WO2017170487A1 - 製剤、製剤用部材およびそれらの製造方法 - Google Patents
製剤、製剤用部材およびそれらの製造方法 Download PDFInfo
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
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- 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
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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- 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
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- 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
- A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
- A61K47/6435—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the peptide or protein in the drug conjugate being a connective tissue peptide, e.g. collagen, fibronectin or gelatin
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- 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
- A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
- A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
- A61K47/6455—Polycationic oligopeptides, polypeptides or polyamino acids, e.g. for complexing nucleic acids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/08—Solutions
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/141—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
- A61K9/146—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers with organic macromolecular compounds
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J3/00—Processes of treating or compounding macromolecular substances
- C08J3/24—Crosslinking, e.g. vulcanising, of macromolecules
- C08J3/244—Stepwise homogeneous crosslinking of one polymer with one crosslinking system, e.g. partial curing
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J3/00—Processes of treating or compounding macromolecular substances
- C08J3/24—Crosslinking, e.g. vulcanising, of macromolecules
- C08J3/247—Heating methods
Definitions
- the present invention relates to a preparation containing an anionic polypeptide, a cationic polypeptide and a cationic drug, and a preparation member containing an anionic polypeptide and a cationic polypeptide.
- the present invention further relates to a method for producing the preparation and a method for producing the preparation member.
- the preparation and the preparation member of the present invention can be used for medicines.
- Patent Document 1 describes a fibrous member, a member capable of gradual release of a cell growth factor, and a vascular occlusion composition containing the cell growth factor.
- Patent Document 2 describes a sustained-release pharmaceutical composition containing a three-component complex containing interleukin-11, an anionic polymer and a cationic polymer.
- Patent Document 2 describes that hyaluronic acid or the like is used as an anionic polymer and protamine or the like is used as a cationic polymer.
- Patent Document 3 describes a method for producing microparticles by simultaneously spraying an active ingredient, a matrix-forming component A, and a matrix-forming component B capable of binding to the matrix-forming component A.
- Patent Document 3 describes that a compound having a cationic dissociation group is used as the matrix-forming component A, and a compound having an anionic dissociation group is used as the matrix-forming component B.
- An object of the present invention is to provide a preparation having both a sufficient loading amount of a drug and a preferable decomposition rate and a method for producing the same.
- Another object of the present invention is to provide a preparation member for use in the preparation of the present invention described above and a method for producing the same.
- the present inventors have used a crosslinked product obtained by crosslinking an anionic polypeptide and a cationic polypeptide as a member for a preparation containing a cationic drug.
- the present inventors have found that a preparation capable of solving the above problems can be provided, and have completed the present invention.
- a preparation comprising a crosslinked product of an anionic polypeptide and a cationic polypeptide, and a cationic drug.
- the preparation according to [1] which is administered locally.
- the preparation according to [1] or [2], wherein the molar ratio of the anionic polypeptide and the cationic polypeptide is 1: 3 to 3: 1.
- Any one of [1] to [3], wherein the anionic polypeptide and the cationic polypeptide are each a gene recombinant having an amino acid sequence derived from a partial amino acid sequence of collagen or a chemically modified product thereof. The formulation described.
- a gene recombinant having an amino acid sequence derived from a partial amino acid sequence of collagen has a repeat of the sequence represented by Gly-XY, X and Y each independently represent any of amino acids, and a plurality of Gly
- -XY may be the same or different and has a molecular weight of 10 kDa or more and 90 kDa or less.
- A represents any amino acid or amino acid sequence
- B represents any amino acid or amino acid sequence
- n Xs independently represent any of the amino acids
- n Ys are Each independently represents any of the amino acids
- n represents an integer of 3 to 100
- m represents an integer of 2 to 10.
- the preparation according to any one of [4] to [6], wherein the n Gly-XY may be the same or different.
- a pharmaceutical preparation member comprising a crosslinked product of an anionic polypeptide and a cationic polypeptide.
- a gene recombinant having an amino acid sequence derived from a partial amino acid sequence of collagen has a repeat of the sequence represented by Gly-XY, X and Y each independently represent any of amino acids, and a plurality of Gly -XY may be the same or different from each other, and the molecular weight is 10 kDa or more and 90 kDa or less, [13] or [14].
- A represents any amino acid or amino acid sequence
- B represents any amino acid or amino acid sequence
- n Xs independently represent any of the amino acids
- n Ys are Each independently represents any of the amino acids
- n represents an integer of 3 to 100
- m represents an integer of 2 to 10.
- the n Gly-XY may be the same or different from each other, and the preparation member according to any one of [13] to [15].
- the preparation of the present invention can sufficiently support a drug and exhibits a preferable degradation rate. According to the preparation member of the present invention and the method of the present invention, it is possible to produce a preparation that can sufficiently support a drug and that exhibits a preferable degradation rate.
- FIG. 1 shows the lysozyme loading of the formulation at each time point.
- the preparation according to the present invention is a preparation containing a crosslinked product of an anionic polypeptide and a cationic polypeptide and a cationic drug.
- Patent Document 1 describes that partially cross-linked gelatin is used as a cell growth factor sustained-release member, but is not a sustained-release member comprising a cationic polypeptide and an anionic polypeptide.
- Patent Document 2 describes a sustained-release preparation containing a three-component complex of interleukin-11, an anionic polymer and a cationic polymer. Not the body.
- Patent Document 3 describes a method for producing microparticles using an active ingredient, a matrix-forming component A, and a matrix-forming component B. A cross-linked product of matrix-forming component A and matrix-forming component B is formed. It is not a thing.
- a crosslinked product of an anionic polypeptide and a cationic polypeptide by using a crosslinked product of an anionic polypeptide and a cationic polypeptide, a preparation exhibiting a sufficiently high acid degradation residual ratio, that is, a preferable degradation rate, can be obtained. It has been demonstrated that it can be provided. In the prior art, the problem of achieving both a sufficient loading amount of a drug and a preferable decomposition rate has not been sufficiently studied.
- the present invention is characterized in that both a sufficient loading amount of the drug and a preferable degradation rate can be achieved by using a crosslinked product of an anionic polypeptide and a cationic polypeptide and a cationic drug.
- Anionic polypeptide means a polypeptide having a property of being negatively charged under physiological conditions (pH 7.4).
- anionic polypeptide examples include alkali-processed gelatin, polyglutamic acid, polyaspartic acid, bovine serum-derived albumin, or a genetically engineered amino acid sequence derived from these partial amino acid sequences.
- a cationic polypeptide eg, acid-processed gelatin, collagen, protamine, polyarginine and a recombinant amino acid sequence derived from those partial amino acid sequences, etc.
- chemical reaction a method in which a cationic amino group is modified to an anionic carboxyl group by treatment with succinic anhydride or maleic anhydride, or acetic anhydride, N-hydroxysuccinimide acetate, N-acetylsuccinimide, phenyl isocyanate or A method of modifying a cationic amino group to an uncharged functional group with acetaldehyde can be mentioned.
- a cationic polypeptide that has been anionically modified by a chemical reaction is referred to as a chemically modified product of the cationic polypeptide.
- the anionic polypeptide is preferably a cationic polypeptide (for example, acid-treated gelatin, collagen, and a gene recombinant of an amino acid sequence derived from a partial amino acid sequence thereof) that has been anionicly modified by a chemical reaction.
- the anionic polypeptide is obtained by anionic modification of a genetically engineered amino acid sequence derived from a partial amino acid sequence of collagen by succinic anhydride treatment.
- the “gene recombinant of an amino acid sequence derived from a partial amino acid sequence of collagen” will be described later.
- the anionic polypeptide may be any of naturally occurring peptides, recombinant peptides, or chemically synthesized peptides.
- a chemically synthesized peptide means an artificially synthesized peptide.
- the peptide such as gelatin may be synthesized by solid phase synthesis or liquid phase synthesis, but is preferably solid phase synthesis.
- Solid-phase synthesis of peptides is known to those skilled in the art. For example, Fmoc group synthesis method using Fmoc group (Fluorenyl-Methoxy-Carbonyl group) as amino group protection, and Boc group (tert-Butyl group) as amino group protection Boc group synthesis method using Oxy Carbonyl group).
- Cationic polypeptide means a polypeptide having a property of being positively charged under physiological conditions (pH 7.4).
- Examples of the cationic polypeptide include acid-modified gelatin, collagen, protamine, polyarginine, or a genetically modified amino acid sequence derived from a partial amino acid sequence thereof, and are derived from acid-treated gelatin, collagen or a partial amino acid sequence thereof.
- a genetically engineered amino acid sequence is preferred, and a genetically engineered amino acid sequence derived from a partial amino acid sequence of collagen is more preferred.
- the cationic polypeptide may be an anionic polypeptide modified to be cationic by a chemical reaction.
- the chemical reaction includes a method of modifying an anionic carboxyl group to a cationic amino group with a diamine such as ethylenediamine, an anionic property with an amino alcohol such as 2-aminoethanethiol or an aminoalcohol such as 2-aminoalcohol.
- a method of modifying an anionic carboxyl group to an uncharged functional group by reductive amination with 5-ethyl-methylpyridine borane or the like. Can be mentioned.
- a product obtained by modifying an anionic polypeptide to be cationic by a chemical reaction is referred to as a chemically modified product of the anionic polypeptide.
- the cationic polypeptide may be a naturally derived peptide, a recombinant peptide, or a chemically synthesized peptide.
- Ratio of anionic polypeptide to cationic polypeptide is not particularly limited as long as the effects of the present invention are not impaired, but preferably 1: 5 to 5: 1. More preferably, it is 1: 3 to 3: 1, more preferably 1: 2 to 2: 1, and particularly preferably 1: 1.
- a genetically engineered amino acid sequence derived from a partial amino acid sequence of collagen has an amino acid sequence similar to collagen or gelatin produced by genetic engineering technology.
- a gene recombinant having an amino acid sequence derived from a partial amino acid sequence of collagen is also referred to as recombinant gelatin in the present specification.
- Recombinant gelatin preferably has a repeating sequence represented by Gly-XY, which is characteristic of collagen (X and Y independently represent any of amino acids).
- Gly-XY is characteristic of collagen (X and Y independently represent any of amino acids).
- the plurality of Gly-XY may be the same or different.
- recombinant gelatin for example, those described in EP1014176, US Pat. No. 6,992,172, International Publication WO2004 / 85473, International Publication WO2008 / 103041, and the like can be used, but the invention is not limited thereto.
- a preferable example of the recombinant gelatin used in the present invention is the recombinant gelatin of the following embodiment.
- Recombinant gelatin has excellent biocompatibility due to the inherent performance of natural gelatin, and is not naturally derived, so there is no concern about bovine spongiform encephalopathy (BSE) and excellent non-infectivity. Recombinant gelatin is more uniform than natural gelatin and its sequence is determined, so that strength and degradability can be precisely designed with less blur due to crosslinking or the like.
- BSE bovine spongiform encephalopathy
- the molecular weight of the recombinant gelatin is not particularly limited, but is preferably 2000 or more and 100000 or less (2 kDa (kilo dalton) or more and 100 kDa or less), more preferably 2500 or more and 95000 or less (2.5 kDa or more and 95 kDa or less), and further preferably. Is from 5,000 to 90,000 (5 kDa to 90 kDa), and most preferably from 10,000 to 90,000 (10 kDa to 90 kDa).
- Recombinant gelatin preferably has a repeating sequence represented by Gly-XY characteristic of collagen.
- the plurality of Gly-XY may be the same or different.
- Gly-XY Gly represents glycine
- X and Y represent any amino acid (preferably any amino acid other than glycine).
- the sequence represented by Gly-XY, which is characteristic of collagen, is a very specific partial structure in the amino acid composition and sequence of gelatin / collagen compared to other proteins. In this part, glycine accounts for about one third of the whole, and in the amino acid sequence, it is one in three repeats.
- Glycine is the simplest amino acid, has few constraints on the arrangement of molecular chains, and greatly contributes to the regeneration of the helix structure upon gelation.
- the amino acids represented by X and Y are rich in imino acids (proline, oxyproline), and preferably account for 10% to 45% of the total.
- 80% or more, more preferably 95% or more, and most preferably 99% or more of the amino acid sequence of the recombinant gelatin is a Gly-XY repeating structure.
- polar amino acids are charged and uncharged at 1: 1.
- the polar amino acid specifically refers to cysteine, aspartic acid, glutamic acid, histidine, lysine, asparagine, glutamine, serine, threonine, tyrosine and arginine, and among these polar uncharged amino acids are cysteine, asparagine, glutamine, serine. Refers to threonine and tyrosine.
- the proportion of polar amino acids is 10 to 40%, preferably 20 to 30%, of all the constituent amino acids.
- the proportion of uncharged amino acids in the polar amino acid is preferably 5% or more and less than 20%, more preferably 5% or more and less than 10%. Furthermore, it is preferable that any one amino acid, preferably two or more amino acids among serine, threonine, asparagine, tyrosine and cysteine are not included in the sequence.
- the minimum amino acid sequence that acts as a cell adhesion signal in a polypeptide is known (for example, “Pathophysiology”, Vol. 9, No. 7 (1990), page 527, published by Nagai Publishing Co., Ltd.).
- the recombinant gelatin used in the present invention may have two or more of these cell adhesion signals in one molecule.
- Specific sequences include RGD sequences, LDV sequences, REDV sequences, YIGSR sequences, PDSGR sequences, RYVVLPR sequences, LTITIPG sequences, RNIAEIIKDI sequences, which are represented by one-letter amino acid notation in that there are many types of cells that adhere.
- IKVAV, LRE, DGEA, and HAV sequences are preferred. More preferred are RGD sequence, YIGSR sequence, PDSGR sequence, LGTIPG sequence, IKVAV sequence and HAV sequence, and particularly preferred is RGD sequence. Of the RGD sequences, an ERGD sequence is preferred.
- the number of amino acids between RGDs is not uniform between 0 and 100, preferably between 25 and 60.
- the content of the minimum amino acid sequence is preferably 3 to 50, more preferably 4 to 30, and particularly preferably 5 to 20 per protein molecule. Most preferably, it is 12.
- the ratio of the RGD motif to the total number of amino acids is preferably at least 0.4%.
- each stretch of 350 amino acids contains at least one RGD motif.
- the ratio of the RGD motif to the total number of amino acids is more preferably at least 0.6%, even more preferably at least 0.8%, even more preferably at least 1.0%, particularly preferably at least 1.2%. %, Most preferably at least 1.5%.
- the number of RGD motifs in the recombinant peptide is preferably at least 4, more preferably 6, more preferably 8, even more preferably 12 or more and 16 or less per 250 amino acids.
- a ratio of 0.4% of the RGD motif corresponds to at least one RGD sequence per 250 amino acids. Since the number of RGD motifs is an integer, a gelatin of 251 amino acids must contain at least two RGD sequences to meet the 0.4% feature.
- the recombinant gelatin of the present invention comprises at least 2 RGD sequences per 250 amino acids, more preferably comprises at least 3 RGD sequences per 250 amino acids, more preferably at least 4 per 250 amino acids. Contains the RGD sequence. As a further aspect of the recombinant gelatin of the present invention, it contains at least 4 RGD motifs, preferably 6, more preferably 8, more preferably 12 or more and 16 or less.
- Recombinant gelatin may be partially hydrolyzed.
- the recombinant gelatin used in the present invention is represented by the formula 1: A-[(Gly-XY) n ] m -B.
- n Xs independently represents any of amino acids
- n Ys independently represents any of amino acids.
- m is preferably an integer of 2 to 10, more preferably an integer of 3 to 5.
- n is preferably an integer of 3 to 100, more preferably an integer of 15 to 70, and most preferably an integer of 50 to 65.
- A represents any amino acid or amino acid sequence
- B represents any amino acid or amino acid sequence.
- the n Gly-XYs may be the same or different.
- the recombinant gelatin used in the present invention has the formula: Gly-Ala-Pro-[(Gly-XY) 63 ] 3 -Gly (wherein 63 X independently represent any of the amino acids). 63 Y's each independently represent any of the amino acids, wherein 63 Gly-XY may be the same or different.
- the naturally occurring collagen referred to here may be any naturally occurring collagen, but is preferably type I, type II, type III, type IV, or type V collagen. More preferred is type I, type II, or type III collagen.
- the collagen origin is preferably human, bovine, porcine, mouse or rat, more preferably human.
- the isoelectric point of the recombinant gelatin used in the present invention is preferably 5 to 10, more preferably 6 to 10, and further preferably 7 to 9.5.
- the measurement of the isoelectric point of recombinant gelatin was described in the isoelectric focusing method (see Maxey, CR (1976; Phitogr. Gelatin 2, Editor Cox, PJ Academic, London, Engl.). Thus, it can be carried out by measuring the pH after passing a 1% by mass gelatin solution through a mixed crystal column of cation and anion exchange resin.
- the recombinant gelatin is not deaminated.
- the recombinant gelatin has no telopeptide.
- the recombinant gelatin is a substantially pure polypeptide prepared with a nucleic acid encoding an amino acid sequence.
- a peptide comprising the amino acid sequence set forth in SEQ ID NO: 1; (2) a peptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence described in SEQ ID NO: 1 and having a function of carrying a cationic drug; or (3) SEQ ID NO: 1 comprising the amino acid sequence having the sequence identity of 80% or more (more preferably 90% or more, particularly preferably 95% or more, most preferably 98% or more) with the amino acid sequence described in 1, and carrying a cationic drug A peptide having an action; Any of
- sequence identity between two amino acid sequences can be determined by any method known to those skilled in the art, and the BLAST ((Basic Local Alignment Search Tool)) program (J. Mol. Biol. 215: 403-410, 1990) Etc. can be used to determine.
- amino acid sequence in which one or several amino acids are deleted, substituted or added is preferably 1 to 20, more preferably 1 to 10, and further preferably 1 to 5. Means, particularly preferably 1 to 3.
- Recombinant gelatin can be produced by genetic recombination techniques known to those skilled in the art. For example, the method described in EP1014176A2, US Pat. No. 6,992,172, International Publication WO2004 / 85473, International Publication WO2008 / 103041, etc. It can be manufactured similarly. Specifically, a gene encoding the amino acid sequence of a predetermined recombinant gelatin is obtained, and this is incorporated into an expression vector to produce a recombinant expression vector, which is introduced into an appropriate host to produce a transformant. . Recombinant gelatin is produced by culturing the obtained transformant in an appropriate medium. Therefore, the recombinant gelatin used in the present invention can be prepared by recovering the recombinant gelatin produced from the culture. .
- the hydrophilicity value “1 / IOB” of the recombinant gelatin is preferably 0 to 1.0. More preferably, it is 0 to 0.6, and still more preferably 0 to 0.4.
- IOB is an index of hydrophilicity / hydrophobicity based on an organic conceptual diagram representing the polarity / non-polarity of an organic compound proposed by Satoshi Fujita. Details thereof can be found in, for example, “Pharmaceutical Bulletin”, vol.2, 2, pp .163-173 (1954), “Area of Chemistry” vol.11, 10, pp.719-725 (1957), “Fragrance Journal”, vol.50, pp.79-82 (1981), etc. Yes.
- methane (CH 4 ) is the source of all organic compounds, and all the other compounds are all methane derivatives, with certain numbers set for their carbon number, substituents, transformations, rings, etc. Then, the score is added to obtain an organic value (OV) and an inorganic value (IV), and these values are plotted on a diagram with the organic value on the X axis and the inorganic value on the Y axis. It is going.
- the IOB in the organic conceptual diagram refers to the ratio of the inorganic value (IV) to the organic value (OV) in the organic conceptual diagram, that is, “inorganic value (IV) / organic value (OV)”.
- hydrophilicity / hydrophobicity is represented by a “1 / IOB” value obtained by taking the reciprocal of IOB. The smaller the “1 / IOB” value (closer to 0), the more hydrophilic it is.
- the hydrophilicity / hydrophobicity index represented by Grand average of hydropathicity (GRAVY) value is preferably 0.3 or less and minus 9.0 or more, preferably 0.0 or less and minus 7.0 or more. Is more preferable.
- Grand average of hydropathicity (GRAVY) values are: Gasteiger E., Hoogland C., Gattiker A., Duvaud S., Wilkins MR, Appel RD, Bairoch A.; Protein Identification and Analysis Tools on the ExPASy Server John M. Walker (ed): The Proteomics Protocols Handbook, Humana Press (2005). Pp.
- the hydrophilicity is high and the water absorption is high.
- crosslinked product of anionic polypeptide and cationic polypeptide In the present invention, the anionic polypeptide and the cationic polypeptide are crosslinked.
- a cross-linked product of an anionic polypeptide and a cationic polypeptide By using a cross-linked product of an anionic polypeptide and a cationic polypeptide, a preparation exhibiting a sufficiently high acid degradation residual ratio, that is, a preferable degradation rate can be provided.
- crosslinking methods include thermal crosslinking, crosslinking with aldehydes (eg, formaldehyde, glutaraldehyde, etc.), crosslinking with condensing agents (carbodiimide, cyanamide, etc.), enzyme crosslinking, photocrosslinking, UV crosslinking, hydrophobic interaction, Hydrogen bonding, ionic interaction, and the like are known, and the above-described crosslinking method can also be used in the present invention.
- the crosslinking method used in the present invention is more preferably thermal crosslinking, ultraviolet crosslinking, or enzyme crosslinking, and particularly preferably thermal crosslinking.
- the enzyme When performing cross-linking with an enzyme, the enzyme is not particularly limited as long as it has a cross-linking action between polymer materials.
- trans-glutaminase and laccase most preferably trans-glutaminase can be used for cross-linking.
- a specific example of a protein that is enzymatically cross-linked with transglutaminase is not particularly limited as long as it has a lysine residue and a glutamine residue.
- the transglutaminase may be derived from a mammal or may be derived from a microorganism. Specifically, transglutaminase derived from a mammal that has been marketed as an Ajinomoto Co., Ltd.
- Human-derived blood coagulation factors Factor XIIIa, Haematologic Technologies, Inc.
- Factor XIIIa Haematologic Technologies, Inc.
- guinea pig liver-derived transglutaminase goat-derived transglutaminase
- rabbit-derived transglutaminase from Oriental Yeast Co., Ltd., Upstate USA Inc., Biodesign Bio International, etc. Etc.
- the reaction temperature at the time of performing crosslinking is not particularly limited as long as crosslinking is possible, but is preferably ⁇ 100 ° C. to 500 ° C., more preferably 0 ° C. to 300 ° C., and still more preferably.
- the temperature is 50 ° C to 300 ° C, more preferably 100 ° C to 250 ° C, and particularly preferably 120 ° C to 200 ° C.
- the reaction time when performing crosslinking is not particularly limited, but is generally 1 hour to 72 hours, preferably 2 hours to 48 hours, more preferably 3 hours to 36 hours. is there.
- the degree of crosslinking (number of crosslinking per molecule) of the crosslinked product in the present invention is not particularly limited, but is preferably 0.5 or more, more preferably 0.5 or more and 30 or less, and still more preferably 1 or more. 25 or less, and particularly preferably 1 or more and 20 or less.
- the method for measuring the degree of cross-linking of the cross-linked product is not particularly limited, but can be measured, for example, by the TNBS (2,4,6-trinitrobenzene sulfonic acid) method.
- crosslinked body a sample was quenched with After reacting for 3 hours at a mixture of aqueous NaHCO 3 and TNBS solution 37 ° C., crosslinked, quenched immediately after mixing aqueous NaHCO 3 and TNBS solution Each blank was prepared, the absorbance (345 nm) of the sample diluted with pure water and the blank was measured, and the degree of cross-linking (number of cross-links per molecule) was calculated from the following (Formula 1) and (Formula 2) can do.
- the cationic drug means a drug having a property of being positively charged under physiological conditions (pH 7.4).
- a polypeptide is preferred as the cationic drug.
- Polypeptides include, but are not limited to, cell growth factors, enzymes, and cytokines.
- a polypeptide for a cationic drug means a substance in which a plurality of amino acids are polymerized by peptide bonds.
- the polypeptide is preferably a substance in which 10 or more amino acids are polymerized.
- Protein is a kind of polypeptide.
- Cell growth factor means an endogenous protein that promotes the proliferation and differentiation of specific cells in an animal body.
- Cell growth factors include platelet-derived growth factor (PDGF), transforming growth factor (TGF), vascular endothelial growth factor (VEGF), connective tissue growth factor (CTGF), hepatocyte growth factor (HGF), brain-derived nerve.
- PDGF platelet-derived growth factor
- TGF transforming growth factor
- VEGF vascular endothelial growth factor
- CGF connective tissue growth factor
- HGF hepatocyte growth factor
- brain-derived nerve examples include trophic factor (BDNF), insulin-like growth factor (IGF), nerve growth factor (NGF), and glial cell line-derived neurotrophic factor (GDNF).
- BDNF trophic factor
- IGF insulin-like growth factor
- NGF nerve growth factor
- GDNF glial cell line-derived neurotrophic factor
- Enzyme means a molecule that functions as a catalyst for chemical reactions occurring in the living body.
- the enzyme include lysozyme, trypsin, ⁇ -chymotrypsin, urokinase, elastase, cytochrome C, bromelain and papain.
- Cytokine is a protein that is secreted from cells of the immune system and carries information that is not specified by the target cell. Cytokines include interferon, interleukin, erythropoietin and the like.
- the content of the cationic drug in the preparation of the present invention is not particularly limited as long as the effects of the present invention are not impaired, but is generally 0 with respect to the mass of the crosslinked product of the anionic polypeptide and the cationic polypeptide. 0.001% by mass to 10% by mass, preferably 0.01% by mass to 10% by mass, and more preferably 0.01% by mass to 5% by mass.
- the preparation of the present invention contains a cationic drug and can be used as a therapeutic agent for diseases as a medicine.
- the preparation of the present invention is preferably a sustained-release preparation and can release the cationic drug at an appropriate rate.
- the sustained-release preparation is generally intended to keep the drug concentration constant over a long period of time, and means a preparation in which the drug is gradually eluted from the preparation.
- the dosage, dosage form, and dosage form of the preparation of the present invention can be appropriately determined according to the purpose of use.
- the preparation of the present invention may be directly administered to a target site in a living body, or distilled water for injection, physiological saline for injection, pH 5-8 buffer (phosphate system, citric acid system, etc.) It may be suspended in a liquid excipient such as an aqueous solvent and administered by, for example, injection or coating.
- the preparation of the present invention may be applied after mixing with an appropriate excipient to form an ointment, gel, cream or the like.
- the dosage form of the preparation of the present invention may be oral administration or parenteral administration (for example, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, etc.) or local administration.
- parenteral administration for example, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, etc.
- local administration for example, for the treatment of heart diseases, direct injection into the cardiac muscle from the intraventricular lumen using a catheter, or local release or application using a catheter to a stenosis or occlusion in a coronary artery Can be mentioned.
- Examples of the dosage form include oral administration agents such as tablets, powders, capsules, granules, extracts and syrups, or injections (for example, intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections). Etc.) and the like.
- oral administration agents such as tablets, powders, capsules, granules, extracts and syrups
- injections for example, intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections). Etc.
- the form of the preparation of the present invention is not particularly limited, and examples thereof include sponges, films, nonwoven fabrics, fibers (tubes), particles, meshes and the like.
- the dosage of the preparation of the present invention is not particularly limited as long as the effects of the present invention are not impaired, but is generally 10 g or less, preferably 5 g or less, more preferably 1 g or less.
- the formulation of the present invention can be formulated according to methods known to those skilled in the art.
- the pharmaceutical carrier when the pharmaceutical carrier is a liquid, it can be dissolved or dispersed, and when the pharmaceutical carrier is a powder, it can be mixed or adsorbed.
- pharmaceutically acceptable additives for example, preservatives, stabilizers, antioxidants, excipients, binders, disintegrants, wetting agents, lubricants, coloring agents, fragrances
- Flavoring agents for example, preservatives, stabilizers, antioxidants, excipients, binders, disintegrants, wetting agents, lubricants, coloring agents, fragrances
- Flavoring agents for example, preservatives, stabilizers, antioxidants, excipients, binders, disintegrants, wetting agents, lubricants, coloring agents, fragrances
- Flavoring agents for example, preservatives, stabilizers, antioxidants, excipients, binders, disintegrants, wetting agents, lubricants, coloring agents
- the preparation of the present invention contains a crosslinked product of an anionic polypeptide and a cationic polypeptide and a cationic drug.
- a crosslinked product of an anionic polypeptide and a cationic polypeptide is used as a member for achieving both a sufficient loading amount of a drug and a preferable degradation rate.
- a preparation member comprising a cross-linked product of an anionic polypeptide and a cationic polypeptide.
- the present invention further includes A freezing step of freezing the solution containing the anionic polypeptide and the cationic polypeptide; A drying step of drying the frozen product obtained in the freezing step; and a crosslinking step of crosslinking the anionic polypeptide and the cationic polypeptide of the dried product obtained in the drying step:
- the manufacturing method of the member for formulation of this invention containing this is provided.
- a freezing process can be performed by a conventional method.
- (A) The difference between the temperature of the highest liquid temperature in the solution and the temperature of the lowest liquid temperature in the solution is 2.5 ° C. or less, and A step of cooling the solution of the anionic polypeptide and the cationic polypeptide to an unfrozen state at a temperature below the melting point of the solvent, and (b) the anionic polypeptide and the cationic polypeptide obtained in step (a)
- the step of freezing the solution can be performed.
- the difference between the temperature at the highest temperature and the temperature at the lowest temperature in the solution is 2.5 ° C or less. (Preferably 2.3 ° C. or lower, more preferably 2.1 ° C. or lower) That is, by reducing the temperature difference, the variation in the size of the porous pores obtained is reduced.
- the lower limit of the difference between the temperature of the highest liquid temperature and the temperature of the lowest liquid temperature in the solution is not particularly limited, and may be 0 ° C. or higher, for example, 0.1 ° C. or higher, 0.5 It may be at least 0 ° C, at least 0.8 ° C, or at least 0.9 ° C.
- the cooling in the step (a) is preferably performed via, for example, a material having a lower thermal conductivity than water, and the portion having the highest liquid temperature in the solution can be assumed to be the portion farthest from the cooling side.
- the portion of the solution having the lowest liquid temperature can be simulated as the liquid temperature of the cooling surface.
- the difference between the temperature of the highest liquid temperature in the solution and the temperature of the lowest liquid temperature in the solution immediately before the generation of heat of solidification is 2.5 ° C. or less, More preferably, it is 2.3 degrees C or less, More preferably, it is 2.1 degrees C or less.
- the “temperature difference immediately before the generation of solidification heat” means a temperature difference when the temperature difference becomes the largest between 1 second and 10 seconds before the generation of solidification heat.
- the temperature of the lowest liquid temperature in the solution is a solvent melting point of ⁇ 5 ° C. or lower, more preferably a solvent melting point of ⁇ 5 ° C. or lower and a solvent melting point of ⁇ 20 ° C. or higher. More preferably, the solvent melting point is ⁇ 6 ° C. or lower and the solvent melting point is ⁇ 16 ° C. or higher.
- the solvent having a solvent melting point is a solvent for the solution of an anionic polypeptide and a cationic polypeptide.
- step (b) the solution of the anionic polypeptide and the cationic polypeptide obtained in step (a) is frozen.
- the cooling temperature for freezing in the step (b) is not particularly limited and depends on the equipment to be cooled, but preferably from 3 ° C. from the temperature of the lowest liquid temperature in the solution.
- the temperature is 30 ° C lower, more preferably 5 ° C to 25 ° C lower, and still more preferably 10 ° C to 20 ° C lower.
- a drying process can be performed by a conventional method, for example, it can dry by performing vacuum drying at the temperature lower than melting
- the drying time is not particularly limited, and can generally be performed for about 12 hours to 144 hours.
- vacuum drying at a temperature lower than the melting point of the solvent and vacuum drying at room temperature (20 ° C.) are performed as described above, for example, vacuum drying at a temperature lower than the melting point of the solvent is performed for 6 hours to 48 hours. And vacuum drying at room temperature (20 ° C.) can be performed for 6 to 96 hours.
- the cross-linking step of cross-linking the dried anionic polypeptide and cationic polypeptide obtained in the drying step can be performed as described above in the present specification.
- the shape of the crosslinked body in the present invention is not particularly limited.
- a fixed shape an indeterminate shape, a spherical shape, a particle shape (granule), a powder shape, a porous shape, a fiber shape, a spindle shape, a flat shape and a sheet shape
- the indefinite shape indicates a shape whose surface shape is not uniform, for example, a shape having irregularities such as rocks.
- illustration of said shape is not different, respectively, For example, it may become an indefinite form as an example of the subordinate concept of a particulate form (granule).
- the present invention further includes A freezing step of freezing the solution containing the anionic polypeptide and the cationic polypeptide; A drying step of drying the frozen material obtained in the freezing step; Cross-linking step of cross-linking the anionic polypeptide and the cationic polypeptide of the dried product obtained in the drying step: and a cationic drug in the cross-linked product of the anionic polypeptide and the cationic polypeptide obtained in the drying step
- the drying step and the crosslinking step can be performed as described above in this specification.
- the drug addition step is not particularly limited as long as the cationic drug can be included in the cross-linked body, but for example, by adding a solution containing a cationic drug to the cross-linked body and immersing the solution in the cross-linked body.
- a cationic drug can be included in the crosslinked product.
- CBE3 described below was prepared as a cationic polypeptide (described in International Publication No. WO2008 / 103041).
- the amino acid sequence of CBE3 does not include serine, threonine, asparagine, tyrosine and cysteine.
- CBE3 has an ERGD sequence. Isoelectric point: 9.34 GRAVY value: -0.682 1 / IOB value: 0.323
- GAP GAPGLQGAPGLQGMPGERGAAGLPGPKGERGDAGPKGADGAPGAPGLQGMPGERGAAGLPGPKGERGDAGPKGADGAPGKDGVRGLAGPIGPPGERGAAGLPGPKGERGDAGPKGADGAPGKDGVRGLAGPIGPPGPAGAPGAPGLQGMPGERGAAGLPGAPG
- the molar ratio of “A” CBE3 to succinylated CBE3 is 1: 3
- the molar ratio of “B” CBE3 to succinylated CBE3 is 1: 1.
- the molar ratio of “C” CBE3 to succinylated CBE3 is 3: 1
- the cylindrical container made of PTFE containing the mixed aqueous solution was cooled from the bottom using a cooling shelf in a vacuum freeze dryer (TF5-85ATNNNN: Takara Seisakusho). Set the shelf temperature to -10 ° C, cool to -10 ° C for 1 hour, then -20 ° C for 2 hours, further -40 ° C for 3 hours, and finally freeze at -50 ° C for 1 hour It was.
- the frozen product obtained above is then dried at ⁇ 20 ° C. for 24 hours after returning the shelf temperature to the ⁇ 20 ° C. setting, and after 24 hours, the shelf temperature is kept at 20 ° C.
- the temperature was raised to 0 ° C., and vacuum drying was further carried out at 20 ° C. for 48 hours until the degree of vacuum was sufficiently reduced (1.9 ⁇ 10 5 Pa), and then taken out from the vacuum freeze dryer.
- the porous body was obtained by the above.
- the obtained porous body was heat-treated at 135 ° C. for 24 hours under a nitrogen atmosphere to be crosslinked.
- the molar ratio of “D” CBE3 to succinylated CBE3 is 1: 3
- the molar ratio of “E” CBE3 to succinylated CBE3 is 1: 1.
- the molar ratio of “F” CBE3 to succinylated CBE3 is 3: 1
- the cylindrical container made of PTFE containing the mixed aqueous solution was cooled from the bottom using a cooling shelf in a vacuum freeze dryer (TF5-85ATNNNN: Takara Seisakusho).
- TF5-85ATNNNN Takara Seisakusho
- Set the shelf temperature to -10 ° C cool to -10 ° C for 1 hour, then -20 ° C for 2 hours, further -40 ° C for 3 hours, and finally freeze at -50 ° C for 1 hour It was.
- the frozen product obtained above is then dried at ⁇ 20 ° C. for 24 hours after returning the shelf temperature to the ⁇ 20 ° C. setting, and after 24 hours, the shelf temperature is kept at 20 ° C.
- the temperature was raised to 0 ° C., and vacuum drying was further carried out at 20 ° C. for 48 hours until the degree of vacuum was sufficiently reduced (1.9 ⁇ 10 5 Pa), and then taken out from the vacuum freeze dryer. Thereby, a porous body was obtained.
- the molar ratio of “G” CBE3 to succinylated CBE3 is 1: 0 (CBE3 only)
- the molar ratio of “H” CBE3 to succinylated CBE3 is 0: 1 (succinylated CBE3 only)
- a cylindrical container made of PTFE containing each aqueous solution was cooled from the bottom using a cooling shelf in a vacuum freeze dryer (TF5-85ATNNNN: Takara Seisakusho).
- TF5-85ATNNNN Takara Seisakusho
- Set the shelf temperature to -10 ° C, cool to -10 ° C for 1 hour, then -20 ° C for 2 hours, further -40 ° C for 3 hours, and finally freeze at -50 ° C for 1 hour It was.
- the frozen product obtained above is then dried at ⁇ 20 ° C. for 24 hours after returning the shelf temperature to the ⁇ 20 ° C. setting, and after 24 hours, the shelf temperature is kept at 20 ° C.
- the temperature was raised to 0 ° C., and vacuum drying was further carried out at 20 ° C. for 48 hours until the degree of vacuum was sufficiently reduced (1.9 ⁇ 10 5 Pa), and then taken out from the vacuum freeze dryer. Thereby, a porous body was obtained.
- the obtained porous body was heat-treated at 135 ° C. for 24 hours under a nitrogen atmosphere to be crosslinked.
- the collected 2.0 mL tube was stirred and then centrifuged at 10,000 g for 1 minute at 4 ° C.
- 1000 ⁇ L of the supernatant was removed, 1000 ⁇ L of water was added and stirred, and then centrifuged at 10,000 g for 1 minute at 4 ° C.
- 900 ⁇ L of the supernatant was removed, 900 ⁇ L of water was added and stirred, followed by centrifugation at 10,000 g for 1 minute at 4 ° C.
- 900 ⁇ L of the supernatant was removed, 900 ⁇ L of water was added and stirred, and then centrifuged at 10,000 g for 1 minute at 4 ° C.
- With the lid open a 2.0 mL tube was placed on a tube rack and frozen at ⁇ 80 ° C.
- the 2.0 mL tube was transferred to the vacuum dryer (FDU-1000, EYELA) together with the tube rack and dried in a vacuum overnight.
- the 2.0 mL tube was taken out and the mass was measured accurately.
- the measured mass is the “total mass after reaction” in the following formula.
- the acid decomposition residual ratio was calculated by the following formula. “Tare mass” in the following formula is the mass of the tube.
- the decomposition rate of the member in the living body can be predicted from the acid decomposition residual rate.
- the residual rate of acid decomposition is large (hard to decompose).
- an acid decomposition residual ratio of 50% or more was evaluated as good and less than 50% was evaluated as defective.
- Table 1 Table 2, and Table 3 show the evaluation results of the acid decomposability of the members produced in Example 1, Comparative Example 1, and Comparative Example 2.
- the preparation member composed of a cross-linked product of CBE3 and succinylated CBE3 had a sufficiently high acid decomposition residual rate, and exhibited favorable physical properties.
- the crosslinking step was not included, the residual rate of acid decomposition was low and did not show preferable physical properties.
- the preparation member consisting only of succinylated CBE3 has a low acid decomposition residual rate, and did not show favorable physical properties.
- the preparation member consisting only of CBE3 has a high acid decomposition residual rate, and exhibits favorable physical properties.
- Formulation members “D”, “E”, “F” composed of non-crosslinked anionic polypeptide and cationic polypeptide produced in Comparative Example 1 and the molar ratio of CBE3 and succinylated CBE3 of Comparative Example 2
- the member “H” having a ratio of 0: 1 was not evaluated because the acid decomposition residual ratio did not satisfy the standard and was poor.
- FIG. 1 shows the lysozyme loading ratio of each preparation at each time point. Since the lysozyme loading was stable after 3 hours, the samples were compared based on the lysozyme loading at 3 hours. The results are shown in Tables 4 and 5.
- Lysozyme is a form of cationic drug. From the lysozyme loading rate, the interaction between each formulation member and the cationic drug can be predicted. A high lysozyme loading indicates that the interaction with the cationic drug is strong, which is preferable as a sustained release member of the cationic drug. Here, 30% or more was considered good and less than 30% was evaluated as bad.
- the preparation member composed of a crosslinked product of CBE3 and succinylated CBE3 has a high lysozyme loading rate, and exhibited preferable physical properties as a sustained release member of a cationic drug.
- the preparation member consisting of CBE3 alone had a low lysozyme loading, and did not exhibit desirable physical properties as a sustained release member of a cationic drug.
- the acid decomposition residual rate was evaluated as A for 90% or more, B for 50% or more and less than 80%, and C for less than 50%.
- the lysozyme loading was evaluated as 50% or more as A, 25% or more and less than 50% as B, and less than 25% as C. It was shown that a good acid decomposition residual rate and a good lysozyme loading rate can be achieved by the constitution of the present invention.
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Abstract
Description
[1] アニオン性ポリペプチドとカチオン性ポリペプチドとの架橋体、およびカチオン性薬物を含む製剤。
[2] 局所投与される、[1]に記載の製剤。
[3] アニオン性ポリペプチドおよびカチオン性ポリペプチドのモル比が1:3~3:1である、[1]または[2]に記載の製剤。
[4] アニオン性ポリペプチドおよびカチオン性ポリペプチドがそれぞれ、コラーゲンの部分アミノ酸配列に由来するアミノ酸配列を有する遺伝子組み換え体またはその化学修飾物である、[1]~[3]のいずれか一に記載の製剤。
[5] アニオン性ポリペプチドがカチオン性ポリペプチドの化学修飾物である、または、カチオン性ポリペプチドがアニオン性ポリペプチドの化学修飾物である、[4]に記載の製剤。
[6] コラーゲンの部分アミノ酸配列に由来するアミノ酸配列を有する遺伝子組み換え体がGly-X-Yで示される配列の繰り返しを有し、XおよびYはそれぞれ独立にアミノ酸の何れかを示し、複数個のGly-X-Yはそれぞれ同一でも異なっていてもよく、分子量が10kDa以上90kDa以下である、[4]または[5]に記載の製剤。
[7] コラーゲンの部分アミノ酸配列に由来するアミノ酸配列を有する遺伝子組み換え体が、
式:A-[(Gly-X-Y)n]m-B
で示され、上記式中、Aは任意のアミノ酸またはアミノ酸配列を示し、Bは任意のアミノ酸またはアミノ酸配列を示し、n個のXはそれぞれ独立にアミノ酸の何れかを示し、n個のYはそれぞれ独立にアミノ酸の何れかを示し、nは3~100の整数を示し、mは2~10の整数を示す。なお、n個のGly-X-Yはそれぞれ同一でも異なっていてもよい、[4]~[6]のいずれか一に記載の製剤。
[8] コラーゲンの部分アミノ酸配列に由来するアミノ酸配列を有する遺伝子組み換え体が、
(1)配列番号1に記載のアミノ酸配列、または
(2)配列番号1に記載のアミノ酸配列と80%以上の配列同一性を有し、カチオン性薬物を担持する作用を有するアミノ酸配列を有する、
[4]~[7]のいずれか一に記載の製剤。
[9] アニオン性ポリペプチドとカチオン性ポリペプチドとの架橋体が、熱処理による架橋体である、[1]~[8]のいずれか一に記載の製剤。
[10] カチオン性薬物が、ポリペプチドである、[1]~[9]のいずれか一に記載の製剤。
[12] アニオン性ポリペプチドおよびカチオン性ポリペプチドのモル比が1:3~3:1である、[11]に記載の製剤用部材。
[13] アニオン性ポリペプチドおよびカチオン性ポリペプチドが、それぞれコラーゲンの部分アミノ酸配列に由来するアミノ酸配列を有する遺伝子組み換え体またはその化学修飾物である、[11]または[12]に記載の製剤用部材。
[14] アニオン性ポリペプチドがカチオン性ポリペプチドの化学修飾物である、または、カチオン性ポリペプチドがアニオン性ポリペプチドの化学修飾物である、[13]に記載の製剤用部材。
[15] コラーゲンの部分アミノ酸配列に由来するアミノ酸配列を有する遺伝子組み換え体がGly-X-Yで示される配列の繰り返しを有し、XおよびYはそれぞれ独立にアミノ酸の何れかを示し、複数個のGly-X-Yはそれぞれ同一でも異なっていてもよく、分子量が10kDa以上90kDa以下である、[13]または[14]に記載の製剤用部材。
[16] コラーゲンの部分アミノ酸配列に由来するアミノ酸配列を有する遺伝子組み換え体が、
式:A-[(Gly-X-Y)n]m-B
で示され、上記式中、Aは任意のアミノ酸またはアミノ酸配列を示し、Bは任意のアミノ酸またはアミノ酸配列を示し、n個のXはそれぞれ独立にアミノ酸の何れかを示し、n個のYはそれぞれ独立にアミノ酸の何れかを示し、nは3~100の整数を示し、mは2~10の整数を示す。なお、n個のGly-X-Yはそれぞれ同一でも異なっていてもよい、[13]~[15]のいずれか一に記載の製剤用部材。
[17] コラーゲンの部分アミノ酸配列に由来するアミノ酸配列を有する遺伝子組み換え体が、
(1)配列番号1に記載のアミノ酸配列、または
(2)配列番号1に記載のアミノ酸配列と80%以上の配列同一性を有し、カチオン性薬物を担持する作用を有するアミノ酸配列を有する、
[13]~[16]のいずれか一に記載の製剤用部材。
[18]アニオン性ポリペプチドとカチオン性ポリペプチドとの架橋体が、熱処理による架橋体である、[11]~[17]のいずれか一に記載の製剤用部材。
上記凍結工程で得られた凍結物を乾燥する乾燥工程;および
上記乾燥工程で得られた乾燥物のアニオン性ポリペプチドおよびカチオン性ポリペプチドを架橋する架橋工程:
を含む、[11]~[18]の何れか一に記載の製剤用部材の製造方法。
[20] アニオン性ポリペプチドおよびカチオン性ポリペプチドを含む溶液を凍結する凍結工程;
上記凍結工程で得られた凍結物を乾燥する乾燥工程;
上記乾燥工程で得られた乾燥物のアニオン性ポリペプチドおよびカチオン性ポリペプチドを架橋する架橋工程:および
上記乾燥工程で得られたアニオン性ポリペプチドとカチオン性ポリペプチドとの架橋体にカチオン性薬物を包含させる薬物添加工程
を含む、[1]~[10]の何れか一に記載の製剤の製造方法。
[製剤]
本発明による製剤は、アニオン性ポリペプチドとカチオン性ポリペプチドとの架橋体、およびカチオン性薬物を含む製剤である。
アニオン性ポリペプチドとは、生理条件(pH7.4)で負に帯電する性質を有するポリペプチドを意味する。
「コラーゲンの部分アミノ酸配列に由来するアミノ酸配列の遺伝子組み換え体」については後記する。
カチオン性ポリペプチドとは、生理条件(pH7.4)で正に帯電する性質を有するポリペプチドを意味する。
アニオン性ポリペプチドとカチオン性ポリペプチドのモル比は、本発明の効果を損なわない限り特に限定されないが、好ましくは1:5~5:1であり、より好ましくは1:3~3:1であり、さらに好ましくは1:2~2:1であり、特に好ましくは1:1である。
コラーゲンの部分アミノ酸配列に由来するアミノ酸配列の遺伝子組み換え体とは、遺伝子組み換え技術により作られたコラーゲンあるいはゼラチン類似のアミノ酸配列を有するポリペプチドもしくは蛋白様物質である。コラーゲンの部分アミノ酸配列に由来するアミノ酸配列の遺伝子組み換え体は、本明細書中においてリコンビナントゼラチンとも称する。
この最小アミノ酸配列の含有量は、タンパク質1分子中3~50個が好ましく、さらに好ましくは4~30個、特に好ましくは5~20個である。最も好ましくは12個である。
好ましくは、リコンビナントゼラチンはテロペプタイドを有さない。
好ましくは、リコンビナントゼラチンは、アミノ酸配列をコードする核酸により調製された実質的に純粋なポリペプチドである。
(1)配列番号1に記載のアミノ酸配列からなるペプチド;
(2)配列番号1に記載のアミノ酸配列において1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつカチオン性薬物を担持する作用を有するペプチド;または
(3)配列番号1に記載のアミノ酸配列と80%以上(さらに好ましくは90%以上、特に好ましくは95%以上、最も好ましくは98%以上)の配列同一性を有するアミノ酸配列からなり、かつカチオン性薬物を担持する作用を有するペプチド;
の何れかである
%配列同一性=[(同一残基数)/(アラインメント長)]×100
2つのアミノ酸配列における配列同一性は当業者に公知の任意の方法で決定することができ、BLAST((Basic Local Alignment Search Tool))プログラム(J.Mol.Biol.215:403-410,1990)等を使用して決定することができる。
本発明においては、アニオン性ポリペプチドおよびカチオン性ポリペプチドは架橋されている。アニオン性ポリペプチドとカチオン性ポリペプチドとの架橋体を使用することにより、十分に高い酸分解残存率、即ち好ましい分解速度を示す製剤を提供することができる。
(式1)は、架橋体1g当たりのリジン量(モル等量)を示す。
(式中、Asはサンプル吸光度、Abはブランク吸光度、Vは反応液量(g)、wは架橋体の質量(mg)を示す。)
(式2)は、1分子あたりの架橋数を示す。
カチオン性薬物とは、生理条件(pH7.4)で正に帯電する性質を有する薬物を意味する。カチオン性薬物としては、ポリペプチドが好ましい。ポリペプチドとしては、細胞増殖因子、酵素、およびサイトカインなどが挙げられるが特に限定されない。
本発明の製剤は、カチオン性薬物を含有するものであり、医薬として疾患の治療剤として使用することができる。本発明の製剤は、好ましくは徐放製剤であり、カチオン性薬物を適切な速度で放出することができる。
上記の通り、本発明の製剤は、アニオン性ポリペプチドとカチオン性ポリペプチドとの架橋体、およびカチオン性薬物を含むものである。本発明の製剤において、アニオン性ポリペプチドとカチオン性ポリペプチドとの架橋体は、薬物の十分な担持量と好ましい分解速度とを両立するための部材として使用されている。即ち、本発明によればさらに、アニオン性ポリペプチドとカチオン性ポリペプチドとの架橋体からなる製剤用部材が提供される。本発明の製剤用部材を構成するアニオン性ポリペプチドとカチオン性ポリペプチドとの架橋体の詳細、および好ましい範囲については、本明細書中上記した通りである。
本発明はさらに、
アニオン性ポリペプチドおよびカチオン性ポリペプチドを含む溶液を凍結する凍結工程;
上記凍結工程で得られた凍結物を乾燥する乾燥工程;および
上記乾燥工程で得られた乾燥物のアニオン性ポリペプチドおよびカチオン性ポリペプチドを架橋する架橋工程:
を含む、本発明の製剤用部材の製造方法を提供する。
凍結工程の一例としては、
(a)溶液内で最も液温の高い部分の温度と溶液内で最も液温の低い部分の温度との差が2.5℃以下であり、かつ、溶液内で最も液温の高い部分の温度が溶媒の融点以下で、アニオン性ポリペプチドおよびカチオン性ポリペプチドの溶液を、未凍結状態に冷却する工程、および
(b)工程(a)で得られたアニオン性ポリペプチドおよびカチオン性ポリペプチドの溶液を凍結する工程
により行うことができる。
乾燥時間は特に限定されず、一般的には12時間~144時間程度行うことができる。上記のように溶媒の融点より低い温度での真空乾燥と、室温(20℃)での真空乾燥とを行う場合には、例えば、溶媒の融点より低い温度での真空乾燥を6時間~48時間行い、室温(20℃)での真空乾燥を、6時間~96時間行うことができる。
本発明はさらに、
アニオン性ポリペプチドおよびカチオン性ポリペプチドを含む溶液を凍結する凍結工程;
上記凍結工程で得られた凍結物を乾燥する乾燥工程;
上記乾燥工程で得られた乾燥物のアニオン性ポリペプチドおよびカチオン性ポリペプチドを架橋する架橋工程:および
上記乾燥工程で得られたアニオン性ポリペプチドとカチオン性ポリペプチドとの架橋体にカチオン性薬物を包含させる薬物添加工程
を含む、本発明の製剤の製造方法を提供する。
薬物添加工程は、架橋体にカチオン性薬物を包含させることができる限り特に限定されないが、例えば、架橋体にカチオン性薬物を含む溶液を添加し、架橋体に上記溶液を浸み込ませることにより、架橋体にカチオン性薬物を包含させることができる。
カチオン性ポリペプチドとして以下に記載のCBE3を用意した(国際公開WO2008/103041号公報に記載)。
CBE3
分子量:51.6kDa
構造:GAP[(GXY)63]3G
アミノ酸数:571個
RGD配列:12個
イミノ酸含量:33%
ほぼ100%のアミノ酸がGXYの繰り返し構造である。CBE3のアミノ酸配列には、セリン、スレオニン、アスパラギン、チロシンおよびシステインは含まれていない。CBE3はERGD配列を有している。
等電点:9.34
GRAVY値:-0.682
1/IOB値:0.323
GAP(GAPGLQGAPGLQGMPGERGAAGLPGPKGERGDAGPKGADGAPGAPGLQGMPGERGAAGLPGPKGERGDAGPKGADGAPGKDGVRGLAGPIGPPGERGAAGLPGPKGERGDAGPKGADGAPGKDGVRGLAGPIGPPGPAGAPGAPGLQGMPGERGAAGLPGPKGERGDAGPKGADGAPGKDGVRGLAGPP)3G
6.3質量%のCBE3および1.5質量%のホウ酸を含み、pHを10に調整した水溶液を作製し、上記水溶液100mLを容量300mLの3つ口フラスコに投入した。上記3つ口フラスコ中の水溶液に無水コハク酸を0.8質量%となるように添加し、混合物を37℃で攪拌しながら2時間反応させた。分画分子量(Molecular Weight Cut Off : MWCO)が3000の透析膜を用い、150倍量の蒸留水に対して透析を3回行い、反応生成物を精製し、コハク化CBE3を得た。以後の実験においては、アニオン性ポリペプチドとしてコハク化CBE3を用いた。
CBE3およびコハク化CBE3の混合水溶液を調整し、ポリテトラフルオロエチレン(PTFE)製の円筒容器に8mL投入した。この時、ポリペプチドの濃度(CBE3およびコハク化CBE3の合計濃度)は4質量%となるようにした。
この際の水溶液中のCBE3およびコハク化CBE3の割合の組み合わせは、以下に記載の通り用意した。
「A」CBE3とコハク化CBE3のモル比が1:3
「B」CBE3とコハク化CBE3のモル比が1:1
「C」CBE3とコハク化CBE3のモル比が3:1
CBE3およびコハク化CBE3の混合水溶液を調整し、PTFE製の円筒容器に8mL投入した。この時、ポリペプチドの濃度(CBE3およびコハク化CBE3の合計濃度)は4質量%となるようにした。
この際の混合水溶液中のCBE3およびコハク化CBE3の割合の組み合わせは、以下に記載の通りで用意した。
「D」CBE3とコハク化CBE3のモル比が1:3
「E」CBE3とコハク化CBE3のモル比が1:1
「F」CBE3とコハク化CBE3のモル比が3:1
CBE3の水溶液、およびコハク化CBE3の水溶液を調整し、それぞれを別々に、PTFE製の円筒容器に8mL投入した。この時、ポリペプチドの濃度(CBE3の濃度、またはコハク化CBE3の濃度)は4質量%となるようにした。
この際の水溶液中のCBE3およびコハク化CBE3の組み合わせは、以下に記載の通り用意した。
「G」CBE3とコハク化CBE3のモル比が1:0(CBE3のみ)
「H」CBE3とコハク化CBE3のモル比が0:1(コハク化CBE3のみ)
実施例1、比較例1および比較例2で製造した製剤用部材について、直径5mm、厚さ2mmに成形した。
成形した部材を、予め質量を測った2.0mLのプラスチックチューブに2個ずつ加え、質量を精密に測った。測定された質量から、チューブの質量を差し引くことにより、反応前の検体質量を求めた。その後、1mol/Lの塩酸を1.7mL加え、よく混合した。さらに、2.0mLチューブを37℃に加温したヒートブロック(DTU-2B:タイテック製)に差し込み、3時間反応させた。反応終了後、氷の入った発泡スチロール容器に2.0mLチューブを回収した。
蓋を開けた状態で2.0mLチューブをチューブ用ラックに立て、-80℃で凍結させた。
2.0mLチューブを取り出し、質量を精密に測った。測定された質量が、下記の式における「反応後の総質量」である。
下記の式で、酸分解残存率を算出した。下記の式における「風袋質量」とは、チューブの質量である。
表2の結果が示すように、架橋工程を含まない場合、酸分解残存率は低く、好ましい物性を示さなかった。
表3の結果が示すように、コハク化CBE3のみからなる製剤用部材は酸分解残存率が低く、好ましい物性を示さなかった。一方で、CBE3のみからなる製剤用部材は酸分解残存率が高く、好ましい物性を示した。
Alexa Fluor 488 Protein Labeling Kit(ThermoFisher Scientific)を用い、リゾチーム1mgを蛍光標識した。
実施例2で製造したアニオン性ポリペプチドとカチオン性ポリペプチドとの架橋体からなる製剤用部材「A」、「B」、「C」、および、比較例2で製造したCBE3とコハク化CBE3のモル比が1:0の製剤用部材「G」を直径5mmの生検トレパンでくり抜き、上下を削ぐことで成形し、2.5mgのスポンジ試料を得た。
その後、各試料に超純水で10倍希釈したリン酸緩衝生理食塩水を500μl添加した。
添加直後、1時間後、3時間後、7時間後に各試料から上清10μlを分取し、96穴黒色プレートに添加した。
分取試料に、超純水で10倍希釈したリン酸緩衝生理食塩水を190μl添加し、プレートリーダーを用いて蛍光量を評価した。
蛍光リゾチームの検量線を作成し、分取試料中の蛍光リゾチーム量を算出した。
下記の計算を行い、各時点でのリゾチーム担持率を計算した。
3時間後にはリゾチームの担持率が安定したことから、3時間時点でのリゾチーム担持率で各試料を比較し、結果を表4および表5に示した。
表5の結果が示すように、CBE3のみからなる製剤用部材はリゾチーム担持率が低く、カチオン性薬物の徐放部材として好ましい物性を示さなかった。
上記した酸分解残存率とリゾチーム担持率の評価の結果のまとめを以下に記載する。
リゾチーム担持率は、50%以上をA、25%以上50%未満をB、25%未満をCと評価した。
本発明の構成により、良好な酸分解残存率と良好なリゾチーム担持率とを両立できることが示された。
Claims (20)
- アニオン性ポリペプチドとカチオン性ポリペプチドとの架橋体、およびカチオン性薬物を含む製剤。
- 局所投与される、請求項1に記載の製剤。
- アニオン性ポリペプチドおよびカチオン性ポリペプチドのモル比が1:3~3:1である、請求項1または2に記載の製剤。
- アニオン性ポリペプチドおよびカチオン性ポリペプチドがそれぞれ、コラーゲンの部分アミノ酸配列に由来するアミノ酸配列を有する遺伝子組み換え体またはその化学修飾物である、請求項1~3のいずれか一項に記載の製剤。
- アニオン性ポリペプチドがカチオン性ポリペプチドの化学修飾物である、または、カチオン性ポリペプチドがアニオン性ポリペプチドの化学修飾物である、請求項4に記載の製剤。
- コラーゲンの部分アミノ酸配列に由来するアミノ酸配列を有する遺伝子組み換え体がGly-X-Yで示される配列の繰り返しを有し、XおよびYはそれぞれ独立にアミノ酸の何れかを示し、複数個のGly-X-Yはそれぞれ同一でも異なっていてもよく、分子量が10kDa以上90kDa以下である、請求項4または5に記載の製剤。
- コラーゲンの部分アミノ酸配列に由来するアミノ酸配列を有する遺伝子組み換え体が、
式:A-[(Gly-X-Y)n]m-B
で示され、上記式中、Aは任意のアミノ酸またはアミノ酸配列を示し、Bは任意のアミノ酸またはアミノ酸配列を示し、n個のXはそれぞれ独立にアミノ酸の何れかを示し、n個のYはそれぞれ独立にアミノ酸の何れかを示し、nは3~100の整数を示し、mは2~10の整数を示す。なお、n個のGly-X-Yはそれぞれ同一でも異なっていてもよい、請求項4~6のいずれか一項に記載の製剤。 - コラーゲンの部分アミノ酸配列に由来するアミノ酸配列を有する遺伝子組み換え体が、
(1)配列番号1に記載のアミノ酸配列、または
(2)配列番号1に記載のアミノ酸配列と80%以上の配列同一性を有し、カチオン性薬物を担持する作用を有するアミノ酸配列を有する、
請求項4~7のいずれか一項に記載の製剤。 - アニオン性ポリペプチドとカチオン性ポリペプチドとの架橋体が、熱処理による架橋体である、請求項1~8のいずれか一項に記載の製剤。
- カチオン性薬物が、ポリペプチドである、請求項1~9のいずれか一項に記載の製剤。
- アニオン性ポリペプチドとカチオン性ポリペプチドとの架橋体からなる製剤用部材。
- アニオン性ポリペプチドおよびカチオン性ポリペプチドのモル比が1:3~3:1である、請求項11に記載の製剤用部材。
- アニオン性ポリペプチドおよびカチオン性ポリペプチドが、それぞれコラーゲンの部分アミノ酸配列に由来するアミノ酸配列を有する遺伝子組み換え体またはその化学修飾物である、請求項11または12に記載の製剤用部材。
- アニオン性ポリペプチドがカチオン性ポリペプチドの化学修飾物である、または、カチオン性ポリペプチドがアニオン性ポリペプチドの化学修飾物である、請求項13に記載の製剤用部材。
- コラーゲンの部分アミノ酸配列に由来するアミノ酸配列を有する遺伝子組み換え体がGly-X-Yで示される配列の繰り返しを有し、XおよびYはそれぞれ独立にアミノ酸の何れかを示し、複数個のGly-X-Yはそれぞれ同一でも異なっていてもよく、分子量が10kDa以上90kDa以下である、請求項13または14に記載の製剤用部材。
- コラーゲンの部分アミノ酸配列に由来するアミノ酸配列を有する遺伝子組み換え体が、
式:A-[(Gly-X-Y)n]m-B
で示され、上記式中、Aは任意のアミノ酸またはアミノ酸配列を示し、Bは任意のアミノ酸またはアミノ酸配列を示し、n個のXはそれぞれ独立にアミノ酸の何れかを示し、n個のYはそれぞれ独立にアミノ酸の何れかを示し、nは3~100の整数を示し、mは2~10の整数を示す。なお、n個のGly-X-Yはそれぞれ同一でも異なっていてもよい、請求項13~15のいずれか一項に記載の製剤用部材。 - コラーゲンの部分アミノ酸配列に由来するアミノ酸配列を有する遺伝子組み換え体が、
(1)配列番号1に記載のアミノ酸配列、または
(2)配列番号1に記載のアミノ酸配列と80%以上の配列同一性を有し、カチオン性薬物を担持する作用を有するアミノ酸配列を有する、
請求項13~16のいずれか一項に記載の製剤用部材。 - アニオン性ポリペプチドとカチオン性ポリペプチドとの架橋体が、熱処理による架橋体である、請求項11~17のいずれか一項に記載の製剤用部材。
- アニオン性ポリペプチドおよびカチオン性ポリペプチドを含む溶液を凍結する凍結工程;
前記凍結工程で得られた凍結物を乾燥する乾燥工程;および
前記乾燥工程で得られた乾燥物のアニオン性ポリペプチドおよびカチオン性ポリペプチドを架橋する架橋工程:
を含む、請求項11~18の何れか一項に記載の製剤用部材の製造方法。 - アニオン性ポリペプチドおよびカチオン性ポリペプチドを含む溶液を凍結する凍結工程;
前記凍結工程で得られた凍結物を乾燥する乾燥工程;
前記乾燥工程で得られた乾燥物のアニオン性ポリペプチドおよびカチオン性ポリペプチドを架橋する架橋工程:および
前記乾燥工程で得られたアニオン性ポリペプチドとカチオン性ポリペプチドとの架橋体にカチオン性薬物を包含させる薬物添加工程
を含む、請求項1~10の何れか一項に記載の製剤の製造方法。
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