Pharmacological Characterization of the RPMI 2650 Model as a

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Pharmacological characterization of the RPMI 2650 model as a relevant tool for assessing the permeability of intranasal drugs Clement Mercier, Sophie Hodin, Zhiguo He, Nathalie Perek, and Xavier Delavenne Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00087 • Publication Date (Web): 30 Apr 2018 Downloaded from http://pubs.acs.org on May 2, 2018

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Molecular Pharmaceutics

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Pharmacological characterization of the RPMI 2650 model as a

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relevant tool for assessing the permeability of intranasal drugs

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Clément Mercier†,b, Sophie Hodin†,‡, Zhiguo He‡,§, Nathalie Perek†,‡, Xavier Delavenne†,‡,║

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†

INSERM, U1059, Dysfonction Vasculaire et Hémostase, Saint-Etienne, France

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‡

Université de Lyon, Saint-Etienne, F-42023, France

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§

Laboratoire de biologie, d’ingénierie et d’imagerie de la greffe de cornée, BiiGC, EA2521, Saint-Etienne,

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France

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║

Laboratoire de Pharmacologie Toxicologie Gaz du sang, CHU de Saint-Etienne, Saint-Etienne, France

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Clément Mercier* : [email protected]

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Sophie Hodin: [email protected]

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Zhiguo He: [email protected]

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Nathalie Perek: [email protected]

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Xavier Delavenne: [email protected]

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*Corresponding author:

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Clément Mercier:

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Address: 10 rue de la Marandière, Faculté de Médecine, Saint-Priest-en-Jarez, France.

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E-mail: [email protected]

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Phone: +33 (0) 4 77 42 14 43

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Graphical abstract

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Abstract

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The RPMI 2650 cell line has been described as a potent model of the human nasal

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mucosa. Nevertheless, pharmacological data are still insufficient and the role of drug efflux

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transporters has not been fully elucidated. We therefore pursued the pharmacological

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characterization of this model, initially investigating the expression of four well-known

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adenosine triphosphate [ATP]-binding cassette (ABC) transporters (P-glycoprotein (P-gp),

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multidrug resistance associated protein (MRP)1, MRP2 and breast cancer resistance protein

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(BCRP)) by means of ELISA and immunofluorescence staining. The functional activity of the

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selected transporters was assessed by accumulation studies based on specific substrates and

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inhibitors. We then performed standardized bidirectional transport experiments under air-

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liquid interface (ALI) culture conditions, using four therapeutic compounds of local intranasal

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relevance in upper airway diseases. Protein expression of P-gp, MRP1, MRP2 and BCRP was

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detected at the membrane of the RPMI 2650 cells. In addition, all four transporters exhibited

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functional activity at the cellular level. In the bidirectional transport experiments, the RPMI

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2650 model was able to accurately discriminate the four therapeutic compounds according to

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their physicochemical properties. The ABC transporters tested did not play a major role in the

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efflux of these compounds at the barrier level. In conclusion, the RPMI 2650 model

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represents a promising tool for assessing the nasal absorption of drugs on the basis of pre-

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clinical pharmacological data.

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Keywords: ABC transporters, bidirectional transport, efflux ratio, permeability, RPMI 2650

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model.

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1. Introduction

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The nasal cavity offers an attractive environment for non-invasive drug delivery. The

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extensive vascularization and high permeability of the sizeable nasal mucosa promote quick

4

absorption of drugs while avoiding both early drug degradation due to first-pass hepatic

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metabolism and limiting side-effects.1,2 Thanks to these properties, the nasal mucosa is

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already a target site for the delivery of several drug classes, including corticosteroids and

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antihistamines.3 Moreover, a wide range of novel nasal formulations is in development and

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numerous investigations are currently focusing on the suitability of the nasal route for vaccine

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and hormone administration.4

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The prediction of drug absorption represents a major goal in the optimization and

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selection of new drug candidates designed for local non-invasive delivery. The evaluation of

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compounds for intranasal administration necessitates the development of in vitro models to

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investigate their transmucosal transport and bioavailability. The selection of well

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characterized and reliable models that closely mimic the physiology of the nasal mucosa is

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therefore crucial.5 Immortalized cell lines are particularly valuable for drug transport studies,

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offering unlimited cell material, genetic stability and reproducibility at affordable cost

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RPMI 2650 is a nasal cell line of human origin isolated from an anaplastic squamous

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cell carcinoma of the nasal septum.6 The RPMI 2650 model has attracted renewed interest in

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nasal permeability studies over the last 10 years thanks to the discovery of specific air-liquid

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interface (ALI) culture conditions.7 Recent data suggest that RPMI 2650 cells grown under

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ALI conditions are able to form a leaky multilayered epithelium having a thickness similar to

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that of human nasal mucosa and establishing a transepithelial electrical resistance (TEER)

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within the same range.8. In addition, RPMI 2650 cells express a wide range of tight junction

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proteins and produce mucoid material with substantial physiological secretion of goblet cells

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such as those found in the nasal mucosa.9 Since these initial findings, further studies have

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focused on optimizing the RPMI 2650 model with a view to its validation as a relevant

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physiological and pharmacological model of the human nasal mucosa for drug transport

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studies.10

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Among the various pharmacological aspects investigated, the effect of ATP binding

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cassette (ABC) transporters on drug disposition has become a specific field of research. These

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primarily active proteins are involved in the efflux of exogenous substrates out of cells and

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are related to drug resistance.11 The expression and functionality of several ABC transporters, 4 ACS Paragon Plus Environment

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such as P-glycoprotein (P-gp), Multidrug Resistance associated Proteins (MRPs) and Breast

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Cancer Resistance Protein (BCRP) have already been demonstrated in multiple physiological

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barriers including those of the intestines,12 lungs13 and brain.14 Conversely, the nasal mucosa

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has been poorly investigated. However, recent data have revealed the expression of a wide

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range of ABC transporters15 that could lead to local drug resistance in upper airway diseases.16

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The identification of ABC transporters in the corresponding in vitro models of nasal mucosa

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is therefore essential.17 The expression and functionality of drug transporters have been

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recently investigated in RPMI 2650 cells. Initial reports suggest that all the major ABC

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transporters are expressed at the gene level.18 Recently, the functional expression of P-gp and

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several MRPs has been demonstrated 8,19. Despite these promising results, the current lack of

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standardized pharmacological data based on the permeability of therapeutic products reduces

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the relevance of the RPMI 2650 model for predicting the nasal absorption of these compounds

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and their potential efflux by drug transporters.10

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Based on these observations, the present study focused on the further pharmacological

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characterization of the RPMI 2650 model. In this context, we initially investigated the protein

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expression and the localization of the ABC transporters P-gp, MRP1, MRP2 and BCRP. We

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then investigated the functional activity of the selected transporters at the cellular level by

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accumulation studies using appropriate substrates and inhibitors. Finally, we performed

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bidirectional transport experiments to determine the permeability profile of four therapeutic

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products with distinct physicochemical properties and to investigate the involvement of ABC

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transporters in RPMI 2650 cells cultured under ALI conditions.

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2. Materials and methods

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2.1 Materials

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ThinCert™ polyethylene terephthalate (PET) culture inserts (1.13 cm² growth area and

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1 µm pore size) were obtained from Greiner Bio-One (Kremsmünster, Austria). Falcon™

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companion plates for culture inserts, tissue culture plates, and tissue culture flasks were

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purchased from Corning (New York, USA).

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Rabbit polyclonal anti-ZO-1 antibodies for immunofluorescence (IF) and ELISA,

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rabbit polyclonal anti-E-cadherin for IF and ELISA, rabbit polyclonal anti-occludin for

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ELISA, mouse monoclonal anti-P-gp for IF, rabbit polyclonal anti-MRP1 for ELISA, rabbit

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polyclonal anti-MRP2 for ELISA, mouse monoclonal anti-BCRP for IF, rabbit polyclonal

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anti-BCRP for ELISA and mouse monoclonal anti-rabbit IgG-biotin for ELISA were

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purchased from Santa Cruz Biotechnology (Dallas, USA).

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Rabbit polyclonal anti-occludin antibodies for IF, rabbit polyclonal anti-claudin 1 for

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IF and ELISA, rabbit polyclonal anti-P-gp for ELISA, rabbit polyclonal anti-MRP1 for IF,

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rabbit polyclonal anti-MPR2 for IF and phalloidin-iFluor 555 were purchased from Abcam

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(Cambridge, United Kingdom).

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Hoechst 33342, rhodamine 123, goat-anti rabbit antibodies (AlexaFluor™ 555), goat-

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anti-mouse antibodies (AlexaFluor™ 488) and SuperFrost™ microscope slides were obtained

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from Thermo Fisher Scientific (Waltham, USA). The “CytoTox-ONE™ Homogeneous

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Membrane Integrity Assay” kit for lactate dehydrogenase (LDH) quantification was

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purchased from Promega (Wisconsin, USA). Normal Goat Serum (NGS) was purchased from

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Millipore (Burlington, USA).

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All other reagents were purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France).

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2.2 Cell culture

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RPMI 2650 nasal epithelial cells were obtained from the American Type Culture

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Collection (ATCC) and cultured in minimum essential medium (MEM) supplemented with

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10% fetal bovine serum (FBS), 1% L-glutamine, 1% non-essential amino acids (NEAA) and

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1% antibiotics/antimycotic (penicillin-streptomycin, amphotericin B) solution at 37°C and 5%

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CO2. The Caco-2 cell line was purchased from ATCC and routinely cultured in DMEM

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supplemented with 20% FBS, 1% NEAA and 1% antibiotics/antimycotic solution. 6 ACS Paragon Plus Environment

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RMPI 2650 cells were seeded at a density of 2 x 105 cells.cm² on porous ThinCert™

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PET culture inserts with a 1.13 cm² growth area and 1 µm pore size. The cultures were

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submerged for two days in culture medium filled in both apical and basal compartments. The

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apical medium was then removed and the inserts were raised to the air-liquid interface for an

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additional 19 days as previously described.8,10 The inserts were used in bidirectional drug

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transport studies after a total of 21 days of culture.

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2.3 Barrier properties of the RPMI 2650 model

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Transepithelial electrical resistance (TEER) was evaluated as a standard indicator of

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epithelium tightness. TEER was measured every two days using an EVOM® resistance meter

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and STX 2 electrodes (World Precision Instruments, Sarasota, USA). Blank filter values were

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subtracted and the corrected values were calculated based on the surface area of the inserts

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(1.13 cm²). Only inserts displaying a TEER above 60 Ω.cm² were used in the transport

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studies.

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The paracellular permeability of the ALI RPMI 2650 model was assessed using the

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hydrophilic compounds sodium fluorescein (NaF) and 4.4 kDa fluorescein isothiocyanate-

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dextran (FITC-Dextran) after 21 days of culture. The compounds were dissolved in Hank’s

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balanced salt solution (HBSS) with 1% HEPES (v/v) to a final concentration of 10 µg/mL and

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filled into the apical donor compartments, while HBSS with 1% HEPES (v/v) alone was

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introduced into the basal receiver compartments. The inserts were then incubated for 2 h at

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37°C and the amount of the fluorescent marker compound reaching the basal compartment

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was evaluated using a fluorometer (Fluoroskan Ascent™, Thermo Fisher Scientific) with

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wavelengths set to 485/535 nm. The apparent permeability (Papp) was calculated according to

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the FDA approved20 equation:

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= × × (

) (Equation 1)

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where V is the volume of the acceptor compartment (in cm3), [Ci] is the initial concentration

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of the molecule (in g/L or mol/L), A is the area of the insert (in cm²), [Cf] is the final

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concentration of the compound in the acceptor compartment (in g/L or mol/L), and t is the

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duration of the experiment (in s).

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2.4 Cytotoxicity assays

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RPMI 2650 cells were seeded in 96-well plates at a density of 50.000 cells per well

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and allowed to growth during three days until subconfluence. Then, the cells were treated

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with increased concentrations of molecules during two hours at 37°C. Following incubation,

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the cytotoxicity of the tested compounds was evaluated by both LDH and MTT assays. LDH

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was quantified using the “CytoTox-ONE™ Homogeneous Membrane Integrity Assay” and

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the MTT assay was performed using the “In Vitro Toxicology Assay Kit, MTT based”

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according to the manufacturer’s guidance.

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2.5 Cell-based ELISA

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Protein expression of the tight junction proteins ZO-1, E-cadherin, occludin, claudin-1

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and the ABC transporters P-gp, MRP1, MRP2 and BCRP was investigated in both the RPMI

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2650 and Caco-2 models. Sub-confluent cells were fixed with 3.7% formaldehyde and

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permeabilized with 0.3% Triton for 20 min. The blocking step was achieved by the addition

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of 10% FBS to the cell culture medium during 30 min. The cells were then further incubated

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for 1 h with the respective primary antibody diluted in 10% FBS to a final concentration of 8

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µg/mL and subsequently for 1 h with the secondary antibody diluted 1/500 in the blocking

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buffer. Non-specific binding was evaluated using the secondary antibody alone. The cells

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were then incubated for 30 min with streptavidin-peroxidase. Protein expression was revealed

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by spectrometry (Multiskan™ RC, Thermo Fisher Scientific, France) on the basis of 3,3’,5,5’-

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tetramethylbenzidine (TMB) absorption at 450 nm. Data were normalized to the whole cell

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protein content of each well using the “QuantiPro™ BCA assay” kit according to the

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manufacturer’s instructions. Expression ratios were calculated by dividing the experimental

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data by the non-specific binding values.

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2.6 Immunofluorescence staining

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ABC transporters were localized by immunofluorescence studies using the mature ALI

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RPMI 2650 model. Briefly, cells were fixed in pure methanol or 1% paraformaldehyde (PFA)

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for 30 min. The non-specific binding sites were then blocked by incubating the inserts in PBS

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containing 2% goat serum and 2% bovine serum albumin (BSA) for 1 h at 37°C. The

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membranes were then cut up and placed in 24-well plates. The primary antibodies were 8 ACS Paragon Plus Environment

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diluted 1/200 in the blocking buffer and incubated for 1 h at 37°C. The secondary antibodies

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were diluted 1/500 in the blocking buffer and incubated for 45 min at 37°C. The nuclei were

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finally counterstained with 5 µg/mL of Hoechst 33342 in PBS for 5 min. An epifluorescence

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inverted microscope IX81 (Olympus, Tokyo, Japan) equipped with Cell^P software (Soft

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Imaging System GmbH, Munster, Germany) was used for image acquisition.

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2.7 ABC transporter functionality studies

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The functional activity of the selected transporters was evaluated at the cellular level

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by targeted accumulation studies based on three fluorescent substrates, 2′,7′-bis(2-

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carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM (2 µM)), rhodamine

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123 (10 µM) and Hoechst 33342 (20 µM), as broad spectrum substrate of MRPs and specific

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substrates of P-gp and BCRP, respectively.

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RPMI 2650 cells were pre-treated for 15 min at 37°C with a range of four inhibitors

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comprising verapamil (100 µM), probenecid (100 µM), cyclosporin A (100 µM) and Ko143

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(10 µM). The substrates were then diluted independently in each inhibitor solution prior to

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further incubation for 1 h at 37°C. Finally, the cells were lysed at room temperature during 30

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min in a solution of sodium dodecyl sulfate (SDS) containing 1% of 10 mM sodium borate.

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The amounts of BCECF and rhodamine 123 inside the cells were determined using a

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fluorometer with wavelengths set to 485/535 nm. The quantity of intracellular Hoechst 33342

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was determined at 350/460 nm. The accumulation of each compound was expressed as a

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percentage of untreated cells and the data were normalized to the whole cell protein content of

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each well using the “QuantiPro™ BCA assay” kit.

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2.8 Bidirectional transport studies

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We investigated the permeability of both therapeutic compounds and specific

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substrates of ABC transporters using a standardized bidirectional transport protocol according

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to FDA guidelines.20 Rhodamine 123 (10 µM) was assessed as a P-gp substrate with

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verapamil as a selective inhibitor. BCECF (10 µM) was evaluated as a broad spectrum MRPs

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substrate with probenecid as a general MRP inhibitor. Chlorothiazide (50 µM) was assessed

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as a BCRP substrate with Ko143 as a specific inhibitor of BCRP. The therapeutic products

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clarithromycin (CLA), azithromycin (AZM), gentamicin (GEN) and budesonide (BUD) were 9 ACS Paragon Plus Environment


Page 10 of 30

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evaluated over a range of five concentrations. The broad-spectrum inhibitor cyclosporin A

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was used at 10 µM for all four compounds.

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Bidirectional transport assays were performed from the apical to the basal

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compartment (Papp A=>B) and from the basal to the apical compartment (Papp B=>A) for 2 h at

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37°C. Following incubation, the amount of drug permeating through the model in both

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directions was quantified by liquid chromatography-mass spectrometry (LC-MS) for

7

chlorothiazide, CLA, AZM, GEN and BUD. Rhodamine 123 and BCECF were quantified

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using a fluorometer at wavelengths of 485/535 nm. The bidirectional permeability was

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calculated according to Equation 1.

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Finally, to determine whether the tested compound underwent active efflux by ABCs

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transporters, the efflux ratio (ER) of each compound was computed according to the equation: ER =

12

( ) ( )

(Equation 2)

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2.9 LC-MS analysis

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CLA, AZM, GEN, BUD and chlorothiazide were quantified using an Aquity

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ultraperformance liquid chromatography (UPLC) system coupled with a Xevo TQ-D triple

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quadrupole mass spectrometer. Positive ionization conditions were used for analysis of AZM

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(m/z 749.75→591.51), BUD (m/z 431.35→413.39), CLA (m/z 748.64→590.51) and GEN

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(m/z 450.34→322.21 + 464.33→322.21 + 478.39→322.21). Negative ionization conditions

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were used for chlorothiazide (293.98→214.16). The internal standards (IS) were [2H5]-

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nordiazepam (276.20→140.12) for AZM, BUD and CLA, kanamycin (484.34→324.20) for

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GEN and [2H6]-salicylic acid (141.14→97.10) for chlorothiazide. The mobile phase

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comprised a mixture of (A) 0.1% formic acid (FA) in water and (B) 0.1% FA in acetonitrile.

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Chromatography was performed by gradient elution on an ethylene-bridged hybrid (BEH)

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amide column (50 mm × 2.1 mm × 1.7 µm) for GEN and on a BEH C18 column (50 mm ×

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2.1 mm × 1.7 µm) for the other drugs. The peak area ratios of the drugs and their respective IS

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were used as [Ci] and [Cf] in Equation 1.

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3. Results

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3.1 Barrier properties of the RPMI 2650 model

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The ALI cultured RPMI 2650 cells showed a regular and homogeneous increase in TEER

4

over 21 days of culture suggesting the formation of tight junctions (Figure 1A). The observed

5

values increased from 25 Ω.cm² at day 8 to reach a maximal plateau of around 80 Ω.cm² after

6

21 days of culture. The paracellular permeability of RPMI 2650 model was also evaluated

7

using the hydrophilic marker NaF and high molecular weight (4.4 kDa) FITC-Dextran (Figure

8

1B). The average Papp (A=>B) for NaF was around 6.0 x 10-6 cm/s after 2 h of incubation. As

9

expected, the average permeability of 4.4 kDa FITC-Dextran (2.5 x 10-6 cm/s) was

10

approximately three times lower than that of NaF. These results were in accordance with the

11

molecular weights of the two compounds and highlighted the functional integrity of the ALI

12

RPMI 2650 model.

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In addition, cell-based ELISA demonstrated the expression of ZO-1, occludin, and E-

14

cadherin (Figure 1C). As expected, these three tight junction proteins were localized at the

15

cell membrane with faint and heterogeneous staining throughout the whole cell layer (Figure

16

1D) suggesting the formation of a leaky epithelium. Taken together, these data emphasized

17

the barrier properties of the ALI cultured RPMI 2650 model.

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3.2 Expression of ABC transporters

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The expression of P-gp, MRP1, MRP2, and BCRP in RPMI 2650 cells was

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investigated using cell-based ELISA (Figure 2). The Caco-2 cell line was used as a positive

22

control in view of its previous characterization as a model for the expression of ABC

23

transporters. Expression ratios ranged from 3.88 (P-gp) to 4.75 (BCRP) for RPMI 2650 cells

24

(Figure 2A) and from 3.12 (MRP1) to 4.79 (P-gp) for Caco-2 cells (Figure 2B), demonstrating

25

protein expression of the four selected transporters in both cell lines. The Caco-2 cell line

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exhibited a greater expression of P-gp and MRP2 compared to the RPMI 2650 cell line.

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Conversely, the amounts of MRP1 and BCRP were found to be higher in RPMI 2650 cells

28

than in Caco-2 cells.

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Immunofluorescence staining showed a membrane localization for P-gp, BCRP and

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MRP1 in the mature ALI RPMI 2650 model (Figure 2C). MRP1 was stained strongly and

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homogeneously over the whole surface of the epithelium whereas MRP2 showed a faint and 11 ACS Paragon Plus Environment


Page 12 of 30

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heterogeneous signal suggesting weaker expression. P-gp exhibited a clear FITC signal

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around the counterstained nuclei indicating consistent expression of the transporter. Finally,

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BCRP displayed a weaker immunofluorescence than P-gp, localized at the edge of the cell

4

membrane. Overall, the RPMI 2650 model expressed well the four ABC transporters studied,

5

namely P-gp, MRP 1 and 2, and BCRP, all these transporters being localized at the plasma

6

membrane except MRP2 showing a diffuse expression.

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3.3 ABC transporter functionality studies

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The functionality of the ABC transporters investigated was assessed at the cellular

10

level by means of intracellular accumulation assays. These experiments were based on the use

11

of three fluorescent substrates, namely rhodamine 123 (P-gp), BCECF (MRPs) and Hoechst

12

33342 (BCRP), incubated with or without well-characterized inhibitors comprising the P-gp

13

inhibitor verapamil, the MRPs inhibitor probenecid, the BCRP inhibitor Ko143 and the broad

14

spectrum inhibitor cyclosporin A (Figure 3).

15

As expected, the accumulation of intracellular BCECF in the cells treated with

16

probenecid was increased approximately three-fold (311 ± 29%) compared to the untreated

17

control (Figure 3A). The addition of cyclosporin A resulted in a similar intracellular

18

accumulation of 319 ± 28%. The presence of either verapamil or Ko143 also led to a four-fold

19

increased accumulation of BCECF within RPMI 2650 cells demonstrating involvement of the

20

ABC transporters P-gp and BCRP. Cells incubated with verapamil showed a three-fold higher

21

(290 ± 40%) accumulation of rhodamine 123 than control cells (Figure 3B). The addition of

22

cyclosporin A led to a similar accumulation of 273 ± 38%. Conversely, the presence of either

23

probenecid (118 ± 5.8%) or Ko143 (109 ± 16%) only slightly increased the percentage of

24

entrapped substrate, highlighting the specific activity of P-gp. Incubation with Hoechst 33342

25

resulted in a reverse accumulation profile compared to that observed with rhodamine 123. The

26

intracellular quantity of dye determined in cells incubated with Ko143 or cyclosporin A was

27

twice as high as in untreated cells (Figure 3C), whereas the incubation of cells with either

28

verapamil or probenecid did not affect the retention of Hoechst 33342 compared to untreated

29

cells. BCRP was therefore functional. Overall, RPMI 2650 cells functionally expressed all

30

four ABC transporters at the cellular level.

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3.4 Bidirectional transport studies

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In the final part of our study, we investigated the permeability profile of the four

3

therapeutic molecules AZM, CLA, GEN and BUD using the ALI RPMI 2650 model by

4

means of bidirectional transport experiments (Figure 4). Reference selective substrates of P-gp

5

(rhodamine 123), BCRP (chlorothiazide) and broad spectrum substrate of MRPs (BCECF-

6

AM) and were also assessed with and without their associated inhibitors in order to precisely

7

determine the activity of each ABC transporter in ALI RPMI 2650 (Figure 5). Previous

8

cytotoxicity assays had demonstrated that these compounds did not adversely affect the

9

viability of RPMI 2650 cells (data not shown). CLA and AZM displayed a similar permeability profile with the lowest average Papp

10

values of the four compounds (Figure 4A, B). The observed Papp (A=>B) values ranged

11

(A=>B)

12

from 5.12 x 10-6 to 4.01 x 10-6 cm/s and 5.62 x 10-6 to 2.96 x 10-6 cm/s for CLA and AZM,

13

respectively. For both compounds, maximal Papp

14

Overall, the Papp

15

manner showing saturatable transport of these macrolides. The Papp (B=>A) values of AZM were

16

slightly higher than CLA, resulting in ER ranging from 1.47 (100 µM) to 1.39 (10 µM). The

17

highest values of Papp (B=>A) were reached at 10 µM, leading to a maximal ER of 1.96 and 2.15

18

for AZM and CLA, respectively. Comparatively, GEN displayed the highest Papp (A=>B) values

19

at the three lowest concentrations with an average of 1.5 x 10-5 cm/s revealing the greatest

20

permeability (Figure 4C). As with the macrolides, a strong decrease in both Papp (A=>B) and Papp

21

(B=>A)

22

compound. However, Papp (B=>A) remained almost identical to Papp (A=>B), resulting in lower ER

23

with values ranging from 1.36 (at 10 µM) to 0.93 (at 1000 µM). BUD displayed a

24

permeability profile differing from that of the other compounds. High and homogeneous

25

permeability values were observed with an average of 1.25 x 10-5 cm/s and 1 x 10-5 cm/s for

26

Papp (A=>B) and Papp (B=>A), respectively (Figure 4D). In contrast to the values determined for the

27

other compounds, Papp (A=>B) values were mostly higher than Papp (B=>A) values, revealing the

28

presence of a facilitated and also saturatable permeability of BUD at the highest concentration

29

(100 µM). Thus, the calculated ERs were below 1. Overall, the four compounds tested

30

displayed ER values below 2. The addition of cyclosporin A did not modulate the ER for any

31

of the compounds.

(A=>B)

(A=>B)

values were observed at 10 µM.

values of CLA and AZM decreased in a concentration-dependent

was observed at increasing concentrations, showing saturatable transport of this

32

In a second phase, we evaluated the bidirectional transport of three substrates of ABC

33

transporters in the presence of the appropriate specific inhibitor. Rhodamine 123, BCECF13 ACS Paragon Plus Environment


Page 14 of 30

1

AM and chlorothiazide exhibited ER of 2.51, 1.64 and 1.93, respectively (Figure 5A, B, C).

2

Addition of their respective inhibitors, verapamil, probenecid and Ko143, did not significantly

3

modulate the corresponding ER. Finally, our results clearly demonstrate that ABC

4

transporters were not involved in the efflux of either reference substrates or therapeutic

5

products at the barrier level in the ALI RPMI 2650 model.

6 7 8

4. Discussion

9

The absorption of locally delivered drugs depends on various pharmaceutical factors,

10

including their physicochemical properties and potential efflux from cells by ABC

11

transporters.21 In preclinical screening studies, in vitro models are used to assess drugs

12

permeability. However, very few pharmacological data have been reported concerning the use

13

of RPMI 2650 cells as a model of the nasal mucosa. This study therefore focused on the

14

further pharmacological characterization of RPMI 2650 model. We investigated the

15

permeability profile of four drugs with local therapeutic potential and distinct

16

physicochemical properties (Table 1). AZM and CLA are macrolide antibiotics used to treat

17

intracellular pathogens.22 Both these compounds have been clinically investigated in the

18

context of local delivery in patients suffering from chronic rhinosinusitis.23 GEN is an

19

antibiotic of the aminoglycoside class and is used to treat lower respiratory tract infections.24

20

The benefit of nasal irrigation with GEN in pediatric sinusitis has already been

21

demonstrated.25 BUD is a corticosteroid already approved in nebulized form and used to

22

alleviate inflammatory disorders.26 All four compounds were assessed according to a FDA-

23

approved bidirectional transport protocol.20,27

24

Prior to conducting transport experiments, we confirmed the barrier properties of the ALI

25

RPMI 2650 model. The TEER values determined in this phase of our study were around 80

26

Ω·cm² after 21 days of culture on PET inserts, similar to the values recorded using a previous

27

model with the same culture conditions. Moreover, the values reported here were close to

28

those reported for excised nasal mucosa, supporting the correlation between the RPMI 2650

29

model and the in vivo situation.28 In addition, we performed permeability studies using the

30

hydrophilic markers NaF and 4.4 kDa FITC-Dextran. The data obtained for NaF and 4.4 kDa

31

FITC-Dextran were higher than previous studies29,30 likely due to a larger pore diameter.

32

Nevertheless, the permeability of both compounds remained in the same range (10-6 cm/s) as

33

those previously reported. 29,30 As expected, the permeability of 4.4 kDa FITC-Dextran was 14 ACS Paragon Plus Environment

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1

lower than that of NaF and higher than that of 10 kDa FITC-Dextran.18 The expression of

2

tight junction proteins and their effective membrane localization reinforce the functional

3

integrity of the ALI RPMI 2650 model as a leaky multilayered epithelium.29

4

RPMI 2650 cells express a wide range of well-known ABC transporters, the protein

5

expression of P-gp, MRP1, MRP2, MRP3, MRP4 and MRP5 having already been

6

demonstrated in RPMI 2650 cells using the Western blot technique.8,19 We confirmed the

7

protein expression of P-gp, MRP1 and MRP2 in both RPMI 2650 cells and in the Caco-2 cell

8

line, used as a positive control for the expression of both P-gp and MRPs.31,32 In addition, our

9

study is the first to report the protein expression of BCRP in RPMI 2650 cells.

10

Immunofluorescence staining corroborated protein expression with a typical membrane

11

localization of P-gp, MRP1 and BCRP that was consistent with previous characterization

12

studies showing an apical expression of P-gp and MRPs by immunofluorescence.8,19 The

13

functionality of the selected ABC transporters at the cellular level was subsequently assessed

14

in three independent accumulation experiments. RPMI 2650 cells exhibited an intracellular

15

retention of rhodamine 123 three-fold higher than that observed in untreated cells in the

16

presence of the P-gp inhibitor verapamil, confirming the specific activity of P-gp in RPMI

17

2650 cells as previously described in efflux experiments.8 The intracellular accumulation of

18

BCECF-AM in RPMI 2650 cells was considerably increased in the presence of probenecid,

19

verapamil and Ko143, suggesting simultaneous involvement of MRPs, P-gp and BCRP in the

20

efflux of BCECF-AM.33 The functionality of MRP1, MRP3 and MRP5 was recently

21

confirmed in RPMI 2650 cells using an efflux assay based on CDCF-AM, an analogue of

22

BCECF-AM.19 Finally, the increased intracellular retention of Hoechst 33342 in the presence

23

of the specific BCRP inhibitor Ko143 revealed the functional expression of BCRP in RPMI

24

2650 cells, as previously demonstrated in enterocyte-like cells.34

25

This validation of the barrier properties of the ALI RPMI 2650 model allowed us to

26

investigate the bidirectional permeability of four therapeutic compounds through the model.

27

AZM and CLA shared a similar permeability profile, the apical-to-basolateral permeability

28

values recorded for these compounds being the lowest among those of the four compounds

29

tested. This finding could be explained by both the strongly lipophilic nature and the high

30

molecular weight (> 700 g/mol) of these macrolides,35 resulting in preferential use of the

31

transcellular pathway to cross the multilayered epithelium and consequently a low

32

permeability. The decrease in permeability for high concentrations could be explained by two

33

assumptions. Firstly, an intracellular accumulation of macrolides may have occurred. This 15 ACS Paragon Plus Environment


Page 16 of 30

1

phenomenon is commonly observed for highly lipophilic compounds for which a sizeable

2

fraction could be trapped inside the cellular barrier during permeability experiments.36 This

3

hypothesis is strengthen by the multilayered properties of ALI RPMI 2650 model that could

4

promote and increase the intracellular retention of CLA and AZM leading to a decrease in

5

permeability for high concentrations. Secondly, macrolides were found to be potent substrates

6

of uptake organic anion transporting polypeptide transporters (OATPs).37,38,39 The saturation

7

of OATPs by high concentrations of macrolides could also explained the observed decrease in

8

permeability. Conversely to macrolides, GEN displayed the highest Papp

9

predominantly due to the hydrophilic properties of this aminoglycoside, coupled with its low

10

molecular weight (477.596 g/mol). This compound therefore tended to quickly diffuse

11

through the leaky epithelium following the paracellular pathway. This observation is

12

supported by the strongly decreased Papp

13

suggesting a saturatable process. Moreover, the permeability values of GEN were found to be

14

lower in 16HBE140 cells than in the RPMI 2650 model, with reported TEER values almost

15

fourfold higher (300 Ω.cm²), suggesting involvement of the paracellular route for this

16

compound.40 The transport of gentamicin may also have been facilitated by the presence of

17

organic cation transporters (OCTs), especially OCT1 and OCT2

18

expressed at the apical side of ciliated cells in human nasal epithelium.42 The observed Papp

19

(A=>B) values

20

previous study conducted in tighter bronchial Calu-3 cells, emphasizing a diffusion driven by

21

the paracellular pathway.43 In addition, BUD exhibited a higher absorptive than secretory

22

permeability suggesting a facilitated diffusion that might involve organic cation transporters.44

23

Taken together, these permeability data highlight the ability of the RPMI 2650 model to

24

accurately discriminate compounds according to their physicochemical properties. This

25

feature is currently being exploited in investigations of the impact of different formulations

26

aiming to enhance the permeation and uptake of intranasal drugs.45

(A=>B)

(A=>B)

values,

values seen at higher concentrations,

39 41

, that were found to be

for BUD were fivefold higher than those reported at identical concentrations in a

27

None of the compounds tested displayed active efflux. As reported in the literature, a

28

compound may be considered as a substrate of ABC transporters if its ER is above 3 and

29

decreases in the presence of the corresponding inhibitor.46 In this study, all the compounds

30

tested exhibited an ER below 2 regardless of their concentration and the ER was not

31

decreased by addition of the broad-spectrum inhibitor cyclosporin A. Although GEN has

32

never been described as a substrate of ABC transporters,40 the three other compounds are all

33

well-known ABC transporter substrates. Both CLA and AZM exhibited high ER in Caco-2 16 ACS Paragon Plus Environment

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1

cells (4.8 and 75, respectively), consistent with a potent involvement of P-gp in the efflux of

2

these macrolides.47 Another study described an asymmetric transport of CLA and AZM in

3

Calu-3 cells resulting in ER of 7.9 and 4.0, respectively.48 BUD displayed an ER of 8.2 in

4

Caco-2 cells, reduced to 2.2 in the presence of the P-gp inhibitor PSC-833.49

5

In addition to therapeutic compounds, specific substrates of each ABC transporter were

6

investigated in bidirectional transport experiments. In this context, rhodamine 123 was

7

evaluated as a selective P-gp substrate, active efflux of this dye having already been

8

demonstrated in Caco-2,50 MDCK,51 and Calu-3 cells.52 MRP activity had been previously

9

assessed using BCECF-AM.33 This dye displayed an asymmetric transport in bovine brain

10

microvascular endothelial cells (BBMEC) with an ER of 3.52, decreased in the presence of

11

the MRP inhibitor indomethacin.53 The functionality of BCRP was evaluated using

12

chlorothiazide. The bidirectional transport of this diuretic was recently investigated in Caco-2

13

cells54 and displayed an ER above 3 that was reduced by Ko143, a specific BCRP inhibitor.55

14

In our study, all three substrates exhibited an ER below 3. Addition of the inhibitors

15

verapamil, probenecid and Ko143 did not decrease the respective ER of rhodamine 123,

16

BCECF-AM and chlorothiazide. Taken together, these data suggest that ABC transporters are

17

not involved in the active efflux of the reference substrates tested in our ALI cultured RPMI

18

2650 model.

19

Finally, our data as a whole concerning ABC transporters revealed a discrepancy.

20

Although we successfully demonstrated the functional expression of P-gp, MRP1, MRP2 and

21

BCRP in RPMI 2650 cells, these transporters were not involved in the efflux of the substrates

22

tested in bidirectional transport experiments. This could be explained by two hypotheses. The

23

first hypothesis is based on the barrier properties of the RPMI 2650 model. RPMI 2650 cells

24

usually display TEER below 100 Ω.cm² and are consequently categorized as forming a leaky

25

multilayered epithelium.10 In contrast, Caco-2 or Calu-3 cells are described as forming tight

26

monolayers in view of their TEER typically above 300 Ω.cm².56,57 Based on these

27

observations, the leaky properties of the multilayered RPMI 2650 model might promote the

28

paracellular permeation of compounds at the expense of ABC transporter-mediated efflux.

29

Hence, ABC transporters might have only a weak effect on drug transport in view of the

30

predominantly paracellular pathway. The second hypothesis is that the RPMI 2650 model

31

might suffer from a low level of transporter expression. This hypothesis is supported by the

32

findings of a recent study that described weaker expression of both P-gp mRNA and P-gp

33

protein in the ALI RPMI 2650 model than in Caco-2 cells.8 Moreover, another study showed 17 ACS Paragon Plus Environment


Page 18 of 30

1

that the expression of ABC transporters in the RPMI 2650 model was nearly identical in

2

pattern to that in nasal primary cells, but was nevertheless weaker.9 Thus, the expression of

3

ABC transporters could be quantitatively insufficient in the RPMI 2650 model to provide a

4

measurable efflux of substrates in bidirectional transport experiments.

5

Our study nevertheless indicated the potential of the RPMI 2650 model as a relevant

6

tool for investigate the permeability of locally administered therapeutic compounds in

7

preclinical screening studies. Further studies should focus on the evaluation of the

8

permeability profile of nebulized drugs directly on ALI RPMI 2650 model to reproduce a

9

current mode of administration of intranasally delivered drugs.58 With regard to ABC drug

10

transporters, further bidirectional transport studies directly comparing the ALI RPMI 2650

11

model to excised nasal mucosa should be performed to accurately clarify the involvement of

12

these transporters in the bioavailability of local drugs. The characterization of SLC uptake

13

transporters in RPMI 2650 model represents another point of future interest for targeted drug

14

delivery.17 The use of nasal primary cells59 or 3D models60 could also represent alternative

15

means of studying drug interactions with drug transporters.

16

Conclusion

17

The present study is the first to investigate the bidirectional permeability of four

18

compounds with relevant local therapeutic potential. The results show that the tested

19

compounds displayed different permeability profiles and permeated through the RPMI 2650

20

model following different pathways in accordance with their physicochemical properties. The

21

ABC transporters P-gp, MRP1, MRP2 and BCRP were expressed in this model and exhibited

22

distinct immunofluorescence membrane staining patterns. Accumulation studies with

23

selective substrates revealed a functional and specific activity of P-gp, MRPs and BCRP at the

24

cellular level. However, these transporters were not involved in the active efflux of the

25

reference substrates or therapeutic compounds tested in bidirectional transport experiments

26

using the ALI RPMI 2650 model. The results of our study nevertheless suggest that the ALI

27

cultured RPMI 2650 model could be used as a relevant tool in preclinical screening studies for

28

assessing the permeability of intranasal drugs.

29 30 31

Acknowledgments 18 ACS Paragon Plus Environment

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1

No funding sources were used for this study.

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

References 19 ACS Paragon Plus Environment


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1 2

(1) Türker, S.; Onur, E.; Ózer, Y. Nasal route and drug delivery systems. Pharm. World. Sci. 2004, 26, 137–142.

3 4 5 6

(2) Juel-Berg, N.; Darling, P.; Bolvig, J.; Foss-Skiftesvik, M. H.; Halken, S.; Winther, L.; Hansen, K. S; Askjaer, N.; Heegaard, S.; Madsen, A. R.; Opstrup, M. S. Intranasal corticosteroids compared with oral antihistamines in allergic rhinitis: A systematic review and meta-analysis. Am. J. Rhinol. Allergy. 2017, 31, 19–28.

7 8

(3) Pires, A.; Fortuna, A.; Alves, G.; Falcão, A. Intranasal drug delivery: how, why and what for? J. Pharm. Pharm. Sci. 2009, 12, 288–311.

9 10

(4) Illum, L. Nasal drug delivery—possibilities, problems and solutions. J. Control. Release. 2003, 87, 187–198.

11 12

(5) Zhang, D.; Luo, G.; Ding, X.; Lu, C. Preclinical experimental models of drug metabolism and disposition in drug discovery and development. Acta. Pharm. Sin. B. 2012, 2, 549–561.

13 14

(6) Moorhead, PS. Human tumor cell line with a quasi-diploid karyotype (RPMI 2650). Exp. Cell. Res. 1965, 39, 190–196.

15 16 17

(7) Bai, S.; Yang, T.; Abbruscato, T. J.; Ahsan, F. Evaluation of human nasal RPMI 2650 cells grown at an air–liquid interface as a model for nasal drug transport studies. J. Pharm. Sci. 2008, 97, 1165–1178.

18 19

(8) Dolberg, A. M.; Reichl, S. Expression of P-glycoprotein in excised human nasal mucosa and optimized models of RPMI 2650 cells. Int. J. Pharm. 2016, 508, 22–33.

20 21 22

(9) Pozzoli, M.; Ong, H. X.; Morgan, L.; Sukkar, M.; Traini, D.; Young P. M.; Sonvico, F. Application of RPMI 2650 nasal cell model to a 3D printed apparatus for the testing of drug deposition and permeation of nasal products. Eur. J. Pharm. Biopharm. 2016, 107, 223–233.

23 24 25

(10) Mercier, C.; Perek, N.; Delavenne, X. Is RPMI 2650 a suitable in vitro nasal model for drug transport studies? Eur. J. Drug. Metab. Pharmacokinet. 2017, DOI: 10.1007/s13318017-0426-x.

26 27

(11) Gottesman, M. M. Mechanisms of cancer drug resistance. Annu. Rev. Med. 2002, 53, 615–627.

28 29

(12) Estudante, M.; Morais, J. G.; Soveral, G.; Benet L. Z. Intestinal drug transporters: An overview. Adv. Drug Deliv. Rev. 2013, 65, 1340–1356.

30 31 32

(13) Nickel, S.; Clerkin, C. G.; Selo, M. A.; Ehrhardt, S. Transport mechanisms at the pulmonary mucosa: implications for drug delivery. Expert. Opin. Drug. Deliv. 2016, 13, 667690.

33 34

(14) Sun, H.; Dai, H.; Shaik, H.; Elmquist W. F. Drug efflux transporters in the CNS. Adv. Drug Deliv. Rev. 2003, 55, 83–105.

35 36 37

(15) Al-Ghabeish, M.; Scheetz, T.; Assem, M.; Donovan M. D. Microarray determination of the expression of drug transporters in humans and animal species used for the investigation of nasal absorption. Mol. Pharm. 2015, 12, 2742–2754.


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


1 2 3

(16) Kocharyan, A.; Feldman, R.; Singleton, A.; Han, X.; Bleier, B. S. P-glycoprotein inhibition promotes prednisone retention in human sinonasal polyp explants. Allergy. Rhinol. 2014, 4, 734–738.

4 5

(17) Anand, U.; Parikh, A.; Ugwu M. C.; Agu R. U. Drug transporters in the nasal epithelium: an overview of strategies in targeted drug delivery. Future. Med. Chem. 2014, 6, 1381–1397.

6 7 8 9

(18) Kreft M. E.; Jerman U. D.; Lasič, E.; LanišnikRižner, T.; Hevir-Kene, L.; Peternel, L.; Kristan, K. The characterization of the human nasal epithelial cell line RPMI 2650 under different culture conditions and their optimization for an appropriate in vitro nasal model. Pharm. Res. 2015, 32, 665–679.

10 11 12

(19) Dolberg A. M.; Reichl, S. Activity of multidrug resistance-associated proteins 1–5 (MRP1–5) in the RPMI 2650 cell line and explants of human nasal turbinate. Mol. Pharm. 2017, 14, 1577–1590.

13 14 15 16

(20) Food and Drug Administration. Guidance for Industry. Drug Interaction Studies – Study Design, Data Analysis, and Implications for Dosing and Labeling. Available from https://www.fda.gov/OHRMS/DOCKETS/98fr/06d-0344-gdl0001.pdf Accessed April 4, 2018.

17 18 19 20

(21) Grassin-Delyle, S.; Buenestado, A.; Naline, E.; Faisy, C.; Blouquit-Laye, S.; Couderc, L. J.; Le Guen, M.; Fischler, M.; Devillier, P. Intranasal drug delivery: an efficient and noninvasive route for systemic administration: focus on opioids. Pharmacol. Ther. 2012, 134, 366–379.

21 22

(22) Tulkens P. M. Intracellular distribution and activity of antibiotics. Eur. J. Clin. Microbiol. Infect. Dis. 1991, 10, 100–106.

23 24

(23) Pynnonen M. A.; Venkatraman, G.; Davis, G. E. Macrolide therapy for chronic rhinosinusitis: a meta-analysis. Otolaryngol. Head. Neck. Surg. 2013, 148, 366–373.

25 26 27

(24) Chen, J. K.; Martin-McNew, B. L.; Lubsch, L. M. Nebulized gentamicin as an alternative to nebulized tobramycin for tracheitis in pediatric patients. J. Pediatr. Pharmacol. Ther. 2017, 22, 9–14.

28 29 30

(25) Wei, J. L.; Sykes, K. J.; Johnson, P.; He, J.; Mayo, M. S. Safety and efficacy of oncedaily nasal irrigation for the treatment of pediatric chronic rhinosinusitis. Laryngoscope. 2011, 121, 1989–2000.

31 32 33

(26) Rudmik, L.; Hoy, M.; Schlosser, R. J.; Harvey, R. J; Welch, K. C.; Lund, V.; Smith, T. L. Topical therapies in the management of chronic rhinosinusitis: an evidence-based review with recommendations. Allergy. Rhinol. 2013, 3, 281–298.

34 35

(27) Hubatsch, I.; Ragnarsson, E. G. E.; Artursson, P. Determination of drug permeability and prediction of drug absorption in Caco-2 monolayers. Nat. Protoc. 2007, 2, 2111–2119.

36 37 38

(28) Boucher, R. C.; Yankaskas, J. R.; Cotton C. U.; Knowles M. R.; Stutts, M. J. Cell culture approaches to the investigation of human airway ion transport. Eur. J. Respir. Dis. 1987, 71, 59–67.

39 40 41

(29) Wengst, A.; Reichl, S. RPMI 2650 epithelial model and three-dimensional reconstructed human nasal mucosa as in vitro models for nasal permeation studies. Eur. J. Pharm. Biopharm. 2010, 74, 290–297. 21 ACS Paragon Plus Environment


Page 22 of 30

1 2 3

(30) Reichl, S.; Becker, K. Cultivation of RPMI 2650 cells as an in vitro model for human transmucosal nasal drug absorption studies: optimization of selected culture conditions. J. Pharm. Pharmacol. 2012, 64, 1621–1630.

4 5 6 7

(31) Anderle, A.; Niederer, E.; Rubas, W.; Hilgendrof, C.; Spahn-Langguth, H.; WunderliAllenspach, H.; Merkle, H. P.; Langguth, P. P-glycoprotein (P-gp) mediated efflux in Caco-2 cell monolayers: The influence of culturing conditions and drug exposure on P-gp expression levels. J. Pharm. Sci. 1998, 87, 757–762.

8 9 10

(32) Hirohashi, T.; Suzuki, H.; Chu, X. Y.; Tamai, I.; Tsuji, A.; Sugiyama, Y. Function and expression of multidrug resistance-associated protein family in human colon adenocarcinoma cells (Caco-2). J. Pharmcol. Exp. Ther. 2000, 292, 265–270.

11 12 13

(33) Bachmeier, C. J.; Trickler, W. J.; Miller, D. W. Drug efflux transport properties of 2′,7′Bis(2-carboxyethyl)-5(6)-carboxyfluoresceinacetoxymethyl ester (BCECF-AM) and its fluorescent free acid, BCECF. J. Pharm. Sci. 2004, 93, 932–942.

14 15 16

(34) Kodama, N.; Iwao, T.; Katano, T.; Ohta, K.; Yuasa, H.; Matsunaga, T. Characteristic analysis of intestinal transport in enterocyte-like cells differentiated from human induced pluripotent stem cells. Drug. Metab. Dispos. 2016. DOI: 10.1124/dmd.116.069336.

17 18 19 20

(35) Koštrun, S.; Munic Kos, V.; Matanović Škugor, M.; Palej Jakopović, I.; Malnar, I.; Dragojević, S.; Ralić, J.; Alihodžić, S. Around the macrolide – Impact of 3D structure of macrocycles on lipophilicity and cellular accumulation. Eur. J. Med. Chem. 2017, 133, 351– 364.

21 22

(36) Youdim, K. A.; Avdeef, A.; Abbott N. J. In vitro trans-monolayer permeability calculations: often forgotten assumptions. Drug Discov. Today. 2003, 8, 997-1003.

23 24 25

(37) Kindla, J.; Fromm, M. F.; König, J. In vitro evidence for the role of OATP and OCT uptake transporters in drug-drug interactions. Expert Opin. Drug Metab. Toxicol. 2009, 5, 489-500.

26 27 28 29

(38) Seithel, A.; Eberl, S.; Singer, K.; Auge, D.; Heinkele, G.; Wolf, N. B.; Dörge, F.; Fromm, M. F.; König, J. The influence of macrolide antibiotics on the uptake of organic anions and drugs mediated by OATP1B1 and OATP1B3. Drug Metab. Dispos. 2007, 35, 779786.

30 31 32 33

(39) Lu, X.; Chan, T.; Zhu, L.; Bao, X.; Velkov, T.; Zhou, Q. T.; Li, J.; Chan, H. K.; Zhou, F. The inhibitory effects of eighteen front-line antibiotics on the substrate uptake mediated by human Organic anion/cation transporters, Organic anion transporting polypeptides and Oligopeptide transporters in in vitro models. Eur. J. Pharm. Sci. 2018, 30, 132-143.

34 35 36

(40) Lim, S. T.; Forbes, B.; Berry, D. J.; Martin, G. P.; Brown, M. B. In vivo evaluation of novel hyaluronan/chitosan microparticulate delivery systems for the nasal delivery of gentamicin in rabbits. Int. J. Pharm. 2002, 231, 73–82.

37 38 39

(41) Gai, Z.; Visentin, M.; Hiller, C.; Krajnc, E.; Tongzhou, L.; Zhen, J.; Kullak-Ublick, G. A. Organic Cation Transporter 2 Overexpression May Confer an Increased Risk of Gentamicin-Induced Nephrotoxicity. Antimicrob. Agents Chemother. 2016, 60, 5573-5580. 22 ACS Paragon Plus Environment

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


1 2 3

(42) Shao, D.; Massoud, E.; Anand, U.; Parikh, A.; Cowley, E.; Clarke, D.; Agu, R. U. Organic cation transporters in human nasal primary culture: expression and functional activity. Ther Deliv. 2013, 4, 439-451.

4 5 6

(43) Borchard, G.; Cassará, M. L.; Roemelé, P. E. H.; Florea, B. I.; Junginger, H. E. Transport and local metabolism of budesonide and fluticasone propionate in a human bronchial epithelial cell line (Calu-3). J. Pharm. Sci. 2002, 91, 1561–1567.

7 8 9

(44) Mukherjee, M.; Pritchard, D. I.; Bosquillon, C. Evaluation of air-interfaced Calu-3 cell layers for investigation of inhaled drug interactions with organic cation transporters in vitro. Int. J. Pharm. 2012, 426, 7–14.

10 11 12

(45) Gonçalves, V. S. S.; Matias, A. A.; Poejo, J.; Serra, A. T.; Duarte, C. M. M. Application of RPMI 2650 as a cell model to evaluate solid formulations for intranasal delivery of drugs. Int. J. Pharm. 2016, 515, 1–10.

13 14

(46) Kerns, E. H.; Di, L. Physicochemical profiling: overview of the screens. Drug. Discov. Today. Technol. 2004, 1, 343–348.

15 16 17

(47) Nožinić, D.; Milić, A.; Mikac, L.; Ralić, J.; Padovan, J.; Antolovićc, R. Assessment of macrolide transport using PAMPA, Caco-2 and MDCKII-hMDR1 assay. Croat. Chem. Acta. 2010, 83, 323–331.

18 19 20 21

(48) Togami, K.; Chono, S.; Morimoto, K. Distribution characteristics of clarithromycin and azithromycin, macrolide antimicrobial agents used for treatment of respiratory infections, in lung epithelial lining fluid and alveolar macrophages. Biopharm. Drug. Dispos. 2011, 32, 389–397.

22 23 24

(49) Dilger, K.; Schwab, M.; Fromm M. F. Identification of budesonide and prednisone as substrates of the intestinal drug efflux pump P-glycoprotein. Inflamm. Bowel. Dis. 2004, 10, 578–583.

25 26 27 28

(50) Zastre, J.; Jackson, J.; Bajwa, M.; Liggins, R.; Iqbal, F.; Burt, H. Enhanced cellular accumulation of a P-glycoprotein substrate, rhodamine-123, by caco-2 cells using low molecular weight methoxypolyethylene glycol-block-polycaprolactone diblock copolymers. Eur. J. Pharm. Biopharm. 2002, 54, 299–309.

29 30 31

(51) Tang, F.; Ouyang, H.; Yang, J. Z.; Borchardt, R. T. Bidirectional transport of rhodamine 123 and Hoechst 33342, fluorescence probes of the binding sites on P-glycoprotein, across MDCK–MDR1 cell monolayers. J. Pharm. Sci. 2004, 93, 1185–1164.

32 33

(52) Hamilton, K. O.; Backstorm, G.; Yazdanian, M. A.; Audus, K.L. P-glycoprotein efflux pump expression and activity in Calu-3 cells. J. Pharm. Sci. 2001, 90, 647–658.

34 35 36

(53) Draper, M. P.; Martell, R. L.; Levy, S. B. Indomethacin-mediated reversal of multidrug resistance and drug efflux in human and murine cell lines overexpressing MRP, but not Pglycoprotein. Br. J. Cancer. 1997, 75, 810–815.

37 38 39 40

(54) Beéry, E.; Rajnai, Z.; Abonyi, T.; Makai, I.; Bánsághi, S.; Erdő, F.; Sziráki, I.; HerédiSzabó, K.; Kis, E.; Jani, M.; Márki-Zay, J.; Tóth G. K.; Krajcsi, P. ABCG2 modulates chlorothiazide permeability-in vitro-characterization of its interactions. Drug. Metab. Pharmacokinet. 2012, 27, 349–353. 23 ACS Paragon Plus Environment


Page 24 of 30

1 2 3 4

(55) Allen J. D.; van Loevezijn, A.; Lakhai, J. M.; van der Valk, M.; van Tellingen, O.; Reid, G.; Schellens J. H.; Koomen G. J.; Schinkel, A. H. Potent and specific inhibition of the breast cancer resistance protein multidrug transporter in vitro and in mouse intestine by a novel analogue of fumitremorgin C. Mol. Cancer. Ther. 2002, 1, 417–425.

5 6 7

(56) Hidalgo, I. J.; Raub, T. J.; Borchardt, R. T. Characterization of the human colon carcinoma cell line (Caco-2) as a model system for intestinal epithelial permeability. Gastroenterol. 1989, 96, 736–749.

8 9

(57) Foster, K. A.; Avery, M. L.; Yazdanian, M.; Audus, K. L. Characterization of the Calu-3 cell line as a tool to screen pulmonary drug delivery. Int. J. Pharm. 2000, 208, 1–11.

10 11 12 13

(58) Lenz, A.G.; Stoeger, T.; Cei, D.; Schmidmeir, M.; Semren, N.; Burgstaller, G.; Lentner, B.; Eickelberg, O.; Meiners, S.; Schmid, O. Efficient bioactive delivery of aerosolized drugs to human pulmonary epithelial cells cultured in air-liquid interface conditions. Am. J. Respir. Cell. Mol. Biol. 2014, 51, 526–535.

14 15 16

(59) Lee, M. K.; Yoo, J. W.; Lin, H.; Kim, Y. S.; Kim, D. D.; Choi, Y. M.; Park, S. K.; Lee, C. H.; Roh, H. J. Air-liquid interface culture of serially passaged human nasal epithelial cell monolayer for in vitro drug transport studies. Drug. Deliv. 2005, 12, 305–311.

17 18 19

(60) Huang, S.; Wiszniewski, L.; Constant, S.; Roggen, E. Potential of in vitro reconstituted 3D human airway epithelia (MucilAir™) to assess respiratory sensitizers. Toxicol. In. Vitro. 2013, 27, 1151–1156.

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(61) DrugBank website. Available from https://www.drugbank.ca/. Accessed April 4, 2018.

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1

Tables

2 3 4

5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35

Table 1. Main physicochemical properties of therapeutic molecules assessed in bidirectional transport experiments. Class

Molecular weight (g/mol)

logP

Known drug transporters interactions P-gp46,47, OATP1B138, OATB2B138 46,47 P-gp , OATP1B137, OATP1B337

Azithromycin

Macrolide

748.98

4.02a

Clarithromycin

Macrolide

747.95

3.16a

Gentamicin

Aminoglycoside

477.60

-1.6b

OCT138, OCT238,40

Budesonide

Glucocorticoid

450.53

2.42b

P-gp47, OCTs42

logP values were obtained from DrugBank website from experimental properties.61 b : logP values were obtained from DrugBank website from predicted properties.61 a:



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Figure 1. Evolution of the TEER of the ALI RPMI 2650 model (A), permeability of NaF/4.4 kDa Dextran following 2 h of incubation (B), expression of tight junction proteins (C) and immunofluorescence staining of ZO-1 (1), E-cadherin (2) and occludin (3) (D)

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(A) N=6 for each point. (B) N=3 for each compound. (C) N=5 for each protein. The negative control comprised use of the secondary antibody alone. Expression ratios were calculated as the experimental values divided by negative control values. Data are expressed as the mean ± standard deviation. Scale bars 20 µm. 26 ACS Paragon Plus Environment

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1

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Figure 2. Protein expression of the ABC transporters P-gp, MPR1, MRP2, BCRP in RPMI 2650 cells (A) and Caco-2 cells (B). Immunofluorescence staining of P-gp (1), BCRP (2), MRP1 (3) and MRP2 (4) (C) in the ALI RPMI 2650 model.

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N=5 for each condition. The negative control comprised use of the secondary antibody alone. Expression ratios were calculated as the experimental values divided by the negative control values. Scale bars 20 µm. 27 ACS Paragon Plus Environment


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Figure 3. Intracellular accumulation of BCECF (A), rhodamine 123 (B) and Hoechst 33342 (C) inside RPMI 2650 cells treated with probenecid, verapamil, Ko143 and cyclosporin A after 60 min of incubation.

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N=5 for each condition. Intracellular accumulation was normalized to the whole cell protein content. Data are expressed as the mean ± standard deviation.


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Figure 4. Bidirectional permeability of clarithromycin (A), azithromycin (B), gentamicine (C) and budesonide (D) through the RPMI 2650 model after 2 h of incubation.

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N=3 for each concentration. CsA: cyclosporin A. Data are expressed as the mean ± standard deviation. 29 ACS Paragon Plus Environment


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Figure 5. Bidirectional transport of rhodamine 123 (A), BCECF (B) and chlorothiazide (C) through the RPMI 2650 model after 2 h of incubation.

4

N=3 for each concentration. Data are expressed as the mean ± standard deviation.


Pharmacological Characterization of the RPMI 2650 Model as a

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