Skeletal muscle enhancing agent

Abstract

The present invention provides a means for enhancing skeletal muscles. Specifically, spermidine or a pharmaceutically acceptable salt thereof is intramuscularly administered as an active ingredient.

Description

Skeletal muscle strengthening agent

[0001] The present invention relates to a skeletal muscle-building agent containing spermidine or a pharmaceutically acceptable salt thereof, which is administered intramuscularly.

[0002] Skeletal muscle homeostasis is controlled by the synthesis and degradation systems (Non-Patent Document 1). When this homeostasis is disrupted and the degradation system becomes dominant, a decrease in skeletal muscle mass (hereinafter also referred to as "muscle atrophy") occurs. Known causes of muscle atrophy include aging, immobility, neurodegenerative diseases, and cachexia (malnutrition due to cancer, etc.). Age-related muscle atrophy, which frequently occurs in the elderly, is also called sarcopenia (Non-Patent Document 2). When sarcopenia causes progressive muscle atrophy throughout the body, daily activities decline, requiring care and assistance, making it a major problem in aging societies. Sarcopenia is also thought to be a cause of chronic lower back pain (Non-Patent Document 11). Muscle atrophy caused by immobility is also called disuse muscle atrophy. Disuse muscle atrophy can occur due to factors such as the use of plaster casts to treat fractures caused by injury or osteoporosis, or prolonged bedriddenness due to senility. Exercise therapy to increase skeletal muscle mass is effective against muscle atrophy, but the development of treatments (e.g., drug therapy) that can be applied to individuals who have difficulty exercising is desired. The mechanisms of muscle atrophy are known to involve increased activity of the muscle-specific ubiquitin-proteasome system and the involvement of myogenic inhibitors (Non-Patent Document 1). Tirm63 (MuRF1) is a representative muscle-specific ubiquitin ligase involved in muscle atrophy, and its inhibition has been reported to suppress muscle atrophy (Non-Patent Document 3). Myostatin (Mstn) is a myogenic inhibitor, and its inhibition has been reported to significantly increase muscle mass (Non-Patent Document 4).

[0003] Skeletal muscle injury (hereinafter also referred to as "muscle injury") is a type of skeletal muscle mass loss caused by factors other than homeostasis. Clinically, muscle injury is classified as muscle contusion (strained muscle), high-energy trauma, or surgery. Muscle injury is known to cause various complications, including functional impairment, muscle atrophy, and localized pain (Non-Patent Document 9). Muscle contusion caused by blunt external force (contusion) is the most common sports injury (Non-Patent Document 10). Although accurate statistics are unavailable, the number of patients with muscle injury in Japan is estimated to be in the tens of thousands per year. RICE (rest, icing, compression, and elevation) treatment has been recommended as first aid for muscle injury. Following first aid, symptomatic treatment for pain and rehabilitation are performed. Skeletal muscle tissue has a mechanism for regenerating injury. Muscle satellite cells are known to be essential for muscle regeneration. Muscle satellite cells located near the basement membrane of muscle fibers are normally quiescent. However, upon muscle injury, they are activated and differentiate into myoblasts, which then undergo cell fusion to form muscle fibers. Once muscle regeneration is complete, the remaining muscle satellite cells return to quiescence (Non-Patent Documents 5 and 6). Factors known to promote muscle regeneration include hepatocyte growth factor (HGF) (Non-Patent Document 7) and insulin-like growth factor 1 (IGF-1) (Non-Patent Document 8). It has also been reported that muscle hypertrophy can be induced by suppressing factors that inhibit muscle regeneration (Patent Document 1). However, muscle regeneration via this mechanism requires a long time. Therefore, conventional treatments that do not specifically promote muscle regeneration result in muscle weakness, delaying the return of muscle injury patients to daily activities and sports activities. Furthermore, if muscle regeneration takes a long time, scar tissue derived from collagen that remains in the muscle tissue for a long period of time may replace some of the muscle tissue. Scar tissue reduces the strength of plastic muscle, increasing the risk of recurrence of muscle injury (Non-Patent Document 9). It is known that muscle regeneration is promoted when skeletal muscle homeostasis becomes synthetically dominated and muscle fibers are formed (Non-Patent Documents 5 and 6).

[0004] International Publication No. 2013 / 039244

[0005] Physiology, vol.23, no.3, pp.160-170, 2008Sarcopenia Muscle, vol.10, no.5, pp.956-961, 2019Science (80-.)., vol.294, no.5547, pp.1704-1708, 2001Cell. Mol. Life Sci., J. Sports Med., vol.33, no.5, pp.745-64, May 2005J. Bone Joint Surg. Am., vol.84-A, no.5, pp.822-32, May 2002Dev. Biol., vol. 194, no.1, pp.114-128, 1998J. Cell. Physiol., vol.138, no.2, pp.311-5, Feb. 1989J. Appl. Physiol., vol.95, no.2, pp.771-780, 2003Am. J. Sports Med., vol.27, no.1, pp.2-9, 1999 Spinal Cord, vol.30, no.5, pp.573-578, 2017

[0006] Conventional treatments for muscle atrophy and muscle damage have limited applicability, require long recovery times, and are prone to muscle weakness, resulting in issues such as a decline in patients' motor function, a shortened healthy lifespan, and long-term cessation of athletic activity in athletes. Furthermore, although the molecular mechanisms of muscle atrophy and regeneration have been extensively studied, no effective drugs have yet been developed. Therefore, there has been a strong demand for the development of drugs that can strengthen skeletal muscle.

[0007] As a result of intensive research aimed at solving the above-mentioned problems, the present inventors have found that intramuscular administration of spermidine to a subject suffering from muscle atrophy or muscle damage inhibits the degradation system or promotes the synthesis system in skeletal muscle homeostasis, resulting in an increase in skeletal muscle weight (i.e., skeletal muscle strengthening), and have completed the present invention. Specifically, the present invention relates to the following [1] to

[10] . [1] A skeletal muscle strengthening agent containing spermidine or a pharmaceutically acceptable salt thereof, characterized by being administered intramuscularly. [2] The skeletal muscle strengthening agent according to [1] above, which is administered to a subject suffering from skeletal muscle atrophy. [3] The skeletal muscle strengthening agent according to [1] or [2] above, wherein the skeletal muscle atrophy is sarcopenia or disuse muscle atrophy. [4] The skeletal muscle strengthening agent according to [1] above, which is administered to a subject suffering from skeletal muscle damage. [5] The skeletal muscle strengthening agent according to [4] above, wherein the skeletal muscle damage is a muscle contusion. [6] The skeletal muscle strengthening agent according to [4] above, wherein the skeletal muscle damage is caused by a myogenic disease. [7] The skeletal muscle strengthening agent according to any one of [1] to [6] above, which strengthens skeletal muscle by inhibiting skeletal muscle degradation or promoting skeletal muscle synthesis. [8] A therapeutic agent for skeletal muscle atrophy, comprising spermidine or a pharmaceutically acceptable salt thereof, characterized by being administered intramuscularly. [9] A therapeutic agent for skeletal muscle damage, comprising spermidine or a pharmaceutically acceptable salt thereof, characterized by being administered intramuscularly.

[10] An agent for restoring skeletal muscle homeostasis, comprising spermidine or a pharmaceutically acceptable salt thereof, characterized by being administered intramuscularly.

[0008] As will be shown in the examples below, the skeletal muscle-building agent of the present invention can build skeletal muscle.

[0009] Figure 1 shows the effect of intramuscular administration of spermidine trihydrochloride (SPD) on skeletal muscle weight in a muscle atrophy model. Figure 2 shows the effect of intramuscular administration of SPD on muscle fiber area in a muscle atrophy model. Figure 3 shows the effect of SPD administration on Trim63 gene expression in a muscle atrophy model. Figure 4 shows the effect of SPD administration on Mstn gene expression in a muscle atrophy model. Figure 5 shows the effect of SPD administration on skeletal muscle weight in a muscle injury model. Figure 6 shows the effect of intramuscular administration of SPD on regenerated muscle area in a muscle injury model. Figure 7 shows the effect of intraperitoneal administration of SPD on regenerated muscle area in a muscle injury model. Figure 8 shows the effect of oral administration of SPD on regenerated muscle area in a muscle injury model.

[0010] [Active ingredient] The skeletal muscle-building agent of the present invention contains spermidine or a pharmaceutically acceptable salt thereof as an active ingredient. Spermidine (H2N(CH2)4NH(CH2)3NH2) is a type of polyamine present in many organisms. Pharmaceutically acceptable salts of spermidine (hereinafter also referred to as "salts thereof") include hydrochloride and phosphate salts. Salts of spermidine also include solvates with pharmaceutically acceptable solvents such as water or ethanol. Spermidine and its salts are known substances and are readily available on the market or can be easily produced by known means.

[0011] [Skeletal Muscle Enhancer] "Skeletal muscle enhancement" refers to increasing skeletal muscle mass that has been reduced due to muscle atrophy, muscle damage, etc. Although the present invention is not limited by a particular theory, it is believed that an increase in skeletal muscle mass is achieved by suppressing the degradation system or promoting the synthesis system in skeletal muscle homeostasis, causing the synthesis system to become dominant (promoting muscle regeneration), increasing the number of individual muscle fibers that make up skeletal muscle, and / or hypertrophy of the individual muscle fibers themselves.

[0012] "Muscle atrophy" refers to a decrease in skeletal muscle mass caused by the dominance of the catabolic system, which is the system that maintains skeletal muscle homeostasis. Causes of muscle atrophy include aging, immobility, neurodegenerative diseases (such as amyotrophic lateral sclerosis and spinal muscular atrophy), and cachexia.

[0013] "Muscle damage" refers to a decrease in skeletal muscle mass caused by causes other than homeostasis (such as muscle fiber necrosis due to external forces or myogenic diseases). Examples of muscle damage caused by external forces include muscle contusion (caused by external forces (e.g., bruising)), muscle strain (caused by internal forces such as sudden muscle contraction), and cervical sprain (also known as whiplash). Examples of myogenic diseases that cause muscle damage include muscular dystrophy and distal myopathy. Note that muscle damage caused by external forces and muscle damage caused by myogenic diseases have in common the fact that compensatory muscle regeneration occurs after muscle fiber necrosis (muscle damage).

[0014] Skeletal muscle strengthening agents used in drug therapy are suitable for treating subjects for whom exercise therapy is difficult, particularly subjects with muscle atrophy due to aging (sarcopenia), muscle atrophy due to disuse muscle atrophy, muscle damage due to external forces (particularly muscle contusion (strained muscle)), or muscle damage due to myogenic diseases.

[0015] The concentration of spermidine or a salt thereof in the skeletal muscle strengthening agent can be appropriately set depending on the degree of muscle atrophy or muscle damage, etc.

[0016] The skeletal muscle-building agent can be applied to any animal with skeletal muscles. The target of application is preferably mammals (humans and non-human mammals (e.g., horses and cows)), more preferably humans. Furthermore, the target of application is not limited to sex or age.

[0017] [Preparation for intramuscular administration] The skeletal muscle enhancer is administered intramuscularly to skeletal muscles. Intramuscular administration exhibits higher effects than other administration routes. Preparations for intramuscular administration include, for example, sterilized liquid preparations such as solutions or suspensions, specifically injections. General formulation techniques can be used for preparation.

[0018] In addition to the active ingredient, intramuscular formulations may contain a pharmaceutically acceptable solvent or diluent. Examples of "pharmaceutically acceptable solvents or diluents" include distilled water for injection, aqueous lidocaine hydrochloride solution (for intramuscular injection), physiological saline, aqueous glucose solution, ethanol, polyethylene glycol, propylene glycol, intravenous injection liquids (e.g., aqueous solutions of citric acid or sodium citrate), electrolyte solutions (for intravenous drip infusion and intravenous injection), and mixtures thereof. Injectable preparations may contain the active ingredient pre-dissolved, or may be in the form of a powder of the active ingredient or the active ingredient with an appropriate carrier, which is dissolved immediately before use. Injectable preparations may contain, for example, 0.005 to 25% by mass of the active ingredient based on the mass of the entire formulation.

[0019] [Treatment agent for skeletal muscle atrophy or damage] Spermidine and its salts can treat muscle atrophy or muscle damage by strengthening skeletal muscle. Therefore, the skeletal muscle strengthening agent of the present invention can also be understood as a treatment agent for skeletal muscle atrophy or damage. The active ingredients and formulation of the treatment agent are the same as those described above for the skeletal muscle strengthening agent.

[0020] [Skeletal muscle homeostasis recovery agent] Spermidine and its salts can recover homeostasis, which tends toward degradation during muscle atrophy, by suppressing the degradation system or promoting the synthesis system. Therefore, the skeletal muscle enhancer of the present invention can also be understood as a skeletal muscle homeostasis recovery agent. The active ingredients and formulation of the recovery agent are the same as those described above for the skeletal muscle enhancer.

[0021] Next, the effects of the present invention will be specifically explained using examples, but the present invention is not limited to these examples.

[0022] [Experimental Method] 1. Evaluation Compound Spermidine trihydrochloride (Nacalai Tesque, Inc.)

[0023] 2. Test Animals Seven-week-old C57BL / 6 male mice (CLEA Japan, Inc.) were used in the experiment at the age of eight weeks.

[0024] 3. Muscle Atrophy Model We used an animal model in which muscle atrophy was induced by immobilizing the hindlimbs (J Appl Physiol, vol. 106, pp. 2049-2059, 2009). Under isoflurane anesthesia, the left hindlimb of a mouse was flexed and immobilized using a surgical stapler (Kaiser Plus, Keisei Medical Industries). The right hindlimb of the same mouse served as a sham treatment and was not immobilized. Three or seven days after the start of immobilization, the tibialis anterior muscle (a skeletal muscle) was dissected and collected. The sample collected after three days was used for muscle atrophy-related gene expression analysis, and the sample collected after seven days was used for muscle weight measurement and muscle tissue section preparation.

[0025] 4. Muscle Atrophy-Related Gene Expression Analysis. The tibialis anterior muscle was homogenized using Sepasol RNA I Super G (Nacalai Tesque, Inc.), vortexed with 1-bromo-3-chloropropane, and centrifuged. RNA was extracted from the supernatant. The extracted RNA was recovered using 2-propanol and 75% ethanol and then subjected to reverse transcription (using ReverTra Ace qPCR RT Master Mix (Toyobo) according to the attached protocol) to synthesize cDNA. The synthesized cDNA was subjected to real-time PCR (using an Applied Biosystems 7500 Real-Time PCR System (Thermo Fisher Scientific)) to measure the mRNA expression of three genes: Trim63 (MuRF1), myostatin (Mstn), and Gapdh. The primer sequences used are as follows: Inhibition of Tirm63 (MuRF1) expression has been reported to suppress muscle atrophy (Non-Patent Document 3), and it is a gene that acts to enhance the degradation system in skeletal muscle homeostasis. Myostatin (Mstn) has been reported to be a myogenesis inhibitor (Non-Patent Document 4), and it is a gene that acts to suppress the synthesis system (in other words, enhance the degradation system) in skeletal muscle homeostasis. Gapdh was used as a control to standardize the expression levels of muscle atrophy-related genes. The expression levels of Trim63 and Mstn mRNA (mTrim63 and mMstn) were calculated as the ratio to the expression level of Gapdh mRNA (mGapdh). Evaluation was based on the percentage of expression levels of other treatment groups, with the expression level in the immobilized left hind limb of the vehicle-treated group (details described below) set at 100%.

[0026] 5. Muscle Injury Model We used an animal model in which muscle injury was induced by administration of snake venom cardiotoxin (CTX). This animal model is widely used in research on muscle regeneration from muscle injury. Under isoflurane anesthesia, 50 μL of 10 μM CTX was administered to the tibialis anterior muscle of the right hind limb of mice. 50 μL of phosphate buffer solution (PBS) was administered to the tibialis anterior muscle of the left hind limb of the same mice as the vehicle. Seven days after CTX administration, the tibialis anterior muscle was dissected and used for muscle weight measurement and muscle tissue section preparation.

[0027] 6. Measurement of muscle weight The body weight of each mouse was measured before harvesting the tibialis anterior muscle. The weight of the harvested tibialis anterior muscle was also measured. From these measurements, the ratio (%) of skeletal muscle weight to mouse body weight (weight of tibialis anterior muscle / body weight of mouse) was calculated.

[0028] Immediately after harvesting, the tibialis anterior muscle was rapidly frozen in isopentane cooled with liquid nitrogen. The frozen muscle tissue was sliced ​​into 10 μm-thick slices using a cryostat (Leica Biosystems) and attached to anti-peel coated slides (Matsunami Glass Industry Co., Ltd.).

[0029] 8. Immunofluorescent Staining of Muscle Tissue Sections. Muscle tissue sections were thoroughly air-dried at room temperature for 30 minutes. Then, they were immersed in acetone cooled to -30°C and fixed at -30°C for 20 minutes. The fixed sections were air-dried once, washed with PBS, and then blocked for 1 hour with a blocking reagent (Blocking One, Nacalai Tesque, Inc.). Next, a primary antibody (monoclonal anti-laminin-2 (α-2 chain) antibody, rat host antibody (Sigma-Aldrich)) diluted 500-fold with the blocking reagent was added and incubated overnight at 4°C. Laminin, which binds to the primary antibody, is a protein expressed in the basement membrane of all muscle cells. Therefore, in this experiment, it was used as an indicator for measuring the area of ​​individual muscle cells in the sections. After reacting with the primary antibody, the muscle tissue sections were washed with PBS and then reacted for 1 hour with a secondary antibody (CF 488A Goat Anti-Rat IgG(H+L) (Biotium)) diluted 500-fold with blocking reagent. The secondary antibody, an anti-rat antibody conjugated with a fluorescent dye, binds to the primary antibody and stains laminin. After reacting with the secondary antibody, the muscle tissue sections were washed with PBS, mounted using "VECTASHIELD Hard Set with DAPI" (Vector), and observed under an inverted microscope FSX100 (Olympus).

[0030] 9. Measurement of Muscle Fiber Cross-Sectional Area Muscle fiber cross-sectional area was measured based on image data acquired by fluorescence observation. After importing the image data into the image analysis software ImageJ (NIH), the cross-sectional area of ​​individual cells was measured based on the laminin-stained cell basement membrane. The area measurement was performed using "Analyze Particles," an analysis program built into ImageJ. Measurement results were presented as a distribution diagram (histogram) of the area and number of single muscle fibers, and the average cross-sectional area of ​​all single muscle fibers (average muscle fiber area). In the muscle injury model (Example 3), regenerated muscle was measured. Centrally nucleated fibers (single muscle fibers with a central nucleus) with a nucleus stained with DAPI contained in the mounting medium at the center of the cell were determined to be regenerated muscle and distinguished from muscle that existed before regeneration (without the central nucleus). In the muscle atrophy model (Examples 1 and 2), no distinction based on the central nucleus was made, and the entire muscle was measured.

[0031] Example 1: Spermidine trihydrochloride (SPD) was administered intramuscularly to muscle atrophy model animals, and changes in skeletal muscle weight and skeletal muscle fiber cross-sectional area were evaluated. For the intramuscular administration group, a formulation prepared by dissolving SPD in PBS (SPD concentration: 196 mM) was used. This formulation (volume: 10 μL) was injected into the tibialis anterior muscles of both legs of the model once daily from the day before immobilization of the left hind limb until the day before dissection. As a control, PBS (vehicle) was administered once daily from the day before immobilization of the left hind limb until the day before dissection. Seven days after the start of immobilization, the tibialis anterior muscles were harvested, and muscle weight and the number and area of ​​single muscle fibers were measured using the methods described above. The results are shown in Figure 1 (muscle weight) and Figure 2 (single muscle fiber area). Regarding the effect of hind limb immobilization on muscle weight, a significant weight loss was observed in the vehicle-administered group (control) (Figure 1). Regarding the effects of SPD administration on muscle weight, the intramuscularly administered group showed a significant increase in muscle weight compared with the vehicle-administered group (control) in both the right hindlimb (sham) and the left hindlimb (immobilized) (Fig. 1). The intramuscularly administered group also showed a significant suppression of muscle weight loss due to hindlimb immobilization (Fig. 1, **P<0.01). When assessed based on single muscle fiber area, the histogram of the intramuscularly administered SPD group shifted to the right (toward larger area) compared with the vehicle-administered group, and a significant increase in mean muscle fiber area was observed (Fig. 2, ***P<0.001). These results indicate that intramuscular administration of SPD inhibited the degradation pathway or promoted the synthesis pathway (enhancing skeletal muscle) during skeletal muscle homeostasis in subjects with normal skeletal muscle and subjects with disuse muscle atrophy.

[0032] Example 2: Spermidine trihydrochloride (SPD) was administered to muscle atrophy model animals using three different administration methods, and the expression of muscle atrophy-related genes Trim63 and Mstn was measured. For the intramuscular administration group, a formulation prepared by dissolving SPD in PBS (SPD concentration: 196 mM) was used. This formulation (volume: 10 μL) was injected into the tibialis anterior muscles of both legs of the model once daily from the day before immobilization of the left hind limb until the day before dissection. For the intraperitoneal administration group, a formulation prepared by dissolving SPD in PBS (SPD concentration: 19.6 mM) was used. This formulation (volume: 10 mL / kg) was injected into the intraperitoneal cavity of the model once daily from the day before immobilization of the left hind limb until the day before dissection. For the oral administration group, a formulation prepared by dissolving SPD in water (SPD concentration: 30 mM) was used. This formulation was orally administered with free access to water from the day before immobilization of the left hind limb until just before dissection. The average water intake during the oral administration period was 2.26 mL / day per mouse. PBS (vehicle) was administered once daily to the intramuscular and intraperitoneal groups as a control from the day before left hindlimb immobilization until the day before dissection. Water was used as a control for the oral administration group. Trim63 and Mstn mRNA expression was measured in the tibialis anterior muscles of the left hindlimb (immobilized: Staple) and right hindlimb (sham: Sham) collected 3 days after the start of immobilization. The results are shown in Figures 3 and 4. Regarding the effect of hindlimb immobilization on Trim63 expression, a significant increase in expression was observed in the vehicle-administered group (control) regardless of the administration method (Figure 3). Regarding the effect of SPD administration on Trim63 expression, the intramuscular administration groups (Spermidine, Staple) significantly suppressed expression by 65.6% compared to the vehicle-administered group (Spermidine, Vehicle) (Figure 3, *P<0.05). On the other hand, the intraperitoneal administration group (Staple, Spermidine) only suppressed expression by 42.3% compared to the vehicle administration group (Staple, Vehicle), and the oral administration group showed no suppression of expression (Fig. 3, **P<0.01). Regarding the effect of hindlimb immobilization on Mstn expression, a significant increase in expression was observed in the vehicle administration group (control) regardless of the administration method.The effect of SPD administration on Mstn expression was significant, with a 61.1% reduction in expression in the intramuscularly administered group (Staple, Spermidine) compared with the vehicle-administered group (Staple, Vehicle) (Figure 4, **P<0.01). In contrast, the intraperitoneal and oral administration groups showed no significant suppression of expression (Figure 4). These results suggest that intramuscular administration of SPD can enhance skeletal muscle mass by suppressing the expression of genes that promote the degradation pathway in skeletal muscle homeostasis, thereby favoring the synthesis pathway.

[0033] Example 3: Spermidine trihydrochloride (SPD) was administered to a muscle injury model using three different administration methods, and changes in skeletal muscle weight and regenerated skeletal muscle fiber cross-sectional area were evaluated. For the intramuscular administration group, a formulation prepared by dissolving SPD in PBS (SPD concentration: 196 mM) was used. This formulation (volume: 10 μL) was injected into the tibialis anterior muscles of both legs of the model once daily from the day before CTX administration until the day before autopsy. For the intraperitoneal administration group, a formulation prepared by dissolving SPD in PBS (SPD concentration: 19.6 mM) was used. This formulation (volume: 10 mL / kg) was injected into the peritoneal cavity of the model once daily from the day before CTX administration until the day before autopsy. For the oral administration group, a formulation prepared by dissolving SPD in water (SPD concentration: 30 mM) was used. This formulation was orally administered ad libitum from the day before CTX administration until just before autopsy. The average water intake during the oral administration period was 2.32 mL / day per mouse. Control rats in the intramuscular and intraperitoneal groups received PBS (vehicle) once daily from the day before CTX administration until the day before dissection. Water was used as a control for the oral administration group. Seven days after CTX administration, tibialis anterior muscles were harvested, and muscle weight and the number and area of ​​regenerated single muscle fibers were measured using the methods described above. The results are shown in Figure 5 (muscle weight) and Figures 6–8 (area of ​​regenerated single muscle fibers). Regarding the effect of CTX administration on muscle weight, the vehicle-administered group (control) showed a significant weight loss regardless of the administration method (Figure 5, **P<0.01). Regarding the effect of SPD administration on muscle weight, the intramuscular administration group showed a significant suppression of muscle weight loss, but the intraperitoneal and oral administration groups showed no significant suppression (Figure 5). When muscle regeneration was evaluated based on single muscle fiber area, the histogram of the intramuscularly administered SPD group shifted to the right (toward larger area) compared to the histogram of the vehicle-administered group, and a significant increase in mean muscle fiber area was observed (Figure 6, *** P<0.001). In contrast, the intraperitoneal and oral administration groups did not exhibit a shift in the histogram due to SPD administration, nor did they exhibit a significant increase in mean muscle fiber area (Figures 7 and 8). These results indicate that intramuscular administration of SPD promoted skeletal muscle regeneration (strengthening skeletal muscle) in subjects with muscle damage.

[0034] By intramuscularly administering the skeletal muscle-enhancing agent of the present invention, subjects with muscle atrophy or muscle damage can return to daily life or sports activities more quickly. Therefore, the present invention can be used to treat muscle atrophy or muscle damage.

[0035] [Sequence Listing Free Text] SEQ ID NO: 1: Primer for the sense strand of Trim63 SEQ ID NO: 2: Primer for the antisense strand of Trim63 SEQ ID NO: 3: Primer for the sense strand of Mstn SEQ ID NO: 4: Primer for the antisense strand of Mstn SEQ ID NO: 5: Primer for the sense strand of Gapdh SEQ ID NO: 6: Primer for the antisense strand of Gapdh

Claims

1. A skeletal muscle enhancer containing spermidine or a pharmaceutically acceptable salt thereof, characterized by being administered intramuscularly.

2. A skeletal muscle enhancer according to claim 1, administered to a subject having skeletal muscle atrophy.

3. The skeletal muscle enhancer according to claim 1 or 2, wherein the skeletal muscle atrophy is sarcopenia or disuse muscle atrophy.

4. A skeletal muscle enhancer according to claim 1, administered to a subject having skeletal muscle damage.

5. The skeletal muscle strengthening agent according to claim 4, wherein the skeletal muscle injury is a muscle contusion.

6. The skeletal muscle enhancer according to claim 4, wherein the skeletal muscle injury is due to a myogenic disease.

7. A skeletal muscle enhancer according to any one of claims 1, 2, 4, 5, or 6, which enhances skeletal muscle by inhibiting the breakdown of skeletal muscle or promoting its synthesis.

8. A therapeutic agent for skeletal muscle atrophy containing spermidine or a pharmaceutically acceptable salt thereof, characterized by being administered intramuscularly.

9. A therapeutic agent for skeletal muscle injury containing spermidine or a pharmaceutically acceptable salt thereof, characterized by being administered intramuscularly.

10. A skeletal muscle homeostasis restorer containing spermidine or a pharmaceutically acceptable salt thereof, characterized by being administered intramuscularly.

11. The skeletal muscle enhancer according to claim 3, which enhances skeletal muscle by inhibiting the breakdown of skeletal muscle or promoting its synthesis.