TRIMETHADIONE N-DEMETHYLATION BY RAT-LIVER CYP2E1 IN-VITRO

Trimethadione (TMO) is a model drug utilized for estimation of hepatic metabolism in clinical studies, and it was reported that TMO N-demeth ylase activity was inhibited by CYP2E1 inhibitors and substrates in ra t in vivo. This study was performed to investigate the involvement of the CYP2E1 subfamily on TMO N-demethylation in vitro and to clarify th ese inhibitory mechanisms. The effects of acetone (AC), imidazole (IM) and N-nitrosodimethylamine (NDA) on TMO N-demethylation were studied in vitro. Rat hepatic microsomal fractions were employed as the enzyme source of TMO N-demethylase and the activity was determined by the pr oduction of dimethadione (DMO). DMO was analyzed by a GC/FTD equipped with a narrow-bore capillary column. TMO N-demethylation was biphasic by the graphic analysis of Eadie-Hofstee plots; this suggests the invo lvement of at least two enzymes in TMO metabolism in the rat. The kine tic parameters for the formation of DMO were analyzed graphically usin g double-reciprocal plots. The apparent K-m1, K-m2 and V-max1, V-max2 values for DMO formation were 4, 20 mM and 182, 595 pmol/mg protein/mi n, respectively. AC and IM inhibited TMO N-demethylase activity compet etively. However, mixed inhibition kinetics was observed by NDA. Furth ermore, TMO N-demethylase activity was inhibited by antiserum to CYP2E 1 by 62% and CYP3A2 by 46%. These results indicate that the CYP2E1 sub family is the major enzyme involved in TMO N-demethylation in rat in v itro although the CYP3A2 is also involved in this transformation.