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 an advanced sleep-phase type circadian rhythm disturbance. A composition according to the present disclosure includes at least one substance selected from the group consisting of maleic acid, salts thereof, and anhydrides 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 advanced sleep phase 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 fluctuations in daily rhythms caused by shift work, differences in wake-up / bedtimes between weekends and weekdays, and differences in sleep duration. 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 lead to mental and physical disorders such as exacerbation of metabolic syndrome symptoms, increased depression, and sleep 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 of sleep and the time of wake-up differ each day. 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):3-27

[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 advanced sleep phase 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 the circadian rhythm period) 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 maleic acid, a low-molecular-weight organic acid, has the effect of regulating the circadian rhythm, particularly by delaying the phase of the circadian rhythm and extending the period of the circadian rhythm.

[0010] The present disclosure was completed based on these findings and through further research, and includes the following embodiments: (I) Composition for regulating circadian rhythm (I-1) A composition for regulating circadian rhythm, comprising as an active ingredient at least one compound selected from the group consisting of maleic acid, its salts, and its anhydrides. (I-2) The composition for regulating circadian rhythm described in (I-1), which is used to delay the phase of a circadian rhythm and / or to extend the period of a circadian rhythm. (I-3) The composition for regulating circadian rhythm described in (I-1) or (I-2), which is a composition for regulating daily rhythms. (I-4) The composition for regulating circadian rhythm described in any of (I-1) to (I-3), which is in the form of a food or drink, a quasi-drug, a pharmaceutical, or an additive therefor. (I-5) A composition for regulating circadian rhythm according to any one of (I-1) to (I-4), which does not contain the following compounds (1) and / or (2): (1) (1R,2S)-2-(((2,4-dimethylpyrimidin-5-yl)oxy)methyl)-2-(3-fluorophenyl)-N-(5-fluoropyridin-2-yl)cyclopropanecarboxamide maleate, (2) ornithine maleate.

[0011] (II) Use for producing a composition for regulating circadian rhythm (II-1) Use of at least one compound selected from the group consisting of maleic acid, its salts, and its anhydrides, or a composition containing same, for producing a composition for regulating circadian rhythm. (II-2) The use according to (II-1), wherein the composition for regulating circadian rhythm is used to delay the phase of a circadian rhythm and / or to extend the period of a circadian rhythm. (II-3) The use according to (II-1) or (II-2), 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-4) The method for adjusting circadian rhythm described in (II-3), wherein the healthy or pre-illness human is a human having at least one symptom selected from the group consisting of 1) to 5) below: 1) a person who may develop a circadian rhythm disorder, 2) a person who has a circadian rhythm sleep disorder, 3) a person whose circadian rhythm is desynchronized, 4) an elderly person who is prone to advancement of the phase of the circadian rhythm and / or shortening of the period due to aging, 5) a person who is in poor health due to a disruption of the circadian rhythm. (II-5) The use according to any one of (II-1) to (II-4), wherein the compound and composition do not contain the following compounds (1) and / or (2): (1) (1R,2S)-2-(((2,4-dimethylpyrimidin-5-yl)oxy)methyl)-2-(3-fluorophenyl)-N-(5-fluoropyridin-2-yl)cyclopropanecarboxamide maleate, (2) ornithine maleate.

[0012] (III) Method for adjusting circadian rhythm (III-1) A method for adjusting the circadian rhythm of a mammal, comprising administering to the mammal at least one compound selected from the group consisting of maleic acid, its salts, and its anhydrides, or a composition containing the same. (III-2) The method according to (III-1), wherein the adjustment of the circadian rhythm is an adjustment that delays the phase of the circadian rhythm and / or an adjustment that extends the period of the circadian rhythm. (III-3) The method for adjusting the circadian rhythm according to (III-1) or (III-2), wherein the mammal is a human or non-human animal in a healthy or pre-disease state, and the method is a non-therapeutic method for the mammal. (III-4) A method for adjusting circadian rhythm according to any one of (III-1) to (III-3), wherein the mammal is a human having at least one symptom selected from the group consisting of 1) to 5) below: 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) an elderly person who is prone to the advancement of the phase of the circadian rhythm and / or the shortening of the period due to aging, and 5) a person who is in poor health due to a disruption of the circadian rhythm. (III-5) A method for regulating circadian rhythm according to any one of (III-1) to (III-4), wherein the compound and composition do not contain the following compounds (1) and / or (2): (1) (1R,2S)-2-(((2,4-dimethylpyrimidin-5-yl)oxy)methyl)-2-(3-fluorophenyl)-N-(5-fluoropyridin-2-yl)cyclopropanecarboxamide maleate, (2) ornithine maleate.

[0013] 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 disorders caused by advanced sleep phase by delaying the phase of the circadian rhythm and / or lengthening the period of the circadian rhythm. Therefore, according to the present disclosure, it is expected that circadian rhythm sleep disorders such as advanced sleep phase syndrome caused by circadian rhythm disorders, lifestyle rhythm disorders caused by circadian rhythm disorders, and poor physical condition caused by circadian rhythm or lifestyle rhythm disorders can be improved.

[0014] In Experimental Example 1, the luciferase luminescence intensity was measured over time using per2 reporter cells with or without added maleic acid (indicated as "Maleic acid" in the figure) and the results of evaluating changes in circadian rhythm are shown.

[0015] (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 maleic acid, its salts, and its anhydrides. Hereinafter, maleic acid, its salts, and its anhydrides are also collectively referred to as "maleic acids."

[0016] The salt of maleic 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 maleic acid with a metal ion, such as a metal maleate (e.g., a combined salt of maleic acid with an alkali metal such as sodium, potassium, or lithium; an alkaline earth metal maleate (e.g., a combined salt of maleic acid with an alkaline earth metal such as magnesium or calcium); a hydrogen metal maleate (e.g., a combined salt of maleic acid with a hydrogen metal such as sodium hydrogen or potassium hydrogen); and a combined salt of maleic acid with an ammonium salt (e.g., an ammonium salt, a tetramethylammonium salt).

[0017] However, the salts of maleic acid disclosed herein do not include the following compound: maleate salt of (1R,2S)-2-(((2,4-dimethylpyrimidin-5-yl)oxy)methyl)-2-(3-fluorophenyl)-N-(5-fluoropyridin-2-yl)cyclopropanecarboxamide. The above compound is the fumarate salt of lemborexant (generic name) represented by the following formula (I):

[0018]

[0019] The anhydride of maleic acid, i.e., maleic anhydride, is rapidly hydrolyzed to maleic acid in water and can be used as an active ingredient in the present compositions in the same manner as maleic acid.

[0020] The present composition is used to regulate the circadian rhythm of mammals, including humans. In this disclosure, the term "regulation" also includes the meanings of "adjustment" and "control." 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.), Bmal (Bmal genes: e.g., Bmal1, etc.), Cryptochrome (cry genes: e.g., cry1, cry2, etc.), and Clock, Dec, and Casein Kinase 1 (CK1 genes: CK1ε, CK1δ, etc.).

[0021] The clock gene targeted by the present disclosure is preferably the per2 gene. Circadian rhythms are based on a negative feedback loop mechanism between the clock genes, with the per2 gene playing a central role in this mechanism (Non-Patent Document 1). 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.

[0022] 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."

[0023] "Adjusting the phase of a circadian rhythm" includes adjusting the phase of the expression rhythm of a clock gene to adjust the light-dark phase of the circadian rhythm. The clock gene referred to here is preferably the per2 gene. "Adjusting the phase of a clock gene expression rhythm" refers to adjusting the temporal change in the phase of the periodic expression rhythm of the clock gene, which is controlled by the activation and / or repression of the expression rhythm at regular intervals. As shown in Experimental Example 1 described below, maleic acids, the active ingredients of the present composition, have the effect of adjusting the phase of the expression rhythm of a clock gene (per2 gene) by delaying the phase of the expression rhythm of the gene chronologically (i.e., along the time axis). This is also referred to as "phase delay effect." Based on the phase delay effect of maleic acid or a salt thereof, the present composition can delay (delay) the phase of the expression rhythm of the clock gene (per2 gene) along the time axis, thereby delaying (delaying) the light-dark phase of the circadian rhythm along the time axis, thereby adjusting the circadian rhythm.

[0024] "Adjusting the period of the circadian rhythm" includes adjusting the light-dark cycle of the circadian rhythm by adjusting the length of each period of the expression rhythm of the clock gene. The clock gene referred to here is preferably the per2 gene. "Period" refers to the time or period (repetition time interval) until a cyclical phenomenon or behavior completes one cycle and returns to the same state as before.

[0025] "Adjusting the period 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, whose expression is controlled by activating and / or repressing the expression at regular intervals. As shown in Experimental Example 1 described below, maleic acids, which are the active ingredients of the present composition, have the effect of adjusting the period of the expression rhythm by lengthening the length of one cycle of the expression rhythm of the clock gene (per2 gene). This is also referred to as "period-extending effect." Based on the period-extending effect of maleic acids, the present composition can extend the period of the expression rhythm of the clock gene (per2 gene), thereby extending the light-dark cycle of the circadian rhythm, thereby adjusting the circadian rhythm.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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 night owls or other reasons; 2) individuals with circadian rhythm sleep disorders, such as advanced sleep phase syndrome; 3) individuals whose circadian rhythms are desynchronized due to blindness or other reasons; 4) elderly individuals prone to aging-related circadian rhythm phase advancement and / or period shortening; and 5) individuals experiencing physical discomfort due to circadian rhythm disturbances, particularly circadian rhythm phase advancement and / or period shortening. The physical discomforts in 5) include physical discomforts such as drowsiness, headache, dull headache, loss of appetite, fatigue, lethargy, metabolic disturbances, endocrine disorders, and / or gastrointestinal dysfunction. These discomforts also include mental discomforts such as decreased concentration or motivation, depression, anxiety, and / or gloominess. These humans include those who are healthy or in a non-diseased state.

[0030] 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.

[0031] The origin of the maleic acids used as the active ingredient of the present composition is not particularly limited, and they may be chemically synthesized products or products extracted and isolated from natural products (animals, plants, microorganisms, and products produced by them). Furthermore, the maleic acids incorporated into the present composition may be crudely purified, regardless of their purity, as long as the effects of the present disclosure are not impaired. For example, as long as the effects of the present disclosure are not impaired, a maleic acid-containing product prepared using a microorganism can be used as is as the active ingredient of the circadian rhythm regulation composition of the present disclosure.

[0032] The content of maleic acids in the composition can be appropriately selected, for example, from a range of 0.1 to 99% by mass, as long as the composition still exhibits its circadian rhythm-regulating effect. The circadian rhythm-regulating effect of the composition can be confirmed by the method described in Experimental Example 1 below. Specifically, the effect of a candidate composition on the expression cycle of a reporter gene (e.g., a luciferase gene) whose expression is controlled by a clock gene promoter (e.g., the per2 promoter) can be evaluated using cultured cells (e.g., human osteosarcoma-derived cells) transfected with the candidate composition. If the expression phase of the reporter gene is delayed and / or the expression cycle is lengthened 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 effect.

[0033] (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 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 which specific ingredients (maleic acids) are concentrated or formulated. Supplements include tablets produced by kneading maleic acids with excipients, binders, etc., and then tableting, as well as powders, pills, capsules, jellies, granules, and other forms. In addition to maleic acids, the present composition may contain, as appropriate, food-grade (edible) carbohydrates, proteins, lipids, vitamins, minerals, sugars (glucose, etc.), natural or artificial sweeteners, citric acid, carbonated water, fruit juice, stabilizers, preservatives, binders, thickeners, and / or emulsifiers.

[0034] 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.

[0035] 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.

[0036] In the case of foods and beverages, the daily intake per adult can be adjusted to a range of 0.53 mg / kg or less, converted into the intake of maleic acid, for example, a range of 1.7 to 30 mg for an adult weighing 60 kg. The same applies when the present composition is formulated in a container divided into daily meals, and the amount of the present composition formulated in the container can be, for example, an amount that contains 1.7 to 30 mg of maleic acid, converted into the intake of maleic acid per daily meal for an adult weighing 60 kg.

[0037] The present composition in the form of a food or beverage can be produced according to standard production methods for various foods and beverages, except that it includes a step of adding maleic acids or a composition containing maleic acids as one of the ingredients (e.g., as an additive for foods and beverages).

[0038] (B) Application to Quasi-drugs, Pharmaceuticals, or Additives Thereof Examples of use of the present composition include quasi-drugs, pharmaceuticals (hereinafter collectively referred to as "pharmaceuticals, etc."), and additives therefor. These contain, for example, maleic 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, and the like. Parenteral administration forms include injections, infusions, nasal administration preparations, enteral administration (feeding tube administration), and the like, but are not limited thereto. Oral administration forms are preferred.

[0039] 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.

[0040] 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 delaying the phase of the circadian rhythm and / or an effect of lengthening the period.

[0041] 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, 1.7 to 30 mg, converted to the dosage of maleic acid, for an adult weighing 60 kg. The dosage may be appropriately selected depending on various conditions, such as the route of administration. The dosage may be administered to the patient once a day or in multiple divided doses. The target patient is typically a human, but also includes non-human mammals, such as pet animals such as dogs. The non-human animal is preferably a diurnal mammal, like humans.

[0042] (II) Use for producing a composition for circadian rhythm regulation The present disclosure includes the use of maleic 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 foods and beverages, quasi-drugs, and pharmaceuticals. It also includes additives for preparing the foods and beverages, quasi-drugs, or pharmaceuticals. The "maleic acids" and "compositions containing the same" are as described above, and the descriptions in (I) above can be used.

[0043] (III) Circadian rhythm regulation method The present disclosure includes a method for regulating the circadian rhythm of a mammal, which comprises administering a maleic acid or a composition containing the same to the mammal. The terms "mammal," "maleic acid," "composition containing the same," "administration," and "regulation of circadian rhythm" are as defined above, and the descriptions in (I) above can be used. As described in (I), circadian rhythm regulation includes regulation of daily rhythm.

[0044] As described above, in this specification, the terms "comprise" and "contain" encompass the meanings of "consist of" and "consist essentially of."

[0045] 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.

[0046] The materials used in the following experimental examples and their sources are as follows: Maleic acid: Wako Special Grade Maleic Acid (Fujifilm Wako Pure Chemical Industries, Ltd.: Product Code 130-00492) 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) (Fujifilm Wako Pure Chemical Industries, Ltd.: product code 044-29765) ・High glucose medium 2: D-MEM (high glucose) (containing L-glutamine and HEPES) (Fujifilm Wako Pure Chemical Industries, Ltd.: product code 044-32955) ・1% penicillin-streptomycin solution (GIBCO 15140): 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) (Ikeda Rika Co., Ltd.)

[0047] 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 maleic acid on circadian rhythm.

[0048] (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.

[0049] 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.

[0050] 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.

[0051] (2) Evaluation of circadian rhythm of maleic 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.

[0052] 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.

[0053] Next, maleic 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 to evaluate changes in circadian rhythms. As a control, the luciferase luminescence intensity of per2 reporter cells cultured in measurement medium without maleic acid was also measured over time.

[0054] 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, T2, and P3. 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 and P3) after T1.

[0055] From the measurement results shown in Figure 1, the phase difference (hours) between the circadian rhythm of maleic acid-added per2 reporter cells (maleic acid-added cells) and the circadian rhythm of control per2 reporter cells (control) is summarized in Table 1.

[0056]

[0057] As shown in Figure 1 and Table 1, it was observed that the addition of maleic acid delayed the phase of the circadian rhythm compared to the control. This confirmed that maleic acid has the effect of regulating the circadian rhythm by delaying the phase of the circadian rhythm.

[0058] The results of comparing the circadian rhythm period (hours) of maleic acid-treated cells with that of the control are shown in Table 2. The period was calculated from the phase difference between T1 and T2 (T2-T1) and the phase difference between P2 and P3 (P3-P2).

[0059]

[0060] As shown in Figure 1 and Table 2, it was found that the period of the circadian rhythm was lengthened by the addition of maleic acid compared to the control. This confirmed that maleic acid has the effect of regulating the circadian rhythm by extending the period of the circadian rhythm.

[0061] 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 compound selected from the group consisting of maleic acid, its salts, and its anhydrides.

2. A composition for regulating circadian rhythm according to claim 1, which is used to delay the phase of a circadian rhythm and / or to extend the period of a circadian rhythm.

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

4. 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.

5. 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.

6. A composition for regulating circadian rhythm according to claim 1 or 2, which does not include a composition containing a maleate salt of (1R,2S)-2-(((2,4-dimethylpyrimidin-5-yl)oxy)methyl)-2-(3-fluorophenyl)-N-(5-fluoropyridin-2-yl)cyclopropanecarboxamide.