WO2020067014A1 - Composition pour la régénération osseuse, kit de composition pour la régénération osseuse, élément pour la régénération osseuse et procédé de régénération osseuse - Google Patents

Composition pour la régénération osseuse, kit de composition pour la régénération osseuse, élément pour la régénération osseuse et procédé de régénération osseuse Download PDF

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WO2020067014A1
WO2020067014A1 PCT/JP2019/037277 JP2019037277W WO2020067014A1 WO 2020067014 A1 WO2020067014 A1 WO 2020067014A1 JP 2019037277 W JP2019037277 W JP 2019037277W WO 2020067014 A1 WO2020067014 A1 WO 2020067014A1
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bone regeneration
composition
bone
component
solution
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PCT/JP2019/037277
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English (en)
Japanese (ja)
Inventor
林　英治
愛 舟▲崎▼
純 諫山
健太郎 内田
亘 齋藤
真太郎 庄司
晶士 高相
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Jsr株式会社
学校法人北里研究所
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Priority to JP2020549215A priority Critical patent/JP7313370B2/ja
Publication of WO2020067014A1 publication Critical patent/WO2020067014A1/fr

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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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
    • A61K31/728—Hyaluronic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • 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
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to a bone regeneration composition, a bone regeneration composition kit, a bone regeneration member, and a bone regeneration method.
  • Regeneration of bone after fracture involves an inflammatory phase in which the fracture site is inflamed due to hematoma occupation of the fracture gap, callus formation due to the conversion of the hematoma to granulocytes, and new bone formation due to osteogenic cells present in the periosteum It is known that the process progresses during a bone repair period, in which the bone is deformed due to repeated bone resorption and formation of new bone, and a remodeling period in which the correction of bone deformation progresses.
  • Patent Documents 1 and 2 Conventionally, a method of using a bone regeneration member containing a growth factor such as a bone morphogenetic protein (BMP) for bone regeneration treatment for a fracture has been known (see Patent Documents 1 and 2).
  • a growth factor such as a bone morphogenetic protein (BMP)
  • the present invention provides a bone regeneration composition, a bone regeneration composition kit, and a bone regeneration member useful for intractable fractures including periosteal damage and the like, and bone regeneration using the composition and the like.
  • the aim is to provide a method.
  • the present inventors have studied to solve the above problems. As a result, the inventors have found that the following problems can be solved by the following bone regeneration composition and the like, and have completed the present invention.
  • the present invention relates to, for example, the following [1] to [10].
  • a composition for bone regeneration comprising at least one component (C) selected from c1) and a substance (c2) that produces peroxide, an osteogenic protein (D), and water (E).
  • the hydroxyaryl group is a group derived from tyramine.
  • [3] The bone regeneration composition according to the above [1] or [2], wherein the degree of substitution of the component (A) with the hydroxyaryl group is 0.1 to 50.
  • [4] The composition for bone regeneration according to any of [1] to [3], wherein the oxidative coupling enzyme (B) is horseradish peroxidase.
  • [5] The composition for bone regeneration according to any of [1] to [4], wherein the component (C) is the peroxide (c1).
  • [6] The composition for bone regeneration according to any of [1] to [5], which is used for intractable fractures.
  • a bone regeneration composition kit having a first solution, a second solution, and a third solution, wherein the first solution is a modified hyaluronic acid in which a hydroxyaryl group is introduced into hyaluronic acid. It contains at least one component (A) selected from an acid and a salt thereof and water (E), and contains the oxidative coupling enzyme (B) for the hydroxyaryl group, and the peroxide (c1) and the peroxide.
  • the second solution contains at least one component (C) selected from the substance (c2) to be formed, but not both, and the second solution contains the oxidative coupling enzyme (B) and the component (C).
  • a composition kit for bone regeneration comprising the component (C) that is not contained in the first solution, wherein the third solution contains an osteogenic protein (D).
  • a bone regeneration method for regenerating bone using a regeneration member is regenerating bone using a regeneration member.
  • a bone regeneration method can be provided.
  • FIG. 1 is a scheme of a bone regeneration process using the bone regeneration composition of the present invention.
  • FIG. 2 is a microCT image measured in Examples and the like.
  • composition for bone regeneration of the present invention comprises: At least one component (A) selected from a modified hyaluronic acid in which a hydroxyaryl group is introduced into hyaluronic acid and a salt thereof; Said hydroxyaryl group oxidative coupling enzyme (B); At least one component (C) selected from a peroxide (c1) and a substance (c2) that generates peroxide; Osteogenic protein (D), Water (E).
  • the component (A) is at least one selected from a modified hyaluronic acid in which a hydroxyaryl group has been introduced into hyaluronic acid, and a salt of the modified hyaluronic acid. Since the bone morphogenetic protein (D) is easily circulated by body fluids, it is generally difficult to maintain its effect at the administration site of the bone morphogenetic protein (D) for a long period of time. In the present embodiment, by using the component (A) together with the bone morphogenetic protein (D), the bone morphogenetic protein (D) can be stably present at the administration site of the composition of the present embodiment, and the bone morphogenetic protein (D) Is expected to be maintained for a long time. Therefore, the composition of the present embodiment has a high bone regeneration effect. Specifically, in the present embodiment, it is considered that by using the component (A) and the bone morphogenetic protein (D), new bone formation can be promoted and bone regeneration speed can be improved.
  • the composition of the present embodiment is useful for treating intractable fractures, particularly for treating periosteal injury-type fractures, and by using the composition of the present embodiment, it is possible to increase the union rate of a fractured bone. .
  • the reason for this is that the affinity between the gel formed from the component (A) and the bone morphogenetic protein (D), which is the active ingredient, is moderately excellent, so that the sustained release rate of the active ingredient from the gel decreases in the fracture. This is probably because the speed is suitable for bone regeneration.
  • the intractable fracture is a fracture of a tissue important in bone formation, such as a damage of a periosteum, or a fracture having impaired blood flow accompanying the damage, and may be accompanied by an infection. Intractable fractures include, for example, open fractures.
  • Hyaluronic acid is a polysaccharide having a disaccharide unit of N-acetylglucosamine and glucuronic acid as a repeating structural unit.
  • Examples of the salt of the modified hyaluronic acid include physiologically or pharmaceutically acceptable salts, and include alkali metal salts such as sodium salt and potassium salt, and ammonium salts.
  • Component (A) has a hydroxyaryl group.
  • the number of carbon atoms of the hydroxyaryl group is usually 6 to 50, preferably 6 to 30.
  • the number of hydroxyl groups bonded to the benzene nucleus is usually 1 or more, preferably 1 to 3, and more preferably 1 or 2.
  • the hydroxyaryl group is not limited to a monohydroxyaryl group.
  • the hydroxyaryl group examples include a hydroxyphenyl group, a hydroxynaphthyl group, and a hydroxyanthracenyl group.
  • the hydroxyaryl group can have a substituent such as an alkyl group.
  • a hydrogen atom is bonded to a carbon atom at least one ortho position to the phenolic hydroxyl group. Since it is considered that the bone morphogenetic protein (D) can be stably present at the administration site of the composition of the present embodiment and can maintain its effective concentration, the hydroxyaryl group is a group derived from tyramine. preferable.
  • Modified hyaluronic acid is preferably a compound in which a hydroxyaryl group is bonded to hyaluronic acid by an ester bond, a urethane bond or an ether bond, directly or via an organic group.
  • the ester bond, urethane bond and ether bond are preferably bonded to a carbon atom to which a hydroxyl group contained in hyaluronic acid has been bonded.
  • the organic group is, for example, an alkanediyl group having 1 to 20 carbon atoms, or a substitution in which at least one selected from an ether bond and a divalent amino group (such as —NH—) is added or inserted to the alkanediyl group.
  • Examples include an alkanediyl group and an organic group having a peptide bond (specifically, a group derived from an oligopeptide such as a dipeptide or an ester thereof).
  • Examples of the carbonylating reagent include nitrophenyl chloroformate, phenyl chloroformate, trichloromethyl chloroformate, and bis (trichloromethyl) carbonate.
  • the amount of the carbonylation reagent is usually 1 to 100 parts by mass based on 100 parts by mass of hyaluronic acid.
  • Examples of the amine compound having a hydroxyaryl group include tyramine.
  • the amount of the amine compound having a hydroxyaryl group is usually 1 to 100 parts by mass relative to 100 parts by mass of the carbonate derivative of hyaluronic acid.
  • the conversion reaction is preferably performed in the presence of a basic compound.
  • the basic compound include trimethylamine, triethylamine, tributylamine, pyridine, and piperidine.
  • the conversion reaction and the hydroxyaryl group introduction reaction are preferably performed in a solvent.
  • the solvent include amide solvents such as dimethylformamide and dimethylacetamide; sulfoxide solvents such as dimethylsulfoxide; halogenated hydrocarbon solvents such as dichloromethane, 1,2-dichloroethane and chloroform; and aromatic hydrocarbons such as benzene, toluene and xylene. Hydrogen solvents; ether solvents such as diethyl ether, tetrahydrofuran, dioxane and the like.
  • an alkali metal salt such as lithium chloride can be used.
  • hyaluronic acid and a functional group capable of reacting with an alcoholic hydroxyl group in hyaluronic acid eg, a carboxy group, an acid halide thereof, an epoxy group, an oxetanyl group
  • a compound having a hydroxyaryl group for example, hyaluronic acid and a functional group capable of reacting with an alcoholic hydroxyl group in hyaluronic acid (eg, a carboxy group, an acid halide thereof, an epoxy group, an oxetanyl group) ) And a compound having a hydroxyaryl group.
  • Examples of the compound having a carboxy group capable of reacting with an alcoholic hydroxyl group in hyaluronic acid or an acid halide thereof and a hydroxyaryl group include, for example, 4-hydroxyphenylacetic acid, 4-hydroxyphenylpropionic acid, 4-hydroxyphenylbutane Acids, 2- (2,5-dihydroxyphenyl) acetic acid, and acid halides thereof.
  • the degree of substitution (DS) of the component (A) with the hydroxyaryl group is usually 0.1 to 50, preferably 1 to 40, more preferably 2 to 30.
  • the degree of substitution can be determined by 1 H NMR.
  • the substitution degree of the component (A) indicates the average number of hydroxyaryl groups introduced per 100 units of the constituent monosaccharide of the component (A).
  • the component (A) having such a degree of substitution improves the gelation rate.
  • the number average molecular weight of the component (A) as measured by absolute molecular weight measurement using a multi-angle light scattering detector is usually 1,000 to 10,000,000, preferably 3,000 to 1,000,000, more preferably 5,000 to 500,000.
  • the molecular weight distribution of the component (A) is usually 1 to 20, preferably 1 to 10, and more preferably 1 to 5.
  • the component (A) one type or two or more types can be used.
  • the content ratio of the component (A) in the composition of the present embodiment is usually 0.05 to 49% by mass, preferably 0.5 to 30% by mass, and more preferably 3 to 15% by mass. Such an embodiment is preferable from the viewpoint of the mechanical strength and handleability of the gel.
  • the composition of the present embodiment contains an oxidative coupling enzyme (B) for a hydroxyaryl group (hereinafter also referred to as “enzyme (B)”).
  • the enzyme (B) is an enzyme capable of directly or indirectly catalyzing an oxidative coupling reaction of a hydroxyaryl group.
  • Examples of the enzyme (B) include phenol oxidase such as laccase and tyrosinase, catalase, and peroxidase.
  • the composition of the present embodiment can be gelled by using peroxidase in combination with a peroxide (c1) such as hydrogen peroxide.
  • a peroxide (c1) such as hydrogen peroxide.
  • the origin of copper enzymes such as laccase and tyrosinase includes, for example, sorghum, mushrooms (Acacia fulica, mushroom), and mold (Polyporus vericolor).
  • Catalase the origin of peroxidase, for example, bovine liver, horse blood cells, human blood cells, M.
  • tyrosinase is preferably derived from mushroom
  • peroxidase is preferably derived from horseradish, that is, horseradish peroxidase.
  • the component (C) is used, and at least one selected from peroxidase and catalase is used as the enzyme (B). More preferably, component (C) is used, and horseradish peroxidase is used as enzyme (B).
  • One or more enzymes (B) can be used.
  • the amount of the enzyme (B) is preferably at least 0.01 U / mL, more preferably at least 0.1 U / mL; preferably at most 1,000 U / mL, and more preferably at most 500 U / mL. preferable.
  • U represents a unit of enzyme activity, and is an amount of enzyme capable of changing 1 ⁇ mol of substrate per minute at a temperature of 30 ° C. under optimum conditions.
  • the composition of the present embodiment contains at least one component (C) selected from a peroxide (c1) and a substance (c2) that generates a peroxide.
  • the peroxide (c1) include hydrogen peroxide.
  • the amount of the peroxide (c1) is usually based on 1 mol of the hydroxyaryl group in the component (A). The amount is 0.01 to 1000 mol, preferably 0.1 to 500 mol, and more preferably 0.5 to 200 mol.
  • the content of the peroxide (c1) in the composition of the present embodiment is preferably from 10 to 1,000 mM, more preferably from 100 to 800 mM, from the viewpoint of reactivity.
  • the peroxide-generating substance (c2) is usually used together with the oxidase (c3).
  • the oxidase (c3) is an enzyme that catalyzes a reaction that consumes the substance (c2) to generate a peroxide such as hydrogen peroxide.
  • Examples of the oxidase (c3) include glucose oxidase, choline oxidase, amino acid oxidase, alcohol oxidase, pyruvate oxidase, and cholesterol oxidase. These enzymes can be appropriately selected depending on the application. For example, glucose oxidase is preferable because the gelation rate is greatly improved.
  • the substance (c2) is consumed by, for example, the oxidase (c3) to generate a peroxide such as hydrogen peroxide, and is selected according to the oxidase (c3).
  • hydrogen peroxide can be generated by the enzyme treatment of the substance (c2) by the oxidase (c3) in the presence of oxygen (eg, oxygen in the air).
  • oxygen eg, oxygen in the air
  • the oxidase (c3) is glucose oxidase, choline oxidase, amino acid oxidase, alcohol oxidase, pyruvate oxidase or cholesterol oxidase
  • the substance (c2) is glucose, choline, amino acid, alcohol, pyruvate or Cholesterol.
  • the content of the oxidase (c3) in the composition of the present embodiment is preferably 0.01 U / mL or more, and more preferably 1 U / mL or more from the viewpoint of reactivity. Is more preferable; 1,000 U / mL or less is preferable, and 500 U / mL or less is more preferable. Further, the content of the substance (c2) is appropriately selected such that the peroxide is appropriately generated.
  • One or more components (C) can be used.
  • Bone morphogenetic protein (D) is a protein that promotes bone formation and repair of fractures. Specifically, it is a factor that enhances the activity of osteoblasts, and a component of mesenchymal stem cells. It is a factor that induces differentiation into osteoblasts.
  • Preferred examples of the bone morphogenetic protein (D) include, for example, BMP-2, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, and BMP-9.
  • the bone morphogenetic protein (D) may be derived from a human or a non-human animal.
  • the bone morphogenetic protein (D) has an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence of naturally occurring BMP, and has the same amino acid sequence as naturally occurring BMP. And a protein having the activity of Thus, the bone morphogenetic protein (D) may be recombinant.
  • BMP-2 is preferable.
  • BMP-2 is a type of factor that controls and / or promotes cell growth.
  • BMP-2 is excellent in bone formation ability and promotes differentiation of mesenchymal stem cells present in periosteum and bone marrow.
  • bone morphogenetic protein (D) for example, bone morphogenetic proteins prepared by various methods can be used as long as the bone morphogenetic protein (D) is purified to a degree that can be used as a medicine.
  • One or more bone morphogenetic proteins (D) can be used.
  • the content of the bone morphogenetic protein (D) is usually 0.01 to 10 parts by mass, preferably 0.05 to 5 parts by mass with respect to 100 parts by mass of the component (A). is there.
  • the bone morphogenetic protein (D) is favorably retained in the hydrogel formed from the composition of the present embodiment.
  • the composition of the present embodiment contains water (E).
  • the content ratio of water (E) in the composition of the present embodiment is usually 50 to 99.9% by mass, preferably 65 to 99% by mass, and more preferably 70 to 97% by mass.
  • the composition can be gelled under conditions of low invasiveness to a living body, and the resulting gel tends to have excellent mechanical strength.
  • the composition of the present embodiment can further contain a polysaccharide other than the component (A).
  • the polysaccharide include, for example, unmodified hyaluronic acid, pullulan, xanthan gum, cellulose, guar gum, fructan, mannan, carrageenan, chitin, chitosan, pectin, starch, amylose, amylopectin, glycogen, dextran into which a hydroxyaryl group has not been introduced. Dextrin, gellan gum and alginic acid.
  • the composition of the present embodiment may further contain one or more selected from phosphoric acid, polyphosphoric acid, a pH adjuster, an ultraviolet absorber, a thickener, a coloring agent and a filler, It may contain a substance expressing function such as a drug or a cell. Further, the composition of the present embodiment can further contain sodium chloride. For example, the composition of the present embodiment can contain a buffer such as a phosphate buffered saline.
  • the composition kit for bone regeneration of the present invention includes a first solution, a second solution, and a third solution described below. And a solution.
  • the first solution contains at least one component (A) selected from a modified hyaluronic acid in which a hydroxyaryl group has been introduced into hyaluronic acid and a salt thereof, and water (E). It contains the ring enzyme (B) and at least one component (C) selected from the peroxide (c1) and the peroxide-generating substance (c2), but not both.
  • the second solution contains one of the oxidative coupling enzyme (B) and the component (C) that is not contained in the first solution.
  • the third solution contains the bone morphogenetic protein (D).
  • Each of the first to third solutions is preferably an aqueous solution. Details of the components (A) to (E) in the first to third solutions are as described above. Further, the amount ratio of the first to third solutions and the content of each component contained in each of the first to third solutions are determined in the composition obtained by mixing the first to third solutions. The amount ratio and content of each of the components (A) to (E) are appropriately set so as to fall within the ranges described in the [Composition for bone regeneration] column.
  • the composition of the present embodiment can be prepared.
  • the order of mixing the respective solutions is not particularly limited, but a mode in which the second solution is mixed with the mixture obtained by mixing the first solution and the third solution, or the second solution and the third solution are mixed. And a solution obtained by mixing the first solution with the first solution.
  • the bone regeneration member of the present invention is formed using the bone regeneration composition or the bone regeneration composition kit of the present embodiment, and specifically, the bone regeneration composition or the bone regeneration composition A gel formed from a composition composition kit.
  • the gel has a crosslinked structure by oxidative coupling of the hydroxyaryl group of component (A), that is, contains a crosslinked product derived from component (A).
  • component (A) contains a crosslinked product derived from component (A).
  • the gel is a hydrogel, specifically a hydrogel containing usually 50 to 99.9% by mass, preferably 70 to 97% by mass of water.
  • the hydrogel generally contains 50 to 0.1% by mass, preferably 30 to 3% by mass of a crosslinked product derived from the component (A).
  • the hydrogel has excellent mechanical strength and flexibility.
  • a xerogel can be obtained by removing water by appropriately drying the hydrogel.
  • the gel is formed by irradiating light with a photosensitizer such as eosin Y, methylene blue, or rose bengal together with the component (A) as described in Macromolecules (2015) 2624-2630, for example. You can also get it.
  • An example of a method for manufacturing a bone regeneration member is as follows.
  • the components (A) to (E) in the composition of the present embodiment or the first to third solutions in the kit of the present embodiment are mixed, and the aqueous solution temperature (crosslinking temperature) is usually 4 to 50 ° C.
  • Gelation can be carried out under the conditions of preferably 10 to 45 ° C., more preferably 20 to 40 ° C., and a reaction time of usually 1 minute to 48 hours, preferably 5 minutes to 30 hours.
  • Each of the components (A) to (D) can be used as an aqueous solution of each component.
  • the formation of the hydrogel can be performed under any of pressurized, normal pressure (atmospheric pressure), and reduced pressure, but is preferably performed under normal pressure.
  • a mold having a cavity having a desired shape is prepared, and the components (A) to (E) of the composition of the present embodiment or the first to third solutions in the kit of the present embodiment are added to the mold.
  • a hydrogel having the above shape can be formed.
  • a film-like hydrogel can be obtained by casting a composition of the present embodiment or a mixture of the first to third solutions in the kit of the present embodiment on a predetermined substrate.
  • the film-shaped hydrogel can be punched into, for example, a columnar shape, a polygonal column shape, or the like.
  • the shape of the bone regeneration member is not particularly limited, and examples thereof include a film shape, a cylindrical shape, a polygonal column shape, a nonwoven fabric shape, a fiber shape, a tube shape, a particle shape, and a mesh shape.
  • the bone morphogenetic protein (D) is carried. Therefore, in the present embodiment, the bone regeneration component is applied to a site to which the bone regeneration member is applied (eg, a bone defect site or a fracture site). It is thought that protein (D) can be maintained for a long period of time.
  • the hydrogel functions as an extracellular matrix in the affected area, and the extracellular matrix is considered to be a scaffold for bone-regenerating cells. Therefore, the bone regeneration member of the present embodiment has a high bone regeneration effect.
  • the composition for bone regeneration, the composition kit for bone regeneration, and the member for bone regeneration of the present embodiment are used for bone regeneration.
  • a bone regeneration composition is administered to an affected area such as a bone defect site or a fracture site of a subject requiring bone regeneration (for example, a human or a mammal other than a human), or a bone regeneration composition kit.
  • a bone regeneration member is formed in an affected part, or by applying the bone regeneration member to the affected part, bone regeneration is induced, and bone regeneration is induced. Can improve the fusion rate.
  • FIG. 1 shows an example of a scheme for estimating bone regeneration when the composition for bone regeneration of the present embodiment is used.
  • the composition for bone regeneration administered to the affected area 30 forms a hydrogel 40, and the bone morphogenetic protein 50 is favorably retained in the hydrogel 40.
  • the bone morphogenetic protein 50 promotes differentiation or proliferation of a mesenchymal stem cell, which is a precursor cell of the bone cell, and the osteoblast 20. As a result, a large new bone (callus) 70 is formed.
  • FIG. 1 bone cells are omitted.
  • the bone to be regenerated in the present embodiment is formed by depositing hydroxyapatite on a collagen fiber formed in a network, and most of the organic matter of the bone is collagen.
  • the usage, dosage, and shape of the composition for bone regeneration, the composition kit for bone regeneration, or the member for bone regeneration of the present embodiment can be appropriately determined according to the purpose of use.
  • the composition for bone regeneration or the mixture of the first to third solutions of the composition kit for bone regeneration is directly applied to an affected part such as a bone defect site or a fracture site in a living body by, for example, injection, application, or the like. Can be administered.
  • the composition or mixture may be mixed with an appropriate excipient to form an ointment, gel, or cream, and then applied to the affected area.
  • the target disease of the bone regeneration composition, the bone regeneration composition kit and the bone regeneration member of the present embodiment includes, as described above, intractable fracture.
  • the obtained solution was cooled to 0 ° C, 9.2 ml of pyridine and 23.8 g of nitrophenyl chloroformate were added, and the mixture was stirred at 0 ° C for 1 hour. Then, 2000 ml of cooled ethanol was added to the flask, the precipitate was separated by filtration, and the precipitate was washed with cooled ethanol and dried.
  • a hydroxyphenyl group-modified hyaluronic acid (A1) was obtained.
  • the degree of substitution (DS) with the hydroxyphenyl group was 3.2.
  • Preparation Example 2 Preparation of First Solution (c1 ) 0.2 g of the hydroxyphenyl group-modified dextran (AR1) obtained in Synthesis Example 2 was mixed with phosphate buffered saline (product name "PBS (-)", The polymer solution was prepared by dissolving in 0.8 g of Kojun Pharmaceutical Co., Ltd. 1.0 g of the above polymer solution and 1.0 g of an aqueous solution of horseradish peroxidase (manufactured by Wako Pure Chemical, product code "169-10791”) (concentration: 0.8 U / ml) were uniformly mixed to form a first solution ( c1) was prepared.
  • PBS (-) phosphate buffered saline
  • composition for bone regeneration was prepared in the same manner as in Example 1 except that the first solution (c1) was used instead of the first solution (1).
  • the composition for bone regeneration was administered to the fracture site of the mouse in the same manner as in Example 1, and after 6 weeks, the mouse was sacrificed and the bone fusion rate was measured by microCT.
  • the bone union rate was 10% (1/10).
  • the microCT image after 6 weeks is shown in FIG.
  • Example 1 was the same as Example 1 except that the third solution (1) was replaced with a phosphate buffered saline (product name “PBS ( ⁇ )”, manufactured by Wako Pure Chemical Industries, Ltd.).
  • a composition for bone regeneration was prepared.
  • the composition for bone regeneration was administered to the fracture site of the mouse in the same manner as in Example 1, and after 6 weeks, the mouse was sacrificed and the bone fusion rate was measured by microCT.
  • the bone union rate was 10% (1/10).
  • the microCT image after 6 weeks is shown in FIG.
  • control A C57BL / 6J mouse (male, 9 weeks old) in which the femur was fractured after cauterizing the periosteum of the femur with an electric scalpel was used as control.
  • the bone union rate was 10% (1/10).
  • the microCT image after 6 weeks is shown in FIG.
  • Example 1 has a higher bone union rate than Comparative Examples 1 and 2 and Experimental Example 1, and the composition for bone regeneration of this embodiment is useful for intractable fractures including periosteal damage. It was revealed.
  • Bone 15 bone marrow 20: mesenchymal stem cells, osteoblasts (periosteum) 30: Affected area (bone defect site, fracture site) 40: Hydrogel 50: Bone morphogenetic protein 60: Proliferated osteoblast 70: New bone 80: Neoperiosteum

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Abstract

L'invention concerne une composition pour la régénération osseuse qui comprend : au moins un composant (A) choisi parmi un acide hyaluronique modifié ayant un groupe hydroxyaryle introduit dans de l'acide hyaluronique et un sel de celui-ci; une enzyme de couplage oxydatif (B) pour le groupe hydroxyaryle; au moins un composant (C) choisi parmi un peroxyde (c1) et une substance (c2) formant un peroxyde; une protéine de formation osseuse (D); et de l'eau (E).
PCT/JP2019/037277 2018-09-28 2019-09-24 Composition pour la régénération osseuse, kit de composition pour la régénération osseuse, élément pour la régénération osseuse et procédé de régénération osseuse WO2020067014A1 (fr)

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