JP2022080694A - Growth factor gene expression promoter - Google Patents

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

There is an application for application of Article 30, Paragraph 2 of the Patent Law. ▲ 1 ▼ Website address: https: // www. ifscc2020. com / virtual_congress. html website publication date: October 21, 2nd year of Reiwa

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 containing ceramides as active ingredients.

Growth factors are endogenous proteins that promote the proliferation and differentiation of specific cells in the animal body. It has been studied that growth factors act on the regulation of various cytological and physiological processes, and the action may be utilized to be compounded as an active ingredient of external medicines and the like.

Various growth factors are known, and examples thereof include fibroblast growth factor (FGF) and transforming growth factor (TGF).

FGF is a multifunctional protein showing a wide range of effects, among which FGF1 and FGF2 have an important function of promoting angiogenesis (growth of new blood vessels from an existing vascular system), and the effect is vascular endothelial growth. It is said to be higher than angiogenic factors such as factor (VEGF) and platelet-derived growth factor (PDGF) (Non-Patent Document 1). On the other hand, FGF1 and FGF2 have no signal sequence for being released extracellularly and are rarely released (Non-Patent Document 2). In addition, FGF2 is marketed as a drug having indications for pressure ulcers, ulcers, and periodontitis in the form of recombinant human basic fibroblast growth factor (Non-Patent Document 3).

TGF, like many other signaling pathways, plays a vital role in tissue development, cell differentiation, and embryonic development. TGF is classified into TGF-α and TGF-β, and TGF-β is said to physiologically suppress angiogenesis (Non-Patent Document 4).

Nature Med 9 (5): 604-13 (2003) Folia Pharmacol. Jpn. 107, 99-107 (1996) Pharmacia Vol.37 No.8, 738-739 (2001) Cell Engineering 14, 398-402 (1995)

It is possible to add the growth factor itself as an active ingredient for the purpose of positively enjoying the action and effect of the growth factor, but considering that the active ingredient is not easily available, it is easy to obtain. A component capable of promoting the production of these growth factors is desired.

Therefore, an object of the present invention is to provide a component capable of promoting the production of growth factors and the like.

As a result of diligent studies, the present inventor unexpectedly found that ceramides have an action of promoting the production of growth factors and the like. The present invention has been completed by further studies based on this finding.

That is, the present invention provides the inventions of the following aspects.
Item 1. A growth factor gene expression promoter containing ceramides as an active ingredient.
Item 2 The growth factor gene expression promoter according to Item 1, wherein the ceramides are human ceramides.
Item 3. Item 2. 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 an active ingredient.
Item 5. An extracellular release promoter of growth factors containing ceramides as an active ingredient.
Item 6. An angiogenesis-promoting agent containing ceramides as an active ingredient.

According to the present invention, a component capable of promoting the production of growth factors and the like is provided.

The amount of FGF2 released per cell number is shown. The amount of TGF-β2 released per cell number is shown.

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 collectively referred to as "growth factor gene expression promoter, etc." Described) is characterized in that ceramides are used as an active ingredient. Hereinafter, the growth factor gene expression promoter and the like of the present invention will be described in detail.

1. 1. The active ingredient ceramides include ceramides and pseudo-ceramides. As the ceramides, either ceramide or pseudo-ceramide may be used alone, or ceramide and pseudo-ceramide may be used in combination.

Ceramide is a kind of sphingolipid, and is a known component as a general term for a group of compounds in which a long-chain fatty acid is amide-bonded to an amino group of sphingosine. Ceramide is ceramide 1, ceramide 2, ceramide 3, ceramide 4, ceramide 5, ceramide 6I, ceramide 6II, ceramide 7, ceramide due to the difference in the structure of sphingosine (structure of N-acyl group, etc.) and the structure of long-chain fatty acid. 8, ceramide 9, ceramide 10, and the like can be mentioned.

Moreover, as a specific example of the ceramide used in this invention, a human type (optically active body) ceramide can be mentioned.

The origin of the ceramide used in the present invention is not particularly limited and may be appropriately set according to the type of ceramide and the like, for example, those extracted from animals (animal ceramide) and those extracted from plants. It may be (vegetable ceramide), one obtained by a microbial fermentation method, one chemically synthesized, or the like.

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

Pseudo-ceramide is a ceramide analog having a structure and properties similar to those of ceramide, and specifically, a compound selected from the compound represented by the following general formula (I) and the compound represented by the general formula (II). Can be mentioned.

In formula (I), R ¹¹ represents a hydrocarbon group having 10 to 26 carbon atoms, R ¹² represents a hydrocarbon group having 9 to 25 carbon atoms, X represents − (CH ₂ ) _n −, and n represents − (CH 2) n −. Represents an integer of 2-6. The hydrocarbon group preferably includes an alkyl group or an alkenyl group. Specific examples of the pseudo-ceramide represented by the formula (I) include N- (hexadecyloxyhydroxypropyl) -N-hydroxyethylhexadecanamid.

In formula (II), R ²¹ and R ²² represent a hydroxylated hydrocarbon group having 1 to 40 carbon atoms which may be the same or different, and R ²³ is an alkylene group or a simple substance having 1 to 6 carbon atoms. Representing a bond, R ²⁴ represents a hydrogen atom, an alkoxy group having 1 to 12 carbon atoms or a 2,3-dihydroxypropyloxy group. However, when R ²³ is a single bond, R ²⁴ is a hydrogen atom. The hydrocarbon group preferably includes an alkyl group or an alkenyl group.

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

Among the above ceramides, the human type (optically active substance) is preferable from the viewpoint of further enhancing the growth factor gene expression promoting effect, the growth factor production promoting effect, the extracellular release promoting effect of the growth factor, and / or the angiogenesis promoting effect. ) Ceramide, more preferably a ceramide selected from the group consisting of ceramide 1, ceramide 2, and ceramide 3, and further preferably 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, preferably 0.002 to 0.2: 10 to 200: 1.

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

Other Ingredients The growth factor gene expression promoter and the like of the present invention may contain other pharmacological components in addition to the active ingredient, if necessary. Examples of such pharmacological components include anti-histamine agents (dipotassium glycyrrhizinate, glycyrrhizinic acid, diphenhydramine, diphenhydramine hydrochloride, etc.), local anesthetics (procaine, tetrakine, bupipacaine, mepipacaine, chloroprocine, proparacaine, meprilcaine, or salts thereof. , Orthokine, oxesazein, oxypolyentoxydecane, funnel extract, percaminpase, tesitdecitin, etc.), anti-inflammatory agents (alantin, indomethacin, fervinac, diclofenac sodium, loxoprofen sodium, etc.), skin protectants (corodion, castor oil, etc.), blood circulation promotion Ingredients (nonyl acid vanillyl amide, nicotinic acid benzyl ester, capsaicin, capsicum extract, etc.), refreshing agents (menthol, camphor, etc.), vitamins (vitamin A, etc.), mucopolysaccharides (chondroitin sodium sulfate, glucosamine, heparin-like substances, etc.) ) Etc. can be mentioned.

In addition, the growth factor gene expression promoter or the like of the present invention may contain a base or an additive, if necessary, in order to obtain a desired pharmaceutical form. Such bases and additives are not particularly limited as long as they are pharmaceutically acceptable, but are, for example, aqueous bases such as water, lower alcohols, polyhydric alcohols; natural oils, mineral oils, waxes, etc.・ Oil-based bases such as waxes and ester oils; surfactants; refreshing agents, preservatives, flavoring agents, coloring agents, viscous agents, pH adjusters, wetting agents, stabilizers, antioxidants, ultraviolet rays Additives such as absorbents, chelating agents, pressure-sensitive adhesives, buffers, solubilizing agents, solubilizers, preservatives and the like can be mentioned.

Formulation form The growth factor gene expression-promoting agent of the present invention may be in any form such as an external skin preparation or an internal preparation, but has a growth factor gene expression-promoting effect, a growth factor production-promoting effect, and a growth factor cell. From the viewpoint of further enhancing the external release promoting effect and / or the angiogenesis promoting effect, a skin external preparation is preferable.

When the growth factor gene expression promoter of the present invention is used as an external skin preparation, its shape is not particularly limited as long as it can be applied transdermally, but for example, it is liquid, solid, or semi-solid (gel-like, semi-solid). Ointment-like, paste-like) and the like.

When the growth factor gene expression promoter of the present invention is used as a skin external preparation, the pharmaceutical form thereof is not particularly limited as long as it can be applied transdermally, but for example, a skin external medicine, a skin external medicine external product, etc. Examples include cosmetics and skin cleaning agents. Specific examples of the formulation form when the ceramide synthesis promoter of the present invention is used as an external skin preparation include creams, lotions, gels, emulsions, liquids, patches, aerosols, ointments, packs and the like. External skin medicines; creams, lotions, gels, emulsions, liquids, patches, aerosols, ointments, packs and other external medicines for skin; creams, lotions, gels, emulsions , Liquids, ointments, packs and the like; skin lotions such as body shampoos, hair shampoos, rinses and the like.

Further, the growth factor gene expression promoter or the like of the present invention may have a form in which ceramides efficiently permeate deep into the skin (dermis layer). Such a form is not particularly limited, and examples thereof include a liposome form, a microemulsion form, a microneedle form, and the like.

Use The growth factor gene expression promoter or the like of the present invention is excellent in the effect of promoting the expression of the growth factor gene and / or the effect of promoting the production of the growth factor. And / or can be used for the purpose of promoting the production of growth factors. Further, the growth factor gene expression promoter and the like of the present invention not only promote the expression of the growth factor gene and the production of the growth factor, but also have the effect of promoting the release of the growth factor to the outside of the cell. It can also be used for the purpose of promoting release to the outside of the cell. Furthermore, the growth factor gene expression promoter and the like of the present invention can also be used for the purpose of promoting angiogenesis by utilizing the action and effect of the growth factor. When used as an angiogenesis-promoting agent, it can also be used for the purpose of wound healing by utilizing the angiogenesis-promoting action.

The growth factor gene expression-promoting agent of the present invention has the FGF2 gene and / or TGF- from the viewpoint of further enhancing the growth factor gene expression-promoting effect, the growth factor production-promoting effect, and / or the extracellular release-promoting effect of the growth factor. It is preferably a gene expression promoter of β gene, a production promoter of FGF2 and / or TGF-β, an extracellular release promoter of FGF2 and / or TGF-β, and a gene of FGF2 gene and / or TGF-β2 gene. More preferably, it is an expression promoter, a FGF2 and / or TGF-β2 production promoter, and an extracellular release promoter of FGF2 and / or TGF-β2.

Usage method When the growth factor gene expression promoter or the like of the present invention is used as an external skin preparation, the method of applying to the skin is not only the method of applying or affixing as it is, but also the ceramides to the deep part (dermis layer) of the skin. May be a physical method that allows for efficient penetration. Such physical supplementation method is not particularly limited, and examples thereof include closed sealing method (ODT: Occulusive Dressing Therapy), electroporation, and sonophoresis.

Further, the growth factor gene expression promoter and the like of the present invention may be used in a regenerative medicine method using fibroblasts. As a specific example of such a method, a regenerative medicine material containing the growth factor gene expression promoter of the present invention and fibroblasts (for example, self-cultured one) is prepared and introduced into the dermis of a patient by injection or the like. The method can be mentioned.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

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

(2) Test method Place a 6-well plate on ice, 400 μL of 5 × MEM (added glutamine and serum), 1,450 μL of natural collagen acidic solution (3 mg / mL, I-AC30; Koken, Tokyo, Tokyo, Ltd.) Japan), 50 μL of 1M NaHCO ₃ , 100 μL of human fibroblasts (2 million cells / mL, TIG-111, 34-year-old female, 17PDL, JCRB Cell Bank, Osaka, Japan) were added sequentially. After gently stirring and incubating at 37 ° C. for 1 hour to gel, 2 mL DMEM supplemented with 2% fetal bovine serum (FBS) was added and cultured for 1 day. The medium was replaced with 2 mL DMEM (2% FBS) containing a growth factor gene expression promoter or its solvent (as a control). After culturing for 48 hours, total RNA was extracted using an RNA extraction kit (RNeasy mini kit, Qiagen GmbH, Hilden, Germany) and analyzed for Affymetrix GeneChip Expression Array. The relative gene expression level was determined with the gene expression level in the control as 1.

(3) Results It was confirmed that the FGF2 gene expression level increased 1.87 times that of the control and the TGF-β2 gene expression also increased 1.87 times that of the control in the group to which the growth factor gene expression promoter containing ceramide was added. did.

Test Example 2: Analysis of extracellularly released protein in human dermal fibroblasts (1) Preparation of growth factor production promoter (growth factor extracellular release promoter) DMSO and PBS were mixed at a ratio of 1: 1 (volume ratio). A 0.02: 80: 1 (weight ratio) mixture of ceramide 1, ceramide 2 and ceramide 3 was dissolved in a solvent at a concentration of 10 mg / mL, and a growth factor production promoter (growth factor extracellular release promoter) was added. Prepared.

(2) Test method Human dermal fibroblasts (2 million cells / mL, TIG-111, 34-year-old woman, 17PDL, JCRB Cell Bank, Osaka, Japan) were subjected to 12-well plates (Corning ^(R) Costar ^(R) 12). 100,000 cells / well seeded in Well Cell Culture Plate), and cultured for 1 day in a medium containing GlutaMAX-I (GIBCO 10566-016) in DMEM containing 2 mL of 2% Fetal Bovine Serum (BLG BI 04-111-1E). did. The medium was replaced with a medium containing GlutaMAX I in 2 mL of DMEM containing 0.2% Fetal Bovine Serum (BLG BI 04-111-1E), and a growth factor production promoter (growth factor extracellular release promoter) was added. , The final concentration of ceramide was 6.25 μg / mL, 12.5 μg / mL, or 25 μg / mL (n = 4 respectively). As a control, a solvent of a growth factor production promoter (growth factor extracellular release promoter) was used (n = 4). The amounts of FGF2 and TGF-β2 per number of cells released into the medium were measured at 1 day, 2 days, 3 days, and 4 days after culturing. Cell counting kit-8 (Dojin Kagaku Kenkyusho) was used for the cell number, TGF-β2 Human, ELISA Kit, and Quantikine (RSDDKG 00) from R & D were used for the amount of TGF-β2, and R &D's for the amount of FGF2. Measurements were made using FGF Basic, Human, ELISA Kit, and Quantikine (DFB 50). In the statistical analysis, an unpaired t-test was performed between the control value for each culture day and the value in the order of storage consent, and p <0.05 was considered to be significantly different in the two-sided test.

(3) Results The amount of FGF2 released per cell number is shown in FIG. 1, and the amount of TGF-β2 released per number of cells is shown in FIG. As shown in these figures, the amount of FGF2 released showed a significant increase in all ceramide concentrations compared to the control (all P <0.01). In addition, 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 (6)

A growth factor gene expression promoter containing ceramides as an active ingredient. The growth factor gene expression promoter according to claim 1, wherein the ceramides are human ceramides. The growth factor gene expression promoter according to claim 1 or 2, which is an expression promoter for the FGF2 gene and / or the TGF-β gene. A growth factor production promoter containing ceramides as an active ingredient. An extracellular release promoter of growth factors containing ceramides as an active ingredient. An angiogenesis-promoting agent containing ceramides as an active ingredient.

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