Absolute Quantification and Differential Expression of Drug Transporters, Cytochrome P450 Enzymes, and UDP-Glucuronosyltransferases in Cultured Primary Human Hepatocytes

The levels of metabolizing enzymes and transporters expressed in hepatocytes are decisive factors for hepatobiliary disposition of most drugs. Induction via nuclear receptor activation can significantly alter those levels, with the coregulation of multiple enzymes and transporters occurring to different extents. Here, we report the use of a targeted liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) method for concurrent quantification of multiple cytochrome P450 (P450), UDP-glucuronosyltransferase (UGT), and transporter proteins in cultured primary human hepatocytes. The effects of culture format (i.e., sandwich culture versus conventional culture) and of dexamethasone (DEX) media concentrations on mRNA, protein, and activity levels were determined for three donors, and protein expression was compared with that in liver. In general, P450 and UGT expression was lower in hepatocyte cultures than that in liver, and CYP2C9 was found to be the most abundant P450 isoform expressed in cultured hepatocytes. The sandwich culture format and 0.1 μM DEX in media retained the protein expression in the hepatocytes closest to the levels found in liver. However, higher in vitro expression was observed for drug transporters, especially for multidrug resistance protein 1 and breast cancer resistance protein. Direct protein quantification was applied successfully to study in vitro induction in sandwich cultured primary hepatocytes in a 24-well format using the prototypical inducers rifampicin, omeprazole, and phenobarbital. We conclude that targeted absolute LC-MS/MS quantification of drug-metabolizing enzymes and transporters can broaden the scope and significantly increase the impact of in vitro drug metabolism studies, such as induction, as an important supplement or future alternative to mRNA and activity data.

[1]  A. Hsu,et al.  Ritonavir. Clinical pharmacokinetics and interactions with other anti-HIV agents. , 1998, Clinical pharmacokinetics.

[2]  R. Obach,et al.  Cytochrome P450 3A4 mRNA Is a More Reliable Marker than CYP3A4 Activity for Detecting Pregnane X Receptor-Activated Induction of Drug-Metabolizing Enzymes , 2010, Drug Metabolism and Disposition.

[3]  Yuichi Sugiyama,et al.  Transporters as a determinant of drug clearance and tissue distribution. , 2006, European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences.

[4]  C. Lindley,et al.  In vivo and in vitro induction of human cytochrome P4503A4 by dexamethasone , 2000, Clinical pharmacology and therapeutics.

[5]  A. D. Rodrigues,et al.  GLUCURONIDATION CONVERTS GEMFIBROZIL TO A POTENT, METABOLISM-DEPENDENT INHIBITOR OF CYP2C8: IMPLICATIONS FOR DRUG-DRUG INTERACTIONS , 2006, Drug Metabolism and Disposition.

[6]  T. Terasaki,et al.  Quantitative targeted absolute proteomics-based ADME research as a new path to drug discovery and development: methodology, advantages, strategy, and prospects. , 2011, Journal of pharmaceutical sciences.

[7]  Sean Kim,et al.  CYP3A4 induction by drugs: correlation between a pregnane X receptor reporter gene assay and CYP3A4 expression in human hepatocytes. , 2002, Drug metabolism and disposition: the biological fate of chemicals.

[8]  K. Shakesheff,et al.  Efficient assessment of the utility of immortalized Fa2N-4 cells for cytochrome P450 (CYP) induction studies using multiplex quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and substrate cassette methodologies , 2008, Xenobiotica; the fate of foreign compounds in biological systems.

[9]  J. Pascussi,et al.  Dual effect of dexamethasone on CYP3A4 gene expression in human hepatocytes. Sequential role of glucocorticoid receptor and pregnane X receptor. , 2001, European journal of biochemistry.

[10]  Tetsuya Terasaki,et al.  Reliability and robustness of simultaneous absolute quantification of drug transporters, cytochrome P450 enzymes, and Udp-glucuronosyltransferases in human liver tissue by multiplexed MRM/selected reaction monitoring mode tandem mass spectrometry with nano-liquid chromatography. , 2011, Journal of pharmaceutical sciences.

[11]  Vikram Sinha,et al.  In Vitro and in Vivo Induction of Cytochrome P450: A Survey of the Current Practices and Recommendations: A Pharmaceutical Research and Manufacturers of America Perspective , 2009, Drug Metabolism and Disposition.

[12]  M Jamei,et al.  Towards a quantitative framework for the prediction of DDIs arising from cytochrome P450 induction. , 2009, Current drug metabolism.

[13]  N. Hewitt,et al.  Induction of hepatic cytochrome P450 enzymes: methods, mechanisms, recommendations, and in vitro–in vivo correlations , 2007, Xenobiotica; the fate of foreign compounds in biological systems.

[14]  E L LeCluyse,et al.  Human hepatocyte culture systems for the in vitro evaluation of cytochrome P450 expression and regulation. , 2001, European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences.

[15]  Tetsuya Terasaki,et al.  Simultaneous absolute quantification of 11 cytochrome P450 isoforms in human liver microsomes by liquid chromatography tandem mass spectrometry with in silico target peptide selection. , 2011, Journal of pharmaceutical sciences.

[16]  J. Sahi,et al.  Current Industrial Practices in Assessing CYP450 Enzyme Induction: Preclinical and Clinical , 2008, The AAPS Journal.

[17]  E. Dratz,et al.  Absolute quantification of the G protein-coupled receptor rhodopsin by LC/MS/MS using proteolysis product peptides and synthetic peptide standards. , 2003, Analytical chemistry.

[18]  Monica H. Elliott,et al.  Current trends in quantitative proteomics. , 2009, Journal of mass spectrometry : JMS.

[19]  J. Sahi,et al.  Hepatocytes as a tool in drug metabolism, transport and safety evaluations in drug discovery. , 2010, Current drug discovery technologies.

[20]  K. Audus,et al.  Formation of extensive canalicular networks by rat hepatocytes cultured in collagen-sandwich configuration. , 1994, The American journal of physiology.

[21]  G. Hamilton,et al.  Isolation and culture of primary human hepatocytes. , 2005, Methods in molecular biology.

[22]  Tetsuya Terasaki,et al.  Quantitative Atlas of Membrane Transporter Proteins: Development and Application of a Highly Sensitive Simultaneous LC/MS/MS Method Combined with Novel In-silico Peptide Selection Criteria , 2008, Pharmaceutical Research.

[23]  Olivier Heudi,et al.  Towards absolute quantification of therapeutic monoclonal antibody in serum by LC-MS/MS using isotope-labeled antibody standard and protein cleavage isotope dilution mass spectrometry. , 2008, Analytical chemistry.

[24]  R. Riley,et al.  EVALUATION OF TIME-DEPENDENT CYTOCHROME P450 INHIBITION USING CULTURED HUMAN HEPATOCYTES , 2006, Drug Metabolism and Disposition.

[25]  Tetsuya Terasaki,et al.  Simultaneous Absolute Protein Quantification of Transporters, Cytochromes P450, and UDP-Glucuronosyltransferases as a Novel Approach for the Characterization of Individual Human Liver: Comparison with mRNA Levels and Activities , 2012, Drug Metabolism and Disposition.

[26]  Liang-Shang Gan,et al.  Effects of prototypical microsomal enzyme inducers on cytochrome P450 expression in cultured human hepatocytes. , 2003, Drug metabolism and disposition: the biological fate of chemicals.

[27]  M. Niemi,et al.  Membrane transporters in drug development , 2010, Nature Reviews Drug Discovery.

[28]  K. Brouwer,et al.  Sandwich-cultured hepatocytes: an in vitro model to evaluate hepatobiliary transporter-based drug interactions and hepatotoxicity , 2010, Drug metabolism reviews.