Stratum corneum barrier function improving agent and pharmaceutical composition

Abstract

The present invention addresses the problem of providing an improving agent for improving a stratum corneum barrier function without external replenishment with any moisturizing substance and a pharmaceutical composition containing said improving agent. The present invention provides: a stratum corneum barrier function improving agent which contains a glycolytic inhibitor as an active ingredient; the stratum corneum barrier function improving agent in which the glycolytic inhibitor is at least one selected from the group consisting of a glucose transporter inhibitor and a glucose competitive inhibitor; either of said stratum corneum barrier function improving agents having at least one selected from the group consisting of an inhibitory effect on the loss of skin moisture retention capacity and an inhibitory effect on skin permeability enhancement; and a pharmaceutical composition which contains any of said stratum corneum barrier function improving agents.

Description

Stratum corneum barrier function improving agent and pharmaceutical composition

[0001] The present invention relates to an agent for improving the stratum corneum barrier function without externally supplementing moisturizing substances, and a pharmaceutical composition containing the agent. This application claims priority to Japanese Patent Application No. 2023-209294, filed on December 12, 2023, the contents of which are incorporated herein by reference.

[0002] The skin has an important barrier function (skin barrier function) that prevents foreign substances from entering the body from the outside and prevents moisture evaporation from the body. If this skin barrier function is disrupted due to genetic or environmental factors, it can lead to various inflammations, itching, skin aging, and allergies, so maintaining the skin barrier function is extremely important for maintaining homeostasis and health.

[0003] The stratum corneum barrier plays an important role in skin barrier function. It is composed of three components: keratinocytes, located in the outermost layer of the skin, intercellular lipids, sebum on the stratum corneum surface, and natural moisturizing factors within the stratum corneum. Various moisturizers have been developed to maintain stratum corneum barrier function, but they essentially achieve their moisturizing function by replenishing these three components. For example, petrolatum replenishes sebum on the stratum corneum surface, ceramide-containing ointments replenish intercellular lipids, and urea ointments replenish natural moisturizing factors, respectively. Hyaluronic acid and heparinoids are also widely used moisturizers, but they exert their moisturizing power through their water adsorption ability. Thus, conventional moisturizers are designed to externally add moisturizing substances. While these moisturizers are important for maintaining the stratum corneum barrier, they are merely artificial external supplements and do not strengthen the physiological stratum corneum barrier itself.

[0004] On the other hand, in recent years, it has become clear that intracellular energy metabolism plays a major role in the regulation of various cellular functions. It has been found that glucose transporters (molecules that take up sugar into cells), particularly GLUT1 (glucose transporter 1), play a major role in the glycolytic pathway of epidermal keratinocytes (Non-Patent Document 1).

[0005] Cibrian et al., Trends in Molecular Medicine, 2020, vol.26(11), p.975-986.Liu et al., Frontiers in Pharmacology, 2021, vol.12, Article 765790.

[0006] An object of the present invention is to provide an improving agent for improving the stratum corneum barrier function without externally supplementing moisturizing substances, and a pharmaceutical composition containing the improving agent.

[0007] The present inventors discovered that GLUT1 expression in epidermal keratinocytes is significantly increased in a mouse model of stratum corneum barrier destruction (tape stripping), suggesting that control of the glycolytic pathway via GLUT1 is involved in maintaining stratum corneum barrier function, and thus completed the present invention.

[0008] That is, the present invention provides the following. [1] A stratum corneum barrier function improver containing a glycolytic inhibitor as an active ingredient. [2] The stratum corneum barrier function improver of [1] above, wherein the glycolytic inhibitor is one or more selected from the group consisting of a glucose transporter inhibitor and a glucose-competitive inhibitor. [3] The stratum corneum barrier function improver of [2] above, wherein the glucose transporter inhibitor is a GLUT1 inhibitor. [4] The stratum corneum barrier function improver of any of [1] to [3] above, which has one or more inhibitory effects selected from the group consisting of an inhibitory effect on a decrease in skin moisture retention ability and an inhibitory effect on an increase in skin permeability. [5] A pharmaceutical composition comprising the stratum corneum barrier function improver of any of [1] to [4] above. [6] The pharmaceutical composition of [5] above, which is used for the prevention or treatment of a skin disease. [7] The pharmaceutical composition of [5] or [6] above, which is administered topically or orally.

[0009] The stratum corneum barrier function improving agent according to the present invention can improve the stratum corneum barrier function without externally supplementing a moisturizing substance, and therefore the stratum corneum barrier function improving agent according to the present invention and a pharmaceutical composition containing the same are suitable for treating and preventing various diseases, such as inflammation, caused by disorders of the stratum corneum barrier function.

[0010] 1 is a diagram showing the results of measuring the amount of transepidermal water loss in both ears of each mouse before tape stripping on day 0 and after tape stripping on day 2 of the stratum corneum barrier damage test in Reference Example 1. FIG. 2 is a diagram showing the results of measuring the amount of dye infiltrated into the ears of each mouse after tape stripping on day 2 of the stratum corneum barrier damage test in Reference Example 1. FIG. 3 is a diagram showing HE-stained images and anti-GLUT1 antibody-stained images of ear tissue sections of each mouse after tape stripping on day 7 of the stratum corneum barrier damage test in Reference Example 1. FIG. 4 is a diagram showing the results of measuring the amount of transepidermal water loss in the ears of mice to which a glycolytic inhibitor (WZB-117, 2-DG, or BAY-876) was applied before tape stripping on day 0 (pre-treatment) and after tape stripping on day 2 (post-treatment) of the stratum corneum barrier damage test in Example 1. This figure shows the results of measuring the amount of dye that penetrated into the ears of mice after tape stripping on day 2 of a stratum corneum barrier damage test in the ears of mice to which a glycolytic inhibitor (WZB-117, 2-DG, or BAY-876) was applied in Example 1. This figure shows the results of measuring the amount of transepidermal water loss on day 4 of a stratum corneum barrier damage test in the ears of mice to which a glycolytic inhibitor (2-DG) was intraperitoneally administered in Example 2. This figure shows HE-stained images of mouse ear tissue sections from a solvent (acetone)-applied group and a glycolytic inhibitor (WZB-117)-applied group before tape stripping (day -1 from the start of the experiment) and after tape stripping (day 5 from the start of the experiment) in Example 3. 1 shows the results of measuring the mRNA levels of filaggrin (Flg) and Tjp3 (ZO-3) in ear skin tissue on days 1, 2, 3, and 5 after the start of the experiment for the solvent (acetone)-applied group and the glycolytic inhibitor (WZB-117)-applied group in Example 3. These are HE-stained images of ear tissue sections in which solvent (acetone) or a glycolytic inhibitor (WZB-117) was applied to normal skin tissue that had not been subjected to tape stripping in Example 3. These are the results of measuring the mRNA levels of filaggrin (Flg), involucrin (IVL), and loricrin (LOR) in epidermal keratinocytes cultured in a WZB-117-containing medium in Example 4.

[0011] <Stratum corneum barrier function improving agent> The stratum corneum barrier function improving agent according to this embodiment contains a glycolysis inhibitor as an active ingredient. As shown in the examples below, the stratum corneum is physically peeled, and the stratum corneum barrier function is impaired by treating the skin surface with a glycolysis inhibitor, thereby restoring the stratum corneum barrier function. That is, the glycolysis inhibitor has one or more inhibitors selected from the group consisting of an inhibitory effect on the reduction of skin moisture retention capacity and an inhibitory effect on the enhancement of skin permeability. The mechanism by which glycolysis inhibitors improve stratum corneum barrier function is not yet clear, but it is speculated that they may be able to maintain the stratum corneum barrier endogenously by controlling the function of epidermal keratinocytes themselves via the glycolysis pathway. Furthermore, as shown in the examples below, glycolysis inhibitors can enhance the gene expression of stratum corneum barrier formation-related factors in damaged skin tissue, which is speculated to be one of the mechanisms by which glycolysis inhibitors repair stratum corneum barrier damage.

[0012] In the present invention and this specification, the term "glycolytic inhibitor" means "a substance that targets a biomolecule that is involved in or affects the glycolytic metabolic pathway and inhibits ATP production by the glycolytic pathway by suppressing or enhancing the function of the biomolecule." The glycolytic inhibitor that is the active ingredient of the stratum corneum barrier function improving agent according to this embodiment is not particularly limited as long as it is a substance that can inhibit the glycolytic metabolic pathway.

[0013] The glycolysis inhibitor used in this embodiment is preferably one or more selected from the group consisting of glucose transporter inhibitors and glucose-competitive inhibitors. By inhibiting glucose uptake into epidermal keratinocytes or suppressing the metabolism of glucose into epidermal keratinocytes, a greater effect can be expected.

[0014] The glucose transporter inhibitor used in this embodiment may be an inhibitor of any glucose transporter present in the cell membrane of epidermal keratinocytes. It may also be an inhibitor specific to a particular glucose transporter, or an inhibitor that exerts an inhibitory effect on various glucose transporters. The active ingredient of the stratum corneum barrier function improver according to this embodiment is preferably a GLUT1 inhibitor (an inhibitor that exerts an inhibitory effect on GLUT1), and particularly preferably a GLUT1-specific inhibitor.

[0015] The glucose transporter inhibitor used in this embodiment is not particularly limited as long as it has glucose transporter inhibitory activity. It may be a low molecular weight compound, a nucleic acid oligomer, a peptide, or a protein. GLUT1 inhibitors consisting of low molecular weight compounds include various molecules known to have inhibitory effects on GLUT1, such as WZB-117 (CAS No.: 1223397-11-2; inhibitor of GLUT1, GLUT3, and GLUT4), Ritonavir (CAS No.: 155213-67-5; inhibitor of GLUT1 and GLUT4), STF-31 (CAS No.: 724741-75-7; inhibitor of GLUT1), and BAY-876 (CAS No.: 1799753-84-6; inhibitor of GLUT1).

[0016] The glucose-competitive inhibitor used in this embodiment is a so-called non-metabolizable glucose analogue, which is structurally similar to glucose but is not metabolized. Examples of the non-metabolizable glucose analogue include various known non-metabolizable glucose analogues such as 2-deoxy-D-glucose (CAS No.: 154-17-6) and D-mannoheptulose (CAS No.: 3615-44-9).

[0017] The stratum corneum barrier function improving agent according to this embodiment may contain the glycolytic inhibitor as it is or as a pharmacologically acceptable salt thereof. The pharmacologically acceptable salt may be an inorganic acid salt, an organic acid salt having a basic moiety such as an amine, an alkali salt, or an organic salt.

[0018] The stratum corneum barrier function improving agent according to this embodiment may contain the glycolytic inhibitor as a free glycolytic inhibitor, or as a solvate or a solvate of a pharmacologically acceptable salt thereof. Examples of solvents that form the solvate include water and ethanol.

[0019] <Pharmaceutical Composition> The pharmaceutical composition according to this embodiment contains the stratum corneum barrier function improving agent according to this embodiment. As shown in the examples below, the stratum corneum barrier function improving agent according to this embodiment can improve the function of an impaired stratum corneum barrier, suppress a decrease in the skin's moisture retention ability, and suppress increased skin permeability. Therefore, the stratum corneum barrier function improving agent according to this embodiment is useful as an active ingredient of a pharmaceutical composition.

[0020] The pharmaceutical composition according to this embodiment is useful as a pharmaceutical composition used for the prevention or treatment of diseases whose pathology can be expected to improve by maintaining or improving the stratum corneum barrier function, and is particularly expected to be effective in suppressing skin inflammation, dryness, invasion of foreign substances, allergic reactions, skin aging, and the onset of infectious diseases, which are caused by impaired stratum corneum barrier function. That is, the pharmaceutical composition according to this embodiment can be used for the prevention or treatment of skin diseases. Examples of such skin diseases include diseases accompanied by symptoms such as skin inflammation, dryness, itching, allergic symptoms, and skin aging. The pharmaceutical composition according to this embodiment is preferably a pharmaceutical composition used for the prevention or treatment of atopic dermatitis, psoriasis, and the like.

[0021] The amount of the stratum corneum barrier function improver to be contained in the pharmaceutical composition of the present invention is not particularly limited as long as it is a therapeutically effective amount that can achieve the effect of improving the stratum corneum barrier function, i.e., an amount sufficient to achieve a therapeutic effect against skin diseases, etc., and is determined appropriately taking into consideration the type of skin disease, administration route, administration form (dosage form), number of doses per day, administration interval, organism species, sex, age, body weight, etc. of the subject to be administered.

[0022] When the pharmaceutical composition of the present invention contains as an active ingredient a drug that is currently or has been approved as a pharmaceutical product in the past, the dosage regimen, such as the route of administration, dosage form, amount of the active ingredient per administration, and administration interval, may be similar to those of therapeutic drugs for other diseases for which such drugs have already been approved, or these may be improved.

[0023] The pharmaceutical composition of the present invention may be a pharmaceutical composition for topical administration or a pharmaceutical composition for oral administration. The route of administration of the pharmaceutical composition of the present invention is not particularly limited and can be appropriately selected from various routes of administration, such as oral administration, intravenous administration, transdermal administration, enteral administration, and nasal administration. The pharmaceutical composition of the present invention can be formulated into oral solid preparations such as powders, granules, capsules, tablets, chewable tablets, and sustained-release preparations; oral liquid preparations such as solutions and syrups; external preparations such as patches, ointments, gels, creams, sprays, lotions, and liniments; injections, suppositories, and the like, by conventional methods, taking into account the desired route of administration.

[0024] Examples of other components contained in the pharmaceutical composition of the present invention include pharmacologically acceptable additives such as excipients, binders, lubricants, disintegrants, fluidizing agents, solvents, solubilizing agents, buffers, suspending agents, emulsifiers, isotonicity agents, stabilizers, preservatives, antioxidants, flavoring agents, coloring agents, etc. Furthermore, the pharmaceutical composition may contain other active ingredients in addition to the stratum corneum barrier function improving agent.

[0025] Examples of excipients include sugars such as lactose, glucose, and D-mannitol, celluloses such as starch and crystalline cellulose, sugar alcohols such as erythritol, sorbitol, and xylitol, dicalcium phosphate, calcium carbonate, and kaolin. Examples of binders include pregelatinized starch, gelatin, gum arabic, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, crystalline cellulose, D-mannitol, trehalose, hydroxypropyl cellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, and polyvinyl alcohol. Examples of lubricants include stearic acid, calcium stearate, talc, sucrose fatty acid esters, and polyethylene glycol. Examples of disintegrants include crospovidone (cross-linked polyvinylpyrrolidone), low-substituted hydroxypropyl cellulose, starch, alginic acid, and sodium alginate. Examples of fluidizers include silicic acid, silicic anhydride, aluminum silicate, calcium silicate, magnesium aluminometasilicate compounds, aluminum oxide, aluminum hydroxide, magnesium oxide, and magnesium hydroxide. Examples of solvents include purified water and physiological saline. Examples of solubilizers include dextran, polyvinylpyrrolidone, sodium benzoate, ethylenediamine, salicylic acid amide, nicotinic acid amide, and polyoxyethylene hydrogenated castor oil derivatives. Examples of buffers include sodium citrate hydrate, sodium acetate hydrate, sodium bicarbonate, trometamol, boric acid, borax, sodium hydrogen phosphate hydrate, and sodium dihydrogen phosphate. Examples of suspending or emulsifying agents include sodium lauryl sulfate, gum arabic, gelatin, lecithin, glycerin monostearate, polyvinyl alcohol, polyvinylpyrrolidone, celluloses such as sodium carboxymethylcellulose, polysorbates, and polyoxyethylene hydrogenated castor oil. Examples of isotonic agents include sugars such as lactose, glucose, and D-mannitol, sodium chloride, potassium chloride, glycerin, propylene glycol, polyethylene glycol, and urea.Examples of stabilizers include polyethylene glycol, dextran sodium sulfate, and sodium sulfite. Examples of preservatives include parahydroxybenzoates, chlorobutanol, benzyl alcohol, phenethyl alcohol, chlorocresol, dehydroacetic acid, and sorbic acid. Examples of antioxidants include sulfites and ascorbic acid. Examples of flavoring agents include sweeteners and fragrances commonly used in the pharmaceutical and food fields. Examples of coloring agents include coloring agents commonly used in the pharmaceutical and food fields.

[0026] The pharmaceutical composition of the present invention is administered in an effective amount to an animal having or suspected of having impaired stratum corneum barrier function. The pharmaceutical composition is preferably administered to mammals, more preferably to humans, or domestic animals or laboratory animals such as mice, rats, rabbits, guinea pigs, hamsters, monkeys, sheep, horses, cattle, pigs, donkeys, dogs, and cats, and even more preferably to humans.

[0027] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0028] [Reference Example 1] The stratum corneum barrier was damaged by tape stripping, and the functional changes were evaluated. Tape stripping is a method of physically peeling the stratum corneum by removing adhesive tape attached to the skin, thereby damaging the stratum corneum barrier function. Tape stripping is a commonly used method for inducing functional and histological changes similar to those observed when the skin is scratched.

[0029] <Stratum corneum barrier damage test by tape stripping method> On the first day (day 0) and the second day of the experiment, both sides (front and back) of the left ear of mice (strain name: C57BL / 6NCrS1c, obtained from Japan SLC Co., Ltd.) (n=5) were tape stripped three times (treated ears). The right ears of the same mice were not tape stripped (untreated control ears).

[0030] <Measurement of transepidermal water loss> The transepidermal water loss of both ears of each mouse was measured using a Tevameter (Courage and Khazaka) before tape stripping on day 0 and after tape stripping on day 2 of the stratum corneum barrier damage test. The measurement results are shown in Figure 1. As a result, in the tape-stripped treated ear, the transepidermal water loss on day 2 from the start of the experiment was significantly increased compared to the untreated ear. This result indicates that when the stratum corneum barrier is damaged by tape stripping, the skin's moisture retention ability is reduced.

[0031] Dye penetration test: Mice were subjected to a dye penetration test on the second day after the start of the stratum corneum barrier damage test. Specifically, each mouse was euthanized, fixed with methanol, and then immersed in a toluidine blue solution for 25 minutes. The mice were then washed in phosphate-buffered saline (PBS). The results showed that the toluidine blue dye had penetrated into the tape-stripped treated ears of all mice, but little penetration of the dye was observed in the untreated ears.

[0032] After washing each mouse's ear with PBS, ear tissue was collected using a 6 mm diameter biopsy trephine (KAI MEDICAL). The collected ear tissue was immersed in formamide and incubated overnight at 63°C to elute the dye. The dye-eluted solution was then collected, and the absorbance at 620 nm was measured to calculate the amount of dye. The amount of dye was calculated based on a standard curve obtained from standard solutions containing known concentrations of dye. Figure 2 shows the results of measuring the amount of dye in each ear. As a result, the amount of dye was significantly higher in the tape-stripped ear compared to the untreated ear (Figure 2).

[0033] These results confirmed that when the stratum corneum barrier function is impaired by tape stripping, the skin's ability to retain moisture decreases and foreign substances become more easily able to penetrate through the skin.

[0034] <GLUT1 Expression Measurement> Mice were tape-stripped on the first day (day 0), 2, 4, and 6 of the experiment, and ear tissue was harvested on day 7. The harvested ear tissue was formalin-fixed and then paraffin-embedded. The tissue sections were stained with hematoxylin and eosin (HE) and immunostained using an anti-GLUT1 antibody (EPR3915, Abcam). Images of the stained sections are shown in Figure 3. As shown in Figure 3, the stratum corneum of the tape-stripped ears was stained for GLUT1, whereas GLUT1 staining was only observed in a small area of ​​the untreated ears. This indicates that disruption of the stratum corneum barrier significantly increased GLUT1 expression in epidermal keratinocytes (Figure 3, arrow). These results suggest that GLUT1-mediated regulation of glycolysis is involved in maintaining stratum corneum barrier function.

[0035] Example 1 The effect of glycolytic inhibitors on stratum corneum barrier dysfunction was evaluated using a stratum corneum barrier impairment test similar to that in Reference Example 1. Three glycolytic inhibitors were used: WZB-117 (Sigma-Aldrich), a pan-glucose transporter inhibitor; BAY-876 (Sigma-Aldrich), a GLUT1-specific inhibitor; and 2-deoxy-D-glucose (2-DG) (Sigma-Aldrich), a glucose-competitive inhibitor. WZB-117 was dissolved in acetone, BAY-876 was dissolved in dimethyl sulfoxide-containing methanol (99.9% methanol, 0.1% dimethyl sulfoxide), and 2-DG was dissolved in a mixed solvent of PBS and acetone (50% PBS, 50% acetone).

[0036] On the day before the first tape stripping (day -1 of the experiment), the day of the first tape stripping (day 0 of the experiment), and the day of the second tape stripping (day 2 of the experiment), each mouse (strain name: C57BL / 6NCrSlc, obtained from Japan SLC, Inc.) (n=5-6) received a glycolytic inhibitor application to the left ear (treated ear) and an equal amount of solvent application to the right ear (untreated ear). Each glycolytic inhibitor was applied at a dose of 20 μg / ear for WZB-117, 0.2 μg / ear for BAY-876, and 1 mg / ear for 2-DG. On days 0 and 2 of the experiment, tape stripping was performed 1 hour after application of the glycolytic inhibitor.

[0037] The mice after the second tape stripping were subjected to a transepidermal water loss measurement and a dye permeation test in the same manner as in Reference Example 1. The results of the transepidermal water loss measurement test are shown in FIG. 4, and the results of the dye permeation test are shown in FIG. 5. In FIGS. 4 and 5, (A) shows the results for mice to which WZB-117 was applied, (B) shows the results for mice to which 2-DG was applied, and (C) shows the results for mice to which BAY-876 was applied. In FIG. 4, "Before treatment" shows the measurement results before tape stripping on the day of the first tape stripping, and "After treatment" shows the measurement results after the second tape stripping.

[0038] As shown in Figure 4, in the right ear where the solvent was applied, the transepidermal water loss after tape stripping was 60 g / m ２ / h, whereas in the left ears treated with WZB-117, 2-DG, or BAY-876, the transepidermal water loss was reduced to about 1 / 2 to 1 / 3 of that in the untreated ear, indicating a significant suppression of the increase in transepidermal water loss. Furthermore, when the stratum corneum barrier function was evaluated by a dye permeation test, the amount of dye permeated transdermally in the left ear (treated ear) treated with a glycolytic inhibitor was reduced to about 1 / 2 to 1 / 3 of that in the right ear (untreated ear) treated with a solvent, regardless of the three glycolytic inhibitors used. The treated ear with a glycolytic inhibitor was significantly lower than the untreated ear (Figure 5). These results demonstrate that tape-stripping-induced stratum corneum barrier disruption (increased transepidermal water loss and dye permeability) was significantly suppressed in the treatment with any of the various glycolytic inhibitors, demonstrating that glycolytic inhibitors suppress stratum corneum barrier dysfunction.

[0039] [Example 2] Animal experiments have shown that the glycolytic inhibitor used in Example 1 can suppress the glycolytic pathway in the skin not only by topical administration (application) but also by systemic administration (Non-Patent Document 2). Therefore, the glycolytic inhibitor was administered systemically to examine its inhibitory effect on stratum corneum barrier dysfunction.

[0040] Specifically, mice (strain name: C57BL / 6NCrSlc, obtained from Japan SLC) (n=7) were intraperitoneally administered 2-DG (12.5 mg or 500 mg / kg body weight) for four consecutive days, starting from the day before the first tape stripping of the stratum corneum barrier damage test (day -1 from the start of the experiment) (treated group). Control mice (n=7) were intraperitoneally administered an equal volume of solvent (PBS) instead of 2-DG (untreated group). On days 0 and 2 from the start of the experiment, tape stripping was performed 1 hour after 2-DG administration, as in Reference Example 1.

[0041] On day 4 of the experiment, the transepidermal water loss of each mouse's ear was measured in the same manner as in Reference Example 1. The measurement results are shown in Figure 6. As shown in Figure 6, the transepidermal water loss was significantly lower in the 2-DG-treated group compared to the untreated group. These results confirmed that glycolytic inhibitors, even when administered systemically, are effective in suppressing stratum corneum barrier dysfunction.

[0042] Example 3 The effect of a glycolytic inhibitor on stratum corneum barrier dysfunction was evaluated in the same manner as in Example 1. WZB-117 (Sigma-Aldrich) was used as the glycolytic inhibitor. WZB-117 was used as a solution dissolved in acetone.

[0043] On the day before the first tape stripping (day -1), the day of the first tape stripping (day 0), the day of the second tape stripping (day 2), and the day of the third tape stripping (day 4), mice (strain name: C57BL / 6NCrSlc, obtained from Japan SLC, Inc.) (n = 5-10) received a treatment with WZB-117 on the left ear (treated ear) and an equal volume of solvent on the right ear (untreated ear). WZB-117 was applied at a dose of 20 μg (54.3 nmol / 20 mL) per ear. On days 0, 2, and 4, tape stripping was performed 1 hour after WZB-117 application.

[0044] Ear tissue was collected from mice before the first tape-stripping (day -1 from the start of the experiment) and after the third tape-stripping (day 5 from the start of the experiment) and examined for histological changes. Specifically, the collected ear tissue was fixed in formalin and then paraffin-embedded tissue sections were prepared. The obtained tissue sections were stained with HE. The results are shown in Figure 7. In the group that did not receive the glycolytic inhibitor WZB-117 (solvent-applied group), the skin (epidermis and dermis) thickened after tape-stripping (top panel of Figure 7). On the other hand, in the group that received the glycolytic inhibitor WZB-117, the morphology of the skin tissue after tape-stripping remained almost unchanged from the skin tissue before tape-stripping (bottom panel of Figure 7).

[0045] Additionally, ear skin tissue was collected from mice in the glycolysis inhibitor WZB-117 group and the solvent (acetone) application group on days 2, 3, and 5 after the start of the stratum corneum barrier impairment test, and time-dependent changes in gene expression of filaggrin (Flg) and Tjp3 (ZO-3), factors related to stratum corneum barrier formation, were examined. Filaggrin is a major protein that constitutes the cells of the stratum corneum, the outermost layer of the skin, and has the function of aggregating and bundling keratin fibers in stratum corneum cells, strengthening the stratum corneum barrier and retaining moisture as a natural moisturizing factor. Tjp3 is a protein that constitutes tight junctions (a skin barrier structure that plays an essential role in moisture retention).

[0046] Specifically, total RNA was extracted from the ear skin tissue, and quantitative PCR was performed using the cDNA obtained after reverse transcription as a template. The relative expression level of each gene was measured using β-actin as an endogenous control, and 2 －ΔＣｔ Based on the method, it was calculated using the following formula: In the formula, "Ct(T)" is the Ct value of the gene to be measured, and "Ct(C)" is the Ct value of the β-actin gene.

[0047]

[0048] The results of measuring the relative expression levels of each gene are shown in Figure 8. As shown in Figure 8, the WZB-117-application group had higher mRNA levels of both filaggrin and Tjp3 than the solvent-application group. These results suggest that glycolysis inhibitors enhance gene expression of stratum corneum barrier formation-related factors, and that this enhanced expression of stratum corneum barrier formation-related factors is one of the mechanisms by which glycolysis inhibitors repair stratum corneum barrier damage.

[0049] To examine the effects of glycolytic inhibitor application on normal skin tissue, mice (strain: C57BL / 6NCrSlc, obtained from Japan SLC) were treated without tape stripping. On days 0, 1, 3, 5, and 7, WZB-117 was applied to the left ear (treated ear) and an equal volume of solvent (acetone) was applied to the right ear (untreated ear). WZB-117 was applied at a dose of 20 μg (54.3 nmol / 20 mL) per ear. Ear tissue was collected from mice on day 8 of the experiment and from untreated control mice. The collected ear tissue was fixed in formalin and then paraffin-embedded tissue sections were prepared. The obtained tissue sections were stained with HE. The results are shown in Figure 9. As shown in Figure 9, the tissues of the WZB-117-applied group were similar to those of the untreated group and the solvent-applied group, and no notable changes were observed macroscopically or histologically when WZB-117 was applied to normal skin.

[0050] Example 4: The effect of a glycolytic inhibitor on the gene expression of factors related to stratum corneum barrier formation was investigated. WZB-117 (Sigma-Aldrich) was used as the glycolytic inhibitor. WZB-117 was used as a solution dissolved in acetone.

[0051] Filaggrin, involucrin (IVL), and loricrin (LOR) were investigated as factors related to stratum corneum barrier formation. Involucrin is one of the major proteins that make up the cornified envelope (lining structure) of stratum corneum cells. Loricrin is a hydrophobic protein rich in glycine, serine, cysteine, etc., and is one of the major proteins that make up the cornified envelope. The proteins that make up the cornified envelope are produced by gene expression in association with the differentiation of epidermal keratinocytes, and their molecular cross-linking forms an extremely strong structure, contributing to the physical and chemical toughness of the stratum corneum together with keratin fibers. For this reason, both involucrin and loricrin are considered indicators of the differentiation of epidermal keratinocytes into keratinocytes.

[0052] Cultured epidermal keratinocytes (Kurabo Industries, Ltd.) were cultured overnight in serum-free liquid growth medium (Humedia-KG2, Kurabo Industries, Ltd.), then the medium was replaced with a basal medium containing only antibiotics (Humedia-KB2, Kurabo Industries, Ltd.) and cultured overnight to starve the cells. WZB-117 was then added to the medium and the cells were cultured for 46 hours.

[0053] After culturing, the cells were collected and the gene expression levels of filaggrin, involucrin, and loricrin were examined. Specifically, total RNA was extracted from the cells after culturing, and quantitative PCR was performed using the cDNA obtained after reverse transcription as a template. The relative expression levels of each gene were measured in the same manner as above, using β-actin as an endogenous control.

[0054] The results of measuring the relative expression levels of each gene are shown in Figure 10. As shown in Figure 10, gene expression of filaggrin, involucrin, and loricrin was enhanced in a manner dependent on the WZB-117 concentration in the medium. This enhanced expression of factors related to stratum corneum barrier formation is thought to be one of the mechanisms by which glycolysis inhibitors repair stratum corneum barrier damage.

Claims

1. A stratum corneum barrier function improver that contains a glycolytic inhibitor as an active ingredient.

2. The stratum corneum barrier function improver according to claim 1, wherein the glycolysis inhibitor is one or more selected from the group consisting of glucose transporter inhibitors and glucose competitive inhibitors.

3. The stratum corneum barrier function improver according to claim 2, wherein the glucose transporter inhibitor is a GLUT1 inhibitor.

4. The stratum corneum barrier function improving agent according to claim 1, having one or more effects selected from the group consisting of an effect of inhibiting a decrease in the skin's moisture retention ability and an effect of inhibiting an increase in skin permeability.

5. A pharmaceutical composition comprising the stratum corneum barrier function improving agent according to any one of claims 1 to 4.

6. The pharmaceutical composition according to claim 5, which is used for the prevention or treatment of skin diseases.

7. The pharmaceutical composition according to claim 5, which is administered topically or orally.

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