Targeted quantitative proteomic analysis of drug metabolizing enzymes and transporters by nano LC-MS/MS in the sandwich cultured human hepatocyte model

Abstract

Introduction: Sandwich-cultured human hepatocytes (SCHHs) are the most common in vitro hepatocyte model used for studying hepatic drug disposition and hepatotoxicity. Targeted quantification of key DME and transporter protein expression is useful for in vitro-in vivo extrapolation of drug and xenobiotic clearance and developing corresponding PBPK models. However, established methods for comprehensive quantification of drug metabolizing enzyme (DMEs) and transporter expression in SCHHs are lacking. In this study, a targeted quantitative proteomic isotope dilution nanoLC-MS/MS method developed in our laboratory was adapted to quantify a panel of phase I & II DMEs and transporter proteins in SCHHs under basal and induced conditions.

Methods: SCHHs were treated with known inducers of DMEs (Rifampin: PXR activator, CITCO: CAR activator) and transporters (CDCA: FXR activator) or with vehicle control (DMSO) for 72 h. Membrane protein was isolated from the SCHHs using a membrane extraction kit and 30 μg membrane protein was digested with trypsin. The resulting peptides were analyzed by isotope dilution nanoLC-MS/MS to quantify the DMEs and transporters.

Results: Using the method, we could quantify fourteen phase I and ten phase II DMEs, and twelve uptake/efflux transporters, under basal and induced conditions in the SCHHs. Analysis showed donor to donor variation in basal protein levels of CYP450s, UGTs and transporters, and that basal protein expression of CYP450s and UGTs was higher than that of transporters. In addition, induction of key proteins in response to rifampin, CITCO and CDCA was observed.

Discussion: We have successfully quantified protein abundance of multiple phase I and II DMEs and uptake and efflux transporters in SCHHs using a method previously developed in our laboratory. Our method is sufficiently sensitive to quantify inter-donor differences in protein concentrations at the basal level as well as changes in protein expression in response to endogenous and exogenous stimuli.

Keywords: Drug metabolizing enzymes; Hepatocytes; Proteomics; Quantification; SCHH model; Targeted; Transporters.

Fig. 1.. Representative total ion chromatogram for transporter peptides in trypsin digested membrane protein isolated from SCHHs (0.1 % DMSO treated sample).

Time is on the x-axis and response (cps) on the y-axis. 30 μg of total membrane protein was digested for 20 h and 0.1 μL (0.06 μg, 0.2% of the total) of the digest was injected onto the nanoAcquity-QTRAP 5500 system. Most of the visible peaks in the chromatogram represent heavy labeled proteotypic tryptic peptide standards and so the chromatograms for the UGT/CYP450, and CESs and Various, MRM methods look similar. Two MRMs were acquired per peptide. Heavy labeled and unlabeled peptides co elute. This is a zoom of the total ion chromatogram (8 to 23 min) for the transporters MRM method. Examples of extracted ion chromatograms for one peptide are shown in Figure S1.

Fig. 2.. Absolute protein quantification of key phase I DMEs at basal level in SCHHs.

Protein expression of 12 CYP isoforms and the membrane protein marker Na⁺/K⁺-ATPase (A), and carboxylesterase isoforms (B) under basal conditions are presented. Protein concentration was quantified in membrane protein fractionated from SCHHs from three donors, and in HLM QC samples analyzed in duplicate with each donor. Each SCHH data point represents DME protein concentration for an individual donor (n=2 biological replicates). Data are plotted on a log scale, and the dotted line indicates the lower limit of quantification.

Fig. 3.. Absolute protein quantification of UGTs at basal level in SCHHs.

Protein expression of 10 UGT isoforms and the membrane protein marker Na⁺/K⁺-ATPase under basal conditions are presented. Protein concentration was quantified in membrane protein fractionated from SCHHs from three donors, and in HLM QC samples analyzed in duplicate with each donor. Each SCHH data point represents DME protein concentration for an individual donor (n=2 biological replicates) and is plotted on a log scale. Dotted line indicates the lower limit of quantification.

Fig. 4.. Protein expression of uptake and efflux transporters at basal level in SCHHs.

Protein expression of 6 uptake and 6 efflux transporters and the membrane protein marker Na⁺/K⁺-ATPase under basal conditions are presented. Protein concentration was quantified in membrane protein fractionated from SCHHs from three donors, and in HLM QC samples analyzed in duplicate with each donor. Each SCHH data point represents transporter protein concentration for an individual donor (n=2 biological replicates) and is plotted on a log scale. Dotted line indicates the lower limit of quantification. Protein concentrations below 0.02 pmol/mg protein are identified by the dashed line.

Fig. 5.. Induction of key DME and transport proteins in SCHHs.

SCHHs from three donors were treated with vehicle control (0.1% DMSO) or known inducers for 72 h, and membrane protein was isolated. (A) Protein concentrations of CYP3A4, UGTs 1A1 and 1A4, and P-gp following treatment with rifampin (10 μM). (B). Protein concentration of CYP2B6 following treatment with CITCO (1.0 μM). (C) Protein concentration of BSEP following treatment with CDCA (100 μM). Data are expressed as the mean ± SEM fold change relative to vehicle control (n=6 per group; n=2 per donor). Student’s t-test, *p<0.05 versus DMSO. Inter-donor differences in induction are shown in Figure S2.