JP7638759B2 - Pharmaceutical or cosmetic compositions - Google Patents

Description

The present invention relates to a caspase-14 production promoter.

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

The epidermis, the outermost layer of the skin, is composed of the stratum corneum, stratum granulosum, stratum spinosum, and stratum basale. 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 broken down by proteolytic enzymes in the stratum corneum and becomes a natural moisturizing factor (NMF), which plays an important role in forming the stratum corneum barrier function and retaining moisture.

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.

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

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

JP 2015-63480 A JP 2016-65048 A

Therefore, the objective of the present invention is to provide a composition that has excellent ability to promote the production of caspase-14.

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

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

The weight average molecular weight of the polysulfated chondroitin sulfate and/or its salt is preferably 8,000 to 10,000,000.

The polysulfated chondroitin sulfate and/or its salt have the following physicochemical properties: a) sulfate group content: 25.8 to 37.3% by weight
b) Intrinsic viscosity: 0.09 to 0.18
It is preferred that the compound has the formula:

1 is a graph showing the relative amounts of caspase-14 mRNA by 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). FIG. 1 is a diagram for explaining the administration schedule of steroids and heparinoids in Example 2. 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), in which (A) is a graph showing the TEWL recovery rate, (B) is a graph showing the stratum corneum moisture content (expressed as high-frequency conductivity), and (C) is a graph showing skin thickness. 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).

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).

Polysulfated chondroitin sulfate can be easily produced by a known method in which chondroitin components such as chondroitin and chondroitin sulfate (A, C, D, E) are reacted with a sulfating agent such as chlorosulfuric acid, concentrated sulfuric acid, or sulfur trioxide-pyridine complex. 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 at 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.

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 to 0.18

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

Examples of such base salts include alkali metal salts such as sodium and potassium, alkaline earth metal salts such as calcium, and magnesium salts.

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.

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 caspase-14 production promoter (however, when the caspase-14 production promoter consists of a concentrate and a propellant, the content is based on the total weight of the concentrate). Polysulfated chondroitin sulfate is highly safe, so when applied to the skin, the above concentrations cause almost no side effects.

Polysulfated chondroitin sulfate or a salt thereof has a high production-promoting effect on caspase-14, and therefore has an 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, such as rough skin, wrinkles, skin aging, dry skin, atopic dermatitis, ichthyosis (particularly ichthyosis vulgaris), psoriasis, xerosis, keratosis, contact dermatitis, and hand eczema.

In addition, when corticosteroids (hereinafter referred to as steroids) were applied to the skin to suppress skin inflammation, etc., it was observed that the concentration of caspase-14 in the skin was reduced even one week after the application was discontinued. 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 steroids, compared to the untreated group after applying steroids. Therefore, the caspase-14 production promoter of the present invention can restore the production of caspase-14 that is reduced by steroids by applying it to the same site as the steroids before, after, or simultaneously with applying the steroids to the skin.

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 "Law 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.

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., as described in the Japanese Pharmacopoeia, and can be used as a pharmaceutical composition or cosmetic composition together with pharmaceutically acceptable additives.

Examples of the additives include, but are not limited to, bases, surfactants, preservatives, pH adjusters, thickeners, etc.

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); and silicone oil. One or more of these may be used.

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 one or more types may be used. 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, and polyoxyethylene sorbitan fatty acid esters such as polyoxyethylene sorbitan monostearate and polyoxyethylene sorbitan tristearate. Examples of amphoteric surfactants include, but are not limited to, N-alkyl-N,N-dimethylammonium betaine, imidazoline-type amphoteric surfactants, etc.

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

Examples of pH adjusters include, but are not limited to, diisopropanolamine, triisopropanolamine, triethanolamine, potassium hydroxide, and sodium hydroxide, and one or more types may be used.

Examples of thickeners include, but are not limited to, sodium alginate, gelatin, carboxyvinyl polymer, and carboxymethylcellulose, and one or more types may be used.

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 the concentrate containing polysulfated chondroitin sulfate and/or its salt.

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

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, 0.03 μg to 30 mg, preferably 0.3 μg to 3 mg, of polysulfated chondroitin sulfate per ^cm2 of skin may be applied once or several times a day.

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

As polysulfated chondroitin sulfate or its salt (abbreviation: MPS), a heparinoid substance listed in the Japanese Pharmacopoeia (organic sulfate group content: 25.8 to 37.3% w/w, glucuronic acid content: 19.0 to 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: 15-17% w/w, Maruho Co., Ltd.) were used.

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), and the cell suspension was seeded on a 24-well microplate at 1.25 x ¹⁰⁵ cells/well and cultured at 37°C, 5% _CO2 , and 95% air. The next day, MPS and control substance-containing medium (0.1, 1, 10, 100 μg/mL) or non-containing medium (HuMedia-KG2 (Kurabo) supplemented with gentamicin/amphotericin (Kurabo) B) were added to the wells at 500 μL/well, and the cells were cultured at 37°C, 5% _CO2 , and 95% air for 24 hours. The culture supernatant was removed, and the cells were washed with PBS, and then 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). In addition, 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).

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.

Example 2: Effect of MPS on reduction in skin physiological function and caspase-14 amount in skin due to repeated administration of steroid The effect of MPS on reduction in skin physiological function due to repeated administration of steroid was evaluated. Dermovate cream 0.05% (containing 0.05% clobetasol propionate, GlaxoSmithKline KK, hereafter ^{referred to} as steroid) was used as the steroid, and Hirudoid® Soft Ointment 0.3% (containing 0.3% heparinoid, Maruho Co., Ltd., hereafter referred to as heparinoid) was used as the MPS.
The hair on the backs of mice (C57BL/6, male, 8 weeks old) was removed, and a steroid was repeatedly administered percutaneously 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 administration 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) had 5 cases (n = 5).

上式において、ＭＰＳはヘパリン類似物質を、ＣＰはステロイド（クロベタゾールプロピオン酸エステル）を意味する。 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 skin barrier function recovery effect, was measured on the 15th and 18th days, and stratum corneum moisture content, which is an indicator of the stratum corneum's enhanced moisture retention effect, 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). The TEWL recovery rate was calculated using the following formula. Note that the TEWL value of the untreated group during the test period was not calculated because it did not change from before steroid administration.

In the above formula, MPS means heparinoid and CP means steroid (clobetasol propionate).

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).

The effects of heparinoids on the decline in skin physiological functions (TEWL value, stratum corneum moisture content, and skin thickness) caused by 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-administered + heparinoid-administered group was significantly higher than that in the steroid-administered + untreated group on day 18 (FIG. 3A).
The stratum corneum moisture content and skin thickness of the steroid-administered + non-treated group were significantly lower than those of the non-treated group. The stratum corneum moisture content of the steroid-administered + heparinoid group was higher on the 15th and 18th days than those of the steroid-administered + non-treated group (Fig. 3B). The skin thickness of the steroid-administered + heparinoid group was significantly higher on the 15th and 18th days than those of the steroid-administered + non-treated group (Fig. 3C). The reason why the skin thickness of the non-treated group increases over time in Fig. 3C is that 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-treated group, the hair cycle is delayed by the application of steroids, so that the variation in skin thickness due to the hair cycle is hardly observed, and the skin atrophy caused by steroids is also maintained even after withdrawal of medication. In fact, the skin thickness in the "steroid-administered + untreated 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-administered + heparinoid-administered 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.

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). 350 μL of homogenate buffer (a mixture of 10 mL of PBS (Thermo Fisher Scientific), 20 μL of Tween 20 (Nacalai Tesque), and 200 μL of Protease Inhibitor Cocktail Set 3 (Merck)) 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 and 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)). The total protein amount was measured using the CBQCA Protein Quantitation Kit (Thermo Fisher Scientific), and the caspase-14 concentration was calculated by correcting for the total protein amount.

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 (3)

A caspase-14 production promoter containing polysulfated chondroitin sulfate and/or its salt as an active ingredient. The caspase-14 production promoter according to claim 1, wherein the weight-average molecular weight of the polysulfated chondroitin sulfate and/or its salt is 8,000 to 10,000,000. The polysulfated chondroitin sulfate and/or its salt have the following physicochemical properties: a) sulfate group content: 25.8 to 37.3% by weight
b) Intrinsic viscosity: 0.09 to 0.18
The caspase-14 production promoter according to claim 1 or 2, comprising:

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