Development of an in-vitro test system for the evaluation of cyclooxygenase-2 inhibitors

Abstract.

Objective and Design: The aim of our study was to establish an in-vitro test system, capable of fast and efficient screening of Cyclooxygenase-2 (COX-2) inhibitors.¶Materials: Mononuclear cells were isolated out of human whole blood, in a one-step centrifugation procedure.¶Treatment and methods: The time- and concentration-dependent induction of COX-2 expression in the blood monocytes (1 × 10⁶ cells/ml) was evalulated by a kinetic profile. The optimal test conditions were fixed at an LPS concentration of 10 μg/ml and a 5 hour incubation time. The test compounds (10⁻⁵ to 10⁻⁸ mol/l) were set at t = 0 into the assay and were co-incubated for the whole period of COX-2 expression (5 hr).¶Results: The following are representative examples of inhibitors with different distinct selectivity for COX-1/2. Indomethacin as a COX-1 selective compound inhibited PGHS-1 (IC₅₀: 0,002 μM) 200 times stronger than PGHS-2 (IC₅₀: 0,43 μM). Diclofenac had an almost equipotent efficacy on PGHS-1 (IC₅₀: 0,05 μM) and PGHS-2 (IC₅₀: 0,03 μM). NS-398 inhibited highly selective COX-2 (IC₅₀ PGHS-1: 10,75 μM vs IC₅₀ PGHS-2: 0,16 μM).¶Conclusions: The model reached the set targets with regard to the differentiation of COX-2 selective compounds, the reproducibility of results and practicability of the assay. In contrast to previous propounded theories, we could demonstrate, that mononuclear cells are not unusually sensitive to NSAIDs and apparently possess no further COX isoforms.