title: Effects of organic solvents on the time-dependent inhibition of CYP3A4 by diazepam.
abstract: The effects of organic solvents, acetonitrile, dimethyl sulfoxide (DMSO), and methanol, which are used to dissolve lipophilic test compounds and cytochrome P450 (P450) substrates, and carried into pre-incubation at 1% (v/v), on time-dependent inhibition of CYP3A4 by diazepam, were evaluated using human liver microsomes (HLM) and recombinant human P450 expressed microsomes (rCYPs). The inactivation kinetics of CYP3A4 by diazepam dissolved in acetonitrile and methanol were almost equal with k(inact)/K(I) values, 0.095 and 0.15 min(-1) mM(-1) for HLM and 1.1 and 1.4 min(-1) mM(-1) for rCYP3A4, respectively. In contrast, the inactivation by diazepam dissolved in 1% DMSO significantly decreased and the kinetic parameter could not be calculated. The formation rate of nordiazepam and temazepam metabolized from diazepam dissolved in DMSO were approximately half of those using substrate dissolved in acetonitrile and methanol in both HLM and rCYP3A4. Dixon plots revealed that the metabolism of diazepam in rCYP3A4 were inhibited by DMSO in a competitive or mixed-type manner with K(i) (inhibition constant) values of 6 and 24 mM for nordiazepam and temazepam, respectively. In conclusion, the time-dependent inhibition of CYP3A4 by diazepam was attenuated by DMSO, while acetonitrile and methanol had no effect. The metabolite formation profile under the conditions tested suggested that DMSO competitively inhibit the formation of the reactive metabolites of diazepam by CYP3A4. The effect of organic solvents should be taken into consideration when evaluating the in vitro time-dependent inhibition of new chemical entities.
authors.affiliation: Drug Metabolism & Pharmacokinetics Research Laboratories, Daiichi Sankyo Co., Ltd, Tokyo 140-8710, Japan. nishiya.yumi.m4@daiichisankyo.co.jp
authors.forename: Y
authors.initials: Y
authors.lastname: Nishiya
authors.forename: K
authors.initials: K
authors.lastname: Nakamura
authors.forename: N
authors.initials: N
authors.lastname: Okudaira
authors.forename: K
authors.initials: K
authors.lastname: Abe
authors.forename: N
authors.initials: N
authors.lastname: Kobayashi
authors.forename: O
authors.initials: O
authors.lastname: Okazaki
mesh_terms: D000097:Acetonitriles / Q000494:pharmacology; D051544:Cytochrome P-450 CYP3A; D065692:Cytochrome P-450 CYP3A Inhibitors; D003975:Diazepam / Q000494:pharmacology*; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D018757:GABA Modulators / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D000432:Methanol / Q000494:pharmacology; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology*; D003708:Nordazepam / Q000494:pharmacology; D012997:Solvents / Q000494:pharmacology*; D013693:Temazepam / Q000494:pharmacology
chemical_list: D000097:Acetonitriles; D065692:Cytochrome P-450 CYP3A Inhibitors; D018757:GABA Modulators; D012997:Solvents; D003708:Nordazepam; D013693:Temazepam; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D003975:Diazepam; D000432:Methanol; D004121:Dimethyl Sulfoxide; C032159:acetonitrile