RT Journal Article SR Electronic T1 Impact of Organic Solvents on Cytochrome P450 Probe Reactions: Filling the Gap with (S)-Warfarin and Midazolam Hydroxylation JF Drug Metabolism and Disposition JO Drug Metab Dispos FD American Society for Pharmacology and Experimental Therapeutics SP 2136 OP 2142 DO 10.1124/dmd.112.047134 VO 40 IS 11 A1 Vanessa González-Pérez A1 Elizabeth A. Connolly A1 Arlene S. Bridges A1 Larry C. Wienkers A1 Mary F. Paine YR 2012 UL http://dmd.aspetjournals.org/content/40/11/2136.abstract 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.