Permeability and metabolism of potential prodrugs for the antimicrobial agent 2,5 bis(4-amidinophenyl)furan (DB75)

DB75 is an effective antimicrobial agent against African trypanosomiasis, Pneumocystis pneumonia and malaria. However, the oral potency of DB75 is poor due to its protonated amidine moieties. For that reason, unprotonated and more lipophilic DB289 was synthesized as a prodrug of DB75 to improve oral potency. The permeability of DB289 across Caco-2 cell monolayers was significantly enhanced compared to DB75, and this increase in transport correlated to improved oral activity of DB289 in rodent models. DB289 is transformed to DB75 via sequential O-demethylation and N-dehydroxylation reactions with four intermediate phase I metabolites (M1, M2, M3 and M4) formed. However, until now, the enzymes catalyzing metabolic conversion of DB289 to DB75 had not been determined. Moreover, permeability assessments of the intermediate metabolites, for their potential to serve as alternative prodrugs for 131375, had not been assessed.; In the current studies, detailed in vitro studies have been performed to further evaluate the permeability and metabolism of DB289 and its metabolites. Apparent permeability of DB289 and M1 across MDCK and Caco-2 cell monolayers were greater than 100-fold compared to those observed for DB75 by HPLC/UV/fluorescence analysis. This data is consistent with increased oral potency of these compounds compared to DB75. M2 demonstrated intracellular metabolism and displayed limited apparent permeability. M3 exhibited moderate permeability, but was shown to be a substrate for the efflux protein, P-glycoprotein. Thus, permeability results for M2 and M3 suggest that these compounds are not likely to serve as alternative prodrugs for DB75. The DB289 analog, M1, however, may be a more efficient prodrug for DB75.; Incubations with human liver microsomes, expressed cytochrome P450 isoforms or expressed cytochrome b5 were used to identify the enzymes catalyzing the metabolic conversions of DB289 to DB75. Based on kinetic analyses and results from reaction phenotyping experiments quantitated by HPLC/MS, CYP450s (CYPs 1A1, 1A2, 1B1 or 3A4) primarily catalyzed the oxidative O-demethylation reactions. However, cytochrome b5 and b5 reductase primarily mediated the reductive N-dehydroxylation reactions. Identification of the enzymes involved in DB289 metabolic activation is important for our understanding of this new candidate drug in vivo.