JP7475597B2 - FGF21 production promoter - Google Patents

Description

The present invention relates to an FGF21 production promoter. More specifically, the present invention relates to a new use of oleanolic acid and its derivatives as FGF21 production promoters.

FGF (fibroblast growth factor) is a multifunctional intercellular signaling factor that exhibits a variety of effects, including proliferation activity and differentiation induction, on various cells including fibroblasts (Non-Patent Document 1). FGF21, one of the FGFs, is a secreted cytokine that exerts various effects on the body, and research is being conducted with the aim of applying it to medical treatment.

In recent years, several ingredients that promote FGF21 production have been reported. Specifically, berberine, a major ingredient of the herbal medicine Coptis japonica (Non-Patent Documents 2 and 3), and a specific alga (Patent Document 1) have been found to promote FGF21 production.

Meanwhile, triterpenes are terpenes with a basic skeleton of 30 carbon atoms, and are expected to have various physiological activities. Regarding oleanolic acid, which is one type of triterpene, it has been reported that it has an anti-caries effect (Patent Document 2), an anti-cancer effect (Patent Document 3), an antioxidant effect (Non-Patent Document 4), an osteoporosis prevention effect (Patent Document 4), an effect of maintaining the undifferentiated state of stem cells (Patent Document 5), a moisturizing effect (Patent Document 6), etc. On the other hand, there have been no reports of a relationship between oleanolic acid and FGF21 production.

Biochemistry Vol. 88, No. 1, pp. 86-93 (2016) "Berberine stimulates FGF21 expression via activation of AMP-activated protein kinase in brown and beige adipocytes", Journal of the Aichi Gakuin University Pharmaceutical Society, Vol. 11, December 2018, pp. 18-19 "Search for natural products that control the metabolic regulator FGF21", Journal of the Aichi Gakuin University Pharmaceutical Society, Vol. 11, December 2018, pp. 22-23 Food Chemistry 2005, 92(4), 721-727

JP 2018-135311 A Japanese Patent Application Laid-Open No. 1-290619 Japanese Patent Application Laid-Open No. 8-119866 JP 2014-141444 A JP 2016-169172 A JP 2018-058804 A

Only a few ingredients that promote FGF21 production have been reported so far. For example, berberine may have specific side effects (e.g., constipation), and certain algae are unstandardized natural products, making it difficult to ensure a consistent quality. Considering these factors, more options for ingredients that promote FGF21 production are desired.

The object of the present invention is to provide a new component that promotes FGF21 production.

After extensive research, the inventors discovered that oleanolic acid has the effect of promoting FGF21 production. Further research revealed that the effect of promoting FGF21 production can be further improved by derivatizing oleanolic acid. The present invention was completed based on these findings and through further research.

（式中、Ｒは、水素原子、炭素数２～３０のアシル基、又は炭素数１～６のアルキル基である。）
項２． 前記Ｒが炭素数２～７の脂肪族アシル基である、項１に記載のＦＧＦ２１産生促進剤。
項３． 脂質代謝低下又は肥満の予防又は改善に用いられる、項１又は２に記載のＦＧＦ２１産生促進剤。 That is, the present invention provides the following aspects.
Item 1. An FGF21 production promoter comprising a compound represented by the following formula (I) and/or a pharma- ceutically acceptable salt thereof:

(In the formula, R is a hydrogen atom, an acyl group having 2 to 30 carbon atoms, or an alkyl group having 1 to 6 carbon atoms.)
Item 2. The FGF21 production promoter according to Item 1, wherein the R is an aliphatic acyl group having 2 to 7 carbon atoms.
Item 3. The FGF21 production promoter according to Item 1 or 2, which is used for preventing or ameliorating impaired lipid metabolism or obesity.

The present invention provides a new component that promotes FGF21 production.

The FGF21 production promoter of the present invention is characterized by containing oleanolic acid and a specific derivative thereof. The FGF21 production promoter of the present invention is described in detail below.

The oleanolic acid and specific derivatives thereof contained in the FGF21 production promoter of the present invention are compounds represented by the following formula (I) and/or pharma- ceutical acceptable salts thereof.

In formula (I), R is a hydrogen atom, an acyl group having 2 to 30 carbon atoms, or an alkyl group having 1 to 6 carbon atoms. When R is a hydrogen atom, compound (I) is oleanolic acid. When R is the above acyl group or the above alkyl group, compound (I) is a derivative in which the hydroxyl group at the 3-position of oleanolic acid is modified to an ester or ether (ester-type derivative or ether-type derivative). In the FGF21 production promoter of the present invention, oleanolic acid, ester-type derivatives of oleanolic acid, and ether-type derivatives of oleanolic acid may be used alone or in combination.

When compound (I) is an ester derivative in which R is an acyl group having 2 to 30 carbon atoms, the acyl group may be either an aliphatic acyl group or an aromatic acyl group. Examples of the aliphatic acyl group include aliphatic acyl groups having 2 to 7 carbon atoms, specifically, linear or branched, saturated or unsaturated acyl groups having 2 to 7 carbon atoms, preferably 2 to 4 carbon atoms, more specifically, alkanoyl groups such as acetyl group, n-propionyl group, isopropionyl group, n-butyryl group, and t-butyryl group; and alkenoyl groups such as acryloyl group (propenoyl group), methacryloyl group, and n-butenoyl group.

Examples of aromatic acyl groups include aromatic acyl groups having 7 to 30 carbon atoms, preferably 7 to 14 carbon atoms. More specifically, examples include a group in which the hydrogen atom of a formyl group is substituted with an aromatic group, and a group in which any hydrogen atom of the above-mentioned aliphatic acyl group is substituted with an aromatic group. Examples of groups in which the hydrogen atom of a formyl group is substituted with an aromatic group include a benzoyl group and a naphthoyl group. Examples of groups in which any hydrogen atom of the above-mentioned aliphatic acyl group is substituted with an aromatic group include preferably the above-mentioned aliphatic acyl groups, preferably groups in which the terminal hydrogen atom of an alkenoyl group is substituted with an aromatic group (benzyl group, naphthyl group, etc.), more preferably groups in which the terminal hydrogen atom of an acryloyl group (propenoyl group) or methacryloyl group is substituted with an aromatic group (benzyl group, naphthyl group, etc.), and even more preferably a cinnamoyl group (3-phenylpropenoyl group).

These ester derivatives may be used alone or in combination.

When compound (I) is an ether derivative in which R is an alkyl group having 1 to 6 carbon atoms, the alkyl group may be either linear or branched, and specific examples thereof include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or t-butyl, n-pentyl, isopentyl, neopentyl, t-pentyl, 2-methylbutyl, n-hexyl, isohexyl, 3-methylpentyl, and ethylbutyl groups, and preferably methyl, ethyl, n-propyl, and isopropyl groups.

These ether derivatives may be used alone or in combination.

Pharmaceutically acceptable salts of the compound represented by formula (I) above include carboxylates, specifically alkali metal salts (sodium salt, potassium salt, etc.); alkaline earth metal salts (calcium salt, magnesium salt, barium salt, etc.); salts with organic bases (salts with triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N,N-dibenzylethylenediamine, etc.), etc.

In the FGF21 production promoter of the present invention, from the viewpoint of obtaining a higher FGF21 production promoting effect, among the compounds represented by the above formula (I) and/or pharma- ceutically acceptable salts thereof, the compounds represented by the above formula (I) are preferred, derivatives of oleanolic acid are more preferred, ester derivatives of oleanolic acid are even more preferred, ester derivatives in which R is an aliphatic acyl group having 2 to 7 carbon atoms are even more preferred, and ester derivatives in which R is an acetyl group (i.e., 3-acetyloleanolic acid) are particularly preferred.

As the oleanolic acid, a commercially available product (for example, available from Fujifilm Wako Pure Chemical Industries, Ltd.) may be used, or it may be obtained by a known manufacturing method. Known manufacturing methods include a method of extracting, isolating and purifying from a plant material containing oleanolic acid, and a method of isolating and purifying from a culture of a microorganism that produces oleanolic acid. Examples of plant materials containing oleanolic acid include olives, grapes, beets, jujubes, almonds, banaba leaves, olive leaves, sage, hawthorn, raspberries, quince, rosemary leaves, guava, shiso leaves, blueberries, prunes, loquats, pomegranates, lemon balm, basil, rose hips, persimmons, and Swertia japonica. Microorganisms that produce oleanolic acid include algae related to the genus Aurantiochytrium, specifically, strain AJ7866 (FERM P-22289), strain AJ7868 (FERM P-22290), strain AJ7867 (FERM P-22304), and their derived strains.

Derivatives of oleanolic acid can be synthesized using oleanolic acid by a known method of derivatizing the hydroxyl group at the 3-position.

For example, an ester derivative of oleanolic acid can be synthesized by applying a known esterification method to the hydroxyl group at the 3-position of oleanolic acid. Specifically, an ester derivative of oleanolic acid can be obtained by reacting oleanolic acid with an acid halide (ROX ¹ ), an active ester (ROX ² ), an acid anhydride (ROR), etc. of a carboxylic acid (ROH) corresponding to the acyl group R in the above formula (I). In the above carboxylic acid, acid halide, active ester, and acid anhydride, R is the same as the acyl group R in the above formula (I). In the above acid halide, X ¹ represents a halogen such as Cl, Br, or I. In the above active ester, ^X2 represents a group derived from an active esterifying agent ^X2OH , and examples of the active esterifying agent include N-hydroxy compounds such as N-hydroxysuccinimide, 1-hydroxybenzotriazole, and N-hydroxy-5-norbornene-2,3-dicarboxylimide; disulfide compounds such as dipyridyl disulfide; and carbodiimides such as dicyclohexylcarbodiimide.

In addition, the ether derivative of oleanolic acid can be synthesized by applying a known etherification method to the hydroxyl group at the 3-position of oleanolic acid. Specifically, the ether derivative of oleanolic acid can be obtained by reacting oleanolic acid with an alkyl halide (RX ¹ ) corresponding to the alkyl group R in the above formula (I). In the above alkyl halide, R is the same as the alkyl group R in the above formula (I). In the above alkyl halide, X ¹ represents a halogen such as Cl, Br, or I.

The oleanolic acid derivative may also be obtained by a method of extraction, isolation, and purification from a plant material containing the oleanolic acid derivative. For example, examples of plant materials containing 3-acetyloleanolic acid include Forsythia spp. (herbal medicine) and red adzuki beans.

Other components The FGF21 production promoter of the present invention may consist of only the above-mentioned compounds as active ingredients, or may contain additives and bases according to the formulation. Such additives and bases are not particularly limited as long as they are pharmacologic acceptable, and examples thereof include excipients, binders, disintegrants, lubricants, isotonicity agents, plasticizers, dispersants, emulsifiers, solubilizers, wetting agents, stabilizers, suspending agents, adhesives, coating agents, glossing agents, water, oils and fats, waxes, hydrocarbons, fatty acids, higher alcohols, esters, water-soluble polymers, surfactants, metal soaps, lower alcohols, polyhydric alcohols, pH adjusters, buffers, antioxidants, UV inhibitors, preservatives, flavorings, fragrances, powders, thickeners, dyes, chelating agents, etc. These additives may be used alone or in combination of two or more. The content of these additives and bases is appropriately set according to the type of additives and bases used, the formulation form of the FGF21 production promoter, etc.

In addition, the FGF21 production promoter of the present invention may contain other nutritional components or pharmacological components as necessary, in addition to the above-mentioned compounds as active ingredients. Such nutritional components and pharmacological components are not particularly limited as long as they are pharmacologic acceptable, and examples thereof include antacids, stomachic agents, digestive agents, intestinal regulators, antispasmodics, mucosal repair agents, anti-inflammatory agents, astringents, antiemetics, antitussives, expectorants, anti-inflammatory enzymes, sedatives, hypnotics, antihistamines, caffeine, cardiac diuretics, antibacterial agents, vasoconstrictors, vasodilators, local anesthetics, herbal extracts, vitamins, and menthols. These nutritional components and pharmacological components may be used alone or in combination of two or more. The content of these components is appropriately set depending on the type of components used, the formulation form of the FGF21 production promoter, and the like.

Formulations The formulation of the FGF21 production promoter of the present invention is not particularly limited as long as it can be administered orally, and examples thereof include solid formulations such as powders, fine granules, granules (including dry syrup), tablets, pills, capsules (soft capsules, hard capsules), etc.; semi-solid formulations such as jellies; and liquid formulations such as liquids, suspensions, syrups, etc. To prepare the FGF21 production promoter of the present invention in these formulations, the above-mentioned compounds as active ingredients, and additives, bases, and pharmacological ingredients added as necessary, may be formulated according to conventional formulation techniques.

Uses The FGF21 production promoter of the present invention is used for the purpose of promoting the production of FGF21. The FGF21 production promoter increases the expression level of FGF21 in the living body by administering it to the living body. As an action based on FGF21, the preventive and therapeutic effects against diabetes, dyslipidemia, obesity, cardiovascular disease, cardiac disorders (hypertrophy, etc.), sepsis, rheumatoid arthritis, non-alcoholic fatty liver disease (NAFLD), and metabolic syndrome (insulin resistance, dyslipidemia, visceral obesity, and hypertension) are known. Therefore, the FGF21 production promoter of the present invention can be used for the purpose of obtaining the above-mentioned action based on FGF21 by increasing the expression level of FGF21.

Dosage and Usage: The FGF21 production promoter of the present invention is used by oral administration. The dosage of the FGF21 production promoter of the present invention is appropriately set depending on the type of active ingredient, the age, sex, constitution, etc. of the subject of administration, but for example, the amount of the compound as the active ingredient per person per day is, for example, about 0.1 to 500 mg/kg body weight, preferably about 1 to 300 mg/kg body weight, more preferably about 2 to 100 mg/kg body weight, and may be taken 1 to 3 times a day, preferably 2 or 3 times a day.

The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

Test Example 1
(1) Oleanolic Acid Commercially available oleanolic acid (Fujifilm Wako Pure Chemical Industries, Ltd.) was prepared.

(2) Synthesis of 3-acetyloleanolic acid Oleanolic acid (38.4 mmol) was dissolved in 500 mL of dichloromethane (super dehydrated), and then acetic anhydride (15.81 g), triethylamine (24.15 mL), and dimethylaminopyridine (465 mg) were added in that order, and the mixture was stirred at room temperature for 12 hours. After the reaction was completed, the reaction solution was diluted with 500 mL of diethyl ether, and then washed with 500 mL of 0.1 M hydrochloric acid (once), 500 mL of purified water (twice), and 500 mL of saturated saline (once) to obtain an ether phase. An appropriate amount of anhydrous magnesium sulfate was added to the ether phase, and filtration was performed. The filtrate was concentrated under reduced pressure, and the obtained residue (25.44 g) was purified by silica gel column chromatography (n-hexane:acetone=15:1). Fractions containing 3-acetyloleanolic acid were collected by comparison with a standard sample in thin layer chromatography, concentrated, and recrystallized from n-hexane/dichloromethane to obtain 3-acetyloleanolic acid (5.3 g) as a white powder.

(3) Isolation and purification of 3-acetyloleanolic acid Forsythia spp. (Tsumura; 2 kg) was immersed in methanol (8 L) at room temperature for 24 hours and extracted three times to obtain a Forsythia spp. extract. The obtained extract was concentrated under reduced pressure to obtain a Forsythia spp. extract (175.3 g). When the obtained Forsythia spp. extract was redissolved in methanol, insoluble matter (34.4 g) precipitated, and the insoluble matter was removed by filtration. The obtained filtrate was subjected to liquid-liquid distribution using ethyl acetate (1 L) and purified water (1 L) to obtain an ethyl acetate fraction (111.3 g). The ethyl acetate fraction was subjected to silica gel column chromatography and eluted with a chloroform/methanol mixed solvent system to obtain 27 fractions. Since activity was observed in the first to sixth fractions obtained, these fractions were concentrated and purified by silica gel column chromatography using a n-hexane/acetone mixed solvent as the mobile phase to obtain 36 fractions. The 27th to 35th fractions were concentrated and recrystallized from n-hexane/dichloromethane to obtain a white powder (21.2 mg). The ^1H -NMR and ^13C -NMR data of the obtained white powder were obtained and compared with the NMR data of 3-acetyloleanolic acid, a known compound, and the obtained white powder was identified as 3-acetyloleanolic acid.

(2) Cell test C2C12 cells were seeded in a culture dish (48-well plate, Corning) containing a growth medium [DMEM (high glucose), penicillin-streptomycin (Wako), and 10% fetal bovine serum (FBS; Wako)] at 25,000 cells per ^cm2 of bottom area. After 24 hours, the medium was replaced with a differentiation medium [DMEM (high glucose), penicillin-streptomycin, 1% HS (horse serum; Thermo Fisher Scientific)] (day 0), and then the cells were cultured in the differentiation medium for 4 days.

On the fourth day of culture, the 50 μM aqueous solution of oleanolic acid (1) or the 50 μM aqueous solution of 3-acetyloleanolic acid prepared in (2) was added to C2C12 cells and incubated for 20 hours. As a control, the same procedure was performed except that 3-acetyloleanolic acid was not added. The medium was collected and FGF21 was measured by ELISA. Mouse/Rat FGF-21 Quantikine ELISA Kit (R&D system) was used to measure FGF21. The relative amount (%) of FGF21 due to the addition of 3-acetyloleanolic acid was calculated when the amount of FGF21 in the control was taken as 100%. The results are shown in Table 1.

As is clear from Table 1, it was confirmed that oleanolic acid (Example 1) and 3-acetyloleanolic acid (Example 2) have the effect of promoting the production of FGF21. Furthermore, it was also confirmed that this effect was more pronounced in 3-acetyloleanolic acid (Example 2).

Test Example 2
Using the 3-acetyloleanolic acid prepared in Test Example 1(2), an inhibition test was carried out using SB203580, a p38MAP kinase inhibitor.

C2C12 cells were prepared on the fourth day of culture in the same manner as in Test Example 1, and a 30 μM aqueous solution of 3-acetyloleanolic acid was added, followed by culture for six hours (Example 3). Separately, a 10 μM aqueous solution of SB203580, a p38 MAP kinase inhibitor, was added to C2C12 cells on the fourth day of culture 30 minutes before the addition of the 30 μM aqueous solution of 3-acetyloleanolic acid, and the cells were cultured for six hours after the addition of 3-acetyloleanolic acid (Reference Example 3). As a control, the same procedure as in Example 3 was performed, except that 3-acetyloleanolic acid was not added (Reference Example 2).

After the culture, total RNA was collected using RNAiso Plus (Takara). cDNA was synthesized from 1 μg of total RNA using ReverTra Ace ^(R) qPCR RT Master Mix with gDNA Remover (Toyobo). The synthesized cDNA was diluted 10-fold with sterile water and then used for real-time PCR. After preparing Realtime PCR Master Mix (THUNDERBIRD SYBR qPCR Mix; Toyobo), primers, and cDNA, the reaction was carried out using TP800 Thermal cycler Dice ^(R) Real-time System (Takara). The relative amounts (%) of FGF21 mRNA in Example 3 and Reference Example 3 were calculated when the amount of FGF21 mRNA in the control was taken as 100%. The results are shown in Table 2.

As is clear from Table 2, it was confirmed that the effect of 3-acetyloleanolic acid, an FGF21 production promoter, in promoting FGF21 production (Example 3) was suppressed by the p38MAP kinase inhibitor SB203580 (10 μM) (Reference Example 3). In other words, it was confirmed that p38MAP kinase is involved in the effect of 3-acetyloleanolic acid in promoting FGF21 production.

Test Example 3
Using the 3-acetyloleanolic acid prepared in Test Example 1(2), an animal experiment was carried out to confirm the FGF21 production promoting effect.

Male 6-week-old ddy mice (Japan SLC Co., Ltd.) that had been pre-bred for 14 days were used as experimental animals, and were kept in a breeding room at room temperature of 24 ± 1°C (lights 8:00 to 20:00) throughout the pre-bred and experimental periods. 3-acetyloleanolic acid was administered intraperitoneally to the mice at a dose of 30 mg/kg. Four hours after administration, blood was collected using a syringe containing sodium heparin. As a control, the same procedure was performed on ddy mice that had been pre-bred in the same way, except that 3-acetyloleanolic acid was not administered. The collected blood was centrifuged (3000 rpm, 15 minutes) to separate plasma, and FGF21 in the plasma was measured by ELISA. Mouse/Rat FGF-21 Quantikine ELISA Kit (R&D system) was used to measure FGF21.

In addition, soleus muscles were excised from mice 4 hours after administration of 3-acetyloleanolic acid. RNAiso Plus (Takara) was added in an amount 10 times that of the excised tissue, and the tissue was homogenized, after which total RNA was extracted. cDNA was synthesized from 1 μg total RNA using ReverTra Ace ^(R) qPCR RT Master Mix with gDNA Remover (Toyobo). The synthesized cDNA was diluted 10-fold with sterilized water and then used in real-time PCR. After preparing Realtime PCR Master Mix (THUNDERBIRD SYBR qPCR Mix; Toyobo), primers, and cDNA, the reaction was carried out using a TP800 Thermal cycler ^Dice® Real-time System (Takara).

The relative amount (%) of plasma FGF21 following administration of 3-acetyloleanolic acid was calculated when the amount of plasma FGF21 in the control was taken as 100%. In addition, the relative amount (%) of FGF21 mRNA in the soleus muscle following administration of 3-acetyloleanolic acid was calculated when the amount of FGF21 mRNA in the soleus muscle in the control was taken as 100%. The results are shown in Table 3.

As is clear from Table 3, it was confirmed that 3-acetyloleanolic acid has the effect of promoting the production of FGF21.

Test Example 4
Using the 3-acetyloleanolic acid prepared in Test Example 1(2), an animal experiment was carried out to confirm the fat reducing effect.

As experimental animals, 6-week-old male ddy mice (Japan SLC Co., Ltd.) that had been pre-bred for 14 days were used, and were bred in a breeding room at room temperature of 24±1°C (lights on from 8:00 to 20:00) throughout the pre-bred and experimental periods. The mice were divided into three groups: a normal diet group, a high-fat diet group, and a high-fat diet + FGF21 production promoter group (10-12 mice per group). The normal diet group was fed normal feed (D12450B 35 kcal%; Research Diets) for three weeks, and the high-fat diet group and the high-fat diet + FGF21 production promoter group were fed high-fat feed (D12492, 60 kcal%; Research Diets) for three weeks. The feed was replaced twice a week (Monday and Friday) at 150 g per replacement, and sterilized distilled water was provided ad libitum in a water bottle. In addition, the high-fat diet + FGF21 production promoter group was intraperitoneally administered 3-acetyloleanolic acid at a dose of 30 mg/kg/day, five times a week (every day from Monday to Friday) for three weeks, together with the high-fat diet. At the end of the study, the body weight and the weight of the perigonal adipose tissue were measured for each group, and the average values were calculated. The results are shown in Table 4.

As is clear from Table 4, it was confirmed that the administration of 3-acetyloleanolic acid, an FGF21 production promoter (Example 5), suppressed weight gain and fat mass gain (Reference Example 6 compared to Reference Example 5) due to a high-fat diet. It was confirmed that 3-acetyloleanolic acid has the effect of suppressing or reducing fat gain. From these results, it is recognized that 3-acetyloleanolic acid, an FGF21 production promoter, is effective in preventing or improving impaired lipid metabolism or obesity.

Claims (2)

An FGF21 production promoter comprising 3-acetyloleanolic acid and/or a pharma- ceutical acceptable salt thereof. The FGF21 production promoter according to claim 1 , which is used for preventing or ameliorating impaired lipid metabolism or obesity.