Beta-2 Adrenergic Agonists Are Substrates and Inhibitors of Human

Mar 9, 2015 - *School of Pharmacy and Pharmaceutical Sciences, Trinity College Dublin, .... Glenn A. Jacobson , Sharanne Raidal , Kate Robson , Christ...
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Beta‑2 Adrenergic Agonists Are Substrates and Inhibitors of Human Organic Cation Transporter 1 Johanna J. Salomon,†,⊥ Yohannes Hagos,‡,§ Sören Petzke,‡ Annett Kühne,§ Julia C. Gausterer,† Ken-ichi Hosoya,∥ and Carsten Ehrhardt*,† †

School of Pharmacy and Pharmaceutical Sciences and Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin 2, Ireland ‡ Zentrum für Physiologie und Pathophysiologie, Georg-August-Universität, 37073 Göttingen, Germany § PortaCellTec Biosciences GmbH, 37073 Göttingen, Germany ∥ Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 930-0887 Toyama, Japan ABSTRACT: Beta-2-adrenergic agonists are first line therapeutics in the treatment of asthma and chronic obstructive pulmonary disease (COPD). Upon inhalation, bronchodilation is achieved after binding to β2-receptors, which are primarily localized on airway smooth muscle cells. Given that β2-adrenergic agonists chemically are bases, they carry net positive charge at physiologic pH value in the lungs (i.e., pH 7.4). Here, we studied whether β2-agonists interact with organic cation transporters (OCT) and whether this interaction exerted an influence on their passage across the respiratory epithelium to their target receptors. [14C]-TEA uptake into proximal (i.e., Calu-3) and distal (i.e., A549 and NCI-H441) lung epithelial cells was significantly reduced in the presence of salbutamol sulfate, formoterol fumarate, and salmeterol xinafoate in vitro. Expression of all five members of the OCT/N family has been confirmed in human pulmonary epithelial cells in situ and in vitro, which makes the identification of the transporter(s) responsible for the β2-agonist interaction challenging. Thus, additional experiments were carried out in HEK-293 cells transfected with hOCT1−3. The most pronounced inhibition of organic cation uptake by β2-agonists was observed in hOCT1 overexpressing HEK-293 cells. hOCT3 transfected HEK-293 cells were affected to a lesser extent, and in hOCT2 transfectants only marginal inhibition of organic cation uptake by β2-agonists was observed. Bidirectional transport studies across confluent NCI-H441 cell monolayers revealed a net absorptive transport of [3H]-salbutamol, which was sensitive to inhibition by the OCT1 modulator, verapamil. Accordingly, salbutamol uptake into hOCT1 overexpressing HEK-293 cells was time- and concentration-dependent and could be completely blocked by decynium-22. Taken together, our data suggest that β2-agonists are specific substrates and inhibitors of OCT1 in human respiratory epithelial cells and that this transporter might play a role in the pulmonary disposition of drugs of this class. KEYWORDS: respiratory epithelium, inhalation biopharmaceutics, pulmonary drug disposition, competitive inhibition, bronchodilators

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INTRODUCTION The influence of drug transporter proteins on the disposition of inhaled therapeutics is still poorly understood.1,2 Early in vitro studies presented data consistent with the idea that drug absorption from the lungs is not exclusively mediated by passive diffusion.5 These pioneering studies showed the expression of drug transporters such as P-glycoprotein in lung tissues in situ or demonstrated transporter-related effects in cultured cells.3,4 Subsequently, studies conducted in experimental animal models and human volunteers confirmed that drug transporters indeed can modulate access of drugs to intracellular targets and submucosal lung tissues and thus potentially influence drug absorption profiles.6−8 Many bronchodilators that are inhaled as pharmaceutical aerosols carry a net positive charge at physiological lung pH because they are either nitrogenous bases (e.g., β2-receptor agonists) or permanent cations (e.g., M3-muscarinergic © XXXX American Chemical Society

antagonists). This accepted fact led us and others to hypothesize that transporters associated with organic cation translocation in other organs, such as organic cation transporters (OCTs and OCTNs; SLC22A1-A5), might also be involved in absorption and clearance processes of cationic drugs in the lung.9−11 A number of studies have reported an interaction between bronchodilators and cation transporters in the lung. Originally, data from our laboratory suggested that the β2-receptor agonist, salbutamol, exhibited concentration- and temperature-dependent net absorption across 16HBE14o- and Calu-3 human Special Issue: Advances in Respiratory and Nasal Drug Delivery Received: December 21, 2014 Revised: February 21, 2015 Accepted: March 9, 2015

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bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL streptomycin. Calu-3 cells were cultured in minimum essential medium (MEM) supplemented with 10% (v/v) FBS, 1% (v/v) nonessential amino acids, 1% (v/v) sodium pyruvate solution, 0.5% (v/v) glucose solution, 100 U/mL penicillin, and 100 μg/ mL streptomycin. NCl-H441 cells were routinely cultured in RPMI-1640 medium supplemented with 5% (v/v) FBS, 1% (v/ v) sodium pyruvate, 100 U/mL penicillin, and 100 μg/mL streptomycin. Twenty-four hours postseeding, the medium was replaced with RPMI-1640 medium, which contained dexamethasone (200 nM) and insulin-transferrin-sodium selenite (ITS) supplement (Roche Diagnostics Limited, West Sussex, U.K.) in addition to the supplements mentioned above. All cell lines were maintained at 37 °C in 5% CO2 atmosphere, and the medium was exchanged every 48 h, until confluent monolayers were reached. For transport studies, cells were grown under air-interfaced culture (AIC) conditions on Transwell Clear inserts (12 mm in diameter, pore size 0.4 μm; Corning, VWR, Dublin, Ireland), i.e., the apical fluid volume was completely removed, and the volume in the basolateral compartment was adjusted to 700 μL 48 h postseeding. Organic Cation Uptake Studies in the Presence of β2Agonists. Uptake experiments were carried out using cells grown in 24-well plates in freshly prepared, bicarbonated Krebs-Ringer buffer (KRB) composed of 15 mM HEPES, 116.4 mM NaCl, 5.4 mM KCl, 0.78 mM NaH2PO4, 25 mM NaHCO3, 1.8 mM CaCl2, 0.81 mM MgSO4, and 5.55 mM glucose; pH 7.4. Cells were grown to confluent monolayers for 5 (A549), 8 (NCI-H441), or 12 (Calu-3) days. Prior to uptake experiments, cell monolayers were washed three-times with prewarmed KRB solution. The inhibitory potency of salbutamol sulfate (500 μM), formoterol fumarate (500 μM), and salmeterol xinafoate (100 μM) on [14C]-TEA uptake was measured for 10 (Calu-3, NCIH441) or 30 min (A549 cells). [3H]-acetylcarnitine uptake was studied in the presence of β2-agonists for 20 min in all cell types. These studies were carried out at 4 and 37 °C, whereby the values obtained at 4 °C were subtracted from the 37 °C values in order to account for passive diffusion. For kinetic studies in A549 cells, salbutamol (1 mM), formoterol (500 μM), and salmeterol (100 μM) were applied simultaneously with [14C]-TEA and unlabeled TEA (in the concentration range from 0.25 to 2 mM), and uptake was measured after 30 min of parallel incubation. Moreover, in Calu-3 cells [3H]-acetylcarnitine uptake was measured in the presence of a constant concentration of 500 μM formoterol and unlabeled acetylcarnitine in the range from 50 to 200 μM for 20 min. At the indicated time points, the uptake was stopped by washing the cell monolayers three-times with ice-cold buffer, and then 400 μL of 1 N NaOH was added to the cells. Monolayers were left to lyse for at least 12 h, before 400 μL of 1 N HCl was added to neutralize the cell lysate. Five-hundred microliters of lysate was used to measure the cell-associated radioactivity in a liquid scintillation counter (Tri Carb TR2100 Packard Scintillation Counter, Ireland). In parallel, total cell protein content was quantified using a protein assay kit (BioRad, Hercules, CA) according to the manufacturer’s instructions. Uptake by OCT Overexpressing HEK-293 Cells. HEK293 cells stably expressing hOCT1, hOCT2, or hOCT3 were selected by hygromycine (10 μg/mL) and grown in tissue culture flasks in high-glucose DMEM medium (Invitrogen, Darmstadt, Germany) supplemented with 10% (v/v) FBS

bronchial epithelial cell line monolayers.9 Subsequent studies provided further evidence on β2-agonist inhibition of OCTmediated pathways in respiratory epithelial and smooth muscle cells in vitro.10,12 In this context, Horvath and colleagues reported formoterol as substrate for hOCT3 in airway smooth muscle cells.13 In a related study, Chiappori et al. observed that salbutamol uptake into bronchial smooth muscle cells was also OCT3 mediated.14 Of late, Mamlouk and colleagues confirmed our initial findings by demonstrating net absorptive salbutamol sulfate transport across Calu-3 cell monolayers and, using an isolated perfused human lung lobe model, Gnadt and coworkers reported cation transporter interactions during the pulmonary absorption of β2-agonists ex vivo.15,16 Nevertheless, while clinically relevant data are starting to emerge, the molecular identity of the transporter(s) responsible for β2receptor agonist transport in lung epithelium remains unclear. Identifying this transporter might be clinically important because mutations of organic cation transporter subtypes have been suggested to contribute to interindividual variations in drug disposition in other organs.17,18 Iwata et al., for example, reported genetic variants in the OCT2 gene on cisplatininduced adverse events in the kidneys.19 Besides, although the proximal airways are assumed to be the primary target for β2receptor agonists, it is pivotal to analyze the absorption pathways of bronchodilators in the distal lung, where absorption of significant portions of inhaled β2-agonists across the air−blood barrier can cause systemic adverse effects.20 In the present study, we investigated whether short- and long acting β2-adrenergic drugs (i.e., salbutamol, formoterol, and salbutamol) interfere with the uptake of the OCT/N substrates, i.e., [14C]-TEA and [3H]-acetylcarnitine, into cellular in vitro models of human distal (A549, NCI-H441) and proximal (Calu-3) lung epithelium. We also studied to what extent organic cation uptake into hOCT1, hOCT2, and hOCT3 overexpressing HEK-293 cells was affected by β2-receptor agonists. Lastly, we determined whether [3H]-salbutamol was a substrate of hOCT1-mediated transport in addition to being an inhibitor.

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EXPERIMENTAL SECTION Materials. [Ethyl-1-14C]-tetraethylammonium chloride 14 ([ C]-TEA; 55 mCi/mmol), [n-methyl-3H] acetyl-L-carnitine hydrochloride ([3H]-acetylcarnitine; 85 Ci/mmol), and [ring-3H]-salbutamol (salbutamol; 20 Ci/mmol) were purchased from American Radiolabeled Chemicals (Herts, U.K.). Unlabeled TEA, acetylcarnitine, 1,1-Diethyl-2,2-cyanine (decynium-22), 1-methyl-4-phenylpyridinium (MPP+), salbutamol sulfate, verapamil, and all cell culture media and supplements were obtained from Sigma-Aldrich (Dublin, Ireland). Formoterol fumarate and salmeterol xinafoate were obtained from Santa Cruz Biotechnology (Heidelberg, Germany). Cell culture plastics were purchased from Greiner BioOne (Frickenhausen, Germany). Cell Culture. A549 (American Type Culture Collection, ATCC CCL-185) cells were obtained from the European Collection of Animal Cell Cultures (Salisbury, U.K.), whereas Calu-3 (ATCC HTB-55) and NCI-H441 (American Type Culture Collection, HTB-174) cells were purchased from LGC Promochem (Teddington, U.K.). Cells were cultured at a seeding density of 40,000 (A549), 75,000 (Calu-3) or 100,000 cells/cm2 (NCI-H441). A549 cells were grown in Dulbecco’s modified Eagle’s medium/Ham’s F12 (1:1 mix) (DMEM/F12) supplemented with 5% (v/v) fetal B

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Molecular Pharmaceutics (Invitrogen), 100 U/mL penicillin, and 100 μg/mL streptomycin. For uptake studies, HEK-293 cells were seeded in 24well plates (2 × 105 cells/well) and grown for 3 days until 90− 100% confluence. Initially, cells were washed with 1 mL of prewarmed mammalian Ringer solution (130 mM NaCl, 4 mM KCl, 1 mM CaCl2, 1 mM Mg2SO4, 1 mM NaH2PO4, 20 mM HEPES, and 20 mM D-glucose; pH 7.4). Cells were then incubated with prewarmed mammalian Ringer solution, containing radioactively labeled 1 μM MPP+ (composed of 20 nM [3H]-MPP+ plus 980 nM unlabeled MPP+) for 5 min. Uptake of [3H]-MPP+ was measured in the presence and absence of salbutamol sulfate, formoterol fumarate, and salmeterol xinafoate (all compounds were used at concentrations of 100 and 500 μM). Moreover, in concentrationdependent inhibition studies, [3H]-MPP+ uptake was performed in hOCT1-transfected HEK-293 cells in the presence of various concentrations of formoterol fumarate and salmeterol xinafoate (i.e., 20, 40, 60, 80, 100, 120, and 140 μM). In the case of salbutamol uptake, time-dependent studies were performed using a concentration of 0.03 μM [3H]-salbutamol. In concentration-dependent studies, concentrations of 0.03, 0.1, and 10 μM were assessed for 5 min in the absence or presence of 20 μM decynium-22. To stop the reaction, cells were washed with ice-cold PBS and lysed with 500 μL of 1 N NaOH solution for 20 min. The solution was then neutralized with 1 N HCl, transferred to scintillation vials and 2.5 mL of Lumasafe scintillation solution (PerkinElmer, Dublin, Ireland) was added to each sample. Incorporated radioactivity was determined by using a scintillation counter (TriCarb 1500 Packard, Meriden, CT). IC50 values were calculated by SigmaPlot 10 Nonlinear Regression Equation: Hyperbola; Hyperbolic Decay, 3 Parameter [f = y0 + (a × b)/(b + x)]. Each experiment was conducted at least in triplicate from two different passages (n ≥ 6). [3H]-Salbutamol Transport Studies. NCI-H441 cells were grown on Transwell Clear filter inserts for at least 10 days (transepithelial electrical resistance (TEER) >350 Ohm × cm2). [3H]-salbutamol transport was studied at an air−liquid interface, i.e., in the case of apical-to-basolateral (a-to-b) transport, 5 μL of tracer solution was applied to the cell monolayer. Seven-hundred microliters of prewarmed KRB solution were added to the basolateral compartment. The cells were kept at 37 °C during the experiment, and 200 μL samples were collected from the receiver compartment and replaced with an equal amount of fresh, prewarmed KRB solution. Basolateral-to-apical (b-to-a) transport was performed by adding 705 μL of [3H]-salbutamol solution to the basolateral (i.e., donor) compartment. A 5 μL sample was taken directly to assess the initial concentration. The transported amount of [3H]-salbutamol was collected by washing the apical surface of the cell monolayers six times with 100 μL of KRB solution each and combining the washings. At the end of transport studies, another 5 μL sample was collected to calculate mass balance. Each experiment was conducted at least in triplicate. TEER values were recorded before and after the flux studies, in order to confirm cell layer integrity, and were 441 ± 52 and 404 ± 20 Ω × cm2, respectively. The radioactivity of samples was determined as described above with a Tri Carb TR2100 Packard Scintillation Counter. The following equation was used to calculate the apparent permeability coefficient (Papp):

Papp = (ΔQ /Δt )/(A × C0)

(1)

where ΔQ was the change in quantity of the compound over a designated period of time (Δt), A was the nominal surface area of the cell layers (1.13 cm2), and C0 was the initial concentration of the drug in the donor fluid used in this study. Data Analysis. The uptake of [14C]-TEA, [3H]-MPP+, 3 [ H]-acetylcarnitine, and [3H]-salbutamol was expressed as the cell-to-medium ratio calculated by the following equations: cell/medium ratio = [3H]dpm per cell protein/[3H] dpm per μL buffer

(2)

cell/medium ratio = [14C] dpm per cell protein/[14C] dpm per mL buffer

(3)

Results were expressed as means ± SD. The significance of differences between means was determined by unpaired, twotailed Student’s t-test. Statistical significance of differences between groups was determined by one-way analysis of variance (ANOVA), followed by the modified Fisher’s leastsquares difference method. All experiments were carried out at least in triplicate. Representative Ki values were estimated by using the Cheng−Prusoff equation with the assumption of onecomponent nature of inhibitors according to Eadie−Hofstee plots and Lineweaver−Burk plot analysis under the chosen conditions.21,22

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RESULTS Effect of β2-Agonists on Organic Cation Uptake by Human Respiratory Epithelial Cells. We investigated whether the β2-agonists, salbutamol, formoterol, and salmeterol, influenced OCT and/or OCTN function in human alveolar (A549), bronchial (Calu-3), and bronchiolar (NCI-H441) epithelial cells. As shown in Table 1A, all three compounds significantly reduced [14C]-TEA uptake into A549 cells to 32.6 ± 6.9% (salbutamol sulfate), 8.8 ± 2.1% (formoterol fumarate), and 17.7 ± 0.5% (salmeterol xinafoate) of control. The β2agonists, however, did not have an effect on [ 3 H]acetylcarnitine uptake in A549 cells (Table 1B). In Calu-3 cells, formoterol and salmeterol decreased uptake of [14C]-TEA and [3H]-acetylcarnitine similarly to approximately 50−60% of control, whereas salbutamol had almost no effect. The uptake of [14C]-TEA into NCI-H441 cells in the presence of all β2agonists was strongly reduced to 35.5 ± 7.5% (salbutamol), 19.1 ± 7.6% (formoterol), and 8.4 ± 14.5% (salmeterol). [3H]Acetylcarnitine uptake into NCI-H441 cells was more affected by β2-agonists in comparison to A549 cells, in the case of formoterol (73.3 ± 3.7% of control) and salmeterol (72.3 ± 7.2% of control), respectively. Kinetic Analysis of Inhibitory Effects on Organic Cation Uptake in A549 and Calu-3 Cells. The kinetics of the β2-agonists, salbutamol, formoterol, and salmeterol, on [14C]-TEA and [3H]-acetylcarnitine uptake were calculated in alveolar and bronchial epithelial cell models (Figures 1 and 2). In A549 cells, [14C]-TEA uptake was significantly (*P < 0.01) decreased in the presence of 1 mM salbutamol (Figure 1A), 500 μM formoterol (Figure 1B), and 100 μM salmeterol (Figure 1C). The evaluated Km and Vmax for [14C]-TEA uptake (i.e., control) were 1108.3 ± 203.1 μM and 0.31 ± 0.03 nmol/ min/mg protein, respectively. In the presence of salbutamol, the apparent Km increased approximately 4-fold (4.6 ± 0.4 C

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Molecular Pharmaceutics Table 1. Effects of β2-Agonists on the Uptake of [14C]-TEA (A) and [3H]-Acetylcarnitine (B) by Human Respiratory Epithelial Cell Layers (A549, Calu-3 and NCI-H441 cells)a

MPP+ and decynium-22 were used as controls for control inhibition of OCTs. Salbutamol showed the weakest inhibitory potential of the three drugs investigated. No decrease in [3H]-MMP+ uptake was observed with 100 μM salbutamol in any of the OCTclones (Figure 3A). When used at 500 μM, uptake of [3H]MPP+ into OCT1-overexpresssing cells was reduced to 58.1 ± 6.8% (salbutamol sulfate). At 500 μM still no significant effects were observed in OCT2- and OCT3-transfected cells. When formoterol was studied, 100 μM of the compound reduced [3H]-MPP+ uptake to 22.9 ± 3.0% in hOCT1transfected cells (Figure 3B). In hOCT2-transfected cells, formoterol used at 100 μM did not cause OCT inhibition; however, when used at 500 μM, [3H]-MPP+ uptake was reduced to 77.1 ± 5.3%. In hOCT3-HEK-293 cells formoterol decreased [3H]-MPP+ uptake to 85.1 ± 1.4% (100 μM) and 28.3 ± 1.4% (500 μM), respectively. Consistent with what was observed with formoterol, salmeterol showed high inhibition levels of [3H]-MPP+ uptake in hOCT1-transfected cells (28.0 ± 3.1% of control at 100 μM, Figure 3C). [3H]-MPP+ uptake was lesser affected in hOCT2HEK-293 cells, i.e., 83.1 ± 2.2% (100 μM) and 62.4 ± 0.4% (500 μM). In hOCT3 clones respective values of 77.1 ± 2.8% (100 μM) and 19.4 ± 0.1% (500 μM) were measured. As hOCT1 was much more susceptible to inhibition by the β2agonists than the other transporters, in a follow-up study, IC50 values of formoterol and salmeterol were determined using hOCT1-HEK-293 clones. Formoterol fumarate inhibited hOCT1 function with an IC50 of 22.3 ± 1.6 μM (Figure 3D) and salmeterol produced an IC50 value of 47.8 ± 7.8 μM (Figure 3E). Salbutamol Transport Across NCI-H441 Cell Monolayers. The bidirectional transepithelial transport of salbutamol across NCI-H441 bronchiolar epithelial cell monolayers was measured at an air−liquid interface in order to mimic physiological conditions, i.e., only a very small volume of apical donor solution was used to avoid artifacts caused by engaged water removal pathways.23 Transport of salbutamol was significantly (P < 0.05) higher in absorptive direction, i.e., the a-to-b permeability coefficient was 1.19 ± 0.23 × 10−6 cm/s, while a Papp value of 0.48 ± 0.23 × 10−6 cm/s was observed in b-to-a direction. When experiments were carried out in the presence of the OCT1 inhibitor, verapamil (50 μM), transport in the a-to-b direction was significantly (P < 0.05) decreased to 0.50 ± 0.04 × 10−6 cm/s, whereas no changes were observed in the b-to-a direction. Salbutamol Uptake by hOCT1-Transfected Cells. To determine if β2-agonists are also substrates for organic cation transporters, [3H]-salbutamol uptake was studied in stably transfected hOCT1-HEK-293 cells. Salbutamol was taken up in a time-dependent fashion with approximately 2-fold higher intracellular concentration in hOCT1-transfected cells, compared to mock-transfected cells (Figure 4A). In the presence of decynium-22 (20 μM), the uptake of salbutamol was diminished to control levels at each time point. Additionally, concentration-dependent uptake was evaluated by using concentrations of 0.03, 0.1, and 10 μM [3H]-salbutamol (Figure 4B). Compared to mock-transfected cells, uptake values were substantially increased in hOCT1-transfected cells (about 2-fold at concentrations of 0.03 and 0.1 μM, and up to 8-fold in the case of 10 μM). Salbutamol uptake, however, did not reach saturation at the concentration range tested.

A [14C]TEA uptake

uptake (% of control) in the presence of salbutamol 500 μM

Uptake (% of control) in the presence of formoterol 500 μM

uptake (% of control) in the presence of salmeterol 100 μM

32.6 ± 6.9** 78.1 ± 34.2* 35.5 ± 7.5

8.8 ± 2.1** 52.4 ± 14.5** 19.3 ± 7.6**

17.7 ± 0.5** 52.9 ± 6.3** 8.4 ± 14.5**

A549 Calu-3 NCIH441

B

[3H]acetylcarnitine uptake

uptake (% of control) in the presence of salbutamol 500 μM

uptake (% of control) in the presence of formoterol 500 μM

uptake (% of control) in the presence of salmeterol 100 μM

A549 Calu-3 NCI-H441

100.0 ± 12.4 103.8 ± 1.8* 129.8 ± 9.0*

107.6 ± 14.2 52.3 ± 2.8* 73.3 ± 3.7*

85.0 ± 4.8* 60.0 ± 3.0** 72.3 ± 7.2*

a

Uptake of [14C]-TEA was measured for 10 min (Calu-3, NCI-H441) and 30 min (A549), whilst uptake of [3H]-acetylcarnitine was studied over 20 min in all cell models in the presence of salbutamol sulfate (500 μM), formoterol fumarate (500 μM) and salmeterol xinafoate (100 μM). Values were obtained by calculating the difference between uptake at 37 °C and 4 °C. Each value represents the means ± SD (n = 3−6). **P < 0.01, *P < 0.05 significantly different from control.

mM). Vmax did not change (0.35 ± 0.02 nmol/min/mg protein). The presence of formoterol also led to a 4-fold increase in the Km value to 4.33 ± 3.22 mM. Again, no significant changes in Vmax were observed (0.20 ± 0.14 nmol/ min/mg protein). A Km value of 12.07 ± 1.33 mM in the presence of salmeterol was observed (i.e., an 11.5-fold increase). In line with the other observations, Vmax remained at constant levels of 0.34 ± 0.03 nmol/min/mg protein. Eadie− Hofstee analysis [shown in Figure 1D (salbutamol), 1E (formoterol), and 1F (salmeterol)] suggested competitive inhibitory kinetics for all three β2-agonists. A substrate specific Ki value of 276.6 ± 90.4 μM for [14C]-TEA uptake was calculated for salbutamol. The Ki value of formoterol for [14C]TEA uptake was estimated to be 92.3 ± 20.3 μM. A Ki value of 16.6 ± 0.5 μM was obtained for salmeterol. The Ki values of all compounds were significantly lower than the respective Km value of [14C]-TEA. When the effect of formoterol on [3H]-acetylcarnitine uptake in Calu-3 cells was studied, the kinetic analyses revealed quite the opposite. A constant concentration of 500 μM formoterol significantly reduced [3H]-acetylcarnitine uptake (Figure 2A), however, Eadie-Hofstee transformation showed noncompetitive inhibition characteristics (Figure 2B). In this case, Km and Vmax were both decreased: Km values of 614.3 ± 108.5 μM and 441.2 ± 175.2 μM in the absence and presence of formoterol were found; Vmax was reduced to 0.06 ± 0.01 nmol/min/mg protein in the presence of formoterol compared to Vmax= 0.10 ± 0.01 nmol/min/mg protein in the absence of the compound. Beta-2 Adrenergic Agonist Inhibition by OCT Subtype. The specificity of β2-agonist interaction with OCTs was studied by measuring [3H]-MPP+ uptake into HEK-293 cells, stably overexpressing hOCT1, hOCT2, and hOCT3 in the presence of the bronchodilators. hOCT-transfected HEK-293 cells exhibited an at least 10-times higher [3H]-MPP+ uptake, when compared to mock-transfected cells (data not shown). D

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Figure 1. Organic cation uptake into A549 cells in the presence (○) or absence (●) of β2-agonists. [14C]-TEA uptake was measured for 30 min at constant concentrations of salbutamol (A; 1 mM), formoterol (B; 500 μM), and salmeterol (C; 100 μM) in A549 cells. The results of inhibition of β2-agonists on [14C]-TEA uptake into A549 cell monolayers were analyzed by Eadie−Hofstee transformation (D, salbutamol; E, formoterol; F, salmeterol). Each data point represents means ± SD (n = 3). *P < 0.01 significantly different from control.

Figure 2. [3H]-acetylcarnitine uptake into Calu-3 cells was studied in the presence (○) or absence (●) of a constant concentration of formoterol (A; 500 μM) for 20 min. Results were analyzed by Eadie−Hofstee transformation (B). Each data point represents means ± SD (n = 3). **P < 0.01, *P < 0.05 significantly different from control.

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DISCUSSION Inhaled salbutamol, formoterol, and salmeterol are routine treatments of asthma and COPD, both in single and combination therapy with other drugs. In this work, we investigated to what extent these β2-agonists interact with

organic cation transporters at the respiratory epithelium. We provided evidence that all three β2-receptor agonists competitively inhibit the uptake of [14C]-TEA, but not that of [3H]acetylcarnitine. Moreover, we demonstrated in hOCT overexpressing HEK-293 cells that β2-agonists exhibited selective E

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Figure 3. Inhibition of [3H]-MPP+ uptake by β2-agonists in hOCT-overexpressing HEK-293 cells. Uptake of [3H]-MPP+ was measured for 5 min at 37 °C in the presence and absence of 100 μM MPP+ or salbutamol (100 and 500 μM) in hOCT1-, hOCT2-, and hOCT3-transfected HEK-293 cells (A). Uptake of [3H]-MPP+ was performed for 5 min at 37 °C in the presence and absence of 20 μM decynium-22 (DEC-22), formoterol (B) and salmeterol (C) (both at concentrations of 100 and 500 μM) in hOCT-transfected HEK-293 cells. Data are presented as percentage of [3H]-MPP+ uptake corrected for uptake into mock-transfected cells. [3H]-MPP+ uptake was also measured in the presence of increasing concentrations (0−140 μM) of formoterol (D) and salmeterol (E). Uptake into mock-transfected cells (○) is also shown. Each data point represents means ± SD (n = 6). **P < 0.01, *P < 0.05 significantly different from uptake into the relevant mock-transfected cells.

Figure 4. Salbutamol uptake in hOCT1-overexpressing HEK-293 cells. Uptake of [3H]-salbutamol (0.03 μM) was measured for 1, 3, and 5 min at 37 °C in the presence and absence of 20 μM decynium-22 (DEC-22) in hOCT1-transfected HEK-293 cells (A). Uptake of [3H]-salbutamol was also measured at various concentrations (0.03, 0.1, and 10 μM) at 37 °C for 5 min in the presence and absence of 20 μM decynium-22 (B). Data represent means ± SD (n = 3−6). *P < 0.001 significantly different from uptake into the relevant mock-transfected cells.

Beta-2 adrenergic agonists are cations at lung physiological pH value. Thus, the question arose, whether this class of drugs is absorbed by passive diffusion only or whether absorption is facilitated by drug transporters. A number of candidate transporters potentially involved in organic cation processing has been identified in the pulmonary epithelium,11 but

inhibition on hOCT1-mediated uptake with IC50 values of 22.3 μM for formoterol fumarate and of 47.8 μM for salmeterol xinafoate. In addition, salbutamol net absorption across bronchiolar epithelial cells was observed, and supporting studies in HEK-293 cells strongly suggested that this process was mediated, at least in part, by OCT1. F

DOI: 10.1021/mp500854e Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics molecular evidence of β2-agonist transport is still elusive. Several reports presented data of, e.g., a concentrationdependent inhibitory effect on the uptake of the organic cation, ASP+, by β2-agonists.10,12 Moreover, a recent study published by Nakanishi et al. provided in vivo evidence of organic cation transporter-mediated tracheal accumulation of the anticholinergic agent ipratropium in mice.24 Here, we showed in A549 and NCI-H441 distal lung epithelial cells that β2-agonists significantly decreased [14C]TEA uptake, whereas no comparable inhibitory effects on [3H]acetylcarnitine uptake was observed. These data strongly suggest that β2-agonists inhibit OCT, but not OCTN2 transporters in the distal lung. These experiments were confirmed in OCT-transfected HEK-293 cells, revealing that hOCT1 was the main target for β2-agonists, followed by hOCT3, while hOCT2 was only marginally affected. OCT2, however, appears to be absent in human bronchial epithelial cells such as Calu-3.12,25 In Calu-3 cells, β2-agonists inhibited both pathways, OCT and OCTN2, in a comparable manner. However, the inhibition kinetics showed that formoterol noncompetitively inhibited the uptake process of [3H]acetylcarnitine, as both Km and Vmax values were decreased in the presence of the β2-agonist by approximately 30%. When investigating the uptake kinetics of [14C]-TEA into A549 cells in the absence and presence of formoterol, salbutamol, and salmeterol, we found competitive inhibition. The Ki values of salmeterol (17 μM), formoterol (92 μM), and salbutamol (277 μM) were about four to 11 times lower than the Km value of TEA itself. The inhibitory potential of the β2agonists in the organotypic cell models followed the compounds’ lipophilicity, an observation that was different from what was found in the heterologous expression systems, where IC50 values were found to be rather in line with the compounds’ binding affinity for the β2-adrenoceptor.26 On the basis of these findings, it can be speculated that the binding site for β2-agonists is located at the same transporter shared with TEA. Organic cation transporters have previously been identified to be polyspecific and bidirectional.27−29 They have a large binding pocket containing partially overlapping binding domains for different substrates.27,30 Prior to the work presented here, it was shown by Dr. Tamai’s laboratory that OCTN1-HEK-293 and OCTN2-HEK293 were both involved in the transport of ipratropium.31 Recently, data from the same group further revealed that the tracheal accumulation of iptratropium in vivo was inhibited by both carnitine and MPP+ and hence suggested an involvement of OCTN2 and OCT2 in the process.24 Direct proof for this hypothesis, however, is still outstanding. Active transport mechanisms of inhaled medicines such as salbutamol have been first discussed in 2005 based on the discovery of a net absorption of the drug across monolayers of bronchial epithelial cells.9 Ever since, the identification of the transporter for β2receptor-agonist translocation was attempted. In Calu-3 cells, a net absorptive direction of salbutamol transport was shown by Mamlouk and colleagues, which was inhibited in the presence of TEA.15 Salbutamol uptake was investigated in bronchial smooth muscle cells by Chiappori et al.14 They discussed a transporter-independent uptake mechanism for salbutamol. In line with these findings, Umwallah and co-workers reported that salbutamol was mainly taken up by paracellular mechanisms in airway smooth muscle cells.32 In the same study, however, the cellular uptake of salbutamol was found to be saturable and inhibitable by lysine and histidine. The uptake

and transport of [3H]-formoterol into Calu-3 cells was previously investigated by Mukherjee et al.33 A net secretive direction of formoterol transport was demonstrated across Calu-3 cell monolayers. It was concluded by the authors that the higher proportion of neutral formoterol at pH 7.4 was most likely the reason for this effect. In our work, salbutamol was found to be a substrate for hOCT1 and taken up in a time- and concentration-dependent manner. Decynium-22, a bona f ide OCT substrate/inhibitor, substantially inhibited the uptake of salbutamol. Transport studies across NCI-H441 cells were carried out under airinterface conditions to represent physiologically relevant conditions.34,35 These studies revealed a net absorption of salbutamol, which was affected by the presence of the OCT1modulator, verapamil.36

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CONCLUSIONS Our study revealed that β2-adrenergic agonists interact with organic cation transporters. The polyspecific organic cation transporter, OCT1 (and to a lesser extent OCT3), can be inhibited by inhaled β2-agonists. Moreover, we found that salbutamol uptake into hOCT1-HEK-293 cells and the drug’s net absorptive transport across bronchiolar epithelial cell monolayers was sensitive to OCT1 modulators. This influence of drug transporters on the bronchodilator passage across the respiratory epithelium to their target receptors raises the question whether drug−drug interactions and genetic variants involving organic cation transporters might have an influence on the clinical outcomes of β2-adrenergic agonist therapy.

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AUTHOR INFORMATION

Corresponding Author

*School of Pharmacy and Pharmaceutical Sciences, Trinity College Dublin, Panoz Institute, Dublin 2, Ireland. Tel: +353-1896-2441. Fax: +353-1-896-2810. E-mail: [email protected]. Present Address ⊥

(J.J.S.) Department of Translational Pulmonology, Translational Lung Research Center Heidelberg (TLCR), Member of the German Center for Lung Research (DZL), University of Heidelberg, 69120 Heidelberg, Germany.

Author Contributions

J.J.S., C.E., and K.I.H. designed the experiments. J.J.S., J.C.G., S.P., and A.K. performed the experiments. J.J.S., C.E., and Y.H. analyzed the data. J.J.S. and C.E. wrote the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work has been funded in part by a Strategic Research Cluster grant (07/SRC/B1154) under the National Development Plan cofunded by EU Structural Funds and SFI. J.C.G. is the recipient of a TOP Stipendium Ausland (Bundesland Niederösterreich).

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ABBREVIATIONS ASP , 4-[4-(dimethylamino)styryl]-1-methylpyridinium iodide; DEC-22, decynium-22:1,1-diethyl-2,2-cyanine; FBS, fetal bovine serum; Ki, inhibitor constant; Km, Michaelis−Menten constant; KRB, Krebs−Ringer buffer; MPP+, 1-methyl-4phenylpyridinium; OCT, organic cation transporter; OCTN, novel organic cation transporter; PBS, phosphate-buffered G

+

DOI: 10.1021/mp500854e Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics

competition for organic cation/carnitine transporters. Pulm. Pharmacol. Ther. 2012, 25, 124−134. (17) Chen, L.; Pawlikowski, B.; Schlessinger, A.; More, S. S.; Stryke, D.; Johns, S. J.; Portman, M. A.; Chen, E.; Ferrin, T. E.; Sali, A.; Giacomini, K. M. Role of organic cation transporter 3 (SLC22A3) and its missense variants in the pharmacologic action of metformin. Pharmacogenet. Genomics 2010, 20, 687−699. (18) Herraez, E.; Lozano, E.; Macias, R. I.; Vaquero, J.; Bujanda, L.; Banales, J. M.; Marin, J. J.; Briz, O. Expression of SLC22A1 variants may affect the response of hepatocellular carcinoma and cholangiocarcinoma to sorafenib. Hepatology 2013, 58, 1065−1073. (19) Iwata, K.; Aizawa, K.; Kamitsu, S.; Jingami, S.; Fukunaga, E.; Yoshida, M.; Yoshimura, M.; Hamada, A.; Saito, H. Effects of genetic variants in SLC22A2 organic cation transporter 2 and SLC47A1 multidrug and toxin extrusion 1 transporter on cisplatin-induced adverse events. Clin. Exp. Nephrol. 2012, 16, 843−851. (20) Morgan, D. J. Clinical pharmacokinetics of beta-agonists. Clin. Pharmacokinet. 1990, 18, 270−294. (21) Cheng, Y.; Prusoff, W. H. Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50% inhibition (I50) of an enzymatic reaction. Biochem. Pharmacol. 1973, 22, 3099−3108. (22) Krohn, K. A.; Link, J. M. Interpreting enzyme and receptor kinetics: keeping it simple, but not too simple. Nucl. Med. Biol. 2003, 30, 819−826. (23) Tan, C. D.; Selvanathar, I. A.; Baines, D. L. Cleavage of endogenous γENaC and elevated abundance of αENaC are associated with increased Na+ transport in response to apical fluid volume expansion in human H441 airway epithelial cells. Pflugers. Arch. 2011, 462, 431−441. (24) Nakanishi, T.; Hasegawa, Y.; Haruta, T.; Wakayama, T.; Tamai, I. In vivo evidence of organic cation transporter-mediated tracheal accumulation of the anticholinergic agent ipratropium in mice. J. Pharm. Sci. 2013, 102, 3373−3381. (25) Macdonald, C.; Shao, D.; Oli, A.; Agu, R. U. Characterization of Calu-3 cell monolayers as a model of bronchial epithelial transport: organic cation interaction studies. J. Drug Targeting 2013, 21, 97−106. (26) Anderson, G. P.; Lindén, A.; Rabe, K. F. Why are long-acting beta-adrenoceptor agonists long-acting? Eur. Respir. J. 1994, 7, 569− 578. (27) Ciarimboli, G. Organic cation transporters. Xenobiotica 2010, 38, 936−971. (28) Koepsell, H.; Lips, K.; Volk, C. Polyspecific organic cation transporters: structure, function, physiological roles, and biopharmaceutical implications. Pharm. Res. 2007, 24, 1227−1251. (29) 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. Handb. Exp. Pharmacol. 2011, 201, 105−167. (30) Biermann, J.; Lang, D.; Gorboulev, V.; Koepsell, H.; Sindic, A.; Schröter, R.; Zvirbliene, A.; Pavenstädt, H.; Schlatter, E.; Ciarimboli, G. Characterization of regulatory mechanisms and states of human organic cation transporter 2. Am. J. Physiol. Cell. Physiol. 2006, 290, C1521−C1531. (31) Nakamura, T.; Nakanishi, T.; Haruta, T.; Shirasaka, Y.; Keogh, J. P.; Tamai, I. Transport of ipratropium, an anti-chronic obstructive pulmonary disease drug, is mediated by organic cation/carnitine transporters in human bronchial epithelial cells: implications for carrier-mediated pulmonary absorption. Mol. Pharmaceutics 2010, 7, 187−195. (32) Unwalla, H. J.; Horvath, G.; Roth, F. D.; Conner, G. E.; Salathe, M. Albuterol modulates its own transepithelial flux via changes in paracellular permeability. Am. J. Respir. Cell Mol. Biol. 2012, 46, 551− 558. (33) Mukherjee, M.; Pritchard, D. I.; Bosquillon, C. Evaluation of airinterfaced Calu-3 cell layers for investigation of inhaled drug interactions with organic cation transporters in vitro. Int. J. Pharm. 2012, 426, 7−14.

saline; TEA, tetraethylammonium; TEER, transepithelial electrical resistance

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DOI: 10.1021/mp500854e Mol. Pharmaceutics XXXX, XXX, XXX−XXX