Metabolic profile, enzyme kinetics, and reaction phenotyping of β-lapachone metabolism in human liver and intestine in vitro.

β-Lapachone (β-Lap) is an NAD(P)H: quinone oxidoreductase 1 (NQO1) target antitumor drug candidate in phase II clinical trials. The present study aimed to uncover the metabolic profile, enzyme kinetics, and enzyme isoforms for the metabolism of β-Lap in human liver and intestine in vitro. NQO1-mediated quinone reduction and subsequent glucuronidation is the predominant metabolic pathway for β-Lap in humans; a pair of regioisomers (M1 and M2) of reduced β-Lap glucuronides were the major metabolites found from human S9 incubations. The overall glucuronidation clearance of β-Lap in human liver S9 was 4754.90 μL/min/mg of protein and was 8.1-fold of that in human intestinal S9. Recombinant UDP-glucuronosyltransferase (UGT) screening, correlation analysis, enzyme kinetics, and chemical inhibition study were performed to determine the UGT isoforms involved in β-Lap metabolism. UGT1A7, UGT1A8, and UGT1A9 are the predominant isoforms responsible for the formation of M2 while UGT2B7 is the main isoform for M1, suggesting a regioselective glucuronidation of reduced quinone by UGTs. It was of interest to find that β-Lap underwent nonenzymatic two-electron reduction, providing a novel explanation for the toxicities of β-Lap to NQO1-negative cells at high concentration and with long-time incubation. In conclusion, this study contributes to a better understanding of not only β-Lap metabolism but its antitumor property as well.