In Vitro Profiling and Mass Balance of the Anti-Cancer Agent Laromustine [14C]-VNP40101M by Rat, Dog, Monkey and Human Liver Microsomes

Abstract

Laromustine (VNP40101M; 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-(methylamino) carbonylhydrazine) is a novel sulfonylhydrazine alkylating agent. For the first time In vitro profiling and mass balance of ( 14 C)-VNP40101M in rat, dog, monkey and human liver microsomes was investigated. Also, the role of human cytochrome P450 (CYP) and flavin-containing monooxygenase (FMO) enzymes in the conversion of ( 14 C)-VNP40101M by NADPH-fortified human liver microsomes was determined. In this study, ( 14 C)-VNP40101M was converted to five radioactive components (C-1, C-2, C-3, C-4 and C-7) after 60 min of incubation with dog, monkey and human liver microsomes. With the exception of C-3, the same components were detected with rat liver microsomes. In the presence of NADPH, after 60 min of incuba- tion, the loss of substrate for rat, dog, monkey and human was 63, 82, 76 and 64%, respectively and mass balance ranged from 91.0 - 99.3%. In the absence of NADPH, after 60 min of incubation with ( 14 C)-VNP40101M (100 �M), the loss of substrate for rat, dog, monkey and human liver microsomes was 59, 53, 61 and 59%, respectively and mass balance ranged from 100.6 - 116.4%. The profiles of metabolites were similar. The relative abundance of individual metabolites was not species dependent. The formation of C-7 was not observed in zero-cofactor (no NADPH) or zero-protein samples, suggesting that its formation was enzymatic. The formation of C-1, C-2, C-3, and C-4 increased with respect to incubation time. Using a panel of CYP enzymes including rCYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4, it was shown that C- 7 formation was catalyzed by CYP2B6 and CYP3A4/5. The results of this study suggest that (1) P450 plays a role in C-7 formation but plays little or no role in the conversion of ( 14 C)-VNP40101M to C-1 through C-4, and (2) the relative abun- dance of individual degradation/metabolite products were not species dependent. These findings provide a comprehensive understanding of the metabolism of this new agent.