University of Southern Denmark

The permeation of acamprosate is predominantly caused by paracellular diffusion across Caco-2 cell monolayers A Paracellular Modeling Approach Antonescu, Irina E; Rasmussen, Karina F; Neuhoff, Sibylle; Frette, Xavier; Karlgren, Maria; Bergström, Christel A S; Nielsen, Carsten Uhd; Steffansen, Bente

Published in: Molecular Pharmaceutics

DOI: 10.1021/acs.molpharmaceut.9b00733

Publication date: 2019

Document version: Accepted manuscript

Citation for pulished version (APA): Antonescu, I. E., Rasmussen, K. F., Neuhoff, S., Frette, X., Karlgren, M., Bergström, C. A. S., Nielsen, C. U., & Steffansen, B. (2019). The permeation of acamprosate is predominantly caused by paracellular diffusion across Caco-2 cell monolayers: A Paracellular Modeling Approach. Molecular Pharmaceutics, 16(11), 4636-4650. https://doi.org/10.1021/acs.molpharmaceut.9b00733

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Download date: 04. Oct. 2021 Subscriber access provided by University Library of Southern Denmark Article The permeation of acamprosate is predominantly caused by paracellular diffusion across Caco-2 cell monolayers: A paracellular modelling approach Irina E Antonescu, Karina F Rasmussen, Sibylle Neuhoff, Xavier Frette, Maria Karlgren, Christel A.S. Bergström, Carsten Uhd Nielsen, and Bente Steffansen Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.9b00733 • Publication Date (Web): 27 Sep 2019 Downloaded from pubs.acs.org on September 29, 2019

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1 2 3 4 Manuscript draft 5 6 7 8 9 10 The permeation of acamprosate is predominantly caused by paracellular 11 12 diffusion across Caco-2 cell monolayers: A paracellular modelling 13 14 15 approach 16 17 Irina E. Antonescu1, Karina F. Rasmussen2††, Sibylle Neuhoff3, Xavier Fretté4, Maria Karlgren5, 18 19 Christel A. S. Bergström5, Carsten Uhd Nielsen1 and Bente Steffansen*1† 20 21 1 Department of Physics, Chemistry & Pharmacy, Faculty of Science, University of Southern Denmark, DK-5230 Odense, 22 23 Denmark 24 2 Department of Pharmacy, University of Copenhagen 2100 Copenhagen Denmark 25 26 3 Certara UK Ltd., Simcyp Division, Sheffield, UK 27 28 4 Department of Chemical Engineering, Biotechnology and Environmental Technology, Faculty of Engineering, University 29 of Southern Denmark, DK-5230 Odense, Denmark 30 31 5 Department of Pharmacy, Uppsala University, Sweden 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 1 57 58 59 60 ACS Paragon Plus Environment Molecular Pharmaceutics Page 2 of 29

1 1 ABSTRACT: In drug development, estimating fraction absorbed (Fa) in man for permeability limited compounds in important but 2 3 2 challenging. To model Fa of such compounds from apparent permeabilities (Papp) across filter-grown Caco-2 cell monolayers, it is 4 3 central to elucidate the intestinal permeation mechanism(s) of the compound. The present study aims to refine a computational 5 6 4 permeability model in order to investigate the relative contribution of paracellular and transcellular routes to the Papp across Caco-2 7 5 monolayers of the permeability limited compound acamprosate having a bioavailability of ~11%. The Papp of acamprosate and of 8 6 several paracellular marker molecules were measured. These Papp values were used to refine system-specific parameters of the Caco-2 9 10 7 monolayers, i.e. paracellular pore radius, pore capacity and potential drop. The refined parameters were subsequently used as input

11 8 in modelling the permeability (Pmodelled) of the tested compounds using mathematical models collected from two published 12 9 permeability models. The experimental data shows that acamprosate Papp across Caco-2 monolayers is low and similar in both 13 -7 -1 14 10 transport directions. The obtained acamprosate Papp, 1.56 ± 0.28 × 10 cm∙s , is similar to the Papp of molecular markers for 15 11 paracellular permeability namely mannitol, 2.72 ± 0.24 × 10-7 cm∙s-1, lucifer yellow, 1.80 ± 0.35 × 10-7 cm∙s-1 and fluorescein, 2.10 16 17 12 ± 0.28 × 10-7 cm∙s-1 and lower than that of atenolol 7.32 ± 0.60 × 10-7 cm∙s-1 (mean ± SEM, n = 3-6), while the end-point amount

18 13 of acamprosate internalized by the cell monolayer, Qmonolayer, was lower than that of mannitol. Acamprosate did not influence the 19 14 barrier function of the monolayers since it neither altered the Papp of the three paracellular markers, nor the transepithelial electrical 20 21 15 resistance (TEER) of the cell monolayer. The Pmodelled for all the paracellular markers and acamprosate was dominated by the Ppara 22 16 component and matched the experimentally obtained Papp. Furthermore, acamprosate did not inhibit the uptake of probe substrates for 23 the solute carriers PEPT1, TAUT, PAT1, EAAT1, B0,+AT/rBAT, OATP2B1 and ASBT expressed in Caco-2 cells. Thus, the P 24 17 modelled 25 18 estimated well Ppara and the paracellular route appears to be the predominant mechanism for acamprosate Papp across Caco-2 26 19 monolayers, while the alternative transcellular routes, mediated by passive diffusion or carriers, are suggested to only play 27 28 20 insignificant roles. 29 30 31 32 21 KEYWORDS: paracellular modelling, acamprosate, permeability-limited absorption, Caco-2, paracellular permeability, 33 22 transcellular permeability, permeability modelling 34 35 ABBREVIATIONS: BCS, biopharmaceutical classification system; IVIVE, in vitro-in vivo extrapolations; Caco-2, human 36 23 37 24 colorectal adeno-carcinoma cells; Papp, apparent permeability; n, number of cell passages; PBPK, physiologically-based 38 25 pharmacokinetic (modelling); F, bioavailability; Fa, fraction absorbed; Ptrans, transcellular permeability; TJs, tight junctions; Ppara, 39 40 26 paracellular permeability; PCM, carrier-mediated permeability; TEER, transepithelial electrical resistance; A-B, apical-to-basolateral;

41 27 B-A, basolateral-to-apical; PABL, aqueous boundary layer permeability; Pf, filter permeability; Pmodelled, modelled apparent 42 28 permeability; SDU, University of Southern Denmark; UU, Uppsala University; DSMZ, Deutsche Sammlung für Mikroorganismen 43 44 29 und Zellkulturen; ATCC, American Type Culture Collection; SLC, solute carriers; ER, efflux ratio; LLOQ, lower limit of 45 30 quantification; SD, standard deviation; SEM, standard error of means; QC, quality control. 46 47 31 48 49 32 50 33 51 52 53 54 55 56 2 57 58 59 60 ACS Paragon Plus Environment Page 3 of 29 Molecular Pharmaceutics

1 1 1. INTRODUCTION 2 3 4 2 Understanding the in vitro permeation mechanisms for permeability-limited compounds is crucial for estimating their

5 3 fraction absorbed (Fa) in man. Estimated human Fa, coupled to IVIVE of metabolism, distribution, and excretion mechanisms, can be 6 4 used for building physiologically-based pharmacokinetic (PBPK) prediction models to evaluate drug safety and efficacy 1. Knowledge 7 8 5 of permeation mechanisms is also important for designing effective permeation enhancing strategies to improve oral bioavailability 9 2-4 6 of compounds with unfavorable intestinal absorption . Caco-2 cells is a generally accepted in vitro model for studying Papp of various 10 compounds. Caco-2 cell based P can be used to estimate human F and the Caco-2 cell model has also been used to study paracellular 11 7 app a 5, 6 1 12 8 transport . For paracellular transported compounds it is, however, challenging to estimate Fa from Caco-2 cell based Papp . 13 9 Elucidating the permeability mechanisms that drive the Fa of such compounds by a combined experimental and modelling approach 14 15 10 may be a way forward.

16 11 Acamprosate is an example of a low permeability compound, i.e. the rate determining step for acamprosate fa is its poor 17 12 permeation across the intestinal epithelia and it is thus a biopharmaceutics classification system (BCS) class III drug compound 7-9. 18 19 13 Acamprosate is widely prescribed for the treatment of alcohol addiction due to its ability to prevent alcohol craving and relapse 7. Up 20 14 to 2 g day-1 is commonly prescribed, divided into three doses 10, 11. The acamprosate salt has a high aqueous solubility (Table I) and 21 15 the dose dissolves in intestinal fluids, after which it dissociates completely to acetyl-homotaurine and calcium moieties, as illustrated 22 23 16 in Figure 2 11, 12. The compound is administered as a calcium salt with a stoichiometry of 2 acetylhomotaurines to 1 calcium ion 13. 24 17 The molecular structure is shown in Figure 1a. A clinically-relevant concentration of dissociated acamprosate in any intestinal 25 segment after administration of a 300-600 mg dose is difficult to estimate. This is due to high interindividual variability of intestinal 26 18 27 19 fluid volume14 water volume administered with the dose and the composition of chyme in the lumen. If acamprosate is administered 28 20 as an immediate release formulation, a rough luminal concentration of 6.6-13.2 mM (dose/250mL co-administered water15) can be 29 30 21 estimated. If administered as an enteric-coated tablet, the luminal concentration becomes even more uncertain and can be roughly 31 22 estimated to be in a range from 20-40 mM (dose/80 mL, the average total small intestine water content14) and up to the aqueous 32 23 solubility of acamprosate of 800 mM. In this study, concentrations of acamprosate in the range of 10-20 mM were applied, as they 33 34 24 are in the estimated clinically-relevant concentration interval and we have demonstrated that up to this concentration acamprosate 35 25 does not affect the barrier properties of the Caco-2 monolayers used as a model system. Despite estimated to reach high luminal 36 concentrations, the oral bioavailability (F) of acamprosate is only 11 ± 1% when administered as enteric coated tablets 11, 16. Since 37 26 38 27 acamprosate is stable in biological fluids 12 and not metabolized neither in the intestine nor in the liver 10, 11, 17, it can be assumed that 39 28 the acamprosate fraction absorbed (Fa) is similar to its oral F. 40 41 29 In this study, we used acamprosate as an example of a low permeability compound to investigate permeation mechanisms 42 30 across Caco-2 monolayers for this class of compounds. To do so, we evaluated the relative contributions of each possible permeation 43 31 route, i.e. carrier-mediated, paracellular, transcellular, aqueous boundary layer (ABL)-limited and filter-limited, to the overall 44 45 32 permeability. Firstly, we measured the Papp of acamprosate in parallel with a range of paracellular marker molecules, i.e. mannitol, 46 33 lucifer yellow, fluorescein and atenolol. Secondly, we evaluated the contribution of carrier-mediated permeability to the observed 47 34 Papp, by measuring the intracellular accumulation of acamprosate and its possible inhibition at a range of carriers expressed in Caco-2 48 49 35 cells. Thirdly, we estimated the paracellular permeability component of the model, by building a paracellular permeation model that 50 36 compiles information on the physicochemical properties of the investigated compounds, monolayer physiology and assay properties. 51 We performed a refinement of the paracellular physiology parameters of our own Caco-2 monolayers (pore radius, pore capacities, 52 37 53 38 potential drop) by using the empirical Papp data for the 5 investigated compounds. Lastly, we estimated the remaining possible 54 55 56 3 57 58 59 60 ACS Paragon Plus Environment Molecular Pharmaceutics Page 4 of 29

1 1 contributions from the passive transcellular, aqueous boundary layer and filter components. The modelled permeabilities from all the 2 3 2 permeation routes were compiled into a permeability model (Pmodelled) that aims to describe their contribution of the overall permeation 4 3 mechanism. 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 4 28 5 Figure 1. Molecular structures of the dominating ionic species expected to permeate across Caco-2 monolayers or human 29 6 intestinal epithelia at pH 6.0-7.4 a) acamprosate (acetylhomotaurine); b) [14C]-mannitol; c) lucifer yellow; d1) fluorescein dianion, 30 31 7 main ionic species at pH 7.4; d2) fluorescein monoanion, main ionic species at pH 6.0; e) atenolol. 32 8 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 4 57 58 59 60 ACS Paragon Plus Environment Page 5 of 29 Molecular Pharmaceutics

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 1 28 2 Figure 2. Compound dissociation and the investigated permeation routes for acamprosate in filter-grown Caco-2 29 30 3 monolayers. a) specialized carrier-mediated transcellular permeability across the lipid bilayers of the enterocytes’ membranes via 31 4 transporter or carrier proteins, PCM; b) diffusion-driven paracellular permeability across the size- and/or charge-restricted pores of the 32 33 5 tight junctions (TJs) and size- and charged- unrestricted pores formed in between the enterocytes, Ppara; c) passive diffusion-driven 34 6 transcellular permeability across the lipid bilayers of the enterocytes’ apical and basal membranes, Ptrans; d) permeability across the 35 7 aqueous boundary layer adjacent to the enterocytes, PABL and e) permeability across the polycarbonate filter on which the Caco-2 cell 36 37 8 monolayers are grown, Pf.. 38 39 9 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 5 57 58 59 60 ACS Paragon Plus Environment Molecular Pharmaceutics Page 6 of 29

1 1 2. MATERIALS AND METHODS 2 3 2 2. 1. Chemicals. Acamprosate calcium (purity >98%) was from Santa Cruz Biotechnology (Heidelberg, Germany) except 4 3 the acamprosate used in the apical uptake studies, which was from bioKEMIX (London, UK). Atenolol (purity ≥ 98%), dilithium 6- 5 6 4 Amino-2-(hydrazinecarbonyl)-1,3-dioxobenzo[de]isoquinoline-5,8-disulfonate (lucifer yellow) (purity ≥ 95%), fluorescein sodium 7 5 (purity 95.3%), Dulbecco’s Modified Eagle’s Medium (DMEM), penicillin/streptomycin (Pen/strep, 100x), L-glutamine (L-glu, 200 8 mM), non-essential amino acids (NEAA), Fetal Bovine Serum (FBS), Triton X-100 10% and trypsin-EDTA (10x) were from Sigma 9 6 10 7 Aldrich (St. Louis, MO, USA). Hank’s Buffered Saline Solution (HBSS) (10x) and sodium bicarbonate (7.5%) were purchased from 11 8 Gibco, Invitrogen (Pairsley, UK). 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (purity >99.5%) and 2-(N- 12 14 13 9 morpholino-)ethanesulfonic acid (MES) (purity ≥99%) were from AppliChem GmbH (Darmstadt, Germany). [ C]-Gly-Sar (56 14 10 mCi/mmol), [3H]-taurine (13.3 Ci/mmol), [3H]-L-proline (75.0 Ci/mmol), [3H]-L-glutamate (51.1 Ci/mmol), [3H]-L-Lysine (17.9 15 14 3 3 3 3 11 Ci/mmol, [ C]-mannitol (57.2 mCi/mmol), [ H]-estrone-3-sulfate ([ H]-E1S) (54.3 Ci/mmol) and [ H]-taurocholic acid ([ H]-TCA) 16 17 12 (4.6 Ci/mmol) and Ultima Gold scintillation liquid were purchased from Perkin Elmer (Boston, MA, USA). All the isotopes had a 18 13 radiochemical purity of >97%. Cell culture plasticware was obtained from Corning Life Science (Wilkes Barre, PA, USA) and part 19 14 of it purchased through Sigma (Copenhagen, Denmark). Water for the experiments was from a Milli-Q water purification system with 20 21 15 a 0.22 µM Millipak filter, both from Millipore (Massachusetts, US). Stock solutions of compounds were prepared in Milli-Q water 22 16 and stored at -20°C. 23 24 17 2.2. Caco-2 cell lines and culture conditions. The transepithelial permeability of acamprosate was measured in two 25 18 laboratories, at the University of Southern Denmark (SDU) and Uppsala University (UU). Similar culturing protocol and assay 26 19 settings were employed, but the Caco-2 cell lines were obtained from different cell banks: Deutsche Sammlung für Mikroorganismen 27 28 20 und Zellkulturen (DSMZ) (Braunschweig, Germany) at SDU and American Type Culture Collection (ATCC) (Manassas, Virginia, 29 21 USA) at UU. The transepithelial permeability of [14C]-mannitol, fluorescein, lucifer yellow and atenolol and the apical uptake of 30 22 [14C]-Gly-Sar, [3H]-taurine, [3H]-proline, [3H]-glutamate and [3H]-lysine was investigated in the DSMZ Caco-2 cell line. The 31 32 23 intracellular accumulation of acamprosate was measured in the ATCC Caco-2 cells. DSMZ Caco-2 cells have been routinely cultured 33 24 as described by Grandvuinet and co-workers 18, while ATCC Caco-2 cells were cultured as described by Hubatsch and co-workers 19. 34 For transepithelial permeability experiments, cells were seeded onto Transwell polycarbonate filters (pore size 0.4 µm, 1.12 cm2 35 25 36 26 growth area), whereas for apical uptake studies cells were seeded on the bottom of 24-well plates (1.9 cm2 growth area). The seeding 37 27 density was 8.93 × 104 cells/cm2 (105 cells/ Transwell insert or 1.78 x 105 cells/well on 24-well plate). DSMZ Caco-2 cells were used 38 39 28 in passages 2-13 and 21-23 days post-seeding for transepithelial permeability studies or 11 days post-seeding for apical uptake studies. 40 29 ATCC Caco-2 cells were used in passages 95-105 and 21-23 days post-seeding for transepithelial permeability studies. Before 41 30 initiating the transepithelial permeability studies, the barrier integrity of the Caco-2 monolayers was estimated by measuring the 42 43 31 transepithelial electrical resistance (TEER) at room temperature (20°C). This was performed in a tissue resistance measurement 44 32 chamber (Endohm-12) for the DSMZ Caco-2 monolayers or an STX2 chopstick electrode for the ATCC Caco-2 monolayers, 45 connected to an Epithelial Voltohmmeter (EVOM 2), all from World Precision Instruments (Sarasota, FL, USA). At the start of the 46 33 47 34 experiment, the Caco-2 DSMZ cells, and ATCC cells showed similar monolayer TEER values (measured at 20°C, mean ± SEM), of 48 35 806 ± 22 (n=12, 6 monolayers in each passage) and 691 ± 107 (n=3, 12 monolayers in each passage) Ω∙cm2 , respectively. 49 20, 21 50 36 2.3. Initial apical uptake studies. The inhibition of range of carriers expressed in Caco-2 cells by acamprosate was 51 37 tested using established carrier substrates. The uptake of 8.9 µM [14C]-Gly-Sar (substrate for the peptide transporter (PEPT) 1, 52 3 3 53 38 SLC15A1), 37.6 nM [ H]-taurine (substrate for the taurine transporter (TAUT), SLC6A6), 6.7 nM [ H]-proline (substrate for the 54 55 56 6 57 58 59 60 ACS Paragon Plus Environment Page 7 of 29 Molecular Pharmaceutics

1 1 proton-coupled amino acid transporter (PAT) 1, SLC36A1), 9.8 nM [3H]-glutamate (substrate for excitatory amino acid transporter 2 3 0,+ 3 2 (EAAT) 1, SLC1A3, and EAAT3, SLC1A1), [ H]-lysine (substrate for the heterodimer between the B amino acid transporter 0,+ 3 4 3 (B AT) and the neutral and basic amino acid transport protein (rBAT), SLC7A9/SLC3A1), 9 nM [ H]-estrone-3-sulfate (E1S) 5 4 (substrate for the organic anion transporting polypeptide (OATP) 2B1, SLCO2B1) and 100 nM [3H]-taurocholic acid (substrate for 6 7 5 the apical sodium dependent bile acid transporter (ASBT), SLC10A2) was studied in DSMZ Caco-2 cells grown on the bottom of 8 6 tissue culture plates. Before the uptake was initiated, cells were pre-incubated with pre-warmed HBSS containing 10 mM MES 9 adjusted to pH 6.00 ± 0.05 with 1 M NaOH (uptake buffer), for 15 minutes at 37°C and 90 rpm on a plate shaker (Edmund Buhler, 10 7 11 8 Germany). After preincubation, the uptake buffer was removed and pre-warmed donor solutions containing 0.5 µCi/mL isotope with 12 9 or without 13 mM cold acamprosate were applied. All acamprosate concentrations and doses are henceforth reported as acetyl- 13 14 10 homotaurinate (not acamprosate calcium). The cells were incubated with the donor solutions for 5 minutes at 37°C and 90 rpm on a 15 11 plate shaker (Edmund Buhler, Germany). At the end of the incubation time, the donor solutions were removed, and the cells were 16 12 rinsed with 3 x 500 µL ice-cold uptake buffer per well. The cells were detached from the plates using 200 µL/well 0.1% Triton X and 17 18 13 incubating for 20 minutes at 37°C and 90 rpm. After the detachment of the cells, the cell lysates from each well were transferred to 19 14 scintillation vials and 2 mL Ultima Gold scintillation liquid was added, and the total radioactivity was quantified by liquid scintillation 20 15 counting on a Packard TriCarb 21000 TR (Meriden, CT, USA). 21 22 16 2.4. Bi-directional transepithelial permeability studies. The apical-to-basolateral (A-B) and basolateral-to-apical (B-A) 23 17 permeability of 20 µM (3.6 µg/mL), 200 µM (36 µg/mL) or 20 mM (3.6 mg/mL) acamprosate, 7 µM (0.4 µCi/mL or 1.28 µg/mL) 24 [14C]-mannitol, 100 µM lucifer yellow (45.7 µg/mL), 26.5 µM fluorescein (10 µg/mL) and 1.5 mM atenolol (0.4 mg/mL) was 25 18 26 19 measured for 120 min (acamprosate, [14C]-mannitol, atenolol) or 240 min (lucifer yellow, fluorescein). The possible influence of 20 27 20 µM or 20 mM acamprosate on the permeability of 7 µM [14C]-mannitol was investigated. For the experiments using 20 mM 28 29 21 acamprosate, the cells were either not treated or pre-treated for 2, 4, 8 or 24 hours at 37°C with 20 mM acamprosate in the cell culture 30 22 medium. Furthermore, the effect of 20 mM acamprosate on the permeability of lucifer yellow and fluorescein and on the monolayer 31 23 TEER was studied. The concentrations of acamprosate and atenolol were chosen to be in the range of luminal concentrations attainable 32 33 24 after oral administration of the most common dose. The concentration of the paracellular markers ([14C]-mannitol, lucifer yellow, 34 25 fluorescein) was chosen as the minimal concentration that can be quantified using the available bioanalysis methods. 35 For all permeability studies, the compounds were dissolved in HBSS supplemented with 10 mM HEPES adjusted to pH 36 26 37 27 7.40 ± 0.05 with 1 M NaOH (termed permeability buffer). All applied solutions henceforth referred to as donor solutions, had a final 38 28 osmolality of 291-311 mOsm/kg and a pH of 7.38-7.42. Prior to the experiment, the monolayers were equilibrated with permeability 39 40 29 buffer for 10 minutes on a plate shaker (Talboys Professional Incubating Microplate Shaker, 37°C, 220 rpm). The permeability studies 41 30 were initiated by removing the permeability buffer from the donor chamber and applying 500 or 1000 µL pre-warmed (37°C) donor 42 31 solutions to either the apical or basolateral chamber, respectively, and add permeability buffer to the receiver side. The cells were 43 44 32 placed immediately on a plate shaker (Talboys Professional Incubating Microplate Shaker, 37°C, 220 rpm). For the fluorescent 45 33 compounds lucifer yellow and fluorescein, the shaking plate was covered with aluminum foil and dark containers were used for 46 34 storing the donor solutions and experiment aliquots. Samples of 100 or 50 µL were aliquoted from the basolateral or apical chamber, 47 48 35 for A-B or B-A permeability, respectively, after 15, 30, 45, 60, 90, and 120 minutes (acamprosate and atenolol), 5, 15, 25, 50, 80, and 49 36 120 min ([14C]-mannitol) or 60, 90, 120, 150, 180, 210, and 240 min (lucifer yellow and fluorescein). The aliquoted sample volumes 50 were replaced with the same volume of pre-warmed permeability buffer. Donor samples of 20 µL ([14C]-mannitol permeability) or 51 37 52 38 100 µL (acamprosate, lucifer yellow, fluorescein, atenolol) were collected both before the donor solutions were added to the donor 53 39 chamber (t=0 min) and at the end of the experiment (t=120 min or 240 min) and were used to calculate the compound mass balance. 54 55 56 7 57 58 59 60 ACS Paragon Plus Environment Molecular Pharmaceutics Page 8 of 29

1 1 All permeability experiments were terminated by rinsing the monolayers with 3 x 500 µL ice-cold permeability buffer. In the case of 2 14 3 2 acamprosate and [ C]-mannitol studies, the polycarbonate filters containing the cells were cut with a scalpel from the Transwell 4 3 supports into Eppendorf tubes. For acamprosate, the filters with cells were mixed with 200 µL 60:40 ACN: water (LC/MS-MS mobile 5 4 phase) and vortexed for at least 2 minutes. This was followed by centrifugation at 14000 rpm, 5 °C for 10 minutes analysis of the 6 7 5 supernatant for acamprosate end-point concentrations. The filters with cells from the [14C]-mannitol study were mixed thoroughly 8 6 with 2 mL scintillation liquid and analyzed directly by scintillation counting. In the case of lucifer yellow and fluorescein permeability 9 studies, the TEER of the monolayers was measured again after the monolayers were re-equilibrated for 10 minutes in permeability 10 7 11 8 buffer at room temperature (20°C). 12 9 2.5. Sample treatment and bioanalysis. Acamprosate samples from the DSMZ Caco-2 study were analyzed with a tandem 13 14 10 6460 Triple Quad MS-MS system (Agilent Technologies) with electrospray ionization (ESI) and an orthogonal ion source design, 15 11 attached to a 1200 Series Infinity LC system (Agilent Technologies). The chromatographic separation was carried on an XBridge™ 16 12 Amide column (2.1x100 mm, 3.5 µm). In the samples from the ATCC Caco-2 study, acamprosate was quantified using a Waters 17 18 13 Xevo Triple-Quadrupole MS with electrospray ionization (Zspray) coupled to an Acquity UPLC (Waters, Milford, MA). The 19 14 chromatographic separation was carried out on an Acquity UPLC HSS T3 column, 2.1x50 mm (1.8 µm). Warfarin was used as an 20 15 internal standard. [14C]-mannitol was quantified by scintillation counting on a Perkin Elmer TriCarb 4910 TR (Boston, MA, USA). 21 22 16 Lucifer yellow and fluorescein samples were transferred in to 96-well black NUNC plates with a transparent bottom and quantified 23 17 with a FLUOStar Omega plate reader (BMG Labtech), excitation 485±12 nm, emission 520 nm. Atenolol samples were analyzed by 24 chromatographic separation with an XTerra™ MS C18 column (2.1x100 mm, 3.4 µm) followed by quantification of atenolol with a 25 18 26 19 Shimadzu RF-20A XS fluorescence detector, excitation 229 nm, emission 309 nm. Full details regarding sample preparation, 27 20 quantification and bioanalytical method validation for all the compounds can be found in Supporting Information, Section S1. 28 29 21 2.6. Data treatment. The accumulated amount of compound in the receiver compartment was normalized to monolayer 30 22 area and plotted as a function of time, then analyzed by linear regression analysis to obtain the steady-state flux (the slope of the best 31 23 fit line). When the analysis showed that the compound uptake as a function of time was linear (R2 > 0.9), i.e. constant flux, the 32 33 24 resulting Papp was calculated using Equation 1. Thus, the apparent permeability (Papp) of acamprosate, mannitol, lucifer yellow, 34 25 fluorescein and atenolol was calculated as the ratio between the steady-state flux J (pmol ∙ cm-2 ∙ min-1) and the initial concentration 35 c (t = 0) of the substance in the donor chamber (µmol/L): 36 26 0 ―1 37 퐽 푚푟 ∙ 퐴 27 푃푎푝푝 = = Equation 1 38 푐0 ∆푡 ∙ 푐0

39 28 Where mt is the total amount (µmol) of acamprosate, mannitol, lucifer yellow, fluorescein or atenolol permeated to the 40 29 receiver compartment across the cell monolayer, A is the monolayer area (1.12 cm2), during the defined time interval (Δt, seconds). 41 42 30 The efflux ratio (ER) was determined by dividing the Papp obtained in the B-A direction with the Papp obtained in the A-B 43 31 direction, as in Equation 2. An efflux ratio ≥ 2 suggests efflux transporters expressed in the cell line are likely to be involved in the 44 32 permeation of the compound in the B-A direction 15. 45 46 푃푎푝푝, 퐵 ― 퐴 33 퐸푅 = 푃 Equation 2 47 푎푝푝, 퐴 ― 퐵 48 34 Compound mass balance (%) after permeability experiments was calculated according to Equation 3:

49 (푚푑, 푒푛푑 + 푚푟, 푒푛푑 + 푚푚, 푒푛푑) 푀푎푠푠 푏푎푙푎푛푐푒 % = × 100 Equation 3 50 35 푚푑, 푠푡푎푟푡 51 36 Where md, end, mr, end and mm, end are the amounts (µmol) of compound present at the end of the permeability assay in the 52 53 37 donor, receiver and monolayer compartments, respectively, while md, start is the amount (µmol) compound present in the donor 54 38 compartment at the start of the assay. 55 56 8 57 58 59 60 ACS Paragon Plus Environment Page 9 of 29 Molecular Pharmaceutics

1 1 2.7. Mathematical modelling of the in vitro permeability of acamprosate, Pmodelled. A semi-mechanistic permeability 2 6, 22 3 2 model, termed Pmodelled, was adapted with minor changes from permeability models described in literature to evaluate the relative

4 3 contribution of each available permeation route, i.e. Ppara, Ptrans, PABL and Pf to the Papp obtained for the five investigated compounds. 5 4 The mathematical model accounts for a) compound-, b) system- and c) assay-specific parameters that contribute to or limit solute 6 7 5 permeation across filter-grown Caco-2 monolayers, as depicted in Figure 3. Pmodelled consists of four permeability components, Ppara, 8 6 Ptrans, PABL and Pf, representing the possible compound permeation routes, each calculated individually as described below. 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 7 34 8 Figure 3. Input parameters and workflow for calculating Pmodelled from its components Ppara, Ptrans, PABL and Pf : a) compound-

35 9 specific parameters: molecular weight (MW), acid dissociation constant (pKa), lipophilicity (logP or logD) and aqueous solubility; b) 36 system-specific properties: paracellular pore radius (R), pore capacity factor for size- and charge-dependent (ε/δ) and independent 37 10 38 11 (ε2/δ2) paracellular pores, potential drop across the cell monolayer (Δφ), villus-fold area expansion factor (kVF) and c) assay-specific 39 12 properties: permeation buffer viscosity (η) and pH, temperature (t°C), plate rotation speed (rpm), filter support area, filter porosity 40 41 13 and filter pore pathlength. 42 43 14 Table I. Compound-specific parameters used as input for Pmodelled 44 a c c d Compound MW Compound type and pKa log P Aqueous fu,ABL 45 (g/mol) major ionic species b solubility 46 (mg/mL) b, c 47 Acamprosate 181.21 23 Sulfonic acid, monoprotic 23 1.83* -3.57 ± 0.17 24* 18.8 25, 26* 1 48 Anion (n = 10) 49 [14C]-mannitol 184.17 27 Radiolabelled saccharide, 13.14 28** -3.10 29** ≥ 100 27** 1 50 27 51 Neutral 30 30 31* 32** 33 52 Lucifer yellow 442.24 Sulfonic acid, diprotic 1.40; 5.70 -2.57 1 * 1 53 Dianion 54 55 56 9 57 58 59 60 ACS Paragon Plus Environment Molecular Pharmaceutics Page 10 of 29

1 Fluorescein 332.31 34 Carboxylic acid, diprotic 34 2.30; 4.35; 2.68 ± 0.68 28** 4.3 1 2 Dianion 6.70 35* (2.3 at pH=6 )36* 3 4 Atenolol 266.34 37 Secondary amine 37, 9.54 38* 0.22 38* 1328** 1 5 Cation 6 1 a molecular weight of the permeating ionic species; b at pH 6.0 - 7.4; c at 25°C; d unbound compound fraction in the ABL, estimated 7 2 to 1 for all compounds; * measured experimentally; **calculated using Advanced Chemistry Development (ACD/Labs) Software 8 3 V11.02 28; 9 10 4 Table II. Assay-specific parameters used as input for Pmodelled 11 Assay-specific parameters 12 Temperature (°C / K) 37 / 310.15 13 Apical: basolateral pH 7.4: 7.4 14 Plate rotation speed (rpm) 220 (well- 15 stirred) 16 6, 39, 40 17 Aqueous boundary layer height, hABL (µm) 500 18 Filter Area (cm2) / radius (mm) 1.12 / 6 41 19 parameters Pore diameter, Df / radius, Rf (µm) 0.4 / 0.2 20 Pore path length, hf (µm) 10

21 Porosity, εf 0.12566 22 5 23 6 2.7.1 Paracellular permeability (Ppara). The permeability across the junctional paracellular pores (Ppara) was calculated 24 25 7 using the extended Adson paracellular diffusion model42 proposed by Avdeef and Tam (Equation 4)39 to which the contributions of 26 8 fractions permeating as dianions were added to the E(Δφ) component of Ppara, as in the paracellular diffusion model reported by 27 Bittermann & Goss22. The paracellular model is a biophysical model which describes the diffusion of molecules and ions under sink 28 9 29 10 boundary conditions across two types of paracellular pores: i) primary, high capacity (ε/δ), size- and charge-restricted cylindrical 30 11 water channels, coated with negative charges and ii) secondary, low capacity (ε2/δ2), size-unrestricted and non-charge-selective 31 32 12 cylindrical water channels: 33 휀 푟퐻푌퐷 휀2 13 푃푝푎푟푎 = × 퐷푎푞 × 퐹 × 퐸(훥휑) + × 퐷푎푞 Equation 4 34 훿 (푅퐶푎푐표 ― 2) 훿2 35 2 14 where 퐷푎푞 is the aqueous diffusion coefficient (cm /s) at the assay temperature; ε/δ and ε2/δ2 are the capacity factors of the primary 36 37 15 and secondary pores, respectively, expressed as the ratio between the porosities ε and ε2 and pore pathlengths δ and δ2 of each pore 38 16 type. The term F (rHYD / RCaco-2) represents the hydrodynamic Renkin sieving function for cylindrical water channels, as described 39 17 below in Equation 5. The equation is a function of rHYD, the hydrodynamic molecular radius of the tested compound and RCaco-2, the 40 41 18 average pore radius the Caco-2 cell monolayers. 2 3 5 42 푟퐻푌퐷 푟퐻푌퐷 푟퐻푌퐷 푟퐻푌퐷 푟퐻푌퐷 19 퐹 = 1 ― × 1 – 2.104 + 2.09 ( ) – 0.95 ( ) Equation 5 43 (푅퐶푎푐표 ― 2) [ (푅퐶푎푐표 ― 2)] [ (푅퐶푎푐표 ― 2) 푅퐶푎푐표 ― 2 푅퐶푎푐표 ― 2 ] 44 45 20 The E(Δφ) term in Equation 4 is a function of the average potential drop Δφ (mV) across the Caco-2 monolayer, caused by 46 21 the electric field generated by the negative charges coating the paracellular junctional pores 6, 22. This creates a charge bias in the 47 22 model: cations are predicted to permeate at a higher rate across paracellular pores as compared to molecules with a net charge equal 48 49 23 to zero, which in turn permeate at higher rates than anions: 50 휅 |훥휑| 휅 |훥휑| 휅 |훥휑| 휅 |훥휑| 24 퐸(훥휑) = 푓(0) + 푓( + ) × 휅|훥휑| + 푓( ― ) × +휅|훥휑| + 푓( +2) × 2휅|훥휑| + 푓( ― 2) × +2휅|훥휑| Equation 6 51 1 ― 푒 ― 푒 ― 1 1 ― 푒 ― 푒 ― 1 52 25 where f(0), f(+), f(-), f(+2) and f(-2) are the concentration fractions of the compound in the uncharged, cationic, anionic dicationic and 53 54 26 dianionic forms, respectively. The model assumes that the electric field created by the negative charge residues coating the paracellular 55 56 10 57 58 59 60 ACS Paragon Plus Environment Page 11 of 29 Molecular Pharmaceutics

1 22 1 channel affects dianionic and dicationic compounds twice as much as anions/cations . The constant κ is described as F / NA kB T = 2 -1 -1 2 -2 3 2 0.037414 mV at 37 °C, where F is Faraday constant (C mol ), NA is Avogadro number (mol ), kB is Boltzmann constant (cm kg s 4 3 K-1) and T is absolute temperature (K). 5 4 Daq, rHYD, f(0), f(+), f(-), f(+2), f(-2) were calculated for each compound using the compound-specific parameters (Table I) at the 6 7 5 assay-specific parameters (Table II). Daq was calculated using Equation 7, which represents an empirical correlation between 8 6 molecular weight (MW, g/mol) of 147 predominantly drug-like compounds and their respective aqueous diffusion coefficients (Daq, 9 cm2/s) reported by Avdeef 43. The equation is valid for compounds with log P < 4 and MW between 30 and 1200 Da, at 25°C. 10 7 O: W 11 8 푙표푔퐷푎푞 = ― 4.13 ― 0.453 × 푙표푔푚푤 Equation 7 12 43 9 The calculated Daq values at 25°C were corrected with a multiplication factor of 1.339 to match the temperature of the assay, 37°C. 13 14 10 rHYD was calculated from the Sutherland-Stokes-Einstein equation for spherical particles, Equation 8, where kB is the Boltzmann 15 11 constant, with a value of 1.38 x 10-16 (cm2 ∙ kg ∙ s-2 ∙ K-1), T is the absolute temperature (K) and η is the permeability buffer viscosity 16 12 with a value of 0.006962 cm2 s-1 at 37 °C 39. 17 +8 18 21.8 10 푘퐵푇 13 푟퐻푌퐷 = (0.92 + ) × Equation 8 19 푀푊 6휋휂퐷푎푞

20 14 The compound fractions f(0), f(+), f(-), f(+2), f(-2) were calculated by inputting the compound pKa and assay pH in the Henderson- 21 15 Hasselbalch Equation 9 to obtain the neutral fraction. 22 1 23 푓 = Equation 9 16 0 |10 ―푝퐾푎| 24 1 + ( |10 ―푝퐻| ) 25 26 17 The ionized fraction for each compound was calculated by subtracting the neutral fraction from 1. Where >2 ionic species 27 18 were present at assay pH, i.e. in the case of fluorescein, the compound fractions were extracted from published Bjerrum diagrams44 28 29 19 using the GetData graph digitizer (getdata-graph-digitizer.com). 30 20 The system-specific parameters depending on the physiology of the Caco-2 monolayers employed in the study could not be 31 43 21 estimated theoretically or collected from other studies as they show high interlaboratory variability . Thus, empirical Papp data 32 33 22 obtained for the 5 investigated compounds in each cell passage was used to refine 4 system parameters in Equation 4: the paracellular

34 23 pore radius (RCaco-2), the capacity factors of primary (ε/δ) and secondary (ε2/δ2) paracellular pores, and the potential drop (Δφ) across 35 the monolayers, at values which best fit the experimental P data. The parameter refinement procedure is described in Section 2.8. 36 24 app 37 25 2.7.2. Passive transcellular permeability (Ptrans). Ptrans was calculated using Equation 10, which is an empirical correlation 38 43 26 between the measured log DO:W at pH 7.4 and log Ptrans for a range of paracellular markers . 39 40 27 푙표푔푃 푡푟푎푛푠 = ―6.4 + 0.54 × 푙표푔퐷 푂:푊 Equation 10 41 28 Log P / log D and pKa values were measured in our laboratory or collected from literature or chemistry databases. Where 42 29 log D (P) was unknown for a compound, it was estimated from log P (D), pKa (Table I) and assay pH (Table II) using the log P/log 43 44 30 D calculator embedded in Simcyp v.18 release 1. The transmembrane permeation of the ionized compound fractions was not

45 31 considered in the calculation of Ptrans as the lipid membrane permeation rate of ions is several orders of magnitude lower than that of 46 uncharged molecules, thus would have an insignificant contribution to P . 47 32 trans 48 33 2.7.3. Aqueous boundary layer permeability (PABL). The stagnant water layer adjacent to the cell monolayer can often 49 34 constitute a limiting step to the permeation of some drug compounds, especially in the case lipophilic compounds with high 50 51 35 membrane permeability. Even though the compounds modelled in the present study are hydrophilic, low permeability compounds, 52 36 it was important to quantify the contribution of PABL to the overall permeability. PABL was calculated using Equation 11, where hABL 53 54 55 56 11 57 58 59 60 ACS Paragon Plus Environment Molecular Pharmaceutics Page 12 of 29

1 1 is the height of the aqueous boundary layer (sum of apical and basolateral ABL). The ABL height was estimated (Table II) as the 2 3 2 maximum value reported in the literature in the case of well-stirred solutions. 4 퐷푎푞 3 푃퐴퐵퐿 = Equation 11 5 ℎ퐴퐵퐿

6 4 2.7.4. Filter permeability (Pf). The permeability across the polycarbonate Transwell filters on which the cell monolayers 7 were seeded was calculated using Equation 12 6 where δ is the filter pore pathlength (µm); Ɛ is the filter porosity and F(푟 / 푅 ) 8 5 f f 퐻푌퐷 푓 9 6 represents the hydrodynamic Renkin sieving function described in Equation 6, where RCaco-2 is replaced with filter pore radius (Rf).

10 퐷푎푞 푟퐻푌퐷 P = [ ] [휀푓 F ( )] Equation 12 11 7 f 훿푓 푅푓 12 8 As the ratio between the radii of the permeating molecules (< 0.0006 µm) and the filter pore radius (0.2 µm) is very low, 13 14 9 the Renkin sieving function across the filter pores has been approximated to a value of 1. By inputting the filter parameters obtained 41 15 10 from the filter producer (Table II) into Equation 12, the filter permeability for our specific assay settings was calculated to Pf = 16 125.66 ∙ D . 17 11 aq 18 12 2.7.5. Modelled in vitro permeability (Pmodelled). The calculated Ppara, Ptrans, PABL and Pf were compiled to Pmodelled according 19 13 to Equation 13, where KVF is the villus-fold monolayer surface area expansion factor and fu, ABL is the fraction of unbound compound 20 21 14 molecules in the ABL: 22 1 1 1 1 15 = ( ) + + Equation 13 23 푃푚표푑푒푙푙푒푑 푃푡푟푎푛푠 × 푓(0) + 푃푝푎푟푎 × 퐾푉퐹 × 푓푢, 퐴퐵퐿 푃퐴퐵퐿 푃푓

24 16 Pmodelled does not include the carrier-mediated permeability sub-component PCM (depicted in Figure 2), as no 25 17 carrier/transporter proteins have been identified in mammalian cells/tissues for any of the 5 investigated compounds at the assay 26 27 18 settings considered by the model. The relative contribution of PABL to Pmodelled was calculated in the form of how much it contributed

28 19 to the resistance to permeation, according to Equation 14. Pf contribution was evaluated similarly.

29 1/푃퐴퐵퐿 % 푃퐴퐵퐿 = × 100 Equation 14 30 20 1/푃푚표푑푒푙푙푒푑 31 After subtracting the contributions from P and P from P , a “clean” P was obtained which only represents 32 21 ABL f modelled monolayer 33 22 the permeation across the Caco-2 cell monolayer. The relative contribution of Ppara to Pmonolayer was calculated using Equation 15:

34 푃푝푎푟푎 % 푃푝푎푟푎 = × 100 Equation 15 35 23 푃푚표푛표푙푎푦푒푟 36 24 For calculating % Ptrans, the same equation was used where Ppara is replaced with Ptrans × f(0). 37 38 2.8. Statistical analysis. All data fitting and statistical analyses were performed using GraphPad Prism (version 7.04 or 39 25 40 26 version 8; San Diego, CA, USA) and Microsoft Excel 2016. Experimental data obtained from several cell passages (n) are presented 41 27 as passage mean ± standard error of means (SEM). The differences between means have been evaluated using one-way ANOVA, 42 43 28 followed by multiple comparisons tests. A post-hoc Tukey’s test was used for the comparison of individual groups. A p-value < 0.05 44 29 was considered statistically significant (*) while a p-value ≥ 0.05 was not statistically significant. 45 30 The refinement of the paracellular parameters RCaco-2, ε/δ, ε2/δ2, Δφ was done by weighted nonlinear regression analysis, 46 47 31 similar to as reported by Avdeef and Tam39. The regression function (Equation 16) was based on Equation 13 in logarithmic form

48 32 expanded with Equations 4-12 which describe the Pmodelled subcomponents:

49 ε ε2 1 ℎ퐴퐵퐿 1 50 33 log 푃푚표푑푒푙푙푒푑(푅퐶푎푐표 ― 2, δ,δ , Δφ) = ―푙표푔[125.66 × 퐷 + 퐷 + 푟 ε ] Equation 2 푎푞 푎푞 0.54 ε 퐻푌퐷 2 ( ― 6.4) × 푙표푔퐷 × 푓(0) + × 퐷푎푞 × 퐹( ) × 퐸(Δφ) + × 퐷푎푞 51 푂:푊 δ 푅 δ2 52 34 16 53 54 55 56 12 57 58 59 60 ACS Paragon Plus Environment Page 13 of 29 Molecular Pharmaceutics

1 1 Initial Pmodelled values were calculated for each compound using as input for the four dependent variables RCaco-2, ε/δ, ε2/δ2, Δφ average 2 43 3 2 values from literature as refined by Avdeef and Tam for several sets of published Caco-2 Papp data: RCaco-2 = 7.42 Å, ε/δ = 10.55 -1 -1 4 3 cm , ε2/δ2 = 0.01 cm , and Δφ = - 51.5 mV. The initial squared sums of errors (SSE) were calculated according to Equation 17 and 5 4 the function was minimized using the Solver Add-On in Microsoft Excel 2016 for the range of Papp obtained for each compound from 6 7 5 several cell passages (for acamprosate, only Papp obtained from the DSMZ Caco-2 cells were used in the refinement). The applied 8 6 constraint was that RCaco-2, ε/δ, ε2/δ2 should give positive values, while Δφ should give negative values. The dependent variables RCaco-2, 9 ε/δ, ε /δ , Δφ were then back-calculated from the minimized SSE. 10 7 2 2 11 ∑푛 표푏푠 푐푎푙푐 2 8 푆푆퐸 = 푖 (푙표푔 푃푎푝푝,푖 ― 푙표푔푃푚표푑푒푙푙푒푑,푖) Equation 17 12 13 9 where n=5 number of compounds used in the model. A goodness of fit (GOF) of the refinement was calculated according to Equation

14 10 18 to evaluate how effective was the concurrent fitting of nv= 4 variables. 15 1/2 푙표푔 푃표푏푠 ― 푙표푔 푃푐푎푙푐 2 16 ∑푛 푎푝푝,푖 푚표푑푒푙푙푒푑,푖 11 퐺푂퐹 = [ 푖 ( 푆퐸푀(푙표푔 푃 ) ) / (푛 ― 푛푣) ] Equation 18 17 푎푝푝,푖 18 12 Where SEM(logPapp,i) is the SEM of the logarithm of each experimental Papp used in the refinement. The equation should return a 19 13 value close to 1 if the model is suitable for the experimental data. 20 21 14 22 23 15 24 16 . 25 26 17 27 18 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 13 57 58 59 60 ACS Paragon Plus Environment Molecular Pharmaceutics Page 14 of 29

1 1 3. RESULTS 2 3 2 3.1. The apparent permeability of acamprosate is low and equal in both transport directions. The bidirectional i.e. 4 A-B and B-A P of acamprosate across Caco-2 monolayers were 1.51 ± 0.40 and 2.31 ± 0.59 × 10-7 cm/s, respectively, when 5 3 app 6 4 measured in the DSMZ and 1.62 ± 0.68 and 1.02 ± 0.49 × 10-7 cm/s, respectively, in the ATCC cells. These data are depicted in 7 5 Figure 4 together with the calculated efflux ratios. In filter-grown Caco-2 cells from both DSMZ and ATCC, 0.1 - 0.5% of the amount 8 9 6 applied on the donor side appeared in the receiver chamber at the end of the permeation assay. For donor concentration of acamprosate 10 7 at 20 mM acamprosate concentration in the receiver chamber was generally above the lower limit of quantification (LLOQ) of the 11 8 applied bioanalysis method. However, when acamprosate donor concentration was 20 µM or 200 µM, the acamprosate concentration 12 13 9 in the receiver chamber was below LLOQ for the first 120 minutes (20 µM) or 90 minutes (200 µM) in the ATCC Caco-2 cells.

14 10 Nevertheless, for the 200 µM investigations, rough Papp values were estimated from end-point accumulation (120 minutes after 15 × -7 16 11 application) to 0.5 ± 0.3 and 0.2 ± 0.1 10 cm/s after apical and basolateral addition, respectively. No statistically significant 17 12 difference was found between acamprosate Papp across monolayers from the two different cell banks, in A-B (P value = 0.8861) and 18 13 B-A (P value = 0.1675) transport directions. No statistically significant difference has been observed between the obtained efflux 19 20 14 ratios (P value = 0.0627) in DSMZ and ATCC Caco-2 monolayers. 21 22 ER=1.570.37 ER=0.630.51

23 ] -1 410 -7

24 s  25 26 310 -7 [cm A-B 27

app B-A 28 -7 29 210 30 31 110 -7 32 33 0

34 P acamprosate 35 DSMZ cells ATCC cells 36 15

37 16 Figure 4. Apparent permeability (Papp) of 20 mM acamprosate across DSMZ and ATCC Caco-2 cells. Filled bars denote 38 the apical-to- basolateral (A-B) P , while empty bars represent the basolateral-to-apical (B-A) P . Results are presented as mean ± 39 17 app app 40 18 SEM, n=3. 41 42 19 In a second study setup, acamprosate Papp ’s were compared to Papp‘s of the paracellular markers mannitol, lucifer yellow, 43 -7 20 and fluorescein. The pooled Papp‘s in the respective A-B and B-A directions were 1.45 ± 0.38 and 1.66 ± 0.45 × 10 cm/s for 44 -7 -7 45 21 acamprosate, 2.64 ± 0.35 and 2.84 ± 0.31 × 10 cm/s for mannitol, 1.31 ± 0.35 and 2.29 ± 0.50 × 10 cm/s for lucifer yellow, 2.17 46 22 ± 0.47 and 2.03 ± 0.38 × 10-7 for fluorescein and 6.18 ± 0.69 and 8.47 ± 0.20 × 10-7 for atenolol, as depicted in Figure 5a. No 47 23 statistically significant difference was found between the A-B vs. B-A Papp for each compound, nor between the Papp of acamprosate 48 49 24 and of mannitol, lucifer yellow and fluorescein. The Papp of atenolol was statistically significantly higher than that of all the other 50 25 compounds, in both A-B and B-A transport directions. Figure 5b shows the calculated efflux ratios (ER) obtained for the compounds 51 in Figure 5. There was no statistically significant difference between the obtained efflux ratios for the 5 investigated compounds. 52 26 53 54 55 56 14 57 58 59 60 ACS Paragon Plus Environment Page 15 of 29 Molecular Pharmaceutics

1 2 A-B 3 B-A 4 5 9.010 -7 * 2.0 6 ] -7

-1 7.510 7 *

s 1.5 8  6.010 -7 9 -7 10 [cm 4.510 1.0

11 app  -7

3.0 10 ratio Efflux P 12 0.5 13 1.510 -7 14 15 0 Acamprosate Mannitol Lucifer Fluorescein Atenolol Acamprosate Mannitol Lucifer Fluorescein Atenolol 16 Yellow Yellow 17 1 18 2 Figure 5. (a) Apparent permeability (Papp) of 20 mM acamprosate (pooled data from DSMZ and ATCC Caco-2 monolayers), 19 3 7 µM [14C]-mannitol, 100 µM lucifer yellow, 26.55 µM fluorescein and 1.5 mM atenolol across filter-grown DSMZ Caco-2 20 4 monolayers. Filled bars denote the permeation in apical-to-basolateral (A-B) transport direction, while empty bars represent 21 22 5 permeation in the basolateral-to-apical (B-A) transport direction. Results are presented as mean ± SEM, n = 3-5. (b) Efflux ratio of

23 6 the Papp from Figure 4a. The dotted lines represent the cut-off values above or below which an active transport process is suspected 24 to contribute to the net P of the compound 15, 45. * (P<0.05) between the P of the compound compared to the P of the other 25 7 app app app 26 8 compounds. 27 9 A parallel study verified whether acamprosate, a compound with relatively high acidity (pKa = 1.83) alters the barrier 28 29 10 function i.e. the Papp of paracellular markers and TEER of the cell monolayers at the high concentrations applied to the monolayers 14 30 11 (10-20 mM). The obtained results are presented in Supporting Information, Figure S2. Papp of [ C]-mannitol across Caco-2 cell 31 12 monolayers was not significantly changed in the presence of 20 µM or 20 mM acamprosate or after pre-incubation of Caco-2 cell 32 33 13 monolayers with 20 mM acamprosate for 2, 4, 8 or 24h, as depicted in Figure S2a. The Papp of Lucifer yellow and fluorescein was 34 14 not affected by 20 mM acamprosate (Figure S2b). The TEER across Caco-2 cell monolayers did not decrease significantly after the 35 15 permeability experiments when lucifer yellow or fluorescein was applied alone or when applied together with 20 mM acamprosate 36 37 16 (Figure S2c). 38 17 3.2. Acamprosate accumulates to a low degree and does not inhibit intestinal carriers in Caco-2 cell monolayers. The 39 end-point intracellular accumulations Q (pmol ∙ cm-2) represents the amount of [14C]-mannitol or acamprosate associated with 40 18 monolayer 41 19 the DSMZ or ATCC Caco-2 cell monolayers, respectively, at the end of the permeability assay. These amounts are plotted in Figure 42 20 6 versus the respective donor concentrations. For both mannitol and acamprosate, similar compound amounts were internalized via 43 44 21 the apical (A-B) and basolateral membrane (B-A). 45 46 47 48 49 50 51 52 53 54 55 56 15 57 58 59 60 ACS Paragon Plus Environment Molecular Pharmaceutics Page 16 of 29

1 2 3 10000 Mannitol AB 4 1000 Mannitol BA 5 ] 6 -2 100

7 cm  10 8

9 monolayer 1

10 Q Acamprosate AB [pmol 0.1 11 Acamprosate BA 12 0.01 13 14 0.001 15 1 10 100 1000 10000 16 Applied concentration [µM] 17 1 2 14 18 2 Figure 6. Qmonolayer (pmol∙cm ), after 120-minute permeability assay at 37°C for 7 and 18 µM [ C]-mannitol and for 20 µM, 19 3 200 µM and 20 mM acamprosate in Caco-2 cell monolayers. Results represent mean ± SEM, n=3-12. 20 21 4 The mass balance of acamprosate from the donor, receiver and intracellular compartments for the 20 mM concentration in 22 5 ATCC Caco-2 cells was 96 ± 15 and 104 ± 14 % in the A-B and B-A transport directions, respectively. 23 6 The uptake rates of [14C]-Gly-Sar, [3H]-taurine, [3H]-proline, [3H]-lysine, [3H]-estrone-3-sulfate (3H-E1S), or [3H]- 24 3 25 7 taurocholic acid ( H-TCA) in the presence of acamprosate are depicted in Figure 6 as a percent of the uptake in the absence of 26 8 acamprosate. No significant difference was observed for the uptake of the substrates in the presence vs. absence of acamprosate. 27 28 29 30 100 31 32 33 34 35 36 50 37

38 (% of control)

39 AT/rBAT 0,+

40 EAAT1

ASBT OATP2B1

of 10-13 mM acamprosate PAT1 B PEPT1 41 TAUT 42 0 Substrate uptake in the presence 43 44 E1S TCA Lysine 45 Gly-SarTaurine Proline 46 9 Glutamate 47 10 Figure 7. Relative uptake of radiolabelled [14C]-Gly-Sar, [3H]-taurine, [3H]-proline, [3H]-glutamate, [3H]-lysine, [3H]- 48 11 estrone-3-sulfate (3H-E1S), or [3H]-taurocholic acid (3H-TCA) at pH=6 in Caco-2 cells seeded on the bottom of the plate in the 49 50 12 presence of 13 mM acamprosate except for 3H-E1S and TCA in which 10 mM acamprosate was applied. Results are presented as % 51 13 of the substrate uptake in the absence of acamprosate (control). The bars represent mean (± S.E.M.), n=1-4. 52 53 54 55 56 16 57 58 59 60 ACS Paragon Plus Environment Page 17 of 29 Molecular Pharmaceutics

1 1 3.3. The apparent permeability of acamprosate is modelled to be predominantly paracellular. The results for the calculated 2 3 2 Daq, rHYD and f(charge) used as input in Pmodelled are listed in Table III. The model refinement yielded the system-specific parameters R,

4 3 ε/δ, ε2/δ2 and Δφ (table IV) characteristic for the DSMZ Caco-2 monolayers employed in the study. The GOF for the model was 2.35 5 4 for the n = 5 investigated compounds. When predicting Pmodelled with refined system parameters from observed Papp data from different 6 7 5 compounds measured in Caco-2 monolayers grown in a different laboratory, the GOF of the present observed vs. predicted 8 6 permeability increases to 44.5. Figure 8a depicts the Pmodelled (predicted) in comparison to Papp (observed) for the investigated 9 compounds. The average P values are within 0.77- and 1.27-fold and within the between-passage S.E.M of the observed average 10 7 modelled 11 8 Papp for all studied compounds. Ppara is suggested to be the major permeation route for all compounds, covering > 99% of Pmodelled for 12 9 acamprosate, lucifer yellow, fluorescein and atenolol and 97% for mannitol (Figures 8b-8e). Ptrans has a minor contribution (3%) to 13 14 10 the Pmodelled of mannitol. Table III. Calculated compound parameters used as input for the prediction of Pmodelled . a b c d d d d d 15 Compound log D Daq rHYD f (-2) f (-1) f (0) f (+1) f (+2) 16 Acamprosate -7.56 94.15 3.61 0.00 1.00 0.00 0.00 0.00 17 18 Mannitol -3.26 93.46 3.63 0.00 0.00 1.00 0.00 0.00 19 Lucifer Yellow -7.50 71.54 4.50 0.98 0.02 0.00 0.00 0.00 20 Fluorescein -1.08 62.85 5.03 0.92 0.08 0.00 0.00 0.00 21 22 Atenolol -1.87 79.08 4.13 0.00 0.00 0.01 0.99 0.00 23 11 a octanol:water distribution coefficient at pH 7.40, calculated from log P (Table I); b aqueous diffusion coefficient at 37°C, × 107 24 12 cm2/s; calculated with Equation 7; c molecular radius, in Å, calculated with Equation 8; d at pH 7.4; the major ionic species is marked 25 13 in bold font; calculated with Equation 9 or extracted from Bjerrum diagrams44. 26 27 14 Table IV. System-specific parameters refined in the paracellular analysis and used as input for Pmodelled 28 29 System-specific parameter Parameters refined with Parameters refined by Avdeef 2010 6 46 30 Caco-2 Papp data from the present study with Tavelin et al. 2003 Papp data 31 Type I pore radius R (Å) 9.9 ± 1.1 7.0 ± 3.1 32 33 Type I pore capacity factor ε/δ (cm-1) 0.020 ± 0.011 1.4 ± 4

34 -1 Type II pore capacity factor ε2/δ2 (cm ) 0.025 ± 0.004 Not refined 35 36 Potential drop Δφ (mV) - 1021 ± 336 - 82 ± 8 a b b 37 Villus-fold area expansion factor kVF 1 1 38 c 39 n 5 9 40 GOF d 2.35 44.50 e 41 P (mannitol) × 107 cm/s d 2.72 ± 0.18 5.03 42 para 43 15 a the model does not discriminate between the effect of the negative charges coating the paracellular pores on monoanions vs. dianions; b c d 44 16 kept fixed; number of Papp values used to build the model; Goodness-of-fit calculated with to Equation 18 using the observed Papp 45 17 data in DSMZ cells for the 5 investigated compounds; e calculated using the refined system parameters listed above in the same 46 18 column. 47 48 49 50 51 52 53 54 55 56 17 57 58 59 60 ACS Paragon Plus Environment Molecular Pharmaceutics Page 18 of 29

1 2 a Charge of major -1 0 -2 -2 +1 3 ionic species 110 -6 4 5 -7 6 810 7 8 610 -7 Observed 9 (cm/s) 10 410 -7 Predicted

11 app 12 P 210 -7 13 14 0 15 16 ow 17 18 Mannitol Atenolol Fluorescein 19 Acamprosate 20 Lucifer Yell 21 3% 22 b c d 23 24 > 99% 97% > 99% 25 26 27 28 29 Acamprosate Mannitol Lucifer Yellow 30 31 Pmodelled = 2.42 P = 1.84 32 Pmodelled = 2.72 modelled 33 e f 34 35 > 99% > 99% 36 37 Ppara 38 39 Ptrans 40 41 Fluorescein Atenolol 42 43 Pmodelled = 1.62 Pmodelled = 7.32 44 45 46 -1 47 Figure 8. (a) Experimentally determined Papp (cm∙s ) expressed as mean ± SEM (N=6-12, pooled data from both A-B and 14 48 B-A) of Papp (white bars, observed) for 20 mM acamprosate, 7 µM [ C]-mannitol, 100 µM lucifer yellow 26.55 µM fluorescein and 49 1.5 mM atenolol compared to the P (grey bars, predicted) for the same compounds. The refinement was done using experimental 50 modelled 51 Papp from different cell passages, thus Pmodelled is expressed as mean ± SEM (n=3). For acamprosate, only Papp from the DSMZ data set 52 7 was used for refinement (b-f). The relative contribution (%) of Ppara (black) and Ptrans (orange), PABL and Pf to Pmodelled (× 10 cm/s) 53 54 55 56 18 57 58 59 60 ACS Paragon Plus Environment Page 19 of 29 Molecular Pharmaceutics

1 predicted for (b) acamprosate, (c) mannitol, (d) lucifer yellow, (e) fluorescein and (f) atenolol. They were calculated using Equations 2 3 14 and 15. PABL and Pf are plotted in the same pie chart, but they are not visible due to their very low contribution to Pmodelled. 4 5 4. DISCUSSION 6 In this study, we used experimental permeability data for a range of compounds to refine a paracellular model that can 7 8 describe the passive in vitro permeability (Pmodelled) of poorly permeable compounds across Caco-2 cells. The obtained Pmodelled for 9 acamprosate was then combined with carrier inhibition and cellular uptake studies to decipher the in vitro permeability mechanism 10 for acamprosate across Caco-2 cells. The data collected in the present work were obtained from Caco-2 cells cultured in two different 11 12 laboratories and with two different cell bank origins. Therefore, it was important to compare the Papp obtained in each laboratory, as 13 data collected from cell monolayers grown in different laboratories may vary due to differences in culture conditions and experimental 14 47 15 procedures . This has been related to differences in cell passage, cell origin, age and TEER of the monolayers, seeding density or 16 type or strength of permeability buffer used 48. In the present studies, similar assay settings were used in each of the laboratories, and 17 the experiments were performed and analyzed by the same researcher. The obtained acamprosate P (Figure 4) using the two 18 app 19 different cell culture vendors are not statistically significantly different, thus the data obtained in the two laboratories are assumed 20 comparable. 21 The obtained P values (Figure 5) in the present study support classifying all the five investigated compounds as having 22 app -7 -1 49 23 permeability-limited Fa (Papp values < 20 × 10 cm∙s ). The obtained permeabilities match with those of other compounds with

24 -7 -1 50 similar oral bioavailability such as sulfasalazine, with a reported Papp of 4 × 10 cm∙s and oral bioavailability of 11-13% or 25 -7 -1 51, 52 26 mannitol with reported Papp values of around 0.3 × 10 cm ∙ s and oral BA of 16% . The oral bioavailability of lucifer yellow 27 has to our knowledge not been measured in human, but the compound shows very low F (%) in the rat (0.89 ± 0.11%) when 100 µM 28 lucifer yellow is administered and in described by FDA to have zero permeability 53. For fluorescein, the obtained P at pH 7.40 29 app 30 could not be directly compared with oral bioavailability in human, as fluorescein is suggested to be transported by the monocarboxylic 31 acid transporter (MCT) 1 at pH 6.0 54 and this might explain why it has high oral bioavailability (99%) in humans 55. As discussed 32 33 below, acamprosate does not have the structural characteristics necessary for being a substrate for the MCT1 transporter. The Papp 34 obtained for atenolol was approx. 4-fold higher than that of acamprosate, and this matches the ratio between the oral bioavailabilities 35 of atenolol and acamprosate (~50% 52 vs. ~11%). 36 37 Since acamprosate Papp was equal in both transport directions, i.e. has similar A-B and B-A permeabilities, we can speculate

38 that either no carrier/transporter-mediated absorptive/secretion mechanisms contribute to acamprosate Papp or that SLCs are saturated 39 at the concentration investigated. P of acamprosate was additionally investigated at 100-fold (200 µM) and 1000-fold (20 µM) 40 app 41 lower concentration, to exclude the existence of polarized carrier- or transporter- mediated permeability that may be saturated at 20 42 mM concentrations. However, only for the end-point (120 minutes) of the 200 µM condition acamprosate amounts in the receiver 43 compartment were above LLOQ. The estimated P was very low and variable between cell passages, but similar in the A-B and B- 44 app 45 A directions. This finding is in agreement with the results obtained by Zornoza et al. 8, where only low concentrations of 4.5 µM 46 14 [ C]-acamprosate was applied to ATCC Caco-2 monolayers of similar TEER and days in culture. They reported a Papp of 10.33 ± 47 -7 -1 48 1.58 × 10 cm∙s equal in both transport directions (ER ~1), suggesting as well that the transport of acamprosate across Caco-2 49 monolayers is not polarized in A-B or B-A transport directions even when acamprosate is applied at low concentrations. 50 The P of mannitol, lucifer yellow or fluorescein is routinely measured in parallel with other investigated compounds to 51 app 52 evaluate whether the barrier function of epithelial cell monolayers is maintained during the permeability assay. All of these are 53 predominantly transported via the paracellular route across intestinal epithelia at pH 7.4 54, 56. Atenolol is also known to be absorbed 54 55 56 19 57 58 59 60 ACS Paragon Plus Environment Molecular Pharmaceutics Page 20 of 29

1 predominantly passively57 via the paracellular route across the intestine6, while it is partly excreted by an organic cation transporter 2 58 3 (OCT) 2 in tandem with the multidrug and toxin extrusion protein (MATE) 1 carrier in the kidney . The Papp of mannitol, lucifer

4 yellow and fluorescein measured in parallel in our cell monolayers are similar to that of acamprosate, while the Papp of atenolol is 5 statistically significantly higher than of all the other tested compounds. Mannitol has additionally a similar molecular hydrodynamic 6 7 radius with acamprosate but is neutral rather than negatively charged. The neutral charge of mannitol might explain the slightly higher

8 Papp and intracellular accumulation obtained for mannitol compared to acamprosate, as it is known that the negative charges that coat 9 the TJs regulating the paracellular permeation of solutes, allow cations and neutral compounds to permeate faster than anionic 10 11 compounds. The paracellular permeation of solutes across paracellular pores is known to be charge-selective, preferentially allowing 12 solutes in the order (from high to low) dications > monocations > neutral molecules/zwitterions> monoanions > dianions 59, 60. The 13 14 present results suggest that the DSMZ Caco-2 monolayers employed in the study are indeed charge-selective, as the Papp of the

15 investigated compounds maintains the ranking for charge-selectivity: Papp values were in the order atenolol (monocation) > mannitol 16 (neutral) > acamprosate (monoanion) > lucifer yellow and fluorescein (dianions). 17 18 It was as well relevant for the scope of our study to demonstrate that such high applied concentrations of acamprosate (up 19 to 20 mM), and the low pKa of 1.83 do not damage the normal barrier function of the Caco-2 monolayer. As acamprosate did not 20 affect the integrity or paracellular permeability of the Caco-2 cell monolayers, it can be assumed it does not self-enhance its junctional 21 22 permeability. This is especially important in the context of providing a robust modelling of Ppara, where the radius of the paracellular 23 pores is a rate-limiting component. Furthermore, acamprosate at high concentrations showed very limited influence the osmotic 24 pressure in the donor solution, therefore it is not likely acamprosate affect its own paracellular permeability in Caco-2 monolayers 61. 25 26 Thus, the obtained Ppara for acamprosate is solely caused by concentration gradient-driven paracellular diffusion. 27 Acamprosate shows very low uptake in the Caco-2 monolayer (Figure 6) and similar uptake via the apical and basolateral 28 29 membrane. This suggests the transcellular permeability component to be limited for acamprosate, therefore the paracellular route 30 must be the main permeation pathway. Cellular acamprosate uptake was even lower than the uptake of mannitol, and since no carrier- 31 mediated component is contributing to the uptake of low concentrations of mannitol, the same scenario should apply for acamprosate. 32 33 The present findings furtherly suggest that at pH 6.0 acamprosate does not inhibit the apical PEPT1, TAUT, PAT1, EAAT1, 34 B0,+AT/rBAT, OATP2B1 or ASBT carriers (Figure 7) expressed in Caco-2 cells 20. The tested carriers have been selected due to the 35 resemblance between the chemical structures of their substrates and that of acamprosate. All the studied carriers are found as well in 36 37 the human intestine 47, 62-65. Therefore, it is not likely that the above carriers would be involved in the in vivo absorption of acamprosate. 38 Positive controls 10 mM Gly-Sar-Sar (PEPT1), 10 mM imidazole-4-acetic acid (TAUT), 30 mM sarcosine (PAT1), 5 mM glutamate 39 0,+ 40 (EAAT1), 20 mM arginine (B AT/rBAT), 1 mM taurocholic acid (OATP2B1) and 1 mM taurolithocholic acid (ASBT) inhibited in 41 other Caco-2 studies the substrate uptake to 14.5 ± 1.7 66, 10.1 ± 2.2 67, 14.5 ± 2.0 66, 1.8 ± 0.04 (this study), 3.9 ± 0.0 68, 23.3 ± 11.3 42 69 and 21.2 ± 2.4 69 % of control, respectively. The inhibition of acamprosate at some carriers present in both Caco-2 and human 43 44 intestine were not tested as acamprosate does not have the structural characteristics of the typical substrate for those carriers. For 45 example, OCTs, i.e. the basolateral OCT2 or the apical OCTN1 have an affinity for various cationic and zwitterionic compounds and 46 seldom for small organic anions 70. The monocarboxylic acid transporter (MCT) 1, which has an affinity for substrates with a 47 48 carboxylic group71, would also not be a good candidate as a carrier for acamprosate. Many other OATP carriers that might be 49 expressed in enterocytes, i.e. OATP1A2, OATP4C1 also have E1S as substrate, thus if these were expressed in Caco-2 and 50 acamprosate was an inhibitor or substrate for these carriers, an effect by acamprosate on E1S transport would have been observed 70. 51 52 Moreover, the low amount of acamprosate taken up by the Caco-2 monolayer and the lack of polarized permeability in either transport 53 54 55 56 20 57 58 59 60 ACS Paragon Plus Environment Page 21 of 29 Molecular Pharmaceutics

1 direction and at both high and low acamprosate concentration suggest there is a limited possibility that any apical carrier could 2 3 facilitate the permeation of acamprosate across the Caco-2 monolayers.

4 Several authors have attempted to describe the relative contribution to Fa of the remaining two possible permeability 5 mechanisms i.e. paracellular and carrier-mediated mechanisms. For acamprosate, these descriptions of permeability were investigated 6 7 either in vitro 8-10, 12 or ex vivo 8 but report conflicting results. Some authors suggest acamprosate is absorbed paracellularly 8, 10, 8 however, they either lack experimental evidence 10 or use acamprosate concentrations several orders of magnitude lower (4.5 µM) 9 than a rough estimation of luminal concentration (from 6.6 mM and up to 800 mM) that can be achieved clinically after the oral 10 11 administration of a common dose of acamprosate (300-600 mg) 8. Chebenat et al. 12 suggest that an unknown carrier or formation of 12 ion-pairs facilitates the Papp of acamprosate, however, these suggestions are based solely on the physical-chemical properties of 13 9 14 acamprosate and not supported by transporter or carrier affinity or inhibition studies. Mas-Serrano et al. provide experimental ex 15 vivo data obtained in rat jejunum to suggest that permeation mechanism of acamprosate across rat epithelia is dominated by passive 16 diffusion and probably a minor saturable process involving an imino-acid carrier. The authors have reported a slight inhibition of 0.1 17 18 mM acamprosate permeation by high concentrations (40 mM) of GABA and taurine, to 78 ± 18 and 68 ± 16%, respectively. The 19 transport of acamprosate was not Na+ dependent though, which would be expected for substrates of imino-acid carriers 9. The same 20 study reports a lack of inhibition by 40 mM GABA or glycine when 1 mM acamprosate was applied, and additionally a lack of 21 22 inhibition by 40 mM taurine or proline, which is in accordance to the results obtained in this study. 23 6, 22, 42, 72, 73 Pmodelled (Equation 13) is an extension of other biophysical permeability models developed to estimate the passive 24 apparent permeability (P ) of compounds across cultured epithelial cell monolayers or rodent small intestine. It was necessary that 25 app 26 system-specific parameters used in the modelling are refined for the current set of compounds and Caco-2 monolayers, as by simply 27 43 applying parameters refined in other studies the goodness-of-fit (GOF) of the Pmodelled (predicted) vs. Papp (observed) is less than 28 29 optimal. Due to parameter refinement, the GOF dropped from a value of 44.5 to 2.35, a value that suggesting a very good fit of the 30 predicted vs. observed data (Table IV and Figure 8a). The paracellular radius was refined to 9.9 ± 1.1 Å, which fits well to the radius 31 refined using experimental data from other Caco-2 studies (4-12 Å). The pore capacity for the size- and charge- selective (Type I) 32 33 pores (ε/δ) was much lower (0.020 ± 0.011 cm-1) than the ε/δ obtained in other refinements (0.78-31 cm-1), suggesting the Caco-2 34 monolayers have a lower porosity (ε) or higher pore pathlength (δ) that other monolayers. Another reason for the difference is that 35 our model refinement considered charge- and size- independent (Type II) paracellular pores as an additional component. The Type II 36 -1 37 pore capacity (ε2/δ2) refined in our monolayers (0.025 ± 0.004 cm ) is similar to the one obtained from a refinement of paracellular 38 transport of poly-ethylene glycols (0.007-0.0011 cm-1)6. Our refinement also suggests that the type I and type II pores have a similar 39 40 capacity in our Caco-2 monolayers. The potential drop Δφ across our monolayers was estimated to a value much lower value (-1021 41 ± 336 mV) than the Δφ refined other studies (-30 to -82 mV). However, the parameter estimation Δφ has shown high variability for 42 the other refinements as well. Nevertheless, a lower Δφ might suggest that the charge-discrimination of compounds permeating via 43 44 the Type I paracellular pores is much more accentuated in our Caco-2 monolayers as compared to other monolayers. The ranking of

45 Pmodelled according to charge-selectivity is the same as the ranking of Papp and matches the theories related to charge-selectivity. Pmodelled 46 only considers the passive components of permeation, therefore a value for the carrier component, P , is not included in the model, 47 CM 48 as for all the studied compounds there is no evidence suggesting an interaction with an intestinal transporter or carrier at the 49 concentrations and pH used for the in vitro assay. Only for fluorescein, an MCT was found to be involved in its permeability at pH 50 6. The same study shows that at pH 7.4, which is the pH used in the present in vitro permeability studies, the P of fluorescein is not 51 app 52 saturable and not mediated by any carriers 54. This might be because MCT1 is not active at pH 7.4 since it needs a proton gradient in 53 54 55 56 21 57 58 59 60 ACS Paragon Plus Environment Molecular Pharmaceutics Page 22 of 29

1 order to be functional. Also, fluorescein is mainly present as dianion at pH > 6.7 and as a monoanion at pH < 6.7, so the monoanion 2 54 3 might have more affinity for the carrier .

4 Modelling each permeability sub-component contributing to Pmodelled (Figure 8b-g) is useful for providing information 5 regarding the relative contribution of the available permeation routes across Caco-2 monolayers. P is the main contributor to the 6 para 7 overall permeability of acamprosate across Caco-2 monolayers, suggesting that the paracellular pathway is the main in vitro 8 permeation route for acamprosate, lucifer yellow, fluorescein, atenolol (> 99%) and mannitol (97%). P only has a minor 9 trans 10 11 contribution (3%) to the Pmodelled of mannitol and an insignificant contribution for the other compounds. This minor 12 13 contribution might explain why the cellular uptake of mannitol was higher than that of acamprosate in Figure 6. PABL and 14 P , have an insignificant contribution to P as both these estimated permeabilities were several orders of magnitude higher than 15 f modelled 16 Pmonolayer, thus not rate-limiting for the permeability of the investigated. PABL or Pf are still relevant to include in the Pmodelled, especially 17 if the model would be applied for estimating the passive permeability component of high-permeability compounds (BCS class I and 18 II), where P or P they might have values in the same range as P . A next step would be to measure a range of different 19 ABL f monolayer 20 paracellular markers in the same Caco-2 system and use them to validate the model, so it can become useful in predicting the passive 21 permeability component for the Papp of compounds in our in vitro assay. Extrapolating the obtained results to the conditions of the 22 23 human intestine would be as well highly relevant and necessary. This would come with a series of challenges, as many parameters 24 kept fixed or refined in our Caco-2 assay are not well defined in literature for each segment of the human intestine (pore radius, pore 25 capacity, pore pathlength, potential drop, mucus thickness etc.) and might as well pose high interindividual variability. Using clinical 26 27 Fa for a dataset of low-permeability/high solubility/non metabolised compounds would be one approach to refine these parameters.

28 However, since they are at least 4 parameters to refine for each segment of the intestine, reliable Fa data for at least 33 compounds 29 would be necessary. Further variability in drug solubility and/or absorption can also be triggered by the presence and composition of 30 31 luminal chyme or intestinal motility at the absorption site(s). A better understanding of the role these factors play in the permeation 32 of low permeability compounds would bring further improvement to intestinal absorption models. 33 The in vitro absorption mechanism for acamprosate described in this study can be useful for several aims. First, it can be 34 35 used to develop reliable PBPK absorption models that can predict the disposition of acamprosate and other BCS class III compounds 36 in human populations, for example by refining the human intestinal paracellular pathway parameters using clinical Fa data for a range 37 38 of non-metabolized, paracellularly absorbed compounds. Second, it could be used to explore strategies for increasing the intestinal 39 absorption and oral bioavailability of acamprosate. For example, if a penetration enhancing agent to be administered concomitantly 40 with acamprosate or other compounds whose Fa is driven by Ppara, the enhancing agent should increase the paracellular permeability 41 74 42 (such as e.g.chitosan ). And third, it provides insight into the investigation of the pathways that acamprosate might use to permeate 43 other barriers in the human body. Acamprosate is not metabolised and is eliminated mainly via renal excretion, by a mechanism that 44 we recently described to involve the organic anion transporter (OAT) 1, with a lower contribution from OAT3 75. Neither OAT1 nor 45 46 OAT3 is expressed in the human intestine, but OAT3 is expressed in the blood-brain barrier 76. For reaching the brain, where it exerts 47 its pharmacological action, acamprosate needs to permeate the blood-brain barrier (BBB), by a mechanism that has yet to be described. 48 A study has shown that acamprosate is not detectable in the rat brain24, but no study has investigated acamprosate distribution in the 49 50 human brain or to what extent acamprosate is able to cross the human BBB and exert its therapeutic effect. Both the renal epithelium 51 and blood-brain barrier have very limited possibility of permeation via the paracellular pathway75, 77, therefore in those instances, 52 53 carrier proteins may be the only alternative for permeation. 54 55 56 22 57 58 59 60 ACS Paragon Plus Environment Page 23 of 29 Molecular Pharmaceutics

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1 5. CONCLUSIONS 2 3 Acamprosate permeability across Caco-2 monolayers is low and similar in both transport directions. It is comparable to the 4 5 permeabilities of mannitol, lucifer yellow, and fluorescein. We build and refined a permeability model that fits very well the observed 6 permeability for all the investigated compounds. The modelled permeability describes that the in vitro permeability of acamprosate 7 8 is mainly due to paracellular diffusion since the paracellular diffusion component accounts for > 99% of acamprosate permeability. 9 This was substantiated by a low cellular uptake and a lack of interaction with major apical uptake carriers in Caco-2 cells. We propose 10 that the paracellular pathway is the predominant route for the in vitro absorption of acamprosate. The approach can be useful for 11 12 estimating Fa and absorption mechanisms of poorly permeable compounds. This can contribute to the long term challenge of building 13 PBPK models for permeability-limited compounds. 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 24 57 58 59 60 ACS Paragon Plus Environment Page 25 of 29 Molecular Pharmaceutics

1 6. ASSOCIATED CONTENT 2 3 Supporting Information 4 The Supporting Information is available free of charge via the Internet at http://pubs.acs.org and contains: 5 6 S1. Analytical method details for quantification of mannitol, acamprosate, lucifer yellow, fluorescein, atenolol. 7 S2. Effect of acamprosate on the barrier integrity of filter-grown Caco-2 monolayers 8 9 7. AUTHOR INFORMATION 10 11 Corresponding Author 12 *E-mail: [email protected]. Phone: +45 53593240 13 Present Addresses 14 15 † Present address: Leo Pharma, Industriparken 55, 2750 Ballerup, Denmark 16 †† Present address: Lundbeck Pharma A/S, Ottiliavej 9, 2500 Valby, Denmark 17 18 ORCID ID 19 Irina-Elena Antonescu: 0000-0001-5239-947X 20 Sibylle Neuhoff 0000-0001-8809-1960 21 22 Carsten Uhd Nielsen: 0000-0001-5776-6865 23 Bente Steffansen 0000-0002-5016-1246 24 25 Author Contributions 26 27 The manuscript was written through the contributions of all authors. All authors have given approval to the final version 28 of the manuscript. 29 30 Funding Sources 31 This research was funded by the University of Southern Denmark and by NordForsk through the Nordic POP consortium. 32 33 Notes 34 The authors declare no competing financial interest. 35 36 37 8. ACKNOWLEDGMENTS 38 This study was supported by University of Southern Denmark (SDU). NordForsk/Nordic POP partially funded the Caco-2 39 40 studies on ATCC cells performed at Uppsala University (UU). We would like to acknowledge Maria Læssøe Pedersen from SDU for 41 cultivating part of the DSMZ Caco-2 cells used in the study. We thank Ahmed Abdulkareem Abdulhussein Al-Ali for providing one 42 data point for figure 6. Janneke Keemink and Maria Mastej are thanked for initial seeding of ATCC Caco-2 cells at UU. Christine 43 44 Wegler is acknowledged for valuable support with LC/MS-MS method development at UU. We thank Patrik Lundquist for useful 45 discussions regarding the Caco-2 experiments at UU. Certara UK (Simcyp Division) is acknowledged for granting free access to the 46 47 Simcyp Simulator through an academic licence (subject to conditions). 48 49 50 51 52 53 54 55 56 25 57 58 59 60 ACS Paragon Plus Environment Molecular Pharmaceutics Page 26 of 29

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1 2 3 4 5 For Table of Contents use only 6 7 8 9 GRAPHICAL ABSTRACT 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 29 57 58 59 60 ACS Paragon Plus Environment