Diphenyl furans and aza analogs: effects of structural modification on in vitro activity, DNA binding, and accumulation and distribution in trypanosomes.

Human African trypanosomiasis is a devastating disease with only a few treatment options, including pentamidine. Diamidine compounds such as pentamidine, DB75, and DB820 are potent antitrypanosomal compounds. Previous investigations have shown that diamidines accumulate to high concentrations in trypanosomes. However, the mechanism of action of this class of compounds remains unknown. A long-hypothesized mechanism of action has been binding to DNA and interference with DNA-associated enzymes. The fluorescent diamidines, DB75 and DB820, have been shown to localize not only in the DNA-containing nucleus and kinetoplast of trypanosomes but also to the acidocalcisomes. Here we investigate two series of analogs of DB75 and DB820 with various levels of in vitro antitrypanosomal activity to determine whether any correlation exists between trypanosome accumulation, distribution, and in vitro activity. Despite wide ranges of in vitro antitrypanosomal activity, all of the compounds investigated accumulated to millimolar concentrations in trypanosomes over a period of 8 h. Interestingly, some of the less potent compounds accumulated to concentrations much higher than those of more potent compounds. All of the compounds were localized to the DNA-containing nucleus and/or kinetoplast, and many were also found in the acidocalcisomes. Accumulation in the nucleus and kinetoplast should be important to the mechanism of action of these compounds. The acidocalcisomes may also play a role in the mechanism of action of these compounds. This investigation suggests that the extent of accumulation alone is not responsible for killing trypanosomes and that organelle-specific accumulation may not predict in vitro activity.

CYP4F enzymes are the major enzymes in human liver microsomes that catalyze the O-demethylation of the antiparasitic prodrug DB289 [2,5-bis(4-amidinophenyl)furan-bis-O-methylamidoxime].

DB289 [2,5-bis(4-amidinophenyl)furan-bis-O-methylamidoxime] is biotransformed to the potent antiparasitic diamidine DB75 [2,5-bis(4-amidinophenyl) furan] by sequential oxidative O-demethylation and reductive N-dehydroxylation reactions. Previous work demonstrated that the N-dehydroxylation reactions are catalyzed by cytochrome b5/NADH-cytochrome b5 reductase. Enzymes responsible for catalyzing the DB289 O-demethylation pathway have not been identified. We report an in vitro metabolism study to characterize enzymes in human liver microsomes (HLMs) that catalyze the initial O-demethylation of DB289 (M1 formation). Potent inhibition by 1-aminobenzotriazole confirmed that M1 formation is catalyzed by P450 enzymes. M1 formation by HLMs was NADPH-dependent, with a Km and Vmax of 0.5 microM and 3.8 nmol/min/mg protein, respectively. Initial screening showed that recombinant CYP1A1, CYP1A2, and CYP1B1 were efficient catalysts of M1 formation. However, none of these three enzymes was responsible for M1 formation by HLMs. Further screening showed that recombinant CYP2J2, CYP4F2, and CYP4F3B could also catalyze M1 formation. An antibody against CYP4F2, which inhibited both CYP4F2 and CYP4F3B, inhibited 91% of M1 formation by HLMs. Two inhibitors of P450-mediated arachidonic acid metabolism, HET0016 (N-hydroxy-N'-(4-n-butyl-2-methylphenyl)formamidine) and 17-octadecynoic acid, effectively inhibited M1 formation by HLMs. Inhibition studies with ebastine and antibodies against CYP2J2 suggested that CYP2J2 was not involved in M1 formation by HLMs. Additionally, ketoconazole preferentially inhibited CYP4F2, but not CYP4F3B, and partially inhibited M1 formation by HLMs. We conclude that CYP4F enzymes (e.g., CYP4F2, CYP4F3B) are the major enzymes responsible for M1 formation by HLMs. These findings indicate that, in human liver, members of the CYP4F subfamily biotransform not only endogenous compounds but also xenobiotics.

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 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.