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 K_m 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 K_m 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 K_m values for the CLZ 6-hydroxylation in HLMs.

4. The K_m 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.

• Chlorzoxazone
• CYP1A2
• CYP2E1
• HLMs
• kinetics
• KOH
• sucrose

Acknowledgements

We thank Dr Chie Emoto, Dr Hiroyuki Sasabe, and Dr Masayuki Furukawa of Tokushima Research Institute, Otsuka Pharmaceutical Co., Ltd. for the helping with scientific discussion.

Declaration of interest

The authors declare no conflicts of interests. The authors alone are responsible for the content and writing of this article.