Abstract
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. M1 formation in HIM was catalyzed by cytochrome P450 (P450) enzymes, as evidenced by potent inhibition by 1-aminobenzotriazole and the requirement for NADPH. Apparent Km and Vmax values ranged from 0.6 to 2.4 μM and from 0.02 to 0.89 nmol/min/mg protein, respectively (n = 9). Of the P450 chemical inhibitors evaluated, ketoconazole was the most potent, inhibiting M1 formation by 66%. Two inhibitors of P450-mediated arachidonic acid metabolism, HET0016 (N-hydroxy-N′-(4-n-butyl-2-methylphenyl)formamidine) and 17-octadecynoic acid, inhibited M1 formation in a concentration-dependent manner (up to 95%). Immunoinhibition with an antibody raised against CYP4F2 showed concentration-dependent inhibition of M1 formation (up to 92%), whereas antibodies against CYP3A4/5 and CYP2J2 had negligible to modest effects. M1 formation rates correlated strongly with arachidonic acid ω-hydroxylation rates (r2 = 0.94, P < 0.0001, n = 12) in a panel of HIM that lacked detectable CYP4A11 protein expression. Quantitative Western blot analysis revealed appreciable CYP4F expression in these HIM, with a mean (range) of 7 (3-18) pmol/mg protein. We conclude that enteric CYP4F enzymes could play a role in the first-pass biotransformation of DB289 and other xenobiotics.
Footnotes
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This study was supported by grants from the Bill and Melinda Gates Foundation, the Medicines for Malaria Venture, and the Division of Intramural Research of the National Institutes of Health, National Institute of Environmental Health Sciences.
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doi:10.1124/dmd.107.016428.
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ABBREVIATIONS: DB75/furamidine, 2,5-bis(4-amidinophenyl) furan; DB289/pafuramidine, 2,5-bis(4-amidinophenyl)furan-bis-O-methylamidoxime; P450, cytochrome P450; HLM, human liver microsome(s); HIM, human intestinal microsome(s); DB289-d8, deuterium-labeled DB289; HETE, hydroxyeicosatetraenoic acid(s); 20-HETE, 20-hydroxy-5Z,8Z,11Z,14Z-eicosatetraenoic acid; EET, epoxyeicosatrienoic acid(s); 20-HETE-d6, deuterium-labeled 20-HETE; 17-ODYA, 17-octadecynoic acid; HET0016, N-hydroxy-N′-(4-n-butyl-2-methylphenyl)formamidine; HPLC, high-performance liquid chromatography; α-NF, α-naphthoflavone; ABT, 1-aminobenzotriazole; MS/MS, tandem mass spectrometry; SRM, selected reaction monitoring; CV, coefficient of variation; DHET, dihydroxyeicosatrienoic acid.
- Received April 30, 2007.
- Accepted August 14, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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