Peptides with muscle loss and inhibition and muscle mass improvement activity and uses thereof

Abstract

The present application relates to a peptide having muscle loss inhibiting and muscle mass improving activity and use thereof, which provides a peptide consisting of the amino acid sequence of SEQ ID NO: 1, and a pharmaceutical composition for preventing or treating a muscle disease including the peptide as an effective ingredient and a food composition for preventing or improving a muscle disease including the peptide as an effective ingredient.

Description

Technical Field

[0001] This application relates to peptides having muscle loss inhibition and muscle mass enhancement activities, and their uses. This application claims priority to Korean Patent Application No. 10-2022-0071033, filed on June 10, 2022, the disclosures of which are incorporated herein by reference. Background Technology

[0002] In recent years, with the rapid increase in the global aging population, a United Nations report projects that by 2050, the population aged 60 and over will exceed 2 billion. Skeletal muscle is the largest organ, accounting for 50% of body weight, and muscle loss and weakness are among the major physical changes that occur due to aging. Therefore, after age 30, skeletal muscle tends to decrease by about 1% per year, and this decline is dramatic after age 65. Such a rapid decrease in muscle mass can significantly impact quality of life, such as reducing physical fitness and increasing the risk of falls and fractures. In light of these concerns, the World Health Organization (WHO) recognized sarcopenia as an official disease in 2017.

[0003] Sarcopenia, caused by the degeneration of spinal nerves, motor nerves, or skeletal muscle fibers associated with muscle diseases, is a representative intractable disease whose pathogenesis remains unclear. Current research suggests that due to the degeneration of motor nerves that induce skeletal muscle contraction, skeletal muscle contraction ceases, or the expression of proteins involved in muscle contraction is reduced or modified, preventing normal skeletal muscle contraction. In the long term, it is known that the motor nerves or skeletal muscles will degenerate into fibrous tissue. The underlying cause of sarcopenia remains unknown, and because no methods have been developed to prevent or reverse the degeneration of motor nerves or skeletal muscles, research is actively underway to develop methods to slow its progression. While exercise, protein, and calorie supplementation are known to be helpful for sarcopenia, their benefits are limited in the majority of patients, the elderly, thus necessitating a therapeutic agent for sarcopenia.

[0004] On the other hand, muscle size or mass is regulated by intracellular signal transduction processes that induce anabolism or catabolism within the muscle. Specifically, when the signal transduction response inducing muscle protein synthesis is dominant over muscle protein breakdown, muscle protein synthesis increases, manifesting as hypertrophy or hyperplasia. Furthermore, muscle cell differentiation and myogenesis are regulated by various muscle regulatory factors. MyoD initiates the expression of muscle-specific genes and induces the differentiation of muscle satellite cells into myoblasts. The induction of myogenin expression through MyoD activity is a crucial factor in myoblast fusion, participating in myotube formation. The myofibrous bundles formed through this process ultimately form muscle.

[0005] Against this technological backdrop, various studies are underway to suppress muscle loss caused by external or endogenous factors and promote muscle protein synthesis (Korean Patent No. 10-2064387), but these studies are still incomplete. Summary of the Invention

[0006] [Technical Issues]

[0007] On the one hand, it provides a peptide consisting of the amino acid sequence of SEQ ID NO: 1.

[0008] On the other hand, a composition for inhibiting muscle loss and improving muscle mass is provided, comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0009] Other objects and advantages of this application will become more apparent from the following detailed description taken in conjunction with the appended claims and drawings. For matters not described herein, those skilled in the art or closely related to the art will readily recognize and infer them, and therefore such description is omitted.

[0010] [Technical Solution]

[0011] Every description and embodiment disclosed in this application is applicable to every other description and embodiment. In other words, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the scope of this application should not be construed as limited to the following specific description.

[0012] On the one hand, it provides a peptide consisting of the amino acid sequence of SEQ ID NO: 1.

[0013] As used in this specification, the term "peptide" can refer to a linear molecule formed by linking amino acid residues together through peptide bonds. The peptide can be manufactured according to chemical synthesis methods known in the art, particularly solid-phase or liquid-phase synthesis techniques (U.S. Patent No. 5,516,891). As a result of persistent efforts to develop peptides with biologically effective activity, a peptide consisting of the amino acid sequence of SEQ ID NO: 1 has been identified. Here, the biologically effective activity can be manifested by any one or more of the following characteristics: (a) increased expression of myogenin (MyoG) and myogenic factor 5 (Myf5), which are markers of early myoblast differentiation; (b) increased expression of myosin heavy chain (MyHC) and α-actinin, which are markers of late myoblast differentiation or fibrous constituent proteins; and (c) decreased expression of α-smooth muscle actin (α-SMA) and interleukin-6 (IL-6), which are inflammatory proteins. Therefore, the peptide can be used for the prevention, improvement, or treatment of muscle diseases.

[0014] To achieve chemical stability, enhanced pharmacological properties (half-life, absorption profile, efficacy, potency, etc.), altered specificity (e.g., broad bioactivity profile), or reduced antigenicity, a protecting group may be attached to the N- or C-terminus of the peptide. In one specific embodiment, the N-terminus of the peptide may be bound to any one of the following protecting groups selected from the group consisting of: acetyl, fluoreonylmeethoxycarbonyl, formyl, palmitoyl, myristyl, stearyl, butoxycarbonyl, allyloxycarbonyl, and polyethylene glycol (PEG), and / or the C-terminus of the peptide may be bound to any one of the following protecting groups selected from the group consisting of: amino (NH2), tertiary alkyl, and hydrazinyl (NHNH2). Alternatively, the peptide may further include a targeting sequence, a tag, a labeled defect, or an amino acid sequence manufactured for a specific purpose of increasing half-life or peptide stability.

[0015] The peptide is artificially synthesized or non-naturally occurring or engineered, where "non-naturally occurring or engineered" means generated through artificial modification, rather than existing in its natural state. Here, artificial modification may include artificially synthesizing an amino acid sequence by mimicking the structure of multiple amino acids, or, as described above, performing operations to obtain chemical stability, enhanced pharmacological properties, altered specificity, or reduced antigenicity.

[0016] The term “stability” as used in this specification may refer to storage stability (e.g., room temperature storage stability) and in vivo stability that protects the peptide from attack by proteolytic enzymes in vivo.

[0017] On the other hand, a pharmaceutical composition for the prevention or treatment of muscle diseases is provided, comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0018] In the description of the peptide, the same terms or elements mentioned are as described above.

[0019] As used in this specification, the term "prevention" refers to any action that inhibits or delays the onset of a disease by applying the composition.

[0020] As used in this specification, the term "treatment" refers to any form of therapy that provides an individual with a disease or at risk of developing a disease, including effects such as improving the individual's condition (e.g., one or more symptoms), slowing disease progression, or alleviating symptom progression. Therefore, "treatment" and "prevention" do not imply a cure or complete elimination of symptoms.

[0021] As used in this specification, the term "muscle disease" refers to a muscle disease, disorder, or condition associated with reduced proliferation or differentiation of myoblasts, and may collectively refer to muscle diseases caused by, for example, decreased muscle function, muscle wasting, or muscle degeneration. Such muscle diseases may include, for example, sarcopenia, atony, muscular dystrophy, muscular atrophy, muscle degeneration, myotonia, amyotrophic sclerosis, myasthenia gravis, and cachexia, but are not limited thereto.

[0022] In one specific embodiment, the pharmaceutical composition according to one embodiment can improve muscle mass or muscle strength or improve muscle function by promoting the proliferation and differentiation of myoblasts.

[0023] While existing functional peptides possess potent biological activity, their size limits their effective influx into target tissues or cells, or their short half-life leads to their rapid disappearance from the body. In contrast, a pharmaceutical composition according to one embodiment comprises a peptide consisting of about 10 or fewer amino acids as the active ingredient. Therefore, the active ingredient exhibits excellent cell penetration, etc., and can achieve effective treatment of muscle diseases, for example, in the case of topical application.

[0024] According to one embodiment, the peptide of this application can significantly increase the expression of early differentiation markers (e.g., MyoG and Myf5) and late differentiation markers or myofiber constituent proteins (e.g., myosin heavy chain, α-actin), thereby promoting differentiation into muscle cells or muscle fibers. Furthermore, the peptide of this application can not only improve muscle quality recovery / regeneration when muscle injury is caused by exogenous factors, but also reduce the infiltration of immune cells and the accumulation of collagen in damaged muscle tissue, thereby alleviating the symptoms of muscle diseases and inhibiting disease progression. Therefore, the peptide can be used as an effective ingredient in pharmaceutical compositions for treating muscle diseases.

[0025] The pharmaceutical composition may include, but is not limited to, a pharmaceutically effective amount of the peptide and / or a pharmaceutically acceptable carrier.

[0026] The term "effective amount" as used in this specification may mean an amount sufficient to achieve the therapeutic efficacy of the pharmaceutical composition for bone diseases.

[0027] The weight ratio between the peptide and the pharmaceutically acceptable carrier can be, for example, 500:1 to 1:500, such as 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.

[0028] Pharmaceutically acceptable carriers are those commonly used in formulation manufacturing, including, but not limited to, lactose, glucose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, [unspecified substance], calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, fossil fuels, magnesium stearate, and mineral oil. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th edition, 1995).

[0029] In addition to the above-mentioned components, the pharmaceutical composition may also include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc., but is not limited thereto.

[0030] The pharmaceutical composition can be administered orally or via the enteral route. In the case of enteral administration, it can be administered via intramuscular injection, intravenous injection, subcutaneous injection, intraperitoneal injection, local application, or direct application, but is not limited thereto.

[0031] The dosage of the pharmaceutical composition may be 0.0001 to 1000 μg, 0.001 to 1000 μg, 0.01 to 1000 μg, 0.1 to 1000 μg or 1.0 to 1000 μg per day, but is not limited thereto. Various methods of administration may be used depending on factors such as formulation method, administration method, patient's age, weight, gender, pathological condition, diet, administration time, route of administration, excretion rate and reaction sensitivity.

[0032] The pharmaceutical composition is formulated using pharmaceutically acceptable carriers and / or excipients according to methods readily practiced by those skilled in the art, so that it can be manufactured in unit dosage form or prepared in multi-dosage containers.

[0033] The dosage form may be a solution, suspension, or emulsion in an oily or aqueous ring, or may be an ointment, cream, gel, transdermal absorbent, poultice, patch, paste, extract, powder, granule, tablet, or capsule, and may additionally include dispersants and / or stabilizers.

[0034] The peptide may be contained, for example, in nanobody or nanobody particles to further improve cell permeability or stability. For example, the nanobody may be manufactured using lecithin as a raw material via a microfluidic apparatus, and may be contained within lecithin particles. Any method known can be used to manufacture the nanobody. The size of the nanobody 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.

[0035] The pharmaceutical composition may also include an ingredient beneficial for the prevention or treatment of muscle diseases, which may be, for example, selected from any one of 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 mixtures thereof, but is not limited thereto.

[0036] In the description of the peptides and pharmaceutical compositions, the same terms or elements mentioned are as described above.

[0037] The term “improvement” as used in this specification may refer to all behaviors that at least reduce parameters related to the mitigation or treatment of the condition, such as the degree of symptoms.

[0038] On the other hand, a method for preventing or treating muscle diseases is provided, which includes administering to an individual a composition comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0039] In the description of the peptide, pharmaceutical composition, etc., the same terms or elements mentioned are as described above.

[0040] The terms “application” or “treatment” as used in this specification are used interchangeably and can mean the delivery of a predetermined substance to an individual or patient by any suitable method. Additionally, the terms can also indicate that a composition according to an embodiment is at least partially localized to a desired area, or that a composition according to an embodiment is administered into an individual via an administration route.

[0041] As used in this specification, the term "individual" refers to an object whose skin condition needs improvement, and more specifically, to mammals or humans or non-human primates, mice, dogs, cats, horses, and cattle.

[0042] The composition may be, for example, a pharmaceutical composition comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0043] The content of the peptide as an active ingredient in the composition can be appropriately selected without limitation according to the form of the drug, the intended use, etc. For example, it can be added in an amount of 0.01 to 15% by weight of the total weight of the composition. In addition, for example, a health beverage composition can be added at a rate of 0.02 to 10g per 100 ml, preferably 0.3 to 10g, but not limited thereto.

[0044] [Beneficial Effects]

[0045] Based on one type of peptide, differentiation into myocytes or myofibrils can be promoted by enhancing the expression of factors involved in myoblast differentiation.

[0046] According to one type of peptide, in the case of muscle damage caused by exogenous factors, it can not only improve the recovery / regeneration of muscle quality, but also reduce the infiltration of immune cells and the accumulation of collagen tissue in the damaged muscle tissue, thereby alleviating the symptoms of muscle disease and inhibiting the progression of the disease.

[0047] Therefore, peptides, according to one aspect, can be used as an effective ingredient in compositions for the prevention, improvement, or treatment of muscle diseases. Attached Figure Description

[0048] Figure 1 To confirm, by Western ink dot assay, the increased expression of myogenin and Myf5, markers of early differentiation of myoblasts, after the addition of the peptide consisting of the amino acid sequence of SEQ ID NO: 1 to C2C12 cells.

[0049] Figure 2 This is a graph illustrating the quantitative assessment of changes in the expression of early differentiation markers in myoblasts after the addition of a peptide consisting of the amino acid sequence of SEQ ID NO: 1 to C2C12 cells. Figure 2 (a) shows the results confirming the expression level of myopoietin, and Figure 2 (b) is the result confirming the expression level of Myf5.

[0050] Figure 3 The results were obtained by immunofluorescence assay to confirm the expression pattern of myopoietin (MyoG), an initial marker of myoblasts, after the addition of a peptide consisting of the amino acid sequence of SEQ ID NO: 1 to C2C12 cells.

[0051] Figure 4This is the result of quantitatively assessing the expression level of myocyte cytopoietin (MyoG) after adding a peptide consisting of the amino acid sequence of SEQ ID NO: 1 to C2C12 cells.

[0052] Figure 5 This study aims to confirm, using Western ink dot assay, the increased expression of MyHC, a marker of late differentiation of myoblasts, after the addition of a peptide consisting of the amino acid sequence of SEQ ID NO: 1 to C2C12 cells.

[0053] Figure 6 This is the result of quantitatively assessing the changes in the expression of late differentiation markers of myoblasts after the peptide consisting of the amino acid sequence of SEQ ID NO: 1 was added to C2C12 cells, and the result is to confirm the expression level of MyHC.

[0054] Figure 7 The results were obtained by immunofluorescence assay to confirm the expression pattern of α-actinin, a late marker of myoblasts, after the addition of a peptide consisting of the amino acid sequence of SEQ ID NO: 1 to C2C12 cells.

[0055] Figure 8 This is the result of quantitatively assessing the expression level of α-actin after adding a peptide consisting of the amino acid sequence of SEQ ID NO: 1 to C2C12 cells.

[0056] Figure 9 This is based on the results of visually confirmed changes in the tibialis anterior (TA) muscle in an animal model of muscle injury induced by BaCl2, based on treatment with a peptide consisting of the amino acid sequence of SEQ ID NO: 1.

[0057] Figure 10 This is a graph that quantitatively assesses changes in muscle mass of TA muscle treated with a peptide based on the amino acid sequence of SEQ ID NO: 1 in an animal model of BaCl2-induced muscle injury, where... Figure 10 (a) shows the results confirming the weight (g) of the TA muscle, and Figure 10 (b) is the result confirming TA muscle (%).

[0058] Figure 11 The results were obtained by H&E staining to confirm changes in the area of ​​immune cell infiltration in muscle tissue of an animal model of BaCl2-induced muscle injury, based on the amino acid sequence of peptide SEQ ID NO: 1.

[0059] Figure 12To quantitatively assess the changes in immune cell infiltration area in muscle tissue of an animal model of BaCl2-derived muscle injury based on treatment with a peptide consisting of the amino acid sequence of SEQ ID NO: 1.

[0060] Figure 13 This study aimed to confirm, by means of Sirius red staining, the changes in collagen accumulation levels in muscle tissue of an animal model of BaCl2-induced muscle injury based on treatment with a peptide consisting of the amino acid sequence of SEQ ID NO: 1.

[0061] Figure 14 To quantitatively assess the changes in collagen accumulation levels in muscle tissue of an animal model of BaCl2-induced muscle injury based on treatment with a peptide consisting of the amino acid sequence of SEQ ID NO: 1.

[0062] Figure 15 This study aims to confirm, by means of Western ink dot assay, the increased expression of MyHC and α-actin, which are myofiber component proteins, in muscle tissue of an animal model of BaCl2-induced muscle injury, based on treatment with a peptide consisting of the amino acid sequence of SEQ ID NO: 1.

[0063] Figure 16 To quantitatively assess the expression levels of MyHC and α-actin, which are myofiber component proteins, in muscle tissue of an animal model of BaCl2-induced muscle injury, based on the peptide consisting of the amino acid sequence of SEQ ID NO: 1.

[0064] Figure 17 This study aimed to confirm, by means of Western ink dot assay, the reduced expression of α-SMA and IL-6, inflammatory proteins, in muscle tissue of an animal model of BaCl2-induced muscle injury, based on treatment with a peptide consisting of the amino acid sequence of SEQ ID NO: 1.

[0065] Figure 18 To quantitatively assess the expression levels of α-SMA and IL-6, inflammatory proteins, in muscle tissue of an animal model of BaCl2-induced muscle injury, based on treatment with a peptide consisting of the amino acid sequence of SEQ ID NO: 1. Detailed Implementation

[0066] The invention will be described in more detail below through embodiments. However, these embodiments are for illustrative purposes only, and the scope of the invention is not limited to these embodiments.

[0067] [Example 1. Peptide Synthesis]

[0068] Peptides having the amino acid sequence of SEQ ID NO: 1 as described in Table 1 below were synthesized using an automated peptide synthesizer (Milligen 9050, Millipore, USA), and the synthesized peptides were purified and separated using C18 reversed-phase high-performance liquid chromatography (HPLC) (Waters Associates, USA). An ACQUITY UPLC BEH300 C18 column (2.1 mm × 100 mm, 1.7 μm, Waters Co, USA) was used as the chromatographic column.

[0069] Table 1

[0070]

[0071] [Example 2. Confirmation of the differentiation-promoting effect of myoblasts in the initial differentiation stage]

[0072] In this embodiment, the effect of peptides according to one embodiment on the differentiation of myoblasts into myoblasts / myofibroblasts during the initial differentiation step was confirmed by evaluating the expression changes of C2C12 cells, which are the initial differentiation marker proteins of mouse myoblasts.

[0073] [2-1. Evaluation through protein expression analysis]

[0074] After culturing C2C12 cells in DMEM medium containing 1% P / S and 2% BCS, they were seeded into 6-well plates when cell confluence reached approximately 70% to 80%. Then, when cell confluence in the 6-well plates reached 100%, the medium was replaced with differentiation medium (1% P / S, 2% horse serum), with 5, 50, or 100 μg / ml of the peptide of SEQ ID NO: 1 added. Thereafter, the same amount of the peptide of SEQ ID NO: 1 was added and replaced every two days. Three days after the first medium replacement, the culture was dissolved in dissolution buffer, centrifuged at 4°C and 12,000 rpm for 30 minutes to obtain proteins, which were then quantified using a BCA kit. Samples containing these proteins were subjected to SDS-PAGE to separate the proteins, followed by electrotransfer to a membrane. The membrane containing the protein was treated and blocked with 5% skim milk, and then incubated with primary antibody overnight at 4°C. After washing with PBS-T, the membrane was incubated with secondary antibody for 1 hour at room temperature, followed by washing with PBS-T again, and then detected using Gel Doc (Bio-Rad, Hercules, CA, USA) and Western blotting assay (Elpis Biotech, Daejeon, Korea). Anti-MyoG (Santa Cruz, USA), Anti-Myf5 (Abcam, UK), 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.

[0075] As a result, Figure 1 and Figure 2 As shown, treatment with the peptide of SEQ ID NO: 1 confirmed an increase in the expression of myopoietin (MyoG) and Myf5, which are markers of early differentiation of myoblasts.

[0076] [2-2. Evaluation by immunofluorescence assay]

[0077] After culturing C2C12 cells in DMEM medium containing 1% P / S and 2% BCS, they were seeded into 6-well plates when cell confluence reached approximately 70% to 80%. Then, when cell confluence in the 6-well plates reached 100%, the medium was replaced with differentiation medium (1% P / S, 2% horse serum), and 100 μg / ml of the peptide of SEQ ID NO: 1 was added. Subsequently, the differentiation medium and 100 μg / ml of the peptide of SEQ ID NO: 1 were added 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 then permeabilized in 0.1% Triton X-100 for 5 minutes. Then, the fixed cells were blocked for 1 hour at room temperature with PBS, 10% FBS, and 0.1% Triton X-100, treated with primary antibody (MyoG, Santa Cruz, USA), and cultured at room temperature for 1 hour. The cells were then washed and cultured again at room temperature for 30 minutes with secondary antibody (goat anti-mouse IgG-TR, Santa Cruz, USA). Subsequently, the cell nuclei were stained with DAPI (Santa Cruz, USA) and observed using a LEICA fluorescence microscope (TCS SP8, LEICA, Germany).

[0078] As a result, Figure 3 and Figure 4 As shown, treatment with the peptide of SEQ ID NO: 1 confirmed that not only was the expression of myopoietin (MyoG), a marker of early differentiation of myoblasts, increased, but the expression in the nuclear region of myoblasts was also increased.

[0079] In summary, the experimental results show that the peptide according to one embodiment helps promote the initial differentiation from myoblasts to myocytes.

[0080] [Example 3. Confirmation of the differentiation-promoting effect of myoblasts in the later differentiation stage]

[0081] In this embodiment, the expression changes of C2C12 cells, which serve as markers of early differentiation in mouse myoblasts, were evaluated to confirm the effect of peptides according to one embodiment on the differentiation of myoblasts into myoblasts / myofibroblasts in later differentiation steps.

[0082] [3-1. Evaluation through protein expression analysis]

[0083] Except for the addition time of the dissolution buffer (7 days after the first change of medium), the procedure was performed in the same manner as in Example 2-1 to confirm the expression level of myosin heavy chain (MyHC) protein, a marker of late differentiation of myoblasts. 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 with differentiation medium only (CM) was used as the comparison group.

[0084] If, as Figure 5 and Figure 6 As shown, treatment with the peptide of SEQ ID NO: 1 confirmed an increase in the expression of myosin heavy chain (MyHC), a marker of late differentiation of myoblasts.

[0085] [3-2. Evaluation by immunofluorescence assay]

[0086] Except for the immobilization time (7 days after the first change of medium), the same procedure as in Example 3-1 was performed to confirm the expression level of myosin heavy chain (MyHC) protein, a marker of late differentiation of myoblasts. 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 group treated with differentiation medium (CM) alone served as the control group.

[0087] As a result, Figure 7 and Figure 8 As shown, treatment with the peptide of SEQ ID NO: 1 confirmed an increase in the expression of α-actin, a marker of late differentiation of myoblasts.

[0088] In summary, the experimental results show that the peptide according to one embodiment helps promote late-stage differentiation from myoblasts to myocytes.

[0089] [Example 4. Confirming the muscle loss inhibition effect using an animal model]

[0090] In this embodiment, an animal model of muscle damage induced by BaCl2 was used as the subject to evaluate changes in muscle mass, the infiltration area of ​​immune cells in muscle tissue, the accumulation level of collagen, and the expression levels of myofibril tissue proteins and inflammatory proteins, in order to confirm the effect of the peptide according to one embodiment on inhibiting muscle loss.

[0091] 【4-1. Muscle Mass Assessment】

[0092] 50 μl of 1.2% (w / v) BaCl2 was injected into the tibialis anterior (TA) muscle of 8-week-old male C57BL / 6 mice to induce muscle injury within one day. Then, 20 mg of the peptide of SEQ ID NO: 1 was orally administered to the mice with induced muscle injury at one-day intervals for a total of six times. For the positive control group, 0.5 μg of IGF-1 was injected intramuscularly at a concentration of 0.01 μg / μl. On day 7 from the induction of muscle injury, mice were sacrificed, and changes in the TA muscle were observed visually and their weight was measured. An untreated group (Non) was used as the control group, a group with muscle injury induced by BaCl2 alone (NC) was used as the negative control group, and a group treated with IGF-1 in the BaCl2-induced muscle injury group (PC) was used as the positive control group.

[0093] As a result, Figure 9 and Figure 10 As shown, treatment with the peptide in SEQ ID NO: 1 confirmed that the muscle mass of the TA muscle in mice with muscle damage induced by BaCl2 increased, thus indicating that it can promote the regeneration of damaged muscle.

[0094] [4-2. Assessment through muscle tissue staining]

[0095] Muscle tissue obtained from mice with induced muscle injury 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 using a gradient ethanol series (from 70% to 100%). The muscle tissue samples were sectioned to a size of 4 μm. Tissue slides containing the sectioned specimens were dewaxed and hydrated using a gradient ethanol series, stained in hematoxylin solution for 1 minute, and stained in eosin solution for 10 seconds. After staining, the tissue sections were successively immersed in 90% and 100% ethanol (ethyl alcohol), and finally immersed twice in xylene for 5 minutes each time before mounting on tissue slides (H&E staining).

[0096] In addition, after dewaxing and hydrating the tissue slides including the sectioned specimens as described above, they were stained in Picro Sirius Red Solution for 60 minutes, then immersed twice in 1% acetic acid solution for 2 minutes each time, and washed with DW. The tissue slides were then immersed in ethyl alcohol, and finally in xylene twice for 5 minutes each time, before being sectioned onto the tissue slides (Sirius Red staining).

[0097] As a result, Figure 11 and Figure 12 As shown, treatment with the peptide in SEQ ID NO: 1 confirmed a reduction in the area of ​​immune cell infiltration in BaCl2-induced muscle damage tissue. Additionally, as... Figure 13 and Figure 14 As shown, it was confirmed that peptide treatment with SEQ ID NO: 1 reduced collagen accumulation in tissues with BaCl2-induced muscle damage.

[0098] [4-3. Evaluation through protein expression analysis in muscle tissue]

[0099] Muscle tissue obtained from mice with induced muscle injury as described in Example 4-1 was lysed using a homogenizer. After adding dissolution buffer to dissolve the lysate, the tissue was centrifuged at 12,000 rpm for 30 minutes at 4°C to obtain proteins, which were then quantified using a BCA kit. Samples containing the proteins were subjected to SDS-PAGE to separate the proteins, followed by electrotransfer to a membrane. The membranes containing the proteins were treated with 5% skim milk to block them, and then allowed to react with primary antibody overnight at 4°C. After washing with PBS-T, the membranes were allowed to react with secondary antibody at room temperature for 1 hour, followed by another wash with PBS-T. The results were then detected using a Gel Doc (Bio-Rad, Hercules, CA, USA) and Western blotting assay (Elpis Biotech, Daejeon, Korea). Anti-MyCH (Santa Cruz, USA), anti-α-actin (Santa Cruz, USA), anti-α-SMA (Abcam, UK), anti-IL-6 (Santa Cruz, USA), and anti-α-tubulin (Santa Cruz, USA) were used as primary antibodies.

[0100] As a result, Figure 15 and Figure 16As shown, treatment with the peptide in SEQ ID NO: 1 confirmed an increase in the expression of MyHC and α-actin, myofiber components, in BaCl2-induced muscle injury tissue. Additionally, as... Figure 17 and Figure 18 As shown, peptide treatment with SEQ ID NO: 1 confirmed that the expression of α-SMA and IL-6, inflammatory proteins, was reduced in tissues with BaCl2-induced muscle damage.

[0101] In summary, the experimental results above indicate that the peptide according to one embodiment not only regenerates or restores damaged muscles, but also helps to inhibit the progression of muscle damage, such as the inflammatory response that causes muscle damage.

[0102] [Dosage Form Example 1. Fabrication of Peptide Nanobody]

[0103] 50 mg of the peptide from Example 1 was dissolved in 500 ml of distilled water by thorough stirring. The conjugate solution was then mixed with 5 g of lecithin, 0.3 ml of sodium oleate, 50 ml of ethanol, and some oil phase. The volume was adjusted to 1 L with distilled water, and then emulsified under high pressure using a microfluidic apparatus to produce peptide nanobody sizes of approximately 100 nm.

[0104] [Dosage Form Example 2. Pharmaceutical Preparation]

[0105] 【2-1. Manufacturing of Powders】

[0106] Mix the following ingredients and fill them into a sealed bag to make a powder.

[0107] The peptide of the present invention, 20 mg

[0108] Lactose 100 mg

[0109] 10 mg of talc

[0110] 【2-2. Tablet Manufacturing】

[0111] Mix the following ingredients and then compress them into tablets according to conventional tablet manufacturing methods.

[0112] The peptide of the present invention, 10 mg

[0113] 100 mg of corn starch

[0114] Lactose 100 mg

[0115] Magnesium stearate 2 mg

[0116] [2-3. Manufacturing of Capsules]

[0117] According to conventional capsule manufacturing methods, the following ingredients are mixed and filled into gelatin capsules to manufacture capsules.

[0118] The peptide of the present invention, 10 mg

[0119] 3 mg of crystalline cellulose

[0120] Lactose 14.8 mg

[0121] Magnesium stearate 0.2 mg

[0122] [2-4. Manufacturing of Injectables]

[0123] According to the conventional manufacturing method for injectable drugs, each ampoule (2 ml) is made with the following ingredient content.

[0124] The peptide of the present invention, 10 mg

[0125] Mannitol 180 mg

[0126] 2974 mg of sterile distilled water for injection

[0127] Na2HPO4·2H2O 26 mg

[0128] 【2-5. Manufacturing of Liquids】

[0129] According to the conventional liquid manufacturing method, each component is dissolved in purified water, and after mixing the following components, purified water is added to adjust the total volume to 100 ml. Then, it is filled into a brown bottle and sterilized to manufacture the liquid.

[0130] The peptide of the present invention, 10 mg

[0131] 10 g of isomers

[0132] Mannitol 5 g

[0133] Appropriate amount of purified water

[0134] The above description of the present invention is for illustrative purposes only. Those skilled in the art will understand that it can be easily modified into other specific forms without changing the technical concept or essential features of the invention. Therefore, the above embodiments should be understood as illustrative in all respects and not restrictive.

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 bound to any one of the following protecting groups selected from the group consisting of: acetyl, fluorenylmethoxycarbonyl, formyl, palmitoyl, myristyl, stearyl, butoxycarbonyl, allyloxycarbonyl, and polyethylene glycol.

3. The peptide according to claim 1, wherein, The C-terminus of the peptide is bound to any one of the following protecting groups selected from the group consisting of: amino (NH2), tertiary alkyl, and hydrazine (NHNH2).

4. A pharmaceutical composition for the prevention or treatment of muscle diseases, comprising the peptide as an active ingredient as described in any one of claims 1 to 3.

5. Use of the peptide comprising the amino acid sequence of SEQ ID NO: 1 in the preparation of compositions for the prevention or treatment of muscle diseases, wherein, The muscle disease mentioned is any one of sarcopenia, muscular dystrophy, and amyotrophic lateral sclerosis.

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