WO2013038740A1 - Method for screening for substance or factor capable of promoting muscle hypertrophy - Google Patents

Abstract

The present invention provides: a method for treating muscular atrophy; and a method and a means both for promoting muscle hypertrophy. Specifically, the present invention provides a method for screening for a substance or factor capable of promoting muscle hypertrophy, comprising: (a) a step of measuring the concentration of superoxide and/or peroxynitrous acid and/or calcium in a muscle cell collected from an animal which has been received a treatment with a substance or factor to be tested; and (b) a step of identifying the tested substance or factor as a candidate for a substance or factor capable of promoting muscle hypertrophy on the basis of the results obtained in step (a).

Description

Screening method for substances or factors that promote muscle hypertrophy

The present invention relates to a method for screening a substance or factor that promotes muscle hypertrophy and a muscle hypertrophy promoter.

Skeletal muscle weight is controlled by the balance between protein synthesis and degradation (Non-patent Document 1). In muscle inactive states such as cast casts and bed rest, the protein content is decreased due to the inhibition of protein synthesis and the promotion of proteolysis, resulting in a decrease in muscle mass and muscle atrophy (disused muscle atrophy). 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).

Sandri, M., Physiology (Bethesda), 2008. 23: 160-170 Nikawa, T., et al., FASEB J, 2004. 18 (3): 522-524 Suzuki, N., et al., J Clin Invest, 2007. 117 (9): 2468-2476 Zhou, X., et al., Cell, 2010. 142 (4): 531-543 Altun, M., et al., J Biol Chem, 2010. 285 (51): 39597-39608 Shimizu, N., et al., Cell Metab, 2011. 13 (2): 170-182 Suzuki, N., et al., J Neurol Sci, 2010. 294 (1-2): 95-101 Wehling, M., et al., J Cell Biol, 2001. 155 (1): 123-131 Rommel, C., et al., Nat Cell Biol, 2001. 3 (11): 1009-1013

Developing treatments for muscle atrophy is one of the major challenges in Japan, which has faced an aging society. If a means for inducing muscle hypertrophy or reducing muscle atrophy can be obtained, amyotrophic lateral sclerosis (Non-patent Document 7) that causes neurogenic muscle atrophy, or molecular pathology in the sense accompanying muscle atrophy, It is expected to lead to the development of effective means for muscular dystrophy (Non-patent Document 8) that may be common.

Inducing muscle hypertrophy by activating protein synthesis is expected as a treatment for muscle atrophy. It is very difficult to use general exercise therapy for patients with severe muscle atrophy and bedridden elderly people, and drug treatment is desired. So far, studies have been conducted to activate protein synthesis by administering Igf-1 (Non-patent Document 9) that activates the PI3K / Akt pathway, but its effect has been questioned.

The inventor's research group has clarified that neuronal nitric oxide synthase (nNOS) is deeply involved in muscle atrophy caused by muscle immobilization (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. Subsequent studies also showed that nNOS was involved in recovery from muscle wasting during reloading. This suggests that nNOS controls not only muscle atrophy but also muscle hypertrophy.

Therefore, 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.

As a result of intensive studies to solve the above-mentioned problems, 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.

That is, 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. .
[4] When the concentration of superoxide and / or peroxynitrite and / or calcium in muscle cells is higher than that in untreated muscle cells, the test substance or factor is a substance or factor that promotes muscle hypertrophy. The method according to any one of [1] to [3], which is identified as a candidate.
[5] The animal according to any one of [1] to [4], wherein the animal is a muscle-atrophic animal, an nNOS-deficient animal, or an animal that has developed muscular dystrophy, or the muscle cell is a muscle cell derived from the animal. The method described.
[6] 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.
[7] The muscle hypertrophy promoter according to [6], wherein the peroxynitrite donor is SIN-1 or molsidomine.
[8] The muscle hypertrophy promoter according to [6], wherein the TRP channel agonist is Hyp9 or olvanil.
[9] 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.

It is the graph (A) which shows the muscle weight of a nNOS wild type and deficient mouse | mouth 7 days after a synergistic muscle excision, and a muscle HE dyeing | staining image (B). It is a graph which shows the relationship between NO after joint muscle resection, and muscle hypertrophy promotion. It is a graph which shows the relationship between NO and muscle hypertrophy promotion by nNOS after cooperative muscle resection. It is a graph which shows the activation of nNOS after overload in a nNOS wild type and a deficient mouse | mouth. It is the photograph (A) and graph (B) which show the control of the activation of mTOR by nNOS after overload in a nNOS wild type and a deficient mouse | mouth. 2 is a graph showing muscle hypertrophy in the presence of a NOX inhibitor. It is a graph which shows superoxide production before and after overload. 2 is a photograph showing the expression of NOX1, NOX2, NOX3 and NOX4 in plantar muscles. It is a graph which shows the change of muscle hypertrophy by administration of a superoxide scavenger or a peroxynitrite scavenger. It is a graph which shows the recovery | restoration of the phenotype of a nNOS deficient mouse | mouth by a peroxy nitrite donor. It is the photograph (A) and graph (B) which show suppression of the activation of mTOR immediately after overload by administration of a peroxynitrite scavenger. It is the photograph (A) and graph (B) which show activation of mTOR by the raise of intracellular calcium concentration by administration of Thapsigargin. It is a graph which shows suppression of muscle hypertrophy by a calcium chelating agent and a TRP channel inhibitor. It is a photograph which shows suppression of activation of mTOR by the overload by a calcium chelator and a TRP channel inhibitor. It is a graph which shows the recovery | restoration of the phenotype of a nNOS deficient mouse | mouth by a NO donor and a peroxynitrite donor, and the influence by a calcium chelator and a TRP channel inhibitor. It is a graph which shows recovery | restoration of the phenotype of a nNOS deficient mouse | mouth by administration of a TRP channel agonist. It is a figure which shows the promotion mechanism of muscle hypertrophy through control of intracellular calcium concentration by peroxynitrite. It is a fluorescence image which shows that the calcium concentration in a myoblast rises by NO / peroxynitrite donor (SIN-1). The time-dependent change of the fluorescence intensity of FIG. 18A is represented as a graph. It is a graph which shows that the calcium concentration in a myoblast rises depending on the density | concentration of SIN-1. It is a graph which shows that the raise of the calcium concentration in the myoblast by SIN-1 is inhibited by the peroxynitrite elimination agent (FeTPPS). It is a fluorescence image which shows that NO / peroxynitrite donor (SIN-1) induces calcium release from sarcoplasmic reticulum. The peak of the fluorescence intensity in FIG. 19A is represented as a graph.

Hereinafter, the present invention will be described in detail. This application claims the priority of Japanese Patent Application No. 2011-200716 filed on September 14, 2011, and includes the contents described in the specification and / or drawings of the above patent application. .

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.

In order to elucidate the function of nNOS 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. As a result of measuring the muscle weight of the mice on the 7th day after the operation, the wild type increased the muscle weight by about 40% compared to the control group, but the deficient type increased only about 20%. In order to clarify when nNOS functions during muscle hypertrophy, NOS activity was measured. As a result, nNOS was activated 3 minutes after overloading and decreased to a steady level after 1 hour. As a result of pharmacological inhibition of nNOS activity, it was found that NO produced during 1 hour of overload promoted muscle hypertrophy. In the nNOS wild type, mTOR was activated 3 minutes after overload, and the protein synthesis pathway was activated, but in the deficient type, the activation was not observed (Example 1, and Ito et al., Sakai 33rd Japan Molecule). Biology Society Presentation 2T9-11).

However, administration of 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).

In order to elucidate the function of NOX, the main supply molecule of superoxide in skeletal muscle, in muscle hypertrophy, NOX activity was pharmacologically inhibited. As a result, the progression of muscle hypertrophy was suppressed as in the case of the nNOS deficient type. In order to clarify the function of peroxynitrite, which is a reaction product of NO and superoxide, in hypertrophy, as a result of administering superoxide elimination agent or peroxynitrite elimination agent, the progression of muscle hypertrophy was suppressed as in the case of nNOS deficient type. Moreover, as a result of investigating the activation of the protein synthesis pathway in the peroxynitrite scavenger administration group, mTOR activation was not observed immediately after overloading, as in the case of the nNOS deficient type. From these results, it was found that peroxynitrite consisting of nNOS-derived NO and NOX-derived superoxide was produced immediately after overloading, and promoted muscle hypertrophy by activating the protein synthesis pathway controlled by mTOR.

Furthermore, since superoxide derived from NOX and peroxynitrite can control the intracellular calcium concentration, we thought that peroxynitrite regulates mTOR through the control of intracellular calcium concentration and promotes muscle hypertrophy (Adachi T et al. Nat Med 2004; 10: 1200-1207; Martins AS et al. J Physiol 2008; 286: 197-210). 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 . In order to clarify the relationship between intracellular calcium concentration and muscle hypertrophy, BAPTA-AM and EGTA, calcium chelators, were injected intramuscularly. As a result, muscle weight was significantly suppressed on the 7th postoperative day. In addition, as a result of intramuscular injection of ryanodine receptor, DHPR, and TRP channel inhibitors (dantrolene, nifedipine, BTP2), which are typical calcium channels in skeletal muscle, the muscle weight on the 7th day after surgery was significant due to the TRP channel inhibitor BTP2. However, it was not suppressed by dantrolene or nifedipine. BTP2 also suppressed mTOR activation immediately after overload. Moreover, the 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.

From these results, it became clear that control of superoxide, peroxynitrite and its downstream intracellular calcium concentration is important for the activation of protein synthesis pathway by overload. Peroxynitrite was also found to promote muscle hypertrophy through the regulation of intracellular calcium concentration (especially sarcoplasmic reticulum calcium concentration) by TRP channels. Therefore, it is considered possible to artificially activate protein synthesis by increasing the intracellular calcium concentration using an agonist of TRP channel.

In the present invention, 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.

In the context of the present invention, “muscular 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, and “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.

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.

The type of test substance or factor that is the subject of this screening method is not particularly limited. For example, 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. Furthermore, the test substance or factor may be an environmental factor such as radiation, ultraviolet rays, carbon concentration, or temperature.

In addition, as a 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. Examples of 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).

When an animal is treated with a test substance or factor, 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. For example, when administering a test substance to an animal, 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. Pass the supernatant through an 80 μm filter to remove cell clumps, transfer the cell suspension to a 50 ml conical bottom tube, and add growth medium to the tube treated with Dispase2 (2.4 IU / ml) -Collagenase XI (0.2%). In addition, the supernatant is similarly collected through a filter. Add growth medium to a total volume of 30 ml, pipette several times, and centrifuge at 1,000 rpm for 5 minutes at 4 ° C. Discard the supernatant, add 20 ml of growth medium to the precipitated cells, resuspend, and centrifuge at 1,000 rpm for 5 minutes at 4 ° C. 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.

When the muscle cell is brought into contact with the test substance or factor, the contact condition varies depending on the type of the substance or factor, but can be easily determined by those skilled in the art. For example, 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. Alternatively, it can be performed by culturing myocytes in the presence of a test factor.

Also, 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. For example, 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. For example, the treatment can be performed over a period of 1 day to several weeks, months, and years.

Furthermore, when examining the additive action, synergistic action, etc. of a plurality of substances and / or factors, the test substances and / or factors may be used in combination.

Subsequently, after the animal or muscle cell is treated with the test substance or factor, 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. For example, 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.

In addition, the calcium concentration in muscle cells can also be measured by a method known in the art. For example, the fluorescence intensity is measured using Fluo-4 that emits fluorescence when bound to calcium. Can be performed.

A 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. As a control, animals or muscle cells that have not been treated with the test substance or factor can be used.

Moreover, in the present invention, as a primary screening, a 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. 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.

Further, in screening for a substance or factor that promotes muscle hypertrophy, 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. May be. As the 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.

As described above, by this screening method, 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. Specifically, 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.

As the superoxide donor which is an active ingredient, any drug can be used as long as it is a drug known to donate superoxide in this technical field.

As the peroxynitrite donating agent that is an active ingredient, any drug can be used as long as it is a drug known to donate peroxynitrite in this technical field. For example, SIN-1, molsidomine, etc. are included and all are commercially available. As the active ingredient 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. For example, 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. As long as 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. . Examples of such carriers and additives 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.

When this muscle hypertrophy promoter is administered orally, 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. In addition, when the muscle hypertrophy promoter is administered parenterally, for example, intravenous injection (including infusion), intramuscular injection, intraperitoneal injection and subcutaneous injection (eg, solution, emulsion, suspension), ointment Preparations such as pills, creams, suppositories, cataplasms, inhalants, liniments, aerosols and other external preparations can be selected. In the case of injections, they are provided in unit dose ampoules or multi-dose containers Is done.

These various preparations 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.

In addition, 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. . For example, 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. For example, 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.

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.

When adding this muscle hypertrophy promoter to food, it can be added to various forms of food such as solid food, jelly food, liquid food, and capsule food. Here, 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. Examples of the jelly-like food include fruit jelly and coffee jelly. Furthermore, examples of 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.). Examples of capsule foods include hard capsules and soft capsules.

When the muscle hypertrophy promoter is added to food, 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 | blend as follows. 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.

Hereinafter, the present invention will be described more specifically with reference to examples and drawings. However, the following examples do not limit the present invention.

[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) and NOX inhibitor DPI (2 mg / kg, Sigma-Aldrich) were administered intraperitoneally 30 minutes before or 60 minutes after the start of overload. In addition, 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, DOJINDO; Example 2 and 3) SNAP (20 mg / kg, Calbiochem; Example 3) was administered intraperitoneally 30 minutes before the start of overload. BAPTA-AM (50 μM, Calbiochem; Example 3), EGTA (1 mM, Dojindo; Example 3), dantrolene (10 μM, Sigma-Aldrich; Example 3), nifedipine (10 μM, Sigma-Aldrich; Example 3) , BTP2 (10 μM, Calbiochem; Example 3), Gd ³⁺ (50 μm, Sigma-Aldrich; Example 3), GsMTx-4 (10 μM, Wako; Example 3), thapsigargin (20 μM, Calbiochem; Example 3) Hyp9 (10 μM, Sigma-Aldrich; Example 3) and olvanil (10 μM, Sigma-Aldrich; Example 3) were intramuscularly injected 30 minutes before the start of overload.

After euthanasia, the plantar muscle was frozen and fixed, sliced to 8 μm, and stained with hematoxylin and eosin.

For western blotting, frozen and thinned plantar muscles were added to 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. After measuring protein concentration using Coomassie Brilliant Blue G-250 (BioRad, CA, USA), an equal volume of 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 μg 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. 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). After washing the transfer membrane with Tris buffered saline (TBST) containing 0.1% Tween 20, incubate with secondary antibody (rabbit specific HRP-labeled antibody, GE Healthcare), wash again with TBST, and ECL Western Blotting Detection Detected with System (GE HealthCare, Buckinghamshire, UK).

In addition, the activity was measured by measuring the amount of citrulline which is a by-product during NO production. After freezing, 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). Mix 30 μl supernatant with 60 μl reaction buffer ([ ³ H] arginine (53.0 Ci / mmol; GE Healthcare), 1 mM NADPH, 750 μM CaCl ₂ , 1 μM calmodulin, 3 μM tetrahydrobiopterin, 3 μM flavin-adenine dinucleotide and 3 μM flavin mononucleotide) And incubated at 37 ° C. for 20 minutes. 1 ml of 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.

As a result of measuring the muscle weight on the 7th day after the operation of the same mouse, 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. As a result, muscle hypertrophy was significantly suppressed in the group administered before overload, but no effect was seen in the group administered after overload (FIG. 3). From these results, it was suggested that NO production by nNOS produced during 1 hour of overload promotes muscle hypertrophy.

As a result of measuring NOS activity 3 minutes, 1, 6 and 12 hours after the start of overload, NOS activity increased 3 minutes after the start of overload, and decreased to a steady level or lower after 1 hour (FIG. 4). A). This increase was not observed in nNOS-deficient mice (FIG. 4B). From these results, it was found that nNOS activated immediately after overload and NO produced thereafter promote muscle hypertrophy.

In the nNOS wild type, 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).

From these results, it was found that nNOS activated immediately after overload promotes muscle hypertrophy through activation of protein synthesis pathway controlled by mTOR.

In addition, administration of rotenone and allopurinol, inhibitors of mitochondrial respiratory chain and xanthine oxidase, did not reduce muscle weight on the 7th postoperative day, but the NOX inhibitor apocynin and DPI administration groups were the same as the deficient type The progression of muscle hypertrophy was suppressed (Fig. 6).

However, administration of the vasodilators tadalafil, sildenafil, and 8Br-cGMP, which activates the cGMP pathway, did not restore the defective phenotype (data not shown) and were produced by nNOS NO is not mediated by vasodilation controlled by the classical NO-cGMP pathway (Kemp-Harper, B. and R. Feil, Meeting report: cGMP matters. Sci Signal, 2008. 1 (9): pe12) It was suggested to promote hypertrophy.

Therefore, it is a cGMP-independent pathway, and peroxynitrite (Heck, DE, Antioxid Redox Signal, 2001. 3 (2): 249-60), which is a reaction product of NO and superoxide, may promote muscle hypertrophy. Thus, experiments for clarifying the functions of superoxide and peroxynitrite immediately after overload were conducted in Examples 2 and 3 below.

[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.

To examine whether NOX activity increases due to overload, superoxide production was measured. 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 μM lucigenin (Sigma-Aldrich)). 200 μM 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).

Also, the expression of 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.

In order to clarify the function of superoxide in muscle hypertrophy and the function of peroxynitrite, a reaction product of NO and superoxide, in the muscle hypertrophy, as described in Example 1, superoxide and peroxynitrite scavenger ebselen As a result of administration of uric acid and FeTPPS, which are peroxynitrite scavengers, the progression of muscle hypertrophy was suppressed as in the deficient type (FIG. 9). Moreover, as a result of administration of molsidomine and SIN-1 which are peroxynitrite donors to the deficient type, muscle weight on the 7th day after the operation recovered to the same level as the wild type, and this recovery was inhibited by co-administration with FeTPPS (FIG. 10). . In addition, as a result of investigating the activation of the protein synthesis pathway in the FeTPPS administration group, as in the deficient form, phosphorylation of p70S6K was not observed immediately after overload, and mTOR was not activated (A and B in FIG. 11). From these results, it was found that peroxynitrite consisting of nNOS-derived NO and NOX-derived superoxide was produced immediately after overloading, and promoted muscle hypertrophy by activating the protein synthesis pathway controlled by mTOR.

[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).

In order to clarify the relationship between intracellular calcium concentration and muscle hypertrophy, BAPTA-AM and EGTA, which are calcium chelating agents, were injected intramuscularly. As a result, the muscle weight on the 7th day after the operation was significantly suppressed (FIG. 13). In addition, 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. As a result, 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). In addition, even when Gd ³⁺ and GsMTx-4, which are inhibitors of the stretch-activated channel, were administered, muscle weight was significantly suppressed (FIG. 13). BAPTA-AM and BTP2 also suppressed mTOR activation immediately after overload, but dantrolene and nifedipine had no effect (FIG. 14).

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).

These results revealed that NO / peroxynitrite produced immediately after overload promoted muscle hypertrophy through the control of intracellular calcium concentration by TRP channels (FIG. 17).

[Reference Example 1]
Changes in intracellular calcium concentration in myoblasts were analyzed in vitro. Specifically, myoblast cell line C2C12 (RCB0987, RIKEN BioResource Center Cell Bank) was cultured in growth medium (DMEM, 10% fetal bovine serum, 1% penicillin-streptomycin) at 37 ° C and 5% CO ₂ concentration. . Thereafter, the cells were cultured in a differentiation medium (DMEM, 2% horse serum, 1% penicillin-streptomycin) for 2 days to induce muscle differentiation. Changes in intracellular calcium concentration were measured using a calcium indicator Fluo-4 (DOJINDO). 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 μM 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). 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). In addition, during treatment with SIN-1, FeTPPS (calbiochem), a peroxynitrite scavenger, was added together with SIN-1 at a concentration of 100 μM. To deplete sarcoplasmic reticulum calcium, thapsigargin (Calbiochem, 2 μM) was added along with Fluo-4.

As a result, the intracellular calcium concentration increased immediately after the cells were treated with SIN-1 which is a NO / peroxynitrite donor (FIGS. 18A and 18B). This increase in intracellular calcium concentration was concentration-dependent (FIG. 18C) and was inhibited by FeTPPS, a peroxynitrite scavenger (FIG. 18D). From these results, it was found that peroxynitrite increases intracellular calcium concentration.

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. As a result, the increase in intracellular calcium concentration by SIN-1 was suppressed by depletion of sarcoplasmic reticulum calcium (FIGS. 19A and 19B). These results indicate that peroxynitrite induces calcium release from the sarcoplasmic reticulum.

All publications, patents and patent applications cited in this specification are incorporated herein by reference in their entirety.

Sequence numbers 1 to 10: Artificial sequence (synthetic oligonucleotide)

Claims (8)

A method for screening a substance or factor that promotes muscle hypertrophy, comprising:
(A) measuring the concentration of peroxynitrite and / or superoxide 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). A method for screening a substance or factor that promotes muscle hypertrophy, comprising:
(A) treating muscle cells with a test substance or factor;
(B) measuring the concentration of peroxynitrite and / or superoxide 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). The method according to claim 1 or 2, further comprising the step of measuring the concentration of peroxynitrite and / or superoxide and / or calcium in the myocytes prior to treatment with the test substance or factor. If the concentration of peroxynitrite and / or superoxide and / or calcium in muscle cells is higher than that in untreated muscle cells, the test substance or factor is identified as a candidate substance or factor that promotes muscle hypertrophy The method according to any one of claims 1 to 3, wherein: The method according to any one of claims 1 to 4, wherein the animal is a muscle-atrophic animal, an nNOS-deficient animal, or an animal that has developed muscular dystrophy, or the muscle cell is a muscle cell derived from the animal. . A muscle hypertrophy promoter comprising at least one drug selected from the group consisting of a peroxynitrite donor, a superoxide donor, and a TRP channel agonist. The muscle hypertrophy promoter according to claim 6, wherein the peroxynitrite donor is SIN-1 or molsidomine. The muscle hypertrophy promoter according to claim 6, wherein the TRP channel agonist is Hyp9 or olbanil.

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