Tight junction relaxation agent containing surfactin and / or sodium salt thereof

Abstract

To provide a novel tight junction relaxation agent, and to provide a compound absorption promoter that contains the relaxation agent.SOLUTION: The present invention provides a tight junction relaxation agent containing surfactin and / or a sodium salt thereof, and a compound absorption promoter containing such a tight junction relaxation agent as an active ingredient.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a tight junction relaxing agent and an absorption enhancer for a compound containing the relaxing agent. [Background technology]

[0002] The skin, especially the epidermis, is known to have a barrier function. This barrier function prevents excessive water loss from the body and the invasion of foreign substances such as bacteria from the outside. A decrease in this barrier function is thought to be a cause of rough skin and dermatitis (xerosis and atopic dermatitis).

[0003] The epidermis is the outermost membrane of the skin, approximately 0.1 to 0.2 mm thick. New cells (keratinocytes) are constantly being born in the basal layer of the epidermis. These keratinocytes differentiate and progress through the spinous layer, granular layer, and stratum corneum to become corneocytes, which are finally exfoliated as dandruff. This process is called turnover. The epidermis undergoes turnover in a cycle of approximately one month.

[0004] During the turnover process, a cornified envelope (CE), primarily composed of keratin fibers, is formed to encase keratinocytes. The CE is formed when multiple CE precursor proteins produced in keratinocytes as they differentiate are crosslinked and insolubilized by the enzyme transglutaminase. Such CE is essential for the function of a healthy stratum corneum. Therefore, the formation and maturation of CE play an important role in improving skin barrier function.

[0005] It is also known that fluids and substances that pass through the epidermis pass through two pathways: the transcellular pathway, which is mediated by transport proteins on the cell membrane, and the paracellular pathway, which is mediated through the intercellular spaces.

[0006] Tight junctions are intercellular adhesion mechanisms (one type of intercellular bond) that seal the gaps between cells in cell sheets in the granular layer. Specifically, tight junctions control the transport of fluids and substances through intercellular pathways. Tight junctions possess diverse skin barrier functions depending on the physiological functions of the epidermis (Patent Document 1). Furthermore, the skin barrier function of tight junctions can be said to regulate the permeability of intercellular pathways. Thus, tight junctions are particularly important for moisturizing the skin and for the blood-brain barrier, which prevents foreign substances from entering the brain. In other words, when drugs are to penetrate the skin and exert their effects, the strong barrier function of tight junctions hinders their penetration. Medical procedures such as intradermal injection of high-molecular-weight functional substances such as collagen, hyaluronic acid, and Botox to reduce or eliminate wrinkles, or irradiation with laser light of a specific wavelength to activate metabolic functions within the dermis, restore skin firmness and elasticity, and eliminate wrinkles, are sometimes used. However, injection of such high-molecular-weight functional substances and laser treatments are painful, and controlling the injection depth within approximately 2 mm is technically difficult, especially when injecting functional substances, and the results depend on the skill of the practitioner. Relaxing the tight junctions in the epidermis may allow drugs that are difficult to penetrate the skin to penetrate more easily and exert their effects. Therefore, substances that relax tight junctions are also attracting attention as targets for the development of cosmetics and medical products. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Patent Publication No. 2012-121871 [Non-patent literature]

[0008] [Non-Patent Document 1] J Pharm.Pharmacol., 2010, 62(6), 702-708 Summary of the Invention [Problem to be solved by the invention]

[0009] An object of the present invention is to provide a novel tight junction relaxing agent and an absorption enhancer for compounds containing such a relaxing agent. [Means for solving the problem]

[0010] The present inventors screened various substances that are expected to have a tight junction relaxing effect and found that surfactin exhibits such a tight junction relaxing effect, leading to the completion of the present invention.

[0011] Therefore, the present application includes the following inventions. (1) A tight junction relaxing agent comprising surfactin and / or its sodium salt. (2) A tight junction relaxant (1) that relaxes tight junctions in the epidermis. (3) A tight junction relaxant of (1) or (2), which relaxes tight junctions through the suppression of cellular claudin expression. (4) A compound absorption enhancer comprising any one of the tight junction relaxants (1) to (3) as an active ingredient. (5) The absorption enhancer of (4), wherein the compound has a medium molecular weight. (6) The absorption enhancer of (4) or (5), wherein the molecular weight of the compound is 4000 or more. (7) The absorption enhancer according to any one of (4) to (6), wherein the compound is hyaluronic acid or its sodium salt. [Effects of the Invention]

[0012] The tight junction relaxant of the present invention relaxes skin tight junctions, thereby facilitating the penetration of various compounds and drugs, ranging from low to high molecular weight compounds, into the skin, thereby achieving, for example, the prevention and improvement of skin conditions such as wrinkles and sagging, as well as the prevention and treatment of skin diseases and rough skin. [Brief explanation of the drawings]

[0013] [Figure 1] We demonstrate that surfactin suppresses CLDN4 expression, thereby inhibiting claudin expression and functioning as a tight junction relaxant. [Figure 2] By suppressing CLDN7 expression, surfactin has claudin expression inhibitory activity and functions as a tight junction relaxant. [Figure 3] This shows that surfactin enhances the penetration of dextran into skin cells. [Figure 4] This shows that surfactin increases the amount of sodium hyaluronate permeated into the skin in a skin model. DETAILED DESCRIPTION OF THE INVENTION

[0014] The present invention provides a tight junction relaxing agent comprising surfactin and / or its sodium salt. Surfactin is known to have a variety of functions, including high surface activity, antibacterial activity, hemolysis, and inhibition of protein denaturation. Surfactin is known to be produced by microorganisms of the genus Bacillus, and industrial mass production of surfactin is possible by culturing these microorganisms. Surfactin or a salt thereof is represented by the following formula: [ka] (In the formula, * represents an optically active site. X represents an amino acid selected from leucine, isoleucine, and valine. R represents an alkyl group or branched alkyl group having 9 to 13 carbon atoms. M represents an alkali metal, alkaline earth metal, substituted or unsubstituted amine, or the like. Preferably, M is sodium.)

[0015] Although surfactin is known to have surface-active properties, there is no information about its effect on tight junctions. Rather, Nicolo et al., J. Pharm. Pharmacol., 2010, 62(6), 702-708 (Non-Patent Document 1) describes that surfactin hinders the skin penetration of asilclovir.

[0016] Tight junctions exist in a band-like structure around cells, sealing the gaps between adjacent cells and maintaining a continuous connection between cells. Tight junctions, which are primarily found in the granular layer of the epidermis and connect cells to each other, prevent water and substances from passing through the intercellular spaces and play an important role in maintaining the barrier function. Furthermore, research into the presence and function of proteins that constitute tight junctions in the granular layer of the epidermis, such as occludin and claudins 1, 4, and 7, is progressing, and the mechanisms underlying skin barrier function are becoming more detailed. The state of tight junctions can also be indirectly examined by examining the presence or absence or enhancement of the expression of these tight junction-constituting proteins, such as claudins. As shown in the Examples, the tight junction relaxing agent of the present invention exerts its tight junction relaxing function by suppressing the expression of claudin 4 and / or claudin 7 in epidermal keratinocytes.

[0017] The present invention also provides a tight junction relaxing agent containing surfactin and / or its sodium salt as a compound absorption enhancer, which comprises the tight junction relaxing agent of the present invention as an active ingredient. The tight junction relaxing agent of the present invention can be applied to the skin together with a compound, specifically a drug, to be penetrated into the skin, or can be applied to the skin in advance to relax the tight junctions in the skin, and then another drug can be applied to the same skin site, thereby promoting the penetration of the drug into the skin. Therefore, in another aspect, the present invention provides a method for allowing a compound to penetrate the skin by relaxing tight junctions in the skin, the method comprising applying the compound to the skin of a subject to whom it is desired that the compound penetrate the skin together with a tight junction relaxing agent of the present invention, or alternatively, applying the tight junction relaxing agent of the present invention to the skin in advance to relax the tight junctions in the skin, and then applying the compound to the skin site.

[0018] The other compound or drug that is to be penetrated is not particularly limited, and it can be medicinally or cosmetically acceptable, and can be low molecular weight compound or medium to high molecular weight compound such as polysaccharide, peptide, protein.Medicinal drug can be antiviral agent, antifungal agent, antibiotic, growth factor, medicine, etc., and cosmetic drug can be cosmetic drug, for example, low molecular weight compound such as vitamin C, vitamin C derivative, vitamin A derivative, vitamin E derivative, inositol, xylitol, glycerin, tranexamic acid, niacinamide, glycylglycine, 4-methoxysalicylate, salicylic acid, hydroxyethyl imidazolidinone, amino acid, urea, etc., hyaluronic acid or its sodium salt, glucosamine, elastin, collagen, polylactic acid, dextrin, etc., and high molecular weight compound such as Botox, etc.

[0019] In this specification, low molecular weight compounds refer to compounds with a molecular weight of 1,000 or less, medium molecular weight compounds refer to compounds with a molecular weight of 1,000 or more and 10,000 or less, and high molecular weight compounds refer to compounds with a molecular weight of more than 10,000.

[0020] Other compounds to be penetrated into the skin in the present invention may be extracts, such as yeast extract, cherry leaf extract, cinnamon bark extract, pine extract, Bulgarian rose water, okra extract, musk extract, burnet extract, dipotassium glycyrrhizinate, ginkgo leaf extract, Chinese angelica tree extract, coix seed extract, licorice extract, Panax ginseng extract, Sophora flavescens extract, star fruit leaf extract, carrot extract, Herb extract, neem leaf extract, saffron extract, cinchona extract, comfrey extract, thyme extract, winter strawberry, yomena, Mube, Ryukyu strawberry, Indian yomena, Lithocarpus edulis, Scutellaria root extract, rosemary extract, apricot kernel, gentian extract, peppermint powder, taiso extract, hop extract, kiwi extract, Roman chamomile extract, apple extract, hawthorn extract, turmeric extract, bayberry, myrtaceae, oak, etc. These may be used alone or in combination.

[0021] A particularly preferred drug for penetration in the present invention is hyaluronic acid or its sodium salt. Hyaluronic acid or its sodium salt has many functions, including retaining moisture in intercellular spaces, maintaining cells by forming a jelly-like matrix within tissues, maintaining tissue lubrication and flexibility, resisting external forces such as mechanical damage, and preventing bacterial infection. For example, the amount of hyaluronic acid or its sodium salt in the skin is said to decrease with age, resulting in signs of skin aging such as fine wrinkles and dryness. Therefore, many cosmetics containing hyaluronic acid or its sodium salt have been proposed as an agent for improving such aging skin. The molecular weight of hyaluronic acid or its sodium salt varies widely, ranging from a minimum of approximately 1,500 to 5,000 when hydrolyzed by proteases to an upper limit of approximately 800,000 to 4,000,000. In the present invention, it is believed that hyaluronic acid or its sodium salt can penetrate the skin with a molecular weight of at least 4,000, a maximum of approximately 2,000,000, or a maximum of approximately 4,000,000.

[0022] The tight junction relaxing agents of the present invention can mean, for example, that the skin penetration of other compounds desired to penetrate the skin is increased by, for example, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 100% or more, 200% or more, 300% or more, 400% or more, or 500% or more compared to the absence of the tight junction relaxing agents of the present invention.

[0023] The tight junction relaxant of the present invention can be used in drug absorption aids, pharmaceutical compositions, quasi-drugs, cosmetics, etc. Examples of dosage forms include thin films, powders, pills, tablets, injections, suppositories, emulsions, capsules, granules, and liquids (including tinctures, liquid extracts, spirits, suspensions, and lemonades). Furthermore, various ingredients commonly used in quasi-drugs, pharmaceuticals, cosmetics, etc., such as oily ingredients, emulsifiers, moisturizers, thickeners, medicinal ingredients, preservatives, powders, pH adjusters, UV absorbers, and antioxidants, can be appropriately blended within limits that do not impair the effects of the present invention.

[0024] All documents mentioned herein are incorporated by reference in their entirety.

[0025] The following examples of the present invention are for illustrative purposes only and do not limit the technical scope of the present invention. The technical scope of the present invention is limited only by the claims. The present invention may be modified, for example, by adding, deleting, or substituting components of the present invention, provided that the modifications do not depart from the spirit of the present invention. [Example]

[0026] The present invention will be described in more detail below with reference to examples, but these examples are not intended to limit the present invention in any way.

[0027] Experiment 1: Effects of various compounds on the expression of tight junction component proteins Experimental method: Western blotting Normal human epidermal keratinocytes (NHEK) were cultured in a 6-well plate using KGM-Gold containing supplements. TM Cells were seeded in medium (Lonza Verviers, Belgium) and incubated for 9 days. The medium was then changed to a calcium concentration of 1.3 mM and cultured for 4 days. After that, they were cultured in a mixed medium supplemented with 1 ng / mL growth factors for 16 days. Then, medium containing various compounds (1.3 mM calcium + 1 ng / mL growth factors) was added and treated for 24 hours. After treatment, cells were washed twice with serum-free α-MEM medium, sample buffer for electrophoresis was added, and cells were harvested using a cell scraper. The samples were sonicated, subjected to SDS-page, and then Western blotting. Antibodies for the tight junction proteins claudin 4 (CLDN4) and claudin 7 (CLDN7) were used for detection. β-Actin was used as an internal standard.

[0028] The results are shown in Figures 1 and 2. It was suggested that surfactin suppresses the expression of CLDN4 and CLDN7, thereby inhibiting claudin expression and functioning as a tight junction relaxant.

[0029] Experiment 2: Effects of various compounds on barrier function Experimental method: Permeation assay Normal human epidermal keratinocytes (NHEK) were placed in the upper chamber of a collagen-coated 24-well plate, and supplemented with KGM-Gold TMThe cells were seeded in 100μL HBSS (Hank's Balanced Salt Solution) containing various compounds and 2μM FITC-dextran and incubated for 11 days. The medium was then changed to a calcium concentration of 1.3mM and cultured for 2 days. The medium in the upper chamber was then removed and dried, and the mixed medium in the lower chamber was replaced every two days for 12 days. Then, 100μL of HBSS (Hank's Balanced Salt Solution) containing various compounds and 2μM FITC-dextran was added to the upper chamber. The cells were then incubated in an incubator for 3 hours, after which the HBSS was removed. After 24 hours, 100μL of the medium in the lower chamber was sampled, and the FITC fluorescence intensity was measured using a fluorescent plate reader.

[0030] The results are shown in Figure 3. Surfactin, which was suggested to function as a tight junction relaxant, was shown to increase the amount of dextran permeation compared to the control (medium). Therefore, it was demonstrated that surfactin functions as a tight junction relaxant and reduces the epidermal barrier function.

[0031] Experiment 3: Effect of surfactin on the permeation of sodium hyaluronate Using EPI-200X (Kurabo) as a skin model, we investigated the effect of surfactin on the penetration of sodium hyaluronate. 1. As a receiver solution, 0.9 mL of Dulbecco's phosphate buffer solution (D-PBS) or D-PBS containing 0.005% surfactin warmed to 37°C was placed in each well of a 6-well plate. 2. Epi Derm EPI-200X (Kurabo, hereafter referred to as the skin model) was placed in a diffusion chamber (EPI-FIX, KURABO) and placed on the 6-well plate from step 1. The plate was allowed to acclimate in a CO2 incubator for 15 minutes. 3. 0.35 mL of 0.1% aqueous solution of fluoresceinamine-labeled sodium hyaluronate (average molecular weight 1.2-1.6 million, FAHA-H2, PG research, hereafter referred to as FA-HA) was added to each donor. 4. The skin models were incubated in a CO2 incubator and collected after 24 hours. 5. The surface of the skin model was thoroughly washed with MQ water, cut with a scalpel, and placed in an Eppendorf tube. 6. Freeze the sample in liquid nitrogen, crush it for 30 seconds, add D-PBS, and then sonicate it for 15 minutes. Then, centrifuge it for 20 minutes (100 rpm). The supernatant was transferred to another Eppendorf tube. 7. Measurement was carried out using a microplate reader (excitation wavelength 490 nm, fluorescence wavelength 520 nm).

[0032] The results are shown in Figure 4. Surfactin, which acts as a tight junction relaxant and reduces the epidermal barrier function, was shown to increase the amount of sodium hyaluronate that penetrates the skin. [Industrial Applicability]

[0033] Surfactin and / or its sodium salt can relax tight junctions, and therefore can be used as an absorption enhancer for compounds, as well as in pharmaceutical, quasi-drug, cosmetic, and food compositions, making it useful in the medical, cosmetic, and food industries.

Claims

1. A tight junction relaxing agent comprising surfactin and / or its sodium salt.

2. The tight junction relaxing agent according to claim 1, which relaxes tight junctions in the epidermis.

3. The tight junction relaxing agent of claim 1, which relaxes tight junctions by suppressing cellular claudin expression.

4. A compound absorption enhancer comprising the tight junction relaxing agent according to claim 1 as an active ingredient.

5. The absorption enhancer of claim 4 , wherein the compound has a medium molecular weight.

6. The absorption enhancer according to claim 4, wherein the molecular weight of the compound is 4000 or more.

7. The absorption enhancer of claim 4, wherein the compound is hyaluronic acid or its sodium salt.

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