JP7759181B2 - Growth factor gene expression promoter - Google Patents

Description

Article 30, paragraph 2 of the Patent Act applies. 1. Website address: https://www.ifscc2020.com/virtual_congress.html Date of website publication: October 21, 2020

The present invention relates to growth factor gene expression promoters, growth factor production promoters, growth factor extracellular release promoters, and angiogenesis promoters, each of which contains ceramides as an active ingredient.

Growth factors are endogenous proteins that promote the proliferation and differentiation of specific cells in animal bodies. Research has shown that growth factors play a role in regulating various cellular and physiological processes, and this effect is utilized in some cases, leading to their inclusion as active ingredients in topical pharmaceuticals.

Various growth factors are known, including fibroblast growth factor (FGF) and transforming growth factor (TGF).

FGFs are multifunctional proteins that exhibit a wide range of effects. In particular, 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 do not have 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).

TGF, like many other signaling pathways, plays a crucial role in tissue development, cell differentiation, and embryonic development. TGFs are classified into TGF-α and TGF-β, and TGF-β is thought 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)

In order to actively enjoy the effects of growth factors, it is possible to incorporate the growth factors themselves as active ingredients, but given that active ingredients are not easily available, there is a need for ingredients that are easily available yet can promote the production of these growth factors.

The present invention therefore aims to provide ingredients that can promote the production of growth factors, etc.

After extensive research, the inventors unexpectedly discovered that ceramides have the ability to promote the production of growth factors, among other things. The present invention was completed through further research based on this finding.

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 active ingredients.
Item 5. An agent for promoting extracellular release of growth factors, which comprises ceramides as active ingredients.
Item 6. An angiogenesis promoter containing ceramides as active ingredients.

The present invention provides ingredients that can promote the production of growth factors, etc.

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 also collectively referred to as "growth factor gene expression promoters, etc.") are characterized by containing ceramides as active ingredients. The growth factor gene expression promoters, etc. of the present invention are described in detail below.

1. The active ingredient ceramides include ceramides and pseudoceramides. Either ceramide or pseudoceramide may be used alone, or ceramide and pseudoceramide may be used in combination.

Ceramide is a type of sphingolipid, a well-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 the sphingosine (such as the structure of the N-acyl group) and the structure of the long-chain fatty acid.

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

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

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

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).

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 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.

In formula (II), R ²¹ and R ²² may be the same or different and represent an optionally hydroxylated hydrocarbon group having 1 to 40 carbon atoms, 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. However, when R ²³ is a single bond, R ²⁴ is a hydrogen atom. The hydrocarbon group is preferably an alkyl group or an alkenyl group.

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

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

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 effects of promoting growth factor gene expression, promoting growth factor production, promoting extracellular release of growth factors, and/or promoting angiogenesis, but examples include 0.0001 to 50% by weight, preferably 0.001 to 30% by weight, and more preferably 0.01 to 10% by weight.

Other Components The growth factor gene expression promoter of the present invention may contain, in addition to the active ingredient, other pharmacological components as needed. Examples of such pharmacological components 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 ( Examples of effective active ingredients include: 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.), refreshing agents (menthol, camphor, etc.), vitamins (vitamin A, etc.), mucopolysaccharides (sodium chondroitin sulfate, glucosamine, heparinoids, etc.), etc.

The growth factor gene expression promoters of the present invention may also contain bases and additives as necessary to form the desired formulation. Such bases and additives are not particularly limited as long as they are pharmaceutically acceptable, and examples 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.

Formulation The growth factor gene expression promoter of the present invention may be in the form of any of topical skin preparations, oral preparations, etc., 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.

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, but examples include liquid, solid, and semi-solid forms (gel, ointment, paste, etc.).

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

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 forms are not particularly limited, but examples include liposomes, microemulsions, and microneedles.

Uses: 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.

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 promoting agent, etc. of the present invention is preferably a gene expression promoting agent for the FGF2 gene and/or TGF-β gene, a FGF2 and/or TGF-β production promoting agent, or an FGF2 and/or TGF-β extracellular release promoting agent, and more preferably a gene expression promoting agent for the FGF2 gene and/or TGF-β2 gene, a FGF2 and/or TGF-β2 production promoting agent, or an FGF2 and/or TGF-β2 extracellular release promoting agent.

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

The growth factor gene expression promoters 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 promoters of the present invention and fibrofibroblasts (e.g., autologous cultured fibroblasts) and introducing the material into the patient's dermis by injection or other means.

The present invention will be explained in more detail below using examples, but the present invention is not limited to these examples.

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 solvent prepared by mixing DMSO and PBS in a 1:1 (volume ratio) mixture to a concentration of 50 μg/mL to prepare a growth factor gene expression promoter.

(2) Test Method: A 6-well plate was placed on ice, and 400 μL of 5xMEM (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 NaHCO ₃ , 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 stirring 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 plate was cultured for 1 day. The medium was then replaced with 2 mL of DMEM (2% FBS) containing a growth factor gene expression promoter or its 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 subjected to Affymetrix GeneChip Expression Array analysis. The relative gene expression level was calculated, with the gene expression level in the control set at 1.

(3) Results In the group to which the 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.

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

(2) Test Method Human dermal fibroblasts (2 million cells/mL, TIG-111, 34-year-old female, 17PDL, JCRB Cell Bank, Osaka, Japan) were seeded at 100,000 cells/well in a 12-well plate ( ^{Corning® Costar®} 12 Well Cell Culture Plate) and cultured for 1 day in a medium consisting of 2 mL of 2% fetal bovine serum (BLG BI 04-111-1E)-containing DMEM supplemented with GlutaMAX-I (GIBCO 10566-016). The medium was replaced with 2 mL of 0.2% Fetal Bovine Serum (BLG BI 04-111-1E)-containing DMEM supplemented with GlutaMAX I, and a growth factor production promoter (a growth factor extracellular release promoter) was added to 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 (growth factor extracellular release promoter) was used as a control (n=4). The amounts of FGF2 and TGF-β2 released per cell were measured after 1, 2, 3, and 4 days of culture. Cell counting kit-8 (Dojindo Laboratories) was used to measure cell numbers, 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 by unpaired t-test between the control values and the stored consent order values for each culture day, with a two-tailed p<0.05 indicating a significant difference.

(3) Results Figure 1 shows the amount of FGF2 released per cell, and Figure 2 shows the amount of TGF-β2 released per cell. As shown in these figures, the amount of FGF2 released was significantly increased compared to the control at all ceramide concentrations (all P<0.01). Furthermore, the amount of TGF-β2 released was significantly increased compared to the control at all ceramide concentrations, especially after 3 days (P<0.01).

Claims (5)

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. The FGF2 gene and/or TGF-β2 gene expression promoter according to claim 1, wherein the ceramide and/or pseudoceramide is human ceramide. An agent for promoting the production of FGF2 and/or TGF-β2, which comprises ceramide and/or pseudoceramide as an active ingredient. An agent for promoting the extracellular release of FGF2 and/or TGF-β2, which comprises ceramide and/or pseudoceramide as an active ingredient. The enhancer according to any one of claims 1 to 4, wherein the ceramide and/or pseudoceramide is ceramide 1, ceramide 2 and/or ceramide 3.