WO2022131750A1 - Composition pour le traitement ou la prévention de la myopathie, de l'obésité ou du diabète - Google Patents

Composition pour le traitement ou la prévention de la myopathie, de l'obésité ou du diabète Download PDF

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WO2022131750A1
WO2022131750A1 PCT/KR2021/018973 KR2021018973W WO2022131750A1 WO 2022131750 A1 WO2022131750 A1 WO 2022131750A1 KR 2021018973 W KR2021018973 W KR 2021018973W WO 2022131750 A1 WO2022131750 A1 WO 2022131750A1
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peptide
muscle
mutant
present
amino acids
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Korean (ko)
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최봉근
이성권
이상협
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주식회사 뉴온바이오
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6489—Metalloendopeptidases (3.4.24)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/4886—Metalloendopeptidases (3.4.24), e.g. collagenase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue

Definitions

  • the present invention relates to an L1 mutant peptide and a composition for alleviating, inhibiting, preventing, or treating muscle disease, obesity or diabetes comprising the same as an active ingredient, and a method for alleviating, inhibiting, preventing, or treating muscle disease, obesity or diabetes using the L1 mutant peptide , and to the alleviation, inhibition, prevention or treatment of muscle disease, obesity or diabetes of the L1 mutant peptide.
  • Muscle atrophy refers to a decrease in muscle volume, which can be clearly seen in patients suffering from cancer or various immunodeficiency diseases, and patients with extremely limited physical activity due to long-term bed life It is more pronounced in people with limited mobility and the elderly.
  • the progress of aging causes a sarcopenia phenomenon in which muscle strength decreases due to a continuous decrease in muscle volume, which leads to a dangerous result in which the elderly may be exposed to various life accidents.
  • An increase in fat mass along with a decrease in muscle mass is called sarcopenic obesity. If the increase in body fat and muscle atrophy act synergistically, it is estimated that the risk of functional disorders and metabolic disorders in the body will further increase.
  • body mass index BMI
  • WHR waist circumference
  • the present inventors screened for peptides that have the potential to show an effect on muscle reduction in order to develop a therapeutic agent for alleviation, inhibition, prevention or treatment of muscle disease related to muscle loss, and significant inhibition of muscle reduction of the double L1 mutant peptide
  • the activity was found, and the active protein was isolated and specified, and it was confirmed that the specified mutant peptide had significantly superior activity than the currently used therapeutic agent for muscle disease related to muscle loss, and completed the present invention.
  • Another object of the present invention is to provide a pharmaceutical composition for alleviating, inhibiting, preventing, or treating muscle disease comprising an L1 mutant peptide as an active ingredient.
  • Another object of the present invention is to provide a method for alleviating, inhibiting, preventing or treating muscle loss by administering an L1 mutant peptide to a subject in need thereof in an amount effective to alleviate, inhibit, prevent or treat muscle disease.
  • Another object of the present invention is to provide a use of the L1 mutant peptide used for alleviation, inhibition, prevention or treatment of muscle disease.
  • Another object of the present invention is to provide a method for alleviating, inhibiting, preventing or treating obesity by administering an L1 mutant peptide to a subject in need thereof in an amount effective to alleviate, suppress, prevent, or treat obesity.
  • Another object of the present invention is to provide a use of the L1 mutant peptide for use in alleviating, suppressing, preventing or treating obesity.
  • Another object of the present invention is to provide a method for alleviating, inhibiting, preventing or treating diabetes by administering an L1 mutant peptide to a subject in need thereof in an amount effective to alleviate, inhibit, prevent, or treat diabetes.
  • Another object of the present invention is to provide a use of the L1 mutant peptide for use in alleviating, suppressing, preventing or treating diabetes.
  • the present invention provides an L1 mutant peptide and a composition for alleviating, inhibiting, preventing, or treating muscle disease, obesity or diabetes comprising the same as an active ingredient, and alleviating, inhibiting, preventing, or treating muscle disease, obesity or diabetes using the L1 mutant peptide it's about how
  • the present invention provides an L1 mutant peptide.
  • the L1 mutant peptide of the present invention is a mutant peptide of wild-type ADAMTS1 (A disintegrin and metalloproteinase with thrombospondin motifs 1).
  • ADAMTS1 is an enzyme belonging to a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS), a multidomain extracellular protease family, and was first discovered among 19 ADAMTS family enzymes found in humans. ADAMTS1 is known to inhibit angiogenesis by interacting with vascular endothelial growth factor A.
  • the L1 mutant peptide may be one in which a part of the peptide consisting of the amino acid sequence of SEQ ID NO: 1 is deleted.
  • the L1 mutant peptide of the present invention is a peptide comprising a deletion of one or more amino acids selected from the group consisting of amino acids at positions 295 to 300 of the peptide consisting of the amino acid sequence of SEQ ID NO: 1 can
  • the L1 mutant peptide of the present invention is histidine at position 295, proline at position 296, serine at position 297 of the peptide consisting of the amino acid sequence of SEQ ID NO: 1 ), position 298 isoleucine (Isoleucine), position 299 arginine (Arginine) and position 300 asparagine (Asparagine) may be a peptide comprising a deletion of one or more positions selected from the group consisting of, for example, 295 to It may be a peptide containing a deletion at position 300.
  • the L1 mutant peptide of the present invention is a peptide further comprising a deletion of one or more amino acids selected from the group consisting of amino acids at positions 301 to 312 of the peptide consisting of the amino acid sequence of SEQ ID NO: 1 can be
  • the L1 mutant peptide of the present invention is a peptide further comprising a deletion of one or more amino acids selected from the group consisting of amino acids at positions 291 to 294 of the peptide consisting of the amino acid sequence of SEQ ID NO: 1 can be
  • the L1 mutant peptide of the present invention has a deletion from aspartic acid at position 413 to serine at position 967 of the peptide consisting of the amino acid sequence of SEQ ID NO: 1 It may be a peptide comprising additionally.
  • the L1 mutant peptide of the present invention may be a peptide further comprising a deletion of amino acids at positions 1 to 252 of the peptide consisting of the amino acid sequence of SEQ ID NO: 1.
  • the L1 mutant peptide of the present invention may be a peptide consisting of the amino acid sequence of SEQ ID NO: 2.
  • the present invention provides a pharmaceutical composition for alleviating, inhibiting or treating muscle disease, comprising the L1 mutant peptide as an active ingredient.
  • the L1 mutant peptide is the same as described above.
  • glucocorticoids such as cortisol, a natural hormone, or many synthetic cortisol analogues (including, for example, prednisone, hydrocortisone and dexamethasone) act on the body through the glucocorticoid receptor (GR).
  • Glucocorticoids play an important role in regulating differentiation decisions both in vivo and ex vivo, promoting adipogenesis and inhibiting muscle formation. Androgen administration has been shown to increase muscle mass while decreasing fat mass by affecting body composition.
  • Muscle insufficiency or weakness is one of the most fatal health problems in children. These muscle disorders affect more than 1 in 3000 people with various congenital myopathy and muscular dystrophy, such as Duchenne Muscular Dystrophy (DMD). These disorders are usually associated with genetic or spontaneous gene mutations. Children with these disorders suffer from a wide range of complications. Currently, due to the lack of effective treatment, the mortality rate is high, and there is a need to develop a new treatment strategy for these muscle diseases.
  • DMD Duchenne Muscular Dystrophy
  • Muscle tissue of adult vertebrates is regenerated from stem cells known as satellite cells or muscle stem cells (MuSC). Satellite cells are distributed throughout the muscle tissue, are similarly stationary in the absence of injury or disease, and are located in anatomically defined crevices. In addition to satellite cells, cell types that may contribute to muscle regeneration include, but are not limited to, mesenchymal cells, bone marrow-derived cells, muscle stromal cells, and mesenchymal stem cells.
  • Tissue engineering seeks to repair or replace damaged or diseased tissue in the body by implanting a combination of cells, biomaterial scaffolds, biologically active molecules and genes.
  • the basic premise of this approach is that exogenously introduced cells will improve the rate and extent of tissue repair.
  • muscle diseases include sarcopenia, amyotrophy, cancer cachexia, muscle damage, muscular dystrophy, cardiac atrophy, atony, muscular dystrophy, and muscle degeneration. It may be selected from the group consisting of myasthenia gravis and myasthenia gravis, but is not limited thereto, and includes all muscle diseases related to muscle loss.
  • sarcopenia was first named by Rosenborg in 1989, and the etymology of sarcopenia is derived from the Greek word "sarco” meaning muscle and "penia” meaning reduced.
  • WHO World Health Organization assigned a disease classification code to sarcopenia, and recognized that muscle mass less than normal was an official disease. It refers to the loss of skeletal muscle mass mainly distributed in the extremities, and is a result of the gradual decrease in skeletal muscle mass associated with aging.
  • Amyotrophy is defined as a loss of muscle mass. It is common when a patient is resting, such as when admitted to a hospital, or when movement is restricted. That is, muscle atrophy often occurs in cachexia, which is a state of being inactive or suffering from two or more diseases such as cancer, AIDS, congestive heart failure, chronic obstructive pulmonary disease, renal failure, and severe burns at the same time. This increases the risk of falls and falls, and is known to be associated with a decrease in energy expenditure, which in turn increases the risk of obesity and metabolic diseases.
  • muscular atrophy the results of serious diseases such as cancer, a metabolic disease, are also the causes of muscular atrophy, which has been demonstrated to be induced by an increase in inflammatory cytokines and activation of inflammatory signaling pathways.
  • cytokines the causes of muscular atrophy
  • metabolic changes such as an increase in the influx of fatty acids in muscle tissue and a decrease in fatty acid oxidation occur, and mitochondrial biosynthesis, structure and dysfunction occurs.
  • the oxidative stress and the inflow of fatty acids in the muscle also cause inflammation, which can lead to muscular atrophy.
  • the present invention provides a method for alleviating, inhibiting or treating muscle disease, comprising administering to a subject a pharmaceutical composition comprising an L1 mutant peptide as an active ingredient.
  • the present invention relates to a composition for alleviating, suppressing, preventing, or treating obesity comprising an L1 mutant peptide as an active ingredient.
  • the L1 mutant peptide is the same as described above.
  • the term “obesity” refers to a state in which adipose tissue is excessively accumulated in the body to the extent that it causes an abnormality in health.
  • the increase in adipose tissue mass in the development of obesity may be due to an increase in the size and number of adipocytes.
  • the increase in cell number may be the result of recruitment of pre-adipose cells from a multipotent stem cell population or from a subpopulation of cells resident in mature white adipose tissue (WAT).
  • WAT white adipose tissue
  • Bone marrow-derived mesenchymal stem cells can differentiate into various cell types including fat, muscle, cartilage and bone. With aging, bone marrow adipogenesis accelerates in vivo, while the bone-forming ability of MSCs decreases. It has been suggested that MSC precursors may be differentiated into adipose rather than bone with interrelationships, contributing to age-related body composition changes.
  • Fat redistribution in the elderly is associated with an increased risk of metabolic syndrome, including diabetes, hypertension, dyslipidemia, atherosclerosis, and relatively increased intra-abdominal fat. There is also a decline in muscle performance associated with muscle aging and normal aging, often with a gradual onset of sarcopenia. Although skeletal muscle has the ability to self-renew, this process is not activated in the elderly. Age-related changes within skeletal muscle tissue and the host environment are known to affect the proliferation and fusion of myoblasts in response to injury in elderly animals.
  • the present invention provides a method for alleviating, suppressing or treating obesity, comprising administering to a subject a pharmaceutical composition comprising an L1 mutant peptide as an active ingredient.
  • the present invention provides a composition for alleviating, inhibiting, preventing, or treating diabetes comprising an L1 mutant peptide as an active ingredient.
  • the L1 mutant peptide is the same as described above.
  • diabetes in the present invention includes all types of diabetes, for example, type 1 diabetes, type 2 diabetes, and hereditary diabetes.
  • Type 1 diabetes is insulin-dependent diabetes mellitus, mainly caused by destruction of ⁇ -cells.
  • Type 2 diabetes is non-insulin-dependent diabetes mellitus, caused by insufficient insulin secretion after a meal or by insulin resistance.
  • the present invention provides a method for alleviating, inhibiting or treating diabetes, comprising administering to a subject a pharmaceutical composition comprising an L1 mutant peptide as an active ingredient.
  • the term 'comprising as an active ingredient' means including an amount sufficient to achieve efficacy or activity of the L1 mutant peptide of the present invention.
  • the content of the peptide as an active ingredient in the composition according to the present invention can be appropriately adjusted depending on the type and purpose of use, the patient's condition, the type and severity of symptoms, etc. 1 to 90.9% by weight, 0.001 to 99% by weight, 0.1 to 99% by weight, 1 to 99% by weight, 0.001 to 90% by weight, 0.1 to 90% by weight, 1 to 90% by weight, 0.001 to 80% by weight, 0.1 to 80% by weight, 1 to 80% by weight, 0.001 to 70% by weight or 0.1 to 70% by weight, for example, may be 1 to 70% by weight, but is not limited thereto. can do.
  • composition according to the present invention may be administered to mammals, including humans, by various routes.
  • the administration method may be any method commonly used, for example, may be administered by oral, dermal, intravenous, intramuscular, subcutaneous, etc. routes, and preferably intravenously.
  • composition of the present invention can be administered in oral dosage forms such as powders, granules, tablets, capsules, ointments, suspensions, emulsions, syrups, and aerosols, or parenteral dosage forms in the form of transdermal preparations, suppositories, and sterile injection solutions according to conventional methods, respectively. It can be formulated and used as such.
  • composition of the present invention may further contain adjuvants such as pharmaceutically suitable and physiologically acceptable carriers, excipients, and diluents in addition to the mixed extract.
  • adjuvants such as pharmaceutically suitable and physiologically acceptable carriers, excipients, and diluents in addition to the mixed extract.
  • Carriers, excipients and diluents that may be included in the composition of the present invention include dextrin, crystalline cellulose, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
  • Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in the extract, for example, starch, calcium carbonate, sucrose ( sucrose) or lactose, gelatin, etc. may be mixed and prepared.
  • lubricants such as magnesium stearate and talc are also used.
  • Formulations for oral use include suspensions, solutions, emulsions, syrups, etc.
  • various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included.
  • Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, transdermal preparations, and the like.
  • Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.
  • witepsol macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like can be used.
  • the pharmaceutical composition of the present invention may be administered alone, but in general, a pharmaceutical carrier selected in consideration of the mode of administration and standard pharmaceutical practice. They may be mixed and administered.
  • the pharmaceutical composition of the present invention may be in the form of a tablet containing starch or lactose, or in the form of a capsule containing alone or an excipient, or an elixir or suspension containing a flavoring or coloring chemical agent. It may be administered orally, orally or sublingually in the form.
  • Such liquid formulations may contain suspending agents (eg, methylcellulose, semisynthetic glycerides such as witepsol or a mixture of apricot kernel oil and PEG-6 esters or PEG-8 and caprylic/capric glyceride mixtures, such as mixtures of glycerides).
  • suspending agents eg, methylcellulose, semisynthetic glycerides such as witepsol or a mixture of apricot kernel oil and PEG-6 esters or PEG-8 and caprylic/capric glyceride mixtures, such as mixtures of glycerides).
  • the dosage of the pharmaceutical composition of the present invention may vary depending on the patient's age, weight, sex, dosage form, health status and disease level, and may be administered in divided doses at regular time intervals according to the judgment of a doctor or pharmacist.
  • the daily dose is 0.1 to 1000 mg/kg, 0.1 to 900 mg/kg, 0.1 to 800 mg/kg, 0.1 to 700 mg/kg, 0.1 to 600 mg/kg, e.g. For example, it may be 0.5 to 500 mg/kg.
  • the above dosage is an example of an average case, and the dosage may be higher or lower depending on individual differences.
  • the daily dosage of the pharmaceutical composition of the present invention is less than the above dosage, a significant effect cannot be obtained, and when it exceeds that, it is not only uneconomical but also undesirable side effects may occur because it is outside the range of the normal dosage. Therefore, it is good to set it as the above range.
  • the present invention relates to a composition for alleviating, inhibiting, preventing, or treating muscle disease, obesity or diabetes comprising an L1 mutant peptide as an active ingredient; a method for alleviating, inhibiting, preventing, or treating muscle disease, obesity or diabetes using an L1 mutant peptide; And it relates to the alleviation, inhibition, prevention or treatment of muscle disease, obesity or diabetes of the L1 mutant peptide.
  • Figure 1a is a photograph showing the result of confirming the expression of the mutant peptide through western blot according to an embodiment of the present invention.
  • Figure 1b is a schematic diagram of a mutant peptide according to an embodiment of the present invention.
  • FIG. 2 is a photograph showing the result of confirming the length of the differentiation-induced myotubes in the control group through Zener staining according to an embodiment of the present invention.
  • FIG. 3 is a photograph showing the result of confirming the length of wild-type differentiation-induced myotubes through Zener staining according to an embodiment of the present invention.
  • FIG. 4 is a photograph showing the result of confirming the length of the L1 mutant differentiation-induced myotube through Zener staining according to an embodiment of the present invention.
  • FIG. 5 is a photograph showing the result of confirming the length of the L3 mutant differentiation-induced myotube through Zener staining according to an embodiment of the present invention.
  • FIG. 6 is a photograph showing the result of confirming the length of the L4 mutant differentiation-induced myotube through Zener staining according to an embodiment of the present invention.
  • FIG. 7 is a photograph showing the result of confirming the length of the differentiation-induced myotube in the control group through immunofluorescence staining according to an embodiment of the present invention.
  • FIG. 8 is a photograph showing the result of confirming the length of the wild-type differentiation-induced myotube through immunofluorescence staining according to an embodiment of the present invention.
  • FIG. 9 is a photograph showing the result of confirming the length of the L1 mutant differentiation-induced myotube through immunofluorescence staining according to an embodiment of the present invention.
  • FIG. 10 is a graph showing the result of confirming the length of differentiation-induced myotubes through immunofluorescence staining according to an embodiment of the present invention.
  • FIG. 11 is a graph showing the results of confirming the thickness of myotubes induced to differentiate through immunofluorescence staining according to an embodiment of the present invention.
  • FIG. 12 is a diagram showing the differentiation result of adipose-derived mesenchymal stem cells into adipocytes according to an embodiment of the present invention.
  • FIG. 13 is a graph showing the result of confirming the degree of adipogenesis according to an embodiment of the present invention.
  • FIG. 14 is a graph showing the measurement result of intracellular glucose uptake according to an embodiment of the present invention.
  • 15 is a graph showing changes in body weight in an animal model with reduced muscle mass according to an embodiment of the present invention.
  • 16 is a graph showing changes in the weight of GA muscle and TA muscle tissue in an animal model with reduced muscle mass according to an embodiment of the present invention.
  • 17 is a graph showing changes in weight of fat and muscle in an animal model with reduced muscle mass according to an embodiment of the present invention.
  • FIG. 18 is a graph showing the results of checking the gene expression levels of MyoD, MyoG, Pax7, and MRF4, which are genes related to muscle differentiation according to an embodiment of the present invention.
  • FIG. 19 is a graph showing the results of confirming the expression levels of MuRF1, Atrogin1, Hes1 genes, which are genes associated with inhibition of muscle differentiation according to an embodiment of the present invention.
  • 20 is a graph showing the result of confirming the length of the myotube differentiation-induced through Zener staining according to an embodiment of the present invention.
  • 21 is a graph showing the results of confirming the activity level of muscle cells according to an embodiment of the present invention.
  • Example 1 C2C12 cell line culture and differentiation induction
  • the myoblast cell line C2C12 cell line (ATCC, CRL-1772), which is mainly used for myocyte differentiation studies, was cultured in DMEM supplemented with 10% fetal bovine serum (FBS), penicillin 100 U/ml and streptomycin 100 ⁇ g/ml. It was cultured in a cell incubator at 37° C. supplied with 5% CO 2 as a medium for use.
  • FBS fetal bovine serum
  • the C2C12 cell line was inoculated in a 24-well cell culture plate at 5 X 10 4 cells/well and cultured in a culture medium.
  • DMEM medium containing 2% horse serum which is a medium for differentiation, to induce differentiation.
  • the differentiation medium was replaced every 3 days, and differentiation was induced in a cell culture medium at 37° C. supplied with 5% CO 2 for a total of 8 days.
  • ADAMTS1 and mutant ADAMTS1 recombinant proteins produced in the CHO-K1 cell line were separated and purified by the method of Example 2 below, and treated together with the induction of differentiation of the C2C12 cell line. It was treated once daily at a concentration of 10, 100 ng/ml.
  • an ADAMTS1 overexpression vector was constructed so that 6x His was placed at the C-terminus of the recombinant protein.
  • the pEF6/V5-His A vector (Invitrogen, V96120) was digested using KpnI and XhoI restriction enzymes.
  • KpnI was inserted at the 5' end of the CDS part of the ADAMTS1 sequence (NM_006988.5) and a sequence recognized as XhoI was inserted at the 3' end, and then T4 DNA ligase was applied to the previously cut vector. It was manufactured by bonding.
  • L1, L2, L3, L4 mutant ADAMTS1 uses wild-type ADAMTS1 as a template, L1 is cleaved from 883 to 900, L2 from 937 to 951, L3 from 1021 to 1038, and L4 from 1084 to 1098 nucleotides and all the mutations were made by removing all sequences after nucleotide 1237.
  • the CHO-K1 cell line is a 6-well cell culture plate using RPMI1640 medium supplemented with 10% Fetal bovine serum (FBS), penicillin 100 U/ml, streptomycin 100 ⁇ g/ml and 25 mM Hepes as a culture medium. was inoculated, and cultured in a 37° C. cell incubator supplied with 5% CO 2 . The next day, the vector was introduced into the cells by mixing Lipofectamine 2000 (Invitrogen, 11668019) with the prepared ADAMTS1 or ADAMTS1 mutant expression vector and treating CHO-K1 cells.
  • FBS Fetal bovine serum
  • blasticidin-S Bovine serum-S
  • blasticidin-S TM blasticidin-S
  • blasticidin-S was first treated to a concentration of 10 ⁇ g/ml. Thereafter, blasticidin-S was mixed and replaced in a new culture medium to a concentration of 10 ⁇ g/ml over a total of 3 times at 4-day intervals.
  • each colony was inoculated into a new cell culture plate and cultured. After confirming whether each colony expresses ADAMTS1 or ADAMTS1 mutant protein through western blot, it was used as a stabilizing cell line.
  • the CHO-K1 stabilized cell line was inoculated into a 100 mm dish, and when the cell density reached 100%, it was exchanged with RPMI1640 medium and cultured for 2 days. After 2 days, only a portion of the upper culture medium was taken from the cultured dish, purified with Ni-NTA agarose (Qiagen, 30210), and recovered, and used in the differentiation induction experiment of the C2C12 cell line. Specifically, the cell culture medium was passed through a gravity chromatography column (Qiagen, 34964) equipped with Ni-NTA agarose and washed with 10 mM imidazole buffer. Washing was repeated 3 times, and then the protein was purified by eluting with 250 mM imidazole buffer.
  • a gravity chromatography column Qiagen, 34964
  • FIGS. 1A and FIG. 1b Taking the culture medium of the ADAMTS1 mutant-overexpressing CHO-K1 stabilized cell line prepared by the method of Example 2, and confirming the expression of the recombinant protein produced by western blotting using an antibody that recognizes 6x-His, the results are shown in FIGS. 1A and FIG. 1b.
  • the L1, L2, L3, and L4 mutations showed a pattern in which the part including the prodomain disappeared and the mature domain was mainly expressed.
  • the remaining mutations except for the L1 mutation showed a significant decrease in protein expression (see FIG. 1a ).
  • the C2C12 cell line was treated with ADAMTS1 or ADAMTS1 mutant recombinant protein while inducing differentiation for 8 days. After the differentiation-induced cells were fixed with 4% paraformaldehyde for 10 minutes, cell membrane permeabilization was performed for 10 minutes using 0.25% Triton X-100.
  • myosin heavy chain (MHC) antibody was mixed with 2% BSA buffer at a ratio of 1:500 and treated for 1 hour.
  • Alexa Fluor® 555 fluorescent antibody was mixed with 2% BSA buffer at a ratio of 1:200 and treated for 1 hour, and the nucleus was stained by DAPI staining.
  • the plate after the staining process was photographed using a fluorescence microscope (Nikon Ts2-FL), and the length of the myotube was measured and statistically processed using the analysis program (Nikon, NIS Elements) provided by the manufacturer. After the analysis was completed, the plate was refrigerated after blocking the light. The results are shown in FIGS. 7 to 11 and Tables 1 to 2 .
  • the C2C12 cell line in which differentiation was induced by treatment with ADAMTS1 wild-type or mutant was longer in thickness and length. It was confirmed that differentiation into Otube. In addition, it was confirmed that the length of the myotube became longer and thicker in the cell line treated with ADAMTS1 L1 mutation compared to the wild type. However, L3 or L4 mutants did not show a better effect than wild-type ADAMTS1 on the length or thickness of myotubes.
  • An adipose-derived mesenchymal stem cell line (ATCC, PCS-500-011) was prepared using 2% fetal bovine serum sold by ATCC, 5 ng/ml of recombinant human epithelial cell factor, and 5 ng/ml of recombinant human fibroblast growth factor. It was cultured in the included mesenchymal stem cell line-only medium (ATCC, PCS-500-030). At the time of cell differentiation, the medium mixed with StemPro medium (Gibco, A10410-01) and the additive (Gibco, A10065-01) provided by the manufacturer was changed and replaced with a new StemPro medium every 3 days for a total of 14 days. Differentiation was induced. While inducing differentiation, ADAMTS1 wild-type or L1 mutant recombinant protein was treated once daily at a concentration of 1 ng/ml or 100 ng/ml.
  • the Oil Red O staining method was used. Specifically, cells that have been differentiated were fixed with 10% formalin and stained with Oil Red O staining solution. After the stained cells were observed under a microscope, the deposited dye was extracted using 100% isopropanol. The extracted dye was quantified by measuring absorbance at a wavelength of 520 nm, and the results are shown in FIGS. 12 and 13 and Table 3.
  • WT 1 (ng/ml) WT 100 (ng/ml) L1 1 (ng/ml) L1 100 (ng/ml) 0.13439 One 0.71642 0.75039 0.67123 0.67372 0.04509 0 0.0447 0.03343 0.15397 0.08999
  • the degree of glucose uptake of the differentiation-induced C2C12 cell line increases under insulin treatment.
  • the C2C12 cell line in which myocyte differentiation was induced by treatment with L1 mutant ADAMTS1 was treated with the wild-type cell line to induce differentiation. It was confirmed that the glucose uptake was further increased compared to .
  • Example 8 Observation of weight recovery in an animal model of muscle loss
  • dexamethasone was administered via intraperitoneal injection at a concentration of 50 mg/kg daily for 12 days.
  • L1 mutant ADAMTS1 was administered by intraperitoneal injection at a concentration of 0.02 ⁇ g/ ⁇ l (Low), 0.2 ⁇ g/ ⁇ l (Mid), and 2 ⁇ g/ ⁇ l (High), respectively. It was administered for a total of 12 days, and the degree of body weight recovery was observed in animal models.
  • Example 9 Observation of TA, GA muscle tissue weight recovery in sarcopenia animal model
  • Example 10 Observation of fat and muscle recovery in muscle loss animal model
  • mice To 12-week-old C57BL/6 mice, dexamethasone was administered by intraperitoneal injection at a concentration of 50 mg/kg every day for 12 days, and at the same time, L1 mutant ADAMTS1 was administered at a concentration of 2 ⁇ g/ ⁇ l by 100 ⁇ l by intraperitoneal injection. After completion of administration, mice were anesthetized through inhalation anesthesia, and muscle mass and body fat mass were analyzed using a dual-energy X-ray absorptiometry (DXA) device.
  • DXA dual-energy X-ray absorptiometry
  • Example 11 Confirmation of increased expression of genes associated with muscle differentiation in an animal model of sarcopenia
  • dexamethasone was administered at a concentration of 50 mg/kg daily for 12 days via intraperitoneal injection, and at the same time, L1 mutant ADAMTS1 was administered at 0.02 ⁇ g/ ⁇ l (Low), 0.2 ⁇ g/ ⁇ l (Mid), and 2 ⁇ g, respectively. It was administered by intraperitoneal injection by 100 ⁇ l at a concentration of / ⁇ l (High).
  • the GA muscle tissue was excised at an autopsy. GA muscle tissue was subjected to homogenization using a pestle in a frozen state, and then RNA was extracted (Qiagen, 74104).
  • the extracted RNA was prepared as cDNA using cDNA synthesis reagent (Promega, A5000).
  • the synthesized cDNA was compared to the relative mRNA expression levels of MyoD, MyoG, Pax7, and MRF4 genes related to muscle differentiation using real-time polymerase chain reaction equipment (Applied Biosystems, Quantstudio3).
  • Example 12 Confirmation of reduced expression of genes associated with inhibition of muscle differentiation in an animal model of muscle loss
  • dexamethasone was administered at a concentration of 50 mg/kg daily for 12 days via intraperitoneal injection, and at the same time, L1 mutant ADAMTS1 was administered at 0.02 ⁇ g/ ⁇ l (Low), 0.2 ⁇ g/ ⁇ l (Mid), and 2 ⁇ g, respectively. It was administered by intraperitoneal injection by 100 ⁇ l at a concentration of / ⁇ l (High).
  • the GA muscle tissue was excised at an autopsy. GA muscle tissue was subjected to homogenization using a pestle in a frozen state, and then RNA was extracted (Qiagen, 74104).
  • cDNA synthesis reagent Promega, A5000.
  • MuRF1, Atrogin1, and Hes1 which are genes related to inhibition of muscle differentiation, were compared using real-time polymerase chain reaction equipment (Applied Biosystems, Quantstudio3).
  • Example 13 Confirmation of increased muscle differentiation in cells (Cancer cachexia model) inducing muscle differentiation inhibition by anticancer agents
  • the C2C12 cell line In a 24-well cell culture plate, the C2C12 cell line, a myoblast cell line, was treated with a cisplatin anticancer drug at a concentration of 50 ⁇ M and a L1 ADAMTS1 mutant recombinant protein at a concentration of 0.1, 1, and 10 ng/ml, respectively, while inducing differentiation for 8 days. Differentiation-induced cells were fixed with methanol for 10 minutes. After sufficient washing with PBS three times, a Zener dyeing reagent dissolved in methanol at a concentration of 2.5 mg/ml was mixed with distilled water 1:1 and dyed for 10 minutes. The stained plate was washed with distilled water, dried and observed under a microscope.
  • the medium was replaced with a serum-free medium and further cultured for 24 hours.
  • the L1 ADAMTS1 mutant protein was treated with 10 ⁇ M of BrdU at concentrations of 0.01, 0.1, 1, 10, and 100 ng/ml, respectively, and reacted for 24 hours.
  • the absorbance was measured at 450 nm using a BrdU fluorescence measurement ELISA kit (Cell signaling technology, 6813S). When the amount of newly synthesized DNA in muscle cells increases, the measured absorbance increases, which can be seen as an increase in muscle cell activity.

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Abstract

La présente invention concerne : un peptide mutant L1 ; une composition destinée à soulager, à inhiber, à prévenir ou à traiter la myopathie, l'obésité ou le diabète, contenant celui-ci utilisé en tant que gradient actif ; une méthode destinée à soulager, à inhiber, à prévenir ou à traiter la myopathie, l'obésité ou le diabète en utilisant le peptide mutant L1 ; et une utilisation, du peptide mutant L1, destinée à soulager, à inhiber, à prévenir ou à traiter la myopathie, l'obésité ou le diabète.
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Citations (3)

* Cited by examiner, † Cited by third party
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US20020090373A1 (en) * 2000-03-22 2002-07-11 Leonard Buckbinder ADAMTS polypeptides, nucleic acids encoding them, and uses thereof
US20030032168A1 (en) * 1997-06-03 2003-02-13 Kunitaka Hirose Human ADAMTS-1 protein, gene encoding the same, pharmaceutical composition, and method for immunologically analyzing human ADAMTS-1 protein
WO2007147497A2 (fr) * 2006-06-17 2007-12-27 Bayer Healthcare Ag Utilisation d'un domaine du type désintégrine et métalloprotéinase à motif de thrombospondine de type 1 (adamts1) comme cible thérapeutique et diagnostique

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US9950043B2 (en) * 2012-11-15 2018-04-24 The Board Of Trustees Of The Leland Stanford Junior University Modulation of muscle and adipocyte distribution and fate

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Publication number Priority date Publication date Assignee Title
US20030032168A1 (en) * 1997-06-03 2003-02-13 Kunitaka Hirose Human ADAMTS-1 protein, gene encoding the same, pharmaceutical composition, and method for immunologically analyzing human ADAMTS-1 protein
US20020090373A1 (en) * 2000-03-22 2002-07-11 Leonard Buckbinder ADAMTS polypeptides, nucleic acids encoding them, and uses thereof
WO2007147497A2 (fr) * 2006-06-17 2007-12-27 Bayer Healthcare Ag Utilisation d'un domaine du type désintégrine et métalloprotéinase à motif de thrombospondine de type 1 (adamts1) comme cible thérapeutique et diagnostique

Non-Patent Citations (2)

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Title
DATABASE Protein 26 June 2022 (2022-06-26), ANONYMOUS : "A disintegrin and metalloproteinase with thrombospondin motifs 1 preproprotein [Homo sapiens]", XP055942839, retrieved from Genbank Database accession no. NP_008919 *
PETERS BAS J.M., RODIN ANDREI S., KLUNGEL OLAF H., STRICKER BRUNO H.CH., DE BOER ANTHONIUS, MAITLAND-VAN DER ZEE ANKE-HILSE: "Variants of ADAMTS1 modify the effectiveness of statins in reducing the risk of myocardial infarction", PHARMACOGENETICS AND GENOMICS, vol. 20, no. 12, 1 December 2010 (2010-12-01), US , pages 766 - 774, XP009537608, ISSN: 1744-6872, DOI: 10.1097/FPC.0b013e328340aded *

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