Evaluation of a novel high-throughput assay for cytochrome P450 2D6 using 7-methoxy-4-(aminomethyl)-coumarin

• Published in: European journal of pharmaceutical sciences Vol. 12; no. 2; pp. 151 - 158
• Main Authors: Venhorst, Jennifer, Onderwater, Rob C.A, Meerman, John H.N, Vermeulen, Nico P.E, Commandeur, Jan N.M
• Format: Journal Article
• Language: English
• Published: Shannon Elsevier B.V 01.12.2000; Elsevier
• Subjects: Analysis; Biological and medical sciences; Calibration; Cell Line; Coumarins - pharmacokinetics; Cytochrome P-450 CYP2D6 - analysis; Cytochrome P-450 CYP2D6 - metabolism; Dextromethorphan - pharmacokinetics; Flow injection analysis; General pharmacology; High-throughput screening; Human CYP2D6; Humans; Inhibition; Kinetics; Medical sciences; Microplate reader; Microsomes - enzymology; Microsomes, Liver - enzymology; Pharmacology. Drug treatments; Recombinant Proteins - analysis; Recombinant Proteins - metabolism; Sensitivity and Specificity; Spectrometry, Fluorescence - methods; Substrate Specificity; Validation; Validation; High-throughput screening; Flow injection analysis; Inhibition; Human CYP2D6; Microplate reader; Human; Fluorescent tracer; Microtiter plate; Digestive system; Coumarine derivatives; Cytochrome P450; Liver; Microsome; Microassay; Demethylation; Flow injection
• ISSN: 0928-0987; 1879-0720
• DOI: 10.1016/S0928-0987(00)00150-0

We recently reported on the design, synthesis and characterisation of a novel and selective substrate of human cytochrome P450 2D6 (CYP2D6), 7-methoxy-4-(aminomethyl)-coumarin (MAMC). Here, we describe a high-throughput microplate reader assay, which makes use of MAMC as a fluorescent probe for determining the inhibition and activity of CYP2D6 in heterologously expressed systems and human liver microsomes. The high-throughput screening (HTS) assay can be used both in an end-point and real-time configuration, and is easy to use, rapid and sensitive. In addition, end-point measurements by means of flow injection analysis have also successfully been performed. The HTS-assay was validated by performing inhibition experiments for several low- and high-affinity ligands ( n=6) of CYP2D6, and comparing the findings to those obtained with the standard O-demethylation assay of dextromethorphan. The results indicate that all compounds tested display competitive inhibition in both the MAMC and dextromethorphan assay, and the K i values reveal a very good correlation ( R 2=0.984) between the two assays. To further demonstrate the usefulness of the HTS-assay, IC 50 values of a series of five N-substituted analogs of 3,4-methylenedioxyamphetamine for CYP2D6 have been determined. The results obtained demonstrate that the current HTS-assay represents a significant improvement over previous assays, with a higher turnover of MAMC and a higher selectivity for CYP2D6.