title: Comprehensive kinetic analysis and influence of reaction components for chlorzoxazone 6-hydroxylation in human liver microsomes with CYP antibodies.
abstract: 1. Chlorzoxazone (CLZ) is currently being used as a marker substrate in vitro/vivo studies to quantify cytochrome P450 2E1 (CYP2E1) activity in humans. Although in CLZ 6-hydroxylation several CYPs are responsible, previous studies have presented the monophasicity of the reaction in human liver microsomes (HLMs). Furthermore, the Km values of CYP2E1 for the 6-hydroxylation in HLMs were reported to be lower than those of its recombinant enzymes. 2. This study aimed to provide the comprehensive Km values for the CLZ 6-hydroxylation in HLMs using CYP antibodies. The Eadie-Hofstee plots revealed a biphasic profile and indicate that the reaction was mainly mediated by CYP1A2 as well as CYP2E1. The formation of 6-hydroxychlorzoxazone was more specific for CYP2E1 activity at higher substrate concentration in HLMs. 3. Moreover, KOH as a vehicle for substrate or sucrose included in HLMs preparation had some effect on the activity of CLZ 6-hydroxylase. These constituents seemed to be casually related to the apparent monophasic kinetics and variability in Km values for the CLZ 6-hydroxylation in HLMs. 4. The Km of CYP1A2 and CYP2E1 in HLMs was 3.8 µmol/L and 410 µmol/L, respectively, and the value of CYP2E1 was close to that of recombinant CYP2E1.
authors: Department of Drug Metabolism, Drug Safety Research Center, Tokushima Research Institute , Otsuka Pharmaceutical Co., Ltd., Tokushima , Japan.
authors: Yoshiya
authors: Y
authors: Yamamura
authors: Noriyuki
authors: N
authors: Koyama
authors: Ken
authors: K
authors: Umehara
mesh_terms: D002753:Chlorzoxazone / Q000031:analogs & derivatives* / Q000493:pharmacokinetics; D002853:Chromatography, Liquid; D019388:Cytochrome P-450 CYP1A2 / Q000378:metabolism*; D019392:Cytochrome P-450 CYP2E1 / Q000378:metabolism*; D006801:Humans; D006900:Hydroxylation; D008862:Microsomes, Liver / Q000187:drug effects* / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D053719:Tandem Mass Spectrometry
chemical_list: D011994:Recombinant Proteins; C039289:6-hydroxychlorzoxazone; D019392:Cytochrome P-450 CYP2E1; C504573:CYP1A2 protein, human; D019388:Cytochrome P-450 CYP1A2; D002753:Chlorzoxazone
keywords: CYP1A2; CYP2E1; Chlorzoxazone; HLMs; KOH; kinetics; sucrose