Dextromethorphan (DMO), a
cough suppressing synthetic analog of
codeine, undergoes parallel O-demethylation to
dextrorphan (DOP), and N-demethylation to
3-methoxymorphinan (MEM), in humans.
3-hydroxymorphinan, a didemethylated metabolite, is formed secondarily. O-demethylation activity is well established as an index reaction for
CYP2D6. However, this pathway appears to be mediated by at least two different
enzymes in vitro. N-demethylation activity has recently been proposed to reflect CYP3A3/4 activity. We investigated both pathways in vitro with microsomal preparations from three human livers to assess the value of DMO as a probe
drug for
CYP2D6 and CYP3A3/4, DMO O-demethylation displayed a biphasic pattern with a high-affinity site reflecting
CYP2D6 activity (mean Ki for
quinidine, 0.1 +/- 0.13 microM). Kinetic parameters for the two O-demethylation mediating
enzymes predict an average relative intrinsic clearance (Vmax/K(m) ratio) of 96% of total O-demethylation mediated via the high-affinity
enzyme. Thus, in vitro data confirms the usefulness of DMO O-demethylation as an index reaction to monitor
CYP2D6 activity. The Eadie-Hofstee plot of DMO N-demethylation was consistent with single-
enzyme Michaelis-Menten kinetics (Vmax varying from 3.3 to 6.8 nmol mg-1 min-1, K(m) from 231 to 322 microM). However,
ketoconazole, a CYP3A3/4 inhibitor, reduced N-demethylation only by 60% and had a mean Ki an order of magnitude higher (0.37 microM) compared to other pure CYP3A3/4 mediated reactions.
Troleandomycin, a mechanism based CYP3A3/4 inhibitor, inhibited MEM formation by an average maximum of 46%, with an IC50 varying from 1 to 2.6 microM. A polyclonal rat liver CYP3A1 antibody inhibited MEM formation only by approximately 50%.
Diethyldithiocarbamate (DDC), a mechanism based
CYP2E1 inhibitor, reduced MEM formation at concentrations up to 150 microM between 33 and 43%. Chemical inhibitors of
CYP2d6 (
quinidine),
CYP1A1/2 (
alpha-naphthoflavone), and
CYP2C9 (
sulfaphenazole), as well as a goat rat liver CYP2C11 polyclonal antibody (inhibitory against human
CYP2C9 and
CYP2C19), had minimal effect on MEM formation rate, thus excluding an involvement of any of these
enzymes. DMO N-demethylation is only partly mediated by CYP3A3/4, and therefore is not a reliable index reaction for CYP3A3/4 activity either in vitro or probably in vivo.