![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Department of Pharmacokinetics and Non-Clinical Safety, Kawanishi Pharma Research Institute, Nippon Boehringer Ingelheim Co., Yato, Kawanishi, Hyogo, Japan (N.I., A.S., W.K., T.I.); Department of Molecular Pharmacokinetics, Graduate School of Pharmaceutical Sciences, the University of Tokyo, Hongo, Bunkyo-ku, Tokyo, Japan (K.M., S.M., Y.S.); and Department of Pharmacokinetics and Drug Metabolism, Boehringer Ingelheim Pharma KG, Biberach on der Riss, Germany (T.E., W.R.)
(Received September 19, 2007; Accepted January 2, 2008)
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
; Hsiang et al., 1999; König et al., 2000a) and OATP1B3 (König et al., 2000b) are responsible for the hepatocellular uptake of organic anions and that several kinds of ATP-binding cassette (ABC) transporters such as multidrug resistance 1 (MDR1), multidrug resistance-associated protein 2 (MRP2), and breast cancer resistance protein (BCRP) play an important role in the biliary excretion of many compounds including clinically important drugs.
The hepatic export of organic anions is generally believed to be mediated predominantly by MRP2 (Evers et al., 1998; Cui et al., 1999; König et al., 1999). However, recent reports suggested that some anionic compounds such as morphine 6-glucuronide (Huwyler et al., 1996) and fexofenadine (Cvetkovic et al., 1999) could be transported by MDR1. Hirano et al. (2005) showed that biliary excretion of pitavastatin is accounted for largely by BCRP in mice. Matsushima et al. (2005) established a set of double-transfected MDCKII cells coexpressing OATP1B1 and MRP2, MDR1, or BCRP and demonstrated that not only MRP2 but also BCRP and MDR1 are involved in the biliary excretion of several organic anions.
In the uptake process of anions, the broad substrate specificity of OATP1B3 commonly overlaps that of OATP1B1, so several compounds can be bisubstrates of both OATP1B1 and OATP1B3 (Vavricka et al., 2002; Hirano et al., 2004). It has recently been reported that some anions such as cholecystokinin octapeptide (CCK-8), telmisartan, and fexofenadine are taken up into hepatocytes mainly by OATP1B3 rather than OATP1B1 (Ismair et al., 2001; Shimizu et al., 2005; Ishiguro et al., 2006). Thus, OATP1B3 and OATP1B1 could be important transporters for the hepatocellular uptake of anionic compounds, and experimental methods to distinguish their contributions have been established (Hirano et al., 2004; Ishiguro et al., 2006).
For the characterization of efflux transport, there are several methods such as transcellular transport study using single ABC transporter-expressing cells and double transfectants in which uptake and efflux transporters are expressed and uptake study into membrane vesicles prepared from transporter-expressing cells. Using single ABC transporter-expressing cells, we can evaluate the transcellular transport of lipophilic compounds with high membrane permeability but not that of hydrophilic compounds including several organic anions such as pravastatin because of their limited access to efflux transporters from intracellular compartments (Sasaki et al., 2002, 2004). For the low membrane-permeable compounds, a membrane vesicle study may be a useful alternative. However, this experiment cannot be applied to compounds that easily adsorb to the membrane filter because it is difficult to distinguish between transporter-mediated uptake into membrane vesicles and nonspecific adsorption. In the transcellular transport assay using double transfectants, we measure the drug concentration in the buffer, so the effect of nonspecific adsorption to the cells and labware is basically negligible in the quantification of ligand concentration. We can also detect the efflux more sensitively because of the intracellular accumulation of ligands by a basolateral uptake transporter. To date, several kinds of double transfectants have been constructed (Cui et al., 2001; Sasaki et al., 2002, 2004; Letschert et al., 2004, 2005; Kopplow et al., 2005; Matsushima et al., 2005). However, there was no good tool for the identification of transporters involved in the biliary excretion of OATP1B3-specific substrates. Thus, this study was performed to demonstrate the usefulness of double transfectants expressing OATP1B3 for the investigation of the transport of tel-glu.
After oral administration of telmisartan, a part of the oral dose is conjugated with glucuronate in intestine and accumulated selectively in liver. Telmisartan is extensively glucuronidated in liver and excreted into bile as tel-glu. (Wienen et al., 2000; Stangier et al., 2000a,b). Thus, the identification of transporters to the hepatobiliary transport of telmisartan and tel-glu is important for predicting the pharmacokinetics and subsequent pharmacological effect of telmisartan. We have reported that telmisartan is taken up into liver mainly via OATP1B3, and the efflux of tel-glu into bile in rats is mediated by MRP2 and another unknown transporter(s) (Nishino et al., 2000; Ishiguro et al., 2006).
In the present study, we show that tel-glu is recognized mainly by OATP1B3 rather than by OATP1B1 using transporter expression systems and human hepatocytes. We also establish a novel set of double transfectants coexpressing OATP1B3 and MDR1, MRP2, or BCRP and characterize the transcellular transport of estradiol 17β-D-glucuronide (E217βG), CCK-8, and tel-glu, which are substrates of OATP1B3, to identify the efflux transporters responsible for their biliary excretion.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
) and MDR1 were kindly provided by Dr. Piet Borst (The Netherlands Cancer Institute, Amsterdam, The Netherlands). All other chemicals and reagents were commercial products of reagent grade.
Cell Culture of Transporter-Expressing HEK293 Cells. OATP1B1-, OATP1B3-, and OATP2B1-expressing or vector-transfected HEK293 cells were established previously (Hirano et al., 2004; Shimizu et al., 2005). HEK293 cells were grown in Dulbecco's modified Eagle's medium (low glucose) (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Invitrogen) and 1% antibiotic-antimycotic solution (Invitrogen) at 37°C with 5% CO2 and 95% humidity. Cells were then seeded in 12-well plates at a density of 1.5 x 105 cells/well. For the transport study, the cell culture medium was replaced with culture medium supplemented with 5 mM sodium butyrate for 24 h before the transport assay to induce the expression of OATP1B1, 1B3, and 2B1.
Transport Study Using Transporter-Expressing HEK293 Cells. The transport study was carried out as described previously (Hirano et al., 2004). Uptake was initiated by adding Krebs-Henseleit buffer containing radiolabeled and unlabeled compounds after cells had been washed twice and preincubated with Krebs-Henseleit buffer at 37°C for 15 min. The uptake was terminated at a designated time by adding ice-cold Krebs-Henseleit buffer after removal of the incubation buffer. Then cells were washed twice with 1 ml of ice-cold Krebs-Henseleit buffer, solubilized in 1 N NaOH, and kept for 1 h at 37°C. During this incubation period, tel-glu was completely converted to telmisartan. Aliquots were transferred to scintillation vials or sample tubes after adding 
volume of 2 N HCl. The radioactivity associated with the cells and incubation buffer was measured by a liquid scintillation counter (Tri-Carb 2500TR, PerkinElmer Life and Analytical Sciences) after transfer to 2-ml scintillation vials. The aliquots for tel-glu were stored at –20°C until HPLC analysis. The remaining 50 µl of cell lysate was used to determine the protein concentration by the method of Lowry et al. (1951) with bovine serum albumin as a standard.
Preparation of Rat and Human Hepatocytes before Transport Assay. Isolated rat hepatocytes were prepared from Sprague-Dawley rats weighing 200 to 300 g by the collagenase perfusion method described previously (Yamazaki et al., 1993). Isolated hepatocytes (viability >80%) were suspended in Krebs-Henseleit buffer, adjusted to 2.0 x 106 cells/ml, and stored on ice before the uptake experiment. Cryopreserved human hepatocytes (lots HH-OCF, HH-094, and HH-TDH) were purchased from Celsis In Vitro Technologies (Baltimore, MD). The hepatocytes were treated as described previously (Shitara et al., 2003). The cryopreserved human hepatocytes were resuspended in Krebs-Henseleit buffer to give a final cell density of 1.0 x 106 viable cells/ml for the uptake study. The number of viable cells was determined by trypan blue staining. To measure the uptake in the absence of Na+, sodium chloride and sodium bicarbonate in Krebs-Henseleit buffer were replaced with choline chloride and choline bicarbonate, respectively.
Transport Study Using Isolated Hepatocytes. Before the uptake studies, cell suspensions were prewarmed in an incubator at 37°C for 3 min. The uptake studies were initiated by adding an equal volume of buffer containing radiolabeled and unlabeled compounds to the cell suspension. After incubation at 37°C for 0.5, 2, or 5 min, the reaction was terminated by separating the cells from the substrate solution. For this purpose, an aliquot of 80 µl of incubation mixture was collected and placed in a centrifuge tube (450 µl) containing 50 µl of 2 N NaOH under a layer of 100 µl of oil (density of 1.015, a mixture of silicone oil and mineral oil; Sigma-Aldrich), and subsequently the sample tube was centrifuged for 15 s in a centrifuge (15,000 rpm, MX-100; Tomy Seiko, Tokyo, Japan). During this process, hepatocytes passed through the oil layer into the alkaline solution. After an overnight incubation in alkali to dissolve the hepatocytes and to allow the conversion of tel-glu into telmisartan, the centrifuge tube was cut, and each compartment was transferred to a scintillation vial or experimental tube. The compartment containing the dissolved cells was neutralized with 50 µl of 2 N HCl. The aliquots for the radioactivity determination were mixed with scintillation cocktail, and the radioactivity was measured by a liquid scintillation counter. The aliquots for tel-glu were stored at –20°C until HPLC analysis.
Estimation of Protein Unbound Concentration of Tel-Glu in the Presence of Human Serum Albumin. The protein unbound concentration of tel-glu in the presence of human serum albumin (0, 0.1, 0.3, 1, 3, and 5%) was determined after a 2-h incubation at 37°C by equilibrium dialysis (DIANORM; Dainippon Pharmaceutical Co. Ltd., Osaka, Japan).
Determination of Tel-Glu Concentration by HPLC. Telmisartan and tel-glu concentrations were determined by HPLC. A Waters alliance HPLC system combined with a fluorescence detector (Waters 474; Waters, Milford, MA) was used. After adding 
volume of acetonitrile, 10-µl aliquots were separated on an XTerra RP18 column (3.5 µm, 4.6 x 150 mm; Waters) using a mobile phase (acetonitrile-water-pyridine, 234:800:0.16) at a flow rate of 1.0 ml/min. The analyte peaks (telmisartan 9 min and tel-glu 15 min) were detected by fluorescence (excitation 300 nm and emission 385 nm). The stability of telmisartan and tel-glu in the Krebs-Henseleit buffer supplemented with 0.3% HSA was confirmed up to 1 h of incubation by this HPLC analysis.
Construction of Stably Transporter-Transfected MDCKII Cell Lines. MDCKII cells expressing MDR1 and MRP2 and parental MDCKII cells were kindly provided by Dr. Piet Borst (The Netherlands Cancer Institute) (Evers et al., 1998; Tang et al., 2002). MDCKII cells were cultured at 37°C and 5% CO2 in Dulbecco's modified Eagle's medium (low glucose) supplemented with 10% fetal bovine serum and 1% antibiotic-antimycotic (Invitrogen). For construction of the BCRP-expressing MDCKII cells, parental MDCKII cells were transfected with the expression vector pcDNA3.1(+) containing human BCRP cDNA (Kondo et al., 2004) by using FuGENE6 reagent (Roche Diagnostics, Indianapolis, IN). At 50% confluence, cells on six-well plates were exposed to serum-free Opti-MEM I (Invitrogen) containing plasmid and FuGENE6 according to the manufacturer's instructions. At 6 h after the initiation of transfection, the plasmid-FuGENE6 solution was replaced with the normal culture medium. The transfected MDCKII cells were selected with neomycin (500 µg/ml; Invitrogen). Expression of BCRP was screened by the detection of BCRP mRNA in cells of each clone. For construction of double-transfected MDCKII cells, MRP2-, BCRP-, and MDR1-expressing MDCKII cells were transfected with the expression vector pcDNA3.1/Zeo(+) containing SLCO1B3 cDNA (Hirano et al., 2004; Iwai et al., 2004), and selection was performed with Zeocin (700 µg/ml; Invitrogen).
Western Blot Analysis. For Western blot analysis, a crude membrane was prepared from MDCKII cells according to the method described in a previous report (Gant et al., 1991). After the crude membrane was suspended in PBS, it was frozen in liquid N2 and stored at –80°C until use. The protein concentrations in the crude membranes prepared from MDCKII cells were determined by the method of Lowry et al. (1951) with bovine serum albumin as a standard. The membrane fraction was dissolved in 3x SDS sample buffer (New England Biolabs, Beverly, MA) with 0.125 M dithiothreitol and loaded onto a 7.5% SDS-polyacrylamide electrophoresis gel (Daiichi Pure Chemical Co. Ltd., Tokyo, Japan). The molecular weight was determined using a prestained protein marker (New England Biolabs). Proteins were transferred electrophoretically to a polyvinylidene difluoride membrane (Pall, East Hills, NY) using a blotter (Trans-blot; Bio-Rad, Hercules, CA) at 15 V for 1 h. The membrane was blocked with Tris-buffered saline with 0.05% Tween 20 (TTBS) and 5% skimmed milk overnight at 4°C. After washing with TTBS, the membrane was incubated at room temperature in TTBS with 1000-fold diluted anti-OATP1B3 polyclonal antiserum, which was raised in rabbits against the 21 amino acids at the carboxyl terminus of the deduced OATP1B3 sequence (Hirano et al., 2004), for 1 h, 125-fold diluted monoclonal antibody against MRP2 (M2III-6; Biochemicals, Gruenberg, Germany) for 2 h, 100-fold diluted monoclonal antibody against MDR1 (C219; Signet Laboratories, Inc., Dedhem, MA) for 1 h, or 200-fold diluted monoclonal antibody against BCRP (BXP-21; Kamiya Biomedical Company, Seattle, WA) for 2 h. For the detection of each transporter, the membrane was placed in contact with 2500-fold diluted donkey anti-rabbit (OATP1B3) or anti-mouse IgG (MRP2, MDR1, and BCRP) conjugated with horseradish peroxidase (GE Healthcare) for 1 h in TTBS. The band was detected using an ECL Plus Western blotting starter kit (GE Healthcare).
Immunocytochemical Staining. For immunocytochemical staining, transfectants were plated at a density of 5.4 x 105 cells in 12-well plates 96 h before the experiments. Sodium butyrate (5 mM) was added to the culture medium 1 day before the experiments. After fixation with methanol at –20°C for 10 min and permeabilization with 1% Triton X-100 in PBS at room temperature for 5 min, cells were incubated for 1 h at room temperature with 50-fold diluted anti-OATP1B3 antiserum, 40-fold diluted monoclonal antibody against MRP2 (M2III-6), 40-fold diluted monoclonal antibody against MDR1 (C219), or 40-fold diluted monoclonal antibody against BCRP (BXP-21). Then the cells were washed with PBS three times and incubated for 1 h at room temperature with 250-fold diluted goat anti-rabbit IgG Alexa 488 (Invitrogen) for OATP1B3 or 250-fold diluted goat anti-mouse IgG Alexa 568 (Invitrogen) for MRP2, MDR1, and BCRP. Nuclei were stained with 250-fold-diluted TO-PRO-3 iodide (Invitrogen). The localization was visualized by confocal laser microscopy (Zeiss LSM-510; Carl Zeiss Inc., Thornwood, NY).
Transcelluar Transport Study Using Double-Transfected MDCKII Cells. MDCKII cells coexpressing OATP1B1 and each hepatic efflux transporter (MDR1, MRP2, and BCRP) (Evers et al., 1998; Tang et al., 2002; Matsushima et al., 2005) and coexpressing OATP1B3 and each hepatic efflux transporter (MDR1, MRP2, and BCRP) were used for transcellular transport studies. The transcellular transport study was performed as reported previously (Sasaki et al., 2002). Briefly, MDCKII cells were seeded on the Transwell (6.5-mm diameter, 0.4 µm pore size; Corning Coster, Bodenheim, Germany) at a cell density of 1.4 x 105 cells/well and grown on Transwell membrane inserts at confluence for 7 days, and the expression of transporters was induced by replacing the culture medium with medium containing 5 mM sodium butyrate for 48 h before the transport study. Cells were first washed with Krebs-Henseleit buffer (118 mM NaCl, 23.8 mM NaHCO3, 4.8 mM KCl, 1.0 mM KH2PO4, 1.2 mM MgSO4, 12.5 mM HEPES, 5.0 mM glucose, and 1.5 mM CaCl2 adjusted to pH 7.4) and preincubated with Krebs-Henseleit buffer at 37°C for 20 min. Subsequently, the transport study was initiated by adding Krebs-Henseleit buffer containing test compounds to either the apical compartments (250 µl) or the basolateral compartments (1 ml) after removal of Krebs-Henseleit buffer used for preincubation. In the case of tel-glu, the incubation volume was changed to 100 µl for the apical compartments and to 600 µl for the basolateral compartments, and 0.3% HSA was supplemented in Krebs-Henseleit buffer. After a designated period, for the radioactive compounds, the radioactivity in 100 µl of medium in the opposite compartments was measured by a liquid scintillation counter (Tri-Carb 2500TR; PerkinElmer Life and Analytical Sciences). For the measurement of tel-glu, a sample of 50 µl was removed from the opposite compartments, and 10-µl aliquots were mixed with an equal volume of 1 N NaOH and stored for 1 h at 37°C for the complete conversion from tel-glu to telmisartan. The mixtures were neutralized with 5 µl of 2 N HCl, and then the tel-glu concentration in the medium was measured by HPLC fluorescence as described above after addition of 6.25 µl of acetonitrile. Additionally, the tel-glu concentration in a 20-µl sample was measured after adding 30 µl of distilled water and 12.5 µl of acetonitrile to assess the decomposition from tel-glu to telmisartan during the incubation period. At the end of the experiments, the cells were washed three times with 1.5 ml of ice-cold Krebs-Henseleit buffer and solubilized in 150 µl of 1 N NaOH. After addition of 75 µl of 2 N HCl and 56 µl of acetonitrile, the tel-glu concentration in the cells was determined by HPLC fluorescence. Twentymicroliter aliquots of cell lysate were used to determine protein concentrations by the method of Lowry et al. (1951) with bovine serum albumin as a standard.
Kinetic Analysis. Ligand uptake was expressed as the uptake volume (microliters per milligram of protein), given as the radioactivity associated with the cells (disintegrations per minute per milligram of protein) divided by its initial concentration in the incubation medium (disintegrations per minute per microliter). Specific uptake was obtained by subtracting the uptake into vector-transfected cells from the uptake into cDNA-transfected cells. Kinetic parameters were obtained using eq. 1:
 | (1) |
). The half-inhibitory concentration (IC50) of inhibitors was obtained by examining their inhibitory effects on the uptake of CCK-8, E217βG, and tel-glu based on eq. 2:
 | (2) |
). To determine the saturable hepatic uptake clearance in human hepatocytes, we first determined the hepatic uptake clearance [CL(2 min–0.5 min)] (microliters per minute per 106 cells) by calculating the slope of the uptake volume (Vd) (microliters per 106 cells) between 0.5 and 2 min (eq. 3). The saturable hepatic uptake clearance (CLhep) was determined by subtracting CL(2 min–0.5 min) in the presence of an excess of cold substrate (excess) from that in the presence of tracer amount of substrate (tracer) (eq. 4).
 | (3) |
 | (4) |
|
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
), we checked the effect of HSA on tel-glu uptake and measured the protein unbound fraction of tel-glu in incubation media in each condition before the uptake experiment with tel-glu. Tel-glu exhibited a very high apparent uptake, which may include nonspecific adsorption to cells, transporter-mediated uptake, and passive diffusion in the absence of HSA (tracer, 5 µM: 268 ± 5 µl/2 min/106 cells; excess, 200 µM: 200 ± 3 µl/2 min/106 cells). The apparent uptake was reduced with an increase in HSA concentration (Fig. 1). The protein unbound fractions of tel-glu in the incubation media with 0.3, 1, 3, and 5% HSA were reduced to 9.9, 3.5, 1.1, and 0.7%, respectively (Fig. 1). Both the apparent uptake into hepatocytes and the unbound fraction of tel-glu were reduced in parallel with increasing concentrations of HSA. This observation suggested that the saturable uptake of tel-glu followed the "free" hypothesis according to which only unbound ligand can be recognized by transporters (Fig. 1). Thus, the tel-glu uptake was assessed with at least 0.3% HSA in the incubation media to prevent the extensive adsorption of tel-glu to the cells and/or culture materials and to use the same experimental conditions as in the telmisartan study (Ishiguro et al., 2006). In the presence of 1% HSA, tel-glu was taken up into isolated rat hepatocytes in a linear fashion for up to 2 min. The uptake was reduced by the presence of an excess of tel-glu (200 µM), indicating that transporter(s) are involved in the tel-glu uptake by isolated rat hepatocytes (Fig. 2A). The concentration dependence of the uptake of tel-glu was studied over the concentration range of 10 to 200 µM in the presence of 1% HSA. An Eadie-Hofstee plot showed one saturable component (Fig. 2B), and the apparent Km and Vmax values for tel-glu uptake in the presence of 1% HSA were 118 ± 4 µM and 754 ± 12 pmol/min/106 cells, respectively. To evaluate the nonsaturable uptake of tel-glu, a tel-glu concentration of 200 µM was used, which is not high enough to saturate the transporter-mediated transport completely, but represents the maximum solubility of tel-glu in the incubation media. Depletion of Na+ in the incubation media did not reduce the uptake of tel-glu (Fig. 3A). Although 10 µM pravastatin slightly stimulated the uptake of tel-glu, the uptake was inhibited by pravastatin, digoxin, and TCA, which are substrates and inhibitors of Oatp isoforms (Noe et al., 1997; Kouzuki et al., 1999; Tokui et al., 1999; Cattori et al., 2000; Sasaki et al., 2002, 2004), with IC50 values of 58.9 ± 26.7, 22.9 ± 9.8, and 7.12 ± 3.18 µM, respectively. An excess amount of TEA (1 mM), a typical substrate of organic cation transporters, caused at most a 30% reduction in the uptake of tel-glu (Fig. 3B). On the basis of these results, Oatp isoforms are considered to be involved in the tel-glu transport by isolated rat hepatocytes.
|
|
Uptake of Tel-Glu in Transporter-Expressing HEK293 Cells. To identify which transporters are important for the hepatic uptake of tel-glu in humans, uptake assays were performed using OATP1B1-, OATP1B3- and OATP2B1-expressing HEK293 cells. For these studies the HSA concentration was reduced from 1 to 0.3% in the incubation media. This was done because only minimal transport activity of E217βG, which is a typical ligand for OATP1B1, was detected in OATP1B1-expressing cells in the presence of 1% HSA owing to the significant reduction in its unbound concentration by binding to HSA (data not shown). Tel-glu was transported by OATP1B3 and OATP2B1 but not by OATP1B1 (Fig. 4A). The concentration dependence of the initial uptake of tel-glu by OATP1B3 and OATP2B1 was studied over the concentration ranges of 0.2 to 20 µM and 0.2 to 50 µM, respectively (Fig. 4B). The Km and Vmax values of OATP1B3- and OATP2B1-mediated tel-glu uptake were 3.40 ± 0.16 µM and 124 ± 4 pmol/min/mg of protein and 1.09 ± 0.10 µM and 22.3 ± 1.0 pmol/min/mg of protein, respectively.
|
Inhibitory Effect of E-sul on OATP1B3- and OATP2B1-Mediated Uptake of Tel-Glu in Transporter Expression Systems. We previously reported that E-sul is a selective inhibitor against OATP1B1 and that 30 µM E-sul completely inhibited OATP1B1-mediated E217βG uptake but did not inhibit OATP1B3-mediated CCK-8 uptake in transporter-expressing HEK293 cells (Ishiguro et al., 2006). In this study, we investigated the effects of E-sul on OATP1B3- and OATP2B1-mediated tel-glu uptake using OATP1B3- and OATP2B1-expressing HEK293 cells. E-sul hardly inhibited the OATP1B3- and OATP2B1-mediated tel-glu uptake with high IC50 values of 216 ± 28 and 223 ± 37 µM, respectively (Fig. 5).
|
Estimation of Relative Contributions of OATP1B1 and OATP1B3 to the Hepatic Uptake of Tel-Glu in Cryopreserved Human Hepatocytes. To confirm the minor contribution of OATP1B1 to overall hepatic tel-glu transport, three batches of cryopreserved human hepatocytes (Lots HH-OCF, HH-094, and HH-TDH) were used for the inhibition study. The uptake of 1 µM E217βG and 2 µM tel-glu by three different batches of cryopreserved human hepatocytes in the presence of 0.3% HSA was increased from 0.5 to 2 min [uptake of E217βG and tel-glu by cryopreserved human hepatocytes (HH-TDH): 3.40 ± 0.49 and 15.4 ± 2.5 µl/min/106 cells, respectively]. The uptake was reduced in the presence of an excess of unlabeled E217βG (200 µM) and tel-glu (50 µM) to 1.33 ± 0.30 and 4.05 ± 0.47 µl/min/106 cells, respectively (HH-TDH). The uptake of E217βG into human hepatocytes was inhibited by more than 50% by an E-sul concentration of 30 µM, whereas the uptake of tel-glu was hardly inhibited by 30 µM E-sul (Table. 1).
|
Expression of Human OATP1B3, MRP2, MDR1, and BCRP in MDCKII Cells. The expression of OATP1B3, MRP2, MDR1, and BCRP in the double-transfected MDCKII cell lines was confirmed by Western blot analysis (Fig. 6). In the parental MDCKII cells, no expression of OATP1B3, MRP2, MDR1, and BCRP was observed. The major band, which appeared at approximately 120 kDa, was detected in all kinds of OATP1B3-transfected cells (Fig. 6A), as shown previously (Hirano et al., 2004). The expression level of OATP1B3 in OATP1B3-transfected MDCKII cells per unit of protein was lower than that in cryopreserved human hepatocytes (Lot HH-OCF). We easily detected human MRP2, MDR1, and BCRP with apparent molecular masses of approximately 190, 170, and 70 kDa, respectively (Fig. 6, B–D). The MRP2, MDR1, and BCRP expression levels in double transfectants per unit of protein were almost comparable with those in single transporter-expressing cells and were much higher than those in cryopreserved human hepatocytes (Lot HH-OCF).
|
Localization of Recombinant Human OATP1B3, MRP2, MDR1, and BCRP. The cellular localization of the recombinant transporters in each transfectant was confirmed by confocal laser scanning microscopy (Fig. 7). OATP1B3 was correctly localized in the basolateral membrane of each transfectant expressing OATP1B3 (Fig. 7, D–I), whereas MDR1 and MRP2 were localized in the apical membrane (Fig. 7, H and I). BCRP was detected mainly in the apical membrane, but some fractions were also detected in the basolateral membrane (Fig. 7G).
|
Transcellular Transport of E217βG, CCK-8, and Tel-Glu across the MDCKII Monolayer Expressing Uptake and Efflux Transporters. To characterize the double-transfected MDCKII cells coexpressing OATP1B3 and MRP2, MDR1, and BCRP, we evaluated the transcellular transport of E217βG (OATP1B1/OATP1B3 bisubstrates) and CCK-8 and tel-glu (specific substrates of OATP1B3) across the MDCKII monolayer expressing uptake and efflux transporters (Figs. 8, 9, and 10). Significantly higher basal-to-apical transport of E217βG was observed in OATP1B1/MRP2, OATP1B1/MDR1, OATP1B1/BCRP, and OATP1B3/MRP2 double transfectants. However, such transport was not observed in parental MDCKII cells, single transfectants (OATP1B1, OATP1B3, MRP2, MDR1, and BCRP) and OATP1B3/MDR1 and OATP1B3/BCRP double transfectants (Fig. 8). Transcellular transport of the OATP1B3-selective substrate of CCK-8 and tel-glu was also determined in the transporter-expressing MD-CKII cells. The basal-to-apical vectorial transport of CCK-8 was significantly higher in the OATP1B3/MRP2 double transfectant than in the OATP1B3 single transfectant. However, no significant difference in vectorial transport was seen between OATP1B3-expressing cells and double transfectants expressing OATP1B3/MDR1 and OATP1B3/BCRP. In cell lines expressing OATP1B1 as an uptake transporter, no transcellular transport of CCK-8 was observed (Fig. 9). Before the transcellular transport study using tel-glu, the stability of tel-glu during the incubation was examined, and the decomposition from tel-glu into tel was negligible up to 3 h in the presence of 0.3% HSA. A higher basal-to-apical transport of tel-glu was found in all three cell lines expressing OATP1B3 and in the OATP1B1/MRP2 double transfectant. The vectorial transport in the double transfectants expressing OATP1B3 was higher than that in OATP1B3-expressing cells (Fig. 10).
|
|
|
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
; Cui et al., 1999; König et al., 1999; Vavricka et al., 2002; Hirano et al., 2004), the importance of OATP1B3, BCRP, and MDR1 has also recently been indicated. Previously we showed that double transfectants coexpressing OATP1B1 and an efflux transporter are a useful system to identify efflux transporters of organic anions in human liver (Matsushima et al., 2005). However, we found that some compounds such as fexofenadine and telmisartan are taken up into human hepatocytes mainly via OATP1B3 (Shimizu et al., 2005; Ishiguro et al., 2006). Telmisartan is excreted into bile predominantly as tel-glu (Wienen et al., 2000). To understand the pharmacokinetics of telmisartan, it is important to clarify the transport mechanisms of tel-glu as well as that of telmisartan. In the present study, we constructed a novel set of double-transfected cells expressing OATP1B3 and MDR1, MRP2, or BCRP and examined the transport mechanisms of tel-glu using rat and human hepatocytes, OATP transporter-expressing HEK293 cells, and double transfectants.
Previously, there were substantial difficulties to overcome in evaluating the transport of lipophilic telmisartan because of its extensive adsorption to cells (Ishiguro et al., 2006). Therefore, we examined whether tel-glu also exhibits this unfavorable property as does telmisartan. In the absence of HSA, the apparent uptake of tel-glu by isolated rat hepatocytes was very high and almost the same as that of telmisartan. To avoid the extensive adsorption of tel-glu to cells by HSA, we decided to use the same experimental conditions as for telmisartan, 1 and 0.3% HSA in the incubation media, for the further evaluation of tel-glu uptake by rat and human hepatocytes, respectively.
Because the involvement of Oatp isoforms on the hepatic uptake of tel-glu into isolated rat hepatocytes was anticipated by uptake experiments using isolated rat hepatocytes, we assessed which OATP transporters were involved in the human hepatic uptake of tel-glu by using cryopreserved human hepatocytes and transporter expression systems. Tel-glu was taken up by OATP1B3 and OATP2B1 but not by OATP1B1 (Fig. 4A). To confirm the minor contribution of OATP1B1 to the hepatic uptake of tel-glu, we performed an inhibition study using three batches of cryopreserved human hepatocytes and an OATP1B1-selective inhibitor, E-sul. Previously we reported that 30 µM E-sul can selectively inhibit the OATP1B1-mediated uptake in the presence of 0.3% HSA compared with OATP1B3 (Ishiguro et al., 2006). As a result, tel-glu was taken up into cryopreserved human hepatocytes in a saturable manner, and 30 µM E-sul did not significantly inhibit the uptake of tel-glu into all batches of cryopreserved human hepatocytes that we tested and OATP1B3- and OATP2B1-expressing HEK293 cells (Table 1; Fig. 5, A and B). These results confirmed the minor contribution of OATP1B1 to tel-glu uptake in cryopreserved human hepatocytes. The transport clearance (Vmax/Km) of tel-glu by OATP1B3-expressing cells was approximately 2-fold higher than that by OATP2B1-expressing cells. Hirano et al. (2004, 2006) reported that the ratio of the protein expression level of OATP2B1 in human hepatocytes to that in our expression system was less than 0.2, whereas for OATP1B3, that ratio was almost 1 by Western blot analysis. Our results suggested that the contribution of OATP2B1 to the hepatic uptake of tel-glu into human hepatocytes was at most one-tenth that of OATP1B3. Therefore, we concluded that tel-glu is taken up into human hepatocytes predominantly by OATP1B3 as telmisartan.
Regarding the biliary excretion of tel-glu, we previously showed that tel-glu is transported by both MRP2 and another transporter(s) that are also expressed in Eisai hyperbilirubinemic rats (Nishino et al., 2000). Because of its extensive adsorption of tel-glu, it was not possible to assess its transport into canalicular membrane vesicles. CCK-8 is a recognized substrate of OATP1B3 and MRP2 (Ismair et al., 2001; Letschert et al., 2004, 2005), but the involvement of BCRP and MDR1 in its efflux remains to be investigated. To identify the efflux transporters of OATP1B3-selective substrates, CCK-8 and tel-glu, we established a set of novel double-transfected MDCKII cells coexpressing OATP1B3 and MDR1, MRP2, or BCRP and characterized their transcellular transports using several kinds of double transfectants.
Western blot and immunocytochemical analyses revealed that OATP1B3, MRP2, and MDR1 were expressed in MDCKII cells and localized correctly on the basolateral (OATP1B3) and apical membrane (MRP2 and MDR1), respectively. However, BCRP was localized mainly on the apical membrane and partially on the basolateral membrane (Figs. 6 and 7). This phenomenon was also observed in OATP1B1/BCRP double transfectants, but basal-to-apical transcellular transport of several compounds could be observed in these cell lines. This finding suggested that a minor distribution of BCRP on the basolateral side may not become a major concern to characterize efflux transport processes (Matsushima et al., 2005).
E217βG is a bisubstrate of OATP1B1 and OATP1B3, and its efflux is mediated by MRP2, MDR1, and BCRP (Hirano et al., 2004; Matsushima et al., 2005). No vectorial transport of E217βG was observed in MDCKII cells expressing only efflux transporter, whereas higher basal-to-apical transport was seen in double transfectants expressing OATP1B1 (Fig. 8) as had been reported previously (Matsushima et al., 2005). This result was most likely due to the limited access of E217βG inside the cells without the action of a suitable uptake transporter such as OATP1B1. Our findings showed that the transcellular transport of E217βG was significantly enhanced only in OATP1B3/MRP2 double transfectants among the three kinds of double transfectants expressing OATP1B3. This result can be explained by the previous findings that the transport clearance of E217βG by OATP1B3 is 7-fold lower than that by OATP1B1 (Hirano et al., 2004) and that E217βG is a good substrate of MRP2 compared with BCRP and MDR1 (Matsushima et al., 2005). In the case of CCK-8, a higher vectorial transport compared with the OATP1B3 single transfectant was observed only in OATP1B3/MRP2 double transfectants (Fig. 9). This finding indicated that CCK-8 was preferably transported by MRP2 rather than by MDR1 and BCRP, which was also in line with a previous report (Letschert et al., 2005).
In a following set of experiments, the vectorial basal-to-apical transcellular transport of tel-glu was observed in all kinds of double transfectants expressing OATP1B3, indicating that tel-glu was a substrate of MRP2, MDR1, and BCRP (Fig. 10). This result implies that other transporter(s), as was predicted by the previous study using Eisai hyperbilirubinemic rats (Nishino et al., 2000), might be MDR1 and/or BCRP. The vectorial transport of tel-glu was also seen in OATP1B1/MRP2 double transfectants (Fig. 10J) but not in MRP2-expressing cells (Fig. 10B), which was apparently inconsistent with the result that tel-glu was a specific substrate of OATP1B3. In our experiment, only a very small uptake of tel-glu by OATP1B1-expressing cells was observed, compared with that by vector-transfected cells in the absence of HSA (vector: 21.5 ± 0.9 µl/0.5 min/mg; OATP1B3: 193 ± 8 µl/0.5 min/mg; and OATP1B1: 36.2 ± 0.9 µl/0.5 min/mg). Because of the high sensitivity of the detection of transport in double-transfected cell lines, this observation may explain the apparent discrepancy.
Taken together, the results of this study using transporter expression systems and human hepatocytes suggested that tel-glu is taken up into human liver mainly via OATP1B3. For the more general purpose of the identification of efflux transporters of OATP1B3-selective substrates, we constructed a novel set of double transfectants coexpressing OATP1B3 and MDR1, MRP2, or BCRP. Telmisartan 1-O-acylglucuronide was excreted via MDR1, MRP2, and BCRP, although the relative quantitative contribution of each transporter has not been determined yet.
In consideration of the fact that OATP1B1 and OATP1B3 are responsible for the hepatic uptake of organic anions in human liver, a set of double transfectants expressing OATP1B3 combined with double transfectants expressing OATP1B1 can be used as a powerful tool for the rapid identification of efflux transporters of many organic anions that are substrates of OATP1B1 or OATP1B3.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
ABBREVIATIONS: OATP/Oatp, organic anion transporting polypeptide; ABC, ATP-binding cassette; MDR1, multidrug resistance 1; MRP2, multidrug resistance-associated protein 2; BCRP, breast cancer resistance protein; CCK-8, cholecystokinin octapeptide; tel-glu, telmisartan acylglucuronide; E217βG, estradiol 17β-D-glucuronide; E-sul, estrone-3-sulfate; TCA, taurocholate; TEA, tetraethylammonium; MDCKII, Madin-Darby canine kidney strain II; HPLC, high-performance liquid chromatography; HSA, human serum albumin; PBS, phosphate-buffered saline.
Address correspondence to: Dr. Yuichi Sugiyama, Department of Molecular Pharmacokinetics, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. E-mail: sugiyama{at}mol.f.u-tokyo.ac.jp
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
|
|
 |
|
  M. Hirouchi, H. Kusuhara, R. Onuki, B. W. Ogilvie, A. Parkinson, and Y. Sugiyama Construction of Triple-Transfected Cells [Organic Anion-Transporting Polypeptide (OATP) 1B1/Multidrug Resistance-Associated Protein (MRP) 2/MRP3 and OATP1B1/MRP2/MRP4] for Analysis of the Sinusoidal Function of MRP3 and MRP4 Drug Metab. Dispos., October 1, 2009; 37(10): 2103 - 2111. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S.-C. Tzou, S. Roffler, K.-H. Chuang, H.-P. Yeh, C.-H. Kao, Y.-C. Su, C.-M. Cheng, W.-L. Tseng, J. Shiea, I-H. Harm, et al. Micro-PET Imaging of {beta}-Glucuronidase Activity by the Hydrophobic Conversion of a Glucuronide Probe Radiology, September 1, 2009; 252(3): 754 - 762. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Evers and X.-Y. Chu Role of the Murine Organic Anion-Transporting Polypeptide 1b2 (Oatp1b2) in Drug Disposition and Hepatotoxicity Mol. Pharmacol., August 1, 2008; 74(2): 309 - 311. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Matsushima, K. Maeda, H. Hayashi, Y. Debori, A. H. Schinkel, J. D. Schuetz, H. Kusuhara, and Y. Sugiyama Involvement of Multiple Efflux Transporters in Hepatic Disposition of Fexofenadine Mol. Pharmacol., May 1, 2008; 73(5): 1474 - 1483. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
and 
represent the uptake of tel-glu into isolated rat hepatocytes (microliters per minute per 106 cells) and protein unbound fraction of tel-glu in the incubation media, respectively. Uptake of tel-glu was measured by incubating cells with 5 µM or 200 µM tel-glu, and the saturable uptake of tel-glu by isolated rat hepatocytes was determined by using eqs. 3 and 4. HSA concentrations used were 0.3, 1, 3, and 5%. Each point with vertical bar shows the mean ± S.E. of three separate determinations.
) and 200 µM (
, 
represent the uptake of tel-glu in the presence of digoxin, taurocholate, pravastatin, and TEA, respectively. Solid lines represent the fitted curves obtained by nonlinear regression analysis. Each bar and vertical bar represents the mean ± S.E. of three separate determinations.
, 
, 
