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


METHODS
Mononuclear cells were isolated out of human whole blood, in a one-step centrifugation procedure.


METHODS
The time- and concentration-dependent induction of COX-2 expression in the blood monocytes (1 x 10(6) cells/ml) was evaluated by a kinetic profile. The optimal test conditions were fixed at an LPS concentration of 10 micrograms/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 microM) 200 times stronger than PGHS-2 (IC50: 0.43 microM). Diclofenac had an almost equipotent efficacy on PGHS-1 (IC50: 0.05 microM) and PGHS-2 (IC50: 0.03 microM). NS-398 inhibited highly selective COX-2 (IC50 PGHS-1: 10.75 microM vs IC50 PGHS-2: 0.16 microM).


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.
Laufer
S
S
Department Drug Research, Merckle GmbH, Blaubeuren, Germany.
Zechmeister
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P
Klein
T
T
D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology; D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology; D015153:Blotting, Western; D002469:Cell Separation; D051546:Cyclooxygenase 2; D052246:Cyclooxygenase 2 Inhibitors; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology*; D015232:Dinoprostone / Q000378:metabolism; D005260:Female; D006801:Humans; D066298:In Vitro Techniques; D007213:Indomethacin / Q000494:pharmacology; D007527:Isoenzymes / Q000096:biosynthesis / Q000378:metabolism*; D007700:Kinetics; D008070:Lipopolysaccharides / Q000494:pharmacology; D008297:Male; D008565:Membrane Proteins; D009000:Monocytes / Q000187:drug effects / Q000201:enzymology; D011451:Prostaglandin-Endoperoxide Synthases / Q000096:biosynthesis / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D000894:Anti-Inflammatory Agents, Non-Steroidal; D052246:Cyclooxygenase 2 Inhibitors; D016861:Cyclooxygenase Inhibitors; D007527:Isoenzymes; D008070:Lipopolysaccharides; D008565:Membrane Proteins; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases; D015232:Dinoprostone; D007213:Indomethacin