Epidermal basal niche protecting agent, pharmaceutical composition for protecting epidermal basal niche, and cosmetic for protecting epidermal basal niche

Abstract

To provide epidermal basal niche protectants, epidermal basal niche protective pharmaceutical compositions, and epidermal basal niche protective cosmetics, containing a laminin fragment.SOLUTION: The present invention contains as an active ingredient a laminin fragment containing an integrin binding site, or a derivative thereof. As the laminin fragment, for example the E8 fragment of laminin 511 can be preferably used. The E8 fragment has skin permeability and protects an epidermal basal niche, increases the number of epidermal stem cells even during skin damage caused by UV irradiation, can maintain the undifferentiated state of epidermal cells, and induces metabolic maintenance and recovery of damaged skin.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an agent for protecting an epidermal basal niche, a pharmaceutical composition for protecting an epidermal basal niche, and a cosmetic for protecting an epidermal basal niche, each of which contains, as an active ingredient, a laminin fragment or the like that contains an integrin binding site. [Background technology]

[0002] Human skin is composed of the epidermis and dermis, and the epidermal basement membrane exists at the junction between the epidermis and dermis. The epidermal basement membrane is a special assembly of connective tissue fibers and matrix, and is a structure in which extracellular matrix proteins are arranged in a sheet shape. The epidermis is a multi-layered structure in which keratinocytes of different morphologies are arranged in layers depending on the stage of maturation, and is called the basal layer, spinous layer, granular layer, and stratum corneum from the deepest part. Epidermal stem cells are distributed in the basal layer in contact with the epidermal basement membrane, mixed with epidermal basal cells. In the body, the basement membrane, its neighboring cells, and the extracellular matrix near the basement membrane interact with each other to form a microenvironment around the cells that is necessary to maintain the properties of epidermal stem cells (called the epidermal basal niche). The epidermal basement membrane influences the mechanical strength and the expression and maintenance of cell differentiation characteristics. For example, the basal cells can maintain the trait of proliferating as basal cells by the signal that they are bound to the epidermal basement membrane, and the detachment of the basal cells from the epidermal basement membrane is one of the signals that induce differentiation of the basal cells. The epidermal basement membrane and the connective tissue directly below undergo structural changes with age. When the skin is damaged by ultraviolet radiation or the like, not only does photoaging accelerate, but the daily turnover of the epidermis is disturbed, causing rough skin and the like. Thus, it is essential for the expression of normal skin function that the epidermal basal cells are regularly bound to the epidermal basement membrane, and the cellular environment near the basement membrane (epidermal basal niche), which consists of the interaction between the cells and the microenvironment surrounding the cells, is stably and sufficiently maintained.

[0003] Laminin is a major component of the epidermal basement membrane extracellular matrix. Non-Patent Document 1 reports that laminin has an activity of promoting the formation of epidermal basement membrane, and that the addition of purified laminin to a culture medium promotes the formation of epidermal basement membrane, and that the epidermal basement membrane structure with high electron density observed under an electron microscope immediately below the epidermal basal cells is observed more frequently (Non-Patent Document 1).

[0004] Laminin 511 binds to integrin α6β1 on the cell surface and is known to be closely involved in the survival of undifferentiated cells as a component of the epidermal basement membrane during development. It has also been suggested that in the skin, laminin 511 may be involved in the homeostasis of epidermal stem cells present at the boundary between the epidermis and dermis. It has been reported that when epidermal basal cells were visualized using an antibody against MCSP (Melanoma-associated chondroitin sulfate proteoglycan), an epidermal basal stem cell marker, the number of MCSP-positive epidermal basal stem cells correlates with the expression level of laminin 511 present near the epidermal basement membrane (Patent Document 1). Figure 4 in Patent Document 1 shows that laminin 511 is reduced by human skin tissue culture, but is enhanced by the addition of an MMP inhibitor (N-hydroxy-2(R)-[[(4-methoxy-phenyl)sulfonyl](3-picolyl)amino]-3-methylbutanamide hydrochloride) and a heparanase inhibitor. By culturing epidermal cells in a culture medium containing a candidate drug, measuring the expression level of laminin 511 in the epidermal cells, and comparing it with the expression level of laminin 511 in a control, it is possible to determine whether the candidate drug has an effect of promoting the expression of laminin 511. In Example 6 of Patent Document 1, when a human abdominal skin sample was cultured for five days in a medium containing 1-(2-hydroxyethyl)-2-imidazolidinone (HEI), the amount of laminin 511 and the number of MCSP-positive cells decreased in the control without HEI administration, but the amount of laminin 511 and the number of MCSP-positive cells on the epidermal basement membrane were maintained in the HEI administration system.

[0005] As shown in Figure 10A, laminin is a cross-shaped molecule in which α, β, and γ chains are associated at the coiled-coil region, and since there are several types of each chain, it is named by combining five types of α chains (LAMA1-5), four types of β chains (LAMB1-4), and three types of γ chains (LAMC1-3), and laminin 511 means laminin α5β1γ1. Integrins are the ligands of laminins, and they form a ligand recognition spot on the head side of the extracellular domain that penetrates the membrane. Binding of laminin to this ligand recognition spot causes basal cells to adhere to the epidermal basement membrane, and signals that promote survival and proliferation are transmitted to the cells through binding with integrins.

[0006] The E8 fragment is known as an integrin-binding region of human laminin α5β1γ1. The main receptor by which cells recognize laminin 511 is α6β1 integrin, and for α6β1 integrin to recognize laminin 511, the globular domain in the laminin α5 chain and glutamic acid in the carboxyl terminal side of the laminin γ1 chain are important sites. Although the E8 fragment is a fragment, it has these important sites, and thus has the ability to bind to α6β1 integrin and transmit various signals to cells, just like the full-length molecule of laminin 511. It has also been reported that the E8 fragment maintains the undifferentiated state of cells and induces proliferation in the culture of ES / iPS cells (Non-Patent Document 2). Applying this fact, there is a culture substrate for culturing human pluripotent stem cells using the E8 fragment (Patent Document 2). According to the culture substrate coated with the E8 fragment, human pluripotent stem cells can be maintained and cultured while retaining their differentiation pluripotency in a culture environment that satisfies feeder-free conditions. The effectiveness of E8 fragments in the culture of iPS cells and ES cells has been reported previously (Non-Patent Document 3).

[0007] Laminin and its fragments are also used to control the differentiation of pluripotent stem cells. For example, there is a method for controlling the differentiation of pluripotent stem cells, characterized by inducing differentiation of pluripotent stem cells in the presence of laminin or its fragments (Patent Document 3). It is said that the ratio of differentiated cells in the resulting cell population can be controlled by changing the type of laminin present during differentiation induction. In the working examples, a human iPS cell line (201B7) is seeded on a culture vessel coated with the E8 fragment of laminin 111, and differentiation is induced by culturing in a differentiation medium and a corneal epithelium maintenance medium. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] International Publication No. 2018 / 074606 [Patent Document 2] JP 2011-78370 A [Patent Document 3] International Publication No. 2018 / 143312 [Non-patent literature]

[0009] [Non-Patent Document 1] Satoshi Amano, "Epidermal basement membrane damage as a sign of early aging and laminin 5* as a key substance in epidermal basement membrane care," J. Soc. Cosmet. Chem. Japan. Vol. 35, No. 1 2001) [Non-Patent Document 2] Taniguchi Y, Ido H, Sanzen N, Hayashi M, Sato-Nishiuchi R, Futaki S, Sekiguchi K.”The C-terminal region of laminin beta chains modulates the integrin binding affinities of laminins” J. Biol Chem. 284, 7820-7831 (2009) [Non-Patent Document 3] Miyazaki T, Futaki S, Suemori H, Taniguchi Y, Yamada M, Kawasaki M, Hayashi M, Kumagai H, Nakatsuji N, Sekiguchi K, Kawase E. “Laminin E8 fragments support efficient adhesion and expansion of dissociated human pluripotent stem cells” Nat Commun. 3, 1236 (2012) Summary of the Invention [Problem to be solved by the invention]

[0010] Damage from external stress, such as rupture or multiplexing of the epidermal basement membrane structure due to exposure, accumulates with aging, and long-term damage to the epidermal basement membrane accumulates structural abnormalities in the dermis, inducing wrinkles and sagging. Patent Document 1 describes that drugs with laminin 511 production promoting effects were screened, and that seaweed extract (Algerex) has the effect of significantly reducing the expression of heparanase gene. However, in addition to seaweed extract, it is desirable to develop an epidermal basal niche protector that can maintain normal skin tissue, especially when damaged by ultraviolet rays, suppress the decrease in the number of epidermal stem cells, maintain the undifferentiation of epidermal cells, and exert skin regeneration functions.

[0011] In view of the above-mentioned current situation, an objective of the present invention is to provide an epidermal basal niche protecting agent, a pharmaceutical composition for protecting the epidermal basal niche, and a cosmetic for protecting the epidermal basal niche that can suppress the decrease in epidermal stem cells and maintain and improve skin function. [Means for solving the problem]

[0012] The inventors discovered that when a laminin fragment containing an integrin binding site is applied to the surface of UV-irradiated skin, it penetrates from the epidermis to the interior, suppressing or increasing the decrease in the number of epidermal stem cells, and that undifferentiated cells maintain their undifferentiated ability even when cultured under aging conditions, thereby ensuring skin elasticity and suppressing skin anisotropy, and thus completed the present invention.

[0013] That is, the present invention provides an agent for protecting the epidermal basal niche, which comprises, as an active ingredient, a laminin fragment containing an integrin binding site, or a derivative thereof.

[0014] The present invention also provides the above-mentioned epidermal basal niche protective agent, characterized in that the laminin fragment is any one of an E8 fragment of laminin-511, an E8 fragment of laminin-521, an E8 fragment of laminin-332, an E8 fragment of laminin-311, and an E8 fragment of laminin-411.

[0015] The present invention also provides the above-mentioned epidermal basal niche protecting agent, wherein the derivative is one in which an alkyl group which may have a substituent having 1 to 10 carbon atoms, an ester or ether which may have a substituent having 1 to 10 carbon atoms, or a fragment of an extracellular matrix component is bound to any one of the α chain, β chain, or γ chain.

[0016] The present invention further provides a pharmaceutical composition for protecting the epidermal basal niche, which contains the above-mentioned epidermal basal niche protecting agent.

[0017] The present invention also provides a cosmetic composition for protecting the epidermal basal niche, which contains the above-mentioned epidermal basal niche protecting agent. Effect of the Invention

[0018] According to the present invention, there is provided an agent for protecting the epidermal basal niche, which comprises, as an active ingredient, a laminin fragment containing an integrin binding site, or a derivative thereof. [Brief description of the drawings]

[0019] [Figure 1] 1 shows the results of Example 1. A is a diagram explaining the experimental procedure of Example 1, and B is a diagram showing the time-dependent surface state of skin tissues that were organ-cultured after adding laminin 511E8 fragment (LN511E8 or LN) to the surface of abdominal skin samples irradiated with UVB. [Diagram 2] 2 shows the results of Example 2. A is a micrograph of a cross section of a culture sample cultured with and without application of laminin 511E8 fragment (LN) to the abdominal skin surface after UVB irradiation. B is a graph showing the results of a statistical analysis of the thickness of the epidermis layer depending on whether or not laminin 511E8 fragment (LN-511E8) was added. [Diagram 3] Figure 3 shows the results of Example 3. A is a cross-sectional view of the abdominal skin surface to which RPE-labeled laminin 511E8 fragment (pre-labeled LN-511E8) was added, followed by organ culture of the abdominal skin, which was then fixed and sliced ​​and stained with an epidermal stem cell marker, and B is a graph showing the results of a statistical analysis of the changes over time in the epidermal stem cell marker with and without the addition of laminin 511E8 fragment (LN). [Figure 4] 4 shows the results of Example 4. The figures show cross-sectional views of samples stained with an epidermal stem cell marker, in which eyelid skin was organ-cultured with and without the addition of RPE-labeled laminin 511 E8 fragment (LN-511E8) to the surface of a UVB-unirradiated eyelid skin sample. [Diagram 5] 5 shows the results of Example 5. A is a photograph of a sample obtained by adding laminin 511E8 fragment (LN511E8) to the surface of abdominal skin, organ culturing the abdominal skin, and then fixing and sectioning the sample, in which the epidermal basement membrane was stained with type IV collagen antibody, and B is a photograph of the same section sample stained with an epidermal stem cell marker. [Figure 6]6 shows the results of Example 6. The figure shows tissue cross-sections observing the epidermal penetration of RPE-labeled laminin-511 (pre-labeled laminin511) (800 kDa) and laminin-511 E8 fragment (pre-labeled laminin511E8) (150 kDa). [Figure 7] 7 shows the results of Example 7. This figure shows the results of a statistical analysis of the number of undifferentiated epidermal cell markers with or without the addition of laminin 511E8 fragment (LN511E8) when undifferentiated HPEKp cells were cultured in a senescence medium. [Figure 8] FIG. 8 shows the results of Example 8 with or without the addition of laminin 511E8 fragment (laminin E8). [Figure 9] FIG. 9 is a diagram showing the results of Example 9, which is a result of a human test, and shows the results with or without addition of laminin 511E8 fragment (LN-511E8) (placebo). [Figure 10] FIG. 1A is a diagram illustrating integrins, which are laminin receptors on the cell surface, and laminins that bind to integrins, and FIG. 1B is a diagram illustrating the integrin-binding site of laminin. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] The first aspect of the present invention is an agent for protecting the epidermal basal niche, which comprises, as an active ingredient, a laminin fragment containing an integrin binding site, or a derivative thereof.

[0021] The human epidermal basement membrane exists in the form of a sheet at the junction between the epidermis and the dermis, and basal cells are arranged on the epidermal basement membrane to form the dermis-epidermis junction. When the basal cells and epidermal basement membrane that constitute the basal layer are damaged by ultraviolet rays, aging, or other stresses, the maintenance of epidermal undifferentiated stem cells present in the basal cells becomes insufficient, causing disruption of the epidermal basement membrane structure and metabolic failure of the epidermal cell layer. In the present disclosure, the "epidermal basal niche" refers to the microenvironment of cells and their surroundings, including epidermal stem cells.

[0022] The epidermal basal niche protective agent of the present disclosure is characterized in that it contains a laminin fragment or a derivative thereof containing an integrin binding site as an active ingredient. It has been known that laminin interacts with integrins, which are cell surface receptors, and contributes to maintaining the function of epithelial cells and controlling the induction of proliferation and differentiation. However, as shown in the Examples described below, even when 800 kDa laminin 511 was administered to the epidermis, it could not penetrate into the basal layer of the epidermis. In contrast, it was found that a laminin fragment containing a 150 kDa integrin binding site penetrates from the epidermis to the inside, reaches the basal layer of the epidermis, and contributes to the proliferation of epidermal stem cells and the maintenance of undifferentiation ability under aging conditions. Note that the above Patent Document 1 measures the effect of promoting laminin 511 expression by administering a drug, but does not administer laminin 511. In addition, as shown in Patent Document 2, the E8 fragment of human laminin 511 is known as a laminin fragment containing an integrin binding site, but it is used by coating a culture vessel, and there has been no example of direct use on living tissue.

[0023] As shown in Figure 10A, laminin-511 is a cruciform molecule in which the α5 chain, β1 chain, and γ1 chain are associated at the coiled-coil region, and contains three globular domains (LG1-3) and two globular domains (LG4-5) on the C-terminal side of the α5 chain. It is believed that the three globular domains (LG1-3) function as integrin binding sites, and that they associate in a trefoil shape and bind to integrins on their bottom side.

[0024] The laminin fragment containing an integrin-binding site that can be used in the present disclosure may be any laminin fragment having integrin-binding ability, and is not limited to being derived from an animal, and may be one prepared by genetic recombination, etc. A conventional laminin fragment containing an integrin-binding site is the E8 fragment, which is obtained by digesting mouse laminin-111 with elastase and forms a heterotrimer (Edgar D et al., J. Cell Biol., 105:589-598, 1987).

[0025] As shown in FIG. 10B, the laminin 511 E8 fragment is a trimer composed of the C-terminus of the α5 chain, the C-terminus of the β1 chain, and the C-terminus of the γ1 chain, to which the globular domains 1-3 are bound. The globular domains 1-3 correspond to the integrin binding site. For convenience, in this disclosure, a fragment that includes an integrin binding site and is composed of a trimer of the α-chain C-terminus, the β-chain C-terminus, and the γ-chain C-terminus of laminin is referred to as the "E8 fragment" regardless of the type of laminin. When specifying a laminin isoform, the laminin isoform is described before the E8 fragment. For example, the E8 fragment of laminin 332 is referred to as the laminin 332 E8 fragment. In addition, the C-terminal fragment of the α chain containing the globular domains 1-3 at the C-terminus is referred to as the "α chain E8", the C-terminal fragment of the β chain is referred to as the "β chain E8", and the C-terminal fragment of the γ chain is referred to as the "γ chain E8".

[0026] Each laminin isoform exhibits different integrin binding specificity, and can exert strong adhesive activity to cells expressing the corresponding integrin. In the present disclosure, the E8 fragment can preferably be one that can bind to α6β1 integrin, α6β4 integrin, α7β1 integrin, and α3β1 integrin. In terms of having the ability to bind to such integrins, the E8 fragment preferably uses E8 fragments of laminin 511, 521, 411, 421, 332, 311, 321, 211, 221, 213, 111, and 121. In the present disclosure, the term "laminin isoform" refers to the name of human laminin, unless otherwise specified. However, the E8 fragment may be an E8 fragment of an isoform of an animal other than human, provided that it exerts substantially the same action as the human laminin isoform.

[0027] The E8 fragment used in the present disclosure can be produced by digesting the E8 fragment from mammals such as human, mouse, rat, cow, and pig with elastase or other enzymes. It may also be produced by appropriately using known gene recombination techniques. The laminin 511 E8 fragment can be produced, for example, by obtaining DNA encoding each of the α-chain, β-chain, and γ-chain proteins of human laminin 511, cutting them into a predetermined length, inserting them into an expression vector, co-introducing the three obtained expression vectors into an appropriate host cell to express them, and purifying the trimer-forming protein by a known method. The E8 fragment can be produced, for example, by the method of Ido et al. (Hiroyuki Ido et al., The Journal of Biological Chemistry, 282, 11144-11154, 2007), but is not limited thereto. In the present disclosure, the E8 fragment may be a modified type in which one or more amino acid residues are modified while maintaining its biological activity. The nucleotide sequence information of the genes encoding the α, β, and γ chains that constitute the laminins of major mammals, and the amino acid sequence information of each chain, can be obtained from publicly known databases (GenBank, etc.).

[0028] The molecular weight of the E8 fragment used in the present disclosure is not limited, but is 100 to 600 kDa, preferably 120 to 400 kDa, and more preferably 120 to 300 kDa. The molecular weight can be adjusted by cutting out the DNA encoding each protein of the laminin α-chain, β-chain, and γ-chain to a predetermined length, preparing DNA encoding the α-chain E8, β-chain E8, and γ-chain E8 to have a desired molecular weight, and producing the E8 fragment by genetic recombination. Laminin 511 is about 800 kDa, but as shown in the Examples below, even when laminin 511 was administered to the epidermis, it could not penetrate the basal layer of the epidermis. In contrast, the 150 kDa laminin 511 E8 fragment penetrated from the epidermis to the inside, reached the basal layer of the epidermis, and acted on basal stem cells, etc.

[0029] Examples of E8 fragments include "iMatrix-511" (150 kDa), "iMatrix-511MG" (150 kDa), "iMatrix-511 silk" (150 kDa), "iMatrix-411" (150 kDa), and "iMatrix-221" (150 kDa), all manufactured by Nippi Corporation.

[0030] The E8 fragment used in the present disclosure may be a derivative of the E8 fragment. For example, by introducing a lipophilic group into the E8 fragment, tissue affinity can be improved and penetration into the vicinity of the epidermal basement membrane can be promoted. In addition, affinity with the epidermal basal niche can be increased. The lipophilic derivatives include alkyl groups having 1 to 10 carbon atoms which may have a substituent, and esters and ethers which may have a substituent having 1 to 10 carbon atoms. Examples of the substituent include carbonate esters, amides, thioethers, thioesters, organic acids, oximes, and imines. Examples of the lipophilic derivatives include those bound to amino acids constituting the α chain E8 region, β chain E8 region, or γ chain E8 region constituting the E8 fragment via a covalent bond, an ether bond, a thioether bond, an ester bond, a thioester bond, a carbonate bond, a carbamic acid bond, a phosphate bond, and an oxime bond. The derivative of the E8 fragment may be an induced protein to which a fragment of an extracellular matrix component is bound. Such fragments of extracellular matrix components include a functional domain of collagen, a functional domain of BMP2 (Bone Morphogenetic Protein-2), a functional domain of BMP4 (Bone Morphogenetic Protein-4), a functional domain of bFGF (basic Fibroblast Growth Factor), a functional domain of nidogen, and a GAG domain of perlecan. A fusion protein in which a functional domain of an extracellular matrix is ​​added to the E8 fragment can also be used as a derivative of the E8 fragment. These can be produced by known genetic engineering.

[0031] The epidermal basal niche protective agent of the present disclosure contains the above-mentioned E8 fragment or a derivative thereof as an active ingredient. In addition, as a component adjacent to the epidermal basement membrane, for example, type IV collagen, type V collagen, type VI collagen, type VII collagen, type XII collagen, type XIV collagen, type XVII collagen, type XVIII collagen, BMP2, BMP4, bFGF, perlecan, nidogen, etc. can be blended within a range that does not impair the effect of the epidermal basal niche protective agent of the present disclosure.

[0032] Furthermore, as an additive component, components such as excipients, pH adjusters, colorants, and flavoring agents that are normally used in formulations can be used. In addition, the composition can be formulated into a desired dosage form depending on the application method. For example, the composition can be formulated into a liquid, spray, ointment, patch, etc. The dosage of the epidermal basal niche protective agent of the present disclosure can be appropriately set depending on the dosage form, method of use, age, sex, degree of disease, and other conditions of the patient.

[0033] As shown in the examples below, the epidermal basal niche protective agent of the present disclosure can protect the epidermal basal niche in skin damaged by UV irradiation, thereby recovering damaged epidermal stem cells, repairing the microenvironment surrounding the cells near the epidermal basement membrane, restoring the metabolism of the epidermal cell layer, suppressing thinning of the epidermal layer of the skin, and recovering darkening of the stratum corneum. Skin is also damaged by UV rays from sunlight, but the epidermal basal niche protective agent of the present disclosure can restore the epidermal basement membrane, prevent thinning of the epidermal layer, and restore skin barrier function through the recovery of epidermal stem cell damage caused by exposure to sunlight. The effect of the epidermal basal niche protective agent on epidermal stem cells has been found to be effective in maintaining the undifferentiated ability of stem cells against cell aging in cell culture experiments.

[0034] Although the details of the mechanism of action are unknown, E8 fragment penetrates the epidermis into the epidermal basement membrane and restores and increases epidermal stem cells that have deteriorated and decreased under damaging environments such as UV irradiation and aging. Since epidermal stem cells differentiate and proliferate into keratinocytes and sequentially form the basal layer, spinous layer, granular layer, and stratum corneum, it is presumed that the recovery of epidermal stem cells suppresses epidermal thinning under UV damage environments. At the same time, it is presumed that the ability to maintain the undifferentiated ability of undifferentiated cells under damaging environments such as aging also suppresses epidermal thinning. In this way, it is presumed that the effect of E8 fragment on epidermal stem cells produces metabolic activation and recovery of epidermal cell groups, leading to the prevention and recovery of skin damage caused by skin irritation such as ultraviolet rays. Furthermore, even in the epidermal basement membrane that has collapsed due to the activation of proteases caused by UV damage, the supply of epidermal basement membrane constituent proteins secreted by stem cell-derived epidermal basal cells is restored, and the epidermal basement membrane is repaired. As a result, when administered to human skin, it can suppress the decline in skin elasticity and maintain a fine, uniform texture.

[0035] Therefore, the epidermal basal niche protecting agent of the present disclosure can be used as an agent for increasing the number of epidermal stem cells, an agent for stabilizing the epidermal basal niche, an agent for preventing skin aging, an agent for promoting skin regeneration, etc.

[0036] The second aspect of the present disclosure is a pharmaceutical composition for protecting the epidermal basal niche, containing the above-mentioned epidermal basal niche protective agent. As described above, the epidermal basal niche protective agent of the present disclosure can prevent skin damage caused by skin irritation due to ultraviolet rays and the like, increase the number of epidermal stem cells in skin tissue, and suppress thinning of the epidermal layer to maintain the skin barrier. For this reason, a pharmaceutical composition can be prepared by containing the above-mentioned epidermal basal niche protective agent.

[0037] The pharmaceutical composition for protecting the epidermal basal niche of the present disclosure can be administered by any route, such as transdermal, intramuscular, or intravenous, but from the viewpoint of direct action on the skin, it is preferable to administer it by transdermal administration. For administration by transdermal administration, it is preferable to formulate it into a skin topical agent, skin patch, etc. In particular, it can be incorporated into a skin topical agent. These pharmaceutical compositions may contain, for example, antioxidants such as sodium bisulfite, ascorbic acid, tocopherol, dibutylhydroxytoluene, sodium edetate hydrate, and benzotriazole; surfactants such as glyceryl monostearate, sorbitan monostearate, polyoxyethylene hydrogenated castor oil 60, and polysorbate 60; preservatives such as methyl parahydroxybenzoate, propyl parahydroxybenzoate, phenoxyethanol, and thymol; pH adjusters such as citric acid hydrate, sodium citrate hydrate, lactic acid, diisopropanolamine, acetic acid, and sodium acetate hydrate; thickeners, alcohols, colorants, fragrances, water, solvents, oily components, UV absorbers, gelling agents, moisturizers, whitening agents, cell activators, various skin nutrients, and other components.

[0038] The third aspect of the present disclosure is a cosmetic for protecting the epidermal basal niche, containing the above-mentioned epidermal basal niche protective agent. A cosmetic containing the epidermal basal niche protective agent of the present disclosure protects and stabilizes the epidermal basal niche, inhibits the decrease in epidermal stem cells, or promotes their increase. It can also maintain the undifferentiated ability of epidermal stem cells and exert anti-aging or anti-ultraviolet effects. As a cosmetic, it can be blended into any cosmetic, and can be used, for example, in beauty serums, lotions, milky lotions, creams, body milks, bath additives, sunscreens, makeup bases, makeup products, lotions, aftershave creams, etc.

[0039] The cosmetic composition for protecting the epidermal basal niche of the present disclosure may contain the above-mentioned ingredients that are blended in ordinary cosmetics, quasi-drugs, pharmaceuticals, etc., so long as the effects of the cosmetic composition are not impaired.

[0040] The cosmetic composition for protecting the epidermal basal niche of the present disclosure suppresses the decrease in skin elasticity and the increase in the anisotropy index of texture by administering the E8 fragment, as shown in the examples described below. This is thought to be due to the protective effect of the epidermal basal niche by the E8 fragment, which increases epidermal stem cells and maintains the undifferentiated ability of epidermal cells. The decrease in epidermal stem cells is related to the aging phenomenon of the epidermis, and epidermal aging causes a decrease in moisture, rough skin, uneven skin tone, and a decrease in skin firmness. The cosmetic composition for protecting the epidermal basal niche of the present disclosure can suppress these aging phenomena by suppressing the decrease or promoting the increase in the number of epidermal stem cells. EXAMPLES

[0041] The present invention will now be described in detail with reference to examples, but these examples are not intended to limit the present invention in any way.

[0042] Example 1 Abdominal skin samples from live tissue samples (ages: 40s, 50s, 60s, and 70s) were exposed to UVB (20 mJ / cm 2 ), and then organ culture was performed. All organ culture was performed using air-liquid interface culture in a human skin tissue maintenance medium (BPI Skin Culture Medium "MIL218C") at 37°C in a 5% CO2 environment. During culture, laminin 511E8 fragment (Nippi Corporation, product name "iMatrix-511", hereafter abbreviated as LN511-E8) was added to the skin sample at a concentration of 1 μg / cm 2 / day.

[0043] LN511-E8 was administered at 1μg / cm to a skin sample from the abdomen of a woman in her 40s who had been exposed to UVB radiation. 2The process of culturing the samples after adding LN511-E8 / day is shown in Figure 1A. The surface condition of the abdominal skin samples on the 1st, 3rd, 10th, and 14th days after culturing is shown in Figure 1B. The results of a control example in which the same amount of physiological saline was added without adding LN511-E8 are also shown in Figure 1B. In the control abdominal skin sample (ad), darkening due to UVB irradiation damage accumulated in the stratum corneum over time. On the other hand, in the abdominal skin sample (eh) to which LN511-E8 was added, darkening of the stratum corneum due to UVB irradiation damage was reduced compared to the comparative example. Although not shown, similar results were obtained in abdominal skin samples from people in their 50s, 60s, and 70s.

[0044] LN511-E8 had the effect of reducing damage to abdominal skin caused by UVB irradiation and suppressing darkening of the stratum corneum.

[0045] Example 2 A cross section of the abdominal skin sample of a woman in her 40s used in Example 1 was observed under a microscope. The results are shown in FIG. 2. The upper part of FIG. 2A is a control example without the addition of LN511-E8, and the lower part of FIG. 2A is the result of the LN511-E8 addition example. In the control example, the thickness of the epidermis layer, which is indicated by a dark color, tended to become thinner over time. On the other hand, in the LN511-E8 addition example, the thickness of the epidermis layer was maintained. The thickness of the layer was measured by specifying the area of ​​the surface layer using image analysis software BZ-X800 (Keyence Corporation). The results were statistically graphed with n=3. The results are shown in FIG. 2B. In the LN511-E8 addition example, a statistically significant difference was detected by t-test from the 3rd to 14th day of culture.

[0046] In abdominal skin organ culture after UVB irradiation, thinning of the epidermal layer was observed over time, but by applying LN511-E8 to the skin surface, thinning of the epidermal layer was suppressed and the epidermal layer thickness was maintained. This suggests that LN511-E8 application reduced damage to abdominal skin caused by UVB irradiation. After skin organ culture with LN511-E8 applied to the skin surface, thinning of the epidermal layer was suppressed with a statistically significant difference on the third day compared to the control, and this effect was maintained on the 14th day of culture.

[0047] Example 3 An abdominal skin sample from a woman in her 40s was used, and cultured in the same manner as in Example 1, except that LN511-E8 pre-labeled with RPE (fluorescent molecule, R-phycoerythrin) was used instead of LN511-E8, and tissue was collected, fixed, and sliced ​​in the same manner as in Example 2. The sample slice was stained with an antibody to an epidermal stem cell marker (MCSP), and then the cell nuclei were stained with DAPI and observed under a confocal laser microscope. The results of the administration of pre-labeled LN511-E8 are shown in Figure 3A. As shown in Figure 3A a on the third day of culture, pre-labeled LN511-E8 was absorbed from the surface of the abdominal skin sample and penetrated into the inside (white, cell nuclei are dark gray), and accumulated in the epidermal basement membrane part (white, indicated by white arrow) over time (Figure 3A a, b, c). It was also observed that epidermal stem cells also increased over time (Figure 3A d, e, f, bright white part, cell nuclei are dark gray).

[0048] After acquiring fluorescent images using a confocal laser scanning microscope, FV1000 (Olympus), the fluorescence intensity of each image was analyzed using ImageJ software, and the proliferation of epidermal stem cells was evaluated in triplicate. Figure 3B shows the results of time-dependent changes in the number of MSCP-positive cells in the epidermis before culture that was not exposed to UV, calculated as 100%. The number of MSCP-positive cells in the RPE-labeled LN511-E8-added group showed a statistically significant increase by t-test compared to the control group that was not administered RPE-labeled LN511-E8, after 7, 10, and 14 days of culture. This demonstrated that LN511-E8 has the effect of increasing the number of epidermal stem cells in skin tissue irradiated with UVB.

[0049] Example 4 An organ culture was performed in the same manner as in Example 1 using an eyelid skin sample (age 70s) that was airlifted alive, except that UVB treatment was not performed. During the culture, RPE-labeled LN511-E8 was applied to the eyelid skin sample at a concentration of 1 μg / cm 2 / day. As a control, the same amount of physiological saline was administered instead of RPE-labeled LN511-E8. Next, sections were taken in the same manner as in Example 3, stained with E-cadherin antibody, and observed with a confocal laser microscope. E-cadherin is a protein that constitutes an intercellular adhesion structure, and the disappearance of E-cadherin indicates the disappearance of the epidermal barrier function. The results are shown in Figure 4. The top two rows of Figure 4 are micrographs of E-cadherin staining, and the bottom two rows of Figure 4 are micrographs of RPE-labeled LN511-E8 addition, with a, b, c, g, h, and i being control examples, and d, e, f, j, k, and l being RPE-labeled LN511-E8 addition examples.

[0050] As shown in FIG. 4, in the control case, destruction of the epidermal barrier was observed as indicated by E-cadherin antibody staining (ac white). On the other hand, in the case of administration of RPE-labeled LN511-E8, the destruction of the epidermal barrier that occurred in the control case was suppressed, and rather, recovery of the epidermal barrier as indicated by E-cadherin antibody staining was observed (FIG. 4d-f). In addition, as the culture progressed, RPE-labeled LN511-E8 penetrated through the epidermis, and accumulation in the epidermal basement membrane as indicated by the arrow in FIG. 4 l was observed (FIG. 4j-l). Unlike Example 1, the eyelid skin sample was not treated with UVB, but the eyelid is a site exposed to natural light (sunlight). Therefore, Example 4 can be said to evaluate the inhibition of skin deterioration due to sunlight exposure by LN511-E8. As shown in FIG. 4, LN511-E8 was thought to have a skin barrier recovery function by preventing thinning of the epidermal layer due to sunlight exposure.

[0051] Example 5 Abdominal skin samples from a woman in her 40s were used and cultured as in Example 1. Sections were taken as in Example 3, and the epidermal basement membrane was stained with type IV collagen antibody and then observed under a confocal laser microscope. The results are shown in FIG. 5A. The arrows in FIG. 5A (a) and FIG. 5A (e) indicate the epidermal basement membrane. In the control example, UVB irradiation caused the epidermal basement membrane to break down over the course of the culture (FIG. 5A, ad), but in the LN511-E8 administration example, recovery of the epidermal basement membrane, indicated by the white triangle, was confirmed over the course of the culture (FIG. 5A, eh).

[0052] In addition, adjacent sections of the tissue shown in Figure 5A were cut out and stained with an antibody against the epidermal stem cell marker (MCSP) and an antibody against E-cadherin, and the epidermal barrier was observed by confocal laser microscope. The results are shown in Figure 5B. B1 is an image stained with an E-cadherin antibody, and B2 is an image stained with an MCSP antibody. The upper row is a control case, and the lower row is an LN511-E8 administration case. As shown in the control case, the epidermal barrier, as shown by the E-cadherin antibody staining, collapsed over time in the UVB-irradiated tissue (Figure 5B1, ad), whereas in the LN511-E8-added case, the epidermal barrier, as shown by the E-cadherin antibody staining, was observed to be maintained without collapse (Figure 5B1, eh). In addition, in the control case, the number of stem cells decreased over the course of culture (Figure 5B2, ad), whereas in the LN511-E8-added case, an increase in the number of stem cells was observed (Figure 5B2, eh).

[0053] Example 6 In the same manner as in Example 3, abdominal skin samples from women in their 40s were used, and RPE-labeled LN511-E8 was added and cultured in the same manner. As a control, the same amount (6.67 pmol) of RPE-labeled Laminin-511, which was RPE-labeled to full-length Laminin-511 (800 kDa), was added and cultured. Tissue pieces were cut out from the cultured tissue over time, fixed, frozen sections were prepared, and then observed with a confocal laser microscope. The results are shown in Figure 6. Figure 6 a shows an example of RPE-labeled Laminin-511 addition, and Figure 6 b shows an example of RPE-labeled LN511-E8 addition. As shown in Figure 6 a, in the example of RPE-labeled Laminin-511 addition, Laminin-511 (800 kDa) remained on the epidermis surface, but penetration into the inside of the epidermis was not observed. In contrast, in the RPE-labeled LN511-E8 example, epidermal penetration of LN511-E8 (150 kDa) was observed (bright white area in Figure 6a).

[0054] Example 7 1 × 10 HPEKp cells (passage 6) were placed on a 10-well slide coated with poly-D-lysine. 6HPEKp cells were seeded and cultured overnight to allow adhesion. Senescence-inducing medium (VitroAge medium) was dropped onto the slide glass, and the cells were cultured with or without LN511-E8 and at different calcium concentrations. The results are shown in Figure 7A. In the control case where LN511-E8 was not added, HPEKp cells were unable to maintain their undifferentiated morphology (round shape) and showed flattened and cobblestone morphology. Furthermore, HPEKp cells showed differentiated morphology such as stronger process extension and flattening depending on calcium concentration. On the other hand, in the case where LN511-E8 was added, HPEKp cells showed round undifferentiated morphology compared to the control at all calcium concentrations. Thus, LN511-E8 suppressed cell differentiation of HPEKp cells, such as flattening and cobblestone morphology, caused by senescence medium and calcium.

[0055] In addition, 1 × 10 HPEKp cells (passage 6) were placed on a 10-well slide coated with poly-D-lysine. 6 Cells were seeded and cultured for 48 hours in senescence induction medium (VitroAge medium) with 1.6 μg / ml LN511-E8 and 0 or 1 mM calcium. As a control, the same amount of saline was added instead of LN511-E8. After culture, the cells were fixed with 4% PFA and stained with MCSP antibody. The results are shown in Figure 7B. In Figure 7B, MCSP-positive cells are shown in white and MCSP-negative cells in black. When observing the ratio of white cells to black cells in one field, the more areas where MCSP-positive cells, an epidermal stem cell marker, and cell nucleus staining colocalize, the more undifferentiated the cells are maintained. The lower column in Figure 7B shows the group without LN511-E8, but at a calcium concentration of 1 mM, the number of white cells decreased and the number of black cells increased. This tendency was also observed when LN511-E8 was added. On the other hand, as shown in the upper column of Figure 7B, when LN511-E8 was added, black cells appeared at 1 mM calcium compared to the control, but their appearance was lower than that of white cells, and it was observed that the degree of undifferentiation of HPEKp cells was maintained at a high level.

[0056] Example 8 1 × 10 HPEKp cells (passage 6) were placed on a 10-well slide coated with poly-D-lysine. 6The cells were seeded and cultured in senescence induction medium (VitroAge medium, containing 1 mM calcium) with 1.6 μg / ml LN511-E8 for 48 hours. After culture, the cells were fixed with 4% PFA and stained with P63 antibody. Three fields of view were randomly selected from each sample well and photographed, and the p63 positive cell rate was quantitatively evaluated by image analysis using ImageJ software. Note that p63 is a basal cell specific marker.

[0057] The results are shown in Figure 8. A statistically significant difference was detected by t-test in the proportion of p63-positive cells by the addition of LN511-E8 compared to before the addition. These precursor cell culture experiments showed that LN511-E8 induces the maintenance of an undifferentiated state when undifferentiated HPEKp cells are cultured under aging conditions. Stem cells have the property of maintaining the ability to remain undifferentiated while differentiating and proliferating. Laminin-511 binds to integrin α6β1 on the cell surface and is known to be closely related to the survival of undifferentiated cells as a component of the epidermal basement membrane. It was suggested that LN511-E8, a laminin fragment containing an integrin-binding site, helps maintain the undifferentiated state of cells even under aging conditions by migrating to the vicinity of the epidermal basement membrane when administered to the epidermis.

[0058] Example 9 The efficacy of LN511-E8 on the skin was evaluated in a human test. The test was conducted on a total of 33 subjects, including 11 human women in their 30s, 40s, and 50s. The purpose of the test was to evaluate the skin improvement effect of long-term use of the test sample on dry skin, dullness, spots, lack of firmness, wrinkles, etc. The test samples used were a test compound formulation containing laminin 511E8 fragment (Nippi Co., Ltd., product name "iMatrix-511") with the following composition, and a placebo formulation with the following composition. The test compound formulation and placebo formulation were prepared by Cosmos Technical Center Co., Ltd. The final concentration of LN511-E8 contained in the test compound formulation was 5 μg / mL.

[0059] [Table 1]

[0060] [Table 2]

[0061] The subjects were those who provided written consent to participate in this study in advance. The study period was 12 weeks from February 25, 2020 to May 20, 2020. Each subject used the test formulation or placebo formulation for 12 consecutive weeks after washing their face twice a day (morning and evening). After washing their face, they took one push (about 0.5 g) of the test formulation or placebo formulation in their hand and applied it to the entire designated side (right or left). They then performed their usual skin care routine. The measurements were carried out in the measurement room of Cosmos Technical Center Co., Ltd. After washing the test area with the designated cleanser, the subjects were allowed to acclimate for 20 minutes in an evaluation room maintained at a room temperature of 20 to 22 ° C and a relative humidity of 40 to 60%, and the measurements were then started. The test areas were the entire left and right faces assigned to them.

[0062] FIG. 9A shows the results of skin viscoelasticity measured with a Cutometer. After administering the test formulation or placebo formulation for four weeks, the skin viscoelasticity of the test site (mouth) was measured on each measurement day using a Cutometer MPA580 (C+K electronic GmbH.). The higher the skin viscoelasticity, the better the texture of the skin. After four weeks, the test formulation showed a statistically significant difference in skin viscoelasticity compared to the placebo formulation.

[0063] Figure 9B shows the results of skin anisotropy measurements. For skin anisotropy (moisture content and texture), a 1.2 × 1.5 cm area near the cheekbones on both sides was measured once on each measurement day using an Epsilon E100 (Biox Systems Ltd, England) and images were taken. The change in stratum corneum moisture content of each test site was measured using the measurement parameter ε. For texture, the anisotropy index (%) was used, and the distribution value for each specific size was used, and the skin texture was evaluated based on the change in the anisotropy index. The texture size was divided into five levels (0-29, 30-59, 60-89, 90-119, 120<, unit: pixels), and the number of zones in each zone was counted and evaluated. The more small zones there were, the better the texture was evaluated to be. A large anisotropy index indicates irregularity in the texture, and a small anisotropy index indicates a finer skin texture. As shown in FIG. 9B, after 4 weeks of use of the test formulation, the anisotropy index was smaller with statistical significance (p<0.05) when compared with the placebo formulation.

[0064] Table 3 shows the results of measuring the skin anisotropy 4 and 12 weeks after administration. For the placebo formulation, the anisotropy index increased with statistical significance after 4 and 12 weeks compared to before administration, whereas for the test formulation, no change in the anisotropy index was observed either 4 or 12 weeks after administration. On the other hand, when the anisotropy index of the test formulation compared to the placebo formulation was calculated, a decrease in the anisotropy index was observed with statistical significance (p<0.05) after 12 weeks of administration compared to the placebo formulation.

[0065] [Table 3] [Industrial Applicability]

[0066] The epidermal basal niche protective agent of the present invention contains a laminin fragment containing an integrin binding site or a derivative thereof as an active ingredient, and is therefore capable of suppressing the increase in epidermal stem cells and thinning of the epidermal layer, thereby restoring skin barrier function, and is also useful as a pharmaceutical composition for protecting the epidermal basal niche or a cosmetic for protecting the epidermal basal niche.

Claims

1. The composition contains a laminin fragment or a derivative thereof containing an integrin binding site as an active ingredient, the laminin fragment is an E8 fragment of laminin 511, The molecular weight of the laminin fragment is between 120 kDa and 300 kDa; The epidermal basal niche protecting agent, wherein the derivative is an alkyl group which may have a substituent having 1 to 10 carbon atoms, an ester or ether which may have a substituent having 1 to 10 carbon atoms, or a fragment of an extracellular matrix component, bound to any one of the α chain, β chain, or γ chain.

2. A pharmaceutical composition for protecting the epidermal basal niche, comprising the epidermal basal niche protecting agent according to claim 1.

3. A cosmetic for protecting the epidermal basal niche, comprising the epidermal basal niche protecting agent according to claim 1.

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