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

Abstract

AbstractObjective 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 (TNFα, IL-1β) 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.Material and methods Human peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation from human EDTA-potassium whole blood. Cells were adjusted at 1×106 cells/ml. PBMCs were stimulated with lipopolysaccharide (LPS; E. coli serotype 026:B6: 1μg/ml) in the presence of test compound (10−5–10−8M) for 4h at 37°C in a 5% CO2-incubator. Induced TNFα and IL-1β protein were measured by ELISA.Results The following are representative examples of inhibitors which effect cytokine synthesis. Corticoid Dexamethasone inhibits IL-1β and TNFα synthesis at IC50 of 38nM and 25nM, respectively. ERK1/ERK2 inhibitor U0126 effects cytokine synthesis at IC50 of 0.34μM for IL-1β production and 0.26μM for TNFα synthesis.p38-MAP kinase inhibitor SB 203580 inhibits IL-1β- and TNF-α-synthesis (IC50sof 0.052μM and 0.46μM) in the same degree as p38-MAP kinase activity (IC50: 0.34μM). Same results could be shown for SB 210313, which had same efficacy on IL-1β and TNFα biosynthesis (IC50's: 1.88μM and 1.01μM) and on p38-MAP kinase (IC50: 6.85μM). Also for SB 202190 this correlation in inhibition of IL-1β and TNFα synthesis (IC50's: 0.055μM and 1.01μM) and p38-MAP kinase inhibition (IC50: 0.088μM) could be shown.Conclusion This study shows the screening assay using PBMCs stimulated with LPS for IL-1β and TNFα synthesis is a reliable test system for the quantification of the effectiveness of new drugs modulating IL-1β and TNFα synthesis which is mainly mediated by p38-MAP Kinase. These assay allows fast detection of IL-1β and TNFα 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. Copyright 2002 OsteoArthritis Research Society International. Published by Elsevier Science Ltd. All rights reserved