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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

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Department of Clinical Pharmacology, Department of Pharmacology of the Center of Drug Absorption and Transport (C_DAT), and §Department of Urology, University Medicine, Greifswald, Germany
Institute of Experimental and Clinical Pharmacology and Toxicology, University of Erlangen-Nuremberg, Erlangen, Germany
Dr. R. Pfleger, GmbH, Bamberg, Germany
*Phone: +49 (0)3834 86-5640. Fax: +49 (0)3834 86-5631. E-mail: [email protected]
Cite this: Mol. Pharmaceutics 2015, 12, 1, 171–178
Publication Date (Web):December 3, 2014
Copyright © 2014 American Chemical Society

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    The cationic, water-soluble quaternary trospium chloride (TC) is incompletely absorbed from the gut and 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. 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 was measured. 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. 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; 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 and 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) μmol/L) and verapamil (IC50 = 4.6 (2.8; 7.5) μmol/L), inhibitors of OATP1A2 and OCT1, respectively. Affinity of TC to OCT1 and P-glycoprotein may be the reason for incomplete oral absorption, wide distribution into liver and kidneys, and 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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    Characterization of stably transfected cells expressing P-gp, OCT1, ASBT, and 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-time-PCR; 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

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