PT - JOURNAL ARTICLE AU - Vanessa González-Pérez AU - Elizabeth A. Connolly AU - Arlene S. Bridges AU - Larry C. Wienkers AU - Mary F. Paine TI - Impact of Organic Solvents on Cytochrome P450 Probe Reactions: Filling the Gap with (<em>S</em>)-Warfarin and Midazolam Hydroxylation AID - 10.1124/dmd.112.047134 DP - 2012 Nov 01 TA - Drug Metabolism and Disposition PG - 2136--2142 VI - 40 IP - 11 4099 - http://dmd.aspetjournals.org/content/40/11/2136.short 4100 - http://dmd.aspetjournals.org/content/40/11/2136.full SO - Drug Metab Dispos2012 Nov 01; 40 AB - (S)-Warfarin 7-hydroxylation and midazolam 1′-hydroxylation are among the preferred probe substrate reactions for CYP2C9 and CYP3A4/5, respectively. The impact of solvents on enzyme activity, kinetic parameters, and predicted in vivo hepatic clearance (ClH) associated with each reaction has not been evaluated. The effects of increasing concentrations [0.1–2% (v/v)] of six organic solvents (acetonitrile, methanol, ethanol, dimethyl sulfoxide, acetone, isopropanol) were first tested on each reaction using human liver microsomes (HLMs), human intestinal microsomes (midazolam 1′-hydroxylation only), and recombinant enzymes. Across enzyme sources, relative to water, acetonitrile and methanol had the least inhibitory effect on (S)-warfarin 7-hydroxylation (0–58 and 9–96%, respectively); acetonitrile, methanol, and ethanol had the least inhibitory effect on midazolam 1′-hydroxylation (0–29, 0–22, and 0–20%, respectively). Using HLMs, both acetonitrile and methanol (0.1–2%) decreased the Vmax (32–60 and 24–65%, respectively) whereas methanol (2%) increased the Km (100%) of (S)-warfarin-hydroxylation. (S)-Warfarin ClH was underpredicted by 21–65% (acetonitrile) and 13–84% (methanol). Acetonitrile, methanol, and ethanol had minimal to modest impact on both the kinetics of midazolam 1′-hydroxylation (10–24%) and predicted midazolam ClH (2–20%). In conclusion, either acetonitrile or methanol at ≤0.1% is recommended as the primary organic solvent for the (S)-warfarin 7-hydroxylation reaction; acetonitrile is preferred if higher solvent concentrations are required. Acetonitrile, methanol, and ethanol at ≤2% are recommended as primary organic solvents for the midazolam 1′-hydroxylation reaction. This information should facilitate optimization of experimental conditions and improve the interpretation and accuracy of in vitro-in vivo predictions involving these two preferred cytochrome P450 probe substrate reactions.