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Vol. 29, Issue 11, 1362-1365, November 2001
Department of Basic Pharmaceutical Sciences, The O-demethylation of dextromethorphan to
dextrorphan in humans is catalyzed primarily by cytochrome P450 2D6
(CYP2D6). However, contrary to conventional wisdom, preparations of
recombinant cytochrome P450 (P450) expressed from
CYP2D6*1 cDNA also appear to produce significant amounts
of 3-methoxymorphinan, the N-demethylated metabolite of
dextromethorphan, when assayed in vitro. We hypothesized that both
pathways were intrinsic to 2D6 and here further examine the kinetics of
formation using a highly purified preparation of CYP2D6 in a
reconstituted lipid system. Purified CYP2D6 protein with a measured
molecular weight of 55772.0 (55769.6 Da predicted) was
reconstituted into an active, lipid-vesicle environment with purified
rat cytochrome P450 reductase before the addition of substrate and
NADPH. Reaction kinetics were followed, and apparent Michaelis-Menten
constants were determined for the appearance of each metabolite by
high-pressure liquid chromatography, using both UV and fluorescence
detection. In a 2-min assay, purified 2D6 catalyzed the formation of
dextrorphan with an apparent Km value of
1.9 ± 0.2 µM and a Vmax value of
8.5 ± 0.2 nmol/nmol of P450/min and measured simultaneously the
formation of 3-methoxymorphinan with an apparent
Km value of 5000 ± 700 µM and
Vmax value of 176 ± 12 nmol (nmol of
P450)
School of
Pharmacy,
West Virginia University,
Morgantown, West Virginia
(A.Y., R.L.H.);
Department of Drug Metabolism
and
Pharmacokinetics,
Boehringer Ingelheim Pharmaceuticals,
Inc.,
Ridgefield, Connecticut (H.D.);
Bayer AG,
Wuppertal,
Germany (D.L.)
1 min1. These results indicate that at
least two distinct binding orientations exist for dextromethorphan
within the active site of CYP2D6.
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