Use of abt199 in the manufacture of a medicament for treating muscle atrophy

Abstract

The application provides application of ABT199 in preparation of a drug for treating muscle atrophy. Compared with the prior art, the application provides a new use of ABT199 in treating muscle atrophy. Experimental research proves that ABT199 administration can significantly restore the exercise capacity of a cachexia-induced muscle atrophy mouse, improve the muscle cross-sectional area of the mouse, restore the reduced glutathione content of the mouse, and improve the superoxide dismutase activity, so as to improve the muscle atrophy phenotype of the mouse and muscle atrophy-related biochemical indexes. ABT199 can significantly restore cell muscle atrophy-related gene and protein expression, and improve the cachexia-induced muscle atrophy phenotype by reducing related inflammatory mediators; ABT199 can significantly improve the mitochondrial membrane potential, increase the expression of mitochondrial neogenesis-related genes, and reduce the production of active oxygen.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of new uses of drugs, and particularly relates to application of ABT199 in preparation of a drug for treating muscle atrophy. BACKGROUND

[0002] Myatrophy (myophagism) refers to muscle volume reduction caused by muscle fiber thinning or even disappearing, etc. Causes of muscle atrophy mainly include neurogenic muscle atrophy, myogenic muscle atrophy and disuse muscle atrophy.

[0003] Symptoms of muscle atrophy are various, mainly including: 1. Muscle volume reduction: patients can usually observe that the affected muscles are obviously thinner than the normal side, and the limb circumference is reduced. For example, the limbs become thin, the trapezius muscle atrophy appears square shoulder, the hand muscle atrophy shows that the palm becomes thin and flat, and the bone morphology is prominent, etc.; 2. Muscle weakness: the muscle strength of the patient with muscle atrophy will be significantly reduced, resulting in the inability to complete daily activities or the need to spend more effort to complete. For example, the main manifestation of calf muscle atrophy is squatting and standing difficulty; small muscle atrophy first shows fine motor dysfunction, such as writing difficulty caused by interosseous muscle atrophy of the hand; head and facial muscle atrophy mainly shows weak chewing, swallowing and coughing, weak tongue extension, etc.; neck and proximal muscle atrophy of the limbs shows weak lifting of the head, the scapula protrudes outward, and the limbs are difficult to lift. 3. Movement dysfunction: due to muscle atrophy and strength reduction, the patient may show symptoms such as gait abnormality, unstable standing, easy to fall, etc. Running, jumping and climbing stairs are also more difficult, and some people cannot complete these actions. 4. Muscle tremor: part of the patients will have involuntary tremor when the muscle contracts, which may be caused by muscle fiber lesions or abnormal excitation of the nervous system. 5. Sensory abnormalities: patients with muscle atrophy may have numbness, tingling or pain, especially in the atrophied muscle area. This sensory abnormality may be caused by nerve damage or muscle lesions.

[0004] At present, the treatment methods of muscle atrophy are different due to causes and conditions, mainly including physical therapy, drug therapy, muscle injection therapy, nutritional support and surgical treatment, etc. Among them, there are many treatment drugs, but further expansion is still needed. SUMMARY

[0005] The application provides application of ABT199 in preparation of a drug for treating muscle atrophy.

[0006] Technical scheme: In order to achieve the above application purpose, the application adopts the following technical scheme:

[0007] In a first aspect, the present application provides use of ABT199, or a composition comprising ABT199 in the preparation of a medicament for treating muscle atrophy.

[0008] As a specific embodiment, the muscle atrophy is cachexia-induced muscle atrophy.

[0009] In a second aspect, the present application provides use of ABT199, or a composition comprising ABT199 in the preparation of a medicament for increasing reduced glutathione content, improving superoxide dismutase and creatine kinase activities.

[0010] In a third aspect, the present application provides use of ABT199, or a composition comprising ABT199 in the preparation of a medicament for inhibiting expression of genes Fbxo32 and Trim63.

[0011] In a fourth aspect, the present application provides use of ABT199, or a composition comprising ABT199 in the preparation of a medicament for inhibiting expression of inflammatory mediators Il6, Cxcr1 and Illb genes.

[0012] In a fifth aspect, the present application provides use of ABT199, or a composition comprising ABT199 in the preparation of a medicament for up-regulating expression of mitochondrial biogenesis-related genes Sirt1, Nrf1 and Tfam1.

[0013] In a sixth aspect, the present application provides use of ABT199, or a composition comprising ABT199 in the preparation of a medicament for improving mitochondrial membrane potential.

[0014] As a specific embodiment, in the composition comprising ABT199, ABT199 is the only active ingredient, or ABT199 is one of the active ingredients.

[0015] As a specific embodiment, in the composition comprising ABT199, pharmaceutically acceptable adjuvants or carriers are further included.

[0016] Beneficial effects: Compared with the prior art, the present application provides a new use of ABT199 in treating muscle atrophy. Experimental studies have confirmed that ABT199 administration can significantly restore the exercise capacity of cachexia-induced muscle atrophy mice, improve the muscle cross-sectional area of mice, restore the reduced glutathione content of mice, and improve the superoxide dismutase activity, thereby improving the muscle atrophy phenotype and muscle atrophy-related biochemical indicators of mice. ABT199 can significantly restore cell muscle atrophy-related gene and protein expression, and improve cachexia-induced muscle atrophy phenotype by reducing related inflammatory mediators; ABT199 can significantly improve mitochondrial membrane potential, increase mitochondrial biogenesis-related gene expression and reduce reactive oxygen species production. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 HE staining pictures (A) and gastrocnemius cross-sectional area statistics and quantification graph (B) of normal mice, CT26 tumor-bearing mice and ABT199 administered mice.

[0018] Figure 2 Gastrocnemius and soleus muscle mass ratio and fatigue tolerance statistics and quantification column chart of normal mice, CT26 tumor-bearing mice and ABT199 administered mice.

[0019] Figure 3 Reduced glutathione, superoxide dismutase and creatine kinase statistics and quantification column chart of normal mice, CT26 tumor-bearing mice and ABT199 administered mice.

[0020] Figure 4 Muscle atrophy related protein banding (A) and mRNA expression statistics and quantification column chart (B) of normal mouse myoblast C2C12 cells, TNFα constructed muscle atrophy cells and ABT199 treated cells.

[0021] Figure 5 HE staining (A) and myotube length and width statistics and quantification column chart (B) of normal mouse myoblast C2C12 cells, TNFα constructed muscle atrophy cells and ABT199 treated cells.

[0022] Figure 6 Inflammatory mediator related gene expression statistics and quantification column chart of normal mouse myoblast C2C12 cells, TNFα constructed muscle atrophy cells and ABT199 treated cells.

[0023] Figure 7 Mitochondrial membrane potential fluorescence staining (A) and reactive oxygen species statistics and quantification column chart (B) of normal mouse myoblast C2C12 cells, TNFα constructed muscle atrophy cells and ABT199 treated cells.

[0024] Figure 8 Mitochondrial biogenesis related gene expression statistics and quantification column chart of normal mouse myoblast C2C12 cells, TNFα constructed muscle atrophy cells and ABT199 treated cells. DETAILED DESCRIPTION

[0025] The technical solutions of the present application will be further described in detail below in combination with the drawings and examples.

[0026] ABT199 used in the following examples, Chinese name is Venetoclax, also known as Venetoclax, is a selective, potent, oral small molecule Bcl-2 inhibitor. Its molecular formula is C 45 H 50 ClN7O7S, the chemical formula structure is:

[0027]

[0028] Example 1

[0029] This experiment revealed that ABT199 can restore the fatigue tolerance of muscle atrophy mouse model, increase the cross section of muscle fiber tissue of mice, and restore the content of reduced glutathione, superoxide dismutase and creatine kinase activity in the blood of mice.

[0030] Experimental method:

[0031] 1. Experimental materials

[0032] 1) Experimental animals: 6-8 week old male Balb / C mice, weighing about 22 grams, purchased from the Experimental Animal Department of Peking University Medical Department. The temperature (21-25°C), humidity (60±5%), light (12 / 12h light / dark cycle), and free drinking water were controlled.

[0033] 2) Experimental drugs: ABT199, dose 50mg / kg

[0034] 3) Experimental materials: CT26 colon cancer cell line

[0035] 2. Experimental content

[0036] 1) Cachexia-induced muscle atrophy model: 6-week-old model mice were injected subcutaneously on the back with mouse colon cancer cells CT26, with 1×10 6 cells injected per mouse. The sham operation group was injected with the same volume of 1×PBS liquid. CT26 cells were prepared by digesting cells in the logarithmic growth phase and in good growth condition, centrifuging at 800rpm for 5min, washing the cells with 1×PBS, resuspending the cells, and counting the cells to adjust the concentration of CT26 cells to 1×10 6 / 100μL. After about 7 days, when the mice showed significant decrease in mobility and the subcutaneous injection mass could be roughly felt on the back, ABT199 was administered by gavage injection at a dose of 50mg / kg, and the administration time was about 1-2 weeks. The subcutaneous tumor was measured by measuring the tumor diameter (including the long and short diameters) of the mice subcutaneously injected with CT26 cells every day, and the tumor volume was calculated according to

[0037] the formula:

[0038] V(mm 3 )=1 / 2×long diameter×short diameter 2

[0039] Euthanasia criteria for tumor-bearing mice: the tumor weight of the mice was more than 1 / 10 of the body weight, or the longest diameter of the tumor was more than 15mm. Behavioral evaluation was performed 2-3 days before euthanasia to determine the behavioral indicators of the mice and to determine the experimental measurements of histochemistry, etc.

[0040] 2) Fatigue tolerance test: Before the formal experiment, BALB / C mice were placed in the fatigue rotarod and given a pre-experiment at a constant speed of 15 rpm to familiarize the mice with the environment. In the formal experiment, the time of falling off the rotarod was recorded when the mice moved. Each mouse was tested three times, and the average falling time was calculated.

[0041] 3) Histological test: The mouse gastrocnemius muscle was isolated, fixed, embedded, sectioned, and HE stained. 4) Reduced glutathione content, superoxide dismutase and creatine kinase activity: The mouse blood was collected,

[0042] placed at room temperature for 30 min, centrifuged at 3500 rpm for 10 min, and the upper serum was taken. The serum was subjected to enzyme activity determination using glutathione (GSH), superoxide dismutase (SOD) and creatine kinase (CK) kits.

[0043] 5) Statistical analysis: Image J was used to measure the muscle cross-sectional area of the muscle tissue HE staining picture. GraphPad Prism 9.0 was used for statistical analysis, and more than two groups of results were subjected to single factor analysis of variance for significance test, with P<0.05 as the difference having statistical significance.

[0044] 3, Experimental results

[0045] The CT26 tumor-bearing mouse model showed a significant decrease in muscle cross-sectional area, and ABT199 could restore the muscle cross-sectional area of the mice Figure 1 ). In addition, the statistical quantification results showed that the gastrocnemius muscle cross-sectional area of the CT26 tumor-bearing mice was 1000-1500 μm 2 in quantity, while ABT199 could restore the gastrocnemius muscle cross-sectional area to 2000-2500 μm 2 and 3000 μm 2 in quantity Figure 1 . In addition, ABT199 significantly restored the mass ratio of the mouse soleus muscle to the mouse body weight, and there was a trend to restore the mass ratio of the gastrocnemius muscle to the mouse body weight Figure 2 . In addition, the results of the behavioral experiment showed that the fatigue tolerance of the CT26 tumor-bearing mice was reduced, and ABT199 had a trend to restore the fatigue tolerance of the mice Figure 2 . In addition, the mouse serum was extracted for biochemical index detection. The results showed that the reduced glutathione content, superoxide dismutase and creatine kinase activity of the CT26 tumor-bearing mice were significantly reduced, while ABT199 treatment could significantly improve the reduced glutathione content of the mice and increase the superoxide dismutase activity, in addition, ABT199 had a trend to increase the creatine kinase activity Figure 3). The above results show that ABT199 administration can significantly restore the exercise capacity of cachexia-induced muscle atrophy mice, improve the cross-sectional area of mice, restore the reduced glutathione content of mice, and improve the superoxide dismutase activity, thereby improving the muscle atrophy phenotype and muscle atrophy-related biochemical indicators of mice.

[0046] Example 2

[0047] This experiment shows that ABT199 reduces the expression of muscle atrophy-related molecular markers in mouse myoblast C2C12 cells.

[0048] Experimental method

[0049] 1. Experimental materials

[0050] 1) Experimental cells: mouse myoblast cells (Pronova, CL-0044)

[0051] 2) Experimental reagents: special horse serum (Solabio, S9050), high-sugar DMEM cell culture medium (Pronova), 0.25% trypsin (Zhongkemaichen), TNFα (Coastal Protein, CF09), tissue cell RNA micro-extraction kit (Meiji Biology, R4012), StarScript III one-pot degenerate reverse transcription premix (Kangrun Biology, A230), Hieff Universal Blue qPCR SYBR Green Master Mix (Yixing Biology, 11184ES), Fbx32 primary antibody (Abeam, ab168372), Trim63 primary antibody (ABclonal, A3101), hematoxylin and eosin (HE) staining kit (Biuntian, C0105S)

[0052] 2. Experimental content

[0053] 1) Cell culture: mouse myoblast C2C12 cells were cultured in high-sugar DMEM medium (10% FBS + 1% penicillin-streptomycin mixture) and placed in a 37°C and 5% carbon dioxide environment for culture and passage. Cells were passaged

[0054] Cells within 10 passages can be used for subsequent experiments

[0055] 2) Cell differentiation: mouse myoblast C2C12 cells cultured in 6-well plates were then subjected to myotube differentiation to simulate an in vitro muscle atrophy model. When the cell density reached 100%, myotube differentiation medium (high-sugar DMEM medium containing 2% special horse serum and 1% penicillin-streptomycin mixture) was added, and the medium was changed every 48 h, for a total of two times of induction and differentiation.

[0056] 3) Construction of an in vitro muscular atrophy cell model and drug treatment: After mouse myoblast differentiation was completed, muscular atrophy was induced and drugs were administered. This experiment consisted of three groups: control group (cells treated with DMSO), model group (cells treated with 20 ng / ml TNFα), and drug treatment group (cells treated with 20 ng / ml TNFα and...

[0057] (Treatment with 10 μM ABT199). Cells were harvested after 24 hours of treatment and used for subsequent experiments.

[0058] 4) RNA extraction and qRT-PCR detection of muscular dystrophy gene and inflammatory mediator expression:

[0059] a) RNA Extraction: RNA extraction was performed according to the instructions of the Tissue Cell RNA Micro-Extraction Kit (MegBio). The 12-well cell culture plates were washed once with PBS, and 100 μL of RTL Lysis Buffer and 100 μL of RNA Binding Buffer were added. The mixture was transferred to a purification column and centrifuged at 13000 rpm for 1 min. 350 μL of Buffer RW1 was added to the column and centrifuged at 13000 rpm for 1 min.

[0060] Add 500 μL of Buffer RW2 (diluted with ethanol) to the column, centrifuge at 13000 rpm for 1 min, and repeat once. Transfer the column to a 1.5 ml centrifuge tube. Add 30 μL of RNase-free water to the center of the column membrane. Let stand for 1 min. Centrifuge at 13000 rpm for 1 min. The resulting solution contains cellular RNA.

[0061] b) cDNA synthesis: Prepare the reverse transcription reaction system according to the following components.

[0062]

[0063] The reaction conditions are as follows:

[0064]

[0065] The cDNA obtained from the reaction was used for subsequent qPCR experiments.

[0066] c) qPCR reaction: cDNA was diluted in deionized water at a ratio of 1:5 and mixed thoroughly with a vortex mixer.

[0067] The following components were used to prepare a qPCR reaction system (20 μL):

[0068]

[0069] The qPCR reaction program is set as follows:

[0070]

[0071] Tbp as internal reference gene, the relative quantification method of gene expression adopts 2 -ΔΔCT

[0072] 5) Western blot detection of dystrophin expression:

[0073] a) Cell lysis: the cells in the 12-well cell culture plate were washed twice with PBS, 100 μL of RIPA lysis buffer (containing protease inhibitors and phosphatase inhibitors) was added to each well, and the cells were thoroughly ground on ice to ensure complete cell lysis.

[0074] b) Protein quantification: the protein quantification process was performed according to the instructions of the BCA protein concentration determination kit (Bi Yun Tian Bio). Take 25 mg / ml protein standard, dilute to a final concentration of 2 mg / ml protein standard. At the same time, configure BCA working solution according to the ratio of 50:1. Add 0, 1, 2, 4, 8, 12, 16, 20 μL of protein standard to the standard wells of the 96-well plate, and add PBS buffer to make up to 20 μL. Dilute the protein sample to be tested 10 times with PBS, add 20 μL to each well; add 200 μL of BCA working solution to all wells, mix gently, and incubate in a 37°C incubator for 20 h. Read the absorbance of each well at 562 nm wavelength on a microplate reader, draw a standard curve, and calculate the protein concentration of the sample to be tested. Then add

[0075] 25 μL of 5xLoading Buffer to the protein lysis solution, and boil in a metal bath at 100°C for 10 min, and store in a -20°C refrigerator.

[0076] c) Protein electrophoresis and membrane transfer: configure 10% separation gel and 5% concentrated gel, load 10 μg per well, and electrophorese at a constant voltage of 80 V for about 2 h. Then transfer the membrane to the NC membrane at a constant current of 200 mA for 80 min.

[0077] d) Antibody incubation: the antibody for the target protein is diluted 1:1000, the internal reference protein antibody is diluted 1:5000, and incubated at 4°C overnight. Then wash the membrane with TBST for 6 times, each time for 5 min. Dilute the corresponding species HRP-labeled secondary antibody 1:

[0078] 5000, incubate at room temperature for 1 h, and develop after washing the membrane with TBST.

[0079] e) Data analysis: the Western Blot color development results are statistically analyzed by Image J software.

[0080] 6) C2C12 myotube hematoxylin-eosin (HE) staining:

[0081] ​a) Cell fixation: fix with fixative for 10 min, wash with distilled water for 2 min, change to fresh distilled water and wash again for 2 min.

[0082] b) Hematoxylin staining: stain with hematoxylin staining solution for 5 min, and wash with tap water for 10 min to remove excess staining solution.

[0083] d) Eosin staining: stain with eosin staining solution for 3 min, and then wash twice with 70% ethanol, and then observe under a microscope and take photos.

[0084] e) Data analysis: Image J software was used to analyze the length and diameter of myotubes.

[0085] 3. Experimental results

[0086] To construct a muscle atrophy cell model, mouse myoblast cells C2C12 were differentiated into myotubes by horse serum, and then treated with 20 ng / ml TNFα. The experimental results showed that TNFα treatment significantly up-regulated the expression of muscle atrophy genes Fbxo32 and Trim63, while ABT199 administration significantly down-regulated the expression of the two genes and proteins. Figure 4 ) At the same time, TNFα treatment significantly reduced the length and diameter of C2C12 myotubes, and ABT199 treatment significantly restored the length and diameter of myotubes. Figure 5 ) In addition, ABT199 significantly improved the expression of inflammatory mediators Il6, Cxcr1 and Il1b, suggesting that ABT199 has an anti-inflammatory effect on the TNFα in vitro myotube atrophy model. Figure 6 ) In summary, the results showed that ABT199 can significantly restore the expression of cell muscle atrophy-related genes and proteins, and improve the cachexia-induced muscle atrophy phenotype by reducing related inflammatory mediators.

[0087] Experimental Example 3

[0088] This experiment showed that ABT199 significantly improved mitochondrial membrane potential, reduced reactive oxygen species production, and up-regulated mitochondrial biogenesis-related gene expression.

[0089] Experimental methods:

[0090] 1. Experimental materials

[0091] 1) Experimental cells: mouse myoblast cells (Pronova, CL-0044)

[0092] 2) Experimental reagents: trick horse serum (Solebo, S9050), high-sugar DMEM cell culture medium (Punoxie), 0.25% trypsin (Zhongkemaichen), TNFα (Taoshu biology, T1076), tissue cell RNA micro-extraction kit (Mei base biology, R4012), StarScript III one-pot degenerate reverse transcription premix (Kangrun biology, A230), Hieff Universal Blue qPCR SYBR Green Master Mix (Yisheng biology, 11184ES), mitochondrial membrane potential detection kit (JC-1) (Biuntian, C2006), reactive oxygen species detection kit (Biuntian, S0033S)

[0093] 2, Experimental content

[0094] 1) Cell culture, differentiation, construction of in vitro muscle atrophy model and drug administration Same as experimental example 2 (1)-(3) experiment

[0095] Content

[0096] 2) RNA extraction and qRT-PCR detection of muscle atrophy gene expression Same as experimental example 2 (4)

[0097] 3) Cell mitochondrial membrane potential staining

[0098] a) JC1 staining solution preparation: JC-1 (200x), ultrapure water, JC-1 staining buffer (50x) according to 1:

[0099] 160:40 ratio to prepare JC-1 staining working solution;

[0100] b) JC-1 staining: cells were cultured in six-well plates, washed with PBS, and 1ml of JC-1 staining working solution was added to each well, and mixed thoroughly. Incubate in a cell incubator at 37°C for 20 minutes.

[0101] c) Washing: JC-1 staining buffer (5x) and distilled water were prepared according to a ratio of 1:4 to prepare the washing solution. After staining, wash twice with the washing solution, and then add 2ml of cell culture medium to each well.

[0102] d) Photograph: take a photo with a fluorescence microscope.

[0103] e) Data analysis: fluorescence intensity was statistically analyzed using Image J software.

[0104] 4) Cell reactive oxygen species (ROS) group detection

[0105] a) DCFH-DA probe staining: Cells were cultured in 96-well dark plate, after washing with PBS, 0.1 ml fresh culture medium mixed with DCFH-DA probe was added to each well, the final concentration was 10 μmmol / L, and incubated in 37 °C incubator for 20 min.

[0106] b) Detection: Fluorescence microplate reader detection, 488 nm excitation wavelength and 525 nm emission wavelength parameters were used.

[0107] 3. Experimental results

[0108] The results of this part of the experiment are used to show that ABT199 can improve mitochondrial membrane potential and related oxidative stress results in muscle atrophy cell model. TNFα treatment reduces mitochondrial staining intensity, and ABT199 administration significantly restores fluorescence intensity ( Figure 7 ). At the same time, TNFα treatment significantly increases ROS staining intensity, and ABT199 administration significantly reduces staining intensity. In addition, ABT199 significantly up-regulates mitochondrial biogenesis-related genes (Sirt1, Nrf1 and Tfam1) Figure 8 ). The above results show that ABT199 can significantly improve mitochondrial membrane potential, increase mitochondrial biogenesis-related gene expression and reduce the production of reactive oxygen species.

[0109] The embodiments of the application are described in detail above with reference to specific embodiments, but the application is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the purpose of the application.

Claims

1. Use of ABT199 or a composition comprising ABT199 in the manufacture of a medicament for the treatment of muscle atrophy, characterized in that, The ABT199 has a chemical formula structure as follows: 。 2. Use according to claim 1, characterized in that, The muscle atrophy is caused by cachexia.

3. Use according to claim 1, characterized in that, The ABT199 or the composition containing the ABT199 can improve superoxide dismutase and creatine kinase activities.

4. Use according to claim 1, characterized in that, The ABT199 or the composition containing the ABT199 can inhibit expressions of genes Fbxo32 and Trim63.

5. The use according to claim 1, characterized in that, The ABT199 or the composition containing the ABT199 can inhibit expressions of inflammatory mediators Il6, Cxcr1 and Il1b genes.

6. Use according to claim 1, characterized in that, The ABT199 or the composition containing the ABT199 can up-regulate expressions of mitochondrial biogenesis-related genes Sirt1, Nrf1 and Tfam1.

7. Use according to claim 1, characterized in that, The ABT199 or the composition containing the ABT199 can improve mitochondrial membrane potential.

8. Use according to any one of claims 1 to 7, characterized in that, In the composition containing the ABT199, the ABT199 serves as the only active ingredient, or the ABT199 serves as one of the active ingredients.

9. Use according to any one of claims 1 to 7, characterized in that, In the composition containing the ABT199, pharmaceutically acceptable adjuvants or carriers are further included.

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