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Activity of Multidrug Resistance-associated Proteins 1 – 5 (MRP1 – 5) in the RPMI 2650 Cell Line and Explants of Human Nasal Turbinate Anne M. Dolberg, and Stephan Reichl Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00838 • Publication Date (Web): 14 Mar 2017 Downloaded from http://pubs.acs.org on March 19, 2017

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Molecular Pharmaceutics

Activity of Multidrug Resistance-associated Proteins 1 – 5 (MRP1 – 5) in the RPMI 2650 Cell Line and Explants of Human Nasal Turbinate Anne M. Dolberg 1, Stephan Reichl 1, 2 * 1

Institut

für

Pharmazeutische

Technologie,

Technische

Universität

Braunschweig,

Braunschweig, Germany 2

Zentrum für Pharmaverfahrenstechnik, Technische Universität Braunschweig, Braunschweig,

Germany

KEYWORDS human nasal mucosa, RPMI 2650 cells, in vitro model, drug delivery, active efflux, ABC transporters

ACS Paragon Plus Environment

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ABSTRACT

The profound influence of ATP-binding cassette (ABC) transporters on the disposition of numerous drugs has led to increased interest in characterizing their expression profiles in various epithelial and endothelial barriers. The present work examined the presence and functional activity of five ABC efflux proteins, i.e. MRP 1 – 5, in freshly isolated human nasal epithelial cells and two in vitro models based on the human RPMI 2650 cell line. To evaluate the expression patterns of MRP1, MRP2, MRP3, MRP4 and MRP5 at the mRNA and protein levels in the ex vivo model and the differently cultured RPMI 2650 cells, reverse transcriptase polymerase chain reaction (RT-PCR), Western blot analysis and indirect immunofluorescence staining were used. The functionality of the MRP transporters in the three models was assessed using efflux experiments and accumulation assays with the respective substrates and inhibitors. The mRNA and protein expression of all selected ABC transporters was detected in excised human nasal mucosa as well as in the corresponding cell culture models. Moreover, the functional expression of the MRP transport proteins was demonstrated in the three models for the first time. Therefore, the potential impact of multidrug resistance-associated proteins 1 – 5 on drug disposition after intranasal administration may be taken into consideration for future developments. The specimens of human nasal turbinate exhibited slightly lower efflux capacities of MRP1, MRP3 and MRP5 in relation to the submerged and ALI-cultured RPMI 2650 cells, but showed a promising comparability to the both in vitro models concerning the activity of MRP2 and MRP4. In this regard, the different RPMI 2650 cell culture models will be able to provide useful experimental data in the preclinical phase to estimate the interaction of particular efflux transporters with drug candidates for nasal application.


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INTRODUCTION In addition to the local treatment of allergic or infectious diseases, the nasal mucosa is also an interesting site for the administration of drugs with systemic action, as it has a number of advantages over other routes of drug delivery, such as peroral and parenteral. The easy accessibility of the nasal cavity allows the possibility of essentially painless self-medication as well as non-invasive drug application to special patient groups such as children, disabled people and elderly individuals. The nasal epithelium is a leaky barrier compared to other mucosal tissues and provides a relatively large surface area (approximately 150 cm2), which is additionally magnified by numerous microvilli and cilia 1. Together with the highly vascularized submucosa, it offers direct blood transportation into the systemic circulation and therefore a rapid onset of therapeutic effects. Because of these characteristics, the nasal mucosa is an ideal application site for opioids such as fentanyl (Instanyl®, PecFent®) to relieve breakthrough pain 2, antiemetic drugs [e.g., metoclopramide (Pramidin®) and ondansetron] to prevent nausea and emesis 3, and triptans (AscoTop®, Imigran®, Zomig®) to treat migraine and cluster headaches 4. Intranasal administration circumvents gastrointestinal degradation and is therefore suitable for the delivery of therapeutic peptides like buserelin (Profact®, Suprecur®), calcitonin (Fortical®, Miacalcin®), oxytocin (Syntocinon®) and desmopressin (Minirin®, Nocutil®, Stimate®). Moreover, the avoidance of hepatic first-pass metabolism is an advantage for pharmaceutical actives such as midazolam. Due to relatively low enzymatic activity

5

and the presence of

sufficient immunocompetent cells, the respiratory epithelium is also attractive for immunization by nasally administered vaccines (Fluenz®, FluMist®)

6,7

. Current investigations focus on the

suitability of other medical compounds for nasal delivery, including diazepam (Plumiaz®),


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human growth hormone and naloxone (Narcan®), as well as the possibility of direct entrance to the central nervous system via the olfactory epithelium 8. Another field of research of rapidly growing interest is that of drug disposition by transporter proteins belonging to the solute carrier (SLC) and the ATP binding cassette (ABC) superfamilies. SLC proteins represent the largest group of membrane-bound transporters found in nearly every cellular membrane and are mainly responsible for the cellular uptake of endogenous and exogenous substrates 9. The expression of ABC transporters, i.e. the most prominent representative of subfamily B, P-glycoprotein (P-gp; synonym: MDR1; gene symbol: ABCB1), as well as certain members of subfamily C, also known as multidrug resistanceassociated proteins (MRPs), is widespread in mammalian tissues and provides for the efflux of numerous organic molecules

10,11

. In comparison with other epithelial and endothelial barriers

(such as the intestine, liver, lung and brain), only limited data concerning the presence of these ATP-powered pumps in the human nasal mucosa have been published. However, the available studies indicate the existence of a broad range of transporters in the upper respiratory system that may appreciably affect the absorption and distribution of intranasally administered drugs

12,13

.

Furthermore, many potential drug candidates for nasal application and their metabolic products are recognized as substrates by various ABC transporters or act as inhibitors of these efflux proteins (Table 1).


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Table 1. Potential drug candidates and their metabolites, respectively, for intranasal application, which were identified as either substrates or inhibitors of different ABC transport proteins. drug candidate/ metabolite

substrate or inhibitor of ABC transporters

studies for intranasal delivery/ drug product

estradiol-17β-glucuronide

substrate of MRP2, MRP3 14–16 and MRP4 17

in vivo (humans) 18; Aerodiol®

substrate of MRP2 and MRP3

in vivo (rats) 20

fexofenadine morphine-3-glucuronide

15,19

substrate of MRP2 and MRP3

in vivo (humans) 2,22; Rylomine®

21

propranolol

substrate of MRP2 23

in vivo (humans) 24 and in vitro (RPMI 2650 cells) 25

resveratrol

substrate of MRP2 and MRP3 26

in vitro (RPMI 2650 cells) 27

sildenafil citrate

inhibitor of MRP4 and MRP5 28

in vivo (rabbits and rats) 29,30

Recently, we determined the molecular and functional expression of P-gp in different models of the human nasal mucosa for drug absorption studies and found a similar expression pattern in the ex vivo and in vitro conditions, substantiating a potential influence on drug disposition

31

. The

excised specimens of human nasal turbinate provide the most physiological approach to the situation in vivo, but the poor availability as well as high inter- and intra-individual differences restrict their usage in high-throughput experiments

32

. The in vitro models are based on

continuously growing RPMI 2650 cells using the improved culture conditions reported previously by our laboratory

32,33

. Despite some limitations due to the tumorous source

34

, the

RPMI 2650 cell line represents nearly physiological properties of the nasal respiratory epithelium and offers diverse advantages over animal studies and primary cultured human nasal


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epithelial (HNE) cells 35. In the present study, we examined the expression and activity of MRP1, MRP2, MRP3, MRP4 and MRP5 (gene symbols: ABCC1, ABCC2, ABCC3, ABCC4 and ABCC5, respectively) in explants of human nasal mucosa as well as differently cultured RPMI 2650 cells to evaluate the influence of these efflux pumps concerning intranasal drug delivery. For this purpose, MRP expression profiles were explored at the mRNA and protein levels using reverse transcriptase polymerase chain reaction (RT-PCR), Western blot analysis and indirect immunofluorescence staining. The functional activity of the selected ABC transporter was investigated by performing efflux experiments and accumulation assays with appropriate substrates and inhibitors. MATERIAL AND METHODS MATERIALS ThinCert™ inserts (with polyethylene terephthalate membrane, 1.13 cm² growth area and 1.0 µm pore size) were obtained from Greiner Bio-One (Frickenhausen, Germany). Polystyrene tissue culture test plates (with 24 wells and 1.86 cm2 growth surface) and cell scrapers were purchased from TPP (Trasadingen, Switzerland). Polystyrene tissue culture flasks were purchased from Sarstedt (Nümbrecht, Germany), and PCR mycoplasma test kit I/C was obtained from PromoCell (Heidelberg, Germany). Acrylamide/bis-acrylamide solution, ammonium persulfate (APS), bovine serum albumin (BSA), calcium chloride dihydrate (CaCl2 · 2H2O), 5(6)-carboxy-2’,7’-dichlorofluorescein diacetate (CDCFDA), dithiaoctanoic

5-(3-(2-(7-chloroquinolin-2-yl)ethenyl)phenyl)-8-dimethylcarbamyl-4,6acid

(MK571),

4',6-diamidino-2-phenylindole

dihydrochloride

(DAPI),

dithiothreitol (DTT), ethylenediaminetetraacetic acid (EDTA), ethidium bromide, glycine,


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indomethacin, 2x sample Laemmli buffer, probenecid, protease inhibitor cocktail (Cat. No. P8340), skim milk powder (Cat. No. 70166), sodium bicarbonate (NaHCO3), sodium deoxycholate (C24H39NaO4), tetramethylethylenediamine (TEMED), Tris base, Tris-HCl, Triton X-100, Tween® 20, 4-nonylphenyl-polyethylene glycol (Nonidet™ P40), verapamil, monoclonal anti-actin antibody produced in mouse (clone AC-40; Cat. No. A3853), anti-mouse, anti-rabbit and anti-rat IgG-fluorescein isothiocyanate (FITC) antibodies all produced in goat (Cat. No. F9137, F9887 and F6258, respectively), monoclonal anti-MRP3 antibody produced in mouse (clone M3II-21; Cat. No. M6567) for indirect immunofluorescence staining, and polyclonal antiMRP3 antibody produced in rabbit (Cat. No. M0318) for Western blotting were purchased from Sigma (Deisenhofen, Germany). Fetal bovine serum (FBS), L-glutamine, minimum essential medium (MEM), non-essential amino acids (NEAA) and phosphate buffered saline (PBS) were obtained from Biochrom (Berlin, Germany). Amphotericin B, penicillin G sodium salt, streptomycin sulfate and trypsinEDTA were purchased from GE Healthcare (Freiburg, Germany). Sodium dodecyl sulfate (SDS), sodium chloride (NaCl), 4-(2-hydroxyethyl)piperazine-1ethanesulfonic acid (HEPES), and ᴅ-glucose monohydrate were obtained from Roth (Karlsruhe, Germany). EDTA disodium salt solution was obtained from MP Biomedicals (Solon, OH, USA), and sodium dihydrogen phosphate monohydrate (NaH2PO4 · H2O) was purchased from Merck (Darmstadt, Germany). Magnesium sulfate heptahydrate (MgSO4 · 7H2O) and potassium chloride (KCl) were obtained from Acros Organics (Geel, Belgium). Polyclonal anti-MRP1 antibody produced in rabbit (Cat. No. ab99531) for Western blotting and for indirect immunostaining, monoclonal anti-MRP2 antibody produced in mouse (clone M2III-


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6; Cat. No. ab3373) for indirect immunostaining, monoclonal anti-MRP2 antibody produced in rabbit (clone EPR10998; Cat. No. ab172630) for Western blotting, monoclonal anti-MRP4 antibody produced in rat (clone M4I-10; Cat. No. ab15602) for Western blotting and for indirect immunostaining, monoclonal anti-MRP5 antibody produced in rat (clone M5II-54; Cat. No. ab24107) for immunofluorescence staining, and polyclonal anti-MRP5 antibody produced in rabbit (Cat. No. ab180724) for Western blotting were purchased from Abcam (Cambridge, UK). TRIzol® reagent was obtained from Invitrogen (Karlsruhe, Germany), and universal agarose was purchased from Peqlab (Erlangen, Germany). Diethylpyrocarbonate (DEPC)-treated water, DreamTaq™ DNA polymerase and buffer, GeneRuler™ low range DNA ladder (25 – 700 bp), RevertAid™ first strand cDNA synthesis kit, chamber slides, Neg-50™ frozen section medium, Superfrost™ Ultra Plus adhesion slides, and Spectra™ multicolor broad range protein ladder (10 – 260 kDa) were obtained from Thermo Scientific (St. Leon-Rot, Germany). Scintillation liquid Optiphase Supermix® was purchased from Perkin Elmer (Waltham, MA, USA), and [3H]erythromycin and [3H]adefovir dipivoxil were obtained from Biotrend (Cologne, Germany). Horseradish peroxidase-conjugated anti-mouse, anti-rabbit and anti-rat antibodies, all produced in goat (Cat. No. DC02L, DC03L and DC01L, respectively), as well as polyvinylidene fluoride (PVDF) blotting membranes (0.45-µm pore size) were purchased from Millipore (Schwalbach, Germany). DAKO REAL™ antibody diluent was obtained from DAKO (Hamburg, Germany), and Lumi-LightPLUS Western blotting substrate was purchased from Roche (Mannheim, Germany). All other chemicals used were analytical grade.


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The Krebs-Ringer Buffer (KRB) was used for functionality assays and contained the following compounds in 1,000 mL of double-distilled water: 6.8 g NaCl, 3.575 g HEPES, 2.1 g NaHCO3, 1.1 g ᴅ-glucose monohydrate, 0.4 g KCl, 0.26 g CaCl2 · 2H2O, 0.2 g MgSO4 · 7H2O and 0.14 g NaH2PO4 · H2O. If necessary, the pH of the KRB was adjusted to 7.4 and the osmolarity to 275 mosmol. All other buffers and solutions were formulated using double-distilled water.


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METHODS Cell Culture The Caco-2 and RPMI 2650 cell lines were purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany, Cat. No. ACC 169 and Cat. No. ACC 287, respectively) and were routinely tested for the absence of mycoplasma infection using the PCR mycoplasma test kit I/C. Cultures used for experiments included passages between 11 and 80 for Caco-2 cells and between 6 and 52 for RPMI 2650 cells. Caco-2 cell line Caco-2 cells were derived from a human epithelial colon adenocarcinoma 36 and are widely used as an in vitro model to predict intestinal drug absorption

37

. In the present work, Caco-2 cells

were utilized as positive controls for all imaging techniques (RT-PCR, Western blot and immunohistochemical staining), as the selected MRPs have been determined for this cell line 38. The activity of the various efflux pumps in Caco-2 cells using appropriate transport experiments was also demonstrated in our previous studies 39,40. The cells were maintained in culture medium consisting of MEM, 20% FBS, 1% NEAA, 100 U/mL penicillin G sodium salt, 100 µg/mL streptomycin sulfate and 0.25 µg/mL amphotericin B. Caco-2 cells were routinely seeded in 25 cm2 tissue culture flasks at a density of 5,000 cells per cm2 and cultivated at 37 °C in a humidified atmosphere containing 5% CO2 for 7 d with medium changes three times per week. RPMI 2650 cell line RPMI 2650 cells isolated from a squamous cell carcinoma of the nasal septum in 1962 are the only commercially available cells simulating the human nasal epithelium

41

. The RPMI 2650


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cells were routinely maintained in polystyrene tissue culture flasks (growth surface: 25 cm2) at 37 °C in a humidified atmosphere containing 5% CO2 for 7 d with three medium changes during the cultivation period. The standard culture medium contained MEM, 10% FBS, 1% Lglutamine, 1% NEAA, 100 U/mL penicillin G sodium salt, 100 µg/mL streptomycin sulfate and 0.25 µg/mL amphotericin B. After 7 d, the cells were washed with EDTA solution and detached from the surface by treating with trypsin-EDTA at 37 °C for 6 min. All cells were collected and counted using a Z2 Coulter Counter (Beckman Coulter, Krefeld, Germany). Subsequently, the cells were resuspended and seeded at a density of 45,000 cells per cm2 in a new tissue culture flask. Liquid-covered culture (LCC) of RPMI 2650 cells For the first in vitro model of human nasal mucosa, RPMI 2650 cells were seeded onto 24-well tissue culture test plates at a density of 100,000 cells per cm2 31. This model was grown as liquidcovered culture for 7 d (subsequently called the LCC model), and the culture medium was replaced three times during the cultivation period. The growth status of every well was examined with an inverted photomicroscope IX50 (Olympus Europe, Hamburg, Germany), and only wells showing approximately 100% confluency were used for transport experiments. RPMI 2650 cells cultured at air-liquid interface (ALI) For the second in vitro model of human nasal mucosa, RPMI 2650 cells were seeded on permeable filter inserts with polyethylene terephthalate (PET) membranes (ThinCert™)

33

characterized by a pore size of 1.0 µm and pore density of 2 · 106 per cm2. As previously described

31

, a seeding density of 200,000 cells per cm2 was used, and RPMI 2650 cells were

submerged and cultivated for 2 days. Afterwards, the cultures were raised to the air-liquid


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interface to facilitate the formation of tight junction proteins 32. Following cultivation at the ALI for an additional 19 days with medium changes three times per week, the model was used for experiments (hereinafter called the ALI model). Nasal Tissue Explants of healthy human nasal mucosa were received from turbinectomy surgeries according to ethical regulations. All experiments included smoking or non-smoking female and male donors aged between 21 and 68 years. The specimens were placed into MEM supplemented with 10% FBS, 1% L-glutamine, 1% NEAA and 1% antibiotic/antimycotic solution immediately after removal and used for experiments within two hours. For efflux and accumulation assays, the excised mucosa was clamped in vertical Ussing diffusion chambers (Harvard Apparatus, Holliston, MA, USA) with an exposed tissue area of 0.13 cm2. TEER Measurement The transepithelial electrical resistance (TEER) was determined before and after each transport experiment to ensure the epithelial integrity of the in vitro and ex vivo models. For RPMI 2650 cells grown on ThinCert™ filter inserts, the EVOM® epithelial volt/ohm meter with EndOhm® chamber (World Precision Instruments, Sarasota, FL, USA) was used. Values were corrected for the background caused by the blank filter and subsequently calculated corresponding to the surface area of ThinCert™ filter inserts (1.13 cm2). For specimens of human nasal mucosa, the TEER was measured using the EVOM® with silver/silver chloride electrodes (both World Precision Instruments, Sarasota, FL, USA). Values were calculated corresponding to the surface area of Ussing diffusion chambers (0.13 cm2). TEER values below 50 Ω·cm² implied either


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damage from pipette tips during the cultivation period or from scalpels through surgical resection; samples with these values were not used for further experiments. Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) The mRNA expression of the selected ABC transporters in the different models of nasal epithelium was investigated utilizing reverse transcriptase polymerase chain reaction (RT-PCR) as previously described

40

. Primers for human multidrug resistance associated proteins 1 – 5

(GenBank accession numbers NM_004996 for MRP1, NM_000392 for MRP2, NM 003786.3 for MRP3, NM_005845 for MRP4 and NM_005688 for MRP5) were custom-made using the OligoPerfect™ Designer (Invitrogen, Carlsbad, CA, USA), and specificity was controlled with the NucleotideBLAST® program (National Center for Biotechnology Information, Bethesda, MD, USA). The extraction of total RNA was performed using TRIzol® Reagent and processed in accordance with the manufacturer’s protocol. Due to the tissue strength of the excised human nasal mucosa, a prehomogenization step was required prior to RNA isolation. This step was carried out with a MM301 Ball Mill (Retsch, Haan, Germany) at 30 Hz for 10 min using glass beads of approximately 0.50 – 0.75 mm in diameter. The quality and quantity of all RNA samples was determined spectrophotometrically using a Spekol 1300 UV spectrometer (Analytik Jena, Jena, Germany). To convert 5 µg RNA into cDNA, the RevertAid™ first strand cDNA synthesis kit and a Labcycler thermocycler (SensoQuest, Göttingen, Germany) were used. A three-step PCR protocol using DreamTaq™ DNA polymerase and buffer was performed after the reverse transcription. Each PCR run consisted of 30 cycles, using different primers and temperatures for the annealing phase depending on the transporter (Table 2).


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Table 2. Sequences of forward (F) and reverse (R) primers, annealing temperature and resulting RT-PCR product size for the different MRPs. ABC transporter

PCR primers

annealing temperature

RT-PCR product size

59.1 °C

183 bp

59.1 °C

211 bp

61.1 °C

187 bp

53.0 °C

238 bp

63.3 °C

529 bp

F 5’ – AGG TGG ACC TGT TTC GTG AC – 3’ MRP1 R 5’ – ACC CTG TGA TCC ACC AGA AGG – 3’ F 5’ – TGC TTC CTG GGG ATA ATC AGC – 3’ MRP2 R 5’ – CAC GGA TAA CTG GCA AAC CTG – 3’ F 5’ – GGC GTC TAT GCT GCT TTA GG – 3’ MRP3 R 5’ – CCT TGG AGA AGC AGT TCA GG – 3’ F 5’ – CCA TTG AAG ATC TTC CTG G – 3’ MRP4 R 5’ – GGT GTT CAA TCT GTG TGC – 3’ F 5’ – CTT CCC GTG GTT CCT TGT GG – 3’ MRP5 R 5’ – GTC AGG GGA GGG AGC CTT GT – 3’

The program started with an initial phase at 94 °C for 5 min, followed by a denaturation phase at 94 °C for 30 sec. After an annealing phase at the primer-specific temperature for 30 sec, a final elongation period at 72 °C for 1 min was performed. Each run included a negative control (DEPC-treated water instead of mRNA) and a positive control (mRNA of Caco-2 cells). The PCR products were separated via 2% agarose gel electrophoresis in a PerfectBlue Mini S gel system (Peqlab, Erlangen, Germany) and visualized with ethidium bromide staining under UV light using the AlphaImager® 1220 gel documentation system (Alpha Innotech, San Leandro, CA, USA). A GeneRuler™ low range DNA ladder (25 – 700 bp) was loaded for the sizing of the resulting PCR products.


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Western Blot Analysis For protein extraction, Caco-2 cells and RPMI 2650 cells were collected from culture flasks and filter inserts, respectively, using a cell scraper, and excised human nasal tissue was cut into small pieces of 1 mm2 in size. The samples were washed twice with ice-cold PBS and then centrifuged using an Allegra™ 64R centrifuge (Beckman Coulter, Brea, CA, USA) at 2,000 x g and 4 °C for 5 min. The cell pellets and mucosa slices were resuspended in a buffer consisting of 150 mM sodium chloride, 50 mM Tris-HCl, 1% Nonidet™ P40, 0.5% sodium deoxycholate and 0.1% SDS in double-distilled water and additional protease inhibitor cocktail. Prior to the incubation phase, the specimens of nasal tissue were sonicated with the ultrasonic disintegrator Soniprep 150 (MSE, London, UK) at 10 Hz for 8 cycles (10 sec on/ 20 sec off). All samples were incubated at 4 °C for 30 min with constant mixing and centrifuged at 4 °C and 16,000 x g for 15 min. The supernatants containing the whole protein fraction were collected and stored at -80 °C 42

until further examination. To quantify the protein content, the Bradford colorimetric method

using a microplate spectrophotometer PowerWave™ XS (BioTek Instruments, Bad Friedrichshall, Germany) was applied. The protein solutions were mixed with 2x Laemmli sample buffer consisting of 4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.004% bromophenol blue and 0.125 M Tris-HCl in a ratio of 1:1 and equal protein amounts of 90 µg were loaded on sodium dodecyl sulfate polyacrylamide gels containing 10% acrylamide. Protein separation was carried out by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in accordance with the method of Laemmli

43

using a Mini-PROTEAN® Tetra Cell unit (Bio-Rad, Munich, Germany). Following the 1-D vertical electrophoresis, the SDS-PA gels were equilibrated in Towbin transfer buffer consisting of 192 mM glycine and 25 mM Tris base in double-distilled water. The separated protein


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samples were transferred onto PVDF blotting membranes using a tank blot system (PerfectBlue tank electro blotter, Peqlab, Erlangen, Germany). Afterwards, the PVDF membranes were blocked with MPBST buffer containing 5% skim milk powder and 0.05% Tween® 20 in PBS for 1 h at room temperature. The primary antibodies were diluted in MPBST buffer (1:2,000 for βactin, 1:200 for MRP1, 1:1,000 for MRP2, 1:200 for MRP3, 1:50 for MRP4 and 1:500 for MRP5) and incubated overnight at 4 °C. The membranes were washed three times in PBST and incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (diluted at a ratio of 1:5,000 with MPBST) for 1 h at room temperature. Peroxidase activity was visualized by chemiluminescence detection using LumiLightPLUS Western blotting substrate under a ChemiLux imager (INTAS, Göttingen, Germany). Indirect Immunohistochemistry For immunostaining, Caco-2 cells and the LCC model were cultivated on chamber slides. The ALI model was routinely cultivated, followed by cutting the filter membrane out of the insert. Excised nasal tissue was embedded in a water-soluble frozen section medium Neg-50™, and frozen specimens of 15 µm were cut utilizing a cryostat microtome HM550 (Thermo Scientific, St. Leon-Rot, Germany). The mucosa slices were placed on Superfrost™ Ultra Plus adhesion slides and air-dried. Prior to antibody staining, the samples were fixed with ice-cold methanol for 10 min and blocked with 2% BSA in PBS for 60 min at room temperature. The primary antibodies were diluted with DAKO REAL™ antibody diluent (1:50 for MRP1, 1:50 for MRP2, 1:30 for MRP3, 1:20 for MRP4 and 1:30 for MRP5) and incubated overnight at 4 °C. Following three washing steps with PBS, the specimens were incubated with the appropriate FITCconjugated secondary antibody (diluted at a ratio of 1:100 in DAKO REAL™ antibody diluent) at room temperature for 1 h. The cell nuclei were counterstained by incubation with DAPI (300


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nM in DAKO REAL™ antibody diluent) at room temperature for 10 min, followed by washing with PBS. Negative controls were prepared using the same procedure but excluding the primary antibodies. Finally, the slides were examined using a confocal laser scanning microscope (CLSM) A1R+ and NIS-Elements imaging software (both Nikon Europe, Kingston, UK). The settings were adjusted so that no FITC signal was observed in the negative control samples (images not shown). Transport Experiments Prior to efflux and accumulation studies, all samples were equilibrated in KRB, pH 7.4, at 37 °C for 1 h and the following assays were also performed at 37 °C. With RPMI 2650 cells, the experiments were performed either in 24-well plates (LCC model) or on ThinCert™ filter inserts (ALI model), and both models were agitated constantly on an orbital shaker. For the explants of human nasal mucosa mounted in a vertical Ussing diffusion system, the donor and acceptor solutions were mixed continuously with carbogen gas. Efflux of 5(6)-Carboxy-2’,7’-Dichlorofluorescein (CDCF) To detect the functional expression of MRP1, MRP3 and MRP5 transporters, an efflux assay was performed using CDCFDA (40 µM) as a substrate and probenecid (10 mM) as a non-specific MRP inhibitor. CDCFDA, a diacetate ester of CDCF, permeates passively through intact cell membranes and then is hydrolyzed by intracellular esterases. The produced CDCF is polar and thus unable to cross the cell membranes by passive diffusion, but cellular efflux can be enabled via active transport mediated by MRP1, MRP3 or MRP5. The addition of probenecid inhibits the function of the ABC proteins and thereby disables the transport of CDCF out of the cells. A CDCFDA solution was added to the apical side of epithelial layers (1,000 µL for the LCC


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model, 500 µL for the ALI model and 2,000 µL for the ex vivo model), followed by incubation for 60 min. Afterwards, the samples were rinsed twice with ice-cold KRB to remove the residue of unpenetrated marker. Defined volumes of tempered KRB with or without probenecid were added to the cell culture models and excised tissues. For the LCC model, 1,000 µL was given on the apical side of epithelial cells, whereas for the ALI model, the apical volume was 500 µL and the basolateral compartment contained 1,500 µL. In the Ussing chambers, both the apical and basolateral compartment contained 3,000 µL. Sample aliquots (200 µL) were withdrawn at 30 and 90 min from the apical compartments, and replaced with the same volume of tempered KRB with or without probenecid. The amounts of emitted CDCF were determined using the GENiosBasic fluorescence microplate reader (Tecan, Männedorf, Switzerland) with a 485 nm excitation filter and 535 nm emission filter. The efflux efficiency was evaluated by dividing the transported substrate amount without inhibitor from the substrate amount under the influence of probenecid according to Equation 1. The resulting quotient, subsequently called the efflux ratio (EfR

CDCF),

was calculated for each time point.

EfR CDCF =

effluxed amount w ithout inhibitor effluxed amount w ith probenecid

Equation 1.

Accumulation of Erythromycin An accumulation experiment was implemented using [3H]erythromycin (0.25 µCi/mL) as a substrate and MK571 (100 µM) as an inhibitor to determine the activity of MRP2. After passive diffusion of erythromycin into intact cells, the molecule is actively pumped out in the extracellular space by MRP2 and P-glycoprotein. To prevent the collateral substrate efflux by MDR1, verapamil (100 µM) was added to block P-gp but not MRP2 in all samples. Under the


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influence of both inhibitors, erythromycin is accumulated intracellularly due to the obstruction of the efflux pumps. In these experiments, 500 µL for both in vitro models and 1,000 µL for the ex vivo model of an [3H]erythromycin solution was added to the apical compartments. After an incubation period of 60 min, the samples were rinsed twice with ice-cold KRB to remove any unpermeated substrate. Epithelial cells were lysed using 1% Triton X-100 (2,000 µL for all models) for 16 h with constant shaking. The quantity of [3H]erythromycin in 500-µL aliquots was determined using an LS 6500 liquid scintillation counter (Beckman Coulter, Brea, CA, USA) following the addition of each sample to 2,000 µL of scintillation liquid. To assess the functional activity of MRP2, an accumulation ratio (hereinafter called AcR

erythromycin)

was

calculated in accordance with Equation 2. For each model, the accumulated substrate amount in the presence of both inhibitors was divided by the substrate amount in the presence of only verapamil.

AcR erythromycin =

accumulated amount

w ith verapamil + MK571

accumulated amount

w ith verapamil

Equation 2.

Accumulation of Adefovir Dipivoxil For the investigation of MRP4-mediated efflux, an accumulation assay was carried out using [3H]adefovir dipivoxil (1 µCi/mL for both in vitro models and 0.5 µCi/mL for the ex vivo model) as a substrate and indomethacin (50 µM) as an inhibitor. The prodrug adefovir dipivoxil diffuses passively into intact cells and then is rapidly and completely hydrolyzed to adefovir and to the monoester metabolite. Adefovir is very polar at physiological pH, and cellular efflux can only be mediated by active transport regulated by MRP4. The addition of indomethacin inhibits the functional activity of MRP4 and therefore increases the intracellular accumulation of adefovir.


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The apical side of the epithelial layers was incubated with 500 µL (for both in vitro models) or 1,000 µL (for the ex vivo model) of an [3H]adefovir dipivoxil solution with or without inhibitor for 120 min. Afterwards, the samples were rinsed twice with ice-cold KRB to remove the residual amounts of substrate, followed by cell lysis with 2,000 µL 1% Triton X-100 for 16 h under continuous agitation. The quantity of adefovir present in aliquots of 500 µL was examined by liquid scintillation counting, as described above. The efflux capacity of MRP4 was also evaluated by calculation of an accumulation ratio (consequently called AcR

adefovir).

The

absorbed substrate amount in the presence of indomethacin was divided by the substrate amount in the absence of inhibitor according to Equation 3.

AcR adefovir =

accumulated amount

w ith indomethacin

accumulated amount

w ithout inhibitor

Equation 3.

Statistical Analysis Data for the transport studies are depicted as the mean + standard deviation (SD). Statistical analysis of the results was performed by a two-tailed, two sample F-test followed by a twotailed, two sample T-test using SPSS software 22 (IBM, Armonk, NY, USA). P values less than 0.05 (* p < 0.05, ** p < 0.01, *** p < 0.001) were considered statistically significant.


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RESULTS mRNA Expression The results of the PCR analysis are given in Figures 1A – 1E. Caco-2 cells were used as positive controls and showed distinct positive signals for all of the tested multidrug resistance-associated proteins. Figure 1A clearly demonstrates that MRP1 mRNA expression was observed in all models, with the strongest band was found for the ALI model. The mRNA expression pattern of MRP2 in the different models is depicted in Figure 1B and showed a distinct signal for the ALI model, but only weak bands were observed in the LCC model and the human nasal turbinate. A similar result was found for mRNA expression of MRP3 (Figure 1C), which was detectable in all models with comparable differences in signal strength. For MRP4 and MRP5, we observed a strong similarity in the mRNA expression pattern (Figure 1D and Figure 1E, respectively). Both in vitro models based on the RPMI 2650 cell line resulted in clear bands of the respective transporter. The excised nasal tissue had slightly weaker signals for both efflux proteins. The mRNA of all tested MRPs was present in the different models of human nasal mucosa, and only deviated in terms of signal strength. For the in vitro models, the expression levels seemed to depend on the cell culturing and revealed stronger band intensities for the ALI model compared to the submerged RPMI 2650 cells.


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Figure 1. PCR amplification products of MRP1 (1A; 183 bp), MRP2 (1B; 211 bp), MRP3 (1C; 187 bp), MRP4 (1D; 238 bp) and MRP5 (1E; 529 bp) in Caco-2 cells (lane 2), RPMI 2650 cells from LCC (lane 3) and ALI (lane 4), and excised human nasal mucosa (lane 5). Lane 1 represents the GeneRuler™ low range DNA ladder (25 – 700 bp). Protein Expression Western Blot Analysis The transporter expression of selected MRPs at the protein level was analyzed by Western blotting, and the results are presented in Figure 2. Caco-2 cells used as positive control produced


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the weakest band for MRP1, while the ALI model showed the strongest signal. For RPMI 2650 cells grown as LCC and specimens of nasal mucosa, clear but slightly weaker bands were detected. For MRP2, only faint signals were found in the different in vitro and ex vivo models of the human nasal mucosa. The positive controls showed the strongest band for this transporter by far. The expression analysis of MRP3 resulted in clear bands in both the LCC model and the ALI model. The Caco-2 cells again exhibited the strongest signal, whereas a slightly weaker band was observed for the excised nasal tissue. The MRP4 protein bands appeared distinctly in all tested samples, suggesting high expression levels in the positive control as well as in our epithelial models. Differing from the results of the RT-PCR analysis, the expression pattern generated for MRP5 showed similar weak signals in the Caco-2 cells, the differently cultured RPMI 2650 cells and the explants of human nasal turbinate. This observation may be caused by a low protein level in all samples or by a suboptimal dilution chosen for the polyclonal primary antibody. The effectiveness of protein transfer during Western blotting was demonstrated by clear β-actin bands for every sample.


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Figure 2. Western blot analysis of MRP1 (171 kDa), MRP2 (174 kDa), MRP3 (180 kDa), MRP4 (159 kDa) and MRP5 (161 kDa) in Caco-2 cells (lane 1), RPMI 2650 cells grown as LCC (lane 2) and at ALI (lane 3), as well as excised human nasal mucosa (lane 4). β-actin (42 kDa) served as the loading control and was detected in every sample. Indirect Immunohistochemistry To examine the localization of the different efflux transporters within the used models, indirect immunofluorescence staining was applied. Caco-2 cells again served as positive controls and are included in every figure. The positive signals of FITC-labeled secondary antibodies indicate MRP expression at the protein level (green channel). To ensure the cell orientation, counterstaining with DAPI (red channel) is given in the respective overlay image. Both in vitro models based on the RPMI 2650 cell line and the Caco-2 cells are shown in the top view; the slices of excised nasal mucosa are presented in the lateral view. All tested samples showed distinct fluorescence signals for MRP1 in the cell membranes (Figures 3A – 3F) and on the apical side of the epithelial layer (Figures 3G and 3H), whereas the strongest immunofluorescence was detected for the RPMI 2650 cells grown at ALI (Figures 3E and 3F).


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For MRP2, the specimens of human nasal mucosa exhibited the clearest fluorescence signal in the apical cell membrane (Figures 4G and 4H). The samples of Caco-2 cells and the differently cultured RPMI 2650 cells showed comparatively faint immunofluorescence (Figures 4A – 4F). Only poor signals were found for the positive control (Figures 5A and 5B) and the ALI model (Figures 5E and 5F) for the immunolocalization of MRP3. Immunofluorescence staining of submerged cultured RPMI 2650 cells (Figures 5C and 5D) and human nasal tissue (Figures 5G and 5H) resulted in very strong signals indicating a consistent expression of this transporter. The most distinct fluorescence signal for MRP4 was observed in Caco-2 cells, as expected (Figures 6A and 6B), followed by a clear apical immunolocalization in the explants of human nasal turbinate (Figures 6G and 6H). In addition, positive but weaker signals were detected in the different in vitro models based on the RPMI 2650 cell line (Figures 6C – 6F). For MRP5, the positive control (Figures 7A and 7B), the LCC model (Figures 7C and 7D) and the ex vivo model (Figures 7G and 7H) showed clear fluorescence signals. The RPMI 2650 cells grown at ALI (Figures 7E and 7F) exhibited a slightly diffuse immunofluorescence for this ABC protein.


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Figure 3. Double-stained samples of Caco-2 cells (3A and 3B; top view), RPMI 2650 cells as LCC (3C and 3D; top view) and at ALI (3E and 3F; top view), as well as excised human nasal mucosa (3G and 3H; lateral view) with DAPI (red channel) and FITC (green channel) indicating the membrane expression of MRP1.


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Functional Activity Table 3. Calculated efflux (EfR) and accumulation ratios (AcR) for the activity tests with CDCFDA, [3H]erythromycin and [3H]adefovir dipivoxil in differently cultured RPMI 2650 cells and specimens of human nasal mucosa.

ratios

LCC model

ALI model

nasal mucosa

EfR (after 30 minutes)

2.68

1.89

1.36

EfR (after 90 minutes)

3.60

3.46

1.39

2.09

5.33

1.68

1.96

2.35

1.79

efflux of CDCF

accumulation of erythromycin AcR (after 60 minutes) accumulation of adefovir AcR (after 120 minutes)

MRP1, MRP3 and MRP5 An efflux study was performed using fluorescent CDCF, and the results given by both in vitro models and the ex vivo model of the human nasal mucosa are presented in Figure 8. Following the uptake and intracellular conversion of nonpolar CDCFDA, the hydrolyzed form is actively transported out of the cells via ABC transporter proteins, in particular MRP1, MRP3 and MRP5. In the presence of the inhibitor probenecid, substrate efflux was blocked and the calculation of the respective efflux ratios (EfR CDCF) yielded values higher than 1, as shown in Table 3. For the submerged cultured RPMI 2650 cells, the mean amounts of CDCF detected in the absence of


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probenecid were significantly higher (p < 0.001) than those in the presence of the inhibitor at both time points. The corresponding efflux ratios were 2.7 after 30 min and 3.6 after 90 min, indicating a considerable activity of one or more of the eligible multidrug resistance-associated proteins in this model. The ALI model had the highest statistically significant differences (p < 0.001) between transported CDCF amounts with and without inhibitor in the apical compartment. The calculated EfR increased from 1.9 at the first measuring point up to 3.5 at the second measuring point, suggesting a pronounced functional expression of MRP transporters in the RPMI 2650 cells grown at ALI as well. In the explants of human nasal turbinate, the differences between emitted substrate quantities depending on the absence or presence of probenecid were statistically significant (p < 0.01) after 30 min as well as after 90 min. The determination of the efflux ratio yielded a value of only 1.4 at both time points, implying a relatively minor functionality of MRP1, MRP3 and MRP5.

Figure 8. Mean amounts of effluxed 5(6)-carboxy-2’,7’-dichlorofluorescein from RPMI 2650 cells grown as LCC (n=9) and at ALI (n=18), as well as excised nasal mucosa (n=8) ± standard


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deviation after 30 and 90 min. Significant differences (** p < 0.01, *** p < 0.001) of substrate quantities found in the respective apical compartments are marked at each time point. MRP2 To analyze the functional expression of MRP2, an accumulation assay using radiolabeled erythromycin as a substrate and MK571 as a nonspecific MRP inhibitor was performed. Because this macrolide antibiotic is also a well-known P-gp substrate, all experiments were implemented under the influence of the MDR1 inhibitor verapamil. The intracellular retention of erythromycin was evaluated depending on the presence or absence of MK571 (Figure 9) and additionally expressed by the corresponding accumulation ratio (AcR

erythromycin;

Table 3). The LCC model

based on the RPMI 2650 cell line showed significantly higher concentrations of erythromycin in the presence of MK571 and verapamil compared to the erythromycin concentrations in the absence of MK571 (p < 0.01). Furthermore, an MRP2-mediated efflux in the submerged cultured RPMI 2650 cells was verified by the calculated AcR of 2.1. For the RPMI 2650 cells grown at ALI, the accumulation study yielded the highest significant differences (p < 0.001) between intracellular erythromycin amounts depending on the absence or presence of the MRP inhibitor. The retained substrate amounts increased more than fivefold when both inhibitors were added, suggesting a distinct efflux activity of apically located ABC transporters in the ALI model. The addition of MK571 and verapamil led to significantly higher substrate quantities in the lysed epithelium of human nasal mucosa in comparison with the intracellular concentration of samples lacking the MRP inhibitor (p < 0.01). For the accumulation ratio, a mean value of 1.7 was achieved, substantiating the functional expression of the ABCC2 gene in the nasal explants.


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Figure 9. Mean amounts of radiolabeled erythromycin accumulated in RPMI 2650 cells grown as LCC (n=10) and at ALI (n=6), as well as specimens of human nasal mucosa (n=16) ± standard deviation. Significant differences (** p < 0.01, *** p < 0.001) of substrate quantities found in the respective epithelium are shown for each model. MRP4 Another accumulation study using tritium-labelled adefovir dipivoxil (synonym: 9(2[bis(pivaloyloxymethoxy)phosphorylmethoxy]ethyl)adenine; bis-POM-PMEA) examined the active efflux by MRP4 in the different models (Figure 10). After the intracellular diester hydrolysis of this compound, the effective antiviral form adefovir (synonym: 9-(2phosphonylmethoxyethyl)adenine; PMEA) acted as an MRP4 substrate and was pumped out of the epithelial cells. Under the influence of the inhibitor indomethacin, the intracellular retention increased and the generated accumulation ratios (AcR

adefovir)

achieved values higher than 1

(Table 3). For the LCC model, the accumulation assay with adefovir dipivoxil resulted in the highest significant differences (p < 0.001) between retained substrate quantities depending on the


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presence or absence of indomethacin. After cell lysis, the detected amounts of adefovir with inhibitor were twice as high as the substrate amount without inhibitor. The RPMI 2650 cells grown on PET filter inserts showed significantly higher concentrations of accumulated adefovir when indomethacin was added (p < 0.001), indicating a relevant functionality of the ABCC4 gene product in the second in vitro model as well. The calculation of the accumulation ratio yielded a value of 2.4 for the ALI model. The differences between retained adefovir quantities with or without inhibitor were also statistically significant (p < 0.01) in the epithelial layer of human nasal turbinate. The corresponding AcR was 1.8 for the ex vivo model, implying the functional activity of apically located MRP4. In accordance with the results generated by RTPCR, Western blot analysis and immunohistochemical staining, accumulation ratios (AcR adefovir) for all samples were in the same range of approximately 2.

Figure 10. Mean amounts of accumulated [3H]adefovir dipivoxil in submerged (n=21) and at ALI cultured RPMI 2650 cells (n=36), as well as excised nasal mucosa (n=19) ± standard deviation. Significant differences (** p < 0.01, *** p < 0.001) of substrate quantities found in the respective cell layer are highlighted for each model.


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DISCUSSION The major influence of transport proteins on the absorption, distribution and elimination of numerous drugs has led to increased interest over the last two decades in characterizing the transporter expression of different epithelial and endothelial barriers

44

. While carrier-mediated

processes are well understood in renal, hepatic and intestinal tissues and a growing amount of data on the human lung is being published

45

, the physiological and pharmacological roles of

transporters in the upper respiratory tract is mostly unknown. Following our investigations regarding the functionality of P-glycoprotein in different in vitro and ex vivo models of the human nasal mucosa

31

, the present work focuses on the expression pattern of particular MRP

transporters in the respiratory nasal epithelium. The mRNA profile of several drug transporters in differently cultured RPMI 2650 cells has been recently examined using quantitative RT-PCR analysis. These studies observed a high expression of MRP1, moderate expression of MRP4 and MRP5, and low expression of MRP2 and MRP3. Moreover, no influence of culture conditions and growth period was observed, with the exception of MRP3, which showed a more pronounced gene expression after a three-week culturing at the air-liquid interface 46. Taking into account that our qualitative PCR method only provided a limited validity concerning the expression level, we found a similar ranking for the mRNA profile of the selected ABC family members within all tested samples. The mRNA of MRP1 – 3 was also detected in parental RPMI 2650 cells as well as melphalan- and paclitaxelresistant variants of the RPMI 2650 cell line by RT-PCR. Surprisingly, no bands for MRP4 and MRP5 were found in any of these cell lines

47

. For human nasal respiratory epithelial cells

obtained from healthy patients, the semi-quantitative PCR analysis resulted in moderate to low expression of MRP1, MRP5 and MRP4. Amplification products of MRP2 and MRP3 were not


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detectable in these samples of the study 48. Corresponding to our findings, the presence of MRP1 – 5 was supported by DNA microarray data for human nasal tissue. Except for MRP2, high expression levels of the drug transporter genes were observed in human as well as bovine specimens of the nasal mucosa. Further examination of Abcc genes in rodents provided deviant expression patterns with positive results for Mrp1, 3, 4 and 5 in mice and only detectable signals for Mrp1 and Mrp5 in rats

13

. The mRNA presence of Abcc1 in olfactory epithelium obtained

from rats was confirmed in a different publication

49

. Nonetheless, another work group showed

the mRNA expression of Mrp1, 2, 3 and 5 genes in rat olfactory epithelial cells by RT-PCR 50. The presence of Mrp1 and Mrp4 was also reported in nasal respiratory as well as olfactory epithelium of rats 51. To date, the only published Western blot analyses of multidrug resistance-associated proteins in nasal tissues were performed on specimens of rat olfactory bulb and epithelium. Investigations of rat olfactory epithelial cells by different research groups revealed high expression for Mrp1 and moderate expression for Mrp3 as well as Mrp5 at the protein level. The protein expression of the Abcc2 and Abcc4 genes in these rodent epithelia was either not found or not investigated by Western blotting

49,50

. For RPMI 2650 cells, the expression of MRP1 and MRP2 at the protein

level was shown in the parental and mephalan-resistant cell lines. Despite positive PCR results in the RPMI 2650 cell line and its mephalan-resistant variant, MRP3 was not detected at the protein level 47. In almost the same manner, we observed a distinct expression at the protein level for a few MRP transporters in the excised epithelium and the LCC model, although only relatively faint band intensities representing mRNA expression were visible. This discrepancy between potentially low mRNA levels and moderate protein levels may be caused by a slow turnover rate


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of MRPs in the respiratory cells or posttranscriptional modifications that may decelerate mRNA degradation and facilitate translation 52. In terms of the RPMI 2650 cell line, these are the first results displaying the cellular distribution of various multidrug resistance-associated proteins with immunohistochemistry. For human nasal mucosa, the only available data on this issue include the immunohistochemical analysis of MRP1 in the nasal respiratory tissue. The study describes the apical expression of MRP1 in ciliated cells of the epithelium as well as in serous cells of the glands, confirming our results regarding the localization of the ABCC1 gene product

53

. In nasal respiratory as well as olfactory epithelial

cells of rats, Mrp1 was likewise found on the apical side, although conflicting results for the expression in respiratory cells were depicted

49–51

. Kudo et al.

49

detected Mrp1 only in rat

olfactory epithelium but not in respiratory epithelium, whereas Genter et al.

51

showed even

higher expression levels in the respiratory cells of rat nasal epithelium compared to the olfactory cells. However, MRP1 is present on the apical as well as basal plasma membranes of different polarized cells

11

, although only basolateral expression was observed for bronchial epithelial

layers of the human lung

54

. In contrast, the ABCC2 gene product is consistently expressed in

apical membranes, including the bronchial and bronchiolar epithelium of humans and mice

55

,

substantiating the currently presented apical localization in the respiratory epithelium of the human nose. The multidrug resistance-associated protein 3, which is predominantly localized in the basolateral membrane of polarized cells, was previously detected in the olfactory neuron layer of rats

50

and in normal human lung cells as well as tumor cell cultures, although the

localization was not described 56. Unlike most other tissues, the ABCC3 gene product is mainly expressed at the apical side of fetal blood vessel endothelia and seems to protect the fetus from toxic materials in the maternal circulation

57

. Similar to the blood-placental barrier, the unusual


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apical expression of MRP3 found in specimens of human nasal turbinate may be explained by its physiological role in detoxification mechanisms together with other MRP-related efflux pumps in the apical plasma membrane. As for the localization of MRP3, the cellular distribution of MRP4 and MRP5 in the human lung is unknown

58

, whereas both ABC proteins are found

apically and basolaterally in diverse mammalian tissues 11,59. Neither for the olfactory epithelium nor the respiratory nasal cells, the localization of MRP4 was described until now. Albeit the expression of the Abcc5 gene product was previously shown in the olfactory neuron layer of rats 50

, this is the first investigation of the cellular distribution of MRP5 in the respiratory epithelium

of human nasal mucosa. Despite the relatively high number of studies describing gene expression in nasal tissues, descriptions of the functional activity of the selected multidrug resistance-associated proteins 1 – 5 in any human or animal model of the nasal mucosa and corresponding cell cultures have not been published. For the efflux assay, the transport of CDCF, which is not recognized by Pglycoprotein but is a well-known substrate of MRP1 60, MRP3 61 and MRP5 62, was determined. If only considering the first measuring point, RPMI 2650 cells cultured at the air-liquid interface seemed to provide appropriate data for the evaluation of MRP-mediated transport in specimens of human nasal tissue, since both yielded an EfR value below 2. The finally effluxed substrate amounts by the both in vitro models based on RPMI 2650 cell line were about threefold higher in absence of probenecid than in presence of the inhibitor, respectively, and thus in the same range as previously shown for Caco-2 cells 40. Besides a less pronounced MRP activity of the excised nasal epithelium, this disparity might be explained by a passive leakage of not completely ionized CDCF from these respiratory cells

63

or by simultaneous uptake processes of CDCF via

several organic anion transporters (OATP; synonym: SLCO; formerly: SLC21) of the solute


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carrier superfamily 64, recently identified in samples of the human nasal mucosa 13. Furthermore, it has to be taken into account that CDCF and probenecid are also recognized by MRP2 as substrate and inhibitor, respectively, which necessitated a more detailed investigation of the MRP2 expression levels in the different nasal models 65,66. For that reason, a functionality assay using erythromycin with the application of two inhibitor substances was implemented, since the macrolide antibiotic serves as a substrate for Pglycoprotein as well as for MRP2

67,68

. In accordance with the results of the mRNA and protein

expression analyses, for the ABCC2 gene product appeared a similar activity level in the LCC model and the excised human nasal mucosa. Noticeably high amounts of retained substrate were found in the post-confluent cell layers of the ALI model when both inhibitors were added, indicating an appreciably higher efflux capacity in the RPMI 2650 cells cultured on ThinCert™ filter inserts. The expression level is hence similar to that of monolayers of MDCK-MRP2 cells cultured on Transwells®, showing a fivefold increase of transported erythromycin in the basolateral to apical (B → A) direction compared to the apical to basolateral (A → B) direction 69

. Therefore, the submerged cultured 2650 cells seemed to be a more suitable model for studies

addressing the potential impact of MRP2 on the disposition of intranasally administered drugs. The immediate uptake and conversion of bis-POM-PMEA to the parent compound PMEA by various cell types makes this prodrug an appropriate candidate to study the cellular efflux by multidrug resistance-associated protein 4

70

. The MRP4 activity was almost at the same level

within the tested samples, but slightly more pronounced in both in vitro models compared to the ex vivo model, which may be caused by extracellular enzymatic degradation of the ester prodrug at the apical side of mucosal tissue

71

. Nonetheless, the differently cultured RPMI 2650 cells

could represent a helpful tool to mimic the human nasal respiratory epithelium with respect to


40

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the functional expression of the ABCC4 gene product. As already mentioned for CDCF and erythromycin, several substrates are transported by two or more of the ABC and SLC proteins. In the case of adefovir, the recognition by the human organic anion transporter 1 (OAT1; synonym: SLC22A6) of the SLC superfamily was described

72

. A concurrent uptake by this SLC protein

may possibly affect the MRP4-mediated efflux of PMEA, but we did not consider this interaction because no gene expression of any OATs in the RPMI 2650 cell line

46

or in human

nasal tissue 13 have been reported so far. Nevertheless, the possible impact of further efflux and uptake proteins that may be unaffected by the applied inhibitors cannot be excluded for most functionality experiments. As shown in Table 1, most candidates for intranasal drug delivery would be used in clinical situations where an intermittent or rapid effect of the drug is desired. In such cases, transporter-mediated efflux of the active compound might delay or prohibit the onset of therapeutic action. Therefore, knowledge of ABC transporters, such as the multidrug resistance-associated proteins, should be a crucial aspect for the investigation of drug absorption by human nasal mucosa and the corresponding models. To date, the impact of these efflux proteins on intranasal drug disposition in vivo remains undefined due to the relatively short residence time of medical compounds on the nasal tissue caused by mucociliary clearance 2. However, in view of the increasing interest in the exploration of pharmaceutical excipients for mucoadhesion as well as the development of special devices to achieve prolonged retention times of the active agent on the nasal mucosa

73,74

, the

characterization of transporter expression profiles in nasal tissues and suitable in vitro models will be certainly emphasized. In this regard, the co-administration of compatible inhibitors might to be taken into account to enhance the drug delivery 12.


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Besides the influence of transporter proteins on the absorption and efficacy of intranasally administered drugs, their physiological roles in the nasal mucosa have yet to be investigated as well. First results suggest the involvement in cell detoxification and, i.e. for MRP1, in the transport of glutathione conjugates 53. In this context, future research should additionally address the identification of factors possibly altering the MRP expression pattern in humans, e.g. smoking, environmental pollution, age or gender. Regarding the use of in vitro models based on RPMI 2650 cell line for the preclinical phase of drug development, further improvement of experimental setups for functionality studies as well as the implementation of specific substrates and inhibitors are needed. Up to now, the RPMI 2650 cells are namely the only available cell line representing the physiological barrier properties of the nasal respiratory epithelium, i.e. the TEER and passive drug diffusion (apparent permeability coefficient) expression of P-glycoprotein

31

33,46

, as well as the

. For the purpose of MRP efflux activity experiments, the

differently cultured RPMI 2650 cells seemed to be appropriate to study the nasal functionality of MRP2 (only LCC model) and MRP4 (both in vitro models), but not yet optimal for the evaluation of MRP1, MRP3 and MRP5 activity (only ALI model in a strictly limited scope). CONCLUSION Regarding the human nasal mucosa, there is still a limited knowledge about the impact of most ABC transporters on the disposition of intranasally administered compounds and a lack of appropriate in vitro models to better predict the nasal drug delivery. The present investigation reveals the gene and protein expression as well as the functionality of five MRP transporters in excised nasal epithelial cells and two differently cultured RPMI 2650 cell culture models. A promising comparability was found between specimens of human nasal tissue and submerged cultured RPMI 2650 cells with respect to the expression of MRP2 as well as between all models


42

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concerning the presence and activity of MRP4. Although the expression of MRP1, MRP3 and MRP5 at the mRNA level as well as protein level was roughly comparable between the excised nasal epithelium and the differently cultured RPMI 2650 cells, a less functionality of eligible multidrug resistance-associated proteins was detected in the explants of human nasal tissue. Therefore, the RPMI 2650 cell line cannot use without restrictions to mimic the nasal MRP activity so far and further research is required to obtain more reliable in vitro-in vivo correlations of efflux experiments for intranasal drug application from these continuously growing cells.


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AUTHOR INFORMATION Corresponding Author *Corresponding author: Stephan Reichl, Tel.: 0049 531 391 5651; Fax: 0049 531 391 8108. E-mail address: [email protected] Present Address Institut für Pharmazeutische Technologie, Technische Universität Braunschweig, Mendelssohnstr. 1, 38106 Braunschweig, Germany. ACKNOWLEDGMENT The authors gratefully acknowledge the German Federal Institute for Risk Assessment (BfR), Berlin, Germany, which funded this work under grant numbers 3-1329-469 and 3-1328-508. For the supply of human nasal tissue specimens, we are grateful to Dr. Reintjes and Dr. Köllisch, HNO Praxis Schlosscarree Braunschweig, Germany, as well as Prof. Schroeder and Dr. Schmidt, Städtisches Klinikum Braunschweig, Germany. Furthermore, we would like to thank the Institute of Pharmacology and Toxicology, Technische Universität Braunschweig, Germany, for the opportunity in using the confocal laser scanning microscope.


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(66) Kong, L.-L.; Shen, G.-L.; Wang, Z.-Y.; Zhuang, X.-M.; Xiao, W.-B.; Yuan, M.; Gong, Z.H.; Li, H. Inhibition of P-Glycoprotein and Multidrug Resistance-Associated Protein 2 Regulates the Hepatobiliary Excretion and Plasma Exposure of Thienorphine and Its Glucuronide Conjugate. Frontiers in Pharmacology 2016, 7, 242, DOI: 10.3389/fphar.2016.00242. (67) Hariharan, S.; Gunda, S.; Mishra, G. P.; Pal, D.; Mitra, A. K. Enhanced corneal absorption of erythromycin by modulating P-glycoprotein and MRP mediated efflux with corticosteroids. Pharmaceutical Research 2009, 26, 1270–1282, DOI: 10.1007/s11095-008-9741-x. (68) Vellonen, K.-S.; Honkakoski, P.; Urtti, A. Substrates and inhibitors of efflux proteins interfere with the MTT assay in cells and may lead to underestimation of drug toxicity. European Journal of Pharmaceutical Sciences 2004, 23, 181–188, DOI: 10.1016/j.ejps.2004.07.006. (69) Vadlapatla, R. K.; Vadlapudi, A. D.; Kwatra, D.; Pal, D.; Mitra, A. K. Differential effect of P-gp and MRP2 on cellular translocation of gemifloxacin. International Journal of Pharmaceutics 2011, 420, 26–33, DOI: 10.1016/j.ijpharm.2011.08.009. (70) Dallas, S.; Schlichter, L.; Bendayan, R. Multidrug resistance protein (MRP) 4- and MRP 5mediated

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TABLE OF CONTENTS


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Figure 1A 82x89mm (300 x 300 DPI)



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Figure 1B 82x89mm (300 x 300 DPI)


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Figure 1C 82x89mm (300 x 300 DPI)



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Figure 1D 82x89mm (300 x 300 DPI)


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Figure 1E 82x89mm (300 x 300 DPI)



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Figure 2 82x62mm (300 x 300 DPI)


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Figure 3F 39x39mm (300 x 300 DPI)


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Figure 3G 39x39mm (300 x 300 DPI)



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Figure 3H 39x39mm (300 x 300 DPI)


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4F 39x39mm (300 x 300 DPI)


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Figure 8 104x72mm (300 x 300 DPI)



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Figure 9 104x72mm (300 x 300 DPI)


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Figure 10 104x72mm (300 x 300 DPI)


Activity of Multidrug Resistance-Associated ... - ACS Publications

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