Application of RO 5-3335 in anti-aging

Abstract

To provide application of RO 5-3335 in anti-aging.SOLUTION: RO 5-3335 is a core binding factor (CBF) leukemia inhibitor, the RO 5-3335 is a benzodiazepine compound, and the RO 5-3335 is applied to anti-aging drugs. The invention applies the core binding factor (CBF) leukemia inhibitor to delaying, preventing and / or treating senescence, provides a new treatment choice for relieving senescence, and has important practical application value in the fields of medicine and anti-senescence.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of RO 5-3335 in anti-aging. [Background technology]

[0002] As the global population ages, the incidence of various aging-related chronic diseases is increasing. Therefore, it is extremely urgent and important to deeply study the mechanisms of aging and explore effective anti-aging strategies. Currently, the field of research on anti-aging drugs is constantly developing. Drugs such as rapamycin, acarbose, metformin, and dasatinib have been shown to have some anti-aging and health-maintaining effects, and may indirectly slow the progression of aging.

[0003] Because aging is a long and complex biological process, research into anti-aging drugs requires a comprehensive consideration of multiple factors, and it is unlikely that a single drug can completely reverse aging. Future research will likely require greater attention to the combination of multiple drugs and methods. Current anti-aging drugs still have safety issues associated with long-term use, and there is a lack of sufficient clinical evidence to prove their anti-aging effects and their effectiveness in preventing and treating aging-related diseases. Therefore, the constant search for and research into ingredients with anti-aging effects is of great significance for research into aging delay. Summary of the Invention

[0004] In view of this, the present invention discloses the application of RO 5-3335 in anti-aging.RO 5-3335 is a benzodiazepine compound.

[0005] The technical solutions of the present invention are as follows:

[0006] The application of RO 5-3335 in anti-aging, wherein RO 5-3335 is a core binding factor (CBF) leukemia inhibitor, RO 5-3335 is a benzodiazepine compound, and RO 5-3335 is used in anti-aging drugs.

[0007] Furthermore, the dose of RO 5-3335 used in the anti-aging drug may be 18 mg / kg / d.

[0008] Furthermore, the dosage form of the drug may include a tablet, a capsule, or an oral liquid.

[0009] Furthermore, RO 5-3335 may be used as a raw material for a formulation for reducing β-galactosidase activity in senescent cells.

[0010] Furthermore, RO 5-3335 may be used as a raw material for a preparation for restoring the proliferative ability of senescent cells.

[0011] Furthermore, RO 5-3335 may be used as a raw material for a preparation for restoring the DNA synthesis ability of senescent cells.

[0012] Additionally, the RO 5-3335 may be used as an ingredient in a preparation for extending lifespan, or the RO 5-3335 may be used as an ingredient in a preparation for reducing anxiety levels.

[0013] Furthermore, the RO 5-3335 may be used as an ingredient in a preparation for improving the ability to recognize novel objects, or the RO 5-3335 may be used as an ingredient in a preparation for improving non-spatial memory ability.

[0014] Furthermore, the RO 5-3335 may be used as an ingredient in a preparation for improving athletic performance, or the RO 5-3335 may be used as an ingredient in a preparation for improving spatial memory ability.

[0015] beneficial effects The present invention applies the leukemia inhibitor RO 5-3335 to anti-aging and lifespan extension in individuals, providing a new use for RO 5-3335. The present invention not only provides more options for preventing and treating age-related diseases, but also contributes to further research and development in the field of human health, and is of great significance to the development of the global health industry. A deeper understanding of the mechanisms of aging and the search for more effective methods to slow aging will enable us to better address the challenges of an aging society and make greater contributions to human health and welfare. [Brief explanation of the drawings]

[0016] In order to more clearly describe the technical solutions in the embodiments of the present invention, the drawings used in the embodiments will be briefly described below. The drawings in the following description are only some embodiments of the present invention, and those skilled in the art can further derive other drawings based on these drawings without any creative efforts. [Figure 1] FIG. 1 shows the change in SA-β-Gal activity after treating bleomycin-induced senescent cells with RO 5-3335, in the application of RO 5-3335 in anti-aging of the present invention. [Figure 2] FIG. 2 shows the change in growth and proliferation ability of bleomycin-induced senescent cells after treatment with RO 5-3335, in the application of RO 5-3335 in anti-aging of the present invention. [Figure 3] FIG. 3 shows the change in EdU incorporation rate after treating bleomycin-induced senescent cells with RO 5-3335, in the application of RO 5-3335 in anti-aging of the present invention. [Figure 4] FIG. 4 shows the survival analysis of senescence-accelerated mice treated with RO 5-3335, demonstrating the application of RO 5-3335 in anti-aging according to the present invention. [Figure 5]FIG. 5 is a schematic diagram showing the age-related anxiety level of senescence-accelerated mice treated with RO 5-3335 in the open field test, in accordance with the application of RO 5-3335 in anti-aging of the present invention. [Figure 6] FIG. 6 is a schematic diagram showing the motor performance of senescence-accelerated mice treated with RO 5-3335 in the rotarod test, in accordance with the application of RO 5-3335 in anti-aging of the present invention. [Figure 7] FIG. 7 is a schematic diagram showing the novel object recognition ability and non-spatial memory ability of senescence-accelerated mice treated with RO 5-3335 in a novel object recognition test, in the application of RO 5-3335 in anti-aging of the present invention. [Figure 8] FIG. 8 is a schematic diagram showing the spatial memory ability of senescence-accelerated mice treated with RO 5-3335 in the water maze test, demonstrating the application of RO 5-3335 in anti-aging according to the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0017] In order to better understand the technical solution of the present invention, the following detailed description of the embodiments of the present invention will be given with reference to the drawings.

[0018] It is clear that the described embodiments are only some of the embodiments of the present invention, and do not represent all of the embodiments. Based on the embodiments of the present invention, all other embodiments that can be obtained by those skilled in the art without making creative efforts are all included in the scope of protection of the present invention.

[0019] The application of RO 5-3335 in anti-aging, wherein RO 5-3335 is a core binding factor (CBF) leukemia inhibitor, RO 5-3335 is a benzodiazepine compound, and RO 5-3335 is used in anti-aging drugs.

[0020] Furthermore, the dose of RO 5-3335 used in the anti-aging drug may be 18 mg / kg / d.

[0021] Furthermore, the dosage form of the drug may include a tablet, a capsule, or an oral liquid.

[0022] Furthermore, RO 5-3335 may be used as a raw material for a formulation for reducing β-galactosidase activity in senescent cells.

[0023] Furthermore, RO 5-3335 may be used as a raw material for a preparation for restoring the proliferative ability of senescent cells.

[0024] Furthermore, RO 5-3335 may be used as a raw material for a preparation for restoring the DNA synthesis ability of senescent cells.

[0025] Additionally, the RO 5-3335 may be used as an ingredient in a formulation to extend lifespan, or the RO 5-3335 may be used as an ingredient in a formulation to reduce anxiety levels.

[0026] Furthermore, the RO 5-3335 may be used as an ingredient in a preparation for improving the ability to recognize novel objects, or the RO 5-3335 may be used as an ingredient in a preparation for improving non-spatial memory ability.

[0027] Furthermore, the RO 5-3335 may be used as an ingredient in a preparation for improving athletic performance, or the RO 5-3335 may be used as an ingredient in a preparation for improving spatial memory ability.

[0028] Senescence-Associated β-Galactosidase (SA-β-Gal) staining Using the BEYOTIME Cell Senescence β-Galactosidase Staining Kit, we measured the SA-β-Gal activity level in target cells using X-Gal as a substrate. Target cells were cultured in 6-well cell culture plates and tested at approximately 60% confluence. The test kit was removed from the refrigerated / freezer cryopreservation box, allowed to warm to room temperature, and a water bath preheated to 37°C before starting the test. The cell culture medium was aspirated and removed with a Pasteur pipette. The cells were washed twice with Dulbecco's phosphate-buffered saline (DPBS) and fixed for 15 minutes at room temperature with 1 mL of β-galactosidase fixative. After fixation, the fixative was discarded and 1 mL of working staining solution (preparation method for working staining solution is shown in Table 1) was added to each well. The edges of the 6-well cell culture plate were then sealed with parafilm, placed in a water bath, and incubated overnight at 37°C. After incubation, the staining solution was discarded, 1 mL of DPBS was added, and the cells were observed and photographed under a conventional optical microscope. If crystal formation was observed during photography or observation, the stained cells were treated with 1 mL of 70% ethanol, then washed with DPBS before observation.

[0029] [Table 1]

[0030] As shown in Figure 1, the dark blue product was clearly reduced in senescent cells treated with RO 5-3335 compared to control cells, indicating that RO 5-3335 significantly reduces β-galactosidase activity, which is involved in senescence, in senescent cells.

[0031] CCK8 (Cell Counting Kit 8) test Using the MCE CCK8 kit, cell growth and proliferation were measured 12, 24, 36, 48, and 60 hours after cell inoculation. After adding DPBS to the outer periphery of a 96-well cell culture plate, an appropriate number of cells (100 μL in volume) were inoculated into the inner periphery of the plate, ensuring approximately 10% cell confluence after 24 hours. Each set of cells was inoculated into five 96-well cell culture plates, with three replicate wells per plate. Prior to the test, the CCK8 solution was removed from the cryopreservation box and allowed to warm to room temperature. 10 μL of CCK8 solution was added to each well (avoiding air bubbles), and the 96-well cell culture plate was incubated in an incubator for 1 hour. The absorbance at 450 nm was measured and recorded using a microplate reader.

[0032] As shown in Figure 2, the absorbance at 450 nm of RO 5-3335-treated senescent cells was significantly elevated compared to control senescent cells 12, 24, 36, 48, and 60 hours after inoculation into a 96-well cell culture plate, indicating that RO 5-3335 partially restores the proliferation ability of senescent cells.

[0033] 5-ethynyl-2'-deoxyuridine (EdU) cell proliferation assay BeyoClick TMThe EdU Cell Proliferation Assay Kit was used to measure the incorporation rate of the thymidine analog EdU 24 hours after inoculation of target cells, thereby assessing their DNA synthesis ability. After adding DPBS to the outer periphery of a 96-well cell culture plate, an appropriate number of cells (100 μL in volume) were inoculated into the inner periphery of the plate. Cell confluence was controlled to be approximately 10% 24 hours after inoculation. Three replicate wells were prepared for each set of cells. Prior to testing, the EdU Cell Proliferation Assay Kit was removed from the refrigerated / freezer cryopreservation box and allowed to return to room temperature. A 1x EdU working solution (10 μM) was prepared according to Table 2-3-6, pre-warmed to 37°C, and 200 μL was aspirated and added to a 96-well cell culture plate. The cells were then incubated for 2 hours to label with EdU. The culture medium in the 96-well cell culture plate was removed, and 200 μL of 4% paraformaldehyde was added and fixed for 15 minutes at room temperature. After removing the fixative, 200 μL of PBS was added to each well, and the cells were washed three times for 5 minutes each. After removing the PBS, 200 μL of permeabilization solution (preparation method for permeabilization solution is shown in Table 2) was added to each well and incubated for 15 minutes at room temperature. After incubation with the permeabilization solution, the cells were washed twice with 200 μL of PBS for 5 minutes each. Furthermore, one drop of peroxidase blocking solution was added to each well and incubated for 20 minutes at room temperature to inactivate endogenous peroxidase in the cells. The cells were then washed three times with 200 μL of PBS for 2 minutes each. After preparing the Click Additive Solution according to Table 3, the Click reaction solution was prepared according to Table 4. (The order and volume of the components in the table must be strictly adhered to during preparation, and the Click reaction solution must be used within 15 minutes of preparation.) After removing the wash solution from the wells, 50 μL of Click reaction solution was added to each well, and the 96-well cell culture plate was gently shaken to ensure that the reaction solution evenly covered the sample wells. After incubation, the cells were washed three times with PBS for 5 minutes each.20 μL of Streptavidin-HRP working solution (preparation method shown in Table 5) was added to the sample and incubated at room temperature for 30 minutes. During incubation, water was added to the gaps of the 96-well cell culture plate to moisten it, thereby preventing evaporation of the Streptavidin-HRP working solution. After incubation, the cells were washed three times with PBS for 2 minutes each. 100 μL of 3,3',5,5'-tetramethylbenzidine color developer solution was added to the sample wells and incubated at room temperature for 30 minutes. The absorbance of each well at 370 nm was measured and recorded.

[0034] [Table 2]

[0035] [Table 3]

[0036] [Table 4]

[0037] [Table 5]

[0038] As shown in Figure 3, 24 hours after seeding into a 96-well cell culture plate, RO 5-3335-treated senescent cells showed a significant increase in absorbance at 370 nm and in the incorporation rate of the thymidine analog Edu compared to control senescent cells, indicating that RO 5-3335 partially restores the DNA synthesis ability of senescent cells.

[0039] RO 5-3335 intervention study in Senescence-Accelerated Mouse-Prone (SAMP8) mice A CMC-Na solution was prepared by homogenously mixing 0.5 g of CMC-Na and 99.5 g of NaCl injection solution. A RO 5-3335 suspension was prepared by dissolving 120 mg of RO 5-3335 in 60 mL of this 0.5% CMC-Na solution. Next, the weights of the SAMP8 mice required for the study were measured. The test group (n = 12) received 9 μL of the RO 5-3335 suspension and 1 μL of NaCl injection solution per gram of body weight (i.e., 18 mg / kg / day), while the control group (n = 12) received 9 μL of the 0.5% CMC-Na solution and 1 μL of NaCl injection solution per gram of body weight. After 30 days of continuous administration, the mice's condition was observed and recorded.

[0040] As shown in Figure 4, after administration of RO 5-3335 to senescence-accelerated mice, the lifespan of the mice was significantly extended, and the survival rate of the senescence-accelerated mice was improved.

[0041] Rotarod test To accurately assess the differences in motor abilities between the test and control groups, mice were subjected to a rotarod test. The mice's motor coordination and balance were assessed based on the time it took for the mice to fall off the rotarod. The test was conducted using a 3.5 cm diameter rotarod and a 6-channel rat / mouse rotarod. The test environment for each mouse was strictly maintained constant in terms of ventilation, noise level, temperature, humidity, and lighting intensity. Two days prior to the start of the test, the mice were first trained on the rotarod. The rotarod speed that the majority of mice were able to handle was monitored and recorded, and this was the final rotation speed. On the day of the test, the mice were tested in order according to their group and number. The mice were placed on the rotarod, which rotated at a constant speed, and the time it took for each mouse to fall off was recorded. Each mouse was required to complete three rotarod tests, with a 1-hour interval between each test.

[0042] As shown in Figure 5, after administration of RO 5-3335 to senescence-accelerated mice, the anxiety level of the mice could be significantly reduced.

[0043] Novel Object Recognition (NOR) To accurately assess the non-spatial learning and memory abilities of test and control mice, mice were subjected to NOR. Their cognitive memory abilities were assessed based on the number of times, time, and distance explored by the mice to new and old objects. An open field with a blue base, a 50 x 50 cm square, and a 40 cm blue fence was used as the NOR field. Experimental mice were placed in order based on their group and number, and allowed to move freely in the open field for 10 minutes. After the mice became accustomed to the test field, the environment, and the operator, a fixed odorless object A was placed in the open field, and the mice were allowed to move freely in the open field for 10 minutes. After 1 hour, another object B was placed in the open field without removing object A. At this time, the mice were placed in the open field with their backs to object A, and the operator quickly left the test field. The mice were allowed to move freely in the open field for 10 minutes, and the distance traveled, the time spent exploring the novel object, and the total exploration time were recorded. When the distance traveled by the mice was consistent, the Recognition Index (RI) was used as an index for determining NOR, and its calculation method is as follows: RI = time spent exploring the novel object / total exploration time × 100%. A large RI is considered to indicate a high level of non-spatial learning and memory ability in the mice.

[0044] As shown in Figure 6, after administration of RO 5-3335 to senescence-accelerated mice, the exercise capacity of the mice was enhanced.

[0045] Open field test To accurately assess the anxiety levels of mice in the test and control groups, an open field test was conducted on the mice. Their behavior in the central area of ​​the open field was used to evaluate their spontaneous behavior and nervous state in a new environment. A 50 x 50 cm square open field with a white base and a 40 cm blue fence was used. Using dedicated software, the central area, accounting for half of the total area of ​​the open field, was designated as the central zone. After the experimental mice became accustomed to the operator and the environment, they were gently removed from their cages in order according to their group and number and placed in the central area of ​​the open field with their backs to the operator. At this time, the operator quickly removed themselves from the testing site. The mice were allowed to move freely in the open field for 15 minutes, and the distance traveled, resting time, and the time and number of times they entered the central area were recorded. Longer periods of time and distance spent in the central area of ​​the open field were considered to indicate low levels of anxiety.

[0046] As shown in FIG. 7, after RO 5-3335 was administered to senescence-accelerated mice, the mice's ability to recognize novel objects was significantly improved, and the mice's non-spatial memory ability was also improved.

[0047] Water maze test To accurately assess the spatial learning and memory abilities of test and control mice, a water maze test was conducted on mice. After multiple training sessions, the mice's cognitive abilities were assessed based on their ability to find a platform hidden at a specific location. A 120-cm diameter water maze tank and an 8-cm diameter platform were used. The water temperature was controlled within a range of 20–22°C. The water maze light source was adjusted to ensure uniform distribution and to eliminate shadows on the water surface. In addition, three black-on-white spatial cues—a circle, a triangle, and a square—were fixed to the three enclosures of the water maze. Experimental mice underwent water maze training for six days prior to the start of formal testing. The specific training method was as follows: If the mouse did not reach the platform within 60 seconds of the water maze latency, the operator guided the mouse to the platform and allowed it to stay there for 20 seconds to familiarize itself with the surrounding environment before removing it from the platform. If the mouse reached the platform within 60 seconds and stayed there for 5 seconds, it was allowed to remain there for 15 seconds, allowing it to acclimate to the environment, after which it was released from the platform. According to the mouse group and number, the experimental mice were slowly placed into the water, facing the wall of the tank and parallel to the water surface, in turn, and their latency was recorded. After all mice completed one training session, the next training session was held every 30 minutes, for a total of four training sessions per day, for a total of six days. After training, the water maze test was performed. At this time, the platform was removed from the water maze, and according to the mouse group and number, the experimental mice were slowly placed into the water, facing the wall of the tank and parallel to the water surface, in turn, and their movement trajectories, the percentage of time spent in the destination quadrant, the average movement speed, and the number of times they crossed the platform were recorded. A clear and accurate movement trajectory, a long percentage of time spent in the destination quadrant, a fast average movement speed, and a high number of times they crossed the platform were considered to indicate high spatial learning and memory abilities.

[0048] As shown in FIG. 8, after administration of RO 5-3335 to senescence-accelerated mice, the spatial memory ability of the mice could be significantly improved.

[0049] Although the present invention has been described in detail above by way of examples, and the principles and embodiments of the present invention have been described herein using specific examples, the explanation of the above examples is intended to help understand the method and technical essence of the present invention. However, those skilled in the art may make modifications to the specific embodiments and application scope based on the spirit of the present invention, and the contents described herein should not be construed as limiting the scope of the present invention.

Claims

1. A drug for improving aging-related learning and memory abilities, comprising RO 5-3335 as an active ingredient, wherein RO 5-3335 is a core-binding factor (CBF) leukemia inhibitor, and RO 5-3335 is a benzodiazepine compound.

2. The drug for improving aging-related learning and memory ability according to claim 1, wherein the dose of RO 5-3335 used in the drug is 18 mg / kg / d.

3. The drug for improving aging-related learning and memory ability according to claim 1, characterized in that the dosage form of the drug comprises a tablet, a capsule, or an oral liquid.

4. The agent for improving aging-related learning and memory abilities according to claim 1, wherein said RO 5-3335 is used to reduce β-galactosidase activity in senescent cells.

5. The agent for improving aging-related learning and memory ability according to claim 1, wherein said RO 5-3335 is used to restore the proliferative ability of senescent cells.

6. The drug for improving aging-related learning and memory ability according to claim 1, wherein RO 5-3335 is used to restore the DNA synthesis ability of senescent cells.

7. The drug for improving aging-related learning and memory abilities according to claim 1, wherein said RO 5-3335 is used to reduce anxiety levels.

8. The agent for improving aging-related learning and memory ability according to claim 1, wherein said RO 5-3335 is used to improve novel object recognition ability or non-spatial memory ability.

9. The agent for improving aging-related learning and memory ability according to claim 1, wherein said RO 5-3335 is used to improve motor ability or spatial memory ability.

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