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

Abstract

The purpose of the present invention is to provide a novel tight junction relaxation agent, and to provide a compound absorption promoter that contains the relaxation agent.　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.

Description

Tight junction relaxant containing surfactin and / or its sodium salt

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

[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 cause rough skin and dermatitis (xerosis and atopic dermatitis).

[0003] The epidermis is the outermost membrane of the skin, and is 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 become corneocytes through the spinous layer, granular layer, and stratum corneum, and finally become dandruff and fall off. This process is called turnover. The epidermis undergoes turnover in a cycle of about one month.

[0004] During the turnover process, a cornified envelope (CE), primarily composed of keratin fibers, is formed to encase keratinocytes. 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 travel 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 function of the epidermis (Patent Document 1). Furthermore, the skin barrier function of tight junctions can also be described as 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.

[0007] JP 2012-121871 A

[0008] J Pharm.Pharmacol., 2010, 62(6), 702-708

[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.

[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] Accordingly, the present application encompasses the following inventions: (1) A tight junction softening agent comprising surfactin and / or its sodium salt. (2) The tight junction softening agent of (1), which softens epidermal tight junctions. (3) The tight junction softening agent of (1) or (2), which softens tight junctions by suppressing cellular claudin expression. (4) A compound absorption enhancer comprising, as an active ingredient, a tight junction softening agent of any of (1) to (3). (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 of any of (4) to (6), wherein the compound is hyaluronic acid or its sodium salt.

[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 molecular weight to high molecular weight, 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.

[0013] This shows that surfactin inhibits claudin expression by suppressing CLDN4 expression, thereby functioning as a tight junction relaxant. This shows that surfactin inhibits claudin expression by suppressing CLDN7 expression, thereby functioning as a tight junction relaxant. This shows that surfactin increases the permeation of dextran into skin cells. This shows that surfactin increases the permeation of sodium hyaluronate into the skin in a skin model.

[0014] The present invention provides a tight junction relaxation agent comprising surfactin and / or its sodium salt. Surfactin is known to have a variety of functions, including high surfactant activity, antibacterial activity, hemolysis, and protein denaturation inhibition. Surfactin is known to be produced by microorganisms of the genus Bacillus, and industrial mass production of surfactin is possible by culturing these microorganisms of the genus Bacillus. Surfactin or its salt is represented by the following formula: (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; and 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 surfactant properties, there is no information on 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, tightly sealing adjacent cells and sealing gaps between them while also continuously connecting cells. Tight junctions, which are primarily present in the granular layer of the epidermis and connect cells to each other, prevent water and substances from penetrating intercellular spaces and play an important role in maintaining 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, claudins 1, 4, and 7, has progressed, and the mechanisms underlying skin barrier function have been elucidated in more detail. 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 relaxant of the present invention exerts its tight junction relaxant 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 comprising surfactin and / or its sodium salt, a compound absorption enhancer, and the tight junction relaxing agent of the present invention as an active ingredient. The tight junction relaxing agent of the present invention can enhance the skin penetration of a compound, specifically a drug, by applying it to the skin together with the compound to be penetrated into the skin, or by applying it to the skin in advance to relax the skin's tight junctions, followed by applying another drug to the skin site. Therefore, in another aspect, the present invention provides a method for relaxing skin's tight junctions to allow a compound to penetrate the skin, the method comprising applying the compound to the skin of a subject to whom skin penetration of the compound is desired together with the tight junction relaxing agent of the present invention, or by applying the tight junction relaxing agent of the present invention to the skin in advance to relax the skin's tight junctions, followed by applying the compound to the skin site.

[0018] The other compounds or drugs to be penetrated are not particularly limited, and can be medicinally or cosmetically acceptable, and can be low molecular weight compounds or medium to high molecular weight compounds such as polysaccharides, peptides, proteins, etc. Medicinal drugs include antiviral agents, antifungal agents, antibiotics, growth factors, medicines, etc., and cosmetic drugs include cosmetic drugs, such as low molecular weight compounds such as vitamin C, vitamin C derivatives, vitamin A derivatives, vitamin E derivatives, inositol, xylitol, glycerin, tranexamic acid, niacinamide, glycylglycine, 4-methoxysalicylate, salicylic acid, hydroxyethylimidazolidinone, amino acids, urea, etc., medium molecular weight compounds such as hyaluronic acid or its sodium salt, glucosamine, elastin, collagen, polylactic acid, dextrin, etc., and high molecular weight compounds such as Botox, etc.

[0019] As used herein, a low molecular weight compound refers to, for example, a compound having a molecular weight of less than 1,000, a medium molecular weight compound refers to, for example, a compound having a molecular weight of 1,000 or more and 10,000 or less, and a high molecular weight compound refers to, for example, a compound having a molecular weight of more than 10,000. Therefore, a medium molecular weight compound may be, for example, a compound having a molecular weight of 1,000 or more, 2,000 or more, 3,000 or more, or 4,000 or more, or 4 million or less, 3 million or less, or 2 million or less.

[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 the appearance of skin aging such as fine wrinkles and dryness. Therefore, numerous 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 with a molecular weight of at least 4,000, a maximum of approximately 2,000,000, or a maximum of approximately 4,000,000 can be penetrated into the skin.

[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 a range that does 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.

[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: Effect of various compounds on the expression of tight junction component proteins Experimental method: Western blotting Normal human epidermal keratinocytes (NHEK) were placed on a 6-well plate and incubated with 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 for 16 days in a mixed medium supplemented with 1 ng / mL growth factors. 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, electrophoresis sample buffer 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. β-Actin was used as an internal standard.

[0028] The results are shown in Figures 1 and 2. It was suggested that surfactin has claudin expression inhibitory activity by suppressing the expression of CLDN4 and CLDN7, and functions as a tight junction relaxant.

[0029] Experiment 2: Effect 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-transwell plate. KGM-Gold containing supplements was placed in the upper chamber. ＴＭ The 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 Sodium Hyaluronate Permeation. Using EPI-200X (Kurabo) as a skin model, we investigated the effect of surfactin on sodium hyaluronate permeation. Specifically, 1. 0.9 mL of Dulbecco's phosphate buffer solution (D-PBS) or 0.005% surfactin dissolved in D-PBS preheated to 37°C was added to a 6-well plate as a receiver solution. 2. An 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. The plate was allowed to acclimate for 15 minutes in a CO2 incubator. 3. 0.35 mL of a 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 model was 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. The skin model was frozen in liquid nitrogen, crushed for 30 seconds, and then D-PBS was added. After 15 minutes of sonication, the model was centrifuged (100 rpm) for 20 minutes, and the supernatant was transferred to another Eppendorf tube. 7. Measurements were taken using a microplate reader (excitation wavelength 490 nm, emission 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.

[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 according to claim 1 or 2, which relaxes tight junctions by suppressing cellular claudin expression.

4. A compound absorption enhancer comprising the tight junction relaxing agent according to any one of claims 1 to 3 as an active ingredient.

5. The absorption enhancer of claim 4, wherein the compound is a medium-molecular compound.

6. The absorption enhancer according to claim 4 or 5, wherein the molecular weight of the compound is 4000 or more.

7. The absorption enhancer according to any one of claims 4 to 6, wherein the compound is hyaluronic acid or its sodium salt.

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