A comprehensive analysis was performed to investigate how inhibition of the human bile salt export pump (BSEP/ABCB11) relates to clinically observed drug-induced liver injury (DILI). Inhibition of taurocholate (TA) transport was investigated in BSEP membrane vesicles for a data set of 250 compounds, and 86 BSEP inhibitors were identified. Structure-activity modeling identified BSEP inhibition to correlate strongly with compound lipophilicity, whereas positive molecular charge was associated with a lack of inhibition. All approved drugs in the data set (n = 182) were categorized according to DILI warnings in drug labels issued by the Food and Drug Administration, and a strong correlation between BSEP inhibition and DILI was identified. As many as 38 of the 61 identified BSEP inhibitors were associated with severe DILI, including 9 drugs not previously linked to BSEP inhibition. Further, among the tested compounds, every second drug associated with severe DILI was a BSEP inhibitor. Finally, sandwich-cultured human hepatocytes (SCHH) were used to investigate the relationship between BSEP inhibition, TA transport, and clinically observed DILI in detail. BSEP inhibitors associated with severe DILI greatly reduced the TA canalicular efflux, whereas BSEP inhibitors with less severe or no DILI resulted in weak or no reduction of TA efflux in SCHH. This distinction illustrates the usefulness of SCHH in refined analysis of BSEP inhibition. In conclusion, BSEP inhibition in membrane vesicles was found to correlate to DILI severity, and altered disposition of TA in SCHH was shown to separate BSEP inhibitors associated with severe DILI from those with no or mild DILI.

Hepatocytes play a pivotal role in human homeostasis. They are essential in regulation of glucose and lipid levels in blood and play a central role in metabolism of amino acids, lipids, drugs and xenobiotic-compounds. In addition, hepatocytes produce a major portion of proteins circulating in the blood. Hepatocytes were isolated from liver tissue obtained from surgical resections. Proteins were extracted and processed using filter aided sample preparation protocol and were analyzed by LC-MS/MS using high accuracy mass spectrometry. Proteins were quantified by the 'Total Protein Approach' and 'Proteomic Ruler'. We report a comprehensive proteomic analysis of purified human hepatocytes and the human hepatoma cell line HepG2. The complete dataset comprises 9400 proteins and provides a comprehensive and quantitative depiction of the proteomes of hepatocytes and HepG2 cells at the protein titer and copy number dimensions. We describe basic cell organization and in detail energy metabolism pathways and metabolite transport. We provide quantitative insights into protein synthesis and drug and xenobiotics catabolism. Our data delineate differences between the native human hepatocytes and HepG2 cells by providing for the first time quantitative data at protein concentrations and copy numbers.

Many different methods are used for mass-spectrometry-based protein quantification in pharmacokinetics and systems pharmacology. It has not been established to what extent the results from these various methods are comparable. Here, we compared six different mass spectrometry-based proteomics methods by measuring the expression of clinically relevant drug transporters and metabolizing enzymes in human liver. Mean protein concentrations were in general quantified to similar levels by methods using whole tissue lysates. Methods using subcellular membrane fractionation gave incomplete enrichment of the proteins. When the enriched proteins were adjusted to levels in whole tissue lysates, they were on average 4-fold lower than those quantified directly in whole tissue lysates. The differences in protein levels were propagated into differences in predictions of hepatic clearance. In conclusion, caution is needed when comparing and applying quantitative proteomics data obtained with different methods, especially since membrane fractionation is common practice for protein quantification used in drug clearance predictions.