Sleep-improving agent

Abstract

To provide a sleep improving agent useful for improving the amount of sleep and the quality of sleep.SOLUTION: The sleep improving agent contains at least one kind selected from metformin, 3,3' - diindolylmethane and their salts as an active ingredient.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a sleep-improving agent. [Background technology]

[0002] Humans spend approximately one-third of their lives sleeping, making it one of the most important human activities. However, many people in modern society suffer from sleep problems. Sleep deprivation has a wide range of effects, including daytime sleepiness and fatigue, increased psychosomatic complaints such as headaches, emotional instability, reduced work efficiency related to impaired attention and judgment, and poor academic performance, and may even lead to serious consequences such as accidents. Furthermore, when various sleep problems, including sleep deprivation, become chronic, they can increase the risk of developing heart disease, cerebrovascular disorders, and other conditions, and worsen their symptoms. Therefore, improving the quantity (sleep time) and quality of sleep is an important health issue.

[0003] A wide variety of sleep-improving materials have been developed to improve sleep. Examples include glycine (Patent Document 1), 3-hydroxybutyric acid (β-hydroxybutyric acid) (Patent Document 2), α-lipoic acid (Patent Document 3), and butyric acid (Patent Document 4). Among these, glycine has been reported to increase sole blood flow and decrease core body temperature in animals (Non-Patent Document 1). It is believed that glycine intake increases peripheral blood flow, enhances heat dissipation, decreases core body temperature, and improves sleep quality. However, the mechanism by which sleep-improving materials exert their effects on sleep remains largely unknown. Sleep-improving drugs such as benzodiazepines and antihistamines act on the central nervous system, and common treatments for improving sleep quality rely on controlling sleep- and wake-related nerves.

[0004] Metformin is an oral drug used to treat type 2 diabetes, and one of the molecular mechanisms underlying its antidiabetic effect is known to be activation of AMP (adenosine monophosphate) kinase (AMPK). AMPK is a key regulator of intracellular energy metabolism and is activated when intracellular energy levels decrease and the intracellular AMP / ATP ratio increases. Known AMPK activators include 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) and 3,3'-diindolylmethane (DIM). Previous reports (Non-Patent Document 1) suggest that increased sleep appetite (the brain state that desires deep sleep, also known as sleep pressure) increases the phosphorylation level of AMPK substrates. This suggests that the AMPK phosphorylation pathway is activated. This suggests that activation of the AMPK phosphorylation pathway may induce deep sleep. However, it has been reported that AICAR does not increase sleep quantity (Non-Patent Document 2), and there have been no reports to date of the sleep-improving effects of other AMPK activators, such as metformin. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Laid-Open No. 2006-333872 [Patent Document 2] Japanese Patent Application Publication No. 2019-172642 [Patent Document 3] Patent No. 6154029 [Patent Document 4] Japanese Patent Application Laid-Open No. 2013-82671 [Non-patent literature]

[0006] [Non-Patent Document 1] Nature 612, 2018, pp.512-518 [Non-patent document 2] Neuropharmacology 57, 2009, p.369-374 Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention relates to providing a sleep-improving agent that is useful for improving the quantity and quality of sleep. [Means for solving the problem]

[0008] The present inventors have found that metformin and 3,3'-diindolylmethane have the effect of improving the quantity and quality of sleep.

[0009] That is, the present invention provides a sleep-improving agent containing at least one active ingredient selected from metformin, 3,3'-diindolylmethane, and salts thereof. [Effects of the Invention]

[0010] According to the present invention, the quantity and quality of sleep can be improved, and therefore the sleep-improving agent of the present invention is useful for maintaining physical and mental health. [Brief explanation of the drawings]

[0011] [Figure 1] Effects of metformin and AICAR exposure on sleep-like behavior in zebrafish larvae. (a) Time course of sleep-like behavior in larvae. The shaded area in the background indicates the dark period (D). The unshaded area indicates the light period (L). (b) The average amount of sleep-like behavior in larvae during the light and dark periods on each day. N=8, Error bars: SEM, †: P<0.1, *: P<0.05, **: P<0.01, Student's T test. [Figure 2]Effects of simultaneous exposure to metformin and dorsomorphin. (a) Time course of sleep-like activity in each group exposed to metformin and dorsomorphin (CC), respectively, and simultaneously. The shaded area in the background indicates the dark period (D). The unshaded area indicates the light period (L). (b) Average sleep-like activity during the light period (L) and dark period (D) on each day. N=8, Error bars: SEM, *: P<0.05, **: P<0.01, Student's T test. [Figure 3] Effect of metformin in a mouse model. A) Transition of non-REM sleep amount up to 8 hours after metformin administration. B) Comparison of non-REM sleep amount, REM sleep amount, and wakefulness amount up to 1-2 hours after administration (*: P<0.05, Welch's t-test). C) Comparison of the time from administration to the appearance of the first non-REM sleep episode (sleep latency) (control N=10, metformin N=12, *: P<0.05, Welch's t-test). DETAILED DESCRIPTION OF THE INVENTION

[0012] In the present invention, at least one compound selected from metformin, 3,3'-diindolylmethane, and salts thereof (hereinafter also referred to as "the compound of the present invention") is used as an active ingredient for improving sleep.

[0013] Metformin used in the present invention is represented by the following formula:

[0014] [ka]

[0015] The 3,3'-diindolylmethane used in the present invention is represented by the following formula:

[0016] [ka]

[0017] The salt of metformin or 3,3′-diindolylmethane is not particularly limited as long as it is a pharmaceutically acceptable salt, and examples thereof include inorganic acid salts such as hydrochloride, sulfate, nitrate, hydrofluoride, and hydrobromide; organic acid salts such as acetate, tartrate, lactate, citrate, fumarate, maleate, succinate, methanesulfonate, ethanesulfonate, benzenesulfonate, toluenesulfonate, naphthalenesulfonate, and camphorsulfonate; metal salts such as sodium salt, potassium salt, lithium salt, calcium salt, and magnesium salt; amine salts such as ammonia, trimethylamine, triethylamine, pyridine, collidine, and lutidine; and organic base salts such as lysine and arginine.

[0018] The compound of the present invention may be a solvate or a non-solvate, and both are included. Preferred examples of solvates include hydrates, alcoholates, acetone solvates, etc. These may be used alone or in combination of two or more.

[0019] The compounds of the present invention are not particularly limited and can be obtained by chemical synthesis or by extraction and purification from natural products containing them, particularly plants, etc. Commercially available reagents can also be used.

[0020] As shown in the Examples below, when sleep evaluation was performed using zebrafish, total sleep time and average sleep duration were calculated, and it was confirmed that the compound of the present invention extended total sleep time and average sleep duration. Furthermore, it was shown that metformin may have a sleep-promoting effect, and that this effect may be mediated by activation of the AMPK pathway. The sleep-promoting effect of metformin was higher than that of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), which is also an AMPK activator like metformin. Furthermore, when metformin was administered intragastrically to mice, sleep quality was evaluated using electroencephalogram (EEG) and electromyogram (EMG) measurements. It was confirmed that the proportion of total sleep time during non-REM sleep (deep sleep), the amount of wakefulness, and sleep latency were reduced. In other words, the compound of the present invention has the effect of improving both the quantity and quality of sleep.

[0021] Therefore, the compound of the present invention can be a sleep improver and can be used to improve the quantity and quality of sleep, and can also be used to produce a sleep improver. Here, "use" may be for use on humans or non-human animals, and may be therapeutic or non-therapeutic use. "Non-therapeutic" is a concept that does not include medical procedures, i.e., methods of surgery, therapy, or diagnosis on humans, and more specifically, does not include methods of surgery, therapy, or diagnosis on humans by physicians, medical professionals, or those under the direction of a physician.

[0022] In this specification, "amount of sleep" means the total amount of sleep (the sum of non-REM sleep and REM sleep), and "improvement in the amount of sleep" means an increase in the total amount of sleep.

[0023] "Sleep quality" refers to overall satisfaction with sleep, and "improved sleep quality" refers to qualitatively improving sleep. Preferably, this refers to observing one or more, preferably two or more, of the following phenomena: an increase in the proportion of non-REM sleep in total sleep, an increase in the duration of non-REM sleep, a decrease in mid-sleep awakenings, and a shortening of sleep latency. "Sleep duration" refers to the continuous time spent in the same sleep stage, "mid-sleep awakenings" refers to the state of waking up between falling asleep and waking up, and "sleep latency" refers to the time it takes to enter sleep, preferably non-REM sleep. "Amelioration" refers to the improvement of a symptom or condition, the prevention or slowing of the worsening of a symptom or condition, or the reversal, prevention, or slowing of the progression of a symptom or condition.

[0024] Methods for assessing the quantity and quality of sleep include the sleep assessment method using zebrafish shown in the Examples below, a method in which electrodes for measuring electroencephalograms and electromyograms are attached to the skull of experimental animals such as rats and mice and measured using wired cables, and a method using a telemetry system in which a transmitter is implanted in the body and information on physiological indices is transmitted wirelessly.In the case of humans, examples include objective assessment methods using non-invasive measurements such as an electroencephalograph, and subjective assessment methods using questionnaires such as a visual analog scale (VAS) or OSA sleep questionnaire (Yamamoto Yukari et al., Brain and Psychiatry Medicine 10:401-409, 1999).

[0025] The sleep-improving agent of the present invention may itself be a pharmaceutical, quasi-drug, cosmetic, food, or feed for improving the quantity and quality of sleep, or it may be a material or preparation to be incorporated into such a pharmaceutical, quasi-drug, cosmetic, food, or feed.

[0026] The pharmaceuticals (including quasi-drugs) contain the compound of the present invention as an active ingredient for improving the quantity and quality of sleep. Furthermore, the pharmaceuticals may contain pharmaceutically acceptable carriers or other active ingredients, medicinal ingredients, etc., as needed, as long as the function of the active ingredient is not impaired.

[0027] Examples of dosage forms of the pharmaceuticals (including quasi-drugs) include oral solid preparations such as tablets (including chewable tablets), capsules, granules, powders, and lozenges; oral liquid preparations such as oral liquids and syrups; and parenteral preparations such as injections, suppositories, inhalants, transdermal agents, and topical agents. The preferred administration form is oral administration.

[0028] The cosmetic contains the compound of the present invention as an active ingredient for improving the quantity and quality of sleep. Furthermore, the cosmetic may contain a cosmetically acceptable carrier, other active ingredients, cosmetic ingredients, etc., as needed, as long as the function of the active ingredient is not impaired. Preferred examples of cosmetics include face and body cosmetics (for example, lotions, gels, creams, packs, etc.), make-up cosmetics, and face or body cleansers.

[0029] Such pharmaceutical and cosmetic preparations can be produced according to conventional methods by combining the compound of the present invention with a pharmaceutically or cosmetically acceptable carrier, the above-mentioned other active ingredients, medicinal ingredients, cosmetic ingredients, etc., as necessary. Examples of the pharmaceutically or cosmetically acceptable carrier include various oils, surfactants, gelling agents, buffers, preservatives, antioxidants, solvents, dispersants, chelating agents, thickeners, UV absorbers, emulsion stabilizers, pH adjusters, pigments, fragrances, etc. Examples of such other active ingredients, medicinal ingredients, and cosmetic ingredients include plant extracts, disinfectants, moisturizers, anti-inflammatory agents, antibacterial agents, keratolytic agents, cooling agents, antiseborrheic agents, cleansers, and makeup ingredients.

[0030] The food contains the compound of the present invention as an active ingredient for improving the quantity and quality of sleep. The food includes foods that claim to improve sleep and have been approved or notified as such (foods for specified health uses, foods with functional claims) as needed. An example of such a claim is "improves sleep quality." Foods approved or notified as functional foods can be distinguished from general foods. The food product may be in the form of a solid, semi-solid, or liquid (e.g., a beverage). Examples include various food compositions (breads, cakes, noodles, confectioneries, frozen foods, ice creams, candies, toppings, soups, dairy products, shakes, beverages, seasonings, etc.), as well as nutritional supplement compositions in the same form as the oral preparations described above (solid preparations such as granules, powders, tablets, capsules, microcapsules, and lozenges).

[0031] Foods in various forms can be prepared according to standard methods by appropriately combining the compound of the present invention with any food material, other active ingredient, or additives acceptable for food (e.g., solvents, softeners, oils, emulsifiers, preservatives, acidulants, sweeteners, bittering agents, pH adjusters, stabilizers, colorants, UV absorbers, antioxidants, moisturizers, thickeners, adhesives, dispersants, flow improvers, humectants, aromatics, seasonings, flavor adjusters), etc.

[0032] The feed contains the compound of the present invention as an active ingredient for improving the quantity and quality of sleep. The feed is preferably in the form of pellets, flakes, mash, or liquid, and examples thereof include livestock feed for cows, pigs, chickens, sheep, horses, etc., small animal feed for rabbits, rats, mice, etc., and pet food for dogs, cats, small birds, etc. The feed can be prepared according to a standard method by appropriately combining the compound of the present invention with other feed ingredients such as meat, protein, grains, bran, lees, sugars, vegetables, vitamins, minerals, gelling agents, shape-retaining agents, pH adjusters, seasonings, preservatives, nutritional supplements, etc.

[0033] The content of the compound of the present invention in the sleep-improving agent of the present invention may vary depending on the dosage form or shape, but can be appropriately set taking into consideration ease of administration or ingestion, etc. For example, in the case of an orally administered formulation, the total amount of the compound of the present invention is preferably 0.5% by mass or more and 99.8% by mass or less, based on the total amount of the formulation.

[0034] The dosage or intake of the sleep-improving agent of the present invention may vary depending on the species, body weight, sex, age, condition, or other factors of the subject to be administered or ingested. The dosage, route, and interval of administration, as well as the amount and interval of intake, can be appropriately determined by those skilled in the art. In the case of oral administration or oral intake, the total amount of the compound of the present invention per day for an adult (body weight 60 kg) is preferably 0.3 g to 1.8 g, more preferably 0.5 g to 1.2 g.

[0035] In the present invention, the above-mentioned dose is preferably administered or taken, for example, once, twice, or three or more times a day. The administration or intake period is not particularly limited, but is preferably continuous, more preferably one week or more, and even more preferably two weeks or more. The timing of administration or ingestion is preferably between after dinner and bedtime, more preferably 30 minutes to 3 hours, and even more preferably 1 to 2 hours before the desired time to sleep.

[0036] The subjects to be administered or ingested with the sleep-improving agent of the present invention are preferably humans or non-human animals who need or desire to improve the amount and quality of sleep. More preferably, the agent is effective for ingestion or administration to humans or non-human animals whose amount or quality of sleep is reduced, healthy humans or non-human animals who need better quality sleep, or humans or non-human animals with sleep disorders. [Example]

[0037] Example 1 - Screening method - Animals used and breeding After collecting fertilized eggs, RW strain zebrafish were reared in an incubator at 28°C in egg water under a 14-h light / 10-h dark cycle (LD cycle 9 am / 7 pm). Five-day-postfertilization (5 dpf) fry, which had established a day-night rhythm, were used for the experiments.

[0038] Examination of conditions for measuring sleep in zebrafish Five-dpf juvenile fish were placed in a 48-well plate, one per well, and their behavior was observed in 500 μL of egg water per well. The fish were placed in a DanioVision (Noldus Information Technology) camera between 3:00 PM and 5:00 PM. Images were continuously captured from 9:00 PM on the day (5 dpf) until 9:00 PM (5-dpf light period), through the dark period (5-dpf dark period), the next day's light period (6-dpf light period), and the subsequent dark period (6-dpf dark period) at a sampling rate of 15 frames / sec and an illuminance of 900 lux during the light period. The fish's movements were detected using a background subtraction object detection algorithm. The tracking data of the detected juvenile centroids were analyzed under the following conditions to determine sleepiness. The minimum distance movement (MDM) limit for fry tracking data was set to 1 mm, and distance fluctuations below the MDM were not detected as movement. The movement detection threshold for detecting fry movement was set at 2 mm / s for the start velocity of the fry's center of mass and 0.1 mm / s for the end velocity. When the center of mass of the fry moved at a velocity of 2 mm / s or greater, the fry were deemed to be moving. When the velocity subsequently decelerated to 0.1 mm / s or less, the fry were deemed to have stopped moving (immobile). The data acquisition time was divided into epochs, and the number of times fry movement was detected per minute was tallied. The criteria for determining whether fry were awake or asleep were set as follows: if movement was detected at least once per minute, the fry were deemed to be awake during that epoch; if no movement was detected, the fry were deemed to be asleep during that epoch. The number of epochs judged as sleep per hour was defined as the total sleep time, and the average total sleep time was calculated for each of the 5-dpf dark period, 6-dpf light period, and 6-dpf dark period. In addition, the average number of consecutive epochs determined to be sleep was calculated, and this was taken as the average sleep duration.

[0039] Compound exposure screening Five-dpf larvae were placed one per well in a 48-well plate and exposed to the compounds by replacing the water with 500 μL of the following compounds (0.04% sodium chloride) per well: 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR, Acadesine), adenosine, α-lipoic acid, 3,3'-diindolylmethane (DIM), glycine, metformin (Fujifilm Wako Pure Chemical Industries, Ltd., 136-18662), and caffeine (negative control). The exposed larvae were placed in a DanioVision (Noldus) monitor set at 28°C between 3:00 PM and 5:00 PM. The light period was defined as 9:00 PM on the day of placement, and tracking data were collected from 9:00 PM onward during a 10-h dark, 14-h light, and 10-h dark period. The tracking sampling rate was 15.00 samples / sec, and the light intensity was 900 lux. For compounds diluted with DMSO, the DMSO concentration was set to 0.1% (vol / vol) or less when dissolved in the larval rearing water. Compounds were evaluated based on the total sleep time (min / h) calculated from the number of epochs judged as sleep during the dark and light periods, and the average sleep duration (min) calculated from the number of consecutive epochs judged as sleep. Two tests with n = 4 for each compound were conducted for a total of n = 8, and the sleep-promoting effect of each test was examined relative to the control group (solvent control).

[0040] As a result, as shown in Table 1, α-lipoic acid, 3,3'-diindolylmethane (DIM), glycine, and metformin were found to extend total sleep time or average duration.

[0041] [Table 1]

[0042] Example 2 - Difference between metformin and AICAR - method -Animals used and their care Wild-type zebrafish (RW strain) were used for the experiment. Mature zebrafish were mated, and fertilized eggs were collected. They were then reared in larval rearing water (0.04% sodium chloride) under a 14-h light (ZT0-14) and 10-h dark (ZT14-24 (ZT0)) cycle at 28°C.

[0043] - Compound exposure Five-dpf larvae were placed in a 48-well plate with their rearing water, one per well. The rearing water was replaced with 500 μL / well of rearing water containing the following compounds at the desired concentrations. 5 mM metformin and 500 μM AICAR were used as AMPK pathway promoters, and their effects on sleep-like behavior in larvae were examined. DMSO was added to each test solution, including the control solution, to a final concentration of 0.5% (vol / vol). Eight larvae were used per group. Larvae were exposed to the compounds between 4 and 2 hours before the onset of the dark period, and then subjected to behavioral analysis.

[0044] -Behavior analysis The 48-well plate containing the larvae was placed in a DanioVision (Noldus Information Technology) camera and continuously photographed under the same light-dark cycle and temperature conditions as those used during rearing, with a sampling rate of 15 frames / sec and 900 lux illuminance during the light period. Larval movement was detected using a background subtraction object detection algorithm. The detected larval centroid tracking data was analyzed under the following conditions: the minimum distance movement (MDM) was set to 1 mm, and distance fluctuations below the MDM were not detected as movement. Movement was determined to begin when the larval centroid moved at a speed of 2.0 mm / s or faster, and to cease when the speed subsequently decelerated to 1.0 mm / s or slower. The analysis period spanned from the start of the first dark period (Day 1 D) set in the camera to 6 hours of the light period three days later (Day 4 L). The analysis range was divided into 1-minute epochs, and epochs in which no movement was detected were defined as sleep bouts, which were sleep-like behaviors of zebrafish larvae.

[0045] result As shown in Figure 1, when we examined the effects of metformin and AICAR exposure on sleep-like behavior in larvae, metformin alone increased sleep-like behavior in larvae, particularly during the dark period. During the dark period on the day of exposure (Day 1 D), the metformin-exposed group had significantly higher mean sleep-like behavior than the DMSO-only control group and the AICAR-exposed group. These results suggest that metformin may have a sleep-promoting effect.

[0046] Example 3 - Effect of combined use of metformin and AMPK inhibitor - method -Animals used and their care Wild-type zebrafish of the RW strain were used in the experiment and were reared in the same manner as in Example 1.

[0047] - Compound exposure Five-dpf larvae were placed in a 48-well plate with rearing water at one fish per well, and compound exposure was performed by replacing the rearing water with 500 μL / well of the compound at the desired concentration. Single or mixed solutions were prepared with metformin at a final concentration of 5 mM and dorsomorphin (CC), an AMPK inhibitor, at a final concentration of 2 μM to examine the effects on sleep-like behavior in larvae. DMSO in each test solution, including the control, was adjusted to a final concentration of 0.2% (vol / vol). Eight fish per group were tested. Larvae were exposed to the compound between 4 and 2 hours before the onset of the dark period, and then subjected to behavioral analysis.

[0048] -Behavior analysis The photographing of the larvae and the analysis of sleep-like behavior were carried out in the same manner as in Example 1.

[0049] result As shown in Figure 2, similar to Example 1, exposure to metformin alone promoted sleep-like behavior in zebrafish larvae, particularly during the dark phase. Compared with the metformin-only exposure group, the groups exposed to the AMPK inhibitor dorsomorphin alone and those exposed simultaneously with metformin showed significantly lower mean sleep-like behavior during the dark phase. However, no significant difference in mean sleep-like behavior was observed between the dorsomorphin-exposed group and the control group. These results suggest that the sleep-promoting effect of metformin may be mediated by activation of the AMPK pathway.

[0050] Example 4 - Effect of metformin in mice - Animals used and breeding Male C57BL / 6J (Japan SLC) mice aged 7-15 weeks were used in this study. Mice were housed at a room temperature of 23±2°C, humidity of 55±10%, and under a 12-hour light-dark cycle (light period: 7:00 am - 7:00 pm). During a pre-breeding period of at least one week, mice had free access to water and CE-2 solid animal diet (Japan CLEA). The study was approved by the Animal Care and Use Committee of Kao Corporation's Tochigi Research Center, which strictly adheres to Japan's Animal Protection Law.

[0051] Placement of EEG and EMG electrodes Mice under deep isoflurane anesthesia were secured in a stereotaxic frame (Stoeling). The hair on the parietal region was shaved and the skin incised. Stainless steel screw electrodes (Biotex) for measuring electrical potential were implanted at coordinates 6.5 mm caudal to the Bregma (the intersection of the sagittal and coronal sutures) (AP, -6.5 mm; ML, ±0 mm) and 1 mm rostral and 1.5 mm rostral to the Lambda (the intersection of the parietal and occipital bones) (AP, approximately -3.0-3.4 mm; ML, +1.5 mm). A Teflon-coated stainless steel wire electrode was placed in the trapezius muscle. The electrode bases were fixed to the head with dental cement (Super-Bond C&B, Sun Medical).

[0052] EEG recording and sleep analysis Mice were transferred to a sleep chamber, and measurement cables (Biotex) were attached to the electrodes. The chamber was set up for a 12-hour light / dark cycle of 9:00 am / 9:00 pm, and allowed to acclimate for at least 5 days. During acclimatization and measurement, the mice were housed on wooden chips (Aspen Chip, Japan Claire) as bedding. Electroencephalograms (EEG) and electromyograms (EMG) were recorded using a VitalRecorder (Kissei Comtec) at a sampling rate of 256 Hz for 24 hours. Data were imported into SleepSign (Kissei Comtec), and EEG signals were subjected to fast Fourier transform (FFT) with a Hanning window, 1024 sampling points, and 4 averaging. The absolute delta (0.5-4 Hz) spectral power (μV²), theta (6-10 Hz) power ratio to delta power (%), and the integrated EMG signal (μV·s) for each 4-second epoch were used to manually classify subjects into REM sleep, non-REM sleep, and wakefulness. The recorded EEG / EMG data were analyzed for 24 hours, starting immediately after oral administration. Sleep latency was calculated as the time from immediately after administration to the onset of the first non-REM sleep.

[0053] Administration Metformin was dissolved in a 0.1% carboxymethylcellulose solution (039-01335, Fujifilm Wako Pure Chemical Industries, Ltd.) and administered intragastrically to mice at 250 mg / 10 mL / kg body weight using an oral gavage. A 0.1% carboxymethylcellulose solution was also administered intragastrically as a control. The test substances were administered between 08:30 and 09:00, just before the light phase began. The same mice were repeatedly administered either the control or metformin, with a three-day washout period between administrations. The EEG measurement cable was disconnected from the measurement device during administration and reconnected after administration.

[0054] result We analyzed the time course of non-REM sleep, REM sleep, and total sleep (the sum of non-REM and REM sleep) every hour after metformin administration to assess sleep quality. The results showed that metformin significantly increased non-REM sleep and total sleep 1-2 hours after administration compared with the vehicle control (Figure 3A). No change was observed in REM sleep quality. Next, we calculated the amount of sleep and wakefulness over the 24 hours after administration. The results showed that metformin significantly increased total non-REM sleep compared to the control group (Figure 3B). Accordingly, the amount of wakefulness also significantly decreased with metformin administration. However, no significant differences were observed in total REM sleep between the two groups. We also calculated the sleep latency (the time from administration to the onset of non-REM sleep) and found that metformin administration significantly shortened sleep latency compared to the control group (Figure 3C).

Claims

1. A sleep-improving agent containing at least one active ingredient selected from metformin, 3,3'-diindolylmethane, and salts thereof.

2. 2. The sleep-improving agent according to claim 1, wherein the improvement in sleep is an increase in total sleep amount, an increase in the proportion of non-REM sleep amount in total sleep amount, an extension of non-REM sleep duration, a decrease in mid-sleep awakenings, or a shortening of sleep latency.

3. The sleep-improving agent according to claim 1 or 2, wherein the improvement of sleep is mediated by activation and modification of the AMPK phosphorylation pathway.

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