Rapid determination of enzyme activities of recombinant human cytochromes P450, human liver microsomes and hepatocytes

Abstract Cytochrome P450 (CYP) substrates that yield fluorescent metabolites were used for rapid screening of drug metabolism activities of 13 recombinant human cytochromes P450, human liver microsomes and human hepatocytes. Reproducible results were obtained using a fluorescent plate reader (CytoFluor) more expediently than those generated using conventional HPLC methods. Typically, results for 96 samples were obtained with the plate reader in less than 10 min as opposed to 15–35 min/sample required by conventional HPLC. The fluorescent substrates used to measure CYP activities were as follows: 3‐cyano‐7‐ethoxycoumarin (CEC) for CYP1A1, CYP1A2, CYP2C9 and CYP2C19; 7‐ethoxyresorufin (7‐ER) for CYP1A1, CYP1A2 and CYP1B1; 3‐[2‐( N , N ‐diethyl‐ N ‐methylammonium)ethyl]‐7‐methoxy‐4‐methylcoumarin (AMMC) for CYP2D6; dibenzylfluorescein (DBF) for CYP3A4, CYP3A5 and CYP2C8; 7‐methoxy‐4‐trifluoromethylcoumarin (7‐MFC) for CYP2E1, CYP2B6 and CYP2C18; and coumarin for CYP2A6. The chemical inhibition and correlation data indicated that the following substrates can be used as specific functional probes for individual cytochrome P450 present in human liver microsomes: coumarin for CYP2A6 ( r =0.82), AMMC for CYP2D6 ( r =0.83) and DBF for CYP3A4 ( r =0.92). The fluorescent plate reader was found to be useful for the rapid assessment of CYP activities (positive control) in both intact cells and subcellular fractions. Copyright © 2003 John Wiley & Sons, Ltd.