IGF-1 production promoter and application thereof

Abstract

To provide a compound that achieves a high promoting ability for expression of the IGF-1 gene.SOLUTION: The present invention relates to an IGF-1 production promoter comprising at least one compound selected from the group consisting of compounds represented by formula (1), for example, and pharmacologically acceptable salts thereof. The present invention further relates to a therapeutic agent for IGF-1-related diseases and a food or beverage, each comprising the IGF-1 production promoter.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an IGF-1 production promoter, a therapeutic agent for IGF-1-related diseases, foods and drinks containing the IGF-1 production promoter, and a method for promoting IGF-1 secretion. [Background technology]

[0002] Insulin-like growth factor-1 (IGF-1) is a peptide hormone with a molecular weight of approximately 7,500 that has a structure and action very similar to insulin. IGF-1 is a peptide hormone produced in the liver, skeletal muscle, skin, and brain, and is known to have growth and metabolism, hair growth promotion, and muscle strengthening effects (Non-Patent Documents 1 and 2). In addition, due to its insulin-like effects, it is a candidate drug for treating diabetes (Non-Patent Document 3), and due to its ability to maintain normal neural activity, it is also a candidate drug for treating autism spectrum disorder (ASD) (Non-Patent Document 4).

[0003] For example, Patent Document 1 describes an insulin-like growth factor-1 production promoter containing α-D-glucopyranosylglycerol. Patent Document 2 describes an IGF-1 production promoter containing swerthiamarin as an active ingredient. Patent Document 3 describes an insulin-like growth factor-1 secretion promoter containing a polyphenol as an active ingredient. Patent Document 4 describes an IGF-1 level-elevating agent containing collagen peptides extracted by treating collagen derived from fish scales and / or fish skin with a protease. Patent Document 5 describes an insulin-like growth factor-1 (IGF-1) expression promoter containing an extract of licorice leaves as an active ingredient. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Laid-Open No. 2009-161475 [Patent Document 2] Japanese Patent Application Laid-Open No. 2009-263262 [Patent Document 3] Japanese Patent Application Laid-Open No. 2011-93807 [Patent Document 4] Japanese Patent Application Laid-Open No. 2012-116773 [Patent Document 5] Japanese Patent Application Laid-Open No. 2013-107907 [Non-patent literature]

[0005] [Non-Patent Document 1] Riia K. Junnila et al, Nat Rev Endocrinol. 2013 Jun;9(6):366-376. [Non-patent document 2] Peter Klover et al, FASEB J. 2009 Sep;23(9):3140-8. [Non-patent document 3] H Kuzuya et al, Diabetes. 1993 May;42(5):696-705. [Non-patent document 4] Ozlem Bozdagi et al, Mol Autism. 2013 Apr 27;4(1):9. Summary of the Invention [Problem to be solved by the invention]

[0006] As mentioned above, IGF-1 gene expression promoters are known, but many of the known IGF-1 gene expression promoters are derived from natural products, and some do not have the properties of pharmaceuticals. Furthermore, the known IGF-1 gene expression promoters have a limited effect in promoting IGF-1 gene expression, which has been an issue.

[0007] Therefore, in order to solve such problems of the conventional techniques, the present inventors have carried out investigations with the aim of providing a compound that has a high ability to promote the expression of the IGF-1 gene. [Means for solving the problem]

[0008] Examples of specific embodiments of the present invention are given below.

[0009] [1] An IGF-1 production promoter comprising at least one compound selected from the group consisting of compounds represented by the following formulas (1) to (3) and pharmacologically acceptable salts thereof: [ka] (In formula (1), R 1 ~R 4 each independently represents a hydrogen atom or a substituent containing at least one atom selected from the group consisting of a carbon atom, an oxygen atom, and a nitrogen atom. [ka] (In formula (2), R 11 ~R 13 each independently represents a hydrogen atom or a substituent containing at least one atom selected from the group consisting of a carbon atom, an oxygen atom, and a nitrogen atom. [ka] (In formula (3), R 21 ~R 23 each independently represents a hydrogen atom or a substituent containing at least one atom selected from the group consisting of a carbon atom, an oxygen atom, and a nitrogen atom. [2] A compound of the formula (1) or a pharmacologically acceptable salt thereof, In the formula (1), R 1 represents a hydroxy group or an alkyl group having 1 to 6 carbon atoms, and R 2 represents a substituent having 1 to 6 carbon atoms containing a carbon atom and an oxygen atom, and R 3 represents an alkyl group having 1 to 6 carbon atoms, and R 4 represents an aliphatic hydrocarbon group having 1 to 12 carbon atoms. [3] In the formula (1), R 1 represents a hydroxy group, and R 2 represents an acyl group, and R 3represents an alkyl group having 1 to 3 carbon atoms, and R 4 represents a cycloalkyl group having 3 to 12 carbon atoms. [4] A compound of the formula (2) or a pharmacologically acceptable salt thereof, In the formula (2), R 11 represents a substituent having 1 to 6 carbon atoms containing a carbon atom and an oxygen atom, and R 12 and R 13 and each independently represent a substituent containing a nitrogen atom, the IGF-1 production promoter according to any one of [1] to [3]. [5] In the formula (2), R 11 represents an alkoxy group, and R 12 and R 13 The IGF-1 production promoter according to [4], wherein each independently represents an amino group. [6] A compound of the formula (3) or a pharmacologically acceptable salt thereof, In the formula (3), R 21 represents a substituent having 1 to 6 carbon atoms and containing a carbon atom and a nitrogen atom, and R 22 represents a substituent containing a nitrogen atom, and R 23 The IGF-1 production promoter according to any one of [1] to [5], wherein represents a substituent having 1 to 6 carbon atoms and containing a carbon atom, an oxygen atom, and a nitrogen atom. [7] In the formula (3), R 21 represents -NRR' (wherein R and R' are alkyl groups having 1 to 3 carbon atoms), and R 22 represents an amino group, and R 23 represents an amide group. [8] A therapeutic agent for IGF-1-related diseases, comprising the IGF-1 production promoter according to any one of [1] to [7]. [9] A food or drink containing the IGF-1 production promoter according to any one of [1] to [7].

[10] A method for promoting secretion of IGF-1 in the blood or organs by orally ingesting the food or beverage described in [9] to a mammal.

[11] Use of at least one compound selected from the group consisting of compounds represented by the following formulas (1) to (3) and pharmacologically acceptable salts thereof for the production of an IGF-1 production promoter: [ka] (In formula (1), R 1 ~R 4 each independently represents a hydrogen atom or a substituent containing at least one atom selected from the group consisting of a carbon atom, an oxygen atom, and a nitrogen atom. [ka] (In formula (2), R 11 ~R 13 each independently represents a hydrogen atom or a substituent containing at least one atom selected from the group consisting of a carbon atom, an oxygen atom, and a nitrogen atom. [ka] (In formula (3), R 21 ~R 23 each independently represents a hydrogen atom or a substituent containing at least one atom selected from the group consisting of a carbon atom, an oxygen atom, and a nitrogen atom. [Effects of the Invention]

[0010] According to the present invention, a compound having a high ability to promote the expression of the IGF-1 gene can be provided. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 is a graph showing the concentration dependence of luciferase activity of compounds D, G and J. [Figure 2] Figure 2 shows the results of confirming the commonality between human and mouse IGF-1 gene expression levels. [Figure 3] FIG. 3 shows the results of MTT assay for compounds D, G and J. [Figure 4]FIG. 4 shows the results of whole transcript analysis for compounds D, G, and J. [Figure 5] FIG. 5 shows the results of pathway analysis of genes that satisfy a P value of <0.05 in the total transcript analysis for compounds D and G. [Figure 6] FIG. 6 shows the results of Western blotting analysis of the expression level of IGF-1 protein in cells. [Figure 7] FIG. 7 shows the results of ELISA measurement of the amount of IGF-1 protein secreted from the cells into the medium. DETAILED DESCRIPTION OF THE INVENTION

[0012] The present invention will be described in detail below. The following description may be based on representative embodiments and specific examples, but the present invention is not limited to such embodiments. In this specification, a numerical range expressed using "to" means a range that includes the numerical values ​​before and after "to" as the lower and upper limits.

[0013] (IGF-1 production promoter) The present embodiment relates to an IGF-1 production promoter comprising at least one compound selected from the group consisting of compounds represented by the following formulas (1) to (3) and pharmacologically acceptable salts thereof. The present embodiment also relates to the use of at least one compound selected from the group consisting of compounds represented by the following formulas (1) to (3) and pharmacologically acceptable salts thereof for the production of an IGF-1 production promoter.

[0014] [ka]

[0015] In formula (1), R 1 ~R 4 each independently represents a hydrogen atom or a substituent containing at least one atom selected from the group consisting of a carbon atom, an oxygen atom, and a nitrogen atom.

[0016] [ka]

[0017] In equation (2), R 11 ~R 13 each independently represents a hydrogen atom or a substituent containing at least one atom selected from the group consisting of a carbon atom, an oxygen atom, and a nitrogen atom.

[0018] [ka]

[0019] In equation (3), R 21 ~R 23 each independently represents a hydrogen atom or a substituent containing at least one atom selected from the group consisting of a carbon atom, an oxygen atom, and a nitrogen atom.

[0020] The compounds represented by the above formulas (1) to (3) each have a heterocyclic fused ring structure such as an indole skeleton, a quinazoline skeleton, or a thienopyridine skeleton, and have physical properties in accordance with Lipinski's five principles, providing excellent properties, for example, as an oral drug. Furthermore, the compounds represented by the above formulas (1) to (3) are non-cytotoxic. In this embodiment, the IGF-1 production promoter can be administered to humans or non-human mammals via either oral or parenteral administration to promote IGF-1 gene expression. Furthermore, in this embodiment, the IGF-1 production promoter can be contacted with cells in vitro to promote IGF-1 gene expression.

[0021] In formula (1), R 1 preferably represents a substituent containing at least one selected from a carbon atom and an oxygen atom, more preferably represents a hydroxy group, an alkyl group having 1 to 6 carbon atoms, or an alkoxy group having 1 to 6 carbon atoms, still more preferably represents a hydroxy group, an alkyl group having 1 to 3 carbon atoms, or an alkoxy group having 1 to 3 carbon atoms, and particularly preferably represents a hydroxy group.

[0022] In formula (1), R 2 represents preferably a substituent containing at least one selected from a carbon atom and an oxygen atom, more preferably a substituent containing a carbon atom and an oxygen atom and having 1 to 6 carbon atoms, even more preferably a substituent containing a carbon atom and an oxygen atom and having 1 to 3 carbon atoms, still more preferably an acyl group, and particularly preferably an acetyl group.

[0023] In formula (1), R 3 preferably represents an alkyl group having 1 to 6 carbon atoms, more preferably represents an alkyl group having 1 to 3 carbon atoms, and particularly preferably represents a methyl group.

[0024] In formula (1), R 4 represents preferably an aliphatic hydrocarbon group having 1 to 12 carbon atoms, more preferably an aliphatic saturated hydrocarbon group having 1 to 12 carbon atoms, more preferably a cycloalkyl group having 3 to 12 carbon atoms, even more preferably a cycloalkyl group having 3 to 6 carbon atoms, and particularly preferably a cyclohexyl group.

[0025] Examples of the compound represented by formula (1) include the following compounds.

[0026] [ka]

[0027] In equation (2), R 11 represents preferably a substituent containing at least one selected from a carbon atom and an oxygen atom, more preferably a hydroxy group or a substituent containing a carbon atom and an oxygen atom and having 1 to 6 carbon atoms, even more preferably a hydroxy group or a substituent containing a carbon atom and an oxygen atom and having 1 to 3 carbon atoms, still more preferably a hydroxy group, an alkyl group having 1 to 3 carbon atoms or an alkoxy group having 1 to 3 carbon atoms, and particularly preferably an alkoxy group having 1 to 3 carbon atoms.

[0028] In equation (2), R 12 and R 13 preferably each independently represents a substituent containing a nitrogen atom, and R 12 and R 13 It is particularly preferred that each independently represents an amino group.

[0029] Examples of the compound represented by formula (2) include the following compounds.

[0030] [ka]

[0031] In equation (3), R 21 preferably represents a substituent containing a carbon atom and a nitrogen atom and having 1 to 6 carbon atoms, more preferably represents -NRR' (where R and R' are alkyl groups having 1 to 3 carbon atoms), and even more preferably represents -NRR' (where R and R' are methyl groups).

[0032] In equation (3), R 22 preferably represents a substituent containing a nitrogen atom, and particularly preferably represents an amino group.

[0033] In equation (3), R 23 represents preferably a substituent containing a carbon atom, an oxygen atom, and a nitrogen atom and having 1 to 6 carbon atoms, more preferably a substituent containing a carbon atom, an oxygen atom, and a nitrogen atom and having 1 to 3 carbon atoms, and particularly preferably an amide group.

[0034] Examples of the compound represented by formula (3) include the following compounds.

[0035] [ka]

[0036] In particular, the IGF-1 production promoter of this embodiment preferably contains the compounds represented by the above-mentioned formulas (1) and (3) or pharmacologically acceptable salts thereof, and particularly preferably contains the above-mentioned compounds G and D or pharmacologically acceptable salts thereof. The compounds represented by the above-mentioned formulas (1) and (3) or pharmacologically acceptable salts thereof are preferably used because they have a particularly high ability to promote expression of the IGF-1 gene while having little effect on the expression of genes other than the IGF-1 gene.

[0037] In particular, the IGF-1 production promoter of this embodiment preferably contains the compound represented by the above formula (1) or a pharmacologically acceptable salt thereof, and particularly preferably contains the above compound G or a pharmacologically acceptable salt thereof. The compound represented by the above formula (1) or a pharmacologically acceptable salt thereof is particularly effective in promoting the expression of the IGF-1 gene, while having little effect on the expression of genes other than the IGF-1 gene. Furthermore, the compound represented by the above formula (1) or a pharmacologically acceptable salt thereof is more preferably used because it does not promote inflammation-related pathways or pathways leading to various diseases.

[0038] The pharmacologically acceptable salt is not particularly limited and can be appropriately selected depending on the purpose. Examples of pharmacologically acceptable salts include hydrochloride, sulfate, hydrobromide, nitrate, hydrogensulfate, phosphate, acetate, lactate, succinate, citrate, maleate, hydroxymaleate, tartrate, fumarate, methanesulfonate, p-toluenesulfonate, camphorsulfonate, sulfamate, mandelate, propionate, glycolate, stearate, malate, ascorbate, pamoate, phenylacetate, glutamate, benzoate, salicylate, and the like. Examples of the salts include thylate, sulfanilate, 2-acetoxybenzoate, ethanedisulfonate, oxalate, isethionate, formate, trifluoroacetate, ethylsuccinate, lactobionate, gluconate, glucoheptonate, 2-hydroxyethanesulfonate, benzenesulfonate, paratoluenesulfonate, lauryl sulfate, aspartate, adipate, hydroiodide, nicotinate, oxalate, picrate, thiocyanate, and undecanoate.

[0039] The IGF-1 production enhancer of this embodiment may be in any form, such as a solid (e.g., tablet, granule, powder, capsule, etc.), sol, gel, or liquid. The content of the above-mentioned compound or a pharmacologically acceptable salt thereof in the IGF-1 production enhancer of this embodiment is not particularly limited and can be appropriately selected depending on the purpose.

[0040] The IGF-1 production promoter of this embodiment may contain various additives, such as excipients (lactose, sucrose, starch syrup, dextrin, cornstarch, crystalline cellulose, etc.), lubricants (magnesium stearate, sucrose fatty acid esters, glycerin fatty acid esters, etc.), disintegrants (carboxymethylcellulose calcium, anhydrous calcium hydrogen phosphate, calcium carbonate, etc.), binders (starch paste solution, hydroxypropyl cellulose solution, gum arabic solution, etc.), solubilizers (gum arabic, polysorbate 80, etc.), sweeteners (sugar, fructose-glucose liquid sugar, honey, aspartame, etc.), coloring agents (β-carotene, food tar dyes, riboflavin, etc.), preservatives (sorbic acid, Examples of additives include: antioxidants (e.g., methyl parahydroxybenzoate, sodium sulfite), thickeners (e.g., sodium alginate, sodium carboxymethylcellulose, sodium polyacrylate), antioxidants (e.g., BHT, BHA, ascorbic acid, tocopherol), flavorings (e.g., peppermint, strawberry flavoring), acidulants (e.g., citric acid, lactic acid, DL-malic acid), seasonings (e.g., DL-alanine, 5'-sodium inosinate, L-sodium glutamate), emulsifiers (e.g., glycerin fatty acid esters, sucrose fatty acid esters), pH adjusters (e.g., citric acid, trisodium citrate), vitamins, minerals, amino acids, etc.

[0041] (Treatment for IGF-1 related diseases) The IGF-1 production enhancer of this embodiment can also be used as a pharmaceutical preparation. This embodiment may also relate to a therapeutic agent for an IGF-1-related disease, which comprises the above-mentioned IGF-1 production enhancer as an active ingredient. The therapeutic agent for an IGF-1-related disease of this embodiment can be used for preventing, treating, or ameliorating pathologies such as improving skin radiance and firmness, restoring flexibility, increasing bone density, growing muscle cells, anabolism, improving glucose metabolism, lowering blood pressure, suppressing inflammation, improving cognitive function, growing hair, activating immune function, and the aging phenomenon associated with physiological aging.

[0042] The therapeutic agent for IGF-1-related diseases may be an external preparation or an internal preparation. When the IGF-1 production enhancer of the present embodiment is used as a pharmaceutical preparation, the dosage of the active ingredient can be adjusted appropriately depending on the gender, age, health condition, etc. of the person taking it.

[0043] When the therapeutic agent for IGF-1-related diseases is a topical agent, the dosage of the active ingredient can be selected appropriately depending on the area of ​​the skin to be treated. Generally, the dosage is about 1 cm2. 2 The daily dose is preferably 0.001 to 1000 mg, more preferably about 0.01 to 500 mg, and even more preferably about 0.1 to 300 mg.

[0044] When the therapeutic agent for IGF-1-related diseases is an internal agent, it may be administered subcutaneously, intramuscularly, intravenously, orally, or transdermally, and the method most suitable for prevention, treatment, etc. may be selected depending on the type of disease. When the therapeutic agent for IGF-1-related diseases is an internal agent, the daily dose of the active ingredient per kg of body weight is preferably 0.001 to 1000 (mg / kg / day), more preferably 0.01 to 500 (mg / kg / day), and even more preferably 0.1 to 300 (mg / kg / day).

[0045] Although therapeutic agents for IGF-1-related diseases are preferably applied to humans, they may also be applied to mammals other than humans as long as their respective functional effects are achieved.

[0046] (food and drink) This embodiment may also relate to foods and beverages containing the IGF-1 production enhancer described above. Examples of foods and beverages include soft drinks, drops, candy, chewing gum, chocolate, gummy candies, yogurt, ice cream, pudding, jelly confectionery, cookies, bread, biscuits, noodles, margarine, shortening, mayonnaise, and dressings. The enhancer may be added as appropriate during a suitable manufacturing process depending on the characteristics and purpose of each food and beverage.

[0047] This embodiment may relate to a method for promoting IGF-1 secretion in the blood or organs by orally ingesting the above-described food or drink to a mammal. By administering the IGF-1 production promoter to a mammal via oral administration, it is possible to promote IGF-1 gene expression. Examples of mammals include humans and non-human mammals.

[0048] (External skin preparation) The present embodiment may also relate to a food or beverage containing the above-mentioned IGF-1 production promoter. The IGF-1 production promoter of the present embodiment can be used as an additive in non-oral cosmetics such as lotions (e.g., lotions, gels, etc.), cosmetic creams, emulsions, foundations, lipsticks, hair styling products, hair growth agents, hair tonics, shampoos, rinses, and bath additives, as well as toothpastes, mouthwashes, gargles, and oral flavorings. [Example]

[0049] The features of the present invention will be explained in more detail below with reference to examples. The materials, amounts used, ratios, treatment contents, treatment procedures, etc. shown in the following examples can be changed as appropriate without departing from the spirit of the present invention. Therefore, the scope of the present invention should not be construed as being limited by the specific examples shown below.

[0050] (Example) <Culture of microglial cell line BV-2> The microglial cell line BV-2 was purchased from Banca Biologicae Cell Factory (San Martino, Italy, RRID: CVCL_0182). Cells were maintained in Dulbecco's modified Eagle's medium (Shimadzu Diagnostics Co., Ltd., Tokyo, Japan, #05915) containing 10% fetal bovine serum (Cytiva, Tokyo, Japan, #SH30396.03) and antibiotics (ThermoFisher Scientific, Waltham, MA, USA, #15140122) and cultured at 37°C in a 5% CO2 atmosphere.

[0051] <Establishment of human pIGF-1 / NanoLuc reporter cell line> The human IGF-1 gene promoter (gene expression regulatory region) of approximately 6112 base pairs was cloned from human genomic DNA (Roche Diagnostics, Mannheim, Germany, #11 691 112 001) using the primer set F: 5'-CGGAAGCTTTGCTTCTGAAGTACAAAG-3' (SEQ ID NO: 1) and R: 5'-CCAGTGAGCGCACCTTTTGAGAG-3' (SEQ ID NO: 2). The cloned fragment was then inserted into the pNL2.1[Nluc / Hygro] Vector (Promega, #N1061) containing NanoLuc as a reporter gene. The resulting vector was linearized by restriction enzyme digestion and then transfected into the microglial cell line BV-2. After 2 days, the medium was replaced with a selective medium containing 500 μg / ml hygromycin B (Fujifilm Wako Pure Chemical Industries, Osaka, Japan). The cells were cultured for 2 weeks, and surviving cells were selected and analyzed by 96% genomic DNA sequencing. The cells were seeded at 0.5 cells / well on a 100-well plate. They were then cultured in selective medium for two weeks, and several stable lines were selected. They were then stimulated with a 100 ng / ml solution of lipopolysaccharide (LPS), a known IGF-1 gene expression-stimulating compound, for 24 hours. The cell line with the highest signal-to-noise ratio (promoted IGF-1 gene expression) was established as a stable line. This established an experimental system for indirect and simple detection of IGF-1 gene expression.

[0052] <Compound screening> The compounds for screening were obtained from the Drug Discovery Institute, Graduate School of Pharmaceutical Sciences, The University of Tokyo. For high-throughput screening (HTS), 9,600 samples (Core Library) were selected from approximately 250,000 samples (Full Library) taking into account the structural diversity of the compounds. The established cell lines were treated with 5 μM compounds for 24 hours, and IGF-1 gene expression was indirectly assessed by luciferase assay (measurement of Nanoluc fluorescence intensity). The procedure was performed according to the manufacturer's instructions. Luminescence was measured using a SpectraMaxParadigm™ DTX800 / 880 (Molecular Devices, San Jose, CA, USA). The luciferase activity of the compounds was expressed as a percentage (T / C %) relative to the negative control (1% DMSO). The mean values ​​for the compound-treated groups were calculated, and 92 compounds that met or exceeded the mean value + 3 × (standard deviation, SD) were selected as primary hit compounds. The primary hit compounds were then subjected to secondary screening to confirm reproducibility and select compounds with higher relative activity values. In the secondary screening, 32 compounds meeting or exceeded the mean value + 3 × SD were selected. In a third screening, compounds with higher activity and reproducibility were further selected. Based on the results of the third screening, dose dependency was confirmed at 0.05, 0.1, 0.5, 1, and 5 μM for 12 compounds with high reproducibility and comparative activity.

[0053] Furthermore, three compounds (compounds D, G, and J) that showed concentration-dependent effects (Figure 1) were examined for their ability to promote Igf-1 gene expression in mouse cultured BV-2 cells. The expression level of Igf-1 in mouse cultured BV-2 cells was measured using real-time quantitative reverse transcription PCR (qRT-PCR), with the negative control (1% DMSO) group set as 1 (Figure 2). RNA isolation, cDNA synthesis, and qRT-PCR were performed as previously described. Using Gapdh as an internal control, the expression level of each transcript was measured using the standard curve method. The following primers were used in qRT-PCR. F: 5'-GTGGATGCTCTTCAGTTCGTGTG-3' (SEQ ID NO: 3) R: 5'-TCCAGTCTCCTCAGATCACAGC-3' (SEQ ID NO: 4)

[0054] [Chemical] [Chemical] [Chemical]

[0055] Furthermore, when the MTT assay (MTT Cell Viability Assay kit, biotium, #30006) was performed with these three compounds, none of them showed toxicity or abnormal growth ability (Figure 3).

[0056] To select those that do not affect genes other than IGF-1, whole transcriptome analysis was performed using a microarray (Clariom D assay, mouse, ThermoFisher Scientific, #902513). For the microarray, after allowing various compounds to act at 5 μM for 24 hours, RNA that had been extracted and purified was used, and the procedure was carried out according to the manufacturer's instructions. In this experiment, those satisfying P value < 0.05, fold change ≥ 1.5 or ≤ 1.5 were defined as the differentially expressed gene group (DEGs). As a result of the whole transcriptome analysis, it was confirmed that compounds D and G did not affect the expression of a large number of genes (Figure 4).

[0057] Genes satisfying P value < 0.05 in the above-mentioned whole transcriptome analysis were subjected to Ingenuity Pathway Analysis (IPA, QIAGEN, Hilden Germany). As a result, it was suggested that compound D might activate pathways that induce diseases and inflammation (Figure 5).

[0058] [[ID=3G]]<Analysis of the expression level of IGF-1 protein> After treating the cells with various compounds, a cell lysis reagent (10 mM HEPES pH 7.9, 0.15 M KCl, 5 mM MgCl2, 1 mM EDTA pH 7.9, 0.5% Triton X-100, 0.5 mM dithiothreitol, Complete Protease Inhibitor Cocktail, PhosSTOP) was added to the recovered cells. After incubation on ice for 20 minutes, the cells were disrupted using an ultrasonic homogenizer. After centrifugation at 12,000 × g for 20 minutes at 4°C, the supernatant was used as the total protein extract. The concentration of the total protein extract was measured using the Bradford method, and equal amounts of protein were prepared and denatured in SDS-PAGE sample buffer. Each sample was subjected to SDS-PAGE and then subjected to Western blotting analysis. After blocking with 5% skim milk for 1 hour at room temperature, primary and secondary antibody reactions were performed. The antibodies used were: anti-IGF-1 (Abcam, Cambridge, UK, #ab9572, 1:2,000), anti-GAPDH (ThermoFisher Scientific, #MA5-15738, 1:10,000), goat anti-rabbit IgG HRP-conjugated (Agilent, Santa Clara, CA, USA, #P044801-2, 1:5,000), and rabbit anti-mouse IgG HRP-conjugated (Agilent, #P026002-2, 1:5,000). Target proteins were visualized using chemiluminescence, and their expression intensity was analyzed using ImageJ software (http: / / imagej.nih.gov / ij / ). GAPDH was used as an internal control (Figure 6). It was confirmed that compounds D and G promote IGF-1 production not only at the gene expression level but also at the protein level.

[0059] The culture supernatant of the cells subjected to Western blotting analysis was collected and centrifuged at 300 × g for 5 minutes at 4°C. The supernatant after centrifugation was collected, and protein was quantified by the Bradford method. Equal amounts of protein were then subjected to ELISA. Secreted IGF-1 was analyzed by ELISA using the Mouse / Rat IGF-1 / IGF-1 Quantikine ELISA Kit (R & D SYSTEMS, Minneapolis, MN, USA, #MG100). All procedures were performed according to the manufacturer's instructions. Absorbance was measured at 450 nm (signal) and 540 nm (background) using a plate reader (Figure 7). Compounds D and G were confirmed to promote IGF-1 production not only at the gene expression level but also at the protein level.

Claims

1. An IGF-1 production promoter comprising at least one compound selected from the group consisting of compounds represented by the following formulas (1) to (3) and pharmacologically acceptable salts thereof: 【Chemistry 1】 (In formula (1), R １ ~R ４ each independently represents a hydrogen atom or a substituent containing at least one atom selected from the group consisting of a carbon atom, an oxygen atom, and a nitrogen atom. 【Chemistry 2】 (In formula (2), R １１ ~R １３ each independently represents a hydrogen atom or a substituent containing at least one atom selected from the group consisting of a carbon atom, an oxygen atom, and a nitrogen atom. 【Transformation 3】 (In formula (3), R ２１ ~R ２３ each independently represents a hydrogen atom or a substituent containing at least one atom selected from the group consisting of a carbon atom, an oxygen atom, and a nitrogen atom.

2. The compound of formula (1) or a pharmacologically acceptable salt thereof is included, In the formula (1), R １ represents a hydroxy group or an alkyl group having 1 to 6 carbon atoms, and R ２ represents a substituent having 1 to 6 carbon atoms containing a carbon atom and an oxygen atom, R ３ represents an alkyl group having 1 to 6 carbon atoms, and R ４ The IGF-1 production enhancer according to claim 1, wherein represents an aliphatic hydrocarbon group having 1 to 12 carbon atoms.

3. In the formula (1), R １ represents a hydroxy group, and R ２ represents an acyl group, R ３ represents an alkyl group having 1 to 3 carbon atoms, and R ４ The IGF-1 production promoter according to claim 2, wherein represents a cycloalkyl group having 3 to 12 carbon atoms.

4. The compound of formula (2) or a pharmacologically acceptable salt thereof is included, In the formula (2), R １１ represents a substituent having 1 to 6 carbon atoms containing a carbon atom and an oxygen atom, R １２ and R １３ The IGF-1 production enhancer according to claim 1, wherein each independently represents a substituent containing a nitrogen atom.

5. In the formula (2), R １１ represents an alkoxy group, and R １２ and R １３ The IGF-1 production enhancer according to claim 4, wherein each independently represents an amino group.

6. The compound of formula (3) or a pharmacologically acceptable salt thereof is included, In the formula (3), R ２１ represents a substituent having 1 to 6 carbon atoms and containing a carbon atom and a nitrogen atom, R ２２ represents a substituent containing a nitrogen atom, and R ２３ The IGF-1 production promoter according to claim 1, wherein represents a substituent having 1 to 6 carbon atoms and containing a carbon atom, an oxygen atom, and a nitrogen atom.

7. In the formula (3), R ２１ represents -NRR' (wherein R and R' are alkyl groups having 1 to 3 carbon atoms), and R ２２ represents an amino group, and R ２３ The IGF-1 production promoter according to claim 6, wherein represents an amide group.

8. A therapeutic agent for IGF-1-related diseases, comprising the IGF-1 production promoter according to any one of claims 1 to 7.

9. A food or drink comprising the IGF-1 production promoter according to any one of claims 1 to 7.

10. A method for promoting secretion of IGF-1 in blood or organs, comprising orally ingesting the food or drink according to claim 9 to a mammal.

11. Use of at least one compound selected from the group consisting of compounds represented by the following formulas (1) to (3) and pharmacologically acceptable salts thereof for the production of an IGF-1 production promoter: 【Chemistry 4】 (In formula (1), R １ ~R ４ each independently represents a hydrogen atom or a substituent containing at least one atom selected from the group consisting of a carbon atom, an oxygen atom, and a nitrogen atom. 【Transformation 5】 (In formula (2), R １１ ~R １３ each independently represents a hydrogen atom or a substituent containing at least one atom selected from the group consisting of a carbon atom, an oxygen atom, and a nitrogen atom. 【Transformation 6】 (In formula (3), R ２１ ~R ２３ each independently represents a hydrogen atom or a substituent containing at least one atom selected from the group consisting of a carbon atom, an oxygen atom, and a nitrogen atom.