Use of a d-glucosamine compound in the regulation of circadian rhythms

Abstract

The present application relates to a kind of D-glucosamine compound in the application of adjusting circadian rhythm.The D-glucosamine compound of the present application shows obvious rapid adjustment circadian rhythm disorder, reduces the synchronization time of circadian rhythm phase shift, and regulates the expression of P-AMPK / AMPK, P-mTOR / mTOR, P70S6K, BMAL1 and P-BMAL1 in U2OS cell, mouse hypothalamus and mouse liver in vitro and in vivo experiment, so it can be used for the treatment of jet lag, shift work, continuous multi-day and night work or sleep disorders related to circadian rhythm, while having the characteristics of high efficiency and low toxicity, can improve and guarantee the ability of work, it has important significance for the personnel of circadian rhythm disorder engaged in night shift, shift work and long-distance trans-zone flight.

Description

Technical Field

[0001] This invention relates to the application of a D-glucosamine compound, and particularly to its application in regulating circadian rhythms. Background Technology

[0002] The Earth's rotation causes 24-hour diurnal rhythms in light, temperature, humidity, and the availability of food. As a result, life on Earth must adjust its physiological behaviors accordingly to adapt to the environment. In this process, most organisms have developed 24-hour behavioral and physiological-biochemical rhythms. For example, human sleep, wakefulness, eating, excretion, hormone secretion, body temperature, blood pressure, and other physiological and metabolic activities have a certain rhythm to adapt to these periodic changes in the external environment.

[0003] Research has found that the mammalian circadian rhythm system is mainly concentrated in a specific brain region within the central nervous system. The circadian rhythm process primarily occurs in the suprachiasmatic nucleus (SCN) and its adjacent structures in the anterior hypothalamus. The SCN is the most important circadian rhythm center in mammals, participating in the control of various rhythmic activities such as sleep and wakefulness cycles. The self-generation and maintenance of SCN activity rhythms depend on a spontaneous transcriptional feedback loop formed by a few genes, primarily including Bmal1, Clock, Per, Cry, and CKIε. Mutations or deletions of any of these clock genes will lead to a prolonged or shortened free-running cycle. The SCN's own rhythm is influenced by both the external environment and internal factors. Disruptions in the rhythms of the core and peripheral biological clock systems caused by the external environment are a key reason for circadian rhythm disorders and other complications in humans.

[0004] Based on the needs of personnel with disrupted circadian rhythms working night shifts, rotating shifts, and long-haul, cross-time zone flights, current domestic and international efforts primarily focus on adjusting sleep patterns according to job requirements, controlling work intensity and duration, and reducing shift work to improve and correct circadian rhythm disorders in complex work environments. However, these methods are relatively limited or passive, lacking proactive and effective measures, especially effective targeted drugs with significant effects, minimal side effects, and convenient application. In recent years, various countries have conducted research on a series of conventional sleep management drugs. Caffeine, modafinil, and amorofemide can be used as wakefulness promoters to improve drowsiness symptoms and enhance alertness; melatonin, melatonin receptor agonists, and hypnotic sedatives can be used to promote sleep; and hormone-like drugs such as caffeine, melatonin, cordycepin, and dexamethasone can also be used for jet lag adjustment. However, all of these drugs suffer from low efficacy, high tolerability, and certain adverse reactions, making them unsuitable for long-term use and failing to meet the needs of regulating circadian rhythms, improving the work capacity of personnel in special environments, and providing medical support. Therefore, developing highly effective and low-toxic drugs to improve circadian rhythm disorders is of great significance.

[0005] Compound (I) is a common D-glucosamine with immunomodulatory activity. It is commonly used as a food antioxidant and infant formula additive, and also as a drug for the treatment and prevention of osteoarthritis in various parts of the body, including the knee, hip, spine, shoulder, hand and wrist, and ankle. There are currently no reports of its application in regulating circadian rhythm disorders. Summary of the Invention:

[0006] The purpose of this invention is to provide an application of a D-glucosamine compound, wherein the D-glucosamine compound is a 2-amino-2-deoxy-D-glucose compound as shown in formula (I), and the application of the compound shown in formula (I), its pharmaceutical salt, or its pharmaceutical composition in improving circadian rhythm disorders, wherein the compound has the following specific structure:

[0007]

[0008] The present invention includes pharmaceutical salts of compounds of formula (I), including inorganic acid salts and organic acid salts, such as hydrochloride, phosphate, sulfate, acetate, maleate, methanesulfonate, fumarate, citrate, benzenesulfonate, methylbenzenesulfonate or tartrate.

[0009] The dosage of the compound of formula (I) or its pharmaceutical salt is as follows. Although the dosage varies depending on the patient, route of administration, symptoms, and other factors, the usual dosage for oral or non-oral administration to adults intended to regulate circadian rhythms is about 0.1 mg / kg to 1000 mg / kg body weight, preferably about 0.1 mg / kg to 800 mg / kg body weight, more preferably 0.5 mg / kg to 500 mg / kg body weight, and can be administered once or several times daily. The present invention also discloses a pharmaceutical composition for regulating circadian rhythms, comprising a 2-amino-2-deoxy-D-pyranose compound of formula (I) or its pharmaceutical salt, and pharmaceutically acceptable excipients.

[0010] The compounds of formula (I) of the present invention, and their pharmaceutical salts, can be safely administered orally or non-orally, or, when combined with pharmaceutically acceptable excipients such as carriers, excipients, and other additives, to form pharmaceutical compositions such as tablets, sustained-release formulations, sugar-coated formulations, capsules, injections, solutions, etc., can be safely administered orally or non-orally. For the preparation of oral pharmaceutical compositions, lactose or starch can be used as carriers, and gelatin, sodium carboxymethyl cellulose, methylcellulose, polyvinylpyrrolidone, etc., are suitable binders or granulating agents. Starch or microcrystalline cellulose can be used as disintegrants, and talc, colloidal silica gel, glyceryl stearate, calcium stearate, or magnesium stearate are commonly used as suitable anti-adhesion agents and lubricants. For example, tablets can be prepared by compressing wet granules. The active ingredient is mixed with a carrier and, selectively, a disintegrant additive. This mixture is then granulated with an aqueous, alcoholic, or aqueous-alcoholic solution of a binder in a suitable device. The dried granules are then compressed into tablets by adding other disintegrants, lubricants, and anti-adhesion agents.

[0011] The compound of formula (I) of this invention is readily soluble in water. To increase solubility, heterocyclic derivatives can be freed and converted into pharmaceutically acceptable inorganic acids and organic acid salts, preferably salts of maleic acid, mesylate, and fumarate, to facilitate parenteral administration in injectable form. When preparing the injectable dosage form, the active ingredient, compound of formula (I) or its pharmaceutical salt, is dissolved in distilled water or various organic solvents. Co-solvents such as sorbitol monolaurate, monostearate, or monooleate may also be added. The injectable may also contain various commonly used additives and preservatives. Before filling ampoules, the injectable must be filtered, and sterilized after filling.

[0012] In different dosage forms, the content of compound (I) or its pharmaceutical salt can be adjusted as needed, and in oral dosage forms such as tablets or capsules, the weight concentration can be 10%-30%. The effect of the compound of the present invention in regulating circadian rhythm has been verified experimentally.

[0013] The application of compound (I) of this invention has been verified through experiments. In vitro experiments confirmed that compound (I) of this invention can promote the phase shift of the circadian rhythm in U2OS cells. In vivo experiments in mice confirmed that compound (I) has a significant promoting effect on the synchronization of circadian rhythms 8 hours after light exposure shift, can significantly reduce the synchronization time of circadian rhythm phase shift, and can regulate the expression of hypothalamic circadian clock proteins Per2, Bmal1 and related phosphorylated proteins P-Per2, P-CaMKII, which play an important role in regulating circadian rhythms and improving circadian rhythm disorders. In summary, compound (I) of this invention or its pharmaceutical salts show a significant effect in improving circadian rhythm disorders, and therefore can be used to regulate circadian rhythms, regulate circadian rhythm disorders caused by time difference, shift work, continuous multi-day and multi-night work, and circadian rhythm-related sleep disorders.

[0014] The present invention also discloses the use of pharmaceutical compositions containing a compound of formula (I) or a pharmaceutical salt thereof in the preparation of drugs for regulating circadian rhythms, wherein regulating circadian rhythms includes adjusting circadian rhythm disorders caused by time difference, shift work, continuous multi-day and multi-night work, and circadian rhythm-related sleep disorders. Attached image description:

[0015] Figure 1 To evaluate the effect of drug administration on the phase shift of the U2OS cell circadian rhythm by administering drugs to U2OS cells after synchronization treatment at CT0 (CT is a standard time unit).

[0016] Figure 2 After synchronizing U2OS cells, drugs were administered at CT0, CT4, CT8, CT12, CT16, CT20, and CT24 to evaluate the effect of drug administration at different times on the phase shift of the U2OS cell circadian rhythm.

[0017] Figure 3 To evaluate the effects of drug administration twice at the original circadian rhythm time ZT15 (Zeitgeber Time, a time unit based on time difference cycle, where ZT0 is the time when the light is on and activity begins), on the phase synchronization and instantaneous advance of the circadian rhythm in mice after a shift in light exposure time by 8 hours.

[0018] Figure 4 To evaluate the effect of drugs on the expression levels of circadian clock proteins in U2OS cells, mouse liver, and mouse hypothalamus 12 hours after administration of CT0 or ZT0 at the circadian rhythm. Detailed implementation method:

[0019] To more fully explain the implementation of the present invention, formulation preparation examples one through four are provided. These examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0020] Example 1: Preparation of tablets containing compound (I) of the present invention

[0021]

[0022]

[0023] The active ingredients, starch, and cellulose were sieved and thoroughly mixed. A polyvinylpyrrolidone solution was mixed with the powders and sieved again to obtain wet granules, which were then dried at 50-60°C. Sodium carboxymethyl starch, magnesium stearate, and talc were pre-sieved and then added to the granules for tableting. Compound (I) was provided by Tianfang Pharmaceutical Co., Ltd., and all other reagents were commercially available. The following examples are similar.

[0024] Example 2: Preparation of sustained-release tablets containing compound (I) of the present invention

[0025] composition Dosage / tablet (mg) Weight concentration (%) Compound of formula (I) 10 10.0 Utch RS-PO 10 10.0 lactose 40 40.0 Calcium hydrogen phosphate 20 20.0 total 100 100.0

[0026] The compound of formula (I), lactose, and dicalcium phosphate were sieved and thoroughly mixed. The Eutechi RS-PO was then mixed with the powder and tableted using a wet granulation process.

[0027] Example 3: Preparation of capsules containing compound (I) of the present invention

[0028] The preparation of capsules containing 100mg of active ingredient per capsule is as follows:

[0029]

[0030] The above active ingredients are thoroughly mixed, sieved, and the resulting granules are dried at 50-60℃. The granule content is measured, the fill weight is calculated, and the granules are then filled into capsules.

[0031] Example 4: Preparation of an injectable formulation comprising compound (I) of the present invention

[0032] composition Dosage Compound of formula (I) 200mg Mannitol 700mg PEG3000 10mg distilled water 100ml

[0033] Adjust the pH to 7.0-7.5, filter the solution to a concentration of 3 mg / ml, dispense 2 ml per ampoule, and freeze-dry to obtain the injection solution.

[0034] Example 5: After U2OS cells were synchronized, CTO was administered to evaluate the effect of the drug on the phase shift of the U2OS cell circadian rhythm.

[0035] 1. Experimental Materials

[0036] Experimental subject: Per2-dLucU2OS cells.

[0037] Experimental equipment: CO2 incubator, microscope, Lumicycle instrument (Actimetrics).

[0038] Experimental reagents: DMEM medium (GIBCO), Australian serum (GIBCO), PBS, DMEM powder, sterile water, HEPES, penicillin and streptomycin, sodium bicarbonate sulfate, gentamicin, DMSO, dexamethasone, etc.

[0039] Experimental drug: Formula (I) D-glucosamine compound was purchased from Sigma-Aldrich.

[0040] 2. Experimental Methods

[0041] Prepare 2×DMEM culture medium: Add one packet of DMEM powder to 470 ml of sterile water containing 10 ml of 1 M HEPES, 10 ml of penicillin and streptomycin, 5 ml of 7.5% sodium bicarbonate solution and 500 mg of gentamicin sulfate, stir thoroughly, adjust the pH value to between 7.2 and 7.4, and filter through a 0.22 μm filter.

[0042] Preparation of XM medium: Add 25 ml of 2×DMEM medium to 25 ml of sterile water containing 1 ml of 100 mM Luciferin and 1 ml of B27.

[0043] Lumicycle monitoring of biological rhythms: Per2-LucU2OS cells or Bmal1-LucU2OS cells were administered at a rate of 5 × 10⁻⁶. 5 Cells were seeded in 3.5 cm culture dishes and reached 100% confluence after 24 hours. XM medium containing 5 mM, 10 mM and 15 mM of compound (I) was prepared. After treating the cells with dexamethasone for 2 hours, the old culture dishes were discarded, and the cells were washed twice with 1 ml PBS and then replaced with 2 ml of XM medium containing compound (I). The cells were sealed with high vacuum grease and sealing film and then placed in a Lumicycle instrument for real-time monitoring of fluorescence intensity.

[0044] Data analysis: Real-time monitoring data was analyzed using LumicycleAnalysis software to examine phase changes and compared with a control group.

[0045] 3. Experimental Results

[0046] Schematic diagrams of phase shifts and their statistical results after Per2-LucU2OS cells were administered with 5mM, 10mM, and 15mM of compound (I) are shown below. Figure 1As shown, compared with the control group, the phase shift was 5.57 ± 0.62 h after administration of 5 mM compound (I) (P < 0.0001, statistically significant), 13.23 ± 0.96 h after administration of 10 mM compound (I) (P < 0.0001, statistically significant), and 17.44 ± 1.02 h after administration of 15 mM compound (I) (P < 0.0001, statistically significant).

[0047] 4. Experimental Conclusions

[0048] Compound (I) can promote a phase shift in the U2OS cell rhythm, and this effect is dose-dependent.

[0049] Example 6: After U2OS cells were synchronized, drugs were administered at different times to evaluate the effect of the drugs on the phase shift of the U2OS cell circadian rhythm.

[0050] 1. Experimental Materials

[0051] Experimental subject: Per2-dLucU2OS cells.

[0052] Experimental equipment: CO2 incubator, microscope, Lumicycle instrument (Actimetrics).

[0053] Experimental reagents: DMEM medium (GIBCO), Australian serum (GIBCO), PBS, DMEM powder, sterile water, HEPES, penicillin and streptomycin, sodium bicarbonate sulfate, gentamicin, DMSO, dexamethasone, etc.

[0054] Experimental drug: Formula (I) D-glucosamine compound was purchased from Sigma-Aldrich.

[0055] 2. Experimental Methods

[0056] Prepare 2×DMEM culture medium: Add one packet of DMEM powder to 470 ml of sterile water containing 10 ml of 1 M HEPES, 10 ml of penicillin and streptomycin, 5 ml of 7.5% sodium bicarbonate solution and 500 mg of gentamicin sulfate, stir thoroughly, adjust the pH value to between 7.2 and 7.4, and filter through a 0.22 μm filter.

[0057] Preparation of XM medium: Add 25 ml of 2×DMEM medium to 25 ml of sterile water containing 1 ml of 100 mM Luciferin and 1 ml of B27.

[0058] Lumicycle monitoring of biological rhythms: Per2-LucU2OS cells or Bmal1-LucU2OS cells were administered at a rate of 5 × 10⁻⁶. 5Cells were seeded in 3.5 cm culture dishes and reached 100% confluence after 24 hours. After 2 hours of dexamethasone treatment, the old culture dishes were discarded, and the cells were washed twice with 1 ml PBS and replaced with 2 ml XM medium. The time at this point was recorded as CT0. 5 mM of compound (I) was administered at 0 hours (CT0), 4 hours (CT4), 8 hours (CT8), 12 hours (CT12), 16 hours (CT16), 20 hours (CT20), and 24 hours (CT24). The cells were sealed with high-vacuum sealing grease and sealing film, and then placed in a Lumicycle instrument for real-time monitoring of fluorescence intensity.

[0059] Data analysis: Real-time monitoring data was analyzed using LumicycleAnalysis software to examine phase changes and compared with a control group.

[0060] 3. Experimental Results

[0061] A schematic diagram of phase shifts and statistical results of phase shifts in Per2-LucU2OS cells after administration of 5 mM compound of formula (I) at CT0, CT4, CT8, CT12, CT16, CT20, and CT24 are shown below. Figure 2 As shown, compared with the control group, the phase shift was 8.69 ± 0.23 hours after administration of 5 mM compound (I) to CT0 (P < 0.0001, statistically significant); 5.50 ± 0.46 hours after administration of 5 mM compound (I) to CT4 (P < 0.0001, statistically significant); 5.33 ± 1.01 hours after administration of 5 mM compound (I) to CT8 (P < 0.0001, statistically significant); 0.82 ± 0.30 hours after administration of 5 mM compound (I) to CT12 (P = 0.3678, no statistically significant); and 3.80 ± 1.23 hours after administration of 5 mM compound (I) to CT16 (P < 0.0001, statistically significant). After administration of 5 mM compound of formula (I) to CT20, the phase shift was 5.73 ± 0.47 hours (P < 0.0001, statistically significant). After administration of 5 mM compound of formula (I) to CT24, the phase shift was 7.78 ± 0.79 hours (P < 0.0001, statistically significant).

[0062] 4. Experimental Conclusions

[0063] Administration of compound (I) at different times promoted a phase shift in the U2OS cell rhythm to varying degrees, demonstrating that this effect is time-dependent. At CT0 or CT24, administration of compound (I) resulted in the longest phase shift time of the U2OS cell circadian rhythm; at CT12, administration of compound (I) resulted in the shortest phase shift time of the U2OS cell circadian rhythm.

[0064] Example 7: 8-hour light exposure followed by a shift to evaluate the effect of the drug on the phase synchronization of the diurnal rhythm in mice.

[0065] 1. Experimental Materials

[0066] Experimental animals: C57BL / 6J male mice (Speford Biotechnology Co., Ltd.), weighing (25±2)g.

[0067] Experimental apparatus: CLOCKLAB rhythmic biology system (Wuhan Pubai Technology Co., Ltd., model: ACT-556B)

[0068] Experimental drugs: Formula (I) D-glucosamine compound was purchased from Sigma-Aldrich, and melatonin was purchased from MedChemExpress.

[0069] 2. Experimental Methods

[0070] Screening for spontaneous activity in mice: Male C57BL / 6J mice were housed individually in cages equipped with running wheels, each cage receiving approximately 200 lux of light. The running wheel activity of each animal was continuously recorded using a digital system that tracks wheel rotations, with data stored every 6 minutes for further analysis. Mice lived in a normal 12:12 hour light cycle environment for approximately 14 days (light on at CT8, light off at CT20). On day 14, a circadian rhythm phase shift was implemented, delaying the light exposure by 8 hours (i.e., shifting the light on / off time 8 hours later, light on at CT16, light off at CT4). Using the Clocklab circadian rhythm biology system, the animals' adaptation to the new light cycle was observed. Mice with relatively uniform diurnal activity were selected for subsequent experiments, while mice with excessively rapid rhythm adjustments or irregular activity were excluded. Thirty-five mice selected from the initial screening were randomly assigned to four groups: a control group (n=5), a 20 mg / kg melatonin group (n=4), a 150 mg / kg compound (I) group (n=6), a 300 mg / kg compound (I) group (n=6), and a 600 mg / kg compound (I) group (n=5). The mice were administered the drug one hour before the next light adjustment. After administration, the light adjustment was delayed by 8 hours. Administration was given at CT15, and the light adjustment was performed at CT16 (light on at CT0, light off at CT12). A second administration was given 24 hours later, for a total of two administrations, administered via gavage. The mice's movement was continuously recorded and observed using the Clocklab circadian rhythm biology system after administration.

[0071] 3. Experimental Results

[0072] Control group, 20 mg / kg melatonin group, 150 mg / kg compound (I) group, 300 mg / kg compound (I) group, 600 mg / kg compound (I) group, phase adjustment as follows Figure 3As shown in the figure, compared with the control group mice, which took 7.40 ± 0.58 days to adapt to the light shifted 8 hours later, the 20 mg / kg melatonin group required 4.00 ± 0.82 days (P = 0.0004, statistically significant), the 150 mg / kg compound (I) group required 6.67 ± 0.52 days (P > 0.9999, no statistically significant), the 300 mg / kg compound (I) group required 5.50 ± 0.55 days (P = 0.046, statistically significant), and the 600 mg / kg compound (I) group required 5.00 ± 0.00 days (P = 0.0061, statistically significant).

[0073] 4. Conclusion

[0074] After delaying the light-on / off time by 8 hours, the original circadian rhythm was disrupted. Mice treated with 300 mg / kg and 600 mg / kg of compound (I) showed accelerated adaptation to light adjustment, while the effect of 150 mg / kg of compound (I) was not significant. Figure 3 B. Figure 3 As shown in Figure C, under the influence of compound (I) at 300 mg / kg and 600 mg / kg, it is evident that the effect of 600 mg / kg is more pronounced than that of 300 mg / kg. Therefore, the speed at which mice adapt to changes in light intensity increases with increasing dose of compound (I), demonstrating a dose-dependent effect. It can also be seen that the effects of 300 mg / kg and 600 mg / kg of compound (I) in accelerating light adaptation in mice are weaker than that of 20 mg / kg melatonin.

[0075] Example 8 evaluates the effect of the drug on the expression levels of circadian clock proteins in U2OS cells, mouse hypothalamus, and mouse liver.

[0076] 1. Experimental Materials

[0077] Experimental animals: C57 / B6J male mice (Speford Biotechnology Co., Ltd.), weighing (25±2) g.

[0078] Experimental instruments: tissue homogenizer (Shanghai Jingxin Technology), microplate reader, electrophoresis apparatus (Bio-Rad Laboratories, USA), Amersham Imager 680 (GE).

[0079] Experimental reagents: RIPA lysis buffer (Beyotime), protease inhibitor mixture (Solepro), phosphatase inhibitor (Beyotime), BCA protein concentration assay kit (Beyotime), 5× protein loading buffer (Beyotime), skim milk powder (BD), 1.5M Tris-HCl buffer (Solepro), 1.0M Tris-HCl buffer (Solepro), 10% SDS (Solepro), AP (ThermoScientific), Tris (Biotopped), glycine (Biotopped), P-AMPK antibody (CST), AMPK antibody (CST), P-mTOR antibody (CST), mTOR antibody (CST), P70S6K antibody (CST), Bmal1 antibody (CST), P-Bmal1 antibody (CST), ACTB antibody (Sangon Biotech), ECL luminescent solution (Mice).

[0080] Experimental drugs: Compound (I) and N-acetylglucosamine (GlcNAc, negative control) were purchased from Sigma-Aldrich.

[0081] 2. Experimental Methods

[0082] Cell protein extraction: U2OS cells were seeded into six-well plates and synchronized with dexamethasone. Protein lysis buffer was added at a rate of 100 μl / well, cells were scraped off, and transferred to 1.5 ml EP tubes. The cells were centrifuged at 12000 rpm for 10 min at 4°C. The supernatant was transferred to a clean EP tube. Protein concentration was determined using a BCA protein assay kit combined with a microplate reader. Once the protein concentration was determined, the appropriate volume of 5× protein loading buffer was added, mixed well, and boiled at 100°C for 10 minutes, followed by cooling in ice water.

[0083] Tissue protein extraction: Mice with normal circadian rhythms were administered ZT12 via gavage at a dose of 300 mg / kg (control solvent, compound of formula I, GlcNAc). Twelve hours later, the hypothalamus and liver were harvested and rapidly frozen in liquid nitrogen. Protein lysis buffer (RIPA:phosphatase inhibitor:protease inhibitor = 970:29:10) was then added at a ratio of 10 mg / 100 μl, and the samples were homogenized using a tissue homogenizer at 60 Hz for 20 seconds. After homogenization, all samples were centrifuged at 12000 rpm for 10 minutes at 4°C, and the supernatant was transferred to clean EP tubes. Protein concentration was determined using a BCA protein assay kit combined with an ELISA reader. Once the protein concentration was determined, the appropriate volume of 5× protein loading buffer was added, mixed, and boiled at 100°C for 10 minutes, followed by ice water cooling.

[0084] Western Blot experiment: 30 μg of cellular protein and 100 μg of animal tissue protein were loaded. Electrophoresis conditions were 100 V for 80 minutes, and transfer conditions were 200 mA for 90 minutes. After transfer, the PVDF membrane was blocked with TBST containing 5% skim milk and slowly shaken at room temperature for 1 hour. After blocking, the PVDF membrane was transferred to primary antibody and slowly shaken at 4°C overnight. The next day, the PVDF membrane was washed three times with TBST for 10 minutes each time. Then, the PVDF membrane was transferred to HRP-conjugated secondary antibody and shaken at room temperature for 1 hour. After shaking at room temperature with the secondary antibody, the PVDF membrane was washed three times with TBST for 10 minutes each time. ECL chemiluminescence buffer was prepared at a 1:1 ratio and evenly added to the PVDF membrane. Imaging was performed on an AI680 system, with ACTB used as an internal control protein.

[0085] 3. Experimental Results

[0086] Drug administration was performed at circadian rhythm times CT0 or ZT0 (control solvent, compound I, N-acetylglucosamine), and cells and tissues were harvested 12 hours later for protein analysis. Results showed that, compared to the solvent control group and the N-acetylglucosamine group, compound (I) significantly upregulated P-AMPK / AMPK in U2OS cells, mouse hypothalamus, and mouse liver 12 hours after administration, and downregulated P-mTOR / mTOR, P70S6K, BMAL1, and P-BMAL1 (…). Figure 4 ).

[0087] 4. Conclusion

[0088] Twelve hours after administration of CT0 or ZT0 at the circadian rhythm, compound (I) significantly upregulated P-AMPK / AMPK in U2OS cells, mouse hypothalamus, and mouse liver at the protein level, and downregulated the expression of P-mTOR / mTOR, P70S6K, BMAL1, and P-BMAL1.

Claims

1. Use of a D-glucosamine compound or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for modulating circadian rhythm, wherein the D-glucosamine compound is a 2-amino-2-deoxy-D-glucopyranose compound of formula (I) ###0001### wherein modulating circadian rhythm includes modulating circadian rhythm disorders or circadian rhythm-related sleep disorders caused by jet lag, shift work, or continuous multiple-day work. （I） 2. The use according to claim 1, wherein the pharmaceutically acceptable salt of the compound of formula (I) is a pharmaceutically acceptable inorganic or organic acid salt.

3. The use according to claim 1 or 2, wherein the pharmaceutically acceptable salt of the compound of formula (I) is a hydrochloride, a phosphate, a sulfate, an acetate, a maleate, a mesylate, a fumarate, a citrate, a besylate, a methylbesylate, or a tartrate.

4. Use of a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for modulating circadian rhythm, wherein modulating circadian rhythm includes modulating circadian rhythm disorders or circadian rhythm-related sleep disorders caused by jet lag, shift work, or continuous multiple-day work.

5. The use according to claim 4, wherein the compound of formula (I) or a pharmaceutically acceptable salt thereof is contained in an amount of 10% to 30%. The pharmaceutical composition is in the form of a tablet, a sustained-release tablet, a capsule, or an injection.

6. Use according to claim 4, characterized in that 6. The use according to any one of claims 1 to 5, wherein the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in an amount of 0.1 to 10 mg per day per 1 kg of body weight.

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