Abstract

Furamidine is an effective antimicrobial agent; however, oral potency of furamidine is poor. A prodrug of furamidine, 2,5-bis(4-amidinophenyl)furan-bis-O-methylamidoxime (DB289), has greatly improved oral potency. DB289 is transformed to furamidine via O-demethylation, and N-dehydroxylation reactions with four intermediate metabolites formed. The O-demethylation reactions have been shown to be catalyzed by cytochrome P450. The enzymes catalyzing the reductive N-dehydroxylation reactions have not been determined. The objective of this study was to identify the enzymes that catalyze N-dehydroxylation of metabolites M1, a monoamidoxime, and M2, a diamidoxime, formed during generation of furamidine. M1 and M2 metabolism was investigated using human liver microsomes and human soluble cytochrome b₅ and NAD cytochrome b₅ reductase, expressed in Escherichia coli. Kinetics of M1 and M2 reduction by human liver microsomes exhibited high affinity and moderate capacity. Metabolism was significantly inhibited by antibodies to cytochrome b₅ and b₅ reductase and by chemical inhibitors of b₅ reductase. The amidoximes were efficiently metabolized by liver mitochondria, which contain cytochrome b₅/b₅ reductase, but not by liver cytosol, which contains minimal amounts of these proteins. Expressed cytochrome b₅/b₅ reductase, in the absence of any other proteins, efficiently catalyzed reduction of both amidoximes. K_m values were similar to those for microsomes, and V_max values were 33- to 36-fold higher in the recombinant system compared with microsomes. Minimal activity was seen with cytochrome b₅ or b₅ reductase alone or with cytochrome P450 reductase alone or with cytochrome b₅. These results indicate that cytochrome b₅ and b₅ reductase play a direct role in metabolic activation of DB289 to furamidine.