WO2025010788A1 - Use of indanyl levulinate in prevention and/or treatment of muscle atrophy-related diseases - Google Patents

Abstract

The present invention relates to a use of indanyl levulinate in the prevention and/or treatment of muscle atrophy-related diseases. Specifically, the present invention relates to a use of an indanyl levulinate compound as represented by formula (I) or a pharmaceutical composition comprising same in the prevention and/or treatment of muscle atrophy-related diseases including myogenic muscle atrophy, disuse muscle atrophy, senile muscle atrophy, and neurogenic muscle atrophy. The compound of the present invention has significant effects in the prevention and/or treatment of muscle atrophy-related diseases by promoting asymmetric division of skeletal muscle stem cells.

Description

Use of indanyl levulinate in preventing and/or treating diseases related to muscle atrophy Technical Field

The present invention relates to use of a novel indan levulinate compound or a pharmaceutical composition containing the same in preventing and/or treating diseases related to muscle atrophy.

Background Art

Muscle atrophy and wasting ^[1,2] refers to the reduction in the size of striated muscles compared to normal due to various reasons, with thinning or even disappearance of muscle fibers. The main clinical manifestations are muscle weakness, hypotonia or rigidity, muscle atrophy or hypertrophy, decreased or absent tendon reflexes, without sensory impairment and fasciculations. Diseases related to muscle atrophy include myogenic muscle atrophy, disuse muscle atrophy, senile muscle atrophy, and neurogenic muscle atrophy.

Myogenic muscular atrophy mainly refers to muscle atrophy caused by muscle pathology, including progressive muscular dystrophy, polymyositis, myotonia atrophica, and secretory myopathy. Progressive muscular dystrophy is the most common. Muscular dystrophy (MD) is a group of genetic diseases that originate in muscle tissue, and most of them have a family history. The clinical characteristics are slow onset, progressive muscle atrophy and weakness. It mainly affects the proximal muscles of the limbs, and rarely the distal muscles. Tendon reflexes disappear, and muscles are pseudo-hypertrophied ^[3] . Serum creatine phosphokinase (CK) levels are significantly increased in MD patients. The electromyography of MD patients shows myogenic damage: spontaneous potentials and fibrillation potentials appear in the insertion potential, positive sharp waves increase, or myotonic potentials appear; during mild muscle contraction, the average duration of the motor unit potential is shortened, the multiphasic potential increases, and the amplitude decreases; during severe muscle contraction, interference-type discharges are present, but the amplitude is low. In muscle tissue biopsy of MD, it can be observed that: the transverse striations of muscle fibers disappear, the thickness varies, the shape changes from polygonal to round, and the atrophied small fibers are mixed in the normal-sized or hypertrophic muscle fibers in a mosaic distribution; the number of sarcolemmal nuclei increases, is densely dark stained, arranged in chains, and the nuclei move inward; the collagen in the muscle fiber interstitium increases; there is adipocyte infiltration; and inflammatory cell infiltration is rare ^[4] . According to the affected part of the patient's muscles, it can be divided into many types ^[5] : Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), Emery-Dreifuss muscular dystrophy (EDMD), limb-girdle muscular dystrophy (LGMD), facioscapulohumeral muscular dystrophy (FSHD), acrofacial muscular dystrophy (DM), oculopharyngeal muscular dystrophy (OPMD), etc. In addition, congenital muscular dystrophy (CMD) is a condition that occurs after birth. Myotonic dystrophy (MMD) is a myotonic disease with progressive muscle weakness and wasting. Distal limb and facial muscle weakness are the main clinical manifestations. It is a multi-system myopathy with gonadal atrophy, alopecia, heart and mental disorders. Although candidate genes for various types of muscular dystrophy have been cloned, their pathogenesis is still unclear. Based on the functional characteristics of proteins encoded by pathogenic genes, it is speculated that the mechanism of MD may include the following aspects ^[6] : destruction of sarcolemma integrity, loss of connection between extracellular matrix and cytoskeleton, cytoskeleton organization defects, muscle fiber movement and contraction disorders, structural protein glycosylation blockage, protein degradation abnormalities, weakened muscle fiber regeneration ability, abnormal apoptosis of muscle cells, interruption of specific information transduction pathways in cells, etc. Faced with such a group of myopathies with complex pathogenesis and high genetic heterogeneity, it is more difficult to find effective therapeutic targets.

Sarcopenia, also known as sarcopenia, refers to the progressive loss of skeletal muscle mass, muscle strength and motor function that occurs with the aging process. Skeletal muscle mass and strength reach their peak in young adults. With age, they begin to decline around the age of 40, and skeletal muscle mass and strength decrease significantly after the age of 50. After the age of 80, skeletal muscle mass and strength are reduced to less than 50% of their youth ^[7,8] . The etiology of senile muscular atrophy is related to changes in hormone levels, imbalance in protein synthesis and degradation, neuromuscular function decline and motor unit reorganization, mitochondrial chromosome damage, free radical oxidative damage and impaired skeletal muscle repair mechanism, cell apoptosis, calcium homeostasis imbalance, and changes in calorie and protein intake. Recent studies have shown that senile muscular atrophy is closely related to the decrease in the number and functional changes of skeletal muscle stem cells. Defects in skeletal muscle regeneration in the elderly are related to skeletal muscle stem cell dysfunction ^[9] . During the aging process of skeletal muscle, skeletal muscle stem cells enter the pre-senescence stage from a quiescent state, and their aging process is accelerated under the pressure of regeneration and proliferation ^[10] . Two-thirds of skeletal muscle stem cells in old mice are defective, with low ability to repair muscle fibers and regenerate. This defect is associated with increased activity of the p38α and p38β MAPK pathways. By inhibiting p38α and p38β, the remaining functional stem cells rapidly proliferate, restoring their ability to regenerate and repair damaged skeletal muscle ^[11] . Other studies have found that as skeletal muscle stem cells age, the activity of the JAK/STAT signaling pathway gradually increases, eventually leading to functional decline in stem cells. The JAK-STAT signal in old mice is significantly higher than that in young mice. Reducing the activity of Jak2 or Stat3 can significantly stimulate muscle stem cell proliferation in vitro and in vivo, and improve muscle regeneration ^[12] . Therefore, regulating the function of skeletal muscle stem cells offers hope for intervention and treatment of senile muscle atrophy.

Disuse muscular atrophy is mainly caused by fractures, upper motor neuron system lesions or other chronic diseases that cause patients to be bedridden for a long time, and muscles do not exercise or exercise very little for a long time, leading to muscle degeneration and atrophy.

Neurogenic muscular atrophy is a group of muscular atrophy caused by lesions of motor neurons and peripheral nerves that control muscles. It mainly refers to muscular atrophy caused by lesions of lower motor neurons such as the anterior horn cells of the spinal cord and their axons. The main clinical manifestations are muscle weakness and muscular atrophy, and serum creatine phosphokinase (CK) and lactate dehydrogenase (LDH) are normal. Electromyography examination shows abnormal spontaneous units, widening of motor unit potential duration, increased amplitude, increased phase, and decreased recruitment ^[13] . Neurogenic muscular atrophy mainly includes: amyotrophic lateral sclerosis (ALS), Hirayama disease (atrophy of the affected forearm and palm muscles), spinal muscular atrophy (bilateral lower limb muscle atrophy), Charcot-Marie-Tooth disease (bilateral calf atrophy), and myasthenia gravis (MG). Amyotrophic lateral sclerosis is a motor neuron disease that may be related to gene mutations. The clinical characteristics are asymmetric atrophy and weakness of the limbs or the muscles of pronunciation and swallowing. Electromyography examination shows extensive lesions of the anterior horn cells of the spinal cord. Unilateral distal upper limb muscular atrophy in adolescents, also known as "Hirayama disease", has an unknown cause and may be related to cervical spinal cord lesions. Clinically, it is mainly manifested as atrophy of the distal muscles of one or both upper limbs, with atrophy of small muscles in the hands (interosseous muscles and thenar muscles). Charcot-Marie-Tooth disease and spinal muscular atrophy are also motor neuron diseases, both of which are related to genetic factors. They both manifest as muscle atrophy in the lower limbs, and the diseased muscle fibers are replaced by adipose tissue. Myasthenia gravis is an autoimmune disease that mainly affects the acetylcholine receptors on the postsynaptic membrane of the neuromuscular junction.

Patients with muscular atrophy lose the ability to take care of themselves due to muscle atrophy and weakness ^[14] . Their limb movements become progressively worse. Some patients experience symptoms of bulbar paralysis, and some patients experience respiratory failure and cardiac dysfunction, which seriously threatens the lives of patients and also causes serious economic losses to society. There is still a lack of effective drug treatment for this type of disease, and there is a strong unmet clinical need.

Summary of the invention

The inventors have discovered and proved through a series of experiments that the small molecule compound of indanyl levulinate can promote the repair of skeletal muscle damage, improve muscular dystrophy in mdx mice and improve the physiological function of skeletal muscle in aged mice. This series of functions is achieved by promoting the asymmetric division of skeletal muscle stem cells.

Therefore, the purpose of the present invention is to provide the use of the compound represented by formula (I) or a pharmaceutical composition containing the same in the preparation of a drug for preventing and/or treating diseases related to muscle atrophy.

In a preferred embodiment, according to the use of the present invention, the muscle atrophy-related diseases include myogenic muscle atrophy, disuse muscle atrophy, senile muscle atrophy, and neurogenic muscle atrophy, preferably myogenic muscle atrophy and senile muscle atrophy.

In another preferred embodiment, according to the use of the present invention, the myogenic muscular atrophy is progressive muscular dystrophy (MD), congenital muscular dystrophy (CMD) or myotonic dystrophy (MMD), wherein the progressive muscular dystrophy is such as Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), Emery-Dreifuss muscular dystrophy (EDMD), limb-girdle muscular dystrophy (LGMD), facioscapulohumeral muscular dystrophy (FSHD), acrofacial muscular dystrophy (DM), oculopharyngeal muscular dystrophy (OPMD).

In another preferred embodiment, according to the use of the present invention, the compound promotes the asymmetric division of skeletal muscle stem cells and accelerates the repair of skeletal muscle under damage and disease, thereby preventing and/or treating the muscle atrophy-related diseases.

In another preferred embodiment, according to the use of the present invention, the pharmaceutical composition contains an effective amount of the compound represented by formula (I) as an active ingredient and a pharmaceutically acceptable carrier or excipient.

The present invention also provides a health product for increasing muscle or resisting muscle atrophy, which comprises the compound represented by the above formula (I) and an excipient for health products.

In the present invention, myogenic muscular atrophy refers to muscular atrophy caused by muscle pathology itself, including various types of muscular dystrophy: Duchenne/Becker muscular dystrophy (DMD/BMD), Emery-Dreifuss muscular dystrophy (EDMD), limb-girdle muscular dystrophy (LGMD), facioscapulohumeral muscular dystrophy (FSHD), acrofacial muscular dystrophy (DM), oculopharyngeal muscular dystrophy (OPMD), congenital muscular dystrophy (CMD), myotonic dystrophy.

In the present invention, disuse muscle atrophy refers to the condition in which the patient is bedridden for a long time due to fracture, upper motor neuron system lesions or other chronic diseases, and the muscles are not exercised for a long time or are rarely exercised. Movement causes muscle degeneration and atrophy.

In the present invention, senile muscle atrophy refers to the progressive reduction of skeletal muscle mass, muscle strength and motor function that occurs with the aging process. Skeletal muscle mass and strength reach their peak in young adult individuals. With age, there is a gradual decline around the age of 40, and skeletal muscle mass and strength decrease significantly after the age of 50. After the age of 80, skeletal muscle mass and strength are almost reduced to less than 50% of the youth.

In the present invention, neurogenic muscular atrophy refers to a group of muscular atrophy caused by lesions of motor neurons and peripheral nerves that control muscles, mainly including amyotrophic lateral sclerosis (ALS), Hirayama disease (atrophy of the affected forearm and palm muscles), spinal muscular atrophy (atrophy of the muscles of the bilateral lower limbs), Charcot-Marie-Tooth disease (atrophy of the bilateral lower legs), etc.

The compound represented by formula (I) of the present invention can be prepared into a pharmaceutical composition or a health care product composition together with a pharmaceutically or physiologically acceptable carrier or excipient.

"Pharmaceutical composition" means a mixture containing the compound represented by formula (I) described herein and other chemical components, as well as other components such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitate the absorption of the active ingredient, and thus exert biological activity.

"Health products" are foods with specific health functions, that is, they are suitable for use by specific groups of people, have the function of regulating body functions, and are not for therapeutic purposes.

Pharmaceutical composition or health product of the present invention can be suitable for oral form, for example tablet, lozenge, lozenge, water or oil suspension, powder or granule, emulsion, hard or soft capsule, or syrup or elixir. Such composition can contain one or more components selected from the following: sweetener, flavoring agent, coloring agent and preservative, to provide pleasing and palatable preparation. Tablet contains active ingredient and nontoxic pharmaceutically acceptable excipient suitable for preparing tablet for mixing. These excipients can be inert excipient, granulating agent, disintegrant, adhesive and lubricant. These tablets can be uncoated, or can be coated by known technology that can provide sustained release effect in a long time by covering the taste of medicine or delaying disintegration and absorption in gastrointestinal tract.

The pharmaceutical composition or health product of the present invention may also be in the form of a sterile injection aqueous solution. Acceptable solvents or solvents that may be used include water, Ringer's solution and isotonic sodium chloride solution. The sterile injection preparation may be a sterile injection water-in-oil in which the active ingredient is dissolved in the oil phase. Microemulsions, injectable solutions or microemulsions can be injected into the patient's bloodstream by local bolus injection. Alternatively, solutions and microemulsions can be administered in a manner that maintains a constant circulating concentration of the compound of the invention.

Pharmaceutical composition of the present invention or health products can also be the form of sterile injection water or oil suspension for intramuscular and subcutaneous administration. Aseptic injection preparation can also be aseptic injection solution or suspension prepared in parenteral acceptable nontoxic diluent or solvent. In addition, sterile fixed oil can be used as solvent or suspension medium conveniently. For this purpose, any blending fixed oil can be used. In addition, fatty acid can also prepare injection.

The pharmaceutical composition or health product of the present invention may be added with other active ingredients, nutrients, carriers and other arbitrary components as needed. As arbitrary components, various formulation ingredients such as crystalline cellulose, gelatin, lactose, starch, magnesium stearate, talc, vegetable and animal fats, oils, gums, polyalkylene glycol and other pharmaceutically acceptable carriers, binders, stabilizers, solvents, dispersion media, brighteners, excipients, diluents, pH buffers, disintegrants, solubilizers, dissolution aids, isotonic agents and the like may be added.

As is well known to those skilled in the art, the dosage of a drug depends on a variety of factors, including but not limited to the following factors: the activity of the specific compound used, the age of the patient, the weight of the patient, the health status of the patient, the behavior of the patient, the diet of the patient, the time of administration, the mode of administration, the rate of excretion, the combination of drugs, etc.; in addition, the best treatment method such as the mode of treatment, the daily dosage of the compound of the present invention or the type of pharmaceutically acceptable salt can be verified according to traditional treatment regimens.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the statistical effect of different doses of the compound of the present invention (75, 100, 150uM) on the asymmetric division of skeletal muscle stem cells. "-" is the control group. ** represents P<0.01, indicating a significant statistical difference.

Figure 2A shows representative fields of view of HE staining and Laminin staining 7 days after skeletal muscle injury.

Figure 2 B shows the detection of skeletal muscle injury regeneration process 7 days after skeletal muscle injury. * represents P<0.05, ** represents P<0.01, indicating significant statistical difference.

Figure 3 A shows the statistical effect of 100uM of the compound on the asymmetric division ratio of skeletal muscle stem cells in mdx mice. ** indicates P<0.01, indicating a significant statistical difference.

Figure 3B shows the effect of 100uM of the compound on the asymmetry of skeletal muscle stem cells in sarcopenic mice Split ratio statistics. ** represents P < 0.01, indicating a significant statistical difference.

Figure 4 A is a graph showing the results of the detection of serum creatine kinase levels in the Mdx mice treated with the compound of the present invention and the control group 2 months after intraperitoneal injection of 5 mg/kg of the compound of the present invention. ** represents P<0.01, indicating a significant statistical difference. mdx represents a muscular dystrophy mouse with a dystrophin gene mutation.

FIG4B is a statistical graph of the single contraction muscle tension of the extensor digitorum longus (EDL) of mice in the compound-administered group and the control group. ** represents P<0.01, indicating a significant statistical difference.

Figure 4 C is a statistical graph showing the muscle tension of the extensor digitorum longus (EDL) of mice in the group treated with the compound of the present invention and the control group. ** represents P<0.01, indicating a significant statistical difference.

Figure 5 A is a graph showing the gripping force test results of mice in the compound-administered group and the control group 10 months after 5 mg/kg of the compound was intraperitoneally injected into old mice. ** represents P<0.01, indicating a significant statistical difference.

FIG5B is a graph showing the running performance test results of mice in the compound-administered group and the control group 10 months after 5 mg/kg of the compound was intraperitoneally injected into old mice. *** represents P<0.001, indicating a significant statistical difference.

Figure 5 C is a graph showing the results of single contraction tension test of mice in the compound-administered group and the control group 10 months after intraperitoneal injection of 5 mg/kg of the compound of the present invention into old mice. * indicates P<0.05, indicating statistically significant difference.

D of Figure 5 is a graph showing the results of tetanic contraction tension test of mice in the compound-administered group and the control group 10 months after intraperitoneal injection of 5 mg/kg of the compound of the present invention into old mice. ** represents P<0.01, indicating a significant statistical difference.

FIG5E is a graph showing the survival rate of mice in the compound-administered group and the control group 10 months after 5 mg/kg of the compound was intraperitoneally injected into old mice.

FIG. 6 is a graph showing the results of the survival rate test of mice in the group administered with the compound of the present invention and the control group in Test Example 6.

DETAILED DESCRIPTION

The following examples are for illustrative purposes only and are not intended to, nor should they be construed as, limiting the present invention in any way. Those skilled in the art will appreciate that routine changes and modifications may be made to the following examples without exceeding the spirit or scope of the present invention.

Preparation Example 1: Preparation of 2,3-dihydro-1H-inden-5-yl 4-oxopentanoate (I)

In an ice-water bath, 2,3-dihydro-1H-inden-5-ol (800 mg, 5.97 mmol, 1 eq), 4-oxopentanoic acid (831 mg, 7.16 mmol, 1.2 eq.), EDC.HCl (1.482 g, 7.76 mmol, 1.3 eq), DMAP (146 mg, 1.19 mmol, 0.2 eq), triethylamine (1.808 g, 17.91 mmol, 3 eq) and DMF (20 mL) were added to a 100 ml round-bottom single-necked flask in sequence and stirred at room temperature overnight. Ethyl acetate (300 mL) was added to the reaction solution, washed with saturated brine 6 times, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (ethyl acetate/petroleum ether) to obtain 1.05 g of a light yellow oil with a yield of 76%.

¹ H NMR (400MHz, CDCl ₃ )δ7.20(d,J＝8.1Hz,1H),7.00–6.92(m,1H),6.83(dd,J＝8.1,2.2Hz,1H),3.01–2.77(m,8H),2.25(s ,3H),2.11(p,J=7.4Hz,2H).

LC-MS: m/z (ES+) 296 (M+Na ⁺ CH ₃ CN) ⁺ ; 255 (M+Na) ⁺ ; Purity: 95.9%.

Experimental Example 1: Effects of the compounds of the present invention on asymmetric division of skeletal muscle stem cells

Single muscle fibers were isolated from wild-type C57BL/6 male mice aged 8-10 weeks, and asymmetric division of skeletal muscle stem cells could be observed after 42 hours of culture.

Eight-week-old C57BL/6 mice (purchased from Jicui Pharmaceuticals) were killed by cervical dislocation, and the extensor digitorum longus muscle was stripped and rinsed twice with phosphate buffered saline (PBS) (8 g NaCl, 0.2 g KCl, 0.24 g KH ₂ PO ₄ , 2.94 g Na ₂ HPO ₄ .12H ₂ O, dissolved in 1 L deionized water, pH adjusted to 7.4). Freshly prepared 1 mg/ml digestion solution (collagenase I (Gibico) dissolved in DMEM medium (Gibico)) was added and placed in an incubator (37°C, 5% CO ₂ ) for digestion for 60 minutes. Single muscle fibers were picked under a stereomicroscope.

The isolated single muscle fibers were cultured in DMEM medium (Gibico) containing 10% fetal bovine serum (Ausbian), 1% penicillin (biotopped) and 1% streptomycin (amresco). At the same time, they were treated with different concentrations of the compounds of the present invention for 42 hours. PBS treatment was used as a control. Control group. Preparation method of the compound of the present invention: using phosphate buffer (PBS) (8g NaCl, 0.2g KCl, 0.24g _{KH2PO4 , 3.58g Na2HPO4.12H2O} , dissolved in 1L deionized water, adjusting pH to 7.4) to dilute the compound stock solution (the compound is dissolved in PBS to obtain the stock solution) to 100mM, adjusting _pH to 7.4, filtering with a 0.22μm filter membrane for sterilization and then storing at 4°C. After 42 hours of compound treatment, Pax7 immunofluorescence staining was performed. The asymmetric division ratio of skeletal muscle adult stem cells was counted.

Conclusion: As shown in Figure 1, the compounds of the present invention promote the asymmetric division of skeletal muscle stem cells in a dose-dependent manner. The effective dose range of the compounds of the present invention in promoting the asymmetric division of skeletal muscle stem cells is 75 μM to 100 μM (Figure 1).

Test Example 2: Effects of the compounds of the present invention on the repair and regeneration of acute skeletal muscle injury

Wild-type C57BL/6 male mice aged 8-10 weeks were divided into two groups, with 5 mice in each group. The control group CTX solution was injected into the left tibialis anterior muscle, and the drug group CTX solution was injected into the right tibialis anterior muscle to create a skeletal muscle acute injury regeneration and repair model.

7 days after skeletal muscle injury, mice were killed by neck dislocation, and the fur of the left and right legs of the hind limbs was cut with scissors to find the tibia of the calf. The fascia on the surface of the tibialis anterior muscle was torn open with blunt forceps, and the entire tibialis anterior muscle was cut off from the tendon. The removed tibialis anterior muscle was quickly placed in muscle fixative (4% paraformaldehyde) for use (HE staining) and quickly embedded in OCT (SAKURA) and frozen in liquid nitrogen for use (frozen section immunofluorescence staining). Muscles fixed with 4% paraformaldehyde are mainly used for HE staining. HE staining shows that the nuclei of cells are stained bright blue by hematoxylin, collagen fibers are light pink, elastic fibers are bright pink, red blood cells are orange-red, and proteinaceous fluid is pink. 7 days after CTX injury of the tibialis anterior muscle, HE staining was performed to preliminarily evaluate whether the compound of the present invention can promote skeletal muscle regeneration function. OCT (SAKURA) embedded frozen section samples are mainly used for immunofluorescence staining of laminin. Laminin is mainly present in the basal lamina structure and is a non-collagen glycoprotein unique to the basal lamina. The central nuclear fiber area after skeletal muscle regeneration can be measured based on the basal lamina contour stained with laminin, thereby evaluating muscle regeneration function after injury.

Conclusion: The regeneration process of skeletal muscle injury was detected 7 days after injury. The cross-sectional area of the central nucleus muscle fibers in the group treated with the compound of the present invention (A and B in FIG2 ) increased significantly, which proved that the compound of the present invention accelerated the regeneration process of skeletal muscle injury.

Experimental Example 3: Effects of the compounds of the present invention on asymmetric division of skeletal muscle stem cells in disease states

The present invention uses mdx mice and 24-month-old aged mice as model mice for DMD disease and sarcopenia disease. Single muscle fibers are isolated from these two types of mice to detect the effect of the compound of the present invention on the asymmetric division of skeletal muscle stem cells under disease conditions.

8-week-old mdx mice (purchased from Jicui Pharmaceuticals) and 24-month-old C57BL/6 mice (purchased from Jicui Pharmaceuticals) were killed by cervical dislocation, and the extensor digitorum longus muscle was stripped and rinsed twice with phosphate buffered saline (PBS) (8 g NaCl, 0.2 g KCl, 0.24 g KH ₂ PO ₄ , 2.94 g Na ₂ HPO ₄ .12H ₂ O, dissolved in 1 L deionized water, pH adjusted to 7.4). Freshly prepared 1 mg/ml digestion solution (collagenase I (Gibico) dissolved in DMEM medium (Gibico)) was added and placed in an incubator (37°C) for digestion for 60 minutes. Single muscle fibers were picked under a stereomicroscope.

Single muscle fibers isolated from diseased mice were cultured in DMEM medium (Gibico) containing 10% fetal bovine serum (Ausbian), 1% penicillin (biotopped) and 1% streptomycin (amresco). At the same time, 100 μM of the compound of the present invention was used for 42 hours. PBS treatment was used as a control group. Preparation method of the compound of the present invention: phosphate buffered saline (PBS) (8g NaCl, 0.2g KCl, 0.24g KH ₂ PO ₄ , 3.58g Na ₂ HPO ₄ .12H ₂ O, dissolved in 1L deionized water, pH adjusted to 7.4) was used to dilute the compound stock solution (the compound was dissolved in PBS to obtain the stock solution) to 100mM, the pH was adjusted to 7.4, and the solution was sterilized by filtering with a 0.22μm filter membrane and stored at 4°C. After 42 hours of compound treatment, Pax7 immunofluorescence staining was performed. The asymmetric division ratio of skeletal muscle adult stem cells was counted.

Conclusion: As shown in A and B of FIG. 3 , the compounds of the present invention significantly promote the asymmetric division of skeletal muscle stem cells in mdx mice (A) and the asymmetric division of skeletal muscle stem cells in sarcopenia mice (B).

Test Example 4: Effect of the compounds of the present invention on significantly improving the pathological phenotype of muscular dystrophy Mdx mice

DMD muscular dystrophy is a very serious muscle disease with X-linked recessive inheritance. Clinically, it develops slowly and manifests as progressive skeletal muscle atrophy and weakness. The prognosis is poor, and death is usually caused by myocardial failure or dyspnea at the age of 20-30.

Mdx mice are a commonly used DMD muscular dystrophy model. The disease in Mdx mice is milder than that in humans. They have a mutation in the dystrophin gene and suffer from severe muscle damage three weeks after birth, followed by a periodic repair of muscle damage.

8-week-old Mdx mice were purchased from Jicui Pharmaceutical Co., Ltd. and bred to obtain Mdx and wild-type (WT) offspring mice in the same cage. At 8 weeks of age, 5 mg/kg of the compound of the present invention (PBS diluted to 500 mM, pH adjusted to 7.4) was intraperitoneally injected daily. PBS (8 g NaCl, 0.2 g KCl, 0.24 g KH ₂ PO ₄ , 3.58 g Na ₂ HPO ₄ ·12H2O, dissolved in 1 liter of deionized water, pH adjusted to 7.4) was used as the control group, with 8-10 Mdx mice in each group. The injection was maintained for 2 months. After the administration, the integrity of skeletal muscle fibers and the physiological function of skeletal muscles were detected.

Measurement of creatine kinase: After the Mdx mice were administered with the drug, blood was collected from the tail vein after running for 4 hours. The whole blood was kept at 4°C for 1 hour, centrifuged at 4000 rpm for 5 minutes, and the supernatant was removed with a pipette and transferred to a new tube, which was the serum. The creatine kinase content in the serum was measured using the CK kit (Abnova).

Conclusion: Figure 4A shows that the serum creatine kinase level in the group treated with the compound of the present invention is significantly lower than that in the control group. Therefore, the integrity of the sarcolemma of the muscle tissue of Mdx mice was significantly improved after daily intraperitoneal injection of the compound of the present invention for 2 months.

Mouse skeletal muscle tension measurement: Prepare electrolyte in advance (NaCl 6.925g, KCl 0.35g, CaCl ₂ 0.266g, MgCl ₂ 0.63g, NaHCO ₃ 2.1g, NaH ₂ PO ₄ 0.312g, D-glucose 0.991g, dissolved in 1L deionized water, pH value adjusted to 7.4, all the above reagents were purchased from Sigma). Add the electrolyte into a 37°C constant temperature bath (Chengdu Instrument Factory) and introduce oxygen. The mice were killed by vertebral dissection, and the extensor digitorum longus muscle with intact tendons was removed. The tendons at both ends were fixed with surgical sutures, and the extensor digitorum longus muscle was fixed in a constant temperature bath and suspended in the middle of the electric shock circle of the S88X dual-channel square wave stimulator (GRASS). The computer controlled the adjustment parameters of the electric stimulation: single contraction muscle strength: voltage: 20 volts, stimulation time 0.3 milliseconds, repeated three times; tetanic contraction muscle strength: voltage: 20 volts, stimulation time: 0.2 milliseconds, stimulation frequency: 200, and the measured tension values were recorded.

Conclusion: As shown in Figure 4B and Figure 4C, the treatment with the compound of the present invention significantly increased the tetanic force and single contraction force of mice. These results show that the compound of the present invention can significantly improve the morphological structure and function of the muscles of Mdx mice, indicating that the compound of the present invention has the potential to treat human muscular dystrophy.

Test Example 5: The compound of the present invention significantly improves the muscle function of elderly mice

The experiment was started when C57BL/6 mice (purchased from Jicui Pharmaceutical Co., Ltd.) were grown to 14 months old. The compound of the present invention (5 mg/kg) (diluted to 500 mM with PBS, adjusted to pH = 7.4) was injected intraperitoneally. PBS (8g NaCl, 0.2g KCl, 0.24g KH ₂ PO ₄ , 3.58g Na ₂ HPO ₄ ·12H ₂ O, dissolved in 1 liter of deionized water, pH adjusted to 7.4) was used as the control group, with 8-10 mice in each group, and the injection was maintained for 10 months. After the administration, the skeletal muscle physiological function and the mortality rate of the aged mice were detected.

Forelimb grip strength measurement: install the grid accessory onto the grip force sensor (BioSEB GS3), place the grip force meter in a stable position, press and hold the start button, and start the experiment when the instrument self-checks and 0.0 is displayed on the screen; place the mouse's forelimbs on the grid, grab the mouse's tail, and pull the mouse backwards along the grid at a steady speed until the limbs are out of the grid, record the value and press the ZERO button to reset it; measure the force of each mouse ten times a day for three days, and take the maximum value as the grip strength value of the mouse for comparison between groups.

Mouse running experiment: connect the mouse running instrument (Columbus Exer-3/6) to the computer and turn on the power supply, and put the mouse in the running cabin channel; open the connection software on the computer, set the conveyor belt rotation, that is, the mouse running speed program: balance at a speed of 10m/min for 3 minutes, then accelerate from 1m/min to 20m/min, and finally move at a constant speed of 20m/min; after the program is set, turn on the electrical stimulation switch at one end of the running cabin channel, and start recording the running time of the mouse. The standard of mouse fatigue is that the mouse still does not jump onto the conveyor belt under the electrical stimulation condition.

Mouse skeletal muscle tension measurement: Prepare electrolyte (NaCl 6.925g, KCl 0.35g, CaCl ₂ 0.266g, MgCl ₂ 0.63g, NaHCO ₃ 2.1g, NaH ₂ PO ₄ 0.312 g, D-glucose 0.991 g, dissolved in 1 L of deionized water, adjusted to pH 7.4, all the above reagents were purchased from Sigma); the electrolyte was added to a 37°C constant temperature bath (Chengdu Instrument Factory), and oxygen was introduced; the mice were killed by vertebral dissection, the extensor digitorum longus with intact tendons were removed, the tendons at both ends were fixed with surgical sutures, the extensor digitorum longus was fixed in a constant temperature bath, and suspended in the middle of the electric shock circle of the S88X dual-channel square wave stimulator (GRASS); the computer controlled electrical stimulation adjustment parameters: single contraction muscle strength: voltage: 20 volts, stimulation time: 0.3 milliseconds, repeated three times; tetanic contraction muscle strength: voltage: 20 volts, stimulation time: 0.2 milliseconds, stimulation frequency: 200, and the measured tension values were recorded.

Conclusion: As shown in FIG5 , the treatment with the compound of the present invention significantly improved the forelimb gripping force (A in FIG5 ) and running performance (B in FIG5 ), single contraction force (C in FIG5 ) and tetanic contraction force (D in FIG5 ) of mice. At the same time, the treatment with the compound of the present invention significantly improved the survival rate of aged mice (E in FIG5 ). These experimental results show that the treatment with the compound of the present invention significantly improved the muscle physiological function and mortality rate of aged mice.

Test Example 6: Acute toxicity test of the compounds of the present invention

49 8-week-old C57BL/6 mice (purchased from Jicui Pharmaceutical Co., Ltd.) were administered with the compound of the present invention by oral gavage at doses of 0.05 g/kg, 0.1 g/kg, 0.5 g/kg, 1 g/kg, 2 g/kg, and 5 g/kg (PBS diluted to 500 mM, pH adjusted to 7.4), and PBS (8 g NaCl, 0.2 g KCl, 0.24 g KH ₂ PO ₄ , 3.58 g Na ₂ HPO ₄ ·12H ₂ O, dissolved in 1 liter of deionized water, pH adjusted to 7.4) as a control group, 7 mice in each group. After the administration, the mice were observed for 4 hours, and the vocalization and convulsion conditions were recorded. The number of deaths was recorded for 7 consecutive days.

Conclusion: The compound of the present invention has a relatively high biological safety. The administration method is adopted. After the administration, the groups with the dosage of 1g/kg, 2g/kg and 5g/kg showed vocalization and convulsion within 4 hours. The death within 7 days after administration was observed. First, there was no death in the control group; in the group with the dosage of 5g/kg, the number of deaths within 7 days was 4 (57.14%); in the group with the dosage of 2g/kg, the number of deaths within 7 days was 1 (14.29%); in the group with the dosage of 1g/kg, the number of deaths within 7 days was 1 (14.29%); in the group with the dosage of 0.5g/kg, 0.2g/kg, 0.1g/kg and 0.05g/kg, the number of deaths within 7 days was 0 (0.00%) (see Figure 6).

These experimental results show that the compounds of the present invention are between Grade 2 (actually non-toxic) and Grade 3 (low toxicity) in the acute toxicity classification standard (reference compound oral acute toxicity classification standard), indicating that the present invention has relatively high biosafety.

References

1.Thakur SS,Swiderski K,Ryall JG,Lynch GS.Therapeutic potential of heat shock protein induction for muscular dys trophy and other muscle wasting conditions.Philos Trans R Soc Lond B Biol Sci.2018 Jan 19;373(1738).pii:20160528.

2.McNally EM and MacLeod H.Therapy insight:cardiovascular complications associated with muscular dystrophies. Nat Clin Pract Cardiovasc Med, 2005.2(6):p.301-8.

3. Liang Xiuling (chief editor), Chen Rong, Zhang Cheng, Xu Pingyi, Li Xunhua (co-editor). Hereditary Diseases of the Nervous System, People's Military Medical Publishing House, July 2001.

4. Chen Bifen, Pathology of Skeletal Muscle Diseases, Fujian Science and Technology Press, 1993.10.

5.Alan EHEmery.Muscular dystrophy into the new millennium.Neuromuscular Disorders 2002,12:343-349.

6.Allen DG,Whitehead NP,Froehner SC.Absence of Dystrophin Disrupts Skeletal Muscle Signaling:Roles of Ca2+,React ive Oxygen Species,and Nitric Oxide in the Development of Muscular Dystrophy.Physiol Rev.2016 Jan;96(1):253-305.

7.Dodds R, Sayer AA. Sarcopenia and frailty: new challenges for clinical practice. Clin Med (Lond). 2015 Dec; 15 Suppl 6:s88-91.

8. Dennison EM, Sayer AA, Cooper C. Epidemiology of sarcopenia and insight into possible therapeutic targets. Nat Rev Rheumatol. 2017 Jun; 13(6):340-347.

9.Brack AS,Bildsoe H,Hughes SM.Evidence that satellite cell decrement contributions to preferential decline in nuc Lear number from large fibers during murine age-related muscle atrophy.J Cell Sci.2005 Oct 15;118(Pt 20):4813-21.

10.Sousa-Victor P,Gutarra S,García-Prat L,Rodriguez-Ubreva J,Ortet L,Ruiz-Bonilla V,JardíM,Ballestar E,González S,Serrano AL,Perdiguero E, P.Geriatric muscle stem cells switch reversible quiescence into senescence.Nature.2014 Feb 20;506(7488):316-21.

11.Cosgrove BD,Gilbert PM,Porpiglia E,Mourkioti F,Lee SP,Corbel SY,Llewellyn ME,Delp SL,Blau HM.Rejuvenation of the muscle stem cell population restores strength to injured aged muscles. Nat Med. 2014 Mar; 20(3):255-64.

12.Price FD, von Maltzahn J, Bentzinger CF, Dumont NA, Yin H, Chang NC, Wilson DH, Frenette J, Rudnicki MA.Inh ibition of JAK-STAT signaling stimulates adult satellite cell function. Nat Med.2014 Oct; 20(10):1174-81.

13.Preston DC,Shapiro BE.Approach to Nerve Conduction Studies and Electromyography-Electromyography and Neuromuscular Disorders(Third Edition)

14.Sandri M.Signaling in muscle atrophy and hypertrophy.Physiology(Bethesda), 2008.23:p.160-70.

15. Finucane MM, Stevens GA, Cowan MJ, Danaei G, Lin JK, Paciorek CJ, Singh GM, Gutierrez HR, Lu Y, Bahalim AN, Farzadfar F, Riley LM, Ezzati M. National, regional, and global trends in body -mass index since 1980:systematic analysis of health examination surveys and Epidemiological studies with 960 country-years and 9·1 million participants. Lancet. 2011 Feb 12;377(9765):557-67.

16.Kelly T1,Yang W,Chen CS,Reynolds K,He J.Global burden of obesity in 2005 and projections to 2030.Int J Obes(Lond).2008 Sep;32(9):1431-7.

17. Guidelines for the prevention and control of overweight and obesity in Chinese adults (trial version). Department of Disease Control, Ministry of Health of the People’s Republic of China. April 2003.

18. Finkelstein E, Trogdon J, Cohen JW, Dietz W. Annual medical spending attributable to obesity:payer-and service-specific estimates. Health Affairs 2009; 28:822-831.

19. 2013-2018 China's Weight Loss Drug Market Analysis and Industry Operation Status Report. Bosi Data Research Center. August 2013.

20. Nikolich-Zugich J. The twilight of immunity: emerging concepts in aging of the immune system. Nat Immunol, 2018.19(1):p.10-19.

21. Sanchis-Gomar F, Gomez-Cabrera MC, Vina J. The loss of muscle mass and sarcopenia:non hormonal intervention. Exp Gerontol, 2011.46(12):p.967-9.

Claims (6)

Use of the compound represented by formula (I) or a pharmaceutical composition containing the same in the preparation of a drug for preventing and/or treating diseases related to muscle atrophy,
The use according to claim 1, wherein the muscle atrophy-related disease is myogenic muscle atrophy, disuse muscle atrophy, senile muscle atrophy, neurogenic muscle atrophy, preferably myogenic muscle atrophy and senile muscle atrophy. The use according to claim 1 or 2, wherein the myogenic muscular atrophy is progressive muscular dystrophy, congenital muscular dystrophy or myotonic dystrophy, wherein the progressive muscular dystrophy is such as Duchenne muscular dystrophy, Becker muscular dystrophy, Emery-Dreifuss muscular dystrophy, limb-girdle muscular dystrophy, facioscapulohumeral muscular dystrophy, acromuscular dystrophy, oculopharyngeal muscular dystrophy. The use according to any one of claims 1 to 3, wherein the compound promotes asymmetric division of skeletal muscle stem cells and accelerates the repair of skeletal muscle under damage and disease, thereby preventing and/or treating the muscle atrophy-related diseases. The use according to any one of claims 1 to 4, wherein the pharmaceutical composition contains an effective amount of the compound represented by formula (I) as an active ingredient and a pharmaceutically acceptable carrier or excipient. A health product for increasing muscle or combating muscle atrophy, comprising a compound represented by formula (I) and an excipient for health products,