title: Measurement of cyclooxygenase inhibition using liquid chromatography-tandem mass spectrometry.
abstract: 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 IC(50) 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 IC(50) determinations.
authors.affiliation: Department of Medicinal Chemistry and Pharmacognosy, University of Illinois College of Pharmacy, 833 S. Wood Street, Chicago, IL 60612, USA.
authors.forename: Hongmei
authors.initials: H
authors.lastname: Cao
authors.forename: Rui
authors.initials: R
authors.lastname: Yu
authors.forename: Yi
authors.initials: Y
authors.lastname: Tao
authors.forename: Dejan
authors.initials: D
authors.lastname: Nikolic
authors.forename: Richard B
authors.initials: RB
authors.lastname: van Breemen
mesh_terms: D000818:Animals; D002623:Chemistry Techniques, Analytical; D002626:Chemistry, Pharmaceutical / Q000379:methods; D002853:Chromatography, Liquid / Q000379:methods*; D051545:Cyclooxygenase 1 / Q000378:metabolism; D051546:Cyclooxygenase 2 / Q000032:analysis* / Q000097:blood; D015232:Dinoprostone / Q000032:analysis*; D006801:Humans; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008024:Ligands; D015203:Reproducibility of Results; D012756:Sheep; D013045:Species Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D008024:Ligands; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; D015232:Dinoprostone
references.citation: Bioorg Med Chem. 2009 Feb 1;17(3):1044-54
references.pmid: 18487053
references.citation: Biochem Biophys Res Commun. 1989 Dec 15;165(2):888-94
references.pmid: 2512924
references.citation: Biochem Pharmacol. 1996 Dec 13;52(11):1777-85
references.pmid: 8986141
references.citation: Prostaglandins. 1987 Aug;34(2):225-40
references.pmid: 3118417
references.citation: Biochem J. 1999 May 1;339 ( Pt 3):607-14
references.pmid: 10215599
references.citation: Nat Protoc. 2006;1(4):1915-21
references.pmid: 17487176
references.citation: Inflamm Res. 2001 May;50(5):262-9
references.pmid: 11409489
references.citation: Genome Biol. 2004;5(9):241
references.pmid: 15345041
references.citation: J Med Chem. 1997 Apr 25;40(9):1347-65
references.pmid: 9135032
references.citation: Prostaglandins. 1974 Jul 25;7(2):123-9
references.pmid: 4850301
references.citation: J Biol Chem. 1999 Jul 16;274(29):20301-6
references.pmid: 10400651
references.citation: Anal Biochem. 2008 Jan 1;372(1):41-51
references.pmid: 17976507
references.citation: Trends Pharmacol Sci. 1999 Nov;20(11):465-9
references.pmid: 10542447
references.citation: J Biol Chem. 1997 May 9;272(19):12393-8
references.pmid: 9139685
references.citation: J Nat Prod. 1998 Jan;61(1):2-7
references.pmid: 9461646
references.citation: Rheumatology (Oxford). 2005 Jul;44(7):846-59
references.pmid: 15855183
references.citation: Proc Natl Acad Sci U S A. 1994 Nov 8;91(23):11202-6
references.pmid: 7972034
references.citation: J Biol Chem. 2007 Jun 1;282(22):16379-90
references.pmid: 17434872