title: Human enteric microsomal CYP4F enzymes O-demethylate the antiparasitic prodrug pafuramidine.
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.
authors: School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
authors: Michael Zhuo
authors: MZ
authors: Wang
authors: Judy Qiju
authors: JQ
authors: Wu
authors: Arlene S
authors: AS
authors: Bridges
authors: Darryl C
authors: DC
authors: Zeldin
authors: Sally
authors: S
authors: Kornbluth
authors: Richard R
authors: RR
authors: Tidwell
authors: James Edwin
authors: JE
authors: Hall
authors: Mary F
authors: MF
authors: Paine
mesh_terms: D000578:Amidines / Q000494:pharmacology; D000906:Antibodies / Q000494:pharmacology; D000977:Antiparasitic Agents / Q000737:chemistry / Q000378:metabolism / Q000493:pharmacokinetics; D016718:Arachidonic Acid / Q000378:metabolism / Q000494:pharmacology; D001550:Benzamidines / Q000737:chemistry / Q000378:metabolism* / Q000493:pharmacokinetics; D001571:Benzoflavones / Q000494:pharmacology; D002090:Butyrophenones / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D000090762:Cytochrome P-450 CYP2J2; D051544:Cytochrome P-450 CYP3A; D042926:Cytochrome P-450 CYP4A; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000276:immunology / Q000378:metabolism*; D000072470:Cytochrome P450 Family 4; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D006801:Humans; D006893:Hydroxyeicosatetraenoic Acids / Q000378:metabolism; D007413:Intestinal Mucosa / Q000378:metabolism; D007422:Intestines / Q000201:enzymology*; D007700:Kinetics; D008745:Methylation / Q000187:drug effects; D008861:Microsomes / Q000201:enzymology* / Q000378:metabolism; D010105:Oxygenases / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism; D010880:Piperidines / Q000494:pharmacology; D011355:Prodrugs / Q000737:chemistry / Q000378:metabolism* / Q000493:pharmacokinetics; D011994:Recombinant Proteins / Q000378:metabolism; D013237:Stereoisomerism
chemical_list: D000578:Amidines; D000906:Antibodies; D000977:Antiparasitic Agents; D001550:Benzamidines; D001571:Benzoflavones; D002090:Butyrophenones; C000717350:CYP2J2 protein, human; D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D005231:Fatty Acids, Unsaturated; C000708209:HET0016; D006893:Hydroxyeicosatetraenoic Acids; D010880:Piperidines; D011355:Prodrugs; D011994:Recombinant Proteins; D016718:Arachidonic Acid; C047008:17-octadecynoic acid; C011512:alpha-naphthoflavone; C033286:furamidine; C055987:20-hydroxy-5,8,11,14-eicosatetraenoic acid; D003577:Cytochrome P-450 Enzyme System; D010105:Oxygenases; C510164:CYP3A5 protein, human; D000090762:Cytochrome P-450 CYP2J2; D051544:Cytochrome P-450 CYP3A; D000072470:Cytochrome P450 Family 4; C510163:CYP3A4 protein, human; C117480:CYP4F2 protein, human; C465478:CYP4A11 protein, human; D042926:Cytochrome P-450 CYP4A; C458513:pafuramidine; C058249:ebastine
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