WO2019189330A1 - 新規反応性アルギン酸誘導体 - Google Patents
新規反応性アルギン酸誘導体 Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0084—Guluromannuronans, e.g. alginic acid, i.e. D-mannuronic acid and D-guluronic acid units linked with alternating alpha- and beta-1,4-glycosidic bonds; Derivatives thereof, e.g. alginates
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
- A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
- A61L15/22—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing macromolecular materials
- A61L15/28—Polysaccharides or their derivatives
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
- A61L26/0009—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form containing macromolecular materials
- A61L26/0023—Polysaccharides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
- A61L31/04—Macromolecular materials
- A61L31/042—Polysaccharides
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
- C08L5/00—Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
- C08L5/04—Alginic acid; Derivatives thereof
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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
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
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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
- A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
Definitions
- the present invention relates to an alginic acid derivative.
- Alginic acid is a bioabsorbable polysaccharide extracted from brown algae such as lessonia, macrocystis, laminaria, ascophyllum, dabilia, kazka, alame, and kombu.
- a polymer obtained by linearly polymerizing two types of uronic acids called acids (G). More specifically, D-mannuronic acid homopolymer fraction (MM fraction), L-guluronic acid homopolymer fraction (GG fraction), and D-mannuronic acid and L-guluronic acid are randomly arranged. This is a block copolymer in which the fractions (M / G fraction) are arbitrarily bound.
- alginic acid is used in a wide range of fields such as food, medicine, cosmetics, textiles and papermaking.
- Patent Documents 1 to 3 attempts have been made to modify alginic acid to a more suitable one for each application.
- Patent Documents 4 to 7 polysaccharide derivatives describing maleimide and / or thiol as a crosslinking group are known.
- JP 2010-209130 A JP 2007-99902 A International Publication No. 2004/099259 Special table 2003-516519 gazette JP-T-2015-502957 FR2967678 International Publication No. 2014/058359
- the present inventors have found that a predetermined alginic acid derivative into which a predetermined cross-linking group is introduced improves the stability after cross-linking and completes the present invention. It came to do. That is, the present invention is as the following modes [1-1] to [22b].
- a part of at least one carboxyl group selected from the group consisting of alginic acid, its ester and its salt is represented by the following formula (I) (excluding the right side of the broken line): (Where -A 1 -is represented by the following formula (excluding the outside of both broken lines): A linker selected from the group consisting of;
- Each R 1 is independently a hydrogen atom, methyl group, isopropyl group, isobutyl group, sec-butyl group, hydroxymethyl group, 2-hydroxyethyl group, thiolmethyl group, methylthioethyl group, carboxymethyl group, carboxyethyl, Group, aminocarbonylmethyl group, aminocarbonylethyl group, aminobutyl group, guanidinopropyl group, benzyl group, 4-hydroxybenzyl group, 3-indolylmethyl group, 4-imidazolylmethyl group, and carbon to which R 1 is bonded.
- -A 1 represents the following formula (in the formula, excluding the outside of the broken lines):
- the group represented by the formula (I) is represented by the following formula (excluding the right side of the broken line):
- [5] The alginic acid derivative according to any one of [1-1] to [4], wherein the weight average molecular weight of the alginic acid derivative measured by gel filtration chromatography is 100,000 Da to 3 million Da.
- a part of at least one carboxyl group selected from the group consisting of alginic acid, its ester and its salt is represented by the following formula (I) (excluding the right side of the broken line):
- Each R 1 is independently a hydrogen atom, methyl group, isopropyl group, isobutyl group, sec-butyl group, hydroxymethyl group, 2-hydroxyethyl group, thiolmethyl group, methylthioethyl group, carboxymethyl group, carboxyethyl, Group, aminocarbonylmethyl group, aminocarbonylethyl group, aminobutyl group, guanidinopropyl group, benzyl group, 4-hydroxybenzyl group, 3-indolylmethyl group, 4-imidazolylmethyl group and carbon atom to which R 1 is bonded
- -A 1 represents the following formula (in the formula, excluding the outside of the broken lines):
- -A 1 - is represented by the following formula (wherein the outside of the broken lines is excluded):
- the group represented by the formula (I) is represented by the following formula (excluding the right side of the broken line):
- the alginic acid derivative according to any one of [1a-1] to [2a] selected from the group consisting of: [3a-1]
- the group represented by the formula (I) is represented by the following formula (wherein the right side of the broken line is excluded):
- a group selected from the group consisting of [4a] The alginic acid derivative according to any one of [1a
- a part of at least one carboxyl group selected from the group consisting of alginic acid, its ester and its salt is represented by the following formula (I) (excluding the right side of the broken line):
- Each R 1 is independently a hydrogen atom, methyl group, isopropyl group, isobutyl group, sec-butyl group, hydroxymethyl group, 2-hydroxyethyl group, thiolmethyl group, methylthioethyl group, carboxymethyl group, carboxyethyl, Group, aminocarbonylmethyl group, aminocarbonylethyl group, aminobutyl group, guanidinopropyl group, benzyl group, 4-hydroxybenzyl group, 3-indolylmethyl group, 4-imidazolylmethyl group and carbon atom to which R 1 is bonded
- -A 1 represents the following formula (in the formula, excluding the outside of both broken lines):
- a part of at least one carboxyl group selected from the group consisting of alginic acid, an ester thereof and a salt thereof has the following formula (II) (excluding the right side of the broken line in the formula):
- P 1 is a hydrogen atom or a protecting group for a thiol group (—SH group)
- -A 2 - is represented by the following formula (excluding both wavy lines outside):
- Ar is a phenylene group which may be substituted with a water-soluble substituent (for example, 1 to 2); n4 is an integer from 0 to 10; m4 is an integer from 0 to 10; p is an integer of 0 to 10)
- the group represented by the formula (II) is represented by the following formula (excluding the right side of the broken line):
- the group represented by the formula (II) is represented by the following formula (excluding the right side of the broken line in each formula):
- the alginic acid derivative according to any one of [6] to [8-1] selected from the group consisting of:
- [12] A composition comprising the alginic acid derivative according to any one of [1-1] to [5] and the alginic acid derivative according to any one of [6] to [11].
- [12a] A composition comprising the alginic acid derivative according to any one of [1a-1] to [5a] and the alginic acid derivative according to any one of [6] to [11].
- [12b] A composition comprising the alginic acid derivative according to any one of [1b-1] to [5b] and the alginic acid derivative according to any one of [6] to [11].
- a crosslinking reaction is performed on the alginic acid derivative according to any one of [1-1] to [5] and the alginic acid derivative according to any one of [6] to [11].
- a crosslinking reaction is performed on the alginic acid derivative according to any one of [1a-1] to [5a] and the alginic acid derivative according to any one of [6] to [11].
- [13b-1] The alginic acid derivative according to any one of [1b-1] to [5b] and the alginic acid derivative according to any one of [6] to [11] are subjected to a crosslinking reaction.
- [13-2] A solution of the alginic acid derivative according to any one of the above [1-1] to [5] is dropped into a solution containing calcium ions as the crosslinked alginic acid structure, and the obtained gel is added to the gel.
- [13a-2] A solution of the alginic acid derivative according to any one of the above [1a-1] to [5a] is dropped into a solution containing calcium ions, and the crosslinked alginate structure is added to the gel obtained.
- [13b-2] The solution of the alginic acid derivative according to any one of the above [1b-1] to [5b] is dropped into a solution containing calcium ions, and the crosslinked alginate structure is added to the gel obtained.
- [13-3] A solution of the alginic acid derivative according to any one of [6] to [11], wherein the crosslinked alginic acid structure is dropped into a solution containing calcium ions.
- [13a-3] A solution of the alginic acid derivative according to any one of the above [6] to [11] is dropped into a solution containing calcium ions, and the crosslinked gel alginate structure is added to the obtained gel.
- the crosslinked alginic acid structure according to the above [13a-1] obtained by performing a crosslinking reaction in a solution of the alginic acid derivative according to any one of [1a-1] to [5a].
- [13b-3] A solution of the alginic acid derivative according to any one of the above [6] to [11] is dropped into a solution containing calcium ions as the crosslinked alginic acid structure.
- the crosslinked alginic acid structure according to [13b-1] which is obtained by performing a crosslinking reaction in a solution of the alginic acid derivative according to any one of [1b-1] to [5b].
- a medical material comprising the crosslinked alginic acid structure according to any one of [13-1] to [13-5].
- [18] A method for producing a crosslinked alginic acid structure, which comprises dropping a solution of the composition according to [12] above into a solution containing calcium ions.
- [18a] A method for producing a crosslinked alginic acid structure, which comprises dropping the solution of the composition according to [12a] above into a solution containing calcium ions.
- [18b] A method for producing a crosslinked alginic acid structure, which comprises dropping a solution of the composition according to [12b] above into a solution containing calcium ions.
- [21] A method for producing a crosslinked alginate structure, comprising partially crosslinking the composition according to [12] with a divalent metal ion.
- [21a] A method for producing a crosslinked alginic acid structure comprising partially crosslinking the composition according to [12a] with a divalent metal ion.
- [21b] A method for producing a crosslinked alginate structure, comprising partially crosslinking the composition according to [12b] with a divalent metal ion.
- the present invention provides a new alginic acid derivative.
- the alginic acid derivative has improved stability after crosslinking.
- Cross-linked alginate structure (AL-EX-8 / AL-EX-7-1-2, AL-EX-9 / AL-EX-7-1-2, AL-EX-10 / AL-EX-7-1 -2 or AL-EX-2-1 / AL-EX-7-1-2) is a graph showing evaluation of gel stability after EDTA treatment.
- Cross-linked alginate structure (AL-EX-8 / AL-EX-7-1-2, AL-EX-9 / AL-EX-7-1-2, AL-EX-10 / AL-EX-7-1 -2 or AL-EX-2-1 / AL-EX-7-1-2).
- alginic acid derivatives are provided.
- a part of the carboxyl group of alginic acid is substituted with a bridging group (also referred to as “reactive group”) via a linker.
- a linker that is, for example, one having one or more carboxyl groups of alginic acid and a linker (-L-) having a bridging group (Z) and an amino group at both ends in an amide bond (formula AL-1 below) (Wherein Z is a bridging group; -L- is a linker.
- -L- is, for example, -A 1- in the above formula (I) or -A in the above formula (II). 2- , etc.).
- the crosslinking group is, for example, an acrylic acid residue or a thiol residue. Both crosslinking groups of acrylic acid residues or thiol residues can form a covalent bond more easily than the Michael addition reaction.
- acrylic acid residue examples include a residue capable of forming a Michael adduct by reaction with a thiol residue. Specific examples include acrylic acid, maleic acid, maleimide, fumaric acid, and the like. It is done.
- Preferred examples of the thiol residue include benzylthiol or thiophenol.
- the cross-linking group is preferably one that can easily form a Michael adduct by a Michael addition reaction.
- the acrylic acid residue is an acryloyl group
- the thiol residue is a thiol group; more preferably
- the acrylic acid residue is a maleimide group
- the thiol residue is a benzylthiol group.
- the crosslinking group may be bonded to a linker (spacer) for bonding to both the crosslinking group and alginic acid and keeping the both at a certain distance.
- a linker spacer
- An alginic acid derivative in which maleimide, or benzylthiol or a thiol-protected form of benzylthiol is bonded via a linker as a crosslinking group is preferable.
- alginic acid derivatives are provided: A part of the carboxyl group of at least one alginic acid selected from the group consisting of alginic acid, its ester and its salt is represented by the following formula (I) (excluding the right side of the broken line): (In the formula, -A 1 -represents the following formula (excluding the outside of both broken lines): A linker selected from the group consisting of; Each R 1 is independently a hydrogen atom, methyl group, isopropyl group, isobutyl group, sec-butyl group, hydroxymethyl group, 2-hydroxyethyl group, thiolmethyl group, methylthioethyl group, carboxymethyl group, carboxyethyl, Group, aminocarbonylmethyl group, aminocarbonylethyl group, aminobutyl group, guanidinopropyl group, benzyl group, 4-hydroxybenzyl group, 3-indolylmethyl group, 4-imidazolylmethyl group and carbon
- alginic acid derivatives A part of the carboxyl group of at least one alginic acid selected from the group consisting of alginic acid, its ester and its salt is represented by the following formula (II) (excluding the right side of the broken line): (Wherein P 1 is a hydrogen atom or a protecting group for a thiol group (—SH group); -A 2 -is represented by the following formula (in the formula, excluding both broken lines): A linker which is In the above -A 2- , Ar is a phenylene group which may be substituted with a water-soluble substituent (for example, 1 to 3); n4 is an integer from 0 to 10; m4 is an integer from 0 to 10; p is an integer of 0 to 10) An alginic acid derivative having a group represented by:
- the alginic acid derivative is more specifically, The following formula (AL-1-I) in which any one or more carboxyl groups of alginic acids form an amide bond with the crosslinking group represented by the formula (I): [In the formula (AL-1-I), the definition of the linker (—A 1 —) is as defined above], or any one or more carboxyl groups of alginic acids Is forming the amide bond with the bridging group represented by the formula (II), the following formula (AL-1-II): [In the formula (AL-1-II), the definition of P 1 and the linker (—A 2 —) is as defined above].
- “having a group of formula (I)”, “substituted by a group of formula (I)”, “having a group of formula (II)” or “substituted by a group of formula (II)” Is a group of formula (I) or a group of formula (II) (that is, a spacer bonded to a bridging group) in at least one selected from the group consisting of alginic acid, esters thereof and salts thereof.
- a spacer bonded to a bridging group in at least one selected from the group consisting of alginic acid, esters thereof and salts thereof.
- the group of formula (I) and / or formula (II) is one of the carboxyl groups of at least one alginic acid selected from the group consisting of alginic acid, its ester and its salt (sometimes referred to as “alginic acid”). It is introduced into alginic acids by replacing the part.
- the weight average molecular weight of the alginic acid derivative represented by the formula (AL-1-I) or the formula (AL-1-II) is preferably 100,000 Da to 3 million Da, more preferably 300,000 Da to 2.5 million Da. More preferably, it is 500,000 Da to 2 million Da.
- the molecular weight of the alginic acid derivative can be determined by the same method as that for the aforementioned alginic acids.
- the bridging group represented by the formula (I) and the alginic acid derivative represented by the formula (AL-1-II) are represented by the formula (II).
- the bridging groups do not have to be bonded to all the carboxyl groups of the structural units of the alginic acids.
- the introduction rate of the group of the formula (I) and the group of the formula (II) in each alginic acid derivative is preferably 1% to 30%, more preferably 2% to 15%, and still more preferably 3% to 10%.
- the introduction rate of the group of the formula (I) and the group of the formula (II) in each alginic acid derivative is a value expressed as a percentage of the number of uronic acid monosaccharide units into which a cross-linking group has been introduced among the uronic acid monosaccharide units that are repeating units of alginic acids.
- the% used for the introduction rate of the group of formula (I) or formula (II) in the alginic acid derivative (formula (AL-1-I) or formula (AL-1-II)) is mol%. means.
- the introduction rate of the group of the formula (I) or the formula (II) can be determined by the method described in Examples described later.
- the maleimide group in the formula (AL-1-I) and the thiol group in the formula (AL-1-II) form a covalent bond (sulfide bond) by the Michael addition reaction, thereby forming a bridge. Can be done.
- crosslinking group and linker in the formula (I), the partial structural formula (except the right side of the broken line in the formula): May be referred to as “crosslinking group” or “reactive group”
- -A 1 - may be referred to as “spacer” or “linker”.
- n is preferably an integer of 1 to 10, more preferably an integer of 1 to 8, and further preferably an integer of 3 to 6.
- m is preferably an integer of 1 to 7, more preferably an integer of 1 to 5, still more preferably an integer of 1 to 3, and particularly preferably 1 or 2.
- j is preferably an integer of 0 to 8, more preferably an integer of 1 to 6, still more preferably an integer of 2 to 4, and particularly preferably 0 or 1. .
- the linker -A 1- in the formula (I) is represented by the following formula (AL-A1-1-a) (wherein both sides of the broken line are not included):
- the configuration of the carbon substituted by the benzyl group is the S-form (AL-A1-1-aS) and the configuration of the carbon substituted by the benzyl group is the R-form (AL-A1) -1-aR) (in any of the formulas, not including both sides of the broken line): Is included.
- one of the formulas (I), the formula (Ix) (where the right side of the broken line is not included): Includes optical isomers, and unless otherwise specified, includes S-isomer (formula (IxS)) or R-isomer (formula (IxR)) Means that.
- an amine derivative (AM-I) corresponding to the formula (I) is synthesized.
- the process it is possible to separate the racemic form into each optically active form by a conventional optical resolution means (separation method), and to synthesize an amine derivative (AM-I) corresponding to the formula (I).
- one of the optical isomers can be selectively synthesized by using asymmetric synthesis, and each optically active substance can be synthesized.
- an alginic acid derivative into which the (optically active) group of the formula (I) having an asymmetric carbon can be introduced.
- Fractionation recrystallization method An optical resolution agent is ion-bonded to the racemate to obtain a crystalline diastereomer, and then the crystalline diastereomer is separated by a fractional recrystallization method and optionally optically resolved. In this method, an optically pure compound is obtained through the agent removal step.
- Optical resolution agents include, for example, (+)-mandelic acid, ( ⁇ )-mandelic acid, (+)-tartaric acid, ( ⁇ )-tartaric acid, (+)-1-phenethylamine, ( ⁇ )-1-phenethylamine, cinchonine , ( ⁇ )-Cinchonidine, brucine and the like.
- Diastereomer method An optical resolution agent is covalently bonded to a racemic mixture to obtain a mixture of diastereomers, and then by a conventional separation means (for example, fractional recrystallization, silica gel column chromatography, HPLC, etc.) In this reaction, an optically pure optical isomer is obtained through separation into an optically pure diastereomer, followed by a step of removing an optical resolution agent by a chemical reaction (hydrolysis reaction or the like).
- a conventional separation means for example, fractional recrystallization, silica gel column chromatography, HPLC, etc.
- the compound of the present invention or the intermediate compound has a hydroxyl group or an amino group (primary or secondary), the compound and an optically active organic acid (for example, ⁇ -methoxy- ⁇ - (trifluoromethyl) phenyl)
- an optically active organic acid for example, ⁇ -methoxy- ⁇ - (trifluoromethyl) phenyl
- ester or amide diastereomers are obtained from each.
- the compound of this invention has a carboxy group
- the diastereomer of an amide body or an ester body is obtained from each by the condensation reaction of the said compound, an optically active amine, or an optically active alcohol.
- the diastereomers obtained by the condensation reaction are separated, and each diastereomer is subjected to a hydrolysis reaction with an acid or a base to be converted into an optically pure optical isomer of the original compound.
- Chiral column method A method in which a racemate or a salt thereof is subjected to direct optical resolution by subjecting it to chromatography using a chiral column (column for separating optical isomers).
- a chiral column for example, CHIRAL series manufactured by Daicel Corporation
- an elution solvent water, various buffer solutions (for example, phosphate buffer) is added.
- Liquid Liquid
- organic solvents eg, single solvents such as ethanol, methanol, isopropanol, acetonitrile, trifluoroacetic acid, and diethylamine, or mixed solvents thereof
- Separation is possible.
- optical isomers can be separated using a chiral column (for example, CP-Chirasil-DeX CB (manufactured by GL Science)).
- a mixture of optical isomers is added to a chiral column (for example, CHIRAL series manufactured by Daicel), and carbon dioxide and an appropriate organic solvent (for example, , Methanol, ethanol, isopropanol, trifluoroacetic acid, and diethylamine, etc.) can be used to separate optical isomers.
- a chiral column for example, CHIRAL series manufactured by Daicel
- carbon dioxide and an appropriate organic solvent for example, Methanol, ethanol, isopropanol, trifluoroacetic acid, and diethylamine, etc.
- Asymmetric synthesis for selectively synthesizing one of the optical isomers includes (1) an asymmetric synthesis reaction in which a racemic compound is enantioselectively reacted to give an optically active substance, and (2) a naturally occurring optically active compound. And a method of synthesizing diastereoselectively from (sugar, amino acid, etc.).
- the group represented by the formula (I) has the following formula (in each formula, excluding the right outer side of the broken line): (In some embodiments, the following formulas (in each formula, excluding the right outside of the dashed line): Except for the group).
- the group represented by the formula (I) is represented by the following formula (in each formula, excluding the right outer side of the broken line): (In some embodiments, the following formulas (in each formula, excluding the right outside of the dashed line): Except for the group).
- the partial structure May be referred to as a “crosslinking group” or “reactive group”, and —A 2 — may be referred to as a “spacer” or “linker”.
- P 1 is a protecting group for a hydrogen atom or a thiol group (—SH group).
- the protective group include protective groups such as an acetyl group, a benzoyl group, a triphenylmethyl group, a methoxymethyl group, and an N-ethylcarbamate group, preferably an acetyl group and a benzoyl group, and more Preferably, it is an acetyl group.
- P 1 is preferably a hydrogen atom, an acetyl group or a benzoyl group, and more preferably a hydrogen atom or an acetyl group.
- n4 is preferably an integer of 0 to 8, more preferably an integer of 0 to 6, still more preferably an integer of 0 to 2, and particularly preferably 0 or 2.
- m4 is preferably an integer of 0 to 8, more preferably an integer of 0 to 6, even more preferably an integer of 0 to 2, and particularly preferably 1.
- p is preferably an integer of 0 to 8, more preferably an integer of 1 to 6, still more preferably an integer of 2 to 4, and particularly preferably 2 or 3.
- Ar is a phenylene group that may be substituted with a water-soluble substituent, such as an o-phenylene group, an m-phenylene group, or a p-phenylene group, and preferably a p-phenylene group. is there.
- the phenylene group may be independently substituted with, for example, 1 to 4, preferably 1 to 3, more preferably 1 to 2 water-soluble substituents.
- a phenylene group is a polyvalent group formed by removing two hydrogen atoms from a benzene ring, and is represented by —C 6 H 4 —.
- the phenylene group includes, for example, an orthophenylene group (o-phenylene group) obtained by removing two hydrogen atoms in the ortho position from each other, a metaphenylene group (m-phenylene group) obtained by removing two hydrogen atoms in the meta position from each other, Examples thereof include a paraphenylene group (p-phenylene group) obtained by removing two hydrogen atoms in the para position from each other.
- the water-soluble substituent includes, for example, a hydroxyl group (—OH), a carboxyl group (—COOH), an amino group (—NH 2 ), a thiol group (—SH), or a sulfo group (—SO 2 OH). And the like, preferably a hydroxyl group and an amino group.
- Ar is an unsubstituted phenylene group (for example, an o-phenylene group, an m-phenylene group, or a p-phenylene group, preferably a p-phenylene group).
- —A 2 — is more preferably the following formula (in each formula, excluding the outside of both broken lines):
- a linker selected from the group consisting of; —Ar— is a p-phenylene group.
- the group represented by the formula (II) has the following formula (in each formula, excluding the right side of the broken line): Selected from the group consisting of
- formula (II) is represented by the following formula (in each formula, excluding the right side of the broken line): Selected from the group consisting of
- the crosslinking group (reactive group) of formula (I) and / or formula (II) may be a group that forms a Michael adduct by the Michael reaction and thus proceeds to the crosslinking reaction. Further, by introducing a spacer (linker), the crosslinking reaction proceeds even when the introduction rate of the crosslinking group is low. Due to the crosslinking reaction, the alginic acid derivative forms a three-dimensional network structure via the crosslinking. Preferred alginic acid derivatives have improved stability after crosslinking compared to before crosslinking.
- Alginic acids used may be naturally derived or synthetic, but are preferably naturally derived.
- Alginic acids preferably used are bioabsorbable polysaccharides extracted from brown algae such as Lessonia, Macrocystis, Laminaria, Ascofilum, Davilia, Kajika, Alame, Kombu, etc., and D-mannuronic acid (M) And L-guluronic acid (G) are polymers in which two types of uronic acids are polymerized in a straight chain. More specifically, D-mannuronic acid homopolymer fraction (MM fraction), L-guluronic acid homopolymer fraction (GG fraction), and D-mannuronic acid and L-guluronic acid are randomly arranged.
- alginic acid means at least one alginic acid selected from the group consisting of alginic acid, alginic acid esters, and salts thereof (for example, sodium alginate).
- Da Dallton
- alginic acid derivatives crosslinked alginic acid, and crosslinked alginic acid.
- the composition ratio (M / G ratio) of D-mannuronic acid and L-guluronic acid of alginic acids varies depending on the type of organisms that are mainly derived from seaweeds, etc., and is also affected by the location and season of the organism. , Ranging from a high G type with an M / G ratio of about 0.2 to a high M type with an M / G ratio of about 5. It is known that the gelation ability of alginic acids and the properties of the generated gel are affected by the M / G ratio, and generally the gel strength increases when the G ratio is high. In addition, the M / G ratio affects the hardness, brittleness, water absorption, flexibility, and the like of the gel.
- the M / G ratio of the alginic acids and / or salts thereof used is usually 0.2 to 4.0, more preferably 0.4 to 3.0, still more preferably 0.5 to 3.0. is there.
- a numerical range indicated using “to” indicates a range including the numerical values described before and after “to” as the minimum value and the maximum value, respectively.
- Alginic acid ester and “alginate” to be used are not particularly limited. However, in order to react with a cross-linking agent, it is necessary not to have a functional group that inhibits the cross-linking reaction.
- the alginic acid ester is preferably propylene glycol alginate.
- alginates include monovalent salts of alginic acid and divalent salts of alginic acid.
- sodium alginate, potassium alginate, ammonium alginate and the like are preferable, sodium alginate or potassium alginate is more preferable, and sodium alginate is particularly preferable.
- Preferred examples of the divalent salt of alginic acid include calcium alginate, magnesium alginate, barium alginate, strontium alginate, and the like.
- Alginic acids are high molecular polysaccharides, and it is difficult to accurately determine the molecular weight, but generally the weight average molecular weight is 10 to 10 million, preferably 10,000 to 8 million, more preferably 20,000 to 300. It is in the range of 10,000.
- the value may vary depending on the measurement method.
- the weight average molecular weight measured by gel permeation chromatography (GPC) or gel filtration chromatography (also referred to as size exclusion chromatography) is preferably 100,000 or more, more preferably 500,000 or more, Preferably, it is 5 million or less, more preferably 3 million or less.
- the preferable range is 100,000 to 5,000,000, more preferably 150,000 to 3,000,000.
- the absolute weight average molecular weight can be measured.
- the weight average molecular weight (absolute molecular weight) measured by GPC-MALS method is preferably 10,000 or more, more preferably 50,000 or more, still more preferably 60,000 or more, and preferably 1,000,000 or less, more preferably 80 10,000 or less, more preferably 700,000 or less, and particularly preferably 500,000 or less.
- the preferable range is 10,000 to 1,000,000, more preferably 50,000 to 800,000, still more preferably 60,000 to 700,000, and particularly preferably 60,000 to 500,000.
- a measurement error of 10% to 20% may occur.
- the value may vary within the range of about 320,000 to 480,000 for 400,000, 400,000 to 600,000 for 500,000, and about 800,000 to 1.2 million for 1,000,000.
- the molecular weight of alginic acids can be measured according to a conventional method.
- Typical conditions when gel filtration chromatography is used for molecular weight measurement are as described in the examples of the present specification to be described later.
- the column for example, Superose 6 Increase 10/300 GL column (GE Healthcare Science) can be used, and as a developing solvent, for example, 10 mmol / L phosphate buffer (pH 7.4) containing 0.15 mol / L NaCl.
- Blue dextran, thyroglobulin, ferritin, aldolase, conalbumin, ovalbumin, ribonuclease A and aprotinin can be used as molecular weight standards.
- the viscosity of the alginic acid used is not particularly limited, but is preferably 10 mPa ⁇ s to 1000 mPa ⁇ s, more preferably 50 mPa ⁇ s to 800 mPa ⁇ s when the viscosity is measured as an aqueous solution of 1 w / w% alginic acid. s.
- the viscosity of an aqueous solution of alginic acid can be measured according to a conventional method.
- a rotational viscometer method such as a coaxial double cylindrical rotational viscometer, a single cylindrical rotational viscometer (Brookfield viscometer), a cone-plate rotational viscometer (cone plate viscometer), etc.
- a rotational viscometer method such as a coaxial double cylindrical rotational viscometer, a single cylindrical rotational viscometer (Brookfield viscometer), a cone-plate rotational viscometer (cone plate viscometer), etc.
- a cone plate viscometer is used.
- Alginic acids are initially high in molecular weight and high in viscosity when extracted from brown algae, but in the process of drying and purification by heat, the molecular weight decreases and the viscosity decreases.
- Alginic acids having different molecular weights can be produced by techniques such as temperature control in the production process, selection of brown algae as a raw material, and molecular weight fractionation in the production process. Furthermore, it is possible to obtain alginic acids having a target molecular weight by mixing with another lot of alginic acids having different molecular weights or viscosities.
- the alginic acid used is not low endotoxin in some embodiments.
- the alginate is of low endotoxin.
- Low endotoxin refers to a low endotoxin level that does not substantially cause inflammation or fever. More preferably, it is desirable to use alginic acids treated with low endotoxin.
- the low endotoxin treatment can be performed by a known method or a method analogous thereto.
- the method of Takada et al. See, for example, JP-A-9-32001 for purifying sodium hyaluronate, and the method of Yoshida et al. (Eg, JP-A-8-269102) for purifying ⁇ 1,3-glucan. Etc.)
- a method of William et al. (For example, see JP-T-2002-530440), a method of purifying a biopolymer salt such as alginate, gellan gum, etc., a method of James et al.
- Low endotoxin treatment is not limited to these, and uses washing, filtration (endotoxin removal filter, charged filter, etc.), ultrafiltration, column (endotoxin adsorption affinity column, gel filtration column, ion exchange resin column, etc.) Purification, adsorption to hydrophobic substances, resin or activated carbon, organic solvent treatment (extraction with organic solvent, precipitation / precipitation by addition of organic solvent, etc.), surfactant treatment (for example, see JP-A-2005-036036) ) Or other known methods, or a combination thereof. These processing steps may be appropriately combined with known methods such as centrifugation. It is desirable to select appropriately according to the type of alginic acid.
- the endotoxin level can be confirmed by a known method, and can be measured, for example, by a method using Limulus reagent (LAL), a method using Enspercy (registered trademark) ES-24S set (Seikagaku Corporation), or the like. .
- LAL Limulus reagent
- Enspercy registered trademark
- ES-24S set Seikagaku Corporation
- the endotoxin treatment method to be used is not particularly limited.
- the endotoxin content of alginic acids is 500 endotoxin units (EU) / g or less when the endotoxin measurement is performed with Limulus reagent (LAL).
- LAL Limulus reagent
- EU endotoxin units
- Low endotoxin-treated sodium alginate can be obtained from commercially available products such as Sea Matrix (registered trademark) (Mochida Pharmaceutical Co., Ltd.), PRONOVA TM UP LVG (FMCBioPolymer), and the like.
- any one or more carboxyl groups of alginic acids are represented by the following formula (AL-1-I) in which an amide bond is formed with the crosslinking group represented by the formula (I).
- A-1-II at least one kind of alginic acid derivative and any one or more carboxyl groups of the alginic acid form an amide bond with the bridging group represented by the formula (II).
- Compositions comprising at least one alginate derivative and body represented are provided.
- each embodiment of the linker (—A 1 —) of the formula (AL-1-I), and each embodiment of the linker (—A 2 —) of the formula (AL-1-II) and P 1 are as described above. Street.
- the weight ratio of the alginic acid derivative of formula (AL-1-I) to the alginic acid derivative of formula (AL-1-II) (the alginic acid derivative of formula (AL-1-I):
- the alginic acid derivative of AL-1-II) is, for example, 1: 1 to 1.5, preferably 1: 1.2 to 1.5, or 1: 1 to 1.2, more preferably 1: 1. It is.
- the weight ratio of the alginic acid derivative of formula (AL-1-II) to the alginic acid derivative of formula (AL-1-I) is, for example, 1: 1 to 1.5, preferably 1: 1.2 to 1.5, or 1: 1 to 1.2, more preferably 1: 1. It is.
- the mixing ratio of the alginic acid derivative of formula (AL-1-I) and the alginic acid derivative of formula (AL-1-II) is equal to the alginic acid derivative of formula (AL-1-I) and the formula
- the ratio of introduction of the crosslinking group (reactive group) (mol%) of the alginic acid derivative of (AL-1-II) is, for example, 1: 1 to 1.5, preferably 1: 1.2 to 1. 5, or 1: 1 to 1.2, more preferably 1: 1.
- the mixing ratio of the alginic acid derivative of the formula (AL-1-II) and the alginic acid derivative of the formula (AL-1-I) is equal to the alginic acid derivative of the formula (AL-1-II) and the formula
- the introduction ratio (mol%) of the crosslinking group (reactive group) of the alginic acid derivative of (AL-1-I) for example, 1: 1 to 1.5, preferably 1: 1.2 to 1. 5, or 1: 1 to 1.2, more preferably 1: 1.
- the cross-linked alginic acid structure is composed of an alginic acid derivative of the formula (AL-1-I), an alginic acid derivative of the formula (AL-1-II), or a mixture thereof (hereinafter simply referred to as “alginic acid derivative”). Described) is a three-dimensional network structure formed via a crosslinking group.
- the crosslinked alginic acid structure can be obtained by subjecting an alginic acid derivative having a crosslinking group to a crosslinking reaction.
- the cross-linking reaction can be carried out, for example, as described below, or an appropriate combination thereof, but is not limited thereto.
- A a crosslinking reaction (covalent crosslinking reaction) by reacting a composition containing an alginic acid derivative of the formula (AL-1-I) with a composition containing an alginic acid derivative of the formula (AL-1-II); Or (b) a composition containing an alginic acid derivative of the formula (AL-1-I) or an alginic acid derivative of the formula (AL-1-II) containing a divalent metal ion (for example, calcium ion, barium ion, etc.) Cross-linking reaction by reacting in solution (ion bond cross-linking reaction).
- a crosslinking reaction covalent crosslinking reaction
- (C) a solution containing an alginic acid derivative of the formula (AL-1-I) and an alginic acid derivative of the formula (AL-1-II) containing a divalent metal ion (for example, calcium ion, barium ion, etc.)
- a divalent metal ion for example, calcium ion, barium ion, etc.
- the method for preparing the crosslinked alginic acid structure is described in 2.2 below.
- the shape of the cross-linked alginate structure is not particularly limited, and examples thereof include a tube-like structure, a fibrous structure, a fiber, a bead, a gel, a substantially spherical gel, a microcapsule, and the like, and a fiber, a bead or a substantially spherical gel. Is preferred.
- cross-linked alginate structures have improved stability.
- the crosslinked alginic acid structure may have an ability to retain the content (content retention).
- the stability of the crosslinked alginic acid structure can be confirmed, for example, by measuring the gel stability or measuring the gel leakage rate.
- Gel stability can be determined as follows. Phosphate buffered saline (PBS) is added to the crosslinked alginate structure gel placed in the container, and the concentration ( ⁇ g / mL) of alginic acid eluted in PBS is measured. The value obtained by dividing the measured alginic acid concentration by the total alginic acid concentration obtained by decomposing the crosslinked alginic acid structure gel is shown as a disintegration rate. Specifically, the gel stability can be determined by the method described in Examples described later. The gel disintegration rate of the crosslinked alginic acid structure is preferably 0% to 90%, more preferably 0% to 70%, and further preferably 0% to 50%. The stability of the crosslinked alginic acid structure increases as the concentration of alginic acid eluted in the aqueous solution decreases, that is, the gel disintegration rate decreases.
- PBS Phosphate buffered saline
- the gel leakage rate can be determined as follows. A fluorescein isothiocyanate-dextran-encapsulated crosslinked alginate structure gel is prepared, phosphate buffered saline (PBS) is added to the gel placed in a container, and the concentration of dextran leaked into the PBS is measured. The value obtained by dividing the measured dextran concentration by the total dextran concentration obtained by decomposing the fluorescein isothiocyanate-dextran-encapsulated cross-linked alginate structure gel is a gel leakage rate. Specifically, the gel leakage rate can be determined by the method described in Examples described later.
- PBS phosphate buffered saline
- the gel leakage rate of the crosslinked alginic acid derivative 48 hours after addition of PBS is preferably 0% to 90%, more preferably 0% to 70%, and further preferably 0% to 50%.
- the stability of the crosslinked alginic acid derivative increases as the gel leakage rate decreases.
- the fluorescein isothiocyanate-dextran-encapsulated crosslinked alginate structure gel was prepared as follows. A solution of an alginic acid derivative having a cross-linking group and a fluorescein isothiocyanate-dextran solution are mixed, this mixed solution is dropped into a solution containing calcium ions, and the obtained gel is allowed to stand at 37 ° C. for 10 minutes in the solution. By causing a crosslinking reaction, a fluorescein isothiocyanate-dextran-encapsulated crosslinked alginate structure gel can be obtained.
- Alginic acid derivatives can be obtained by a condensation reaction of a linker terminal amino group into which a crosslinking group (reactive group) has been introduced and a carboxyl group of alginic acids.
- the alginic acid derivative represented by the formula (AL-1-I) or the formula (AL-1-II) is an amine derivative represented by the formula (AM-I) (wherein —A 1 — Is the same as defined in some embodiments described above) or an amine derivative represented by formula (AM-II) (wherein P 1 and -A 2 -are Can be produced by a condensation reaction using an arbitrary carboxyl group of alginic acids with a condensing agent.
- Carbodiimide (DCC), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (WSC ⁇ HCl), benzotriazol-1-yloxytris (dimethylamino) phosphonium hexafluorophosphate (BOP reagent), bis (2-Oxo-3-oxazolidinyl) phosphinic chloride (BOP-Cl 2-chloro-1,3-dimethylimidazolinium hexafluorophosphate (CIP), or 4- (4,6-dimethoxy-1,3,5-triazin-2-yl) -4-methylmorpholinium
- a condensing agent selected from chloride (DMT-MM), etc.
- an alcoholic solvent such as tetrahydrofuran, 1,4-dioxane, etc., methanol, ethanol, 2-propanol, etc., to the extent that alginic acid does not precipitate
- an inorganic base such as sodium hydrogen carbonate or sodium carbonate
- an organic base such as triethylamine or pyridine
- a mixed solvent of water and a solvent selected from solvents, polar solvents such as N, N-dimethylformamide, and the like a solvent selected from solvents, polar solvents such as N, N-dimethylformamide, and the like.
- An alginic acid derivative of the formula (AL-1-I) by conducting a condensation reaction in the presence at a temperature between 0 ° C. and 50 ° C. Can be manufactured.
- the introduction rate of the amino derivative of the formula (AM-I) or the formula (AM-II) is:
- the adjustment can be made by appropriately selecting and combining reaction conditions such as the following (i) to (v).
- P 1 of the formula (AL-1-II) is a thiol protector (acetyl group, benzoyl group, etc.)
- 0.5 wt% to 1 wt% of an aqueous solution of the thiol protected alginic acid derivative is introduced.
- the protected thiol group was hydrolyzed at 0 ° C. to 30 ° C. using an excess amount of an inorganic base such as sodium hydroxide or potassium hydroxide to obtain a thiol group-introduced alginic acid derivative (formula (AL-1-II )
- Can produce P 1 is a hydrogen atom, neutralize excess base, and use it in the crosslinking reaction in solution.
- reaction of maleic anhydride with an amine represented by the formula (III) in a solvent such as an alcohol solvent such as methanol or ethanol in the presence or absence of a base such as triethylamine can also be performed by the reaction of the formula (III).
- a solvent such as an alcohol solvent such as methanol or ethanol
- a base such as triethylamine
- reaction Formula A (Reaction Formula A) ⁇ Step 2>
- a solvent that does not affect the reaction such as an ether solvent such as 1,4-dioxane, a hydrocarbon solvent such as toluene, and a halogen solvent such as 1,2-dichloroethane
- the compound of the formula (VI) can be produced by using a base such as sodium acetate in an acetic anhydride solvent and heating from 40 ° C. to a temperature at which the solvent is refluxed (eg, 100 ° C.).
- the compound of formula (VI) can also be produced by carrying out a cyclization treatment using a base such as sodium acetate in acetic anhydride without isolating the monoamide after the operation of 1>.
- the compound of the formula (VI) can also be produced by using an appropriate condensing agent and leading to an active ester.
- P 2 in the process for producing the amine derivative of the formula (AM-I) is —C (O) O-tertBu group, —C (O) O—Bn group, —C (O) CH 3 group, —C ( O) A protecting group for an amino group selected from CF 3 group, —SO 2 Ph, —SO 2 PhMe group, —SO 2 Ph (NO 2 ) group, and the like.
- P 1 is tert-butoxycarbonyl (—C (O) O-tertBu group)
- deprotection can be achieved by using an acid such as hydrogen chloride or trifluoroacetic acid.
- 1,4-dioxane containing hydrogen chloride, cyclopentyl methyl ether, ethyl acetate or the like can be used.
- trifluoroacetic acid a solvent such as methylene chloride and toluene which are solventless or inert to acids can be used.
- the amine of the formula (AM-I) can be obtained as a salt such as hydrochloride, trifluoroacetate, etc., if necessary.
- Inactive solvents such as ether solvents such as 1,4-dioxane and halogen solvents such as methylene chloride in the presence or absence of organic bases such as triethylamine and pyridine, and inorganic bases such as potassium bicarbonate. React inside, or By reaction with a carboxylic acid derivative suitable condensing agent or acid catalyst, it is possible to produce a thiol-protecting of formula (IX).
- N-protected form represented by the formula (IX) can be prepared by methods known in the literature, such as (Experimental Chemistry Course 5th Edition 16, Synthesis of Organic Compounds IV, Amino Acids / Peptides, 258-283, 2007, Maruzen), etc. In accordance with the method described in the literature, and methods known in the literature, such as “Protective Groups in Organic Synthesis 4th Edition”, 4th edition, 2007, John Producing the compound of formula (AM-II) by deprotection according to the deprotection method described in the book of John Wiley & Sons, Green et al. Can do.
- P 2 in the process for producing the amine derivative of the formula (AM-II) is —C (O) O-tertBu group, —C (O) O—Bn group, —C (O) CH 3 group, —C ( O) A protecting group for an amino group selected from CF 3 group, —SO 2 Ph, —SO 2 PhMe group, —SO 2 Ph (NO 2 ) group, and the like.
- P 1 is tert-butoxycarbonyl (—C (O) O-tertBu group)
- deprotection can be achieved by using an acid such as hydrogen chloride or trifluoroacetic acid.
- 1,4-dioxane containing hydrogen chloride, cyclopentyl methyl ether, ethyl acetate or the like can be used.
- a solvent such as methylene chloride and toluene which are solventless or inert to acids can be used.
- the amine of the formula (AM-II) can be obtained as a salt of hydrochloride, trifluoroacetate, etc., if necessary.
- a crosslinked alginic acid structure can be obtained by a method including subjecting the alginic acid derivative having a crosslinking group to the above-described crosslinking reaction. Specifically, for example, it can be prepared by the following method, but is not limited thereto.
- a solution containing the alginate derivative of formula (AL-1-I) described above is partially cross-linked, for example, by dropping it into a solution containing a divalent metal ion to obtain a specific structure.
- a solution containing the alginic acid derivative of the above formula (AL-1-II) By adding the structure such as a gel obtained above to a solution containing the alginic acid derivative of the above formula (AL-1-II), the surface of the structure is subjected to a further crosslinking reaction. A crosslinked alginic acid structure can be obtained.
- the alginic acid derivative of the formula (AL-1-I) is converted into the alginic acid derivative of the formula (AL-1-II), and the alginic acid derivative of the formula (AL-1-II) is converted into the formula (AL-1-I). It is also possible to carry out by substituting the alginic acid derivative of
- a solution containing the alginic acid derivative of formula (AL-1-I) is mixed with a solution containing the alginic acid derivative of formula (AL-1-II).
- a crosslinked alginate structure which is a specific structure, can be obtained by partially crosslinking, for example, by dropping into a solution containing a valent metal ion.
- the divalent metal ion used in the above method include calcium ion, magnesium ion, barium ion, strontium ion, zinc ion and the like, and calcium ion is preferable.
- the calcium ion concentration of the solution containing calcium ions is not particularly limited, and examples thereof include 1 mM to 1 M, preferably 5 mM to 500 mM, more preferably 10 mM to 300 mM.
- the solvent or solution used for the crosslinking reaction is not particularly limited, and examples thereof include ultrapure water, cell culture medium, phosphate buffered saline (PBS), and physiological saline. preferable.
- the specific structure include a tubular structure, a fibrous structure, a fiber, a bead, a gel, a substantially spherical gel, and a microcapsule.
- Alginic Acid derivatives can be used in place of conventional alginic acid in a wide range of fields such as food, medicine, cosmetics, textiles and papermaking.
- the preferred use of the alginic acid derivative or the crosslinked alginic acid structure is specifically for medical use such as a wound dressing, a postoperative adhesion-preventing material, a drug sustained-release substrate, a cell culture substrate, and a cell transplant substrate. Materials.
- Examples of the shape of the crosslinked alginate structure when used as a medical material include a tube shape, a fiber shape, a fiber, a bead, a gel, a substantially spherical gel, and the like, preferably a bead, a gel, or a substantially spherical gel. It is more preferable to use a substantially spherical gel.
- JEOL JNM-ECX400 FT-NMR was used for nuclear magnetic resonance spectrum (NMR) measurement.
- Root temperature in the examples generally indicates a temperature of about 0 ° C. to about 35 ° C.
- the “introduction rate” in the examples is determined by measuring 1H-NMR in D 2 O and calculating from the ratio of the proton integral value of the maleimide group or aromatic ring of the reactive substituent and alginic acid to “mol% (NMR integration ratio)”. It was described in.
- Example 1 To the compound (500 mg) obtained in ⁇ Step 1> was added 4N-ethyl hydrogen chloride solution (5.0 mL), and the mixture was stirred at room temperature for 1.5 hours. After adding ethyl acetate (5.0 mL), the precipitate was collected by filtration and washed with ethyl acetate. The obtained hygroscopic solid was suspended in ethyl acetate, and ethyl acetate was distilled off under reduced pressure, followed by drying under reduced pressure to obtain the title compound (328 mg) as a white solid.
- the introduction rate of the reactive group is 5.3 mol% (NMR integration ratio).
- the molecular weight was broadly eluted from 2.61 million Da to 19,000 Da, and the weight average molecular weight was calculated to be 1.46 million Da.
- Example 2 Trifluoroacetic acid (2.3 mL) was added to the compound (0.5 g) obtained in ⁇ Step 1> under ice-water cooling, and the mixture was stirred at room temperature for 1 hour. Diisopropyl ether (11.3 mL) was added and stirred at room temperature for 30 minutes, and then the precipitated solid was collected by filtration and washed with diisopropyl ether. The obtained hygroscopic solid was suspended in diisopropyl ether, the solvent was distilled off, and the residue was dried under reduced pressure to obtain the title compound (0.3 g) as a pale yellow solid.
- the introduction ratio of the reactive group is 4.4 mol% (NMR integration ratio).
- the molecular weight was broadly eluted from 2.73 million Da to 11,000 Da, and the weight average molecular weight was calculated to be 1.44 million Da.
- Example 3 The compound obtained in ⁇ Step 1> (500 mg) and maleic anhydride (217 mg) were suspended in ethanol (5.0 mL) and stirred at room temperature for 30 minutes. Ethanol was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (heptane-ethyl acetate) to obtain an amide compound (423 mg). Acetic anhydride (4.2 mL) was added to the obtained colorless oil and sodium acetate (100 mg), stirred at 40 ° C for 1 hour, then at 60 ° C for 1 hour, at 80 ° C for 1.5 hours, at 100 ° C. Stir for 2 hours.
- Example 3 Trifluoroacetic acid (1.9 mL) was added to the compound (275 mg) obtained in ⁇ Step 2> under ice-water cooling, and the mixture was stirred at room temperature for 15 minutes. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate-30% methanol / ethyl acetate) to give the title compound (231 mg) as a colorless oil.
- the introduction ratio of the reactive group is 3.7 mol% (NMR integration ratio).
- the molecular weight was broadly eluted from 2.72 million Da to 11,000 Da, and the weight average molecular weight was calculated to be 1.44 million Da.
- Example 4 The compound (1.20 g) obtained in ⁇ Step 1> was suspended in acetonitrile (24.0 mL). Potassium thioacetate (0.53 g) was added, and the mixture was stirred at room temperature for 30 minutes. Ethyl acetate (50 mL) and water (20 mL) were added to the reaction solution to separate it. The organic layer was washed successively with water (20 mL) and saturated brine (10 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was triturated with tert-butyl methyl ether, and the solid was collected by filtration and washed with tert-butyl methyl ether. The obtained solid was dried under reduced pressure at 40 ° C. to obtain the title compound (1.27 g) as a white solid.
- Example 4 4N-hydrogen chloride / 1,4-dioxane (4.2 mL) was added to the compound (0.60 g) obtained in ⁇ Step 2> under ice-water cooling, and 30 at room temperature. Stir for minutes. 4N-hydrogen chloride / 1,4-dioxane (2.1 mL) was added, and the mixture was further stirred at room temperature for 30 minutes. Diisopropyl ether (12.6 mL) was added to the reaction solution, and the resulting precipitate was collected by filtration, washed with diisopropyl ether and dried under reduced pressure to obtain the title compound (0.46 g) as a white solid.
- the introduction ratio of the reactive group is 5.6 mol% (NMR integration ratio).
- the molecular weight was broadly eluted from 2.77 million Da to 14,000 Da, and the weight average molecular weight was calculated to be 1.42 million Da.
- Example 5 The compound (560 mg) obtained in ⁇ Step 1> was dissolved in tetrahydrofuran (11.2 mL). Lithium aluminum hydride (146 mg) was added over 5 minutes, and the mixture was stirred at room temperature for 1 hour. A saturated aqueous sodium sulfate solution (50 drops) was added under ice water cooling, followed by stirring at the same temperature for 1 hour. The precipitated insoluble material was removed by filtration and washed with tetrahydrofuran. The filtrate was concentrated under reduced pressure to obtain the title compound (569 mg) as a colorless oil.
- Example 5 The compound (400 mg) obtained in ⁇ Step 2> was dissolved in tetrahydrofuran (8.0 mL). p-Toluenesulfonyl chloride (272 mg), N, N-dimethyl-4-aminopyridine (15 mg) and triethylamine (0.33 mL) were added, and the mixture was stirred at 70 ° C. for 6 hours. Ethyl acetate (25 mL) and water (10 mL) were added to the reaction solution, liquid separation was performed, and the aqueous layer was extracted with ethyl acetate (5 mL).
- Example 5 The compound (224 mg) obtained in ⁇ Step 3> was suspended in acetonitrile (4.5 mL). Potassium thioacetate (87 mg) was added and stirred at room temperature for 30 minutes. Ethyl acetate (20 mL) and water (10 mL) were added to the reaction solution to separate the layers. The organic layer was washed successively with water (10 mL) and saturated brine (5 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (10% ethyl acetate / heptane to ethyl acetate) to obtain the title compound (189 mg) as a white solid.
- Example 5 Using the compound (189 mg) obtained in ⁇ Step 4>, the same operation as in (Example 4) ⁇ Step 3> was performed to obtain the title compound (140 mg) as a white solid.
- the introduction ratio of the reactive group is 4.2 mol% (NMR integration ratio).
- the molecular weight was broadly eluted from 2.61 million Da to 21,000 Da, and the weight average molecular weight was calculated to be 1.42 million Da.
- Example 4 The compound (160 mg) obtained in ⁇ Step 4> is dissolved in water (8.0 mL), 1N-aqueous sodium hydroxide solution (112 ⁇ L) is added, and the mixture is heated at 25 ° C. for 2 hours. Stir to prepare a 2 wt% solution of the title compound. When ethanol was precipitated, it became a gel, and the solution was used for the test. The ethanol was partially treated, and disappearance of the acetyl group was confirmed by NMR.
- Example 8 To a mixture of the compound obtained in ⁇ Step 1> (0.074 g) and dichloromethane (0.22 mL), trifluoroacetic acid (0.52 mL) was added under ice-cooling and stirring. Stir at room temperature for 2 hours. After completion of the reaction, the reaction solution was concentrated and diisopropyl ether (20 mL) was added. Since a gummy compound was formed, the mixture was concentrated under reduced pressure and dried to obtain the title crude compound (0.097 g) as a pale yellow gummy compound.
- the introduction rate of the crosslinking group was 4.8 mol% (NMR integration ratio).
- the molecular weight was broadly eluted from 2.73 million Da to 2,000 Da, and the weight average molecular weight was calculated to be 1.36 million Da.
- reaction mixture was diluted with ethyl acetate (20 mL), and the suspension was filtered.
- the assembled product was purified by silica gel column chromatography (12% ethyl acetate / heptane to 100% ethyl acetate) to obtain the title compound (108 mg) as a white amorphous.
- Example 9 To a mixture of the compound (0.1 g) obtained in ⁇ Step 1> and dichloromethane (1.3 mL), trifluoroacetic acid (0.7 mL) was added under ice-cooling and stirring. And stirred at room temperature for 30 minutes. After completion of the reaction, the reaction solution was concentrated under reduced pressure, and diisopropyl ether (20 mL) was added. The suspension was filtered to obtain the title compound (0.12 g) as a white solid.
- Example 8 Using a sodium alginate (ALG-2 manufactured by Kimika Co., Ltd.) aqueous solution (29.7 mL) adjusted to 1% by weight and the compound (27.5 mg) obtained in (Example 9) ⁇ Step 2>, ( Example 8) The same operation as in ⁇ Step 3> was performed to obtain the title compound (264.8 mg) as a white cotton-like compound.
- AAG-2 sodium alginate manufactured by Kimika Co., Ltd.
- the introduction rate of the crosslinking group was 6.0 mol% (NMR integration ratio).
- the molecular weight was broadly eluted from 2.77 million Da to 6,000 Da, and the weight average molecular weight was calculated to be 1.46 million Da.
- the crude product was purified by silica gel column chromatography (25% ethyl acetate / heptane to 100% ethyl acetate, ethyl acetate to 20% methanol / ethyl acetate) to obtain the title compound (0.21 g) as a white amorphous. .
- the crude product was purified by silica gel column chromatography (25% ethyl acetate / heptane to 100% ethyl acetate, ethyl acetate to 20% methanol / ethyl acetate).
- the collected fraction was concentrated under reduced pressure and dissolved in tert-butyl methyl ether (20 mL). This solution was washed successively with saturated aqueous sodium hydrogen carbonate (5 mL), water (5 mL) twice and saturated brine (5 mL), and dried over anhydrous sodium sulfate.
- the organic layer was concentrated under reduced pressure to obtain the title compound (220 mg) as a white amorphous.
- Example 10 Using the compound (0.22 g) obtained in ⁇ Step 2>, the same operation as in (Example 9) ⁇ Step 2> was performed to give the title compound (0.25 g) as a white solid. Got as.
- Example 8 Using a sodium alginate (ALG-2 manufactured by Kimika Co., Ltd.) aqueous solution (49.4 mL) adjusted to 1% by weight and the compound (52.4 mg) obtained in (Example 10) ⁇ Step 3>, ( Example 8) The same operation as in ⁇ Step 3> was performed to obtain the title compound (485 mg) as a white cotton-like compound.
- AAG-2 sodium alginate manufactured by Kimika Co., Ltd.
- the introduction rate of the crosslinking group was 5.3 mol% (NMR integration ratio).
- the molecular weight was broadly eluted from 2.87 million Da to 20,000 Da, and the weight average molecular weight was calculated to be 1.43 million Da.
- the introduction rate of the reactive group means a value expressed as a percentage of the number of reactive groups introduced per uronic acid monosaccharide unit which is a repeating unit of alginic acid.
- the amount of alginic acid required for calculating the introduction rate was measured by the carbazole sulfate method using a calibration curve, and the amount of reactive group was measured by the absorbance measurement method using the calibration curve.
- the molecular weight of the cross-linked group-introduced alginic acid is blue dextran (molecular weight 2 million Da, SIGMA), thyroglobulin (molecular weight 669,000 Da, GE Healthcare Science), ferritin (molecular weight 440,000 Da, GE Healthcare Science) , Aldolase (molecular weight 158,000 Da, GE Healthcare Science), conalbumin (molecular weight 75,000 Da, GE Healthcare Science), ovalbumin (molecular weight 44,000 Da, GE Healthcare Science)
- ribonuclease A molecular weight: 1.37,000 Da, GE Healthcare Science
- aprotinin molecular weight: 6500 Da, GE Healthcare Science
- the molecular weight of alginic acid before introduction of the crosslinking group was determined as follows. That is, each alginic acid was weighed in consideration of loss on drying, and ultrapure water was added to prepare a 1% aqueous solution. Next, the solution was diluted to a 10 mmol / L phosphate buffer (pH 7.4) containing NaCl having an alginic acid concentration of 0.2% and a solution composition of 0.15 mol / L.
- Insoluble matter was removed by a hydrophilic PVDF Mylex GV33 filter (MERCK-Millipore) having a pore size of 0.22 ⁇ m, and then 200 ⁇ L was subjected to gel filtration, and gel filtration was carried out under the same conditions as for the crosslinkable group-introduced alginic acid. Detection was performed with a differential refractometer. Alternatively, insoluble matters were removed by passing through a polyethersulphone Miniisart High Flow filter (SARTORUS) having a pore diameter of 0.45 ⁇ m.
- SARTORUS polyethersulphone Miniisart High Flow filter
- the weight average molecular weight of the alginic acid before the introduction of the crosslinking group was determined by the same method as the calculation method of the molecular weight of the crosslinking group-introduced alginic acid.
- Hi was calculated from differential refractometer data.
- the molecular weight of alginic acid (ALG-2) used in Examples 1 to 5 and Example 7-1 before introduction of the crosslinking group was eluted broadly from 2.6 million Da to 145,000 Da, and the weight average The molecular weight was calculated to be 1.46 million Da.
- alginic acid used in Examples 8 to 10 before the introduction of the crosslinking group was broadly eluted from 9600 Da to 25,110,000 Da, and the weight average molecular weight was calculated to be 1.38 million Da.
- aqueous alginate solution (2) and aqueous alginic acid solution (7-1) were mixed, and the mixed aqueous solution was placed in a syringe equipped with an 18-gauge syringe needle.
- the syringe was set at a flow rate of 1 mL / min.
- the alginate gel was obtained by installing in a pump, dripping for 30 seconds in a calcium chloride solution having a concentration of 30 mmol / L, and stirring for 5 minutes. This gel was washed once with 10 mL of PBS and then allowed to stand at 37 ° C. for 10 minutes in PBS for chemical crosslinking to obtain a chemically crosslinked alginate gel.
- the concentration of alginic acid in the recovered aqueous solution was measured by the carbazole sulfate method, and the values corrected for the alginic acid concentration in the aqueous solution at each time point with the alginic acid concentration already recovered were calculated from the alginic acid concentration at all time points and the alginic acid concentration after the end of the test.
- a value obtained by dividing the value by the alginic acid concentration as a percentage was taken as the disintegration rate, and was used as an index of gel stability.
- the gel stability of the alginate gel obtained by the above method using the alginate aqueous solution (3) and the alginate aqueous solution (7-1) was also measured.
- the results are shown in FIG.
- the alginic acid gel prepared from the alginic acid (ALG-2) used as a control almost dissolved in 8 hours, whereas the crosslinked alginic acid structure (alginic acid derivative) obtained by crosslinking the alginate derivative introduced with the crosslinking group of this example was used.
- (AL-EX-2) / alginic acid derivative (AL-EX-7-1) cross-linked alginic acid structure obtained by cross-linking, and alginic acid derivative (AL-EX-3) / alginic acid derivative (AL-EX- All of the crosslinked alginic acid structures obtained by crosslinking 7-1) have improved stability.
- PBS buffered saline
- Alginic acid aqueous solution (8), alginic acid aqueous solution (9), and alginic acid aqueous solution (10) each alginate aqueous solution and alginic acid aqueous solution (7-1-2) are mixed in an equal amount of 250 ⁇ L each, and the concentration is 30 mmol / L. 40 mL of calcium solution was added and stirred for 5 minutes to obtain an alginate gel. This gel was washed once with 10 mL of PBS to obtain a chemically crosslinked alginate gel.
- the concentration of alginic acid in the recovered aqueous solution was measured by the carbazole sulfate method, and the values corrected for the alginic acid concentration in the aqueous solution at each time point with the alginic acid concentration already recovered were calculated from the alginic acid concentration at all time points and the alginic acid concentration after the end of the test.
- a value obtained by dividing the value by the alginic acid concentration as a percentage was taken as the disintegration rate, and was used as an index of gel stability.
- the results are shown in FIG.
- the crosslinked alginic acid structure obtained by crosslinking the alginic acid derivative (AL-EX-9) / alginic acid derivative (AL-EX-7-1-2) had a decay rate of about 39% after 96 hours.
- the crosslinked alginic acid structure obtained by crosslinking (AL-EX-8) / alginic acid derivative (AL-EX-7-1-2) has a disintegration rate of about 40% after 96 hours, and the alginic acid derivative (AL- EX-10) / alginic acid derivative (AL-EX-7-1-2) cross-linked, the cross-linked alginate structure had a disintegration rate of about 55% after 96 hours, and the gel stability measurement (1 It was suggested that the stability was improved as compared with the alginic acid gel prepared from alginic acid (ALG-2).
- Example 2 Introduction rate (NMR integration ratio) produced by the same method as in ⁇ Step 3> 3.4 mol% cross-linked group-introduced alginic acid derivative (AL-EX-2-1), obtained in Example 8.
- the alginate derivative (AL-EX-8) obtained in Example 9, the alginate derivative obtained in Example 9 (AL-EX-9), and the alginate derivative obtained in Example 10 (AL-EX-10) It was dissolved in water to 0.5% to obtain an alginic acid aqueous solution (2-1), an alginic acid aqueous solution (8), an alginic acid aqueous solution (9), and an alginic acid aqueous solution (10).
- the 2% cross-linked group-introduced alginic acid derivative (AL-EX-7-1-2) (introduction rate (NMR integration ratio) 5.1 mol%) obtained by the same method as in Example 7-1 was added to phosphoric acid. 3 volumes of buffered saline (PBS) was added to 0.5% to obtain an aqueous alginate solution (7-1-2).
- PBS buffered saline
- Alginic acid aqueous solution (2-1), alginic acid aqueous solution (8), alginic acid aqueous solution (9), and alginic acid aqueous solution (10) were mixed in an equal amount of 250 ⁇ L each of alginic acid aqueous solution and alginic acid aqueous solution (7-1-2). 40 mL of a calcium chloride solution having a concentration of 30 mmol / L was added and stirred for 5 minutes to obtain an alginate gel.
- the concentration of alginic acid in the recovered aqueous solution was measured by the carbazole sulfate method, and the values corrected for the alginic acid concentration in the aqueous solution at each time point with the alginic acid concentration already recovered were calculated from the alginic acid concentration at all time points and the alginic acid concentration after the end of the test.
- a value obtained by dividing the value by the alginic acid concentration as a percentage was taken as the disintegration rate, and was used as an index of gel stability.
- the results are shown in FIG.
- the crosslinked alginic acid structure obtained by crosslinking the alginic acid derivative (AL-EX-2-1) / alginic acid derivative (AL-EX-7-1-2) has a decay rate of about 49% after 24 hours.
- the crosslinked alginic acid structure obtained by crosslinking the alginic acid derivative (AL-EX-9) / alginic acid derivative (AL-EX-7-1-2) has a decay rate of about 28% after 24 hours.
- the crosslinked alginic acid structure obtained by crosslinking (AL-EX-8) / alginic acid derivative (AL-EX-7-1-2) has a disintegration rate of about 32% after 24 hours
- the alginic acid derivative (AL- EX-10) / Alginic acid derivative (AL-EX-7-1-2) cross-linked alginic acid structure obtained by cross-linking has a disintegration rate of about 32% after 24 hours
- the alginic acid structure excluding calcium cross-linking Even on the body Shift was also able to confirm that there is stability.
- Example 2 Alginic acid derivative (AL-EX-2) obtained in ⁇ Step 3>, or (Example 3) Alginic acid derivative (AL-EX-3) obtained in ⁇ Step 4>, or raw material alginate Alginate (ALG-2; control) into which no reactive group is introduced is dissolved in water to a concentration of 1%, 1/100 volume of 1N sodium bicarbonate aqueous solution is added, and alginate aqueous solution is added.
- Alginic acid aqueous solution (3) and alginic acid aqueous solution (ALG-2-aq) were obtained.
- fluorescein having a molecular weight of 2 million was prepared to 1 mg / mL with 2% by weight alginate derivative (AL-EX-7-1) solution obtained in (Example 7-1) with phosphate buffered saline (PBS).
- Isothiocyanate-dextran Sigma Aldrich, FD2000S was added in an equal amount to 1% by weight to obtain an alginate aqueous solution (7-1).
- Alginic acid aqueous solution (2) and alginic acid aqueous solution (7-1), or alginic acid aqueous solution (3) and alginic acid aqueous solution (7-1) are mixed in equal amounts, and the mixed aqueous solution is a syringe equipped with an 18 gauge needle.
- the syringe was placed in a syringe pump set at a flow rate of 1 mL / min, dropped into a calcium chloride solution having a concentration of 30 mmol / L for 30 seconds, and stirred for about 20 minutes to obtain an alginate gel.
- This gel was washed once with 10 mL of PBS to obtain a fluorescein isothiocyanate-dextran-encapsulated chemically crosslinked alginate gel.
- 20 mL of PBS was added and shaken at 37 ° C., and the aqueous solution was collected over time.
- 5 ⁇ L of alginate lyase (Nippon Gene, 319-08261) was added to the test solution and shaken at 37 ° C. for 2 hours to completely disintegrate the gel, and an aqueous solution was recovered.
- the concentration of dextran in the collected aqueous solution was measured by a fluorometric method (excitation light: 485 nm, fluorescence: 535 nm), and the value obtained by dividing the dextran concentration at each time point by the dextran concentration after the end of the test was expressed as a percentage. And used as an index of gel stability.
- the results are shown in FIG.
- the gel prepared from alginic acid (ALG-2) used as a control showed a leakage rate of less than 40% in 24 hours and about 70% in 48 hours, whereas the cross-linked group-introduced alginic acid derivative of this example was crosslinked.
- a crosslinked alginic acid structure obtained by crosslinking the alginic acid derivative (AL-EX-2) / alginic acid derivative (AL-EX-7-1), and an alginic acid derivative (AL- EX-3) / alginic acid derivative (cross-linked alginic acid structure obtained by cross-linking alginic acid derivative (AL-EX-7-1)) has a leakage rate of about 10% for 24 hours and a leakage rate of 10 to 15% for 48 hours. The stability was improved in both cases.
- the alginate derivative (AL-EX-8) obtained in Example 9, the alginate derivative obtained in Example 9 (AL-EX-9), and the alginate derivative obtained in Example 10 (AL-EX-10) Aqueous alginic acid aqueous solution was prepared by dissolving in water to 2.0%, and fluorescein isothiocyanate-dextran (Sigma Aldrich, FD150S) having a molecular weight of 150,000 prepared to 1 mg / mL of 4/5 volume in this aqueous alginate solution, And 2.2 volumes of water, and 0.2 mg / mL fluorescein isothiocyanate-dextran-containing 0.5% aqueous alginate solution (2-1), aqueous alginate solution Liquid (8), aqueous alginic acid (9), and was obtained alginate solution (10).
- the 2% cross-linked group-introduced alginic acid derivative (AL-EX-7-1-2) (introduction rate (NMR integration ratio) 5.1 mol%) obtained by the same method as in Example 7-1 was added to phosphoric acid. 3 volumes of buffered saline (PBS) was added to 0.5% to obtain an aqueous alginate solution (7-1-2).
- Alginic acid aqueous solution (2-1), alginic acid aqueous solution (8), alginic acid aqueous solution (9), and alginic acid aqueous solution (10) were mixed in an equal amount of 250 ⁇ L each of alginic acid aqueous solution and alginic acid aqueous solution (7-1-1-2).
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Abstract
Description
すなわち、本発明は、以下の態様[1-1]~[22b]の通りである。
-A1-は、下記式(式中、両破線外側は除く):
R1は、それぞれ独立して、水素原子、メチル基、イソプロピル基、イソブチル基、sec-ブチル基、ヒドロキシメチル基、2-ヒドロキシエチル基、チオールメチル基、メチルチオエチル基、カルボキシメチル基、カルボキシエチル基、アミノカルボニルメチル基、アミノカルボニルエチル基、アミノブチル基、グアニジノプロピル基、ベンジル基、4-ヒドロキシベンジル基、3-インドリルメチル基、4-イミダゾイルメチル基、およびR1が結合する炭素原子と当該炭素原子が結合する窒素原子と共に環を形成するプロパン-1,3-ジイル基からなる群より選択される基であり; R2は、それぞれ独立して、水素原子、メチル基、イソプロピル基、イソブチル基、sec-ブチル基、ヒドロキシメチル基、2-ヒドロキシエチル基、チオールメチル基、メチルチオエチル基、カルボキシメチル基、カルボキシエチル基、アミノカルボニルメチル基、アミノカルボニルエチル基、アミノブチル基、グアニジノプロピル基、ベンジル基、4-ヒドロキシベンジル基、3-インドリルメチル基、4-イミダゾイルメチル基、およびR2が結合する炭素原子と当該炭素原子が結合する窒素原子と共に環を形成するプロパン-1,3-ジイル基からなる群より選択される基であり;
nは、1~18の整数であり;
mは、1~9の整数であり;
jは、0~9の整数である)
で表される基を有する、アルギン酸誘導体。
[1-2]式(I)において、-A1-が、下記式(式中、両破線外側は除く):
nが、1~18の整数であり;
mが、1~9の整数である、前記[1-1]に記載のアルギン酸誘導体。
[2]式(I)において、-A1-が、下記式(式中、両破線外側は除く):
[3]式(I)で表される基が、下記式(式中、破線右側は除く):
[4]架橋基の導入率が、1%~30%である、前記[1-1]~[3]のいずれか1項に記載のアルギン酸誘導体。
[5]アルギン酸誘導体のゲルろ過クロマトグラフィー法により測定した重量平均分子量が、10万Da~300万Daである、前記[1-1]~[4]のいずれか1項に記載のアルギン酸誘導体。
R1は、それぞれ独立して、水素原子、メチル基、イソプロピル基、イソブチル基、sec-ブチル基、ヒドロキシメチル基、2-ヒドロキシエチル基、チオールメチル基、メチルチオエチル基、カルボキシメチル基、カルボキシエチル基、アミノカルボニルメチル基、アミノカルボニルエチル基、アミノブチル基、グアニジノプロピル基、ベンジル基、4-ヒドロキシベンジル基、3-インドリルメチル基、4-イミダゾイルメチル基およびR1が結合する炭素原子と当該炭素原子が結合する窒素原子と共に環を形成するプロパン-1,3-ジイル基からなる群より選択される基であり;
R2は、それぞれ独立して、水素原子、メチル基、イソプロピル基、イソブチル基、sec-ブチル基、ヒドロキシメチル基、2-ヒドロキシエチル基、チオールメチル基、メチルチオエチル基、カルボキシメチル基、カルボキシエチル基、アミノカルボニルメチル基、アミノカルボニルエチル基、アミノブチル基、グアニジノプロピル基、ベンジル基、4-ヒドロキシベンジル基、3-インドリルメチル基、4-イミダゾイルメチル基およびR2が結合する炭素原子と当該炭素原子が結合する窒素原子と共に環を形成するプロパン-1,3-ジイル基からなる群より選択される基であり;
nは、1~18の整数であり;
mは、1~9の整数であり;
jは、0~9の整数である)
で表される基を有する、アルギン酸誘導体。
[1a-2]式(I)において、-A1-は、下記式(式中、両破線外側は除く):
nが、1~18の整数であり;
mが、1~9の整数であり;
jが、0~9の整数である)で表される全記[1a-1]に記載のアルギン酸誘導体。
[2a]式(I)において、-A1-が、下記式(式中、両破線外側は除く):
[2a-1]前記態様[2a]において、好ましくは、-A1-が、下記式(式中、両破線外側は除く):
[3a]式(I)で表される基が、下記式(式中、破線右側は除く):
[3a-1]前記態様[3a]において、好ましくは、式(I)で表される基が、下記式(式中、破線右側は除く):
[4a]式(I)で表される基の導入率が、1%~30%である、前記[1a-1]~[3a-1]のいずれか1項に記載のアルギン酸誘導体。
[5a]アルギン酸誘導体のゲルろ過クロマトグラフィー法により測定した重量平均分子量が、10万Da~300万Daである、前記[1a-1]~[4a]のいずれか1項に記載のアルギン酸誘導体。
R1は、それぞれ独立して、水素原子、メチル基、イソプロピル基、イソブチル基、sec-ブチル基、ヒドロキシメチル基、2-ヒドロキシエチル基、チオールメチル基、メチルチオエチル基、カルボキシメチル基、カルボキシエチル基、アミノカルボニルメチル基、アミノカルボニルエチル基、アミノブチル基、グアニジノプロピル基、ベンジル基、4-ヒドロキシベンジル基、3-インドリルメチル基、4-イミダゾイルメチル基およびR1が結合する炭素原子と当該炭素原子が結合する窒素原子と共に環を形成するプロパン-1,3-ジイル基からなる群より選択される基であり;
R2は、それぞれ独立して、水素原子、メチル基、イソプロピル基、イソブチル基、sec-ブチル基、ヒドロキシメチル基、2-ヒドロキシエチル基、チオールメチル基、メチルチオエチル基、カルボキシメチル基、カルボキシエチル基、アミノカルボニルメチル基、アミノカルボニルエチル基、アミノブチル基、グアニジノプロピル基、ベンジル基、4-ヒドロキシベンジル基、3-インドリルメチル基、4-イミダゾイルメチル基およびR2が結合する炭素原子と当該炭素原子が結合する窒素原子と共に環を形成するプロパン-1,3-ジイル基からなる群より選択される基であり;
nは、1~18の整数であり;
mは、1~9の整数であり;
jは、0~9の整数である)
で表される基を有する、アルギン酸誘導体(但し、-A1-=-CH2CH2-は除く)。
[1b-2]式(I)において、-A1-は、下記式(式中、両破線外側は除く):
nが、1~18の整数であり;
mが、1~9の整数であり;
jが、0~9の整数である)で表される全記[1b-1]に記載のアルギン酸誘導体(但し、-A1-=-CH2CH2-は除く)。
[2b]式(I)において、-A1-が、下記式(式中、両破線外側は除く):
[2b-1]前記態様[2b]において、好ましくは、-A1-が、下記式(式中、両破線外側は除く):
[3b]式(I)で表される基が、下記式(式中、破線右側は除く):
[3b-1]前記態様[3b]において、好ましくは、式(I)で表される基が、下記式(式中、破線右側は除く):
[4b]式(I)で表される基の導入率が、1%~30%である、前記[1b-1]~[3b-1]のいずれか1項に記載のアルギン酸誘導体。
[5b]アルギン酸誘導体のゲルろ過クロマトグラフィー法により測定した重量平均分子量が、10万Da~300万Daである、前記[1b-1]~[4b]のいずれか1項に記載のアルギン酸誘導体。
P1は、水素原子またはチオール基(-SH基)の保護基であり、
-A2-は、下記式(式中、両波線外側は除く):
前記-A2-において、Arは、水溶性置換基で(例えば、1~2個)置換されていてよいフェニレン基であり;
n4は、0~10の整数であり;
m4は、0~10の整数であり;
pは、0~10の整数である)
で表される架橋基を有する、アルギン酸誘導体。
[7-1]式(II)において、P1が、水素原子、またはアセチル基である、前記[6]に記載のアルギン酸誘導体。
[8-1]-A2-が、下記式(各式中、両破線外側は除く):
Arが、p-フェニレン基である、前記[6]~[7-1]のいずれか1項に記載のアルギン酸誘導体。
[9-1]式(II)で表される基が、下記式(各式中、破線右側は除く):
[12a]前記[1a-1]~[5a]のいずれか1項に記載のアルギン酸誘導体および、前記[6]~[11]のいずれか1項に記載のアルギン酸誘導体を含有する、組成物。
[12b]前記[1b-1]~[5b]のいずれか1項に記載のアルギン酸誘導体および、前記[6]~[11]のいずれか1項に記載のアルギン酸誘導体を含有する、組成物。
[13a-1]前記[1a-1]~[5a]のいずれか1項に記載のアルギン酸誘導体および、前記[6]~[11]のいずれか1項に記載のアルギン酸誘導体に、架橋反応を施すことにより得られる架橋アルギン酸構造体。
[13b-1]前記[1b-1]~[5b]のいずれか1項に記載のアルギン酸誘導体および、前記[6]~[11]のいずれか1項に記載のアルギン酸誘導体に、架橋反応を施すことにより得られる架橋アルギン酸構造体。
[13a-2]架橋アルギン酸構造体が、前記[1a-1]~[5a]のいずれか1項に記載のアルギン酸誘導体の溶液を、カルシウムイオンを含む溶液中に滴下し、得られたゲルに、前記[6]~[11]のいずれか1項に記載のアルギン酸誘導体の溶液中において架橋反応を施すことにより得られる、前記[13a-1]に記載の架橋アルギン酸構造体。
[13b-2]架橋アルギン酸構造体が、前記[1b-1]~[5b]のいずれか1項に記載のアルギン酸誘導体の溶液を、カルシウムイオンを含む溶液中に滴下し、得られたゲルに、前記[6]~[11]のいずれか1項に記載のアルギン酸誘導体の溶液中において架橋反応を施すことにより得られる、前記[13b-1]に記載の架橋アルギン酸構造体。
[13a-3]架橋アルギン酸構造体が、前記[6]~[11]のいずれか1項に記載のアルギン酸誘導体の溶液を、カルシウムイオンを含む溶液中に滴下し、得られたゲルに、前記[1a-1]~[5a]のいずれか1項に記載のアルギン酸誘導体の溶液中において架橋反応を施すことにより得られる、前記[13a-1]に記載の架橋アルギン酸構造体。
[13b-3]架橋アルギン酸構造体が、前記[6]~[11]のいずれか1項に記載のアルギン酸誘導体の溶液を、カルシウムイオンを含む溶液中に滴下し、得られたゲルに、前記[1b-1]~[5b]のいずれか1項に記載のアルギン酸誘導体の溶液中において架橋反応を施すことにより得られる、前記[13b-1]に記載の架橋アルギン酸構造体。
[13a-4]架橋アルギン酸構造体が、前記[12a]に記載の組成物の溶液を、カルシウムイオンを含む溶液中に滴下することにより得られる、前記[13a-1]に記載の架橋アルギン酸構造体。
[13b-4]架橋アルギン酸構造体が、前記[12b]に記載の組成物の溶液を、カルシウムイオンを含む溶液中に滴下することにより得られる、前記[13b-1]に記載の架橋アルギン酸構造体。
[13a-5]架橋アルギン酸構造体が、ファイバー、ビーズ、略球形のゲルまたはマイクロカプセルである、前記[13a-1]~[13a-4]のいずれか1項に記載の架橋アルギン酸構造体。
[13b-5]架橋アルギン酸構造体が、ファイバー、ビーズ、略球形のゲルまたはマイクロカプセルである、前記[13b-1]~[13b-4]のいずれか1項に記載の架橋アルギン酸構造体。
[14a]前記[13a-1]~[13a-5]のいずれか1項に記載の架橋アルギン酸構造体を含む医療用材料。
[14b]前記[13b-1]~[13b-5]のいずれか1項に記載の架橋アルギン酸構造体を含む医療用材料。
[15a]架橋アルギン酸構造体が、ビーズまたは略球形のゲルである、前記[14a]に記載の医療用材料。
[15b]架橋アルギン酸構造体が、ビーズまたは略球形のゲルである、前記[14b]に記載の医療用材料。
[16a]前記[1a-1]~[5a]のいずれか1項に記載のアルギン酸誘導体の溶液を、カルシウムイオンを含む溶液中に滴下し、得られたゲルに、前記[6]~[11]のいずれか1項に記載のアルギン酸誘導体の溶液中において架橋反応を施すことを含む、架橋アルギン酸構造体の製造方法。
[16b]前記[1b-1]~[5b]のいずれか1項に記載のアルギン酸誘導体の溶液を、カルシウムイオンを含む溶液中に滴下し、得られたゲルに、前記[6]~[11]のいずれか1項に記載のアルギン酸誘導体の溶液中において架橋反応を施すことを含む、架橋アルギン酸構造体の製造方法。
[17a]前記[6]~[11]のいずれか1項に記載のアルギン酸誘導体の溶液を、カルシウムイオンを含む溶液中に滴下し、得られたゲルに、前記[1a-1]~[5a]のいずれか1項に記載のアルギン酸誘導体の溶液中において架橋反応を施すことを含む、架橋アルギン酸構造体の製造方法。
[17b]前記[6]~[11]のいずれか1項に記載のアルギン酸誘導体の溶液を、カルシウムイオンを含む溶液中に滴下し、得られたゲルに、前記[1b-1]~[5b]のいずれか1項に記載のアルギン酸誘導体の溶液中において架橋反応を施すことを含む、架橋アルギン酸構造体の製造方法。
[18a]前記[12a]に記載の組成物の溶液を、カルシウムイオンを含む溶液中に滴下することを含む、架橋アルギン酸構造体の製造方法。
[18b]前記[12b]に記載の組成物の溶液を、カルシウムイオンを含む溶液中に滴下することを含む、架橋アルギン酸構造体の製造方法。
[19a]前記[1a-1]~[5a]のいずれか1項に記載のアルギン酸誘導体を、部分的に2価金属イオンで架橋し、特定の構造体を得た後に、前記[6]~[11]のいずれか1項に記載のアルギン酸誘導体により架橋反応を施すことを含む、架橋アルギン酸構造体の製造方法。
[19b]前記[1b-1]~[5b]のいずれか1項に記載のアルギン酸誘導体を、部分的に2価金属イオンで架橋し、特定の構造体を得た後に、前記[6]~[11]のいずれか1項に記載のアルギン酸誘導体により架橋反応を施すことを含む、架橋アルギン酸構造体の製造方法。
[20a]前記[6]~[11]のいずれか1項に記載のアルギン酸誘導体を、部分的に2価金属イオンで架橋し、特定の構造体を得た後に、前記[1a-1]~[5a]のいずれか1項に記載のアルギン酸誘導体により架橋反応を施すことを含む、架橋アルギン酸構造体の製造方法。
[20b]前記[6]~[11]のいずれか1項に記載のアルギン酸誘導体を、部分的に2価金属イオンで架橋し、特定の構造体を得た後に、前記[1b-1]~[5b]のいずれか1項に記載のアルギン酸誘導体により架橋反応を施すことを含む、架橋アルギン酸構造体の製造方法。
[21a]前記[12a]に記載の組成物を、部分的に2価金属イオンで架橋することを含む、架橋アルギン酸構造体の製造方法。
[21b]前記[12b]に記載の組成物を、部分的に2価金属イオンで架橋することを含む、架橋アルギン酸構造体の製造方法。
[22a]前記[1a-1]~[5a]のいずれか1項に記載のアルギン酸誘導体、前記[6]~[11]のいずれか1項に記載のアルギン酸誘導体、および、2価金属イオンを用いることを含む、架橋反応を施すことにより得られる、内容物の保持性を有する架橋アルギン酸構造体。
[22b]前記[1b-1]~[5b]のいずれか1項に記載のアルギン酸誘導体、前記[6]~[11]のいずれか1項に記載のアルギン酸誘導体、および、2価金属イオンを用いることを含む、架橋反応を施すことにより得られる、内容物の保持性を有する架橋アルギン酸構造体。
1.アルギン酸誘導体
ここでは、アルギン酸誘導体が提供される。アルギン酸誘導体は、アルギン酸のカルボキシル基の一部が、リンカーを介して架橋基(「反応性基」ともいう)が置換されたものである。すなわち、例えば、アルギン酸の任意の1つ以上のカルボキシル基と、架橋基(Z)及びアミノ基を両末端に有するリンカー(-L-)がアミド結合をしたもの(下記式AL-1)が挙げられる(式中、Zは、架橋基であり;-L-は、リンカーである。-L-は、例えば、前記式(I)における-A1-、又は、前記式(II)における-A2-、等が挙げられる)。
アルギン酸、そのエステルおよびその塩からなる群から選択される少なくとも1種のアルギン酸類のカルボキシル基の一部に、下記式(I)(式中、破線右側は除く):
R1は、それぞれ独立して、水素原子、メチル基、イソプロピル基、イソブチル基、sec-ブチル基、ヒドロキシメチル基、2-ヒドロキシエチル基、チオールメチル基、メチルチオエチル基、カルボキシメチル基、カルボキシエチル基、アミノカルボニルメチル基、アミノカルボニルエチル基、アミノブチル基、グアニジノプロピル基、ベンジル基、4-ヒドロキシベンジル基、3-インドリルメチル基、4-イミダゾイルメチル基およびR1が結合する炭素原子と当該炭素原子が結合する窒素原子と共に環を形成するプロパン-1,3-ジイル基からなる群より選択される基であり;
R2は、それぞれ独立して、水素原子、メチル基、イソプロピル基、イソブチル基、sec-ブチル基、ヒドロキシメチル基、2-ヒドロキシエチル基、チオールメチル基、メチルチオエチル基、カルボキシメチル基、カルボキシエチル基、アミノカルボニルメチル基、アミノカルボニルエチル基、アミノブチル基、グアニジノプロピル基、ベンジル基、4-ヒドロキシベンジル基、3-インドリルメチル基、4-イミダゾイルメチル基およびR2が結合する炭素原子と当該炭素原子が結合する窒素原子と共に環を形成するプロパン-1,3-ジイル基からなる群より選択される基であり;
nは、1~18の整数であり;
mは、1~9の整数であり:
jは、0~9の整数である)
で表される架橋基(反応性基)を有する、アルギン酸誘導体。いくつかの態様のアルギン酸誘導体では、-A1-=-CH2CH2-である場合を除く。
アルギン酸、そのエステルおよびその塩からなる群から選択される少なくとも1種のアルギン酸類のカルボキシル基の一部に、下記式(II)(式中、破線右側は除く):
-A2-は、下記式(式中、両破線外側は除く):
前記-A2-において、Arは、水溶性置換基で(例えば、1~3個)置換されていてよいフェニレン基であり;
n4は、0~10の整数であり;
m4は、0~10の整数であり;
pは、0~10の整数である)
で表される基を有する、アルギン酸誘導体。
アルギン酸類の任意の1つ以上のカルボキシル基が、前記式(I)で表わされる架橋基とアミド結合を形成している下記式(AL-1-I):
アルギン酸類の任意の1つ以上のカルボキシル基が、前記式(II)で表わされる架橋基とアミド結合を形成している下記式(AL-1-II):
ここで、式(I)中、部分構造式(式中破線右側を除く):
また、-A1-を「スペーサー」または「リンカー」という場合がある。
より好ましくは、下記式(各式中、破線右側は除く):
(2)メチル基(アラニンの側鎖)
(3)イソプロピル基(バリンの側鎖)
(4)イソブチル基(ロイシンの側鎖)
(5)sec-ブチル基(イソロイシンの側鎖)
(6)ヒドロキシメチル基(セリンの側鎖)
(7)2-ヒドロキシエチル基(トレオニンの側鎖)
(8)チオールメチル基(システインの側鎖)
(9)メチルチオエチル基(メチオニンの側鎖)
(10)カルボキシメチル基(アスパラギン酸の側鎖)
(11)カルボキシエチル基(グルタミン酸の側鎖)
(12)アミノカルボニルメチル基(アスパラギンの側鎖)
(13)アミノカルボニルエチル基(グルタミンの側鎖)
(14)アミノブチル基(リシンの側鎖)
(15)グアニジノプロピル基(アルギニンの側鎖)
(16)ベンジル基(フェニルアラニンの側鎖)
(17)4-ヒドロキシベンジル基(チロシンの側鎖)
(18)3-インドリルメチル基(トリプトファンの側鎖)
(19)4-イミダゾイルメチル基(ヒスチジンの側鎖)
(20)R1が結合する炭素原子と当該炭素原子が結合する窒素原子と共に環を形成するプロパン-1,3-ジイル基(プロリンの側鎖)
分別再結晶法:ラセミ体に対して光学分割剤をイオン結合させ、結晶性のジアステレオマーを得た後、其の結晶性のジアステレオマーを分別再結晶法によって分離し、所望により光学分割剤の除去工程を経て、光学的に純粋な化合物を得る方法である。光学分割剤は、例えば、(+)-マンデル酸、(-)-マンデル酸、(+)-酒石酸、(-)-酒石酸、(+)-1-フェネチルアミン、(-)-1-フェネチルアミン、シンコニン、(-)-シンコニジン、及びブルシン等が挙げられる。
ジアステレオマー法:ラセミ体の混合物に光学分割剤を共有結合させ、ジアステレオマーの混合物を得、次に、通常の分離手段(例えば、分別再結晶、シリカゲルカラムクロマトグラフィー、及びHPLC等)により光学的に純粋なジアステレオマーへ分離し、その後、化学反応(加水分解反応等)による光学分割剤の除去工程を経て、光学的に純粋な光学異性体を得る反応である。
例えば、高速液体クロマトグラフィー(HPLC)の場合には、キラルカラム(例えば、ダイセル社製CHIRALシリーズ等)に光学異性体の混合物を添加し、溶出溶媒(水、種々の緩衝液(例えば、リン酸緩衝液)、及び有機溶媒(例えば、エタノール、メタノール、イソプロパノール、アセトニトリル、トリフルオロ酢酸、及びジエチルアミン等)等の単独溶媒、又は其れらの混合溶媒)を用いて展開することで、光学異性体の分離が可能である。又、例えば、ガスクロマトグラフィーの場合、キラルカラム(例えば、CP-Chirasil-DeX CB(ジーエルサイエンス社製)等)を使用して、光学異性体の分離が可能である。又、例えば、超臨界流体クロマトグラフィー(SFC)の場合には、キラルカラム(例えば、ダイセル社製CHIRALシリーズ等)に光学異性体の混合物を添加し、溶出溶媒に二酸化炭素及び適当な有機溶媒(例えば、メタノール、エタノール、イソプロパノール、トリフルオロ酢酸、及びジエチルアミン等)を使用して、光学異性体の分離が可能である。
P1は、好ましくは、水素原子、アセチル基またはベンゾイル基であり、より好ましくは、水素原子、またはアセチル基である。
本明細書中、水溶性置換基とは、例えば、水酸基(-OH)、カルボキシル基(-COOH)、アミノ基(-NH2)、チオール基(-SH)又はスルホ基(-SO2OH)等の置換基が挙げられ、好ましくは、水酸基およびアミノ基が挙げられる。
用いられるアルギン酸類は、天然由来でも合成物であってもよいが、天然由来であるものが好ましい。好ましく用いられるアルギン酸類は、レッソニア、マクロシスティス、ラミナリア、アスコフィラム、ダービリア、カジカ、アラメ、コンブなどの褐藻類から抽出される生体内吸収性の多糖類であって、D-マンヌロン酸(M)とL-グルロン酸(G)という2種類のウロン酸が直鎖状に重合したポリマーである。より具体的には、D-マンヌロン酸のホモポリマー画分(MM画分)、L-グルロン酸のホモポリマー画分(GG画分)、およびD-マンヌロン酸とL-グルロン酸がランダムに配列した画分(M/G画分)が任意に結合したブロック共重合体である。本明細書中、アルギン酸類と記載する場合、アルギン酸、アルギン酸エステル、及びそれらの塩(例えば、アルギン酸ナトリウム)からなる群から選択される少なくとも1種のアルギン酸を意味する。
本明細書中、アルギン酸、アルギン酸誘導体、架橋アルギン酸、及び架橋アルギン酸の分子量において、単位としてDa(ダルトン)を付記する場合がある。
ここでは、アルギン酸類の任意の1つ以上のカルボキシル基が、前記式(I)で表わされる架橋基とアミド結合を形成している下記式(AL-1-I)で表わされる少なくとも1種のアルギン酸誘導体、及びアルギン酸類の任意の1つ以上のカルボキシル基が、前記式(II)で表わされる架橋基とアミド結合を形成している下記式(AL-1-II)で表わされる少なくとも1種のアルギン酸誘導と体を含む組成物が提供される。尚、式(AL-1-I)のリンカー(-A1-)の各態様、及び、式(AL-1-II)のリンカー(-A2-)及びP1の各態様は、前記の通りである。
架橋アルギン酸構造体は、式(AL-1-I)のアルギン酸誘導体、式(AL-1-II)のアルギン酸誘導体、またはこれらの混合物(以下、単に「アルギン酸誘導体」と記載する)が架橋基を介して三次元の網目構造を形成したものである。架橋アルギン酸構造体は、架橋基を有するアルギン酸誘導体に、架橋反応を施すことにより得ることができる。架橋反応は、例えば、次に記載のもの、あるいはこれらを適宜組み合わせて実施できるが、これらに限定されない。
(b)式(AL-1-I)のアルギン酸誘導体または式(AL-1-II)のアルギン酸誘導体を含む組成物を、2価金属イオン(例えば、カルシウムイオン、バリウムイオン、等)を含む溶液中で反応させることによる架橋反応(イオン結合架橋反応)。
(c)式(AL-1-I)のアルギン酸誘導体および式(AL-1-II)のアルギン酸誘導体を含む組成物を、2価金属イオン(例えば、カルシウムイオン、バリウムイオン、等)を含む溶液中で反応させることによる架橋反応(共有結合架橋反応+イオン結合架橋反応)。
架橋アルギン酸構造体の形状は特に限定されないが、例えば、チューブ状構造体、繊維状構造体、ファイバー、ビーズ、ゲル、略球形のゲル、マイクロカプセル等が挙げられ、ファイバー、ビーズまたは略球形のゲルが好ましい。
アルギン酸誘導体は、架橋基(反応性基)が導入されたリンカー末端アミノ基とアルギン酸類のカルボキシル基を縮合反応させることにより得ることができる。
具体的には、式(AL-1-I)又は式(AL-1-II)で表わされるアルギン酸誘導体は、各々、式(AM-I)で表わされるアミン誘導体(式中、-A1-は、前述のいくつかの態様中の定義と同じである)、又は、式(AM-II)で表わされるアミン誘導体(式中、P1及び-A2-は、前述のいくつかの態様中の定義と同じである)を、アルギン酸類の任意のカルボキシル基とを、縮合剤を用いる縮合反応により製造することができる。
0.5重量%~1重量%のアルギン酸水溶液及び式(AM-I)で表わされるアミノ誘導体を用いて、文献公知の方法、例えば、『実験化学講座 第5版 16、有機化合物の合成IV、カルボン酸および誘導体、エステル類、p35-70、酸アミドおよび酸イミド、p118-154、アミノ酸・ペプチド、p258-283、2007年、丸善』等に記載された方法に準じて、1,3-ジシクロヘキシルカルボジイミド(DCC)、1-エチル-3-(3-ジメチルアミノプロピル)カルボジイミド塩酸塩(WSC・HCl)、ベンゾトリアゾール-1-イルオキシトリス(ジメチルアミノ)ホスホニウムヘキサフルオロホスフェイト(BOP試薬)、ビス(2-オキソ-3-オキサゾリジニル)ホスフィニッククロリド(BOP-Cl)、2-クロロ-1,3-ジメチルイミダゾリニウムヘキサフルオロホスフェイト(CIP)、又は4-(4,6-ジメトキシ-1,3,5-トリアジン-2-イル)-4-メチルモルホリニウムクロリド(DMT-MM)、等から選択される縮合剤の存在下、アルギン酸が析出しない程度の、テトラヒドロフラン、1、4-ジオキサン等のエーテル系溶媒、メタノール、エタノール、2-プロパノール、等のアルコール系溶媒、N,N-ジメチルホルムアミド等の極性溶媒等から選択される溶媒と水との混合溶媒中、炭酸水素ナトリウム、炭酸ナトリウム等の無機塩基、又はトリエチルアミン、ピリジン等の有機塩基の存在下又は非存在下にて、0℃から50℃間の温度で縮合反応を行うことにより、式(AL-1-I)のアルギン酸誘導体を製造することができる。
0.5重量%~1重量%のアルギン酸水溶液及び式(AM-II)で表わされるアミノ誘導体を用いて、前述の[式(AL-1-I)のアルギン酸誘導体の製法]に準じて反応をおこなうことにより、式(AL-1-II)のアルギン酸誘導体を製造することができる。
式(III)[式(III)の化合物は市販化合物又は市販化合物から文献公知の製造方法により製造できる化合物である。式中P2は、アミノ基の保護基であり、適宜選択できる]で表されるアミンを用い、文献公知の方法、たとえば(実験化学講座 第5版 16、有機化合物の合成IV、カルボン酸および誘導体、酸アミドおよび酸イミド、146-154頁、2007年、丸善)等に記載された方法に準じて、マレイン酸と、1,3-ジシクロヘキシルカルボジイミド(DCC)、1-エチル-3-(3’-ジメチルアミノプロピル)カルボジイミド塩酸塩(WSC・HCl)、ベンゾトリアゾール-1-イルオキシトリス(ジメチルアミノ)ホスホニウムヘキサフルオロホスフェイト(BOP試薬)、ビス(2-オキソ-3-オキサゾリジニル)ホスフィニッククロリド(BOP-Cl)、2-クロロ-1,3-ジメチルイミダゾリニウムヘキサフルオロホスフェイト(CIP)、4-(4,6-ジメトキシ-1,3,5-トリアジン-2-イル)-4-メチルモルホリニウムクロリド(DMT-MM)等の縮合剤の存在下、1,4-ジオキサン等のエーテル系溶媒、塩化メチレン等のハロゲン系溶媒、N,N-ジメチルホルムアミド等の極性溶媒等の溶媒中、炭酸水素ナトリウム、炭酸ナトリウム等の無機塩基やトリエチルアミン、ピリジン等の有機塩基の存在下または非存在下、0℃から50℃の温度で反応させることにより、式(IV)の化合物を製造することができる。
式(IV)で表されるモノアミドを、1,4-ジオキサン等のエーテル系溶媒、トルエン等の炭化水素系溶媒、1,2-ジクロロエタン等のハロゲン系溶媒等の、反応に影響しない溶媒中、あるいは無水酢酸溶媒中で酢酸ナトリウム等の塩基を用い、40℃から溶媒が還流する温度(例えば、100℃)で加熱することによって、式(VI)の化合物を製造することができるが、<工程1>の操作実施後にモノアミド体を単離することなく、無水酢酸中、酢酸ナトリウム等の塩基を用い、環化処理を行うことによっても式(VI)の化合物を製造することができる。
式(V)[式(V)の化合物は市販化合物又は市販化合物から文献公知の製造方法により製造できる化合物である]で表されるアルコールとマレイミド(1H-ピロール-2,5-ジオン)をトリフェニルホスフィン等のホスフィン試薬存在下、テトラヒドロフラン等のエーテル系溶媒、トルエン等の炭化水素系溶媒等の溶媒中、アゾジカルボン酸ジエチル、アゾジカルボン酸ジイソプロピル等の光延試薬を-78℃から室温程度の温度で反応させることによっても、式(VI)の化合物を製造することができる。
式(VI)で表される保護体を、文献公知の方法、例えば(実験化学講座 第5版 16、有機化合物の合成IV、アミノ酸・ペプチド、258-283頁、2007年、丸善)等に記載された方法に準じて脱保護することにより、又、文献公知の方法、例えば、『プロテクティブ・グループス・イン・オーガニック・シンセシス(Protective Groups in Organic Synthesis 4thEdition) 第4版、2007年、ジョン ウィリー アンド サンズ(John Wiley & Sons)、グリーン(Greene)ら』の成書に記載された脱保護の方法に準じて、脱保護を行うことで、式(AM-I)の化合物を製造することができる。
たとえば、P1がtert-ブトキシカルボニル(-C(O)O-tertBu基)である場合、塩化水素やトリフルオロ酢酸等の酸を用いることで脱保護ができる。又、塩化水素を含んだ1,4-ジオキサン、シクロペンチルメチルエーテル、酢酸エチル等を用いることができる。又、トリフルオロ酢酸は、無溶媒または酸に不活性な塩化メチレン、トルエン等の溶媒を用いることもできる。
式(AM-I)のアミンは、必要に応じて、塩酸塩、トリフルオロ酢酸塩、等の塩として、得ることができる。
式(VIII)[式(VIII)の化合物は市販化合物又は市販化合物から文献公知の製造方法により製造できる化合物である。式中P2は、アミノ基の保護基であり、適宜選択できる]で表されるチオール体を用い、文献公知の方法、例えば、(Protective Groups in Organic Synthesis 第3版 PROTECTION FOR THE THIOL GROUP、457-486頁、1999年)等に記載された方法に準じて、アセチルクロリド、ベンゾイルクロリド、等の酸ハライド、又はトリフェニルメチルクロリド、等のアルキルハライド、又は、エチルイソシアネート、等のイソシアネート、等をトリエチルアミン、ピリジン等の有機塩基、炭酸水素カリウム等の無機塩基存在下または非存在下で、1,4-ジオキサン等のエーテル系溶媒、塩化メチレン等のハロゲン系溶媒等の、反応に不活性な溶媒中反応させる、あるいはカルボン酸誘導体と適当な縮合剤や酸触媒中で反応させることにより、式(IX)のチオール保護体を製造することができる。
式(XI)[式(XI)の化合物は市販化合物又は市販化合物から文献公知の製造方法により製造できる化合物である。式中P2は、アミノ基の保護基であり、適宜選択できる]で表されるハロゲン置換体(X=Cl、Br、I)を用い、チオ安息香酸、チオ酢酸、チオ酢酸カリウム等のアシルチオ誘導体等をアセトニトリル、塩化メチレン、N、N-ジメチルホルムアミド等の、反応に不活性な溶媒中で、炭酸カリウム等の塩基存在下または非存在下で反応させることにより、式(IX)の化合物を製造することができる。
式(IX)で表されるN-保護体を、文献公知の方法、例えば(実験化学講座 第5版 16、有機化合物の合成IV、アミノ酸・ペプチド、258-283頁、2007年、丸善)等に記載された方法に準じて脱保護することにより、又、文献公知の方法、例えば、『プロテクティブ・グループス・イン・オーガニック・シンセシス(Protective Groups in Organic Synthesis 4thEdition) 第4版、2007年、ジョン ウィリー アンド サンズ(John Wiley & Sons)、グリーン(Greene)ら』の成書に記載された脱保護の方法に準じて、脱保護を行うことで、式(AM-II)の化合物を製造することができる。
たとえば、P1がtert-ブトキシカルボニル(-C(O)O-tertBu基)である場合、塩化水素やトリフルオロ酢酸等の酸を用いることで脱保護ができる。又、塩化水素を含んだ1,4-ジオキサン、シクロペンチルメチルエーテル、酢酸エチル等を用いることができる。又、トリフルオロ酢酸は、無溶媒または酸に不活性な塩化メチレン、トルエン等の溶媒を用いることもできる。
架橋アルギン酸構造体は、架橋基を有するアルギン酸誘導体に前述の架橋反応を施すことを含む方法により得ることができる。具体的には、例えば、以下の方法により調製できるが、これらに限定されない。
前述の式(AL-1-I)のアルギン酸誘導体を含む溶液を、2価金属イオンを含む溶液中に滴下するなどして部分的に架橋し、特定の構造体を得る。前記で得られた、例えばゲル等の構造体を、前述の式(AL-1-II)のアルギン酸誘導体を含む溶液に添加することにより、前記構造体の表面等にさらなる架橋反応を施すことにより、架橋アルギン酸構造体を得ることができる。なお、この方法は、式(AL-1-I)のアルギン酸誘導体を式(AL-1-II)のアルギン酸誘導体に、式(AL-1-II)のアルギン酸誘導体を式(AL-1-I)のアルギン酸誘導体に、それぞれ置き換えて実施することも可能である。
前述の式(AL-1-I)のアルギン酸誘導体を含む溶液と、前述の式(AL-1-II)のアルギン酸誘導体を含む溶液とを混和し、当該混合溶液を、2価金属イオンを含む溶液中に滴下するなどして部分的に架橋し、特定の構造体である、架橋アルギン酸構造体を得ることができる。
上記方法に用いる2価金属イオンとして、具体的には、例えば、カルシウムイオン、マグネシウムイオン、バリウムイオン、ストロンチウムイオン、亜鉛イオン等が挙げられ、カルシウムイオンが好ましい。前記カルシウムイオンを含む溶液のカルシウムイオン濃度は特に限定されないが、例えば、1mM~1Mが挙げられ、5mM~500mMが好ましく、10mM~300mMがより好ましい。
アルギン酸誘導体は、食品、医療、化粧品、繊維、製紙などの幅広い分野で、従来のアルギン酸の代わりに用いることができる。アルギン酸誘導体または架橋アルギン酸構造体の好ましい用途としては、具体的には、創傷被覆材、術後癒着防止材、薬剤徐放用基材、細胞培養用基材、細胞移植用基材等の医療用材料が挙げられる。
2-(2,5-ジオキソ-2,5-ジヒドロ-1H-ピロール-1-イル)エチルアミノ基導入アルギン酸(AL-EX-1)の合成
分子量は、261万Daから1万9千Daまでブロードに溶出され、重量平均分子量は、146万Daと計算された。
2-(2-(2,5-ジオキソ-2,5-ジヒドロ-1H-ピロール-1-イル)エトキシ)エチルアミノ基導入アルギン酸(AL-EX-2)の合成
分子量は、273万Daから1万1千Daまでブロードに溶出され、重量平均分子量は、144万Daと計算された。
2-(2-(2-(2,5-ジオキソ-2,5-ジヒドロ-1H-ピロール-1-イル)エトキシ)エトキシ)エチルアミノ基導入アルギン酸(AL-EX-3)の合成
分子量は、272万Daから1万1千Daまでブロードに溶出され、重量平均分子量は、144万Daと計算された。
S-(4-(2-アミノエチル)カルバモイル)ベンジル)エタンチオエート基導入アルギン酸(AL-EX-4)の合成
分子量は、277万Daから1万4千Daまでブロードに溶出され、重量平均分子量は、142万Daと計算された。
S-(4-(3-((3-アミノプロピル)アミノ)-3-オキソプロピル)ベンジル)エタンチオエート基導入アルギン酸(AL-EX-5)の合成
分子量は、261万Daから2万1千Daまでブロードに溶出され、重量平均分子量は、142万Daと計算された。
2-(N-(4-(メルカプトメチル)ベンザミド))エチルアミノ基導入アルギン酸(AL-EX-7-1)水溶液の調製
分子量は、273万Daから2千Daまでブロードに溶出され、重量平均分子量は、136万Daと計算された。
(S)-2-(2-アミノアセトアミド)-N-(2-(2、5-ジオキソ-2、5-ジヒドロ-1H-ピロール-1-イル)エチル)-3-フェニルプロパンアミド基導入アルギン酸(AL-EX-10)の合成
tert-ブチル (S)-(2-((1-((2-(2,5-ジオキソ-2,5-ジヒドロ-1H-ピロール-1-イル)エチル)アミノ)-1-オキソ-3-フェニルプロパン-2-イル)アミノ)-2-オキソエチル)カルバメートの合成
(S)-2-(2-アミノアセトアミド)-N-(2-(2、5-ジオキソ-2、5-ジヒドロ-1H-ピロール-1-イル)エチル)-3-フェニルプロパンアミド基導入アルギン酸(AL-EX-10)の合成
分子量は、287万Daから2万Daまでブロードに溶出され、重量平均分子量は、143万Daと計算された。
反応性基の導入率は、アルギン酸の繰り返し単位であるウロン酸単糖単位あたりに導入
された反応性基の数を百分率で表した値を意味する。導入率の算出に必要なアルギン酸の
量は、検量線を利用したカルバゾール硫酸法により測定し、反応性基の量は、検量線を利
用した吸光度測定法により測定した。
実施例で得られた架橋基導入アルギン酸固体を秤量し、0.15mol/LのNaClを含む10mmol/Lリン酸緩衝液(pH7.4)に添加し、室温で1時間以上攪拌して溶解し、0.2%溶液を調製した。この溶液を孔径0.45μmのポリエーテルスルフォン製Minisart High Flowフィルター(SARTORIUS社)に通し不溶物を除いた後、この200μLをSuperose6 Increase 10/300 GLカラム(GEヘルスケアサイエンス社)に供し、ゲルろ過を実施した。ゲルろ過は、クロマトグラフ装置としてAKTA Explorer 10Sを、展開溶媒として0.15mol/L NaClを含む10mmol/Lリン酸緩衝液(pH7.4)を使用し、室温で流速0.8mL/mimの条件で実施した。各試料のクロマトグラムは、220nm若しくは240nmの吸収をモニターし作製した。また別法として215 nmの吸収をモニターした。得られたクロマトグラムのピーク解析は、Unicorn5.31ソフトウエア(GEヘルスケアサイエンス社)にて行った。
(実施例2)<工程3>で得られたアルギン酸誘導体(AL-EX-2)、または(実施例3)<工程4>で得られたアルギン酸誘導体(AL-EX-3)を、各々、濃度が1%となるよう水に溶かしてアルギン酸水溶液(2)及びアルギン酸水溶液(3)を得た。さらに(実施例7-1)で得た2重量%のアルギン酸誘導体(AL-EX-7-1)溶液にリン酸緩衝生理食塩水(PBS)を等量加えて1重量%とし、アルギン酸水溶液(7-1)を得た。
対照として用いたアルギン酸(ALG-2)から作成した、アルギン酸ゲルが8時間でほぼ溶解したのに対し、本実施例の架橋基導入アルギン酸誘導体を架橋して得られた架橋アルギン酸構造体(アルギン酸誘導体(AL-EX-2)/アルギン酸誘導体(AL-EX-7-1)を架橋して得られた架橋アルギン酸構造体、および、アルギン酸誘導体(AL-EX-3)/アルギン酸誘導体(AL-EX-7-1)を架橋して得られた架橋アルギン酸構造体)は、いずれも安定性が向上した。
実施例8で得られたアルギン酸誘導体(AL-EX-8)、実施例9で得られたアルギン酸誘導体(AL-EX-9)、および実施例10で得られたアルギン酸誘導体(AL-EX-10)を、濃度が0.5%となるよう水に溶かしてアルギン酸水溶液(8)、アルギン酸水溶液(9)、及びアルギン酸水溶液(10)を得た。さらに実施例7-1と同様の方法で得た2%の架橋基導入アルギン酸誘導体(AL-EX-7-1-2)(導入率(NMR積分比)=5.1 mol%)にリン酸緩衝生理食塩水(PBS)を3容量加えて0.5%とし、アルギン酸水溶液(7-1-2)を得た。
アルギン酸誘導体(AL-EX-9)/アルギン酸誘導体(AL-EX-7-1-2)を架橋して得られた架橋アルギン酸構造体は96時間後で崩壊率が約39%であり、アルギン酸誘導体(AL-EX-8)/アルギン酸誘導体(AL-EX-7-1-2)を架橋して得られた架橋アルギン酸構造体は96時間後で崩壊率が約40%、及びアルギン酸誘導体(AL-EX-10)/アルギン酸誘導体(AL-EX-7-1-2)を架橋して得られた架橋アルギン酸構造体は96時間後で崩壊率が約55%であり、ゲル安定性の測定(1)のアルギン酸(ALG-2)から作成したアルギン酸ゲルに比べて、安定性が向上していることが示唆された。
(実施例2)<工程3>と同様の方法で製造した導入率(NMR積分比)=3.4 mol%の架橋基導入アルギン酸誘導体(AL-EX-2-1)、実施例8で得られたアルギン酸誘導体(AL-EX-8)、実施例9で得られたアルギン酸誘導体(AL-EX-9)、および実施例10で得られたアルギン酸誘導体(AL-EX-10)を、濃度が0.5%となるよう水に溶かしてアルギン酸水溶液(2‐1)、アルギン酸水溶液(8)、アルギン酸水溶液(9)、及びアルギン酸水溶液(10)を得た。さらに実施例7-1と同様の方法で得た2%の架橋基導入アルギン酸誘導体(AL-EX-7-1-2)(導入率(NMR積分比)=5.1 mol%)にリン酸緩衝生理食塩水(PBS)を3容量加えて0.5%とし、アルギン酸水溶液(7-1-2)を得た。
アルギン酸誘導体(AL-EX-2-1)/アルギン酸誘導体(AL-EX-7-1-2)を架橋して得られた架橋アルギン酸構造体は24時間後で崩壊率が約49%であり、アルギン酸誘導体(AL-EX-9)/アルギン酸誘導体(AL-EX-7-1-2)を架橋して得られた架橋アルギン酸構造体は24時間後で崩壊率が約28%であり、アルギン酸誘導体(AL-EX-8)/アルギン酸誘導体(AL-EX-7-1-2)を架橋して得られた架橋アルギン酸構造体は24時間後で崩壊率が約32%、及びアルギン酸誘導体(AL-EX-10)/アルギン酸誘導体(AL-EX-7-1-2)を架橋して得られた架橋アルギン酸構造体は24時間後で崩壊率が約32%であり、カルシウム架橋を除いたアルギン酸構造体にても、いずれも安定性があることが確認できた。
(実施例2)<工程3>で得られたアルギン酸誘導体(AL-EX-2)、または(実施例3)<工程4>で得られたアルギン酸誘導体(AL-EX-3)、あるいは原料アルギン酸である反応性基を導入していないアルギン酸(ALG-2;対照)を、濃度が1%となるよう水に溶かして、1/100容量の1規定-炭酸水素ナトリウム水溶液を添加し、アルギン酸水溶液(2)、アルギン酸水溶液(3)及びアルギン酸水溶液(ALG-2-aq)を得た。さらに(実施例7-1)で得た2重量%のアルギン酸誘導体(AL-EX-7-1)溶液にリン酸緩衝生理食塩水(PBS)で1 mg/mLに調製した分子量200万のフルオレセインイソチオシアナート-デキストラン(シグマアルドリッチ、FD2000S)を等量加えて1重量%とし、アルギン酸水溶液(7-1)を得た。
アルギン酸水溶液(2)およびアルギン酸水溶液(7-1)、又は、アルギン酸水溶液(3)およびアルギン酸水溶液(7-1)を等量混和し、この混和した水溶液を18ゲージの注射針を装着した注射筒に入れ、この注射筒を流速1 mL/分に設定したシリンジポンプに設置し、濃度が30 mmol/Lの塩化カルシウム溶液に30秒間滴下し、約20分間撹拌してアルギン酸ゲルを得た。このゲルを10 mLのPBSで1度洗浄し、フルオレセインイソチオシアナート-デキストラン内包化学架橋アルギン酸ゲルを得た。このゲルに20 mLのPBSを添加し、37℃で振盪して、経時的に水溶液を回収した。試験終了後、試験溶液にアルギン酸リアーゼ(ニッポンジーン、319-08261)を5μL添加し、37℃で2時間振盪させてゲルを全て崩壊させ、水溶液を回収した。回収した水溶液中のデキストラン濃度を蛍光定量法(励起光:485nm、蛍光:535nm)により測定し、各時点のデキストラン濃度を試験終了後のデキストラン濃度で除した値を百分率で表した値を漏出率とし、ゲル安定性の指標とした。
対照として用いたアルギン酸(ALG-2)から調製されたゲルが24時間で40%弱、48時間で約70%の漏出率を示したのに対し、本実施例の架橋基導入アルギン酸誘導体を架橋して得られた架橋アルギン酸構造体(アルギン酸誘導体(AL-EX-2)/アルギン酸誘導体(AL-EX-7-1)を架橋して得られた架橋アルギン酸構造体、および、アルギン酸誘導体(AL-EX-3)/アルギン酸誘導体(AL-EX-7-1)を架橋して得られた架橋アルギン酸構造体)は、24時間の漏出率が約10%、48時間の漏出率が10~15%程度であり、いずれも安定性が向上した。
(実施例2)<工程3>と同様の方法で製造した導入率(NMR積分比)=3.4 mol%の架橋基導入アルギン酸誘導体(AL-EX-2-1)、実施例8で得られたアルギン酸誘導体(AL-EX-8)、実施例9で得られたアルギン酸誘導体(AL-EX-9)、および実施例10で得られたアルギン酸誘導体(AL-EX-10)を、濃度が2.0%となるよう水に溶かしてアルギン酸水溶液を調製し、このアルギン酸水溶液に4/5容量の1 mg/mLに調製した分子量15万のフルオレセインイソチオシアナート-デキストラン(シグマアルドリッチ、FD150S)、及び2.2容量の水を加え、0.2 mg/mLフルオレセインイソチオシアナート-デキストラン含有0.5%アルギン酸水溶液(2‐1)、アルギン酸水溶液(8)、アルギン酸水溶液(9)、及びアルギン酸水溶液(10)を得た。さらに実施例7-1と同様の方法で得た2%の架橋基導入アルギン酸誘導体(AL-EX-7-1-2)(導入率(NMR積分比)=5.1 mol%)にリン酸緩衝生理食塩水(PBS)を3容量加えて0.5%とし、アルギン酸水溶液(7-1-2)を得た。
アルギン酸水溶液(2‐1)、アルギン酸水溶液(8)、アルギン酸水溶液(9)、及びアルギン酸水溶液(10)の各アルギン酸水溶液とアルギン酸水溶液(7-1-1-2)を各々250 μLずつ等量混和し、濃度が30 mmol/Lの塩化カルシウム溶液を40 mL添加し、5分間撹拌してアルギン酸ゲルを得た。このゲルを10 mLの生理食塩水で1度洗浄し、フルオレセインイソチオシアナート-デキストラン内包化学架橋アルギン酸ゲルを得た。このゲルに19.5 mLの生理食塩水を添加し、37℃で振盪して、経時的に水溶液を回収し、回収した量と同量のPBSを補充した。試験終了後、試験溶液にアルギン酸リアーゼ(ニッポンジーン、319-08261)を10 μL添加し、37℃で3時間以上振盪させてゲルを全て崩壊させ、水溶液を回収した。回収した水溶液中のデキストラン濃度を蛍光定量法(励起光:485nm、蛍光:535nm)により測定し、各時点のデキストラン濃度を試験終了後のデキストラン濃度で除した値を百分率で表した値を透過率とした。
アルギン酸誘導体(AL-EX-2―1)/アルギン酸誘導体(AL-EX-7-1-2)を架橋して得られた架橋アルギン酸構造体、アルギン酸誘導体(AL-EX-9)/アルギン酸誘導体(AL-EX-7-1-2)を架橋して得られた架橋アルギン酸構造体、アルギン酸誘導体(AL-EX-8)/アルギン酸誘導体(AL-EX-7-1-2)を架橋して得られた架橋アルギン酸構造体、及びアルギン酸誘導体(AL-EX-10)/アルギン酸誘導体(AL-EX-7-1-2)を架橋して得られた架橋アルギン酸構造体は、いずれも3時間後で約20%弱、24時間で約50%の漏出率を示した。
Claims (24)
- アルギン酸、そのエステルおよびその塩からなる群から選択される少なくとも1種のカルボキシル基の一部に、下記式(I)(式中、破線右外側は除く):
R1は、それぞれ独立して、水素原子、メチル基、イソプロピル基、イソブチル基、sec-ブチル基、ヒドロキシメチル基、2-ヒドロキシエチル基、チオールメチル基、メチルチオエチル基、カルボキシメチル基、カルボキシエチル基、アミノカルボニルメチル基、アミノカルボニルエチル基、アミノブチル基、グアニジノプロピル基、ベンジル基、4-ヒドロキシベンジル基、3-インドリルメチル基、4-イミダゾイルメチル基およびR1が結合する炭素原子と当該炭素原子が結合する窒素原子と共に環を形成するプロパン-1,3-ジイル基からなる群より選択される基であり;
R2は、それぞれ独立して、水素原子、メチル基、イソプロピル基、イソブチル基、sec-ブチル基、ヒドロキシメチル基、2-ヒドロキシエチル基、チオールメチル基、メチルチオエチル基、カルボキシメチル基、カルボキシエチル基、アミノカルボニルメチル基、アミノカルボニルエチル基、アミノブチル基、グアニジノプロピル基、ベンジル基、4-ヒドロキシベンジル基、3-インドリルメチル基、4-イミダゾイルメチル基およびR2が結合する炭素原子と当該炭素原子が結合する窒素原子と共に環を形成するプロパン-1,3-ジイル基からなる群より選択される基であり;
nは、1~18の整数であり;
mは、1~9の整数であり;
jは、0~9の整数である)
で表される基を有する、アルギン酸誘導体。 - アルギン酸、そのエステルおよびその塩からなる群から選択される少なくとも1種のカルボキシル基の一部に、下記式(I)(式中、破線右外側は除く):
R1は、それぞれ独立して、水素原子、メチル基、イソプロピル基、イソブチル基、sec-ブチル基、ヒドロキシメチル基、2-ヒドロキシエチル基、チオールメチル基、メチルチオエチル基、カルボキシメチル基、カルボキシエチル基、アミノカルボニルメチル基、アミノカルボニルエチル基、アミノブチル基、グアニジノプロピル基、ベンジル基、4-ヒドロキシベンジル基、3-インドリルメチル基、4-イミダゾイルメチル基およびR1が結合する炭素原子と当該炭素原子が結合する窒素原子と共に環を形成するプロパン-1,3-ジイル基からなる群より選択される基であり;
R2は、それぞれ独立して、水素原子、メチル基、イソプロピル基、イソブチル基、sec-ブチル基、ヒドロキシメチル基、2-ヒドロキシエチル基、チオールメチル基、メチルチオエチル基、カルボキシメチル基、カルボキシエチル基、アミノカルボニルメチル基、アミノカルボニルエチル基、アミノブチル基、グアニジノプロピル基、ベンジル基、4-ヒドロキシベンジル基、3-インドリルメチル基、4-イミダゾイルメチル基およびR2が結合する炭素原子と当該炭素原子が結合する窒素原子と共に環を形成するプロパン-1,3-ジイル基からなる群より選択される基であり;
nは、1~18の整数であり;
mは、1~9の整数であり;
jは、0~9の整数である)
で表される基を有するアルギン酸誘導体(但し、-A1-=-CH2CH2-は除く)。 - 式(I)で表される基の導入率が、1%~30%である、請求項1~8のいずれか1項に記載のアルギン酸誘導体。
- アルギン酸誘導体のゲルろ過クロマトグラフィー法により測定した重量平均分子量が、10万Da~300万Daである、請求項1~9のいずれか1項に記載のアルギン酸誘導体。
- 式(II)において、P1が、水素原子、アセチル基またはベンゾイル基である、請求項11に記載のアルギン酸誘導体。
- 式(II)で表される基の導入率が、1%~30%である、請求項11~15のいずれか1項に記載のアルギン酸誘導体。
- アルギン酸誘導体のゲルろ過クロマトグラフィー法により測定した重量平均分子量が、10万Da~300万Daである、請求項11~16のいずれか1項に記載のアルギン酸誘導体。
- 請求項1~10のいずれか1項に記載のアルギン酸誘導体および、請求項11~17のいずれか1項に記載のアルギン酸誘導体を含有する、組成物。
- 請求項1~10のいずれか1項に記載のアルギン酸誘導体および、請求項11~17のいずれか1項に記載のアルギン酸誘導体に、架橋反応を施すことにより得られる架橋アルギン酸構造体。
- 請求項19に記載の架橋アルギン酸構造体を含む医療用材料。
- 架橋アルギン酸構造体が、ビーズまたは略球形のゲルである、請求項20に記載の医療用材料。
- 請求項1~10のいずれか1項に記載のアルギン酸誘導体の溶液を、カルシウムイオンを含む溶液中に滴下し、得られたゲルに、請求項11~17のいずれか1項に記載のアルギン酸誘導体の溶液中において架橋反応を施すことを含む、架橋アルギン酸構造体の製造方法。
- 請求項11~17のいずれか1項に記載のアルギン酸誘導体の溶液を、カルシウムイオンを含む溶液中に滴下し、得られたゲルに、請求項1~10のいずれか1項に記載のアルギン酸誘導体の溶液中において架橋反応を施すことを含む、架橋アルギン酸構造体の製造方法。
- 請求項18に記載の組成物の溶液を、カルシウムイオンを含む溶液中に滴下することを含む、架橋アルギン酸構造体の製造方法。
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WO2022034889A1 (ja) | 2020-08-14 | 2022-02-17 | 国立大学法人東京大学 | 多糖誘導体、多糖誘導体-薬物コンジュゲート、その製造方法 |
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US11472892B2 (en) | 2022-10-18 |
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