Fluorescence-based assays for screening nine cytochrome P450 (P450) activities in intact cells expressing individual human P450 enzymes.

In this study we describe a battery of fluorescence assays for rapid measurement in intact cells of the activity of nine cytochromes P450 (P450s) involved in drug metabolism. The assays are based on the direct incubation of monolayers of cells expressing individual P450 enzymes with a fluorogenic substrate followed by fluorimetric quantification of the product formed and released into incubation medium. For each individual P450 activity, different fluorescence probes were examined, and the one showing the best properties (highest metabolic rates, lowest background fluorescence) was selected: 3-cyano-7-ethoxycoumarin for CYP1A2 and CYP2C19, coumarin for CYP2A6, 7-ethoxy-4-trifluoromethylcoumarin for CYP2B6, dibenzylfluorescein for CYP2C8, 7-methoxy-4-trifluoromethylcoumarin (MFC) for CYP2C9 and CYP2E1, 3-[2-(N,N-diethyl-N-methylammonium)ethyl]-7-methoxy-4-methylcoumarin for CYP2D6, and 7-benzyloxy-4-trifluoromethylcoumarin for CYP3A4. Fluorescence-based assays are highly sensitive and allow the simultaneous measurement of a large number of samples using plate readers, thus enhancing sample throughput. Major advantages over high-throughput assays in subcellular fractions are that, as living cells are used, manual handling and enzyme damage are minimized, the endoplasmic reticulum of the cells remains intact, exogenous cofactors or NADPH-regenerating systems are not required, and transport processes are maintained. These assays can be applied to preliminary screening of inhibitory effects of new drugs on individual P450 enzymes. After comparison of the results obtained using the fluorescent probes in intact P450-expressing cells and those obtained using the high-performance liquid chromatography-based selective assays in the same cells, in primary human hepatocytes or in human liver microsomes, a fairly good agreement was found.

[1]  A. Li,et al.  Applications of primary human hepatocytes in the evaluation of pharmacokinetic drug-drug interactions: Evaluation of model drugs terfenadine and rifampin , 1997, Cell Biology and Toxicology.

[2]  R. Laufer,et al.  Demethylation of radiolabelled dextromethorphan in rat microsomes and intact hepatocytes. , 2003, European journal of biochemistry.

[3]  David Raunig,et al.  In vitro drug interactions of cytochrome p450: an evaluation of fluorogenic to conventional substrates. , 2003, Drug metabolism and disposition: the biological fate of chemicals.

[4]  Y. Ohno,et al.  In vitro activation of 7-benzyloxyresorufin O-debenzylation and nifedipine oxidation in human liver microsomes , 2003, Xenobiotica; the fate of foreign compounds in biological systems.

[5]  S. D. Turner,et al.  Cytochrome P450 fluorometric substrates: identification of isoform-selective probes for rat CYP2D2 and human CYP3A4. , 2002, Drug metabolism and disposition: the biological fate of chemicals.

[6]  Y. Kohno,et al.  Application of microtiter plate assay to evaluate inhibitory effects of various compounds on nine cytochrome P450 isoforms and to estimate their inhibition patterns. , 2002, Drug metabolism and pharmacokinetics.

[7]  K. Bateman,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. , 2001, Drug metabolism and disposition: the biological fate of chemicals.

[8]  M. Namba,et al.  Establishment of the transformants expressing human cytochrome P450 subtypes in HepG2, and their applications on drug metabolism and toxicology. , 2001, Toxicology in vitro : an international journal published in association with BIBRA.

[9]  A. Nomeir,et al.  Inhibition of CYP3A4 in a rapid microtiter plate assay using recombinant enzyme and in human liver microsomes using conventional substrates. , 2001, Drug metabolism and disposition: the biological fate of chemicals.

[10]  C. Masimirembwa,et al.  Application of higher throughput screening (HTS) inhibition assays to evaluate the interaction of antiparasitic drugs with cytochrome P450s. , 2001, Drug metabolism and disposition: the biological fate of chemicals.

[11]  S. D. Turner,et al.  Substrate-dependent modulation of CYP3A4 catalytic activity: analysis of 27 test compounds with four fluorometric substrates. , 2000, Drug metabolism and disposition: the biological fate of chemicals.

[12]  J. Venhorst,et al.  Evaluation of a novel high-throughput assay for cytochrome P450 2D6 using 7-methoxy-4-(aminomethyl)-coumarin. , 2000, European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences.

[13]  A. Y. Lu,et al.  Human cytochrome P-450 3A4: in vitro drug-drug interaction patterns are substrate-dependent. , 2000, Drug metabolism and disposition: the biological fate of chemicals.

[14]  R. J. Price,et al.  CYP isoform induction screening in 96-well plates: use of 7-benzyloxy-4-trifluoromethylcoumarin as a substrate for studies with rat hepatocytes , 2000, Xenobiotica; the fate of foreign compounds in biological systems.

[15]  Metabolism of 7-benzyloxy-4-trifluoromethylcoumarin by human hepatic cytochrome P450 isoforms , 2000, Xenobiotica; the fate of foreign compounds in biological systems.

[16]  J. Marois,et al.  Description of a 96-well plate assay to measure cytochrome P4503A inhibition in human liver microsomes using a selective fluorescent probe. , 1999, Analytical biochemistry.

[17]  J B Houston,et al.  CYP3A4 drug interactions: correlation of 10 in vitro probe substrates. , 1999, British journal of clinical pharmacology.

[18]  C. Masimirembwa,et al.  Heterologous expression and kinetic characterization of human cytochromes P-450: validation of a pharmaceutical tool for drug metabolism research. , 1999, Drug metabolism and disposition: the biological fate of chemicals.

[19]  M. T. Donato,et al.  Characterization of drug metabolizing activities in pig hepatocytes for use in bioartificial liver devices: comparison with other hepatic cellular models. , 1999, Journal of hepatology.

[20]  A. Pfeifer,et al.  High Expression of Human CYP2C in Immortalized Human Liver Epithelial Cells. , 1999, Toxicology in vitro : an international journal published in association with BIBRA.

[21]  Rodrigues Ad,et al.  Integrated Cytochrome P450 Reaction Phenotyping: Attempting to Bridge the Gap Between cDNA-expressed Cytochromes P450 and Native Human Liver Microsomes , 1999 .

[22]  A. D. Rodrigues,et al.  Integrated cytochrome P450 reaction phenotyping: attempting to bridge the gap between cDNA-expressed cytochromes P450 and native human liver microsomes. , 1999, Biochemical pharmacology.

[23]  A. Nomeir,et al.  Validation of a rapid microtiter plate assay to conduct cytochrome P450 2D6 enzyme inhibition studies , 1998 .

[24]  Y. Sugiyama,et al.  Prediction of pharmacokinetic alterations caused by drug-drug interactions: metabolic interaction in the liver. , 1998, Pharmacological reviews.

[25]  M. T. Donato,et al.  The Coumarin 7-Hydroxylation Microassay in Living Hepatic Cells in Culture , 1998, Alternatives to laboratory animals : ATLA.

[26]  D. Waxman,et al.  Human cytochrome P4502B6: interindividual hepatic expression, substrate specificity, and role in procarcinogen activation. , 1997, Drug metabolism and disposition: the biological fate of chemicals.

[27]  S. Ekins,et al.  Examination of purported probes of human CYP2B6. , 1997, Pharmacogenetics.

[28]  D. Acosta,et al.  Metabolism of ketoconazole and deacetylated ketoconazole by rat hepatic microsomes and flavin-containing monooxygenases. , 1997, Drug metabolism and disposition: the biological fate of chemicals.

[29]  B. W. Penman,et al.  Microtiter plate assays for inhibition of human, drug-metabolizing cytochromes P450. , 1997, Analytical biochemistry.

[30]  J. Cashman,et al.  Role of hepatic flavin-containing monooxygenase 3 in drug and chemical metabolism in adult humans. , 1995, Chemico-biological interactions.

[31]  K. Korzekwa,et al.  Cytochromes P450 expression systems. , 1995, Annual review of pharmacology and toxicology.

[32]  J. Buters,et al.  A highly sensitive tool for the assay of cytochrome P450 enzyme activity in rat, dog and man. Direct fluorescence monitoring of the deethylation of 7-ethoxy-4-trifluoromethylcoumarin. , 1993, Biochemical pharmacology.

[33]  P. Thomas,et al.  Methoxyresorufin and benzyloxyresorufin: substrates preferentially metabolized by cytochromes P4501A2 and 2B, respectively, in the rat and mouse. , 1993, Biochemical pharmacology.

[34]  J V Castell,et al.  A microassay for measuring cytochrome P450IA1 and P450IIB1 activities in intact human and rat hepatocytes cultured on 96-well plates. , 1993, Analytical biochemistry.

[35]  A. Pfeifer,et al.  Simian virus 40 large tumor antigen-immortalized normal human liver epithelial cells express hepatocyte characteristics and metabolize chemical carcinogens. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[36]  T. Shimada,et al.  Purification and characterization of human liver microsomal cytochrome P-450 2A6. , 1991, Molecular pharmacology.

[37]  I. White.  A continuous fluorometric assay for cytochrome P-450-dependent mixed function oxidases using 3-cyano-7-ethoxycoumarin. , 1988, Analytical biochemistry.

[38]  H Babich,et al.  Comparisons of two in vitro cytotoxicity assays-The neutral red (NR) and tetrazolium MTT tests. , 1988, Toxicology in vitro : an international journal published in association with BIBRA.

[39]  R. Mayer,et al.  Ethoxy-, pentoxy- and benzyloxyphenoxazones and homologues: a series of substrates to distinguish between different induced cytochromes P-450. , 1985, Biochemical pharmacology.

[40]  R. Tallarida,et al.  Manual of Pharmacologic Calculations: With Computer Programs , 1984 .

[41]  R. H. Walmsley,et al.  Manual of pharmacologic calculations with computer programs , 1982 .

[42]  Joseph F. Williams.  Annual Review of Pharmacology , 1975 .

[43]  O. H. Lowry,et al.  Protein measurement with the Folin phenol reagent. , 1951, The Journal of biological chemistry.