Application of dimethyl succinate in preparation of medicine for treating meningeal lymphatic vessel drainage dysfunction related diseases caused by sleep disorder

Abstract

The invention discloses an application of dimethyl succinate in preparation of a medicine for treating meningeal lymphatic vessel drainage dysfunction related diseases caused by sleep disorders. Dimethyl succinate is injected into the stomach of a mouse with sleep disorder, the meningeal lymphatic vessel drainage function of the mouse is obviously improved, and dimethyl succinate can penetrate through the blood brain barrier to directly act on the meningeal lymphatic vessel, so that the problem that the treatment means in the prior art is difficult to surmount the blood brain barrier is solved; new possibility is provided for treating meningeal lymphatic vessel drainage dysfunction; the dimethyl succinate achieves a remarkable treatment effect only through oral administration and intragastric administration, so that the operation difficulty and the infection risk are reduced, and the administration convenience is realized.

Description

Technical Field:

[0001] The present invention relates to the technical field of drug research and development, and particularly to the use of dimethyl succinate in the preparation of a drug for treating diseases related to meningeal lymphatic drainage dysfunction caused by sleep disorders. Background Art:

[0002] Aging has led to cerebrovascular diseases becoming one of the main causes of death, especially vascular cognitive impairment and dementia. Research shows that the brain's ability to clear waste weakens with age, which may accelerate neurodegenerative diseases. Therefore, studying the cerebral vascular system is crucial for the prevention and treatment of neurological diseases. In recent years, progress has been made in cerebrovascular hydrodynamics research, revealing the core role of neural activity in regulating the movement of brain tissue fluid, especially during slow-wave sleep. The synergistic effect of neural activity and fluid flow emphasizes the importance of sleep for brain health, and aging may affect this process.

[0003] The lymphatic system is crucial for maintaining fluid balance and internal environmental homeostasis. Meningeal lymphatic vessels (MLVs) are responsible for draining macromolecules, clearing waste, and transporting immune cells. MLVs are associated with various central nervous system diseases, and their function weakens with age. However, by injecting VEGF-C protein, the drainage and clearance ability of MLVs can be enhanced, and cognitive impairment can be slowed down. In 2021, research at the University of Virginia found a close link between MLVs and Alzheimer's disease. After enhancing the function of MLVs, the pathology and cognitive function of model mice were improved. In 2024, research at Hainan University found that near-infrared light therapy can improve the structure and function of MLVs, having a positive impact on Alzheimer's disease mice. As an emerging technology, lymphaticovenular anastomosis also shows potential in the treatment of Alzheimer's disease. These findings indicate that targeting lymphatic drainage function is a potential method for treating central nervous system diseases.

[0004] Currently, there is still a lack of definite treatment means for the changes in meningeal lymphatic drainage function caused by sleep disorders. Although intracisternal injection of recombinant VEGF-C protein is a potential treatment method, this operation has high technical requirements and there is a risk of infection. Therefore, there is a need to find a method that can improve meningeal lymphatic drainage under sleep disorders and reduce the accumulation of metabolic waste in the brain, in order to improve the resulting decline in short-term memory and learning ability. Dimethyl succinate is widely used in the field of pharmaceutical synthesis. It can be used as a raw material or intermediate for various pharmaceutical syntheses. For example, miglitol analogs for lowering blood sugar can be synthesized using it as a raw material. However, there is currently no report that dimethyl succinate can improve nervous system degenerative diseases related to meningeal lymphatic dysfunction caused by sleep disorders. Summary of the Invention:

[0005] The present invention provides the use of dimethyl succinate in the preparation of a drug for treating neurological degenerative diseases associated with meningeal lymphatic drainage dysfunction caused by sleep disorders. By intragastric administration of a low dose of dimethyl succinate, the meningeal lymphatic drainage function can be effectively improved, solving the problems in the prior art of ineffective passage through the blood-brain barrier, extremely high operation difficulty, and easy infection when using recombinant VEGF-C protein injection to improve meningeal lymphatic drainage dysfunction, and providing a new approach for the improvement and treatment of meningeal lymphatic drainage dysfunction.

[0006] The present invention is achieved by the following technical solutions:

[0007] Use of dimethyl succinate in the preparation of a drug for treating neurological degenerative diseases associated with meningeal lymphatic drainage dysfunction.

[0008] Preferably, the neurological degenerative disease includes Alzheimer's disease.

[0009] Preferably, the meningeal lymphatic drainage dysfunction is caused by sleep disorders.

[0010] Preferably, the drug exerts its effect by increasing the meningeal lymphatic drainage function and the efflux of intracranial metabolites.

[0011] Preferably, the drug uses dimethyl succinate as the active ingredient and is made into any pharmaceutically acceptable dosage form.

[0012] Preferably, the dosage form is an oral preparation.

[0013] In sleep-disordered mice, there was a significant decline in short-term memory and learning ability, and this cognitive decline was directly related to the drainage function of meningeal lymphatic vessels (MLVs). Therefore, treatment targeting MLVs is expected to be an effective strategy for alleviating memory and learning disorders caused by sleep disorders. Preliminary studies showed that succinate dehydrogenase in meningeal lymphatic endothelial cells in a sleep-disordered mouse model is an effective therapeutic target and can improve the drainage dysfunction caused by sleep disorders. Based on this, it is speculated that succinate, as a metabolic substrate, may have similar efficacy.

[0014] Sleep-disordered mice were treated by intragastric administration of dimethyl succinate according to the body weight ratio. The results showed that after continuous intragastric administration of dimethyl succinate for seven days, the meningeal lymphatic drainage function of the mice was significantly improved, and the effect was consistent with that of intracisternal injection of VEGF-C adeno-associated virus, and was particularly obvious compared with the control group that did not receive intragastric treatment. This confirmed the effectiveness of dimethyl succinate in improving the meningeal lymphatic drainage function, and at the same time verified the safety of dimethyl succinate to cells through a cell model. This discovery provides a new approach for treating cognitive decline related to sleep disorders.

[0015] The present invention has the following beneficial effects:

[0016] 1. The present invention provides the use of dimethyl succinate in the preparation of a drug for treating neurological degenerative diseases related to meningeal lymphatic drainage dysfunction caused by sleep disorders. By intragastric administration of dimethyl succinate to sleep-disordered mice, it was found that the meningeal lymphatic drainage function of the mice was significantly improved, indicating that dimethyl succinate has the potential to become a drug for treating or improving meningeal lymphatic dysfunction.

[0017] 2. In the present invention, dimethyl succinate can penetrate the blood-brain barrier and directly act on the meningeal lymphatics, which is difficult to achieve by many existing treatment means. It effectively solves the problem that it is difficult for treatment means such as recombinant VEGF-C protein and AAV to cross the blood-brain barrier, providing new possibilities for treating meningeal lymphatic drainage dysfunction.

[0018] 3. In the present invention, dimethyl succinate achieved a significant therapeutic effect only through oral gavage. Compared with the operation of injecting recombinant VEGF-C protein into the cisterna magna, it has the convenience of drug administration, greatly reducing the operation difficulty and infection risk, and improving the feasibility of treatment and patient compliance. Description of the Drawings:

[0019] Figure 1 It is the result and statistical chart of the reduction of short-term learning and memory ability in mice after 7 days of sleep deprivation in the embodiment;

[0020] Figure 2 It is the result and statistical chart of the reduction of meningeal lymphatic drainage function in mice after 7 days of sleep deprivation in the embodiment;

[0021] Figure 3 It is the result and statistical chart of the improvement of the reduction of short-term memory and learning ability in mice caused by sleep deprivation after 3 weeks of injection of AAV9-VEGF-C adeno-associated virus into the cisterna magna in the embodiment;

[0022] Figure 4 It is the result and statistical chart of the improvement of the reduction of meningeal lymphatic drainage function in mice caused by sleep deprivation after 3 weeks of injection of AAV9-VEGF-C adeno-associated virus into the cisterna magna in the embodiment;

[0023] Figure 5 It is the result diagram of the improvement of the reduction of meningeal lymphatic drainage function caused by sleep deprivation after intragastric administration of dimethyl succinate in the embodiment. Detailed Embodiments:

[0024] The following embodiments are further descriptions of the present invention, rather than limitations on the present invention.

[0025] Unless otherwise defined, the technical terms used in the following examples have the same meanings as commonly understood by those skilled in the art to which the present invention pertains. The test reagents used in the following examples are all conventional biochemical reagents unless otherwise specified; the experimental methods are all conventional methods unless otherwise specified.

[0026] Example 1 The drainage function of meningeal lymphatics is closely related to the short-term memory ability of mice

[0027] I. Experimental methods

[0028] 1. Experimental animals

[0029] In this study, half of the male and female C57BL / 6J background wild-type (WT) mice aged 8 to 12 weeks were purchased from Jiangsu Jicui Biotechnology Co., Ltd., China. The production license number of experimental animals was SCXK (Su) 2015-0001. The mice were raised in an environment with a 12-hour light-dark cycle, and food and water were freely available: SPF-class barrier environment (temperature 22 ± 1 °C, humidity 50 ± 10%). A 12h:12h artificial light-dark cycle regulation was adopted (light period 08:00-20:00). Standardized laboratory rodent feed and sterilized drinking water were freely available through an automatic supply system. The bedding in the cages was changed weekly, and pathogenic microorganism monitoring was implemented. All animal experiment operations were approved by the Experimental Animal Ethics Committee of Zhongshan Ophthalmic Center, Sun Yat-sen University, and strictly followed the experimental animal research specifications.

[0030] 2. Modeling method

[0031] After the mice were placed in the sleep deprivation instrument for one week to adapt, the sleep deprivation instrument was started, and sleep deprivation was carried out at a speed of 7 revolutions per minute (see the literature: Vaccaro A, Kaplan Dor Y, Nambara K, Pollina EA, Lin C, Greenberg ME, Rogulja D. Sleep Loss Can Cause Death through Accumulation of Reactive Oxygen Species in the Gut. Cell. 2020 Jun 11;181(6):1307-1328.e15.doi:10.1016 / j.cell.2020.04.049.Epub 2020 Jun 4.PMID:32502393.). In this experiment, the mice were sleep-deprived for seven days. If there were any accidentally dead mice in the experiment, we used new mice for replacement.

[0032] 3. Experimental design

[0033] Experiment A: To detect the relationship between 7-day sleep deprivation and the short-term memory learning ability of mice, a completely random stratified grouping strategy was adopted. Thirty-two C57BL / 6J mice (half male and half female, 10±2 weeks old) were divided according to the principle of balanced gender ratio into: (1) Mice with normal sleep schedule were used as the baseline control group (Ctrl, 16 mice in each group); (2) According to the above modeling method, mice were sleep deprived for 7 days and used as the 7-day sleep deprivation group (SD7, 16 mice in each group). After 7 days of sleep deprivation, we performed standardized behavioral tests on the two groups of mice and randomly selected 6 mice from each group for cisterna magna injection of fluorescent microglobulin tracer (Beads) to detect the meningeal lymphatic drainage function.

[0034] Experiment B: To confirm that increasing the meningeal lymphatic drainage function can improve the effects of sleep deprivation on the short-term memory and learning ability of mice, we injected AAV9-VEGF-C adeno-associated virus into the cisterna magna of mice while sleep depriving them for 7 days, and detected the behavioral ability and meningeal lymphatic drainage function of mice 3 weeks after virus injection. Our experiment used the following grouping strategy: (1) The group injected with control virus into the cisterna magna while sleep depriving for 7 days (AAV-Ctrl+SD7, as the control), 5-8 mice in each group; (2) The group injected with AAV9-VEGF-C adeno-associated virus into the cisterna magna while sleep depriving for 7 days (AAV-VEGF-C+SD7), 5-8 mice in each group; Both AAV9-VEGF-C adeno-associated virus and control virus are serotype AAV9, carrying the CMV promoter. The control virus is a nonsense sequence and will not affect the biological functions of mice. Both viruses were purchased from Shandong Vigene Biosciences Co., Ltd.; The virus titer is 1×1013 vg / mL, the injection volume is 3 μL, and the rate is 0.1 μL / min.

[0035] The specific steps for injecting virus into the cisterna magna are as follows: Pentobarbital sodium (concentration of 1.5%) was intraperitoneally injected to achieve surgical anesthesia level. The mice were fixed on a digital stereotaxic apparatus for the brain, and the incisor hook was adjusted so that the bregma and lambda were on the same horizontal plane. The atlanto-occipital membrane was exposed under a surgical microscope, and a 34G ultra-fine puncture needle was used to establish an injection channel, and then 3 μL of AAV9-VEGF-C adeno-associated virus or control virus was injected. The needle was withdrawn 10 minutes later.

[0036] Experiment C: To detect the effects of intragastric administration of dimethyl succinate on the short-term memory and learning ability of sleep-deprived mice, the mice were grouped as follows: (1) The group that was intragastrically administered dimethyl sulfoxide phosphate solution and sleep-deprived for seven days (SD7+DMSO, as a control), with 6 mice in each group; (2) The group that was intragastrically administered dimethyl succinate solution and sleep-deprived for 7 days (SD7+Succinate), with 6 mice in each group. The dimethyl succinate solution was prepared by dissolving 1 g of dimethyl succinate in 0.5 mL of dimethyl sulfoxide and then diluting it to 50 mL with phosphate buffer solution (PBS). The dimethyl sulfoxide phosphate solution was prepared by dissolving 0.5 mL of dimethyl sulfoxide in 49.5 mL of phosphate buffer solution (PBS); the dosing dose was 60 mg / kg, and intragastric administration was carried out at a fixed time period (09:00) every day for seven consecutive days. Then, the two groups of mice were injected with fluorescent microglobulin tracer (Beads) into the cisterna magna to detect the meningeal lymphatic drainage function.

[0037] 4. Detection methods

[0038] 4.1 Behavioral tests

[0039] The behavioral test was novel object recognition. The specific steps were as follows: One week before the experiment, the mice were stroked for 1-2 minutes every day to prevent stress in the mice. Before the experiment, the novel object recognition box without objects was sprayed with 75% alcohol and wiped clean with a tissue. Before the experiment, the experimental mice were transferred to the behavioral laboratory to adapt for 6 hours, and then the objects were prepared for the experiment. Subsequently, the mice were gently taken out of the breeding cage and placed in the lower right corner of the novel object recognition box without objects to allow them to freely explore to familiarize themselves with the environment. Then, two identical objects were placed on the empty diagonal space and fixed with double-sided solid glue to prevent them from moving. Then, the mice were taken out and placed in the lower right corner to allow the mice to freely explore for 10 minutes, and video recording was carried out. Trajectory tracking was performed using ANY-maze6.3 software. After 10 minutes of free exploration, the mice were returned to the cage, and the box was wiped with 75% alcohol again to eliminate the odor inside the box. One hour later, the short-term memory and learning ability of the mice in the next stage were measured. One of the objects was replaced with another object and placed in the mice that had undergone the experiment one hour ago. After the experimenter left the current space, recording was carried out for 10 minutes, and the box was wiped with 75% alcohol again to eliminate the odor inside the box. The ANY-maze behavioral software was used to statistically analyze the short-term memory ability of the mice, and the cognitive index calculation formula was used for statistics. The cognitive index calculation formula was (time around the new object - time around the old object) / (time around the new object + time around the old object)×100%. The larger the cognitive index value, the stronger the short-term memory and learning ability of the mice.

[0040] 4.2 Detection of meningeal lymphatic drainage function by injecting fluorescent microglobulin tracer into the cisterna magna

[0041] The specific steps are as follows: After anesthetizing the mice with 1.5% sodium pentobarbital, expose the cisterna magna. Fix the mice using a stereotaxic apparatus for the brain. Draw 3 μL of fluorescent microspheres with a Hamilton 10 μL syringe, and then inject them into the cisterna magna of the mice (injection rate: 0.5 μL / min). After 30 minutes, remove the deep cervical lymph nodes to observe the meningeal lymphatic vessel drainage function in the mice. Quantitatively analyze the distribution of fluorescent signals in the deep cervical lymph nodes through a laser confocal microscopy imaging system (take pictures under fixed conditions, compare the fluorescent intensity of the lymph nodes per unit area. For specific references, see the literature: Ahn JH, Cho H, Kim JH, Kim SH, Ham JS, Park I, Suh SH, Hong SP, Song JH, Hong YK, Jeong Y, Park SH, Koh GY. Meningeal lymphatic vessels at the skull base drain cerebrospinal fluid. Nature. 2019 Aug;572(7767):62 - 66. doi:10.1038 / s41586 - 019 - 1419 - 5. Epub 2019 Jul 24. PMID:31341278.). Since the main drainage pathway of the meningeal lymphatic vessels is the deep cervical lymph nodes, observing the drainage area of the fluorescent microspheres in the deep cervical lymph nodes can reflect the drainage function of the meningeal lymphatic vessels.

[0042] 5. Data analysis

[0043] All data are expressed as mean ± SEM. The number of samples shown in the legend refers to biological replicates. Statistical analysis is performed using Prism 9.0 software. For comparison between two groups, when the data are normally distributed and the variances are homogeneous, an unpaired Student’s t - test is used for analysis. When the data are normally distributed but the variances are heterogeneous, an unpaired Welch’s t - test is used for analysis. For comparison among multiple groups, one - way analysis of variance or two - way analysis of variance is used, followed by Tukey's post - hoc test. A p - value less than 0.05 is considered statistically significant, and the p - value is marked in the figure, keeping four decimal places.

[0044] II. Experimental results

[0045] 1. In the group of mice sleep - deprived for 7 days, the short - term learning and memory abilities are reduced, and the meningeal lymphatic vessel drainage function is reduced

[0046] Place the two groups of mice in a sleep - deprivation instrument for 7 days of sleep deprivation or maintain normal rest, and then conduct behavioral tests for novel object recognition and tests for meningeal lymphatic vessel drainage function in the mice (Experiment A in the experimental design). The results of the behavioral tests are as Figure 1As shown, the cognitive index of mice in the 7-day sleep deprivation group (SD7) for novel object recognition was significantly lower than that of the baseline control group (Ctrl), indicating that the short-term memory and learning ability of mice were significantly reduced after 7 days of sleep deprivation. The results of the meningeal lymphatic vessel drainage function detection were as follows Figure 2 As shown, the area of fluorescent microspheres drained from the meningeal lymphatic vessels to the deep cervical lymph nodes in the 7-day sleep deprivation group (SD7) of mice accounted for approximately 4.99788% of the total area of the lymph nodes, which was lower than that of the baseline control group (Ctrl). It can be seen that 7 days of sleep deprivation can significantly affect the meningeal lymphatic vessel drainage function and lead to a decline in the short-term learning and memory ability of mice.

[0047] 2. Intracisternal injection of AAV9-VEGF-C adeno-associated virus can rescue the reduction of short-term memory and learning ability in mice caused by sleep deprivation

[0048] Since AAV9-related adenovirus can bind well to the endothelial cells of meningeal lymphatic vessels and significantly promote the significant growth of lymphatic endothelial cells, we injected AAV9-VEGF-C adeno-associated virus or control virus into two groups of mice while sleep-depriving them for 7 days, and then performed behavioral tests and intracisternal injection of fluorescent microspheres to detect the meningeal lymphatic vessel drainage 3 weeks after virus injection (Experiment B in the experimental design). The results of the behavioral tests were as follows Figure 3 As shown, the recognition time of novel objects in the AAV-VEGF-C+SD7 group of mice was improved compared with that in the AAV-Ctrl+SD7 group 3 weeks after injection. The results of the meningeal lymphatic vessel drainage function detection were as follows Figure 4 As shown, the drainage area of fluorescent microspheres in the deep cervical lymph nodes of mice in the AAV-VEGF-C+SD7 group was significantly improved compared with that in the AAV-Ctrl+SD7 group, which also indicates that improving the meningeal lymphatic vessel drainage function can improve the decline of short-term memory and learning ability in mice caused by sleep deprivation. Therefore, in summary, we can see that sleep deprivation can reduce the drainage function of meningeal lymphatic vessels, thereby reducing the short-term memory and learning ability of mice. And improving the meningeal lymphatic vessel drainage function can be used as an effective potential treatment method.

[0049] 3. Dimethyl succinate improves the reduction of meningeal lymphatic vessel drainage function caused by sleep deprivation

[0050] We gavaged two groups of mice with dimethyl succinate or DMSO at a dose of 60 mg / kg while sleep-depriving them for 7 days, and then detected the changes in the meningeal lymphatic vessel drainage function (Experiment C in the experimental design). The results were as follows Figure 5 As shown, the drainage area of fluorescent microspheres in the deep cervical lymph nodes of mice in the SD7+Succinate group was significantly improved compared with that in the SD7+DMSO group, indicating that dimethyl succinate can improve the meningeal lymphatic vessel drainage function.

[0051] In summary, it can be seen that intragastric administration of dimethyl succinate to mice can significantly improve the reduced meningeal lymphatic drainage function caused by sleep deprivation and improve the decline in short-term learning and memory ability of mice caused by sleep disorders.

Claims

1. Application of dimethyl succinate in the preparation of drugs for treating neurodegenerative diseases associated with meningeal lymphatic drainage dysfunction.

2. The use according to claim 1, characterized in that: The neurodegenerative diseases include Alzheimer's disease.

3. The use according to claim 1, characterized in that: The meningeal lymphatic drainage dysfunction is caused by sleep disorders.

4. The use according to claim 1, characterized in that: The drug works by increasing the drainage function of the meningeal lymphatic vessels and the efflux of intracranial metabolites.

5. The use according to claim 1, characterized in that: The medicine uses dimethyl succinate as an active ingredient and is prepared into any pharmaceutically acceptable dosage form.

6. The use according to claim 5, characterized in that: The dosage form is an oral dosage form.

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