JP2024080456A - Muscle mass maintenance agent and its use - Google Patents

Abstract

[Problem] To provide a muscle mass maintenance agent capable of suppressing loss of muscle mass. [Solution] The active ingredient of the muscle mass maintenance agent for suppressing loss of muscle mass is R-3-hydroxybutyric acid and/or a salt thereof. The active ingredient may include a salt of R-3-hydroxybutyric acid. The muscle mass maintenance agent may be capable of preventing and/or treating sarcopenia. The muscle mass maintenance agent may be capable of suppressing loss of muscle mass without dietary therapy and/or exercise therapy. The muscle mass maintenance agent may be a muscle mass maintenance agent capable of suppressing loss of muscle mass in elderly people and/or diabetic patients. The muscle mass maintenance agent may be capable of suppressing loss of skeletal muscle mass (particularly skeletal muscle mass of the lower limbs). [Selected Figures] None

Description

The present invention relates to a muscle mass maintenance agent containing R-3-hydroxybutyric acid and/or its salt as an active ingredient, and its use.

In recent years, aging has progressed rapidly in Japan and other developed countries, and in this aging society, there is an urgent need to maintain the health of the elderly, promote their participation in social activities, and reduce the burden on medical facilities. Under these circumstances, in order to contribute to the health and longevity of the nation's citizens, the Japan Society of Sarcopenia and Frailty was established in September 2016 as a general incorporated association, and sarcopenia and frailty instructors are engaged in activities related to sarcopenia therapy.

The word frailty comes from the English word "Frailty," which is used in the field of geriatrics overseas and means "weakness," "senility," or "vulnerability." Xue QL et al., Journal Gerontology: MEDICAL SCIENCE (2008) (Non-Patent Document 1) and other publications report on research into frailty, and according to these publications, frailty can be defined as follows. First, when muscle strength and muscle mass decrease due to aging, the amount of activity in daily life decreases, and energy consumption decreases. Furthermore, in such a state, appetite is lost, so food intake decreases, and the body becomes malnourished due to insufficient intake of nutrients such as protein. If the state of malnutrition continues, weight will decrease, and muscle strength and muscle mass will decrease. This vicious cycle is called the frailty cycle, and the possibility of becoming in need of care increases due to falls, fractures, or worsening chronic diseases.

Meanwhile, sarcopenia was proposed by Rosenberg in 1989 as a term meaning "loss of muscle mass due to aging." Subsequently, the European Working Group on Sarcopenia in Older People (EWGSOP) developed a practical clinical definition and a unified view of diagnostic criteria for sarcopenia due to aging, and sarcopenia now refers to a state in which muscle mass is reduced in the frailty cycle, resulting in reduced muscle strength and physical function.

In addition, the number of patients with diabetes, a lifestyle-related disease, is increasing in relation to the aging society. Various research reports, such as Kashima, S. et al., Scientific Reports 11, 15167 (2021) (Non-Patent Document 2), have reported a bidirectional relationship in which chronic hyperglycemia and insufficient insulin action cause a decrease in skeletal muscle mass throughout the body in diabetes, and the decrease in skeletal muscle mass worsens glucose metabolism, thereby increasing the risk of diabetes. Therefore, in addition to the decrease in skeletal muscle mass due to aging and senescence, the decrease in skeletal muscle mass due to lifestyle-related diseases such as diabetes, which are closely related to aging, is also an important cause of the development of sarcopenia.

In contrast, there are currently no pharmaceutical treatments for sarcopenia, and only dietary and exercise therapies are known. However, as in the frailty cycle, once sarcopenia develops, food intake decreases and exercise therapy is difficult, so there is a need for treatments other than dietary and exercise therapy. In addition, in developed countries, metabolic syndrome, as typified by diabetes, is also a problem, and there are an increasing number of cases in which dietary restrictions are required to eliminate obesity. In other words, there is a fundamental trade-off between preventing lifestyle-related diseases caused by excessive nutritional intake and preventing sarcopenia, so from this perspective, there is a long awaited method of preventing and treating sarcopenia other than dietary therapy.

Regarding active ingredients related to muscle strength, JP2015-514104A (Patent Document 1) discloses ketone bodies or ketone body esters for use in maintaining or improving a subject's muscle power output. This document states that the subject is particularly useful for healthy individuals, such as athletes and military personnel, who have a high level of fitness. Furthermore, in the Examples, the skeletal muscle power output (W) of athletes who consumed a beverage containing (R)-3-hydroxybutyric acid-R-1,3-butanediol ester was measured.

JP 2015-514104 A

Xue QL, Bandeen-Roche K, Varadhan R, et al. Initial manifestations of frailty criteria and the development of frailty phenotype in the Women’s Health and Aging Study II, Journal Gerontology: MEDICAL SCIENCE 2008, Vol. 63A, No.9, 984-990 Kashima, S., Inoue, K. & Matsumoto, M. Low creatinine levels in diabetes mellitus among older individuals: the Yuport Medical Checkup Center Study. Scientific Reports 11, 15167 (2021), https://doi.org/10.1038/s41598-021-94441-9

However, Patent Document 1 does not mention muscle mass. In addition, Patent Document 1 aims to maintain or improve muscle power output in healthy individuals, and does not mention sarcopenia.

Therefore, the object of the present invention is to provide a muscle mass maintenance agent that can suppress muscle mass loss and its use.

Another object of the present invention is to provide a muscle mass maintenance agent capable of preventing and/or treating sarcopenia and uses thereof.

As a result of intensive research into solving the above problems, the inventors discovered that administering R-3-hydroxybutyric acid and/or its salts as an active ingredient can effectively inhibit muscle mass loss, leading to the completion of the present invention.

That is, the muscle mass maintenance agent (or muscle strength decline inhibitor) according to embodiment [1] of the present invention is a muscle mass maintenance agent for inhibiting muscle mass loss, and contains R-3-hydroxybutyric acid and/or a salt thereof as an active ingredient.

Aspect [2] of the present invention is the aspect [1] described above, in which the active ingredient comprises a salt of R-3-hydroxybutyric acid.

Aspect [3] of the present invention is an aspect in which the muscle mass maintenance agent of aspect [1] or [2] can prevent and/or treat sarcopenia.

Aspect [4] of the present invention is an aspect in which the muscle mass maintenance agent according to any one of aspects [1] to [3] can suppress the loss of muscle mass without dietary therapy and/or exercise therapy.

Aspect [5] of the present invention is an aspect in which the muscle mass maintenance agent according to any one of aspects [1] to [4] above is capable of suppressing the loss of muscle mass in elderly people and/or diabetic patients.

Aspect [6] of the present invention is any of aspects [1] to [5] above, in which the muscle mass is skeletal muscle mass.

Aspect [7] of the present invention is aspect [6], in which the skeletal muscle is a skeletal muscle of the lower limb.

Aspect [8] of the present invention is an aspect in which the muscle mass maintenance agent according to any one of aspects [1] to [7] above is capable of suppressing a decrease in the expression level of insulin-like growth factor and/or collagen genes.

Aspect [9] of the present invention is an aspect in which the muscle mass maintenance agent of any of aspects [1] to [8] above is a liquid composition, a semi-solid composition, or a solid composition.

Aspect [10] of the present invention is an aspect in which the muscle mass maintenance agent of any of aspects [1] to [9] above is a physiologically active composition or pharmaceutical composition for oral ingestion.

The present invention also includes, as aspect [11], a drug for preventing and/or treating sarcopenia, which contains R-3-hydroxybutyric acid and/or a salt thereof as an active ingredient.

The present invention also includes, as aspect [12], an inhibitor for inhibiting a decrease in skeletal muscle mass and/or skeletal muscle fiber mass, the inhibitor containing R-3-hydroxybutyric acid and/or a salt thereof as an active ingredient.

The present invention also includes, as aspect [13], a muscle mass promoter that increases skeletal muscle mass and/or skeletal muscle fiber mass in at least one patient selected from the group consisting of elderly people, diabetic patients, and sarcopenic patients, the muscle mass promoter containing R-3-hydroxybutyric acid and/or a salt thereof as an active ingredient.

The present invention also includes, as aspect [14], an inhibitor for inhibiting a decrease in the expression level of an insulin-like growth factor and/or collagen gene, the inhibitor containing R-3-hydroxybutyric acid and/or a salt thereof as an active ingredient.

The present invention also includes, as aspect [15], a promoter for increasing the expression level of insulin-like growth factor and/or collagen gene in at least one selected from the group consisting of elderly people, diabetic patients, and sarcopenic patients, the promoter containing R-3-hydroxybutyric acid and/or a salt thereof as an active ingredient.

In the present invention, the active ingredient of the muscle mass maintenance agent is R-3-hydroxybutyric acid [(R)3HB or BHB] and/or a salt thereof, and therefore the loss of muscle mass can be suppressed. In particular, the loss of muscle mass can be suppressed without dietary therapy and/or exercise therapy, and therefore the agent is effective in preventing and/or treating sarcopenia.

FIG. 1 is a graph showing the relationship between a skeletal muscle mass reduction inhibitor and skeletal muscle mass in an example. FIG. 2 is a graph showing the relationship between an inhibitor of skeletal muscle mass decrease and the average area of muscle fibers of the tibialis anterior muscle in an example. FIG. 3 is a graph showing the relationship between an inhibitor of a decrease in skeletal muscle mass and the expression level of IGF1 in an example. FIG. 4 is a graph showing the relationship between an inhibitor of a decrease in skeletal muscle mass and the expression level of Col1a1 in an example. FIG. 5 is a graph showing the relationship between an inhibitor of a decrease in skeletal muscle mass and the expression level of Col3a1 in an example.

The muscle mass maintenance agent of the present invention contains R-3-hydroxybutyric acid and/or a salt thereof [hereinafter sometimes collectively referred to as "(R)3HB (salt)"] as an active ingredient. The muscle mass maintenance agent of the present invention may be formed of the active ingredient alone, or may be formed of a composition containing the active ingredient. The muscle mass maintenance agent of the present invention inhibits the decrease in striated muscle and/or muscle fibers (slow muscle fibers, fast muscle fibers) (particularly promotes the increase in elderly people, diabetic patients, and sarcopenia patients), and can therefore be used as an agent for inhibiting or preventing muscle weakness in humans, and may be, for example, a drug for preventing and/or treating sarcopenia.

[(R)3HB (salt) as active ingredient]
In the muscle mass maintaining agent of the present invention, the active ingredient (first active ingredient) comprises R-3-hydroxybutyric acid and/or a salt thereof.

The (R)3HB (salt) may be in a crystalline form or an amorphous form. Of these, the (R)3HB (salt) in the solid composition or semi-solid composition may be in a crystalline form.

Examples of salt types include alkali metal salts such as sodium salt, potassium salt, and lithium salt; alkaline earth metal salts such as magnesium salt and calcium salt; and ammonium salt. These salts of (R)3HB can be used alone or in combination of two or more. Of these, alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as magnesium salt and calcium salt are preferred, with alkali metal salts being more preferred; sodium salt, magnesium salt, and calcium salt are more preferred in terms of their relatively low deliquescence and excellent handleability; and sodium salt is most preferred in terms of the ease of adjusting the balance of various properties.

The (R)3HB (salt) used as a muscle mass maintenance agent may be (R)3HB alone, a mixture of (R)3HB and a salt of (R)3HB, or a salt of (R)3HB alone.

In its acid form, (R)3HB (salt) has a strong sour taste and is poorly palatable and easy to take, whereas in its salt form, it is highly palatable and easy to take. For this reason, when taken orally, it is preferable to reduce the proportion of (R)3HB in the active ingredient and include a salt of (R)3HB as the main ingredient. Specifically, the proportion of the salt of (R)3HB may be 50% by mass or more of the active ingredient, preferably 80% by mass or more, even more preferably 90% by mass or more, even more preferably 95% by mass or more, and most preferably 100% by mass.

As the (R)3HB (salt), at least one selected from the group consisting of (R)3HB, the sodium salt of (R)3HB, the magnesium salt of (R)3HB, and the calcium salt of (R)3HB is preferred, and a combination of (R)3HB and the sodium salt of (R)3HB, or the sodium salt of (R)3HB alone is more preferred, with the sodium salt of (R)3HB alone being even more preferred.

The proportion of the first active ingredient [(R)3HB (salt)], calculated as the free form of (R)3HB, in the muscle mass maintenance agent may be 0.01 to 100% by mass, for example 0.05 to 100% by mass, preferably 0.1 to 100% by mass (particularly 0.2 to 100% by mass, etc.).

In this specification and claims, "converted to (R)3HB in free form" means that the salt of (R)3HB is converted to R-3-hydroxybutyric acid in acid form. For example, when the active ingredient contains a salt of (R)3HB such as sodium R-3-hydroxybutyrate, the mass of sodium R-3-hydroxybutyrate is converted to the mass of R-3-hydroxybutyric acid to calculate the proportion of the active ingredient [i.e., the total proportion of (R)3HB and salt of (R)3HB].

[(S)3HB (salt)]
The muscle mass maintenance agent of the present invention may further contain, in addition to the R-form of the active ingredient, the S-form, which is an optical isomer of the R-form, i.e., S-3-hydroxybutyric acid and/or a salt thereof [sometimes collectively referred to as (S)3HB (salt)].

In the muscle mass maintenance agent of the present invention, 3HB (salt) may contain (R)3HB (salt) and may be racemic, but if the proportion of (S)3HB (salt) is too high, biocompatibility decreases, so a low proportion of (S)3HB (salt) is preferable, and it is particularly preferable that it is unavoidably contained. The mass proportion of the R form in 3HB (salt) may be 10% by mass or more, preferably 50% by mass or more, more preferably 80% by mass or more, more preferably 90% by mass or more, and most preferably 100% by mass. The optical purity (enantiomer or optical isomer excess) of the R form in 3HB (salt) is, for example, 50% e.e. or more (e.g. 80% e.e. or more), preferably 90% e.e. or more (e.g. 95 to 100% e.e.), and more preferably 98 to 100% e.e. (e.g. 99 to 100% e.e., particularly substantially 100% e.e.).

3HB (salt), including (R) 3HB (salt), may be a commercially available product. Examples of commercially available products include chemically synthesized 3HB (salt) and 3HB (salt) produced by fermentation using microorganisms. Of these, fermentation-produced 3HB (salt) is preferred because of the high purity of the R-isomer, and fermentation-produced 3HB (salt) using biomass raw materials (resources derived from living organisms) is particularly preferred.

[3HB Oligomer]
The muscle mass maintenance agent of the present invention may further contain a 3HB oligomer. The average degree of polymerization of the 3HB oligomer may be 2 or more, for example, 2 to 100, preferably 2 to 10, further preferably 2 to 5, more preferably 2 to 4.5, and most preferably 2 to 4. The 3HB oligomer may be an oligomer that is inevitably mixed in during the production process of 3HB, etc.

The proportion of 3HB oligomer may be 10 parts by mass or less, preferably 5 parts by mass or less, more preferably 3 parts by mass or less, and most preferably 1 part by mass or less, per 100 parts by mass of the first active ingredient. The muscle mass maintenance agent of the present invention may not substantially contain 3HB oligomer, and it is particularly preferable that it does not contain 3HB oligomer.

[Characteristics of muscle mass maintenance agent]
The muscle mass maintenance agent of the present invention can be used to suppress the loss of muscle mass (muscle mass and volume) in mammals such as humans, and is preferably used to suppress the loss of muscle mass in medium-sized or large mammals (especially humans). In particular, the muscle mass maintenance agent of the present invention can suppress the loss of muscle mass without dietary therapy and/or exercise therapy, so it is preferably used for the prevention and/or treatment of sarcopenia. It can also be used as a promoter for increasing muscle mass or muscle in elderly people, diabetic patients, and sarcopenia patients.

The muscle mass maintenance agent of the present invention is effective for muscles throughout the body, particularly striated muscles, and is particularly effective for skeletal muscles. Representative target skeletal muscles include, for example, skeletal muscles of the head (facial muscles, occipital muscles, masticatory muscles, etc.), skeletal muscles of the neck (sternocleidomastoid muscles, serratus anterior muscles, etc.), skeletal muscles of the chest (trapezius muscles, latissimus dorsi muscles, long dorsi muscles, erector spinae muscles, etc.), skeletal muscles of the abdomen (rectus abdominis muscles, external oblique muscles, etc.), skeletal muscles of the upper limbs (deltoid muscles, biceps brachii muscles, triceps brachii muscles, etc.), and skeletal muscles of the lower limbs (gluteus maximus muscles, adductor longus muscles, sartorius muscles, quadriceps muscles, tibialis anterior muscles, extensor digitorum longus muscles, gastrocnemius muscles, soleus muscles, adductor magnus muscles, biceps femoris muscles, semitendinosus muscles, semimembranosus muscles, plantar muscles, etc.). Among these, the muscle mass maintenance agent of the present invention can be suitably used for the skeletal muscles of the upper and/or lower limbs, which have the greatest impact on activities of daily life, and is particularly suitable for the skeletal muscles of the lower limbs, which are most important for preventing the progression of sarcopenia and the need for nursing care. The skeletal muscles of the lower limbs for which the muscle mass maintenance agent of the present invention is effective are preferably the quadriceps (rectus femoris, vastus medialis, vastus lateralis, vastus intermedius, etc.), the extensors of the legs (tibialis anterior, extensor digitorum longus, peroneus third, extensor hallucis longus, etc.), and the flexors of the legs (plantaris; triceps surae such as gastrocnemius lateralis, gastrocnemius medialis, and soleus; flexor digitorum longus; flexor posterioris; flexor hallucis longus, etc.), and the quadriceps and gastrocnemius are particularly preferred.

The muscle mass maintenance agent of the present invention can suppress the decrease in the expression level of insulin-like growth factors such as IGF1 and IGF2 (particularly IGF1), and can suppress the decrease in the expression level of insulin-like growth factors such as IGF1, particularly in elderly people and/or diabetic patients, and can therefore be used as an agent for maintaining (inhibiting decrease) the expression level of insulin-like growth factors in people with reduced muscle mass or muscle strength, for example, in sarcopenia. It can also be used as a promoter for increasing the expression level of insulin-like growth factors in elderly people, diabetic patients, and sarcopenic patients.

The muscle mass maintenance agent of the present invention can suppress the decrease in the expression level of collagen genes such as Col1a1 and Col3a1, which express collagens such as type I collagen and type III collagen, and can suppress the decrease in the expression level of collagen genes, particularly in elderly people and/or diabetic patients, and can therefore be used as an agent for maintaining (inhibiting decrease) the expression level of collagen genes in people with reduced muscle mass or muscle strength, for example, sarcopenia patients. It can also be used as a promoter for increasing the expression level of collagen genes in elderly people, diabetic patients, and sarcopenia patients.

The muscle mass maintenance agent of the present invention may be administered orally or parenterally. Oral administration may be, for example, in the form of a liquid, solid, or semi-solid agent. Parenteral administration may be, for example, by inhalation, injection, transdermal, or nasal administration. Of these, oral administration is preferred because (R)3HB (salt), especially (R)3HB salt, is more palatable.

The muscle mass maintenance agent of the present invention can suppress the loss of muscle mass in medium-sized or large mammals such as humans even at small doses. In particular, by using the R-isomer of 3HB (salt), it is possible to efficiently suppress the loss of muscle mass at small doses.

The muscle mass maintenance agent of the present invention can be administered in a daily dose of, for example, about 0.01 to 50 g (particularly 0.05 to 30 g) per kg of body weight, calculated as R-3-hydroxybutyric acid, which is the free form of the first active ingredient, for example, 0.1 to 20 g (particularly 0.3 to 10 g, etc.), preferably 0.5 to 8 g, and more preferably 1 to 5 g (particularly 2 to 4 g, etc.).

The administration schedule (number of times per day) of the muscle mass maintenance agent of the present invention is not particularly limited, and may be once or multiple times per day, for example, 1 to 5 times, preferably 1 to 3 times, more preferably 1 to 2 times, and even more preferably once. When taking multiple times per day, it is preferable to take the agent at intervals of 3 hours, 6 hours, 8 hours, or 12 hours depending on the number of times taken.

When the muscle mass maintenance agent of the present invention is a composition containing a first active ingredient [i.e., (R)3HB (salt)], the proportion of the first active ingredient, calculated as free (R)3HB, in the composition can be selected from a range of about 0.01 to 99% by mass, preferably 0.1 to 95% by mass, and more preferably 0.5 to 90% by mass. The proportion of the first active ingredient can be selected depending on the form of the composition, as described below.

When the muscle mass maintenance agent of the present invention is a composition containing a first active ingredient, it may be either a physiologically active composition (functional beverage, nutritional supplement beverage, nutritional supplement, functional food composition, etc.) or a pharmaceutical composition, and is preferably a physiologically active composition or pharmaceutical composition for oral ingestion, and is particularly preferably a physiologically active composition or pharmaceutical composition that can prevent and/or treat sarcopenia while undergoing dietary therapy and/or exercise therapy, or without dietary therapy and/or exercise therapy.

When the muscle mass maintenance agent of the present invention is a composition containing a first active ingredient, the composition may be in the form of a liquid composition (liquid preparation), a solid composition (solid preparation), or a semi-solid composition (semi-solid preparation). Of these, liquid compositions and semi-solid compositions are preferred, and liquid compositions are particularly preferred. Of these, liquid compositions and semi-solid compositions are preferred, and liquid compositions are particularly preferred, in that they can take advantage of the excellent palatability characteristics.

[Liquid composition]
When the muscle mass maintenance agent of the present invention is a liquid composition, examples of the liquid composition (liquid preparation) include drinks (beverages), suspensions, emulsions, elixirs, syrups, jellies, extracts, spirits, injections, etc. The method of ingestion or administration of the liquid composition may be a parenteral administration method such as injection administration, but oral ingestion (administration) in the form of a drink or the like is preferred from the viewpoint of making use of the highly palatable characteristic.

Drink preparations include various beverages, such as lactic acid bacteria beverages; milk and dairy product beverages; nutritional drinks; carbonated beverages, fruit beverages, coffee beverages, tea beverages, soy milk beverages, vegetable beverages, sports and functional beverages, dairy beverages and other soft drinks; alcoholic beverages, etc.

(First active ingredient)
In the liquid composition, the first active ingredient preferably contains a salt of (R)3HB, and may be a mixture of (R)3HB and a salt of (R)3HB, or may be a salt of (R)3HB alone, with a salt of (R)3HB being particularly preferred. Also, by making the first active ingredient a mixture of (R)3HB and a salt of (R)3HB, it is possible to maintain the ease of administration and suppress salt intake.

The proportion of the first active ingredient, calculated as the free form of (R)3HB, in the liquid composition can be selected from a range of about 0.01 to 50% by mass, for example 0.05 to 30% by mass, preferably 0.1 to 20% by mass, further preferably 0.5 to 10% by mass, more preferably 1 to 8% by mass, and most preferably 2 to 5% by mass. If the proportion of the first active ingredient is too low, there is a risk that the effect of maintaining muscle mass will be reduced, and conversely, if it is too high, there is a risk that palatability will be reduced.

(Second Active Ingredient)
The liquid composition may further contain a second active ingredient. Examples of the second active ingredient include therapeutic agents for lifestyle-related diseases (e.g., obesity, diabetes, hypertension, collagen disease, impaired glucose tolerance, hypercholesterolemia, hypertriglyceridemia, low HDL cholesterol, metabolic syndrome, infectious diseases, sepsis, and chronic kidney disease). These second active ingredients may be used alone or in combination of two or more.

The proportion of the second active ingredient may be 100 parts by mass or less per 100 parts by mass of the first active ingredient, and is preferably 50 parts by mass or less (e.g., 0.1 to 50 parts by mass), more preferably 30 parts by mass or less, and even more preferably 10 parts by mass or less.

(Sugars)
The liquid composition may further contain a saccharide. In particular, when the first active ingredient contains (R)3HB in the form of an acid, it is preferable to contain a saccharide in order to suppress a strong sour taste and improve the ease of administration.

Examples of sugars include pentoses such as arabinose and xylose; hexoses such as glucose, fructose, galactose, mannose, and sorbose; rare sugars such as psicose; and monosaccharides such as honey; disaccharides such as sucrose (e.g., white sugar, refined white sugar, powdered sugar, granulated sugar, cane sugar, brown sugar, and brown sugar), lactose, isomerized lactose, maltose, isomaltose, and trehalose; oligosaccharides (trisaccharides or more) such as maltotriose, isomaltotriose, panose, and starch hydrolysates (dextrins); and sugar alcohols such as xylitol, erythritol, sorbitol, mannitol, reduced maltose syrup (maltitol), reduced starch syrup, reduced palatinose, and reduced lactose (lactitol).

These sugars can be used alone or in combination of two or more. Of these, monosaccharides such as glucose and fructose; disaccharides such as sucrose and lactose; and sugar alcohols such as xylitol and erythritol are preferred. Furthermore, when sugars are added mainly to improve ease of administration, sugar alcohols such as erythritol are preferred, and when sugars are added as an energy source in addition to ease of administration, monosaccharides and disaccharides are preferred.

The proportion of sugars may be 1000 parts by mass or less per 100 parts by mass of the first active ingredient, for example, 10 to 1000 parts by mass, preferably 30 to 500 parts by mass, even more preferably 50 to 300 parts by mass, even more preferably 100 to 250 parts by mass, and most preferably 150 to 200 parts by mass.

(Sweetening Agent)
In order to improve the ease of administration, the liquid composition may further contain a sweetener (non-sugar sweetener) in addition to or instead of the above-mentioned saccharides which also function as sweeteners.

Examples of sweeteners include natural sweeteners such as stevia, licorice, and amacha; and artificial sweeteners such as saccharin, saccharin sodium, aspartame, acesulfame potassium, sucralose, and neotame. These sweeteners can be used alone or in combination of two or more. Among these, artificial sweeteners such as acesulfame potassium and sucralose are preferred because they are easy to improve intake. Furthermore, the sweeteners are preferably used in combination with the sugars because they are easy to adjust intake.

The proportion of the sweetener is, for example, 0.01 to 20 parts by mass, preferably 0.1 to 15 parts by mass, even more preferably 0.5 to 10 parts by mass, even more preferably 1 to 8 parts by mass, and most preferably 2 to 5 parts by mass, per 100 parts by mass of the first active ingredient.

(Acidulant)
The liquid composition may further contain an acidulant (acidulant). As the acidulant, an organic acid is usually used. Examples of the organic acid include saturated monocarboxylic acids such as acetic acid; saturated dicarboxylic acids such as malonic acid, succinic acid, succinic anhydride, and glutaric acid; unsaturated polycarboxylic acids such as maleic acid and fumaric acid; hydroxymonocarboxylic acids such as lactic acid; hydroxypolycarboxylic acids such as malic acid, tartaric acid, and citric acid; ascorbic acid, gluconic acid, and the like. These organic acids can be used alone or in combination of two or more.

These organic acids may be in the form of a salt. Examples of the salt include alkali metal salts such as sodium salts, potassium salts, and lithium salts; alkaline earth metal salts such as magnesium salts and calcium salts; and ammonium salts. These salts can be used alone or in combination of two or more.

The acidulants can be used alone or in combination of two or more kinds. Among the acidulants, saturated carboxylic acids such as acetic acid or their salts; hydroxypolycarboxylic acids such as malic acid, tartaric acid, and citric acid or their salts; and ascorbic acid or their salts are preferred, and saturated monocarboxylic acid salts such as sodium acetate; and hydroxypolycarboxylic acid salts such as potassium hydrogen tartrate, sodium hydrogen tartrate, and sodium citrate are more preferred. Furthermore, when the muscle mass maintenance agent of the present invention is a liquid composition, alkali metal salts or alkaline earth metal salts of organic acids such as sodium citrate are preferred from the viewpoint of adjusting the acidity, and alkali metal salts of hydroxypolycarboxylic acids are particularly preferred.

The proportion of the acidulant is, for example, 1 to 50 parts by mass, preferably 3 to 40 parts by mass, even more preferably 5 to 30 parts by mass, even more preferably 8 to 20 parts by mass, and most preferably 10 to 15 parts by mass, per 100 parts by mass of the first active ingredient.

(amino acid)
The liquid composition may further contain an amino acid to act as an energy source or to impart taste masking or functionality.

Examples of amino acids include neutral amino acids such as glycine, alanine, valine, isoleucine, proline, methionine, tryptophan, tyrosine, and glutamine; acidic amino acids such as aspartic acid and glutamic acid; and basic amino acids such as lysine, arginine, and ornithine.

These amino acids may be in the form of a salt. Examples of the salt include alkali metal salts such as sodium salt, potassium salt, and lithium salt; and alkaline earth metal salts such as magnesium salt and calcium salt. These salts can be used alone or in combination of two or more.

These amino acids can be used alone or in combination of two or more. Among these amino acids, neutral amino acids such as alanine are preferred because they function as an energy source and also have excellent taste-masking properties.

The proportion of the amino acid is, for example, 1 to 100 parts by mass, preferably 5 to 80 parts by mass, more preferably 10 to 50 parts by mass, even more preferably 15 to 40 parts by mass, and most preferably 20 to 30 parts by mass, per 100 parts by mass of the first active ingredient.

(Fragrance)
The liquid composition may further contain a flavoring to improve the ease of administration. The flavoring may be a natural flavoring or a synthetic flavoring.

Examples of natural flavors include fruit essences or oils such as strawberry, blueberry, apple, plum, lime vanilla, and pepper; peel essences or oils such as orange, white grape, grapefruit, and lemon; bark essences or oils such as cinnamon; bark powders such as cinnamon powder; root vegetable essences or oils such as ginger; root vegetable powders such as ginger powder; seed powders such as vanilla beans and cocoa powder; leafy essences or oils such as peppermint, spearmint, and rosemary; leafy powders such as peppermint powder; and flower essences or oils such as jasmine, lavender, rose, rosemary, and hyacinth.

Examples of synthetic fragrances include benzyl acetate, linalyl acetate, citral, citronellal, citronellol, cis-jasmine, cis-3-hexenol, and menthol.

These fragrances can be used alone or in combination depending on the application.

The proportion of the fragrance is, for example, 1 to 50 parts by mass, preferably 3 to 40 parts by mass, even more preferably 5 to 30 parts by mass, even more preferably 8 to 20 parts by mass, and most preferably 10 to 15 parts by mass, per 100 parts by mass of the first active ingredient.

(Oils and fats)
The liquid composition may further contain fats and oils. The fats and oils are not particularly limited as long as they are edible fats and oils, and may be any of vegetable fats and oils, animal fats and oils, and processed fats and oils.

Examples of vegetable oils include soybean oil, cottonseed oil, linseed oil, castor oil, safflower oil, rice oil, germ rice oil, corn oil, sesame oil, sunflower oil, rice sugar oil, rapeseed oil such as canola oil, vegetable oils (salad oil, white refined oil) such as peanut oil, palm kernel oil, olive oil, grapeseed oil; palm oils such as coconut oil and carotino oil, vegetable oils (vegetable fats) such as cocoa butter, etc.

Examples of animal fats and oils include butter, beef tallow, milk fat, lard, and fish oil.

Examples of processed fats and oils include margarine, shortening, coconut milk, and medium chain triglycerides (MCT) such as _C8-10 fatty acid triglycerides.

These oils and fats may be fractionated oils, interesterified oils, or hydrogenated oils. These oils and fats may be used alone or in combination of two or more.

The proportion of oils and fats may be 100 parts by mass or less per 100 parts by mass of the first active ingredient, preferably 30 parts by mass or less, even more preferably 10 parts by mass or less, even more preferably 5 parts by mass or less, and most preferably 1 part by mass or less.

(protein)
The liquid composition may further contain a protein, such as whey protein, casein protein, soy protein, pea protein, wheat protein, egg protein, rice protein, etc. These proteins may be used alone or in combination.

The proportion of protein may be, for example, 3000 parts by mass or less, for example 1000 parts by mass or less (e.g., 1 to 1000 parts by mass), preferably 800 parts by mass or less (e.g., 10 to 800 parts by mass), and more preferably 500 parts by mass or less (e.g., 100 to 500 parts by mass) per 100 parts by mass of the first active ingredient.

(3HB oligomer)
The liquid composition may further contain a 3HB oligomer. The average degree of polymerization of the 3HB oligomer may be 2 or more, for example, 2 to 100, preferably 2 to 10, more preferably 2 to 5, more preferably 2 to 4.5, and most preferably 2 to 4. The 3HB oligomer may be an oligomer that is inevitably mixed in during the production process of the composition. The proportion of the 3HB oligomer may be 10 parts by mass or less, preferably 5 parts by mass or less, more preferably 3 parts by mass or less, and most preferably 1 part by mass or less, relative to 100 parts by mass of the first active ingredient.

(Conventional Additives)
The liquid composition may further contain other components, such as additives commonly used in the fields of health foods (functional foods) and pharmaceuticals.

Examples of conventional additives include ketone esters (e.g., ketone esters of 3HB and 1,3-butanediol), vitamins (e.g., vitamin A, vitamin D, vitamin E, vitamin B ₁ , vitamin B _{2 , vitamin B 6 , vitamin C, vitamin B 1 , vitamin B 2 , vitamin B 3 , vitamin B 4 , vitamin B 5 , vitamin B 6 , vitamin B 7 , vitamin B 8 , vitamin B 9 , vitamin B 10 , vitamin B 11 , vitamin B 12 , vitamin B 13} , vitamin B 14 , vitamin B 15 , vitamin B 16 , vitamin B 17 , vitamin B 18 , vitamin B 19 , vitamin B 20 , vitamin B 21 , vitamin B 22 , vitamin B ₂₃ , vitamin B 24 , vitamin B 25 , vitamin B 26 , vitamin B 27 ₁₂ , etc.), dietary fiber (e.g., wheat bran, corn bran, oat bran, corn fiber, soybean dietary fiber, beet fiber, crystalline cellulose, agar, chitosan, chitin, hemicellulose, lignin, glucan, etc.), coloring agents (edible dyes such as food yellow No. 5, food red No. 2, and food blue No. 2; edible lake dye; red iron oxide, etc.), seasonings, leavening agents or foaming agents (such as baking soda), fluidizing agents (e.g., talc, light anhydrous silicic acid, hydrated silicon dioxide, magnesium aluminometasilicate, etc.), thickening stabilizers or water-retaining emulsion stabilizers (thickening polysaccharides such as pectin and cellulose), pH adjusters (inorganic salts such as baking soda, trisodium phosphate, and potassium dihydrogen phosphate), preservatives (antiseptics, antibacterial agents, etc.), antifoaming agents, surfactants, emulsifiers, antioxidants, light stabilizers, brewing agents, etc. These additives can be used alone or in combination of two or more.

The proportion (total proportion) of conventional additives can be selected from the range of about 0.01 to 50 parts by mass per 100 parts by mass of the first active ingredient, for example, 0.01 to 10 parts by mass, preferably 0.1 to 5 parts by mass, and more preferably 0.2 to 3 parts by mass.

(water)
The liquid composition preferably further contains water. The proportion of water is, for example, 100 to 10,000 parts by mass, preferably 500 to 8,000 parts by mass, more preferably 1,000 to 5,000 parts by mass, and even more preferably 2,000 to 4,000 parts by mass, relative to 100 parts by mass of the first active ingredient.

(Characteristics of the liquid composition)
The pH of the liquid composition at a temperature of 25° C. may be 7 or less, for example, from 3 to 7, and from the viewpoint of productivity, may be 4.5 or less, preferably from 3 to 4.5, and more preferably from 3 to 4. If the pH of the liquid composition is too low, there is a risk that the acidity will be too strong.

In this specification and claims, pH can be measured by conventional methods, for example, using a commercially available pH meter.

The acidity of the liquid composition (converted to the mass of citric acid) is, for example, 1 to 20% by mass, preferably 1 to 10% by mass, and more preferably 1 to 5% by mass.

[Solid Composition]
When the muscle mass maintenance agent of the present invention is a solid composition, examples of the solid composition (solid agent) include powders, fine granules, granules, pills, tablets, flakes, cakes, gummies, nougat (candy), films, capsules, etc. The solid composition may be a sustained release formulation, an orally disintegrating tablet, etc.

(First active ingredient)
In the solid composition, the first active ingredient (R)3HB (salt) may be in the form of a salt. The salt of (R)3HB in the form of a salt may be granular (or powdered). Examples of the shape of the granular 3HB salt include spherical, ellipsoidal, polyhedral, plate-like, fibrous, and irregular shapes. Among these shapes, spherical shapes such as approximately spherical are preferred.

The average particle size (effective diameter) of the granular (R) 3HB salt is, for example, 0.1 to 1000 μm, preferably 0.3 to 500 μm, more preferably 0.5 to 100 μm, and even more preferably 1 to 50 μm. If the average particle size of the granular 3HB salt is too small, there is a risk that handling properties will decrease, and conversely, if it is too large, there is a risk that productivity will decrease.

In this specification and claims, the average particle size (effective diameter) is the median particle size ( _D50 : the measured value of the particle size at 50% of the cumulative value in the particle size distribution), and can be measured by wet measurement on a volume basis in accordance with the laser diffraction scattering method (ISO133201 and ISO9276-1) using a particle size distribution measuring device (manufactured by Nikkiso Co., Ltd., device name: Microtrac MT3300ExII).

The proportion of the first active ingredient, calculated as the free form of (R)3HB, in the solid composition can be selected from a range of about 0.1 to 99% by mass, for example 1 to 90% by mass, preferably 3 to 80% by mass, further preferably 5 to 70% by mass, more preferably 8 to 50% by mass, and most preferably 10 to 30% by mass. If the proportion of the first active ingredient is too low, there is a risk that the effect of maintaining muscle mass will be reduced, and conversely, if it is too high, there is a risk that palatability will be reduced.

(Second Active Ingredient)
The solid composition may further contain a second active ingredient. Examples of the second active ingredient include the second active ingredients exemplified in the section on the liquid composition. The second active ingredient may be used alone or in combination of two or more kinds.

The proportion of the second active ingredient may be 100 parts by mass or less per 100 parts by mass of the first active ingredient, and is preferably 50 parts by mass or less (e.g., 0.1 to 50 parts by mass), more preferably 30 parts by mass or less, and even more preferably 10 parts by mass or less.

(Excipients)
The solid composition may further contain an excipient. The excipient may be an organic excipient and/or an inorganic excipient. Examples of organic excipients include crystalline cellulose; sugar alcohols such as D-mannitol, sorbitol, xylitol, maltitol, erythritol, lactitol, and reducing starch syrup; sugars (monosaccharides or disaccharides) such as lactose (including anhydrous lactose and lactose hydrate), sucrose, glucose, fructose, and maltose; and starches (or polysaccharides) such as oligosaccharides, corn starch, potato starch, wheat starch, rice starch, partially pregelatinized starch, and pregelatinized starch. Examples of inorganic excipients include light anhydrous silicic acid, anhydrous calcium hydrogen phosphate, anhydrous calcium phosphate, calcium silicate, calcium carbonate, and calcium sulfate. These excipients may be used alone or in combination of two or more. Among these, crystalline cellulose, sugars (sugar alcohols, monosaccharides and disaccharides), light anhydrous silicic acid, anhydrous calcium hydrogen phosphate, etc. are preferred.

The proportion of the excipient in the solid composition is 1 to 90% by mass, preferably 2 to 80% by mass, more preferably 10 to 70% by mass, and particularly preferably 20 to 65% by mass.

(Binder)
The solid composition may further contain a binder. Examples of binders include cellulose ethers such as hydroxypropyl cellulose (HPC), hydroxypropyl methyl cellulose (HPMC), methyl cellulose (MC), sodium carboxymethyl cellulose (CMC-Na), and carmellose sodium; soluble starches such as carboxymethyl starch, pregelatinized starch, and partially pregelatinized starch; polysaccharides such as dextrin (including resistant dextrin), pullulan, gum arabic, agar, gelatin, tragacanth, and sodium alginate; and synthetic polymers such as povidone (polyvinylpyrrolidone), copolyvidone, carboxyvinyl polymer, polyacrylic acid polymer, polylactic acid, polyvinyl alcohol, and polyethylene glycol. These binders can be used alone or in combination of two or more. Among these, HPMC, carmellose sodium, and povidone are preferred.

The proportion of the binder in the solid composition is 0.5 to 20% by mass, preferably 1 to 15% by mass, and more preferably 2 to 10% by mass.

(Disintegrant)
The solid composition may further contain a disintegrant. Examples of disintegrants include cornstarch, hydroxypropyl starch, sodium carboxymethyl starch, carmellose, carmellose sodium, carmellose calcium, croscarmellose sodium, crospovidone, low-substituted hydroxypropyl cellulose, and crystalline cellulose. These disintegrants can be used alone or in combination of two or more. Among these, sodium carboxymethyl starch, carmellose calcium, croscarmellose sodium, and crospovidone are preferred.

The proportion of the disintegrant in the solid composition is 1 to 20% by mass, preferably 2 to 15% by mass.

(lubricant)
The solid composition may further contain a lubricant. Examples of the lubricant include stearic acid, magnesium stearate, calcium stearate, sucrose fatty acid ester, talc, waxes, sodium lauryl sulfate, magnesium lauryl sulfate, and macrogol 6000. Among these, magnesium stearate is preferred. In the solid composition (particularly, tablets), the proportion of the lubricant is 0.1 to 5% by mass, preferably 0.2 to 3% by mass, in the solid composition.

(Coating Base)
The solid composition may further contain a coating base, such as a sugar coating base, a water-soluble coating base, an enteric coating base, or a sustained-release coating base.

Examples of sugar-coating bases include monosaccharides such as sucrose and sugar alcohols.

Examples of water-soluble coating bases include cellulose ethers such as hydroxypropyl cellulose, hydroxypropylmethyl cellulose, hydroxyethyl cellulose, and methylhydroxyethyl cellulose; synthetic polymers such as polyacrylic acid polymers and povidone; and polysaccharides such as pullulan.

Examples of enteric coating bases include cellulose polymers such as hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate, carboxymethylethylcellulose, and acetylcellulose phthalate; methacrylic acid copolymers; and natural products such as shellac.

Examples of sustained-release coating bases include cellulose-based polymers such as ethyl cellulose; and acrylic acid-based polymers such as aminoalkyl methacrylate copolymer RS and ethyl acrylate-methyl methacrylate copolymer suspensions.

Additives for coating bases include, for example, light-blocking agents such as titanium oxide; flow agents such as talc; colorants; plasticizers; and organic acids.

These coating bases and additives can be used alone or in combination.

The proportion of the coating base in the solid composition is 0.1 to 80% by mass, preferably 1 to 50% by mass, and more preferably 3 to 30% by mass.

(Other ingredients)
The solid composition may further contain sugars, sweeteners, acidulants, amino acids, flavors, oils and fats, proteins, 3HB oligomers, and conventional additives exemplified in the section on the liquid composition. The proportions are also the same as in the liquid composition.

[Semi-solid composition]
When the muscle mass maintenance agent of the present invention is a semi-solid composition, examples of the semi-solid composition (semi-solid preparation) include ointments (oil-soluble ointments, water-soluble ointments), gels (suppositories, patches, etc.), creams, slurries, pastes, jellies, puddings, mousses, sherbets, etc. The semi-solid composition may be filled or packaged in a container by a conventional method, or may be filled or packaged in a container in a state of being sterilized or sterilized by a method such as heat treatment.

(First active ingredient)
In the semi-solid composition, the first active ingredient (R)3HB (salt) may be in the form of a salt. The salt of (R)3HB in the form of a salt may be granular (or powdered). Examples of the shape of the granular 3HB salt include spherical, ellipsoidal, polyhedral, plate-like, fibrous, and irregular shapes. Among these shapes, spherical shapes such as approximately spherical are preferred.

The average particle size (effective diameter) of the granular (R) 3HB salt is, for example, 0.1 to 1000 μm, preferably 0.3 to 500 μm, more preferably 0.5 to 100 μm, and even more preferably 1 to 50 μm. If the average particle size of the granular 3HB salt is too small, there is a risk that handling properties will decrease, and conversely, if it is too large, there is a risk that productivity will decrease.

The proportion of the first active ingredient, calculated as the free form of (R)3HB, in the semi-solid composition can be selected from a range of about 0.1 to 99% by mass, for example 1 to 90% by mass, preferably 3 to 80% by mass, further preferably 5 to 70% by mass, more preferably 8 to 50% by mass, and most preferably 10 to 30% by mass. If the proportion of the first active ingredient is too low, the effect of maintaining muscle mass may be reduced, and conversely, if it is too high, palatability may be reduced.

(Second Active Ingredient)
The semi-solid composition may further contain a second active ingredient. Examples of the second active ingredient include the active ingredients exemplified in the liquid composition section. The second active ingredient may be used alone or in combination of two or more kinds.

The proportion of the second active ingredient may be 100 parts by mass or less per 100 parts by mass of the first active ingredient, and is preferably 50 parts by mass or less (e.g., 0.1 to 50 parts by mass), more preferably 30 parts by mass or less, and even more preferably 10 parts by mass or less.

(Liquid base)
The semi-solid composition may further contain a liquid base. The liquid base includes an aqueous base and an oil base.

Examples of aqueous bases include water, lower aliphatic alcohols (eg, C _1-4 alkyl alcohols such as ethanol and isopropanol), polyethylene glycols (Macrogol 400, Macrogol 4000, etc.), and glycerin.

Examples of oily bases include fats and oils (vegetable oils such as cacao butter, castor oil, and olive oil, and animal oils such as lard), waxes (beeswax, etc.), hydrocarbons (vaseline, squalane, liquid paraffin, plastibase, etc.), higher alcohols (stearyl alcohol, behenyl alcohol, etc.), higher fatty acid esters (isopropyl myristate, cetyl ethylhexanoate, etc.), and silicone oils.

The liquid base may be an emulsion base (W/O type, O/W type) or a gel, and the gel may be a gelled hydrogel base, a lyogel (FAPG) base containing a fatty alcohol and propylene glycol, etc.

The proportion of the liquid base in the composition is 10 to 95% by mass, preferably 30 to 90% by mass, and more preferably 50 to 85% by mass.

(Gelling Agent)
The semi-solid composition may further contain a gelling agent. Examples of the gelling agent include water-soluble or water-swellable polysaccharides such as dextran, pullulan, agar, pectin, alginic acid, sodium alginate, carrageenan, galactomannan, glucomannan, curdlan, gum arabic, tragacanth gum, gellan gum, karaya gum, guar gum, xanthan gum, locust bean gum, tara gum, tamarind seed gum, and psyllium seed gum; water-soluble cellulose ethers such as methylcellulose, hydroxypropylcellulose, methylhydroxypropylcellulose, hydroxyethylcellulose, and sodium carboxymethylcellulose; Examples of gelling agents include water-soluble synthetic polymers such as carboxyvinyl polymers, polyacrylic acid or sodium polyacrylate, polyethylene glycol, polyoxyethylene-polyoxypropylene copolymers, polyacrylamide, and poly(N,N-dimethylacrylamide); gluten (a complex of gliadin and glutenin) derived from wheat flour; fish protein (myofibrillar protein) used to form the legs of fish paste products; proteins such as collagen, casein, albumin, gelatin, and royal jelly; and inorganic water-soluble polymers such as bentonite, organically modified bentonite, magnesium aluminum silicate, and silicic anhydride. These gelling agents can be used alone or in combination of two or more.

The proportion of the gelling agent is, for example, 0.01 to 100 parts by mass, preferably 0.03 to 10 parts by mass, and more preferably 0.05 to 1 part by mass, per 100 parts by mass of the liquid base.

(Other ingredients)
The semi-solid composition may further contain sugars, sweeteners, acidulants, amino acids, flavors, oils and fats, proteins, 3HB oligomers, and conventional additives exemplified in the section on liquid compositions. The proportions are also the same as those of the liquid compositions.

[Method of manufacturing muscle mass maintenance agent]
The muscle mass maintenance agent of the present invention can be produced by a conventional production method depending on the form. When the muscle mass maintenance agent of the present invention is a liquid composition, if (R)3HB (salt) is in the form of an acid, the acidity is too strong, so it is preferable to produce the liquid composition by mixing (R)3HB with an alkaline component. In this production method, the acidity can be suppressed by increasing the pH of the liquid composition or converting (R)3HB into a salt form by using an alkaline component as a raw material. The alkaline component is not particularly limited as long as it is an alkaline component commonly used in foods and medicines, but alkali metal compounds, alkaline earth metal compounds, basic amino acids, etc. are commonly used, and alkali metal compounds or alkaline earth metal compounds are preferred as acidulants or pH adjusters, and alkali metal compounds are particularly preferred as acidulants.

When the muscle mass maintenance agent of the present invention is a semi-solid composition or a solid composition, it may be manufactured through a conventional method, such as granulation, mixing, tableting, sterilization, etc., depending on the form (dosage form) of the composition.

The present invention will be described in more detail below based on examples, but the present invention is not limited to these examples. The raw materials and evaluation methods used are as follows.

Example 1, Reference Example 1 and Comparative Example 1
[Comparison method between the presence and absence of a skeletal muscle mass reduction inhibitor]
Eight-week-old male C57BL/6J mice were intraperitoneally administered 125 mg/kg of streptozotocin (STZ) for two consecutive days to induce type 1 diabetes. Three days after STZ administration, the body weight and blood glucose level were measured, and the mice were divided into groups so that their blood glucose levels were similar.

In Comparative Example 1, DM mice administered STZ were used as the diabetic group (DM group), and were allowed to drink 0.9% by mass NaCl aqueous solution ad libitum and were kept on a normal diet (n=6).

On the other hand, in Example 1, DM mice administered with STZ were used as the 3HB-administered group (KB group), and were allowed to drink an aqueous solution containing 2.3% by mass of 3HB (equivalent to 0.9% by mass NaCl) ad libitum and were kept on a normal diet. Crystalline (R) 3HB sodium (OKETOA (registered trademark) D-BHB, manufactured by Osaka Gas Chemicals Co., Ltd.) was used as the 3HB (n=6).

In addition, in Reference Example 1, the mice in the control group that were not administered STZ were allowed to drink 0.9% by mass NaCl aqueous solution ad libitum and were kept on a normal diet (n=5).

[Evaluation method]
(Skeletal Muscle Mass)
After the three groups, DM group, KB group and control group, were kept for 4 weeks, blood was collected from the abdominal vena cava under anesthesia, and skeletal muscle was excised from each tissue to measure the skeletal muscle mass.

(IGF1, Col1a1 and Col3a1)
The collected blood was mixed with heparin (Mochida Pharmaceutical Co., Ltd.), centrifuged at 3,000g for 5 minutes at 4°C, and the supernatant was stored at -80°C. After thawing and centrifugation, the supernatant was subjected to analysis of blood biochemical concentrations. For the analysis, an automatic serum analyzer (Beckman Coulter) was used to analyze the expression levels of mRNA for IGF1, a growth hormone that causes muscle hypertrophy like insulin, and Col1a1 and Col3a1, proteins that can prevent collagen loss. The method for analyzing blood biochemical concentrations is as follows.

Total RNA was prepared using RNeasy Lipid Tissue Mini kit (Qiagen). A part of the excised gastrocnemius muscle was homogenized in QIAZOL reagent (Qiagen) and incubated at room temperature for 5 minutes, after which chloroform was added. The mixture was then stirred in a vortex mixer for 20 seconds, incubated at room temperature for 3 minutes, and centrifuged at 4°C and 12,000g for 15 minutes. The supernatant was mixed with an equal amount of 70% ethanol by volume, dispensed into the kit's column, and centrifuged at 4°C and 12,000 rpm for 5 seconds to remove the flow-through compartment. 500 μL of RW buffer (Qiagen) was poured into the column, and the column was washed by centrifuging at 4°C and 8,000g for 5 seconds. Next, the column was washed in the same manner with 500 μL of RPE Buffer (Qiagen) previously mixed with ethanol, and washing with this RPE Buffer was repeated once more. Finally, the empty column was centrifuged at 4 ° C. and 8,000 g for 2 minutes to complete column washing. The washed column was set in a 1.5 mL tube, RNase free water was added in 50 μL portions, and the column was centrifuged at 4 ° C. and 8,000 g for 1 minute to elute the RNA, and the RNA concentration was measured using Nano drop (registered trademark) 2000 (Thermo Fisher Scientific Co., Ltd.). Next, 5 × First-Strand buffer, 10 mM dNTP mix, Random Primer, and ReverTra Ace RT were mixed, and reverse transcription reaction was performed. After incubation at 30°C for 10 minutes, the mixture was incubated at 42°C for 60 minutes, and then heat-treated at 95°C for 10 minutes to stop the reaction. The cDNA after the reverse transcription reaction was diluted with dH ₂ O and then subjected to gene expression analysis by the qPCR method. THUNDERBIRD (registered trademark) SYBR (registered trademark) qPCR Mix (manufactured by Toyobo Co., Ltd.) was used for the qPCR reaction. A primer set containing both sense and antisense and having a final concentration of 5 μM was used as the primer. The template cDNA was added to the reaction solution, set in a StepOne real-time PCR system (manufactured by Applied biosystems), and after thermal denaturation at 95°C for 2 minutes, 40 cycles were performed with 95°C for 15 seconds → 60°C for 1 minute as one cycle, and the calculated Ct value was used for analysis. The L19 gene was used for correction of gene expression analysis.

(muscle fiber area)
The excised tibialis anterior muscle was frozen in isopentane cooled with liquid nitrogen, and frozen sections were prepared using a cryostat (Leica) and stored at -80°C. After thawing, the thin sections were dried and stained with Hematoxylin Eosin (HE). After mounting on a glass plate, images were taken using an upright microscope (Olympus BX53, Olympus Corporation). The cross-sectional area of 120 muscle fibers per mouse was measured using Nikon Elements imaging software (Nikon Corporation).

[Evaluation results]
The results of comparing skeletal muscle mass for Example 1, Reference Example 1, and Comparative Example 1 are shown in Figure 1. As is clear from Figure 1, weight loss was suppressed by ingestion of the skeletal muscle mass loss inhibitor in the TA (tibialis anterior muscle), EDL (extensor digitorum longus muscle), gastro (gastrocnemius muscle), soleus (soleus muscle), and quads (quadriceps muscle).

Figure 1 shows the most clear effect of maintaining skeletal muscle mass, particularly in the quadriceps (quads), with Example 1 showing a significant decrease in skeletal muscle mass compared to Reference Example 1 (p<0.0001), and Comparative Example 1 showing a significant decrease in skeletal muscle mass compared to Reference Example 1 (p<0.0001), but Example 1 showing a significant increase in skeletal muscle mass compared to Comparative Example 1 (p<0.05).

In Figures 1 to 5, "*" means a p-value less than 0.05 (p<0.05), "**" means a p-value less than 0.01 (p<0.01), "***" means a p-value less than 0.001 (p<0.001), "****" means a p-value less than 0.0001 (p<0.0001), and "ns" means not significant.

The average quadriceps mass (quads) in Reference Example 1 was 0.221 g with a standard deviation of 0.0201 g, the average quadriceps mass in Comparative Example 1 was 0.100 g with a standard deviation of 0.0360 g, and the average quadriceps mass in Example 1 was 0.1343 g with a standard deviation of 0.0522 g.

Figure 1 also shows the effect of maintaining skeletal muscle mass in the gastrocnemius muscle, with Example 1 showing a significant decrease in skeletal muscle mass compared to Reference Example 1 (p<0.0001), and Comparative Example 1 showing a significant decrease in skeletal muscle mass compared to Reference Example 1 (p<0.0001), but Example 1 showing an increase in skeletal muscle mass compared to Comparative Example 1.

The average mass of the gastrocnemius muscle in Reference Example 1 was 0.153 g with a standard deviation of 0.0123 g, the average mass of the quadriceps muscle in Comparative Example 1 was 0.0758 g with a standard deviation of 0.0218 g, and the average mass of the quadriceps muscle in Example 1 was 0.0987 g with a standard deviation of 0.0334 g.

Figure 2 shows the results of comparing the average muscle fiber area of the tibialis anterior (TA) muscle for Example 1, Reference Example 1, and Comparative Example 1. As is clear from Figure 2, it was confirmed that the ingestion of a skeletal muscle mass reducer inhibited the reduction of muscle fibers.

In Figure 2, the area of muscle fibers was significantly reduced in Comparative Example 1 compared to Reference Example 1 (p<0.001), but the area of muscle fibers was significantly increased in Example 1 compared to Comparative Example 1 (p<0.05).

The average value of the average area of the muscle fibers of the tibialis anterior (TA) in Reference Example 1 was 2377.4 μm ² with a standard deviation of 58.80 μm ² , the average value of the average area of the muscle fibers of the tibialis anterior in Comparative Example 1 was 1337.1 μm ² with a standard deviation of 121.7 μm ² , and the average value of the average area of the muscle fibers of the tibialis anterior in Example 1 was 2105.6 μm ² with a standard deviation of 625.1 μm ² .

The results of comparing the expression levels of IGF1, Col1a1, and Col3a1 for Example 1, Reference Example 1, and Comparative Example 1 are shown in Figures 3 to 5.

As is clear from Figure 3, the expression level of IGF1, a growth hormone that, like insulin, causes muscle hypertrophy, was suppressed by taking an inhibitor of skeletal muscle mass loss, which is presumably also involved in the suppression of skeletal muscle weight.

In Figure 3, Comparative Example 1 significantly reduced the expression level of IGF1 compared to Reference Example 1 (p<0.001), while Example 1 significantly increased the expression level of IGF1 compared to Comparative Example 1 (p<0.01).

The average value of the relative mRNA expression level of IGF1 in Reference Example 1 was 0.808, with a standard deviation of 0.199. The average value of the relative mRNA expression level of IGF1 in Comparative Example 1 was 0.233, with a standard deviation of 0.078. The average value of the relative mRNA expression level of IGF1 in Example 1 was 0.512, with a standard deviation of 0.149.

As is clear from Figures 4 and 5, the decrease in the production of Col1a1 and Col3a1, which are proteins that can prevent collagen loss, was suppressed by taking an inhibitor of skeletal muscle mass loss. It can be assumed that the suppression of muscle protein loss is also involved in the suppression of skeletal muscle weight.

In Figure 4, the amount of Col1a1 produced in Comparative Example 1 was significantly reduced compared to Reference Example 1 (p<0.01), while Example 1 significantly increased the amount of Col1a1 expression compared to Comparative Example 1 (p<0.05). In Figure 5, the amount of Col3a1 produced in Comparative Example 1 was significantly reduced compared to Reference Example 1 (p<0.01), while Example 1 significantly increased the amount of Col3a1 expression compared to Comparative Example 1 (p<0.05).

The average value of the relative mRNA expression level of Col1a1 in Reference Example 1 was 1.079, with a standard deviation of 0.515. The average value of the relative mRNA expression level of Col1a1 in Comparative Example 1 was 0.080, with a standard deviation of 0.058. The average value of the relative mRNA expression level of Col1a1 in Example 1 was 0.617, with a standard deviation of 0.366.

The average value of the relative mRNA expression level of Col3a1 in Reference Example 1 was 0.827, with a standard deviation of 0.321. The average value of the relative mRNA expression level of Col3a1 in Comparative Example 1 was 0.053, with a standard deviation of 0.040. The average value of the relative mRNA expression level of Col3a1 in Example 1 was 0.358, with a standard deviation of 0.215.

These results demonstrate that even if sarcopenia develops due to diabetes, loss of skeletal muscle weight can be suppressed by taking the skeletal muscle mass reduction inhibitor of the present invention without dietary or exercise therapy.

The muscle mass maintenance agent of the present invention is used in the field of functional foods and medicines to inhibit loss of muscle mass, and can be used as a functional food (such as a nutritional supplement or supplement) for healthy individuals, elderly individuals, and sick individuals, and is particularly useful as a functional food or medicine for elderly individuals or sick individuals (particularly sarcopenia patients and diabetes patients) for whom dietary therapy and exercise therapy are difficult. In particular, the muscle mass maintenance agent of the present invention is suitable as a functional food or medicine for preventing and/or treating sarcopenia, and is particularly suitable as a functional food or medicine for preventing and/or treating sarcopenia caused by diabetes and/or aging.

Claims (15)

A muscle mass maintenance agent for preventing muscle mass loss, the agent containing R-3-hydroxybutyric acid and/or its salt as an active ingredient. The muscle mass maintenance agent according to claim 1, wherein the active ingredient comprises a salt of R-3-hydroxybutyric acid. The muscle mass maintenance agent according to claim 1 or 2, which is capable of preventing and/or treating sarcopenia. The muscle mass maintenance agent according to claim 1 or 2, which is capable of suppressing loss of muscle mass without dietary therapy and/or exercise therapy. The muscle mass maintenance agent according to claim 1 or 2, which is capable of suppressing the loss of muscle mass in elderly people and/or diabetic patients. The muscle mass maintenance agent according to claim 1 or 2, wherein the muscle mass is skeletal muscle mass. The muscle mass maintenance agent according to claim 1 or 2, wherein the skeletal muscle is a skeletal muscle of the lower limbs. The muscle mass maintenance agent according to claim 1 or 2, which is capable of suppressing a decrease in the expression level of insulin-like growth factor and/or collagen genes. The muscle mass maintenance agent according to claim 1 or 2, which is a liquid composition, a semi-solid composition or a solid composition. The muscle mass maintenance agent according to claim 1 or 2, which is a physiologically active composition or pharmaceutical composition for oral intake. A drug for preventing and/or treating sarcopenia, the drug containing R-3-hydroxybutyric acid and/or its salt as an active ingredient. An inhibitor for inhibiting a decrease in skeletal muscle mass and/or skeletal muscle fiber mass, the inhibitor containing R-3-hydroxybutyric acid and/or a salt thereof as an active ingredient. A muscle mass promoter that increases skeletal muscle mass and/or skeletal muscle fiber mass in at least one patient selected from the group consisting of elderly people, diabetic patients, and sarcopenia patients, the muscle mass promoter comprising R-3-hydroxybutyric acid and/or a salt thereof as an active ingredient. An inhibitor for suppressing a decrease in the expression level of insulin-like growth factor and/or collagen genes, the inhibitor containing R-3-hydroxybutyric acid and/or its salt as an active ingredient. A promoter for increasing the expression level of insulin-like growth factor and/or collagen gene in at least one patient selected from the group consisting of elderly people, diabetic patients, and sarcopenic patients, the promoter comprising R-3-hydroxybutyric acid and/or a salt thereof as an active ingredient.

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