Growth factor gene expression promoter

Abstract

To provide a component that can promote production or the like of a growth factor.SOLUTION: Ceramides are an active ingredient of a growth factor gene expression promoter, a growth factor production promoter, an extracellular release promoter of a growth factor, and an angiogenesis promoter.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a growth factor gene expression promoter, a growth factor production promoter, a growth factor extracellular release promoter, and an angiogenesis promoter, each of which contains ceramides as an active ingredient. [Background technology]

[0002] Growth factors are endogenous proteins that promote the proliferation and differentiation of specific cells in animals. Research has shown that growth factors play a role in regulating various cellular and physiological processes, and they are sometimes used as active ingredients in topical medicines and other products.

[0003] There are various known growth factors, including, for example, fibroblast growth factor (FGF) and transforming growth factor (TGF).

[0004] FGFs are multifunctional proteins that exhibit a wide range of effects. Among them, FGF1 and FGF2 have the important function of promoting angiogenesis (the growth of new blood vessels from existing vasculature), and this effect is said to be greater than that of angiogenic factors such as vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) (Non-Patent Document 1). On the other hand, FGF1 and FGF2 lack a signal sequence for extracellular release and are therefore rarely released (Non-Patent Document 2). Furthermore, FGF2 is sold in the form of recombinant human basic fibroblast growth factor as a pharmaceutical indicated for the treatment of pressure ulcers, ulcers, and periodontitis (Non-Patent Document 3).

[0005] TGFs, like many other signaling pathways, play crucial roles in tissue development, cell differentiation, and embryonic development. TGFs are classified into TGF-α and TGF-β, and TGF-β is known to physiologically suppress angiogenesis (Non-Patent Document 4). [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Nature Med 9 (5): 604-13 (2003) [Non-patent document 2] Folia Pharmacol. Jpn. 107, 99-107 (1996) [Non-patent document 3] Pharmacia Vol.37 No.8, 738-739 (2001) [Non-patent document 4] Cell Engineering 14, 398-402 (1995) Summary of the Invention [Problem to be solved by the invention]

[0007] In order to actively enjoy the effects of growth factors, it is possible to incorporate growth factors themselves as active ingredients. However, in view of the fact that active ingredients are not easily available, it is desirable to have ingredients that are easily available yet can promote the production of these growth factors.

[0008] Therefore, an object of the present invention is to provide a component that can promote the production of growth factors, etc. [Means for solving the problem]

[0009] The present inventors have conducted extensive research and have unexpectedly found that ceramides have the ability to promote the production of growth factors, etc. The present invention was completed through further research based on this finding.

[0010] That is, the present invention provides the following aspects. Item 1. A growth factor gene expression promoter containing ceramides as active ingredients. Item 2. The growth factor gene expression promoter according to Item 1, wherein the ceramide is a human ceramide. Item 3. The growth factor gene expression promoter according to Item 1 or 2, which is an expression promoter for the FGF2 gene and / or the TGF-β gene. Item 4. A growth factor production promoter containing ceramides as the active ingredient. Item 5. An agent for promoting the extracellular release of growth factors, containing ceramides as active ingredients. Item 6. An angiogenesis promoter containing ceramides as active ingredients. [Effects of the Invention]

[0011] According to the present invention, a component capable of promoting the production of growth factors and the like is provided. [Brief explanation of the drawings]

[0012] [Figure 1] The amount of FGF2 released per cell number is shown. [Figure 2] The amount of TGF-β2 released per cell number is shown. DETAILED DESCRIPTION OF THE INVENTION

[0013] The growth factor gene expression promoter, growth factor production promoter, growth factor extracellular release promoter, and angiogenesis promoter of the present invention (hereinafter, these agents are also collectively referred to as "growth factor gene expression promoter, etc.") are characterized by containing ceramides as active ingredients. The growth factor gene expression promoter, etc. of the present invention will be described in detail below.

[0014] 1. Active ingredient The ceramides include ceramides and pseudoceramides. Either ceramide or pseudoceramide may be used alone, or ceramide and pseudoceramide may be used in combination.

[0015] Ceramide is a type of sphingolipid, a known component that collectively refers to a group of compounds in which a long-chain fatty acid is amide-bonded to the amino group of sphingosine. Ceramides include ceramide 1, ceramide 2, ceramide 3, ceramide 4, ceramide 5, ceramide 6I, ceramide 6II, ceramide 7, ceramide 8, ceramide 9, and ceramide 10, depending on the structure of sphingosine (e.g., the structure of the N-acyl group) and the structure of the long-chain fatty acid.

[0016] Specific examples of ceramides used in the present invention include human (optically active) ceramides.

[0017] Furthermore, the origin of the ceramide used in the present invention is not particularly limited and may be appropriately determined depending on the type of ceramide, etc. For example, it may be one extracted from an animal (animal ceramide), one extracted from a plant (plant ceramide), one obtained by microbial fermentation, or one chemically synthesized.

[0018] These ceramides may be used alone or in combination of two or more.

[0019] Pseudoceramides are ceramide analogs similar in structure and properties to ceramide, and specific examples include compounds selected from compounds represented by the following general formula (I) and compounds represented by the following general formula (II).

[0020] [ka]

[0021] In formula (I), R 11 represents a hydrocarbon group having 10 to 26 carbon atoms, and R 12 represents a hydrocarbon group having 9 to 25 carbon atoms, and X is -(CH2) n -, and n is an integer of 2 to 6. The hydrocarbon group is preferably an alkyl group or an alkenyl group. A specific example of the pseudoceramide represented by formula (I) is N-(hexadecyloxyhydroxypropyl)-N-hydroxyethylhexadecanamide.

[0022] [ka]

[0023] In formula (II), R 21 and R 22represent hydrocarbon groups having 1 to 40 carbon atoms which may be the same or different and which may be hydroxylated, and R 23 represents an alkylene group having 1 to 6 carbon atoms or a single bond, and R 24 represents a hydrogen atom, an alkoxy group having 1 to 12 carbon atoms, or a 2,3-dihydroxypropyloxy group, provided that R 23 When is a single bond, R 24 is a hydrogen atom. The hydrocarbon group is preferably an alkyl group or an alkenyl group.

[0024] These pseudo-ceramides may be used alone or in combination of two or more.

[0025] Among the above ceramides, from the viewpoint of further enhancing the growth factor gene expression promoting effect, growth factor production promoting effect, extracellular release promoting effect of growth factors, and / or angiogenesis promoting effect, preferred is human (optically active) ceramide, more preferred is a ceramide selected from the group consisting of ceramide 1, ceramide 2, and ceramide 3, and even more preferred is a combination of ceramide 1, ceramide 2, and ceramide 3. When ceramide 1, ceramide 2, and ceramide 3 are combined, the weight ratio thereof is 0.0002 to 1:1 to 1000:1, and preferably 0.002 to 0.2:10 to 200:1.

[0026] The content of the above-mentioned active ingredient in the growth factor gene expression promoter of the present invention is not particularly limited as long as it exhibits the growth factor gene expression promoting effect, growth factor production promoting effect, extracellular release of growth factors promoting effect, and / or angiogenesis promoting effect, but can be, for example, 0.0001 to 50% by weight, preferably 0.001 to 30% by weight, and more preferably 0.01 to 10% by weight.

[0027] Other ingredients The growth factor gene expression promoter of the present invention may contain, in addition to the active ingredient, other pharmacological ingredients as needed. Examples of such pharmacological ingredients include antihistamines (dipotassium glycyrrhizinate, glycyrrhizinic acid, diphenhydramine, diphenhydramine hydrochloride, etc.), local anesthetics (procaine, tetracaine, bupivacaine, mepipacaine, chloroprocaine, proparacaine, meprylcaine or salts thereof, orthocaine, oxethazaine, oxypolyethoxydecane, Scopolia extract, percaminpase, tesitdesitin, etc.), anti-inflammatory agents ( Allantoin, indomethacin, felbinac, diclofenac sodium, loxoprofen sodium, etc.), skin protectants (collodion, castor oil, etc.), blood circulation promoting ingredients (vanillyl nonyl amide, nicotinic acid benzyl ester, capsaicin, chili pepper extract, etc.), cooling agents (menthol, camphor, etc.), vitamins (vitamin A, etc.), mucopolysaccharides (sodium chondroitin sulfate, glucosamine, heparinoids, etc.).

[0028] Furthermore, the growth factor gene expression promoter of the present invention may contain bases or additives as necessary to obtain a desired formulation. Such bases and additives are not particularly limited as long as they are pharmaceutically acceptable, and examples thereof include aqueous bases such as water, lower alcohols, and polyhydric alcohols; oily bases such as natural oils, mineral oils, waxes, and ester oils; surfactants; and additives such as cooling agents, preservatives, flavoring agents, colorants, thickeners, pH adjusters, humectants, stabilizers, antioxidants, UV absorbers, chelating agents, adhesives, buffers, solubilizers, solubilizers, and preservatives.

[0029] Formulation The growth factor gene expression promoter of the present invention may be in any dosage form, such as a topical skin preparation or an oral preparation, but from the viewpoint of further enhancing the growth factor gene expression promoting effect, growth factor production promoting effect, extracellular release of growth factors promoting effect, and / or angiogenesis promoting effect, topical skin preparations are preferred.

[0030] When the growth factor gene expression promoter of the present invention is used as an external preparation for skin, its form is not particularly limited as long as it can be applied transdermally, and examples thereof include liquid, solid, and semi-solid forms (gel, ointment, paste, etc.).

[0031] When the growth factor gene expression promoter of the present invention is used as an external preparation for skin, the formulation form is not particularly limited as long as it can be applied transdermally, and examples thereof include external skin medicines, external skin quasi-drugs, cosmetics, skin cleansers, etc. Specific examples of the formulation form when the ceramide synthesis promoter of the present invention is used as an external skin preparation include external skin medicines such as creams, lotions, gels, emulsions, liquids, patches, aerosols, ointments, packs, etc.; external skin quasi-drugs such as creams, lotions, gels, emulsions, liquids, patches, aerosols, ointments, packs, etc.; cosmetics such as creams, lotions, gels, emulsions, liquids, ointments, packs, etc.; skin cleansers such as body shampoos, hair shampoos, rinses, etc.

[0032] Furthermore, the growth factor gene expression promoter of the present invention may have a form that allows ceramides to efficiently penetrate deep into the skin (dermis layer). Such a form is not particularly limited, but examples thereof include a liposome form, a microemulsion form, and a microneedle form.

[0033] Purpose The growth factor gene expression promoters of the present invention are excellent in promoting the expression of growth factor genes and / or the production of growth factors, and therefore can be used for the purpose of promoting the expression of growth factor genes and / or the production of growth factors. Furthermore, the growth factor gene expression promoters of the present invention not only promote the expression of growth factor genes and the production of growth factors, but also have the effect of promoting the extracellular release of growth factors, and therefore can be used for the purpose of promoting the extracellular release of growth factors. Furthermore, the growth factor gene expression promoters of the present invention can also be used for the purpose of promoting angiogenesis by utilizing the action and effect of growth factors. When used as an angiogenesis promoter, they can also be used for the purpose of wound healing by utilizing the angiogenesis-promoting effect.

[0034] From the viewpoint of further enhancing the growth factor gene expression promoting effect, growth factor production promoting effect, and / or extracellular release promoting effect of growth factors, the growth factor gene expression promoter etc. of the present invention is preferably a gene expression promoter of the FGF2 gene and / or TGF-β gene, a production promoter of FGF2 and / or TGF-β, or an extracellular release promoter of FGF2 and / or TGF-β, and more preferably a gene expression promoter of the FGF2 gene and / or TGF-β2 gene, a production promoter of FGF2 and / or TGF-β2, or an extracellular release promoter of FGF2 and / or TGF-β2.

[0035] How to use When the growth factor gene expression promoter of the present invention is used as a topical agent for skin, the method of application to the skin may be not only a direct application or application by patching, but also a physical method that efficiently penetrates the ceramides deep into the skin (dermis layer). Such physical methods include, but are not limited to, occlusive dressing therapy (ODT), electroporation, sonophoresis, etc.

[0036] The growth factor gene expression promoter of the present invention may also be used in regenerative medicine methods using fibroblasts. A specific example of such a method involves preparing a regenerative medicine material containing the growth factor gene expression promoter of the present invention and fibroblasts (e.g., autologous cultured fibroblasts) and introducing the material into the dermis of a patient by injection or other means. [Example]

[0037] The present invention will be explained in more detail below by showing examples, but the present invention is not limited to these examples.

[0038] Test Example 1: Gene expression analysis in human dermal fibroblasts (1) Preparation of growth factor gene expression promoter A 0.02:80:1 (weight ratio) mixture of ceramide 1, ceramide 2, and ceramide 3 was dissolved in a 1:1 (volume ratio) mixture of DMSO and PBS to a concentration of 50 μg / mL to prepare a growth factor gene expression promoter.

[0039] (2) Test method A 6-well plate was placed on ice, and 400 μL of 5x MEM (supplemented with glutamine and serum), 1,450 μL of natural collagen acidic solution (3 mg / mL, I-AC30; Koken Co., Ltd., Tokyo, Japan), 50 μL of 1 M NaHCO3, and 100 μL of human fibroblasts (2 million cells / mL, TIG-111, 34-year-old female, 17PDL, JCRB Cell Bank, Osaka, Japan) were sequentially added. After gentle mixing and incubation at 37°C for 1 hour to allow gelation, 2 mL of DMEM supplemented with 2% fetal bovine serum (FBS) was added and the medium was cultured for 1 day. The medium was then replaced with 2 mL of DMEM (2% FBS) containing growth factor gene expression promoters or their solvent (as a control). After 48 hours of culture, total RNA was extracted using an RNA extraction kit (RNeasy Mini Kit, Qiagen GmbH, Hilden, Germany) and analyzed using Affymetrix GeneChip Expression Array. The relative gene expression level was calculated, with the gene expression level in the control set at 1.

[0040] (3) Results In the group to which a growth factor gene expression promoter containing ceramide was added, it was confirmed that the amount of FGF2 gene expression increased to 1.87 times that of the control, and that TGF-β2 gene expression also increased to 1.87 times that of the control.

[0041] Test Example 2: Analysis of extracellularly released proteins in human dermal fibroblasts (1) Preparation of growth factor production promoter (growth factor extracellular release promoter) A 0.02:80:1 (weight ratio) mixture of ceramide 1, ceramide 2, and ceramide 3 was dissolved in a 1:1 (volume ratio) mixture of DMSO and PBS to a concentration of 10 mg / mL to prepare a growth factor production promoter (an agent for promoting the extracellular release of growth factors).

[0042] (2) Test method Human dermal fibroblasts (2 million cells / mL, TIG-111, 34-year-old female, 17PDL, JCRB Cell Bank, Osaka, Japan) were cultured in a 12-well plate (Corning (R) Costar (R)Cells were seeded at 100,000 cells / well in a 12-well cell culture plate and cultured for 1 day in 2 mL of DMEM containing 2% fetal bovine serum (BLG BI 04-111-1E) supplemented with GlutaMAX-I (GIBCO 10566-016). The medium was replaced with 2 mL of DMEM containing 0.2% fetal bovine serum (BLG BI 04-111-1E) supplemented with GlutaMAX-I, and a growth factor production promoter (a promoter of extracellular release of growth factors) was added to achieve a final ceramide concentration of 6.25 μg / mL, 12.5 μg / mL, or 25 μg / mL (n=4 for each). The solvent for the growth factor production promoter (a promoter of extracellular release of growth factors) was used as a control (n=4). The amounts of FGF2 and TGF-β2 released into the medium per cell were measured after 1, 2, 3, and 4 days of culture. Cell counts were measured using Cell Counting Kit-8 (Dojindo Laboratories), TGF-β2 levels were measured using R&D's TGF-β2 Human ELISA Kit, Quantikine (RSDDKG 00), and FGF2 levels were measured using R&D's FGF Basic Human ELISA Kit, Quantikine (DFB 50). Statistical analysis was performed using unpaired t-tests between the control values ​​and the stored values ​​at each culture day, with a p<0.05 value indicated a significant difference.

[0043] (3) Results The amount of FGF2 released per cell is shown in Figure 1, and the amount of TGF-β2 released per cell is shown in Figure 2. As shown in these figures, the amount of FGF2 released showed a significant increase compared to the control at all ceramide concentrations (all P<0.01). Furthermore, the amount of TGF-β2 released showed a significant increase compared to the control at all ceramide concentrations, especially after 3 days (P<0.01).

Claims

1. An agent for promoting the expression of FGF2 gene and / or TGF-β2 gene, which comprises ceramide and / or pseudoceramide as an active ingredient.

2. 2. The FGF2 gene and / or TGF-β2 gene expression promoter according to claim 1, wherein the ceramide and / or pseudoceramide is human ceramide.

3. An agent for promoting the production of FGF2 and / or TGF-β2, which comprises ceramide and / or pseudoceramide as an active ingredient.

4. An agent for promoting the extracellular release of FGF2 and / or TGF-β2, which comprises ceramide and / or pseudoceramide as an active ingredient.

5. An enhancer described in any of claims 1 to 4, wherein the ceramide and / or pseudo-ceramide is ceramide 1, ceramide 2 and / or ceramide 3.

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