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-demethyl-ation and reductive N-dehydroxylation reactions. Previous work demonstrated that the N-dehydroxylation reactions are catalyzed by cytochrome b 5 /NADH-cytochrome b 5 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 NADPHdependent, with a K m and V max of 0.5 M 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 (Nhydroxy-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.As part of our search for new lead compounds for the treatment of African trypanosomiasis (African sleeping sickness) and other parasitic infections, aromatic dicationic compounds, such as DB75 [2,5-bis(4-amidinophenyl) furan], have been evaluated for efficacy in multiple models of infection. These diamidine-type compounds are effective against a broad range of pathogens in vitro, including Trypanosoma brucei, Leishmania spp., Pneumocystis carinii, and

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.Diamidine compounds, such as pentamidine, propamidine, and diminazene, have been used for many years as chemotherapeutic agents for infections caused by a variety of microbes, including parasites and fungi. Pentamidine has been used for almost 60 years as a treatment for human African trypanosomiasis and is also used to treat leishmaniasis and the opportunistic infection Pneumocystis pneumonia (32). Diminazene has been used widely for treatment of animal trypanosomiasis (9) and has also been used in humans (25). Recently, pafuramidine, or DB289, a methamidoxime prodrug of the diamidine DB75 (furamidine), has been developed as an oral treatment for early-stage sleeping sickness caused by Trypanosoma brucei gambiense. DB289 is currently in phase III clinical trials in sub-Saharan Africa (6). In addition to DB75 and DB289, a library of diamidines and prodrugs has been synthesized, with various activities against many parasites (1-3, 14-18, 32).Although diamidines have been used therapeutically for over half a century, their mechanism of action is not well understood. Many mechanisms of action have been proposed (32), but one mechanism of action of diamidines that has often been hypothesized is binding to DNA in the nucleus or kinetoplast, leading to interference of DNA-associated enzymes, such as topoisomerase II (27, 34)....

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 2,5-Bis(4-amidinophenyl)furan-bis-O-methylamidoxime (DB289) is an antimicrobial prodrug developed for the treatment of a variety of microbial infections. DB289 has completed phase II clinical trials for African trypanosomiasis in Angola and the Democratic Republic of Congo and is currently enrolled in phase II trials for malaria in Thailand and for Pneumocystis pneumonia in Peru. In the phase II clinical trials involving patients with primary-stage African trypanosomiasis, treatment with DB289 achieved cure rates of approximately 95%. Moreover, DB289 was found to be well tolerated, with no significant side effects (J. Allen, unpublished).The study of drug metabolism is a key component in the drug discovery process. A compound's metabolic pathway can provide valuable information, including the identification of metabolites, the rate and extent of metabolism, the enzymes responsible for catalyzing metabolism, and potentially dangerous drug-drug interactions. More importantly for DB289 is the role of drug metabolism for activation of this inactive prodrug. The phase I metabolic pathway for DB289 conversion to the active dicationic compound 2,5-bis(4-amidinophenyl)furan (furamidine; DB75) has been determined in vitro using freshly isolated rat hepatocytes (Zhou, 2001). DB289 uptake and metabolism by rat liver hepatocytes was rapid, with furamidine detectable inside the cells within 30 min. Further investigations resulted in the detection of four other intermediate phase I metabolites as shown in Fig. 1. The metabolic conversion of DB289 to furamidine is complex with two different metabolic routes that converge on M4. Efficient transformation and enzymatic activation through this metabolic pathway are required for sufficient quantities of furamidine to reach its target. Therefore, characterizing the enzymes involved in the metabolic conversion is important for evaluating the metabolic disposition of these compounds in vivo.Preliminary results using rat hepatocytes with 1-aminobenzotria- Article, publication date, and citation information can be found at http://dmd.aspetjournals.org. doi:10.1124/dmd.105.005017.ABBREVIATIONS: DB289, 2,5-bis(4-amidinophenyl)furan-bis-O-methylamidoxime; ABT, 1-aminobenzotriazole; P450, cytochr...

The synthesis and characterization of maleate type crosslinkable nonlinear optical (NLO) polymers derived from maleic anhydride and fumaryl chloride is described. Preliminary results demonstrated this is a convenient, inexpensive, and versatile method of fabricating crosslinked NLO polymer thin films. These maleate type polyesters containing NLO chromophores such as Disperse Red 1 9 are capable of crosslinking to form a hardened lattice under either thermal or highenergy radiation conditions. Crosslinking is a critical nano-scale technique for second order nonlinear optical as well as other potential photonic applications where molecular orientations need to be aligned and be stabilized against molecular thermal motions. Photolithographic techniques may be readily employed in this system to fabricate patterned polymer waveguide.

No abstract