Measurement of cyclooxygenase inhibition using liquid chromatography-tandem mass spectrometry

Because cyclooxygenases (COX) convert arachidonic acid into pro-inflammatory cyclic endoperoxides, inhibition of these enzymes and especially the inducible COX-2 form is an important therapeutic approach to manage inflammatory diseases and possibly prevent cancer. Due to side effects of existing non-selective and COX-2 selective non-steroidal anti-inflammatory agents, the discovery of new COX inhibitors continues to be an area of active investigation. Since existing assays are slow or lack specificity, a liquid chromatography-tandem mass spectrometry (LC-MS-MS) based COX inhibition assay was developed and validated for the rapid and accurate quantitative analysis of the COX product prostaglandin E-2. The assay was validated using four COX inhibitors, celecoxib, indomethacin, resveratrol, and diclofenac that exhibit different selectivities towards COX-1 and COX-2. The IC50 values of celecoxib and resveratrol for ovine and human COX-2 were compared, and the K-m values were determined. Since considerable inter-species variation was observed, human COX-2 should be used for the discovery of COX inhibitors intended for human use. This sensitive and accurate LC-MS-MS based assay is suitable for the rapid screening of ligands for COX-1 and COX-2 inhibition and for IC50 determinations. (c) 2010 Elsevier B.V. All rights reserved.