Stimulation of tolbutamide hydroxylation by acetone and acetonitrile in human liver microsomes and in a cytochrome P-450 2C9-reconstituted system.
Organic solvents are often used to solubilize lipophilic new chemical entities before their addition to in vitro test systems such as microsomal stability or cytochrome P-450 (CYP) inhibition. However, the effect of these organic solvents on the test systems is not usually characterized. This study was initiated to evaluate the effect of acetonitrile and acetone, in addition to other organic solvents, on the tolbutamide hydroxylation activity of CYP2C9 in both human liver microsomes and a CYP2C9-reconstituted system. Both acetonitrile and acetone significantly stimulated the NADPH-dependent tolbutamide hydroxylation by nearly 2- to 3-fold in human liver microsomes and CYP2C9-reconstituted system when incubated at 2 and 4% final solvent concentrations. When cumene hydroperoxide was used instead of NADPH, both acetone 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 lengths and various functional groups, including hydroxyl, ketone, and aldehyde. Unlike acetone, two other ketone-containing solvents, methyl ethyl ketone (2-butanone) and diethyl ketone (3-pentanone) failed to significantly enhance tolbutamide hydroxylation. Other solvents tested, including methanol, ethanol, propanol, 1-butanol, 2-butanol, 1-pentanol, 2-pentanol, acetaldehyde, and dimethyl sulfoxide significantly inhibited NADPH-dependent tolbutamide hydroxylation. Overall, the stimulatory effect of both acetonitrile and acetone on tolbutamide hydroxylation was found to be primarily due to a consistent increase in V(max), whereas K(m) was unchanged in both human liver microsomes and the reconstituted CYP2C9 system. These data suggest that acetone and acetonitrile stimulate NADPH-mediated tolbutamide hydroxylation via the CYP reductase and not by modifying the affinity of tolbutamide for the CYP2C9 enzyme.
Palamanda
J
J
Department of Drug Metabolism and Pharmacokinetics, Schering-Plough Research Institute, Kenilworth, NJ 07033, USA.
Feng
W W
WW
Lin
C C
CC
Nomeir
A A
AA
D000096:Acetone / Q000494:pharmacology*; D000097:Acetonitriles / Q000494:pharmacology*; D001189:Aryl Hydrocarbon Hydroxylases; D002851:Chromatography, High Pressure Liquid; D065729:Cytochrome P-450 CYP2C9; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D005260:Female; D006801:Humans; D006900:Hydroxylation / Q000187:drug effects; D007004:Hypoglycemic Agents / Q000378:metabolism*; D008297:Male; D008862:Microsomes, Liver / Q000187:drug effects / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D012997:Solvents / Q000494:pharmacology*; D039181:Steroid 16-alpha-Hydroxylase; D013250:Steroid Hydroxylases / Q000378:metabolism*; D014044:Tolbutamide / Q000378:metabolism*
D000097:Acetonitriles; D007004:Hypoglycemic Agents; D012997:Solvents; D000096:Acetone; D009249:NADP; D003577:Cytochrome P-450 Enzyme System; D014044:Tolbutamide; D013250:Steroid Hydroxylases; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; D039181:Steroid 16-alpha-Hydroxylase; C032159:acetonitrile