Received for publication May 1, 2007.
Revised August 13, 2007.
Accepted for publication August 14, 2007.

HUMAN ENTERIC MICROSOMAL CYP4F ENZYMES O-DEMETHYLATE THE ANTIPARASITIC PRODRUG PAFURAMIDINE

Abstract

CYP4F enzymes, including CYP4F2 and CYP4F3B, were recently demonstrated as 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 (HIMs), we characterized the enteric enzymes that catalyze the initial O-demethylation of DB289 to the intermediate metabolite, M1. M1 formation in HIMs was catalyzed by P450 enzymes, as evidenced by potent inhibition by 1-aminobenzotriazole and the requirement for NADPH. Apparent K_m and V_max values ranged from 0.6-2.4 µM and from 0.02-0.89 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 -hydroxylation rates (r²=0.94, P<0.0001; n=12) in a panel of HIMs that lacked detectable CYP4A11 protein expression. Quantitative Western blot analysis revealed appreciable CYP4F expression in these HIMs, 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.

Key words: CYP4, extrahepatic cytochrome P450, extrahepatic drug metabolism, first-pass metabolism, human CYP enzymes, prodrugs