WO2022131750A1 - Composition for treating or preventing myopathy, obesity or diabetes - Google Patents

Abstract

The present invention relates to: an L1 mutant peptide; a composition for alleviating, inhibiting, preventing or treating myopathy, obesity or diabetes, containing same as an active gradient; a method for alleviating, inhibiting, preventing or treating myopathy, obesity or diabetes by using the L1 mutant peptide; and a use, of the L1 mutant peptide, for alleviating, inhibiting, preventing or treating myopathy, obesity or diabetes.

Description

Composition for treating or preventing muscle disease, obesity or diabetes

This patent application claims priority to Korean Patent Application No. 10-2020-0174844 filed with the Korean Intellectual Property Office on December 14, 2020, the disclosure of which is incorporated herein by reference.

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.

The decrease in human physical activity is a result of lifestyle changes caused by industrialization in modern society, and the seriousness of the negative impact on physical and mental health has been raised for quite some time. Lack of exercise due to decreased physical activity is a major cause of obesity and various lifestyle-related diseases, including metabolic syndrome, resulting in a decrease in muscle mass. Muscle mass in the body plays a very important role in survival, and a decrease in skeletal muscle volume leads to a decrease in basal metabolic rate and a decrease in muscle strength, leading to considerable difficulties in maintaining daily physical activity.

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.

As people age, changes in body composition occur in both men and women, and these changes occur irrespective of changes in body weight. In general, the change in body composition is judged to be due to a change in energy balance according to weight gain. In addition, the decrease in muscle mass gradually progresses from about 30 years of age, and sarcopenia in the elderly has a serious impact on dysfunction, quality of life, medical costs, and the like.

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.

Due to their ease of access in clinical practice, body mass index (BMI) and waist circumference (WHR), which are used as indicators of obesity, have limitations in that they do not reflect well the effects of age, exercise, and changes in body composition due to weight loss. In particular, muscle mass among body composition is reported to be related to metabolic syndrome, and is suggested as a predictor of the cause of all mortality. Recently, as factors more related to insulin resistance, glucose metabolism, lipid concentration and blood pressure in relation to health, there is a need to pay attention to increase in body fat percentage and muscle loss.

Numerous papers and patent documents are referenced throughout this specification and their citations are indicated. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to more clearly describe the level of the technical field to which the present invention pertains and the content of the present invention.

Accordingly, 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.

Accordingly, it is an object of the present invention to provide an L1 mutant peptide.

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.

It is an object of the present invention to provide a pharmaceutical composition for alleviating, suppressing, preventing or treating obesity 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 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.

It is an object of the present invention to provide a pharmaceutical composition for alleviating, inhibiting, preventing or treating diabetes 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 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.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

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

Hereinafter, the present invention will be described in more detail.

According to one aspect of the present invention, 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.

In the present invention, 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.

In a specific embodiment of the present invention, 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

In a specific embodiment of the present invention, 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.

In a specific embodiment of the present invention, 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

In a specific embodiment of the present invention, 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

In a specific embodiment of the present invention, 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.

In a specific embodiment of the present invention, 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.

In a specific embodiment of the present invention, the L1 mutant peptide of the present invention may be a peptide consisting of the amino acid sequence of SEQ ID NO: 2.

According to another aspect of the present invention, the present invention provides a pharmaceutical composition for alleviating, inhibiting or treating muscle disease, comprising the L1 mutant peptide as an active ingredient.

In the present invention, the L1 mutant peptide is the same as described above.

The distribution of body mass in muscle and adipose tissue has a significant impact on an individual's health. It is known that many factors are involved in the balance of adipocyte and muscle cell differentiation. For example, 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.

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.

Adult MuSCs could be transplanted into damaged or defective skeletal muscle to reconstruct muscle fibers and improve function, potentially providing therapeutic applications for MuSCs. However, a major obstacle to translating this technique is the lack of understanding of how differentiation decisions are determined and the lack of developed tools to control and facilitate these decisions of therapeutic benefit. Advancement in these areas could lead to a variety of new treatments for individuals with impaired muscle mass or function, or to address imbalances between muscle and adipose tissue.

In the present invention, 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.

The name 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. In early 2017, the World Health Organization (WHO) 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. Among the causes of 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. Similarly, in an obese environment, the generation of reactive oxygen species that induce oxidative stress increases, and 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.

According to another aspect of the present invention, 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.

According to another aspect of the present invention, the present invention relates to a composition for alleviating, suppressing, preventing, or treating obesity comprising an L1 mutant peptide as an active ingredient.

In the present invention, the L1 mutant peptide is the same as described above.

As used herein, 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). Bone marrow-derived mesenchymal stem cells (BM-MSCs) 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.

According to another aspect of the present invention, 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.

According to another aspect of the present invention, the present invention provides a composition for alleviating, inhibiting, preventing, or treating diabetes comprising an L1 mutant peptide as an active ingredient.

In the present invention, the L1 mutant peptide is the same as described above.

As used herein, the term 'diabetes' refers to a chronic disease characterized by a relative or absolute lack of insulin resulting in glucose-intolerance. The term 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 β-cells. Type 2 diabetes is non-insulin-dependent diabetes mellitus, caused by insufficient insulin secretion after a meal or by insulin resistance.

According to another aspect of the present invention, 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.

As used herein, 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.

The 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.

The 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.

The 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.

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.

In the case of formulation, diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants may be used. 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. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

Formulations for oral use include suspensions, solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, 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.

As the preparation of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like can be used.

In the embodiment of applying the composition of the present invention to humans, 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.

For example, 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).

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.

For example, based on the active ingredient content, 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.

If 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

12 is a diagram showing the differentiation result of adipose-derived mesenchymal stem cells into adipocytes according to an embodiment of the present invention.

13 is a graph showing the result of confirming the degree of adipogenesis according to an embodiment of the present invention.

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.

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.

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.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

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 μg/ml. It was cultured in a cell incubator at 37° C. supplied with 5% CO ₂ as a medium for use.

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. When the cells in the cell culture plate reached a density of 90%, they were replaced with 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.

Example 2. Production of Recombinant Proteins

For the isolation and purification of the recombinant protein produced in the cell, an ADAMTS1 overexpression vector was constructed so that 6x His was placed at the C-terminus of the recombinant protein. Specifically, the pEF6/V5-His A vector (Invitrogen, V96120) was digested using KpnI and XhoI restriction enzymes. Here, a sequence recognized as 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 μg/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. After culturing for 24 hours, the cells were diluted 10-fold, transferred to a 100 mm cell culture dish, and then blasticidin-S (Blasticidin-S ^TM ) was first treated to a concentration of 10 μg/ml. Thereafter, blasticidin-S was mixed and replaced in a new culture medium to a concentration of 10 μg/ml over a total of 3 times at 4-day intervals. After independent colonies were formed by blasticidin-S, 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.

Example 3. Confirmation of recombinant protein expression

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.

As can be seen in 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. On the other hand, the remaining mutations except for the L1 mutation showed a significant decrease in protein expression (see FIG. 1a ).

Example 4. Jenner's staining

Differentiation was induced in the C2C12 cell line in the same manner as in Example 3, and the differentiated cell line was 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, and the results are shown in FIGS. 2 to 6 .

As can be seen in FIGS. 2 to 6 , it was confirmed that the length of the myotube was increased when ADAMTS1 was treated with the C2C12 cell line when differentiation was induced. It was confirmed that the length of the O-tube was increased and the thickness was increased.

Example 5. Immunofluorescence staining

In a 24-well cell culture plate, 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.

Then, after blocking with 2% BSA for 30 minutes, myosin heavy chain (MHC) antibody was mixed with 2% BSA buffer at a ratio of 1:500 and treated for 1 hour. Then, 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 .

division	ng/ml	average (um)	Sd
Ctrl	-	707.937	119.37
WT	0.01	1354.07	168.904
	One	1498.86	48.3997
	100	1558.95	117.736
L1 mutation	0.01	1618.18	136.365
	One	1676.78	111.361
	100	1750.25	126.443
L3 mutation	0.01	1311.13	217.574
	One	1253.49	204.303
	100	1198.5	168.134
L4 mutation	0.01	1128.15	89.4677
	One	1170.02	70.0004
	100	1129.45	69.5174

	ng/ml	average (um)	Sd
Ctrl	-	31.4956	5.74572
WT	0.01	40.0933	6.15254
	One	44.0056	4.55134
	100	42.46	4.14553
L1 mutation	0.01	40.2333	5.33697
	One	47.04	6.05918
	100	47.6722	3.60209
L3 mutation	0.01	33.54	4.98323
	One	37.4922	7.18262
	100	38.2789	6.7859
L4 mutation	0.01	36.5778	3.06914
	One	31.7889	5.00286
	100	31.8011	5.26334

As can be seen in FIGS. 7 to 11 and Tables 1 to 2, compared to the C2C12 cell line in which differentiation was induced by treatment with a differentiation medium, 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.

Example 6. Confirmation of adipogenesis level

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.

In order to confirm the degree of adipogenesis of differentiation-induced adipose-derived mesenchymal stem cells, 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.

Diff. -	Diff. +	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

As can be seen in FIGS. 12 and 13 and Table 3, it was confirmed that both the wild-type and L1 mutants of ADAMTS1 inhibit the differentiation of adipose-derived mesenchymal stem cells into adipocytes.

Example 7. Measurement of uptake of intracellular glucose

Cells that change according to the activity of increasing muscle differentiation using 2-NBDG (2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino-2-deoxygluocose), a fluorescent glucose analogue) Glucose uptake was measured.C2C12 cell line was treated with ADAMTS1 or L1 mutant recombinant protein at a concentration of 100 ng/ml, respectively, once daily while inducing differentiation for 8 days.The differentiation-induced cells were treated with DMEM medium without glucose 2 After treatment with 100 nM insulin for 15 minutes, 2-NBDG was added to a final concentration of 100 μg/ml, glucose uptake, excitation 485 nm, emission 535 It was measured in nm, and the results are shown in FIG. 14 and Table 4.

division	Ins-			Ins+
	-	WT	L1	-	WT	L1
#One	One	1.30543	1.604692	1.703563	1.912419	2.461015
#2	0.896475	1.33754	1.913586	1.634735	1.954727	2.110051
#3	0.982087	1.272934	1.61764	1.693908	1.797068	2.530066
#4	0.890083	1.294861	1.593617	1.664756	1.831639	2.46592
Mean	0.942161	1.302691	1.682383	1.674241	1.873963	2.391763
SD	0.056976	0.026887	0.154447	0.031077	0.072355	0.190425

As can be seen in Figure 14 and Table 4, the degree of glucose uptake of the differentiation-induced C2C12 cell line increases under insulin treatment. In particular, 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

To 12-week-old C57BL/6 mice, dexamethasone was administered via intraperitoneal injection at a concentration of 50 mg/kg daily for 12 days. At the same time, L1 mutant ADAMTS1 was administered by intraperitoneal injection at a concentration of 0.02 μg/μl (Low), 0.2 μg/μl (Mid), and 2 μg/μl (High), respectively. It was administered for a total of 12 days, and the degree of body weight recovery was observed in animal models.

As can be seen in Figure 15, in the case of the animal model inducing muscle loss by administration of dexamethasone, a clear weight loss was confirmed compared to the normal control group, and when the L1 mutant ADAMTS1 was treated in the animal model, Low, Mid and High dose groups In all, it was confirmed that the body weight was recovered significantly, and it was observed that the body weight was recovered depending on the dose of the administered L1 mutant ADAMTS1.

Example 9: Observation of TA, GA muscle tissue weight recovery in sarcopenia animal model

In order to confirm the change in muscle tissue weight in a sarcopenic animal model in which dexamethasone was administered to 12-week-old C57BL/6 mice for 12 days, an autopsy was performed after administration to extract TA and GA muscle tissue. The extracted muscle tissue was compared by measuring the weight.

As can be seen in FIG. 16 , in the case of the animal model induced by dexamethasone administration, a significant decrease in tissue weight was confirmed in both the TA muscle and the GA muscle compared to the normal control group, and the L1 mutant ADAMTS1 was treated in the muscle reduction induced animal model. In one case, it was confirmed that the weight of TA muscle and GA muscle tissue was significantly recovered in the Mid and High dose groups.

Example 10: Observation of fat and muscle recovery in muscle loss animal model

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 μg/μl by 100 μl 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.

As can be seen in FIG. 17 , in the case of an animal model in which muscle loss was induced by administration of dexamethasone, both fat and muscle were significantly reduced. When the L1 mutant ADAMTS1 was treated in the muscle loss induced animal model, it was confirmed that both fat and muscle were significantly recovered. In particular, it was confirmed that the muscle was restored to a normal level.

Example 11: Confirmation of increased expression of genes associated with muscle differentiation in an animal model of sarcopenia

To 12-week-old C57BL/6 mice, 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 μg/μl (Low), 0.2 μg/μl (Mid), and 2 μg, respectively. It was administered by intraperitoneal injection by 100 μl at a concentration of /μl (High). After the administration of dexamethasone and L1 mutant ADAMTS1 was completed, 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).

As can be seen in FIG. 18 , when L1 mutant ADAMTS1 was treated with respect to the dexamethasone treatment-induced muscle loss induction animal model, the expression levels of MyoD, MyoG, Pax7, and MRF4 genes were all reduced, and in the Low and High dose groups It was confirmed that all gene expression was significantly increased compared to the dexamethasone treatment group in a dose-dependent manner.

This shows that the L1 mutant ADAMTS1 increases muscle differentiation by increasing the expression of genes associated with muscle differentiation.

Example 12: Confirmation of reduced expression of genes associated with inhibition of muscle differentiation in an animal model of muscle loss

To 12-week-old C57BL/6 mice, 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 μg/μl (Low), 0.2 μg/μl (Mid), and 2 μg, respectively. It was administered by intraperitoneal injection by 100 μl at a concentration of /μl (High). After the administration of dexamethasone and L1 mutant ADAMTS1 was completed, 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). For the synthesized cDNA, the relative mRNA expression levels of 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).

As can be seen in Figure 19, when L1 mutant ADAMTS1 was treated with respect to the dexamethasone treatment-induced muscle loss inducing animal model, the expression of MuRF1, Atrogin1, Hes1 genes was significantly reduced in both the Low dose group and the High dose group. did. This shows that the L1 mutant ADAMTS1 can increase muscle differentiation by suppressing the genes associated with the inhibition of muscle differentiation.

Example 13: Confirmation of increased muscle differentiation in cells (Cancer cachexia model) inducing muscle differentiation inhibition by anticancer agents

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 μM 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.

As can be seen in FIG. 20 , for a cancer cachexia cell model in which muscle differentiation was inhibited by treatment with cisplatin, when L1 mutant ADAMTS1 was treated, the length of the myotube, which was reduced, was significantly increased in a dose-dependent manner. .

Example 14: Confirmation of increased activity of primary muscle cells

After culturing the primary muscle cells in a 96-well plate for 24 hours, the medium was replaced with a serum-free medium and further cultured for 24 hours. Then, the L1 ADAMTS1 mutant protein was treated with 10 μM of BrdU at concentrations of 0.01, 0.1, 1, 10, and 100 ng/ml, respectively, and reacted for 24 hours. Then, 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.

As can be seen in FIG. 21 , it was confirmed that the activity of the primary muscle cells was significantly increased in a dose-dependent manner when the L1 mutant ADAMTS1 was treated.

As the specific parts of the present invention have been described in detail above, for those of ordinary skill in the art, these specific descriptions are only preferred embodiments, and it is clear that the scope of the present invention is not limited thereto.

Claims (12)

1. L1 mutant ADAMTS1 (A Disintegrin and Metalloprotease with ThromboSpondin motifs) 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.
2. According to claim 1, wherein the L1 mutant peptide is a peptide consisting of the amino acid sequence of SEQ ID NO: 1, histidine at position 295, proline at position 296, proline at position 296, serine at position 297, iso at position 298 Leucine (Isoleucine), position 299 arginine (Arginine), and position 300 asparagine (Asparagine) that comprises a deletion of amino acids at one or more positions selected from the group consisting of, the peptide.
3. The peptide of claim 1, wherein the L1 mutant peptide comprises a deletion of amino acids at positions 295 to 300 of the peptide consisting of the amino acid sequence of SEQ ID NO: 1.
4. The peptide of claim 1, wherein the L1 mutant peptide further comprises 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.
5. The peptide of claim 1, wherein the L1 mutant peptide further comprises 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.
6. According to claim 1, wherein the L1 mutant peptide further comprises a deletion of amino acids 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 Phosphorus, peptide.
7. The peptide of claim 1, wherein the L1 mutant peptide further comprises a deletion of amino acids at positions 1 to 252 of the peptide consisting of the amino acid sequence of SEQ ID NO: 1.
8. According to claim 7, wherein the L1 mutant peptide is a peptide consisting of the amino acid sequence of SEQ ID NO: 2, the peptide.
9. A pharmaceutical composition for alleviating, inhibiting, preventing or treating muscle disease comprising the peptide of any one of claims 1 to 8.
10. 10. The method of claim 9, wherein the muscle disease is sarcopenia, muscular atrophy (Amyotrophy), cancer cachexia (Cancer cachexia), muscle damage, muscular dystrophy, cardiac atrophy, dystonia (atony), muscular dystrophy (muscular dystrophy) , which will be selected from the group consisting of muscle degeneration and myasthenia gravis, a pharmaceutical composition for alleviating, inhibiting, preventing or treating muscle disease.
11. A pharmaceutical composition for alleviating, suppressing, preventing or treating obesity comprising the peptide of any one of claims 1 to 8.
12. A pharmaceutical composition for alleviating, inhibiting, preventing or treating diabetes, comprising the peptide of any one of claims 1 to 8.

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