Composition for preventing or treating sarcopenia comprising an ALOX5 inhibitor

Abstract

The present invention relates to a composition for preventing or treating sarcopenia, comprising an Alox5 inhibitor, which contains a substance that targets Alox5 and inhibits its expression or activity, thereby improving muscle performance, increasing muscle mass and cross-sectional area of ​​muscle fibers, and reducing the expression of muscle atrophy regulatory factors, thereby effectively preventing, improving and treating sarcopenia.

Description

[Technical field]

[0001] The present invention relates to a composition for preventing or treating sarcopenia, comprising an Alox5 inhibitor. [Background technology]

[0002] Sarcopenia is a concept that began when Irwin Rosenberg introduced the term "Sarcopenia" in 1989. It is a compound word of the Greek words "sarco" meaning muscle and "penia" meaning decrease. Sarcopenia is associated with aging and refers to a decrease in muscle strength due to a decrease in muscle mass. Here, "muscle" refers to skeletal muscle and has nothing to do with smooth muscle. In other words, sarcopenia refers to a decrease in skeletal muscle distributed mainly in the limbs, and is distinguished from cachexia, a state of significant muscle loss that appears in the terminal stage of malignant tumors, muscle wasting due to acute diseases such as influenza, or primary muscle disease.

[0003] In recent years, as the population of people aged 65 and over has rapidly increased, the prevalence of osteoporosis and sarcopenia has also risen sharply. It is estimated that gradual loss of muscle mass occurs from the 40s to the 70s, decreasing by 8% per decade, and then decreases more rapidly, reaching up to 15% per decade. Numerous follow-up studies have revealed that there are a variety of physiological changes that occur in the elderly, and that muscle mass and bone density generally decrease simultaneously with aging.

[0004] Meanwhile, various researches are being conducted on methods for effectively controlling sarcopenia. For example, growth hormone (GH) has been developed to increase muscle mass, but it is very expensive and has some undesirable side effects such as shortening the average age. Mitochondrial stabilizing drugs have also not shown any effect on sarcopenia. In addition, it is a very inappropriate method for patients or bedridden patients, so the development of therapeutic drugs and techniques for sarcopenia that can induce muscle regeneration and differentiation has become a major issue, and although researches on this topic are being conducted, they are still insufficient.

[0005] Therefore, in an aging society, it is expected that the demand for pharmaceutical compositions and health functional foods that are effective against sarcopenia will continue to increase, and research into new drugs that are effective against sarcopenia is required. Summary of the Invention [Problem to be solved by the invention]

[0006] The technical problem to be solved by the present invention is to provide a pharmaceutical composition for preventing or treating sarcopenia.

[0007] In addition, the technical problem to be solved by the present invention is to provide a health functional food for preventing or improving sarcopenia.

[0008] The technical problems that the present invention aims to solve are not limited to the above-mentioned technical problems, and other technical problems will be clearly understood by those skilled in the art from the following description of the invention. [Means for solving the problem]

[0009] In order to achieve the above technical objectives, one aspect of the present invention provides a pharmaceutical composition for preventing or treating sarcopenia, comprising an Alox5 (arachidonate 5-lipoxygenase) inhibitor.

[0010] In an embodiment of the present invention, the Alox5 inhibitor may be a nucleic acid, an antibody, an aptamer, a peptide, a protein, a compound or a natural product.

[0011] In an embodiment of the present invention, the Alox5 inhibitor may be one selected from the group consisting of malotilate, wedelolactone, zileuton, nordihydroguaiaretic acid, psoralidin, docebenone, licofelone, lonapalene, enazadrem, cirsiliol, picrinine, atreleuton, and mixtures thereof.

[0012] In an embodiment of the present invention, the prevention or treatment of sarcopenia may be by reducing the production of Leukotriene B4 (LTB4).

[0013] In an embodiment of the present invention, the prevention or treatment of sarcopenia may be by decreasing the expression of Atrogin-1 or MuRF-1; or by increasing the expression of one or more genes selected from Efna5 (Ephrin A5), Fut4 (Fucosyltransferase 4), Igf1 (Insulin-like growth factor 1), Sod3 (Superoxide dismutase 3), and Plk1 (Polo like kinase 1).

[0014] In an embodiment of the present invention, the sarcopenia may be due to aging.

[0015] In order to achieve the above technical objectives, another aspect of the present invention provides a health functional food for preventing or improving sarcopenia, comprising an Alox5 inhibitor.

[0016] In an embodiment of the present invention, the Alox5 inhibitor may be a nucleic acid, an antibody, an aptamer, a peptide, a protein, a compound or a natural product.

[0017] In an embodiment of the present invention, the Alox5 inhibitor may be one selected from the group consisting of malotilate, wedelolactone, zileuton, nordihydroguaiaretic acid, psoralidin, docebenone, licofelone, lonapalene, enazadrem, cirsiliol, picrinine, atreleuton, and mixtures thereof.

[0018] In an embodiment of the present invention, the prevention or amelioration of sarcopenia may be due to a decrease in the production of leukotriene B4 (LTB4).

[0019] In an embodiment of the invention, the prevention or amelioration of sarcopenia may be due to a decrease in expression of atrogin-1 or MuRF-1; or an increase in expression of one or more genes selected from Efna5 (ephrin A5), Fut4 (fucosyltransferase 4), Igf1 (insulin-like growth factor 1), Sod3 (superoxide dismutase 3) and Plk1 (polo-like kinase 1).

[0020] In an embodiment of the present invention, the sarcopenia may be due to aging. Effect of the Invention

[0021] The present invention relates to a composition for preventing or treating sarcopenia, comprising an Alox5 inhibitor, which contains a substance that targets Alox5 and inhibits its expression or activity, thereby improving muscle performance, increasing muscle mass and cross-sectional area of ​​muscle fibers, and reducing the expression of muscle atrophy regulatory factors, thereby effectively preventing, improving and treating sarcopenia.

[0022] It should be understood that the effects of the present invention are not limited to the effects described above, but include all effects that can be inferred from the configuration of the invention described in the description of the invention or the claims. [Brief description of the drawings]

[0023] [Figure 1a] 1a to 1g are diagrams confirming the atrophy-suppressing effect of malotilate treatment in myotubes from dexamethasone (Dex)-treated mice. Fig. 1a shows C2C12 muscle fibers stained by myosin heavy chain immunochemical staining. [Figure 1b] FIG. 1 shows the average diameter of muscle fibers. [Figure 1c] FIG. 1 shows the distribution of muscle fiber diameter relative to the total number of muscle fibers. [Figure 1d] FIG. 1 shows the expression levels of atrogin-1 and Murf-1 at the mRNA level analyzed through qPCR analysis. [Figure 1e] FIG. 1 shows a comparative analysis of protein synthesis rates through the SUnSET assay. [Figure 1f] This is a diagram confirming the degree of phosphorylation of FoxO3a and the expression of FoxO3a and atrogin-1 through Western blotting, and the expression level of the corresponding gene is quantified through the expression level of α-tubulin. [Figure 1g] This figure shows the expression levels of LC3bI, LC3bII, and p62 confirmed by Western blotting, and the expression levels of the corresponding genes were quantified through the expression level of α-tubulin (*=p<0.05, **=p<0.01, ***=p<0.001 are statistically significant compared to the untreated group, #=p<0.05, ##=p<0.01, ###=p<0.001 are statistically significant compared to the Dex group). [Figure 2a]Figures 2a to 2c show the atrophy-suppressing effect of zileuton and malotilate treatment on myotubes from glucocorticoid-treated mice. Figure 2a shows a representative image of C2C12 myofibers stained by myosin heavy chain immunochemical staining. The cells were cultured as follows: (1) cultured in differentiation medium for 120 hours (untreated), (2) cultured in differentiation medium for 96 hours followed by additional treatment with DM and 10 μM Dex for 24 hours (Dex), and (3) cultured in differentiation medium for 96 hours followed by additional treatment with DM, 10 μM Dex, and 10 μM zileuton for 24 hours (Mal) (scale bar = 150 μm). [Figure 2b] FIG. 1 shows the average diameter of muscle fibers. [Figure 2c] This figure shows the expression levels of atrogin-1 and Murf-1 at the mRNA level analyzed by qPCR (*=p<0.05, **=p<0.01, ***=p<0.001 are statistically significant compared to the untreated group, #=p<0.05, ##=p<0.01, ###=p<0.001 are statistically significant compared to the Dex-treated control group). [Figure 3a] Figures 3a to 3c show the atrophy-suppressing effect of wedelolactone and malotilate treatment on myotubes from glucocorticoid-treated mice. Figure 3a shows a representative image of C2C12 myofibers stained by myosin heavy chain immunochemical staining. The cells were cultured as follows: (1) cultured in differentiation medium for 120 hours (untreated), (2) cultured in differentiation medium for 96 hours followed by additional treatment with DM and 10 μM Dex for 24 hours (Dex), and (3) cultured in differentiation medium for 96 hours followed by additional treatment with DM, 10 μM Dex, and 20 μM wedelolactone for 24 hours (Mal) (scale bar = 150 μm). [Figure 3b] FIG. 1 shows the average diameter of muscle fibers. [Figure 3c]This figure shows the expression levels of atrogin-1 and Murf-1 at the mRNA level analyzed by qPCR (*=p<0.05, **=p<0.01, ***=p<0.001 are statistically significant compared to the untreated group, #=p<0.05, ##=p<0.01, ###=p<0.001 are statistically significant compared to the Dex-treated control group). [Figure 4a] Figures 4a to 4d are figures confirming the myotube atrophy suppression effect of siAlox5. Figure 4a shows C2C12 muscle fibers stained by myosin heavy chain immunochemical staining. [Figure 4b] The mean diameter of muscle fibers is shown. [Figure 4c] FIG. 1 shows the distribution of muscle fiber diameter relative to the total number of muscle fibers. [Figure 4d] FIG. 13 is a graph showing the expression level of atrogin-1 at the mRNA level through qPCR analysis. [Figure 4e] FIG. 13 shows the decrease in the expression level of Alox5 by siAlox5 confirmed through qPCR analysis. [Figure 4f] FIG. 13 shows a decrease in the expression level of Alox5 by siAlox5, confirmed by Western blotting. [Figure 4g] Figures 4g and 4h show the effect of malotilate treatment on Alox5 expression in Dex-treated myotubes. Figure 4g shows the expression of Alox5 by Western blotting, and the expression level of the corresponding gene was quantified through the expression level of α-tubulin. [Figure 4h] FIG. 1 shows the concentration of LTB4 accumulated in C2C12 muscle fibers confirmed through ELISA analysis. [Figure 4i] Figures 4i to 4k show the effects of exogenous LTB4 treatment in Dex-treated myotubes, and Figure 4i shows C2C12 muscle fibers stained by myosin heavy chain immunochemical staining. [Figure 4j] FIG. 1 shows the average diameter of muscle fibers. [Figure 4k] FIG. 13 is a graph showing the expression level of atrogin-1 at the mRNA level through qPCR analysis. [Figure 5a] Figures 5a to 5d are graphs confirming the effect of malotilate treatment on myogenesis. Figure 5a is a graph confirming the expression of Myh2 through Western blotting. [Figure 5b] FIG. 1 shows Myh2 expression confirmed by Western blotting. [Figure 5c] FIG. 1 shows the levels of Myh2 and phosphorylated Akt confirmed by Western blotting. [Figure 5d] Expression levels of Pax7, Myf5, Myog, and Myh2 at the mRNA level were analyzed through qPCR analysis (*=p<0.05, **=p<0.01, ***=p<0.001 are statistically significant compared to the untreated group, #=p<0.05, ##=p<0.01, ###=p<0.001 are statistically significant compared to the Dex group). [Figure 6a] Figures 6a to 6l are graphs confirming the inhibitory effect of malotilate treatment on skeletal muscle atrophy in a Dex-treated sarcopenic mouse model, and Figure 6a shows the results of measuring the body weight of the mice. [Figure 6b] The mouse body weight was measured on the final day of drug treatment. [Figure 6c] The graph shows the results of measuring the grip strength of the mice on the final day of drug treatment. [Figure 6d] This shows the results of measuring the maximum duration of movement of mice on the final day of drug treatment. [Figure 6e] This shows the results of measuring the maximum sustainable speed of movement of mice on the final day of drug treatment. [Figure 6f] This is the value obtained by measuring the weight of the quadriceps muscle of the mouse. [Figure 6g] This is the value obtained by measuring the weight of the mouse soleus muscle. [Figure 6h] Representative photographs of mouse quadriceps muscle after hematoxylin-eosin staining. [Figure 6i] These are the results of measuring the average cross-sectional area of ​​muscle fibers. [Figure 6j]The cross-sectional area of ​​muscle fibers is distributed relative to the total number of muscle fibers. [Figure 6k] The concentration of LTB4 accumulated in the quadriceps muscle of mice was confirmed through ELISA analysis. [Figure 6l] The expression levels of atrogin-1 and Murf-1 in the quadriceps of mice were confirmed at the mRNA level (*=p<0.05, **=p<0.01, ***=p<0.001 are statistically significant compared to the untreated group, #=p<0.05, ##=p<0.01, ###=p<0.001 are statistically significant compared to the Dex group). [Figure 7a] 7a to 7l are graphs confirming the inhibitory effect of malotilate treatment on skeletal muscle atrophy in a mouse model of sarcopenia due to aging. Fig. 7a shows the results of measuring the body weight of mice over a 4-week experimental period. [Figure 7b] The mouse body weight was measured on the final day of drug treatment. [Figure 7c] The graph shows the results of measuring the grip strength of the mice on the final day of drug treatment. [Figure 7d] This shows the results of measuring the maximum duration of movement of mice on the final day of drug treatment. [Figure 7e] This is the value obtained by measuring the weight of the quadriceps muscle of the mouse. [Figure 7f] This is the value obtained by measuring the weight of the mouse soleus muscle. [Figure 7g] Representative photographs of mouse quadriceps muscle after hematoxylin-eosin staining. [Figure 7h] These are the results of measuring the average cross-sectional area of ​​muscle fibers. [Figure 7i] The results show the cross-sectional area of ​​muscle fibers distributed relative to the total number of muscle fibers. [Figure 7j] Representative photographs after immunohistochemical staining using myosin heavy chain 2A, 2B, and laminin antibodies. [Figure 7k] The results are the average cross-sectional area of ​​muscle fibers stained with myosin heavy chains 2A and 2B. [Figure 7l]The cross-sectional area of ​​myosin heavy chain 2A and 2B stained muscle fibers is shown as a distribution relative to the total number of muscle fibers (*=p<0.05, **=p<0.01, ***=p<0.001 are statistically significant compared to the untreated group, #=p<0.05, ##=p<0.01, ###=p<0.001 are statistically significant compared to the Dex group). [Figure 8a] Figures 8a to 8g show the results of analysis of the cell transcriptome of myotubes atrophied by Dex treatment treated with malotilate. Figure 8a shows genes that were up-regulated or down-regulated through the intracellular transcriptome analysis of C2C12 muscle fibers. [Figure 8b] FIG. 1 shows the changes in gene expression between the Dex and Dex_Mal groups in a volcano plot. [Figure 8c] FIG. 1 shows changes in gene expression between the Dex and Dex_Mal groups in a single hierarchical heat map. [Figure 8d] FIG. 1 shows gene expression changes between Dex and Dex_Mal groups through correlation between genes and functional roles. [Figure 8e] This figure shows gene expression changes between the Dex and Dex_Mal groups through KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis. [Figure 8f] 1 is a heat map quantitatively showing changes in gene expression levels between Dex / Un and Dex_Mal / Dex. [Figure 8g] This is a summary of sarcopenia-related genes from heat maps of Dex / Un and Dex_Mal / Dex. [Figure 9a] 9a to 9c are diagrams confirming the atrophy-preventing effect of malotilate treatment in cultured human skeletal muscles. Fig. 9a shows myofibers of human-derived myoblasts stained with hematoxylin and eosin. [Figure 9b] FIG. 1 shows the average diameter of muscle fibers and the distribution of muscle fibers. [Figure 9c]This is a graph showing the expression levels of atrogin-1 and Murf-1 at the mRNA level through qPCR analysis (*=p<0.05, **=p<0.01, ***=p<0.001 are statistically significant compared to the untreated group, #=p<0.05, ##=p<0.01, ###=p<0.001 are statistically significant compared to the Dex group). [Figure 10] FIG. 1 shows the link between Alox5 activity and sarcopenia, and the mechanism by which malotilate alleviates sarcopenia. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] The present invention will be described in detail below.

[0025] The present invention relates to a pharmaceutical composition for preventing or treating sarcopenia, comprising an Alox5 (arachidonate 5-lipoxygenase) inhibitor.

[0026] The Alox5 inhibitor can be a substance that inhibits the expression or activity of the Alox5 gene or protein.

[0027] The inhibitor may be a nucleic acid, an antibody, an aptamer, a peptide, a protein, a compound, or a natural product, and is not particularly limited as long as it inhibits the expression or activity of the Alox5 gene or protein.

[0028] For example, the nucleic acid may be an antisense nucleotide, a small interfering RNA (siRNA), a short hairpin RNA (shRNA), a microRNA, or a recombinant RNA that binds complementarily to Alox5 mRNA, or an antibody, an aptamer, a peptide, a protein, a compound, or a natural product that specifically binds to Alox5.

[0029] The Alox5 inhibitor may be one selected from the group consisting of malotilate, wedelolactone, zileuton, nordihydroguaiaretic acid, psoralidin, docebenone, licofelone, lonapalene, enazadrem, cirsiliol, picrinine, atreleuton, and mixtures thereof.

[0030] Preferably, the Alox5 inhibitor may be malotilate.

[0031] The malotylate may be represented by the following structural formula (1): [ka]

[0032] The prevention or treatment of sarcopenia may be by reducing the production of Leukotriene B4 (LTB4).

[0033] LTB4 is an active product of Alox5, and LTB4 increases in atrophic skeletal muscle. In an embodiment of the present invention, when myotubes atrophied by dexamethasone (Dex) treatment are treated with the composition of the present invention, it was confirmed that the production of LTB4 is reduced, and the atrophy of myotubes caused by Dex is inhibited (FIG. 4h). More specifically, LTB4 is involved in the dephosphorylation of FoxO3, which is a key regulator of the ubiquitin / proteasome pathway of skeletal muscle atrophy, through the catabolic signal pathway, and if the production of LTB4 is reduced, the dephosphorylation of FoxO3 is inhibited, thereby preventing or treating sarcopenia.

[0034] In addition, the reduction in LTB4 production can reduce unregulated autophagy and inhibit skeletal muscle atrophy. In an embodiment of the present invention, when Dex-treated myotubes were treated with an Alox5 inhibitor (e.g., malotilate), the ratio of LC3bII to LC3bI was reduced, and the expression of p62 was increased, thereby reducing unregulated autophagy (Figure 1g).

[0035] The present invention can prevent or treat sarcopenia by decreasing the expression of muscle atrophy regulating factors (atrogenes) or increasing the expression of anti-atrophy factors.

[0036] The muscle atrophy regulating factor may be, for example, but is not limited to, atrogin-1 or MuRF-1.

[0037] The anti-atrophy factor may be, for example, but is not limited to, Efna5 (Ephrin A5), Fut4 (Fucosyltransferase 4), Igf1 (Insulin-like growth factor 1), Sod3 (Superoxide dismutase 3), or Plk1 (Polo like kinase 1).

[0038] The sarcopenia may include sarcopenia due to any cause, such as aging, sepsis, starvation, chronic obstructive pulmonary disease, diabetes, cancer, etc., but preferably, the sarcopenia may be due to aging.

[0039] In an embodiment of the present invention, it was confirmed that skeletal muscle atrophy was suppressed through an increase in muscle mass and cross-sectional area of ​​muscle fibers by treatment with an Alox5 inhibitor (e.g., malotilate) in an animal model of sarcopenia caused by Dex treatment or aging (Figures 6f to 6j and 7e to 7i). Therefore, the composition of the present invention has excellent efficacy in preventing or treating sarcopenia.

[0040] The pharmaceutical composition of the present invention further comprises a pharma- ceutically acceptable carrier, which is a carrier that is commonly used in formulation, and includes, but is not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above-mentioned components, the pharmaceutical composition of the present invention may further comprise a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharma-ceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995). The suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as the formulation method, administration method, age, body weight, sex, severity of disease symptoms, diet, administration time, administration route, excretion rate, and reaction sensitivity of the patient, and an ordinary skilled physician can easily determine and prescribe an effective dosage for the intended treatment. Meanwhile, the dosage of the pharmaceutical composition of the present invention may be 0.01 to 2000 mg / kg (body weight) per day, but is not limited thereto.

[0041] The pharmaceutical composition of the present invention may be administered orally or parenterally, and when administered parenterally, it may be administered intravenously, subcutaneously, intramuscularly, intraperitoneally, transdermally, etc. Preferably, the administration route of the pharmaceutical composition of the present invention is determined according to the type of disease to which it is applied.

[0042] The pharmaceutical composition of the present invention may be prepared in a unit dose form by formulating it with pharma- ceutical acceptable carriers and / or excipients, or may be filled in a multi-dose container, by a method easily practicable by a person of ordinary skill in the art to which the present invention pertains. In this case, the dosage form may be in the form of a solution, suspension, emulsion in an oily or aqueous medium, extract, powder, granule, tablet, or capsule, and may further contain a dispersant or stabilizer.

[0043] The present invention relates to a health functional food containing an Alox5 inhibitor for preventing or improving sarcopenia.

[0044] The Alox5 inhibitor contained in the health functional food for preventing or improving sarcopenia according to the present invention, its mechanism of action, and the cause of sarcopenia are as described above.

[0045] The health functional food of the present invention can be manufactured and processed in the form of tablets, capsules, powder, granules, liquid phase, pills, etc. for the purpose of preventing or improving sarcopenia.

[0046] In the present invention, functional health food means food manufactured and processed using raw materials or ingredients having useful functionalities for the human body, and means food that is ingested for the purpose of obtaining useful effects for health purposes, such as regulating nutrients for the structure and functions of the human body or physiological actions.

[0047] The health functional food of the present invention may contain ordinary food additives, and unless otherwise specified, the suitability of such food additives shall be determined based on the specifications and standards for the relevant item in accordance with the general provisions and general test methods of the Food Additives Code approved by the Food and Drug Administration.

[0048] Examples of items listed in the Food Additives Code include, but are not limited to, chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; natural additives such as persimmon color, licorice extract, crystalline cellulose, high-quality color, and guar gum; and mixed preparations such as sodium L-glutamate preparations, noodle additive alkali agents, preservative preparations, and tar color preparations.

[0049] For example, the health functional food in tablet form may be prepared by granulating the mixture of the extract with excipients, binders, disintegrants and other additives in a conventional manner, and then compressing the mixture by adding a lubricant, etc., or by directly compressing the mixture. The health functional food in tablet form may also contain sweeteners, etc., as necessary.

[0050] Among the capsule-type health functional foods, hard capsules can be prepared by filling a mixture of the extract and additives such as excipients into a normal hard capsule, and soft capsules can be prepared by filling a mixture of the extract and additives such as excipients into a capsule base such as gelatin. The soft capsules may contain plasticizers such as glycerin or sorbitol, colorants, preservatives, etc. as necessary.

[0051] The round-shaped health functional food can be prepared by mixing the extract with excipients, binders, disintegrants, etc., and molding the mixture in a conventional manner. If necessary, the mixture can be covered with white sugar or other coating agents, or the surface can be coated with substances such as starch and talc.

[0052] The health functional food in granular form can be produced by mixing the extract with excipients, binders, disintegrants, etc., in granular form by a conventionally known method, and may contain flavoring agents, sweeteners, etc., as necessary.

[0053] The health functional foods may be beverages, meats, chocolates, food, snacks, pizza, ramen, other noodles, gums, candies, ice cream, alcoholic beverages, vitamin complexes, and dietary supplements.

[0054] The health functional food can be orally administered as a nutrient, and the application form is not particularly limited.For example, when administered orally, the daily intake is preferably 5000mg or less, more preferably 2000mg or less, and most preferably 500-1500mg or 650mg.When formulated as a capsule or tablet, one tablet can be administered once a day with water.

[0055] The present invention will be described in detail below with reference to examples.

[0056] <Example: Confirmation of preventive or therapeutic effect of skeletal muscle atrophy using Alox5 inhibitor> Materials and Methods 1. Reagents and antibodies Dexamethasone (Dex) was purchased from Santa Cruz Biotechnology (TX, USA). Malotilate was purchased from Selleckchem (TX, USA). Leukotriene B4 (LTB4) was purchased from Tocris (Bristol, UK).

[0057] 2.Cell culture C2C12 mouse myoblasts were obtained from Koram Biothec (Korea). The cells were maintained in DMEM growth medium supplemented with 10% FBS, 1% penicillin and streptomycin. When the myoblasts reached approximately 90% proliferation, they were treated with DMEM differentiation medium supplemented with 2% HS, 1% penicillin and streptomycin to differentiate into myofibers for 96 hours. The differentiated myofibers were treated with 10 μM Dex for 24 hours to induce sarcopenia. To evaluate the degree of differentiation of muscle cells, myoblasts differentiating in the differentiation medium were treated with the corresponding compounds for 24 or 48 hours.

[0058] 3. Immunochemical staining method The myofibers were stained by immunochemical staining using myosin heavy chain antibody. Specifically, the myofibers were washed three times with PBS and fixed with 3.7% formaldehyde solution. Then, they were washed with PBS, cleared with 0.2% Triton® X-100 solution, and washed again with PBS. They were blocked with 1% bovine serum albumin solution (1% BSA in PBS-T) and incubated with primary antibody overnight at 4°C. After washing with PBS, they were treated with secondary antibody for 1 hour. Then, the myotubes were counterstained with 1 μM DAPI in PBS. Fluorescent images of stained myotubes from five different areas were obtained using a DMI3000B microscope (Leica, Germany) and analyzed using ImageJ 1.52 software (National Institutes of Health, Bethesda, MD, USA). Myotubes were classified as myosin heavy chain-positive cells containing four or more nuclei, and the diameter of muscle fibers containing four or more nuclei was measured.

[0059] 4. Western Blotting The concentration of intracellular proteins was measured using a Bradford assay (Bio-Rad, CA, USA) and loaded onto 10% and 12% polyacrylamide gels for electrophoresis. The separated proteins were then transferred to a PVDF membrane, and the primary antibodies used were diluted 1:1000 in TBS-T containing 5% skim milk and incubated overnight at 4°C. The secondary antibodies were diluted 1:2000 and incubated for 35 min at room temperature (RT). Band intensity was measured using ImageJ 1.52 software (National Institutes of Health, USA) and normalized to α-tubulin for intracellular proteins. The primary and secondary antibodies used are summarized in Tables 1 and 2, respectively.

[0060] [Table 1]

[0061] [Table 2]

[0062] 5. RT-PCR and quantitative real-time PCR For quantitative real-time PCR, the transcript levels of the genes of interest were analyzed using a StepOnePlus® Real-Time PCR System (Applied Biosystems, UK). Total RNA was reverse transcribed to prepare cDNA using AccuPower® RT PreMix (Bioneer Corporation). The obtained cDNA was submitted to real-time PCR according to the manufacturer's instructions with the following modifications: PCR was performed in triplicate in a total volume of 20 μL 2X Power SYBR Green PCR Master Mix (Enzynomics, Korea) containing 200 nM (final concentration) of each specific primer and 1 μL of cDNA synthesized from 1 μg of total RNA. PCR amplification consisted of 40 cycles of denaturation, annealing, and extension after incubating the mixture at 95°C for 10 min. Denaturation was performed at 95°C for 15 s, annealing at 60°C for 1 min, and extension was performed at 72°C for 20 s with fluorescence detection at 72°C after each cycle. After the final cycle, melting point analysis of all samples was performed within the range of 60°C to 95°C using continuous fluorescent detection. A specific cDNA sample was included in each run to serve as a reference for comparison between runs. The expression level of GAPDH was used to calculate and normalize the expression levels of all other genes. Results are presented as relative expression levels for each gene. The synthesized cDNA was amplified by quantitative real-time PCR (qRT-PCR) or standard PCR. The PCR primers used in this experiment are listed in Table 3 below.

[0063] [Table 3]

[0064] 6. LTB4 Enzyme-linked Immunoprecipitation LTB4 ELISA kit was purchased from My Biosource (CA, USA), and the concentration of LTB4 in mouse leg muscle and cells was measured by immunoenzyme-linked immunoprecipitation according to the manufacturer's instructions. Samples (100 μL) were stored at room temperature (RT) for 1 hour in a 96-well plate containing polyclonal LTB4 antibody. After this, an enzyme reaction was performed to stop the process, and the absorbance at 405 nm was measured.

[0065] 7. Animal Experiments Animal studies were performed in accordance with the Institute of Laboratory Animals' Guide for the Care and Use of Laboratory Animals. In addition, the animal studies were approved by the Animal Care and Use Committee of the Gwangju Institute of Science and Technology (approval number: GIST-2021-059). Twelve-week-old male C57BL / 6 mice were purchased from Damool Science (Daejeon, Korea).

[0066] 8. Dexamethasone-induced Sarcopenia Mouse Model Twelve-week-old male C57BL / 6J mice were divided into three groups of five mice each and treated with drugs as follows: 1) PBS containing 5% DMSO and 5% Tween 80 (vehicle group), 2) 15 mg / kg dexamethasone (15 mg / kg concentration, PBS containing 5% DMSO and 5% Tween 80) (dexamethasone group), and 3) 10 mg / kg malotilate (10 mg / kg concentration, PBS containing 15 mg / kg dexamethasone, 5% DMSO, and 5% Tween 80) (malotilate group) by intraperitoneal injection (solution volume = 200 μL) for 2 weeks. After that, the mice's muscle function and condition were analyzed.

[0067] 9. Sarcopenia model using aged mice Male C57BL / 6J mice aged 21 months or older were divided into two groups of 5 mice each and treated with drugs as follows: 1) 0.5% carboxymethylcellulose containing 6.73% DMSO (vehicle group) or 2) 50 mg / kg malotilate (50 mg / kg concentration, 0.5% carboxymethylcellulose containing 6.73% DMSO) (malotilate group) orally administered daily for 4 weeks. Muscle function and condition were then analyzed.

[0068] 10. Grip strength measurement Grip strength was measured using a BIO-GS3 grip strength measuring device (Bioseb, FL, USA). The mouse was placed on a grid and gently pulled backwards with all four paws until the mouse fell off the grid. Trials were performed with a 30-s time interval, and the maximum value of three trials was selected as the representative muscle strength.

[0069] 11. Measurement of muscle fatigue Muscle fatigue was measured using two models using a rotarod (Ugo Basile, Italy). The first model was a constant speed model that measured how long the mouse could continue exercising on a cylinder rotating at a constant speed, and the second model was an accelerating model that measured the speed at which the mouse could sustain exercising when the speed of the cylinder increased at regular time intervals. One day before the measurements with the two models, the mice were trained to adapt to the speed from 5 rpm to 15 rpm. When the mouse was unable to continue exercising more than four times per minute and fell off, it was determined that the mouse could not continue exercising any more and the training was terminated.

[0070] 12. Muscle Dissection and Histological Analysis Mice were anesthetized with 22 mg / kg ketamine (Yuhan, Korea) and 10 mg / kg xylazine (Bayer, Korea) administered intraperitoneally via IP injection before sacrifice. Quadriceps, gastrocnemius, tibialis anterior, and soleus muscles were sampled and weighed. For immunohistochemical staining, the samples were fixed in 4% paraformaldehyde overnight at 4°C, embedded in paraffin solution, and stored at −80°C. Sectioning and hematoxylin-eosin staining were performed at the Laboratory Animal Resource Center of Gwangju Institute of Science and Technology. Hematoxylin-eosin staining was performed using a kit (Merck, Germany). The cross-sectional area of ​​muscle fibers was measured using ImageJ 1.48 software (National Institutes of Health, USA).

[0071] 13. Immunohistochemical staining method Immunohistochemical staining was performed using myosin heavy chain type 2A and type 2B (DSHB, IA, USA) and anti-laminin antibody (Abcam, Cambridge, UK). Counterstaining was performed with 1 μM DAPI solution. The cross-sectional area of ​​muscle fibers was visualized using a LEICA DM 2500 (Leica, Germany) and then measured using ImageJ 1.48 software (National Institutes of Health, USA).

[0072] 14.RNA Sequencing The total RNA concentration was measured using Quant-IT RiboGreen (Invitrogen, #R11490). TapeStation RNA screentape (Agilent, #5067-5576) was used to assess the total RNA integrity. To prepare high-purity RNA, only RNA with a RIN value of 7.0 or higher was used for RNA library construction.

[0073] Only 0.5 μg of total RNA from each sample was used for library construction using the Illumina TruSeq Standard Total RNA Library Construction Gold Kit (Illumina Inc., San Diego, CA, USA, #20020599). The first step of the workflow was to remove rRNA from the total RNA. The remaining mRNA was then fragmented into small fragments using divalent cations at high temperature. The cleaved RNA fragments were cloned into double-stranded cDNA using SuperScript II reverse transcriptase (Invitrogen, #18064014) with random primers DNA polymerase I, RNase H, and dUTP. These cDNA fragments were then subjected to a final repair step, single "A" base addition, and adaptor ligation, and the products were purified and enriched by PCR to generate the final cDNA libraries.

[0074] Libraries were quantified using the KAPA Library Quantification Kit for Illumina Sequencing Platforms according to the qPCR Quantification Protocol Guide (KAPA BIOSYSTEMS, #KK4854) and qualified using a TapeStation D1000 ScreenTape (Agilent Technologies, #5067-5582). Indexed libraries were then submitted to Illumina NovaSeq (Illumina Inc., San Diego, CA, USA) for paired-end (2 × 100 bp) sequencing at Macrogen Incorporated.

[0075] 15. Culture and experiment of human myoblast cells Human myoblasts were purchased from Thermo-Fisher Scientific (USA). Myoblasts were thawed using a water bath and washed with 20 mL of DM by centrifugation at 180 g for 5 min at room temperature. Myoblasts were then resuspended in DM to a concentration of 4.8 × 10 4 Cells were seeded in 12-well plates at a density of 1000 cells / well. After 48 h, myoblasts were treated with compounds of interest for 24 h. Myotubes were stained with H&E. Myotube diameters were measured using ImageJ 1.48 software (National Institutes of Health, USA) after light microscopic analysis of DIC-captured images (Olympus CKX41).

[0076] 16. Statistical analysis Statistical significance was determined using Student's t-test. A p value of less than 0.05 was considered to have statistical significance. Unless otherwise stated, all data are shown from three or more repeated experiments, and error bars on graphs are standard deviations.

[0077] <Result> 1. Alox5 inhibitors (malotilate, zileuton, wedelolactone) inhibit atrophy in myotubes from glucocorticoid-treated mice. A glucocorticoid-treated model was used to investigate the effect of Alox5 inhibitors on myotube atrophy. The Alox5 inhibitors used were malotilate, zileuton, and wedelolactone. This model was chosen because glucocorticoid levels are increased by various types of skeletal muscle atrophy, such as aging, sepsis, starvation, chronic obstructive pulmonary disease, diabetes, and cancer. Mice treated with the glucocorticoid Dex showed myotube atrophy, with a decreased diameter and an increased proportion of thin myotubes. Malotilate treatment inhibited the effect of Dex on myotube atrophy (Figures 1a-c). Atrogin-1 and MuRF-1 are important atrogenes that are up-regulated in skeletal muscle wasting. Malotilate treatment inhibited the increase in atrogin-1 and MuRF-1 in Dex-treated myotubes (Figure 1d). Furthermore, total protein synthesis was decreased by Dex treatment and restored by malotilate treatment (Fig. 1e). The transcription factor FoxO3a (forkhead box O3) is a key regulator of the ubiquitin / proteasome pathway in skeletal muscle atrophy. Dephosphorylation of FoxO3a by catabolic signaling pathways induced its translocation from the cytoplasm to the nucleus and an increase in target muscle atrophy regulators. FoxO3a phosphorylation levels were decreased in Dex-treated myotubes and increased by malotilate treatment (Fig. 1f), accompanied by a decrease in atrogin-1 expression. Unregulated autophagy is a major feature of skeletal muscle atrophy. Dex treatment increased autophagy in myotubes (increased ratio of LC3bII to LC3bI and decreased expression of p62), as mentioned above. Autophagy in Dex-treated myotubes was decreased by malotilate treatment (Fig. 1g).

[0078] In addition, referring to Figures 2 and 3, it can be confirmed that the diameter of C2C12 myotubes decreases upon dexamethasone treatment, and that the diameter of muscle fibers is restored as a result of treatment with Alox5 inhibitors zileuton, wedelolactone, and malotilate. It can also be confirmed that the expression levels of UPS-linked enzymes atrogin-1 and Murf-1 increase upon dexamethasone treatment, but the expression levels of atrogin-1 and Murf-1 decrease as a result of treatment with Alox5 inhibitors zileuton, wedelolactone, and malotilate.

[0079] 2. Alox5 siRNA gene knockdown prevents myotube atrophy associated with increased levels of the Alox5 product, leukotriene B4. Malotilate is characterized as a selective inhibitor of Alox5. The effect of Alox5 gene knockdown on myotube atrophy was assessed in Dex-treated myotubes. Alox5 siRNA reduced gene expression and inhibited myotube atrophy in Dex-treated myotubes (Figure 4a-c). Gene knockdown was accompanied by a decrease in atrogin-1 expression (Figure 4d). The ability of siRNA to reduce Alox5 expression was confirmed by qPCR and Western blotting (Figure 4e and Figure 4f).

[0080] Treatment with malotilate, an Alox5 enzyme inhibitor, did not affect the expression of Alox5 in Dex-treated myotubes (Fig. 4g). Leukotriene B4 (LTB4) is an Alox5 product involved in inflammatory responses and catabolic pathways such as NF-κ and FoxO signaling. LTB4 levels were increased in Dex-treated myotubes and were lowered by malotilate (Fig. 4h). Thus, application of exogenous LTB4 did not affect Dex-treated myotubes or the expression of atrogin-1 (Fig. 4i-k).

[0081] 3. Malotilate does not significantly affect the myogenic program. To assess the effect of malotilate treatment on myogenesis, mouse myotubes were induced to differentiate in the presence of malotilate. Expression of the differentiation marker myosin heavy chain type II (Myh2) was not affected by malotilate treatment during myogenesis (Figure 5a and 5b). Although increased phosphorylation of Akt is a major signaling event during myogenesis, the phosphorylation level of Akt in differentiated myotubes was not significantly altered by malotilate treatment (Figure 5c). Expression analysis of master gene regulators of the myogenic program (Pax7, Myf5, MyoG, and Myh2) showed no significant changes 24 h after malotilate treatment (Figure 5d).

[0082] 4. Malotilate inhibits skeletal muscle atrophy in glucocorticoid-treated models. To investigate the effect of malotilate to prevent skeletal muscle atrophy in vivo, the Dex glucocorticoid treatment model was chosen as the first test. Malotilate treatment for 14 days did not significantly affect the body weight of Dex-treated mice (Figure 6a and 6b). Malotilate treatment significantly increased the grip strength of Dex-treated mice (Figure 6c). Dex treatment reduced the time to fall and maximum velocity in the rotarod performance test, both of which were significantly increased by malotilate treatment (Figure 6d and 6e). Evaluation of skeletal muscle mass shows that malotilate treatment increases the mass of quadriceps and soleus muscles in dexamethasone-treated mice (Figure 6f and 6g). The mean cross sectional area (CSA) of muscle fibers and the proportion of fibers with larger CSA were increased by malotilate treatment (Figure 6h-j). ELISA analysis showed that levels of the Alox5 product LTB4 were increased in Dex-treated muscles and reduced by malotilate treatment (Fig. 6k), and malotilate treatment also reduced the expression of muscle atrophy regulators atrogin-1 and MuRF-1 in Dex-treated mice (Fig. 6l).

[0083] 5. Malotilate inhibits skeletal muscle atrophy in aging mice. To evaluate the potential of malotilate to treat aging-associated skeletal muscle atrophy (referred to as sarcopenia), aged mice were treated with oral delivery for 4 weeks. Malotilate treatment did not affect the body weight of aged mice (Figure 7a and Figure 7b). Grip strength was significantly increased by malotilate treatment (Figure 7c). The mean time to fall was increased in malotilate-treated mice, but did not reach statistical significance (Figure 7d). Muscle mass of the quadriceps and soleus muscles was significantly increased in malotilate-treated aged mice (Figure 7e and Figure 7f). Histological analysis of the quadriceps shows that the mean cross-sectional area of ​​muscle fibers was increased by malotilate treatment (Figure 7h). The distribution of muscle fiber diameters showed that giant muscle fibers (>5000 μm) were more prevalent in malotilate-treated aged mice. 2) account for a much higher proportion of CSA in the fast-twitch fibers (Fig. 7i). Immunohistochemical analysis of fast-twitch fibers types IIA and IIB, which preferentially atrophy during aging compared with slow-twitch fibers type I, indicates that malotilate treatment preserves the CSA of fast-twitch fibers (Fig. 7j-l).

[0084] 6. Analysis of the Cellular Transcriptome of Malothioate-treated Myotubes To obtain an overview of the mechanism by which Alox5 suppression by malotilate inhibits muscle atrophy, myotubes atrophied by Dex were treated with malotilate and the cellular transcriptome was investigated using RNA sequencing. Expression patterns were compared between untreated and Dex-treated myotubes and normal or malotilate-treated myotubes (Fig. 8a). Heatmap and volcano plots showed a total of 139 differentially expressed genes (DEGs) between myotubes treated with Dex or Dex and malotilate (Fig. 8b and Fig. 8c). KEGG (Kyoto Encyclopedia of Genes and Genome) pathway analysis showed that metabolic pathways were highly upregulated by malotilate treatment (Fig. 8d). Gene Ontology (GO) functional analysis showed that organ development was the highest biological process increased in myotubes treated with Dex and malotilate compared to Dex alone (Fig. 8e). A heatmap of genes differentially expressed in Dex, malotilate, and Dex alone compared to malotilate and untreated is shown in Figure 8f. The expression of many genes known to be associated with skeletal muscle atrophy was modulated by malotilate treatment. Genes known to be associated with skeletal muscle atrophy are shown in Figure 8g.

[0085] 7. Malotilate prevents atrophy in human skeletal muscle cultures. Human donor skeletal muscle precursor cells were differentiated into myotubes and induced to undergo atrophy by dexamethasone treatment. Histological analysis showed that co-treatment with malotilate could prevent atrophy of human myotubes, as shown by an increase in the mean diameter of myotubes and a higher proportion of large-sized myotubes (Figure 9a and 9b). Malotilate treatment also significantly reduced the expression of muscle atrophy regulators atrogin-1 and MuRF-1 in human myotubes (Figure 9c).

Claims

1. A pharmaceutical composition for preventing or treating sarcopenia, comprising an Alox5 (arachidonic acid-5-lipoxygenase) inhibitor.

2. The pharmaceutical composition for preventing or treating sarcopenia according to claim 1 , wherein the Alox5 inhibitor is a nucleic acid, an antibody, an aptamer, a peptide, a protein, a compound or a natural product.

3. The pharmaceutical composition for preventing or treating sarcopenia according to claim 1, wherein the Alox5 inhibitor is one selected from the group consisting of malotilate, wedelolactone, zileuton, nordihydroguaiaretic acid, psoralidin, docebenone, licofelone, lonaparen, enazadrem, cirsiliol, picrinine, atreleuton, and mixtures thereof.

4. The pharmaceutical composition for preventing or treating sarcopenia according to claim 1, wherein the prevention or treatment of sarcopenia is achieved by reducing the production of leukotriene B4 (LTB4).

5. The pharmaceutical composition for preventing or treating sarcopenia according to claim 1, wherein the prevention or treatment of sarcopenia is due to a decrease in expression of atrogin-1 or MuRF-1; or an increase in expression of one or more genes selected from Efna5 (ephrin A5), Fut4 (fucosyltransferase 4), Igf1 (insulin-like growth factor 1), Sod3 (superoxide dismutase 3) and Plk1 (polo-like kinase 1).

6. The pharmaceutical composition for preventing or treating sarcopenia according to claim 1 , wherein the sarcopenia is caused by aging.

7. A health functional food containing an Alox5 inhibitor for preventing or improving sarcopenia.

8. The health functional food for preventing or improving sarcopenia according to claim 7, wherein the Alox5 inhibitor is a nucleic acid, an antibody, an aptamer, a peptide, a protein, a compound or a natural product.

9. The health functional food for preventing or improving sarcopenia according to claim 7, wherein the Alox5 inhibitor is one selected from the group consisting of malotilate, wedelolactone, zileuton, nordihydroguaiaretic acid, psoralidin, docebenone, licofelone, lonapalene, enazadrem, cirsiliol, picrinine, atreleuton, and mixtures thereof.

10. The health functional food for preventing or ameliorating sarcopenia according to claim 7, wherein the prevention or amelioration of sarcopenia is due to a decrease in the production of leukotriene B4 (LTB4).

11. The health functional food for preventing or ameliorating sarcopenia according to claim 7, wherein the prevention or amelioration of sarcopenia is due to a decrease in the expression of atrogin-1 or MuRF-1; or an increase in the expression of one or more genes selected from Efna5 (ephrin A5), Fut4 (fucosyltransferase 4), Igf1 (insulin-like growth factor 1), Sod3 (superoxide dismutase 3), and Plk1 (polo-like kinase 1).

12. The health functional food for preventing or improving sarcopenia according to claim 7, wherein the sarcopenia is caused by aging.

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