An in-vitro screening assay for the detection of inhibitors of proinflammatory cytokine synthesis: a useful tool for the development of new antiarthritic and disease modifying drugs.
OBJECTIVE
This work targets the development of a new tool to help develop new anticytokine drugs that prevent or reduce the progression of arthritic diseases. The specific aim of our study was to establish a fast and reliable in vitro screening assay of cytokine synthesis inhibitors (TNFalpha, IL-1beta) which shows better correlation with enzyme assays than previously reported in vitro assays. The test system should be able to detect p38-MAP kinase inhibitors.


METHODS
Human peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation from human EDTA-potassium whole blood. Cells were adjusted at 1 x 10(6) cells/ml. PBMCs were stimulated with lipopolysaccharide (LPS; E. coli serotype 026:B6: 1 microg/ml) in the presence of test compound (10(-5)-10(-8)M) for 4h at 37 degrees C in a 5% CO(2)-incubator. Induced TNFalpha and IL-1beta protein were measured by ELISA.


RESULTS
The following are representative examples of inhibitors which effect cytokine synthesis. Corticoid Dexamethasone inhibits IL-1beta and TNFalpha synthesis at IC(50) of 38 nM and 25 nM, respectively. ERK1/ERK2 inhibitor U0126 effects cytokine synthesis at IC(50) of 0.34 microM for IL-1beta production and 0.26 microM for TNFalpha synthesis.p38-MAP kinase inhibitor SB 203580 inhibits IL-1beta- and TNF-alpha-synthesis (IC(50)sof 0.052 microM and 0.46 microM) in the same degree as p38-MAP kinase activity (IC(50): 0.34 microM). Same results could be shown for SB 210313, which had same efficacy on IL-1beta and TNFalpha biosynthesis (IC(50)'s: 1.88 microM and 1.01 microM) and on p38-MAP kinase (IC(50): 6.85 microM). Also for SB 202190 this correlation in inhibition of IL-1beta and TNFalpha synthesis (IC(50)'s: 0.055 microM and 1.01 microM) and p38-MAP kinase inhibition (IC(50): 0.088 microM) could be shown.


CONCLUSIONS
This study shows the screening assay using PBMCs stimulated with LPS for IL-1beta and TNFalpha synthesis is a reliable test system for the quantification of the effectiveness of new drugs modulating IL-1beta and TNFalpha synthesis which is mainly mediated by p38-MAP Kinase. These assay allows fast detection of IL-1beta and TNFalpha synthesis inhibitors with different modes of action, including p38-MAP kinase inhibitors. The results obtained with our in-vitro screening assay show good correlation with results from enzyme assays.
Laufer
S
S
Institute of Pharmacy, Department of Pharmaceutical and Medicinal Chemistry, Eberhard-Karls-University Tübingen, A. d. Morgenstelle 8, D-72076 Tübingen, Germany. Stefan.Laufer@uni-tuebingen.de
Greim
C
C
Bertsche
T
T
D015153:Blotting, Western; D016207:Cytokines / Q000037:antagonists & inhibitors* / Q000096:biosynthesis; D005260:Female; D006801:Humans; D007375:Interleukin-1 / Q000096:biosynthesis; D008070:Lipopolysaccharides / Q000494:pharmacology; D008297:Male; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009000:Monocytes / Q000187:drug effects; D012333:RNA, Messenger / Q000032:analysis; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis; D048051:p38 Mitogen-Activated Protein Kinases
D016207:Cytokines; D007375:Interleukin-1; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases