Human enteric microsomal CYP4F enzymes O-demethylate the antiparasitic prodrug pafuramidine

Drug Metab Dispos. 2007 Nov;35(11):2067-75. doi: 10.1124/dmd.107.016428. Epub 2007 Aug 20.

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

  • Research Support, N.I.H., Intramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amidines / pharmacology
  • Antibodies / pharmacology
  • Antiparasitic Agents / chemistry
  • Antiparasitic Agents / metabolism
  • Antiparasitic Agents / pharmacokinetics
  • Arachidonic Acid / metabolism
  • Arachidonic Acid / pharmacology
  • Benzamidines / chemistry
  • Benzamidines / metabolism*
  • Benzamidines / pharmacokinetics
  • Benzoflavones / pharmacology
  • Butyrophenones / pharmacology
  • Chromatography, High Pressure Liquid
  • Cytochrome P-450 CYP3A
  • Cytochrome P-450 CYP4A
  • Cytochrome P-450 Enzyme Inhibitors
  • Cytochrome P-450 Enzyme System / immunology
  • Cytochrome P-450 Enzyme System / metabolism*
  • Cytochrome P450 Family 4
  • Enzyme Inhibitors / pharmacology
  • Fatty Acids, Unsaturated / pharmacology
  • Humans
  • Hydroxyeicosatetraenoic Acids / metabolism
  • Intestinal Mucosa / metabolism
  • Intestines / enzymology*
  • Kinetics
  • Methylation / drug effects
  • Microsomes / enzymology*
  • Microsomes / metabolism
  • Oxygenases / antagonists & inhibitors
  • Oxygenases / immunology
  • Oxygenases / metabolism
  • Piperidines / pharmacology
  • Prodrugs / chemistry
  • Prodrugs / metabolism*
  • Prodrugs / pharmacokinetics
  • Recombinant Proteins / metabolism
  • Stereoisomerism

Substances

  • Amidines
  • Antibodies
  • Antiparasitic Agents
  • Benzamidines
  • Benzoflavones
  • Butyrophenones
  • Cytochrome P-450 Enzyme Inhibitors
  • Enzyme Inhibitors
  • Fatty Acids, Unsaturated
  • Hydroxyeicosatetraenoic Acids
  • N-hydroxy-N'-(4-butyl-2-methylphenyl)formamidine
  • Piperidines
  • Prodrugs
  • Recombinant Proteins
  • Arachidonic Acid
  • 17-octadecynoic acid
  • alpha-naphthoflavone
  • furamidine
  • 20-hydroxy-5,8,11,14-eicosatetraenoic acid
  • Cytochrome P-450 Enzyme System
  • Oxygenases
  • CYP3A5 protein, human
  • Cytochrome P-450 CYP3A
  • Cytochrome P450 Family 4
  • arachidonate epoxygenase
  • CYP3A4 protein, human
  • CYP4F2 protein, human
  • CYP4A11 protein, human
  • Cytochrome P-450 CYP4A
  • pafuramidine
  • ebastine