Abstract
The cytochromes P450 (CYPs) responsible for the oxidative metabolism of tacrolimus and its in vitro major metabolite M-I, the 13-O-mono-demethylated metabolite, were characterized in human liver microsomes. Human liver microsomes and ten human CYPs expressed in Hep G2 cells were used in the experiments.
1. When 14C-labeled tacrolimus (14C-tacrolimus) was incubated with human liver microsomes, M-I formation was mainly observed in the early stage of incubation, while many peaks of the more polar metabolites, including M-VII, the 13, 15-O-di-demethylated metabolite, were detected in the late stage of incubation. When the microsomes were incubated with 14C-M-I, formation of M-VII and unidentified metabolites were observed. The rates of formation of M-I from 14C-tacrolimus in 10 different human liver microsomes correlated well with the activities of testosterone 6β-hydroxylation and tolbutamide methyl-hydroxylation, but not with the marker enzyme activities of other CYPs. The metabolism of both tacrolimus and M-I by human liver microsomes were inhibited by ketoconazole and anti-CYP3A4 antiserum.
2. After incubation of 14C-tacrolimus with Hep G2 cell lysates containing expressed human CYPs, M-I formation was observed in the reaction system with CYP3A3, 3A4 and 3A5, but not with CYP1A2, 2A6, 2B6, 2C8, 2C9, 2D6 or 2E1. After incubation of the lysates with 14C-M-I, M-VII formation was again observed only in the system with the CYP3A subfamily. Substrate disappearance of both tacrolimus and M-I was the most efficiently catalyzed by CYP3A4.
3. These results suggest that tacrolimus is metabolized to polar metabolites through M-I by human liver microsomes, and that the metabolism of both tacrolimus and M-I are mainly catalyzed by CYP3A4.
