Fibroblast growth factor 21 inducer and composition for suppressing alcohol or simple carbohydrate preference

Abstract

A novel substance is provided which can suppress alcohol preference and / or simple sugar preference.　This fibroblast growth factor 21 inducer contains oligosaccharides, including at least one sugar selected from D-sorbitol, D-psicose and D-tagatose, and / or at least one sugar residue selected from D-sorbitol, D-psicose and D-tagatose.

Description

[Technical Field]

[0001] The present invention relates to a fibroblast growth factor 21 (hereinafter referred to as "FGF21") inducer. More specifically, the present invention relates to an FGF21 inducer containing either or both of at least one carbohydrate selected from the group consisting of D-sorbitol, D-psicose, and D-tagatose, and an oligosaccharide containing a sugar residue of the at least one carbohydrate. The present invention also relates to a composition for suppressing alcohol preference or simple carbohydrate preference in a mammal, comprising the FGF21 inducer. [Background technology]

[0002] FGF21 is a hormone that regulates energy homeostasis, and its expression is induced in the liver in response to various metabolic stresses, including starvation and a high-carbohydrate diet. FGF21 acts via a complex between the cell membrane FGF receptor and the single-pass transmembrane protein β-Klotho. FGF21 is known to regulate alcohol preference and sweetness preference (Non-Patent Document 1). The present inventors have found that the suppression of simple carbohydrate preference is caused by the activation of oxytocin (Oxt) neurons in the brain due to the complex formation between secreted FGF21 and β-Klotho (Non-Patent Document 2).

[0003] Alcoholic beverages are a familiar part of life and culture. However, alcohol is addictive, and chronic exposure can cause organ damage (e.g., liver disease or stroke). It is estimated that there are approximately 1.1 million alcoholics in Japan, approximately 3 million at risk, and approximately 10 million high-risk drinkers. Drugs available for controlling alcohol consumption include disulfiram, cyanamide solution, acamprosate, and nalmefene.

[0004] Glucose is used as almost the sole energy source in the brain and central nervous system and plays an important role in vital activities. However, excessive intake of carbohydrates accumulates as fat in adipose tissue, causing obesity. It is reported that there are approximately 20 million obese people and those at risk of becoming obese in Japan. Mazindol and gastrointestinal lipase inhibitors are commercially available as drugs for treating obesity.

[0005] The existence of sugar craving, an inability to stop craving sugar or sugar-rich foods, has been pointed out. This sugar craving is a type of addiction symptom and is also known as sugar addiction. It is believed that sugar is what causes this addiction. Thus, while simple carbohydrates such as glucose and sugar (sucrose) are used as an energy source in the body, they can also cause addiction symptoms or lead to obesity. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Talukdar S et al., “FGF21 Regulates Sweet and Alcohol Preference.”, Cell Metab.2016 Feb 9;23(2):344-349 [Non-patent document 2] Sho Matsui and Tsutomu Sasaki et al., “Neuronal SIRT1 regulates macronutrient-based diet selection through FGF21 and oxytocin signaling in mice”, NATURE COMMUNICATIONS (2018) 9:4604 Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present disclosure is to provide a novel composition that can suppress either or both of alcohol preference and simple carbohydrate preference. [Means for solving the problem]

[0008] The present inventors focused on FGF21 as a target that can suppress either or both of alcohol preference and simple carbohydrate preference. The present inventors investigated various orally ingestible carbohydrates and found that specific rare sugars or sugar alcohols increase the secretion of FGF21.

[0009] Exemplary aspects of the present disclosure provide an FGF21 inducer, a composition for suppressing alcohol preference, and a composition for suppressing simple carbohydrate preference. More specifically, exemplary aspects of the present disclosure provide the following embodiments: [Item 1] An FGF21 inducer comprising at least one carbohydrate selected from the group consisting of D-sorbitol, D-psicose, and D-tagatose, and / or an oligosaccharide containing at least one sugar residue selected from the group consisting of D-sorbitol, D-psicose, and D-tagatose. [Item 2] The FGF21 inducer according to Item 1, wherein the at least one carbohydrate comprises D-sorbitol or D-tagatose. [Item 3] The FGF21 inducer according to Item 1, wherein the at least one carbohydrate is D-sorbitol. [Item 4] The FGF21 inducer according to Item 1, wherein the at least one carbohydrate is D-tagatose. [Item 5] The FGF21 inducer according to Item 1, which contains at least two types of carbohydrates selected from the group consisting of D-sorbitol, D-psicose, and D-tagatose as carbohydrates and / or sugar residues. [Item 6] A composition for suppressing alcohol preference in a mammal, comprising the FGF21 inducer according to any one of Items 1 to 5. [Item 7] A composition for suppressing simple carbohydrate preference in a mammal, comprising the FGF21 inducer according to any one of Items 1 to 5. [Item 8] The composition according to Item 6 or Item 7, which is a supplement. [Item 9] The pharmaceutical composition according to Item 6 for treating alcoholism. [Item 10] The pharmaceutical composition according to Item 7 for treating obesity. [Brief explanation of the drawings]

[0010] [Figure 1] Figure 1(a) is a boxplot showing the preference for 4% ethanol in β-Klotho Flox mice (white) and Oxt neuron-specific Klb knockout mice (gray). Figure 1(b) is a boxplot showing the preference for 8% ethanol in β-Klotho Flox mice (white). Figure 1(c) is a boxplot showing the preference for 16% ethanol in β-Klotho Flox mice (gray). [Figure 2] Figure 2(a) is a bar graph showing the preference for 16% aqueous ethanol for mice fed a normal diet (NC: open) and mice fed a high-sucrose diet (HSD: gray). Figure 2(b) is a bar graph showing the preference for 8% aqueous ethanol. Figure 2(c) is a bar graph showing the preference for 4% aqueous ethanol. Figures 2(d)-(f) are bar graphs showing the plasma FGF21 concentrations for mice fed a normal diet (open) and mice fed a high-sucrose diet (gray) shown in Figures 2(a)-(c). Figure 2(g) is a scatter plot showing the results of the preference for 16% aqueous ethanol and the plasma FGF21 concentrations shown in Figures 2(a) and (d). Figure 2(h) is a scatter plot showing the results for Figures 2(b) and (e). Figure 2(i) is a scatter plot showing the results for Figures 2(c) and (f). [Figure 3] Figure 3(a) is a boxplot showing the preference for 100 mM sucrose solution in β-Klotho Flox mice (white) and Oxt neuron-specific Klb knockout mice (gray). Figure 3(b) is a boxplot showing the preference for 0.2% saccharin solution. Figure 3(c) is a boxplot showing the preference for 2% dextrin solution. [Figure 4] This is a box plot showing the relative expression level of the Fgf21 gene in primary cultured hepatocytes cultured with the addition of each of six types of carbohydrates to the culture medium. [Figure 5] FIG. 1 is a box plot showing the concentration of FGF21 in the culture medium secreted from primary hepatocytes cultured in culture medium containing a range of concentrations of D-tagatose. [Figure 6] This is a box plot showing the concentration of FGF21 in the culture medium secreted from primary cultured hepatocytes cultured with the addition of each of 48 types of carbohydrates. [Figure 7] 1 is a bar graph showing the concentration of FGF21 in the culture medium secreted from primary hepatocytes cultured in culture medium containing a series of concentrations of D-sorbitol, D-psicose, or D-tagatose. [Figure 8] This is a box plot showing the time course of the relative expression level of the Fgf21 gene in primary hepatocytes cultured with the addition of four types of carbohydrates, including D-sorbitol. [Figure 9] This is a box plot showing the time-dependent change in the relative expression level of the Fgf21 gene in primary hepatocytes cultured with the addition of five types of carbohydrates, including D-Psicose. [Figure 10] FIG. 1 is a box plot showing the concentration of FGF21 in the culture medium secreted from primary hepatocytes cultured in a culture medium containing a single carbohydrate or a combination of two carbohydrates. [Figure 11] Figure 11(a) is a box plot showing FGF21 concentrations in the plasma of mice administered with D-sorbitol, D-psicose, or D-tagatose, and Figure 11(b) is a bar graph showing the area under the FGF21 concentration-time curve (AUC) in mouse plasma. [Figure 12] 1 is a bar graph showing that preference for a 50 mM sucrose aqueous solution is suppressed. [Figure 13] 1 is a bar graph showing that preference for an 8% aqueous ethanol solution is suppressed. DETAILED DESCRIPTION OF THE INVENTION

[0011] As used herein, "sugar" refers to carbohydrates including sugars or sugar alcohols. The sugar according to the present disclosure refers to at least one compound selected from the group consisting of D-sorbitol, D-psicose, and D-tagatose. The sugar may be, for example, at least two compounds selected from the group consisting of D-sorbitol, D-psicose, and D-tagatose. In one embodiment, the sugar may be a single compound selected from D-sorbitol, D-psicose, or D-tagatose. In one embodiment, the sugar may be a combination of D-sorbitol and D-psicose, a combination of D-psicose and D-tagatose, a combination of D-sorbitol and D-tagatose, or a combination of D-sorbitol, D-psicose, and D-tagatose.

[0012] As used herein, "sugar" or "simple carbohydrate" refers to a monosaccharide or disaccharide. Examples of simple carbohydrates include, but are not limited to, glucose, fructose, sucrose, lactose, and maltose. As used herein, "polysaccharide" refers to a sugar compound in which multiple sugars are linked together, and which contains 20 or more sugars. A polysaccharide consisting of only one type of monosaccharide is called a homoglycan, and a polysaccharide consisting of multiple types of monosaccharides is called a heteroglycan. Examples of polysaccharides include, but are not limited to, amylose, amylopectin, dextrin, starch, and glycogen.

[0013] As used herein, the term "synthetic sweetener" refers to a compound that does not exist in nature but is produced by chemical synthesis, and that, when orally ingested by a mammal, causes the mammal to perceive a sweet taste. Examples of synthetic sweeteners include, but are not limited to, aspartame, sucralose, neotame, advantame, saccharin, and acesulfame potassium.

[0014] As used herein, "oligosaccharide" refers to a sugar compound in which multiple sugar residues are linked, and which contains 3 to 20 sugars. The oligosaccharide according to the present disclosure preferably contains 3 to 10 sugars. The oligosaccharide has a molecular weight of, for example, 300 to 3,000. The oligosaccharide may be composed of one type of sugar residue or two types of sugar residues. Oligosaccharides can be synthesized from at least one sugar selected from the group consisting of D-sorbitol, D-psicose, and D-tagatose by organic chemical synthesis or enzymatic chemical synthesis, but are not limited thereto. Oligosaccharides can be synthesized, for example, by a method utilizing a glycosyltransferase (glucosyltransferase) and / or a method utilizing the reverse reaction of a glycolytic enzyme (glycosidase).

[0015] As used herein, "D-sorbitol" or "D-Sorbitol" refers to a sugar alcohol represented by formula 1: [ka] (Formula 1) D-sorbitol has approximately 75% fewer calories than sucrose and is therefore used as a sweetener in low-calorie foods. D-sorbitol can be produced by known methods and is also commercially available. Examples of known production methods include, but are not limited to, the reduction of D-glucose with the enzyme aldose reductase. D-sorbitol is commercially available, for example, from Tokyo Chemical Industry Co., Ltd.

[0016] As used herein, "D-psicose" or "D-Psicose" refers to a hexose sugar represented by formula 2: [ka] (Formula 2) D-psicose, also known as allulose, provides only 0.3% of the calories of sucrose as energy. D-psicose can be produced by known methods and is commercially available. Such known production methods include, but are not limited to, production from fructose using the enzyme D-tagatose 3-epimerase, or extraction from plants of the genus Zea mays. D-psicose is commercially available, for example, from Tokyo Chemical Industry Co., Ltd.

[0017] As used herein, "D-tagatose" refers to a hexose sugar represented by formula 3: [ka] (Formula 3) D-tagatose has 92% of the sweetness of sucrose, but only 38% of the calories are utilized as energy. D-tagatose can be produced according to known methods and is also commercially available. Known production methods include, but are not limited to, hydrolyzing lactose, separating the galactose from glucose, and isomerizing the resulting galactose with calcium hydroxide in an alkaline environment. Solid D-tagatose can also be obtained by purifying and crystallizing the tagatose mixture obtained by the above method. D-tagatose is commercially available, for example, from Tokyo Chemical Industry Co., Ltd.

[0018] As used herein, "enantiomer" refers to a stereoisomer that is a non-superimposable mirror image. In this specification, the stereoisomer of a sugar is defined as the D-isomer, and its enantiomer is defined as the L-isomer, based on the configuration of d-glyceraldehyde. D-sorbitol, D-psicose, and D-tagatose are all D-isomers corresponding to the configuration of d-glyceraldehyde. The desired enantiomer can be separated from a mixture of different enantiomers according to known methods, thereby increasing its purity. Examples of such known methods include, but are not limited to, chiral HPLC.

[0019] As used herein, "FGF21" refers to a hormone protein encoded by the Fgf21 gene in mammals and secreted from the liver. FGF21 belongs to the endocrine FGF class of fibroblast growth factors (FGFs) and acts via a cell membrane FGF receptor that forms a complex with the single-pass transmembrane protein β-Klotho. The complex formed by FGF21 and β-Klotho activates oxytocin (Oxt) neurons in the brain and can suppress alcohol and simple carbohydrate preferences. An increase in blood FGF21 levels can suppress alcohol and simple carbohydrate preferences.

[0020] The amount of FGF21 in blood may be the FGF21 concentration (e.g., ng / ml, nM) in mammalian blood (whole blood, plasma, or serum) or the weight of FGF21 (e.g., ng, mg) contained in a given amount of blood. The amount of FGF21 in blood can be measured according to known methods. Known methods include, but are not limited to, ELISA using an anti-FGF21 antibody.

[0021] As used herein, the term "inducing agent" refers to a combination of substances that induce the expression of a desired product in a mammal. The combination may consist of a single component or may be a composition containing two or more components. The combination may be in, but is not limited to, a solid or liquid form. The inducing agent may, for example, include a substance that causes the production of a desired product in the cells of a mammal, which is secreted into the blood. In this example, the inducing agent may result in an increase in the amount of the desired product in the blood of the mammal.

[0022] As used herein, "FGF21 inducer" refers to a compound containing a substance that induces the expression of FGF21 in the body of a mammal. FGF21 inducers include, but are not limited to, substances that induce the expression of FGF21 secreted into the blood in mammalian hepatocytes. The FGF21 inducer of the present invention contains, as a substance that induces the expression of FGF21 in the body of a mammal, either one or both of at least one carbohydrate selected from the group consisting of D-sorbitol, D-psicose, and D-tagatose, and an oligosaccharide containing a sugar residue of the at least one carbohydrate.

[0023] In one embodiment, the FGF21 inducer comprises a carbohydrate selected from D-sorbitol, D-psicose, and D-tagatose, and / or an oligosaccharide containing a sugar residue selected from D-sorbitol, D-psicose, and D-tagatose. An FGF21 inducer containing only D-psicose, only an oligosaccharide containing a sugar residue of D-psicose, or both as an FGF21-inducing component can increase blood FGF21 concentrations relatively quickly. An FGF21 inducer containing only one of the carbohydrates D-sorbitol and D-tagatose as a carbohydrate and / or sugar residue can maintain high blood FGF21 concentrations for a relatively long period of time.

[0024] In one embodiment, the FGF21 inducer comprises at least two carbohydrates selected from the group consisting of D-sorbitol, D-psicose, and D-tagatose as carbohydrates and / or sugar residues. The FGF21 inducer comprises at least two carbohydrates selected from the group consisting of D-sorbitol, D-psicose, and D-tagatose, and / or an oligosaccharide comprising at least two carbohydrate residues selected from the group consisting of D-sorbitol, D-psicose, and D-tagatose. The at least two carbohydrates or sugar residues may be a combination of D-sorbitol and D-psicose, a combination of D-sorbitol and D-tagatose, a combination of D-psicose and D-tagatose, or a combination of D-sorbitol, D-psicose, and D-tagatose.

[0025] The at least two types of carbohydrates and / or the oligosaccharides may exert a synergistic or additive effect on FGF21 induction. An FGF21 inducer containing the at least two types of carbohydrates and / or the oligosaccharides may exert a high FGF21-inducing effect while reducing its intake. An FGF21 inducer containing D-tagatose and D-sorbitol or D-psicose as carbohydrates and / or sugar residues may exert a high FGF21-inducing effect while reducing its intake. An FGF21 inducer containing D-tagatose and D-psicose as carbohydrates and / or sugar residues may increase blood FGF21 concentrations relatively quickly and maintain high FGF21 concentrations for a relatively long period of time while reducing its intake.

[0026] An FGF21 inducer containing D-psicose and D-tagatose or D-sorbitol as a carbohydrate and / or sugar residue can increase blood FGF21 relatively quickly and maintain a high FGF21 concentration for a relatively long period of time. An FGF21 inducer containing D-sorbitol and D-tagatose as a carbohydrate and / or sugar residue can maintain a high FGF21 concentration for a relatively long period of time. An FGF21 inducer containing D-sorbitol, D-psicose, and D-tagatose as a carbohydrate and / or sugar residue can increase blood FGF21 relatively quickly and maintain a high FGF21 concentration for a relatively long period of time.

[0027] The FGF21 inducer may be in an orally ingestible form, such as, but not limited to, tablets, capsules, granules, powders, syrups, or liquids. The orally acceptable additives may be, but are not limited to, excipients, disintegrants, binders, humectants, stabilizers, buffers, lubricants, preservatives, seasonings, flavorings, fragrances, acidulants, or colorants, or combinations thereof. The FGF21 inducer may be produced according to known methods appropriate to its form. When the FGF21 inducer is in the form of a powder, it can be produced, for example, by blending the at least one carbohydrate and / or an oligosaccharide containing a sugar residue of the at least one carbohydrate in powder form with, as needed, an orally acceptable additive in powder form. When the FGF21 inducer is in the form of a liquid, it can be produced, for example, by blending the at least one carbohydrate and / or an oligosaccharide containing a sugar residue of the at least one carbohydrate with an aqueous liquid.

[0028] The amount of at least one carbohydrate selected from the group consisting of D-sorbitol, D-psicose, and D-tagatose, and / or an oligosaccharide containing a sugar residue of said at least one carbohydrate, in an FGF21 inducer, is appropriately determined depending on the formulation. The daily intake of said at least one carbohydrate and / or an oligosaccharide containing a sugar residue of said at least one carbohydrate, to be incorporated into an FGF21 inducer, is, but is not limited to, an amount that significantly increases the amount of FGF21 in the blood of a mammal after one week of intake of the FGF21 inducer, compared to the amount of FGF21 in the mammal's blood before intake of the FGF21 inducer. A significant difference test can be performed using, but is not limited to, Tukey's test. The mammal may be, for example, a mouse. The amount that significantly increases the amount of FGF21 in the blood of a mouse is, for example, 1 to 10 g / kg body weight, 3 to 7 g / kg body weight, or 5 g / kg body weight. The amount that significantly increases the blood FGF21 level in mammals other than mice can be determined appropriately by those skilled in the art in light of the disclosures of the present specification. The intake amount of carbohydrates according to the present disclosure for mammals other than mice can be determined, for example, according to the human equivalent dose (HED) based on body surface area. The amount that significantly increases the blood FGF21 level in humans is, for example, 100 mg to 1 g / kg body weight, 200 to 700 mg / kg body weight, 300 to 500 mg / kg body weight, or 300 to 400 mg / kg body weight.

[0029] The number of times an FGF21 inducer is taken per day may be, for example, a single dose of the daily amount, or it may be taken in multiple divided doses. An FGF21 inducer taken once per day may, for example, contain, as an FGF21-inducing component, only one of the carbohydrates D-sorbitol and D-tagatose, only an oligosaccharide containing the sugar residue of the carbohydrate, or both of them as the carbohydrate or sugar residue, or may contain at least two carbohydrates selected from the group consisting of D-sorbitol, D-psicose, and D-tagatose, an oligosaccharide containing the sugar residue of the at least two carbohydrates, or both of them. The FGF21 inducer may be taken directly as a supplement, or may be added to a food or drink and taken, without limitation.

[0030] As used herein, the term "composition" may be in a solid or liquid form, but is not limited thereto. The composition may be, for example, a formulation containing a substance capable of suppressing alcohol preference in a mammal. In this example, the composition may, for example, cause an increase in a product capable of suppressing alcohol preference in the blood of the mammal.

[0031] As used herein, the term "supplement" refers to a non-pharmaceutical composition that is orally ingested to prevent the onset of a specific symptom in a mammal and to maintain or improve the physical condition or health of the mammal. In one embodiment, the supplement is a food with functional claims.

[0032] As used herein, "treatment" refers to the prevention of the onset of a given symptom or condition, or the cure of a given symptom or condition. As used herein, "treatment" refers to the alleviation, mitigation, or complete cure of a given disease state or condition.

[0033] As used herein, a "composition for suppressing either or both of alcohol preference and simple carbohydrate preference in a mammal" refers to a substance capable of suppressing either or both of alcohol preference and simple carbohydrate preference in a mammal, comprising at least one carbohydrate selected from the group consisting of D-sorbitol, D-psicose, and D-tagatose, and / or an oligosaccharide containing a sugar residue of said at least one carbohydrate. The composition may contain, but is not limited to, an FGF21 inducer described herein. In one embodiment, the composition is for suppressing alcohol preference in a mammal. In another embodiment, the composition is for suppressing simple carbohydrate preference in a mammal. In another embodiment, the composition is for suppressing alcohol preference and simple carbohydrate preference in a mammal. The form of the composition may be any of the forms described for FGF21 inducers, as appropriate.

[0034] In one embodiment, the composition for reducing simple carbohydrate preference is a composition for reducing preference for glucose, fructose, sucrose, lactose, and maltose. In another embodiment, the composition for reducing simple carbohydrate preference is a composition for reducing preference for glucose, fructose, and sucrose. In another embodiment, the composition for reducing simple carbohydrate preference is a composition that reduces preference for simple carbohydrates but does not substantially change preference for either or both of synthetic sweeteners and polysaccharides. In one embodiment, the composition for reducing simple carbohydrate preference comprises at least one carbohydrate (e.g., one or two) selected from the group consisting of D-sorbitol and D-tagatose, and / or an oligosaccharide containing a sugar residue of the at least one carbohydrate.

[0035] The synthetic sweetener is at least one compound selected from the group consisting of, but not limited to, aspartame, sucralose, neotame, advantame, saccharin, and acesulfame potassium. An example of the synthetic sweetener is saccharin. The polysaccharide is at least one compound selected from the group consisting of, but not limited to, amylose, amylopectin, dextrin, starch, and glycogen. An example of the polysaccharide is dextrin. In one embodiment, the composition for suppressing simple carbohydrate preference is a composition that suppresses preference for simple carbohydrates but does not substantially change preference for saccharin and dextrin.

[0036] In one embodiment, the composition for reducing alcohol preference is a composition for reducing preference for an aqueous composition (e.g., alcoholic beverage) containing a substance in which a hydrocarbon hydrogen atom is replaced with a hydroxyl group (e.g., ethanol). In another embodiment, the composition for reducing alcohol preference comprises at least one carbohydrate (e.g., one or two carbohydrates) selected from the group consisting of D-psicose and D-tagatose, and / or an oligosaccharide containing a sugar residue of the at least one carbohydrate.

[0037] The daily intake amount of the at least one carbohydrate and / or oligosaccharide containing a sugar residue of the at least one carbohydrate incorporated into the composition for suppressing either or both of alcohol preference and simple carbohydrate preference described herein is, but is not limited to, an amount that significantly reduces either or both of the alcohol preference and simple carbohydrate preference of a mammal after ingesting the composition for one week compared to either or both of the mammal's alcohol preference and simple carbohydrate preference before ingesting the composition. A significant difference test can be performed using, but is not limited to, Tukey's test. The mammal is, for example, a mouse. The amount that significantly increases blood FGF21 levels in mice is, for example, 1 to 10 g / kg body weight, 3 to 7 g / kg body weight, or 5 g / kg body weight. The amount that significantly increases blood FGF21 levels in mammals other than mice can be determined appropriately by those skilled in the art in light of the disclosures herein. The amount that significantly increases the amount of FGF21 in human blood is, for example, 100 mg to 1 g / kg body weight, 200 to 700 mg / kg body weight, 300 to 500 mg / kg body weight, or 300 to 400 mg / kg body weight.

[0038] As used herein, a "medicament" or "pharmaceutical composition" refers to a formulation of substances for treating a given symptom or sign in a mammal. The formulation may be, but is not limited to, a solid or liquid form. An example of a medicament or pharmaceutical composition is a formulation of substances for treating alcoholism in a mammal. In this example, the medicament or pharmaceutical composition may provide a preventive, therapeutic, or alleviating effect on alcoholism.

[0039] As used herein, a "medicine for treating obesity" or a "medicine for treating alcoholism" refers to a substance for treating obesity or alcoholism, which comprises at least one carbohydrate selected from the group consisting of D-sorbitol, D-psicose, and D-tagatose, and / or an oligosaccharide comprising a sugar residue of said at least one carbohydrate. The medicament may comprise, but is not limited to, an FGF21 inducer described herein. In one embodiment, the medicament is a pharmaceutical composition for treating obesity. A pharmaceutical composition for treating obesity is administered, for example, for the purpose of reducing the intake of simple carbohydrates. In another embodiment, the medicament is a pharmaceutical composition for treating alcoholism. A pharmaceutical composition for treating alcoholism is administered, for example, for the purpose of reducing the intake of alcoholic beverages (alcoholic beverages). In one embodiment, the pharmaceutical composition for treating alcoholism comprises at least one carbohydrate selected from the group consisting of D-psicose and D-tagatose, and / or an oligosaccharide comprising a sugar residue of said at least one carbohydrate.

[0040] The pharmaceutical form may be, but is not limited to, an orally ingestible form, such as a tablet, capsule, granule, powder, dust, syrup, or liquid. Pharmaceutically acceptable orally ingestible additives may be, for example, additives known in the pharmaceutical field and additives described for FGF21 inducers. Pharmaceuticals can be manufactured according to, but are not limited to, known methods appropriate for the form.

[0041] The number of times the composition or medicament disclosed herein is to be taken per day may be, for example, a single daily dose or multiple divided doses. The composition or medicament disclosed herein to be taken once per day may, for example, contain, as an FGF21-inducing component, only one of the carbohydrates D-sorbitol and D-tagatose, only an oligosaccharide containing a sugar residue of the carbohydrate, or both, or at least two carbohydrates selected from the group consisting of D-sorbitol, D-psicose, and D-tagatose, an oligosaccharide containing a sugar residue of the at least two carbohydrates, or both.

[0042] In one embodiment, the FGF21 inducer or medicament described herein contains 1 to 99% by mass, 5 to 80% by mass, or 10 to 50% by mass of at least one carbohydrate selected from the group consisting of D-sorbitol, D-psicose, and D-tagatose, and / or an oligosaccharide containing a sugar residue of said at least one carbohydrate, based on its total amount being 100% by mass. In one embodiment, the FGF21 inducer or medicament described herein contains 1 to 99% by mass, 5 to 80% by mass, or 10 to 50% by mass of D-sorbitol, D-psicose, or D-tagatose, based on its total amount being 100% by mass.

[0043] As used herein, "alcoholism" refers to a group of physiological, behavioral, and / or cognitive phenomena in which drinking alcohol is of great value to the subject and takes priority over other behaviors. Physiological conditions in alcoholism include alcohol withdrawal symptoms, including seizures and hallucinations, that occur when drinking is stopped or reduced. Cognitive phenomena in alcoholism include an intense desire to drink alcohol, a sense of compulsion (craving), loss of control over drinking or the amount of alcohol consumed, and reduced or lost interest in recreational activities other than drinking. Alcoholism can be accompanied by the development of alcohol tolerance. Alcohol tolerance refers to a state in which a person must drink larger amounts of alcohol than before to achieve the same level of intoxication.

[0044] As used herein, "obesity" refers to a pathological condition accompanied by health disorders caused by or related to obesity (hereinafter also referred to as "obesity-related diseases") and requiring weight loss. Obesity-related diseases include impaired glucose tolerance (e.g., type 2 diabetes, impaired glucose tolerance), dyslipidemia, hypertension, hyperuricemia, or gout, coronary artery disease (e.g., myocardial infarction or angina pectoris), cerebral infarction (e.g., cerebral thrombosis or transient ischemic attack), fatty liver, menstrual disorders or pregnancy complications (e.g., pregnancy-induced hypertension, gestational diabetes, or hearing loss), sleep apnea syndrome or obesity-hypoventilation syndrome, orthopedic diseases (e.g., osteoarthritis, spondylosis osteoarthritis), and obesity-related kidney disease.

[0045] The FGF21 inducer is, for example, orally ingested by a mammal. The medicament is administered to a mammal. The mammal may be, but is not limited to, a human, a primate (monkey, chimpanzee), a livestock animal (cow, horse, pig, sheep), or a pet animal (dog, cat). The mammal is preferably a human. The medicament is preferably orally administered to a human.

[0046] In one embodiment, the composition for reducing alcohol preference is orally taken by an alcoholic, pre-alcoholic, or high-risk drinker. In one embodiment, the composition for reducing simple carbohydrate preference is orally taken by an obese or pre-obese individual.

[0047] In one embodiment, the medicament for treating alcoholism is orally administered to a patient suffering from alcoholism. In one embodiment, the medicament for treating obesity is orally administered to a patient suffering from obesity.

[0048] In one embodiment, there is provided use of at least one carbohydrate selected from the group consisting of D-sorbitol, D-psicose, and D-tagatose, and / or an oligosaccharide containing a sugar residue of said at least one carbohydrate, for the manufacture of a medicament for treating alcoholism or obesity in a mammal. The features described herein for the FGF21 inducer, composition, or medicament also apply to this embodiment.

[0049] In one embodiment, there is provided a method for treating alcoholism or obesity, comprising orally administering to a mammal in need thereof a medicament for treating alcoholism or obesity, wherein the medicament contains at least one carbohydrate selected from the group consisting of D-sorbitol, D-psicose, and D-tagatose, and / or an oligosaccharide containing a sugar residue of the at least one carbohydrate. The features described herein for the FGF21 inducer, composition, or medicament also apply to this embodiment.

[0050] In one embodiment, there is provided a use of at least one carbohydrate selected from the group consisting of D-sorbitol, D-psicose, and D-tagatose, and / or an oligosaccharide containing a sugar residue of said at least one carbohydrate, in a mammal to induce FGF21 in the mammal. For example, the amount of FGF21 in the blood of the mammal increases compared to the amount of FGF21 in the blood before use of the carbohydrate and / or the oligosaccharide. The features described herein for the FGF21 inducer, composition, or medicament are also included in this embodiment.

[0051] In one embodiment, a method for inducing FGF21 in a mammal is provided, comprising administering to the mammal at least one carbohydrate selected from the group consisting of D-sorbitol, D-psicose, and D-tagatose, and / or an oligosaccharide containing a sugar residue of said at least one carbohydrate. For example, the amount of FGF21 in the mammal's blood increases compared to the amount of FGF21 in the blood before administration of the carbohydrate and / or the oligosaccharide. The features described herein for FGF21 inducers, compositions, and medicaments are also included in this embodiment.

[0052] Specific examples are provided below that illustrate exemplary embodiments of the present disclosure and are not intended to limit the scope of the invention as set forth in the appended claims in any way.

[0053] [Example 1] (mouse) Nine-week-old male β-Klotho Flox mice served as the control group. Nine-week-old male oxytocin (Oxt) neuron-specific β-Klotho knockout mice, which lack the FGF21 receptor specifically in Oxt neurons (Cell Metab. 2012 Sep 5;16(3):387-93), served as the experimental group. (Breeding conditions) Mice were housed individually in polycarbonate cages (CLEA Japan, Inc.). Each mouse was allowed free access to solid food (CE-2; CLEA Japan, Inc.). The temperature of the breeding room and test solution was maintained at 25±1°C. The light-dark cycle in the breeding room was maintained at 12 hours light and 12 hours dark (switching between 06:00 and 18:00).

[0054] (Test solution) A 4% aqueous ethanol solution, an 8% aqueous ethanol solution, and a 16% aqueous ethanol solution were used as test solutions. (2-bottle choice test) First period (habituation to the experimental conditions): Two water bottles for measuring trace drinking volume, each containing sterilized water, were presented 24 hours a day for four days, allowing the rats to learn to drink from the two water bottles. Period 2 (measurement of preference for the baseline test solution): Two micro-drinking bottles, one containing sterile water and the other containing the test solution, were presented 24 hours a day for 4 days. To eliminate the influence of location preference, the two micro-drinking bottles were swapped every 24 hours.

[0055] (Measurement of the amount of solution ingested) The amount of solution ingested by the mice was measured using a water bottle for measuring minute amounts of drinking water for mice and rats (Drink-O-Measurer, manufactured by Shin Factory). (Preference calculation) Preference for the test solution was calculated using the following formula: Preference = (total amount of drinking water for the test solution over 4 days) / (total amount of drinking water for the sterile water over 4 days + total amount of drinking water for the test solution over 4 days) × 100

[0056] (Test results) The preference for each test solution was tested for significant differences between the control and experimental groups using Student's t-test. The results obtained for each test solution are summarized in Figure 1. Figure 1(a)-(c) show that alcohol preference was increased in oxytocin (Oxt) neuron-specific β-Klotho knockout mice (gray) compared with β-Klotho Flox mice (white). These results suggest that the action of FGF21 via β-Klotho, a co-receptor of FGF21, is necessary for the suppression of alcohol preference in mice.

[0057] [Example 2] (mouse) Nine-week-old male C57BL / 6JJcl mice (CLEA Japan, Inc.) were used. (Breeding conditions) Mice were individually housed under essentially the same conditions as in Example 1, except that the solid food (CE-2) was replaced with powdered food (CE-2). During the following test periods, the food was replaced with new food as appropriate. During the fourth period described below, the corresponding groups were given powdered food (CE-2) and high-sucrose powdered food (high-sucrose diet; Oriental Bioservices Co., Ltd.), respectively. (Test solution) An aqueous ethanol solution with the same concentration as in Example 1 was used. (Measurement of the amount of solution ingested and calculation of palatability) The same method as in Example 1 was used.

[0058] (2-bottle choice test) First period (habituation to the experimental conditions): Two water bottles for measuring trace drinking volume, each containing sterilized water, were presented 24 hours a day for four days, allowing the rats to learn to drink from the two water bottles. Period 2 (measurement of preference for the baseline test solution): Two micro-drinking bottles, one containing sterile water and the other containing the test solution, were presented 24 hours a day for 4 days. To eliminate the influence of location preference, the two micro-drinking bottles were swapped every 24 hours. - Period 3 (a cool-off period to eliminate the influence of alcohol in Period 2): Two water bottles containing sterile water for measuring micro-drinking were presented 24 hours a day for 7 days.

[0059] Period 4 (main experiment): Mice consuming powdered diet (CE-2) served as the control group. Two groups of mice consuming high-sucrose diet (HSD) powdered diet (high-sucrose diet; Oriental Bioservices Co., Ltd.) served as experimental groups. Food was provided in a mouse multi-feeder, and fresh powdered food was replaced every 24 hours. Two water bottles for measuring micro-drinking volume, one containing sterilized water and the other containing the test solution, were presented 24 hours a day for four days. To eliminate the influence of location preference, the positions of the two water bottles for measuring micro-drinking volume were swapped every 24 hours. Period 5 (blood sampling): After Period 4, subjects were given a water bottle containing sterile water for 24 hours, after which blood was collected from the heart under anesthesia. The blood was treated with heparin (final concentration 10 μg / ml; Fujifilm Wako Pure Chemical Industries, Ltd.) and centrifuged at 2000 rpm at 4°C for 20 minutes to collect plasma. Plasma FGF21 concentrations were measured using the Fibroblast Growth Factor 21 Mouse / Rat ELISA (BioVendor) according to the manufacturer's instructions.

[0060] (Test results) The preference for each test solution during the fourth period was tested using Student's t-test to determine whether there was a significant difference between the control group (CE-2 intake group) and the experimental group (high-sucrose diet intake group) (*: p<0.05). The results obtained for each test solution are summarized in Figures 2(a)-(c). Figures 2(a) and (b) show that the preference for alcohol was significantly reduced in the experimental group (gray) that consumed the high-sucrose diet compared to the control group (open) that consumed the normal diet (CE-2).

[0061] The plasma FGF21 concentrations measured during the fifth period were tested for significant differences between the control group (CE-2 intake group) and the experimental group (high-sucrose diet intake group) using Student's t-test (*: p<0.05). The results obtained from each group corresponding to each test solution are summarized in Figure 2(d)-(f). Figure 2(d)-(f) show that plasma FGF21 concentrations were significantly increased in the experimental group (gray) that consumed the high-sucrose diet compared to the control group (open) that consumed the normal diet (CE-2).

[0062] We investigated whether there was a correlation between alcohol preference and plasma FGF21 concentration. Figures 2(g)-(i) show a negative correlation between alcohol preference and plasma FGF21 concentration. These results suggest that alcohol preference is suppressed as blood FGF21 concentration increases.

[0063] [Example 3] (mouse) Nine-week-old male β-Klotho Flox mice were used as the control group (n=9). Nine-week-old male oxytocin (Oxt) neuron-specific β-Klotho knockout mice were used as the experimental group (n=9). (Breeding conditions) The same rearing conditions as in Example 1 were used.

[0064] (Test solution) 0.2% saccharin (Fujifilm Wako Pure Chemical Industries, Ltd.), 100 mM sucrose (Fujifilm Wako Pure Chemical Industries, Ltd.), and 2% dextrin (Fujifilm Wako Pure Chemical Industries, Ltd.), adjusted to the specified concentrations using sterilized water, were used as test solutions. (2-bottle choice test) First period (habituation to the experimental conditions): Two water bottles for measuring trace drinking volume, each containing sterilized water, were presented 24 hours a day for four days, allowing the rats to learn to drink from the two water bottles. Period 2 (measurement of preference for the test solution): Two water bottles for measuring micro-drinking volume, one containing sterilized water and the other containing the test solution, were presented 24 hours a day for three days. To eliminate the influence of location preference, the positions of the two water bottles for measuring micro-drinking volume were swapped every 24 hours.

[0065] (Measurement of the amount of solution ingested and calculation of palatability) The amount of solution ingested was measured and the palatability was calculated in substantially the same manner as in Example 1.

[0066] (Test results) We used Welch's t test to test for significant differences between the control and experimental groups in the preference for each test solution (*: p<0.05). The results for each test solution are summarized in Figure 3. Figure 3(a) shows that the preference for the simple sugar sucrose was significantly increased in oxytocin (Oxt) neuron-specific β-Klotho knockout mice (gray) compared with β-Klotho Flox mice (open). On the other hand, Figures 3(b) and (c) show that the preference for the synthetic sweetener saccharin or the polysaccharide dextrin was not significantly increased or decreased in oxytocin (Oxt) neuron-specific β-Klotho knockout mice (gray) compared with β-Klotho Flox mice (open). These results suggest that the action of FGF21 via β-Klotho is required for the suppression of simple carbohydrate preference in mice.

[0067] [Example 4] (Preparation of primary cultured hepatocytes) Primary cultures of hepatocytes were prepared using 9-week-old male C57BL / 6JJcl mice (CLEA Japan, Inc.) according to the following procedure. Under anesthesia, a silicone tube was inserted into the portal vein of the mouse, and the abdominal aorta was cut. Simultaneously with the aorta cutting, EGTA solution (37°C) was infused through the silicone tube inserted into the portal vein, followed by collagenase solution (37°C). The liver was then dissected and transferred to a petri dish. Hepatocytes were liberated from the dissected liver by pipetting. Hanks' solution (4°C) was added to the liberated hepatocytes, and the suspension containing the recovered hepatocytes was filtered through a 100-μm cell strainer (Corning Falcon). The resulting filtrate was centrifuged at low speed (50 g, 3 minutes). The sediment was redispersed in Hanks' solution (4°C) and centrifuged at low speed. This redispersion and centrifugation process was repeated three times. The resulting sediment was dispersed in culture medium (37°C) and collected at a concentration of 1 × 10 5 The cells were seeded at 1000 cells / well onto a 24-well collagen Type I coated microplate. Three hours after seeding, the culture medium was replaced with fresh medium, and after culturing for 24 hours, the obtained hepatocytes were used as primary cultured hepatocytes. The culture medium used was Williams' medium E (Sigma-Aldrich) supplemented with fetal bovine serum (10%; ThermoFischer), ITS-G supplement (x100) (1 μM; Fujifilm Wako Pure Chemical Industries, Ltd.), and dexamethasone (1 μM; Fujifilm Wako Pure Chemical Industries, Ltd.).

[0068] (Sugar or sugar alcohol addition experiments to primary cultured hepatocytes and gene expression analysis) The prepared primary cultured hepatocytes were cultured for 24 hours in a culture medium containing test solutions of each of carbohydrates 1 to 6 shown in the table below at 25 mM or in a culture medium containing the same volume of vehicle (distilled water). [Table 1]

[0069] After 24 hours of culture, the culture supernatant was removed, and the wells containing the cultured hepatocytes were washed with D-PBS(-). Total RNA was extracted from the cultured hepatocytes using RNAiso Plus (Takara Bio Inc.). cDNA was prepared from 1 μg of total RNA using GoScript® Reverse Transcriptase (Promega). Real-time PCR was performed using the prepared cDNA to measure gene expression levels (Light Cycler 480 SYBR Green I; ROCHE). The amount of FGF21 mRNA was normalized with the amount of β-actin mRNA. The sequences of the primers used for real-time PCR are shown in the table below.

[0070] [Table 2]

[0071] (Test results) The amount of FGF21 mRNA in cultured hepatocytes was tested for significant differences between hepatocyte groups cultured in a medium supplemented with vehicle and hepatocyte groups cultured in a medium supplemented with test solutions of each of carbohydrates 1 to 6 using Tukey's test (n = 3-4) (#: p < 0.01). The results obtained for each hepatocyte group are summarized in Figure 4. Figure 4 shows that the expression level of the FGF21 gene was significantly increased by D-glucose and D-tagatose. Furthermore, it shows that the degree of increase in FGF21 gene expression was greater with the addition of D-tagatose than with the addition of D-glucose.

[0072] [Example 5] (Measurement of FGF21 secretion amount by adding D-tagatose) Primary cultured hepatocytes were cultured for 24 hours and the culture supernatants were collected (n=4) in the same manner as in Example 4, except that the concentrations of D-tagatose added were varied from 5 mM to 25 mM. The amount of FGF21 in the collected culture supernatants was measured using Fibroblast Growth Factor 21 Mouse / Rat ELISA, as in Example 2.

[0073] (Test results) The results of measuring FGF21 in the culture supernatant are summarized in Figure 5. Statistical analysis was performed using Tukey's test. Figure 5 shows that vehicle and 25 mM D-mannitol were classified into group A, and the amount of FGF21 secreted was significantly different from that of 25 mM D-glucose or 5 mM D-tagatose, which were classified into group B. Figure 5 also shows that the amount of FGF21 secreted by the addition of D-tagatose significantly increased in a dose-dependent manner. The level of Fgf21 gene expression was examined in the same manner as in Example 4, and the amount of Fgf21 gene expression also increased in a dose-dependent manner with D-tagatose. These results suggest a positive correlation between the increase in Fgf21 gene expression by D-tagatose and the increase in FGF21 secretion.

[0074] [Example 6] (Sugar or sugar alcohol addition experiment to primary cultured hepatocytes and measurement of FGF21 secretion) Primary hepatocytes prepared in the same manner as in Example 4 were cultured for 24 hours in a culture medium containing test solutions of each of the sugars or sugar alcohols 1 to 48 shown in the table below at 25 mM, or in a culture medium containing the same volume of vehicle (distilled water). [Table 3]

[0075] After 24 hours of culture, the culture supernatant was collected and stored at −30° C. until the amount of FGF21 in the culture supernatant was measured. The amount of FGF21 in the culture supernatant was measured using Fibroblast Growth Factor 21 Mouse / Rat ELISA as in Example 2 (n=3).

[0076] (Test results) The results of the FGF21 levels in the culture supernatants containing each test solution are summarized in Figure 6. A test using a culture medium containing a vehicle (distilled water) without added carbohydrates served as a negative control, and a test using a culture medium containing the monosaccharide D-glucose served as a positive control. The average value of the positive control is indicated by a dashed line in Figure 6. Figure 6 shows that the addition of D-fructose, D-sorbitol, D-xylitol, D-arabitol, D-psicose, and D-tagatose to the culture medium resulted in FGF21 expression levels exceeding those of the positive control. D-xylitol is known to increase FGF21 gene expression (Uebanso T et al. PLoS One. 2011;6(8):e22976). In this test, three sugars or sugar alcohols showed FGF21 expression levels exceeding those induced by D-xylitol: D-sorbitol, D-psicose, and D-tagatose.

[0077] Three types of sugars or sugar alcohols, D-sorbitol, D-psicose, and D-tagatose, were added to the culture medium at concentrations of 5 mM, 10 mM, 15 mM, and 25 mM, and the amount of FGF21 in the culture supernatant was measured in the same manner as above (n=4). The results showed that the expression level of FGF21 increased in a D-sorbitol dose-dependent manner (Figure 7). As with Example 5, the expression level of FGF21 increased in a D-tagatose dose-dependent manner, with the exception that the expression level was highest at 15 mM. At the same concentration of 25 mM, D-sorbitol and D-tagatose had a higher ability to stimulate FGF21 secretion than D-glucose.

[0078] [Example 7] The amount of FGF21 mRNA was measured in the same manner as in Example 4, except that test solutions of sugars or sugar alcohols 1 to 4 shown in the table below were added to a concentration of 25 mM instead of the carbohydrates (Table 1) used in Example 4. The amount of FGF21 mRNA was measured 4 hours, 8 hours, 12 hours, and 24 hours after the addition of the sugars or sugar alcohols. [Table 4]

[0079] (Test results) Regarding FGF21 mRNA in cultured stem cells, a significant difference was examined using Tukey's test between hepatocyte groups cultured in a culture medium supplemented with vehicle (distilled water) and hepatocyte groups cultured in a culture medium supplemented with each test solution of sugar or sugar alcohol 1 to 4 (n = 4) (*: p < 0.05, **: p < 0.01). The results obtained for each hepatocyte group are summarized in Figure 8. Figure 8 shows that the expression level of the FGF21 gene significantly increased 4 to 24 hours after the addition of D-sorbitol, and that the expression level peaked 12 hours after addition.

[0080] [Example 8] The amount of FGF21 mRNA was measured in the same manner as in Example 4, except that test solutions of sugars or sugar alcohols 1 to 5 shown in the table below were added to a concentration of 25 mM instead of the carbohydrates (Table 1) used in Example 4. The amount of FGF21 mRNA was measured 4 hours, 8 hours, 12 hours, and 24 hours after the addition of the sugars or sugar alcohols. [Table 5]

[0081] (Test results) Tukey's test was used to determine whether there was a significant difference in FGF21 mRNA levels in cultured stem cells between hepatocytes cultured in a culture medium supplemented with vehicle (distilled water) and hepatocytes cultured in a culture medium supplemented with each of the test solutions of sugars or sugar alcohols 1 to 5 (n = 4) (*: p < 0.05, **: p < 0.01). The results obtained for each hepatocyte group are summarized in Figure 9. Figure 9 shows that the expression level of the FGF21 gene significantly increased 4 to 12 hours after the addition of D-Psicose. Figure 9 also shows that the expression level peaked 4 or 8 hours after the addition of D-Psicose and then decreased to the same level as vehicle by 24 hours.

[0082] [Example 9] Primary cultured hepatocytes were cultured for 24 hours and the culture supernatant was collected (n=3) in the same manner as in Example 4, except that test solutions of sugars, sugar alcohols, or combinations thereof shown in the following table were used instead of the carbohydrates (Table 1) used in Example 4. The amount of FGF21 in the collected culture supernatant was measured using Fibroblast Growth Factor 21 Mouse / Rat ELISA, as in Example 2. [Table 6]

[0083] (Test results) The results of measuring FGF21 in the culture supernatant are summarized in Figure 10. Statistical analysis was performed using Tukey's test. Figure 10 shows that the expression level of the Fgf21 gene was significantly increased (P<0.01) when all of the carbohydrate combinations used in Example 9, including the use of D-fructose, D-tagatose, D-sorbitol, and D-xylitol alone, were used. The expression level of the Fgf21 gene was greatest when a combination of D-tagatose and D-sorbitol was used. The increase in the expression level of the Fgf21 gene from the total value when each carbohydrate was used was greatest when a combination of D-tagatose and D-psicose was used.

[0084] The combination of D-tagatose and D-sorbitol, and the combination of D-tagatose and D-psicose, resulted in a higher Fgf21 gene expression level than the sum of the Fgf21 gene expression levels of each of the two carbohydrates constituting each combination (Figure 10). These results indicate that the combinations exert a synergistic effect on Fgf21 gene expression. The Fgf21 gene expression level when using the combination of D-sorbitol and D-psicose was approximately equal to the sum of the Fgf21 gene expression levels of each of the two carbohydrates constituting the combination (Figure 10). These results indicate that the combinations exert an additive effect on Fgf21 gene expression.

[0085] On the other hand, when the combination of D-xylitol and D-tagatose, D-xylitol and D-sorbitol, or D-xylitol and D-psicose was used, the Fgf21 gene expression level was less than or almost equal to the sum of the Fgf21 gene expression levels of the two carbohydrates that make up each combination (Figure 10). These results indicate that the combinations do not have a synergistic effect on Fgf21 gene expression.

[0086] [Example 10] (mouse) Nine-week-old male C57BL / 6JJcl mice (CLEA Japan) were used. (Breeding conditions) The same rearing conditions as in Example 1 were used.

[0087] (Carbohydrate administration and blood sampling) Mice were orally administered 5 g / kg body weight of distilled water or various carbohydrates (D-glucose, D-tagatose, D-psicose, or D-sorbitol). 4, 6, 8, 12, and 24 hours after oral administration, 80 μL of blood was collected from the tail vein of each mouse using hematocrit capillary tubes (heparinized; AS ONE). The collected blood was centrifuged at 2,000 g for 20 minutes at 4°C, and plasma was collected and stored at -30°C until analysis. (Measurement of FGF21 levels in plasma) The amount of FGF21 in the collected plasma was measured using Fibroblast Growth Factor 21 Mouse / Rat ELISA, as in Example 2.

[0088] (Test results) The significance of plasma FGF21 levels between mice administered distilled water and those administered various carbohydrates was examined using Tukey's multiple comparison test (n = 4) (*: p < 0.05, **: p < 0.01). In the D-Psicose group, plasma FGF21 levels peaked 4 hours after administration (Fig. 11a, △). In the D-Tagatose group, plasma FGF21 levels peaked 6 hours after administration (Fig. 11a, □). In the D-Sorbitol group, plasma FGF21 levels peaked 8 hours after administration (Fig. 11a, ◇). These results suggest that the timing of blood FGF21 elevation can be adjusted by the type or combination of sugars or sugar alcohols administered. For example, the combination of D-Psicose and D-Sorbitol appears to rapidly increase blood FGF21 levels and maintain these levels for a long period of time.

[0089] In vivo (in mice), FGF21 levels peaked earlier with D-psicose administration than with D-sorbitol administration (Figure 11a, ◇: 8 hours after administration) (Figure 11a, △: 4 hours after administration). This result is consistent with the in vitro (in primary liver cell cultures) results, in which FGF21 gene expression was earlier with D-psicose administration than with D-sorbitol administration (Figure 8, 12 hours after administration) (Figure 9, 4 hours after administration). These results suggest that in vivo FGF21 expression profiles (e.g., levels and time course) and FGF21-mediated effects (e.g., suppression of simple sugar / alcohol preference) can be predicted from in vitro studies.

[0090] The AUC for plasma FGF21 levels was calculated for each mouse group (Fig. 11b). Fig. 11b shows that D-Psicose, D-Tagatose, and D-Sorbitol all significantly increased plasma FGF21 levels, and that the AUC values ​​for each carbohydrate were nearly equivalent.

[0091] [Example 11] (mouse) Nine-week-old male C57BL / 6JJcl mice (CLEA Japan) were used. (Breeding conditions) The same rearing conditions as in Example 1 were used. (Test solution) A 50 mM sucrose solution in water was used as the test solution.

[0092] (2-bottle choice test) First period (habituation to the experimental conditions): Two water bottles for measuring trace drinking volume, each containing sterilized water, were presented 24 hours a day for four days, allowing the rats to learn to drink from the two water bottles. Period 2 (baseline measurement): Mice were orally administered 5g / kg of distilled water and then presented with two water bottles for measuring micro-drinking volume, one containing sterile water and the other containing 50mM sucrose solution, for 24 hours a day for 2 days. To eliminate the influence of location preference, the positions of the two water bottles for measuring micro-drinking volume were swapped every 24 hours. Third period (administration experiment): Mice were orally administered 5g / kg body weight of vehicle (distilled water) or various carbohydrates, D-tagatose, D-psicose, or D-sorbitol, and the same experiment as in the second period was carried out. (Measurement of the amount of solution ingested) The amount of solution ingested by the mice was measured using a water bottle for measuring minute amounts of drinking water for mice and rats (Drink-O-Measurer, manufactured by Shin Factory).

[0093] (Test results) A Student's t-test was used to test for significant differences in the amount of 50 mM sucrose solution consumed between the second period (pretrial) and the third period (posttrial) (n = 6). The results are summarized in Figure 12. Figure 12 shows that the amount of 50 mM sucrose solution consumed was significantly reduced in the groups of mice administered D-sorbitol or D-tagatose (*: p < 0.05). Figure 12 also shows that the amount of 50 mM sucrose solution consumed tended to be reduced in the group of mice administered D-psicose (p = 0.068). These results suggest that D-sorbitol, D-tagatose, and D-psicose have the effect of suppressing simple carbohydrate preference.

[0094] [Example 12] (mouse) Nine-week-old C57BL / 6JJcl male mice (CLEA Japan) were used. (Breeding conditions) The same rearing conditions as in Example 1 were used. (Test solution) The test solution used was 8% ethanol, the ethanol concentration of which was adjusted using sterile water.

[0095] (2-bottle choice test) First period (habituation to the experimental conditions): Two water bottles for measuring trace drinking volume, each containing sterilized water, were presented 24 hours a day for two days, allowing the rats to learn to drink from the two water bottles. Period 2 (measurement of preference for the baseline test solution): Two micro-drinking bottles, one containing sterile water and the other containing the test solution, were presented 24 hours a day for 6–7 days. To eliminate the influence of location preference, the two micro-drinking bottles were swapped every 24 hours. The amount of solution ingested by the mice was measured using a water bottle for measuring minute amounts of drinking water for mice and rats (Drink-O-Measurer, manufactured by Shin Factory). Preference for the test solution was calculated using the following formula: Preference = (total amount of test solution consumed over the last 2 days) / (total amount of sterile water consumed over the last 2 days + total amount of test solution consumed over the last 2 days) x 100

[0096] Period 3 (measurement of changes in preference due to carbohydrate administration): Mice that showed a preference for alcohol of 66% or higher in Period 2 were presented with two water bottles for measuring micro-drinking volume: one containing sterilized water and the other containing the test solution, 24 hours a day for two days. During Period 3, 5g / kg body weight of vehicle (distilled water) or various carbohydrates, D-tagatose, D-sorbitol, or D-psicose, were orally administered (once a day at 2pm) and preference was measured. Preference for the test solution was calculated using the following formula: Preference = (total amount of drinking water for the test solution over two days) / (total amount of drinking water for the sterile water over two days + total amount of drinking water for the test solution over two days) × 100

[0097] (Test results) Preference for the test solution was examined using a paired t-test to determine whether there was a significant difference between the groups of mice administered distilled water and those administered various carbohydrates (n = 5–7). The results are summarized in Figure 13. Figure 13 shows that preference for 8% ethanol aqueous solution was significantly reduced in the groups of mice administered D-tagatose or D-psicose (*: p < 0.05). In the group of mice administered D-sorbitol, data for individual mice varied widely, and no significant decrease in the estimated mean was observed. In this group, mice were observed to have a reduced preference for 8% ethanol aqueous solution. This indicates that D-sorbitol suppressed the preference of mice that showed a preference for 8% ethanol. These results suggest that D-sorbitol, D-tagatose, or D-psicose have the effect of suppressing alcohol preference.

Claims

1. A fibroblast growth factor 21 (hereinafter referred to as "FGF21") inducer containing at least one carbohydrate selected from the group consisting of D-sorbitol, D-psicose, and D-tagatose, and / or an oligosaccharide containing at least one sugar residue selected from the group consisting of D-sorbitol, D-psicose, and D-tagatose.

2. The FGF21 inducer according to claim 1, wherein the at least one carbohydrate comprises D-sorbitol or D-tagatose.

3. The FGF21 inducer according to claim 1, wherein the at least one carbohydrate is D-psicose.

4. The FGF21 inducer according to claim 1, wherein the at least one carbohydrate is D-sorbitol.

5. The FGF21 inducer according to claim 1, wherein the at least one carbohydrate is D-tagatose.

6. 2. The FGF21 inducer according to claim 1, which comprises at least two carbohydrates selected from the group consisting of D-sorbitol, D-psicose, and D-tagatose as carbohydrates and / or sugar residues.

7. A composition for suppressing alcohol preference in a mammal, comprising an FGF21 inducer, the FGF21 inducer contains at least one carbohydrate selected from the group consisting of D-sorbitol, D-psicose, and D-tagatose, and / or an oligosaccharide containing at least one sugar residue selected from the group consisting of D-sorbitol, D-psicose, and D-tagatose; A composition wherein the FGF21 inducer is the only active ingredient for suppressing alcohol preference.

8. The composition of claim 7, which is a supplement.

9. 8. The pharmaceutical composition according to claim 7 for treating alcoholism.

Images