Pharmaceutical composition for prevention or treatment of sarcopenia in companion animals

Abstract

The present invention relates to a pharmaceutical composition for the prevention or treatment of sarcopenia in companion animals. The pharmaceutical composition inhibits an increase in the production and mRNA expression of myostatin protein, which directly affects muscle loss and reduced muscle strength, and thus can exhibit the effect of more fundamentally preventing or treating sarcopenia in companion animals.

Description

Pharmaceutical composition for preventing or treating sarcopenia in companion animals

[0001] The present invention relates to a pharmaceutical composition for preventing or treating sarcopenia in companion animals.

[0002] The concept of sarcopenia began in 1989 when Irwin Rosenberg introduced the term 'sarcopenia'. Its origin is a combination of the Greek words "sarco" meaning muscle and "penia" meaning decreased. Sarcopenia refers to a decline in muscle strength due to a decrease in muscle mass associated with aging. Here, "muscle" refers to skeletal muscle and has no relation to smooth muscle. In other words, sarcopenia refers to the loss of skeletal muscle mass mainly distributed in the limbs, and is distinguished from cachexia, a state of significant muscle loss that occurs in the terminal stages of malignant tumors, muscle wasting due to acute diseases such as influenza, or primary muscle disease.

[0003] There are three main treatments for sarcopenia. The first is exercise. Exercise has been reported to increase skeletal muscle protein synthesis in the short term and enhance muscle strength and mobility in older adults. However, it is not suitable for long-term treatment. The second option is pharmacological treatment. Testosterone or anabolic steroids can be used, but these can induce masculinization in women and cause side effects such as prostate symptoms in men. Other approved treatments include dehydroepiandrosterone (DHEA) and growth hormone, although studies have shown that these can be effective in areas including Selective Androgen Receptor Modulators (SARMs). Dietary therapy is also considered a treatment option, but nutritional assessments have shown that malnutrition and modern dietary habits are inadequate for maintaining adequate total body mass.

[0004] Myostatin is a polypeptide growth factor belonging to the TGF-β superfamily. TGF-β has numerous isoforms, and these are known to be involved in cell proliferation, apoptosis, differentiation, and bone formation and maintenance (Massague & Chen, 2000). Myostatin, a member of growth differentiation factor (GDF) type 8, is involved in tissue growth and development and acts by activating the Smad signaling pathway. Furthermore, it has been reported to influence bone formation and regeneration by inhibiting cell cycle and proliferation of progenitor cells via the p21 gene. Myostatin is primarily produced in skeletal muscle cells and acts autocrine to cause muscle loss and decreased strength. It is known to inhibit protein synthesis and cell proliferation in myoblasts by suppressing the expression of IGF-1 and Follistatin, which are involved in muscle hypertrophy.

[0005] Meanwhile, with the rise of single-person households and nuclear families, the number of pet-owning households is rapidly increasing. Advances in medical technology and improvements in living environments are also leading to an increase in the life expectancy of pets. Consequently, sarcopenia in pets, a condition characterized by a decline in muscle mass and function, is a chronic and life-threatening condition that causes inconveniences in daily life. However, there is currently no effective treatment.

[0006] Against this backdrop, the inventors of the present invention conducted research to discover a substance capable of treating sarcopenia in companion animals by suppressing myostatin expression and promoter activity, which cause muscle loss and muscle strength loss, and completed the present invention.

[0007] One object of the present invention is to provide a pharmaceutical composition for preventing or treating sarcopenia in animals, containing D-Leu-OMe-HCl as an active ingredient.

[0008] Another object of the present invention is to provide an animal feed composition for preventing or improving sarcopenia containing D-Leu-OMe-HCl as an active ingredient.

[0009] One aspect of the present invention provides a pharmaceutical composition for preventing or treating sarcopenia in animals, containing D-Leu-OMe-HCl as an active ingredient.

[0010] The above D-Leu-OMe-HCl may correspond to a form in which D-Leu-Ome is combined with or mixed with HCl.

[0011] According to one specific example of the present invention, the composition may inhibit myostatin mRNA or protein expression.

[0012] According to one specific example of the present invention, the composition may reduce myostatin promoter activity.

[0013] According to one specific example of the present invention, the sarcopenia may be due to aging.

[0014] The pharmaceutical composition of the present invention can be manufactured by a method known in the pharmaceutical field for use as a medicament, and can be manufactured and used in the form of a powder, granule, tablet, capsule, or injection by mixing with a pharmaceutically acceptable carrier, excipient, diluent, stabilizer, preservative, etc. In addition, the composition can be manufactured as a sustained-release preparation by including a base used for sustained-release purposes in addition to the active ingredient so that the active ingredient is slowly released.

[0015] Pharmaceutically acceptable carriers may further include, for example, carriers for oral administration or carriers for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, etc. In addition, various drug delivery materials used for oral administration of peptide preparations may be included. In addition, carriers for parenteral administration may include water, suitable oils, saline solution, aqueous glucose, and glycols, etc., and may further include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite, or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol. In addition to the above components, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweetening agents, flavoring agents, emulsifiers, suspending agents, etc.

[0016] Examples of the diluent include, but are not limited to, non-aqueous solvents such as propylene glycol, polyethylene glycol, vegetable oils such as olive oil and peanut oil, and aqueous solvents such as saline (preferably 0.8% saline), water containing a buffer medium (preferably 0.05 M phosphate buffer).

[0017] The above excipients include, but are not limited to, starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, anhydrous skim milk, glycerol, propylene, glycol, water, ethanol, and the like.

[0018] The above stabilizers include, but are not limited to, carbohydrates such as sorbitol, mannitol, starch, sucrose, dextran, glutamate, and glucose, and proteins such as animal, vegetable, or microbial proteins such as milk powder, serum albumin, and casein.

[0019] The above preservatives include, but are not limited to, thimerosal, merthiolate, gentamicin, neomycin, nystatin, amphotericin B, tetracycline, penicillin, streptomycin, and polymyxin B.

[0020] The pharmaceutical composition of the present invention may be administered to mammals, including humans, by any method, for example, orally or parenterally. Parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal administration.

[0021] The pharmaceutical composition of the present invention can be formulated as a preparation for oral administration or parenteral administration according to the administration route as described above.

[0022] The total effective dose of the pharmaceutical composition of the present invention can be administered to a patient as a single dose, or can be administered as a split treatment regimen with long-term administration as multiple doses. The pharmaceutical composition of the present invention can vary the content of the active ingredient depending on the severity of the disease. This can be determined by considering various factors such as the formulation method, administration route, and number of treatments, as well as the patient's age, weight, health condition, symptoms of the disease, and administration time and method. Taking these points into account, a person of ordinary skill in the art will be able to determine an appropriate effective dosage of the composition of the present invention. The pharmaceutical composition according to the present invention is not particularly limited in its formulation, administration route, or administration method as long as it exhibits the effects of the present invention.

[0023] Another aspect of the present invention provides an animal feed composition for preventing or improving sarcopenia containing D-Leu-OMe-HCl as an active ingredient.

[0024] The feed composition of the present invention may refer to any natural or artificial diet, meal, etc., or ingredients of the meal, which are suitable for animals to eat, ingest, and digest, and may be manufactured in various forms known in the art. In this case, the type of feed is not particularly limited, and feed commonly used in the relevant technical field may be used. Non-limiting examples of the feed include plant feeds such as grains, roots, fruits, food processing by-products, algae, fibers, pharmaceutical by-products, oils and fats, starches, meal, or grain by-products; and animal feeds such as proteins, inorganic substances, oils and fats, mineral substances, oils and fats, single-cell proteins, zooplankton, or food. These may be used alone or in combination of two or more.

[0025] The feed composition of the present invention may additionally contain substances that exhibit various effects, such as nutrient supplementation and weight loss prevention, increased digestibility and availability of fiber in feed, improved milk quality, prevention of reproductive disorders and improved conception rate, and prevention of summer heat stress. For example, the feed composition may additionally contain mineral preparations such as sodium bicarbonate, bentonite, magnesium oxide, and complex minerals; trace mineral preparations such as zinc, copper, cobalt, and selenium; vitamins such as carotene, vitamin E, vitamins A, D, E, nicotinic acid, and vitamin B complex; protected amino acids such as methionine and lysate; protected fatty acids such as fatty acid calcium salts; live bacteria such as probiotics (lactic acid bacteria), yeast cultures, and mold fermentations; and yeast agents.

[0026] The pharmaceutical composition for preventing or treating sarcopenia in companion animals of the present invention can exhibit a more fundamental preventive or therapeutic effect on sarcopenia in companion animals by inhibiting the increase in the production and mRNA expression of myostatin protein, which directly affects muscle loss and muscle strength reduction.

[0027] Figure 1 schematically illustrates the synthetic process of leucine derivatives (L-leucine, D-leucine, DL-leucine, D-Leu-N-Ac, D-Leu-Ododecyl, D-Leu-N-Fmoc, and D-Leu-OMe).

[0028] Figure 2 shows the results of a cytotoxicity test of a leucine derivative using MTT analysis.

[0029] Figure 3 compares the differentiation of mouse skeletal muscle cells C2C12 after giving medium containing L-leucine to mouse skeletal muscle cells grown in medium without L-leucine.

[0030] Figure 4 shows the degree of differentiation of mouse skeletal muscle cells C2C12 and the number of nuclei in differentiated cells measured and compared on days 1, 6, and 9 after giving medium containing L-leucine to mouse skeletal muscle cells grown in medium without L-leucine.

[0031] Figure 5 compares the expression levels of skeletal muscle differentiation markers (MyoD, MyoG, MyhC1, and Myf5) of mouse skeletal muscle cells C2C12 in a medium without L-leucine and in a medium treated with L-leucine.

[0032] Figure 6 compares the myostatin promoter activity of mouse skeletal muscle cells C2C12 treated with leucine derivatives.

[0033] Figure 7 compares the expression levels of myostatin mRNA in mouse skeletal muscle cells C2C12 according to the concentration of leucine derivatives.

[0034] Figure 8 compares the amount of myostatin mRNA expression in mouse skeletal muscle cells C2C12 according to the elapsed time after treatment with a leucine derivative.

[0035] Figure 9 compares the expression levels of myostatin mRNA in mouse skeletal muscle cells C2C12 treated with 1 mM of L-leucine and 0.01, 0.1, and 1 mM of D-Leu-OMe-HCl.

[0036] Figure 10 compares the expression levels of myostatin mRNA in mouse skeletal muscle cells C2C12 according to the elapsed time after treatment with L-leucine and D-Leu-OMe-HCl.

[0037] Figure 11 shows the degree of differentiation of mouse skeletal muscle cells C2C12 grown in a medium without L-leucine, and the cell number was measured and compared on days 1, 3, and 5 after giving media containing 1 mM L-leucine and D-Leu-OMe-HCl to mouse skeletal muscle cells.

[0038] Figure 12 shows the degree of differentiation of mouse skeletal muscle cells C2C12 grown in a medium without L-leucine, and compared the number of nuclei in differentiated cells by observing the degree of differentiation on days 1, 6, and 9 after giving media containing 1 mM L-leucine and D-Leu-OMe-HCl to mouse skeletal muscle cells grown in a medium without L-leucine.

[0039] Figure 13 compares the expression levels of skeletal muscle differentiation markers (MyoD, MyoG, MyhC1, and Myf5) in mouse skeletal muscle cells C2C12 after treatment with L-leucine and D-Leu-OMe-HCl.

[0040] Figure 14 shows the results of observing the effects of FOXO3 siRNA and SMAD3 siRNA on the mRNA expression of myostatin, FOXO3, SMAD3, and MuRF1.

[0041] Figure 15 shows the results of observing the effects of L-leucine and D-Leu-OMe-HCl treatment on the mRNA expression of FOXO1, FOXO3, SMAD2, SMAD3, MuRF1, and MAFbx in mouse skeletal muscle cells C2C12.

[0042] Figure 16 shows the results of observing the effect on the activity level of FOXO and the expression of AKT-mTOR signaling proteins in mouse skeletal muscle cells C2C12 after treatment with D-Leu-OMe-HCl.

[0043] Figure 17 shows the results of observing the effects of D-Leu-OMe-HCl treatment on the mRNA expression of myostatin, FOXO1, FOXO3, SMAD2, SMAD3, NF-kB, MuRF1, and MAFbx in mouse skeletal muscle cells C2C12.

[0044] Figure 18 shows the results of observing the effect on the expression of myostatin signal-related proteins and AKT-mTOR signaling proteins in mouse skeletal muscle cells C2C12 after treatment with D-Leu-OMe-HCl.

[0045] Figure 19 shows the results of measuring body weight changes, grip strength, and muscle weight after a muscle loss inhibition efficacy experiment conducted on 20-month-old aged mice.

[0046] Figure 20 shows the results of blood analysis performed after the muscle loss inhibition efficacy experiment.

[0047] Figure 21 shows the results of cross-sectional area analysis using H&E staining after the muscle reduction inhibition efficacy experiment.

[0048] The present invention will be described in more detail below through one or more examples. However, these examples are intended to exemplify the present invention and the scope of the present invention is not limited to these examples.

[0049]

[0050] Example 1. Synthesis of leucine derivatives

[0051] In order to select a candidate substance capable of suppressing the promoter activity and mRNA expression of myostatin (MSTN), a muscle loss marker, leucine derivatives in Table 1 were synthesized using the method in Figure 1. L-leucine in Table 1 is L (Levo) leucine, D-leucine is D (Dextro) leucine, and DL-leucine is a racemate or racemic mixture containing both L and D leucine.

[0052]

[0053]

[0054] 1-1. Synthesis of D-Leu-N-Ac and D-Leu-N-Fmoc

[0055] In a 100 mL 2-neck round bottom flask, 5 g of D-leucine and 25 mL of distilled water were dissolved, and 25 mL of alcohol was added and stirred. After lowering the temperature to 5 ℃, 13.2 mL of alkyl chloride was added dropwise, followed by 30.7 g of NaOH, and stirred rapidly for 6 hours. After filtering the white solid, it was washed three times with 500 mL of 5% hydrochloric acid (35% aq.) / 85% t-Butyl alcohol / 10% distilled water, and filtered to obtain D-Leu-N-Ac (D-Leucine-N-Acetyl) and D-Leu-N-Fmoc (D-Leucine-N-fluorenylmethoxycarbonyl).

[0056]

[0057] 1-2. Synthesis of D-Leu-Ododecyl and D-Leu-OMe

[0058] In a 1000 mL round-bottom flask, 122 mL of R-OH, 50 g of D-Lucine, and 127.64 g of NaCl were added and dissolved at room temperature. The mixture was stirred for 30 minutes, then 97 g of Na2SO4 were added, and 77.8 mL of H2SO4 were slowly added. The mixture was stirred for 13 hours while maintaining the temperature at 65 ℃. The reaction mixture was cooled to room temperature, filtered, and concentrated. 100 mL of EA was added and dissolved, and the organic layer was washed once with a saturated aqueous solution of NaHCO3 to obtain D-Leu-Ododecyl (D-Leucine-O-dodecyl) and D-Leu-OMe (D-Leucine methyl ester). In the examples below, D-Leu-Ododecyl-HCl and D-Leu-OMe-HCl, which are in the form of D-Leu-Ododecyl and D-Leu-Ome combined with HCl or mixed with HCl, were used.

[0059]

[0060] Example 2. Cytotoxicity test of leucine derivatives using MTT assay

[0061] To confirm the cytotoxicity of the derivative synthesized in Example 1, an MTT assay was performed. Specifically, 3X10 mouse skeletal muscle cells C2C12 (ATCC, US) were seeded in a 6-well culture plate. 5 After seeding the cells, the following day, complete medium (0.8 mM L-leucine) or medium without L-leucine was treated with L-leucine, D-leucine, DL-leucine, D-Leu-N-Ac, D-Leu-Ododecyl-HCl, D-Leu-N-Fmoc, and D-Leu-OMe-HCl at concentrations of 0.001, 0.01, 0.1, 1, and 10 mM, respectively. Cell viability was checked after 24, 48, and 72 hours, and is shown in Fig. 2.

[0062] As a result, treatment with D-Leu-OMe-HCl at concentrations up to 0.1 mM did not affect cell viability compared to the control group, and no cytotoxicity was observed at any concentration for up to 48 hours.

[0063] Through these results, it was confirmed that D-Leu-OMe-HCl is non-cytotoxic and can be safely applied as a pharmaceutical composition or feed composition.

[0064]

[0065] Example 3. Cell growth effect of L-leucine

[0066] 3-1. Measurement of the degree of differentiation of mouse skeletal muscle cells C2C12 by L-leucine and the number of nuclei in differentiated cells.

[0067] To determine the effect of L-leucine on the differentiation of mouse skeletal muscle cells C2C12, differentiation media containing 1 mM L-leucine were given to mouse skeletal muscle cells C2C12 continuously grown in a medium without L-leucine, and the number of cells was measured on days 1, 3, and 5 (Fig. 3), and the degree of differentiation of mouse skeletal muscle cells and the number of nuclei present in differentiated cells were measured on days 1, 6, and 9 (Fig. 4).

[0068] As a result, cell growth was significantly promoted in the medium containing L-leucine compared to the control group without L-leucine, and the number of cells with six or more nuclei was significantly increased.

[0069]

[0070] 3-2. Effect of L-leucine on the promotion of mouse skeletal muscle differentiation marker expression

[0071] To determine the effect of L-leucine on the expression of skeletal muscle differentiation markers, 3X10 mouse skeletal muscle cells C2C12 were cultured in 6-well culture plates. 5After dividing the cells, they were differentiated for 4 days and then treated with L-leucine. After 3, 6, and 9 days, qRT-PCR was performed to determine the expression levels of skeletal muscle differentiation markers MyoD, MyoG, MyhC1, and Myf5, and after 48 hours, Western blot was performed to determine the expression levels of differentiation markers. The results are shown in Figure 5.

[0072] As a result, it was confirmed that when L-leucine was treated in C2C12, the expression of skeletal muscle differentiation markers MyoD, MyoG, and MyhC1 significantly increased, while the expression of Myf5 decreased.

[0073]

[0074] Example 4. Inhibitory effect on myostatin promoter activity

[0075] In order to confirm the effect of the leucine derivative of Example 1 on the activity of the myostatin (MSTN) promoter, a muscle loss marker, 3X10 mouse skeletal muscle cells C2C12 were cultured in a 6-well culture plate. 5 After cell division, the next day, the pGL4.15 empty vector and the pGL4.15-MSTN vector containing the myostatin promoter were transfected using Lipofectamin 2000 for 4 hours. After 4 hours, L-leucine, D-leucine, DL-leucine, D-Leu-N-Ac, D-Leu-Ododecyl-HCl, D-Leu-N-Fmoc, and D-Leu-OMe-HCl were treated at concentrations of 0.1 and 1 mM, respectively, in a medium without L-leucine. After 1 day, the myostatin promoter activity was measured using a Dual luciferase assay kit (Promega Inc.), and the results are shown in Fig. 6.

[0076] As a result, L-leucine, D-leucine, DL-leucine, and D-Leu-OMe-HCl were found to inhibit the promoter activity of myostatin, and the inhibitory effects of L-leucine, D-leucine, and D-Leu-OMe-HCl were particularly excellent at a concentration of 1 mM.

[0077]

[0078] Example 5. Inhibitory effect on myostatin mRNA expression

[0079] 5-1. Experiment on the inhibitory effect of leucine derivatives on myostatin mRNA expression according to concentration

[0080] 3X10 mouse skeletal muscle cells C2C12 in a 6-well culture plate 5 After 24 hours of seeding, cells were treated with L-leucine, D-leucine, DL-leucine, D-Leu-N-Ac, D-Leu-Ododecyl-HCl, D-Leu-N-Fmoc, and D-Leu-OMe-HCl at concentrations of 0.1 and 1 mM, respectively, in a medium without L-leucine. After 12 hours, the level of myostatin mRNA expression was confirmed using qRT-PCR, and the myostatin mRNA expression value was corrected using the expression value of beta-actin, and is shown in Fig. 7.

[0081] As a result, among leucine derivatives, L-leucine, D-leucine, DL-leucine, and D-Leu-OMe-HCl were found to inhibit the expression of myostatin mRNA, and the inhibitory effect of D-Leu-OMe-HCl was particularly excellent at a concentration of 1 mM.

[0082]

[0083] 5-2. Experimental study on the time-dependent inhibition of myostatin mRNA expression by leucine derivatives.

[0084] 3X10 mouse skeletal muscle cells C2C12 in a 6-well culture plate 5After 24 h of seeding, cells were treated with L-leucine, D-leucine, DL-leucine, D-Leu-N-Ac, D-Leu-Ododecyl-HCl, D-Leu-N-Fmoc, and D-Leu-OMe-HCl at a concentration of 1 mM each in a medium without L-leucine. After 12, 24, and 48 h, the level of myostatin mRNA expression was confirmed using qRT-PCR, and the myostatin mRNA expression value was corrected using the expression value of beta-actin, and is shown in Fig. 8.

[0085] As a result, among the leucine derivatives, D-leu-OMe-HCl was confirmed to have the highest inhibitory effect on myostatin mRNA expression at all elapsed times. Furthermore, the inhibitory effect on myostatin mRNA expression was confirmed to be evident from 12 hours and maintained for up to 48 hours.

[0086]

[0087] 5-3. Experiment and comparison of the inhibitory effects of L-leucine and D-Leu-OMe-HCl on myostatin mRNA expression.

[0088] 3X10 mouse skeletal muscle cells C2C12 in a 6-well culture plate 5 Cells were seeded and 24 hours later, 1 mM L-leucine and 0.01, 0.1, and 1 mM D-Leu-OMe-HCl were treated in a medium without L-leucine. After 12, 24, and 48 hours, the level of myostatin mRNA expression was confirmed using qRT-PCR, and the myostatin mRNA expression value was corrected using the expression value of beta-actin, and is shown in Figures 9 and 10.

[0089] Comparing the mRNA expression levels after 24 hours, D-Leu-OMe-HCl was found to suppress myostatin mRNA expression in a concentration-dependent manner and to have a superior inhibitory effect compared to L-leucine (Fig. 9). Furthermore, comparing the mRNA expression levels after 12, 24, and 48 hours at 1 mM, D-Leu-OMe-HCl was found to suppress myostatin mRNA expression in a time-dependent manner and to have a superior inhibitory effect compared to L-leucine (Fig. 10).

[0090]

[0091] Example 6. Measurement of the degree of differentiation of mouse skeletal muscle cells C2C12 by L-leucine and D-Leu-OMe-HCl and the number of nuclei in differentiated cells.

[0092] To determine the effects of L-leucine and D-Leu-OMe-HCl on the differentiation of mouse skeletal muscle cells C2C12, differentiation media containing 1 mM L-leucine and D-Leu-OMe-HCl were given to mouse skeletal muscle cells C2C12 continuously grown in a medium without L-leucine, and the number of cells was measured on days 1, 3, and 5 (Fig. 11), and the degree of differentiation of mouse skeletal muscle cells and the number of nuclei present in differentiated cells were measured on days 1, 6, and 9 (Fig. 12).

[0093] As a result, the growth rates were similar in cells to which L-leucine and D-Leu-OMe-HCl were added until the third day, but from the fifth day onwards, the growth rate of cells to which D-Leu-OMe-HCl was added was found to be faster than that of L-leucine, and the number of cells containing six or more nuclei was also confirmed to be statistically significantly higher in cells to which D-Leu-OMe-Hcl was added.

[0094]

[0095] Example 7. Effect of L-leucine and D-Leu-OMe-HCl on the expression of mouse skeletal muscle differentiation markers.

[0096] 3X10 mouse skeletal muscle cells C2C12 in a 6-well culture plate 5 After dividing the cells, they were differentiated for 4 days and then treated with 0.1 mM L-leucine and D-Leu-OMe-HCl. After 3, 6, and 9 days, qRT-PCR was performed to confirm the expression levels of the differentiation markers MyoD, MyoG, and MyhC1, and after 48 hours, Western blot was performed to confirm the expression levels of the differentiation markers. The results are shown in Figure 13.

[0097] As a result, it was confirmed that D-leu-OMe-HCl significantly increased the expression of MyoD, MyoG, and MyhC1 compared to L-leucine at all elapsed times.

[0098]

[0099] Example 8. Inhibitory effect on the expression of factors involved in the activation of the myostatin promoter.

[0100] 8-1. Inhibitory effects of myostatin expression by FOXO3 siRNA and SMAD3 siRNA

[0101] To determine the effects of FOXO3 siRNA and SMAD3 siRNA on myostatin promoter activity, 3X10 mouse skeletal muscle cells C2C12 were seeded in a 6-well culture plate. 5 After cell division, the next day, the pGL4.15 empty vector and the pGL4.15-MSTN vector containing the myostatin promoter were transfected using Lipofectamin 2000 for 4 hours. After 4 hours, FOXO3 siRNA and SMAD3 siRNA were treated at concentrations of 1, 10, 25, and 50 nM, respectively, in medium lacking L-leucine. After 1 day, the myostatin promoter activity was measured using the Dual luciferase assay kit (Promega Inc.), and the results are shown in Figure 14.

[0102] In addition, to determine the effects of FOXO3 siRNA and SMAD3 siRNA on the mRNA expression of FOXO1, FOXO3, SMAD2, and SMAD3 involved in myostatin promoter activation and the mRNA expression of MuRF1 and MAFbx, muscle atrophy markers regulated by myostatin, 3X10 mouse muscle cells (TA, tibialis anterior) were seeded in a 6-well culture plate. 5 After dividing the cells, they were differentiated for 4 days and then treated with 50 nM of FOXO3 siRNA and SMAD3 siRNA. After 24 hours, qRT-PCR was performed to confirm the mRNA expression levels of FOXO3, FOXO1, MSTN MuRF1, and MAFbx, and after 48 hours, Western blot was performed to confirm the expression levels of differentiation markers, which are shown in Figure 14.

[0103] As a result, it was confirmed that the mRNA expression of FOXO3, MSTN, and MuRF1 was suppressed by FOXO3 siRNA, and the mRNA expression of SMAD3, MSTN, and MuRF1 was suppressed by SMAD3 siRNA. Accordingly, it was confirmed that the expression of myostatin is regulated by FOXO3 and SMAD3.

[0104]

[0105] 8-2. Inhibitory effects of L-leucine and D-Leu-OMe-HCl on the expression of factors involved in the activation of the myostatin promoter.

[0106] 3X10 mouse skeletal muscle cells C2C12 in a 6-well culture plate 5After dividing the cells, they were differentiated for 4 days and then treated with 1 mM L-leucine and 0.01, 0.1, and 1 mM D-Leu-OMe-HCl. After 24 hours, qRT-PCR was performed to confirm the mRNA expression levels of FOXO1, FOXO3, SMAD2, SMAD3, MuRF1, and MAFbx, and after 48 hours, Western blot was performed to confirm the expression levels of differentiation markers. The results are shown in Figure 15.

[0107] As a result, it was confirmed that the mRNA expression of FOXO1, FOXO3, SMAD2, SMAD3, MuRF1, and MAFbx was inhibited at all concentrations, and for FOXO1, FOXO3, SMAD2, MuRF1, and MAFbx1, the inhibitory effect of D-Leu-OMe-HCl at a concentration of 1 mM was confirmed to be superior to the inhibitory effect by L-leucine.

[0108]

[0109] 8-3. Confirmation of the molecular mechanism of D-Leu-OMe-HCl

[0110] It is known that the expression of muscle atrophy markers MuRF1 and MAfbx32 is induced by the [PI3K / Akt → FOXO activation and GSK3 inactivation] mechanism. In order to confirm the molecular mechanism by which D-Leu-OMe acts on myotube cells, D-Leu-OMe-HCl was treated under the same C2C12 culture medium conditions as in Example 8-2, and after 48 hours, the activity level of FOXO and the expression of AKT-mTOR signaling proteins were confirmed by Western blotting, and the results are shown in Figure 16.

[0111] As a result, it was found that treatment with D-Leu-OMe-HCl inhibited the phosphorylation and protein expression of FOXO3a and SMAD3. In addition, the protein expression of MuRF1 and MAFbx was suppressed, and the expression of AKT-mTOR signaling proteins involved in myocyte differentiation was increased.

[0112]

[0113] Example 9. Effect of D-Leu-OMe-HCl on mouse muscle cells

[0114] 9-1. Inhibitory effect on mRNA expression of myostatin and related factors

[0115] 3X10 mouse muscle cells (TA, tibialis anterior) in a 6-well culture plate 5 After dividing the cells, they were differentiated for 4 days and then treated with D-Leu-OMe-HCl. After 24 hours, qRT-PCR was performed to determine the mRNA expression levels of myostatin, FOXO1, FOXO3, SMAD2, SMAD3, NF-kB, MuRF1, and MAFbx, and after 48 hours, Western blot was performed to determine the expression levels of differentiation markers, which are shown in Figure 17.

[0116] As a result, it was confirmed that the mRNAs of myostatin, SMAD2, SMAD3, MuRF1, and MAFbx were significantly reduced compared to the control group when treated with D-Leu-OMe-HCl.

[0117]

[0118] 9-2. Inhibition of myostatin-related factor expression and promotion of AKT-mTOR signaling protein expression

[0119] The results of performing Western blot to confirm the expression of myostatin signal-related proteins and AKT-mTOR signaling proteins 48 hours after treatment with D-Leu-OMe-HCl under the same C2C12 culture medium conditions as in Example 9-1 are shown in Figure 18.

[0120] As a result, it was confirmed that the expression of myostatin signaling-related proteins decreased compared to the control group when treated with D-Leu-OMe-HCl, while the expression of AKT-mTOR signaling proteins increased.

[0121]

[0122] Example 10. Experimental study on the efficacy of D-Leu-OMe-HCl in suppressing muscle loss in aged mice.

[0123] 10-1. Changes in body weight, grip strength, and muscle mass

[0124] Typically, the life cycle of mice is classified as mature adults at 3-6 months of age, middle-aged at 10-15 months of age, and old at 18-24 months of age (Flurkey K, Currer JM, Harrison DE. 2007). In order to confirm the inhibitory effect of D-Leu-OMe-HCl on muscle loss, 24-month-old mice were divided into two groups: a group that consumed 20 mg / kg of water daily (experimental group) and a group that consumed normal PBS in water (control group). After maintaining the group for 4 months, body weight changes, grip strength, and muscle weight were measured.

[0125] Specifically, to determine the effects of D-Leu-OMe-HCl on mouse muscle strength and mass, the grip strength of 24-month-old male mice (N=5, old group) was measured as the maximum muscle strength. The mice grasped a steel bar connected to a force gauge and then pulled the tail of the mice against the steel bar until the forelimbs and hindlimbs released the steel bar. Grip strength (g) was measured three times during the experimental period (17 weeks) using a grip dynamometer (Bioseb, Vitrolles Cedex, France), and the maximum grip strength value was used for analysis. After measuring the grip strength, the TA muscle of each mouse (experimental and control groups) was excised and weighed.

[0126] As a result, no decrease in body weight was observed in the experimental group compared to the control group (Fig. 19A), and grip strength was confirmed to remain higher than in the control group (Fig. 19B). In addition, the weight of the TA muscle was similar to that of the control group (Fig. 19C).

[0127]

[0128] 10-2. Blood analysis

[0129] To determine the effect of D-Leu-OMe-HCl on muscle cells, blood analysis was performed after sacrificing 24-month-old mice of Example 10-1, and the results are shown in Fig. 20 and Table 2.

[0130] Myoglobin is an oxygen-binding protein found in muscle cells, C-reactive protein (CRP) is an acute reactant that increases in the blood when acute inflammation or tissue damage occurs in the body, and creatine kinase (CK) is an enzyme found in large quantities in muscles, and its blood level increases when muscles are damaged. The analysis results showed that the concentrations of myostatin and myoglobin in the blood of the experimental group that consumed D-Leu-OMe-HCl for 4 months significantly decreased compared to the control group. Meanwhile, the concentrations of CRP and CK tended to decrease in the experimental group compared to the control group, but did not show a statistically significant change (Figure 20).

[0131] Additionally, blood analysis results showed no significant changes in most values, and the level of lactate dehydrogenase, an indicator of muscle damage, was confirmed to have significantly decreased.

[0132]

[0133]

[0134] 10-3. Muscle cell H&E staining results

[0135] To confirm the effect of D-Leu-OMe-HCl on muscle cells, the 24-month-old mice of Example 10-1 were sacrificed, and the mouse muscle cells were stained with H&E staining and the cross-sectional area analysis results are shown in Figure 21.

[0136] As a result, it was confirmed that the experimental group that consumed D-Leu-OMe-HCl for 4 months had more thick bundle muscles compared to the control group.

[0137]

[0138] These results confirmed that D-Leu-OMe-HCl had an excellent effect in suppressing muscle loss in mice.

[0139]

[0140] The present invention has been described above, focusing on specific embodiments thereof. Those skilled in the art will appreciate that the present invention can be implemented in modified forms without departing from its essential characteristics. Therefore, the disclosed embodiments should be considered illustrative rather than limiting. The scope of the present invention is set forth in the claims, not the foregoing description, and all differences within the scope equivalent thereto should be construed as being encompassed by the present invention.

Claims

1. A pharmaceutical composition for preventing or treating sarcopenia in animals, containing D-Leu-OMe-HCl as an active ingredient.

2. In the first paragraph, the composition is a pharmaceutical composition for preventing or treating sarcopenia in animals that suppresses myostatin mRNA or protein expression.

3. In the first paragraph, the composition is a pharmaceutical composition for preventing or treating sarcopenia in animals that reduces myostatin promoter activity.

4. A pharmaceutical composition for preventing or treating sarcopenia in animals, wherein the sarcopenia in paragraph 1 is caused by aging.

5. Animal feed composition for preventing or improving sarcopenia containing D-Leu-OMe-HCl as an active ingredient.

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