Composition for preventing or treating muscle loss-related diseases comprising peroxisome functional activation inducer or peroxisome or peroxisome mimetic as active ingredient, and composition for preventing, ameliorating or treating hair loss comprising peroxisome as active ingredient

Abstract

The present invention relates to a composition for preventing or treating muscle loss-related diseases, comprising a peroxisome functional activation inducer as an active ingredient, and it has been confirmed that, when acyl CoA is not oxidized by the β oxidation system of peroxisome and / or when peroxisome biosynthetic factors are knocked down, muscle differentiation is inhibited, muscle atrophy is induced, and the progression rate and development of the induced muscle atrophy deteriorate. On the other hand, the present invention can be effectively used as a preventive or therapeutic agent for muscle loss-related diseases, in that the muscle atrophy effect is recovered as the function of peroxisome is restored. The present invention relates to a pharmaceutical composition for preventing or treating muscle loss-related diseases, comprising peroxisome or a peroxisome mimetic as an active ingredient, and through investigation of the relationship between peroxisomal β-oxidation dysregulation and sarcopenia, it was confirmed that peroxisomal dysfunction indirectly impairs mitochondrial efficacy, interferes with metabolic equilibrium, and can contribute to muscle damage through altered lipid metabolism, oxidative stress, and inflammation development. It was also confirmed that peroxisome or a peroxisome mimetic had a remarkably excellent treatment effect when administered to a muscle damage model, and thus the present invention can be used as an innovative and promising strategy for preventing or treating muscle loss. The present invention relates to a composition for preventing, ameliorating or treating hair loss, comprising peroxisome as an active ingredient, and provides a peroxisome complex to which is conjugated hyaluronic acid into which a thiol group has been introduced. The complex of the present invention induces peroxisomal activation, thereby exhibiting the effects of increasing the total number and size of hair follicles, increasing the proportion of hair follicles in the anagen phase, and increasing the area of hair follicles per unit area. Moreover, by having confirmed all of the effects of increasing the number and size of blood vessels, increasing skin fibroblast proliferation and migration activity, and increasing hair growth, the present invention can be effectively used as an excellent composition for preventing, ameliorating or treating hair loss.

Description

A composition for preventing or treating a disease related to muscle loss, comprising a peroxisome function inducer, or a peroxisome or a peroxisome mimic as an active ingredient, and a composition for preventing, improving, or treating hair loss, comprising a peroxisome as an active ingredient

[0001] The present invention relates to a composition for preventing or treating a disease related to muscle loss, comprising a peroxisome function activation inducer, or a peroxisome or a peroxisome mimic as an active ingredient, and a composition for preventing, improving, or treating hair loss, comprising a peroxisome as an active ingredient.

[0002] This application claims priority to Korean Patent Application No. 10-2023-0187681, filed December 20, 2023, the entire disclosure of which is incorporated herein by reference.

[0003] This application claims priority to Korean Patent Application No. 10-2024-0068902, filed May 27, 2024, the entire disclosure of which is incorporated herein by reference.

[0004] This application claims priority to Korean Patent Application No. 10-2024-0085337, filed June 28, 2024, the entire disclosure of which is incorporated herein by reference.

[0005] [Composition for preventing or treating muscle-loss-related diseases containing a peroxisome function inducer as an active ingredient and pharmaceutical composition for preventing or treating muscle-loss-related diseases containing peroxisome or peroxisome mimetic as an active ingredient]

[0006] Sarcopenia is a condition characterized by a decline in skeletal muscle mass and function, encompassing both atrophy (wasting) and a decrease in the number (hypoplasia) of muscle fibers. Sarcopenia is known to be caused by various factors, including aging, hormonal imbalances, nutritional deficiencies, lack of physical activity, inflammation, and degenerative diseases. Advances in medical technology and the development of various treatments have led to increased life expectancy worldwide, leading to a growing aging population. Consequently, the demand for sarcopenia treatment is expected to continue to grow.

[0007] Muscle atrophy is a pathological phenomenon characterized by a decrease in muscle fiber diameter due to a decrease in muscle cell proteins, microtubules, and cytoplasm. This type of muscle atrophy causes muscle weakness and increased muscle fatigue, and is known to contribute to musculoskeletal damage, joint immobility, ligament and joint damage, joint inflammation, sepsis, cancer, and aging.

[0008] At this time, quantitative and qualitative decline and reduction of skeletal muscle mass lead to a decline in quality of life, such as a decline in exercise ability, and this leads to a vicious cycle of worsening muscle atrophy due to lack of exercise.

[0009] A representative example of a muscle wasting disease is muscular dystrophy. A disease characterized by the gradual loss of muscle mass throughout the body, muscular atrophy can be caused by a variety of factors, including lack of mechanical stimulation, starvation, and cancer. Atrophy can be defined as the loss of muscle tissue resulting from disuse, disease of the muscle itself, or damage to the nerves that control it. Typically, disuse can lead to a significant loss of muscle strength, which can gradually progress to muscular atrophy. Muscle atrophy caused by disease of the muscle itself includes myasthenia gravis, muscular dystrophy (progressive muscular dystrophy, myotonic dystrophy, Duchenne, Becker, limb-girdle, facioscapulohumeral), and inflammation that occurs in the muscle itself. Muscle atrophy caused by damage to the nerves that control the muscles includes spinal muscular amyotrophy (Berardnig-Hoffmann, Kugelberg-Welander disease), amyotrophic lateral sclerosis (ALS): Lou Gehrig's disease, and spinobular muscular atrophy (Kennedy's disease).

[0010] Muscular dystrophy is classified into skeletal and spinal muscular atrophy, and in the case of skeletal muscular dystrophy, the number of patients is rapidly increasing due to the aging population and rapid changes in eating habits, and it is emerging as a serious social problem.

[0011] Representative skeletal muscle dystrophies include sarcopenia and disease-related muscle wasting. Patients suffering from these conditions experience a gradual loss of muscle function and mass due to failure of normal muscle regeneration following muscle wasting. Currently, only symptomatic treatment and exercise therapy are available, and no effective treatments are available. Although sarcopenia received an FDA disease code in 2016, there is an urgent need for effective treatment, necessitating the development of new drugs.

[0012] Despite this technical background, no agent using peroxisome function inducer has been reported for the treatment of muscle atrophy-related diseases.

[0013]

[0014] [Composition for preventing, improving, or treating hair loss containing peroxisome as an active ingredient]

[0015] Hair loss refers to the absence of hair in areas where hair normally grows, and refers to the loss of terminal hair on the scalp. Hair loss can be categorized into androgenetic alopecia, female pattern baldness, telogen effluvium, alopecia areata, and M-shaped alopecia. Causes of hair loss include genetics, stress, environmental pollution, poor eating habits, excessive dieting, drinking, smoking, and ultraviolet rays, but the exact cause of hair loss has not been identified. Hair loss can cause inconvenience in daily life and, in severe cases, can lead to extreme stress, making hair loss treatment crucial in modern society.

[0016] Treatment options for hair loss include surgical options, such as hair transplants, and non-surgical options, such as medication. Currently, medications approved for hair loss treatment in Korea and abroad include minoxidil, Propecia, and dutasteride. However, commercially available formulations of Propecia and dutasteride have been reported to cause side effects such as erectile dysfunction, decreased libido, ejaculation disorders, and gynecomastia, including breast tenderness and enlargement. Furthermore, pregnant women can cause abnormalities in the external genitalia of a male fetus, making them contraindicated for pregnant women or women of childbearing potential.

[0017] In addition, when taking minoxidil orally, side effects such as fluid retention, tachycardia, nausea, dyspnea, and gynecomastia in men have been reported. When applied to the affected area for a long period of time, side effects such as scalp irritation symptoms such as scalp dryness, itching, and erythema, and allergic contact dermatitis have also been reported. Although minoxidil slows down hair loss, it is difficult to expect the effect of thick hair growing long and thick, and it is difficult to expect satisfactory results in people who have completely lost hair.

[0018] Despite the growing interest in hair loss prevention, there are few reports of hair loss treatments that can replace drugs with problematic side effects while still being effective.

[0019] [Composition for preventing or treating diseases related to muscle loss, containing a peroxisome function inducer as an active ingredient]

[0020] One object of the present invention is to provide a pharmaceutical composition for preventing or treating a disease related to muscle loss, comprising a peroxisome function activation inducer as an active ingredient.

[0021] Another object of the present invention is to provide a kit for preventing or treating sarcopenia or muscular dystrophy, comprising a peroxisome function activation inducer and an instruction manual.

[0022]

[0023] [Pharmaceutical composition for preventing or treating muscle wasting-related diseases containing peroxisome or peroxisome mimic as an active ingredient]

[0024] The purpose of the present invention is to provide a pharmaceutical composition for preventing or treating a disease related to muscle loss, comprising at least one selected from the group consisting of peroxisomes and peroxisome mimics as an active ingredient.

[0025] Another object of the present invention is to provide a kit for preventing or treating a disease related to muscle loss, comprising a composition comprising at least one active ingredient selected from the group consisting of peroxisomes and peroxisome mimics, and an instruction manual.

[0026]

[0027] [Composition for preventing, improving, or treating hair loss containing peroxisome as an active ingredient]

[0028] The purpose of the present invention is to provide a pharmaceutical composition for preventing or treating hair loss, which contains peroxisome as an active ingredient.

[0029] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating hair loss, which comprises at least one of peroxisome and peroxisome bound to hyaluronic acid with a thiol group as an active ingredient.

[0030] Another object of the present invention is to provide a kit for preventing or treating hair loss, comprising a composition comprising at least one of peroxisome and peroxisome bound to hyaluronic acid with a thiol group introduced therein as an active ingredient, and an instruction manual.

[0031] Another object of the present invention is to provide a health functional food composition for improving hair loss, promoting hair growth, promoting hair growth, or improving scalp condition, which comprises at least one of peroxisome or peroxisome combined with hyaluronic acid having a thiol group introduced therein as an active ingredient.

[0032] Another object of the present invention is to provide a cosmetic composition for improving hair loss, promoting hair growth, or improving scalp condition, which comprises at least one of peroxisome and peroxisome combined with hyaluronic acid having a thiol group introduced therein as an effective ingredient.

[0033] Another object of the present invention is to provide a method for producing a peroxisome bound to hyaluronic acid having a thiol group introduced therein, comprising the following steps:

[0034] (S1) A step of producing hyaluronic acid (HA-SH) with a thiol group introduced therein by reacting a solution containing dissolved hyaluronic acid with a solution containing dissolved EDC, HOBT, and cystamine; and

[0035] (S2) A step of producing a peroxisome in which hyaluronic acid with a thiol group introduced is combined by mixing the peroxisome and the above HA-SH.

[0036]

[0037] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by a person having ordinary skill in the technical field to which the present invention belongs from the description below.

[0038] [Composition for preventing or treating diseases related to muscle loss, containing a peroxisome function inducer as an active ingredient]

[0039] The present invention provides a pharmaceutical composition for preventing or treating a disease related to muscle loss, comprising a peroxisome function activation inducer as an active ingredient.

[0040] In one embodiment of the present invention, the peroxisome function activation inducer may include, but is not limited to, a peroxisome β oxidation activation inducer.

[0041] In one embodiment of the present invention, the peroxisome β-oxidation activity inducer may include, but is not limited to, an activity inducer for at least one of acyl CoA thioesterase and nudix hydrolase.

[0042] In one embodiment of the present invention,

[0043] The above acyl CoA thioesterase may be at least one selected from the group consisting of ACOT 1, ACOT 2, ACOT 3, ACOT 4, ACOT 5, ACOT 6, ACOT 7, ACOT 8, ACOT 9, ACOT 10, ACOT 11, and ACOT 12, but is not limited thereto.

[0044] In one embodiment of the present invention, the nudix hydrolase may be at least one selected from the group consisting of NUDT7, NUDT19, and NUDT8, but is not limited thereto.

[0045] In one embodiment of the present invention, the peroxisome β oxidation activity inducer may further include, but is not limited to, an acyl CoA oxidase (ACOX) activity inducer.

[0046] In one embodiment of the present invention, the acyl CoA oxidase may be at least one selected from the group consisting of ACOX1, ACOX2, ACOX3, and ACOX4, but is not limited thereto.

[0047] In one embodiment of the present invention, the peroxisome β oxidation activity inducer further includes an acyl CoA oxidase (ACOX) activity inducer,

[0048] The above acyl CoA oxidase may be at least one selected from the group consisting of ACOX1, ACOX2, ACOX3, and ACOX4, but is not limited thereto.

[0049] In one embodiment of the present invention, the peroxisome function activation inducer may include, but is not limited to, a peroxisomal biogenesis factor (PEX) activation inducer.

[0050] In one embodiment of the present invention, the peroxisome biosynthesis factor may be at least one selected from the group consisting of Pex2, Pex5, Pex7, Pex11, Pex13, Pex16, and Pex19, but is not limited thereto.

[0051] In one embodiment of the present invention, the peroxisome function activation inducer further includes a peroxisomal biogenesis factor (PEX) activation inducer,

[0052] The above peroxisome biosynthesis factor may be at least one selected from the group consisting of Pex2, Pex5, Pex7, Pex11, Pex13, Pex16, and Pex19, but is not limited thereto.

[0053] In one embodiment of the present invention, the activation inducer may be, but is not limited to, one or more of an overexpression agent or an activating agent.

[0054] In one embodiment of the present invention, the overexpression agent or activation agent may be any one selected from the group consisting of a recombinant vector for overexpressing a protein to be overexpressed or activated or a gene encoding the same, a compound that specifically binds to the gene, a peptide, an aptamer, a primer, a probe, and an antibody, but is not limited thereto.

[0055] In one embodiment of the present invention, the muscle loss-related disease may be at least one selected from the group consisting of sarcopenia, muscular atrophy, myasthenia, muscular dystrophy, myotonia, hypotonia, muscular weakness, amyotrophic lateral sclerosis (ALS), and muscular dystrophy, but is not limited thereto.

[0056] In one embodiment of the present invention, the muscle wasting related disease may be induced by, but is not limited to, fatty acids, dexamethasone, BaCl2, denervation, and myocardial infarction.

[0057] In one embodiment of the present invention, the composition may be characterized by one or more of the following, but is not limited thereto:

[0058] Increases root canal length and formation; and

[0059] Increases the area of ​​muscle fibers.

[0060] The present invention provides a kit for preventing or treating sarcopenia or muscular dystrophy, comprising a peroxisome function activation inducer and an instruction manual.

[0061] In one embodiment of the present invention, the peroxisome function activation inducer may include, but is not limited to, a peroxisome β oxidation activation inducer.

[0062] In one embodiment of the present invention, the muscle loss-related disease may be at least one selected from the group consisting of sarcopenia, muscular atrophy, myasthenia, muscular dystrophy, myotonia, hypotonia, muscular weakness, amyotrophic lateral sclerosis (ALS), and muscular dystrophy, but is not limited thereto.

[0063]

[0064] In addition, the present invention provides a method for preventing or treating a disease related to muscle loss, comprising a step of administering a composition containing a peroxisome function activation inducer as an active ingredient in a pharmaceutically effective amount to a subject in need thereof.

[0065] In addition, the present invention provides a composition comprising a peroxisome function activation inducer as an active ingredient for use in preventing or treating diseases related to muscle loss.

[0066] In addition, the present invention provides the use of a composition comprising a peroxisome function inducer as an active ingredient for preparing a preparation for preventing or treating a disease related to muscle loss.

[0067]

[0068] [Pharmaceutical composition for preventing or treating muscle wasting-related diseases containing peroxisome or peroxisome mimic as an active ingredient]

[0069] The present invention provides a pharmaceutical composition for preventing or treating a disease related to muscle loss, comprising at least one selected from the group consisting of peroxisomes and peroxisome mimics as an active ingredient.

[0070] In one embodiment of the present invention, the peroxisome may be additionally combined with chitosan oligosaccharide, but is not limited thereto.

[0071] In one embodiment of the present invention, the peroxisome mimic may be, but is not limited to, a nanozyme.

[0072] In one embodiment of the present invention, the nanozyme may be, but is not limited to, hyaluronic acid-adipic acid dihydrazide-hemin (HA-ADH-hemin).

[0073] In one embodiment of the present invention, the muscle loss-related disease may be at least one selected from the group consisting of sarcopenia, muscular atrophy, myasthenia, muscular dystrophy, myotonia, hypotonia, muscular weakness, amyotrophic lateral sclerosis (ALS), and muscular dystrophy, but is not limited thereto.

[0074] In one embodiment of the present invention, the peroxisome may be included at a concentration of more than 0 to 900 μg / ml based on the entire composition, but is not limited thereto.

[0075] In one embodiment of the present invention, the peroxisome mimic may be included at a concentration of more than 0 to 1 mg / ml based on the total composition, but is not limited thereto.

[0076] In one embodiment of the present invention,

[0077] The composition may be characterized by one or more selected from the group consisting of, but not limited to:

[0078] (a) Recovery of reduced root canal diameter and length;

[0079] (b) muscle fiber size is restored;

[0080] (c) root canal is formed;

[0081] (d) reduced inflammation and lipid accumulation; and

[0082] (e) Increased expression of mitochondrial and antioxidant markers.

[0083] The present invention provides a kit for preventing or treating a disease related to muscle loss, comprising a composition comprising at least one active ingredient selected from the group consisting of peroxisomes and peroxisome mimics, and an instruction manual.

[0084]

[0085] In addition, the present invention provides a method for preventing or treating a disease related to muscle loss, comprising administering to a subject in need thereof a pharmaceutically effective amount of a composition comprising at least one active ingredient selected from the group consisting of peroxisomes and peroxisome mimics.

[0086] In addition, the present invention provides a composition comprising at least one selected from the group consisting of peroxisomes and peroxisome mimics as an active ingredient, for use in preventing or treating diseases related to muscle loss.

[0087] In addition, the present invention provides a use for manufacturing a preparation for preventing or treating a disease related to muscle loss, the preparation comprising a composition comprising at least one selected from the group consisting of peroxisomes and peroxisome mimics as an active ingredient.

[0088]

[0089] [Composition for preventing, improving, or treating hair loss containing peroxisome as an active ingredient]

[0090] The present invention provides a pharmaceutical composition for preventing or treating hair loss, comprising peroxisome as an active ingredient.

[0091] In one embodiment of the present invention, the peroxisome may be bound to hyaluronic acid having a thiol group introduced thereto, but is not limited thereto.

[0092] In one embodiment of the present invention, the degree of thiolation of the hyaluronic acid into which the thiol group is introduced may be 40% to 80%, but is not limited thereto.

[0093] In one embodiment of the present invention, the peroxisome may be characterized by at least one selected from the group consisting of, but not limited to:

[0094] Expresses PM70;

[0095] Does not express TOMM20; and

[0096] Contains catalase.

[0097] In one embodiment of the present invention, the composition may be characterized by at least one selected from the group consisting of, but not limited to:

[0098] Activates peroxisomes;

[0099] Increases the number and size of hair follicles;

[0100] Increases the proportion of hair follicles in the growth phase;

[0101] Increases the area of ​​hair follicles per unit area;

[0102] Increase the number and size of blood vessels;

[0103] Increases skin fibroblast proliferation and migration activity; and

[0104] Increases hair growth.

[0105] The present invention provides a pharmaceutical composition for preventing or treating hair loss, comprising at least one of a peroxisome and a peroxisome bound to hyaluronic acid having a thiol group introduced therein as an active ingredient.

[0106] The present invention provides a kit for preventing or treating hair loss, comprising a composition comprising at least one of peroxisome and peroxisome bound to hyaluronic acid with a thiol group introduced therein as an active ingredient, and an instruction manual.

[0107] The present invention provides a health functional food composition for improving hair loss, promoting hair growth, promoting hair growth, or improving scalp condition, which comprises at least one of peroxisome and peroxisome combined with hyaluronic acid having a thiol group introduced therein as an active ingredient.

[0108] The present invention provides a cosmetic composition for improving hair loss, promoting hair growth, or improving scalp condition, comprising at least one of peroxisome and peroxisome combined with hyaluronic acid having a thiol group introduced therein as an effective ingredient.

[0109] The present invention provides a method for producing a peroxisome bound to hyaluronic acid having a thiol group introduced therein, comprising the following steps:

[0110] (S1) A step of producing hyaluronic acid (HA-SH) with a thiol group introduced therein by reacting a solution containing dissolved hyaluronic acid with a solution containing dissolved EDC, HOBT, and cystamine; and

[0111] (S2) A step of producing a peroxisome in which hyaluronic acid with a thiol group introduced is combined by mixing the peroxisome and the above HA-SH.

[0112]

[0113] In addition, the present invention provides a method for preventing, improving, treating, promoting hair growth, promoting hair growth, or improving scalp condition, which comprises administering to a subject in need thereof a pharmaceutically effective amount of a composition comprising at least one of peroxisome and peroxisome bound to hyaluronic acid with a thiol group introduced therein as an active ingredient.

[0114] In addition, the present invention provides a method for preventing hair loss, a method for improving hair loss, a method for promoting hair growth, a method for promoting hair growth, or a method for improving scalp condition, which comprises a step of administering to a subject in need thereof a composition comprising at least one of peroxisome and peroxisome bound to hyaluronic acid having a thiol group introduced therein as an active ingredient in a food or cosmetically effective amount thereof.

[0115] In addition, the present invention provides a composition comprising at least one of peroxisome or peroxisome bound to hyaluronic acid having a thiol group introduced therein as an active ingredient for use in preventing, improving, treating, promoting hair growth, promoting hair growth, or improving scalp condition.

[0116] The present invention provides a use of a composition comprising at least one of peroxisome and peroxisome combined with hyaluronic acid having a thiol group as an active ingredient for manufacturing a preparation for preventing, improving, treating, promoting hair growth, promoting hair growth, or improving scalp condition.

[0117]

[0118] [Composition for preventing or treating diseases related to muscle loss, containing a peroxisome function inducer as an active ingredient]

[0119] According to a composition for preventing or treating a disease related to muscle loss, which contains a peroxisome function inducer as an active ingredient, it was confirmed that when acyl CoA is not oxidized by the β-oxidation system of the peroxisome and / or when a peroxisome biosynthesis factor is knocked down, muscle differentiation is inhibited, muscle atrophy is induced, and the rate and course of the induced muscle atrophy are worsened. On the other hand, since the muscle atrophy effect is restored as the function of the peroxisome is restored, the present invention can be usefully utilized as a preventive or therapeutic agent for a disease related to muscle loss.

[0120]

[0121] [Pharmaceutical composition for preventing or treating muscle wasting-related diseases containing peroxisome or peroxisome mimic as an active ingredient]

[0122] According to a pharmaceutical composition for preventing or treating diseases related to muscle loss, which comprises peroxisomes or peroxisome mimetics as active ingredients, the association between dysregulation of peroxisomal β-oxidation and sarcopenia was investigated, and it was confirmed that peroxisome dysfunction can indirectly impair mitochondrial efficiency, disrupt metabolic balance, and contribute to muscle damage through altered lipid metabolism, oxidative stress, and inflammation. In addition, since it was confirmed that the administration of peroxisomes or peroxisome mimetics to a muscle damage model had a significantly superior therapeutic effect, it can be utilized as an innovative and promising strategy for preventing or treating muscle loss.

[0123]

[0124] [Composition for preventing, improving, or treating hair loss containing peroxisome as an active ingredient]

[0125] According to a composition for preventing, improving, or treating hair loss, which comprises peroxisomes as an active ingredient, a peroxisome complex is provided, wherein hyaluronic acid having a thiol group is combined. The complex of the present invention exhibits the effects of inducing peroxisome activation, thereby increasing the number and size of total hair follicles, increasing the proportion of hair follicles in the anagen phase, and increasing the area of ​​hair follicles per unit area. In addition, it has been confirmed that the complex increases the number and size of blood vessels, increases the proliferation and migration activity of skin fibroblasts, and increases hair growth, and therefore, it can be usefully utilized as an excellent composition for preventing, improving, and treating hair loss.

[0126] [Composition for preventing or treating diseases related to muscle loss, containing a peroxisome function inducer as an active ingredient]

[0127] Figures 1a and 1b show that knockdown of Pex5 and Pex7 induces myotube atrophy.

[0128] Figure 1a shows the bright-field images, cell myotube diameter length, and the results of RT-PCR analysis of mRNA levels of Pex5, MyoD, McK, Mrf5, MYHC, MyoG, Myh-4, and Myh-7 after infecting C2C12 cells with siRNA specific for Pex5 (siPex5).

[0129] Figure 1b shows the results of an experiment performed after infecting C2C12 cells with siRNA (siPex5) specific to Pex7, which is the same experiment as Figure 1a.

[0130] Figures 2 to 3g are experimental results showing that peroxisomal lipid metabolism, particularly fatty acid β-oxidation, plays an important role in muscle atrophy.

[0131] Figure 2 illustrates the general pathological signaling pathways in amyotrophic lateral sclerosis (ALS) patients, Duchenne muscular dystrophy (DMD) and dexamethasone (DEX)-induced atrophy mice.

[0132] Figure 3a shows the results of confirming the infection efficiency through RT-PCR after infecting C2C12 cells with shRNA specifically expressing Acox1.

[0133] Figure 3b shows brightfield images and myotube analyzer images after C2C12 cell differentiation and infection with or without shRNA specific for ACox1 (shACox1) in a time-dependent manner.

[0134] Figure 3c shows the results of measuring the cell myotube diameter and length of C2C12 cells treated with shACox1.

[0135] Figure 3d shows the results of analyzing the mRNA levels of MYHC and MyoG in shACox1-treated C2C12 cells.

[0136] Figure 3e shows bright field images and myotube analyzer images after treatment of C2C12 cells with palmitic acid (50 μM), dicarboxylic acid (50 μM), and phytol (50 μM).

[0137] Figure 3f shows the results of measuring the diameter and length of myotubes of C2C12 cells treated with palmitic acid (50 μM), dicarboxylic acid (50 μM), and phytol (50 μM).

[0138] Figure 3g shows the results of analyzing the mRNA levels of MyHC, MyoG, Myh-4, and Myh-7 in C2C12 cells treated with palmitic acid (50 μM), dicarboxylic acid (50 μM), and phytol (50 μM).

[0139] Figures 4a to 4e are experimental results showing that peroxisomal acyl-CoA metabolism contributes to muscle atrophy and loss of muscle function.

[0140] Figure 4a shows the intramuscular peroxisome-related gene profiles of ALS patients, DMD mice, and DEX-induced atrophy mouse models.

[0141] Figures 4b to 4d are H&E images and transcript levels of atrogenes (Atrogin-1 and MuRF-1) in the muscles of DEN-, MI-, and BaCl2-induced muscle atrophy models.

[0142] Figure 4e shows the results of analyzing peroxisome-related genes and possible pathological signaling pathways in muscles of DEN-, MI-, and BaCl2-induced muscle atrophy models.

[0143] Figure 4f shows the results of immunocytochemical analysis of MyHc.

[0144] Figure 4g shows changes in the expression levels of Atrogin-1, MuRF-1, and Myostatin depending on whether Dex (50 μM) was treated.

[0145] Figure 4h shows the effect of DEX treatment on inducing myotube atrophy in C2C12 cells, and the expression levels of Atrogin-1, MuRF-1, and Mstn were confirmed.

[0146] Figure 4i shows the results of profile analysis of peroxisome-related genes according to treatment with or without Dex (50 μM).

[0147] Figure 5a shows the inhibitory effect of myotube formation according to the knockdown of Acot12 and Nudt7.

[0148] Figure 5b shows the method for producing Acot4 knockout (KO) mice and the results of genotyping.

[0149] Figure 5c shows the expression levels of myogenic differentiation genes in mice deficient in Acot12, Acot4, and Nudt7, or in WT mice.

[0150] Figure 5d shows bright-field images of primary myoblasts isolated from muscles of mice deficient in Acot12, Acot4, and Nudt7, or WT mice.

[0151] Figure 5e shows the results of an experiment using a myotube analyzer in mice deficient in Acot12, Acot4, and Nudt7, or in WT mice.

[0152] Figure 5f shows the results of myotube diameter analysis of mice deficient in Acot12, Acot4, and Nudt7, or WT mice.

[0153] Figure 6a shows the H&E staining results for the tibialis anterior (TA) muscles of 6-month-old and 12-month-old WT and Acot12- / -, Acot4- / -, and Nudt7- / - mice.

[0154] Figure 6b shows the results of analyzing the size and lipid accumulation area of ​​muscle fibers in 6-month-old and 12-month-old WT and Acot12- / -, Acot4- / -, and Nudt7- / - mice.

[0155] Figure 6c shows the results of Oil-Red O staining of the tibialis anterior (TA) muscles of 6-month-old and 12-month-old WT and Acot12- / -, Acot4- / -, and Nudt7- / - mice.

[0156] Figure 6d shows the results of analyzing the correlation between muscle fiber size and lipid accumulation.

[0157] Figures 7a to 7d are experimental results showing that deficiency of Acot12, Acot4, and Nudt7 worsens muscle atrophy caused by BaCl2.

[0158] Figure 7a shows the H&E staining results of the tibialis anterior (TA) muscle 1 day after BaCl2 injury.

[0159] Figure 7b shows the results of analyzing the expression levels of Acot12, Acot4, and Nudt7 by RT PCR by quantifying the muscle fiber size.

[0160] Figure 7c shows representative gross morphology and H&E staining results of tibialis anterior (TA) muscles 1 day after BaCl2 injury in Acot12- / -, Acot4- / -, and Nudt7- / - mice.

[0161] Figure 7d shows the muscle fibers colored differently according to their classified sizes analyzed by Image Pro and quantified the muscle length.

[0162] Figure 8a shows the results of analyzing the introduction efficiency of Acot12, Acot4, and Nudt7.

[0163] Figures 8b to 8g are experimental results showing that muscle atrophy induced by BaCl2 injection was recovered when Acot12, Acot4, and Nudt7 were overexpressed.

[0164] Figure 8b shows bright-field images of muscle cells overexpressing Acot12, Acot4, and Nudt7, immunocytochemical images of MyHC, and myotube analyzer images.

[0165] Figure 8c shows the myotube length and fusion index when Acot12, Acot4, and Nudt7 were overexpressed.

[0166] Figure 8d shows representative gross morphology and H&E staining results of tibialis anterior (TA) muscles injected with lentiviruses containing Acot12 (A12OE), Acot4 (A4OE), and Nudt7 (N7OE) constructs.

[0167] Figure 8e is a bar graph that displays the muscle fibers identified in Figure 8d in different colors according to their size and quantifies the percentage.

[0168] Figure 8f quantifies the size of the muscle fibers identified in Figure 8d.

[0169] Figure 8g shows the expression levels of Acot12, Acot4, and Nudt7 in the muscle fibers identified in Figure 8d confirmed through immunocytochemistry.

[0170]

[0171] [Pharmaceutical composition for preventing or treating muscle wasting-related diseases containing peroxisome or peroxisome mimic as an active ingredient]

[0172] Figure 9a shows the improvement of skeletal muscle atrophy and peroxisome biogenesis in aged mice through heterochronic parabiosis, and shows H&E images of the heterochronic parabiosis model and TA muscle.

[0173] Figure 9b shows the results of muscle fiber size analysis performed on tibialis anterior (TA) muscles of mice across different age groups (young, old, and parabiosis) regarding the effect of decreased peroxisome biogenesis with aging.

[0174] Figure 9c shows the expression levels of peroxisome-related genes in the TA muscle of old mice.

[0175] Figure 9d shows the improvement of skeletal muscle atrophy and peroxisome biogenesis in aged mice through heterochronic parabiosis, and the results of IF staining of PMP70 in TA muscle (scale bar, 100 μm).

[0176] Figures 10a and 10c show the effects of Pex5 and Pex7 knockdown on myotube atrophy and the effects of Pex5 and Pex7 knockdown on muscle cell integrity, respectively (scrambled siRNA used as control (Con). Bright-field images: Scale bar: 200 μm).

[0177] Figure 10b shows the effect of peroxisome dysfunction on myotube atrophy in C2C12 cells, and is the result of myotube characterization of C2C12 cells transfected with siRNA specific for Pex5 (siPex5) and Pex7 (siPex7).

[0178] Figure 10d shows histological and IHC staining results of human skeletal muscle samples obtained from individuals of different ages (44-year-old male (Lt. wrist ORIF), 50-year-old male (Rt. extensor digitorum branch [EDB]), 66-year-old female (Rt. wrist), 76-year-old female (hip), 81-year-old female (quadriceps)) regarding improvement of skeletal muscle atrophy and peroxisome biogenesis in aged mice through heterochronic parabiosis.

[0179] Figures 11a to 11c show the effects of fatty acids and phytol on C2C12 root canals.

[0180] Figures 11a and 11b are bright field images when C2C12 was treated with palmitic acid (50 μM), dicarboxylic acid (50 μM), and phytol (50 μM), and Figure 11c is the result of root canal diameter and length analysis using Image Pro software.

[0181] Figure 11d shows the effect of lipid accumulation on muscle cell differentiation, with the results of analyzing the mRNA levels of MyHC, MyoG, Myh-4, and Myh-7 when C2C12 cells were treated with palmitic acid (50 μM), dicarboxylic acid (50 μM), and phytol (50 μM).

[0182] Figure 11e shows the bright-field image (left) and the results of myotube diameter and length analysis (right) of C2C12 myotubes treated with phytol (50 μM) using Image Pro software regarding muscle damage induced by a phytol diet in the TA muscle.

[0183] Figure 11f shows the effects of fatty acids and phytol on C2C12 myotubes, showing the expression levels of peroxisome-related genes in the tibialis anterior (TA) muscle of phytol-diet mice when C2C12 were treated with palmitic acid (50 μM), dicarboxylic acid (50 μM), and phytol (50 μM).

[0184] Figure 11g shows the effect of lipid accumulation on muscle cell differentiation, showing the expression levels of Acox1 and lipid ROS when C2C12 was treated with palmitic acid (50 μM), dicarboxylic acid (50 μM), and phytol (50 μM) (data are expressed as mean ± standard error (SE), Con, control. *P<0.05, ***P<0.001).

[0185] Figures 11h and 11i relate to muscle damage caused by a phytol diet in the TA muscle.

[0186] Figure 11h shows the expression levels of peroxisome markers PMP70 and ACOX1 in the tibialis anterior (TA) muscle of mice fed a phytol-rich diet.

[0187] Figure 11i shows the H&E image of Figure 11h, the expression level of Atrogin-1, an indicator of muscle atrophy, and the results of muscle fiber size analyzer analysis of TA muscles (data are expressed as mean ± standard error (SE), n=3 per group, CON, control group. *P<0.05, ***P<0.001).

[0188] Figure 12a shows the mRNA levels of catalase, MyoD, McK, Mrf5, MYHC, MyoG, Myh-4, and Myh-7, which are related to the induction of myotube atrophy by knockdown of catalase and Acox1.

[0189] Figures 12b and 12c show the effect of peroxisome dysfunction on myotube atrophy in C2C12 cells.

[0190] Figure 12b shows the results of cell morphology examination, including changes in fiber diameter and length, in C2C12 cells treated with siRNA specific for catalase (siCat).

[0191] Figure 12c shows the time-dependent effect of transfecting differentiated C2C12 cells with shRNA specific for Acox1 (shAcox1).

[0192] Figures 12d to 12g relate to the induction of myotube atrophy by knockdown of catalase and Acox1.

[0193] Figures 12d and 12f show the transfection efficiency of shAcox1.

[0194] Figure 12e shows the mRNA levels of MYHC and MyoG following knockdown of Acox1.

[0195] Figure 12g shows the levels of lipid accumulation (BODIPY™ 493 / 503, green) and lipid ROS (BODIPY™ 581 / 591, red) regardless of the presence or absence of shAcox1(D4-D6) transfection (*P<0.05, **P<0.01, ***P<0.001).

[0196] Figures 13a to 13d relate to the potential role of peroxisomal fatty acid β-oxidation in myotube atrophy.

[0197] Figure 13a shows common pathological signaling pathways between amyotrophic lateral sclerosis (ALS) patients, Duchenne muscular dystrophy (DMD) and dexamethasone (DEX)-induced atrophy mice, and peroxisome-related gene profiles in muscles of ALS patients, DMD mice and DEX-induced atrophy mouse models (right).

[0198] Figure 13b shows intramuscular H&E images (left panel) and transcript levels of Atrogin-1 and MuRF-1 in DEN- (n=5), MI- (n=3), and BaCl2-induced (n=3) muscle atrophy models (right panel) (scale bar: 200 μm).

[0199] Figure 13c shows the results of immunocytochemical analysis of MyHC and the results of analyzing the expression levels of Atrogin-1, MuRF-1, and Mstn (50 μM, 24 hours) regardless of the presence or absence of DEX.

[0200] Figure 13d shows the profile of peroxisome-related genes regardless of Dex treatment (n=3 per group. Data are expressed as mean ± standard error (SE), *P<0.05, **P<0.01, ***P<0.001. con: control; DEN, denervation; MI, muscle ischemia; Mstn, myostatin.)

[0201] Figures 14a and 14b show the effect of peroxisome dysfunction on myotube atrophy in C2C12 cells.

[0202] Figure 14a shows peroxisome-related genes and potential pathological signaling pathways in muscle tissues of DEN-, MI-, and BaCl2-induced muscle atrophy models.

[0203] Figure 14b shows the results of immunohistochemical staining analysis for ACOT12, ACOT4, and NUDT7 in quadriceps femoris tissues from 67- and 81-year-old patients (scale bar, 200 μm. Data are expressed as mean ± SE, n=3 per group, CON, control group. ***P<0.001).

[0204] Figure 14c shows the induction of muscle damage by excessive coenzyme A (CoA), acetyl coenzyme A (acetyl CoA), and succinyl coenzyme A (succinyl CoA). The left image is a bright-field image, and the right graph shows the mRNA expression level of MuRF-1 (data are mean ± standard error (SE), *P<0.05, **P<0.01).

[0205] Figure 14d shows the generation of Acot4 knockout (KO) mice, showing the DNA sequence of the Acot4 locus of living F0 mice - deleted nucleotides and the results of genotyping of Acot4 KO mice.

[0206] Figures 14e to 14j show the aggravating effect of Dex-induced myotube atrophy in Acot12, Acot4, and Nudt7 deficient mice.

[0207] Figure 14e shows a bright field image (scale bar: 200 μm) and the results of root canal analysis in the above mouse.

[0208] Figure 14f shows the expression levels of myogenic differentiation genes in primary myoblasts isolated from the muscles of Acot12- / -, Acot4- / -, and Nudt7- / - mice.

[0209] Figure 14g shows the expression levels of Atrogin-1, MuRF-1, and Mstn in DEX-treated C2C12 cells.

[0210] Figure 14h is a bright field and root canal analysis image for Figure 15g, and Figure 14i is a root canal diameter analysis result.

[0211] Figure 14j shows the expression levels of Atrogin-1 and MuRF-1 in primary myoblasts isolated from muscles of Acot12- / -, Acot4- / -, and Nudt7- / - mice compared to WT mice (data are presented as mean ± standard error (SE). n=3 per group. *P<0.05, **P<0.01, ***P<0.001.).

[0212] Figures 14k to 14l show the effects of aggravating muscle atrophy in Acot12, Acot4, and Nudt7 deficient mice.

[0213] Figure 14k shows H&E staining of the above mouse.

[0214] Figure 14l shows the results of Oil Red O staining of TA muscles from 6- and 12-month-old WT and Acot12- / -, Acot4- / -, and Nudt7- / - mice (myofiber size and lipid accumulation area were measured, and the correlation between myofiber size and lipid accumulation was analyzed (right panel). Original magnification, 100×. n=5 per group.).

[0215] Figures 15a and 15b show the effects of exacerbating muscle atrophy after inducing BaCl2 damage in Acot12, Acot4, and Nudt7 deficient mice.

[0216] Figure 15a shows the results of H&E staining of TA muscle one day (next day) after BaCl2 injury (muscle fibers were sorted by size and analyzed using Image Pro software (lower panel)).

[0217] Figure 15b shows a quantification graph of muscle fiber size and expression levels of Acot12, Acot4, and Nudt7 (right panel) in the mouse of Figure 15a (data are expressed as mean ± standard error (SE). *P<0.05, ***P<0.001.).

[0218] Figures 15c and 15d relate to the role of peroxisomal Acyl-CoA metabolism in muscle atrophy and muscle function impairment.

[0219] Figure 15c shows the results of immunocytochemical analysis of MyHC using Acot12-, Acot4-, or Nudt7-overexpressing myoblasts treated with or without dexamethasone DEX (50 μM) (myotube and fiber size were analyzed and presented as brightfield images (scale bar: 200 μm)).

[0220] Figure 15d shows representative gross morphology and H&E staining analysis results of TA muscles after injection of lentivirus containing Acot12 (A12OE), Acot4 (A4OE), or Nudt7 (N7OE) constructs (top photo and graph). Also shown are H&E staining of TA muscles 1 day after BaCl2-induced injury in wild-type (WT), Acot12- / -, Acot4- / -, and Nudt7- / - mice (photos and graphs below) (Muscle fibers are color-coded according to size categories determined using Image Pro software, and muscle lengths quantified. A12KO, Acot12- / -; A4KO, Acot4, Acot4- / -; N7KO, Nudt7- / -. *P < 0.05 and ***P < 0.001 vs. Con, #P < 0.05 and ###P < 0.001 vs. Dex. n=3 per group.).

[0221] Figures 15e to 15h show the restorative effects of Acot12, Acot4, and Nudt7 on Bacl2-damaged TA muscle atrophy.

[0222] Figure 15e shows a bright-field image of a primary root canal and the results of root canal diameter analysis.

[0223] Figure 15f shows the expression levels of mitochondrial-related genes.

[0224] Figure 15g shows the results of immunohistochemical analysis.

[0225] Figure 15h shows the results of RT-PCR analysis (data are expressed as mean ± standard error (SE) n=3 per group. ***P<0.001.).

[0226] Figures 16a and 16b relate to peroxisome-COS-FITC complexes as a therapeutic strategy for BaCl2-induced muscle atrophy.

[0227] Figure 16a is a schematic diagram showing the therapeutic effect of peroxisome-COS-FITC complex on muscle tissue.

[0228] Figure 16b shows a schematic diagram and scanning electron microscope (SEM) image of the synthesis and chemical structure of the COS-FITC complex.

[0229] Figures 16c to 16e relate to the evaluation of isolated peroxisomes.

[0230] Figure 16c shows the results of immunoblot analysis of peroxisomal membrane protein 70 (PMP70) and catalase. In addition, the catalase activity of isolated peroxisomes and peroxisome-COS-FITC complexes is shown.

[0231] Figure 16d shows an immunofluorescence image of C2C12 cells treated with peroxisome-COS-FITC.

[0232] Figure 16e shows bright-field images and fiber diameter and length analysis results of DEX-induced C2C12 cells with or without peroxisome-COS-FITC treatment (data are expressed as mean ± standard error (SE). **P<0.01, ***P<0.001.)

[0233] Figures 16f and 16g relate to peroxisome-COS-FITC complexes as a therapeutic strategy for BaCl2-induced muscle atrophy.

[0234] Figure 16f shows bioluminescence and fluorescence images of peroxisome-COS-FITC complexes in mice (top) and muscle tissue (bottom) 24 hours after injection of peroxisome-COS-FITC complexes.

[0235] Figure 16g shows representative gross morphology and H&E staining results of TA muscles injected with peroxisome-COS-FITC complex (different colors are used according to the classified size of muscle fibers analyzed by Image Pro. Muscle length is quantified. *P < 0.05, **P < 0.01. n=3 per group.).

[0236] Figures 17a to 17d relate to the effect of nanozyme treatment on BaCl2-induced muscle atrophy.

[0237] Figure 17a shows a schematic representation of a nanozyme-based therapeutic approach targeting muscle atrophy, and Figure 17b shows a schematic of nanozyme synthesis.

[0238] Figure 17c shows a HA-hemin conjugate forming a nanoparticle, which is a polymer network containing a heme group.

[0239] Figure 17d shows the UV-Vis spectrum analysis results of nanozymes, with HA (black, third graph from the top on the Y-axis), hemin (0.05 mg / mL, red, second graph from the top on the Y-axis), HA-ADH-hemin (0.05 mg / mL hemin basis, blue, first graph from the top on the Y-axis), and HA-ADH-hemin (0.05 mg / mL, green, fourth graph from the top on the Y-axis) color-coded respectively.

[0240] Figure 17e shows photographic images of HA-ADH-hemin (0.1 and 0.01 mg / mL hemin) in a pH 7.4 PBS solution (first) and after 2 weeks (second). Hemin in a pH 12 (third) and pH 7.4 PBS solution (fourth) are shown, respectively.

[0241] Figure 17f shows photographic images of HA-ADH-hemin solutions (pH 6, pH 7, pH 8) and HA-ADH-hemin solution (pH 7.4) two weeks after nanozyme synthesis.

[0242] Figure 17g shows the UV-Vis spectra of hemin and HA-ADH-hemin in PBS solution at pH 7.4. Hemin was either dissolved directly at pH 7.4 (black, the third graph from the top on the Y-axis) or dissolved at pH 12 and the pH of the solution was adjusted to pH 7.4 (red, the second graph from the top on the Y-axis). HA-ADH-hemin was dissolved at pH 7.4 (blue, the first graph from the top on the Y-axis). The final hemin standard of the samples was 0.05 mg / mL.

[0243] Figure 17h shows the EDS spectrum of HA-ADH powder.

[0244] Figure 17i shows the particle size distribution of HA-ADH-hemin.

[0245] Figure 17j shows the particle size distribution of HA-ADH-hemin (0.05 and 0.01 mg / mL).

[0246] Figure 17k shows the results of measuring the solubility and turbidity of HA-ADH-hemin over a wide pH range.

[0247] Figure 17l shows the relative activity of HA-ADH-hemin (0.01 to 0.5 mg / ml) in the presence of various H2O2 concentrations (10 mM, 40 mM, 70 mM, and 100 mM).

[0248] Figure 17m shows the effect of nanozyme on preventing or treating myotube atrophy induced by DEX or phytol in a dose-dependent manner in C2C12 cells treated with various doses (1 ug / ml, 5 ug / ml, 10 ug / ml, and 100 ug / ml) of C2C12 cells.

[0249] Figure 17n shows the results of H&E staining and immunocytochemical analysis of ACOX1, PMP70, and PLN2 staining of TA muscle 3 days after BaCl2 injury, after BaCl2 treatment of muscle tissue and injection of nanozyme at various doses (10 ug / ml, 50 ug / ml, 200 ug / ml, and 300 ug / ml).

[0250] Figure 17o shows the expression of peroxisome-related genes in TA muscles damaged by BaCl2, regardless of whether nanozyme was treated.

[0251] Figures 17p to 17s show the effect of nanozyme on improving mitochondrial function of TA muscle induced by BaCl2.

[0252] Figure 17p shows the expression levels of mitochondrial-related genes in TA muscles induced with BaCl2, regardless of nanozyme treatment.

[0253] Figure 17q shows the results of immunohistochemical staining analysis of carnitine palmitoyltransferase 1 (CPT1) in TA muscle.

[0254] Figure 17r shows the results of fluorescence visualization of mitochondria using MitoTracker.

[0255] Figure 17s shows the results of measuring ATP production (data are expressed as mean ± standard error (SE). *P<0.05, **P<0.01, ***P<0.001.).

[0256]

[0257] [Composition for preventing, improving, or treating hair loss containing peroxisome as an active ingredient]

[0258] Figure 18 shows that the separated and extracted peroxisomes express the peroxisome marker PMP70 and do not express the mitochondrial marker TOMM20.

[0259] Figure 19a shows the HA-SH synthesis process.

[0260] Figure 19b shows the nuclear magnetic resonance spectra (1H NMR spectra) of HA and HA-SH.

[0261] Figure 19c shows an experimental method for analyzing the hyaluronic acid decomposition enzyme resistance of HA-SH, and Figure 19d shows the results of analyzing the hyaluronic acid decomposition enzyme resistance of HA-SH.

[0262] Figures 19e and 19f show that the HA-SH-PERO of the present invention maintains the function of peroxisomes even after binding to HA-SH.

[0263] Figure 20 shows the results of staining hair follicles when treated with HA-SH and HA-SH-PERO, respectively.

[0264] Figure 21 shows the results of staining skin tissue when treated with HA-SH and HA-SH-PERO, respectively.

[0265] Figure 22 shows the results of analyzing the growth stage, number, and area of ​​hair follicles per unit area when treated with HA-SH and HA-SH-PERO, respectively.

[0266] Figures 23a and 23b show the results confirming that the number and size of blood vessels increased when HA-SH and HA-SH-PERO were treated, respectively.

[0267] Figures 24a and 24b show the results of confirming the increased activity of fibroblast proliferation and migration when treated with HA-SH and HA-SH-PERO, respectively.

[0268] Figures 25a and 25b show the results of confirming the hair growth effect when treated with HA-SH and HA-SH-PERO, respectively.

[0269] [Composition for preventing or treating diseases related to muscle loss, containing a peroxisome function inducer as an active ingredient]

[0270] The present invention confirmed that when peroxisome function activity was inhibited, the length and number of myotubes decreased, and the size and length of muscle fibers decreased. This is because not only the peroxisome function itself but also fat is excessively accumulated due to the inhibition of function, and it was proven that beta-oxidation, which is a fatty acid oxidation mechanism of peroxisomes, is related and muscle loss occurs when beta-oxidation is inhibited. A peroxisome function activity inducer includes a substance that induces the activity of at least one of the peroxisome function itself and the beta-oxidation function of peroxisomes. In this case, the peroxisome function relates to a peroxisome biosynthesis factor. That is, in the present invention, the peroxisome function activator may be at least one of 1) a peroxisome acyl CoA metabolism activator or 2) a peroxisome biosynthesis factor activator, but is not limited thereto. The beta-oxidation function activation inducer may include an acyl CoA thioesterase or nudix hydrolase / acyl CoA oxidase (ACOX) activity inducer. In one embodiment of the present invention, the present invention was completed by confirming that muscle loss and atrophy were restored when acyl CoA thioesterase or nudix hydrolase was overexpressed.

[0271] The present invention provides a pharmaceutical composition for preventing or treating a disease related to muscle loss, comprising a peroxisome function activation inducer as an active ingredient.

[0272] In the present invention, "peroxisomes" are lysosomes distributed in large numbers in kidney and liver cells. In mammals, they are mainly distributed in the liver and kidney, where they break down toxins, and in plants, they appear in photosynthetic cells and germinating seeds. They are known to perform different functions depending on the cell in which they are present. They have a diameter of approximately 0.4 to 1.3 micrometers, but they contain lattice-shaped granules containing oxidizing enzymes, and the role of these enzymes is to produce hydrogen peroxide (secondary lysosomal). Peroxisomes contain an enzyme called catalase, which breaks down hydrogen peroxide, a substance harmful to the human body, into water and oxygen.

[0273] In particular, peroxisomes are reported to be involved in, but not limited to, the preprocessing of fatty acids for oxidation in mitochondria, with the function of precisely cleaving the chain length of fatty acids, especially long-length fatty acids.

[0274] In the present invention, "beta-oxidation (hereinafter, β-oxidation)" is a reaction that occurs in the matrix of prokaryotic cells or mitochondria, and is a catabolic process in which fatty acids are broken down. During this process, acetyl CoA, which enters the TCA cycle, and NADH and FADH2, which are used in the electron transport chain, are generated. It is called beta-oxidation because the beta carbon of the fatty acid is oxidized to a carbonyl group. Beta-oxidation is mainly promoted by mitochondrial trifunctional proteins and enzyme complexes in the inner mitochondrial membrane. However, fatty acids with excessively long chains are oxidized in peroxisomes.

[0275] In one embodiment of the present invention, the peroxisome function activation inducer may include, but is not limited to, a peroxisome β oxidation activation inducer.

[0276] In the present invention, the peroxisome β-oxidation activity inducer may include a peroxisome acyl CoA metabolism activator, and may mean an agent that decomposes long-chain and very long-chain fatty acyl CoAs by the β-oxidation activator or the acyl CoA metabolism activator, or an agent that increases the decomposition activity of acyl CoAs.

[0277] In one embodiment of the present invention, the peroxisome β-oxidation activity inducer may include, but is not limited to, an activity inducer for at least one of acyl CoA thioesterase and nudix hydrolase.

[0278] In the present invention, “acyl CoA thioesterase (compatible with ACOT)” refers to an enzyme that converts acyl CoA into a fatty acid, and is not excluded by some structural characteristics such as WT (wild type), exogenous, endogenous, recombinant, or mutant, and has a broad meaning that includes any enzyme that can function as a thioesterase capable of converting CoA into a fatty acid. Therefore, in the present invention, all substances in the process of converting acyl CoA into a fatty acid by a biosynthetic enzyme having thioesterase activity may be included.

[0279] In one embodiment of the present invention, the acyl CoA thioesterase may be at least one selected from the group consisting of ACOT 1, ACOT 2, ACOT 3, ACOT 4, ACOT 5, ACOT 6, ACOT 7, ACOT 8, ACOT 9, ACOT 10, ACOT 11, and ACOT 12, but is not limited thereto.

[0280] In the present invention, “nudix hydrolase” refers to a superfamily of hydrolases capable of cleaving a nucleoside diphosphate attached to x (any residue). This reaction can produce nucleoside monophosphate (NMP) and XP. Substrates hydrolyzed by nudix enzymes can include a wide range of organic pyrophosphates with varying degrees of substrate specificity, including nucleoside diphosphates and triphosphates, dinucleosides and diphosphoinositol polyphosphates, nucleotide sugars, and RNA caps.

[0281] In the present invention, the nudix hydrolase may be NUDT15, NUDT21, NUDT6, or NUDT1, and in one embodiment of the present invention, the nudix hydrolase may be at least one selected from the group consisting of NUDT7, NUDT19, and NUDT8, preferably NUDT7, but is not limited thereto.

[0282] In the present invention, the NUDT7 enzyme is one of nudix hydrolases, and the substrate of NUDT7 may be short- and medium-chain acyl-CoAs, but is not limited thereto.

[0283] In the present invention, NUDT19 is known to be involved in peroxisomal lipid metabolism, etc., and NUDT7 and NUDT19 are known to exist primarily in peroxisomes. The substrates of NUDT19 may be, but are not limited to, CoA-SH, short- and medium-chain acyl-CoAs.

[0284] It is known that both NUDT7 and NUDT19 exist within peroxisomes and have the same type of substrate, but their binding sites for CoA are different.

[0285] In the present invention, the NUDT8 enzyme is a nudic hydrolase that mainly exists in mitochondria, and can use CoA-SH, short- and medium-chain acyl-CoAs as substrates, but is not limited thereto.

[0286] In one embodiment of the present invention, the peroxisome β oxidation activity inducer may further include, but is not limited to, an acyl CoA oxidase (ACOX) activity inducer.

[0287] In the present invention, “acyl CoA oxidase (ACOX)” may refer to an enzyme that mediates the following reaction formula:

[0288] .

[0289] This enzyme belongs to the oxidoreductase family, and may be an enzyme that acts specifically on the CH-CH group of a donor that uses oxygen as an acceptor. Acyl CoA oxidase can be commonly used interchangeably with fatty acyl-CoA oxidase, acyl coenzyme A oxidase, or fatty acyl-coenzyme A oxidase. This enzyme is known to be involved in three metabolic pathways: fatty acid metabolism, polyunsaturated fatty acid biosynthesis, and the ppar signaling pathway, and utilizes a single cofactor, FAD.

[0290] In one embodiment of the present invention, the acyl CoA oxidase may be at least one selected from the group consisting of ACOX1, ACOX2, ACOX3, and ACOX4, but is not limited thereto.

[0291] In one embodiment of the present invention, the peroxisome function activation inducer may include, but is not limited to, a peroxisomal biogenesis factor (PEX) activation inducer.

[0292] In one embodiment of the present invention, the peroxisome biosynthesis factor may be at least one selected from the group consisting of Pex2, Pex5, Pex7, Pex11, Pex13, Pex16, and Pex19, but is not limited thereto.

[0293] In the present invention, PEX5 or PEX7 acts as a receptor for matrix protein import and can recognize the peroxisome targeting signal (PTS). PEX5 recognizes the C-terminal PTS1 as Ser-Lys-Leu (SKL), and PEX7 can recognize the N-terminal PTS2 as (R / K)(L / V / I)X5(H / Q)(L / A). Deficiency of Pex5 or Pex7 can result in, but is not limited to, defects in matrix protein import and defects in peroxisomal membrane ghosts.

[0294] Because peroxisomes lack nucleic acids, peroxisomal matrix proteins, including peroxisomal enzymes, are encoded by nuclear genes and synthesized in the cytoplasm. These proteins are then translated and imported across the peroxisomal membrane. This unique mechanism makes accurate protein synthesis for peroxisomal function crucial, as well as their import into the peroxisome. This process may be facilitated by “peroxisomal biogenesis factors.” Proteins imported into the peroxisome typically possess a peroxisome targeting signal (PTS), typically PTS1 at the C-terminus or PTS2 near the N-terminus. PTS1 and PTS2 can be recognized by the PEX5 and PEX7 receptors, respectively. These receptors can transport enzymes into the peroxisome. This process may be facilitated, but is not limited, by the docking factor PEX14.

[0295] In this regard, in one embodiment of the present invention, when PEX5 or PEX7 was knocked down, the expression of the myosin heavy chain gene in myotube cells decreased, the expression of muscle differentiation markers also decreased, and the expression level of myogenic regulatory factors decreased, thereby proving that muscle loss occurs when peroxisome function related to peroxisome biosynthesis factors is inhibited.

[0296] In one embodiment of the present invention, the activation inducer may be, but is not limited to, one or more of an overexpression agent or an activating agent.

[0297] In the present invention, “activation inducer” can be defined in a broad sense to include all substances that activate the expression of a protein related to the peroxisome function of the present invention to increase the level of the protein, for example, an enzyme, etc., in a cell, or to enhance the activity of the protein.

[0298] In the present invention, the term "overexpressing agent" may refer to a substance capable of overexpressing a protein associated with peroxisome function, thereby enhancing the level of a protein associated with peroxisome function within a cell. In this case, "overexpression" or "overenhancement" does not imply a specific, fixed value, but rather refers to a level generally considered to be overexpressed or enhanced in the art, taking into account the type of biological sample being analyzed, the condition of the subject of the sample, and other factors.

[0299] In the present invention, “activating agent” means any substance that enhances the activity of a protein related to peroxisome function.

[0300] In one embodiment of the present invention, the overexpression agent or activation agent may be any one selected from the group consisting of a recombinant vector for overexpressing a protein to be overexpressed or activated or a gene encoding the same, a compound that specifically binds to the gene, a peptide, a protein, a drug, an exosome, a nucleic acid, an aptamer, a primer, a probe, and an antibody, but is not limited thereto.

[0301] In addition, vectors, exosomes, proteins, drugs, etc. may be used as delivery systems for delivering the above-mentioned preparation to target cells, but are not limited thereto, and all general methods that can be applied depending on the concentration of the preparation, single dose, frequency, etc. may be applied.

[0302] In the present invention, “protein” is used interchangeably with “polypeptide” or “peptide” and refers to a polymer of amino acid residues, such as those commonly found in proteins in their natural state.

[0303] In the present invention, “polynucleotide” or “nucleic acid” refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in the form of single or double strands. Unless otherwise limited, known analogs of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides are also included.

[0304] In the present invention, a “vector” is a substance capable of delivering a preparation according to the present invention into a living body, and may be a viral vector or a non-viral vector.

[0305] The above viral vectors include lentiviral vectors, retroviral vectors, adeno-associated virus vectors, etc., and the above non-viral vectors include naked DNA and liposomes, but are not limited thereto.

[0306] The above retroviral vector may include any one that can be applied by a person skilled in the art to deliver the preparation of the present invention, and a representative example thereof is lentivirus, and may be any one or more vectors selected from the group consisting of, for example, HIV-1 lentiviral vector, HIV-2 lentiviral vector, alpharetroviral vector, equine infectious anemia virus (EIAV) lentiviral vector, MoMLV vector, X-MLV vector, P-MLV vector, A-MLV vector, GALV vector, HEV-W vector, SIV-1 vector, FIV-1 vector, and SERV-1-5 vector, but is not limited thereto.

[0307] Methods for delivering the vector of the present invention into a host cell include, but are not limited to, microinjection (Capecchi, MR, Cell, 22:479(1980)), calcium phosphate precipitation (Graham, FL et al., Virology, 52:456(1973)), electroporation (Neumann, E. et al., EMBO J., 1:841(1982)), liposome-mediated transfection (Wong, TK et al., Gene, 10:87(1980)), DEAE-dextran treatment (Gopal, Mol. Cell Biol., 5:1188-1190(1985)), and gene bombardment (Yang et al., Proc. Natl. Acad. Sci., 87:9568-9572(1990)), when the host cell is a eukaryotic cell. No, any method that can be generally applied by a person skilled in the art can be applied.

[0308] In the present invention, “exosome” is a concept that includes exosome analogues, which are small-sized lipid bilayer vesicles, and can contain a wide variety of “cargoes” including, but not limited to, peptides, proteins, lipids, transcriptional regulators, messenger RNA (mRNA), non-coding RNA (ncRNA), and double-stranded DNA (dsDNA).

[0309] The exosomes may originate from a variety of biological fluids, including, but not limited to, saliva, urine, blood, blood plasma, milk, synovial fluid, ascites fluid, sap, and fruit extracts.

[0310] The above exosomes may refer to exosomes naturally secreted from cells, or may include exosomes secreted artificially stimulated through compounds, electrical stimulation, etc. for the purpose of enhancing productivity or physiological activity, but are not limited thereto.

[0311] The size of the above exosomes is not limited, and may include, but is not limited to, exosomes of a general size as well as exosomes of a size manipulated to fit the size of the cargo.

[0312] In one embodiment of the present invention, a recombinant vector containing a gene encoding each of the enzymes, for example, Acot12 (A4OE), Acot4 (A12OE), or Nudt7 (N7OE), was used to overexpress acyl CoA thioesterase or nudix hydrolase, but is not limited thereto, and any method or material for overexpressing or enhancing the activity of the enzyme can exhibit the muscle loss recovery and muscle increase effects confirmed in the present invention.

[0313] In one embodiment of the present invention, the muscle loss-related disease may be at least one selected from the group consisting of sarcopenia, muscular atrophy, myasthenia, muscular dystrophy, myotonia, hypotonia, muscular weakness, amyotrophic lateral sclerosis (ALS), and muscular dystrophy, but is not limited thereto.

[0314] In the present invention, muscle loss may refer to a decrease in muscle mass, and a decrease in muscle mass includes all symptoms and diseases that may appear as a decrease in at least one of a decrease in the size or number of muscle fibers. If the size of muscle fibers decreases, muscular dystrophy may be induced, and if the number of muscle fibers decreases, sarcopenia or muscle hypoplasia may be induced. In addition, in the present invention, muscle loss may include, but is not limited to, a qualitative decrease in which muscle cells are unable to perform the functions that general muscle cells are responsible for.

[0315] In one embodiment of the present invention, the muscle wasting related disease may be induced by, but is not limited to, fatty acids, dexamethasone, BaCl2, denervation, and myocardial infarction.

[0316] In one embodiment of the present invention, the composition may be characterized by one or more of the following, but is not limited thereto:

[0317] Increases root canal length and formation; and

[0318] Increases the area of ​​muscle fibers.

[0319] The pharmaceutical composition according to the present invention may be formulated and used in the form of external preparations such as powders, granules, sustained-release granules, enteric-coated granules, liquids, eye drops, ellipsoids, emulsions, suspensions, alcohols, troches, aromatic waters, limonades, tablets, sustained-release tablets, enteric-coated tablets, sublingual tablets, hard capsules, soft capsules, sustained-release capsules, enteric capsules, pills, tinctures, soft extracts, dry extracts, fluid extracts, injections, capsules, irrigation solutions, ointments, lotions, pastes, sprays, inhalants, patches, sterile injection solutions, or aerosols, according to a conventional method, and the external preparations may have formulations such as creams, gels, patches, sprays, ointments, ointments, lotions, liniments, pastes, or cataplasmas.

[0320] Carriers, excipients and diluents that may be included in the pharmaceutical composition according to the present invention include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0321] When formulating, it is usually prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants.

[0322] The additives of the tablets, powders, granules, capsules, pills, and troches according to the present invention include excipients such as corn starch, potato starch, wheat starch, lactose, sucrose, glucose, fructose, D-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, calcium hydrogen phosphate, calcium sulfate, sodium chloride, sodium bicarbonate, purified lanolin, microcrystalline cellulose, dextrin, sodium alginate, methylcellulose, sodium carboxymethylcellulose, kaolin, urea, colloidal silica gel, hydroxypropyl starch, hydroxypropyl methylcellulose (HPMC), HPMC 1928, HPMC 2208, HPMC 2906, HPMC 2910, propylene glycol, casein, calcium lactate, and Primogel; Gelatin, gum arabic, ethanol, agar powder, cellulose acetate phthalate, carboxymethylcellulose, calcium carboxymethylcellulose, glucose, purified water, sodium caseinate, glycerin, stearic acid, sodium carboxymethylcellulose, sodium methylcellulose, methylcellulose, microcrystalline cellulose, dextrin, hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, refined shellac, starch starch, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl alcohol, polyvinyl pyrrolidone, and binders such as hydroxypropyl methylcellulose, corn starch, agar powder, methylcellulose, bentonite, hydroxypropyl starch, sodium carboxymethylcellulose, sodium alginate, Disintegrants such as carboxymethylcellulose calcium, calcium citrate, sodium lauryl sulfate, anhydrous silicic acid, 1-hydroxypropyl cellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginic acid, amylose, guar gum, baking soda, polyvinylpyrrolidone, calcium phosphate, gelled starch, gum arabic, amylopectin, pectin, sodium polyphosphate, ethylcellulose, sucrose, magnesium aluminum silicate, di-sorbitol solution, and light anhydrous silicic acid;Lubricants such as calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopodium dentata, kaolin, petrolatum, sodium stearate, cacao butter, sodium salicylate, magnesium salicylate, polyethylene glycol (PEG) 4000, PEG 6000, liquid paraffin, hydrogenated soybean oil (Lubri wax), aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, macrogol, synthetic aluminum silicate, anhydrous silicic acid, higher fatty acids, higher alcohols, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, and light anhydrous silicic acid can be used.

[0323] As additives of the liquid formulation according to the present invention, water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, monostearate sucrose, polyoxyethylene sorbitol fatty acid esters (twin esters), polyoxyethylene monoalkyl ethers, lanolin ethers, lanolin esters, acetic acid, hydrochloric acid, ammonia water, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethylcellulose, sodium carboxymethylcellulose, etc. can be used.

[0324] The syrup according to the present invention may include a solution of white sugar, other sugars, or sweeteners, and may also include a fragrance, a coloring agent, a preservative, a stabilizer, a suspending agent, an emulsifier, a viscosity modifier, and the like, as needed.

[0325] Purified water may be used in the emulsion according to the present invention, and emulsifiers, preservatives, stabilizers, fragrances, etc. may be used as needed.

[0326] The suspension according to the present invention may include suspending agents such as acacia, tragacanth, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose (HPMC), HPMC 1828, HPMC 2906, and HPMC 2910, and surfactants, preservatives, stabilizers, colorants, and fragrances may be used as needed.

[0327] The injection according to the present invention includes a solvent such as distilled water for injection, 0.9% sodium chloride injection, Ringer's injection, dextrose injection, dextrose + sodium chloride injection, PEG, lactated Ringer's injection, ethanol, propylene glycol, non-volatile oils - sesame oil, cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate, and benzene benzoate; a solubilizing agent such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidine, propylene glycol, tween, nitrile acid amide, hexamine, and dimethylacetamide; a buffer such as weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, albumins, peptones, and gums; It may include isotonic agents such as sodium chloride; stabilizers such as sodium bisulfite (NaHSO3), carbon dioxide gas, sodium metabisulfite (Na2S2O5), sodium sulfite (Na2SO3), nitrogen gas (N2), and ethylenediaminetetraacetic acid; oxidizing agents such as sodium bisulfite 0.1%, sodium formaldehyde sulfoxylate, thiourea, disodium ethylenediaminetetraacetic acid disodium, and acetone sodium bisulfite; analgesics such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; and suspending agents such as sodium cis-methylenediamine, sodium alginate, Tween 80, and aluminum monostearate.

[0328] The suppository according to the present invention comprises cocoa butter, lanolin, withepsol, polyethylene glycol, glycerogelatin, methylcellulose, carboxymethylcellulose, a mixture of stearic acid and oleic acid, Subanal, cottonseed oil, peanut oil, palm oil, cocoa butter + cholesterol, lecithin, ranet wax, glycerol monostearate, Tween or Span, Imhausen, monolene (propylene glycol monostearate), glycerin, Adeps solidus, Buytyrum Tego-G, Cebes Pharma 16, hexalide base 95, Cotomar, Hydroxocote SP, S-70-XXA, S-70-XX75 (S-70-XX95), Mechanisms such as Hydrokote 25, Hydrokote 711, Idropostal, Massa estrarium (A, AS, B, C, D, E, I, T), Massa-MF, Masupol, Masupol-15, Neosupostal-N, Paramound-B, Suposiro (OSI, OSIX, A, B, C, D, H, L), Suppository type IV (AB, B, A, BC, BBG, E, BGF, C, D, 299), Supostal (N, Es), Wecovi (W, R, S, M, Fs), and Tezester triglyceride basis (TG-95, MA, 57) can be used.

[0329] Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid dosage forms are prepared by mixing the extract with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

[0330] Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

[0331] The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment. The effective dosage level can be determined based on factors including the type and severity of the patient's disease, drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment period, concurrently used drugs, and other factors well known in the medical field.

[0332] The pharmaceutical composition according to the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents. It can be administered sequentially or simultaneously with conventional therapeutic agents, or in single or multiple doses. Taking all of the above factors into account, it is important to administer an amount that achieves maximum efficacy with minimal side effects. This amount can be readily determined by those skilled in the art to which the present invention pertains.

[0333] The pharmaceutical composition of the present invention can be administered to a subject via various routes. All modes of administration are conceivable, including oral ingestion, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, intrathecal injection, sublingual administration, buccal mucosal administration, rectal insertion, vaginal insertion, ocular administration, otic administration, nasal administration, inhalation, oral or nasal spraying, dermal administration, and transdermal administration.

[0334] The pharmaceutical composition of the present invention is determined according to the type of drug as an active ingredient along with various related factors such as the disease to be treated, route of administration, age, sex, weight, and severity of the disease of the patient.

[0335] In the present invention, “subject” means a subject requiring treatment for a disease, and more specifically, means a mammal such as a human or non-human primate, mouse, rat, dog, cat, horse, and cow.

[0336] In the present invention, “administration” means providing a predetermined composition of the present invention to an individual by any appropriate method.

[0337] In the present invention, “prevention” means any action that suppresses or delays the onset of a target disease, “treatment” means any action that improves or beneficially changes a target disease and its metabolic abnormality symptoms by administering a pharmaceutical composition according to the present invention, and “improvement” means any action that reduces a parameter related to a target disease, for example, the severity of a symptom, by administering a composition according to the present invention.

[0338]

[0339] The present invention provides a kit for preventing or treating sarcopenia or muscular dystrophy, comprising a peroxisome function activation inducer and an instruction manual.

[0340] In one embodiment of the present invention, the peroxisome function activation inducer may include, but is not limited to, a peroxisome β oxidation activation inducer.

[0341] In one embodiment of the present invention, the muscle loss-related disease may be at least one selected from the group consisting of sarcopenia, muscular atrophy, myasthenia, muscular dystrophy, myotonia, hypotonia, muscular weakness, amyotrophic lateral sclerosis (ALS), and muscular dystrophy, but is not limited thereto.

[0342] In the present invention, the “kit” refers to a tool that can prevent or treat diseases related to muscle loss by restoring muscle loss or exhibiting the effect of increasing the quantity and / or quality of muscles, including a preparation for activating the peroxisome function of the present invention. In addition to the above substances, the kit of the present invention may include other components, compositions, solutions, devices, etc. that are usually required for methods of storing and processing them. As a specific example, in this case, each component can be applied once or more without limitation in the number of times, there is no limitation on the order in which each substance is applied, and the application of each substance can be performed simultaneously or microscopically.

[0343] In the present invention, the kit may include a container; instructions; and the like. The container may serve to package the substance, and may also serve to store and fix the substance. The material of the container may take the form of, for example, a bottle, a tub, a sachet, an envelope, a tube, an ampoule, and the like, and these may be formed partially or wholly from plastic, glass, paper, foil, wax, and the like. The container may be initially equipped with a completely or partially detachable stopper, which may be part of the container or may be attached to the container by mechanical, adhesive, or other means, and may also be equipped with a stopper for allowing access to the contents by means of a syringe needle. The kit may include an outer package, and the outer package may include instructions for the use of the components.

[0344]

[0345] [Pharmaceutical composition for preventing or treating muscle wasting-related diseases containing peroxisome or peroxisome mimic as an active ingredient]

[0346] The present invention provides a pharmaceutical composition for preventing or treating a disease related to muscle loss, comprising at least one selected from the group consisting of peroxisomes and peroxisome mimics as an active ingredient.

[0347] In the present invention, "peroxisomes" are lysosomes distributed in large numbers in kidney and liver cells. In mammals, they are mainly distributed in the liver and kidney, where they break down toxins, and in plants, they appear in photosynthetic cells and germinating seeds. They are known to perform different functions depending on the cell in which they are present. They have a diameter of approximately 0.4 to 1.3 micrometers, but they contain lattice-shaped granules containing oxidizing enzymes, and the role of these enzymes is to produce hydrogen peroxide (secondary lysosomal). Peroxisomes contain an enzyme called catalase, which breaks down hydrogen peroxide, a substance harmful to the human body, into water and oxygen.

[0348] In particular, peroxisomes are reported to be involved in, but not limited to, the preprocessing of fatty acids for oxidation in mitochondria, with the function of precisely cleaving the chain length of fatty acids, especially long-length fatty acids.

[0349] In the present invention, "beta-oxidation (hereinafter, β-oxidation)" is a reaction that occurs in the matrix of prokaryotic cells or mitochondria, and is a catabolic process in which fatty acids are broken down. During this process, acetyl CoA, which enters the TCA cycle, and NADH and FADH2, which are used in the electron transport chain, are generated. It is called beta-oxidation because the beta carbon of the fatty acid is oxidized to a carbonyl group. Beta-oxidation is mainly promoted by mitochondrial trifunctional proteins and enzyme complexes in the inner mitochondrial membrane. However, fatty acids with excessively long chains are oxidized in peroxisomes.

[0350] Because peroxisomes lack nucleic acids, peroxisomal matrix proteins, including peroxisomal enzymes, are encoded by nuclear genes and synthesized in the cytoplasm. These proteins are then translated and imported across the peroxisomal membrane. This unique mechanism makes accurate protein synthesis for peroxisomal function crucial, as well as their import into the peroxisome. This process may be facilitated by “peroxisomal biogenesis factors.” Proteins imported into the peroxisome typically possess a peroxisome targeting signal (PTS), typically PTS1 at the C-terminus or PTS2 near the N-terminus. PTS1 and PTS2 can be recognized by the PEX5 and PEX7 receptors, respectively. These receptors can transport enzymes into the peroxisome. This process may be facilitated, but is not limited, by the docking factor PEX14.

[0351] In the present invention, the term "peroxisome mimetic" may refer to a broad substance that includes all substances that mimic the function of the peroxisome of the present invention. For example, as exemplarily described, peroxisomes degrade fatty acids by participating in beta-oxidation, which is the first step in the catabolism of fatty acids. In one embodiment of the present invention, it was demonstrated through specific data that peroxisomes can indirectly enhance mitochondrial function and further perform functions for preventing, improving, or treating diseases related to muscle loss. Therefore, in the present invention, the term "peroxisome mimetic" may refer to a preparation that mimics all of the functions of the peroxisome described above, and in particular, may include all functions necessary for producing a therapeutic effect on diseases related to muscle loss. Furthermore, although nanozymes were used as the peroxisome mimetic, the present invention is not limited thereto, and it is obvious to those skilled in the art that if the function of the peroxisome can be mimicked, the therapeutic effect on diseases related to muscle loss observed in one embodiment of the present invention when treating peroxisomes and nanozymes is applied will be exhibited.

[0352] In one embodiment of the present invention, the peroxisome may be additionally combined with, but is not limited to, chitosan oligosaccharide.

[0353] In the present invention, it was confirmed that when peroxisomes were additionally combined with chitosan oligosaccharides, the cartilage-related therapeutic effect increased.

[0354] In the present invention, chitosan oligosaccharides may be expressed by the following structural formula, but are not limited thereto, and may include all compounds judged to be chitosan oligosaccharides in the art, or salts thereof in a pharmaceutically acceptable form.

[0355]

[0356] In the present invention, x:y may be (0.45 to 3) : (7 to 9.55), specifically (0.45 to 1) : (9 to 9.55), and more specifically, 1:9 or 0.45:9.55, but is not limited thereto. According to the art, it may be common to use a ratio of x:y of 1:9 in drug or gene delivery. At this time, the higher the ratio of y, the easier the delivery may be in that the higher the ratio of y, the more amine groups there are, and the better the delivery efficiency of genes, etc. In the sense that x:y of the present invention includes all ratios generally used in chitosan oligosaccharides used to deliver specific substances in the art, and is not limited to a specific ratio.

[0357] In the present invention, peroxisomes may be additionally combined with chitosan oligosaccharides. In this case, peroxisomes may be encapsulated and combined with chitosan oligosaccharides. In one embodiment of the present invention, FITC is combined with chitosan oligosaccharides to encapsulate peroxisomes for observation of peroxisome-chitosan oligosaccharide interactions. However, this is for analysis purposes only and is not limited thereto.

[0358] In one embodiment of the present invention, the peroxisome mimic may be, but is not limited to, a nanozyme.

[0359] In the present invention, "nanozyme" refers to a nanomaterial exhibiting enzyme-like properties. Nanozymes are nanomaterials with unique enzyme-like properties and are known to overcome the limitations of natural enzymes, such as low stability, high cost, and storage difficulties.

[0360] In the present invention, nanozyme was used as a peroxisome mimic, i.e., an artificial peroxisome, and it was confirmed that it exhibited therapeutic activity against muscle loss similar to or greater than that of peroxisomes and modified peroxisomes, thereby proving that it can be utilized as a therapeutic agent for diseases related to muscle loss, including sarcopenia.

[0361] In one embodiment of the present invention, the nanozyme may be, but is not limited to, hyaluronic acid-adipic acid dihydrazide-hemin (HA-ADH-hemin).

[0362] In the present invention, “hyaluronic acid” refers to an anionic, non-sulfated glycosaminoglycan that is widely distributed throughout connective, epithelial, and neural tissues. Hyaluronic acid is known to be one of the major components of the extracellular matrix and to significantly contribute to cell proliferation and migration. It can be expressed by the structural formula below, but is not limited thereto, and includes all pharmaceutically acceptable salts thereof. In addition, in the structural formula below, n can be 1 to 10,000, for example, 20 to 6,000, specifically 100 to 1,000, and more specifically 400 to 600, but is not limited thereto, and can include any n number generally used in the art within the range of 1 to 10,000. In one embodiment of the present invention, 400 to 600 was used as the n value. That is, the number of hyaluronic acids was used in a range of about 400 to 700, specifically 450 to 600, and more specifically about 530, but is not limited thereto, and a range of n (the number of hyaluronic acids) generally used in the art can be used.

[0363]

[0364] In the present invention, “adipic acid dihydrazide” is a chemical substance used for crosslinking an aqueous emulsion, and may be expressed by the following structural formula, but is not limited thereto, and includes all pharmaceutically acceptable salts thereof.

[0365]

[0366] In the present invention, “hemin” means iron (Fe) with a coordination chloride ligand. 3+) is protoporphyrin IX containing the ion. Chemically, hemin is known to differ from the related heme compound hematin in that the coordination ion is primarily the chloride ion of hemin, whereas the coordination ion is the hydroxide ion of hematin. Furthermore, hemin can be produced endogenously in the human body during the replacement of old red blood cells, and several proteins in human blood (hemopexin, serum albumin, etc.) are known to bind to hemin. Hemin can be represented by the following chemical structural formula, but is not limited thereto, and is meant to include all pharmaceutically acceptable salts thereof.

[0367]

[0368] In the present invention, “HA-ADH-hemin” refers to a nanozyme manufactured as a peroxisome mimic, which is a substance manufactured by composing hyaluronic acid, adipic acid dihydrazide, and hemin, and can be expressed by the following structural formula, but is not limited thereto.

[0369]

[0370] In the present invention, x:y may be 1:99 to 50:50, specifically 8:92 to 15:85, but is not limited thereto. In addition, in one embodiment of the present invention, the hemin introduction rate during the production of hyaluronic acid-hemin may be about 8 to 15%, but is not limited thereto.

[0371] In one embodiment of the present invention, the muscle loss-related disease may be at least one selected from the group consisting of sarcopenia, muscular atrophy, myasthenia, muscular dystrophy, myotonia, hypotonia, muscular weakness, amyotrophic lateral sclerosis (ALS), and muscular dystrophy, but is not limited thereto.

[0372] In the present invention, muscle loss may refer to a decrease in muscle mass, and a decrease in muscle mass includes all symptoms and diseases that may appear as a decrease in at least one of a decrease in the size or number of muscle fibers. If the size of muscle fibers decreases, muscular dystrophy may be induced, and if the number of muscle fibers decreases, sarcopenia or muscle hypoplasia may be induced. In addition, in the present invention, muscle loss may include, but is not limited to, a qualitative decrease in which muscle cells are unable to perform the functions that general muscle cells are responsible for.

[0373] In general, there are cases where severe muscle strength loss occurs due to muscle disuse, which gradually progresses to muscle atrophy. Muscle atrophy due to diseases of the muscle itself includes myasthenia gravis, muscular dystrophy (progressive muscular dystrophy, myotonic dystrophy, Duchenne type, Becker type, limb-girdle type, facioscapulohumeral type), and inflammation that occurs in the muscle itself, and muscle atrophy due to damage to the nerves that control the muscles includes spinal muscular amyotrophy (Berardnig-Hoffmann type, Kugelberg-Welander disease), amyotrophic lateral sclerosis (ALS): Lou Gehrig's disease, and spinobular muscular atrophy (Kennedy's disease).

[0374] In one embodiment of the present invention, the peroxisome may be included at a concentration of more than 0 to 900 μg / ml based on the entire composition, but is not limited thereto.

[0375] In the present invention, the peroxisome is greater than 0 and 800 μg / ml, greater than 0 and 700 μg / ml, greater than 0 and 600 μg / ml, greater than 0 and 500 μg / ml, greater than 0 and 400 μg / ml, greater than 0 and 300 μg / ml, greater than 0 and 200 μg / ml, greater than 0 and 100 μg / ml, 10 to 900 μg / ml, 10 to 800 μg / ml, 10 to 700 μg / ml, 10 to 600 μg / ml, 10 to 500 μg / ml, 10 to 400 μg / ml, 10 to 300 μg / ml, 10 to 200 μg / ml, 10 to 100 μg / ml, 20 to 900 μg / ml, 20 to It may be, but is not limited to, 800 μg / ml, 20 to 700 μg / ml, 20 to 600 μg / ml, 20 to 500 μg / ml, 20 to 400 μg / ml, 20 to 300 μg / ml, 20 to 200 μg / ml, 20 to 100 μg / ml, 25 to 900 μg / ml, 25 to 800 μg / ml, 25 to 700 μg / ml, 25 to 600 μg / ml, 25 to 500 μg / ml, 25 to 400 μg / ml, 25 to 300 μg / ml, or 25 to 200 μg / ml.

[0376] In the present invention, excellent therapeutic activity against muscle loss was confirmed when various concentrations of peroxisomes were treated, and for example, concentrations of 25 μg / ml to 100 μg / ml were treated, but the present invention is not limited thereto.

[0377] In one embodiment of the present invention, the peroxisome mimic may be included at a concentration of greater than 0 to 1 mg / ml based on the total composition, but is not limited thereto.

[0378] In the present invention, the peroxisome mimic may be greater than 0 and 0.9 mg / ml, greater than 0 and 0.8 mg / ml, greater than 0 and 0.7 mg / ml, greater than 0 and 0.6 mg / ml, greater than 0 and 0.5 mg / ml, greater than 0 and 0.4 mg / ml, greater than 0 and 0.3 mg / ml, greater than 0 and 0.2 mg / ml, greater than 0 and 0.1 mg / ml, 0.01 to 1 mg / ml, 0.01 to 0.9 mg / ml, 0.01 to 0.8 mg / ml, 0.01 to 0.7 mg / ml, 0.01 to 0.6 mg / ml, 0.01 to 0.5 mg / ml, 0.01 to 0.4 mg / ml, 0.01 to 0.3 mg / ml, or 0.01 to 0.2 mg / ml based on the total composition, but It is not limited.

[0379] In the present invention, we confirmed excellent therapeutic activity against muscle loss when treated with various concentrations of the peroxisome mimetic HA-ADH-hemin. At concentrations ranging from 0.01 mg / ml to 0.1 mg / ml, the peroxisome mimetic exhibited uniform size.

[0380] In one embodiment of the present invention, the composition may be characterized by at least one selected from the group consisting of, but not limited to:

[0381] (a) Recovery of reduced root canal diameter and length;

[0382] (b) muscle fiber size is restored;

[0383] (c) root canal is formed;

[0384] (d) reduced inflammation and lipid accumulation; and

[0385] (e) Increased expression of mitochondrial and antioxidant markers.

[0386] The pharmaceutical composition according to the present invention may be formulated and used in the form of external preparations such as powders, granules, sustained-release granules, enteric-coated granules, liquids, eye drops, ellipsoids, emulsions, suspensions, alcohols, troches, aromatic waters, limonades, tablets, sustained-release tablets, enteric-coated tablets, sublingual tablets, hard capsules, soft capsules, sustained-release capsules, enteric capsules, pills, tinctures, soft extracts, dry extracts, fluid extracts, injections, capsules, irrigation solutions, ointments, lotions, pastes, sprays, inhalants, patches, sterile injection solutions, or aerosols, according to a conventional method, and the external preparations may have formulations such as creams, gels, patches, sprays, ointments, ointments, lotions, liniments, pastes, or cataplasmas.

[0387] Carriers, excipients and diluents that may be included in the pharmaceutical composition according to the present invention include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0388] When formulating, it is usually prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants.

[0389] The additives of the tablets, powders, granules, capsules, pills, and troches according to the present invention include excipients such as corn starch, potato starch, wheat starch, lactose, sucrose, glucose, fructose, D-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, calcium hydrogen phosphate, calcium sulfate, sodium chloride, sodium bicarbonate, purified lanolin, microcrystalline cellulose, dextrin, sodium alginate, methylcellulose, sodium carboxymethylcellulose, kaolin, urea, colloidal silica gel, hydroxypropyl starch, hydroxypropyl methylcellulose (HPMC), HPMC 1928, HPMC 2208, HPMC 2906, HPMC 2910, propylene glycol, casein, calcium lactate, and Primogel; Gelatin, gum arabic, ethanol, agar powder, cellulose acetate phthalate, carboxymethylcellulose, calcium carboxymethylcellulose, glucose, purified water, sodium caseinate, glycerin, stearic acid, sodium carboxymethylcellulose, sodium methylcellulose, methylcellulose, microcrystalline cellulose, dextrin, hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, refined shellac, starch starch, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl alcohol, polyvinyl pyrrolidone, and binders such as hydroxypropyl methylcellulose, corn starch, agar powder, methylcellulose, bentonite, hydroxypropyl starch, sodium carboxymethylcellulose, sodium alginate, Disintegrants such as carboxymethylcellulose calcium, calcium citrate, sodium lauryl sulfate, anhydrous silicic acid, 1-hydroxypropyl cellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginic acid, amylose, guar gum, baking soda, polyvinylpyrrolidone, calcium phosphate, gelled starch, gum arabic, amylopectin, pectin, sodium polyphosphate, ethylcellulose, sucrose, magnesium aluminum silicate, di-sorbitol solution, and light anhydrous silicic acid;Lubricants such as calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopodium dentata, kaolin, petrolatum, sodium stearate, cacao butter, sodium salicylate, magnesium salicylate, polyethylene glycol (PEG) 4000, PEG 6000, liquid paraffin, hydrogenated soybean oil (Lubri wax), aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, macrogol, synthetic aluminum silicate, anhydrous silicic acid, higher fatty acids, higher alcohols, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, and light anhydrous silicic acid can be used.

[0390] As additives of the liquid formulation according to the present invention, water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, monostearate sucrose, polyoxyethylene sorbitol fatty acid esters (twin esters), polyoxyethylene monoalkyl ethers, lanolin ethers, lanolin esters, acetic acid, hydrochloric acid, ammonia water, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethylcellulose, sodium carboxymethylcellulose, etc. can be used.

[0391] The syrup according to the present invention may include a solution of white sugar, other sugars, or sweeteners, and may also include a fragrance, a coloring agent, a preservative, a stabilizer, a suspending agent, an emulsifier, a viscosity modifier, and the like, as needed.

[0392] Purified water may be used in the emulsion according to the present invention, and emulsifiers, preservatives, stabilizers, fragrances, etc. may be used as needed.

[0393] The suspension according to the present invention may include suspending agents such as acacia, tragacanth, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose (HPMC), HPMC 1828, HPMC 2906, and HPMC 2910, and surfactants, preservatives, stabilizers, colorants, and fragrances may be used as needed.

[0394] The injection according to the present invention includes a solvent such as distilled water for injection, 0.9% sodium chloride injection, Ringer's injection, dextrose injection, dextrose + sodium chloride injection, PEG, lactated Ringer's injection, ethanol, propylene glycol, non-volatile oils - sesame oil, cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate, and benzene benzoate; a solubilizing agent such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidine, propylene glycol, tween, nitrile acid amide, hexamine, and dimethylacetamide; a buffer such as weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, albumins, peptones, and gums; It may include isotonic agents such as sodium chloride; stabilizers such as sodium bisulfite (NaHSO3), carbon dioxide gas, sodium metabisulfite (Na2S2O5), sodium sulfite (Na2SO3), nitrogen gas (N2), and ethylenediaminetetraacetic acid; oxidizing agents such as sodium bisulfite 0.1%, sodium formaldehyde sulfoxylate, thiourea, disodium ethylenediaminetetraacetic acid disodium, and acetone sodium bisulfite; analgesics such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; and suspending agents such as sodium cis-methylenediamine, sodium alginate, Tween 80, and aluminum monostearate.

[0395] The suppository according to the present invention comprises cocoa butter, lanolin, withepsol, polyethylene glycol, glycerogelatin, methylcellulose, carboxymethylcellulose, a mixture of stearic acid and oleic acid, Subanal, cottonseed oil, peanut oil, palm oil, cocoa butter + cholesterol, lecithin, ranet wax, glycerol monostearate, Tween or Span, Imhausen, monolene (propylene glycol monostearate), glycerin, Adeps solidus, Buytyrum Tego-G, Cebes Pharma 16, hexalide base 95, Cotomar, Hydroxocote SP, S-70-XXA, S-70-XX75 (S-70-XX95), Mechanisms such as Hydrokote 25, Hydrokote 711, Idropostal, Massa estrarium (A, AS, B, C, D, E, I, T), Massa-MF, Masupol, Masupol-15, Neosupostal-N, Paramound-B, Suposiro (OSI, OSIX, A, B, C, D, H, L), Suppository type IV (AB, B, A, BC, BBG, E, BGF, C, D, 299), Supostal (N, Es), Wecovi (W, R, S, M, Fs), and Tezester triglyceride basis (TG-95, MA, 57) can be used.

[0396] Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid dosage forms are prepared by mixing the extract with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

[0397] Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

[0398] The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment. The effective dosage level can be determined based on factors including the type and severity of the patient's disease, drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment period, concurrently used drugs, and other factors well known in the medical field.

[0399] The pharmaceutical composition according to the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents. It can be administered sequentially or simultaneously with conventional therapeutic agents, or in single or multiple doses. Taking all of the above factors into account, it is important to administer an amount that achieves maximum efficacy with minimal side effects. This amount can be readily determined by those skilled in the art to which the present invention pertains.

[0400] The pharmaceutical composition of the present invention can be administered to a subject via various routes. All modes of administration are conceivable, including oral ingestion, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, intrathecal injection, sublingual administration, buccal mucosal administration, rectal insertion, vaginal insertion, ocular administration, otic administration, nasal administration, inhalation, oral or nasal spraying, dermal administration, and transdermal administration.

[0401] The pharmaceutical composition of the present invention is determined according to the type of drug as an active ingredient along with various related factors such as the disease to be treated, route of administration, age, sex, weight, and severity of the disease of the patient.

[0402] In the present invention, “subject” means a subject requiring treatment for a disease, and more specifically, means a mammal such as a human or non-human primate, mouse, rat, dog, cat, horse, and cow.

[0403] In the present invention, “administration” means providing a predetermined composition of the present invention to an individual by any appropriate method.

[0404] In the present invention, “prevention” means any action that suppresses or delays the onset of a target disease, “treatment” means any action that improves or beneficially changes a target disease and its metabolic abnormality symptoms by administering a pharmaceutical composition according to the present invention, and “improvement” means any action that reduces a parameter related to a target disease, for example, the severity of a symptom, by administering a composition according to the present invention.

[0405] The present invention provides a kit for preventing or treating a disease related to muscle loss, comprising a composition comprising at least one active ingredient selected from the group consisting of peroxisomes and peroxisome mimics, and an instruction manual.

[0406] In the present invention, the “kit” refers to a tool that can prevent or treat a disease related to muscle loss by including a preparation for treating a disease related to muscle loss, including the composition of the present invention, and thereby restoring muscle loss or exhibiting the effect of increasing the quantity and / or quality of muscles. In addition to the above substances, the kit of the present invention may include other components, compositions, solutions, devices, etc. that are usually required for methods of storing and processing them. As a specific example, in this case, each component may be applied once or more without limitation in the number of times, there is no limitation on the order in which each substance is applied, and the application of each substance may be performed simultaneously or microscopically.

[0407] In the present invention, the kit may include a container; instructions; and the like. The container may serve to package the substance, and may also serve to store and fix the substance. The material of the container may take the form of, for example, a bottle, a tub, a sachet, an envelope, a tube, an ampoule, and the like, and these may be formed partially or wholly from plastic, glass, paper, foil, wax, and the like. The container may be initially equipped with a completely or partially detachable stopper, which may be part of the container or may be attached to the container by mechanical, adhesive, or other means, and may also be equipped with a stopper for allowing access to the contents by means of a syringe needle. The kit may include an outer package, and the outer package may include instructions for the use of the components.

[0408]

[0409] [Composition for preventing, improving, or treating hair loss containing peroxisome as an active ingredient]

[0410] The present invention relates to a therapeutic agent for controlling hair loss and promoting hair growth through restoration of peroxisome function, and more particularly, to a method for efficiently separating peroxisomes from cells and a method for dispersing such peroxisomes in a hyaluronic acid polymer network having a thiol group introduced therein, thereby controlling hair loss and promoting hair growth through restoration of peroxisome function, and to a peroxisome-based hair loss control / treatment agent and a method for producing the same. In the present invention, a peroxisome / hyaluronic acid-thiol complex was developed based on a hyaluronic acid polymer having a thiol group introduced therein for efficient delivery of peroxisomes, and finally, the hair loss control and hair growth promoting effects of the peroxisome / hyaluronic acid-thiol complex were confirmed through an animal model, thereby completing the present invention.

[0411] Most existing hair loss treatments utilize drugs like minoxidil or Propecia, primarily focusing on hormonal regulation or blood flow enhancement. However, the present invention proposes a novel mechanism for hair loss treatment by focusing on managing cellular oxidative stress and improving metabolic function through restoration or enhancement of peroxisome function. Therefore, the present invention discloses the excellent hair loss treatment effects of peroxisomes, hyaluronic acid with thiol groups, and other peroxisomes combined with thiol-containing hyaluronic acid.

[0412] Furthermore, the present invention utilizes a method for effectively targeting peroxisomes using hyaluronic acid. While hyaluronic acid itself can have positive effects on skin and hair health, binding to peroxisomes may enable more efficient delivery to specific cells. Furthermore, hyaluronic acid is generally recognized as a safe substance and exhibits a significantly lower risk of side effects, offering significant advantages over chemical / hormonal drugs.

[0413] Accordingly, the present invention provides a pharmaceutical composition for preventing or treating hair loss, which contains peroxisome as an active ingredient.

[0414] In the present invention, "peroxisomes" are lysosomes distributed in large numbers in kidney and liver cells. In mammals, they are primarily distributed in the liver and kidney, where they decompose toxins. In plants, they appear in photosynthetic cells and germinating seeds. They are known to perform different functions depending on the cell in which they are present. They have a diameter of approximately 0.4 to 1.3 micrometers, but contain lattice-shaped granules containing oxidizing enzymes, which produce hydrogen peroxide (secondary lysosomes).

[0415] Peroxisomes contain an enzyme called catalase, which breaks down hydrogen peroxide, a substance harmful to the human body, into water and oxygen. Furthermore, peroxisomes have been reported to be involved in, but not limited to, the preprocessing of fatty acids for oxidation in mitochondria, through their ability to precisely cleave the chain length of fatty acids, particularly long-chain fatty acids.

[0416] In the present invention, peroxisomes were extracted from the liver or kidney of male mice, and were found to express PMP70, one of the membrane proteins of peroxisomes, but TOMM20, a mitochondrial protein marker, was not detected, confirming that they were peroxisomes.

[0417] In one embodiment of the present invention, the peroxisome may be bound to hyaluronic acid having a thiol group introduced thereto, but is not limited thereto.

[0418] In the present invention, “hyaluronic acid” refers to an anionic, non-sulfated glycosaminoglycan that is widely distributed throughout connective, epithelial, and neural tissues. Hyaluronic acid is known to be one of the major components of the extracellular matrix and to significantly contribute to cell proliferation and migration. It can be expressed by the structural formula of the following chemical formula 1, but is not limited thereto, and includes all pharmaceutically acceptable salts thereof. In addition, in the following structural formula 1, n may be 1 to 10,000, for example, 20 to 6,000, specifically 100 to 1,000, and more specifically 400 to 600, but is not limited thereto, and may include any n number generally used in the art within the range of 1 to 10,000. In one embodiment of the present invention, 400 to 600 was used as the n value. That is, the number of hyaluronic acids was used in a range of about 400 to 700, specifically 450 to 600, and more specifically about 530, but is not limited thereto, and a range of n (the number of hyaluronic acids) generally used in the art can be used.

[0419] [Chemical Formula 1]

[0420]

[0421] In the present invention, the “thiol group” refers to an organosulfur compound with a -SH functional group, which can be used interchangeably with a thiol. When the sulfur (S) of the thiol group is replaced with oxygen (O), it becomes an alcohol, and organosulfur compounds containing a thiol group, such as CoA and cysteine, are known to play a very important role and mechanism in relation to detoxification in metabolism.

[0422] In the present invention, the peroxisome may comprise a complex formed by binding hyaluronic acid to which a thiol group has been introduced. This complex may be referred to as "hyaluronic acid to which a thiol group has been introduced (hereinafter, HA-SH)" and may be represented by the following chemical formula 2.

[0423] [Chemical Formula 2]

[0424]

[0425] A thiol group is a substance having a -SH group, and has the property of reversibly combining with -SH in the body and then dissociating again. In addition, the thiol group can form a chelate bond or a coordination bond with metals having a divalent cation, and can also combine with cysteine ​​having a thiol group in proteins or amino acids. Therefore, in the present invention, the thiol group can form a three-dimensional structure that stably positions the peroxisome, and can act as a component that maintains or activates the function of the peroxisome, but is not limited thereto. In the present invention, according to Examples 1 to 8, the excellent hair loss treatment effect of not only the peroxisome but also the peroxisome bound to hyaluronic acid having a thiol group was confirmed.

[0426] In general, research on polymers that have introduced thiol groups has shown that the above characteristics appear when the introduction ratio of thiol groups is 10% or more, and that there is no significant difference when the introduction ratio is 20% to 30% or more.

[0427] Therefore, in the present invention, x:y in HA-SH may be 9:1 to 2:8, specifically, 7:3 to 4:6, and more specifically, in one embodiment of the present invention, HA-SH in a ratio of 4:6 was used, but is not limited thereto.

[0428] In one embodiment of the present invention, the degree of thiolation of the hyaluronic acid into which the thiol group is introduced may be 40% to 80%, and the degree of thiolation of the HA-SH used in the present invention was 60%, but is not limited thereto.

[0429] Accordingly, the thiol group substitution rate of the HA-SH of the present invention may be 10% or more, for example, less than 100%, 95% or more, 90% or more, 85% or more, 80% or more, 75% or more, 70% or more, 65% or more, 60% or more, 55% or more, 50% or more, 45% or more, 40% or more, 39% or more, 38% or more, 37% or more, 36% or more, 35% or more, 34% or more, 33% or more, 32% or more, 31% or more, 30% or more, 29% or more, 28% or more, 27% or more, 26% or more, 25% or more, 24% or more, 23% or more, 22% or more, 21% or more, 20% or more, 19% or more, 18% or more, 17% It may be, but is not limited to, 16% or more, 15% or more, 14% or more, 13% or more, 12% or more, 11% or more, or 10% or more.

[0430] In the present invention, the thiol group substitution rate is analyzed by Ellman's assay, and may mean the number or concentration of thiol groups in the entire sample.

[0431] Ellman's assay is a well-known method for quantifying free thiol groups (unbound thiol groups). In this method, cysteamine is used to analyze the absorbance at 412 nm, and a curve is created based on the absorbance (x-axis) versus the concentration (y-axis). Afterwards, the unknown sample, HA-SH, is measured to quantify the thiol groups. The degree of thiol group substitution is calculated based on the hyaluronic acid monomer. For example, if the degree of thiolation is 60%, it means that 40% of the monomers in the entire hyaluronic acid polymer have remaining carboxyl groups, and 60% are substituted with thiol groups.

[0432] In the present invention, cysteine ​​was replaced with cysteamine, and the unknown sample was considered HA-SH. Therefore, the thiol group substitution rate of HA-SH of the present invention can be reproduced by any method modified within the scope of common sense of those skilled in the art, as long as the method of the known Ellmans's assay is modified.

[0433] In the present invention, the peroxisome bound to the hyaluronic acid with the thiol group introduced therein was produced through a step of first preparing the hyaluronic acid with the thiol group introduced therein and then mixing it with the peroxisome. Therefore, the complex of the present invention, the peroxisome bound to the hyaluronic acid with the thiol group introduced therein, can be used interchangeably as a hyaluronic acid-thiol group-peroxisome (HA-SH-PERO), but is not limited thereto.

[0434] In one embodiment of the present invention, the peroxisome may be characterized by at least one selected from the group consisting of, but not limited to:

[0435] Expresses PM70;

[0436] Does not express TOMM20; and

[0437] Contains catalase.

[0438] In the present invention, it was confirmed that the peroxisome bound to hyaluronic acid having a thiol group is maintained in its unique characteristics and functions of peroxisome in that it expresses PM70, a peroxisome marker, contains catalase, and does not express TOMM20, a mitochondrial marker, despite being bound to hyaluronic acid having a thiol group.

[0439] In one embodiment of the present invention, the composition may be characterized by at least one selected from the group consisting of, but not limited to:

[0440] Activates peroxisomes;

[0441] Increases the number and size of hair follicles;

[0442] Increases the proportion of hair follicles in the growth phase;

[0443] Increases the area of ​​hair follicles per unit area;

[0444] Increase the number and size of blood vessels;

[0445] Increases skin fibroblast proliferation and migration activity; and

[0446] Increases hair growth.

[0447] In general, peroxisomes may lose their function after 24 hours of extraction. Peroxisomes that have been thawed after freezing have differences from peroxisomes that are directly extracted, such as a decrease in size. However, in the present invention, it was confirmed that when peroxisomes are combined with HA-SH, they become relatively stable and maintain their function. That is, in the present invention, when peroxisomes are combined with HA-SH and the complex is processed, it was confirmed that not only do they maintain the function of the peroxisomes, but also that they activate their function. In addition, peroxisomes combined with HA-SH not only maintain their function stably for a long period of time due to their resistance to degradation, but also replace, maintain, or restore the ROS scavenging function of peroxisomes, thereby exhibiting effects of inhibiting, treating, improving, and preventing hair loss.

[0448] When the HA-SH-PERO of the present invention was treated, it was confirmed that the overall number of hair follicles significantly increased, and among them, the number of hair follicles in the growth phase significantly increased.

[0449] The pharmaceutical composition according to the present invention may further comprise suitable carriers, excipients, and diluents commonly used in the manufacture of pharmaceutical compositions. The excipients may be, for example, one or more selected from the group consisting of diluents, binders, disintegrants, lubricants, adsorbents, moisturizers, film-coating materials, and controlled-release additives.

[0450] The pharmaceutical composition according to the present invention may be formulated and used in the form of external preparations such as powders, granules, sustained-release granules, enteric-coated granules, liquids, eye drops, ellipsoids, emulsions, suspensions, alcohols, troches, aromatic waters, limonades, tablets, sustained-release tablets, enteric-coated tablets, sublingual tablets, hard capsules, soft capsules, sustained-release capsules, enteric capsules, pills, tinctures, soft extracts, dry extracts, fluid extracts, injections, capsules, irrigation solutions, ointments, lotions, pastes, sprays, inhalants, patches, sterile injection solutions, or aerosols, according to a conventional method, and the external preparations may have formulations such as creams, gels, patches, sprays, ointments, ointments, lotions, liniments, pastes, or cataplasmas.

[0451] Carriers, excipients and diluents that may be included in the pharmaceutical composition according to the present invention include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

[0452] When formulating, it is usually prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants.

[0453] The additives of the tablets, powders, granules, capsules, pills, and troches according to the present invention include excipients such as corn starch, potato starch, wheat starch, lactose, sucrose, glucose, fructose, D-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, calcium hydrogen phosphate, calcium sulfate, sodium chloride, sodium bicarbonate, purified lanolin, microcrystalline cellulose, dextrin, sodium alginate, methylcellulose, sodium carboxymethylcellulose, kaolin, urea, colloidal silica gel, hydroxypropyl starch, hydroxypropyl methylcellulose (HPMC) 1928, HPMC 2208, HPMC 2906, HPMC 2910, propylene glycol, casein, calcium lactate, and Primogel; Gelatin, gum arabic, ethanol, agar powder, cellulose acetate phthalate, carboxymethylcellulose, calcium carboxymethylcellulose, glucose, purified water, sodium caseinate, glycerin, stearic acid, sodium carboxymethylcellulose, sodium methylcellulose, methylcellulose, microcrystalline cellulose, dextrin, hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, refined shellac, starch starch, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl alcohol, polyvinyl pyrrolidone, and binders such as hydroxypropyl methylcellulose, corn starch, agar powder, methylcellulose, bentonite, hydroxypropyl starch, sodium carboxymethylcellulose, sodium alginate, Disintegrants such as carboxymethylcellulose calcium, calcium citrate, sodium lauryl sulfate, anhydrous silicic acid, 1-hydroxypropyl cellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginic acid, amylose, guar gum, baking soda, polyvinylpyrrolidone, calcium phosphate, gelled starch, gum arabic, amylopectin, pectin, sodium polyphosphate, ethylcellulose, sucrose, magnesium aluminum silicate, di-sorbitol solution, and light anhydrous silicic acid;Lubricants such as calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopodium dentata, kaolin, petrolatum, sodium stearate, cacao butter, sodium salicylate, magnesium salicylate, polyethylene glycol (PEG) 4000, PEG 6000, liquid paraffin, hydrogenated soybean oil (Lubri wax), aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, macrogol, synthetic aluminum silicate, anhydrous silicic acid, higher fatty acids, higher alcohols, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, and light anhydrous silicic acid can be used.

[0454] As additives of the liquid formulation according to the present invention, water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, monostearate sucrose, polyoxyethylene sorbitol fatty acid esters (twin esters), polyoxyethylene monoalkyl ethers, lanolin ethers, lanolin esters, acetic acid, hydrochloric acid, ammonia water, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethylcellulose, sodium carboxymethylcellulose, etc. can be used.

[0455] The syrup according to the present invention may include a solution of white sugar, other sugars, or sweeteners, and may also include a fragrance, a coloring agent, a preservative, a stabilizer, a suspending agent, an emulsifier, a viscosity modifier, and the like, as needed.

[0456] Purified water may be used in the emulsion according to the present invention, and emulsifiers, preservatives, stabilizers, fragrances, etc. may be used as needed.

[0457] The suspension according to the present invention may include suspending agents such as acacia, tragacanth, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose (HPMC), HPMC 1828, HPMC 2906, and HPMC 2910, and surfactants, preservatives, stabilizers, colorants, and fragrances may be used as needed.

[0458] The injection according to the present invention includes a solvent such as distilled water for injection, 0.9% sodium chloride injection, Ringer's injection, dextrose injection, dextrose + sodium chloride injection, PEG, lactated Ringer's injection, ethanol, propylene glycol, non-volatile oils - sesame oil, cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate, and benzene benzoate; a solubilizing agent such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidine, propylene glycol, tween, nitrile acid amide, hexamine, and dimethylacetamide; a buffer such as weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, albumin, peptone, and gums; It may include isotonic agents such as sodium chloride; stabilizers such as sodium bisulfite (NaHSO3), carbon dioxide gas, sodium metabisulfite (Na2S2O5), sodium sulfite (Na2SO3), nitrogen gas (N2), and ethylenediaminetetraacetic acid; oxidizing agents such as sodium bisulfite 0.1%, sodium formaldehyde sulfoxylate, thiourea, disodium ethylenediaminetetraacetic acid, and acetone sodium bisulfite; analgesics such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; and suspending agents such as sodium cis-methylenediamine, sodium alginate, Tween 80, and aluminum monostearate.

[0459] The suppository according to the present invention comprises cocoa butter, lanolin, withepsol, polyethylene glycol, glycerogelatin, methylcellulose, carboxymethylcellulose, a mixture of stearic acid and oleic acid, Subanal, cottonseed oil, peanut oil, palm oil, cocoa butter + cholesterol, lecithin, ranet wax, glycerol monostearate, Tween or Span, Imhausen, monolene (propylene glycol monostearate), glycerin, Adeps solidus, Buytyrum Tego-G, Cebes Pharma 16, hexalide base 95, Cotomar, Hydroxocote SP, S-70-XXA, S-70-XX75 (S-70-XX95), Mechanisms such as Hydrokote 25, Hydrokote 711, Idropostal, Massa estrarium (A, AS, B, C, D, E, I, T), Massa-MF, Masupol, Masupol-15, Neosupostal-N, Paramound-B, Suposiro (OSI, OSIX, A, B, C, D, H, L), Suppository type IV (AB, B, A, BC, BBG, E, BGF, C, D, 299), Supostal (N, Es), Wecovi (W, R, S, M, Fs), and Tezester triglyceride basis (TG-95, MA, 57) can be used.

[0460] Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid dosage forms are prepared by mixing the extract with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

[0461] Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

[0462] The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment. The effective dosage level can be determined based on factors including the type and severity of the patient's disease, drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment period, concurrently used drugs, and other factors well known in the medical field.

[0463] The pharmaceutical composition according to the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents. It can be administered sequentially or simultaneously with conventional therapeutic agents, or in single or multiple doses. Taking all of the above factors into account, it is important to administer an amount that achieves maximum efficacy with minimal side effects. This amount can be readily determined by those skilled in the art to which the present invention pertains.

[0464] The pharmaceutical composition of the present invention can be administered to a subject via various routes. All modes of administration are conceivable, including oral ingestion, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, intrathecal injection, sublingual administration, buccal mucosal administration, rectal insertion, vaginal insertion, ocular administration, otic administration, nasal administration, inhalation, oral or nasal spraying, dermal administration, and transdermal administration.

[0465] The pharmaceutical composition of the present invention is determined according to the type of drug as an active ingredient, along with various related factors such as the disease to be treated, the route of administration, the patient's age, sex, weight, and the severity of the disease. Specifically, the effective dose of the composition according to the present invention may vary depending on the patient's age, sex, and weight, and is generally 0.001 to 150 mg per 1 kg of body weight, preferably 0.01 to 100 mg, administered daily or every other day, or divided into 1 to 3 times a day. However, since the dosage may increase or decrease depending on the route of administration, the severity of the disease, sex, weight, age, etc., the above dosage does not limit the scope of the present invention in any way.

[0466] In the present invention, “subject” means a subject requiring treatment for a disease, and more specifically, means a mammal such as a human or non-human primate, mouse, rat, dog, cat, horse, and cow.

[0467] In the present invention, “administration” means providing a predetermined composition of the present invention to an individual by any appropriate method.

[0468] In the present invention, “prevention” means any action that suppresses or delays the onset of a target disease, “treatment” means any action that improves or beneficially changes a target disease and its metabolic abnormality symptoms by administering a pharmaceutical composition according to the present invention, and “improvement” means any action that reduces a parameter related to a target disease, for example, the severity of a symptom, by administering a composition according to the present invention.

[0469]

[0470] The present invention provides a pharmaceutical composition for preventing or treating hair loss, comprising at least one of a peroxisome and a peroxisome bound to hyaluronic acid having a thiol group introduced therein as an active ingredient.

[0471] As used herein, the term "quasi-drug" refers to preparations used for sterilization, insecticide, and similar purposes to prevent infectious diseases as defined in Article 2, Paragraph 7, Subparagraph D of the Pharmaceutical Affairs Act. It may also refer to products with milder effects than pharmaceuticals, among those used for the purpose of diagnosing, treating, improving, alleviating, managing, or preventing human or animal diseases. Furthermore, the term "quasi-drug" may include external skin preparations and personal hygiene products.

[0472] The above skin may include, but is not limited to, any skin area of ​​the body, including the face, hands, arms, legs, feet, chest, stomach, back, buttocks, and scalp.

[0473] The type or formulation of the pharmaceutical composition of the present invention is not particularly limited, but may be a bandage, gauze, cotton, adhesive bandage, disinfectant, shower foam, gargle, wet tissue, detergent soap, hand wash, humidifier filler, mask, or filter filler.

[0474] When the composition of the present invention is included in an over-the-counter drug for the purpose of improving skin condition, the composition may be used as is or in combination with other over-the-counter drug ingredients, and may be used appropriately according to a conventional method. The amount of active ingredients mixed may be appropriately determined depending on the intended use, and the over-the-counter drug composition according to the present invention may contain 0.01 wt% to 20 wt% of microorganisms, their lysates, culture solutions, or mixtures thereof, based on the total weight of the composition.

[0475] The above-mentioned external preparation for skin may be a cream, a gel, an ointment, a skin emulsifier, a skin suspension, a transdermal patch, a lotion, or a combination thereof. The above-mentioned external preparation for skin may be appropriately mixed with a combination of ingredients commonly used in external preparations for skin such as cosmetics or medicines, and as needed. The above-mentioned external preparation for skin may also appropriately mix with metal sequestering agents such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, and gluconic acid, caffeine, tannin, bellapamil, licorice extract, glabridin, hot water extract of the fruit of Kalin, various herbal medicines, drugs such as tocopheryl acetate, glycyrrhizic acid, tranexamic acid and derivatives or salts thereof, vitamin C, magnesium ascorbic acid phosphate, ascorbic acid glucoside, arbutin, kojic acid, glucose, fructose, and trehalose, and sugars such as the above-mentioned external preparation for skin.

[0476] The skin external application composition according to the present invention may contain 0.00001 wt% to 80 wt% of microorganisms, their lysates, culture solutions, or mixtures thereof based on the total weight of the composition, but is not limited thereto, and the added content such as the wt% and the type of the added material may include all of the amounts, mixing ratios, and materials generally added in the art.

[0477]

[0478] The present invention provides a kit for preventing or treating hair loss, comprising a composition comprising at least one of peroxisome and peroxisome bound to hyaluronic acid with a thiol group introduced therein as an active ingredient, and an instruction manual.

[0479] In the present invention, the “kit” means a tool that can produce a peroxisome bound to hyaluronic acid having a thiol group introduced therein using the peroxisome, thiol group, hyaluronic acid, etc. of the present invention, or a tool that can deliver the peroxisome to prevent, improve, or treat hair loss, etc. In addition to the above substances, the kit of the present invention may include other components, compositions, solutions, devices, etc. that are usually necessary for methods of storing and processing them. As a specific example, in this case, each component may be applied once or more without limitation in the number of times, there is no limitation in the order in which each substance is applied, and the application of each substance may be performed simultaneously or microscopically.

[0480] In the present invention, the kit may include a container; instructions; and the like. The container may serve to package the substance, and may also serve to store and fix the substance. The material of the container may take the form of, for example, a bottle, a tub, a sachet, an envelope, a tube, an ampoule, and the like, and these may be formed partially or wholly from plastic, glass, paper, foil, wax, and the like. The container may be initially equipped with a completely or partially detachable stopper, which may be part of the container or may be attached to the container by mechanical, adhesive, or other means, and may also be equipped with a stopper for allowing access to the contents by means of a syringe needle. The kit may include an outer package, and the outer package may include instructions for the use of the components.

[0481]

[0482] The present invention provides a health functional food composition for improving hair loss, promoting hair growth, promoting hair growth, or improving scalp condition, which comprises at least one of peroxisome and peroxisome combined with hyaluronic acid having a thiol group introduced therein as an active ingredient.

[0483] In the present invention, "hair loss improvement" refers to the action of preventing hair loss by weakening the regression phase of hair follicles and inducing the growth phase. Therefore, the term "hair loss alleviation," "hair loss prevention," and "hair loss treatment" can be used interchangeably, referring to suppressing hair loss caused by a decrease in the proportion of hair in the growth phase.

[0484] In the present invention, "promoting hair growth" refers to promoting hair growth, ultimately increasing the proportion of hair in the anagen phase among all hair types. Therefore, the term can have the same meaning as "improving hair growth," meaning it suppresses hair loss caused by a decrease in the proportion of hair in the anagen phase.

[0485] In the present invention, “hair growth promotion” means a hair growth function that generates new hair, or a function that promotes hair growth, as well as a function that promotes delay from the growth phase to the regression phase, thickens hair, improves hair density, and helps existing hair grow healthily.

[0486] In the present invention, “prevention” means all actions of the composition of the present invention that suppress or delay hair loss by weakening the regression phase of hair follicles and inducing the growth phase.

[0487] In the present invention, “treatment” means any action of the composition of the present invention to suppress or delay hair loss by weakening the regression phase of hair follicles and inducing the growth phase.

[0488] In the present invention, “food” means a natural product or processed product containing one or more nutrients, preferably a product that has gone through a certain degree of processing to become directly edible, and in its usual sense, includes all health functional foods, beverages, food additives, and beverage additives.

[0489] In the present invention, the term “functional food” is the same as food for special health use (FoSHU), and means a food with high medical or therapeutic effects that is processed to efficiently exhibit a bioregulatory function in addition to providing nutrition, and can be manufactured in the form of tablets, capsules, pills, granules, powders, liquids, flakes, pastes, syrups, gels, jellies, bars, or films.

[0490] Here, "functionality" means regulating nutrients for the structure and function of the human body or obtaining a beneficial effect for health purposes, such as physiological functions. The health functional food of the present invention can be manufactured by a method commonly used in the art, and during the manufacturing process, raw materials and ingredients commonly added in the art can be added.

[0491] In addition, the formulation of the above health functional food can be manufactured without limitation as long as it is a formulation recognized as a health functional food. The health functional food of the present invention can be manufactured in various forms of formulation, and unlike general drugs, it has the advantage of not having side effects that can occur with long-term use of drugs because it uses food as a raw material, and it is highly portable, so the health functional food of the present invention can be consumed as a supplement to enhance the effects of promoting hair growth and alleviating hair loss.

[0492] The health functional food of the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients. The natural carbohydrates mentioned above include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As sweeteners, natural sweeteners such as thaumatin and stevia extract, or synthetic sweeteners such as saccharin and aspartame, may be used.

[0493] In addition to the above, the health functional food of the present invention may contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. These ingredients may be used independently or in combination. The proportion of these additives is not particularly important, but is generally selected from the range of 1 to 100 parts by weight per 100 parts by weight of the health functional food of the present invention, but is not particularly limited thereto.

[0494] In the present invention, there is no particular limitation on the type of the health functional food. Specifically, examples of foods to which the composition of the present invention can be added include dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and particularly, all foods designed to sufficiently exert the body's regulatory functions, such as regulating the biological defense rhythm, disease prevention, and recovery, of the food group or food composition that has added value so that the function of the food in the conventional sense can be performed and expressed for a specific purpose.

[0495] In the present invention, “food additive” refers to a substance that is added to, mixed with, infiltrated into, or used in any other way in the manufacture, processing, or preservation of food, and must be harmless to the human body when consumed over a long period of time, such as a health functional food.

[0496] When the composition of the present invention is used as a food additive, the food additive may be added as is or used together with other foods or food ingredients, and may be used appropriately according to a conventional method.

[0497] The amount of active ingredients mixed can be appropriately determined depending on the intended use (prevention, health, or therapeutic treatment). Generally, when manufacturing a food or beverage, the composition of the present invention can be added in an amount of 15% by weight or less, or 10% by weight or less, based on the raw material. However, for long-term intake for health and hygiene purposes or health control purposes, the amount may be less than the above range, and since there are no safety issues, the active ingredients can also be used in amounts greater than the above range.

[0498] In the present invention, the composition may include various food additives that are food-related and acceptable, and may further include appropriate carriers, excipients, and diluents commonly used in the manufacture of foods.

[0499] In addition to the above, the composition of the present invention may contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the composition of the present invention may contain fruit pulp for the production of natural fruit juice, fruit juice drinks, and vegetable drinks. These ingredients may be used independently or in combination. The ratio of these additives is not particularly important, but is generally selected in the range of 0.01-0.20 parts by weight per 100 parts by weight of the composition of the present invention, but is not limited thereto, and may be an optimal or arbitrary amount depending on the type, function, etc. of the product utilized.

[0500] In the present invention, there is no particular limitation on the type of food. Examples of foods to which the substance can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and all health functional foods in the conventional sense can be included, but are not limited thereto.

[0501] In addition, the composition according to the present invention can be added to health drinks, and, like conventional beverages, can contain various flavoring agents or natural carbohydrates as additional ingredients. The natural carbohydrates mentioned above are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As sweeteners, natural sweeteners such as thaumatin and stevia extract, or synthetic sweeteners such as saccharin and aspartame can be used. The proportion of the above natural carbohydrates may be generally about 0.01-0.20 g, or about 0.04-0.10 g per 100 mL of the composition of the present invention, but is not limited thereto, and may be a general amount added in the art, or may include the maximum range to enhance the efficacy of the composition of the present invention, and may include an optimal, arbitrary amount considering the synergistic effect with other substances added together.

[0502]

[0503] The present invention provides a cosmetic composition for improving hair loss, promoting hair growth, or improving scalp condition, comprising at least one of peroxisome and peroxisome combined with hyaluronic acid having a thiol group introduced therein as an effective ingredient.

[0504] In the present invention, the composition may be characterized by at least one selected from the group consisting of a softening toner, astringent toner, nourishing toner, nourishing cream, massage cream, essence, eye cream, eye essence, cleansing cream, cleansing foam, cleansing water, pack, powder, body lotion, body cream, body oil, body essence, makeup base, foundation, hair dye, shampoo, rinse, and body cleanser, but is not limited thereto.

[0505] In the present invention, “cosmetics” may be a concept that includes all items that beautify the appearance of the human body, but is not limited thereto, and may include the broadest meaning used in the art.

[0506] The cosmetic composition of the present invention can be used in cosmetics or as a cosmetic additive. When the composition of the present invention is used as a cosmetic additive, it can be used in the manufacture of a cosmetic composition for maintaining the cleanliness of hands or feet. Examples thereof include, but are not limited to, soap (solid soap, liquid soap, foam soap, body soap, hand soap, etc.), cleansing foam, shampoo (hair shampoo, dry shampoo, etc.). Examples of specific formulations include, but are not limited to, formulations of hair tonic, hair conditioner, hair essence, hair ampoule, hair lotion, hair nourishing lotion, hair shampoo, hair rinse, hair treatment, hair cream, hair nourishing cream, hair moisture cream, hair massage cream, hair wax, hair aerosol, hair pack, hair nourishing pack, hair soap, hair cleansing foam, hair oil, hair drying agent, hair preservative, hair dye, hair waving agent, hair bleaching agent, hair gel, hair glaze, hair dressing agent, hair lacquer, hair moisturizer, hair mousse or hair spray.

[0507] The cosmetic composition of the present invention can be manufactured into any formulation commonly manufactured in the art, and for example, can be formulated into a longevity (skin lotion), skin, skin softener, skin toner, astringent, solution, suspension, emulsion, paste, powder, gel, cream, hand cream, hand sanitizer, lotion, milk lotion, moisture lotion, nourishing lotion, body lotion, body cleanser, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation, spray, and sheet, but is not limited thereto. More specifically, it can be manufactured into a formulation of a flexible toner, a nourishing toner, a nourishing cream, a massage cream, a moisture cream, an essence, a nourishing essence, an eye cream, a cleansing cream, a cleansing foam, cleansing water, a pack, a spray, or a powder.

[0508] Compositions of this type can be prepared using methods conventional in the art. Those skilled in the art can easily select the amount of additional ingredients, such as the moisturizer, as long as it does not impair the purpose and effects of the present invention.

[0509] In the present invention, the composition can be prepared in the form of a general emulsified formulation and a solubilized formulation. Cosmetics in the form of an emulsified formulation include a nourishing toner, a cream, an essence, etc., and cosmetics in the form of a solubilized formulation include an emulsified toner. More specifically, the composition of the present invention can be provided in the form of a solution, a gel, an anhydrous product in the form of a solid or paste, an emulsion obtained by dispersing an oil phase in an aqueous phase, a suspension, an emulsion, a microemulsion, a microcapsule, a mask pack, a microgranule or an ionic (liposome) or non-ionic vesicle dispersion, a cream, a skin, a lotion, a powder, an ointment, a spray, a paste, a pack, a cleanser, a soap, a surfactant-containing cleanser, an oil, a powder foundation, a bath powder, an emulsion foundation, a wax, a foundation, or a concealer stick. In addition, the composition can be prepared in the form of a foam or an aerosol composition further containing a compressed propellant.

[0510] When the formulation of the above composition is a paste, cream or gel, the carrier component may include animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, etc.

[0511] If the formulation of the above composition is a powder or spray, the carrier component may include lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, etc., and especially in the case of a spray, the composition may additionally include a propellant such as chlorofluorohydrocarbon, propane / butane, dimethyl ether, etc.

[0512] When the formulation of the above composition is a solution or emulsion, the carrier component may include a solvent, a solubilizer, an emulsifier, etc., and specifically, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol, fatty acid ester of sorbitan, etc.

[0513] When the formulation of the above composition is a suspension, the carrier component may include a liquid diluent such as water, ethanol, or propylene glycol; a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, or polyoxyethylene sorbitan ester; microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, or tragacanth.

[0514] When the formulation of the above composition is a surfactant-containing cleansing, the carrier component may include fatty alcohol sulfate, fatty alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide ether sulfate, alkyl amidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanol amide, vegetable oil, lanolin derivative, ethoxylated glycerol fatty acid ester, etc.

[0515] In addition, the compounding components that may be added are not limited to the examples above, and any of the above components may be compounded within a range that does not impair the purpose and effect of the present invention, but are preferably compounded in an amount of 0.01-10% by weight, and more preferably 0.01-5% by weight, relative to the total weight.

[0516] The above cosmetic composition may further include functional additives and components included in general cosmetic compositions in addition to the components disclosed herein, and may further include purified water, thickeners, preservatives, stabilizers, solubilizers, surfactants, carriers, fragrances, or combinations thereof that are commonly used.

[0517] Examples of the above carriers include alcohol, oil, surfactant, fatty acid, silicone oil, humectant, moisturizer, viscosity modifier, emulsion, stabilizer, ultraviolet scattering agent, ultraviolet absorber, colorant, fragrance, etc. Compounds / compositions that can be used as the above alcohol, oil, surfactant, fatty acid, silicone oil, humectant, moisturizer, viscosity modifier, emulsion, stabilizer, ultraviolet scattering agent, ultraviolet absorber, colorant, fragrance, etc. are already known in the art, so a person skilled in the art can select and use an appropriate corresponding substance / composition. In addition, the cosmetic composition may further contain, as necessary, ingredients such as sunscreens, antioxidants (butylated hydroxyanisole, propyl gallate, ellisorbic acid, tocopheryl acetate, butylated hydroxytoluene, etc.), preservatives (methylparaben, butylparaben, propylparaben, phenoxyethanol, imidazolidinyl urea, chlorphenesin, etc.), colorants, pH adjusters (triethanolamine, citric acid, citric acid, sodium citrate, malic acid, sodium malate, formaldehyde, sodium formaldehyde, succinic acid, sodium succinate, sodium hydroxide, sodium hydrogen phosphate, etc.), moisturizers (glycerin, sorbitol, propylene glycol, butylene glycol, hexylene glycol, diglycerin, betaine, glycereth-26, methylgluceth-20, etc.), and lubricants.

[0518] In the present invention, the composition may further comprise at least one known ingredient having an effect of improving hair loss, promoting hair growth, promoting hair growth, or improving scalp condition, and furthermore, may further comprise at least one substance commonly used in cosmetic compositions. Specifically, the composition may further comprise an auxiliary agent commonly used in the field of cosmetics or dermatology, such as a fatty substance, an organic solvent, a solubilizer, a thickening and gelling agent, an emollient, an antioxidant, a suspending agent, a stabilizer, a foaming agent, a fragrance, a surfactant, water, an ionic or nonionic emulsifier, a filler, a metal ion sequestering agent and a chelating agent, a preservative, a vitamin, a blocking agent, a humectant, an essential oil, a dye, a pigment, a hydrophilic or lipophilic active agent, a lipid vesicle, or any other ingredient commonly used in cosmetics. In addition, the above ingredients may be introduced in an amount commonly used in the field of dermatology.

[0519] In the present invention, the composition may further include a composition selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, polymeric peptides, polymeric polysaccharides, and sphingolipids.

[0520] The cosmetic composition of the present invention may contain, in addition to the above essential ingredients, other ingredients commonly incorporated into cosmetics as needed, and may include, for example, a fat component, a moisturizer, an emollient, a surfactant, organic and inorganic pigments, organic powders, ultraviolet absorbers, preservatives, sterilizers, antioxidants, plant extracts, pH adjusters, alcohols, pigments, fragrances, blood circulation promoters, cooling agents, antiperspirants, purified water, etc.

[0521] The present invention provides a method for producing a peroxisome bound to hyaluronic acid having a thiol group introduced therein, comprising the following steps:

[0522] (S1) A step of producing hyaluronic acid (HA-SH) with a thiol group introduced therein by reacting a solution containing dissolved hyaluronic acid with a solution containing dissolved EDC, HOBT, and cystamine; and

[0523] (S2) A step of producing a peroxisome in which hyaluronic acid with a thiol group introduced is combined by mixing the peroxisome and the above HA-SH.

[0524] Hereinafter, preferred examples are presented to aid in understanding the present invention. However, the following examples are provided solely to facilitate a better understanding of the present invention, and the scope of the present invention is not limited by the following examples.

[0525]

[0526] [Composition for preventing or treating diseases related to muscle loss, containing a peroxisome function inducer as an active ingredient]

[0527]

[0528] [Example]

[0529]

[0530] [Experimental Materials and Methods]

[0531]

[0532] Animal experiments

[0533] All animal studies were approved by the Wonkwang University Institutional Animal Care and Use Committee and followed institutional guidelines (WKU22-58). All mice were housed at 23 ± 1°C, with a 12-h light / dark cycle and a relative humidity of 50 ± 5%, with access to food and water.

[0534]

[0535] Muscle injury procedure

[0536] All muscle lesion procedures were performed under isoflurane anesthesia inhaled with a mixture of oxygen and room air. Muscle lesions were induced by injecting 50 μl of a 1.2% BaCl2 solution in phosphate-buffered saline (PBS) into the tibialis anterior (TA) muscle of sedated 9-10-week-old rats. The control group received an equal volume of PBS, and the contralateral hindlimb served as an untreated control.

[0537]

[0538] Generation of Acot4- / - mice

[0539] Total Acot4 knockout (KO) mice were generated by germline transmission of an RGEN-induced mutant allele. Acot4-specific guide RNA and Cas9 protein (50 ng / μl each) were injected into the cytoplasm of C57BL / 6N mouse oocytes and transferred into pseudopregnant foster female mice. The absence of Acot4 in the mutants was confirmed using routine tail DNA genotyping and Western blotting. The absence of Acot4 was confirmed by PCR genotyping. The following genotyping primers were designed to distinguish between Acot4+ / +, Acot4+ / -, or Acot4- / - alleles: Nested 1st PCR Forward: 5'-gaggcctccagctcctaatg-3' (SEQ. 213); Reverse: 5'-cacctctcaaccacccaact-3' (SEQ. 214). Nested 2nd PCR Forward: 5'-tgcgcgacgagaaggg-3'(SEQ.215); Reverse: 5'-tgatcaggcgccagaaagg-3'(SEQ.216). PCR products were separated by polyacrylamide gel electrophoresis and stained with ethidium bromide.

[0540]

[0541] C2C12 cell culture and differentiation

[0542] C2C12 cells were purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured in high-glucose Dulbecco's modified Eagle's medium (DMEM; Gibco-Invitrogen, Grand Island, NY) supplemented with 10% fetal bovine serum (Gibco-Invitrogen, Grand Island, NY) and 1% penicillin-streptomycin (Thermo Fisher Scientific Korea Ltd., Seoul, Korea). Cells that reached 80–90% confluence were differentiated in DMEM supplemented with 2% horse serum (Gibco-Invitrogen). Cells were cultured at 37°C in the presence of 5% CO2, and the medium was changed every other day. Differentiated myoblasts were treated with 500 μM dexamethasone (DEX, Sigma-Aldrich, St. Louis, MI, USA), 50 μM palmitic acid (PA, Cayman), 50 μM phytol (Sigma-Aldrich), or 50 μM docosanoic acid (DCA, Cayman) or transfected with pLKO.1 shacox1.

[0543]

[0544] Primary murine myoblast isolation and culture

[0545] Primary myoblast culture and differentiation were performed according to the guidelines of the Journal of Visualized Experiments. Muscle samples were obtained from hindlimb muscles of 7-10-week-old wild-type (WT), Acot12- / -, Acot4- / -, and Nudt7- / - mice. Muscle samples were minced in digestion medium containing collagenase type II (10 mg / ml, Worthington, #LS004177) and dephase II (2.4 U / ml, Roche Applied Science, Penzberg, Germany, #0494207001). After 30 min of digestion, the cell suspension was passed through a 70-μm strainer (Falcon, #352350), and the filtered cell solution was centrifuged and cultured in growth medium for 3 days. On the fourth day of culture, small fibers were trypsinized, and primary myoblasts were cultured on collagen type I-coated plates.

[0546]

[0547] Morphometric evaluations

[0548] Root canal diameter and length were measured using Image-Pro Plus software (Media Cybernetics, Rockville, MD, USA) and a myotube analyzer (https: / / github.com / SimonNoe / myotube-analyzer-app). The fiber index was calculated as follows: (number of nuclei in myotubes containing at least 3 nuclei / total number of nuclei) × 100 (from 5 randomly selected fields per well). Root canal diameter was measured at the thickest part of each root canal. The average length and diameter of at least 200 root canals from 10 randomly selected fields were determined for each condition.

[0549]

[0550] Histological & oil-red O staining

[0551] For histological analysis, sacrificed muscles were fixed in 10% neutral buffered formalin before embedding in paraffin or OCT compound. H&E staining was performed on 5-μm paraffin-embedded muscle sections, and frozen tibialis anterior muscle sections were stained with Oil Red O (Sigma-Aldrich) in 60% isopropanol, and the staining intensity was quantified using Image-Pro Plus software.

[0552]

[0553] Lentiviral constructs packaging and delivery

[0554] To inject lentiviral particles, 293T cells were transfected with pLenti-GIII-CMV, pLenti-GIII-CMV-mAcot4-c-term-HA, pLenti-GIII-CMV-mAcot12-c-term-HA, or pLenti-GIII-CMV-mNudt7-c-term-HA using the 3rd Generation Packaging System Mix (Applied Biosystems, #LV053) according to the manufacturer's protocol. Specifically, Lenti-X 293 T cells (Clontech, #632180) were transfected with plasmids using Lentifectin (ABM, G074) in OPTI-MEMI medium (Thermo Fisher Scientific, #31985062), the supernatant was collected, and the lentiviral particles were concentrated using a Lenti-X concentrator (Clontech, #631232). Cells were infected with lentiviral supernatant for 2 days in a humidified incubator at 37°C in the presence of 5% CO2. For lentivirus injection, lentivirus was injected into the BaCl2-induced tibialis anterior muscle for 3 days.

[0555]

[0556]

[0557]

[0558]

[0559] Immunofluorescence staining

[0560] Cells grown on coverslips were fixed with 4% paraformaldehyde (PFA; Sigma-Aldrich) for 10 min. After washing three times in cold 1 × PBS, cells were blocked with 3% normal goat serum (NGS; Vector Laboratories, Newark, CA, USA) and incubated with antimyosin scaffold heavy chain (MYHC, 1:100, R&D Systems, Minneapolis, MN, USA, #MAB4470) and a fluorescently conjugated secondary antibody. Nuclei were stained with mounting medium containing 4,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories).

[0561]

[0562] qRT-PCR (quantitative real-time PCR)

[0563] Total RNA was isolated using RNAiso Plus (TaKaRa, #9109) according to the manufacturer's instructions. Then, 1 μg of RNA was reverse transcribed using 5x All-In-One RT Master Mix (ABM, #G492). Real-time PCR was performed on an ABI StepOnePlus instrument (Applied Biosystems, Waltham, MA, USA) using AMIGENE qPCR Green Mix Hi-ROX (Enzo Biochem, Inc., Fammingdale, NY, USA, #ENZ-NUC103). The cycling protocol was 40 cycles of 95°C for 10 s, 57°C for 15 s, and 72°C for 10 s, and all qRT-PCR reactions were performed in triplicate. The relative expression levels of each gene were normalized to 18S rRNA expression levels. Forward and reverse primer sequences are shown in Table 2 below.

[0564]

[0565]

[0566]

[0567]

[0568] Statistical analyses

[0569] All results are expressed as the mean ± standard error of the mean (SE) of at least three independent experiments. Counts were performed by three independent operators who were unaware of the treatments. Representative experiments and images are shown unless otherwise noted. Unpaired, two-tailed Student's t-tests were used for two groups. Differences between multiple groups were evaluated using analysis of variance, followed by Fisher's least significant difference test.

[0570]

[0571] Example 1. Confirmation of the relationship between peroxidase biosynthesis factor dysfunction and muscle function.

[0572] To investigate the relationship between peroxisome function and muscle function, we knocked down peroxisomal biogenesis factor (PEX) function and examined the effects in muscle cells. To this end, we transplanted interfering RNAs for PEX5 and PEX7 into C2C12 myoblasts and confirmed the effects. PEX5 and PEX7 act as receptors for matrix protein import and recognize the peroxisome targeting signal (PTS). PEX5 is known to recognize the C-terminal PTS1 as Ser-Lys-Leu (SKL), and PEX7 is known to recognize the N-terminal PTS2 as (R / K)(L / V / I)X5(H / Q)(L / A).

[0573] Specifically, C2C12 myoblasts were differentiated into contractile myotubes and small interfering RNAs specific for PEX5 or PEX7 were transfected. For this purpose, C2C12 cells were infected with Pex5-specific siRNA (siPex5) and scrambled siRNA was used as a control (Con). After infection, the diameter and length of cell myotubes were measured using Image Pro software, and the mRNA levels of Pex5, MyoD, McK, Mrf5, MYHC, MyoG, Myh-4, and Myh-7 were analyzed by RT-PCR. Pex7 knockdown was performed in the same manner as Pex5.

[0574]

[0575] As a result (Fig. 1a and Fig. 1b), the introduction of siPex5 into C2C12 cells after 4 days of differentiation resulted in a decrease in the expression of myosin heavy chain (MyHC), Myh4 (encoding MHC-IIb), and Myh7 (encoding MHC-I). In addition, muscle creatine kinase (Mck), a muscle differentiation marker, was decreased by the knockdown of Pex5. Since the expression of the MyHC gene is regulated by myogenesis regulatory factor (MRF), the expression levels of Mrf5 and myognin (Myog) also decreased on the 6th day of culture. Correspondingly, the introduction of siPex7 into C2C12 cells after the 4th day of differentiation resulted in a significant decrease in the expression levels of muscle differentiation markers and muscle regulatory factors (Fig. 1b).

[0576] These results indicate that peroxisome function is involved in muscle differentiation and myogenesis, and further suggest that dysregulation of peroxisome function may affect muscle integrity and function.

[0577]

[0578] Example 2. Confirmation of the relationship between peroxisomal lipid metabolism and muscle loss.

[0579] We analyzed the association between peroxisomal lipid metabolism and muscle loss. Specifically, to identify regulatory pathways responsible for muscle health, we analyzed muscle samples from patients with amyotrophic lateral sclerosis (ALS; GSE41414) (n = 7, mean age: control, 64 ± 4; ALS, 64 ± 8) and mouse models of dexamethasone (DEX; GSE209528)-induced and Duchenne muscular dystrophy (DMD; GSE7187).

[0580]

[0581] As a result (Fig. 2), the co-downregulated genes extracted from ALS patients and dexamethasone-induced and DMD mouse models suggested that dysregulation of lipid metabolism, including fatty acid metabolism, long-chain fatty acid transport, and lipid biosynthesis, is closely related to the development and progression of muscle loss.

[0582]

[0583] Example 3. Confirmation of the association between dysregulation of peroxisomal β-oxidation and muscle loss.

[0584] In Example 2, the relationship between peroxisomal lipid metabolism and muscle atrophy was confirmed. Accordingly, in Example 3, the relationship between the β-oxidation function of peroxisomes and muscle loss was confirmed.

[0585]

[0586] Example 3-1. Confirmation of the effect of introducing Acox1-specific shRNA on muscle atrophy caused by fatty acid β-oxidation damage.

[0587]

[0588] First, we introduced shRNA (shAcox1) specific for acyl-CoA oxidase 1 (Acox1), the first enzyme involved in the peroxisomal β-oxidation system, to catalyze the desaturation of acyl-CoA to 2-trans-enoyl-CoA, thereby impairing peroxisomal fatty acid β-oxidation. The infection efficiency of shAcox1 is shown in Fig. 3a.

[0589]

[0590] As a result, C2C12 myoblasts were differentiated in the absence or presence of shAcox1 over time (i.e., days 0–2, days 2–4, and days 4–6). The diameter and length of myofibers decreased in the presence of shAcox1, with the greatest decrease in the diameter and length of shAcox1-infected myofibers on days 0–2 (Fig. 3b and 3c). RT-PCR analysis confirmed that the expression levels of Myog and MyHC were significantly reduced following Acox1 knockdown (Fig. 3d).

[0591]

[0592] Example 3-2. Confirmation of muscle atrophy caused by fatty acid β-oxidation damage following fatty acid treatment.

[0593] The same assay as in Example 3-1 was performed by exposing C2C12 cells to specific fatty acid subsets, such as palmitic acid (C16:0), dicarboxylic acid (C18:2, DCA), and phytol, which impair peroxisomal β-oxidation.

[0594]

[0595] As a result, differentiation of C2C12 myoblasts was inhibited, resulting in a decrease in the diameter and length of myotubes (Fig. 3e and Fig. 3f), and the expression levels of Myog and MyHC (Fig. 3g) were confirmed to be consistent with the experimental results when Acox1 was knocked down.

[0596]

[0597] The results of Examples 3-1 and 3-2 confirmed that peroxisomal fatty acid β-oxidation plays an important role in the degeneration of muscle cell mass and function as well as in the differentiation of C2C12 myoblasts.

[0598]

[0599] Example 4. Confirmation of the role of peroxisomes in a muscle atrophy model.

[0600] Example 4-1. Confirmation of decreased gene expression related to peroxisome function in human and mouse muscle atrophy subjects.

[0601] To further investigate the role of peroxisomes in the pathogenesis of sarcopenia, we analyzed peroxisomal genes extracted from GEO data (human muscular atrophy, GSE41414; mouse muscular atrophy, GSE209528; mouse muscular atrophy, GSE7187).

[0602]

[0603] As a result, among the genes related to peroxisome function, Pex19 and Pex7, which are peroxisome biogenesis factors (Pex), were found to be decreased in both humans and mice (Fig. 4a). In ALS patients, the expression levels of Pex2, Pex11a, Pex12, Pex16, ATP-binding cassette subfamily D member 3 (ABCD3), and acyl-CoA thioesterase 8 (Acot8) were downregulated, and in DEX-damaged mouse muscles, the expression levels of Pex2, Pex5, Abcd2, Abcd3, Nudt7, and Acox1 were decreased.

[0604]

[0605] Example 4-2. Confirmation of the role of peroxisomes in models of denervation, myocardial infarction, and BaCl2-induced muscle atrophy.

[0606] In this example, histological analysis and expression analysis of ubiquitin ligases were performed using three muscle atrophy models: denervation (DEN), myocardial infarction (MI), and BaCl2-induced muscle atrophy animal models.

[0607]

[0608] As a result, histological analysis of the tibialis anterior muscle showed that muscle atrophy was significantly worsened, and fiber size analysis showed that the fiber diameter was significantly reduced in the DEN-, MI-, and BaCl2-induced animal models (Figs. 4b to 4d).

[0609] The expression levels of Atrogin-1 and MuRF1, ubiquitin ligases associated with skeletal muscle atrophy, were significantly increased.

[0610]

[0611] Furthermore, common genes whose expression was significantly downregulated in DEN-, MI-, and BaCl2-induced animal models among peroxisomal genes were extracted and the related signaling pathways were analyzed. As a result, CoA hydrolase, LCFA transporter, fatty acid ligase, and CoA ligase activities were identified as commonly downregulated signaling pathways (Fig. 4e).

[0612]

[0613] Example 4-3. Confirmation of the role of peroxisomes in a dexamethasone-induced muscle atrophy model.

[0614] Exposure of C2C12 to dexamethasone resulted in a decrease in muscle fiber diameter and expression levels of Atrogin-1, MuRF1, and myostatin (MSTN) (Fig. 4f, Fig. 4g, and Fig. 4h).

[0615]

[0616] Additionally, peroxisome profiles were significantly reduced. Among them, Abcd3, Acox1, Acot4, Pex7, Pex11, Pex13, Pex16, and Pex19 were significantly reduced in C2C12 cells treated with dexamethasone compared to the control group (Fig. 4i).

[0617]

[0618] Example 5. Confirmation of activity in exacerbating myotube atrophy due to peroxisomal acyl-CoA metabolism disorder.

[0619] Peroxisomes are involved in the β-oxidation of VLCFAs and branched-chain fatty acids, oxidizing transported acyl-CoA to acetyl-CoA. Acyl-CoA metabolism plays a crucial role in peroxisomes, and dysfunction in the regulation of metabolic mechanisms for peroxisomal acyl-CoA homeostasis can lead to peroxisomal dysfunction. Among the genes involved in acyl-CoA metabolism, two acyl-CoA thioesterases, Acot4 and Acot12, are involved in the degradation of acyl-CoAs, particularly long-chain and very-long-chain fatty acyl-CoAs. Furthermore, the nudix hydrolase Nudt7 also functions in peroxisomal acyl-CoA metabolism.

[0620] Therefore, the following Acot4, Acot12, and Nudt7 knockdowns were performed to induce peroxisome dysfunction and to confirm myotube formation, atrophy effect, and muscle atrophy activity, respectively.

[0621]

[0622] Example 5-1. Confirmation of increased myotube formation inhibitory activity following knockdown of Acot12 and Nudt7.

[0623] First, we analyzed the inhibition of myotube formation following knockdown of Acot12 and Nudt7 in vitro. Specifically, C2C12 cells were infected with shCon, shAcot12, and shNudt7 and treated with or without dexamethasone.

[0624]

[0625] As a result, it was confirmed that myotube formation was significantly reduced in dexamethasone-treated myoblasts following knockdown of Acot12 and Nudt7 (Fig. 5a).

[0626]

[0627] Example 5-2. Confirmation of increased myotube atrophy activity due to deficiency of Acot12, Acot4, and Nudt7.

[0628] To investigate the functional role of peroxisomal acyl-CoA metabolism in the pathogenesis of muscle atrophy, Acot4 knockout (KO) mice were generated via germline transmission of a mutant Acot4 allele induced by RNA-guided endonuclease (RGEN) (Fig. 5b). Furthermore, various analyses were performed using KO mice for Acot12 and Nudt7, previously generated in our laboratory.

[0629]

[0630] As a result (Fig. 5c), it was confirmed that the expression of Pax3, Pax7, and Myt5 in primary myoblasts isolated from the tibialis anterior muscles of WT mice significantly decreased as myoblast differentiation progressed, whereas the expression of Myhc, Mck, and Myh4 significantly increased. On the other hand, the expression levels of Myhc, Mck, and Myh4 in primary myoblasts isolated from the tibialis anterior muscles of Acot12- / -, Acot4- / -, and Nudt7- / - mice were significantly lower than those in the control myoblasts.

[0631]

[0632] Additionally (Figs. 5d-5f), the diameter of myotubes was significantly reduced in the Acot12- / -, Acot4- / -, and Nudt7- / - models. Exposure of primary myoblasts from Acot12- / -, Acot4- / -, and Nudt7- / - myoblasts to dexamethasone significantly reduced myotube formation and increased the expression levels of Atrogin-1 and MuRF1 compared to WT myoblasts.

[0633]

[0634] Example 6. Confirmation of muscle loss due to peroxisomal acyl-CoA metabolism disorder.

[0635] Example 6-1. Confirmation of the effects of Acot12, Acot4, and Nudt7 deficiency on reduced muscle fiber size and increased lipid accumulation.

[0636] To further investigate the role of Acot12, Acot4, and Nudt7 in muscle loss, tibialis anterior muscles from 6- and 12-month-old WT, Acot12- / -, Acot4- / -, and Nudt7- / - mice were analyzed.

[0637]

[0638] As a result, the size of Acot12- / -, Acot4- / -, and Nudt7- / - tibialis anterior muscle fibers was significantly reduced compared to WT mice (Fig. 6a and 6b). Lipid accumulation was also increased in Acot12- / -, Acot4- / -, and Nudt7- / - tibialis anterior muscles (Fig. 6c). A negative linear correlation was confirmed between this lipid accumulation and muscle fiber size (Fig. 6d). In silico analysis using GSE19674 (a mouse model of spinal muscular atrophy) revealed a positive correlation between SCD1 and Atrogin-1 expression.

[0639]

[0640] Example 6-2. Confirmation of the activity of promoting muscle atrophy progression due to deficiency of Acot12, Acot4, and Nudt7.

[0641] Next, we investigated whether deficiency of Acot12, Acot4, and Nudt7 contributes to the progression of muscle atrophy. Specifically, muscle damage induced by BaCl2 depolarized the sarcomere membrane, ruptured the membrane, and damaged myofibers.

[0642]

[0643] H&E staining revealed that injection of BaCl2 into the tibialis anterior muscle of mice resulted in a decrease in the area and diameter of muscle fibers (Fig. 7a). Analysis using Image Pro revealed that muscle fibers were color-coded according to their size, with large fibers in red and small fibers in blue. Injection of BaCl2 increased the number of bluish muscle fibers, confirming an increase in the number of small fibers in the tibialis anterior muscle injected with BaCl2 (Figs. 7a and 7b).

[0644] Furthermore, injection of BaCl2 into the tibialis anterior muscles of Acot12- / -, Acot4- / -, and Nudt7- / - mice resulted in a significant decrease in muscle fiber length compared to WT mice (Fig. 7c). In the tibialis anterior muscles of Acot12- / -, Acot4- / -, and Nudt7- / - mice injected with BaCl2, the number of small, bluish muscle fibers increased (Fig. 7d).

[0645]

[0646] Example 7. Confirmation of muscle loss recovery activity through overexpression of Acot12, Acot4, and Nudt7.

[0647] To determine whether restoration of acyl-CoA metabolism could reverse the atrophic effects of dexamethasone, C2C12 cells were transfected with expression vectors encoding Acot12 (A4OE), Acot4 (A12OE), or Nudt7 (N7OE), and transduction efficiency was determined (Fig. 8a). Subsequently, myotube morphology, the percentage of nuclei within differentiated myotubes, and the fusion index were analyzed.

[0648]

[0649] As a result, it was confirmed that the reduced myotube length and fusion index induced by dexamethasone were restored by introducing A4OE, A12OE, or N7OE (Figs. 8b and 8c). In addition, it was confirmed that the area and length of muscle fibers reduced by BaCl2 injection were significantly restored by in vivo injection of lentivirus A4OE, A12OE, or N7OE (Figs. 8d to 8g).

[0650]

[0651] [Pharmaceutical composition for preventing or treating muscle wasting-related diseases containing peroxisome or peroxisome mimic as an active ingredient]

[0652]

[0653] [Example]

[0654]

[0655] Ethics Statement for Human Muscle Samples

[0656] This study was approved by the Public Clinical Trials Committee of the Ministry of Health and Welfare (IRB number P01-202104-31-009). Written informed consent was obtained from all participating patients. The inventors complied with all relevant ethical regulations for work with human participants, including the guidelines of the National Institutes of Health.

[0657]

[0658] Acquisition of subjects and human tissues

[0659] Thirty patients who underwent total knee replacement (TKR) for knee osteoarthritis, had not received steroid injections within the 3 months prior to surgery, and had no history of knee surgery, were selected. During TKR surgery, a small portion (5 x 5 mm) of the quadriceps femoris muscle was directly excised using a surgical blade. The collected muscle samples were immediately frozen at -80°C for histological analysis.

[0660]

[0661] animal testing

[0662] All animal studies were approved by the Institutional Animal Care and Use Committee of Wonkwang University and followed institutional guidelines (WKU22-58). All mice were housed at 23 ± 1°C with a 12-h light / dark cycle and a relative humidity of 50 ± 5%, with free access to food and water.

[0663]

[0664] Parabiosis

[0665] C57BL / 6 WT mice were used for parabiosis surgery (anatomically joining two individuals) to connect younger (2 months old) and older (16 months old) mice. To study aging and regeneration, the animals' elbow and knee joints were surgically connected to promote shared blood circulation, and the skin was sutured together. Eight weeks after surgery, the tibialis anterior (TA) muscle was harvested from the mice for histological and biological analysis.

[0666]

[0667] Muscle injury procedure

[0668] All muscle lesion procedures were performed under inhaled isoflurane anesthesia using a mixture of oxygen and room air. Fifty microliters of a 1.2% BaCl2 solution dissolved in phosphate-buffered saline (PBS) was injected into the TA muscle of sedated 9- to 10-week-old mice. The control group received an identical volume of PBS. The contralateral hind limb served as an untreated control.

[0669]

[0670] Generation of Acot4- / - mice

[0671] Global Acot4 KO mice were generated by germline transmission of a mutant allele induced by RGEN. Acot4-specific guide RNA and Cas9 protein (each 50 ng / μl) were injected into the cytoplasm of C57BL / 6N mouse eggs and transferred into pseudopregnant foster female mice. The absence of Acot4 in the mutants was confirmed using routine tail DNA genotyping and Western blotting. The absence of Acot4 was confirmed by PCR genotyping. Genotyping primers were designed to distinguish between Acot4+ / +, Acot4+ / -, or Acot4- / - alleles: Nested primary PCR Forward: 5'-gaggcctccagctcctaatg-3' (SEQ. 213); Reverse: 5'-cacctctcaaccacccaact-3' (SEQ. 214). Nested secondary PCR Forward: 5'-tgcgcgacgagaaggg-3' (SEQ. 215); Reverse: 5'-tgatcaggcgccagaaagg-3' (SEQ. 216). PCR products were separated by polyacrylamide gel electrophoresis and stained with ethidium bromide.

[0672]

[0673] C2C12 cell culture and differentiation

[0674] C2C12 cells were purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured in high-glucose Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (Gibco-Invitrogen) and 1% penicillin-streptomycin (Thermo Fisher Scientific Korea Ltd., Seoul, Korea). Cells at 80–90% confluence were differentiated in DMEM supplemented with 2% horse serum (Gibco-Invitrogen). Cells were cultured at 37°C in the presence of 5% CO2, and the culture medium was changed every 2 days. Differentiated myoblasts were treated with 50 μM dexamethasone (DEX; Sigma-Aldrich, St. Louis, MI, USA), 50 μM palmitic acid (PA, Cayman), 50 μM phytol (Sigma-Aldrich), or 50 μM. Transfected with docosahexanoic acid (DCA, Cayman) or pLKO.1 shAcox1.

[0675]

[0676] BODIPY 493 / 503 and BODIPY 581 / 591 staining

[0677] Two days after transfection with shAcox1, C2C12 cells were washed with PBS and cultured in culture medium supplemented with BODIPY 493 / 503 (1:500, Thermo Fisher Scientific Korea Ltd., D3922) and BODIPY 581 / 591 (1:1000, Thermo Fisher Scientific Korea Ltd., D3861) for 30 min at 37°C. Nuclei were stained using DAPI-containing mounting medium (Vector Laboratories).

[0678]

[0679] Isolation and culture of primary rat myoblasts

[0680] Primary myoblast culture and differentiation were performed according to the guidelines of the Journal of Visualized Experiments. Muscle samples were collected from the hindlimb muscles of 7- to 10-week-old wild-type (WT), Acot12- / -28, Acot4- / -, and Nudt7- / -29 mice. Muscle samples were minced into small pieces in digestion medium containing collagenase type II (10 mg / ml, Worthington, #LS004177) and dispase II (2.4 U / ml, Roche Applied Science, Penzberg, Germany, #04942078001). After 30 min of digestion, the cell suspension was passed through a 70 μm strainer (Falcon, #352350). The filtered cell solution was centrifuged and cultured in growth medium for 3 days. On the fourth day of culture, small fibers were trypsinized and primary myoblasts were cultured on type I collagen-coated plates.

[0681]

[0682] Synthesis of fluorescein isothiocyanate (FITC)-labeled chitosan oligosaccharide (COS-FITC)

[0683] 50 mg of chitosan oligosaccharide (Mn 5 kDa, COS) (Sigma-Aldrich, Milwaukee, WI, USA) was dissolved in 1% (v / v) acetic acid solution (2.5 ml), and 10.4 mg of FITC (Sigma-Aldrich, Milwaukee, WI, USA) was dissolved in 0.5 ml of methanol, respectively. After complete dissolution, FITC dissolved in methanol was slowly added to the COS solution and reacted in a darkroom for 4 h. The obtained product was dialyzed against HCl solution (pH ~5) for 2 days using a membrane (MWCO: 12-14 kDa, SpectraPor, USA), and then against distilled water and deionized water (DDW) for 2 days in a darkroom. The final product was lyophilized.

[0684]

[0685] Preparation of COS-FITC-coated peroxisomes

[0686] To prepare COS-FITC-coated peroxisomes, COS-FITC was dissolved in various concentrations in a pH 7.4 PBS solution. The COS-FITC solution was slowly added to the peroxisome solution (100 μg / ml) and reacted in a darkroom for 2 h. The final concentrations of the COS-FITC coating solution were adjusted to 25 μg / ml, 50 μg / ml, and 100 μg / ml.

[0687]

[0688] Synthesis of hemin- and ADH-linked hyaluronic acid (HA-ADH-hemin)

[0689] HA-ADH-hemin was synthesized via a two-step reaction: introduction of ADH into the HA backbone to synthesize HA-ADH, and coupling of HA-ADH and hemin with standard EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, (TCI, Tokyo, Japan)). Briefly, 500 mg of sodium hyaluronate (Mn 200 kDa, HA) (Contipro, Dolni Dobrouc, Czech Republic) was dissolved in distilled and deionized water (DDW, 50 ml). EDC (76 mg) was slowly added to the HA solution. After 30 min of reaction, ADH (459 mg) was added to the HA solution and reacted for 12 h. The pH of the reaction solution was maintained at 5 during the reaction. The HA-ADH product was purified using a dialysis membrane (MWCO 3.5 kDa, SpectraPor, USA) for 3 days and then lyophilized. HA-ADH was further reacted with hemin using a standard carbodiimide coupling agent. Briefly, HA-ADH (500 mg) was dissolved in DDW (30 ml) and hemin (258 mg) was dissolved in DMSO (20 ml), respectively. After complete dissolution of HA-ADH in DDW and hemin in DMSO, the hemin solution was added to the HA-ADH solution. After homogeneous mixing, EDC (152 mg) and NHS (91 mg) were slowly added to the HA-ADH / hemin solution. The reaction time was 12 h. The product was dialyzed using a membrane (MWCO 3.5 kDa, SpectraPor, USA) for 3 days. The final product was freeze-dried and stored in a moisture-free desiccator before use.

[0690]

[0691] Characteristics of HA-ADH-hemin

[0692] The conjugation of hemin groups to the HA backbone was confirmed using a UV-Vis spectrophotometer (UV-2450, Shimadzu, Kyoto, Japan) at the Core Facility for Biomedical Materials Analysis and Imaging Support at Wonkwang University, supported by the National Research Facilities and Equipment Center. Briefly, HA-ADH-hemin (0.1 mg / ml) was dissolved in phosphate-buffered saline (PBS) solution at pH 12, and the UV-vis spectrum of HA-ADH-hemin was obtained. The particle size distribution of HA-ADH-hemin was determined using dynamic light scattering (DLS, ELSZneo, Otsuka Electronics, Japan). The chemical composition of HA-ADH-hemin was analyzed using a scanning electron microscope equipped with energy-dispersive spectroscopy (SEM-EDS, S-4800, Hitachi Ltd., Tokyo, Japan) at the Core Facility for Biomedical Materials Analysis and Imaging Support.

[0693]

[0694] Catalytic activity of HA-ADH-hemin

[0695] The relative catalytic activity of HA-ADH-hemin in a pH 7.4 PBS solution was measured using pyrogallol. Briefly, a mixed solution (0.9 ml) of HA-ADH-hemin (0.01 mg / ml, 0.05 mg / ml, 0.1 mg / ml, and 0.5 mg / ml) and pyrogallol (5 mM) was prepared. An increase in absorbance at 420 nm was observed when hydrogen peroxide (H2O2) was added to the mixed solution. The concentration of H2O2 was varied from 0 to 100 mM (0 mM, 10 mM, 40 mM, 70 mM, and 100 mM). The relative catalytic activity was calculated by dividing the change in A420 by the A385 of HA-ADH-hemin. All experiments were performed in triplicate.

[0696]

[0697] Morphological evaluation

[0698] Root canal diameter and length were determined using Image-Pro Plus software (Media Cybernetics, Rockville, MD, USA) and a myotube analyzer (https: / / github.com / SimonNoe / myotube-analyzer-app). The fiber index was calculated as (number of nuclei in a root canal containing at least 3 nuclei / total number of nuclei) × 100 in five randomly selected fields per well. Root canal diameter was measured as the thickest part of each root canal. The average length and diameter of at least 200 root canals in 10 randomly selected fields were determined for each condition. Root canal length and diameter represent the maximum length and width, respectively.

[0699]

[0700] Lentiviral constructs packaging and delivery

[0701] To prepare lentiviral particles for injection, 293T cells were transfected with pLenti-GIII-CMV, pLenti-GIII-CMV-Acot4, pLenti-GIII-CMV-Acot12, or pLenti-GIII-CMV-Nudt7 using the 3rd Generation Packaging System Mix (Applied Biosystems, #LV053) according to the manufacturer's protocol. Briefly, plasmids were transfected into Lenti-X 293 T cells (Clontech, #632180) using Lentifectin (ABM, G074) in OPTI-MEM I medium (Thermo Fisher Scientific, #31985062), the supernatant was collected, and the lentiviral particles were concentrated using a Lenti-X Concentrator (Clontech, #631232). Cells were infected with lentiviral supernatant for 2 days in a humidified incubator at 37°C in the presence of 5% CO2. Lentivirus was injected into BaCl2-induced TA muscles for 3 days for lentiviral injection.

[0702]

[0703] Histology & Oil Red O staining

[0704] For histological analysis, sacrificed muscles were fixed in 10% neutral buffered formalin before embedding in paraffin or OCT compound. 5-μm paraffin-embedded muscle sections were used for H&E staining. Frozen TA muscle sections were stained with Oil Red O (Sigma-Aldrich) dissolved in 60% isopropanol. Staining intensity was quantified using Image-Pro Plus software.

[0705]

[0706] Immunofluorescence (IF) staining

[0707] Cells grown on coverslips were fixed with 4% paraformaldehyde (PFA, Sigma-Aldrich) for 10 min. After washing three times with ice-cold 1× PBS, cells were blocked with 3% normal goat serum (NGS; Vector Laboratories, Newark, CA, USA) and incubated with anti-myosin skeletal heavy chain (MYHC, 1:100, R&D Systems, Minneapolis, MN, USA, #MAB4470) and fluorescently conjugated secondary antibodies. Cryosections of TA muscles were blocked with 3% NGS and incubated with anti-PMP70 antibody (Thermo Fisher Scientific Korea Ltd., #PA1-650). Nuclei were stained with DAPI-containing mounting medium (Vector Laboratories).

[0708]

[0709] Immunohistochemical (IHC) staining

[0710] After deparaffinization and hydration, 5 μm paraffin-embedded muscle sections were blocked with 2% horse serum for 1 h. Tissue sections were incubated overnight with anti-ACOX1 (Cusabio, CSB-PA617998), anti-PMP70 (Thermo Fisher Scientific Korea Ltd., #PA1-650), anti-PLN2 (Abcam, ab52356), and anti-ACOT4 (Thermo Fisher Scientific) and anti-ACOT12 (Mybiosource; #MBS273137). For mouse NUDT7 IHC detection, a NUDT7 C-terminal-specific peptide (6–20 amino acids, GLPEPVRNNLIDDAK-C) was synthesized by Ab frontier (Seoul, Korea). An enzyme-linked secondary antibody (anti-rabbit-HRP) was used.

[0711]

[0712] quantitative real-time (qRT)-PCR

[0713] Total RNA was isolated using RNAiso Plus (TaKaRa, #9109) according to the manufacturer's instructions. 1 μg of RNA was then reverse-transcribed using 5X All-In-One RT Master Mix (ABM, #G492). Real-time PCR was performed on an ABI StepOnePlus instrument (Applied Biosystems, Waltham, MA, USA) using AMPIGENE qPCR Green Mix Hi-ROX (Enzo Biochem, Inc., Fammingdale, NY, USA, #ENZ-NUC103). The cycling protocol was 40 cycles of 95°C for 10 s, 57°C for 15 s, and 72°C for 10 s. All qRT-PCR reactions were performed in triplicate. The relative expression levels of each gene were normalized to the 18S rRNA expression levels. Forward and reverse primer sequences are listed in Tables 1 and 3.

[0714]

[0715]

[0716]

[0717] Statistical analysis

[0718] All results are expressed as the mean ± standard error (SE) of at least three independent experiments. Counts were performed by three independent operators who were unaware of the treatments. Representative experiments and images are shown unless otherwise specified. For two groups, an unpaired two-tailed Student's t-test was used. Analysis of variance was used to evaluate differences between multiple groups, followed by Fisher's least significant difference test.

[0719]

[0720] Example 8. Confirmation of muscle-related changes in the tibialis anterior muscle

[0721] Aged mice exposed to the systemic environment of young mice exhibited increased muscle fiber size and rejuvenated myotubes in the tibialis anterior (TA) muscle (Figs. 9a and 9b). Notably, aged mice receiving parabiosis showed significantly improved expression of peroxisome genes, including peroxisome biosynthesis factors (Pex3, Pex5, Pex12, Pex14, and Pex26), as well as Abcd3, Acot2, and Acot4. This was accompanied by a restoration of peroxisome abundance (Figs. 9c and 9d).

[0722]

[0723] Example 9. Confirmation of the correlation between muscle cell differentiation and peroxisome biosynthesis factors.

[0724] We analyzed the role of peroxisome biogenesis factors in myocytes. Specifically, we investigated the effects of peroxisome biogenesis factors on muscle cell differentiation by applying siRNA to silence Pex5 (siPex5) and Pex7 (siPex7) in C2C12 myoblasts.

[0725]

[0726] As a result, we confirmed that the deficiency of Pex5 or Pex7 significantly inhibited the influx of matrix proteins into peroxisomes, resulting in the formation of peroxisomal membrane ghosts. Introduction of siPex5 into C2C12 cells significantly reduced myotube size and downregulated the expression of key muscle proteins and genes, including myosin heavy chain (MyHC), Myh4 (encoding MHC-IIb), Myh7 (encoding MHC-I), and muscle creatine kinase (Mck) (Fig. 10a). Furthermore, this resulted in smaller myotube diameter and length (Fig. 10b). Similarly, inhibition of Pex7 significantly reduced markers of muscle differentiation and myogenicity regulators (Fig. 10c), highlighting the crucial role of peroxisome biogenesis in myoblast differentiation and myotube formation. Furthermore, the levels of ACOX1 and PMP70 proteins were also found to be lower in the elderly (Fig. 10d), which is consistent with the experimental results described above.

[0727]

[0728] In Examples 8 and 9, heterologous parabiosis comprising surgical combinations of young and old rats was demonstrated to significantly counteract age-related muscle wasting.

[0729]

[0730] Example 10. Confirmation of the inhibitory effect of fatty acids on peroxisomal β-oxidation and myoblast differentiation.

[0731] The effects of different fatty acid types on peroxisomal β-oxidation and myoblast differentiation were further investigated.

[0732]

[0733] As a result, it was observed that exposure to specific fatty acids such as palmitic acid, dicarboxylic acid and phytol had detrimental effects on cell differentiation as evidenced by a decrease in myotube size and inhibition of myogenic marker expression (Figs. 11a to 11e).

[0734] Specifically, a phytol-rich diet in mice was shown to downregulate Acox1 and Abcd3 expression, leading to inhibition of peroxisomal β-oxidation (Figs. 11f-11h). Furthermore, this inhibition was found to be associated with decreased muscle fiber size and increased Atrogin-1 expression (Fig. 11i).

[0735]

[0736] Example 11. Inhibitory effects on peroxisomal β-oxidation and myoblast differentiation induced by oxidative stress.

[0737] Inhibition of catalase (siCAT), an important enzyme that alleviates oxidative stress, in C2C12 cells resulted in decreased expression of myogenic regulatory factors and reduced myotube size (Fig. 12a and Fig. 12b).

[0738]

[0739] Knockdown of Acox1 in C2C12 myoblasts significantly altered myotube morphology, especially when performed during early differentiation stages (Fig. 12c-f), and increased lipid accumulation and lipid ROS production (Fig. 12g), confirming the important role of peroxisomal β-oxidation in maintaining muscle cell mass and function.

[0740]

[0741] Example 12. Confirmation of decreased peroxisome gene expression in a muscle atrophy model.

[0742] We observed changes in peroxisomal gene expression associated with muscle atrophy across diverse data sets (including human muscular atrophy, mouse muscular atrophy, amyotrophic lateral sclerosis (ALS), dexamethasone (DEX)-induced muscular dystrophy, and Duchenne muscular dystrophy (DMD)) and in three distinct models of muscle atrophy (denervation, myocardial infarction, and BaCl2-induced).

[0743]

[0744] As a result, the results were confirmed as shown in Figure 13a (various data sets) and Figure 13b (separate muscle atrophy models), respectively. Furthermore, introduction of DEX into C2C12 cells resulted in a decrease in myotube diameter and a marked decrease in the peroxisome profile (Figures 13c and 13d).

[0745]

[0746] Example 13. Confirmation of decreased lipid metabolism in a muscle atrophy model.

[0747] The importance of peroxisomal β-oxidation in acyl-CoA metabolism was reaffirmed by the consistent observation of downregulation of lipid metabolism genes in both human and mouse models of muscle atrophy (Figs. 14a and 14b). Direct exposure to CoA, acetyl-CoA, or succinyl-CoA significantly inhibited myotube formation (Fig. 14c).

[0748]

[0749] To further investigate the functional effects of peroxisomal acyl-CoA metabolism on muscle atrophy, Acot4 knockout (KO) mice were generated through germline transmission of an RNA-guided endonuclease-induced mutation, and muscle-related functions were analyzed from various perspectives (Fig. 14d).

[0750]

[0751] As a result, primary myoblasts isolated from Acot12- / - (A12KO), Acot4- / - (A4KO), and Nudt7- / - (N7KO) mice exhibited significantly impaired myotube formation (Fig. 14e) and altered expression levels of myogenic markers (Fig. 14f). Treatment with DEX further exacerbated this condition by reducing myotube formation and increasing the expression of atrophy-related markers in A12KO, A4KO, and N7KO myoblasts (Figs. 14g-j). Studies in Acot4, Acot12, and Nudt7 KO mice demonstrated an essential role for peroxisomal acyl-CoA metabolism in muscle atrophy, as evidenced by decreased muscle fiber size and increased lipid accumulation (Figs. 14k and 14l).

[0752]

[0753] Example 14. Confirming the correlation between muscle damage and mitochondrial function.

[0754] Muscle cells were analyzed after muscle damage was induced by BaCl2 injection. Results showed a decrease in muscle fiber area and diameter, along with an increase in smaller myotubes after BaCl2 injection (Figs. 15a and 15b). In vivo experiments further emphasized the importance of peroxisomal genes in strategies to maintain muscle health and prevent or treat muscle atrophy without negatively impacting mitochondrial function (Figs. 15c to 15h).

[0755]

[0756] Example 15. Confirmation of the therapeutic effect of peroxisome administration on muscle atrophy.

[0757] Example 15-1. Production of peroxisome-cos-FITC

[0758] To further investigate the central role of peroxisome activity in age-related muscle atrophy and its treatment, we analyzed whether restoring peroxisome function in aged muscle fibers through peroxisome delivery could be an effective therapeutic approach. This strategy was expected to have the potential to halt or even reverse the progression of sarcopenia, as shown in Figure 16a.

[0759]

[0760] First, to track cell binding and behavior after peroxisome injection, fluorescein-5-isothiocyanate-labeled chitosan oligosaccharide (COS-FITC) was synthesized according to a previously established method (Fig. 16b). Peroxisomes were encapsulated with COS-FITC at various concentrations (25–100 μg / ml). The morphology and activity of isolated peroxisomes were confirmed (Fig. 16c), and detailed fluorescence imaging confirmed the successful internalization of COS-bound peroxisomes into C2C12 cells (Fig. 16d).

[0761]

[0762] Example 15-2. Confirmation of excellent muscle atrophy treatment activity following peroxisome-cos-FITC treatment.

[0763] When peroxisome-cos-FITC prepared in Example 15-1 was introduced into DEX-treated C2C12 cells, the decrease in myotube diameter and length caused by DEX treatment was alleviated (Fig. 16e).

[0764] Furthermore, in vivo imaging system (IVIS) analysis confirmed the effective delivery of peroxisome-COS-FITC complexes to TA muscles affected by BaCl2, and the persistence of COS-FITC fluorescence signals indicated the localization and maintenance of peroxisomes within the muscle tissue (Fig. 16f). Histological evaluation of TA muscles showed that direct administration of peroxisomes after BaCl2 injection moderately improved fiber size and reduced inflammation. In particular, muscles treated with peroxisome-COS-FITC complexes showed significantly preserved muscle fiber size and structure with minimal inflammatory response compared to the other groups (Fig. 16g). This indicates that COS-FITC conjugation significantly enhances the therapeutic effect of peroxisome delivery in combating muscle atrophy.

[0765]

[0766] Example 16. Peroxisome mimetic: Confirmation of excellent muscle atrophy treatment effect following nanozyme HA-ADH-hemin treatment

[0767] After confirming the excellent muscle atrophy treatment effect according to peroxisome treatment confirmed in Example 15, a peroxisome mimetic nanozyme was produced and investigated whether the muscle atrophy treatment activity was maintained (Fig. 17a).

[0768]

[0769] Example 16-1. Preparation and Characterization of Nanozyme HA-ADH-Hemin

[0770] HA-ADH-hemin was prepared as a peroxisome mimic. The HA-ADH-hemin conjugate was synthesized to form nanoparticles that catalyze H2O2-mediated reactions, effectively reducing H2O2-induced oxidative stress and cartilage damage.

[0771] To synthesize HA-ADH-hemin, adipic acid dihydrazide was first introduced into the HA backbone to form HA-ADH (Figure 17b, first to second). Hemin was then conjugated to HA-ADH using standard carbodiimide chemistry, forming an amide bond between the amine group of HA-ADH and the carboxylic acid group of ADH (Figure 17b, second to third).

[0772]

[0773] Figure 17c shows the schematic of HA-ADH-hemin, which features multiple hemin groups along the HA backbone. To confirm hemin binding, UV-Vis spectra were acquired for both HA-ADH-hemin and hemin in a pH 12 PBS solution, and a characteristic absorbance peak (~350 nm) representing hemin was observed (Figure 17d). The absorbances at wavelengths of ~400 nm and 400–500 nm are attributed to the π–π* transition of porphyrin mediated by iron ions. In HA-ADH-hemin, a characteristic splitting indicating hemin dimerization was observed near 400 nm. The degree of hemin substitution of HA-ADH-hemin was calculated to be 3.5% by comparing the absorbance of HA-ADH-hemin at 385 nm with a standard curve of hemin concentrations.

[0774]

[0775] The solubility and appearance of HA-ADH-hemin in various pH solutions showed excellent solubility at neutral pH, which appeared as a transparent, brownish color without significant precipitation (Fig. 17e and Fig. 17f). In fact, hemin is practically insoluble in water, but if hemin is dissolved in a basic pH solution and the pH is adjusted to neutral, it can be dissolved in a neutral pH solution. In contrast, HA-ADH-hemin showed excellent solubility in a neutral pH solution. At 0.1 mg / mL and 0.01 mg / mL hemin standards, HA-ADH-hemin was readily dissolved in a pH 7.4 PBS solution without any treatment (Fig. 17e, first). Furthermore, HA-ADH-hemin in a pH 7.4 PBS solution was found to remain stable for at least 2 weeks (Fig. 17e, second).

[0776]

[0777] Additionally, hemin in a pH 12 solution was confirmed to have a similar brown color without precipitate (Fig. 17e, third). However, hemin directly dissolved in a pH 7.4 PBS solution showed significant aggregates (Fig. 17e, fourth). The UV-Vis spectrum also supported the solubility of HA-ADH-hemin. Hemin directly dissolved in a pH 7.4 PBS solution did not exhibit a UV-Vis spectrum due to hemin's insolubility (Fig. 17g).

[0778]

[0779] Additionally, the absorbance value of HA-ADH-hemin at 385 nm in a pH 7.4 PBS solution was significantly higher than that of the hemin solution adjusted to pH 7.4. Considering the same hemin base of hemin and HA-ADH-hemin, it appears that a small portion of hemin is insoluble at pH 7.4.

[0780]

[0781] EDS spectral analysis confirmed the presence of iron-containing porphyrin structures in the polymer network (Fig. 17h). HA-ADH-hemin (0.1 mg / mL) formed nano-sized aggregates (280–425 nm) due to the hydrophobic nature of hemin in the polymer chain (Fig. 17i). Furthermore, HA-ADH-hemin at concentrations of 0.05 mg / mL and 0.01 mg / mL also exhibited particle size distributions similar to those of other HA-ADH-hemins (Fig. 17j).

[0782]

[0783] The solubility and turbidity of HA-ADH-hemin were further evaluated, highlighting its solubility over a wide pH range and increased turbidity in acidic solutions (Fig. 17k). The catalytic activity of HA-ADH-hemin was verified using pyrogallol, and the addition of HA-ADH-hemin and hydrogen peroxide increased the activity (Fig. 17l).

[0784]

[0785] Example 16-2. Confirmation of excellent muscle atrophy treatment activity following nanozyme HA-ADH-hemin treatment.

[0786] When the nanozyme manufactured in Example 16-1 was administered to C2C12 cells, it was shown that myotube formation damaged by DEX and phytol was restored in a dose-dependent manner (Fig. 17m).

[0787]

[0788] Furthermore, histological staining confirmed that muscle fiber size was improved in a dose-dependent manner after BaCl2 injection with nanozyme, and inflammatory cell infiltration and lipid accumulation were also significantly reduced. Furthermore, ACOX1 and PMP70 staining levels, along with peroxisome gene profiles, were decreased in a dose-dependent manner, with the highest effect observed at the highest concentration (Figs. 17n and 17o). Furthermore, nanozyme treatment upregulated essential mitochondrial and antioxidant markers (Figs. 17p to 17s).

[0789]

[0790] These experimental results demonstrate that the nanozyme of the present invention is a peroxisome substitute capable of mimicking the feasible peroxisome function, as it was confirmed to restore peroxisome activity and provide a new treatment method for muscle atrophy.

[0791]

[0792] [Composition for preventing, improving, or treating hair loss containing peroxisome as an active ingredient]

[0793]

[0794] [Example]

[0795]

[0796] Example 17. Isolation and characterization of peroxisomes

[0797] Peroxisomes were isolated and characterized. Specifically, peroxisomes were isolated from the livers and kidneys of four to six eight-week-old male mice, and Western blot analysis was performed. The extracted peroxisomes were frozen in pellet form at -60°C and then resuspended in cold PBS for use when needed.

[0798]

[0799] As a result, PMP70, a membrane protein of the peroxisome, was detected. Conversely, TOMM20, a mitochondrial protein marker, was not detected (Figure 18). This confirmed that peroxisomes had been successfully isolated from the liver and kidney.

[0800]

[0801] Example 18. Production of HA-SH and HA-SH-PERO

[0802] Example 18-1. Production of HA-SH

[0803] Thiolated hyaluronic acid (HA-SH) with a thiol group was produced. Specifically, the HA-SH of the present invention was synthesized by the following method. First (Fig. 19a), a hyaluronic acid polymer (500 mg) was dissolved in 25 ml of distilled and deionized water (DDW), and then EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 479 mg, TCI, CAS: 25952-53-8, product number D1601), HOBT (1-Hydroxybenzotriazole hydrate, 385 mg, Sigma-Aldrich, product number: 54802), and cysteamine (384 mg) were dissolved in 10 ml of DDW and added, and the pH was maintained at 6-7 during the reaction. The reaction was carried out for 12 hours. After the reaction was completed, non-reactants were removed through membrane dialysis for three days. First, membrane dialysis was performed using a 10 mM NaCl solution for two days, and then using DDW for one day. After this, the final HA-SH was obtained through freeze-drying.

[0804]

[0805] Example 18-2. Confirmation of the characteristics of HA-SH

[0806] The characteristics of HA-SH manufactured in Example 18-1 were analyzed. Nuclear magnetic resonance spectroscopy confirmed the introduction of thiol groups into the hyaluronic acid polymer. Furthermore, analysis using Ellman's assay revealed that the degree of thiolation of the hyaluronic acid polymer was 59.6%, approximately 60%.

[0807]

[0808] In addition, HA-SH was shown to have hyaluronidase (HAdase) resistance compared to hyaluronic acid. To analyze this, the amount of hyaluronic acid degraded was measured using the uronic acid carbazole assay. Specifically, hyaluronic acid degrading enzyme (HAdase) was added together with HA-SH inside the dialysis membrane, and the dialysis membrane was placed on top of 5 mL of PBS solution in a 15 mL tube. After incubation for 2 days, the PBS solution outside the dialysis membrane was recovered, and the amount of hyaluronic acid released from the dialysis membrane was measured using the uronic acid carbazole assay (Fig. 19c).

[0809]

[0810] As a result, it was confirmed that HA-SH has a higher resistance to degradation by hyaluronic acid decomposition enzyme than HA and can maintain its function for a longer period of time (Fig. 19d).

[0811]

[0812] Example 18-3. Production and characterization of HA-SH-PERO.

[0813] The peroxisomes extracted in Example 17 were mixed with the thiol-group-introduced hyaluronic acid (HA-SH) produced in Example 18-1 to produce peroxisomes bound to the thiol-group-introduced hyaluronic acid (HA-SH) (hereinafter, HA-SH-PERO). Specifically, HA-SH was dissolved in distilled water (dH2O) at a concentration of 4 mg / ml and then placed in a stirrer for more than one day to completely dissolve all HA-SH. Thereafter, a 5 mg / ml peroxisome solution dissolved in PBS was prepared. The HA-SH solution and the peroxisome solution were mixed at a ratio of 5:1, and the mixture was placed in a stirrer at a refrigerated temperature for more than 1 hour to bind the HA-SH and peroxisomes.

[0814]

[0815] We analyzed whether HA-SH-PERO maintained peroxisomal activity after binding to HA-SH. As a result, PMP70, a peroxisomal membrane protein, and catalase, a peroxisomal enzyme, were detected (Figs. 19e and 19f). Therefore, it was confirmed that HA-SH-PERO maintained peroxisome shape and function regardless of binding to HA-SH.

[0816]

[0817] Example 19. Confirmation of hair loss activity of HA-SH-PERO: Increase in hair follicle size, confirmation of characteristics of hair follicles in the growth phase

[0818] In order to analyze the hair loss activity of HA-SH-PERO produced in Example 18, the characteristics of hair follicles when treated with HA-SH-PERO were analyzed. Specifically, after removing the back hair of 6-week-old male mice, the mice were divided into two groups and injected subcutaneously with HA-SH and HA-SH-Pero. Specifically, HA-SH was mixed with PBS in a 5:1 ratio and injected into the backs of the control group mice 6 times with 10 μl each, for a total of 60 μl. The mixed solution of HA-SH and peroxisome produced in Examples 17 and 18 was injected into the backs of the experimental group mice 6 times with 10 μl each, for a total of 60 μl. The injections were performed subcutaneously on the backs of the mice. Then, the skin tissue was observed through H&E staining and connective tissue staining (Masson's Trichrome stain), and the shape, size, and location of the hair follicles were compared.

[0819]

[0820] As a result, it was confirmed that the size of the hair follicles was significantly larger when treated with HA-SH-PERO compared to when treated with HA-SH. In addition, the characteristics of hair follicles in the growth phase were observed, such as hair starting to grow or being located deep in the hypodermis (Fig. 20).

[0821]

[0822] Example 20. Confirmation of hair loss activity of HA-SH-PERO: Confirmation of cell division and growth phase hair follicles

[0823] In order to analyze the hair loss activity of HA-SH-PERO produced in Example 18, its effect on skin tissue was analyzed. At this time, the experimental conditions were the same as in Example 19. Immunohistochemical staining was performed on skin tissue treated with HA-SH and HA-SH-PERO using Ki-67 antibody.

[0824]

[0825] As a result, it was confirmed that it corresponds to the result of H&E of Example 19. Specifically, it was shown that Ki-67 staining was strongly developed in the hair follicles, especially in the hair bulb area, of the tissue treated with HA-SH-PERO. Ki-67 is expressed in the proliferation phase (G1, S, G2, M phase) of the cell cycle, and is expressed in dividing cells, so Ki67 antibody stains dividing cells. Therefore, through these experimental results, it was confirmed that the hair follicles corresponding to anagen in the skin tissue treated with HA-SH-PERO had many actively dividing cells (Fig. 21).

[0826]

[0827] Example 21. Confirmation of hair loss activity of HA-SH-PERO: Increase in the number of hair follicles in the growth phase and the area of ​​hair follicles

[0828] In order to analyze the hair loss activity of HA-SH-PERO produced in Example 18, its effect on the growth phase of hair follicles was analyzed. Specifically, hair follicle stages were distinguished and analyzed through H&E staining and Masson's trichrome staining, and the experimental conditions were the same as in Example 19.

[0829]

[0830] As a result, in the group treated with HA-SH, the ratio corresponding to anagen, which corresponds to the growth phase of the hair follicle growth cycle, was significantly higher, and the ratio corresponding to telogen, which corresponds to the resting phase of the hair follicle, was not observed. On the other hand, in the group treated with HA-SH-PERO, the anagen ratio was over 80%, meaning that most hair follicles were in the hair growth phase, and some hair follicles were in the catagen or telogen phase, which correspond to the regression phase after hair growth was completed (Fig. 22, first graph). In addition, the number of hair follicles itself was significantly higher in the group treated with HA-SH-PERO than in the group treated with HA-SH, and a large number of hair follicles in the growth phase (anagen) were observed (Fig. 22, second graph). Table 4 below shows the ratio of hair follicles corresponding to each growth phase in the groups treated with each substance.

[0831]

[0832]

[0833]

[0834] In addition, the area of ​​hair follicles per unit area was found to be wider in the group treated with HA-SH-PERO, and this was confirmed to be statistically significant (Fig. 22, third graph).

[0835]

[0836] Example 22. Confirmation of hair loss activity of HA-SH-PERO: Effect on blood vessels

[0837] In order to analyze the hair loss activity of HA-SH-PERO produced in Example 18, its effect on blood vessels was investigated. As a specific experimental method, HA-SH and HA-SH-Pero were injected subcutaneously into the area after removing some of the hair on the back of the mouse in the same manner as in Example 19. After confirming hair growth, the back skin was collected, fixed and dehydrated, and made into paraffin tissue. Then, 5-μm-thick sections were made to prepare tissue slice slides. The slides were made into serial sections and stained with H&E and immunohistochemical staining. The skin of the mouse is composed of layers of the epidermis, dermis, and hypodermis. Hair follicles are spread throughout the dermis and hypodermis, and anagen hair follicles are mainly located deep in the hypodermis, so observation was focused on the hypodermis.

[0838]

[0839] First, immunohistochemical staining using CD29 antibody showed that the number and size of blood vessels were greater in the tissue treated with HA-SH-PERO (Fig. 23a).

[0840] In addition, to confirm the hair loss activity of HA-SH-PERO and the effect of peroxisomes, immunohistochemical staining was performed using PMP70 antibody, and it was found that more peroxisomes were observed in tissues treated with HA-SH-PERO (Fig. 23b).

[0841]

[0842] Example 23. Confirmation of the effect of HA-SH-PERO on increasing proliferation and migration of human skin fibroblasts.

[0843] The human skin fibroblast proliferation and migration activity of HA-SH-PERO produced in Example 18 was analyzed.

[0844]

[0845] Example 23-1. Effect of HA-SH-PERO on human skin fibroblast proliferation

[0846] First, a viability (proliferation) assay was performed to analyze the proliferation effect of HA-SH-PERO on human dermal fibroblasts (HDFs). Specifically, 2 x 10 human dermal fibroblasts were seeded in a 24-well culture plate. 4 Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) + 10% FBS (fetal bovine serum) + Penicillin / Streptomycin medium at a concentration of 10 cells / well at 37°C and 5% CO2 for 24 hours. 2 μl, 5 μl, and 10 μl of the manufactured HA-SH-PERO (Prx) were added to the cultured cells, respectively, and phosphate-buffered saline (PBS) was used as a control. Cell viability (proliferation) was measured using the EZ-Cytox Enhanced Cell Viability Assay Kit (Itsbio, Korea) according to the manufacturer's instructions.

[0847]

[0848] As a result, it was confirmed that the proliferation of human skin fibroblasts significantly increased in a concentration-dependent manner as peroxisome activity increased (Fig. 24a).

[0849]

[0850] Example 23-2. Effect of HA-SH-PERO on increasing migration of human skin fibroblasts

[0851] Next, a cell migration assay (scratch wound healing) was performed to analyze the effect of HA-SH-PERO on increasing the proliferation of human dermal fibroblasts. Specifically, human dermal fibroblasts were seeded at 1 x 10 in a 24-well culture plate. 5Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) + 10% FBS (fetal bovine serum) + Penicillin / Streptomycin medium at a concentration of 10 cells / well at 37°C and 5% CO2 for 24 hours to 90-100% confluency. The cultured cells were scratched with a plastic tip to create a scratch wound, and 2 μl, 5 μl, and 10 μl of the manufactured HA-SH-PERO (Prx) were added to the cultured cells, respectively. As a control, phosphate-buffered saline (PBS) was added, and the cells that had migrated to the scratch wound after 8 and 22 hours were measured using a Leica light microscope (Wetzlar, Germany).

[0852]

[0853] As a result, it was found that the migration of human dermal fibroblasts (HDFs) increased in a concentration-dependent manner as peroxisome activity increased, and this was confirmed to be statistically significant (Fig. 24b).

[0854]

[0855] Example 24. Confirmation of the hair growth effect of HA-SH-PERO

[0856] The hair growth effect of HA-SH-PERO produced in Example 18 was analyzed. As a specific experimental method, the back hair of mice was removed in the same manner as in Example 19, and photographs of the hair-removed area were taken every day to compare the effects of HA-SH and HA-SH-PERO.

[0857]

[0858] As a result, it was confirmed through experiments using an in vivo animal model that hair growth significantly increased compared to the control group as peroxisome activity increased.

[0859] First, a significant increase in the overall number of hair follicles was observed. The catagen phase of hair follicles is a period in which hair growth slowly stops or hair that has stopped growing is maintained. During this period, treatment with HA-SH-Pero showed an increase in the number of hairs in the catagen phase as hair follicles recovered and grew. According to Figures 25a and 25b, compared to HA-SH, HA-SH-Pero showed a faster hair growth rate and a faster rate at which hair follicles reached the catagen phase.

[0860]

[0861] Upon entering the anagen stage, the skin begins to turn gray, and soon after turning dark gray, hair begins to grow. According to Figure 25a, in the HA-SH-PERO-treated group, the back began to turn gray from day 9, hair began to grow from day 12, and by day 19, most of the back was covered with hair.

[0862]

[0863] Analysis of the area of ​​the black area using the black-and-white photographs showed that in the HA-SH-PERO-treated group, more than 10% of the shaved area was covered with hair on the 12th day, which increased to 91% on the 19th day. In contrast, in the HA-SH-treated group, hair began to grow on the 12th day, 10% was covered with hair on the 15th day, and only about 50% was covered with hair on the 19th day (Fig. 25b). These results suggest that HA-SH-PERO induces the transition from the telogen to the anagen phase of hair follicles more quickly.

[0864]

[0865] The foregoing description of the present invention is provided for illustrative purposes only. Those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

[0866] [Composition for preventing or treating diseases related to muscle loss, containing a peroxisome function inducer as an active ingredient]

[0867] According to a composition for preventing or treating a disease related to muscle loss, which contains a peroxisome function inducer as an active ingredient, it was confirmed that when acyl CoA is not oxidized by the β-oxidation system of the peroxisome and / or when a peroxisome biosynthesis factor is knocked down, muscle differentiation is inhibited, muscle atrophy is induced, and the rate and course of the induced muscle atrophy are worsened. On the other hand, since the muscle atrophy effect is restored as the function of the peroxisome is restored, the present invention can be usefully utilized as a preventive or therapeutic agent for a disease related to muscle loss, and thus its industrial applicability is recognized.

[0868]

[0869] [Pharmaceutical composition for preventing or treating muscle wasting-related diseases containing peroxisome or peroxisome mimic as an active ingredient]

[0870] According to a pharmaceutical composition for preventing or treating diseases related to muscle loss, which comprises peroxisomes or peroxisome mimetics as active ingredients, the association between peroxisome β-oxidation dysregulation and sarcopenia was investigated, and it was confirmed that peroxisome dysfunction can indirectly impair mitochondrial efficiency, disrupt metabolic balance, and contribute to muscle damage through altered lipid metabolism, oxidative stress, and inflammation. In addition, since a significantly superior therapeutic effect was confirmed when peroxisomes or peroxisome mimetics were administered to a muscle damage model, it can be utilized as an innovative and promising strategy for preventing or treating muscle loss, and thus its industrial applicability is recognized.

[0871]

[0872]

[0873] [Composition for preventing, improving, or treating hair loss containing peroxisome as an active ingredient]

[0874] According to a composition for preventing, improving, or treating hair loss, which comprises peroxisomes as an active ingredient, a peroxisome complex is provided, in which hyaluronic acid having a thiol group introduced therein is combined. The complex of the present invention exhibits the effects of inducing the activation of peroxisomes, thereby increasing the number and size of total hair follicles, increasing the proportion of hair follicles in the anagen phase, and increasing the area of ​​hair follicles per unit area. In addition, the complex has been confirmed to increase the number and size of blood vessels, increase the proliferation and migration activity of skin fibroblasts, and increase hair growth. Therefore, the complex can be usefully utilized as an excellent composition for preventing, improving, and treating hair loss, and its industrial applicability is recognized.

Claims

1. A pharmaceutical composition for preventing or treating a disease related to muscle loss, comprising a peroxisome function activation inducer as an active ingredient.

2. In paragraph 1, A pharmaceutical composition wherein the peroxisome function activation inducer comprises a peroxisome β oxidation activation inducer.

3. In paragraph 2, A pharmaceutical composition, wherein the peroxisome β-oxidation activity inducer comprises an activity inducer for at least one of acyl CoA thioesterase and nudix hydrolase.

4. In paragraph 3, A pharmaceutical composition, wherein the acyl CoA thioesterase is at least one selected from the group consisting of ACOT 1, ACOT 2, ACOT 3, ACOT 4, ACOT 5, ACOT 6, ACOT 7, ACOT 8, ACOT 9, ACOT 10, ACOT 11, and ACOT 12.

5. In paragraph 3, A pharmaceutical composition, wherein the nudix hydrolase is at least one selected from the group consisting of NUDT7, NUDT19, and NUDT8.

6. In paragraph 2, The above peroxisome β oxidation activity inducer further includes an acyl CoA oxidase (ACOX) activity inducer, A pharmaceutical composition, wherein the acyl CoA oxidase is at least one selected from the group consisting of ACOX1, ACOX2, ACOX3, and ACOX4.

7. In paragraph 1, The above peroxisome function activation inducer further includes a peroxisomal biogenesis factor (PEX) activation inducer, A pharmaceutical composition, wherein the peroxisome biosynthesis factor is at least one selected from the group consisting of Pex2, Pex5, Pex7, Pex11, Pex13, Pex16, and Pex19.

8. In paragraph 1, A pharmaceutical composition, wherein the above-mentioned activity inducer is at least one of an overexpression agent or an activating agent.

9. In paragraph 8, A pharmaceutical composition wherein the above overexpression agent or activation agent is any one selected from the group consisting of a recombinant vector for overexpressing a protein to be overexpressed or activated or a gene encoding the same, a compound, a peptide, an aptamer, a primer, a probe, and an antibody that specifically binds to the gene.

10. In any one of paragraphs 1 to 9, A pharmaceutical composition, wherein the disease related to muscle loss is at least one selected from the group consisting of sarcopenia, muscular atrophy, myasthenia, muscular dystrophy, myotonia, hypotonia, muscular weakness, amyotrophic lateral sclerosis (ALS), and muscular dystrophy.

11. A method for preventing or treating a disease related to muscle loss, comprising a step of administering a composition containing a peroxisome function activation inducer as an active ingredient in a pharmaceutically effective amount to a subject in need thereof.

12. Use of a composition containing a peroxisome function inducer as an active ingredient for preventing or treating diseases related to muscle loss.

13. Use of a composition containing a peroxisome function activation inducer as an active ingredient for manufacturing a preparation for preventing or treating a disease related to muscle loss.

14. A pharmaceutical composition for preventing or treating a disease related to muscle loss, comprising at least one active ingredient selected from the group consisting of peroxisomes and peroxisome mimics.

15. In paragraph 14, A pharmaceutical composition wherein the peroxisome is additionally combined with chitosan oligosaccharide.

16. In paragraph 14, A pharmaceutical composition, wherein the peroxisome mimic is a nanozyme.

17. In paragraph 16, A pharmaceutical composition wherein the above nanozyme is hyaluronic acid-adipic acid dihydrazide-hemin (HA-ADH-hemin).

18. In any one of paragraphs 14 to 17, A pharmaceutical composition, wherein the disease related to muscle loss is at least one selected from the group consisting of sarcopenia, muscular atrophy, myasthenia, muscular dystrophy, myotonia, hypotonia, muscular weakness, amyotrophic lateral sclerosis (ALS), and muscular dystrophy.

19. In paragraph 14, A pharmaceutical composition, wherein the peroxisome is included in a concentration of more than 0 to 900 μg / ml based on the entire composition.

20. In paragraph 14, A pharmaceutical composition, wherein the peroxisome mimetic is included in a concentration of more than 0 to 1 mg / ml based on the total composition.

21. In paragraph 14, A pharmaceutical composition characterized in that the composition comprises at least one selected from the group consisting of: (a) Recovery of reduced root canal diameter and length; (b) muscle fiber size is restored; (c) root canal is formed; (d) reduced inflammation and lipid accumulation; and (e) Increased expression of mitochondrial and antioxidant markers.

22. A kit for preventing or treating a disease related to muscle loss, comprising a composition comprising at least one active ingredient selected from the group consisting of peroxisomes and peroxisome mimics, and an instruction manual.

23. A method for preventing or treating a disease related to muscle loss, comprising a step of administering to a subject in need thereof a pharmaceutically effective amount of a composition containing at least one active ingredient selected from the group consisting of peroxisomes and peroxisome mimics.

24. A composition comprising at least one active ingredient selected from the group consisting of peroxisomes and peroxisome mimics, for use in preventing or treating diseases related to muscle loss.

25. A use for producing a preparation for preventing or treating a disease related to muscle loss, comprising a composition comprising at least one active ingredient selected from the group consisting of peroxisomes and peroxisome mimics.

26. A pharmaceutical composition for preventing or treating hair loss, containing peroxisome as an active ingredient.

27. In paragraph 26, A pharmaceutical composition in which the above peroxisome is combined with hyaluronic acid having a thiol group introduced therein.

28. In paragraph 27, A pharmaceutical composition, wherein the degree of thiolation of the hyaluronic acid into which the thiol group is introduced is 40% to 80%.

29. In paragraph 26 or 27, A pharmaceutical composition, wherein the peroxisome is characterized by at least one selected from the group consisting of: Expresses PM70; Does not express TOMM20; and Contains catalase.

30. In any one of paragraphs 26 to 28, A pharmaceutical composition characterized by at least one selected from the group consisting of the above compositions: Activates peroxisomes; Increases the number and size of hair follicles; Increases the proportion of hair follicles in the growth phase; Increases the area of ​​hair follicles per unit area; Increases the number and size of blood vessels; Increases skin fibroblast proliferation and migration activity; and Increases hair growth.

31. A pharmaceutical composition for preventing or treating hair loss, comprising at least one of peroxisome or peroxisome combined with hyaluronic acid having a thiol group introduced therein as an active ingredient.

32. A kit for preventing or treating hair loss, comprising a composition comprising at least one of peroxisome and peroxisome combined with hyaluronic acid having a thiol group introduced therein as an active ingredient, and an instruction manual.

33. A health functional food composition for improving hair loss, promoting hair growth, promoting hair growth, or improving scalp condition, comprising at least one of peroxisome or peroxisome combined with hyaluronic acid having a thiol group introduced therein as an effective ingredient.

34. A cosmetic composition for improving hair loss, promoting hair growth, or improving scalp condition, comprising at least one of peroxisome or peroxisome combined with hyaluronic acid having a thiol group introduced therein as an effective ingredient.

35. A method for producing a peroxisome bound to hyaluronic acid having a thiol group, comprising the following steps: (S1) a step of producing hyaluronic acid (HA-SH) with a thiol group introduced therein by reacting a solution containing dissolved hyaluronic acid with a solution containing dissolved EDC, HOBT, and cystamine; and (S2) A step of producing a peroxisome combined with hyaluronic acid having a thiol group introduced therein by mixing peroxisome and the above HA-SH.

36. A method for preventing, improving, treating, promoting hair growth, promoting hair growth, or improving scalp condition, comprising a step of administering to a subject in need thereof a pharmaceutically effective amount of a composition comprising at least one of peroxisome, or peroxisome combined with hyaluronic acid having a thiol group introduced therein, as an active ingredient.

37. A composition comprising at least one of peroxisome or peroxisome combined with hyaluronic acid having a thiol group introduced as an active ingredient for use in preventing, improving, treating, promoting hair growth, promoting hair growth, or improving scalp condition.

38. Use of a composition comprising at least one of peroxisome or peroxisome combined with hyaluronic acid having a thiol group introduced therein as an active ingredient for manufacturing a preparation for preventing, improving, treating, promoting hair growth, promoting hair growth, or improving scalp condition.

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