Composition for inhibiting the degradation of laminin 511

Abstract

To provide a novel composition for application on the skin of human or the like that prevents the deterioration of skin function.SOLUTION: The present invention provides a composition that comprises tranexamic acid for inhibiting the degradation of laminin 511 in the skin.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to, for example, a composition containing tranexamic acid for use on the skin of humans or the like, which inhibits the degradation of laminin 511 in the skin, and the like.

[0002] The epidermis of the skin is a skin tissue present in the outermost layer of the skin. The epidermis is mainly composed of the stratum corneum, stratum granulosum, stratum spinosum, and stratum basale. The basal cells present in the stratum basale divide and move to the outer layer. During this movement, enucleation occurs in the cells, and they flatten and differentiate into the stratum corneum, which eventually peels off. This turnover period is said to be about 45 days. However, in aging skin, the turnover rate slows down, and the entire epidermis becomes thinner. As a result, it is known that skin functions such as a decrease in barrier function and a decrease in moisture content occur (Patent Document 1).

[0003] Laminin is an important protein that constitutes the basement membrane such as the skin epidermis. Laminin is one of the major extracellular matrices and is a protein involved in cell adhesion, migration, proliferation, etc. Laminin is a heterotrimeric molecule having three different subunits (α-chain, β-chain, γ-chain). To date, five types of α-chains (α1, α2, α3, α4, α5), three types of β-chains (β1, β2, β3), and three types of γ-chains (γ1, γ2, γ3) have been found, and due to differences in their combinations, there are many isoforms of laminin. Members of the laminin family are named according to the types of subunits. For example, laminin consisting of α5-chain, β1-chain, and γ1-chain is called laminin 511 (Patent Document 2). Laminin controls the development of epithelium across the boundary between epithelium and stroma and is essential for the expression of epithelial functions (Patent Document 3).

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

[0005] [Non-Patent Document 1] Journal of the Japanese Society on Thrombosis and Hemostasis, Vol. 31, No. 4, 2020, pp. 381-387, The Role of the Plasminogen Activator-Plasmin System in the Nervous System: From Neurophysiology to Pathophysiology. [Overview of the project] [Problems that the invention aims to solve]

[0006] The problems that this invention aims to solve include providing a novel composition for preventing the deterioration of skin function for use on human skin, etc. [Means for solving the problem]

[0007] Therefore, after diligent research, the inventors of the present invention have newly confirmed that by adding tranexamic acid to a pseudo-epidermal tissue consisting of a pseudo-basement membrane that mimics the basement membrane of human epidermis (for example, one containing laminin 511) and a culture medium of human epidermal cells, the degradation of the pseudo-basement membrane can be suppressed (for example, the basement membrane and basal layer can be protected), and thus the present invention has been completed.

[0008] The present invention includes the following items. [Item 1] A composition containing tranexamic acid for inhibiting the degradation of laminin 511 in the skin (such as tissues including the basement membrane and specific cells in human epidermis). [Item 2] The composition according to [Item 1], in the form of a composition for external use on the skin. [Effects of the Invention]

[0009] The present invention provides novel compositions for preventing the deterioration of skin function for use on human skin, etc. [Brief explanation of the drawing]

[0010] [Figure 1] The results of the Western blot performed in Experiment 1 are shown. [Modes for carrying out the invention]

[0011] The following describes embodiments for carrying out the present invention.

[0012] (Tranexamic acid) Tranexamic acid is an abbreviation for trans-4-aminomethylcyclohexane-1-carboxylic acid. Tranexamic acids include tranexamic acid, tranexamic acid salts, tranexamic acid esters, tranexamic acid amides, and polymers of tranexamic acid. Examples of salts in tranexamic acid salts include metal salts such as sodium salts, potassium salts, and magnesium salts; and inorganic salts such as hydrochloride salts, phosphate salts, and sulfate salts. Examples of esters in tranexamic acid esters include vitamin esters such as vitamin A esters, vitamin E esters, and vitamin C esters, and alkyl esters. Examples of amides in tranexamic acid amides include methylamide.

[0013] In the present invention, tranexamic acid also includes derivatives of tranexamic acid. Examples of derivatives of tranexamic acid include tranexamic acid dimers (trans-4-(trans-4-aminomethylcyclohexanecarbonyl)aminomethylcyclohexanecarboxylic acid hydrochloride), esters of tranexamic acid and hydroquinone (trans-4-aminomethylcyclohexanecarboxylic acid 4'-hydroxyphenyl ester), esters of tranexamic acid and gentisic acid (2-(trans-4-aminomethylcyclohexylcarbonyloxy)-5-hydroxybenzoic acid and its salts), amides of tranexamic acid (trans-4-aminomethylcyclohexanecarboxylic acid methylamide and its salts, trans-4-acetylaminomethylcyclohexanecarboxylic acid and its salts, trans-4-(p-methoxybenzoyl)aminomethylcyclohexanecarboxylic acid and its salts, trans-4-guanidinomethylcyclohexanecarboxylic acid and its salts, etc.).

[0014] There are no particular limitations on the amount of one or more tranexamic acid (including derivatives of tranexamic acid) selected from those contained in the composition of the present invention. However, in order to achieve the desired effect, the amount of tranexamic acid relative to the total amount of the composition is generally preferably 0.001% by weight or more, more preferably 0.01% by weight or more, and even more preferably 0.1% by weight or more. Furthermore, considering the desired effect, etc. (for example, the fact that even if a certain amount is added, a substantial increase in effect cannot be expected, and that it tends to become difficult to incorporate in the formulation of topical skin preparations), the amount of tranexamic acid relative to the total amount of the composition is generally preferably 20% by weight or less, more preferably 10% by weight or less, and even more preferably 7% by weight or less.

[0015] (Inhibition of laminin 511 degradation) Laminin 511 is degraded by ultraviolet light. Damage to the basement membrane caused by ultraviolet light leads to the formation of wrinkles and age spots. The factors involved in the degradation of laminin 511 are largely unknown. However, the following has been elucidated regarding its role in regulating brain nerve function (Non-Patent Literature 1).

[0016] The plasminogen activator-plasmin (PA-Plm) system is a reaction in which plasminogen (PLG) is selectively cleaved by tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) to produce plasmin. Plasmin (Plm) degrades extracellular matrix components such as laminin, which serves as a scaffold for nerve cells, and neurocan, a component of the perineuronal network. However, this degradation of the extracellular matrix promotes neurite formation and dendritic spine motility, suggesting that plasmin enhances neuronal plasticity. Plasmin activity has been reported to impair long-term potentiation (LTP), induce neurodegeneration and neuronal death, and disrupt axonal guidance in hippocampal mossy fibers. The mechanism of this disruption is suggested to be plasmin-mediated degradation of laminin and activation of monocyte chemotactic protein-1, a monocyte migration factor. These plasmin-mediated neurotoxic effects have been demonstrated in neuroexcitotoxicity models that induce the expression of tPA (tissue plasminogen activator) and plasmin, as well as in experimental systems involving the addition of plasmin to cultured cells, suggesting that they are due to plasmin's excessive extracellular protein degradation. Furthermore, it has been shown that excessive activation of the tPA-Plm system in the brain suppresses dendritic formation and synapse formation in Purkinje cells of the cerebellum. The fact that tPA or Plm-deficient mice show tolerance to restraint stress-induced memory and learning impairment, and that restraint stress increases tPA activity, at least in the amygdala, suggests that stress-induced enhancement of the tPA-Plm system impairs synaptic activity during the memory and learning process. Additionally, the fact that inhibition of plasmin activity in the basolateral amygdala reduces social avoidance due to social defeat stress suggests that plasmin is also involved in the formation or reorganization of neural circuits that induce depression-related behaviors.

[0017] The following examples demonstrate that in a pseudo-epidermal basement membrane containing components that constitute the human epidermal basement membrane (e.g., laminin 511), the addition of tranexamic acid suppressed the degradation of laminin 511 by uPA and PLG.

[0018] (Form of the composition) The composition according to the present invention (such as a composition for external use on the skin) can be in a form suitable for use, such as an ampoule, capsule, powder, granule, liquid, gel, foam, emulsion, sheet, mist, spray, etc. It includes 1) pharmaceuticals, 2) quasi-drugs, 3) topical or systemic external skin preparations (for example, basic cosmetics such as lotion, emulsion, cream, ointment, lotion, oil, pack, etc., cleansing agents for the face and skin such as solid soap, liquid soap, hand wash, skin cleansing agents, massage agents, cleansing agents, depilatory agents, hair removal agents, beard shaving treatment agents, after-shave lotion, pre-shave lotion, shaving cream, foundation, lipstick, blush, eye shadow, eyeliner, mascara, etc. makeup cosmetics, perfumes, nail cosmetics, nail enamel, nail enamel remover, patch materials, plaster materials, tape agents, sheet materials, adhesive agents, aerosol agents, etc.), 4) pharmaceutical or / and cosmetic preparations for application to the scalp and hair (for example, shampoo, rinse, hair treatment, pre-hair treatment, permanent solution, hair dye, hair styling agent, hair tonic, hair growth and nourishing agent, patch, plaster, tape, sheet, aerosol, etc.), 5) bath agents to be added to bath water, 6) and others, such as axillary odor preventives, deodorants, antiperspirants, sanitary products, sanitary napkins, wet tissues, etc.

[0019] In addition, such a composition may, if necessary, contain, within a range that does not impair the effects of the present invention, for example, components commonly used in cosmetics, that is, oily components, lower alcohols, polyhydric alcohols, aqueous components such as humectants, ultraviolet absorbers, antioxidants, beauty components, preservatives, oil-soluble resins, dyes, cooling agents, pigments, fragrances, etc., as appropriate within a range that does not interfere with the effects of the present invention.

[0020] Next, examples will be given to explain the present invention in more detail, but the present invention is not limited to these examples in any way. In the following examples, the unit % of the numerical values indicating the addition amounts of various components means mass %.

Examples

[0021] The following describes embodiments of the present invention. The experimental materials used in the experiments described in the following embodiments are as follows. ·KGM medium: KGMtm Gold Keratinocyte Growth Medium BulletKittm, Calcium Free, Lonza, 00195769 • PLG: Plasminogen, Sigma-Aldrich, 528180 • uPA: Urokinase-type plasminogen activator, R&D systems, 1310-SE-010 • TXA: Tranexamic acid, Japanese Pharmacopoeia Tranexamic Acid • iMatrix-511: A model (scaffold) of the human epidermal basement membrane, a recombinant protein with the same sequence as the laminin 511-E8 fragment, Nippi Corporation (892011, 892012) • Bradford method: Protein quantification was performed using the Bradford Protein Assay Kit (BIORAD). Following the instructions for this kit, the sample solution (containing protein) and Coomassie blue reagent were thoroughly mixed, and the difference in absorbance at a wavelength of 595 nm between the sample solution and a protein-free background was measured using a spectrophotometer. The protein concentration was determined from the standard straight line drawn from the absorbance of a standard sample (standard; in Experiment 1 below, BSA: bovine serum albumin was used) with a known protein concentration.

[0022] (Experiment 1: Confirmation of whether or not the addition of tranexamic acid inhibits the degradation of laminin 511) In a human epidermal basement membrane model (iMatrix-511), the presence or absence of laminin 511 (a factor constituting the basement membrane) was confirmed by observing the inhibitory effect of adding tranexamic acid on its degradation.

[0023] (Experimental method) (1) Incubation of a pseudo-basement membrane (iMatrix511) with the addition of a predetermined sample. The following samples, as shown in Table 1, were mixed with KGM medium, a specialized culture medium used for epidermal cell culture, and added to dishes coated with iMatrix-511. The addition of the samples shown in Table 1 was performed to create the following groups.

[0024] [Table 1]

[0025] After adding the samples listed in Table 1, the cultures were incubated for 16 hours under conditions of 5% CO2 and 37°C.

[0026] (2) Preparation of samples for use in Western blotting After 16 hours of incubation of (1), the iMatrix was recovered using an extraction solution containing Passive Lysis Buffer 5x (Promega), cOmplete™, Mini Protease Inhibitor Cocktail (Roche), and phenylmethylsulfonyl fluoride (PMSF). After vortexing and freeze-thawing, the supernatant obtained by centrifugation was reduced with β-mercaptoethanol and further denatured by heat, and this was used as the sample for Western blotting.

[0027] (3) Western blot The proteins from each group (Test Examples 1 to 5) recovered in (2) were separated on an SDS-polyacrylamide gel. After electrophoresis by conventional polyacrylamide electrophoresis (SDS-PAGE), the samples were blotted onto a PVDF membrane (TransBlot Turbo Transfer Pack (BioRad)). The membrane was blocked with blocking buffer PVDF Blocking Reagent (TOYOBO) for 1 hour. The membrane was then washed with washing buffer (Tris buffer solution containing 0.1% Tween). Subsequently, the membrane was incubated overnight at 4°C in Can Get Signal Solution 1 (TOYOBO) with anti-Lamininα5 antibody (abcam, ab14509). The blot was then washed with washing buffer and incubated with secondary antibody (cytiva) at room temperature for 1 hour. After binding of the secondary antibody, the sample was washed with a specified washing buffer, and Lamininα5 was detected using ECL-PLUS Western blotting detection reagent (Amersham). The band intensity was analyzed using densitometry and compared with the control group.

[0028] (Experimental results) The experimental results are shown in Figure 1. In Figure 1, the marker bands are shown in the leftmost lane. The band shown around a molecular weight of 100 kDa represents Laminin α5. The intensity of the bands in each group, with Test Example 1 set to 1.000, was 0.303 for Test Example 2, 0.400 for Test Example 3, 0.499 for Test Example 4, and 0.853 for Test Example 5. These experimental results indicate that, in a model of the human epidermal basement membrane, the addition of tranexamic acid suppresses the degradation of laminin 511 by uPA and PLG.

[0029] Although embodiments of the present invention (including examples) have been described above with reference to the drawings, the specific configuration of the present invention is not limited thereto, and any design changes, etc., that do not depart from the spirit of the present invention are still included. [Industrial applicability]

[0030] The present invention can be used as a composition for preventing the deterioration of skin function for use on the skin of humans, etc.

Claims

[Claim 1] A composition containing tranexamic acid for inhibiting the degradation of laminin 511 in the epidermal basement membrane.

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