Adjustment of circadian rhythm

Abstract

The present disclosure provides a compound, composition, and method effective for adjusting a circadian rhythm. More specifically, the present disclosure provides a compound, composition, or method that is useful for ameliorating a delayed sleep-phase type circadian rhythm disturbance. A composition according to the present disclosure includes at least one substance selected from the group consisting of fumaric acid and salts thereof as an active component.

Description

Circadian rhythm regulation

[0001] The present disclosure relates to regulating circadian rhythms. More particularly, the present disclosure relates to compounds useful for improving sleep-delay-type circadian rhythm disturbances, and compositions containing the same.

[0002] Living organisms actively change their internal environment on a roughly 24-hour (daily) cycle in synchronization with the 24-hour day-night cycle caused by the Earth's rotation. Basic bodily functions, such as body temperature and hormone secretion, are known to exhibit roughly 24-hour rhythms in humans, a periodic rhythm known as the "circadian rhythm." Because circadian rhythms are observed even in resting states without light or temperature changes, living organisms possess a clock mechanism known as the circadian clock. The circadian clock of mammals, including humans, is known to reside in the suprachiasmatic nucleus of the hypothalamus, located in the underside of the central brain. Furthermore, recent molecular biology research has revealed that clock proteins are synthesized in circadian clock cells by multiple genes known as clock genes, which then bind and decompose in a roughly 24-hour cycle. The circadian rhythm of the circadian clock is determined by this genetic activity. In addition to the sleep-wake rhythm, various biological rhythms are known as examples of biological phenomena that follow a circadian rhythm, such as appetite, sexual desire, blood pressure and body temperature rhythms, endocrine system rhythms (e.g., secretion of melatonin, adrenal cortical hormones, growth hormone, etc.), metabolic rhythms, immune system rhythms, and autonomic nervous system rhythms.

[0003] Because the cycle of the human body clock is slightly longer than 24 hours (though a small number of people have a shorter cycle), the human circadian rhythm is designed to synchronize the timing of the body clock with the 24-hour light-dark cycle of the external world (this is called the "entrainment mechanism"). This entrainment mechanism allows humans to synchronize their body clock with seasonal changes in daylength due to the Earth's revolution and changes in the light-dark cycle that occur with rapid travel to different time zones. For example, when traveling long distances by airplane in a short period of time, such as on an international business trip or vacation, a phase difference (time difference) occurs between the departure and arrival points, often resulting in the so-called "jet lag." When jet lag occurs, sleep-wake rhythms are disrupted during the period until the circadian rhythm gradually adjusts to the time zone of the destination, which can lead to temporary physical discomfort, such as reduced attention and concentration, as well as the various biological rhythm disruptions mentioned above.

[0004] Recently, a symptom similar to jet lag known as "social jet lag" has become a problem. Social jet lag is a jet lag phenomenon that occurs in everyday life due to changes in daily rhythms such as shift work, or differences in wake-up / bedtimes and sleep duration between weekends and weekdays. Social jet lag is caused in part by circadian rhythm disruption, and has been reported to not only cause poor physical health such as daytime sleepiness, decreased mental function, and lethargy, but also to exacerbate metabolic syndrome symptoms, increase depression, sleep disorders, and other mental and physical disorders.

[0005] Circadian rhythm sleep-wake disorders, a type of sleep disorder, are primarily classified into three types. One is "delayed sleep phase type," in which patients are unable to fall asleep from late at night until dawn and are unable to wake up until midday or early evening the next day. The second is "advanced sleep phase type," in which patients become sleepy in the evening or early evening and wake up early in the morning. The third is "non-24-hour sleep-wake type," in which the time they fall asleep and wake up differ each day. Of these, "delayed sleep phase type" is the most common. Light therapy is currently a common treatment for these symptoms. Administration of melatonin, an endogenous hormone that regulates circadian rhythms, and its precursors (serotonin, tryptophan, etc.) is also being investigated. In addition, ornithine (Patent Document 1), substances that inhibit the vasopressin receptors V1a and V1b (Patent Document 2), cinobufagins contained in the herbal medicine Senso (collected and dried secretions from the parotid or sebaceous glands of toads and the like) (Patent Document 3), cinnamic acids contained in the herbal medicine Cinnamon Bark (Patent Document 4), and (1R,2S)-2-(((2,4-dimethylpyrimidin-5-yl)oxy)methyl)-2-(3-fluorophenyl)-N-(5-fluoropyridin-2-yl)cyclopropanecarboxamide and a pharmaceutically acceptable salt thereof (Patent Document 5) have been described as having an effect of regulating circadian rhythm, and are expected to be useful in improving circadian rhythm sleep-wake disorders.

[0006] Japanese Patent Application Laid-Open No. 2019-182775 International Publication No. WO2019 / 176220 Japanese Patent Application Laid-Open No. 2016-204280 Japanese Patent Application Laid-Open No. 2016-204281 Japanese Patent Application Laid-Open No. 2021-120410

[0007] Molecular components of the Mammalian circadian clock. Buhr ED, Tkahashi JS. Handb Exp Pharmacol. 2013;(217):3-27Tahara Y, et al., Gut Microbiota-Derived Short Chain Fatty Acids Induce Circadian Clock Entrainment in Mouse Peripheral Tissue. Sci Rep. 2018;8(1):1395.Yamajuku D, et al., Real-time monitoring in three-dimensional hepatocytes reveals that insulin acts as a synchronizer for liver clock. Sci Rep. 2012;2:439. doi: 10.1038 / srep00439E. Yamamoto et. al., Effect of fumaric acid on the growth of Lactobacillus delbrueckii ssp. bulgaricus during yogurt fermentation, J Dairy Sci.2021 Sep;104(9):9617-9626.V. Gokmen & J. Acar, Fumaric acid in apple juice: a potential indicator of microbial spoilage of apples used as raw material: Food Additives & Contaminants: Vol 21, No 7, 2004, p.626-631 (tandfonline.com)

[0008] An objective of the present disclosure is to provide compounds, compositions, and methods for use in regulating circadian rhythms, and also to provide compounds, compositions, and methods for effective use in improving sleep-phase delayed circadian rhythm disorders.

[0009] In recent molecular biological research on circadian clocks, focusing on clock genes, instead of animal experiments, in vitro testing methods have been widely used to examine the effects on circadian clocks (control of circadian rhythms) by monitoring the circadian rhythm of luciferase activity using mammalian cells transfected with a reporter plasmid in which a luciferase gene is linked to the promoter of a clock gene such as Period 1 (Per1), Period 2 (Per2), or Bmal1. To solve the above-mentioned problems, the present inventors conducted extensive research using established in vitro testing methods and found that fumaric acid, a low-molecular-weight organic acid, has the effect of regulating the circadian rhythm, particularly by advancing the phase of the circadian rhythm and shortening its period.

[0010] Furthermore, the present inventors have found that fumaric acid selectively enhances gene expression of the short-chain fatty acid receptor GPR41 in enteroendocrine cells, which is known to be involved in the secretion of the anorectic hormone peptide YY (PYY) and the insulin secretion-promoting hormone GLP-1. This suggests that fumaric acid may not only regulate circadian rhythms by acting on clock genes in the central nervous system, but also stimulate GLP-1 secretion via GPR41 in enteric cells, thereby regulating circadian rhythms in peripheral tissues via insulin.

[0011] The present disclosure was completed based on these findings and further studies, and includes the following embodiments: (I) Composition for regulating circadian rhythm (I-1) A composition for regulating circadian rhythm, containing at least one active ingredient selected from the group consisting of fumaric acid and salts thereof. (I-2) The composition for regulating circadian rhythm described in (I-1), which is used to advance the phase of a circadian rhythm and / or shorten the period of a circadian rhythm. (I-3) The composition for regulating circadian rhythm described in (I-1) or (I-2), which contains a fermentation product of a fumaric acid-producing bacterium or a processed product thereof, which contains at least one active ingredient selected from the group consisting of fumaric acid and salts thereof. (I-4) The composition for regulating circadian rhythm described in (I-3), wherein the fumaric acid-producing bacterium is at least one lactic acid bacterium selected from the group consisting of Streptococcus thermophilus and Lactiplantibacillus plantarum. (I-5) A composition for regulating circadian rhythm according to any one of (I-1) to (I-4), which is a composition for regulating daily rhythm. (I-6) A composition for regulating circadian rhythm according to any one of (I-1) to (I-5), which is a food or drink, a quasi-drug, a pharmaceutical, or an additive therefor. (I-7) A composition for regulating circadian rhythm according to any one of (I-1) to (I-6), which does not contain the following compounds (1) and (2): (1) a fumarate salt of (1R,2S)-2-(((2,4-dimethylpyrimidin-5-yl)oxy)methyl)-2-(3-fluorophenyl)-N-(5-fluoropyridin-2-yl)cyclopropanecarboxamide, (2) a fumarate salt of ornithine.

[0012] (II) Use for producing a composition for regulating circadian rhythm (II-1) Use of at least one compound selected from the group consisting of fumaric acid and its salts, or a composition containing the same, for producing a composition for regulating circadian rhythm. (II-2) The use according to (II-1), wherein the composition containing at least one compound selected from the group consisting of fumaric acid and its salts is a fermentation product of a fumaric acid-producing bacterium or a processed product thereof. (II-3) The use according to (II-2), wherein the fumaric acid-producing bacterium is at least one lactic acid bacterium selected from the group consisting of Streptococcus thermophilus and Lactiplantibacillus plantarum. (II-4) The use according to any of (II-1) to (II-3), wherein the composition for regulating circadian rhythm is a quasi-drug, a pharmaceutical product, a non-therapeutic food or drink for healthy or pre-illness humans or mammals other than humans, or an additive therefor. (II-5) The use according to (II-4), wherein the healthy or pre-illness human is a human having at least one symptom selected from the group consisting of the following 1) to 4): 1) a person who may suffer from a circadian rhythm disorder, 2) a person who has a circadian rhythm sleep disorder, 3) a person whose circadian rhythm is desynchronized, 4) a person who is suffering from poor health due to a disruption of the circadian rhythm. (II-6) The use according to any of (II-1) to (II-5), wherein the compound and composition do not contain the compounds of the following (1) and (2): (1) a fumarate salt of (1R,2S)-2-(((2,4-dimethylpyrimidin-5-yl)oxy)methyl)-2-(3-fluorophenyl)-N-(5-fluoropyridin-2-yl)cyclopropanecarboxamide, (2) a fumarate salt of ornithine.

[0013] (III) Compound for use in regulating circadian rhythm and composition containing same (III-1) At least one compound selected from the group consisting of fumaric acid and salts thereof, or a composition containing same, for use in regulating circadian rhythm. (III-2) A compound for use according to (III-1) or a composition containing same, wherein the composition is a fermentation product of a fumaric acid-producing bacterium or a processed product thereof. (III-3) A compound for use according to (III-2) or a composition containing same, wherein the fumaric acid-producing bacterium is at least one lactic acid bacterium selected from the group consisting of Streptococcus thermophilus and Lactiplantibacillus plantarum. (III-4) A compound for use according to any one of (III-1) to (III-3) or a composition containing the same, wherein the compound and composition do not include the following compounds (1) and (2): (1) a fumarate salt of (1R,2S)-2-(((2,4-dimethylpyrimidin-5-yl)oxy)methyl)-2-(3-fluorophenyl)-N-(5-fluoropyridin-2-yl)cyclopropanecarboxamide, (2) a fumarate salt of ornithine.

[0014] (IV) Method for regulating circadian rhythm (IV-1) A method for regulating the circadian rhythm of a mammal, comprising administering to the mammal at least one compound selected from the group consisting of fumaric acid and salts thereof, or a composition containing the same. (IV-2) The method for regulating circadian rhythm according to (IV-1), wherein the composition is a fermentation product of a fumaric acid-producing bacterium or a processed product thereof. (IV-3) The method for regulating circadian rhythm according to (IV-2), wherein the fumaric acid-producing bacterium is at least one lactic acid bacterium selected from the group consisting of Streptococcus thermophilus and Lactiplantibacillus plantarum. (IV-4) The method for regulating circadian rhythm according to any one of (IV-1) to (IV-3), wherein the mammal is a human or non-human animal in a healthy or pre-diseased state, and the method is a non-therapeutic method for the mammal. (IV-5) The method for regulating circadian rhythm according to any one of (IV-1) to (IV-4), wherein the mammal is a human having at least one symptom selected from the group consisting of the following 1) to 4): 1) a person who may suffer from a circadian rhythm disorder, 2) a person who has a circadian rhythm sleep disorder, 3) a person whose circadian rhythm is desynchronized, or 4) a person who suffers from poor health due to a disruption of the circadian rhythm. (IV-6) The method for regulating circadian rhythm according to any one of (III-1) to (IV-5), wherein the compound and composition do not contain the compounds of the following (1) and (2): (1) a fumarate salt of (1R,2S)-2-(((2,4-dimethylpyrimidin-5-yl)oxy)methyl)-2-(3-fluorophenyl)-N-(5-fluoropyridin-2-yl)cyclopropanecarboxamide, (2) a fumarate salt of ornithine.

[0015] According to the present disclosure, circadian rhythm can be adjusted. More preferably, the compound of the present disclosure, a composition containing the compound, or a method using the compound or the composition can improve circadian rhythm disturbances caused by delayed sleep phase by advancing the phase of the circadian rhythm and / or shortening the period of the circadian rhythm. Therefore, according to the present disclosure, it is expected that circadian rhythm sleep disorders such as delayed sleep phase syndrome caused by circadian rhythm disturbances, lifestyle rhythm disturbances caused by circadian rhythm disturbances, and poor physical condition caused by circadian rhythm or lifestyle rhythm disturbances can be improved.

[0016] In Experimental Example 1, luciferase luminescence intensity was measured over time using per2 reporter cells with or without fumaric acid (fumaric acid) added (control), and the results of evaluating changes in circadian rhythm are shown. In Experimental Example 2, the effect of fumaric acid on the expression of the GPR41 gene in Caco-2 cells (human colon cancer-derived cells) is shown.

[0017] (I) Circadian rhythm regulation composition The circadian rhythm regulation composition of the present disclosure (hereinafter also simply referred to as "the composition") is characterized by containing, as an active ingredient, at least one compound selected from the group consisting of fumaric acid and its salts. Hereinafter, fumaric acid and its salts are also collectively referred to as "fumaric acids."

[0018] The salt of fumaric acid may be any salt that provides the effects of the present disclosure and is pharmaceutically acceptable or ingestible, and includes a combined salt of fumaric acid with a metal ion, such as a metal fumarate (e.g., a combined salt of fumaric acid with an alkali metal such as sodium, potassium, or lithium; an alkaline earth metal fumarate (e.g., a combined salt of fumaric acid with an alkaline earth metal such as magnesium or calcium); a hydrogen metal fumarate (e.g., a combined salt of fumaric acid with a hydrogen metal such as sodium hydrogen or potassium hydrogen); and a combined salt of fumaric acid with an ammonium salt (e.g., an ammonium salt, a tetramethylammonium salt).

[0019] However, the salts of fumaric acid disclosed herein do not include the following compounds: (1) (1R,2S)-2-(((2,4-dimethylpyrimidin-5-yl)oxy)methyl)-2-(3-fluorophenyl)-N-(5-fluoropyridin-2-yl)cyclopropanecarboxamide fumarate, and (2) ornithine fumarate. The compound (1) is the fumarate of lemborexant (generic name) represented by the following formula (I):

[0020]

[0021] The present composition is used to regulate the circadian rhythm of mammals, including humans. The "circadian rhythm" includes the expression rhythm of genes, including clock genes. Therefore, the present composition also includes compositions used to regulate the expression rhythm of genes, including clock genes. Clock genes are genes that control biological rhythms, particularly circadian rhythms, and are also called circadian rhythm control genes. Known clock genes found in mammals, including humans, include Period (per genes: e.g., per1, per2, etc.), Cryptochrome (cry genes: e.g., cry1, cry2, etc.), Clock, Dec, Casein Kinase 1 (CK1 genes: CK1ε, CK1δ, etc.), and Bmal (Bmal genes: e.g., Bmal1, etc.).

[0022] The clock gene targeted by the present disclosure is preferably the per2 gene. Circadian rhythms are based on a negative feedback loop mechanism between these clock genes, with the per2 gene playing a central role in this mechanism (Non-Patent Document 1: Molecular components of the Mammalian circadian clock. Buhr ED, Takahashi JS. Handb Exp Pharmacol. 2013;(217):3-27). Therefore, by controlling the expression rhythm of the per2 gene, the expression of a series of clock genes is controlled, thereby enabling the adjustment of the circadian rhythm as a biological rhythm. Therefore, the present composition may be used to adjust the expression rhythm of a series of clock genes through the adjustment of the expression rhythm of the per2 gene. Alternatively, the composition may be used to adjust the circadian rhythm as a biological rhythm by adjusting the expression rhythm of a series of clock genes through the adjustment of the expression rhythm of the per2 gene.

[0023] In the present disclosure, "adjusting the circadian rhythm" includes the meaning of "adjusting the phase of the circadian rhythm" and / or "adjusting the period of the circadian rhythm."

[0024] "Adjusting the phase of the circadian rhythm" includes adjusting the light-dark phase of the circadian rhythm by adjusting the phase of the expression rhythm of a clock gene. The clock gene referred to here is preferably the per2 gene. "Adjusting the phase of the expression rhythm of a clock gene" means adjusting the temporal change in the phase of the periodic expression rhythm of the gene, which is controlled by the activation and / or repression of the expression of the clock gene at regular intervals. As shown in Experimental Example 1 described below, fumaric acids, the active ingredients of the present composition, have the effect of adjusting the phase of the expression rhythm of the clock gene (per2 gene) by advancing the phase of the expression rhythm of the gene chronologically (i.e., along the time axis). This is also referred to as "phase advancement." Based on the phase advancement effect of fumaric acids, the present composition can advance (advance) the phase of the expression rhythm of the clock gene (per2 gene) along the time axis, thereby advancing (advancing) the light-dark phase of the circadian rhythm along the time axis, thereby adjusting the circadian rhythm.

[0025] "Adjusting the period of a circadian rhythm" includes adjusting the length of one cycle of the expression rhythm of a clock gene to adjust the light-dark cycle of the circadian rhythm. The clock gene referred to here is preferably the per2 gene. "Period" refers to the time or period (repetition interval) until a cyclical phenomenon or behavior completes one cycle and returns to the previous state. "Adjusting the period of one cycle of the expression rhythm of a clock gene" means adjusting the length of the periodic expression rhythm (length of one cycle: period) of the gene, which is controlled by the activation and / or repression of the expression of the clock gene at regular intervals. As shown in Experimental Example 1 below, fumaric acids, the active ingredients of the present composition, shorten the period of the expression rhythm of the clock gene (per2 gene) by shortening the length of one cycle of the expression rhythm. This is also referred to as "period-shortening effect." Based on the period-shortening effect of fumaric acids, the present composition can shorten the light-dark cycle of the circadian rhythm by shortening the period of the expression rhythm of the clock gene (per2 gene), thereby adjusting the circadian rhythm.

[0026] Furthermore, as shown in Experimental Example 2 below, fumaric acids, the active ingredient of the present composition, exert the effect of inducing gene expression of GPR41, a short-chain fatty acid receptor present in intestinal cells. GPR41 is known to be involved in the secretion of energy metabolism-related intestinal hormones such as the insulin secretion-promoting hormone GLP-1 (Non-Patent Document 2). Therefore, fumaric acids are thought to exert their effect of promoting GLP-1 secretion and insulin secretion in peripheral tissues through the induction of GPR41 gene expression. Dietary hormones such as insulin are known to function as circadian rhythm synchronizers in peripheral tissues (Non-Patent Document 3). Therefore, the present composition containing fumaric acids can regulate circadian rhythms by promoting insulin secretion in peripheral tissues through the induction of GPR41 gene expression.

[0027] Thus, "regulating circadian rhythm" as used herein includes regulation in central tissues and / or regulation in peripheral tissues. According to the present composition, circadian rhythm can be regulated through regulation in both central and peripheral tissues based on the effects of fumaric acids as shown in Experimental Examples 1 and 2. The term "regulation" as used herein also includes the meanings of "adjustment" and "control."

[0028] This composition allows for the adjustment of daily rhythms through the regulation of circadian rhythms. Therefore, this composition can also be used as a composition for regulating daily rhythms. Here, "daily rhythm" refers to the rhythm of daily life based on a person's unique circadian rhythm, i.e., the way of living (behavioral rhythm). Specifically, this refers to a rhythm in which each element of life, such as sleep, wakefulness, activity, rest, and eating, repeats at a regular cycle while being influenced by the environment, based on the circadian rhythm.

[0029] The present compositions include foods and beverages, quasi-drugs, and pharmaceuticals used to regulate circadian or circadian rhythms. Here, foods and beverages include non-therapeutic foods and beverages for humans. Furthermore, foods and beverages include not only foods and beverages for humans but also foods for non-human animals such as pets and livestock (i.e., including bait, feed, and laboratory feed). Furthermore, pharmaceuticals and quasi-drugs include not only those for humans but also those for non-human animals.

[0030] The subjects to which the foods, beverages, quasi-drugs, and pharmaceuticals used for adjusting circadian rhythms or life rhythms are applied are mammals including humans, and preferably humans. In the present disclosure, humans include humans in good health and humans in a pre-illness state who are not yet seriously ill but who are experiencing physical or mental discomfort.

[0031] The present composition can be suitably applied to, but is not limited to, individuals with at least one of the following symptoms: 1) individuals at risk of circadian rhythm disorders, such as shift workers, long-hour workers, international travelers, or individuals with irregular schedules due to factors such as late-night sleepiness; 2) individuals with circadian rhythm sleep disorders, such as delayed sleep phase syndrome; 3) individuals whose circadian rhythms are desynchronized due to blindness or other conditions; and 4) individuals experiencing poor physical health due to circadian rhythm disorders, particularly delayed circadian rhythm phases and / or lengthened periods. The poor physical health conditions listed in 4) above include physical disorders such as drowsiness, headache, dullness, loss of appetite, fatigue, lethargy, metabolic rhythm disorders, endocrine disorders, and / or gastrointestinal dysfunction. These individuals also include mental disorders such as decreased concentration or motivation, depression, anxiety, and / or depression. These individuals include individuals who are healthy or in a pre-symptomatic state.

[0032] The composition also includes an additive for preparing a food, drink, quasi-drug, or pharmaceutical product used to regulate circadian rhythms or life rhythms. By preparing a food, drink, quasi-drug, or pharmaceutical product using the circadian rhythm-regulating composition as an additive, the food, drink, quasi-drug, or pharmaceutical product can be imparted with a circadian rhythm-regulating effect. As a result, a food, drink, quasi-drug, or pharmaceutical product for circadian rhythm regulation that has a circadian rhythm-regulating effect can be prepared.

[0033] The origin of the fumaric acids used as the active ingredient of the present composition is not particularly limited, and they may be chemically synthesized or extracted and isolated from natural products (animals, plants, microorganisms, and products produced by them). They may also be extracted and isolated from a fermentation product prepared using a microorganism (fumaric acid-producing bacteria). Furthermore, the fumaric acids incorporated into the present composition may be crudely purified, regardless of their purity, as long as the effects of the present invention are not impaired. For example, as long as the effects of the present disclosure are not impaired, the fumaric acid-containing fermentation product itself prepared using a microorganism or a processed product thereof can be used as is as the active ingredient of a composition for circadian rhythm regulation.

[0034] Here, examples of microorganisms include bacteria capable of producing fumaric acids. In the present disclosure, bacteria capable of producing fumaric acids are collectively referred to as "fumaric acid-producing bacteria." Examples of such fumaric acid-producing bacteria include, but are not limited to, lactic acid bacteria, as long as they have the ability to produce fumaric acids. Examples of such lactic acid bacteria include, but are not limited to, lactic acid bacteria belonging to the Streptococcus genus and Lactiplantibacillus genus. Examples of lactic acid bacteria belonging to the Streptococcus genus include S. thermophilus, and examples of lactic acid bacteria belonging to the Lactiplantibacillus genus include L. plantarum (see, for example, Non-Patent Documents 4 and 5). Fermented products prepared by culturing these lactic acid bacteria contain fumaric acids. Therefore, these fermented products or processed products thereof (collectively referred to as "fumaric acid-containing fermented products") can be used as active ingredients in the present composition, or they can be used as the present composition as they are. The processed product is a processed product of a fermentation product prepared using a fumaric acid-producing bacterium, and includes, without limitation, processed products that have been subjected to various treatments (e.g., concentration treatment, purification treatment, drying treatment, etc.) so that they contain fumaric acids at a high concentration or with a high purity.

[0035] The content of fumaric acids contained in the present composition can be appropriately selected, for example, within the range of 0.1 to 99% by mass, as long as the present composition exerts its circadian rhythm regulating effect.

[0036] The circadian rhythm regulating activity of the present composition can be confirmed by the method described in Experimental Example 1 below. Specifically, the effect of the candidate composition on the expression cycle of a reporter gene (e.g., luciferase gene) whose expression is controlled by a clock gene promoter (e.g., per2 promoter) can be assessed by using cultured cells (e.g., human osteosarcoma-derived cells) into which the reporter gene (e.g., luciferase gene) has been introduced. If this test shows that the expression phase of the reporter gene advances and / or the expression cycle shortens in the presence of the candidate composition compared to the absence of the candidate composition (control), the composition can be determined to have a circadian rhythm regulating activity.

[0037] Alternatively, or in addition to this evaluation method, the method described in Experimental Example 2 below can also be used for evaluation. Specifically, evaluation can be performed by examining the effect of a candidate composition on the expression of the GPR41 gene using cultured cells of peripheral tissues capable of expressing the gene (e.g., cells derived from human colon cancer). If this test shows that the expression of the GPR41 gene increases in the presence of the candidate composition compared to the absence of the candidate composition (control), it can be determined that the composition has a circadian rhythm regulating effect.

[0038] (A) Application to Foods and Beverages, or Food and Beverage Additives One use form of the present composition includes foods and beverages, and food and beverage additives. The form of the foods and beverages and food and beverage additives is not particularly limited, and may be liquid, semi-liquid (including paste, gel, and sol), or solid, including beverage forms. Foods and beverages also include health foods (including functional foods, nutritional supplements, dietary supplements, health foods with disease risk reduction claims (foods for specified health uses, nutritional functional foods, and foods with functional claims), and foods for patients), as well as other general foods and beverages. Health foods are foods and beverages consumed with the expectation of maintaining or improving health. Supplements are foods and beverages in a formulation in which specific ingredients (fumaric acids) are concentrated. All of these foods and beverages are non-therapeutic foods and beverages not intended for therapeutic purposes.

[0039] Foods and beverages may include, for example, dairy products such as yogurt and cheese, which are produced by subjecting food materials such as milk to microbial fermentation to produce fumaric acids, and koji-fermented foods. Furthermore, supplements may be produced by adding excipients, binders, etc. to the composition, kneading the mixture, and then tableting the mixture; powders (pulverized), pills, capsules, jellies, granules, etc. In addition to fumaric acids, the composition may also contain, as appropriate, food-grade (edible) carbohydrates, proteins, lipids, vitamins, minerals, sugars (e.g., glucose), natural or artificial sweeteners, citric acid, carbonated water, fruit juice, stabilizers, preservatives, binders, thickeners, and / or emulsifiers.

[0040] When the food or drink is a beverage, examples of non-alcoholic beverages include, but are not limited to, mineral water, near water, sports drinks, tea drinks, milk drinks, coffee drinks, fruit juice drinks, vegetable juice drinks, fruit and vegetable juice drinks, carbonated drinks, etc. Beer drinks with an alcohol content of less than 1%, such as non-alcoholic beer, may also be used.

[0041] The term "tea beverage" in the above-mentioned non-alcoholic beverages refers to beverages made by brewing leaves (tea leaves) from the tea plant, an evergreen tree of the Theaceae family, or leaves of plants other than tea plants, or grains, etc., and includes all types of fermented tea, semi-fermented tea, and unfermented tea. Specific examples of tea beverages include Japanese tea (e.g., green tea, barley tea), black tea, herbal tea (e.g., jasmine tea), Chinese tea (e.g., Chinese green tea, oolong tea), roasted green tea, etc. "Milk beverage" refers to beverages made primarily from raw milk, cow's milk, etc., or foods produced using these as raw materials, and includes not only those made from cow's milk, etc., but also those made from processed milk, such as nutrient-enriched milk, flavored milk, and sweetened hydrolyzed milk.

[0042] In the case of foods and beverages, the daily intake per adult can be adjusted to a range of 6 mg / kg or less, converted to the intake of fumaric acid, for example, a range of 20 to 360 mg for an adult weighing 60 kg. The same applies when the composition is formulated in a container divided into daily meals, and the amount of the composition formulated in the container can be, for example, an amount of fumaric acid in the range of 20 to 360 mg for an adult weighing 60 kg, converted to the intake of fumaric acid per daily meal.

[0043] The present composition in the form of a food or beverage can be produced according to standard manufacturing methods for various foods and beverages, except that it includes a step of adding and blending fumaric acids or a composition containing fumaric acids as one of the ingredients (e.g., as an additive for foods and beverages).

[0044] (B) Application to Quasi-drugs, Pharmaceuticals, or Their Additives One use form of the composition includes quasi-drugs, pharmaceuticals (hereinafter collectively referred to as "pharmaceuticals, etc."), and their additives. These contain, for example, fumaric acids as active ingredients and can be produced by mixing pharmaceutically acceptable carriers or additives such as carriers, excipients, binders, and diluents according to standard methods. These may be in the form of oral or parenteral administration. Oral administration forms include granules, powders, tablets, pills, capsules, syrups, etc. Parenteral administration forms include injections, infusions, nasal administration preparations, and enteral administration (feeding tube administration). However, they are not limited to these. Oral administration forms are preferred.

[0045] Pharmaceutically acceptable carriers include excipients or diluents. In addition to the carrier, additives such as preservatives, stabilizers, binders, pH adjusters, buffers, thickeners, gelling agents, and antioxidants may be added as needed. These additives are preferably those used in pharmaceutical preparations.

[0046] The pharmaceutical agent etc. of the present disclosure may be used in combination with other ingredients that have a circadian rhythm adjusting effect, in particular an effect of advancing the phase of the circadian rhythm and / or an effect of shortening the period.

[0047] The dosage of the pharmaceuticals and the like disclosed herein varies depending on factors such as the dosage form, the patient's age and body weight, and the nature or severity of the symptoms to be treated. However, a preferred range of dosage for a typical adult per day for oral administration is, for example, 20 to 360 mg, converted to the dose of fumaric acid, for an adult weighing 60 kg. The dosage may be appropriately selected depending on various conditions, such as the route of administration. The drug may be administered to a patient once or multiple times daily. The target patient is typically a human, but may also include non-human mammals, such as pet animals such as dogs. The non-human animal is preferably a diurnal mammal, similar to humans.

[0048] (II) Use for producing a composition for circadian rhythm regulation The present disclosure includes the use of fumaric acids or compositions containing the same for producing a composition for circadian rhythm regulation. As described above, a "composition for circadian rhythm regulation" is a composition used to regulate the circadian rhythm or life rhythm of mammals, including humans, and includes non-therapeutic foods and beverages, quasi-drugs, or pharmaceuticals. It also includes additives for preparing the non-therapeutic foods and beverages, quasi-drugs, or pharmaceuticals. "Fumaric acids" and "compositions containing the same" are as described above, and the descriptions in (I) above may be used.

[0049] (III) Compounds for use in circadian rhythm regulation and compositions containing the same The present disclosure includes fumaric acids or compositions containing the same for use in circadian rhythm regulation. "Regulation of circadian rhythm," "fumaric acids," and "compositions containing the same" are as described above, and the descriptions in (I) above can be used. As described in (I), regulation of circadian rhythm includes regulation of daily rhythm.

[0050] (IV) Circadian rhythm adjustment method The present disclosure includes a method for adjusting the circadian rhythm of a mammal, which comprises administering a fumaric acid or a composition containing the same to the mammal. The terms "mammal," "fumaric acid," "composition containing the same," "administration," and "adjustment of circadian rhythm" are as defined above, and the descriptions in (I) above can be used. As described in (I), adjustment of circadian rhythm includes adjustment of daily rhythm.

[0051] As described above, in this specification, the terms "comprise" and "contain" encompass the meanings of "consist of" and "consist essentially of."

[0052] The present invention will be described below using experimental examples to aid in understanding the configuration and effects of the present disclosure. However, the present disclosure is not limited by these experimental examples. Unless otherwise specified, the following experiments were carried out at room temperature (25±5°C) and atmospheric pressure. Unless otherwise specified, "%" and "parts" in the following descriptions mean "% by mass" and "parts by mass," respectively.

[0053] The materials used in the following experimental examples and their sources are as follows: Fumaric acid: Fumaric acid Wako Reagent (Fujifilm Wako Pure Chemical Industries, Ltd.: Product code 069-00652) NanoLuc (registered trademark) luciferase vector: A vector for expressing luciferase derived from Pleurotus erythrorhizus. This is the pNL2.2 NlucP Hygro Vector, which has hygromycin resistance and a pest sequence (Promega Corporation). Transfection reagent 1: ViaFect (trademark) Transfection Reagent (Promega Corporation). U-2 OS cells: An osteosarcoma-derived cell line obtained from a 15-year-old Caucasian female (ATCC (Cat. No. HTB-96)). Endurazine: A substrate for luciferase reporter assays using the NanoLuc (registered trademark) luciferase vector (Promega Corporation).・High glucose medium 1: D-MEM (high glucose) (containing L-glutamine and phenol red) (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.: product code 044-29765) ・High glucose medium 2: D-MEM (high glucose) (containing L-glutamine and HEPES) (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.: product code 044-32955) ・1% penicillin-streptomycin solution (GIBCO 15140): manufactured by Thermo Fisher Scientific ・Long-term real-time luminescence measurement system for cultured cells: WSL-1565, Chronos HT (measurement method: photon counting method using a photomultiplier tube to measure weak luminescence) (manufactured by Ikeda Rika Co., Ltd.) ・Caco-2 cells: epithelial cell line derived from human colon cancer (Abcam)

[0054] Experimental Example 1: Evaluation of circadian rhythm using per2 reporter cells With reference to the "Cell-based circadian assays" described in the paper by H. Oike et al. (Non-Patent Document 1), a circadian rhythm assay (luciferase reporter assay) was performed using per2 reporter cells to evaluate the effect of fumaric acid on circadian rhythm.

[0055] (1) Preparation of per2 reporter cells. A vector incorporating the per2 promoter region into the NanoLuc® luciferase vector was transfected into U-2 OS cells using Transfection Reagent 1 according to standard procedures. After approximately 24 hours, the medium was replaced, and dexamethasone was added to a final concentration of 100 nM for synchronization. After 2 hours, 1% substrate (Endurazine) was added, and luciferase luminescence intensity was measured every 30 minutes using a plate reader with CO2 and temperature control.

[0056] In the above measurements, cells from wells that showed a consistent rhythm in luminescence intensity were grown in a 24-well plate and then seeded (5 × 10 4 The cells were cultured at 1 × 10 cells / 10 cm dish and hygromycin-resistant colonies were formed by hygromycin selection. After 25 days, the colonies formed were collected and seeded onto a 96-well plate (1 × 10 3 Three days later, the luminescence intensity of luciferase was measured every 60 minutes using a plate reader with CO2 control and temperature control, as described above.

[0057] Based on this measurement, cell lines that showed a stable circadian rhythm with stable fermentation intensity were further subjected to limiting dilution and cloning. After that, cell lines with little variation in the circadian rhythm period and a stable period were selected and used as "per2 reporter cells" in the experiments described below.

[0058] (2) Evaluation of circadian rhythm of fumaric acid using per2 reporter cells. The per2 reporter cells prepared above were cultured at 5 × 10 3 The cells were seeded into a 96-well plate at 100 cells / well and cultured in high-glucose medium 1 supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, and hygromycin at 37°C under 5% CO2 conditions.

[0059] After 2 days of incubation, 100 nM dexamethasone was added and the cells were incubated for 2 hours. Then, 1% endurazine was added and the luciferase luminescence intensity was measured every 12 minutes in high-glucose medium 2 supplemented with 5% FBS and 1% penicillin-streptomycin.

[0060] Next, fumaric acid was added to the measurement medium to a final concentration of 5 mM, and the luciferase luminescence intensity was measured over time using a long-term real-time luminescence measurement system for cultured cells (WSL-1565, Chronos HT) to evaluate changes in circadian rhythms. As a control, the luciferase luminescence intensity of per2 reporter cells cultured in measurement medium without fumaric acid was also measured over time.

[0061] The measurement results are shown in Figure 1. In the luminescence intensity rhythm, the troughs are represented by T and the peaks by P, and the order of appearance is P1, T1, P2, and T2. Because the rhythm of P1 is unstable, the circadian rhythm of per2 reporter cells was evaluated from the phases of the troughs (T1 and T2) and peaks (P2) after T1.

[0062] From the measurement results shown in Figure 1, the phase difference (hours) between the circadian rhythm of fumaric acid-treated per2 reporter cells (fumaric acid-treated cells) and the circadian rhythm of control per2 reporter cells (control) is summarized in Table 1.

[0063]

[0064] As shown in Figure 1 and Table 1, it was observed that the addition of fumaric acid advanced the phase of the circadian rhythm compared to the control. This confirmed that fumaric acid has the effect of regulating the circadian rhythm by advancing the phase of the circadian rhythm.

[0065] The results of comparing the circadian rhythm period (hours) of the fumaric acid-treated cells and the control are shown in Table 2. The period was calculated from the phase difference between T1 and T2 (T2-T1).

[0066]

[0067] As shown in Figure 1 and Table 2, it was found that the addition of fumaric acid shortened the period of the circadian rhythm compared to the control. This confirmed that fumaric acid has the effect of regulating the circadian rhythm by shortening the period of the circadian rhythm.

[0068] Experimental Example 2: Induction of GPR41 expression by fumaric acid. Caco-2 cells were cultured at 2 x 10 5 Cells were seeded at 1000 cells / well in a 24-well plate. After 3 weeks of differentiation induction, 5 mM fumaric acid was added, and 60 minutes later, the cells were harvested, RNA was extracted, and GPR41 gene expression was measured by real-time PCR. The results are shown in Figure 2.

[0069] As shown in Figure 2, the addition of fumaric acid increased the expression level of the GPR41 gene. This suggests that fumaric acid may promote GLP-1 secretion through the expression of the GPR41 gene and regulate the circadian rhythm of peripheral tissues mediated by insulin.

[0070] This disclosure provides circadian rhythm regulators that are derived from highly safe food ingredients or have been confirmed to be safe, thereby preventing or improving physical conditions caused by disruption of circadian rhythms, and supporting the maintenance and improvement of health and work efficiency in people who are not yet ill.

Claims

1. A composition for regulating circadian rhythm, comprising as an active ingredient at least one member selected from the group consisting of fumaric acid and its salts.

2. A composition for regulating circadian rhythm according to claim 1, wherein the composition for regulating circadian rhythm is used to advance the phase of the circadian rhythm and / or to shorten the period of the circadian rhythm.

3. A composition for regulating circadian rhythm according to claim 1 or 2, which contains a fermentation product of a fumaric acid producing bacterium or a processed product thereof, containing at least one selected from the group consisting of fumaric acid and its salts.

4. The composition for regulating circadian rhythm according to claim 3, wherein the fumaric acid producing bacterium is at least one lactic acid bacterium selected from the group consisting of Streptococcus thermophilus and Lactiplantibacillus plantarum.

5. A composition for regulating circadian rhythm according to claim 1 or 2, which is a composition for regulating life rhythm.

6. A composition for regulating circadian rhythm according to claim 1 or 2, which is a food or drink, a quasi-drug, a medicine, or an additive therefor.

7. The composition for regulating circadian rhythm according to claim 3, which is a food or drink, a quasi-drug, a medicine, or an additive therefor.

8. A composition for regulating circadian rhythm according to claim 1 or 2, which does not include a composition containing either one or both of the following compounds (1) and (2): (1) a fumarate salt of (1R,2S)-2-(((2,4-dimethylpyrimidin-5-yl)oxy)methyl)-2-(3-fluorophenyl)-N-(5-fluoropyridin-2-yl)cyclopropanecarboxamide, (2) a fumarate salt of ornithine.

Images