WO2025127077A1 - Stratum corneum barrier function improving agent and pharmaceutical composition - Google Patents

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

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

The skin has an important barrier function (skin barrier function) that prevents foreign substances from entering the body from the outside world and prevents water from evaporating from within 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 the onset of allergies, so maintaining the skin barrier function is extremely important for maintaining homeostasis and health.

The "stratum corneum barrier" is a structure that plays an important role in the skin barrier function. The stratum corneum barrier is composed of three factors: keratinocytes located in the outermost layer of the skin, lipids between keratinocytes, sebum on the surface of the stratum corneum, and natural moisturizing factors in the stratum corneum. Various moisturizers have been developed to maintain the stratum corneum barrier function, but they basically exert their moisturizing function by supplementing the above three constituent factors. For example, petrolatum exerts its moisturizing ability by supplementing sebum on the surface of the stratum corneum, ceramide-containing ointments supplement intercellular lipids, and urea ointments supplement natural moisturizing factors. In addition, hyaluronic acid and heparin-like substances are also widely used as moisturizers, and they exert their moisturizing ability through their water adsorption ability. Thus, conventional moisturizers are made with the idea of adding moisturizing-related substances externally. Although these moisturizers are important for maintaining the stratum corneum barrier, they have the problem that they merely supplement artificial substances externally and do not strengthen the physiological stratum corneum barrier itself.

On the other hand, in recent years, it has become clear that intracellular energy metabolism plays a major role in controlling the functions of various cells. It has been found that glucose transporters (molecules that take up sugar into cells), in particular GLUT1 (glucose transporter 1), play a major role in the glycolysis system of epidermal keratinocytes (Non-Patent Document 1).

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.

The present invention aims to provide an improving agent for improving the stratum corneum barrier function without externally supplementing moisturizing substances, and a pharmaceutical composition containing said improving agent.

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

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 agent for improving stratum corneum barrier function according to [1] above, 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 improving agent according to [2], wherein the glucose transporter inhibitor is a GLUT1 inhibitor.
[4] The agent for improving stratum corneum barrier function according to any one of the above [1] to [3], which has one or more 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 improving agent according to any one of [1] to [4] above.
[6] The pharmaceutical composition according to the above [5], which is used for the prevention or treatment of a skin disease.
[7] The pharmaceutical composition according to [5] or [6] above, which is administered topically or orally.

The stratum corneum barrier function improver of the present invention can improve the stratum corneum barrier function without externally supplementing moisturizing substances. Therefore, the stratum corneum barrier function improver of 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.

FIG. 1 shows 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 from the start of the stratum corneum barrier damage test in Reference Example 1. FIG. 1 shows the results of measuring the amount of dye infiltrated into the ears of each mouse after tape stripping on the second day of the stratum corneum barrier damage test in Reference Example 1. 1 shows HE stained images and anti-GLUT1 antibody stained images of ear tissue sections of each mouse after tape stripping on the 7th day of the stratum corneum barrier damage test in Reference Example 1. FIG. 1 shows the results of measuring transepidermal water loss in mouse ears to which a glycolytic inhibitor (WZB-117, 2-DG or BAY-876) was applied before tape stripping on day 0 (before treatment) and after tape stripping on day 2 (after treatment) in a stratum corneum barrier damage test in Example 1. FIG. 1 shows the results of measuring the amount of dye that had penetrated into the ears of mice after tape stripping on the second day of a stratum corneum barrier damage test in Example 1, in which a glycolytic inhibitor (WZB-117, 2-DG or BAY-876) was applied to the ears of the mice. FIG. 1 shows the results of measuring transepidermal water loss on the fourth day after the start of a stratum corneum barrier impairment test in the ears of mice administered a glycolysis inhibitor (2-DG) intraperitoneally in Example 2. In Example 3, HE stained images of mouse ear tissue sections from a solvent (acetone) application group and a glycolytic inhibitor (WZB-117) application group before tape stripping (day -1 from the start of the experiment) and after tape stripping (day 5 from the start of the experiment) are shown. FIG. 13 shows the results of measuring the amounts of filaggrin (Flg) and Tjp3 (ZO-3) mRNA in the auricular skin tissue on days 1, 2, 3, and 5 from the start of the experiment in the solvent (acetone) application group and the glycolytic inhibitor (WZB-117) application group in Example 3. In Example 3, HE stained images of ear tissue sections were obtained by applying a solvent (acetone) or a glycolytic inhibitor (WZB-117) to normal skin tissue that had not been subjected to tape stripping. FIG. 13 shows the results of measuring the amounts of mRNA of filaggrin (Flg), involucrin (IVL) and loricrin (LOR) in epidermal keratinocytes cultured in a WZB-117-containing medium in Example 4.

<Stratum corneum barrier function improver>
The stratum corneum barrier function improving agent according to this embodiment has a glycolysis inhibitor as an active ingredient. As shown in the following examples, the stratum corneum is physically peeled off, and the surface of the skin with impaired stratum corneum barrier function is treated with a glycolysis inhibitor, thereby recovering the stratum corneum barrier function. That is, the glycolysis inhibitor has at least one selected from the group consisting of an inhibitory effect on the decrease in skin moisture retention ability and an inhibitory effect on the increase in skin permeability. The mechanism of action by which glycolysis inhibitors improve stratum corneum barrier function is not yet clear, but it is speculated that the stratum corneum barrier can be maintained endogenously by controlling the function of epidermal keratinocytes themselves through glycolysis. In addition, as shown in the following examples, in damaged skin tissue, glycolysis inhibitors can enhance gene expression of stratum corneum barrier formation-related factors, which is speculated to be one of the mechanisms by which glycolysis inhibitors repair stratum corneum barrier damage.

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 or enhances the function of the biomolecule, thereby inhibiting ATP production by the glycolytic pathway." 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.

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 the uptake of glucose into epidermal keratinocytes or suppressing the metabolism of glucose itself within epidermal keratinocytes, greater effects can be expected.

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. As the active ingredient of the stratum corneum barrier function improver according to this embodiment, a GLUT1 inhibitor (an inhibitor that exerts an inhibitory effect on GLUT1) is preferred, and a GLUT1-specific inhibitor is particularly preferred.

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. As a GLUT1 inhibitor consisting of a low molecular weight compound, various molecules that are known to have an inhibitory effect on GLUT1, such as WZB-117 (CAS No.: 1223397-11-2; inhibitor for GLUT1, GLUT3, and GLUT4), Ritonavir (CAS No.: 155213-67-5; inhibitor for GLUT1 and GLUT4), STF-31 (CAS No.: 724741-75-7; inhibitor for GLUT1), and BAY-876 (CAS No.: 1799753-84-6; inhibitor for GLUT1), can be used.

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. As the non-metabolizable glucose analogue, for example, 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) can be used.

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

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

<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 following examples, the stratum corneum barrier function improving agent according to this embodiment can improve the function of the impaired stratum corneum barrier, suppress the decrease in the moisture retention ability of the skin, and suppress the increase in skin permeability.Therefore, the stratum corneum barrier function improving agent according to this embodiment is useful as an active ingredient of the pharmaceutical composition.

The pharmaceutical composition according to this embodiment is useful as a pharmaceutical composition used for the prevention or treatment of diseases in which the pathology can be expected to improve by maintaining or improving the barrier function of the stratum corneum, and is particularly expected to be effective in suppressing inflammation, dryness, invasion of foreign matter, allergic reactions, skin aging, and the onset of infectious diseases caused by impaired barrier function of the stratum corneum. In other words, 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 inflammation or dryness of the skin, 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.

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

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

The pharmaceutical composition according to the present invention may be a pharmaceutical composition for topical administration or may be a pharmaceutical composition for oral administration. The route of administration of the pharmaceutical composition according to the present invention is not particularly limited and may be appropriately selected from various routes of administration such as oral administration, intravenous administration, transdermal administration, enteral administration, and nasal administration. The pharmaceutical composition according to the present invention may be formulated into oral solid preparations such as powders, granules, capsules, tablets, chewable preparations, 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 a conventional method, taking into consideration the intended route of administration, etc.

Other components contained in the pharmaceutical composition of the present invention include, for example, pharmacologically acceptable additives such as excipients, binders, lubricants, disintegrants, flow agents, solvents, solubilizers, buffers, suspending agents, emulsifiers, isotonicity agents, stabilizers, preservatives, antioxidants, flavoring agents, and colorants. The pharmaceutical composition may also contain other active ingredients in addition to the stratum corneum barrier function improving agent.

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. Binders include pregelatinized starch, gelatin, gum arabic, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, crystalline cellulose, D-mannitol, trehalose, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, and polyvinyl alcohol. Lubricants include stearic acid, calcium stearate, talc, sucrose fatty acid esters, and polyethylene glycol. Disintegrants include crospovidone (crosslinked polyvinylpyrrolidone), low-substituted hydroxypropylcellulose, starch, alginic acid, and sodium alginate. Fluidizers include silicic acid, anhydrous silicic acid, aluminum silicate, calcium silicate, magnesium aluminometasilicate compound, aluminum oxide, aluminum hydroxide, magnesium oxide, and magnesium hydroxide. Examples of the solvent include purified water and physiological saline. Examples of the solubilizing agent include dextran, polyvinylpyrrolidone, sodium benzoate, ethylenediamine, salicylic acid amide, nicotinic acid amide, polyoxyethylene hydrogenated castor oil derivatives, etc. Examples of the buffer include sodium citrate hydrate, sodium acetate hydrate, sodium hydrogen carbonate, trometamol, boric acid, borax, sodium hydrogen phosphate hydrate, sodium dihydrogen phosphate, etc. Examples of the suspending agent or emulsifying agent include sodium lauryl sulfate, gum arabic, gelatin, lecithin, glycerin monostearate, polyvinyl alcohol, polyvinylpyrrolidone, celluloses such as sodium carboxymethylcellulose, polysorbates, polyoxyethylene hydrogenated castor oil, etc. Examples of the isotonicity agent include sugars such as lactose, glucose, D-mannitol, sodium chloride, potassium chloride, glycerin, propylene glycol, polyethylene glycol, urea, etc. Stabilizers include polyethylene glycol, sodium dextran sulfate, sodium sulfite, etc. Preservatives include paraoxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenethyl alcohol, chlorocresol, dehydroacetic acid, sorbic acid, etc. Antioxidants include sulfites, ascorbic acid, etc. Flavoring agents include sweeteners and fragrances commonly used in the pharmaceutical and food fields. Coloring agents include coloring agents commonly used in the pharmaceutical and food fields.

The pharmaceutical composition of the present invention is administered in an effective amount to an animal having or suspected of having a disorder in the stratum corneum barrier function. The pharmaceutical composition is preferably administered to a mammal, more preferably to a human or a livestock or laboratory animal such as a mouse, rat, rabbit, guinea pig, hamster, monkey, sheep, horse, cow, pig, donkey, dog, or cat, and even more preferably to a human.

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.

[Reference example 1]
The stratum corneum barrier was damaged by tape stripping, and the functional changes were evaluated. Tape stripping is a method in which the stratum corneum is physically peeled off by removing adhesive tape applied to the skin, damaging the barrier function of the stratum corneum. Tape stripping is a widely used method that induces functional and histological changes similar to those observed when the skin is scratched.

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

<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 treated ears that were subjected to tape stripping, the transepidermal water loss on day 2 from the start of the experiment was significantly increased compared to the untreated ears. This result means that when the stratum corneum barrier is damaged by tape stripping, the moisture retention ability of the skin is reduced.

<Dye permeation test>
The dye permeation test was performed on mice 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 mouse was then washed in PBS (phosphate buffered saline). As a result, in all mice, the toluidine blue dye had permeated into the treated ears that had been tape stripped, but little dye permeation was observed in the untreated ears.

Ear tissue was collected from the ears of each mouse after washing with PBS 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 immersion solution in which the dye had been eluted was then collected, and the absorbance at a wavelength of 620 nm was measured to calculate the amount of dye. The amount of dye was calculated based on a standard curve determined from a standard solution containing dye of known concentration. The measurement results of the amount of dye in each ear are shown in Figure 2. As a result, the amount of pigment was significantly higher in the treated ears that had been subjected to tape stripping compared to the untreated ears (Figure 2).

These results confirm that impairing the stratum corneum barrier function by tape stripping reduces the skin's ability to retain moisture and makes it easier for foreign bodies to penetrate through the skin.

<Measurement of GLUT1 expression>
Mice were tape-stripped on the first day (day 0), 2, 4, and 6 of the experiment, and ear tissue was collected on the 7th day. The collected ear tissue was fixed in formalin and then paraffin-embedded tissue sections were prepared. The obtained tissue sections were stained with hematoxylin and eosin (HE) and immunostained using anti-GLUT1 antibody (EPR3915, Abcam). The stained images are shown in Figure 3. As shown in Figure 3, the stratum corneum of the treated ears that had been tape-stripped was stained with GLUT1, whereas GLUT1 staining was observed only in a very small area of the untreated ears. In other words, it was confirmed that when the stratum corneum barrier was destroyed, the expression of GLUT1 in epidermal keratinocytes was significantly enhanced (Figure 3, arrow). These results suggest that the control of glycolysis via GLUT1 is involved in the maintenance of the stratum corneum barrier function.

[Example 1]
The effect of glycolysis inhibitors on stratum corneum barrier dysfunction was evaluated using the same stratum corneum barrier damage test as in Reference Example 1. As glycolysis inhibitors, three types 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 dimethylsulfoxide-containing methanol (99.9% methanol, 0.1% dimethylsulfoxide), and 2-DG was dissolved in a mixed solvent of PBS and acetone (50% PBS, 50% acetone).

　On the day before the first tape stripping in the stratum corneum barrier damage test (day -1 from the start of the experiment), the day of the first tape stripping (day 0 from the start of the experiment), and the day of the second tape stripping (day 2 from the start of the experiment), each mouse (strain name: C57BL/6NCrSlc, obtained from Japan SLC Co., Ltd.) (n=5-6) had a glycolytic inhibitor applied to the left ear (treated ear) and an equal amount of solvent applied 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 from the start of the experiment, tape stripping was performed 1 hour after application of the glycolytic inhibitor.

　Transepidermal water loss was measured and a dye permeation test was performed on the mice after the second tape stripping in the same manner as in Reference Example 1. The results of the transepidermal water loss measurement test are shown in Figure 4, and the results of the dye permeation test are shown in Figure 5. In Figures 4 and 5, (A) shows the results of mice applied with WZB-117, (B) shows the results of mice applied with 2-DG, and (C) shows the results of mice applied with BAY-876. In Figure 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.

As shown in Figure 4, in the right ear to which the solvent was applied, the transepidermal water loss increased to about 60 g/ ^m2 /h after tape stripping, whereas in the left ear to which WZB-117, 2-DG or BAY-876 was applied, the transepidermal water loss was suppressed to about 1/2 to 1/3 of that in the untreated ear, and the increase in the transepidermal water loss was significantly suppressed. In addition, when the stratum corneum barrier function was evaluated by a dye permeation test, the amount of dye that permeated transdermally in the left ear to which the glycolytic inhibitor was applied (treated ear) was suppressed to about 1/2 to 1/3 of that in the right ear to which the solvent was applied (untreated ear), regardless of which of the three glycolytic inhibitors was used, and the amount in the treated ear to which the glycolytic inhibitor was applied was significantly lower than that in the untreated ear (Figure 5). As these results show, the destruction of the stratum corneum barrier (increased transepidermal water loss and pigment permeability) induced by tape stripping was significantly suppressed by the application of any of the various glycolysis inhibitors, demonstrating that glycolysis inhibitors suppress stratum corneum barrier dysfunction.

[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.

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 4 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). Control mice (n=7) were intraperitoneally administered an equal amount 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 administration of 2-DG in the same manner as in Reference Example 1.

On the fourth day after the start of the experiment, the amount of transepidermal water loss in the ears of each mouse was measured in the same manner as in Reference Example 1. The results are shown in Figure 6. As shown in Figure 6, the amount of transepidermal water loss was significantly lower in the treatment group administered 2-DG compared to the untreated group. These results confirmed that glycolysis inhibitors, even when administered systemically, are effective in suppressing stratum corneum barrier dysfunction.

[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.

　On the day before the first tape stripping in the stratum corneum barrier damage test (day -1 from the start of the experiment), the day of the first tape stripping (day 0 from the start of the experiment), the day of the second tape stripping (day 2 from the start of the experiment), and the day of the third tape stripping (day 4 from the start of the experiment), each mouse (strain name: C57BL/6NCrSlc, obtained from Japan SLC Co., Ltd.) (n=5-10) had WZB-117 applied to the left ear (treated ear) and an equal amount of solvent applied to 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 from the start of the experiment, tape stripping was performed 1 hour after WZB-117 application.

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 histological changes were examined. 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 was not applied with the glycolytic inhibitor WZB-117 (solvent application group), the skin (epidermis, dermis) was thickened after tape stripping (top of Figure 7). On the other hand, in the group that was applied with the glycolytic inhibitor WZB-117, the morphology of the skin tissue after tape stripping was almost the same as that of the skin tissue before tape stripping (bottom of Figure 7).

In addition, ear skin tissue was collected from mice in the glycolysis inhibitor WZB-117 group and the solvent (acetone) application group on the second, third, and fifth days after the start of the stratum corneum barrier damage test, and the time-dependent changes in gene expression of filaggrin (Flg) and Tjp3 (ZO-3), which are factors related to stratum corneum barrier formation, were examined. Filaggrin is the main protein that makes up 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 constituent protein of tight junctions (a barrier structure in the skin that plays an essential role in retaining moisture).

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 calculated using the following formula based on the 2 ^-ΔCt method, with β-actin as an endogenous control. 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.

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 increase gene expression of stratum corneum barrier formation-related factors, and that this increased expression of stratum corneum barrier formation-related factors is one of the mechanisms by which glycolysis inhibitors repair damage to the stratum corneum.

To examine the effect of applying a glycolytic inhibitor to normal skin tissue, mice (strain: C57BL/6NCrSlc, obtained from Japan SLC) were given WZB-117 on the left ear (treated ear) and an equal volume of the solvent (acetone) on the right ear (untreated ear) on days 0, 1, 3, 5, and 7 of the experiment without tape stripping. 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 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 in the WZB-117-applied group were similar to those in the untreated and solvent-applied groups, and no notable changes were observed macroscopically or histologically when WZB-117 was applied to normal skin.

[Example 4]
The effect of glycolysis inhibitors on gene expression of stratum corneum barrier formation-related factors was examined. WZB-117 (Sigma-Aldrich) was used as a glycolysis inhibitor. WZB-117 was dissolved in acetone and used as a solution.

Filaggrin, involucrin (IVL), and loricrin (LOR) were investigated as factors related to the formation of the stratum corneum barrier. Involucrin is one of the main proteins that make up the cornified envelope (backing structure) of keratinocytes. Loricrin is a hydrophobic protein that is rich in glycine, serine, cysteine, etc., and is one of the main proteins that make up the cornified envelope. The proteins that make up the cornified envelope are produced by genetic expression in association with the differentiation of epidermal keratinocytes, and become an extremely strong structure due to cross-linking between these molecules, contributing to the physical and chemical toughness of the stratum corneum together with keratin fibers. For this reason, both involucrin and loricrin are considered to be indicators of the differentiation of epidermal keratinocytes into keratinocytes.

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.

After culture, the cells were harvested and the gene expression levels of filaggrin, involucrin and loricrin were examined. Specifically, total RNA was extracted from the cells after culture, 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.

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 increased in a manner dependent on the concentration of WZB-117 in the medium. This increased expression of factors related to stratum corneum barrier formation was thought to be one of the mechanisms by which glycolysis inhibitors repair damage to the stratum corneum barrier.

Claims (7)

An agent that improves the barrier function of the stratum corneum, containing a glycolytic inhibitor as an active ingredient. 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. The stratum corneum barrier function improver according to claim 2, wherein the glucose transporter inhibitor is a GLUT1 inhibitor. The stratum corneum barrier function improving agent according to claim 1, which has 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. A pharmaceutical composition comprising the stratum corneum barrier function improving agent according to any one of claims 1 to 4. The pharmaceutical composition according to claim 5, which is used for the prevention or treatment of skin diseases. The pharmaceutical composition according to claim 5, which is administered topically or orally.

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