CYP1A1 and CYP1B1-mediated biotransformation of the antitrypanosomal methamidoxime prodrug DB844 forms novel metabolites through intramolecular rearrangement.

DB844 (CPD-594-12), N-methoxy-6-{5-[4-(N-methoxyamidino)phenyl]-furan-2-yl}-nicotinamidine, is an oral prodrug that has shown promising efficacy in both mouse and monkey models of second stage human African trypanosomiasis. However, gastrointestinal (GI) toxicity was observed with high doses in a vervet monkey safety study. In the current study, we compared the metabolism of DB844 by hepatic and extrahepatic cytochrome P450s to determine whether differences in metabolite formation underlie the observed GI toxicity.

Safety, pharmacokinetic, and efficacy studies of oral DB868 in a first stage vervet monkey model of human African trypanosomiasis.

There are no oral drugs for human African trypanosomiasis (HAT, sleeping sickness). A successful oral drug would have the potential to reduce or eliminate the need for patient hospitalization, thus reducing healthcare costs of HAT. The development of oral medications is a key objective of the Consortium for Parasitic Drug Development (CPDD).

Novel arylimidamides for treatment of visceral leishmaniasis.

Arylimidamides (AIAs) represent a new class of molecules that exhibit potent antileishmanial activity (50% inhibitory concentration [IC(50)], <1 microM) against both Leishmania donovani axenic amastigotes and intracellular Leishmania, the causative agent for human visceral leishmaniasis (VL). A systematic lead discovery program was employed to characterize in vitro and in vivo antileishmanial activities, pharmacokinetics, mutagenicities, and toxicities of two novel AIAs, DB745 and DB766. They were exceptionally active (IC(50) < or = 0.

New treatment option for second-stage African sleeping sickness: in vitro and in vivo efficacy of aza analogs of DB289.

African sleeping sickness is a fatal parasitic disease, and all drugs currently in use for treatment have strong liabilities. It is essential to find new, effective, and less toxic drugs, ideally with oral application, to control the disease. In this study, the aromatic diamidine DB75 (furamidine) and two aza analogs, DB820 and DB829 (CPD-0801), as well as their methoxyamidine prodrugs and amidoxime metabolites, were evaluated against African trypanosomes.

A gel-free MS-based quantitative proteomic approach accurately measures cytochrome P450 protein concentrations in human liver microsomes.

The human cytochrome P450 (P450) superfamily consists of membrane-bound proteins that metabolize a myriad of xenobiotics and endogenous compounds. Quantification of P450 expression in various tissues under normal and induced conditions has an important role in drug safety and efficacy. Conventional immunoquantification methods have poor dynamic range, low throughput, and a limited number of specific antibodies.

Antiparasitic compounds that target DNA.

Designed, synthetic heterocyclic diamidines have excellent activity against eukaryotic parasites that cause diseases such as sleeping sickness and leishmania and adversely affect millions of people each year. The most active compounds bind specifically and strongly in the DNA minor groove at AT sequences. The compounds enter parasite cells rapidly and appear first in the kinetoplast that contains the mitochondrial DNA of the parasite.

Synthesis and in vitro antiprotozoal activity of bisbenzofuran cations.

Forty three cationic bisbenzofurans were synthesized either by interaction of o-hydroxyaldehydes with alpha-halogenated ketones followed by intramolecular ring closure or by a copper- or palladium-mediated heteroannulation of substituted o-iodophenols with terminal acetylenes. In vitro antiprotozoal activities of compounds 1-43 against Trypanosoma brucei rhodesiense, Plasmodium falciparum, and Leishmania donovani and cytotoxicity against mammalian cells were influenced by the position and the type of cationic substituents as well as the length of the carbon linker between aromatic moieties. One bisamidine displayed an antitrypanosomal efficacy comparable to that of pentamidine and melarsoprol.

Human enteric microsomal CYP4F enzymes O-demethylate the antiparasitic prodrug pafuramidine.

CYP4F enzymes, including CYP4F2 and CYP4F3B, were recently shown to be the major enzymes catalyzing the initial oxidative O-demethylation of the antiparasitic prodrug pafuramidine (DB289) by human liver microsomes. As suggested by a low oral bioavailability, DB289 could undergo first-pass biotransformation in the intestine, as well as in the liver. Using human intestinal microsomes (HIM), we characterized the enteric enzymes that catalyze the initial O-demethylation of DB289 to the intermediate metabolite, M1.

Synthesis and in vitro antiprotozoal activities of dicationic 3,5-diphenylisoxazoles.

3,5-bis(4-amidinophenyl)isoxazole (3)-an analogue of 2,5-bis(4-amidinophenyl)furan (furamidine) in which the central furan ring is replaced by isoxazole-and 42 novel analogues were prepared by two general synthetic pathways. The 43 isoxazole derivatives were assayed against Trypanosoma brucei rhodesiense (T. brucei rhodesiense) STIB900, Plasmodium falciparum (P.

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.

Accumulation and intracellular distribution of antitrypanosomal diamidine compounds DB75 and DB820 in African trypanosomes.

The aromatic diamidine pentamidine has long been used to treat early-stage human African trypanosomiasis (HAT). Two analogs of pentamidine, DB75 and DB820, have been shown to be more potent and less toxic than pentamidine in murine models of trypanosomiasis. The diphenyl furan diamidine, DB75, is the active metabolite of the prodrug DB289, which is currently in phase III clinical trials as a new orally active candidate drug to treat first-stage HAT.

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.

Dications that target the DNA minor groove: compound design and preparation, DNA interactions, cellular distribution and biological activity.

Fluorescence microscopy of trypanosomes from drug treated mice shows that biologically active heterocyclic diamidines that target the DNA minor groove bind rapidly and specifically to parasite kinetoplast DNA (k-DNA). The observation that the kinetoplast is destroyed, generally within 24 hours, after drug treatment is very important for understanding the biological mechanism, and suggests that the diamidines may be inhibiting some critical opening/closing step of circular k-DNA. Given the uncertainties in the biological mechanism, we have taken an empirical approach to generating a variety of synthetic compounds and DNA minor groove interactions for development of improved and new biological activities.

O-alkoxyamidine prodrugs of furamidine: in vitro transport and microsomal metabolism as indicators of in vivo efficacy in a mouse model of Trypanosoma brucei rhodesiense infection.

Five O-alkoxyamidine analogues of the prodrug 2,5-bis[4-methoxyamidinophenyl]furan were synthesized and evaluated against Trypanosoma brucei rhodesiense in the STIB900 mouse model by oral administration. The observed in vivo activity of these prodrugs demonstrates that compounds with an O-methoxyamidine or O-ethoxyamidine group effectively cured all trypanosome-infected mice, whereas prodrugs with larger side-chains did not completely cure the mice. Permeability across Caco-2 cell monolayers and microsomal metabolism were used to identify the underlying mechanisms of prodrug efficacy.