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
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.


METHODS
Bovine aortic endothelial cells (BAECs).


METHODS
Unstimulated bovine aortic coronary endothelial cells (BAECs) were used as a source of COX-1 and BAECs pretreated with ASA (100 microM) 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 microM) and COX-2 (IC50: 0.03/0.6 microM). Indomethacin inhibited COX-1 more potently than COX-2 (IC50: 0.008/0.04 microM). Aceclofenac inhibited COX-2 more potently than COX-1 (IC50: 3.0/7.3 microM). DFU and Cl-SC57666 [16] inhibited COX-2 (IC50: 0.04/0.001 microM) highly selectively but did not inhibit COX-1 (IC50: >100 microM).


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.
Dannhardt
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G
Institute of Pharmacy, University of Mainz, Germany. dannhrdt@mail.uni-mainz.de
Ulbrich
H
H
D000818:Animals; D001013:Aorta, Thoracic / Q000166:cytology / Q000201:enzymology; D015153:Blotting, Western; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; D052246:Cyclooxygenase 2 Inhibitors; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology*; D004353:Drug Evaluation, Preclinical; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology*; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007700:Kinetics; D016859:Lipoxygenase Inhibitors; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D052246:Cyclooxygenase 2 Inhibitors; D016861:Cyclooxygenase Inhibitors; D007527:Isoenzymes; D016859:Lipoxygenase Inhibitors; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; D011451:Prostaglandin-Endoperoxide Synthases; D013755:Tetradecanoylphorbol Acetate