The use of 3-[2-(N,N-diethyl-N-methylammonium)ethyl]-7-methoxy-4-methylcoumarin (AMMC) as a specific CYP2D6 probe in human liver microsomes.
Recently, a novel nonfluorescent probe 3-[2-(N,N-diethyl-N-methylammonium)-ethyl]-7-methoxy-4-methylcoumarin (AMMC), which produces a fluorescent metabolite AMHC (3-[2-(N,N-diethyl-N-methylammonium)ethyl]-7-hydroxy-4-methylcoumarin) was used with microsomes containing recombinant enzymes (rCYP) to monitor CYP2D6 inhibition in a microtiter plate assay. This article describes the studies that were performed in human liver microsomes (HLM) to establish the selectivity of AMMC toward CYP2D6. Metabolism studies in HLM showed that AMMC was converted to one metabolite identified by mass spectrometry as AMHC. Kinetic studies indicated an apparent K(m) of 3 microM with a V(max) of 20 pmol/min. mg of protein for the O-demethylation reaction. The O-demethylation of AMMC in HLM was inhibited significantly in the presence of a CYP2D6 inhibitory antibody. Using a panel of various HLM preparations (n = 12), a good correlation (r(2) = 0.95) was obtained between AMMC O-demethylation and bufuralol metabolism, a known CYP2D6 substrate, but not with probes for the other major xenobiotic metabolizing CYPs. Finally, only rCYP2D6 showed detectable metabolism in experiments conducted with rCYPs using AMMC at a concentration of 1.5 microM (near K(m)). However, at a concentration of 25 microM AMMC, rCYP1A also contributed significantly to the formation of AMHC. Knowing the experimental conditions under which AMMC was selective for CYP2D6, a microtiter assay was developed to study the inhibition of various compounds in HLM using the fluorescence of AMHC as an indication of CYP2D6 activity. The inhibition potential of various chemicals was found to be comparable to those determined using the standard CYP2D6 probe, bufuralol, which requires high-performance liquid chromatography separation for the analysis of its CYP2D6-mediated 1'-hydoxylated metabolite.
Chauret
N
N
Merck Frosst Center for Therapeutic Research, C.P. 1005, Pointe-Claire-Dorval, Kirkland, Quebec, Canada H9R 4P8. nathalie_chauret@merck.com
Dobbs
B
B
Lackman
R L
RL
Bateman
K
K
Nicoll-Griffith
D A
DA
Stresser
D M
DM
Ackermann
J M
JM
Turner
S D
SD
Miller
V P
VP
Crespi
C L
CL
D003374:Coumarins / Q000494:pharmacology*; D065690:Cytochrome P-450 CYP2D6 Inhibitors; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005456:Fluorescent Dyes / Q000494:pharmacology*; D006801:Humans; D008862:Microsomes, Liver / Q000378:metabolism*; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology*
C437963:3-(2-(N,N-diethyl-N-methylammonium)ethyl)-7-methoxy-4-methylcoumarin; D003374:Coumarins; D065690:Cytochrome P-450 CYP2D6 Inhibitors; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D000644:Quaternary Ammonium Compounds