WO2019240126A1 - Pharmaceutical composition containing mesenchymal stem cells as monoamine production promoter - Google Patents

Pharmaceutical composition containing mesenchymal stem cells as monoamine production promoter Download PDF

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WO2019240126A1
WO2019240126A1 PCT/JP2019/023079 JP2019023079W WO2019240126A1 WO 2019240126 A1 WO2019240126 A1 WO 2019240126A1 JP 2019023079 W JP2019023079 W JP 2019023079W WO 2019240126 A1 WO2019240126 A1 WO 2019240126A1
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pharmaceutical composition
monoamine
mesenchymal stem
cells
stem cells
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PCT/JP2019/023079
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French (fr)
Japanese (ja)
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漆畑　直樹
勝幸 隠岐
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株式会社バイオミメティクスシンパシーズ
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Priority to CN201980006018.3A priority Critical patent/CN111479576A/en
Publication of WO2019240126A1 publication Critical patent/WO2019240126A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/35—Fat tissue; Adipocytes; Stromal cells; Connective tissues
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48—Reproductive organs
    • A61K35/51—Umbilical cord; Umbilical cord blood; Umbilical stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/04—Centrally acting analgesics, e.g. opioids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • 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.
  • Japanese Patent Application Laid-Open No. 2008-61569 describes a method for maintaining mesenchymal stem cell differentiation using a stem cell growth medium containing serotonin and a p38 MAP kinase inhibitor.
  • 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.
  • Japanese 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.
  • a pharmaceutical composition containing mesenchymal stem cells as an active ingredient has the effect of promoting the production of monoamine neurotransmitters and their main metabolites in vivo such as blood and cerebrospinal fluid. Based on findings from examples.
  • the present invention relates to a pharmaceutical composition.
  • 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 a monoamine that 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 that are neurotransmitters are serotonin and / or noradrenaline.
  • monoamine metabolites are 5-hydroxyindoleacetic acid (5-HIAA) and 3-methoxy-4-hydroxyphenylethylene glycol (MHPG) or both.
  • 5-Hydroxyindoleacetic acid (5-HIAA) is the main metabolite of serotonin.
  • 3-methoxy-4-hydroxyphenylethylene glycol (MHPG) is the main 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 a biological function enhancer due to an increase in monoamine. Specific examples of use are analgesics and muscle potentiators.
  • the pharmaceutical composition of the present invention has the effect of increasing the amount of serotonin and noradrenaline, or their main metabolites. 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 a pharmaceutical composition.
  • 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 placenta, umbilical cord, umbilical cord blood, and various fetal cells 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 cell may be a human mesenchymal stem cell or a mesenchymal stem cell derived from a non-human animal such as a mouse, rat, cat, or dog.
  • a method for isolating mesenchymal stem cells from each tissue a known method can be adopted.
  • 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 the production of a monoamine that 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.
  • Examples of monoamine metabolites are 5-hydroxyindoleacetic acid (5-HIAA) and 3-methoxy-4-hydroxyphenylethylene glycol (MHPG) or both.
  • 5-Hydroxyindoleacetic acid (5-HIAA) is the main metabolite of serotonin.
  • 3-methoxy-4-hydroxyphenylethylene glycol (MHPG) is the main metabolite of noradrenaline.
  • the neurotransmitter called monoamine is a substance having one amino group in its structure, and is typified by serotonin having an indole skeleton, and dopamine, noradrenaline, and adrenaline having a catechol group.
  • dopamine is related to feelings of pleasure and joy
  • noradrenaline is related to feelings of anger and anxiety
  • serotonin has the function of controlling the functions of these monoamines.
  • the receptors for these monoamines that function as neurotransmitters are G protein-coupled receptors in addition to 5-HT3 type receptors which are ion channel types.
  • 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.
  • 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.
  • 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.
  • 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 a biological function enhancer 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 orally administered drug, a drink, a tablet, or 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.
  • 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.
  • the pharmaceutical composition of the present invention is an injection or drink, in addition to mesenchymal stem cells, water, physiological saline, solvent, culture supernatant, pH adjuster, antioxidant (vitamin C), trehalose, etc.
  • Various materials may be included.
  • 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 depending on the symptom and purpose of the patient or administration subject, or may be administered twice, or three or more times until the effect appears. Good.
  • the pharmaceutical composition of the present invention is a general-purpose drug, it is generally administered to a patient who is allogeneic with respect to the origin of mesenchymal stem cells contained as a main component.
  • the pharmaceutical composition of the present invention produced as a general-purpose product after being proliferated by culture is not precluded from being administered to patients (eg, monozygotic twins from which mesenchymal stem cells are collected). It does not prevent administration to humans from which mesenchymal stem cells were collected.
  • the density of human mesenchymal stem cells in the pharmaceutical composition of the present invention at the time of administration 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 cells administered to humans is usually in the range of 1 ⁇ 10 5 to 1 ⁇ 10 7 cells / kg body weight per administration, although it depends on the intended frequency of administration. However, it is not limited to this, and can be appropriately increased or decreased according to the degree of symptoms.
  • the present specification describes a mesenchymal stem cell, or a monoamine that is a neurotransmitter, or a monoamine in a subject's living body, including a step of administering a mesenchymal stem cell or a medicinal composition of the present invention containing a mesenchymal stem cell as an active ingredient. Also provided is a method of promoting the production of metabolites.
  • 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.
  • AD-MSC Adipose tissue-derived mesenchymal stem cells
  • P0 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.
  • the adipose tissue 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 ⁇ m 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.
  • serum-free medium Procul AD; Rohto Pharmaceutical Co., Ltd.
  • 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.
  • the umbilical cord tissue 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 ⁇ m 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.
  • CellBIND registered trademark
  • Corning Corning
  • 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 Marie) 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.
  • 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.
  • Plasma and cerebrospinal fluid stored after collection were analyzed quantitatively by HPLC-MS / MS.
  • the substances to be measured were 3-methoxy-4-hydroxyphenylethylene glycol (MHPG), the main metabolite of noradrenaline, and 5-hydroxyindoleacetic acid (5-HIAA), the main metabolite of serotonin.
  • MHPG 3-methoxy-4-hydroxyphenylethylene glycol
  • 5-HIAA 5-hydroxyindoleacetic acid
  • the sample was washed with ultrapure water (200 ⁇ L) and eluted with 2% formic acid (200 ⁇ L) diluted with methanol to obtain a measurement sample.
  • 2% formic acid 200 ⁇ L
  • methanol 2% formic acid
  • 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
  • the internal standard 5- HIAA-d5 was carried out from 197.0969 ⁇ 151.085-151.095
  • MHPG was carried out from 167.0703 ⁇ 135.043-135.045.
  • 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.
  • 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).
  • 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.
  • an increase in 5-HIAA 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).
  • 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.
  • an increase in 5-HIAA 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).
  • adipose tissue-derived mesenchymal stem cells or umbilical cord tissue-derived mesenchymal stem cells reveals that MHPG and 5-HIAA in peripheral blood and cerebrospinal fluid increase. It was found that mesenchymal stem cells can be used as a pharmaceutical composition having an effect of promoting monoamine production.
  • the pharmaceutical composition of the present invention increases the production of neurotransmitters called monoamines such as serotonin or noradrenaline in the body of an administered subject, so that it can be used as a psychiatric disorder therapeutic agent, analgesic agent, muscle strength enhancer, etc. Can be used.
  • the present invention can be used in the pharmaceutical industry.

Abstract

[Problem] To provide a pharmaceutical composition that promotes the production of monoamine neurotransmitters in a living body having blood and cerebral fluid. [Solution] This pharmaceutical composition containing mesenchymal stem cells as an effective component has a function of promoting the production of monoamine neurotransmitters and main metabolic products thereof in blood, cerebral fluid, etc. in a living body. Examples of the monoamine include serotonin and noradrenalin, and examples of the metabolic products of monoamine include 5-hydroxyindoleacetic acid (5-HIAA) and 3-methoxy-4-hydroxyphenylethylene glycol (MHPG).

Description

モノアミン産生増加剤としての間葉系幹細胞を含む医薬組成物Pharmaceutical composition comprising mesenchymal stem cells as monoamine production increasing agent
本発明は，セロトニン，ノルアドレナリン，それらの主要代謝産物のいずれかもしくは複数の濃度を，生体内で上昇させるための医薬組成物に関する。 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.
　特開2008-61569号公報には，セロトニン及びｐ３８ＭＡＰキナーゼ阻害剤を含む幹細胞増殖培地により間葉系幹細胞分化維持をはかる方法が記載されている。この公報では，セロトニンは，培地に添加される組成のひとつとして記載されている。 Japanese Patent Application Laid-Open No. 2008-61569 describes a method for maintaining mesenchymal stem cell differentiation 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.
　特許第5541845号公報には，間葉系幹細胞を含むアトピー性皮膚炎の治療剤が記載されている。 Japanese Patent No. 5541845 describes a therapeutic agent for atopic dermatitis containing mesenchymal stem cells.
　特許第6191694号公報には，間葉系幹細胞を含む軟骨損傷の治療剤が記載されている。 Japanese Patent No. 6191694 describes a therapeutic agent for cartilage damage including mesenchymal stem cells.
特開2008-61569号公報JP 2008-61569 A 特許第5541845号公報Japanese Patent No. 5541845 特許第5541845号公報Japanese Patent No. 5541845
　本発明は，血液および脳脊髄液を含む生体において，モノアミン系神経伝達物質の産生を促進する医薬組成物を提供することを目的とする。この組成物は，例えば，セロトニン，ノルアドレナリン，またはそれらの主要代謝産物のうちいずれかまたは複数の量を増加させる作用を有する。 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.
　上記の課題は，間葉系幹細胞を有効成分として含む医薬組成物が，血液および脳脊髄液などの生体内における，モノアミン系神経伝達物質やその主要代謝産物の産生を促進する作用を有するという実施例による知見に基づく。 The above problem is that a pharmaceutical composition containing mesenchymal stem cells as an active ingredient has the effect of promoting the production of monoamine neurotransmitters and their main metabolites in vivo such as blood and cerebrospinal fluid. Based on findings from examples.
　本発明は，医薬組成物に関する。これを本発明の医薬組成物とよぶ。この医薬組成物は，医薬又は薬剤として機能する。この医薬組成物は，間葉系幹細胞を有効成分として含む。間葉系幹細胞の由来組織の例は，脂肪組織又は臍帯組織である。この医薬組成物は，治療や予防を含めた各種の目的（例えば筋肉増強，精神安定，鎮痛）を達成するために必要とされる有効量の間葉系幹細胞を含んでもよい。 The present invention relates to a pharmaceutical composition. 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).
　本発明の医薬組成物は，神経伝達物質であるモノアミン，又はそのモノアミンの代謝産物の産生を促進するための医薬組成物である。この医薬組成物は，モノアミンが低下することによる諸症状や疾患の治療剤として利用できるほか，モノアミンが増加することによる生体機能の亢進剤としても利用できる。 The pharmaceutical composition of the present invention is a pharmaceutical composition for promoting the production of a monoamine that 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.
　神経伝達物質であるモノアミンの例は，セロトニン及びノルアドレナリンのいずれか又は両方である。そして，モノアミンの代謝産物の例は，５－ハイドロキシインドール酢酸（５－ＨＩＡＡ）及び３－メトキシ－４－ハイドロキシフェニルエチレングリコール（MHPG）のいずれか又は両方である。５－ハイドロキシインドール酢酸（５－ＨＩＡＡ）は，セロトニンの主要代謝産物である。一方，３－メトキシ－４－ハイドロキシフェニルエチレングリコール（MHPG）は，ノルアドレナリンの主要代謝産物である。 Examples of monoamines that are neurotransmitters are serotonin and / or noradrenaline. Examples of monoamine metabolites are 5-hydroxyindoleacetic acid (5-HIAA) and 3-methoxy-4-hydroxyphenylethylene glycol (MHPG) or both. 5-Hydroxyindoleacetic acid (5-HIAA) is the main metabolite of serotonin. On the other hand, 3-methoxy-4-hydroxyphenylethylene glycol (MHPG) is the main metabolite of noradrenaline.
　この医薬組成物は，モノアミンが低下することによる諸症状や疾患の治療剤として利用できるほか，モノアミンが増加することによる生体機能の亢進剤としても利用できる。具体的な用途例は，鎮痛剤及び筋肉増強剤である。 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 a biological function enhancer due to an increase in monoamine. Specific examples of use are analgesics and muscle potentiators.
　本発明の医薬組成物は，セロトニンおよびノルアドレナリン，またはそれらの主要代謝産物の量を増加させる作用を有する。このため，この医薬組成物は，モノアミン低下による諸症状や疾患の治療剤として，または，モノアミン増加による生体機能の亢進剤として，有用である。 The pharmaceutical composition of the present invention has the effect of increasing the amount of serotonin and noradrenaline, or their main metabolites. 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.
図１は，モノアミン測定の概略を示す概念図である。FIG. 1 is a conceptual diagram showing an outline of monoamine measurement. 図２は，モノアミン定量結果を示す図面に代わるグラフである。FIG. 2 is a graph instead of a drawing showing the monoamine determination results.
　本発明は，医薬組成物に関する。これを本発明の医薬組成物ともよぶ。この医薬組成物は，医薬，薬剤として機能する。この医薬組成物は，間葉系幹細胞を有効成分として含む。この医薬組成物は，治療や予防を含めた各種の目的（例えば筋肉増強，精神安定，鎮痛）を達成するために求められる有効量の間葉系幹細胞を含んでもよい。 The present invention relates to a pharmaceutical composition. 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).
　間葉系幹細胞には，滑膜細胞，脂肪細胞，骨髄，歯髄，及び歯根膜等の成人の組織からだけでなく，胎盤，臍帯，臍帯血，及び胎児の種々の細胞等から単離されるものも含まれる。増殖工程において増殖させる間葉系幹細胞は，滑膜，臍帯，臍帯血，羊膜，骨髄，及び，脂肪組織からなる群より選択される組織由来であってもよい。間葉系幹細胞の由来組織が，脂肪組織又は臍帯組織であることが好ましい。 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 placenta, umbilical cord, umbilical cord blood, and various fetal cells 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.
間葉系幹細胞は，医薬組成物を投与する患者の自家細胞であることが好ましいが，同種細胞であってもよい。また，間葉系幹細胞は，ヒト間葉系幹細胞であってもよいし，マウス，ラット，ネコ，イヌ等の非ヒト動物由来間葉系幹細胞であってもよい。各組織から間葉系幹細胞を単離する方法は，公知の方法を採用することが可能であり，例えば，コラゲナーゼ法によって組織から間葉系幹細胞を好適に分離することができる。 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 cell may be a human mesenchymal stem cell or a mesenchymal stem cell derived from a non-human animal such as a mouse, rat, cat, or dog. 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.
　本発明の医薬組成物は，神経伝達物質であるモノアミン，又は前記モノアミンの代謝産物の産生を促進するための医薬組成物である。この医薬組成物は，モノアミンが低下することによる諸症状や疾患の治療剤として利用できるほか，モノアミンが増加することによる生体機能の亢進剤としても利用できる。 The pharmaceutical composition of the present invention is a pharmaceutical composition for promoting the production of a monoamine that 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.
　モノアミンの例は，セロトニン及びノルアドレナリンのいずれか又は両方である。そして，モノアミンの代謝産物の例は，５－ハイドロキシインドール酢酸（５－ＨＩＡＡ）及び３－メトキシ－４－ハイドロキシフェニルエチレングリコール（MHPG）のいずれか又は両方である。５－ハイドロキシインドール酢酸（５－ＨＩＡＡ）は，セロトニンの主要代謝産物である。一方，３－メトキシ－４－ハイドロキシフェニルエチレングリコール（MHPG）は，ノルアドレナリンの主要代謝産物である。 Examples of monoamines are serotonin and / or noradrenaline. Examples of monoamine metabolites are 5-hydroxyindoleacetic acid (5-HIAA) and 3-methoxy-4-hydroxyphenylethylene glycol (MHPG) or both. 5-Hydroxyindoleacetic acid (5-HIAA) is the main metabolite of serotonin. On the other hand, 3-methoxy-4-hydroxyphenylethylene glycol (MHPG) is the main metabolite of noradrenaline.
　モノアミンと称される神経伝達物質は，構造にアミノ基を１つ有する物質であり，インドール骨格を有するセロトニン，そしてカテコール基を有するドーパミン，ノルアドレナリン，アドレナリンに代表される。一般的に，ドーパミンは快楽や喜びの感情に関連し，ノルアドレナリンは怒りや不安の感情に関連し，セロトニンはこれらのモノアミンの機能を制御する機能があるとされる。神経伝達物質として機能するこれらのモノアミンの受容体は，イオンチャネル型である５－ＨＴ３型受容体の他は，Gタンパク質共役型受容体である。 The neurotransmitter called monoamine is a substance having one amino group in its structure, and is typified 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. The receptors for these monoamines that function as neurotransmitters are G protein-coupled receptors in addition to 5-HT3 type receptors which are ion channel types.
　セロトニンは，必須アミノ酸のトリプトファンから合成され，消化管粘膜，血小板，及び脳にそれぞれ，90%，8%，2%程度存在する。腸においては，内分泌細胞の一種である，腸クロム親和性細胞がオータコイドとして分泌し，腸の蠕動運動を促進するため，腸におけるセロトニンの分泌低下は，便秘につながる。消化管で分泌されたセロトニンの一部は血小板に取り込まれる。一方で，脳内でセロトニンを合成するセロトニン神経の細胞体は，脳幹の縫線核に集中するが，その神経線維は中枢神経系に広く投射される。そのため，中枢神経系でセロトニンに制御される機能としては，体温調節，情動，認知機能，覚醒と睡眠，摂食行動など，多岐に渡る。 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.
セロトニンをターゲットとした医薬品としては，選択的セロトニン再取込阻害薬（SSRI）や，セロトニン・ノルアドレナリン再取込阻害薬（SNRI）が存在する。これらは，セロトニントランスポーターを阻害することにより，シナプス間隙のセロトニン濃度を上昇させる抗鬱薬として使用される。鬱病患者の約３８％においては，セロトニン濃度の低下が認められ，これが鬱病の原因の１つとされている。これをモノアミン仮説という。 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.
　本発明の医薬組成物は，モノアミンが低下することによる諸症状や疾患の治療剤として利用できるほか，モノアミンが増加することによる生体機能の亢進剤としても利用できる。具体的な用途例は，鎮痛剤及び筋肉増強剤である。セロトニンは，覚醒作用，鎮痛作用，抗うつ作用などがあり，慢性疲労症候群や疼痛，抑うつなどに対して，本医薬組成物の有用性が考えられる。ノルアドレナリンは，心身の覚醒や興奮，交感神経を活性化する作用，昇圧作用，記憶を固定することに重要な，海馬におけるシナプス長期増強の正の制御，判断力や集中力の上昇，心筋の興奮性亢進，鎮痛作用，筋力増強作用などがあるため，疼痛，恐怖，不安，抑うつ，心不全，判断力低下，記憶力低下，筋力低下，副交感神経亢進などに対して，本医薬組成物の有用性が考えられる。 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 a biological function enhancer 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.
  本発明の医薬組成物は，経口投与剤，ドリンク剤，錠剤，又は注射剤として投与できる。注射剤は，静脈内，筋肉内または皮下等に有効成分である医薬組成物を投与することができ，これらのうち静脈内投与がより好ましい。静脈内投与を行う場合，注射剤は，注射器から直接患者に投与してもよく，また点滴バッグ中で点滴液に先ず添加して患者に点滴静注を行ってもよい。 医 薬 The pharmaceutical composition of the present invention can be administered as an orally administered drug, 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.
  本発明の医薬組成物が注射剤又はドリンク剤の場合，間葉系幹細胞の他に，水，生理食塩水，溶媒，培養上清，ｐＨ調整剤，酸化防止剤（ビタミンＣ），及びトレハロースといった各種素材を含んでもよい。本発明の医薬組成物が錠剤の場合は，賦形剤，担体といった各種素材を含んでもよい。 When the pharmaceutical composition of the present invention is an injection or drink, in addition to mesenchymal stem cells, water, physiological saline, solvent, culture supernatant, pH adjuster, antioxidant (vitamin C), trehalose, etc. 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.
  本発明の医薬組成物の投与方式は，患者や投与対象の症状や目的の程度に応じて，単回投与でもよく，効果が現れるまで２回，又は３回以上の複数回投与を行ってもよい。 The administration method of the pharmaceutical composition of the present invention may be a single administration depending on the symptom and purpose of the patient or administration subject, or may be administered twice, or three or more times until the effect appears. Good.
  本発明の医薬組成物は，汎用の薬剤であることから，主成分として含有される間葉系幹細胞の起源に対して同種（allogeneic）である患者に投与されるのが一般であるが，同系である患者（例えば，間葉系幹細胞の採取源の一卵性双生児）に投与することを妨げるものではなく，また，培養による増殖を経て汎用のものとして製造された本発明の医薬組成物が，その間葉系幹細胞の採取源となったヒトに対して投与されることを妨げるものでもない。 Since the pharmaceutical composition of the present invention is a general-purpose drug, it is generally administered to a patient who is allogeneic with respect to the origin of mesenchymal stem cells contained as a main component. The pharmaceutical composition of the present invention produced as a general-purpose product after being proliferated by culture is not precluded from being administered to patients (eg, monozygotic twins from which mesenchymal stem cells are collected). It does not prevent administration to humans from which mesenchymal stem cells were collected.
  上記投与時における，本発明の医薬組成物中のヒト間葉系幹細胞の密度は１×１０2～１×１０9個／ｍＬとすることが好ましく，2×１０6～2×１０7個／ｍＬとすることが更に好ましい。また，ヒトに対する同細胞の投与個数は，意図する投与回数にもよるが，通常，１回の投与につき１×１０5～１×１０7個／ｋｇ体重の範囲であることが好ましい。但しこれに限られることなく，症状の程度に応じて適宜増減することができる。 The density of human mesenchymal stem cells in the pharmaceutical composition of the present invention at the time of administration 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 cells administered to humans is usually in the range of 1 × 10 5 to 1 × 10 7 cells / kg body weight per administration, although it depends on the intended frequency of administration. However, it is not limited to this, and can be appropriately increased or decreased according to the degree of symptoms.
　本明細書は，間葉系幹細胞，又は間葉系幹細胞を有効成分として含む本発明の医薬組成物を対象に投与する工程を含む，対象の生体内において，神経伝達物質であるモノアミン，又はモノアミンの代謝産物の産生を促進する方法をも提供する。神経伝達物質であるモノアミン，又はモノアミンの代謝産物の産生が促進されたことは，例えば，医薬組成物を対象に投与する前後の対象の血液から分離した血漿，または脳脊髄液から，モノアミン，又はモノアミンの代謝産物の含有量（血中濃度）を測定して，比較することで確認すればよい。 The present specification describes a mesenchymal stem cell, or a monoamine that is a neurotransmitter, or a monoamine in a subject's living body, including a step of administering a mesenchymal stem cell or a medicinal composition of the present invention containing a mesenchymal stem cell as an active ingredient. Also provided is a method of promoting the production of metabolites. 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． 間葉系幹細胞移植によるラット生体中モノアミン濃度定量試験
１－１．間葉系幹細胞の調製：脂肪組織由来間葉系幹細胞（AD-MSC）
（１）初代培養（P0）
脂肪組織由来間葉系幹細胞を用いた再生医療を受ける患者より，投与用細胞の調製に必要な原料となる皮下脂肪組織を分取した後の余剰組織について，研究用利用用途の同意取得の後に皮下脂肪の提供を受け，初代培養に供した。皮下脂肪組織は遠心分離（４００×gで５分間）に供し，上から順に脂質画分，脂肪組織画分，および水性画分の３層に分離した。中層の脂肪組織画分を残して，上層と下層を破棄した。残した脂肪組織画分に対して，組織重量当たり４倍量の０．１５％コラゲナーゼ酵素溶液を添加し，３７℃で１時間浸透させ，酵素処理を行った。脂肪組織が分散された後, 遠心分離（４００×gで５分間）に供し，間葉系幹細胞を含む間質血管細胞画分として，沈殿画分を30 mLのPBS（-）溶液で懸濁した。その後，セルストレーナー（７０μm径）に懸濁液を通液し，通液画分を再度遠心分離（４００×gで５分間）に供し，セルストレーナーに捕捉された組織残渣等は破棄した。沈殿画分を６ mLの無血清培養液（Procul AD; ロート製薬）で懸濁し，T-25フラスコ（CellBIND（登録商標）; Corning）に全量を播種し，インキュベーター内（３７℃,５％ CO2）に静置して初代培養を開始した。 1. 1. Monoamine concentration quantitative test 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）継代培養（P0→P1→P2→P3）
　３日に１回の頻度で培地全交換を実施し，上澄みは破棄して，フラスコ底面上で増殖する細胞を選択的に増殖した。セミコンフルエントまで増殖したT-25フラスコの細胞に対して，2 mLの酵素溶液（TrypLE Secelt（登録商標）；Thermo Fisher Scientific）を添加し剥離した（３７℃，５分間静置）。細胞をPBS（-）で希釈し，遠心分離（４００×gで５分間）に供した。沈殿した細胞を培養液で懸濁し，トリパンブルー染色法による細胞数計測を行った結果，1.36×106個の生細胞が回収できたため，その全量をT-150フラスコ（CellBIND（登録商標）; Corning）２枚に等分して無血清培養液（Procul AD; ロート製薬）で全量を播種し，インキュベーター内（３７℃,５％ CO2）に静置して継代培養を行った（P0→P1）。
　３日に１回の頻度で培地全交換を実施し，上澄みは破棄して，フラスコ底面上で増殖する細胞を選択的に増殖した。播種から3日目にセミコンフルエントまで増殖した細胞に対して，6 mLの酵素溶液（TrypLE Select（登録商標）；Thermo Fisher Scientific）を添加して剥離した（３７℃，５分間静置）。細胞をPBS（-）で希釈し，遠心分離（４００×gで５分間）に供した。沈殿した細胞を無血清培養液（Procul AD; ロート製薬）で懸濁し，トリパンブルー染色法による細胞数計測を行った結果，1.17×107個の生細胞を回収できたため，その一部を3.9×103/cm2細胞濃度でT-150フラスコ８枚に播種を行い継代した（P1→P2）。播種から3日目にセミコンフルエントまで増殖したことを確認後，同様の手順で細胞の回収を行った。その結果，生存率が95.2％であり，生細胞数を5.27×107個回収したことを確認し，凍結保存液（STEMCELL BANKER;日本全薬工業）に懸濁してラットへの投与日まで液体窒素タンクで保管した。 (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．間葉系幹細胞の調製：臍帯組織由来間葉系幹細胞（UC-MSC）
（１）初代培養（P0）
通常分娩を行う産婦より同意を得て取得した臍帯組織を，採取の翌日に初代培養に供した。約10 cmの臍帯組織から臍帯血を除去した後，医療用メスで細断し，0.15 % Collagenase溶液に浸して37℃で16時間，シェーカーで緩やかに攪拌させながら酵素処理を行った。臍帯組織の分散を目視で確認した後，PBS(-)で10倍希釈を行った後，1,000×g, 5分の遠心分離に供し，沈殿画分を残して上層を破棄した。その後，沈殿画分をPBS(-)で懸濁した後，70μmのフィルターに供し，通液画分をコニカルチューブ２本に等分した後，400×g, 5分の遠心分離に供し，細胞を含む沈殿画分を，24 mLの無血清培養液（Procul AD; ロート製薬）培地で懸濁した。その後，T-150フラスコ（CellBIND（登録商標）; Corning）1枚に全量を播種し，インキュベーター内（３７℃,５％ CO2）に静置して初代培養を開始した。 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）継代培養（P0→P1→P2）
　2日に１回の頻度で培地全交換を実施し，上澄みは破棄して，フラスコ底面上で増殖する細胞を選択的に増殖した。セミコンフルエントまで増殖したT-150フラスコの細胞に対して，6mLの酵素溶液（TrypLE Select（登録商標）；Thermo Fisher Scientific）を添加し剥離した（３７℃，５分間静置）。細胞をPBS（-）で希釈し，遠心分離（４００×gで５分間）に供した。沈殿した細胞を培養液で懸濁し，トリパンブルー染色法による細胞数計測を行った結果，4.33×106個の生細胞が回収できたため，その全量をT-150フラスコ（CellBIND（登録商標）; Corning）5枚に等分して無血清培養液（Procul AD; ロート製薬）で全量を播種し，インキュベーター内（３７℃,５％ CO2）に静置して継代培養を行った（P0→P1）。2日に１回の頻度で培地全交換を実施し，播種から４日目にセミコンフルエントまで増殖したことを確認後，同様の手順で細胞の回収を行った。
P2において，2.83×107個の生細胞を回収できたため，その一部を6.7×103/cm2細胞濃度でT-150フラスコ10枚に播種を行い継代した（P1→P2）。2日に１回の頻度で培地全交換を実施し，播種から４日目にセミコンフルエントまで増殖したことを確認後，同様の手順で細胞の回収を行った。その結果，生存率が90.5％であり，生細胞数を4.43×107個回収したことを確認し，凍結保存液（STEMCELL BANKER;日本全薬工業）に懸濁して，ラットへの投与日まで液体窒素タンクで保管した。 (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．　投与する細胞懸濁液の調製
ラットへの細胞投与日に，液体窒素タンクに保管された脂肪組織由来間葉系幹細胞，及び臍帯由来間葉系幹細胞を室温で解凍し，解凍後速やかにPBS（-）で希釈した後，400×g, 5分の遠心分離に供した。細胞を含む沈殿画分をPBS(-)で懸濁し，トリパンブルー染色法による計測を行ったところ，脂肪組織由来間葉系幹細胞及び臍帯由来間葉系幹細胞の生存率がそれぞれ91.5%と87.1％であった。これにより，投与に用いる細胞として十分な生存率を維持していることを確認した。次に，再度400×g, 5分の遠心分離に供し，上澄みを破棄して細胞を含む沈殿画分をPBS(-)で懸濁し，ラットへの投与に用いる細胞懸濁液の細胞濃度が5×106 cells/mLに調整した。 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．　ラットへの投与と測定検体の採取
　（１）投与　
SDラット（雄性，４週齢，体重180 g前後；日本クレア）に対して，脂肪組織由来間葉系幹細胞，及び臍帯由来間葉系幹細胞の懸濁液を，尾静脈より１匹あたり5×106 cells（1mL）の割合で単回投与した。投与には3０Gの注射針を使用した。また，コントロールとして，PBS(-)投与群も設定し，細胞と同様に尾静脈より1mLを単回投与した。なお，脂肪組織由来間葉系幹細胞投与群，臍帯由来間葉系幹細胞投与群，及びPBS(-)投与群はそれぞれラットの数を６匹と設定した（n=6／群）。細胞またはPBS(-)の投与直後から投与１時間後までの観察において，ラットの運動機能の低下や呼吸困難，振戦等の症状の他，一般状態に異常は確認されなかった。そのため，投与量として安全な範囲で投与されたことを確認した。 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. In the adipose tissue-derived mesenchymal stem cell administration group, the umbilical cord-derived mesenchymal stem cell administration group, and the PBS (−) administration group, the number of rats was set to 6 (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）血漿の調整　
細胞またはPBS(-)の投与から７日後に，各実験群３匹ずつから約3mLずつ採血を行い，末梢血に10μg／mLのヘパリンを添加して緩やかに混和した後，1,200×g/10分間の遠心分離により血漿画分を得た後，速やかに－８０℃で保管した。 (2) Plasma adjustment
Seven days after administration of cells or PBS (-), about 3 mL of blood was collected from 3 animals in each experimental group, and 10 μg / mL heparin was added to the peripheral blood and mixed gently, and then 1,200 × g / 10 Plasma fractions were obtained by centrifugation for 1 minute and then immediately stored at -80 ° C.
（３）脳脊髄液の調整
細胞またはPBS(-)の投与から７日後に，小脳延髄槽に穿刺を行うことにより，各実験群３匹ずつから脳脊髄液を約70μLずつ採取した。採取後，速やかに－８０℃で保管した。 (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 (−). Immediately after collection, it was stored at -80 ° C.
１－５．　モノアミン定量
　採取後に保管した血漿及び脳脊髄液について，HPLC-MS/MSによるモノアミンの定量解析を行った。測定対象物質は，ノルアドレナリンの主要代謝産物の3-メトキシ-4-ハイドロキシフェニルエチレングリコール（MHPG）と，セロトニンの主要代謝産物である５－ハイドロキシインドール酢酸（5-HIAA）とした。これらを測定することにより，その前駆物質であるノルアドレナリンとセロトニンの量も把握することができるため，臨床検査にもMHPGと５－HIAAは活用される物質である。 1-5. Monoamine determination Plasma and cerebrospinal fluid stored after collection were analyzed quantitatively by HPLC-MS / MS. The substances to be measured were 3-methoxy-4-hydroxyphenylethylene glycol (MHPG), the main metabolite of noradrenaline, and 5-hydroxyindoleacetic acid (5-HIAA), the main metabolite of serotonin. By measuring these, the amount of precursors noradrenaline and serotonin can be ascertained, so MHPG and 5-HIAA are also used in clinical tests.
（１）前処理
サンプルの前処理として，血漿サンプルの場合，各検体200μLに，0.4mol/L過塩素酸を200μL加え，攪拌後，遠心分離（17,500×g，4℃，5min）に供し，上澄み画分を固相抽出に供した（Oasis HLB uElution 96-wells Plate 30um；Waters）。固相抽出の手順として，メタノール（200μL），超純水（200μL），検体（200μL），5-HIAA測定時には内部標準物質として5-HIAA-d5（10μL）の順にカラムに供した。次に超純水（200μL）で洗浄し，メタノールで希釈した2% ギ酸（200μL）で溶出し，測定サンプルとした。脳脊髄液サンプルの場合，0.4mol/L過塩素酸を７０μLとして，それ以降は血漿サンプルと同様とした。 (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）定量解析
測定にはUltimate 3000 Rapid Separation with Q Exactiveシステム（Thermo Fisher Scientific）を用い，カラムはScherzo SS C18，100×3.0mm，3um（Imtakt）を用いた。液体クロマトグラフィーの測定条件として，カラム温度は30℃，グラジエントを掛けるための移動相Aとしてメタノール/水/ギ酸=35/65/0.5を，移動相Bとしてメタノール/100mM ギ酸アンモニウム=40/60を，グラジエントは表１の通りとし，測定サンプルのインジェクト量は５μLとした。質量分析は，イオン化モードはポジティブモード，分解能は17,500，キャピラリー温度は263℃，スプレー電圧は2.5V，定量における質量レンジは，5-HIAAは192.0655→146.055-146.065にて，内部標準物質の5-HIAA-d5は197.0969→151.085-151.095にて，MHPGは167.0703→135.043-135.045にて実施した。 (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.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　（３）定量結果
MHPGと5-HIAA定量の結果を図２に示す。
「血漿－MHPG」
図２（ａ）に示される通り，血漿サンプルにおけるMHPGは，コントロールのPBS(-)投与群が4.081 ng/mL，AD-MSC投与群が7.125 ng/mL，UC-MSC投与群が6.391 ng/mLであり，有意差検定の結果，PBS(-)投与群に対してAD-MSC投与群及びUC-MSC投与群は，MHPG濃度の上昇が確認された（p値＜0.05）。 (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).
　「脳脊髄液－MHPG」
図２（ｂ）に示される通り，血漿サンプルにおけるMHPGは，コントロールのPBS(-)投与群が7.352 ng/mL，AD-MSC投与群が11.602 ng/mL，UC-MSC投与群が19.678 ng/mLであり，有意差検定の結果，PBS(-)投与群に対してAD-MSC投与群及びUC-MSC投与群は，MHPG濃度の上昇が確認された（p値＜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).
　「血漿－5-HIAA」
図２（c）に示される通り，血漿サンプルにおける５－ＨＩＡＡは，PBS(-)投与群が12.746 ng/mL，AD-MSC投与群が22.450 ng/mL，UC-MSC投与群が19.317 ng/mLであり，有意差検定の結果，PBS(-)投与群に対してAD-MSC投与群及びUC-MSC投与群は，５－ＨＩＡＡ濃度の上昇が確認された（p値＜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, an increase in 5-HIAA 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).
　「脳脊髄液－5-HIAA」
図２（ｄ）に示される通り，血漿サンプルにおける５－ＨＩＡＡは，PBS(-)投与群が4.633 ng/mL，AD-MSC投与群が9.265 ng/mL，UC-MSC投与群が9.519 ng/mLであり，有意差検定の結果，PBS(-)投与群に対してAD-MSC投与群及びUC-MSC投与群は，５－ＨＩＡＡ濃度の上昇が確認された（p値＜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, an increase in 5-HIAA 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).
　以上の結果の通り，脂肪組織由来間葉系幹細胞または臍帯組織由来間葉系幹細胞の全身性投与により，末梢血中及び脳脊髄液中のMHPG及び5－HIAAが増加することが明らかとなり，生体におけるモノアミン産生促進作用を有する医薬組成物として，間葉系幹細胞を活用できることを見出した。 As shown above, systemic administration of adipose tissue-derived mesenchymal stem cells or umbilical cord tissue-derived mesenchymal stem cells reveals that MHPG and 5-HIAA in peripheral blood and cerebrospinal fluid increase. It was found that mesenchymal stem cells can be used as a pharmaceutical composition having an effect of promoting monoamine production.
　本発明の医薬組成物は，投与された対象の体内において，セロトニン又はノルアドレナリンといったモノアミンと称される神経伝達物質の産生量を増加させるため，精神疾患の治療剤，鎮痛剤，筋力増強剤などとして利用されうる。このため，本発明は，医薬産業において利用されうる。
  The pharmaceutical composition of the present invention increases the production of neurotransmitters called monoamines such as serotonin or noradrenaline in the body of an administered subject, so that it can be used as a psychiatric disorder therapeutic agent, analgesic agent, muscle strength enhancer, etc. Can be used. Thus, the present invention can be used in the pharmaceutical industry.

Claims (5)

  1. 　間葉系幹細胞を有効成分として含む，神経伝達物質であるモノアミン，又は前記モノアミンの代謝産物の産生を促進するための医薬組成物。 A pharmaceutical composition for promoting the production of a monoamine, which is a neurotransmitter, or a metabolite of the monoamine, comprising mesenchymal stem cells as an active ingredient.
  2. 　請求項１に記載の医薬組成物であって，前記モノアミンが，セロトニン又はノルアドレナリンであり，前記モノアミンの代謝産物が５－ハイドロキシインドール酢酸（５－ＨＩＡＡ）又は３－メトキシ－４－ハイドロキシフェニルエチレングリコール（MHPG）である，医薬組成物。 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. A pharmaceutical composition which is (MHPG).
  3. 　請求項１又は２に記載の医薬組成物であって，前記間葉系幹細胞の由来組織が，脂肪組織又は臍帯組織である，医薬組成物。 The pharmaceutical composition according to claim 1 or 2, wherein the mesenchymal stem cell-derived tissue is adipose tissue or umbilical cord tissue.
  4. 　請求項３に記載の医薬組成物であって，鎮痛剤である，医薬組成物。 The pharmaceutical composition according to claim 3, which is an analgesic.
  5. 　請求項３に記載の医薬組成物であって，筋肉増強剤である，医薬組成物。 The pharmaceutical composition according to claim 3, which is a muscle strengthening agent.
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