C198s mutant adamts1 peptide and pharmaceutical composition comprising same for treating muscle diseases

Abstract

The present invention relates to a mutant ADAMTS1 peptide and a pharmaceutical composition comprising same for preventing, alleviating, or treating muscle diseases. If the mutant ADAMTS1 peptide of the present invention is used, the differentiation of muscle fibers can be promoted, and effective prevention or treatment of muscle diseases is possible.

Description

C198S mutant ADAMTS1 peptide and pharmaceutical composition for treating muscle disease comprising the same

[0001] The present invention was made with the support of the Ministry of Science and ICT, the Ministry of Health and Welfare, and the Ministry of Trade, Industry and Energy of the Republic of Korea under project number 00165803. The research management specialized organization of the project is the National Drug Development Foundation, the research project name is "National New Drug Development Project (Ministry of Science and ICT, Ministry of Welfare, Ministry of Trade, Industry and Energy)", the research project name is "Development of Lead Substance and Production Process for NB-101 Sarcopenia Treatment with NOTCH1 Inhibitory Mechanism", the lead organization is NewOnBio Co., Ltd., and the research period is 2022.08.01-2024.07.31.

[0002] This patent application claims priority to Republic of Korea Patent Application No. 10-2024-0053621, filed with the Korean Intellectual Property Office on April 22, 2024, the disclosure of which is incorporated herein by reference.

[0003] The present invention relates to a C198S mutant ADAMTS1 peptide and a pharmaceutical composition comprising the same for preventing or treating muscle diseases.

[0004]

[0005] Muscles are the organs responsible for movement within the human body and are divided into skeletal muscle, cardiac muscle, and visceral muscle. Skeletal muscle induces movement through contraction and relaxation, while cardiac muscle regulates the heartbeat and circulates blood throughout the body. Visceral muscle facilitates digestion in the digestive system, helping to absorb nutrients and maintain bodily functions. Therefore, muscles play an essential role in all human activities.

[0006] Muscle cell differentiation and muscle formation are regulated by various muscle regulatory factors. Among these, MyoD (myoblast determination protein 1) initiates the expression of muscle-specific genes and differentiates satellite cells (myosatellitocytes) into myoblasts. MyoD activity induces the expression of myogenin, which plays a crucial role in the fusion of myoblasts and the formation of myotubes. The muscle fibers formed through this process form bundles, ultimately forming muscles.

[0007] As muscle mass decreases, muscle strength for physical activity declines, and the musculoskeletal system begins to decline. Slower walking speed and weakened grip strength are key symptoms of muscle mass loss, which can increase the risk of falls, fractures, joint damage, metabolic disorders, and cardiovascular disease. Muscle mass loss primarily manifests as a decrease in muscle fiber volume and can be caused by aging, muscle disuse, or lack of exercise. Other pathological conditions, such as cachexia, sepsis, starvation, cancer treatment, and excessive exposure to stress hormones, can also contribute.

[0008] Sarcopenia is associated with the degeneration of motor neurons that drive skeletal muscle contraction, decreased or altered expression of proteins involved in muscle contraction within skeletal muscle, and changes in the fiber structure of motor neurons or skeletal muscle. However, the underlying cause of sarcopenia remains unclear, necessitating the development of effective methods to slow its progression.

[0009]

[0010] The present inventors screened peptides that are likely to be effective in treating muscle loss in order to develop a therapeutic agent for alleviating, suppressing, preventing, or treating muscle diseases associated with sarcopenia, and among them, they identified the C198S mutant ADAMTS1 peptide as having a significant muscle fiber length and thickness increasing activity, thereby completing the present invention.

[0011] Accordingly, the purpose of the present invention is to provide a mutant ADAMTS1 peptide comprising an amino acid sequence in which cysteine ​​at position 198 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with serine and at least one amino acid among amino acids at positions 222 to 251 is deleted.

[0012] Another object of the present invention is to provide a nucleic acid molecule encoding the peptide.

[0013] Another object of the present invention is to provide a recombinant vector comprising the nucleic acid molecule.

[0014] Another object of the present invention is to provide a host cell comprising the recombinant vector.

[0015] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating muscle disease comprising the peptide.

[0016] Other objects and advantages of the present invention will become more apparent from the detailed description, claims and drawings below.

[0017]

[0018] According to one aspect of the present invention, the present invention provides a mutant ADAMTS1 peptide comprising an amino acid sequence in which cysteine ​​at position 198 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with serine and at least one amino acid among amino acids at positions 222 to 251 is deleted.

[0019] In order to develop a drug for alleviating, suppressing, preventing or treating muscle diseases associated with sarcopenia, the present inventors screened peptides that are likely to be effective in treating sarcopenia, and among them, the C198S mutant ADAMTS1 peptide was found to have a significant ability to increase muscle fiber length and thickness.

[0020] In the present invention, "ADAMTS1 (A disintegrin and metalloproteinase with thrombospondin motifs 1)" means a peptide encoded by the human ADAMTS1 gene, wherein the ADAMTS1 protein comprises a propeptide region, a metalloproteinase region, a disintegrin-like region, and a thrombospondin (TS) type 1 motif. The ADAMTS1 protein contains two disintegrin loops and three C-terminal TS motifs and has antiangiogenic properties. Expression of the ADAMTS1 gene may be associated with various inflammatory processes as well as the development of cancer cachexia, and is known to be highly likely to be required for normal growth, fertility, organ morphology, and function.

[0021] In the present invention, a "peptide" is a polymer in which amino acid units are artificially or naturally linked, and the function of the peptide varies depending on the combination of amino acids, and each amino acid is linked by a covalent bond called a peptide bond. A peptide is a substance in which 2 to 50 amino acids are linked, and a peptide having 2 amino acid residues is called a dipeptide, a peptide having 3 amino acid residues is called a tripeptide, a peptide having 2 to 10 amino acid residues is called an oligopeptide, and a peptide having about 10 to 50 amino acid residues is called a polypeptide.

[0022] In the present invention, the peptide may comprise at least one additional amino acid residue at the C-terminus and / or N-terminus of the peptide. The additional amino acid residues may be added individually or collectively for purposes such as improving productivity, purification, stabilization in vivo or ex vivo, coupling, or detection of the complex.

[0023] For example, the peptide may additionally comprise a cysteine ​​residue at the C-terminus and / or N-terminus of the peptide. The additional amino acid residue may also provide a "tag" for purification or detection of the peptide, for example, for interaction of the tag with a specific peptide. In the case of a His6 tag, a tag such as a His6 tag, a (HisGlu)3 tag ("HEHEHE" tag), a "myc" (c-myc) tag, or a "FLAG" tag may be provided for immobilized metal affinity chromatography (IMAC).

[0024] In one embodiment of the present invention, the C198S mutant peptide of the present invention may be a peptide comprising a deletion of one or more amino acids selected from the group consisting of arginine at position 249, lysine at position 250, lysine at position 251, and arginine at position 252 of a peptide consisting of an amino acid sequence of SEQ ID NO: 1, and for example, may be a peptide comprising a deletion of amino acids at positions 249 to 251.

[0025] In one specific embodiment of the present invention, the C198S mutant peptide of the present invention may be a peptide comprising a deletion of at least one amino acid selected from the group consisting of arginine at position 249, lysine at position 250, lysine at position 251, and arginine at position 252 of a peptide consisting of an amino acid sequence of SEQ ID NO: 1, and a deletion of at least one amino acid selected from the group consisting of amino acids at positions 223 to 248 and 253 to 281, for example, a peptide comprising an amino acid sequence of SEQ ID NO: 1, and a deletion of at least one amino acid selected from the group consisting of arginine at position 249, lysine at position 250, lysine at position 251, and arginine at position 252, and a deletion of at least one amino acid selected from the group consisting of amino acids at positions 223 to 248. It may be a peptide containing a deletion of one or more amino acids.

[0026] In the present invention, the deletion of one or more amino acids selected from the group consisting of amino acids at positions 223 to 248 is selected from the group consisting of amino acids at positions 223, 223 to 224, 223 to 225, 223 to 226, 223 to 227, 223 to 228, 223 to 229, 223 to 230, 223 to 231, 223 to 232, 223 to 233, 223 to 234, 223 to 235, 223 to 236, 223 to 237, 223 to 238, 223 to 239, 223 to 240, 223 to 241, 223 to It may be an amino acid deletion at positions 242, 223 to 243, 223 to 244, 223 to 245, 223 to 246, 223 to 247, or 223 to 248.

[0027] In the present invention, deletion of one or more amino acids selected from the group consisting of amino acids at positions 253 to 281 is selected from the group consisting of amino acids at positions 253, 253 to 254, 253 to 255, 253 to 256, 253 to 257, 253 to 258, 253 to 259, 253 to 260, 253 to 261, 253 to 262, 253 to 263, 253 to 264, 253 to 265, 253 to 266, 253 to 267, 253 to 268, 253 to 269, 253 to 270, It may be an amino acid deletion at positions 253 to 271, 253 to 272, 253 to 273, 253 to 274, 253 to 275, 253 to 276, 253 to 277, 253 to 278, 253 to 279, 253 to 280, or 253 to 281.

[0028] In one embodiment of the present invention, the mutant ADAMTS1 peptide comprises an amino acid sequence additionally including a deletion of amino acids at positions 468 to 967 in the amino acid sequence represented by SEQ ID NO: 1.

[0029] In one embodiment of the present invention, positions 1 to 252 of the amino acid sequence represented by SEQ ID NO: 1 are a propeptide region, and positions 253 to 467 are a metalloproteinase region. In the present invention, the "propeptide region" refers to a region that is cleaved when ADAMTS1 is activated and secreted into the extracellular matrix, and is known to play a role in inhibiting enzyme activity by binding to the active site of the metalloproteinase region.

[0030] In the present invention, the "metalloproteinase domain" refers to a zinc ion (Zn 2+ ) refers to a region with protein hydrolase activity dependent on extracellular matrix, and is known to play a role in decomposing and regulating proteins constituting the extracellular matrix.

[0031] In one embodiment of the present invention, the peptide comprises an amino acid sequence of SEQ ID NO: 3.

[0032] In one embodiment of the present invention, the peptide comprises an amino acid sequence of SEQ ID NO: 4.

[0033] The above peptide may include amino acid sequence variants that exhibit biological activity equivalent to that of the peptide comprising the amino acid sequence of SEQ ID NO: 3 of the present invention, as will be appreciated by those skilled in the art. For example, the amino acid sequence may be altered to improve biological properties. Such alterations include, for example, deletions, insertions, and / or substitutions of amino acid sequence residues.

[0034] The variants are said to have "substantial similarity", meaning that the two peptide sequences share at least about 90% sequence identity, and more preferably at least about 95%, 98%, or 99% sequence identity, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights. Preferably, the non-identical residue positions differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functionality of the protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percent or degree of similarity may be adjusted upward to compensate for the conservative nature of the substitutions.

[0035] These amino acid mutations are based on the relative similarity of amino acid side-chain substituents, such as hydrophobicity, hydrophilicity, charge, and size. For example, arginine, lysine, and histidine are all positively charged residues; alanine, glycine, and serine have similar sizes; and phenylalanine, tryptophan, and tyrosine have similar shapes. Therefore, based on the above considerations, arginine, lysine, and histidine; alanine, glycine, and serine; and phenylalanine, tryptophan, and tyrosine can be considered biologically functional equivalents.

[0036] When introducing mutations, the hydrophobicity index of amino acids can be considered. Each amino acid is assigned a hydrophobicity index based on its hydrophobicity and charge: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine ​​(+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); asphaltene (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0037] The hydrophobic amino acid index is crucial for imparting interactive biological functions to proteins. It is well known that amino acids with similar hydrophobic indices must be substituted to retain similar biological activity. When introducing mutations based on hydrophobic indices, substitutions are preferably made between amino acids with a difference in hydrophobicity index of ±2, more preferably ±1, or even ±0.5.

[0038] Meanwhile, it is also well known that substitutions between amino acids having similar hydrophilicity values ​​result in proteins with equivalent biological activity. As disclosed in U.S. Patent No. 4,554,101, the following hydrophilicity values ​​are assigned to each amino acid residue: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5±1); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); Phenylalanine (-2.5); Tryptophan (-3.4).

[0039] When introducing a mutation with reference to a hydrophilicity value, substitution is preferably made between amino acids showing a difference in hydrophilicity value within ± 2, more preferably within ± 1, and even more preferably within ± 0.5.

[0040] Meanwhile, amino acid exchanges in proteins that do not alter the overall activity of the molecule are well known in the art. The most common exchanges are between amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0041] Considering the above variants having biologically equivalent activity, the peptide can also be interpreted as including a sequence showing substantial identity with the sequence described in the sequence number.

[0042] The above substantial identity means a sequence that preferably exhibits at least 80% homology, more preferably at least 85% homology, even more preferably at least 90% homology, and most preferably at least 95% homology when the sequence of the present invention and any other sequence are aligned to the greatest extent possible and the aligned sequence is analyzed using an algorithm commonly used in the art.

[0043] Alignment methods for sequence comparison are well known in the art. Various alignment methods and algorithms are described in Smith and Waterman, Adv. Appl. Math. 2:482 (1981); Needleman and Wunsch, J. Mol. Bio. 48:443 (1970); Pearson and Lipman, Methods in Mol. Biol. 24: 307-31 (1988); Higgins and Sharp, Gene 73:237-44 (1988); Higgins and Sharp, CABIOS 5:151-3 (1989); Corpet et al., Nuc. Acids Res. 16:10881-90 (1988); Huang et al., Comp. Appl. BioSci. 8:155-65 (1992) and Pearson et al., Meth. Mol. Biol. 24:307-31(1994). NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403-10(1990)) is available at NCBI (National Center for Biological Information) and can be used in conjunction with sequence analysis programs such as blastp, blastn, blastx, tblastn, and tblastx on the Internet.

[0044] In one embodiment of the present invention, the peptide is characterized by promoting differentiation of muscle cells.

[0045] In the present invention, the term "muscle cell (myocyte)", also called muscle fiber, is a type of cell that creates muscle tissue, and has a long, tube-like shape that differentiates from myoblasts. Myofibrils are very thin fibers that form the cytoplasm of muscle fibers and are cylindrical cell organelles. Myofibrils are made up of filaments that start at one end of the cell and are connected to the other end, with each end attached to the cell membrane.

[0046] Myogenic cells differentiate into myocytes through transcription factors (Myf5, MyoD, Myogenin, and Myf6) that induce muscle differentiation. Initially, Myf5 and MyoD are expressed in myogenic cells. Later, when differentiation into myocytes begins, division and proliferation cease, and the expression of Myf5 and MyoD decreases, while the expression of myogenin increases, promoting myocyte differentiation. Finally, the expression of Myf6 completes differentiation into myocytes.

[0047] In one embodiment of the present invention, the mutant ADAMTS1 peptide has improved protein purity compared to the NM3 ADAMTS1 peptide.

[0048] In one specific example of the present invention, the fact that the protein purity is improved compared to the NM3 ADAMTS1 peptide means that when purifying a recombinant protein, a dimer is formed by cysteine, which lowers the purity, and the purity is improved by substituting cysteine ​​with serine to suppress the formation of new dimers.

[0049] According to one aspect of the present invention, the present invention provides a nucleic acid molecule encoding the peptide.

[0050] In one embodiment of the present invention, the nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 15.

[0051] The term "nucleic acid molecule" as used in the present invention comprehensively refers to DNA (gDNA and cDNA) and RNA molecules. Nucleotides, the basic structural units of nucleic acid molecules, include not only natural nucleotides but also analogues with modified sugar or base moieties. The nucleotides include not only the nucleotide sequence encoding the amino acid sequence of the peptide, but also complementary sequences to that sequence.

[0052] According to a specific embodiment of the present invention, the sequence of a peptide comprising the amino acid sequence of the mutant ADMATS1 peptide of the present invention is included in the attached sequence listing of the present invention.

[0053] Considering the mutations having the biological equivalent activity described above, the nucleic acid molecule of the present invention encoding the amino acid sequence constituting the peptide is interpreted to also include a sequence showing substantial identity therewith. The above substantial identity means that when the sequence of the present invention and any other sequence are aligned to the greatest extent possible and the aligned sequence is analyzed using an algorithm commonly used in the art, at least 60% homology (e.g., 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, or 69%), more specifically 70% homology (e.g., 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%), even more specifically 80% homology (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%), and even more specifically 90% homology (e.g., 90% or more homology) By "homology" we mean sequences that exhibit a homology of at least 95% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%), most specifically, a homology of at least 95% (e.g., 95%, 96%, 97%, 98%, or 99%). All integers greater than or equal to 60% and less than or equal to 100%, and decimals therebetween, are encompassed within the scope of the present invention with respect to % homology.

[0054] According to one aspect of the present invention, the present invention provides a recombinant vector comprising the nucleic acid molecule.

[0055] In the present invention, the term "vector" refers to a means for expressing a target gene in a host cell, and includes plasmid vectors, bacteriophage vectors, phagemid vectors, cosmid vectors, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), adenovirus vectors; retrovirus vectors and lentivirus vectors.

[0056] According to one embodiment of the present invention, a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of the mutant ADAMTS1 peptide in the vector of the present invention is operatively linked to a promoter of the vector.

[0057] The expression "operatively linked" in the present invention means that a nucleic acid expression control sequence and a nucleic acid sequence encoding a target protein or RNA are functionally linked. That is, the expression means that a nucleic acid sequence encoding a protein or RNA is linked in such a way that gene expression is enabled by the expression control sequence. For example, a promoter and a nucleic acid sequence encoding a protein or RNA must be operably linked to affect the expression of the encoding nucleic acid sequence. The operative linkage with a recombinant vector can be produced using a genetic recombination technique well known in the art, and site-specific DNA cleavage and ligation uses enzymes generally known in the art.

[0058] The recombinant vector system of the present invention can be constructed through various methods known in the art, and specific methods thereof are disclosed in Sambrook et al., Molecular Cloning, Laboratory Manual, Cold Spring Harbor Laboratory Press (2001), which is incorporated herein by reference.

[0059] The vector of the present invention can typically be constructed as a vector for cloning or as a vector for expression. In addition, the vector of the present invention can be constructed using a prokaryotic cell or a eukaryotic cell as a host.

[0060] For example, when the vector of the present invention is an expression vector and uses a eukaryotic cell as a host, a promoter derived from the genome of a mammalian cell (e.g., metallothionine promoter, beta-actin promoter, human hemoglobin promoter, and human muscle creatine promoter) or a promoter derived from a mammalian virus (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, tk promoter of HSV, mouse mammary tumor virus (MMTV) promoter, LTR promoter of HIV, promoter of Moloney virus, promoter of Epstein-Barr virus (EBV), and promoter of Rous sarcoma virus (RSV)) can be used, and generally has a polyadenylation sequence as a transcription termination sequence.

[0061] The vector of the present invention may be fused with other sequences to facilitate the purification of peptides or proteins expressed therefrom. Examples of such fused sequences include glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA), and 6-His (hexahistidine; Qiagen, USA).

[0062] Meanwhile, the expression vector of the present invention includes an antibiotic resistance gene commonly used in the art as a selectable marker, for example, a resistance gene for ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and tetracycline.

[0063] According to one aspect of the present invention, the present invention provides a host cell comprising the recombinant vector.

[0064] Any host cell capable of stably and continuously cloning and expressing the vector of the present invention is known in the art and can be used. For example, suitable eukaryotic host cells for the vector include, but are not limited to, monkey kidney cells 7 (COS7), NSO cells, SP2 / 0, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, MDCK, myeloma cell lines, HuT 78 cells, and HEK-293 cells.

[0065] In the present invention, "transformed", "transduced" or "transfected" refers to a process by which an exogenous nucleic acid is transferred or introduced into a host cell. A "transformed", "transduced" or "transfected" cell is a cell that has been transformed, transduced or transfected with an exogenous nucleic acid, and the cell includes the cell and progeny cells resulting from passage thereof.

[0066] According to one aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating muscle disease, comprising the peptide.

[0067] The term “prevention” used in the present invention means any act of inhibiting the onset or progressing of the disease by administering the pharmaceutical composition according to the present invention.

[0068] The term "treatment" used in the present invention includes suppression of occurrence or recurrence of the disease, alleviation of symptoms, reduction of any direct or indirect pathological consequences of the disease, reduction of the rate of disease progression, improvement, alleviation, palliation or improved prognosis of the disease state, etc., by administration of the pharmaceutical composition according to the present invention.

[0069] In the present invention, “subject” or “patient” refers to an animal, preferably a mammal, including a human, pig, chimpanzee, dog, cat, cow, mouse, rabbit or rat.

[0070] The pharmaceutical composition of the present invention may be formulated as a powder, granule, tablet, coated tablet, pill, dragee, capsule, liquid, suspension, gel, syrup, slurry, suppository, enema, emulsion, paste, ointment, cream, lotion, powder, spray or suspension.

[0071] The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier and diluent. The pharmaceutically acceptable carrier and diluent may be biologically and physiologically compatible with the subject. The pharmaceutically acceptable carrier is one commonly used in the formulation and includes, but is not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. Examples of the pharmaceutically acceptable diluent include, but are not limited to, saline, an aqueous buffer, a solvent, or a dispersion media.

[0072] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective amount may be determined based on the type and severity of the patient's disease, the activity and sensitivity of the drug to the drug, the time of administration, the route of administration and the excretion rate, the treatment period, concurrently used drugs, and other factors well known in the medical field. The amount of the composition used may vary depending on the patient's age, sex, and weight, but the peptide may be administered once or several times a day in an amount sufficient to achieve a blood concentration useful for treating muscle diseases.

[0073] The dosage of the pharmaceutical composition of the present invention is preferably 0.001 to 100 mg / kg (body weight) per day, and the pharmaceutical composition at the dosage described above can be applied locally to the desired area, depending on the purpose of application.

[0074] The pharmaceutical composition of the present invention can be administered to a subject via various routes. All modes of administration are conceivable, including intracerebral administration, oral ingestion, subcutaneous injection, intraperitoneal injection, intravenous injection, intramuscular injection, intrathecal injection, sublingual administration, buccal mucosal administration, rectal insertion, vaginal insertion, ocular administration, otic administration, nasal administration, inhalation, oral or nasal spraying, dermal administration, and transdermal administration. Preferably, the method is intravenous injection.

[0075] The dosage of the pharmaceutical composition of the present invention may vary depending on the patient's condition and weight, the severity of the disease, the drug form, the route of administration, and the time of administration, but can be appropriately selected by those skilled in the art. Therefore, the above dosage does not limit the scope of the present invention in any way.

[0076] The pharmaceutical composition according to the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents. It can be administered sequentially or simultaneously with conventional therapeutic agents, or in single or multiple doses. Taking all of the above factors into account, it is important to administer an amount that achieves maximum efficacy with the minimum amount possible without causing side effects. This can be readily determined by those skilled in the art.

[0077] In one embodiment of the present invention, the muscle disease may be selected from the group consisting of sarcopenia, amyotrophy, cancer cachexia, muscle damage, muscular dystrophy, cardiac atrophy, atony, muscular dystrophy, muscular atrophy, and myasthenia gravis, but is not limited thereto.

[0078] In this invention, "sarcopenia" refers to a general term for a condition in which the number of muscle fibers that make up a muscle decreases. Causes include chronic inflammation, hormonal imbalances, and nutritional deficiencies, but the primary cause is considered to be muscle wasting due to decreased muscle movement. Recently, various countries have been registering sarcopenia as an official disease. The United States assigned sarcopenia a disease code (M63.84) in 2016, and Japan added it to its list of diseases in 2018. Korea also included sarcopenia in the 8th revision of the Korean Classification of Diseases (KCD).

[0079] In the present invention, "amyotrophy" refers to a general term for a disease in which muscles shrink and weaken. It is caused by mutations in genes necessary for normal muscle development and maintenance. Most cases are progressive, with symptoms continuing to worsen after onset.

[0080] In one embodiment of the present invention, the muscle disease may be a disease caused by muscle wasting or degeneration, wherein muscle wasting is characterized by a gradual loss of muscle mass and the weakening and degeneration of voluntary muscles, such as skeletal muscles, or involuntary muscles, such as cardiac muscles. Muscle wasting and degeneration are caused by genetic factors, acquired factors, aging, etc.

[0081] In one aspect of the present invention, the present invention provides a method for preventing or treating muscle disease, comprising administering the mutant ADMATS1 peptide or a composition comprising the same to a subject in need of treatment or prevention.

[0082] The method for preventing or treating the above muscle disease is common to the above-described ADAMTS1 peptide or composition in that it includes a mutant ADAMTS1 peptide or a composition containing the same, and description of the common parts is omitted to avoid excessive redundant description in the specification.

[0083]

[0084] The features and advantages of the present invention are summarized as follows:

[0085] (a) The present invention provides a mutant ADAMTS1 peptide comprising an amino acid sequence in which cysteine ​​at position 198 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with serine and at least one amino acid among amino acids at positions 222 to 251 is deleted.

[0086] (b) The present invention provides a nucleic acid molecule encoding the peptide.

[0087] (c) The present invention provides a recombinant vector comprising the nucleic acid molecule.

[0088] (d) The present invention provides a host cell comprising the recombinant vector.

[0089] (e) The present invention provides a pharmaceutical composition for preventing or treating muscle disease comprising the peptide.

[0090] (f) When the mutant ADAMTS1 polypeptide of the present invention is used, differentiation of muscle fibers can be promoted, and effective prevention or treatment of muscle diseases is possible.

[0091]

[0092] Figure 1 shows a schematic diagram of NM3, NM4, NM5, and NM6 mutant ADAMTS1.

[0093] Figure 2 shows the comparative results of muscle cell differentiation when treated with NM3 to NM6 peptides at a concentration of 10 ng / mL.

[0094] Figure 3 shows a schematic diagram of NM3, NM3a, NM3b, NM3c, and NM3d mutant ADAMTS1.

[0095] Figure 4 shows the comparative results of muscle cell differentiation when treated with NM3 to NM6 peptides at a concentration of 10 ng / mL.

[0096] Figure 5 shows a comparative schematic diagram of the previously developed M3 mutant ADAMTS1 recombinant peptide (M3), NM3 mutant ADAMTS1 recombinant peptide (NM3), and C198S mutant ADAMTS1 recombinant peptide (C198S).

[0097] Figures 6a and 6b show the results of detecting dimers of the NM3 peptide under the following conditions: (1) immediately after purification; (2) after standing at room temperature for 2 days; (3) after concentration; (4) when 1 M ammonium sulfate was treated to the cell culture medium for purification; and (5) when 1 M ammonium sulfate was treated to the supernatant after the first purification for purification.

[0098] Figures 7a and 7b show the results of confirming the dimer form of the NM3 peptide through LC-MS analysis.

[0099] Figure 8 shows the results of detection of dimers of NM3 peptide through peptide mapping.

[0100] Figure 9a shows the results of measuring C198S protein expression by transfecting CHO cells with cysteine ​​(Cys) at position 198 of the NM3 peptide replaced with serine (Ser).

[0101] Figure 9b shows the results of dimer detection of the C198S recombinant protein.

[0102] Figure 10 shows the LC-MS analysis results of the C198S peptide.

[0103] Figures 11a and 11b show the results of measuring muscle weight recovery when C198S peptide is administered in a mouse animal model in which muscle damage is induced.

[0104] Figures 12a to 12c show the effect of C198S peptide administration on the expression of eMyHC (embryonic myosin heavy chain), an early expression factor of muscle differentiation, in a mouse animal model in which muscle damage is induced.

[0105] Figures 13a to 13c show the effects of C198S peptide administration on the mRNA expression of MyHC, NICD, and Hes1 in a mouse animal model in which muscle damage is induced.

[0106] Figures 14a to 14d show the results of measuring muscle cross-sectional area using H&E staining in a mouse animal model in which muscle damage was induced.

[0107] Figure 15 shows the results of measuring grip strength in a mouse animal model in which muscle damage was induced.

[0108]

[0109] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples, in accordance with the gist of the present invention.

[0110]

[0111] Example

[0112]

[0113] Throughout this specification, “%” used to indicate the concentration of a particular substance is (weight / weight) % for solid / solid, (weight / volume) % for solid / liquid, and (volume / volume) % for liquid / liquid, unless otherwise stated.

[0114]

[0115] Example 1: Production of recombinant proteins

[0116] 1-1: Vector Creation

[0117] The present inventors constructed an ADAMTS1 overexpression vector in which 6X His was attached to the C-terminus of the recombinant protein to isolate and purify the mutant ADAMTS1 recombinant protein produced in cells.

[0118] Specifically, pEF6 / V5-His A vector (Invitrogen, V96120) was digested using Kpnl and Xhol restriction enzymes. Here, a sequence recognized by Kpnl was inserted at the 5' end of the CDS portion of the ADAMTS1 sequence (NM_006988.5), and a sequence recognized by Xhol was inserted at the 3' end, and then ligated to the previously digested vector using T4 DNA ligase to produce the construct.

[0119]

[0120] 1-2: Production of M1, M2, M3, M4, M5, and M6 mutant ADAMTS1 sequences

[0121] M1, M2, M3, M4, M5, and M6 mutant ADAMTS1 were constructed by using wild-type ADAMTS1 as a template and truncating it from nucleotide 664 to nucleotide 693 for M1, 723 for M2, 753 for M3, 783 for M4, 813 for M5, and 843 for M6, respectively. Additionally, all mutants were constructed by deleting all sequences after nucleotide 1237.

[0122]

[0123] 1-3: Construction of NM3, NM4, NM5, and NM6 mutant ADAMTS1 sequences

[0124] NM3, NM4, NM5, and NM6 mutant ADAMTS1 were produced by using wild-type ADAMTS1 as a template and truncating it to nucleotide 753 for NM3, 783 for NM4, 813 for NM5, and 843 for NM6, respectively, starting from nucleotide 664, and removing all sequences after nucleotide 1402.

[0125] NM3, NM4, NM5, and NM6 mutant ADAMTS1 are forms in which the c-term length is extended from nucleotide 1237 to nucleotide 1402 of M3, M4, M5, and M6 mutant ADAMTS1.

[0126] Schematic diagrams of NM3, NM4, NM5, and NM6 mutant ADAMTS1 are shown in Figure 1.

[0127] To confirm the muscle differentiation ability of NM3, NM4, NM5, and NM6 mutant ADAMTS1, each was treated at a concentration of 10 ng / mL, and the muscle differentiation length was measured. The results are shown in Fig. 2 and Table 1.

[0128] As shown in Fig. 2 and Table 1, the muscle differentiation length increased when treated with NM3, NM4, NM5, and NM6 mutant ADAMTS1 proteins compared to the control group, and among them, the muscle differentiation ability of the NM3 mutant protein was confirmed to be the most effective.

[0129]

[0130] -Mean (um)SDControl868.74335.518NM31982.868137.969NM41614.22582.709NM51676.79531.553NM61639.93367.975

[0131]

[0132] 1-4: Construction of NM3a, NM3b, NM3c, and NM3d mutant ADAMTS1 sequences

[0133] NM3a, NM3b, NM3c, and NM3d mutant ADAMTS1 were constructed using NM3 mutant ADAMTS1 as a template and the RKKR sequence at the very end of the precursor domain of ADAMTS1 as a starting point. NM3a deleted R from the RKKR sequence, NM3b deleted RK from the RKKR sequence, NM3c added one amino acid after the RKKR sequence, and NM3d added three amino acids after the RKKR sequence.

[0134] Schematic diagrams of NM3a, NM3b, NM3c, and NM3d mutant ADAMTS1 are shown in Figure 3.

[0135] To confirm the muscle differentiation ability of NM3a, NM3b, NM3c, and NM3d mutant ADAMTS1, each was treated at a concentration of 10 ng / mL, and the muscle differentiation length was measured. The results are shown in Fig. 4 and Table 2.

[0136] -Mean (um)SDControl892.3617.622NM31997.06898.200NM3a1838.34358.362NM3b1880.13845.427NM3c1870.35250.771NM3d1873.93857.421

[0137] As shown in Fig. 4 and Table 2, the muscle differentiation length increased when treated with NM3, NM4, NM5, and NM6 mutant ADAMTS1 proteins compared to the control group, and among them, the muscle differentiation ability of the NM3 mutant protein was confirmed to be the most effective.

[0138]

[0139] 1-5: Construction of C198S mutant ADAMTS1 sequence

[0140] The C198S mutant ADAMTS1 was created by a point mutation from G to C at nucleotide 593 of the NM3 mutant ADAMTS1. When the C198S mutant ADAMTS1 protein was synthesized due to the point mutation, amino acid 198 was changed from Cys to Ser.

[0141] A schematic diagram of the C198S mutant ADAMTS1 is shown in Figure 5.

[0142]

[0143] 1-6: Transformation

[0144] CHO-K1 cells were seeded in 6-well cell culture plates using RPMI1640 medium supplemented with 10% FBS, 100 U / mL penicillin, 100 μg / mL streptomycin, and 25 mM Hepes, and cultured in a 5% CO2, 37°C cell incubator. The next day, Lipofectamine 2000 (Invitrogen, 116680199) was mixed with the ADAMTS1 mutant expression vector and injected to introduce the vector into CHO-K1 cells.

[0145] After culturing for 24 hours, the cells were diluted 10-fold and transferred to 100 mm cell culture dishes, and then treated with blasticidin-S at a concentration of 10 μg / mL for the first time. Thereafter, the culture medium was replaced with new blasticidin-S at a concentration of 10 μg / mL three times at 4-day intervals. After independent colonies were formed by blasticidin-S, each colony was inoculated onto a new cell culture plate and cultured.

[0146]

[0147] 1-7: Purification of recombinant proteins

[0148] After confirming the expression of ADAMTS or ADAMTS1 mutant proteins through Western blotting, the stabilized cell line was used. The CHO-K1 stabilized cell line was adapted to serum-free medium, made into suspension cells, and then cultured at 1X10 6 The cells were added to a 250 mL Erlenmeyer flask in an amount of 100 mL to obtain a concentration of 100 cells / mL. The cells were cultured for a total of 9 days in a shaking cell incubator at 37°C with 5% CO2 supply and 110 rpm. After culture, fed-batch culture was performed on the 5th and 7th days by adding 10% of the total medium with serum-free medium. After 9 days of culture, the supernatant was purified with Ni-NTA agarose (Qiagen, 30210), recovered, and used in a differentiation induction experiment of the C2C12 cell line.

[0149] Specifically, the Ni-NTA agarose purification method involved passing the cell culture solution through a gravity chromatography column (Qiagen, 34964) onto which Ni-NTA agarose had been deposited, washing with 10 mM imidazole buffer, repeating the washing three times, and then eluting with 250 mM imidazole buffer to purify the protein.

[0150]

[0151] Example 2: High-purity purification of recombinant proteins

[0152] The cell culture medium producing the NH3 mutant ADAMTS1 recombinant protein was harvested and impurities were removed using a 0.22 μm filter. Subsequently, primary column purification was performed using a nickel chelating resin. The primary purification was performed 2–3 times while confirming the expression level, and then dialysis was performed with PBS. For the secondary purification, column purification was performed using a gel filtration resin. The secondary column purification eluate was collected by fraction, and only the fractions showing a purity of 92% or higher were used after HPLC analysis. The amount of protein detected in the fractions was confirmed, and the process was repeated 8–9 times to concentrate the fractions to a final concentration of 5–10 mg / mL.

[0153]

[0154] Example 3: HPLC analysis

[0155] HPLC analysis was performed on the NM3 mutant ADAMTS1 recombinant protein and the C198S mutant ADAMTS1 recombinant protein produced from Example 2 above. The analytical equipment and detailed conditions for HPLC analysis were as follows.

[0156] - HPLC: Waters, e2695

[0157] - Column: Tosoh, TSKgel G3000SWXL

[0158] - Flow rate: 1 mL / min

[0159] - Mobile phase: Sodium phosphate buffer, pH 6.8

[0160] - Injection conc.: 1 mg / mL

[0161] - Injection amount: 20-50 μg

[0162] - Wavelength: 280 nm

[0163] To characterize the NM3 mutant ADAMTS1 recombinant protein, the NM3 recombinant protein was reacted under five conditions, and protein expression was confirmed by SDS-PAGE and HPLC analysis was performed. The five conditions are as follows: (1) immediately after purification; (2) when the purified solution was left at room temperature for 2 days; (3) immediately after concentrating the purified solution; (4) when the cell culture medium was treated with 1 M ammonium sulfate for purification; and (5) when the primary purified solution was treated with 1 M ammonium sulfate for purification.

[0164] The results of reducing the protein and performing SDS-PAGE are shown in Figure 6a.

[0165] As shown in Fig. 6a, it was confirmed that the NM3 mutant ADAMTS1 recombinant protein exists as a monomer, but when confirmed under non-reduced conditions, it was confirmed to exist in two forms, a dimer and a monomer.

[0166] In addition, the results confirmed by HPLC are shown in Fig. 6b, and as shown in Fig. 6b, it was confirmed that two main peaks appeared.

[0167]

[0168] Example 4: LC-MS analysis and peptide mapping

[0169] The present inventors attempted to perform LC-MS to confirm the form in which the NM3 dimer is formed.

[0170] Specifically, LC-MS analysis was performed on a UPLC (Waters, ACQUITY PREMIERE) instrument using 50 μg of protein sample via a C4 column (Waters, ACQUITY UPLC BEH C4 column). Mass spectrometry was performed using a Waters Synapt XS instrument in the mass-to-charge ratio (m / z) range of 400–3500. Subsequently, molecular weight calculations were performed using Waters MassLynx software, and the results are shown in Figure 7.

[0171] As shown in Fig. 7, peaks were observed within the 44-49 kDa and 91-97 kD ranges. Specifically, in the 44-49 kDa range, which corresponds to the size of the NM3 mutant ADAMTS1 protein, peaks appeared in various forms, and were judged to be variants of protein modification, but it was confirmed that the exact type of modification was difficult to identify. In addition, in the 91-97 kD range, which corresponds to the protein size in which dimers are formed, various types of peaks appeared. These appear to be various combinations of peaks 1 to 4 indicated in Fig. 7, and among these, only peaks 1+1, 2+2, and 3+4 had predictable protein sizes.

[0172] Additionally, the inventors performed peptide mapping to analyze the dimer formation site of the protein.

[0173] Protein samples were denatured with urea and then reacted with trypsin at a ratio of 1:50 at 37°C for 16 hours. After the reaction, the samples were analyzed using a UPLC device (Waters, ACQUITY PREMIERE) and a C18 column (Waters, ACQUITY UPLC BEH C18 column). Analysis was performed using a Waters Synapt XS mass spectrometer in the mass-to-change ratio (m / z) range of 50-2000. The results of the mass spectrometry analysis were used to identify the location of dimer formation using Biopharmalynx software (Waters).

[0174] The peptide mapping results are shown in Figure 8.

[0175] When the dimer formation location was confirmed based on the peptide mapping analysis results shown in Figure 8 and the sequence information of NM3 in the Uniport database, cysteine ​​(Cys) at position 198 was identified as a cysteine-switch motif, and Cys at position 198 was confirmed to have no fixedly paired Cys.

[0176] A plasmid that was constructed to form a 198-Cys-to-Ser substitution was transfected into a CHO-K1 cell line to confirm protein expression, and the results are shown in Figure 9.

[0177] As shown in Figure 9, the Western blot analysis results confirmed that protein expression was normal even when amino acid 198 was substituted with Ser.

[0178] When the C198S mutant ADAMTS1 protein was purified and confirmed by SDS-PAGE, protein expression was confirmed only in the monomeric form under both reducing and non-reducing conditions, and both the purified solution and the concentrated solution showed a single peak in HPLC analysis.

[0179] Additionally, the present inventors analyzed the C198S mutant ADAMTS1 recombinant protein using LC-MS, and the results are shown in Fig. 10.

[0180] As shown in Fig. 10, unlike the NM3 mutant ADAMTS1 recombinant protein, a peak showing a uniform protein size difference was confirmed. This showed a uniform size difference of 291 to 292 Da depending on the number of sialic acids, and a peak showing a uniform size difference was also confirmed for the dimer formed before purification. Unlike NM3, the C198S mutant ADAMTS1 recombinant protein was confirmed to not form new dimers after removing dimers through initial purification.

[0181]

[0182] Example 5: Observation of muscle recovery in an animal model of muscle damage.

[0183] To conduct an experiment to observe muscle recovery in an animal model of muscle damage, the present inventors first induced muscle damage by intramuscularly injecting 250 μL of 1.2% BaCl into the left TA (Tibialis Anterior) muscle of 7-week-old C57BL6 mice. Simultaneously, 10 mg / kg of the C198S mutant ADAMTS1 recombinant protein was administered intraperitoneally for a total of 10 days, and the degree of muscle recovery in the animal model was observed. After the administration was complete, the weight of the TA muscle tissue was measured, and the weight of the muscle damage-induced tissue (Left TA muscle) was compared as a ratio to the weight of the normal tissue (Right TA muscle). The experimental results are shown in Figures 11a to 11b and Table 3.

[0184]

[0185] Comparison of the weight of tissues induced by muscle damage compared to normal tissue - TA muscle weight Left / Right (%) Control BaCl2 BaCl2+ C198 Savage-1.01-16.56-6.31 STDEV6.322.862.31

[0186] As shown in Figures 11a to 11b and Table 3, when muscle damage occurred, a decrease in muscle weight of approximately 16% was observed, but when C198S was administered, it was confirmed that the degree of muscle weight decrease was reduced to -6%.

[0187] Therefore, these results suggest that the C198S mutant ADAMTS1 recombinant protein can repair damaged muscles.

[0188]

[0189] Example 6: Confirmation of expression of eMyHC, an early muscle differentiation factor, in an animal model induced by muscle damage.

[0190] The present inventors sought to determine the effect of administration of C198S mutant ADAMTS1 recombinant protein on the expression of eMyHC (embryonic myosin heavy chain) in an animal model of muscle damage.

[0191] First, administration of the C198S mutant ADAMTS1 recombinant protein was performed for 10 days, and on the third day, one group was autopsied to isolate TA muscle tissue. The isolated TA tissue was fixed with 4% paraformaldehyde, embedded in paraffin, and tissue sections were obtained from paraffin blocks using a microtome. The tissue sections were washed with xylene, deparaffinized three times with 100%, 95%, and 70% ethanol, and then rehydrated to perform the antigen exposure step. Afterwards, the sections were blocked with TBS blocking buffer containing 10% normal serum and 1% BSA for 2 hours at room temperature.

[0192] Next, the eMyHC (embryonic myosin heavy chain) primary antibody was diluted in TBS buffer containing 1% BSA and reacted at 4℃ for 16 hours. The next day, the slides were washed twice with TBS buffer containing 0.025% Triton X-100, and then the HRP-conjugated secondary antibody was diluted in TBS buffer containing 1% BSA and reacted at room temperature for 1 hour. After the secondary antibody reaction was completed, the slides were washed three times with TBS buffer and then reacted at room temperature for 10 minutes using 3,3'-diaminobenzidine (DAB) chromogen to develop color. After the color reaction was completed, the slides were washed in running water for 5 minutes, dehydrated and mounted, and then analyzed under a microscope.

[0193] The analysis results are shown in Figures 12a to 12c.

[0194] As shown in Figures 12a to 12c, eMyHC, a protein known to be expressed early in muscle differentiation, showed a pattern of increased expression as natural recovery progressed following muscle damage. In the group co-administered with the C198S mutant ADAMTS1 recombinant protein, eMyHC protein expression was observed to increase throughout muscle tissue.

[0195] Therefore, these results imply that the C198S mutant ADAMTS1 recombinant protein can help in muscle damage recovery by promoting the expression of eMyHC.

[0196]

[0197] Example 7: Confirmation of reduction in muscle differentiation inhibitors in an animal model of muscle damage

[0198] In order to confirm the tendency of reduction of muscle differentiation inhibitory factors in a muscle damage-induced animal model, the present inventors administered the C198S mutant ADAMTS1 recombinant protein to a muscle damage animal model for 10 days, and then isolated TA muscle tissue from the animals and confirmed the protein expression of muscle differentiation-related factors through Western blot analysis. The results are shown in Fig. 13.

[0199] As shown in Fig. 13, the expression of MyHC (Myosin heavy chain) expressed from myotubes in which muscle differentiation occurred was confirmed to increase when the C198S mutant ADAMTS1 recombinant protein was administered. In addition, the expression of NICD (Notch intracellular domain) produced in the NOTCH1 signaling pathway was confirmed to decrease in the group administered the C198S mutant ADAMTS1 recombinant protein. In the case of Hes1, a downstream factor of NOTCH1 signaling, it was confirmed that muscle differentiation was partially promoted in the BaCl2-treated group compared to the control group due to natural muscle recovery, and in the group administered the C198S mutant ADAMTS1 recombinant protein, the BaCl 2 - It was confirmed that the expression of Hes1 protein was further reduced compared to the treatment group.

[0200] These results imply that the C198S mutant ADAMTS1 recombinant protein can restore muscle damage by inhibiting muscle differentiation inhibitors MyHC, NICD, and Hes1.

[0201]

[0202] Example 8: Measurement of muscle cross-sectional area in an animal model of muscle damage

[0203] The present inventors sought to determine the effect of C198S peptide administration on muscle cross-sectional area in a mouse animal model in which muscle damage was induced.

[0204] Specifically, after administering the C198S mutant ADAMTS1 recombinant protein for 10 days to a muscle damage animal model, TA muscle tissue was isolated from the animal, and the CSA (Cross sectional area) was measured through H&E staining, and the results are shown in Figures 14a to 14d and Table 4.

[0205]

[0206] Comparison of cross-sectional area size according to peptide administration - CSA (μm 2 )ControlBaCl2BaCl2+ C198Saverage1810.32990.171719.89STDEV173.37155.7479.20

[0207] As shown in Figures 14a to 14d and Table 4, the average cross-sectional area of ​​TA muscle tissue was decreased by BaCl2, but it was confirmed that it was restored to a level close to the normal group when the C198S mutant ADAMTS1 recombinant protein was co-administered.

[0208] Therefore, these results suggest that the C198S mutant ADAMTS1 recombinant protein may aid in the recovery of damaged muscles.

[0209]

[0210] Example 9: Evaluation of the degree of improvement in exercise performance in an animal model of muscle damage

[0211] To confirm the change in grip strength when C198S peptide is administered to a mouse animal model induced with muscle damage, the inventors administered C198S mutant ADAMTS1 recombinant protein to the animal model with muscle damage for 10 days, and measured grip strength at the starting point, on the 3rd day, on the 6th day, and on the 10th day to evaluate the degree of improvement in exercise performance.

[0212] The evaluation results are shown in Figure 15.

[0213] As shown in Figure 15, it was confirmed that the grip strength decreased due to muscle damage induced by BaCl2 increased again when the C198S mutant ADAMTS1 recombinant protein was co-administered.

[0214] Therefore, these results suggest that the C198S mutant ADAMTS1 recombinant protein may help improve exercise performance.

[0215]

[0216] While specific aspects of the present invention have been described in detail above, it is clear to those skilled in the art that these specific descriptions are merely preferred implementation examples and that the scope of the present invention is not limited thereto.

Claims

1. A mutant ADAMTS1 peptide comprising an amino acid sequence in which cysteine ​​at position 198 is substituted with serine and at least one amino acid among amino acids at positions 222 to 251 is deleted in the amino acid sequence indicated by sequence number 1.

2. A mutant ADAMTS1 peptide according to claim 1, wherein the mutant peptide comprises a deletion of at least one amino acid selected from the group consisting of arginine at position 249, lysine at position 250, lysine at position 251, and arginine at position 252 of a peptide having an amino acid sequence of SEQ ID NO:

1.

3. A mutant ADAMTS1 peptide according to claim 1, wherein the mutant peptide comprises a deletion of amino acids at positions 249 to 251 of a peptide consisting of the amino acid sequence of SEQ ID NO:

1.

4. A mutant ADAMTS1 peptide according to claim 1, wherein the mutant peptide comprises a deletion of at least one amino acid selected from the group consisting of arginine at position 249, lysine at position 250, lysine at position 251, and arginine at position 252 of a peptide having an amino acid sequence of SEQ ID NO: 1, and a deletion of at least one amino acid selected from the group consisting of amino acids at positions 223 to 248 and 253 to 281.

5. A mutant ADAMTS1 peptide according to claim 1, wherein the mutant ADAMTS1 peptide comprises an amino acid sequence additionally including a deletion of amino acids at positions 468 to 967 in the amino acid sequence represented by SEQ ID NO:

1.

6. A mutant ADAMTS1 peptide according to claim 1, wherein the peptide comprises an amino acid sequence of SEQ ID NO:

3.

7. A mutant ADAMTS1 peptide according to claim 1, wherein the peptide comprises an amino acid sequence of SEQ ID NO:

4.

8. A mutant ADAMTS1 peptide according to claim 1, characterized in that the peptide promotes differentiation of muscle cells.

9. A nucleic acid molecule encoding a peptide of any one of claims 1 to 8.

10. In the 9th paragraph, the nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO:

15.

11. A recombinant vector comprising the nucleic acid molecule of paragraph 9.

12. A host cell containing the recombinant vector of paragraph 11.

13. A pharmaceutical composition for preventing or treating muscle disease, comprising the peptide of any one of claims 1 to 8.

14. A pharmaceutical composition for preventing or treating muscle disease, wherein the muscle disease in claim 13 is selected from the group consisting of sarcopenia, amyotrophy, cancer cachexia, muscle damage, muscular dystrophy, cardiac atrophy, atony, muscular dystrophy, muscle atrophy, and myasthenia gravis.

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