WO2013137382A1 - Bone metabolism improving agent - Google Patents

Bone metabolism improving agent Download PDF

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Publication number
WO2013137382A1
WO2013137382A1 PCT/JP2013/057160 JP2013057160W WO2013137382A1 WO 2013137382 A1 WO2013137382 A1 WO 2013137382A1 JP 2013057160 W JP2013057160 W JP 2013057160W WO 2013137382 A1 WO2013137382 A1 WO 2013137382A1
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bone
hydroxyproline
bone metabolism
salt
improving
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PCT/JP2013/057160
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French (fr)
Japanese (ja)
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義宏 野村
一貴 上原
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協和発酵バイオ株式会社
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    • 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
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/401—Proline; Derivatives thereof, e.g. captopril
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis

Definitions

  • the present invention relates to a bone metabolism improving agent containing an N-acylated derivative of hydroxyproline or a salt thereof as an active ingredient.
  • osteoporosis is a disease that is characterized by loss of bone mass and strength and causes fragile fractures. In osteoporosis, bone formation does not catch up with excessive bone resorption (because the balance between bone formation by osteoblasts and bone resorption by osteoclasts is lost), and it is known that bone loss is caused. ing.
  • N-acylated derivatives of hydroxyproline or salts thereof are effective for improving arthritis (patent document 1), pressure ulcer (patent document 2), dry skin (patent document 3), liver damage (patent document 4), etc.
  • the effect of improving rough skin (Patent Document 5) has been reported.
  • N-acetylhydroxyproline, an N-acylated derivative of hydroxyproline is used as a cosmetic raw material and quasi-drug additive in Japan, and as a drug for wound healing (burn treatment) in Europe as Oxaceprol. Used as a raw material.
  • An object of the present invention is to provide an agent for improving bone metabolism that is effective in improving bone-related diseases.
  • the present invention relates to the following (1) to (5): (1) An agent for improving bone metabolism comprising an N-acylated derivative of hydroxyproline or a salt thereof as an active ingredient. (2) A bone resorption inhibitor containing an N-acylated derivative of hydroxyproline or a salt thereof as an active ingredient. (3) An osteoclast differentiation inhibitor containing an N-acylated derivative of hydroxyproline or a salt thereof as an active ingredient. (4) Use of a salt of an N-acylated derivative of hydroxyproline for the production of a bone metabolism improving agent. (5) A method for improving bone metabolism, wherein a subject in need of improving bone metabolism is provided with a sufficient amount of a salt of an N-acylated derivative of hydroxyproline to improve bone metabolism of the subject. A method for improving bone metabolism comprising the step of administering.
  • a safe and effective bone metabolism improving agent containing an N-acylated derivative of hydroxyproline or a salt thereof as an active ingredient can be provided.
  • the bone metabolism improving agent of the present invention can improve bone metabolism.
  • “improving bone metabolism” means improving the balance between bone formation by osteoblasts and bone resorption by osteoclasts performed in vivo.
  • improving the balance between bone formation and bone resorption means suppressing bone resorption.
  • inhibiting bone resorption means inhibiting osteoclast differentiation.
  • Hydroxyproline an N-acylated derivative of hydroxyproline used in the present invention, is widely present in nature as a major constituent amino acid component in collagen and as a constituent amino acid component of elastin.
  • Naturally-occurring hydroxyproline depends on whether proline is D-form or L-form, whether the position of hydroxyl group is 3-position or 4-position, and whether the stereoisomer is cis-form or trans-form. Eight types of stereoisomers are known.
  • cis-4-hydroxy-L-proline cis-4-hydroxy-D-proline, cis-4-hydroxy-D-proline, cis-3-hydroxy-L-proline, cis-3-hydroxy-D-proline, trans-4-hydroxy -L-proline, trans-4-hydroxy-D-proline, trans-3-hydroxy-L-proline, trans-3-hydroxy-D-proline and the like.
  • any hydroxyproline can be used, but trans-4-hydroxy-L-proline is preferably used.
  • Hydroxyproline can be obtained by acid hydrolysis of animal-derived collagen such as pigs and cows and purification by conventional methods, but hydroxyproline produced using microorganisms is more preferably used.
  • the microorganism includes a microorganism belonging to the genus selected from the genus Amycolatopsis, the genus Dactylosporangium and the genus Streptomyces, or the proline 3-hydroxylase or proline 4-position derived from the microorganism.
  • a microorganism into which a hydroxylase gene has been introduced can be used.
  • proline 3-hydroxylase or proline 4-hydroxylase gene derived from a microorganism belonging to the genus selected from the genus Amycolatopsis, Dactyrosporandium and Streptomyces to Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989), Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997), etc.
  • trans-4-hydroxy-L-proline is obtained by using proline 4-hydroxylase (Japanese Patent Laid-Open No. 7-313179) isolated from a microorganism belonging to the genus Amycolatopsis or Dactyrosporandium.
  • Cis-3-hydroxy-L-proline can be produced using a proline 3-hydroxylase isolated from a microorganism belonging to the genus Streptomyces (Japanese Patent Laid-Open No. 7-322885).
  • cis-4-hydroxyproline can be produced using proline 4-hydroxylase (WO2009 / 139365) isolated by a microorganism belonging to the genus Mesozobium.
  • acyl moiety of the N-acylated derivative of hydroxyproline used in the present invention examples include linear or branched acyl having 2 to 23 carbon atoms, and more specifically, acetyl, propionyl, butyryl, isobutyryl. Valeryl, hexanoyl, heptanoyl, octanoyl, decanoyl, eicosanoyl, etc., among which acetyl and propionyl are preferred.
  • the N-acylated derivative of hydroxyproline can be produced by a known method. That is, it is preferably prepared by N-acylation of hydroxyproline in an aqueous medium or an organic solvent using an active derivative (an acid anhydride, acid chloride, etc.) of a fatty acid having an alkyl group having 1 to 22 carbon atoms. it can.
  • the obtained N-acylated derivative of hydroxyproline can be purified using a conventional purification method such as crystallization or chromatography.
  • salts of N-acylated derivatives of hydroxyproline include alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as magnesium salt and calcium salt, ammonium salts such as ammonium and tetramethylammonium, morpholine And organic amine addition salts such as piperidine.
  • a salt of an N-acylated derivative of hydroxyproline can be administered as it is, but it is usually desirable to provide it as various preparations.
  • Preparations can be produced by any method well known in the technical field of pharmaceutics by mixing the active ingredient with one or more pharmacologically acceptable carriers.
  • the preparation may further contain any other active ingredient for treatment.
  • additives such as excipients, binders, disintegrants, lubricants, dispersants, suspending agents, emulsifiers, diluents, buffers, antioxidants, and bacterial inhibitors should be used. it can.
  • dosage forms include tablets, powders, granules, pills, suspensions, emulsions, soaking and decoction, capsules, syrups, solutions, elixirs, extracts, tinctures, fluid extracts, and other oral preparations. Any of parenteral agents such as injections, drops, creams, suppositories and the like may be used.
  • the dosage forms suitable for oral administration are tablets, powders and granules, sugars such as lactose, glucose, sucrose, mannitol, sorbitol, starches such as potato, wheat, corn, calcium carbonate, calcium sulfate , Inorganic substances such as sodium hydrogen carbonate, sodium chloride, excipients such as crystalline cellulose, licorice powder, gentian powder, etc., starch, agar, gelatin powder, crystalline cellulose, carmellose sodium, carmellose calcium, calcium carbonate, Disintegrants such as sodium bicarbonate and sodium alginate, magnesium stearate, talc, hydrogenated vegetable oil, macrogol, silicone oil and other lubricants, polyvinyl alcohol, hydroxypropylcellulose, methylcellulose, ethylcellulose, carmellose, gelatin Binders starch glue solution and the like, surfactants such as fatty acid esters can be formulated by adding a plasticizer such as glycerin.
  • a plasticizer
  • the dosage form suitable for oral administration is a liquid preparation such as syrup, water, sucrose, sorbitol, sugars such as fructose, glycols such as polyethylene glycol, propylene glycol, sesame oil, olive oil, soybean oil, etc. Oils, p-hydroxybenzoic acid esters and other preservatives, paraoxybenzoic acid derivatives such as methyl paraoxybenzoate, preservatives such as sodium benzoate, and flavors such as strawberry flavor and peppermint. can do.
  • a liquid preparation such as syrup, water, sucrose, sorbitol, sugars such as fructose, glycols such as polyethylene glycol, propylene glycol, sesame oil, olive oil, soybean oil, etc.
  • Oils, p-hydroxybenzoic acid esters and other preservatives, paraoxybenzoic acid derivatives such as methyl paraoxybenzoate, preservatives such as sodium benzoate, and flavors such as strawberry flavor and peppermint. can do.
  • the dosage form suitable for parenteral administration is an injection
  • it preferably comprises a sterile aqueous preparation containing an N-acylated derivative of hydroxyproline or a salt thereof that is isotonic with the blood of the recipient.
  • a solution for injection can be prepared using a carrier comprising a salt solution, a glucose solution, or a mixture of a salt solution and a glucose solution.
  • preparations suitable for oral administration include additives generally used in foods and drinks such as sweeteners, coloring agents, preservatives, thickening stabilizers, antioxidants, color formers, bleaches, fungicides, gum bases.
  • Sweeteners such as sweeteners, coloring agents, preservatives, thickening stabilizers, antioxidants, color formers, bleaches, fungicides, gum bases.
  • Bitterings enzymes, brighteners, acidulants, seasonings, emulsifiers, fortifiers, manufacturing agents, fragrances, spice extracts and the like may be added.
  • the ingestion form of the preparation is desirably the most effective in improving bone metabolism, and examples thereof include oral administration and parenteral administration such as intravenous, intraperitoneal or subcutaneous administration.
  • the concentration of the salt of the hydroxyproline N-acylated derivative in the bone metabolism-improving agent of the present invention is appropriately selected according to the type of preparation, the effect expected by administration of the preparation, etc.
  • the salt of the N-acylated derivative of hydroxyproline is usually 0.1 to 100% by weight, preferably 0.5 to 80% by weight, particularly preferably 1 to 70% by weight.
  • the dose and frequency of administration of the bone metabolism-improving agent of the present invention vary depending on the administration form, patient age, body weight, nature or severity of the condition to be treated, but usually N-acyl of hydroxyproline per adult day.
  • the salt of the derivatized derivative is usually administered once to several times a day so as to be 50 mg to 30 g, preferably 100 mg to 10 g, particularly preferably 200 mg to 3 g.
  • the administration period is not particularly limited, but is usually 1 day to 2 years, preferably 1 month to 12 months.
  • improvement means that by taking the above-mentioned improving agent, the onset of bone metabolism is prevented, the onset rate is reduced, or the symptoms at the time of onset are suppressed or treated.
  • the bone metabolism improving agent of the present invention can be used for improving bone metabolism.
  • the bone metabolism improving agent of the present invention can be used for the effects expected from the improvement of bone metabolism.
  • diseases related to bone include, for example, osteoporosis, juvenile osteoporosis, bone dysplasia, hypercalcemia, decreased bone mass associated with hyperparathyroidism, osteomalacia, osteocalcinosis, osteolytic bone disease, bone Necrosis, Paget's disease of bone, osteoarthritis, fracture, bone loss due to prosthesis fixation, periodontal bone loss, metastatic bone disease, bone loss due to cancer, bone loss due to aging, Examples include bone loss due to side effects of other medications (such as steroids), bone loss due to weightlessness, and other conditions involving bone abnormalities or bone damage.
  • other medications such as steroids
  • a salt of an N-acylated derivative of hydroxyproline can be used for producing a bone metabolism improving agent.
  • the present invention includes a method for improving bone metabolism.
  • the method of the invention comprises administering to a subject in need of improving bone metabolism an amount of a salt of an N-acylated derivative of hydroxyproline sufficient to improve the subject's bone metabolism.
  • the following is a test example for examining the bone metabolism improving effect of a salt of an N-acylated derivative of hydroxyproline.
  • Osteoclast Differentiation Inhibition RAW264 cells an ascites-derived macrophage cell line of BALB / c mice, which are osteoclast-like cells, were treated with 10% fetal bovine serum (FBS: GIBCO) and 1% antibiotics (PNS Antibiotic Mixture). : GIBCO) in an ⁇ -MEM medium (SIGMA) and used for the experiment. The cells were cultured in a 37 ° C., 5% CO 2 incubator.
  • RAW264 cells were seeded in a 96-well plate by 8 ⁇ 10 2 cells / well and cultured in a medium of 200 ⁇ L / Well.
  • RANKL receptor activator of NF- ⁇ B ligand: SIGMA
  • SIGMA receptor activator of NF- ⁇ B ligand
  • N-acetylhydroxyproline with a final concentration of 1.15 ⁇ 10-2 mol / L (Kyowa Hakko Bio) was added.
  • the control group was examined in three groups: RANKL-stimulated, RANKL-stimulated group, and RANKL-stimulated + N-acetylhydroxyproline group. Thereafter, the medium of 100 ⁇ L / well was changed every other day and cultured for 6 days.
  • TRAP thyroid-resistant acid phosphatase
  • Primary Cell a staining kit (Primary Cell). The number of mature osteoclasts, which are multinucleated osteoclasts having three or more nuclei, was counted among the stained osteoclasts.
  • N-acetylhydroxyproline has an osteoclast differentiation inhibitory action, and it was shown to be a bone resorption inhibitor in osteoclasts and a bone metabolism improver in vivo.
  • N-acetylhydroxyproline was manufactured by Kyowa Hakko Bio.
  • composition (component; blending ratio% by weight) N-acetylhydroxyproline; 20%, lactose; 40%, calcium lactate; 10%, magnesium stearate; 25%, calcium carbonate; 5%
  • composition (component; blending g) N-acetylhydroxyproline: 30 g, vitamin C: 1 g, vitamin B1: 0.005 g, vitamin B2: 0.01 g, vitamin B6: 0.025 g, liquid sugar: 150 g, citric acid: 3 g , Fragrance; 1 g
  • hard capsules 360 mg per capsule
  • Composition component; formulation mg
  • N-acetylhydroxyproline 250 mg
  • lactose 60 mg
  • corn starch 30 mg
  • hydroxypropylcellulose 20 mg
  • Lactose and corn starch are added to N-acetylhydroxyproline and mixed, and an aqueous solution of hydroxypropylcellulose is added thereto and kneaded.
  • granules are produced by an ordinary method using an extrusion granulator.
  • a hard capsule is produced by filling the granule into a gelatin hard capsule.
  • soft capsules (170 mg per capsule) are produced by the following method.
  • Composition component; compounding mg) N-acetylhydroxyproline; 25 mg, soybean oil; 120 mg, glucosamine; 25 mg
  • soft capsules are produced by filling the soft capsules using a rotary soybean automatic molding machine according to a conventional method.
  • cream phase component Squalane 5.0 g, olive oil 3.0 g, hydrogenated lanolin 2.0 g, beeswax 2.5 g, monoglyceryl stearate 2.0 g, polyoxyethylene stearyl ether 2.5 g, propylparaben 1 .5 g, 1,3-butylene glycol 5.0 g, perfume, (water phase component) N-acetyl-trans-4-hydroxy-L-proline 5.0 g, methylparaben 0.5 g, carbopol 9400.03 g, tri Ethanolamine 0.3g, refined starch syrup 70.97g (Preparation method) Heat the oil phase component and water phase component to 80 ° C to homogenize them, add the water phase to the oil phase with stirring, emulsify and cool to obtain cream It was.
  • ointment 0.2 g white petrolatum, 1.0 g stearyl alcohol, 0.3 g sodium lauryl sulfate, (aqueous phase component) 5.0 g N-acetyl-trans-4-hydroxy-L-proline, propylene Glycol 5.0 g ⁇ ⁇ ⁇ ethyl paraoxybenzoate, 0.02 g ⁇ ⁇ ⁇ butyl paraoxybenzoate 1.0 g purified water tank 81.45 g (preparation method) The phase component and the aqueous phase component were each heated to 80 ° C. to make the water phase oil phase While stirring, the mixture was cooled after emulsification to obtain an ointment.
  • tape agent adheresive solvent

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Abstract

Provided is a bone metabolism improving agent which comprises, as the active ingredient, an N-acylated derivative of hydroxyproline that is efficacious in improving bone-related diseases or a salt thereof. Examples of the bone-related diseases include osteoporosis, juvenile osteoporosis, dysostosis, hypercalcemia, bone loss associated with hyperparathyroidism, osteomalacia, osteohalisteresis, osteolytic diseases, osteonecrosis, bone Paget's disease, osteoarthritis, bone fracture, bone loss caused by prosthesis implantation, periodontal bone loss, metastatic bone diseases, bone loss caused by cancer, bone loss caused by aging, bone loss as a side effect of therapy with other drugs (such as steroids), bone loss in gravityless environment and other symptoms associated with bone abnormality or bone injury.

Description

骨代謝改善剤Bone metabolism improver
　本発明は、ヒドロキシプロリンのＮ－アシル化誘導体またはその塩を有効成分として含有する骨代謝改善剤に関する。 The present invention relates to a bone metabolism improving agent containing an N-acylated derivative of hydroxyproline or a salt thereof as an active ingredient.
　近年、高齢者人口の急激な増加やカルシウム摂取量の低下により、骨粗鬆症をはじめとする骨に関する疾患を発症する患者が増加している。従って、骨に関する疾患に対する安全で且つ有効な予防・治療法の開発が望まれている。例えば骨粗鬆症は、骨質量と骨強度の損失を特徴とし、脆弱性骨折の原因となる疾患である。骨粗鬆症においては、過剰な骨吸収に対して骨形成が追いつかないことから（骨芽細胞による骨形成と破骨細胞による骨吸収とのバランスが崩れることから）骨量減少が引き起こされることが知られている。 In recent years, the number of patients who develop bone-related diseases such as osteoporosis is increasing due to a rapid increase in the elderly population and a decrease in calcium intake. Therefore, development of a safe and effective preventive / therapeutic method for bone related diseases is desired. For example, osteoporosis is a disease that is characterized by loss of bone mass and strength and causes fragile fractures. In osteoporosis, bone formation does not catch up with excessive bone resorption (because the balance between bone formation by osteoblasts and bone resorption by osteoclasts is lost), and it is known that bone loss is caused. ing.
　ヒドロキシプロリンのＮ－アシル化誘導体またはその塩には関節炎（特許文献１）、褥瘡（特許文献２）、皮膚の乾燥（特許文献３）、肝障害（特許文献４）などの改善効果やエタノールによる肌荒れの改善効果（特許文献５）などが報告されている。ヒドロキシプロリンのＮ－アシル化誘導体である、Ｎ－アセチルヒドロキシプロリンは、日本国内では化粧品原料および医薬部外品添加剤として、欧州ではオキサセプロール (Ｏｘａｃｅｐｒｏｌ)として、創傷治癒（火傷治療）の医薬品原料として使用されている。 N-acylated derivatives of hydroxyproline or salts thereof are effective for improving arthritis (patent document 1), pressure ulcer (patent document 2), dry skin (patent document 3), liver damage (patent document 4), etc. The effect of improving rough skin (Patent Document 5) has been reported. N-acetylhydroxyproline, an N-acylated derivative of hydroxyproline, is used as a cosmetic raw material and quasi-drug additive in Japan, and as a drug for wound healing (burn treatment) in Europe as Oxaceprol. Used as a raw material.
　しかしながら、ヒドロキシプロリンのＮ－アシル化誘導体またはその塩により骨代謝が改善することは知られていない。 However, it is not known that bone metabolism is improved by N-acylated derivatives of hydroxyproline or salts thereof.
ＷＯ２００２／０５５０７３WO2002 / 055073 ＷＯ２００４／０２８５３１WO2004 / 028531 ＷＯ２００６／０３３３５５WO2006 / 033355 ＷＯ２００７／１２９７２５WO2007 / 129725 ＷＯ２００８／１２６７８２WO2008 / 126782
　本発明の目的は、骨に関する疾患の改善に有効な骨代謝改善剤を提供することにある。 An object of the present invention is to provide an agent for improving bone metabolism that is effective in improving bone-related diseases.
　本発明は、下記の（１）～（５）に関する
（１）ヒドロキシプロリンのＮ－アシル化誘導体またはその塩を有効成分として含有する骨代謝改善剤。
（２）ヒドロキシプロリンのＮ－アシル化誘導体またはその塩を有効成分として含有する骨吸収抑制剤。
（３）ヒドロキシプロリンのＮ－アシル化誘導体またはその塩を有効成分として含有する破骨細胞分化抑制剤。
（４）骨代謝改善剤の製造のためのヒドロキシプロリンのＮ－アシル化誘導体の塩の使用。
（５）骨代謝を改善する方法であって、骨代謝を改善させる必要のある被験体に該被験体の骨代謝を改善させるのに十分な量のヒドロキシプロリンのＮ－アシル化誘導体の塩を投与する工程を含む、骨代謝を改善する方法。 The present invention relates to the following (1) to (5): (1) An agent for improving bone metabolism comprising an N-acylated derivative of hydroxyproline or a salt thereof as an active ingredient.
(2) A bone resorption inhibitor containing an N-acylated derivative of hydroxyproline or a salt thereof as an active ingredient.
(3) An osteoclast differentiation inhibitor containing an N-acylated derivative of hydroxyproline or a salt thereof as an active ingredient.
(4) Use of a salt of an N-acylated derivative of hydroxyproline for the production of a bone metabolism improving agent.
(5) A method for improving bone metabolism, wherein a subject in need of improving bone metabolism is provided with a sufficient amount of a salt of an N-acylated derivative of hydroxyproline to improve bone metabolism of the subject. A method for improving bone metabolism comprising the step of administering.
　本発明により、ヒドロキシプロリンのＮ－アシル化誘導体またはその塩を有効成分として含有する、安全で効果的な骨代謝改善剤を提供することができる。 According to the present invention, a safe and effective bone metabolism improving agent containing an N-acylated derivative of hydroxyproline or a salt thereof as an active ingredient can be provided.
図１は、ＲＡＷ２６４細胞を各サンプルで刺激し、６日間培養後の１ウェルあたりの成熟破骨細胞数を示したものである。平均±ＳD、Ｎ＝６、＊＊＊はＰ＜０．００１の有意差を示す。FIG. 1 shows the number of mature osteoclasts per well after RAW264 cells were stimulated with each sample and cultured for 6 days. Mean ± SD, N = 6, *** indicates a significant difference of P <0.001.
　本発明の骨代謝改善剤は、骨代謝を改善させることができる。本発明において「骨代謝を改善させる」とは、生体内で行われている骨芽細胞による骨形成と破骨細胞による骨吸収とのバランスを改善させることを意味する。また、骨形成と骨吸収とのバランスを改善させるとは、骨吸収を抑制することを含んだものを意味する。なお、骨吸収を抑制するとは、破骨細胞の分化を抑制することを含んだものを意味する。 The bone metabolism improving agent of the present invention can improve bone metabolism. In the present invention, “improving bone metabolism” means improving the balance between bone formation by osteoblasts and bone resorption by osteoclasts performed in vivo. In addition, improving the balance between bone formation and bone resorption means suppressing bone resorption. Note that inhibiting bone resorption means inhibiting osteoclast differentiation.
　本発明で用いられるヒドロキシプロリンのＮ－アシル化誘導体のヒドロキシプロリンは、コラーゲン中の主要構成アミノ酸成分として、また、エラスチンの構成アミノ酸成分として自然界に広く存在する。天然に存在するヒドロキシプロリンとしては、プロリンがＤ体であるかＬ体であるか、水酸基の位置が３位か４位か、およびその立体異性体がシス体であるかトランス体であるかによって、８種類の立体異性体が知られている。具体的には、シス－４－ヒドロキシ－Ｌ－プロリン、シス－４－ヒドロキシ－Ｄ－プロリン、シス－３－ヒドロキシ－Ｌ－プロリン、シス－３－ヒドロキシ－Ｄ－プロリン、トランス－４－ヒドロキシ－Ｌ－プロリン、トランス－４－ヒドロキシ－Ｄ－プロリン、トランス－３－ヒドロキシ－Ｌ－プロリンおよびトランス－３－ヒドロキシ－Ｄ－プロリン等があげられる。本発明では、いずれのヒドロキシプロリンも用いることができるが、トランス－４－ヒドロキシ－Ｌ－プロリンが好ましく用いられる。 Hydroxyproline, an N-acylated derivative of hydroxyproline used in the present invention, is widely present in nature as a major constituent amino acid component in collagen and as a constituent amino acid component of elastin. Naturally-occurring hydroxyproline depends on whether proline is D-form or L-form, whether the position of hydroxyl group is 3-position or 4-position, and whether the stereoisomer is cis-form or trans-form. Eight types of stereoisomers are known. Specifically, cis-4-hydroxy-L-proline, cis-4-hydroxy-D-proline, cis-3-hydroxy-L-proline, cis-3-hydroxy-D-proline, trans-4-hydroxy -L-proline, trans-4-hydroxy-D-proline, trans-3-hydroxy-L-proline, trans-3-hydroxy-D-proline and the like. In the present invention, any hydroxyproline can be used, but trans-4-hydroxy-L-proline is preferably used.
　ヒドロキシプロリンは、ブタやウシ等の動物由来のコラーゲンを酸加水分解し、常法により精製して取得することができるが、微生物を用いて製造したヒドロキシプロリンがより好適に用いられる。 Hydroxyproline can be obtained by acid hydrolysis of animal-derived collagen such as pigs and cows and purification by conventional methods, but hydroxyproline produced using microorganisms is more preferably used.
　微生物としては、アミコラトプシス（Ａｍｙｃｏｌａｔｏｐｓｉｓ）属、ダクチロスポランジウム（Ｄａｃｔｙｌｏｓｐｏｒａｎｇｉｕｍ）属およびストレプトマイセス（Ｓｔｒｅｐｔｏｍｙｃｅｓ）属から選ばれる属に属する微生物または該微生物由来のプロリン３位水酸化酵素またはプロリン４位水酸化酵素遺伝子を導入された微生物等を用いることができる。 The microorganism includes a microorganism belonging to the genus selected from the genus Amycolatopsis, the genus Dactylosporangium and the genus Streptomyces, or the proline 3-hydroxylase or proline 4-position derived from the microorganism. A microorganism into which a hydroxylase gene has been introduced can be used.
　アミコラトプシス属、ダクチロスポランジウム属およびストレプトマイセス属から選ばれる属に属する微生物由来のプロリン３位水酸化酵素またはプロリン４位水酸化酵素遺伝子の微生物への導入は、Ｍｏｌｅｃｕｌａｒ　Ｃｌｏｎｉｎｇ，Ａ　Ｌａｂｏｒａｔｏｒｙ　Ｍａｎｕａｌ，Ｓｅｃｏｎｄ　Ｅｄｉｔｉｏｎ，Ｃｏｌｄ　Ｓｐｒｉｎｇ　Ｈａｒｂｏｒ　Ｌａｂｏｒａｔｏｒｙ　Ｐｒｅｓｓ（１９８９）、Ｃｕｒｒｅｎｔ　Ｐｒｏｔｏｃｏｌｓ　ｉｎ　Ｍｏｌｅｃｕｌａｒ　Ｂｉｏｌｏｇｙ，Ｊｏｈｎ　Ｗｉｌｅｙ　＆　Ｓｏｎｓ（１９８７－１９９７）等に記載された方法に準じて行うことができる。 Introduction of a proline 3-hydroxylase or proline 4-hydroxylase gene derived from a microorganism belonging to the genus selected from the genus Amycolatopsis, Dactyrosporandium and Streptomyces to Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989), Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997), etc.
　また、例えば、トランス－４－ヒドロキシ－Ｌ－プロリンは、アミコラトプシス属またはダクチロスポランジウム属に属する微生物より単離したプロリン４位水酸化酵素（特開平７－３１３１７９号公報）を用いて製造することができ、シス－３－ヒドロキシ－Ｌ－プロリンは、ストレプトマイセス属に属する微生物より単離したプロリン３位水酸化酵素（特開平７－３２２８８５号公報）を用いて製造することができ〔バイオインダストリー，１４，３１（１９９７）〕、シス－４－ヒドロキシプロリンはメソリゾビウム属に属する微生物により単離したプロリン４位水酸化酵素（ＷＯ２００９／１３９３６５）を用いて製造することができる。 Further, for example, trans-4-hydroxy-L-proline is obtained by using proline 4-hydroxylase (Japanese Patent Laid-Open No. 7-313179) isolated from a microorganism belonging to the genus Amycolatopsis or Dactyrosporandium. Cis-3-hydroxy-L-proline can be produced using a proline 3-hydroxylase isolated from a microorganism belonging to the genus Streptomyces (Japanese Patent Laid-Open No. 7-322885). [Bioindustry, 14, 31 (1997)], and cis-4-hydroxyproline can be produced using proline 4-hydroxylase (WO2009 / 139365) isolated by a microorganism belonging to the genus Mesozobium.
　本発明で用いるヒドロキシプロリンのＮ－アシル化誘導体のアシル部分としては、例えば、炭素数２～２３の直鎖または分岐状のアシルがあげられ、より具体的には、アセチル、プロピオニル、ブチリル、イソブチリル、バレリル、ヘキサノイル、ヘプタノイル、オクタノイル、デカノイル、エイコサノイル等があげられるが、中でも、アセチル、プロピオニルが好ましい。 Examples of the acyl moiety of the N-acylated derivative of hydroxyproline used in the present invention include linear or branched acyl having 2 to 23 carbon atoms, and more specifically, acetyl, propionyl, butyryl, isobutyryl. Valeryl, hexanoyl, heptanoyl, octanoyl, decanoyl, eicosanoyl, etc., among which acetyl and propionyl are preferred.
　ヒドロキシプロリンのＮ－アシル化誘導体は、公知の方法により製造することができる。即ち、好ましくは炭素数１～２２のアルキル基を有する脂肪酸の活性誘導体（酸無水物、酸塩化物等）を用い、ヒドロキシプロリンを水性媒体中または有機溶媒中でＮ－アシル化することにより調製できる。得られたヒドロキシプロリンのＮ－アシル化誘導体は、結晶化、クロマトグラフィー等の通常の精製法を用いて精製することができる。 The N-acylated derivative of hydroxyproline can be produced by a known method. That is, it is preferably prepared by N-acylation of hydroxyproline in an aqueous medium or an organic solvent using an active derivative (an acid anhydride, acid chloride, etc.) of a fatty acid having an alkyl group having 1 to 22 carbon atoms. it can. The obtained N-acylated derivative of hydroxyproline can be purified using a conventional purification method such as crystallization or chromatography.
　ヒドロキシプロリンのＮ－アシル化誘導体の塩における塩としては、ナトリウム塩、カリウム塩等のアルカリ金属塩、マグネシウム塩、カルシウム塩等のアルカリ土類金属塩、アンモニウム、テトラメチルアンモニウム等のアンモニウム塩、モルホリン、ピペリジン等の付加した有機アミン付加塩等があげられる。 Examples of salts of N-acylated derivatives of hydroxyproline include alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as magnesium salt and calcium salt, ammonium salts such as ammonium and tetramethylammonium, morpholine And organic amine addition salts such as piperidine.
　本発明の骨代謝改善剤としては、ヒドロキシプロリンのＮ－アシル化誘導体の塩をそのまま投与することも可能であるが、通常各種の製剤として提供するのが望ましい。 As the bone metabolism improving agent of the present invention, a salt of an N-acylated derivative of hydroxyproline can be administered as it is, but it is usually desirable to provide it as various preparations.
　製剤は、有効成分を薬理学的に許容される一種またはそれ以上の担体と一緒に混合し、製剤学の技術分野においてよく知られている任意の方法により製造することができる。また、該製剤は更に任意の他の治療のための有効成分を含有していてもよい。 Preparations can be produced by any method well known in the technical field of pharmaceutics by mixing the active ingredient with one or more pharmacologically acceptable carriers. The preparation may further contain any other active ingredient for treatment.
　製剤化する際には、賦形剤、結合剤、崩壊剤、潤沢剤、分散剤、懸濁剤、乳化剤、希釈剤、緩衝剤、抗酸化剤、細菌抑制剤等の添加剤を用いることができる。 When formulating, additives such as excipients, binders, disintegrants, lubricants, dispersants, suspending agents, emulsifiers, diluents, buffers, antioxidants, and bacterial inhibitors should be used. it can.
　剤形としては、例えば錠剤、散剤、顆粒剤、丸剤、懸濁剤、乳剤、浸剤・煎剤、カプセル剤、シロップ剤、液剤、エリキシル剤、エキス剤、チンキ剤、流エキス剤等の経口剤、注射剤、点滴剤、クリーム剤、坐剤等の非経口剤のいずれでもよい。 Examples of dosage forms include tablets, powders, granules, pills, suspensions, emulsions, soaking and decoction, capsules, syrups, solutions, elixirs, extracts, tinctures, fluid extracts, and other oral preparations. Any of parenteral agents such as injections, drops, creams, suppositories and the like may be used.
　例えば、経口投与に適当な剤形が、錠剤、散剤および顆粒剤等の場合には、乳糖、ブドウ糖、蔗糖、マンニトール、ソルビトール等の糖類、バレイショ、コムギ、トウモロコシ等の澱粉、炭酸カルシウム、硫酸カルシウム、炭酸水素ナトリウム、塩化ナトリウム等の無機物、結晶セルロース、カンゾウ末、ゲンチアナ末等の植物末等の賦形剤、澱粉、寒天、ゼラチン末、結晶セルロース、カルメロースナトリウム、カルメロースカルシウム、炭酸カルシウム、炭酸水素ナトリウム、アルギン酸ナトリウム等の崩壊剤、ステアリン酸マグネシウム、タルク、水素添加植物油、マクロゴール、シリコーン油等の滑沢剤、ポリビニールアルコール、ヒドロキシプロピルセルロース、メチルセルロース、エチルセルロース、カルメロース、ゼラチン、澱粉のり液等の結合剤、脂肪酸エステル等の界面活性剤、グリセリン等の可塑剤などを添加して製剤化することができる。 For example, when the dosage forms suitable for oral administration are tablets, powders and granules, sugars such as lactose, glucose, sucrose, mannitol, sorbitol, starches such as potato, wheat, corn, calcium carbonate, calcium sulfate , Inorganic substances such as sodium hydrogen carbonate, sodium chloride, excipients such as crystalline cellulose, licorice powder, gentian powder, etc., starch, agar, gelatin powder, crystalline cellulose, carmellose sodium, carmellose calcium, calcium carbonate, Disintegrants such as sodium bicarbonate and sodium alginate, magnesium stearate, talc, hydrogenated vegetable oil, macrogol, silicone oil and other lubricants, polyvinyl alcohol, hydroxypropylcellulose, methylcellulose, ethylcellulose, carmellose, gelatin Binders starch glue solution and the like, surfactants such as fatty acid esters can be formulated by adding a plasticizer such as glycerin.
　経口投与に適当な剤形が、シロップ剤のような液体調製物である場合は、水、蔗糖、ソルビトール、果糖等の糖類、ポリエチレングリコール、プロピレングリコール等のグリコール類、ごま油、オリーブ油、大豆油等の油類、ｐ－ヒドロキシ安息香酸エステル類等の防腐剤、パラオキシ安息香酸メチル等のパラオキシ安息香酸誘導体、安息香酸ナトリウム等の保存剤、ストロベリーフレーバー、ペパーミント等のフレーバー類などを添加して製剤化することができる。 If the dosage form suitable for oral administration is a liquid preparation such as syrup, water, sucrose, sorbitol, sugars such as fructose, glycols such as polyethylene glycol, propylene glycol, sesame oil, olive oil, soybean oil, etc. Oils, p-hydroxybenzoic acid esters and other preservatives, paraoxybenzoic acid derivatives such as methyl paraoxybenzoate, preservatives such as sodium benzoate, and flavors such as strawberry flavor and peppermint. can do.
　非経口投与に適当な剤形が、注射剤の場合には、好ましくは受容者の血液と等張であるヒドロキシプロリンのＮ－アシル化誘導体またはその塩を含む滅菌水性剤からなる。例えば、塩溶液、ブドウ糖溶液または塩溶液とブドウ糖溶液の混合物からなる担体等を用いて注射用の溶液を調製することができる。 When the dosage form suitable for parenteral administration is an injection, it preferably comprises a sterile aqueous preparation containing an N-acylated derivative of hydroxyproline or a salt thereof that is isotonic with the blood of the recipient. For example, a solution for injection can be prepared using a carrier comprising a salt solution, a glucose solution, or a mixture of a salt solution and a glucose solution.
　また、経口投与に適当な製剤には、一般に飲食品に用いられる添加剤、例えば甘味料、着色料、保存料、増粘安定剤、酸化防止剤、発色剤、漂白剤、防かび剤、ガムベース、苦味料、酵素、光沢剤、酸味料、調味料、乳化剤、強化剤、製造用剤、香料、香辛料抽出物等が添加されてもよい。 In addition, preparations suitable for oral administration include additives generally used in foods and drinks such as sweeteners, coloring agents, preservatives, thickening stabilizers, antioxidants, color formers, bleaches, fungicides, gum bases. Bitterings, enzymes, brighteners, acidulants, seasonings, emulsifiers, fortifiers, manufacturing agents, fragrances, spice extracts and the like may be added.
　製剤の摂取形態は、骨代謝の改善に際し最も効果的なものを使用するのが望ましく、経口投与または、例えば静脈内、腹膜内もしくは皮下投与等の非経口投与をあげることができる。 The ingestion form of the preparation is desirably the most effective in improving bone metabolism, and examples thereof include oral administration and parenteral administration such as intravenous, intraperitoneal or subcutaneous administration.
　本発明の骨代謝改善剤中のヒドロキシプロリンのＮ－アシル化誘導体の塩の濃度は、製剤の種類、該製剤の投与により期待する効果等に応じて適宜選択されるが、例えば経口剤の場合、ヒドロキシプロリンのＮ－アシル化誘導体の塩として、通常は０．１～１００重量％、好ましくは０．５～８０重量％、特に好ましくは１～７０重量％である。 The concentration of the salt of the hydroxyproline N-acylated derivative in the bone metabolism-improving agent of the present invention is appropriately selected according to the type of preparation, the effect expected by administration of the preparation, etc. The salt of the N-acylated derivative of hydroxyproline is usually 0.1 to 100% by weight, preferably 0.5 to 80% by weight, particularly preferably 1 to 70% by weight.
　本発明の骨代謝改善剤の投与量および投与回数は、投与形態、患者の年齢、体重、治療すべき症状の性質もしくは重篤度により異なるが、通常、成人一日当り、ヒドロキシプロリンのＮ－アシル化誘導体の塩として通常は５０ｍｇ～３０ｇ、好ましくは１００ｍｇ～１０ｇ、特に好ましくは２００ｍｇ～３ｇとなるように、一日一回ないし数回投与する。投与期間は、特に限定されないが、通常は１日間～２年間、好ましくは１ヶ月間～１２ヶ月間である。 The dose and frequency of administration of the bone metabolism-improving agent of the present invention vary depending on the administration form, patient age, body weight, nature or severity of the condition to be treated, but usually N-acyl of hydroxyproline per adult day. The salt of the derivatized derivative is usually administered once to several times a day so as to be 50 mg to 30 g, preferably 100 mg to 10 g, particularly preferably 200 mg to 3 g. The administration period is not particularly limited, but is usually 1 day to 2 years, preferably 1 month to 12 months.
　本発明において「改善」とは、上記改善剤を摂取することで、骨代謝の発症を予防する、発症率を低減させる、または発症時の症状を抑制、治療する、などの効果を及ぼすことをいう。 In the present invention, “improvement” means that by taking the above-mentioned improving agent, the onset of bone metabolism is prevented, the onset rate is reduced, or the symptoms at the time of onset are suppressed or treated. Say.
　本発明の骨代謝改善剤は、骨代謝を改善するために使用することがきる。本発明の骨代謝改善剤は、骨代謝の改善から期待される効果に使用することができる。骨代謝の改善から期待される効果として、骨に関する疾患に使用することができる。骨に関する疾患は例えば骨粗鬆症、若年性骨粗鬆症、骨形成不全、高カルシウム血症、上皮小体機能亢進症に伴う骨量の減少、骨軟化症、骨石灰脱失症、骨溶解性骨疾患、骨壊死、骨パジェット病、骨関節症、骨折、人工補装具固定による骨量の減少、歯周の骨の喪失、転移性の骨疾患、癌による骨量の減少、加齢による骨量の減少、他の薬物療法(ステロイドのような)の副作用による骨量の減少、無重力による骨量の減少、その他骨異常または骨損傷を伴う状態などがあげられる。 The bone metabolism improving agent of the present invention can be used for improving bone metabolism. The bone metabolism improving agent of the present invention can be used for the effects expected from the improvement of bone metabolism. As an effect expected from improvement of bone metabolism, it can be used for diseases related to bone. Diseases related to bones include, for example, osteoporosis, juvenile osteoporosis, bone dysplasia, hypercalcemia, decreased bone mass associated with hyperparathyroidism, osteomalacia, osteocalcinosis, osteolytic bone disease, bone Necrosis, Paget's disease of bone, osteoarthritis, fracture, bone loss due to prosthesis fixation, periodontal bone loss, metastatic bone disease, bone loss due to cancer, bone loss due to aging, Examples include bone loss due to side effects of other medications (such as steroids), bone loss due to weightlessness, and other conditions involving bone abnormalities or bone damage.
　さらに本発明においては、骨代謝改善剤を製造するために、ヒドロキシプロリンのＮ－アシル化誘導体の塩を使用され得る。 Furthermore, in the present invention, a salt of an N-acylated derivative of hydroxyproline can be used for producing a bone metabolism improving agent.
　さらにまた本発明は、骨代謝を改善させるための方法を包含する。本発明の方法は、骨代謝を改善させる必要のある被験体に、該被験体の骨代謝を改善させるのに十分な量のヒドロキシプロリンのＮ－アシル化誘導体の塩を投与する工程を含む。 Furthermore, the present invention includes a method for improving bone metabolism. The method of the invention comprises administering to a subject in need of improving bone metabolism an amount of a salt of an N-acylated derivative of hydroxyproline sufficient to improve the subject's bone metabolism.
　以下に、ヒドロキシプロリンのＮ－アシル化誘導体の塩の骨代謝改善効果を調べた試験例を示す。 The following is a test example for examining the bone metabolism improving effect of a salt of an N-acylated derivative of hydroxyproline.
試験例 Test example
　破骨細胞分化抑制作用の検討
　破骨細胞様細胞であるＢＡＬＢ/ｃマウスの腹水由来マクロファージ細胞株であるＲＡＷ２６４細胞を１０％ｆｅｔａｌ　ｂｏｖｉｎｅ　ｓｅｒｕｍ（ＦＢＳ：ＧＩＢＣＯ社）および１％抗生物質（ＰＮＳ　Ａｎｔｉｂｉｏｔｉｃ　Ｍｉｘｔｕｒｅ：ＧＩＢＣＯ社）を含むα―ＭＥＭ培地（ＳＩＧＭＡ社）中で培養し、実験に供した。細胞の培養は３７℃、５％ＣＯ２インキュベータ内で行った。 Examination of Osteoclast Differentiation Inhibition RAW264 cells, an ascites-derived macrophage cell line of BALB / c mice, which are osteoclast-like cells, were treated with 10% fetal bovine serum (FBS: GIBCO) and 1% antibiotics (PNS Antibiotic Mixture). : GIBCO) in an α-MEM medium (SIGMA) and used for the experiment. The cells were cultured in a 37 ° C., 5% CO 2 incubator.
　ＲＡＷ２６４細胞を９６ウェルプレートに８Ｘ１０２ｃｅｌｌｓ／ｗｅｌｌずつ播種し、２００μＬ／Ｗｅｌｌの培地で培養した。２４時間の培養後、終濃度５０ｎｇ／ｍＬの破骨細胞分化誘導因子であるＲＡＮＫＬ（ｒｅｃｅｐｔｏｒ　ａｃｔｉｖａｔｏｒ　ｏｆ　ＮＦ－κＢ　ｌｉｇａｎｄ：ＳＩＧＭＡ社）、および終濃度１．１５Ｘ１０－２ｍｏｌ／ＬのＮ－アセチルヒドロキシプロリン（協和発酵バイオ社）を添加した。対照群：ＲＡＮＫＬ刺激なし、ＲＡＮＫＬ刺激群、およびＲＡＮＫＬ刺激＋Ｎ－アセチルヒドロキシプロリン群、の３群で検討した。その後、１日おきに１００μＬ／ｗｅｌｌの培地を交換し、６日間培養した。 RAW264 cells were seeded in a 96-well plate by 8 × 10 2 cells / well and cultured in a medium of 200 μL / Well. After culturing for 24 hours, RANKL (receptor activator of NF-κB ligand: SIGMA), which is an osteoclast differentiation inducing factor with a final concentration of 50 ng / mL, and N-acetylhydroxyproline with a final concentration of 1.15 × 10-2 mol / L (Kyowa Hakko Bio) was added. The control group was examined in three groups: RANKL-stimulated, RANKL-stimulated group, and RANKL-stimulated + N-acetylhydroxyproline group. Thereafter, the medium of 100 μL / well was changed every other day and cultured for 6 days.
　培養後、破骨細胞のマーカー酵素であるＴＲＡＰ（酒石酸耐性酸ホスファターゼ）染色を染色キット（プライマリーセル社）を用いて行った。染色された破骨細胞の中で核を３核以上有する多核化した破骨細胞である成熟破骨細胞数をカウントした。 After culturing, staining with TRAP (tartrate-resistant acid phosphatase), which is an osteoclast marker enzyme, was performed using a staining kit (Primary Cell). The number of mature osteoclasts, which are multinucleated osteoclasts having three or more nuclei, was counted among the stained osteoclasts.
　その結果、図１に示すように対照群では成熟破骨細胞はカウントされなかったのに対し、ＲＡＮＫＬ刺激により有意に成熟破骨細胞数が増加し、破骨細胞の分化が誘導された。Ｎ－アセチルヒドロキシプロリン添加群では、ＲＡＮＫＬ刺激による成熟破骨細胞数が有意に減少し、破骨細胞の分化が抑制された。この結果から、Ｎ－アセチルヒドロキシプロリンの破骨細胞分化抑制作用が明らかとなり、破骨細胞における骨吸収抑制剤、および生体内における骨代謝改善剤となることが示された。 As a result, mature osteoclasts were not counted in the control group as shown in FIG. 1, whereas the number of mature osteoclasts was significantly increased by RANKL stimulation, and osteoclast differentiation was induced. In the N-acetylhydroxyproline addition group, the number of mature osteoclasts by RANKL stimulation was significantly reduced, and osteoclast differentiation was suppressed. From this result, it was revealed that N-acetylhydroxyproline has an osteoclast differentiation inhibitory action, and it was shown to be a bone resorption inhibitor in osteoclasts and a bone metabolism improver in vivo.
　以下に、本発明の実施例を示す。 Examples of the present invention are shown below.
　下記実施例ではＮ－アセチルヒドロキシプロリンは、それぞれ協和発酵バイオ社製のものを用いた。 In the following examples, N-acetylhydroxyproline was manufactured by Kyowa Hakko Bio.
　以下に示した組成に従い成分を混合した後、打錠機で打錠し、直径８ｍｍ、重量２００ｍｇの錠剤を製造する。組成（成分；配合割合　重量％）　Ｎ－アセチルヒドロキシプロリン；２０％、乳糖；４０％、乳酸カルシウム；１０％、ステアリン酸マグネシウム；２５％、炭酸カルシウム；５％ After mixing the components according to the composition shown below, the mixture is compressed with a tableting machine to produce a tablet having a diameter of 8 mm and a weight of 200 mg. Composition (component; blending ratio% by weight) N-acetylhydroxyproline; 20%, lactose; 40%, calcium lactate; 10%, magnesium stearate; 25%, calcium carbonate; 5%
　以下に示した配合により飲料を製造する。組成（成分；配合ｇ）　Ｎ－アセチルヒドロキシプロリン；３０ｇ、ビタミンＣ；１ｇ、ビタミンＢ１；０．００５ｇ、ビタミンＢ２；０．０１ｇ、ビタミンＢ６；０．０２５ｇ、液糖；１５０ｇ、クエン酸；３ｇ、香料；１ｇ Beverages are produced according to the formulation shown below. Composition (component; blending g) N-acetylhydroxyproline: 30 g, vitamin C: 1 g, vitamin B1: 0.005 g, vitamin B2: 0.01 g, vitamin B6: 0.025 g, liquid sugar: 150 g, citric acid: 3 g , Fragrance; 1 g
　以下に示した処方で、下記の方法によりハードカプセル剤（１カプセルあたり３６０ｍｇ）を製造する。組成（成分；配合ｍｇ）　Ｎ－アセチルヒドロキシプロリン；２５０ｍｇ、乳糖；６０ｍｇ、コーンスターチ；３０ｍｇ、ヒドロキシプロピルセルロース；２０ｍｇ With the formulation shown below, hard capsules (360 mg per capsule) are produced by the following method. Composition (component; formulation mg) N-acetylhydroxyproline; 250 mg, lactose; 60 mg, corn starch; 30 mg, hydroxypropylcellulose; 20 mg
　Ｎ－アセチルヒドロキシプロリンに乳糖およびコーンスターチを添加して混合し、これにヒドロキシプロピルセルロースの水溶液を添加して練合する。次いで、押し出し造粒機を用いて、常法により顆粒を製造する。この顆粒をゼラチンハードカプセルに充填することにより、ハードカプセル剤を製造する。 Lactose and corn starch are added to N-acetylhydroxyproline and mixed, and an aqueous solution of hydroxypropylcellulose is added thereto and kneaded. Next, granules are produced by an ordinary method using an extrusion granulator. A hard capsule is produced by filling the granule into a gelatin hard capsule.
　以下に示した処方で、下記の方法によりソフトカプセル剤（１カプセルあたり１７０ｍｇ）を製造する。組成（成分；配合ｍｇ）　Ｎ－アセチルヒドロキシプロリン；２５ｍｇ、大豆油；１２０ｍｇ、グルコサミン；２５ｍｇ With the formulation shown below, soft capsules (170 mg per capsule) are produced by the following method. Composition (component; compounding mg) N-acetylhydroxyproline; 25 mg, soybean oil; 120 mg, glucosamine; 25 mg
　大豆油にＮ－アセチルヒドロキシプロリンおよびグルコサミンを添加して混合する。次いで、ロータリー・ダイズ式自動成型機を用いて、常法に従い、ソフトカプセルに充填することにより、ソフトカプセル剤を製造する。 Add N-acetylhydroxyproline and glucosamine to soybean oil and mix. Next, soft capsules are produced by filling the soft capsules using a rotary soybean automatic molding machine according to a conventional method.
　クリームの調製（油相成分）スクワラン５．０ｇ、オリーブ油３．０ｇ、水添ラノリン２．０ｇ、ミツロウ２．５ｇ、モノグリセリルステアレート２．０ｇ、ポリオキシエチレンステアリルエーテル２．５ｇ、プロピルパラベン１．５ｇ、１，３－ブチレングリコール５．０ｇ、香料微量、（水相成分）Ｎ－アセチル－トランス－４－ヒドロキシ－Ｌ－プロリン５．０ｇ、メチルパラベン０．５ｇ、カーボポール９４００．０３ｇ、トリエタノールアミン０．３ｇ、精製水 ７０．９７ｇ（調製法）油相成分および水相成分をそれぞれ８０℃に熱して均一にし、水相を油相に攪拌しながら加え、乳化後冷却しクリームを得た。 Preparation of cream (oil phase component) Squalane 5.0 g, olive oil 3.0 g, hydrogenated lanolin 2.0 g, beeswax 2.5 g, monoglyceryl stearate 2.0 g, polyoxyethylene stearyl ether 2.5 g, propylparaben 1 .5 g, 1,3-butylene glycol 5.0 g, perfume, (water phase component) N-acetyl-trans-4-hydroxy-L-proline 5.0 g, methylparaben 0.5 g, carbopol 9400.03 g, tri Ethanolamine 0.3g, refined starch syrup 70.97g (Preparation method) Heat the oil phase component and water phase component to 80 ° C to homogenize them, add the water phase to the oil phase with stirring, emulsify and cool to obtain cream It was.
　軟膏の調製（油相成分）白色ワセリン０．２ｇ、ステアリルアルコール１．０ｇ、ラウリル硫酸ナトリウム０．３ｇ、（水相成分）Ｎ－アセチル－トランス－４－ヒドロキシ－Ｌ－プロリン５．０ｇ、プロピレングリコール５．０ｇ パラオキシ安息香酸エチル、０．０２ｇ パラオキシ安息香酸ブチル１．０ｇ 精製水 ８１．４５ｇ（調製法）相成分および水相成分をそれぞれ８０℃に熱して均一にし、水相を油相に攪拌しながら、乳化後冷却し軟膏を得た。 Preparation of ointment (oil phase component) 0.2 g white petrolatum, 1.0 g stearyl alcohol, 0.3 g sodium lauryl sulfate, (aqueous phase component) 5.0 g N-acetyl-trans-4-hydroxy-L-proline, propylene Glycol 5.0 g エ チ ル ethyl paraoxybenzoate, 0.02 g ブ チ ル butyl paraoxybenzoate 1.0 g purified water tank 81.45 g (preparation method) The phase component and the aqueous phase component were each heated to 80 ° C. to make the water phase oil phase While stirring, the mixture was cooled after emulsification to obtain an ointment.
　パックの調製（油相成分）エタノール８．０ｇ、ポリオキシエチレンオレイルエーテル１．０ｇ、パラオキシ安息香酸メチル ０．２ｇ（水相成分）Ｎ－アセチル－トランス－４－ヒドロキシ－Ｌ－プロリン５．０ｇ、プロピレングリコール ４．０ｇ、グリセリン５．０ｇ、ポリビニルアルコール１５．０ｇ、精製水 ６１．６ｇ、香料０．２ｇ（調製法）水相成分、油相成分およびそれぞれ８０℃に熱して均一にし、水相を油相に攪拌しながら、パックを得た。 Preparation of pack (oil phase component) Ethanol 8.0 g, polyoxyethylene oleyl ether 1.0 g, paraoxybenzoic acid methyl ester 0.2 g (aqueous phase component) N-acetyl-trans-4-hydroxy-L-proline 5.0 g 4.0 g of propylene glycol, 5.0 g of glycerin, 15.0 g of polyvinyl alcohol, 61.6 g of purified syrup, 0.2 g of fragrance (preparation method) water phase component, oil phase component and each heated to 80 ° C. to be uniform, water A pack was obtained while stirring the phase into the oil phase.
　テープ剤の調製（粘着剤溶剤）　スチレン－イソプロピレン－スチレンブロック共重合体７．０ｇ、エステルガム２５．０ｇ、イソプロピレンゴム５．０ｇ、トルエン１５．０ｇ、酢酸エチル１４．２ｇ、ヘキサン２５．０ｇ、（薬効成分）Ｎ－アセチル－トランス－４－ヒドロキシ－Ｌ－プロリン３．０ｇ、エタノール５．０ｇ、（経皮吸収促進剤）オレイルアルコール０．８ｇ、（調製法）粘着剤溶剤、薬効成分をそれぞれ均一にし、薬効成分、経皮吸収促進剤を粘着剤溶剤に加え、室温で攪拌し組成物を得た。この組成物をシリコーン処理したポリエステルフィルム上に延展し、１２０℃で乾燥させ冷却後、ポリエチレンフィルムへ粘着剤層を転写させ、テープ剤を得た。 Preparation of tape agent (adhesive solvent) 7.0 g of styrene-isopropylene-styrene block copolymer, 25.0 g of ester gum, 5.0 g of isopropylene rubber, 15.0 g of toluene, 14.2 g of ethyl acetate, 25. 0 g, (medicinal ingredient) N-acetyl-trans-4-hydroxy-L-proline 3.0 g, ethanol 5.0 g, (transdermal absorption enhancer) oleyl alcohol 0.8 g, (preparation method) adhesive solvent, medicinal effect The ingredients were made uniform, a medicinal ingredient and a transdermal absorption accelerator were added to the adhesive solvent, and the mixture was stirred at room temperature to obtain a composition. This composition was spread on a silicone-treated polyester film, dried at 120 ° C. and cooled, and then the pressure-sensitive adhesive layer was transferred to a polyethylene film to obtain a tape agent.

Claims (5)

  1. ヒドロキシプロリンのＮ－アシル化誘導体またはその塩を有効成分として含有する骨代謝改善剤。 An agent for improving bone metabolism comprising an N-acylated derivative of hydroxyproline or a salt thereof as an active ingredient.
  2. ヒドロキシプロリンのＮ－アシル化誘導体またはその塩を有効成分として含有する骨吸収抑制剤。 A bone resorption inhibitor comprising an N-acylated derivative of hydroxyproline or a salt thereof as an active ingredient.
  3. ヒドロキシプロリンのＮ－アシル化誘導体またはその塩を有効成分として含有する破骨細胞分化抑制剤。 An osteoclast differentiation inhibitor containing an N-acylated derivative of hydroxyproline or a salt thereof as an active ingredient.
  4. 骨代謝改善剤の製造のためのヒドロキシプロリンのＮ－アシル化誘導体の塩の使用。 Use of a salt of an N-acylated derivative of hydroxyproline for the production of an agent for improving bone metabolism.
  5. 骨代謝を改善する方法であって、骨代謝を改善させる必要のある被験体に該被験体の骨代謝を改善させるのに十分な量のヒドロキシプロリンのＮ－アシル化誘導体の塩を投与する工程を含む、骨代謝を改善する方法。 A method for improving bone metabolism, comprising administering to a subject in need of improving bone metabolism a sufficient amount of a salt of an N-acylated derivative of hydroxyproline to improve bone metabolism in said subject. A method for improving bone metabolism, comprising:
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