Substrate-dependent effect of acetonitrile on human liver microsomal cytochrome P450 2C9 (CYP2C9) activity.
Acetonitrile is an organic solvent commonly used to increase the solubility of lipophilic substrates for in vitro studies. In this study, we examined its effect on four reactions (diclofenac hydroxylation, tolbutamide methyl hydroxylation, phenytoin hydroxylation, and celecoxib methyl hydroxylation) catalyzed by human liver microsomes and by the recombinant CYP2C9. In both cases, the effect of acetonitrile on activity was found to be substrate-dependent. Namely, it increased diclofenac 4'-hydroxylase and tolbutamide methyl hydroxylase activities, but decreased celecoxib methyl hydroxylase activity in a concentration-dependent manner. By comparison, hydroxylation of phenytoin was resistant to its effect. The presence of acetonitrile (3%, v/v) gave rise to a lower K(m) and a higher V(max) for diclofenac hydroxylase in both liver microsomes and recombinant CYP2C9 preparations (87 and 52% increase in V(max)/K(m) ratio, respectively). On the other hand, the inhibitory effect of the solvent (1%, v/v) toward celecoxib hydroxylase was characterized by a decrease in V(max) (human liver microsomes) or a change in both K(m) and V(max) (rCYP2C9), leading to 25 and 46% decrease in V(max)/K(m) for both systems. The results of this study underscore the need for careful evaluation of solvent effects before initiation of inhibition or cytochrome P450 reaction phenotyping studies.
Tang
C
C
Department of Drug Metabolism, Merck Research Laboratories, West Point, Pennsylvania 19486-0004, USA. cuyue_tang@merck.com
Shou
M
M
Rodrigues
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D000097:Acetonitriles / Q000494:pharmacology*; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000493:pharmacokinetics; D019138:Antibodies, Blocking / Q000494:pharmacology; D000927:Anticonvulsants / Q000493:pharmacokinetics; D001189:Aryl Hydrocarbon Hydroxylases; D000068579:Celecoxib; D065729:Cytochrome P-450 CYP2C9; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004008:Diclofenac / Q000493:pharmacokinetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007004:Hypoglycemic Agents / Q000493:pharmacokinetics; D066298:In Vitro Techniques; D008862:Microsomes, Liver / Q000187:drug effects* / Q000201:enzymology*; D010641:Phenotype; D010672:Phenytoin / Q000493:pharmacokinetics; D011720:Pyrazoles; D039181:Steroid 16-alpha-Hydroxylase; D013250:Steroid Hydroxylases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013449:Sulfonamides / Q000493:pharmacokinetics; D014044:Tolbutamide / Q000493:pharmacokinetics
D000097:Acetonitriles; D000894:Anti-Inflammatory Agents, Non-Steroidal; D019138:Antibodies, Blocking; D000927:Anticonvulsants; D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D007004:Hypoglycemic Agents; D011720:Pyrazoles; D013449:Sulfonamides; D004008:Diclofenac; D010672:Phenytoin; 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; D000068579:Celecoxib; C032159:acetonitrile