WO2016031264A1 - Skeletal muscle bulking agent and use thereof - Google Patents

Skeletal muscle bulking agent and use thereof Download PDF

Info

Publication number
WO2016031264A1
WO2016031264A1 PCT/JP2015/053580 JP2015053580W WO2016031264A1 WO 2016031264 A1 WO2016031264 A1 WO 2016031264A1 JP 2015053580 W JP2015053580 W JP 2015053580W WO 2016031264 A1 WO2016031264 A1 WO 2016031264A1
Authority
WO
WIPO (PCT)
Prior art keywords
meclizine
congenital
syndrome
muscle
skeletal muscle
Prior art date
Application number
PCT/JP2015/053580
Other languages
French (fr)
Japanese (ja)
Inventor
欽司 大野
石黒　直樹
哲朗 飛田
松下　雅樹
Original Assignee
国立大学法人名古屋大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 国立大学法人名古屋大学 filed Critical 国立大学法人名古屋大学
Publication of WO2016031264A1 publication Critical patent/WO2016031264A1/en

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines

Definitions

  • the present invention relates to a skeletal muscle bulking agent and use thereof.
  • the skeletal muscle bulking agent of the present invention is used, for example, for the treatment of sarcopenia.
  • Muscle loss such as sarcopenia
  • Muscle loss that causes a decrease in skeletal muscle mass is considered to be one of the causes of reduced mobility, falling fractures, and vulnerability in the elderly.
  • the treatment is seen as a trump card for maintaining a healthy life expectancy.
  • There is no fundamental cure for sarcopenia and exercise therapy, nutrition therapy, and pharmacotherapy have been attempted, but there are still no effective treatments due to adherence and side effects.
  • the causes of sarcopenia include various factors such as changes in age-related hormone balance, age-related changes in muscles and nerves, changes in disuse due to other diseases and social factors, and inadequate intake of amino acids and vitamins due to undernutrition. Predisposing factors are considered to be involved in multiple ways. Therefore, the fundamental treatment is difficult, and the development of a new treatment method is required.
  • skeletal muscle loss is the main cause or cause of various diseases in addition to sarcopenia and other muscle loss. Many of these diseases have not yet been established as appropriate treatments, and there is a need to provide new treatments.
  • a main problem of the present invention is to provide a novel treatment strategy for a disease whose main cause or part of the etiology is skeletal muscle loss.
  • the present inventors tried to find a low molecular weight compound effective for increasing the amount of skeletal muscle from among already approved drugs.
  • FDA-approved drug library Prestwick ⁇ Chemicals
  • 320 types that can be clinically applied in Japan and that can be administered for a long period of time are selected, and a sub-library is created to screen for effective compounds. did.
  • meclizine has a cell proliferation promoting effect and a differentiation inhibiting effect. It was. That is, meclizine was selected as a very promising compound.
  • the therapeutic agent according to [3], wherein the disease is sarcopenia.
  • the above-mentioned diseases include muscle damage due to trauma or surgery, disuse muscular atrophy, disuse syndrome, easy fall, neurogenic muscular atrophy, osteoporosis, osteoarthritis, osteoarthritis, scoliosis and posterior Spinal deformity such as mania, obesity, rheumatoid arthritis, dermatomyositis, polymyositis, myositis associated with autoimmune diseases, locomotive syndrome, metabolic syndrome, motor organ instability, dynapenia, flail, rhabdomyolysis, It is a disease selected from the group consisting of cachexia due to muscular dystrophy, congenital myopathy, congenital myasthenia syndrome, congenital hypoplasia, congenital metabolic disorders, impaired glucose tolerance or diabetes or cancer, [3 ] The therapeutic agent as described in.
  • a method for treating bone system diseases comprising a step of administering to a patient with cachexia due to cancer or
  • a method for treating muscular dystrophy comprising the step of administering meclizine or a pharmaceutically acceptable salt thereof in a therapeutically effective amount to a patient with muscular dystrophy.
  • a physical training or physical exercise supplement containing a meclizine or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the present invention relates to a skeletal muscle bulking agent and use thereof.
  • the present invention is based on a result of finding a novel effect of meclizine, that is, an effect of increasing skeletal muscle mass.
  • meclizine promotes myoblast proliferation while reversibly inhibiting its differentiation is worthy of special mention.
  • the skeletal muscle bulking agent of the present invention contains meclizine or a pharmaceutically acceptable salt thereof as an active ingredient.
  • Meclizine is a compound of the substance name 1-[(4-chlorophenyl) phenylmethyl] -4-[(3-methylphenyl) methyl] piperazine and is known as a kind of antihistamine.
  • Meclizine is sold as an over-the-counter (OTC) as a motion sickness medicine.
  • OTC over-the-counter
  • Meclizine is sometimes referred to as meclozine.
  • meclizine and the term “meclozin” are used interchangeably.
  • a pharmacologically acceptable salt of meclizine may be used as the active ingredient of the skeletal muscle bulking agent of the present invention.
  • Commercial meclizine preparations are often formulated in the form of meclizine hydrochloride. Therefore, the hydrochloride is also preferably used in the present invention.
  • the “pharmacologically acceptable salt” is not limited to this, and various salts such as an acid addition salt and an amino acid addition salt are assumed.
  • acid addition salts include inorganic acid salts such as hydrochloride, sulfate, nitrate, phosphate, hydrobromide, acetate, maleate, fumarate, citrate, benzenesulfonate, Organic acid salts such as benzoate, malate, oxalate, methanesulfonate, and tartrate are listed.
  • amino acid addition salts include glycine addition salts, phenylalanine addition salts, lysine addition salts, aspartic acid addition salts, and glutamic acid addition salts.
  • Muscles are roughly classified into skeletal muscles, smooth muscles and myocardium. Skeletal muscle is composed of muscle cells (muscle fibers) and connective tissue. Skeletal muscles are also called voluntary muscles and are histologically striated muscles. In addition to the role of moving the body, skeletal muscle has various roles such as maintaining posture, stabilizing joints, generating heat, and protecting blood vessels and organs.
  • the skeletal muscle bulking agent of the present invention can be applied to the treatment or prevention of diseases whose main cause or part of the pathogenesis is skeletal muscle loss. That is, the present invention also provides a therapeutic agent for the disease (containing a skeletal muscle bulking agent).
  • “Therapeutic agent” refers to a drug that exhibits a therapeutic or prophylactic effect on a target disease or condition.
  • Therapeutic effects include alleviation of symptoms or concomitant symptoms characteristic of the target disease / pathology (lightening), prevention or delay of worsening of symptoms, and the like.
  • the latter can be regarded as one of the preventive effects in terms of preventing the seriousness.
  • the therapeutic effect and the preventive effect are partially overlapping concepts, and it is difficult to clearly distinguish them from each other, and there is little benefit in doing so.
  • a typical preventive effect is to prevent or delay the recurrence of symptoms characteristic of the target disease / pathology.
  • it shows some therapeutic effect or preventive effect with respect to a target disease / pathology, or both, it corresponds to the therapeutic agent with respect to a target disease / pathology.
  • a disease in which a decrease in skeletal muscle is the main cause or a part of the etiology is, for example, sarcopenia typified by sarcopenia.
  • Other spinal deformities such as muscle damage due to trauma and surgery, disuse muscle atrophy, disuse syndrome, easy fall, neurogenic muscle atrophy, osteoporosis, osteoarthritis, osteoarthritis, scoliosis and kyphosis , Obesity, rheumatoid arthritis, dermatomyositis, polymyositis, myositis associated with autoimmune disease, locomotive syndrome, metabolic syndrome, motor organ instability, dynapenia, flail, rhabdomyolysis, muscular dystrophy, congenital
  • the therapeutic agent of the present invention can also be applied to myopathy, congenital myasthenia syndrome, congenital hypoplasia, congenital metabolic disorders, impaired glucose tolerance or cachexia due to diabetes or cancer.
  • the preparation of the therapeutic agent of the present invention can be performed according to a conventional method.
  • other pharmaceutically acceptable ingredients for example, carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, preservatives, physiological Saline solution and the like.
  • excipient lactose, starch, sorbitol, D-mannitol, sucrose and the like can be used.
  • disintegrant starch, carboxymethylcellulose, calcium carbonate and the like can be used. Phosphate, citrate, acetate, etc. can be used as the buffer.
  • emulsifier gum arabic, sodium alginate, tragacanth and the like can be used.
  • suspending agent glyceryl monostearate, aluminum monostearate, methyl cellulose, carboxymethyl cellulose, hydroxymethyl cellulose, sodium lauryl sulfate and the like can be used.
  • soothing agent benzyl alcohol, chlorobutanol, sorbitol and the like can be used.
  • stabilizer propylene glycol, ascorbic acid or the like can be used.
  • preservatives phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, and the like can be used.
  • preservatives benzalkonium chloride, paraoxybenzoic acid, chlorobutanol and the like can be used.
  • the dosage form for formulation is not particularly limited. Examples of dosage forms are tablets, powders, fine granules, granules, capsules, syrups, injections, external preparations, and suppositories.
  • the therapeutic agent of the present invention is administered orally or parenterally (intravenous, intraarterial, subcutaneous, intradermal, intramuscular, or intraperitoneal injection, transdermal, nasal, transmucosal, etc.) depending on the dosage form.
  • Applies to Systemic and local administration are also indicated by the subject. These administration routes are not mutually exclusive, and two or more arbitrarily selected can be used in combination (for example, intravenous injection or the like is performed simultaneously with oral administration or after a predetermined time has elapsed).
  • the therapeutic agent of the present invention contains an active ingredient in an amount necessary for obtaining an expected therapeutic effect (that is, a therapeutically effective amount).
  • the amount of the active ingredient in the therapeutic agent of the present invention generally varies depending on the dosage form, but the amount of the active ingredient is set, for example, within the range of about 0.1 wt% to about 99 wt% so as to achieve a desired dose.
  • the dosage of the therapeutic agent of the present invention is set so as to obtain the expected therapeutic effect.
  • symptoms symptoms, patient age, sex, weight, etc. are generally considered.
  • a person skilled in the art can set an appropriate dose in consideration of these matters.
  • the dose can be set so that the amount of active ingredient per day is 1 mg to 500 mg, preferably 5 mg to 300 mg, particularly preferably 10 mg to 200 mg.
  • the administration schedule for example, once to several times a day, once every two days, or once every three days can be adopted. In preparing the administration schedule, the patient's medical condition and the duration of effect of the active ingredient can be taken into consideration.
  • treatment with another medicine for example, existing therapeutic agent
  • the existing therapeutic technique may be combined with the treatment with the therapeutic agent of the present invention.
  • the present application is intended for diseases mainly caused by skeletal muscle loss (muscle loss such as sarcopenia, muscle damage due to trauma or surgery, disuse muscle atrophy, disuse syndrome, Falls, neurogenic muscle atrophy, osteoporosis, osteoarthritis, osteoarthritis, spinal deformity such as scoliosis and kyphosis, obesity, rheumatoid arthritis, dermatomyositis, polymyositis, myositis associated with autoimmune diseases, Locomotive syndrome, metabolic syndrome, motor organ instability, dynapenia, flail, rhabdomyolysis, muscular dystrophy, congenital myopathy, congenital myasthenia syndrome, congenital hypoplasia, congenital metabolic disorders, There is also provided a therapeutic method characterized by administering a therapeutically effective amount of the therapeutic agent of the present invention to a patient with impaired glucose tolerance or cachexia due to diabetes or cancer.
  • skeletal muscle loss muscle loss
  • disuse muscle atrophy disuse muscle
  • the skeletal muscle bulking agent of the present invention can also be used as a supplementary nutrient for physical training / physical exercise for the purpose of increasing or strengthening muscles.
  • the “supplementary nutrient” used herein is used to promote the effects of physical training / physical exercise, and has a higher effect than when it is not used.
  • a supplementary nutritional supplement it can also be provided in the form of a food composition containing it in addition to the form (dosage form) similar to the therapeutic agent.
  • a food composition for example, it is provided in the form of powder, granule powder, tablet, paste, liquid or the like as a dietary supplement (supplement, nutrition drink, etc.).
  • the food composition of the present invention preferably contains an active ingredient in such an amount that a desired effect, that is, an increase in muscle can be expected.
  • the addition amount can be determined in consideration of the age, physique (height, weight, etc.), gender, etc. of the subject (user) in which it is used.
  • the skeletal muscle bulking agent of the present invention is also useful in the fields of animal husbandry, pets (animals) and aquaculture.
  • livestock cattle, pig, horse, sheep, etc.
  • poultry chicken, duck, goose, turkey, quail
  • Pheasants, etc. fish
  • fish una, red sea bream, yellowtail, etc.
  • the present invention can also be used as a meat volume increasing agent. It is also possible to use the skeletal muscle bulking agent of the present invention for maintaining the health and body shape of pets.
  • Tannic acid facilitates expression of the polypyrimidine tract binding protein and alleviates deleterious inclusion of CHRNA1 exon P3A due to an hnRNP H-disrupting mutation in congenital myasthenic syndrome. Hum. Mol. Genet., 18, 1229-1237.).
  • Hu5 / KD3 cell line (distributed by National Longevity Medical Research Center, Director Yasuhiro Hashimoto) is a cell line in which telomerase is continuously expressed in human myoblasts. Differentiate into myotubes with induction medium (Shiomi K et al. Gene Ther. 2011; 18: 857-866). Two types of media are used for culturing the Hu5 / KD3 cell line.
  • pmGM (20% fetal bovine serum (FBS, Thermo Scientific), 2% Ultroser G (Biosepra, PALL), penicillin G (100 u / ml) and streptomycin sulfate (100 ⁇ g / ml)
  • Penstrep Dulbecco's Modified Eagle's Medium
  • pmDM 2% horse serum, 1% insulin-transferrin-selenate (ITS, Invitrogen)
  • penicillin G 100 u / ml
  • streptomycin sulfate 100 ⁇ g / ml
  • the cultured cells were buffered (50 mM HEPES pH 7.0, 150 mM NaCl, 10% glycerol, 1% TritonX-100, 1.5 mM MgCl2, 1 mM EGTA, 100 mM NaF, 10 mM sodium pyrophosphate, 1 ⁇ g / ⁇ l aprotinin, 1 ⁇ g / ⁇ l leupeptin, 1 ⁇ g / ⁇ l pepstatin A, 1 mM PMSF, 1 mM sodium orthovanadate). All proteins were dissolved in 1 ⁇ laemmli buffer, separated by SDS-PAGE (10% or 7.5% gel), and transferred to a PVDF membrane (Immobilon-P, Millipore).
  • All proteins were dissolved in 1 ⁇ laemmli buffer, separated by SDS-PAGE (10% or 7.5% gel), and transferred to a PVDF membrane (Immobilon-P, Millipore).
  • the transferred membrane was washed with a Tris buffer containing 0.05% Tween 20 (TBS-T) and then blocked with TBS-T containing 3% bovine serum albumin (room temperature, 1 hour). Subsequently, the treated membrane was reacted with mouse anti-MYH monoclonal antibody (H-300, Santa Cruz Biotechnology, dilution 1: 200) or anti-GAPDH antibody (G9545, Sigma-Aldrich, dilution 1: 600) (4 ° C overnight. The membrane was washed three times and then reacted with a secondary antibody (HRP-labeled goat anti-rabbit IgG antibody (GE Healthcare, 1: 6000)) (room temperature, 1 hour). Detection was performed using Amersham ECL Western blotting detection reagents (GE Healthcare), and quantification was performed using the ImageJ program.
  • TBS-T Tris buffer containing 0.05% Tween 20
  • TBS-T Tris buffer containing 0.05% bovine serum albumin
  • the BrdU assay is a method for quantifying cell division and is used to evaluate cell proliferation in the same manner as the MTS assay. Also in the BrdU assay, meclizine showed a cell growth promoting action (FIG. 3).
  • the morphology of cells on differentiation induction day 7 was compared with and without meclizine (control) (FIG. 4).
  • control meclizine
  • formation of myotube myotube
  • myotubes are not observed in cells administered with meclizine.
  • Differentiation is suppressed by meclizine, which is a morphologically undifferentiated state.
  • MYH myosin heavy chain protein
  • Myosin heavy chain protein is a structural protein of muscle expressed at the end of differentiation.
  • DAPI nuclear staining was used as a control for cell number.
  • meclizine suppressed the expression of myosin heavy chain protein (FIG. 5).
  • Western blot analysis also showed that meclizine suppressed the expression of myosin heavy chain protein (MYH) in cells on day 7 of differentiation induction (FIG. 6).
  • the gene expression level of myosin heavy chain protein (MYH) was compared by quantitative RT-PCR. Evaluation by quantitative RT-PCR was performed as follows. First, total RNA was isolated from cells using Trizol (Life Technologies). First strand cDNA was synthesized with ReverTra Ace (Toyobo). Using LightCycler 480 Real-Time PCR (Roche) and SYBR Green (Takara), mRNA expression levels of myosin heavy chain 1 (MYH1), myosin heavy chain 7 (MYH7), myogenin (MYOG), Pax7 and dystrophin (DMD) It was measured. The mRNA level was corrected by the expression level of GAPDH.
  • MYH1 myosin heavy chain 1
  • MYH7 myosin heavy chain 7
  • MYOG myogenin
  • DMD dystrophin
  • Method C57BL / 6J mice were fed a special feed (meclizine-containing feed (4 g / 10 kg) or control feed) from the 16th day of birth (0w) and reared in a cage. On the 23rd day after birth (1w), the 30th day after birth (2w) and the 37th day after birth (3w), body weight was measured. The animals were sacrificed on the 37th day after birth, and X-ray photography and micro CT measurement were performed. Micro CT measurement was performed according to the following procedure. First, the whole body of the mouse was scanned with a 35 ⁇ m wide slice in a cross section perpendicular to the body axis.
  • the mouse sacroiliac joint was Merckmar, and the vertebral body with the sacroiliac joint was the first sacrum.
  • the fourth lumbar vertebral body was identified starting from the first sacrum.
  • the body axis cross-sectional image data at the fourth and fifth lumbar intervertebral intervertebral discs were taken into the image analysis software.
  • the muscle cross-sectional area of the paraspinal muscles (vertical spine and multifidus) was measured with image analysis software (FIG. 16). Three slice images were measured per intervertebral space.
  • MDX mice muscle dystrophy model mice
  • a special feed meclizine-containing feed (4g / 10kg) or control feed
  • 21st day (0w) to the 49th day (4w) after birth. Reared in The body weight was measured on the 28th day after birth (1w), the 35th day after birth (2w), the 42nd day after birth (3w), and the 49th day after birth (4w).
  • the meclizine-administered mouse had a larger paraspinal muscle cross-sectional area than the control (FIG. 17).
  • the results are shown as a ratio to the control.
  • meclizine was found to increase body weight and paraspinal muscle cross-sectional area in normal mice.
  • ⁇ Summary> Meclizine inhibited human myoblast differentiation and promoted proliferation in a dose-dependent manner. ⁇ The differentiation inhibitory effect of meclizine was reversible. • Mice receiving meclizine increased body weight and muscle cross-sectional area. ⁇ Meclizine also increased body weight in muscular dystrophy model mice. For muscular dystrophy, meclodin administration is expected to increase muscle mass and weight.
  • the skeletal muscle bulking agent of the present invention contains meclizine, which is an already approved drug, as an active ingredient. Meclizine has an antihistamine effect and is marketed as an OTC (over-the-counter). It has been used safely for more than 50 years and has established safety such as optimal dose, side effects and contraindications. This fact is a great merit for clinical application.
  • the therapeutic strategy using the skeletal muscle bulking agent of the present invention is not limited to sarcopenia and other disease groups (muscle damage caused by trauma or surgery, disuse).

Abstract

The present invention addresses the problem of providing a new therapeutic strategy for an ailment wherein skeletal muscle loss is the main cause or partial cause of the ailment. Provided is a skeletal muscle bulking agent containing meclizine or a pharmaceutically acceptable salt thereof as an active ingredient.

Description

骨格筋増量剤及びその用途Skeletal muscle bulking agent and use thereof
　本発明は骨格筋増量剤及びその用途に関する。本発明の骨格筋増量剤は、例えば、筋肉減少症の治療に利用される。本出願は、２０１４年８月２９日に出願された日本国特許出願第２０１４－１７６２４８号に基づく優先権を主張するものであり、当該特許出願の全内容は参照により援用される。 The present invention relates to a skeletal muscle bulking agent and use thereof. The skeletal muscle bulking agent of the present invention is used, for example, for the treatment of sarcopenia. This application claims priority based on Japanese Patent Application No. 2014-176248 filed on Aug. 29, 2014, the entire contents of which are incorporated by reference.
　サルコペニアなど、骨格筋量の低下をきたす筋肉減少症は、高齢者の移動能力の低下、転倒骨折、脆弱性の要因の一つと考えられている。その治療は健康寿命維持の切り札と目されている。筋肉減少症に対する根本的治療法はなく、運動療法や栄養療法、薬物療法が試みられているが、アドヒアランスや副作用の問題のため、いまだに有効な治療法は存在しない。尚、筋肉減少症に対する試みのいくつかを以下に示す（特許文献１～３）。 Muscle loss, such as sarcopenia, that causes a decrease in skeletal muscle mass is considered to be one of the causes of reduced mobility, falling fractures, and vulnerability in the elderly. The treatment is seen as a trump card for maintaining a healthy life expectancy. There is no fundamental cure for sarcopenia, and exercise therapy, nutrition therapy, and pharmacotherapy have been attempted, but there are still no effective treatments due to adherence and side effects. Some of the trials for sarcopenia are shown below (Patent Documents 1 to 3).
特開２００８－０１３４７３号公報JP 2008-013473 A 特開２０１３－０４８６２２号公報JP 2013-048622 A 国際公開第２０１１／０９８４４９International Publication No. 2011/098449
　サルコペニアの原因として、加齢性のホルモンバランスの変化、筋肉、神経の加齢性変化、他の疾患の合併や社会的要因による廃用性変化、低栄養によるアミノ酸やビタミンの摂取不足などの多岐にわたる素因が複合的に関与するとされている。その為根本的な治療は困難であり、新たな治療法の開発が求められている。 The causes of sarcopenia include various factors such as changes in age-related hormone balance, age-related changes in muscles and nerves, changes in disuse due to other diseases and social factors, and inadequate intake of amino acids and vitamins due to undernutrition. Predisposing factors are considered to be involved in multiple ways. Therefore, the fundamental treatment is difficult, and the development of a new treatment method is required.
　一方、骨格筋の減少は、サルコペニアなどの筋肉減少症の他にも各種疾患の主因又は一因となる。それらの疾患の中には、適切な治療法が確立していなものも多く、新たな治療法の提供が求められている。 On the other hand, skeletal muscle loss is the main cause or cause of various diseases in addition to sarcopenia and other muscle loss. Many of these diseases have not yet been established as appropriate treatments, and there is a need to provide new treatments.
　そこで、本発明の主たる課題は、骨格筋の減少を主因又は病因の一部とする疾患に対する、新規な治療戦略を提供することにある。 Therefore, a main problem of the present invention is to provide a novel treatment strategy for a disease whose main cause or part of the etiology is skeletal muscle loss.
　上記課題を解決すべく本発明者らは、骨格筋の増量に有効な低分子化合物を既認可薬の中から見出すことを試みた。具体的には、FDA認可薬ライブラリー（Prestwick Chemicals社）のうち、日本国内で臨床応用可能、かつ長期投与が可能な320種を選びサブライブラリーを作成し、その中から有効な化合物をスクリーニングした。ヒト由来の筋芽細胞にテロメラーゼ等を持続的に発現させた株細胞（Hu5/KD3細胞株）を利用した増殖アッセイ及び分化誘導アッセイの結果、メクリジンに細胞増殖促進効果と分化抑制効果が認められた。即ち、極めて有望な化合物としてメクリジンが選抜された。更なる検討を加えたところ、メクリジンの分化抑制効果は可逆的であり、薬剤の成分として望ましい特性を備えることが明らかとなった。また、メクリジンの効果に用量依存性も認められた。動物モデルを用いた実験においても、メクリジンに筋肉を増加させる効果が確認された。 In order to solve the above-mentioned problems, the present inventors tried to find a low molecular weight compound effective for increasing the amount of skeletal muscle from among already approved drugs. Specifically, among the FDA-approved drug library (Prestwick 社 Chemicals), 320 types that can be clinically applied in Japan and that can be administered for a long period of time are selected, and a sub-library is created to screen for effective compounds. did. As a result of proliferation assay and differentiation induction assay using cell lines (Hu5 / KD3 cell lines) that continuously expressed telomerase etc. in human myoblasts, meclizine has a cell proliferation promoting effect and a differentiation inhibiting effect. It was. That is, meclizine was selected as a very promising compound. As a result of further studies, it became clear that the differentiation inhibitory effect of meclizine is reversible and has desirable characteristics as a drug component. In addition, dose-dependence was observed in the effect of meclizine. In an experiment using an animal model, the effect of increasing muscle on meclizine was also confirmed.
　以上のように、本発明者らの詳細な検討によって、メクリジンが骨格筋の増量に有効であることが判明した。以下に示す本願発明は、主として上記知見及び考察に基づく。
　［１］メクリジン又はその薬学的に許容される塩を有効成分として含有する、骨格筋増量剤。
　［２］有効成分が塩酸メクリジンである、［１］に記載の治療薬。
　［３］［１］又は［２］に記載の骨格筋増量剤を含有する、骨格筋の減少を主因又は病因の一部とする疾患用の治療薬。
　［４］前記疾患が筋肉減少症である、［３］に記載の治療薬。
　［５］前記疾患がサルコペニアである、［３］に記載の治療薬。
　［６］前記疾患が、外傷や手術による筋損傷、廃用性筋萎縮、廃用症候群、易転倒性、神経原性筋萎縮、骨粗鬆症、変形性関節症、変形性脊椎症、側弯症や後弯症等の脊柱変形、肥満症、関節リウマチ、皮膚筋炎、多発筋炎、自己免疫疾患に伴う筋炎、ロコモーティブ症候群、メタボリック症候群、運動器不安定症、ダイナペニア（dynapenia）、フレイル、横紋筋融解症、筋ジストロフィー症、先天性筋症、先天性筋無力症候群、先天的筋低形成症、先天代謝障害、耐糖能異常ないし糖尿病若しくは癌等による悪液質からなる群より選択される疾患である、［３］に記載の治療薬。
　［７］前記疾患が筋ジストロフィー症である、［３］に記載の治療薬。
　［８］メクリジン又はその薬学的に許容される塩を治療上有効量、サルコペニア等の筋肉減少症、外傷や手術による筋損傷、廃用性筋萎縮、廃用症候群、易転倒性、神経原性筋萎縮、骨粗鬆症、変形性関節症、変形性脊椎症、側弯症や後弯症等の脊柱変形、肥満症、関節リウマチ、皮膚筋炎、多発筋炎、自己免疫疾患に伴う筋炎、ロコモーティブ症候群、メタボリック症候群、運動器不安定症、ダイナペニア（dynapenia）、フレイル、横紋筋融解症、筋ジストロフィー症、先天性筋症、先天性筋無力症候群、先天的筋低形成症、先天代謝障害、又は耐糖能異常ないし糖尿病若しくは癌等による悪液質の患者に投与するステップを含む、骨系統疾患の治療法。
　［９］メクリジン又はその薬学的に許容される塩を治療上有効量、筋ジストロフィー症の患者に投与するステップを含む、筋ジストロフィー症の治療法。
　［１０］メクリジン又はその薬学的に許容される塩を有効成分として含有する、フィジカルトレーニング又はフィジカルエクササイズの補助栄養剤。
　［１１］メクリジン又はその薬学的に許容される塩を有効成分として含有する、畜産、愛玩動物又は養殖用の肉量増加剤。 As described above, detailed investigations by the present inventors have revealed that meclizine is effective for increasing the amount of skeletal muscle. The present invention shown below is mainly based on the above knowledge and consideration.
[1] A skeletal muscle bulking agent containing meclizine or a pharmaceutically acceptable salt thereof as an active ingredient.
[2] The therapeutic agent according to [1], wherein the active ingredient is meclizine hydrochloride.
[3] A therapeutic agent for a disease mainly comprising a decrease in skeletal muscle or a part of the etiology, comprising the skeletal muscle bulking agent according to [1] or [2].
[4] The therapeutic agent according to [3], wherein the disease is sarcopenia.
[5] The therapeutic agent according to [3], wherein the disease is sarcopenia.
[6] The above-mentioned diseases include muscle damage due to trauma or surgery, disuse muscular atrophy, disuse syndrome, easy fall, neurogenic muscular atrophy, osteoporosis, osteoarthritis, osteoarthritis, scoliosis and posterior Spinal deformity such as mania, obesity, rheumatoid arthritis, dermatomyositis, polymyositis, myositis associated with autoimmune diseases, locomotive syndrome, metabolic syndrome, motor organ instability, dynapenia, flail, rhabdomyolysis, It is a disease selected from the group consisting of cachexia due to muscular dystrophy, congenital myopathy, congenital myasthenia syndrome, congenital hypoplasia, congenital metabolic disorders, impaired glucose tolerance or diabetes or cancer, [3 ] The therapeutic agent as described in.
[7] The therapeutic agent according to [3], wherein the disease is muscular dystrophy.
[8] A therapeutically effective amount of meclizine or a pharmaceutically acceptable salt thereof, sarcopenia or other muscle loss, muscle damage due to trauma or surgery, disuse muscle atrophy, disuse syndrome, easy fall, neurogenicity Muscle atrophy, osteoporosis, osteoarthritis, osteoarthritis, spinal deformity such as scoliosis and kyphosis, obesity, rheumatoid arthritis, dermatomyositis, polymyositis, myositis associated with autoimmune disease, locomotive syndrome, metabolic syndrome, Motor organ instability, dynapenia, flail, rhabdomyolysis, muscular dystrophy, congenital myopathy, congenital myasthenia syndrome, congenital hypoplasia, congenital metabolic disorders, or impaired glucose tolerance or diabetes Alternatively, a method for treating bone system diseases, comprising a step of administering to a patient with cachexia due to cancer or the like.
[9] A method for treating muscular dystrophy comprising the step of administering meclizine or a pharmaceutically acceptable salt thereof in a therapeutically effective amount to a patient with muscular dystrophy.
[10] A physical training or physical exercise supplement containing a meclizine or a pharmaceutically acceptable salt thereof as an active ingredient.
[11] A meat volume increasing agent for livestock, pets or aquaculture, containing meclizine or a pharmaceutically acceptable salt thereof as an active ingredient.
MTSアッセイの結果。メクリジンに細胞増殖促進効果を認めた（n=9）。コントロールに対する相対値で示した。Results of MTS assay. Meclizine was found to have a cell growth promoting effect (n = 9). The values are shown relative to the control. 実験プロトコール。サンプルD0とサンプルD7をウエスタンブロット解析及び免疫染色に供した。Experimental protocol. Samples D0 and D7 were subjected to Western blot analysis and immunostaining. BrdUアッセイの結果。メクリジンに細胞増殖促進効果を認めた。サンプルD0、D7及びD14のいずれについても細胞の増殖促進が認められる。コントロールに対する相対値で示した。Results of BrdU assay. Meclizine was observed to promote cell growth. In any of samples D0, D7, and D14, cell proliferation was promoted. The values are shown relative to the control. 形態観察の結果。サンプルD7の細胞を観察した。＊は筋管を示す。Results of morphological observation. The cells of sample D7 were observed. * Indicates myotubes. 免疫染色の結果。サンプルD7の細胞を免疫染色で評価した。細胞数のコントロールとして、DAPIで核染色した。MYHはミオシン重鎖タンパクを表す。Results of immunostaining. Sample D7 cells were evaluated by immunostaining. As a control of the cell number, nuclear staining was performed with DAPI. MYH represents myosin heavy chain protein. ウエスタンブロットの結果。サンプルD0及びD7について評価した。MYHはミオシン重鎖タンパクを表す。グラフ（右）はコントロールに対する相対値で示した（n=3）。＊　p<0.05Western blot results. Samples D0 and D7 were evaluated. MYH represents myosin heavy chain protein. The graph (right) is shown relative to the control (n = 3). * P <0.05 可逆性の検討に使用した培養方法（上）とサンプルD0とD7における細胞の形態（下）。Culture method used for studying reversibility (top) and cell morphology in samples D0 and D7 (bottom). 形態観察の結果。サンプルD14の細胞を観察した。＊は筋管を示す。Results of morphological observation. The cells of sample D14 were observed. * Indicates myotubes. 免疫染色の結果。サンプルD14の細胞を免疫染色で評価した。細胞数のコントロールとして、DAPIで核染色した。Results of immunostaining. Sample D14 cells were evaluated by immunostaining. As a control of the cell number, nuclear staining was performed with DAPI. ウエスタンブロットの結果。サンプルD14について評価した。MYHはミオシン重鎖タンパクを表す。Western blot results. Sample D14 was evaluated. MYH represents myosin heavy chain protein. 定量RT-PCRの結果。サンプルD14の細胞からmRNAを単離し、ミオシン重鎖遺伝子の発現量を調べた。コントロールに対する相対値で示した。＊　p<0.001Results of quantitative RT-PCR. MRNA was isolated from the cells of sample D14, and the expression level of the myosin heavy chain gene was examined. The values are shown relative to the control. * P <0.001 BrdUアッセイの結果。サンプルD0及びD7を用い、メクリジンの細胞増殖促進効果の用量依存性を調べた。＊　p<0.05Results of BrdU assay. Samples D0 and D7 were used to examine the dose dependence of meclizine's cell growth promoting effect. * P <0.05 ウエスタンブロットの結果。サンプルD7を用い、メクリジンの分化抑制効果の用量依存性を調べた。コントロールに対する相対値で示した。Western blot results. Using sample D7, the dose dependency of the differentiation inhibitory effect of meclizine was examined. The values are shown relative to the control. 生後37日目のマウスの外観とレントゲン撮影の結果。The appearance of the mouse on the 37th day after birth and the results of X-ray photography. マウスの体重の推移（成長曲線）。正常マウス（C57BL/6Jマウス）を用い、メクリジンの効果を検討した。左：オス（n=7）、右：メス（n=9）。Transition of mouse body weight (growth curve). Normal mice (C57BL / 6J mice) were used to examine the effect of meclizine. Left: male (n = 7), right: female (n = 9). マイクロCT測定の結果。傍脊柱筋（脊柱起立筋および多裂筋）の筋断面積を画像解析ソフトにて計測した。Results of micro CT measurement. The muscle cross-sectional area of the paraspinal muscles (vertical standing muscle and multifidus muscle) was measured with image analysis software. 傍脊柱筋断面積の計測結果。左：オス（n=7）、右：メス（n=9）。Results of paraspinal muscle cross-sectional area measurement. Left: male (n = 7), right: female (n = 9). マウスの体重の推移（成長曲線）。筋ジストロフィーモデルマウス（MDXマウス）。を用い、メクリジンの効果を検討した。左：オス（n=8）、右：メス（n=5）。Transition of mouse body weight (growth curve). Muscular dystrophy model mouse (MDX mouse). The effect of meclizine was examined. Left: male (n = 8), right: female (n = 5).
　本発明は骨格筋増量剤及びその用途に関する。本発明はメクリジンの新規な効果、即ち、骨格筋を増量させる効果を見出した成果に基づく。本発明の意義及び重要性を理解する上で、メクリジンが筋芽細胞の増殖を促進する一方で、その分化を可逆的に抑制した事実は特筆に値する。 The present invention relates to a skeletal muscle bulking agent and use thereof. The present invention is based on a result of finding a novel effect of meclizine, that is, an effect of increasing skeletal muscle mass. In understanding the significance and importance of the present invention, the fact that meclizine promotes myoblast proliferation while reversibly inhibiting its differentiation is worthy of special mention.
　本発明の骨格筋増量剤はメクリジン（meclizine）又はその薬学的に許容される塩を有効成分として含有する。メクリジンは、物質名1-[(4-クロロフェニル)フェニルメチル]-4-[(3-メチルフェニル)メチル]ピペラジンの化合物であり、抗ヒスタミン薬の一種として知られている。メクリジンは乗り物酔い止め薬としてOTC（over the counter）販売されている。メクリジンはメクロジン（meclozine）と呼称されることもある。本明細書では用語「メクリジン」と用語「メクロジン」を交換可能に使用する。 The skeletal muscle bulking agent of the present invention contains meclizine or a pharmaceutically acceptable salt thereof as an active ingredient. Meclizine is a compound of the substance name 1-[(4-chlorophenyl) phenylmethyl] -4-[(3-methylphenyl) methyl] piperazine and is known as a kind of antihistamine. Meclizine is sold as an over-the-counter (OTC) as a motion sickness medicine. Meclizine is sometimes referred to as meclozine. In this specification, the term “meclizine” and the term “meclozin” are used interchangeably.
　本発明の骨格筋増量剤の有効成分として、メクリジンの薬理学的に許容される塩を用いても良い。市販されているメクリジン製剤では、多くの場合、メクリジンの塩酸塩の形で処方されている。そこで、本発明においても好ましくは塩酸塩を用いる。但し、「薬理学的に許容される塩」はこれに限定されるものではなく、様々な塩、例えば、酸付加塩、アミノ酸付加塩等の利用が想定される。酸付加塩の例としては塩酸塩、硫酸塩、硝酸塩、リン酸塩、臭化水素酸塩などの無機酸塩、酢酸塩、マレイン酸塩、フマル酸塩、クエン酸塩、ベンゼンスルホン酸塩、安息香酸塩、リンゴ酸塩、シュウ酸塩、メタンスルホン酸塩、酒石酸塩などの有機酸塩が挙げられる。アミノ酸付加塩の例としてはグリシン付加塩、フェニルアラニン付加塩、リジン付加塩、アスパラギン酸付加塩、グルタミン酸付加塩が挙げられる。 As the active ingredient of the skeletal muscle bulking agent of the present invention, a pharmacologically acceptable salt of meclizine may be used. Commercial meclizine preparations are often formulated in the form of meclizine hydrochloride. Therefore, the hydrochloride is also preferably used in the present invention. However, the “pharmacologically acceptable salt” is not limited to this, and various salts such as an acid addition salt and an amino acid addition salt are assumed. Examples of acid addition salts include inorganic acid salts such as hydrochloride, sulfate, nitrate, phosphate, hydrobromide, acetate, maleate, fumarate, citrate, benzenesulfonate, Organic acid salts such as benzoate, malate, oxalate, methanesulfonate, and tartrate are listed. Examples of amino acid addition salts include glycine addition salts, phenylalanine addition salts, lysine addition salts, aspartic acid addition salts, and glutamic acid addition salts.
　筋肉は、骨格筋、平滑筋及び心筋に大別される。骨格筋は筋細胞（筋繊維）及び結合組織から構成される。骨格筋は随意筋とも呼ばれ、組織学的には横紋筋である。骨格筋には身体を動かすという役割の他、姿勢の保持、関節の安定、熱の発生、血管・臓器の保護等、様々な役割がある。本発明の骨格筋増量剤は、骨格筋の減少を主因又は病因の一部とする疾患の治療又は予防に適用可能である。即ち、本発明は、当該疾患用の治療薬（骨格筋増量剤を含有する）も提供する。「治療薬」とは、標的の疾病ないし病態に対する治療的又は予防的効果を示す医薬のことをいう。治療的効果には、標的疾患／病態に特徴的な症状又は随伴症状を緩和すること（軽症化）、症状の悪化を阻止ないし遅延すること等が含まれる。後者については、重症化を予防するという点において予防的効果の一つと捉えることができる。このように、治療的効果と予防的効果は一部において重複する概念であり、明確に区別して捉えることは困難であり、またそうすることの実益は少ない。尚、予防的効果の典型的なものは、標的疾患／病態に特徴的な症状の再発を阻止ないし遅延することである。尚、標的疾患／病態に対して何らかの治療的効果又は予防的効果、或いはこの両者を示す限り、標的疾患／病態に対する治療薬に該当する。 Muscles are roughly classified into skeletal muscles, smooth muscles and myocardium. Skeletal muscle is composed of muscle cells (muscle fibers) and connective tissue. Skeletal muscles are also called voluntary muscles and are histologically striated muscles. In addition to the role of moving the body, skeletal muscle has various roles such as maintaining posture, stabilizing joints, generating heat, and protecting blood vessels and organs. The skeletal muscle bulking agent of the present invention can be applied to the treatment or prevention of diseases whose main cause or part of the pathogenesis is skeletal muscle loss. That is, the present invention also provides a therapeutic agent for the disease (containing a skeletal muscle bulking agent). “Therapeutic agent” refers to a drug that exhibits a therapeutic or prophylactic effect on a target disease or condition. Therapeutic effects include alleviation of symptoms or concomitant symptoms characteristic of the target disease / pathology (lightening), prevention or delay of worsening of symptoms, and the like. The latter can be regarded as one of the preventive effects in terms of preventing the seriousness. In this way, the therapeutic effect and the preventive effect are partially overlapping concepts, and it is difficult to clearly distinguish them from each other, and there is little benefit in doing so. A typical preventive effect is to prevent or delay the recurrence of symptoms characteristic of the target disease / pathology. In addition, as long as it shows some therapeutic effect or preventive effect with respect to a target disease / pathology, or both, it corresponds to the therapeutic agent with respect to a target disease / pathology.
　「骨格筋の減少を主因又は病因の一部とする疾患」に該当するものは、例えば、サルコペニアに代表される筋肉減少症である。その他、外傷や手術による筋損傷、廃用性筋萎縮、廃用症候群、易転倒性、神経原性筋萎縮、骨粗鬆症、変形性関節症、変形性脊椎症、側弯症や後弯症等の脊柱変形、肥満症、関節リウマチ、皮膚筋炎、多発筋炎、自己免疫疾患に伴う筋炎、ロコモーティブ症候群、メタボリック症候群、運動器不安定症、ダイナペニア（dynapenia）、フレイル、横紋筋融解症、筋ジストロフィー症、先天性筋症、先天性筋無力症候群、先天的筋低形成症、先天代謝障害、耐糖能異常ないし糖尿病若しくは癌等による悪液質等にも、本発明の治療薬を適用可能である。 “A disease in which a decrease in skeletal muscle is the main cause or a part of the etiology” is, for example, sarcopenia typified by sarcopenia. Other spinal deformities such as muscle damage due to trauma and surgery, disuse muscle atrophy, disuse syndrome, easy fall, neurogenic muscle atrophy, osteoporosis, osteoarthritis, osteoarthritis, scoliosis and kyphosis , Obesity, rheumatoid arthritis, dermatomyositis, polymyositis, myositis associated with autoimmune disease, locomotive syndrome, metabolic syndrome, motor organ instability, dynapenia, flail, rhabdomyolysis, muscular dystrophy, congenital The therapeutic agent of the present invention can also be applied to myopathy, congenital myasthenia syndrome, congenital hypoplasia, congenital metabolic disorders, impaired glucose tolerance or cachexia due to diabetes or cancer.
　本発明の治療薬の製剤化は常法に従って行うことができる。製剤化する場合には、製剤上許容される他の成分（例えば、担体、賦形剤、崩壊剤、緩衝剤、乳化剤、懸濁剤、無痛化剤、安定剤、保存剤、防腐剤、生理食塩水など）を含有させることができる。賦形剤としては乳糖、デンプン、ソルビトール、D-マンニトール、白糖等を用いることができる。崩壊剤としてはデンプン、カルボキシメチルセルロース、炭酸カルシウム等を用いることができる。緩衝剤としてはリン酸塩、クエン酸塩、酢酸塩等を用いることができる。乳化剤としてはアラビアゴム、アルギン酸ナトリウム、トラガント等を用いることができる。懸濁剤としてはモノステアリン酸グリセリン、モノステアリン酸アルミニウム、メチルセルロース、カルボキシメチルセルロース、ヒドロキシメチルセルロース、ラウリル硫酸ナトリウム等を用いることができる。無痛化剤としてはベンジルアルコール、クロロブタノール、ソルビトール等を用いることができる。安定剤としてはプロピレングリコール、アスコルビン酸等を用いることができる。保存剤としてはフェノール、塩化ベンザルコニウム、ベンジルアルコール、クロロブタノール、メチルパラベン等を用いることができる。防腐剤としては塩化ベンザルコニウム、パラオキシ安息香酸、クロロブタノール等と用いることができる。 The preparation of the therapeutic agent of the present invention can be performed according to a conventional method. In the case of formulating, other pharmaceutically acceptable ingredients (for example, carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, preservatives, physiological Saline solution and the like). As the excipient, lactose, starch, sorbitol, D-mannitol, sucrose and the like can be used. As the disintegrant, starch, carboxymethylcellulose, calcium carbonate and the like can be used. Phosphate, citrate, acetate, etc. can be used as the buffer. As the emulsifier, gum arabic, sodium alginate, tragacanth and the like can be used. As the suspending agent, glyceryl monostearate, aluminum monostearate, methyl cellulose, carboxymethyl cellulose, hydroxymethyl cellulose, sodium lauryl sulfate and the like can be used. As the soothing agent, benzyl alcohol, chlorobutanol, sorbitol and the like can be used. As the stabilizer, propylene glycol, ascorbic acid or the like can be used. As preservatives, phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, and the like can be used. As preservatives, benzalkonium chloride, paraoxybenzoic acid, chlorobutanol and the like can be used.
　製剤化する場合の剤形も特に限定されない。剤形の例は錠剤、散剤、細粒剤、顆粒剤、カプセル剤、シロップ剤、注射剤、外用剤、及び座剤である。本発明の治療薬はその剤形に応じて経口投与又は非経口投与（静脈内、動脈内、皮下、皮内、筋肉内、又は腹腔内注射、経皮、経鼻、経粘膜など）によって対象に適用される。また、全身的な投与と局所的な投与も対象により適応される。これらの投与経路は互いに排他的なものではなく、任意に選択される二つ以上を併用することもできる（例えば、経口投与と同時に又は所定時間経過後に静脈注射等を行う等）。本発明の治療薬には、期待される治療効果を得るために必要な量（即ち治療上有効量）の有効成分が含有される。本発明の治療薬中の有効成分量は一般に剤形によって異なるが、所望の投与量を達成できるように有効成分量を例えば約0.1重量％～約99重量％の範囲内で設定する。 The dosage form for formulation is not particularly limited. Examples of dosage forms are tablets, powders, fine granules, granules, capsules, syrups, injections, external preparations, and suppositories. The therapeutic agent of the present invention is administered orally or parenterally (intravenous, intraarterial, subcutaneous, intradermal, intramuscular, or intraperitoneal injection, transdermal, nasal, transmucosal, etc.) depending on the dosage form. Applies to Systemic and local administration are also indicated by the subject. These administration routes are not mutually exclusive, and two or more arbitrarily selected can be used in combination (for example, intravenous injection or the like is performed simultaneously with oral administration or after a predetermined time has elapsed). The therapeutic agent of the present invention contains an active ingredient in an amount necessary for obtaining an expected therapeutic effect (that is, a therapeutically effective amount). The amount of the active ingredient in the therapeutic agent of the present invention generally varies depending on the dosage form, but the amount of the active ingredient is set, for example, within the range of about 0.1 wt% to about 99 wt% so as to achieve a desired dose.
　本発明の治療薬の投与量は、期待される治療効果が得られるように設定される。治療上有効な投与量の設定においては一般に症状、患者の年齢、性別、及び体重などが考慮される。当業者であればこれらの事項を考慮して適当な投与量を設定することが可能である。例えば、成人（体重約60kg）を対象として一日当たりの有効成分量が1mg～500mg、好ましくは5mg～300mg、特に好ましくは10mg～200mgとなるよう投与量を設定することができる。投与スケジュールとしては例えば一日一回～数回、二日に一回、或いは三日に一回などを採用できる。投与スケジュールの作成においては、患者の病状や有効成分の効果持続時間などを考慮することができる。 The dosage of the therapeutic agent of the present invention is set so as to obtain the expected therapeutic effect. In setting a therapeutically effective dose, symptoms, patient age, sex, weight, etc. are generally considered. A person skilled in the art can set an appropriate dose in consideration of these matters. For example, for an adult (body weight of about 60 kg), the dose can be set so that the amount of active ingredient per day is 1 mg to 500 mg, preferably 5 mg to 300 mg, particularly preferably 10 mg to 200 mg. As the administration schedule, for example, once to several times a day, once every two days, or once every three days can be adopted. In preparing the administration schedule, the patient's medical condition and the duration of effect of the active ingredient can be taken into consideration.
　本発明の治療薬による治療に並行して他の医薬（例えば既存の治療薬）による治療を行ったり、既存の治療手技に対して本発明の治療薬による治療を組み合わせたりしてもよい。 In parallel with the treatment with the therapeutic agent of the present invention, treatment with another medicine (for example, existing therapeutic agent) may be performed, or the existing therapeutic technique may be combined with the treatment with the therapeutic agent of the present invention.
　以上の記述から明らかな通り本願は、骨格筋の減少を主因又は病因の一部とする疾患（サルコペニア等の筋肉減少症、外傷や手術による筋損傷、廃用性筋萎縮、廃用症候群、易転倒性、神経原性筋萎縮、骨粗鬆症、変形性関節症、変形性脊椎症、側弯症や後弯症等の脊柱変形、肥満症、関節リウマチ、皮膚筋炎、多発筋炎、自己免疫疾患に伴う筋炎、ロコモーティブ症候群、メタボリック症候群、運動器不安定症、ダイナペニア（dynapenia）、フレイル、横紋筋融解症、筋ジストロフィー症、先天性筋症、先天性筋無力症候群、先天的筋低形成症、先天代謝障害、耐糖能異常ないし糖尿病若しくは癌等による悪液質等）の患者に対して本発明の治療薬を治療上有効量投与することを特徴とする治療法も提供する。 As is clear from the above description, the present application is intended for diseases mainly caused by skeletal muscle loss (muscle loss such as sarcopenia, muscle damage due to trauma or surgery, disuse muscle atrophy, disuse syndrome, Falls, neurogenic muscle atrophy, osteoporosis, osteoarthritis, osteoarthritis, spinal deformity such as scoliosis and kyphosis, obesity, rheumatoid arthritis, dermatomyositis, polymyositis, myositis associated with autoimmune diseases, Locomotive syndrome, metabolic syndrome, motor organ instability, dynapenia, flail, rhabdomyolysis, muscular dystrophy, congenital myopathy, congenital myasthenia syndrome, congenital hypoplasia, congenital metabolic disorders, There is also provided a therapeutic method characterized by administering a therapeutically effective amount of the therapeutic agent of the present invention to a patient with impaired glucose tolerance or cachexia due to diabetes or cancer.
　本発明の骨格筋増量剤は、筋肉の増量ないし強化を目的としたフィジカルトレーニング／フィジカルエクササイズの補助栄養剤としても利用できる。ここでの「補助栄養剤」とは、フィジカルトレーニング／フィジカルエクササイズの効果を促進するために使用されるものであって、それを使用しない場合に比較して高い効果をもたらすものをいう。補助栄養剤の場合、治療薬と同様の形態（剤型）の他、それを含有させた食品組成物の形態で提供することもできる。食品組成物の場合、例えば、栄養補助食品（サプリメント、栄養ドリンク等）として粉末、顆粒末、タブレット、ペースト、液体等の形状で提供される。食品組成物の形態で提供することによって、本発明の補助栄養剤を日常的に摂取したり、継続的に摂取したりすることが容易となる。本発明の食品組成物には、所望の効果、即ち筋肉の増加が期待できる量の有効成分が含有されることが好ましい。添加量は、それが使用される対象（利用者）の年齢、体格（身長、体重など）、性別などを考慮して定めることができる。 The skeletal muscle bulking agent of the present invention can also be used as a supplementary nutrient for physical training / physical exercise for the purpose of increasing or strengthening muscles. The “supplementary nutrient” used herein is used to promote the effects of physical training / physical exercise, and has a higher effect than when it is not used. In the case of a supplementary nutritional supplement, it can also be provided in the form of a food composition containing it in addition to the form (dosage form) similar to the therapeutic agent. In the case of a food composition, for example, it is provided in the form of powder, granule powder, tablet, paste, liquid or the like as a dietary supplement (supplement, nutrition drink, etc.). By providing in the form of a food composition, it is easy to ingest the supplementary nutrient of the present invention on a daily basis or continuously. The food composition of the present invention preferably contains an active ingredient in such an amount that a desired effect, that is, an increase in muscle can be expected. The addition amount can be determined in consideration of the age, physique (height, weight, etc.), gender, etc. of the subject (user) in which it is used.
　本発明の骨格筋増量剤は畜産、愛玩動物（ペット）、養殖の分野でも有用である。本発明の骨格筋増量剤を飼料に混合して摂食させたり、或いは注射により投与したりすれば、家畜（ウシ、ブタ、ウマ、ヒツジ等）、家禽（ニワトリ、アヒル、ガチョウ、七面鳥、ウズラ、キジ等）、魚類（マグロ、マダイ、ブリなど）の肉量の増加を図ることができる。このように、本発明は肉量増加剤としても利用できる。また、ペットの健康維持、体型維持などに本発明の骨格筋増量剤を用いることも可能である。 The skeletal muscle bulking agent of the present invention is also useful in the fields of animal husbandry, pets (animals) and aquaculture. When the skeletal muscle bulking agent of the present invention is mixed with feed and fed or administered by injection, livestock (cattle, pig, horse, sheep, etc.), poultry (chicken, duck, goose, turkey, quail) , Pheasants, etc.) and fish (tuna, red sea bream, yellowtail, etc.). Thus, the present invention can also be used as a meat volume increasing agent. It is also possible to use the skeletal muscle bulking agent of the present invention for maintaining the health and body shape of pets.
　近年、既存薬の新たな効能を探索し、適用拡大を図ること、即ちドラッグ・リポジショニング（drug repositioning）戦略が注目されている（Abbott, A. (2002) Neurologists strike gold in drug screen effort. Nature, 417, 109.；Bian, Y., Masuda, A., Matsuura, T., Ito, M., Okushin, K., Engel, A.G. and Ohno, K. (2009) Tannic acid facilitates expression of the polypyrimidine tract binding protein and alleviates deleterious inclusion of CHRNA1 exon P3A due to an hnRNP H-disrupting mutation in congenital myasthenic syndrome. Hum. Mol. Genet., 18, 1229-1237.）。この戦略のメリットの一つは、同定された薬剤は至適服用量や副作用などが既に確立していることから、直ちに臨床応用できることである。以下では、サルコペニアに代表される筋肉減少症の治療に有効な薬剤を同定することを目指し、1,186種類のFDA認可薬をスクリーニングすることにした。 In recent years, drug-repositioning strategies have been attracting attention by exploring new effects of existing drugs and expanding their application (Abbott, A. (2002) Neurologists strike gold in drug screen effort. Nature , 417, 109 .; Bian, Y., Masuda, A., Matsuura, T., Ito, M., Okushin, K., Engel, AG and Ohno, K. (2009) Tannic acid facilitates expression of the polypyrimidine tract binding protein and alleviates deleterious inclusion of CHRNA1 exon P3A due to an hnRNP H-disrupting mutation in congenital myasthenic syndrome. Hum. Mol. Genet., 18, 1229-1237.). One of the merits of this strategy is that the identified drug can be immediately applied clinically because the optimal dose and side effects have already been established. In the following, we decided to screen 1,186 FDA-approved drugs with the aim of identifying effective drugs for the treatment of sarcopenia, such as sarcopenia.
Ａ．Hu5/KD3細胞株を用いた、1,186種類のFDA認可薬からのスクリーニング
１．材料と方法
（１）MTSアッセイ
　Hu5/KD3細胞株(国立長寿医療研究センター・橋本有弘部長より分与)はヒト由来の筋芽細胞にテロメラーゼ等を持続的に発現させた株細胞で、分化誘導培地により筋管細胞に分化する(Shiomi K et al. Gene Ther. 2011; 18:857-866)。Hu5/KD3細胞株の培養には２種類の培地を用いる。即ち、増殖用には、pmGM（20% ウシ胎児血清(FBS, Thermo Scientific)、2% Ultroser G (Biosepra, PALL)、ペニシリンG (100 u/ml)及びストレプトマイシン硫酸塩(100μg/ml)(Penstrep, Life Technologies )を添加したDulbecco’s Modified Eagle’s Medium (DMEM, Invitrogen)）が、分化誘導用にはpmDM（2% ウマ血清、1% インスリン－トランスフェリン－亜セレン酸塩(ITS, Invitrogen)、ペニシリンG (100 u/ml)及びストレプトマイシン硫酸塩(100μg/ml)(Penstrep, Life Technologies )を添加したDMEM）が使用される。 A. Screening from 1,186 FDA approved drugs using Hu5 / KD3 cell line Materials and Methods (1) MTS assay Hu5 / KD3 cell line (distributed by National Longevity Medical Research Center, Director Yasuhiro Hashimoto) is a cell line in which telomerase is continuously expressed in human myoblasts. Differentiate into myotubes with induction medium (Shiomi K et al. Gene Ther. 2011; 18: 857-866). Two types of media are used for culturing the Hu5 / KD3 cell line. That is, for growth, pmGM (20% fetal bovine serum (FBS, Thermo Scientific), 2% Ultroser G (Biosepra, PALL), penicillin G (100 u / ml) and streptomycin sulfate (100 μg / ml) (Penstrep , Life Technologies) added Dulbecco's Modified Eagle's Medium (DMEM, Invitrogen)) for differentiation induction, pmDM (2% horse serum, 1% insulin-transferrin-selenate (ITS, Invitrogen), penicillin G ( 100 u / ml) and streptomycin sulfate (100 μg / ml) (DMEM supplemented with Penstrep, Life Technologies) are used.
　Prestwick Chemicals社の1,186種のFDA認可薬ライブラリーのうち、日本国内で臨床応用可能、かつ長期投与が可能な320種を選びサブライブラリーを作成した。サブライブラリー薬剤10μMを添加した培地で未分化Hu5/KD3細胞を培養し、MTSアッセイ(Cell 96 AQueus One Solution Cell Proliferation Assay, Promega)により細胞の増殖を定量した。操作は添付のマニュアルに従った。 Of the 1,186 FDA-approved drug libraries from Prestwick® Chemicals, we selected 320 types that can be clinically applied in Japan and can be administered for a long time, and created a sub-library. Undifferentiated Hu5 / KD3 cells were cultured in a medium supplemented with 10 μM of the sublibrary drug, and cell proliferation was quantified by MTS assay (CellTS96 AQueus One Solution Cell Proliferation Assay, Promega). The operation followed the attached manual.
（２）BrdUアッセイ
　Hu5/KD3細胞株を分化誘導した場合における、細胞増殖に与える各薬剤の影響を調べた。薬剤を添加したpmGM中でHu5/KD3細胞株（0.5×105個／培養皿）を3日間培養した。この時点のサンプルをサンプルD0とした。薬剤を含有したpmDMに培地を交換し、分化誘導した。分化誘導条件で7日間培養したものをサンプルD7、14日間培養したものをサンプルD14とした。BrdU ELISA（細胞増殖 ELISA、BrdU 発色キット（ロシュ・ダイアグノスティックス株式会社））で細胞の増殖を定量した。操作は添付のマニュアルに従った。 (2) BrdU assay When the Hu5 / KD3 cell line was induced to differentiate, the effect of each drug on cell proliferation was examined. The Hu5 / KD3 cell line (0.5 × 10 5 cells / culture dish) was cultured for 3 days in pmGM to which the drug was added. The sample at this time was designated as sample D0. The medium was changed to pmDM containing the drug to induce differentiation. Sample D7 was cultured for 7 days under differentiation induction conditions, and sample D14 was cultured for 14 days. Cell proliferation was quantified by BrdU ELISA (Cell proliferation ELISA, BrdU color development kit (Roche Diagnostics Inc.)). The operation followed the attached manual.
（３）形態観察
　3×105個のHu5/KD3細胞を6ウェルプレートに播種し、メクリジン（10μM）を添加した増殖用培地（pmGM）中で3日間培養後、メクリジン（10μM）を添加した分化用培地（pmDM）に変更し、さらに7日間培養（分化誘導）を行った。コントロールにはメクリジンを添加しない培地を使用した。培養後の細胞の形態を撮影した（SZ61SZ61 microscope (Olympus)、XZ-1 digital camera (Olympus)）。 (3) Morphological observation 3 × 10 5 Hu5 / KD3 cells were seeded in a 6-well plate, cultured in a growth medium (pmGM) supplemented with meclizine (10 μM) for 3 days, and then added meclizine (10 μM). The medium was changed to differentiation medium (pmDM) and further cultured for 7 days (differentiation induction). A medium without the addition of meclizine was used as a control. The cell morphology after culture was photographed (SZ61SZ61 microscope (Olympus), XZ-1 digital camera (Olympus)).
（４）免疫染色（図２）
　6ウェルコラーゲンコートプレート（セルタイトC-1プレート、住友ベークライト株式会社）にHu5/KD3細胞（3×105 個／ウェル）を播種し、メクリジンを添加（10μM）したpmGMで3日間培養した。メクリジン（10μM）を添加した分化用培地（pmDM）に変更し、さらに7日間培養（分化誘導）を行った。コントロールにはメクリジンを添加しない培地を使用した。細胞を3.7%ホルムアルデヒ含有PBSで10分処理した後、0.05% Triton X-100で10分処理し、固定化した。10%ヤギ血清アルブミン含有PBSでブロッキングした後、抗MYH抗体 (H-300, Santa Cruz Biotechnology、希釈率1:200)とインキュベートした（4℃、終夜）。洗浄後、二次抗体を反応させた。その後、包埋処理し、蛍光を検出した。 (4) Immunostaining (Figure 2)
Hu5 / KD3 cells (3 × 10 5 cells / well) were seeded on a 6-well collagen-coated plate (Celtite C-1 plate, Sumitomo Bakelite Co., Ltd.), and cultured for 3 days in pmGM supplemented with meclizine (10 μM). The medium was changed to differentiation medium (pmDM) supplemented with meclizine (10 μM), and further cultured for 7 days (differentiation induction). A medium without the addition of meclizine was used as a control. The cells were treated with PBS containing 3.7% formaldehyde for 10 minutes, and then fixed with 0.05% Triton X-100 for 10 minutes. After blocking with PBS containing 10% goat serum albumin, it was incubated with anti-MYH antibody (H-300, Santa Cruz Biotechnology, dilution ratio 1: 200) (4 ° C., overnight). After washing, the secondary antibody was reacted. Thereafter, embedding was performed, and fluorescence was detected.
（５）ウエスタンブロット解析（図２）
　6ウェルコラーゲンコートプレート（セルタイトC-1プレート、住友ベークライト株式会社）にHu5/KD3細胞（3×105 個／ウェル）を播種し、メクリジンを添加（10μM）したpmGMで3日間培養した。メクリジン（10μM）を添加した分化用培地（pmDM）に変更し、さらに7日間培養（分化誘導）を行った。コントロールにはメクリジンを添加しない培地を使用した。培養後の細胞をバッファー（50 mM HEPES pH 7.0, 150 mM NaCl, 10% glycerol, 1% TritonX-100, 1.5 mM MgCl2, 1 mM EGTA, 100 mM NaF, 10 mM sodium pyrophosphate, 1 μg/μl aprotinin, 1 μg/μl leupeptin, 1 μg/μl pepstatin A, 1 mM PMSF, 1 mM sodium orthovanadate）で溶解した。全タンパク質を1×laemmliバッファーに溶解し、SDS-PAGE（10%又は7.5%ゲル）で分離し、PVDF膜(Immobilon-P, Millipore)に転写した。転写後の膜を0.05% Tween 20 (TBS-T)含有のトリスバッファーで洗浄した後、3%ウシ血清アルブミン含有TBS-Tでブロッキングした（室温、１時間）。続いて、処理後の膜をマウス抗MYHモノクローナル抗体(H-300, Santa Cruz Biotechnology, dilution 1:200)又は抗-GAPDH抗体(G9545, Sigma-Aldrich, dilution 1:600)と反応させた（4℃、終夜）。膜を３回洗浄した後、二次抗体（HRP標識ヤギ抗ウサギIgG抗体 (GE Healthcare, 1:6000)）と反応させた（室温、１時間）。Amersham ECL Western blotting detection reagents (GE Healthcare)を用いて検出し、ImageJ programで定量化した。 (5) Western blot analysis (Figure 2)
Hu5 / KD3 cells (3 × 10 5 cells / well) were seeded on a 6-well collagen-coated plate (Celtite C-1 plate, Sumitomo Bakelite Co., Ltd.), and cultured for 3 days in pmGM supplemented with meclizine (10 μM). The medium was changed to differentiation medium (pmDM) supplemented with meclizine (10 μM), and further cultured for 7 days (differentiation induction). A medium without the addition of meclizine was used as a control. The cultured cells were buffered (50 mM HEPES pH 7.0, 150 mM NaCl, 10% glycerol, 1% TritonX-100, 1.5 mM MgCl2, 1 mM EGTA, 100 mM NaF, 10 mM sodium pyrophosphate, 1 μg / μl aprotinin, 1 μg / μl leupeptin, 1 μg / μl pepstatin A, 1 mM PMSF, 1 mM sodium orthovanadate). All proteins were dissolved in 1 × laemmli buffer, separated by SDS-PAGE (10% or 7.5% gel), and transferred to a PVDF membrane (Immobilon-P, Millipore). The transferred membrane was washed with a Tris buffer containing 0.05% Tween 20 (TBS-T) and then blocked with TBS-T containing 3% bovine serum albumin (room temperature, 1 hour). Subsequently, the treated membrane was reacted with mouse anti-MYH monoclonal antibody (H-300, Santa Cruz Biotechnology, dilution 1: 200) or anti-GAPDH antibody (G9545, Sigma-Aldrich, dilution 1: 600) (4 ° C overnight. The membrane was washed three times and then reacted with a secondary antibody (HRP-labeled goat anti-rabbit IgG antibody (GE Healthcare, 1: 6000)) (room temperature, 1 hour). Detection was performed using Amersham ECL Western blotting detection reagents (GE Healthcare), and quantification was performed using the ImageJ program.
２．結果
　320種の薬剤を候補化合物としてMTSアッセイを繰り返し行った。9種類の薬剤に細胞増殖促進効果が認められた（図１）。これらの薬剤の内、メクリジンに注目し、以降の検討を行った。 2. Results The MTS assay was repeated using 320 drugs as candidate compounds. Nine kinds of drugs were found to promote cell proliferation (Fig. 1). Of these drugs, we focused on meclizine and conducted further studies.
　BrdUアッセイは細胞分裂を定量する方法であり、MTSアッセイと同様に細胞増殖の評価に利用される。BrdUアッセイにおいても、メクリジンは細胞増殖促進作用を示した（図３）。 The BrdU assay is a method for quantifying cell division and is used to evaluate cell proliferation in the same manner as the MTS assay. Also in the BrdU assay, meclizine showed a cell growth promoting action (FIG. 3).
　分化誘導7日目の細胞についてメクリジンを投与した場合と投与しない場合（コントロール）で形態を比較した（図４）。コントロールでは形態的にmyotube（筋管）の形成が認められるが、メクリジンを投与した細胞では筋管が認められない。メクリジンにより分化が抑制され、形態的に未分化な状態である。 The morphology of cells on differentiation induction day 7 was compared with and without meclizine (control) (FIG. 4). In the control, formation of myotube (myotube) is observed morphologically, but myotubes are not observed in cells administered with meclizine. Differentiation is suppressed by meclizine, which is a morphologically undifferentiated state.
　分化誘導7日目の細胞についてMYH（ミオシン重鎖タンパク）の免疫染色を行った。ミオシン重鎖タンパクは分化末期に発現する筋肉の構造タンパクである。細胞数のコントロールとしてDAPIの核染色を用いた。分化誘導7日目で、メクリジンはミオシン重鎖タンパクの発現を抑制していることがわかる（図５）。ウエスタンブロット解析でも、分化誘導7日目の細胞でメクリジンがミオシン重鎖タンパク(MYH)の発現を抑制していることが示された（図６）。 The cells on day 7 of differentiation induction were immunostained with MYH (myosin heavy chain protein). Myosin heavy chain protein is a structural protein of muscle expressed at the end of differentiation. DAPI nuclear staining was used as a control for cell number. On day 7 of differentiation induction, it was found that meclizine suppressed the expression of myosin heavy chain protein (FIG. 5). Western blot analysis also showed that meclizine suppressed the expression of myosin heavy chain protein (MYH) in cells on day 7 of differentiation induction (FIG. 6).
Ｂ．メクリジンの効果の可逆性
　メクリジンの分化抑制効果が可逆的であるか検討した。
１．方法（図７上）
　Hu5/KD3細胞をメクリジン含有pmGM中で3日間培養した（サンプルD0）。その後、メクリジン含有pmDMで7日間培養し、分化誘導を行った（サンプルD7）。さらに7日間メクリジン含有pmGMで分化誘導を行ったサンプル（D14、メクリジン）と、メクリジンをウォシュアウト（除去）し、メクリジンを含まないpmGMで分化誘導を行ったサンプル（D14、メクリジン除去コントロール）を形態観察、免疫染色及びウエスタンブロットで比較した。操作及び評価方法は上記Ａの実験に準じた。また、ミオシン重鎖タンパク(MYH)の遺伝子発現量を定量RT-PCRで比較した。定量RT-PCRでの評価は以下の通り行った。まず、Trizol(Life Technologies)を用い、細胞から全RNAを単離した。第１鎖cDNAをReverTra Ace(Toyobo)で合成した。LightCycler 480 Real-Time PCR (Roche)及びSYBR Green (Takara)を用い、ミオシン重鎖1(MYH1)、ミオシン重鎖7(MYH7)、myogenin (MYOG)、Pax7及びジストロフィン(DMD)のmRNA発現量を測定した。mRNAレベルはGAPDHの発現量で補正した。 B. Reversibility of the effect of meclizine It was investigated whether the effect of inhibiting the differentiation of meclizine was reversible.
1. Method (upper figure 7)
Hu5 / KD3 cells were cultured for 3 days in pmGM containing meclizine (sample D0). Thereafter, the cells were cultured in meclizine-containing pmDM for 7 days to induce differentiation (sample D7). Furthermore, a sample (D14, meclizine) in which differentiation was induced with meclizine-containing pmGM for 7 days and a sample (D14, meclizin removal control) in which differentiation was induced with pmGM that was washed out (removed) and meclizine was not included. Comparison was made by observation, immunostaining and Western blotting. The operation and evaluation method were in accordance with the above experiment A. The gene expression level of myosin heavy chain protein (MYH) was compared by quantitative RT-PCR. Evaluation by quantitative RT-PCR was performed as follows. First, total RNA was isolated from cells using Trizol (Life Technologies). First strand cDNA was synthesized with ReverTra Ace (Toyobo). Using LightCycler 480 Real-Time PCR (Roche) and SYBR Green (Takara), mRNA expression levels of myosin heavy chain 1 (MYH1), myosin heavy chain 7 (MYH7), myogenin (MYOG), Pax7 and dystrophin (DMD) It was measured. The mRNA level was corrected by the expression level of GAPDH.
２．結果
　実験系の妥当性を確認するため、メクリジン含有pmDM（分化誘導用培地）で培養した後の細胞の形態を観察した。D0、D7ともに未分化な形態を維持しており、これまでの結果と同様にメクリジンが分化誘導を抑制する作用を示しており、実験の条件に問題がないことが確認された（図７下）。 2. Results In order to confirm the validity of the experimental system, the morphology of the cells after culturing in meclizine-containing pmDM (medium for differentiation induction) was observed. Both D0 and D7 are maintained in an undifferentiated form, and like the results so far, meclizine has shown an action of suppressing differentiation induction, and it was confirmed that there is no problem in the experimental conditions (bottom of FIG. 7). ).
　分化誘導の7日目～14日目までを、メクリジンを含まないpmGMで分化誘導を行った場合（D14、メクリジン除去コントロール）では筋管が認められた（図８左）。メクリジンを投与し続けた細胞では筋管は認められなかった（図８右）。免疫染色では、メクリジン除去コントロールにMYHを認めたのに対し（図９左）、メクリジンを投与し続けた細胞はMYHが認められない（図９右）。ウエスタンブロット解析においても、メクリジンを投与し続けた細胞はMYHの発現が乏しかったのに対して、メクリジン除去コントロールではMYHの発現を認めた（図１０）。また、リアルタイムPCRによる発現解析では、メクリジンを投与し続けた細胞はMYH1、MYH7の発現が乏しかったのに対して、メクリジン除去コントロールでは、より多くのMYHの発現を認めた（図１１）。 When differentiation was induced with pmGM without meclizine from day 7 to day 14 of differentiation induction (D14, meclizine removal control), myotubes were observed (FIG. 8 left). Myotubes were not observed in cells that continued to be administered meclizine (FIG. 8 right). In the immunostaining, MYH was observed in the meclizine removal control (FIG. 9 left), whereas MYH was not observed in the cells continuously administered with meclizine (FIG. 9 right). Also in Western blot analysis, cells that continued to be administered meclizine had poor MYH expression, whereas the meclizine removal control showed MYH expression (FIG. 10). In addition, in the expression analysis by real-time PCR, cells that continued to be administered meclizine had poor expression of MYH1 and MYH7, whereas in the meclizine removal control, more MYH expression was observed (FIG. 11).
　以上の通り、メクリジンを除去することにより筋芽細胞の分化が誘導された。従って、メクリジンの分化抑制効果は永続的なものでは無く可逆的なものだと考えられた。 As described above, myoblast differentiation was induced by removing meclizine. Therefore, it was considered that the differentiation inhibitory effect of meclizine is not permanent but reversible.
Ｃ．メクリジンの効果の用量依存性
　メクリジンの細胞増殖促進効果及び分化抑制効果の用量依存性を調べた。
１．方法
　細胞増殖促進効果の用量依存性はBrdUアッセイによって検討した。実験方法は上記Ａ．１．（２）に準じ、メクリジン濃度0μM（コントロール）、2.5μM、5μM、10μM、20μMでの細胞増殖促進効果を比較した。分化抑制効果の用量依存性はウエスタンブロットで検討した。実験方法は上記Ａ．１．（５）に準じ、メクリジン濃度0μM（コントロール）、2.5μM、5μM、10μM、20μMでのMYH発現抑制効果をサンプルD7で比較した。 C. Dose dependency of meclizine effect The dose dependency of meclizine on cell growth promoting effect and differentiation inhibiting effect was examined.
1. Method The dose dependence of the cell growth promoting effect was examined by BrdU assay. The experimental method is as described in A. above. 1. According to (2), the cell proliferation promoting effects were compared at meclizine concentrations of 0 μM (control), 2.5 μM, 5 μM, 10 μM, and 20 μM. The dose dependence of the differentiation inhibitory effect was examined by Western blot. The experimental method is as described in A. above. 1. According to (5), the MYH expression inhibitory effect at a meclizine concentration of 0 μM (control), 2.5 μM, 5 μM, 10 μM, and 20 μM was compared in sample D7.
２．結果
　BrdUアッセイでは、メクリジンは、サンプルD0及びサンプルD7において、5μMをピークに用量依存的に細胞増殖効果を示した（図１２）。また、ウエスタンブロット解析でも、メクリジンは20μMまで用量依存的にMYHの発現を抑制した（図１３）。これらの結果より、メクリジンは用量依存的に分化を抑制する効果があると考えられた。 2. Results In the BrdU assay, meclizine showed a cell growth effect in a dose-dependent manner with a peak at 5 μM in sample D0 and sample D7 (FIG. 12). In Western blot analysis, meclizine suppressed MYH expression in a dose-dependent manner up to 20 μM (FIG. 13). From these results, meclizine was considered to have an effect of suppressing differentiation in a dose-dependent manner.
Ｄ．動物実験モデルでの検討
　メクリジンの効果を動物実験モデルで検討した。
１．方法
　C57BL/6Jマウスに生後16日目（0w）から専用の飼料（メクリジン含有飼料（4g/10kg）又はコントロール飼料）を摂食させ、ケージ内で飼育した。生後23日目(1w)、生後30日目(2w)、生後37日目(3w)に体重を測定した。生後37日目に屠殺し、レントゲン撮影とマイクロCT測定を行った。マイクロCT測定は以下の手順で行った。まず、マウスの全身を体軸に直行する断面で35μm幅スライスでスキャンした。マウスの仙腸関節をメルクマールにし、仙腸関節のある椎体を第1仙骨とした。第一仙骨を起点にして第４腰椎椎体を同定した。第四第五腰椎椎間板高位における体軸断面像画像データを画像解析ソフトに取り込んだ。傍脊柱筋（脊柱起立筋および多裂筋）の筋断面積を、画像解析ソフトにて計測した（図１６）。１椎間につき３スライスの画像を計測した。 D. Examination in animal experiment model The effect of meclizine was examined in an animal experiment model.
1. Method C57BL / 6J mice were fed a special feed (meclizine-containing feed (4 g / 10 kg) or control feed) from the 16th day of birth (0w) and reared in a cage. On the 23rd day after birth (1w), the 30th day after birth (2w) and the 37th day after birth (3w), body weight was measured. The animals were sacrificed on the 37th day after birth, and X-ray photography and micro CT measurement were performed. Micro CT measurement was performed according to the following procedure. First, the whole body of the mouse was scanned with a 35 μm wide slice in a cross section perpendicular to the body axis. The mouse sacroiliac joint was Merckmar, and the vertebral body with the sacroiliac joint was the first sacrum. The fourth lumbar vertebral body was identified starting from the first sacrum. The body axis cross-sectional image data at the fourth and fifth lumbar intervertebral intervertebral discs were taken into the image analysis software. The muscle cross-sectional area of the paraspinal muscles (vertical spine and multifidus) was measured with image analysis software (FIG. 16). Three slice images were measured per intervertebral space.
　一方、MDXマウス（筋ジストロフィーモデルマウス）に生後21日目（0w）から生後49日目（4w）まで、専用の飼料（メクリジン含有飼料（4g/10kg）又はコントロール飼料）を摂食させ、ケージ内で飼育した。生後28日目(1w)、生後35日目(2w)、生後42日目(3w)、生後49日目（4w）に体重を測定した。 On the other hand, MDX mice (muscular dystrophy model mice) were fed a special feed (meclizine-containing feed (4g / 10kg) or control feed) from the 21st day (0w) to the 49th day (4w) after birth. Reared in The body weight was measured on the 28th day after birth (1w), the 35th day after birth (2w), the 42nd day after birth (3w), and the 49th day after birth (4w).
２．結果
　C57BL/6Jマウスについて生後37日目で外観撮影とレントゲン撮影を行った。外観上、メクリジン投与マウスとコントロールのプロポーションは同じであり、メクリジン投与マウスの方が体格が大きかった（図１４）。薬剤投与前（生後16日目（0w））では、メクリジン投与マウスとコントロールに体重差はなかった（図１５）。一方、生後23日目(1w)、生後30日目(2w)及び生後37日目(3w)において、オス（n=7）、メス（n=9）ともにコントロールに比べメクリジン投与マウスの方が体重が多かった（図１５）。マイクロCT測定の結果では、コントロールに比べメクリジン投与マウスの方が傍脊柱筋断面積が大きかった（図１７）。結果はコントロールとの比で示した。以上の通り、正常マウスに対して体重及び傍脊柱筋断面積を増加させる作用がメクリジンに認められた。 2. Results Appearance photography and X-ray photography were performed on the C57BL / 6J mouse on the 37th day after birth. In appearance, the proportions of meclizine-administered mice and controls were the same, and meclizine-administered mice were larger in size (FIG. 14). Before drug administration (16 days after birth (0w)), there was no difference in body weight between mice administered with meclizine and controls (FIG. 15). On the other hand, at 23 days after birth (1w), 30 days after birth (2w) and 37 days after birth (3w), both male (n = 7) and female (n = 9) meclizine-treated mice He was heavy (Figure 15). As a result of micro CT measurement, the meclizine-administered mouse had a larger paraspinal muscle cross-sectional area than the control (FIG. 17). The results are shown as a ratio to the control. As described above, meclizine was found to increase body weight and paraspinal muscle cross-sectional area in normal mice.
　MDXマウスを用いた実験においても、オス（n=8）、メス（n=5）ともにコントロールに比べメクロジン投与マウスの方が体重増加量が多かった（図１８）。 Also in experiments using MDX mice, both male (n = 8) and female (n = 5) had more weight gain in mice treated with meclozin than in controls (FIG. 18).
＜まとめ＞
・メクリジンは用量依存的にヒト筋芽細胞の分化を抑制し、増殖を促進した。
・メクリジンの分化抑制効果は可逆的であった。
・メクリジンを投与したマウスは体重と筋肉断面積が増加した。
・筋ジストロフィーモデルマウスにおいてもメクリジンは体重を増加させた。筋ジストロフィー症に対し、メクロジン投与により筋量増加、体重増加の効果が期待される。 <Summary>
• Meclizine inhibited human myoblast differentiation and promoted proliferation in a dose-dependent manner.
・ The differentiation inhibitory effect of meclizine was reversible.
• Mice receiving meclizine increased body weight and muscle cross-sectional area.
・ Meclizine also increased body weight in muscular dystrophy model mice. For muscular dystrophy, meclodin administration is expected to increase muscle mass and weight.
　本発明の骨格筋増量剤は、既認可薬であるメクリジンを有効成分とする。メクリジンは抗ヒスタミン作用を有し、乗り物酔い止め薬としてOTC(over the counter)販売されている。50年以上安全に使用されてきた実績があり、至適服用量・副作用・禁忌など、安全性が確立されている。この事実は、臨床応用上の大きなメリットとなる。本発明の骨格筋増量剤を用いた治療戦略は、サルコペニアに代表される筋肉減少症の他、筋肉減少を主因または病因の一部とする他の疾患群（外傷や手術による筋損傷、廃用性筋萎縮、廃用症候群、易転倒性、神経原性筋萎縮、骨粗鬆症、変形性関節症、変形性脊椎症、側弯症や後弯症等の脊柱変形、肥満症、関節リウマチ、皮膚筋炎、多発筋炎、自己免疫疾患に伴う筋炎、ロコモーティブ症候群、メタボリック症候群、運動器不安定症、ダイナペニア（dynapenia）、フレイル、横紋筋融解症、筋ジストロフィー症、先天性筋症、先天性筋無力症候群、先天的筋低形成症、先天代謝障害、耐糖能異常ないし糖尿病若しくは癌等による悪液質等）に対する根本的治療法となり得る。また、若齢正常マウスにおいても筋肉増加効果を認めたことから、健常人における筋肉増量剤やフィジカルトレーニングの補助的薬剤となり得る。更には、畜産や養殖の分野への適用、応用も期待される。 The skeletal muscle bulking agent of the present invention contains meclizine, which is an already approved drug, as an active ingredient. Meclizine has an antihistamine effect and is marketed as an OTC (over-the-counter). It has been used safely for more than 50 years and has established safety such as optimal dose, side effects and contraindications. This fact is a great merit for clinical application. The therapeutic strategy using the skeletal muscle bulking agent of the present invention is not limited to sarcopenia and other disease groups (muscle damage caused by trauma or surgery, disuse). Muscular atrophy, disuse syndrome, easy fall, neurogenic muscular atrophy, osteoporosis, osteoarthritis, osteoarthritis, spinal deformity such as scoliosis and kyphosis, obesity, rheumatoid arthritis, dermatomyositis, frequent occurrence Myositis, myositis associated with autoimmune disease, locomotive syndrome, metabolic syndrome, motility instability, dynapenia, flail, rhabdomyolysis, muscular dystrophy, congenital myopathy, congenital myasthenia syndrome, congenital It can be a fundamental treatment for hypoxia, inborn errors of metabolism, glucose intolerance or cachexia due to diabetes or cancer). Moreover, since the muscle increase effect was recognized also in the young normal mouse | mouth, it can become an auxiliary | assistant medicine of the muscle bulking agent and physical training in a healthy person. Furthermore, application and application in the fields of livestock and aquaculture are also expected.
　この発明は、上記発明の実施の形態及び実施例の説明に何ら限定されるものではない。特許請求の範囲の記載を逸脱せず、当業者が容易に想到できる範囲で種々の変形態様もこの発明に含まれる。本明細書の中で明示した論文、公開特許公報、及び特許公報などの内容は、その全ての内容を援用によって引用することとする。 The present invention is not limited to the description of the embodiments and examples of the above invention. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims. The contents of papers, published patent gazettes, patent gazettes, and the like specified in this specification are incorporated by reference in their entirety.

Claims (11)

  1. 　メクリジン又はその薬学的に許容される塩を有効成分として含有する、骨格筋増量剤。 A skeletal muscle bulking agent containing meclizine or a pharmaceutically acceptable salt thereof as an active ingredient.
  2. 　有効成分が塩酸メクリジンである、請求項１に記載の治療薬。 The therapeutic agent according to claim 1, wherein the active ingredient is meclizine hydrochloride.
  3. 　請求項１又は２に記載の骨格筋増量剤を含有する、骨格筋の減少を主因又は病因の一部とする疾患用の治療薬。 A therapeutic agent for diseases mainly comprising a decrease in skeletal muscle or a part of the etiology, comprising the skeletal muscle bulking agent according to claim 1 or 2.
  4. 　前記疾患が筋肉減少症である、請求項３に記載の治療薬。 The therapeutic agent according to claim 3, wherein the disease is sarcopenia.
  5. 　前記疾患がサルコペニアである、請求項３に記載の治療薬。 The therapeutic agent according to claim 3, wherein the disease is sarcopenia.
  6. 　前記疾患が、外傷や手術による筋損傷、廃用性筋萎縮、廃用症候群、易転倒性、神経原性筋萎縮、骨粗鬆症、変形性関節症、変形性脊椎症、側弯症や後弯症等の脊柱変形、肥満症、関節リウマチ、皮膚筋炎、多発筋炎、自己免疫疾患に伴う筋炎、ロコモーティブ症候群、メタボリック症候群、運動器不安定症、ダイナペニア（dynapenia）、フレイル、横紋筋融解症、筋ジストロフィー症、先天性筋症、先天性筋無力症候群、先天的筋低形成症、先天代謝障害、耐糖能異常ないし糖尿病若しくは癌等による悪液質からなる群より選択される疾患である、請求項３に記載の治療薬。 The diseases include muscle damage due to trauma or surgery, disuse muscle atrophy, disuse syndrome, easy fall, neurogenic muscle atrophy, osteoporosis, osteoarthritis, osteoarthritis, scoliosis, kyphosis, etc. Spinal deformity, obesity, rheumatoid arthritis, dermatomyositis, polymyositis, myositis associated with autoimmune disease, locomotive syndrome, metabolic syndrome, motor organ instability, dynapenia, flail, rhabdomyolysis, muscular dystrophy, 4. The disease selected from the group consisting of congenital myopathy, congenital myasthenia syndrome, congenital hypoplasia, congenital metabolic disorders, impaired glucose tolerance or diabetes or cancer and cachexia. Remedy.
  7. 　前記疾患が筋ジストロフィー症である、請求項３に記載の治療薬。 The therapeutic agent according to claim 3, wherein the disease is muscular dystrophy.
  8. 　メクリジン又はその薬学的に許容される塩を治療上有効量、サルコペニア等の筋肉減少症、外傷や手術による筋損傷、廃用性筋萎縮、廃用症候群、易転倒性、神経原性筋萎縮、骨粗鬆症、変形性関節症、変形性脊椎症、側弯症や後弯症等の脊柱変形、肥満症、関節リウマチ、皮膚筋炎、多発筋炎、自己免疫疾患に伴う筋炎、ロコモーティブ症候群、メタボリック症候群、運動器不安定症、ダイナペニア（dynapenia）、フレイル、横紋筋融解症、筋ジストロフィー症、先天性筋症、先天性筋無力症候群、先天的筋低形成症、先天代謝障害、又は耐糖能異常ないし糖尿病若しくは癌等による悪液質の患者に投与するステップを含む、骨系統疾患の治療法。 A therapeutically effective amount of meclizine or a pharmaceutically acceptable salt thereof, sarcopenia or other muscle loss, trauma or surgical muscle damage, disuse muscle atrophy, disuse syndrome, easy fall, neurogenic muscle atrophy, Osteoporosis, osteoarthritis, osteoarthritis, spinal deformities such as scoliosis and kyphosis, obesity, rheumatoid arthritis, dermatomyositis, polymyositis, myositis associated with autoimmune diseases, locomotive syndrome, metabolic syndrome, motor organ failure Stabilization, dynapenia, flail, rhabdomyolysis, muscular dystrophy, congenital myopathy, congenital myasthenia syndrome, congenital hypoplasia, congenital metabolic disorders, glucose intolerance, diabetes or cancer A method for treating bone system diseases comprising the step of administering to a patient with cachexia.
  9. 　メクリジン又はその薬学的に許容される塩を治療上有効量、筋ジストロフィー症の患者に投与するステップを含む、筋ジストロフィー症の治療法。 A method for treating muscular dystrophy, comprising the step of administering meclizine or a pharmaceutically acceptable salt thereof in a therapeutically effective amount to a patient with muscular dystrophy.
  10. 　メクリジン又はその薬学的に許容される塩を有効成分として含有する、フィジカルトレーニング又はフィジカルエクササイズの補助栄養剤。 Physical training or physical exercise supplement containing meclizine or a pharmaceutically acceptable salt thereof as an active ingredient.
  11. 　メクリジン又はその薬学的に許容される塩を有効成分として含有する、畜産、愛玩動物又は養殖用の肉量増加剤。 A meat volume increasing agent for livestock, pets or aquaculture containing meclizine or a pharmaceutically acceptable salt thereof as an active ingredient.
PCT/JP2015/053580 2014-08-29 2015-02-10 Skeletal muscle bulking agent and use thereof WO2016031264A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2014176248 2014-08-29
JP2014-176248 2014-08-29

Publications (1)

Publication Number Publication Date
WO2016031264A1 true WO2016031264A1 (en) 2016-03-03

Family

ID=55399172

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2015/053580 WO2016031264A1 (en) 2014-08-29 2015-02-10 Skeletal muscle bulking agent and use thereof

Country Status (1)

Country Link
WO (1) WO2016031264A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024029173A1 (en) * 2022-08-03 2024-02-08 国立大学法人東海国立大学機構 Agent for preventing or ameliorating osteoporosis

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013032761A1 (en) * 2011-09-01 2013-03-07 Kraft Foods Global Brands Llc Degradable chewing gum and method of making the same

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013032761A1 (en) * 2011-09-01 2013-03-07 Kraft Foods Global Brands Llc Degradable chewing gum and method of making the same

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
MATSUSHITA M ET AL., PLOS ONE, vol. 8, no. 12, 2013, pages e81569 *
MUCHIR A ET AL., SKELETAL MUSCLE, vol. 3, no. 17, 2013 *
PRADO CMM ET AL., BRITISH JOURNAL OF CANCER, vol. 106, 2012, pages 1583 - 1586 *
ROBERTS PJ ET AL., ONCOGENE, vol. 26, 2007, pages 3291 - 3310 *
STROBEL S ET AL., ARTHRITIS RESEARCH & THERAPY, vol. 12, no. 2, 2010, pages R34 *
TANNAPFEL A ET AL., GUT, vol. 52, 2003, pages 706 - 712 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024029173A1 (en) * 2022-08-03 2024-02-08 国立大学法人東海国立大学機構 Agent for preventing or ameliorating osteoporosis

Similar Documents

Publication Publication Date Title
CN1759834B (en) Application of berberine or associated with Simvastatin in preparing product for preventing or curing disease or symptom related to blood fat
TWI314053B (en) Use of compounds that are effective as selective opiate receptor modulators
KR101802411B1 (en) Composition for preventing or treating of obesity comprising FAM19A5 and screening method for agent for treatment of obesity using the same
JPH07507569A (en) How to promote nitrogen retention in humans
JP4234439B2 (en) IL-12 expression regulator
US20160256529A1 (en) Combination therapy
CN114126599A (en) Methods and compositions for altering senescence-associated secretory phenotypes
WO2016031264A1 (en) Skeletal muscle bulking agent and use thereof
US8088799B2 (en) Pharmaceutical composition comprising a sodium hydrogen exchange inhibitor and an angiotensin converting enzyme inhibitor
CN111217885B (en) Medicinal cocrystal of akebia saponin D-organic acid and use thereof
JP6716330B2 (en) Uroplakin expression promoter
US20210338700A1 (en) Pharmaceutical use of anemoside b4 against acute gouty arthritis
CN104114165B (en) For treating the method and composition of diverticulosis
JPH03135918A (en) Immune-activating agent
US20180030102A1 (en) Application of metrnl protein in preparing hypolipidemic, hypoglycemic medicine
JP3738268B2 (en) Interleukin-6 production inhibitor
JP6566520B2 (en) Pharmaceutical composition for the treatment or prevention of muscle disease
CN116019801B (en) Amino acid composition for promoting secretion of endogenous growth hormone
JP6977979B2 (en) Nerve injury treatment or preventive medicine
US10463645B2 (en) Method for delaying the onset of pulmonary fibrosis or treating pulmonary fibrosis
WO2024029173A1 (en) Agent for preventing or ameliorating osteoporosis
CN117503789A (en) Application of CMP-Neu5Ac in preparation of food and medicine for promoting bone growth
WO2021106760A1 (en) Composition for preventing decrease in muscle mass, preventing weakness of muscular power, increasing muscle mass or strengthening muscular power
JP2015512948A (en) Method for treating hyperuricemia in patients with gout using halofenate or halofenic acid and anti-inflammatory agents
US20240115530A1 (en) Administration of baiba to increase benefit of losing weight of intermittent fasting

Legal Events

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

Ref document number: 15835933

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

NENP Non-entry into the national phase

Ref country code: JP

122 Ep: pct application non-entry in european phase

Ref document number: 15835933

Country of ref document: EP

Kind code of ref document: A1