Application of ABT199 in preparation of medicine for treating muscular atrophy

Abstract

The invention provides an application of ABT199 in preparation of a medicine for treating amyotrophy. Compared with the prior art, the invention provides a new application of the ABT199 in treating amyotrophy. Experimental studies prove that the ABT199 administration can significantly recover the exercise ability of mice with cachexia-induced amyotrophy, improve the cross sectional area of the muscles of the mice, recover the content of reduced glutathione in the mice, and improve the activity of superoxide dismutase, so that the phenotype of the amyotrophy of the mice and the related biochemical indexes of the amyotrophy are improved. The ABT199 can significantly restore cell amyotrophy related gene and protein expression, and improve cachexia-induced amyotrophy phenotype by reducing related inflammatory mediators; the ABT199 can significantly improve the mitochondrial membrane potential, increase the expression of mitochondrial neogenesis related genes and reduce the generation of active oxygen.

Description

Technical Field

[0001] The present invention belongs to the technical field of new drug applications, and in particular relates to the application of ABT199 in the preparation of drugs for treating muscular atrophy. Background Art

[0002] Muscular atrophy (myatrophy; myophagism) refers to the reduction of muscle volume caused by skeletal muscle nutritional disorders, thinning or even disappearance of muscle fibers, etc. The causes of muscular atrophy are mainly divided into neurogenic muscular atrophy, myogenic muscular atrophy and disuse muscular atrophy.

[0003] The symptoms of muscle atrophy are diverse, mainly including: 1. Reduced muscle volume: Patients can usually observe that the affected muscles are significantly thinner than the normal side, and the limb circumference is reduced. For example, the limbs become thinner, the deltoid muscle atrophy appears square shoulders, the hand muscle atrophy is manifested as thinning and flattening of the palm, and the bone morphology is prominent; 2. Muscle weakness: The muscle strength of patients with muscle atrophy will be significantly reduced, resulting in the inability to complete daily activities or requiring greater effort to complete. For example, calf muscle atrophy is mainly manifested as difficulty in squatting and standing; small muscle atrophy first manifests as fine motor disorders, such as interosseous muscle atrophy in the hands will cause difficulty in writing; head and facial muscle atrophy is mainly manifested as weak chewing, swallowing choking, and weak tongue extension; neck and limb proximal muscle atrophy is manifested as weak head lifting, scapula protruding outward, and difficulty lifting the limbs. 3. Motor dysfunction: Due to muscle atrophy and reduced strength, patients may show symptoms such as abnormal gait, unstable standing, and easy falling. Actions such as running, jumping, and climbing stairs will also become more strenuous, and some people may not even be able to complete these actions. 4. Muscle tremor: Some patients experience involuntary tremors when their muscles contract, which may be caused by muscle fiber lesions or abnormal excitement of the nervous system. 5. Abnormal sensation: Patients with muscular atrophy may experience numbness, tingling or pain, especially in the atrophied muscle area. This abnormal sensation may be caused by nerve damage or muscle lesions.

[0004] At present, the treatment methods for muscular atrophy vary according to the cause and condition, mainly including physical therapy, drug therapy, intramuscular injection therapy, nutritional support and surgical treatment. Among them, there are many therapeutic drugs, but there is still a need for further expansion. Summary of the invention

[0005] Purpose of the invention: In view of the problems existing in the prior art, the present invention provides the use of ABT199 in the preparation of drugs for treating muscular atrophy.

[0006] Technical solution: To achieve the above-mentioned invention object, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides use of ABT199, or a composition comprising ABT199, in the preparation of a drug for treating muscular atrophy.

[0008] As a specific embodiment, the muscle atrophy is muscle atrophy caused by cachexia.

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

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

[0011] In a fourth aspect, the present invention provides use of ABT199, or a composition comprising ABT199, in the preparation of a drug for inhibiting the gene expression of inflammatory mediators Il6, Cxcr1 and Il1b.

[0012] In a fifth aspect, the present invention provides use of ABT199, or a composition comprising ABT199, in the preparation of a drug for upregulating the expression of mitochondrial neogenesis-related genes Sirt1, Nrf1, and Tfam1.

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

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

[0015] As a specific embodiment, the composition comprising ABT199 further comprises a pharmaceutically acceptable excipient or carrier.

[0016] Beneficial effects: Compared with the prior art, the present invention provides a new use of ABT199 for the treatment of muscular atrophy. Experimental studies have confirmed that the administration of ABT199 can significantly restore the motor ability of mice with cachexia-induced muscular atrophy, improve the cross-sectional area of ​​mouse muscle, restore the reduced glutathione content of mice, and enhance the activity of superoxide dismutase, thereby improving the muscular atrophy phenotype and muscular atrophy-related biochemical indicators of mice. ABT199 can significantly restore the expression of cellular muscular atrophy-related genes and proteins, and improve the cachexia-induced muscular atrophy phenotype by reducing related inflammatory mediators; ABT199 can significantly increase mitochondrial membrane potential, increase the expression of mitochondrial neogenesis-related genes and reduce the production of reactive oxygen species. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0018] Figure 2 This is a statistical quantitative bar graph of the gastrocnemius and soleus muscle mass ratio and fatigue tolerance in normal mice, CT26 tumor-bearing mice and ABT199-administered mice.

[0019] Figure 3 The bar graphs are the statistical quantification of reduced glutathione, superoxide dismutase and creatine kinase in normal mice, CT26 tumor-bearing mice and ABT199-administered mice.

[0020] Figure 4 The muscular atrophy-related protein development bands (A) and mRNA expression statistical quantitative bar graphs (B) were constructed for normal mouse myoblasts C2C12, TNFα and ABT199-treated cells.

[0021] Figure 5 HE staining of normal mouse myoblasts C2C12, TNFα-induced myotrophic cells and ABT199 treatment (A) and statistical quantitative bar graph of myotube length and width (B).

[0022] Figure 6 Statistical quantitative bar graphs of gene expression related to inflammatory mediators in normal mouse myoblasts C2C12 and TNFα were constructed for myoblasts and ABT199-treated cells.

[0023] Figure 7 Fluorescence staining of mitochondrial membrane potential (A) and quantitative bar graph of reactive oxygen species (B) for normal mouse myoblasts C2C12, TNFα-induced muscular atrophy cells and ABT199-treated cells.

[0024] Figure 8 Statistical quantitative bar graphs of mitochondrial neogenesis-related gene expression in normal mouse myoblasts C2C12, TNFα, and ABT199-treated cells were constructed. DETAILED DESCRIPTION

[0025] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments.

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

[0027]

[0028] Example 1

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

[0030] Experimental methods:

[0031] 1. Experimental Materials

[0032] 1) Experimental animals: 6-8 week old male Balb / C mice, weighing about 22 g, were purchased from the Laboratory Animal Department of Peking University Health Science Center. The mice were kept under controlled temperature (21-25°C), humidity (60±5%), and light (12 / 12h light / dark cycle), and had free access to food and water.

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

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

[0035] 2. Experimental content

[0036] 1) Establishment of cachexia-induced muscle atrophy model: Mouse colon cancer cells CT26 were injected subcutaneously on the back of mice in the model group from 6 weeks of age, with 1×10 cells injected per mouse. 6 The sham operation group was injected with the same volume of 1× PBS liquid; CT26 cell preparation: CT26 cells in the logarithmic growth phase and in good growth state were digested, centrifuged at 800 rpm for 5 min, washed with 1× PBS and resuspended, and the cells were counted. The CT26 cell concentration was adjusted to 1×10 6 / 100μL; mice were given the drug. After about 7 days, when the mice showed a significant decrease in mobility and the subcutaneous injection mass could be slightly felt on the back, ABT199 was injected intragastrically at a dose of 50mg / kg for about 1-2 weeks; subcutaneous tumor measurement: mice were subcutaneously injected with CT26 cells and the tumor diameter (including the long and short diameters) was measured every day, and the tumor diameter was measured according to the

[0037] The tumor volume was calculated by the following formula:

[0038] V(mm 3 ) = 1 / 2 × major diameter × minor diameter 2

[0039] Euthanasia criteria for tumor-bearing mice: The weight of the mouse tumor exceeds 1 / 10 of the body weight or the longest diameter of the mouse tumor exceeds 15mm. Behavioral evaluation is performed 2-3 days before euthanasia to measure the behavioral indicators of mice and histochemical and other experimental measurements.

[0040] 2) Fatigue tolerance test of cachexia mice: Before the formal experiment, BALB / C mice were placed in a fatigue rotarod and rotated at a constant speed of 15 rpm for a pre-experiment to familiarize the mice with the environment. During the formal experiment, the time it took for the mice to fall while exercising on the fatigue rotarod was recorded. Each mouse was tested three times, and the average time to fall was calculated.

[0041] 3) Histochemical determination: The gastrocnemius muscle of mice was isolated, fixed, embedded, sliced ​​and HE stained. 4) Reduced glutathione content, superoxide dismutase and creatine kinase activity: Blood of mice was collected,

[0042] Place at room temperature for 30 minutes, centrifuge at 3500rpm for 10 minutes, and take the upper serum. Serum enzyme activity was determined using chimpanzee superoxide dismutase (GSH), superoxide dismutase (SOD) and creatine kinase (CK) kits.

[0043] 5) Statistical analysis: The muscle cross-sectional area was measured from the HE staining images of muscle tissue using Image J. Statistical analysis was performed using GraphPad Prism 9.0. One-way analysis of variance was used for significance test for results of more than two groups, with P < 0.05 considered statistically significant.

[0044] 3. Experimental results

[0045] The muscle cross-sectional area of ​​CT26 tumor-bearing mice was significantly reduced, and ABT199 was able to restore the muscle cross-sectional area of ​​mice ( Figure 1 In addition, the results of statistical quantitative graphs showed that the cross-sectional area of ​​the gastrocnemius muscle of CT26 tumor-bearing mice was between 1000 and 1500 μm 2 ABT199 can restore the cross-sectional area of ​​gastrocnemius muscle to 2000-2500 μm 2 and 3000μm 2 The above quantity ( Figure 1 ). In addition, ABT199 significantly restored the ratio of soleus muscle to body weight of mice, and there was a trend of restoring the ratio of gastrocnemius muscle to body weight of mice ( Figure 2 ). In addition, behavioral experimental results showed that the fatigue tolerance of CT26 tumor-bearing mice was reduced, while ABT199 showed a trend of restoring the fatigue tolerance of 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 activities of CT26 tumor-bearing mice were significantly reduced, while ABT199 treatment could significantly improve the reduced glutathione content of mice and increase the superoxide dismutase activity. In addition, ABT199 showed a trend of increasing creatine kinase activity ( Figure 3). The above results show that ABT199 administration can significantly restore the motor ability of mice with cachexia-induced muscle atrophy, improve the muscle cross-sectional area of ​​mice, restore the content of reduced glutathione in mice, and enhance the activity of superoxide dismutase, thereby improving the muscle atrophy phenotype and muscle atrophy-related biochemical indicators of mice.

[0046] Example 2

[0047] This experiment showed that ABT199 reduced the expression of muscular atrophy-related molecular markers in mouse myoblasts C2C12 cells.

[0048] Experimental methods

[0049] 1. Experimental Materials

[0050] 1) Experimental cells: mouse myoblasts (Pnosai, CL-0044)

[0051] 2) Experimental reagents: Special grade horse serum (Solaibo, S9050), high glucose DMEM cell culture medium (Punosai), 0.25% trypsin (Zhongke Maichen), TNFα (Jiang'an Protein, CF09), tissue cell RNA micro-extraction kit (Meiji Bio, R4012), StarScript III one-tube de-genomic reverse transcription premix (Kangrun Bio, A230), Hieff Universal Blue qPCRSYBR Green Master Mix (Yisheng Biotechnology, 11184ES), Fbx32 primary antibody (Abcam, ab168372), Trim63 primary antibody (ABclonal, A3101), hematoxylin and eosin (HE) staining kit (Biyuntian, C0105S)

[0052] 2. Experimental content

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

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

[0055] 2) Cell differentiation: Mouse myoblast C2C12 cells were cultured in 6-well plates and then differentiated into myotubes to simulate the in vitro muscle atrophy model. When the cell density reached 100%, myotube differentiation medium (high-glucose DMEM medium containing 2% special horse serum and 1% penicillin-streptomycin mixture) was added, and the medium was changed every 48 hours, and differentiation was induced twice in total.

[0056] 3) Construction of in vitro muscular atrophy cell model and drug treatment: After the mouse myoblasts were differentiated, muscular atrophy was induced and drug treatment was performed. There were three groups in this experiment: control group (cells were treated with DMSO), model group (cells were treated with 20ng / ml TNFα), drug treatment group (cells were treated with 20ng / ml TNFα and

[0057] 10 μM ABT199 treatment). The cells were treated for 24 h and then harvested for subsequent experiments.

[0058] 4) RNA extraction and qRT-PCR detection of muscular atrophy genes and inflammatory mediators expression:

[0059] a) RNA extraction: RNA extraction was performed according to the instructions of the tissue cell RNA micro-extraction kit (Meiji Bio). The 12-well cell culture plate was washed once with PBS, and 100 μL RTL Lysis Buffer was added, followed by 100 μL RNA Binding Buffer. The mixture was transferred to the purification column and centrifuged at 13000 rpm for 1 min. 350 μL Buffer RW1 was added to the column and centrifuged at 13000 rpm for 1 min.

[0060] Add 500μL Buffer RW2 (diluted with ethanol) to the column and centrifuge at 13000rpm for 1min. Repeat once. Transfer the column to a 1.5ml centrifuge tube. Add 30μL RNase Free Water to the center of the column membrane. Let stand for 1min. Centrifuge at 13000rpm for 1min. The resulting solution is 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: dilute cDNA in deionized water at a ratio of 1:5 and vortex mix.

[0067] The qPCR reaction system (20 μL) was prepared with the following components:

[0068]

[0069] The qPCR reaction program was set up as follows:

[0070]

[0071] Tbp was used as the internal reference gene, and the relative quantification method of gene expression was 2 -ΔΔCT Way.

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

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

[0074] b) Protein quantification: The protein quantification process is carried out according to the instructions for use of the BCA protein concentration determination kit (Biyuntian Biotechnology). Take 25mg / ml protein standard and dilute it to a final concentration of 2mg / ml protein standard. At the same time, prepare the BCA working solution at a ratio of 50:1. Add 0, 1, 2, 4, 8, 12, 16, and 20μL of protein standard to the standard wells of the 96-well plate, and add PBS buffer to make up to 20μL. The protein sample to be tested is diluted 10 times with PBS, and 20μL is added to each well; 200μL of BCA working solution is added to all wells, gently aspirate to mix, and incubate at 37°C for 20 hours. Read the absorbance of each well at a wavelength of 562nm on the microplate reader, draw a standard curve, and calculate the protein concentration of the sample to be tested. Then add the protein lysate.

[0075] 25 μL 5× Loading Buffer, boil in metal bath at 100°C for 10 min, and store in a -20°C refrigerator.

[0076] c) Protein electrophoresis and membrane transfer: Prepare 10% separation gel and 5% concentration gel, load 10 μg of sample into each well, and conduct constant electrophoresis.

[0077] Electrophoresis was performed at 80V for about 2 hours. Then the membrane was transferred to NC membrane at a constant current of 200mA for 80 minutes. d) Antibody incubation: Target protein antibody was diluted 1:1000, internal reference protein antibody was diluted 1:5000 and incubated at 4°C overnight. Then the membrane was washed 6 times with TBST, 5 minutes each time. Corresponding species HRP-labeled secondary antibody 1:

[0078] Dilute to 5000, incubate at room temperature for 1 hour, wash the membrane with TBST and then develop.

[0079] e) Data analysis: The grayscale values ​​of Western Blot results were statistically analyzed using Image J software.

[0080] 6) Hematoxylin and eosin (HE) staining of C2C12 myotubes:

[0081] a) Cell fixation: Fix with fixative for 10 min, wash with distilled water for 2 min, then replace with fresh distilled water and wash again for 2 min.

[0082] b) Hematoxylin staining: stain with hematoxylin solution for 5 minutes, and rinse with tap water for 10 minutes. c) Differentiation: differentiate with hematoxylin solution for 15 seconds, then rinse with tap water for 10 minutes.

[0083] d) Eosin staining: After staining with eosin solution for 3 minutes, the cells were washed twice with 70% ethanol and then observed under a microscope and photographed.

[0084] e) Data analysis: The imaging results were analyzed using Image J software to calculate the length and diameter of myotubes.

[0085] 3. Experimental results

[0086] To construct a muscle atrophy cell model, mouse myoblasts C2C12 were differentiated into myotubes by horse serum and then treated with 20ng / ml TNFα. The experimental results showed that TNFα treatment significantly upregulated the expression of muscle atrophy genes Fbxo32 and Trim63, while ABT199 administration significantly downregulated the expression of the two genes and proteins ( Figure 4 ). Meanwhile, TNFα treatment significantly reduced the length and diameter of C2C12 myotube cells, while ABT199 treatment significantly restored the length and diameter of myotube cells ( Figure 5 In addition, ABT199 significantly improved the gene expression of inflammatory mediators Il6, Cxcr1, and Il1b, suggesting that ABT199 has anti-inflammatory effects in the TNFα in vitro myotube atrophy model ( Figure 6 ). The above results show that ABT199 can significantly restore the expression of muscular atrophy-related genes and proteins in cells, and improve the cachexia-induced muscular atrophy phenotype by reducing related inflammatory mediators.

[0087] Experimental Example 3

[0088] This experiment showed that ABT199 significantly increased mitochondrial membrane potential, reduced reactive oxygen production and upregulated the expression of genes related to mitochondrial neogenesis.

[0089] Experimental methods:

[0090] 1. Experimental Materials

[0091] 1) Experimental cells: mouse myoblasts (Pnosai, CL-0044)

[0092] 2) Experimental reagents: special horse serum (Solebo, S9050), high-glucose DMEM cell culture medium (Punosai), 0.25% trypsin (Zhongke Maichen), TNFα (Taoshu Biotechnology, T1076), tissue cell RNA micro-extraction kit (Meiji Biotechnology, R4012), StarScript III one-tube de-genomic reverse transcription premix (Kangrun Biotechnology, A230), Hieff Universal Blue qPCRSYBR Green Master Mix (Yisheng Biotechnology, 11184ES), Mitochondrial Membrane Potential Detection Kit (JC-1) (Biyuntian, C2006), Reactive Oxygen Species Detection Kit (Biyuntian, S0033S)

[0093] 2. Experimental content

[0094] 1) Cell culture, differentiation, construction of in vitro muscle atrophy model and drug administration were the same as those in Experimental Example 2 (1)-(3)

[0095] content

[0096] 2) RNA extraction and qRT-PCR detection of muscular atrophy gene expression were the same as in Experimental Example 2 (4)

[0097] 3) Cell mitochondrial membrane potential staining

[0098] a) Preparation of JC1 staining solution: JC-1 (200×), ultrapure water, and JC-1 staining buffer (50×) according to 1:

[0099] Prepare JC-1 staining working solution in a ratio of 160:40;

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

[0101] c) Washing: Prepare washing solution with JC-1 staining buffer (5×) and distilled water in a ratio of 1:4. After washing twice with the staining solution, add 2 ml of cell culture medium to each well.

[0102] d) Photography: Take photos using a fluorescence microscope.

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

[0104] 4) Detection of cellular reactive oxygen species (ROS)

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

[0106] b) Detection: Detection was performed using a fluorescence microplate reader with an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

[0107] 3. Experimental results

[0108] The experimental results in this section are used to demonstrate that ABT199 can improve mitochondrial membrane potential and related oxidative stress results in muscle atrophy cell models. TNFα treatment reduced mitochondrial staining intensity, while ABT199 administration significantly restored fluorescence intensity ( Figure 7 ). At the same time, TNFα treatment significantly increased the ROS staining intensity, while ABT199 administration significantly reduced the staining intensity. In addition, ABT199 significantly upregulated mitochondrial neogenesis-related genes (Sirt1, Nrf1, and Tfam1) ( Figure 8 ). The above results show that ABT199 can significantly enhance mitochondrial membrane potential, increase the expression of mitochondrial neogenesis-related genes and reduce the production of reactive oxygen species.

[0109] The implementation modes of the present invention have been described in detail above in conjunction with specific embodiments, but the present invention is not limited to the above-mentioned implementation modes, and various changes can be made within the knowledge scope of ordinary technicians in the relevant technical field without departing from the purpose of the present invention.

Claims

1. Use of ABT199 or a composition comprising ABT199 in the preparation of a drug for treating muscular atrophy.

2. The use according to claim 1, characterized in that: The muscle atrophy is muscle atrophy caused by cachexia.

3. Use of ABT199 or a composition comprising ABT199 in the preparation of a drug for increasing the content of reduced glutathione and enhancing the activities of superoxide dismutase and creatine kinase.

4. Use of ABT199, or a composition comprising ABT199, in the preparation of a drug for inhibiting the expression of genes Fbxo32 and Trim63.

5. Use of ABT199 or a composition comprising ABT199 in the preparation of a drug for inhibiting the gene expression of inflammatory mediators Il6, Cxcr1 and Il1b.

6. Use of ABT199, or a composition comprising ABT199, in the preparation of a drug for upregulating the expression of mitochondrial neogenesis-related genes Sirt1, Nrf1 and Tfam1.

7. Use of ABT199 or a composition comprising ABT199 in the preparation of a drug for increasing mitochondrial membrane potential.

8. The use according to any one of claims 1 to 7, characterized in that: In the composition containing ABT199, ABT199 is used as the only active ingredient, or ABT199 is used as one of the active ingredients.

9. The use according to any one of claims 1 to 7, characterized in that: The composition comprising ABT199 also includes a pharmaceutically acceptable excipient or carrier.

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