Validated method for rapid inhibition screening of six cytochrome P450 enzymes by liquid chromatography-tandem mass spectrometry.
In vitro drug interaction data can be used in guiding clinical interaction studies, or, the design of new candidates. To make such a claim, it must be assured that the in vitro data obtained is confident. To meet this need, a rapid liquid chromatography-tandem mass spectrometry (LC/MS/MS) method has been validated and employed for routine screening of new chemical entities for inhibition of six major human cytochrome P450 (CYP) isoforms using cDNA-expressed CYPs. Probe substrates were used near the Michaelis-Menten constant (K(m)) concentration values for CYP1A2 (phenacetin), CYP2C9 (tolbutamide), CYP2C19 (S-mephenytoin), CYP2D6 (dextromethorphan) and CYP3A4 (midazolam and dextromethorphan). The major metabolites of CYP-specific probe substrates were quantified. The LC/MS/MS method was found to be accurate and precise within the linear range of 1.0-2000 ng/ml for each analyte in enzyme incubation mixture. The lower limit of quantification (LLOQ) was 1.0 ng/ml. The limit of detection (LOD) for the tested analytes was 0.48 ng/ml or better based on signal-to-noise ratio >3. The inhibition potential of the six CYP isoforms has been evaluated using their known selective inhibitors. The 50% inhibitory concentrations (IC(50) values) measured by this method demonstrated high precision and are consistent with the literature values.
Li
Xiaoyan
X
Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 646 Songtao Road, Shanghai 201203, China.
Chen
Xiaoyan
X
Li
Qiang
Q
Wang
Linling
L
Zhong
Dafang
D
D002138:Calibration; D002853:Chromatography, Liquid / Q000379:methods*; D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*
D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors