Composition comprising probiotic bacteria and use thereof for the treatment of insomnia

Abstract

The present application provides a composition comprising probiotics and its use for treating insomnia. Further, the present application also provides a dual orexin receptor antagonist polypeptide O-59 polypeptide, which can inhibit the over-activation of the orexin system by antagonizing the orexin 1 receptor (OX1R) and the orexin 2 receptor (OX2R), thereby improving sleep. After the polypeptide is combined with the probiotic composition, the insomnia symptoms of the insomnia mice can be significantly improved, and the application prospect is good.

Description

Technical Field

[0001] This application relates to the biological field, and more specifically to compositions containing probiotics and their use in treating insomnia. Background Technology

[0002] With continuous social development and a faster pace of life, the body's internal and external pressures are increasing, leading to a growing number of people suffering from insomnia across a wider age range. Sleep disorders are recognized as the second most common mental disorder. As a chronic, non-communicable disease, insomnia significantly disrupts people's lives. Fatigue, depression, daytime sleepiness, and many other related problems caused by insomnia severely impact quality of life and may even lead to diabetes, hypertension, and cardiovascular disease. Recommended medications for insomnia include benzodiazepines, non-benzodiazepine hypnotics ("Z-drugs"), melatonin receptor agonists like Ramettene, and first-generation histamine antagonists like doxepin. However, these medications have significant side effects, potentially including lingering drowsiness the next day, poor motor coordination, frequent falls, memory and cognitive impairment, as well as strong addictive properties and severe withdrawal symptoms. Furthermore, existing treatment options face significant limitations in terms of drug abuse, drug tolerance, drug discontinuation, and safety.

[0003] Research has found that orexin antagonists are an important direction for drug development in the treatment of insomnia. Since orexin OX-A and OX-B correspond to orexin receptors OX1R and OX2R, respectively, research on orexin receptor antagonists has been divided into two main categories from the outset: one is selective orexin receptor antagonists (SORAs), which have significantly different affinities for orexin receptors OX1R and OX2R; the other is called dual orexin receptor antagonists (DORAs), which have similar affinities for orexin receptors OX1R and OX2R and are not selective.

[0004] Smart and Porter et al. of GlaxoSmithKline (GSK) were the first to discover that quinolinium compounds could act as 1-SORAs, especially compound 1 (SB-334867, considered the first 1-SORA). It can cross the blood-brain barrier and has a strong affinity for OX1R. Unfortunately, it lacks oral bioavailability. It also has strong affinity for two 5-hydroxytryptamine receptor (5-HT) subtypes (5-HT2B and 5-HT2C), and its binding to OX1R is reversible. In experiments, it caused modified behavior in rats. It did not have significant sedative-hypnotic effects, resulting in a lack of drug-like properties. However, it has played a crucial role in orexin pharmacological research, and to date, more than 200 publications have used it as a reference compound. The synthesized compounds 2 (SB-408124) and 3 (SB-410220) have good oral bioavailability and a strong affinity for OX1R. A series of analogues of the compound were synthesized, and structure-activity relationship studies showed that similar physiological activities could be obtained when the 2-methylbenzoxazol group was replaced with a 2,4-disubstituted aniline group. Merck synthesized more than 260 4,4-difluoropiperidine amide compounds, of which 8 showed a strong affinity for OX1R, with an IC50 as low as 1.3 nmol·L⁻¹ (IC50 for OX2R = 512 nmol·L⁻¹), and is expected to enter clinical trials. Merck discovered that compounds with a tetrahydroisoquinoline backbone are excellent selective orexin-1 receptor (OX1R) antagonists. Lead compound 9 has an IC50 of 119 nmol·L⁻¹ against OX1R and an IC50 of 8,152 nmol·L⁻¹ against OX2R, with insufficient selectivity. However, compound 10 has an IC50 as low as 17 nmol·L⁻¹ against OX1R and an IC50 greater than 10,000 nmol·L⁻¹ against OX2R, making it an excellent 1-SORA. Based on the above research, Actelion developed the oral drug 12 (ACT-335827). Pharmacological studies on rats have confirmed that 12 can selectively bind to OX1R (IC50 for OX1R = 6 nmol·L⁻¹, IC50 for OX2R = 417 nmol·L⁻¹). However, 12 does not have a hypnotic effect, but rather helps relieve fear stress and anxiety symptoms in rats, making it a very promising anti-anxiety drug.

[0005] Since the inception of orexin receptor antagonist research, the debate between selective orexin receptor antagonists (SORAs) and dual orexin receptor antagonists (DORAs) has never ceased, with many pharmaceutical giants simultaneously conducting research on both. In August 2014, the US FDA approved Merck's suvorexant (29, MK-4305) for marketing, making it the first orexin receptor antagonist used to treat insomnia. Pharmacologically, suvorexant belongs to the DORA category. With the high-resolution (2.5 Å) crystal structure resolution of the protein crystals showing the binding of suvorexant to the human orexin-2 receptor (hOX2R), the binding site and optimal conformation of the two were clearly observed for the first time, ushering in a golden age for DORA research.

[0006] Studies have shown that orexin receptor antagonists can effectively increase sleep without affecting sleep structure; simultaneously, downregulation of both OX1R and OX2R receptors can alleviate the damage to hippocampal learning and memory caused by long-term insomnia. Furthermore, the high selectivity of dual orexin receptor antagonists may overcome the current problems of strong addiction and severe withdrawal reactions in clinical insomnia drug treatments, offering hope for long-term safe medication regimens for insomnia. Currently, the three orexin receptor antagonists approved by the FDA still have some issues that require further research and overcoming. In my country, domestically developed orexin receptor antagonists YZJ1139 from Yangtze River Pharmaceutical Group and HEC83518 from Dongyangguang Pharmaceutical Group are undergoing clinical trials, still in phase III. Although many drugs are currently under research, highly effective drugs for treating insomnia still need further development. Summary of the Invention

[0007] This invention provides a composition containing probiotics and its use in treating insomnia.

[0008] Specifically, this invention identified a dual orexin receptor antagonist peptide through high-throughput screening. By antagonizing orexin 1 receptor (OX1R) and orexin 2 receptor (OX2R), it inhibits the overactivation of the orexin system, thereby improving sleep.

[0009] The polypeptide is the O-59 polypeptide, and its amino acid sequence is shown in SEQ ID NO: 1. The polypeptide has been shown to have good safety and no significant nephrotoxicity in mice after high-dose gavage experiments.

[0010] Furthermore, the polypeptides of the present invention can be modified or have amino acids replaced, but the biological activity of the polypeptides is still retained.

[0011] Specifically, the polypeptide can be prepared for use as a pharmaceutical treatment for insomnia.

[0012] In one embodiment, the polypeptide disclosed in this invention, or a pharmaceutical composition comprising the polypeptide of this invention, can be administered once, or, depending on the dosing regimen, administered several times at different time intervals within a specified period. For example, administered once, twice, three times, or four times daily. In one embodiment, administered once daily. In another embodiment, administered twice daily. Administration can continue until the desired therapeutic effect is achieved or to maintain the desired therapeutic effect indefinitely. A suitable dosing regimen for the pharmaceutical composition disclosed in this invention depends on the pharmacokinetic properties of the polypeptide, such as dilution, distribution, and half-life, which can be determined by a person skilled in the art. Furthermore, a suitable dosing regimen for the composition disclosed in this invention includes the duration of administration, depending on factors within the knowledge and experience of a person skilled in the art, such as the disease being treated, the severity of the disease being treated, the age and physical condition of the patient being treated, the patient's medical history, the nature of concurrent therapies, and the desired therapeutic effect. Such a person skilled in the art should also understand that adjustments to the dosing regimen may be required depending on the individual patient's response to the dosing regimen, or if the individual patient requires adjustments over time.

[0013] The present invention discloses polypeptides that can be administered simultaneously with, before, or after one or more other therapeutic agents. The polypeptides of the present invention can be administered separately to other therapeutic agents via the same or different routes of administration, or administered together with them in the form of a pharmaceutical composition.

[0014] For an individual weighing approximately 50-70 kg, the pharmaceutical compositions and combinations disclosed in this invention may be in unit dose form containing approximately 1-1000 mg, or approximately 1-500 mg, or approximately 1-250 mg, or approximately 1-150 mg, or approximately 0.5-100 mg, or approximately 1-50 mg of active ingredient. The therapeutically effective amount of the polypeptide, pharmaceutical composition, or combination thereof depends on the individual's species, weight, age, individual condition, the disorder or disease being treated, or its severity. A physician, clinician, or veterinarian with common skills can readily determine the effective amount of each active ingredient required to prevent, treat, or inhibit the development of a disorder or disease.

[0015] The dosage characteristics cited above have been demonstrated in in vitro and in vivo studies using advantageous mammals (e.g., mice, rats, dogs, monkeys) or their isolated organs, tissues, and specimens. This invention discloses peptides for in vitro use in solution, such as an aqueous solution, and also for in vivo use in intestinal, parenteral, and especially intravenous, form, such as a suspension or aqueous solution.

[0016] In one embodiment, the therapeutically effective dose of the disclosed polypeptide is from about 0.1 mg to about 2,000 mg daily. The pharmaceutical composition thereof should provide a dose of the polypeptide from about 0.1 mg to about 2,000 mg. In a particular embodiment, the prepared pharmaceutical dosage unit form can provide about 1 mg to about 2,000 mg, about 10 mg to about 1,000 mg, about 20 mg to about 500 mg, or about 25 mg to about 250 mg of the main active ingredient or a combination of the main ingredients in each dosage unit form. In a particular embodiment, the prepared pharmaceutical dosage unit form can provide about 10 mg, 20 mg, 25 mg, 50 mg, 100 mg, 250 mg, 500 mg, 1000 mg, or 2000 mg of the main active ingredient.

[0017] Furthermore, the drug of the present invention may also be supplemented with a second therapeutic agent for treating insomnia.

[0018] Specifically, the second treatment agent is selected from the following groups: for example, modafinil, amoronin, doxepin, alprazolam, bromazepam, clonazan, clonazepam, clozadiazepam, flunitrazepam, flurazepam, lorazepam, midazolam, nitrazepam, oxazepam, temazepam, triazolam, indeprom, zopiclone, eszopiclone, zaleplon, zolpidem, gaboxard, vigabatrin, tiagabine, EVT 201 (Evotec Pharmaceuticals), estazolam, ketocelin, risperidone, escelin, flurisserin (Sanofi-Aventis, France), profancelin, MDL 100907 (Sanofi-Aventis, France), HY10275 (Eli Lilly), APD125 (Arena). Pharmaceuticals, San Diego, CA), AVE8488 (Sanofi-Aventis, France), Repinolone, Salizotan, Itaspirone, Buspirone, MN-305 (MediciNova, San Diego, CA), Melatonin, Rametamide (Takeda Pharmaceuticals, Japan), VEC-162 (Vanda Pharmaceuticals, Rockville, Maryland), PD-6735 (Phase II, Discovery), Agomelatine, Lamotrigine, Gabapentin, Pregabalin, Appetite Peptide, 1,3-Diarylurea, SB-334867-a (GlaxoSmithKline, UK), GW649868 (GlaxoSmithKline), Benzamide Derivatives, Org 50081 (Organon-Netherlands), Ritanserine, Nefazodone, Serzone, Trazodone, Cassospiran (GlaxoSmithKline), Amitriptyline, Amoxapine, Bupropion, Citalopram, Clomipramine, Desipramine, Doxepin, Duloxetine, Etapril, Fluoxetine, Fluvoxamine, Imipramine, Iscarbohydrazine, Maprotiline, Mirtazapine, Nefazodone, Desipramine Metriptyline, paroxetine, phenelzine sulfate, protriptyline, sertraline, transphenylcyclopropionamide, trazodone, trimethoprim, venlafaxine, chlorpromazine, haloperidol, fluphenazine, fluphenazine, loxapine, mesoridazine, indole, perphenazine, pimozide, prochlorperazine, thioridazine, tevothixol, trifluoperazine, clozapine, aripiprazole, olanzapine, quetiapine, risperidone, ziprasidone, and palpanidone.

[0019] Furthermore, the drug or drug composition further contains a probiotic composition.

[0020] Specifically, the probiotic strains can be Lactobacillus or Bifidobacterium. Examples of preferred Lactobacillus species include *Lactobacillus rhamnosus*, *Lactobacillus paracasei*, and *Lactobacillus reuteri*. Particularly preferred strains are *Lactobacillus rhamnosus* ATCC 53103, *Lactobacillus rhamnosus* CGMCC 1.3724, *Lactobacillus reuteri* ATCC 55730, and *Lactobacillus reuteri* DSM 17938. Examples of preferred Bifidobacterium species include *Bifidobacterium lactis*, *Bifidobacterium longum*, *Bifidobacterium breve*, and *Bifidobacterium infantis*. Specially selected strains include *Bifidobacterium lactis* CNCMI-3446, specially sold by Christian Hansen, Denmark, under the trademark Bb12; *Bifidobacterium longum* NCC3001, ATCC BAA-999, sold by Morinaga Milk Industry Co., Ltd., Japan, under the trademark BB536; *Bifidobacterium breve* strain, sold by Danisco, under the trademark Bb-03; *Bifidobacterium breve* strain, sold by Morinaga, under the trademark M-16V; *Bifidobacterium breve* strain, sold by Institut Rosell (Lallemand), under the trademark R0070; and *Bifidobacterium infantis* strain, sold by Procter & Gamble Co., under the trademark B.Infantis. Probiotics can be selected from the following list.It includes: Bifidobacteria, Lactobacillus, Lactococcus, Enterococcus, Streptococcus, Propionibacterium, Pediococci, Escherichia coli, Saccharomyces de Barry, Kluyveromyces, yeast, Schizosomalosomal, Zygomyces, Yersinia, Candida, Bifidobacterium longum, Bifidobacterium lactis, Bifidobacterium animalis, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium bifidum, Bifidobacterium adolescentis, Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus fermentum, and Lactobacillus johnsonii. *Lactobacillus johnsonii*, *Lactobacillus gasseri*, *Lactobacillus rhamnosus*, *Lactococcus* subspecies such as *Lactococcus lactis*, *Lactococcus cremoris*, *Lactococcus diacetylactis*, *Enterococcus faecium*, *Enterococcus facealis*, *Saccharomyces cerevisiae*, *Saccharomyces boulardii*, *Schizosaccharomyces pombe*, *Kluyveromyces lactis*, *Yarrowia lypolitica*, or mixtures thereof.Preferred strains include *Lactobacillus johnsonii* (NCC533; CNCM I-1225), *Bifidobacterium longum* (NCC490; CNCM I-2170), *Bifidobacterium longum* (NCC2705; CNCM I-2618), *Bifidobacterium longum* (NCC3001; ATCCBAA-999), *Bifidobacterium lactis* (NCC2818; CNCM I-3446), *Bifidobacterium breve* (strain A), *Lactobacillus paracasei* (NCC2461; CNCM I-2116), *Lactobacillus rhamnosus* GG (ATCC53103), *Lactobacillus rhamnosus* LPR (NCC4007; CGMCC 1.3724), *Lactobacillus reuteri* (ATCC 55730), *Lactobacillus reuteri* (DSM 17938), *Enterococcus faecium* SF 68 (NCIMB10415), *Saccharomyces boulardii*, and mixtures thereof.

[0021] Selected probiotic strains can be cultured using any suitable method known in the art, and prepared by, for example, freeze-drying or spray-drying, for use in the pharmaceutical or nutritional compositions of the present invention. Optionally, bacterial strains already prepared in suitable forms for use in nutritional compositions such as infant formula can be purchased from specialized suppliers such as Christian Hansen and Morinaga.

[0022] Furthermore, the present invention has obtained a better probiotic composition through screening. Specifically, the probiotic composition contains Lactobacillus reuteri DSM 17938 and Lactobacillus helveticus R0052.

[0023] Specifically, the preferred daily dose of probiotics is 10e3 to 10e12 CFU. The amount of probiotics included in the supplement will be selected depending on how the supplement is administered. For example, if the supplement is administered twice daily, each supplement may contain 5 x 10e2 to 5 x 10e11 CFU of probiotics. The supplement may be in the form of, for example, tablets, capsules, lozenges, suppositories, chewing gum, or liquid. The supplement may also contain protective aqueous gels (e.g., gums, proteins, modified starches), binders, film-forming agents, encapsulating agents / materials, wall / shell materials, matrix compounds, coatings, emulsifiers, surfactants, solubilizers (oils, fats, waxes, lecithin, etc.), adsorbents, carriers, fillers, co-compounds, dispersants, wetting agents, processing aids (solvents), flow agents, flavor masking agents, weighting agents, gelling agents, and gelling agents. Supplements may also contain traditional pharmaceutical additives and adjuvants, excipients and diluents, including but not limited to water, gelatin, dextrin, lignin sulfonates, talc, sugar, starch, gum arabic, vegetable oil, polyalkylene glycols, flavoring agents, preservatives, stabilizers, emulsifiers, buffers, lubricants, colorings, humectants, fillers, etc.

[0024] Beneficial effects This invention provides a composition containing probiotics and its use in treating insomnia. Furthermore, this invention also provides a dual orexin receptor antagonist peptide, O-59, which can improve sleep by inhibiting the overactivation of the orexin system through antagonism of orexin 1 receptor (OX1R) and orexin 2 receptor (OX2R). Combining the peptide with the probiotic composition significantly improves insomnia symptoms in insomniac mice, showing promising application prospects. Attached Figure Description

[0025] Figure 1 Figure 1 shows the effect of each group on the sleep rate of mice. Figure 2 Figure 1 shows the effect of each group on mouse body weight. Detailed Implementation Specific embodiments of the invention will now be described in more detail with reference to the accompanying drawings. While specific embodiments of the invention are shown in the drawings, it should be understood that the invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the invention and to fully convey the scope of the invention to those skilled in the art.

[0026] Example 1: Screening of candidate peptides antagonizing orexin 1 receptor (OX1R) and orexin 2 receptor (OX2R) Based on the three-dimensional structures of OX1R and OX2R, a deep generative model was used to simulate and design candidate peptides that can specifically bind to the key residues of the OX1R binding pocket (Ser103, Pro123, Gln126, Ala127, His344) and the OX2R binding pocket (Thr111, Pro131, Gln134, Thr135, His350). Ten candidate peptides with higher scores were selected for subsequent identification experiments. The ten candidate peptides are O-4, O-11, O-29, O-33, O-59, O-63, O-78, O-92, O-112, and O-156.

[0027] Example 2: Identification of antagonistic activity of candidate peptides against OX1R and OX2R Chinese hamster ovary (CHO) cells expressing human orexin-1 and human orexin-2 receptors, respectively, were grown in Ham F-12 medium containing 300 μg / mL G418, 100 U / mL penicillin, 100 μg / mL streptomycin, and 10% heat-inactivated fetal bovine serum (FCS). Cells were seeded at 10,000 cells / well in 96-well plates. The seeded plates were incubated overnight at 37°C in 5% CO2. Human orexin-A, as an agonist, was prepared as a 1 mM methanol:water (1:1) stock solution, diluted in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g / L, and 20 mM HEPES for experiments, with a final concentration of 3 nM.

[0028] Each peptide group was prepared as a 10 mM DMSO stock solution, then diluted in a 96-well plate with DMSO. The diluted solution was then transferred to HBSS containing 0.1% bovine serum albumin (BSA), 0.375 g / L NaHCO3, and 20 mM HEPES. 50 μL of staining buffer (HBSS containing 1% FCS, 20 mM HEPES, 0.375 g / L NaHCO3, 5 mM probenecid (Sigma), and 3 μM fluo-4AM fluorescent calcium indicator (1 mM DMSO stock solution containing 10% pluronic acid)) was added to each well. The 96-well cell plate was incubated at 37°C for 50 min in 5% CO2, and then equilibrated at room temperature for 30 min before measurement. In the fluorescence imaging plate reader, each peptide group was added to the plate at a volume of 10 μL / well, incubated for 120 min, and finally, 10 μL / well of agonist was added. Fluorescence was measured at 1-second intervals in each well, and the height of each fluorescence peak was compared to the height of the fluorescence peak induced by 3 nM orexin-A with the medium instead of the antagonist. IC50 values ​​(the concentration of the compound required to inhibit a 50% agonistic response) were determined and normalized using the IC50 values ​​of the obtained plate reference compounds. Optimized conditions were achieved by adjusting pipetting speed and cell division protocols. As is known to those skilled in the art, the calculated IC50 values ​​fluctuate based on daily cell assay performance. Geometric mean values ​​are given for multiple determinations of the IC50 values ​​for the same compound. The antagonistic activities of the peptide groups are shown in Table 1.

[0029] Table 1. Antagonistic activity results of each group of peptides As can be seen from the results in Table 1, candidate peptide O-59 has a good dual-target inhibition effect. Subsequent experiments will be conducted using this peptide. The amino acid sequence of peptide O-59 is shown in SEQ ID NO: 1.

[0030] Example 3 Preparation of Probiotic Composition Lactobacillus reuteri DSM 17938 and Lactobacillus helveticus R0052 were purchased from XKbio.

[0031] After activating the preserved Lactobacillus reuteri DSM 17938, it was inoculated into MRS liquid medium and cultured at 37°C for 24 h. Then, it was centrifuged at 4°C and 12,000 r / min for 15 min to collect the bacterial sludge. An appropriate amount of protective agent was added and stored frozen at -80°C for later use. The composition of the MRS medium was: peptone 10 g / L, beef extract 10 g / L, yeast extract 5 g / L, glucose 20 g / L, dipotassium hydrogen phosphate 2 g / L, Tween 80 1 g / L, ammonium citrate 2 g / L, sodium acetate 5 g / L, magnesium sulfate 0.1 g / L, manganese sulfate 0.05 g / L. The pH was adjusted to 6.5 and sterilized at 121°C for 20 min.

[0032] The cryopreserved Lactobacillus helveticus R0052 was inoculated into MRS medium, shaken and mixed evenly, and cultured at a constant temperature of 37°C for 24 h to obtain a bacterial suspension. According to an inoculation amount of 3% by weight of the MRS medium, the obtained bacterial suspension was added to the MRS medium and cultured in an incubator at a constant temperature of 37°C for 16 - 20 h to obtain the Lactobacillus helveticus R0052 culture solution. It was centrifuged at 4°C and 12,000 r / min for 15 min to collect the bacterial sludge. An appropriate amount of protective agent was added and stored frozen at -80°C for later use.

[0033] 6 g of Lactobacillus reuteri DSM 17938, 3 g of Lactobacillus helveticus R0052, 20 mg of sodium alginate, 40 mg of chitosan, and 20 mg of carboxymethyl cellulose were taken. After mixing the components evenly, probiotic composition A was obtained.

[0034] Furthermore, 6 g of Lactobacillus reuteri DSM 17938, 3 g of Lactobacillus helveticus R0052, 0.5 g of O - 59 polypeptide, 20 mg of sodium alginate, 40 mg of chitosan, and 20 mg of carboxymethyl cellulose were taken. After mixing the components evenly, probiotic composition B was obtained.

[0035] Example 4 Verification of the Anti - insomnia Effect of O - 59 Polypeptide and / or Probiotic Composition The animals used in this study were SPF - grade male ICR mice, weighing (20 ± 2) g, provided by Beijing Spearf Bio - technology Co., Ltd. The animal production license number was SCXK (Beijing) 2019 - 0010, and the certificate number was 10011230101930023. After the animals were purchased, they were first adaptively fed in a clean environment for 5 d, and the feeding environment temperature was (22 ± 1)°C. During the feeding period, they were fed with ordinary feed, allowed to drink water freely, and the bedding was kept dry and the environment was quiet.

[0036] Except for the control group, all other groups of mice were intraperitoneally injected with a suspension of p-chlorophenylalanine (PCPA, used in this invention to induce an insomnia mouse model) (450 mg / kg, dissolved in gum arabic, pH=7), once daily for 5 consecutive days. Mouse behavior was observed during the modeling period. Compared with the control group, the other groups of mice exhibited significant behavioral changes, including circadian rhythm disorder, increased daytime activity, disheveled fur, increased aggression, significant weight loss, and grayish-white feces, indicating successful establishment of the insomnia model. After successful modeling, medication was administered according to the following regimen; Polypeptide treatment group: O-59 polypeptide, 0.1g / kg, administered orally; Probiotic composition A: 0.5 g / kg orally, probiotic composition A orally; Probiotic composition B: 0.5 g / kg orally, probiotic composition B; Positive control group: Lemborexant (Dayvigo®) administered by gavage, 0.1 g / kg; Control group and model group: administered equal volume of physiological saline by gavage; Each group was given the drug once daily (10 mice per group).

[0037] Mice in each group were administered oral gavage for 12 days. Each group of mice was also intraperitoneally injected with 30 mg / kg sodium pentobarbital (this dose is the validated hypnotic dose of sodium pentobarbital for mice). Within 30 minutes, the number of mice falling asleep in each group was recorded. Sleep was defined as the disappearance of the righting reflex. When a mouse was placed in a dorsal position, it would immediately right itself. If a mouse could not right itself within 1 minute, the righting reflex was considered to have disappeared, and the mouse had entered sleep. The sleep rate was calculated based on the number of mice falling asleep (sleep rate = number of mice falling asleep / total number of mice × 100%). Results are as follows: Figure 1 As shown.

[0038] Depend on Figure 1 It was found that the number of mice falling asleep in each treatment group was significantly increased compared with the blank group and the model group (P<0.05). The sleep rate of probiotic composition B reached 100%, which was significantly higher than the 80% of the positive control group. Probiotic composition B had a higher sleep rate than probiotic composition A, indicating that the combination of O-59 peptide and probiotics can synergistically enhance the effect and significantly improve the sleep rate of mice.

[0039] After 15 days of continuous drug administration, all mice in each group were intraperitoneally injected with sodium pentobarbital at a dose of 50 mg / kg (this dose was determined by the minimum threshold dose required to induce sleep in 100.00% of mice in a preliminary experiment). The sleep latency and duration were observed and recorded for each group of mice, using the disappearance of the righting reflex as the criterion. The sleep latency was defined as the time from the start of injection to the disappearance of the righting reflex. The results are shown in Table 2.

[0040] Table 2. Effects of sleep latency and sleep duration on each group Table 2 shows that, compared with the control group, the sleep latency of the model group was significantly prolonged (P<0.05). Compared with the model group, the sleep latency of each treatment group was significantly shortened (P<0.05); and the sleep duration of each treatment group was significantly prolonged (P<0.05). In particular, the probiotic composition B significantly reduced sleep latency and increased sleep duration, indicating that the combination of O-59 peptide and probiotics can significantly shorten sleep latency and increase sleep duration.

[0041] After 20 days of continuous drug administration, the average weight of mice in each group was measured, and the results are as follows: Figure 2 As shown.

[0042] from Figure 2 As can be seen, compared with the blank group, the weight gain of mice in the model group was significantly reduced, which was statistically significant (P < 0.01). Compared with the model group, the weight gain of mice in probiotic composition A, probiotic composition B, and the positive group was significantly higher than that in the model group. This fully demonstrates that the peptides of the present invention can improve the weight of mice by improving their sleep quality, and can significantly improve the quality of life of mice, showing good efficacy.

[0043] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0044] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. An O-59 polypeptide, the amino acid sequence of which is shown in SEQ ID NO: 1, wherein the polypeptide is a dual orexin receptor antagonist polypeptide that improves sleep by inhibiting the overactivation of the orexin system by antagonizing orexin 1 receptor (OX1R) and orexin 2 receptor (OX2R).

2. Use of the O-59 polypeptide as described in claim 1 in the preparation of a medicament for treating insomnia.

3. The use as described in claim 2, characterized in that... The drug also includes a second therapeutic agent, which is a probiotic composition consisting of Lactobacillus reuteri DSM 17938 and Lactobacillus helveticus R0052 in a 2:1 mass ratio.

4. The use as described in claim 3, wherein the medicament comprises a pharmaceutically acceptable carrier.

5. A pharmaceutical composition for treating insomnia, characterized in that... The pharmaceutical composition is composed of the following components: Lactobacillus reuteri DSM 17938, Lactobacillus helveticus R0052, the O-59 polypeptide of claim 1, sodium alginate, chitosan, and carboxymethyl cellulose. The components are mixed to obtain the pharmaceutical composition; wherein Lactobacillus reuteri DSM 17938 6g, Lactobacillus helveticus R0052 3g, and O-59 polypeptide 0.5g.

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