Interplay of the Organic Cation Transporters OCT1 and OCT2 with the

Article Cite This: Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Interplay of the Organic Cation Transporters OCT1 and OCT2 with the Apically Localized Export Protein MATE1 for the Polarized Transport of Trospium Birgit Deutsch,† Claudia Neumeister,‡ Ulrich Schwantes,‡ Martin F. Fromm,† and Jörg König*,† †

Mol. Pharmaceutics Downloaded from pubs.acs.org by UNIV OF WINNIPEG on 01/19/19. For personal use only.

Institute of Experimental and Clinical Pharmacology and Toxicology, Friedrich-Alexander-Universität Erlangen-Nürnberg, 91054 Erlangen, Germany ‡ Dr. R. Pfleger GmbH, 96052 Bamberg, Germany ABSTRACT: The anticholinergic drug trospium is secreted into urine and, to a smaller extent, into bile. Chemically, it is an organic cation, and it is a substrate of the uptake transporters OCT1 and OCT2 as well as for the export proteins MATE1 and MATE2-K as determined in uptake studies using HEK293 cells. So far, neither MATE-mediated export nor the interplay of OCTmediated uptake and MATE-mediated export have been investigated. Therefore, we used polarized monolayers of singleand double-transfected MDCKII cells (MDCK-OCT1, MDCKOCT2, MDCK-MATE1, MDCK-OCT1-MATE1, and MDCKOCT2-MATE1) and the respective control cells (MDCK-Co) for transcellular transport assays. We demonstrate that the transcellular, basal-to-apical transport of trospium is significantly higher in all cell lines compared to control cells over nearly the complete concentration range tested. The transcellular transport mediated by double-transfected MDCK-OCT1-MATE1 and MDCK-OCT2-MATE1 exceeded that in the single-transfected cells (MDCK-OCT1-MATE1 vs MDCK-OCT1: 2.2-fold; MDCK-OCT1-MATE1 vs MDCK-MATE1: 1.7-fold; MDCK-OCT2-MATE1 vs MDCK-OCT2: 6.1-fold; MDCK-OCT2MATE1 vs MDCK-MATE1: 1.8-fold at a trospium concentration of 1.0 μM; p < 0.001 each). Thus, we show that MATE1 does not only mediate the uptake of trospium into HEK293 cells but also the efflux of trospium out of polarized MDCKII-cells. Furthermore, our results indicate that OCT1 or OCT2 as uptake transporters and MATE1 as an export protein contribute to the transcellular transport of trospium at concentrations normally reached during trospium therapy. These data suggest that both, OCT-mediated uptake as well as MATE1-mediated efflux may contribute to trospium renal and biliary elimination. KEYWORDS: trospium, OCT1, OCT2, MATE1, transcellular drug transport, drug excretion

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Wenge et al.28 showed that trospium is a substrate of organic cation transporters (OCTs). OCTs are members of the SLC22 family of solute carriers and are expressed in the basolateral membrane of cells where they mediate the uptake of substrates from blood into the cells. OCT1 (SLC22A1) is expressed in the basolateral membrane of hepatocytes, whereas OCT2 (SLC22A2) is located in the basolateral membrane of renal proximal tubule cells.11−17 As multidrug and toxin extrusion proteins (MATEs) have a quite similar substrate spectrum as OCTs, trospium was also tested and characterized as a substrate for these transporters by Chen et al.18 MATEs are expressed in the apical membrane of cells. MATE1 (SLC47A1) is expressed in both, the canalicular membrane of hepatocytes and the luminal membrane of proximal tubule cells, whereas

INTRODUCTION Trospium is a quaternary amine, which belongs to the drug family of anticholinergics. It is used to treat the symptoms of overactive bladder syndrome.1,2 Due to its chemical structure as quaternary amine, trospium is a completely positively charged molecule with hydrophilic properties under physiological conditions. Therefore, trospium can hardly diffuse through plasma membranes and needs to be transported across them by transport proteins. Only a small amount is absorbed in the intestine, leading to an oral bioavailability of about 10%. The plasma protein binding is about 40−60% depending on the dosage.3 Intracellularly, trospium is only partially metabolized and excreted mainly unchanged, preferentially by the kidneys (about 70−80% of intravenously administered dose) and, to a smaller extent (about 20−30% of intravenously administered dose), by the bile and the gut.1−10 Its renal clearance is about 540 mL/min,10 which considerably exceeds glomerular filtration rate. This indicates that trospium must be additionally secreted into urine by transport proteins.3 © XXXX American Chemical Society

Received: July 24, 2018 Revised: October 10, 2018 Accepted: January 3, 2019

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DOI: 10.1021/acs.molpharmaceut.8b00779 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics

added as selection antibiotics. Routine subculturing was performed twice a week by using trypsin (0.05%)−EDTA (0.02%) solution. Concentration-Dependent Transcellular Transport Studies. Transcellular transport assays were conducted as previously described.29 Cells were seeded on ThinCert-TC inserts at densities appropriate for each cell line to get a tight monolayer at the day of experiment (MDCK-Co and MDCKMATE1: 500 000 cells per filter insert; MDCK-OCT1, MDCK-OCT2, MDCK-OCT1-MATE1 and MDCK-OCT2MATE1: 600 000 cells per filter insert). Tightness was routinely tested with [3H]inulin. After 48 h, cells were incubated with sodium butyrate (10 mM) to enhance protein expression.37 Transport experiments were performed 72 h after seeding. Incubation medium was composed of 142 mM NaCl, 5 mM KCl, 1 mM K2HPO4, 1.2 mM MgSO4, 1.5 mM CaCl2, 5 mM glucose, and 12.5 mM HEPES buffer (pH 6.0−8.0). The pH in the basal compartment was 7.3 and in the apical compartment 6.5 to enhance MATE1 export activity. Radiolabeled ([3H]trospium) and unlabeled substrate were dissolved in incubation medium to get incubation medium with concentrations of 0.1, 1, 10, or 50 μM trospium, respectively. First, cells were washed once with prewarmed incubation medium. Then, culture medium was exchanged against incubation medium containing unlabeled and radiolabeled trospium in the basal compartment and against incubation medium without substrate in the apical compartment. Afterwards, the cells were incubated at 37 °C and 5% CO2 for 60 min. At the end of the incubation period, aliquots (400 μL) were taken from the apical compartment, cells were washed thrice with ice-cold incubation buffer (pH 7.3) to stop transport and then lysed with 600 μL sodium dodecyl sulfate (0.2%). Aliquots (400 μL) of the lysate were taken to measure intracellular accumulation of trospium. Radioactivity in the aliquots was determined by liquid scintillation counting (Tricarb2800, PerkinElmer Life Sciences GmbH, Germany). Protein concentrations were determined by bicinchoninic acid assay (Pierce BCA protein assay kit, ThermoFisher Scientific, Waltham, MA, USA). pH-Dependent Transcellular Transport Studies. To study the pH-dependency of MATE-mediated transport, the same experimental setup was used with minor modifications. In contrast to prior experiments, trospium was used at a concentration of 1.0 μM, and the pH in the apical compartment was adjusted to 6.0, 6.5, 7.0, 7.5, and 8.0 to achieve different proton concentrations in the apical compartment. Time-Dependent Transcellular Transport Studies. Experimental setup for investigating the time dependency of trospium transport was also comparable to that described for concentration-dependent transcellular transport studies with pH values of 7.3 in the basal compartment and 6.5 in the apical compartment. Trospium was used at a concentration of 1 μM, and incubation time was set to 30, 60, and 120 min. Data Analysis. Transport experiments were done in two independent experiments at two different days with three separate wells per day for each cell line (n = 6). Transport values were normalized to the respective protein concentration. All data are presented as mean ± standard deviation of the mean (SD), only Km values are presented as mean ± standard error of the mean (SEM). Multiple comparisons were analyzed by one-way ANOVA followed by Tukey’s multiple comparison test or Dunnett’s multiple comparison test as applicable using

MATE2-K (SLC47A2) is only expressed in the apical membrane of proximal tubule cells.19 MATEs are protoncoupled export proteins and, therefore, are believed to act as efflux transporters in vivo.12−15,19−27 Studies concerning OCT-mediated trospium transport revealed a Km value of 17.0 μM for OCT1 and 8.0 μM for OCT2.28 For OCT1, this Km value was confirmed by Chen et al., but they calculated a considerably lower Km value, 0.6 μM, for OCT2.18 Interestingly, Bexten et al. showed that trospium is a substrate for OCT1 (with a much higher Km value of 106 μM), but found no time-dependent uptake of trospium into HEK-OCT2 cells.10 Transport of trospium by the export proteins MATE1 and MATE2-K was studied by Chen et al. using HEK293 cells recombinantly overexpressing these transporters. In uptake experiments, they showed that MATE1 transports trospium with a high capacity, but relatively low affinity (Km= 15.4 μM) compared to MATE2-K, which has a low capacity, but a slightly higher affinity (Km= 8.2 μM).18 Taken together, available data suggest that the transcellular transport of trospium during its hepatobiliary and renal elimination could be mediated by the interplay of OCTs and MATEs, with OCTs being responsible for the uptake of trospium into the cells and MATEs subsequently facilitating the efflux of trospium out of the cells. As previous studies18,28 were only performed as uptake studies with nonpolarized HEK293 cells overexpressing either OCTs or MATEs, the aim of our current work was to examine the transcellular, basal-toapical transport of trospium by using monolayers of polarized Madin−Darby canine kidney II (MDCKII) cells singletransfected with OCT1 (MDCK-OCT1), OCT2 (MDCKOCT2), or MATE1 (MDCK-MATE1) or double-transfected with OCT1 and MATE1 or OCT2 and MATE1 (MDCKOCT1-MATE1 or MDCK-OCT2-MATE1).

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EXPERIMENTAL SECTION Material. [3H]Trospium trifluoroacetate (24.6 Ci/mmol, RC Tritec AG (Teufen, Switzerland)) and unlabeled trospium chloride were kindly provided by Dr. R. Pfleger GmbH (Bamberg, Germany). [3H]Inulin (2.25 Ci/mmol) was obtained from PerkinElmer (Boston, MA, USA). Cell culture media, selection antibiotics (hygromycin, G418), and trypsin (0.05%)−EDTA (0.2%) solution were obtained from LifeTechnologies (Carlsbad, CA, USA). Cellstar 12-well cell culture plates were from Greiner BioOne (Frickenhausen, Germany), and ThinCert-TC inserts (12 well, 0.4 μm pore size, translucent) were obtained from Sarstedt (Nümbrecht, Germany). Sodium butyrate was bought from Merck KGaA (Darmstadt, Germany). All other chemicals were purchased from Carl Roth GmbH & Co. KG (Karlsruhe, Germany). Cell Lines. Experiments were conducted with Madin-Darby canine kidney II (MDCKII) cells. Single-transfected cell lines stably overexpressing OCT1 (MDCK-OCT1), OCT2 (MDCK-OCT2) or MATE1 (MDCK-MATE1), double-transfected cell lines stably overexpressing OCT1 or OCT2 together with MATE1 (MDCK-OCT1-MATE1 and MDCKOCT2-MATE1) and vector-transfected control cells (MDCKCo) have previously been established and characterized.18,29−36 Cell Culture. Cells were cultured in minimal essential medium (including 10% heat-inactivated fetal bovine serum, 2 mM nonessential amino acids, 100 μg/mL streptomycin and 100 IU/mL penicillin) at 37 °C and 5% CO2. Hygromycin B (250 μg/mL) and/or G418 (800 μg/mL) were regularly B


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Figure 1. Transcellular, basal-to-apical transport and intracellular accumulation of trospium studied in monolayers of MDCKII cells overexpressing OCT1 and/or MATE1. pH was adjusted to 7.3 in the basal compartment and to 6.5 in the apical compartment. Different concentrations of trospium (0.1, 1, 10, and 50 μM) were added to the basal compartment and accumulation of trospium in the apical compartment and intracellularly was measured after 60 min. (A) Transcellular transport of trospium in MDCK-OCT1, MDCK-MATE1, MDCK-OCT1-MATE1, and MDCK-Co cells after 60 min (*** p < 0.001 vs MDCK-Co, ### p < 0.001 vs MDCK-OCT1, § p < 0.05 vs MDCK-MATE1, §§§ p < 0.001 vs MDCK-MATE1). (B) Intracellular accumulation of trospium in MDCK-OCT1, MDCK-MATE1, MDCK-OCT1-MATE1 and MDCK-Co cells after 60 min (*** p < 0.001 vs MDCK-Co, ### p < 0.001 vs MDCK-OCT1-MATE1). (C) Concentration dependency of transcellular transport and intracellular accumulation in MDCK-OCT1, MDCK-MATE1, MDCK-OCT1-MATE1, and MDCK-Co cells after 60 min. Data are shown as mean ± SD (n = 6).

cells (Figure 1A,C). Highest transport ratios compared to MDCK-Co were detected at a concentration of 1 μM. At this concentration, transcellular transport by MDCK-OCT1MATE1 cells was 24.5-fold higher compared to MDCK-Co cells and 2.2- and 1.7-fold higher compared to MDCK-OCT1 cells or MDCK-MATE1 cells, respectively. In line with these results, intracellular accumulation of trospium by MDCKOCT1-MATE1 cells was lower than that in MDCK-OCT1 cells, but higher than that in MDCK-MATE1 and MDCK-Co cells (Figure 1B,C). Based on the results obtained with singletransfected MDCK-OCT1, a Km for OCT1-mediated trospium transport of 22.0 ± 3.0 μM could be calculated. pH-Dependency of Transcellular Trospium Transport by MDCK-OCT1-MATE1 Cells. pH-dependency of transcellular transport and intracellular accumulation of trospium was investigated by varying the pH value in the apical

GraphPad Prism 5.01 (GraphPad Software, San Diego, CA, USA). A value of p < 0.05 was defined as statistically significant.

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RESULTS Concentration-Dependency of Transcellular Trospium Transport by MDCK-OCT1-MATE1 Cells. First, we investigated the concentration-dependency of transcellular, basal-to-apical trospium transport mediated by the hepatic uptake transporter OCT1 and the apically localized export protein MATE1 by using polarized monolayers of MDCK-OCT1-MATE1. MDCK-OCT1, MDCK-MATE1 and MDCK-Co cells were used as control cells. Over the complete concentration range tested, transcellular transport by MDCKOCT1-MATE1 cells was significantly higher compared to the transcellular transport by single-transfected and MDCK-Co C


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Molecular Pharmaceutics compartment. Transcellular transport in MDCK-OCT1MATE1 cells (Figure 2A) was highest at pH 6.5 (649.1 ±

Figure 3. Time-dependency of transcellular, basal-to-apical transport and intracellular accumulation of trospium studied in monolayers of MDCKII cells overexpressing OCT1 and/or MATE1. pH was adjusted to 7.3 in the basal compartment and to 6.5 in the apical compartment. Trospium (1 μM) was added to the basal compartment and incubation time was varied between 30 and 120 min (** p < 0.01 MDCK-OCT1 vs MDCK-Co, *** p < 0.001 MDCK-OCT1 vs MDCK-Co, ### p < 0.001 MDCK-MATE1 vs MDCK-Co, §§ p < 0.01 MDCK-OCT1-MATE1 vs MDCK-Co, §§§ p < 0.001 MDCK-OCT1MATE1 vs MDCK-Co). Data are shown as mean ± SD (n = 6).

Figure 2. pH-dependency of transcellular, basal-to-apical transport and intracellular accumulation of trospium studied in monolayers of MDCKII cells overexpressing OCT1 and/or MATE1. Trospium (1 μM) was added to the basal compartment (pH = 7.3) and pH in the apical compartment was varied between 6.0 and 8.0. Incubation time was 60 min (*** p < 0.001 MDCK-MATE1 vs MDCK-Co, ** p < 0.01 MDCK-MATE1 vs MDCK-Co, * p < 0.05 MDCK-MATE1 vs MDCK-Co, ### p < 0.001 MDCK-OCT1-MATE1 vs MDCK-Co). Data are shown as mean ± SD (n = 6).

relatively low Km value for OCT2-mediated trospium transport of 2.1 ± 1.8 μM was calculated. Time-Dependency of Transcellular Trospium Transport by MDCK-OCT2-MATE1 Cells. Time-dependency of OCT2- and MATE1-mediated transcellular trospium transport was also investigated by using different incubation periods. Transport of trospium into the apical compartment mediated by MDCK-OCT2-MATE1 increased over time (Figure 5A), whereas the intracellular accumulation in these cells tended to slightly decline over time (Figure 5B).

43.9 pmol × mg protein−1 × h−1 vs 316.3 ± 40.1 pmol × mg protein−1 × h−1 at pH 8.0). Intracellular accumulation in MDCK-OCT1-MATE1 (Figure 2B) was lowest at pH 6.5 (17.1 ± 2.3 pmol × mg protein−1 × h−1 vs 39.9 ± 7.7 pmol × mg protein−1 × h−1 at pH 8.0) (Figure 2). Time-Dependency of Transcellular Trospium Transport by MDCK-OCT1-MATE1 Cells. Time-dependency of transcellular trospium transport by OCT1 and MATE1 was tested by using different incubation periods. Figure 3A shows that the transcellular transport of trospium into the apical compartment mediated by MDCK-OCT1-MATE1 increased over time. The intracellular accumulation in these cells was constant from 60 min on (Figure 3B). Concentration-Dependency of Transcellular Trospium Transport by MDCK-OCT2-MATE1 Cells. Next, the transporter combination of OCT2 and MATE1, expressed in renal proximal tubule cells, was investigated using polarized monolayers of MDCK-OCT2-MATE1. MDCKOCT2, MDCK-MATE1 and MDCK-Co cells were used as control cells. In line with the experiments with MDCK-OCT1MATE1, transcellular, basal-to-apical transport by MDCKOCT2-MATE1 cells was significantly higher compared to that in all other cell lines over the complete concentration range (Figure 4A,C) with transport ratios at 1 μM of 34.2-fold, 6.1fold, and 1.8-fold compared to MDCK-Co, MDCK-OCT2, and MDCK-MATE1, respectively. Intracellular accumulation of trospium in MDCK-OCT2-MATE1 cells was lower than that in MDCK-OCT2 cells at low trospium concentrations of 0.1 and 1 μM but higher when concentrations of 10 or 50 μM were used (Figure 4B,C). In line with these observations, a

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DISCUSSION The aim of this study was to investigate the interplay of the basolateral uptake transporters OCT1 and OCT2 with the apically localized export protein MATE1 in the transcellular transport of trospium. In our experiments, we detected a significantly higher transcellular transport of trospium by all cell lines compared to the control cell line at nearly all concentrations tested. The relatively high transcellular transport mediated by the single-transfected cells MDCK-OCT1, MDCK-OCT2, and MDCK-MATE1 could be explained by endogenously expressed transporters, e.g. canine P-glycoprotein or Oct1/Oct2.38 Nevertheless, the transcellular transport mediated by double-transfected MDCK-OCT1-MATE1 and MDCK-OCT2-MATE1 cells was always significantly higher than the transport mediated by the single-transfected cells (Figures 1A,C and 4A,C). Thus, we showed for the first time, that both, uptake and efflux transporters are important for the transcellular transport of trospium. So far, trospium was only tested as a substrate for MATE-proteins in uptake experiments with single-transfected HEK293 cells,18 but MATE-mediated D


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Figure 4. Transcellular, basal-to-apical transport and intracellular accumulation of trospium studied in monolayers of MDCKII cells overexpressing OCT2 and/or MATE1. pH was adjusted to 7.3 in the basal compartment and to 6.5 in the apical compartment. Different concentrations of trospium (0.1, 1, 10, and 50 μM) were added to the basal compartment and accumulation of trospium in the apical compartment and intracellularly was measured after 60 min. (A) Transcellular transport of trospium in MDCK-OCT2, MDCK-MATE1, MDCK-OCT2-MATE1 and MDCK-Co cells after 60 min (* p < 0.05 vs MDCK-Co, *** p < 0.001 vs MDCK-Co, ### p < 0.001 vs MDCK-OCT2, §§§ p < 0.001 vs MDCK-MATE1). (B) Intracellular accumulation of trospium in MDCK-OCT2, MDCK-MATE1, MDCK-OCT2-MATE1 and MDCK-Co cells after 60 min (*** p < 0.001 vs MDCK-Co, ### p < 0.001 MDCK-OCT2 vs MDCK-OCT2-MATE1). (C) Concentration dependency of transcellular transport and intracellular accumulation in MDCK-OCT2, MDCK-MATE1, MDCK-OCT2-MATE1, and MDCK-Co cells after 60 min. Data are shown as mean ± SD (n = 6).

In the transcellular transport experiments with MDCKOCT1-MATE1 cells and the respective single- and vectortransfected cells, we detected that the intracellular accumulation of trospium was always highest in MDCK-OCT1 cells over the complete concentration range tested (Figure 1B). This accumulation pattern was expected, as MDCK-OCT1 cells only overexpress the basolateral uptake transporter OCT1 which is described to mediate the uptake of trospium into cells.18,28 Based on the data gained with the single-transfected cells, we determined a preliminary Km value of 22.0 μM for OCT1 which is comparable to previously published Km values of 17.028 or 15.1 μM,18 indicating that OCT1 is a transporter with a rather low affinity and high capacity for trospium. Considering the estimated peak concentrations of trospium of

export out of the cells, a mechanism that could be involved in trospium secretion into bile and urine, has not been investigated so far. As recently demonstrated by Gessner et al.,36 uptake experiments with HEK293 cells overexpressing MATE1 may result in different conclusions on the transport properties of a substance than transcellular transport experiments using MDCK-MATE1 cells. Different substrate affinities on the intracellular and extracellular moieties of the transport protein have been suggested as a possible underlying reason.36 Our experiments now show for the first time that MATE1 facilitates the efflux of trospium out of the cells, i.e. in the transport direction that is relevant for renal and hepatobiliary clearance mechanisms. E


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range of the previously published value of 0.6 μM.18 This indicates that OCT2 is a high affinity but low capacity transporter for trospium and implies saturation of OCT2mediated trospium transport at concentrations of 10 and 50 μM. As these concentrations relevantly exceed the low Km value, OCT2 possibly starts to mediate an efflux of trospium. The ability of OCT2 to act also as an efflux transporter has been demonstrated in several publications. Studies with different concentrations of the standard OCT2-substrate choline showed that OCT2 mediates an uptake at low concentrations but a bidirectional transport at higher concentrations.40 In line, time-dependent uptake studies with the cardiovascular risk factor TMAO showed an OCT2mediated uptake at an incubation time of 1 or 2 min but an OCT2-mediated efflux of TMAO at 5 and 10 min.36 Therefore, it was anticipated that the transport direction of OCT2 is reversible and dependent on the electrochemical gradient of its substrate,16,41−43 suggesting that OCT2mediated trospium uptake is particularly important at low substrate concentrations. Interestingly, the intracellular accumulation in double-transfected MDCK-OCT2-MATE1 cells increased continuously when higher trospium concentrations were used. The underlying mechanisms behind this observation remain unclear. Possibly, the additionally overexpressed MATE1, which is supposed to be a low affinity and high capacity transporter for trospium (Km = 15.4 μM)18, is able to provide an intracellular substrate flow sufficient to reduce the OCT2-mediated trospium efflux. This possibly results in significantly higher intracellular accumulation at high concentrations compared to the accumulation in MDCK-OCT2 cells. Considering the peak plasma concentrations of trospium reached after oral administration of 20 mg trospium, which is about 7.7 nM3, it is likely that this effect is negligible in vivo and that OCT2 and MATE1 contribute equally to the transport of trospium into urine. In comparison to MDCKOCT1-MATE1 cells, transcellular trospium transport mediated by MDCK-OCT2-MATE1 cells seems to be more efficient. This is in line with observed trospium tissue concentrations in mice, which are higher in kidneys than in liver,5 and with the fact that the physiological pH of the bile is higher than the pH of the primary urine. Both might reduce the transport mediated by MATE1 in the liver and might lead to a lower transport rate of OCT1-MATE1 in vivo. Furthermore, this could be one of the reasons why trospium is predominantly eliminated into urine.3,4 However, it has to be considered that the efflux transporters P-glycoprotein and MATE2-K, the second member of the SLC47 family, may also play a role in the pharmacokinetics of trospium in vivo due to their expression patterns19,26,44,45 and their transport characteristics for trospium.10,18 Our observation that OCT1- or OCT2-mediated uptake as well as MATE1-mediated efflux play a crucial role in the transcellular transport of trospium at low concentrations is in line with the results for the anticholinergic drug ipratropium investigated by Chen et al.18 This compound is also an organic cation and characterized as a substrate of OCTs and MATEs. Comparable to our experiments with low concentrations of trospium, transcellular transport experiments with ipratropium demonstrated a strongly increased intracellular accumulation in single-transfected cells overexpressing OCT1 or OCT2 compared to all other cell lines. The transcellular transport of ipratropium was strongly increased in the double-transfected cell lines overexpressing OCT1 or OCT2 and MATE1

Figure 5. Time-dependency of transcellular, basal-to-apical transport and intracellular accumulation of trospium studied in monolayers of MDCKII cells overexpressing OCT2 and/or MATE1. pH was adjusted to 7.3 in the basal compartment and to 6.5 in the apical compartment. Trospium (1 μM) was added to the basal compartment and incubation time was varied between 30 and 120 min (* p < 0.05 MDCK-OCT2 vs MDCK-Co, ** p < 0.01 MDCK-OCT2 vs MDCKCo, *** p < 0.001 MDCK-OCT2 vs MDCK-Co, ### p < 0.001 MDCK-MATE1 vs MDCK-Co, §§§ p < 0.001 MDCK-OCT1-MATE1 vs MDCK-Co). Data are shown as mean ± SD (n = 6).

about 300 nM reached in the portal vein after administration of a standard dose of 20 mg trospium orally,3,39 transport mediated by OCT1 seems not to be saturated in vivo. In line with these findings for the intracellular accumulation of trospium, the transcellular transport of trospium measured in the apical compartment was always significantly higher in double-transfected MDCK-OCT1-MATE1 cells overexpressing the uptake transporter OCT1 and the efflux transporter MATE1 simultaneously, compared to the single-transfected cells either overexpressing OCT1 or MATE1. Thus, we showed that both, OCT1 and MATE1 contribute considerably to the transcellular transport of trospium as already supposed in literature.18 The results with MDCK-OCT2-MATE1 cells, however, were more difficult to interpret. As expected, at low trospium concentrations of 0.1 or 1 μM, the intracellular accumulation was highest in the single-transfected cells only overexpressing the uptake transporter OCT2 and the transcellular transport was significantly highest in the double-transfected MDCKOCT2-MATE1 cells, additionally overexpressing the efflux transporter MATE1 (Figure 4A,B). When higher concentrations of trospium were used, the transcellular transport remained highest in MDCK-OCT2-MATE1 cells, but the accumulation ratio of trospium in MDCK-OCT2 compared to MDCK-Co cells decreased with higher substrate concentrations. At 50 μM substrate concentration, accumulation of trospium was not significantly different between MDCKOCT2 and MDCK-Co cells (Figure 4A,B). An explanation for this observation could be the low Km value of OCT2 determined for trospium. Based on our experiments, we calculated a preliminary Km value of 2.1 μM, which is in the F


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(2) Pak, R. W.; Petrou, S. P.; Staskin, D. R. Trospium chloride: a quaternary amine with unique pharmacologic properties. Curr. Urol. Rep. 2003, 4 (6), 436−40. (3) Doroshyenko, O.; Jetter, A.; Odenthal, K. P.; Fuhr, U. Clinical pharmacokinetics of trospium chloride. Clin. Pharmacokinet. 2005, 44 (7), 701−20. (4) Füsgen, I.; Hauri, D. Trospium chloride: an effective option for medical treatment of bladder overactivity. Int. J. Clin. Pharmacol. Ther. 2000, 38 (5), 223−34. (5) Geyer, J.; Gavrilova, O.; Petzinger, E. The role of P-glycoprotein in limiting brain penetration of the peripherally acting anticholinergic overactive bladder drug trospium chloride. Drug Metab. Dispos. 2009, 37 (7), 1371−4. (6) Todorova, A.; Vonderheid-Guth, B.; Dimpfel, W. Effects of tolterodine, trospium chloride, and oxybutynin on the central nervous system. J. Clin. Pharmacol. 2001, 41 (6), 636−44. (7) Staskin, D. R. Trospium chloride: Distinct among other anticholinergic agents available for the treatment of overactive bladder. Urol. Clin. N. Am. 2006, 33 (4), 465−73. (8) Staskin, D.; Kay, G.; Tannenbaum, C.; Goldman, H. B.; Bhashi, K.; Ling, J.; Oefelein, M. G. Trospium chloride is undetectable in the older human central nervous system. J. Am. Geriatr. Soc. 2010, 58 (8), 1618−9. (9) Staskin, D.; Kay, G.; Tannenbaum, C.; Goldman, H. B.; Bhashi, K.; Ling, J.; Oefelein, M. G. Trospium chloride has no effect on memory testing and is assay undetectable in the central nervous system of older patients with overactive bladder. Int. J. Clin. Pract. 2010, 64 (9), 1294−300. (10) Bexten, M.; Oswald, S.; Grube, M.; Jia, J.; Graf, T.; Zimmermann, U.; Rodewald, K.; Zolk, O.; Schwantes, U.; Siegmund, W.; Keiser, M. Expression of drug transporters and drug metabolizing enzymes in the bladder urothelium in man and affinity of the bladder spasmolytic trospium chloride to transporters likely involved in its pharmacokinetics. Mol. Pharm. 2015, 12 (1), 171−8. (11) Koepsell, H. The SLC22 family with transporters of organic cations, anions and zwitterions. Mol. Aspects Med. 2013, 34 (2−3), 413−35. (12) Motohashi, H.; Inui, K. Organic cation transporter OCTs (SLC22) and MATEs (SLC47) in the human kidney. AAPS J. 2013, 15 (2), 581−8. (13) Koepsell, H. Polyspecific organic cation transporters and their biomedical relevance in kidney. Curr. Opin. Nephrol. Hypertens. 2013, 22 (5), 533−8. (14) Nies, A. T.; Koepsell, H.; Damme, K.; Schwab, M. Organic cation transporters (OCTs, MATEs), in vitro and in vivo evidence for the importance in drug therapy. In Handbook of Experimental Pharmacology, Fromm, M. F., Kim, R. B., Eds.; Springer: Berlin, 2011; Vol. 201, pp 105−67. (15) Koepsell, H.; Lips, K.; Volk, C. Polyspecific organic cation transporters: structure, function, physiological roles, and biopharmaceutical implications. Pharm. Res. 2007, 24 (7), 1227−51. (16) Koepsell, H.; Endou, H. The SLC22 drug transporter family. Pflug. Arch. Eur. J. Phy. 2004, 447 (5), 666−76. (17) Koepsell, H.; Schmitt, B. M.; Gorboulev, V. Organic cation transporters. Rev. Physiol. Biochem. Pharmacol. 2003, 150, 36−90. (18) Chen, J.; Brockmöller, J.; Seitz, T.; König, J.; Chen, X.; Tzvetkov, M. V. Tropane alkaloids as substrates and inhibitors of human organic cation transporters of the SLC22 (OCT) and the SLC47 (MATE) families. Biol. Chem. 2017, 398 (2), 237−249. (19) Masuda, S.; Terada, T.; Yonezawa, A.; Tanihara, Y.; Kishimoto, K.; Katsura, T.; Ogawa, O.; Inui, K. Identification and functional characterization of a new human kidney-specific H+/organic cation antiporter, kidney-specific multidrug and toxin extrusion 2. J. Am. Soc. Nephrol. 2006, 17 (8), 2127−35. (20) Motohashi, H.; Inui, K. Multidrug and toxin extrusion family SLC47: physiological, pharmacokinetic and toxicokinetic importance of MATE1 and MATE2-K. Mol. Aspects Med. 2013, 34 (2−3), 661−8. (21) Tanihara, Y.; Masuda, S.; Sato, T.; Katsura, T.; Ogawa, O.; Inui, K. Substrate specificity of MATE1 and MATE2-K, human multidrug

simultaneously compared to the single-transfected cells. These findings indicate that both, uptake and efflux transporters are necessary for transcellular transport of this compound. In contrast, the rate-limiting step for the transcellular transport of lamivudine33 or chloroquine32 seems to be the transport across the apical membrane mediated by MATE1. In these studies, no significant difference between the transcellular transport mediated by MDCK-OCT2-MATE1 cells and the transcellular transport mediated by MDCK-MATE1 cells could be detected.32,33 In the case of cimetidine, another OCT- and MATE-substrate,21,46 OCT2 seems to be important for the uptake into the cells, but transcellular transport seems to be mainly facilitated by MATE1, as a considerably higher transcellular transport could only be detected for MDCKMATE1 and MDCK-OCT2-MATE1 cells.47 However, the amount of intracellular cimetidine appears to be important for drug−drug interactions at the intracellular binding site of the apically localized efflux transporter MATE1, so that the role of OCT2 for this compound should not be neglected.47 This observation shows the importance of a precise understanding of the molecular transport mechanisms behind the cellular uptake and transcellular transport of drugs for evaluation of drug−drug interactions. So far, data investigating drug interactions with trospium are rare. In one study, the OCTand MATE-substrate metformin (500 mg, twice daily) was given together with trospium (60 mg, once daily). Whereas no significant change in pharmacokinetics could be detected for metformin, Cmax and AUC24 of trospium were reduced by 34 and 29%, respectively.48 Considering the results of our present work, further in vitro and in vivo studies concerning this topic might be reasonable using additional OCT/MATE-inhibitors. Taken together, our work demonstrates the important role of OCT1 and OCT2 as well as of MATE1 in the transcellular transport of trospium. Thus, we could contribute to a better understanding of the pharmacokinetics of trospium during hepatobiliary and renal elimination.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +49 9131 85 22077; Fax: +49 9131 85 22773; E-mail: [email protected]. ORCID

Birgit Deutsch: 0000-0002-1647-6762 Martin F. Fromm: 0000-0002-0334-7478 Jörg König: 0000-0001-7016-5482 Notes

The authors declare the following competing financial interest(s): The authors affirm to have no conflict of interest concerning this project apart from the mentioned support by Dr. R. Pfleger GmbH, Bamberg, Germany.

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ACKNOWLEDGMENTS This work was supported by a research grant of Dr. R. Pfleger GmbH, Bamberg, Germany. We thank Eva Hoier and Katrin Singer for excellent technical assistance and Fabian Müller for carefully reading the manuscript.

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REFERENCES

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Interplay of the Organic Cation Transporters OCT1 and OCT2 with the

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