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]

Abstract

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

Pharmacokinetics and Metabolism of the Prodrug DB289 (2,5-Bis[4-(N-methoxyamidino)phenyl]furan Monomaleate) in Rat and Monkey and Its Conversion to the Antiprotozoal/Antifungal Drug DB75 (2,5-Bis(4-guanylphenyl)furan Dihydrochloride)

“…It should be noted that the reverse biotransformations are also possible, at least in vitro, since the dicationic amidine, diminazene (which also possesses antitrypanocidal properties) is known to undergo two successive N-oxidation reactions: first to the mono-oxime and then to the di-oxime (Clement et al, 1992). The metabolic conversion of DB289 to DB75 in vitro using isolated rat hepatocytes consists of sequential oxidative O-demethylation and reductive N-dehydroxylation reactions (Zhou et al, 2004), and it has been demonstrated recently that hepatic CYP4F enzymes, including CYP4F2 and CYP4F3B, catalyze the initial O-demethylation of DB289 in humans (Wang et al, 2006). Pentamidine is metabolized in vitro mainly by multiple phase I cytochrome P450-induced side-chain C-hydroxylation reactions, but its minor biotransformation pathways involve N-hydroxylation to the oximes (Berger et al, 1990a(Berger et al, ,b, 1991(Berger et al, , 1992Clement and Jung, 1994).…”

Human Enteric Microsomal CYP4F EnzymesO-Demethylate the Antiparasitic Prodrug Pafuramidine

“…Results indicated that members of the CYP4F subfamily, including CYP4F2 and CYP4F3B, were the primary catalysts of this reaction (Wang et al, 2006). However, M1 formation rates in the panel of HLM varied only 4-fold (Wang et al, 2006). Thus, if this reaction is the rate-limiting step in the biotransformation of DB289, then variation in hepatic metabolism may have a minor contribution to the variation in the systemic exposure of DB289.…”

“…Results indicated that members of the CYP4F subfamily, including CYP4F2 and CYP4F3B, were the primary catalysts of this reaction (Wang et al, 2006). However, M1 formation rates in the panel of HLM varied only 4-fold (Wang et al, 2006).…”

“…DB289 is biotransformed to DB75 in rats, monkeys, and humans by sequential O-demethylation and N-dehydroxylation reactions (Zhou et al, 2004;Saulter et al, 2005;Wang et al, 2006;Midgley et al, 2007). Recent studies in rats and monkeys showed that 14 C-DB289 was well absorbed after p.o.…”

“…We recently characterized the hepatic enzymes responsible for the initial O-demethylation of DB289 (to M1) using recombinant cytochrome P450 (P450) and a panel of human liver microsomes (HLM) (Wang et al, 2006). Results indicated that members of the CYP4F subfamily, including CYP4F2 and CYP4F3B, were the primary catalysts of this reaction (Wang et al, 2006).…”