JP2019214534A - Pharmaceutical composition containing mesenchymal stem cells as monoamine production increasing agent - Google Patents

Abstract

To provide pharmaceutical compositions that promote the production of monoamine neurotransmitter in living organisms including blood and cerebrospinal fluid.SOLUTION: Provided is a pharmaceutical composition for promoting monoamine or monoamine metabolite production, comprising mesenchymal stem cells as an active ingredient. Monoamine whose production is promoted is, for example, serotonin or noradrenaline, and monoamine metabolite production is, for example, 5-hydroxyindoleacetic acid (5-HIAA) or 3-methoxy-4-hydroxyphenylethylene glycol (MHPG).SELECTED DRAWING: Figure 1

Description

The present invention relates to a pharmaceutical composition for increasing the concentration of any one or more of serotonin, noradrenaline, and their main metabolites in vivo.

  JP-A-2008-61569 describes a method for maintaining differentiation of mesenchymal stem cells using a stem cell growth medium containing serotonin and a p38 MAP kinase inhibitor. In this publication, serotonin is described as one of the compositions added to the medium.

  Japanese Patent No. 5541845 describes a therapeutic agent for atopic dermatitis containing mesenchymal stem cells.

  Patent No. 6191694 describes a therapeutic agent for cartilage damage including mesenchymal stem cells.

JP 2008-61569 A Japanese Patent No. 5541845 Japanese Patent No. 5541845

  An object of the present invention is to provide a pharmaceutical composition that promotes production of a monoamine neurotransmitter in a living body including blood and cerebrospinal fluid. This composition has the effect of increasing the amount of one or more of, for example, serotonin, noradrenaline, or their main metabolites.

  The above-mentioned problem is caused by the fact that a pharmaceutical composition containing mesenchymal stem cells as an active ingredient has an effect of promoting the production of monoamine neurotransmitters and major metabolites thereof in a living body such as blood and cerebrospinal fluid. Based on findings from examples.

  The present invention relates to pharmaceutical compositions. This is called the pharmaceutical composition of the present invention. This pharmaceutical composition functions as a medicine or a drug. This pharmaceutical composition contains mesenchymal stem cells as an active ingredient. Examples of tissue derived from mesenchymal stem cells are adipose tissue or umbilical cord tissue. The pharmaceutical composition may comprise an effective amount of mesenchymal stem cells required to achieve various purposes including treatment and prevention (eg, muscle enhancement, mental stability, analgesia).

  The pharmaceutical composition of the present invention is a pharmaceutical composition for promoting the production of monoamine, which is a neurotransmitter, or a metabolite of the monoamine. This pharmaceutical composition can be used as a therapeutic agent for various symptoms and diseases caused by a decrease in monoamine, and can also be used as an agent for enhancing biological functions due to an increase in monoamine.

  Examples of neurotransmitter monoamines are serotonin and / or noradrenaline. And examples of metabolites of monoamines are either or both of 5-hydroxyindoleacetic acid (5-HIAA) and 3-methoxy-4-hydroxyphenylethylene glycol (MHPG). 5-Hydroxyindoleacetic acid (5-HIAA) is the major metabolite of serotonin. On the other hand, 3-methoxy-4-hydroxyphenylethylene glycol (MHPG) is a major metabolite of noradrenaline.

  This pharmaceutical composition can be used as a therapeutic agent for various symptoms and diseases caused by a decrease in monoamine, and can also be used as an agent for enhancing biological functions due to an increase in monoamine. Specific examples of use are analgesics and muscle potentiators.

  The pharmaceutical composition of the present invention has an effect of increasing the amount of serotonin and noradrenaline, or a major metabolite thereof. Therefore, this pharmaceutical composition is useful as a therapeutic agent for various symptoms and diseases caused by monoamine lowering, or as a biological function enhancer by increasing monoamine.

FIG. 1 is a conceptual diagram showing an outline of monoamine measurement. FIG. 2 is a graph instead of a drawing showing the monoamine determination results.

  The present invention relates to pharmaceutical compositions. This is also called the pharmaceutical composition of the present invention. This pharmaceutical composition functions as a medicine and a medicine. This pharmaceutical composition contains mesenchymal stem cells as an active ingredient. This pharmaceutical composition may contain an effective amount of mesenchymal stem cells required to achieve various purposes including treatment and prevention (for example, muscle enhancement, mental stability, analgesia).

  Mesenchymal stem cells are isolated not only from adult tissues such as synovial cells, adipocytes, bone marrow, dental pulp, and periodontal ligament, but also from various cells of placenta, umbilical cord, umbilical cord blood, and fetus. Is also included. The mesenchymal stem cells grown in the growth step may be derived from a tissue selected from the group consisting of synovium, umbilical cord, umbilical cord blood, amniotic membrane, bone marrow, and adipose tissue. The tissue derived from mesenchymal stem cells is preferably adipose tissue or umbilical cord tissue.

The mesenchymal stem cells are preferably autologous cells of the patient to whom the pharmaceutical composition is administered, but may be allogeneic cells. The mesenchymal stem cells may be human mesenchymal stem cells, or may be mesenchymal stem cells derived from non-human animals such as mice, rats, cats, and dogs. As a method for isolating mesenchymal stem cells from each tissue, a known method can be adopted. For example, mesenchymal stem cells can be suitably separated from the tissue by the collagenase method.

  The pharmaceutical composition of the present invention is a pharmaceutical composition for promoting production of monoamine, which is a neurotransmitter, or a metabolite of the monoamine. This pharmaceutical composition can be used as a therapeutic agent for various symptoms and diseases caused by a decrease in monoamine, and can also be used as an agent for enhancing biological functions due to an increase in monoamine.

  Examples of monoamines are serotonin and / or noradrenaline. And examples of metabolites of monoamines are either or both of 5-hydroxyindoleacetic acid (5-HIAA) and 3-methoxy-4-hydroxyphenylethylene glycol (MHPG). 5-Hydroxyindoleacetic acid (5-HIAA) is the major metabolite of serotonin. On the other hand, 3-methoxy-4-hydroxyphenylethylene glycol (MHPG) is a major metabolite of noradrenaline.

  A neurotransmitter called a monoamine is a substance having one amino group in its structure, and is represented by serotonin having an indole skeleton, and dopamine, noradrenaline and adrenaline having a catechol group. In general, dopamine is related to feelings of pleasure and joy, noradrenaline is related to feelings of anger and anxiety, and serotonin has the function of controlling the functions of these monoamines. Receptors for these monoamines that function as neurotransmitters are G protein-coupled receptors in addition to the ion channel type 5-HT3-type receptors.

  Serotonin is synthesized from the essential amino acid tryptophan and is present in the gastrointestinal mucosa, platelets, and brain at about 90%, 8%, and 2%, respectively. In the intestines, enterochromophilic cells, a type of endocrine cell, are secreted as an otacoid and promote intestinal peristaltic movement. Therefore, decreased serotonin secretion in the intestine leads to constipation. Part of serotonin secreted by the digestive tract is taken up by platelets. On the other hand, the cell bodies of serotonergic neurons that synthesize serotonin in the brain are concentrated in the raphe nucleus of the brainstem, but the nerve fibers are widely projected to the central nervous system. Therefore, the functions controlled by serotonin in the central nervous system are diverse, including body temperature regulation, emotion, cognitive function, arousal and sleep, and eating behavior.

Examples of drugs targeting serotonin include selective serotonin reuptake inhibitors (SSRI) and serotonin / noradrenaline reuptake inhibitors (SNRI). They are used as antidepressants to increase serotonin concentration in the synaptic cleft by inhibiting the serotonin transporter. In about 38% of depressed patients, a decrease in serotonin levels is observed, which is one of the causes of depression. This is called the monoamine hypothesis.

  The pharmaceutical composition of the present invention can be used as a therapeutic agent for various symptoms and diseases caused by a decrease in monoamine, and can also be used as an enhancer for a biological function due to an increase in monoamine. Specific examples of use are analgesics and muscle potentiators. Serotonin has wakefulness, analgesic action, antidepressant action, etc., and the usefulness of this pharmaceutical composition is considered for chronic fatigue syndrome, pain, and depression. Noradrenaline is important for mental and physical arousal and excitement, sympathetic activation, pressor action, and memory fixation, positive control of synaptic long-term potentiation in the hippocampus, increased judgment and concentration, myocardial excitement This pharmaceutical composition is useful for pain, fear, anxiety, depression, heart failure, poor judgment, memory weakness, muscle weakness, parasympathetic nerve enhancement, etc. Conceivable.

  The pharmaceutical composition of the present invention can be administered as an oral preparation, a drink, a tablet, or an injection. As an injection, a pharmaceutical composition which is an active ingredient can be administered intravenously, intramuscularly or subcutaneously, and among these, intravenous administration is more preferable. When administered intravenously, the injection may be administered directly to the patient from a syringe, or may be first added to the infusion solution in an infusion bag and administered intravenously to the patient.

  When the pharmaceutical composition of the present invention is an injection or a drink, in addition to mesenchymal stem cells, water, physiological saline, a solvent, a culture supernatant, a pH adjuster, an antioxidant (vitamin C), and trehalose Various materials may be included. When the pharmaceutical composition of the present invention is a tablet, it may contain various materials such as excipients and carriers.

  The administration method of the pharmaceutical composition of the present invention may be a single administration, or may be administered twice, or three or more times until the effect appears, depending on the condition of the patient or administration subject and the purpose. Good.

  Since the pharmaceutical composition of the present invention is a general-purpose drug, it is generally administered to patients who are allogeneic to the origin of the mesenchymal stem cells contained as the main component. It does not prevent administration to patients (eg, identical twins from which mesenchymal stem cells are collected), and the pharmaceutical composition of the present invention, which is manufactured as a general-purpose product through propagation by culture, is useful. It does not prevent administration to the human from which the mesenchymal stem cells were collected.

At the time of the above administration, the density of human mesenchymal stem cells in the pharmaceutical composition of the present invention is preferably 1 × 10 ² to 1 × 10 ⁹ cells / mL, and 2 × 10 ⁶ to 2 × 10 ⁷ cells / mL. More preferably, it is mL. The number of the cells to be administered to a human depends on the intended number of administrations, but is usually preferably in the range of 1 × 10 ⁵ to 1 × 10 ⁷ cells / kg body weight per administration. However, it is not limited to this, and can be appropriately increased or decreased according to the degree of symptoms.

  The present specification provides a neurotransmitter monoamine or monoamine in a living body of a subject, which comprises a step of administering to the subject a mesenchymal stem cell or a pharmaceutical composition of the present invention containing the mesenchymal stem cell as an active ingredient. And methods for promoting the production of metabolites of The production of monoamines, which are neurotransmitters, or metabolites of monoamines has been promoted by, for example, plasma separated from the subject's blood before and after administration of the pharmaceutical composition to the subject, or cerebrospinal fluid, monoamine, or The content (blood concentration) of monoamine metabolites may be measured and compared.

1. Quantitative test of monoamine concentration in rat living body by mesenchymal stem cell transplantation 1-1. Preparation of mesenchymal stem cells: Adipose tissue-derived mesenchymal stem cells (AD-MSC)
(1) Primary culture (P0)
After obtaining consent for research use from surplus tissue after separating subcutaneous adipose tissue, which is a raw material necessary for preparing cells for administration, from patients undergoing regenerative medicine using adipose tissue-derived mesenchymal stem cells Subcutaneous fat was provided for primary culture. The subcutaneous adipose tissue was subjected to centrifugation (400 × g for 5 minutes) and separated into three layers from the top in the order of lipid fraction, adipose tissue fraction, and aqueous fraction. The upper and lower layers were discarded, leaving the middle adipose tissue fraction. To the remaining adipose tissue fraction, 4 times the amount of 0.15% collagenase enzyme solution per tissue weight was added and permeated at 37 ° C. for 1 hour for enzyme treatment. After the adipose tissue is dispersed, it is subjected to centrifugation (400 × g for 5 minutes), and the precipitated fraction is suspended in 30 mL of PBS (−) solution as a stromal vascular cell fraction containing mesenchymal stem cells. did. Thereafter, the suspension was passed through a cell strainer (70 μm diameter), and the passed fraction was subjected to centrifugation again (400 × g for 5 minutes), and the tissue residue captured by the cell strainer was discarded. The precipitate fraction is suspended in 6 mL of serum-free medium (Procul AD; Rohto Pharmaceutical Co., Ltd.), and the whole volume is seeded in a T-25 flask (CellBIND (registered trademark); Corning), and then in an incubator (37 ° C, 5% CO ₂ ) It left still and the primary culture was started.

(2) Subculture (P0 → P1 → P2 → P3)
The whole medium was changed once every three days, the supernatant was discarded, and the cells that grew on the bottom of the flask were selectively grown. 2 mL of enzyme solution (TrypLE Secelt (registered trademark); Thermo Fisher Scientific) was added to the cells in the T-25 flask grown to semi-confluent, and detached (37 ° C., left for 5 minutes). Cells were diluted with PBS (−) and subjected to centrifugation (400 × g for 5 minutes). As a result of suspending the precipitated cells in the culture solution and counting the number of cells by trypan blue staining, 1.36 × 10 ⁶ viable cells were recovered, so the total amount was T-150 flask (CellBIND (registered trademark); Corning) Equally divided into two plates, seeded with serum-free medium (Procul AD; Rohto Pharmaceutical), and left in an incubator (37 ° C, 5% CO ₂ ) for subculture (P0 → P1).
The whole medium was changed once every three days, the supernatant was discarded, and the cells that grew on the bottom of the flask were selectively grown. 6 mL of enzyme solution (TrypLE Select (registered trademark); Thermo Fisher Scientific) was added to the cells grown to semi-confluent on the third day after seeding, and detached (37 ° C., left for 5 minutes). Cells were diluted with PBS (−) and subjected to centrifugation (400 × g for 5 minutes). The precipitated cells were suspended in a serum-free medium (Procul AD; Rohto Pharmaceutical Co., Ltd.), and the number of cells was counted by trypan blue staining. As a result, 1.17 × 10 ⁷ viable cells were recovered. 8 T-150 flasks were seeded and passaged at a concentration of 10 ³ / cm ² cells (P1 → P2). After confirming that the cells had grown to semi-confluent on the third day after seeding, cells were collected in the same manner. As a result, it was confirmed that the survival rate was 95.2%, and 5.27 × 10 ⁷ viable cells were collected. Stored in a nitrogen tank.

1-2. Preparation of mesenchymal stem cells: Umbilical cord tissue-derived mesenchymal stem cells (UC-MSC)
(1) Primary culture (P0)
Umbilical cord tissue obtained with consent from a normal parturition mother was subjected to primary culture the day after collection. Umbilical cord blood was removed from about 10 cm of umbilical cord tissue, and then cut into pieces with a medical scalpel, immersed in 0.15% Collagenase solution, and subjected to enzyme treatment with gentle stirring on a shaker at 37 ° C. for 16 hours. After visually confirming the dispersion of the umbilical cord tissue, it was diluted 10-fold with PBS (−) and then centrifuged at 1,000 × g for 5 minutes, and the upper layer was discarded leaving the precipitate fraction. After that, the precipitate fraction is suspended in PBS (-), then applied to a 70 μm filter, the flow-through fraction is equally divided into two conical tubes, and then centrifuged at 400 × g for 5 minutes to obtain cells. The precipitate fraction containing was suspended in 24 mL of serum-free medium (Procul AD; Rohto Pharmaceutical). Thereafter, the entire amount was seeded in one T-150 flask (CellBIND (registered trademark); Corning), and left in an incubator (37 ° C., 5% CO ₂ ) to initiate primary culture.

(2) Subculture (P0 → P1 → P2)
The medium was completely changed once every two days, the supernatant was discarded, and the cells that grew on the bottom of the flask were selectively grown. 6 mL of enzyme solution (TrypLE Select (registered trademark); Thermo Fisher Scientific) was added to the cells in the T-150 flask grown to semi-confluent, and detached (37 ° C., left for 5 minutes). Cells were diluted with PBS (−) and subjected to centrifugation (400 × g for 5 minutes). As a result of suspending the precipitated cells in the culture solution and counting the number of cells by trypan blue staining, 4.33 × 10 ⁶ viable cells were recovered, so that the total amount was T-150 flask (CellBIND (registered trademark); Corning) Equally divided into 5 plates, seeded with serum-free medium (Procul AD; Rohto Pharmaceutical), and left in an incubator (37 ° C, 5% CO ₂ ) for subculture (P0 → P1). The medium was completely exchanged once every two days, and after confirming that the cells had grown to semi-confluent on the 4th day after seeding, the cells were collected in the same procedure.
Since 2.83 × 10 ⁷ viable cells were recovered in P2, a portion of the cells were seeded into 10 T-150 flasks at a concentration of 6.7 × 10 ³ / cm ² and subcultured (P1 → P2). The medium was completely exchanged once every two days, and after confirming that the cells had grown to semi-confluent on the 4th day after seeding, the cells were collected in the same procedure. As a result, the survival rate was 90.5%, and it was confirmed that 4.43 × 10 ⁷ viable cells were collected. Suspended in a cryopreservation solution (STEMCELL BANKER; Nippon Zenyaku Kogyo) until the day of administration to rats Stored in a liquid nitrogen tank.

1-3. Preparation of cell suspension for administration On the day of cell administration to rats, adipose tissue-derived mesenchymal stem cells and umbilical cord-derived mesenchymal stem cells stored in a liquid nitrogen tank are thawed at room temperature, and immediately after thawing, PBS ( After dilution with-), it was centrifuged at 400 xg for 5 minutes. When the precipitate fraction containing cells was suspended in PBS (-) and measured by trypan blue staining, the survival rates of adipose tissue-derived mesenchymal stem cells and umbilical cord-derived mesenchymal stem cells were 91.5% and 87.1%, respectively. Met. Thus, it was confirmed that a sufficient survival rate was maintained as a cell used for administration. Next, centrifuge again at 400 xg for 5 minutes. Discard the supernatant and suspend the precipitate fraction containing cells in PBS (-). The cell concentration of the cell suspension used for administration to rats will be The concentration was adjusted to 5 × 10 ⁶ cells / mL.

1-4. Rat administration and measurement sample collection (1) Administration
A suspension of adipose tissue-derived mesenchymal stem cells and umbilical cord-derived mesenchymal stem cells is applied to SD rats (male, 4 weeks of age, body weight around 180 g; Japan Claire) at a rate of 5 × per mouse from the tail vein. A single dose of 10 ⁶ cells (1 mL) was given. A 30 G needle was used for administration. In addition, a PBS (-) administration group was set as a control, and 1 mL was administered once from the tail vein in the same manner as the cells. The number of rats was set to 6 in each of the adipose tissue-derived mesenchymal stem cell administration group, the umbilical cord-derived mesenchymal stem cell administration group, and the PBS (-) administration group (n = 6 / group). In observations from immediately after administration of cells or PBS (-) to 1 hour after administration, no abnormalities were observed in the general state in addition to symptoms such as decreased motor function, dyspnea, and tremor. Therefore, it was confirmed that the dose was administered within a safe range.

(2) Plasma adjustment
Seven days after the administration of the cells or PBS (-), about 3 mL of blood was collected from three animals in each experimental group, and 10 µg / mL heparin was added to peripheral blood and gently mixed, and then 1,200 × g / 10 After obtaining the plasma fraction by centrifugation for 1 minute, it was immediately stored at -80 ° C.

(3) Preparation of cerebrospinal fluid About 70 μL of cerebrospinal fluid was collected from 3 mice in each experimental group by puncturing the cerebellar medullary tub 7 days after administration of the cells or PBS (−). After collection, they were immediately stored at -80 ° C.

1-5. Monoamine determination Plasma and cerebrospinal fluid stored after collection were analyzed quantitatively by HPLC-MS / MS. The analytes were 3-methoxy-4-hydroxyphenylethylene glycol (MHPG), a major metabolite of noradrenaline, and 5-hydroxyindoleacetic acid (5-HIAA), a major metabolite of serotonin. By measuring these, the amounts of their precursors, noradrenaline and serotonin, can also be ascertained, so MHPG and 5-HIAA are also used in clinical tests.

(1) As a pretreatment of the pretreated sample, in the case of a plasma sample, 200 μL of 0.4 mol / L perchloric acid is added to 200 μL of each specimen, stirred, and then centrifuged (17,500 × g, 4 ° C., 5 min) The supernatant fraction was subjected to solid phase extraction (Oasis HLB uElution 96-wells Plate 30um; Waters). As the solid phase extraction procedure, methanol (200 μL), ultrapure water (200 μL), specimen (200 μL), and 5-HIAA-d5 (10 μL) were applied to the column in this order as an internal standard when measuring 5-HIAA. Next, the sample was washed with ultrapure water (200 μL) and eluted with 2% formic acid (200 μL) diluted with methanol to obtain a measurement sample. In the case of cerebrospinal fluid samples, 0.4 mol / L perchloric acid was adjusted to 70 μL, and thereafter the same as the plasma samples.

(2) The Ultimate 3000 Rapid Separation with Q Exactive system (Thermo Fisher Scientific) was used for quantitative analysis measurement, and the column was Scherzo SS C18, 100 × 3.0 mm, 3 um (Imtakt). The measurement conditions for liquid chromatography are as follows: the column temperature is 30 ° C., methanol / water / formic acid = 35/65 / 0.5 is used as mobile phase A to apply the gradient, and methanol / 100 mM ammonium formate = 40/60 is used as mobile phase B. The gradient was as shown in Table 1, and the injection amount of the measurement sample was 5 μL. In mass spectrometry, the ionization mode is positive mode, the resolution is 17,500, the capillary temperature is 263 ° C, the spray voltage is 2.5V, the mass range for quantitation is 192.0655 → 146.055-146.065, and the internal standard 5- HIAA-d5 was carried out from 197.0969 → 151.085-151.095, and MHPG was carried out from 167.0703 → 135.043-135.045.

(3) Quantitative results
The results of quantification of MHPG and 5-HIAA are shown in FIG.
"Plasma-MHPG"
As shown in FIG. 2 (a), MHPG in the plasma sample was 4.081 ng / mL in the control PBS (-) administration group, 7.125 ng / mL in the AD-MSC administration group, and 6.391 ng / mL in the UC-MSC administration group. As a result of a significant difference test, an increase in MHPG concentration was confirmed in the AD-MSC administration group and the UC-MSC administration group compared to the PBS (−) administration group (p value <0.05).

`` Cerebrospinal fluid-MHPG ''
As shown in FIG. 2 (b), MHPG in the plasma sample was 7.352 ng / mL in the control PBS (-) administration group, 11.602 ng / mL in the AD-MSC administration group, and 19.678 ng / mL in the UC-MSC administration group. As a result of a significant difference test, an increase in MHPG concentration was confirmed in the AD-MSC administration group and the UC-MSC administration group compared to the PBS (−) administration group (p value <0.05).

`` Plasma-5-HIAA ''
As shown in FIG. 2 (c), 5-HIAA in the plasma sample was 12.746 ng / mL in the PBS (-) administration group, 22.450 ng / mL in the AD-MSC administration group, and 19.317 ng / mL in the UC-MSC administration group. As a result of a significant difference test, 5-HIAA concentration was confirmed to increase in the AD-MSC group and the UC-MSC group compared to the PBS (-) group (p value <0.05).

`` Cerebrospinal fluid-5-HIAA ''
As shown in FIG. 2 (d), 5-HIAA in the plasma sample was 4.633 ng / mL in the PBS (-) administration group, 9.265 ng / mL in the AD-MSC administration group, and 9.519 ng / mL in the UC-MSC administration group. As a result of a significant difference test, 5-HIAA concentration was confirmed to increase in the AD-MSC group and the UC-MSC group compared to the PBS (-) group (p value <0.05).

  These results indicate that systemic administration of adipose tissue-derived mesenchymal stem cells or umbilical cord tissue-derived mesenchymal stem cells increases MHPG and 5-HIAA in peripheral blood and cerebrospinal fluid. It has been found that mesenchymal stem cells can be utilized as a pharmaceutical composition having a monoamine production promoting action in E. coli.

  The pharmaceutical composition of the present invention increases the production of a neurotransmitter called a monoamine such as serotonin or noradrenaline in the body of a subject to which the composition is administered. Can be used. Thus, the present invention can be used in the pharmaceutical industry.

Claims (5)

  A pharmaceutical composition for promoting the production of monoamine, which is a neurotransmitter, or a metabolite of the monoamine, comprising a mesenchymal stem cell as an active ingredient.   The pharmaceutical composition according to claim 1, wherein the monoamine is serotonin or noradrenaline, and the metabolite of the monoamine is 5-hydroxyindoleacetic acid (5-HIAA) or 3-methoxy-4-hydroxyphenylethylene glycol. (MHPG).   The pharmaceutical composition according to claim 1 or 2, wherein the mesenchymal stem cell-derived tissue is an adipose tissue or an umbilical cord tissue.   4. The pharmaceutical composition according to claim 3, which is an analgesic.   The pharmaceutical composition according to claim 3, which is a muscle-building agent.

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