WO2015093901A1 - Composition pharmaceutique contenant un composant d'induction d'activation de la protéine taz pour différenciation musculaire et régénération musculaire - Google Patents

Composition pharmaceutique contenant un composant d'induction d'activation de la protéine taz pour différenciation musculaire et régénération musculaire Download PDF

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WO2015093901A1
WO2015093901A1 PCT/KR2014/012600 KR2014012600W WO2015093901A1 WO 2015093901 A1 WO2015093901 A1 WO 2015093901A1 KR 2014012600 W KR2014012600 W KR 2014012600W WO 2015093901 A1 WO2015093901 A1 WO 2015093901A1
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muscle
formula
pharmaceutical composition
expression
compound represented
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PCT/KR2014/012600
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English (en)
Korean (ko)
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황은숙
홍정호
김낙정
배명애
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이화여자대학교 산학협력단
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Publication of WO2015093901A1 publication Critical patent/WO2015093901A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4184—1,3-Diazoles condensed with carbocyclic rings, e.g. benzimidazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system

Definitions

  • composition for muscle differentiation and muscle regeneration comprising a TAZ protein activation induction component
  • the present invention relates to 2-butyl-6-styryl-1_ [2 '-(1H-tetrazol-5-yl) -bisphenyl-4-ylmethyl] -1H-benzoimidazole-5-carboxylic acid
  • Methyl ester (2-butyl-6-styryl-l- [2 '-(lH-tetrazol-5-yl) -biphenyl-4-ylmeth ⁇ - ⁇ -benzo imi dazo 1 e-5-car boxy 1 ic acid methyl ester, TM-53) and 2-butyl-5-styryl-l- [2 '-(1H-tetrazol-5-yl) -bisphenyl-4-ylmethyl] -1H-benzoimidazole-6 -Carboxylic acid.
  • the present invention relates to a pharmaceutical composition for inducing muscle cell differentiation, a pharmaceutical composition for muscle regeneration, and a pharmaceutical composition for treating or preventing muscle disorders.
  • Transcriptional coactivator with PDZ binding motif (TAZ) is expressed as a bovine protein that binds to 14-3-3 proteins in the cytoplasm (Kanai F et al. (2000). EMBO J 19 6778) -6791).
  • TAZ protein is sequestered in the cytoplasm by 14-3-3 protein, while TAZ dephosphorylated by protein phosphatase I is located in the nucleus and various transcription Promotes transcriptional transcriptional activity (Hong W, Guan KL ⁇ 2Q ⁇ 2).
  • TAZ is RUNX2 To bind to a variety of PPXY motif-containing transcription factors such as run-related transcription factor 2 (RUNX2) and peroxisome prol if erators-act ivated receptor ⁇ (PPARy), and in the nucleus To promote their transcriptional activity (Cui CB et al, (2003). Mol Cell Bio J 23 1004-1013).
  • RUNX2 run-related transcription factor 2
  • PPARy peroxisome prol if erators-act ivated receptor ⁇
  • TAZ promotes osteogenic differentiation involving RUNX2, and PPARy derived from mesenchymal stem cells inhibits induction of adipocyte differentiation. Differentiation from mesenchymal stem cells to myocytes is also promoted as MyoD binds directly to TAZ and is transcribed (Arnold HH, Winter B (1998). Curr Opin Genet Dev 8 539—544;
  • TAZ MyoD dependent myocyte differentiation control gene expression and non-muscle cell conversion into myocytes.
  • MRFs muscle regulatory factors
  • muscle damage can lead to genetic disorders, cancer, It has been reported that it can be caused by common diseases, including inflammation (0 ⁇ 13 ⁇ ⁇ 1011), infection (infect ion) or a number of medical effects.
  • inflammation (0 ⁇ 13 ⁇ ⁇ 1011)
  • infection infect ion
  • skeletal muscles are known to have regenerative capacity and form new myotubes within a few days after severe muscle injury, but these skeletal muscles are slow, inefficient, and incomplete, thus regenerating and restoring skeletal muscles. Research to increase facilitation is needed.
  • muscle cell differentiation and muscle regeneration are usually induced through the expression of inflammatory cytokines such as TNF-a, IL- ⁇ , and IL-6, and the myocyte factor myogenin ( Tidball JG (2005) .Am J Physio J Regul Integr Cowp Physiol 288 R345-353).
  • cytokines such as TNF-a, IL- ⁇ , and IL-6
  • myocyte factor myogenin Tidball JG (2005) .Am J Physio J Regul Integr Cowp Physiol 288 R345-353.
  • severe bone loss results in the breakdown of important proteins in muscle, muscle atrophy F-box (MAFbx) and muscle specific ubiquitin li gases proteins.
  • the ring finger protein Kmuscle-specific RING finger protein 1, MuRFl is activated (Bonaldo P, Sandr i M (2013).
  • Methyl ester (2-butyl-6-styryl-l- [2 '-(lH-tetrazol-5-yl) -biphenyl-4-y nethyl] -lH-benzoimidazole-5-carboxyl ic acid methyl ester, hereinafter TM— It is represented by 53.) or
  • Methyl ester (2-butyl_5-styryl-l- [2 '-(lH-tetrazol-5-yl) -bipheny ⁇ 4-ylmethyl] -lH-benzoimidazole-6-car boxy lie acid methyl-ester, hereinafter TM—54 ), In vitro (/ / iro) to the TAZ protein .
  • TM-53 and TM- 54 of the present invention is a pharmaceutical for inducing muscle cell differentiation
  • the present invention was completed by confirming that the composition, the pharmaceutical composition for muscle regeneration, and the pharmaceutical composition for the treatment or prevention of muscle disorders could be usefully used.
  • An object of the present invention is a 2-butyl-6-styryl-1_ [2 '-(1H-tetrazol-5-yl) -bisphenyl-4-ylmethyl] -1H-benzo as a substance for promoting muscle cell differentiation.
  • Methyl ester (2-buty ⁇ 5-styryl-l- [2 '-(lH-tetrazol-5-yl) -biphenyl-4-ylmethyl] -lH-benzoimidazole-6-carboxyl ic acid methyl ester, TM ⁇ 54)
  • a pharmaceutical composition for inducing muscle cell differentiation comprising the same as an active ingredient—water
  • pharmaceutical composition for muscle regeneration and to provide a pharmaceutical composition for the treatment or prevention of muscle disorders will be.
  • the present invention provides a pharmaceutical composition for inducing muscle cell differentiation containing the compound represented by the following [Formula 1] or a pharmaceutically acceptable salt thereof as an active ingredient;
  • the present invention provides a pharmaceutical composition for regeneration of meat containing the compound represented by the above [Formula 1] or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the present invention provides a pharmaceutical composition for treating or preventing muscle disorders containing the compound represented by the above [Formula 1] or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the present invention provides a health food for inducing muscle cell differentiation, containing the compound represented by the following [Formula 1] or a pharmaceutically acceptable salt thereof as an active ingredient;
  • the present invention provides a health food for improving or preventing muscle disorders containing the compound represented by the above [Formula 1] or a pharmaceutically acceptable salt thereof as an active ingredient. '
  • the present invention provides a compound represented by the above [Formula 1] or a pharmaceutically acceptable salt thereof for use as a pharmaceutical composition for inducing muscle cell differentiation or muscle regeneration.
  • the present invention provides a compound represented by the above [Formula 1] or a pharmaceutically acceptable salt thereof for use as a health food for muscle cell differentiation induction or muscle regeneration.
  • the present invention provides a compound represented by the above [Formula 1] or a pharmaceutically acceptable salt thereof for use as a pharmaceutical composition for treating and preventing muscle disorders.
  • the present invention also provides a compound represented by the above [Formula 1] or a pharmaceutically acceptable salt thereof for use as a health food for improving or preventing muscle disorders.
  • the present invention comprises the step of administering a pharmaceutical composition containing a pharmaceutically effective amount of the compound represented by the above [Formula 1] or a pharmaceutically acceptable salt thereof as an active ingredient to an individual suffering from muscle disorders Provides a method for treating muscle disorders.
  • the present invention is a method for preventing muscle disorders comprising administering to a subject a pharmaceutical composition containing a pharmaceutically effective amount of the compound represented by the above [Formula 1] or a pharmaceutically acceptable salt thereof as an active ingredient.
  • a pharmaceutical composition containing a pharmaceutically effective amount of the compound represented by the above [Formula 1] or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the TM-53 and TM-54 of the present invention are differentiated from myoblast and non-myoblasts to myocytes and transdifferentiated depending on the TAZ protein in vitro cell conditions (/ y? W ' ro). It promotes transdi f ferent i at i on) and decreases the expression of muscle atrophy related genes in muscle cells treated with muscle atrophy inducers, thereby increasing the expression of muscle cell marker proteins and in vivo (/ wVo).
  • TM-53 and TM-54 of the present invention is a pharmaceutical composition for inducing muscle cell differentiation, a pharmaceutical composition for muscle regeneration, and a pharmaceutical for treating or preventing muscle disorders It can be usefully used as an active ingredient of a red composition.
  • FIG. 2 shows quantitatively an increase in the degree of nuclear placement of TAZ protein in cells dependent on the concentration of TM-53 in isotonic or hyposonic conditions. .
  • Figure 3 shows the intranuclear location of Endogenous TAZ proteins by TM-53 and TM-54 in myoblasts via i ⁇ unostaining.
  • Figure 4 shows the relative position of the endogenous TAZ protein nucleus, cytoplasmic c and the whole cell (pancel lular) by TM-53 and TM-54 in myoblasts.
  • Figure 5 shows the morphologi cal change of muscle cells induced differentiation from myoblasts treated with TM-53 or TM-54.
  • Figure 6 shows the expression of MyHC, a marker protein of muscle cells in muscle cells induced differentiation from myoblasts by treatment with TM-53 or TM-54.
  • Figure 7 shows quantitatively the expression of MyHC (MyHC + cel l), a marker protein of muscle cells in muscle cells induced differentiation from myoblasts by treatment with TM-53 or TM-54;
  • MyHC MyHC + cel l
  • the results are expressed as the mean standard error of the means (SEM), and after the analysis using the Student's t-test, the P value is less than 0.05.
  • the significant results were expressed as p ⁇ 0.05O), P ⁇ 0.005 (**), and P ⁇ 0.0005 **).
  • FIG. 9 shows relative expression of differentiation marker proteins from myoblasts to myocytes according to TM-53 and TM-54 throughputs.
  • TAZ and MyoD the differentiation marker genes from myoblasts to myocytes according to TM-53 and TM-54 throughput.
  • TAZ knockout with or without TM-53 or TM-54.
  • Loss of myocyte differentiation capacity in myoblasts (TAZi).
  • Figure 13 shows reduced expression of TAZ in TAZi cells with or without treatment with TM-53 or TM-54.
  • FIG. 15 shows expression confirmation of MyoD through the activity of the MCK promoter induced by MyoD in mouse embryonic fibroblasts (MEF) knocked out TAZ.
  • Figure 16 shows the effect of differentiation into muscle cells through the myocyte conversion differentiation according to MyoD expression in TAZ knocked out MEF (KO-MEF).
  • Figure 17 shows the confirmation of myocyte marker gene expression according to MyoD expression in myocytes induced differentiation induced muscle cells from TAZ knocked out MEF;
  • the results are represented by the mean ⁇ SEM, and analyzed using the Student's t-test, and confirmed that the P value is less than 0.05, showing a significant result, P ⁇ 0.05 (*) and P ⁇ 0.0005 (***).
  • Figure 19 shows the synergistic effect of the expression of myocyte marker proteins according to TM-53 and TM-54 when differentiating into myocytes from KO + TAZ cells that restored the expression of TAZ in K0-MEF.
  • Figure 20 shows the synergistic effect of the expression of myocyte marker genes according to TM-53 and TM-54 when differentiated from K0 + TAZ cells that restored TAZ expression in K0-MEF into myocytes;
  • the results are expressed in mean SEM SEM, and analyzed using the Student's t-test, and confirmed that the P value is less than 0.05, indicating a significant result, P ⁇ 0.050), P ⁇ 0.005 ( **) and P ⁇ 0.0005 **).
  • Figure 21 shows the muscle atrophy improvement effect of TM-53 and TM-54 on the muscle atrophy induced by dihydrochloride (DEX) in muscle cells through the expression level of MyHC protein.
  • FIG. 22 shows the effect of TM-53 and TM-54 on muscular dystrophy on DEX-induced muscular atrophy in muscle cells through reduction in the expression level of muscle atrophy related genes; In the analysis of the expression level, the results are expressed as mean ⁇ SEM, and analyzed using the Student's t-test, and confirmed that the P value is less than 0.05, showing a significant result, P ⁇ 0.05 (*) and P ⁇ 0.005O *).
  • Figure 23 shows the effect of improving the momentum by TM-53 or TM-54 in the muscle injury mouse model.
  • 25 shows the inhibitory effect of MyRF and desmin expression of Myocytes and desmin and muscle atrophy related protein MuRFl by TM-53 and TM-54 in muscle impaired mouse model.
  • the present invention is a compound represented by the following [Formula 1] or
  • the compound represented by [Formula 1] is 2-butyl-6-styryl-1- [2 '-(1H-tetrazol-5-yl) —bisphenyl-4-yl represented by the following [Formula 2]: Methyl] -1H-benzoimidazol-5-carboxylic acid
  • Methyl ester (2-buty ⁇ 6-styryl-l- [2 '-(lH-tetrazol-5-yl) -biphenyl-4-ylmethyl] -lH-benzoimidazole-5-carboxyl ic acid methyl ester, TM-53) Is preferably, but is not limited thereto.
  • the compound represented by [Formula 1] is 2-butyl-5-styryl -1- [2 '-(1H-tetrazol-5-yl) -bisphenyl-4-yl represented by the following [Formula 3] Methyl] -1H-benzoimidazole-6-carboxylic acid
  • Methyl ester (2-butyl-5-styryl-l- [2 '— (lH-tetrazol-5-yl) -biphenyl-4-ylme1; hyl] -IH-benzo i mi dazo 1 e ⁇ 6-car boxy 1 that the ic acid methyl ester, TM_54) is not limited, a preferable thereto.
  • the compound represented by the above [Formula 1] is myoblast differentiation from myoblasts (myobl ast) or muscle cells derived from non-myocytes It is preferred, but not limited to, to promote either or both transdi f ferent iat ions.
  • the compound represented by the above [Formula 1] is preferably to reduce the expression of any one or both of muRFl or MAFbx, muscle atrophy related genes in differentiated muscle cells, but is not limited thereto.
  • the compound represented by [Formula 1] is preferably to increase the expression of myocyte marker gene MCK, myogenin (Myogenin) or MyoD, or myocyte marker protein MyHC in differentiated muscle cells, but is not limited thereto. .
  • the present inventors as a TAZ regulator
  • TM-53 and TM-54 confirmed the effect of TAZ on nuclear local izat ion regulation by TM-53 and TM-54. As the concentration of TM-53 increases, Cos7 cells and sources It was confirmed that the degree of localization of the TAZ protein in the nucleus (myoblast) increased (see FIGS. 1 to 4).
  • TM-53 and TM-54 In order to confirm the myogenic st ill latory effects exhibited by TM-53 and TM-54, the source cells were induced to differentiate into muscle cells in the presence of TM-53 or TM-54. Treatment with 53 or TM-54 induces differentiation of muscle cells, showing an effective muscle cell and differentiation effect (see FIGS. 5 to 7), which was confirmed to be TM-53 or TM-54 concentration dependent (FIG. 8). To FIG. 11).
  • TAZi cells and TAZ knockout MEFs K0 MEF
  • TAZ depends on the presence or absence of MyoD expression in non-muscle cells.
  • myoD turnover differentiation according to MyoD expression in TAZ knocked out MEF showed that KO + MyoD not expressing TAZ did not show differentiation into muscle cells. It was confirmed (see FIGS. 16 to 17), and even when treated with TM-53 or TM-54, TAZ and MyoD-dependent muscle cell conversion differentiation was observed (see FIGS. 18 to 20).
  • the TM-53 and TM-54 is the source cell and the non-exhibits a concentration, dependent differentiation promoting effect on differentiation, and conversion differentiated from the source cell to muscle cell significantly, the compounds of the invention or a pharmaceutical thereof, Alternatively, the acceptable salt may be usefully used as an active ingredient of the pharmaceutical composition for inducing muscle cell differentiation.
  • the compounds of the present invention may be used in the form of pharmaceutically acceptable salts, and acid salts formed by pharmaceutically acceptable free acids are useful as salts. "Acid addition salts are hydrochloric acid.
  • Inorganic acids such as nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphite, aliphatic mono and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and alkanedioates, aromatic acids, Obtained from non-toxic organic acids such as aliphatic and aromatic sulfonic acids.
  • These pharmaceutically toxic salts include sulfate, pyrosulfate, bisulfate, sulfite bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide and iodide , Fluoride, acetate, propionate, decanoate, caprylate, ' acrylate, formate, isobutyrate, caprate, heptanoate, propyrate, oxalate, malonate, succinate, suverate, Sebacate, fumarate, maleate, butyne-1, 4-dioate, nucleic acid-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitro benzoate, hydroxybenzoate, mexicic Benzoate, phthalate, terephthalate, Benzenesulfonate, toluenesulfonate, chlor
  • the acid addition salt according to the present invention is a conventional method, for example, by dissolving the compound of the formula [1] in an excess of aqueous acid solution, the salt is a miscible organic solvent, for example methanol, ethanol, acetone or aceto It can be prepared by precipitation using nitrile.
  • the same amount of the compound of [Formula 1] and acid or alcohol in water may be heated, and then the mixture may be evaporated to dryness, or the precipitated salt may be prepared by inhalation subfiltration.
  • Bases can also be used to make pharmaceutically acceptable metal salts.
  • Alkali metal or alkaline earth metal salts are obtained, for example, by dissolving the compound in an excess of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the compound salt at no cost, and evaporating and drying the filtrate. At this time, it is pharmaceutically suitable to prepare sodium, potassium or calcium salt as the metal salt.
  • Silver salts are also obtained by reacting alkali or alkaline earth metal salts with a suitable negative salt (eg silver nitrate).
  • the compound of [Formula 1] of the present invention includes not only pharmaceutically acceptable salts, but also all salts, hydrates, and solvates that can be prepared by conventional methods.
  • the addition salt according to the present invention can be prepared by a conventional method, for example, by dissolving the compound of [Formula 1] in a water-miscible organic solvent, for example acetone, methanol, ethanol, or acetonitrile and the excess organic acid It may be prepared by addition or precipitation of an acidic aqueous solution of an inorganic acid followed by precipitation or crystallization. This mixture can then be evaporated and dried to evaporate the solvent or excess acid to obtain additional salts or to precipitate prepared salts by suction filtration.
  • a water-miscible organic solvent for example acetone, methanol, ethanol, or acetonitrile
  • This mixture can then be evaporated and dried to evaporate the solvent or excess acid to obtain additional salts or to precipitate prepared salts by suction filtration.
  • composition of the present invention When using the composition of the present invention as a medicine, a pharmaceutical containing the compound of Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient
  • the composition may be formulated and administered in various oral or parenteral dosage forms as described below, but is not limited thereto.
  • Formulations for oral administration include, for example, tablets, pills, hard / soft capsules, solutions, suspensions, emulsifiers, syrups, granules, elixirs, etc. These formulations may contain diluents (e.g., lactose, dex) It contains, but is not limited to, troz, sucrose, manny, sorbbi, cellulose and / or glycine, lubricants such as silica, talc, stearic acid and its magnesium or calcium salts and / or polyethylene glycols.
  • diluents e.g., lactose, dex
  • lubricants such as silica, talc, stearic acid and its magnesium or calcium salts and / or polyethylene glycols.
  • Tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, methylcellose, sodium carboxymethylcellose and / or polyvinylpyridine, optionally starch, agar, alginic acid or its sodium salt Disintegrating or boiling mixtures such as and / or absorbents, colorants, flavors, and sweeteners.
  • binders such as magnesium aluminum silicate, starch paste, gelatin, methylcellose, sodium carboxymethylcellose and / or polyvinylpyridine, optionally starch, agar, alginic acid or its sodium salt Disintegrating or boiling mixtures such as and / or absorbents, colorants, flavors, and sweeteners.
  • compositions comprising the compound of formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient may be parenterally administered.
  • Parenteral administration may be performed by subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection. It depends on how to inject.
  • the compound of Formula 1 or a pharmaceutically acceptable salt thereof is mixed with water with a stabilizer or a buffer to prepare a formulation for parenteral administration. It may be prepared in a dosage form.
  • the composition may be sterilized and / or contain preservatives, stabilizers, hydrating or emulsifying accelerators, auxiliaries such as salts and / or buffers for the control of osmotic pressure, and other therapeutically useful substances, and common methods, It may be formulated according to the granulation or coating method.
  • the dosage of the compound of the present invention to the human body may vary depending on the age, weight, sex, dosage form, health condition and degree of disease of the patient, and generally based on an adult patient having a weight of 60 kg. 0.001 to 1,000 mg / day, preferably 0.01 to 500 mg / day, and may be administered once or several times a day at regular intervals according to the judgment of a doctor or pharmacist.
  • the present invention also provides a pharmaceutical composition, muscle regeneration containing the Formula 1 compound or a pharmaceutically acceptable salt thereof represented by, as an active ingredient.
  • the compound represented by the above [Formula 1] is preferably contained in a concentration of 1 to 100 ⁇ , more preferably contained in a concentration of 2 to 50 ⁇ , more specifically 2.5 to 10 ⁇ It is most preferably included in the concentration of ⁇ , but is not limited thereto.
  • the muscle regeneration is regeneration of damaged muscle cells, and the damage may include all of genetic factors including surgery, cancer, inflammation and infection, and environmental factors, but is not limited thereto.
  • the present inventors induce the differentiation of muscle cells to determine the improvement effect of ⁇ -53 and ⁇ -54 on muscle atrophy in atrophic muscle cells, and then dexamethasone. The treatment induced muscle atrophy and the degree of muscle atrophy in the presence of TM-53 or TM-54 was compared. As a result, the muscle cells differentiated with TM-53 or TM-54 were confirmed to have reduced MyHc expression due to DEX treatment, and there was no decrease in MyHC protein in TM-53 and TM-54 treated groups.
  • the present inventors in order to confirm the regeneration effect of the muscles damaged by TM-53 and TM-54 in vivo, the effect of improving the momentum and histological effects of TM-53 or TM-54 in a mouse model that caused muscle damage
  • the mouse model showed improvement of muscle activity (see FIG. 23), muscle regeneration in muscle tissue, Accordingly, the increase in the expression level of the muscle cell marker protein and the decrease in the expression level of muscle atrophy related protein were confirmed (see FIGS. 24 and 25).
  • TM-53 and TM-54 not only increase the expression of muscle cell marker protein while reducing the expression of muscle atrophy related genes in muscle cells treated with muscle atrophy inducing substances, but also the muscles of muscle injury models in vivo. Since the regeneration effect is significant, the compound of the present invention or a pharmaceutically acceptable salt thereof may be usefully used as an active ingredient of a pharmaceutical composition for muscle regeneration.
  • the present invention provides a pharmaceutical composition for treating or preventing muscle disorders containing the compound represented by the above [Formula 1] or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the compound represented by the above [Formula 1] is preferably contained in a concentration of 1 to 100 ⁇ , more preferably in a concentration of 2 to 50 ⁇ , more specifically 2.5 to 10 ⁇ It is most preferably included in the concentration of ⁇ , but is not limited thereto.
  • the 'muscle disorder are internal factors, such as bruises (contus ion), lacerations (lacerat ion) or apjwa (crush) muscle tension of external factors, sudden severe falls or sports activities, such as (musc le strain) May be at least one selected from the group consisting of muscle damage that occurs when blood supply that may occur during treatment, muscle wasting due to genetic or environmental causes, and loss of muscle function.
  • muscular atrophy muscle le atrophy
  • myopathy myopathy
  • muscle damage muscle damage
  • muscle dystrophy muscle dystrophy
  • muscle thrombocytopenia sarcopenia
  • the compound of the present invention or a pharmaceutically acceptable salt thereof is useful as an active ingredient of the pharmaceutical composition for the treatment or prevention of muscle disorders Can be used.
  • the present invention provides a health food for inducing muscle cell differentiation containing the compound represented by the following [Formula 1] or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the present invention provides a health food for muscle regeneration, containing the compound represented by the above [Formula 1] or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the present invention provides a health food for improving or preventing muscle disorders containing the compound represented by the above [Formula 1] or a pharmaceutically acceptable salt thereof as an active ingredient.
  • Methyl ester (2-butyl-6—styryl-l- [2 '-(lH-tetrazo ⁇ 5-yl) -biphenyl-4-ylmethyl] -lH-benzoimidazole-5-carboxyl ic acid methyl ester (TM_53) Preferred but not limited thereto.
  • the compound represented by [Formula 1] is 2-butyl-5-styryl-1- [2 '-(1H-tetrazol-5-yl) -bisphenyl-4-yl represented by the following [Formula 3]: Methyl] -1H-benzoimidazol-6-carboxylic acid
  • Methyl ester (2-bu ty 1 -5- st yr y 1-1- [2 '-(1H-1 etr azo 1 -5-y 1) -bi pheny 1 -4-y 1 me thyl] -lH- benzoimidazole-6-carboxylic acid methyl ester, TM ′ 54), but is not limited thereto.
  • the compound represented by the above [Formula 1] preferably promotes any one or both of myocyte-derived muscle cell differentiation or non-myodermal cell-derived differentiation, but is not limited thereto.
  • the compound represented by the above [Formula 1] is preferably to reduce the expression of any one or both of MuRFl or MFAbx, muscle atrophy related genes in differentiated muscle cells, but is not limited thereto.
  • Compound represented by the above [Formula 1] is a muscle cell marker gene MCK in differentiated muscle cells. It is desirable to increase the expression of myogenin or MyoD, or MyHC, a muscle cell marker protein, but is not limited thereto.
  • the compound represented by the above [Formula 1] is preferably contained in a concentration of 1 to 100 ⁇ , more preferably contained in a concentration of 2 to 50 ⁇ , more specifically 2.5 to 10 y It is most preferably included in the concentration of M, but is not limited thereto.
  • the muscle regeneration is regeneration of damaged muscle cells, and the damage may include all genetic factors including surgery, cancer, inflammation and infection, and environmental factors, but is not limited thereto. .
  • the muscle disorder ' may be caused by external factors such as contusions, l acerat ions or crushes, sudden or severe falls or sports. Internal factors such as muscle strain during activity, blood that can occur during treatment, muscle damage that occurs when supply is interrupted, muscle wasting due to genetic or environmental causes, and loss of muscle function It may be one or more selected from the group consisting of, specifically, muscle atrophy (musc le atrophy), myopathy, muscle injury (muscle le), muscular dystrophy (musc le dystrophy), sarcopeni a, It is more preferred, but not limited to, one or more selected from the group consisting of myoneural conduct ive di seases and nerve injury.
  • TM-53 and TM-54 of the present invention significantly show a concentration-dependent differentiation promoting effect on the differentiation and conversion of myocytes and non-myoblasts to myocytes, and muscle cells treated with muscle atrophy-inducing substances
  • the compounds of the present invention or pharmaceuticals thereof have a significant effect on the muscle regeneration effect of the muscle injury model in vivo (/ wVo).
  • 'acceptable salts is muscle cell differentiation inducing or promoting the health food, health food muscle regeneration, or treatment of a muscle disorder, or may be useful as an active ingredient of a pharmaceutical composition for preventing, for ⁇
  • the compound represented by [Formula 1] of the present invention or a pharmaceutically acceptable salt thereof may be added to a food as it is, used with other foods or food ingredients, or may be appropriately used according to a conventional method.
  • the mixing amount of the active ingredient can be suitably determined according to the purpose of use (prevention or improvement). In general, the amount of the compound in the health food is equal to the total food weight. 0.1 to 90 parts by weight may be added. However, in the case of long-term intake for health and hygiene or health control, the amount may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety. .
  • the health functional beverage composition of the present invention has no particular limitation on other ingredients except for containing the compound of the present invention as essential ingredients in the indicated ratios, and various flavoring agents or natural carbohydrates as additional ingredients such as ordinary drinks It may contain.
  • natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And sugars such as conventional sugars such as polysaccharides such as dextrin, cyclotextin and the like, and xylyl, sorbyl, and erythritol.
  • natural flavoring agents tautin, stevia extract (e.g., Rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used.
  • the proportion of natural carbohydrates is generally from about 1 to 20 g, preferably from about 5 to 12 g per 100 compositions of the present invention.
  • the compound represented by [Formula 1] of the present invention or a pharmaceutically acceptable salt thereof Flavors, colorants and neutralizing agents such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors ( Cheeses, chocolates, and the like), pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, and carbonation agents used in alcoholic beverages.
  • the compound represented by [Formula 1] of the present invention or a pharmaceutically acceptable salt thereof may contain a flesh for preparing natural fruit juice and fruit juice beverage and vegetable beverage. These components can be used independently or in combination.
  • the present invention is a pharmaceutical for inducing muscle cell differentiation or muscle regeneration It provides a compound represented by the above [Formula 1] or a pharmaceutically acceptable salt thereof for use as a composition.
  • the present invention provides a compound represented by the above [Formula 1] or a pharmaceutically acceptable salt thereof for use as a health food for muscle cell differentiation induction or muscle regeneration.
  • the present invention provides a compound represented by the above [Formula 1] or a pharmaceutically acceptable salt thereof for use as a pharmaceutical composition for treating and preventing muscle disorders.
  • the present invention provides a compound represented by the above [Formula 1] or a pharmaceutically acceptable salt thereof for use as a health food for improving or preventing muscle disorders.
  • the present invention comprises the step of administering a pharmaceutical composition containing a pharmaceutically effective amount of the compound represented by the above [Formula 1] or a pharmaceutically acceptable salt thereof as an active ingredient to an individual suffering from a muscle disorder It provides the muscle disorder 'way.
  • the present invention is a method for preventing muscle disorders comprising administering to a subject a pharmaceutical composition containing a pharmaceutically effective amount of the compound represented by the above [Formula 1] or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the TAZ regulator 2-butyl-6-styryl-1_ [2 '-(1H-tetrazol-5-yl) -bisphenyl-4-ylmethyl] -1H-benzoimidazole -5-carboxylic acid
  • Methyl ester (2-butyl-5-styry ⁇ l- [2 '-(lH-tetrazol-5-yl) -biphenyl-4-ylmethyl] -lH-benzo imi dazo 1 e-6-car boxy lie acid methyl ester , TM-54) were synthesized (Korean Patent No. 10-1117128).
  • the purity of the synthesized TM-53 and TM-54 was measured by high performance liquid chromatography (HPLC), and dissolved in dimethyl sulfoxide (DMMS0) at a concentration of 10 nig / mi. Stored.
  • TM-53 and TM-54 were synthesized as shown in the following [Formula 2] and [Formula 3], and the purity was confirmed to be 95%.
  • the green fluorescent protein well TAZ-GFP protein Connect the (green fluroescence protein, GFP) to build a vector that encodes', retrovirus for transduction them ( retrovirus) was prepared.
  • the full-length TAZ cDNA was amplified by PCR, and then inserted into a pEGFP vector (Invitrogen, USA), which is an expression vector containing a gene of GFP, to construct a pEGEP-TAZ vector.
  • a pEGFP vector Invitrogen, USA
  • cDNA of full-length histone H2B was amplified by PCR and inserted into an expression vector (Clontech Laboratories, USA) containing a gene of red fluorescence protein (RFP).
  • RFP red fluorescence protein
  • the constructed pEGEP-TAZ and pREP-H2B vectors were transformed into Platinum E (Plat.inum E, plat E; purchased from ATCC, USA), incubated in a 32 ° C incubator, pEGEP-TAZ and PREP-H2B A retrovirus containing a vector was obtained. Obtained Retroviruses were concentrated using PEG Virus Precipitation Kit (BioVision, USA). '
  • GFP-TAZ stabilized cells comprising pEGEP-TAZ and pREP-H2B vectors were prepared. Specifically, after inoculating and cultivating Cos7 cells (ATCC purchase, USA) to 5 ⁇ 10 3 cells per well in a 96-well plate, the retrovirus concentrated in Example ⁇ 2-1> was treated with Cos7 cells, 8 pEGEP-TAZ and PREP-H2B vectors were transfected by incubation for 24 hours in a medium containing ⁇ g / mi polybrene (Sigma Aldrich, USA).
  • Transfected Cos7 cells were screened in a medium containing 2.5 xg / mt puromycin (puromycin; Sigma Aldrich, USA), and then TM-53 prepared in Example 1 was 0, 1, 10 or 100, respectively.
  • GFP-TAZ stabilized cells were prepared by treating the transfected Cos7 cells at a concentration of ⁇ and incubating for 2 hours at an isotonic concentration of 300 mOsm NaCl or hypotonic condition of 150 mOsm NaCl. EJ al. (2012) Br J Pharmacol 165: 1584-1594).
  • the GFP-TAZ stabilized cells prepared above were BD pathway wfo
  • the TAZ protein was identified by fluorescence of GFP with a 3 ⁇ 4 ⁇ system (BD Pathway Bioimaging system; BD Biosciences ⁇ USA).
  • fluorescence of REP coupled with histone H2B was observed and corrected based on the fluorescence signal, and then fluorescence signal of GFP was quantitatively calculated.
  • Example 3 Identification of In Vitro Positioning Effect on TAZ of VII1-53 and TM-54 in Myoblasts
  • the nucleus position of endogenous TAZ protein was confirmed in C2C12 cells.
  • TM-53 or ⁇ -54 in C2C12 cells inoculated into DMEM medium containing 10% fetal bovine serum (FBS) and cultured in myoblasts. Treated for 24 hours, and then treated with phosphate buffered saline (PBS) containing 4% formaldehyde (fixed saline, PBS) for 15 minutes at room temperature, 0,1% Triton X-100 (triton) X-100) was treated to impart permeability to the cell membrane. After treatment, the treated cells were blocked in the silver for 1 hour by adding 4% normal donkey serum, and then incubated overnight at 4 ° C. with anti-TAZ antibody as a primary antibody.
  • PBS phosphate buffered saline
  • Triton X-100 triton
  • Example 4 Identification of Myogenic stimulatory Effects of 11-53 and TM-54 on Muscle Cell Differentiation ⁇ 4-l> Confirmation of differentiation-inducing effect on muscle cells following treatment of ⁇ -53 and lTtf-54 To confirm the differentiation-inducing effect of TM-53 and TM-54 on differentiation from myoblasts to muscle cells Myoblasts were induced to differentiate into myocytes in the presence of TM-53 or TM-54.
  • C2C12 cells which are myoblasts
  • DMEM medium containing 10% FBS
  • C2C12 myoblasts were inoculated in DMEM medium containing 10% FBS and cultured until 100% confluent.
  • C2C12 myoblasts were induced to differentiate into mature muscle cells while incubating for 6 hours with 10 ⁇ M of ⁇ -53 or ⁇ -54.
  • the cell morphology was confirmed by phase contrast / microscopy, followed by fluorescence immunostaining of MyHC, a myocyte marker, in differentiated muscle cells by the same method as in ⁇ Example 3>.
  • the primary antibody for fluorescence immunostaining an anti-MyHC antibody was used, and the nuclei were stained with DAPI to compare the position of the nucleus and the expression level of fluorescence.
  • DMS0 containing no TM-53 or TM-54 was treated in the same manner as above and compared.
  • C2C12 cells which are myoblasts, were inoculated in DMEM medium containing 10% FBS and cultured until 100% saturation, followed by ⁇ -53 at a concentration of 0, 2.5, 5 or 10 ⁇ .
  • DMEM medium containing 10% FBS
  • ⁇ -53 at a concentration of 0, 2.5, 5 or 10 ⁇ .
  • Each process a "or TM-54 and incubated for 6 hours and then induced to differentiate into mature cell origin C2C12 muscle cells.
  • the induced muscle cells were obtained and crushed to obtain a supernatant obtained from each cell as a protein extract, and then the whole protein was separated from SDS-PAGE ge l, and nitrocel membrane (ni trocel). lulose membrane) and blocked with 4% skim milk or 5% bovine serum albumi (BSA).
  • BSA bovine serum albumi
  • the membrane was treated with anti-MyHC antibody (product number: MF-20, DSHB, USA) and anti-myogenin antibody as primary antibody, incubated at 4 ° C. overnight, washed with TBST, Goose-derived anti-mouse IgG antibody (goat ant i-mouse IgG) as a secondary antibody 1) treated with membranes and TBST containing bovine serum albumi (BSA) and incubated for 1 hour. Western blot was performed. As a control for comparing the degree of expression, using the anti- ⁇ -actin antibody (Santa Cruz Biotechnology, USA) as the primary antibody was carried out in the same manner as described above to express the expression of ⁇ -actin Confirmed.
  • anti-MyHC antibody product number: MF-20, DSHB, USA
  • anti-myogenin antibody as primary antibody
  • Induction of differentiation into muscle cells depends on the throughput of TM-53 and TM-54 . To determine whether or not they were treated, various concentrations of TM-53 or TM-54 were used to induce differentiation from myoblasts to myocytes, and the muscle cell marker gene, muscle creatine kinase (MCC), Transcriptional levels of myogenin, MyoD and TAZ were confirmed.
  • MCC muscle creatine kinase
  • C2C12 cells which are myocytes
  • DMEM medium containing 10% FBS
  • ⁇ —53 or ⁇ -54 at a concentration of 0, 2.5, 5 or 10 ⁇ .
  • C2C12 myocytes matured.
  • Induced to differentiate into muscle cells After induction, the induced muscle cells were obtained, suspended in TRIZOL (TRIzol; Invitrogen, USA) reagents, and extracted total RA of muscle cells according to the manufacturer's protocol, and the primers and superscript II kits of Table 1 below.
  • TRIZOL TRIzol; Invitrogen, USA
  • TAZ knockdown cell line (TAZ knockdown, TAZi) which reduced the amount of TAZ expression was prepared by applying virus shRNA technique.
  • the pSRP-shTAZ vector was transformed into platinum E cells and cultured in a 32 ° C. incubator to obtain a retrovirus containing the pSRP-shTAZ vector, which was concentrated using a PEGvirus precipitation kit. Then, the concentrated virus was treated with C2C12 cells, incubated for 24 hours in a medium containing 8 g / m £ polybrene (polybrene; Sigma Aldrich, USA), and C2C12 cells transfected with pSRP-shTAZ vector.
  • polybrene polybrene
  • TAZ-knocked out myocytes were prepared.
  • pAZRP vectors containing no gene knocking out TAZ were transfected into C2C12 cells using the same method as described above to prepare TAZ expressing C2C12 cells (coni).
  • TAZi cells were induced to differentiate into muscle cells in order to confirm the effect of the expression of TAZ on the promoting effect of the differentiation of muscle cells represented by TM-53 and TM-54.
  • TAZi cells were induced to differentiate into mature muscle cells. Then, the same as in the above ⁇ 4-2> Western blot was performed to check the expression of MyHC protein. In order to compare the same amount of protein used in the analysis, the expression of ⁇ -actin (act in) was confirmed.
  • TAZ expressing C2C12 cells (coni) prepared in Example ⁇ 5-1> were used.
  • DMS0 which does not contain TM-53 or TM-54, was treated in the same manner as above, and compared them.
  • TAZ-expressing myoblasts show the effect of promoting the differentiation of muscle cells by TM-53 or TM-54 compared to the solvent control, whereas TAZ knockdown myoblasts (TAZi) did not express MyHC even in TM-53 or TM-54 treatment, confirming that the differentiation of myoblasts did not appear (Fig. 12).
  • TAZi TAZ knockdown myoblast line
  • TAZi cells prepared in the above ⁇ 5-1> inoculated in DMEM medium containing KM FBS and incubated until 100% saturated, ⁇ —53 or ⁇ -54 at a concentration of 10 ⁇ ⁇ , respectively During treatment and incubation for 6 hours, TAZi cells were induced to differentiate into mature muscle cells. After induction, the induced myocytes were obtained, total RNA was extracted using the same method as in Example ⁇ 4-3> to synthesize cDNAs of MCK, myogenin and TAZ genes, respectively, and then real-t ime. RCR was performed to quantitatively confirm the transcriptional expression of the gene.
  • Example ⁇ 5-1> The coni cells prepared in Example ⁇ 5-1> were used as the negative control, and the solvent was used. Control group is to "handle DMS0 that do not contain or TM-53 TM-54 in the same manner as described above, were compared. ⁇
  • TM-53 and TM-54 embryonic fibroblasts (MEFs) of TAZ knockout mice were prepared.
  • mice after removing the head and liver from embryos of 13-14 days old mice of control wild-type mice and TAZ knockout mice (knockout K0), the tissues were subdivided, and then 0.05% Trypsin-0.02% EDTA. The cells were separated by treatment for 30 minutes. Culture of MEF cells was performed using DMEM medium containing 10% FBS and subcultured to remove other tissue cells and prepared by using ⁇ -MEF and KOMEF cell lines.
  • MyoD expression including the pMCK-luciferase (luc if erase, luc) reporter gene in K ()-MEF prepared in Example ⁇ 6-1> A vector (Jeong H et al. (2010) .FASEB J 24: 3310-3320) was transfected in the same manner as in Example ⁇ 5-1> to prepare a KO / MyoD cell line. Then, the prepared KO / MyoD cells were inoculated in DMEM medium containing 10% FBS and incubated until 100% saturated, and then treated with ⁇ -53 or ⁇ -54 at a concentration of 10 ⁇ , respectively.
  • the cultured KO / MyoD cells were suspended in reporter lysi s buf fer to extract cellular proteins.
  • the extracted cellular protein 10 ⁇ was mixed with a luciferase substrate (promega, USA) to measure the luminescence with a luminometer (luminometer; Berthold, Germany) to induce the MCK promoter of MyoD. Expression level was confirmed.
  • a control for calibration the beta-galactosidase activity following the expression of the pCMVbeta-gal gene along with the MyoD expression vector was measured and then the transfect ion efficiency was corrected.
  • the increase in MCK promoter activity was expressed as fold induct ion or percentage (%) compared to the solvent control.
  • a WT / MyoD cell line transfected with MyoD expression vector containing pMCK-luc reporter gene in ⁇ -MEF was confirmed by the same method as above, and the expression level of MyoD was confirmed as a solvent control.
  • DMS0 not containing TM-54 was treated in the same manner as above, and compared.
  • a retrovirus (RV) containing a MyoD expression vector in the same manner as in Example ⁇ 5-1> -MyoD were infected to obtain WT-MEF (WT + MyoD) or K0-MEF (K0 + MyoD) expressing MyoD, respectively. Then, the obtained WT + MyoD or KO + MyoD was inoculated into DMEM medium containing 10% FBS and cultured to induce differentiation into mature muscle cells, and the shape of the cells was confirmed by a phase contrast microscope. As a negative control, retrovirus (RV) containing a MyoD expression vector was performed in the same manner as above. .
  • Example ⁇ 4-3> After performing the same method as in Example ⁇ 7-1> to obtain WT + MyoD or KO + MyoD, and incubating them to induce differentiation into mature muscle cells, and in Example ⁇ 4-3> In the same manner, cDNA of the muscle cells was synthesized, and real-time PCR was performed to quantitatively confirm the transcriptional expression of MCK, myogenin and MyoD genes. As a negative control, retrovirus (RV) containing MyoD expression vector was transfected into WT + MyoD or KO + MyoD to differentiate the muscle cells in the same manner as above.
  • RV retrovirus
  • TM-53 and TM-54 when inducing differentiation from non-muscle cells to muscle cells through TAZ-dependent myocyte turnover differentiation, treatment with TM-53 or TM-54 induced differentiation.
  • Expression of myocyte marker genes was confirmed in muscle cells derived from WT + MyoD and KO + MyoD.
  • retrovirus (RV-MyoD) expressing MyoD in WT-MEF or K0-MEF prepared by performing the same method as Example ⁇ 6-1> in the same manner as in Example ⁇ 5-1> ) was infected to obtain WT-MEF (WT + My is 3) or K0-MEF KO + MyoD expressing MyoD, respectively. Then, the obtained WT + MyoD or KO + MyoD was inoculated in DMEM medium containing 10% FBS, incubated until 100% saturated, and then ⁇ -53 or ⁇ —54 at a concentration of 10 ⁇ , respectively. Treatment and induction led to differentiation into mature muscle cells.
  • Differentiated muscle cells were obtained to synthesize cDNA in the same manner as in Example ⁇ 4-3>, and real-time PCR was performed to confirm the level of transcriptional expression of MCK and myogenin genes.
  • the expression of the ⁇ -actin gene was confirmed by performing the same method as described above, corrected based on this, and compared based on the expression level of MyoD gene.
  • DMS0 which does not contain TM-53 or TM-54, was treated in the same manner as above and compared.
  • TAZ expression was performed on MyoD-induced TAZ gene-removing cells. After recovery, the effect of promoting conversion differentiation from non-muscle cells to muscle cells by TM-53 or TM-54 was confirmed through the expression of muscle cell differentiation marker protein.
  • the cDNA of MyoD or TAZ is amplified by PCR and linked to the expression vector (l igat i on), and then amplified and obtained by plasmid by bacter ia transformat i on, cut with a specific restriction enzyme.
  • MyoD expression vector or TAZ expression vector By identifying the cDNA expression vector const ruct ion through sequencing, MyoD expression vector or TAZ expression vector (Hong JH et al, (2005). Science 309: 1074-1078).
  • TAZ knockout cells by transfecting the MyoD expression vector and the TAZ expression vector in the same manner as in Example ⁇ 5-1> to K0-MEF prepared by performing the same method as in Example ⁇ 6_1>. Restored expression of TAZ at (K0 + TAZ).
  • the obtained K0 + TAZ was inoculated in DMEM medium containing 10% FBS and incubated until 100% saturated, and then treated with ⁇ -53 or ⁇ -54 at a concentration of 10 ⁇ and incubated, respectively. , Induced to differentiate into mature muscle cells. Differentiated muscle cells were obtained and Western blot was performed in the same manner as in Example ⁇ 4-2> to confirm the expression of MyHC, myogenin and MyoD proteins. TAZ knockout cells that did not restore TAZ expression were used as a negative control, and DMS0 containing no TM-53 or TM-54 as a solvent control was treated in the same manner as above and compared.
  • KO + MyoD was evaluated as. After restoring the expression, the effect of promoting the differentiation of non-muscle cells into muscle cells by TM-53 or TM-54 was confirmed through the expression of myocyte marker genes.
  • Example ⁇ 6-1> and a a K0-MEF prepared by performing the same method after the building in the "Example ⁇ 8-2> MyoD expression vector and TAZ expression vector ⁇ 5-1> Transfection was performed in the same manner as in TAZ knockout cells to restore expression of TAZ (K0 + TAZ). Then, the obtained K0 + TAZ was inoculated in a DMEM medium containing 10% FBS and incubated until 100% saturation, followed by treatment and incubation of ⁇ -53 or ⁇ -54 at a concentration of 10 ⁇ , respectively, Induced to differentiate into mature muscle cells.
  • cDNA was synthesized in the same manner as in Example ⁇ 4>3>, and real-t ime PCR was performed to confirm the level of transcriptional expression of MCK and myogenin genes.
  • the expression of the gene of ⁇ -actin was confirmed by performing the same method as described above, and corrected based on this, and as a solvent control, DMS0 without ⁇ -53 or ⁇ -54 was the same as above. Treated by the method, and compared them.
  • Texametason a substance that induces muscle atrophy after treatment with TM-53 and TM-54, induced differentiation ( dexamethasone, DEX) -induced myHC protein expression induction effect.
  • C2C12 cells which are myocytes, were inoculated in DMEM medium containing 1OT FBS and incubated until 10OT saturated, followed by incubation for 6 days by treating ⁇ -53 or ⁇ —54 at a concentration of 10 ⁇ , respectively.
  • C2C12 myoblasts were induced to differentiate into mature muscle cells.
  • muscle atrophy was induced by treating the differentiated muscle cells with 500 ⁇ M dexamethasone (dexmethasone (DEX) for 24 hours to induce muscle atrophy, followed by Western blot in the same manner as in Example ⁇ 4-2>. Expression of MyHC was confirmed.
  • DEX dexamethasone
  • TM-53 and TM-54 In order to confirm the improvement effect of TM-53 and TM-54 on muscle atrophy in atrophic muscle cells, after treatment with TM-53 or TM-54, differentiation was induced. Expression of muscle atrophy related genes in muscle cells that induced muscle atrophy was confirmed.
  • C2C12 cells which are myocytes, were inoculated in DMEM medium containing 1OT FBS and cultured until 100% saturated, followed by incubation for 6 days by treating ⁇ -53 or ⁇ —54 at a concentration of 10 ⁇ , respectively.
  • C2C12 myoblasts were induced to differentiate into mature muscle cells. After induction, induced muscle atrophy by treating 500 ⁇ dexamethasone (dexmethasone (DEX)) for 24 hours on differentiated muscle cells, and then synthesizing cDNA of muscle cells in the same manner as in Example ⁇ 4-3>.
  • Real-t ime PCR was performed to confirm the expression of MuRFl and MAFbx genes, which are muscle atrophy related genes.
  • the sequences of the primers used for cDNA synthesis and real-time PCR are as described in Table 2 below.
  • normal cells without DEX treatment were used for differentiated muscle cells, and as a control group, DMS0 containing no TM-53 or TM-54 was treated in the same manner as above.
  • mice Male C57BL / 6 wild-type mice were prepared, and the guidelines specified by the Institutional Animal Care and Use Co. ittee (IACUC) (IACUC-2011-01-027, 2012-01-035 and 2013-). 01-074) were bred in the experimental animal laboratory of Ewha laboratory animal genetic research center. When the reared mice are 12 weeks old, the mice weighing 25 to 30 g are selected and weakly anesthetized, and then the hip bones are made in small incisions, the sciatic nerves are exposed, and 6-0 silk thread ( The wound was closed with 6-0 silk suture thread) to prepare a nerve injury-induced muscular dystrophy mouse model.
  • IACUC Institutional Animal Care and Use Co. ittee
  • the embodiment to ⁇ 10-1> A muscle damage mouse model for screening prepared in 6, 50mg / kg TM-53 come after intraperitoneal injection to each mouse model, 7days added. Breeding. Then, an electric shock was applied to the tibialis anterior (TA) to induce the mouse model to exercise in a treadmill, and the amount of movement of the muscle-injured mouse model was confirmed through the running speed. As a result, when the TM-53 was administered as shown in FIG. 23, the amount of exercise of the mouse model was increased even without stimulation to the tibia, indicating that muscle activity was improved (FIG. 23).
  • TA tibialis anterior
  • Example ⁇ 10-1> 12 small muscle injury mouse models were selected and divided into 6 groups of 6 mice each, and 50 nig / kg TM-53 or TM-54 intraperitoneally in each mouse model group. After injection, the animals were further bred for 7 days. Then, muscle tissue was obtained at the expense of the mouse model, and then fixed with paraffin according to a known method, staining the muscle tissue with hematoxyHn-Eos in staining to histological damage of the muscle. The degree was confirmed. '
  • Example ⁇ 10-1> 12 muscle-injured mouse models prepared in Example ⁇ 10-1> were divided into 6 groups of 6 mice each, and 50nig / kg TM-53 or TM-54 was intraperitoneally in each mouse model group. After injection, the animals were further bred for 7 days. Then, the mouse model was regenerated to obtain muscle tissue, cell extracts were obtained from the muscle tissue, and then Western blot was performed in the same manner as in Example ⁇ 4-2> to perform MyHC, desmin, and MuRFl. Expression of the protein was confirmed.
  • mice treated with TM-53 or TM-54 as shown in FIG. In the muscle cells of the model, the expression of MyHC and desmin proteins were significantly increased, and muRFl, a muscle atrophy-related protein expressed in the solvent control group, was not expressed. Thus, the effect of TM-53 and TM-54 on muscle atrophy was improved. (Fig. 25).

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Abstract

La présente invention concerne une composition contenant TM-53 et TM-54 en tant que substance active pour induire ou stimuler la différenciation de myocytes, une composition pharmaceutique pour la régénération musculaire, et une composition pharmaceutique pour traiter ou prévenir une dystrophie musculaire. Plus spécifiquement, l'ester méthylique d'acide 2-butyl-styryl-1-[2'-(1H-tétrazole-5-yl)-bisphényl-4-ylméthyl]-1H-benzoimidazole-5-carboxylique (ester méthylique d'acide (2-butyl-6-styryl-1-[2'-(1H-tétrazol-5-yl)-biphényl-4-ylméthyl]-1H-benzoimidazole-5-carboxylique, TM-53) ou l'ester méthylique d'acide 2-butyl-5-styryl-1-[2'-(1H-tétrazole-5-yl)-bisphényl-4-ylméthyl]-1H-benzoimidazole-5-carboxylique (ester méthylique d'acide 2-butyl-5-styryl-1-[2'-(1H-tétrazol-5-yl)-biphényl-4-ylméthyl]-1H-benzoimidazole-6-carboxylique, TM-54) stimule la différenciation et la transdifférenciation en myocytes à partir de myoblastes et de non-myoblastes dépendant de la protéine TAZ in vitro, augmente l'expression de protéine marqueur myogénique tout en réduisant l'expression de gènes associés à une amyotrophie dans des myocytes traités avec un matériau induisant une amyotrophie, et présente significativement un effet de génération de muscles dans le modèle de dommages musculaires in vivo, et peut donc être favorablement utilisé en tant que substance active d'une composition pour induire ou stimuler la différenciation de myocytes, une composition pharmaceutique pour la génération de muscles, et une composition pharmaceutique pour traiter ou prévenir une dystrophie musculaire.
PCT/KR2014/012600 2013-12-19 2014-12-19 Composition pharmaceutique contenant un composant d'induction d'activation de la protéine taz pour différenciation musculaire et régénération musculaire WO2015093901A1 (fr)

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KR101993510B1 (ko) * 2017-01-06 2019-06-26 연세대학교 산학협력단 제라닉산 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 포함하는 근육 질환 예방 또는 치료용 조성물
KR101996124B1 (ko) * 2017-01-06 2019-07-03 연세대학교 산학협력단 수베르산 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 포함하는 근육 질환 예방 또는 치료용 조성물
KR102279751B1 (ko) * 2019-06-28 2021-07-21 경북대학교 산학협력단 근원성 인자 또는 nogo-a 측정 제제를 포함하는 근육병 진단용 조성물과 이를 이용한 근육병 진단 방법

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