JP7748715B2 - Fibroblast growth factor 21 inducer and composition for suppressing alcohol or simple carbohydrate preference - Google Patents

Description

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 said at least one carbohydrate. The present invention also relates to a composition for suppressing alcohol preference or simple carbohydrate preference in a mammal, comprising said FGF21 inducer.

FGF21 is a hormone that regulates energy homeostasis and 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 and sweet taste preferences (Non-Patent Document 1). The present inventors have found that the suppression of simple carbohydrate preference is caused by activation of oxytocin (Oxt) neurons in the brain due to the formation of a complex between secreted FGF21 and β-Klotho (Non-Patent Document 2).

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). In Japan, it is estimated that there are approximately 1.1 million people with alcohol dependence, approximately 3 million at risk, and approximately 10 million high-risk drinkers. Medications available on the market to control alcohol consumption include disulfiram, cyanamide solution, acamprosate, and nalmefene.

Glucose is used as almost the only energy source in the brain and central nervous system, and plays an important role in vital activities. However, excessive carbohydrate intake 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 medications for treating obesity.

It has been noted that sugar cravings exist, which are an inability to stop craving sugar and sugar-rich foods. This sugar craving is a type of addiction symptom and is also known as sugar addiction. It is believed that sugar is what causes these addiction symptoms. 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.

Talukdar S et al., “FGF21 Regulations Sweet and Alcohol Preference.”, Cell Metab. 2016 Feb 9;23(2):344-349 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

One objective of the present disclosure is to provide a novel composition that can suppress either or both alcohol preference and simple carbohydrate preference.

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

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.

Figure 1(a) is a box plot 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 box plot showing the preference for 8% ethanol in ethanol. Figure 1(c) is a box plot showing the preference for 16% ethanol. 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(a) is a box plot showing the preference for 100 mM sucrose solution for β-Klotho Flox mice (white) and Oxt neuron-specific Klb knockout mice (gray). Figure 3(b) is a box plot showing the preference for 0.2% saccharin solution. Figure 3(c) is a box plot showing the preference for 2% dextrin solution. This is a box plot showing the relative expression level of the FGF21 gene in primary cultured hepatocytes cultured with six types of carbohydrates added to the culture medium. 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. FIG. 10 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. 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. This is a box plot showing the change over time in the relative expression level of the FGF21 gene in primary hepatocytes cultured with the addition of four types of carbohydrates, including D-sorbitol. This is a box plot showing the change over time in the relative expression level of the FGF21 gene in primary hepatocytes cultured with the addition of five types of carbohydrates, including D-Psicose. 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(a) is a box plot showing the FGF21 concentration 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. 1 is a bar graph showing that preference for 50 mM sucrose aqueous solution is suppressed. 1 is a bar graph showing that preference for an 8% aqueous ethanol solution is suppressed.

As used herein, "sugar" refers to carbohydrates including sugars or sugar alcohols. A carbohydrate 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 carbohydrate 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 carbohydrate may be D-sorbitol, D-psicose, or D-tagatose alone. In one embodiment, the carbohydrate 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.

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 consisting of 20 or more sugars linked together. A polysaccharide consisting of only one type of monosaccharide is called a homoglycan, while 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.

As used herein, "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.

As used herein, "oligosaccharide" refers to a sugar compound composed of three to fewer than 20 sugar residues. Oligosaccharides according to the present disclosure preferably contain three to 10 sugar residues. Oligosaccharides have a molecular weight of, for example, 300 to 3,000. Oligosaccharides may consist 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, using organic chemical synthesis or enzymatic chemical synthesis. 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).

As used herein, "D-sorbitol" or "D-Sorbitol" refers to a sugar alcohol represented by formula 1:
(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 such known production methods include, but are not limited to, a method in which D-glucose is reduced with the enzyme aldose reductase. D-sorbitol is commercially available, for example, from Tokyo Chemical Industry Co., Ltd.

As used herein, "D-psicose" or "D-Psicose" refers to a hexose sugar represented by formula 2:
(Formula 2)

D-psicose, also known as allulose, provides only 0.3% of the calories of sucrose as energy. D-psicose can be produced according to known methods and is commercially available. Such known production methods include, but are not limited to, a method of producing it from fructose using the enzyme D-tagatose 3-epimerase, or a method of extracting it from plants of the genus Zea mays. D-psicose is commercially available, for example, from Tokyo Chemical Industry Co., Ltd.

As used herein, "D-tagatose" or "D-Tagate" refers to a hexose sugar represented by formula 3:
(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.

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

As used herein, "FGF21" refers to a hormone protein encoded by the FGF21 gene in mammals and secreted by 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.

The amount of FGF21 in blood may be the FGF21 concentration (e.g., ng/ml, nM) in the mammal's 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.

As used herein, "inducer" refers to a formulation containing a substance that induces the expression of a desired product in a mammal. The formulation may consist of a single component or may be a composition containing two or more components. The formulation may be in, but is not limited to, a solid or liquid form. An inducer may include, for example, a substance that causes the cells of a mammal to produce a desired product that is secreted into the blood. In this example, the inducer may result in an increase in the amount of the desired product in the blood of the mammal.

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 said at least one carbohydrate.

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.

In one embodiment, the FGF21 inducer 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. The FGF21 inducer contains at least two types of carbohydrates selected from the group consisting of D-sorbitol, D-psicose, and D-tagatose, and/or an oligosaccharide containing at least two types of carbohydrate residues selected from the group consisting of D-sorbitol, D-psicose, and D-tagatose. The at least two types of 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.

The at least two types of carbohydrates and/or oligosaccharides may have a synergistic or additive effect on FGF21 induction. An FGF21 inducer containing the at least two types of carbohydrates and/or oligosaccharides may exhibit a high FGF21 induction effect while reducing its intake. An FGF21 inducer containing D-tagatose and D-sorbitol or D-psicose as carbohydrates and/or sugar residues may exhibit a high FGF21 induction 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.

FGF21 inducers containing D-psicose and D-tagatose or D-sorbitol as carbohydrates and/or sugar residues can increase blood FGF21 levels relatively quickly and maintain high FGF21 concentrations for a relatively long period of time. FGF21 inducers containing D-sorbitol and D-tagatose as carbohydrates and/or sugar residues can maintain high FGF21 concentrations for a relatively long period of time. FGF21 inducers containing D-sorbitol, D-psicose, and D-tagatose as carbohydrates and/or sugar residues can increase blood FGF21 levels relatively quickly and maintain high FGF21 concentrations for a relatively long period of time.

The FGF21 inducer may be in an orally ingestible form, such as, but not limited to, a tablet, capsule, granule, powder, dust, syrup, or liquid. The orally acceptable additive may be, but is not limited to, an excipient, disintegrant, binder, humectant, stabilizer, buffer, lubricant, preservative, seasoning, flavoring, fragrance, acidulant, colorant, or a combination thereof. The FGF21 inducer may be manufactured according to known methods appropriate to its form. When the FGF21 inducer is in a powder form, it can be manufactured, for example, by blending the at least one carbohydrate and/or 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 a liquid form, it can be manufactured, for example, by blending the at least one carbohydrate and/or oligosaccharide containing a sugar residue of the at least one carbohydrate with an aqueous liquid.

The amount of at least one carbohydrate selected from the group consisting of D-sorbitol, D-psicose, and D-tagatose, and/or oligosaccharides containing sugar residues of said at least one carbohydrate, in an FGF21 inducer, is appropriately determined depending on the formulation. The daily intake amount of said at least one carbohydrate and/or oligosaccharides containing sugar residues of said at least one carbohydrate 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 that has taken an FGF21 inducer for one week, compared to the amount of FGF21 in the mammal's blood before taking the FGF21 inducer. A significant difference test can be performed using, but is not limited to, Tukey's test. The mammal is, for example, a mouse. An amount that significantly increases the amount of FGF21 in the blood of a mouse is, for example, 1-10 g/kg body weight, 3-7 g/kg body weight, or 5 g/kg body weight. The amount that significantly increases the amount of FGF21 in the blood of 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 amount of FGF21 in the blood of 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.

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, 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. The FGF21 inducer may be taken directly as a supplement, or added to a food or beverage.

As used herein, a "composition" may be in, but is not limited to, a solid or liquid form. The composition may be, for example, a formulation containing a substance that can result in the suppression of alcohol preference in a mammal. In this example, the composition may result in, for example, an increase in a product in the blood of a mammal that can result in the suppression of alcohol preference.

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

As used herein, "treatment" means preventing the onset of a specific symptom or pathological condition, or treating a specific symptom or pathological condition. As used herein, "treatment" means alleviating, mitigating, or completely curing a specific disease state or pathological condition.

As used herein, a "composition for suppressing either or both of alcohol preference and simple carbohydrate preference in a mammal" 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 said at least one carbohydrate, as a substance capable of suppressing either or both of alcohol preference and simple carbohydrate preference in a mammal. The composition contains, but is not limited to, an FGF21 inducer described herein. In one embodiment, the composition is a composition for suppressing alcohol preference in a mammal. In another embodiment, the composition is a composition for suppressing simple carbohydrate preference in a mammal. In another embodiment, the composition is a composition 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.

In one embodiment, the composition for suppressing simple carbohydrate preference is a composition for suppressing preference for glucose, fructose, sucrose, lactose, and maltose. In another embodiment, the composition for suppressing simple carbohydrate preference is a composition for suppressing preference for glucose, fructose, and sucrose. In another embodiment, the composition for suppressing simple carbohydrate preference is a composition that suppresses preference for simple carbohydrates but does not substantially alter preference for either or both of synthetic sweeteners and polysaccharides. In one embodiment, the composition for suppressing 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.

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.

In one embodiment, the composition for suppressing alcohol preference is a composition for suppressing preference for an aqueous composition (e.g., alcoholic beverages) containing a substance in which a hydrocarbon hydrogen atom has been replaced with a hydroxyl group (e.g., ethanol). In another embodiment, the composition for suppressing alcohol preference contains at least one carbohydrate (e.g., one or two) 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.

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 that has ingested 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 test for significance can be performed using, but is not limited to, Tukey's test. The mammal is, for example, a mouse. Amounts that significantly increase blood FGF21 levels in mice are, for example, 1 to 10 g/kg body weight, 3 to 7 g/kg body weight, or 5 g/kg body weight. Amounts that significantly increase 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.

As used herein, "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 mitigating effect on alcoholism.

As used herein, a "medicine for treating obesity" or a "medicine for treating alcoholism" 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, as a substance for treating obesity or alcoholism. The pharmaceutical comprises, but is not limited to, an FGF21 inducer described herein. In one embodiment, the pharmaceutical 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 pharmaceutical 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.

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 with respect to FGF21 inducers. Pharmaceuticals may be manufactured according to known methods appropriate for the form, but are not limited to these.

The compositions or medicaments disclosed herein may be taken daily in a single dose, or in multiple divided doses. A composition or medicament disclosed herein that is taken once daily may contain, for example, 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.

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.

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 precedence over other behaviors. Physiological symptoms of alcoholism include alcohol withdrawal symptoms, including seizures and hallucinations, that occur upon cessation of drinking or reduced alcohol consumption. Cognitive symptoms of 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 also involve the development of alcohol tolerance, which refers to the need to drink larger amounts of alcohol to achieve the same level of intoxication.

As used herein, "obesity" refers to a pathological condition accompanied by health problems 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.

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 administered orally to a human.

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

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.

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.

In one embodiment, there is provided a method for treating alcoholism or obesity, comprising orally administering to a mammal in need thereof a pharmaceutical agent for treating alcoholism or obesity, wherein the pharmaceutical agent 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 pharmaceutical agent also apply to this embodiment.

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, 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 FGF21 inducers, compositions, or pharmaceuticals are also included in this embodiment.

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 said carbohydrate and/or said oligosaccharide. The features described herein for FGF21 inducers, compositions, and pharmaceuticals are also included in this embodiment.

Specific examples are described below, which illustrate exemplary embodiments of the present disclosure and are not intended to limit in any way the scope of the invention as set forth in the appended claims.

[Example 1]
(mouse)
Nine-week-old male β-Klotho Flox mice were used as a control group. Nine-week-old male oxytocin (Oxt) neuron-specific β-Klotho knockout mice (Cell Metab. 2012 Sep 5;16(3):387-93), which lack the FGF21 receptor specifically in Oxt neurons, were used as an 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).

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

(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).
(Calculation of preference)
Preference for the test solution was calculated using the following formula:
Preference = (total amount of drinking water of test solution for 4 days) / (total amount of drinking water of sterilized water for 4 days + total amount of drinking water of test solution for 4 days) x 100

(Test results)
The preference for each test solution was tested using Student's t-test to determine whether there was a significant difference between the control group and the experimental group. The results obtained for each test solution are summarized in Figure 1. Figures 1(a) to 1(c) show that preference for alcohol was increased in oxytocin (Oxt) neuron-specific β-Klotho knockout mice (gray) compared to β-Klotho Flox mice (open). 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.

[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 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 having the same concentration as in Example 1 was used.
(Measurement of Ingested Solution Amount and Calculation of Preference)
The same method as in Example 1 was used.

(Two-bottle choice test)
First period (habituation to experimental conditions): Two water bottles for measuring trace drinking volume containing sterilized water were presented 24 hours a day for four days to allow the rats to learn to drink from the two water bottles for measuring trace drinking volume.
Period 2 (measurement of preference for the baseline test solution): Two water bottles for measuring micro-drinking volume, one containing sterilized water and the other containing the test solution, were presented for 24 hours 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.
-Third period (cool-off period to eliminate the influence of alcohol in the second period): Two water bottles for measuring trace amounts of drinking water containing sterilized water were presented 24 hours a day for 7 days.

- Fourth period (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 using 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.
Fifth period (blood sampling): After the fourth period, 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 4°C, 2000 rpm, and plasma was collected. Plasma FGF21 concentrations were measured using Fibroblast Growth Factor 21 Mouse/Rat ELISA (BioVendor) according to the attached instructions.

(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) to 2(c). Figures 2(a) and 2(b) show that the preference for alcohol was significantly reduced in the experimental group (grey) that had consumed the high-sucrose diet compared to the control group (open) that had consumed the normal diet (CE-2).

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 Figures 2(d)-(f). Figures 2(d)-(f) show that plasma FGF21 concentrations were significantly increased in the experimental group (gray) that consumed a high-sucrose diet compared to the control group (open) that consumed a normal diet (CE-2).

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

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

(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.
(Two-bottle choice test)
First period (habituation to experimental conditions): Two water bottles for measuring trace drinking volume containing sterilized water were presented 24 hours a day for four days to allow the rats to learn to drink from the two water bottles for measuring trace drinking volume.
Second period (measurement of preference for test solution): Two water bottles for measuring micro-drinking volume, one containing sterilized water and the other containing the test solution, were presented for 24 hours 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.

(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.

(Test results)
We used Welch's t test to test whether there was a significant difference between the control and experimental groups in the preference for each test solution (*: p<0.05). The results obtained 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 to 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 to β-Klotho Flox mice (open). These results suggest that the action of FGF21 via β-Klotho is necessary for the simple carbohydrate-specific suppression of simple carbohydrate preference in mice.

[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 transected. Simultaneously with the aorta transecting, 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 a culture medium (37°C) and seeded at 1 x ¹⁰ cells/well onto a 24-well microplate coated with collagen Type I. Three hours after seeding, the culture medium was replaced with fresh medium, and after culturing for 24 hours, the resulting hepatocytes were used as primary cultured hepatocytes.
The culture medium used was prepared by adding 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.) to Williams' medium E (Sigma-Aldrich).

(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 supplemented with test solutions of each of carbohydrates 1 to 6 shown in the table below at 25 mM or in a culture medium supplemented with the same volume of vehicle (distilled water).

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.

(Test results)
The amount of FGF21 mRNA in cultured hepatocytes was examined by Tukey's test to determine whether there was a significant difference 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 (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 in the presence of 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.

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

(Test results)
The measurement results of 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. As in Example 4, the level of expression of the FGF21 gene was examined, and the amount of expression of the FGF21 gene also increased in a dose-dependent manner with D-tagatose. These results suggest that there is a positive correlation between the increase in FGF21 gene expression by D-tagatose and the increase in FGF21 secretion.

[Example 6]
(Experiment of adding sugar or sugar alcohol to primary cultured hepatocytes and measurement of FGF21 secretion amount)
Primary hepatocytes prepared in the same manner as in Example 4 were cultured for 24 hours in a culture medium supplemented with test solutions of each of sugars or sugar alcohols 1 to 48 shown in the table below at 25 mM or in a culture medium supplemented with the same volume of vehicle (distilled water).

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).

(Test results)
The results of the FGF21 levels in the culture supernatants to which each test solution was added are summarized in Figure 6. A test using a culture medium containing a carbohydrate-free vehicle (distilled water) served as a negative control, and a test using a culture medium containing the monosaccharide D-glucose served as a positive control. In Figure 6, the average value of the positive control is indicated by a dashed line. Figure 6 shows that when D-Fructose, D-Sorbitol, D-Xylitol, D-Arabitol, D-Psicose, and D-Tagateose were added to the culture medium, the FGF21 expression level exceeded that of the positive control. Here, D-Xylitol is known to increase FGF21 gene expression (Uebanso T et al. PLoS One. 2011;6(8):e22976.). In the results of this test, three types of sugars or sugar alcohols showed FGF21 expression levels exceeding that of D-Xylitol: D-sorbitol, D-psicose, and D-tagatose.

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 amount of FGF21 expression increased in a D-sorbitol dose-dependent manner (Figure 7). As with Example 5, the amount of FGF21 expression increased in a D-tagatose dose-dependent manner, except that it was greatest at 15 mM. At the same concentration of 25 mM, D-sorbitol and D-tagatose had a greater ability to stimulate FGF21 secretion than D-glucose.

[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.

(Test results)
Tukey's test was used to determine whether there was a significant difference in FGF21 mRNA levels in cultured stem cells between hepatocyte groups cultured in a culture medium supplemented with vehicle (distilled water) and hepatocyte groups cultured in a culture medium supplemented with each of the test solutions of sugars or sugar alcohols 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.

[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.

(Test results)
Tukey's test was used to determine whether there was a significant difference in FGF21 mRNA levels in cultured stem cells between hepatocyte groups cultured in a culture medium supplemented with vehicle (distilled water) and hepatocyte groups 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.

[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 in the same manner as in Example 2.

(Test results)
The measurement results of 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-Tagateose, D-Sorbitol, and D-Xylitol carbohydrates alone, were used. The expression level of the FGF21 gene was greatest when a combination of D-Tagateose 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-Tagateose and D-Psicose was used.

The FGF21 gene expression levels of the combinations of D-Tagateose and D-Sorbitol, and D-Tagateose and D-Psicose were greater than the sum of the FGF21 gene expression levels of each of the two carbohydrates that make up each combination (Figure 10). These results indicate that the combinations have 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 that make up the combination (Figure 10). These results indicate that the combinations have an additive effect on FGF21 gene expression.

On the other hand, when the combination of D-Xylitol and D-Tagateose, the combination of D-Xylitol and D-Sorbitol, or the combination of D-Xylitol and D-Psicose was used, the FGF21 gene expression levels were smaller than or approximately equal to the sum of the FGF21 gene expression levels of each 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.

[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.

(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. Four, six, eight, 12, and 24 hours after oral administration, 80 μL of blood was collected from the tail vein of each mouse using a hematocrit capillary tube (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 amount in plasma)
The amount of FGF21 in the collected plasma was measured using Fibroblast Growth Factor 21 Mouse/Rat ELISA, as in Example 2.

(Test results)
The plasma FGF21 levels were examined using Tukey's multiple comparison test to determine whether there was a significant difference between the distilled water-administered mice and the various carbohydrate-administered mice (n = 4) (*: p < 0.05, **: p < 0.01). In the D-Psicose-administered group, plasma FGF21 levels peaked 4 hours after administration (Fig. 11a, △). In the D-Tagateose-administered group, plasma FGF21 levels peaked 6 hours after administration (Fig. 11a, □). In the D-Sorbitol-administered group, plasma FGF21 levels peaked 8 hours after administration (Fig. 11a, ◇). These results suggest that the timing of increasing blood FGF21 levels can be adjusted by changing the type or combination of sugars or sugar alcohols administered. For example, it is suggested that a combination of D-Psicose and D-sorbitol can relatively quickly increase the blood FGF21 concentration and maintain that concentration for a long period of time.

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 cultured liver cells) results, in which FGF21 gene expression was observed 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., amounts and changes over time) or FGF21 effects (e.g., suppression of simple sugar/alcohol preference) can be predicted from in vitro test results.

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

[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 aqueous sucrose solution was used as the test solution.

(Two-bottle choice test)
First period (habituation to experimental conditions): Two water bottles for measuring trace drinking volume containing sterilized water were presented 24 hours a day for four days to allow the rats to learn to drink from the two water bottles for measuring trace drinking volume.
Second period (baseline measurement): Mice were orally administered 5 g/kg of distilled water and presented with two water bottles for measuring micro-drinking volume, one containing sterile water and the other containing 50 mM sucrose solution, for 24 hours each for two 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 with 5 g/kg body weight of vehicle (distilled water) or various carbohydrates, D-Tagatese, D-Psicose or D-Sorbitol, and the same test 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).

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

[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)
8% ethanol was used as the test solution, and the ethanol concentration was adjusted using sterile water.

(Two-bottle choice test)
First period (habituation to experimental conditions): Two water bottles for measuring trace drinking volume containing sterilized water were presented for 24 hours for two days, and the rats were allowed to learn to drink from the two water bottles for measuring trace drinking volume.
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 for 24 hours 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 two days) / (total amount of sterile water consumed over the last two days + total amount of test solution consumed over the last two days) x 100

- Third period (measurement of changes in preference due to carbohydrate administration): Mice that showed a preference for alcohol of 66% or higher in the second period were presented with two water bottles for measuring micro-drinking volume, one containing sterilized water and the other containing the test solution, for 24 hours a day for two days. During the third period, 5 g/kg body weight of vehicle (distilled water) or various carbohydrates D-Tagatese, D-Sorbitol, or D-Psicose were orally administered (once a day at 2 p.m.), and preference was measured.
Preference for the test solution was calculated using the following formula:
Preference = (total amount of drinking water of test solution for 2 days) / (total amount of drinking water of sterilized water for 2 days + total amount of drinking water of test solution for 2 days) x 100

(Test results)
A paired T test was used to determine whether there was a significant difference in preference for the test solution between the mice administered distilled water and the mice 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 mice administered D-Tagateose or D-Psicose (*: p < 0.05). In the mice administered D-Sorbitol, there was a large variability in the data for individual mice, and no significant decrease in the estimated mean was observed. In this group, mice were observed to have a suppressed 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-Tagateose, or D-Psicose have the effect of suppressing alcohol preference.

Claims (9)

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. The FGF21 inducer according to claim 1, wherein the at least one carbohydrate comprises D-sorbitol or D-tagatose. The FGF21 inducer according to claim 1, wherein the at least one carbohydrate is D-psicose. The FGF21 inducer according to claim 1, wherein the at least one carbohydrate is D-sorbitol. The FGF21 inducer according to claim 1, wherein the at least one carbohydrate is D-tagatose. The FGF21 inducer according to claim 1, which contains at least two carbohydrates selected from the group consisting of D-sorbitol, D-psicose, and D-tagatose as carbohydrates and/or sugar residues. 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. The composition of claim 7, which is a supplement. The pharmaceutical composition of claim 7 for treating alcoholism.