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 K(m) and V(max) values ranged from 0.6 to 2.4 microM 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 omega-hydroxylation rates (r(2) = 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.
Publication types
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Research Support, N.I.H., Intramural
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Research Support, Non-U.S. Gov't
MeSH terms
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Amidines / pharmacology
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Antibodies / pharmacology
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Antiparasitic Agents / chemistry
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Antiparasitic Agents / metabolism
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Antiparasitic Agents / pharmacokinetics
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Arachidonic Acid / metabolism
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Arachidonic Acid / pharmacology
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Benzamidines / chemistry
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Benzamidines / metabolism*
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Benzamidines / pharmacokinetics
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Benzoflavones / pharmacology
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Butyrophenones / pharmacology
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Chromatography, High Pressure Liquid
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Cytochrome P-450 CYP2J2
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Cytochrome P-450 CYP3A
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Cytochrome P-450 CYP4A
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Cytochrome P-450 Enzyme Inhibitors
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Cytochrome P-450 Enzyme System / immunology
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Cytochrome P-450 Enzyme System / metabolism*
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Cytochrome P450 Family 4
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Enzyme Inhibitors / pharmacology
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Fatty Acids, Unsaturated / pharmacology
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Humans
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Hydroxyeicosatetraenoic Acids / metabolism
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Intestinal Mucosa / metabolism
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Intestines / enzymology*
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Kinetics
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Methylation / drug effects
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Microsomes / enzymology*
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Microsomes / metabolism
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Oxygenases / antagonists & inhibitors
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Oxygenases / immunology
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Oxygenases / metabolism
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Piperidines / pharmacology
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Prodrugs / chemistry
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Prodrugs / metabolism*
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Prodrugs / pharmacokinetics
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Recombinant Proteins / metabolism
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Stereoisomerism
Substances
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Amidines
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Antibodies
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Antiparasitic Agents
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Benzamidines
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Benzoflavones
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Butyrophenones
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CYP2J2 protein, human
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Cytochrome P-450 Enzyme Inhibitors
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Enzyme Inhibitors
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Fatty Acids, Unsaturated
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HET0016
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Hydroxyeicosatetraenoic Acids
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Piperidines
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Prodrugs
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Recombinant Proteins
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Arachidonic Acid
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17-octadecynoic acid
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alpha-naphthoflavone
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furamidine
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20-hydroxy-5,8,11,14-eicosatetraenoic acid
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Cytochrome P-450 Enzyme System
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Oxygenases
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CYP3A5 protein, human
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Cytochrome P-450 CYP2J2
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Cytochrome P-450 CYP3A
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Cytochrome P450 Family 4
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CYP3A4 protein, human
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CYP4F2 protein, human
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CYP4A11 protein, human
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Cytochrome P-450 CYP4A
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pafuramidine
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ebastine