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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.
Drug Metab Dispos. 2001 Sep; 29(9):1196-200.DM

Abstract

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.

Authors+Show Affiliations

Merck Frosst Center for Therapeutic Research, C.P. 1005, Pointe-Claire-Dorval, Kirkland, Quebec, Canada H9R 4P8. nathalie_chauret@merck.comNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article

Language

eng

PubMed ID

11502727

Citation

Chauret, N, et al. "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." Drug Metabolism and Disposition: the Biological Fate of Chemicals, vol. 29, no. 9, 2001, pp. 1196-200.
Chauret N, Dobbs B, Lackman RL, et al. 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. Drug Metab Dispos. 2001;29(9):1196-200.
Chauret, N., Dobbs, B., Lackman, R. L., Bateman, K., Nicoll-Griffith, D. A., Stresser, D. M., Ackermann, J. M., Turner, S. D., Miller, V. P., & Crespi, C. L. (2001). 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. Drug Metabolism and Disposition: the Biological Fate of Chemicals, 29(9), 1196-200.
Chauret N, et al. 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. Drug Metab Dispos. 2001;29(9):1196-200. PubMed PMID: 11502727.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - 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. AU - Chauret,N, AU - Dobbs,B, AU - Lackman,R L, AU - Bateman,K, AU - Nicoll-Griffith,D A, AU - Stresser,D M, AU - Ackermann,J M, AU - Turner,S D, AU - Miller,V P, AU - Crespi,C L, PY - 2001/8/15/pubmed PY - 2001/11/3/medline PY - 2001/8/15/entrez SP - 1196 EP - 200 JF - Drug metabolism and disposition: the biological fate of chemicals JO - Drug Metab Dispos VL - 29 IS - 9 N2 - 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. SN - 0090-9556 UR - https://www.unboundmedicine.com/medline/citation/11502727/The_use_of_3_[2__NN_diethyl_N_methylammonium_ethyl]_7_methoxy_4_methylcoumarin__AMMC__as_a_specific_CYP2D6_probe_in_human_liver_microsomes_ L2 - http://dmd.aspetjournals.org/cgi/pmidlookup?view=long&pmid=11502727 DB - PRIME DP - Unbound Medicine ER -