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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 Maria Bexten, Stefan Oswald, Markus Grube, Jia Jia, Tanja Graf, Uwe Zimmermann, Kathrin Rodewald, Oliver Zolk, Ulrich Schwantes, Werner Siegmund, and Markus Keiser Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/mp500532x • Publication Date (Web): 03 Dec 2014 Downloaded from http://pubs.acs.org on December 7, 2014

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Molecular Pharmaceutics

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

Maria Bexten1, Stefan Oswald1, Markus Grube2, Jia Jia1, Tanja Graf1, Uwe Zimmermann3, Kathrin Rodewald1, Oliver Zolk4°, Ulrich Schwantes5, Werner Siegmund1 and Markus Keiser1*

1

Department of Clinical Pharmacology and 2Department of Pharmacology of the Center of

Drug Absorption and Transport (C_DAT), University Medicine, Greifswald, Germany; 3

Department of Urology University Medicine, Greifswald, Germany;

4

Institute of

Experimental and Clinical Pharmacology and Toxicology, University of ErlangenNuremberg, Erlangen, Germany and 5Dr. R. Pfleger GmbH, Bamberg, Germany °Present address: Institute of Pharmacology of Natural Products and Clinical Pharmacology, University of Ulm, Ulm, Germany

Corresponding Author *Department of Clinical Pharmacology, University Medicine Greifswald, Center of Drug Absorption and Transport (C_DAT), Felix-Hausdorff-Str. 3, D-17487 Greifswald, Germany. Phone:

+49 (0)3834 86-5640.

Fax: +49 (0)3834 86-5631.

E-mail:

greifswald.de

1 ACS Paragon Plus Environment

markus.keiser@uni-


Table of Contents/Abstract Graphic

100 TC uptake in HBEP (pmol/mg × min)

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75 50 25

Km = 18.5 ± 4.8 µmol/l Vmax = 106 ± 11.3 pmol/mg × min

0 0

20 40 TC (µmol/l)

60


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Abstract Background: The cationic, water-soluble quaternary trospium chloride (TC) is incompletely absorbed from the gut, undergoes wide distribution but does not pass the blood-brain barrier. It is secreted by the kidneys, liver and intestine. To evaluate potential transport mechanisms for TC, we measured affinity of the drug to the human uptake and efflux transporters known to be of pharmacokinetic relevance. Methods: Affinity of TC to the uptake transporters OATP1A2, -1B1, -1B3, -2B1, OCT1, -2, 3, OCTN2, NTCP, and ASBT and the efflux carriers P-gp, MRP2 and MRP3 transfected in HEK293 and MDCK2 cells. To identify relevant pharmacokinetic mechanisms in the bladder urothelium, mRNA expression of multidrug transporters, drug metabolizing enzymes and nuclear receptors, and the uptake of TC into primary human bladder urothelium (HBU) cells were measured. Results: TC was shown to be a substrate of OATP1A2 (Km = 6.9±1.3 µmol/l, Vmax = 41.6±1.8 pmol/mg×min), OCT1 (Km = 106±16 µmol/l and Vmax = 269±18 pmol/mg×min) and P-gp (Km = 34.9±7.5 µmol/l, Vmax = 105±9.1 pmol/mg×min, lipovesicle assay). The genetic OATP1A2 variants *2 and *3 were loss-of-function transporters for TC. The mRNA expression analysis identified the following transporter proteins in the human urothelium: ABCB1 (P-gp), ABCC1-5 (MRP1-5), ABCG2 (BCRP), SLCO2B1 (OATP2B1), SLCO4A1 (OATP4A1), SLC22A1 (OCT1), SLC22A3 (OCT3), SLC22A4 (OCTN1), SLC22A5 (OCTN2) and SLC47A1 (MATE1). Immuno-reactive P-gp and OATP1A2 were localized to the apical cell layers. Drug metabolizing enzymes CYP3A5, -2B6, -2B7 -2E1, SULT1A1-4, UGT1A1-10 and UGT2B15, and nuclear receptors NR1H3, NR1H4 were also expressed on mRNA level. TC was taken up into HBU cells (Km = 18.5±4.8 µmol/l, Vmax = 106±11.3 pmol/mg×min) by mechanisms that could be synergistically inhibited by naringin (IC50 = 10.8 (8.4; 13.8)



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µmol/l) and verapamil (IC50 = 4.6 (2.8; 7.5) µmol/l), inhibitors of OATP1A2 and OCT1, respectively. Conclusion: Affinity of TC to OCT1 and P-glycoprotein may be the reasons for incomplete oral absorption, wide distribution into liver and kidneys, as well as for substantial intestinal and renal secretions. Absence of brain distribution may result from affinity to P-gp and a low affinity to OATP1A2. The human urothelium expresses many drug transporters and drug metabolizing enzymes that may interact with TC and other drugs eliminated into the urine.


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Keywords Trospium chloride, transporter proteins, metabolizing enzymes, bladder urothelium, pharmacokinetics Abbreviations BSP, bromosulfophthalein; E1S, estrone-3-sulphate; HEK, Human embryonic kidney; HBU, primary human bladder urothelial cells; MIE, maximal inhibitory effect; MDCK, MadinDarby canine kidney; MPP, N-methyl-4-phenylpyridinium; MRP, human multidrug resistance-associated protein; NTCP, Na+/taurocholate cotransporting polypeptide; OATP, Organic anion transporting polypeptide; OCT, Organic cation transporter; P-gp, Pglycoprotein; Rh123, rhodamine-123; TA, taurocholic acid; TC, trospium chloride; TLDA, TaqMan® low density array; VC, vector control



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Introduction Efficient treatment of patients with overactive bladder syndrome (OAB) caused by idiopathic and neurogenic detrusor hyperactivity is of growing challenge for health care systems in the ageing populations of highly developed countries. Prescribed drugs with level Ia evidence from controlled clinical studies are muscarinic receptor blocking agents.1,2 Their clinical use is limited by peripheral anticholinergic side effects at therapeutic doses including dry mouth and constipation, or by cognitive disturbances, headache and blurred vision because muscarinic receptor subtypes are not organ-selectively expressed, and the anticholinergic drugs do not have selective affinity to M1-M5 receptors, with the exception of the so called M2-selective antagonists (e.g. darifenacin).3-7 A dissociation between the desired detrusor relaxation and undesired effects in other organs can be achieved, firstly, by drugs with additional pharmacodynamic action mechanisms such as phosphodiesterase inhibition (e.g. oxybutynin), or calcium and calmodulin antagonism (e.g. propiverine), and secondly, by prescription of the drugs in extended-release or transdermal dosage forms, by which a lower portion of metabolites with high anticholinergic efficacy are generated (e.g. oxybutynin) or, thirdly, by administration of drugs with a preferential distribution pattern e.g. trospium chloride (TC).2,8 TC is a cationic, highly polar quaternary compound with low oral bioavailability (∼ 10 %) which does not pass the blood-brain barrier (BBB) and, therefore, does not cause cognitive disturbances.9,10 This is a major advantage compared to the tertiary drugs oxybutynin, tolterodine or propiverine, particularly in elderly patients at risk of Alzheimer´s disease or vascular dementia.11 After intravenous administration (2 mg, 7 healthy subjects), the watersoluble TC has a high distribution volume (7.12 ± 1.94 L/kg), despite restricted permeation into the brain, and it has high renal clearance (543 ± 104 ml/min), and substantial intestinal clearance (99.7 ± 39.0 ml/min) (own published data). It is as efficient in OAB treatment as the other anticholinergic drugs with level Ia evidence, which are lipid soluble tertiary amines.3,6,7 6 ACS Paragon Plus Environment

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To understand the invasion route of orally administered TC from gut lumen towards to the target site(s) in the urinary bladder, and to the places of undesired action (e.g. salivary glands), affinity of TC to multidrug transporter proteins known to be involved in absorption and distribution of drugs must be evaluated. Candidate transporters in the small intestine, BBB and urinary bladder are in particular members of the solute carrier (SLC) superfamily (e.g. OATPs, OATs, OCTs) and members of the ATP-binding cassette (ABC) superfamily e.g. P-glycoprotein (P-gp), BCRP, and MRPs. It has been already shown that TC is a substrate of OCT1 and OCT2, and an inhibitor of OCT3, which are expressed in several organs including the apical membrane of enterocytes.12-15 However, there are nearly no data available on expression, localization and function of multidrug transporters (and drug metabolizing enzymes) in the human bladder. One exception is OCT1, an uptake carrier for TC, which was detected in the human urothelium with a slight predominance in the intermediate cell layers.14 In our paper, data are provided on affinity of TC to transporters expressed in the small intestine, BBB and the bladder urothelium which might be of functional relevance for absorption and organ distribution of the drug. To identify potential distribution mechanisms for TC into the bladder urothelium after systemic (oral) and local (intravesical) administration, we measured expression of multidrug transporters, drug metabolizing enzymes and nuclear receptors in the human bladder urothelium and characterized the uptake of TC into primary human bladder urothelial (HUB) cells.



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Material and Methods Chemicals: Taurocholate (TA), estrone-3-sulphate (E1S), N-methyl-4-phenylpyridinium (MPP+), bromosulfophthalein (BSP), rhodamine-123 (Rh123), chenodeoxycholate acid, naringin and verapamil were obtained from Sigma-Aldrich (Taufkirchen, Germany). Trospium chloride was kindly provided by Dr. R. Pfleger (Bamberg, Germany). [3H]-BSP (14 Ci/mmol; 1 µCi/µl) was purchased from Hartmann Analytic (Braunschweig, Germany), [3H]-TA (4.6 Ci/mmol; 1 µCi/µl) from PerkinElmer Life and Analytical Sciences (Waltham, USA), [3H]-MPP (85 Ci/mmol; 1 µCi/µl) and [3H]-E1S (50 Ci/mmol; 1 µCi/µl) from Biotrend (Cologne, Germany) and [3H]-TC (37 Ci/mmol; 0.1 µCi/µl) from RC Tritec (Teufen, Switzerland). PSC-833 was kindly provided by Novartis (Basel, Switzerland). Cell culture: HBU cells were purchased from CELLnTEC (Bern, Switzerland) and grown in CnT-58 medium at 37 °C, 95 % humidity, and 5 % CO2 according to the manufacturer’s instructions. Cells were taken from an adjacent, unaffected bladder region during a surgical intervention from one patient with benign prostatic hyperplasia and isolated and characterized by the company as described on their homepage (www.cellntec.com). When growing in CnTPR-D medium, cells are positive for uroplakin IIIa, an urothelial-specific integral membrane protein that is synthesized in terminally differentiated superficial urothelial cells (umbrella cells).

16,17

The cells used in this study were described as “Human Bladder Epithelial

Progenitor (HBEP) cells” by the manufacturer but are referred to here as HBU cells, as it was felt this more accurately described the nature of the cultures. For all experiments, HBU cells reaching confluence were used. Human embryonic kidney 293 (HEK293) and Madin-Darby canine kidney (MDCK2) cells were purchased from the European Collection of Cell Cultures (Salisbury, United Kingdom). HEK293 cells were grown in minimal essential medium supplemented with 10 % fetal bovine serum, 2 mM L-glutamine, 2 mM nonessential amino acids, 100 units/ml penicillin, and 100 µg/ml streptomycin. MDCK2 cells were grown in Dulbecco's modified Eagle's medium 8 ACS Paragon Plus Environment

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supplemented with 10 % fetal bovine serum, 4 mM L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin (PAA, Coelbe, Germany) at 37 °C, 95 % humidity, and 5 % CO2. HEK293 cells stably transfected with human OATP1A2, OATP1B1, OATP1B3, OATP2B1, NTCP, OCT2 or OCT3, and MDCK2 cells stably transfected with OCTN2 or MRP2, and the respective vector-transfected control cells were established as previously described.18-22 Affinity of TC to efflux transporters: Membrane transport of TC mediated by MRP2, MRP3 and P-gp, respectively, was investigated using inside-out vesicles. For all experiments, 30 µg of total vesicle protein were used. The preparation of inside-out vesicles from stably transfected MDCK2-MRP2 and HEK293-P-gp was performed as described previously.23,24 MRP3 vesicles were purchased from Life Technologies (Darmstadt, Germany). Functionality of P-gp lipovesicles was ensured by an Rh123 accumulation assay in the presence or absence of ATP. Fluorescence of Rh123 was measured using a microplate reader (Synergy HT, Biotek, Bad Friedrichshall, Germany). Functionality of MRP2 and MRP3 were ensured as described previously.22,24 ATP-dependent transport of [3H]-TC and unlabeled TC (0-100 µmol/l) into the vesicles was measured by rapid filtration using nitrocellulose filters (0.22 µM pore size; Millipore, Billerica, USA). In control experiments, ATP was replaced by AMP. In a competition assay, P-gp vesicles were incubated with radio-labeled TC and 10 µmol/l of the known P-gp inhibitor PSC-833 25. Affinity to uptake transporters: For uptake and competition assays, cells were seeded in 24well plates and incubated in full growth medium at an initial density of 200,000 cells/well for 3 days. Before each experiment, cells were washed once with 37 °C incubation buffer (142 mmol/l NaCl, 5 mmol/l KCl, 1 mmol/l K2HPO4, 1.2 mmol/l MgSO4, 1. mmol/l CaCl2, 5 mmol/l glucose, 12.5 mmol/l HEPES; pH 7.3). After the respective uptake or competition experiment, cells were washed three times with ice-cold PBS and lysed with 800 µl roomtemperature 0.5 % Triton X-100 (Merck, Darmstadt, Germany) and 0.5 % sodium 9 ACS Paragon Plus Environment


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deoxycholate (Sigma-Aldrich, Steinheim, Germany). 200 µl cell suspension were mixed with 2 ml of scintillation cocktail (Rotiszint ecoplus; Roth, Karlsruhe, Germany) and measured using a scintillation beta counter (type 1409, LKB-Wallac, Turku, Finland). Protein concentration was determined to quantify cell density after the experiments using the BCA assay according to the manufacturer’s instructions (Pierce, Rockford, USA). In competition assays, the reference substrates [3H]-BSP, [3H]-E1S, [3H]-TA, [3H]-MPP+ and [3H]-carnitine were dissolved in incubation buffer and unlabeled BSP, E1S, TA, MPP+ and carnitine were added to reach the respective final concentration of 0.05 µmol/l in OATP1B1, 1 µmol/l in OATP1A2, OATP1B3 and OATP2B1 and 10 µmol/l in OCT1, 2, 3, NTCP and ASBT transfected cells. The competition assays of 10 µM MPP+ with verapamil (0-316 µmol/l) as an inhibitor of OCT1 26, and 1 µM E1S with naringin (0-500 µmol/l) as an inhibitor of OATP1A2

27

, were performed using the respective OCT1- and OATP1A2*1, *2 *3-

transfected cells. Inhibitory effects of TC were investigated in all cell lines using the above mentioned reference substrates and TC at concentrations up to 1000 µmol/l. Uptake activities of OCT1, ASBT, OATP1A2*1, *2 and *3 cells were measured using [3H]MPP+, [3H]-TA and [3H]-E1S dissolved with unlabeled MPP+, TA and E1S to reach final concentrations of 0-100 µmol/l for MPP+ and TA and 0-400 µmol/l for E1S. The cellular uptake of all reference substrates was in the linear range during the incubation time of 10 seconds by OCT1 and 5 minutes by OATP, ASBT, NTCP and OCTN2 transfected cells. Uptake of TC by OATP, NTCP, ASBT, OCT and OCTN2 was first measured in time dependent uptake studies for 15 minutes with [3H]-TC dissolved with unlabeled TC to reach a final concentration of 10 µmol/l. The Michaelis-Menten constant (Km) and the maximal uptake rate (Vmax) values for OCT1, OATP1A2*1, *2 and *3 were calculated using 0-250 µmol TC and an incubation time of 5 minutes. Differences in E1S and TC uptake in OATP1A2*1, *2 and *3 transfected cells was corrected to the percentage of transporter protein in whole cell lysate. For the pH-dependent uptake of TC in OATP1A2- and OCT110 ACS Paragon Plus Environment

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transfected cells, the pH of the incubation buffer was changed to 3.0 and 5.5, respectively. Changes in cell viability as caused by pH changing, and the lack of cytotoxicity by TC, were confirmed using the Presto Blue™ cell viability reagent (Life Technologies, Darmstadt, Germany). Competition assays were performed using OCT1 and OATP1A2-transfected cells with [3H]-TC and unlabeled TC (10 µmol/l) as a substrate, and 0-100 µmol/l of the OCT1 inhibitor verapamil and 0-1,000 µmol/l of the specific OATP1A2 inhibitor naringin

27

,

respectively. Expression of transporters and drug metabolizing enzymes in the human bladder: mRNA was ®

isolated using the NucleoSpin

RNA II kit (Macherey-Nagel, Düren, Germany) from the

human urothelium of six donors. The samples were taken from unaffected regions of urinary bladders that had to be dissected because of urothelial cell carcinoma or voiding dysfunction. The urothelium was carefully scraped off the bladder wall and immediately frozen using liquid nitrogen. The study was approved by the local ethics committee (III UV 35/04) and each patient gave written informed consent. The cDNA was synthesized using the High Capacity RNA-to-cDNA kit (Life Technologies, Darmstadt, Germany) according to the manufacturer’s instructions. High-throughput qPCR (TaqMan® low density array, TLDA) was performed using the 7900HT system to measure gene expression of 40 drug metabolizing enzymes and transporters in human urothelium (Table S1). The TLDA card was configured into the respective gene set (in duplicates). The TLDA array card also contained six endogenous control genes. 500 ng cDNA were mixed with distilled water and 50 μL of 2 x TaqMan® Gene Expression master mix (Life Technologies, Darmstadt, Germany) to a total volume of 100 µl. Each sample was loaded into a port of the TLDA-card, centrifuged twice for 1 minute using a swing rotor, and evaluated using an ABI 7900HT system (Life Technologies, Darmstadt, Germany) at 50 °C for 2 min, at 94.5 °C for 10 min, followed by 40 cycles at 97 °C for 30 seconds and at 59.7 °C for 1 minute. Data analysis was performed using 11 ACS Paragon Plus Environment


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the SDS RQ Manager 1.2 (Life Technologies, Darmstadt, Germany). The expression level of each transporter mRNA was normalized to the mean value of all measured house-keeping genes. To exclude a stromal contamination of the urothelium the mRNA expression level of the stromal marker desmin (DES) was investigated by real-time PCR (Assay ID Hs00157258_m1, Life Technologies, Darmstadt, Germany) and compared to the mRNA expression of desmin in the stroma. Immunoblot analysis and immunofluorescence microscopy in the human urothelium: Localization of P-gp and OATP1A2 in the human urothelium was characterized by immunofluorescence staining using paraffin embedded tissue slides. After deparaffinization and rehydration, the sections were boiled in target retrieval solution using citrate puffer pH 6.0 for P-gp staining and the DAKO target retrieval buffer pH 9.0 (both Dako, Hamburg, Germany) for OATP1A2 staining. After blocking using 5% FCS in PBS, tissue slides were incubated overnight with the respective antibody solutions at 4 °C; C219 for P-gp (Enzo Life Sciences, Lörrach, Germany), 1/20 dilution; SLCO1A2 antibody for OATP1A2 (Abcam, Cambridge, United Kingdom), 1/50 dilution. After additional washing steps using PBS, the slides were incubated with Alexa Fluor 488 labeled secondary antibodies (Life Technologies, Darmstadt, Germany) at room temperature for 1 h. After removal of unbound secondary antibodies by further washing steps (PBS, 3x), slides were embedded with DAPI dye containing fluorescence mounting medium (Life Technologies, Darmstadt, Germany). Immunofluorescence microscopy analysis was performed using the laser scanning confocal microscopy system LSM780 (Carl Zeiss MicroImaging, Jena, Germany). Uptake transport in HBU cells: Time dependent uptake of TC in HBU cells was measured using [3H]-TC and 10 µmol/l unlabeled TC with incubation times of 5 seconds to 5 minutes. Km and Vmax-values of the TC uptake in HBU cells were calculated by using [3H]-TC and TC (0-60 µmol), and an incubation time of 30 seconds. Competition assays were performed using


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[3H]-TC and 10 µmol/l TC as substrates, and verapamil and naringin, respectively, as inhibitors (both 0-500 µmol/l). Pharmacokinetic and statistical evaluation: The transporter-mediated net uptake in the cell experiments was obtained by subtracting the uptake into vector-transfected cells from that in the transporter-transfected cells. Km and Vmax were assessed using Prism 5.01 (GraphPad Software, San Diego, USA). The differences in TC uptake in OATP1A2*1, *2 and *3 uptake were corrected to the transporter protein levels in the whole cell lysate. All experiments were performed at least three times in triplicates. Km and Vmax are presented as arithmetic means ± standard deviation (M±SD). The intrinsic clearance (Clint) was calculated by Vmax/Km. IC50 values are shown as the geometric mean and geometric standard deviation.



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Results Characterization of cells transfected with P-gp, OCT1, ASBT, OATP1A2*2 and *3 HEK-P-gp transfected cells showed the expected band of 170 kDa and immunofluorescence staining which localized the protein in the cell membrane, whereas no band and no fluorescent signal were detectable in vector transfected cells. The Rh123 assay showed an ATP dependent accumulation of Rh123 in P-gp expressing lipovesicles which could be modulated by PSC833, an inhibitor of P-gp (Figure S1).28 The selected cell clones of the HEK293-OCT1 were characterized by a significantly higher expression of SLC22A1 (OCT1) using real-time-PCR and mass spectrometry-based targeted proteomics. The cells transported the standard substrate of MPP+ comparably to previously published data in stably transfected MDCK2 cells.29 The MPP+-uptake was within the linear range within the incubation time of 10 seconds and could be efficiently inhibited by verapamil (Figure S2). HEK293-ASBT cells were characterized by a significantly higher expression of ASBT compared to the vector control cells. TA showed an ASBT mediated transport in stably transfected HEK293-ASBT cells which was comparable to previously published data in transiently transfected HEK293 cells.30 The TA uptake was within the linear range within the incubation time of 5 minutes and could be efficiently inhibited by the standard inhibitor chenodeoxycholate acid (Figure S3). HEK293-OATP1A2*1, *2 and *3 cells showed in Western-blot analysis the expected bands of 60 and 80 kDa of which the lower band represents a deglycosylated form of OATP1A2 (data not shown).

31

Interestingly, protein

expression of OATP1A2*1, *2 and *3 were significantly different in transfected cell lines as shown by protein quantification in the whole cell lysate by LC-MS/MS (389 % for OATP1A2*2 and 248 % for OATP1A2*3, while non-variant protein OATP1A2*1 was set to 100 %). By immunostaining, OATP1A2*1, *2 and *3 were localized to the plasma membrane and to a less extent in the cytosol, whereas staining was not visible in vector transfected 14 ACS Paragon Plus Environment

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control cells. The functional characterization within the incubation time of 2 minutes showed a higher E1S uptake in HEK293-OATP1A2*1 than in *2 and *3 transfected cells with significant differences in Km and Vmax-values. Uptake of E1S was efficiently inhibited by naringin in all three cell lines (Figure S4, Table S3). Transport of TC TC showed no cytotoxic effects on our transfected cell lines or on HBU cells at the concentrations tested (data not shown). The drug was a strong inhibitor of OATP1A2*1, *2 and *3, OATP1B1, OATP1B3, OATP2B1, OCT1 and OCT2. In contrast, OCT3, NTCP and OCTN2 were less influenced at concentrations up to 1,000 µmol/l (Figure S5, Table S4). In our transport studies with HEK293 cells transfected with OATP1A2, OATP1B1, OATP1B3, OATP2B1, NTCP, ASBT, OCT1, OCT2 and OCTN2, it could be shown that TC was only taken up by OATP1A2 and OCT1 in a time-dependent manner within the incubation time up to 15 minutes (Figure S6). The uptake by OATP1A2 and OCT1 was saturable within the incubation time of 5 minutes and could be inhibited by naringin and verapamil, respectively (Figure 1). The Km and Vmax values for the uptake by OATP1A2 and OCT1 were significantly different. Moreover, the genetic variants OATP1A2*2 and *3 showed significant differences in TC uptake compared to the wild type OATP1A2 (Table 1). Changes in pH values of the incubation medium were without effect on cell viability. At pH 5.5, we found an increased uptake of TC by OATP1A2, but decreased transport by OCT1 with a significant influence on Km and Vmax values. TC uptake was not detectable at pH 3.0 (Table S4). In inside-out lipovesicle assays, TC was a substrate of human P-gp, but not for MRP2 and MRP3. The P-gp-mediated uptake of TC in lipovesicles could be inhibited by PSC-833 (Figure 2, Table 1). Gene expression of transporters and drug metabolizing enzymes On mRNA-level, the following efflux carriers of the ABC-family were found to be expressed in the human bladder urothelium with cycle threshold-values of maximum 35: 15 ACS Paragon Plus Environment


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ABCC1 (MRP1) > ABCC4 (MRP4) > ABCC3 (MRP3) > ABCB1 (P-gp) > ABCC5 (MRP5) > ABCG2 (BCRP) > ABCC2 (MRP2) > ABCB11 (BSEP). The following members of the solute carrier family were detected: SLCO2B1 (OATP2B1) > SLC22A5 (OCTN2) > SLCO4A (OATP4A1)

> SLC22A1

(OCT1) = SLC22A3 (OCT3) >

SLCO1B3

(OATP1B3)

>

SLCO1A2 (OATP1A2) > SLCO1B1 (OATP1B1) > SLC22A2 (OCT2). The mRNA expression of the stromal marker desmin was 1.2 % in the urothel compared to the expression level in the stroma, indicating that the mRNA was of urothelial origin. P-gp and OATP1A2 were localized to the apical cells layers of the urothelium, however, OATP1A2 was also expressed at a comparable level in the underlying connective tissue (Figure 3). Apart from multidrug transporters, we found that several drug metabolizing enzymes of the cytochrome P450 (CYP), UDP-glucuronosyltransferase (UGT) and sulfotransferase (SULT) superfamilies were also expressed in the human bladder urothelium.TC was taken up in HBU cells in a time and concentration dependent manner. The uptake could efficiently be inhibited by verapamil and naringin which are inhibitors of OCT1 and OATP1A2, respectively. The inhibitors exerted an additive effect (Figure 4).


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OATP1A2

OCT1

TC (pmol/mg × min)

50

*1 *2

40

250 200

*3 30

150 Km = 6.9 ± 1.3 µmol/l

20

100

Vmax = 41.6 ± 1.8 pmol/mg × min

10

50

0

0

Km = 106 ± 16 µmol/l Vmax = 269 ± 18 pmol/mg × min

0

TC uptake (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


50

100 150 200 250 TC (µmol/l)

0

125

125

100

100

75

75

50

50

25

25 IC50 = 7.6 (2.5; 22.8) µmol/l

0 0

0.1

100 150 200 250 TC (µmol/l)

IC50 = 0.9 (0.7; 1.1) µmol/l

0

1 10 100 1000 naringin (µmol/l)

50

0

0.1

1 10 100 verapamil (µmol/l)

Figure 1. Uptake (M±SD) of trospiumchloride (TC) into stably transfected OATP1A2*1, *2, *3 and OCT1 HEK293-cells and competition of TC with naringin and verapamil, respectively. Km and Vmax-values are only given for OATP1A2*1. All experiments were performed three times in triplicates.



TC (pmol/mg × min)

250

100

AMP ATP

200 150 100

125

50 0 0

20

80

40 60 TC (µmol/l)

80

100

100 TC uptake (%)

TC (pmol/mg × min)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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60 40 Km = 34.9 ± 7.5 µmol/l

20

75 IC50 = 4.8 (3.2; 7.3) µmol/l

50 25

Vmax = 105 ± 9.1 pmol/mg × min

0

0 0

25

50 75 TC (µmol/l)

100

0

0.1

1 10 100 PSC833 (µmol/l)

Figure 2. Transport (M±SD) of trospiumchloride (TC) in P-gp expressing lipovesicles and competition of TC-uptake using PSC-833. The small figure shows the P-gp mediated transport of TC by using AMP or ATP. All experiments were performed three times in triplicates.


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Figure 3. Immunofluorescence staining of P-gp and OATP1A2 in human bladder. P-gp and OATP1A2 are stained in green; nuclei staining was performed using DAPI (blue); scale bar = 20 µm



125 100

75

TC uptake (%)

TC (pmol/mg × min)

100

50 25

Km = 18.5 ± 4.8 µmol/l

75 50 25

Vmax = 106 ± 11.3 pmol/mg × min

IC50 = 10.8 (8.4; 13.8) µmol/l

0

0 0

20 40 TC (µmol/l)

0

60

125

125

100

100 TC uptake (%)

TC uptake (%)

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75 50 25

0.1

1 10 100 1000 naringin (µmol/l)

75 50 25

IC50 = 4.5 (3.0; 6.9) µmol/l

0 0

IC50 = 4.6 (2.8; 7.5) µmol/l

0

0.1 1 10 100 1000 naringin, verapamil (µmol/l)

0

0.1

1 10 100 1000 verapamil (µmol/l)

Figure 4. Transport (M±SD) of trospiumchloride (TC) in HBU cells and competition of TCuptake with naringin, verapamil and both inhibitors. All experiments were performed three times in triplicates.


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Table 1. TC uptake in P-gp expressing inside-out lipovesicle and in stable transfected HEK293 cells expressing OATP1A2*1, *2, *3 and OCT1

Cell line / Transporter

Km (µmol/l)a

Vmax (pmol/mg × min)a

Clint (µl/mg × min)a

HEK-P-gp lipovesicles 34.9 ± 7.5* 105 ± 9.1* 8.9 ± 7.8 HEK293-OATP1A2*1 6.9 ± 1.3 41.6 ± 1.8 4.2 ± 1.1 *2 2.5 ± 0.5 5.0 ± 0.2* 7.9 ± 1.8* *3 63.9 ± 12.8* 11.9 ± 0.9* 0.4 ± 0.3* 269 ± 18* 2.9 ± 0.7* HEK293-OCT1 106 ± 16* a Data represent the means ± SD of Km, Vmax and Clint from three experiments performed in triplicates. *p80 % of the renal blood flow.11 TC is not significantly distributed in the brain to exert clinically relevant anticholinergic (adverse) drug reactions.46-49 One rationale behind that advantage of TC compared to tertiary anticholinergics (e.g. oxybutynin, tolterodine, propiverine) is the function of the efflux carrier P-gp, an integrated part of the blood-brain barrier.34 Absence of P-gp facilitates brain distribution of TC in rats.45 Another explanation could be low affinity of TC to OATP1A2, a major uptake transporter for drugs in the brain.34 However, as the Km-value of TC for HEK293-OATP1A2 was 6.9 ± 1.3 µmol/l and maximum plasma concentrations during chronic treatment range ∼4-8 nmol/l of which ∼50 % is bound to plasma proteins, OATP1A2 cannot be reasonably considered as the transporter which carries TC into human brain. 11 As a



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second consequence, the genetic loss-of-function polymorphisms of OATP1A2, as described in our in-vitro study with transfected HEK293 cells, are most likely not of clinical relevance. From a clinical point of view, distribution of TC to the muscarinic receptor compartment(s) in the urinary bladder is of major interest. Contraction of the normal and hyperactive detrusor is mediated by M3 and M2 receptors localized to the smooth muscle membrane

50

which can

easily be antagonized by TC in the extracellular space, after being filtered from the blood via the fenestrated capillary endothelium. Efficacy of TC absorbed via the blood vessels is undisputed, because, firstly, the plasma levels after chronic treatment with TC are in the range of ∼4-8 nmol/l, secondly, the plasma-protein binding accounts for 40 % to 60 % over the therapeutic concentration range11 whereas, thirdly, the binding affinity of TC to M2 and M3 receptors (pKi) was found to be ∼0.5-0.7 nmol/l.51 An alternative concept to control detrusor hyperactivity is blocking of the afferent sensory nerves which were localized next to the basal urothelial cell layers.52 Experimental evidence exists, that the afferent receptors can be antagonized by TC, and other anticholinergics after intravesical administration, or after being eliminated into the urine after oral dosing.53-55 We have shown in our in-vitro experiments that TC is taken up by HBU cells via a mechanism which is susceptible to additive inhibition by verapamil and naringin, specific inhibitors of OCT1 and OATP1A2, respectively.26,27 Because OCT1 and, to a lower extent OATP1A2, are expressed and located in the human urothelium (our data), and the fact that TC has in-vitro affinity to HEK293-OCT1 and HEK293-OATP1A2, both are putative uptake carriers for TC from the luminal (urinary) site of the bladder.14 However, it cannot be excluded that other transporter proteins expressed in the urothelium are involved in the uptake of TC. Moreover, this concept, is contradicted by the following findings: firstly, the Km-values of TC for OCT1 (106 ± 16 µmol/L) and OATP1A2 (6.9 ± 1.3 µmol/l) are higher than the maximal concentrations of TC in the urine after oral administration of a single therapeutic dose of 30 mg IR in fasting healthy subjects (own unpublished data). Secondly, the efflux carrier P-gp is 24 ACS Paragon Plus Environment

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predominantly expressed in the apical cell layers of the human urothelium to which TC has comparably higher affinity (34.9 ± 7.5 µmol/l) than to OCT1, but lower affinity than to OATP1A2. Finally, the surface of the urothelium is covered by a hydrophobic waxy glycocalyx which serves as a tight barrier to avoid reabsorption of compounds which are excreted by the kidneys.56,57 In addition to the primary scope of our in-vitro studies, it must be considered that the human urothelium keeps a wide spectrum of multidrug transporters and drug metabolizing enzymes which significance for the disposition of drugs and xenobiotic compounds needs to be evaluated in future investigations. Conclusion TC is a substrate of the uptake transporters OATP1A2 and OCT1, as well as of the efflux carrier P-gp. Affinity to these multidrug transporters could be the reason for incomplete absorption, intensive distribution into the liver and kidneys, as well as for substantial intestinal and renal tubular secretion. TC exerts no central anticholinergic effects because of its affinity to P-gp. Uptake into urothelial receptor compartments from the bladder lumen might be possible after a high intraluminal dosage of TC but is not reasonably explained after oral administration of a standard dose of 30 mg IR by OATP1A2 and/or OCT1 affinity.



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Supporting information Characterization of stably transfected cells expressing P-gp, OCT1, ASBT, OATP1A2*1, *2 and *3; determination of expression rates using mass spectrometry-based targeted proteomics; competition of TC in transfected cell lines; time dependent uptake of TC in HBEP cells, stable transfected HEK293 cells and MDCK2 cells; overview of genes used for real-timePCR, influence of pH on TC uptake in HEK293-OATP1A2 and HEK-OCT1 and additional methods. This material is available free of charge via the Internet at http://pubs.acs.org.

Acknowledgment This work was supported by Dr. R. Pfleger GmbH (Bamberg, Germany, supplier of trospium chloride, Spasmex®) and by the German Federal Ministry of Education and Research Grants InnoProfile 03IP612 / 03IPT612X. We are very grateful to Dr. Bruno Stieger (Department of Clinical Pharmacology and Toxicology, Zurich, Switzerland) for providing the coding sequence of NTCP. We also like to express our thanks to Danilo Wegner and Marten Möller for reliable and very helpful assistance. The authors are indebted to Anna Wolf (Dr. R. Pfleger GmbH) for proof reading the English manuscript.


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