Sleep-improving agent

Abstract

This sleep-improving agent contains one peptide among the following (I)-(IV). (I) is a peptide having an amino acid sequence of a prepro-orexin C-terminal peptide. (II) is a peptide that shares an amino acid sequence identity of at least 90% with the amino acid sequence of the (I) prepro-orexin C-terminal peptide. (III) is a peptide that is a part of the amino acid sequence of the (I) prepro-orexin C-terminal peptide. (IV) is a mutant form of the (I) or (III) peptide in which one or two amino acids are deleted, substituted, added, or inserted in the (I) or (III) peptide.

Description

Sleep aids

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

[0002] The sleep-wake cycle is the basis of biological rhythms, and its disruption not only leads to sleep disorders but also to various diseases such as depression and diabetes. The hypothalamic orexin system secretes the neuropeptides orexin A and orexin B, which play an essential role in maintaining wakefulness through activation of orexin-1 and orexin-2 receptors in the brain. Therefore, orexin deficiency leads to hypersomnia (narcolepsy). Therefore, drugs that enhance the wake-maintaining function of orexin may be effective in treating hypersomnia. On the other hand, antagonists that inhibit both orexin-1 and orexin-2 receptors (e.g., suvorexant and lemborexant) suppress wakefulness and induce non-REM and REM sleep, and are therefore clinically used to treat insomnia. Drugs that inhibit orexin activity without orexin receptor involvement (e.g., physiological antagonists) may also be effective in treating insomnia.

[0003] Preproorexin, expressed in hypothalamic neurons in the brain, is fragmented by post-translational modification to produce orexin A, orexin B, and a peptide consisting of 31 amino acids in their C-terminal region (orexin C). In an assay system using HEK293 cells and CHO cells overexpressing orexin receptors, orexin A and orexin B were identified as physiologically active substances (Non-Patent Document 1). This document did not evaluate the activity of orexin C in detail, and it was considered not to be a physiologically active peptide. Previously, the present inventors discovered that the preproorexin C-terminal peptide (orexin C) increases brain-derived neurotrophic factor in cerebral cortical neurons and centrally suppresses feeding in mice, demonstrating that it is an active peptide (Patent Document 1).

[0004] However, there are no reports examining the involvement of orexin C in sleep and wakefulness.

[0005] Japan Patent Application Publication No. 2023-135605

[0006] Cell 1998;92:573-585

[0007] The problem to be solved by the present invention is to provide a sleep-improving agent containing, as an active ingredient, a substance that exhibits the effect of improving sleep.

[0008] The present invention encompasses the following embodiments. Item 1. A sleep-improving agent containing any one of the following peptides (I) to (IV): (I) a peptide having the amino acid sequence of a preproorexin C-terminal peptide; (II) a peptide having 90% or more amino acid sequence identity to the amino acid sequence of the preproorexin C-terminal peptide of (I); (III) a peptide that is a part of the amino acid sequence of the preproorexin C-terminal peptide of (I); (IV) a mutant of the peptide of (I) or (III) in which one or two amino acids have been deleted, substituted, added, or inserted relative to the peptide of (I) or (III). Item 2. The sleep-improving agent of Item 1, wherein the peptide of (I) has the amino acid sequence represented by any one of SEQ ID NOs: 1 to 3. Item 3. The sleep-improving agent of Item 1, which is a sleep-inducing agent. Item 4. The sleep-improving agent of Item 1, wherein the sleep improvement includes improvement in sleep depth or improvement in the ability to fall asleep. Item 5. The sleep-improving agent of Item 1, which is an agent for preventing or treating a sleep disorder. Item 6. Item 6. The sleep-improving agent according to any one of Items 1 to 5, in the form of a pharmaceutical composition. Item 7. The sleep-improving agent according to any one of Items 1 to 5, in the form of a food composition. Item 8. The sleep-improving agent according to any one of Items 1 to 5, in the form of a feed. Item 9. A method for improving sleep (excluding methods for treating humans), comprising administering to a subject any one of the following peptides (I) to (IV): (I) a peptide having the amino acid sequence of a preproorexin C-terminal peptide; (II) a peptide having 90% or more amino acid sequence identity to the amino acid sequence of the preproorexin C-terminal peptide of (I); (III) a peptide that is a part of the amino acid sequence of the preproorexin C-terminal peptide of (I); or (IV) a variant of the peptide of (I) or (III), in which one or two amino acids are deleted, substituted, added, or inserted relative to the peptide of (I) or (III). Item 10. The method according to Item 9, in which the sleep improvement includes prevention or treatment of a sleep disorder.

[0009] According to the sleep-improving agent of the present invention, sleep disorders are improved by containing orexin C, a mutant peptide thereof, or a partial peptide thereof as an active ingredient.

[0010] Sleep-inducing effect of orexin C in mice. Mouse orexin C (mOXC) (filled circles) or phosphate-buffered saline (PBS) as a control (open circles) was administered intracerebroventricularly to C57BL / 6J mice at ZT12. Wakefulness (A, D), REM sleep (B, E), and non-REM sleep (C, F) were measured. (AC) Wakefulness, REM sleep, and non-REM sleep were measured as cumulative values ​​every 2 hours. (D) Total wakefulness over the 12-hour period in (A). (E) Total REM sleep over the 12-hour period in (B). (F) Total non-REM sleep over the 12-hour period in (C). Total n = 7 per group. *P < 0.05, †P < 0.1: Significant differences determined by Student's paired t-test.

[0011] In this specification, the term "comprise" is a concept that encompasses "consist essentially of" and "consist only of."

[0012] As used herein, "administration" to a subject includes ingestion by the subject.

[0013] As used herein, a peptide refers to a molecule in which two or more but less than 50 amino acids are linked together. A molecule in which 10 or more amino acids are linked together is particularly called a polypeptide.

[0014] In this specification, a molecule consisting of 50 or more amino acids is referred to as a protein.

[0015] As used herein, the term "preproorexin C-terminal peptide" refers to a peptide consisting of orexin A, the GKR sequence, or orexin B, and the amino acid sequence from the next amino acid following the GKR sequence or the GRR sequence to the last amino acid in the amino acid sequence of preproorexin. The preproorexin C-terminal peptide is also called orexin gene-related peptide (OGRP) or orexin C (OXC).

[0016] Hereinafter, embodiments for carrying out the present invention will be described. The embodiments described below are examples of typical embodiments of the present invention, and the scope of the present invention should not be construed as being narrow.

[0017] Preproorexin, expressed in hypothalamic neurons in the brain, is fragmented by post-translational modification to produce orexin A, orexin B, and a C-terminal peptide (orexin C) consisting of 31 amino acids in their C-terminal region. Research by the present inventors disclosed in JP 2023-135605 A revealed the possibility that orexin C may exert its physiological activity via a biomolecule other than the orexin 1 receptor or orexin 2 receptor. The present inventors focused on the fact that orexin A and orexin B are factors that maintain wakefulness, and revealed that orexin C is also a factor that affects sleep and / or wakefulness.

[0018] The amino acid sequences of prepro-orexin are Uniprot number O55232 for rat, Uniprot number O55241 for mouse, and Uniprot number O43612 for human. The amino acid sequences of rat, mouse, and human orexin C are as follows: Rat orexin C (rOXC): AGAELEPYPCPGRRCPTATATALAPRGGSRV (SEQ ID NO: 1) Mouse orexin C (mOXC): AGAELEPHPCSGRGCPTVTTTALAPRGGSGV (SEQ ID NO: 2) Human orexin C (hOXC): AGAEPAPRPCLGRRCSAPAAASVAPGGQSGI (SEQ ID NO: 3)

[0019] According to one aspect of the present invention, there is provided a sleep-improving agent containing any one of the following peptides (I) to (IV): (I) a peptide having the amino acid sequence of a preproorexin C-terminal peptide; (II) a peptide having 90% or more amino acid sequence identity to the amino acid sequence of the preproorexin C-terminal peptide of (I); (III) a peptide that is a part of the amino acid sequence of the preproorexin C-terminal peptide of (I); and (IV) a variant of the peptide of (I) or (III) in which one or two amino acids are deleted, substituted, added, or inserted relative to the peptide of (I) or (III).

[0020] The sleep-improving agent of the present invention contains any one of the peptides (I) to (IV) above as an active ingredient. Any one of the peptides (I) to (IV) above is useful for improving sleep. Sleep improvement includes improvements in sleep quality, sleep quantity, or both. Sleep improvement also includes, for example, at least one of sleep induction, sleep onset promotion, suppression of waking during sleep, improvement in sleep depth, and increase in sleep time. Increased sleep time includes an increase in REM sleep time, an increase in non-REM sleep time, or both, and preferably includes an increase in non-REM sleep time.

[0021] The sleep-improving agent of the present invention may contain one type of peptide selected from any one of peptides (I) to (IV), or may contain two or more types of peptides. When the sleep-improving agent of the present invention contains two or more types of peptides selected from any one of peptides (I) to (IV), the amount of peptides refers to the total amount of peptides (I) to (IV).

[0022] In some embodiments, the peptide (I) above is a polypeptide consisting only of the amino acid sequence of a preproorexin C-terminal peptide.

[0023] In some embodiments, the peptide (I) above is a polypeptide having an amino acid sequence represented by any one of SEQ ID NOs: 1 to 3.

[0024] In some embodiments, the peptide (I) above is a polypeptide consisting solely of the amino acid sequence represented by any one of SEQ ID NOs: 1 to 3.

[0025] In some embodiments, the peptide (II) is a polypeptide having 90% or more amino acid sequence identity to the amino acid sequence of the preproorexin C-terminal peptide (I) and having a sleep-improving effect. The sleep-improving effect includes, for example, improved sleep in a subject compared to when the subject is not administered the polypeptide.

[0026] In some embodiments, the peptide (III) above is a peptide, preferably a polypeptide, that is a continuous portion of the amino acid sequence of the preproorexin C-terminal peptide (I) above.

[0027] In some embodiments, the peptide (III) is a peptide that is a continuous portion of the amino acid sequence of the preproorexin C-terminal peptide (I), and is a peptide consisting of 5 to 30 amino acids, and is preferably a polypeptide.

[0028] In some embodiments, the peptide (III) is a peptide that is a continuous portion of the amino acid sequence of the preproorexin C-terminal peptide (I), and is a peptide consisting of 8 to 30 amino acids, and is preferably a polypeptide.

[0029] In some embodiments, the peptide (III) is a peptide, preferably a polypeptide, that is a continuous portion of the amino acid sequence of the preproorexin C-terminal peptide (I) and has a sleep-improving effect. The sleep-improving effect includes, for example, improved sleep in a subject compared to when the subject is not administered the polypeptide.

[0030] In some embodiments, the peptide (IV) is a mutant of the peptide (I) or (III) in which one or two amino acids are deleted, substituted, added, or inserted, and is a peptide, preferably a polypeptide, that has a sleep-improving effect. The sleep-improving effect includes, for example, improved sleep in a subject compared to when the subject is not administered the peptide.

[0031] In some embodiments, the peptide (IV) is a mutant of the peptide (I) or (III) in which one or two amino acids are deleted, substituted, added, or inserted relative to the preproorexin C-terminal peptide (I), and is a peptide, preferably a polypeptide, that has a sleep-improving effect. The sleep-improving effect includes, for example, improved sleep in a subject compared to when the subject is not administered the peptide.

[0032] In some embodiments, the sleep-improving agent of the above aspect is a sleep-inducing agent.

[0033] In some embodiments, the sleep improvement of the sleep-improving agent of the above aspect includes improvement in the depth of sleep or improvement in falling asleep.

[0034] In some embodiments, the sleep-improving agent is an agent for preventing or treating a sleep disorder.

[0035] In some embodiments, the sleep-improving agent of the above aspect is in the form of a pharmaceutical composition. The pharmaceutical composition may contain a pharmaceutically acceptable pharmaceutical carrier, and various organic or inorganic carrier substances commonly used as formulation materials are used as pharmaceutical carriers. Examples of such pharmaceutical carriers include excipients, diluents, additives, disintegrants, binders, coating agents, lubricants, glidants, gliding agents, flavoring agents, sweeteners, solubilizers, etc. Furthermore, the pharmaceutical composition may contain formulation additives such as preservatives, antioxidants, colorants, flavoring agents, odorants, stabilizers, and fragrances, as needed.

[0036] The dosage form of the pharmaceutical composition may be any dosage form that can be used for pharmaceutical preparations, and may be administered orally or parenterally (e.g., intracerebrally, intravenously, intraperitoneally, etc.), including, for example, tablets, granules, powders, injections, etc.

[0037] When the sleep-improving agent is in the form of a pharmaceutical composition, the dosage of any of the polypeptides (I) to (IV) administered to a subject is preferably 0.0001 to 5000 mg / kg / day, more preferably 0.001 to 1000 mg / kg / day, per kg of the subject's body weight.

[0038] The daily dose may be administered orally or parenterally in one dose or in 2 to 4 divided doses at appropriate intervals.

[0039] In some embodiments, the sleep-improving agent of the above aspect is in the form of a food composition.

[0040] The term "food" generally refers to all foods, including beverages, contained in food compositions, but also includes general foods, including so-called health foods, as well as foods for specified health uses and foods with nutrient functions, as defined by the Ministry of Health, Labor and Welfare's Health Function Food System. Food also includes supplements.

[0041] The food composition may contain appropriate food-acceptable carriers, such as various food ingredients, seasonings, flavorings, and sweeteners. The food composition may also be formulated as a powder, tablet, capsule, or drinkable preparation. Food-acceptable carriers include the pharmaceutically acceptable carriers described above.

[0042] When the sleep-improving agent is in the form of a food composition, the dosage of any of the peptides (I) to (IV) above to a subject is preferably 0.0001 to 5000 mg / kg / day, more preferably 0.001 to 1000 mg / kg / day, per kg of the subject's body weight.

[0043] The daily dose can be administered orally in one dose or in 2 to 4 divided doses at appropriate intervals.

[0044] In some embodiments, the food composition contains any of the peptides (I) to (IV) above, preferably in an amount of 0.0001 to 10% by weight, more preferably 0.001 to 5% by weight, and more preferably 0.01 to 1% by weight.

[0045] In some embodiments, the sleep-improving agent of the above aspect is in the form of a feed, and the feed is administered to a non-human animal.

[0046] When the sleep-improving agent is in the form of feed, the dosage of any of the peptides (I) to (IV) above to a subject is preferably 0.0001 to 5000 mg / kg / day, more preferably 0.001 to 1000 mg / kg / day, per kg of subject body weight.

[0047] The daily dose can be administered orally in one dose or in 2 to 4 divided doses at appropriate intervals.

[0048] According to another aspect of the present invention, there is provided a method for producing the sleep-improving agent of the above aspect, the method comprising the step of adding any one of the peptides (I) to (IV) above. For example, the pharmaceutical composition, food composition, or feed can be produced by adding any one of the peptides (I) to (IV) above.

[0049] According to another aspect of the present invention, there is provided a method for improving sleep, comprising administering to a subject any one of the following peptides (I) to (IV): (I) a peptide having the amino acid sequence of a preproorexin C-terminal peptide; (II) a peptide having 90% or more amino acid sequence identity to the amino acid sequence of the preproorexin C-terminal peptide of (I); (III) a peptide that is a part of the amino acid sequence of the preproorexin C-terminal peptide of (I); and (IV) a variant of the peptide of (I) or (III) in which one or two amino acids are deleted, substituted, added, or inserted relative to the peptide of (I) or (III).

[0050] The subject may be a human or a non-human animal, preferably a non-human mammal, including, but not limited to, a mouse, a rat, etc. In some embodiments, the subject is one in need of improved sleep.

[0051] In some embodiments, the peptide (I) above is a polypeptide consisting only of the amino acid sequence of a preproorexin C-terminal peptide.

[0052] In some embodiments, the peptide (I) above is a polypeptide having an amino acid sequence represented by any one of SEQ ID NOs: 1 to 3.

[0053] In some embodiments, the peptide (I) above is a polypeptide consisting solely of the amino acid sequence represented by any one of SEQ ID NOs: 1 to 3.

[0054] In some embodiments, the peptide (II) is a polypeptide having 90% or more amino acid sequence identity to the amino acid sequence of the preproorexin C-terminal peptide (I) and having a sleep-improving effect, preferably a polypeptide. The sleep-improving effect includes, for example, improved sleep in a subject compared to when the subject is not administered the peptide.

[0055] In some embodiments, the peptide (III) is a peptide, preferably a polypeptide, that is a continuous portion of the amino acid sequence of the preproorexin C-terminal peptide (I).

[0056] In some embodiments, the peptide (III) is a peptide that is a continuous portion of the amino acid sequence of the preproorexin C-terminal peptide (I), and is a peptide consisting of 5 to 30 amino acids, more preferably a polypeptide.

[0057] In some embodiments, the peptide (III) is a peptide that is a continuous portion of the amino acid sequence of the preproorexin C-terminal peptide (I), and is a peptide consisting of 8 to 30 amino acids, more preferably a polypeptide.

[0058] In some embodiments, the peptide (III) is a peptide, preferably a polypeptide, that is a continuous portion of the amino acid sequence of the preproorexin C-terminal peptide (I) and has a sleep-improving effect. The sleep-improving effect includes, for example, improved sleep in a subject compared to when the subject is not administered the peptide.

[0059] In some embodiments, the peptide (IV) is a mutant of the peptide (I) or (III) in which one or two amino acids are deleted, substituted, added, or inserted, and is a peptide, preferably a polypeptide, that has a sleep-improving effect. The sleep-improving effect includes, for example, improved sleep in a subject compared to when the subject is not administered the peptide.

[0060] In some embodiments, the peptide (IV) is a mutant of the peptide (I) or (III) in which one or two amino acids are deleted, substituted, added, or inserted relative to the preproorexin C-terminal peptide (I), and is a peptide, preferably a polypeptide, that has a sleep-improving effect. The sleep-improving effect includes, for example, improved sleep in a subject compared to when the subject is not administered the peptide.

[0061] Any of the peptides (I) to (IV) above can be administered in the form of the sleep-improving agent of the above-mentioned embodiment, the pharmaceutical composition, or the food composition.

[0062] In some embodiments, the sleep improvement method excludes medical procedures on humans (eg, methods of treating humans).

[0063] In some embodiments, the method for improving sleep comprises administering any one of the peptides (I) to (IV) to a non-human animal. The improvement of sleep may be the prevention or treatment of a sleep disorder. The method for improving sleep may comprise administering any one of the peptides (I) to (IV) to a non-human animal. Any one of the peptides (I) to (IV) may be administered to the non-human animal in an amount effective for the prevention or treatment of the sleep disorder.

[0064] In some embodiments, the method for improving sleep comprises administering any of the peptides (I) to (IV) to a human. The improvement of sleep may exclude medical treatment for the human (e.g., a method for treating a human). Alternatively, the improvement of sleep may be the prevention or treatment of a sleep disorder in a human. The method for improving sleep may be a method for preventing or treating a sleep disorder comprising administering any of the peptides (I) to (IV) to a human. Any of the peptides (I) to (IV) may be administered to a human in an amount effective for preventing or treating the sleep disorder.

[0065] Because mouse orexin C suppresses wakefulness and induces sleep in mice, orexin C may be effective in treating insomnia. Furthermore, treatments for hypersomnia (narcolepsy), which have been difficult to develop until now, may be achievable by inhibiting the action of orexin C. Thus, orexin C is a novel target for improving sleep-wake disorders.

[0066] The disclosures of all patent applications and publications cited herein are hereby incorporated by reference in their entirety.

[0067] The following examples are intended for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Unless otherwise specified, reagents are commercially available or are obtained or prepared according to conventional techniques or literature procedures in the art.

[0068] 1. Experimental Method: Male C57BL / 6J mice (8 weeks old) were anesthetized with isoflurane via inhalation, implanted with electrodes for EEG analysis (Pinnacle Technology, KS, USA) and a guide cannula for intraventricular administration, and then allowed to recover for 1 week. After 3 days of acclimation to an EEG / EMG data acquisition system (Pinnacle Technology) for sleep / wake analysis, mice were intraventricularly administered mouse orexin C (mOXC, 3 nmol, SEQ ID NO: 2) or phosphate-buffered saline (PBS) as a control into the lateral ventricle (0.3 mm posterior and 0.9 mm lateral to the Bregma, 2.2 mm from the brain surface), and EEG / EMG were recorded for 12 hours. The mOXC polypeptide was synthesized. After a further 3 days, mice were administered PBS or mOXC (3 nmol) in a crossover study, and EEG / EMG were recorded for 12 hours. The results of these two consecutive experiments were combined and evaluated as a two-group comparison between the mOXC-treated and PBS-treated groups. Wake time, REM sleep time, and non-REM sleep time were calculated using Sleepsign software (Kissei Comtech, Nagano, Japan). Statistical significance was determined by Student's paired t-test.

[0069] 2. Results As shown in Figure 1(A)-(F), when mOXC (3 nmol) was intracerebroventricularly administered to mice at ZT12 (the start time of the mouse wakefulness phase), wakefulness time was reduced (Figure 1(A) and (D)), REM sleep time was mildly increased (Figure 1(B) and (E)), and non-REM sleep time was significantly increased (Figure 1(C) and (F)).

Claims

1. A sleep-improving agent containing any one of the following peptides (I) to (IV): (I) a peptide having the amino acid sequence of a preproorexin C-terminal peptide; (II) a peptide having 90% or more amino acid sequence identity to the amino acid sequence of the preproorexin C-terminal peptide of (I); (III) a peptide which is a part of the amino acid sequence of the preproorexin C-terminal peptide of (I); (IV) a mutant form of the peptide of (I) or (III) in which one or two amino acids have been deleted, substituted, added, or inserted in the peptide of (I) or (III).

2. The sleep-improving agent according to claim 1, wherein the peptide (I) is a peptide having an amino acid sequence represented by any one of SEQ ID NOs: 1 to 3.

3. The sleep improving agent according to claim 1, which is a sleep-inducing agent.

4. The sleep improving agent according to claim 1, wherein the sleep improvement includes improvement in depth of sleep or improvement in falling asleep.

5. The sleep-improving agent according to claim 1, which is an agent for preventing or treating sleep disorders.

6. The sleep-improving agent according to any one of claims 1 to 5, which is in the form of a pharmaceutical composition.

7. The sleep-improving agent according to any one of claims 1 to 5, which is in the form of a food composition.

8. The sleep-improving agent according to any one of claims 1 to 5, which is in the form of a feed.

9. A method for improving sleep (excluding methods for treating humans), comprising administering to a subject any of the following peptides (I) to (IV): (I) a peptide having the amino acid sequence of preproorexin C-terminal peptide (II) a peptide having 90% or more amino acid sequence identity to the amino acid sequence of the preproorexin C-terminal peptide of (I) (III) a peptide that is a part of the amino acid sequence of the preproorexin C-terminal peptide of (I) (IV) a mutant form of the peptide of (I) or (III) in which one or two amino acids have been deleted, substituted, added, or inserted relative to the peptide of (I) or (III).

10. The method of claim 9, wherein said improving sleep includes preventing or treating a sleep disorder.