Patent Application
20230404885
The present invention relates to a composition for external use on the skin, and more particularly to a topical skin composition comprising 1-(2-hydroxyethyl)-2-imidazoliclinone or a derivative thereof and pyriclinecarboxamide.
With the arrival of a full-fledged aging society, research and development on aging mechanisms are becoming active. Among them, the skin forms the outermost layer of the human body, plays an important role in bioprotection, and is a particularly important organ in terms of beauty because its appearance can be visually perceived. From the viewpoint of maintaining or improving quality of life (QOL) in terms of both health and beauty, there has been an increase of interests in skin aging prevention in recent years.
For the purpose of skin aging prevention, topical skin preparations such as cosmetics have long been blended with moisturizing agents that are expected to improve the skin's ability to retain moisture, vitamins E and C which are expected to act as antioxidants, and the like. Further, as a result of recent studies, it has become clear that the reduction and degeneration of matrix components in the skin are greatly involved in wrinkles, sagging, and loss of firmness associated with aging.
In particular, the decrease in collagen, which is the main component of the matrix, is related to the sagging and firmness of the skin, and the degradation and degeneration of elastin caused by the expression of the elastin degrading enzyme is thought to be the cause of the decrease in elasticity. The collagen production promoting agents, collagen degrading enzyme-inhibiting agents, elastin degrading enzyme-inhibiting agents, and the like are blended as anti-aging agents. Nevertheless, although these drugs have certain effects, they have not yet shown sufficient effects.
Patent Document 1 discloses an anti-aging skin preparation for external use that is effective in restoring and maintaining skin firmness and elasticity, reducing wrinkles and sagging, and reducing pigmentation, and is capable of restoring and maintaining a youthful skin condition, wherein the anti-aging skin external preparation is characterized by comprising niacin and ubiquinone.
Patent Document 2 discloses a cyclic carboxamide derivative having a specific structure, as a drug effective for preventing and suppressing skin aging from the viewpoint of the connection between heparanase and skin aging, and as a component of a whitening agent effective in preventing and suppressing pigmentation such as spots, freckles, and dullness.
An objective of the present invention is to find a composition that is effective in improving skin conditions from the viewpoint of maintaining or improving quality of life (QOL) in terms of both health and beauty.
As a result of intensive studies, the inventors of the present invention have found that a composition comprising 1-(2-hydroxyethyl)-2-imidazoliclinone or a derivative thereof and pyridinecarboxamide exhibits an effect of promoting epidermal stem cell proliferation.
Accordingly, the disclosure of the present invention provides the embodiments below.
(1) A topical skin composition comprising
1-(2-hydroxyethyl)-2-imidazolidinone or a derivative thereof and pyridinecarboxamide as active ingredients.
(2) The composition according to (1), which is a cosmetic composition.
(3) The composition according to (1) or (2), which is for use in promoting the proliferation of epidermal stem cells.
(4) The composition according to (3), wherein the epidermal stem cells are MCSP-positive cells.
(5) The composition according to any one of (1) to (4), which promotes keratinocyte proliferation.
(6) The composition according to any one of (1) to (5), wherein the 1-(2-hydroxyethyl)-2-imidazolidinone or a derivative thereof is a cyclic carboxamide derivative represented by the general formula (I) below:
wherein, n is an integer of 1 to 3, R1 is a hydrogen atom or a hydrocarbon group having 1 to 6 carbon atoms which may be substituted with a hydroxyl group, and X is —CH2— or a group represented by —N(R2)—, and R2 refers to a hydrogen atom or a hydrocarbon group having 1 to 6 carbon atoms which may be substituted with a hydroxyl group.
(7) The composition according to any one of (1) to (6), wherein the pyridinecarboxamide is selected from the group consisting of 2-pyridinecarboxamide, 3-pyridinecarboxamide, 4-pyridinecarboxamide, and a mixture thereof.
(8) A cosmetic method, comprising applying to the skin a composition containing 1-(2-hydroxyethyl)-2-imidazoliclinone or a derivative thereof and pyridinecarboxamide.
(9) The composition according to any one of (1) to (7), which is for use in promoting the expression of collagen in the papillary dermis.
(10) The composition according to any one of (1) to (7), which is for use in promoting the expression of collagen in the dermis.
Since the composition according to the disclosure of the present invention promotes the increase of epidermal stem cells and facilitates the proliferation of keratinocytes, it can be suitably used, for example, as a cosmetic composition such as cosmetics effective in improving skin conditions.
One embodiment in the disclosure of the present invention relates to a composition for external application to the skin which comprises 1-(2-hydroxyethyl)-2-imidazoliclinone (HEI) or a derivative thereof and pyridinecarboxamide as active ingredients. Such compositions are preferably prepared as cosmetic compositions.
1-(2-hydroxyethyl)-2-imidazolidinone (HEI) is a compound with the structure below. In the present specification, 1-(2-hydroxyethyl)-2-imidazoliclinone (HEI) is sometimes referred to as S173.
A derivative of 1-(2-hydroxyethyl)-2-imidazolidinone can be, for example, a compound represented by the general formula (I) below:
wherein, n is an integer of 1 to 3, R1 is a hydrogen atom or a hydrocarbon group having 1 to 6 carbon atoms which may be substituted with a hydroxyl group, and X is —CH2— or a group represented by —N(R2)—, and R2 refers to a hydrogen atom or a hydrocarbon group having 1 to 6 carbon atoms which may be substituted with a hydroxyl group.
1-(2-hydroxyethyl)-2-imidazoliclinone and derivatives thereof are known to act as inhibitors of the heparanase activity (see JP 2014-111640 (Patent Document 2)). Heparanase is an enzyme that exists in various cells and specifically degrades the heparan sulfate chains of various heparan sulfate proteoglycans. In the skin, epidermal keratinocytes, dermal fibroblasts, vascular endothelial cells, and the like, which constitute the epidermis, are produced. It is known that its production is increased in various cancer cells, and its association with cancer malignancy has been suggested. It is known that cancer cells with high heparanase production have a high metastatic potential and a high ability to induce angiogenesis (Vlodaysky I., et. al., Semin Cancer Biol., 2002; 12(2): 121-129 (see Non-patent Document 1)).
Heparan sulfate proteoglycans function to accumulate heparan sulfate-binding growth factors (bFGF, HGF, VEGF, HB-EGF, etc.) extracellularly. Perlecan, which is a type of heparan sulfate proteoglycan, is also present in the epidermal basement membrane at the boundary between the epidermis and dermis. In the skin, the transfer of growth factors between the epidermis and dermis is controlled by the binding of the heparan sulfate-binding growth factors to the epidermal basement membrane. Further, perlecan present in the epidermal basement membrane also regulates the action of growth factors on epidermal basal cells bound to the basement membrane, which has been shown to be essential for good epidermal proliferation and differentiation.
Further, 1-(2-hydroxyethyl)-2-imidazoliclinone (HEI) is also known to act as an MMP (matrix metalloproteinase) inhibitor. HEI, for example, inhibits MMP-9 but does not affect the activities of MMP-1 and MMP-2.
Pyriclinecarboxamides are compounds with the molecular formula C6H6N2O and include the following compounds with different positions of the carboxamide group: 2-pyriclinecarboxamide (picolinamide), 3-pyriclinecarboxamide (nicotinamide), and 4-pyridinecarboxamide (isonicotinamide). In the embodiments pertaining to the disclosure of the present invention, any of 2-pyridinecarboxamide (picolinamide), 3-pyriclinecarboxamide (nicotinamide), and 4-pyridinecarboxamide (isonicotinamide), or a mixture thereof, may be used. In some embodiments, 3-pyriclinecarboxamide (nicotinamide) is suitably used. The structure of 3-pyriclinecarboxamide (nicotinamide) is shown below.
As detailed in the Examples section, the inventors of the present invention have now found that compositions comprising 1-(2-hydroxyethyl)-2-imidazoliclinone or a derivative thereof and pyriclinecarboxamide are effective in promoting an increase in epidermal stem cells and an increase in keratinocytes. The increase in these cells can be evaluated, for example, by immunohistochemical staining. For example, epidermal stem cells can be identified as MCSP-positive cells. Thus, one embodiment of the disclosure of the present invention relates to compositions characterized in that they comprise 1-(2-hydroxyethyl)-2-imidazoliclinone or a derivative thereof and pyridinecarboxamide as active ingredients for use in promoting an increase in epidermal stem cells. Such compositions can preferably be prepared as topical skin compositions.
In other words, the composition according to the disclosure of the present invention can also be expressed as an epidermal stem cell proliferation promoting agent comprising 1-(2-hydroxyethyl)-2-imidazoliclinone or a derivative thereof and pyridinecarboxamide as active ingredients. Compositions and agents according to the disclosure of the present invention may be used for non-therapeutic cosmetic applications, in particular. Also, from another aspect, an embodiment of the disclosure of the present invention relates to the use of a composition comprising 1-(2-hydroxyethyl)-2-imidazoliclinone or a derivative thereof and pyridinecarboxamide for promoting epidermal stem cell proliferation. In such uses, 1-(2-hydroxyethyl)-2-imidazoliclinone or its derivative and pyridinecarboxamide may be used in the form of a composition in combination with further components. From yet another aspect, one embodiment of the disclosure of the present invention relates to the use of 1-(2-hydroxyethyl)-2-imidazoliclinone or a derivative thereof and pyridinecarboxamide in the manufacture of an epidermal stem cell proliferation promoting agent. From yet another aspect, one embodiment of the disclosure of the present invention relates to a method for promoting the increase of epidermal stem cells, which comprises administering to a subject (for example, a human) a composition comprising 1-(2-hydroxyethyl)-2-imidazoliclinone or a derivative thereof and pyridinecarboxamide.
In addition, one embodiment according to the disclosure of the present invention relates to a composition characterized by comprising 1-(2-hydroxyethyl)-2-imidazoliclinone or a derivative thereof and pyridinecarboxamide as active ingredients for use in facilitating the proliferation of keratinocytes. Such compositions are preferably prepared as skin compositions for external use.
In other words, the composition according to the disclosure of the present invention can also be expressed as a keratinocyte proliferation promoting agent comprising 1-(2-hydroxyethyl)-2-imidazolidinone or a derivative thereof and pyridinecarboxamide as active ingredients. Compositions and agents according to the disclosure of the present invention may be used for non-therapeutic cosmetic applications, in particular. From another aspect, one embodiment of the disclosure of the present invention relates to the use of a composition comprising 1-(2-hydroxyethyl)-2-imidazolidinone or a derivative thereof and pyridinecarboxamide for promoting keratinocyte proliferation. In such uses, 1-(2-hydroxyethyl)-2-imidazolidinone or its derivatives and pyridinecarboxamide may be used in the form of a composition in combination with further components. From yet another aspect, one embodiment of the disclosure of the present invention relates to the use of 1-(2-hydroxyethyl)-2-imidazolidinone or a derivative thereof and pyridinecarboxamide in the manufacture of a keratinocyte proliferation promoting agent. From yet another aspect, one embodiment of the disclosure of the present invention relates to a method of promoting keratinocyte proliferation, comprising administering a composition comprising 1-(2-hydroxyethyl)-2-imidazolidinone or a derivative thereof and pyridinecarboxamide to a subject (for example, a human).
Furthermore, one embodiment according to the disclosure of the present invention relates to a composition characterized by comprising 1-(2-hydroxyethyl)-2-imidazolidinone or a derivative thereof and pyridinecarboxamide as active ingredients for use in the promotion of collagen expression in the papillary dermis. The collagen can be Type I, III, IV, or V collagen. Such compositions are preferably prepared as skin compositions for external use.
In other words, the composition according to the disclosure of the present invention can be expressed as an agent that promotes the expression of collagen in the papillary dermis, comprising 1-(2-hydroxyethyl)-2-imidazolidinone or a derivative thereof and pyridinecarboxamide as active ingredients. Compositions and agents according to the disclosure of the present invention may be used for non-therapeutic cosmetic applications, in particular. Further, from another aspect, one embodiment of the disclosure of the present invention relates to the use of a composition comprising 1-(2-hydroxyethyl)-2-imidazolidinone or a derivative thereof and pyridinecarboxamide for promoting collagen expression in the papillary dermis. In such uses, 1-(2-hydroxyethyl)-2-imidazoliclinone or its derivatives and pyridinecarboxamide may be used in the form of a composition in combination with further components. From yet another aspect, one embodiment of the disclosure of the present invention relates to the use of 1-(2-hydroxyethyl)-2-imidazoliclinone or a derivative thereof and pyridinecarboxamide in the manufacture of an agent for promoting collagen expression in the papillary dermis. From yet another aspect, one embodiment of the disclosure of the present invention relates to a method of promoting collagen expression in the papillary dermis, comprising administering a composition comprising 1-(2-hydroxyethyl)-2-imidazoliclinone or a derivative thereof and pyridinecarboxamide to a subject (for example, a human).
Furthermore, one embodiment according to the disclosure of the present invention comprises 1-(2-hydroxyethyl)-2-imidazoliclinone or a derivative thereof and pyridinecarboxamide as active ingredients for use in promoting collagen expression in the dermis. The collagen can be Type I, Type III, Type IV, or Type V collagen. Such compositions are preferably prepared as skin compositions for external use.
In other words, the composition according to the disclosure of the present invention comprises 1-(2-hydroxyethyl)-2-imidazoliclinone or a derivative thereof and pyridinecarboxamide as active ingredients, and it can also be presented as an agent for promoting collagen expression in the dermis. Compositions and agents according to the disclosure of the present invention may be used for non-therapeutic cosmetic applications, in particular. From another aspect, an embodiment of the disclosure of the present invention relates to a composition comprising 1-(2-hydroxyethyl)-2-imidazolidinone or a derivative thereof and pyridinecarboxamide for promoting collagen expression in the dermis. In such uses, 1-(2-hydroxyethyl)-2-imidazolidinone or its derivatives and pyridinecarboxamide may be used in the form of a composition in combination with further components. From yet another aspect, one embodiment of the disclosure of the present invention relates to the use of 1-(2-hydroxyethyl)-2-imidazolidinone or a derivative thereof and pyridinecarboxamide in the manufacture of an agent for promoting collagen expression in the dermis. From yet another aspect, one embodiment of the disclosure of the present invention relates to a method for promoting the collagen expression in the dermis, comprising administering a composition comprising 1-(2-hydroxyethyl)-2-imidazolidinone or a derivative thereof and pyridinecarboxamide to a subject (for example, a human).
One embodiment of the disclosure of the present invention relates to a cosmetic method characterized in applying to the skin a composition comprising 1-(2-hydroxyethyl)-2-imidazolidinone or a derivative thereof and nicotinamide. The subject can be any animal, and is preferably a human. The cosmetic method according to the disclosure of the present invention is a non-therapeutic method and does not include the so-called medical practice. In the cosmetic or non-therapeutic method according to the disclosure of the present invention, the composition for external use on the skin is applied to areas where desired effects are wished for, such as the face, neck, hands, arms, and legs. Application can be in any manner, including application by hand, applicator, and the like. The number, frequency, amount, and such of application are appropriately set according to the desired effect and the like.
Also, 1-(2-hydroxyethyl)-2-imidazoliclinone or its derivatives and pyridinecarboxamide can be converted into inorganic salts or organic salts by known methods. Salts used in the embodiments of the disclosure of the present invention are not particularly limited, and for example, inorganic salts include hydrochloride, sulfate, phosphate, hydrobromide, sodium salt, potassium salt, magnesium salt, calcium salts, ammonium salts and the like. Organic salts include acetate, lactate, maleate, fumarate, tartrate, citrate, methanesulfonate, p-toluenesulfonate, triethanolamine salt, diethanolamine salt, amino acid salt, and the like.
1-(2-hydroxyethyl)-2-imidazoliclinone and its derivatives with a heparanase activity inhibiting effect and/or an MMP inhibitory effect, especially those with both heparanase activity inhibiting effect and MMP inhibiting effect (especially MMP-9 inhibiting effect), can be suitably used in the agents of the disclosure of the present invention. The composition according to the disclosure of the present invention may comprise only one type of 1-(2-hydroxyethyl)-2-imidazolidinone or a derivative thereof, and two or more types of the above compounds or salts thereof may be used in any optional combination or proportion.
The content of 1-(2-hydroxyethyl)-2-imidazolidinone or a derivative thereof and pyridinecarboxamide or a salt thereof in the compositions of the disclosure of the present invention is not particularly limited as long as the amount is sufficient to effectively produce the desired effect and should be selected according to the application of the agent. In general, however, the ratio of 1-(2-hydroxyethyl)-2-imidazolidinone or its derivative and pyridinecarboxamide or its salt to the total amount of the agent is usually 0.0001% or more by mass, especially 0.0001% or more by mass, and usually less than 1% by mass, especially less than 0.2% by mass. When two or more 1-(2-hydroxyethyl)-2-imidazoliclinone or its derivatives and pyridinecarboxamide or its salt are used, the total amount of them should meet the above range.
The compositions according to the disclosure of the present invention may also comprise any other optional ingredient, in addition to 1-(2-hydroxyethyl)-2-imidazoliclinone or a derivative thereof and pyridinecarboxamide or a salt thereof, provided that the actions of 1-(2-hydroxyethyl)-2-imidazoliclinone or a derivative thereof and pyridinecarboxamide or a salt thereof are not substantially impaired. The additional components include other compounds (CGS27023A, BIPBIPU, or such) having an inhibitory effect of a heparanase activity or any other activity (MMP inhibitory activity, especially MMP-9 inhibitory activity, or such), and pharmaceutically acceptable carriers and/or adjuvants. Examples of the additional ingredients include herbal medicines that act as heparanase inhibitors such as valerian extract, lily extract, chomeisou extract, soapberry extract, chimp extract (see Japanese Patent Application No. 2014-165586), mangosteen extract, turmeric extract, (tormentilla extract), and such (see Japanese Patent Application No. 2018-545781), but are not limited to thereto. Such additional components may be used alone with one single type, or two or more types may be used in any combination and proportion. Accordingly, some embodiments of the present invention relate to topical skin compositions comprising as active ingredients, pyridinecarboxamide and a compound having both heparanase inhibitory activity and MMP (especially MMP-9) inhibitory activity and/or a combination of a heparanase inhibiting agent and an MMP (especially MMP-9) inhibiting agent.
The compositions according to the disclosure of the present invention can be produced according to a conventional method, and they can also be prepared with only 1-(2-hydroxyethyl)-2-imidazolidinone or its derivatives and pyridinecarboxamide, or in addition with one or more of its salts, as ingredients that constitute a topical skin composition. Ingredients normally used in cosmetics, pharmaceuticals, and other topical skin care products, such as oils, surfactants, powders, colorants, water, alcohols, thickening agents, chelating agents, silicones, antioxidants, UV absorbers, moisturizing agents, fragrances, various medicinal ingredients, preservatives, pH adjusting agents, neutralizing agents, and the like are blended as needed.
The dosage form of the composition according to the disclosure of the present invention is not limited, and it may be appropriately selected according to the use thereof. Examples of administration routes include topical administration (external application to the skin). Formulations for topical administration (material for external application to the skin) include solution systems, solubilized systems, emulsified systems, powder dispersion systems, water-oil two-phase systems, water-oil-powder three-phase systems, or such, in patch form, ointment, cream, emulsion, lotion, gel, aerosol, and other forms.
Further, for the compositions according to the disclosure of the present invention, one or more optional components such as carriers and/or adjuvants may be blended in addition to the 1-(2-hydroxyethyl)-2-imidazolidinone or a derivative thereof and pyridinecarboxamide or a salt thereof according to the disclosure of the present invention, as long as the action of 1-(2-hydroxyethyl)-2-imidazolidinone or a derivative thereof and pyridinecarboxamide or a salt thereof is not substantially impaired. The additional ingredients are not particularly limited, and may be appropriately selected according to the use, dosage form, mode of administration of the pharmaceutical composition, and examples include pharmaceutically acceptable carriers and/or adjuvants. The adjuvants include, for example, diluents, binding agents, disintegrants, thickening agents, dispersants, reabsorption enhancers, flavoring agents, buffers, surfactants, solubilizers, preservatives, emulsifiers, isotonic agents, stabilizers, pH adjusters, and the like.
As specific examples, the compositions according to the disclosure of the present invention can comprise ingredients normally used in topical agents as needed, for example, whitening agents, moisturizing agents, antioxidants, oil-based ingredients, UV absorbers, surfactants, thickening agents, alcohols, powder ingredients, colorants, aqueous ingredients, water, various skin nutrition agents or such. In addition, metal ion sequestering agents such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, and gluconic acid; preservatives such as methylparaben, ethylparaben, butylparaben and such; caffeine, tannin, verapamil, tranexamic acid and derivatives thereof, licorice extract, glabridine, hot water extract of the quince fruit, various herbal medicines, pharmaceutical agents such as tocopherol acetate, glycyrrhizic acid and its derivatives or salts, whitening agents such as vitamin C, magnesium ascorbate phosphate, ascorbic acid glucoside, arbutin, and kojic acid; saccharides such as glucose, fructose, mannose, sucrose, trehalose, and such; vitamin A derivatives such as retinoic acid, retinol, retinol acetate, retinol palmitate, and such can also be blended appropriately as needed.
The above ingredients are illustrative and not limiting. Also, these components can be blended in an appropriate combination according to the formulation depending on the desired form.
The dosage form of the topical skin preparations according to the disclosure of the present invention is not particularly limited, and as described above it can be, for example, a solution system, solubilized system, emulsion system, powder dispersion system, water-oil two-phase system, water-oil-powder three-phase system, ointment, gel, aerosol, or any other formulation. The form of use is also not limited particularly, and can take any optional form such as lotion, milky lotion, cream, essence, jelly, gel, ointment, pack, mask, foundation, or such.
By applying a composition according to the disclosure of the present invention to the skin, it can be used as a cosmetic method for improving the skin condition. The usage and dosage of the topical skin preparation according to the disclosure of the present invention in such cosmetic methods are not particularly limited, and they are appropriately determined depending on the dosage form and skin condition to be treated. Typically, a suitable amount, for example, 0.1 ml to 1 ml per square cm2, can be rubbed directly into the skin, or the appropriate amount can be soaked into a gauze and applied to the skin several times per day, for example, one to five times per day.
The above description of the present invention with specific examples is only illustrative, and the invention can be implemented with any modification within the scope not departing from the claims The various features and embodiments of the present invention referred to in various places above may be applied to the description of other parts of the present invention with the necessary changes as appropriate. Thus, features identified in one embodiment may be combined with features identified in the other embodiments as appropriate. All references cited herein, including patents, patent applications, articles, textbooks, and sequence accession numbers, and references cited therein are incorporated herein by reference in their entirety. To the extent that one or more of the incorporated references and similar materials differ from or contradict this application in any respect, including but not limited to defined terms, usage of terms, techniques described, and such, the description in the present application takes precedence.
Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to the examples below.
The three-dimensional epidermal skin model (EPI-200-3S) purchased from MatTek was treated with nicotinamide (final concentration 7.5×10−3 M) and MMP-9 inhibitor CGS27023A (Reference 1) (final concentration 10−5 M), and it was cultured in a special medium (EPI-100 ASY) supplemented with the heparanase inhibitor BIPBIPU (Reference 2) (final concentration 10−5 M). As a control, the cells were cultured in a medium to which an equal volume of the DMSO solvent was added without these drugs. The medium was exchanged once every two days, and the pieces of tissue were collected on the fourth day.
A dermis-containing three-dimensional skin model (EFT-400) purchased from MatTek was treated with nicotinamide (final concentration 7.5×10−3 M) and CGS27023A (final concentration 10−5 M), and it was cultured in a special medium (EPI-100 ASY) supplemented with BIPBIPU (final concentration 10−5 M). As a control, the cells were cultured in a medium to which an equal volume of the DMSO solvent was added without these drugs. The medium was exchanged once every two days, and the pieces of tissue were collected on the fourth day.
Fresh abdominal skin samples from subjects (20's to 30's) who gave informed consents were purchased from KAC Co., Ltd. They were cultured in a mixture medium of equal volumes of 10% FBS-DMEM (Thermo Fisher Science, 11885084) and Humeclia-KG2 (KURABO, KK-2150S), supplemented with nicotinamide (7.5×10−3 M or 1.5×10−2 M final concentration) and 1-(2-hydroxyethyl)-2-imidazoliclinone (Reference 3) (S173, final concentration a proprietary ingredient that inhibits both MMP-9 and heparanase. As a control, the cells were cultured in a medium to which an equal volume of the DMSO solvent was added without these drugs. The medium was changed every day, and pieces of the tissue were collected on the second day.
The recovered skin model and fresh human skin were dehydrated and fixed using cold acetone according to the AMeX method, then substituted with acetone, methyl benzoate and xylene in that order, and embedded in paraffin. Sections were prepared with a thickness of 3 μm, and sections for tissue staining were prepared.
Paraffin sections with a thickness of 3 μm were deparaffinized with xylene and then hydrated with EtOH. Fluorescent immunostaining was performed using an Ki-67(SP6) antibody (Thermo Fisher Scientific, RM-9106-S, rabbit mouse monoclonal antibody) and an MCSP (melanoma-associated chondroitin sulphate proteoglycan) antibody (Millopore, MAB2029, 9.2.27, mouse monoclonal antibody).
For fluorescent immunostaining of various types of collagens, an antibody against Type I collagen (ROCKLAND, 600-401-103-0.5, rabbit polyclonal antibody), an antibody against Type II collagen (ROCKLAND, 600-401-105S, rabbit polyclonal antibody), an antibody against Type IV collagen (Progen, 10760, rabbit polyclonal antibody), and an antibody against Type V collagen (ORIGENE, AM10159PU-N, mouse monoclonal antibody) were used.
The collected fresh human skin was heat-treated on a hot plate at 60° C. for 1 minute and then cooled on ice to separate the epidermis and dermis. The epidermis was placed in a 1 mL Trizol solution containing zirconia balls and was shaken for 3 minutes with a tissue pulverizer to crush the epidermis. RNA was extracted using chloroform and isopropanol, and the RNA was purified using the RNeasy mini kit (QIAGEN). After the RNA concentration was measured using NanoDrop, cDNA was synthesized using SuperScript VILO (Invitrogen). After that, the synthesized cDNA was used for quantitative PCR analysis which was performed using platinum SYBR green (Invitrogen). Table 1 shows the primers for the genes used.
Collagen gene expression analysis was performed in the same manner as described above using the separated dermis. Table 2 shows the primers used.
In addition, the results of investigating the effect of nicotinamide (NAM) and the combination of MMP-9 inhibitor CGS27023A and heparanase inhibitor BIPBIPU on keratinocyte proliferation in a three-dimensional skin model containing dermis are illustrated in
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2) MacPherson L J, Bayburt E K, Capparelli M P, Carroll B J, Goldstein R, Justice M R, et al. Discovery of CGS 27023A, a non-pepticlic, potent, and orally active stromelysin inhibitor that blocks cartilage degradation in rabbits. Journal of medicinal 1997; 40(16): 2525-32.
3) Iriyama S, Yamanishi H, Kunizawa N, Hirao T, Amano S. 1-(2-hydroxyethyl)-2-imidazoliclinone, a heparanase and matrix metalloproteinase inhibitor, improves epidermal basement membrane structure and epidermal barrier function. Experimental dermatology. 2019; 28(3): 247-53.
4) Iriyama S, Yasuda M, Nishikawa S, Takai E, Hosoi J, Amano S. Decrease of laminin-511 in the basement membrane due to photoaging reduces epidermal stem/progenitor cells. Scientific reports. 2020; 10(1): 12592.
Since the compositions according to the disclosure of the present invention promote the proliferation of epidermal stem cells and facilitate the increase of keratinocytes, they can be suitably used, for example, as a cosmetic composition such as cosmetics effective for improving skin conditions.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2020-207552 | Dec 2020 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2021/045725 | 12/13/2021 | WO |
