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Unusual Dehydroxylation of Antimicrobial Amidoxime Prodrugs by Cytochrome b5 and NADH Cytochrome b5 Reductase

2005; American Society for Pharmacology and Experimental Therapeutics; Linguagem: Inglês

10.1124/dmd.105.005017

1521-009X

Janelle Saulter,

Pharmacological Effects and Toxicity Studies

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 b 5 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.