Implantable devices using chemically cross-linked alginate

Abstract

Provided is a transplantation device comprising a hydrogel in which insulin-secreting cells or pancreatic islets are enclosed, wherein the hydrogel is prepared by gelatinizing an alginic acid derivative via chemical crosslinking. Accordingly, a novel transplantation device is provided.

Description

[Technical field]

[0001] The present invention relates to a device for transplanting cells or the like into a living body, and more particularly to a transplant device using chemically crosslinked alginate and a method for producing the same. [Background technology]

[0002] As a treatment for type I diabetes, in addition to conventional insulin injections and pancreas transplantation, pancreatic islet transplantation has been performed both domestically and abroad. However, due to problems such as a shortage of donors, especially in Japan, the number of cases is still limited. Xenotransplantation using porcine islets can be an effective technique to resolve this donor shortage, but in both allotransplantation and xenotransplantation, immune rejection is unavoidable, making it necessary to take immunosuppressants for a long period of time, and the risk of complications and the possibility of adverse effects on the remaining transplanted pancreatic islets have been reported (Non-Patent Document 1: Organ Biology, VOL24, No.1, pp.7-12, 2017).

[0003] To solve this problem, a technology that has been under consideration for some time is bioartificial pancreas (BAP, also called bioartificial islet), in which pancreatic islets are covered (encapsulated) with a polymer gel or semipermeable membrane that can be isolated from the recipient's immune cells, etc., but is permeable to nutrients, insulin, etc., and then transplanted into the body (Patent Document 1: JP 55-157502 A, Patent Document 2: JP 60-258121 A, Patent Document 3: WO 95 / 28480 pamphlet, Patent Document 4: WO 92 / 19195 pamphlet, Patent Document 5: JP 2017-196150 A).

[0004] Bioartificial islets are mainly classified into (1) "microcapsule type" in which each islet is coated with a polymer gel, etc., (2) "macrocapsule type" in which a large number of islets are coated with a polymer gel or a semipermeable membrane, etc., and (3) "blood perfusion type" in which islets are encapsulated in an immunoisolation device or hollow fiber module made of a semipermeable membrane and blood is perfused through the device (Non-Patent Document 1).

[0005] The microencapsulation type is a technology in which individual islets are encapsulated in a polymer gel that can be isolated from immune cells but is permeable to nutrients and insulin, and then transplanted into the body (mainly into the abdominal cavity) in the same way as regular islet transplantation. In addition to being able to isolate them from the recipient's immune cells, the isolation membrane is relatively thin, so the permeation time due to diffusion is short, and there are advantages in that nutrients can be permeated and cells can respond quickly, but it is difficult to recover the islets when their function declines.

[0006] The blood perfusion type is a technology in which blood is perfused through a flow path that isolates the pancreatic islet with a semipermeable membrane. It applies accumulated technologies such as artificial dialysis and bioartificial livers, and a lot of basic research has been conducted on it. However, there are issues such as the large size of the device and the high risk of blood clot formation, and it has the disadvantage that it is prone to forming blood clots and becoming clogged when used for a long period of time, so it has not yet been put to practical use.

[0007] The macrocapsule type is a technology that has been improved to overcome the drawbacks of the microcapsule type, namely, to enable the removal of islets when their function declines. However, no studies have been reported on islet transplantation using macrocapsule type xenogeneic islets that have shown excellent results, and no bioartificial islets using xenogeneic islets have been found that overcome the problems in islet transplantation, such as the shortage of donors, the use of immunosuppressants, and the long-term survival and functional maintenance of islets.

[0008] There is a report on the synthesis of alginate hydrogel capsules using the click reaction and a comparison of their stability, water swelling and diffusion with ionically cross-linked alginate capsules (Non-Patent Document 2: JOURNAL OF BIOMEDICAL MATERIALS RESEARCH, Part B / VOL103B, ISSUE 5, P1120-1132 (2015)). The document states that the alginate capsules formed by the click reaction are ionically cross-linked (C 2+ ) crosslinking is disclosed to be more stable. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] Japanese Patent Publication No. 55-157502 [Patent Document 2] Japanese Patent Application Publication No. 60-258121 [Patent Document 3] International Publication No. 95 / 28480 Brochure [Patent Document 4] International Publication No. 92 / 19195 Brochure [Patent Document 5] JP 2017-196150 A [Non-patent literature]

[0010] [Non-Patent Document 1] Organ Biology, VOL24, No. 1, pages 7-12, 2017 [Non-Patent Document 2] JOURNAL OF BIOMEDICAL MATERIALS RESEARCH,PartB / VOL103B,ISSUE 5,P1120-1132(2015) Summary of the Invention [Problem to be solved by the invention]

[0011] In the circumstances described above, there has been a demand for new and practical implantable devices. [Means for solving the problem]

[0012] Means of the Invention The present inventors have conducted extensive research to solve the above problems and have discovered the following (1) to (10), and have completed the present invention based on these findings. (1) The novel alginate derivatives used herein (e.g., alginate derivatives of formula (HA-I) and formula (HA-II)) are hydrogelled, for example, by chemical cross-linking. When flat alginate gels prepared using the chemically cross-linking alginate derivatives were transplanted into the body (into the abdominal cavity of healthy mice), there was no significant change in the size of the flat gels even after 5 weeks, the gels did not dissolve and maintained their shape, and the gels had excellent in vivo stability. (2) Furthermore, no adhesion or inflammation was observed in the abdominal cavity of the mouse. (3) When Min6 cells were embedded in the flat gel and cultured for 3 to 4 weeks, the survival of Min6 clusters was confirmed, proliferation was good, and there was no cytotoxicity. (4) When a transplant device made of chemically cross-linked alginate gel embedded with porcine pancreatic islets and coated with a semipermeable membrane was transplanted into a diabetic model mouse, it was shown to suppress blood glucose levels for up to 75 days. (5) In (4) above, when the transplant device was extracted 10 weeks after transplantation, no adhesions, angiogenesis, inflammation, or other disorders were found. Furthermore, when the survival of the pancreatic islets in the transplant device was confirmed by dithizone staining, they were found to be fully alive. Furthermore, when the transplant device extracted 10 weeks after transplantation was opened and the alginate gel inside was confirmed, its shape was maintained. (6) Excellent material permeability to the inside and outside of the device. (7) When the hydrogel was a thin device with a thickness of less than 500 μm, the healing rate of animals implanted with the device was higher than when the device was a thin device with a thickness of 500 μm or more. (8) In thin devices with hydrogel thicknesses of less than 500 μm, the ratio of oxygen concentration at the center of the device to the surface is higher than in devices with hydrogel thicknesses of 500 μm or more. (9) When the hydrogel is shaken, it does not collapse or is difficult to collapse. (10) When the hydrogel is shaken, the islets contained in the hydrogel are hardly removed from the gel.

[0013] The novel alginic acid derivatives used herein (e.g., alginic acid derivatives of formula (HA-I) and formula (HA-II)) are, for example, those that can be used for chemical crosslinking, i.e., those that have a reactive group that can be used for chemical crosslinking or a complementary reactive group to the reactive group introduced therein.

[0014] The chemical crosslinking may occur, for example, between an alginic acid derivative having a reactive group that can be used for chemical crosslinking introduced therein and an alginic acid derivative having a reactive group complementary to the reactive group introduced therein. The reactive group and the reactive group complementary to the reactive group may both be introduced into one molecule of alginic acid, or may be introduced separately. The chemical crosslinking may occur within the molecule of the alginic acid derivative, or may occur between molecules, or may occur with another molecule having a reactive group or a reactive group complementary to the reactive group introduced therein.

[0015] The chemical crosslinking is carried out, for example, by a crosslinking reaction via the Huisgen reaction (1,3-dipolar cycloaddition reaction), and may be carried out, for example, between alginic acid derivatives of formula (HA-I) and formula (HA-II), or, for example, between an alginic acid derivative of formula (HA-I) and another molecule having an azide group, or, for example, between an alginic acid derivative of formula (HA-II) and another molecule having an alkyne group. The chemical crosslinking is carried out, for example, by a crosslinking reaction by the Huisgen reaction (1,3-dipolar cycloaddition reaction), and may be carried out, for example, between alginic acid derivatives of formula (HB-I) and formula (HB-II), or between an alginic acid derivative of formula (HB-I) and another molecule having an azide group, or between an alginic acid derivative of formula (HB-II) and another molecule having an alkyne group.

[0016] Here, a device for transplanting cells or the like into a living body, prepared using an alginic acid derivative that is gelled by chemical crosslinking, more specifically, a transplant device including, for example, a chemically crosslinked alginic acid gel in which insulin-secreting cells or pancreatic islets or the like are embedded, and a semipermeable membrane that covers the gel as necessary, and a method for producing the same, are provided. The alginic acid derivative that is gelled by chemical crosslinking is, for example, an alginic acid derivative of formula (HA-I) or formula (HA-II) in which a cyclic alkyne group or an azide group is introduced via an amide bond and a divalent linker to any one or more carboxyl groups of alginic acid, and a novel crosslinked alginic acid is obtained by performing a Huisgen reaction (1,3-dipolar cycloaddition reaction) using the alginic acid derivatives of formula (HA-I) and formula (HA-II). Alternatively, the alginic acid derivative to be gelled by chemical crosslinking is, for example, an alginic acid derivative of formula (HB-I) or formula (HB-II) in which a cyclic alkyne group or azide group is introduced via an amide bond and a divalent linker to any one or more carboxyl groups of alginic acid, and a novel crosslinked alginic acid can be obtained by performing a Huisgen reaction (1,3-dipolar cycloaddition reaction) using the alginic acid derivatives of formula (HB-I) and formula (HB-II). Exemplary embodiments may be as follows: [1-1] to [3-4].

[0017] In this specification, the alginic acid derivatives according to formula (HA-I) and formula (HB-I) have the structure of formula (I) in common. Therefore, the general formula of the alginic acid derivatives according to formula (HA-I) and formula (HB-I) is both represented by formula (I). In the present specification, the alginic acid derivatives according to formula (HA-II) and formula (HB-II) have the structure of formula (II) in common. Therefore, the general formula of the alginic acid derivatives according to formula (HA-II) and formula (HB-II) is both represented by formula (II). In the present specification, the crosslinked alginic acids according to formula (HA-III-L) and formula (HB-III-L) have the structure of formula (III-L) in common. Therefore, the general formula of the crosslinked alginic acids according to formula (HA-III-L) and formula (HB-III-L) is represented by formula (III-L).

[0018] The first aspect of the present invention may be as follows: [1-1] to [1-34].

[0019] [1-1] A transplant device comprising a hydrogel in which insulin-secreting cells or pancreatic islets are encapsulated, the hydrogel being formed by gelling an alginic acid derivative by chemical crosslinking.

[0020] [1-2] The transplant device described in [1-1], wherein the hydrogel contains chemical crosslinks formed between alginic acid derivatives as crosslinks.

[0021] [1-3] The transplant device described in [1-1] or [1-2], wherein the hydrogel contains chemical crosslinks via triazole rings formed by the Huisgen reaction as crosslinks.

[0022] [1-4] The transplant device described in any one of [1-1] to [1-3], wherein the chemical crosslinking is generated by 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.

[0023] [1-5] The implantable device described in [1-4], wherein the chemical crosslinking is generated by a cyclic alkyne group introduced into any one or more carboxyl groups of the first alginic acid and an azide group introduced into any one or more carboxyl groups of the second alginic acid.

[0024] [1-6] The transplant device according to any one of [1-1] to [1-5] above, wherein the chemical crosslinking is a chemical crosslinking by a combination of the alginic acid derivatives described in the following (A) and (B): (A): An amide bond and a divalent linker (-L 1 A cyclic alkyne group (Akn) is introduced via a cyclic alkyne group (Akn) of the following formula (HA-I): [ka] [In formula (HA-I), (ALG) represents alginic acid; -NHCO- represents an amide bond via any carboxyl group of alginic acid; -L 1 - is the following partial structural formula (each formula does not include the areas outside the dashed lines at both ends): [ka] A represents a divalent linker selected from the group consisting of the following partial structural formulas (each formula does not include the right side of the dashed line): [ka] wherein the asterisk represents a chiral center; (B): An amide bond and a divalent linker (-L 2 -), an azide group has been introduced via the following formula (HA-II): [ka] [In formula (HA-II), (ALG) represents alginic acid; -NHCO- represents an amide bond via any carboxyl group of alginic acid; -L 2 - is the following partial structural formula (each formula does not include the areas outside the dashed lines at both ends): [ka] The alginic acid derivative is represented by the following formula:

[0025] [1-7] The chemically crosslinked alginic acid derivative is such that any carboxyl group of the first alginic acid and any carboxyl group of the second alginic acid are represented by the following formula (HA-III-L): [ka] [In formula (HA-III-L), -CONH- and -NHCO- at both ends represent an amide bond via any carboxyl group of alginic acid; -L 1 - is as defined in the above embodiment [1-6]; -L 2 - is as defined in the above embodiment [1-6]; X is the following partial structural formula: [ka] The implantable device according to claim 1-6, wherein the alginate is linked via a cyclic group selected from the group consisting of: (in each formula, the areas outside the dashed lines at both ends are not included; and an asterisk represents a chiral center).

[0026] [1-8] The transplant device according to any one of [1-1] to [1-5] above, wherein the chemical crosslinking is a chemical crosslinking by a combination of the alginic acid derivatives described in the following (A) and (B): (A): An amide bond and a divalent linker (-L 1 A cyclic alkyne group (Akn) is introduced via a cyclic alkyne group (Akn) of the following formula (HB-I): [ka] [In formula (HB-I), (ALG) represents alginic acid; -NHCO- represents an amide bond via any carboxyl group of alginic acid; -L 1 - Refer to the table below [Table 1-1] [Table 1-2] [In each formula, the areas outside the dashed lines at both ends are not included]; Akn is shown in the table below: [Table 2] an alginic acid derivative represented by the partial structural formula (I) shown in the formula (I) (each formula does not include the right side of the dashed line); (B): An amide bond and a divalent linker (- L 2 -), an azide group is introduced via the following formula (HB-II): [ka] [In formula (HB-II), (ALG) represents alginic acid; -NHCO- represents an amide bond via any carboxyl group of alginic acid; -L 2 -See the table below: [Table 3-1] [Table 3-2] [wherein each formula does not include the areas outside the dashed lines at both ends]] (However, in the alginic acid derivative represented by the formula (HB-I), -L 1 - is any one linker selected from the group consisting of (L1-1), (L1-2a), (L1-2b), (L1-11) and (L1-12); and an alginic acid derivative represented by formula (HB-II), in which -L 2 - Excluding chemical crosslinking by combination with a derivative in which (L2-10) is the linker).

[0027] [1-9] The chemically crosslinked alginic acid derivative is such that any carboxyl group of the first alginic acid and any carboxyl group of the second alginic acid are represented by the following formula (HB-III-L): [ka] [In formula (HB-III-L), -CONH- and -NHCO- at both ends represent an amide bond via any carboxyl group of alginic acid; -L 1 - is as defined in [1-8] above; -L 2 - is as defined in [1-8] above; X is as shown in the following table: [Table 4-1] [Table 4-2] (In each formula, the areas outside the dashed lines at both ends are not included.) (However, in the alginic acid derivative represented by formula (HB-I), -L 1 - is any one linker selected from the group consisting of (L1-1), (L1-2a), (L1-2b), (L1-11) and (L1-12); and an alginic acid derivative represented by formula (HB-II), in which -L 2 The implantable device described in [1-8] above, excluding cross-linked alginic acid in combination with a derivative in which - is the linker (L2-10).

[0028] [1-10] The alginic acid derivative of formula (HA-I) is represented by the following formula (EX-1-(I)-A-2): [ka] The alginic acid derivative of formula (HA-II) is represented by the following formula (EX-2-(II)-A-2): [ka] The implantable device described in [1-6] or [1-7].

[0029] [1-11] The alginic acid derivative of the formula (HA-I) is the following formula (EX-3-(I)-A-2): [ka] The alginic acid derivative of formula (HA-II) is represented by the following formula (EX-4-(II)-A-2): [ka] The implantable device described in [1-6] or [1-7].

[0030] [1-12] The alginic acid derivative of the formula (HA-I) is represented by the following formula (EX-1-(I)-A-2): [ka] The alginic acid derivative of formula (HA-II) is represented by the following formula (EX-4-2-(II)-A-2): [ka] The implantable device described in [1-6] or [1-7].

[0031] [1-13] The alginic acid derivative of the formula (HA-I) is the following formula (EX-3-(I)-A-2): [ka] The alginic acid derivative of formula (HA-II) is represented by the following formula (EX-2-(II)-A-2): [ka] The implantable device described in [1-6] or [1-7].

[0032] [1-14] The alginic acid derivative of the formula (HA-I) is the following formula (EX-3-(I)-A-2): [ka] The alginic acid derivative of formula (HA-II) is represented by the following formula (EX-4-2-(II)-A-2): [ka] The implantable device described in [1-6] or [1-7].

[0033] [1-15] The transplant device described in any one of [1-1] to [1-14] above, wherein the pancreatic islet is a human pancreatic islet or a porcine pancreatic islet.

[0034] [1-16] The transplant device described in [1-15], wherein the pancreatic islet is an adult porcine pancreatic islet.

[0035] [1-17] The transplant device described in [1-15], wherein the pancreatic islet is a fetal, neonatal, or perinatal porcine islet.

[0036] [1-18] The transplant device according to any one of [1-1] to [1-14], wherein the insulin-secreting cells or pancreatic islets are pancreatic islets, pancreatic islet cells, or beta cells obtained from a human donor, pancreatic islets, pancreatic islet cells, or beta cells obtained from a pig donor, or pancreatic islets, pancreatic islet cells, or beta cells obtained by differentiation from human-derived stem cells.

[0037] [1-19] The transplant device described in [1-18], wherein the insulin-secreting cells or pancreatic islets are pancreatic islets, pancreatic islet cells, or beta cells obtained from a donor human.

[0038] [1-20] The transplantation device described in any one of [1-1] to [1-19], wherein the hydrogel is further covered with a semipermeable membrane.

[0039] [1-21] The implantable device described in [1-20], wherein the semipermeable membrane is a dialysis membrane formed from a cellulose derivative.

[0040] [1-22] The implantable device described in [1-21], wherein the cellulose derivative is cellulose acetate.

[0041] [1-23] The implantation device according to any one of [1-1] to [1-22], wherein the implantation site of the implantation device is subcutaneous or intraperitoneal.

[0042] [1-24] The implantation device described in any one of [1-1] to [1-23] above, wherein the thickness of the implantation device is 0.1 to 5 mm.

[0043] [1-25] The implantation device described in any one of [1-1] to [1-23] above, wherein the thickness of the implantation device is 100 μm or more and less than 1000 μm.

[0044] [1-26] The transplant device described in any one of [1-1] to [1-23] above, wherein the thickness of the hydrogel is 0.1 to 5 mm.

[0045] [1-27] The transplant device described in any one of [1-1] to [1-23] above, wherein the thickness of the hydrogel is 100 μm or more and less than 500 μm.

[0046] [1-28] The transplant device according to any one of [1-1] to [1-27] above, wherein a hydrogel containing the insulin-secreting cells or pancreatic islets is prepared and then covered with a semipermeable membrane.

[0047] [1-29] The transplant device described in any one of the items [1-1] to [1-28] above, which is obtained by suspending insulin-secreting cells or pancreatic islets in a solution of an alginic acid derivative that is hydrogelled by chemical crosslinking, encapsulating the solution of the suspended insulin-secreting cells or pancreatic islets in a semipermeable membrane, and then contacting the semipermeable membrane with a solution containing a divalent metal ion to gel the alginic acid derivative in the semipermeable membrane.

[0048] [1-30] The implantable device described in [1-29], wherein the solution containing the divalent metal ion is a solution containing calcium ions.

[0049] [1-31] ​​The transplant device described in any one of [1-1] to [1-30], wherein when three pieces of the hydrogel measuring 10 mm in length and 20 mm in width are shaken and stirred in 12 mL of saline solution at 37°C for 96 hours under conditions of reciprocal shaking at an amplitude of 25 mm and a shaking speed of 180 rpm while maintaining the temperature at 37°C, the hydrogel does not disintegrate.

[0050] [1-32] The transplant device described in any one of [1-1] to [1-31], wherein when three pieces of the hydrogel measuring 10 mm in length and 20 mm in width are shaken and stirred in 12 mL of saline solution at 37°C for 96 hours under conditions of reciprocal shaking at an amplitude of 25 mm and a shaking speed of 180 rpm while maintaining the temperature at 37°C, the disintegration rate of the hydrogel is 30% or less.

[0051] [1-33] The transplant device described in any one of [1-1] to [1-32], in which when three pieces of the hydrogel measuring 10 mm in length and 20 mm in width are shaken and stirred in 12 mL of saline solution at 37°C for 24 hours under conditions of reciprocal shaking at an amplitude of 25 mm and a shaking speed of 180 rpm while maintaining the temperature at 37°C, almost no cells fall off from the hydrogel.

[0052] [1-34] The transplant device described in any one of [1-1] to [1-33], wherein when three pieces of the hydrogel measuring 10 mm in length and 20 mm in width are shaken and stirred in 12 mL of saline solution at 37°C for 24 hours under conditions of reciprocal shaking at an amplitude of 25 mm and a shaking speed of 180 rpm while maintaining the temperature at 37°C, the rate of cells falling off from the hydrogel after shaking and stirring is 30% or less, assuming that the number of cells encapsulated in the hydrogel is 100%.

[0053] The second aspect of the present invention may be as follows: [2-1] to [2-34].

[0054] [2-1] A transplantation device comprising a hydrogel in which insulin-secreting cells or pancreatic islets are encapsulated, the hydrogel being formed by gelling an alginic acid derivative by chemical crosslinking; A transplant device, wherein the thickness of the hydrogel is greater than or equal to 100 μm and less than 500 μm.

[0055] [2-2] The transplant device described in [2-1], wherein the thickness of the hydrogel is greater than or equal to 150 μm and less than 500 μm.

[0056] [2-3] The transplant device described in [2-1] or [2-2], wherein the thickness of the hydrogel is 200 μm or more and less than 300 μm.

[0057] [2-4] The transplant device described in any one of [2-1] to [2-3] above, wherein the hydrogel contains chemical crosslinks formed between alginic acid derivatives as crosslinks.

[0058] [2-5] The transplant device described in any one of [2-1] to [2-4] above, wherein the hydrogel contains chemical crosslinks via triazole rings formed by a Huisgen reaction as crosslinks.

[0059] [2-6] The transplant device described in any one of [2-1] to [2-5], wherein the chemical crosslinking is generated by 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.

[0060] [2-7] The implantable device described in [2-6], wherein the chemical crosslinking is generated by a cyclic alkyne group introduced into any one or more carboxyl groups of the first alginic acid and an azide group introduced into any one or more carboxyl groups of the second alginic acid.

[0061] [2-8] The transplant device according to any one of [2-1] to [2-7] above, wherein the chemical crosslinking is a chemical crosslinking by a combination of the alginic acid derivatives described in the following (A) and (B): (A): An amide bond and a divalent linker (-L 1A cyclic alkyne group (Akn) is introduced via a cyclic alkyne group (Akn) of the following formula (HA-I): [ka] [In formula (HA-I), (ALG) represents alginic acid; -NHCO- represents an amide bond via any carboxyl group of alginic acid; -L 1 - is the following partial structural formula (each formula does not include the areas outside the dashed lines at both ends): [ka] A represents a divalent linker selected from the group consisting of the following partial structural formulas (each formula does not include the right side of the dashed line): [ka] wherein the asterisk represents a chiral center; (B): An amide bond and a divalent linker (-L 2 -), an azide group has been introduced via the following formula (HA-II): [ka] [In formula (HA-II), (ALG) represents alginic acid; -NHCO- represents an amide bond via any carboxyl group of alginic acid; -L 2 - is the following partial structural formula (each formula does not include the areas outside the dashed lines at both ends): [ka] The alginic acid derivative is represented by the following formula:

[0062] [2-9] The chemically crosslinked alginic acid derivative is such that any carboxyl group of the first alginic acid and any carboxyl group of the second alginic acid are represented by the following formula (HA-III-L): [ka] [In formula (HA-III-L), -CONH- and -NHCO- at both ends represent an amide bond via any carboxyl group of alginic acid; -L 1 - is as defined in the above embodiment [2-8]; -L 2 - is as defined in the above embodiment [2-8]; X is the following partial structural formula: [ka] The implantable device according to claim 2-8, wherein the alginate is linked via a cyclic group selected from the group consisting of: (in each formula, the areas outside the dashed lines at both ends are not included; and an asterisk represents a chiral center).

[0063] [2-10] The transplant device according to any one of [2-1] to [2-7], wherein the chemical crosslinking is a chemical crosslinking by a combination of the alginic acid derivatives described in the following (A) and (B): (A): An amide bond and a divalent linker (-L 1 A cyclic alkyne group (Akn) is introduced via a cyclic alkyne group (Akn) of the following formula (HB-I): [ka] [In formula (HB-I), (ALG) represents alginic acid; -NHCO- represents an amide bond via any carboxyl group of alginic acid; -L 1 -See the table below: [Table 5-1] [Table 5-2] [In each formula, the areas outside the dashed lines at both ends are not included]; Akn is shown in the table below: [Table 6] an alginic acid derivative represented by the partial structural formula (I) shown in the formula (I) (each formula does not include the right side of the dashed line); (B): An amide bond and a divalent linker (- L 2 -), an azide group is introduced via the following formula (HB-II): [ka] [In formula (HB-II), (ALG) represents alginic acid; -NHCO- represents an amide bond via any carboxyl group of alginic acid; -L 2 -See the table below: [Table 7-1] [Table 7-2] [wherein each formula does not include the areas outside the dashed lines at both ends]] (However, in the alginic acid derivative represented by the formula (HB-I), -L 1 - is any one linker selected from the group consisting of (L1-1), (L1-2a), (L1-2b), (L1-11) and (L1-12); and an alginic acid derivative represented by formula (HB-II), in which -L 2 - Excluding chemical crosslinking by combination with a derivative in which (L2-10) is the linker).

[0064] [2-11] The chemically crosslinked alginic acid derivative is such that any carboxyl group of the first alginic acid and any carboxyl group of the second alginic acid are represented by the following formula (HB-III-L): [ka] [In formula (HB-III-L), -CONH- and -NHCO- at both ends represent an amide bond via any carboxyl group of alginic acid; -L 1 - is as defined in [2-10] above; -L 2 - is as defined in [2-10] above; X is as shown in the following table: [Table 8-1] [Table 8-2] (In each formula, the areas outside the dashed lines at both ends are not included.) (However, in the alginic acid derivative represented by formula (HB-I), -L 1 - is any one linker selected from the group consisting of (L1-1), (L1-2a), (L1-2b), (L1-11) and (L1-12); and an alginic acid derivative represented by formula (HB-II), in which -L 2 The implantable device described in [2-10] above (excluding cross-linked alginic acid in combination with a derivative in which - is the linker of (L2-10)).

[0065] [2-12] The alginic acid derivative of the formula (HA-I) is the following formula (EX-1-(I)-A-2): [ka] The alginic acid derivative of formula (HA-II) is represented by the following formula (EX-2-(II)-A-2): [ka] The implantable device described in [2-8] or [2-9].

[0066] [2-13] The alginic acid derivative of the formula (HA-I) is the following formula (EX-3-(I)-A-2): [ka] The alginic acid derivative of formula (HA-II) is represented by the following formula (EX-4-(II)-A-2): [ka] The implantable device described in [2-8] or [2-9].

[0067] [2-14] The alginic acid derivative of the formula (HA-I) is represented by the following formula (EX-1-(I)-A-2): [ka] The alginic acid derivative of formula (HA-II) is represented by the following formula (EX-4-2-(II)-A-2): [ka] The implantable device described in [2-8] or [2-9].

[0068] [2-15] The alginic acid derivative of the formula (HA-I) is the following formula (EX-3-(I)-A-2): [ka] The alginic acid derivative of formula (HA-II) is represented by the following formula (EX-2-(II)-A-2): [ka] The implantable device described in [2-8] or [2-9].

[0069] [2-16] The alginic acid derivative of the formula (HA-I) is the following formula (EX-3-(I)-A-2): [ka] The alginic acid derivative of formula (HA-II) is represented by the following formula (EX-4-2-(II)-A-2): [ka] The implantable device described in [2-8] or [2-9].

[0070] [2-17] The transplant device described in any one of [2-1] to [2-16] above, wherein the pancreatic islet is a human pancreatic islet or a porcine pancreatic islet.

[0071] [2-18] The transplant device described in [2-17], wherein the pancreatic islet is an adult porcine pancreatic islet.

[0072] [2-19] The transplant device described in [2-17], wherein the pancreatic islet is a fetal, neonatal, or perinatal porcine islet.

[0073] [2-20] The transplant device described in any one of [2-1] to [2-19], wherein the insulin-secreting cells or pancreatic islets are pancreatic islets, pancreatic islet cells, or beta cells obtained from a human donor, pancreatic islets, pancreatic islet cells, or beta cells obtained from a pig donor, or pancreatic islets, pancreatic islet cells, or beta cells obtained by differentiation from human-derived stem cells.

[0074] [2-21] The transplant device described in [2-20], wherein the insulin-secreting cells or pancreatic islets are pancreatic islets, pancreatic islet cells, or beta cells obtained from a donor human.

[0075] [2-22] The transplantation device described in any one of [2-1] to [2-21] above, wherein the hydrogel is further covered with a semipermeable membrane.

[0076] [2-23] The transplant device described in [2-22], wherein the semipermeable membrane is a dialysis membrane formed from a cellulose derivative.

[0077] [2-24] The implantable device described in [2-23], wherein the cellulose derivative is cellulose acetate.

[0078] [2-25] The implantation device according to any one of [2-1] to [2-24], wherein the implantation site of the implantation device is subcutaneous or intraperitoneal.

[0079] [2-26] The transplant device described in any one of [2-1] to [2-25] above, wherein the thickness of the transplant device is 100 μm or more and less than 1000 μm.

[0080] [2-27] The implantable device described in [2-26], wherein the thickness of the implantable device is 150 μm or more and less than 500 μm.

[0081] [2-28] The transplant device according to any one of [2-1] to [2-27] above, wherein a hydrogel containing the insulin-secreting cells or pancreatic islets is prepared and then covered with a semipermeable membrane.

[0082] [2-29] The transplant device described in any one of the items [2-1] to [2-28] above, which is obtained by suspending insulin-secreting cells or pancreatic islets in a solution of an alginic acid derivative that is hydrogelled by chemical crosslinking, encapsulating the solution of the suspended insulin-secreting cells or pancreatic islets in a semipermeable membrane, and then contacting the semipermeable membrane with a solution containing a divalent metal ion to gel the alginic acid derivative in the semipermeable membrane.

[0083] [2-30] The implantable device described in [2-29], wherein the solution containing the divalent metal ion is a solution containing calcium ions.

[0084] [2-31] The transplant device described in any one of [2-1] to [2-30], wherein when three pieces of the hydrogel measuring 10 mm in length and 20 mm in width are shaken and stirred in 12 mL of saline solution at 37°C for 96 hours under conditions of reciprocal shaking at an amplitude of 25 mm and a shaking speed of 180 rpm while maintaining the temperature at 37°C, the hydrogel does not disintegrate.

[0085] [2-32] The transplant device described in any one of [2-1] to [2-31], wherein when three pieces of the hydrogel measuring 10 mm in length and 20 mm in width are shaken and stirred in 12 mL of saline solution at 37°C for 96 hours under conditions of reciprocal shaking at an amplitude of 25 mm and a shaking speed of 180 rpm while maintaining the temperature at 37°C, the disintegration rate of the hydrogel is 30% or less.

[0086] [2-33] The transplant device described in any one of [2-1] to [2-32], in which when three pieces of the hydrogel measuring 10 mm in length and 20 mm in width are shaken and stirred in 12 mL of saline solution at 37°C for 24 hours under conditions of reciprocal shaking at an amplitude of 25 mm and a shaking speed of 180 rpm while maintaining the temperature at 37°C, almost no cells fall off from the hydrogel.

[0087] [2-34] The transplant device described in any one of [2-1] to [2-33], wherein when three pieces of the hydrogel measuring 10 mm in length and 20 mm in width are shaken and stirred in 12 mL of saline solution at 37°C for 24 hours under conditions of reciprocal shaking at an amplitude of 25 mm and a shaking speed of 180 rpm while maintaining the temperature at 37°C, the rate of cells falling off from the hydrogel after shaking and stirring is 30% or less, when the number of cells encapsulated in the hydrogel is taken as 100%.

[0088] The third aspect of the present invention may be as follows: [3-1] to [3-4].

[0089] [3-1] A method for producing a transplant device comprising a hydrogel in which insulin-secreting cells or pancreatic islets are encapsulated, comprising the following steps (a) to (d): Step (a): As an optional step, a step of removing the pancreas from a living body and isolating the pancreatic islets; Step (b): A step of mixing a cell or tissue selected from the group consisting of insulin-secreting cells, pancreatic islets, cultured pancreatic islet cells, and pancreatic islet cells differentiated from stem cells with a solution of an alginic acid derivative that can be hydrogelled by chemical crosslinking; Step (c): contacting the solution of the alginic acid derivative obtained in step (b) with a solution containing a divalent metal ion to prepare a gel having a thickness of 0.1 to 5 mm (100 to 5000 μm); Step (d): As an optional step, coating the gel obtained in step (c) with a semipermeable membrane.

[0090] [3-2] A method for producing a transplant device comprising a hydrogel in which insulin-secreting cells or pancreatic islets are encapsulated, comprising the following steps (a) to (d): Step (a): As an optional step, a step of removing the pancreas from a living body and isolating the pancreatic islets; Step (b): A step of mixing a cell or tissue selected from the group consisting of insulin-secreting cells, pancreatic islets, cultured pancreatic islet cells, and pancreatic islet cells differentiated from stem cells with a solution of an alginic acid derivative that can be hydrogelled by chemical crosslinking; Step (c): contacting the solution of the alginic acid derivative obtained in step (b) with a solution containing a divalent metal ion to prepare a gel having a thickness of 100 μm or more and less than 500 μm; Step (d): As an optional step, coating the gel obtained in step (c) with a semipermeable membrane.

[0091] [3-3] A method for producing a transplant device comprising a hydrogel in which insulin-secreting cells or pancreatic islets are encapsulated, comprising the following steps (a) to (d): Step (a): As an optional step, a step of removing the pancreas from a living body and isolating the pancreatic islets; Step (b): A step of mixing a cell or tissue selected from the group consisting of insulin-secreting cells, pancreatic islets, cultured pancreatic islet cells, and pancreatic islet cells differentiated from stem cells with a solution of an alginic acid derivative that can be hydrogelled by chemical crosslinking; Step (c): encapsulating the solution of the alginic acid derivative obtained in step (b) in a semipermeable membrane; Step (d): A step of contacting the semipermeable membrane obtained in step (c) with a solution containing a divalent metal ion to gel the solution of the alginic acid derivative in the semipermeable membrane.

[0092] [3-4] The method for producing the implantation device according to any one of [3-1] to [3-3] above, wherein the solution containing the divalent metal ion is a solution containing calcium ions.

[0093] [3-5] A method for producing a transplant device comprising a hydrogel in which insulin-secreting cells or pancreatic islets are encapsulated, comprising the following steps: (a) (b) (e) (d) Step (a): As an optional step, a step of removing the pancreas from a living body and isolating the pancreatic islets; Step (b): A step of mixing a cell or tissue selected from the group consisting of insulin-secreting cells, pancreatic islets, cultured pancreatic islet cells, and pancreatic islet cells differentiated from stem cells with a solution of an alginic acid derivative that can be hydrogelled by chemical crosslinking; Step (e): forming a gel from the solution of the alginic acid derivative obtained in step (b) into any desired shape; Step (d): As an optional step, coating the gel obtained in step (e) with a semipermeable membrane.

[0094] [3-6] A method for producing a transplant device comprising a hydrogel in which insulin-secreting cells or pancreatic islets are encapsulated, comprising the following steps (a), (b), (e), and (f): Step (a): As an optional step, a step of removing the pancreas from a living body and isolating the pancreatic islets; Step (b): A step of mixing a cell or tissue selected from the group consisting of insulin-secreting cells, pancreatic islets, cultured pancreatic islet cells, and pancreatic islet cells differentiated from stem cells with a solution of an alginic acid derivative that can be hydrogelled by chemical crosslinking; Step (e): placing the solution of the alginic acid derivative obtained in step (b) in any container or on any surface; Step (f): Gelling the solution of alginic acid derivative in the container or on the surface obtained in step (e).

[0095] [3-7] A method for manufacturing an implantable device described in [3-6], wherein the any container is a semipermeable membrane, a beaker, or a petri dish.

[0096] [3-8] A method for manufacturing an implantable device described in [3-6], wherein the any surface is a semipermeable membrane surface, a plastic surface or a glass surface.

[0097] [3-9] The method for producing the transplantation device described in any one of [3-5] to [3-8] above, wherein the hydrogel has a thickness of 0.1 to 5 mm (100 to 5000 μm).

[0098] [3-10] A method for producing a transplant device described in any one of [3-5] to [3-9] above, wherein the gelation of the alginic acid derivative solution does not include a step of contacting the solution with a divalent metal ion or a solution thereof.

[0099] [3-11] A method for producing a transplant device comprising a hydrogel in which insulin-secreting cells or pancreatic islets are encapsulated, comprising the following steps (a) to (c) and (f): Step (a): As an optional step, a step of removing the pancreas from a living body and isolating the pancreatic islets; Step (b): A step of mixing a cell or tissue selected from the group consisting of insulin-secreting cells, pancreatic islets, cultured pancreatic islet cells, and pancreatic islet cells differentiated from stem cells with a solution of an alginic acid derivative that can be hydrogelled by chemical crosslinking; Step (c): encapsulating the solution of the alginic acid derivative obtained in step (b) in a semipermeable membrane; Step (f): A step of gelling the solution of the alginic acid derivative in the semipermeable membrane obtained in step (c) to prepare a gel having a thickness of 0.1 to 5 mm (100 to 5000 μm). Effect of the Invention

[0100] The present invention provides a novel implantable device, which preferably exhibits at least one of the following advantages: (1) It has excellent biocompatibility and stability, low cytotoxicity, and almost no adhesion or inflammation at the transplant site. (2) The gel dissolves little and its shape is maintained for a long period of time. (3) The blood sugar lowering effect can be sustained for a long period of time, making it possible to regulate blood sugar. (4) Even after long-term use, the alginate gel in the semipermeable membrane does not dissolve and can maintain its shape, and the survival and function of the pancreatic islets can be maintained, making it suitable for long-term use. (5) It is replaceable, capable of immunoisolation, and causes little adhesion or inflammation, making it a highly safe medical material. (6) Excellent material permeability inside and outside the device. (7) When the hydrogel is shaken, the hydrogel does not collapse or collapses only slightly. (8) When the hydrogel is shaken, the islets contained in the hydrogel are hardly removed from the gel. Moreover, in a transplant device in which the thickness of the hydrogel is thin (for example, less than 500 μm), it is preferable that at least one of the effects (1) to (8) and / or one or more of the subordinate effects are exhibited. (9) The healing rate when the device was implanted was higher than that when thicker hydrogels were used. (10) The ratio of oxygen concentration at the center of the device to that at the surface is high.

[0101] A more preferred embodiment of the transplantation device has excellent transplantation results and functionality, is made of a novel material, and can maintain a blood glucose lowering effect and regulate blood glucose for a long period of time by transplanting it into a diabetic patient (particularly type I diabetes and insulin-deficient type II diabetes). In addition, when the function of insulin-secreting cells or pancreatic islets in the hydrogel is reduced, they can be retrieved. Alternatively, periodic replacement or additional transplantation becomes possible. In addition, insulin-secreting cells differentiated from stem cells (such as iPS cells) or human pancreatic islets can be used as the insulin-secreting cells or pancreatic islets to be encapsulated in the hydrogel of the transplantation device. Therefore, the device of a more preferred embodiment is useful. [Brief description of the drawings]

[0102] [Figure 1] Photographs of flat alginate gel: (a) before transplantation, (b) after transplantation. [Diagram 2] Photographs of flat alginate gel: (a) before transplantation, (b) after transplantation. [Diagram 3] Photographs of flat alginate gel: (a) before transplantation, (b) after transplantation. [Figure 4-1] 1 is a photograph of the implantable device produced. [Figure 4-2] 1 is a photograph of the implantable device produced. [Figure 5-1] FIG. 13 is a graph showing blood glucose level fluctuations in mice implanted with an implantable device (up to day 75 after implantation). [Figure 5-2] FIG. 13 is a graph showing blood glucose level fluctuations in mice implanted with an implantable device (up to day 305 after implantation). [Figure 5-3] FIG. 13 is a graph showing blood glucose level fluctuations in mice implanted with an implantable device (up to day 26 after relay implantation). [Figure 6-1] FIG. 13 is a graph showing changes in body weight of mice implanted with an implantable device (up to day 75 after implantation). [Figure 6-2] FIG. 13 is a graph showing changes in body weight of mice implanted with implantation devices (up to day 305 after implantation). [Figure 6-3] FIG. 13 shows changes in body weight of mice implanted with implantable devices (up to day 26 after relay implantation). [Figure 7-1] FIG. 13 is a graph showing blood glucose level fluctuations in mice implanted with an implantable device (up to day 305 after implantation). [Figure 7-2] FIG. 13 is a graph showing blood glucose level fluctuations in mice implanted with an implantable device (up to day 26 after relay implantation). [Figure 8-1] FIG. 13 is a graph showing changes in body weight of mice implanted with implantation devices (up to day 305 after implantation). [Figure 8-2] FIG. 13 shows changes in body weight of mice implanted with implantable devices (up to day 26 after relay implantation). [Figure 9-1] FIG. 13 is a graph showing blood glucose level fluctuations in mice implanted with an implantable device (up to day 305 after implantation). [Figure 9-2] FIG. 13 is a graph showing blood glucose level fluctuations in mice implanted with an implantable device (up to day 26 after relay implantation). [Figure 10-1] FIG. 13 is a graph showing changes in body weight of mice implanted with implantation devices (up to day 305 after implantation). [Figure 10-2]FIG. 13 shows changes in body weight of mice implanted with implantable devices (up to day 26 after relay implantation). [Figure 11-1] FIG. 13 is a graph showing blood glucose level fluctuations in mice implanted with an implantable device (up to day 178 after implantation). [Figure 11-2] FIG. 13 is a graph showing blood glucose level fluctuations in mice implanted with an implantable device (up to day 178 after implantation). [Figure 11-3] FIG. 13 is a graph showing blood glucose level fluctuations in mice implanted with an implantable device (up to day 40 after relay implantation). [Figure 11-4] FIG. 13 is a graph showing blood glucose level fluctuations in mice implanted with an implantable device (up to day 178 after implantation). [Figure 11-5] FIG. 13 is a graph showing blood glucose level fluctuations in mice implanted with an implantable device (up to day 40 after relay implantation). [Figure 12-1] FIG. 13 is a graph showing changes in body weight of mice implanted with a transplantation device (up to day 178 after implantation). [Figure 12-2] FIG. 13 is a graph showing changes in body weight of mice implanted with a transplantation device (up to day 178 after implantation). [Figure 12-3] FIG. 13 shows changes in body weight of mice implanted with implantable devices (up to day 40 after relay implantation). [Figure 12-4] FIG. 13 is a graph showing changes in body weight of mice implanted with a transplantation device (up to day 178 after implantation). [Figure 12-5] FIG. 13 shows changes in body weight of mice implanted with implantable devices (up to day 40 after relay implantation). [Figure 13-1] FIG. 13 is a graph showing blood glucose level fluctuations in mice implanted with an implantable device (up to day 17 after implantation). [Figure 13-2] FIG. 13 is a graph showing blood glucose level fluctuations in mice implanted with an implantable device (up to day 17 after implantation). [Figure 13-3] FIG. 13 is a graph showing blood glucose level fluctuations in mice implanted with an implantable device (up to day 17 after implantation). [Figure 13-4] FIG. 13 is a graph showing blood glucose level fluctuations in mice implanted with an implantable device (up to day 17 after implantation). [Figure 13-5] FIG. 13 is a graph showing blood glucose level fluctuations in mice implanted with an implantable device (up to day 17 after implantation). [Figure 14-1] FIG. 13 is a graph showing changes in body weight of mice implanted with a transplantation device (up to day 17 after implantation). [Figure 14-2] FIG. 13 is a graph showing changes in body weight of mice implanted with a transplantation device (up to day 17 after implantation). [Figure 14-3] FIG. 13 is a graph showing changes in body weight of mice implanted with a transplantation device (up to day 17 after implantation). [Figure 14-4] FIG. 13 is a graph showing changes in body weight of mice implanted with a transplantation device (up to day 17 after implantation). [Figure 14-5] FIG. 13 is a graph showing changes in body weight of mice implanted with a transplantation device (up to day 17 after implantation). [Figure 15-1] FIG. 1 shows evaluation sites for oxygen concentration in a hydrogel. [Figure 15-2] FIG. 1 shows the oxygen concentration in a cross section of a hydrogel. [Figure 15-3] FIG. 1 shows the oxygen concentration in a cross section of a hydrogel. [Figure 15-4] FIG. 1 shows the oxygen concentration in a cross section of a hydrogel. [Figure 16-1] FIG. 1 shows the permeation rate of human insulin from an implantable device. [Figure 16-2] FIG. 1 shows the permeability of glucose from an implantable device. [Figure 17] FIG. 1 shows the disintegration rate of hydrogels upon shaking. [Figure 18-1] This is a micrograph of the external fluid 24 hours after the start of the test (the bar represents 1000 μm). [Figure 18-2] This is a micrograph of the external fluid 24 hours after the start of the test (the bar represents 1000 μm). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0103] Here, a device for transplanting cells or the like into a living body, prepared using an alginic acid derivative that is gelled by chemical crosslinking, more specifically, a transplant device including, for example, a chemically crosslinked alginic acid gel in which insulin-secreting cells or pancreatic islets or the like are embedded, and a semipermeable membrane that covers the gel as necessary, and a method for producing the same, are provided. The alginic acid derivative that is gelled by chemical crosslinking is, for example, an alginic acid derivative of formula (HA-I) or formula (HA-II) in which a cyclic alkyne group or an azide group is introduced via an amide bond and a divalent linker to any one or more carboxyl groups of alginic acid, and a novel crosslinked alginic acid is obtained by performing a Huisgen reaction (1,3-dipolar cycloaddition reaction) using the alginic acid derivatives of formula (HA-I) and formula (HA-II). Alternatively, the alginic acid derivative to be gelled by chemical crosslinking is, for example, an alginic acid derivative of formula (HB-I) or formula (HB-II) in which a cyclic alkyne group or azide group is introduced via an amide bond and a divalent linker to any one or more carboxyl groups of alginic acid, and a novel crosslinked alginic acid can be obtained by performing a Huisgen reaction (1,3-dipolar cycloaddition reaction) using the alginic acid derivatives of formula (HB-I) and formula (HB-II).

[0104] The "transplant device" refers to a device using a hydrogel in which insulin-secreting cells or pancreatic islets are encapsulated. The hydrogel is an alginic acid derivative that has been gelled by chemical crosslinking. Therefore, the alginic acid derivative used is one that can be gelled by chemical crosslinking. The shape of the hydrogel in which the insulin-secreting cells or pancreatic islets are, for example, flat. In the transplant device, the hydrogel may be further covered with a semipermeable membrane, in which case the hydrogel in which the insulin-secreting cells or pancreatic islets are inserted into the semipermeable membrane.

[0105] The term "insulin-secreting cells" used in the transplantation device means cells having the function of secreting insulin, for example, β-cells that secrete insulin among cells constituting pancreatic islets. The term "insulin-secreting cells" may also be cells that have acquired insulin secretion function through differentiation, maturation, modification, etc., and may include, for example, cells having insulin secretion function obtained by differentiating stem cells such as iPS cells, ES cells, or somatic stem cells (e.g., mesenchymal stem cells), cells having insulin secretion function obtained by maturing immature cells or precursor cells, and cells to which insulin secretion function has been imparted by genetic recombination. Here, differentiating or maturing the cells includes culturing the cells, i.e., cells obtained by differentiation or maturation may include cells obtained by culture. A "pancreatic islet" is also known as an islet of Langerhans and is a cell mass composed of an average of about 2000 islet cells. A pancreatic islet is composed of five types of cells: α cells that secrete glucagon, β cells that secrete insulin, δ cells that secrete somatostatin, ε cells that secrete ghrelin, and PP (pancreatic polypeptide) cells that secrete pancreatic polypeptide. "Insulin-secreting cells or pancreatic islets" are also described as cells or tissues capable of secreting a biologically active product.

[0106] As used herein, the term "pancreatic islet cells" refers to cells that contain at least one of the five types of cells described above, and preferably contain at least β cells. In some embodiments, the pancreatic islet cells may be a mixture containing all of α cells, β cells, δ cells, ε cells, and PP cells, or may be cells contained in pancreatic islets. Furthermore, the "pancreatic islet cells" may be cells that have become pancreatic islet cells through differentiation, maturation, modification, etc. In this case, the "pancreatic islet cells" may include, for example, pancreatic islet cells obtained by differentiating stem cells such as iPS cells, ES cells, and somatic stem cells (e.g., mesenchymal stem cells), as well as pancreatic islet cells obtained by maturing immature cells or precursor cells. It is preferable that "insulin-secreting cells or pancreatic islets (including pancreatic islet cells)" have a degree of viability and function that allows them to recover from a pathological condition of a patient when transplanted into the patient. The function of insulin-secreting cells, pancreatic islets, or pancreatic islet cells includes, for example, secreting insulin, and it is preferable that they maintain glucose responsiveness even after transplantation.

[0107] The "implantation device" in some embodiments is also called a bioartificial pancreatic islet, and is an example of a bioartificial organ. The cells contained in the bioartificial pancreatic islet include the above-mentioned "insulin-secreting cells," "pancreatic islets," or "pancreatic islet cells," and include, for example, insulin-secreting cells. The insulin-secreting cells may be either cells contained in pancreatic islets collected from humans or pigs, or pancreatic islets differentiated from stem cells (e.g., ES cells, iPS cells, and somatic stem cells (e.g., mesenchymal stem cells)).

[0108] The transplant device of the present invention may use cells other than "insulin-secreting cells, pancreatic islets, and pancreatic islet cells."

[0109] As for the cells other than the "insulin-secreting cells, pancreatic islets and pancreatic islet cells", any cells that can be used for cell transplantation can be used, and the type is not particularly limited. In addition, one type of cell may be used, or a combination of multiple types of cells may be used. As the cells used, preferably, animal cells, more preferably vertebrate-derived cells, and particularly preferably human-derived cells can be used. The type of vertebrate-derived cells (particularly human-derived cells) may be any of stem cells (e.g., pluripotent cells or somatic stem cells), precursor cells, or mature cells. As the pluripotent cells, for example, embryonic stem (ES) cells, germline stem (GS) cells, or induced pluripotent stem (iPS) cells can be used. As the somatic stem cells, for example, mesenchymal stem cells (MSCs), hematopoietic stem cells, amniotic cells, umbilical cord blood cells, bone marrow-derived cells, myocardial stem cells, adipose-derived stem cells, or neural stem cells can be used.

[0110] As the precursor cells and mature cells, for example, cells derived from skin, dermis, epidermis, muscle, cardiac muscle, nerve, bone, cartilage, endothelium, brain, epithelium, heart, kidney, liver, spleen, oral cavity, cornea, bone marrow, umbilical cord blood, amniotic membrane, or hair can be used. As the human-derived cells, for example, ES cells, iPS cells, MSCs, chondrocytes, osteoblasts, osteoblast precursor cells, mesenchymal cells, myoblasts, cardiac muscle cells, cardiac muscle blasts, nerve cells, hepatocytes, fibroblasts, corneal endothelial cells, vascular endothelial cells, corneal epithelial cells, amniotic cells, umbilical cord blood cells, bone marrow-derived cells, or hematopoietic stem cells can be used. In addition, the origin of the cells may be either autologous or allogeneic. In some embodiments, among the above, for example, ES cells, iPS cells, and mesenchymal stem cells (MSCs) can be used.

[0111] The donor of the "insulin-secreting cells or pancreatic islets (including pancreatic islet cells)" is an animal, preferably a vertebrate, specifically a human, pig, monkey, rat, or mouse, and more preferably a human or pig. In some embodiments, the donor of the "insulin-secreting cells, pancreatic islets, or pancreatic islet cells" is a pig in order to eliminate the shortage of donors. The "insulin-secreting cells or pancreatic islets (including pancreatic islet cells)" may be either a pancreatic islet obtained from a donor animal, or an insulin-secreting cell or pancreatic islet cell obtained from a donor-derived cell, and may be, for example, an insulin-secreting cell or pancreatic islet cell differentiated from a human-derived ES cell or iPS cell.

[0112] When the "insulin-secreting cells or pancreatic islets (including pancreatic islet cells)" are derived from pigs, examples of the insulin-secreting cells include adult porcine islets, and fetal, neonatal, or perinatal porcine islets. The islets may be appropriately cultured before use, or fetal, neonatal, or perinatal porcine islets may be matured.

[0113] The implantation method of the implantable device can be cut and placed, injected, endoscopic, or laparoscopic. The transplantation site is not particularly limited, and examples thereof include subcutaneous, intraperitoneal, intrahepatic, intramuscular, intraomental, and subrenal capsule, with subcutaneous or intraperitoneal transplantation being preferred.

[0114] Here, the term "semipermeable membrane" refers to a membrane that allows only molecules or ions of a certain size or less to pass through. In a system of a solute that does not pass through a semipermeable membrane and a solvent that shows permeability, when two solutions of different concentrations are in contact with each other through the semipermeable membrane, an osmotic pressure is generated at the separation, and only the solvent passes through. The implantation 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 implantation device of some embodiments is a hydrogel alone (e.g., in which insulin-secreting cells or pancreatic islets are encapsulated), i.e., the hydrogel is not coated with a semipermeable membrane. The implantation device in which the hydrogel is not coated with a semipermeable membrane is preferably one that has excellent biocompatibility and stability, has little cytotoxicity, causes almost no adhesion or inflammation at the implantation site, has little gel dissolution, maintains its shape for a long period of time, and more preferably has a long-term hypoglycemic effect and can regulate blood glucose. In another embodiment of the implantation device, the hydrogel is coated with a semipermeable membrane. Examples of semipermeable membranes include membranes or tubes used in dialysis. Dialysis tubes, cotton cellulose dialysis membranes, regenerated cellulose dialysis membranes, cellulose ester dialysis membranes, etc. can also be used. Examples of product names include Cellu-Sep T Tubular Membrane (Membrane Filtration Products), Spectra Biotech Membrane (REPLIGEN (formerly SPECTRUM)), and Spectra / Por CE Dialysis Tube (REPLIGEN (formerly SPECTRUM)). The "semipermeable membrane" is preferably a semipermeable membrane made of cellulose ester. A specific example is a dialysis membrane, Spectra / PorCE dialysis tube (REPLIGEN (formerly SPECTRUM)). The cellulose ester is more preferably a polymer of cellulose acetate.

[0115] The semipermeable membrane used here contains a resin. The semipermeable membrane can be prepared, for example, by dissolving at least one type of resin in a solvent and solidifying the dissolved resin. Such a resin is not particularly limited. Examples of such a resin include ethylene-vinyl alcohol copolymers, polysulfone polymers, polyacrylonitrile polymers, cellulose polymers such as cellulose acetate, polyamide polymers, and polycarbonate polymers. Cellulose polymers such as cellulose acetate are more preferred.

[0116] The semipermeable membrane used herein has a "molecular weight cutoff". The "molecular weight cutoff" refers to the maximum molecular weight that is not substantially blocked. Molecules having a molecular weight above the molecular weight cutoff are substantially prevented from passing through the semipermeable membrane. The "molecular weight cutoff" of the semipermeable membrane used herein is preferably 100 kDa (kilodaltons). For example, Spectra / Por CE dialysis tube (made by REPLIGEN (formerly SPECTRUM)), which is a cellulose ester dialysis membrane, is sold in standards such as 100-500 Da (daltons), 0.5-1 kDa, 3.5-5 kDa, 8-10 kDa, 20 kDa, 50 kDa, 100 kDa, 300 kDa, and 1000 kDa, with the cutoff value being the "MWCO". For example, when the cutoff value has a molecular weight cutoff of greater than about 500,000 daltons, molecules such as IgG and complement can enter these semipermeable membranes, but host cells such as immune cells are prevented from entering the semipermeable membrane, and insulin, cellular nutrients, and oxygen can pass through the semipermeable membrane. The unit dalton symbol is Da, and 1000 Da means 1 kDa.

[0117] Here, the definition of the thickness of the transplantation device is as described below. In some embodiments, the thickness of the transplantation device is 0.1 to 5 mm (100 to 5000 μm), preferably 0.1 to 3 mm (100 to 3000 μm). Alternatively, it is preferably 0.5 to 5 mm (500 to 5000 μm), more preferably 1 to 3 mm (1000 to 3000 μm). When the hydrogel in which insulin-secreting cells or pancreatic islets are encapsulated is covered with a semipermeable membrane, the thickness of the transplantation device is 0.15 to 5 mm (150 to 5000 μm), preferably 0.2 to 3 mm (200 to 3000 μm) in terms of the thickness of the semipermeable membrane. Alternatively, it is preferably 0.5 to 5 mm (500 to 5000 μm), more preferably 1 to 3 mm (1000 to 3000 μm).

[0118] In the case of a thin transplantation device, the thickness of the transplantation device is 100 μm or more and less than 1000 μm, and preferably 100 μm or more and less than 500 μm. Alternatively, it is preferably 150 μm or more and less than 1000 μm, and more preferably 150 μm or more and less than 500 μm. When the hydrogel in which the insulin-secreting cells or pancreatic islets are encapsulated is covered with a semipermeable membrane, the thickness of the transplantation device is preferably 150 μm or more and less than 1000 μm, and more preferably 150 μm or more and less than 500 μm. Alternatively, it is preferably 200 μm or more and less than 1000 μm, and more preferably 200 μm or more and less than 500 μm.

[0119] The definition of the thickness of the hydrogel is as described below. In some embodiments, the thickness of the hydrogel is 0.1 to 5 mm (100 to 5000 μm), preferably 0.1 to 3 mm (100 to 3000 μm), and more preferably 0.1 to 1 mm (100 to 1000 μm). Alternatively, it is 0.15 to 5 mm (150 to 5000 μm), preferably 0.15 to 3 mm (150 to 3000 μm), and more preferably 0.15 to 1 mm (150 to 1000 μm). Alternatively, it is 0.2 to 5 mm (200 to 5000 μm), preferably 0.2 to 3 mm (200 to 3000 μm), and more preferably 0.2 to 1 mm (200 to 1000 μm). Alternatively, it is 0.5 to 5 mm (500 to 5000 μm), preferably 0.5 to 3 mm (500 to 3000 μm), and more preferably 0.5 to 1 mm (500 to 1000 μm). When the transplantation device includes a semipermeable membrane, the thickness of the hydrogel in the semipermeable membrane is 0.1 to 3 mm (100 to 3000 μm), preferably 0.1 to 2 mm (100 to 2000 μm), and more preferably 0.1 to 1 mm (100 to 1000 μm). Alternatively, it is 0.15 to 3 mm (150 to 3000 μm), preferably 0.15 to 2 mm (150 to 2000 μm), and more preferably 0.15 to 1 mm (150 to 1000 μm). Alternatively, it is 0.2 to 3 mm (200 to 3000 μm), preferably 0.2 to 2 mm (200 to 2000 μm), and more preferably 0.2 to 1 mm (200 to 1000 μm). Alternatively, it is preferably 1 to 3 mm (1000 to 3000 μm), and more preferably 1.5 to 2 mm (1500 to 2000 μm). When the transplantation device does not include a semipermeable membrane, the thickness of the hydrogel is 0.1 to 5 mm (100 to 5000 μm), preferably 0.1 to 3 mm (100 to 3000 μm), and more preferably 0.1 to 1 mm (100 to 1000 μm). Alternatively, it is 0.15 to 5 mm (150 to 5000 μm), preferably 0.15 to 3 mm (150 to 3000 μm), and more preferably 0.15 to 1 mm (150 to 1000 μm). Alternatively, it is 0.2 to 5 mm (200 to 5000 μm), preferably 0.2 to 3 mm (200 to 3000 μm), and more preferably 0.2 to 1 mm (200 to 1000 μm). Alternatively, it is 0.5 to 5 mm (500 to 5000 μm), preferably 0.5 to 3 mm (500 to 3000 μm), and more preferably 0.5 to 1 mm (500 to 1000 μm).

[0120] In the case of a thin hydrogel, the thickness of the hydrogel is 100 μm or more and less than 500 μm, preferably 100 μm or more and less than 400 μm, and more preferably 100 μm or more and less than 300 μm. Alternatively, it is 150 μm or more and less than 500 μm, preferably 150 μm or more and less than 400 μm, more preferably 150 μm or more and less than 300 μm, and even more preferably 200 μm or more and less than 300 μm. Such thickness is the same whether the transplant device includes a semipermeable membrane or does not include a semipermeable membrane.

[0121] The shape of the transplantation device is not particularly limited as long as it is flat. A flat plate means a flat plate having a nearly constant thickness and a wide area. Examples of the plate shape include flat plate shapes such as polygons such as triangles, squares, and pentagons, and circles. The transplantation device preferably has the above-mentioned thickness and has a nearly constant thickness throughout the entire plate. The thickness variation of the plate-shaped transplantation device is preferably within ±20%, more preferably within ±10%. The thickness of the transplantation device is the thickness of the maximum thickness of the transplantation device. For example, when the hydrogel is enclosed in a dialysis tube, which is a semipermeable membrane, the shape of the transplantation device may appear like a rugby ball, with both ends being slightly thinner and the center being thicker than both ends, by sealing both ends of the dialysis tube. In such a shape, the thickness of the transplantation device means the thickness near the center, which is the maximum thickness. The shape of the hydrogel is not particularly limited as long as it is flat. A flat plate means a flat plate having a nearly constant thickness and a wide area. Examples of the plate shape include flat plate shapes such as polygons such as triangles, rectangles, and pentagons, and circles. The hydrogel preferably has the above-mentioned thickness and is nearly constant throughout the plate. The thickness variation of the hydrogel is preferably within ±20%, more preferably within ±10%. The thickness of the hydrogel is the thickness of the maximum thickness of the hydrogel. In some embodiments, the flat hydrogel is a crosslinked alginic acid gel having a minor 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 can also be a circle, a rectangle, a hexagon, an octagon, or the like. The area of ​​the flat hydrogel is, for example, 144 to 270 mm. 2 It can also be expressed as:

[0122] As used herein, "IEQ" is an abbreviation for Islet Equivalents, and is an international unit expressing the amount of pancreatic islets, where pancreatic islets are considered to be spherical and an islet with a diameter of 150 μm is defined as 1 IEQ.

[0123] According to the Japanese Society of Pancreas and Islet Transplantation's standard for fresh islet transplantation (islet transplantation manual), one of the conditions for transplanting fresh islets is "islet mass of 5000 IEQ / kg (patient weight) or more," which is also used here as a reference. The transplantation device can be set appropriately to the number of islets calculated to produce the desired therapeutic effect, and it is possible to set an appropriate device depending on the patient's weight, the severity of symptoms, etc. The amount of insulin-secreting cells can also be appropriately determined in accordance with the amount of pancreatic islets.

[0124] A method for manufacturing an implantable device will now be described in more detail. In the method for producing a transplantation device, "step (a): as an optional step, a step of removing a pancreas from a living organism and isolating pancreatic islets" means that step (a) is optional. The "living organism" is, for example, a human or a non-human mammal, and an example of a non-human mammal is a pig. When performing step (a), for example, in the case of isolating porcine islets, a sterile viable pancreas can be obtained from an adult pig under aseptic conditions and islet cells can be isolated according to a known procedure in the art, or the method described in Shimoda et al. (Cell Transplantation, Vol. 21, pp. 501-508, 2012), or the standard Ricordi technique using the Edmonton protocol. Isolation of islets from other non-human mammals or human islets can also be performed according to the isolation of porcine islets. Thereafter, the isolated islets may be used as they are, or may be cultured and used. Pancreatic islets were cultured in a medium (Connaught Medical Research Laboratory (CMRL)-based Miami-defined media #1 (MM1; Mediatech-Cellgro, Herndon, VA)-supplemented with 0.5% human serum albumin) at 5% CO 2The cells can be cultured for one day at 37°C in a humidified atmosphere of 95% air.

[0125] Next, in "step (b): a step of mixing a solution of an alginic acid derivative capable of hydrogelling by chemical crosslinking with cells or tissues selected from the group consisting of insulin-secreting cells, pancreatic islets, pancreatic islet cells obtained by culture, and pancreatic islet cells obtained by differentiation from stem cells", examples of alginic acid derivatives capable of hydrogelling by chemical crosslinking include the alginic acid derivatives represented by the above formulas (I) and (II). In step (b), for example, a 0.1-5 wt % aqueous solution or physiological saline solution of the alginic acid derivative is prepared, and a required amount of cells or tissues selected from the group consisting of insulin-secreting cells, pancreatic islets, pancreatic islet cells obtained by culture, and pancreatic islet cells obtained by differentiation from stem cells (for example, the pancreatic islets obtained in step (a), insulin-secreting cells isolated from the pancreatic islets, or pancreatic islet cells obtained by culturing the pancreatic islet cells isolated from the pancreatic islets) is appropriately suspended in the solution. Here, the "solution of alginic acid derivative capable of hydrogelation by chemical crosslinking" refers to two types of solutions, for example, a solution of an alginic acid derivative represented by the above formula (HA-I) and a solution of an alginic acid derivative represented by the above formula (HA-II). Alternatively, the "solution of alginic acid derivative capable of hydrogelation by chemical crosslinking" refers to two types of solutions, for example, a solution of an alginic acid derivative represented by the above formula (HB-I) and a solution of an alginic acid derivative represented by the above formula (HB-II). In this case, in step (b), these two types of solutions and the solutions in which cells or tissues are mixed therein are prepared separately without being mixed. At this time, cells or tissues may be mixed only in one of the two types of solutions, or may be mixed in both.

[0126] Next, in "step (c): contacting the solution of the alginic acid derivative obtained in step (b) with a solution containing a divalent metal ion to prepare a gel having a thickness of 0.1 to 5 mm (100 to 5000 μm) or a thickness of 100 μm or more and less than 500 μm", the solution of the alginic acid derivative obtained in step (b) in which cells or tissues (e.g., pancreatic islets) are suspended is gelled. At this time, first, a solution of the alginic acid derivative represented by formula (HA-I) or formula (HB-I) and a solution of the alginic acid derivative represented by formula (HA-II) or formula (HB-II) may be mixed in appropriate amounts according to the introduction rate of each chemical crosslinking group. Next, the mixed solution is contacted with a solution containing a divalent metal ion, so that ionic crosslinking and chemical crosslinking proceed at the same time, and a gel can be prepared. More specifically, the gel can be prepared in the same manner as in the <General Preparation Method> of [Production of Plate-Type Alginic Acid Gel] described in Example 5 below. Then, "step (d): as an optional step, a step of covering the gel obtained in step (c) with a semipermeable membrane" means that step (d) is optional. When step (d) is performed, the gel obtained in step (c) is covered with a semipermeable membrane by a method known in the art or a method equivalent thereto. For example, the gel is covered by inserting it into a semipermeable membrane (e.g., a tube of a semipermeable membrane sealed at one end) and sealing the other end.

[0127] Alternatively, in the "step (c): step of encapsulating the solution of the alginic acid derivative obtained in step (b) in a semipermeable membrane", the solution of the alginic acid derivative obtained in step (b) in which cells or tissues (e.g., pancreatic islets) are suspended is covered with a semipermeable membrane by a method known in the art or a method equivalent thereto. At this time, first, a solution of the alginic acid derivative represented by formula (HA-I) or formula (HB-I) and a solution of the alginic acid derivative represented by formula (HA-II) or formula (HB-II) may be mixed in appropriate amounts according to the introduction rate of each chemical crosslinking group. Next, the mixed solution in which the cells or tissues (e.g., pancreatic islets) are suspended is inserted into a semipermeable membrane (e.g., a tube of a semipermeable membrane sealed at one end) and covered by sealing the other end. Next, in "step (d): contacting the semipermeable membrane obtained in step (c) with a solution containing a divalent metal ion to gel the alginic acid solution in the semipermeable membrane", the semipermeable membrane encapsulating the alginic acid solution obtained in step (c) is contacted with a solution containing a divalent metal ion to gel the alginic acid solution in the semipermeable membrane.

[0128] The device obtained in step (d) may be washed with a solvent such as physiological saline, or may be cultured in a medium for a predetermined period of time.

[0129] "Step (e): a step of forming a gel by forming the solution of the alginic acid derivative obtained in step (b) into an arbitrary shape" or "step (e): a step of placing the solution of the alginic acid derivative obtained in step (b) in an arbitrary container or on an arbitrary surface" refers to a step of keeping the solution of the alginic acid derivative obtained in step (b) in which cells or tissues (e.g., pancreatic islets) are suspended in a certain shape using an arbitrary container, surface, etc., by a method known in the art or a method equivalent thereto, and gelling the solution by the progress of chemical crosslinking. At this time, first, a solution of the alginic acid derivative represented by formula (HA-I) or formula (HB-I) and a solution of the alginic acid derivative represented by formula (HA-II) or formula (HB-II) may be mixed in appropriate amounts according to the introduction rate of each chemical crosslinking group. The arbitrary container refers to a container capable of retaining a solution in a certain shape, and examples thereof include a semipermeable membrane, a beaker, and a petri dish, with a semipermeable membrane being preferred. The arbitrary surface refers to a surface onto which the solution can be placed in a certain shape, and examples thereof include a semipermeable membrane surface, a plastic surface, or a glass surface, with a semipermeable membrane surface being preferred.

[0130] "Step (f): gelling the solution of the alginic acid derivative in the container or on the surface obtained in step (e)" or "Step (f): gelling the solution of the alginic acid derivative in the semipermeable membrane obtained in step (c) to produce a gel having a thickness of 0.1 to 5 mm (100 to 5000 μm)" means gelling the solution held in any container, surface, or semipermeable membrane by chemical crosslinking. At this time, the gel may be produced by simultaneously proceeding with ionic crosslinking and chemical crosslinking by contacting with a solution containing a divalent metal ion, or the gel may be produced by proceeding only with chemical crosslinking without contacting with a divalent metal ion or its solution.

[0131] The solution containing "divalent metal ions" used in the implantation device includes solutions containing calcium ions, barium ions, strontium ions, etc. A solution containing calcium ions or barium ions is preferable, and a solution containing calcium ions is more preferable. A solution containing a divalent metal ion can be obtained, for example, by dissolving a salt of the divalent metal ion in a solvent. Examples of the salt of the divalent metal ion include calcium chloride, barium chloride, and strontium chloride. Examples of the solvent include water, physiological saline, and HEPES buffer solution. In some embodiments, the solution containing divalent metal ions is a solution containing calcium ions, preferably an aqueous solution containing calcium chloride. The amount of the solution containing the divalent metal ion to be used is desirably adjusted appropriately depending on the amount and molecular weight of the alginic acid derivative to be used.

[0132] When the transplant device has a semipermeable membrane, the hydrogel in the device may be prepared either by encapsulating a solution of an alginic acid derivative in the semipermeable membrane and then contacting it with a divalent metal ion solution, or by gelling the alginic acid derivative before encapsulating it in the semipermeable membrane and then encapsulating it in the semipermeable membrane. Here, "contact" includes, for example, immersing a semipermeable membrane containing a solution of an alginic acid derivative in a divalent metal ion solution, or pouring a divalent metal ion solution onto a semipermeable membrane containing a solution of an alginic acid derivative.

[0133] The hydrogel used in the implant device refers to a water-insoluble polymer with a three-dimensional network structure and its water-swollen body. Here, hydrogel may be simply called gel.

[0134] By changing the concentration of the polymer used in preparing the hydrogel, the molecular weight of the molecules that can pass through the original mesh structure of the gel can be freely changed. In other words, it is thought that the mesh of the gel will be small when the concentration of the polymer is high, and large when the concentration of the polymer is low. If the mesh of the mesh structure is too large, antibodies, etc. will penetrate into the mesh structure. In this case, a rejection reaction is likely to occur against the insulin-secreting cells or pancreatic islets in the gel. The rejection reaction inhibits the production of necessary substances such as insulin.

[0135] In general, hydrogel materials are made of polymers such as collagen, hyaluronan, gelatin, fibronectin, elastin, tenascin, laminin, vitronectin, polypeptides, heparan sulfate, chondroitin, chondroitin sulfate, keratan, keratan sulfate, dermatan sulfate, carrageenan, heparin, chitin, chitosan, alginate, alginic acid derivatives, agarose, agar, cellulose, methylcellulose, carboxymethylcellulose, glycogen and derivatives thereof, as well as fibrin, fibrinogen, thrombin, polyglutamic acid, polylactic acid, polyglycolic acid, lactic acid-glycolic acid copolymer, vinyl alcohol polymer, gellan gum, xanthan gum, galactomannan, guar gum, locust bean gum, and tara gum.

[0136] Here, as the material for the hydrogel, an alginic acid derivative is preferable from the viewpoints of biocompatibility, long-term engraftment and functional maintenance of pancreatic islets, and the like.

[0137] Each aspect of the alginic acid derivative will be described in more detail below.

[0138] 1. Alginic acid In the present specification, the term alginic acid refers to at least one type of alginic acid (sometimes referred to as "alginic acid derivatives") selected from the group consisting of alginic acid, alginic acid esters, and salts thereof (e.g., sodium alginate). The alginic acid used may be natural or synthetic, but is preferably natural. The alginic acid derivatives preferably used are bioabsorbable polysaccharides extracted from brown algae such as Lessonia, Macrocystis, Laminaria, Ascophyllum, Durvillea, Ecklonia cava, Eisenia bicolor, and Laminae, and are linear polymers of two types of uronic acid, D-mannuronic acid (M) and L-guluronic acid (G). More specifically, they are block copolymers in which a homopolymer fraction of D-mannuronic acid (MM fraction), a homopolymer fraction of L-guluronic acid (GG fraction), and a fraction in which D-mannuronic acid and L-guluronic acid are randomly arranged (M / G fraction) are bonded.

[0139] In this specification, alginic acid may be expressed as (ALG)-COOH, where alginic acid is (ALG) and any one of the carboxyl groups of alginic acid is -COOH.

[0140] In some embodiments, the alginic acid is sodium alginate. Commercially available sodium alginate can be used as the sodium alginate. Here, in the examples described below, sodium alginate A-1, A-2, A-3, B-1, B-2, and B-3 (sold by Mochida Pharmaceutical Co., Ltd.) are used as the sodium alginate. The viscosity, weight average molecular weight, and M / G ratio of a 1 w / w% aqueous solution of each sodium alginate are shown in the table below.

[0141] [Table 9]

[0142] The physical properties of the sodium alginates A-1, A-2, A-3, B-1, B-2, and B-3 were measured by the following methods. The measurement methods are not limited to these methods, but the physical properties may differ from those described above depending on the measurement method.

[0143] [Viscosity measurement of sodium alginate] Measurements were performed using a rotational viscometer (cone-plate type rotational viscometer) according to the viscosity measurement method of the Japanese Pharmacopoeia (16th edition). Specific measurement conditions are as follows. Sample solutions were prepared using MilliQ water. The measurement equipment used was a cone-plate type rotational viscometer (viscosity and viscoelasticity measuring device Rheostress RS600 (Thermo Haake GmbH) sensor: 35 / 1). The rotation speed was 1 rpm when measuring 1 w / w% sodium alginate solution. The reading time was 2 minutes, and the average value from 1 minute to 2 minutes was used. The average value of three measurements was used as the measured value. The measurement temperature was 20°C.

[0144] [Weight-average molecular weight measurement of sodium alginate] The measurements were performed using two methods: (1) gel permeation chromatography (GPC) and (2) GPC-MALS. The measurement conditions were as follows:

[0145] [Pretreatment method] The sample was dissolved in an eluent and then filtered through a 0.45 μm membrane filter to prepare a measurement solution. (1) Gel permeation chromatography (GPC) measurement [Measurement conditions (relative molecular weight distribution measurement)] Column: TSKgel GMPW-XL x 2 + G2500PW-XL (7.8 mm ID x 300 mm x 3) Eluent: 200mM sodium nitrate aqueous solution Flow rate: 1.0mL / min Concentration: 0.05% Detector: RI detector Column temperature: 40℃ Injection volume: 200μL Molecular weight standards: standard pullulan, glucose

[0146] (2)GPC-MALS measurement [Refractive index increment (dn / dc) measurement (measurement conditions)] Differential refractometer: Optilab T-rEX Measurement wavelength: 658nm Measurement temperature: 40℃ Solvent: 200mM sodium nitrate aqueous solution Sample concentration: 0.5-2.5mg / mL (5 concentrations)

[0147] [Measurement conditions (absolute molecular weight distribution measurement)] Column: TSKgel GMPW-XL x 2 + G2500PW-XL (7.8 mm ID x 300 mm x 3) Eluent: 200mM sodium nitrate aqueous solution Flow rate: 1.0mL / min Concentration: 0.05% Detector: RI detector, light scattering detector (MALS) Column temperature: 40℃ Injection volume: 200μL

[0148] In this specification, the molecular weight of alginic acid, alginic acid derivatives, crosslinked alginic acid, and crosslinked alginic acid may be expressed in units of Da (dalton).

[0149] The composition ratio (M / G ratio) of D-mannuronic acid and L-guluronic acid in alginic acids varies mainly depending on the type of organism from which seaweed is derived, and is also influenced by the growing place and season of the organism, and ranges widely 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. The gelling ability of alginic acids and the properties of the gel formed are affected by the M / G ratio, and it is generally known that a high G ratio leads to high gel strength. 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 their salts used is usually 0.2 to 4.0, more preferably 0.4 to 3.0, and even more preferably 0.5 to 3.0.

[0150] In this specification, a numerical range indicated using "~" indicates a range that includes the numerical values ​​before and after "~" as the minimum and maximum values, respectively.

[0151] The term "alginic acid ester" and "alginate salt" used herein is not particularly limited, but it is necessary that they do not have a functional group that inhibits the crosslinking reaction in order to react with a crosslinking agent. Preferred examples of alginic acid esters include propylene glycol alginate.

[0152] In the present specification, examples of alginates include monovalent alginic acid salts and divalent alginic acid salts. Examples of monovalent alginic acid salts include sodium alginate, potassium alginate, ammonium alginate, etc., more preferably sodium alginate or potassium alginate, and particularly preferably sodium alginate. Examples of divalent alginic acid salts include calcium alginate, magnesium alginate, barium alginate, strontium alginate, etc.

[0153] Alginic acid is a polymeric polysaccharide, and although it is difficult to accurately determine its molecular weight, its weight-average molecular weight is generally in the range of 1,000 to 10,000,000, preferably 10,000 to 8,000,000, and more preferably 20,000 to 3,000,000. It is known that when measuring the molecular weight of polymeric substances derived from natural products, the values ​​can vary depending on the measurement method.

[0154] For example, 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 preferably 5 million or less, more preferably 3 million or less, with the preferred range being 100,000 to 5 million, more preferably 150,000 to 3 million.

[0155] Also, for example, the absolute weight average molecular weight can be measured by the GPC-MALS method. 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, even more preferably 60,000 or more, and preferably 1,000,000 or less, more preferably 800,000 or less, even 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 500,000 to 800,000, even more preferably 60,000 to 700,000, and particularly preferably 60,000 to 500,000.

[0156] Usually, when the molecular weight of a polymeric polysaccharide is calculated by the above-mentioned method, a measurement error of 10% to 20% may occur. For example, if the molecular weight is 400,000, the value may vary within a range of 320,000 to 480,000, if the molecular weight is 500,000, the value may vary within a range of 400,000 to 600,000, and if the molecular weight is 1,000,000, the value may vary within a range of 800,000 to 1,200,000.

[0157] The molecular weight of alginic acids can be measured according to a conventional method.

[0158] Representative conditions for using gel filtration chromatography for molecular weight measurement are as described in the Examples of this specification below. For example, a Superose6 Increase10 / 300 GL column (GE Healthcare Sciences) can be used as the column, for example, a 10 mmol / L phosphate buffer (pH 7.4) containing 0.15 mol / L NaCl can be used as the developing solvent, and blue dextran, thyroglobulin, ferritin, aldolase, conalbumin, ovalbumin, ribonuclease A and aprotinin can be used as the molecular weight standards.

[0159] The viscosity of alginic acid used in this specification is not particularly limited, but when the viscosity is measured as a 1 w / w% aqueous solution of alginic acids, it is preferably 10 mPa·s to 1000 mPa·s, and more preferably 50 mPa·s to 800 mPa·s.

[0160] The viscosity of the aqueous solution of alginic acid can be measured according to a conventional method. For example, the viscosity can be measured using a coaxial double cylinder rotational viscometer, a single cylinder rotational viscometer (Brookfield type viscometer), a cone-plate type rotational viscometer, etc., according to the rotational viscometer method. It is preferable to follow the viscosity measurement method of the Japanese Pharmacopoeia (16th edition). More preferably, a cone-plate type viscometer is used.

[0161] When alginic acids are first extracted from brown algae, they have a large molecular weight and a high viscosity, but the molecular weight decreases and the viscosity decreases during processes such as drying by heat and purification. Alginic acids with different molecular weights can be produced by controlling conditions such as temperature during the production process, selecting the brown algae used as raw material, and molecular weight fractionation during the production process. Furthermore, it is possible to produce alginic acids with the desired molecular weight by mixing with other lots of alginic acids with different molecular weights or viscosities.

[0162] The alginic acid used herein is, in some embodiments, alginic acid that has not been treated with low endotoxins, or, in other embodiments, alginic acid that has been treated with low endotoxins. Low endotoxins means that the endotoxin level is low enough to not substantially cause inflammation or fever. More preferably, alginic acids that have been treated with low endotoxins are desirable.

[0163] The endotoxin reduction treatment can be carried out by a known method or a method similar thereto. For example, the purification of sodium hyaluronate can be carried out by the method of Suga et al. (see, for example, Japanese Patent Application Laid-Open No. 9-324001, etc.), the purification of β1,3-glucan by Yoshida et al. (see, for example, Japanese Patent Application Laid-Open No. 8-269102, etc.), the purification of biopolymer salts such as alginate and gellan gum by Williams et al. (see, for example, Japanese Translation of PCT International Publication No. 2002-530440, etc.), the purification of polysaccharides by James et al. (see, for example, the pamphlet of International Publication No. 93 / 13136, etc.), the method of Lewis et al. (see, for example, the specification of U.S. Patent No. 5,589,591, etc.), the purification of alginate by Herman Frank et al. (see, for example, Appl Microbiol Biotechnol (1994) 40:638-643, etc.), etc., or methods similar thereto. The low endotoxin treatment is not limited to these, but may be performed by known methods such as washing, filtration with a filter (endotoxin removal filter, charged filter, etc.), ultrafiltration, purification using a column (endotoxin adsorption affinity column, gel filtration column, column with ion exchange resin, etc.), adsorption to hydrophobic substances, resins or activated carbon, organic solvent treatment (extraction with organic solvent, precipitation / sedimentation by adding organic solvent, etc.), surfactant treatment (see, for example, JP 2005-036036 A, etc.), or by appropriately combining these. These treatment steps may be appropriately combined with known methods such as centrifugation. It is desirable to select an appropriate method according to the type of alginic acid.

[0164] The endotoxin level can be confirmed by known methods, for example, a method using Limulus Alkaline Lympholysate (LAL) or a method using Endospecy (registered trademark) ES-24S set (Seikagaku Corporation).

[0165] The method of endotoxin treatment used is not particularly limited, but as a result, the endotoxin content of alginic acids is preferably 500 endotoxin units (EU) / g or less, more preferably 100 EU / g or less, particularly preferably 50 EU / g or less, and particularly preferably 30 EU / g or less, when endotoxin is measured using a Limulus reagent (LAL). Low-endotoxin sodium alginate is available, for example, under the trade name Sea Matrix (registered trademark) (Mochida Pharmaceutical Co., Ltd.), PRONOVA TM It is available commercially as a product such as UP LVG (FMCBioPolymer).

[0166] 2. Alginic acid derivatives The present specification provides a novel alginic acid derivative, in which a reactive group in the Huisgen reaction or a complementary reactive group to the reactive group is introduced to any one or more carboxyl groups of alginic acid via an amide bond and a divalent linker. More specifically, the compound represented by the following formula (HA-I) or (HB-I): [ka] [In the formula (HA-I) or formula (HB-I), (ALG), -L 1 -, Akn are defined as in the first embodiment above], and an alginic acid derivative represented by the following formula (HA-II) or formula (HB-II): [ka] [In the formula (HA-II) or formula (HB-II), (ALG), -L 2 The definitions of - are the same as those in the first aspect described above.

[0167] The bivalent linker (-L 1 -or-L 2Any straight-chain group can be used as long as it does not inhibit the reaction between the reactive group and a complementary reactive group. Specifically, a straight-chain alkylene group (-(CH 2 ) n -, n = 1 to 30) (-CH 2 - may be replaced by a plurality of (for example, 1 to 10 or 1 to 5) groups such as -C(=O)-, -CONH-, -O-, -NH-, -S-, a benzene ring, or a heterocycle (a 5- or 6-membered aromatic heterocycle or a 5- or 6-membered non-aromatic heterocycle such as a pyridine ring, a piperidine ring, or a piperazine ring), and the -CH 2 The hydrogen atom of - is an oxo group (=O), C 1-6 may be substituted with a plurality of (for example, 1 to 10 or 1 to 5) groups selected from alkyl groups (for example, methyl groups, ethyl groups, n-propyl groups, iso-propyl groups, etc.), halogen atoms (for example, fluorine atoms, chlorine atoms, bromine atoms, iodine atoms, etc.), hydroxyl groups (-OH), etc.).

[0168] The novel alginic acid derivatives represented by formula (HA-I) or formula (HB-I) and formula (HA-II) or formula (HB-II) in this specification can be produced, for example, by the method represented by the following formula.

[0169] [ka]

[0170] The weight-average molecular weight of the alginic acid derivatives represented by formula (HA-I), formula (HA-II), formula (HB-I) or formula (HB-II) in the present specification is 100,000 Da to 3,000,000 Da, preferably 300,000 Da to 2,500,000 Da, and more preferably 500,000 Da to 2,000,000 Da. The molecular weight of both alginic acid derivatives can be determined by the method described below.

[0171] In the present specification, Akn-L of formula (HA-I) or formula (HB-I) 1The -NH- group does not necessarily have to be bonded to all the carboxyl groups of the alginic acid building block, and the N 3 -L 2 It is not necessary for the --NH-- group to be attached to every carboxyl group of the alginic acid building block.

[0172] In the present specification, Akn-L of formula (HA-I) or formula (HB-I) 1 When the -NH- group is referred to as a reactive group, N 3 -L 2 The -NH- group serves as a complementary reactive group. Conversely, the N 3 -L 2 When the -NH- group is referred to as a reactive group, Akn-L of formula (HA-I) or formula (HB-I) 1 The -NH- group serves as a complementary reactive group.

[0173] In this specification, the introduction rate of the reactive group or the complementary reactive group is 0.1% to 30% or 1% to 30%, respectively, preferably 2% to 20%, and more preferably 3% to 10%.

[0174] The introduction rate of the reactive group or the complementary reactive group is the number of uronic acid monosaccharide units into which each reactive group is introduced, expressed as a percentage, among the uronic acid monosaccharide units, which are the repeating units of alginic acids. In this specification, unless otherwise specified, % used for the introduction rate of the reactive group or the complementary reactive group in the alginic acid derivative (formula (HA-I), formula (HA-II), formula (HB-I) or formula (HB-II)) means mol%. The introduction rate of each reactive group or the complementary reactive group can be determined by the method described in the Examples below.

[0175] In the present specification, the cyclic alkyne group (Akn) in formula (HA-I) or formula (HB-I) and the azide group in formula (HA-II) or formula (HB-II) form a triazole ring by Huisgen reaction, thereby forming a crosslink.

[0176] 3. Huisgen reaction The Huisgen reaction (1,3-dipolar cycloaddition reaction) is a condensation reaction between compounds having a terminal azide group and a terminal alkyne group, as shown in the following formula. As a result of the reaction, a disubstituted 1,2,3-triazole ring is obtained in good yield, and it has the characteristic that no unnecessary by-products are generated. Although it is thought that the reaction may produce a 1,4- or 1,5-disubstituted triazole ring, it is possible to obtain the triazole ring regioselectively by using a copper catalyst.

[0177] [ka]

[0178] In addition, the Huisgen reaction without using a copper catalyst has been reported by Wittig and Krebs. In other words, this reaction produces a cycloadduct by simply mixing cyclooctyne and phenyl azide (see the following formula, R 3 = phenyl). In this reaction, the triple bond of cyclooctyne is significantly distorted, so the reaction with phenyl azide removes this distortion, and the reaction proceeds spontaneously, making a catalyst unnecessary.

[0179] [ka]

[0180] As described above, the Huisgen reaction can use azide compounds having substituted primary azides, secondary azides, tertiary azides, aromatic azides, etc., and compounds having terminal or cyclic alkyne groups, which are complementary reactive groups to the azide group. In addition, since the Huisgen reaction reacts almost exclusively with azide groups and alkyne groups, it is possible to substitute various functional groups (e.g., ester groups, carboxyl groups, alkenyl groups, hydroxyl groups, amino groups, etc.) in the reaction substrate.

[0181] In some embodiments, in order to form crosslinks between alginic acid molecules via 1,2,3-triazole rings easily and efficiently in a short time without generating undesirable by-products and without using a copper catalyst to avoid the cytotoxicity of copper catalysts, a cyclic alkyne group (cyclooctyl group), for example, as described in the first embodiment above, is used as the alkyne group in the Huisgen reaction.

[0182] In the preferred embodiment, the cross-linking method of alginic acid derivatives hardly forms any undesirable by-products in the reaction (Huisgen reaction), which allows the creation of novel forms of biocompatible materials using alginic acid and the incorporation of various biologically active molecules in the formation of alginic acid hydrogels, as well as the incorporation of cellular materials in alginic acid hydrogels for reconstructive surgery or gene therapy.

[0183] 4. Cross-linked Alginate Crosslinked alginates can be classified into those (i) via divalent metal ion bonds, (ii) via chemical bonds, or (iii) via both divalent metal ion bonds and chemical bonds. All crosslinked alginates have the property of forming gel-like to semi-solid, and in some cases sponge-like, morphologies.

[0184] In crosslinked alginic acid via divalent metal ion bonds, the reaction proceeds at an ultrahigh speed and is reversible, whereas in crosslinked alginic acid via chemical bonds, the reaction proceeds slowly under relatively mild conditions and is irreversible. The physical properties of crosslinked alginic acid can be adjusted, for example, by changing the concentration of the aqueous solution containing the divalent metal ion (e.g., calcium chloride aqueous solution) used or the introduction rate of reactive groups introduced into alginic acid.

[0185] By utilizing the above-mentioned crosslinking reaction, it is possible to prepare various alginate structures. For example, a specific structure can be instantly produced from an alginate solution by ionic crosslinking reaction, and a crosslinking reaction by chemical bonds can be used to strengthen the structure (e.g., to obtain long-term stability, etc.). In addition, for example, in a crosslinked alginate structure via both divalent metal ion bonds and chemical bonds, it is possible to reversibly release the divalent metal ions incorporated by ionic crosslinking, thereby producing a structure in which only crosslinks by chemical bonds remain. Furthermore, cross-linked alginate structures using preferred embodiments of alginate derivatives are stable because they contain cross-linking by chemical bonds, and are advantageous in that they can maintain their shape for a long period of time compared to cross-linked alginate structures that are only ionic cross-linked using sodium alginate.

[0186] The crosslinked alginic acid of one embodiment can be obtained by mixing the alginic acid derivatives of the formula (HA-I) and the formula (HA-II) and carrying out the Huisgen reaction. The crosslinked alginic acid of one embodiment can be obtained by mixing the alginic acid derivatives of the formula (HB-I) and the formula (HB-II) and carrying out the Huisgen reaction.

[0187] In one embodiment, the crosslinked alginic acid forms a three-dimensional network structure through chemical crosslinking (crosslinking by a triazole ring formed from an alkyne group and an azide group). A preferred alginic acid derivative is one that has improved stability after crosslinking.

[0188] In some embodiments, the crosslinked alginate has a structure represented by the following formula (HA-III-L) or (HB-III-L) between any carboxyl group of the first alginate and any carboxyl group of the second alginate: [ka] [In formula (HA-III-L) or formula (HB-III-L), -CONH- and -NHCO- at both ends represent an amide bond via any carboxyl group of alginic acid; 1 -, -L2 -, and X are each as defined in the first embodiment above.

[0189] In some embodiments, when preparing crosslinked alginic acid, the mixing ratio of the alginic acid derivative of formula (HA-I) or formula (HB-I) to the alginic acid derivative of formula (HA-II) or formula (HB-II) is, for example, 1 to 1.5:1, preferably 1.2 to 1.5:1, or 1 to 1.2:1, and more preferably 1:1, in terms of weight ratio of the derivative of formula (HA-I) or formula (HB-I) to the derivative of formula (HA-II) or formula (HB-II).

[0190] In some embodiments, when preparing crosslinked alginic acid, the mixing ratio of the alginic acid derivative of formula (HA-II) or formula (HB-II) to the alginic acid derivative of formula (HA-I) or formula (HB-I) is, 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, and more preferably 1:1, in terms of weight ratio of the derivative of formula (HA-II) or formula (HB-II) to the derivative of formula (HA-I) or formula (HB-I).

[0191] In some embodiments, when preparing crosslinked alginic acid, the mixing ratio of the alginic acid derivative of formula (HA-I) or formula (HB-I) to the alginic acid derivative of formula (HA-II) or formula (HB-II) is, more preferably, the ratio of the introduction rate (mol%) of the reactive group of the alginic acid derivative of formula (HA-I) or formula (HB-I) to the alginic acid derivative of formula (HA-II) or 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.

[0192] In some embodiments, when preparing crosslinked alginic acid, the mixing ratio of the alginic acid derivative of formula (HA-II) or formula (HB-II) to the alginic acid derivative of formula (HA-I) or formula (HB-I) is, more preferably, the ratio of the introduction rate (mol%) of the reactive group of the alginic acid derivative of formula (HA-II) or formula (HB-II) to the alginic acid derivative of formula (HA-I) or 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.

[0193] Furthermore, in the above-mentioned mixing ratio, it is also possible to replace the alginic acid derivative of formula (HA-I) or formula (HB-I) with the alginic acid derivative of formula (HA-II) or formula (HB-II), and the alginic acid derivative of formula (HA-II) or formula (HB-II) with the derivative of formula (HA-I) or formula (HB-I), respectively.

[0194] In the crosslinked alginic acid, it is not necessary that all carboxyl groups of the alginic acid constituent units have the crosslinks of the above formula (HA-III-L) or formula (HB-III-L). The introduction rate of the crosslinks of the above formula (HA-III-L) or formula (HB-III-L) in the crosslinked alginic acid (also called the crosslinking rate) is, for example, in the range of 0.1 to 80%, 0.3 to 60%, 0.5 to 30%, or 1.0 to 10%.

[0195] The concentration of the alginic acid derivative of formula (HA-I), formula (HA-II), formula (HB-I) or formula (HB-II) in the Huisgen reaction to obtain crosslinked alginic acid is usually in the range of 1 to 500 mg / mL, preferably 5 to 100 mg / mL.

[0196] The reaction temperature for the Huisgen reaction is usually an external temperature in the range of 4 to 60°C, and preferably an external temperature in the range of 15 to 40°C.

[0197] The stirring time for forming crosslinked alginic acid (hydrogel) is, for example, from a few seconds to 24 hours, from a few seconds to 12 hours, from a few seconds to 30 minutes, or from a few seconds to 10 minutes.

[0198] The reaction solvent or reaction solution used in the Huisgen reaction is not particularly limited, but examples thereof include tap water, pure water (e.g., distilled water, ion-exchanged water, RO water, RO-EDI water, etc.), ultrapure water, cell culture medium, phosphate buffered saline (PBS), saline, and HEPES buffer solution, and is preferably ultrapure water.

[0199] The cross-linked alginate in some embodiments is a cross-linked alginate that includes chemical cross-links via triazole rings formed by the Huisgen reaction as cross-links, and ionic cross-links formed in part by calcium ions.

[0200] 5. Cross-linked Alginate Constructs The crosslinked alginate structure can be obtained by a method including subjecting the alginate derivative to a crosslinking reaction. For example, the crosslinked alginate structure can be prepared by the following methods, but is not limited thereto.

[0201] [Mixing method] A mixed solution of alginic acid derivatives obtained by mixing an alginic acid derivative of formula (HA-I) or formula (HB-I) with an alginic acid derivative of formula (HA-II) or formula (HB-II) can be dropped into a solution containing divalent metal ions to obtain a cross-linked alginic acid structure, which is a specific structure in which chemical cross-links (cross-links via triazole rings formed from alkyne groups and azide groups by the Huisgen reaction) and ionic cross-links (cross-links partially formed by divalent metal ions) are formed.

[0202] [Coating method] A solution containing an alginic acid derivative of formula (HA-I) or formula (HB-I) is dropped into a solution containing a divalent metal ion to obtain a specific partially crosslinked structure. The structure obtained above, such as a gel, is added to a solution containing an alginic acid derivative of formula (HA-II) or formula (HB-II) to perform a further crosslinking reaction (Huisgen reaction) on the surface of the structure, thereby obtaining a crosslinked alginic acid structure. This method can also be carried out by replacing the alginic acid derivative of formula (HA-I) or formula (HB-I) with the alginic acid derivative of formula (HA-II) or formula (HB-II), and replacing the alginic acid derivative of formula (HA-II) or formula (HB-II) with the alginic acid derivative of formula (HA-I) or formula (HB-I).

[0203] The divalent metal ion used in the above method is not particularly limited, but examples thereof include calcium ion, magnesium ion, barium ion, strontium ion, zinc ion, etc., and is preferably calcium ion.

[0204] The solution containing calcium ions used in the above method is not particularly limited, but examples thereof include aqueous solutions such as calcium chloride, calcium carbonate, and calcium gluconate, with calcium chloride being preferred.

[0205] The calcium ion concentration of the solution containing calcium ions used in the above method is not particularly limited, but may be, for example, 1 mM to 1 M, preferably 5 mM to 500 mM, and more preferably 10 mM to 300 mM.

[0206] The solvent or solution used in the method is not particularly limited, and examples thereof include tap water, pure water (e.g., distilled water, ion-exchanged water, RO water, RO-EDI water, etc.), ultrapure water, cell culture medium, phosphate buffered saline (PBS), saline, and HEPES buffer, and is preferably ultrapure water.

[0207] Specific examples of the crosslinked alginate structure include fibrous structures, fibers, beads, gels, and roughly spherical gels. A preferred crosslinked alginate structure has improved stability. The crosslinked alginate structure may also have the ability to retain contents therein (content retention).

[0208] The physical properties of the alginate gel can be adjusted by adjusting physical property values ​​such as hardness, elasticity, resilience, breaking strength, and stress at break.

[0209] 6. Biocompatibility of alginate derivatives and cross-linked alginate structures In the present specification, the alginic acid derivative or the crosslinked alginic acid structure has biocompatibility. In the present specification, biocompatibility means a property of not inducing an interaction between a biomaterial (here, for example, the alginic acid derivatives represented by formula (HA-I) and formula (HA-II), and the crosslinked alginic acid structure produced using the two alginic acid derivatives) and a living body, a local reaction of tissue adjacent to the biomaterial, or a systemic reaction.

[0210] In this specification, the biocompatibility of the alginic acid derivatives or cross-linked alginic acid structures will be confirmed in the examples regarding biocompatibility described later.

[0211] 7. Stability of cross-linked alginate structures The stability of the crosslinked alginate structure can be confirmed, for example, by measuring the gel stability, and the permeability can be confirmed by measuring the gel permeability.

[0212] [Method for measuring gel stability] Phosphate buffered saline (PBS) is added to the crosslinked alginate structure gel placed in a container, and the concentration (μg / mL) of alginate leaked into the PBS is measured. The measured alginate concentration is divided by the total alginate concentration obtained by decomposing the crosslinked alginate structure gel, and the value expressed as a percentage is taken as the disintegration rate. Specifically, the gel stability can be determined by the method described in the Examples below.

[0213] In this specification, the gel collapse rate of the crosslinked alginate structure is preferably 0% to 90%, more preferably 0% to 70%, and even more preferably 0% to 50%. The lower the concentration of alginic acid leaked into the aqueous solution, i.e., the lower the gel collapse rate, the higher the stability of the crosslinked alginate structure.

[0214] [Method for measuring gel permeability] A crosslinked alginate structure gel containing fluorescein isothiocyanate-dextran is prepared, and physiological saline is added to the gel placed in a container, and the concentration of dextran leaked into the physiological saline is measured. The measured dextran concentration is divided by the total dextran concentration obtained by decomposing the fluorescein isothiocyanate-dextran-containing crosslinked alginate structure gel, and the value expressed as a percentage is the gel permeability. Specifically, the gel permeability can be determined by the method described in the Examples below.

[0215] The gel permeability of the crosslinked alginate 24 hours after addition of physiological saline is preferably 0% to 90%, more preferably 0% to 70%, and even more preferably 0% to 50%, when it contains dextran with a molecular weight of 2 million. When it contains dextran with a molecular weight of 150,000, for example, if the intended use of the crosslinked alginate structure gel is for the release or production of proteins or antibodies, it is preferably 1% to 100%, more preferably 10% to 100%, and even more preferably 30% to 100%. When it is intended to be used as an immune barrier, it is preferably 0% to 90%, more preferably 0% to 70%, and even more preferably 0% to 50%.

[0216] Regarding the permeability of the cross-linked alginate structure, a lower permeability indicates a lower permeability to the contents and substances outside the gel, and a higher permeability indicates a higher permeability to the contents and substances outside the gel.

[0217] The permeability of the gel can be adjusted by the molecular weight and concentration of the alginic acid used, the type and introduction rate of crosslinking groups introduced into the alginic acid, the type and concentration of divalent metal ions used for gelation, or a combination of these.

[0218] [Method for preparing cross-linked alginate structure gel containing ingredients] For example, a cross-linked alginate structure gel containing fluorescein isothiocyanate-dextran as the content can be prepared by the following method.

[0219] (1) A solution of an alginic acid derivative represented by formula (HA-I) or formula (HB-I) is mixed with a fluorescein isothiocyanate-dextran solution. (2) The mixed solution obtained in (1) is mixed with a solution of an alginic acid derivative represented by formula (HA-II) or formula (HB-II). (When formula (HA-I) or formula (HB-I) in (1) is changed to formula (HA-II) or formula (HB-II), formula (HA-II) or formula (HB-II) in (2) is changed to formula (HA-I) or formula (HB-I).) (3) The mixed solution obtained in (2) is dropped into a solution containing calcium ions, and the resulting gel forms chemical and ionic crosslinks in the solution, thereby producing a crosslinked alginate structure gel containing fluorescein isothiocyanate-dextran.

[0220] 8. Method for synthesizing alginic acid derivatives Alginic acid derivatives represented by formula (HA-I) or formula (HA-II) can be produced by referring to PCT / JP2019 / 023478 (filed June 13, 2019).

[0221] 9. Applications of alginate derivatives and cross-linked alginate structures As described above, the alginic acid derivatives can be used to prepare transplant devices. In addition to transplant devices, the alginic acid derivatives can be used in place of conventional alginic acid in a wide range of fields, such as food, medicine, cosmetics, textiles, and papermaking. Specific preferred applications of the alginic acid derivatives or crosslinked alginic acid structures include medical materials such as wound dressings, postoperative adhesion inhibitors, drug sustained release substrates, cell culture substrates, and cell transplant substrates.

[0222] When used as a medical material, the shape of the cross-linked alginate structure may be tubular, fibrous, fiber, beads, gel, or approximately spherical gel, etc., and is preferably in the form of beads, gel, or approximately spherical gel, and more preferably in the form of an approximately spherical gel.

[0223] A particularly preferred embodiment of a transplant device using an alginic acid derivative has excellent biocompatibility and stability, low cytotoxicity, causes almost no adhesion or inflammation at the transplant site, and maintains its shape for a long period of time with little gel dissolution (regardless of the presence or absence of a semipermeable membrane), thereby enabling the device to maintain its hypoglycemic effect and regulate blood sugar for a long period of time.

[0224] The cure rate in the present invention is expressed as the ratio of the number of treated cases to the number of transplanted cases after a specified period of time has elapsed after transplantation of a hydrogel containing insulin-secreting cells or pancreatic islets, or a transplant device containing said hydrogel, into diabetic patients or diabetic model animals. For example, a hydrogel containing insulin-secreting cells or pancreatic islets, or a transplantation device containing the hydrogel, is transplanted into multiple diabetic model mice obtained by the method of Example 7, and mice whose blood glucose levels are 300 mg / dL or less (however, blood glucose levels exceeding 300 mg / dL up to three times during the period) during a specified period after transplantation are considered to be cured mice, and are expressed as the percentage of cured mice relative to the diabetic model mice. The healing rate is, for example, 20% or more, preferably 35% or more, more preferably 50% or more two weeks after transplantation, or 20% or more, preferably 35% or more, more preferably 50% or more one month after transplantation.

[0225] In the present invention, the oxygen permeability is expressed as the ratio of the oxygen concentration at the center of a hydrogel containing insulin-secreting cells or pancreatic islets, or a transplant device containing said hydrogel, to the surface oxygen concentration of 100%. In the case of a flat gel, the center means approximately the center in the vertical, horizontal and thickness directions. The oxygen permeability may be calculated by measuring the hydrogel or device, or by calculation. When calculating by measurement, the oxygen permeability can be calculated from the measured values ​​of the surface oxygen concentration and the central oxygen concentration using, for example, a needle-type oxygen meter (OXY-1 ST trace) manufactured by PreSens.

[0226] When calculating the oxygen permeability, the reaction-diffusion equation for the oxygen concentration when the hydrogel is placed in a Cartesian coordinate system in space is given as follows:

number

number

number

[0227] In the present invention, the substance permeability refers to the substance permeability from a hydrogel containing insulin-secreting cells or pancreatic islets or a transplantation device containing the hydrogel after a specific substance is encapsulated in the hydrogel or the transplantation device and the hydrogel is left in a stirred solution for a specific period of time. Examples of the substance include glucose and insulin. For example, the insulin permeability or glucose permeability when 500 mg of human insulin or 250 mg of glucose are encapsulated in a hydrogel or a transplant device and stirred in 40 mL of physiological saline containing 0.01 Tween 20 at room temperature for 24 hours can be mentioned. The insulin permeability is 50% or more, preferably 70% or more, more preferably 80% or more, and the glucose permeability is 50% or more, preferably 70% or more, more preferably 80% or more.

[0228] The hydrogel of the present invention is not broken, decomposed or dissolved to a small extent even when shaken under a predetermined condition. 3 Hydrogel, length 10mm x width 20mm x thickness 100μm to less than 500μm or approximately 20mm 3 More than 100mm 3 Hydrogels less than 10 mm long x 20 mm wide x 250 μm thick or approximately 50 mm 3 Three pieces of the hydrogel are prepared and added to 12 mL of 37°C physiological saline solution in a 15 mL conical tube. The hydrogel is shaken at 37°C using a shaking incubator (for example, a medium-sized thermostatic shaking incubator (Tatec Corporation, Bioshaker (registered trademark) BR-43FL MR)) at a reciprocating shaking amplitude of 25 mm and a shaking speed of 180 rpm. There is little or no rupture, decomposition, or dissolution. The term "low decomposition or dissolution of the hydrogel" refers to, for example, the case where the disintegration rate is 40% or less, preferably 20% or less, more preferably 10% or less, when the proportion of alginic acid dissolved in the solution when shaken under the above conditions is taken as 100% alginic acid in the hydrogel. In particular, the disintegration rate is 30% or less, preferably 20% or less, more preferably 10% or less, when the proportion of alginic acid dissolved in the solution when shaken under the above conditions for 24 hours is taken as the disintegration rate. In addition, the disintegration rate is 30% or less, preferably 20% or less, more preferably 10% or less, when the proportion of alginic acid dissolved in the solution when shaken under the above conditions for 96 hours is taken as the disintegration rate.

[0229] The hydrogel of the present invention is such that, even when shaken under a predetermined condition, the cells in the hydrogel do not fall off or fall off only slightly. 3 Hydrogel, length 10mm x width 20mm x thickness 100μm to less than 500μm or approximately 20mm 3 More than 100mm 3 Hydrogels less than 10 mm long x 20 mm wide x 250 μm thick or approximately 50 mm 3 Three pieces of hydrogels (in each case, cells are encapsulated) are prepared and placed in 12 mL of 37°C physiological saline solution in a 15 mL conical tube. The cells are then shaken at 37°C using a shaking incubator (for example, a medium-sized thermostatic shaking incubator (Tatec Corporation, Bioshaker (registered trademark) BR-43FL MR)) at a reciprocating vibration amplitude of 25 mm and a shaking speed of 180 rpm. When the cells are shaken at 37°C, either no cells fall off or only a small amount of cells fall off. The term "low cell loss from the hydrogel" refers to, for example, a loss rate of 30% or less, preferably 20% or less, more preferably 10% or less, when the number of cells in the hydrogel at the start of the test is taken as 100% and the percentage of cells that fall off from the hydrogel when shaken under the above conditions is taken as the loss rate. In particular, the loss rate after shaking for 24 hours under the above conditions is 30% or less, preferably 20% or less, more preferably 10% or less.

[0230] All documents and publications described in this specification are incorporated herein by reference in their entirety regardless of their purpose. In addition, this specification incorporates by reference the disclosure contents of the claims, specifications, and drawings of Japanese Patent Application No. 2019-122063 (filed June 28, 2019) and International Patent Application No. PCT / JP2020 / 25324 (filed June 26, 2020).

[0231] In addition, the objects, features, advantages, and ideas of the present invention will be clear to those skilled in the art from the description of this specification, and those skilled in the art can easily implement the present invention from the description of this specification. The best mode for carrying out the invention and specific examples are shown to show preferred embodiments of the present invention, and are shown for illustrative or explanatory purposes, and do not limit the present invention thereto. It is clear to those skilled in the art that various modifications can be made based on the description of this specification within the intent and scope of the present invention disclosed in this specification. EXAMPLES

[0232] Next, examples and test examples will be given in order to explain the present invention in more detail. However, these examples are merely examples and test examples and do not limit the present invention. In addition, the examples may be modified within the scope of the present invention.

[0233] Nuclear magnetic resonance spectroscopy (NMR) was measured using a JEOL JNM-ECX400 FT-NMR (JEOL). Liquid chromatography-mass spectrometry (LC-Mass) was measured using the following method. [UPLC] A Waters AQUITY UPLC system and a BEH C18 column (2.1 mm x 50 mm, 1.7 μm) (Waters) were used, with a mobile phase and gradient conditions of acetonitrile: 0.05% trifluoroacetic acid aqueous solution = 5:95 (0 min) - 95:5 (1.0 min) - 95:5 (1.6 min) - 5:95 (2.0 min).

[0234] 1In the H-NMR data, s is singlet, d is doublet, t is triplet, q is quartet, m is multiplet, br is broad, J is coupling constant, Hz is hertz, CDCl 3 is deuterated chloroform, DMSO-D 6 is deuterated dimethyl sulfoxide, D 2 O stands for heavy water. 1 In the H-NMR data, hydroxyl groups (OH), amino groups (NH 2 ), and carboxyl group (COOH) protons, etc., which are broadband and therefore cannot be confirmed, are not included in the data.

[0235] In the LC-Mass data, M is the molecular weight, RT is the retention time, [M+H] + ,[M+Na] + means the molecular ion peak.

[0236] In the examples, "room temperature" generally refers to a temperature of about 0°C to about 35°C. The reactive substituent introduction rate (mol%) in the examples is 1 H-NMR(D 2 This indicates the ratio of the number of moles of the introduced reactive substituents to the number of moles of the monosaccharide (guluronic acid and mannuronic acid) units constituting alginic acid, calculated from the above formula (1).

[0237] In the examples, sodium alginate having the physical properties shown in Table 10 was used as the sodium alginate before the reactive groups or complementary reactive groups were introduced.

[0238] Table 12 shows the physical properties (specifically, reactive group introduction rate (mol%), molecular weight, and weight average molecular weight (10,000 Da)) of the alginic acid derivatives (Example 1a, Example 2a) into which reactive groups were introduced, obtained in (Example 1) to (Example 4-2).

[0239] Example 1 Synthesis of dibenzocyclooctyne-amino group-introduced alginic acid (Example 1a, Example 1b, Example 1c, Example 1d, and Example 1e): [ka]

[0240] (Example 1a) Synthesis of dibenzocyclooctyne-amino group-introduced alginic acid (EX1-(I)-A-2a): 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (111.65 mg) and 1 molar sodium bicarbonate solution (403.5 μL) were added to a 1% by weight aqueous solution of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.) (43.6 mL). A commercially available dibenzocyclooctyne-amine [CAS: 1255942-06-3] (EX1-SM, 83.62 mg) in ethanol solution (2 mL) was added dropwise to this solution and stirred at room temperature for 18 hours. Sodium chloride (400 mg) was added, followed by ethanol (87.2 mL) and stirring at room temperature for 30 minutes. The resulting precipitate was collected by filtration, washed with ethanol, and dried under reduced pressure to obtain the title compound EX1-(I)-A-2a (376 mg) as a pale yellow solid.

[0241] The introduction rate of the reactive substituent (dibenzocyclooctyne-amino group) was 6.9 mol% (NMR integral ratio).

[0242] (Example 1b) Synthesis of dibenzocyclooctyne-amino group-introduced alginic acid (EX1-(I)-A-2b): 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (330 mg) and 1 molar sodium bicarbonate solution (300 μL) were added to a 1% by weight aqueous solution of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.) (120 mL). A commercially available dibenzocyclooctyne-amine [CAS: 1255942-06-3] (EX1-SM, 80 mg) in ethanol solution (12 mL) was added dropwise to this solution and stirred at 30°C for 4 hours. Sodium chloride (1.2 g) was added, followed by ethanol (240 mL) and stirring at room temperature for 30 minutes. The resulting precipitate was collected by filtration, washed with ethanol, and then dried under reduced pressure to obtain the title compound EX1-(I)-A-2b (1.19 g) as a white solid. 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 EX1-(I)-A-2b (1.2 g) as a white amorphous solid.

[0243] The introduction rate of reactive substituents (dibenzocyclooctyne-amino groups) was 5.0 mol% (NMR integral ratio).

[0244] (Example 1c) Synthesis of dibenzocyclooctyne-amino group-introduced alginic acid (EX1-(I)-A-2c): 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (167 mg) and 1 molar sodium bicarbonate solution (151 μL) were added to a 1% by weight aqueous solution of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.) (120 mL). A commercially available dibenzocyclooctyne-amine [CAS: 1255942-06-3] (EX1-SM, 42 mg) in ethanol solution (12 mL) was added dropwise to this solution, and the mixture was stirred at 30°C for 3.5 hours. Sodium chloride (1.2 g) was added, followed by ethanol (240 mL) and stirring at room temperature for 30 minutes. The resulting precipitate was collected by filtration, washed with ethanol, and then dried under reduced pressure to obtain the title compound EX1-(I)-A-2c (1.2 g) as a white solid. 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 EX1-(I)-A-2c (1.15 g) as a white amorphous substance.

[0245] The introduction rate of the reactive substituent (dibenzocyclooctyne-amino group) was 2.3 mol% (NMR integral ratio).

[0246] (Example 1d) Synthesis of dibenzocyclooctyne-amino group-introduced alginic acid (EX1-(I)-A-2d): 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (281 mg) and 1 molar sodium bicarbonate solution (253 μL) were added to an aqueous solution (201 mL) of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.) prepared at 1% by weight. To this solution, a commercially available dibenzocyclooctyne-amine [CAS: 1255942-06-3] (EX1-SM, 70 mg) in ethanol solution (20 mL) was added dropwise and stirred at 32°C for 3 hours. Sodium chloride (2.01 g) was added, followed by addition of ethanol (402 mL) and stirring at room temperature for 30 minutes. The resulting precipitate was collected by filtration, washed with ethanol, and then dried under reduced pressure to obtain the title compound EX1-(I)-A-2d (1.94 g) as a white solid. The white solid was dissolved in 130 mL of water, freeze-dried, and then dried at room temperature for 2 hours to obtain the title compound EX1-(I)-A-2d (1.84 g) as a white amorphous solid.

[0247] The introduction rate of the reactive substituent (dibenzocyclooctyne-amino group) was 2.4 mol% (NMR integral ratio).

[0248] (Example 1e) Synthesis of dibenzocyclooctyne-amino group-introduced alginic acid (EX1-(I)-A-2e): 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (307 mg) and 1 molar sodium bicarbonate solution (277 μL) were added to a 1% by weight aqueous solution of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.) (250 mL). A commercially available dibenzocyclooctyne-amine [CAS: 1255942-06-3] (EX1-SM, 77 mg) in ethanol solution (25 mL) was added to this solution and stirred at 32°C for 3 hours. Sodium chloride (2.5 g) was added to the reaction solution, followed by addition of ethanol (500 mL) and stirring at room temperature for 30 minutes. The resulting precipitate was collected by filtration, washed with ethanol, and then dried under reduced pressure to obtain the title compound EX1-(I)-A-2e (2.42 g) as a white solid. The white solid was dissolved in 160 mL of water, freeze-dried, and then dried at room temperature for 2 hours to obtain the title compound EX1-(I)-A-2e (2.34 g) as a white amorphous solid.

[0249] The introduction rate of the reactive substituent (dibenzocyclooctyne-amino group) was 2.2 mol% (NMR integral ratio).

[0250] 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): [ka]

[0251] <Step 1> Synthesis of methyl 4-(2-((tert-butoxycarbonyl)amino)ethoxy)benzoate (compound EX2-IM-1): [ka]

[0252] A solution of diethyl azodicarboxylate (40% toluene solution, 1.92 mL) was added to a solution of triphenylphosphine (0.96 g) in tetrahydrofuran (2.59 mL) under ice-cooling and stirring, and the mixture was stirred at room temperature for 20 minutes. To this solution, a solution of commercially available methyl 4-hydroxybenzoate [CAS: 99-76-3] (compound EX2-SM, 0.37 g) and 2-(tert-butoxycarbonyl)ethanolamine [CAS: 26690-80-2] (0.39 g) in tetrahydrofuran (1.1 mL) was added under ice-cooling and stirring, and the mixture was stirred at room temperature for 17 hours. The reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (5% ethyl acetate / n-heptane to 40% ethyl acetate / n-heptane) to obtain a mixture of compound 1 and compound 2. This mixture was dissolved in methyl tert-butyl ether (20 mL) and washed with 1N aqueous sodium hydroxide solution (5 mL) twice and saturated saline solution (5 mL) in that order. The organic layer was dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain compound EX2-IM-1 (0.45 g) as a pink oil.

[0253] NMR data (CDCl 3 )(δ:ppm):7.98 (2H, d, J = 8.8 Hz), 6.90 (2H, d, J = 8.8 Hz), 4.97 (1H, br s), 4.07 (2H, t, J = 5.2 Hz), 3.88 (3H, s), 3.56 (2H, q, J = 5.2 Hz), 1.45 (9H, s)

[0254] <Step 2> Synthesis of 4-(2-aminoethoxy)-N-(3-azidopropyl)benzamide hydrochloride (compound EX2-IM-3): [ka]

[0255] (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>, and the mixture was stirred at 60°C for 3 hours and 30 minutes. 1N hydrochloric acid (5 mL) was added to the reaction solution, and the mixture was extracted three times with ethyl acetate (10 mL). The organic layer was washed successively with water (5 mL) and saturated saline (5 mL), dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was dissolved in acetonitrile (4.4 mL), and 3-azidopropan-1-amine [CAS: 88192-19-2] (0.15 g) and O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (0.57 g) were added. Next, N,N-diisopropylethylamine (0.52 mL) was added under ice-cooling and stirring, and the mixture was stirred at room temperature for 5 hours. Water (10 mL) was added to the reaction solution, and the mixture was extracted three times with ethyl acetate (15 mL). The organic layer was dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (16% ethyl acetate / n-heptane to 100% ethyl acetate) to obtain a fraction containing compound EX2-IM-2 (0.71 g).

[0256] To the fraction containing compound EX2-IM-2 (0.71 g), 4N hydrogen chloride / 1,4-dioxane (4.9 mL) was added and stirred at room temperature for 20 minutes. Diisopropyl ether was added to the reaction solution, and the precipitate was filtered to obtain the title compound EX2-IM-3 (0.49 g) as a white solid.

[0257] NMR data (CDCl 3 )(δ:ppm):7.60 (2H, d, J = 8.8 Hz), 6.93 (2H, d, J = 8.8 Hz), 4.19 (2H, t, J = 4.8 Hz), 3.31-3.29 (6H, m), 1.77-1.71 (2H, m).LC-MS:M(free amine)=263, RT=0.54(min), [M+H] + =264

[0258] (Example 2a) Synthesis of 4-(2-aminoethoxy)-N-(3-azidopropyl)benzamide group-introduced alginic acid (compound EX2-(II)-A-2a): [ka]

[0259] To a 1% by weight aqueous solution of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.) (19.6 mL), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (50.19 mg), the compound EX2-IM-3 (54.37 mg) obtained in (Example 2) <Step 2>, and 1 molar sodium bicarbonate water (181.4 μL) were added under ice-cooling and stirring, and the mixture was stirred at room temperature for 5 hours. After adding sodium chloride (200 mg), ethanol (39.2 mL) was added, and the mixture was stirred at room temperature for 30 minutes. The resulting precipitate was collected by filtration, washed with ethanol, and dried under reduced pressure to obtain the title compound EX2-(II)-A-2a (198 mg) as a white solid.

[0260] The introduction rate of the reactive substituent (4-(2-aminoethoxy)-N-(3-azidopropyl)benzamide group) was 6.1 mol% (NMR integral ratio).

[0261] (Example 2b) Synthesis of 4-(2-aminoethoxy)-N-(3-azidopropyl)benzamide group-introduced alginic acid (compound EX2-(II)-A-2b): [ka]

[0262] To a 1% by weight aqueous solution of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.) (120 mL), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (330 mg), the compound EX2-IM-3 (90 mg) obtained in (Example 2) <Step 2>, and 1 molar sodium bicarbonate water (450 μL) were added under ice-cooling and stirring, and the mixture was stirred at 30° C. for 4 hours. After adding sodium chloride (1.2 g), ethanol (240 mL) was added, and the mixture was stirred at room temperature for 30 minutes. The resulting precipitate was collected by filtration, washed with ethanol, and dried under reduced pressure to obtain the title compound EX2-(II)-A-2b (1.22 g) as a white solid. 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 solid.

[0263] The introduction rate of the reactive substituent (4-(2-aminoethoxy)-N-(3-azidopropyl)benzamide group) was 4.9 mol% (NMR integral ratio).

[0264] (Example 2c) Synthesis of 4-(2-aminoethoxy)-N-(3-azidopropyl)benzamide group-introduced alginic acid (compound EX2-(II)-A-2c): [ka]

[0265] To a 1% by weight aqueous solution of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.) (120 mL), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (167 mg), the compound EX2-IM-3 (45 mg) obtained in (Example 2) <Step 2>, and 1 molar sodium bicarbonate water (227 μL) were added under ice-cooling and stirring, and the mixture was stirred at 30° C. for 3.5 hours. After adding sodium chloride (1.2 g), ethanol (240 mL) was added and the mixture was stirred at room temperature for 30 minutes. The resulting precipitate was collected by filtration, washed with ethanol, and dried under reduced pressure to obtain the title compound EX2-(II)-A-2c (1.22 g) as a white solid. 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 solid.

[0266] The introduction rate of the reactive substituent (4-(2-aminoethoxy)-N-(3-azidopropyl)benzamide group) was 2.3 mol % (NMR integral ratio).

[0267] (Example 2d) Synthesis of 4-(2-aminoethoxy)-N-(3-azidopropyl)benzamide group-introduced alginic acid (compound EX2-(II)-A-2d): [ka]

[0268] 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (307 mg), compound EX2-IM-3 (83 mg) obtained in (Example 2) <Step 2>, and 1 molar sodium bicarbonate water (416 μL) were added to a 1 wt% aqueous solution of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd.: A-2) (220 mL), and the mixture was stirred at 32° C. for 3 hours. Sodium chloride (2.2 g) was added, followed by ethanol (440 mL), and the mixture was stirred at room temperature for 30 minutes. The resulting precipitate was collected by filtration, washed with ethanol, and then dried under reduced pressure to obtain the title compound EX2-(II)-A-2d (2.13 g) as a white solid. The white solid was dissolved in 130 mL of water, lyophilized, and then dried at room temperature for 2 hours to obtain the title compound EX2-(II)-A-2d (1.99 g) as a white amorphous solid.

[0269] The introduction rate of the reactive substituent (4-(2-aminoethoxy)-N-(3-azidopropyl)benzamide group) was 2.3 mol % (NMR integral ratio).

[0270] (Example 2e) Synthesis of 4-(2-aminoethoxy)-N-(3-azidopropyl)benzamide group-introduced alginic acid (compound EX2-(II)-A-2e): [ka]

[0271] 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (377 mg), compound EX2-IM-3 (102 mg) obtained in (Example 2) <Step 2>, and 1 molar sodium bicarbonate water (511 μL) were added to a 1 wt% aqueous solution of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd.: A-2) (270 mL), and the mixture was stirred at 32° C. for 3 hours. Sodium chloride (2.7 g) was added to the reaction solution, followed by ethanol (540 mL) and stirring at room temperature for 30 minutes. The resulting precipitate was collected by filtration, washed with ethanol, and dried under reduced pressure to obtain the title compound EX2-(II)-A-2e (2.60 g) as a white solid. The white solid was dissolved in 160 mL of water, lyophilized, and then dried at room temperature for 2 hours to obtain the title compound EX2-(II)-A-2e (2.56 g) as a white amorphous solid.

[0272] The introduction rate of the reactive substituent (4-(2-aminoethoxy)-N-(3-azidopropyl)benzamide group) was 2.4 mol% (NMR integral ratio).

[0273] Example 3 Synthesis of N-(4-(aminoethyl)benzyl)-2-(cyclooct-2-yn-1-yloxy)acetamide group-introduced alginic acid (Example 3a, Example 3b, and Example 3c): [ka]

[0274] <Step 1> Synthesis of tert-butyl (4-(4((2,2,2-trifluoroacetamido)methyl)benzyl)carbamate (compound EX3-IM-1): [ka]

[0275] With reference to a method known in the literature (Bioorganic & Medicinal Chemistry (2003) 11: 4189-4206), tert-butyl (4-(aminoethyl)benzyl)carbamate [CAS: 108468-80-4] (EX3-SM1, 0.67 g), triethylamine (0.39 mL) and methanol (6.67 mL) were mixed and cooled with ice to add ethyl trifluoroacetate (0.44 mL) dropwise to the mixture, and the mixture was stirred at the same temperature for 5 hours. The reaction was stopped with water (10 mL) and extracted three times with ethyl acetate (10 mL). The collected organic layer was washed with saturated saline (5 mL) and dried over anhydrous sodium sulfate. The dried organic layer was filtered and concentrated to obtain the crude title compound EX3-IM-1 (0.671 g) as a pale yellow amorphous substance.

[0276] NMR data (CDCl 3 )(δ:ppm): 7.29 (2H, d, J = 8.4 Hz), 7.25 (2H, d, J = 7.6 Hz), 6.51 (1H, br s), 4.86 (1H, br s), 4.51 (2H, d, J = 5.2 Hz), 4.31 (2H, d, J = 6.0 Hz), 1.46 (9H, s).LC-MS:M=332,RT=0.97(min),[M+Na]+=355.

[0277] <Step 2> Synthesis of N-(4-(aminoethyl)benzyl)-2,2,2-trifluoroacetamide hydrochloride (compound EX3-IM-2): [ka]

[0278] To a 1,4-dioxane solution (3.5 mL) of the compound EX3-IM-1 (0.5 g) obtained in (Example 3) <Step 1>, 4N hydrogen chloride / 1,4-dioxane (3.5 mL) was added under stirring in a water-cooled bath, and the mixture was stirred at room temperature for 3 hours. Diisopropyl ether (40 mL) was added to the reaction solution, and the precipitate was filtered to obtain the title compound EX3-IM-2 (0.36 g) as a white solid.

[0279] NMR data (D 2 O)(δ:ppm): 7.29 (2H, d, J = 8.0 Hz), 7.25 (2H, d, J = 8.4 Hz), 4.38 (2H, s), 4.02 (2H, s).LC-MS:M(free amine)=232,RT=0.53(min),[M+H]+=233.

[0280] <Step 3> Synthesis of N-(4-((2-(cyclooct-2-yn-1-yloxy)acetamido)methyl)benzyl)-2,2,2-trifluoroacetamide (compound EX3-IM-3): [ka]

[0281] According to a method known in the literature (Org. Process Res. Dev. (2018) 22: 108-110), a carboxylic acid [CAS: 917756-42-4] (EX3-SM2, 0.17 g) synthesized from cycloheptene [CAS: 628-92-2] and O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (0.26 g) were added dropwise to an acetonitrile (1.7 mL) solution of EX3-IM-2 (0.26 g) obtained in (Example 3) <Step 2> and N,N-diisopropylethylamine (0.51 mL) under ice-cooling and stirring, and the mixture was stirred at room temperature for 1 hour and 30 minutes. After adding water (5 mL) to stop the reaction, the mixture was extracted three times with ethyl acetate (5 mL). The organic layer was washed with saturated saline (3 mL) and then dried over anhydrous sodium sulfate. The dried organic layer was filtered, and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (12% ethyl acetate / n-heptane to 100% ethyl acetate) to obtain the title compound EX3-IM-3 (0.189 g) as a white amorphous substance.

[0282] NMR data (CDCl 3 )(δ:ppm): 7.31 (2H, d, J = 8.4 Hz), 7.26 (2H, d, J = 8.0 Hz, Overlapped with solvent peak.), 6.84 (1H, br s), 6.52 (1H, br s), 4.52 (2H, d, J = 6.0 Hz), 4.49 (2H, d, J = 6.4 Hz), 4.26-4.23 (1H, m), 4.11 (1H, d, J = 15.2 Hz), 3.94 (1H, d, J = 15.2 Hz), 2.26-2.09 (3H, m), 2.00-1.58 (6H, m), 1.48-1.44 (1H, m).LC-MS:M=396,RT=0.99(min),[M+H]+=397.

[0283] <Step 4> Synthesis of N-(4-(aminomethyl)benzyl)-2-(cyclooct-2-yn-1-yloxy)acetamide (compound EX3-IM-4): [ka]

[0284] To a mixture of compound EX3-IM-3 (0.18 g) obtained in (Example 3) <Step 3> and methanol (1.8 mL), potassium carbonate (0.126 g) aqueous solution (0.9 mL) was added dropwise under ice-cooling and stirring, 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 three times with ethyl acetate (5 mL). The organic layer was washed with saturated saline (5 mL) and then dried over anhydrous sodium sulfate. The organic layer was filtered, and 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.

[0285] NMR data (CDCl 3 )(δ:ppm): 7.28-7.28 (4H, m), 6.80 (1H, br s), 4.48 (2H, d, J = 6.0 Hz), 4.26-4.21 (1H, m), 4.11 (1H, d, J = 15.2 Hz), 3.93 (1H, d, J = 15.2 Hz), 3.86 (2H, s), 2.28-2.07 (3H, m), 1.99-1.40 (7H, m, Overlapped with solvent peak.).LC-MS:M=300,RT=0.68(min),[M+H]+=301.

[0286] (Example 3a) Synthesis of N-(4-(aminomethyl)benzyl)-2-(cyclooct-2-yn-1-yloxy)acetamide group-introduced alginic acid (EX3-(I)-A-2a) [ka]

[0287] To an aqueous solution (60 mL) of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.) adjusted to 1% by weight, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (183 mg) was added under stirring at room temperature. Then, a solution of compound EX3-IM-4 (41.2 mg) obtained in (Example 3) <Step 4> in ethanol (5 mL) was added dropwise at the same temperature, and the mixture was stirred at 40 degrees for 4 hours. After cooling to room temperature, sodium chloride (600 mg) was added, followed by ethanol (119 mL), and the mixture was stirred for 30 minutes. The resulting precipitate was collected by filtration, washed three times with ethanol (5 mL), and then dried under reduced pressure to obtain the title compound EX3-(I)-A-2a as a white solid. The white solid was dissolved in 40 mL of water, lyophilized, and then dried at 40° C. for 22 hours to obtain the title compound EX3-(I)-A-2a (597 mg) as a white fluffy material.

[0288] The introduction rate of the reactive substituent (N-(4-(aminomethyl)benzyl)-2-(cyclooct-2-yn-1-yloxy)acetamide group) was 3.7 mol % (NMR integral ratio).

[0289] (Example 3b) Synthesis of N-(4-(aminomethyl)benzyl)-2-(cyclooct-2-yn-1-yloxy)acetamide group-introduced alginic acid (EX3-(I)-A-2b) [ka]

[0290] 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (405 mg), a solution of compound EX3-IM-4 (121 mg) obtained in (Example 3) <Step 4> in ethanol (29 mL), and 1 molar concentration sodium bicarbonate water (366 μL) were added to an aqueous solution (290 mL) of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.) adjusted to 1% by weight, and the mixture was stirred at 32°C for 3 hours and 20 minutes. Sodium chloride (2.9 g) was added, followed by ethanol (580 mL) and stirring at room temperature for 30 minutes. The resulting precipitate was collected by filtration, washed with ethanol, and then dried under reduced pressure to obtain the title compound EX3-(I)-A-2b (2.67 g) as a white solid. The white solid was dissolved in 180 mL of water, freeze-dried, dried at 40° C. for 6 hours, and then refrigerated and stored, and then dried again at 40° C. for 5 hours to obtain the title compound EX3-(I)-A-2b (2.77 g) as a white amorphous solid.

[0291] The introduction rate of the reactive substituent (N-(4-(aminomethyl)benzyl)-2-(cyclooct-2-yn-1-yloxy)acetamide group) was 2.1 mol % (NMR integral ratio).

[0292] (Example 3c) Synthesis of N-(4-(aminomethyl)benzyl)-2-(cyclooct-2-yn-1-yloxy)acetamide group-introduced alginic acid (EX3-(I)-A-2c) [ka]

[0293] 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (363 mg), a solution of compound EX3-IM-4 (109 mg) obtained in (Example 3) <Step 4> in ethanol (26 mL), and 1 molar sodium bicarbonate water (328 μL) were added to an aqueous solution (260 mL) of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.) adjusted to 1% by weight, and the mixture was stirred at 32° C. for 3 hours. Sodium chloride (2.6 g) was added to the reaction solution, followed by addition of ethanol (520 mL), and the mixture was stirred at room temperature for 30 minutes. The resulting precipitate was collected by filtration, washed with ethanol, and then dried under reduced pressure to obtain the title compound EX3-(I)-A-2c (2.69 g) as a white solid. The white solid was dissolved in 180 mL of water, freeze-dried, and then dried at 40° C. for 2.5 hours to obtain the title compound EX3-(I)-A-2c (2.42 g) as a white amorphous solid.

[0294] The introduction rate of the reactive substituent (N-(4-(aminomethyl)benzyl)-2-(cyclooct-2-yn-1-yloxy)acetamide group) was 2.0 mol % (NMR integral ratio).

[0295] Example 4 Synthesis of N-(2-aminoethyl)-4-azidobenzamide group-introduced alginic acid (Example 4): [ka]

[0296] <Step 1> Synthesis of tert-butyl (2-(4-azidobenzamido)ethyl)carbamate (compound EX4-IM-1): [ka]

[0297] To 4-azidobenzoic acid (EX4-SM, [CAS: 6427-66-3] 700 mg), thionyl chloride (783 μL) and N,N-dimethylformamide (3 μL) were added and stirred at 70°C for 1 hour. The reaction solution was concentrated under reduced pressure, and methylene chloride (1 mL) was added to the residue to perform azeotropic distillation, and the same operation was carried out again. To the obtained pale yellow solid in methylene chloride (3 mL), a solution of tert-butyl (2-aminoethyl) carbamate [CAS: 57260-73-8] (825 mg) and pyridine (1.04 mL) in methylene chloride (7.0 mL) was added under ice-water cooling, and the mixture was stirred at room temperature for 1 hour. The reaction mixture was diluted with tert-butyl methyl ether (30 mL) and washed successively with water (10 mL), saturated sodium bicarbonate water (5 mL), 0.5N citric acid (5 mL x 2), water (5 mL), and saturated saline (5 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was triturated with tert-butyl methyl ether / heptane, and the solid was filtered and washed with tert-butyl methyl ether / heptane to obtain the title compound EX4-IM-1 (1.1 g) as a white solid.

[0298] NMR data (CDCl 3 )(δ:ppm): 7.83(2H, d, J = 8 Hz), 7.26(1H, brs), 7.05(2H, d, J = 8 Hz), 4.97(1H, brs), 3.55(2H, q, J = 5 Hz), 3.45-3.37(2H, m), 1.43(9H, s), LC-MS:M=305, RT=0.90(min), [M+H] + =306, [M+Na] + =328

[0299] <Step 2> Synthesis of N-(2-aminoethyl)-4-azidobenzamide hydrochloride (compound EX4-IM-2): [ka]

[0300] The compound (EX4-IM-1, 500 mg) obtained in (Example 4) <Step 1> was suspended in 1,4-dioxane (1.5 mL). 4H2Cl / 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 solution, 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 obtain the title compound EX4-IM-2 (365 mg) as a pale beige solid.

[0301] NMR data (DMSO-d 6 )(δ:ppm): 8.68(1H, t, J = 6 Hz), 7.93(2H, d, J = 9 Hz), 7.82(1H, brs), 7.22(2H, d, J = 9 Hz), 3.49(2H, q, J = 6 Hz), 2.97(2H, t, J = 6 Hz), LC-MS: M(free amine)=205, RT=0.56(min), [M+H] + =206

[0302] <Step 3-1> Synthesis of N-(2-aminoethyl)-4-azidobenzamide group-introduced alginic acid (EX4-(II)-A2): [ka]

[0303] 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (167 mg), compound EX4-IM-2 (37 mg) obtained in (Example 4) <Step 2>, and 1 molar sodium bicarbonate water (227 μL) were added to a 1 wt% aqueous solution of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd.: A-2) (60 mL), and the mixture was stirred at 30° C. for 3.5 hours. Sodium chloride (600 mg) was added, followed by ethanol (120 mL), and the mixture was stirred at room temperature for 30 minutes. The resulting precipitate was collected by filtration, washed with ethanol, and then dried under reduced pressure to obtain the title compound EX4-(II)-A-2 (615 mg) as a white powder. The white solid was dissolved in 40 mL of water, freeze-dried, and then dried at 40° C. for 22 hours to obtain the title compound EX4-(II)-A-2 (583 mg) as a white amorphous solid.

[0304] The introduction rate of the reactive group (N-(2-aminoethyl)-4-azidobenzamide group) was 5.3 mol % (NMR integral ratio).

[0305] (Example 4-2) Synthesis of N-(2-(2-aminoethoxy)ethyl)-4-azidobenzamide group-introduced alginic acid (Example 4-2a and Example 4-2b): [ka]

[0306] <Step 1> Synthesis of tert-butyl (2-(2-(4-azidobenzamido)ethoxy)ethyl)carbamate (compound EX4-2-IM-1): [ka]

[0307] 4-Azidobenzoic acid [CAS: 6427-66-3] (EX4-SM, 300 mg), tert-butyl (2-(2-aminoethoxy)ethyl)carbamate [CAS: 127828-22-2] (376 mg) were dissolved in acetonitrile (6.0 mL). O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (0.77 g) and diisopropylethylamine (707 μL) were added and stirred at room temperature for 16 hours. Ethyl acetate (20 mL) and water (10 mL) were added to the reaction solution, and the layers were separated. The organic layer was washed with water (10 mL) and saturated saline (5 mL) in that order, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (20% ethyl acetate / n-heptane to 70% ethyl acetate / n-heptane) to obtain the title compound EX4-2-IM-1 (673 mg) as a pale yellow gum.

[0308] NMR data (CDCl 3 )(δ:ppm): 7.83(2H, d, J = 9 Hz), 7.08(2H, d, J = 9 Hz), 6.61(1H, brs), 4.84(1H, brs), 3.68-3.64(4H, m), 3.56(2H, t, J = 5 Hz), 3.34(2H, q, J = 5 Hz), 1.44 (9H, s)

[0309] <Step 2> Synthesis of N-(2-(2-aminoethoxy)ethyl)-4-azidobenzamide hydrochloride (compound EX4-2-IM-2): [ka]

[0310] To the compound (EX4-2-IM-1, 670 mg) obtained in <Step 1> of (Example 4-2), 4N-hydrogen chloride / 1,4-dioxane (4.7 mL) was added under ice-water cooling, and the mixture was stirred at room temperature for 2 hours. Diisopropyl ether (14 mL) was added to the reaction solution, 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 obtain the title compound EX4-2-IM-2 (604 mg) as a pale beige solid.

[0311] NMR data (DMSO-d 6 )(δ:ppm): 8.61(1H, t, J = 6 Hz), 7.95(3H, brs), 7.93(2H, d, J = 9 Hz), 7.20(2H, d, J = 9 Hz), 3.62(2H, t, J = 5 Hz), 3.57(2H, t, J = 6 Hz), 3.46 (2H, q, J = 6 Hz), 3.02-2.93 (2H, m), LC-MS (free amine): RT=0.57 (min), [M+H]+=250

[0312] (Example 4-2a) Synthesis of N-(2-(2-aminoethoxy)ethyl)-4-azidobenzamide group-introduced alginic acid (EX4-2-(II)-A-2a) [ka]

[0313] 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (366.09 mg) and 1 molar sodium bicarbonate solution (274.51 μL) were added to an aqueous solution (118 mL) of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.) prepared to 1% by weight. Then, a solution of compound EX4-2-IM-2 (78.44 mg) obtained in (Example 4-2) <Step 2> in water (1 mL) and ethanol (1 mL) was added at room temperature and stirred at 40° C. for 4 hours. Sodium chloride (1.2 g) was added at room temperature, followed by addition of ethanol (237 mL) and stirring at room temperature for 30 minutes. The resulting precipitate was collected by filtration, washed with ethanol, and dried under reduced pressure to obtain the title compound EX4-2-(II)-A-2a (1.17 g) as a white solid. The white solid was dissolved in 80 mL of water and lyophilized to give the title compound EX4-2-(II)-A-2a (1.14 g) as a white fluffy mass.

[0314] The introduction rate of the reactive group (N-(2-(2-aminoethoxy)ethyl)-4-azidobenzamide group) was 2.72 mol % (NMR integral ratio).

[0315] (Example 4-2b) Synthesis of N-(2-(2-aminoethoxy)ethyl)-4-azidobenzamide group-introduced alginic acid (EX4-2-(II)-A-2b) [ka]

[0316] 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (279 mg) and 1 molar sodium bicarbonate solution (378 μL) were added to an aqueous solution (200 mL) of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.) prepared to 1% by weight. Then, compound EX4-2-IM-2 (77 mg) obtained in (Example 4-2) <Step 2> was added at room temperature and stirred at 32°C for 3.25 hours. Sodium chloride (2.0 g) was added to the reaction solution at room temperature, followed by addition of ethanol (400 mL) and stirring at room temperature for 30 minutes. The resulting precipitate was collected by filtration, washed with ethanol, and dried under reduced pressure to obtain the title compound EX4-2-(II)-A-2b (1.93 g) as a white solid. The white solid was dissolved in 130 mL of water and lyophilized to obtain the title compound EX4-2-(II)-A-2b (1.85 g) as a white amorphous solid.

[0317] The introduction rate of the reactive group (N-(2-(2-aminoethoxy)ethyl)-4-azidobenzamide group) was 2.3 mol % (NMR integral ratio).

[0318] [Table 10]

[0319] [Measurement of introduction rate of reactive group or complementary reactive group] The introduction rate of the reactive group or the complementary reactive group means the number of reactive groups or the complementary reactive groups introduced per uronic acid monosaccharide unit, which is a repeating unit of alginic acid, expressed as a percentage. In this embodiment, the introduction rate (mol%) of the reactive group or the complementary reactive group is 1 The amount of alginic acid required to calculate the introduction rate was measured by the carbazole-sulfuric acid method using a calibration curve, and the amount of reactive groups or complementary reactive groups can also be measured by an absorbance measurement method using a calibration curve.

[0320] [Molecular weight measurement] The alginic acid solid obtained in the examples into which reactive groups or complementary reactive groups have been introduced was dissolved in 10 mmol / L phosphate buffer (pH 7.4) containing 0.15 mol / L NaCl to prepare a 0.1% solution, which was then passed through a polyethersulfone filter (Minisart High Flow Filter, Sartorius) with a pore size of 0.22 μm to remove insoluble matter, and used as a sample for gel filtration. The spectrum of each sample was measured using a spectrophotometer DU-800 (Beckman-Coulter), and the measurement wavelength in gel filtration for each compound was determined. For compounds that did not have a specific absorption wavelength, a differential refractometer was used.

[0321] 200 μL of the gel filtration sample was applied to a Superose6 Increase10 / 300 GL column (GE Healthcare Sciences). Gel filtration was performed at room temperature with a flow rate of 0.8 mL / min using an AKTA Explorer 10S chromatograph and 10 mmol / L phosphate buffer (pH 7.4) containing 0.15 mol / L NaCl as the developing solvent. The elution profile of the sample was created by monitoring the absorption at the wavelength determined for each compound. The obtained chromatogram was analyzed with Unicorn5.31 software (GE Healthcare Sciences) to determine the peak range.

[0322] The molecular weight of alginic acid into which a reactive group or a complementary reactive group has been introduced was determined by gel filtration under the same conditions as for alginic acid into which a reactive group or a complementary reactive group has been introduced, using blue dextran (molecular weight 2 million Da, SIGMA), thyroglobulin (molecular weight 669,000 Da, GE Healthcare Science), ferritin (molecular weight 440,000 Da, GE Healthcare Science), aldolase (molecular weight 158,000 Da, GE Healthcare Science), conalbumin (molecular weight 75,000 Da, GE Healthcare Science), ovalbumin (molecular weight 44,000 Da, GE Healthcare Science), ribonuclease A (molecular weight 13,700 Da, GE Healthcare Science), and aprotinin (molecular weight 6500 Da, GE Healthcare Science) as standards, and the elution volume of each component was determined using Unicorn software. The elution volume of each component was plotted on the horizontal axis and the logarithm of the molecular weight on the vertical axis, respectively, and linear regression was performed to create a calibration curve. Two calibration curves were prepared, one from blue dextran to ferritin and the other from ferritin to aprotinin.

[0323] Using this calibration curve, the molecular weight (Mi) at elution time i of the previously obtained chromatogram was calculated. Next, the absorbance at elution time i was read and designated as Hi. From these data, the weight average molecular weight (Mw) was calculated using the following formula.

[0324]

number

[0325] Example 5 Preparation of flat alginate gel

[0326] [Preparation of alginic acid solution] Using the alginic acid derivative obtained in each Example, a 1.6% by weight or 3.3% by weight aqueous solution was prepared and sterilized by filtration using Mini Sart High Flow (Sartorius, 0.2 μm, Cat. 16532GUK). The salt concentration of the aqueous solution after sterilization by filtration was adjusted to a 1.5% by weight or 3.0% by weight physiological saline solution. Alginic acid of Example 1(a, b, c): 1.5 wt% saline solution (Solutions of Example 5-1a, Example 5-1b, 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% saline solution (Solution of Example 5-3a) Alginic acid of Example 4: 3.0 wt% saline solution (Solution of Example 5-4) Alternatively, a 1.0% by weight solution of the alginic acid derivative obtained in each Example is prepared in physiological saline, and sterilized by filtration using MILLEX GV 0.22 μm (Millipore, 0.22 μm, Cat. SLGV033RS). Alginic acid of Example 1(d): 1.0 wt% saline solution (Solution of Example 5-1d) Alginic acid of Example 2(d): 1.0 wt% saline solution (Solution of Example 5-2d) Alginic acid of Example 3(b): 1.0 wt% saline solution (Solution of Example 5-3b) Alginic acid of Example 4-2(a): 1.0 wt% saline solution (Solution of Example 5-5a) Alginic acid of Example 1(e): 0.5 wt% saline solution (Solution of Example 5-1e) Alginic acid of Example 2(e): 0.5 wt% saline solution (Solution of Example 5-2e) Alginic acid of Example 3(c): 0.5 wt% saline solution (solution of Example 5-3b) Alginic acid of Example 4-2(b): 0.5 wt% saline solution (Solution of Example 5-5b) In the following examples, each of these alginic acid saline solutions was used in the tests after adjusting the concentration appropriately. The alginic acid prepared in Example 1(a, b, c) or Example 3(a) is combined with the alginic acid prepared in Example 2(a, b, c) or Example 4 to produce a chemically crosslinked alginic acid gel. More specifically, the solution of Example 5-1(a, b, c) is combined with the solution of Example 5-2(a, b, c), and the solution of Example 5-3a is combined with the solution of Example 5-4. In addition, the alginic acid prepared in Example 1(d) or Example 3(b) is combined with the alginic acid prepared in Example 2(d) or Example 4-2(a) to produce a chemically crosslinked alginic acid gel. More specifically, the solution of Example 5-1d is combined with the solution of Example 5-2d, the solution of Example 5-1d is combined with the solution of Example 5-5a, the solution of Example 5-3b is combined with the solution of Example 5-2d, and the solution of Example 5-3b is combined with the solution of Example 5-5a. In addition, the alginic acid prepared in Example 1(e) or Example 3(c) is combined with the alginic acid prepared in Example 2(e) or Example 4-2(b) to produce a chemically crosslinked alginic acid gel. More specifically, the solution of Example 5-1e is combined with the solution of Example 5-2e, the solution of Example 5-1e is combined with the solution of Example 5-5b, the solution of Example 5-3c is combined with the solution of Example 5-2e, and the solution of Example 5-3c is combined with the solution of Example 5-5b.

[0327] [Production of flat alginate gel] <General preparation method> 3.5 cm culture dish with 55 mmol / L calcium chloride (CaCl 2 Add 2 mL of the aqueous solution, drop 400 μL of alginate solution using a P1000 pipette, and let stand for 10 minutes. If the alginate starts to solidify after 5 minutes or more, shake the culture dish by hand to solidify the edges of the alginate. After 10 minutes, add an additional 4 mL of calcium chloride solution and let stand for 5 minutes. Wash three times with saline to obtain a flat alginate gel. Here, the flat gel refers to an alginate gel having a size of, for example, a minor diameter of 12 to 15 mm, a major diameter of 12 to 18 mm, and a thickness of about 0.5 to 5 mm, and can have a circular, rectangular, hexagonal, octagonal, or other shape, and is not particularly limited.

[0328] Example 6 Biocompatibility testing of alginate gel

[0329] [Example 6-1: Preparation of flat alginate gel for transplantation] According to the procedure of "Preparation of plate-type alginic acid gel" described in Example 5, plate-type alginic acid gel was prepared using a 55 mmol / L aqueous calcium chloride solution. The following flat plate-type alginate gels were prepared using the following alginate solutions: an aqueous solution of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd.: B-2) prepared at 1% by weight; an aqueous solution of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd.: A-2); a solution obtained by mixing solutions of Examples 5-1b and 5-2b in a 2:1 (volume ratio); a solution obtained by mixing solutions of Examples 5-1d and 5-2d in a 1:1 (volume ratio); a solution obtained by mixing solutions of Examples 5-1d and 5-5 in a 1:1 (volume ratio); a solution obtained by mixing solutions of Examples 5-3b and 5-2d in a 1:1 (volume ratio); and a solution obtained by mixing solutions of Examples 5-3b and 5-5a in a 1:1 (volume ratio). The prepared alginate gel on the plate was cultured overnight in D-MEM medium. The next day, the medium was replaced with serum-free D-MEM medium, and then with physiological saline. The medium was left to stand for at least 1 hour to obtain an alginate gel for transplantation into animals.

[0330] Example 6-1a: Using an aqueous solution of sodium alginate (B-2, manufactured by Mochida Pharmaceutical Co., Ltd.) prepared at 1% by weight, a flat alginate gel with a short diameter (12 mm), long diameter (15 mm), and thickness (5 mm) was obtained. A photograph of this flat alginate gel is shown in Figure 1(a).

[0331] Example 6-1b: A flat alginate gel with a short diameter (12 mm), long diameter (12 mm), and thickness (4 mm) was obtained using a solution obtained by mixing the solutions of Example 5-1b and Example 5-2b at a volume ratio of 2:1. The chemical crosslinking group was prepared at a concentration of 1%. A photograph of this flat alginate gel is shown in Figure 2(a).

[0332] Example 6-1c: A flat alginate gel with a short diameter (12 mm), long diameter (12 mm), and thickness (4 mm) was obtained using a solution obtained by mixing the solutions of Example 5-1b and Example 5-2b at a volume ratio of 2:1. The chemical crosslinking group was prepared at a concentration of 2%. A photograph of this flat alginate gel is shown in Figure 3(a).

[0333] Example 6-1d: Using an aqueous solution of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.) adjusted to 1% by weight, a flat alginate gel with a short diameter (approximately 12 mm), long diameter (approximately 12 mm), and thickness (approximately 4 mm) was obtained.

[0334] Example 6-1e: Using a solution obtained by mixing the solutions of Example 5-1d and Example 5-2d at a ratio of 1:1 (volume ratio), a flat alginate gel with a short diameter (about 12 mm), a long diameter (about 12 mm), and a thickness (about 4 mm) was obtained. The chemical crosslinking group was prepared at a concentration of 1%.

[0335] Example 6-1f: Using a solution obtained by mixing the solution of Example 5-1d and the solution of Example 5-5a at a ratio of 1:1 (volume ratio), a flat alginate gel with a short diameter (about 12 mm), a long diameter (about 12 mm), and a thickness (about 4 mm) was obtained. The chemical crosslinking group was prepared at a concentration of 1%.

[0336] Example 6-1g: Using a solution obtained by mixing the solution of Example 5-3b and the solution of Example 5-2d at a ratio of 1:1 (volume ratio), a flat alginate gel with a short diameter (about 12 mm), a long diameter (about 12 mm), and a thickness (about 4 mm) was obtained. The chemical crosslinking group was prepared at a concentration of 1%.

[0337] Example 6-1h: Using a solution obtained by mixing the solution of Example 5-3b and the solution of Example 5-5a at a ratio of 1:1 (volume ratio), a flat alginate gel with a short diameter (about 12 mm), a long diameter (about 12 mm), and a thickness (about 4 mm) was obtained. The chemical crosslinking group was prepared at a concentration of 1%.

[0338] [Example 6-2: Implantation test of flat alginate gel into animals] Each of the alginate gels prepared in Examples 6-1a to 6-1c was transplanted into the abdominal cavity of a healthy mouse C57BL / 6NCr. After 5 weeks, the abdomen was opened to extract the gel, and the condition of the gel was confirmed. Intraperitoneal adhesions and inflammation were also confirmed. In addition, each of the alginate gels prepared in Examples 6-1d to 6-1h was transplanted into the abdominal cavity of a healthy mouse C57BL / 6NCr. After 1, 2 or 4 weeks, the abdomen was opened to extract the gel, and the condition of the gel was confirmed. Intraperitoneal adhesions and inflammation were also confirmed.

[0339] Alginate gel of Example 6-1a: The excised alginate gel did not maintain its original shape, but was in pieces, and the amount of gel that could be confirmed as remaining was small. A photograph of this state is shown in Figure 1(b). Alginate gel of Example 6-1b: The size of the excised alginate gel did not change. A photograph of this state is shown in Figure 2(b). Alginate gel of Example 6-1c: The excised alginate gel was broken, but it maintained almost its original shape and there was no change in size of the gel. A photograph of this state is shown in Figure 3(b).

[0340] Alginate gel of Example 6-1d: The alginate gel extracted one week later did not maintain its original shape and was in pieces, and only a small amount of remaining gel was recovered. Alginate gel of Example 6-1f: There was no change in size of the alginate gels extracted after 1 week, 2 weeks, and 4 weeks. Alginate gel of Example 6-1g: The alginate gels extracted after 1 week and 2 weeks showed no change in size. The alginate gels extracted after 4 weeks were cracked but maintained almost the original shape and showed no change in size. Alginate gel of Example 6-1h: The alginate gels extracted after 1 week and 2 weeks showed no change in size. The alginate gels extracted after 4 weeks were cracked but maintained almost the original shape and showed no change in size.

[0341] Regarding the alginate gels of Examples 6-1a, 6-1b, and 6-1c, when the abdomen was opened 5 weeks after transplantation, no adhesion or inflammation was found between the intraperitoneal organs. There was also no adhesion or inflammation in the omentum or mesenteric membrane in which the gel was embedded. There was also no adhesion or inflammation in the liver to which the broken gel had been attached.

[0342] Regarding the alginate gels of Examples 6-1d, 6-1f, 6-1g, and 6-1h, when the abdomen was opened 1 week, 2 weeks, and 4 weeks after transplantation, no adhesion or inflammation was found between the intraperitoneal organs. There was also no adhesion or inflammation in the omentum or mesenteric membrane in which the gel was embedded. There was no adhesion or inflammation in the liver to which the fragmented gel had been attached.

[0343] [Example 6-3: Cell viability confirmation test for flat alginate gel] MIN6 cells (5 × 10 6 cells) was added to each of the alginate solutions used in the preparation of the alginate gels of Examples 6-1a, 6-1b, 6-1c, 6-1d, 6-1e, 6-1f, 6-1g, and 6-1h, and then alginate gels were produced and cultured in D-MEM medium for 3 to 4 weeks, and the survival of the MIN6 cells was confirmed under a microscope. Cell proliferation in the alginate gels of Examples 6-1a, 6-1b, 6-1c, 6-1d, 6-1e, 6-1f, 6-1g, and 6-1h was good, and sufficient cell survival was observed under a microscope.

[0344] Example 7 Evaluation of implantable devices by intraperitoneal implantation into diabetic model mice

[0345] [Isolation of porcine islets] Viable, sterile pancreases were obtained from adult pigs under aseptic conditions and islet cells were isolated according to procedures known in the art, or according to the method described by Shimoda et al. (Cell Transplantation, Vol. 21, pp. 501-508, 2012), or the standard Ricordi technique using the Edmonton protocol.

[0346] [Method for culturing isolated pancreatic islets] The isolated islets were then cultured in a medium (Connaught Medical Research Laboratory (CMRL)-based Miami-defined media #1 (MM1; Mediatech-Cellgro, Herndon, VA)-supplemented with 0.5% human serum albumin) at 37 °C under 5% CO in accordance with the method described by Noguchi et al. (Transplantation Proceedings, 42, 2084-2086 (2010)). 2 The cells were cultured in a humidified atmosphere of 95% air at 37°C for 1 day. The cultured islets were used to prepare a transplant device.

[0347] Preparation of implantable devices Using Example 1b with a crosslinking group introduction rate of 5.0 mol % and Example 2b with a crosslinking group introduction rate of 4.9 mol %, a 1.5 wt % saline solution of Example 5-1b and a 3.0 wt % saline solution of Example 5-2b are prepared, respectively. By mixing the solution of Example 5-1b and the solution of Example 5-2b in a 2:1 (volume ratio), a 2 wt % alginic acid solution with an introduction rate of crosslinking groups equivalent to 5 mol % can be prepared. The solutions of Example 5-1b and Example 5-2b are diluted 2-fold and 4-fold to prepare solutions, which are then mixed in a 2:1 (volume ratio) mixture to prepare solutions. The mixture of the two-fold diluted solutions is designated as the solution of Example 7-1, and the mixture of the four-fold diluted solutions is designated as the solution of Example 7-2. In addition, in a similar manner, the solution of Example 5-1c and the solution of Example 5-2c are mixed in a 2:1 (volume ratio) and diluted 4-fold to obtain a mixed solution, which is used as the solution of Example 7-3.

[0348] The implantation devices prepared using the solutions of Example 7-1 and Example 7-2 (100 μL, 200 μL) and the solution of Example 7-3 (100 μL, 200 μL) were as follows. <Implantable Device> Example 7-1a: Implantable device prepared using 100 μL of the solution of Example 7-1 Example 7-1b: Implantable device prepared using 200 μL of the solution of Example 7-1 Example 7-2a: Implantable device prepared using 100 μL of the solution of Example 7-2 Example 7-2b: Implantable device prepared using 200 μL of the solution of Example 7-2 Example 7-3a: Implantable device prepared using 100 μL of the solution of Example 7-3 Example 7-3b: Implantable device prepared using 200 μL of the solution of Example 7-3

[0349] In order to prepare 100μL or 200μL of porcine islet-containing 0.5wt% to 1.0wt% alginic acid solution, the islet pellets dispensed into one device were suspended in the alginic acid solution (B1) of Example 5-1b, and then mixed with the alginic acid solution (C1) of Example 5-2b to obtain the solutions of Examples 7-1a, 7-1b, 7-2a, and 7-2b in which the porcine islets were suspended. The alginic acid solution (B1) of Example 5-1b and the alginic acid solution (C1) of Example 5-2b were adjusted in concentration with physiological saline according to the pellet amount (10 to 30μL) of 10000IEQ of porcine islet per device. In addition, in order to prepare 100μL or 200μL of 0.5wt% to 1.0wt% alginic acid solution containing porcine islets, the islet pellets dispensed into one device were suspended in the alginic acid solution (B1) of Example 5-1c, and then 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 islets were suspended. The alginic acid solution (B1) of Example 5-1c and the alginic acid solution (C1) of Example 5-2c were adjusted in concentration with physiological saline according to the pellet amount (10 to 30μL) of 10000IEQ of porcine islets per device.

[0350] The porcine islet-containing alginic acid solutions prepared in Examples 7-1a, 7-2a, 7-2b, and 7-3b were quickly sealed in a semipermeable membrane (Spectrum dialysis tube "Spectra / Pore CE (molecular weight cutoff: 100,000)") (one end of the semipermeable membrane was heat sealed, the alginic acid solution was poured in, and the membrane was sealed with a titanium clip) to obtain a 55 mmol / L CaCl 2 The device was immersed in the solution for 10 to 15 minutes to gel the alginate solution in the device.

[0351] After gelation, the transplantation device was washed with physiological saline for 3 minutes and cultured overnight in transplantation medium (M199-nicotinamide-FBS+P / S). Next, it was immersed in serum-free transplantation medium (M199+P / S) for 30 minutes, and then immersed in physiological saline+P / S for pre-transplant washing for 30 minutes to prepare a device for transplantation into a mouse. A photograph of the prepared transplantation device is shown in Figure 4-1.

[0352] - Dimensions of implanted device: length 10 mm x width 26 mm x thickness approx. 2 mm ·Culture conditions: M199+Nicotinamide+Fetal bovine serum+ Penicillin / Streptomycin (P / S), O / N Washing conditions: 1) Serum-free medium for transplantation (M199+P / S), 30 min, rt 2) Physiological saline (saline+P / S), 30 min, rt

[0353] [Table 11]

[0354] [Evaluation of implantable devices (implantation testing)] Animals used: Diabetic model mice of wild-type immunocompetent mice (C57BL / 6NCr). 13-17 weeks old, male, 25-35 g. A diabetic model was created by a single injection of streptozocin solution at 110 mg / kg via tail vein in about one week. Individuals with casual blood glucose levels of 300 mg / dL or more and 600 mg / dL or less were used as diabetic models.

[0355] Method of administration / implantation: The mice were anesthetized with 0.25-0.3 mL of a triple anesthetic mixture (Domitor / Midazolam / Betorfal) administered intraperitoneally. Under anesthesia, the abdomen was shaved and disinfected, and then a 2 cm midline incision was made. After washing, the device was simply placed in the abdominal cavity and implanted without fixation. After implantation, the abdomen was closed and 0.25-0.3 mL of a medetomidine antagonist (Antisedan) was injected subcutaneously to awaken the mice. The surgery was performed by keeping the mice warm on a heat pad. No immunosuppressants were administered. No fluid replacement, antibiotics, or anti-inflammatory drugs were administered.

[0356] Blood glucose and weight measurement: Random blood glucose levels were measured at regular times during the day before transplantation and every few days from day 1 after transplantation. Blood glucose levels were measured using a drop of blood from a scalpel incision on the tail using the Glutest Neo Alpha and Glutest Neo sensors. Body weight was measured on an electronic balance immediately before random blood glucose level measurements. Mice with device implants were considered cured of diabetes when their random blood glucose levels were less than 300 mg / dL. 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 up to day 305 after transplantation are shown in FIG. 5-2, and the body weight fluctuations up to day 305 after transplantation are shown in FIG. 6-2. Furthermore, the transplantation device was removed on day 305 after transplantation and transplanted into another diabetic model mouse (here, transplantation of a device into a diabetic model mouse, removal of the transplanted device after a predetermined period of time, and transplantation of the removed device into another diabetic model mouse is called "relay transplantation"), and the blood glucose level fluctuations up to day 26 after relay transplantation are shown in FIG. 5-3, and the body weight fluctuations up to day 26 after relay transplantation are shown in FIG. 6-3. In FIG. 5-1 to 3 and FIG. 6-1 to 3, #1 and #2 respectively mean numbers that identify the individual mice that were transplanted. Body weight fluctuations were normal and blood glucose levels remained normal for 75 days, and for 305 days after transplantation and for 26 days after a further relay transplant.

[0357] When the implantation device of Example 7-2b was used, there was no abnormality in body weight fluctuation, and the blood glucose level was maintained at a normal level for 75 days, similar to the case of Example 7-2a. FIG. 7-1 shows the blood glucose level fluctuations and FIG. 8-1 shows the body weight fluctuations up to day 305 after implantation when the implantation device of Example 7-2b was used. Furthermore, the implantation device was removed on day 305 after implantation and implanted into another diabetic model mouse, and FIG. 7-2 shows the blood glucose level fluctuations and FIG. 8-2 shows the body weight fluctuations up to day 26 after relay implantation. Body weight fluctuations remained normal and blood glucose levels remained normal for 305 days after transplantation and for 26 days after a further relay transplant.

[0358] When the implantation device of Example 7-3b was used, there was no abnormality in body weight fluctuation, and the blood glucose level was maintained at a normal level for 75 days, similar to Example 7-2a. FIG. 9-1 shows blood glucose level fluctuations and FIG. 10-1 shows body weight fluctuations up to day 305 after implantation when the implantation device of Example 7-3b was used. Furthermore, the implantation device was removed on day 305 after implantation and implanted into another diabetic model mouse, and FIG. 9-2 shows blood glucose level fluctuations and FIG. 10-2 shows body weight fluctuations up to day 26 after relay implantation. In addition, in #2 and #3, the device was removed midway and the test was terminated. Body weight fluctuations remained normal and blood glucose levels remained normal for 305 days after transplantation and for 26 days after a further relay transplant.

[0359] In the preparation of the transplantation device, a transplantation device was prepared in which pancreatic islets were encapsulated in a semipermeable membrane without using an alginic acid derivative. When the device was transplanted into mice in the same manner as in the above-mentioned [Evaluation of transplantation device (transplantation test)], no blood glucose suppression effect was observed in diabetic mice.

[0360] Assessment of tissue reactivity: The tissue reactivity was evaluated as follows. A few weeks after implantation, or after blood glucose levels were increased at any time, the device-implanted mice were anesthetized with a triple anesthetic mixture, the abdomen was disinfected under anesthesia, an approximately 4 cm midline incision was made, and the implanted device was searched for between the intraperitoneal organs. If a part of the device was found between the organs, it was slowly removed with tweezers to see if the device could be removed alone. The surface condition of the removed device was observed. <Observation items> 1. Check whether angiogenesis is present on the surface of the device. If angiogenesis is present, observe whether it is at the capillary level or at the large blood vessel level. 2. Next, observe whether there is adhesion or connection to organs, the peritoneum, the omentum, etc. Check whether the organs can be dissected bluntly or whether sharp dissection is required. 3. If there is direct adhesion to an organ, confirm which organ is adhered to which part of the device (whether the entire surface, part, edge, fold or sealing part of the device, etc.). 4. Check whether there is any inflammation on the organ side. * After the device is removed, the abdomen is closed. The mouse is awakened by subcutaneous injection of an antagonist. The surgery is performed by keeping the mouse warm on a heat pad.

[0361] When the transplantation device of Example 7-1a was used, the device was removed 10 weeks after transplantation and the tissue reactivity was observed. As a result, (1) no angiogenesis was observed on the surface of the device, (2) the device was not adhered to organs, peritoneum, omentum, etc., (3) the organs could be bluntly peeled off and the device was not directly adhered to the organs, and (4) no inflammation was observed on the organ side.

[0362] The viable islet cells in the extracted device were stained as follows: (a) staining of islet cells with Dithizone, (b) staining of islet cells with Dithizone, (c) fluorescent staining of live cells with FDA, (d) fluorescent staining of dead cells with PI, and then observed under a microscope after staining unstained islet cells dispersed in alginate gel. As a result, it was confirmed that the islet cells were sufficiently viable in the device.

[0363] The device was removed 10 weeks after implantation, and the shape of the alginate gel in the device was confirmed. The results showed that the shape of the alginate gel in the implant device was maintained after it was placed in the body for a long period of time.

[0364] Example 8 Evaluation of the implantable device by intraperitoneal implantation into diabetic model mice 2

[0365] [Method for isolating porcine islets and culturing isolated islets] The pancreatic islets used to prepare the transplant device were obtained by the isolation and culture methods described in Example 7.

[0366] Preparation of implantable devices In the same manner as in Example 7, the solution of Example 5-1c and the solution of Example 5-2c are mixed in a 2:1 (volume ratio) and the mixture is diluted 4 times to give the solution of Example 8.

[0367] The implantable devices prepared using the solution of Example 8 (100 μL, 75 μL, 50 μL) were as follows. <Implantable Device> Example 8-1: Implantable device prepared using 100 μL of the solution of Example 8 Example 8-2: Implantable device prepared using 75 μL of the solution of Example 8 Example 8-3: Implantable device prepared using 50 μL of the solution of Example 8

[0368] In order to prepare 100 μL, 75 μL or 50 μL of 0.5 wt% alginic acid solution containing porcine islets, the islet pellets dispensed into one device were suspended in the alginic acid solution (B1) of Example 5-1c, and then mixed with the alginic acid solution (C1) of Example 5-2c to prepare the solution of Example 8 in which the porcine islets were suspended. The alginic acid solution (B1) of Example 5-1b and the alginic acid solution (C1) of Example 5-2b were adjusted in concentration with physiological saline according to the pellet amount (10 to 30 μL) of 10,000 IEQ of porcine islets per device.

[0369] The porcine islet-containing alginic acid solution prepared in Example 8 was quickly filled into a semipermeable membrane (Spectrum dialysis tube "Spectra / Pore CE (molecular weight cutoff: 100,000)") (one end of the semipermeable membrane was heat-sealed, the alginic acid solution was poured in, and the other end was heat-sealed to seal it) and 55 mmol / L CaCl 2 The device was immersed in the solution for 10 to 15 minutes to gel the alginate solution in the device.

[0370] After gelation, the transplantation device was washed with physiological saline for 3 minutes and cultured overnight in transplantation medium (M199-nicotinamide-FBS+P / S). Next, it was immersed in serum-free transplantation medium (M199+P / S) for 30 minutes, and then immersed in physiological saline+P / S for pre-transplant washing for 30 minutes to prepare a device for transplantation into a mouse. A photograph of the prepared transplantation device is shown in Figure 4-2.

[0371] - Dimensions of implant device: length 10 mm x width 26 mm x thickness approx. 0.25-0.8 mm Size of hydrogel in implantable device: Example 8-1: Length approx. 10 mm x width approx. 20 mm x thickness approx. 0.5 mm (500 μm) Example 8-2: Length: approx. 10 mm x width: approx. 20 mm x thickness: approx. 0.375 mm (375 μm) Example 8-3: Length: approx. 10 mm x width: approx. 20 mm x thickness: approx. 0.25 mm (250 μm) ·Culture conditions: M199+Nicotinamide+Fetal bovine serum+ Penicillin / Streptomycin (P / S), O / N Washing conditions: 1) Serum-free medium for transplantation (M199+P / S), 30 min, rt 2) Physiological saline (saline+P / S), 30 min, rt

[0372] [Evaluation of implantable devices (implantation testing)] The experiment was carried out in accordance with Example 7. The changes in blood glucose level up to day 178 after implantation when the implantation device of Example 8-1 was used are shown in FIG. 11-1, and the changes in body weight are shown in FIG. 12-1. FIG. 11-2 shows the blood glucose level change and FIG. 12-2 shows the body weight change up to day 178 after implantation when the implantation device of Example 8-2 was used. Furthermore, the implantation device was removed on day 178 after implantation, and FIG. 11-3 shows the blood glucose level change and FIG. 12-3 shows the body weight change up to day 40 after relay implantation. FIG. 11-4 shows blood glucose level fluctuations and FIG. 12-4 shows body weight fluctuations up to day 178 after implantation when the implantation device of Example 8-3 was used. Furthermore, the implantation device was removed on day 178 after implantation, and FIG. 11-5 shows blood glucose level fluctuations and FIG. 12-5 shows body weight fluctuations up to day 40 after relay implantation. In Figures 11-1 to 11-5 and Figures 12-1 to 12-5, the symbols indicated by # and numbers each represent a number for identifying an individual transplanted mouse. Body weight fluctuations were normal and blood glucose levels remained normal for 178 days, and for another 40 days after relay transplantation, body weight fluctuations were normal and blood glucose levels remained normal.

[0373] The excised device was subjected to GsIs to confirm that the device had insulin secretion ability.

[0374] Example 9 Evaluation of the implantable device by intraperitoneal implantation into diabetic model mice 3 According to the method of Example 7, the following devices were prepared. <Implantable Device> Example 9-1: An implantable device prepared using 200 μL of an aqueous solution of sodium alginate (B-2, manufactured by Mochida Pharmaceutical Co., Ltd.) adjusted to 1% by weight. Example 9-2: An implantable device prepared using 200 μL of a solution produced in the same manner as in Example 7-1 Example 9-3: An implantable device prepared using 200 μL of a solution produced in the same manner as in Example 7-2 Example 9-4: An implantable device prepared using 100 μL of a solution produced in the same manner as in Example 7-2 Example 9-5: An implantable device prepared using 200 μL of a solution produced in the same manner as in Example 7-3 Example 9-6: An implantable device prepared using 100 μL of a solution produced in the same manner as in Example 7-3 Example 9-7: An implantable device prepared using 50 μL of a solution produced in the same manner as in Example 7-3

[0375] - Dimensions of implanted device: length 10 mm x width 26 mm x thickness approx. 0.25~1.3 mm Size of hydrogel in implantable device: Example 9-1, Example 9-2, Example 9-3, and Example 9-5: Length: about 10 mm x width: about 20 mm x thickness: about 1 mm (1000 μm) Example 9-4, Example 9-6: Length about 10 mm × width about 20 mm × thickness about 0.5 mm (500 μm) Example 9-7: Length: approx. 10 mm x width: approx. 20 mm x thickness: approx. 0.25 mm (250 μm)

[0376] The transplantation test was carried out in accordance with Example 7. The devices of Examples 9-1 to 9-7 were each transplanted into a number of diabetic model mice. Mice whose blood glucose levels were 300 mg / dL or less for 178 days after transplantation (however, blood glucose levels exceeding 300 mg / dL up to three times during the period were allowed) were defined as cured mice, and the number of cured mice relative to the number of transplanted diabetic model mice was calculated as the cure rate, with the following results.

[0377] [Table 12]

[0378] It was confirmed that the healing rate of diabetic model mice was higher in the transplantation device containing the hydrogel produced from the alginic acid derivative of the present invention than in the transplantation device containing the hydrogel produced from natural alginic acid. In addition, it was confirmed that the thinner the thickness of the hydrogel, the higher the healing rate, and in this example, the healing rate was about 40% at a thickness of 500 μm or more, while the healing rate improved to about 62% at a thickness of 250 μm. Note that the healing rate of Example 9-2 is 0.0%, but this is due to the small number of subjects, and it is expected that the healing rate will be about the same as that described in Example 9-3 by increasing the number of subjects.

[0379] Example 10 Evaluation of the implantable device by intraperitoneal implantation into diabetic model mice 4

[0380] Preparation of implantable devices As pancreatic islets, MIN6 cells (2.5 × 10 6 A device was produced using the same method as in Example 7. The 2% alginic acid solution of A-2 was diluted 4 times to prepare the solution of Example 10-1. The solution of Example 5-1e and the solution of Example 5-2e were mixed at a 1:1 (volume ratio) to prepare a solution of Example 10-2. Moreover, the solution of Example 5-1e and the solution of Example 5-5b were mixed at 1:1 (volume ratio) to obtain a solution of Example 10-3. Moreover, a solution obtained by mixing the solution of Example 5-3c and the solution of Example 5-2e at a 1:1 (volume ratio) was used as a solution of Example 10-4. Moreover, a solution obtained by mixing the solution of Example 5-3c and the solution of Example 5-5b at a 1:1 (volume ratio) was used as a solution of Example 10-5. In all of Examples 10-2 to 10-5, the concentration of the chemical crosslinking group was 0.5%.

[0381] The implantation devices prepared using the solutions of Examples 10-1 to 10-5 (each 50 μL) are as follows. <Implantable Device> Example 10-1: Implantable device prepared using 50 μL of the solution of Example 10-1 Example 10-2: Implantable device prepared using 50 μL of the solution of Example 10-2 Example 10-3: Implantable device prepared using 50 μL of the solution of Example 10-3 Example 10-4: Implantable device prepared using 50 μL of the solution of Example 10-4 Example 10-5: Implantable device prepared using 50 μL of the solution of Example 10-5

[0382] - Dimensions of implanted device: length 10 mm x width 26 mm x thickness approx. 0.4 mm - Size of hydrogel in implant device: Length approx. 10mm x Width approx. 20mm x Thickness approx. 0.25mm (250μm)

[0383] [Evaluation of implantable devices (implantation testing)] The experiment was carried out in accordance with Example 7. The changes in blood glucose level up to day 17 after implantation when the implantation device of Example 10-1 was used are shown in FIG. 13-1, and the changes in body weight are shown in FIG. 14-1. The changes in blood glucose level up to day 17 after implantation when the implantation device of Example 10-2 was used are shown in FIG. 13-2, and the changes in body weight are shown in FIG. 14-2. The changes in blood glucose level up to day 17 after implantation when the implantation device of Example 10-3 was used are shown in FIG. 13-3, and the changes in body weight are shown in FIG. 14-3. The changes in blood glucose level up to day 17 after implantation when the implantation device of Example 10-4 was used are shown in FIG. 13-4, and the changes in body weight are shown in FIG. 14-4. The changes in blood glucose level up to day 17 after implantation when the implantation device of Example 10-5 was used are shown in FIG. 13-5, and the changes in body weight are shown in FIG. 14-5. In Figures 13-1 to 13-5 and Figures 14-1 to 14-5, the symbols indicated by # and numbers each represent the number for identifying an individual transplanted mouse. There were no abnormal changes in body weight, and blood glucose levels remained normal for 12 to 17 days.

[0384] Assessment of tissue reactivity: The tissue reactivity was evaluated in accordance with Example 7.

[0385] When the transplant devices of Examples 10-1 to 10-5 were used, the devices were removed two weeks after transplantation and the tissue reactivity was observed. As a result, (1) no angiogenesis was observed on the device surface, (2) the device was not adhered to organs, peritoneum, omentum, etc., (3) the organs could be bluntly peeled off and the device was not directly adhered to the organs, and (4) no inflammation was observed on the organ side.

[0386] The device was then opened two weeks after implantation to confirm the shape of the alginate gel in the device. The results showed that the shape of the alginate gel in the implant device was maintained after it had been placed in the body for a long period of time.

[0387] Example 11 Evaluating oxygen permeability of implantable devices

[0388] [Simulation of oxygen permeability] A simulation was performed under the following conditions for oxygen permeability for a hydrogel measuring 10 mm in length and 20 mm in width, with a thickness of 1 mm for Example 11-1, 0.5 mm for Example 11-2, and 0.25 mm for Example 11-3. In the simulation, it was assumed that the oxygen concentration on the hydrogel surface was uniform and always constant, that the specific respiration rate of the cells was always constant, that the cell volume was ignored and oxygen was consumed uniformly within the hydrogel, and that oxygen diffused evenly from the entire surface of the gel to the center.

[0389] First, the reaction-diffusion equation for the oxygen concentration when the hydrogel is placed in a Cartesian coordinate system in space is set as follows:

number

number

number

[0390] [Table 13]

[0391] FIG. 15-1 shows a hydrogel, and FIG. 15-2 to FIG. 15-4 show the oxygen permeability in the cross section of the part indicated by the black arrow. The legend is shown in FIG. 15-2, and the display contents are the same in FIG. 15-3 to FIG. 15-4. FIG. 15-2 shows a 1 mm thick hydrogel of Example 11-1, and the oxygen permeability of the surface is 100 to 75%, whereas the oxygen permeability of the center is 25% or less. In FIG. 15-3 showing Example 11-2, the oxygen permeability of the center is improved to 50 to 25% by making the thickness 0.5 mm, and in FIG. 15-4 showing Example 11-3, the oxygen permeability of the center is 75 to 50%, which is more than half of the oxygen concentration of the surface, by making the thickness 0.25 mm.

[0392] Example 12 Assessment of substance permeability through implantable devices Preparation of implantable device Using Example 1e with a crosslinking group introduction rate of 2.2 mol% and Example 2e with a crosslinking group introduction rate of 2.4 mol%, an aqueous solution of Example 12-1e with a crosslinking group introduction rate of 2.2 mol% and an aqueous solution of Example 12-2e with a crosslinking group introduction rate of 2.4 mol% were prepared, respectively. Note that physiological saline was used to prepare the aqueous solutions. A 2 wt% alginic acid solution can be prepared by mixing the solution of Example 12-1e and the solution of Example 12-2e at 1:1 (volume ratio). 25 μL of this solution mixed with physiological saline at 1:1 (volume ratio) was mixed with 25 μL of 0.01% Tween 20-containing physiological saline solution containing 500 ng of human insulin or 250 μg of glucose to make 50 μL. This solution was quickly sealed in a semipermeable membrane (Spectrum's dialysis tube "Spectra / Pore CE (molecular weight cutoff 100,000)") (one end of the semipermeable membrane was heat-sealed, then the alginic acid solution was poured in, and the other end was heat-sealed and sealed), and the device was immersed in a 55 mmol / LCaCl2 solution for 10 to 15 minutes to gel the alginic acid solution in the device. The final concentration of alginic acid was 0.5%. The implantable devices prepared with such solutions were hydrogels 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

[0393] [Test Method] The hydrogel of Example 12-1 or Example 12-2 was stirred in 40 mL of saline solution containing 0.01% Tween 20 at room temperature for 24 hours, and the insulin concentration or glucose concentration in the external solution was measured. The human insulin permeability or glucose permeability was calculated, taking the value when the human insulin or glucose encapsulated in the hydrogel was diffused into the saline solution as 100%.

[0394] Figure 16-1 shows the insulin concentration in the external solution and Figure 16-2 shows the glucose concentration up to 24 hours after the start of the test when the implantable device of Example 12 was used. Both human insulin and glucose were rapidly permeated, and it was confirmed that 100% had permeated after 24 hours of stirring, confirming that the implantable device of the present invention has excellent permeability to human insulin and glucose.

[0395] (Example 13) Evaluation of hydrogel disintegration Preparation of implantable devices First, the following physiological saline solutions of Examples 13-1 to 13-5 were prepared. [Table 14]

[0396] The implantation devices prepared using the solutions of Examples 13-1 to 13-5 are as follows. <Implantable Device> Example 13-1: After preparing 50 μL of the solution of Example 13-1, it was quickly sealed in a semipermeable membrane (Spectrum dialysis tube "Spectra / Pore CE (molecular weight cutoff 100,000)") (after heat sealing one end of the semipermeable membrane, an alginic acid solution was poured in, and the other side was also heat sealed to seal it), and 55 mmol / L CaCl 2 The device was immersed in the solution for 10 to 15 minutes to gel the alginate solution in the device. After that, the alginate gel was removed from the encapsulation membrane. Example 13-2: An implantable device was prepared using 200 μL of the solution of Example 13-2, and then prepared in the same manner as in Example 13-1. Example 13-3a: An implantable device prepared in the same manner as in Example 13-1 after preparation using 50 μL of the solution of Example 13-3. Example 13-3b: An implantable device prepared in the same manner as in Example 13-1 after preparation using 100 μL of the solution of Example 13-3. Example 13-3c: An implantable device prepared in the same manner as in Example 13-1 after preparation using 200 μL of the solution of Example 13-3. Example 13-4: An implantable device was prepared in the same manner as in Example 13-1 after preparation using 200 μL of the solution of Example 13-4. Example 13-5a: An implantable device prepared in the same manner as in Example 13-1 after preparation using 50 μL of the solution of Example 13-5. Example 13-5b: An implantable device prepared in the same manner as in Example 13-1 after preparation using 100 μL of the solution of Example 13-5. Example 13-5c: An implantable device prepared in the same manner as in Example 13-1 after preparation using 200 μL of the solution of Example 13-5.

[0397] [Test Method] Three implantation devices of any one of Examples 13-1 to 13-5c were added to 12 mL of physiological saline solution at 37 ° C. in a 15 mL conical tube, and the device was shaken at 37 ° C. with a medium-sized constant temperature shaking incubator (Taitec Co., Ltd., Bioshaker (registered trademark) BR-43FL·MR) using a reciprocating shaking method at an amplitude of 25 mm and a shaking speed of 180 rpm. The external solution was collected 1.5 hours, 6 hours, 24 hours, and 96 hours after the start of the test, and the alginic acid concentration was quantified by the carbazole sulfate method. After the external solution was collected 96 hours after the start of the test, the implantation device was collected and treated with lyase to dissolve the alginic acid gel, and the alginic acid concentration in the remaining gel was quantified. The alginic acid content in the device was calculated from the alginic acid concentration in the external solution and the alginic acid concentration in the remaining gel at the end of the test, and the disintegration rate was calculated as the content of alginic acid leaked from the device, calculated from the alginic acid concentration confirmed in the external solution after the start of each test, assuming this to be 100%.

[0398] The disintegration rate up to 96 hours after the start of the test when the implantable device of Example 13 was used is shown in Figure 17. The disintegration rates 24 hours and 96 hours after the start of the test are shown in Table 15.

[0399] [Table 15]

[0400] In the transplant devices using natural alginic acid in Examples 13-1 and 13-2, the alginic acid in the hydrogel quickly dissolved and the hydrogel collapsed, but in the transplant devices of the present invention in Examples 13-3 to 13-5, almost no alginic acid was dissolved and the hydrogel did not collapse, confirming that they are highly stable.

[0401] Example 14 Model study of cell detachment from alginate gels. Preparation of implantable device The implantable devices prepared were as follows: <Implantable Device> Example 14-1: 5 μL of a solution of polystyrene beads (106-125 μm, Polysciences, cat No. 19824) was added to 45 μL of the solution of Example 13-1 as a substitute for islet cells to prepare a solution, which was then quickly sealed in a semipermeable membrane (Spectrum dialysis tube "Spectra / Pore CE (molecular weight cutoff: 100,000)") (one end of the semipermeable membrane was heat sealed, and then an alginic acid solution was poured in, and the other end was also heat sealed to seal the membrane), and 55 mmol / L CaCl 2 The device was immersed in the solution for 10 to 15 minutes to gel the alginate solution in the device. After that, the alginate gel was removed from the encapsulation membrane. Example 14-2: A transplant device prepared in the same manner as in Example 14-1, using 45 μL of the solution of Example 13-3a and 5 μL of polystyrene beads instead of islet cells.

[0402] [Test Method] Three implantation devices of any of Examples 14-1 and 14-2 were added to 12 mL of physiological saline solution at 37° C. in a 15 mL conical tube, and the solution was shaken at 37° C. by a reciprocating shaking method using a medium-sized thermostatic shaking incubator (Taitec Corporation, Bioshaker (registered trademark) BR-43FL·MR) at an amplitude of 25 mm and a shaking speed of 180 rpm. The external solution was collected 24 hours after the start of the test, and the polystyrene beads that had fallen into the external solution were observed under a microscope.

[0403] Figure 18-1 shows a photograph of external fluid observed 24 hours after the start of the test when the implantable device of Example 14-1 was used, and Figure 18-2 shows a photograph of external fluid observed 24 hours after the start of the test when the implantable device of Example 14-2 was used. In the case of the transplant device using natural alginic acid in Example 14-1, many beads were observed in the external solution, and it was confirmed that most of the microbeads had fallen off 24 hours after the start of the test. On the other hand, in the case of the transplant device of the present invention in Example 14-2, almost no microbeads were found to fall off (the falling rate is thought to be 10% or less), and it was confirmed that the encapsulated cells do not fall off for a long period of time, and that the device has excellent stability.

[0404] From the above, it has become clear that the implantable device according to a preferred embodiment exhibits at least one of the following effects. (1) It has excellent biocompatibility and stability, low cytotoxicity, and almost no adhesion or inflammation at the transplant site. (2) The gel dissolves little and its shape is maintained for a long period of time. (3) The blood sugar lowering effect can be sustained for a long period of time, making it possible to regulate blood sugar. (4) Even after long-term use, the alginate gel in the semipermeable membrane does not dissolve and can maintain its shape, and the survival and function of the pancreatic islets can be maintained, making it suitable for long-term use. (5) It is replaceable, capable of immunoisolation, and causes little adhesion or inflammation, making it a highly safe medical material. (6) Excellent material permeability to the inside and outside of the device. (7) When the hydrogel was a thin device with a thickness of less than 500 μm, the healing rate of animals implanted with the device was higher than when the device was a thin device with a thickness of 500 μm or more. (8) When the hydrogel is a thin device with a thickness of less than 500 μm, the ratio of oxygen concentration at the center to the surface of the device is higher than when the hydrogel is 500 μm or thicker. (9) When the hydrogel is shaken, the hydrogel does not collapse or collapses little. (10) When the hydrogel is shaken, the islets contained in the hydrogel are less likely to fall off from the gel.

[0405] A more preferred embodiment of the transplantation device has excellent transplantation results and functionality, is made of a novel material, and can maintain a blood glucose lowering effect and regulate blood glucose for a long period of time by transplanting it into a diabetic patient (particularly type I diabetes and insulin-deficient type II diabetes). In addition, when the function of insulin-secreting cells or pancreatic islets in the hydrogel is reduced, they can be retrieved. Alternatively, periodic replacement or additional transplantation becomes possible. In addition, insulin-secreting cells differentiated from stem cells (such as iPS cells) or human pancreatic islets can be used as the insulin-secreting cells or pancreatic islets to be encapsulated in the hydrogel of the transplantation device. Therefore, the more preferred embodiment of the transplantation device is useful.

Claims

1. A transplant device comprising a hydrogel in which insulin-secreting cells or pancreatic islets are encapsulated, the hydrogel being gelled by chemical crosslinking formed by a combination of alginic acid derivatives described in (A) and (B) below, and the thickness of the hydrogel being 100 μm or more and less than 500 μm. (A): An amide bond and a divalent linker (-L １ A cyclic alkyne group (Akn) is introduced via a cyclic alkyne group (Akn) represented by the following formula (I): 【Chemistry 1】 [In formula (I), (ALG) represents alginic acid; -NHCO- represents an amide bond via any carboxyl group of alginic acid; -L １ - is the following partial structural formula (each formula does not include the areas outside the dashed lines at both ends): 【Chemistry 2】 A represents a divalent linker selected from the group consisting of the following partial structural formulas (each formula does not include the right side of the dashed line): 【Chemistry 3】 wherein the asterisk represents a chiral center; (B): An amide bond and a divalent linker (-L ２ -), an azide group is introduced via the following formula (II): 【Chemistry 4】 [In formula (II), (ALG) represents alginic acid; -NHCO- represents an amide bond via any carboxyl group of alginic acid; -L ２ - is the following partial structural formula (each formula does not include the areas outside the dashed lines at both ends): 【Chemistry 5】 The alginic acid derivative is represented by the following formula:

2. The chemically crosslinked alginic acid derivative is such that any carboxyl group of the first alginic acid and any carboxyl group of the second alginic acid are represented by the following formula (III-L): 【Chemistry 6】 [In formula (III-L), -CONH- and -NHCO- at both ends represent an amide bond via any carboxyl group of alginic acid; -L １ - is as defined in claim 1; -L ２ - is as defined in claim 1; X is the following partial structural formula: 【Chemistry 7】 The implantable device of claim 1, wherein the alginate is linked via a cyclic group selected from the group consisting of:

3. The following formula (III-L): 【Chemistry 8】 No-L ２ -XL １ - combination is represented by the following partial structural formula: 【Chemistry 9】 The implantable device of claim 2, wherein the group is selected from the group consisting of the following: (each formula does not include the areas outside the dashed lines at both ends):

4. A transplant device comprising a hydrogel in which insulin-secreting cells or pancreatic islets are encapsulated, the hydrogel being gelled by chemical crosslinking formed by a combination of alginic acid derivatives described in (A) and (B) below, and the thickness of the hydrogel being 100 μm or more and less than 500 μm. (A): An amide bond and a divalent linker (-L １ A cyclic alkyne group (Akn) is introduced via a cyclic alkyne group (Akn) represented by the following formula (I): 【Chemistry 10】 [In formula (I), (ALG) represents alginic acid; -NHCO- represents an amide bond via any carboxyl group of alginic acid; -L １ - Refer to the table below: 【Table 1-1】 【Table 1-2】 [In each formula, the areas outside the dashed lines at both ends are not included]; Akn is shown in the following table: 【Table 2】 [In each formula, the right side of the dashed line is not included], and a cyclic alkyne group selected from the group consisting of the partial structural formulas shown in (B): An amide bond and a divalent linker (-L) are bonded to any one or more carboxyl groups of alginic acid. ２ -), an azide group is introduced via the following formula (II): 【Chemistry 11】 [In formula (II), (ALG) represents alginic acid; -NHCO- represents an amide bond via any carboxyl group of alginic acid; -L ２ - Refer to the table below: 【Table 3-1】 【Table 3-2】 [In each formula, the outside of the broken lines at both ends is not included] (provided that in the alginic acid derivative represented by formula (I), -L １ - is any one linker selected from the group consisting of (L1-1), (L1-2a), (L1-2b), (L1-11) and (L1-12); and an alginic acid derivative represented by formula (II), in which -L ２ (Excluding chemical crosslinking by combination with a derivative in which - is the linker of (L2-10)).

5. The chemically crosslinked alginic acid derivative is such that any carboxyl group of the first alginic acid and any carboxyl group of the second alginic acid are represented by the following formula (III-L): 【Chemistry 12】 [In formula (III-L), -CONH- and -NHCO- at both ends represent an amide bond via any carboxyl group of alginic acid; -L １ - is as defined in claim 4; -L ２ - is as defined in claim 4; X is as shown in the following table: 【Table 4-1】 【Table 4-2】 (In each formula, the areas outside the dashed lines at both ends are not included.) (However, in the alginic acid derivative represented by formula (I), -L １ - is any one linker selected from the group consisting of (L1-1), (L1-2a), (L1-2b), (L1-11) and (L1-12); and an alginic acid derivative represented by formula (II), in which -L ２ The implantable device of claim 4, excluding cross-linked alginic acid in combination with a derivative in which - is the linker of (L2-10).

6. The alginic acid derivative of formula (I) has the following formula: 【Chemistry 13】 The implantable device of claim 1, wherein the -NHCO- bonded to (ALG) represents an amide bond via any carboxyl group of alginic acid.

7. The alginic acid derivative of formula (II) has the following formula: 【Chemistry 14】 The implantable device of claim 1, wherein the -NHCO- bonded to (ALG) represents an amide bond via any carboxyl group of alginic acid.

8. The alginic acid derivative of formula (I) has the following formula: 【Chemistry 15】 [wherein, (ALG) represents alginic acid; -NHCO- bonded to (ALG) represents an amide bond via any carboxyl group of alginic acid], The alginic acid derivative of formula (II) has the following formula: 【Chemistry 16】 The implantable device of claim 1, wherein the -NHCO- bonded to (ALG) represents an amide bond via any carboxyl group of alginic acid.

9. The alginic acid derivative of formula (I) has the following formula: 【Chemistry 17】 [In the formula, (ALG) represents alginic acid; -NHCO- bonded to (ALG) represents an amide bond via any carboxyl group of alginic acid], The alginic acid derivative of formula (II) has the following formula: 【Chemistry 18】 The implantable device according to claim 1, wherein (ALG) represents alginic acid; and -NHCO- bonded to (ALG) represents an amide bond via any carboxyl group of alginic acid.

10. The implantation device according to any one of claims 1 to 9, wherein the pancreatic islet is a human pancreatic islet or a porcine pancreatic islet.

11. The implant device of claim 10, wherein the pancreatic islets are adult porcine pancreatic islets.

12. The implantation device of claim 10, wherein the pancreatic islets are fetal, neonatal, or perinatal porcine islets.

13. The implantable device according to any one of claims 1 to 12, wherein the hydrogel is further covered with a semipermeable membrane.

14. 14. The implantable device of claim 13, wherein the semipermeable membrane is a dialysis membrane formed from a cellulose ester.

15. The implantable device of claim 14, wherein the cellulose ester is cellulose acetate.

16. The implantable device of claim 1 or 4, wherein the hydrogel further comprises ionic crosslinks formed in part by divalent metal ions.

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