TY - JOUR T1 - Defining the Selectivity of Chemical Inhibitors Used for Cytochrome P450 Reaction Phenotyping: Overcoming Selectivity Limitations with a Six-Parameter Inhibition Curve-Fitting Approach JF - Drug Metabolism and Disposition JO - Drug Metab Dispos SP - 1259 LP - 1271 DO - 10.1124/dmd.122.000884 VL - 50 IS - 9 AU - Angela C. Doran AU - Woodrow Burchett AU - Connor Landers AU - Gabrielle M. Gualtieri AU - Amanda Balesano AU - Heather Eng AU - Alyssa L Dantonio AU - Theunis C. Goosen AU - R. Scott Obach Y1 - 2022/09/01 UR - http://dmd.aspetjournals.org/content/50/9/1259.abstract N2 - The utility of chemical inhibitors in cytochrome P450 (CYP) reaction phenotyping is highly dependent on their selectivity and potency for their target CYP isoforms. In the present study, 17 inhibitors of CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4/5 commonly used in reaction phenotyping were evaluated for their cross-enzyme selectivity in pooled human liver microsomes. The data were evaluated using a statistical desirability analysis to identify (1) inhibitors of superior selectivity for reaction phenotyping and (2) optimal concentrations for each. Among the inhibitors evaluated, α-naphthoflavone, furafylline, sulfaphenazole, tienilic acid, N-benzylnirvanol, and quinidine were most selective, such that their respective target enzymes were inhibited by ∼95% without inhibiting any other CYP enzyme by more than 10%. Other commonly employed inhibitors, such as ketoconazole and montelukast, among others, were of insufficient selectivity to yield a concentration that could adequately inhibit their target enzymes without affecting other CYP enzymes. To overcome these shortcomings, an experimental design was developed wherein dose response data from a densely sampled multi-concentration inhibition curve are analyzed by a six-parameter inhibition curve function, allowing accounting of the inhibition of off-target CYP isoforms inhibition and more reliable determination of maximum targeted enzyme inhibition. The approach was exemplified using rosiglitazone N-demethylation, catalyzed by both CYP2C8 and 3A4, and was able to discern the off-target inhibition by ketoconazole and montelukast from the inhibition of the targeted enzyme. This methodology yields more accurate estimates of CYP contributions in reaction phenotyping.SIGNIFICANCE STATEMENT Isoform-selective chemical inhibitors are important tools for identifying and quantifying enzyme contributions as part of a cytochrome P450 (CYP) reaction phenotyping assessment for projecting drug-drug interactions. However, currently employed practices fail to adequately compensate for shortcomings in inhibitor selectivity and the resulting confounding impact on estimates of the CYP enzyme contribution to drug clearance. In this report, we describe a detailed half maximal inhibitory concentration (IC50) study design with 6-parameter modeling approach that yields more accurate estimates of enzyme contribution. ER -