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Verinurad is a selective uric acid transporter 1 (URAT1) inhibitor in development for the treatment of chronic kidney disease and heart failure. In humans, two major acyl glucuronide metabolites have been identified: direct glucuronide M1 and N-oxide glucuronide M8. Using in vitro systems recommended by regulatory agencies, we evaluated the interactions of verinurad, M1, and M8 with major drug-metabolizing enzymes and transporters and the potential for clinically relevant drug-drug interactions (DDIs). The IC50 for inhibition of CYP2C8, CYP2C9, and CYP3A4/5 for verinurad was ≥14.5 µM, and maximum free plasma concentration (Iu,max)/IC50 was

Human hepatocytes show marked differences in cell size, gene expression, and function throughout the liver lobules, an arrangement termed liver zonation. However, it is not clear if these zonal size differences, and the associated phenotypic differences, are retained in isolated human hepatocytes, the "gold standard" for in vitro studies of human liver function. Here, we therefore explored size differences among isolated human hepatocytes and investigated whether separation by size can be used to study liver zonation in vitro. We used counterflow centrifugal elutriation to separate cells into different size fractions and analyzed them with label-free quantitative proteomics, which revealed an enrichment of 151 and 758 proteins (out of 5163) in small and large hepatocytes, respectively. Further analysis showed that protein abundances in different hepatocyte size fractions recapitulated the in vivo expression patterns of previously described zonal markers and biological processes. We also found that the expression of zone-specific cytochrome P450 enzymes correlated with their metabolic activity in the different fractions. In summary, our results show that differences in hepatocyte size matches zonal expression patterns, and that our size fractionation approach can be used to study zone-specific liver functions in vitro.

3D cultures of primary human hepatocytes (PHH) are emerging as a more in vivo-like culture system than previously available hepatic models. This work describes the characterisation of drug metabolism in 3D PHH spheroids. Spheroids were formed from three different donors of PHH and the expression and activities of important cytochrome P450 enzymes (CYP1A2, 2B6, 2C9, 2D6, and 3A4) were maintained for up to 21 days after seeding. The activity of CYP2B6 and 3A4 decreased, while the activity of CYP2C9 and 2D6 increased over time (P 0.05). For six test compounds, that are metabolised by multiple enzymes, intrinsic clearance (CL

Disease progression to nonalcoholic steatohepatitis (NASH) has profound effects on the expression and function of drug-metabolizing enzymes and transporters, which provide a mechanistic basis for variable drug response. Breast cancer resistance protein (BCRP), a biliary efflux transporter, exhibits increased liver mRNA expression in NASH patients and preclinical NASH models, but the impact on function is unknown. It was shown that the transport capacity of multidrug resistance protein 2 (MRP2) is decreased in NASH. SN-38, the active irinotecan metabolite, is reported to be a substrate for Bcrp, whereas SN-38 glucuronide (SN-38G) is a Mrp2 substrate. The purpose of this study was to determine the function of Bcrp in NASH through alterations in the disposition of SN-38 and SN-38G in a Bcrp knockout (Bcrp(−/−) KO) and methionine- and choline-deficient (MCD) model of NASH. Sprague Dawley [wild-type (WT)] rats and Bcrp(−/−) rats were fed either a methionine- and choline-sufficient (control) or MCD diet for 8 weeks to induce NASH. SN-38 (10 mg/kg) was administered i.v., and blood and bile were collected for quantification by liquid chromatography–tandem mass spectrometry. In Bcrp(−/−) rats on the MCD diet, biliary efflux of SN-38 decreased to 31.9%, and efflux of SN-38G decreased to 38.7% of control, but WT-MCD and KO-Control were unaffected. These data indicate that Bcrp is not solely responsible for SN-38 biliary efflux, but rather implicate a combined role for BCRP and MRP2. Furthermore, the disposition of SN-38 and SN-38G is altered by Bcrp(−/−) and NASH in a gene-by-environment interaction and may result in variable drug response to irinotecan therapy in polymorphic patients.

Organic anion transporter (OAT) 2 and OAT7 were recently shown to be involved in the hepatic uptake of drugs; however, there is limited understanding of the population variability in the expression of these transporters in liver. There is also a need to derive relative expression-based scaling factors (REFs) that can be used to bridge in vitro functional data to the in vivo drug disposition. To this end, we quantified OAT2 and OAT7 surrogate peptide abundance in a large number of human liver tissue samples ( n = 52), as well as several single-donor cryopreserved human hepatocyte lots ( n = 30) by a novel, validated liquid chromatography tandem mass spectrometry (LC-MS/MS) method. The average surrogate peptide expression of OAT2 and OAT7 in the liver samples was 1.52 ± 0.57 and 4.63 ± 1.58 fmol/μg membrane protein, respectively. While we noted statistically significant differences ( p < 0.05) in hepatocyte and liver tissue abundances for both OAT2 and OAT7, the differences were relatively small (1.8- and 1.5-fold difference in median values, respectively). Large interindividual variability was noted in the hepatic expression of OAT2 (16-fold in liver tissue and 23-fold in hepatocytes). OAT7, on the other hand, showed less interindividual variability (4-fold) in the livers, but high variability for the hepatocyte lots (27-fold). A significant positive correlation in OAT2 and OAT7 expression was observed, but expression levels were neither associated with age nor sex. In conclusion, our data suggest marked interindividual variability in the hepatic expression of OAT2/7, which may contribute to the pharmacokinetic variability of their substrates. Because both transporters were less abundant in hepatocytes than livers, a REF-based approach is recommended when scaling in vitro hepatocyte transport data to predict hepatic drug clearance and liver exposure of OAT2/7 substrates.

Targeted protein quantification using liquid chromatography-tandem mass spectrometry with stable isotope-labeled standards is recognized as the gold standard of practice for protein quantification. Such assays, however, can only cover a limited number of proteins, and developing targeted methods for larger numbers of proteins requires substantial investment. Alternatively, large-scale global proteomic experiments along with computational methods such as the "total protein approach" (TPA) have the potential to provide extensive protein quantification. In this study, we compared the TPA-based quantitation of seven major hepatic uptake transporters in four human liver tissue samples using global proteomic data obtained from two multiplexed tandem mass tag experiments (performed in two independent laboratories) to the quantitative data from targeted proteomic assays. The TPA-based quantitation of these hepatic transporters [sodium-taurocholate cotransporting polypeptide (NTCP/SLC10A1), organic anion transporter 2 (OAT2/SLC22A7), OAT7/SLC22A9, organic anion-transporting polypeptide 1B1 (OATP1B1/SLCO1B1), OATP1B3/SLCO1B3, OATP2B1/SLCO2B1, and organic cation transporter (OCT1/SLC22A1)] showed good-to-excellent correlations (Pearson r = 0.74-1.00) to the targeted data. In addition, the values were similar to those measured by targeted proteomics with 71% and 86% of the data sets falling within 3-fold of the targeted data. A comparison of the TPA-based quantifications of enzyme abundances to available literature data showed that the majority of the enzyme quantifications fell within the reference data intervals. In conclusion, these results demonstrate the capability of multiplexed global proteomic experiments to detect differences in protein expression between samples and provide reasonable estimations of protein expression levels.

Tolbutamide is primarily metabolized by CYP2C9, and, thus, is frequently applied as a clinical probe substrate for CYP2C9 activity. However, there is a marked discrepancy in the in vitro-in vivo extrapolation of its metabolic clearance, implying a potential for additional clearance mechanisms. The goal of this study was to evaluate the role of hepatic uptake transport in the pharmacokinetics of tolbutamide and to identify the molecular mechanism thereof. Transport studies using singly transfected cells expressing six major hepatic uptake transporters showed that tolbutamide is a substrate to organic anion transporter 2 (OAT2) alone with transporter affinity [Michaelis-Menten constant (Km)] of 19.5 ± 4.3 µM. Additionally, OAT2-specific transport was inhibited by ketoprofen (an OAT2 inhibitor) and 1 mM rifamycin SV (pan inhibitor), but not by cyclosporine and rifampicin (OAT polypeptides/Na+-taurocholate cotransporting polypeptide inhibitors). Uptake studies in primary human hepatocytes confirmed the predominant role of OAT2 in the active uptake with significant inhibition by rifamycin SV and ketoprofen, but not by the other inhibitors. Concentration-dependent uptake was noted in human hepatocytes with active transport characterized by Km and Vmax values of 39.3 ± 6.6 µM and 426 ± 30 pmol/min per milligram protein, respectively. Bottom-up physiologically based pharmacokinetic modeling was employed to verify the proposed role of OAT2-mediated hepatic uptake. In contrast to the rapid equilibrium (CYP2C9-only) model, the permeability-limited (OAT2-CYP2C9 interplay) model better described the plasma concentration-time profiles of tolbutamide. Additionally, the latter well described tolbutamide pharmacokinetics in carriers of CYP2C9 genetic variants and quantitatively rationalized its known drug-drug interactions. Our results provide first-line evidence for the role of OAT2-mediated hepatic uptake in the pharmacokinetics of tolbutamide, and imply the need for additional clinical studies in this direction.

Understanding transporter-mediated drug disposition and pharmacokinetics (PK) in patients with nonalcoholic fatty liver disease (NAFLD) is critical in developing treatment options. Here, we quantified the expression levels of major drug transporters in healthy, steatosis, and nonalcoholic steatohepatitis (NASH) liver samples, via liquid-chromatography tandem mass spectrometry-based proteomics, and used the data to predict the PK of substrate drugs in the disease state. Expression of organic anion transporting polypeptides (OATPs) and multidrug resistance-associated protein (MRP)2 is significantly lower in NASH livers; whereas MRP3 is induced while no change was observed for organic cation transporter (OCT)1. Physiologically-based pharmacokinetic models verified with PK data from healthy subjects well recovered the PK in NASH subjects for morphine (involving OCT1) and its glucuronide metabolites (MRP2/MRP3/OATP1B), 99m TC-mebrofenen (OATP1B/MRP2/MRP3), and rosuvastatin (OATP1B/breast cancer resistance protein). Overall, considerations to altered protein expression can enable quantitative prediction of PK changes in subjects with NAFLD.

Freshly isolated human hepatocytes are considered the gold standard for in vitro studies of liver functions, including drug transport, metabolism, and toxicity. For accurate predictions of the in vivo outcome, the isolated hepatocytes should reflect the phenotype of their in vivo counterpart, i.e., hepatocytes in human liver tissue. Here, we quantified and compared the membrane proteomes of freshly isolated hepatocytes and human liver tissue using a label-free shotgun proteomics approach. A total of 5144 unique proteins were identified, spanning over 6 orders of magnitude in abundance. There was a good global correlation in protein abundance. However, the expression of many plasma membrane proteins was lower in the isolated hepatocytes than in the liver tissue. This included transport proteins that determine hepatocyte exposure to many drugs and endogenous compounds. Pathway analysis of the differentially expressed proteins confirmed that hepatocytes are exposed to oxidative stress during isolation and suggested that plasma membrane proteins were degraded via the protein ubiquitination pathway. Finally, using pitavastatin as an example, we show how protein quantifications can improve in vitro predictions of in vivo liver clearance. We tentatively conclude that our data set will be a useful resource for improved hepatocyte predictions of the in vivo outcome.

Differences in the expression and function of the organic anion transporting polypeptide (OATP) transporters contribute to interindividual variability in atorvastatin clearance. However, the importance of the bile acid transporter sodium taurocholate cotransporting polypeptide (NTCP, SLC10A1) in atorvastatin uptake clearance (CLupt) is not yet clarified. To elucidate this issue, we investigated the relative contribution of NTCP, OATP1B1, OATP1B3, and OATP2B1 to atorvastatin CLupt in 12 human liver samples. The impact of inhibition on atorvastatin CLupt was also studied, using inhibitors of different isoform specificities. Expression levels of the four transport proteins were quantified by liquid chromatography tandem mass spectrometry. These data, together with atorvastatin in vitro kinetics, were used to predict the maximal transport activity (MTA) and interindividual differences in CLupt of each transporter in vivo. Subsequently, hepatic uptake impairment on coadministration of five clinically interacting drugs was predicted using in vitro inhibitory potencies. NTCP and OATP protein expression varied 3.7- to 32-fold among the 12 sample donors. The rank order in expression was OATP1B1 > OATP1B3 ≈ NTCP ≈ OATP2B1. NTCP was found to be of minor importance in atorvastatin disposition. Instead, OATP1B1 and OATP1B3 were confirmed as the major atorvastatin uptake transporters. The average contribution to atorvastatin uptake was OATP1B1 > OATP1B3 >> OATP2B1 > NTCP, although this rank order varied among individuals. The interindividual differences in transporter expression and CLupt resulted in marked differences in drug-drug interactions due to isoform-specific inhibition. We conclude that this variation should be considered in in vitro to in vivo extrapolations.

1. Penciclovir, ganciclovir, creatinine, para-aminohippuric acid (PAH), ketoprofen, estrone 3-O-sulfate (E3S), dehydroepiandrosterone 3-O-sulfate (DHEAS) and cyclic guanosine monophosphate (cGMP) were screened as substrates of human liver organic anion transporters OAT2 and OAT7. 2. For OAT7, high uptake ratios (versus mock transfected HEK293 cells) of 29.6 and 15.3 were obtained with E3S and DHEAS. Less robust uptake ratios (≤3.6) were evident with the other substrates. OAT2 (transcript variant 1, OAT2-tv1) presented high uptake ratios of 30, 13, ∼35, ∼25, 8.5 and 9 with cGMP, PAH, penciclovir, ganciclovir, creatinine and E3S, respectively. No uptake was observed with DHEAS. 3. Although not a substrate of either transporter, ketoprofen did inhibit transfected OAT2-tv1 (IC50 of 17, 22, 23, 24, 35 and 586 μM; creatinine, ganciclovir, penciclovir, cGMP, E3S and prostaglandin F2α, respectively) and penciclovir uptake (IC50 = 27 µM; >90% inhibition) by plated human hepatocytes (PHH). 4. It is concluded that penciclovir and ketoprofen may serve as useful tools for the assessment of OAT2 activity in PHH. However, measurement of OAT7 activity therein will prove more challenging, as high uptake rates are evident with E3S and DHEAS only and both sulfoconjugates are known to be substrates of organic anion transporting polypeptides.

Glyburide is widely used for the treatment of type 2 diabetes. We studied the mechanisms involved in the disposition of glyburide and its pharmacologically active hydroxy metabolites M1 and M2b and evaluated their clinical pharmacokinetics and the potential role in glyburide-induced cholestasis employing physiologically based pharmacokinetic (PBPK) modeling. Transport studies of parent and metabolites in human hepatocytes and transfected cell systems imply hepatic uptake mediated by organic anion-transporting polypeptides. Metabolites are also subjected to basolateral and biliary efflux by P-glycoprotein, breast cancer resistance protein, and multidrug resistance-associated proteins, and are substrates to renal organic anion transporter 3. A PBPK model in combination with a Bayesian approach was developed considering the identified disposition mechanisms. The model reasonably described plasma concentration time profiles and urinary recoveries of glyburide and the metabolites, implying the role of multiple transport processes in their pharmacokinetics. Predicted free liver concentrations of the parent (∼30-fold) and metabolites (∼4-fold) were higher than their free plasma concentrations. Finally, all three compounds showed bile salt export pump inhibition in vitro; however, significant in vivo inhibition was not apparent for any compound on the basis of a predicted unbound liver exposure-response effect model using measured in vitro IC50 values. In conclusion, this study demonstrates the important role of multiple drug transporters in the disposition of glyburide and its active metabolites, suggesting that variability in the function of these processes may lead to pharmacokinetic variability in the parent and the metabolites, potentially translating to pharmacodynamic variability.

Transporter-mediated hepatic uptake is proven to be the rate-determining step in the systemic clearance of several drugs. Therefore, accurate measurement of active and passive uptake clearances in vitro is critical to facilitate pharmacokinetics and drug-drug interaction predictions. Here, we evaluated the plated human hepatocytes (PHH) and studied the effect of incubation temperature and inhibitor concentration on uptake measurements, in order to reliably estimate hepatic uptake components. Uptake rates measured using PHH, at 37°C without and with rifamycin SV, were comparable with those obtained from suspension hepatocytes and sandwich-cultured hepatocytes for a set of 10–13 compounds. Apparent permeability across monolayers of low-efflux Madin-Darby canine kidney cells was measured at 4, 10, and 37°C. Of the 23 compounds evaluated, 13 compounds showed >2-fold reduction in passive permeability at 4°C compared to 37°C, inferring that low-temperature incubations may underestimate passive uptake. Inhibition studies using transporter-transfected cells suggested that ∼20 μM rifamycin SV completely inhibited organic anion-transporting polypeptides (OATPs), while no significant inhibition was noted for other hepatic uptake transporters. On the basis of inhibition profiles, the contribution of active versus passive and OATP versus non-OATP transport to the PHH uptake was discerned for various endogenous substrates and statins. With the exception of fluvastatin, the statins studied were predominantly transported by OATPs in PHH and the non-OATP transporters, such as Na+-taurocholate co-transporting polypeptide, played a minimal role. In conclusion, PHH is useful for uptake measurements, and rifamycin SV employed at different concentrations can reliably estimate active and passive uptake and characterize OATP-dependent active uptake.

(1) Oxaliplatin-based chemotherapy for colorectal cancer liver metastasis is associated with sinusoidal injury of liver parenchyma. The effects of oxaliplatin-induced liver injury on the protein level remain unknown. (2) Protein expression in liver tissue was analyzed-from eight patients treated with FOLFOX (combination of fluorouracil, leucovorin, and oxaliplatin) and seven controls-by label-free liquid chromatography mass spectrometry. Recursive feature elimination-support vector machine and Welch t-test were used to identify classifying and relevantly changed proteins, respectively. Resulting proteins were analyzed for associations with gene ontology categories and pathways. (3) A total of 5891 proteins were detected. A set of 184 (3.1%) proteins classified the groups with a 20% error rate, but relevant change was observed only in 55 (0.9%) proteins. The classifying proteins were associated with changes in DNA replication (p < 0.05) through upregulation of the minichromosome maintenance complex and with the innate immune response (p < 0.05). The importance of DNA replication changes was supported by the results of Welch t-test (p < 0.05). (4) Six weeks after FOLFOX treatment, less than 1% of identified proteins showed changes in expression associated with DNA replication, cell cycle entry, and innate immune response. We hypothesize that the changes remain after recovery from FOLFOX treatment injury.

The hepatic organic anion transporting polypeptides (OATPs) influence the pharmacokinetics of several drug classes and are involved in many clinical drug–drug interactions. Predicting potential interactions with OATPs is, therefore, of value. Here, we developed in vitro and in silico models for identification and prediction of specific and general inhibitors of OATP1B1, OATP1B3, and OATP2B1. The maximal transport activity (MTA) of each OATP in human liver was predicted from transport kinetics and protein quantification. We then used MTA to predict the effects of a subset of inhibitors on atorvastatin uptake in vivo. Using a data set of 225 drug-like compounds, 91 OATP inhibitors were identified. In silico models indicated that lipophilicity and polar surface area are key molecular features of OATP inhibition. MTA predictions identified OATP1B1 and OATP1B3 as major determinants of atorvastatin uptake in vivo. The relative contributions to overall hepatic uptake varied with isoform specificities of the inhibitors.

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. (C) 2016 The Authors. Published by Elsevier B.V.

Isolated human hepatocytes are commonly used to predict transporter-mediated clearance in vivo. Such predictions, however, do not provide estimations of transporter contributions and consequently do not allow predictions of the outcome resulting from a change in the activity of a certain transporter, for example, by inhibition or a genetic variant with reduced function. Pitavastatin is a drug that is heavily dependent on hepatic transporters for its elimination, and it is excreted mainly as unchanged drug in the bile. For this reason, pitavastatin was used as a model drug to demonstrate the applicability of a bottom-up approach to predict transporter-mediated disposition in sandwich-cultured human hepatocytes (SCHHs), allowing for the estimation of transporter contributions. Transport experiments in transfected human embryonic kidney cells (HEK293 cell lines) and inverted membrane vesicles overexpressing each of the relevant transport proteins were used to generate parameter estimates for the mechanistic model. By adjusting for differences in transporter abundance between the in vitro systems and individual SCHH batches, the model successfully predicted time profiles of medium and intracellular accumulation. Our predictions of pitavastatin bile accumulation could not be confirmed, however, because of a very low biliary excretion of pitavastatin in relation to the hepatic uptake in our SCHHs. This study is, to our knowledge, the first to successfully simulate transporter-mediated processes in a complex system such as SCHHs at the level of individual transport proteins using a bottom-up approach.