Fucose-containing composition and use thereof

Abstract

The present invention addresses the problem of providing a composition, particularly a pharmaceutical composition and / or a food / beverage composition, for inducing the differentiation of a skeletal muscle and / or promoting the regeneration of a skeletal muscle. More specifically, the present invention addresses the problem of developing a component for promoting the differentiation of a satellite cell or a myoblast into a muscle fiber and promoting the formation of a skeletal muscle. A composition containing fucose or a fucose-releasing compound as an active ingredient has an excellent activity to promote the induction of skeletal muscle differentiation and / or the regeneration of a skeletal muscle, and is therefore useful for various applications in which the formation of a skeletal muscle is desired.

Description

Fucose-containing composition and its use

[0001] The present invention relates to a fucose-containing composition and uses thereof. More particularly, the present invention relates to a composition containing fucose or a fucose-releasing compound and uses thereof.

[0002] Skeletal muscle tissue is composed of muscle fibers, which are giant multinucleated cells. Between the cell membrane and basement membrane of muscle fibers, tissue stem cells called mononucleated satellite cells exist. Several to several dozen satellite cells are attached to each muscle fiber. Muscle fibers themselves cannot undergo cell division, and skeletal muscle regenerates through the action of satellite cells, which are tissue stem cells. Normally, satellite cells are quiescent and do not proliferate. However, when skeletal muscle receives a stimulus such as injury, satellite cells are activated and become precursor cells called myoblasts. Myoblasts proliferate through repeated cell division and become aligned in a certain direction. These aligned myoblasts then fuse with adjacent cells to differentiate and mature into multinucleated muscle cells (muscle fibers) called myotubes.

[0003] Skeletal muscle regeneration involves the activation of satellite cells, which are tissue stem cells, followed by their differentiation and proliferation into myoblasts, and then their differentiation and maturation into muscle fibers. It is known that the ability to regenerate muscle is reduced when any of these processes is inhibited by factors such as aging (Non-Patent Document 1).

[0004] Recent research has reported that Fut8 (fucosyltransferase 8) is an enzyme that plays an important role in muscle differentiation (Non-Patent Document 2). Fut8 is an enzyme that adds core fucose to proteins. Fut8 activity promotes muscle differentiation, and its deficiency inhibits muscle cell maturation. For this reason, it has been suggested that Fut8 dysfunction may be associated with muscle diseases and muscle atrophy.

[0005] M. Fukuoka,Commun Biol. 2021;4(1):427N. Hayashiji, Cells. 2022;12(1):144

[0006] The present invention aims to provide a composition, particularly a pharmaceutical composition and / or a food or beverage composition, for inducing differentiation and / or promoting regeneration of skeletal muscle. More specifically, the present invention aims to develop a component that promotes differentiation of muscle satellite cells and / or myoblasts into muscle fibers and promotes skeletal muscle formation.

[0007] In light of the above-mentioned problems, the present inventors focused on fucosyltransferase 8 (Fut8), which they had been studying extensively. Fut8 is a glycosyltransferase that performs N-glycosylation. There are 13 families of fucosyltransferases, each of which functions complements the others, but Fut8 is the only enzyme capable of producing a structure known as core fucose, in which fucose is attached via an α1,6 bond to the N-acetylglucosamine at the base of the N-glycan. Because proteins with core fucose are abundant in brain tissue and cancer cells, Fut8 has been studied in the fields of the brain and nervous system and cancer. The present inventors discovered that the addition of fucose during muscle differentiation induction of the established skeletal muscle progenitor cell line C2C12 significantly increases the fusion index in a concentration-dependent manner, and that the addition of L-fucose promotes myotube formation. Furthermore, when L-fucose was administered to mice in vivo that had been administered cardiotoxin, a cytotoxin that specifically inhibits protein kinase C and has the effect of depolarizing skeletal muscle fibers, muscle regeneration was promoted and muscle fiber thickness and wet muscle weight were restored, demonstrating that fucose is useful for inducing differentiation and / or promoting regeneration of skeletal muscle, leading to the completion of the present invention. Fucose is a monosaccharide discovered in fucoidan, a cell wall polysaccharide of the seaweed Fucus. In recent years, its effects on reducing abdominal visceral fat and improving bowel movements have been reported, and it is commercially available as a product with functional claims. However, there have been no reports of its effect on skeletal muscle.

[0008] That is, the present invention provides the following: [1] A composition for inducing differentiation and / or promoting regeneration of skeletal muscle, comprising fucose or a fucose-releasing compound as an active ingredient. [2] The composition according to [1], wherein the induction of skeletal muscle differentiation is myotube formation. [3] The composition according to [1] or [2], which is used to increase muscle strength and / or muscle mass. [4] The composition according to any of [1] to [3], which is a pharmaceutical composition. [5] The composition according to [4], which is used for preventing or treating a disease caused by muscle damage or muscle dysfunction. [6] The composition according to [5], wherein the disease is selected from muscle damage, muscle contusion, muscle atrophy, and muscular dystrophy. [7] The composition according to any of [1] to [3], which is a food or drink composition. [8] The composition according to [7], which is selected from a food for specified health uses, a food with functional claims, and a food with nutrient claims. [9] The composition according to [7], which is a supplement.

[10] Fucose or a fucose-releasing compound used for inducing differentiation and / or promoting regeneration of skeletal muscle.

[11] The fucose or fucose-releasing compound according to

[10] , wherein the induction of skeletal muscle differentiation is myotube formation.

[12] The fucose or fucose-releasing compound according to

[10] or

[11] , wherein the fucose or fucose-releasing compound is for use in increasing muscle strength and / or muscle mass.

[13] The fucose or fucose-releasing compound according to any of

[10] to

[12] , which is contained in a pharmaceutical composition.

[14] The composition according to

[13] , wherein the pharmaceutical composition is for preventing or treating a disease caused by muscle damage or muscle dysfunction.

[15] The composition according to

[14] , wherein the disease is selected from muscle damage, muscle contusion, muscle atrophy, and muscular dystrophy.

[16] The fucose or fucose-releasing compound according to any of

[10] to

[12] , which is contained in a food or beverage composition.

[17] A method for inducing skeletal muscle differentiation and / or promoting regeneration, comprising administering fucose or a fucose-releasing compound to a subject.

[18] The method according to

[17] , wherein the induction of skeletal muscle differentiation is myotube formation.

[19] A method for enhancing muscle strength and / or increasing muscle mass, comprising administering fucose or a fucose-releasing compound to a subject.

[20] A method for preventing or treating a disease caused by muscle damage or muscle dysfunction, comprising administering fucose or a fucose-releasing compound to a subject.

[21] The method of

[20] , wherein the disease is selected from muscle damage, muscle contusion, muscle atrophy, and muscular dystrophy.

[0009] Fucose has the effect of inducing differentiation and / or promoting regeneration of skeletal muscle. Therefore, the composition of the present invention containing fucose or a fucose-releasing compound as an active ingredient can promote myotube formation and regenerate damaged or atrophied skeletal muscle, and can be used not only as a pharmaceutical composition for preventing and treating muscle injury, muscle contusion, muscle atrophy, muscular dystrophy, etc., but also for promoting skeletal muscle formation during muscle training and in infants. Furthermore, since fucose is a monosaccharide widely present in animals and plants, the composition of the present invention is highly safe and can be widely used.

[0010] 1A and 1B are graphs showing that L-fucose administration increases the differentiation index. A: Images obtained by double staining myoblasts (C2C12 cells) with MY32 antibody and DAPI after adding L-fucose (0, 50, 100, and 200 μM) and culturing for 4 days. B: Results (graph) obtained by calculating the differentiation index using the stained image. 1B: Images (graph) showing that L-fucose administration increases the length of myotubes. A: Bright-field observation images of myoblasts obtained by primary culture after adding L-fucose (0 and 100 μM) and culturing for 1, 2, and 3 days (MD Day 1, Day 2, and Day 3). B: Results (graph) of measuring the length of myotubes formed using bright-field observation. 1B: Images (graph) showing that L-fucose administration increases the proportion of beating myotubes. A: Images of myoblasts obtained by primary culture, which were cultured for four days after the addition of L-fucose (0, 100, 200 μM), showing the pulsation of the cells. In the image, the darker areas indicate areas with stronger pulsation. B: Results of quantification of the proportion of pulsating myotube cells using the photographed images. This figure shows that L-fucose administration promotes sarcomere formation in skeletal muscle and leads to uniform sarcomere length. A: Electron microscope images of myoblasts obtained by primary culture, which were cultured for six days after the addition of L-fucose (0, 200 μM). B: Graph showing the results of sarcomere length measurement using ImageJ using the obtained electron microscope images. This figure shows that L-fucose administration promotes muscle fiber regeneration in mice with muscle injury. A mouse model of muscle injury was prepared by injection of cardiotoxin (CTX). This figure shows images of thin sections prepared from the tibialis anterior muscle of a model mouse that received oral or intraperitoneal administration (i.p.) of L-fucose for 7 days after muscle injury, triple-stained with F1.652, laminin a2, and DAPI. Physiological saline was used as a control. This figure demonstrates that L-fucose administration promotes muscle fiber regeneration and increases muscle wet weight in muscle-injured mice. Top: Graph quantifying the percentage of F1.652-positive muscle fibers using the fluorescent staining images in Figure 5. The vertical axis of the graph indicates the percentage of F1.652-positive muscle fibers among the total number of muscle fibers.Bottom: Graph showing the weight of the tibialis anterior (TA) muscle collected at the same time, corrected for body weight (BW), to calculate the wet muscle weight. Graph showing that L-fucose administration promotes muscle fiber regeneration and improves grip strength in muscle-injured mice. Grip tests were performed 3, 5, and 7 days after cardiotoxin (CTX) injury. Graph showing that L-fucose administration promotes weight gain in mdx Duchenne muscular dystrophy model mice. A: Photographs showing the lower limb muscles of 8-week-old mdx mice administered with or without L-fucose. B: Graph showing the change over time in body weight of mdx mice administered with or without L-fucose. Graph showing that L-fucose administration increases the wet muscle weight of the tibialis anterior (TA), soleus (Sole), gastrocnemius (Gas), and quadriceps (Quad) in mdx Duchenne muscular dystrophy model mice. 1A and 1B are diagrams showing that L-fucose administration promotes muscle regeneration in the tibialis anterior muscle of mdx Duchenne muscular dystrophy model mice. A: HE stained image of a cross section of the tibialis anterior muscle of mdx mice administered or not administered with L-fucose. B: A graph showing the results of measuring the maximum muscle circumference and the total number of muscle fibers of the tibialis anterior muscle of mdx mice administered or not administered with L-fucose. 1B: A graph showing the results of measuring the diaphragm diameter of mdx mice administered or not administered with L-fucose. 1C: A diagram showing that L-fucose administration promotes muscle hypertrophy in tenotomy model mice. A: Photographs of the plantaris muscle of mice administered or not administered with L-fucose (soleus muscle, gastrocnemius tenotomy, non-tenotomy). B: Graph showing the wet muscle weight of the plantar muscle of mice (tenotomy, non-tenotomy) administered with or without L-fucose. Figure showing that L-fucose administration promotes muscle hypertrophy in tenotomy model mice. A: HE stained image of the plantar muscle of tenotomy mice administered with or without L-fucose. B: Graph showing the distribution of muscle fiber diameters in the plantar muscle of tenotomy mice administered with or without L-fucose.

[0011] The present invention will now be described. Terms used in this specification have the meanings commonly used in the art unless otherwise specified.

[0012] In the present invention, "skeletal muscle differentiation induction" refers to the process by which precursor cells transform into mature skeletal muscle cells. This process can be multistep. First, precursor cells called myoblasts proliferate, followed by a stage called myotube formation, in which they fuse to form multinucleated myotubes. Ultimately, myotubes mature into muscle fibers and acquire functional contractile capacity. It is known that muscle-specific genes specific to each stage are expressed sequentially at each stage of differentiation, and the progress of skeletal muscle differentiation induction can be assessed using such gene expression as an indicator. Alternatively, the progress of skeletal muscle differentiation induction can be assessed histochemically using the ability to form muscle fibers as an indicator of maturation into muscle fibers. "Skeletal muscle regeneration" refers to the process by which damaged muscle tissue is repaired, in which tissue (muscle) stem cells called muscle satellite cells play a central role. After injury, muscle satellite cells are activated, proliferate, and differentiate into myoblasts. These myoblasts migrate to the damaged site and fuse to form new muscle fibers. In addition to inducing skeletal muscle differentiation, the composition of the present invention also promotes regeneration.

[0013] Fucose, included as an active ingredient in the present invention, is a monosaccharide classified as a deoxyhexose and is also known as 6-deoxy-L-galactose. Fucose is widely present as a component of glycoproteins and glycolipids on the cell surfaces of many organisms, including mammals, plants, and fungi. The fucose used in the present invention may be extracted and purified from naturally occurring animals and plants, or obtained by chemical synthesis, enzymatic methods, or other methods. While any of the L-fucose (L-fucose), D-fucose (D-fucose), and DL-fucose (DL-fucose) forms can be used, L-fucose is preferred. Commercially available fucose is preferred because of its simplicity and ability to maintain consistent quality. In the present invention, fucose may be provided as a fucose-releasing compound, as long as it can ultimately act on myoblasts or muscle satellite cells. A "fucose-releasing compound" refers to a compound capable of liberating fucose in the digestive tract or in vivo. Examples of fucose-releasing compounds include GDP-fucose, saccharides containing fucose as a constituent sugar, such as fucose-containing oligosaccharides and fucose-containing polysaccharides, etc. Fucoidan, which can release fucose by being decomposed by enzymes or microorganisms in the digestive tract or in the body, is also a fucose-releasing compound.

[0014] Fucose or a fucose-releasing compound may be used as a salt thereof. Such salts are not particularly limited, but are preferably salts acceptable for use as pharmaceuticals or foods, and examples thereof include salts with inorganic bases (e.g., alkali metals such as sodium and potassium; alkaline earth metals such as calcium and magnesium; aluminum, ammonium), organic bases (e.g., trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, N,N-dibenzylethylenediamine), inorganic acids (e.g., hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid), organic acids (e.g., formic acid, acetic acid, trifluoroacetic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid), basic amino acids (e.g., arginine, lysine, ornithine), or acidic amino acids (e.g., aspartic acid, glutamic acid).

[0015] Because fucose is useful for inducing skeletal muscle differentiation and / or promoting regeneration, in the present invention, fucose or a fucose-releasing compound can be used as an agent for inducing skeletal muscle differentiation or promoting skeletal muscle regeneration (hereinafter, these may also be referred to as the agent of the present invention). The present invention also provides a composition for inducing skeletal muscle differentiation and / or promoting regeneration, which contains fucose or a fucose-releasing compound as an active ingredient (hereinafter, these may also be referred to as the composition of the present invention). The composition of the present invention can be prepared as a pharmaceutical composition by blending fucose or a fucose-releasing compound with a pharmaceutically acceptable carrier (hereinafter, these may also be referred to as the pharmaceutical composition of the present invention). Furthermore, the composition of the present invention can be prepared as a food or beverage composition by adding various additives to fucose or a fucose-releasing compound (hereinafter, these may also be referred to as the food or beverage composition of the present invention). The composition of the present invention, the pharmaceutical composition of the present invention, and the food or beverage composition of the present invention may be collectively referred to as the composition of the present invention, etc. The compositions of the present invention can be applied to, for example, laboratory animals such as rodents (e.g., mice, rats, hamsters, guinea pigs) and rabbits, pets (e.g., dogs and cats), livestock and poultry (e.g., cows, pigs, goats, horses, sheep, and chickens), primates (e.g., monkeys, orangutans, and chimpanzees), and humans, with humans being particularly preferred. When applied to animals other than humans, the dosage of the compositions of the present invention may be adjusted appropriately based on the general dosage for humans described herein, and further depending on the body weight or size of the animal, or the physical condition and sensitivity of the subject at the time of administration.

[0016] As shown by the data in the Examples, fucose has (1) the effect of promoting muscle differentiation, (2) the effect of promoting muscle regeneration, and (3) the effect of improving muscle function. Due to these excellent pharmacological effects, fucose is useful as a pharmaceutical composition for preventing or treating various diseases caused by muscle damage or muscle dysfunction. Furthermore, due to the above-mentioned pharmacological effects that fucose can exert, fucose has the effect of increasing muscle strength and / or muscle mass, and can also be used as a muscle strength and / or muscle mass enhancing agent (composition).

[0017] The diseases to which the pharmaceutical composition of the present invention is applicable are diseases for which induction of skeletal muscle differentiation and / or promotion of regeneration is effective, specifically, muscle damage, muscle dysfunction, and diseases resulting therefrom, such as muscular dystrophy, myopathy, muscle fatigue syndrome, rhabdomilysis, chronic fatigue syndrome, muscle sprain, muscle contusion, muscle rupture, muscle atrophy, muscle bruise, muscle pull, muscle injury (e.g., muscle injury due to sports), etc. The pharmaceutical composition of the present invention is preferably applied to muscle injury, muscle contusion, muscle atrophy, and muscular dystrophy.

[0018] The dosage of the pharmaceutical composition of the present invention is not particularly limited as long as the effects of the present invention are achieved, and can be appropriately determined depending on the type and severity of the disease of the subject, the species of animal to be administered, the drug tolerance, body weight, age, etc. of the subject. For oral or parenteral administration, the active ingredient fucose is typically administered to an adult at a daily dose of about 0.1 to 2000 mg / kg body weight in the case of oral administration, and about 0.1 to 2000 mg / kg body weight in the case of parenteral administration. It may be administered in divided doses several times a day.

[0019] The pharmaceutical composition of the present invention may contain, in addition to the active ingredient fucose, pharmaceutically acceptable carriers and additives. Examples of pharmaceutically acceptable carriers and additives include excipients such as sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate, and calcium carbonate; binders such as cellulose, methylcellulose, hydroxypropyl cellulose, polypropylpyrrolidone, gelatin, gum arabic, polyethylene glycol, sucrose, and starch; disintegrants such as starch, carboxymethylcellulose, hydroxypropyl starch, sodium glycol-starch, sodium bicarbonate, calcium phosphate, and calcium citrate; and magnesium stearate. Examples of suitable additives include, but are not limited to, lubricants such as ammonium, aerosil, talc, and sodium lauryl sulfate; fragrances such as citric acid, menthol, glycyrrhizin ammonium salt, glycine, and orange powder; preservatives such as sodium benzoate, sodium hydrogen sulfite, methylparaben, and propylparaben; stabilizers such as citric acid, sodium citrate, and acetic acid; suspending agents such as methylcellulose, polyvinylpyrrolidone, and aluminum stearate; dispersing agents such as surfactants; diluents such as water, saline, and orange juice; and base waxes such as cocoa butter, polyethylene glycol, and white kerosene.

[0020] In one embodiment, the pharmaceutical composition of the present invention may be formulated as a preparation suitable for oral administration, such as a liquid preparation in which an effective amount of a substance is dissolved in a diluent such as water or physiological saline, a capsule, granule, powder, or tablet containing an effective amount of a substance as a solid or granule, a suspension in which an effective amount of a substance is suspended in a suitable dispersion medium, or an emulsion in which a solution in which an effective amount of a substance is dissolved is dispersed and emulsified in a suitable dispersion medium.

[0021] In another embodiment, the pharmaceutical composition of the present invention may be formulated as a preparation suitable for parenteral administration. Preparations suitable for parenteral administration (e.g., intravenous injection, subcutaneous injection, intramuscular injection, local injection, etc.) include aqueous and non-aqueous isotonic sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, isotonicity agents, etc. Also included are aqueous and non-aqueous sterile suspensions, which may contain suspending agents, solubilizers, thickeners, stabilizers, preservatives, etc. Such preparations may be packaged in unit-dose or multi-dose containers, such as ampoules or vials. Alternatively, the active ingredient and a pharmaceutically acceptable carrier may be lyophilized and stored in a state that requires only dissolving or suspending in an appropriate sterile vehicle immediately before use.

[0022] The pharmaceutical composition of the present invention may be one in which a unit intake or a fraction thereof of the pharmaceutical composition is individually packaged or filled, or one in which multiple unit intakes or fractions thereof are collectively packaged or filled.

[0023] Examples of pharmaceutical compositions in which unit intakes or fractions thereof are individually packaged or filled include those in which unit intakes or fractions thereof are individually packaged or filled in conventional packaging (e.g., PTP (press through packing) sheets, paper containers, film (e.g., plastic film) containers, glass containers, plastic containers). Medicaments individually packaged or filled in this manner may be further combined and packaged or filled together in a single container (e.g., paper containers, film (e.g., plastic film) containers, glass containers, plastic containers). Examples of pharmaceutical compositions in which multiple unit intakes or fractions thereof are collectively packaged or filled include those in which multiple tablets or capsules are packaged or filled in a single container (e.g., paper containers, film (e.g., plastic film) containers, glass containers, plastic containers) without being separated. The pharmaceutical composition of the present invention may also contain a sufficient number of unit intakes or fractions thereof for long-term intake.

[0024] As shown by the data in the Examples, fucose has (1) the effect of promoting muscle differentiation, (2) the effect of promoting muscle regeneration, and (3) the effect of improving muscle function. Due to these excellent pharmacological effects, it is also useful as a food or beverage composition for the purposes of, for example, myotube formation (muscle differentiation), increasing muscle mass (muscle regeneration), and strengthening muscle strength. Here, food or beverage compositions are processed for human or animal consumption, and include general foods and health functional foods (foods with functional claims, foods with nutrient claims, and foods for specified health uses). The form of the food or beverage composition is not particularly limited, but examples include liquid, paste, solid, powder, etc. Examples of general foods include wheat flour products (e.g., bread, macaroni, spaghetti, noodles, cake mix, fried chicken flour, breadcrumbs, etc.), instant foods (e.g., instant noodles, cup noodles, retort / prepared foods, canned foods, microwave foods, instant soup / stew, instant miso soup / cleaning soup, canned soup, freeze-dried foods, etc.), processed agricultural products (e.g., canned agricultural products, canned fruit, jams / marmalades, pickles, etc.), , boiled beans, dried agricultural products, cereals (processed grain products), etc.), processed seafood products (e.g., canned seafood, fish ham and sausage, fish paste products, seafood delicacies, tsukudani (prepared stew), etc.), processed livestock products (e.g., canned livestock products and pastes, livestock ham and sausage), milk and dairy products (e.g., fermented milk, milk drinks, lactic acid bacteria drinks, sweetened condensed milk, skim milk powder, sweetened milk powder, modified milk powder, cream, cheese, butter, ice cream, etc.), fats and oils (e.g., butter, margarines, vegetable oils, etc.), basic seasonings (e.g., soy sauce, miso, sauces, tomato-processed seasonings, mirin, vinegars, etc.), complex seasonings and foods (e.g., cooking mixes, curry bases, sauces, dressings, noodle soups, spices, etc.), frozen foods (e.g., frozen ingredients, semi-cooked frozen foods, cooked frozen foods, etc.), confectioneries (e.g., caramel, candy, chewing gum, chocolate, cookies, biscuits, cakes, pies, snacks, crackers, Japanese sweets, rice snacks, bean snacks, dessert sweets, etc.), beverages (e.g., carbonated drinks, natural fruit juices, fruit juice drinks, soft drinks with fruit juice, fruit pulp drinks, fruit drinks with fruit particles, vegetable drinks, soy milk, soy milk drinks, coffee drinks, tea drinks, powdered drinks, concentrated drinks, sports drinks, energy drinks, alcoholic drinks, other beverages, etc.), other commercially available foods and beverages, etc.The definition and classification of health functional foods are stipulated in Japan's Health Promotion Act and Food Sanitation Act. The use claims of health functional foods are made in accordance with the Consumer Affairs Agency's guidelines based on Japan's Food Labeling Act and Health Promotion Act. Outside of Japan, for example, regulations based on guidelines are imposed by the European Food Safety Authority (EFSA) in Europe and the Food and Drug Administration (FDA) in the United States. Foods for specified health uses (FOSHU) are labeled with the health benefits approved by the Consumer Affairs Agency, functional foods are labeled with specific functions, and foods with nutritional claims are labeled with the inclusion of specific nutrients and the functions of those nutrients. In the present invention, food and beverage compositions for the purpose of muscle differentiation and muscle regeneration may be, and are recommended to be, labeled with claims such as "promoting skeletal muscle formation during muscle training," "promoting skeletal muscle formation in infants," "improving walking ability," "increasing muscle mass and strength," and "maintaining muscle mass and strength."

[0025] In addition to the foods and beverages having the above-mentioned forms, the food and beverage compositions of the present invention also include supplement-form food and beverage compositions and foods for patients (including foods for those requiring nursing care and foods for those with swallowing difficulties). When preparing such supplement-form food and beverage compositions or foods for patients, it is preferable to prepare them in the form of a liquid (drinkable), syrup, dry syrup, jelly preparation (including those prepared at the time of use; the same applies below), granules, powder, pills, tablets, capsules (hard capsules, soft capsules), lozenges, chewable tablets, etc., so as to facilitate continuous intake. Preferred are liquids (drinkable), jelly preparations, granules, tablets, and capsules (hard capsules, soft capsules), and more preferably liquids (drinkable) and jelly preparations. Such formulations can be prepared according to conventional methods using pharmaceutically acceptable carriers and additives (described above) depending on the form of each formulation.

[0026] The food and drink composition of the present invention may be a food and drink (pet food) for pets (dogs, cats, hamsters, rabbits, birds, etc.), or a food and drink (feed composition) for livestock (cows, pigs, poultry).

[0027] The dosage of the food and drink composition of the present invention is not particularly limited as long as the effects of the present invention are achieved, and varies depending on the purpose of administration, the species, body weight, age, etc. of the animal to be administered, but typically, an amount of fucose of about 1 to 2000 mg / kg body weight per day is administered to the subject for an adult.

[0028] The contents of all publications, including patents and patent applications, cited in this specification are hereby incorporated by reference in their entirety to the same extent as if fully set forth herein.

[0029] The present invention will be described in detail below using examples, but the present invention is not limited thereto. Furthermore, the reagents and materials used are commercially available unless otherwise specified. Abbreviations used in this specification are the same as those commonly used in the art unless otherwise specified.

[0030] Example 1: Addition of L-fucose promotes muscle differentiation (1) C2C12 cells were cultured at 3.0 × 10 in 10% FBS / DMEM (Sigma D6429). ４ / cm ２ The cells were seeded onto 12-well plates at 100°C. The next day, L-fucose was added to 2% HS / DMEM at concentrations of 0, 50, 100, or 200 μM and cultured for 4 days (medium change on day 2). On day 4, the cells were washed with PBS and fixed with cold 4% paraformaldehyde (PFA) for 5 minutes, followed by blocking. MY32 antibody (Sigma: A4335) was added as the primary antibody at a 1:300 dilution and left overnight. The following day, a secondary antibody was added at a 1:1000 dilution and left for 2 hours. After washing with PBS, the cells were stained with DAPI and observed under a fluorescence microscope (Figure 1A). The differentiation index represents the number of MY32-positive nuclei divided by the total number of nuclei (Figure 1B). The MY32 antibody is used to stain skeletal muscle-specific myosin heavy chain (MHC). DAPI is used for nuclear staining. L-fucose increased the Differentiation Index in a concentration-dependent manner, indicating that fucose promotes muscle differentiation.

[0031] Example 2: Addition of L-fucose promotes muscle differentiation (2) Mouse myoblasts (K. Yoshioka et al., Front Cell Dev Biol. 2020 Aug 13;8:793) obtained by primary culture in 15% FBS, 10 ng / ml bFGF, 1% CEE / DMEM (Sigma D6429) were cultured at 2.0 × 10 ４ / cm ２ The cells were seeded onto a 6-well plate at 100 μM. The next day, L-fucose was added to 2% HS / DMEM at a concentration of 0 (control in Figure 2) or 100 μM, and the cells were cultured for 3 days (the medium was replaced on the second day). The length of myotubes was measured under bright field observation (Figure 2A), and significance was examined using an unpaired t-test (p<0.01, t-test, each 3) (Figure 2B). The addition of L-fucose increased the length of myotubes, indicating that fucose promotes muscle differentiation.

[0032] Example 3: Addition of L-fucose promotes muscle differentiation (3) Mouse myoblasts (as in Example 2) obtained by primary culture in 15% FBS, 10 ng / ml bFGF, 1% CEE / DMEM (Sigma D6429) were cultured at a concentration of 2.0 × 10 ４ / cm ２ The cells were seeded onto a 6-well plate at 100 μM. The next day, L-fucose was added to 2% HS / DMEM at concentrations of 0, 100, or 200 μM, and cultured for 4 days (the medium was replaced on day 2). On day 4, videos of myotube pulsation were taken using a Sony SI8000 (Figure 3A). Analysis was performed using the SI8000 analysis software to quantify the percentage of beating myotubes (p<0.01, One-way ANOVA, each 3) (Figure 3B). The vertical axis of the graph indicates the percentage of beating myotubes per area. L-fucose concentration-dependently increased the percentage of beating myotubes, demonstrating that fucose promotes muscle differentiation.

[0033] Example 4: Addition of L-fucose promotes muscle differentiation (4) Mouse myoblasts (as in Example 2) obtained by primary culture in 15% FBS, 10 ng / ml bFGF, 1% CEE / DMEM (Sigma D6429) were cultured at a concentration of 2.0 × 10 ４ / cm ２The cells were seeded onto a 6-well plate. The next day, L-fucose was added to 2% HS / DMEM at a concentration of 0 or 200 μM, and cultured for 6 days (medium was replaced on days 2 and 4). On day 6, the cells were washed with PBS, fixed with glutaraldehyde, and then sliced ​​into blocks for electron microscopy (Figure 4A). Sarcomere length was measured using ImageJ, and a significance test was performed (p<0.05, Unpaired t-test, each 3) (Figure 4B). Clear striations were observed in cells cultured with L-fucose. Furthermore, sarcomere length became uniform and fell within the optimal range, demonstrating that fucose promotes muscle differentiation.

[0034] The results of Examples 1 to 4 demonstrate that adding fucose to myoblast culture can promote muscle differentiation. This effect can be applied to, for example, the culture of artificial meat and the differentiation culture of stem cells such as iPS cells.

[0035] Example 5: Addition of L-fucose promotes muscle regeneration (1) Cardiotoxin (CTX) was injected at 10 μM into the tibialis anterior muscle of 8-week-old male C57B6 / J mice to create a muscle injury model mouse. Cardiotoxin causes muscle damage by depolarizing the muscle membrane. For 7 days after muscle injury, 1 mg / g (body weight) / day of L-fucose was administered intraperitoneally and orally via a probe. As a control, saline in an amount equal to the dose of L-fucose was administered orally via a probe. On the 7th day of regeneration, the mice were euthanized by cervical dislocation, and the tibialis anterior muscle was collected, frozen, and thin sections 10 μm thick were prepared. Thin sections were fixed in cold acetone for 15 minutes, blocked, and then incubated overnight with primary antibodies F1.652 (DSHB) at 50x dilution and Laminin a2 (Sigma L9393) at 500x dilution. The following day, secondary antibodies were added at 1:1000 dilution and incubated for 2 hours. After washing with PBS, the sections were stained with DAPI and observed under a fluorescent microscope. F1.652 is a monoclonal antibody used for immunostaining fetal myosin heavy chain (Fetal MHC), a marker of regenerating muscle fibers. The addition of L-fucose increased the number of muscle fibers stained with F1.652, indicating that fucose promotes muscle regeneration. The results are shown in Figure 5.

[0036] Example 6: Addition of L-fucose Promotes Muscle Regeneration (2) Data obtained from the fluorescent staining images in Example 5 were quantified. The percentage of F1.652-positive muscle fibers among all muscle fibers was quantified (Figure 6A). Muscle wet weight was calculated by correcting the weight of the tibialis anterior (TA) muscle collected in Example 5 by body weight (BW) (Figure 6B). All data were subjected to a significance test (p<0.05, p<0.01; one-way ANOVA, each 3). Muscle wet weight refers to the weight of muscle measured without removing the water contained in the muscle tissue. Because muscle wet weight measures the weight of muscle containing its inherent water content, it can be an important indicator for assessing muscle condition and health. Addition of L-fucose increased the number of muscle fibers stained with F1.652, indicating that fucose promotes muscle regeneration. Furthermore, addition of fucose also increased muscle wet weight.

[0037] Example 7: Addition of L-fucose improves muscle function. 10 μM cardiotoxin (CTX) was injected into the tibialis anterior (TA), gastrocnemius (GAS), and quadriceps (QUAD) muscles of 8-week-old male C57B6 / J mice to create a muscle injury model mouse. 1 mg / g (body weight) / day of L-fucose was administered intraperitoneally for 7 days after muscle injury. As a control, saline was administered intraperitoneally in an amount equal to the L-fucose dose. Hindlimb grip strength was measured on days 3, 5, and 7 after CTX injury using a grip strength tester for small animals. Grip strength without muscle injury was set to 1, and the ratios of grip strength on days 3, 5, and 7 after CTX injury were shown (p<0.05, p<0.01. Unpaired t-test, each 5). The results are shown in Figure 7. The addition of L-fucose was observed to improve grip strength, indicating that fucose significantly improves muscle function.

[0038] Example 8: Treatment of Muscular Dystrophy with L-Fucose Duchenne muscular dystrophy (DMD) is a genetic mutation that occurs in one in 3,500 newborn boys. The lack of dystrophin causes progressive muscle damage. No established treatment exists, and most patients die by the age of 30. To evaluate the effect of L-fucose on DMD, we used Duchenne muscular dystrophy model mice (mdx, obtained from the Central Institute for Experimental Animals, a public interest incorporated foundation). Like patients, mdx mice have a dystrophin mutation, which causes muscle damage. However, they are a mild model mouse with high regenerative capacity and no impact on lifespan. Male mdx mice were intraperitoneally administered 1 mg / g (body weight) / day of L-fucose and an equal amount of saline daily from 3 to 8 weeks of age. Figure 8A shows the lower limb muscles of an 8-week-old mdx mouse. Figure 8B shows the change in body weight over time for each week of age (p<0.05, Unpaired t-test, each 4). It was found that the addition of L-fucose promoted weight gain in mdx mice.

[0039] Example 9: Addition of L-fucose increases muscle wet weight The wet muscle weights of the tibialis anterior (TA), soleus (Sole), gastrocnemius (Gas), and quadriceps (Quad) muscles of mdx mice treated with L-fucose or as controls were measured in the same manner as in Example 8. The results are shown in Figure 9. The muscle wet weights were calculated by correcting each muscle for body weight and multiplying by 1000 (p<0.05. Unpaired t-test, each 4). It was found that addition of L-fucose increased the wet weight of each muscle in mdx mice. In particular, the wet muscle weights of the gastrocnemius and quadriceps muscles increased significantly.

[0040] Example 10: Addition of L-fucose promotes muscle regeneration (3) HE staining was performed on cross sections of the tibialis anterior muscle from mdx mice treated with L-fucose or from the control group as in Example 8 (Figure 10A). Bar indicates 200 μm. Figure 10B shows the results of measuring the maximum muscle circumference and total number of muscle fibers in the tibialis anterior muscle (p<0.05. Unpaired t-test, each 3).

[0041] Example 11: Addition of L-fucose promotes diaphragm regeneration. As in Example 8, HE staining was performed on the diaphragms of mdx mice treated with L-fucose or as a control (FIG. 11A). Bar indicates 100 μm. FIG. 11B shows the results of measuring the diaphragm diameter of each mouse (p<0.01, Unpaired t-test, each 4).

[0042] Example 12: Addition of L-fucose Promotes Muscle Regeneration (4) The tenotomy model induces muscle hypertrophy by resecting the gastrocnemius and soleus tendons and increasing the load on the plantaris muscle. Tenotomy models were created by resecting the tendons in the left foot of 8-week-old C57B6 / J male mice under anesthesia. The sutured right foot served as a control. Starting on the day of resection, L-fucose was orally administered at a dose of 0.5 mg / g body weight and an equivalent volume of saline for two weeks to sacrifice the plantaris muscle. Figure 12A shows a photograph of the sacrificed plantaris muscle. Figure 12B shows the plantaris muscle mass corrected for body weight. Tenotomy-induced hypertrophy was significantly greater with L-fucose intake than with the control. (p<0.05, Unpaired t-test, each 5-6). Figure 13A shows an HE-stained image of the sacrificed plantaris muscle. The bar indicates 100 μm. Figure 13B shows the distribution of muscle fiber diameters in the plantaris muscle. Administration of L-fucose decreased the proportion of small muscle fibers and increased the proportion of large muscle fibers compared to saline.

[0043] The composition of the present invention containing fucose or a fucose-releasing compound as an active ingredient can promote myotube formation and regenerate damaged or atrophied skeletal muscle, and can be used not only as a pharmaceutical composition for the prevention and treatment of muscle injury, muscle contusion, muscle atrophy, muscular dystrophy, etc., but also for promoting skeletal muscle formation during muscle training, promoting skeletal muscle formation in infants, etc. Furthermore, because fucose is a monosaccharide that is widely present in animals and plants, the composition of the present invention is highly safe and can be widely used.

Claims

1. A composition for inducing differentiation and / or promoting regeneration of skeletal muscle, comprising fucose or a fucose-releasing compound as an active ingredient.

2. The composition according to claim 1, wherein the induction of skeletal muscle differentiation is myotube formation.

3. The composition of claim 1, which is a pharmaceutical composition.

4. The composition according to claim 3, which is used for the prevention or treatment of diseases caused by muscle damage or muscle dysfunction.

5. The composition of claim 4, wherein the disease is selected from muscle injury, muscle strain, muscle atrophy, and muscular dystrophy.

6. The composition according to claim 1, which is a food or beverage composition.

7. The composition according to claim 6, which is selected from a food for specified health uses, a food with functional claims, and a food with nutrient function claims.

8. The composition according to claim 6, which is a supplement.

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