Stimulation of tolbutamide hydroxylation by acetone and acetonitrile in human liver microsomes and in a cytochrome P-4502C9-reconstituted system

Organic solvents are often used to solubilize lipophilic new chemical entit ies before their addition to in vitro test systems such as microsomal stabi lity or cytochrome P-450 (CYP) inhibition. However, the effect of these org anic solvents on the test systems is not usually characterized. This study was initiated to evaluate the effect of acetonitrile and acetone, in additi on to other organic solvents, on the tolbutamide hydroxylation activity of CYP2C9 in both human liver microsomes and a CYP2C9-reconstituted system. Bo th acetonitrile and acetone significantly stimulated the NADPH-dependent to lbutamide hydroxylation by nearly 2- to 3-fold in human liver microsomes an d CYP2C9-reconstituted system when incubated at 2 and 4% final solvent conc entrations. When cumene hydroperoxide was used instead of NADPH, both aceto ne and acetonitrile significantly inhibited tolbutamide hydroxylation. This NADPH-dependent stimulatory effect was further evaluated by examining the effect of a series of other organic solvents with different carbon chain le ngths and various functional groups, including hydroxyl, ketone, and aldehy de. Unlike acetone, two other ketone-containing solvents, methyl ethyl keto ne (2-butanone) and diethyl ketone (3-pentanone) failed to significantly en hance tolbutamide hydroxylation. Other solvents tested, including methanol, ethanol, propanol, 1-butanol, 2-butanol, 1-pentanol, 2-pentanol, acetaldeh yde, and dimethyl sulfoxide significantly inhibited NADPH-dependent tolbuta mide hydroxylation. Overall, the stimulatory effect of both acetonitrile an d acetone on tolbutamide hydroxylation was found to be primarily due to a c onsistent increase in V-max, whereas K-m was unchanged in both human liver microsomes and the reconstituted CYP2C9 system. These data suggest that ace tone and acetonitrile stimulate NADPH-mediated tolbutamide hydroxylation vi a the CYP reductase and not by modifying the affinity of tolbutamide for th e CYP2C9 enzyme.