Tight junctions (TJs) are complex biochemical structures that seal the intercellular space and prevent the free movement of solutes across epithelial cell sheets. Modulating the TJ seal is a promising option for increasing the transdermal absorption of drugs. Within TJs, the binding of the claudin (CLDN) family of tetratransmembrane proteins through cis- and trans-interactions is an integral part of seal formation. Because epidermal TJs contain CLDN-1 and CLDN-4, a binder for these CLDNs may be a useful modulator of the permeability of the epidermal barrier. Here, we investigated whether m19, which can bind to CLDN-1/-4 (also CLDN-2/-5), modulates the integrity of epidermal TJs and the permeability of cell sheets to solutes. Treatment of normal human epidermal keratinocytes (NHEKs) with the CLDN binder reduced the integrity of TJs. A CLDN-1–specific binder (a monoclonal antibody, clone 7A5) also weakened the TJ seal in NHEKs. Although m19 attenuated the TJ barrier in human intestinal epithelial cells (Caco-2), 7A5 did not. Treatment of NHEKs with 7A5 enhanced permeation of a paracellular permeation marker. These findings indicate that CLDN-1 is a potential target for modulating the permeability of the epidermis, and that our CLDN-1 binder is a promising candidate molecule for development as a dermal absorption enhancer.
The administration of pharmaceutical agents across the skin is advantageous because it is noninvasive, can easily be stopped, and avoids first-pass metabolism (Tran, 2013). Skin covers the majority of the body’s surface and acts as a barrier to the passage of xenobiotics, such as drugs and pathologic micro-organisms, into the body. Skin comprises two layers: the upper epidermis and the lower dermis, which contains blood vessels. For drugs to be successfully administered transdermally, they must cross the epidermis and reach the blood vessels.
The epidermis itself is also a multilayer structure comprising four layers: the outermost layer is called the stratum corneum, which is followed by the stratum granulosum, stratum spinosum, and stratum basale. The stratum corneum and stratum granulosum play critical roles in preventing the free movement of solutes between the outside and inside of the body (Tsukita and Furuse, 2002; Prausnitz et al., 2004; Karande et al., 2005).
The stratum corneum consists mainly of cells called corneocytes, which are dead cells filled with keratin. These corneocytes are connected by specialized cell structures called corneodesmosomes, which stabilize the structure of the stratum corneum. The stratum corneum also contains lipids, such as ceramides, cholesterol, and fatty acids, which form multilamellar bilayers and contribute to the barrier function of the skin (Prausnitz et al., 2004). Modulation of the lipid bilayers of the stratum corneum represents a potential strategy for improving dermal drug delivery, and several chemical permeation enhancers that alter the structure of the lipid bilayers have already been developed, which include surfactants (Tween and SDS), fatty acid esters (oleic acid), terpenes (limonene), and solvents (dimethylsulfoxide and ethanol) (Prausnitz et al., 2004; Karande et al., 2005). However, methods to enhance the permeability of the stratum granulosum are yet to be developed because the function of the stratum granulosum as a permeation barrier is not yet fully understood.
The free movement of solutes across epithelial cell sheets is prevented by an anastomosing meshwork of complex biochemical structures called tight junctions (TJs) that eliminate the intercellular spaces between adjacent cells (Staehelin, 1974). TJs contain a variety of scaffold (zonula occludens protein) and transmembrane (occludin, junctional adhesion molecules, claudins) proteins (Van Itallie and Anderson, 2014). The claudin (CLDN) family of tetratransmembrane proteins, which comprises more than 20 members, are key components of TJs (Furuse et al., 1998; Furuse and Tsukita, 2006), and their expression profiles and sealing functions differ depending on the tissue in which they are present. Given that trans-interactions between CLDNs in adjacent TJs seal the paracellular space (Suzuki et al., 2014, 2015), disruption of these trans-interactions by CLDN binders is a potential strategy for improving drug absorption across the epidermis.
The interspace between the stratum corneum and stratum granulosum is also sealed by TJs (Hashimoto, 1971). TJs in the stratum granulosum contain CLDN-1 and -4 (Furuse et al., 2002; Brandner, 2009). In CLDN-1–deficient mice, solutes approximately 600 Da in size are able to permeate the stratum granulosum (Furuse et al., 2002). Furthermore, in human keratinocytes, which are the predominant cell type in the epidermis, reduced CLDN-4 expression is associated with reduced TJ integrity (Yuki et al., 2007).
Clostridium perfringens enterotoxin (CPE) is a 35-kDa polypeptide that is a major cause of food poisoning in humans (McClane and Chakrabarti, 2004). CLDN-3 and -4 are CPE receptors (Sonoda et al., 1999), and the C-terminal receptor-binding polypeptide of CPE (C-CPE) was the first CLDN-3/-4 binder (Sonoda et al., 1999). C-CPE enhances the mucosal absorption of drugs by interacting with CLDN-4 (Kondoh et al., 2005).
We previously developed several CLDN-binding molecules. One, a C-CPE mutant polypeptide (m19), bound to several CLDNs, including CLDN-1, -2, -4, and -5, and reduced TJ integrity in the intestinal epithelium more than did C-CPE (Takahashi et al., 2012). We also created four anti–CLDN-1 monoclonal antibodies (mAbs; clones 2C1, 3A2, 5F2, 7A5) and have shown that they all bound CLDN-1, but not CLDN-2–9, and prevented CLDN-1–mediated hepatitis C virus infection in a human hepatocyte cell line (Fukasawa et al., 2015). Furthermore, we developed the anti–CLDN-4 mAb (clone 3B11), which bound CLDN-4 but not CLDN-1–3 or CLDN-5–9 (Kondoh et al., 2014). In the present study, we investigated whether these CLDN binders can be used to modulate the permeability of the epidermal barrier in a human keratinocyte model.
Materials and Methods
Normal human epidermal keratinocytes (NHEKs; Takara Bio Inc., Shiga, Japan) were grown under a 5% CO2 atmosphere at 37°C in keratinocyte growth medium 2 containing 0.06 mM Ca2+ and the following additives: 0.4% (v/v) bovine pituitary extract, 0.125 ng/ml epidermal growth factor, 5 µg/ml insulin, 0.33 µg/ml hydrocortisone, 0.39 µg/ml of epinephrine, and 10 µg/ml of transferrin. Human intestinal epithelial cells (Caco-2; American Type Culture Collection, Manassas, VA) were grown under a 5% CO2 atmosphere at 37°C in modified Eagle’s medium containing 10% fetal bovine serum. Human embryonic kidney 293T cells (American Type Culture Collection) and HT1080 cells stably transfected with mock treatment, human CLDN-1, CLDN-2, CLDN-4, or CLDN-5 (mock/HT1080 cells, CLDN-1/HT1080 cells, CLDN-2/HT1080 cells, CLDN-4/HT1080 cells, or CLDN-5/HT1080 cells) were grown under a 5% CO2 atmosphere at 37°C in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (Li et al., 2014).
We previously created m19, which can bind to CLDN-1/-2/-4/-5, by using the CLDN-3/-4–binding polypeptide C-CPE as a template (Takahashi et al., 2012). m19 tagged with histidine at the N terminus was prepared as described. In brief, pET16b plasmids encoding m19 were transduced into Escherichia coli (BL21), and the production of the recombinant proteins was induced by the addition of isopropyl-d-thiogalactopyranoside. Harvested cells were lysed in buffer A [10 mM Tris-HCl (pH 8.0), 400 mM NaCl, 5 mM MgCl2, 0.1 mM phenylmethanesulfonyl fluoride, 1 mM 2-mercaptoethanol, and 10% glycerol]. Lysates were applied to a HiTrap Chelating HP column (GE Healthcare, Little Chalfont, UK), and the recombinant proteins were eluted with buffer A containing imidazole. The solvent was exchanged with phosphate-buffered saline (PBS) by using a PD-10 column (GE Healthcare), and the purified protein was stored at −20°C until use. Purification of the recombinant protein was confirmed by means of SDS-PAGE followed by staining with Coomassie Brilliant Blue. Protein was quantified with a BCA protein assay kit (Pierce Biotechnology, Waltham, MA) using bovine serum albumin as a standard.
We previously developed mouse anti-human CLDN-1 mAbs (clones 2C1, 3A2, and 7A5) and one rat anti-human CLDN-4 mAb (clone 3B11) (Kondoh et al., 2014; Fukasawa et al., 2015). Antibodies were purified from the ascitic fluid produced by BALB/c mice inoculated intraperitoneally with hybridoma cells by using immobilized protein G columns. Eluted antibodies were dialyzed in PBS, and the protein concentration was determined by measuring absorbance at 280 nm. The purity of the mAbs was confirmed by means of SDS-PAGE followed by staining with Coomassie Brilliant Blue.
Mock/HT1080 cells, CLDN/HT1080 cells, NHEKs, or Caco-2 cells were collected with a cell scraper and lysed in radioimmunoprecipitation assay buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% SDS, 1% NP40, 0.5% sodium deoxycholate). The resultant supernatants were recovered by centrifugation. Lysates (10 µg/sample) were subjected to SDS-PAGE on a 15% polyacrylamide gel followed by blotting of the separated proteins onto a polyvinylidene difluoride membrane. Membranes were incubated with Tris-buffered saline containing 0.1% Tween 20 (T-TBS) containing 2% bovine serum albumin for 2 hours and then incubated with the primary antibodies for 1 hour [anti–CLDN-1, 0.25 µg/ml (Invitrogen, Waltham, MA); anti–CLDN-2, 0.5 µg/ml (Invitrogen); anti–CLDN-3, 0.25 µg/ml (Invitrogen); anti–CLDN-4, 0.5 µg/ml (Invitrogen); anti–CLDN-5, 0.4 µg/ml (Sigma-Aldrich, St. Louis, MO); and anti–β-actin, 0.4 µg/ml (Sigma-Aldrich)]. After washing with T-TBS, the membrane was incubated with horseradish peroxidase–conjugated secondary antibodies [goat anti-rabbit IgG, 0.5 µg/ml (EMD Millipore, Billerica, MA) or goat anti-mouse IgG, 0.5 µg/ml (EMD Millipore)] for 1 hour. Immunoreactive bands were detected with a chemiluminescence reagent (Nacalai Tesque, Kyoto, Japan) under an LAS 4010 image analyzer (GE Healthcare).
Flow Cytometric Analysis.
CLDN/HT1080 cells, NHEKs, or Caco-2 cells (5 × 105 cells) were incubated with PBS containing 1% bovine serum albumin, m19 (10 µg/ml), or one of the mAbs (5 µg/ml) for 1 hour at 4°C. Cells treated with m19 were then treated with anti-histidine–tagged mAb (5 µg/ml; Thermo Fisher Scientific, Waltham, MA). The cells were then incubated with fluorescein isothiocyanate–conjugated antibody (10 µg/ml; Jackson ImmunoResearch Laboratories, West Grove, PA). Binder-bound cells were detected with a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ), and data were analyzed with the CellQuest software (Becton Dickinson).
Measurement of Transepithelial Electrical Resistance.
Transepithelial electrical resistance (TEER) values gradually increased in NHEKs after the cells reached confluence. Attenuation of the increase in TEER over time was assessed as an index of the influence of the epidermal absorption enhancers on a layer of NHEKs seeded into culture plate inserts (Yuki et al., 2007; Kirschner et al., 2013). NHEKs (3.0 × 104 cells) were seeded into culture plate inserts (Transwell inserts, growth area = 0.33 cm2; Corning, NY). When the cultures became confluent, the medium was changed to growth medium containing 1.8 mM Ca2+, and the cells were cultured for an additional 2 days (Gonzalez-Mariscal et al., 1990; Nigam et al., 1992; Yuki et al., 2007). Cells were then basolaterally treated with one of the mAbs (50 µg/ml) or m19 (20 µg/ml). TEER was measured during treatment using a Millicell-ERS epithelial voltohmmeter (EMD Millipore). The background TEER value of a well containing only medium was subtracted from the recorded TEER; these values were then normalized to the surface area of the well.
Caco-2 cells were recovered by gentle pipetting with trypsin until a single-cell suspension was obtained, and the cells were seeded into 24-well Transwell inserts (6.0 × 104 cells/0.33-cm2 well). The Caco-2 monolayers were then cultured for 10 days before being basolaterally treated with one of the mAbs (50 μg/ml) or m19 (20 μg/ml). TEER values were measured as described earlier.
Paracellular Flux of Dextran.
Fluorescein isothiocyanate–conjugated dextran with an average molecular mass of 4 kDa (FD-4) is a tracer molecule that can be used to evaluate paracellular permeability (Balda et al., 1996; Yuki et al., 2007). We evaluated the paracellular flux of FD-4 in NHEKs as described previously (Balda et al., 1996). In brief, when NHEKs reached confluence, they were transferred to growth medium containing 1.8 mM Ca2+ and cultured for an additional 2 days. The cells were then treated with 7A5 (50 µg/ml) or m19 (20 µg/ml) for 48 hours. FD-4 was dissolved in P buffer [10 mM HEPES (pH 7.4), 1 mM sodium pyruvate, 10 mM glucose, 3 mM CaCl2, and 145 mM NaCl] without dialysis at a concentration of 100 µg/ml. The media in the apical and basal compartments of Transwell plates on which the keratinocyte sheets were grown were replaced with 100 µl of P buffer containing FD-4 and 600 µl of P buffer, respectively. The keratinocyte sheet was then incubated at 37°C for an additional 2 hours. The flux of FD-4 from the apical side to the basal side of the cell sheets was measured using a fluorometer (SpectraMax Gemini; Molecular Devices, Sunnyvale, CA).
A plasmid vector encoding an alanine-substituted CLDN-1 mutant was prepared using substituted site-specific primers and pcDNA3.1-Hyg-(+) encoding FLAG–CLDN-1 as a template by means of polymerase chain reaction using a KOD-Plus Mutagenesis Kit (Toyobo, Osaka, Japan).
293T cells (2.5 × 106 cells) were seeded in a 100-mm2 plate. One day after seeding, 5.0 µg of plasmid vector encoding CLDN-1 or one of the CLDN-1 mutants was transduced with 293T cells using the transfection reagent X-treme GENE HP in accordance with the manufacturer’s protocol (Roche, Mannheim, Germany). Two days after transfection, the cells were recovered and used for analysis.
Synthesis of Peptides.
SPOT peptides were synthesized with a MultiPep automatic peptide synthesizer (Intavis AG, Köln, Germany) on amino-polyethylene glycol–functionalized cellulose membranes. To cover the whole length of the CLDN-1 polypeptide chain, we designed 67 peptides, each 15 amino acids in length starting at the N terminus and shifting by 3 amino acids to the C terminus (Supplemental Table 1).
CLDN-1 peptides were synthesized on TentaGel amide resin (Intavis) following the standard Fmoc solid-phase peptide synthesis method using a MultiPep automatic peptide synthesizer. The synthetic peptides were purified by means of reverse-phase high-performance liquid chromatography by using a YMC-PAC ODS-A column (YMC Co. Ltd., Tokyo, Japan). Peptides of approximately more than 90% purity were used for TEER assay.
Peptide SPOT Array Analysis.
The SPOT array membrane was incubated with 7A5 (0.5 µg/ml) in T-TBS containing 2.0% skim milk for 2 hours at room temperature. After washing with T-TBS, the membrane was incubated with horseradish peroxidase–conjugated anti-mouse IgG antibodies (Jackson ImmunoResearch Laboratories) for 1 hour at room temperature. The 7A5-bound spots were detected with ECL Prime Western Blotting Detection Reagent (GE Healthcare).
Competition Analysis of Synthetic Peptides Containing the Second Extracellular Loop Domain of CLDN-1.
Cells (5 × 105 cells) were incubated with clone 7A5 (0.5 μg/ml) or control IgG (0.5 μg/ml) for 1 hour at 4°C. The cells were incubated with fluorescein isothiocyanate–conjugated anti-mouse IgG antibody (Rockland Immunochemicals, Gilbertsville, PA), and the antibody-bound cells were detected and analyzed with a flow cytometer (FACSCalibur). In a competition assay, 7A5 (0.5 μg/ml) was incubated with competitors (peptides shown in Supplemental Table 2, 1.0 mg/ml) for 2 hours at room temperature before treating the cells as described earlier.
Statistical difference was assessed using a two-way repeated measures analysis of variance or Student’s t test. Statistical significance was set at P < 0.05.
Effects of m19 on TJ Integrity in NHEKs.
We evaluated the effects of five CLDN binders on the permeability of the epidermal barrier by using NHEKs, which are a widely used in vitro model of the human epidermis (Karande et al., 2005; Yuki et al., 2007; Rachakonda et al., 2008; Wato et al., 2012). Calcium depletion has been shown to reduce TJ integrity in NHEKs, which is accompanied by decreased expression of CLDN-1 and -4 (Yuki et al., 2007). Knockdown of CLDN-1 or -4 expression has also been shown to decrease TJ integrity in NHEKs (Kirschner et al., 2013). Therefore, here, we targeted CLDN-1 and -4.
Previously, we created the C-CPE mutant polypeptide m19 and showed that it bound to CLDN-1 and -4 (also CLDN-2 and -5) and that it decreased TJ integrity in a Caco-2 cell monolayer (Takahashi et al., 2012). In the present study, immunoblot analysis showed that both NHEKs and Caco-2 cells expressed CLDN-1, -2, and -4 (the expression of CLDN-2 and -4 in NHEKs was faint but detected) (Fig. 1A). m19 bound to CLDN-1/HT1080 cells, CLDN-2/HT1080 cells, and CLDN-4/HT1080 cells (Fig. 1B). Flow cytometric analysis confirmed that m19 bound to both NHEKs and Caco-2 cells (Fig. 1C). Treatment of NHEKs with m19 (20 μg/ml) attenuated the increase in TEER, and the values reduced to 80.4% and 57.9% of that in vehicle-treated cells at 12 and 36 hours after treatment, respectively (Fig. 2A), indicating that m19 decreased TJ integrity. Treatment with m19 (20 μg/ml) also decreased TJ integrity in Caco-2 cells (Fig. 2B; TEER values reduced to 15.7% and 9.1% of that in vehicle-treated cells at 12 and 36 hours after treatment, respectively).
Effects of Anti–CLDN-1 or -4 mAbs on TJ Integrity.
Because solutes (∼600 Da) can permeate the epidermis of CLDN-1–deficient mice but not CLDN-4–deficient mice (Furuse et al., 2002; Fujita et al., 2012), we next investigated whether treatment with CLDN-1 binder was enough to loosen the integrity of the epidermal barrier. We recently developed mouse anti–CLDN-1 mAbs (Fukasawa et al., 2015). Clone 7A5 is an mAb specific to human CLDN-1 (Fig. 3A). 7A5 was associated with both NHEKs and Caco-2 cells (Fig. 3B). Treatment of NHEKs with 7A5 attenuated the increase in TEER (Fig. 3C). Although m19 disrupted TJ integrity in Caco-2 cells, 7A5 did not (Fig. 3D). Furthermore, 3B11, an anti–CLDN-4 mAb, also showed TJ integrity–modulating activity similar to that of 7A5 in NHEKs (Fig. 4). However, 3B11 also reduced TJ integrity in Caco-2 cells (Supplemental Fig. 1). These results show that CLDN-1 may be a more suitable target for improving the epidermal absorption of drugs than CLDN-4 in terms of tissue specificity of permeation-enhancing activity.
Next, we investigated whether 7A5 enhanced the epidermal absorption of drugs by using the paracellular tracer FD-4 as a model drug (Balda et al., 1996; Yuki et al., 2007). Treatment with 7A5 increased the permeation of FD-4 in NHEKs (Fig. 5; vehicle, 1.07 × 10−5 cm/min; 7A5, 2.11 × 10−5 cm/min), suggesting that CLDN-1 is a potent target for development of epidermal absorption enhancer.
7A5 recognized the primary structure of CLDN-1 in addition to the tertiary one (e.g., amino acid sequence) (Fukasawa et al., 2015). Peptide SPOT array analysis of CLDN-1 showed that the peptide C2 (amino acids 148–162), which mostly overlapped with the second extracellular loop domain (amino acids 146–160), contained an epitope of 7A5 (Fig. 6A). Alanine scanning analysis of the second extracellular loop domain revealed that aspartic acid and methionine at amino acid positions 150 and 152, respectively, were involved in the binding of 7A5 to CLDN-1 (Fig. 6B). A synthetic peptide containing the second extracellular loop domain (amino acids 148–162) prevented the interaction of 7A5 with CLDN-1–expressing HT1080 cells (Fig. 7). However, the peptide did not decrease TJ integrity in NHEKs (Fig. 8).
Dead cells containing lipids and proteins in the stratum corneum and the TJs between epithelial cells in the stratum granulosum are two physiologic barriers to the dermal absorption of drugs (Tsukita and Furuse, 2002). In the present study, by using two different types of CLDN-1–binding molecules, a polypeptide (m19) and a mAb (clone 7A5), we showed that CLDN-1 is a potential target for modulation of the epithelial TJ barrier (Takahashi et al., 2012; Fukasawa et al., 2015).
CLDNs laterally associate with each other via cis-interactions to form CLDN strands, which then associate via trans-interactions to eliminate the intercellular space (Furuse et al., 1999; Angelow et al., 2008). The structures of the CLDN extracellular loop domains differ among the CLDN members (Suzuki et al., 2014, 2015). Trans-interactions at the second extracellular loop are critical for intercellular sealing (Krause et al., 2008; Piontek et al., 2008). m19 might interact with the second extracellular loop domain (Fujita et al., 2000; Takahashi et al., 2012). Previously, we showed that 7A5, which attenuated the epidermal TJ integrity and enhanced the epidermal permeation of FD-4 in this study (Figs. 3–5), recognized the second extracellular loop domain of CLDN-1 (Fukasawa et al., 2015). 2C1 and 3A2, which also bind to the second extracellular loop domain of CLDN-1 as well as 7A5 (Fukasawa et al., 2015; Supplemental Fig. 2A), attenuated the increase in TEER in NHEKs (Supplemental Fig. 2B). Taken together, these results suggest that CLDN-1 binders may modulate the permeability of the epidermal barrier by targeting the second extracellular loop domain.
Mrsny et al. (2008) reported that synthetic peptides corresponding to the first extracellular loop domain (amino acids 53–80) of CLDN-1, but not the second extracellular loop domain (amino acids 146–160), reduced TJ integrity in a human intestinal cell line (T84). Treatment of the Caco-2 monolayer with a first loop peptide has also been shown to decrease TJ integrity (Zwanziger et al., 2012); however, in the present study, 7A5 treatment did not decrease TJ integrity in Caco-2 cells (Fig. 3), which may be due to the different molecular sizes of the peptides and mAbs. In the present study, the treatment of Caco-2 cells with 3B11, an anti–CLDN-4 mAb, decreased TJ integrity (Supplemental Fig. 1), and the expression profiles and TJ barrier functions of CLDNs have been shown to differ among tissues (Furuse and Tsukita, 2006). Furthermore, the mosaic expression patterns of the CLDNs have led to a variety of cis- and trans-interaction profiles of the CLDN family being elucidated. Therefore, a second possible explanation for these different effects of anti-CLDN molecules is that the patterns of CLDN expression in the TJs are different between intestinal cells and epidermal cells. The amounts of the CLDNs present and affinity of anti-CLDN molecules to CLDN-1 may also differ between these cells. A detailed analysis, therefore, is needed to better understand the mechanisms underlying the modulation of TJ integrity by CLDN-1 binders. We also should investigate TJ-modulating activity of a peptide mimetic containing the first loop domain in an epidermal model.
Antibody-type CLDN-1 binders are difficult to use as epidermal absorption enhancers because of their high cost of production. Therefore, small molecules, such as peptides, are more appropriate for long-term clinical use. A series of epitope mapping of 7A5 indicated that the second extracellular loop domain may be an epitope of 7A5 (Fig. 6). The peptide did not decrease TJ integrity in NHEKs (Fig. 8). The peptide may be unable to adopt the three-dimensional structure of the second extracellular loop domain of CLDN-1 because it only contains one loop domain and one β-sheet domain. The peptide may be too small to form the structure of the second loop domain. Indeed, whereas 7A5 prevented in vitro infection of hepatitis C virus in a human hepatocyte cell line, the peptide did not (data not shown). The structure of CLDN-15 indicates that the first β-sheet of the first loop and fifth β-sheet of the second loop form an antiparallel β-sheet structure (Suzuki et al., 2014). A peptide mimetic containing this antiparallel structure may therefore represent a small molecule that binds to CLDN-1.
In conclusion, CLDN-1 is a potent target for enhancing epidermal absorption, and we show here that a CLDN-1 binder attenuated the epidermal barrier in an in vitro model of human skin. A combination of modulators for the stratum corneum, such as fatty acids, esters, and alcohols, and modulators for the stratum granulosum, such as CLDN-1 binders, may provide a breakthrough in improving the dermal absorption of drugs.
The authors thank all the members of their laboratory for technical support and useful comments.
Participated in research design: Nakajima, Nagase, S. Takeda, Fukasawa, Kondoh.
Conducted experiments: Nakajima, Nagase, Iida.
Contributed new reagents or analytic tools: Yamashita, Shirasago, Watari, H. Takeda, Sawasaki, Fukasawa.
Performed data analysis: Nakajima, Nagase, S. Takeda, Fukasawa, Yagi, Kondoh.
Wrote or contributed to the writing of the manuscript: Nakajima, Nagase, S. Takeda, Fukasawa, Kondoh.
- Received April 18, 2015.
- Accepted July 1, 2015.
M.N. and S.N. contributed equally to this work.
This work was supported in part by a Health and Labour Sciences Research Grant from the Ministry of Health, Labour, and Welfare of Japan (to M.K. and M.F.); a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan [Grant 24390042]; the Adaptable and Seamless Technology Transfer Program through Target-driven R&D, Japan Science and Technology Agency [Grants AS242Z01694Q and AS251Z00905Q]; the Takeda Science Foundation; and the Platform for Drug Discovery, Informatics, and Structural Life Science of the Ministry of Education, Culture, Sports, Science and Technology, Japan (T.S. and M.K.).
- C-terminal receptor-binding domain of Clostridium perfringens enterotoxin
- Clostridium perfringens enterotoxin
- fluorescein isothiocyanate–conjugated dextran with an average molecular mass of 4 kDa
- monoclonal antibody
- normal human epidermal keratinocyte
- phosphate-buffered saline
- transepithelial electric resistance
- tight junction
- Tris-buffered saline containing 0.1% Tween 20
- Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics