[go: up one dir, main page]

WO2013038740A1 - Procédé de criblage pour identifier une substance ou un facteur capable de favoriser l'hypertrophie musculaire - Google Patents

Procédé de criblage pour identifier une substance ou un facteur capable de favoriser l'hypertrophie musculaire Download PDF

Info

Publication number
WO2013038740A1
WO2013038740A1 PCT/JP2012/057128 JP2012057128W WO2013038740A1 WO 2013038740 A1 WO2013038740 A1 WO 2013038740A1 JP 2012057128 W JP2012057128 W JP 2012057128W WO 2013038740 A1 WO2013038740 A1 WO 2013038740A1
Authority
WO
WIPO (PCT)
Prior art keywords
muscle
factor
peroxynitrite
muscle hypertrophy
superoxide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP2012/057128
Other languages
English (en)
Japanese (ja)
Inventor
武田　伸一
尚基 伊藤
ウルス ルーグ
友子 鈴木
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
National Center of Neurology and Psychiatry
Original Assignee
National Center of Neurology and Psychiatry
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by National Center of Neurology and Psychiatry filed Critical National Center of Neurology and Psychiatry
Publication of WO2013038740A1 publication Critical patent/WO2013038740A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5061—Muscle cells
    • 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
    • A61P21/04—Drugs for disorders of the muscular or neuromuscular system for myasthenia gravis

Definitions

  • the present invention relates to a method for screening a substance or factor that promotes muscle hypertrophy and a muscle hypertrophy promoter.
  • Non-patent Document 1 Skeletal muscle weight is controlled by the balance between protein synthesis and degradation (Non-patent Document 1).
  • Muscle atrophy is not only immobilized by space flight under microgravity (Non-Patent Document 2) or tail suspension (Non-Patent Document 3), but also casheia (Non-Patent Document 4), aging (Sarkopair) (Non-Patent Document) 5), also caused by steroid administration (Non-patent Document 6).
  • Non-patent Document 7 amyotrophic lateral sclerosis
  • Neurogenic muscle atrophy or molecular pathology in the sense accompanying muscle atrophy
  • Non-patent Document 8 amyotrophic lateral sclerosis
  • nNOS neuronal nitric oxide synthase
  • Non-patent Document 3 NNOS released from the muscle cell membrane during mouse tail suspension moves to the cytoplasm, produces nitric oxide (NO), inhibits phosphorylation of Forkheadkbox O (Foxo), and activates expression of E3 ubiquitin ligase Caused muscle atrophy.
  • nNOS was involved in recovery from muscle wasting during reloading. This suggests that nNOS controls not only muscle atrophy but also muscle hypertrophy.
  • the present invention aims to clarify the function of nNOS during muscle hypertrophy and to establish a new treatment method for muscle atrophy based on its molecular basis, that is, a method and means for promoting muscle hypertrophy. To do.
  • the present inventors have found that peroxynitrite produced by NO and superoxide controls intracellular calcium concentration via TRP channels, thereby causing muscle hypertrophy. It was found that it was promoted, and it was found that muscle hypertrophy can be promoted by increasing the concentration of superoxide and / or peroxynitrite and / or calcium in muscle cells, leading to the completion of the present invention. It was.
  • the present invention includes the following [1] to [9].
  • [1] A screening method for substances or factors that promote muscle hypertrophy, (A) measuring the concentration of superoxide and / or peroxynitrite and / or calcium in muscle cells obtained from an animal treated with a test substance or factor; (B) A method comprising identifying a test substance or factor as a candidate substance or factor that promotes muscle hypertrophy based on the result of (a).
  • [2] A screening method for substances or factors that promote muscle hypertrophy, (A) treating muscle cells with a test substance or factor; (B) measuring the concentration of superoxide and / or peroxynitrite and / or calcium in the muscle cells; (C) A method comprising identifying a test substance or factor as a candidate substance or factor that promotes muscle hypertrophy based on the result of (b). [3] The method according to [1] or [2], further comprising a step of measuring superoxide and / or peroxynitrite and / or calcium concentration in muscle cells before treatment with the test substance or factor. .
  • the test substance or factor is a substance or factor that promotes muscle hypertrophy.
  • a muscle hypertrophy promoter comprising at least one drug selected from the group consisting of a superoxide donor, a peroxynitrite donor, and a TRP channel agonist.
  • a method for promoting muscle hypertrophy comprising administering to a subject an effective amount of at least one drug selected from the group consisting of a superoxide donor, a peroxynitrite donor, and a TRP channel agonist.
  • the screening method according to the present invention can identify substances or factors that promote muscle hypertrophy and is useful for the development of therapeutic methods such as muscle atrophy.
  • the muscle hypertrophy promoter according to the present invention can effectively promote muscle hypertrophy and is useful for prevention or treatment of muscle atrophy.
  • the present invention provides a method and a muscle hypertrophy promoter for screening for substances and factors that promote muscle hypertrophy based on the involvement of superoxide, peroxynitrite and intracellular calcium in muscle hypertrophy.
  • the present inventor performed hindlimb cooperative muscle excision in nNOS wild-type and deficient mice to induce compensatory muscle hypertrophy due to overload.
  • the wild type increased the muscle weight by about 40% compared to the control group, but the deficient type increased only about 20%.
  • NOS activity was measured.
  • nNOS was activated 3 minutes after overloading and decreased to a steady level after 1 hour.
  • pharmacological inhibition of nNOS activity it was found that NO produced during 1 hour of overload promoted muscle hypertrophy.
  • vasodilators such as 8Br-cGMP cannot restore the defective phenotype, and NO produced by nNOS is mediated by vasodilation controlled by the classical NO-cGMP pathway. It was suggested that muscle hypertrophy was promoted. Therefore, it is a cGMP-independent pathway, and it is thought that peroxynitrite, which is a reaction product of NO and superoxide, may promote muscle hypertrophy. Superoxide and peroxynitrite immediately after overload An experiment was conducted to clarify the function of (Example 2).
  • BAPTA-AM and EGTA calcium chelators
  • nNOS-deficient phenotype was restored by NO donor and peroxynitrite donor, but the effect was completely suppressed by co-administration with BAPTA-AM and BTP2. Furthermore, administration of a TRP channel agonist (Hyp9, olvanil) restored the nNOS-deficient phenotype and increased muscle weight. Furthermore, it was found that peroxynitrite is involved in the increase of calcium concentration in myoblasts, specifically, inducing calcium release in the sarcoplasmic reticulum.
  • a substance or factor that promotes or inhibits muscle hypertrophy is identified based on the concentration of superoxide and / or peroxynitrite and / or calcium in muscle cells. That is, by treating an animal or muscle cell with a test substance or factor and measuring the concentration of superoxide and / or peroxynitrite and / or calcium in the animal or muscle cell, the test substance or factor becomes superoxide. And / or whether it affects peroxynitrite and / or calcium concentrations, ie muscle hypertrophy.
  • muscle hypertrophy refers to an increase in muscle weight due to an increase in the weight of a single muscle fiber or cross-sectional area accompanying an increase in the amount of endogenous protein
  • promotes muscle hypertrophy By promoting the increase in amount, it means promoting the increase in muscle weight due to the increase in single muscle fiber weight or cross-sectional area.
  • the screening method in the screening method according to the present invention (hereinafter, also referred to as “the screening method”), muscle cells obtained from an animal treated with a test substance or factor are prepared, or muscle cells are treated with the test substance or factor. Then, the superoxide and / or peroxynitrite and / or calcium concentration in the muscle cells is measured. Preferably, prior to treatment with a test substance or factor, superoxide and / or peroxynitrite and / or calcium concentrations in muscle cells or muscle cells obtained from animals are measured.
  • the target animal is not particularly limited as long as it has muscles, and mammals such as humans, primates (monkeys, chimpanzees, etc.), livestock animals (cattle, horses, pigs, etc.), pets ( Dogs, cats, etc.), laboratory animals (mouse, rats, monkeys, etc.), amphibians, reptiles and birds. Further, the animal may be a normal animal, an muscularly atrophic animal, an nNOS-deficient animal, or an animal that has developed muscular dystrophy. In general, after the effectiveness of a test substance or factor is confirmed at the cellular level, the effectiveness is evaluated in a laboratory animal, and further in a human, for example, by a clinical test.
  • test substance or factor that is the subject of this screening method is not particularly limited.
  • the test substance or factor can be any substance, specifically a naturally occurring molecule such as an amino acid, peptide, oligopeptide, polypeptide, protein, nucleic acid, lipid, carbohydrate (such as sugar), steroid, glycopeptide Synthetic analogs or derivatives of naturally occurring molecules, such as peptidomimetics, nucleic acid molecules (aptamers, antisense nucleic acids, double-stranded RNA (RNAi), etc.), etc .; non-naturally occurring molecules, such as And low molecular organic compounds (inorganic and organic compound libraries, combinatorial libraries, etc.) produced using combinatorial chemistry techniques and the like; and mixtures thereof.
  • the test substance or factor may be a single substance, a complex composed of a plurality of substances, a transcription factor, or the like.
  • the test substance or factor may be an environmental factor such as radiation, ultraviolet rays, carbon concentration, or temperature.
  • test substance or factor a single test substance or factor may be tested independently, or a mixture of several candidate test substances or factors (including a library or the like) may be tested.
  • the library containing a plurality of test substances or factors include a synthetic compound library (such as a combinatorial library) and a peptide library (such as a combinatorial library).
  • the treatment conditions such as the treatment amount, treatment period, treatment route, etc. vary depending on the type of test substance or factor, but those skilled in the art can easily determine it. it can.
  • the route of administration is intramuscular injection, oral administration, intravenous injection, intraperitoneal injection, transdermal administration, subcutaneous injection, depending on the type of test substance and the type of animal used.
  • Such administration forms can be appropriately used.
  • Muscle cells can be collected by methods known in the art. Specifically, for example, after chopping skeletal muscle and transferring it to a 50 ml conical bottom tube, 4 ml of Dispase2 (2.4 IU / ml) -Collagenase XI (0.2%) solution per 1 g of muscle weight is added, Incubate at 37 ° C for 45-60 minutes, pipetting with a pipette every 15 minutes. Thereafter, the tissue piece is crushed by passing several times through an 18 G injection needle, and the supernatant is collected.
  • the precipitated cells are suspended in 25 ml of growth medium, transferred to an uncoated 15 cm culture dish, added with bFGF, and cultured for 90 minutes at 37 ° C., 5% CO 2 concentration, and the supernatant containing unattached cells is collected. Using this supernatant, wash the bottom of the culture dish, change the direction 180 degrees, incubate again at 37 ° C, 5% CO 2 concentration for 90 minutes, and collect the supernatant. Transfer the collected supernatant to a 15 cm collagen-coated culture dish, add bFGF, and culture overnight at 37 ° C. and 5% CO 2 concentration. The next day, if the myoblasts are more than 30-40% confluent, passaging, and if less, change the medium. In order to prevent myogenic differentiation, subculture or medium exchange is performed every day thereafter. Alternatively, commercially available muscle cells or publicly available muscle cells can be used.
  • the contact condition varies depending on the type of the substance or factor, but can be easily determined by those skilled in the art.
  • such contact can be achieved by culturing myocytes in a medium supplemented with a test substance, immersing the myocytes in a solution containing the test substance, and laminating the test substance on the muscle cells.
  • it can be performed by culturing myocytes in the presence of a test factor.
  • the effect and effectiveness of the test substance or factor can be examined under several conditions. Such conditions include time or duration, amount (large or small), number of times, etc. of treatment with the test substance or factor.
  • a plurality of doses can be set by preparing a dilution series of the test substance.
  • the treatment period of the test substance or factor can also be set as appropriate.
  • the treatment can be performed over a period of 1 day to several weeks, months, and years.
  • test substances and / or factors may be used in combination.
  • the concentration of superoxide and / or peroxynitrite and / or calcium in the muscle cell obtained from the animal or the muscle cell is appropriately determined. taking measurement. For example, immediately after treatment, 30 minutes, 1 hour, 3 hours, 5 hours, 10 hours, 15 hours, 20 hours, 24 hours (1 day), 2-10 days, 10-20 Measurements are taken after 20-30 days, 1-6 months later.
  • the measurement of superoxide can be performed by a method known in the art, for example, by using lucigenin that emits light by reacting with superoxide, and measuring the emission intensity.
  • Peroxynitrite can be measured by a method known in the art.
  • the peroxynitrite is measured by measuring the fluorescence intensity of a fluorescent substance emitted by reacting peroxynitrite with an active oxygen species or an active nitrogen species. This can be done by calculating the difference from the control group treated with the nitric acid scavenger.
  • the calcium concentration in muscle cells can also be measured by a method known in the art.
  • the fluorescence intensity is measured using Fluo-4 that emits fluorescence when bound to calcium. Can be performed.
  • test substance or factor that increases or decreases the superoxide and / or peroxynitrite and / or calcium concentration relative to the control after measuring the superoxide and / or peroxynitrite and / or calcium concentration in muscle cells Selected as a substance or factor that promotes or inhibits muscle hypertrophy.
  • animals or muscle cells that have not been treated with the test substance or factor can be used.
  • test substance or factor that has been treated with a test substance or factor and that has increased the superoxide and / or peroxynitrite and / or calcium concentration in the myocyte is obtained.
  • test substance or factor Selected and then as a secondary screen, animals were treated with the selected test substance or factor and the concentrations of superoxide and / or peroxynitrite and / or calcium in the muscle cells of the animals were measured to show increased concentrations
  • a test substance or factor may be selected.
  • the selected test substance or factor is administered to an experimental animal to determine whether the test substance or factor promotes muscle hypertrophy in the experimental animal.
  • an experimental animal a model animal, preferably a mouse, in which muscle atrophy has been induced by hindlimb suspension, denervation, dexamethasone administration, or the like can be used.
  • Whether or not a test substance or factor promotes muscle hypertrophy in an experimental animal depends on the type of experimental animal and the like, but can be appropriately determined by those skilled in the art by methods known in the art. For example, muscle tissue can be collected from an animal and the muscle weight can be measured.
  • a candidate substance or factor that promotes muscle hypertrophy can be identified, and further, the effectiveness of the substance or factor that promotes muscle hypertrophy can be confirmed.
  • the muscle hypertrophy promoter according to the present invention contains a substance or factor capable of increasing the superoxide and / or peroxynitrite and / or calcium concentration.
  • the muscle hypertrophy promoter according to the present invention includes at least one drug selected from the group consisting of a superoxide donor, a peroxynitrite donor, and a TRP channel agonist.
  • the muscle hypertrophy promoter according to the present invention is useful for the prevention or treatment of diseases or disorders such as muscle atrophy, especially for the treatment of patients with severe muscle atrophy and difficulty in exercise therapy such as rehabilitation and for the increase in muscle mass of bedridden patients. It is.
  • any drug can be used as long as it is a drug known to donate superoxide in this technical field.
  • any drug can be used as long as it is a drug known to donate peroxynitrite in this technical field.
  • SIN-1, molsidomine, etc. are included and all are commercially available.
  • TRP channel agonist any drug can be used as long as it is a drug known in the art to have agonist activity with respect to the TRP channel.
  • Hyp9, olvanil and the like are included, and commercially available products can be used.
  • the muscle hypertrophy promoter may contain, as an active ingredient, one superoxide donor and / or a peroxynitrite donor and / or a TRP channel agonist, or two or more superoxide donors And / or a combination of a peroxynitrite donating agent and / or a TRP channel agonist.
  • this muscle hypertrophy promoter contains as an active ingredient at least one drug selected from the group consisting of a superoxide donor, a peroxynitrite donor, and a TRP channel agonist, it exerts the above-described muscle hypertrophy promoter action. .
  • the muscle hypertrophy promoter may contain a pharmaceutically acceptable carrier or additive in addition to a superoxide donor and / or a peroxynitrite donor and / or a TRP channel agonist as active ingredients.
  • a pharmaceutically acceptable carrier or additive include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymer, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, xanthan gum, Examples include gum arabic, casein, gelatin, agar, glycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, and lactose.
  • the additive to be used is appropriately or in combination selected from the above depending on the dosage form.
  • tablets, capsules (hard capsules, soft capsules, microcapsules, etc.), granules, powders, pills, troches, liquids for internal use, liquids, suspensions, Any of emulsion, syrup, etc. may be used, and it may be a dry preparation re-dissolved when used.
  • intravenous injection including infusion
  • intramuscular injection intraperitoneal injection
  • subcutaneous injection eg, solution, emulsion, suspension
  • ointment Preparations such as pills, creams, suppositories, cataplasms, inhalants, liniments, aerosols and other external preparations can be selected.
  • injections they are provided in unit dose ampoules or multi-dose containers Is done.
  • compositions are excipients, extenders, binders, wetting agents, disintegrating agents, lubricants, surfactants, dispersants, buffering agents, pH adjusting agents, preservatives, solubilizers commonly used in medicine.
  • Agents, preservatives, flavoring agents, absorption promoters, soothing agents, stabilizers, tonicity agents and the like can be appropriately selected and produced by conventional methods.
  • the superoxide donor and / or peroxynitrite donor and / or TRP channel agonist to be added to the muscle hypertrophy promoter varies depending on the type of active ingredient, application, dosage form, route of administration, etc. 0.01 to 90% by weight, preferably 1 to 50% by weight.
  • the effective amount (dose or intake) of the muscle hypertrophy promoter varies depending on the type of active ingredient contained in the agent, the age and weight of the subject, the administration route, and the number of administrations, and can be varied over a wide range.
  • the effective amount of a superoxide donor and / or peroxynitrite donor and / or TRP channel agonist per day for an adult is usually about 1 week to about 1 year, preferably once or several times a day. Can be administered for about 1 month to about 12 months.
  • This muscle hypertrophy promoter does not particularly limit the target (subject) to be used.
  • it can be administered to or ingested by subjects such as humans, domestic animals (such as cows), pets (such as dogs and cats), and laboratory animals (such as monkeys).
  • This muscle hypertrophy promoter can be combined with other muscle hypertrophy promoters known in the art or methods effective for promoting muscle hypertrophy. For example, it is combined with administration of protein that is a nutrient necessary for muscle hypertrophy, administration of hormones (growth hormone, etc.) that assimilate the nutrient to the muscle, load on the muscle (eg, mild exercise, strength training, pressure training), etc. be able to.
  • hormones growth hormone, etc.
  • the present muscle hypertrophy promoter is not limited to the use as a pharmaceutical composition, and may be blended in other products such as food or feed.
  • Food and “feed” refer to natural products containing one or more nutrients and processed products thereof, including all food and drink.
  • a food or feed containing the present muscle hypertrophy promoter is useful as a health supplement product for promoting muscle hypertrophy.
  • solid food includes bread dough; dough for baked confectionery such as rice crackers, biscuits and cookies; noodles such as buckwheat and udon; fish products such as kamaboko and chikuwa; livestock products such as ham and sausage; powdered milk and the like It is done.
  • jelly-like food include fruit jelly and coffee jelly.
  • liquid foods include beverages such as soft drinks and fruit beverages (tea, coffee, tea, fermented milk, lactic acid bacteria beverages, etc.), seasonings (mayonnaise, dressings, seasoning seasonings, etc.).
  • capsule foods include hard capsules and soft capsules.
  • the amount added is 0.01 to 90% by weight of the superoxide donor and / or peroxynitrite donor and / or TRP channel agonist with respect to the whole food. It can mix
  • the intake that can be expected to be effective is appropriately determined according to the individual case, taking into account the age, weight, sex, and degree of symptoms.
  • the number of intakes can be divided into several times a day, in which case the amount can be divided according to the number of times. In addition, it can be taken continuously over a long period of time.
  • Example 1 In this example, in order to clarify the contribution of nNOS in muscle hypertrophy, cooperative muscle excision was performed on nNOS wild-type and deficient mice to induce compensatory muscle hypertrophy due to overload. Subjects include Jackson Laboratories (Wilmington, NNOS-deficient mice purchased from MA) and C57BL / 6 mice purchased from CLEA Japan (Japan) were used.
  • Compensatory muscle hypertrophy induction and drug administration by synergistic muscle resection were performed as follows. That is, the gastrocnemius and soleus tendons of 12-week-old mice were excised under anesthesia (Adams, GR and F. Haddad, J Appl Physiol, 1996. 81 (6): 2509-16). The control group underwent sham surgery to cut the skin. After surgery, 1 mg / ml ampicillin was added to drinking water. The NOS inhibitor L-NAME (1 mg / ml, Sigma-Aldrich) was administered as drinking water and the drinking water was changed daily.
  • nNOS inhibitor 7-NI 50 mg / kg, Calbiochem
  • NOX inhibitor DPI 2 mg / kg, Sigma-Aldrich
  • apocynin (10 mg / kg, Sigma-Aldrich)
  • tadalafil (10 mg / kg, Toronto Research Chemicals)
  • sildenafil (10 mg / kg, Sigma-Aldrich)
  • 8-Br-cGMP 10 mg / kg, Calbiochem
  • Rotenone (5 mg / kg, Sigma-Aldrich), allopurinol (10 mg / kg, Cayman), uric acid (200 mg / kg Sigma-Aldrich; Example 2), FeTPPS (30 mg / kg, Calbiochem; Example) 2), ebselen (30 mg / kg, Sigma-Aldrich; Example 2), molsidomine (50 mg / kg, Sigma-Aldrich; Example 2), SIN-1 (10 mg / kg
  • BAPTA-AM 50 ⁇ M, Calbiochem; Example 3
  • EGTA 1 mM, Dojindo; Example 3
  • dantrolene 10 ⁇ M, Sigma-Aldrich; Example 3
  • nifedipine 10 ⁇ M, Sigma-Aldrich; Example 3)
  • BTP2 10 ⁇ M, Calbiochem; Example 3
  • Gd 3+ 50 ⁇ m, Sigma-Aldrich; Example 3
  • GsMTx-4 10 ⁇ M, Wako; Example 3
  • Hyp9 10 ⁇ M, Sigma-Aldrich; Example 3)
  • olvanil 10 ⁇ M, Sigma-Aldrich; Example 3) were intramuscularly injected 30 minutes before the start of overload.
  • the plantar muscle was frozen and fixed, sliced to 8 ⁇ m, and stained with hematoxylin and eosin.
  • sample buffer (0.1% Triton X-100, 50 mM HEPES (pH7.4), 4 mM EGTA, 10 mM EDTA, 15 mM Na 4 P 2 O 7 , 100 mM glycerophosphate, 25 mM NaF, 5 mM Na 2 VO 4 , and complete protease inhibitor cocktail (Roche)) and centrifuged (15,000 g, 10 minutes), and the supernatant was collected.
  • sample buffer 0.1% Triton X-100, 50 mM HEPES (pH7.4), 4 mM EGTA, 10 mM EDTA, 15 mM Na 4 P 2 O 7 , 100 mM glycerophosphate, 25 mM NaF, 5 mM Na 2 VO 4 , and complete protease inhibitor cocktail (Roche)
  • centrifuged (15,000 g, 10 minutes), and the supernatant was collected.
  • sample loading buffer (30% glycerol, 5% 2-mercaptoethanol, 2.3% SDS, 62.5 mM Tris-HCl ( pH 6.8), 0.05% bromophenol blue), heat-denatured at 60 ° C. for 15 minutes, and 30 ⁇ g was subjected to Western blotting.
  • the PVDF transfer membrane was blocked with Tris-buffered saline (TBS) + 5% skim milk (snow mark), and incubated for 16 hours at 4 ° C. using the primary antibody.
  • TBS Tris-buffered saline
  • Primary antibodies include Akt (# 9272, Cell Signaling Technology), p-Akt (Ser473) (# 9271, Cell Signaling Technology), p70S6K (# 9202, Cell Signaling Technology), and p-p70S6K (Thr389) (# 9205 , Cell Signaling Technology).
  • the activity was measured by measuring the amount of citrulline which is a by-product during NO production.
  • the thinned plantar muscle is homogenized with 10 times the amount of measurement buffer (25 mM Tris-HCl (pH 7.4), 1 mM EDTA, 1 mM EGTA, and 0.1 mM NaCl) and centrifuged (1,000 g, The supernatant was collected after 10 minutes).
  • reaction stop buffer (20 mM HEPES (pH 5.5) containing 2 mM EDTA) was added, and then mixed with AG50WX-8 columns (Na + form; Dowex) to recover unreacted [ 3 H] arginine Thereafter, the amount of [ 3 H] citrulline was measured with a liquid scintillation counter.
  • the student-t test was used to compare the data between the two groups. For comparison between multiple groups, ANOVA test was performed and then multiple group test by Tukey's method was performed. Data are shown using mean ⁇ standard error, and p ⁇ 0.05 was determined to be significant.
  • the wild type increased the muscle weight by about 40% compared to the control group, but the deficient type increased only about 20% (Fig. 1).
  • a and B In order to clarify the contribution of NO in muscle hypertrophy, the NOS inhibitor L-NAME was administered as described above. In the group administered L-NAME from 3 days before to 7 days after surgery, and from 3 days before surgery to 1 day after surgery, muscle weight was significantly reduced on the 7th day after surgery, but in the group administered after 1 day after surgery, No effect was seen (Figure 2). To examine whether NO production was due to nNOS, 7-NI, an nNOS-specific inhibitor, was administered 30 minutes before or 60 minutes after overload.
  • phosphorylation of p70S6K threonine 389 (Zoncu, R., A. Efeyan, and DM Sabatini, Nat Rev Mol Cell Biol, 2011. 12 (1): 21-: 35) increased, mTOR was activated, and the protein synthesis pathway was activated, but the activation was not observed in the defective type (A and B in FIG. 5).
  • nNOS activated immediately after overload promotes muscle hypertrophy through activation of protein synthesis pathway controlled by mTOR.
  • vasodilators tadalafil, sildenafil, and 8Br-cGMP which activates the cGMP pathway
  • Example 2 In this example, the contribution of mitochondrial respiratory chain, xanthine oxidase, and NADPH oxidase (NOX), which are main supply molecules of superoxide in skeletal muscle, to muscle hypertrophy was examined.
  • Superoxide production was measured by measuring luminescence by reaction between superoxide and lucigenin. After freezing, homogenize sliced plantar muscle with measurement buffer (150 mM NaCl, 50 mM Tris-HCl (pH 7.4), 25 mM EGTA, 25 mM EDTA, and complete protease inhibitor cocktail (Roche)), The supernatant was collected after 1,000 g for 10 minutes. The supernatant was mixed with measurement buffer (100 mM KH 2 PO 4 , (pH 7.0) and 10 ⁇ M lucigenin (Sigma-Aldrich)). 200 ⁇ M NADPH was added to the reaction solution immediately before the measurement and measured with a liquid scintillation counter. As a result, production of superoxide did not increase immediately after overload (FIG. 7).
  • NOX1, NOX2, NOX3 and NOX4 in the plantar muscle was examined by RT-PCR using a primer set having the following sequence.
  • NOX2 and NOX4 are expressed in the plantar muscles (FIG. 8), and NOX4 is a constantly activated enzyme (Bedard, K. and KH Krause, Physiol Rev, 2007. 87 (1): 245-313) It has been reported that NOX4 promotes muscle hypertrophy.
  • Example 3 Peroxynitrite can control intracellular calcium concentration (Trebak, M., et al., Antioxid Redox Signal, 2010. 12 (5): 657-74). It was thought that mTOR is controlled via control and promotes muscle hypertrophy. In order to clarify the relationship between intracellular calcium concentration and mTOR activation, intramuscular injection of thapsigargin that inhibits calcium uptake by sarcoplasmic reticulum and raises intracellular calcium concentration resulted in activation of mTOR (A and B in FIG. 12).
  • BAPTA-AM and EGTA which are calcium chelating agents, were injected intramuscularly.
  • the muscle weight on the 7th day after the operation was significantly suppressed (FIG. 13).
  • inhibitors of ryanodine receptor, L-type calcium channel, and TRP channel (dantrolene, nifedipine, BTP2), which are typical calcium channels in skeletal muscle, were injected intramuscularly.
  • the muscle weight on the 7th day after the operation was significantly suppressed by the TRP channel inhibitor BTP2, but not by dantrolene and nifedipine (FIG. 13).
  • the nNOS deficient phenotype was recovered by NO donor (SNAP) and peroxynitrite donor (SIN-1), but the effect was suppressed by co-administration with BAPTA-AM and BTP2 (FIG. 15).
  • the deficient phenotypes are TRP channel agonists, ie, TRPC6 channel agonists Hyp9 (Leuner, K., et al., Mol Pharmacol, 2010. 77 (3): 368-77) and TRPV1 channel agonists It was recovered by administration of olvanil (FIG. 16).
  • C2C12 that induced myogenic differentiation was cultured in PSS solution (140 mM NaCl, 5 mM KCl, 2.5 mM CaCl 2 , 1 mM MgCl 2 , 10 mM HEPES, 10 mM glucose pH 7.0) for 6 hours or more, and then 8 ⁇ M Fluo-4 was added. And incubated at room temperature for 30 minutes. After removing excess Fluo-4 and culturing at 37 ° C. for 5 minutes, changes in fluorescence intensity due to SIN-1 (DOJINDO) were measured every 3 seconds using an inverted fluorescence microscope (Olympus).
  • PSS solution 140 mM NaCl, 5 mM KCl, 2.5 mM CaCl 2 , 1 mM MgCl 2 , 10 mM HEPES, 10 mM glucose pH 7.0
  • SIN-1 When extracellular calcium was removed, SIN-1 was added to the cells using 0 Ca2 + solution (140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 10 mM HEPES, 10 mM glucose, 2 mM EGTA pH 7.0).
  • FeTPPS calbiochem
  • thapsigargin a peroxynitrite scavenger
  • the increase in intracellular calcium concentration occurs due to the uptake of extracellular calcium or the release of calcium stored in the intracellular sarcoplasmic reticulum.
  • Extracellular calcium was removed (0Ca2 +), or calcium in the sarcoplasmic reticulum was depleted by thapsigargin.
  • SIN-1 the increase in intracellular calcium concentration by SIN-1 was suppressed by depletion of sarcoplasmic reticulum calcium (FIGS. 19A and 19B).
  • Sequence numbers 1 to 10 Artificial sequence (synthetic oligonucleotide)

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Biomedical Technology (AREA)
  • Cell Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Organic Chemistry (AREA)
  • Physical Education & Sports Medicine (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Neurology (AREA)
  • Toxicology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Microbiology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

Cette invention concerne : une méthode de traitement de l'atrophie musculaire; et un procédé et un moyen qui tous deux favorisent l'hypertrophie musculaire. Plus spécifiquement, cette invention concerne un procédé de criblage pour identifier une substance ou un facteur capable de favoriser l'hypertrophie musculaire, ledit procédé comprenant : (a) une étape consistant à mesurer la concentration de superoxyde et/ou d'acide peroxynitreux et/ou de calcium dans une cellule musculaire prélevée sur un animal ayant reçu un traitement basé sur la substance ou le facteur à tester; et (b) une étape consistant à identifier la substance ou le facteur testé comme substance ou facteur candidat à titre de substance ou de facteur capable de favoriser l'hypertrophie musculaire sur la base des résultats obtenus à l'étape (a).
PCT/JP2012/057128 2011-09-14 2012-03-21 Procédé de criblage pour identifier une substance ou un facteur capable de favoriser l'hypertrophie musculaire Ceased WO2013038740A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2011-200716 2011-09-14
JP2011200716 2011-09-14

Publications (1)

Publication Number Publication Date
WO2013038740A1 true WO2013038740A1 (fr) 2013-03-21

Family

ID=47882986

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2012/057128 Ceased WO2013038740A1 (fr) 2011-09-14 2012-03-21 Procédé de criblage pour identifier une substance ou un facteur capable de favoriser l'hypertrophie musculaire

Country Status (2)

Country Link
JP (1) JPWO2013038740A1 (fr)
WO (1) WO2013038740A1 (fr)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006162346A (ja) * 2004-12-03 2006-06-22 Biomarker Science:Kk 筋萎縮の診断方法、抗筋萎縮作用の評価方法、及び抗筋萎縮物質のスクリーニング方法

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006162346A (ja) * 2004-12-03 2006-06-22 Biomarker Science:Kk 筋萎縮の診断方法、抗筋萎縮作用の評価方法、及び抗筋萎縮物質のスクリーニング方法

Non-Patent Citations (11)

* Cited by examiner, † Cited by third party
Title
BODINE, S. C. ET AL.: "Akt/mTOR pathway is a crucial regulator of skeletal muscle hypertrophy and can prevent muscle atrophy in vivo", NATURE CELL BIOLOGY, vol. 3, no. 11, 2001, pages 1014 - 1019 *
ITO, N. ET AL.: "Neuronal nitric oxide synthase (nNOS) is an essential mediator for early stage of muscle hypertrophy", 33RD ANNUAL MEETING OF THE MOLECULAR BIOLOGY SOCIETY OF JAPAN, 83RD ANNUAL MEETING OF THE JAPANESE BIOCHEMICAL SOCIETY GODO TAIKAI KOEN YOSHISHU, 2010 *
ITO, N. ET AL.: "Neuronal nitric oxide synthase regulates muscle hypertrophy through Akt signaling pathway", 81ST ANNUAL MEETING OF THE JAPANESE BIOCHEMICAL SOCIETY, 31ST ANNUAL MEETING OF THE MOLECULAR BIOLOGY SOCIETY OF JAPAN GODO TAIKAI KOEN YOSHISHU, 2008 *
NIKAWA, T.: "Development of ubiquitin ligase inhibitor as a drug against unloading-mediated muscle atrophy", JOURNAL OF JAPANESE BIOCHEMICAL SOCIETY, vol. 81, no. 7, 2009, pages 614 - 618 *
NISHIDA, M. ET AL.: "TRP channels : formation of signal complex and regulation of cellular functions", FOLIA PHARMACOL, vol. 121, 2003, JPN., pages 223 - 232 *
NUMATA, T. ET AL.: "Structures and variable functions of TRP channels", JOURNAL OF JAPANESE BIOCHEMICAL SOCIETY, vol. 81, no. 11, 2009, pages 962 - 983 *
ROMMEL, C. ET AL.: "Mediation of IGF-1-induced skeletal myotube hypertrophy by PI(3)K/Akt/mTOR and PI(3)K/Akt/GSK3 pathways", NATURE CELL BIOLOGY, vol. 3, no. 11, 2001, pages 1009 - 1013 *
SHIMIZU, N. ET AL.: "Crosstalk between glucocorticoid receptor and nutritional sensor mTOR in skeletal muscle", CELL METABOLISM, vol. 13, February 2011 (2011-02-01), pages 170 - 182, XP028144015, DOI: doi:10.1016/j.cmet.2011.01.001 *
SUZUKI, N. ET AL.: "No production results in suspension-induced muscle atrophy through dislocation of neuronal NOS", THE JOURNAL OF CLINICAL INVESTIGATION, vol. 117, no. 9, September 2007 (2007-09-01), pages 2468 - 2476 *
TAKEDA, S.: "The role of neuronal nitric oxide synthase (nNOS) in molecular mechanism of muscle atrophy and hypertrophy", 81ST ANNUAL MEETING OF THE JAPANESE BIOCHEMICAL SOCIETY, 31ST ANNUAL MEETING OF THE MOLECULAR BIOLOGY SOCIETY OF JAPAN GODO TAIKAI KOEN YOSHISHU, vol. 4S18-3, 2008 *
ZANOU, N. ET AL.: "Role of TRPC1 channel in skeletal muscle function", AM J PHYSIOL CELL PHYSIOL, vol. 298, 2010, pages C149 - C162 *

Also Published As

Publication number Publication date
JPWO2013038740A1 (ja) 2015-03-23

Similar Documents

Publication Publication Date Title
Ma et al. Leucine and isoleucine have similar effects on reducing lipid accumulation, improving insulin sensitivity and increasing the browning of WAT in high-fat diet-induced obese mice
US10772862B2 (en) Lipid scavenging in Ras cancers
Li et al. Berberine improves pressure overload-induced cardiac hypertrophy and dysfunction through enhanced autophagy
Jiang et al. ATF4 protects against sorafenib-induced cardiotoxicity by suppressing ferroptosis
US20170027897A1 (en) mTORC1 MODULATION BY AMINO ACIDS AND USES THEREOF
JP2022116259A (ja) プロスタグランジンｅ２を用いる、筋再生のための組成物および方法
US20140011761A1 (en) Modulating Endoplasmic Reticulum Stress in the Treatment of Tuberous Sclerosis
Peters et al. Dose-dependent effects of leucine supplementation on preservation of muscle mass in cancer cachectic mice
Wan et al. Erythromycin has therapeutic efficacy on muscle fatigue acting specifically on orosomucoid to increase muscle bioenergetics and physiological parameters of endurance
Gong et al. Different roles of insulin receptor a and b in maintaining blood glucose homeostasis in zebrafish
US12472159B2 (en) Compositions comprising amino acids for use in the prevention and treatment of chemotherapy side effects
US9693994B2 (en) Class IIa HDAC inhibitors for the treatment of infection
US9072778B2 (en) Treatment regimen for N-MYC, c-MYC, and L-MYC amplified and overexpressed tumors
Dai et al. Piperlongumine, a natural alkaloid from Piper longum L. ameliorates metabolic-associated fatty liver disease by antagonizing the thromboxane A2 receptor
WO2013038740A1 (fr) Procédé de criblage pour identifier une substance ou un facteur capable de favoriser l'hypertrophie musculaire
WO2013141202A1 (fr) Agent de construction musculaire et procédé de criblage pour une substance de construction musculaire
KR20120058047A (ko) 스핑고신-1-포스페이트（ｓｐｈｉｎｇｏｓｉｎｅ－１－ｐｈｏｓｐｈａｔｅ） 또는 이의 약학적으로 허용가능한 염을 유효성분으로 포함하는 비만 예방 및 치료용 약학적 조성물
Wang et al. MEK1/2 Inhibitors Induce Class 1 Alcoholic Dehydrogenase (ADH1) Expression by Regulating Farnesoid X Receptor
RU2558792C2 (ru) Композиция для улучшения состояния при гипоальбуминемии
JP2020513232A (ja) 糖代謝異常の検出方法と予防及び治療
Lee et al. Muscle extracts from Antarctic fishes Trematomus bernacchii and T. Newnesi enhance myofiber regeneration and muscle function through mTOR signaling
TW202327566A (zh) 包含藻褐素及小分子褐藻醣膠之組合物用於製備治療或改善慢性腎病之醫藥品的用途及包含藻褐素、小分子褐藻醣膠及左旋肉鹼之組合物及包含其的醫藥品
Dhanani Disruption of Branched-Chain Amino Acid Catabolism Impairs Rat Myoblast Survival and Differentiation
Ohyama Studies on the food compounds showing anti-obesity effect and their mechanism to suppress obesity
Chen Role of autophagy in cancer

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 12831023

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2013533540

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 12831023

Country of ref document: EP

Kind code of ref document: A1