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

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 (<i>K</i>_m = 6.9 ± 1.3 μmol/L; <i>V</i>_max = 41.6 ± 1.8 pmol/mg·min), OCT1 (<i>K</i>_m = 106 ± 16 μmol/L; <i>V</i>_max = 269 ± 18 pmol/mg·min), and P-gp (<i>K</i>_m = 34.9 ± 7.5 μmol/L; <i>V</i>_max = 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: <i>ABCB1</i> (P-gp), <i>ABCC1–5</i> (MRP1–5), <i>ABCG2</i> (BCRP), <i>SLCO2B1</i> (OATP2B1), <i>SLCO4A1</i> (OATP4A1), <i>SLC22A1</i> (OCT1), <i>SLC22A3</i> (OCT3), <i>SLC22A4</i> (OCTN1), <i>SLC22A5</i> (OCTN2), and <i>SLC47A1</i> (MATE1). Immuno-reactive P-gp and OATP1A2 were localized to the apical cell layers. Drug metabolizing enzymes <i>CYP3A5, </i>-<i>2B6</i>, -<i>2B7</i> -<i>2E1</i>, <i>SULT1A1–4, UGT1A1–10</i>, and <i>UGT2B15,</i> and nuclear receptors <i>NR1H3</i> and <i>NR1H4</i> were also expressed on mRNA level. TC was taken up into HBU cells (<i>K</i>_m = 18.5 ± 4.8 μmol/L; <i>V</i>_max = 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

Perfusion model and experimental design.

<p>Panel (A) shows a diagram of the <i>ex vivo</i> placenta perfusion model as performed in the study. Details of the assay are described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007986#s2" target="_blank">methods</a> section. <b>Of note 3 probes on the maternal side cannulate the intervillous space and on the fetal side one probe each cannulate the artery and vein</b>. Panel (B) shows the experimental design for each of the 6 placentas studied. A complete experiment comprised 3 phases. Phase I involved addition of rMSP1₄₂ to the maternal circulation with samples removed from the maternal and fetal circulation every 30 minutes until the completion of the phase. Phase II and phase III involved addition of rMSP1₄₂ previously mixed with 10% or 25% human plasma containing anti-MSP1 antibodies to form ICs. The perfusion pressures recorded throughout the experiment are shown. A significant rise in perfusion pressure indicates poor placental flow of medium through the vasculature. If pressures rose too quickly as in placenta 2, the experiment was terminated. Placenta 4 was terminated early to examine whether MSP1₄₂ without specific antibody could be detected by immunohistochemistry in the villous stroma. Placenta 6 included MSP1₄₂ with 25% human plasma from individuals that have never resided in a malaria endemic area and did not have a phase with lower serum concentrations.</p

Assays used to detect MSP1 and antibodies to MSP1.

a<p>Polyclonal anti-MSP1₄₂ was resuspended in carbonated-bicarbonated buffer (pH 9.6) and <b>used to coat the ELISA plates as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007986#s2" target="_blank">materials and methods</a> section. As a standard, recombinant MSP1₄₂ was resuspended in the same media used for the test samples (i.e. plasma from N. American subjects or perfusates) and added to the wells coated with anti-MSP1₄₂ at serial two-fold dilutions starting at 4 ng/ml</b>. Sensitivity of the assay was approximately 200 pg/ml.</p>b<p>Plates were coated with rMSP1₄₂ in PBS.</p>c<p>Plates were coated with streptavidin in PBS (Pierce Biotechnology Inc). Standards used for this experiment were bMSP1₄₂ or pooled human plasma containing anti-MSP1 (25%) combined with bMSP1₄₂ as described in the text. The assay had a sensitivity ∼175–200 pg/ml <b>to detect the bMSP1₄₂ in the samples</b>.</p>d<p>Perfusate was added at 1∶5 dilution to wells coated with streptavidin and incubated overnight at 4°C. Plates were washed thoroughly (6 times) with 0.1% Tween as described in the text to remove any unbound antibody prior to adding goat anti-human IgG. Control experiments using MSP1₄₂ incubated with normal serum had <0.15 OD and the mean+2 SD was subtracted from background described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007986#pone-0007986-g004" target="_blank">figure 4</a>.</p

Immunohistological localization of MSP1₄₂ in the villous stroma before and after perfusion.

<p>Panel (A,C) show before and panels (B,D) after perfusion with MSP1₄₂ in the absence of anti-MSP1 (No. 4; A,B) and following addition of anti-MSP1 (No. 5; C,D). The syncytiotrophoblastic membrane (STM) is shown in light green with strings of nuclei (blue). MSP1₄₂ has intense green staining only in the villous stroma of No. 5 following perfusion (indicated by arrows, D). <b>Panel E shows a hematoxylin and eosin stained section from the same placenta for reference. The labels are: IVS – intervillous space, FC – fetal capillary, STM - syncytiotrophoblastic membrane, FVS – fetal villous stroma. All sections are at 200× magnification</b>. The staining with pre-bleed sera from rats used for making the anti-rMSP1₄₂ polyclonal sera was identical to that shown in panels A and C (not shown).</p

Permeability of antipyrine, creatinine, erythropoietin and viability characteristics of perfused placentas used in the studya.

a<p>All values shown fall within the range of values for previous placental perfusion studies without any experimental interventions <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007986#pone.0007986-Linnemann1" target="_blank">[38]</a>.</p>b<p>Experiment number (each represents a complete perfusion experiment with different placentas.</p>c<p>Means±SD are shown for all phases of the experiment. For placenta 4 only one phase is shown.</p

MSP1₄₂ does not co-localize with placental macrophages (Hofbauer cells).

<p>Panel <b>(A)</b> shows MSP1₄₂ localizes to fetal vascular structures (green staining, indicated by green arrows) and not with placental Hofbauer cells (CD68 positive cells, red staining indicated by red arrow). Panel <b>(B)</b> shows a greater magnification of the villous stroma. <b>The results are shown for placenta 3</b>. No co-localization of CD68 positive cells with MSP1₄₂ was observed for all 4 placentas examined. <b>The left had panel at 400× and right at 800×</b>.</p

Measurement of MSP1₄₂ and free anti-MSP1 antibodies in maternal and fetal circulation with the placental perfusion model.

<p>The levels of MSP1 in the fetal perfusate were measured before (nondissociated) and after treatment with dissociation buffer (dissociated) during the 3 phases of the experiment. <b>The differences in levels of MSP1₄₂ detected in the fetal perfusate between phases II and III (placentas 3 and 5) correlated with percentage of plasma containing anti-MSP1 antibodies added to the maternal compartment</b>. Each point represents the mean value performed in triplicate. The variation between replicate samples was <10%. <b>Assay 1 was used to measure free MSP1 and assay 2 for free anti-MSP1 antibodies (see </b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007986#pone-0007986-t001" target="_blank"><b>table 1</b></a><b>)</b>.</p

The presence of human IgG complexed to rMSP1₄₂ in fetal perfusate.

<p>rMSP1₄₂ was biotinylated (bMSP1₄₂) and thus the configuration of ELISA differed from that described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007986#pone-0007986-g003" target="_blank">Fig. 3</a>, e.g. streptavidin was used to capture bMSP1₄₂ followed by anti-human IgG (assay 3, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007986#pone-0007986-t001" target="_blank">table 1</a>, squares). Nondissociated MSP1₄₂ levels were also measured (assay 2, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007986#pone-0007986-t002" target="_blank">table 2</a>, triangles). The assay was performed for placenta 5. Values indicate means±SD of replicate assays (N = 3). Asterisks indicate significant differences from P<0.05 to P<0.001 between samples. <b>Assay 3 was used to measure non-dissociated bMSP1₄₂ and assay 4 for human IgG complexed to rMSP1₄₂ (see </b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007986#pone-0007986-t001" target="_blank"><b>table 1</b></a><b>)</b>.</p

MSP1₄₂ co-localizes with fetal endothelial cells following perfusion.

<p>MSP1₄₂ (green staining) co-localizes with CD31 (PECAM-1, red staining) indicating its presence in and around the fetal vascular endothelial cells (upper and lower panels for placentas 1 and 5 respectively). Similar co-localization was observed for placentas 2 and 3 (not shown). <b>The upper panels are at 400× and lower at 200× magnification</b>.</p

MSP1₄₂ co-localizes with human IgG in structures consistent with fetal endothelial cells.

<p>This suggests MSP1₄₂ may be complexed with IgG. <b>The results are shown for placenta 3</b>. <b>Sections are at 400× magnification</b>.</p