Epidermal cell transporter-related gene expression amplifier

Abstract

To utilize the effect of a diacylglycerol PEG adduct in amplifying the expression of transporter-related genes in epidermal cells.SOLUTION: A agent for amplifying the expression of transporter-related genes in epidermal cells comprises a diacylglycerol PEG adduct as an active ingredient, wherein the diacylglycerol PEG adduct has a structural formula specified herein, where the carbon number of R in the long-chain fatty acid falls within the range of 11-23 and n in the polyethylene glycol chain is within the range of 11-46.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to an epidermal cell transporter-related gene expression amplifying agent and an expression amplifying method.

Background Art

[0002] Patent Document 1 discloses a preparation method in which a diacylglycerol polyethylene glycol adduct (hereinafter referred to as "diacylglycerol PEG adduct") is used as a lipid molecule and spontaneously forms vesicles by mixing with water or a surfactant. Such vesicles are used in a drug delivery system for encapsulating or binding target substances such as proteins and drugs inside or on their surface and delivering them to cells in the living body. Vesicles composed of diacylglycerol PEG adducts have a form in which their surface is covered with hydrophilic PEG chains, and have good permeability into the living body and stability in blood.

[0003] Patent Document 2 describes that by binding a charged element to the surface of a vesicle composed of a diacylglycerol PEG adduct to make it positively charged, the permeability and retention in the stratum corneum of the epidermis can be improved.

[0004] Vesicles in a drug delivery system have been simply recognized as carriers of drugs, which are target substances. Recently, it has been known that the molecules of diacylglycerol PEG adducts derived from vesicles decomposed in the living body also exert useful effects in the living body. Patent Document 3 discloses that diacylglycerol PEG adducts contribute to enhancing the expression of moisturizing-related substances such as profilaggrin, filaggrin, and natural moisturizing factor NMF in the epidermis. Further, Patent Document 4 discloses that diacylglycerol PEG adducts contribute to enhancing the expression of antioxidant-related substances such as Nrf2 and PPARG, which are oxidative stress response genes in the epidermis, and antioxidant enzymes such as NQO-1, CAT, and HMOX1.

[0005] On the other hand, various transport proteins (so-called transporters) present in the cell membrane play a role in the uptake and excretion of various substances into epidermal cells. For example, sodium-dependent vitamin C transporters (SVCTs) that transport vitamin C into cells have been discovered, and two types, type 1 (SVCT1) and type 2 (SVCT2), are known (Patent Document 5). Furthermore, for example, xCT proteins are known that are involved in the xCT mechanism, which involves taking in cystine, one of the amino acids, into cells and releasing glutamate from within the cell to the extracellular space. Cystine taken into cells is involved in glutathione production via cysteine ​​(Patent Document 6). For example, the ABCC1 protein is known as one of the ABC transporters that has the function of expelling various substances from inside the cell to outside the cell (Patent Document 7). Furthermore, aquaporin proteins that function as water channels to regulate the amount of water in cells are known, and aquaporin 3 (AQP3) in particular is abundant in the epidermis (Patent Document 8). [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Japanese Patent Publication No. 4497765 (Self-forming thermodynamically stable liposome and its application) [Patent Document 2] Japanese Patent Publication No. 6297737 (Method for preparing positively charged niosomes and charged niosomes) [Patent Document 3] Japanese Patent Publication No. 6805385 (An agent for enhancing the expression of moisturizing substances in the epidermis) [Patent Document 4] Japanese Patent Publication No. 6860739 (Antioxidant expression enhancer in the epidermis) [Patent Document 5] International Publication No. 2007 / 094312 (Vitamin C transporter production promoter) [Patent Document 6] Japanese Patent Publication No. 2010-280675 (Composition for Glutathione Enhancement) [Patent Document 7] Japanese Patent Publication No. 2022-514669 (Composition containing Lactobacillus rhamnosus extract) [Patent Document 8] Japanese Patent Publication No. 2011-32191 (Aquaporin 3 expression regulator) [Overview of the project] [Problems that the invention aims to solve]

[0007] As mentioned above, various epidermal cell transporters are known, but there are few reports of substances that enhance the expression of these transport proteins. Regarding diacylglycerol PEG adducts, little is known about their relationship to the various mechanisms of drug uptake or efflux by epidermal cells.

[0008] The objective of the present invention is to utilize newly discovered properties of diacylglycerol PEG adducts related to epidermal cell transporters, and in particular to amplify the expression of epidermal cell transporter-related genes. [Means for solving the problem]

[0009] To achieve the above objectives, the present invention provides the following configuration. Aspects of the present invention are: An expression amplification agent for epidermal cell transporter-related genes, The active ingredient is a diacylglycerol PEG adduct, wherein the diacylglycerol PEG adduct has the following structural formula, the number of carbon atoms in the long-chain fatty acid R is in the range of 11 to 23, and n in the polyethylene glycol chain is in the range of 11 to 46. the law of nature, The aforementioned epidermal cell transporter-related gene is SVCT2. ru.

[0011] [ka]

[0012] goodAlternatively, the diacylglycerol PEG adduct is at least one selected from the group consisting of glycerol dimyristate PEG-12 (GDM12), glycerol distearate PEG-12 (GDS12), glycerol distearate PEG-23 (GDS23), glycerol dipalmitate PEG-23 (GDP23), and glycerol dioleate PEG-12 (GDO12). Preferably, the diacylglycerol PEG adduct penetrates into the epidermis in a solution state or a vesicle state.

[0013] Yet another aspect of the present invention is a cosmetic containing the above-described epidermal cell transporter-related gene expression amplifying agent. Yet another aspect of the present invention is an external preparation for skin containing the above-described epidermal cell transporter-related gene expression amplifying agent. The above cosmetic or the above external preparation for skin may further contain or ascorbic acid derivatives body and may further contain hydroquinone in that case. <​​​​​​​​​​​​​​​​​​​​​​Figure 4 is a graph showing the relative expression amplification levels of the SLC7A11 gene related to GDM12. [Figure 5] Figure 5 is a graph showing the relative expression amplification levels of the GSR gene related to GDS23. [Figure 6] Figure 6 is a graph showing the relative expression amplification levels of the GSR gene related to GDM12. [Figure 7] Figure 7 is a graph showing the relative expression amplification levels of the ABCC1 gene related to GDS23. [Figure 8] Figure 8 is a graph showing the relative expression amplification levels of the ABCC1 gene related to GDM12. [Figure 9] Figure 9 is a graph showing the relative expression amplification levels of the AQP3 gene related to GDS23. [Figure 10] Figure 10 is a graph showing the relative expression amplification levels of the AQP3 gene related to GDM12. [Figure 11] Figure 11 shows the band image of the SVCT2 protein in a Western blot related to GDS23. [Figure 12] Figure 12 is a graph showing the expression levels of the SVCT2 protein. [Figure 13] Figure 13 is a graph showing the results of an evaluation test on the cell uptake effect of vitamin C. [Figure 14] Figure 14 is a graph showing the results of an evaluation test of the cystine uptake effect of GDS23. [Modes for carrying out the invention]

[0016] Embodiments of the present invention will be described below with reference to the drawings. This invention was created by utilizing a newly discovered property of diacylglycerol polyethylene glycol adduct (diacylglycerol PEG adduct). The newly discovered property is the ability to amplify the expression of human epidermal cell transporter genes and related genes.

[0017] The structural formula of the diacylglycerol PEG adduct, a lipid molecule related to the present invention, is schematically shown.

[0018] [ka]

[0019] Diacylglycerol PEG adducts consist of a glycerol skeleton (CH2CHCH2) with three carbon atoms, a PEG chain which is a linear polyethylene glycol bonded to one of the terminal carbon atoms of the skeleton, and identical long-chain fatty acids (COOR) bonded to the other two carbon atoms of the skeleton. The PEG chain portion is hydrophilic, while the long-chain fatty acid portion is hydrophobic.

[0020] In the following explanation, when referring to a specific diacylglycerol PEG adduct, it will be denoted as "[di] + [name of long-chain fatty acid] + [glycerol] + [PEG-n]" based on the type of long-chain fatty acid and the number of n in the PEG chain. For example, if the long-chain fatty acid is myristic acid and the number of n in the PEG chain is 12, it will be referred to as "dimrylistate PEG-12". In addition, specific diacylglycerol PEG adducts may also be referred to by abbreviations.

[0021] The number of carbon atoms in R in long-chain fatty acids can be in the range of 11 to 23. Examples of long-chain fatty acids in this range include myristic acid, palmitic acid, stearic acid, or oleic acid. The number of n atoms in the PEG chain can be in the range of 11 to 46. The following are examples of diacylglycerol PEG adducts related to the present invention. The melting point and abbreviation are shown in parentheses. • Glycerol dimyristate PEG-12 (25.0℃: GDM12) Glycerol distearate PEG-12 (40.0℃: GDS12) Glycerol distearate PEG-23 (39.8℃: GDS23) Glycerol dipalmitate PEG-23 (31.2℃:GDP23) Glycerol dioleate PEG-12 (25.0℃: GDO12)

[0022] Various transport proteins (so-called transporters) present in the cell membrane of human epidermal cells are responsible for the uptake and excretion of various substances into and out of epidermal cells. In this invention, the epidermal cell transporters and related genes whose expression amplification by diacylglycerol PEG adduct has been confirmed are SVCT2, SLC7A11, GSR, ABCC1, and aquaporin 3. In this specification, these are collectively referred to as "epidermal cell transporter-related genes." Each of these genes codes for a corresponding protein or enzyme.

[0023] The SVCT2 protein, encoded by the SVCT (sodium-dependent vitamin C transporter)2 gene, forms the pathway necessary for vitamin C (ascorbic acid) to pass through cells. Vitamin C inhibits melanin production in epidermal cells, contributing to skin whitening. Ascorbic acid can be administered to the skin, for example, as an ingredient in cosmetics. Because ascorbic acid has insufficient stability and skin penetration, ascorbic acid derivatives with improved properties are also used as a substitute for or in combination with ascorbic acid. Ascorbic acid derivatives are either converted to ascorbic acid in the skin or act in a similar manner to ascorbic acid.

[0024] The xCT (cystine / glutamate transporter) protein encoded by the SLC7A11 gene takes cystine, an amino acid, into cells. Once inside the cell, cystine is reduced to cysteine, which, along with glutamic acid and glycine, produces the tripeptide glutathione (reduced glutathione). Reduced glutathione is an antioxidant that protects cells from oxidation by being oxidized itself (oxidized glutathione). Although cystine can be synthesized in the body, it can also be administered to the skin as an ingredient in cosmetics, for example. When cystine is administered externally, it is converted into dimer cystine in the skin and taken up into cells by xCT transporters.

[0025] The glutathione-disulfide reductase (GSR) gene encodes glutathione reductase, an enzyme that reduces oxidized glutathione back to reduced glutathione. Although GSR is not a transporter itself, it acts in conjunction with the xCT and ABCC1 transporters, and therefore, in this invention, it is considered a transporter-related gene. Glutathione is an amino acid that can be synthesized in the body, but it can also be administered to the skin as an ingredient in cosmetics, for example. Glutathione administered externally (reduced glutathione) is also reduced by glutathione reductase when oxidized in the skin.

[0026] The ABCC1 protein, encoded by the ABC (ATP binding cassette) C1 gene, is one of the proteins in the ABC transporter family and is also known as MRP1. The ABCC1 protein expels glutathione, to which harmful substances such as toxins have been attached, from the cell to the outside.

[0027] The aquaporin 3 protein, encoded by the aquaporin 3 gene, functions as a water channel in the epidermis that regulates the amount of water in cells. The aquaporin 3 protein is responsible for distributing water throughout the epidermis, thereby maintaining skin hydration and elasticity.

[0028] The inventors discovered that applying diacylglycerol PEG adducts to human epidermis amplifies the expression of epidermal cell transporter-related genes. This amplification of epidermal cell transporter-related gene expression leads to increased production of the proteins and enzymes they encode. This is a novel effect of diacylglycerol PEG adducts on human epidermis, particularly in relation to membrane transport in the epidermal cell membrane, and represents a new property of diacylglycerol PEG adducts. This property can provide effects on the epidermis such as skin whitening, antioxidant, detoxification, and moisturizing. This is not merely a physical protective effect on the epidermal surface, but an effect obtained within the epidermal cells themselves.

[0029] This invention provides an expression amplification agent for epidermal cell transporter-related genes, utilizing newly discovered properties of the diacylglycerol PEG adduct, with the diacylglycerol PEG adduct as an active ingredient. Furthermore, this invention provides a method for amplifying the expression of epidermal cell transporter-related genes using the diacylglycerol PEG adduct as an active ingredient.

[0030] In this invention, when applying diacylglycerol PEG adducts to human epidermis, one type may be used alone, or multiple types may be used in combination.

[0031] According to the present invention, diacylglycerol PEG adducts that reach the epidermis can amplify the expression of epidermal cell transporter-related genes and increase the production of corresponding proteins and enzymes compared to the absence of diacylglycerol PEG adducts. As a result, not only the condition within the epidermis but also the condition of the epidermal surface is improved. Therefore, the present invention can provide cosmetics or topical skin preparations containing diacylglycerol PEG adducts as an active ingredient, acting as an expression amplifier for epidermal cell transporter-related genes. Both cosmetics and topical skin preparations are applied to the surface of the epidermis. Here, all substances other than cosmetics are referred to as topical skin preparations, such as ointments. Such cosmetics or topical skin preparations can be provided in various forms, such as aqueous solutions, emulsions, gels, and creams. In addition to diacylglycerol PEG adducts, these cosmetics or topical skin preparations may contain other active ingredients and / or various commonly included components.

[0032] One method for delivering diacylglycerol PEG adducts to the human epidermis is to deliver them to the epidermis in a solution form dissolved in water or a predetermined solvent. For example, a solution of diacylglycerol PEG adducts at a predetermined concentration using phosphate-buffered saline (PBS(-)) as a solvent can be prepared and applied to the epidermal surface to allow penetration into the epidermis. The applied solution penetrates, for example, into the stratum corneum, the uppermost layer, then into the granular layer below the stratum corneum, and further into the layers below that. The diacylglycerol PEG adducts then amplify the expression of epidermal cell transporter-related genes in each layer of the epidermis into which they penetrate.

[0033] Another method for delivering diacylglycerol PEG adducts to the epidermis is to deliver them in vesicle form. Such vesicles are formed as closed spherical shells consisting of a bilayer of diacylglycerol PEG adducts, or multiple layers of bilayers, with hydrophilic PEG chains positioned on the outermost surface. By preparing diacylglycerol PEG adduct vesicles and applying them to the epidermal surface, they can penetrate into the epidermis. After reaching the epidermis, the vesicles decompose and separate into individual molecules, allowing the diacylglycerol PEG adducts themselves to exert their effects.

[0034] In conventional drug delivery systems, diacylglycerol PEG adducts, which are the material for vesicles, have been considered merely carriers of the target substance. However, in this invention, the diacylglycerol PEG adduct itself is used as the active ingredient. Therefore, in this invention, the target substance incorporated into the vesicle, which is typically required in conventional drug delivery systems, is essentially unnecessary. In this invention, by permeating vesicles formed by mixing only water and diacylglycerol PEG adducts into the epidermis, the diacylglycerol PEG adduct itself can function as an expression amplifier for epidermal cell transporter-related genes.

[0035] Some diacylglycerol PEG adducts spontaneously form vesicles when mixed with water at a predetermined temperature (see Patent Documents 1 and 2). For example, a suspension of GDM12 or GDO12 vesicles can be obtained by mixing 2% by mass of GDM12 or GDO12 with 98% by mass of deionized water at room temperature and stirring. Another example is to dissolve 2% by mass of GDS12 or GDS23 at 45-55°C, then mix with 98% by mass of deionized water at 45-55°C and stir to obtain a suspension of GDS12 or GDS23 vesicles. Yet another example is to dissolve 2% by mass of GDP23 at 37°C, then mix with 98% by mass of deionized water at 37°C and stir to obtain a suspension of GDP23 vesicles. The vesicles remain stable even when the suspension obtained at a temperature higher than room temperature is cooled to room temperature.

[0036] As another example, the present invention also includes the use of vesicles formed by mixing and stirring aqueous solutions of various substances with diacylglycerol PEG adducts, instead of the water described above. In this case, the substances contained in the aqueous solution may be given other functions.

[0037] As yet another example, the present invention also includes cases where the surface of vesicles formed by mixing and stirring water or an aqueous solution with a diacylglycerol PEG adduct is modified with a charged element such as a cationic surfactant. Patent Document 2 describes that positively charged vesicles have particularly excellent permeability and retention properties in the epidermis.

[0038] The following shows the relationship between the application of diacylglycerol PEG adducts to epidermal cells and epidermal cell transporter-related genes, based on test data.

[0039] (1) Tests on the amplification of expression of epidermal cell transporter-related genes We conducted tests to confirm the amplification of mRNA expression in each epidermal cell transporter-related gene.

[0040] (1-1) Test Method Normal human epidermal keratinocytes (NHEKs) 2.0 × 10⁻⁶ 4 Cells were seeded in HuMedia-KG2 medium (Kurabo Industries Ltd.) at a cell density of cells / well into 96-well plates and cultured for 24 hours at 37°C under 5% CO2 conditions. Subsequently, diacylglycerol PEG adducts were added to HuMedia-KB2 medium (Kurabo Industries Ltd.), and each cell was cultured at 37°C under 5% CO2 conditions for a predetermined time. Table 1 shows the target gene, the type of diacylglycerol PEG adduct added, the amount added, and the culture time. The control (no adduct) was cultured under the same conditions.

[0041] [Table 1]

[0042] After culturing each sample for a predetermined time, RNA was extracted from each sample and control cell. The extracted RNA was reverse transcribed to prepare cDNA, and the mRNA of the target gene was quantified by quantitative real-time PCR expression analysis. GAPDH (glyceraldehyde 3-phosphate dehydrogenase) was used as the internal standard.

[0043] The analysis involved correcting the mRNA expression level of each target gene using the expression level of GAPDH, an internal standard in the same sample. Furthermore, the corrected value of the sample was calculated as the relative expression level, with the control's corrected value set to 1.

[0044] (1-2) Test results Figure 1 shows the relative expression levels of the SVCT2 gene in GDS23, and Figure 2 shows the relative expression levels in GDM12. In GDS23, the expression level was approximately 1.3 to 1.7 times higher than that of the control. In GDM12, the expression level was approximately 1.4 times higher than that of the control.

[0045] Figure 3 shows the relative expression levels of the SLC7A11 gene in GDS23, and Figure 4 shows the relative expression levels in GDM12. In GDS23, the expression level was approximately 4 to 12 times higher than that of the control. In GDM12, the expression level was approximately 1.4 times higher than that of the control.

[0046] Figure 5 shows the relative expression levels of the GSR gene in GDS23, and Figure 6 shows the relative expression levels in GDM12. In GDS23, the expression level was approximately 1.8 times higher than that of the control. In GDM12, the expression level was approximately 1.5 times higher than that of the control.

[0047] Figure 7 shows the relative expression levels of the ABCC1 gene in GDS23, and Figure 8 shows the relative expression levels in GDM12. In GDS23, the expression level was approximately 2.1 times higher than that of the control. In GDM12, the expression level was approximately 1.3 times higher than that of the control.

[0048] Figure 9 shows the relative expression levels of the aquaporin 3 gene in GDS23, and Figure 10 shows the relative expression levels in GDM12. In GDS23, the expression level was approximately 1.6 to 3.5 times higher than that of the control. In GDM12, the expression level was approximately 1.6 to 2.2 times higher than that of the control.

[0049] (2) Tests on increased SVCT2 protein production A Western blotting test was performed to confirm increased production of the protein encoded by the SVCT2 gene.

[0050] (2-1) Test Method Normal human epidermal keratinocytes (NHEKs) are divided into 1.0 × 10⁻⁶ cells. 5 Cells were seeded in HuMedia-KG2 medium at a cell density of cells / well into 24-well plates and cultured for 24 hours at 37°C under 5% CO2 conditions. Subsequently, 50 μM glycerol distearate PEG-23 (GDS23) was added to HuMedia-KB2 medium, and the cells were incubated at 37°C under 5% CO2 conditions for 24 hours. The control (no additive) was cultured in HuMedia-KB2 medium alone.

[0051] After culturing, Western blotting was performed. First, proteins were extracted from the cells using SDS-PAGE containing 10% mercaptoethanol. Electrophoresis of the protein extract was performed using a polyacrylamide gel, and the proteins were transferred to a membrane using a semi-dry method. SVCT2 protein was quantified using the predetermined primary and secondary antibodies shown in Table 2. The amount of SVCT2 protein was corrected by the amount of GAPDH, an internal standard in the same sample, and the corrected value of the sample was calculated with the control's corrected value set to 1.

[0052] [Table 2]

[0053] (2-2) Test Results Figure 11 shows the band image of the SVCT2 protein in a Western blot related to GDS23. Figure 12 is a graph showing the expression level of the SVCT2 protein.

[0054] As shown in Figure 11, an increase in SVCT2 protein was observed in the GDS23-treated sample. As shown in Figure 12, the amount of SVCT2 protein in the GDS23-treated sample was approximately 1.4 times that of the control.

[0055] (3) Evaluation test of the effect of increased SVCT2 on cellular uptake of vitamin C (ascorbic acid) The results of the tests in (1) and (2) above confirmed that the application of diacylglycerol PEG adducts to epidermal cells increased the expression of the SVCT2 gene, and therefore increased the production of the vitamin C transporter protein encoded by it. Therefore, the cellular uptake effect of vitamin C was evaluated. This evaluation was performed by quantifying the cytotoxicity of hydroquinone (HQ) when diacylglycerol PEG adducts, vitamin C, and hydroquinone (HQ) were added to epidermal cells. Hydroquinone itself does not have cytotoxic effects and has a whitening effect, but when it is oxidized to benzoquinone, it exhibits cytotoxicity (see Patent Document 4). On the other hand, if hydroquinone is added to the epidermis together with an oxidation inhibitor, the hydroquinone should not be oxidized, and its cytotoxicity should be suppressed (the same applies to the evaluation test in (4) below).

[0056] (3-1) Test Method Normal human epidermal keratinocytes (NHEKs) 2.0 × 10⁻⁶ 4 Cells were seeded in HuMedia-KG2 medium at a cell density of cells / well into a 96-well plate and cultured for 24 hours at 37°C under 5% CO2 conditions. Subsequently, samples with 50 μM glycerol distearate PEG-23 (GDS23) added and samples without GDS23 were cultured in HuMedia-KB2 medium at 37°C under 5% CO2 conditions for 24 hours.

[0057] Next, the samples were washed with PBS(-), and the GDS23-added and GDS23-free samples were cultured for 24 hours in the following six different culture media. HuMedia-KB2 medium containing 0 μM ascorbic acid (AsA) HuMedia-KB2 medium containing 1 μM ascorbic acid (AsA) HuMedia-KB2 medium containing 5 μM ascorbic acid (AsA) HuMedia-KB2 medium containing 10 μM ascorbic acid (AsA) HuMedia-KB2 medium containing 50 μM ascorbic acid (AsA) HuMedia-KB2 medium containing 100 μM ascorbic acid (AsA)

[0058] The samples were then washed with PBS(-) and incubated for 24 hours in HuMedia-KB2 medium containing 400 μM hydroquinone (HQ).

[0059] The control group (without GDS23, AsA, or HQ additives) was cultured in HuMedia-KB2 medium only.

[0060] Subsequently, cell viability was measured using a neutral red assay. The cell viability of the control sample was set to 100%, and the cell viability of the GDS23-treated sample and the GDS23-untreated sample were calculated separately.

[0061] (3-2) Test Results Figure 13 is a graph showing the results of an evaluation test of the cellular uptake effect of vitamin C with respect to GDS23. Excluding the control on the far left, the six samples on the left are samples without GDS23, and the six samples on the right are samples with GDS23 added.

[0062] Of the six GDS23-free samples, the leftmost one is untreated with ascorbic acid, while the other five are treated with ascorbic acid. Comparing these, it can be seen that there is no difference in cell viability between the untreated and untreated samples when treated with low concentrations of ascorbic acid (5-100 μM), indicating that ascorbic acid has almost no effect on hydroquinone-induced cell damage.

[0063] In contrast, the GDS23-treated samples showed a higher cell viability compared to the GDS23-free samples, indicating a significant reduction in hydroquinone-induced cell damage. Among the six GDS23-treated samples, the ascorbic acid-treated sample (the leftmost sample) showed a higher cell viability than the ascorbic acid-free sample. This is thought to be because the increase in SVCT2 due to GDS23 increased the uptake of ascorbic acid into cells, leading to an increase in intracellular ascorbic acid concentration and suppression of hydroquinone oxidation.

[0064] Furthermore, among the GDS23-added samples, the cell viability was increased even in the ascorbic acid-free sample compared to the GDS23-free sample. This is thought to be due to the HQ cytotoxicity reduction effect of GDS23 itself, as disclosed in Patent Document 4, and is considered to be due to a mechanism different from the vitamin C transporter-increasing effect of the present invention.

[0065] (4) Evaluation test of cystine uptake effect by increasing SLC7A11 The results of the tests in (1) and (2) above confirmed that the application of diacylglycerol PEG adducts to epidermal cells increased the expression of the SLC7A11 gene, and therefore increased the production of the xCT transporter protein encoded by it. Therefore, the cytotoxic effect of cystine was evaluated. This evaluation was performed by quantifying the cytotoxicity of hydroquinone (HQ) when diacylglycerol PEG adducts, cystine, and hydroquinone (HQ) were added to epidermal cells.

[0066] (4-1) Test Method Normal human epidermal keratinocytes (NHEKs) 2.0 × 10⁻⁶ 4 Cells were seeded in HuMedia-KG2 medium at a cell density of cells / well into a 96-well plate and cultured for 24 hours at 37°C under 5% CO2 conditions. Subsequently, samples with 50 μM glycerol distearate PEG-23 (GDS23) added and samples without GDS23 were cultured in HuMedia-KB2 medium at 37°C under 5% CO2 conditions for 24 hours.

[0067] Next, the samples were washed with PBS(-), and the GDS23-added and GDS23-unadded samples were cultured for 30 hours in the following three different culture media. Note that the cysteine ​​added to the culture medium is oxidized in the medium or between cells to be converted to its dimer, cystine, which is then taken up by the cells. HuMedia-KB2 medium containing 0 μM cysteine ​​(Cys). HuMedia-KB2 medium containing 10 μM cysteine ​​(Cys) HuMedia-KB2 medium containing 50 μM cysteine ​​(Cys)

[0068] The samples were then washed with PBS(-) and incubated for 24 hours in HuMedia-KB2 medium containing 400 μM hydroquinone (HQ).

[0069] The control group (without GDS23, Cys, or HQ) was cultured in HuMedia-KB2 medium only.

[0070] Subsequently, cell viability was measured using a neutral red assay. The cell viability of the control sample was set to 100%, and the cell viability of the GDS23-treated sample and the GDS23-untreated sample were calculated separately.

[0071] (4-2) Test Results Figure 14 is a graph showing the results of an evaluation test of the cystine uptake effect of GDS23. Excluding the control on the far left, the three samples on the left are GDS23-free samples, and the three samples on the right are GDS23-added samples.

[0072] Of the three GDS23-free samples, the leftmost one is cysteine-free, while the other two are cysteine-treated. Comparing these, the 10 μM and 50 μM cysteine-treated samples showed no difference in cell viability compared to the cysteine-free samples, indicating that cysteine ​​(cystine) has almost no effect on hydroquinone-induced cell damage. In other words, it can be seen that cystine uptake does not occur in the GDS23-free samples.

[0073] In contrast, the GDS23-treated sample showed a higher cell viability compared to the GDS23-free sample, indicating a significant reduction in hydroquinone-induced cell damage. Among the three GDS23-treated samples, the other two cysteine-treated samples showed even greater cell viability than the leftmost cysteine-free sample. This is thought to be because the increase in SLC7A11 due to GDS23 increased the uptake of cystine into cells, leading to an increase in intracellular cystine concentration and enhanced glutathione production, which in turn suppressed hydroquinone oxidation.

[0074] Furthermore, even in the leftmost cysteine-free sample among the GDS23-added samples, the cell viability is increased compared to the GDS23-free sample. This is thought to be due to the HQ cytotoxicity reduction effect of GDS23 itself, as disclosed in Patent Document 4, and is considered to be due to a mechanism different from the xCT transporter-enhancing effect of the present invention.

[0075] (5) Preparation of cosmetics and topical skin preparations The following are examples of preparations for cosmetics and topical skin preparations containing an epidermal cell transporter-related gene expression amplifier with diacylglycerol PEG adduct as the active ingredient. Each of the following preparations includes an ascorbic acid derivative. The ascorbic acid derivative has improved stability and skin permeability compared to ascorbic acid. After penetration into the skin, the ascorbic acid derivative is converted to ascorbic acid by decomposition or enzymatic action and acts accordingly, or it acts in the same way as ascorbic acid without modification. Examples of ascorbic acid derivatives include 3-O-ethyl ascorbic acid, 3-laurylglyceryl ascorbic acid, and myristyl 3-glyceryl ascorbic acid. The cosmetics and other preparations of the present invention may contain either or both ascorbic acid and / or ascorbic acid derivatives.

[0076] (5-1) Example of lotion preparation The lotion in Preparation Example 1 contains glycerol dimyristate PEG-12, glycerol distearate PEG-23, ascorbic acid and its derivatives, and glutathione.

[0077] [Table 3]

[0078] (5-2) Example of emulsion preparation The emulsion of Preparation Example 2 contains glycerol dimyristate PEG-12, glycerol distearate PEG-23, ascorbic acid and its derivatives, cysteine, and glutathione.

[0079] [Table 4]

[0080] (5-3) Example of preparation of water-based gel The aqueous gel in Preparation Example 3 contains glycerol dimyristate PEG-12, glycerol distearate PEG-23, ascorbic acid and its derivatives, and glutathione.

[0081] [Table 5]

[0082] (5-4) Examples of preparations for hydroquinone-containing creams The hydroquinone-containing cream of Preparation Example 4 contains hydroquinone, as well as glycerol dimyristate PEG-12, glycerol distearate PEG-23, ascorbic acid and its derivatives, and glutathione. The antioxidant effects of these substances suppress the cytotoxicity of hydroquinone. As a result, the cosmetic effects of hydroquinone are exerted.

[0083] [Table 6]

[0084] (5-5) Example of preparation of hydroquinone-containing ointment The hydroquinone-containing ointment of Preparation Example 5 contains hydroquinone, as well as glycerol distearate PEG-23, ascorbic acid, glutathione, and cysteine.

[0085] [Table 7]

[0086] Although not exemplified, there are many other combinations of ingredients besides diacylglycerol PEG adducts. One or more ingredients from the group consisting of ascorbic acid, ascorbic acid derivatives, glutathione, and cysteine ​​are selected as needed. In such cases, hydroquinone may also be included. These points are also true for topical skin preparations other than cosmetics.

[0087] The present invention has been described above with reference to the examples, but the present invention is not limited to these examples, and obvious modifications therefrom are also included in the present invention.

Claims

1. An agent for amplifying the expression of epidermal cell transporter-related genes, The active ingredient is a diacylglycerol PEG adduct, wherein the diacylglycerol PEG adduct has the following structural formula, the number of carbon atoms in R in the long-chain fatty acid is in the range of 11 to 23, and n in the polyethylene glycol chain is in the range of 11 to 46. An epidermal cell transporter-related gene expression amplifier wherein the epidermal cell transporter-related gene is SVCT2. 【Chemistry 1】

2. The epidermal cell transporter-related gene expression amplification agent according to claim 1, wherein the diacylglycerol PEG adduct is at least one from the group consisting of glycerol dimyristate PEG-12 (GDM12), glycerol distearate PEG-12 (GDS12), glycerol distearate PEG-23 (GDS23), glycerol dipalmitate PEG-23 (GDP23), and glycerol dioleate PEG-12 (GDO12).

3. The epidermal cell transporter-related gene expression amplification agent according to claim 1, wherein the diacylglycerol PEG adduct penetrates into the epidermis in a solution state or in a vesicle state.

4. A cosmetic composition comprising the epidermal cell transporter-related gene expression amplification agent described in claim 1.

5. The cosmetic composition according to claim 4, further comprising ascorbic acid or an ascorbic acid derivative.

6. The cosmetic composition according to claim 5, further comprising hydroquinone.

7. A topical skin preparation comprising the epidermal cell transporter-related gene expression amplification agent described in claim 1.

8. The topical skin preparation according to claim 7, further comprising ascorbic acid or an ascorbic acid derivative.

9. The topical skin preparation according to claim 8, further comprising hydroquinone.

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