Dodecylmaltoside Modulates Bicellular Tight Junction Contacts To

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Dodecylmaltoside Modulates Bicellular Tight Junction Contacts To Promote Enhanced Permeability K. Gradauer,†,‡ M. Iida,§ A. Watari,§ M. Kataoka,‡ S. Yamashita,‡ M. Kondoh,§ and S. T. Buckley*,† †

Global Research, Novo Nordisk A/S, DK-2760 Måløv, Denmark Faculty of Pharmaceutical Sciences, Setsunan University, Osaka 573-0101, Japan § Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871, Japan

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‡

ABSTRACT: Intestinal permeation enhancers are a crucial component of many oral formulations, without which many drugs would show an insufficient absorption in the gut. The present study sought to provide a better understanding of the molecular interaction of such absorption enhancers with the intestine, by investigating the effect of the surfactant-like permeation enhancer dodecylmaltoside (DDM) on Caco-2 cells. The extent to which the action of DDM is apportioned between the para- and transcellular routes was addressed by examining the transport of relevant marker compounds ([3H]mannitol and [3H]-propranolol, respectively). In the case of [3H]-mannitol, a robust permeation enhancement was achieved with 0.5 mM DDM (∼6-fold), whereas little effect was seen on the permeation of [3H]-propranolol. Concomitantly measured TEER values revealed a rapid onset of action of DDM with a swift recovery and complete restitution (>90%) within 4 h after washout. To localize the site(s) of action of DDM at the absorptive surface of Caco-2 cells, sulfo-NHS-SS-biotin, a membraneimpermeable compound, was applied apically. In the presence of 0.5 mM DDM, translocated biotin was found to be accumulated toward bicellular contacts, whereas no biotin permeation was observed in untreated control cells. Western blot analysis of DDMtreated and untreated Caco-2 cells revealed an interaction of DDM with specific tight junction associated proteins, resulting in a reduction of claudin-3 and -4 and also occludin, as well as a depletion of claudin-2 from lipid rafts. Collectively, the results presented provide a more in depth understanding of the molecular mechanism(s) underlying the permeation-enhancing actions of DDM. KEYWORDS: maltoside, permeation enhancement, tight junctions, claudins, lipid rafts

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INTRODUCTION The gastrointestinal (GI) barrier is composed of a network of epithelial cells interconnected by tight junctions, which severely hinders the uptake of drugs and, in particular, that of large, hydrophilic ones (e.g., peptides and proteins). Thus, to effectively overcome these impediments and thereby achieve acceptable bioavailability upon oral administration, employment of an absorption enhancer is often necessary.1 The manner by which increased absorption of an orally delivered peptide is achieved is governed to a great extent by the behavior and mechanism of action of the coformulated absorption enhancer. A large proportion of the intestinal permeation enhancers reported in the literature exhibit surfactant (-like) properties.1,2 On account of this physicochemical trait, many have been reported to enhance permeation via membrane perturbation.3,4 However, especially at lower concentrations, some have been observed to increase permeability primarily through paracellular pathways.5,6 One such example that has been employed in a number of peptide formulations is maltosides a family of nonionic surfactants.7 Although several studies point toward paracellular permeation enhancement as the predominate mechanism underlying the effect of maltosides,4,8 there still remains a dearth of information pertaining to the precise mode © 2017 American Chemical Society

of action and, in particular, the manner in which they interact with tight junctions. Paracellular permeability is regulated by a junctional complex located in the apicolateral membrane of epithelial cells, termed tight junctions (TJs).9 They consist of transmembrane proteins, e.g., claudins (CL), occludin (OCLN), and junctional adhesion molecules (JAMs); and peripheral proteins, e.g., zonula occludens proteins (ZO). Even though the knockout of specific claudins leads to a loss of barrier function,10 claudins are not the only structural component important for TJ integrity. In fact, rather than solely focusing on the protein element of TJs, several groups have been investigating the role of the lipid bilayer itself on the function of TJs.11,12 Nusrat et al. were the first to demonstrate a colocalization of TJ-associated proteins with cholesterol- and glycolipid-rich, detergent-resistant membrane domains, termed lipid rafts.13 Later, it was observed that a disruption of such lipid rafts invoked the loss of barrier function.14,15 Following this path, recent studies have investigated the effect of the permeation enhancers sodium Received: Revised: Accepted: Published: 4734

April 11, 2017 August 29, 2017 October 6, 2017 October 6, 2017 DOI: 10.1021/acs.molpharmaceut.7b00297 Mol. Pharmaceutics 2017, 14, 4734−4740

Brief Article

Molecular Pharmaceutics

the flux of [3H]-mannitol and [3H]-propranolol in human intestinal cell monolayers. Hanks balanced salt solution (HBSS) containing 5.36 mM KCl, 136.89 mM NaCl, 0.34 mM Na2HPO4, 0.44 mM KH2PO4, 4.17 mM NaHCO3, 1.26 mM CaCl2, 0.49 mM MgCl2, 0.41 mM MgSO4, and 25 mM glucose was used as standard buffer solution in this study (transport medium, TM). The apical (AP) and basolateral (BL) sides were filled with 0.4 mL of TM pH 6.5 and 1 mL of TM pH 7.4, respectively. After allowing the cells to equilibrate for 20 min, the AP TM was removed and replaced by 0.4 mL of test solution. Test solutions contained 0.32 μCi of either [3H]mannitol or [3H]-propranolol in the presence or absence of 0.16 mM DDM or 0.5 mM DDM. All plates were incubated at 37 °C and 5% CO2 with gentle shaking for 2 h. The permeation of [3H]-mannitol or [3H]-propranolol was monitored by taking samples from the BL compartment after 15, 30, 45, 60, 90, and 120 min, which were subsequently analyzed in a scintillation counter (Packard TopCount) after a 1:1 dilution with scintillation fluid. Flux (J [mol/s]) was determined from steady-state appearance rates of compound in the receiver fluid. The apparent permeability coefficient, Papp [cm/s], was calculated according to eq 1,

caprate (C10) and acylcarnitines on the distribution of TJassociated proteins within the cell membrane.16,17 However, the number of studies investigating this hypothetical mode of action remains relatively limited. The present work sought to provide a deeper mechanistic understanding of the molecular events that drive and direct the permeation enhancing effects of dodecylmaltoside (DDM), and thereby clarify to what extent lipid rafts might be involved. The extent to which the action of DDM is apportioned between the para- and transcellular routes was also addressed by examining the transport of relevant marker compounds ([3H]-mannitol and [3H]-propranolol, respectively) in Caco-2 cells, together with monitoring of alterations in barrier properties via transepithelial electrical resistance (TEER) measurements and the subsequent rate of recovery. In addition, visualization of the nature of the effect of DDM on the intestinal epithelial barrier was performed using confocal laser scanning microscopy (CLSM), whereby the translocation of a hydrophilic, cell membrane impermeable compound, sulfo-NHS-SS-biotin, was investigated. Moreover, the effect of DDM on the integrity of TJ-associated proteins was investigated, as well as its impact on their distribution between cholesterol-rich lipid rafts and nonraft regions of the membrane.

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Papp = J /(AC i)

MATERIALS AND METHODS Materials. Rabbit anti-CL-1 polyclonal antibody (pAb), mouse anti-CL-2 monoclonal antibody (mAb), rabbit anti-CL-3 pAb, mouse anti-CL-4 mAb, mouse anti-OCLN mAb, and rabbit anti-ZO-1 pAb were purchased from Invitrogen (Carlsbad, CA, USA). Mouse anti-E-cadherin mAb and mouse anti-β-actin mAb were purchased from BD Biosciences (Franklin Lakes, NJ, USA) and Sigma-Aldrich (St. Louis, MO, USA), respectively. Goat anti-rabbit IgG peroxidase-conjugated antibody and goat anti-mouse IgG peroxidase-conjugated antibody were purchased from Millipore (Bedford, MA, USA). Sulfosuccinimidyl-2-[biotinamido]-ethyl-1,3-dithiopropionate (sulfo-NHS-SS-biotin) and Alexa-555 conjugated streptavidin were purchased from Thermo Fisher Scientific (Waltham, MA, USA). FITC-conjugated anti-rabbit IgG antibody, 4′,6-diamidino-2-phenylindole (DAPI), and Ndodecyl-β-D-maltopyranoside (dodecylmaltoside, DDM) were purchased from Affymetrix (Santa Clara, CA, USA). All other reagents used were of the highest purity available (95% or higher). Caco-2 Cell Culture. Caco-2 cells were cultured in DMEM supplemented with 10% (v/v) FBS, 1% (v/v) nonessential amino acids, and 0.5% (v/v) antibiotic−antimycotic mixture in a flask of adequate volume (Nippon Becton Dickinson, Tokyo, Japan) at 95% humidity and 37 °C in a 95% air/5% CO2 atmosphere. Cells (passages 47−60) were harvested using trypsin−EDTA and seeded on cell culture inserts with a polyethylene terephthalate membrane (1.13 cm2 growth area for permeation studies; 4.2 cm2 growth area for Western blotting and the biotin assay). The medium was changed every 48 h for 14−21 days, transepithelial electrical resistance (TEER) was measured with a chopstick electrode (MillicellERS, Millipore, Billerica, MA) prior to testing, and monolayers 90%) being achieved within 2 and 4 h following exposure to 0.16 and 0.5 mM DDM, respectively. Plotting the Papp of [3H]-mannitol versus the membrane conductance (Gt) revealed a strong linear relationship (r2 = 0.96) (Figure 1F). Biotin Assay. To provide further insight into specific facets of the molecular mechanism(s) underpinning the permeation enhancing effect of DDM, sulfo-NHS-SS-biotin, a membraneimpermeable compound, was applied apically to localize the site(s) of action of DDM at the absorptive surface of Caco-2 cells. Following application and subsequent washing, translocated biotin (red; Figure 2) was found to be accumulated toward bicellular contacts (green; Figure 2) as revealed by colocalization of both signals in the lateral (z) axis scan of the cell monolayer. In contrast, no biotin permeation was observed in untreated control cells. Effect of DDM on the Integrity and Distribution of Tight Junction Associated Proteins. To investigate the effect of DDM on the integrity and distribution of CL-1, -2, -3, -4, OCLN, and E-Cad, Caco-2 cells were incubated for 2 h with either 0.16 or 0.5 mM DDM or in the absence of DDM to serve as a control (Figure 3). Examining expression levels in the soluble and insoluble fractions, clear differences were observed between the different proteins. When compared with untreated cells, CL-1 and E-Cad did not show any difference in their distribution following exposure to 0.16 or 0.5 mM DDM. In contrast, the amount of CL-4 and OCLN was decreased in both the soluble and insoluble fractions, and also in the whole cell lysate, after incubation with 0.5 mM DDM. The pattern of distribution of CL-2 and -3 revealed a reduction in the insoluble fraction upon incubation with DDM. However, although the amount in the soluble fraction increased for CL-2, it remained unchanged for CL-3. Furthermore, differences were also observed for these two claudins in the whole cell lysate whereby DDM gave rise to a reduction in the amount of CL-3, while CL-2 levels remained unaffected.

slight permeation enhancement effect was observed for the transcellular marker, [3H]-propranolol. This lack of transcellular permeation enhancement as well as the transient effect on TEER upon exposure and subsequent removal of DDM strongly supports the notion of a predominantly paracellular permeation enhancement effect. Consistent with our findings, a number of other groups investigating the effect of maltosides on various cell lines report primarily paracellular effects.8,19,20 However, maltosides should not be considered as one homogeneous group as it relates to their permeation enhancing properties. Eley et al. compared in their work 13 alkylglycosides with different chain lengths and carbohydrate moieties and found that they differ significantly in their impact on tight junctions and cell integrity. DDM and octylglucoside were the only two showing activity at low concentrations, a rapid onset of their permeation enhancement, and a short cell recovery time, indicating a negligible disruption of cell membranes and a mainly paracellular permeation enhancement effect.4 To investigate the impact of DDM on the permeation of macromolecules, sulfo-NHS-SS-biotin was used as a model compound. Adding it to the apical side of Caco-2 monolayers in the presence of DDM clearly demonstrated the presence of sulfo-NHS-SS-biotin within the paracellular space. This provides visualize evidence to support the paracellular-mediated permeation enhancing effect of DDM. Moreover, it identified the bicellular junctions as its primary target. Intriguingly, this finding differs from that reported in analogous studies with other intestinal permeation enhancers. Sodium caprate (C10), for example, is a medium chain fatty acid, which has previously been marketed in a rectal suppository in Sweden and Japan21 and is currently in clinical trials as a component of oral peptide formulations.22 In contrast to DDM, C10 induced the translocation of sulfo-NHS-SS-biotin through tricellular tight junctions by removing tricellulin from tight junctions.18 Interestingly, sodium laurate (C12), which differs in structure from sodium caprate by just two carbon atoms, is reported to exhibit a distinctly different effect as it relates to drug permeation. Studies carried out in the intestinal epithelial cell model HT-29/B6 revealed an enhanced paracellular permeation of fluorescein (330 Da) while permeabilities for FITCdextran (4 kDa) and sulpho-NHS-SS-biotin (607 Da) remained unaltered.23 These results highlight the complexity of the mechanisms of intestinal permeation enhancement and emphasize the importance of differentiating between individual compounds, despite apparent structural similarities. For a deeper insight into the mechanism by which DDM increases paracellular drug absorption, both concentrations were employed in investigations to examine their impact on tight junction associated proteins and on E-cadherin, a protein associated with adherens junctions.24 To investigate the distribution of those proteins within the plasma membrane, cells were solubilized using Triton X-100 to separate lipid rafts from other parts of the cell followed by an analysis of the proteins in both fractions. The results observed indicate different effects of DDM on different TJ-associated proteins (Figure 3). While no effect was seen on CL-1 and E-Cad, different effects were seen for CL-2, -3, -4, and OLCN. This apparent selectivity was not entirely unexpected and is consistent with that reported for other molecules.25−28 Similar to DDM, C10, for example, showed a predominant effect on CL-4, -5, and OCLN.16 However, while C10 seemed to only change the protein distribution from the insoluble to the soluble fraction, DDM lowered the protein content in both

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DISCUSSION The aim of the present study was to expand the molecular understanding of the mode of action of maltosides as it relates to their action as intestinal permeation enhancers. DDM concentrations of 0.16 mM and 0.5 mM were used throughout, whereby 0.16 mM corresponded to a concentration just below the reported critical micelle concentration (CMC) of 0.18 mM. While the lower concentration provided a mild enhancing effect on [3H]-mannitol permeation, the higher concentration of 0.5 mM elicited a much more pronounced effect (Figure 1). In contrast to the paracellular marker, [3H]-mannitol, only a very 4738

DOI: 10.1021/acs.molpharmaceut.7b00297 Mol. Pharmaceutics 2017, 14, 4734−4740

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Molecular Pharmaceutics Notes

fractions, as well as in the whole cell lysate. A similar observation was made by Doi et al.,27 who found that palmitoylcarnitine reduced the protein levels of CL-1,-4, and -5 in Caco-2 monolayers, which was attributed to cholesterol leakage from the cell membrane into the apical media. In addition, it was shown that the reductions of CL-4 protein level were restored after removal of palmitoylcarnitine. Given that DDM is a surfactant, and thus likely to interact with the membrane, a similar mechanism might underlie the permeation enhancing effect of DDM. Disrupting the lipid rafts in the membrane might lead to a delocalization of specific TJassociated proteins from lipid rafts followed by internalization and degradation of TJ-associated proteins triggered by the change in phospholipid composition surrounding the protein.11 In the case of CL-2 the protein seemed to be only delocalized from the lipid rafts, but without any subsequent degradation. Another observation reported for several permeation enhancers, including maltosides, is a differential permeation enhancing effect depending on the region of the intestine.8,29 In a previous study carried out in our lab, DDM demonstrated a pronounced permeation enhancement for FD4 in colonic but not jejunal in situ instillation studies in rats.30 Knowing now that DDM interacts with CL-2, -3, -4, and OCLN, it could be hypothesized as to whether differences in the expression of tight junction associated proteins in different segments of the intestine could be the reason for this observation. Although earlier studies did not find differences in the expression pattern of TJ proteins,31 more recent studies contradict this. For example, Markov et al. found a much higher expression of “tightening” claudins, namely, CL-1, -3, -4, -5, and -8 in the colonic than in the small intestinal tissue of rats,32 and Lameris et al. investigated the expression of different claudins in human intestinal tissue, reporting an increase in CL-3, -4, -7, and -8 from proximal to distal areas.33 Summarizing the results from the present study, we were able to provide a more detailed picture of the permeation enhancing mechanism of dodecylmaltoside. DDM induced a rapid and reversible decrease in TEER, and an enhanced permeation of the paracellular marker [3H]-mannitol via opening of tight junctions. Moreover, it promoted the paracellular passage of the hydrophilic macromolecule, sulfo-NHS-SS-biotin, which was shown to permeate through bicellular tight junctions. These effects were found to be attributed to the interaction of DDM with specific tight junction associated proteins, resulting in a reduction of the tightening claudins -3 and -4 and also occludin, as well as a depletion of claudin-2 from lipid rafts. Even though tight junctions are highly complex structures and the effect of permeation enhancers on TJ-associated proteins is difficult to predict, the present study provides a further step toward a better understanding of the molecular mechanism(s) of DDM, and potentially, to some extent, that of other maltosides and related nonionic surfactants.

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The authors declare the following competing financial interest(s): S.T.B. is an employee of and holds stock in Novo Nordisk A/S.

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*Global Research, Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Måløv, Denmark. Tel: +45 3079 4609. E-mail: spby@ novonordisk.com. ORCID

S. T. Buckley: 0000-0001-9804-7242 4739

DOI: 10.1021/acs.molpharmaceut.7b00297 Mol. Pharmaceutics 2017, 14, 4734−4740

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DOI: 10.1021/acs.molpharmaceut.7b00297 Mol. Pharmaceutics 2017, 14, 4734−4740