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. 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.
Wang
Michael Zhuo
MZ
School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
Wu
Judy Qiju
JQ
Bridges
Arlene S
AS
Zeldin
Darryl C
DC
Kornbluth
Sally
S
Tidwell
Richard R
RR
Hall
James Edwin
JE
Paine
Mary F
MF
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
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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