Abstract

Artemether (AM) is one of the most effective anti-malarial drugs. The elimination half-life of AM is very short and it shows large interindividual variability in pharmacokinetic parameters. The aim of this study was to identify cytochrome P450 (CYP) isozymes responsible for the demethylation of AM, and to evaluate functional differences between 26 CYP2B6 allelic variants in vitro. Of 14 recombinant CYPs examined in this study, CYP2B6 and CYP3A4 were primarily responsible for production of the desmethyl metabolite dihydroartemisinin. The intrinsic clearance ( V_max / K_m) of CYP2B6 was 6-fold higher than that of CYP3A4. AM demethylation by human liver microsomes was more closely correlated to immunoquantified CYP2B6 protein content (r2 = 0.548) than CYP3A4 protein content (r2 = 0.109). Wild-type CYP2B6.1 and 25 CYP2B6 allelic variants (CYP2B6.2-CYP2B6.21 and CYP2B6.23-CYP2B6.27) were heterologously expressed in COS-7 cells. In vitro analysis revealed no enzymatic activity in 5 variants (CYP2B6.8, CYP2B6.12, CYP2B6.18, CYP2B6.21, and CYP2B6.24), lower activity in 7 variants (CYP2B6.10, CYP2B6.11, CYP2B6.14, CYP2B6.15, CYP2B6.16, CYP2B6.20, and CYP2B6.27), and higher activity in 4 variants (CYP2B6.2, CYP2B6.4, CYP2B6.6, and CYP2B6.19), compared to wild-type CYP2B6.1. In kinetic analysis, 3 variants (CYP2B6.2, CYP2B6.4, and CYP2B6.6) exhibited significantly higher V_max, and 3 variants (CYP2B6.14, CYP2B6.20 and CYP2B6.27) exhibited significantly lower V_max, compared with CYP2B6.1. This functional analysis of CYP2B6 variants could provide useful information for individualization of anti-malarial drug therapy.