Pharmaceutical composition for preventing or treating muscular dystrophy comprising zaltoprofen as active ingredient

Abstract

The present invention provides a composition for preventing or treating muscular dystrophy, the composition comprising Zaltoprofen as an active ingredient. The composition according to the present invention increases the expression of utrophin, reduces the expression of myostatin, MAFbx, and MuRF1, and can inhibit muscle loss, increase muscle, and improve muscle function by inducing muscle differentiation and increasing the diameter of myotubes. Thus, the composition is expected to be used in various ways as medicines, quasi-medicines, fragrance materials, functional biomaterials, and functional food materials for alleviating, preventing, or treating muscular dystrophy.

Description

Pharmaceutical composition for preventing or treating muscular dystrophy containing zaltoprofen as an active ingredient

[0001] The present invention provides Zaltoprofen, a drug among NSAIDs, for the treatment of muscular dystrophy, an intractable muscular disease.

[0002] Muscular dystrophy (MD) is one of the five severe, progressive, rare, and incurable diseases designated by the United Nations, and Duchenne muscular dystrophy accounts for approximately 50% of muscular dystrophy cases.

[0003] Duchenne muscular dystrophy (DMD) is caused by a mutation in the dystrophin gene located on p21 of the X chromosome, resulting in a deficiency of the dystrophin protein. 65% of all patients are caused by deletions in the dystrophin gene, 5-10% by duplications of the gene, and the remainder by point mutations or microdeletions. Deficiency of the dystrophin protein mainly causes progressive degeneration of skeletal muscles, where the muscles themselves are replaced by connective tissue or fat, resulting in pseudohypertrophy of the muscles and muscle weakness. Therefore, clinical symptoms of Duchenne muscular dystrophy are known to include muscle weakness, muscle hypertrophy, muscle atrophy, loss of cardiac muscle fibers, and mild mental retardation.

[0004] The utrophin gene shares significant structural similarities with the dystrophin gene. Therefore, the potential use of utrophin as a replacement for dystrophin has been suggested. However, no therapeutics based on utrophin have been developed to date.

[0005] Myostatin is a polypeptide growth factor belonging to the TGF-β superfamily. TGF-β has numerous isoforms, and these are known to be involved in cell proliferation, apoptosis, differentiation, and bone formation and maintenance (Massague & Chen, 2000). Myostatin, a member of growth differentiation factor (GDF) type 8, is involved in tissue growth and development and acts by activating the Smad signaling pathway. Furthermore, it has been reported to influence bone formation and regeneration by inhibiting cell cycle and proliferation of progenitor cells via the p21 gene. Myostatin is primarily produced in skeletal muscle cells and acts autocrine to cause muscle loss and decreased strength. It is known to inhibit protein synthesis and cell proliferation in myoblasts by suppressing the expression of IGF-1 and Follistatin, which are involved in muscle hypertrophy.

[0006] In addition, the 'PI3K / Akt → FOXO activation and GSK3 inactivation' mechanism induces muscle atrophy by causing inhibition of muscle synthesis and protein degradation. This mechanism induces protein degradation represented by the ubiquitin-proteasome system through a sub-mechanism of inducing the expression of atrogene (MuRF-1, etc.), a gene that induces muscle atrophy.

[0007] The present inventors conducted extensive research to develop a control substance targeting utrophin transcription for the development of a treatment for Duchenne muscular dystrophy, and confirmed that zaltoprofen, a NSNID compound, can effectively control muscle atrophy, and completed the present invention.

[0008] The technical problem to be achieved by the present invention is to provide zaltoprofen for use in preventing and treating muscular dystrophy.

[0009] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below.

[0010] To solve the above problem, the present invention provides a pharmaceutical composition for preventing or treating muscular dystrophy containing zaltoprofen as an active ingredient.

[0011] As one embodiment of the present invention, the zaltoprofen may inhibit myostatin mRNA or protein expression in muscle cells.

[0012] As another embodiment of the present invention, the zaltoprofen may inhibit the expression of FOXO3 in muscle cells.

[0013] As another embodiment of the present invention, the zaltoprofen may increase the expression of utropin in muscle cells.

[0014] As another embodiment of the present invention, the zaltoprofen may reduce the expression of MAFbx and MuRF1 in muscle cells.

[0015] As another embodiment of the present invention, the muscle cell may have a mutation in the dystrophin gene, as long as the mutation alters the function of wild-type dystrophin.

[0016] As another embodiment of the present invention, the zaltoprofen may induce or promote muscle differentiation.

[0017] As another embodiment of the present invention, the zaltoprofen may increase the diameter of the root canal.

[0018] The present invention also provides a method for preventing or treating muscular dystrophy, comprising administering to a subject a pharmaceutically effective amount of zaltoprofen.

[0019] In one embodiment of the present invention, the subject may be a patient exhibiting symptoms of muscle dysfunction, muscle loss, muscle atrophy, muscle wasting, and / or muscle degeneration, preferably a patient with muscular dystrophy, and more preferably a patient with Duchenne muscular dystrophy.

[0020] As another embodiment of the present invention, the method may further include a step of measuring muscle strength of the subject before and after administration, wherein the measurement of muscle strength may be performed through grip strength measurement.

[0021] The present invention also provides the use of zaltoprofen for the manufacture of a medicament for preventing or treating muscular dystrophy.

[0022] In addition, the present invention provides a food composition for preventing or improving muscular dystrophy, which contains zaltoprofen as an active ingredient.

[0023] The present invention provides a composition for preventing or treating muscular dystrophy, comprising zaltoprofen as an active ingredient. The composition of the present invention can suppress muscle loss and improve muscle growth and function by increasing the expression of utrophin, decreasing the expression of myostatin, MAFbx, and MuRF1, and inducing muscle differentiation and increasing the diameter of myotubes.

[0024] In Figure 1, A is the result of measuring the cytotoxicity of zaltoprofen.

[0025] Figure 1B shows the mRNA expression level of utrophin in the root canal treated with zaltoprofen.

[0026] Figure 1 C shows the increase in utropin expression following zaltoprofen treatment at the protein level.

[0027] Figure 1D shows the increase in utrophin expression following zaltoprofen treatment confirmed through immunofluorescence staining.

[0028]

[0029] In Figure 2, A shows the expression levels of syntrophin and dystrobrevin in wild-type mice and mdx mice.

[0030] In Figure 2, B confirms the increase in the expression levels of syntropin and dystrobrevin according to zaltoprofen treatment.

[0031] Figures 2C to E confirm that zaltoprofen increases the expression of α-syntrophin, α-dystrobrevin, and dystroglycan in a time-dependent manner.

[0032]

[0033] Figure 3A shows CaMKII phosphorylation according to the concentration of zaltoprofen treatment in myoblasts.

[0034] Figure 3B is an image showing intracellular Ca2+ after treating dystrophin knockout myoblasts with zaltoprofen.

[0035] Figure 3C shows the results of treating dystrophin knockout myoblasts with zaltoprofen and confirming an increase in the expression of utrophin, and confirming that this effect of zaltoprofen was reduced by STO609 treatment.

[0036] Figure 3D shows the co-immunoprecipitation results confirming the direct binding of utrophin and α-syntrophin after zaltoprofen treatment and that the binding was inhibited by STO609.

[0037] Figures 3E and F show the results confirming the increase in expression of MEF2A and PGC1α according to the concentration of zaltoprofen treatment.

[0038]

[0039] In Figure 4, A confirms the decrease in myostatin, MAFbx, and MuRF1 expression at the gene level following zaltoprofen treatment.

[0040] In Fig. 4, B is the result of confirming A in Fig. 4 at the protein level.

[0041] In Figure 4, C shows the phosphorylation level of Foxo3a according to zaltoprofen treatment and treatment concentration.

[0042] In Figure 4, D shows the phosphorylation level of p70S6K according to zaltoprofen treatment and treatment concentration.

[0043] Figure 4E shows changes in ADP and ATP levels according to zaltoprofen treatment.

[0044]

[0045] In Figure 5, A confirms the change in MHC level according to zaltoprofen treatment.

[0046] Figure 5B shows differentially expressed genes related to muscle differentiation following zaltoprofen treatment.

[0047] In Figure 5, C is an image confirming an increase in the diameter of the root canal according to zaltoprofen treatment.

[0048] Figure 5D is a graph that quantifies the results of Figure 5C.

[0049]

[0050] In Figure 6, A is a graph confirming the change in forelimb grip strength of mdx mice treated with zaltoprofen.

[0051] Figure 6B is a graph confirming the level of utrophin expression at the gene level in the TA muscle of mdx mice treated with zaltoprofen.

[0052] Figure 6C shows the decrease in expression of MuRF1 and MAFbx in the TA muscle of mdx mice treated with zaltoprofen at the gene level.

[0053] Figure 6D shows the level of creatine kinase in the serum of experimental animals.

[0054]

[0055] Figure 7 shows the results of comparing the myostatin expression inhibition and utrophin expression increase activities of zaltoprofen with loxoprofen, pelubiprofen, and celecoxib to confirm whether the NSAIDs (nonsteroidal anti-inflammatory drugs) to which zaltoprofen belongs exhibit the same activity as zaltoprofen.

[0056] Muscular dystrophy is a disease characterized by muscle atrophy and weakness caused by a genetic mutation that results in the absence of the dystrophin-glycoprotein complex, a component of the striated muscle plasma membrane. The utrophin gene has a high structural similarity to the dystrophin gene, suggesting the possibility of replacing dystrophin in cases of dystrophin deficiency. The present inventors conducted extensive research to develop a drug for treating muscular dystrophy and confirmed that among NSAIDs, zaltoprofen can increase the expression of utrophin, prevent muscle loss, and improve the symptoms of muscular dystrophy, thereby completing the present invention.

[0057] Accordingly, the present inventor provides a pharmaceutical composition for preventing or treating muscular dystrophy containing zaltoprofen as an active ingredient.

[0058] As used herein, the term "prevention" refers to any action that inhibits or delays the onset of muscular dystrophy by administering a composition. As used herein, the term "treatment" refers to any action that improves or beneficially alters the symptoms of muscular dystrophy by administering a composition.

[0059] The above pharmaceutical composition can reduce myostatin promoter activity, thereby more fundamentally preventing and treating muscle wasting and muscle strength loss.

[0060] The above pharmaceutical composition increases the expression of utrophin and simultaneously improves the symptoms of muscular dystrophy, so the disease to be treated by the present invention may be Duchenne muscular dystrophy among muscular dystrophies.

[0061] Meanwhile, zaltoprofen is a drug belonging to the nonsteroidal anti-inflammatory drug (NSAID) class and is known as a COX-2 inhibitor. The present inventors compared the myostatin expression inhibitory activity and utrophin expression promoting effect of loxoprofen, pelubiprofen, and celecoxib with zaltoprofen to determine whether the activity of zaltoprofen confirmed in muscle cells can be obtained in other NSAID drugs. As a result, the effect of increasing utrophin expression was confirmed with loxoprofen and celecoxib, but this effect was not as effective as that of zaltoprofen. It was confirmed that not all NSAID drugs inhibit myostatin expression, and some drugs rather increase it. In other words, it can be seen that not all NSAIDs can be used to treat muscular dystrophy, and among NSAIDs, zaltoprofen in particular can be an effective drug for treating muscular dystrophy.

[0062] A pharmaceutical composition for preventing or treating muscle atrophy according to one embodiment of the present invention may contain zaltoprofen at a concentration of 0.001 mM or more, and specifically, may contain it at a concentration of 0.001 mM to 1000 mM.

[0063] The pharmaceutical composition of the present invention can be manufactured by a method known in the pharmaceutical field for use as a medicament, and can be manufactured and used in the form of a powder, granule, tablet, capsule, or injection by mixing with a pharmaceutically acceptable carrier, excipient, diluent, stabilizer, preservative, etc. In addition, the composition can be manufactured as a sustained-release preparation by including a base used for sustained-release purposes in addition to the active ingredient so that the active ingredient is slowly released.

[0064] Pharmaceutically acceptable carriers may further include, for example, carriers for oral administration or carriers for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, etc. In addition, various drug delivery materials used for oral administration of peptide preparations may be included. In addition, carriers for parenteral administration may include water, suitable oils, saline solution, aqueous glucose, and glycols, etc., and may further include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite, or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol. In addition to the above components, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, etc.

[0065] Examples of the diluent include, but are not limited to, non-aqueous solvents such as propylene glycol, polyethylene glycol, vegetable oils such as olive oil and peanut oil, and aqueous solvents such as saline solution (preferably 0.8% saline solution) and water containing a buffer medium (preferably 0.05 M phosphate buffer).

[0066] The above excipients include, but are not limited to, starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, anhydrous skim milk, glycerol, propylene, glycol, water, ethanol, and the like.

[0067] The above stabilizers include, but are not limited to, carbohydrates such as sorbitol, mannitol, starch, sucrose, dextran, glutamate, and glucose, and proteins such as animal, vegetable, or microbial proteins such as milk powder, serum albumin, and casein.

[0068] The above preservatives include, but are not limited to, thimerosal, merthiolate, gentamicin, neomycin, nystatin, amphotericin B, tetracycline, penicillin, streptomycin, and polymyxin B.

[0069] The pharmaceutical composition of the present invention can be administered to mammals, including humans, by any method, and can be administered, for example, orally or parenterally. Parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal administration.

[0070] The pharmaceutical composition of the present invention can be formulated as a preparation for oral administration or parenteral administration according to the administration route as described above.

[0071] The total effective dose of the pharmaceutical composition of the present invention can be administered to a patient as a single dose, or can be administered by a split treatment regimen in which multiple doses are administered over a long period of time. The pharmaceutical composition of the present invention can vary the content of the active ingredient depending on the severity of the disease. This can be determined by considering various factors such as the formulation method, administration route, and number of treatments, as well as the patient's age, weight, health condition, symptoms of the disease, and administration time and method. Taking these points into account, a person of ordinary skill in the art will be able to determine an appropriate effective dosage of the composition of the present invention. The pharmaceutical composition according to the present invention is not particularly limited in its formulation, administration route, or administration method as long as it exhibits the effects of the present invention.

[0072] The composition of the present invention can be added to a food for the purpose of preventing or improving muscle atrophy, and the food can be a health functional food. In the present invention, a health functional food refers to a food having a bioregulatory function such as disease prevention or improvement, biodefense, immunity, and recovery after disease, and must be harmless to the human body when consumed over a long period of time.

[0073] When the composition of the present invention is used as a food additive, the zaltoprofen may be added as is or used together with other foods or food ingredients, and may be used appropriately according to a conventional method. The amount of the active ingredient mixed may be appropriately determined depending on the purpose of use (prevention, health, or therapeutic treatment). Generally, when manufacturing a food or beverage, the composition of the present invention is added in an amount of 15% by weight or less, preferably 10% by weight or less, based on the raw material. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be below the above range, and since there is no problem in terms of safety, the active ingredient may also be used in an amount above the above range.

[0074] There are no specific restrictions on the types of foods mentioned above. Examples of foods to which the above substances can be added include meat, sausages, bread, chocolate, candy, snacks, confectionery, pizza, ramen and other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, including all health foods in the conventional sense.

[0075] The health beverage composition of the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients, similar to conventional beverages. The natural carbohydrates mentioned above may include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, natural sweeteners such as dextrin and cyclodextrin, or synthetic sweeteners such as saccharin and aspartame. The proportion of the natural carbohydrate is generally about 0.01 to 10 g, preferably about 0.01 to 0.1 g, per 100 ml of the composition of the present invention.

[0076] In addition to the above, the composition of the present invention may include various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the composition of the present invention may include fruit pulp for the production of natural fruit juice, fruit juice drinks, and vegetable drinks. These components may be used independently or in combination. The proportion of these additives is not particularly critical, but is typically selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

[0077]

[0078] The present invention is susceptible to various modifications and embodiments. Specific embodiments are illustrated in the drawings and described in detail in the following detailed description. However, this is not intended to limit the present invention to specific embodiments, but rather to encompass all modifications, equivalents, and alternatives falling within the spirit and technical scope of the present invention. In describing the present invention, detailed descriptions of related known technologies will be omitted if they are deemed to obscure the gist of the present invention.

[0079] [Experimental Method]

[0080] 1. Primary myoblast culture

[0081] Wild-type control mice (C57BL / 6) and C57BL / 6-derived DMD knockout mice were used. A stop codon was expressed in place of the glutamine codon of exon 20 of the dystrophin gene using a cytosine-based editing method. Primary myoblasts were isolated from the hindlimb skeletal muscles of 1-week-old wild-type and DMD knockout mice. Wild-type and DMD knockout mouse primary myoblasts were cultured in Ham's F-10 medium (WELGENE, LM009-02) containing 10% cosmic calf serum (Hyclone, SH30087.03), 10 ng / ml human basic fibroblast growth factor (Human bFGF; Peprotech, 100-18B), and 1% antibiotics at 37°C in a humidified atmosphere of 5% CO2 and 95% air. Primary myoblasts from wild-type and DMD knockout mice were differentiated in high-glucose DMEM supplemented with 5% horse serum and 1% antibiotics. Culture plates were coated with collagen before use (Sigma, C8919).

[0082]

[0083] 2. Cell viability test

[0084] Primary myoblasts were prepared in 96-well plates. Cells were treated with various concentrations of zaltoprofen for 24 h. MTT ((3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reagent was added to each well. After incubation for 4 h at 37°C in a CO2 incubator, the medium was removed, and DMSO was added to each well. Absorbance was measured at 540 nm using a Synergy H1 multi-mode microplate reader (BioTek).

[0085]

[0086] 3. Western Blotting

[0087] Total protein extracts were prepared by lysing cells in lysis buffer (lysis buffer supplemented with 1 M Tris-HCl (pH 7.4), 5 M NaCl, 0.5 M EDTA, 10% glycerol, 1% TritonX100, 1 mM PMSF, 1 mM Na3Vo4, and 50 mM NaF). Protein extracts were diluted with 5X sample buffer and denatured at 95°C for 5 min. Lysates were loaded onto 8–10% SDS-PAGE gels or 4–15% gradient gels (BMS, #4561084) and transferred to 0.45 μm nitrocellulose membranes (GE healthcare). After transfer, the membranes were stained with Ponceau S and cut into strips according to the molecular weight of each protein studied. The membranes were blocked with 5% skim milk for 1 h at room temperature and then washed with TBS-T. The membrane was incubated overnight at 4°C with primary antibodies. Subsequently, the membrane was incubated with the appropriate HRP-conjugated secondary antibody for 1 hour at room temperature and then washed three times with TBST. The membrane was used for chemiluminescence-based detection. Band intensities were analyzed using the ChemiDoc system and iBright Analysis software (Invitrogen, CL1000).

[0088]

[0089] 4. RNA extraction and real-time PCR

[0090] Total RNA was extracted from primary myoblasts from wild-type and DMD knockout mice using Qiazol (Qiagen). RNA concentration was measured using a Nanodrop spectrophotometer. mRNA expression was assessed by real-time PCR (qRT-PCR) (QuantStudio 3 Real-Time PCR, Thermo Fisher Scientific) using the SYBR Green system (TOPreal™ qPCR 2X Pre-MIX, Enzynomics). Primers used are listed in Table 1:

[0091] ASSOCIATED PHYSIOLOGY GAPDHforwardGTG TTC CTA CCC CCA ATG TG1reverseCCT GCT TCA CCA CCT TCT TG2UtrophinforwardCGT GAT TGG TGA CCT GGA CA3reverseTGA GCG ACG TGT CCA TCA AA4α-SyntrophinforwardATC TTC AAG GGA CTG GCA GC5reverseCCT GTC TTC TTG AGG GCC TG6β-DystrobrevinforwardCAC AAC TAC CAG CTC TGC CA7reverseTAT TGG TCA GAG GGC GAG GA8DystroglycanforwardGTG CCT ACG CCT ACA TCT CC9reverseCGG CTG TGG GCT CTA CAT AG10MyostatinforwardGCG ATG AGC ACT CCA CGG AA11reverseGCC TCT GGG GTT TGC TTG GT12MAFbxforwardATG CAC ACT GGT GCA GAG AG13reverseTGT AAG CAC ACA GGC AGG TC14MuRF1forwardGTC CAT GTC TGG AGG TCG TT15reverseAGG AGC AAG TAG GCA CCT CA16MEF2AforwardGAG CCT CAT GAA AGC AGG AC17reverseCAA GTT CTG AGG TGG CAA GC18PGC1αforwardGCG GTA TTC ATC CCT CTT GA19reverseATG TGT CGC CTT CTT GCT CT20

[0092]

[0093] 5. Immunostaining

[0094] Cells were cultured on gelatin-coated 18-mm glass plates, fixed with freshly prepared 4% paraformaldehyde (PFA) for 15 min, permeabilized with 0.1% Triton X-100 for 5 min, blocked with 4% BSA for 30 min, and then stained with primary antibodies overnight at 4°C. The following primary antibody dilution was used: mouse anti-utrophin (1:200, Santa). After washing, cells were stained with secondary fluorescent antibodies (Alexa488 mouse) for 1 h. Finally, cells were mounted with mounting solution containing DAPI (Invitrogen). Images were acquired with CLSM (Carl Zeiss, LSM800 with Airyscan).

[0095]

[0096] 6. Confocal Laser Scanning Microscopy (CLMS)

[0097] Total 7 x 10 4 Primary myoblasts from wild-type and DMD knockout mice were seeded onto collagen-coated confocal dishes (SPL, 101350) and cultured at 37°C for 24 h. Primary myoblasts from wild-type and DMD knockout mice were labeled with Fluo-3 / AM (Invitrogen, F1241) to monitor [Ca2+]i. Fluorescence intensity in living cells was observed using CLSM (Carl Zeiss, LSM800 with Airyscan).

[0098]

[0099] 7. Co-immunoprecipitation

[0100] Cell proteins (1 mg) were incubated overnight at 4°C with anti-syntrophin a1 (mouse monoclonal antibody) or anti-IgG (normal mouse antibody). Immune complexes were captured with protein A-Sepharose (Amersham, Uppsala, Sweden) after 3 h of incubation. The precipitated immune complexes were washed three times with washing buffer (25 mM HEPES, 5 mM EDTA, 1% Triton X-100, 50 mM NaF, 150 mM NaCl, 10 mM PMSF). The washed samples were denatured with SDS sample buffer (125 mM Tris-HCl (pH 6.8), 20% (v / v) glycerol, 4% (w / v) SDS, 100 mM DTT, 0.1% (w / v) bromophenol blue) and heated at 100°C for 5 min.

[0101]

[0102] 8. ATP / ADP / AMP Analysis

[0103] ATP and ADP levels were measured using the ATP / ADP / AMP assay kit (#A-125) from the Biomedical Research Service Center. Cells were deproteinized using the boiling water method. The amounts of ATP and ADP in the cell lysates were measured in relative luminescence units (RLU). Luminescence was measured using a Synergy H1 multimode microplate reader (BioTek). Calculations were performed according to the manufacturer's instructions.

[0104]

[0105] 9. Myogenic differentiation and determination of myotube cell morphology

[0106] Primary myoblasts from DMD knockout mice were differentiated in high-glucose DMEM supplemented with 5% horse serum and 1% antibiotics. Culture plates were coated with collagen (Sigma, C8919) before use. Zaltoprofen was treated in differentiation medium for 72 h. Myoblast differentiation was determined by analyzing myotube cell diameters using hematoxylin and eosin (H&E) staining. Images were captured and processed using a CKX53 microscope (Olympus). All diameters were calculated from 10 random fields in each image using ImageJ software.

[0107]

[0108] 10. Microarray analysis

[0109] Data were summarized and normalized using the robust multiple averaging (RMA) method implemented in Affymetrix® Power Tools (APT). Results were exported to gene-level RMA analysis, and differentially expressed gene (DEG) analysis was performed. Statistical significance of expression data was determined using the LPE test and fold change, where the null hypothesis is that there is no difference between groups. False discovery rate (FDR) was controlled by adjusting the p value using the Benjamini-Hochberg algorithm. For the differentially expressed gene set, hierarchical clustering analysis was performed using complete linkage and Euclidean distance as measures of similarity. All data analyses of differentially expressed genes were performed using R 3.3.2. KEGG pathways were downloaded and processed using Morepheus software.

[0110]

[0111] 11. Animal testing

[0112] In vivo experiments were performed using C57BL / 10ScSn-Dmdmdx / J (mdx) mice from the Jackson Laboratory. Seven-week-old male mdx mice were treated with zaltoprofen (0.1 mg / kg) or control (PBS) by intraperitoneal injection every other day for 4 weeks (n=6 each group). Animal experiments were approved by the Korea University Institutional Animal Care and Use Committee and were performed in compliance with relevant guidelines and regulations.

[0113]

[0114] 12. In vivo forelimb grip strength test

[0115] Forearm grip strength tests were performed twice a week using a calibrated grip strength tester (Jeung Do Bio, JD-A-22) to assess forearm grip strength. Results are presented as the average of three replicates.

[0116]

[0117] 13. Enzyme-linked immunosorbent assay

[0118] Enzyme-linked immunosorbent assay (ELISA) was performed using a standard sandwich procedure. Serum creatine kinase levels were measured using a sandwich ELISA kit (MyBioSource, MBS261027) according to the manufacturer's instructions.

[0119]

[0120] [Experimental Results]

[0121] Example 1. Confirmation of increased utropin expression due to zaltoprofen treatment

[0122] Based on primary myoblasts and screening data, we evaluated the therapeutic potential of zaltoprofen for the treatment of dystrophic DMD muscles. As a first step in investigating the effects of zaltoprofen on DMD, we used primary myoblasts derived from skeletal muscles of male C57BL / 6 DMD knockout mice (DMD KO primary myoblasts). To determine the appropriate dose of zaltoprofen, cells were treated with various doses of zaltoprofen (3 μM–1 mM) for 24 hours and then cell viability was measured (Fig. 1A). Cell viability in cells treated with 300 μM zaltoprofen decreased to 79.96% compared to untreated cells (P<0.01).

[0123] We harvested myotubes treated with zaltoprofen for 12 hours, and the mRNA level of utrophin significantly increased in a dose-dependent manner (Fig. 1B). Zaltoprofen also upregulated the protein level of utrophin during myoblast differentiation (Fig. 1C). Utrophin is localized at the NMJ and sarcolemma in DMD. Immunofluorescence analysis revealed an increase in utrophin in zaltoprofen-treated myotubes (Fig. 1D). An increase in utrophin is a therapeutic marker for DMD treatment. Therefore, these results suggest that zaltoprofen can compensate for the absence of dystrophin by increasing utrophin.

[0124]

[0125] Example 2. Confirmation of increased adhesive protein components in the sarcolemma by zaltoprofen treatment.

[0126] As described above, loss of dystrophin and the dystrophin glycoprotein complex in DMD patients leads to sarcolemmal destruction and instability induced by muscle contraction. Similar to DMD patients, mdx mice exhibit loss of the dystrophin-glycoprotein complex (DGC), which includes dystrophin protein, α-syntrophin, and α-dystrobrevin (Fig. 2A). Furthermore, we previously reported that α-syntrophin is reduced in the sarcolemma of mdx mice. The alleviation of the DMD phenotype may be accompanied by increased sarcolemmal protein content.

[0127] As shown in Figure 2B, zaltoprofen increased not only utrophin but also sarcolemmal proteins such as α-syntrophin, α-dystrobrevin, and dystroglycan. To confirm the increasing effect of zaltoprofen on sarcolemmal proteins, we performed qRT-PCR. We found that zaltoprofen increased α-syntrophin protein expression at the mRNA level in a time-dependent manner (Figure 2C). These results suggest that zaltoprofen treatment is associated with the restoration of sarcolemmal protein components in DMD skeletal muscle, thereby increasing membrane stability.

[0128]

[0129] Example 3. Confirmation that zaltoprofen treatment induces an increase in utrophin transcription factor through calcium signaling.

[0130] Myoblasts treated with zaltoprofen for 24 hours were harvested, and phosphorylation of CaMKII increased in a dose-dependent manner (Fig. 3A). To investigate whether calcium signaling was involved in the zaltoprofen-induced increase in utrophin, myoblasts were treated with the cytosolic calcium (Ca2+) indicator Fluo-3 / AM. We observed a rapid increase in intracellular Ca2+ concentration after zaltoprofen treatment in DMD knockout (KO) myoblasts (Fig. 3B). To confirm this result, we pretreated DMD knockout (KO) myoblasts with the CaMKK inhibitor STO609 and then treated them with zaltoprofen. qRT-PCR revealed that STO609 suppressed the zaltoprofen-induced increase in utrophin (Fig. 3C). We then performed co-immunoprecipitation between utrophin and α-syntrophin. The results of the above co-immunoprecipitation showed that utrophin directly binds to α-syntrophin after zaltoprofen treatment and that STO609 inhibits the binding (Fig. 3D).

[0131] Cytosolic calcium is involved in both myogenic differentiation and the induction of contraction. Furthermore, CaMK signaling appears to be involved in the calcium- and contraction-mediated upregulation of peroxisome proliferator-activated receptor gamma coactivator 1alpha (PGC1α) in skeletal muscle. One role of PGC1α is to regulate utrophin transcription. The correlation between the CaMK pathway and utrophin upregulation was predicted to be PGC1α, and qRT-PCR analysis revealed that zaltoprofen upregulates MEF2A and PGC1α in a dose-dependent manner (Figures 3E and 3F).

[0132]

[0133] Example 4. Confirmation of decreased myostatin expression in DMD KO myotubes treated with zaltoprofen.

[0134] We further tested whether zaltoprofen modulates muscle atrophy markers. To test this, we measured the expression levels of muscle atrophy markers, including myostatin (MSTN), MAFbx32, and MuRF1. First, myostatin promoter activity was measured using a luciferase activity assay (Figure 4A). The effects of zaltoprofen on muscle atrophy were confirmed using qRT-PCR and Western blotting. As a result, we confirmed that zaltoprofen decreased myostatin mRNA levels in a dose-dependent manner (Figure 4B).

[0135] Furthermore, zaltoprofen dose-dependently downregulated the expression of atrophy-related proteins MSTN, MAFbx32, and MuRF1 (Fig. 4C). Because FoxO3a induces the expression of atrophy-related MuRF1 and MAFbx32, we hypothesized that FoxO3a is involved in the downregulated atrophy-related genes. We found that zaltoprofen-treated myotubes exhibited increased FoxO3a expression at the protein level, along with increased phosphorylation. These results suggest that zaltoprofen can regulate muscle atrophy-related genes by downregulating FoxO3a. Previous studies have shown that PGC1α regulates muscle size by inhibiting FoxO3 function and protecting skeletal muscle from atrophy.

[0136] Furthermore, downregulation of MuRF1 and MAFbx expression was accompanied by activation of positive regulators of protein synthesis. Zaltoprofen increased p70S6K phosphorylation at Thr389 (Fig. 4D). Taken together, these results suggest that zaltoprofen can reduce muscle atrophy by downregulating muscle atrophy markers and increasing protein synthesis in DMD conditions.

[0137]

[0138] Example 5. Confirmation of the effect of zaltoprofen on regulating myogenic differentiation in DMD KO myotubes.

[0139] Based on the results of other studies suggesting that enhancing muscle differentiation may help combat the dystrophic pathology of DMD, as well as the results in Figure 1, we hypothesized that zaltoprofen alleviates DMD through muscle differentiation. To confirm this hypothesis, we evaluated the effect of zaltoprofen on muscle differentiation. DMD myoblasts were differentiated from DMD with zaltoprofen treatment for 72 hours. As a result, zaltoprofen treatment increased myosin heavy chain levels (Figure 5A).

[0140] These results were also confirmed by H&E staining of myotubes. DMD myoblasts were cultured in differentiation medium with or without zaltoprofen until cell fusion and multinucleated myotube formation were achieved. H&E staining and muscle diameter measurements showed that zaltoprofen significantly increased myotube diameter (Figures 5C and 5D). Zaltoprofen was confirmed to increase membrane stability by upregulating sarcomere adhesion components. Therefore, zaltoprofen-induced myogenic differentiation may also prolong membrane stabilization.

[0141]

[0142] Example 6. Confirmation of phenotypic alleviation in mdx mice by zaltoprofen treatment.

[0143] No differences in body weight were observed in experimental animals after zaltoprofen treatment. As a first step to investigate the effects of zaltoprofen on mdx mice, forelimb grip strength was assessed in the control and zaltoprofen-treated groups (Figure 6A). Four weeks after zaltoprofen injection, zaltoprofen-treated mice exhibited increased muscle strength compared to control mdx mice.

[0144] Additionally, we observed an increase in utrophin levels in the TA muscle after zaltoprofen treatment (Fig. 6B). In terms of muscle atrophy, the expression of MuRF1 and MAFbx in the TA muscle was decreased in the zaltoprofen-treated group (Fig. 6C). To further investigate the effects of zaltoprofen, we measured serum creatine kinase (CK) levels (Fig. 6D). CK levels are known to be higher in DMD patients and are therefore used as a diagnostic marker. After 4 weeks of zaltoprofen treatment, CK levels decreased.

[0145]

[0146] Although the embodiments described above have been described with limited drawings, those skilled in the art will appreciate that various technical modifications and variations can be applied based on the above. For example, appropriate results can still be achieved even if the described techniques are performed in a different order than described, and / or components of the described systems, structures, devices, circuits, etc. are combined or combined in a different manner than described, or are replaced or substituted with other components or equivalents.

[0147] Therefore, other implementations, other embodiments, and equivalents to the claims also fall within the scope of the claims described below.

Claims

1. A pharmaceutical composition for preventing or treating muscular dystrophy containing zaltoprofen as an active ingredient.

2. In paragraph 1, A pharmaceutical composition, wherein the above muscular dystrophy is Duchenne muscular dystrophy.

3. In paragraph 1, A pharmaceutical composition wherein the composition inhibits myostatin mRNA or protein expression in muscle cells.

4. In paragraph 1, A pharmaceutical composition, wherein the composition increases the expression of utropin.

5. In paragraph 1, A pharmaceutical composition wherein the composition inhibits the expression of MAFbx and MuRF1.

6. In paragraph 1, A pharmaceutical composition wherein the composition promotes muscle differentiation.

7. In paragraph 1, A pharmaceutical composition, wherein the composition increases the diameter of a root canal.

8. A food composition for preventing or improving muscular dystrophy, comprising zaltoprofen as an active ingredient.

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