Evaluation of drug permeation under fed state conditions using mucus-covered Caco-2 cell epithelium

Accepted Manuscript Evaluation of drug permeation under fed state conditions using mucus-covered Caco-2 cell epithelium

Ditlev Birch, Ragna G. Diedrichsen, Philip C. Christophersen, Huiling Mu, Hanne M. Nielsen PII: DOI: Reference:

S0928-0987(18)30109-X doi:10.1016/j.ejps.2018.02.032 PHASCI 4429

To appear in:

European Journal of Pharmaceutical Sciences

Received date: Revised date: Accepted date:

20 January 2018 26 February 2018 27 February 2018

Please cite this article as: Ditlev Birch, Ragna G. Diedrichsen, Philip C. Christophersen, Huiling Mu, Hanne M. Nielsen , Evaluation of drug permeation under fed state conditions using mucus-covered Caco-2 cell epithelium. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Phasci(2017), doi:10.1016/j.ejps.2018.02.032

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ACCEPTED MANUSCRIPT

Evaluation of drug permeation under fed state conditions using mucus-covered Caco-2 cell epithelium

Ditlev Birch1, Ragna G. Diedrichsen1, Philip C. Christophersen2,3, Huiling Mu2 and Hanne M. Nielsen1*

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Center for Biopharmaceuticals and Biobarriers in Drug Delivery, Department of Pharmacy, Faculty of Health

and Medical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark 2

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Section for Pharmaceutical Design and Drug Delivery, Department of Pharmacy, Faculty of Health and

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Medical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark 3

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Present address: Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Måløv, Denmark

Corresponding author

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Hanne Mørck Nielsen

University of Copenhagen Universitetsparken 2

PHONE +45 3533 6346

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2100 Copenhagen, Denmark

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Faculty of Health and Medical Sciences

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Department of Pharmacy

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E-MAIL [email protected]

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ABSTRACT The absence of a surface-lining mucus layer is a major pitfall for the Caco-2 epithelial model. However, this can be alleviated by applying biosimilar mucus (BM) to the apical surface of the cell monolayer, thereby constructing a mucosa mimicking in vivo conditions. This study aims to elucidate the influence of BM as a

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barrier towards exogenic compounds such as permeation enhancers, and components of fed state simulated intestinal fluid (FeSSIF). Caco-2 cell monolayers surface-lined with BM were exposed to several

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compounds with distinct physicochemical properties, and the cell viability and permeability of the cell

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monolayer was compared to that of cell monolayers without BM and well-established mucus-secreting epithelial models (HT29 monolayers and HT29/Caco-2 co-culture monolayers). Exposure of BM-covered

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cells to constituents from FeSSIF revealed that it comprised a strong, hydrophilic barrier effect as 90% of BM-covered cells remained viable for > 4 h, and the permeation rate of hydrophobic drugs was reduced. In

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contrast, the permeation rate of hydrophilic drugs was largely unaffected. Control monolayers displayed a loss of barrier function and < 10% viable cells. The efficacy of fatty acid permeation enhancers were altered

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when investigated in BM-covered cells as compared to all the other studied epithelial models. Thus, Caco-2

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cell monolayers surface-lined with BM constitute a valuable in vitro model that makes it possible to mimic

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intestinal fed state conditions when studying drug permeation.

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KEYWORDS Caco-2 cell culture model; biosimilar mucus; fed state simulated intestinal fluid; fatty acids; drug delivery;

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permeation enhancers

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ABBREVIATIONS BM, Biosimilar mucus; C10, sodium caprate; C12, sodium laurate; NaTC, sodium taurocholate; PC, phosphatidylcholine; MO, monoolein; OA, oleic acid; TEER, transepithelial electrical resistance; HBSS, Hank’s Balanced Salt Solution; PAA, polyacrylic acid; FeSSIF, fed state simulated intestinal fluid; Papp,

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apparent permeation coefficient

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1. INTRODUCTION The Caco-2 cell culture model was first described in the 1980’s as a model for the human intestinal epithelium expressing tight junctions as well as biorelevant levels of metabolic enzymes and active transporters (1,2). Since then, it has been widely used as an in vitro tool for investigating the uptake and

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transport of drug candidates in the small intestine, testing drug delivery systems with a variety of properties (3–5) and combinations of excipients which can potentially enhance the transepithelial

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permeation of some drugs (4,6,7). Additionally, the Caco-2 model has been applied in combination with

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other in vitro models to improve the biorelevance of the model (8–10). Despite the versatility of the Caco-2 cell culture epithelium, a significant limitation is the absence of a surface-lining mucus layer (11). The

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absence of mucus limits the use of in vivo relevant levels of exogenous constituents in the incubation medium during an experiment. In addition, as mucus constitutes a steric and interactive barrier towards

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the constituents of the intestinal fluid (12), the presence (or absence) of mucus may affect the drug permeation rate and the degree of interaction between compounds and the epithelium (10). Despite being

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proven to affect the permeation of some drugs (13), experimental work with fed state simulated intestinal

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fluid (FeSSIF, (14)) has so far been limited. In the rare examples from previous studies, it is often concluded that the medium is incompatible with the cells (8,15,16), or it is being used as modified versions containing

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reduced concentrations of bile salts and/or lipids compared to fed state levels (9,17). Therefore, significant efforts have been put into developing models, which combines the versatility of the Caco-2 cell culture

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model with a mucus layer, and co-culturing of Caco-2 cells with mucus-producing cells has been the primary focus (18). Although such models have been investigated for more than twenty years, a model providing the desired protective effect combined with a tight epithelial barrier has yet to be established (19). An alternative to a co-culture model is including mucus aspirates on the apical surface of the Caco-2 cell epithelium. However, the applicability of this approach is often limited due to the high osmolality (> 350 mOsm/kg) caused by the mucus isolated from the intestines of animals, and this proves to be detrimental to the Caco-2 cells (10). Porcine mucins have been examined as constituents in the incubation medium

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applied to Caco-2 cells. In these studies, the aim were to investigate the protective effects of mucins towards damage induced by intestinal components during the fasted state (20), bacterial translocation (21) as well as norovirus particles (22). This approach may provide some protective effect, but does not encompass the viscoelastic properties of in vivo mucus. Recently, the applicability of an isotonic biosimilar

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mucus (BM) matrix that exhibits similar rheological properties as mucus collected from pig intestines has been characterized and evaluated (10,23,24) as an alternative to using animal aspirates and co-culture

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models. The BM reproducibly provides the desired steric and interactive barrier properties without

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compromising the epithelial integrity or decreasing the viability of the exposed cells (24). Therefore, this study aimed to evaluate the utility of Caco-2 cell epithelium surface-lined with BM (hereafter denoted

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Caco-2BM). Specifically, the barrier capabilities of the mucosa model were related to effects on transmucosal drug permeation when applying solutions containing bile salts and lipids, mimicking the

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composition of fed state intestinal fluid. In addition, the effect of BM and fed state simulated intestinal fluid on the potency of the permeation enhancers sodium caprate (C10) and sodium laurate (C12) (6,25) was

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investigated. Drug permeation and tolerance towards test compounds of the Caco-2BM model were

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compared to that of the standard Caco-2 cell epithelium, as well as to that of monolayers of mucusproducing cells (HT29 cells) and of the Caco-2/HT29 co-culture model. The examined compounds included

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in this study were selected to accommodate the physico-chemical properties of multiple BCS classes of

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compounds as well as routes of epithelial permeation and observed oral bioavailability (Table 1).

2. MATERIALS AND METHODS 2.1 Materials

Caco-2 cells (ACC 169) were purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen (Braunschweig, Germany). HT29-MTX-E12 cells were kindly donated by Professor David Brayden, University College Dublin, originally sourced from Professor Per Artursson, Uppsala University. 14C-D-mannitol (57.2 Ci/mmol) and Ultima Gold™ scintillation fluid were obtained from Perkin Elmer (Boston, MA, USA). 3H-L-

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cyclosporine (20 Ci/mmol), 3H-metoprolol (20 and 80 Ci/mmol) and 3H-L-ovalbumin (40 Ci/g) were from American Radiolabeled Chemicals (St. Louis, MO, USA). Oleic acid (Pharma grade), N-2-

hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES, ≥ 99.5%) and 2-(N-morpholino)ethanesulfonic acid hydrate (MES, ≥ 99.5%) were from PanReac AppliChem (Darmstadt, Germany). Fenofibrate

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was generously donated by LifeCyclePharma (originally manufactured by ChemAgis, Beer-Sheva, Israel). Monoolein (MO, ≥ 90%) was generously donated by Danisco (DuPont Nutrition Biosciences, Grindsted,

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Denmark). Phosphatidylcholine from soybean (PC, ≥ 99.0%) was purchased from Lipoid (Ludwigshafen,

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Germany). Polyacrylic acid, Mw 29,400-39,400 (PAA) was purchased from Lubrizol (Brussels, Belgium). Sodium caprate (C10, ≥ 98.0%), sodium laurate (C12, ≥ 98.0%), albumin from chicken egg white (ovalbumin,

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≥ 98.0%), cyclosporine (≥ 99.0%), sodium taurocholate hydrate (NaTC, ≥ 95.0%), bovine serum albumin (BSA, ≥ 98.0%), cholesterol (≥ 99.0%), mucin (type II from porcine stomach) and phenazine methosulfate

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(PMS, ≥ 90.0%) were from Sigma-Aldrich (St. Louis, MO, USA). Hank’s Balanced Salt Solution (HBSS) and fetal bovine serum were from Gibco Life Technologies (Paisley, UK). MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-

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carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) was purchased from Promega (Madison, WI,

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USA). All other reagents, including cell culture materials, were from Sigma-Aldrich (St. Louis, MO, USA). Ultrapure water from a Barnstead NanoPure system (Thermo Scientific, Waltham, MA, USA) was used for

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2.2 Methods

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all studies. All reagents and chemicals were of analytical grade unless otherwise specified.

2.2.1 Cell culture models

Caco-2 cells and HT29 cells were maintained in cell culture flasks (175 cm2 surface area, Sigma-Aldrich) in Dulbecco’s Modified Eagles Medium supplemented with 10% v/v fetal bovine serum, penicillin (100 U/mL), streptomycin (100 μg/mL), L-glutamine (1% v/v) and non-essential amino acids (1% v/v). The cells were subcultured by trypsinization on a weekly basis. Cells for experiments were seeded onto polycarbonate Transwell® filter inserts (1.12 cm2 surface area, 0.4 μm pore size, Corning Costar from Sigma-Aldrich) at a

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total cell seeding density of 9.0 × 104 cells/cm2 for Caco-2 and HT29 cell monolayers. Caco-2 and HT29 cell suspensions were mixed at a ratio of 1:1 for a final concentration of 9.0 × 10 4 cells/cm2 of both cell types. The monolayers were kept in a humidified incubator at 37 °C and 5% CO2. The growth medium was replaced every second or third day. Permeability studies were performed after 18-20 days of culturing for

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Caco-2 cells, and after 25-27 days for HT29 cells and Caco-2/HT29 cell co-cultures, in all cases 18-24 h after the last medium change. All experiments were performed on two passages of cells unless otherwise

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specified, using nine and three different passages of Caco-2 or HT29 cells, respectively.

2.2.2 Biosimilar mucus

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The preparation of BM was slightly modified from Boegh et al. (24) by omitting linoleic acid from the original recipe. This approach did not affect the rheological properties of the mucus, and was carried out to

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prevent any contribution by other fatty acids than the one included in the test solutions. Briefly, solution A containing cholesterol (0.36% w/v), polysorbate 80 (0.163% w/v) and PC (0.18% w/v) were mixed in isotonic

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buffer (10 mM MES, 1.30 mM CaCl2, 1.00 mM MgSO4 and 137 mM NaCl dissolved in ultrapure water and

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adjusted to pH 6.5). Simultaneously, solution B containing mucin (5% w/v), BSA, (3.1% w/v) and PAA (0.9% w/v) was prepared in a similar buffer, but without NaCl. The solutions were mixed at a volume ratio of 1:9

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the use.

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(A:B) and the pH was adjusted to 6.5 with NaOH. The mucus was left to equilibrate overnight at 4 °C before

2.2.3 Epithelial integrity

The transepithelial electrical resistance (TEER) was measured in all cell monolayers before the permeability experiments. The cell monolayers were washed twice with hHBSS (10 mM HEPES, 0.05% w/v BSA dissolved in Hank’s Balanced Salt Solution (HBSS) and adjusted to pH 7.4) and equilibrated for 20 min at room temperature (RT) in the second wash medium followed by measurement of TEER using a resistance chamber connected to a voltmeter (Endohm and EVOM, respectively, both from World Precision

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Instruments, Sarasota, FL, USA). Monolayers displaying an initial TEER below 200 Ω × cm2 were discarded. This procedure was repeated after the permeability experiments; albeit the cell monolayers were washed three times to ensure complete removal of the BM.

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2.2.4 Epithelial cell viability The MTS/PMS assay was used to verify the effect on the cellular viability for all cell models, and performed

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immediately after the permeability and integrity assays by adding 330 µL of MTS/PMS in buffer (240 µg/mL

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MTS and 2.4 µg/mL PMS dissolved in hHBSS) and 1000 µL of hHBSS to the apical and basolateral side of the cell monolayers, respectively. The cell monolayers were subsequently kept on a shaking table (37 °C, 50

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rpm, Thermo MaxQ 2000, Thermo Fischer Scientific, West Palm Beach, FL, USA) for 1 h. Samples of the MTS/PMS buffer were collected in a 96-well plate and the absorbance measured at 492 nm using a plate

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reader (POLARstar OPTIMA, BMG LABTECH, Ortenberg, Germany).

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2.2.5 Membrane permeability

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Before applying the test compounds, the Caco-2 cell monolayers were washed twice and covered with 250 µL BM on the apical side and 1000 µL hHBSS on the basolateral side, and kept at 37 °C for 10 min. Caco-2

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cell monolayers, HT29 cell monolayers, and Caco-2/HT29 co-culture monolayers were treated similarly using mHBSS (10 mM MES, 0.05% w/v BSA dissolved in HBSS and adjusted to pH 6.5) instead of BM. After

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equilibration, 100 µL test solutions were added to the apical side of the cell layer. The test solutions consisted of mHBSS, mHBSSFeSSIF (concentration in the 350 µL apical volume: 10 mM NaTC, 2 mM PC, 0.8 mM OA, 5 mM MO dissolved in mHBSS and adjusted to pH 6.5), C10 (concentration in the 350 µL apical volume: 10-100 mM in mHBSS or mHBSSFeSSIF) or C12 (concentration in the 350 µL apical volume: 1-10 mM in mHBSS or mHBSSFeSSIF). All test solutions were spiked with 3H-metoprolol (0.5 µCi/mL), 14C-mannitol (1 µCi/mL), fenofibrate (1 µg/mL or 50 µg/mL in mHBSS or mHBSSFeSSIF, respectively), desmopressin (1 mg/mL), cyclosporine (1 µg/mL or 50 µg/mL in mHBSS or mHBSSFeSSIF, respectively, both spiked with 1 µCi/mL 3H-

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cyclosporine) or ovalbumin (1 mg/mL, spiked with 0.25 µCi/mL 3H-ovalbumin). Further, Ca2+ and Mg2+ were omitted from the test solution medium for the test solutions containing C10 or C12 to prevent precipitation of the lipids. The cells were then kept on a shaking table (37 °C, 50 rpm, Thermo MaxQ 2000, Thermo Fischer Scientific) for 4 h, and 100 µL samples were collected from the basolateral chamber at 15-30 min

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intervals, with replenishment of the collected sample volume with hHBSS (37 °C). 2 mL scintillation fluid was mixed with each radioactive sample that was subsequently quantified by liquid scintillation (Perkin-

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Elmer Tri-Carb 2910 TR, Perkin Elmer, Boston, MA, USA). Desmopressin, fenofibrate and fenofibric acid

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(degraded fenofibrate) were quantified by LC-MS as described below.

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2.2.6 Desmopressin, fenofibrate, and fenofibric acid quantitative analysis Desmopressin, fenofibrate, and fenofibric acid was quantified using HPLC-MS on an Agilent HPLC equipped

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with a binary pump system, column oven and autosampler, all from Agilent Technologies, the 1200 series (Agilent Technologies, Colorado Springs, CO, USA). The separation of all compounds was performed by

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injecting 10 µL of sample kept at 4 °C, and elution were performed through a Phenomenex Aeris Peptide

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column (Xb-C18; 100 Å, 100 × 2.1 mm, 3.6 μm) kept at 40 °C. For desmopressin, a mobile phase consisting of 0.1% v/v formic acid in ultrapure water as solvent A and 0.1% v/v formic acid in acetonitrile as solvent B

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was used with a gradient of 10% B  50% B over 2 min, followed by 2 min at 50% B. For fenofibrate and fenofibric acid, a mobile phase consisting of 0.33% v/v formic acid in ultrapure water as solvent A, and

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methanol as solvent B was used with a gradient of 40% B  95% B over 3.5 min followed by 1 min at 95%. The flow rate was 0.5 mL/min for separation of desmopressin, and 0.7 mL/min for fenofibrate and fenofibric acid. The quantification was performed using an Agilent Technologies 6140 Quadrupole. The molecular ion was detected using selective ion monitoring at a mass-to-charge ratio (m/z) of 1069.4, 361.1 and 317.0 for desmopressin, fenofibrate and fenofibric acid, respectively. Data were recorded and analyzed by Agilent ChemStation. The limit of detection (LOD) for all compounds was < 10 ng/mL.

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2.2.7 Data analysis All statistical analysis was performed in GraphPad Prism 7 (GraphPad Software, La Jolla, CA, USA) using a one-way ANOVA combined with a Tukey’s multiple comparison analysis for comparison of the mean of individual dataset (e.g., different TEER). In addition, a two-way ANOVA combined with a Dunnett’s multiple

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comparisons analysis was employed to compare the means of different concentrations within a treatment as well as between treatments (e.g., different concentrations of FA with different incubation medium). The

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calculation of the apparent permeability coefficients (Papp) and the relative viability of the cells are

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described elsewhere (27,55). Lag phases were determined as the intersection of the steady-state flux curve

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with the initial flux curve (during the lag phase).

3. RESULTS

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3.1 Effect of simulated fed state intestinal fluid on the integrity and viability of cell monolayers surface-lined with biosimilar mucus

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To evaluate the ability of BM to protect the Caco-2 cell epithelium and thus prevent previously reported

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detrimental effects to cells caused by constituents in mHBSSFeSSIF (8,15,16), the monolayers with and without BM were exposed to mHBSSFeSSIF. Established mucus-secreting cell culture models, namely mucus-

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producing HT29 monocultures and Caco-2/HT29 co-cultures were also used for comparison. When comparing the cellular viability (i.e. metabolic activity) and epithelial integrity (i.e. TEER) in Caco-2

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monolayers with and without BM, and when exposed to mHBSSFeSSIF for 4 h, it was observed that the cell epithelia covered with BM displayed only an insignificant decrease in viability as compared to the controls (Fig. 1A), and no decrease in TEER (Fig. 1B). In comparison, cells exposed to mHBSSFeSSIF in the absence of BM displayed < 10% cell viability and a significant reduction in TEER (10% of initial value) as compared to mHBSS-exposed cells. Notably, the control cells also exhibited a decrease in TEER (55.7% of initial value). HT29-cell monocultures and co-cultured Caco-2/HT29-cells did also display a decrease in viability (43.6% and 35.0% compared to mHBSS, for HT29 cells and Caco-2/HT29 cell co-cultures, respectively, Fig. 1) and in

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TEER (to 5-10% of the initial value for both cell lines). The barrier integrity as measured by the Papp value, total permeated amount, lag phase and time span for steady-state flux of the paracellular marker mannitol was compared for Caco-2BM cells incubated with mHBSS and mHBSSFeSSIF (Table 2). It was observed that the flux of mannitol across BM-covered Caco-2 monolayers exposed to mHBSSFeSSIF was comparable to that of

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BM-covered cells exposed to mHBSS, displaying similar Papp values (approximately 0.1 × 10-6 cm/s) and a total permeation of < 1% of the added dose for both media. In addition, no lag phase was observed, and

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the flux remained at steady-state for the entire experiment. In comparison, Caco-2 monolayers without BM

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exposed to mHBSSFeSSIF displayed a significantly higher permeability, and correspondingly the total permeated amount of hydrophilic marker increased to 19.7%. This tendency was the same with the HT29

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cell monolayers (Papp of 3.0 × 10-6 cm/s, 12.9% permeated, Fig. S2) and the co-cultured Caco-2/HT29 monolayers (Papp of 4.0 × 10-6 cm/s, 10.4% permeated), together with the presence of a lag phase and a

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shorter timespan for steady-state flux.

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For metoprolol, it was observed that the permeation across Caco-2BM was not affected by the presence of

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mHBSSFeSSIF (Papp of 1.9 × 10-6 cm/s, 8.2% permeated dose and 2.1 × 10-6 cm/s, 8.5% permeated dose when kept with BM and mHBSS or mHBSSFeSSIF, respectively). Further, a lag phase was absent in both cases. In

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comparison, Caco-2 cell monolayers without BM and co-cultured Caco-2/HT29 cell monolayers both displayed an approximately 1.5-fold increase in permeation rate and relative permeated amount upon

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exposure to mHBSSFeSSIF, as well as a shorter time-span of the steady-state flux. The permeation rate across HT29 cell monolayers was unaffected by exposure to mHBSSFeSSIF. However, the lag phase and timespan of the steady-state flux were both shorter compared to that observed with the mHBSS-exposed HT29 monolayers.

3.2. Role of biosimilar mucus on the permeation enhancing effects of medium-chain fatty acids

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To investigate whether and how BM constitutes a barrier to the effect of fatty acids (FAs), Caco-2 and Caco2BM cell culture monolayers were exposed to C10 or C12, and the effects on integrity, viability and permeability to mannitol and metoprolol was monitored. For Caco-2BM cells, the viability decreased by 1020% upon exposure to C10 (10-20 mM, Fig. 2A) or C12 (1-10 mM, Fig. 2B). However, the viability was only

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significantly lower than the control for C10 at concentrations above 20 mM. The TEER for the same cell monolayers following the 4 h experiments were significantly lower for all C10 (1.9-63.4%) or C12 (43.1-

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89.2%) combinations, compared to the control. The Papp for mannitol was increased by the addition of C10

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at concentrations of 20 mM or higher (Fig. 3A), but it was unaffected by C12 (Fig. 3B). Contrary to this, the Papp for metoprolol was reduced at higher concentrations of both FAs. In comparison, Caco-2 cell

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monocultures, as well as HT29-cell monocultures and co-cultured Caco-2/HT29 cell monolayers exposed to 10 mM C10 all displayed significantly lower cell viabilities (compared to control cells) and TEER values

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(relative to initial values). These observations were corroborated by increased Papp values for mannitol (3.9 × 10-6 cm/s, 3.1 × 10-6 cm/s and 5.1 × 10-6 cm/s for Caco-2 cell monolayers, HT29 cell monolayers and Caco-

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2/HT29 cell monolayers, respectively, Fig. 3A and Fig. S2A); effects that became more significant at higher

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concentrations of FA. The permeation rate of metoprolol was associated with an immediate increase at 10 mM C10 (9.4 × 10-6 cm/s 10.8 × 10-6 cm/s, 12.2 × 10-6 cm/s for Caco-2, HT29, and Caco-2/HT29 cell

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Caco-2BM cells.

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monolayers, respectively); an increase, which decreased at higher concentrations of FA, as observed for

Upon exposure to C12 at concentrations of 2 mM or higher, a similar viability-lowering effect was observed for Caco-2 cell monolayers, HT29 cell monolayers, and Caco-2/HT29 co-cultured monolayers. This observation was also associated with an increase in the Papp for mannitol (4.0 × 10-6 cm/s, 2.1 × 10-6 cm/s and 5.1 × 10-6 cm/s for Caco-2, HT29 and Caco-2/HT29, respectively). In addition, the Papp of metoprolol increased for FA concentrations up to 5 mM C12 (8.2 × 10-6 cm/s, 8.9 × 10-6 cm/s and 9.1 × 10-6 cm/s for Caco-2, HT29, and Caco-2/HT29, respectively) and subsequently decreased as previously reported.

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Replacing mHBSS with mHBSSFeSSIF as a solvent for C10 and C12 for Caco-2BM cell monolayers resulted in a negligible increase in the Papp values for both mannitol and metoprolol (Table S1) and a similar development in TEER (Fig. S4) for most concentrations of FA. However, cell monolayers displayed higher

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cell viability after being exposed to C10 added to mHBSSFeSSIF at a concentration of 20 mM (approximately 100% viability) or 50 mM (approximately 80% viability) compared to when C10 was added to mHBSS

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(approximately 80% and 40% viability at 20 mM or 50 mM, respectively, Fig. S3). This effect was similarly

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observed after exposure to C12; albeit the differences were slightly lower (100% in mHBSSFeSSIF compared to approximately 85% in mHBSS at 2 mM and 5 mM). Furthermore, an increase in the amount of permeated

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metoprolol was observed for both C10 (2.2-4.3% of the initial dose) and C12 (1.9-3.3% of the initial dose), along with a prolonged timespan of the steady-state flux phase and a shorter or absent lag phase. The

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effect on the mannitol permeation was ambiguous, as some combinations displayed higher permeation

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rate and permeated amount in mHBSS whereas other did in mHBSSFeSSIF.

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3.3. Assessment of the biosimilar mucus-covered Caco-2 cell monolayers for transmucosal drug delivery studies

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The permeability properties of Caco-2 cell monolayers and Caco-2BM cell monolayers were compared for selected macromolecular drugs for assessment of the barrier functions of BM, the effect of mHBSSFeSSIF, as

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well as the effect of 5 mM C12 or 20 mM C10 as permeation enhancers in the mucus-covered epithelium. Test concentrations of C10 and C12 were selected by considering their effects on mannitol permeation and cytotoxicity (Fig. 2, Fig. 3). The Papp values obtained for desmopressin and ovalbumin were reduced in Caco2BM cell monolayers (0.04 × 10-6 cm/s and 0.2 × 10-6 cm/s, respectively, compared to 0.4 × 10-6 cm/s and 0.3 × 10-6 cm/s for desmopressin and ovalbumin, respectively, obtained without BM (Table 3). These values correlated with a lower permeated amount in Caco-2BM cell monolayers for desmopressin (0.1%, down from 1.4%) and a negligible amount for ovalbumin (0.7% down from 0.8%). Addition of mHBSSFeSSIF to Caco-2BM

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cell monolayers increased the Papp values for permeation of these two compounds across the BM-covered epithelium (0.1 × 10-6 cm/s and 0.4 × 10-6 cm/s for desmopressin and ovalbumin, respectively). The Papp values and permeated amounts of desmopressin were further increased by the addition of FAs dissolved in mHBSSFeSSIF (approximately 30-fold and 10-fold for C10 and C12, respectively). These values exceeded those

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obtained with the Caco-2 cell monolayers in the absence of BM. The Papp values (at steady-state) for ovalbumin in mHBSSFeSSIF were not affected by the presence of FAs, although the permeated amount was

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significantly increased (to 1.6% and 2.1%, for C10 and C12, respectively). This observation correlates with a

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prolonged duration of the steady-state flux to > 240 min with both C10 and C12, as compared to 115 min without the presence of a FA.

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Similar to the observations with desmopressin and ovalbumin, the Papp value for cyclosporine was reduced (0.3 × 10-6 cm/s) when investigated in Caco-2BM cell monolayers as compared to Caco-2 cell monolayers (0.6

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× 10-6 cm/s). However, contrary to the observations with desmopressin and ovalbumin, and in line with observations for metoprolol, the Papp value was further reduced upon addition of mHBSSFeSSIF, and was

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unaffected by the addition of FAs. Similar trends were observed for the permeated amount of cyclosporine

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and the timespan for the steady-state flux. No permeation of fenofibrate across Caco-2 and Caco-2BM cell monolayers was detectable for any concentration or buffer composition as well as with or without BM, and

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4. DISCUSSION

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the permeation parameters are thus not reported.

It is of utmost importance to study the interaction between exogenic compounds and mucosal membranes when designing and developing new drugs. In vitro cell culture models are used to a great extent in these studies. In excess, exogenous compounds like bile salts, FAs, and permeation enhancers induce detrimental changes to epithelial cell monolayers cultured in vitro (8,15,56). The detrimental effects, which arise upon exposure of cells to solutions such as FeSSIF may be a result of several factors. These factors include hypoor hypertonicity of the incubation solution (15,57) and elevated levels of bile salts and extracellular cations

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such as calcium (49,58). Non-isotonic solutions may lead to swelling or shrinking of the cell, which in turn affects cell homeostasis (57,59). Furthermore, bile salts have been reported to increase plasma membrane fluidity by dissolution of membrane lipids (60), as well as affecting ATP formation (61). The described factors may all lead secondary cytotoxic effects, e.g., by influx of extracellular ions which accumulate and

PT

disrupt the cellular organelles (58,62) or altered activity of intracellular enzymes (61,63). In vivo, the epithelial cells are protected by the mucus barrier against exogenous compounds. The barrier is divided

RI

into the glycocalyx closest to the membrane, the firmly adhered mucus and the loosely adhered mucus,

SC

which faces the lumen (64). Estimating the thickness of the mucus barrier is complicated in particular by the dynamic state of the loosely adhered mucus, which is continuously shedded into the lumen, and is

NU

easily removed upon mechanical agitation (65), e.g., following ingestion. As a consequence, reports on the average thickness of the mucus barrier in the small intestines varies significantly, but typically estimates the

MA

thickness to be 100-200 µm (66,67). For the Caco-2 cell culture model, the absence of a mucus layer surface-lining the epithelium leads to increased interaction between constituents of the test medium and

D

the membrane posing an increased risk of compromising the integrity of the epithelial barrier. The co-

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cultured Caco-2/HT29 cell culture model simulates a mucosal barrier like that of the small intestine, and the presence of mucus is expected to limit the risk of epithelial membrane damage caused by exogenous

CE

compounds (9). Although an increased tolerance towards exogenous compounds is achieved, it was observed in this study that the cell monolayer integrity was still compromised, when exposed to bile salts

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or FAs, at levels present in the small intestine following food intake, i.e., at fed state (68). Although BM cannot mimic the dynamic state of mucus in vivo, i.e. shedding and recycling (65), nor the variety of mucins that are present in human mucus, it may constitute a steric and interactive barrier, which limits detrimental effects induced by exogenous compounds to cell epithelia in vitro. The volumes of BM employed in the current study constitute a mucus barrier of ∼ 225 µm. This is within the range of what is observed in vivo, albeit the volume was a requirement in order to obtain a homogenously distributed mucus barrier which covered the entire cell surface.

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4.1 Effects of fed state intestinal components on Caco-2 monolayers surface-lined with biosimilar mucus The data presented in this study confirms previous observations that Caco-2 cell monolayers do not maintain biological barrier properties or metabolic activities following exposure to the constituents of fed

PT

state intestinal fluid (15,49). Previous approaches to avoid the effect on cell viability resulting from exposure to simulated fed state medium include reducing the concentration of bile salts (17) or increasing

RI

the concentration of PC (69). The latter study by Patel et al. did recognize that even low amounts of bile

SC

salts (approximately 4 mM of taurocholate) resulted in a 50% decrease in Caco-2 cell viability. Moreover, fed state medium incorporating bile salts and phospholipids in a biorelevant ratio resulted in reduced cell

NU

viability (66,68,70). The amount of constituents investigated in this study were based on the FeSSIF-V2 developed by Jantratid et al. (14) for in vitro dissolution studies. The sodium chloride content was reduced

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and the buffer components changed as compared to the original formula to eliminate cellular effects, which would be caused by high osmotic pressure and low pH, respectively. The amount of C10 was based

D

on postprandial levels of FAs, as reported by Vertzoni et al. (68) and Armand et al. (70). In comparison, a

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reduced amount of C12 was used in order to accommodate its lower solubility in the test medium, as well as the higher permeability enhancing effect conferred by C12 compared to that of C10 (6). These

CE

concentrations of FAs resulted in cytotoxic effects, expressed as a decreased metabolic activity of the cells, at all tested concentrations of FAs in Caco-2 cell monolayers. A significant decrease (> 30%) in cell viability

AC

was observed for both C10 and C12 at the lowest tested concentrations, and was even more pronounced at higher concentrations of FA. These data corroborate with reported values on the cytotoxicity of C10 and C12 tested in Caco-2 cells (25,71). By surface-lining Caco-2 cell monolayers with BM, the detrimental effect on cell viability caused by the FAs was reduced when the FA was dosed in mHBSS. The reduced negative effect of the FAs on the epithelial cells was even more prominent when mHBSSFeSSIF was used. This difference suggests an interaction between FAs and the constituents of mHBSSFeSSIF. One explanation could be an increased formation of mixed micelles composed of FAs, PC, and NaTC. The critical micelle

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concentration (CMC) for NaTC and PC were reported as 8 mM (72) and 0.61-0.92 mg/mL (corresponding to 0.8-1.2 mM (73,74), and 26.1 mM an 2.8 mM for C10 and C12, respectively (56). Hence, the CMC of NaTC and PC are lower than the concentrations of both NaTC and PC (10 mM and 2 mM, respectively) used in the present study, whereas the CMC for the fatty acids were examined above and below CMC. These conditions

PT

imply that the free fractions of NaTC and PC, as well as of C10 or C12, are greatly diminished under test conditions applying mHBSSFeSSIF in comparison to those obtained when using mHBSS. Consequently, the

RI

amount available for direct interaction with the membrane will be reduced, as the lipids will be partially

SC

incorporated into micelles. Hence, the improved cell viability observed for Caco-2BM cells kept with lipids in mHBSSFeSSIF compared to mHBSS can be ascribed to a reduction in the free FA concentration and not

NU

directly to the presence of BM. Notably, the concentration of bile salts necessary to form micelles requires BM in order to not be cytotoxic. The formation of mixed micelles may also explain the reduced permeation

MA

rate of metoprolol at high lipid concentrations. As metoprolol is hydrophobic (LogP of 1.9, Table 1), the formation and subsequent partition of drug into mixed micelles will greatly reduce the amount of free drug,

D

thereby reducing Papp. This observation is corroborated in a study by Wuyts et al., wherein several lipophilic

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compounds were reported to exhibit a reduced permeation rate across rat ileum in fed state medium compared to that observed when dosed in fasted state medium, which was attributed to partitioning of the

CE

compounds into micelles (49). These observations are in line with a study by Gradauer et al. (75), who observed a reduced effect of several permeation enhancers upon dissolution in fasted or fed state

AC

simulated intestinal fluid as compared to when buffer was used. In addition, the presence of micelles contributed to the solubilization of the lipid components (68), thereby increasing the physical stability of the dispersed lipids by limiting phase-separation. Preserving a pseudo-homogenous system may reduce the risk of formation of aggregated free FA that can interact locally with the monolayer.

4.2 Barrier properties of biosimilar mucus towards drug-membrane interactions

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The permeability data obtained for cell monolayers suggests that Caco-2 cell monolayers, HT29 cell monolayers, and co-cultured Caco-2/HT29 cell monolayers all displayed an increased permeability for both mannitol and metoprolol, along with a substantial decrease in TEER when exposed to various amounts of C10 or C12, or mHBSSFeSSIF. Both read-out parameters suggest a strong interaction between exogenous

PT

compounds and the epithelial cells. However, this was only observed for cell monolayers, which also displayed decreased cell viability. Since no increase in the permeation of test compounds was observed at

RI

non-cytotoxic concentrations, it was not possible to determine whether the effect on drug permeation and

SC

TEER was due to a reversible effect on the tight junctions. Additionally, an irreversible, cytotoxic effect leading to loss of epithelial integrity could lead to an increase in drug permeation and a decrease in TEER.

NU

The increase in membrane permeability as a result of FA exposure correlates well with reported literature on the permeation enhancing effect of FAs (6,25,50). However, other reports suggest that C10 and C12

MA

induces cytotoxicity in the same concentration range (25,71,76). Therefore, it cannot be ruled out that the enhancing effect result from compromising the epithelial cells. In addition, Whitehead et al. (77) observed

D

that treatment of Caco-2 cell monolayers with 1% v/v Triton™ X-100, thereby effectively dissolving the

PT E

bilayer, resulted in a Papp value for mannitol at 5.5 × 10-6 cm/s, effectively meaning that Papp values close to this suggests unhindered diffusion of mannitol. The same authors also observed that cells exposed to OA, a

CE

fatty acid-based permeation enhancer, displayed a very narrow window of concentrations that elicited a permeation enhancing effect and concentrations that were cytotoxic to Caco-2 cells. As stressed cells are

AC

prone to autophagocytosis which leads to an up-regulation of the metabolic activity (78) , it is likely that the safe concentration threshold for each FA may be even lower than anticipated. Upon surface-lining with BM, the permeability of Caco-2 cell monolayers to a small molecule paracellular marker (mannitol) was not affected when exposed to both mHBSS and mHBSSFeSSIF, as compared to Caco-2 cell monolayers. Contrary to this, the permeation rate of a small molecule transcellular marker (metoprolol) was significantly reduced 2-2.5-fold. These data correlate with the hypothesis that BM constitutes a hydrophilic barrier, which primarily limits the permeation of hydrophobic compounds (10). The data are

19

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supported by the observations of Boegh et al., who used a similar setup and reported a negligible decrease in mannitol permeation, and a significant decrease in testosterone permeation following the surface-lining of Caco-2 cell monolayers with BM (10). Testosterone is a small molecule transcellular marker, which expresses similar physicochemical properties and permeation rate across Caco-2 cell monolayers as

PT

metoprolol (26). The same authors also conducted studies on FITC-BSA, a protein that expresses similar properties as ovalbumin included in this study. Here, the authors observed a significant lag phase

RI

(approximately 90 min) and a 3.3-fold reduction in the Papp value when surface-lined Caco-2 cell monolayers

SC

were used. While the reduction in the Papp value observed for ovalbumin were less than for BSA (approximately 1.5-fold), both compounds displayed Papp values in the range of 1-3 × 10-7 cm/s,

NU

independent of the presence or absence of BM on the surface of the Caco-2 epithelium. These results suggest that BM has little to no effect on the diffusion of large, hydrophilic compounds through the

MA

mucosa. Finally, the 2-3-fold reduction in the Papp value for cyclosporine tested in Caco-2BM cell monolayers overall correlated well with the observed values for metoprolol (Table 1). Upon examining cyclosporine and

D

fenofibrate which are hydrophobic (LogP of 2.9 and 5.1, respectively, table 1), it was observed that the Papp

PT E

of cyclosporine was reduced by substituting mHBSS for mHBSSFeSSIF (Table 2, Fig. 3), whereas fenofibrate was not detected on the basolateral side following incubation with either buffer. The reduced permeation

CE

rate for cyclosporine in mHBSSFeSSIF is consistent with the behavior of metoprolol in media containing lipids and bile salts above CMC. Hence, partitioning of cyclosporine into mixed micelles provide a reasonable

AC

explanation for the reduced permeation rate in mHBSSFeSSIF. Notably, the actual amount of cyclosporine that permeated across the monolayer was increased from 65 ± 2 pmol to 3183 ± 150 pmol when dosed in mHBSSFeSSIF due to the higher dose, which could be dissolved in mHBSSFeSSIF. It should be noted that the dose of cyclosporine dissolved in the mHBSS buffer corresponded to 1 µg/mL, which is somewhat lower than the reported saturation solubility of cyclosporine in aqueous buffer (28.6 µg/mL (37)). However, it is likely that the solubility of cyclosporine in mHBSSFeSSIF exceeded 50 µg/mL based on data reported by Guo et al., (79). In that study, the authors reported solubility values of > 1 mg/mL in various bile salt and PC

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combinations. The apparent absence of permeated fenofibrate as well as fenofibric acid (the major metabolite of fenofibrate) is in contrast to previous permeation studies regarding fenofibrate. Those studies employed fenofibrate in NaTC-supplemented buffer (46), supersaturated solutions (54) or in solid dosage forms (80). Additionally, fenofibrate was confirmed to translocate across Caco-2 monolayers when administered in fasted state simulated intestinal fluid (3 mM NaTC, 0.2 mM PC in mHBSS at pH 6.5),

PT

exhibiting a Papp value of 34.2 × 10-6 cm/s. Notably, this permeation rate is higher as compared to a study by

RI

Berthelsen et al. (2.1 × 10-6 cm/s) who investigated permeation of fenofibrate across Caco-2 monolayers in

SC

a 5 mM NaTC solution. In addition, it is lower than predicted in silico in aqueous buffer, reported by Sjøgren et al. These differences in permeation between the reported studies and the current study are consistent

NU

with the partitioning of fenofibrate into micelles at elevated concentrations of lipid, as was the case for

detection by the employed LC-MS method.

MA

metoprolol and cyclosporine. Consequently, the permeated amount of fenofibrate was inadequate for

Upon introducing FAs as potential permeation enhancers to the mHBSSFeSSIF medium, the permeation rate

D

of the investigated hydrophobic compounds (fenofibrate, metoprolol and cyclosporine) were largely

PT E

unaffected. This fact corroborates our observations on limited cytotoxicity of FA when dosed in mHBSSFeSSIF. It also lends credit to the hypothesis that the interactions between the cell membrane and the lipids and/or

values.

CE

the hydrophobic drugs are impaired at concentrations of bile salt and phospholipid close to their CMC

AC

Contrary to what was observed for the hydrophobic compounds, the permeation of mannitol across the mucosa was increased > 10-fold upon addition of 20 mM C10 to the mHBSS and the mHBSSFeSSIF. A similar, yet less prominent effect was observed with Caco-2 cell monolayers exposed to 5 mM C12 in mHBSSFeSSIF, which elicited a 7-fold increase in the Papp value for mannitol. Neither of the FAs induced a cytotoxic response at 20 mM (C10) or 5 mM (C12) in mHBSSFeSSIF, despite having previously been reported as cytotoxic at these concentrations (56). In addition, all Papp values for mannitol were below that of cells exposed to 100 mM C10, which were employed as positive controls (i.e. monolayers displaying unrestricted

21

ACCEPTED MANUSCRIPT

permeation of mannitol) due to pronounced cytotoxicity. The positive control values were comparable to values reported by Whitehead et al. (77), who dissolved the examined epithelia using Triton™ X-100. The Papp values for ovalbumin displayed only a minor increase in the permeation rate and the total permeated amount upon addition of 20 mM C10 or 5 mM C12, compared to mHBSS and mHBSS FeSSIF. In contrast, the

PT

Papp values for permeation of desmopressin across Caco-2BM cell monolayers were significantly increased (8.5-fold and 3.3-fold when exposed to C10 and C12, respectively, as compared to the permeation across

RI

mHBSS–exposed Caco-2 cell monolayers). This observation was in line with an increase in the relative

SC

permeated amount of desmopressin (7.8-fold and 3.3-fold across for Caco-2 monolayers exposed to C10 and C12, respectively). As described by Lindmark et al. (22), the effect of FAs may be predominantly on the

NU

tight junctions. This process might partly explain the lacking ability to enhance the permeation of ovalbumin, which has been claimed to permeate by both the paracellular as well as the transcellular route

MA

via endocytic pathways (45). Similarly, it correlates well with the increased permeation of desmopressin and mannitol, which both permeates by the paracellular route (41,43). It may prove interesting to use BM

D

as a tool in other aspects of drug delivery, since BM constitutes a barrier to drugs based on their

PT E

physicochemical properties. These aspects would primarily revolve around mucus-drug interactions, e.g., drug diffusion in mucus, barrier capabilities of BM when combined with non-cellular assays (e.g., as

AC

5. CONCLUSION

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performed by Hens et al. (81)) as well as the interplay between mucus and incubation media such as FeSSIF.

This study demonstrated that surface-lining Caco-2 cell monolayers with BM can be used as a tool to avoid detrimental effects to Caco-2 cell monolayers caused by exogenous compounds. The protective effect of BM surpassed the effect of established alternative intestinal in vitro cell models, namely the HT29 cell monolayers, and the co-cultured Caco-2/HT29 cell monolayers. The BM constituted a barrier to diffusion of hydrophobic compounds, since the transmucosal permeation rate and associated parameters were reduced for hydrophobic drugs. But the permeation of hydrophilic drugs was largely unaffected by the

22

ACCEPTED MANUSCRIPT

presence of mucus. In contrast to the HT29 and the Caco-2/HT29 models, the BM surface-lined Caco-2 mucosal model could be used in combination with fed state simulated intestinal fluid without risking detrimental effects on the epithelial cells. Application of mHBSSFeSSIF increased the permeation of both small and large hydrophilic compounds, whereas the permeation of hydrophobic compounds was reduced

PT

probably due to partitioning of the drug into mixed micelles composed of NaTC, PC and FAs in the test medium. The addition of FAs as permeation enhancers increased the permeation rate of mannitol, but not

RI

that of the much larger ovalbumin. In addition, the permeation of metoprolol and cyclosporine was

SC

decreased upon addition of FAs. These occurrences were in line with the expected mechanisms for fatty

lined with BM constitutes a valuable in vitro model.

MA

6. ACKNOWLEDGEMENTS

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acid-based permeation enhancers. In conclusion, this study confirms that Caco-2 cell monolayers surface-

Laboratory technicians Mette Frandsen and Lene Grønne Pedersen are acknowledged for cell culturing.

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Furthermore, the research leading to these results has received support from the Innovative Medicines

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Initiative Joint Undertaking under grant agreement n° 115363 resources, which are composed of financial contribution from the European Union's Seventh Framework Programme (FP7/2007-2013) and EFPIA

CE

companies’ in kind contribution. The Danish Council for Independent Research, Technology and Production Sciences supported the project (grant no. 4005-00455) as well as the Novo Nordisk Foundation (Grand

funding

AC

Challenge Program). The Alfred Benzon Foundation and The Drug Research Academy are acknowledged for the

equipment

used

in

this

project.

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Table 1

Cyclosporine

Ovalbumin

Peptide

Protein

IV 1202 > 2.9 (30–32)

III 44300

28.6 (34)

> 1 × 10

4.7 (30)

4.6 (37) Para- and transcellular (42)

Paracellular (38)

Transcellular (39)

0.2-2 (10,23,24) 16-18 (23,24)

18-27.0 (23,24) > 95 (27,46)

Transcellular

Paracellular (40)

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Permeation route

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Table 1: Physico-chemical properties of the compounds included in this study. Compound Mannitol Metoprolol Fenofibrate Desmopressin Small Small Small Type Peptide molecule molecule molecule BCS class III I II III Mw (Da) 182.2 267.4 360.9 1069 < -2.47 > 1.88 > 5.1 < -1.95 LogP (23,24) (25,26) (27) (28–30) a Aqueous solubilty < 0.25 4 4 4 > 1 × 10 > 1 × 10 > 1 × 10 (µg/mL) (27,33) b pKa/pI 13.5 (35) 9.7(36) None 10.2 (30)

4

e

0.8-8 n/a (15,23,45) f Oral bioavailability 30-70 5-95 n/a (%) (27) (39,49,50) Degraded to P-gp Paracellular Transcellular 385 AA Notes fenofibric 9 AA sequence substrate, 11 marker marker sequence acid (51) AA sequence a b c At 25 °C. pKa for mannitol, metoprolol, pI for desmopressin, cyclosporine and ovalbumin. In Caco-2 cell monolayers. d e f In the presence of 5 mM sodium taurocholic acid. In the absence of Pg-p inhibitors. Dependent on delivery system, see (50) for details on cyclosporine bioavailability. Abbreviations: Mw: Molecular weight, LogP: Octanol:water partition coefficient. Papp: Apparent permeability coefficient. AA: Amino acid d

2.1 (43)

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Papp (× 10 cm/s)

0.1-0.7 (24,28,44) 0.08-0.16 (47,48)

Transcellular (41)

<< 0

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Table 2

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Table 2: Permeability characteristics of the cell culture epithelial models following exposure to fed state simulated intestinal fluid. Parameter Medium Caco-2 Caco-2BM HT29 Caco-2/HT29 mHBSS 0.1 ± 0.0 0.1 ± 0.0 0.2 ± 0.1 0.2 ± 0.0 Mannitol *,† † * * mHBSSFeSSIF 7.8 ± 1.1 0.1 ± 0.0 3.0 ± 0.2 4.0 ± 1.5 -6 Papp (× 10 cm/s) † 1 1 mHBSS 4.7 ± 0.2 1.9 ± 0.2 2.7 ± 0.1 3.3 ± 0.2 Metoprolol *,† 2 2 mHBSSFeSSIF 7.2 ± 0.5 2.1 ± 0.1 2.7 ± 0.1 4.7 ± 0.3 mHBSS 0.6 ± 0.1 0.4 ± 0.0 1.0 ± 0.2 0.8 ± 0.0 Mannitol *,† † * * Permeated mHBSSFeSSIF 19.7 ± 0.6 0.8 ± 0.1 12.9 ± 0.9 10.4 ± 3.4 a amount (%) mHBSS 10.7 ± 0.7 8.2 ± 0.7 10.6 ± 0.2 8.8 ± 0.4 Metoprolol 3 3,4 4 mHBSSFeSSIF 14.2 ± 1.7 8.5 ± 0.2 13.6 ± 0.3 12.9 ± 2.0 mHBSS 0 0 0 0 Mannitol * † mHBSSFeSSIF 31.3 ± 2.8 0 18.6 ± 8.4 26.1 ± 11.7 Lag phase (min) † mHBSS 0 0 17.1 ± 7.7 0 Metoprolol † mHBSSFeSSIF 0 0 0 16.7 ± 7.5 c c c c mHBSS > 240 > 240 > 240 > 240 Timespan for Mannitol c mHBSSFeSSIF 40 ± 5 > 240 215.0 ± 11.4 150.0 ± 40.2 steady state flux c mHBSS 52.5 ± 3.4 > 240 60.0 ± 6.7 90.0 ± 13.4 b (min) Metoprolol mHBSSFeSSIF 45.0 ± 6.7 180.0 ± 26.8 55.0 ± 6.3 67.5 ± 3.4 Abbreviations: BM: Biosimilar mucus. Papp: Apparent permeability coefficient. mHBSS was used as a negative control. a b ) Permeated amount relative to administered dose following 4 h of exposure. ) Timespan following a lag phase for 2 c which steady-state flux was maintained (r > 0.95). ) Steady state flux was observed for the entire experiment. Numbers marked with an asterisk indicate a significant difference between mHBSSFeSSIF and the control (mHBSS) for that setup (p < 0.05). Numbers marked with a cross indicate a significant difference between results for that cell type and all other cell types in the experiment (p < 0.05). Numbers marked with a superscripted, italicized cipher indicate a significant difference between those two experiments (p < 0.05). Mean ± SEM, n = 6-12.

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Table 3

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Table 3: Effect of sodium caprate, sodium laurate and mHBSS FeSSIF on the permeation of model compounds across Caco-2 cell monolayers surface-lined with biosimilar mucus. With BM mHBSS Compound Parameter mHBSSFeSSIF + mHBSSFeSSIF without BM mHBSS mHBSSFeSSIF 20 mM C10 + 5 mM C12 -6 Papp (× 10 cm/s) 0.4 ± 0.1 0.04 ± 0.0 0.1 ± 0.0 3.4 ± 0.7 1.3 ± 0.3 Timespan for steadyDesmopressin > 240 > 240 > 240 > 240 > 240 state flux (min) Amount permeated (%) 1.4 ± 0.6 0.1 ± 0.0 0.6 ± 0.1 10.9 ± 2.6 4.6 ± 1.6 -6 Papp (× 10 cm/s) 0.3 ± 0.0 0.2 ± 0.0 0.4 ± 0.0 0.3 ± 0.1 0.4 ± 0.1 Timespan for steadyOvalbumin 45.0 ± 0 115 ± 5 180 ± 26.8 240.0 ± 0.0 240.0 ± 0.0 state flux (min) Amount permeated (%) 0.8 ± 0.1 0.7 ± 0.1 1.6 ± 0.4 1.6 ± 0.3 2.1 ± 0.4 -6 Papp (× 10 cm/s) 0.6 ± 0.0 0.3 ± 0.0 0.2 ± 0.0 0.2 ± 0.0 0.2 ± 0.0 Timespan for steadyCyclosporine 52.5 ± 3.3 60.0 ± 0.0 52.5 ± 3.3 47.5 ± 2.5 51.0 ± 3.7 state flux (min) Amount permeated (%) 0.9 ± 0.0 0.6 ± 0.0 0.4 ± 0.0 0.5 ± 0.0 0.5 ± 0.0 Abbreviations: BM: Biosimilar mucus. C10: Sodium caprate. C12: Sodium laurate. Papp: Apparent permeability coefficient.

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Fig. 1: The relative viability (A) and TEER (B) of Caco-2 cell monolayers with or without biosimilar mucus (BM, black), HT29 cell monolayers (grey) and Caco-2/HT29 co-cultured monolayers (white) following exposure to mHBSSFeSSIF. Significant differences are marked with asterisks (***p < 0.001). TEER is relative to initial values, whereas viability is relative to control cells exposed to mHBSS. Mean ± SEM, n = 6. Fig. 2: Effect of sodium caprate (C10, A) and sodium laurate (C12, B) on the viability and TEER properties of Caco-2 cell monolayers following 4 h of exposure. Cells were surface-covered with biosimilar mucus (black) or not (white). Viability (squares) is depicted relative to the viability of control cells. TEER (circles) is depicted relative to initial values. Mean ± SEM, n = 6.

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Fig. 3: Effect of sodium caprate (C10, A) and sodium laurate (C12, B) on the apparent permeability coefficient (Papp) for mannitol (circles) and metoprolol (squares) following 4 h of exposure to Caco-2 cell monolayers (white) and monolayers surface-lined with biosimilar mucus (black). Mean ± SEM, n = 6.

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Graphics Abstract

Figure 1

Figure 2

Figure 3