Impact of organic solvents on cytochrome P450 probe reactions: filling the gap with (S)-Warfarin and midazolam hydroxylation.
Enviado por Vanessa Gonzalez-Perez el
Título | Impact of organic solvents on cytochrome P450 probe reactions: filling the gap with (S)-Warfarin and midazolam hydroxylation. |
Publication Type | Journal Article |
Year of Publication | 2012 |
Autores | González-Pérez, V, Connolly, EA, Bridges, AS, Wienkers, LC, Paine, MF |
Journal | Drug Metab Dispos |
Volume | 40 |
Issue | 11 |
Pagination | 2136-42 |
Date Published | 2012 Nov |
ISSN | 1521-009X |
Palabras clave | Aryl Hydrocarbon Hydroxylases, Cytochrome P-450 CYP3A, Cytochrome P-450 Enzyme System, Humans, Hydroxylation, Intestine, Small, Liver, Microsomes, Microsomes, Liver, Midazolam, Solvents, Warfarin |
Abstract | (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. |
DOI | 10.1124/dmd.112.047134 |
Alternate Journal | Drug Metab. Dispos. |
PubMed ID | 22896727 |
PubMed Central ID | PMC3477202 |
Grant List | R01-GM077482 / GM / NIGMS NIH HHS / United States |