Peptides having muscle loss inhibitory and muscle mass increasing activity, and their uses

Abstract

The present application relates to a peptide having muscle loss inhibitory and muscle mass increasing activities, and uses thereof, a peptide consisting of the amino acid sequence of SEQ ID NO: 1, a pharmaceutical composition for preventing or treating muscle diseases containing the peptide as an active ingredient, and a food composition for preventing or improving muscle diseases containing the peptide as an active ingredient are provided.

Description

Technical Field

[0001] The present invention relates to a peptide having muscle loss inhibitory and muscle mass enhancing activities, and uses thereof.

[0002] This application claims priority to Korean Patent Application No. 10-2022-0071033, filed on June 10, 2022, the disclosure of which is incorporated herein by reference.

Background Art

[0003] Recently, as the elderly population is increasing rapidly worldwide, according to a report by the United Nations, it is predicted that the population aged 60 or over will exceed 2 billion by 2050. Skeletal muscle is the largest organ reaching 50% of the total body weight, and muscle reduction and muscle strength weakening are one of the major physical changes caused by aging. Thereby, skeletal muscle tends to decrease by about 1% per year from the human body after the age of 30, and decreases rapidly after the age of 65. Such a rapid reduction in muscle has a great impact on the quality of life such as reducing physical ability and increasing the risk of falls and fractures, and reflecting such concerns, in 2017, the World Health Organization (WHO) recognized sarcopenia as an official disease.

[0004] Myofascial pain syndrome, induced by the degeneration of spinal nerves, motor nerves, or skeletal muscle fibers associated with muscle diseases, is one of the representative intractable diseases whose cause remains unknown. Previous research has shown that in myofascial pain syndrome, motor nerves that induce skeletal muscle contraction degenerate, preventing the progression of skeletal muscle contraction, or that the expression of proteins involved in muscle contraction within the skeletal muscle is reduced, or that these proteins are deformed, preventing the progression of normal skeletal muscle contraction. In the long term, the aforementioned motor nerves or skeletal muscle are deformed into fibrous tissue. Because the fundamental cause of such myofascial pain syndrome has not yet been elucidated, and no methods have been developed to prevent or reverse the degeneration of motor nerves or skeletal muscle, research is currently actively underway to develop methods to slow the progression of myofascial pain syndrome. Currently, exercise, protein, and calorie supplementation are known to be helpful for myofascial pain syndrome, but these are not very effective for the elderly, who make up the majority of myofascial pain syndrome patients, and there is a pressing need for myofascial pain syndrome treatments.

[0005] Muscle size, or muscle mass, is regulated by intracellular signaling processes that induce anabolic and catabolic reactions within the muscle. Specifically, when signaling reactions that induce muscle protein synthesis are dominant over muscle protein breakdown, muscle protein synthesis increases, which manifests as increased muscle size (hypertrophy) or an increase in muscle fiber number (hyperplasia). Furthermore, muscle cell differentiation and muscle formation are regulated by various muscle regulatory factors. MyoD initiates the expression of muscle-specific genes, inducing the differentiation of muscle satellite cells into myoblasts. The induction of myogenin expression by MyoD activity is the most important element in myoblast fusion and is involved in the formation of myotubes. Muscle fibers formed through such processes bundle together and ultimately form muscle.

[0006] Against this technological backdrop, multifaceted research is underway to inhibit muscle loss caused by external or intrinsic factors and to promote muscle protein synthesis (Korean Patent Registration No. 10-2064387), but the reality is that it is still insufficient. [Overview of the project] [Problems that the invention aims to solve]

[0007] One embodiment provides a peptide comprising the amino acid sequence of SEQ ID NO: 1.

[0008] Another embodiment provides a muscle loss inhibitory composition and a muscle mass increasing composition containing a peptide having the amino acid sequence of Sequence ID No. 1 as an active ingredient.

[0009] Other purposes and advantages of this application will become clearer from the detailed description below, along with the attached claims and drawings. Any matters not described herein are readily apparent and can be inferred by any person skilled in the art of this application or a similar art, and therefore are omitted from this description. [Means for solving the problem]

[0010] Each description and embodiment disclosed in this application may also apply to each other description and embodiment. That is, all combinations of the diverse elements disclosed in this application fall within the scope of this application. Furthermore, the specific descriptions described below cannot be considered to limit the scope of this application.

[0011] One embodiment provides a peptide comprising the amino acid sequence of SEQ ID NO: 1.

[0012] As used herein, the term "peptide" may mean a linear molecule formed by the bonding of amino acid residues to one another via peptide bonds. Such peptides can be produced by chemical synthesis methods known in the art, particularly by solid-phase synthesis or liquid-phase synthesis (U.S. Patent No. 5,516,891). The inventors have made diligent efforts to develop a peptide with biologically effective activity and have identified a peptide consisting of the amino acid sequence of Sequence ID No. 1. Here, the biologically effective activity also exhibits one or more of the following characteristics selected from (a) increased expression of Myo G (myogenin) and (Myf5: myogenic factor 5), which are early differentiation markers for myoblasts; (b) increased expression of MyHC (myosin heavy chain) and α-actinin, which are late differentiation markers for myoblasts or muscle fiber constituent proteins; and (c) decreased expression of inflammatory proteins α-SMA (α-smooth muscle actin) and IL-6 (interleukin-6). Therefore, the peptide can be used for the prevention, improvement, or treatment of muscle diseases.

[0013] The peptide may also have a protecting group attached to its N-terminus or C-terminus to acquire chemical stability, enhanced pharmacological properties (half-life, water absorption, potency, efficacy, etc.), altered specificity (e.g., broad biological activity spectrum), or reduced antigenicity. In one specific example, the N-terminus of the peptide may be bound to one protecting group selected from the group consisting of acetyl, fluorenylmethoxycarbonyl, formyl, palmitoyl, myristyl, stearyl, butoxycarbonyl, allyloxycarbonyl, and polyethylene glycol (PEG), and / or the C-terminus of the peptide may be bound to one protecting group selected from the group consisting of amino (-NH2), tertiary alkyl, and azide (-NHNH2). The peptide may also selectively include a targeted sequence, a tag, labeled residues, or an amino acid sequence manufactured for a specific purpose to increase half-life or peptide stability.

[0014] The peptides are artificially synthesized, non-naturally occurring, or engineered, where "non-naturally occurring or engineered" means a state produced by artificial modification rather than the state in which they naturally occur. Here, the artificial modification may include mimicking multiple amino acid structures to artificially synthesize amino acid sequences, or, as described above, being engineered to acquire chemical stability, enhanced pharmacological properties, altered specificity, or reduced antigenicity.

[0015] As used herein, the term “stability” may refer not only to in vivo stability, which protects the peptide from attack by endogenous protein-cleaving enzymes, but also to storage stability (e.g., room temperature storage stability).

[0016] Another embodiment provides a pharmaceutical composition for the prevention or treatment of muscle diseases, comprising a peptide having the amino acid sequence of Sequence ID No. 1 as an active ingredient.

[0017] As stated above, any terms or elements mentioned in the description of the peptides that are the same as those already mentioned are as described above.

[0018] In this specification, the term "prevention" means all actions that suppress or delay the onset of a disease by administering the composition.

[0019] In this specification, the term “treatment” means any form of treatment that provides an effect to an individual suffering from or at risk of developing a disease, including improvement of the individual’s condition (e.g., one or more symptoms), delay of disease progression, delay of symptom onset, or slowing of symptom progression. Accordingly, the terms “treatment” and “prevention” as used herein are not intended to mean a cure or complete elimination of symptoms.

[0020] In this specification, the term "muscle disease" refers to a disease, illness, or condition of the muscle associated with reduced proliferation or differentiation of myoblasts, and also collectively to muscle diseases resulting in, for example, muscle dysfunction, muscle wasting, or muscle degeneration. The muscle diseases include, but are not limited to, sarcopenia, atony, muscular dystrophy, muscular atrophy, muscle degeneration, myoricosis, amyotrophic lateral sclerosis, myasthenia gravis, and cachexia.

[0021] In one specific example, the pharmaceutical composition according to one example may increase muscle mass or muscle strength, or improve muscle function, by promoting the proliferation and differentiation of myoblasts.

[0022] Conventional functional peptides, despite their effective biological activity, suffer from disadvantages such as being unable to effectively enter target tissues or target cells due to their size, or being eliminated from the body in a short period due to their short half-life. On the other hand, the pharmaceutical composition according to one example contains a peptide consisting of approximately 10 or fewer amino acids as an active ingredient, and as a result, it has excellent cell penetration rate for the active ingredient, and for example, when administered topically, it can provide an effective therapeutic effect on muscle diseases.

[0023] According to one example, the peptide of the present invention can significantly increase the expression of early differentiation markers (e.g., Myo G and Myf5) and late differentiation markers of myoblasts, or muscle fiber constituent proteins (e.g., myosin heavy chain, α-actinin), thereby promoting differentiation into muscle cells or muscle fibers. Furthermore, the peptide of the present invention can not only improve muscle mass recovery / regeneration in the event of muscle damage caused by exogenous factors, but also infiltrate immunotherapy cells into damaged muscle tissue, reduce the accumulation of collagen tissue, alleviate the symptoms of muscle diseases, and inhibit disease progression. The peptide can be utilized as an active ingredient in pharmaceutical compositions for the treatment of muscle diseases.

[0024] The pharmaceutical composition may also, but is not limited to, contain a pharmaceutically effective amount of the peptide and / or a pharmaceutically acceptable carrier.

[0025] As used herein, the term "pharmaceutical effective dose" may mean an amount sufficient to achieve the therapeutic efficacy of the pharmaceutical composition for bone disease.

[0026] The weight ratio of the peptide to the pharmaceutically acceptable carrier is, for example, also 500:1 to 1:500. As an example, the weight ratio is 450:1 to 1:450, 400:1 to 1:400, 350:1 to 1:350, 300:1 to 1:300, 250:1 to 1:250, 200:1 to 1:200, 150:1 to 1:150, 100:1 to 1:100, 80:1 to 1:80, 60:1 to 1:60, 40:1 to 1:40, 20:1 to 1:20, 10:1 to 1:10, 8:1 to 1:8, 6:1 to 1:6, 4:1 to 1:4, or 2:1 to 1:2, but is not limited thereto.

[0027] The pharmaceutically acceptable carrier is one generally used in the manufacture of pharmaceuticals and includes, but is not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0028] The pharmaceutical composition may also contain, in addition to the above components, lubricants, wetting agents, sweeteners, flavoring agents, emulsifying agents, suspending agents, preservatives, etc., but is not limited thereto.

[0029] The pharmaceutical composition may be administered orally or parenterally. In the case of parenteral administration, it may be administered by intramuscular injection, intravenous injection, subcutaneous injection, intraperitoneal injection, topical administration, transdermal administration, etc., but is not limited thereto.

[0030] The dosage of the aforementioned pharmaceutical composition may be 0.0001 to 1,000 μg, 0.001 to 1,000 μg, 0.01 to 1,000 μg, 0.1 to 1,000 μg, or 1.0 to 1,000 μg per day, but is not limited to these values. It can be administered in various ways depending on factors such as the formulation method, administration method, the patient's age, weight, sex, medical condition, diet, administration time, route of administration, excretion rate, and response sensitivity.

[0031] The pharmaceutical composition may be manufactured in unit dose form or encapsulated in a multi-dose container by formulating it using pharmaceutically acceptable carriers and / or excipients in a manner readily available to a person with ordinary skill in the art to which the invention pertains.

[0032] The dosage form may be a solution in oil or an aqueous medium, in the form of a suspension or emulsion, or in the form of an ointment, cream, gel, transdermal absorbent, cataplasm, patch, paste, extract, powder, granule, tablet or capsule, and may also contain a dispersant and / or stabilizer.

[0033] The peptide may be contained in nanosomes or nanoparticles, for example, to further improve cell permeability or stability. For example, the nanosomes may be manufactured using lecithin as a raw material by a microfluidizer and contained, for example, within lecithin particles. Any known method for producing the nanosomes may be used. The size of the nanosome particles may be 10 to 200 nm, for example, 10 to 180 nm, 10 to 160 nm, 10 to 140 nm, 10 to 120 nm, 10 to 100 nm, 10 to 80 nm, 10 to 60 nm, 10 to 40 nm, 10 to 20 nm, 50 to 200 nm, 50 to 180 nm, 50 to 160 nm, 50 to 140 nm, 50 to 120 nm, 50 to 100 nm, 50 to 80 nm, or 50 to 60 nm.

[0034] The pharmaceutical composition may also include additional components beneficial for the prevention or treatment of muscle diseases, the components being, for example, one selected from vitamin C, vitamin B1, vitamin B2, vitamin B6, vitamin H, vitamin PP, provitamin B5, vitamin A, vitamin D, vitamin E, vitamin K1, or carotene, or a mixture thereof.

[0035] Another embodiment provides a food composition for the prevention or improvement of muscle diseases, comprising a peptide having the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0036] Of the terms or elements mentioned in the above-mentioned descriptions of peptides and pharmaceutical compositions, those that are the same as those already mentioned are as stated above.

[0037] In this specification, the term "improvement" may mean all actions that at least reduce parameters related to the alleviation or treatment of a condition, such as the severity of symptoms.

[0038] In one specific example, the food composition according to one embodiment may increase muscle mass or muscle strength, or improve muscle function, by promoting the proliferation and differentiation of myoblasts.

[0039] The food composition may be used by adding the peptide as is or in combination with other foods or food ingredients, and can be used appropriately by conventional methods. The food composition may also contain food-grade food additives in addition to the active ingredient, and the amount of the active ingredient may be appropriately determined depending on the intended use (preventive, health-promoting, or therapeutic treatment).

[0040] The food composition may also contain additional components beneficial for the prevention or improvement of muscle diseases, the components being, for example, one selected from vitamin C, vitamin B1, vitamin B2, vitamin B6, vitamin H, vitamin PP, provitamin B5, vitamin A, vitamin D, vitamin E, vitamin K1, or carotene, or a mixture thereof.

[0041] In this specification, the term "food additive" means a component that can be added to food as an auxiliary, and is added to manufacture health functional foods in each dosage form, and can be appropriately selected and used by those skilled in the art. Examples of such food additives include various nutritional supplements, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, colorants and fillers, pectin and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, and carbonating agents used in carbonated beverages, but the types of such food additives are not limited by the examples given above.

[0042] The aforementioned food composition may also include a health functional food. In this specification, the term "health functional food" refers to a food manufactured and processed in the form of tablets, capsules, powders, granules, liquids, and pills using raw materials and ingredients that have functional properties useful to the human body. Here, "functional" means that it modulates nutrients or obtains effects useful for health purposes, such as physiological effects, on the structure and function of the human body. The health functional food can be manufactured by methods commonly used in the ordinary art, and during the aforementioned manufacturing, raw materials and ingredients commonly added in the ordinary art may be added. Furthermore, the dosage form of the health functional food can be manufactured without restriction, as long as it is a dosage form recognized as a health functional food. The aforementioned food composition can be manufactured in a variety of dosage forms, and unlike general medicines, it has the advantage of being made from food as a raw material and not having side effects that can occur with long-term use of medicines, and is highly portable. A health functional food according to one embodiment can be taken as an adjunct to enhance the therapeutic effect of muscle diseases.

[0043] Furthermore, there are no restrictions on the types of health foods to which the composition according to one embodiment may be used / applied. Food compositions containing the peptide as an active ingredient may be produced by mixing it with other suitable auxiliary ingredients that may be contained in health functional foods and known additives, as can be selected by those skilled in the art. Examples of foods to which it may be added include meats, sausages, bread, chocolates, candies, snacks, confectionery, pizzas, ramen and other noodles, gums, dairy products including ice cream, various soups, drinking water, tea, beverages, alcoholic beverages, and vitamin complexes. In addition, it may be added to soups, teas, jellies and juices, etc.

[0044] Another embodiment provides a method for preventing or treating a muscle disease, comprising the step of administering to an individual a composition containing a peptide having the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0045] Regarding the terms or elements mentioned in the descriptions of peptides, pharmaceutical compositions, food compositions, etc., those that are the same as those already mentioned are as stated above.

[0046] In this specification, the terms “administer” and “apply” are used interchangeably and may mean providing a given substance to an individual or patient by any suitable method. The terms may also mean bringing a composition according to one embodiment to at least partial localization to a desired site, or placing a composition according to one embodiment within an individual via a route of administration.

[0047] In this specification, the term "individual" means an object requiring improvement of its skin condition, and more specifically, a primate, whether human or non-human, or a mammal such as a mouse, dog, cat, horse, or cow.

[0048] The composition is, for example, a pharmaceutical composition or food composition containing a peptide having the amino acid sequence of Sequence ID No. 1 as an active ingredient.

[0049] The content of the peptide as an active ingredient contained in the composition can be appropriately and non-restrictively selected depending on the form of the food / pharmaceutical, the desired use, etc., and can be added in amounts of, for example, 0.01 to 15% by weight of the total composition weight. For example, in a health beverage composition, it can be added in a ratio of 0.02 to 10 g, preferably 0.3 to 1 g, based on 100 ml, but is not limited to these amounts. [Effects of the Invention]

[0050] According to one embodiment of the peptide, it may be possible to improve the expression of factors involved in myoblast differentiation and promote differentiation into muscle cells or muscle fibers.

[0051] According to one embodiment of the peptide, when muscle damage is caused by exogenous factors, it not only improves muscle mass recovery / regeneration, but also infiltrates immunotherapy cells into the damaged muscle tissue, reduces the accumulation of collagen tissue, alleviates the symptoms of muscle diseases, and inhibits disease progression.

[0052] Therefore, a peptide according to one embodiment can be used as an active ingredient in compositions for the prevention, improvement, or treatment of muscle diseases. [Brief explanation of the drawing]

[0053] [Figure 1] This is the result of adding a peptide consisting of the amino acid sequence of Sequence ID No. 1 to C2C12 cells, and then confirming the increased expression of myogenin and Myf5, which are early differentiation markers for myoblasts, via Western blotting. [Figure 2] The following shows the quantitative evaluation of changes in the expression of early differentiation markers of myoblasts after adding a peptide consisting of the amino acid sequence of Sequence ID No. 1 to C2C12 cells. (a) shows the results of confirming the expression level of myogenin, and (b) shows the results of confirming the expression level of Myf5. [Figure 3]This is the result of adding a peptide consisting of the amino acid sequence of Sequence ID No. 1 to C2C12 cells, and then confirming the expression pattern of Myo G (myogenin), an early differentiation marker for myoblasts, via immunofluorescence. [Figure 4] This is the result of quantitatively evaluating the expression level of Myo G (myogenin) after adding a peptide consisting of the amino acid sequence of Sequence ID No. 1 to C2C12 cells. [Figure 5] This is the result of adding a peptide consisting of the amino acid sequence of Sequence ID No. 1 to C2C12 cells, and then confirming the increased expression of MyHC, a marker for late differentiation of myoblasts, via Western blotting. [Figure 6] This study quantitatively evaluated the changes in the expression of late differentiation markers in myoblasts after adding a peptide consisting of the amino acid sequence of Sequence ID No. 1 to C2C12 cells, and confirmed the expression level of MyHC. [Figure 7] This is the result of adding a peptide consisting of the amino acid sequence of Sequence ID No. 1 to C2C12 cells, and then confirming the expression pattern of α-actinin, a late differentiation marker for myoblasts, via immunofluorescence. [Figure 8] This is the result of quantitatively evaluating the expression level of α-actinin after adding a peptide consisting of the amino acid sequence of SEQ ID NO: 1 to C2C12 cells. [Figure 9] This is the result of visually confirming changes in TA muscle following treatment with a peptide consisting of the amino acid sequence of SEQ ID NO: 1 in an animal model in which muscle damage was induced by BaCl2. [Figure 10] In an animal model in which muscle damage was induced by BaCl2, the change in muscle mass of TA muscle was quantitatively evaluated by treatment with a peptide consisting of the amino acid sequence of SEQ ID NO: 1. (a) shows the result of confirming the weight (g) of TA muscle, and (b) shows the result of confirming the percentage of TA muscle. [Figure 11]This study confirmed, via H&E staining, changes in the immunocell infiltration area following treatment with a peptide consisting of the amino acid sequence of SEQ ID NO: 1 in muscle tissue derived from an animal model in which muscle damage was induced by BaCl2. [Figure 12] This study quantitatively evaluated changes in the immune cell infiltration area in muscle tissue derived from an animal model in which muscle damage was induced by BaCl2, following treatment with a peptide consisting of the amino acid sequence of SEQ ID NO: 1. [Figure 13] This study confirmed, via Sirius Red staining, changes in collagen accumulation levels in muscle tissue derived from an animal model in which muscle damage was induced by BaCl2, following treatment with a peptide consisting of the amino acid sequence of SEQ ID NO: 1. [Figure 14] This study quantitatively evaluated the changes in collagen accumulation levels in muscle tissue derived from an animal model in which muscle damage was induced by BaCl2, following treatment with a peptide consisting of the amino acid sequence of SEQ ID NO: 1. [Figure 15] This study, conducted via Western blotting, confirmed increased expression of MyHC and α-actin, muscle fiber constituent proteins, in muscle tissue derived from an animal model in which muscle damage was induced by BaCl2, following treatment with a peptide consisting of the amino acid sequence of SEQ ID NO: 1. [Figure 16] This study quantitatively evaluated the expression levels of MyHC and α-actin, muscle fiber constituent proteins, in muscle tissue derived from an animal model in which muscle damage was induced by BaCl2, after treatment with a peptide consisting of the amino acid sequence of SEQ ID NO: 1. [Figure 17] This study, conducted via Western blotting, confirmed a reduction in the expression of the inflammatory proteins α-SMA and IL-6 in muscle tissue derived from an animal model in which muscle damage was induced by BaCl2, following treatment with a peptide consisting of the amino acid sequence of SEQ ID NO: 1. [Figure 18]This study quantitatively evaluated the expression levels of the inflammatory proteins α-SMA and IL-6 in muscle tissue derived from an animal model in which muscle damage was induced by BaCl2, after treatment with a peptide consisting of the amino acid sequence of SEQ ID NO: 1. [Modes for carrying out the invention]

[0054] The present invention will be described in more detail below with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

[0055] Example 1. Synthesis of peptides Using an automated peptide synthesizer (Milligen 9050 (Millipore, USA)), peptides having the amino acid sequence of SEQ ID NO: 1, as shown in Table 1 below, were synthesized. These synthesized peptides were then separated into pure molecules using C18 reversed-phase high-performance liquid chromatography (HPLC) (Waters Associates, USA). The column used was ACQUITY UPLC BEH300 C18 (2.1 mm x 100 mm, 1.7 μm (Waters Co, USA)).

[0056] [Table 1]

[0057] Example 2. Confirmation of the effect of promoting myoblast differentiation in the early differentiation stage. In this example, we evaluate the changes in the expression of early differentiation marker proteins in mouse myoblasts (C2C12 cells) to confirm the effect of the peptide from this example on the differentiation from myoblasts to muscle cells / muscle fibers during the early differentiation stage.

[0058] 2-1. Evaluation via protein expression analysis C2C12 cells were cultured in DMEM medium containing 1% P / S and 2% BCS. When the cell confluency reached approximately 70-80%, the cells were seeded into 6-well culture plates. Subsequently, when the cell confluency of the cells in the 6-well culture plates reached 100%, the culture medium was replaced with differentiation medium (1% P / S, 2% horse serum), and 5, 50, or 100 μg / ml of the peptide of SEQ ID NO: 1 was added. Thereafter, the same amount of peptide of SEQ ID NO: 1 as the differentiation medium was added every two days. Three days after the first medium change, lysis buffer was added to the culture to lysate it, and the culture was centrifuged at 4°C and 12,000 rpm for 30 minutes to obtain the protein, which was then quantified using a BCA kit. SDS-PAGE was performed on the samples containing the protein, and after separating each protein, it was transferred to a membrane by electrophoresis. The membranes to which the aforementioned proteins were attached were blocked by treating them with 5% skim milk powder, and then the primary antibody was reacted overnight at 4°C. After washing with PBS-T, the secondary antibody was reacted at room temperature for 1 hour, and after further washing with PBS-T, it was detected using Gel Doc (Bio-Rad (Hercules (CA, USA))) and Western detection reagent (Elpis Biotech (Daejeon, South Korea))). Anti-Myo G (Santa Cruz (USA)) was used as the primary antibody, along with anti-Myf5 (Abcam (UK)) and anti-GAPDH (Santa Cruz (USA)). A non-treated group (Non) was used as the control group, and a group treated only with differentiation medium (CM) was used as the comparison group.

[0059] As a result, as shown in Figures 1 and 2, we confirmed that treatment with the peptide of Sequence ID No. 1 increased the expression of Myo G (myogenin) and Myf5, which are early differentiation markers for myoblasts.

[0060] 2-2. Evaluation via immunofluorescence C2C12 cells were cultured in DMEM medium containing 1% P / S and 2% BCS. When the cell confluency reached approximately 70-80%, the cells were seeded into 6-well culture plates. Subsequently, when the cell confluency in the 6-well culture plates reached 100%, the culture medium was replaced with differentiation medium (1% P / S, 2% horse serum), and 100 μg / ml of the peptide of Sequence ID No. 1 was added. Thereafter, the differentiation medium and the peptide of Sequence ID No. 1 were alternated every two days. Three days after the first medium change, the cultured cells were fixed with 4% paraformaldehyde (PFA) at room temperature for 10 minutes, and permeabilization was performed in 0.1% Triton X-100 for 5 minutes. Subsequently, the fixed cells were treated with PBS, 10% FBS, and 0.1% Triton X-100, blocked at room temperature for 1 hour, then treated with the primary antibody Myo G (Santa Cruz, USA), and incubated at room temperature for 1 hour. The cells were then washed and incubated with the secondary antibody (goat anti-mouse IgG-TR (Santa Cruz, USA)) at room temperature for 30 minutes. The cell nuclei were then stained with DAPI (Santa Cruz, USA) and observed using a LEICA fluorescence microscope (TCSSP8 (LEICA, Germany)).

[0061] As a result, as shown in Figures 3 and 4, we confirmed that treatment with the peptide of Sequence ID No. 1 not only increased the expression of Myo G (myogenin), an early differentiation marker for myoblasts, but also increased its expression in the nuclear region of myoblasts.

[0062] In summary, the experimental results described above indicate that the peptide from one example contributes to promoting the initial differentiation of myoblasts into muscle cells.

[0063] Example 3. Confirmation of the effect of promoting myoblast differentiation in the late differentiation stage. In this example, we will evaluate the changes in the expression of early differentiation marker proteins in mouse myoblasts (C2C12 cells) to confirm the effect of the peptide from this example on the differentiation from myoblasts to muscle cells / muscle fibers in the later differentiation stage.

[0064] 3-1. Evaluation via protein expression analysis Except for the point at which the lysis buffer was added (7 days after the first change of culture medium), the procedure was carried out in the same manner as in Example 2-1, and the expression level of MyHC (Myosin heavy chain) protein, a marker of late differentiation of myoblasts, was confirmed. Anti-MyHC (Santa Cruz, USA) and anti-GAPDH (Santa Cruz, USA) were used as primary antibodies. An untreated group (Non) was used as the control group, and a group treated only with differentiation medium (CM) was used as the comparison group.

[0065] As a result, as shown in Figures 5 and 6, we confirmed that treatment with the peptide of Sequence ID No. 1 increased the expression of MyHC (Myosin heavy chain), a marker of late differentiation in myoblasts.

[0066] 3-2. Evaluation via immunofluorescence Except for the immobilization stage (7 days after the first change of culture medium), the procedure was carried out in the same manner as in Example 3-1, and the expression level of MyHC (Myosin heavy chain) protein, a late differentiation marker for myoblasts, was confirmed. Anti-α-actinin (Santa Cruz, USA) was used as the primary antibody, goat anti-mouse IgG-TR (Santa Cruz, USA) was used as the secondary antibody, and a control group (CM) treated only with differentiation medium was used.

[0067] As a result, as shown in Figures 7 and 8, we confirmed that treatment with the peptide of Sequence ID No. 1 increased the expression of α-actinin, a marker of late differentiation in myoblasts.

[0068] In summary, the experimental results described above indicate that the peptide from one example contributes to promoting the late differentiation of myoblasts into muscle cells.

[0069] Example 4. Confirmation of muscle loss inhibition effect using an animal model. In this example, the effect of the peptide described in one example on inhibiting muscle loss is confirmed by evaluating changes in muscle mass, the area of ​​immunoepidemic cell infiltration within muscle tissue, the level of accumulated collagen, and the expression levels of muscle fiber constituent proteins and inflammatory proteins in an animal model in which muscle damage is induced by BaCl2.

[0070] 4-1. Muscle Mass Assessment 50 μl of 1.2% (w / v) BaCl2 aqueous solution was injected into the anterior tibial (TA) muscle of 8-week-old male C57BL / 6 mice to induce muscle damage for 1 day. Subsequently, 20 mg of the peptide of Sequence ID No. 1 was orally administered to the mice with induced muscle damage at a concentration of 0.1 mg / μl for a total of 6 doses at 1-day intervals. For the positive control group, 0.5 μg of IGF-I was intramuscularly injected at a concentration of 0.01 μg / μl. Seven days after the induction of muscle damage, the mice were sacrificed, and the changes in the anterior tibial (TA) muscle were visually observed and measured in weight. The control group was the untreated group (Non), the negative control group was the group in which only muscle damage induced by BaCl2 was induced (NC), and the positive control group was the group in which muscle damage induced by BaCl2 was treated with IGF-I (PC).

[0071] As a result, as shown in Figures 9 and 10, we confirmed that treatment with the peptide of Sequence ID No. 1 increased the muscle mass of the entire tibial (TA) muscle in mice in which muscle damage was induced by BaCl2, and thus we were able to learn that it can promote the regeneration of damaged muscle.

[0072] 4-2. Evaluation via muscle tissue staining Muscle tissue obtained from mice in which muscle damage was induced as in Example 4-1 was washed with PBS at room temperature and then fixed with 4% paraformaldehyde (PFA). The fixed muscle tissue was then washed three times with PBS and dehydrated with a gradient ethanol series (70% to 100%). The muscle tissue samples were sectioned to a size of 4 μm. Tissue slides containing the sectioned specimens were dewaxed and dehydrated via a gradient ethanol series, then sequentially stained with hematoxylin solution for 1 minute and then with eosin solution for 10 seconds. After staining, the tissue slides were sequentially immersed in 90% and 100% ethanol, and finally immersed twice in xylene for 5 minutes each, before mounting to the tissue slides (H&E staining).

[0073] Furthermore, the tissue slides containing the aforementioned sectioned specimens were dewaxed and hydrated as described above, then stained with Picro-Sirius Red Solution for 60 minutes, followed by immersion in 1% acetic acid solution for 2 minutes twice, and then washed with DW. After that, the tissue slides were immersed in ethanol, and finally immersed in xylene for 5 minutes twice, before mounting onto the tissue slides (Sirius Red staining). As a result, as shown in Figures 11 and 12, we confirmed that treatment with the peptide of SEQ ID NO: 1 reduced the area of ​​immunoepidemic cell infiltration in tissues affected by BaCl2-induced muscle damage. Furthermore, as shown in Figures 13 and 14, we confirmed that treatment with the peptide of SEQ ID NO: 1 reduced the accumulation of collagen in tissues affected by BaCl2-induced muscle damage.

[0074] 4-3. Evaluation via analysis of protein expression in muscle tissue Muscle tissue obtained from mice in which muscle damage was induced according to Example 4-1 was broken up using a homogenizer, and the broken-up material was dissolved by adding lysis buffer. The tissue was then centrifuged at 4°C and 12,000 rpm for 30 minutes to obtain the protein, which was then quantified using a BCA kit. The sample containing the protein was subjected to SDS-PAGE to separate each protein, and then transferred to a membrane by electrophoresis. The membrane with the attached protein was blocked by treating it with 5% skim milk powder, and then the primary antibody was reacted overnight at 4°C. After washing with PBS-T, the secondary antibody was reacted at room temperature for 1 hour and washed again with PBS-T, and then detected using GelDoc (Bio-Rad, Hercules, CA (USA)) and Western detection reagents (ElpisBiotech, Daejeon, Korea). On the other hand, anti-MyHC (Santa Cruz, USA) was used as the primary antibody, along with anti-α-actinin (Santa Cruz, USA), anti-α-SMA (Abcam, UK), anti-IL-6 (Santa Cruz, USA), and anti-α-tubulin (Santa Cruz, USA). As a result, as shown in Figures 15 and 16, we confirmed that treatment with the peptide of SEQ ID NO: 1 increased the expression of MyHC and α-actinin, which are intra-tissue muscle fiber constituent proteins that are affected by BaCl2-induced muscle damage. Furthermore, as shown in Figures 17 and 18, we confirmed that treatment with the peptide of SEQ ID NO: 1 decreased the expression of α-SMA and IL-6, which are intra-tissue inflammatory proteins that are affected by BaCl2-induced muscle damage.

[0075] In summary, the experimental results suggest that the peptide according to one embodiment can not only promote the regeneration or recovery of damaged muscles, but also contribute to suppressing the progression of muscle damage, such as the inflammatory response that causes muscle damage.

[0076] Dosage Form Example 1: Production of Peptide Nanosomes 50 mg of the peptide from Example 1 was dissolved in 500 ml of distilled water by thorough stirring. The mixture was then mixed with 5 g of lecithin, 0.3 ml of sodium oleate, 50 ml of ethanol, and a small amount of oil. After adjusting the volume with distilled water to a total volume of 1 L, the mixture was emulsified using a microfluidizer under high pressure to produce peptide nanosomes with a size of approximately 100 nm.

[0077] Dosage Form Examples 2. Pharmaceutical Preparations 2-1. Manufacturing of powdered medicines The following ingredients are mixed and filled into airtight packets to produce the powder. 20 mg of the peptide of the present invention Lactose 100mg Talc 10mg

[0078] 2-2. Manufacturing of Tablets After mixing the following ingredients, tablets are manufactured by compressing them using a standard tablet manufacturing method. 10 mg of the peptide of the present invention Corn starch 100mg Lactose 100mg Magnesium stearate 2mg

[0079] 2-3. Manufacturing of Capsules The following ingredients are mixed using a standard capsule manufacturing method, filled into gelatin capsules, and then the capsules are produced. 10 mg of the peptide of the present invention Crystalline cellulose 3 mg Lactose 14.8mg Magnesium stearate 0.2 mg

[0080] 2-4. Manufacturing of injectable drugs The following ingredients are manufactured per ampoule (2 ml) using a standard method for manufacturing injectable drugs. 10 mg of the peptide of the present invention Mannitol 180mg 2,974 mg of sterile distilled water for injection Na2HPO4·2H2O 26mg 2-5. Manufacturing of liquid formulations

[0081] The liquid preparation is manufactured by dissolving each component in purified water using a standard liquid preparation method, mixing the components listed below, adding purified water to adjust the total volume to 100 ml, filling it into a brown bottle, and sterilizing it. 10 mg of the peptide of the present invention 10g of high fructose sugar Mannitol 5g Purified water (appropriate amount)

[0082] The above description of the present invention is illustrative, and a person with ordinary skill in the art to which the present invention pertains will understand that the invention can be easily modified into other specific forms without altering the technical idea or essential features. Therefore, the embodiments described above should be understood to be illustrative and not limiting in all respects.

Claims

1. A peptide consisting of the amino acid sequence of SEQ ID NO:

1.

2. The peptide according to claim 1, wherein the N-terminus of the peptide is bonded to one protecting group selected from the group consisting of an acetyl group, a fluorenylmethoxycarbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group, a butoxycarbonyl group, an allyloxycarbonyl group, and polyethylene glycol (PEG).

3. The C-terminus of the peptide is an amino group (-NH ２ )), tertiary alkyl group and hydrazino (-NHNH ２ The peptide according to claim 1, which is bonded to one protecting group selected from the group consisting of )).

4. The peptide according to claim 1, wherein the peptide exhibits one or more of the following characteristics: (a) Increased expression of myogenin and myogenic factor 5, (b) Enhancement of myosin heavy chain and α-actin expression, (c) Reduction in the expression of α-smooth muscle actin and interleukin-6.

5. A pharmaceutical composition for the prevention or treatment of muscle disease, comprising the peptide described in any one of claims 1 to 4 as an active ingredient.

6. The pharmaceutical composition according to claim 5, wherein the muscle disease is a muscle disease resulting from decreased muscle function, muscle wasting, or muscle degeneration.

7. The pharmacochemical composition according to claim 6, wherein the muscle disease is one of sarcopenia, atony, muscular dystrophy, muscular atrophy, muscle degeneration, myoricosis, amyotrophic lateral sclerosis, myasthenia gravis, and cachexia.

8. A food composition for the prevention or improvement of muscle disease, comprising the peptide described in any one of claims 1 to 4 as an active ingredient.

9. The food composition according to claim 8, wherein the muscle disease is a muscle disease resulting from decreased muscle function, muscle wasting, or muscle degeneration.

10. The food composition according to claim 8, further comprising any one component selected from vitamin C, vitamin B1, vitamin B2, vitamin B6, vitamin H, vitamin PP, provitamin B5, vitamin A, vitamin D, vitamin E, vitamin K1, or carotene, or a mixture thereof.

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