Therapeutic agent for muscular atrophy

Abstract

The purpose of the present invention is to provide a novel agent for the prevention or treatment of muscular atrophy. The present invention provides an agent for the prevention or treatment of muscular atrophy, wherein the agent contains a Dll4 function inhibitor as an active ingredient. The Dll4 function inhibitor is selected from the group consisting of inhibitors of binding between Dll4 and Notch2, inhibitors of the expression of Dll4, or inhibitors of binding of collagen with LL4.

Description

Muscle atrophy treatment

[0001] The present invention relates to a therapeutic agent for muscle atrophy. More specifically, the present invention relates to a novel therapeutic agent for muscle atrophy containing a functional inhibitor of D114 as an active ingredient.

[0002] Muscle atrophy is the atrophy of muscle fibers due to a decrease in myofibrils, resulting in pathological muscle weakness such as impaired movement and mobility. Diseases accompanied by muscle atrophy can be broadly divided into two types based on their cause: neurogenic muscular atrophy, which is caused by damage to motor nerves, and myogenic muscular atrophy, which is caused by damage to the muscle tissue itself.

[0003] Representative neurogenic muscular atrophy diseases include amyotrophic lateral sclerosis (ALS), spinal muscular atrophy, spinal-bulbar muscular atrophy, Guillain-Barré syndrome, etc. Currently, there are no effective treatments or drugs for these diseases, and effective therapeutic agents are desperately needed.

[0004] In addition to genetic muscle diseases such as muscular dystrophy, congenital myopathy, and distal myopathy, myogenic muscle atrophy also includes non-genetic muscle diseases such as age-related muscular atrophy (sarcopenia) and disuse muscular atrophy caused by reduced activity due to illness or being bedridden. Age-related muscular atrophy and disuse muscular atrophy, in particular, are steadily increasing with the aging of society. Currently, there are no effective preventive or therapeutic drugs for these various muscle atrophies, and the development of effective preventive and therapeutic drugs is urgently needed.

[0005] Adult skeletal muscle is a highly plastic tissue, easily losing or increasing its mass in response to mechanical and metabolic stimuli. The transmembrane protein Notch has been recognized as a molecule controlling skeletal muscle size. Notch is composed of the Notch family 1-4, and is known to regulate fate decisions, such as proliferation and differentiation, in tissue stem cells, including satellite cells (muscle satellite cells) in skeletal muscle.

[0006] The present inventors compared the expression levels of Notch2 mRNA and protein in the skeletal muscle of mice in which satellite cells had been specifically killed with those of normal mice, and found that Notch2 is expressed not only in satellite cells but also in muscle fibers. They created transgenic mice (N2ICD-Tg mice) that constitutively express activated Notch2 specifically in skeletal muscle fibers and performed phenotypic analysis. They found that N2ICD-Tg mice exhibited muscle atrophy in fast-twitch muscles, but that there was no significant difference in the number of cell nuclei in muscle fibers between N2ICD-Tg mice and control mice. They also reported that constitutive activation of Notch2 signaling in muscle fibers has a negative effect on the maintenance of muscle mass (Non-Patent Document 1).

[0007] The present inventors have reported that Notch1 and Notch2 are essential for maintaining the resting and undifferentiated state of satellite cells, which are skeletal muscle stem cells (Non-Patent Document 2). Furthermore, the present inventors have generated mice lacking both Notch1 and Notch2 specifically in muscle fibers (Mlc1fCre / +:Notch1f / f:Notch2f / f) and induced muscle atrophy by tail suspension or diabetes. They found that wild-type mice showed significant reductions in muscle weight and muscle fiber cross-sectional area (CSA), while Notch1 / Notch2-deficient mice were resistant to muscle atrophy (Non-Patent Document 3).

[0008] Mammals are known to have five types of Notch ligands: Jagged 1 (Jag1), Jagged 2 (Jag2), Delta-like ligand-1 (Dll1), Delta-like ligand-3 (Dll3), and Delta-like ligand-4 (Dll4) (Non-Patent Documents 4 and 5). Of these, Dll1 and Dll4 have similar structures but have been reported to have different functions (Non-Patent Document 6).

[0009] Dll4 has been reported to be expressed in differentiated myotubes (Non-Patent Document 7). Recently, it has also been reported that Dll4 is expressed in microvascular endothelial cells (ECs) of muscle tissue and in other tissues such as the retina (Non-Patent Document 8, Non-Patent Document 9).

[0010] Physical Fitness Science, Vol. 64 (2015) No. 1, p. 18 Fujimaki et al., Stem Cells 36, 278-285, 2018 Physical Fitness Science, Vol. 69, No. 1, (2020) p. 88Sjoqvist M. et al., Dev Biol 447, 58-70 (2019)Koovall R. et al., Dev Cell 41, 228-241 (2017)Preusse K. et al., PLos genetics 11, e1005328 (2015)Low S. et al., Stem Cells 36(3), 458-466 (2018)Verma M. et al., Cell Stem Cell 23(4), 530-543 e9 (2018)Miloudi K. et al., Proc Natl Acad Sci USA 116, 4538-4547 (2019)eLife 2018;7:e40045. DOI: https: / / doi.org / 10.7554 / eLife.40045Glycobiology, 2020, 1-11 doi: 10.1093 / glycob / cwaa113

[0011] An object of the present invention is to provide a new therapeutic agent for muscle atrophy.

[0012] The present inventors discovered that Delta-Like Protein 4 (Dll4) activates Notch2 in muscle fibers, inducing muscle atrophy, and that muscle atrophy can be suppressed by inhibiting Dll4-mediated Notch2 activation, leading to the completion of the present invention. The present invention includes the following: [1] A preventive or therapeutic agent for muscle atrophy, comprising as an active ingredient a Dll4 function inhibitor (preferably, a function inhibitor of Dll4 secreted from endothelial cells, more preferably a function inhibitor of Dll4 secreted from vascular endothelial cells). [2] The preventive or therapeutic agent for muscle atrophy described in [1] above, wherein the Dll4 function inhibitor inhibits Dll4 expressed in vascular endothelial cells present in muscle tissue. [3] The preventive or therapeutic agent for muscle atrophy described in [1] or [2] above, wherein the Dll4 function inhibitor is an inhibitor of the binding between Dll4 and Notch2. [4] The preventive or therapeutic agent for muscle atrophy according to [3] above, wherein the binding inhibitor is an anti-Dll4 antibody that inhibits the binding of Dll4 to Notch2. [5] The preventive or therapeutic agent for muscle atrophy according to [4] above, wherein the anti-Dll4 antibody is an antibody that recognizes 5 to 10 amino acids selected from the amino acid sequence set forth in the amino acid sequence information for Dll4 (NP_061947.1). [6] The preventive or therapeutic agent for muscle atrophy according to [1] or [2] above, wherein the Dll4 function inhibitor is an expression inhibitor of Dll4. [7] The preventive or therapeutic agent for muscle atrophy according to [6] above, wherein the expression inhibitor of Dll4 is an siRNA or shRNA that induces RNA interference against Dll4 mRNA. [8] The preventive or therapeutic agent for muscle atrophy according to [1] or [2] above, wherein the Dll4 function inhibitor is an inhibitor of collagen binding to Dll4. [9] The agent for preventing or treating muscular atrophy according to any one of [1] to [8] above, wherein the muscular atrophy is a neurogenic muscular atrophy or a myogenic muscular atrophy.

[10] The agent for preventing or treating muscular atrophy according to [9] above, wherein the muscular atrophy is selected from the group consisting of amyotrophic lateral sclerosis (ALS), myasthenia gravis, progressive muscular atrophy, spinal muscular atrophy, spinal-bulbar muscular atrophy, Guillain-Barré syndrome, and muscular atrophy due to physical spinal cord injury.

[11] The preventive or therapeutic agent for muscle atrophy according to [9] above, wherein the muscle atrophy is selected from the group consisting of age-related muscle atrophy (sarcopenia), disuse muscle atrophy, muscular dystrophy, diabetic muscle atrophy, congenital myopathy, distal myopathy, steroid myopathy, drug-induced myopathy, rhabdomyolysis, spinal muscular atrophy, progressive spinal-bulbar muscular atrophy, muscle wasting diseases, Charcot-Marie-Tooth disease, Lambert-Eaton syndrome, muscle atrophy induced by cachexia (e.g., cancer cachexia, myocardial infarction, renal failure, COPD, sepsis, HIV infection), and muscle atrophy associated with burns, etc.

[0013] The present invention provides a preventive or therapeutic agent for muscle atrophy based on a new mechanism of action.

[0014] This figure shows the culture conditions for satellite cells isolated from mouse EDL muscles. The left figure shows the gene expression level of Notch2, the center figure shows body weight, and the right figure shows the results of measuring muscle mass (n=3 (Cont), n=5 (N2-mTg)). Data represent mean ± SEM, and points indicate individual mice (this also applies to the following figures unless otherwise specified). * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001, and n.s. indicates no significant difference (this also applies to the following figures unless otherwise specified). Notch2 flox / flox (N2 f / f ) mouse and Mlc1f Cre / + ;Notch2 - / - (N2 - / - ) The results of qPCR analysis of Notch2 gene expression in individual muscle fibers isolated from mouse EDL muscles (n = 5). a) Overview of the hindlimb unloading (HU) test. b) Analysis of Notch2 expression in muscle fibers isolated from the flexor digitorum superficialis (Plantaris: PLA) (n = 5). c) N2 f / f and N2 - / - The results show that HU-induced muscle weight loss in the soleus (SOL), PLA, and gastrocnemius (GAS) muscles in mice (n=5). The left bar indicates N2 f / f Mouse results, right bar is N2 - / -Figure 1 shows the results of mice. d is a representative image of laminin immunostaining on a tissue cross-section of PLA muscle after HU (scale bar = 100 μm). e shows the myofiber cross-sectional area (CSA) of PLA muscle (n = 5). a shows an overview of the diabetes-induced muscle atrophy model test. b shows blood glucose levels (n = 5-8) 14 days after STZ injection. c shows the results of confirming Notch2 expression in muscle fibers isolated from EDL (n = 6). d shows the results of confirming muscle weight loss in the tibialis anterior (TA), PLA, and GAS 14 days after STZ injection (n = 8). The left bar in each case represents N2 f / f Mouse results, right bar is N2 - / - Results for mice are shown. e) Diabetes-induced N2 f / f and N2 - / - Representative images of laminin immunostaining of mouse TA muscle cross sections (scale bar = 100 μm). f shows the myofiber cross-sectional area (CSA) of TA muscle fibers (n = 5). f / f ;Akita mouse or N2 - / - The results show the relative muscle weight loss in Akita mice (n = 3). The left bar indicates N2 f / f Mouse results, right bar is N2 - / - The results for mice are shown below. Under DB conditions, N2 f / f Akita mouse and N2 - / - These are measurements of muscle force generated by tetanic stimulation in the TA muscle of Akita mice (n = 4). Representative tetanic force recordings (100 Hz) (left panel) and quantified maximum muscle force (right panel) are shown. Representative transmission electron microscopy (TEM) images of longitudinal GAS muscle sections under DB conditions are shown. The magnified images enclosed in boxes on the right show T-tubules (top panel) and sarcomere structures (bottom panel), respectively. Arrows indicate abnormal mitochondria (scale bar = 1 μm). The top panel shows N2 f / f Mice and N2 - / - The number of significantly up-regulated genes common to both HU and DB conditions in GAS muscle of mice (number of genes with fold change >2.0, q<0.05, and TPM>500) is shown. f / fThe top 5 pathways from KEGG pathway analysis of commonly upregulated genes (361 genes) between HU and DB conditions in mice are shown. The left panel shows representative images of myofibers stimulated with each protein (scale bar = 50 μm). The right panel shows quantitative data on myofiber diameter (n = 5 mice per condition, >15 myofibers were measured for each mouse). Gene expression analysis results after 24 hours of culture on plates coated with Dll4 recombinant protein are shown (n = 5). The left bar in each case shows the results for Cont, and the right bar shows the results for rDll4. The top panel is a schematic diagram of the co-culture conditions. The bottom left panel shows representative images after co-culture (scale bar = 50 μm). The bottom right panel shows quantitative data on myofiber diameter (n = 4 mice per condition, >15 myofibers were measured for each mouse). The left panel shows the results of N2 f / f Mice and N2 - / - The right image shows the results of culturing mouse EDL muscle fibers on plates pre-coated with recombinant Dll4 protein. f / f Mouse-derived EDL muscle fibers were cultured on plates pre-coated with recombinant Dll4 protein with the addition of DAPT. The top image shows a representative image of muscle fibers (scale bar = 50 μm). The bottom image shows quantitative data on muscle fiber diameter (n = 4 mice per condition, >15 muscle fibers were measured for each mouse). The left side of each image shows the results for Control, and the right side shows the results for rDll4. The left image shows an overview of the study. The right image shows the results of measuring muscle mass in SOL, PLA, and GAS. The left bar shows the results for Control, and the right bar shows the results for DAPT. The left image shows an overview of the study. The center image shows blood glucose concentration, and the right image shows the results of measuring muscle mass in SOL, PLA, and GAS. The left bar shows the results for Control, and the right bar shows the results for DAPT. The left image shows CD31 derived from freshly isolated muscle fibers and muscle tissue. + The right panel shows the expression of Dll4 in endothelial cells (n=4). The right panel shows the expression of CD31 cells derived from wild-type GAS muscle under HU and DB conditions. +The expression of Notch ligands in endothelial cells is shown (n = 4-6 mice per condition). Representative images of longitudinal sections of TA muscle from wild-type mice under HU and DB conditions, immunohistochemically stained with CD31 and D114 (scale bar = 20 μm), are shown. Representative confocal microscopy images (A) and immunoelectron microscopy images (B) show the distribution of D114 protein in ECs in GAS muscle tissue of DB mice. The confocal microscopy images (scale bar = 20 μm) visualize D114 and CD31, respectively, which are related to D114. ＋ CD31 ＋ EC and Dll4 － CD31 ＋ The white arrows (p) and arrowheads (q) correspond to ECs (Dll4-positive CD31 ＋ EC (Figure B) and Dll4-negative CD31 ＋Figure 1 corresponds to an immunoelectron microscopy image (scale bar = 5 μm) of ECs (Figure C). Figure 1 shows a DAB-stained image of Dll4 protein visualized by immunoelectron microscopy (the right panel of Figure B is a magnified image), demonstrating accumulation of Dll4 protein (red arrow) on the surface of myofibers. In the magnified image, Dll4 signal was detected in collagen-like fibers (green arrowheads). Figure 1 shows a DAB-stained image of Dll4-negative ECs (scale bar = 5 μm) (negative control). Figure 1 shows a schematic diagram of the transwell culture system used in this example. Figure 1 shows the results of measuring Dll4 expression in ECs transfected with siCont and siDll4. Figure 1 shows a representative image of myofibers (scale bar = 50 μm). Figure 2 shows quantitative data of myofiber diameter (n = 4 mice per condition, >20 myofibers were measured for each mouse). The left panel shows representative images of myofibers when ECs were co-cultured with a control antibody (αCont) and an anti-Dll4 neutralizing antibody (scale bar = 50 μm). The right panel shows quantitative data on myofiber diameter (n = 4 mice per condition, >15 myofibers were measured for each mouse). (a) shows an outline of a study in which a neutralizing antibody was administered to a wild-type mouse HU model. (b) shows the number of satellite cells per EDL myofiber in antibody-treated mice on day 7 (n = 4). (c) shows the reduction in muscle weight in SOL, PLA, and GAS muscles (n = 7). The left bar indicates αCont, and the right bar indicates αDll4. (d) shows representative images of laminin immunohistochemical staining of PLA muscle cross sections. (e) shows quantitative data on myofiber CSA (n = 6). (f) shows the expression of FoxO target genes and muscle atrophy-causing genes in GAS muscles (n = 6). Of the four bars, the leftmost bar represents αCont-GC, the second bar represents αCont-HU, the third bar represents αDll4-GC, and the rightmost bar represents αDll4-HU. (a) shows an outline of a study in which neutralizing antibodies were administered to a wild-type mouse DB model. (b) shows blood glucose 14 days after STZ injection (n = 5-7). (c) shows DB-induced muscle weight loss in the TA, PLA, and GAS muscles (n = 5-6). The left bar represents αCont, and the right bar represents αDll4. (d) shows a representative image of laminin immunohistochemical staining of a TA muscle cross section.(e) Quantitative data on muscle fiber CSA (n = 5). (f) Expression of FoxO target genes and muscle atrophy-causing genes in GAS muscle (n = 5). Of the four bars, the leftmost bar represents αCont-Veh, the second bar represents αCont-DB, the third bar represents αDll4-Veh, and the rightmost bar represents αDll4-DB. (a) Summary of a study in which soluble Dll4-Fc recombinant protein was administered to a wild-type mouse DB model. (b) Blood glucose 14 days after STZ injection (n = 5). (c) DB-induced muscle weight loss in TA, PLA, and GAS (n = 5). The left bar represents PBS, and the right bar represents rDll4-Fc. (a) Summary of a study in which a neutralizing antibody was administered to a wild-type mouse STZ-induced severe hyperglycemia model. b) Grip strength on day 7 (left) or day 14 (right) after STZ injection (n=9-10). c) Survival curve (n=10). P values ​​for survival experiments were calculated using the log-rank test. a) Overview of a study in which a neutralizing antibody was administered to a wild-type mouse model of mechanical load-induced muscle hypertrophy. b) Overload-induced PLA muscle weight increase (PLA muscle weight / body weight: OL-treated group (OL) vs. untreated group (sedentary control: Sed)) (n=6). c) Representative images of laminin immunohistochemical staining of overloaded PLA muscle cross sections. d) Quantitative data for muscle fiber CSA (n=6). a) N2. f / f and N2 - / -Figure 1 shows an outline of a study in which mice were subjected to mechanical overload (OL) to induce muscle hypertrophy. Analysis was performed 14 days after tenotomy. Figures b and c show the PLA muscle weight and weight gain induced by overload (PLA muscle weight / body weight: OL-treated group (OL) vs. untreated group (sedentary control: Sed)) (n = 6-8). Figure 1 shows an outline of a study in which DAPT was administered to wild-type mice in a model of mechanical overload (OL)-induced muscle hypertrophy. After tenotomy, DAPT (10 mg / kg) was intraperitoneally administered and analysis was performed 14 days later. Figures b and c show the PLA muscle weight and weight gain induced by overload (PLA muscle weight / body weight: OL-treated group (OL) vs. untreated group (sedentary control: Sed)) (n = 6). Representative TEM images of immunogold labeling of Dll4 in GAS muscle isolated from diabetic mice. Gold particles were localized near myofibers (a), collagen-like fibers (b), or extracellular vesicles (c) in the interstitial space between ECs and myofibers. Scale bar = 200 nm.

[0015] The present invention will be described below, taking as an example exemplary embodiments, along with preferred methods and materials that can be used in carrying out the present invention, but the present invention is not limited to the embodiments described below. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. Furthermore, any materials and methods equivalent or similar to those described herein can also be used in carrying out the present invention. Furthermore, all publications and patents cited in this specification in connection with the present invention are incorporated herein by reference and constitute a part of this specification, for example, to describe methods, materials, and the like that can be used in the present invention.

[0016] In this specification, the expression "A to B" indicating a range of numerical values ​​means a range of numerical values ​​including the endpoints A and B. The same applies to "A to B." In addition, in this specification, the word "about" is used to mean that ±10% is acceptable.

[0017] Herein, a preventive or therapeutic agent for muscular atrophy containing a Dll4 function inhibitor of the present invention as an active ingredient may be referred to as the "preventive or therapeutic agent of the present invention," the "pharmaceutical composition of the present invention," or the "composition of the present invention." Herein, an active ingredient that can be used in a preventive or therapeutic agent of the present invention may be referred to as the "active ingredient of the preventive or therapeutic agent of the present invention," the "active ingredient of the pharmaceutical composition of the present invention," the "active ingredient of the composition of the present invention," or the "active ingredient of the present invention." When the context clearly indicates otherwise, the term "Dll4 function inhibitor" may be simply referred to as the "active ingredient." Herein, the term "Dll4 function inhibitor" is used to encompass any of an inhibitor of Dll4 binding to Notch2, an inhibitor of Dll4 expression, and an inhibitor of collagen binding to Dll4, unless the context clearly indicates otherwise. Herein, the terms Dll4 and Dll4 protein refer to Dll4 as a substance (protein) and are used interchangeably, unless the context clearly indicates otherwise. In this specification, Notch2 and Notch2 protein refer to Notch2 as a substance (protein) and are used interchangeably, unless otherwise clearly indicated by the context.

[0018] The present invention relates to a preventive or therapeutic agent for muscle atrophy, containing a Dll4 function inhibitor as an active ingredient. In the present invention, the term "Dll4 function inhibitor" refers to a substance that can suppress muscle atrophy by inhibiting the function of Dll4, which causes muscle atrophy based on the Dll4-Notch2 signaling axis. The inventors have found that microvascular endothelium in muscle tissue upregulates and secretes Dll4, a Notch2 ligand, and that Dll4 secreted from vascular endothelial cells selectively activates Notch2 by binding to Notch2 present on the surface of muscle fibers without direct cell-to-cell contact, resulting in the induction of muscle atrophy signals and muscle atrophy. The inventors have also found that it is important for Dll4 secreted from endothelial cells, as a ligand, to bind to collagen in order to bind to its receptor, Notch2. The present inventors further found that Dll4 is expressed on the membrane of extracellular vesicles secreted by endothelial cells, and that muscle atrophy due to Dll4-mediated activation of Notch2 occurs via extracellular vesicles. They also found that Dll4 secreted from endothelial cells exists in a cleaved form with a smaller molecular weight than the originally predicted Dll4, and that this cleaved form is important for binding to and activation of Notch2. Therefore, in the present invention, the term "Dll4 function inhibitor" refers to a substance that inhibits the action of Dll4, which induces muscle atrophy via Notch2, and is used to mean, but is not limited to, any of the following: a substance that inhibits the binding between Dll4 and Notch2 (preferably a substance that inhibits the binding between Dll4 secreted from blood vessels and Notch2 in muscle fibers), a substance that inhibits the expression of Dll4 (including, for example, an inhibitor of Dll4 expression in endothelial cells and an inhibitor of Dll expression in extracellular vesicles), and a substance that inhibits the binding of Dll4 to Notch2 by inhibiting the binding of collagen to Dll4.

[0019] Substances that inhibit the binding between Dll4 and Notch2 (hereinafter referred to as "Dll4 and Notch2 binding inhibitors," "Dll4 and Notch2 binding inhibitors," or simply "binding inhibitors" or "binding inhibitors" when clear from the context) inhibit the binding between Dll4 and Notch2, thereby suppressing Notch2 activation and inhibiting the induction of muscle atrophy signals, thereby suppressing muscle atrophy. As a result, they can be used as active ingredients in preventive or therapeutic agents for muscle atrophy. Examples of binding inhibitors include, but are not limited to, substances that bind to Dll4 and inhibit Dll4 from physically binding to Notch2. Specifically, examples include small molecules, medium molecules, antibodies, and the like that bind to the Notch2-binding site of Dll4 (hereinafter sometimes referred to as the "Notch2-binding site of Dll4," the "Notch2-binding site," or the "receptor-binding site"). A preferred example is an antibody that recognizes the binding site of D114 to Notch2. Furthermore, examples of binding inhibitors include, but are not limited to, substances that bind to Notch2 and inhibit the physical binding of Notch2 to D114. Specific examples include small molecules, medium molecules, antibodies, and the like that bind to the Notch2 binding site to D114 (hereinafter sometimes referred to as the "Notch2 binding site to D114," the "D114 binding site," or the "ligand binding site"). A preferred example is an antibody that recognizes the Notch2 binding site to D114.

[0020] Dll4 secreted from blood vessels binds to Notch2 in muscle fibers. Therefore, the binding inhibitor is preferably a substance that inhibits the binding between Dll4 secreted from blood vessels and Notch2 in muscle fibers. Furthermore, substances that inhibit the binding between Dll4 and Notch2 also include binding inhibitors that target protein modifications of Dll4, such as glycosylation of the extracellular domain of Notch2.

[0021] Small or medium-molecular compounds can be screened according to standard methods using as an index the binding activity to Dll4, the binding activity to Notch2, or the activity of inhibiting the binding of Dll4 to Notch2. For example, but not limited to, Dll4 and Notch2 can be allowed to bind to each other in the presence or absence of a test substance, and then the Dll4-Notch2 complex can be detected by ELISA using an anti-Dll4 antibody and an anti-Notch2 antibody, thereby screening for a substance that inhibits the binding of Dll4 to Notch2. Anti-Dll4 antibodies and anti-Notch2 antibodies can be prepared according to standard methods, but commercially available antibodies may also be used.

[0022] Alternatively, a system can be used in which Dll4 molecules are chemically modified to emit fluorescence when bound to Notch2 molecules, isolated muscle fibers are cultured as adherents, and the binding of Dll4 to Notch2 expressed in the muscle fibers is detected by fluorescence. Alternatively, a system can be used in which the binding of Dll4 molecules to Notch2 molecules is detected using a luciferase reporter assay. Furthermore, instead of isolated muscle fibers, similar experiments can be performed using cells in which Notch2 is overexpressed on the cell surface.

[0023] Antibodies that recognize the binding site of Dll4 to Notch2, or antibodies that recognize the binding site of Notch2 to Dll4, can be produced by conventional methods using Dll4 protein or Notch2 protein. Alternatively, they can be produced by conventional methods using genetic engineering techniques with reference to the amino acid sequence information of Dll4 or Notch2. The nucleic acid and amino acid sequences of Dll4 and Notch2 are available from publicly known databases. Human Dll4 is registered as a nucleic acid sequence (NM_019074.4) and an amino acid sequence (NP_061947.1), and human Notch2 is registered as a nucleic acid sequence (NM_024408.4) and an amino acid sequence (NP_077719.2). For example, antibodies against amino acid sequences thought to contain the binding site can be produced with reference to reports on the binding site of Dll4 and Notch2 (e.g., Non-Patent Document 10, Non-Patent Document 11). Further, for example, but not limited to, an anti-Notch2 antibody can include an anti-Notch2 antibody that binds to the EGF repeats 11-13 region of Notch2, since Dll4 has been suggested to bind to the EGF repeats 11-13 region of the extracellular domain of Notch2. The antibody may be full-length (e.g., IgG1, IgG2, or IgG4) or may contain the antigen-binding site and be modified to improve functionality or to remove non-essential effector functions. Examples include disulfide Fvs, dAb fragments, fragments containing the complementarity-determining regions (CDRs), single domains such as isolated CDR fragments, defined FR3-CDR3-FR4 peptides, single-chain antibodies (scFvs), minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), diabodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, bispecific antibodies, multispecific antibodies (e.g., triabodies, tetrabodies), polypeptides comprising at least a portion of an antibody with specific antigen-binding ability, and other engineered molecules such as small modular immunopharmaceuticals. Alternatively, the antibody may be an antigen-binding fragment of an antibody.Antigen-binding fragments of antibodies include any naturally occurring or synthetic, enzymatically or biochemically obtained, or genetically engineered polypeptide or glycoprotein that specifically binds to the Notch2-binding site of the antigen, D114, to form a complex. The above also applies when Notch2 is used as the antigen.

[0024] Antigen-binding fragments of antibodies can be obtained from intact antibody molecules or their amino acid or genetic information using any suitable standard technique, such as, for example, proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding the variable and (optionally) constant domains of the antibody. The DNA can be sequenced and manipulated using chemical or molecular biology techniques, for example, to place one or more variable and / or constant domains in the appropriate positions, or to introduce optional codons to create cysteine ​​residues or to modify, add, or delete amino acids, etc., to produce any antigen-binding fragment.

[0025] Antigen-binding fragments of antibodies typically contain at least one variable domain. Variable domains may be of any size or amino acid composition and generally contain at least one CDR, which is flanked or in-frame by one or more framework sequences. In antigen-binding fragments having a VH domain connected to a VL domain, the VH and VL domains are positioned relative to one another in any suitable configuration. Antigen-binding fragments of antibodies include, for example, dimeric variable regions, such as VH-VH, VH-VL, or VL-VL dimers. Alternatively, antigen-binding fragments of antibodies contain monomeric VH or VL domains.

[0026] In certain embodiments, an antigen-binding fragment of an antibody contains at least one variable domain covalently linked to at least one constant domain. Exemplary arrangements of variable and constant domains within an antigen-binding fragment of an antibody include, but are not limited to, (i) VH-CH1; (ii) VH-CH2; (iii) VH-CH3; (iv) VH-CH1-CH2; (v) VH-CH1-CH2-CH3; (vi) VH-CH2-CH3; (vii) VH-CL; (viii) VL-CH1; (ix) VL-CH2; (x) VL-CH3; (xi) VL-CH1-CH2; (xii) VL-CH1-CH2-CH3; (xiii) VL-CH2-CH3; and (xiv) VL-CL. In any arrangement of variable and constant domains, including any of the exemplary arrangements described above, the variable and constant domains are either directly linked to each other or linked by a linker region. The linker region consists of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids and connects adjacent variable and / or constant domains. Furthermore, antigen-binding fragments of antibodies include homo- or heterodimers (or other multimers) of any of the above variable and constant domain arrangements, in which one or more monomeric VH or VL domains are non-covalently associated.

[0027] Like intact antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies typically contain at least two different variable domains, each capable of specifically binding to a different antigen or a different epitope on the same antigen. Any multispecific antibody, including the bispecific antibody herein, can be produced using conventional techniques available in the art and can be used as an antigen-binding fragment of an antibody for the purposes of the present invention.

[0028] A substance that inhibits Dll4 expression (hereinafter referred to as a "Dll4 expression inhibitor," "Dll4 expression inhibitor," or simply an "expression inhibitor" or "expression inhibitor" if clear from the context) inhibits Dll4 expression, thereby reducing Dll4 expression, for example, in endothelial cells or extracellular vesicles, thereby suppressing Notch2 activation by Dll4 and inhibiting the induction of muscle atrophy signals, thereby suppressing muscle atrophy. Preferably, the substance reduces Dll4 expression in endothelial cells, more preferably in vascular endothelial cells. As a result, the substance can be used as an active ingredient in a preventive or therapeutic agent for muscle atrophy. Examples of expression inhibitors include nucleic acid molecules that specifically inhibit Dll4 expression. Nucleic acid molecules that specifically inhibit Dll4 expression are usually non-naturally occurring nucleic acids, such as RNAi, antisense nucleic acids, or ribozymes that specifically interfere with Dll4 expression. "RNAi" refers to any RNA capable of down-regulating the expression of targeted D114, including siRNA, dsRNA, ssRNA, and shRNA molecules. Preferably, the D114 expression inhibitor is siRNA or shRNA.

[0029] siRNA or shRNA is typically designed against a region 19-50 nucleotides downstream of the translation initiation codon. In a preferred embodiment, the RNAi molecule is an siRNA having a length of at least about 10-40 nucleotides, preferably about 15-30 nucleotides. The siRNA or shRNA may include naturally occurring RNA, synthetic RNA, or recombinantly produced RNA, as well as modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or modification of one or more nucleotides. Such modifications may include modifications that render the siRNA resistant to nuclease digestion, and may include the addition of non-nucleotide material, for example, to the termini of the molecule or to one or more internal nucleotides of the siRNA.

[0030] The expression inhibitor nucleic acid of the present invention has the ability to specifically hybridize to a gene or transcript encoding D114. To specifically hybridize, the expression inhibitor nucleic acid does not need to have 100% complementarity with the target sequence, D114, but preferably has at least about 90% complementarity. More preferably, the complementarity between the expression inhibitor nucleic acid of the present invention and the target sequence (the D114 target sequence) is at least 95%, 96%, 97%, 98%, 99%, or 100%.

[0031] A substance that inhibits collagen binding to D114 (hereinafter referred to as a "substance that inhibits D114 binding to collagen," "D114 binding inhibitor," or simply a "binding inhibitor" or "binding inhibitor" if the context makes clear) inhibits the binding of D114 secreted from vascular endothelial cells to collagen, thereby inhibiting the binding of D114 to Notch2 and suppressing Notch2 activation, thereby suppressing the induction of muscle atrophy signals, thereby suppressing muscle atrophy. As a result, it can be used as an active ingredient in a preventive or therapeutic agent for muscle atrophy. Examples of binding inhibitors include, but are not limited to, substances that bind to D114 and inhibit the physical binding of D114 to collagen. Specific examples include small molecules, medium molecules, antibodies, and the like that bind to the collagen-binding site of D114 (hereinafter sometimes referred to as the "collagen-binding site of D114" or "collagen-binding site"). Preferably, an antibody that recognizes the collagen-binding site of D114 is used. Furthermore, examples of the binding inhibitor include, but are not limited to, substances that bind to collagen and inhibit the physical binding of collagen to D114. When an antibody is used, various antibodies can be used, similar to the antibody that inhibits the binding between D114 and Notch2.

[0032] Other Dll4 function inhibitors include, for example, substances that inhibit the cleavage of the intracellular domain of Notch2 (activation of Notch2) even when Dll4 binds to the extracellular domain of Notch2. Such inhibitors include, but are not limited to, inhibitors of metalloproteases and secretases involved in Notch2 cleavage.

[0033] Compositions containing the Dll4 function inhibitor of the present invention as an active ingredient are used for the prevention or treatment of diseases accompanied by muscle atrophy. The compositions of the present invention can also be used to improve muscle atrophy, and thus muscle mass loss or muscle weakness. Examples include maintaining muscle weakened by aging and improving urinary incontinence. Muscle atrophy refers to muscle wasting, resulting in a decrease in muscle mass. The "muscle atrophy" targeted by the preventive or therapeutic agent of the present invention includes both neurogenic muscular atrophy and myogenic muscular atrophy. Neurogenic muscular atrophy includes, for example, amyotrophic lateral sclerosis (ALS), myasthenia gravis, progressive muscular atrophy, spinal muscular atrophy, spinal-bulbar muscular atrophy, Guillain-Barré syndrome, and muscular atrophy due to physical spinal cord injury. Examples of myogenic muscular atrophy diseases include age-related muscular atrophy (sarcopenia), disuse muscular atrophy, muscular dystrophies (e.g., Duchenne muscular dystrophy, Becker muscular dystrophy, distal muscular dystrophy, congenital muscular dystrophy, Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, myotonic muscular dystrophy type 1 or myotonic muscular dystrophy type 2, and limb-girdle muscular dystrophy), and diabetic muscular atrophy. Examples of such myopathy include atrophy, congenital myopathy, distal myopathy, steroid myopathy, drug-induced myopathy, rhabdomyolysis, spinal muscular atrophy, progressive spinal-bulbar muscular atrophy, muscle wasting diseases (e.g., age-related sarcopenia, cachexia), Charcot-Marie-Tooth disease, Lambert-Eaton syndrome, muscle atrophy induced by cachexia (e.g., cancer cachexia, myocardial infarction, renal failure, COPD, sepsis, HIV infection), muscle atrophy associated with burns, etc.

[0034] The agent for preventing or treating muscle atrophy of the present invention comprises a therapeutically effective amount of a D114 functional inhibitor. A "therapeutically effective amount" refers to an amount sufficient to produce a therapeutic effect when administered to a mammal in need of treatment. The therapeutically effective amount varies depending on the subject and the disease symptoms being treated, the subject's weight and age, the severity of the disease symptoms, the method of administration, etc., and can be easily determined by one skilled in the art. In some embodiments, the D114 functional inhibitor is a small or medium molecule. In some embodiments, the D114 functional inhibitor is an antibody or an antigen-binding fragment thereof, which is used in a substantially purified form. In some embodiments, the D114 functional inhibitor is a nucleic acid, preferably an siRNA or shRNA. In some embodiments, the D114 functional inhibitor of the present invention is administered prophylactically to a subject experiencing muscle atrophy. In some embodiments, the D114 functional inhibitor of the present invention is administered therapeutically to a subject experiencing muscle atrophy.

[0035] The subject to which the prophylactic or therapeutic agent of the present invention is administered is a mammal, preferably a human. In a more specific embodiment, the subject is a human suffering from muscle atrophy. The composition of the present invention can be administered to a subject suffering from mild or moderate muscle atrophy not only for therapeutic purposes but also for prophylactic purposes to prevent the progression of muscle atrophy. The composition of the present invention can be administered to a subject suffering from moderate or severe muscle atrophy primarily for therapeutic purposes.

[0036] The composition of the present invention comprises a therapeutically effective amount of a DDL4 function inhibitor and further comprises a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" refers to a carrier approved for use by a regulatory agency or listed in a commonly accepted pharmacopoeia for use in animals, particularly humans, and includes any and all solvents, dispersion media, coating agents, antioxidants, chelating agents, preservatives (e.g., antibacterial agents, antifungal agents), surfactants, buffers, osmotic pressure adjusting agents, absorption retardants, salts, drug stabilizers, excipients, diluents, binders, disintegrants, sweeteners, flavoring agents, lubricants, dyes, etc., and combinations thereof, as known to those skilled in the art. Unless any carrier is incompatible with the active ingredient of the present invention, it can be used in the composition of the present invention.

[0037] The composition of the present invention can be formulated into a desired dosage form depending on the administration method, such as oral administration or parenteral administration. The dosage form is not particularly limited, and for oral administration, it can be formulated into solid preparations such as powders, granules, tablets, lozenges, and capsules; or liquid preparations such as solutions, syrups, suspensions, and emulsions. For parenteral administration, it can be formulated into suppositories, sprays, inhalants, ointments, patches, injections (including infusions), and the like. The composition of the present invention may also be a lyophilized preparation that is dissolved by adding sterile water or the like when used. The formulation can be carried out by a known method depending on the dosage form.

[0038] In one embodiment, the present invention provides a method for preventing or treating a subject by administering a therapeutically effective amount of a D114 functional inhibitor to the subject. The therapeutically effective amount, administration method, and other details of the D114 functional inhibitor are as described above for the preventive agent and therapeutic agent.

[0039] The dose of the DDL4 function inhibitor, which is an active ingredient of the present invention, is appropriately selected depending on the type of DDL4 function inhibitor, and also depending on the symptoms, age, condition, administration route, etc. of the subject to be administered.

[0040] When a small or medium molecule is used as the DDL4 function inhibitor, which is the active ingredient of the present invention, the composition of the present invention can be administered orally or parenterally as a pharmaceutical, but is preferably administered orally. For oral administration, the composition can be used in the form of solid preparations such as tablets, powders, capsules, and granules, which may contain conventional additives such as excipients, binders, diluents, and lubricants. It can also be administered as a solvent, such as an aqueous or oily suspension, solution, syrup, or elixir. For parenteral administration, the composition can be used as an injection or the like. The dosage cannot be generally determined, as it varies depending on the patient's age, weight, and condition, the type and severity of the disease, and the method of administration, but is typically about 0.1 to about 500 mg / kg per day for an adult.

[0041] When an antibody is used as the DDL4 function inhibitor, which is the active ingredient of the present invention, for the prevention or treatment of disease in adult patients, it is typically administered at a single dose of, but not limited to, about 0.01 mg per kg of body weight, preferably about 0.05 mg, more preferably about 0.1 mg per kg, and about 100 mg per kg of body weight, preferably about 50 mg, more preferably about 20 mg, and even more preferably about 10 mg per kg. When an antigen-binding fragment of an antibody of the present invention is used, a higher dose than the above-mentioned dose is typically administered, for example, but not limited to, about 1.5 to about 50 times, preferably about 1.5 to about 20 times, and more preferably about 1.5 to about 10 times the dose. The frequency and interval of administration are adjusted depending on the severity of the condition. In one embodiment, the antibody or the like is administered as a starting dose of at least about 1 mg to about 1000 mg. In certain embodiments, the initial dose is followed by second or multiple subsequent doses of the antibody or antigen-binding fragment thereof in an amount that is about the same as or less than that of the initial dose, where the subsequent doses are separated by at least 2-5 days; at least 1 week; at least 2 weeks; at least 3 weeks; at least 4 weeks; at least 5 weeks; at least 6 weeks; at least 7 weeks; at least 8 weeks; at least 9 weeks; at least 10 weeks; or at least 12 weeks.

[0042] The compositions of the present invention are prepared in dosage forms prepared in unit doses suitable for the desired dose of the active ingredient, whether for oral or parenteral administration. Dosage forms containing a unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of antibody contained in a unit dose is generally preferably about 5 to about 1,000 mg per dosage form.

[0043] The administration treatment plan, including the administration dose and administration schedule, can be determined by the physician who diagnosed the subject (patient) based on the patient's symptoms, age, condition, administration route, etc., and by referring to guidelines presented by various organizations, as necessary.

[0044] The administration route of the pharmaceutical composition of the present invention can be determined arbitrarily, taking into account the symptoms, condition, and other conditions of the subject. Administration routes include, but are not limited to, intradermal, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The pharmaceutical composition can be administered by any convenient route, for example, by infusion or bolus injection, or by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal, and intestinal mucosa), and may also be administered together with other biologically active agents. Administration can be systemic or local.

[0045] Various delivery systems are known for administering active pharmaceutical ingredients, and the pharmaceutical compositions of the present invention can be administered using these. Examples include encapsulation in liposomes, microparticles, nanoparticles, and microcapsules. In some embodiments, the pharmaceutical compositions can also be delivered in controlled release systems. Controlled release systems place the composition near the target, thus requiring only a small systemic dose.

[0046] When the pharmaceutical composition of the present invention is administered by injection, injectable preparations include intravenous, subcutaneous, intradermal, intraperitoneal, and intramuscular injection forms, or drip infusion forms. These injectable preparations can be prepared with reference to known methods. Injectable preparations can be prepared, for example, by dissolving, suspending, or emulsifying the antibody or salt thereof of the present invention in a sterile aqueous or oily medium commonly used for injections. Aqueous media for injection include, for example, physiological saline, an isotonic solution containing glucose, and other adjuvants. These media can also be used in combination with appropriate solubilizers such as alcohols (e.g., ethanol), polyhydric alcohols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)]. Oily media include, for example, sesame oil and soybean oil, which can be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. The prepared injections are preferably filled into appropriate ampoules.

[0047] When the pharmaceutical composition of the present invention is an injection, it can be prepared as an injection to be prepared at the time of use or as a pre-filled syringe.The pharmaceutical composition of the present invention can be administered subcutaneously or intravenously with a standard needle and syringe.Also, a pen delivery device can be used for subcutaneous administration.The pen delivery device can be reusable or disposable.

[0048] When a nucleic acid, such as siRNA, is used as a DDL4 function inhibitor in the composition of the present invention, it may be a single siRNA or a mixture of multiple siRNAs (so-called cocktail). When the siRNA of the present invention is used in vivo, the siRNA can be directly injected into the affected area, or a vector capable of expressing the siRNA can be used. When the siRNA is directly injected into the affected area, it can be complexed with liposomes, such as lipofectamine, lipofectin, cellfectin, and other positively charged liposomes, and then injected. When a vector capable of expressing the siRNA is used, for example, an expression vector containing a DNA sequence encoding an RNA comprising the sense strand sequence of the siRNA and its complementary antisense strand sequence under the control of a promoter is preferably used.

[0049] To obtain siRNA, shRNA can be used or expressed intracellularly. The shRNA that can be used in the present invention comprises the sense strand sequence, the antisense strand sequence, and a single-stranded loop sequence covalently linking the sense strand sequence and the antisense strand sequence, and is an RNA that is processed by Dicer, an intracellular RNase, to form siRNA. The shRNA may be a single shRNA or a mixture of multiple shRNAs (a so-called cocktail).

[0050] A viral vector may be used to deliver siRNA to the affected area. The viral vector that can be used is not particularly limited, and examples thereof include adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, retroviral vectors (e.g., leukemia viral vectors), and herpes viral vectors. The viral vector is preferably one that lacks self-replication ability so as not to cause disease when used in humans. For example, in the case of an adenoviral vector, a self-replication-deficient adenoviral vector lacking the E1 gene and E3 gene can be used. The viral vector can be constructed according to known methods.

[0051] Since the purpose of the present invention is to suppress the expression of the DDL4 gene in skeletal muscle, adeno-associated virus vectors with selective delivery to skeletal muscle are particularly preferred. Currently, types I to XI of adeno-associated virus vectors are known, and these can be used without limitation. However, vectors developed in the future can also be used without limitation as long as they achieve the objectives of the present invention. Preferred vectors include, but are not limited to, AAV1, AAV2, AAV4, AAV6, AAV7, AAV8, and AAV9. Various adeno-associated virus vectors that can be used preferably are commercially available.

[0052] When using a viral vector, genes can be introduced into cells by directly injecting the vector into the affected area and infecting the cells. In particular, adenoviral vectors have been confirmed to be capable of highly efficient gene transfer into various cell types and have actually been used clinically for gene therapy. This vector also does not integrate into the genome, so its effect is transient and is thought to be safer than other viral vectors.

[0053] The dosage of siRNA or shRNA when administered directly is not particularly limited, and an amount that produces an effect of reducing DDL4 at the protein level is selected. The dosage is a therapeutically effective amount, and can also be administered multiple times at regular time intervals. When actually used, the dosage is determined by the judgment of a specialist depending on the patient's condition, age, sex, severity, etc.

[0054] The vector carrying the siRNA or shRNA is administered to the patient together with a pharmaceutically acceptable carrier, such as physiological saline or a buffer solution. The pharmaceutical composition may further contain stabilizers, preservatives, isotonicity agents, etc. The method of administration of the pharmaceutical composition of the present invention is not particularly limited, and may be either local or non-local administration, with local administration being preferred. In the case of local administration, the vector can be administered directly using a syringe or the like. In the case of non-local administration, it can be administered, for example, intravenously. As for the administration form, for example, a viral vector or a complex of a plasmid and liposome is administered in a form suspended in a pharmaceutically acceptable carrier.

[0055] The present invention will be specifically described below using examples, but the present invention is not limited to the following examples. The methods used in the examples of the present invention are described below. (1) Antibodies and Reagents Various antibodies and reagents used in the examples were purchased from commercial sources.

[0056] (2) Animals All animal experimental procedures were approved by the Institutional Animal Care and Usage Committee (IACUC) of Kumamoto University. Animals were housed in a pathogen-free, temperature- and humidity-controlled environment. They were housed under a 12-hour light cycle with free access to food and water. Commercially available mice were used in the examples. Mice with specific genetic phenotypes were also bred to generate mice with the desired genetic phenotype, as needed.

[0057] (3) In vitro inhibition of Notch signaling. The effects of inhibiting the Notch signaling pathway on muscle in vitro were examined using wild-type mice. Ultra-LEAF™ anti-mouse Dll4 antibody (Dll4 blocking antibody) or Ultra-LEAF™ American hamster IgG control antibody (control antibody) was administered intraperitoneally (10 mg / kg body weight) every four days in each experiment. Recombinant Dll4-Fc chimeric protein (rDll4) was dissolved in phosphate-buffered saline (PBS) and administered every two days (0.5 mg / kg body weight). The γ-secretase inhibitor, DAPT, dissolved in 10% ethanol and corn oil was administered every two days (10 mg / kg body weight). Control mice received an intraperitoneal injection of vehicle at an equivalent volume in the rDll4 or DAPT experiments.

[0058] (4) Tail-Suspension Hindlimb Unloading Model Mice were individually suspended by their tails in each cage using a string tied to one end of the tail and the other end of a custom-made cage lid. The length of the string was adjusted to allow the mice to freely move their forelimbs and access food and drink. Litter-matched, age-matched, terrestrial (non-suspended) mice served as controls. After 7 days of suspension, the mice were killed by cervical dislocation, and the soleus (SOL), flexor digitorum superficialis (PLA), and gastrocnemius (GAS) muscles were harvested.

[0059] (5) STZ-Induced Diabetes Model Mice received a single intraperitoneal injection of STZ (150 mg / kg body weight), a level toxic to insulin-producing pancreatic β cells, or an equal volume of vehicle (0.1 M citrate buffer, pH 4.5) as a control. Hyperglycemia was monitored using an Accu-Chek (Roche) on day 3, and only mice with a casual blood glucose level of >300 mg / dL were used for the experiment. Mice were sacrificed 14 days after STZ injection. For survival experiments, wild-type mice were intraperitoneally injected with a single high dose of STZ (250 mg / kg), which induces severe diabetes. These mice were then administered either a Dll4 neutralizing antibody or a control antibody (10 mg / kg body weight) every four days. The appearance of diabetic mice was monitored daily, and their weights and blood glucose levels were measured weekly. The experiment was terminated when all mice in either group died or were euthanized.

[0060] (6) Tenotomy: To induce muscle hypertrophy, tenotomy surgery was performed on both legs of mice. After removing the hair from anesthetized mice, an incision was made along the midline of the calf. After resection of the tendons belonging to the SOL and GAS, the skin was closed with three or four sutures. After 14 days of overload, the mice were sacrificed and the PLA muscles were harvested.

[0061] (7) Measurement of Grip Strength and Tetanic Muscle Strength. Whole-limb grip strength was measured using a rodent grip strength meter. The peak tension (N) when the mouse released the grip was recorded. The maximum strength determined from 10 consecutive measurements was used for data analysis. Tetanic muscle strength was measured in the tibialis anterior (TA) muscle using a whole-animal muscle strength testing system. Briefly, mice were anesthetized with isoflurane and placed on a 37°C heating plate throughout the procedure. The right foot was fixed to a footplate connected to a servomotor, and the right knee was immobilized with a knee clamp. The TA muscle was stimulated subcutaneously with two needle electrodes at 5 mA. Tetanic contractions were induced by 350 ms stimulation at a frequency of 100 Hz. Each contraction was followed by an interval of at least 2 min, and the maximum muscle strength determined from three consecutive measurements was used for data analysis.

[0062] (8) Muscle fiber isolation and satellite cell culture. Muscle fibers were isolated from the extensor digitorum longus (EDL) or flexor digitorum superficialis (PLA) muscles by enzymatic digestion using type I collagenase, following a slightly modified version of our previous report (Ono et al., Cell Report 10, 1135-1148 (2015)). For gene expression analysis, the isolated muscle fibers were immediately frozen using liquid nitrogen. Satellite cells were obtained from muscle fibers isolated from the EDL muscle. Satellite cells were incubated at 37°C and 5% CO. ２ Myotubes were cultured in GM (DMEM supplemented with 30% fetal bovine serum (FBS), 1% chicken embryo extract, 10 ng / ml basic FGF, and 1% penicillin-streptomycin) in an atmosphere containing 4-hydroxybenzoates (4OH) and 4-hydroxybenzoates (4OH). Myogenic differentiation was induced by replacing the medium with DM (DMEM supplemented with 2% horse serum and 1% penicillin-streptomycin). Cre recombinase expression in differentiated myotubes was induced using 4OH TMX (1 μM).

[0063] (9) EC Isolation and Culture. Primary endothelial cells (ECs) were isolated from muscle tissue of wild-type mice using Dynabeads, following a minor modification of the method described by Muramatsu et al. (Arteriosclerosis, thrombosis, and vascular biology 40, 2425-2439 (2020)). Anesthetized mice were perfused with PBS, and hindlimb muscle tissue was harvested. The muscle tissue was digested with 0.2% type II collagenase, followed by erythrocyte lysis. Cells were incubated with Dynabeads conjugated with anti-CD31 antibody and separated into endothelial and non-endothelial fractions using a magnetic separation system. After primary selection, cells were cultured in DMEM (supplemented with 20% FBS, non-essential amino acids, sodium pyruvate, 25 mM HEPES, 0.1 mg / ml heparin, 0.1 mg / ml endothelial cell growth supplement, and 1% penicillin-streptomycin) for 7 days. For the second selection, expanded cells were collected and incubated with anti-CD102 antibody-conjugated Dynabeads to increase the purity of ECs. Purified primary ECs were cultured until confluent for coculture with myofibers. To reduce Dll4 expression, ECs were transfected with 20 nM Dll4-siRNA (SASI_Mm01_00092621, MilliporeSigma) or control-siRNA (MilliporeSigma) using Lipofectamine RNAiMAX.

[0064] (10) Plasmid transfection. Mouse Dll1 cDNA and mouse Dll4 cDNA were cloned into the pcDNA3.1+P2A-eGFP plasmid vector (GenScript) to generate pcDNA3.1+Dll1-P2A-eGFP (Dll1-GFP) and pcDNA3.1+Dll4-P2A-eGFP (Dll4-GFP). HEK293 cells were transfected with either the Dll1-GFP plasmid, the Dll4-GFP plasmid, or the pcDNA3.1+P2A-eGFP control plasmid using Lipofectamine LTX with Plus reagent according to the manufacturer's instructions. GFP signals were captured using a fluorescence microscope.

[0065] (11) Myofiber Culture and Diameter Measurement. Culture dishes were coated with recombinant Notch ligand proteins according to the report by Sakai et al. (Sakai et al., PLoS One 12, e0177516 (2017)). Culture dishes were first coated with 10 μg / ml of anti-Fc antibody at room temperature for 1 hour, and then coated with Matrigel (Corning) for 20 minutes. The Fc antibody-coated dishes were incubated with 5 ng / μl of Fc fusion protein of each Notch ligand (Dll1, Dll4, Jag1, and Jag2) at room temperature for 1 hour. Individual myofibers isolated from EDL muscles were placed in serum-free DMEM and incubated for 1 hour under CO -incubation. ２ The myofibers were cultured in a CO incubator for 48 hours. DAPT (10 μM) was used to inhibit γ-secretase activity in the myofibers. Experiments of co-culture of myofibers with HEK293 cells were performed as follows. Isolated myofibers were cultured in serum-free DMEM for 48 hours in CO with HEK293 cells transfected with plasmids encoding GFP, Dll1-GFP, and Dll4-GFP. ２ The cells were cultured in an incubator. Transwell Permeable Supports (8 μm pore transwell membrane; Corning) were used for co-culture of myofibers and primary ECs isolated from muscle tissue. Individual myofibers and ECs were placed in plate wells (lower wells) and transwell inserts (upper wells), respectively, and incubated under CO ２ The myofibers were cultured in serum-free DMEM for 48 hours in an incubator. To block Dll4 protein secreted by ECs, anti-Dll4 neutralizing antibody (500 ng / ml) was added to the culture medium, and a control antibody (500 ng / ml) was used as a control. Cultured myofibers were captured using a fluorescence microscope, and the diameters of at least 15 individual myofibers were quantified using ImageJ (NIH).

[0066] (12) Cell Sorting. Muscle-associated endothelial cells were freshly obtained by cell sorting and their gene expression was evaluated. Mouse GAS muscle was minced and digested with 0.2% type II collagenase, followed by erythrocyte lysis. Mononuclear cells were incubated with PE-conjugated CD31 antibody at 4°C for 30 minutes. CD31 ＋ Cells were sorted using an S3e cell sorter (BioRad). Debris and dead cells were excluded by forward and side scatter. Sorted cells were collected into microtubes using ISOGEN II (Nippon Gene).

[0067] (13) RNA Extraction and Quantitative Reverse Transcription-PCR. Total RNA was extracted from muscle tissue, isolated myofibers, cultured cells, and freshly sorted cells using the ISOGEN II and RNeasy Kit (Qiagen) according to the manufacturer's instructions. cDNA was prepared using the ReverTra Ace kit with genomic DNA remover (Toyobo). Quantitative PCR (qPCR) was performed using the THUNDERBIRD SYBR qPCR mix (Toyobo) and a real-time PCR detection system (CFX96; BioRad). The primer sequences used were as follows: TATA box binding protein (Tbp) as a standard, and Pax7, Notch1, Notch2, Rbpj, Hes1, Dll1, Dll4, Jag1, Jag2, Ccng2, Cdkn1b, Rbl2, Bnip3, MuRF1, Atrogin1, Fbxo31, and MUSA1.

[0068] (14) RNA sequencing. Total RNA was obtained from GAS muscle using ISOGEN II and the RNeasy kit. Library construction and RNA sequencing were performed by Novogene (Beijing).

[0069] (15) Immunofluorescence. Isolated PLA or TA muscles were immediately frozen in liquid nitrogen-cooled 2-methylbutane and stored at -80°C until analysis. Frozen tissues were sliced ​​at 10 μm thickness using a cryostat, and the frozen sections were subjected to immunohistochemical analysis. Immunocytochemical analysis of satellite cells and isolated muscle fibers was performed according to our previous publication (Ono et al., Cell Report 10, 1135-1148 (2015)). Samples were incubated with primary antibodies overnight at 4°C. They were then fixed with a 4% paraformaldehyde (PFA) / PBS solution and permeabilized / blocked with PBS containing 0.3% Triton X-100 and 5% normal goat serum for 30 minutes at room temperature. Immunostained specimens were visualized using Alexa Fluor-conjugated secondary antibodies and observed under a fluorescent microscope. Myofiber CSA was quantified from at least 500 myofibers using Image-J (NIH), and the number of satellite cells per myofiber was counted from at least 15 myofibers. Whole-mount staining of muscle tissue was performed for vascular staining. Anesthetized mice were perfused with 4% PFA / PBS solution, and GAS muscles were carefully harvested. The excised tissue was sliced ​​to a thickness of 1 mm using a sharp razor blade and immersed overnight at 4°C in 1% bovine serum albumin / 0.1% PBST blocking solution. The blocked tissue was incubated overnight at 4°C with anti-CD31 and anti-D114 antibodies as primary antibodies. The tissue was washed three times with PBST and stained overnight at 4°C with the appropriate species-specific secondary antibodies and DAPI (nuclear stain). The muscles were washed five times again with PBST and analyzed using a confocal fluorescence microscope.

[0070] (16) Transmission Electron Microscopy (TEM) Under deep anesthesia with isoflurane inhalation, mice were fixed by perfusion through the ascending aorta with 2% PFA and 2.5% glutaraldehyde in 0.1 M phosphate buffer. GAS muscles were cut into cubes (1 mm ３ ) and incubate on ice for 1 hour in 1% OsO ４After post-fixation in 0.1 M phosphate buffer containing α-glucan, the tissue was embedded in epoxy resin, block stained with 1.5% uranyl acetate, dehydrated in ethanol, infiltrated with propylene oxide, and embedded in Araldite. Ultrathin sections (60-70 nm thick) were cut using an ultramicrotome and stained with 1.5% uranyl acetate and lead citrate. Images were taken using a TEM (Hitachi H7700).

[0071] (17) Correlative Confocal Scanning Laser Microscopy (CSLM)-Electron Microscopy (EM). Under deep anesthesia with isoflurane inhalation, mice were fixed with 4% PFA and 0.1% glutaraldehyde in 0.1 M phosphate buffer by perfusion through the ascending aorta. Serial 70 μm-thick sections were cut from the GAS muscle using a vibrating microtome (Dosaka TTK-3000) and observed by correlative CSLM-EM. Specifically, the sections were rapidly frozen and thawed in liquid nitrogen vapor, then incubated with anti-Dll4 antibody (1:200), anti-dystrophin antibody (1:300), and anti-CD31 antibody (1:250) at 4°C for 7 days, followed by overnight incubation with biotinylated donkey anti-rat IgG (1:250). The sections were then incubated overnight with Alexa 488-conjugated anti-mouse IgG (1:200), rhodamine red-conjugated donkey anti-rabbit IgG (1:250), and Alexa 647-conjugated streptavidin (1:500). The longer incubation periods with the primary and secondary antibodies were used to improve antibody penetration into deeper parts of the 70-μm-thick sections. After high-resolution imaging using a confocal laser scanning optical microscope, the Dll4 fluorescent signal was converted to a diaminobenzidine tetrahydrochloride (DAB) signal by overnight incubation with mouse peroxidase-antiperoxidase conjugate (1:500), followed by DAB reaction and 1% O . ｓ O ４ The sections were post-fixed with 1.5% uranyl acetate. The sections were dehydrated and flat-embedded in epoxy resin. Ultrathin sections were cut using an ultramicrotome and stained with uranyl acetate and lead citrate. Images were taken using a TEM (Hitachi H7700).

[0072] (18) Protein Extraction and Immunoblotting. Total protein lysates were obtained from cultured satellite cells or satellite cell-derived myotubes after exposure to RIPA buffer (FUJIFILM-Wako). Protein concentrations in each sample were quantified using the Pierce BCA Protein Assay Kit (ThermoFisher). To quantify protein secretion into the medium during EC culture, the culture medium was concentrated using Amicon Ultra (Merck). Primary antibodies were diluted in CanGetSignal solution A (TOYOBO) and incubated with the membrane containing the electrophoretically migrated protein bands at 4 °C overnight. The membrane was then washed and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies diluted in CanGetSignal solution B (TOYOBO) at room temperature for 1 h and visualized using chemiluminescence.

[0073] (19) Statistics All statistical analyses were performed using GraphPad Prism 8. Statistical comparisons between two conditions were performed using Student's two-tailed unpaired t-test. For comparisons of more than two groups, data were analyzed using one-way ANOVA followed by Dunnett's multiple comparison post-hoc test or two-way ANOVA followed by Tukey's or Sidak's multiple comparison post-hoc test. P values ​​of <0.05 (*), <0.01 (**), and <0.001 (***) were considered statistically significant. All data are expressed as mean ± standard error of mean (SEM). n.s. indicates that the results were not statistically significant.

[0074] (Example 1) Induction of muscle atrophy by Notch2 The present inventors have reported that satellite cells (skeletal muscle stem cells) express Notch1 and Notch2, which cooperatively maintain the quiescent and undifferentiated states of satellite cells in adult mice (Non-Patent Document 2). Therefore, we confirmed that Notch2 is expressed not only in undifferentiated mononuclear satellite cells but also in terminally differentiated multinucleated muscle fibers (muscle fibers). Specifically, we used the method shown in Figure 1 to induce muscle atrophy by using Pax7. CreERT2 / + ;Rosa26 LSL-DTA / +Satellite cells were isolated from mouse EDL muscles and cultured in myoblast growth medium (GM). The medium was then switched to myoblast differentiation medium (DM). Undifferentiated satellite cells were depleted by treatment with 4OH-MTX. Notch2 expression was then confirmed by PCR analysis and Western blotting.

[0075] Next, to examine the effect of constitutive expression of Notch2 on muscle fibers in vivo, we used HSA rtTA ;TR Cre and Rosa26 LSL-N2ICD We generated (N2ICD-mTg) mice. In these mice, the intracellular domain of Notch2 (N2ICD) is specifically induced in muscle fibers by doxycycline (Dox). Tg mice and control mice were administered Dox (2 mg / ml in drinking water) for three weeks, after which Notch2 expression, body weight, and muscle mass were measured. N2ICD-mTg mice showed no difference in body weight compared to controls, but showed a significant decrease in muscle mass. The results are shown in Figure 2. This suggests that forced expression of N2ICD induces muscle atrophy.

[0076] (Example 2) Test using a mouse model of muscle atrophy To investigate the role of Notch2 in muscle fibers, we investigated whether Notch2 expression was related to Mlc1f Cre (Mlc Cre / + ;N2 f / f ,N2 - / - ), which is genetically ablated by the myosin light chain 1f promoter-driven Cre recombinase expression (Mlc Cre / + ) Mouse and Notch2 f / f (N2 f / f ) mice were crossed to generate muscle fiber-specific Notch2 deficiency (N2 - / - As shown in Figure 3, Notch2-deficient mice (N2 - / - ) had reduced Notch2 expression, but Notch2-deficient mice grew normally and had no significant changes in body weight, muscle weight, or myonuclei or Pax7 per extensor digitorum longus (EDL) muscle fiber. + No abnormal phenotype was observed with respect to satellite cell number.

[0077] We tested the effects of Notch2 inactivation in two different muscle atrophy models: disuse-induced muscle atrophy and metabolic disorder-induced muscle atrophy. f / f ) and Notch2-deficient mice (N2 - / - As shown in Figure 4, hindlimb unloading (HU) was performed for 7 days and analyzed. f / f In mice, HU increased Notch2 gene expression in plantar fasciitis (PLA) muscle fibers compared with ground loading control (GC). f / f N2 decreased muscle mass in the soleus (SOL), PLA, and gastrocnemius (GAS) muscles of mice. - / - We found that muscle mass in mice was barely affected. Immunohistochemical analysis confirmed that the cross-sectional area (CSA) of PLA muscles did not change under HU conditions. Furthermore, as shown in d, the thickness of individual muscle fibers was significantly different from that of N2. - / - HU was greater in the skeletal muscle, indicating that Notch2 expressed by muscle fibers is required for mechanical unloading-induced muscle atrophy.

[0078] Diabetes mellitus (DB) causes multiple metabolic abnormalities, including persistent hyperglycemia, and is a high risk factor for muscle weakness. We used a streptozotocin (STZ)-induced DB mouse model, which induces hyperglycemia by ablating pancreatic β cells. Mice were intraperitoneally injected with STZ (150 mg / kg body weight) or vehicle (Veh) and analyzed 14 days later. N2 f / f and N2 - / - Mice showed similar levels of hyperglycemia after STZ injection, and Notch2 gene expression was upregulated in EDL muscle fibers of DB and HU mice. Muscle mass of TA, PLA, and GAS muscles and CSA of TA muscle were significantly higher than those of N2. f / f DB mice showed a significant decrease, while N2 - / - The thickness of individual muscle fibers was not reduced in DB mice. - / - The DB was large (Fig. 5e). The results are shown in Fig. 5.

[0079] Example 3: Test using a type 1 diabetic mouse model (Akita mouse model) We investigated the effect of Notch2 on muscle atrophy using a type 1 diabetic mouse model (Akita mouse model) in which Notch2 deletion was introduced. As a result, we confirmed that muscle atrophy was also reduced in the tibialis anterior (TA), PLA, and GAS muscles in the Akita mouse model. The results are shown in Figure 6.

[0080] (Example 4) N2 f / f DB mice and N2 - / - Comparison with DB mice. When comparing muscle strength in the tibialis anterior (TA), N2 f / f In DB mice, as shown in Figure 7, TA force generation was significantly reduced, whereas N2 - / - The DB mice did not show a decrease in the N2 f / f Abnormal triads and swollen mitochondria were observed in the muscles of DB mice, whereas N2 - / - These were not observed in DB mice.

[0081] These results demonstrate that Notch2 is required for the loss of muscle mass and function due to mechanical unloading or the induction of metabolic disorders.

[0082] (Example 5) Genetic analysis in muscle under HU and DB conditions Under both HU- and DB- conditions, N2 f / f DB mice and N2 - / - Transcriptome analysis was performed on each of the DB mice using RNA sequencing. f / f The expression of many genes was significantly altered in the muscles of DB mice, but N2 - / - Few genes were statistically significantly altered in DB mice, indicating that Notch2 primarily regulates gene expression dynamics in muscle induced by mechanical unloading and metabolic abnormalities. Only eight genes were commonly identified as differentially expressed genes (DEGs) under both HU and DB conditions compared with their respective controls. N2 f / fUsing mice, we performed Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis using common DEGs in HU and DB conditions and found that the FoxO signaling pathway was associated with the top DEGs, as shown in Figure 9. Analysis of the expression of FoxO target genes and atrogenesis genes that induce muscle atrogenesis revealed that N2 f / f In mouse muscle, the expression of FoxO target genes, such as Ccng2, Cdkn1b, Rbl2, and Bnip318, as well as ubiquitin ligands, such as MuRF1, Atrogin1, Fbxo31, and MUSA1, which are known to induce muscle atrophy, were upregulated. Conversely, these genes were upregulated in N2 - / - There was no change in mouse muscle.

[0083] However, myofiber-specific RBP-J-deficient mice and Hes1-deficient mice were not resistant to HU- and DB-induced muscle atrophy. Furthermore, ablation of RBP-J and overexpression of N2ICD in muscle fibers reduced muscle mass, suggesting that forced activation of Notch2 can induce muscle atrophy independently of RBP-J. This finding suggests that, unlike satellite cells, which are regulated by the canonical Notch-RBP-J-Hes / Hey pathway, the canonical Notch-RBP-J signaling pathway is not involved in Notch2-mediated muscle atrophy. These findings suggest that Notch2 acts as a critical mediator of muscle atrophy under both mechanically and metabolically abnormal conditions, governing atrophic transcriptional activity independently of RBP-J.

[0084] (Example 6) Investigation of molecules that regulate Notch2-mediated muscle atrophy We investigated which of the five known Notch ligands activates Notch2 in muscle fibers. We confirmed the expression of each ligand in adult muscle tissue and found that gene expression of four ligands (Jag1, Jag2, Dll1, and Dll4) except for Dll3 could be detected in adult muscle tissue.

[0085] Next, we evaluated the effects of Jag1, Jag2, Dll1, and Dll4 on muscle fibers. Freshly isolated muscle fibers from mouse EDL muscles were stimulated with each of these four recombinant proteins in serum-free DMEM for 48 hours using tissue culture plates coated with the four proteins. The results are shown in Figure 10. Muscle fiber atrophy was induced by Dll4 treatment alone, but not by other ligands. Furthermore, gene expression analysis after 24 hours of culture on plates coated with Dll4 recombinant protein revealed that Dll4-stimulated muscle fibers upregulated FoxO target genes and atrogen, as shown in Figure 11. These results were consistent with the in vivo atrophy model.

[0086] Example 7: Induction of muscle atrophy via cell-cell contact To investigate whether Notch ligands can induce muscle fiber atrophy via cell-cell contact, HEK293 cells were transfected with either Dll1 or Dll4 expression plasmid vectors and cocultured with isolated muscle fibers for 48 hours to confirm expression in muscle fibers. Figure 12 shows the results of coculture of EDL muscle fibers with HEK293 cells transfected with plasmids encoding Dll1-GFP, Dll4-GFP, or GFP (Cont.). There was no change in the size of muscle fibers when cocultured with Dll1-expressing HEK293 cells. Furthermore, compared with coculture with Dll1-expressing HEK293 cells, coculture with Dll4-expressing HEK293 cells significantly reduced muscle fiber size, further demonstrating that Dll4 is the specific ligand that induces muscle fiber atrophy.

[0087] (Example 7) Study of gamma-secretase inhibitors on Dll4-induced muscle atrophy Notch, a transmembrane receptor, undergoes cleavage of its transmembrane domain by gamma-secretase, allowing the intracellular domain to translocate to the nucleus and function as a transcription factor. Therefore, the effect of gamma-secretase activity on Dll4-induced muscle fiber atrophy was examined using the gamma-secretase inhibitor DAPT. N2 cells were cultured for 48 hours on plates pre-coated with recombinant Dll4 protein using a medium containing the gamma-secretase inhibitor DAPT (10 μM) or PBS. f / fEDL muscle fibers derived from mice were cultured. The results are shown in Figure 13. N2 - / - Both muscle fibers and those treated with a gamma-secretase inhibitor (DAPT) were resistant to D114 stimulation, confirming that D114-induced muscle atrophy is mediated by Notch2 and gamma-secretase activity.

[0088] Next, we investigated the effects of γ-secretase inhibitors in two muscle atrophy models, mechanical unloading (HU) and diabetes-induced (DB), by intraperitoneally injecting wild-type mice with the γ-secretase inhibitor DAPT (10 mg / kg body weight) or corn oil (Cont) every other day during the HU or DB induction period. Results from the HU model are shown in Figure 14, and results from the DB model are shown in Figure 15. Treatment with DAPT significantly suppressed muscle atrophy in both the HU and DB models. These results indicate that the Dll4-Notch2 signaling axis induces muscle atrophy via γ-secretase activity.

[0089] (Example 8) Identification of the source of Dll4 Next, the source of the Dll4 ligand was identified. To confirm the site where the Dll4 ligand is expressed, CD31 derived from adult muscle tissue was used. + The expression of Dll4 ligand in endothelial cells (ECs positive for the endothelial cell marker CD31) was measured and compared with that in freshly isolated muscle fibers. The results are shown in the left panel of Figure 16. As a result, CD31 + The Dll4 gene was expressed approximately 400 times more in ECs. Furthermore, using wild-type mice, CD31 derived from GAS muscle was expressed under both HU and DB conditions. +We measured the expression of Notch ligands (Dll1, Dll4, Jag1, and Jag2) in endothelial cells. The results are shown in the right panel of Figure 16. Under both HU and DB conditions, Dll4 was upregulated, whereas other ligands were not. Furthermore, immunohistochemical analysis confirmed the upregulation of Dll4. The results are shown in Figure 17. We also performed immunoelectron microscopy analysis. The results are shown in Figure 18. As shown in Figure 18A, Dll4 was distributed not only on the surface of ECs but also in collagenous fibers in the interstitial space between ECs and myofibers in diabetic muscle tissue. Furthermore, as shown in Figures 18B and 18C, Dll4 was also enriched on the surface of myofibers. These results suggest that in muscle tissue under DB conditions, ECs upregulate and secrete Dll4 into the interstitial space, and that the secreted Dll4 selectively activates the Notch2 receptor expressed on the surface of myofibers.

[0090] The expression of Dll4 in ECs was confirmed by immunoblot analysis. ECs isolated from muscle tissue were cultured under low glucose conditions (5 mM) similar to healthy blood levels (90 mg / dl) and under high glucose conditions (25 mM) mimicking diabetic hyperglycemia (450 mg / dl). Measurement of Dll4 in the supernatant revealed higher expression levels under high glucose conditions (25 mM) than under low glucose conditions (5 mM).

[0091] Example 9: Inhibition of Dll4 Expression The function of Dll4 secreted by ECs in muscle fiber atrophy was evaluated as follows. As shown in Figure 19A, a transwell-based coculture system was established between primary ECs isolated from muscle fibers and muscle fibers. EC-derived secretions passed through the upper chamber and reached the muscle fibers placed on the bottom. Primary ECs were transfected with control siRNA (siCont) or siRNA against Dll4 (siDll4) one day before coculture with muscle fibers. Dll4 expression in ECs transfected with siCont or siDll4 is shown in Figure 19B. As shown in Figures 19C and 19D, in this system, coculture with ECs significantly reduced muscle fiber size, but this muscle fiber atrophy could be completely rescued by siRNA-mediated knockdown of Dll4 in ECs.

[0092] (Example 10) Study of Dll4 Inhibition The effect of anti-Dll4 neutralizing antibody was confirmed using the same co-culture system as in Example 9. The results are shown in Figure 20. Treatment with anti-Dll4 neutralizing antibody (αDll4) (500 ng / ml) completely suppressed muscle fiber atrophy in the co-culture system.

[0093] These results indicate that EC-derived Dll4 can activate Notch2 in muscle fibers without direct cell-cell contact, inducing muscle atrophy.

[0094] Example 11: Suppression of Muscle Atrophy Using Anti-Dll4 Antibody (HU Model) The inhibitory effect of muscle atrophy using a neutralizing antibody against Dll4 protein was examined as follows. Muscle atrophy was induced in wild-type mice under HU conditions. Anti-Dll4 antibody (αDll4) (10 mg / kg body weight) or a control antibody (αCont) was intraperitoneally injected twice (days 0 and 4) during the 7-day HU period. Seven days after the initial antibody administration, the mice were euthanized and analyzed. The results are shown in Figures 21 and 22 . Treatment with αDll4 dramatically reduced the HU-induced loss of muscle mass and CSA in SOL, PLA, and GAS muscles, without affecting the number of satellite cells per muscle fiber. This confirmed that anti-Dll4 antibody can suppress muscle atrophy. As shown in Fig. 1d, the thickness of individual muscle fibers was larger in αDll4 HU. Furthermore, genetic analysis showed that the expression levels of FoxO target genes and atrogin1-excepted genes responsible for muscle atrophy (atrogens) were also suppressed by αDll4.

[0095] Example 12: Suppression of Muscle Atrophy Using Anti-Dll4 Antibody (DB Model) The inhibitory effect of a neutralizing antibody against Dll4 protein on muscle atrophy was examined using a wild-type mouse DB-induced muscle atrophy model. Mice were injected with STZ (150 mg / kg body weight; DB) or vehicle (veh; citrate buffer), followed by three intraperitoneal administrations of αDll4 (10 mg / kg body weight) or a control antibody (αCont) over a two-week period (days 3, 7, and 11 after STZ administration). Analysis was performed 14 days after STZ administration. The results are shown in Figures 23 and 24 . Administration of αDll4 demonstrated a potent preventive effect against DB-induced muscle atrophy without affecting blood glucose levels. As shown in Figure 24d, the thickness of individual muscle fibers was larger in αDll4 DB mice.

[0096] Example 13: Inhibition of Muscle Atrophy Using Soluble Recombinant Anti-Dll4 Protein. Using a wild-type mouse model of DB-induced muscle atrophy, we investigated the inhibitory effect of soluble recombinant Dll4-Fc protein, which blocks the binding of endogenous Dll4 to the Notch receptor. Wild-type mice were intraperitoneally injected with STZ (150 mg / kg body weight; DB) or vehicle (Veh; citrate buffer), followed by four doses (days 7, 9, 11, and 13) of soluble Dll4-Fc recombinant protein (rDll4-Fc) (0.5 mg / kg) over a 14-day period. Analysis was performed 14 days after STZ administration. PBS was used as a control. The results are shown in Figure 25. Administration of soluble recombinant Dll4-Fc protein attenuated DB-induced muscle atrophy without affecting blood glucose levels.

[0097] These results confirm that Dll4 protein-targeting preventive and therapeutic agents can be used to treat muscle weakness due to disuse and diabetes. However, when endothelial cell-specific Dll4 knockout mice were generated and analyzed, muscle mass was slightly reduced and no preventive effect against HU-induced muscle atrophy was observed, suggesting that an appropriate concentration of Dll4 in endothelial cells is essential for maintaining tissue homeostasis.

[0098] Example 14: Confirmation of the Effect of Anti-Dll4 Antibody on Severe Hyperglycemia The effect of anti-Dll4 antibody (αDll4) on mortality in STZ-induced severe hyperglycemia in mice was evaluated. Wild-type mice were administered a very high dose of STZ (250 mg / kg) to induce severe hyperglycemia. Twenty STZ-injected wild-type mice were divided into two groups treated with either αDll4 or αCont (10 mg / kg body weight each). The antibody was administered intraperitoneally every four days and continued until the mice were exhausted or died (maximum of 58 days). The results are shown in Figure 26. Both the αDll4 and αCon groups showed severe hyperglycemia after STZ injection (blood glucose levels <600 mg / dl). Death or euthanasia is shown as a Kaplan-Meier plot over time. While αCon-treated mice had a shorter lifespan (approximately 58 days), αD114-treated mice had a significantly higher survival rate (70%) and increased muscle strength, indicating that attenuation of αD114 activity protects against disruption of whole-body homeostasis caused by hyperglycemia.

[0099] Example 15: Muscle Hypertrophy Effect of Dll4 Inhibition We investigated whether attenuation of the Dll4-Notch2 signaling axis promotes mechanical overload-induced muscle hypertrophy. Muscle hypertrophy was induced in wild-type mice by mechanical overloading (OL). PLA muscle hypertrophy was induced by synergistic ablation (tenotomy) of the SOL and GAS muscles in both legs. Mice without synergistic ablation served as controls. After tenotomy, αDll4 (10 mg / kg body weight) or αCont was intraperitoneally administered twice over a 2-week period (days 5 and 10 after tenotomy), and analysis was performed on day 14. The results are shown in Figure 27. Hypertrophy efficiency, measured by muscle mass and cross-sectional area of ​​the PLA muscle, was significantly higher in αDll4-treated mice compared with αCon-treated mice. Furthermore, as shown in c, the thickness of individual muscle fibers was larger in αD114 OL.

[0100] Using a mechanical overload-induced muscle hypertrophy model, N2 - / -The test was performed on mice treated with DAPT and mice treated with DAPT. The results are shown in Figures 28 and 29. In both cases, muscle hypertrophy was more readily demonstrated compared to control mice.

[0101] These results demonstrate that the endothelial Dll4-myofiber Notch2 axis is important not only for inducing muscle atrophy but also for suppressing muscle hypertrophy.

[0102] Example 16: Confirmation of intramuscular Dll4 distribution The intramuscular Dll4 distribution was confirmed by immunoelectron microscopy. Using an STZ-induced DB mouse model, 60-micrometer-thick vibratome sections were prepared from harvested GAS muscles. The sections were processed overnight for immunohistochemical staining using a primary antibody against Dll4 (1:50), followed by overnight incubation with 10 nm-nanogold goat anti-mouse IgG (1:50, CRL). The sections were then resuspended in 1% OsO ４ The sections were post-fixed in aqueous solution, en bloc stained with 1.5% uranyl acetate, dehydrated, and flat-embedded in epoxy resin. Ultrathin sections were stained with uranyl acetate and lead citrate and examined under a transmission electron microscope (H-7700, Hitachi). The results are shown in Figure 30. Dll4 was confirmed to be distributed in the interstitial space between ECs and myofibers (particularly associated with collagen-like fibers and extracellular vesicles) and on the surface of ECs and myofibers. Therefore, it is suggested that in muscle tissue under DB conditions, ECs suppress Dll4, secrete it into the interstitial space, and selectively activate Notch2 receptors expressed on the surface of myofibers.

[0103] The above detailed description is merely illustrative of the objects and scope of the present invention and is not intended to limit the scope of the appended claims. Various modifications and substitutions to the described embodiments will be apparent to those skilled in the art from the teachings set forth herein, without departing from the scope of the appended claims.

[0104] The present invention provides a preventive or therapeutic agent for muscle atrophy based on a novel mechanism of action. The composition of the present invention is useful for preventing, treating, or ameliorating muscle atrophy or muscle mass loss.

[0105] This application is based on patent application No. 2021-56481 filed in Japan (filing date: March 30, 2021), the contents of which are incorporated in their entirety by reference herein.

Claims

1. A preventive or therapeutic agent for myopathic muscular dystrophy, comprising as an active ingredient a functional inhibitor of Dll4 secreted from endothelial cells of blood vessels present in muscle tissue, said functional inhibitor being selected from the group consisting of a binding inhibitor that inhibits the binding of Notch2 to Dll4, an expression inhibitor of Dll4, and a binding inhibitor of collagen to Dll4.

2. The preventive or therapeutic agent according to claim 1 , wherein the binding inhibitor that inhibits the binding of Notch2 to Dll4 is an anti-Dll4 antibody.

3. The preventive or therapeutic agent according to claim 1, wherein the anti-Dll4 antibody is an antibody that recognizes 5 to 10 amino acids selected from the amino acid sequence described in the amino acid sequence information of Dll4 (NP_061947.1).

4. The preventive or therapeutic agent according to claim 1 , wherein the inhibitor of Dll4 expression is siRNA or shRNA that induces RNA interference against Dll4 mRNA.

5. The preventive or therapeutic agent according to any one of claims 1 to 4, wherein the myogenic muscular atrophy disease is selected from the group consisting of age-related muscular atrophy (sarcopenia), disuse muscular atrophy, muscular dystrophy, diabetic muscular atrophy, congenital myopathy, distal myopathy, steroid myopathy, drug-induced myopathy, rhabdomyolysis, muscle wasting disease, Charcot-Marie-Tooth disease, Lambert-Eaton syndrome, cachexia-induced muscular atrophy, and muscular atrophy associated with burns or the like.