WO2014119634A1 - Muscle-building agent, and pharmaceutical composition containing same - Google Patents

Abstract

The objective of the present invention is to develop and provide a novel muscle-building agent in which a muscle hypertrophy promoter and a muscle atrophy therapeutic agent are active ingredients, which can induce muscle building without exercise, and which has few side effects. For example, provided is a muscle-building agent which contains a P2Y receptor antagonist such as APT and UTP, and which directly or indirectly activates inositol trisphosphate receptors in the sarcoplasmic reticulum membrane, thereby inducing the release of calcium ions from the sarcoplasmic reticulum.

Description

Muscle increasing agent and pharmaceutical composition containing the same

The present invention relates to a muscle increasing agent and a pharmaceutical composition for treating or preventing a disorder or disease associated with muscle atrophy. The present invention also relates to a method for separating a muscle growth factor.

Skeletal muscle weight is controlled by the balance between protein synthesis and degradation (Non-patent Document 1). In muscle inactive states such as casts and bed rest, muscle protein content decreases due to inhibition of protein synthesis and promotion of protein degradation, resulting in a decrease in muscle mass and atrophy of muscles (disused muscles atrophy). Such muscle atrophy is not only immobilized by space flight under microgravity (Non-patent Document 2), tail suspension (Non-patent Document 3), etc., but also cachexia (Non-patent Document 4), aging (Sarcope) (Non-patent document 5), steroid administration (Non-patent document 6), etc.

Developing treatments for muscle atrophy is one of the major challenges in Japan and other developed countries that have an aging society. If the reduction of muscle atrophy or the induction of muscle hypertrophy can be controlled, the muscle atrophy such as amyotrophic lateral sclerosis (Non-patent Document 7) or muscular dystrophy (Non-patent Document 8) that causes neurogenic muscle atrophy. It is expected to lead to the development of effective therapies for diseases associated with the disease.

The method of activating protein synthesis to induce muscle hypertrophy has promise as a treatment for muscle atrophy. However, the method of activating protein synthesis by general exercise therapy is very difficult for patients with severe muscle atrophy and bedridden elderly people. Therefore, a treatment method that induces muscle atrophy reduction or muscle hypertrophy with drugs without exercise is desired.

Non-Patent Document 9 discloses a method of activating protein synthesis by administering Igf-1 that activates the PI3K / Akt pathway. However, Igf-1 has a risk of promoting myocardial hypertrophy and cancer cell growth.

On the other hand, the present inventors have found that neuronal nitric oxide synthase (nNOS) is deeply involved in muscle atrophy caused by immobilization of skeletal muscle (Non-patent Document 3). Subsequent studies revealed that nNOS is also involved in recovery from muscle wasting during reloading. This result suggests that nNOS controls not only muscle atrophy but also muscle hypertrophy.

Furthermore, in the non-patent document 10 and the patent documents 1 and 2, the present inventors, when nNOS is activated by a mechanical load, peroxynitrite generated from NO and superoxide is converted to TRP ( Transient receptor potential） protein) Promotes muscle hypertrophy by regulating intracellular calcium ion concentration through TRPV1, a receptor family, and activation of protein synthesis pathway by mTOR And subsequent muscle hypertrophy, and TRPV1 agonists can promote muscle hypertrophy and reduce muscle atrophy. However, the expression level of TRPV1 in skeletal muscle is not necessarily high, and in the case of oral administration, a large amount of TRPV1 agonist must be administered.

Japanese Patent Application 2011-200716 Japanese Patent Application 2012-063182

Sandri, M., Physiology (Bethesda), 2008, 23: 160-170 Nikawa, T. et al., 2004, FASEB J, 18: 522-524 Suzuki, N. et al., 2007, J Clin Invest, 117: 2468-2476 Zhou, X. et al., 2010, Cell, 142: 531-543 Altun, M. et al., 2010, J Biol Chem, 285: 39597-39608 Shimizu, N. et al., 2011, Cell Metab, 13: 170-182 Suzuki, N. et al., 2010, J Neurol Sci, 294: 95-101 Wehling, M. et al., 2001, J Cell Biol, 155: 123-131 Rommel, C., et al., 2001, Nat Cell Biol, 3 (11): 1009-1013 Ito N., et al., 2013, Nat Med, 19 (1): 101-106

The inventions described in Non-Patent Document 10 and Patent Documents 1 and 2 are all based on activation of mTOR by controlling intracellular calcium ion concentration via TRPV1. If mTOR can be activated in the same manner as TRPV1 by controlling intracellular calcium ion concentration through other calcium ion channels, muscle atrophy treatment using substances other than TRPV1 agonists becomes possible. In addition, an additive or synergistic effect can be expected in the treatment of muscle atrophy by using such a substance in combination with a TRPV1 agonist.

Therefore, the present invention is a search for a new muscle-increasing agent that acts via a calcium ion channel other than TRPV1, development of a pharmaceutical composition for treating or preventing a disorder or disease associated with muscle atrophy, which contains it as an active ingredient, and It is an object of the present invention to develop and provide a new method for isolating muscle growth factors for promoting muscle hypertrophy and treating muscle atrophy.

In order to solve the above-mentioned problems, the present inventors refer to inositol triphosphate receptor (IP3R: inositol trisphosphate receptor, which is often referred to as “IP3R” in this specification), which is a known calcium ion channel existing in cells. ) Was verified. In neurons, the PLC (phospholipaselipC) / IP3R signaling pathway via the P2Y receptor that functions on the cell membrane and functions as a nucleic acid receptor is known (Ralevic, V. and Burnstock, G., 1998, Pharmacol Rev, 50: 413-492). For example, in neurons, as shown in FIG. 1, when the P2Y receptor on the cell membrane is activated by the binding of ATP or UTP, IP3R present on the endoplasmic reticulum membrane is activated by intracellular signal transduction, and calcium ions Is released. On the other hand, in muscle cells, IP3R is known to exist on the sarcoplasmic reticulum membrane (Powell JA, et al., 2001, J Cell Sci, 114: 3673-3683), but via the P2Y receptor. The physiological function of the PLC / IP3R signaling pathway has not been elucidated. However, this time, the present inventors have also demonstrated that, in the soleus and gastrocnemius muscle cells, the PLC / IP3R signaling pathway via the P2Y receptor induces the release of calcium ions from the sarcoplasmic reticulum. The inventors obtained new knowledge that mTOR can be activated and muscle hypertrophy can be promoted, and the present invention has been completed. That is, the present invention provides the following.

(1) It is characterized in that it activates IP3R on the sarcoplasmic reticulum membrane directly or indirectly by acting from outside the muscle cell or inside the muscle cell, and induces the release of calcium ions from the sarcoplasmic reticulum. Muscle increasing agent.

(2) The muscle increasing agent according to (1), wherein the muscle cells are slow muscle cells.

(3) The muscle increasing agent according to (1) or (2), wherein the muscle increasing agent is a P2Y receptor agonist.

(4) The muscle increasing agent according to (3), wherein the P2Y receptor agonist is ATP or UTP or a derivative thereof, a salt thereof, or a combination thereof.

(5) The muscle increasing agent according to (1) or (2), wherein the muscle increasing agent is inositol triphosphate or a salt thereof, an ester thereof or a prodrug thereof.

(6) A pharmaceutical composition for treating or preventing a disorder or disease associated with muscle atrophy, comprising the muscle increasing agent according to any one of (1) to (5) as an active ingredient.

(7) The pharmaceutical composition according to (6), wherein the disorder accompanied by muscle atrophy is selected from the group consisting of disuse muscle atrophy, cachexia, sarcopair, and muscle atrophy under microgravity.

(8) The pharmaceutical composition according to (6), wherein the disease accompanied by muscular atrophy is amyotrophic lateral sclerosis or muscular dystrophy.

(9) A method for separating a muscle increasing agent, comprising: (a) treating a muscle cell with a test substance and / or a test factor; (b) measuring an activity of IP3R in the muscle cell; c) The method comprising the step of isolating a test substance or test factor that increases the activity of IP3R as a muscle increasing agent based on the measurement result of step (b).

(10) The method according to (9), wherein the muscle cell is a slow muscle cell.

(11) The method according to (9) or (10), wherein the change in activation of IP3R is measured by a change in calcium ion concentration in muscle cells or sarcoplasmic reticulum.

This specification includes the contents described in the specification and / or drawings of Japanese Patent Application No. 2013-015927, which is the basis of the priority of the present application.

According to the muscle increasing agent of the present invention, a muscle increasing agent that acts via IP3 / Ca ²⁺ signaling can be provided.

According to the pharmaceutical composition of the present invention, it is possible to provide a pharmaceutical composition for treating or preventing a disorder or disease associated with muscle atrophy, which contains a muscle increasing agent other than a TRPV1 agonist as an active ingredient.

According to the present invention, it is possible to isolate a new muscle increasing factor using IP3 / Ca ²⁺ signaling in muscle cells.

The conceptual diagram of a known P2Y / PLC / IP3R signaling pathway is shown. It is a figure which shows the time-dependent change of the C2C12 intracellular calcium ion concentration after various nucleic acid processing. It is the figure which quantified FIG. 2A. *: P <0.05; ***: p <0.001 (hereinafter the same) It is a figure which shows the relationship between ATP concentration and intracellular calcium ion concentration. It is a figure which shows the relationship between a UTP density | concentration and intracellular calcium ion density | concentration. **: p <0.01 (hereinafter the same) It is a figure which shows the relationship between an ATP analog and C2C12 intracellular calcium ion concentration. It is a figure which shows the raise suppression of the intracellular calcium ion concentration by ATP or UTP by Thapsigargin (tg). It is a figure which shows the time-dependent change of the C2C12 intracellular calcium ion concentration after processing with P2Y receptor inhibitor suramin or PLC inhibitor U73122 in addition to ATP or UTP. It is the figure which quantified FIG. 3A. P2Y shows inhibiting elevation of intracellular calcium concentration by ATP or UTP of ₂ when the siRNA treatment. It is a figure which shows the raise suppression of the intracellular calcium concentration by ATP or UTP at the time of IP3R inhibitor XeC treatment. FIG. 3 is a Western blot diagram showing activation of mTOR in C2C12 cells by ATP or UTP treatment by phosphorylation of mTOR substrate p70S6K. FIG. 4 is a western blot diagram showing the results of verification using mTOR inhibitor rapamycin that phosphorylation of the substrate p70S6K by ATP or UTP treatment is derived from the activity of mTOR. The following shows the results of verifying the relationship between mTOR activated in C2C12 cells by ATP or UTP treatment and the increase in C2C12 intracellular calcium ion concentration using extracellular calcium chelator EGTA or intracellular calcium chelator BAPTA-AM It is a Western blot diagram. FIG. 4 is a Western blot diagram showing that the activity of mTOR by ATP or UTP treatment is via the P2Y / PLC pathway using the P2Y receptor inhibitor suramin or the PLC inhibitor U73122. FIG. 2 is a Western blot diagram showing that mTOR activity by ATP or UTP treatment using P2Y ₂ siRNA is via the P2Y / PLC pathway. FIG. 3 is a Western blot diagram showing that the activity of mTOR by ATP or UTP treatment using an IP3R inhibitor XeC is mediated by IP3R. It is a Western blot figure which shows that MAPK in C2C12 cell is activated by ATP or UTP treatment. It is a figure which shows that the expression level of a JunB gene increases by the exercise | movement (exercise) load or overload (overload) to a mouse | mouth, or ATP or UTP process to C2C12 cell. It is a figure which shows that the raise of the expression of JunB by the ATP process of C2C12 cell is suppressed by the inhibitor U0126 of BAPTA-AM and p44 / 42 * MAPK. It is a western blotting figure which shows that the increase in the amount of JunB protein by ATP process of C2C12 cell is suppressed by BAPTA-AM and rapamycin. It is a figure which shows the change of the muscle weight of the soleus muscle by administration of ATP, BAPTA-AM, or rapamycin. It is a figure which shows the change of the muscle weight of a gastrocnemius by administration of ATP, BAPTA-AM, or rapamycin. It is a figure which shows the change of the muscle weight of the soleus muscle by administration of UTP, BAPTA-AM, or rapamycin. It is a figure which shows the therapeutic effect of ATP or UTP in the muscle atrophy of the soleus muscle induced by the unloading by hind limb suspension. It is a figure which shows the therapeutic effect of ATP or UTP in the muscle atrophy of the gastrocnemius muscle induced by denervation by sciatic nerve resection. It is a conceptual diagram which shows the action mechanism of the muscle hypertrophy in a myocyte through the P2Y / PLC / IP3R signal transduction pathway by ATP or UTP which is the muscle increase agent of this invention disclosed by this specification. It is a figure which shows the time-dependent change of the calcium ion concentration in the single muscle fiber derived from soleus muscle after ATP or UTP processing. It is a figure which shows the time-dependent change of the calcium ion density | concentration in the single muscle fiber derived from a soleus muscle after processing with P2Y receptor inhibitor suramin, PLC inhibitor inhibitor U73122, or IP3R inhibitor XeC in addition to ATP. 0Ca ²⁺ indicates the removal of extracellular calcium. FIG. 4 is a Western blot diagram showing activation of mTOR in single muscle fibers derived from soleus muscle by ATP treatment by phosphorylation of mTOR substrate p70S6K.

1. Muscle increasing agent The 1st mode of the present invention is related with a muscle increasing agent. Hereinafter, the present invention will be specifically described.

1-1. Structure The muscle increasing agent of this embodiment is a substance that acts on muscle cells and induces muscle increase.

In the present invention, “muscle increase” refers to promotion of muscle hypertrophy, suppression of muscle atrophy, increase of muscle strength or maintenance of muscle strength, or a combination thereof.

In 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 associated with an increase in the amount of endogenous protein, and “promotion of muscle hypertrophy” refers to an endogenous protein in a single muscle fiber. By promoting the increase in the amount, it means promoting the increase in the muscle weight due to the increase in the muscle fiber weight or cross-sectional area. In addition, “muscle atrophy” means a state in which the weight or cross-sectional area of a single muscle fiber is partially reduced, accompanied by a decrease in muscle strength, and “reduction of muscle atrophy” means the weight or cross-sectional area of a single muscle fiber. Preventing or decreasing the rate of decrease, or increasing decreased single muscle fiber weight or cross-sectional area. “Muscle strength” is a force (muscle tension) for contracting a muscle, and “increase or maintenance of muscle strength” means that the force is increased or maintained.

The muscle-increasing agent of this aspect acts directly or indirectly on the membrane of the sarcoplasmic reticulum by acting from outside or inside the myocyte and directly activates the inositol triphosphate receptor from the sarcoplasmic reticulum. Muscle gain is induced by the release of calcium ions.

In the present invention, “muscle cell” means a cell constituting skeletal muscle. Although it is substantially synonymous with a myofiber, in the present invention, myoblasts before differentiation into myocytes are also included. Muscle cells can be broadly classified into slow muscle cells (slow muscle fibers) and fast muscle cells (fast muscle fibers). Preferred myocytes in the present invention are slow muscle cells.

“The sarcoplasmic reticulum” is a special smooth endoplasmic reticulum that exists in muscle cells and stores calcium ions inside. The inositol triphosphate receptor described later is distributed on the sarcoplasmic reticulum membrane and contributes to the control of calcium ion concentration in muscle cells.

An inositol triphosphate receptor (IP3R: inositol trisphosphate receptor) is an ion channel built-in receptor that exists on the endoplasmic reticulum membrane or sarcoplasmic reticulum membrane and functions as a calcium ion channel. It has a binding site for the ligand inositol triphosphate (inositol-1,4,5-triphosphate: IP3: inositol trisphosphate, often referred to herein as “IP3”) and is activated by the binding of IP3 And release calcium ions from the endoplasmic reticulum membrane or sarcoplasmic reticulum to the cytoplasm. The target in the present invention is IP3R that exists particularly on the sarcoplasmic reticulum membrane.

The muscle increasing agent of the present invention includes a substance that acts from outside the muscle cell and indirectly activates IP3R, and a substance that acts inside the muscle cell and directly or indirectly activates IP3R.

Substances that act from outside the muscle cells and indirectly activate IP3R include, for example, binding to receptors present on the muscle cell membrane, and the IP3 / Ca ²⁺ signaling pathway (PLC / IP3R in the muscle cell). A ligand molecule capable of positively controlling a signal transduction pathway (including P2Y / PLC / IP3R signal transduction pathway) and increasing intracellular calcium ion concentration, that is, the receptor agonist. The type of receptor is not particularly limited as long as it exists on the muscle cell membrane and can control the downstream IP3 / Ca ²⁺ signaling pathway. Examples thereof include P2Y receptor (including P2Y ₁ receptor, P2Y ₂ receptor, P2Y ₄ receptor and P2Y ₆ receptor), PAR-1, etc., which are G protein-coupled receptors. P2Y receptor is preferable. In the case of the P2Y receptor, the P2Y receptor agonist is not particularly limited as long as it is a substance that activates the P2Y receptor. For example, nucleotides and other natural and artificial low molecular compounds. The P2Y receptor agonist used in the present invention is preferably ATP or UTP, or a derivative having physiological activity equivalent to them, and salts thereof. Examples of ATP derivatives include ATPgammaS (ATPγS: adenosine 5′-0- (3-thiotriphosphate)), 2-MeSATP (2- (methylthio) adenosine-5′-triphosphate) and the like. . ATP and / or UTP are particularly preferable because they are also present in the living body and have a low rate of side effects because of a high metabolic rate in the living body. In addition, it may be a P2Y receptor agonist such as diquafosol or a salt thereof. When treating myocytes with the receptor agonist, two or more different agonists may be used in combination. For example, when a P2Y receptor agonist is used, the ATP or UTP or a derivative thereof, or a salt thereof can be used in combination.

On the other hand, substances that act in muscle cells and indirectly activate IP3R include, for example, active signaling factors that positively regulate the IP3 / Ca ²⁺ signaling pathway in muscle cells, and such Examples thereof include low-molecular compounds, compounds, peptides and the like that activate signal transduction factors. Examples of the substance that acts in muscle cells and directly activates IP3R include IP3 or a salt thereof, or an ester or a prodrug thereof.

Muscle increase by the muscle increasing agent of the present embodiment is caused by the muscle increasing agent directly or indirectly activating IP3R in muscle cells and inducing calcium ion release from the sarcoplasmic reticulum as described above. As shown in Examples described later, mTOR and MAPK are activated by an increase in the concentration of calcium ions in muscle cells via IP3R. From the Examples, activation of MAPK enhances the expression of JunB protein (Raffaello, A., et al., 2010, J Cell Biol 191: 101-113) involved in the promotion of muscle hypertrophy and activation of mTOR Has been suggested to promote JunB protein translation. As described above, the muscle increasing agent of this embodiment promotes muscle hypertrophy and / or suppresses muscle atrophy by positively controlling the calcium ion concentration in muscle cells via the IP3 / Ca ²⁺ signaling pathway. It seems to be.

1-2. Effect According to the muscle increasing agent of this embodiment, muscle hypertrophy can be induced without accompanying exercise by increasing the calcium ion concentration in muscle cells via the IP3 / Ca ²⁺ signaling pathway. In addition, ATP or UTP, which is a muscle increasing agent of this embodiment, is also present in cells and has an advantage of having almost no side effects because of its high metabolic rate.

2. A pharmaceutical composition for treating or preventing a disorder or disease associated with muscle atrophy A second aspect of the present invention is a pharmaceutical composition for treating or preventing a disorder or disease associated with muscle atrophy (hereinafter referred to simply as “pharmaceutical composition”). ”)”. Hereinafter, the present invention will be specifically described.

2-1. Configuration 2-1-1. Active ingredient The pharmaceutical composition of this aspect contains the muscle increasing agent as described in the said 1st aspect as an active ingredient. One pharmaceutical composition of the present invention may contain one or more muscle increasing agents described in the first aspect. Moreover, it can combine with the method effective in inclusion of other well-known muscle increasing agents other than the muscle increasing agent as described in a 1st aspect, or muscle increase. For example, administration of protein which is a nutrient necessary for muscle increase, administration of hormones (growth hormone, etc.) that assimilate the nutrient to muscle, load on muscle (eg, mild exercise, strength training, pressure training), etc. You may combine. Moreover, it can also be included in one pharmaceutical composition in combination with one or two or more muscle increasing agents described in Japanese Patent Application No. 2012-063182.

The amount (content) of the muscle increasing agent according to the first aspect to be blended in the pharmaceutical composition of the present aspect is the type of muscle increasing agent included in the pharmaceutical composition and / or its effective amount (dosage or Intake amount), the type of disorder or disease, the dosage form of the drug composition, and the type of carrier or additive to be described later, and may be appropriately determined in consideration of the respective conditions. In the present specification, the “effective amount” is an amount necessary for the muscle augmenting agent to function as an active ingredient in a pharmaceutical composition, and has almost no adverse side effects on the living body to which it is applied. Or the amount which is not given at all. This effective amount may vary depending on various conditions such as subject information, route of administration, and number of doses. Here, the “subject” refers to a living body to which the pharmaceutical composition is applied. For example, humans, livestock (cattle, horses, sheep, goats, pigs, chickens, ostriches, etc.), racehorses, pets (dogs, cats, rabbits, etc.), laboratory animals (mouse, rats, guinea pigs, monkeys, etc.) Applicable. Preferably, it is a human (in this case, particularly called “subject”). The “subject information” is various individual information of the living body to which the drug composition is applied. For example, in the case of a subject, the subject is suffering from a general health condition, a disease or a disease. Includes progress, severity, age, weight, sex, dietary habits, drug sensitivity, presence or absence of concomitant drugs, and resistance to treatment. The final effective amount of the muscle increasing agent and the application amount calculated based on the effective amount are ultimately determined by the judgment of the doctor, dentist, veterinarian, etc. according to the information of the individual subject. The As described above, the content of the muscle-increasing agent in the pharmaceutical composition of the present invention varies depending on the conditions, but as a specific example of the content of the muscle-increasing agent per dosage unit of the pharmaceutical composition, it is necessary to use other pharmaceuticals in combination. For human adults who do not, 0.01 to 90% by weight, preferably 1 to 50% by weight, based on the total weight. When it is necessary to administer a large amount of the pharmaceutical composition in order to obtain the pharmacological effect of the muscle increasing agent, it can be divided into several times for the purpose of reducing the burden on the subject. For example, an effective amount of muscle augmentation agent per day for an adult may be administered for about 1 week to about 1 year, preferably about 1 month to about 12 months, once or several times a day.

2-1-2. Carrier The pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier as required. “Pharmaceutically acceptable carrier” refers to an additive usually used in the field of pharmaceutical technology. Examples thereof include solvents, excipients, binders, disintegrants, fillers, emulsifiers, fluid addition regulators, lubricants, human serum albumin, and the like.

The solvent may be, for example, water or any other pharmaceutically acceptable aqueous solution, or a pharmaceutically acceptable organic solvent. Examples of the aqueous solution include physiological saline, isotonic solutions containing glucose and other adjuvants, phosphate buffers, and sodium acetate buffers. Examples of adjuvants include D-sorbitol, D-mannose, D-mannitol, sodium chloride, low concentration nonionic surfactants, polyoxyethylene sorbitan fatty acid esters, and the like.

Excipients include, for example, sugars such as monosaccharides, disaccharides, cyclodextrins and polysaccharides, metal salts, citric acid, tartaric acid, glycine, polyethylene glycol, pluronic, kaolin, silicic acid, or combinations thereof. It is done.

Examples of the binder include starch paste using plant starch, pectin, xanthan gum, simple syrup, glucose solution, gelatin, tragacanth, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, shellac, paraffin, polyvinylpyrrolidone, or combinations thereof. Can be mentioned.

Examples of the disintegrant include the starch, lactose, carboxymethyl starch, crosslinked polyvinylpyrrolidone, agar, laminaran powder, sodium bicarbonate, calcium carbonate, alginic acid or sodium alginate, polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfate, stearin. Examples include acid monoglycerides or salts thereof.

Examples of fillers include petrolatum, the sugar and / or calcium phosphate.

Examples of emulsifiers include sorbitan fatty acid esters, glycerin fatty acid esters, sucrose fatty acid esters, and propylene glycol fatty acid esters.

Examples of the flow addition regulator and lubricant include silicate, talc, stearate or polyethylene glycol.

In addition to the above, solubilizers, suspending agents, diluents, dispersants, surfactants, soothing agents, stabilizers, absorption promoters, bulking agents, moisturizers that are commonly used in medicine, if necessary , Moisturizers, wetting agents, adsorbents, flavoring agents, disintegration inhibitors, coating agents, colorants, preservatives, preservatives, antioxidants, fragrances, flavoring agents, sweeteners, buffering agents, tonicity agents, etc. Can be included as appropriate.

Such a carrier is mainly used for facilitating the formation of the dosage form and maintaining the dosage form and the drug effect, and for making the muscle-increasing agent, which is an active ingredient, less susceptible to degradation by in vivo enzymes and the like. Therefore, it may be used as needed.

2-1-3. Dosage Form The dosage form of the pharmaceutical composition of the present embodiment does not inactivate the muscle increasing agent or other additional active ingredient described in the first aspect which is an active ingredient, and the active ingredient is administered in vivo after administration. The form is not particularly limited as long as it can exert a pharmacological effect. The dosage form of the pharmaceutical composition varies depending on the administration method and / or prescription conditions. In general, the administration methods can be broadly classified into oral administration and parenteral administration, but the pharmaceutical composition may be in a dosage form suitable for each administration method. For example, dosage forms suitable for oral administration include solid preparations (including tablets, pills, sublingual tablets, capsules, drops, lozenges), granules, powders, powders, liquids (internal solutions, suspensions). Agents, emulsions, and syrups). The solid preparation can be made into a dosage form with a coating known in the art, for example, a sugar-coated tablet, a gelatin-encapsulated tablet, an enteric tablet, a film-coated tablet, a double tablet, or a multilayer tablet as necessary. . Parenteral administration is subdivided into systemic administration and topical administration, and local administration is further subdivided into tissue administration, transepidermal administration, transmucosal administration, and rectal administration. A suitable dosage form may be used. For example, a dosage form suitable for systemic or intra-tissue administration includes an injection that is a liquid. Suitable dosage forms for transepidermal or transmucosal administration include solutions (including coating agents, eye drops, nasal drops, and inhalants), suspensions (including emulsions and creams), and powders (nasal drops). And a paste agent, a gel agent, an ointment, a plaster, and the like. Suppositories can be mentioned as dosage forms suitable for rectal administration.

Since the pharmaceutical composition of this embodiment is applied for the treatment or prevention of a disorder or disease accompanied by muscle atrophy as described in the section “2-2. Target disorders and diseases” described later, In principle, it is skeletal muscle. Therefore, the administration method is not limited, but local administration directly administered to the target skeletal muscle or systemic administration via the circulatory system can be preferably used. Specifically, for example, local administration by intramuscular injection, intravascular administration such as intravascular injection (including intravenous injection and intraarterial injection) or lymphatic vessel injection may be mentioned. Injectables are combined with the above-mentioned excipients, emulsifiers, suspensions, surfactants, stabilizers, pH adjusters, etc. as appropriate, and mixed in unit dosage forms generally required for pharmaceutical practice. It can be formulated and is provided in unit dose ampoules or multi-dose containers.

The specific shape and size of each dosage form are not particularly limited as long as the dosage form is within the range of dosage forms known in the art for each dosage form.

About the manufacturing method of the pharmaceutical composition of this invention, what is necessary is just to formulate according to the conventional method of the said technical field. For example, the method described in Remington's Pharmaceuticals Sciences (Merck Publishing Co., Easton, Pa.) Can be referred to.

2-2. Target Disorders and Diseases The disorder or disease targeted for treatment or prevention of the pharmaceutical composition of this embodiment is a disorder or disease associated with muscle atrophy. As used herein, “treatment” refers to alleviating or eliminating a disorder or disease affected and / or symptoms associated therewith. In the present specification, “prevention” means prevention of occurrence of a disorder or morbidity of a disease. Disorders associated with muscle atrophy include, for example, disuse muscle atrophy caused by muscle inactivity such as cast fixation and bed rest, malignant tumors, chronic diseases such as respiratory disease, aging (sarcope), and steroid administration Examples include side effects and muscle atrophy under microgravity such as life in outer space. Examples of the disease accompanied by muscular atrophy include amyotrophic lateral sclerosis, muscular dystrophy, and polymyositis.

2-3. Effect Since the pharmaceutical composition of the present aspect contains the muscle increasing agent according to the first aspect as an active ingredient, the muscle strength can be maintained by suppressing the progression of muscle atrophy without exercising and / or the muscle strength can be recovered or strengthened by muscle hypertrophy. Can be augmented. Therefore, it can be a useful therapeutic agent for patients with severe muscle atrophy and those who are difficult to exercise such as rehabilitation such as bedridden elderly people.

3. Food-drinks for muscle increase The 3rd aspect of this invention is related with the food-drinks for muscle increase which added the muscle increasing agent of a 1st aspect. Hereinafter, the present invention will be specifically described.

3-1. Configuration In the present specification, “a food and drink for increasing muscle” refers to foods and beverages intended to increase muscle.

Here, “food and drink” refers to natural products containing one or more nutrients and processed products thereof, and includes health functional foods such as foods for specified health use and nutritional functional foods, and health foods. Further, not only general foods that humans eat, but also all foods and drinks including feeds fed to animals other than humans such as domestic animals, competing horses, pets, and experimental animals are included.

The muscle increasing food / beverage product of this aspect mix | blends the muscle increasing agent as described in a 1st aspect 1 type, or 2 or more types. The blending amount of the muscle increasing agent according to the first aspect in the muscle increasing food or drink is such that the content of the muscle increasing agent according to the first aspect is 0.01 to 90% by weight, for example, with respect to the total amount of the muscle increasing food or drink. What is necessary is just to mix | blend. The intake that can be expected to be effective is appropriately determined according to the individual case in consideration of age, weight, gender, symptom level, and the like. 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, since it has no or little side effects, it can be taken continuously over a long period of time.

When adding the muscle increasing agent described in the first aspect to food and drink, it may be added to various forms of food such as solid food, jelly food, liquid food, and capsule food. Here, solid foods include dough for bread, baked confectionery (rice crackers, biscuits, cookies, etc.), noodles, fish products (kamaboko, chikuwa, etc.), livestock products (ham, sausages, etc.), powdered milk, pet food, fodder Etc. Examples of the jelly-like food include fruit jelly and coffee jelly. Furthermore, examples of liquid foods include tea, coffee, tea, soft drinks, fruit drinks, milk drinks, seasonings (mayonnaise, dressings, seasoning liquids, etc.). Examples of capsule foods include hard capsules and soft capsules.

3-2. Effect The food and drink for muscle increase of this aspect is not only a patient having a disorder or disease accompanied by muscle atrophy, but also a healthy individual, as a health supplement product for daily muscle increase if it is a human, Moreover, if it is livestock for meat, it is useful for the purpose of early growth and early shipment.

4). A method for separating muscle gain factors The fourth aspect of the present invention relates to a method for separating muscle gain factors. As shown in the examples described below, activating the IP3 / Ca ²⁺ signaling pathway via activation of IP3R in muscle cells pharmacologically induces muscle hypertrophy or reduces muscle atrophy. Is possible. Therefore, according to the separation method of the present invention, it is possible to isolate a novel muscle-increasing factor that brings about a pharmacological effect of muscle increase, based on the activation of IP3R in muscle cells by treatment with a test substance.

4-1. Configuration In this embodiment, the “muscle increase factor” is a generic term for various factors that can cause muscle increase. The muscle increasing factor includes substances having a muscle increasing action (muscle increasing agent) and environmental factors having a muscle increasing action such as radiation, ultraviolet rays, carbon concentration, temperature, pressure, vibration and the like.

The method for separating a muscle growth factor of this embodiment includes (1) a test substance treatment step, (2) an IP3R activity measurement step, and (3) a muscle growth factor separation step as essential steps. In addition, the steps from (1) the test substance treatment step to (3) the muscle augmentation factor separation step can be repeated a plurality of times as necessary. Furthermore, if necessary, (4) an in vivo confirmation step may be included as a selection step after the muscle augmentation factor separation step.

(1) Test substance etc. treatment step In this specification, the "test substance etc. treatment step" is a step of treating muscle cells expressing IP3R with a test substance and / or a test factor.

The type of “myocyte expressing IP3R” used in this step is not particularly limited as long as it is a myocyte expressing IP3R on the sarcoplasmic reticulum membrane. Slow muscle cells are preferred. Any of established myocytes, primary cultured myocytes, subcultured myocytes, etc. may be used. Such muscle cells can be obtained and prepared according to methods known in the art, or commercially available cell lines or publicly available cells can be used. For example, C2C12 (RCB0987, RIKEN BioResource Center cell bank: myoblast cell line), HEK-293 cells (known to overexpress human TRPV1), and the like can be used.

In the present specification, the “test substance” refers to a substance that is used in the method for separating muscle augmentation factors of this embodiment and is a candidate substance that is expected as a muscle augmentation factor, that is, a muscle augmentation agent candidate substance.

The type of “test substance” used in this step is not particularly limited. For example, natural substances or non-natural substances can be mentioned. The “natural substance” referred to here is a substance that exists in nature. Examples of the present invention include amino acids, peptides, oligopeptides, polypeptides, proteins, nucleic acids, lipids, carbohydrates (such as sugars), steroids, glycopeptides, glycoproteins, proteoglycans, and the like. “Non-natural substances” are substances that do not exist in nature and are artificially synthesized. In the present invention, synthetic analogs or derivatives of natural substances such as peptidomimetics and nucleic acid molecules (aptamers, antisense nucleic acids, RNAi molecules, etc.), combinatorial chemistry such as inorganic and organic compound libraries or combinatorial libraries, etc. An example is a low molecular weight organic compound produced using a technique or the like. One test substance may be a complex composed of a plurality of substances in addition to one composed of a single molecule. As the test substance, not only a single substance but also two or more different substances can be used in combination. For example, it may be a test substance library composed of a plurality of test substance groups. Examples of such test substance libraries include synthetic compound libraries (combinatorial libraries, etc.), peptide libraries (combinatorial libraries, etc.) and the like.

As used herein, “test factor” refers to an environmental factor that can affect muscle cells. For example, radiation, ultraviolet rays, carbon concentration, temperature, pressure, vibration and the like can be mentioned.

The method of treating muscle cells with a test substance and / or test factor varies depending on the substance or factor. For example, a test substance is usually treated so as to come into contact with muscle cells. The contact condition can be determined based on a known technique in the technical field depending on the type of the test substance. For example, a method of culturing myocytes in a medium containing a test substance, a method of immersing myocytes in a solution containing the test substance, and a method of laminating the test substance on the myocytes. Moreover, if it is a test factor, it will normally treat so that a muscle cell may be exposed to a test factor. For example, a method of culturing myocytes in an environment where the test factor is present can be mentioned.

Conditions such as the amount (amount, concentration) or strength of the test substance and / or test factor, treatment time, number of times, etc. may be determined as necessary. For example, a plurality of doses can be set by preparing a dilution series of the test substance. The treatment time can also be set as appropriate. For example, the treatment can be performed over a period of one day to several weeks, months, and years. When verifying the additive action, synergistic action, etc. of a plurality of substances and / or test factors, a plurality of test substances and / or test factors may be combined and treated.

(2) IP3R activity measurement step In this specification, the “IP3R activity measurement step” is a step of measuring the activity of IP3R in the myocytes treated in the test substance treatment step.

In this step, IP3R activity may be measured at an appropriate time after the test substance treatment step. For example, immediately after the test substance treatment step, 30 minutes, 1 hour, 3 hours, 5 hours, 10 hours, 15 hours, 20 hours, 24 hours (1 day), 2-10 days Measure after 10-20 days, after 20-30 days, and after 1-6 months.

IP3R activation can be measured by methods known in the art. For example, a method of measuring by a change in calcium ion concentration in muscle cells or a change in calcium ion concentration in sarcoplasmic reticulum can be mentioned. As a specific method, for example, by measuring the fluorescence intensity in muscle cells using Fluo-4 that emits fluorescence by binding to calcium ions, the calcium ion concentration in muscle cells is measured. (Gee KR, et al., Cell Calcium. 2000, 27 (2): 97-106).

When measuring the activity of IP3R, for example, IP3R activity in muscle cells before treatment with the test substance and / or test factor, or for a control cultured under the same conditions and not treated with the test substance and / or test factor It is desirable to measure the IP3R activity of myocytes as a control activity. The measurement method of the control activity is the same as the measurement method performed on the myocytes treated in the test substance treatment step, except that the myocytes are not treated with the test substance and / or test factor. That's fine.

(3) Muscle Increase Factor Separation Step In this specification, “muscle increase factor separation step” refers to a test substance and / or test factor that increases IP3R activity based on the measurement result in the IP3R activity measurement step. As a step to separate.

The increase in IP3R activity is determined by the measurement result in the IP3R activity measurement step. The determination can be made based on, for example, a statistically significant difference from the control. Specifically, when the IP3R activity (measurement activity) in the myocytes treated with the test substance and / or the test factor is compared with the control activity, the measurement activity increases statistically significantly with respect to the control activity. If so, it may be determined that the activity of IP3R has increased. Here, “statistically significant” means that there is a significant difference when the measured activity and the control activity are treated statistically. Specifically, for example, the risk rate (significance level) is less than 5%, 1%, or 0.1%. The test method is not particularly limited as long as it is a known method capable of determining the presence or absence of significance. For example, a student-t test method or a multiple comparison test method can be used.

Alternatively, the determination can be performed based on the measurement threshold value. Specifically, if the measured activity is equal to or greater than a predetermined threshold, it may be determined that the activity of IP3R has increased.

When it is determined that the IP3R activity has increased according to the measurement result in the IP3R activity measurement step, the test substance or test factor used for the treatment of muscle cells in the test substance treatment step is separated as a muscle increase factor. At this time, when the test substance or test factor used in the test substance treatment step is a single substance or a single environmental factor, the substance or environmental factor can be recognized as a muscle increasing factor. On the other hand, when treated with a combination of two or more different substances such as a test substance library or test substances and test factors in the test substance treatment step, the test substances and / or test factors separated in this step are , Isolated as a muscle growth factor candidate including at least one muscle growth factor. In this case, the first series of steps in the separation method of the present aspect is a primary separation process, and the test substance or test factor obtained by separating or subtracting a part of the test substance or test factor used in the test substance treatment step of the primary separation process Using the (secondary test substance or the like), a series of steps in the separation method of this embodiment is executed again as a secondary separation step. If it is determined that the IP3R activity has increased in the secondary separation step, the target muscle growth factor is included in the secondary test substance or the like. In this way, when the muscle cells are treated with a combination of two or more different substances or test substances and test factors in the test substance treatment step, the secondary separation step and subsequent steps are repeated as necessary to increase muscle growth factors. By gradually narrowing down the candidates, the target muscle increasing factor can be obtained.

(4) In Vivo Confirmation Step In this specification, the “in vivo confirmation step” means that an animal is treated with a muscle augmentation factor or a muscle augmentation factor candidate separated at a muscle augmentation factor separation step, and IP3R activation or muscle in vivo. This is a step to confirm the increase. This step is a selection step performed after the muscle increase factor separation step, and may be performed as necessary for the purpose of separating a highly effective muscle increase factor.

As the animal, a laboratory animal may be used. 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.

In this step, the muscle growth factor or the muscle growth factor candidate separated in the muscle growth factor separation step is administered to the animal, the activation of IP3R in the muscle cells of the animal is measured, and whether or not the muscle gain has increased in the animal. Can be determined. Whether or not a muscle growth factor or a muscle growth factor candidate has a muscle increasing action in an animal can be appropriately determined by a method known in the art, although it varies depending on the type of animal. For example, after a muscle growth factor or muscle growth factor candidate is administered to an experimental animal, muscle tissue is collected from the animal, and muscle weight, muscle cross-sectional area, muscle tension, etc. are measured to confirm the muscle increasing effect. be able to.

4-2. Effect According to the separation method of the present embodiment, a new muscle growth factor having a pharmacological effect of muscle growth that induces muscle hypertrophy or reduces muscle atrophy via IP3 / Ca ²⁺ signaling pathway in muscle cells is isolated. can do.

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

[Experimental Example 1]
(the purpose)
We examined whether the P2Y / PLC / IP3R pathway also exists in the skeletal muscle cell lineage.

(material)
C2C12 cells, a mouse myoblast cell line, were used. Nucleic acids used as ligands are ATP, UTP, TTP, CTP, GTP, ATPγS, 2-MeS ATP, ADP, AMP and adenosine (all Sigma-Aldrich). 2 μM thapsigargin (tg) (Calbiochem) was used as a calcium depleting agent for sarcoplasmic reticulum.

(Method)
C2C12 myotube was treated with various nucleic acids, and intracellular calcium ion concentration was monitored using calcium indicator Fluo-4. The concentration of each nucleic acid was treated with 100 μM. However, the experiment in which the concentration was varied was performed at 0.1, 1, 10, 100, or 1000 μM (according to the illustrated concentration). The following method was used for measurement of intracellular calcium ion concentration. First, C2C12 cells were cultured in growth medium (DMEM, 10% fetal bovine serum, 1% penicillin-streptomycin) at 37 ° C. under 5% CO ₂ , and then differentiation medium (DMEM, 2% horse serum, 1 % Penicillin-streptomycin) for 2 days to induce muscle differentiation. Subsequently, C2C12 cells in which myogenic differentiation was induced were cultured in serum-free DMEM for 8 hours, and then PSS solution (140 mM NaCl, 5 mM KCl, 2.5 mM CaCl ₂ , 1 mM MgCl ₂ , 10 mM HEPES, 10 After culturing for 6 hours or more in mM glucose, pH 7.0), a calcium indicator Fluo-4 (DOJINDO) (4 μM) 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 treatment with each nucleic acid were measured over time using an inverted fluorescence microscope (Olympus) every 180 seconds until 180 seconds later.

Whether the increase in intracellular calcium ion concentration by ATP or UTP was derived from the influx of extracellular calcium or the release from the sarcoplasmic reticulum in C2C12 cells was verified by removing or depleting the respective calcium. Extracellular calcium was removed by adding 0 Ca ²⁺ solution (140 mM NaCl, 5 mM KCl, 1 mM MgCl ₂ , 10 mM HEPES, 10 mM glucose, 2 mM EGTA, pH 7.0) to the cells. When depleting sarcoplasmic reticulum calcium, thapsigargin was added along with Fluo-4 to deplete sarcoplasmic reticulum calcium.

• 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 the mean value ± standard error, and p <0.05 was determined to be significant.

(result)
Results are shown in FIGS. 2A-F. As shown in FIGS. 2A and 2B, among the main nucleic acids ATP, UTP, CTP, TTP, and GTP, it was revealed that the intracellular calcium ion concentration was increased by ATP and UTP. Further, as shown in FIGS. 2C and 2D, it was also found that the intracellular calcium ion concentration increases depending on the concentration of ATP or UTP, but slightly decreases when treated at a high concentration such as 1000 μM. Furthermore, when treated with ATP analogs such as ATPγS, 2-MeS ATP, ADP, AMP, and adenosine, the intracellular calcium ion concentration increases with ATPγS and 2-MeS ATP, albeit weakly, as shown in FIG. 2E. Was seen. The degree of increase was in the order of ATP>ATPγS> 2-MeS ATP. On the other hand, no increase in concentration was observed in the treatment with ADP, AMP and adenosine. As shown in FIG. 2F, the removal of extracellular calcium did not inhibit the increase in intracellular calcium ion concentration by ATP or UTP, whereas the increase in intracellular calcium ion concentration by addition of thapsigargin. Was inhibited. From the above results, it was found that the release of calcium ions from the sarcoplasmic reticulum was also promoted by ATP or UTP treatment in skeletal muscle cells.

[Experiment 2]
(the purpose)
It was verified that the increase in intracellular calcium ion concentration by ATP or UTP in Experimental Example 1 was via the P2Y receptor, PLC, and IP3R calcium ion channel.

(material)
The basic material conformed to Experimental Example 1. In this experimental example, 100 μM suramin (Sigma-Aldrich) was used as a P2Y receptor inhibitor, and 10 μM U73122 (Sigma-Aldrich) was used as a PLC inhibitor. P2Y ₂ siRNA was purchased from sigma-aldrich. The base sequence of each P2Y ₂ siRNA used is as follows. P2Y ₂ # 1: 5'-caaucauuuguacgugugatt-3 '(sequence number 1), P2Y ₂ # 2: 5'-cuaaggacauucggcuauatt-3' (sequence number 2), P2Y ₂ # 3: 5'-gucuugacccgguacucuatt-3 '(sequence) Number 3). Control siRNA was purchased from Sigma-Aldrich as well. As the IP3R inhibitor, 2 μM xestospongin C (XeC; Sigma-Aldrich) was used.

(Method)
The basic operation was performed according to Experimental Example 1. Here, C2C12 was treated by adding suramin or U73122 to ATP and UTP. In addition, in the P2Y ₂ gene expression suppression using P2Y ₂ siRNA, jetPRIME (Polyplus) was used for transfection of siRNA. The specific method followed the attached protocol.

(result)
Results are shown in FIGS. 3A-D.

As shown in FIGS. 3A and 3B, the increase in intracellular calcium ion concentration by ATP or UTP was suppressed by the addition of a suramin or U73122 inhibitor. In addition, as shown in FIG. 3C, the result of suppressing the expression of P2Y ₂ by siRNA, increase in intracellular calcium ion concentration by ATP or UTP was inhibited. Furthermore, as shown in FIG. 3D, the increase in intracellular calcium ion concentration by ATP or UTP was inhibited by XeC. From these results, it was proved that the intracellular calcium ion concentration was also increased in the skeletal muscle cells by the activation of the P2Y / PLC / IP3R pathway by ATP or UTP.

[Experiment 3]
(the purpose)
In order to examine the effect of increased intracellular calcium ion concentration by ATP or UTP in ex vivo, it was verified that an increase in intracellular calcium ion concentration by ATP or UTP can be confirmed even in isolated muscle fibers derived from skeletal muscle. .

(material)
As the mouse, C57BL / 6 mouse (12 weeks old: CLEA Japan) was used. Nucleic acids used as ligands are ATP and UTP (both Sigma-Aldrich). Instead of the C2C12 cells in Experimental Example 1, single muscle fibers were used in this Experimental Example.

(Method)
The basic method conformed to the method of Experimental Example 1. Single muscle fibers were isolated by removing the soleus muscle from anesthetized mice and then adding 0.2% collagenase 1 in PSS solution (140 mM NaCl, 5 mM KCl, 2.5 mM CaCl ₂ , 1 mM MgCl ₂ , 10 mM HEPES, 10 mM) Treated with glucose, pH 7.0) at 37 ° C for 1.5 hours, and then loosened the muscle fibers by pipetting. Single muscle fibers were isolated from the loosened muscle fibers, and 4 μM Fluo-4 was added to measure calcium concentration. Collagenase 1 was purchased from Worthington biochemical corporation.

(result)
The results are shown in FIG. In the soleus muscle mainly composed of slow muscle cells, an increase in intracellular calcium concentration dependent on ATP or UTP was observed. From this result, it was proved that the intracellular calcium ion concentration was increased by the binding of ATP or UTP not only in vitro by the myoblast cell line but also ex vivo by a single muscle fiber extracted from the mouse individual.

[Experimental Example 4]
(the purpose)
In Experimental Example 3, it was verified that the increase in intracellular calcium ion concentration in single muscle fibers due to ATP or UTP was via P2Y receptor, PLC, and IP3R calcium ion channel.

(material)
The basic material conformed to Experimental Examples 1 to 3. The ligand is ATP, and the P2Y receptor inhibitor, PLC inhibitor, and IP3R inhibitor are 100 μM suramin (Sigma-Aldrich), 10 μM U73122 (Sigma-Aldrich), and 2 μM xestospongin C (XeC; Sigma-Aldrich) was used. Furthermore, 0Ca ²⁺ solution (140 mM NaCl, 5 mM KCl, 1 mM MgCl ₂ , 10 mM HEPES, 10 mM glucose, 2 mM EGTA, pH 7.0) was used to remove extracellular calcium from single muscle fibers. .

(Method)
Single muscle fibers were prepared from soleus muscles extracted from 12-week-old C57BL / 6 mice according to the method of Experimental Example 3. Other basic operations were performed in accordance with Experimental Examples 1 and 2.

(result)
FIG. 9 shows the result. The increase in intracellular calcium concentration due to ATP was suppressed by P2Y receptor inhibitor (suramin), PLC inhibitor (U73122), and IP3R inhibitor (XeC). Also, removal of extracellular calcium did not inhibit the increase in intracellular calcium concentration by ATP (0Ca ²⁺ ). From these results, it was demonstrated that the ATP-dependent increase in intracellular calcium concentration observed in single muscle fibers in Experimental Example 3 occurred via the P2Y / PLC / IP3 receptor pathway.

[Example 1: Activation of mTOR in skeletal muscle cells by P2Y receptor activation]
(the purpose)
It was verified whether mTOR was activated by activation of the P2Y / PLC / IP3R pathway of skeletal muscle cells by ATP or UTP.

(material)
Primary antibodies for Western blot include p70S6K antibody (# 9202, Cell Signaling Technology), p-p70S6K (Thr421 / Ser424) antibody (# 9204, Cell Signaling Technology), p-p70S6K (Thr389) antibody (# 9205, Cell Signaling) Technology), Akt antibody (# 9272, Cell Signaling Technology), p-Akt (Ser473) antibody (# 9271, Cell Signaling Technology) and p-Akt (Thr308) antibody (# 9275, Cell Signaling Technology). As the secondary antibody, a rabbit-specific HRP-labeled antibody (GE Healthcare) was used. Moreover, 0.1 μM rapamycin (Sigma-Aldrich) was used as an mTOR inhibitor. 50 μM BAPTA-AM (Calbiochem) was used as the intracellular calcium chelator, and 2 mM EGTA (Sigma-Aldrich) was used as the extracellular calcium chelator.

(Method)
The treatment of C2C12 cells with ATP or UTP was basically performed according to Experimental Example 1, and cultured using a medium to which additional additives were added as necessary. p70S6K was used as the mTOR substrate.

The Western blot was performed according to the following procedure. First, C2C12 cells were sample buffer (0.1% Triton X-100, 50 mM HEPES (pH 7.4), 4 mM EGTA, 10 mM EDTA, 15 mM Na ₄ P ₂ O ₇ , 100 mM glycerophosphate, 25 mM NaF, 5 mM Homogenized with Na ₂ VO ₄ and complete protease inhibitor cocktail (Roche) and centrifuged (15,000 g, 10 minutes) and the supernatant was collected. After measuring the protein concentration using Coomassie Brilliant Blue G-250 (BioRad), an equal amount of sample loading buffer (30% glycerol, 5% 2-mercaptoethanol, 2.3% SDS, 62.5 mM Tris-HCl (pH 6.8)) , 0.05% bromophenol blue) and heat-denatured at 60 ° C. for 15 minutes. 30 μg was subjected to Western blot. 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. The transfer membrane was washed with Tris buffered saline (TBST) containing 0.1% Tween 20, and then incubated with a secondary antibody. It was washed again with TBST and detected with ECL Western Blotting Detection System (GE HealthCare, Buckinghamshire).

(result)
Results are shown in FIGS. 4A-F.

As shown in FIG. 4A, phosphorylation of Thr at position 389 (start Met is position 1; the same applies hereinafter), Thr at position 421, and Ser at position 424, in p70S6K, which is a mTOR substrate, by ATP or UTP treatment. It rose in a concentration-dependent manner. In addition, as shown in FIG. 4B, phosphorylation of p70S6K by ATP or UTP was inhibited by rapamycin, an mTOR inhibitor. From these results, it became clear that mTOR is also activated by ATP or UTP in skeletal muscle cells.

As a result of examining whether the activation of mTOR by ATP or UTP occurs via an increase in extracellular or intracellular calcium ion concentration, as shown in FIG. 4C, phosphorylation of p70S6K by ATP or UTP is extracellular. It was suppressed to some extent by EGTA, a calcium chelate, and completely suppressed by BAPTA-AM, an intracellular calcium chelate. These results suggest that both calcium ion release from the sarcoplasmic reticulum and extracellular calcium ion influx are involved in mTOR activation.

Furthermore, as shown in FIG. 4D, phosphorylation of p70S6K by ATP or UTP was suppressed by suramin, a P2Y receptor inhibitor, and U73122, a PLC inhibitor. Further, as shown in FIG. 4E, phosphorylation of p70S6K by ATP or UTP by inhibiting expression of the P2Y ₂ by P2Y ₂ siRNA was inhibited. In addition, as shown in FIG. 4F, phosphorylation of p70S6K by ATP or UTP was also suppressed by XeC, an IP3R inhibitor. These results showed that in vitro activation of mTOR in skeletal muscle cells by ATP or UTP is via the P2Y / PLC / IP3R pathway.

[Example 2: Activation of MAPK by P2Y activation and regulation of expression of downstream gene JunB]
(the purpose)
It has been reported that MAPK is activated by an increase in intracellular calcium ion concentration and is involved in various downstream gene expression regulation (Hazzalin CA & Mahadevan LC, 2002, Nat Rev Mol Cell Biol. 3 (1): 30 -40). Therefore, it is verified whether the increase of calcium ion concentration in muscle cells by ATP / UTP activates various signal molecules such as MAPK other than mTOR, and if MAPK is activated, downstream genes are expressed. did.

(material)
C2C12 cells or C57BL / 6 mice (12 weeks old: Japan Claire) were used.

Primary antibodies for Western blot include p-Erk1 / 2 (Thr202 / Tyr204) antibody (# 9101, Cell Signaling Technology) for p44 / 42 MAPK, and p-AMPKα (Thr172) antibody (# 2535, Cell Signaling for AMPKα Technology) and AMPKα antibody (# 2603, Cell Signaling Technology) and JunB antibody (sc-46, Santa Cruz) were used. As the secondary antibody, a rabbit-specific HRP-labeled antibody (GE Healthcare) was used. As an inhibitor of MAPK, 10 μM U0126® (Sigma-Aldrich) was used.

(Method)
The treatment of C2C12 cells with ATP or UTP was basically performed according to Experimental Example 1, and cultured using a medium to which additional additives were added as necessary. The Western blot was performed according to the method described in Example 1.

The treadmill (MK-680S, Muromachi Kikai) was used for the exercise load to the mouse. After running at an initial speed of 5 m / min for 5 minutes, the speed was increased by 1 m / min every minute and increased to a speed of 20 m / min. After running for a total of 30 minutes, the gastrocnemius muscle was excised and collected under anesthesia (Moresi, V. et al., 2010, Cell 143, 35-45). Control group mice underwent sham surgery to cut the skin.

A comprehensive analysis of gene regulation activated by overload, exercise, and administration of ATP or UTP to C2C12 cells, or C57BL / 6 mice, and is controlled by intracellular calcium ion concentration We searched for candidate genes that could promote muscle hypertrophy.

(result)
Results are shown in FIGS. 5A-D.

As shown in FIG. 5A, phosphorylation of Thr at position 202 and Thr at position 204 in p-Erk1 / 2 increased in a concentration-dependent manner by ATP or UTP treatment. From this result, it became clear that MAPK is activated by ATP or UTP also in skeletal muscle cells. On the other hand, no concentration-dependent increase in phosphorylation of AMPK was observed.

Since activation of MAPK is involved in various gene expression controls, activation of MAPK by ATP or UTP treatment in muscle cells is also involved in the transcriptional control of downstream genes. As a result of exhaustively searching for candidate genes that can be controlled by intracellular calcium ion concentration in muscle cells and can promote muscle hypertrophy as described above, it is clear that the expression level of JunB gene increases as shown in FIG. 5B. became. JunB gene expression has been reported to promote muscle hypertrophy (Raffaello, A., et al., 2010, J Cell Biol, 191: 101-113). Moreover, as shown in FIG. 5C, the increase in the expression of JunB by ATP treatment of C2C12 cells was suppressed by U0126, which is an inhibitor of BAPTA-AM and p44 / 42 MAPK. These results indicate that in muscle cells, the MAPK pathway is activated through the increase of intracellular calcium ion concentration due to the activation of the P2Y / PLC / IP3R pathway by ATP or UTP treatment, and the expression of JunB, a downstream gene, is increased. It became clear to do. Furthermore, as shown in FIG. 5D, it was revealed that the increase in the amount of JunB protein due to ATP treatment of myocytes was suppressed by BAPTA-AM and rapamycin, an mTOR inhibitor. These results suggest that mTOR is not involved in the regulation of JunB transcription, but is involved in the subsequent protein translation pathway.

[Example 3: Promotion of muscle hypertrophy by administration of ATP or UTP]
(the purpose)
It was confirmed that muscle hypertrophy was promoted by administering ATP or UTP, which is a muscle increasing agent of the present invention, to mice in which disuse muscle atrophy was induced.

(material)
As the mouse, C57BL / 6 mouse (12 weeks old: CLEA Japan) was used.

10 μM rapamycin (Sigma-Aldrich) was used as an mTOR inhibitor, and 50 μM BAPTA-AM (Calbiochem) was used as an intracellular calcium chelator.

(Method)
For administration of ATP or UTP to mice, 150 μL of 500 μM ATP or UTP solution prepared by dissolving in physiological saline was administered twice a day by intramuscular injection for one week.

In order to examine the therapeutic effect of ATP or UTP in muscle atrophy, muscle atrophy by unloading and denervation was induced by sciatic nerve resection and hindlimb suspension, respectively.

Disuse muscle atrophy in mice was induced by hindlimb suspension (Suzuki, N. et al., 2007, J Clin Invest 117, 2468-2476). Specifically, the mice were reared so that their hind limbs were separated from the floor by 1 mm or more. Two weeks later, the gastrocnemius and soleus were extracted from the mouse, and the body weight and muscle weight were measured. In addition, the gastrocnemius muscle is a muscle having a higher proportion of slow muscles than other skeletal muscles, like the soleus muscle.

Also, neurogenic muscle atrophy in mice was induced by sciatic nerve resection (Moresi, V. et al., 2010, Cell, 143: 35-45). Two weeks after excision of the sciatic nerve, the gastrocnemius and soleus were removed from the mouse, and the muscle weight was measured.

Furthermore, compensatory muscle hypertrophy in mice was induced by synergistic muscle resection. Specifically, the gastrocnemius and soleus tendons of the mouse were excised under anesthesia (Adams, G. R. & Haddad, F., J Appl Physiol, 1996, 81: 2509-2516). The control group received a sham operation to cut the skin.

(result)
The results are shown in FIGS.

The muscle weights of the soleus muscle (FIGS. 6A and 6C) and gastrocnemius muscle (FIG. 6B) were increased by administration of ATP or UTP. This increase in muscle weight by ATP was suppressed by BAPTA-AM or rapamycin (FIGS. 6A, B, and C).

FIG. 6D shows the therapeutic effect of ATP or UTP on soleus muscle atrophy caused by unloading, and FIG. 6E shows the therapeutic effect of ATP or UTP on gastrocnemius muscle atrophy caused by denervation. In either case, muscle atrophy was induced in the control group to which ATP or UTP was not administered, but the decrease in muscle weight was attenuated in the ATP or UTP administration group.

From the above results, as shown in FIG. 7, the muscle increasing agent of the present invention such as ATP or UTP can be administered to muscle cells to control intracellular calcium ion concentration via the P2Y / PLC / IP3R signaling pathway. It has been proved that mTOR and MAPK can be activated by this, and muscle hypertrophy can be promoted and muscle atrophy can be reduced by the expression of JunB protein.

[Example 4: Activation of mTOR in single muscle fiber by P2Y receptor activation]
(the purpose)
It was confirmed that mTOR was also activated by the activation of P2Y / PLC / IP3R pathway by ATP observed in single muscle fibers.

(material)
The reagents and the like basically used those described in Example 1. Specifically, p70S6K antibody (# 9202, Cell Signaling Technology), p-p70S6K (Thr421 / Ser424) antibody (# 9204, Cell Signaling Technology), p-p70S6K (Thr389) antibody (# 9205, Cell Signaling Technology) and Akt antibody (# 9272, Cell Signaling Technology). As the secondary antibody, a rabbit-specific HRP-labeled antibody (GE Healthcare) was used. p70S6K was used as the mTOR substrate.

(Method)
In this example, ATP was used as the ligand. ATP was administered to mice by intramuscular injection according to the method described in Example 3. Isolation of soleus muscle-derived single muscle fibers from ATP-administered mice followed the method described in Experimental Example 3. Other basic operations were performed according to Example 1.

(result)
The results are shown in FIG. When ATP was injected intramuscularly, phosphorylation of Thr at position 389 in p70S6K, a mTOR substrate, increased. These results demonstrated that ATP-dependent increase in intracellular calcium concentration and subsequent activation of mTOR also occur in single muscle fibers.

It should be noted that all publications, patents and patent applications cited in this specification are incorporated herein by reference as they are.

Claims (11)

It is characterized by inducing calcium ion release from the sarcoplasmic reticulum by directly or indirectly activating the inositol triphosphate receptor on the sarcoplasmic reticulum membrane by acting from outside or inside the muscle cell. A muscle increasing agent. The muscle increasing agent according to claim 1, wherein the muscle cells are slow muscle cells. The muscle increasing agent according to claim 1 or 2, which is a P2Y receptor agonist. The muscle increasing agent according to claim 3, wherein the P2Y receptor agonist is ATP or UTP or a derivative thereof, a salt thereof, or a combination thereof. The muscle increasing agent according to claim 1 or 2, which is inositol triphosphate or a salt thereof, an ester thereof or a prodrug thereof. A pharmaceutical composition for treating or preventing a disorder or disease associated with muscle atrophy, comprising the muscle increasing agent according to any one of claims 1 to 5 as an active ingredient. The pharmaceutical composition according to claim 6, wherein the disorder accompanied by muscle atrophy is selected from the group consisting of disuse muscle atrophy, cachexia, sarcopair, and muscle atrophy under microgravity. The pharmaceutical composition according to claim 6, wherein the disease accompanied by muscular atrophy is amyotrophic lateral sclerosis or muscular dystrophy. A method of separating a muscle increasing agent,
(A) treating muscle cells with a test substance and / or a test factor;
(B) a step of measuring the activity of inositol triphosphate receptor in the muscle cell, and a test substance or test for increasing the activity of inositol triphosphate receptor based on the measurement result of (c) (b) step Separating the factor as a muscle augmenting agent. The method according to claim 9, wherein the muscle cells are slow muscle cells. The method according to claim 9 or 10, wherein a change in activation of the inositol triphosphate receptor is measured by a change in calcium ion concentration in muscle cells or sarcoplasmic reticulum.

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