The Nonmetabolized Î²-Blocker Nadolol Is a Substrate of OCT1, OCT2

Article pubs.acs.org/molecularpharmaceutics

The Nonmetabolized β‑Blocker Nadolol Is a Substrate of OCT1, OCT2, MATE1, MATE2-K, and P‑Glycoprotein, but Not of OATP1B1 and OATP1B3 Shingen Misaka,†,‡ Jana Knop,† Katrin Singer,† Eva Hoier,† Markus Keiser,§ Fabian Müller,† Hartmut Glaeser,† Jörg König,† and Martin F. Fromm*,† †

Institute of Experimental and Clinical Pharmacology and Toxicology, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany ‡ Department of Pharmacology, School of Medicine, Fukushima Medical University, Fukushima, Japan § Department of Clinical Pharmacology, Center of Drug Absorption and Transport (C_DAT), University Medicine of Greifswald, Greifswald, Germany ABSTRACT: Nadolol is a nonmetabolized β-adrenoceptor antagonist and is a substrate of OATP1A2, but not of OATP2B1. However, other drug transporters involved in translocation of nadolol have not been characterized in detail. We therefore investigated nadolol as a potential substrate of the hepatic uptake transporters OATP1B1, OATP1B3, and OCT1 and of the renal transporters OCT2, MATE1, and MATE2-K expressed in HEK cells. Moreover, the importance of P-glycoprotein (P-gp) for nadolol transport was studied using double transfected MDCK-OCT1-P-gp cells. Nadolol was not transported by OATP1B1 and OATP1B3. In contrast, a significantly higher nadolol accumulation (at 1 and 10 μM) was found in OCT1, OCT2, MATE1, and MATE2-K cells compared to control cells (P < 0.01). Km values for OCT2-, MATE1-, and MATE2-K-mediated nadolol uptake were 122, 531, and 372 μM, respectively. Cimetidine (100 μM, P < 0.01) and trimethoprim (100 μM, P < 0.001) significantly inhibited OCT1-, OCT2-, MATE1-, and MATE2-K-mediated nadolol transport. The P-gp inhibitor zosuquidar significantly reduced basal to apical nadolol transport in monolayers of MDCK-OCT1-P-gp cells. In summary, nadolol is a substrate of the cation transporters OCT1, OCT2, MATE1, MATE2-K, and of P-gp. These data will aid future in vivo studies on potential transporter-mediated drug−drug or drug−food interactions with involvement of nadolol. KEYWORDS: multidrug and toxin extrusion protein (MATE), metformin, nadolol, organic anion transporting polypeptide (OATP), organic cation transporter (OCT), P-glycoprotein

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canalicular membrane of hepatocytes.6 Renal secretion of organic cations can be mediated by an interplay of OCT2mediated uptake across the basal membrane of proximal tubular cells and efflux via MATE1, MATE2-K, and P-glycoprotein (Pgp).7 Moreover, P-gp localized in the luminal membrane of enterocytes and in the canalicular membrane of hepatocytes could contribute to limited nadolol absorption and to its biliary elimination, respectively.8,9 The objective of this study was therefore to investigate whether nadolol is transported by intestinal, hepatic, and renal uptake and efflux transporters OATP1B1, OATP1B3, OCT1, OCT2, MATE1, MATE2-K, and P-gp using human embryonic kidney 293 (HEK) cells stably or transiently expressing these transporters as well as Caco-2 and OCT1-P-gp doubletransfected Madin−Darby canine kidney (MDCK) II cell

INTRODUCTION Nadolol, a nonselective β-adrenoceptor blocker, is a base with a log D7.4 of 0.93.1 It is not metabolized in humans and 73% and 23% of an intravenous dose is eliminated via the kidneys and the feces, respectively.2 Due to these properties nadolol is a potentially interesting compound for the investigation of transporter-mediated drug−drug interactions in humans. Recently, we showed that green tea substantially reduces nadolol plasma concentrations in humans (−85%) possibly in part by inhibition of OATP1A2-mediated uptake of nadolol in the intestine.3 However, the pharmacokinetic properties as well as the mechanisms underlying the observed drug interactions of nadolol with itraconazole and erythromycin/neomycin4,5 are not fully understood. We therefore investigated nadolol as a potential substrate of the hepatic uptake transporters organic anion transporting polypeptide 1B1 (OATP1B1), 1B3 (OATP1B3), and organic cation transporter 1 (OCT1). A transporter possibly involved in the biliary transport of nadolol could be multidrug and toxin extrusion protein 1 (MATE1), which is expressed in the © 2015 American Chemical Society

Received: Revised: Accepted: Published: 512

September 25, 2015 November 23, 2015 December 24, 2015 December 24, 2015 DOI: 10.1021/acs.molpharmaceut.5b00733 Mol. Pharmaceutics 2016, 13, 512−519

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Molecular Pharmaceutics

Germany). After selection with Geneticin (800 μg/mL) for Pgp [and hygromycin (250 μg/mL) for OCT1], single colonies of the transfectants were screened for ABCB1 and SLC22A1 mRNA expression using RT-PCR and LightCycler-based quantitative RT-PCR (Roche Diagnostics-Applied Science, Mannheim, Germany) to detect the cell clones with the highest expression, as described previously.18 Protein expression of P-gp and OCT1 was verified by immunoblot and immunofluorescence analysis. Immunoblot Analysis. Immunoblot analysis was performed as described previously.18,19 For detection of OCT1 and P-gp 10 μg of total homogenates of all cell lines were diluted with Laemmli buffer (62 mM Tris−HCl, 2% sodium dodecyl sulfate (SDS), 10% glycerol, 0.01% bromphenol blue, and 0.4 mM dithiothreitol) and incubated for 30 min at 37 °C before separation on 10% or 7.5% SDS-polyacrylamide gels. The separated proteins were blotted onto nitrocellulose membranes and incubated either with a rabbit polyclonal antihuman OCT1 antiserum [KEN; 1:10000]20 or with a mouse monoclonal antihuman P-gp antibody (1:4000; SigmaAldrich GmbH) overnight. As secondary antibody, a horseradish peroxidase-conjugated goat antirabbit IgG (GE Healthcare Europe GmbH, Freiburg, Germany) at a 1:10000 dilution and a horseradish peroxidase-conjugated goat antimouse IgG (Jackson Immuno Research, West Grove, PA, USA) diluted 1:1500 were used. Immunoreactive bands were visualized with the ChemiDoc XRS imaging system (Bio-Rad Laboratories GmbH, Munich, Germany) using ECL Western Blotting Detection Reagents (GE Healthcare Europe GmbH). Subsequently, the membranes were stripped and reincubated with a mouse monoclonal antihuman β-actin antibody (1:10000; Sigma-Aldrich GmbH) and detected as described above. Confocal Laser Scanning Immunofluorescence Microscopy. MDCKII cells were grown on Transwell membrane inserts (diameter, 14 mm; pore size, 0.4 μm; Greiner Bio-One GmbH, Frickenhausen, Germany) for 3 days to confluence and induced with 10 mM sodium butyrate for 24 h.21 Cells were fixed with ice-cold 70% methanol, permeabilized for 10 min using TBS/Triton (0.4%), blocked with BSA, and incubated in TBS and 2% BSA with the OCT1 and/or P-gp antibody (dilution 1:200 [KEN] and 1:500 [anti-P-gp]) overnight. The secondary Alexa Flour 568 coupled goat antirabbit IgG (Invitrogen GmbH) and Cy2-coupled goat antimouse antibody (Dianova GmbH, Hamburg, Germany) were used for 30 min at room temperature. Nuclei of MDCK-P-gp single-transfected cells were stained with SYTOX Orange (Molecular Probes, Eugene, OR) at a dilution of 1:30000. Thereafter, the Transwell membranes were cut from the membrane inserts and mounted onto microscope slides using an aqueous mounting medium (Thermo Fisher Scientific, Rockford, IL). The fluorescence was visualized using a confocal laser scanning microscope Axiovert 100M (Carl Zeiss MicroImaging GmbH, Jena, Germany), and images were further processed using the Zeiss LSM Image Browser (version 4.2.0.121). Uptake Studies. Cells were seeded in 12-well plates coated with poly-D-lysine hydrobromide (Greiner Bio-One) at a density of 7.0 × 105 cells/well for HEK-OATP1B1, HEKOATP1B3, HEK-OCT1, HEK-OCT2, and HEK-MATE1 cells. After 24 h, cells were incubated for further 24 h with 10 mM sodium butyrate-containing medium to increase protein expression.21 Prior to the uptake experiments only HEKMATE1 and HEK-MATE2-K cells were preincubated in uptake buffer (142 mM NaCl, 5 mM KCl, 1 mM K2PO4, 1.2 mM

monolayers. In addition, the effects of known inhibitors on nadolol transport were examined.

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MATERIALS AND METHODS Chemicals. [3H]Digoxin (20 Ci/mmol) and [3H]nadolol (25 Ci/mmol) were obtained from American Radiolabeled Chemicals (St. Louis, MO). [14C]Metformin (92.7 mCi/ mmol) was obtained from Biotrend (Cologne, Germany), and [3H]sulphobromophthalein (14 Ci/mmol) was purchased from Hartmann Analytic GmbH (Braunschweig, Germany). Unlabeled sulphobromophthalein was purchased from Applichem GmbH (Darmstadt, Germany). Unlabeled cimetidine, digoxin, metformin, nadolol, poly-D-lysine hydrobromide, and trimethoprim were purchased from Sigma-Aldrich (Taufkirchen, Germany). Zosuquidar trihydrochloride (LY335979) was obtained from Selleck Chemicals (Houston, TX). Minimum essential medium (MEM) and Opti-MEM was obtained from Gibco (Thermo Fischer Scientific, Waltham, MA). Sodium butyrate was purchased from Merck KGaA (Darmstadt, Germany). All cell culture media supplements were obtained from Invitrogen GmbH (Karlsruhe, Germany). All other chemicals and reagents, unless stated otherwise, were obtained from Carl Roth GmbH + Co. KG (Karlsruhe, Germany) and were of the highest grade available. Cell Culture. Stably transfected HEK-OATP1B1, HEKOATP1B3, HEK-OCT1, HEK-OCT2, and HEK-MATE1 as well as transiently transfected HEK-MATE2-K cells and the respective vector control (VC) cells were used as previously described.10−15 HEK-OATP1B1, HEK-MATE1, and the respective VC cells were cultured in MEM containing 10% heat-inactivated fetal bovine serum (FBS), 800 μg/mL Geneticin, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C and 5% CO2, whereas for HEK-OATP1B3, HEKOCT1, HEK-OCT2, and the respective VC cells, 250 μg/mL hygromycin was used instead of Geneticin. Parental HEK cells for transient transfection with a cDNA encoding MATE2-K were cultured in the same medium without the addition of Geneticin.12 HEK-VC cells were established by transfection with a plasmid lacking the insert for expression. Caco-2 cells were obtained from American Type Culture Collection (Rockville, MD) and were cultured in MEM containing 10% FBS, 1% sodium pyruvate, 100 U/mL penicillin, and 100 μg/ mL streptomycin at 37 °C and 5% CO2. The cells were routinely subcultured by trypsinization using trypsin (0.05%)− EDTA (0.02%) solution. Cloning of the Human ABCB1 (P-gp) cDNA. The fulllength cDNA encoding human P-gp was cloned, based on the respective reference sequence (NM_000927.4), from human kidney. The amplified P-gp coding sequence was cloned into the vector pcDNA3.1D/V5-His-TOPO (Invitrogen GmbH) and verified by sequencing (AGOWA GmbH, Berlin, Germany). Subsequently, the ABCB1 cDNA, encoding for a P-gp protein 100% identical to the reference sequence, was cloned into the expression vector pcDNA3.1(+) (Invitrogen GmbH) and the resulting plasmid [pcDNA3.1(+)-P-gp] was used for transfection. Generation of Stably Transfected Cells. Generation and validation of MDCK-VC, MDCK-OCT1, and MDCK-P-gp cells has been described previously.16,17 In order to generate the MDCK-OCT1-P-gp double-transfected cell line, MDCKOCT1 cells were transfected with the plasmid pcDNA3.1(+)P-gp using the Effectene transfection reagent kit according to the manufacturer’s instructions (QIAGEN GmbH, Hilden, 513

DOI: 10.1021/acs.molpharmaceut.5b00733 Mol. Pharmaceutics 2016, 13, 512−519

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counting (for nadolol) or by measuring berberine fluorescence as described.20 The intracellular amount of berberine in the single- and double-transfected cell lines was calculated in the same way. Data Analysis. For cellular uptake assays, the transport mediated by OATP1B1, OATP1B3, OCT1, OCT2, MATE1, and MATE2-K was determined by subtracting the uptake (pmol/min/mg protein) in VC cells from that in the respective transporter-expressing cells. Transport kinetics was calculated by GraphPad Prism (ver. 6.01, GraphPad Software, Inc., San Diego, CA). The calculated parameters were the Michaelis− Menten constant (Km) and maximum transport rate (Vmax). In the transcellular transport experiments apparent permeability coefficients (Papp) were calculated using the following equation:

MgSO4, 1.5 mM CaCl2, 5 mM glucose, and 12.5 mM HEPES, pH 7.3) containing 30 mM ammonium chloride for 20 min at 37 °C. We used this method of intracellular acidification to generate an outwardly directed pH gradient to stimulate the organic cation/H+ antiporters MATE1 and MATE2-K.22 Before the uptake experiment, all cells were washed with prewarmed uptake buffer. Subsequently, cells were incubated with the test solution, containing the substrate drug, i.e., BSP for OATP1B1 (0.05 μM) and OATP1B3 (1 μM) or metformin for OCT1, OCT2, MATE1, and MATE2-K (10 μM for all cell lines) or nadolol (ranging from 1 to 1000 μM) at 37 °C for 5 min (OCT1, OCT2, MATE1, and MATE2-K) or 10 min (OATP1B1 and OATP1B3). Then cells were washed three times with ice-cold uptake buffer and lysed with 0.2% SDS. Intracellular accumulation of radioactivity was measured in 500 μL of cells lysates by liquid scintillation counting (TriCarb 2800; PerkinElmer Life and Analytical Sciences, Bonn, Germany). The protein concentration of each sample was determined in 25 μL of cells lysates by bicinchoninic acid assay (BCA Protein Assay Kit; Thermo Fisher Scientific). For inhibition studies, nadolol (1 μM) or metformin (10 μM) transport was investigated in the presence of known inhibitors cimetidine or trimethoprim at a final concentration of 100 μM for the inhibition of OCT1-, OCT2-, MATE1-, or MATE2-Kmediated transport. Transcellular Transport Studies in Caco-2 Cell Monolayers. Caco-2 cells were plated on Transwell membrane inserts (diameter, 14 mm; pore size, 0.4 μm; Greiner Bio-One GmbH). Transport experiments were performed on day 7 after plating. About 1 h prior to the start of the transport experiment, the medium in each compartment was replaced by Opti-MEM. For transport experiments, OptiMEM in each compartment was then replaced with 800 μL of serum-free medium with addition of a radiolabeled substrate [3H]digoxin (5 μM) or [3H]nadolol (1, 10, and 100 μM) on the basal or the apical side of the monolayer. During the incubation at 37 °C, the amounts of substrate appearing in the opposite compartment (basal or apical) were measured after 1, 2, 3, and 4 h in 50 μL aliquots. Experiments were conducted only with wells that had a transepithelial resistance of >200 Ω after correction for the resistance obtained in control blank wells. The radioactivity was measured in 50 μL samples by liquid scintillation counting (TriCarb 2800; PerkinElmer Life and Analytical Sciences). Transcellular Transport Studies in MDCK-OCT1-P-gp Cell Monolayers. Vectorial transport assays in MDCKOCT1-P-gp double-transfected cells were performed as described previously.13 For the characterization of the double transfectants, MDCK-VC, MDCK-OCT1, and MDCK-P-gp single-transfected cells were used in control experiments. In brief, cells were seeded onto Transwell filters (diameter, 14 mm; pore size, 0.4 μm; Greiner Bio-One GmbH) at an initial density of 4 × 105 cells/well and grown for 3 days. Twenty-four hours before the transport experiments, the cells were induced with 10 mM sodium butyrate. Subsequently, after being washed with prewarmed uptake buffer, cells were incubated with 800 μL uptake buffer containing a mixture of radiolabeled and nonlabeled nadolol (final concentration: 10 μM) in the basal compartment. For the characterization of the double-transfectants, berberine was used in a concentration of 25 μM. After 1 h (for berberine) or at indicated time points (for nadolol), aliquots from the apical compartments were taken for the determination of the radioactivity by liquid scintillation

Papp = dQ /dt × 1/(A × C0) [cm/s × 10−6]

where dQ/dt is the permeability rate, A is the surface of the cell monolayer (1.13 cm2), and C0 is the initial concentration of compound applied to the donor chamber. Statistical Analysis. All transport experiments were performed on two separate days with at least two wells per time point and day (i.e., at least n = 4 per experiment). All data are expressed as mean ± standard error of the mean (SEM). Statistical differences among groups were evaluated by unpaired t test (between two groups) or one-way ANOVA (between multiple groups) with Dunnet’s test using GraphPad Prism (GraphPad Software, Inc.). Differences were regarded as statistically significant with P < 0.05.

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RESULTS Characterization of the Established Double-Transfected MDCK-OCT1-P-gp Cell Line. Transfection of the existing MDCK-OCT1 cell line with the ABCB1 cDNA encoding for human P-gp resulted in the double-transfected MDCK-OCT1-P-gp cell line recombinantly overexpressing the uptake transporter OCT1 simultaneously with the export pump P-gp. Immunoblot analyses demonstrated comparable amounts of OCT1 and P-gp in single-transfected cell lines and in the double-transfected MDCK-OCT1-P-gp cell line (Figure 1A). Immunofluorescence analysis localized OCT1 in the lateral membrane of polarized grown MDCKII cells and P-gp in the apical membrane (Figure 1B). Functionality of both transporters was tested using the model substrate berberine.20 As shown in Figure 1C significant berberine uptake could be detected only in MDCK-OCT1 cells, whereas transcellular transport of berberine could be measured only in doubletransfected MDCK-OCT1-P-gp cells. Involvement of Hepatic Uptake Transporters in the Cellular Accumulation of Nadolol. No significant OATP1B1-mediated nadolol uptake was observed at 1 and 10 μM (Figure 2A). There was also no difference in cellular uptake of nadolol between HEK-OATP1B3 and VC cells (Figure 2B). As a positive control, uptake of the prototypical substrate BSP was measured in HEK-OATP1B1 and HEKOATP1B3 cells and the respective VC cells (Figure 2A,B). BSP uptake in transfected cells was 8.1-fold (OATP1B1) and 5.8fold (OATP1B3) higher compared with VC cells (P < 0.001). Cellular uptake of nadolol at 1 and 10 μM was 5.2- and 6.5-fold higher, respectively, in HEK-OCT1 cells compared with HEKVC cells (P < 0.001, Figure 2C), indicating that nadolol is a substrate of OCT1. OCT1-mediated uptake of the prototypical 514


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Figure 1. Characterization of MDCK-OCT1 and -P-gp single transfectants and the double-transfected cell line MDCK-OCT1-Pgp. (A) OCT1 (left) and P-gp protein (right) expression in MDCKcontrol cells (MDCK-Co), MDCK-OCT1, and MDCK-P-gp single transfectants and the established double-transfected cell line MDCKOCT1-P-gp. (B) Immunolocalization of the subcellular localization of the OCT1 (red fluorescence) and the P-gp (green fluorescence) in the respective single- and double-transfected cells examined by confocal laser scanning microscopy. a, b, and c show top view images of the cell monolayers; a′, b′, and c′ are vertical optical sections through the monolayers at the positions, indicated by the green lines. (C) Uptake and transcellular transport of berberine (25 μM) in the respective single- and double-transfected cells. Berberine was administered at the basal side of monolayers of MDCK-control cells (Co), MDCK-OCT1 cells (OCT1), MDCK-P-gp cells (P-gp), and of MDCK-OCT1-P-gp cells (OCT1-P-gp) and the uptake into the cells (left panel) or the transcellular transport of berberine into the apical compartment (right panel) was measured after 1 h. ***P < 0.001 vs Co.

OCT1 substrate metformin was 9.6-fold higher compared with VC cells (P < 0.001). Kinetic analysis showed that OCT1mediated nadolol uptake (1 to 1000 μM) was not saturated across this concentration range indicating that the affinity of nadolol to OCT1 is low with a Km value higher than 500 μM (Figure 2D). Involvement of Renal Cation Transporters in the Cellular Accumulation of Nadolol. To clarify whether nadolol is a substrate of renal cation transporters, HEK-OCT2, HEK-MATE1, HEK-MATE2-K, and the respective VC cells were incubated with nadolol (1 μM or 10 μM) for 5 min (Figure 3A−C). OCT2-, MATE1-, and MATE2-K-mediated nadolol uptake was 2.4-fold (P < 0.001), 2.4-fold (P < 0.001),

Figure 2. Uptake of nadolol by HEK cells transfected with the hepatic uptake transporters OATP1B1 (A), OATP1B3 (B), and OCT1 (C). Sulfobromophthalein (BSP, for OATP1B1 and OATP1B3) and metformin (for OCT1) served as positive controls. (D) Kinetics of OCT1-mediated uptake of nadolol. Net uptake was calculated as the difference between nadolol uptake into OCT1-expressing cells and uptake into the vector control cells. Uptake was measured for 10 min (OATP1B1 and OATP1B3) or 5 min (OCT1). Inset: Eadie−Hostee plot of OCT1-mediated uptake of nadolol. Data are presented as mean ± SEM (n = 4). ***P < 0.001 vs vector control (VC). 515


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Figure 4. Kinetics of OCT2- (A), MATE1- (B), and MATE2-Kmediated (C) uptake of nadolol. Net uptake was calculated as the difference between nadolol uptake into OCT2-, MATE1-, and MATE2-K-expressing cells and uptake into the respective vector control cells. Uptake was measured for 5 min. Inset: Eadie−Hostee plots of OCT2-, MATE1-, and MATE2-K-mediated uptake of nadolol. Data are presented as mean ± SEM (n = 4).

Figure 3. Uptake of nadolol by HEK cells transfected with the renal transporters OCT2 (A), MATE1 (B), and MATE2-K (C). Metformin served as positive control. Data are presented as mean ± SEM (n = 4). **P < 0.01, ***P < 0.001 vs vector control (VC).

Involvement of P-gp in Transcellular Transport of Nadolol. To investigate whether P-gp plays a role in the efflux of nadolol, we first performed transcellular transport studies (1, 10, and 100 μM) in monolayers of Caco-2 cells. Nadolol translocation from the basal to the apical side of the monolayers was small with less than 2% of administered compound reaching the apical compartment after 4 h (Papp,B‑A = 1.0 ± 0.1 × 10−6 cm/s). At all concentrations, basal to apical transport of nadolol was not higher compared to apical to basal transport (Figure 6A−C). The basal to apical transport of the prototypical P-gp substrate digoxin (5 μM) was considerably higher than the apical to basal transport with efflux ratios higher than 3.5 (P < 0.001) (Figure 6D). Our data with the Caco-2 cells indicate that nadolol has poor membrane permeability. In order to increase access of nadolol into the cells we used MDCK-OCT1-P-gp double-transfected cells, based on our findings described above that nadolol is a OCT1 substrate. Polarized, basal to apical transport of nadolol

and 25.8-fold (P < 0.001), respectively, higher compared to VC cells at 10 μM. These findings indicate that nadolol is a substrate of OCT2, MATE1, and MATE2-K. Km values for OCT2-, MATE1-, and MATE2-K-mediated nadolol uptake were 122 ± 44, 531 ± 170, and 372 ± 84 μM, respectively (Figure 4A−C). Inhibition of Transporter-Mediated Nadolol Uptake by Cimetidine and Trimethoprim. In the presence of cimetidine (100 μM), nadolol uptake by OCT1, OCT2, MATE1, and MATE2-K was significantly inhibited by 34%, 100%, 85%, and 90%, respectively (Figure 5A,C,E,G). In addition, trimethoprim significantly inhibited OCT1-, OCT2-, MATE1-, and MATE2-K-mediated nadolol uptake by 89%, 87%, 88%, and 95%, respectively (Figure 5A,C,E,G). In control experiments, cimetidine and trimethoprim significantly reduced OCT1-, OCT2-, MATE1-, and MATE2-K-mediated metformin uptake (Figure 5B,D,F,H). 516


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Figure 6. Basal to apical and apical to basal translocation of nadolol (A−C) and digoxin (D) across Caco-2 monolayers. Basal to apical, closed circle; apical to basal, open circle. Data are presented as mean ± SEM (n = 6). ***P < 0.001 vs apical to basal transport at the respective time point.

Figure 5. Inhibition of OCT1-, OCT2-, MATE1-, and MATE2-Kmediated uptake of nadolol (A, C, E, G; 1 μM) and metformin (B, D, F, H; 10 μM) by cimetidine (100 μM) and trimethoprim (100 μM). Data are presented as mean ± SEM (n = 4). **P < 0.01, ***P < 0.001 vs uptake without inhibitor (control).

nadolol transport was significantly reduced by zosuquidar indicating that nadolol is a P-gp substrate (Papp,B‑A at 4 h with and without zosuquidar: 0.8 ± 0.1 vs 1.3 ± 0.1 × 10−6 cm/s, Figure 7). In MDCK-VC cells, no difference was observed in basal to apical nadolol transport in the absence or presence of zosuquidar (data not shown).

across monolayers of MDCK-OCT1-P-gp double-transfected cells in the absence and presence of the P-gp inhibitor zosuquidar is shown in Figure 7. At 2, 3, and 4 h basal to apical 517


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in the apical domain of distal nephrons.24 With regard to hypothesis (1) it should be considered that administration of 4 × 300 mg of cimetidine for 2 days did not significantly increase AUC and Cmax of nadolol in healthy volunteers.25 Unfortunately, nadolol renal clearance was not determined in this study. Interestingly, Morrison et al. reported after intravenous administration of nadolol to healthy volunteers a nadolol renal clearance of 131 to 150 mL/min, which points to active renal nadolol secretion.26 The differences in the observed renal clearances between Morrison et al.26 and Misaka et al.3 could be due to differences in study design (iv vs po, sampling periods, differences in plasma concentrations) and ethnicity. As expected,27 nadolol was poorly permeable across Caco-2 monolayers. Basal to apical translocation was not greater than apical to basal translocation in the Caco-2 monolayers. This negative result could be due to a lack of nadolol uptake into the Caco-2 cells. Our findings are in line with transporter expression data in Caco-2 cells determined by mass spectrometry.28 Uchida et al. showed as expected the highest expression for P-gp in Caco-2 cells among a panel of 28 human transporters. In contrast, expression of OCT1, OCT2, and OCT3 in Caco-2 cells was below the limit of quantification. These expression data support our findings that no sufficient transporter-mediated uptake occurs for nadolol in Caco-2 cells yielding in lack of polarized nadolol transcellular translocation. Therefore, we established and characterized a new doubletransfected MDCKII cell line, which expresses both an uptake transporter for cationic drugs and the efflux transporter P-gp. Using this double-transfected cell line we could show that basal to apical translocation of nadolol was moderately, but significantly inhibited by the P-gp inhibitor zosuquidar (Papp = −40%). This indicates that nadolol is transported by P-gp. Our findings are in line with a previous study suggesting that nadolol is a substrate of P-gp.29 Inhibition of intestinal and renal P-gp is one possible underlying mechanism of significantly increased nadolol AUC or Cmax after coadministration of the Pgp inhibitors itraconazole and erythromycin/neomycin, respectively, to healthy volunteers.4,5 Taken together, our in vitro experiments predicted a contribution of OCTs/MATEs to biliary and renal transport of nadolol. In vivo data obtained from studies in humans are not in line with OCT2/MATE-mediated renal secretion of nadolol. Further studies should clarify the reasons for this in vitro−in vivo discrepancy. This aspect should also be relevant for in vitro transporter studies during drug development30,31 because “false” positive in vitro studies will trigger further, potentially unnecessary studies in healthy volunteers. Moreover, our data indicate an involvement of P-gp in nadolol transcellular translocation.

Figure 7. Basal to apical translocation of nadolol across monolayers of MDCK-OCT1-P-gp double-transfected cells. Zosuquidar (1 μM) was used as P-gp inhibitor. With zosuquidar, closed circle; without zosuquidar, open circle. Data are presented as mean ± SEM (n = 6). *P < 0.05, **P < 0.01 vs without zosuquidar.

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DISCUSSION Since nadolol is not metabolized in humans it might be an interesting compound for investigations on transportermediated drug interactions. The goal of the present in vitro studies was to contribute to a better understanding of the drug transporters, which are involved in the transcellular translocation of nadolol. We previously reported that nadolol is a substrate of OATP1A2, but not of OATP2B1.3 Inhibition of OATP1A2mediated intestinal nadolol uptake by catechins could contribute to the substantial decrease in nadolol plasma concentrations after ingestion of green tea.3 Our present data indicate that nadolol uptake into hepatocytes is not mediated by OATP1B1 and OATP1B3. According to our present data hepatic nadolol uptake could be mediated by OCT1, whereas MATE1 and P-gp localized in the canalicular membrane of hepatocytes could be involved in nadolol biliary elimination. We also showed that nadolol is a good substrate of the renal cation transporters (OCT2, MATE1, and MATE2-K). At 10 μM nadolol the uptake ratios for OCT2-, MATE1-, and MATE2-K-mediated nadolol uptake were 2.4-, 2.4-, and 25.8-fold compared to VC. The Km value for OCT2-mediated nadolol uptake was 122 μM, whereas the respective Km values were 531 and 372 μM for MATE1- and MATE2-K-mediated nadolol uptake, respectively. These Km values are considerably higher compared to the unbound nadolol plasma concentrations (approximately 0.2 μM) after an oral administration of nadolol (30 mg),3 indicating that these transporters will not be saturated in the clinical setting. As expected, in vitro transport of nadolol by these cation transporters could be significantly inhibited by prototypical inhibitors such as cimetidine and trimethoprim. From these data one would expect renal secretion of nadolol in humans. However, renal clearance of nadolol was only 82 mL/min in our study in healthy volunteers.3 This number roughly corresponds to the expected clearance by filtration when taking into account a reported serum protein binding of approximately 14%.23 There are at least two potential explanations for this in vitro−in vivo discrepancy. (1) OCT2-mediated nadolol uptake into renal proximal tubular cells is less pronounced in humans than expected from the in vitro experiments. (2) Nadolol is actively secreted by an interplay of OCT2 and MATE1/ MATE2-K, but a pronounced renal reabsorption by transporters occurs more distally. One candidate transporter is OATP1A2, which has been reported to be expressed in humans

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AUTHOR INFORMATION

Corresponding Author

*Tel: +49 9131 85 22772. Fax: +49 9131 85 22773. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Dr. Sabine Klatt for expert advice and Ms. Ingrid Schmidt for excellent technical assistance. This work was supported by the German Federal Ministry of Education and Research Grant InnoProfile 03IPT612X. 518


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The Nonmetabolized Î²-Blocker Nadolol Is a Substrate of OCT1, OCT2

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