WO2022137345A1 - 化学架橋アルギン酸を用いた移植用デバイス - Google Patents

化学架橋アルギン酸を用いた移植用デバイス Download PDF

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WO2022137345A1
WO2022137345A1 PCT/JP2020/047944 JP2020047944W WO2022137345A1 WO 2022137345 A1 WO2022137345 A1 WO 2022137345A1 JP 2020047944 W JP2020047944 W JP 2020047944W WO 2022137345 A1 WO2022137345 A1 WO 2022137345A1
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Prior art keywords
alginic acid
formula
group
transplantation
solution
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English (en)
French (fr)
Japanese (ja)
Inventor
雅之 霜田
久美子 安嶋
正司 古迫
直人 津田
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Mochida Pharmaceutical Co Ltd
National Center for Global Health and Medicine
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Mochida Pharmaceutical Co Ltd
National Center for Global Health and Medicine
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Priority to CN202080108152.7A priority Critical patent/CN117042783A/zh
Priority to CA3202982A priority patent/CA3202982A1/en
Priority to EP20966837.5A priority patent/EP4268855A4/en
Priority to KR1020237020727A priority patent/KR20230123959A/ko
Priority to AU2020482526A priority patent/AU2020482526A1/en
Priority to JP2022570823A priority patent/JP7625014B2/ja
Priority to US18/268,427 priority patent/US20240316248A1/en
Priority to PCT/JP2020/047944 priority patent/WO2022137345A1/ja
Publication of WO2022137345A1 publication Critical patent/WO2022137345A1/ja
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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
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/52—Hydrogels or hydrocolloids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
    • A61F2/022—Artificial gland structures using bioreactors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/37—Digestive system
    • A61K35/39—Pancreas; Islets of Langerhans
    • 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
    • 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
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/20—Polysaccharides
    • 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
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • 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
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • 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
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • 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
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
    • C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
    • C08J3/075—Macromolecular gels
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0676—Pancreatic cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0676—Pancreatic cells
    • C12N5/0677—Three-dimensional culture, tissue culture or organ culture; Encapsulated cells
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2305/00—Characterised by the use of polysaccharides or of their derivatives not provided for in groups C08J2301/00 or C08J2303/00
    • C08J2305/04—Alginic acid; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2405/00—Characterised by the use of polysaccharides or of their derivatives not provided for in groups C08J2401/00 or C08J2403/00
    • C08J2405/04—Alginic acid; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/70—Polysaccharides
    • C12N2533/74—Alginate

Definitions

  • the present invention relates to a device for transplanting cells or the like into a living body. More specifically, the present invention relates to a transplant device using chemically crosslinked alginic acid, and a method for producing the same.
  • Non-Patent Document 1 Organ Biologic, VOL24, No. 1, pp. 7-12, 2017).
  • pancreatic islets are coated (encapsulated) with a polymer gel or semi-permeable membrane that can be isolated from the recipient's immune cells and can permeate nutrients and insulin, and then transplanted into the body.
  • BAP bioartificial pancreas
  • the technique of artificial pancreas has been studied for a long time (Patent Document 1: Japanese Patent Application Laid-Open No. 55-157502, Patent Document 2: Japanese Patent Application Laid-Open No. 60-258121). Gazette, Patent Document 3: International Publication No. 95/28480, Patent Document 4: International Publication No. 92/19195, Patent Document 5: Japanese Patent Application Laid-Open No. 2017-196150).
  • bioartificial islets are mainly (1) "microcapsule type” in which individual islets are coated with polymer gel or the like, and (2) “macro” in which many islets are coated with polymer gel or semipermeable membrane. It is classified into “capsule type” and (3) "blood perfusion type” in which pancreatic islets are encapsulated in an immunoisolation device or hollow thread module made of a semipermeable membrane and blood is perfused into the device (Non-Patent Document 1). ..
  • the microcapsule type encapsulates individual islets using a polymer gel that can be isolated from immune cells and is permeable to nutrients and insulin, and is transplanted into the body (mainly intraperitoneally) in the same way as normal islet transplantation. It is a technology. It can be isolated from the recipient's immune cells, and because the isolation film is relatively thin, it has the advantage that the permeation time due to diffusion is short, and the permeation of nutrients and the response of cells are accelerated. It is difficult to recover.
  • the blood perfusion type is a technique for perfusing blood into a flow path that separates the pancreatic island with a semi-transparent membrane, and has applied the accumulation of techniques such as artificial dialysis and bio-artificial liver, and many basic studies have been conducted.
  • the problems are that the size of the device is large and the risk of blood clot formation is high, and there is a drawback that blood clots are easily formed and clogged during long-term use, and it has not been put into practical use.
  • the macrocapsule type is an improved technique for the purpose of enabling the removal of pancreatic islets when the function of the islets is reduced, which is a drawback of the microcapsule type.
  • studies on islet transplantation using macrocapsule-type heterologous islets have not yet reported excellent results, and islets such as donor shortage, use of immunosuppressants, and long-term engraftment and functional maintenance of islets.
  • Bioartificial islets using heterologous islets that overcome the problems of transplantation have not yet been found.
  • Non-Patent Document 2 JOURNAL OF BIOMEDICAL MATERIALS RESEARCH, PartB / VOL103B, ISSUE). 5, P1120-1132 (2015)).
  • the document discloses that alginate capsules formed by the click reaction are more stable than ionic cross-linking (C 2+ ) cross-linking.
  • the novel alginic acid derivatives used here are hydrogelled by, for example, chemical cross-linking, and the chemistry thereof.
  • an alginate gel prepared into a flat plate using a cross-linking alginic acid derivative was transplanted into a living body (intraperitoneal of a healthy mouse), the size of the flat plate gel did not change significantly even after 5 weeks, and the gel was dissolved. It maintains its shape and has excellent in vivo stability.
  • the islets in the transplantation device were confirmed to be alive by dithizone staining, it was confirmed that they were sufficiently alive.
  • the device for transplantation removed 10 weeks after transplantation was opened and the alginate gel inside was confirmed, the shape state was maintained.
  • the hydrogel is a thin device having a thickness of less than 500 ⁇ m, the healing rate of the animal transplanted with the device is higher than that of the device having a thickness of 500 ⁇ m or more.
  • the thickness of the hydrogel is less than 500 ⁇ m, the ratio of the oxygen concentration in the central part to the surface of the device is higher than that in the case of a thin device having a thickness of 500 ⁇ m or more.
  • the hydrogel does not disintegrate or is difficult to disintegrate.
  • the islets contained in the hydrogel are less likely to fall off from the gel.
  • novel alginic acid derivatives used herein can be used, for example, for chemical cross-linking formation, i.e., chemical cross-linking.
  • a reactive group that can be used for formation or a complementary reactive group of the reactive group is introduced.
  • the chemical cross-linking formation may occur, for example, between an alginic acid derivative into which a reactive group can be used for chemical cross-linking formation and an alginic acid derivative into which a reactive group complementary to the reactive group has been introduced. .. Both the reactive group and the complementary reactive group of the reactive group may be introduced into one molecule of alginic acid, or may be introduced separately. Further, the chemical cross-linking may be carried out within the molecule of the alginic acid derivative or between the molecules, and the reactive group or another reactive group complementary to the reactive group is introduced. May be done with molecules.
  • the chemical cross-linking is carried out, for example, by a cross-linking reaction by a Huisgen reaction (1,3-bipolar addition cyclization reaction), for example, between the alkyne derivatives of the formulas (HA-I) and (HA-II). It may be carried out, or, for example, between an arginic acid derivative of formula (HA-I) and another molecule having an azido group, or an arginic acid derivative of formula (HA-II) and an alkyne group. It may be done between other molecules that have.
  • the chemical cross-linking formation is carried out by, for example, a cross-linking reaction by a Huisgen reaction (1,3-bipolar addition cyclization reaction), for example, alkyne derivatives of the formulas (HB-I) and (HB-II). It may be carried out between, or, for example, between an arginic acid derivative of formula (HB-I) and another molecule having an azido group, or an arginic acid derivative of formula (HB-II) and an alkyne. It may be carried out between other molecules having a group.
  • Huisgen reaction (1,3-bipolar addition cyclization reaction
  • alkyne derivatives of the formulas (HB-I) and (HB-II for example, alkyne derivatives of the formulas (HB-I) and (HB-II). It may be carried out between, or, for example, between an arginic acid derivative of formula (HB-I) and another molecule having an azido group, or an arginic acid derivative of
  • a device for transplanting cells or the like prepared using an alginic acid derivative gelled by chemical cross-linking, more specifically, for example, a chemistry in which insulin-secreting cells or pancreatic islets are embedded.
  • a device for transplantation including a crosslinked islet gel and, if necessary, a semi-permeable membrane covering the gel, a method for producing the same, and the like are provided.
  • the alginic acid derivative gelled by chemical cross-linking is, for example, a formula (HA-I) in which a cyclic alkyne group or an azido group is introduced into any one or more carboxyl groups of alginic acid via an amide bond and a divalent linker.
  • the alkyne derivative of the formula (HA-II), and the Huisgen reaction (1,3-dipolar addition cyclization reaction) is carried out using the alkyne derivative of the formula (HA-I) and the formula (HA-II).
  • a novel cross-linked alkynic acid is obtained.
  • the alginic acid derivative gelled by chemical cross-linking has, for example, a formula (HB) in which a cyclic alkyne group or an azido group is introduced into any one or more carboxyl groups of alginic acid via an amide bond and a divalent linker.
  • the Huisgen reaction (1,3-dipolar addition cyclization reaction) is carried out using the alkyne derivative of the formula (HB-I) or the formula (HB-II) and the alkyne derivative of the formula (HB-I) and the formula (HB-II). By doing so, a novel crosslinked alkynic acid can be obtained.
  • An exemplary embodiment may be as follows [1-1] to [3-4].
  • the alginic acid derivatives according to the formula (HA-I) and the formula (HB-I) both have the structure of the formula (I) in common. Therefore, the general formulas of the alginic acid derivatives according to the formula (HA-I) and the formula (HB-I) are both represented by the formula (I). Further, in the present specification, the alginic acid derivatives according to the formula (HA-II) and the formula (HB-II) both have the structure of the formula (II) in common. Therefore, the general formulas of the alginic acid derivatives according to the formula (HA-II) and the formula (HB-II) are both represented by the formula (II).
  • the crosslinked alginic acid according to the formula (HA-III-L) and the formula (HB-III-L) both have the structure of the formula (III-L) in common. Therefore, the general formulas of the crosslinked alginic acid according to the formula (HA-III-L) and the formula (HB-III-L) are both represented by the formula (III-L).
  • the first aspect of the present invention may be as follows [1-1] to [1-34].
  • the chemical cross-linking results from a cyclic alkyne group introduced into any one or more carboxyl groups of alginic acid and an azide group introduced into any one or more carboxyl groups of alginic acid.
  • the transplant device according to any one of 1-1] to [1-3].
  • the chemical cross-linking is a chemical cross-linking by the combination of the alginic acid derivatives described in the following (A) and (B), according to any one of the above [1-1] to [1-5].
  • the chemically crosslinked alginic acid derivative has an arbitrary carboxyl group of the first alginic acid and an arbitrary carboxyl group of the second alginic acid as the following formula (HA-III-L):.
  • HA-III-L -CONH- and -NHCO- at both ends represent amide bonds mediated by any carboxyl group of alginic acid
  • -L 1- is the same as the definition in the above aspect [1-6]
  • -L 2- is the same as the definition in the above aspect [1-6]
  • X is the following partial structural formula: It is a cyclic group selected from the group of (in each formula, the outside of the broken line at both ends is not included), and the star mark represents the chiral center], which is a cross-linked alginic acid bonded via the above [1-6].
  • Device for porting is a cyclic group selected from the group of (in each formula, the outside of the broken line at both ends is not included), and the star mark represents the chiral center],
  • the chemical cross-linking is a chemical cross-linking by the combination of the alkyne derivatives described in the following (A) and (B), according to any one of the above [1-1] to [1-5].
  • -L1-1 is any one linker selected from the group of (L1-1), (L1-2a), (L1-2b), (L1-11) or (L1-12).
  • the alginic acid derivative represented by the formula (HB-II) the chemical cross-linking by the combination with the derivative in which —L2— is the linker of (L2-10) is excluded).
  • the chemically crosslinked alginic acid derivative has an arbitrary carboxyl group of the first alginic acid and an arbitrary carboxyl group of the second alginic acid having the following formula (HB-III-L): [In formula (HB-III-L), -CONH- and -NHCO- at both ends represent amide bonds mediated by any carboxyl group of alginic acid; -L 1- is the same as the definition in [1-8] above; -L 2- is the same as the definition in [1-8] above; X is the table below: It is a cross-linked alginic acid bonded via a cyclic group selected from the group of partial structural formulas described in (In each formula, the outside of the broken line at both ends is not included)] (however, in the formula (HB-I)).
  • -L1-1- is any one linker selected from the group of (L1-1), (L1-2a), (L1-2b), (L1-11) or (L1-12).
  • the alginic acid derivative of the formula (HA-I) is the following formula (EX-1- (I) -A-2).
  • the alginic acid derivative of the formula (HA-II) is the following formula (EX-2- (II) -A-2).
  • the transplant device according to the above [1-6] or [1-7].
  • the alginic acid derivative of the formula (HA-I) is the following formula (EX-3- (I) -A-2).
  • the alginic acid derivative of the formula (HA-II) is the following formula (EX-4- (II) -A-2).
  • the transplant device according to the above [1-6] or [1-7].
  • the alginic acid derivative of the formula (HA-I) is the following formula (EX-1- (I) -A-2).
  • the alginic acid derivative of the formula (HA-II) is the following formula (EX-4-2- (II) -A-2).
  • the transplant device according to the above [1-6] or [1-7].
  • the alginic acid derivative of the formula (HA-I) is the following formula (EX-3- (I) -A-2).
  • the alginic acid derivative of the formula (HA-II) is the following formula (EX-2- (II) -A-2).
  • the transplant device according to the above [1-6] or [1-7].
  • the alginic acid derivative of the formula (HA-I) is the following formula (EX-3- (I) -A-2).
  • the alginic acid derivative of the formula (HA-II) is the following formula (EX-4-2- (II) -A-2).
  • the transplant device according to the above [1-6] or [1-7].
  • the insulin-secreting cells or islets are pancreatic islets, islet cells, or ⁇ -cells obtained from a human donor, or arelets, islets, or ⁇ -cells obtained from a pig as a donor.
  • transplantation device according to any one of [1-1] to [1-22] above, wherein the transplantation site of the transplantation device is subcutaneous or intraperitoneal.
  • transplant device according to any one of the above [1-1] to [1-23], wherein the transplant device has a thickness of 0.1 to 5 mm.
  • transplant device according to any one of [1-1] to [1-23], wherein the thickness of the transplant device is 100 ⁇ m or more and less than 1000 ⁇ m.
  • Insulin-secreting cells or pancreatic islets are suspended in a solution of an alginic acid derivative hydrogelated by chemical cross-linking, and the solution in which the insulin-secreting cells or pancreatic islets are suspended is encapsulated in a semipermeable membrane.
  • Portable device is obtained by gelling the alginic acid derivative in the semipermeable membrane by contacting the permeable membrane with a solution containing divalent metal ions.
  • the second aspect of the present invention may be as follows [2-1] to [2-34].
  • the chemical cross-linking is a chemical cross-linking by the combination of the alginic acid derivatives described in the following (A) and (B), according to any one of the above [2-1] to [2-7].
  • the chemically crosslinked alginic acid derivative has an arbitrary carboxyl group of the first alginic acid and an arbitrary carboxyl group of the second alginic acid as the following formula (HA-III-L):.
  • HA-III-L -CONH- and -NHCO- at both ends represent amide bonds mediated by any carboxyl group of alginic acid
  • -L 1- is the same as the definition in the above aspect [2-8]
  • -L 2- is the same as the definition in the above aspect [2-8]
  • X is the following partial structural formula: It is a cyclic group selected from the group of (in each formula, the outside of the broken line at both ends is not included), and the star mark represents the chiral center], which is a cross-linked alginic acid bonded via the above [2-8].
  • Device for porting is a cyclic group selected from the group of (in each formula, the outside of the broken line at both ends is not included), and the star mark represents the chir
  • the chemical cross-linking is a chemical cross-linking by the combination of the alkyne derivatives described in the following (A) and (B), according to any one of the above [2-1] to [2-7].
  • -L1-1 is any one linker selected from the group of (L1-1), (L1-2a), (L1-2b), (L1-11) or (L1-12).
  • the alginic acid derivative represented by the formula (HB-II) the chemical cross-linking by the combination with the derivative in which —L2— is the linker of (L2-10) is excluded).
  • the chemically crosslinked alginic acid derivative has an arbitrary carboxyl group of the first alginic acid and an arbitrary carboxyl group of the second alginic acid having the following formula (HB-III-L): [In formula (HB-III-L), -CONH- and -NHCO- at both ends represent amide bonds mediated by any carboxyl group of alginic acid; -L 1- is the same as the definition in [2-10] above; -L 2- is the same as the definition in [2-10] above; X is the table below: It is a crosslinked alginic acid bonded via [a cyclic group selected from the group of partial structural formulas described in (the outside of the broken line at both ends is not included in each formula)] (however, in the formula (HB-I)).
  • -L1-1- is any one linker selected from the group of (L1-1), (L1-2a), (L1-2b), (L1-11) or (L1-12).
  • the alginic acid derivative of the formula (HA-I) is the following formula (EX-1- (I) -A-2).
  • the alginic acid derivative of the formula (HA-II) is the following formula (EX-2- (II) -A-2).
  • the transplant device according to the above [2-8] or [2-9].
  • the alginic acid derivative of the formula (HA-I) is the following formula (EX-3- (I) -A-2).
  • the alginic acid derivative of the formula (HA-II) is the following formula (EX-4- (II) -A-2).
  • the alginic acid derivative of the formula (HA-I) is the following formula (EX-1- (I) -A-2).
  • the alginic acid derivative of the formula (HA-II) is the following formula (EX-4-2- (II) -A-2).
  • the transplant device according to the above [2-8] or [2-9].
  • the alginic acid derivative of the formula (HA-I) is the following formula (EX-3- (I) -A-2).
  • the alginic acid derivative of the formula (HA-II) is the following formula (EX-2- (II) -A-2).
  • the transplant device according to the above [2-8] or [2-9].
  • the alginic acid derivative of the formula (HA-I) is the following formula (EX-3- (I) -A-2).
  • the alginic acid derivative of the formula (HA-II) is the following formula (EX-4-2- (II) -A-2).
  • the insulin-secreting cells or islets are pancreatic islets, islet cells, or ⁇ -cells obtained from a human donor, or arelets, islets, or ⁇ -cells obtained from a pig as a donor.
  • transplantation device according to any one of [2-1] to [2-24] above, wherein the transplantation site of the transplantation device is subcutaneous or intraperitoneal.
  • transplant device according to any one of [2-1] to [2-25], wherein the thickness of the transplant device is 100 ⁇ m or more and less than 1000 ⁇ m.
  • Insulin-secreting cells or pancreatic islets are suspended in a solution of an alginic acid derivative hydrogelated by chemical cross-linking, and the solution in which the insulin-secreting cells or pancreatic islets are suspended is encapsulated in a semipermeable membrane.
  • Portable device is obtained by gelling the alginic acid derivative in the semipermeable membrane by contacting the permeable membrane with a solution containing divalent metal ions.
  • the third aspect of the present invention may be as follows [3-1] to [3-4].
  • a method for producing a transplantation device containing a hydrogel containing insulin-secreting cells or pancreatic islets which comprises the following steps (a) to (d).
  • Step (c) A step of contacting the solution of the alginic acid derivative obtained in the step (b) with a solution containing divalent metal ions to prepare a gel having a thickness of 0.1 to 5 mm (100 to 5000 ⁇ m).
  • Step (a) As an optional step, a step of removing the pancreas from the living body and separating pancreatic islets
  • Step (c) A step of contacting the solution of the alginic acid derivative obtained in the step (b) with a solution containing divalent metal ions to prepare a gel having a thickness of 100 ⁇ m or more and less than 500 ⁇ m.
  • Step (a) As an optional step, a step of removing the pancreas from the living body and separating pancreatic islets
  • Step (c) A step of enclosing the solution of the alginic acid derivative obtained in the step (b) in a semipermeable membrane.
  • Step (a) As an optional step, a step of removing the pancreas from the living body and separating pancreatic islets
  • Step (e) A step of preparing a gel using the solution of the alginic acid derivative obtained in the step (b) as an arbitrary shape.
  • a method for producing a device for transplantation comprising a hydrogel in which insulin-secreting cells or pancreatic islets are encapsulated, which comprises the following steps (a), (b), (e) and (f).
  • Step (a) As an optional step, a step of removing the pancreas from the living body and separating pancreatic islets
  • Step (e) A step of putting the solution of the alginic acid derivative obtained in the step (b) into an arbitrary container or placing it on an arbitrary surface.
  • a method for producing a transplantation device containing a hydrogel containing insulin-secreting cells or pancreatic islets which comprises the following steps (a) to (c) (f).
  • Step (c) A step of enclosing the solution of the alginic acid derivative obtained in the step (b) in a semipermeable membrane.
  • the present invention provides a new implantable device.
  • the implantable device exhibits at least one or more of the following effects: (1) Excellent biocompatibility and stability, low cytotoxicity, and almost no adhesion or inflammation at the transplant site. (2) The gel is less dissolved and the shape is maintained for a long period of time. (3) It becomes possible to maintain the hypoglycemic effect and regulate blood glucose for a long period of time. (4) After long-term use, the alginate gel in the semipermeable membrane can maintain its shape without dissolving, and can maintain the survival and function of pancreatic islets, and can be used for a long period of time. (5) It is a highly safe medical material that can be replaced, immunoisolated, and has less adhesion and inflammation.
  • More preferred embodiments of the transplant device have excellent transplant performance and functionality, are novel in terms of materials, and can be transplanted into diabetic patients (particularly type I diabetes and insulin-depleted type II diabetes) to provide long-term blood glucose. It is possible to maintain the descent effect and regulate blood sugar. In addition, recovery is possible when the function of insulin-secreting cells or islets in the hydrogel is reduced. Alternatively, regular replacement or additional transplantation is possible. Further, as the insulin-secreting cells or islets enclosed in the hydrogel of the transplantation device, insulin-secreting cells differentiated from stem cells (iPS or the like) or human pancreatic islets can also be used. Therefore, a device of a more preferred embodiment is useful.
  • a device for transplanting cells or the like prepared using an alginic acid derivative gelled by chemical cross-linking, more specifically, for example, a chemistry in which insulin-secreting cells or pancreatic islets are embedded.
  • a transplanting device including a crosslinked islet gel and, if necessary, a semi-permeable membrane covering the gel, a method for producing the same, and the like are provided.
  • the alginic acid derivative gelled by chemical cross-linking is, for example, a formula (HA-I) in which a cyclic alkyne group or an azido group is introduced into any one or more carboxyl groups of alginic acid via an amide bond and a divalent linker.
  • the alkyne derivative of the formula (HA-II), and the Huisgen reaction (1,3-dipolar addition cyclization reaction) is carried out using the alkyne derivative of the formula (HA-I) and the formula (HA-II).
  • a novel cross-linked alkynic acid is obtained.
  • the alginic acid derivative gelled by chemical cross-linking has, for example, a formula (HB) in which a cyclic alkyne group or an azido group is introduced into any one or more carboxyl groups of alginic acid via an amide bond and a divalent linker.
  • the Huisgen reaction (1,3-dipolar addition cyclization reaction) is carried out using the alkyne derivative of the formula (HB-I) or the formula (HB-II) and the alkyne derivative of the formula (HB-I) and the formula (HB-II). By doing so, a novel crosslinked alkynic acid can be obtained.
  • the "transplanting device” is a hydrogel in which insulin-secreting cells or pancreatic islets are encapsulated.
  • the hydrogel is a gel of an alginic acid derivative by chemical cross-linking. Therefore, as the alginic acid derivative, one that can be gelled by chemical cross-linking is used.
  • the shape of the hydrogel in which insulin-secreting cells or pancreatic islets are encapsulated is, for example, a flat plate type.
  • the hydrogel may be further coated with a semipermeable membrane, in which case the hydrogel encapsulating insulin-secreting cells or islets is inserted into the semipermeable membrane.
  • the "insulin-secreting cell” used in the transplantation device means a cell having a function of secreting insulin, and for example, in the cells constituting the pancreatic islet, it means a ⁇ cell secreting insulin.
  • the "insulin-secreting cell” may be a cell having an insulin-secreting function due to differentiation, maturation, modification, or the like, and may be, for example, an iPS cell, an ES cell, or a somatic stem cell (for example, a mesenchymal system). Cells having an insulin secretory function obtained by differentiating stem cells such as stem cells), cells having an insulin secretory function obtained by maturing immature cells and precursor cells, and cells having an insulin secretory function obtained by gene recombination.
  • the differentiation or maturation of the cell includes culturing the cell, that is, the cell obtained by differentiation or maturation may include the cell obtained by culturing.
  • the “islets”, also known as Langerhans islets, are cell masses composed of an average of about 2000 islet cells. Pancreatic islands are 5 cells that secrete glucagon, ⁇ cells that secrete insulin, ⁇ cells that secrete somatostatin, ⁇ cells that secrete grelin, and PP (pancreatic polypeptide) cells that secrete pancreatic polypeptide. Consists of seed cells.
  • “Insulin-secreting cells or islets” are also referred to as cells or tissues having the secretory function of biologically active products.
  • the “islet cell” may be any cell containing at least one of the above five types of cells, but preferably contains at least ⁇ cells.
  • the islet cells may be a mixture containing all of ⁇ cells, ⁇ cells, delta cells, ⁇ cells, and PP cells, or may be in a state contained in islets.
  • the “islet cells” may be those that have become islet cells due to differentiation, maturation, modification, or the like.
  • the "islet cells” include, for example, pancreatic islet cells obtained by differentiating stem cells such as iPS cells, ES cells, and somatic stem cells (for example, mesenchymal stem cells), and immature cells and progenitor cells.
  • pancreatic islet cells obtained by maturing.
  • the "insulin-secreting cells or pancreatic islets (including pancreatic islets)" preferably have a viability and a function capable of recovering the pathological condition of the patient when transplanted into the patient.
  • Functions of insulin-secreting cells, islets or islets cells include, for example, secreting insulin, and it is preferable that glucose responsiveness is maintained even after transplantation.
  • bio-artificial islets are examples of bio-artificial organs.
  • the cells included in the bioartificial islets include the above-mentioned “insulin-secreting cells”, “islets” or “islets cells”, and include, for example, insulin-secreting cells.
  • Insulin-secreting cells are either cells contained in islets collected from humans or pigs, or islets differentiated from stem cells (eg, ES cells, iPS cells, and somatic stem cells (eg, mesenchymal stem cells)). But it may be.
  • the transplantation device of the present invention may use cells other than "insulin-secreting cells, islets and islet cells".
  • the cells other than "insulin-secreting cells, islets and islet cells" any cell can be used as long as it can be used for cell transplantation, and the type thereof is not particularly limited.
  • the cells to be used may be one type or a combination of a plurality of types of cells. Examples of the cells to be used include animal cells, more preferably vertebrate-derived cells, and particularly preferably human-derived cells.
  • the type of vertebrate-derived cells (particularly human-derived cells) may be stem cells (eg, pluripotent cells or somatic stem cells), progenitor cells, or mature cells.
  • an embryonic stem (ES) cell for example, an embryonic stem (ES) cell, a reproductive stem (GS) cell, or an induced pluripotent stem (iPS) cell can be used.
  • somatic stem cells for example, mesenchymal stem cells (MSC), hematopoietic stem cells, sheep membrane cells, umbilical cord blood cells, bone marrow-derived cells, myocardial stem cells, adipose-derived stem cells, or nerve stem cells can be used.
  • progenitor cells and mature cells include skin, dermal, epidermis, muscle, myocardium, nerve, bone, cartilage, endothelial, brain, epithelium, heart, kidney, liver, spleen, oral cavity, corneal marrow, bone marrow, and cord blood.
  • Cells derived from amniotic membrane or hair can be used.
  • human-derived cells include ES cells, iPS cells, MSCs, cartilage cells, osteoblasts, osteoblast precursor cells, interstitial cells, myoblasts, myocardial cells, myocardial blast cells, nerve cells, and hepatocytes.
  • Fibroblasts corneal endothelial cells, vascular endothelial cells, corneal epithelial cells, sheep membrane cells, umbilical cord blood cells, bone marrow-derived cells, or hematopoietic stem cells can be used.
  • the origin of the cell may be either an autologous cell or an allogeneic cell.
  • ES cells iPS cells, mesenchymal stem cells (MSCs) can be used, for example.
  • Donors of "insulin-secreting cells or islets (including islet cells)" are animals, preferably vertebrates, and specific examples include humans, pigs, monkeys, rats or mice, more preferably humans. Or islets. Donors of "insulin-secreting cells, islets or islets cells” are, in some embodiments, pigs from the perspective of resolving the donor shortage.
  • the "insulin secreting cell or pancreatic island (including pancreatic island cell)” may be either a pancreatic island obtained from a donor animal, or an insulin secreting cell or a pancreatic island cell obtained from a donor-derived cell, for example, a human. It may be an insulin secretory cell or pancreatic islet cell differentiated from the derived ES cell or iPS cell.
  • pancreatic islets including pancreatic islets
  • pancreatic islets are derived from pigs, adult porcine islets or embryonic, neonatal, or perinatal porcine islets can be mentioned.
  • the pancreatic islets may be appropriately cultured before use, or pancreatic islets matured from embryonic, neonatal, or perinatal porcine islets may be used.
  • transplantation site is not particularly limited, and examples thereof include subcutaneous, intraperitoneal, intrahepatic, intramuscular, intraocular, and subrenal capsule, but subcutaneous or intraperitoneal transplantation is preferable.
  • the "semipermeable membrane (semipermeable membrane)” is a membrane that allows only molecules or ions of a certain size or less to permeate. It is a system of a solute that does not permeate the semipermeable membrane and a solvent that exhibits permeability. When two solutions of two concentrations are brought into contact with each other via the semipermeable membrane, osmotic pressure is generated and only the solvent permeates.
  • the implantable device described herein may include a semipermeable membrane, or the semipermeable membrane is not essential, i.e., it may not include a semipermeable membrane.
  • the implant device is a hydrogel alone (eg, encapsulated with insulin-secreting cells or islets), i.e., the hydrogel is not coated with a semipermeable membrane.
  • Implantable devices in which the hydrogel is not coated with a semipermeable membrane are preferably biocompatible and stable, have less cytotoxicity, have little adhesion or inflammation at the site of implantation, and have less gel dissolution and shape. It is maintained for a long period of time, and more preferably, it is capable of sustaining a hypoglycemic effect and regulating blood glucose for a long period of time.
  • the hydrogel is coated with a semipermeable membrane.
  • semi-permeable membrane examples include membranes or tubes used for dialysis, and dialysis tubes, cotton cellulose dialysis membranes, regenerated cellulose dialysis membranes, cellulose ester dialysis membranes, etc. can also be used, and the trade name is Cellu-. Examples include Sep T Tubular Membrane (Membrane Filtration Products), Spectra Biotech Membrane (REPLICEN (formerly SPECTRUM)), and Spectra / Pore CE dialysis tube (REPLIGEN (formerly SPECTRUM)).
  • the "semipermeable membrane” is preferably a semipermeable membrane made of cellulose ester. Specific examples include a Spectra / Pore CE dialysis tube (REPLIGEN (formerly SPECTRUM)), which is a dialysis membrane. It is more preferable that the cellulose ester is a polymer of cellulose acetate.
  • the semipermeable membrane used here contains a resin.
  • the semipermeable membrane can be produced, for example, by dissolving at least one kind of resin in a solvent and coagulating the dissolved resin.
  • the resin is not particularly limited.
  • a resin for example, a resin such as an ethylene-vinyl alcohol-based copolymer, a polysulfone-based polymer, a polyacrylonitrile-based polymer, a cellulose-based polymer such as cellulose acetate, a polyamide-based polymer, or a polycarbonate-based polymer can be used. Can be done. More preferably, it is a cellulosic polymer such as cellulose acetate.
  • the semipermeable membrane used here has a "molecular weight cutoff".
  • "Molecular weight cutoff” means the magnitude of the maximum molecular weight that is not substantially blocked. Molecules with a molecular weight above the molecular weight cutoff are substantially prevented from entering and exiting the semipermeable membrane.
  • the "molecular weight cutoff" of the semipermeable membrane used here is preferably 100 kDa (kilodalton).
  • the cutoff value is 100 to 500 Da (Dalton) and 0.5 to 1 kDa with the cutoff value as "MWCO".
  • the cutoff value has a molecular weight cutoff greater than about 500,000 daltons, IgG. Molecules such as and dialysis can enter these semi-transparent membranes, but host cells such as immune cells are blocked from entering the semi-transparent membrane, and insulin and cell nutrients and oxygen are translucent. It will be able to pass through the membrane.
  • the unit Dalton symbol means Da, and 1000 Da means 1 kDa.
  • the thickness of the implantable device is preferably 0.1-5 mm (100-5000 ⁇ m) and preferably 0.1-3 mm (100-3000 ⁇ m). Alternatively, it is preferably 0.5 to 5 mm (500 to 5000 ⁇ m), more preferably 1 to 3 mm (1000 to 3000 ⁇ m).
  • the thickness of the implantable device is 0.15-5 mm (150-5000 ⁇ m) in semipermeable membrane thickness when the hydrogel containing insulin-secreting cells or islets is coated with a semipermeable membrane. , 0.2 to 3 mm (200 to 3000 ⁇ m) is preferable. Alternatively, it is preferably 0.5 to 5 mm (500 to 5000 ⁇ m), more preferably 1 to 3 mm (1000 to 3000 ⁇ m).
  • the thickness of the transplant device is preferably 100 ⁇ m or more and less than 1000 ⁇ m, and preferably 100 ⁇ m or more and less than 500 ⁇ m. Alternatively, it is preferably 150 ⁇ m or more and less than 1000 ⁇ m, and more preferably 150 ⁇ m or more and 500 ⁇ m.
  • the thickness of the transplantation device is preferably 150 ⁇ m or more and less than 1000 ⁇ m, preferably 150 ⁇ m or more. It is more preferably less than 500 ⁇ m. Alternatively, it is preferably 200 ⁇ m or more and less than 1000 ⁇ m, and more preferably 200 ⁇ m or more and 500 ⁇ m.
  • the thickness of the hydrogel is 0.1-5 mm (100-5000 ⁇ m), preferably 0.1-3 mm (100-3000 ⁇ m), preferably 0.1-1 mm (100-3000 ⁇ m). 1000 ⁇ m) is more preferable. Alternatively, it is 0.15 to 5 mm (150 to 5000 ⁇ m), preferably 0.15 to 3 mm (150 to 3000 ⁇ m), and more preferably 0.15 to 1 mm (150 to 1000 ⁇ m). Alternatively, it is 0.2 to 5 mm (200 to 5000 ⁇ m), preferably 0.2 to 3 mm (200 to 3000 ⁇ m), and more preferably 0.2 to 1 mm (200 to 1000 ⁇ m).
  • the implantable device contains a semipermeable membrane
  • the thickness of the hydrogel in the semipermeable membrane may be 0.1-3 mm (100-3000 ⁇ m) and 0.1-2 mm (100-2000 ⁇ m). It is preferably 0.1 to 1 mm (100 to 1000 ⁇ m), more preferably 0.1 to 1 mm (100 to 1000 ⁇ m).
  • it is 0.15 to 3 mm (150 to 3000 ⁇ m), preferably 0.15 to 2 mm (150 to 2000 ⁇ m), and more preferably 0.15 to 1 mm (150 to 1000 ⁇ m).
  • it is 0.2 to 3 mm (200 to 3000 ⁇ m), preferably 0.2 to 2 mm (200 to 2000 ⁇ m), and more preferably 0.2 to 1 mm (200 to 1000 ⁇ m).
  • it is preferably 1 to 3 mm (1000 to 3000 ⁇ m), more preferably 1.5 to 2 mm (1500 to 2000 ⁇ m).
  • the thickness of the hydrogel is preferably 0.1 to 5 mm (100 to 5000 ⁇ m), preferably 0.1 to 3 mm (100 to 3000 ⁇ m), 0. It is more preferably 1 to 1 mm (100 to 1000 ⁇ m).
  • it is 0.15 to 5 mm (150 to 5000 ⁇ m), preferably 0.15 to 3 mm (150 to 3000 ⁇ m), and more preferably 0.15 to 1 mm (150 to 1000 ⁇ m).
  • it is 0.2 to 5 mm (200 to 5000 ⁇ m), preferably 0.2 to 3 mm (200 to 3000 ⁇ m), and more preferably 0.2 to 1 mm (200 to 1000 ⁇ m).
  • it is 0.5 to 5 mm (500 to 5000 ⁇ m), preferably 0.5 to 3 mm (500 to 3000 ⁇ m), and more preferably 0.5 to 1 mm (500 to 1000 ⁇ m).
  • the thickness of the hydrogel is preferably 100 ⁇ m or more and less than 500 ⁇ m, preferably 100 ⁇ m or more and less than 400 ⁇ m, and more preferably 100 ⁇ m or more and less than 300 ⁇ m.
  • it is preferably 150 ⁇ m or more and less than 500 ⁇ m, preferably 150 ⁇ m or more and less than 400 ⁇ m, more preferably 150 ⁇ m or more and less than 300 ⁇ m, and further preferably 200 ⁇ m or more and less than 300 ⁇ m.
  • Such a thickness is the same when the device for transplantation contains a semipermeable membrane and when the device does not contain a semipermeable membrane.
  • the shape of the transplant device is not particularly limited as long as it is a flat plate.
  • the flat plate means a flat plate, and indicates a plate shape having a substantially constant thickness and a large area.
  • Examples of the shape of the plate include flat plate shapes such as polygons such as triangles, quadrangles, and pentagons, and circles.
  • the transplanting device has the above-mentioned thickness and a substantially constant thickness in the entire plate shape.
  • the thickness variation in the plate-shaped transplant device is preferably within ⁇ 20%, more preferably within ⁇ 10%.
  • the thickness of the implantable device is the thickness of the thickest portion of the implantable device.
  • the shape of the transplantation device looks like a rugby ball, and both ends are slightly thin.
  • the center may be thicker than both ends.
  • the thickness of the implant device means the thickness near the center, which is the part of the maximum thickness.
  • the shape of the hydrogel is not particularly limited as long as it is a flat plate.
  • the flat plate means a flat plate, and indicates a plate shape having a substantially constant thickness and a large area. Examples of the shape of the plate include flat plate shapes such as polygons such as triangles, quadrangles, and pentagons, and circles.
  • the hydrogel has the above-mentioned thickness and has a substantially constant thickness in the entire plate shape.
  • the variation in thickness of the hydrogel is preferably within ⁇ 20%, more preferably within ⁇ 10%.
  • the thickness of the hydrogel is the thickness of the thickest portion of the hydrogel.
  • the flat plate hydrogel is, for example, a crosslinked alginic acid gel having a short diameter of 12 to 15 mm, a major diameter of 12 to 18 mm, and a thickness of about 0.1 to 5 mm, and is circular. , Square, hexagon, octagon, etc. can also be taken.
  • the flat plate type hydrogel is expressed by the area, it can be expressed as, for example, 144 to 270 mm 2 .
  • IEQ is an abbreviation for Islet Equivalents, and is an international unit representing the amount of islets, in which pancreatic islets are regarded as spherical and islets with a diameter of 150 ⁇ m are defined as 1 IEQ.
  • one of the conditions for transplanting fresh islets is "islet amount 5000 IEQ / kg (patient weight) or more". I will refer to it here as well.
  • the transplantation device can be appropriately set to the number of pancreatic islets calculated so as to produce a desired therapeutic effect, and can be appropriately set to an appropriate device depending on the weight of the patient, the degree of symptoms, and the like.
  • the amount of insulin secreting cells can also be appropriately set according to the pancreatic islets.
  • step (a): a step of removing the pancreas from a living body and separating pancreatic islets as an optional step means that the step (a) is optional. means.
  • the "living body” is, for example, a human or a non-human mammal, and examples of the non-human mammal include pigs.
  • step (a) is performed, for example, in the case of isolation of porcine pancreatic islets, a known procedure of the present technique, or Shimoda et al. (Shimoda; Cell Transplantation, Vol. 21, pp. 501-508, 2012).
  • Aseptic viable pancreas can be obtained from adult pigs under aseptic conditions and islet cells can be isolated according to the method described in 1 or according to a standard Ricordy technique using the Edmonton protocol. Isolation of other non-human mammalian islets or human islets can also be performed according to the isolation of porcine islets. After that, the isolated pancreatic islets may be used as they are, or may be cultured and used. For the culture of pancreatic islands, for example, according to the method of Noguchi et al.
  • an alginic acid derivative that can be hydrogelized by chemical cross-linking.
  • cells or tissues selected from the group consisting of, for example, the alginic acid derivatives represented by the above-mentioned formulas (I) and (II) are mentioned as being able to be hydrogelized by chemical cross-linking. Can be done.
  • step (b) for example, a 0.1 to 5% by weight aqueous solution or a physiological saline aqueous solution of the alginic acid derivative is prepared, and insulin-secreting cells, islets, cultured pancreatic islet cells, and stem cells are prepared in the solution.
  • Cells or tissues selected from the group consisting of more differentiated islet cells eg, islets obtained in step (a), insulin secreting cells isolated from the islets, or islet cells isolated from the islets).
  • the pancreatic islet cells obtained by culturing the cells are appropriately suspended in a required amount.
  • the "solution of the alginic acid derivative that can be hydrogelized by chemical cross-linking” is represented by, for example, the solution of the alginic acid derivative represented by the above-mentioned formula (HA-I) and the above-mentioned formula (HA-II).
  • HA-I the solution of the alginic acid derivative represented by the above-mentioned formula
  • HA-II the solution of the alginic acid derivative represented by the above-mentioned formula
  • HB-I the above-mentioned formula
  • HB-II above-mentioned formula
  • There are two types of solutions of alginic acid derivatives In this case, in step (b), these two solutions and the solution in which cells or tissues are mixed with them are prepared separately without mixing. At this time, the cells or tissues may be miscible with only one of the two solutions, or may be miscible with both.
  • step (c) the solution of the alginic acid derivative obtained in step (b) is brought into contact with a solution containing divalent metal ions to have a thickness of 0.1 to 5 mm (100 to 5000 ⁇ m) or a thickness of 100 ⁇ m.
  • a solution containing divalent metal ions to have a thickness of 0.1 to 5 mm (100 to 5000 ⁇ m) or a thickness of 100 ⁇ m.
  • the solution of the alginic acid derivative obtained in the step (b) in which the cells or tissues (for example, pancreatic islands) are suspended is gelled.
  • the solution of the alginic acid derivative of the formula (HA-I) or the formula (HB-I) and the solution of the alginic acid derivative represented by the formula (HA-II) or the formula (HB-II) are subjected to the respective chemistry.
  • each dose may be appropriately mixed.
  • ionic cross-linking proceeds and chemical cross-linking also proceeds, so that a gel can be prepared. More specifically, the gel can be produced in the same manner as in [Production of flat plate-type alginate gel] ⁇ general preparation method> described in Example 5 described later.
  • step (d): as an optional step of coating the gel obtained in step (c) with a semipermeable membrane means that step (d) is optional.
  • the gel obtained in the step (c) is coated with a semipermeable membrane by a method known in the art or a method similar thereto.
  • the gel is inserted into a semipermeable membrane (eg, a semipermeable membrane tube with one end sealed) and coated by sealing the other end.
  • step (c): the step of encapsulating the solution of the alginic acid derivative obtained in step (b) in a semipermeable membrane is obtained in step (b) in which cells or tissues (for example, pancreatic islands) are suspended.
  • the solution of the alginic acid derivative obtained is coated with a semipermeable membrane by a method known in the art or a method similar thereto.
  • the solution of the alginic acid derivative of the formula (HA-I) or the formula (HB-I) and the solution of the alginic acid derivative represented by the formula (HA-II) or the formula (HB-II) are subjected to the respective chemistry.
  • each dose may be appropriately mixed.
  • the mixed solution in which the cells or tissues (eg, islets) are suspended is then inserted into a semipermeable membrane (eg, a semipermeable membrane tube with one end sealed) and coated by sealing the other end. ..
  • a semipermeable membrane eg, a semipermeable membrane tube with one end sealed
  • the step (d) the step of contacting the semipermeable membrane obtained in the step (c) with a solution containing divalent metal ions to gel the alginic acid solution in the semipermeable membrane
  • the step ( The semipermeable membrane containing the alginic acid solution obtained in c) is brought into contact with a solution containing divalent metal ions to gel the alginic acid solution in the semipermeable membrane.
  • the device obtained in step (d) may be washed with a solvent such as physiological saline. Further, it may be cultured in a medium for a predetermined period of time.
  • Step (e): The solution of the alginic acid derivative obtained in the step (b) is optional.
  • a solution of the alginic acid derivative obtained in step (b) in which cells or tissues (for example, pancreatic islands) are suspended is used as a method known in the art.
  • it is kept in a certain shape using an arbitrary container, surface, etc., and gelled by the progress of chemical cross-linking.
  • the arbitrary container refers to a container capable of holding the solution in a constant shape, and examples thereof include a semipermeable membrane, a beaker, or a petri dish, and a semipermeable membrane is preferable.
  • the arbitrary surface refers to a surface on which the solution can be placed in a fixed shape, and examples thereof include a semipermeable membrane surface, a plastic surface, or a glass surface, and a semipermeable membrane surface is preferable.
  • a solution of an alginic acid derivative to prepare a gel having a thickness of 0.1 to 5 mm (100 to 5000 ⁇ m) the solution held in an arbitrary container, surface or semipermeable membrane is chemically cross-linked. It means to gel.
  • the gel may be prepared by simultaneously advancing ionic cross-linking and chemical cross-linking by contacting with a solution containing divalent metal ions, and advancing only chemical cross-linking without contacting with divalent metal ions or the solution thereof. , You may make a gel.
  • the solution containing "divalent metal ion" used in the transplantation device examples include a solution containing calcium ion, barium ion, strontium ion and the like.
  • a solution containing calcium ions or barium ions is preferable, and a solution containing calcium ions is more preferable.
  • the solution containing the divalent metal ion can be obtained, for example, by dissolving a salt of the divalent metal ion in a solvent.
  • the salt of the divalent metal ion include calcium chloride, barium chloride, strontium chloride and the like.
  • the solvent include water, physiological saline, and HEPES buffer.
  • the solution containing divalent metal ions is a solution containing calcium ions, preferably an aqueous solution containing calcium chloride. It is desirable to appropriately adjust the amount of the solution containing the divalent metal ion according to the amount of the alginic acid derivative used, the molecular weight and the like.
  • the hydrogel in the device can be prepared by encapsulating the solution of the alginic acid derivative in the semipermeable membrane and then contacting it with a divalent metal ion solution. Either it may be gelled before being encapsulated in the membrane and then encapsulated in the semipermeable membrane in the latter half.
  • contact means immersing a semipermeable membrane containing a solution of an alginic acid derivative in a divalent metal ion solution, applying a divalent metal ion solution to the semipermeable membrane containing a solution of an alginic acid derivative, and the like. Can be mentioned.
  • Hydrogel used for transplantation devices refers to a polymer having a three-dimensional network structure that is insoluble in water and a swollen body due to the water.
  • hydrogel may be simply referred to as gel.
  • the molecular weight of the molecule that can pass through the network structure of this gel can be changed greatly and freely. That is, it is conceivable that the mesh structure of the gel has a small mesh when the concentration of the polymer is high, and a large mesh when the concentration of the polymer is low. If the mesh of the network structure is too large, antibodies and the like will invade the network structure. In this case, rejection of insulin-secreting cells or islets in the gel is likely to occur. Rejection inhibits the production of necessary substances such as insulin.
  • the material of hydrogel consists of the following polymers.
  • collagen hyaluronan, gelatin, fibronectin, elastin, tenacin, laminin, bitronectin, polypeptide, heparan sulfate, chondroitin, chondroitin sulfate, keratane, keratane sulfate, dermatin sulfate, carrageenan, heparin, chitin, chitosan, alginate, alginate derivative.
  • an alginic acid derivative is preferable from the viewpoint of biocompatibility, long-term engraftment of pancreatic islets, maintenance of function, and the like.
  • alginic acid in the present specification, when referring to alginic acid, at least one alginic acid (sometimes referred to as "alginic acids") selected from the group consisting of alginic acid, alginic acid esters, and salts thereof (for example, sodium alginate) is referred to as alginic acid.
  • alginic acid used may be of natural or synthetic origin, but is preferably of natural origin.
  • Preferred alginic acids are bioabsorbable polysaccharides extracted from brown algae such as Lessonia, Macrocystis, Laminaria, Ascophyllum, Derbilia, Ecklonia cava, Arame, Combu and D-mannuronic acid (M).
  • It is a polymer in which two types of uronic acid, L-gluuronic acid (G), are linearly polymerized. More specifically, the homopolymer fraction of D-mannuronic acid (MM fraction), the homopolymer fraction of L-gluuronic acid (GG fraction), and the random arrangement of D-mannuronic acid and L-gluuronic acid. It is a block copolymer in which the resulting fractions (M / G fractions) are arbitrarily bonded.
  • alginic acid may be referred to as (ALG) -COOH, with alginic acid as (ALG) and one of any carboxyl groups of alginic acid as -COOH.
  • the alginate is sodium alginate.
  • the sodium alginate commercially available sodium alginate can be used.
  • the sodium alginate is the sodium alginate of A-1, A-2, A-3, B-1, B-2, and B-3 described in the table below (publisher: Mochida Pharmaceutical Co., Ltd.). Co., Ltd.) is used.
  • the viscosity, weight average molecular weight and M / G ratio of each 1 w / w% aqueous solution of sodium alginate are shown in the table below.
  • the physical property values of the sodium alginate A-1, A-2, A-3, B-1, B-2, and B-3 were measured by the following various methods.
  • the measuring method is not limited to the method, but each physical property value may differ from the above depending on the measuring method.
  • Da (Dalton) may be added as a unit in the molecular weights of alginic acid, alginic acid derivatives, crosslinked alginic acid, and crosslinked alginic acid.
  • the composition ratio (M / G ratio) of D-mannuronic acid and L-gluuronic acid of alginic acids differs mainly depending on the type of organism from which seaweeds are derived, and is also affected by the habitat and season of the organism. , 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 gelling ability of alginic acids and the properties of the produced gel are affected by the M / G ratio, and generally, the gel strength increases when the G ratio is high.
  • the M / G ratio also affects the hardness, brittleness, water absorption, flexibility, etc. 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. be.
  • the numerical range indicated by using “-” indicates a range including the numerical values before and after "-" as the minimum value and the maximum value, respectively.
  • alginic acid ester and “alginate” used in the present specification are not particularly limited, but in order to react with a cross-linking agent, it is necessary that they do not have a functional group that inhibits the cross-linking reaction.
  • Preferred examples of the alginic acid ester include propylene glycol alginate.
  • examples of alginate include a monovalent salt of alginic acid and a divalent salt of alginic acid.
  • the monovalent salt of alginic acid is preferably sodium alginate, potassium alginate, ammonium alginate, etc., more preferably sodium alginate or potassium alginate, and particularly preferably sodium alginate.
  • Preferred examples of the divalent salt of alginic acid include calcium alginate, magnesium alginate, barium alginate, strontium alginate, and the like.
  • Alginic acid is a high molecular weight polysaccharide and it is difficult to accurately determine its molecular weight, but it generally has a weight average molecular weight of 10 to 10 million, preferably 10,000 to 8 million, and more preferably 20,000 to 3 million. Is the range of. It is known that in the measurement of the molecular weight of a polymer substance derived from a natural product, the value may differ depending on the measurement method.
  • the weight average molecular weight measured by gel permeation chromatography (GPC) or gel filtration chromatography (collectively referred to as size exclusion chromatography) is preferably 100,000 or more, more preferably 500,000 or more, and also. It is preferably 5 million or less, more preferably 3 million or less. The preferred range is 100,000 to 5 million, more preferably 150,000 to 3 million.
  • the absolute weight average molecular weight can be measured.
  • the weight average molecular weight (absolute molecular weight) measured by the 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 million or less, more preferably 80. It is 10,000 or less, more preferably 700,000 or less, and particularly preferably 500,000 or less.
  • the preferred 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 fluctuate in the range of 320,000 to 480,000 for 400,000, 400,000 to 600,000 for 500,000, and 800,000 to 1.2 million for 1 million.
  • 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 Examples of the present specification described later.
  • the column for example, Superose6 Increase10 / 300 GL column (GE Healthcare Science Co., Ltd.) can be used, and as a developing solvent, for example, a 10 mmol / L phosphate buffer solution (pH 7.4) containing 0.15 mol / L NaCl.
  • bluedextran, tyroglobulin, ferritin, aldolase, conalbumin, obalbumin, ribonuclease A and aprotinin can be used as molecular weight standards.
  • the viscosity of alginic acid used in the present specification is not particularly limited, but when the viscosity is measured as an aqueous solution of 1 w / w% alginic acid, it is preferably 10 mPa ⁇ s to 1000 mPa ⁇ s, more preferably 50 mPa ⁇ s. It is s to 800 mPa ⁇ s.
  • the viscosity of the aqueous solution of alginic acid can be measured according to a conventional method.
  • a co-axis double-cylindrical rotational viscometer, a single cylindrical rotational viscometer (Brookfield type viscometer), a cone-plate type rotational viscometer (cone plate type viscometer), etc. of the rotational viscometer method are used. Can be measured. It is preferable to follow the viscosity measurement method of the Japanese Pharmacopoeia (16th edition). More preferably, a cone plate type viscometer is used.
  • Alginic acids initially have a large molecular weight and high viscosity when extracted from brown algae, but in the process of drying and refining by heat, the molecular weight becomes small and the viscosity becomes low.
  • Alginic acids having different molecular weights can be produced by methods such as controlling conditions such as temperature in the production process, selecting brown algae as raw materials, and fractionating the molecular weight in the production process. Further, it is also possible to obtain alginic acid having a desired molecular weight by mixing with another lot of alginic acid having a different molecular weight or viscosity.
  • the alginic acid used herein is alginic acid that has not been treated with low endotoxin in some embodiments, or alginic acid that has been treated with low endotoxin in some other embodiments.
  • Low endotoxin means that the endotoxin level is low enough not to cause inflammation or fever. More preferably, it is alginic acid treated with low endotoxin.
  • the low endotoxin treatment can be performed by a known method or a method similar thereto.
  • the method of Suga et al. For purifying sodium hyaluronate see, for example, JP-A-9-324001
  • the method of Yoshida et al. For purifying ⁇ 1,3-glucan (eg, JP-A-8-269102).
  • William et al.'S method for purifying biopolymer salts such as alginate and gellan gum (see, eg, JP-A-2002-530440)
  • James et al.'S method for purifying polysaccharides eg, international publication.
  • the method of Lewis et al. See, eg, US Pat. No.
  • Low endotoxin treatment is not limited to these, but for cleaning, filtration with filters (endotoxin removal filter, charged filter, etc.), ultrafiltration, columns (endotoxin adsorption affinity column, gel filtration column, column with ion exchange resin, etc.).
  • the endotoxin level can be confirmed by a known method, and can be measured by, for example, a method using Limulus reagent (LAL), a method using Endospecy (registered trademark) ES-24S set (Seikagaku Corporation), or the like. ..
  • LAL Limulus reagent
  • Endospecy registered trademark
  • ES-24S set Seikagaku Corporation
  • the method for treating endotoxin used is not particularly limited, but as a result, the endotoxin content of alginic acids should be 500 endotoxin units (EU) / g or less when the endotoxin is measured with the Limulus reagent (LAL). Is more preferable, and more preferably 100 EU / g or less, particularly preferably 50 EU / g or less, and particularly preferably 30 EU / g or less.
  • Sodium alginate treated with low endotoxin can be obtained from commercial products such as Sea Matrix (registered trademark) (Mochida Pharmaceutical Co., Ltd.) and PRONOVA TM UP LVG (FMCBioPolymer).
  • alginic Acid Derivatives Provided herein are novel alginic acid derivatives.
  • the alginic acid derivative is a reactive group in the Huisgen reaction or a reactive group complementary to the reactive group via an amide bond and a divalent linker to any one or more carboxyl groups of the alginic acid.
  • the following formula (HA-I) or formula (HB-I): Alginic acid represented by [the definitions of (ALG), -L1- , and Akn in the formula (HA-I) or the formula (HB-I) are the same as the definitions in the first aspect described above, respectively].
  • a plurality of (for example, 1 to 10 or 1 to 5) may be substituted with the selected group.
  • novel alginic acid derivative in the present specification is the formula (HA-I) or the formula (HB-I), and the alginic acid derivative represented by the formula (HA-II) or the formula (HB-II) is, for example, the following formula. It is possible to manufacture by the method of.
  • the weight average molecular weight of the alginic acid derivative represented by the formula (HA-I), the formula (HA-II), the formula (HB-I) or the formula (HB-II) in the present specification is 100,000 Da to 3 million Da. Yes, preferably 300,000 Da to 2.5 million Da, and more preferably 500,000 Da to 2 million Da.
  • the molecular weights of both alginic acid derivatives can be determined by the method described later.
  • the Akn-L1 - NH-group of the formula (HA-I) or the formula (HB-I) does not have to be bonded to all the carboxyl groups of the alginic acid constituent unit, and the formula ( The N3-L2-NH - group of HA-II) or formula (HB- II ) need not be attached to all carboxyl groups of the alginic acid building block.
  • the Akn-L1 - NH-group of the formula (HA-I) or the formula (HB-I) is referred to as a reactive group
  • the N of the formula (HA-II) or the formula (HB-II) is used.
  • the 3 -L2 - NH-group is a complementary reactive group.
  • the Akn of the formula (HA-I) or the formula (HB-I) is referred to as a reactive group
  • the -L1 -NH-group is a complementary reactive group.
  • the introduction rate of the reactive group or the complementary reactive group is 0.1% to 30% or 1% to 30%, preferably 2% to 20%, respectively, more preferably. Is 3% to 10%.
  • the introduction rate of the reactive group or the complementary reactive group is expressed as a percentage of the number of uronic acid monosaccharide units into which each reactive group has been introduced among the uronic acid monosaccharide units which are repeating units of alginic acids. It is the value that was set.
  • Reactive groups or complementary reactions in alginic acid derivatives (formula (HA-I), formula (HA-II), formula (HB-I) or formula (HB-II)) herein, unless otherwise noted.
  • The% used for the introduction rate of the sex group means mol%.
  • the introduction rate of each reactive group or complementary reactive group can be determined by the method described in Examples described later.
  • the cyclic alkyne group (Akn) in the formula (HA-I) or the formula (HB-I) and the azide group in the formula (HA-II) or the formula (HB-II) are triazoled by the Huisgen reaction. It forms a ring, which forms a crosslink.
  • the Huisgen reaction (1,3-dipolar addition cyclization reaction) is a condensation reaction between compounds having a terminal azide group and a terminal alkyne group as shown in the following formula.
  • a disubstituted 1,2,3-triazole ring is obtained in good yield, and it has a feature that no extra by-product is generated. It is considered that a 1,4- or 1,5-disubstituted triazole ring can be formed in the reaction, but a regioselective triazole ring can be obtained by using a copper catalyst.
  • the Huisgen reaction is an azide compound having substituted primary azide, secondary azide, tertiary azide, aromatic azide, etc., and a terminal or cyclic alkyne which is a complementary reactive group of the azide group.
  • a compound having a group can be used.
  • various functional groups for example, ester group, carboxyl group, alkenyl group, hydroxyl group, amino group, etc. should be substituted in the reaction substrate. Is possible.
  • 1,2,3-triazoles are short-term, easily, and efficiently without the use of copper catalysts to produce unwanted by-products and avoid copper-catalyzed cytotoxicity.
  • the cyclic alkyne group (cyclooctyl group) described in the first aspect described above is used as the alkyne group of the Huisgen reaction.
  • Cross-linked alginic acid is mediated by (i) a divalent metal ionic bond, (ii) a chemical bond, or (iii) a divalent metal ionic bond and a chemical bond. There is something. Any cross-linked alginic acid has the property of forming a gel-like to semi-solid, and in some cases, sponge-like morphology.
  • Cross-linked alginic acid via a divalent metal ion bond proceeds at an ultrafast speed and is reversible, whereas cross-linked alginic acid via a chemical bond proceeds slowly under relatively mild conditions. And it is irreversible.
  • the physical properties of the crosslinked alginic acid are adjusted by, for example, changing the concentration of the aqueous solution containing the divalent metal ion to be used (for example, the calcium chloride aqueous solution) or the introduction rate of the reactive group introduced into the alginic acid. Is possible.
  • alginic acid structures can be produced.
  • an ionic cross-linking reaction a specific structure can be instantly formed from an alginic acid solution, and a cross-linking reaction by a chemical bond is used to strengthen the structure of the structure (for example, to obtain long-term stability, etc.). It is possible to do.
  • a crosslinked alginic acid structure via both a divalent metal ionic bond and a chemical bond the divalent metal ion incorporated by the ionic bonding was reversibly released, and only the bridging by the chemical bond remained. It is also possible to create a structure.
  • the crosslinked alginic acid structure using the alginic acid derivative in the preferred embodiment has stability because it contains crosslinks by chemical bonds, and has a longer shape than the crosslinked alginic acid structure having only ion crosslinks using sodium alginate. It can be maintained for a period of time, which is advantageous.
  • the crosslinked alginic acid of a certain aspect can be obtained by mixing the alginic acid derivatives of the above formula (HA-I) and the above formula (HA-II) and carrying out the Huisgen reaction. Further, the crosslinked alginic acid of a certain aspect can be obtained by mixing the alginic acid derivatives of the above formula (HB-I) and the above formula (HB-II) and carrying out a Huisgen reaction.
  • Alginic acid forms a three-dimensional network structure via chemical cross-linking (cross-linking with a triazole ring formed from an alkyne group and an azido group).
  • the preferred alginic acid derivative is one in which the stability of the crosslinked alginic acid after cross-linking is improved.
  • the crosslinked alginic acid has the following formula (HA-III-L) or formula (HB-III-L) between any carboxyl group of the first alginic acid and any carboxyl group of the second alginic acid.
  • formula (HA-III-L) or formula (HB-III-L) -CONH- and -NHCO- at both ends represent amide bonds mediated by any carboxyl group of alginic acid; -L1- , -L 2- and X are the same as the definitions in the first aspect, respectively] to be crosslinked alginic acid amide-bonded.
  • the arginic acid derivative of the formula (HA-I) or the formula (HB-I) and the arginic acid derivative of the formula (HA-II) or the formula (HB-II) in preparing the crosslinked arginic acid is the weight ratio of the derivative of the formula (HA-I) or the formula (HB-I) to the derivative of the formula (HA-II) or the formula (HB-II), for example, 1 to 1.5: 1. , Preferably 1.2 to 1.5: 1, or 1 to 1.2: 1, more preferably 1: 1.
  • the arginic acid derivative of the formula (HA-II) or the formula (HB-II) and the arginic acid derivative of the formula (HA-I) or the formula (HB-I) in preparing the crosslinked arginic acid is the weight ratio of the derivative of the formula (HA-II) or the formula (HB-II) to the derivative of the formula (HA-I) or the formula (HB-I), for example, 1 to 4.0: 1. , Preferably 1.5 to 4.0: 1, or 1.2 to 1.5: 1, or 1 to 1.2: 1, more preferably 1: 1.
  • the arginic acid derivative of the formula (HA-I) or the formula (HB-I) and the arginic acid derivative of the formula (HA-II) or the formula (HB-II) in preparing the crosslinked arginic acid is more preferably the introduction rate (mol%) of the reactive group of the alginic acid derivative of the formula (HA-I) or the formula (HB-I) and the alginic acid derivative of the formula (HA-II) or the formula (HB-II).
  • Ratio for example, 1 to 1.5: 1, preferably 1.2 to 1.5: 1, or 1 to 1.2: 1, more preferably 1: 1.
  • the arginic acid derivative of the formula (HA-II) or the formula (HB-II) and the arginic acid derivative of the formula (HA-I) or the formula (HB-I) in preparing the crosslinked arginic acid is more preferably the introduction rate (mol%) of the reactive group of the alginic acid derivative of the formula (HA-II) or the formula (HB-II) and the alginic acid derivative of the formula (HA-I) or the formula (HB-I).
  • Ratio for example, 1 to 4.0: 1, preferably 1.5 to 4.0: 1, or 1.2 to 1.5: 1, or 1 to 1.2: 1, more preferably. It is 1: 1.
  • the alginic acid derivative of the formula (HA-I) or the formula (HB-I) is converted into the alginic acid derivative of the formula (HA-II) or the formula (HB-II) into the formula (HA-II) or the formula (HA-II). It is also possible to replace the alginic acid derivative of (HB-II) with the derivative of the formula (HA-I) or the formula (HB-I), respectively.
  • Cross-linked alginic acid does not need to have all the carboxyl groups of the constituent units of alginic acid having the above-mentioned formula (HA-III-L) or formula (HB-III-L) cross-linking.
  • the introduction rate (also referred to as the crosslinking rate) of the crosslinking represented by the above formula (HA-III-L) or the above formula (HB-III-L) is, for example, 0.1 to 80%, 0.3 to 0.3 to It ranges from 60%, 0.5 to 30%, or 1.0 to 10%.
  • the concentration of the alginic acid derivative of the formula (HA-I), the formula (HA-II), the formula (HB-I) or the formula (HB-II) in the Huisgen reaction for obtaining the crosslinked alginic acid is usually 1 to 500 mg / mL. Yes, preferably in the range of 5-100 mg / mL.
  • the reaction temperature of the Huisgen reaction is usually an outside temperature of 4 to 60 ° C, preferably an outside temperature of 15 to 40 ° C.
  • the stirring time for forming the crosslinked alginic acid (hydrogel) is, for example, several seconds to 24 hours, several seconds to 12 hours, several seconds to 30 minutes, or several seconds to 10 minutes.
  • the reaction solvent or reaction solution used for the Huisgen reaction is not particularly limited, but is, for example, tap water, pure water (for example, distilled water, ion-exchanged water, RO water, RO-EDI water, etc.), ultrapure water, cells.
  • pure water for example, distilled water, ion-exchanged water, RO water, RO-EDI water, etc.
  • ultrapure water examples thereof include a culture medium, phosphate buffered physiological saline (PBS), physiological saline, HEPES buffer, and the like, and ultrapure water is preferable.
  • the cross-linked alginic acid of some embodiments is a cross-linked alginic acid comprising a chemical cross-linking by a triazole ring formed by the Huisgen reaction as a cross-linking and an ionic cross-linking partially formed by calcium ions.
  • cross-linked alginic acid structure can be obtained by a method including subjecting the alginic acid derivative to a cross-linking reaction.
  • it can be prepared by the following method, but is not limited to these.
  • a mixed solution of an alginic acid derivative of the formula (HA-I) or the formula (HB-I) and an alginic acid derivative of the formula (HA-II) or the formula (HB-II) obtained by mixing the alginic acid derivative is a divalent metal ion.
  • chemical cross-linking cross-linking by a triazole ring formed from an alkin group and an azido group by the Huisgen reaction
  • ionic cross-linking cross-linking partially formed by divalent metal ions
  • a crosslinked alginic acid structure which is a specific structure, can be obtained.
  • a specific structure partially crosslinked can be obtained by dropping a solution containing an alginic acid derivative of the formula (HA-I) or the formula (HB-I) into a solution containing a divalent metal ion.
  • a structure such as a gel obtained above to a solution containing an alginic acid derivative of the above formula (HA-II) or formula (HB-II)
  • further crosslinking is performed on the surface of the structure or the like.
  • Huisgen reaction By carrying out the reaction (Huisgen reaction), a crosslinked alginic acid structure can be obtained.
  • the alginic acid derivative of the formula (HA-I) or the formula (HB-I) is converted into the alginic acid derivative of the formula (HA-II) or the formula (HB-II) into the formula (HA-II) or the formula (HA-II). It is also possible to replace the alginic acid derivative of HB-II) with the alginic acid derivative of the formula (HA-I) or the formula (HB-I), respectively.
  • the divalent metal ion used in the above method is not particularly limited, and examples thereof include calcium ion, magnesium ion, barium ion, strontium ion, zinc ion and the like, and calcium ion is preferable.
  • the solution containing calcium ions used in the above method is not particularly limited, and examples thereof include aqueous solutions such as an aqueous solution of calcium chloride, an aqueous solution of calcium carbonate, and an aqueous solution of calcium gluconate, and an aqueous solution of calcium chloride is preferable.
  • the calcium ion concentration of the solution containing calcium ions used in the above method is not particularly limited, but examples thereof include 1 mM to 1 M, preferably 5 mM to 500 mM, and more preferably 10 mM to 300 mM.
  • the solvent or solution used in the above method is also not particularly limited, but is, for example, tap water, pure water (for example, distilled water, ion-exchanged water, RO water, RO-EDI water, etc.), ultrapure water, cell culture medium. , Phosphoric acid buffered physiological saline (PBS), physiological saline, HEPES buffer and the like, preferably ultrapure water.
  • tap water pure water (for example, distilled water, ion-exchanged water, RO water, RO-EDI water, etc.), ultrapure water, cell culture medium.
  • pure water for example, distilled water, ion-exchanged water, RO water, RO-EDI water, etc.
  • ultrapure water cell culture medium.
  • PBS Phosphoric acid buffered physiological saline
  • HEPES buffer physiological saline
  • ultrapure water preferably ultrapure water.
  • cross-linked alginic acid structures include, for example, fibrous structures, fibers, beads, gels, substantially spherical gels, and the like.
  • the preferred cross-linked alginic acid structure is one with improved stability.
  • the crosslinked alginic acid structure may have an ability to hold the contents inside the crosslinked alginic acid structure (content holding property).
  • the physical characteristics of the alginate gel can be adjusted by the physical characteristics such as hardness, elasticity, repulsive force, tearing force, and stress at break.
  • biocompatibility of alginic acid derivative or cross-linked alginic acid structure has biocompatibility.
  • biocompatibility refers to biocompatibility produced using biomaterials (here, for example, arginic acid derivatives represented by the formulas (HA-I) and (HA-II), and both arginic acid derivatives. It has biocompatibility with the property of not causing a reaction such as an interaction between a cross-linked alginic acid structure) and a living body, a local reaction of a tissue adjacent to the biological material, or a systemic reaction. That is.
  • the biocompatibility of the alginic acid derivative or the crosslinked alginic acid structure will be confirmed in the examples of biocompatibility described later.
  • Stability of the crosslinked alginic acid structure The stability of the crosslinked alginic acid structure can be confirmed by, for example, measuring the gel stability, and the permeability can be confirmed by measuring the gel transmittance.
  • Phosphate buffered saline PBS
  • concentration of alginic acid leaked into the PBS ⁇ g / mL
  • 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 as a percentage is defined as the disintegration rate.
  • 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%, still more preferably 0% to 50%.
  • the stability of the crosslinked alginic acid structure means that the lower the concentration of alginic acid leaked into the aqueous solution, that is, the lower the gel disintegration rate, the higher the stability.
  • a cross-linked alginic acid structure gel containing fluorescein isothiocyanate-dextran is prepared, physiological saline is added to the gel in a container, and the concentration of dextran leaked into the physiological saline is measured.
  • the gel permeability is the value obtained by dividing the measured dextran concentration by the total dextran concentration obtained by decomposing the fluorescein isothiocyanate-dextran-encapsulating cross-linked arginic acid structure gel.
  • the gel transmittance can be determined by the method described in Examples described later.
  • the gel permeability of the crosslinked alginic acid 24 hours after the addition of the physiological saline solution is preferably 0% to 90%, more preferably 0% to 70%, and further preferably 0% to 70% when containing dextran having a molecular weight of 2 million, for example. It is preferably 0% to 50%. Further, when dextran having a molecular weight of 150,000 is included, for example, if the purpose of use of the crosslinked alginic acid structure gel is to release / produce a protein or an antibody, it is preferably 1% to 100%, more preferably 10. % To 100%, more preferably 30% to 100%. If the purpose of use is an immune septum, it is preferably 0% to 90%, more preferably 0% to 70%, and even more preferably 0% to 50%.
  • the permeability of the crosslinked alginic acid structure means that the lower the transmittance, the lower the permeability of the content and the extragel substance, and the higher the transmittance, the higher the permeability of the content and the extragel substance. means.
  • the transmittance of the gel can be adjusted by the molecular weight and concentration of alginic acid used, the type and introduction rate of cross-linking groups to be introduced into alginic acid, the type and concentration of divalent metal ions used for gelation, or a combination thereof. be.
  • a crosslinked alginic acid structure gel containing fluorescein isothiocyanate-dextran as a content can be prepared by the following method.
  • alginic Acid Derivative Synthesis Method The alginic acid derivative represented by the formula (HA-I) or the formula (HA-II) can be produced by referring to PCT / JP2019 / 023478 (filed on June 13, 2019).
  • Alginic Acid derivatives can be used for producing devices for transplantation as described above.
  • alginic acid derivatives can be used in place of traditional alginic acid in a wide range of fields such as food, medicine, cosmetics, textiles and papermaking.
  • Preferred uses of the alginic acid derivative or the crosslinked alginic acid structure are specifically for medical use such as a wound dressing, a postoperative adhesion preventive material, a substrate for sustained release of a drug, a substrate for cell culture, and a substrate for cell transplantation. Materials are mentioned.
  • Examples of the shape of the crosslinked alginic acid structure when used as a medical material include tubular, fibrous, fiber, beads, gel, and substantially spherical gel, and beads, gel, and substantially spherical gel are preferable. It is more preferable to use a substantially spherical gel.
  • a particularly preferred embodiment of the implant device using an alginic acid derivative is excellent in biocompatibility and stability, has low cytotoxicity, has little adhesion or inflammation at the transplant site, and dissolves the gel (with or without a semipermeable membrane). It is possible to maintain the shape for a long period of time, maintain the hypoglycemic effect for a long period of time, and regulate blood glucose.
  • the cure rate in the present invention means that a hydrogel containing insulin-secreting cells or pancreatic islets or a transplantation device containing the hydrogel is transplanted into a diabetic patient or a diabetic model animal, and a predetermined period of time has elapsed after the transplantation. In, it is expressed by the ratio of the number of treated cases to the number of transplanted cases.
  • a hydrogel in which insulin-secreting cells or pancreatic islets are encapsulated or a transplantation device containing the hydrogel is transplanted into a plurality of diabetes model mice obtained by the method of Example 7, and during a predetermined period after transplantation.
  • the case where the blood glucose level is 300 mg / dL or less is defined as a cured mouse, and is represented by the ratio of the cured mouse to the diabetes model mouse.
  • the cure rate is, for example, 20% or more, preferably 35% or more, and more preferably 50% or more two weeks after transplantation. Alternatively, it is 20% or more, preferably 35% or more, more preferably 50% or more one month after transplantation.
  • the oxygen permeability in the present invention is expressed as the ratio of the central oxygen concentration when the surface oxygen concentration of the hydrogel containing insulin-secreting cells or pancreatic islets or the transplantation device containing the hydrogel is 100%. Will be done.
  • the central portion means a substantially central portion in any of the vertical direction, the horizontal direction, and the thickness direction.
  • the oxygen permeability may be calculated by measuring the hydrogel or the device, or may be calculated by calculation. When calculating by measurement, for example, a needle type oxygen meter (OXY-1 ST trace) manufactured by PreSens can be used, and the calculation can be performed from the measured values of the surface oxygen concentration and the central oxygen concentration.
  • the oxygen permeability does not have to be 0%, for example, 1 to 100%, preferably 10 to 100%, more preferably 25 to 100%, still more preferably 50 to 100%, and particularly preferably 75 to 100%. be. Further, it is preferably 25 to 50%, more preferably 50 to 75%, and particularly preferably 75 to 100%.
  • the substance permeability in the present invention means a hydrogel in which insulin-secreting cells or pancreatic islets are encapsulated, or a transplant device containing the hydrogel in which a predetermined substance is encapsulated and left in a stirred solution for a predetermined time. It means the material permeability from the hydrogel or the device for transplantation.
  • the substance include glucose, insulin and the like.
  • insulin permeability or glucose permeability when 500 mg of human insulin or 250 mg of glucose is encapsulated in a hydrogel or a device for transplantation and stirred in 40 mL of 0.01 Tween 20-containing physiological saline at room temperature for 24 hours can be mentioned.
  • the insulin permeability is 50% or more, preferably 70% or more, and more preferably 80% or more.
  • the glucose permeability is 50% or more, preferably 70% or more, and more preferably 80% or more.
  • the hydrogel of the present invention has little or no breakage, decomposition or dissolution even when shaken under predetermined conditions.
  • a shaking incubator for example, medium-sized constant temperature shaking.
  • a tofu incubator Tetec Co., Ltd., Bio-Shaker (registered trademark) BR-43FL / MR
  • the mixture was shaken under the conditions of a reciprocating shaking method with an amplitude of 25 mm and a shaking speed of 180 rpm while maintaining the temperature at 37 ° C. Occasionally, there is little or no breakage, decomposition or dissolution.
  • Low decomposition or dissolution of hydrogel means that, for example, when alginic acid in the hydrogel is 100% and the proportion of alginic acid eluted in the solution when shaken under the above conditions is the disintegration rate, the disintegration rate is 40. % Or less, preferably 20% or less, more preferably 10% or less.
  • the disintegration rate is 30% or less, preferably 20% or less, more preferably 10% or less.
  • the disintegration rate is 30% or less, preferably 20% or less, more preferably 10% or less.
  • the cells in the hydrogel do not fall off or are less likely to fall off even when shaken under predetermined conditions.
  • the number of cells shedding from the hydrogel is small is, for example, when the number of cells in the hydrogel at the start of the test is 100% and the percentage of cells shedding from the hydrogel when shaken under the above conditions is defined as the shedding rate.
  • the dropout rate is 30% or less, preferably 20% or less, and more preferably 10% or less. In particular, it refers to a case where the dropout rate when shaken for 24 hours under the above conditions is 30% or less, preferably 20% or less, and more preferably 10% or less.
  • JEOL JNM-ECX400 FT-NMR (JEOL Ltd.) was used for the measurement of the nuclear magnetic resonance spectrum (NMR).
  • Liquid chromatography-mass spectrometry spectrum (LC-Mass) was measured by the following method.
  • [UPLC] Using a Waters AQUITY UPLC system and a BEH C18 column (2.1 mm x 50 mm, 1.7 ⁇ m) (Waters), acetonitrile: 0.05% trifluoroacetic acid aqueous solution 5:95 (0 minutes) to 95: 5 Mobile phase and gradient conditions from (1.0 min) to 95: 5 (1.6 min) to 5:95 (2.0 min) were used.
  • the pattern of the NMR signal is s for singlet, d for doublet, t for triplet, q for quartet, m for multiplet, br for broad, J for coupling constant, Hz for hertz, CDCl 3 .
  • Means deuterated chloroform, DMSO - D 6 means deuterated dimethyl sulfoxide, and D2 O means heavy water.
  • signals that cannot be confirmed because they are broadband, such as hydroxyl group (OH), amino group (NH 2 ), and carboxyl group (COOH) protons, are not described in the data.
  • M means molecular weight
  • RT means retention time
  • [M + H] + and [M + Na] + mean molecular ion peaks.
  • the "room temperature” in the examples usually indicates a temperature of about 0 ° C to about 35 ° C.
  • the reactive substituent introduction rate (mol%) in the examples was introduced with respect to the number of moles of monosaccharides (gluronic acid and mannuronic acid) constituting alginic acid calculated from 1 H - NMR (D2O). It shall indicate the ratio of the number of moles of the reactive substituent.
  • sodium alginate showing the physical property values shown in Table 10 was used as the sodium alginate before the reactive group or the complementary reactive group was introduced.
  • Table 12 shows the physical property values (specifically, the physical properties of the reactive group-introduced alginic acid derivatives (Example 1a, Example 2a) obtained in (Example 1) to (Example 4-2). , Reactive group introduction rate (mol%), molecular weight, and weight average molecular weight (10,000 Da)).
  • Example 1 Synthesis of dibenzocyclooctyne-amino group-introduced alginic acid (Example 1a, Example 1b, Example 1c, Example 1d, and Example 1e):
  • Example 1a Synthesis of dibenzocyclooctyne-amino group-introduced alginic acid (EX1- (I) -A-2a): 4- (4,6-dimethoxy-1,3,5-triazine-2-yl)-in an aqueous solution (43.6 mL) of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd .: A-2) prepared to 1% by weight. 4-Methylmorpholinium chloride (DMT-MM) (111.65 mg), 1 molar concentration-sodium alginate (403.5 ⁇ L) was added.
  • DMT-MM 4-Methylmorpholinium chloride
  • the introduction rate of the reactive substituent was 6.9 mol% (NMR integration ratio).
  • the introduction rate of the reactive substituent was 5.0 mol% (NMR integration ratio).
  • the introduction rate of the reactive substituent was 2.3 mol% (NMR integration ratio).
  • the introduction rate of the reactive substituent was 2.4 mol% (NMR integration ratio).
  • the introduction rate of the reactive substituent was 2.2 mol% (NMR integration ratio).
  • Example 2 Synthesis of 4- (2-aminoethoxy) -N- (3-azidopropyl) benzamide group-introduced alginic acid (Example 2a, Example 2b, Example 2c, Example 2d, and Example 2e):
  • Example 2 Lithium hydroxide monohydrate (0.25 g) was added to a methanol (4.4 mL) solution of the compound EX2-IM-1 (0.44 g) obtained in ⁇ Step 1>. , 60 ° C. for 3 hours and 30 minutes. 1N-hydrochloric acid (5 mL) was added to the reaction mixture, and the mixture was extracted 3 times with ethyl acetate (10 mL). The organic layer was washed successively with water (5 mL) and saturated brine (5 mL), dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure.
  • the introduction rate of the reactive substituent (4- (2-aminoethoxy) -N- (3-azidopropyl) benzamide group) was 6.1 mol% (NMR integration ratio).
  • the white solid was dissolved in 80 mL of water, freeze-dried, and then dried at 40 ° C. for 23 hours to obtain the title compound EX3- (II) -A-2b (1.19 g) as a white amorphous substance.
  • the white solid was dissolved in 80 mL of water, freeze-dried, and then dried at 40 ° C. for 22 hours to obtain the title compound EX2- (II) -A-2c (1.15 g) as a white amorphous substance.
  • the introduction rate of the reactive substituent (4- (2-aminoethoxy) -N- (3-azidopropyl) benzamide group) was 2.3 mol% (NMR integration ratio).
  • the introduction rate of the reactive substituent (4- (2-aminoethoxy) -N- (3-azidopropyl) benzamide group) was 2.3 mol% (NMR integration ratio).
  • the introduction rate of the reactive substituent (4- (2-aminoethoxy) -N- (3-azidopropyl) benzamide group) was 2.4 mol% (NMR integration ratio).
  • Example 3 To a 1,4-dioxane solution (3.5 mL) of the compound EX3-IM-1 (0.5 g) obtained in ⁇ Step 1> under water-cooled stirring, 4N-hydrogen chloride. / 1,4-dioxane (3.5 mL) was added, and the mixture was stirred at room temperature for 3 hours. After adding diisopropyl ether (40 mL) to the reaction solution, the precipitate was filtered to obtain the title compound EX3-IM-2 (0.36 g) as a white solid.
  • Example 3 With respect to the mixture of the compound EX3-IM-3 (0.18 g) and methanol (1.8 mL) obtained in ⁇ Step 3>, potassium carbonate (0.126) was stirred under ice-cooling. g) An aqueous solution (0.9 mL) was added dropwise, and the mixture was stirred at room temperature for 17 hours and 30 minutes. Methanol was distilled off under reduced pressure, and the mixture was extracted 3 times with ethyl acetate (5 mL). The organic layer was washed with saturated brine (5 mL) and then dried over anhydrous sodium sulfate. After filtering the organic layer, the solvent was distilled off under reduced pressure to obtain the title crude compound EX3-IM-4 (0.13 g) as a pale yellow oil.
  • the introduction rate of the reactive substituent (N- (4- (aminomethyl) benzyl) -2- (cyclooct-2-in-1-yloxy) acetamide group) was 3.7 mol% (NMR integration ratio).
  • the introduction rate of the reactive substituent (N- (4- (aminomethyl) benzyl) -2- (cyclooct-2-in-1-yloxy) acetamide group) was 2.0 mol% (NMR integration ratio).
  • tert-butyl (2-aminoethyl) carbamate [CAS: 57260-73-8] (825 mg) and pyridine (1.04 mL) It was added to a methylene chloride (7.0 mL) solution under ice-water cooling, and stirred at room temperature for 1 hour.
  • the reaction solution is diluted with tert-butyl methyl ether (30 mL), water (10 mL), saturated layered water (5 mL), 0.5N-citric acid (twice with 5 mL), water (5 mL), saturated saline. It was washed sequentially with water (5 mL).
  • Example 4 The compound (EX4-IM-1,500 mg) obtained in ⁇ Step 1> was suspended in 1,4-dioxane (1.5 mL). 4 Predetermined-hydrogen chloride / dioxane solution (3.5 mL) was added under ice-water cooling, and the mixture was stirred at room temperature for 2.5 hours. Diisopropyl ether (10.5 mL) was added to the reaction mixture, and the mixture was stirred at room temperature for 50 minutes. The solid was filtered, washed with diisopropyl ether, and dried under reduced pressure to give the title compound EX4-IM-2 (365 mg) as a light beige solid.
  • the introduction rate of the reactive group was 5.3 mol% (NMR integration ratio).
  • Example 4-2 In the compound (EX4-2-IM-1,670 mg) obtained in ⁇ Step 1>, 4 default-hydrogen chloride / 1,4-dioxane (4.7 mL) under ice-water cooling. ) was added, and the mixture was stirred at room temperature for 2 hours. Diisopropyl ether (14 mL) was added to the reaction mixture, and the mixture was stirred for 30 minutes. The obtained solid was collected by filtration, washed with diisopropyl ether, and dried under reduced pressure to give the title compound EX4-2-IM-2 (604 mg) as a light beige solid.
  • the introduction rate of the reactive group was 2.72 mol% (NMR integration ratio).
  • Reactive group or complementary reactive group introduction rate means the number of reactive groups or complementary reactive groups introduced per uronic acid monosaccharide unit, which is a repeating unit of alginic acid, expressed as a percentage. do.
  • the reactive group or complementary reactive group introduction rate (mol%) was calculated by the integral ratio of 1 H-NMR.
  • the amount of alginic acid required to calculate the introduction rate is measured by the carbazole sulfate method using a calibration curve, and the amount of the reactive group or complementary reactive group is measured by the absorbance measurement method using a calibration curve. You can also do it.
  • the molecular weight of alginic acid into which a reactive group or a complementary reactive group has been introduced is bluedextran (molecular weight 2 million Da, SIGMA), tyroglobulin (molecular weight 66,900 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), Obalbumin (molecular weight 4.4) 10,000 Da, GE Healthcare Science), Ribonuclease A (Molecular Weight 137,000 Da, GE Healthcare Science) and Aprotinin (Molecular Weight 6500 Da, GE Healthcare Science) are used as standard products and are reactive groups or complementary.
  • the molecular weight (Mi) at the elution time i of the previously obtained chromatogram was calculated. Then, the absorbance at the elution time i was read and used as Hi. From these data, the weight average molecular weight (Mw) was calculated from the following formula.
  • Alginic acid of Example 1 (a, b, c): 1.5 wt% physiological saline solution (solutions of Examples 5-1a, 5-1b, and Example 5-1c) Alginic acid of Example 2 (a, b, c): 3.0 wt% physiological saline solution (solutions of Example 5-2a, Example 5-2b, Example 5-2c) Alginic acid of Example 3 (a): 1.5 wt% physiological saline solution (solution of Example 5-3a) Alginic acid of Example 4: 3.0 wt% physiological saline solution (solution of Example 5-4)
  • a 1.0 wt% physiological saline aqueous solution is prepared and sterilized by filtration using MILLEX GV 0.22 ⁇ m (Millipore, 0.22 ⁇ m, Cat.
  • Example 1 Alginic acid of Example 1 (d): 1.0 wt% physiological saline solution (solution of Example 5-1d) Alginic acid of Example 2 (d): 1.0 wt% physiological saline solution (solution of Example 5-2d) Alginic acid of Example 3 (b): 1.0 wt% physiological saline solution (solution of Example 5-3b) Alginic acid of Example 4-2 (a): 1.0 wt% physiological saline solution (solution of Example 5-5a) Alginic acid of Example 1 (e): 0.5 wt% physiological saline solution (solution of Example 5-1e) Alginic acid of Example 2 (e): 0.5 wt% physiological saline solution (solution of Example 5-2e) Alginic acid of Example 3 (c): 0.5 wt% physiological saline solution (solution of Example 5-3b) Alginic acid of Example 4-2 (b): 0.5 wt% physiological saline solution (solution
  • the solution of Example 5-1 (a, b, c) and the solution of Example 5-2 (a, b, c) are combined, and the solution of Example 5-3a and the solution of Example 5 are combined.
  • a chemically crosslinked alginic acid gel is obtained by combining alginic acid prepared in Example 1 (d) or Example 3 (b) with alginic acid prepared in Example 2 (d) or Example 4-2 (a).
  • Example 5-1d and the solution of Example 5-2d are combined, the solution of Example 5-1d and the solution of Example 5-5a are combined, and the solution of Example 5-3b is combined.
  • the solution and the solution of Example 5-2d are combined, and the solution of Example 5-3b and the solution of Example 5-5a are combined.
  • a chemically crosslinked alginic acid gel is obtained by combining alginic acid prepared in Example 1 (e) or Example 3 (c) with alginic acid prepared in Example 2 (e) or Example 4-2 (b).
  • the solution of Example 5-1e and the solution of Example 5-2e are combined, the solution of Example 5-1e and the solution of Example 5-5b are combined, and the solution of Example 5-3c is combined.
  • the solution and the solution of Example 5-2e are combined, and the solution of Example 5-3c and the solution of Example 5-5b are combined.
  • the flat plate gel is, for example, an alginate gel having a short diameter of 12 to 15 mm, a long diameter of 12 to 18 mm, and a thickness of about 0.5 to 5 mm, and is circular, quadrangular, or hexagonal. It is also possible to take an octagon or the like, and it is not particularly limited.
  • Example 6-1 Production of flat plate-type alginate gel for transplantation
  • a flat plate-type alginate gel was produced using a 55 mmol / L calcium chloride aqueous solution according to the "production of a flat plate-type alginate gel" described in Example 5.
  • the alginate solution was prepared to 1% by weight, an aqueous solution of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd .: B-2), an aqueous solution of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd .: A-2), a solution of Example 5-1b and Examples.
  • the following flat plate type alginate gel was prepared using.
  • the prepared alginate gel on a flat plate was cultured overnight in D-MEM medium. The next day, it was replaced with a serum-free D-MEM medium, further replaced with physiological saline, and allowed to stand for 1 hour or longer to obtain an alginate gel for transplantation into animals.
  • Example 6-1a An aqueous solution of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd .: B-2) prepared to 1% by weight was used to obtain an alginate gel on a flat plate having a short diameter (12 mm), a long diameter (15 mm) and a thickness (5 mm). A photograph of this flat plate-type alginate gel is shown as FIG. 1 (a).
  • Example 6-1b Short diameter (12 mm) -long diameter (12 mm) -thickness (4 mm) flat plate using a solution obtained by mixing the solution of Example 5-1b and the solution of Example 5-2b at a ratio of 2: 1 (volume ratio). An upper alginate gel was obtained. The chemical cross-linking group was adjusted to a concentration of 1%. A photograph of this flat plate-type alginate gel is shown as FIG. 2 (a).
  • Example 6-1c Short diameter (12 mm) -long diameter (12 mm) -thickness (4 mm) flat plate using a solution obtained by mixing the solution of Example 5-1b and the solution of Example 5-2b at a ratio of 2: 1 (volume ratio). An upper alginate gel was obtained. The chemical cross-linking group was adjusted to a concentration of 2%. A photograph of this flat plate-type alginate gel is shown as FIG. 3 (a).
  • Example 6-1d Using an aqueous solution of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd .: A-2) prepared to 1% by weight, a short diameter (about 12 mm) -long diameter (about 12 mm) -thickness (about 4 mm) alginate gel on a flat plate was prepared. Obtained.
  • Example 6-1e Short diameter (about 12 mm) -long diameter (about 12 mm) -thickness (about 4 mm) using a solution in which the solution of Example 5-1d and the solution of Example 5-2d were mixed at a ratio of 1: 1 (volume ratio). ) On a flat plate, an alginate gel was obtained. The chemical cross-linking group was adjusted to a concentration of 1%.
  • Example 6-1f Short diameter (about 12 mm) -long diameter (about 12 mm) -thickness (about 4 mm) using a solution in which the solution of Example 5-1d and the solution of Example 5-5a were mixed at a ratio of 1: 1 (volume ratio). ) On a flat plate, an alginate gel was obtained. The chemical cross-linking group was adjusted to a concentration of 1%.
  • Example 6-1g Short diameter (about 12 mm) -long diameter (about 12 mm) -thickness (about 4 mm) using a solution in which the solution of Example 5-3b and the solution of Example 5-2d were mixed at a ratio of 1: 1 (volume ratio). ) On a flat plate, an alginate gel was obtained. The chemical cross-linking group was adjusted to a concentration of 1%.
  • Example 6-1h Short diameter (about 12 mm) -long diameter (about 12 mm) -thickness (about 4 mm) using a solution in which the solution of Example 5-3b and the solution of Example 5-5a were mixed at a ratio of 1: 1 (volume ratio). ) On a flat plate, an alginate gel was obtained. The chemical cross-linking group was adjusted to a concentration of 1%.
  • Example 6-2 Transplantation test of flat plate alginate gel into animals
  • Each alginate gel prepared in Examples 6-1a to 6-1c was transplanted into the abdominal cavity of healthy mouse C57BL / 6NCr. After 5 weeks, the abdomen was opened and the gel was removed, and the state of the gel was confirmed. Intra-abdominal adhesions and inflammation were also confirmed.
  • each alginate gel prepared in Examples 6-1d to h was transplanted into the abdominal cavity of healthy mouse C57BL / 6NCr. After 1, 2, or 4 weeks, the abdomen was opened and the gel was removed, and the state of the gel was confirmed. Intra-abdominal adhesions and inflammation were also confirmed.
  • Alginate gel of Example 6-1a The extracted alginate gel did not maintain its original shape, was disjointed, and the amount of remaining gel that could be confirmed was small. A photograph of that state is shown as FIG. 1 (b). Alginate gel of Example 6-1b: The removed alginate gel did not change the size of the gel. A photograph of that state is shown as FIG. 2 (b). Alginate gel of Example 6-1c: The extracted alginate gel was cracked, but the original shape was almost maintained, and the size of the gel did not change. A photograph of that state is shown as FIG. 3 (b).
  • Example 6-1d alginate gel The alginate gel removed after 1 week did not maintain its original shape, was disjointed, and the amount of remaining gel that could be confirmed was small.
  • Alginate gel of Example 6-1f There was no change in gel size in any of the alginate gels removed after 1 week, 2 weeks, and 4 weeks.
  • Example 6-1 g of alginate gel There was no change in gel size in any of the alginate gels removed after 1 week and 2 weeks. The alginate gel removed after 4 weeks was cracked, but the original shape was almost maintained, and the gel size did not change.
  • Alginate gel of Example 6-1h There was no change in gel size in any of the alginate gels removed after 1 week and 2 weeks. The alginate gel removed after 4 weeks was cracked, but the original shape was almost maintained, and the gel size did not change.
  • the alginate gels of Examples 6-1a, 6-1b, and 6-1c were transplanted, and when the abdomen was opened and confirmed 5 weeks later, there was no adhesion or inflammation between the intra-abdominal organs. There were no adhesions or inflammation in the omentum or intestinal membrane in which the gel was buried. There were no adhesions or inflammation in the liver to which the disjointed gel had adhered.
  • Example 6-1d The alginate gels of Example 6-1d, Example 6-1f, Example 6-1g, and Example 6-1h were transplanted, and the abdomen was opened and confirmed after 1 week, 2 weeks, and 4 weeks. There were no adhesions or inflammation between the intra-abdominal organs. There were no adhesions or inflammation in the omentum or intestinal membrane in which the gel was buried. There were no adhesions or inflammation in the liver to which the disjointed gel had adhered.
  • Example 6-3 Cell survival confirmation test of flat plate-type alginate gel
  • MIN6 cells (5 ⁇ 10 6 cells), which are cell lines of pancreatic islet Langerhans ⁇ cells, were used in Examples 6-1a, 6-1b, 6-1c, 6-1d, and 6-. 1e, Example 6-1f, Example 6-1g, Example 6-1h
  • the arginate gel was prepared, and the arginate gel was prepared and used in D-MEM medium for 3 to 4 weeks. After culturing, the survival of MIN6 cells was confirmed under a microscope.
  • Example 6-1a Alginate gels of Example 6-1a, Example 6-1b, Example 6-1c, Example 6-1d, Example 6-1e, Example 6-1f, Example 6-1g, Example 6-1h Cell proliferation was good in the medium, and it was observed that the cells were sufficiently alive under the microscope.
  • Example 1b with an introduction rate of 5.0 mol% and Example 2b with an introduction rate of 4.9 mol% 1.5% by weight of the physiological saline solution of Example 5-1b and 3.0 were used, respectively. Adjust the aqueous physiological saline solution of Example 5-2b in% by weight.
  • a 2% by weight alginic acid solution having a crosslinking group introduction rate of 5 mol% can be prepared.
  • the solutions of Examples 5-1b and 5-2b are diluted 2-fold and 4-fold to prepare solutions, which are mixed at a ratio of 2: 1 (volume ratio) to prepare a solution.
  • the mixed solution of the 2-fold diluted solution is used as the solution of Example 7-1, and the mixed solution of the 4-fold diluted solution is used as the solution of Example 7-2. Further, in the same manner, the solution of Example 5-1c and the solution of Example 5-2c were mixed at a ratio of 2: 1 (volume ratio), and the mixed solution of the 4-fold diluted solution was used in Example 7-3. Make it a solution.
  • Example 7-1a Transplant device prepared using 100 ⁇ L of the solution of Example 7-1
  • Example 7-1b Transplant device prepared using 200 ⁇ L of the solution of Example 7-1
  • Example 7-1b Transplant device prepared using 100 ⁇ L of the solution of Example 7-2
  • Example 7-2b Transplant device prepared using 200 ⁇ L of the solution of Example 7-2
  • Example 7-3a Example 7-3 Transplant device prepared using 100 ⁇ L of solution
  • Example 7-3b Transplant device prepared using 200 ⁇ L of solution of Example 7-3
  • the pancreatic island pellet dispensed into one device was suspended in the alginic acid solution (B1) of Example 5-1b. Later, the solutions of Examples 7-1a, 7-1b, 7-1b, and 7.2b were mixed with the alginic acid solution (C1) of Example 5-2b to suspend the porcine pancreatic islands. And said.
  • the alginic acid solution (B1) of Example 5-1b and the alginic acid solution (C1) of Example 5-2b were prepared with physiological saline according to the pellet amount (10 to 30 ⁇ L) of the pig pancreatic islet amount of 10000IEQ per device.
  • the concentration was adjusted. Further, in order to prepare a 100 ⁇ L or 200 ⁇ L porcine pancreatic island-containing 0.5% by weight to 1.0% by weight alginic acid solution, the pancreatic island pellet dispensed into the alginic acid solution (B1) of Example 5-1c for one device was added. After suspension, it was mixed with the alginic acid solution (C1) of Example 5-2c to prepare the solutions of Examples 7-3a and 7-3b in which the porcine pancreatic island was suspended.
  • the alginic acid solution (B1) of Example 5-1c and the alginic acid solution (C1) of Example 5-2c were prepared with physiological saline according to the pellet amount (10 to 30 ⁇ L) of the pig pancreatic islet amount of 10000IEQ per device. The concentration was adjusted.
  • the alginic acid solution containing porcine pancreatic islands prepared as Examples 7-1a, 7-2a, 7-2b and 7-3b was rapidly subjected to a semipermeable membrane (Spectrum dialysis tube "Spectra / Pore CE (Spectra / Pore CE)”. Enclosed in (fractional molecular weight 100,000) ”) (after heat-sealing one end of the semipermeable membrane, put an alginic acid solution and enclose it with a titanium clip), and soak it in a 55 mmol / LCaCl 2 solution for 10 to 15 minutes. The alginic acid solution inside was gelled.
  • the transplanting device was washed with physiological saline for 3 minutes and cultured overnight in the transplanting medium (M199-nicotinamide-FBS + P / S). Then, it was soaked in a serum-free medium for transplantation (M199 + P / S) for 30 minutes, and then soaked in physiological saline for pre-transplantation + P / S for 30 minutes and washed to obtain a device for transplantation into mice.
  • a photograph of the prepared transplant device is shown in FIG. 4-1.
  • Administration method / transplantation method Anesthetized by intraperitoneal administration of 0.25 to 0.3 mL of a three-kind mixed anesthetic (domitol / midazolam / betorfar), abdominal shaving and disinfection under anesthesia, a midline incision of about 2 cm in the abdomen, and a device for transplantation after washing. It was simply placed in the abdominal cavity and transplanted without fixation. After transplantation, the abdomen was closed and 0.25 to 0.3 mL of a medetomidine antagonist (antisedan) was subcutaneously injected to awaken the patient. The surgery was performed by keeping the mice warm on a heat pad. No administration of immunosuppressants. No administration of fluid replacement, antibiotics, anti-inflammatory agents, etc.
  • a three-kind mixed anesthetic domitol / midazolam / betorfar
  • abdominal shaving and disinfection under anesthesia a midline incision of about 2 cm in the abdomen
  • a device for transplantation after washing It was simply
  • Blood glucose / weight measurement method Blood glucose levels were measured before and every few days after transplantation at regular times during the day. Blood glucose was measured with a drop of blood from a scalpel cut in the tail using the Glutest Neo Alpha and Glutest Neo sensors. Body weight was measured with an electronic balance immediately before blood glucose measurement. The blood glucose level of the device-transplanted mice was less than 300 mg / dL as an individual whose diabetes was cured. The blood glucose level fluctuations up to day 75 after transplantation when the transplantation device of Example 7-2a was used are shown in FIG. 5-1 and the body weight fluctuations are shown in FIG. 6-1. In addition, changes in blood glucose level up to day 305 after transplantation are shown in Fig. 5-2, and changes in body weight are shown in Fig. 6-2.
  • transplantation device was taken out and transplanted to another diabetes model mouse (here, the device was transplanted to the diabetes model mouse, the transplanted device was taken out after a predetermined period of time, and the removed device was taken out to another.
  • Transplanting into a diabetic model mouse is called "relay transplantation"
  • blood glucose level fluctuations up to day 26 after relay transplantation are shown in Fig. 5-3
  • body weight fluctuations are shown in Fig. 6-3.
  • # 1 and # 2 mean numbers for identifying the transplanted mouse solids, respectively.
  • There was no abnormality in body weight fluctuation and the blood glucose level was maintained at a normal level for 75 days.
  • body weight fluctuation for 305 days after transplantation and 26 days after further relay transplantation and the blood glucose level was maintained normally.
  • Example 7-2b As for the blood glucose level fluctuation when the transplant device of Example 7-2b was used, there was no abnormality in the body weight fluctuation as in Example 7-2a, and the blood glucose level was maintained at a normal value for 75 days.
  • the blood glucose level fluctuations up to day 305 after transplantation when the transplantation device of Example 7-2b was used are shown in FIG. 7-1, and the body weight fluctuations are shown in FIG. 8-1.
  • the transplantation device was taken out on day 305 after transplantation and transplanted to another diabetes model mouse, and the fluctuation of blood glucose level and the fluctuation of body weight up to day 26 after relay transplantation are shown in FIG. 7-2 and the change in body weight in FIG. 8-2.
  • For 305 days after transplantation and 26 days after further relay transplantation there were no abnormalities in body weight fluctuations, and blood glucose levels were maintained normally.
  • Example 7-3b As for the blood glucose level fluctuation when the transplant device of Example 7-3b was used, there was no abnormality in the body weight fluctuation as in Example 7-2a, and the blood glucose level was maintained at a normal value for 75 days.
  • the blood glucose level fluctuations up to day 305 after transplantation when the transplantation device of Example 7-3b was used are shown in FIG. 9-1, and the body weight fluctuations are shown in FIG. 10-1.
  • the transplantation device was taken out and transplanted to another diabetes model mouse, and the blood glucose level fluctuations up to day 26 after the relay transplantation were shown in FIG. 9-2 and the body weight fluctuations were shown in FIG. 10-2.
  • # 2 and # 3 the device was removed in the middle and the test was completed.
  • 305 days after transplantation and 26 days after further relay transplantation there were no abnormalities in body weight fluctuations, and blood glucose levels were maintained normally.
  • transplantation device In the preparation of the transplantation device, a transplantation device in which pancreatic islets were encapsulated in a semipermeable membrane was prepared without using an alginate derivative. When transplanted into mice by the same method as in the above-mentioned [Evaluation of transplantation device (transplantation test)], no hypoglycemic effect was observed in diabetic mice.
  • tissue reactivity was evaluated as follows. A few weeks after the transplant, or after the blood glucose level rises at any time, the device transplanted mouse is anesthetized with a three-kind mixed anesthetic, the abdomen is disinfected under anesthesia, the abdomen is incised about 4 cm in the middle, and the transplanted device is inserted between the intraperitoneal organs. looked for. If a part of the device is seen between the organs, slowly remove it with tweezers and check whether the device can be removed by itself. Observe the surface condition of the removed device. ⁇ Observation items> 1. 1. Examine the surface of the device for angiogenesis.
  • angiogenesis observe whether it is at the level of capillaries or even thick blood vessels. 2. 2. Next, observe whether they are adhered to or connected to organs, peritoneum, omentum, etc. Investigate whether the organ can be exfoliated slowly or sharply. 3. 3. If it is directly adhered to an organ, check which part of the device (whole surface, part, side, device crease, sealing part, etc.) is adhered. 4. Check if there is inflammation on the organ side. * After removing the device, the abdomen is closed. Subcutaneous injection of antagonist is awakened. Surgery is performed by keeping the mouse warm on a heat pad.
  • Example 7-1a When the transplantation device of Example 7-1a was used, the device was removed 10 weeks after the transplantation, and the tissue reactivity was observed. As a result, (1) no angiogenesis was formed on the surface of the device, and (2) the device was found. It did not adhere to the organs, peritoneum, omentum, etc., (3) the organs could be bluntly detached, did not adhere directly to the organs, and (4) no inflammation was observed on the organ side. ..
  • pancreatic islet cells The appearance of the viable pancreatic islet cells in the excised device is stained as follows, that is, (a) staining of pancreatic islet cells with DISZONE, (b) staining of pancreatic islet cells with DISZONE, and (c) fluorescent staining of live cells with FDA. , (D) After fluorescent staining of dead cells with PI, pancreatic islet cells dispersed in alginate gel without staining were observed under a microscope. As a result, it was confirmed that the islet cells were sufficiently alive in the device.
  • the excised device was opened and the shape of the alginate gel in the device was confirmed. As a result, it was clarified that the shape of the alginate gel in the transplant device placed in the living body for a long period of time was maintained.
  • Example 8 [Preparation of transplant device] In the same manner as in Example 7, the solution of Example 5-1c and the solution of Example 5-2c were mixed at a ratio of 2: 1 (volume ratio), and a mixed solution of a 4-fold diluted solution was used in Example 8. Make it a solution.
  • Example 8 The transplantation devices prepared with the solutions of Example 8 (100 ⁇ L, 75 ⁇ L, 50 ⁇ L) are as follows. ⁇ Porting device>
  • Example 8-1 Transplant device prepared using 100 ⁇ L of the solution of Example 8
  • Example 8-2 Transplant device prepared using 75 ⁇ L of the solution of Example 8
  • Example 8-3 Example 8 Transplant device prepared with 50 ⁇ L of solution
  • the pancreatic island pellet dispensed into one device was suspended in the alginic acid solution (B1) of Example 5-1c, and then the example. It was mixed with the alginic acid solution (C1) of 5-2c to obtain the solution of Example 8 in which the porcine pancreatic island was suspended.
  • the alginic acid solution (B1) of Example 5-1b and the alginic acid solution (C1) of Example 5-2b were prepared with physiological saline according to the pellet amount (10 to 30 ⁇ L) of the pig pancreatic islet amount of 10000IEQ per device. The concentration was adjusted.
  • the alginic acid solution containing porcine pancreatic island prepared as Example 8 was rapidly encapsulated in a semipermeable membrane (dialysis tube "Spectra / Pore CE (molecular weight cut-off 100,000)" manufactured by Spectrum Co., Ltd.) (one end of the semipermeable membrane was heat-sealed). After that, an alginic acid solution was added, and the other end was heat-sealed and sealed) and immersed in a 55 mmol / LCaCl 2 solution for 10 to 15 minutes to gel the alginic acid solution in the device.
  • a semipermeable membrane dialysis tube "Spectra / Pore CE (molecular weight cut-off 100,000)" manufactured by Spectrum Co., Ltd.
  • the transplanting device was washed with physiological saline for 3 minutes and cultured overnight in the transplanting medium (M199-nicotinamide-FBS + P / S). Then, it was soaked in a serum-free medium for transplantation (M199 + P / S) for 30 minutes, and then soaked in physiological saline for pre-transplantation + P / S for 30 minutes and washed to obtain a device for transplantation into mice.
  • a photograph of the prepared transplant device is shown in FIG. 4-2.
  • Example 8-1 length about 10 mm ⁇ width about 20 mm ⁇ thickness about 0.5 mm (500 ⁇ m)
  • Example 8-2 length about 10 mm ⁇ width about 20 mm ⁇ thickness about 0.375 mm (375 ⁇ m)
  • Example 8-3 length about 10 mm ⁇ width about 20 mm ⁇ thickness about 0.25 mm (250 ⁇ m)
  • the blood glucose level fluctuations up to day 178 after transplantation when the transplantation device of Example 8-3 was used are shown in FIG. 11-4, and the body weight fluctuations are shown in FIG. 12-4.
  • the device for transplantation was taken out on day 178 after transplantation, and the fluctuation of blood glucose level up to day 40 after relay transplantation was shown in FIG. 11-5, and the fluctuation of body weight was shown in FIG. 12-5.
  • the indications represented by # and numbers mean the numbers that identify the transplanted mouse individuals, respectively.
  • There was no abnormality in body weight fluctuation and the blood glucose level was maintained at a normal level for 178 days.
  • the blood glucose level was maintained normally.
  • GsIs was performed on the removed device, and it was confirmed that the device had insulin secretory capacity.
  • Example 9 Evaluation of transplantation device by intraperitoneal transplantation into diabetes model mice 3
  • the following devices were prepared according to the method of Example 7.
  • Example 9-1 Transplant device prepared using 200 ⁇ L of a sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd .: B-2) aqueous solution prepared in an amount of 1% by weight
  • Example 9-2 The same method as in Example 7-1.
  • Example 9-3 Device for transplantation prepared using 200 ⁇ L of the solution prepared in Example 9-3: Device for transplantation prepared using 200 ⁇ L of the solution prepared in the same manner as in Example 7-2
  • Example 9-4 Example 7-2 Transplant device prepared using 100 ⁇ L of solution prepared in the same manner as in Example 9-5: Transplant device prepared using 200 ⁇ L of solution prepared in the same manner as in Example 7-3
  • Example 9-6 Implementation Transplant device prepared using 100 ⁇ L of solution prepared in the same manner as in Example 7-3
  • Example 9-7 Transplant device prepared using 50 ⁇ L of solution prepared in the same manner as in Example 7-3.
  • Example 9-1 Length about 10 mm ⁇ width about 20 mm ⁇ thickness about 1 mm (1000 ⁇ m)
  • Example 9-4 Length about 10 mm x Width about 20 mm x Thickness about 0.5 mm (500 ⁇ m)
  • Example 9-7 length about 10 mm ⁇ width about 20 mm ⁇ thickness about 0.25 mm (250 ⁇ m)
  • the transplantation test was performed according to Example 7.
  • the devices of Examples 9-1 to 9-7 were transplanted into a plurality of diabetes model mice, respectively.
  • Mice whose blood glucose level was 300 mg / dL or less for 178 days after transplantation (however, it is allowed to exceed 300 mg / dL up to 3 times during the period) are defined as cured mice, and are cured for the number of transplanted diabetes model mice. When the number of mice was calculated as the cure rate, the following results were obtained.
  • the cure rate of the diabetic model mouse was higher in the transplantation device containing the hydrogel produced by the alginic acid derivative of the present invention than in the transplantation device containing the hydrogel produced by the natural alginic acid. Further, it was confirmed that the thinner the thickness of the hydrogel, the higher the healing rate, and in this example, the healing rate is about 40% when the thickness is 500 ⁇ m or more, while the healing rate is improved to about 62% when the thickness is 250 ⁇ m. Was done.
  • the cure rate of Example 9-2 is 0.0%, it is caused by the small number of N, and as the number of N increases, the cure rate reaches the level described in Example 9-3. Is expected to be.
  • MIN6 cells 2.5 ⁇ 10 6 cells
  • pancreatic islet Langerhans ⁇ cells which are cell lines of pancreatic islet Langerhans ⁇ cells
  • a device was produced by a method according to Example 7.
  • a solution of 2% A-2 alginic acid diluted 4-fold was used as the solution of Example 10-1.
  • a solution obtained by mixing the solution of Example 5-1e and the solution of Example 5-2e at a ratio of 1: 1 (volume ratio) was used as the solution of Example 10-2.
  • a solution obtained by mixing the solution of Example 5-1e and the solution of Example 5-5b at a ratio of 1: 1 (volume ratio) was used as the solution of Example 10-3.
  • Example 10-5 a solution obtained by mixing the solution of Example 5-3c and the solution of Example 5-2e at a ratio of 1: 1 (volume ratio) was used as the solution of Example 10-4. Further, a solution obtained by mixing the solution of Example 5-3c and the solution of Example 5-5b at a ratio of 1: 1 (volume ratio) was used as the solution of Example 10-5. All of Examples 10-2 to 10-5 have a concentration of 0.5% as a chemical cross-linking group.
  • the transplantation devices prepared with the solutions of Examples 10-1 to 10-5 are as follows. ⁇ Porting device>
  • Example 10-1 Transplant device prepared using 50 ⁇ L of the solution of Example 10-1
  • Example 10-2 Transplant device prepared using 50 ⁇ L of the solution of Example 10-2
  • Example 10-3 Transplant device prepared using 50 ⁇ L of the solution of Example 10-3
  • Example 10-4 Transplant device prepared using 50 ⁇ L of the solution of Example 10-4
  • Example 10-5 Example 10-5 Transplant device prepared with 50 ⁇ L of solution
  • FIG. 14-4 The blood glucose level fluctuations up to day 17 after transplantation when the transplantation device of Example 10-5 was used are shown in FIG. 13-5, and the body weight fluctuations are shown in FIG. 14-5.
  • the indications represented by # and numbers mean the numbers that identify the transplanted mouse individuals, respectively. There was no abnormality in body weight fluctuation, and the blood glucose level was maintained at a normal level for 12 to 17 days.
  • tissue reactivity was evaluated according to Example 7.
  • the transplantation device of Examples 10-1 to 5 When the transplantation device of Examples 10-1 to 5 was used, the device was removed 2 weeks after the transplantation, and the tissue reactivity was observed. As a result, (1) no angiogenesis was formed on the surface of the device, and (2) The device is not adhered to the organ, peritoneum, omentum, etc., (3) the organ can be bluntly detached, is not directly adhered to the organ, and (4) inflammation is observed on the organ side. I didn't.
  • Example 11-1 For a hydrogel having a length of 10 mm and a width of 20 mm, the oxygen permeability of Example 11-1 is 1 mm, Example 11-2 is 0.5 mm, and Example 11-3 is 0.25 mm under the following conditions.
  • a simulation was performed. In the simulation, the oxygen concentration on the surface of the hydrogel is uniform and always constant, the specific respiration rate of the cells is always constant, the volume of the cells is ignored, and the oxygen is consumed uniformly in the hydrogel. It was assumed that oxygen diffusion would occur evenly from the entire surface of the gel to the center.
  • FIGS. 15-1 represent hydrogels
  • FIGS. 15-2 to 15-4 show oxygen permeability in the cross section indicated by the black arrow.
  • the legend is shown in FIG. 15-2, and the displayed contents are the same in FIGS. 15-3 to 15-4.
  • FIG. 15-2 shows a hydrogel having a thickness of 1 mm according to Example 11-1, and the oxygen permeability of the surface was 100 to 75%, while the oxygen permeability of the central portion was 25% or less. ..
  • FIG. 15-3 shows Example 11-2
  • the oxygen permeability in the central portion is improved to 50 to 25%
  • FIG. 15-4 showing Example 11-3 the oxygen permeability is improved to 50 to 25%.
  • the thickness By setting the thickness to 0.25 mm, the oxygen permeability in the central portion was 75 to 50%, which was more than half of the oxygen concentration on the surface.
  • Example 12 Evaluation of substance permeability from transplantation device [Preparation of transplantation device] Using Example 1e with an introduction rate of 2.2 mol% and Example 2e with an introduction rate of 2.4 mol%, an aqueous solution of 2% by weight of Example 12-1e and 2% by weight of Example 12 were used, respectively. An aqueous solution of -2e was prepared. A physiological saline solution was used to prepare the aqueous solution. By mixing the solution of Example 12-1e and the solution of Example 12-2e at a ratio of 1: 1 (volume ratio), a 2% by weight alginic acid solution can be prepared.
  • the edges were heat-sealed and encapsulated) and soaked in 55 mmol / LCaCl2 solution for 10-15 minutes to gel the alginic acid solution in the device.
  • the final concentration of alginic acid was 0.5%.
  • the transplantation device prepared with such a solution was used as a hydrogel of Example 12-1 (containing insulin) and Example 12-2 (containing glucose).
  • Hydrogel size length 10 mm x width 20 mm x thickness approx. 0.25 mm
  • Example 12-1 or Example 12-2 was stirred in 40 mL of a 0.01% Tween 20-containing physiological saline solution at room temperature for 24 hours, and the insulin concentration or glucose concentration in the external solution was measured to obtain a hydrogel.
  • the human insulin permeability or glucose permeability was determined when the enclosed human insulin or glucose was diffused into a physiological saline solution as 100%.
  • the insulin concentration in the external solution up to 24 hours after the start of the test when the transplant device of Example 12 was used is shown in FIG. 16-1, and the glucose concentration is shown in FIG. 16-2. It was confirmed that both human insulin and glucose were rapidly permeated, and 100% was permeated by stirring for 24 hours, confirming that the implantable device of the present invention was excellent in permeation of human insulin and glucose.
  • Example 13 Evaluation of hydrogel disintegration [Preparation of transplantation device] First, the following physiological saline solutions of Examples 13-1 to 13-5 were prepared.
  • Example 13-1 After preparing a solution of Example 13-1 using 50 ⁇ L, it is rapidly encapsulated in a semipermeable membrane (Spectrum's dialysis tube “Spectra / Pore CE (molecular weight cut off: 100,000)”). After heat-sealing one end of the semipermeable membrane, add an alginic acid solution, heat-seal the other side to enclose it), and soak it in a 55 mmol / LCaCl 2 solution for 10 to 15 minutes to immerse the alginic acid solution in the device. Gelled.
  • a semipermeable membrane Spectrum's dialysis tube “Spectra / Pore CE (molecular weight cut off: 100,000). After heat-sealing one end of the semipermeable membrane, add an alginic acid solution, heat-seal the other side to enclose it), and soak it in a 55 mmol / LCaCl 2 solution for 10 to 15 minutes to immerse the alginic acid solution
  • Example 13-2 A transplant device prepared in the same manner as in Example 13-1 after preparing using 200 ⁇ L of the solution of Example 13-2.
  • 13-3a Transplant device prepared in the same manner as in Example 13-1 after preparing using 50 ⁇ L of the solution of Example 13-3
  • Example 13-3b Using 100 ⁇ L of the solution of Example 13-3.
  • Example 13-3c Prepared in the same manner as in Example 13-1 after preparing using 200 ⁇ L of the solution of Example 13-3.
  • Example 13-4 A device for transplantation prepared in the same manner as in Example 13-1 after preparing using 200 ⁇ L of the solution of Example 13-4
  • Example 13-5a Example 13-5.
  • Example 13-5b After preparing the solution of Example 13-5 using 100 ⁇ L
  • Example 13-1 Transplant device prepared in the same manner as in Example 13-5c: A transplant device prepared in the same manner as in Example 13-1 after preparing using 200 ⁇ L of the solution of Example 13-5.
  • the device for transplantation was recovered and treated with lyase to dissolve the alginic acid gel, and then the concentration of alginic acid in the residual gel was quantified.
  • the alginic acid content in the device from the alginic acid concentration in the external liquid at the end of the test and the alginic acid concentration in the residual gel, and calculate from the alginic acid concentration confirmed from the external liquid after the start of each test when this is set to 100%.
  • the content of alginic acid leaked from the device was calculated as the disintegration rate.
  • FIG. 17 shows the disintegration rate up to 96 hours after the start of the test when the transplantation device of Example 13 was used.
  • Table 15 shows the disintegration rates 24 hours and 96 hours after the start of the test.
  • the alginic acid in the hydrogel was rapidly dissolved and the hydrogel was disintegrated. It was confirmed that the device for transplantation has excellent stability because almost no alginic acid elution was observed and the hydrogel did not disintegrate.
  • Example 14 Model test of cell shedding from alginate gel
  • the prepared transplant devices are as follows. ⁇ Porting device> Example 14-1: Prepared by adding 5 ⁇ L of a solution of polystyrene beads (106-125 ⁇ m, Polyscience, cat No. 19824) as a substitute for pancreatic islet cells to 45 ⁇ L of the solution of Example 13-1, and then rapidly half. Enclosed in a translucent membrane (Spectrum dialysis tube "Spectra / Pore CE (fractional molecular weight 100,000)”) (After heat-sealing one end of the semi-transparent membrane, add an alginic acid solution and heat-seal the other side as well.
  • Spectrum dialysis tube “Spectra / Pore CE (fractional molecular weight 100,000)
  • Example 14-2 Prepared by adding 5 ⁇ L of polystyrene beads instead of pancreatic islet cells to 45 ⁇ L of the solution of Example 13-3a, and then Example 14-. Transplant device made in the same way as 1.
  • ⁇ Test method Add three transplanting devices of any of Examples 14-1 to 2 to 12 mL of a 37 ° C. physiological saline solution in a 15 mL conical tube, and add a medium-sized constant temperature shaking incubator (Tietech Co., Ltd., Bio-Shaker (registered trademark)). Using BR-43FL (MR), the mixture was shaken under the conditions of a reciprocating shaking method with an amplitude of 25 mm and a shaking speed of 180 rpm while maintaining the temperature at 37 ° C. Twenty-four hours after the start of the test, the external liquid was collected, and the polystyrene beads that had fallen into the external liquid were observed under a microscope.
  • a medium-sized constant temperature shaking incubator Tetech Co., Ltd., Bio-Shaker (registered trademark)
  • FIG. 18-1 shows photographs of the external fluid observed 24 hours after the start of the test when the transplantation device of Example 14-1 was used, and 24 hours after the start of the test when the transplantation device of Example 14-2 was used.
  • the later photographs of the external liquid observation are shown in FIG. 18-2, respectively.
  • the transplantation device using the natural alginic acid of Example 14-1 a large number of beads were observed in the external solution, and it was confirmed that most of the microbeads were shed 24 hours after the start of the test.
  • the microbeads were hardly shed (the shedding rate is considered to be 10% or less), and the encapsulated cells were considered not to be shed for a long period of time and were stable. It was confirmed that it was excellent in sex.
  • the transplantation device of the preferred embodiment exhibits at least one or more of the following effects.
  • the gel is less dissolved and the shape is maintained for a long period of time.
  • the alginate gel in the semipermeable membrane can maintain its shape without dissolving, and can maintain the survival and function of pancreatic islets, and can be used for a long period of time.
  • It can be exchanged, can be immunoisolated, has less adhesion, inflammation, etc., and is a highly safe medical material.
  • the hydrogel is a thin device having a thickness of less than 500 ⁇ m
  • the healing rate of the animal transplanted with the device is higher than that of the device having a thickness of 500 ⁇ m or more.
  • the ratio of the oxygen concentration in the central portion to the surface of the device is higher than in the case of a thin device having a thickness of 500 ⁇ m or more.
  • the hydrogel does not disintegrate or is difficult to disintegrate.
  • the islets contained in the hydrogel are less likely to fall off from the gel.
  • More preferred embodiments of the transplant device have excellent transplant performance and functionality, are novel in terms of materials, and can be transplanted into diabetic patients (particularly type I diabetes and insulin-depleted type II diabetes) to provide long-term blood glucose. It is possible to maintain the descent effect and regulate blood sugar. In addition, recovery is possible when the function of insulin-secreting cells or islets in the hydrogel is reduced. Alternatively, regular replacement or additional transplantation is possible. Further, as the insulin-secreting cells or islets enclosed in the hydrogel of the transplantation device, insulin-secreting cells differentiated from stem cells (iPS or the like) or human pancreatic islets can also be used. Therefore, a more preferred embodiment of the implantable device is useful.

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