In vitro metabolism of 2,5-bis(4-amidinophenyl)furan-bis-O-methylamidoxime - An orally active prodrug of the antimicrobial agent 2,5-bis(4-amidinophenyl)furan
Zhou, L. × Thakker, D. Voyksner, R. Annaert, Pieter Stephens, C. Boykin, D. Tidwell, R. Hall, J. #
AAPS PharmSci vol:3 issue:3
Purpose. 2,5-Bis(4-amidinophenyl)furan (DB75) is a promising antimicrobial agent. However, it suffers from poor oral activity. In contrast, a novel prodrug of DB75, 2,5-bis(4-amidinophenyl)furan-bis-O-methylamidoxime (DB289), has excellent oral activity. Metabolism of DB289 was investigated using freshly isolated rat hepatocytes to examine its metabolic transformation to DB75.Methods. Freshly isolated rat hepatocytes were incubated with 25mM DB289 and d8-DB289 (1:1). Aliquots of culture medium and cell lysate supernatant were collected at selected times and analyzed using HPLC/UV/MSn (ion trap) to identify the metabolites. In parallel, 25 mM [14C]DB289 was incubated with freshly isolated rat hepatocytes and the metabolites were quantified using HPLC/UV/radiochemical detection. Recoveries of radioactivity in extracellular medium, cell lysate supernatant and cell lysate pellet were determined by liquid scintillation counting. Results. DB75 was observed at 30 min inside cells and accumulated intracellularly with time. Two other metabolites, M1 and M2, were detected both inside and outside of cells; however, M2 was found predominantly inside the cells. M1 was formed by mono O-demethylation of DB289 and M2 was presumably formed by dehydroxylation of M1. Small quantities of DB290 (bis O-demethylated M1) were produced from M1. M3, the mono amidoxime of DB75, was detected as a transient product of M2 and DB290. Large quantities of M1, M2 and M3 glucuronides were also detected in extracellular medium. Total recovery of radioactivity at all times was greater than 85% with the majority distributed in cell lysate supernatant and lower amount in extracellular medium. The radioactivity retained in the cell lysate pellet increased with time. Conclusion. DB289 was converted to DB75 via a series of stepwise O-demethylations and dehydroxylation of amidoxime. Phase II conjugation reactions also contributed to its metabolism. These metabolic pathways provide important information regarding formation of DB75 from its prodrug, DB289, which is under clinical evaluation as an orally active antitrypanosomal agent.