Guinea pigs are sensitized against egg
albumin.
• Challenge after 3 weeks causes in isolated
organs release of mediators, e.g. histamine,
which induce contraction in isolated ileum
53Dr. Aditi M. Panditrao
55. References
• Mahesh G, Ramkanth S, Saleem MTS. Anti-inflammatory drugs from medicinal plants- A
comprehensive review. International Journal of Review in Life Sciences 2011; 1(1): 1-10.
• Paschapur MS, Patil MB, Kumar R, Patil SR. Evaluation of anti-inflammatory activity of ethanolic
extract of Borassus flabellifer L. male flowers (inforescences) in experimental animals. Journal of
Medicinal Plant Research 2009; 3(2): 49-54.
• Kumar V, Bhat ZA, Kumar D, Khan NA, Chashoo IA. Evaluation of anti-inflammatory potential of leaf
extracts of Skimmia anquetilia. Asian Pacific Journal of Tropical Biomedicine 2012; 627-630.
• Lavanya R, Maheshwari US, Harish G, Raj BJ, Kamali S, Hemamalani D, Varma BJ, Reddy UC.
Investigation of In-vitro anti- inflammatory, anti-platelet and antiarthritic activities in the leaves of
Anisomeles malabarica Linn. Research Journal of Pharmaceutical, Biological and Chemical Sciences
2010; 1(4): 745-752.
• Talhouk RS, El-Jouni W, Baalbaki R, Gali-Muhtasib H, Joanna K, Talhouk SN. Anti- inflammatory
bioactivities in water extract of Centaurea ainetensis. Journal of Medicinal Plants Reserch 2008;
2(2): 24-33.
• Ghani A. Medicinal plants of Bangladesh with chemical constituents and uses. 2nd ed. Dhaka:
Asiatic Society of Bangladesh; 2003, p. 345.
• Kumar S, Kumar D, Deshmukh RR, Lokhande PD, More SN, Rangari VD. Antidiabetic potential of
Phyllanthus reticulatus in alloxan-induced diabetic mice. Fitoterapia 2008; 79: 21-23.
• Winter CA, Risley EA, Nuss GW. Carrageenan induced oedema in hind paws of the rats as an assay
for anti-inflammatory drugs. Proceedings of the Society for Experimental Biology and Medicine;
111: 544-557.
• Okokon JE, Udoh AE, Frank SG, Amazu LU. Anti-inflammatory and analgesic activities of Melanthera
scandens. Asian Pacific Journal of Tropical Biomedicine 2012; 1: 144-148.
• Manning, D. C., Snyder, S. H., Kachur, J. F., Miller, R. J. & Field, M. (1982) Nature (London) 299, 256-
259.
55Dr. Aditi M. Panditrao
56. References
• Das S, Datta R, Nandy S. Phytochemical screening and evaluation of anti-
inflammatory activity of methanolic extract of Abroma augusta Linn. Asian
Pacific Journal of Tropical Disease 2012; S114-S117.
• Georgewill OA, Georgewill UO, Nwankwoala RNP. Anti-inflammatory
effects of Morninga oleifera Lam extract in rats. Asian Pacific Journal of
Tropical Medicine 2010; 3: 133-135.
• Georgewill OA, Georgewill UO. Evaluation of the anti-inflammatory activity
of extract of Vernonia amygdalina. Asian Pacific Journal of Tropical
Medicine 2010; 3: 150-51.
• Rosa MD, Giround JP, Willoghby DA. Studies of the acute inflammatory
response induced in rats in different sites by Carrageenan and turpentine.
Journal of Pathology 1971; 104: 15-29.
• Sheetal S. Chaudhari, Sanjay R. Chaudhari, Machindra J. Chavan. Analgesic,
anti-inflammatory and anti-arthritic activity of Cassia uniflora Mill. Asian
Pacific Journal Tropical Biomedicine 2012; 2: 181-86.
• Khan H, Khan MA, Muhammad N, Ashraf N, Gul F, Tariq SA. Anti-
inflammatory and antioxidant activity of Joshanda partially mediated
through inhibition of lipoxygenase. Phytopharmacology 2012; 3: 19-28
56Dr. Aditi M. Panditrao
