Pharmaceutical or cosmetic composition

Abstract

To provide compositions having a high ability to promote claudin-1 production, and to provide compositions capable of enhancing a tight junction barrier.SOLUTION: The present invention relates to a claudin-1 production promoter containing polysulfated chondroitin sulfate and / or a salt thereof as an active ingredient, and to a tight junction barrier enhancer containing polysulfated chondroitin sulfate and / or a salt thereof as an active ingredient.SELECTED DRAWING: Figure 2

Description

Technical Field

[0001] The present invention relates to a promoter for Claudin-1 production. In addition, the present invention relates to a tight junction barrier enhancer.

Background Art

[0002] The skin is composed of the epidermis, dermis, and underlying subcutaneous tissue from the outside. The epidermis, which is the outermost layer of the skin, plays the most important role in maintaining the skin's barrier function. In addition, the epidermis of the skin is composed of the stratum corneum (horny layer), stratum granulosum, stratum spinosum, and stratum basale. The skin's barrier function is mainly borne by two factors: the stratum corneum barrier derived from the stratum corneum and the tight junction (TJ) barrier derived from the stratum granulosum.

[0003] Among these, in the stratum corneum barrier, natural moisturizing factors (NMF) and intercellular lipids in the stratum corneum are thought to play important roles in the formation of the stratum corneum barrier function and water retention. On the other hand, in the tight junction barrier, tight junctions, which are intercellular adhesion structures present in the stratum granulosum, are thought to play a role in preventing the leakage of body moisture, ions, etc. and preventing the invasion of foreign substances such as pathogens.

[0004] Traditionally, the skin's barrier function was thought to be solely the responsibility of the stratum corneum. However, it is now known that tight junctions in the granular layer also play an important role in the skin's barrier function. Tight junctions are known to be composed of cell membrane proteins such as the claudin family and occludin, as well as the intracellular lining protein ZO-1. Among these constituent proteins, claudin-1 is thought to play a particularly important role in the tight junction barrier function, as mice with claudin-1 knockout die within one day of birth due to water evaporation. Furthermore, when biotin is administered to the dermis of mice with claudin-1 knockout, the biotin passes through the granular layer and leaks into the stratum corneum, suggesting that tight junctions also act as a barrier to substance permeation in the epidermis (Non-Patent Literature 1).

[0005] Furthermore, since claudin-1 is known to be distributed not only in the skin but also in tissues such as the cornea of ​​the eye, testes, brain, kidneys, lungs, and liver (Non-Patent Literature 2), promoting the production of claudin-1 can not only improve the barrier function of the skin but also be expected to have therapeutic or preventive effects on diseases caused by a decrease in claudin-1 in the above-mentioned tissues. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] The Journal of Cell Biology 156:1099-1111 (2002) [Non-Patent Document 2] Fukuoka Medical Journal 99(2):25-31, 2008 [Overview of the project] [Problems that the invention aims to solve]

[0007] Therefore, the object of the present invention is to provide a composition having excellent claudin-1 production promoting ability. Furthermore, the object of the present invention is to provide a composition having excellent tight junction barrier enhancing effect. [Means for solving the problem]

[0008] In order to solve the above problems, the present inventors investigated substances that can promote the production of claudin-1 and found that polysulfated chondroitin sulfate or its salt has excellent claudin-1 production ability and excellent tight junction barrier enhancing effect, thus completing the present invention.

[0009] In other words, the present invention relates to a claudin-1 production promoter containing polysulfated chondroitin sulfate and / or a salt thereof as an active ingredient.

[0010] Furthermore, the present invention relates to a tight junction barrier enhancer containing polysulfated chondroitin sulfate and / or a salt thereof as an active ingredient. [Brief explanation of the drawing]

[0011] [Figure 1] This graph shows the effects of heparinoid (MPS), chondroitin sulfate (ChS), and hyaluronic acid (HA) on the amount of claudin-1 mRNA in human epidermal keratinocytes. In Figure 1, the amount of claudin-1 mRNA is expressed as a relative amount, with the mRNA amount in the group without the evaluation substance added set to 1. [Figure 2] This is a Western blot image showing the effects of heparinoid (MPS), chondroitin sulfate (ChS), and hyaluronic acid (HA) on claudin-1 protein expression levels in human epidermal keratinocytes. [Figure 3]This graph shows the effects of heparinoid (MPS), chondroitin sulfate (ChS), and hyaluronic acid (HA) on transepithelial electrical resistance (TER) in human epidermal keratinocytes. [Figure 4] This graph shows the effects of heparinoid (MPS), chondroitin sulfate (ChS), and hyaluronic acid (HA) on transepithelial electrical resistance (TER) in a histamine-induced tight junction barrier reduction model. [Figure 5] These are Western blot images and graphs showing the effect of heparin-like substances (MPS) on claudin-1 protein expression in a tight junction barrier reduction model (a three-dimensional cultured human skin model with added corticosteroids). [Figure 6] This image shows the effect of heparinoid (MPS) on biotin leakage in a tight junction barrier reduction model (a three-dimensional cultured human skin model supplemented with corticosteroids). [Modes for carrying out the invention]

[0012] The claudin-1 production promoter / tight junction barrier enhancer of the present invention contains polysulfated chondroitin sulfate and / or a salt thereof as an active ingredient. Polysulfated chondroitin sulfate is a polymer that uses a disaccharide composed of N-acetyl-D-galactosamine and D-glucuronic acid as a repeating unit, and contains approximately 2 to 4, preferably 2 to 3, sulfate ester residues per unit (disaccharide).

[0013] Polysulfated chondroitin sulfate can be easily produced by a known method of reacting chondroitin components such as chondroitin and chondroitin sulfate (A, C, D, E) with a sulfating agent such as chlorosulfuric acid, concentrated sulfuric acid, sulfur trioxide-pyridine complex. Chondroitin sulfate A has a sulfate ester residue at the 4-position of N-acetylgalactosamine, chondroitin sulfate C has a sulfate ester residue at the 6-position of N-acetylgalactosamine, chondroitin sulfate D has sulfate ester residues at the 6-position of N-acetylgalactosamine and the 2-position or 3-position of glucuronic acid, and chondroitin sulfate E has sulfate ester residues at both the 4-position and 6-position of N-acetylgalactosamine.

[0014] Examples of preferred polysulfated chondroitin sulfate include heparin-like substances listed in the specifications of pharmaceutical ingredients outside the Japanese Pharmacopoeia. Specifically, it is polysulfated chondroitin sulfate showing the following values as physicochemical properties. a) Sulfate group content: 25.8 to 37.3% by weight b) Intrinsic viscosity: 0.09 to 0.18

[0015] Polysulfated chondroitin sulfate may be used in the form of a free acid derived from a sulfate residue, but usually a base salt is used.

[0016] Examples of the base salt include alkali metal salts such as sodium and potassium, alkaline earth metal salts such as calcium, and magnesium salts. [[ID=s19]]

[0017] The weight average molecular weight of the polysulfated chondroitin sulfate or its salt used in the present invention is not particularly limited, but is usually about 8,000 to 10,000,000, preferably about 8,000 to 1,000,000, more preferably about 10,000 to 100,000, and particularly preferably about 10,000 to 50,000.

[0018] The content of the active ingredient, polysulfated chondroitin sulfate and / or its salt, is preferably 0.01 to 10% by weight, more preferably 0.05 to 5% by weight, and particularly preferably 0.1 to 1.0% by weight, based on the total weight of the claudin-1 production promoter or tight junction barrier enhancer (however, if the claudin-1 production promoter / tight junction barrier enhancer consists of a stock solution and a propellant, the total weight of the stock solution is used as the basis).

[0019] Polysulfated chondroitin sulfate or its salts have a high production-promoting effect on claudin-1. Furthermore, polysulfated chondroitin sulfate or its salts increased TER (which decreases when barrier function is impaired), an indicator used to evaluate the barrier function of tight junctions, and improved the leakage of substances from the granular layer to the stratum corneum. This indicates that polysulfated chondroitin sulfate or its salts have an effect of enhancing the tight junction barrier.

[0020] Therefore, the present invention can be used to prevent and / or treat conditions and / or diseases caused by a decrease in the skin's moisturizing ability. Furthermore, the present invention can be used to treat or prevent diseases caused by a decrease in claudin-1 production in tissues other than the skin where claudin-1 is distributed (such as the cornea, testes, brain, kidneys, lungs, and liver).

[0021] Furthermore, it has been reported that histamine reduces claudin-1 production and inhibits the skin's barrier function (see, for example, Allergy 68, 37-47 (2013)), and experiments conducted by the present inventors also confirmed a tendency for histamine to reduce TER. In contrast, it was observed that when histamine was used in combination with polysulfated chondroitin sulfate or its salt, the TER reduced by histamine increased, meaning that the barrier function by tight junctions was restored. Therefore, the present invention can also be used to restore claudin-1 production or the tight junction barrier that has been reduced by histamine.

[0022] The claudin-1 production promoter / tight junction barrier enhancer of the present invention can be used as a pharmaceutical composition or a cosmetic composition. Examples of pharmaceutical compositions include those that fall under the categories of prescription drugs, over-the-counter drugs, or quasi-drugs as defined in the "Act on Securing Quality, Efficacy and Safety of Pharmaceuticals, Medical Devices, etc." Examples of cosmetic compositions include those that fall under the category of cosmetics and quasi-drugs, which are medicated cosmetics, as defined in the "Act on Securing Quality, Efficacy and Safety of Pharmaceuticals, Medical Devices, etc."

[0023] The claudin-1 production promoter / tight junction barrier enhancer of the present invention can be used by systemic or topical administration. For example, for systemic administration, it can be used as an oral, intravenous, or injectable preparation, or it can be used as a topical preparation (e.g., inhalant, eye drop, nasal spray, suppository, topical skin preparation, etc.) for application / administration to the oral cavity, bronchi, lungs, nose, rectum, skin, eyes, or ears.

[0024] The claudin-1 production promoter / tight junction barrier enhancer of the present invention is particularly preferably for skin application, and the dosage form for this purpose is not particularly limited as long as it is in a form that can be applied to the skin, and examples include ointments, creams, gels, lotions, sprays (including foams), patches, etc., as described in the Japanese Pharmacopoeia, and can be used as a pharmaceutical composition or cosmetic composition together with pharmaceutically acceptable additives.

[0025] Examples of the aforementioned additives are not limited to, but include bases, surfactants, preservatives, pH adjusters, and thickeners.

[0026] Examples of bases, though not particularly limited, include higher hydrocarbons such as white petrolatum, squalane, and light liquid paraffin; waxes such as bleached beeswax, lanolin, and ceresin wax; oils and fats such as olive oil, jojoba oil, triacetin, and hydrogenated castor oil; higher alcohols such as lanolin alcohol, cetanol, myristyl alcohol, stearyl alcohol, and cetostearyl alcohol; fatty acids such as stearic acid; esters such as isopropyl myristate, stearyl myristate, and medium-chain triglyceride; polyhydric alcohols such as glycerin and 1,3-butylene glycol; lower monohydric alcohols such as ethanol and isopropanol; water (purified water); macrogol (polyethylene glycol); and silicone oil. One type or multiple types may be used.

[0027] Examples of surfactants (including those used as foaming agents in addition to emulsifiers) are not particularly limited, but include cationic surfactants, anionic surfactants, nonionic surfactants, and amphoteric surfactants, and one type or multiple types may be used. Examples of cationic surfactants are not particularly limited, but include cetyltrimethylammonium chloride, lauryldimethylbenzylammonium chloride, tetrabutylammonium chloride, dioctadecyldimethylammonium chloride, etc. Examples of anionic surfactants are not particularly limited, but include sodium alkylbenzene sulfonate, sodium dodecyl sulfate, coconut alcohol ethoxysulfate, sodium α-olefin sulfonate, emulsified cetostearyl alcohol (a mixture of cetostearyl alcohol and sodium cetostearyl sulfate), etc. Nonionic surfactants are not particularly limited, but examples include glycerin fatty acid esters such as glyceryl monostearate, polyoxyethylene alkyl ethers such as polyoxyethylene cetyl ether and polyoxyethylene behenyl ether, polyoxyethylene sorbitan fatty acid esters such as polyoxyethylene sorbitan monostearate and polyoxyethylene sorbitan tristearate. Amphoteric surfactants are not particularly limited, but examples include N-alkyl-N,N-dimethylammonium betaine and imidazoline-type amphoteric surfactants.

[0028] Examples of preservatives, though not limited to them, include dibutylhydroxytoluene, sodium edetate hydrate, and parahydroxybenzoic acid esters, and one or more types may be used.

[0029] Examples of pH adjusting agents, though not limited to them, include diisopropanolamine, triisopropanolamine, triethanolamine, potassium hydroxide, and sodium hydroxide, and one or more types may be used.

[0030] Examples of thickening agents, though not particularly limited, include sodium alginate, gelatin, carboxyvinyl polymer, and carboxymethylcellulose, and one or more types may be used.

[0031] Furthermore, if the formulation is a foam, a propellant such as liquefied petroleum gas (LPG) or compressed gas can be used along with the stock solution containing polysulfated chondroitin sulfate and / or its salt.

[0032] The claudin-1 production promoter / tight junction barrier enhancer of the present invention may also contain additives commonly used as additives in pharmaceutical or cosmetic compositions (e.g., buffers, fragrances, colorants, UV absorbers, etc.).

[0033] The dosage and frequency of the claudin-1 production promoter / tight junction barrier enhancer according to the present invention may be appropriately adjusted according to the target disease and the severity of its symptoms, the concentration of polysulfated chondroitin sulfate and / or its salt, age, body weight, etc. For example, when used topically on the skin, polysulfated chondroitin sulfate may be administered to 1 cm of skin. 2 Apply 0.03 μg to 30 mg, preferably 0.3 μg to 3 mg, once or several times a day.

[0034] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. [Examples]

[0035] As polysulfated chondroitin sulfate or its salt (abbreviated as MPS), we used a heparin-like substance listed in the Japanese Pharmacopoeia's Standards for Non-Official Drug Ingredients (organosulfate group content: 25.8-37.3% w / w, glucuronic acid content: 19.0-24.0% w / w, manufactured by Maruho Co., Ltd.). For comparison, hyaluronic acid (abbreviated as HA, Tokyo Chemical Industry Co., Ltd.) and chondroitin sulfate (abbreviated as ChS, organic sulfate group content: 15-17% w / w, Maruho Co., Ltd.) were used as control substances.

[0036] [Example 1] Effect of heparin-like substance (MPS) on claudin-1 mRNA expression in human epidermal keratinocytes Adult human epidermal keratinocytes (purchased from Thermo Fisher Scientific) were seeded in a cell suspension in a 24-well microplate and cultured at 37°C, 5% CO2, and 95% air. Six hours later, 500 μL / well of medium containing MPS or a control substance (0.1, 1, 10, and 100 μg / mL) or a medium without MPS (HuMedia-KG2 (Kurabo Corporation)) was added, and the cells were cultured at 37°C, 5% CO2, and 95% air for 48 hours. After removing the culture supernatant and washing the cells with PBS, total RNA was extracted using the RNeasy Plus Mini Kit (QIAGEN). Subsequently, cDNA was synthesized using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). Synthesized cDNA, TaqMan Gene Expression1 Master Mix (Thermo Fisher Scientific), and TaqMan Gene Expression Assays (Thermo Fisher Scientific) were mixed, and the amount of claudin-1 mRNA was measured using a real-time PCR system (Thermo Fisher Scientific). GAPDH was used as a control gene for correction. Relative expression levels are shown as the ratio of expression levels in each group with the evaluation substance added, with the expression level in the group without the evaluation substance set to 1. Each group with and without the evaluation substance consisted of four subjects.

[0037] The results are shown in Figure 1. *P<0.05 and **P<0.01 in the figure indicate significant differences compared to the group without the evaluation substance (shown as "0" at the left edge of Figure 1). A concentration-dependent increase in claudin-1 mRNA was observed in the MPS-added group. In contrast, no significant difference was observed with the control substance ChS, and the amount of claudin-1 mRNA decreased with the control substance HA. These results suggest that MPS has an effect of increasing claudin-1 expression in epidermal keratinocytes.

[0038] [Example 2] Effect of heparin-like substance (MPS) on claudin-1 protein expression in human epidermal keratinocytes. Adult human epidermal keratinocytes (purchased from Thermo Fisher Scientific) were seeded in 12-well microplates and cultured at 37°C, 5% CO2, and 95% air. After 6 hours, 2 mL / well of medium containing MPS or a comparator substance (0.1, 1, 10, and 100 μg / mL) or a medium without MPS (HuMedia-KG2 (Kurabo Corporation)) was added, and the cells were cultured for a further 48 hours at 37°C, 5% CO2, and 95% air. After culturing was complete, RIPA Buffer (Radio Immunoprecipitation Assay Buffer, Nacalai Tesque Co., Ltd.) was added to lyse the cells, and the supernatant was collected after centrifugation. This was used as the cell extract, and its protein concentration was measured according to the BCA Protein Assay Kit (Thermo Fisher Scientific) manual to prepare a constant protein concentration for SDS-PAGE. XV PANTERA System and Trans-Blot (登録商標) SDS-PAGE and membrane transfer were performed using a Transfer System. After blocking with 5% skim milk, the samples were immersed in primary antibody solution (Claudin-1 Polyclonal Antibody (Thermo Fisher Scientific), β-actin (8H10D10) Mouse mAb (Cell Signaling)) and shaken overnight at 4°C. After washing with TBS-T, the samples were immersed in secondary antibody solution (Anti-rabbit IgG, HRP-linked Antibody (Cell Signaling), Anti-mouse IgG, HRP-linked Antibody (Cell Signaling)) and washed again. Chemiluminescence was detected using SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific).

[0039] The results are shown in Figure 2. As is clear from Figure 2, a concentration-dependent increase in claudin-1 protein levels was observed in the MPS-treated group. In contrast, no such effect was observed with the control substances ChS and HA. These results confirm that MPS has the effect of increasing claudin-1 protein levels in epidermal keratinocytes.

[0040] [Example 3] Effect of heparin-like substance (MPS) on increasing transepithelial electrical resistance (TER) in human epidermal keratinocytes.

[0041] TER values ​​were measured using a real-time cell analyzer (ECIS-Zθ, Applied BioPhysics) and an electrode station 96W (ECIS-96WAS, Applied BioPhysics). A 10 mM L-cysteine ​​aqueous solution (Nacalai Tesque Co., Ltd.) was added to a 96-well plate with electrodes (96W20idf PET, Applied BioPhysics). Each well was then washed with distilled water. A cell suspension of adult human epidermal keratinocytes (purchased from Thermo Fisher Scientific) was added in 1.25 × 10⁻⁶ wells. 5 cells / cm 2 Cells were then seeded in each well, and 6 hours after seeding, 200 μL / well of medium containing MPS or a control substance (0.1, 1, 10, and 100 μg / mL) or a medium without it (HuMedia-KG2 (Kurabo Corporation)) was added. Measurements were then started over time at 37°C, 5% CO2, and 95% air using ECIS-Zθ. The TER value at each measurement point was calculated as the change from the time of addition of the test substance, and measurements were taken up to 72 hours later.

[0042] The results are shown in Figure 3. *P<0.05 and **P<0.01 in the figure indicate a significant difference compared to the group without the evaluation substance (vehicle). As is clear from Figure 3, MPS (Figure 3A) increased TER in human epidermal keratinocytes in a concentration-dependent manner. The control substances ChS and HA (Figures 3B and C) also showed a TER-increasing effect, but MPS was confirmed to have the highest TER-increasing effect. TER is a resistance value that arises from tight junctions limiting ion permeability, and is widely used as an index to evaluate tight junction barrier function. From the TER measurement results in Example 3, it was confirmed that MPS improves tight junction barrier function.

[0043] [Example 4] Effect of heparin-like substance (MPS) on increasing transepithelial electrical resistance (TER) in a histamine-induced tight junction barrier reduction model. Six hours after cell seeding, TER values ​​were measured in the same manner as in Example 3, except that histamine (0.1 mM) alone, histamine (0.1 mM) and MPS-containing medium (0.1, 1, 10, and 100 μg / mL), histamine (0.1 mM) and a control substance-containing medium (0.1, 1, 10, and 100 μg / mL), or a non-containing medium (HuMedia-KG2 (Kurabo Corporation)) were added to a total volume of 200 μL / well, and the measurement time was extended to 48 hours.

[0044] The results are shown in Figure 4. As is clear from Figure 4, histamine significantly reduced TER (tight junction barrier function was reduced). MPS increased the histamine-reduced TER in a concentration-dependent manner (Figure 4A), but the control substances ChS and HA showed almost no increasing effect (Figures 4B and C). From this, it was confirmed that MPS restores the tight junction barrier function that has been reduced by histamine.

[0045] [Example 5] Evaluation of tight junction barriers using a three-dimensional cultured human skin model. Three-dimensional cultured human skin models (EPI-200, MatTek) were transferred to 6-well plates containing either a mixed test solution of various concentrations of MPS and a single concentration of clobetasol propionate (CP, Tokyo Chemical Industry Co., Ltd.) (MPS concentrations: 0, 1, 10, and 100 μg / mL; CP concentration: 30 μg / mL) or 2 mL of culture medium (vehicle). After incubation in a CO2 incubator (set: 37°C, 5% CO2), the samples were transferred to test solutions containing 1 mg / mL biotin and incubated again. After 48 hours of incubation, the samples were divided and used to prepare frozen blocks and for Western blotting.

[0046] Western blot After the culture was complete, RIPA Buffer (Nacalai Tesque) was added to lyse the three-dimensional cultured human skin model, and the supernatant (cell extract) was collected by centrifugation. This was used as the cell extract, and its protein concentration was measured according to the BCA Protein Assay Kit (Thermo Fisher Scientific) manual to prepare a constant protein concentration for SDS-PAGE. XV PANTERA System and Trans-Blot (登録商標) SDS-PAGE and membrane transfer were performed using a Transfer System. After blocking with 5% skim milk, the samples were immersed in primary antibody solution (Claudin-1 Polyclonal Antibody (Thermo Fisher Scientific), β-actin (8H10D10) Mouse mAb (Cell Signaling)) and shaken overnight at 4°C. After washing with TBS-T, the samples were immersed in secondary antibody solution (Anti-rabbit IgG, HRP-linked Antibody (Cell Signaling), Anti-mouse IgG, HRP-linked Antibody (Cell Signaling)) and shaken at room temperature for 1 hour. Chemiluminescence was detected using SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific).

[0047] immunostaining The frozen block was sectioned to 5 μm thickness, fixed with 95% EtOH (-20°C, 30 min), and then blocked with 1% BSA / PBS(-). 400 μL of primary antibodies (Claudin-1 Polyclonal Antibody (Invitrogen) and Anti-Mouse E-cadherin Monoclonal Antibody (Invitrogen)) diluted with 1% BSA / PBS(-) were added, and the mixture was incubated overnight at 4°C in a humidified chamber. After washing with PBS, 400 μL of secondary antibodies (Goat anti-Rabbit IgG(H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Invitrogen), Streptavidin, Alexa Fluor™568 conjugate (Invitrogen), Alexa Fluor 647-conjugated AffiniPure Goat Anti-Rat IgG, Light Chain Specific (Jackson ImmunoResearch), Hoechst 33342 (Invitrogen)) diluted 2-fold with 1% BSA / PBS(-) were added and allowed to stand at room temperature for 1 hour. After washing with PBS, the samples were mounted using Mowiol and observed with a confocal laser scanning microscope (Olympus Corporation).

[0048] Figure 5A shows images obtained from Western blotting. Figure 5B shows the relative amount of claudin-1 to β-actin (n=6). In Figure 5B, ††P<0.01 (compared to the vehicle group with both CP and MPS at 0 μg / mL), *P<0.05, and **P<0.01 (compared to the control group with CP at 30 and MPS at 0 μg / mL). As is clear from Figure 5, the amount of claudin-1 protein decreased significantly with the addition of corticosteroids (CP), but recovered in a concentration-dependent manner with the addition of MPS.

[0049] Figure 6 shows the results of immunohistochemistry. The top panel shows the fluorescence image of claudin-1 (green). The middle and bottom panels show the fluorescence images of biotin (red) and ZO-1 (green), and the bottom panel is a magnified view of a part of the middle panel. SC represents the stratum corneum, and SG represents the granular layer. In a normal 3D cultured skin model with only culture medium added (hereinafter referred to as the normal skin model; labeled "Vehicle" in Figure 6), the expression of claudin-1 is clearly observed, as shown in the upper panel. Furthermore, in the normal skin model, the tracer (biotin) remains in the granular layer (the arrows in the lower image of the normal skin model indicate how biotin remains in the granular layer due to tight junctions), and no leakage to the stratum corneum is observed. These images indicate that the tight junction barrier functions normally in the normal skin model. In contrast, in a 3D cultured skin model with added CP (hereinafter referred to as the CP model), a decrease in claudin-1 was observed, as shown in the upper panel. Furthermore, as shown in the middle and lower panels, the tight junction barrier in the granular layer was destroyed, resulting in biotin leakage from the granular layer to the stratum corneum (the arrows in the lower image of the CP model indicate the points where biotin leaks to the stratum corneum). On the other hand, by adding MPS simultaneously with CP to the CP model, claudin-1 expression was clearly confirmed, similar to the normal skin model, and no leakage of biotin into the stratum corneum was observed. The results from Example 5 confirmed that the addition of MPS increased the expression of claudin-1 protein and improved the tight junction barrier function.

Claims

[Claim 1] A claudin-1 production promoter containing polysulfated chondroitin sulfate and / or a salt thereof as an active ingredient.

Images