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Research ArticleArticle

Prediction of Organic Anion-Transporting Polypeptide 1B1- and 1B3-Mediated Hepatic Uptake of Statins Based on Transporter Protein Expression and Activity Data

Annett Kunze, Jörg Huwyler, Gian Camenisch and Birk Poller
Drug Metabolism and Disposition September 2014, 42 (9) 1514-1521; DOI: https://doi.org/10.1124/dmd.114.058412

Abstract

Organic anion-transporting polypeptides (OATP) 1B1 and OATP1B3 are drug transporters mediating the active hepatic uptake of their substrates. Because they exhibit overlapping substrate specificities, the contribution of each isoform to the net hepatic uptake needs to be considered when predicting drug-drug interactions. The relative contribution of OATP1B1- and OATP1B3-mediated uptake of statins into hepatocytes was estimated based on either relative transporter protein expression data or relative activity data. Therefore, kinetics of eight statins and OATP1B1- and OATP1B3-specific reference substrates was determined in OATP1B1- and OATP1B3-expressing human embryonic kidney 293 cells and in human cryopreserved hepatocytes. Absolute OATP1B1 and OATP1B3 protein abundance was determined by liquid chromatography-tandem mass spectrometry in all expression systems. Transporter activity data generated in recombinant cell lines were extrapolated to hepatocyte values using relative transporter expression factors (REF) or relative activity factors (RAF). Our results showed a pronounced OATP1B1 and comparatively low OATP1B3 protein expression in the investigated hepatocyte lot. Based on REF scaling, we demonstrated that the active hepatic uptake clearances of reference substrates, atorvastatin, pravastatin, rosuvastatin, and simvastatin were well predicted within twofold error, demonstrating that OATP1B1 and OATP1B3 were major contributors. For other statins, the net hepatic uptake clearance was underpredicted, suggesting the involvement of other hepatic uptake transporters. Summarized, we showed that REF- and RAF-based predictions were highly similar, indicating a direct transporter expression-activity relationship. Moreover, we demonstrated that the REF-scaling method provided a powerful tool to quantitatively assess the transporter-specific contributions to the net uptake clearance of statins in hepatocytes

Introduction

Human drug uptake transporters are membrane-bound proteins that facilitate the active cellular uptake of compounds that cannot cross cellular membranes by passive diffusion due to their physiochemical properties. Expressed at the basolateral membrane of hepatocytes, organic anion-transporting polypeptides (OATPs) mediate the uptake of mainly anionic drugs from the blood into the liver. OATP inhibition due to drug-drug interactions (DDI) can lead to increased plasma concentration levels of drugs, thus posing a potential risk for toxicity in peripheral organs. Following comedication involving hydroxymethylglutaryl-coenzyme A reductase inhibitors (statins), muscle toxicity and severe myopathy are reported risks that have been partially attributed to inhibition of OATPs (Staffa et al., 2002; Neuvonen et al., 2006; Shitara and Sugiyama, 2006). Thus, predicting the transporter-mediated DDI risk is a necessity for the development of new molecular entities.

Cryopreserved human hepatocytes are a common tool to assess the hepatic uptake of compounds. Hepatocytes coexpress various uptake transporters, including OATP1B1 (SLCO1B1), OATP1B3 (SLCO1B3), OATP2B1 (SLCO2B1), the organic anion transporter 2 (OAT2; SLC22A7), the organic cation transporter 1 (OCT1; SLC22A1), and the sodium taurocholate cotransporting polypeptide (NTCP; SLC10A1). Because a variety of compounds, including statins, exhibit an overlap of transporter specificity, compound uptake in hepatocytes reflects the sum of all transporter-specific contributions (Neuvonen et al., 2006; Shitara and Sugiyama, 2006; Noe et al., 2007; Kalliokoski and Niemi, 2009; Knauer et al., 2010; Bi et al., 2013; Shitara et al., 2013).

To assess the relative contribution of specific transporters to the net hepatic uptake in vitro, methods based on relative transporter expression and transporter activity have been introduced. Hirano et al. (2004) established a method that allows the estimation of the contribution of OATP1B1- and OATP1B3-mediated uptake in hepatocytes based on relative activity factors (RAF). Determined as ratios of the uptake transporter activity of transporter-specific substrates in hepatocytes relative to the activity in recombinant cell lines, the RAF method has been widely used to estimate the contribution of OATP1B1 and OATP1B3 to the hepatic uptake of various compounds (Hirano et al., 2004; Shimizu et al., 2005; Shitara and Sugiyama, 2006; Kitamura et al., 2008; Williamson et al., 2013). In addition, Hirano et al. (2004, 2006) used protein expression data from Western blot analysis to estimate relative expression factors (REFs) to determine the contribution of OATP1B1, OATP1B3, and OATP2B1 to the net hepatic uptake of pitavastatin and estradiol 17β-D-glucuronide. Although the predicted transporter contributions based on RAFs and REFs were within a comparable range, net hepatic uptake clearances estimated from REFs were significantly overpredicted compared with observed values. Recently, the contribution of OATP1B1-mediated hepatic uptake of five substrates was investigated using a gene knockdown approach (Williamson et al., 2013). Compared with a RAF-based method, highly similar results were obtained in predicting the transporter-specific contributions to the net hepatic uptake. Yet, extending the described approaches to any transport protein of interest is challenging due to practical limitations, such as the need for specific antibodies for Western blots, transporter-specific substrates for RAFs, and gene-specific knockdown. Moreover, the RAF-based and small interfering RNA-based approaches are restricted to investigated cell systems (i.e., hepatocytes) and do not allow the extrapolation of transporter activities to any tissue based on in vitro experiments.

Recently, novel quantitative targeted absolute proteomics (QTAP) methods, based on liquid chromatography-tandem mass spectrometry (LC-MS/MS), have been used to determine the absolute transporter protein abundance in plasma membrane samples of various human tissues, including liver and brain (Kamiie et al., 2008; Sakamoto et al., 2011; Uchida et al., 2011; Ohtsuki et al., 2012; Schaefer et al., 2012). Moreover, REFs determined by LC-MS/MS–based approaches are used in first studies to determine the specific contribution of hepatic uptake transporters in cryopreserved hepatocytes and human liver (Karlgren et al., 2012; Kimoto et al., 2012; Bi et al., 2013; Vildhede et al., 2014).

It was the aim of the present study to determine the contribution of OATP1B1 and OATP1B3 to the net hepatic uptake clearance of atorvastatin, cerivastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin. For this purpose, protein expression levels of OATP1B1 and OATP1B3 were measured in cryopreserved human hepatocytes and in recombinant human embryonic kidney (HEK)293 cell lines. Subsequently, we determined the uptake clearances of statins and used REFs derived from QTAP analysis to extrapolate OATP1B1 and OATP1B3 activities obtained in recombinant cells to hepatocyte values. Finally, to further validate the REF-based scaling method, we assessed the correlation between uptake transporter activity and their relative protein abundance by comparing RAF- and REF-based predictions.

Materials and Methods

Compounds.

[3H]Atorvastatin calcium (0.37 MBq/nmol), [3H]cholecystokinin octapeptide (CCK8; 3.65 MBq/nmol), [3H]cerivastatin sodium (0.185 MBq/nmol), [3H]fluvastatin sodium (0.74 MBq/nmol), [3H]lovastatin acid (0.37 MBq/nmol), [3H]pitavastatin calcium (0.185 MBq/nmol), [3H]pravastatin sodium (0.185 MBq/nmol), [3H]rosuvastatin calcium (0.37 MBq/nmol), and [3H]simvastatin acid (0.37 MBq/nmol) were obtained from American Radiolabeled Chemicals (St. Louis, MO). [3H]Estrone-3-sulfate ammonium (E3S; 1.67 MBq/nmol) was purchased from PerkinElmer (Boston, MA). All other compounds and reagents were of analytical grade and purchased from commercial sources.

Cell Systems.

LiverPool cryopreserved human hepatocytes (lot PQP) were obtained from Celsis, In Vitro Technologies (Brussels, Belgium). The hepatocyte pool was derived from nontransplantable fresh liver tissues of 20 donors (gender, 10 male and 10 female; age, 17–75; average age, 52; ethnic background, 16 Caucasians, 2 Blacks, and 2 Hispanics). A HEK293 cell line stably expressing human OATP1B3 (polybrene transfection method) was purchased from Deutsches Krebsforschungszentrum (Heidelberg, Germany) (König et al., 2000). A recombinant HEK293 cell line with stable expression of human OATP1B1 was generated in-house using the Flp-In system (Invitrogen by Life Technologies, Paisley, United Kingdom), as previously described (Kunze et al., 2012).

All HEK293 cell lines were cultivated in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum, 1% L-glutamine, and 1% penicillin/streptomycin. For HEK293 cells expressing OATP1B1 or OATP1B3, 100 ng/µL hygromycin B or 800 ng/µL geneticin, respectively, was added to the cultivation medium.

The protein content of solubilized cells (solved in 0.2 N NaOH) was determined using the Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA), according to the manufacturer’s recommendations.

Determination of Absolute Transporter Protein Abundance.

Absolute protein expression levels of human OATP1B1 and OATP1B3 in the membrane fractions of recombinant HEK293 cells and human cryopreserved hepatocytes were determined by peptide-based LC-MS/MS. The preparation of membrane fractions as well as the QTAP analysis was performed by BertinPharma (Sakamoto et al., 2011; Ohtsuki et al., 2012).

Three samples, each containing approximately 80 million HEK293-OATP1B1 or HEK293-OATP1B3 cells, were quickly harvested in ice-cold lysis buffer [10 mM Tris-HCl, 250 mM sucrose, and Complete Protease Inhibitor (Roche, Basel, Switzerland)]. Subsequently, the samples were centrifuged (537g, 4°C, 5 minutes) and the supernatant was aspirated. Two samples of human cryopreserved hepatocytes (approximately 30 million cells per sample) were thawed and immediately suspended in InVitroGRO HT medium (BioreclamationIVT; Baltimore, MD). Samples were then centrifuged (50g; 4°C; 5 minutes), and the supernatant was aspirated. All cells were stored as dry pellet at −80°C and were shipped to BertinPharma on dry ice.

All cell samples were processed by BertinPharma following published protocols (Sakamoto et al., 2011; Ohtsuki et al., 2012). The cell pellets were suspended in lysis buffer, followed by homogenization. Thereafter, submitochondrial fractions were isolated by centrifugation (25 minutes; 10,800g; 4°C). The supernatant was collected, and the microsomal fractions were obtained by centrifugation (60 minutes; 100,000g; 4°C). The microsomal pellet was suspended in 10 mM Tris-HCl buffer (pH 7.4; 250 mM sucrose). The plasma membrane fractions were obtained by ultracentrifugation of the microsomal fractions through a 38% (w/v) sucrose solution (Sakamoto et al., 2011; Ohtsuki et al., 2012).

Total protein contents were determined by Lowry’s method before and after each fractionation step. The absolute transporter protein abundance in the respective plasma membrane fractions was determined using simultaneous QTAP based on LC-MS/MS with multiple reactions monitoring (Sakamoto et al., 2011; Ohtsuki et al., 2012). The same reference peptides were selected as previously published by Uchida et al. (2011): LNTVGIAK for OATP1B1; IYNSVFFGR for OATP1B3; VLLQTLR for OATP2B1; NVALLALPR for OAT2; LSPSFADLFR for OCT1; and GIYDGDLK for NTCP.

The respective transporter protein expression (exp) was obtained as the amount of transporter protein (fmol) per amount of plasma membrane protein (µg protmem). To determine the transporter expression per amount of total protein [fmol/(µg prot)], exp was multiplied with the amount of plasma membrane protein obtained per amount of total sample protein.

Uptake Studies in Suspended Human Hepatocytes.

Hepatocyte uptake of reference compounds (E3S; CCK8) and the statins was assessed by the oil spin method, as previously described (Umehara and Camenisch, 2012). Frozen hepatocytes were thawed and directly suspended in InVitroGRO HT medium. After centrifugation (537g, 5 minutes, low brakes), the supernatant was aspirated and the cells were immediately suspended in 1 mL prewarmed Krebs-Henseleit buffer (KHB). Subsequently, cells were counted and the suspension was adjusted to a concentration of 1.0–1.5 × 105 viable cells/mL (viability: 83–95%).

Hepatocyte uptake studies were initiated by adding 50 µL hepatocyte suspension to 100 µL substrate solution (KHB containing a mixture of radiolabeled and nonlabeled study compound at specific concentration). All incubations were carried out at 37°C and at 4°C following preincubation times of 5 minutes and 15 minutes at 37°C and 4°C, respectively. At designated time points, incubations were terminated transferring the sample to a mineral oil/NaOH-containing tube [Hepatocyte Transporter Suspension Assay Kit (BD Biosciences, Woburn MA)], followed by immediate centrifugation (10,000 rpm; 1 minute). The tubes were cut, and the radioactivity in the cell pellets as well as in the supernatants (for mass balance studies) was analyzed by liquid scintillation counting (Packard Tri-Carb 2700TR; PerkinElmer, Waltham, MA).

All hepatocyte incubations were performed for 90 seconds. In time-dependent uptake experiments (1, 2, 3, and 5 minutes) for E3S (0.1 µM), pitavastatin (0.5 µM), CCK8 (0.5 µM), and rosuvastatin (0.5 µM), the rate of uptake was found to lie in a time-linear uptake phase and was consequently applied to all statin incubations. For concentration-dependent kinetic studies, a broad concentration range was defined for all substrates (0.01–300 µM; 5–10 concentration points). To demonstrate uptake transporter activity, E3S (0.03 µM) uptake was measured in the absence and presence of an OATP inhibitor cocktail [a combination of atorvastatin (10 µM) and rifamycin (20 µM)] in all hepatocyte studies.

Uptake Studies in Human HEK293-OATP1B1– and HEK293-OATP1B3–Expressing Cell Lines.

Cellular uptake studies using plated HEK293-OATP1B1, HEK293-OATP1B3, and HEK293 parental cells were performed, as previously described (Kunze et al., 2012). Uptake studies at 37°C were initiated by incubating the cells with the substrate solution at the respective concentrations (mix of radiolabeled and unlabeled study compound in KHB). To determine mass balances of the studied compounds, aliquots of the incubation solution were taken from each well prior to the termination of the incubation. Afterward, the incubation was terminated by aspirating the remaining incubation solution, followed by washing the cells twice with ice-cold phosphate-buffered saline. Subsequently, the cells were lysed in NaOH (0.2 N). The protein contents of the solubilized cells were determined, as described above. The amount of radiolabeled compound in the cell samples and in the incubation solution was quantified using liquid scintillation counting, as described above.

Time-dependent experiments in OATP1B1- and OATP1B3-expressing HEK293 cells were performed to define the time-linear range for subsequent concentration-dependent studies (0.01–300 µM; 6–9 concentration points). An incubation time of 3 minutes was chosen for all cell lines and compounds, except for E3S (1 minute). Uptake of each study compound (0.01 µM) was measured in the absence and presence of an OATP inhibitor cocktail [a combination of atorvastatin (10 µM) and rifamycin (20 µM)]. Functional activity of OATP1B1 and OATP1B3 in the recombinant cell lines was confirmed in each experiment, as previously described (Kunze et al., 2012).

Data Analysis.

The uptake kinetics of the investigated compounds was calculated by normalizing the measured radioactivity to the incubation time and protein content. Consequently, these uptake rates (Vapp; pmol × min−1 × mg−1) were divided by the applied substrate concentrations to obtain the apparent uptake clearances (PSapp; µL × min−1 × mg−1). Results are presented as uptake clearances throughout the whole manuscript to simplify the visualization of kinetic data.

As previously described, PSapp values determined in suspended hepatocytes are potentially affected by nonspecific binding (NSB) of the compound to plastic surfaces of the assay device or to cellular structures (Umehara and Camenisch, 2012). To account for plastic binding, total compound recoveries were calculated for all incubations, and PSapp values were multiplied by a respective correction factor (total theoretical recovery divided by obtained recovery). In a second step, control incubations were performed at 4°C to correct PSapp obtained at 37°C (PSapp,tot,37°C) for saturable NSB processes to cells (Umehara and Camenisch, 2012):Embedded Image(1)where the difference between the apparent uptake clearances determined from 4°C incubations at the lowest and highest substrate concentrations (PSapp,tot,4°C,Cmin and PSapp,tot,4°C,Cmax, respectively) was used to describe saturable nonspecific cell binding. For incubations with OATP1B1- and OATP1B3-expressing HEK293 cells, none of the studied compounds showed significant NSB to plastic (recoveries >90%). Moreover, no relevant saturable NSB to cells was observed in control incubations using HEK293 parental cells. Consequently, no correction of uptake clearances was required for studies with cell lines and PStot equals PSapp. The different experimental setups between HEK293 cells (plated) and hepatocytes (suspension) and the resulting difference in cell surface exposed to the incubation medium might explain the lack of saturable NSB in HEK293 parental cells. Moreover, as discussed previously, a difference in NSB between HEK293 cells and hepatocytes could be a result of different cell membrane compositions (e.g., lipids and protein contents) between HEK293 cells and hepatocytes (Mateus et al., 2013).

The total uptake clearance (PStot) is the sum of the active uptake clearance for saturable, transporter-mediated and passive permeation processes (PSact and PSpas, respectively):Embedded Image(2)where the active transporter-mediated process is following Michaelis-Menten kinetics with Km and S representing the Michaelis-Menten constant (µM) and the substrate concentration (µM), respectively. For initial uptake rates and clearances at very low substrate concentration (S < < Km), PSact can be approximated with PSact,max representing a measure of the intrinsic transporter activity. Kinetic parameters were estimated by fitting eq. 2 to the measured data using a nonlinear least square method.

Estimation of OATP Contribution in Human Hepatocytes.

The relative contribution of OATP1B1 and OATP1B3 to the hepatic uptake of compounds can be assessed by determining RAF or REF for a specific transporter. In brief, RAFs for OATP1B1 and OATP1B3 were determined by comparing the transporter activities of transporter-specific reference substrates (E3S for OATP1B1; CCK8 for OATP1B3) in recombinant cell lines and in hepatocytes, as described by Hirano et al. (2004) and Kimoto et al. (2011):Embedded Image(3)Embedded Image(4)Subsequently, RAF1B1 and RAF1B3 were applied to predict the combined uptake transporter activity of OATP1B1 and OATP1B3 cosubstrates in hepatocytes (PSact,max,HEP) based on the transporter activities measured in recombinant cells (PSact,max,1B1;1B3):Embedded Image(5)Alternatively, we used absolute OATP1B1 and OATP1B3 protein expression data in hepatocytes and recombinant HEK293 cells to derive the REFs for OATP1B1 and OATP1B3:Embedded Image(6)Embedded Image(7)where exp represents the specific transporter expression [fmol/(µg prot)] determined as described above. In analogy to eq. 5, the net transporter activity of compounds into hepatocytes was calculated from recombinant cells using the REF values:

Embedded Image(8)

Statistics.

All incubations for kinetic studies were performed in triplicates (n = 3), in which values are given as the mean and S.D. Statistical significance for the differences in uptake clearances obtained in incubations in the presence and absence of transporter inhibitors was assessed by unpaired, two-tailed Student’s t test. Differences were considered to be statistically significant for P values below 0.05. For parameter estimation based on data fitting, the coefficients of determination (R2) were determined to assess the goodness of fit. Moreover, fold-error deviations between the observed and predicted hepatic PSact,max values were calculated to assess the performance of the REF- and RAF-based prediction methods.

Results

Transporter Abundance.

The transporter protein abundance in plasma membrane fractions of HEK293-OATP1B1 and HEK293-OATP1B3 cells and pooled human cryopreserved hepatocytes (lot PQP) was determined by the QTAP method. Figure 1 and Table 1 show the measured abundances of the uptake transporters OATP1B1, OATP1B3, OATP2B1, NTCP, OAT2, and OCT1 in cryopreserved human hepatocytes. Significant differences in expression levels were found between the specific transporters, ranging from 0.35 fmol/(µg protmem) for OATP1B3 to 15.85 fmol/(µg protmem) for OATP1B1. OATP2B1, NTCP, and OAT2 showed similar expression levels [1.03 to 2.62 fmol/(µg protmem)], whereas the expression of OCT1 was comparatively higher [6.94 fmol/(µg protmem)]. In plasma membranes of recombinant HEK293 cells, the protein expression levels of OATP1B1 and OATP1B3 were measured at 23.97 fmol/(µg protmem) and 1.44 fmol/(µg protmem) (Table 1). Transporter protein expression levels, shown in Table 1, are normalized to either the amount of total plasma membrane (µg protmem) or the total sample protein (µg prot).

Fig. 1.
Fig. 1.

Transporter protein expression in plasma membrane fractions of pooled cryopreserved human hepatocytes. The bars represent mean values of two independent measurements performed in triplicates, and error bars represent S.D.

View this table:
TABLE 1

Absolute transporter protein expression

The absolute transporter protein expression was normalized to the amount of plasma membrane protein (fmol/µg protmem) or total protein (fmol/µg prot) in the respective expression system.

Determination of Pharmacokinetic Parameters.

We performed concentration-dependent incubations in HEK293-OATP1B1– and OATP1B3-overexpressing cells and in suspended hepatocytes to subsequently estimate pharmacokinetic parameters of our study compounds. Control incubations in the presence of the OATP inhibitors atorvastatin and rifamycin resulted in a significant decrease in PStot values in recombinant cell lines and hepatocytes, confirming transporter functionality in all uptake experiments (data not shown).

Figure 2 shows the uptake clearances of the OATP1B1- and OATP1B3-specific reference compounds E3S and CCK8, respectively, into recombinant cell lines and suspended hepatocytes. E3S uptake clearances in HEK293-OATP1B1 were decreased in a concentration-dependent manner, and PStot was significantly reduced in the presence of the OATP inhibitors. Also, HEK293-OATP1B3 cells showed transport activity for E3S, which, however, was significantly lower compared with HEK293-OATP1B1 cells (Fig. 2A). For CCK8, a concentration-dependent decrease in uptake clearances and inhibition in presence of OATP inhibitors were found in recombinant HEK293-OATP1B3, but not in HEK293-OATP1B1 cells (Fig. 2B).

Fig. 2.
Fig. 2.

Uptake kinetics of reference compounds in recombinant HEK293-OATP1B1 or HEK293-OATP1B3 cells (A, B) and human cryopreserved hepatocytes (C, D). The total uptake clearances (PStot) of E3S (A) and CCK8 (B) in recombinant HEK293 cells are shown in the presence and absence of OATP inhibitors atorvastatin and rifamycin (AR). For hepatocytes, PStot, as well as apparent uptake clearances determined from 37°C and 4°C incubations (PSapp,tot,37°C and PSapp,tot,4°C, respectively), are shown for E3S (C) and CCK8 (D). Data are presented as mean values of triplicates, with the error bars representing the S.D. Lines represent the fit to the data according to eq. 2.

Compound recoveries in hepatocyte studies above 85% were obtained for E3S, CCK8, atorvastatin, lovastatin, pitavastatin, and rosuvastatin. In contrast, substantial concentration-dependent NSB to the experimental device was found for cerivastatin (69%), fluvastatin (79%), pravastatin (56%), and simvastatin (71%).

As illustrated in Fig. 2, C and D, experiments with E3S and CCK8 using human hepatocytes showed a concentration-dependent decrease in PSapp values at 4°C incubations. As previously described, such a profile indicates saturation of temperature-independent NSB of the compound to cellular structures. Consequently, uptake clearances determined from 37°C incubations were corrected according to eq. 1. The resulting PStot values showed a concentration-dependent decrease for E3S and CCK8, indicating that both compounds were actively transported into hepatocytes. The observed PStot value for E3S was more than 10-fold higher than the value observed for CCK8.

Figure 3 shows representative kinetic profiles for rosuvastatin and cerivastatin. For both compounds, OATP1B1 or OATP1B3 uptake clearances were significantly reduced in the presence of the OATPs inhibitors (Fig. 3, A and B). Moreover, saturation of transporter activities at high concentrations of both statins was observed in HEK293-OATP1B1 and HEK293-OTAP1B3 cells. However, in both cell lines, the concentration-dependent decrease in PStot was more pronounced for rosuvastatin than for cerivastatin, probably due to the comparatively high passive uptake clearance obtained for the latter compound.

Fig. 3.
Fig. 3.

Uptake kinetics of rosuvastatin and cerivastatin in recombinant HEK293-OATP1B1 or HEK293-OATP1B3 cells (A, B) and human cryopreserved hepatocytes (C, D). The total uptake clearance (PStot) of rosuvastatin (A) and cerivastatin (B) in recombinant cell lines is shown in the presence and absence of OATP inhibitors atorvastatin and rifamycin (AR). Data are presented as mean values of triplicates, with the error bars representing the S.D. Lines represent the fit to the data according to eq. 2.

Atorvastatin, fluvastatin, pravastatin, and pitavastatin were also actively transported by OATP1B1 and OATP1B3, whereas lovastatin was found to be a substrate for only OATP1B1, but not for OATP1B3. The simvastatin uptake clearances decreased in a concentration-dependent manner in OATP1B1 and OATP1B3 cells, but coincubation with the OATP inhibitors did not affect the total uptake clearance in either cell line.

Hepatocyte uptake profiles of rosuvastatin and cerivastatin are shown in Fig. 3, C and D. The uptake clearances of both compounds were decreased in a concentration-dependent manner. Compared with the PStot value of rosuvastatin observed at initial concentrations, a very high PStot of cerivastatin was obtained. However, at high substrate concentrations, PStot of cerivastatin was significantly higher compared with the value determined for rosuvastatin, indicating a high contribution of the passive uptake clearance to the total cerivastatin uptake clearance.

Table 2 summarizes the estimated maximal activities (PSact,max), the ratios between active to passive compound clearances, and the global goodness of fit (R2) for parameter estimations according to eq. 2. In recombinant cell lines, highest PSact,max values were obtained for E3S and CCK8 in HEK293-OATP1B1– and OATP1B3-expressing cells, respectively. Although E3S also exhibited the highest PSact,max value in hepatocytes, active CCK8 transport was comparatively low. For the statins, comparable activities (PSact,max) of OATP1B1 and OATP1B3 were derived from recombinant cells, whereas higher activities were generally obtained in human hepatocytes. The ratios between active and passive uptake clearances (PSact,max/PSpas) represent a measure of the contribution of the transporter-mediated process to the total uptake clearance. E3S exhibited the highest ratios in HEK293-OATP1B1 cells and in hepatocytes. Among the statins, the highest ratio was measured for pravastatin in hepatocytes. Together with atorvastatin and rosuvastatin, pravastatin also showed highest ratios in recombinant cell lines. Ratios below one were obtained for simvastatin, fluvastatin, and cerivastatin in all expression systems, indicating an extensive contribution of passive permeation to the total uptake clearance for these compounds.

View this table:
TABLE 2

Pharmacokinetic parameters of statins and reference compounds

PSact,max refers to the maximal transporter activity in the respective expression system; PSpas refers to the passive permeation; PSact,max/PSpas describes the ratio of the active to passive uptake clearance; and R2 determines the coefficient of determination.

REF- and RAF-Based Prediction of Compound Uptake in Suspended Hepatocytes.

Table 3 lists the observed and predicted hepatic PSact,max values as well as the determined scaling factors. We obtained very similar transporter-specific scaling factors with values of 0.853 and 1.112 for REF1B1 and RAF1B1 (1.3-fold deviation), respectively, and of 0.181 and 0.113 for REF1B3 and RAF1B3 (1.6-fold deviation), respectively. Consequently, the PSact,max values predicted from the RAF and REF methods were highly comparable. Given the high similarity between the two scaling methods, only the results for REF-based scaling are discussed in the following.

View this table:
TABLE 3

Observed and predicted hepatic PSact,max values

PSact,max refers to the observed or predicted maximal transporter activity in hepatocytes. 1B1 and 1B3 determine OATP1B1 and OATP1B3, respectively. According to eqs. 3–7, the following scaling factors were used: relative expression factor (REF)1B1 = 0.853; REF1B3 = 0.181; relative activity factor (RAF)1B1 = 1.112; RAF1B3 = 0.113.

PSact,max values in hepatocytes were predicted from the extrapolated sum of OATP1B1 and OATP1B3 activities (eq. 8). A good resulting prediction accuracy between the observed and predicted values for E3S, CCK8, atorvastatin, pravastatin, rosuvastatin, and simvastatin was obtained with errors below twofold. In contrast, poor predictability was observed for cerivastatin, fluvastatin, lovastatin, and pitavastatin with errors between two- and sixfold, thus indicating a significant underprediction.

Figure 4 illustrates the contribution of OATP1B1 and OATP1B3 to the observed active uptake clearance of statins in suspended hepatocytes predicted from REF scaling. For all statins, OATP1B1-mediated uptake into hepatocytes was significantly higher than active uptake by OATP1B3. Moreover, the contribution of OATP1B1 and OATP1B3 to the hepatic uptake clearance of atorvastatin, pravastatin, rosuvastatin, and simvastatin was above 50%, indicating that both isoforms were the major contributors to the hepatic uptake of these compounds. In contrast, OATP1B1- and OATP1B3-mediated uptake of cerivastatin, fluvastatin, lovastatin, and pitavastatin was not the major determinant of the active hepatic uptake clearance determined in hepatocyte lot PQP.

Fig. 4.
Fig. 4.

Fractional contribution of OATP1B1 (gray) and OATP1B3 (black) to the observed maximal active uptake clearance of statins in suspended hepatocytes (lot PQP), in which PSact,max measured in hepatocytes represents 100%. The transporter contributions were predicted with the REF method based on measured transporter activities in recombinant HEK293-OATP1B1 and HEK293-OATP1B3 cells (Table 2).

Discussion

In the present study, we determined the OATP1B1- and OATP1B3-mediated uptake clearances of statins in single transporter-expressing HEK293 cells. Based on relative transporter protein expression data, we used the transporter activities to estimate the OATP1B1- and OATP1B3-mediated uptake of statins into hepatocytes.

Recently, QTAP methods were established to quantify low abundant proteins, and first expression levels in cryopreserved hepatocytes and liver samples were reported for the major hepatic uptake transporters OATP1B1, OATP1B3, OATP2B1, OAT2, OCT1, and NTCP (Bi et al., 2012; Kimoto et al., 2012; Ohtsuki et al., 2012; Bi et al., 2013). Literature data on OATP transporter expression show high interindividual differences. For OATP1B1, values between 2 and 12 fmol/(µg protmem) in human liver samples and 2 and 7 fmol/(µg protmem) in cryopreserved hepatocytes are reported (Karlgren et al., 2012; Kimoto et al., 2012; Ohtsuki et al., 2012; Vildhede et al., 2014).

For OATP1B3 reported values vary between 1 and 6 fmol/(µg protmem) in human liver samples and 1 and 2 fmol/(µg protmem) in cryopreserved hepatocytes (Karlgren et al., 2012; Ohtsuki et al., 2012; Vildhede et al., 2014). Reported values for other uptake transporters in human liver tissues vary between 1 and 4 (OATP2B1), 1 and 10 (NTCP), 1 and 3 (OAT2), and 3 and 15 fmol/(µg protmem) (OCT1) (Karlgren et al., 2012; Kimoto et al., 2012; Ohtsuki et al., 2012; Vildhede et al., 2014).

In the present study, transporter protein expression was quantified for pooled (20 donors) human cryopreserved hepatocytes. Measured protein expression levels were within the range of reported values, with 16 (OATP1B1), 0.4 (OATP1B3), 2 (OATP2B1), 3 (NTCP), 1 (OAT2), and 7 fmol/(µg protmem) for OCT1 (Table 1). In the tested lot of human hepatocytes, OATP1B1 protein was found to be expressed at a substantially higher level than OATP1B3. Such a pronounced OATP1B1 abundance with a concomitant low OATP1B3 expression has not yet been reported for protein levels in cryopreserved hepatocytes. In contrast, comparatively high differences in OATP1B1 and OATP1B3 expression levels were observed in human liver samples (Vildhede et al., 2014). Thus, the level of OATP1B1 protein expression is in agreement with reported values in human liver samples, whereas generally lower values are reported in human cryopreserved hepatocytes.

On a functional level, high variability in active uptake values determined in different cryopreserved hepatocytes is shown for statins and our reference substrates (Hirano et al., 2004; Watanabe et al., 2010; Kimoto et al., 2012). Hirano et al. (2004) reported maximum transporter activities between 36 and 84 µL × min−1 × mg−1 for E3S and 1 and 5 µL × min−1 × mg−1 for CCK8 in three different cryopreserved human hepatocyte lots. Whereas our CCK8 results were comparable, we obtained a significantly higher maximal activity for E3S uptake in hepatocytes. These findings are in line with the high OATP1B1 protein expression level obtained in the hepatocyte lot PQP.

Differences in reported transporter activities and protein expression levels might be a result of substantial interindividual variation in transporter protein abundances observed in human liver samples (Nies et al., 2013; Vildhede et al., 2014). Furthermore, differences in hepatocyte isolation or transporter protein quantification procedures, as well as in the selection of reference peptides for QTAP analysis, were attributed to impact determined transporter protein abundances (Balogh et al., 2012; Lundquist et al., 2014). All studied statins are reported substrates of OATP1B1, and fluvastatin, rosuvastatin, pravastatin, and pitavastatin are substrates of OATP1B3 (Hirano et al., 2004; Neuvonen et al., 2006; Noe et al., 2007; Kalliokoski and Niemi, 2009). Our results confirmed that all statins were substrates of OATP1B1. Only lovastatin was not identified as an OATP1B3 substrate. Simvastatin demonstrated concentration-dependent uptake kinetics in OATP1B1 and OATP1B3 cells, but no significant inhibition was observed upon coadministration with OATP inhibitors. We assume that its extensive passive permeation might have masked the contribution of the active transport process.

To investigate the correlation between transporter activity and expression level, we predicted the OATP1B1 and OATP1B3 activities in hepatocytes from studies in cell lines expressing the respective transporter, using RAF- and REF-based methods. In the ideal case, in which measured activity of a specific transporter is directly proportional to its protein expression level, the transporter-specific REF would be equal to the respective RAF. Our determined REF and RAF values for OATP1B1 (0.853 and 1.112) and for OATP1B3 (0.181 and 0.113) showed a high similarity indicating a direct correlation between expression levels and activity for OATP1B1 and OATP1B3. These results should neither be affected by individual variation in transporter protein expression, nor by variation in the hepatocyte preparation, as absolute protein abundance measurements, as well as kinetic experiments, were performed for the same batch of human hepatocytes. Thus, even if transporter protein expression in cryopreserved hepatocytes would not represent transporter expression levels in freshly isolated or human liver samples, as indicated in studies by Kimoto et al. (2012) and Lundquist et al. (2014), the correlation between transporter protein expression and activity should not be affected, as protein abundances and transporter activities were determined for the same lot of hepatocytes in the present study. In contrast, given the substantial variation in reported transporter protein abundances between different hepatocyte lots or liver samples, it is crucial to characterize transporter expression in hepatocytes to compare with the respective activities.

Using protein expression-based REF scaling, the observed hepatic PSact,max for the reference compounds E3S and CCK8 were predicted within twofold error. For statins, the REF-based predicted PSact,max values were in good agreement with the observed values, with fold error deviation below two for atorvastatin, pravastatin, rosuvastatin, and simvastatin, indicating that their active hepatic uptake was mainly described by OATP1B1- and OATP1B3-mediated transport. In contrast, the active hepatic uptake of cerivastatin, fluvastatin, lovastatin, and pitavastatin was underestimated.

Within the scope of this study, the hepatic uptake activities were only extrapolated using OATP1B1 and OATP1B3 values. Therefore, a potential explanation could be the involvement of other transporters in the hepatic uptake of these compounds. Recently, Bi et al. (2013) showed that NTCP is significantly involved in the hepatic uptake of fluvastatin, pitavastatin, and rosuvastatin. NTCP protein abundance in our hepatocyte pool was about twofold higher than the reported value by Bi et al. (2013). Hence, a significant contribution of NTCP to the net hepatic uptake of the respective statin is likely to be expected for our investigated hepatocyte lot. Moreover, atorvastatin, fluvastatin, pravastatin, and rosuvastatin, as well as E3S, were shown to be substrates of OATP2B1 (Noe et al., 2007; Kalliokoski and Niemi, 2009; Knauer et al., 2010). OATP2B1 was found to be expressed at a comparable level to reported human liver data (Kimoto et al., 2012; Vildhede et al., 2014). Therefore, we assumed that OATP2B1 might have contributed to the uptake of statins in the studied hepatocyte lot. Thus, comparing REF-based extrapolation of specific hepatic transporter activities with measured hepatic net uptake provides information about the potential involvement of other transporters in the net hepatic uptake activity.

It is expected that REF-based scaling will represent a powerful tool for in vitro–in vivo extrapolation of OATP1B1 and OATP1B3 activities to subsequently predict their contribution to the net hepatic uptake clearance and to assess the impact of OATP1B1- and OATP1B3-mediated DDIs. Recently, Karlgren et al. (2012) and Vildhede et al. (2014) predicted the contribution of OATP1B1, OATP1B3, OATP2B1, and NTCP to the atorvastatin uptake clearance based on protein expression data determined for human liver samples and recombinant cell lines by a LC-MS/MS method. Subsequently, the impact on isoform-specific inhibition to the atorvastatin clearance was assessed. Based on the substantial variation in transporter protein abundance among the investigated liver samples (12 donors), the predicted transporter-specific uptake clearances showed high interindividual variability ranging between 26 and 89% and 1.8 and 60% for OATP1B1 and OATP1B3, respectively. Moreover, Nies et al. (2013) demonstrated that genetic polymorphism significantly contributed to variation in OATP1B1 protein expression and functionality, observed among 82 individuals. The high interindividual variability in transporter-protein expression observed in these studies clearly needs to be considered when REF-based methods are used for in vitro–in vitro extrapolation of transporter activities. This aspect becomes especially important when results from DDI studies with relatively small numbers of subjects are compared with in vitro–in vivo extrapolation approaches.

In summary, we demonstrated as a proof-of-concept that the OATP1B1 and OATP1B3 activity in hepatocytes can be extrapolated from recombinant cell lines based on absolute transporter protein measurements. We further assessed the relative contribution of OATP1B1 and OATP1B3 to the total hepatic uptake. Moreover, in contrast to RAF-based scaling methods, this approach is expected to allow scaling of transporter activities from in vitro incubations in recombinant cell lines to any tissues, given the respective transporter abundance is known. Therefore, future research will be required to strengthen the evidence that scaling of transporter activities based on absolute protein abundance data represents a powerful tool to predict transporter-mediated in vivo clearance processes and DDI effects.

Acknowledgments

The authors thank the many Novartis Drug Metabolism and Pharmacokinetic scientists of Basel, Switzerland, who have supported this work. The authors also thank Francis Heitz for technical assistance and Joel Krauser for critical evaluation of this manuscript.

Authorship Contributions

Participated in research design: Kunze, Huwyler, Camenisch, Poller.

Conducted experiments: Kunze.

Performed data analysis: Kunze, Poller.

Wrote or contributed to the writing of the manuscript: Kunze, Huwyler, Camenisch, Poller.

Footnotes

Abbreviations

CCK8
cholecystokinin octapeptide
DDI
drug-drug interaction
E3S
estrone-3-sulfate
exp
transporter protein expression
HEK
human embryonic kidney
KHB
Krebs-Henseleit buffer
LC-MS/MS
liquid chromatography-tandem mass spectrometry
NSB
nonspecific binding
NTCP
sodium taurocholate cotransporting polypeptide
OATP
organic anion-transporting polypeptide
OCT
organic cation transporter
QTAP
quantitative targeted absolute proteomics
RAF
relative activity factor
REF
relative expression factor

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Prediction of OATP-Mediated Hepatic Uptake of Statins

Annett Kunze, Jörg Huwyler, Gian Camenisch and Birk Poller
Drug Metabolism and Disposition September 1, 2014, 42 (9) 1514-1521; DOI: https://doi.org/10.1124/dmd.114.058412
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