Involvement of cytochrome P450 2C9, 2E1 and 3A4 in trimethadione N-demethylation in human microsomes

BACKGROUND AND OBJECTIVES: Trimethadione (TMO), an antiepileptic drug, may be used as a candidate for estimating hepatic drug-oxidizing activity. While TMO metabolism is mainly catalysed by CYP2C9, CYP2E1 and CYP3A4 the contribution of the different isoforms is unclear. In this study, we determined the percentage contribution of the three CYPs (CYP2C9, CYP2E1 and CYP3A4) to TMO N-demethylation. METHOD: We used human liver microsomes and human recombinant CYPs expressed in human B-lymphoblast cells and baculovirus-infected insect cells. RESULTS: The mean Km, Vmax and Vmax/Km values of TMO N-demethylation in human microsomes were 3.66 (mm), 503 (pmol/min/mg) and 2.61 (mL/h/mg), respectively. In the microsomes from human B-lymphoblast cells or baculovirus-infected insect cells, CYP 2C9, CYP 2E1 and CYP3A4 exhibited similar Km and higher Vmax in baculovirus-infected insect cells than B-lymphoblast cells. In baculovirus-infected insect cells, CYP2C9, CYP2E1 and CYP3A4 exhibited activities of 32, 286 and 77 pmol/min/pmol CYP, respectively. No CYP activity catalysed by CYP1A2 and 2D6 were detected in the two human cDNA expressed CYP isoforms. CONCLUSION: TMO is metabolized not only by CYP2E1 but also CYP3A4 and CYP2C9. The order of this metabolism is as follows: CYP2E1 > CYP3A4 > CYP2C9.