title: Rapid and quantitative measurement of metabolic stability without chromatography or mass spectrometry.
abstract: Metabolic stability measurements are a critical component of preclinical drug development. Available measurement strategies rely on chromatography and mass spectrometry, which are expensive and labor intensive. We have developed a general method to determine the metabolic stability of virtually any compound by quantifying cofactors in the mechanism of cytochrome P450 enzymes using fluorescence intensity measurements. While many previous studies have shown that simple measurements of cofactor depletion do not correlate with substrate conversion (i.e., metabolic stability) in P450 systems, the present work employs a reaction engineering approach to simplify the overall rate equation, thus allowing the accurate and quantitative determination of substrate depletion from simultaneous measurements of NADPH and oxygen depletion. This method combines the accuracy and generality of chromatography with the ease, throughput, and real-time capabilities of fluorescence.
authors.affiliation: Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, New York 12180, USA.
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Traylor
authors.forename: Jessica D
authors.initials: JD
authors.lastname: Ryan
authors.forename: Eric S
authors.initials: ES
authors.lastname: Arnon
authors.forename: Jonathan S
authors.initials: JS
authors.lastname: Dordick
authors.forename: Douglas S
authors.initials: DS
authors.lastname: Clark
mesh_terms: D000818:Animals; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004353:Drug Evaluation, Preclinical / Q000191:economics / Q000379:methods*; D005453:Fluorescence; D009249:NADP / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D004364:Pharmaceutical Preparations / Q000378:metabolism*; D013997:Time Factors
chemical_list: D004364:Pharmaceutical Preparations; D009249:NADP; D003577:Cytochrome P-450 Enzyme System; D010100:Oxygen