Impact of organic solvents on cytochrome P450 probe reactions: filling the gap with (S)-Warfarin and midazolam hydroxylation.
(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 (Cl(H)) 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 V(max) (32-60 and 24-65%, respectively) whereas methanol (2%) increased the K(m) (100%) of (S)-warfarin-hydroxylation. (S)-Warfarin Cl(H) 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 Cl(H) (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.
Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
Vanessa
V
González-Pérez
Elizabeth A
EA
Connolly
Arlene S
AS
Bridges
Larry C
LC
Wienkers
Mary F
MF
Paine
D001189:Aryl Hydrocarbon Hydroxylases / Q000378:metabolism; D065729:Cytochrome P-450 CYP2C9; D051544:Cytochrome P-450 CYP3A / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D006801:Humans; D006900:Hydroxylation / Q000187:drug effects; D007421:Intestine, Small / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D008099:Liver / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D008861:Microsomes / Q000187:drug effects / Q000378:metabolism; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D008874:Midazolam / Q000378:metabolism / Q000493:pharmacokinetics* / Q000494:pharmacology; D012997:Solvents / Q000494:pharmacology*; D014859:Warfarin / Q000378:metabolism / Q000493:pharmacokinetics* / Q000494:pharmacology
D012997:Solvents; D014859:Warfarin; D003577:Cytochrome P-450 Enzyme System; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; C510164:CYP3A5 protein, human; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D008874:Midazolam