Use of VPS-34 inhibitors in the preparation of drugs for improving motor dysfunction

Abstract

The present invention discloses the use of a VPS-34 inhibitor in the preparation of a drug for improving motor dysfunction. The present invention proves through animal experiments that SAR405, a VPS-34 inhibitor, has a significant effect in improving motor function, especially the motor function of elderly patients with motor dysfunction, and improving muscle condition.

Description

Technical Field

[0001] The present invention specifically relates to the use of a VPS-34 inhibitor in preparing a drug for improving motor dysfunction. Background Art

[0002] Elderly people suffer from systemic organ degeneration and multiple chronic diseases (such as stroke, arthritis, etc.), which often lead to motor dysfunction. Although some patients have undergone rehabilitation treatment, they fail to persist for a long time, resulting in varying degrees of joint contracture deformity, limited limb movement, inability to take care of themselves, and decreased quality of life. In order to improve the quality of life of elderly patients with motor dysfunction and reduce the burden on society and family, comprehensive rehabilitation treatments including massage, passive joint movement, muscle strengthening training, sitting and standing balance and transfer training, walking and stair climbing training, ADL training, etc. are also needed in daily life, as well as taking some drugs that relieve neurotrophic nerves, such as compound levodopa and amantadine. These drugs for treating elderly motor dysfunction often have obvious side effects such as mental disorders and abnormal cardiovascular system, so it is necessary to develop new drugs that are beneficial to improving elderly motor dysfunction.

[0003] SAR405 is a low molecular weight PIK3C3 / VPS34 inhibitor with a molecular weight of 443.85 and a molecular formula of C 19 H 21 ClF3N5O2, CAS No. 1523406-39-4, structural formula:

[0004]

[0005] It is highly specific and has low selectivity for other protein kinases and phosphoinositide kinases. SAR405 affects vesicle trafficking from late endosomes to lysosomes and is also a potent autophagy inhibitor that inhibits autophagy in tumor cells and synergizes with MTOR inhibition. There are currently no reports on the efficacy of SAR405 in improving motor dysfunction. Summary of the invention

[0006] In order to solve the above problems, the present invention provides the use of a VPS-34 inhibitor in the preparation of a drug for improving motor dysfunction.

[0007] Furthermore, the VPS-34 inhibitor includes a VSP-34 gene knockout or knockdown agent, SAR405, preferably SAR405.

[0008] Furthermore, the medicine is a medicine for improving motor dysfunction in the elderly.

[0009] Furthermore, the drug is a drug for improving motor function in the elderly.

[0010] Furthermore, the drug is a drug that promotes the release of synaptic vesicles in the aged nervous system.

[0011] Furthermore, the medicine is a medicine for improving the muscle condition of the elderly.

[0012] Furthermore, the drug is a preparation prepared with VPS-34 inhibitor as an active ingredient and pharmaceutically acceptable excipients.

[0013] Furthermore, the preparation is an injection.

[0014] Furthermore, the concentration of the VPS-34 inhibitor in the injection is 5 to 50 μM.

[0015] Furthermore, the concentration of the VPS-34 inhibitor in the injection is 20 μM.

[0016] The VPS-34 inhibitor of the present invention is used in the preparation of a drug for improving motor dysfunction. Animal experiments have shown that SAR405, a VPS-34 inhibitor, has a significant effect in improving motor function, especially the motor function of elderly patients with motor dysfunction, and improving muscle condition.

[0017] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0018] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 SAR405 improves the movement of N2 nematodes. A: Bodybends of N2 nematodes on the 9th day after treatment with different concentrations of SAR405. B: Thrashing of N2 nematodes on the 9th day after treatment with different concentrations of SAR405. C: Bodybends of N2 nematodes on the 11th day after treatment with different concentrations of SAR405. D: Thrashing of N2 nematodes on the 11th day after treatment with different concentrations of SAR405. ns, no significance, not significant; ***P<0.001; ****P<0.0001.

[0020] Figure 2SAR405 improves the motility of N2 nematodes during aging. A: Body bends of N2 nematodes during aging under SAR405 treatment. B: Thrashing of N2 nematodes during aging under SAR405 treatment. SAR405 concentration was 20 μM. **P<0.01; ***P<0.001; ****P<0.0001.

[0021] Figure 3 SAR405 improves the motility of N2 nematodes in the middle and late stages. A: Body bends of N2 nematodes treated with SAR405 from the 5th and 7th days with aging. B: Thrashing of N2 nematodes treated with SAR405 from the 5th and 7th days with aging. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.

[0022] Figure 4 SAR405 promotes synaptic vesicle release in aged nematodes. A: Statistics of the frequency of miniature postsynaptic currents (mPSCs) in young (Day 3) and old (Day 11) N2 nematodes and the old group treated with SAR405. B: Statistics of the amplitude of miniature postsynaptic currents (mPSCs) in young (Day 3) and old (Day 11) N2 nematodes and the old group treated with SAR405. The concentration of SAR405 was 20 μM. **P<0.01; ***P<0.001.

[0023] Figure 5 SAR405 improves exercise capacity and muscle condition in aged mice. A: SAR405 treatment regimen for aged mice. B: Whole-body images of aged mice after treatment; C: Body weight of mice on days 15 and 30 after the start of treatment; D: Distances that aged mice ran to exhaustion on a treadmill at different treatment periods; E: Representative staining images of Laminin protein (green) and DAPI (blue) in cross-sections of tibialis anterior (TA) muscles. Scale bar: 50 μm. F: Average cross-sectional area of ​​TA muscle fibers in control and SAR405-treated aged mice. G: Representative images of succinate dehydrogenase (SDH) staining on TA muscles of young mice and SAR405-treated aged mice. Scale bar: 50 μm. H: Quantification of average SDH intensity in TA muscle fibers. DETAILED DESCRIPTION

[0024] Example 1: Study on the effect of VPS-34 inhibitor on alleviating motor decline in the elderly

[0025] Previous experiments have determined that knocking down vps-34 can improve the locomotor ability of nematodes through gene regulation. Based on this, an inhibitor, SAR405, was found, which competitively binds to the ATP binding site in the VPS-34 structure, resulting in the inability of phosphate in ATP to transfer to PI, resulting in the inhibition of PI(3)P production. The specific effects of the ATP-competitive VPS34 inhibitor SAR405 on locomotor decline are studied as follows:

[0026] 1. Effects of different concentrations of SAR405 on the improvement of nematode movement

[0027] 1.1 Methods

[0028] SAR405 was dissolved in DMSO to prepare SAR405 solutions with gradient concentrations, which were dripped onto the nematode growth medium containing E. coli OP50, and N2 nematodes synchronized to adulthood (day 1) were cultured therein. The body bending frequencies (body bends) and twisting frequencies (thrashing) of the nematodes on days 9 and 11 of culture were counted.

[0029] Among them, the body bends counting method: pick the nematodes on the solid culture medium containing OP50, wait for the nematodes to adapt for about 10 seconds, and then count the total number of nematodes swinging in a sinusoidal shape within 1 minute. A total of 15 nematodes were counted, and the results were repeated 3 times independently.

[0030] Thrashing statistical method: Pick nematodes and place them in M9 buffer. After the nematodes adapt for about 10 seconds, count the total number of C-shaped thrashings within 1 minute. A total of 15 nematodes are counted and repeated 3 times independently.

[0031] 1.2 Results

[0032] For specific results, see Figure 1 ,from Figure 1 It can be seen that on the 9th day, the bodybends of nematodes in the 20μM and 50μM SA405 treatment groups increased significantly ( Figure 1 A), thrashing of nematodes was significantly increased in 10μM, 20μM and 50μM SAR405 ( Figure 1 B); On the 11th day, the body bends of nematodes in the 20μM and 50μM SAR405 treatment groups increased significantly ( Figure 1 C), and thrashing of nematodes was increased at all SAR405 concentrations ( Figure 1 D). This series of results showed that the inhibitor SAR405 significantly improved the movement ability of nematodes. Combining the data of each group, SAR405 at a concentration of 20μM had the best effect on improving the movement of nematodes.

[0033] 2. Effect of the optimal concentration of SAR405 on the improvement of locomotor decline in nematodes during aging

[0034] 2.1 Methods

[0035] SAR405 was dissolved in DMSO to prepare a 20 μM SAR405 solution, which was added dropwise to the nematode growth medium containing Escherichia coli OP50. N2 nematodes synchronized to adulthood (day 1) were cultured therein, and the movement ability, i.e., the body bending frequency (body bends) and twisting (thrashing) frequency of the nematodes, on days 3, 5, 7, 9, 11, and 13 were counted.

[0036] 2.2 Results

[0037] For specific results, see Figure 2 ,from Figure 2 It can be seen that on the 3rd and 5th days after the inhibitor treatment, there was no significant change in body bends and thrashing. Starting from the 7th day, SAR405 significantly increased the body bends and thrashing of N2 nematodes ( Figure 2 A, B). This result indicates that SAR405 can significantly improve the motility of nematodes during the aging process.

[0038] 3. Effect of the optimal concentration of SAR405 on the improvement of motor decline in nematodes in the middle and late stages of aging

[0039] Previous experiments have found that RNAi can improve the movement of nematodes in the middle and late stages of adult nematodes. If SAR405 can also improve movement after treating nematodes in the middle and late stages, it will have encouraging potential in clinical practice.

[0040] 3.1 Methods

[0041] SAR405 was dissolved in DMSO to prepare a 20 μM SAR405 solution, which was added dropwise to the nematode growth medium containing Escherichia coli OP50. N2 nematodes synchronized to the middle-aged and old age period (5th and 7th days) were cultured therein, and the movement ability of the nematodes, i.e., the frequency of body bends and thrashing, was counted every other day.

[0042] 3.2 Results

[0043] For specific results, see Figure 3 ,from Figure 3 It can be seen that when SAR405 was added on the 5th and 7th day after N2 nematodes became adults, until the 13th day, the body bends of nematodes were significantly improved regardless of whether SAR405 treatment was started on the 5th or 7th day ( Figure 3 A). Similarly, it was observed that SAR405 treatment significantly enhanced the thrashing ability of nematodes in the following days ( Figure 3 B) These results indicate that SAR405 treatment of nematodes in the middle and late stages can also significantly improve the motility of nematodes.

[0044] 4. Mechanism of action of SAR405 in improving motor decline

[0045] 4.1 Methods

[0046] Changes in mPSCs mean changes in the number of synaptic released vesicles. The experiment used an electrophysiological instrument to record the amplitude and frequency of electrophysiological indicators mPSCs between nerves and muscles of N2 nematodes that were synchronized and grown to day 3 (young nematodes), day 11 (old nematodes), and day 11 (old nematodes) treated with 20μM SAR405 solution to determine the effect of SAR405 on the changes in mPSCs.

[0047] 4.2 Results

[0048] For specific results, see Figure 4 ,from Figure 4 It can be seen that the frequency of mPSCs in N2 nematodes on the 11th day was significantly lower than that on the 3rd day, and the frequency of mPSCs in N2 nematodes on the 11th day after SAR405 treatment increased significantly ( Figure 4 A). In addition, the amplitude of N2 mPSCs on day 11 was also significantly lower than that on day 3. Similarly, the amplitude of N2 mPSCs increased significantly on day 11 after SAR405 treatment ( Figure 4 B) These results suggest that the release of synaptic vesicles from the nematode nervous system decreases with aging, and that the VPS-34 inhibitor SAR405 promotes the release of synaptic vesicles in aged nematodes.

[0049] 5. Effect of SAR405 on the motor ability of aging mice

[0050] To further determine whether SAR405 can improve the motor ability of aged mice, naturally aged 65-week-old C57 mice were selected for the experiment.

[0051] 5.1 Methods

[0052] The aged mice were intraperitoneally injected with SAR405 at a concentration of 5 mg / kg every day, and the running distance of the mice on the treadmill was counted before the first injection of SAR405 and on the 7th, 14th, 21st and 28th days after the injection.

[0053] 5.2 Results

[0054] For specific results, see Figure 5,from Figure 5 (AH) It can be seen that SAR405 significantly increased the running distance of mice on both the 21st and 28th days ( Figure 5 D). In addition, we detected the changes in the body weight of mice within one month after SAR405 administration and found that SAR405 treatment did not affect the body weight of mice ( Figure 5 C). Testing the size and mitochondrial activity of mouse muscles after administration also showed that SAR405 improved muscle condition ( Figure 5 EH). These results indicate that SAR405 can significantly improve the motor performance of naturally aged mice.

[0055] In summary, the present invention proves through animal experiments that SAR405 has a significant effect in improving motor function, especially in improving motor ability caused by aging. It is applied to motor dysfunction, promotes the recovery of nervous system function, improves muscle state, and thus improves motor ability, which has practical clinical application value.

Claims

1. Use of VPS-34 inhibitors in the preparation of drugs for improving motor dysfunction, Features: The VPS-34 inhibitor is SAR405; The medicine is a medicine for improving motor dysfunction in the elderly.

2. The use according to claim 1, Features: The medicine is a medicine for improving the motor function of the elderly.

3. The use according to claim 2, Features: The drug is a drug that promotes the release of synaptic vesicles in the aged nervous system.

4. The use according to claim 1, Features: The medicine is a medicine for improving the muscle status of the elderly.

5. The use according to any one of claims 1 to 4, Features: The drug is a preparation prepared by using the VPS-34 inhibitor as an active ingredient and adding pharmaceutically acceptable excipients.

6. The use according to claim 5, Features: The preparation is an injection.

7. The use according to claim 6, Features: The concentration of the VPS-34 inhibitor in the injection is 5-50 μM.

8. The use according to claim 7, Features: The concentration of the VPS-34 inhibitor in the injection was 20 μM.

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