CN116617205A - Use of indanyl levulinate in preventing and/or treating diseases related to muscle atrophy - Google Patents

Abstract

The present invention relates to the use of indanyl levulinate for the prevention and/or treatment of muscle wasting-related diseases. In particular, the invention relates to the use of the indanyl levulinate compound shown in the formula (I) or a pharmaceutical composition containing the indanyl levulinate compound in preventing and/or treating muscle-atrophy related diseases including myogenic muscle atrophy, disuse muscle atrophy, senile muscle atrophy and neurogenic muscle atrophy. The compound can have remarkable effect of preventing and/or treating diseases related to muscular atrophy by promoting the asymmetric division of skeletal muscle stem cells.

Description

The role of indanyl levulinate in the prevention and/or treatment of diseases related to muscle atrophy use

technical field

The present invention relates to the use of a novel indanyl levulinate compound or a pharmaceutical composition containing it in the prevention and/or treatment of diseases related to muscle atrophy.

Background technique

Muscle atrophy (Muscle atrophy and wasting) ^[1,2] refers to the reduction in the volume of striated muscles due to various reasons, and the muscle fibers become thinner or even disappear. The clinical manifestations are mainly muscle weakness, muscle hypotonia or rigidity, muscle atrophy or hypertrophy, decreased or absent tendon reflexes, without sensory disturbance and muscle fasciculation. Diseases related to muscular atrophy include myogenic muscular atrophy, disuse muscular atrophy, senile muscular atrophy, and neurogenic muscular atrophy.

Myogenic muscular atrophy mainly refers to muscle atrophy caused by muscle lesions, including progressive muscular dystrophy, polymyositis, atrophic rigidity, and secretory myopathy. Progressive muscular dystrophy is more common. Progressive muscular dystrophy (Muscular Dystrophy, MD) is a group of genetic diseases that originate in the muscle tissue, and most of them have a family history; the clinical features are slow onset, progressive muscle atrophy and weakness; it mainly involves the proximal muscles of the limbs , very few are distal; tendon reflexes disappear, muscle pseudohypertrophy ^[3] . Serum creatine phosphokinase (CK) levels were significantly elevated in MD patients. The EMG of patients with MD showed myogenic damage: spontaneous potentials and fibrillation potentials appeared in insertion potentials, increased positive sharp waves, or myotonic potentials; the average duration of motor unit potentials was shortened during mild muscle contraction, and multiphasic potentials increased , the amplitude is reduced; when the muscle contraction is severe, the discharge is disturbing, but the amplitude is low. Muscle tissue biopsy of MD can be observed: muscle fiber striations disappear, varying in thickness, from polygonal to round, atrophic small fibers mixed in normal volume or hypertrophic muscle fibers in a mosaic distribution; sarcolemma nuclei increase, dense and deep staining, chain arrangement, and inward migration of nuclei; increased interstitial collagen between muscle fibers; adipocyte infiltration; inflammatory cell infiltration is rare ^[4] . Patients can be divided into many types according to the muscle involvement ^[5] : Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), Emery-Dreifuss muscular dystrophy (EDMD), limb -Girdle muscular dystrophy (LGMD), face-shoulder-humeral muscular dystrophy (FSHD), extremity muscular dystrophy (DM), oculopharyngeal muscular dystrophy (OPMD), etc. In addition, congenital muscular dystrophy (CMD) is a type of severe muscular dystrophy with onset after birth; myotonic muscular dystrophy (MMD) is a myotonic disease with progressive muscle weakness and muscle exhaustion. Facial weakness is the main clinical manifestation. It is a myopathy with multisystem damage, accompanied by gonadal atrophy, alopecia, heart and mental disorders. Although candidate genes for various types of muscular dystrophy have been cloned, their pathogenesis remains unclear. According to the functional characteristics of the protein encoded by the disease-causing gene, it is speculated that the mechanism of MD may have the following aspects ^[6] : damage to the integrity of the sarcolemma, loss of the connection between the extracellular matrix and the cytoskeleton, defects in the organization of the cytoskeleton, impairment of muscle fiber movement and contraction, Structural protein glycosylation is blocked, protein degradation is abnormal, muscle fiber regeneration ability is weakened, muscle cells are abnormally apoptosis, and specific information transduction pathways in cells are interrupted. Facing such a myopathy group with complex pathogenesis and highly genetic heterogeneity, it is more difficult to find effective therapeutic targets.

Senile muscular atrophy, also known as sarcopenia, refers to the progressive loss of skeletal muscle mass, muscle strength, and motor function that accompany the aging process. Skeletal muscle mass and strength peak in young adult individuals. With age, there is a gradual decline around the age of 40, and the mass and strength of skeletal muscle decrease significantly after the age of 50. After the age of 80, the mass and strength of skeletal muscle are almost reduced to less than 50% of the youth ^[7,8] . The etiology of senile muscular atrophy and changes in hormone levels, protein synthesis and decomposition imbalance, neuromuscular function decline and motor unit reorganization, mitochondrial chromosome damage, free radical oxidative damage and repair mechanism damage of skeletal muscle, cell apoptosis, calcium homeostasis State imbalance, changes in caloric and protein intake, etc. Recent studies have shown that age-related muscle atrophy is closely related to the decrease in the number and function of skeletal muscle stem cells. Defects in aging skeletal muscle regeneration are associated with skeletal muscle stem cell dysfunction ^[9] . During the aging process of skeletal muscle, skeletal muscle stem cells enter the pre-aging stage from a resting state, and accelerate their aging process under the pressure of regeneration and proliferation ^[10] . Two-thirds of the skeletal muscle stem cells in aged mice were defective and less able to repair muscle fibers and regenerate. This defect is associated with increased activity of the p38α and p38β MAPK pathways. Inhibiting p38α and p38β among them, the stem cells that still have function have undergone rapid expansion and restored the ability to regenerate and repair damaged skeletal muscle ^[11] . Other studies have found that with the aging of skeletal muscle stem cells, the activity of the JAK/STAT signaling pathway will gradually increase, eventually leading to functional decline of stem cells. JAK-STAT signaling was significantly higher in aged mice than in young mice. Reducing the activity of Jak2 or Stat3 can significantly stimulate the proliferation of muscle stem cells in vitro and in vivo, and improve the regeneration ability of muscles ^[12] . Therefore, regulating the function of skeletal muscle stem cells brings hope for the intervention and treatment of senile muscle atrophy.

Disuse muscular atrophy is mainly due to fractures or upper motor neuron system lesions or other chronic diseases that cause patients to stay in bed for a long time, and the muscles do not exercise for a long time or rarely exercise, resulting in muscle degeneration and atrophy.

Neurogenic muscular atrophy is a group of muscle atrophy caused by motor neurons and peripheral neuropathy that innervates muscles. The main clinical manifestations were muscle weakness and muscle atrophy, and serum creatine phosphokinase (CK) and lactate dehydrogenase (LDH) were normal. Needle electromyography showed that abnormal spontaneous units were dispensable, motor unit potential duration was broadened, amplitude increased, phase increased, and recruitment decreased ^[13] . Neurogenic muscular atrophy mainly includes: amyotrophic lateral sclerosis (ALS), Hirayama disease (affected forearm and palm muscle atrophy), spinal muscular atrophy (bilateral lower extremity muscle atrophy), Charcot-Marie-Tooth disease (bilateral calf atrophy ), myasthenia gravis (MG). Amyotrophic lateral sclerosis is a motor neuron disease that may be related to gene mutations. It is clinically characterized by asymmetric onset of limbs or muscle atrophy and weakness of the articulation and swallowing muscles. Electromyography shows extensive anterior horns of the spinal cord Cytopathy. Adolescent unilateral distal upper limb muscular atrophy, also known as "Hirayama disease", has an unknown etiology and may be related to cervical spinal cord lesions. Clinically, it mainly manifests as one or both distal upper limb muscular atrophy, especially small hand muscle atrophy. (Interosseous muscles, thenar muscles) are obvious. Charcot-Marie-Tooth 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 primarily affects acetylcholine receptors on the postsynaptic membranes of the neuromuscular junction.

Patients with muscular atrophy suffer from loss of self-care ability due to muscle atrophy and muscle weakness ^[14] , progressive exacerbation of limb movements, some patients have symptoms of bulbar paralysis, and some patients have respiratory failure and cardiac dysfunction, which seriously threaten the lives of patients and also cause serious economic losses to society. There is still a lack of effective drug treatments for such diseases, and there is a strong unmet clinical need.

Contents of the invention

Through a series of experiments, the inventors found and proved that the small molecular 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. And this series of functions is realized by promoting the asymmetric division of skeletal muscle stem cells.

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

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

In another preferred embodiment, according to the purposes of the present invention, wherein the myogenic muscle atrophy is progressive muscular dystrophy (MD), congenital muscular dystrophy (CMD) or myotonic dystrophy ( MMD), wherein the progressive muscular dystrophy is for example Duchenne muscular dystrophy (DMD), Becker (Becker) muscular dystrophy (BMD), Emery-Dreifuss muscular dystrophy (EDMD), limb-girdle muscular dystrophy (LGMD), face-shoulder-humeral muscular dystrophy (FSHD), acral muscular dystrophy (DM), oculopharyngeal muscular dystrophy (OPMD).

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

In another preferred embodiment, according to the use of the present invention, 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.

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 excipients for health product.

In the present invention, myogenic muscle atrophy refers to muscle atrophy caused by the disease of the muscle itself. Including various types of muscular dystrophies: Duchenne/Becker muscular dystrophy (DMD/BMD), Emery-Dreifuss muscular dystrophy (EDMD), limb-girdle muscular dystrophy (LGMD), Facial-shoulder-brachial muscular dystrophy (FSHD), acral muscular dystrophy (DM), oculopharyngeal muscular dystrophy (OPMD), congenital muscular dystrophy (CMD), myotonic dystrophy.

In the present invention, disuse muscle atrophy refers to the long-term bed rest of the patient due to fracture or upper motor neuron system disease or other chronic diseases, and the muscles do not exercise for a long time or rarely exercise, resulting in muscle degeneration and atrophy.

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

In the present invention, neurogenic muscle atrophy refers to a group of muscle atrophy due to peripheral neuropathy of motor neurons and innervating muscles. Mainly include: amyotrophic lateral sclerosis (ALS), Hirayama disease (affected forearm and palm muscle atrophy), spinal muscular atrophy (bilateral lower extremity muscle atrophy), Charcot-Marie-Tooth disease (bilateral calf atrophy), etc.

The compound represented by the formula (I) of the present invention can be prepared into a pharmaceutical composition or a health 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 physiological/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to promote the administration to the organism, facilitate the absorption of the active ingredient and thus exert biological activity.

"Health care products" are foods with specific health functions, that is, a type of food that is suitable for specific groups of people, has the ability to regulate body functions, and is not for the purpose of treatment.

The pharmaceutical composition or health product of the present invention can be in a form suitable for oral administration, such as tablet, troche, lozenge, water or oil suspension, powder or granule, emulsion, hard or soft capsule , or syrup or elixir. Such compositions may contain one or more ingredients selected from the group consisting of sweetening, flavoring, coloring and preservative agents to provide a pleasing and palatable preparation. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients suitable for the manufacture of tablets. These excipients may be inert excipients, granulating agents, disintegrants, binders, and lubricants. These tablets may be uncoated, or they may be coated by known techniques to mask the taste of the drug or to delay disintegration and absorption in the gastrointestinal tract, thereby providing sustained release over an extended period of time.

The pharmaceutical composition or health care product described in the present invention can also be in the form of sterile injectable aqueous solution. Among the acceptable vehicles or solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. The sterile injectable preparation may be a sterile injectable oil-in-water microemulsion in which the active ingredient is dissolved in an oily phase, and the injection or microemulsion can be injected into the patient's bloodstream by local bulk injection. Alternatively, solutions and microemulsions are administered in such a way as to maintain a constant circulating concentration of the compounds of the invention.

The pharmaceutical composition or health product of the present invention can also be in the form of sterile injection water or oil suspension for intramuscular and subcutaneous administration. The sterile injectable preparation may also be a sterile injectable solution or suspension prepared in a parenterally acceptable non-toxic diluent or solvent. In addition, sterile fixed oils are conveniently employed as a solvent or suspending medium. For this purpose, any blended and fixed oil may be used. In addition, fatty acids are also used in the preparation of injectables.

The pharmaceutical composition or health care product of the present invention can be added with other optional ingredients such as other medicinal ingredients, nutritional agents, carriers, etc. as required. As optional ingredients, pharmaceutically acceptable carriers and binders such as crystalline cellulose, gelatin, lactose, starch, magnesium stearate, talc, vegetable and animal fats, oils, gums, polyalkylene glycols, etc. can be added , Stabilizers, solvents, dispersion media, brighteners, excipients, diluents, pH buffers, disintegrants, solubilizers, dissolution aids, isotonic agents and other formulation ingredients.

As is well known to those skilled in the art, the dosage of the drug to be administered depends on many 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 state of health of the patient, the behavior of the patient , the patient's diet, administration time, administration method, excretion rate, 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 based on Traditional treatment options to validate.

Description of drawings

Fig. 1 is the proportion statistics of asymmetric division of skeletal muscle stem cells treated with different doses of the compound of the present invention (75, 100, 150uM). "-" is the control group. ** stands for P<0.01, indicating significant statistical difference.

A of Fig. 2 is a representative field of HE staining and Laminin staining 7 days after skeletal muscle injury.

B of Fig. 2 is the detection of regeneration process of skeletal muscle injury 7 days after skeletal muscle injury. * stands for P<0.05, ** stands for P<0.01, indicating significant statistical difference.

A of Figure 3 is the proportion statistics of the asymmetric division of skeletal muscle stem cells in mdx mice treated with 100 uM of the compound. ** stands for P<0.01, indicating significant statistical difference.

B of Fig. 3 is the proportion statistics of the asymmetric division of skeletal muscle stem cells in sarcopenic mice treated with 100 uM of the compound. ** stands for P<0.01, indicating significant statistical difference.

A of Fig. 4 is a graph showing the detection results of creatine kinase levels in the serum of the compound-administered group of the present invention and the control group after 5 mg/kg of the compound of the present invention was intraperitoneally injected into Mdx mice for 2 months. ** stands for P<0.01, indicating significant statistical difference. mdx stands for muscular dystrophic mice with mutations in the dystrophin gene.

B of Fig. 4 is a single-contraction muscle tension chart of the extensor digitorum longus (EDL) of the mice administered with the compound of the present invention and the control group. ** stands for P<0.01, indicating significant statistical difference.

Figure 4 C is a statistical chart of the muscle tension of the extensor digitorum longus (EDL) of the mice administered with the compound of the present invention and the control group. ** stands for P<0.01, indicating significant statistical difference.

A of Fig. 5 is a diagram showing the test results of the grip strength of mice in the compound-administered group of the present invention and the control group after 10 months of intraperitoneal injection of the compound of the present invention into aged mice. ** stands for P<0.01, indicating significant statistical difference.

B of Fig. 5 is a graph showing the running performance detection results of mice in the compound-administered group of the present invention and the control group after 10 months of intraperitoneal injection of the compound of the present invention into aged mice. *** stands for P<0.001, indicating significant statistical difference.

C of Fig. 5 is a graph showing the single contraction tension test results of the mice administered with the compound of the present invention and the control group after 10 months of intraperitoneal injection of the compound of the present invention into aged mice. * stands for P<0.05, indicating statistical difference.

D of Figure 5 is a graph showing the test results of tetanic contraction tension of the mice administered with the compound of the present invention and the control group after 10 months of intraperitoneal injection of the compound of the present invention into aged mice. ** stands for P<0.01, indicating significant statistical difference.

E of Fig. 5 is a graph showing the detection results of the survival rate of the mice in the compound-administered group of the present invention and the control group after 10 months of intraperitoneal injection of the compound of the present invention into aged mice.

Fig. 6 is a graph showing the detection results of the survival rate of the mice in the compound administration group of the present invention and the control group in Test Example 6.

Detailed ways

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

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

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

Test example 1: the effect of the compound of the present invention on the asymmetric division of skeletal muscle stem cells

A single muscle fiber was isolated from wild-type C57BL/6 male mice aged 8-10 weeks and cultured for 42 hours to observe asymmetric division of skeletal muscle stem cells.

Eight-week-old C57BL/6 mice (purchased from Jizui Yaokang Co., Ltd.) were sacrificed by neck dislocation, the extensor digitorum longus muscle was stripped, and phosphate-buffered saline (PBS) (8g NaCl, 0.2g KCl, 0.24g KH ₂ PO ₄ , 2.94g Na ₂ HPO ₄ .12H ₂ O, dissolved in 1L deionized water, adjust the pH value to 7.4) rinse twice. Add freshly prepared 1 mg/ml digestion solution (collagenase I (Gibico) dissolved in DMEM medium (Gibico)), and place 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 served as the control group. Compound preparation method of the present invention: use phosphate buffer saline (PBS) (8gNaCl, 0.2g KCl, 0.24g KH ₂ PO ₄ , 3.58g Na ₂ HPO ₄ .12H ₂ O, dissolve in 1L deionized water, adjust the pH value to 7.4) Dilute the stock solution of the compound (prepared by dissolving the compound in PBS) to 100 mM, adjust the pH value to 7.4, filter and sterilize through a 0.22 μm filter membrane, and store in 4°C. Pax7 immunofluorescent staining was performed 42 hours after compound treatment. Statistics of asymmetric division ratio of skeletal muscle adult stem cells.

Conclusion: As shown in Figure 1, the compound of the present invention dose-dependently promotes the asymmetric division of skeletal muscle stem cells. The effective dosage range of the compounds of the present invention for promoting the asymmetric division of skeletal muscle stem cells is 75 μM-100 μM ( FIG. 1 ).

Test example 2: the effect of the compound of the present invention on the repair and regeneration of acute injury of skeletal muscle

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

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

Conclusions: The regeneration process of skeletal muscle injury was detected 7 days after injury. The cross-sectional area of central nucleus muscle fibers (A and B of FIG. 2 ) was significantly increased in the compound-treated group of the present invention. It is proved that the compound of the present invention accelerates the regeneration process of skeletal muscle injury.

Test example 3: the effect of the compound of the present invention on the asymmetric division of skeletal muscle stem cells in disease state

The present invention adopts mdx mice and 24-month old mice as model mice of DMD disease and sarcopenia disease. Single muscle fibers were isolated from these two kinds of mice to detect the effect of the compound of the present invention on the asymmetric division of skeletal muscle stem cells in disease state.

8-week-old mdx mice (purchased from Jizui Pharmaceutical Company) and 24-month-old C57BL/6 mice (purchased from Jizui Pharmaceutical Company) were sacrificed by neck dislocation, the extensor digitorum longus was stripped, and phosphate buffered saline ( PBS) (8g NaCl, 0.2g KCl, 0.24gKH ₂ PO ₄ , 2.94g Na ₂ HPO ₄ .12H ₂ O, dissolved in 1L deionized water, adjusted to pH 7.4) and rinsed twice. Add freshly prepared 1 mg/ml digestion solution (collagenase I (Gibico) dissolved in DMEM medium (Gibico)), and place 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). Simultaneously, they were treated with 100 μM of the compound of the present invention for 42 hours. PBS treatment served as the control group. The preparation method of the compound of the present invention: use phosphate buffer saline (PBS) (8g NaCl, 0.2g KCl, 0.24g KH ₂ PO ₄ , 3.58g Na ₂ HPO ₄ .12H ₂ O, dissolve in 1L deionized water, adjust the pH value To 7.4) Dilute the stock solution of the compound (prepared by dissolving the compound in PBS) to 100 mM, adjust the pH value to 7.4, filter and sterilize through a 0.22 μm filter membrane, and store at 4°C. Pax7 immunofluorescent staining was performed 42 hours after compound treatment. Statistics of asymmetric division ratio of skeletal muscle adult stem cells.

Conclusion: As shown in A and B of Figure 3, the compound of the present invention significantly promotes the asymmetric division of skeletal muscle stem cells in mdx mice (A) and the asymmetric division of skeletal muscle stem cells in sarcopenic mice (B).

Test Example 4: The compound of the present invention significantly improves the pathological phenotype of muscular dystrophy Mdx mice

DMD muscular dystrophy is a very serious muscle disease with X-linked recessive genetic properties, clinically slow onset, manifested as progressive aggravation of skeletal muscle atrophy and weakness. The prognosis is poor, and it usually dies with myocardial failure or dyspnea at the age of 20-30.

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

8-week-old Mdx mice were purchased from Jizui Yaokang Co., Ltd., and were bred to obtain Mdx and wild-type (WT) progeny mice in the same cage. At the age of 8 weeks, daily intraperitoneal injection of the compound of the present invention at a dose of 5 mg/kg (diluted to 500 mM in PBS, adjusted to pH=7.4), 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, adjusted to pH 7.4) as a control group, 8-10 Mdx mice in each group, and the injection was maintained for 2 months. After administration, the integrity of skeletal muscle fibers and the physiological function of skeletal muscle were detected.

Measurement of creatine kinase: Blood was collected from the tail vein of the Mdx mice after running for 4 hours. Let the whole blood stand at 4°C for 1 hour, centrifuge at 4000rpm for 5 minutes, take out the supernatant with a pipette and transfer it to a new tube, which is the serum. Serum creatine kinase content was measured using CK kit (Abnova).

Conclusion: A of Figure 4 shows that the level of creatine kinase in the serum of the compound-treated group of the present invention is significantly lower than that of the control group. Therefore, the sarcolemma integrity of muscle tissue in 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 solution 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, adjust the pH value to 7.4, and the above reagents were purchased from sigma). The electrolyte solution was added to a 37°C constant temperature bath (Chengdu Instrument Factory), and oxygen was introduced. The mice were sacrificed by breaking the vertebrae, and the extensor digitorum longus with intact tendons were taken out, the tendons at both ends were fixed with surgical wires, the extensor digitorum longus was fixed in a constant temperature bath, and hung on the electric shock circle of S88X dual-channel square wave stimulator (GRASS) Middle; 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 recorded The measured tension value.

Conclusion: As shown in Figure 4B and Figure 4C, the compound treatment 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 aged mice

C57BL/6 mice (purchased from Jichui Pharmaceutical Co., Ltd.) were grown to 14 months old to start the experiment. Inject the compound of the present invention (5mg/kg) intraperitoneally (diluted to 500mM in PBS, adjust pH=7.4), 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, adjust the pH value to 7.4) as a control group, with 8-10 rats in each group, and the injection was maintained for 10 months. After administration, the physiological function of skeletal muscle and the mortality rate of aged mice were detected.

Determination of forelimb grasping force: install the grid accessories on the grasping force sensor (BioSEB GS3), place the grasping force instrument in a stable position, press and hold the start button, and the instrument will start the experiment when the instrument self-checks until 0.0 is displayed on the screen; put the mouse Put the forelimbs on the grid, grab the tail of the mouse and pull the mouse backward along the direction of the grid at a constant speed until the limbs are off the grid. After recording the value, press the ZERO key to reset; each mouse is measured ten times a day, Measure for three days, take the maximum value and record it as the value of the mouse's grasping force, and compare between groups.

Mouse running experiment: Connect the mouse running instrument (Columbus Exer-3/6) to the computer and turn on the power, put the mouse in the running manifest channel; open the connection software on the computer, set the conveyor belt to rotate and the mouse will run Speed program: Balance at a speed of 10m/min for 3 minutes, then accelerate to 20m/min at 1m/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 manifest channel, and start recording mice The time of running, taking the mice not jumping on the conveyor belt under the condition of electrical stimulation as the standard of mouse fatigue.

Mouse skeletal muscle tension measurement: prepare electrolyte solution 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 of deionized water, the pH value was adjusted to 7.4, and the above reagents were purchased from sigma); the electrolyte solution was added to a 37°C constant temperature bath (Chengdu Instrument Factory), and oxygen was introduced; For the extensor digitorum longus, the tendons at both ends are fixed with surgical wires, the extensor digitorum longus is 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); computer-controlled electric stimulation adjustment parameters: single contraction Muscle strength: voltage: 20 volts, stimulation time 0.3 milliseconds, repeat three times; tetanic contraction muscle strength: voltage: 20 volts, stimulation time: 0.2 milliseconds, stimulation frequency: 200, and record the measured tension value.

Conclusion: as shown in Figure 5, the compounds of the present invention significantly improve the forelimb grip (A of Figure 5) and running performance (B of Figure 5), the single contraction force of the muscle fibers (C of Figure 5) and the rigidity of the mice Contraction force (Figure 5, D). At the same time, treatment with the compound of the present invention significantly increased the survival rate of aged mice (Fig. 5E). These experimental results show that the compound of the present invention significantly improves the muscle physiological function and mortality of aged mice.

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

Purchased 49 C57BL/6 mice of 8 weeks (purchased from Jicui Yaokang Co., Ltd.), administered the compound of the present invention by intragastric administration, and the doses were 0.05g/kg, 0.1g/kg, 0.5g/kg, 1g/kg kg, 2g/kg, 5g/kg (diluted to 500mM in PBS, adjusted to pH=7.4), 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, adjust the pH value to 7.4) as a control group, 7 rats in each group. Observed for 4 hours after the administration, recorded vocalization and convulsions, observed continuously for 7 days and recorded the number of deaths.

Conclusion: The compound of the present invention has relatively high biological safety. It is administered by intragastric administration. After the administration, vocalization and convulsions occur within 4 hours. Administration observes the death situation in 7 days, at first there is no any death in the control group; Dosage is the administration group of 5g/kg, 4 (57.14%) of dead number in 7 days; Dosage is the administration group of 2g/kg, The number of death within 7 days is 1 (14.29%); the dose is 1g/kg administration group, the number of death is 1 (14.29%) within 7 days; the dose is 0.5g/kg, 0.2g/kg, 0.1g /kg, 0.05g/kg administration group, the number of death within 7 days 0 (0.00%) (see Figure 6).

These experimental results show that the compound of the present invention is between level 2 (actually non-toxic) and level 3 (low toxicity) of the acute toxicity grading standard (reference compound oral acute toxicity grading standard). It shows that the present invention has relatively high biological safety.

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Claims (6)

1. The application of a compound shown in the formula (I) or a pharmaceutical composition containing the compound in preparing a medicament for preventing and/or treating muscular atrophy related diseases,

2. use according to claim 1, wherein the muscle-wasting-related disorder is muscle-wasting, disuse, senile, neurogenic, preferably muscle-wasting and senile.

3. The use according to claim 1 or 2, wherein the myogenic muscular atrophy is progressive muscular dystrophy, congenital muscular dystrophy or tonic muscular dystrophy, wherein the progressive muscular dystrophy is e.g. duchenne muscular dystrophy, becker muscular dystrophy, emery-Dreifuss muscular dystrophy, limb-belted muscular dystrophy, facial-shoulder-brachial muscular dystrophy, acromioclavicular muscular dystrophy.

4. Use according to any one of claims 1 to 3, wherein the compound accelerates repair of skeletal muscle under injury and disease by promoting asymmetric division of skeletal muscle stem cells, thereby preventing and/or treating the muscle wasting-related diseases.

5. The use according to any one of claims 1 to 4, wherein the pharmaceutical composition contains an effective amount of a compound represented by formula (I) as an active ingredient and a pharmaceutically acceptable carrier or excipient.

6. A health product for increasing muscle or resisting muscular atrophy comprises a compound shown in formula (I) and excipient for health product,