Unusual dehydroxylation of antimicrobial amidoxime prodrugs by cytochrome b5 and NADH cytochrome b5 reductase

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 b(5) and NAD cytochrome b(5) 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 b(5) and b5 reductase and by chemical inhibitors of b(5) reductase. The amidoximes were efficiently metabolized by liver mitochondria, which contain cytochrome b(5)/b(5) reductase, but not by liver cytosol, which contains minimal amounts of these proteins. Expressed cytochrome b(5)/b(5) 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 b(5) or b(5) reductase alone or with cytochrome P450 reductase alone or with cytochrome b(5). These results indicate that cytochrome b(5) and b(5) reductase play a direct role in metabolic activation of DB289 to furamidine.