Metabolism of benzamidoxime (N-hydroxyamidine) in human hepatocytes and role of UDP-glucuronosyltransferases

Abstract

N-Hydroxyamidines (amidoximes) can act as pro-drugs of amidines (e.g. ximelagatran, a novel direct thrombin inhibitor). This known pro-drug principle is based on the N-reduction of an oral bioavailable amidoxime to its active form. Previous study of the metabolism of the model substrate benzamidoxime by pig hepatocytes demonstrated the formation of benzamidoxime-O-glucuronide in addition to the well-established N-reduction. The objective of the present work was to investigate the glucuronidation of benzamidoxime by using cultivated cryopreserved human hepatocytes. Furthermore, the involvement of human UDP-glucuronosyltransferases (UGTs) was examined by incubating benzamidoxime in the presence of eight human hepatic recombinant UGT enzymes. Metabolites were analysed by liquid chromatography/mass spectrometry using electrospray ionization and compared with authentic synthetic compounds. For the first time, the O-glucuronidation of benzamidoxime was demonstrated in cultures of human hepatocytes. UGT1A9 is the most efficient enzyme conjugating benzamidoxime, whereas the conversion activities of UGT1A1 and UGT1A3 were 60-fold lower. Human hepatocytes form two non-mutagenic compounds: benzamidine, as the predominating metabolite, and benzamidoxime-O-glucuronide to a lesser extent. N-oxidation of benzamidine was not detected.