Pharmaceutical or cosmetic compositions

Abstract

To provide a composition having a high capability of promoting caspase-14 production.SOLUTION: A caspase-14 production promoter contains oversulfated chondroitin sulfate and / or a salt thereof as an active ingredient.SELECTED DRAWING: Figure 4

Description

[Technical field]

[0001] The present invention relates to an agent for promoting the production of caspase-14. [Background technology]

[0002] From the outside, the skin is composed of the epidermis, dermis, and the subcutaneous tissue below it, and the epidermis, which is the outermost layer of the skin, plays the most important role in maintaining the skin barrier function. The skin barrier function prevents the evaporation of moisture inside the stratum corneum (horny layer) and prevents the intrusion of foreign substances (allergens, bacteria, etc.) from the outside world. Therefore, if the skin barrier function is weakened, problems such as the skin becoming vulnerable to external stimuli and the skin losing moisture easily occur, leading to various skin troubles.

[0003] The epidermis, the outermost layer of the skin, is composed of the stratum corneum, stratum granulosum, stratum spinosum, and stratum basale, and filaggrin is present in the stratum corneum (the outermost layer of the epidermis) as the main protein that fills the cytoplasm of keratinocytes. Filaggrin is decomposed by proteolytic enzymes in the stratum corneum and becomes a natural moisturizing factor (NMF), which plays an important role in the formation of the stratum corneum barrier function and moisture retention.

[0004] Therefore, the presence of filaggrin in the stratum corneum is important for keeping the skin moisturized and maintaining the skin barrier function; however, even if there is sufficient filaggrin, the skin's moisturizing ability will not improve unless it is broken down into natural moisturizing factors. For this reason, research into substances that can promote the production of filaggrin-degrading enzymes is in progress.

[0005] Caspase (Cysteine-ASPartic-acid-protease) is a type of cysteine ​​protease, and 14 types are currently known. Among them, caspase-14 plays an important role as one of the enzymes that breaks down filaggrin and maintains and increases natural moisturizing factors.

[0006] Regarding substances that promote the production of caspase-14, for example, Patent Document 1 discloses a caspase-14 expression inducer containing a phenylethylamine derivative as an active ingredient, and Patent Document 2 discloses a caspase-14 expression promoter containing soluble proteins and / or hydrolysates thereof from the pearl oyster nacre and pearls. However, there is still a demand for substances that can promote the production of caspase-14. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] JP 2015-63480 A [Patent Document 2] JP 2016-65048 A Summary of the Invention [Problem to be solved by the invention]

[0008] Therefore, an objective of the present invention is to provide a composition having excellent ability to promote the production of caspase-14. [Means for solving the problem]

[0009] In order to solve the above problems, the present inventors investigated substances that can promote the production of caspase-14, and as a result, found that polysulfated chondroitin sulfate or a salt thereof has excellent caspase-14 production ability, thereby completing the present invention.

[0010] That is, the present invention relates to a caspase-14 production promoter comprising polysulfated chondroitin sulfate and / or a salt thereof as an active ingredient.

[0011] The weight-average molecular weight of the polysulfated chondroitin sulfate and / or a salt thereof is preferably 8,000 to 10,000,000.

[0012] In addition, the polysulfated chondroitin sulfate and / or its salt have the following physicochemical properties: a) Sulfate group content: 25.8~37.3% by weight b) Intrinsic viscosity: 0.09~0.18 It is preferred that the compound has the formula: [Brief description of the drawings]

[0013] [Figure 1] 1 is a graph showing the relative amounts of caspase-14 mRNA due to heparinoids (MPS), chondroitin sulfate (ChS), and hyaluronic acid (HA) (the amount of mRNA in the group to which no substance was added is taken as 1). [Diagram 2] FIG. 1 is a diagram illustrating the administration schedule of a steroid and a heparinoid in Example 2. [Diagram 3] FIG. 1 shows the results of measurements in a non-treated group (NT), a non-treated group after steroid administration (CP+NT), and a group after steroid administration and heparinoid administration (CP+MPS). (A) is a graph showing the TEWL recovery rate, (B) is the stratum corneum moisture content (expressed as high-frequency conductivity), and (C) is a graph showing skin thickness. [Figure 4] FIG. 1 shows the expression levels of caspase-14 protein in a non-treated group (NT), a non-treated group after steroid administration (CP+NT), and a heparinoid-administered group after steroid administration (CP+MPS). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] The caspase-14 production promoter of the present invention contains polysulfated chondroitin sulfate and / or a salt thereof as an active ingredient. Polysulfated chondroitin sulfate is a polymer that contains a disaccharide consisting of N-acetyl-D-galactosamine and D-glucuronic acid as a repeating unit, and contains about 2 to 4, and preferably about 2 to 3, sulfate ester residues per unit (disaccharide).

[0015] Polysulfated chondroitin sulfate can be easily produced by a known method of reacting a chondroitin component such as chondroitin or chondroitin sulfate (A, C, D, E) with a sulfating agent such as chlorosulfuric acid, concentrated sulfuric acid, sulfur trioxide-pyridine complex, etc. Chondroitin sulfate A has a sulfate ester residue at the 4th position of acetylgalactosamine, chondroitin sulfate C has a sulfate ester residue at the 6th position of acetylgalactosamine, chondroitin sulfate D has a sulfate ester residue at the 6th position of acetylgalactosamine and the 2nd or 3rd position of glucuronic acid, and chondroitin sulfate E has a sulfate ester residue at both the 4th and 6th positions of acetylgalactosamine.

[0016] A preferred example of the polysulfated chondroitin sulfate is a heparinoid listed in the Japanese Pharmacopoeia's Pharmaceutical Ingredients Standards. Specifically, it is a polysulfated chondroitin sulfate exhibiting the following physicochemical properties: a) Sulfate group content: 25.8~37.3% by weight b) Intrinsic viscosity: 0.09~0.18

[0017] Polysulfated chondroitin sulfate may be used in the form of a free acid derived from the sulfate residues, but is usually used in the form of a base salt.

[0018] Examples of the base salt include alkali metal salts such as sodium and potassium, alkaline earth metal salts such as calcium, and magnesium salts.

[0019] The weight average molecular weight of the polysulfated chondroitin sulfate or a salt thereof 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.

[0020] The content of the active ingredient polysulfated chondroitin sulfate and / or a salt thereof 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 caspase-14 production promoter (however, when the caspase-14 production promoter is composed of a concentrate and a propellant, the content is based on the total weight of the concentrate). Polysulfated chondroitin sulfate is highly safe, and therefore when used by applying it to the skin, it hardly causes side effects at the above concentration.

[0021] Polysulfated chondroitin sulfate or a salt thereof has a high production-promoting effect on caspase-14, and therefore has excellent ability to decompose filaggrin and increase natural moisturizing factors, thereby improving the moisturizing ability of the skin. Therefore, the present invention can be used to prevent and / or treat conditions and / or diseases caused by decreased moisturizing ability of the skin. Examples of such conditions and / or diseases include rough skin, wrinkles, skin aging, dry skin, atopic dermatitis, ichthyosis (particularly ichthyosis vulgaris), psoriasis, xerosis, keratosis, contact dermatitis, hand eczema, etc.

[0022] In addition, when a corticosteroid (hereinafter, steroid) was applied to the skin to suppress skin inflammation, etc., it was observed that the concentration of caspase-14 in the skin was decreased even one week after the application was stopped. In contrast, it was observed that the concentration of caspase-14 was increased by applying polysulfated chondroitin sulfate or a salt thereof to the same skin site after applying a steroid, compared with the non-treated group after applying the steroid. Therefore, the caspase-14 production promoter of the present invention can restore the production of caspase-14 decreased by the steroid by applying it to the same site as the steroid before, after, or at the same time as applying the steroid to the skin.

[0023] The caspase-14 production promoter of the present invention is particularly preferably for application to the skin, and can be used as a pharmaceutical composition or cosmetic composition. Examples of pharmaceutical compositions include compositions that fall under the category of medical drugs, prescription drugs, over-the-counter drugs, or quasi-drugs as defined in the Act on Ensuring Quality, Efficacy, and Safety of Pharmaceuticals, Medical Devices, etc. Examples of the cosmetic composition include cosmetics as defined in the "Law for Ensuring Quality, Efficacy and Safety of Pharmaceuticals, Medical Devices, etc." and compositions that fall under the category of quasi-drugs, i.e. medicated cosmetics.

[0024] The dosage form is not particularly limited as long as it is a form that can be applied to the skin, and examples of the dosage form include ointments, creams, gels, lotions, sprays (including foams), patches, etc. described in the Japanese Pharmacopoeia, and the composition can be used as a pharmaceutical composition or cosmetic composition together with pharmaceutically acceptable additives.

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

[0026] Examples of bases include, but are not limited to, higher hydrocarbons such as white petrolatum, squalane, and light liquid paraffin; waxes such as white beeswax, lanolin, and ceresin wax; fats and oils 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 fatty acid triglycerides; polyhydric alcohols such as glycerin and 1,3-butylene glycol; lower monohydric alcohols such as ethanol and isopropanol; water (purified water); macrogol (polyethylene glycol); silicone oil; and the like. Only one type or multiple types may be used.

[0027] Examples of surfactants (including those used as emulsifiers and foaming agents) include, but are not limited to, cationic surfactants, anionic surfactants, nonionic surfactants, and amphoteric surfactants, and may be used alone or in combination. Examples of cationic surfactants include, but are not limited to, cetyltrimethylammonium chloride, lauryldimethylbenzylammonium chloride, tetrabutylammonium chloride, and dioctadecyldimethylammonium chloride. Examples of anionic surfactants include, but are not limited to, sodium alkylbenzenesulfonate, sodium dodecyl sulfate, sodium coconut alcohol ethoxy sulfate, sodium α-olefinsulfonate, and emulsified cetostearyl alcohol (a mixture of cetostearyl alcohol and sodium cetostearyl sulfate). Examples of nonionic surfactants include, but are not limited to, 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, etc. Examples of amphoteric surfactants include, but are not limited to, N-alkyl-N,N-dimethylammonium betaine, imidazoline-type amphoteric surfactants, etc.

[0028] Examples of preservatives include, but are not limited to, dibutylhydroxytoluene, sodium edetate hydrate, and paraoxybenzoic acid esters, and one or more of these may be used.

[0029] Examples of the pH adjuster include, but are not limited to, diisopropanolamine, triisopropanolamine, triethanolamine, potassium hydroxide, sodium hydroxide, and the like, and one or more kinds may be used.

[0030] Examples of the thickening agent include, but are not limited to, sodium alginate, gelatin, carboxyvinyl polymer, carboxymethylcellulose, etc., and one or more kinds may be used.

[0031] In addition, when the dosage form is a foam, a propellant such as a liquefied gas such as liquefied petroleum gas (LPG) or a compressed gas can be used together with a concentrate containing polysulfated chondroitin sulfate and / or a salt thereof.

[0032] In addition to the above, the caspase-14 production promoter of the present invention may contain additives that are generally used as additives for pharmaceutical compositions or cosmetic compositions (e.g., buffers, fragrances, colorants, ultraviolet absorbers, etc.).

[0033] The dosage and frequency of administration of the caspase-14 production promoter according to the present invention may be appropriately adjusted depending on the target disease and the severity of its symptoms, the concentration of polysulfated chondroitin sulfate and / or a salt thereof, age, body weight, etc. For example, the dosage and frequency of administration of the caspase-14 production promoter according to the present invention may be appropriately adjusted depending on the target disease and the severity of its symptoms, the concentration of polysulfated chondroitin sulfate and / or a salt thereof, age, body weight, etc. 2 A dose of 0.03 μg to 30 mg, and preferably 0.3 μg to 3 mg, may be applied 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 (abbreviation: MPS), a heparinoid substance listed in the Japanese Pharmacopoeia Extra Pharmaceutical Ingredients Standards (organic sulfate group content: 25.8-37.3% w / w, glucuronic acid content 19.0-24.0% w / w, Maruho Co., Ltd.) was used. As control substances for comparison, hyaluronic acid (abbreviation: HA, Tokyo Chemical Industry Co., Ltd.) and chondroitin sulfate (abbreviation: Chs, organic sulfate group content: 15 to 17% w / w, Maruho Corporation) were used.

[0036] [Example 1] Effect of MPS on caspase-14 expression in human epidermal keratinocytes Adult human epidermal keratinocytes (purchased from Thermo Fisher Scientific) were suspended in HuMedia-KG2 (Kurabo Industries, Ltd.), and the cell suspension was diluted to 1.25 × 10 5 The cells were seeded in a 24-well microplate at 100 cells / well and cultured at 37°C, 5% CO2, and 95% air. The next day, 500 μL / well of MPS and control substance-containing medium (0.1, 1, 10, and 100 μg / mL) or non-containing medium (HuMedia-KG2 (Kurabo) supplemented with gentamicin / amphotericin (Kurabo) B) was added, and the cells were cultured at 37°C, 5% CO2, and 95% air for 24 hours. The culture supernatant was removed, the cells were washed with PBS, and total RNA was extracted using RNeasy Plus Mini Kit (QIAGEN). Then, cDNA was synthesized using High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). The synthesized cDNA, TaqMan Gene Expression Master Mix (Thermo Fisher Scientific), and TaqMan Gene Expression Assays (Thermo Fisher Scientific) were mixed, and the amount of caspase-14 mRNA was measured using a real-time PCR system (Thermo Fisher Scientific). GAPDH was used as a control gene for correction. The relative expression level indicates the expression level ratio of each evaluation substance addition group when the expression level of the evaluation substance non-addition group is set to 1. Each evaluation substance addition group and non-addition group had 4 cases (n=4).

[0037] The results of the relative expression level of caspase-14 mRNA are shown in Figure 1. In the figure, *P<0.05 and **P<0.01 indicate significant differences compared to the group to which no substance was added (shown as "0" on the left side of Figure 1). In the MPS-added group, a concentration-dependent increase in the expression level of caspase-14 mRNA was confirmed. Furthermore, compared to the control substances HA and ChS, the increase in caspase-14 mRNA expression in the MPS-added group was significant. These results suggest that MPS has the effect of increasing the expression of caspase-14 in epidermal keratinocytes.

[0038] [Example 2] Effect of MPS on the decrease in skin physiological function and caspase-14 amount in the skin caused by repeated steroid administration The effect of MPS on the decline in skin physiological function caused by repeated administration of steroids was evaluated. The steroid was Dermovate Cream 0.05% (containing clobetasol propionate 0.05%, GlaxoSmithKline KK, hereafter referred to as steroids), and the MPS was Hirudoid. [登録商標] Soft ointment 0.3% (containing 0.3% heparinoid, Maruho Co., Ltd., hereafter referred to as heparinoid) was used. The hair on the backs of mice (C57BL / 6, male, 8 weeks old) was removed and steroids were repeatedly administered transdermally once a day for 10 days. Starting the day after the final steroid administration, the mice were divided into an untreated group in which no drug was applied (steroid-administered + untreated group) and a group in which a heparinoid was administered (steroid-administered + heparinoid-administered group). In the steroid + heparinoid group, the heparinoid was repeatedly administered percutaneously once a day for 7 days from the day after the final steroid administration (day 11) (see Figure 2). The group that received neither steroid nor heparinoid was set as the untreated group. Each of the non-treatment group (NT), steroid administration + non-treatment group (CP+NT), and steroid administration + heparinoid administration group (CP+MPS) consisted of 5 cases (n=5).

[0039] As indicators of skin physiological function, the day when the steroid was first administered was counted as day 1, and transepidermal water loss (TEWL), which is an indicator of the recovery of skin barrier function, was measured on the 15th and 18th days, and stratum corneum moisture content, which is an indicator of the enhancement of stratum corneum moisture retention, and skin thickness were measured on the 11th, 15th, and 18th days. TEWL values ​​were measured using a portable water evaporation meter Vapometer (Delphin Technologies). TEWL recovery rates were calculated using the following formula. Note that the TEWL values ​​of the untreated group during the test period were not calculated because they did not change from before steroid administration.

number

[0040] The moisture content of the stratum corneum was measured and evaluated as high-frequency conductivity using a stratum corneum moisture content measuring device SKICON-200EX (Yayoi Co., Ltd.). The skin thickness was measured by pinching the applied site with a Digimatic Thickness Gauge (Mitutoyo Corporation).

[0041] The effects of heparinoids on the decline in skin physiological functions (TEWL value, stratum corneum moisture content, and skin thickness) due to repeated steroid administration are shown in Figures 3A, 3B, and 3C. In Figure 3, * indicates a significant difference between the "steroid administration + no treatment group" and the "no treatment group" (*P<0.05), and † indicates a significant difference between the "steroid administration + heparinoid administration group" and the "steroid administration + no treatment group" (†P<0.05). The TEWL recovery rate in the steroid + heparinoid treatment group was significantly higher than that in the steroid + no treatment group on day 18 (Figure 3A). The stratum corneum moisture content and skin thickness of the steroid-administered non-treatment group were significantly lower than those of the non-treatment group. The stratum corneum moisture content of the steroid-administered non-heparinoid group was higher on days 15 and 18 than those of the steroid-administered non-treatment group (Fig. 3B). The skin thickness of the steroid-administered non-treatment group was significantly higher on days 15 and 18 than those of the steroid-administered non-treatment group (Fig. 3C). The reason why the skin thickness of the non-treatment group increased over time in Fig. 3C is thought to be because the mice used in the test were in the hair growth phase, since the skin thickness of mice varies depending on the hair cycle. In contrast, in the steroid-administered non-treatment group, the hair cycle is delayed by the application of steroids, so the variation in skin thickness due to the hair cycle is hardly observed, and the skin atrophy caused by steroids is thought to have been maintained even after withdrawal of medication. In fact, the skin thickness in the "steroid administration + no treatment group" remained at low values, being 0.48 mm, 0.47 mm, and 0.47 mm on days 11, 15, and 18, respectively. In contrast, the skin thickness in the "steroid administration + heparinoid administration group" was 0.47 mm, 0.51 mm, and 0.53 mm on days 11, 15, and 18, respectively, and a tendency for skin thickness to recover over time was observed.

[0042] On the day after the final administration (day 18), the animals in each group were euthanized, and the skin was collected using a biopsy trephine φ8.0 mm (Kai Industries Co., Ltd.). 350 μL of homogenate buffer (a mixture of 10 mL of PBS (Thermo Fisher Scientific), 20 μL of Tween 20 (Nacalai Tesque Co., Ltd.), and 200 μL of Protease Inhibitor Cocktail Set 3 (Merck Co., Ltd.)) was added to the collected skin pieces, tungsten beads (Biomedical Science Co., Ltd.) were added, and the skin pieces were crushed using Shake Master NEO (Biomedical Science Co., Ltd.). The entire amount of the extracted solution was collected, centrifuged at 1870 g for 5 minutes, and the supernatant was collected. The amount of caspase-14 in the collected supernatant was measured using an ELISA kit (Casp14 ELISA Kit Mouse (Aviva Systems Biology Co., Ltd.)). The total protein amount was measured using a CBQCA Protein Quantitation Kit (Thermo Fisher Scientific), and the caspase-14 concentration was calculated by correcting for the total protein amount.

[0043] The results are shown in Figure 4. In the figure, * indicates a significant difference between the "steroid-administered + no treatment group" and the "no treatment group" (*P<0.05), and † indicates a significant difference between the "steroid-administered + heparinoid-administered group" and the "steroid-administered + no treatment group" (†P<0.05). The caspase-14 concentration in the steroid-administered + untreated group was significantly lower than that in the untreated group, while the caspase-14 concentration in the steroid-administered + heparinoid-administered group was significantly higher than that in the steroid-administered + untreated group. As described above, MPS is expected to have an improving effect on skin symptoms that may be caused by a decrease in the amount of caspase-14 in the skin.

Claims

1. A caspase-14 production promoter comprising polysulfated chondroitin sulfate and / or a salt thereof as an active ingredient.

2. The caspase-14 production promoter according to claim 1, wherein the polysulfated chondroitin sulfate and / or a salt thereof has a weight-average molecular weight of 8,000 to 10,000,000.

3. The polysulfated chondroitin sulfate and / or its salt have the following physicochemical properties: a) Sulfate group content: 25.8-37.3% by weight b) Intrinsic viscosity: 0.09-0.18 The caspase-14 production promoter according to claim 1 or 2, comprising:

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