Antimicrobial peptide expression promoter

Abstract

To provide an antimicrobial peptide expression promoter that promotes the expression of antimicrobial peptides associated with host defense.SOLUTION: An antimicrobial peptide expression promoter contains a late-stage autophagy inhibitor as an active ingredient.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an antimicrobial peptide expression promoter that promotes the expression of antimicrobial peptides involved in host defense. [Background technology]

[0002] Human skin is in contact with the external environment and is constantly exposed to the risk of invasion by foreign substances such as pathogenic microorganisms (bacteria, fungi, viruses, etc.). In addition, the skin has a symbiotic relationship with resident bacteria, and in a healthy state, it also functions as a biological barrier that excludes the invasion of pathogenic microorganisms. However, if resident bacteria abnormally proliferate due to some cause (e.g., weakened immunity) and invade skin tissue, opportunistic infection occurs. The skin protects the body from the dangers of pathogenic microorganisms, and at the same time, it has developed a natural immune mechanism, i.e., a chemical barrier mechanism, using antimicrobial peptides as one of the means to control the excessive proliferation of resident bacteria (Non-Patent Document 1).

[0003] As the name suggests, antimicrobial peptides have antimicrobial activity and exhibit bactericidal and bacteriostatic effects against various bacteria. A large number of antimicrobial peptides related to biological defense have been identified, but the representative antimicrobial peptides that function in the skin are well known to be human beta-defensin-1 (hBD-1), human beta-defensin-2 (hBD-2), human beta-defensin-3 (hBD-3), ribonuclease 7 (RNase 7), S100 calcium-binding protein A7 (S100A7) [also known as psoriasin], and human cathelicidin LL-37 (LL-37).

[0004] For example, hBD-3 exhibits strong antibacterial activity against gram-positive bacteria (e.g., Staphylococcus aureus), gram-negative bacteria (e.g., Pseudomonas aeruginosa), and fungi (e.g., Candida albicans). In addition, hBD-3 has the favorable characteristic that its activity is not affected by salts derived from sweat, etc. (Non-Patent Document 1). Also, in atopic dermatitis, the expression level of hBD-3 in the skin is not sufficiently high to suppress infection, and it has been suggested that this may be related to the onset or severity of atopic dermatitis (Non-Patent Document 2).

[0005] hBD-2 is considered to be one of the inducible antimicrobial peptides whose expression increases mainly when the skin barrier is damaged, such as during skin inflammation (Non-Patent Document 1). Furthermore, it has been pointed out that, like hBD-3, one of the reasons why atopic dermatitis patients are more likely to suffer repeated skin infections than psoriasis patients is that the expression level of hBD-2 is lower than that of psoriasis patients (Non-Patent Document 3).

[0006] hBD-1 exhibits antibacterial activity against various bacteria including Escherichia coli (Non-Patent Document 1). It has also been reported that hBD-1 enhances the expression of proteins that constitute tight junctions in skin epithelial cells and is involved in the physical barrier of the skin (Non-Patent Document 4).

[0007] In recent years, it has been found that one of the causes of the onset of atopic dermatitis is the abnormal proliferation of Staphylococcus aureus (Non-Patent Document 5). Like hBD-3, RNase 7 exhibits high antibacterial activity against Staphylococcus aureus and is considered to be an important antibacterial peptide that constitutes the chemical barrier of the skin (Non-Patent Document 6). In addition, it is known that individuals with high expression levels of RNase 7 in the steady state have higher resistance to Staphylococcus aureus infection than individuals with low expression levels (Non-Patent Document 7).

[0008] As described above, it has been suggested that increasing and maintaining the expression level of antimicrobial peptides at an appropriate state is very important from the viewpoint of strengthening the chemical barrier of the skin and, in turn, skin health. It is known that the expression of hBD-3 is increased by inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) (Non-Patent Document 8), and inflammatory mediators such as 15-d-prostaglandin J2 and prostaglandin D2 (Non-Patent Document 9). However, since these are inflammation-related factors, they cannot be easily and practically used as a means of promoting the expression of hBD-3. It has also been reported that the expression of hBD-2 is also promoted by inflammation-inducing substances such as lipopolysaccharide (LPS) in addition to TNF-α and IL-1β (Non-Patent Document 1). However, in this case, too, it is difficult to say that it can be easily and practically used for the same reasons as in the case of hBD-3. It is known that the expression of hBD-1 increases with the keratinization process of epidermal cells (Non-Patent Document 1), but there are still many unknowns about the factors and mechanisms that control its expression. It has been reported that the expression of RNase 7 is induced in the presence of interleukin-17A (IL-17A) and interferon-γ (IFN-γ) (Non-Patent Document 10) and by Staphylococcus aureus (Non-Patent Document 11), but as in the case of the above-mentioned antimicrobial peptides, it is currently largely unknown what factors and mechanisms control it under non-inflammatory conditions.

[0009] Therefore, given this situation, there was a strong need to develop a method to promote and control the expression of antimicrobial peptides, which act as the skin's chemical barrier, under non-inflammatory conditions (conditions without the use of inflammation-related factors or inflammation-inducing substances).

[0010] Autophagy is a mechanism for maintaining homeostasis within cells, and plays a role in keeping the inside of the cells in a normal state by digesting intracellular organelles and proteins that are damaged by cellular activity or by external stimuli (Non-Patent Document 12). However, tumor cells grow quickly and do not undergo sufficient vascularization, so they are under metabolic stress (low oxygen, low glucose). As a result, they activate autophagy to avoid this, procuring amino acids necessary for survival and maintaining the survival and proliferation of cancer cells. Therefore, as one method of treating cancer, suppressing activated autophagy has attracted attention. For example, clinical research is being conducted with the aim of enhancing the antitumor effect by combining chloroquine and hydroxychloroquine, which are known as representative autophagy inhibitors, with anticancer drugs (Non-Patent Documents 12, 13).

[0011] However, it has not been known that late-stage autophagy inhibitors promote the expression of antimicrobial peptides. [Prior art documents] [Non-patent literature]

[0012] [Non-Patent Document 1] Niyonsaba F et al. Protective roles of the skin against infection: Implication of naturally occurring human antimicrobial agents β-defensins, cathelicidin LL-37 and lysozyme. J Dermatol Sci 40: 157-168, 2005 [Non-Patent Document 2] Nomura I et al. Cytokine milium of atopic dermatitis, as compared to psoriasis, skin prevents induction of innate immune response genes. J Immunol 171: 3262-3269, 2003 [Non-Patent Document 3] Ong PY et al. Endogenous antimicrobial peptides and skin infections in atopic dermatitis. New Engl J Med 347: 1151-1160, 2002 [Non-Patent Document 4] Goto H et al. Human beta defensin-1 regulates the development of tight junctions in cultured human epidermal keratinocytes. J Dermatol Sci 71: 145-148, 2013 [Non-Patent Document 5] Kobayashi T et al. Dysbiosis and Staphylococcus aureus colonization drives inflammation in atopic dermatitis. Immunity 42: 756-766, 2015 [Non-Patent Document 6] Cho JS et al. Lucky number seven: RNase 7 can prevent Staphylococcus aureus skin colonization. J Invest Dermatol 130: 2703-2706, 2010 [Non-Patent Document 7] Zanger P et al. Constitutive expression of the antimicrobial peptide RNase 7 is associated with Staphylococcus aureus infection of the skin. J Infect Dis 200: 1907-1915, 2009 [Non-Patent Document 8] Harder J et al. Isolation and characterization of human β-defensin-3, a novel human inducible peptide antibiotic. J Bio Chem 276: 5707-5713, 2001 [Non-Patent Document 9] Bernard JJ et al. Cycloxygenase-2 enhances antimicrobial peptide expression and killing of Staphylococcus aureus. J Immunol 185: 6535-6544, 2010 [Non-Patent Document 10] Simanski M et al. IL-17A and IFN-γ synergistically induce RNase 7 expression via STAT3 in primary keratinocytes. PLoS One 8: e59531, 2013 [Non-Patent Document 11] Wanke I et al. Skin commensals amplify the innate immune response to pathogens by activation of distinct signaling pathways. J Invest Dermatol 131: 382-390, 2011 [Non-Patent Document 12] "Autophagy - The Cellular Self-Degradation System that Supports Life", edited by Noboru Mizushima and Tamotsu Yoshimori, Kagaku Dojin Co., Ltd., 2012 [Non-Patent Document 13] Buzun K et al. Autophagy modulators in cancer therapy. Int J Mol Sci 22: 5804, 2021 Summary of the Invention [Problem to be solved by the invention]

[0013] The present invention relates to providing an antimicrobial peptide expression promoter that promotes the expression of antimicrobial peptides involved in host defense. [Means for solving the problem]

[0014] The present inventors searched for substances that promote the expression of antimicrobial peptides, and found that autophagy inhibitors that can suppress late autophagy have the effect of promoting the expression of antimicrobial peptides derived from human skin and can therefore serve as agents for promoting the expression of these antimicrobial peptides.

[0015] Specifically, the present invention relates to an antimicrobial peptide expression promoter that contains an autophagy inhibitor as an active ingredient. Effect of the Invention

[0016] According to the present invention, it is possible to enhance the natural immune function (mainly antibacterial action) in the skin, and as a result, it is possible to strengthen the skin barrier function or prevent or improve atopic dermatitis. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] In the present invention, the term "antimicrobial peptide" refers to an antimicrobial peptide that functions in epithelial cells of human skin, and examples thereof include human β-defensins such as hBD-1, hBD-2, and hBD-3, and RNase 7, which belongs to the RNase A superfamily. Human β-defensins are low molecular weight cationic peptides that contain six cysteine ​​residues forming three disulfide bonds.

[0018] hBD-1 is a 3.9 kDa peptide consisting of 36 amino acids, and is a constitutively expressed antimicrobial peptide in the skin. Unlike other hBDs, its expression level is not affected (inducible) by cytokines or bacteria.

[0019] hBD-2 is a 4.3 kDa peptide consisting of 41 amino acids, and is hardly expressed when the skin is in a healthy state (steady state). However, when the skin barrier is damaged due to psoriasis, the expression level of hBD-2 increases dramatically. In addition, the expression level increases when exposed to cytokines or bacteria. Due to these properties, hBD-2 is classified as an inducible antimicrobial peptide.

[0020] hBD-3 is a 5.1 kDa peptide consisting of 45 amino acids, and is a constitutively expressed antimicrobial peptide in the skin. However, it is also known to have inducible properties because its expression is further increased by exposure to cytokines and bacteria (Non-Patent Document 1, Clausen M et al. Measurement of AMPs in stratum corneum of atopic dermatitis and healthy skin-tape stripping technique. Sci Rep 8: 1666, 2018).

[0021] RNase 7 is a cationic 14.5 kDa protein with ribonuclease activity. RNase 7 is considered to be one of the major antimicrobial peptides in the skin because of its high expression in healthy skin. Like hBD-3, it also has inducible properties because its expression level increases upon exposure to cytokines and bacteria (Non-Patent Document 1, Rademacher F et al. The antimicrobial and immunomodulatory function of RNase 7 in skin. Front Immunol 10: 2553, 2019).

[0022] It is well known that the antibacterial peptides described above exhibit a broad antibacterial spectrum against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus faecium, Propionibacterium acnes, and the like. It has also been shown that hBD-2, hBD-3 and RNase 7 exhibit high antifungal activity against tinea fungi, i.e., athlete's foot fungi (e.g., Trichophyton rubrum), and these antimicrobial peptides have attracted increasing attention as host defense factors (Fritz P et al. Inhibition of dermatophytes by the antimicrobial peptides human β-defensin-2, ribonuclease 7 and psoriasin. Med Mycol 50: 579-584, 2012, Firat YH et al. Infection of keratinocytes with Trichophytum rubrum induces epidermal growth factor-dependent RNase 7 and human beta-defensin-3 expression. PloS One, 9: e93941, 2014).

[0023] In the present invention, "promotion of expression of an antimicrobial peptide" means promotion of expression of the mRNA and / or protein of the antimicrobial peptide. Furthermore, the promotion of antimicrobial peptide expression can be evaluated, for example, by using a cell system such as normal human epidermal keratinocytes, extracting mRNA or protein from the cells, and measuring the amount of mRNA or protein encoding the antimicrobial peptide using real-time PCR, Northern blotting, or ELISA or Western blotting, respectively.

[0024] "Inhibition of autophagy" includes inhibition of basal autophagy activity and inhibition of each process of autophagy (for example, inhibition of autophagosome formation), however, in the present invention, a "late autophagy inhibitor" refers to a substance that inhibits the process of autolysosome formation (late autophagy process) through the fusion of autophagosomes and lysosomes.

[0025] In the present invention, examples of late-stage autophagy inhibitors include acid protease inhibitors such as Pepstatin A and E64d; vacuolar ATPase inhibitors such as Bafilomycin A1; lysosomotropic agents such as Chloroquine, Hydroxychloroquine, Lys05, ARN5187, and Compound 30; and compounds known to inhibit late-stage autophagy such as Clomipramine and Lucanthone (Pasquier B. Autophagy inhibitors. Cell Mol Life Sci 73: 985-1001, 2016). Of these, Bafilomycin A1, Chloroquine, and Hydroxychloroquine are preferred because of their ease of availability and the abundance of biological and medical information available.

[0026] Bafilomasin A1 is a novel ATPase inhibitor that inhibits the expression of lysosome-specific ATPase (V-ATPase) and endoplasmic reticulum calcium pump (sarco / endoplasmic reticulum Ca 2+It is known that autophagy is suppressed at a late stage by inhibiting V-ATPase (SERCA) (Mauvezin C et al. Autophagosome-lysosome fusion is independent of V-ATPase-mediated acidification. Nat Commun 6: 7007, 2015). The mechanism by which chloroquine and hydroxychloroquine inhibit late-stage autophagy is currently unknown; however, they do not have the same effect as bafilomycin A1, and so inhibit late-stage autophagy through a different mechanism (Mauthe M et al. Chloroquine inhibits autophagic flux by decreasing autophagosome-lysosome fusion. Autophagy 14: 1435-1455, 2018).

[0027] As shown in the Examples below, when bafilomycin A1, chloroquine, and hydroxychloroquine, which are representative compounds that suppress late autophagy, were added to normal human epidermal keratinocytes, the expression levels of hBD-1, hBD-2, hBD-3, and RNase 7 mRNA significantly increased. As described above, the mechanisms by which bafilomycin A1, chloroquine, and hydroxychloroquine inhibit late-stage autophagy are thought to differ. However, all of them can promote the expression of antimicrobial peptides, so substances that inhibit autophagy are not limited to these, and it is thought that any inhibitor of late-stage autophagy can promote the expression of antimicrobial peptides. In addition, because substances that inhibit late autophagy can promote the expression of antimicrobial peptides, this means that it is possible to screen for new agents that promote the expression of antimicrobial peptides by using the inhibition of late autophagy as an indicator. This indicates that the inhibition of late autophagy can be used as a novel screening method for agents that promote the expression of antimicrobial peptides.

[0028] Therefore, an autophagy inhibitor that suppresses late stage autophagy can be an antimicrobial peptide expression promoter, preferably an antimicrobial peptide expression promoter derived from human skin, and can be used to produce an antimicrobial peptide expression promoter, preferably an antimicrobial peptide expression promoter derived from human skin. That is, the late stage autophagy inhibitor can be used to promote antimicrobial peptide expression, preferably an antimicrobial peptide expression promoter derived from human skin. Here, use on humans may be either therapeutic or non-therapeutic. "Non-therapeutic" refers to a concept that does not include medical procedures, i.e., a concept that does not include a method of surgery, therapy, or diagnosis on humans, more specifically, a concept that does not include a method of surgery, therapy, or diagnosis on humans by a physician or a person under the instruction of a physician.

[0029] The late autophagy inhibitor of the present invention may itself be a cosmetic, quasi-drug, or medicine for promoting the expression of antimicrobial peptides, preferably antimicrobial peptides derived from human skin, or it may be a material or preparation used by being incorporated into such cosmetics, quasi-drugs, medicines, etc.

[0030] Cosmetics, quasi-drugs, or pharmaceuticals containing the late autophagy inhibitor of the present invention can be preferably in the form of topical skin preparations, specifically, in various forms such as ointments, emulsified cosmetics, creams, milks, lotions, gels, and aerosols. Such preparations can be obtained by mixing and dispersing the preparation directly or together with a pharma- ceutically acceptable carrier, such as various oils, surfactants, gelling agents, preservatives, antioxidants, solvents, alcohol, water, chelating agents, thickeners, UV absorbers, emulsion stabilizers, pH adjusters, colorants, fragrances, etc., using a general manufacturing method, and then processing into a desired form. Furthermore, these cosmetics, quasi-drugs, pharmaceuticals, etc. can be appropriately blended with plant extracts, bactericides, moisturizers, anti-inflammatory agents, antibacterial agents, cooling agents, antiseborrheic agents, etc., depending on the respective preparations, within the scope of not interfering with the effects of the present invention.

[0031] The content (w / w) of the active ingredient (late-stage autophagy inhibitor) in the cosmetic, quasi-drug, or pharmaceutical is preferably 0.001% or more, more preferably 0.01% or more, and preferably 100% or less, more preferably 50% or less, more preferably 10% or less, and also preferably 0.001 to 50%, more preferably 0.01 to 20%, and more preferably 0.01 to 10%.

[0032] The dosage of the above-mentioned cosmetics, quasi-drugs, or pharmaceuticals is not particularly limited as long as it is an amount that can obtain an effect, and may vary depending on the condition, weight, sex, age, or other factors of the subject, but is preferably 0.0001 g or more, more preferably 0.0002 g or more, more preferably 0.0005 g or more, and preferably 5 g or less, more preferably 4 g or less, more preferably 3 g or less per adult (60 kg) per day as the active ingredient (late autophagy inhibitor). Also, the dosage is preferably 0.0005 to 5 g, more preferably 0.001 to 2 g per day. The preparation may be ingested or administered according to any intake or administration schedule, but is preferably administered once or several times a day for several weeks to several months, and more preferably three times a day for six weeks or more. Furthermore, the target users of the above-mentioned cosmetics, quasi-drugs, or pharmaceuticals are not particularly limited as long as they are in need thereof, and examples of such users include people who wish to strengthen the natural immune function in epithelial cell organs, including the skin barrier, and people who wish to prevent or improve atopic dermatitis. EXAMPLES

[0033] The present invention will be described more specifically below with reference to examples.

[0034] Example 1 Effect of bafilomycin A1 on promoting expression of antimicrobial peptides 1) Cell culture was performed using purchased normal human epidermal keratinocytes (Life Technologies) in EpiLife serum-free cell culture medium (Life Technologies) supplemented with HuMedia-KG proliferation agent set (Kurabo Industries) (2 mL / well) at 37°C in 5% (v / v) CO2. Cells: 1 x 10 5 The cells were seeded at 1000 cells / well in a 6-well culture plate and cultured until subconfluent (approximately 80%). The cell culture medium was then replaced with a medium containing additives other than human recombinant growth factor G (hEFG) and bovine pituitary extract (BPE) from the HuMedia-KG proliferation agent (5 types), and the cells were cultured for another 24 hours. Subsequent experiments were performed after the medium was replaced again with a medium containing additives other than human recombinant growth factor G (hEFG) and bovine pituitary extract (BPE) from the HuMedia-KG proliferation agent (5 types).

[0035] 2) Bafilomycin A1 (Fujifilm Wako Pure Chemical Industries, Ltd.) dissolved in DMSO was added to the cells prepared in 1) above to final concentrations of 10, 50, and 100 nM, and the cells were cultured for 72 hours. The same amount of DMSO was added to the control.

[0036] 3) Total RNA was extracted from the cultured cells using RNA extraction kits [QIAshredder (Qiagen) and RNeasy Mini Kit (Qiagen)]. Reverse transcription reaction from RNA to cDNA was performed at a scale of 1000 ng using the High Capacity RNA-to-cDNA kit (Applied Biosystems). Real-time PCR analysis was performed using TaqMan primer / probe assay reagent (Applied Biosystems) with a 7500 real-time PCR system (Applied Biosystems). The expression levels of the hBD-1 gene, hBD-2 gene, hBD-3 gene, and RNase 7 gene were normalized with the housekeeping gene, ribosomal protein lateral stalk subunit P0 (RPLP0), and the solvent alone was used as a control, and the expression level was calculated as a relative change of 1.0. Incidentally, including in the following Examples, tests for significant differences between two groups were performed using Student's t-test, and tests for significant differences between three or more groups were performed using Tukey's multiple comparison test after confirming significance using one-way analysis of variance.

[0037] 4) Results As shown in Table 1, bafilomycin A1 significantly increased the expression levels of RNase 7, hBD-3, hBD-1 and hBD-2 compared to the control (n=3).

[0038] [Table 1]

[0039] Example 2: Effect of chloroquine on promoting expression of antimicrobial peptides 1) To cells prepared in the same manner as in 1) of Example 1, chloroquine (Fujifilm Wako Pure Chemical Industries, Ltd.) dissolved in DMSO was added to a final concentration of 100 μM, and the cells were cultured for 72 hours. As a control, the same amount of DMSO was added. After the cultivation was completed, the mixture was treated in the same manner as in Example 1 and subjected to real-time PCR analysis.

[0040] 2) Results As shown in Table 2, chloroquine significantly increased the expression levels of RNase 7 and hBD-3 compared to the control (n=3).

[0041] [Table 2]

[0042] Example 3: Effect of hydroxychloroquine on promoting expression of antimicrobial peptides 1) Hydroxychloroquine (Fujifilm Wako Pure Chemical Industries, Ltd.) dissolved in water was added to cells prepared in the same manner as in 1) of Example 1 to a final concentration of 100 μM, and the cells were cultured for 72 hours. As a control, the same amount of water was added. After the cultivation was completed, the mixture was treated in the same manner as in Example 1 and subjected to real-time PCR analysis.

[0043] 2) Results As shown in Table 3, hydroxychloroquine significantly increased the expression level of RNase 7 compared to the control (n=3).

[0044] [Table 3]

Claims

1. An antimicrobial peptide expression promoter that contains a late autophagy inhibitor as an active ingredient.

2. The antimicrobial peptide expression promoter according to claim 1, wherein the antimicrobial peptide is an antimicrobial peptide derived from human skin.

3. The antimicrobial peptide expression promoter according to claim 1, wherein the late autophagy inhibitor is one or more selected from chloroquine, hydroxychloroquine and bafilomycin A1.

4. The antimicrobial peptide expression promoter according to claim 1 or 2, wherein the antimicrobial peptide is RNase 7, hBD-3, hBD-1 or hBD-2.

Images