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

Abstract

ABSTRACT: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 M 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 drugmetabolizing 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.

Upcyte Human Hepatocytes: a Potent In Vitro Tool for the Prediction of Hepatic Clearance of Metabolically Stable Compounds

“…A quantity amounting to 400 ng total RNA per sample was reverse transcribed for first-strand cDNA synthesis following the protocol for High Capacity cDNA RT Kit (Applied Biosystems, Foster City, CA). Relative mRNA expression of main P450s were analyzed by quantitative real-time PCR, as published previously (Schaefer et al, 2012), using custom-made primers (Biomers GmbH, Ulm, Germany) and inventoried TaqMan gene expression assays (Life Technologies/AB).…”

Interindividual Variability in Hepatic Organic Anion-Transporting Polypeptides and P-Glycoprotein (ABCB1) Protein Expression: Quantification by Liquid Chromatography Tandem Mass Spectroscopy and Influence of Genotype, Age, and Sex

“…We (Deo et al, 2012;Prasad et al, 2013) and others Bi et al, 2012;Kimoto et al, 2012;Ohtsuki et al, 2012;Schaefer et al, 2012;Tucker et al, 2012) have reported data on the expression of some of these hepatic transporters. Here we extended these studies to determine 1) the interindividual variability in expression of OATP1B1 (SLCO1B1), OATP1B3 (SLCO1B3), OATP2B1 (SLCO2B1), and P-gp (ABCB1) in a large set (n = 64) of human liver samples; and 2) the influence of genotype, age, and sex on such expression.…”

Hepatic Basolateral Efflux Contributes Significantly to Rosuvastatin Disposition I: Characterization of Basolateral Versus Biliary Clearance Using a Novel Protocol in Sandwich-Cultured Hepatocytes

“…Therefore, the effects of these changes may have minimal overall impact on RSV CL Bile . Available data suggest that expression of MRP3 and MRP4 in rat and human SCH may be relatively stable over days in culture (Swift et al, 2010a;Ohtsuki et al, 2012;Schaefer et al, 2012). However, robust quantitative proteomics data for transport proteins that mediate CL BL are not yet available.…”