In-vitro test system for the evaluation of cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) inhibitors based on a single HPLC run with UV detection using bovine aortic coronary endothelial cells (BAECs)

Objective and Design: The aim of this study was to develop a new, whole-cell test system which is easy to handle and requires a standard equipment for the parallel screening of COX-1 and COX-2 inhibitors. Materials: Bovine aortic endothelial cells (BAECs). Treatment and methods: Unstimulated bovine aortic coronary endothelial cells (BAECs) were used as a source of COX-1 and BAECs pretreated with ASA (100 muM) and activated with phorbol myristate acetate (PMA) were used as a source of COX-2. The time- and concentration-dependent induction of COX-2 expression in the BAECs was evaluated by a kinetic profile (HPLC analysis) and detected by Western-Blot analysis using polyclonal antibodies against COX-1 and COX-2. Results: In BAECs, diclofenac and meloxicam showed balanced inhibition of COX-1 (IC50: 0.01/0.4 muM) and COX-2 (IC50: 0.03/0.6 muM). Indomethacin inhibited COX-1 more potently than COX-2 (IC50: 0.008/0.04 muM). Aceclofenac inhibited COX-2 more potently than COX-1 (IC50: 3.0/7.3 muM). DFU and CI-SC57666 [16] inhibited COX-2 (IC50: 0.04/0.001 muM) highly selectively but did not inhibit COX-1 (IC50: > 100 muM). Conclusions: In summary an assay has been developed, for the determination of IC50-values for inhibitors of COX-1/2 on cells of the same origin, in line with values in the literature. Moreover, new insights have been gained into the relationship of COX-1/2 and lipoxygenase pathways in BAECs by detecting 15- and 12-HETE: Inhibition of COX-1 by the NSAIDs mostly resulted in an enhancement of 15-HETE and 12-HETE release. In contrast inhibition of COX-2 decreased 15-HETE release.