WO2015020206A1

WO2015020206A1 - Tissue swelling material

Info

Publication number: WO2015020206A1
Application number: PCT/JP2014/071058
Authority: WO
Inventors: 奨舩山
Original assignee: 生化学工業株式会社
Priority date: 2013-08-08
Filing date: 2014-08-08
Publication date: 2015-02-12
Also published as: JP2016172783A

Abstract

　Provided is a crosslinked GAG, wherein the crosslinked GAG has the following crosslinking group bonded by an amide bond derived from a carboxyl group of the GAG molecule -CONH-R¹-X-R²-NHCO-, or -CONH-R³-X-R⁴-X'-R⁵-NHCO- [where the symbols in the formula are defined as indicated in the description].

Description

Tissue swelling material

The present invention relates to a cross-linked glycosaminoglycan (GAG), a raw material composition thereof, and a use thereof.

Glycosaminoglycan composed of amino sugar and uronic acid, that is, GAG is widely used as a medical material because of its excellent biocompatibility. GAG hydrogels obtained by crosslinking GAG are also widely used as medical materials. Among them, Japanese Patent Application Publication No. 2010-502824 discloses that hyaluronic acid in which a crosslinkable group is bonded to a carboxy group of D-glucuronic acid constituting hyaluronic acid is subjected to crosslinking by a click-type reaction. However, the click-type reaction in which an azide group and an alkyne group are reacted to form a 1,2,3-triazole ring requires a copper catalyst, and its use in biological systems is greatly limited due to its toxicity. Therefore, a SPAAC that does not require a copper catalyst and uses a crosslinkable group such as cycloalkynylene that reacts quickly and highly selectively as a reactive group due to the strain of the ring structure, and the crosslink reaction proceeds rapidly due to the strain. It is described in WO2012 / 165462 that a hydrogel of a polysaccharide such as hyaluronic acid is obtained by a (strain-promoted azide-alkyne cycloaddition) type reaction. However, since the hydrogel of WO2012 / 165462 has a crosslinkable group bonded to the hydroxy group of N-acetyl-glucosamine constituting hyaluronic acid via an alkylene chain, the remaining period after gel formation in vivo Was as short as one week, and its use was limited.

On the other hand, vesicoureteral reflux (VUR) is a condition in which urine flowing from the kidney to the ureter and then to the bladder returns from the bladder to the ureter and the kidney when urinating, and the joint between the ureter and the bladder It is thought to arise because the “valve” function is inherently insufficient. The treatment includes antibacterial drugs; laparotomy such as extravesical reconstruction; a high-viscosity gel in which dextranomer beads are mixed with sodium hyaluronate, a carrier gel, at the connection between the bladder and ureter Endoscopic treatment that produces a long-term bulging effect is performed by injecting and surrounding the staying site with connective tissue (Deflux package insert).

Stress urinary incontinence (SUI) refers to a state in which the increase in bladder pressure during abdominal pressure exceeds the urethral resistance due to a decrease in urethral resistance, and urine leaks without bladder contraction. For example, a characteristic symptom is that urine leaks without intention of urine when coughing, sneezing, having a heavy object, running, or climbing stairs. In diagnosis in general practice, symptom evaluation and physical examination are important, but subjective symptoms and pathological conditions may not always match, and various objective tests are required in specialized practice. However, while the importance of QOL evaluation is being pointed out worldwide in severity assessment, treatment selection, and treatment effect assessment, there is also a situation in which a global severity classification has not yet been established (in EBM) Based on urinary incontinence clinical practice guidelines). The treatment of stress urinary incontinence (SUI) can be divided into conservative treatment such as lower urinary tract rehabilitation, drug treatment, vaginal interior fitting, and surgical treatment. Generally, lower urinary tract rehabilitation is indicated for mild to moderate disease, and surgical treatment is indicated for moderate to severe disease. Drug therapy is positioned as an adjunct therapy because its effectiveness is still insufficient. A typical example of lower urinary tract rehabilitation from mild to moderate is pelvic floor muscle training, and in cases of stress urinary incontinence, disappearance of about 30% to 40% has been reported in mild cases. However, it has not been popularized because it requires a certain amount of know-how for guidance and there are few comedic staff who can provide specialized guidance.

Surgical treatment of stress urinary incontinence can be broadly divided into bladder neck lifting such as retropubic surgery, bladder neck sling surgery, and periurethral collagen injection therapy. As for surgical treatment, comparison of treatment results of various surgical procedures has a large variation in the definition of urinary incontinence improvement, few papers have examined long-term results, surgical procedures are not constant, and there are many changes by the surgeon, no It is not easy because there are very few randomized comparative studies. From a study of long-term results by meta-analysis conducted in the guidelines on surgical treatment of female stress urinary incontinence prepared by the American Urological Association in 1997, and from a randomized comparative study, the long-term results of 4 years or more show a retropubic bladder Neck elevation 84%, transvaginal bladder neck elevation 67%, anterior vaginal wall plasticization 61%, sling surgery 83%, retropubic bladder neck elevation and sling surgery, etc. (Female Stress Urinary Incontinence Clinical Guidelines Panel Summary Report on Surgical Management of Female Stress Urinary Incontinence, The Journal of Urology, Volume 158, Issuee 3,880, . However, no surgical treatment that is less invasive and simpler than periurethral collagen injection has been found.

In this periurethral collagen injection therapy, collagen is injected into the bladder neck and proximal urethral mucosa to ensure close contact between the bladder neck and proximal urethra. Injection by needle puncture is performed. Although it is the least invasive and simple method among surgical treatments, the recurrence rate is high, and it is said that more than two injections are often required to obtain a stable effect (urinary incontinence based on EBM) Clinical guidelines). In fact, there were products (such as Coaptite (registered trademark) Injectable Implant) used for periurethral injection therapy using collagen as a material, but the current situation is withdrawing from the market from the viewpoint of long-term therapeutic effects. . Furthermore, in order to obtain this long-term therapeutic effect, products using various materials have been developed and marketed. For example, a product using calcium hydroxyapatite as a material was found, but it had the same effect as that of collagen and tended to disappear with time. Products made of carbon beads did not absorb earlier than collagen, but there is a concern that injection is more difficult than collagen because the needle gauge used for injection is thicker. In addition to this inconvenience, it is known that the suspension gel tends to be injected first, leaving the carbon bead particles. Silicone has a long history as a tissue swelling material in the cosmetic surgery field, but it has been relatively newly approved in the United States as a treatment material for stress urinary incontinence, and has not shown long-term effects. Polytetrafluoroethylene (PTFE) is a plastic material that forms granulation seeds and has not been approved in the United States due to the risk of tissue migration. Ethylene vinyl alcohol has been confirmed to have serious complications including urethral erosion. It is no longer recommended (Injectable Bulking Agents for Incontinence, Author: Raymond Rackley, Chief Editor: Edward David Kim, May 2, 2011).
As described above, exemplified in the clinical use of urology, but as a tissue swelling material, it has the properties of a material that is easy to use, can be injected as one of the minimally invasive methods, and is highly safe, and A substance having long-term durability and shape maintenance has not yet been created.

Special table 2010-502824 WO2012 / 165462

The present invention provides a long-term residual tissue swelling material that is highly safe in the living body and remains in the body for a long time, and is useful for diseases requiring treatment with a long-term residual tissue swelling material such as vesicoureteral reflux disease. Is an issue.

As a result of diligent research to solve the above problems, the present inventor has attached a SPAG reaction to a GAG derivative in which a SPAAC type reactive group is introduced into the carboxy group of GAG represented by hyaluronic acid via an amide bond. It is found that the cross-linked GAG obtained by the above method has excellent safety in the living body and remains stable in the living body for a long period of time, and further requires treatment with a long-term residual tissue swelling material such as vesicoureteral reflux disease. As a result, the present invention has been found to be useful as a material for treating diseases.

In the present invention, the first GAG molecule and the second GAG molecule are represented by the following crosslinking group:
—CONH—R ¹ —XR ² —NHCO—, or —CONH—R ³ —XR ⁴ —X′—R ⁵ —NHCO—
[Where:
-CONH and NHCO- at both ends mean an amide bond derived from the carboxy group of the GAG molecule,
R ¹ to R ⁵ are the same or different and each represents an alkylene group, an alkenylene group, or an alkynylene group, and —CH ₂ — in the group represents> C═O, —CONH—, arylene, —O—, or May be replaced by -S-;
X and X ′ are the same or different and have the formula:

The direction of A and B bonds may be either
Here, A and B each represent a binding site with any of R ¹ to R ⁵ to which X or X ′ is bound;
Y ¹ ~ ^{Y 6} are the same or ^{^{^{different, -CR 6 R 6 '-,}}} - C (-R 6) =, - NR 7 -, = N -, - O-, or -S- represents, - ^{NR 7 -, = N -,} - O-, and -S- are never adjacent;
R ⁶ and R ^{6 ′} are the same or different and are a hydrogen atom, a halogen atom, a hydroxy group, an amino group, an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group or a carboxy group, which may be mono- or di-substituted with alkyl. Or together formed an oxo group, and —CH ₂ — in the alkyl group, alkenyl group, alkynyl group or alkoxy group represents> C═O, —CONH—, arylene, —O—. Or may be replaced by -S-;
R ⁷ represents an alkyl group, an alkenyl group or an alkynyl group, and —CH ₂ — in the alkyl group, alkenyl group or alkynyl group represents> C═O, —CONH—, arylene, —O— or —S—. May be replaced by; or
In two neighboring groups of Y 1 ^- Y ^6, R ⁶ in R ⁶ and the other group of one group is a saturated or 3-unsaturated together with the ring atoms to which each is attached A 6-membered ring can be formed, and the bond B can be bonded to the 3- to 6-membered ring]
The first GAG molecule and the second GAG molecule may be the same molecule bound via a GAG.

The present invention further includes a composition comprising the following (1) GAG derivative A and (2) GAG derivative B or (3) compound C:
(1) GAG derivative A in which a SPAAC type reactive group is introduced into a GAG molecule via an amide bond derived from a carboxy group of the GAG molecule and a divalent spacer group;
(2) GAG derivative B in which a reactive group complementary to the reactive group of GAG derivative A is introduced into the GAG molecule via an amide bond derived from a carboxy group of the GAG molecule and a divalent spacer group;
(3) Compound C defined by the following structure having at least two reactive groups complementary to the reactive group of GAG derivative A;

[Where:
Y is the same or different and is a reactive group complementary to the reactive group of GAG derivative A;
Z is an n-valent spacer group,
n is an integer of 2 or more]
About.

The present invention also relates to a tissue swelling material kit comprising the tissue swelling material comprising the composition; the GAG derivative A and the GAG derivative B or the compound C.
The present invention is also the GAG derivative A or the GAG derivative B,

A GAG derivative in which an amino group of an amine selected from the group consisting of an amino group of an amine and a carboxy group of a GAG molecule is amide-bonded; and an intermediate compound for obtaining the GAG derivative.
following:

And a cyclooctyne derivative amine selected from the group consisting of:

The GAG cross-linked product of the present invention obtained by subjecting the composition of the present invention to a SPAAC type reaction is useful as a material for treating a disease requiring treatment with a long-term residual tissue swelling material such as vesicoureteral reflux. .

FIG. 6 is a graph showing a change with time in viscosity value after mixing according to Example 21. It is a figure which shows the time-dependent change of the viscosity value after mixing by Example 22. It is a figure which shows the relationship between the distortion | strain of gel solid, and stress by Example 23. It is a figure which shows the result of the shape maintenance performance test of Example 24. It is a figure which shows the result of the shape maintenance performance test of Example 25. _{2.2% HA (N 3 + DIMAC} ) is a diagram showing the histology after injection 4 weeks. _{2.2% HA (N 3 + DIMAC} ) is a diagram showing the histology after injection 24 weeks.

In the present specification, an alkyl group means a monovalent linear or branched saturated aliphatic hydrocarbon group having 1 to 14 carbon atoms, preferably 1 to 8 carbon atoms. A divalent linear or branched saturated aliphatic hydrocarbon group having 1 to 14 carbon atoms, preferably 1 to 8 carbon atoms.

An alkenyl group is a monovalent linear or branched unsaturated aliphatic hydrocarbon group having 2 to 14 carbon atoms, preferably 2 to 8 carbon atoms, having at least one double bond. The alkenylene group means a divalent linear or branched unsaturated aliphatic hydrocarbon having 2 to 14 carbon atoms, preferably 2 to 8 carbon atoms, having at least one double bond. Refers to the group.

The alkynyl group refers to a monovalent linear or branched unsaturated aliphatic hydrocarbon group having 2 to 14 carbon atoms, preferably 2 to 8 carbon atoms, having at least one triple bond. The alkynylene group is a divalent linear or branched unsaturated aliphatic hydrocarbon group having 2 to 14 carbon atoms, preferably 2 to 8 carbon atoms, having at least one triple bond. Say.

An alkoxy group refers to a monovalent group formed by an oxygen atom to which the above alkyl group is bonded.

An arylene group refers to a divalent monocyclic or polycyclic aromatic hydrocarbon group having 6 to 20 carbon atoms as ring-constituting atoms. Specific examples include phenylene (1,2-, 1,3- or 1,4-phenylene), naphthylene, and the like.

As used herein, the term “complementary reactive group” refers to a functional group that can react with a reactive functional group to form a chemical bond such as a covalent bond. It refers to any one functional group in a triple bond combination.

In the present specification, the amount of each component in the composition is the total amount of the plurality of substances present in the composition unless there is a specific indication when there are a plurality of substances corresponding to each component in the composition. means.

Hereinafter, the present invention will be described in detail.

GAG cross-linked body As described above, in the present invention, the first GAG molecule and the second GAG molecule have the following cross-linking group:
—CONH—R ¹ —XR ² —NHCO—, or —CONH—R ³ —XR ⁴ —X′—R ⁵ —NHCO—
The GAG cross-linked product is linked via the formula, and each symbol in the formula is also as described above, and the first GAG molecule and the second GAG molecule may be the same molecule.

GAG
In the present invention, GAG, that is, glycosaminoglycan has a repeating structural unit of an amino sugar (glucosamine, galactosamine) and a disaccharide consisting of uronic acid or galactose, and its hydroxy group may be sulfated. It is an acidic polysaccharide of 5 to 2,000 kDa. Examples of such GAGs include hyaluronic acid, chondroitin, chondroitin sulfate, heparin, heparan sulfate, dermatan sulfate and keratan sulfate. In the present invention, among these, at least one selected from the group consisting of hyaluronic acid, chondroitin sulfate, and chondroitin is preferable; at least one selected from the group consisting of hyaluronic acid and chondroitin sulfate is more preferable; hyaluronic acid is particularly preferable preferable. These GAGs may be pharmaceutically acceptable salts thereof. Examples of such salts include sodium salts, potassium salts, magnesium salts and calcium salts, among which sodium salts are preferred.

The origin of GAG is not particularly limited, and may be derived from animals or microorganisms, or may be chemically synthesized. For example, when sodium hyaluronate is used, one derived from chicken crown can be exemplified, and when chondroitin sulfate is used, one derived from shark cartilage can be exemplified. The molecular weight of GAG is not particularly limited, but the weight average molecular weight is preferably 5 to 2,000 kDa, more preferably 10 to 500 kDa. When hyaluronic acid or a pharmaceutically acceptable salt thereof is used as GAG, its weight average molecular weight is preferably 5 to 2,000 kDa, more preferably 10 to 500 kDa. When chondroitin sulfate or a pharmaceutically acceptable salt thereof is used as GAG, its weight average molecular weight is 5 to 200 kDa, more preferably 10 to 100 kDa.

One embodiment of the GAG molecule of the present invention has the formula:

[Where:
R ⁸ and R ⁹ represent a hydrogen atom or a hydroxy group; provided that when R ⁸ is a hydroxy group, R ⁹ is a hydrogen atom, and when R ⁸ is a hydrogen atom, R ⁹ is a hydroxy group;
R ¹⁰ is ONa or OH],
In the case where R ⁸ is a hydroxy group, at least one of the hydroxy groups in the structural unit is —OSO ₃ Na or —OSO ₃ H. GAG can be preferably mentioned.

Cross-linking The GAG cross-linked product of the present invention comprises a first GAG molecule and a second GAG molecule having the following cross-linking group:
—CONH—R ¹ —XR ² —NHCO—, or —CONH—R ³ —XR ⁴ —X′—R ⁵ —NHCO—
The first GAG molecule and the second GAG molecule may be the same molecule.

Here, R ¹ to R ⁵ are the same or different and each represents an alkylene group, an alkenylene group, or an alkynylene group, and —CH ₂ — in the group represents> C═O, —CONH—, arylene, —O -Or -S- may be substituted. Preferred alkylene, alkenylene or alkynylene groups are each a divalent linear or branched aliphatic hydrocarbon as defined above, each consisting of 1 to 14, more preferably 1 to 8 carbon atoms. It is a group.

X and X ′ may be the same or different and have the formula:

The direction of A and B bonds may be either
Here, A and B each represent a binding site with any of R ¹ to R ⁵ to which X or X ′ is bound;
Y ¹ ~ ^{Y 6} are the same or ^{^{^{different, -CR 6 R 6 '-,}}} - C (-R 6) =, - NR 7 -, = N -, - O-, or -S- represents, - ^{NR 7 -, = N -,} - O-, and -S- are never adjacent;
R ⁶ and R ^{6 ′} are the same or different and are a hydrogen atom, a halogen atom, a hydroxy group, an amino group, an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group or a carboxy group, which may be mono- or di-substituted with alkyl. Or together formed an oxo group, and —CH ₂ — in the alkyl group, alkenyl group, alkynyl group or alkoxy group represents> C═O, —CONH—, arylene, —O—. Or may be replaced by -S-;
R ⁷ represents an alkyl group, an alkenyl group or an alkynyl group, and —CH ₂ — in the alkyl group, alkenyl group or alkynyl group represents> C═O, —CONH—, arylene, —O— or —S—. May be replaced by; or
In two neighboring groups of Y 1 ^- Y ^6, R ⁶ in R ⁶ and the other group of one group is a saturated or 3-unsaturated together with the ring atoms to which each is attached A 6-membered ring can be formed, and the bond B can be bonded to the 3- to 6-membered ring. Examples of the 3- to 6-membered ring include a cycloalkyl ring having 3 to 6 members, a phenyl ring, or a 5- to 6-membered heteroaryl ring.

Preferred examples of X and X ′ include the following groups.

More preferable examples of X and X ′ include the following groups.

More preferable examples of the crosslinking group in the GAG crosslinked product of the present invention include the following, wherein m is an integer of 3 to 8 (the spacer actually used in the examples).
—CONH—CH ₂ —CH ₂ —CO—X—CH ₂ —CH ₂ —O—CH ₂ —CH ₂ —NHCO—;
—CONH—CH ₂ —CO—X—CH ₂ —CH ₂ —NHCO—;
—CONH—CH ₂ —CO—X—CH ₂ —CH ₂ —O—CH ₂ —CH ₂ —NHCO—;
—CONH—CH ₂ —CO—X—CH ₂ — (CH ₂ —O—CH ₂ ) _m —CH ₂ —NHCO—;
—CONH—CH ₂ —X—CH ₂ —CH ₂ —NHCO—;
—CONH—CH ₂ —O—X—CH ₂ —CH ₂ —NHCO—

In the above bridging group, X is preferably selected from the group consisting of the following groups.

More preferably, it is the following group:

More preferably, they are the following groups.

In the various preferred embodiments of X and X ′ described above, one of the nitrogen atoms at both ends of the three nitrogen atoms of the triazolyl ring moiety can have a bond.

Introduction rate In the GAG crosslinked product of the present invention, it is not necessary that all carboxy groups of the GAG structural unit have the above-mentioned crosslinking groups. The ratio of the number of moles of linked bridging groups to the number of moles of GAG disaccharide repeating units (hereinafter referred to as “introduction ratio”) is the type of GAG, the viscosity required for use as a tissue swelling material, It can be determined appropriately according to the necessary stress, administration site, and the like. For example, when hyaluronic acid or chondroitin sulfate is used as GAG, the introduction ratio is preferably 0.1 to 80%, and more preferably 0.5 to 20%, relative to the number of moles of GAG disaccharide repeating units. It is.

Composition The present invention also relates to a composition comprising at least one of the following (1) GAG derivative A and (2) at least one of GAG derivative B or (3) compound C.
(1) GAG derivative A in which a SPAAC type reactive group is introduced into a GAG molecule via an amide bond derived from a carboxy group of GAG and a divalent spacer group;
(2) GAG derivative B in which a reactive group complementary to the reactive group of GAG derivative A is introduced into the GAG molecule via an amide bond derived from a carboxy group of the GAG molecule and a divalent spacer group;
(3) Compound C defined by the following structure having at least two reactive groups complementary to the reactive group of GAG derivative A;

[Where:
Y is the same or different and is a reactive group complementary to the reactive group of GAG derivative A;
Z is an n-valent spacer group,
n is an integer of 2 or more].
That is, the composition of the present invention includes (1) a combination of GAG derivative A and (2) GAG derivative B; or (1) a combination of GAG derivative A and (3) compound C.

Type of GAG In the GAG derivatives A and B constituting the composition of the present invention, the same GAG as the GAG described for the above-mentioned GAG cross-linked product can be used as GAG, and consists of hyaluronic acid, chondroitin sulfate and chondroitin. GAG selected from the group can be preferably used, hyaluronic acid or a salt thereof and chondroitin sulfate or a salt thereof can be more preferably used, and hyaluronic acid having higher biocompatibility can be particularly preferably used.

In the divalent GAG derivatives A and B constituting the composition of the spacer group, and n valent spacer groups present invention, an amide bond and SPAAC type reactive group the carboxy group of the GAG form, a divalent spacer group Are connected through. As the divalent spacer group, any chain group can be used as long as it does not inhibit the reaction between the SPAAC type reactive group and the reactive group complementary to the reactive group. As such a divalent spacer group, groups R ¹ to R ⁵ used in the aforementioned GAG crosslinked product, that is, an alkylene group, an alkenylene group, or an alkynylene group can be used, and —CH _{2 in the} group can be used. — May be replaced by> C═O, —CONH—, arylene, —O—, or —S—.

The n-valent spacer group of the compound C constituting the composition of the present invention is the same as the divalent spacer group, and an n-valent group derived from an alkyl group, an alkenyl group, or an alkynyl group can be used. The —CH ₂ — in the group may be replaced by> C═O, —CONH—, arylene, —O—, or —S—. Here, as the arylene, a phenylene group such as 1,2-, 1,3- or 1,4-phenylene can be used, and among them, a 1,4-phenylene group can be preferably used. Here, n is an integer of 2 or more, preferably 2.

In the compositions of SPAAC type reactive groups present invention, GAG derivatives A and B, Compound C has a SPAAC type reactive group, or complementary reactive group and the reactive moiety. The SPAAC-type reaction is a click-type reaction that reacts a triple bond of an azide group and an alkyne to form an 1,2,3-triazole ring quickly and easily without producing an undesirable by-product. The reaction using a compound having a cycloalkynyl group, which does not require a copper catalyst and does not require a copper catalyst, and allows the crosslinking reaction to proceed rapidly and highly selectively due to the distortion of the ring structure. The GAG derivatives A and B and the compound C that can be employed in the composition of the present invention can have any SPAAC type reactive group or a reactive group complementary to the reactive group. Specifically, A combination of a group derived from a cycloalkynyl group having 7 to 9 carbon atoms, preferably 7 or 8 carbon atoms, more preferably 8 carbon atoms, and an azide group can be used. The cycloalkynyl group has the formula:

[Where:
Here, B represents a binding site with a divalent or n-valent spacer group;
Y ¹ ~ ^{Y 6} are the same or ^{^{^{different, -CR 6 R 6 '-,}}} - C (-R 6) =, - NR 7 -, = N -, - O-, or -S- represents, - ^{NR 7 -, = N -,} - O-, and -S- are never adjacent;
R ⁶ and R ^{6 ′} are the same or different and are a hydrogen atom, a halogen atom, a hydroxy group, an amino group, an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group or a carboxy group, which may be mono- or di-substituted with alkyl. Or together formed an oxo group, and —CH ₂ — in the alkyl group, alkenyl group, alkynyl group or alkoxy group represents> C═O, —CONH—, arylene, —O—. Or may be replaced by -S-;
R ⁷ represents an alkyl group, an alkenyl group, or an alkynyl group, and —CH ₂ — in the alkyl group, alkenyl group, or alkynyl group represents> C═O, —CONH—, arylene, —O—, or —S May be replaced by; or
In two neighboring groups of Y 1 ^- Y ^6, R ⁶ in R ⁶ and the other group of one group is a saturated or 3-unsaturated together with the ring atoms to which each is attached A 6-membered ring can be formed, and the bond B can be bonded to the 3- to 6-membered ring]
The cyclic group shown by these can be mentioned. Examples of the 3- to 6-membered ring include a cycloalkyl ring having 3 to 6 members, a phenyl ring, or a 5- to 6-membered heteroaryl ring.

Specific examples of the SPAAC type reactive group or a reactive group complementary to the reactive group preferably include a combination of a reactive group having a skeleton selected from the group consisting of the following skeletons and an azide group. Can do.

Even more preferably, a combination of a group selected from the group consisting of the following groups and an azide group can be mentioned more preferably.

Rate of introduction of reactive group into GAG In the GAG derivatives A and B constituting the composition of the present invention, the SPAAC type reactive group and the reactive group complementary to the reactive group are all of the GAG constituent units. It is not necessary to bind to the carboxy group. The ratio of the number of moles of linked crosslinks relative to the number of moles of GAG disaccharide repeating units, that is, the introduction ratio can be appropriately determined according to the type of GAG, the necessary viscosity, the necessary stress, the administration site, and the like. For example, when hyaluronic acid or chondroitin sulfate is used as GAG, the introduction ratio is preferably 0.1 to 80%, and more preferably 0.5 to 20%, relative to the number of moles of GAG disaccharide repeating units. It is.

GAG derivatives A and B and compound C
Among the GAG derivatives A and B, the GAG derivative having a cycloalkynyl group as a SPAAC type reactive group is preferably the following:
following:

A GAG derivative in which an amino group of an amine and a carboxy group of GAG are amide-bonded is selected from the group consisting of:
Among these, in terms of reactivity and biocompatibility, the following:

More preferred cyclooctyne derivatives GAG derivatives in which the amino group of the amine and the carboxy group of GAG are amide-bonded are more preferred, most preferably:

A GAG derivative in which the amino group of the amine and the carboxy group of GAG are amide-bonded.

Of the GAG derivatives A and B, the GAG derivative having an azide group as a SPAAC type reactive group is preferably the following:
N ₃ —CH ₂ —CH ₂ —NH ₂ ;
N ₃ —CH ₂ —CH ₂ —CH ₂ —NH ₂ ;
N ₃ —CH ₂ —CH ₂ —CH ₂ —CH ₂ —NH ₂ ;
N ₃ —CH ₂ —C (═O) —NH—CH ₂ —CH ₂ —NH ₂ ;
N ₃ —CH ₂ —CH ₂ —O—CH ₂ —CH ₂ —NH ₂ ;
N ₃ —CH ₂ — [CH ₂ —O—CH ₂ ] _2-10 —CH ₂ —NH ₂ ;
A GAG derivative obtained by amide-bonding an amino group of an azidoamine selected from the group consisting of carboxy group of GAG can be given.

As compound C, preferably, the following compounds can be mentioned.

Composition ratio of GAG derivatives A and B and compound C In the composition of the present invention, the SPAAC type reactive group possessed by GAG derivatives A, B and compound C, and the reactive group complementary to the reactive group are 1 The molar ratio is from 1: 1 to 1: 4, preferably from 1: 1 to 1: 2, more preferably 1: 1.
The composition of the present invention may contain a medium. Examples of the medium include water, physiological saline, and phosphate buffer. When the composition includes a medium, the total content of GAG derivatives A and B and compound C is, for example, 0.5% by mass or more, and preferably 1.0% by mass or more in the composition.

Method for Producing GAG Derivatives A and B and Compound C GAG derivatives A and B contained in the composition of the present invention are SPAAC type reactive groups, reactive groups complementary to the reactive groups, and amino groups as spacers. The amino group of the spacer compound bonded via the group can be obtained by condensing with the carboxy group of the GAG molecule.

For example, a spacer compound in which a cycloalkynyl group and an amino group, which are typical SPAAC-type reactive groups, are bonded via a spacer group can be synthesized as shown below.

Methyl-6-bromo-6-deoxy-2,3-di-O-methyl-α, D-glucopyranoside 3 was synthesized by the method described in Organic Letter, 2008, Vol. 10, No. 14, 3097-99. To do. After the zinc reduction / reductive amination reaction using 3 as a raw material, the imino group is protected with a t-butoxycarbonyl (Boc) group to give 4. Subsequently, after azacyclooctene 5 is obtained by olefin metathesis reaction, ketone body 6 is obtained by oxidation using pyridinium dichromate (PDC), and 7 is obtained by hydrogenation of 6 under a palladium carbon catalyst. After adding semicarbazide hydrochloride to 7, treatment with selenium dioxide gives selenodiazole 8. Subsequently, the Boc group of 8 is removed under acidic conditions to convert to imine hydrochloride 9 and condensed with 9-fluorenylmethyloxycarbonyl (Fmoc) glycine to be 10. Finally, it is converted to azacyclooctyne while removing the Fmoc group by treatment with triphenylphosphine under heating under reflux, and DIMAC-amine hydrochloride 11 is synthesized by treatment with hydrochloric acid.

Other spacer compounds in which a SPAAC type reactive group cycloalkynyl group and amino group are bonded via a spacer group are also described in Org. Biomol. Chem., 2009, 7, 635-638, Bioconjugate Chem. 2010, 21, It can be easily synthesized by the method described in 2076-2085 and the like, and a method analogous thereto.

Further, a spacer compound in which an azide group and an amino group, which are SPAAC type reactive groups, are bonded via a spacer group is commercially available or can be easily synthesized from a commercially available compound. For example, the above-mentioned azidoamines are known as Chem. Commun., 2005, 5390-5392; Chem.Commun., 2006, 2012-2014; J. Org. Chem. 2006,71 (17), 6697-6700; Tetrahderon Lett., 2001, 2709-2711; J. Am. Chem. Soc., 2005, 127, 12434-12435; Tetrahderon Lett., 2001, 2709-2711 or a method analogous thereto .

Compound C is also commercially available or can be easily synthesized from commercially available compounds.

The thus obtained spacer compound to which a reactive group is bonded and GAG are mixed with 4- (4,6-dimethoxy-1,3,5-triazin-2-yl) -4-yl in an ethanol / water mixed solution at room temperature. GAG derivatives A and B can be obtained by subjecting to a condensation reaction using a condensing agent such as methylmorpholinium chloride (DMT-MM).

Further, GAG derivatives A and B or GAG derivative A and compound C thus obtained are mixed in water, in a mixed solution of an organic solvent that can be arbitrarily mixed with water, and in a solution containing an arbitrary salt. The GAG crosslinked product of the present invention can also be obtained by subjecting it to a SPAAC type reaction at a temperature of 60 ° C.

Use of the Composition of the Present Invention The composition of the present invention can form the crosslinked GAG of the present invention by subjecting it to a SPAAC type reaction in a solvent such as physiological saline or phosphate buffer as necessary. it can. Since the GAG crosslinked body has excellent safety in vivo and can remain stably in the living body for a long time, it is used as a therapeutic material for various diseases requiring treatment with a long-term residual tissue swelling material. be able to.
Specifically describing such an application, the composition of the present invention can remain in a living body for a long time after injection, for example, so that functional reconstruction of a living tissue lost or removed for some reason is possible. It can be used in a variety of applications, including intended surgical or orthopedic applications, plastic surgery applications that aesthetically repair lost tissue. More specifically, examples include gastroesophageal reflux disease (GERD) and fecal incontinence (FI). As for another more specific use, radical prostatectomy is an operation in which a skin incision of about 7 to 20 cm is made in the lower abdomen, the prostate and seminal vesicle are removed, and the bladder and urethra are anastomosed. After the operation, a catheter is inserted into the bladder, and the catheter is removed after a urethral examination in about one week. In this case, it is known that postoperative complications are accompanied by dysuria in about 70% of patients. If this is a dysuria due to the absence of a tissue gap where the prostate was present, the composition of the present invention can be used as a medium to support the urethra. Furthermore, from the situation where total prostatectomy is possible in laparoscopic surgery, it can also be used for tissue reconstruction to prevent postoperative complications after extraction surgery in minimally invasive surgery.
Thus, the composition of the present invention can be used as a long-term residual tissue swelling material in various regions. Among them, the composition of the present invention is used to treat a disease requiring treatment with a tissue swelling material such as vesicoureteral reflux, stress urinary incontinence, or fecal incontinence in the urological field; tissue after radical prostatectomy It can be used as a long-term residual tissue bulge in reconstruction; tissue reconstruction in the surgical, orthopedic or plastic surgery area.

In use, GAG derivative A and GAG derivative B or compound C, which are constituents of the composition of the present invention, can be administered in vivo as, for example, as a powder or as a solution in a solvent. Preferably, it can be dissolved and mixed in a solvent such as physiological saline or phosphate buffer as necessary to obtain a liquid composition, which can be administered in vivo by means such as injection. The concentration in the case of dissolving in a solvent can be appropriately determined according to the viscosity required for use as a long-term residual tissue swelling material. That is, by mixing GAG derivative A and GAG derivative B or compound C, the formation of a GAG cross-linked product is started by the SPAAC reaction, and the viscosity of the mixture starts to increase. However, the concentration of GAG derivative A and GAG derivative B or compound C When the value is high, the viscosity of the mixture also increases rapidly. Therefore, when the composition of the present invention is used as an abdominal subcutaneous shape maintaining material used in diseases such as vesicoureteral reflux, the time until the viscosity value reaches 1 Pa · s is 12 hours or less (measurement conditions: The concentrations of GAG derivative A and GAG derivative B or compound C can be determined to be an E-type rotational viscometer, standard cone (1 ° 34'xR24), 25 ° C, 5 rpm).

Upon administration, GAG derivative A and GAG derivative B or compound C are each separately in powder form or dissolved and separately administered as a liquid composition, or immediately before administration, GAG derivative A and GAG derivative B Alternatively, compound C may be mixed and used as a liquid composition in the form of a powder or dissolved, and then crosslinked in a living body by a SPAAC reaction to form a long-term residual tissue swelling material.

In addition, the dose may be an amount required as a long-term residual tissue swelling material in diseases such as vesicoureteral reflux.

Kit for tissue swelling material The kit for tissue swelling material contains the following (1) GAG derivative A and (2) GAG derivative B or (3) compound C.
(1) GAG derivative A in which a SPAAC type reactive group is introduced into a GAG molecule via an amide bond derived from a carboxy group of the GAG molecule and a divalent spacer group;
(2) GAG derivative B in which a reactive group complementary to the reactive group of GAG derivative A is introduced into the GAG molecule via an amide bond derived from a carboxy group of the GAG molecule and a divalent spacer group;
(3) Compound C defined by the following structure having at least two reactive groups complementary to the reactive group of GAG derivative A;

[Where:
Y is the same or different and is a reactive group complementary to the reactive group of GAG derivative A;
Z is an n-valent spacer group,
n is an integer of 2 or more].
Examples of the kit of the present invention include a GAG derivative A solution and a GAG derivative B solution or a compound C solution, each of which is filled in separate containers, or a GAG derivative A powder, And a powder of GAG derivative B or a powder of compound C, and water for injection, each of which is filled in separate containers. At the time of use, GAG derivative A and GAG derivative B or compound C are mixed as it is in powder form or separately as a liquid composition, or just before administration, and in powder form or liquid form It can be administered as a composition.

Hereinafter, the present invention will be described in detail by way of examples. However, this should not limit the technical scope of the present invention. The following abbreviations have the following meanings.
HA Hyaluronic acid CS Chondroitin sulfate DIMAC Dimethoxyazacyclooctyne DBCO Dibenzylcyclooctyne CuAAC Copper-catalyzed azide-alkyne cycloaddition SPAAC Azide-cycloalkyne cycloaddition promoted by ring strain

Example 1 Preparation of DIMAC-Amine Hydrochloride

The DIMAC-amine hydrochloride represented by was prepared according to the following procedure.

(Step 1) Synthesis of Compound 4

Compound 3 was synthesized by the method described in Organic Letter, 2008, Vol. 10, No. 14, 3097-99.

Compound 3 24.6 g (1.00 eq., 86.1 mmol) was dissolved in 1-propanol / water = 19/1 (15 mL). 19.0 g (3.50 eq., 302 mmol) of sodium cyanoborohydride (NaBH ₃ CN) dissolved in Zn powder 225 g (40.0 eq., 3.45 mol), tetrahydrofuran (THF) (300 mL) at room temperature, Allylamine 162 mL (25.0 eq., 2.17 mol) was sequentially added, and the mixture was heated to reflux while stirring in an oil bath at 115 ° C. for 1.5 hours. After confirming the disappearance of the raw materials by thin layer chromatography (TLC), the cooled reaction solution was filtered through Celite. The residue was washed with methanol (MeOH), and the filtrate was concentrated under reduced pressure. To the mixture obtained, methanol / methylene chloride / 1N aqueous HCl solution = 6/2/1 (1350 mL) was added, and 1 at room temperature. Stir for hours. The reaction solution was concentrated under reduced pressure to distill off the organic solvent, 20% aqueous sodium hydroxide solution (123 mL) and water (330 mL) were added, liquid separation extraction was performed 5 times with chloroform, and the collected organic layer was sodium sulfate. As a result of drying under reduced pressure and filtration under reduced pressure, an intermediate imine was obtained.

The obtained imine compound was dissolved in 1,4-dioxane (500 mL), 20.7 g (1.10 eq., 94.6 mmol) of di-tert-butyl dicarbonate (Boc ₂ O) was added at room temperature, Stir for 1 hour. After confirming the disappearance of the intermediate by TLC, the reaction solution was distilled off under reduced pressure. The obtained residue was purified by silica gel column chromatography (ethyl acetate / hexane = 1/3) to obtain 21.2 g (67.3 mmol) of compound 4 as a pale yellow transparent viscous liquid (yield 78%).

TLC (ethyl acetate / hexane = 1/1): R _f value = 0.48 (compound 4)
¹ H-NMR (500 MHz, CDCl ₃ , δ) 1.47 (9H, s, Boc), 2.17-2.74 (1H, m), 3.14 (1H, brs), 3.24 (1H, dd. 15 Hz, 8 Hz), 3.44- 3.54 (7H, m), 3.64 (1H, brs), 3.84 (1H, brs), 4.02 (1H, m), 4.31 (1H, brs), 5.12 (2H, m), 5.22 (1H, d, 5Hz) , 5.37 (1H, dt, 18Hz, 2Hz), 5.78 (1H, m), 5.94 (1H, m)
ESI-MS: Calcd for C ₁₆ H ₂₉ NO ₅ [M + H] ⁺ , 316.2; found 316.6

(Step 2) Synthesis of Compound 5

9.00 g (1.00 eq., 28.5 mmol) of Compound 4 was dissolved in anhydrous dichloromethane. In an oil bath at 45 ° C., 1.34 g (0.0550 eq., 1.57 mmol) of Grubbs second generation catalyst (Bis [4- [Bis (tert-butyl) phosphino] -N, N-dimethylbenzenamide] dichloropalladium) was added. The mixture was added and heated to reflux in an oil bath at 50 ° C. for 35 minutes. After confirming disappearance of the starting material by TLC, the reaction solution was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate / hexane = 1/3) to obtain 6.66 g (23.2 mmol) of Compound 5 as a colorless transparent viscous liquid. (Yield 81%)
TLC (ethyl acetate / hexane = 1/1): R _f value = 0.42 (compound 5)
¹ H-NMR (500 MHz, CDCl ₃ , δ) 1.46-1.51 (9H, m, Boc), 2.83-2.95 (2H, m), 3.23-3.31 (1H, m), 3.42-3.77 (9H, m), 4.41-4.44 (1.5 / 2H, m), 4.59 (0.5 / 2H, d, 18Hz), 5.46-5.48 (1H, m), 5.56-5.59 (1H, m)
ESI-MS: Calcd for C ₁₄ H ₂₅ NO ₅ [M + H] ⁺ , 288.2; found 288.6

(Step 3) Synthesis of Compound 6

To 6.90 g (1.00 eq., 24.0 mmol) of Compound 5 was added 10 g of molecular sieves 4A (powder) and anhydrous dichloromethane (500 mL) at room temperature under an argon gas atmosphere to obtain a suspension solution. In an oil bath at 40 ° C., 13.6 g (1.50 eq., 36.0 mmol) of pyridinium dichromate (PDC) was added and stirred for 5 hours. Thereafter, the mixture was further stirred overnight at room temperature. After confirming disappearance of the starting material by TLC, the reaction solution was filtered, and the residue was washed with chloroform (600 mL). After the filtrate was concentrated under reduced pressure, 600 mL of chloroform was added and washed twice with water (300 mL). The organic layer was dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate / hexane = 1/3) to obtain 5.67 g (19.9 mmol) of Compound 6 as a yellow transparent viscous liquid. (Yield 83%)
TLC (ethyl acetate / hexane = 1/1): R _f value = 0.38 (compound 6)
¹ H-NMR (500 MHz, CDCl ₃ , δ) 1.43-1.46 (9H, m, Boc), 3.38-3.55 (8H, m), 3.67-3.94 (3H, m), 4.02-4.06 (1H, m), 5.84-5.95 (2H, m)
ESI-MS: Calcd for C ₁₄ H ₂₃ NO ₅ [M + H] ⁺ , 286.2; found 286.6

(Step 4) Synthesis of Compound 7

5.64 g (1.00 eq., 19.8 mmol) of Compound 6 was dissolved in ethanol (500 mL), 5% palladium / carbon 1.13 g was added, and the reaction system was replaced with hydrogen gas. Stir overnight. After confirming disappearance of the raw material by TLC, the reaction solution was filtered through Celite, and the residue was washed with methanol (700 mL). The filtrate was concentrated under reduced pressure and vacuum-dried to obtain 5.61 g (19.5 mmol) of Compound 7 as a pale yellow transparent viscous liquid. (Yield 99%)
TLC (ethyl acetate / hexane = 1/1): R _f value = 0.29 (compound 7)
¹ H-NMR (500 MHz, CDCl ₃ , δ) 1.45-1.47 (9H, m, Boc), 1.99-2.18 (2H, m), 2.34-2.5 (2H, m), 2.71-2.76 (0.6 / 1H, m ), 2.81-2.89 (1H, m), 2.95 (0.4 / 1H, dt, 14Hz, 5Hz), 3.33-3.34 (3H, m), 3.55-3.57 (3H, m), 3.59-3.64 (0.4 / 1H, m), 3.69-3.89 (3H, m), 3.95-3.96 (0.6 / 1H, m)
ESI-MS: Calcd for C ₁₄ H ₂₅ NO ₅ [M + H] ⁺ , 288.2; found 288.6

(Step 5) Synthesis of Compound 8

3.98 g (1.00 eq., 13.9 mmol) of compound 7 was dissolved in 110 mL (0.789 eq., 11.0 mmol) of a 100 mM aniline solution in ethanol / water = 1/1 to give 15.5 g of semicarbazide hydrochloride. (10.0 eq., 139 mmol) and 11.2 mL (14.0 eq., 195 mmol) of acetic acid were added and stirred at room temperature for 8.5 hours. After confirming the disappearance of the raw material by TLC, the pH of the reaction solution was adjusted to about 6 with 192 mL of 1N aqueous sodium hydroxide solution. Ethanol in the reaction solution was distilled off under reduced pressure, ethyl acetate (400 mL) was added, and the mixture was washed with water (100 mL). The organic layer was dried over sodium sulfate, concentrated under reduced pressure, and dried under vacuum to obtain a serenadiazole intermediate as a crude product.

The obtained intermediate was dissolved in 1,4-dioxane (50 mL), and 7.11 g (4.60 eq., 6.41 mmol) of selenium dioxide was dissolved in 1,4-dioxane / water = 1/1 (30 mL). The resulting solution was slowly added dropwise over 4.5 hours at room temperature. Then, after reacting at room temperature for 4 days, the reaction solution was concentrated under reduced pressure. Ethyl acetate (400 mL) was added to the residue and washed with water (100 mL). The organic layer was dried over sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (ethyl acetate / hexane = 1/1) to obtain 4.49 g (11.9 mmol) of Compound 8 as a brown transparent viscous liquid. Obtained (yield 86%).

TLC (ethyl acetate / hexane = 1/1): R _f value = 0.53 (compound 8)
¹ H-NMR (500 MHz, CDCl ₃ , δ) 1.46-1.49 (9 H, m, Boc), 2.37 (0.4 / 1 H, dd, 14 Hz, 10 Hz), 2.43 (0.6 / 1 H, dd, 15 Hz, 10 Hz), 2.88 -2.98 (1H, m), 3.31-3.41 (5H, m), 3.57 (3H, s), 3.77 (0.6 / 1H, dd, 15Hz, 5Hz), 3.89 (0.4 / 1H, dd, 14Hz, 5Hz), 4.08-4.16 (1H, m), 4.24 (0.6 / 1H, ddd, 14Hz, 5Hz, 4Hz), 4.36-4.39 (0.4 / 1H, m), 5.83-5.41 (1H, m)
ESI-MS: Calcd for C ₁₄ H ₂₃ N ₃ O ₄ Se [M + H] ⁺ , 378.1; found 378.6

(Step 6) Synthesis of Compound 9

4N Hydrogen chloride / 1,4-dioxane (12 mL) was added to 2.99 g (1.00 eq., 7.95 mmol) of Compound 8 in an ice bath, and the mixture was stirred at room temperature for 1.5 hours. The reaction solution became a red-white suspension. After confirming the disappearance of the raw materials by TLC, hexane (80 mL) was added to the reaction solution, and the supernatant was removed four times. Further, diethyl ether (80 mL) was added, and the supernatant was removed three times. Repeatedly, the precipitate was washed. The obtained precipitate was vacuum-dried to obtain 2.59 g (8.28 mmol) of the target product 9 as a red white powder (yield: quantitative).
¹ H-NMR (500 MHz, D ₂ O, δ) 3.34 (3H, s, OMe), 3.39-3.45 (1H, m), 3.48-3.57 (5H, m), 3.67-3.73 (2H, m), 3.91 -3.97 (1H, m), 4.23 (1H, t, 6Hz), 5.58 (1H, d, 6Hz)
ESI-MS: Calcd for C ₉ H ₁₅ N ₃ O ₂ Se [M + H] ⁺ , 278.0; found 278.2

(Step 7) Synthesis of Compound 10

Fmoc-glycine 571 mg (1.20 eq., 1.92 mmol) was dissolved in methanol / water = 9/1 (4 mL), and 5N aqueous sodium hydroxide solution (384 μL, 1.20 eq., 1.92 mmol) was added. . To this solution, a solution of 500 mg (1.00 eq., 1.60 mmol) of Compound 9 in methanol / water = 9/1 (6 mL) was added at room temperature, and methanol / water = 1/1 (500 μL) was further added. . 532 mg (1.20 eq., 9.40 mmol) 4- (4,6-dimethoxy-1,3,5-triazin-2-yl) -4-methylmorpholinium chloride (DMT-MM) and 5N sodium hydroxide The aqueous solution was added until the pH became around 7, and the mixture was stirred at room temperature for 3 hours. The reaction mixture was concentrated under reduced pressure, ethyl acetate (300 mL) was added to the residue, and the mixture was washed twice with 1N hydrochloric acid (100 mL) and further with a saturated aqueous sodium bicarbonate solution (100 mL). The organic layer was dried over sodium sulfate and then purified by silica gel column chromatography (ethyl acetate / hexane = 3/2 to 2/1) to obtain 687 mg (1.24 mmol) of compound 10 as a pale yellow white amorphous (yield). Rate 77%).
TLC (ethyl acetate / hexane = 3/1): R _f value = 0.37 (compound 10)
¹ H-NMR (500MHz, CDCl ₃ , δ) 2.57 (0.4 / 1H, dd, 14Hz, 5Hz), 3.24-3.60 (10H, m), 3.68-3.70 (0.6 / 1H, m), 3.82-4.14 (3H , m), 4.22-4.50 (4H, m), 5.40-5.48 (1H, m, NH), 5.67 (0.6 / 1H, brs, NH) 5.84 (0.4 / 1H, brs, NH), 7.29-7.33 (2H , m), 7.38-7.42 (2H, m), 7.16 (2H, t, 7Hz), 7.76 (2H, dd, 8Hz, 4Hz)
ESI-MS: Calcd for C ₂₆ H ₂₈ N ₄ O ₅ Se [M + H] ⁺ , 557.1; found 557.1

(Step 8) Synthesis of DIMAC-amine hydrochloride 11

2.20 g (1.00 eq., 3.96 mmol) of Compound 10 was dissolved in anhydrous toluene (44 mL), and 1.87 g (1.80 eq., 7.14 mmol) of triphenylphosphine was added at room temperature. After refluxing with heating at 130 ° C. for 20 hours, the reaction solution was concentrated under reduced pressure. The residue was diluted with ethyl acetate (60 mL) and extracted five times with water (30 mL). The aqueous layer was concentrated under reduced pressure and vacuum dried to obtain DIMAC-amine as a yellow viscous liquid.

The obtained amine compound was dissolved in diethyl ether (25 mL), the solution was vigorously stirred, and 4N hydrogen chloride / 1,4-dioxane (1 mL) was added to precipitate a precipitate. The supernatant was removed, washed with diethyl ether (40 mL) three times, and the precipitate was vacuum-dried to obtain 574 mg (2.19 mmol) of DIMAC-amine hydrochloride as a pale red white powder (yield) 55%).
¹ H-NMR (500 MHz, D ₂ O, δ) 2.24-2.39 (1H, m), 2.60-2.81 (1H, m), 3.16 (1H, dd, 15 Hz, 9 Hz), 3.37-3.44 (4H, m) , 3.55-3.60 (3H, m, OMe), 3.69-3.83 (1H, m), 3.93-4.06 (2H, m), 4.09-4.20 (2H, m), 4.27-4.38 (1H, m)
ESI-MS: Calcd for C ₁₁ H ₁₈ N ₂ O ₃ [M + H] ⁺ , 227.1; found 227.1

[Example 2]: Introduction of DIMAC-amine hydrochloride into hyaluronic acid 300 mg of hyaluronic acid (HA: 1,100 kDa, carboxy group 1.00ea, derived from chicken crown, Seikagaku Corporation) and 30 mL of water for injection (WFI) and 19.7 mg (0.100 eq., 75.0 μmol) of DIMAC-amine hydrochloride 11 prepared in Example 1, Step 8 was added after dissolving in 30 mL of ethanol (EtOH). Furthermore, 4- (4,6-dimethoxy-1,3,5-triazin-2-yl) -4-methylmorpholinium chloride n hydrate (DMT-MM, Tokuyama Corporation) 35.1 mg (0 169 eq., 127 μmol) and stirred overnight. Thereafter, 4.50 mL of 5% aqueous sodium hydrogen carbonate solution was added and stirred for 4 hours, and then 129 μL of 50% aqueous acetic acid solution, 1.50 g of sodium chloride / 4.92 mL of water for injection were added in that order, and ethanol was added to form a precipitate. . The supernatant was removed and washed twice with 90% ethanol / water for injection, twice with ethanol, and once with diethyl ether. The obtained precipitate was dried under reduced pressure overnight to obtain 220 mg of target DIMAC-introduced hyaluronic acid. ^When the introduction rate was calculated by ¹ H-NMR (in D ₂ O), it was 10%.
The introduction rate was calculated from the integrated value of the peak derived from the raw material GAG skeleton and the integrated ratio of the peak derived from the compound.

[Example 3]
252 mg of DIMAC-introduced hyaluronic acid was obtained in the same manner as in Example 2 except that the molecular weight of the raw material hyaluronic acid was 800 kDa. The introduction rate was 12%.

[Example 4]
241 mg of DIMAC-introduced hyaluronic acid was obtained in the same manner as in Example 2 except that the molecular weight of the raw material hyaluronic acid was 600 kDa. The introduction rate was 7%.

[Example 5]
237 mg of DIMAC-introduced hyaluronic acid was obtained in the same manner as in Example 2 except that the molecular weight of the raw material hyaluronic acid was 170 kDa. The introduction rate was 10%.

Example 6: Introduction of DBCO-amine hydrochloride to hyaluronic acid The following structural formula:

DBCO-C2-amine (Crick Chemistry Tools) represented by is introduced by forming an amide bond in hyaluronic acid according to the following procedure.

Hyaluronic acid (250 kDa, carboxy group 1.00 ea, derived from chicken crown, Seikagaku Corporation) 500 mg was dissolved in water for injection (WFI) 50 mL and ethanol (EtOH) 50 mL, and DBCO-C2-amine 17.2 mg (.0.1 050 eq., 62.5 μmol) and 62.5 μL of 1N hydrochloric acid (0.050 eq., 62.5 μmol) were added. Further, 29.2 mg (0.085 eq., 106 μmol) of DMT-MM was added and stirred overnight. Thereafter, 7.5 mL of 5% aqueous sodium hydrogen carbonate solution was added and stirred for 4 hours, then neutralized with 50% aqueous acetic acid solution, 2.5 g of sodium chloride / 8.2 mL of water for injection were sequentially added, and ethanol was added to precipitate. Formed. The supernatant was removed, and washing with 90% ethanol / water for injection was performed twice, washing with ethanol twice, and washing with diethyl ether once. The obtained precipitate was dried under reduced pressure overnight to obtain 498 mg of the target DBCO-introduced hyaluronic acid. ^When the introduction rate was calculated by ¹ H-NMR (in D ₂ O), it was 5%.

[Example 7]
The added amount of DBCO-C2-amine was 34.4 mg (0.100 eq., 125 μmol), the added amount of 1N hydrochloric acid was 125 μL (0.100 eq., 125 μmol), and the added amount of DMT-MM was 58.4 mg (0. 169 eq., 211 μmol) In the same manner as in Example 6, 521 mg of the target DBCO-introduced hyaluronic acid was obtained. The introduction rate was 10%.

[Example 8]: Introduction of azido-amine hydrochloride into hyaluronic acid 500 mg of HA (1,100 kDa, carboxy group 1.00 ea, derived from chicken crown, Seikagaku Corporation), 50 mL of water for injection (WFI), and ethanol ( EtOH) was dissolved in 50 mL, and 61.0 mg (0.400 eq., 498 μmol) of 2-azidoethanamine hydrochloride was added. Further, 234 mg (0.678 eq., 845 μmol) of 4- (4,6-dimethoxy-1,3,5-triazin-2-yl) -4-methylmorpholinium chloride n hydrate (DMT-MM) was added. In addition, the mixture was stirred overnight. Thereafter, 7.5 mL of 5% aqueous sodium hydrogen carbonate solution was added and stirred for 4 hours, and then 215 μL of 50% aqueous acetic acid solution, 2.5 g of sodium chloride / 8.2 mL of water for injection were sequentially added, and ethanol was added to form a precipitate. . The supernatant was removed, and washing with 90% ethanol / water for injection was performed twice, and washing with ethanol was performed three times. The resulting precipitate was dried overnight under reduced pressure to obtain 547 mg of the desired azide-introduced hyaluronic acid. The introduction rate was calculated by analyzing a derivative in which the azide group was coumarin-labeled with SPAAC by GPC-HPLC. The introduction rate was 22%.

[Example 9]
531 mg of azide-introduced hyaluronic acid was obtained in the same manner as in Example 8, except that the amount of 2-azidoethanamine hydrochloride added was changed to 45.8 mg (0.300 eq., 374 μmol). The introduction rate was 21%.

[Example 10]
535 mg of azide-introduced hyaluronic acid was obtained in the same manner as in Example 8, except that the amount of 2-azidoethanamine hydrochloride added was 30.5 mg (0.200 eq., 249 μmol). The introduction rate was 16%.

[Example 11]
490 mg of azide-introduced hyaluronic acid was obtained in the same manner as in Example 8, except that the raw material hyaluronic acid was 170 kDa and the amount of 2-azidoethanamine hydrochloride added was 15.3 mg (0.100 eq., 125 μmol). . The introduction rate was 10%.

[Example 12]
The azide group was introduced in the same manner as in Example 11 except that the amine was changed to 12.7 mg (0.100 eq., 50.0 μmol) of 11-azido-3,6,9-trioxaundecan-1-amine hydrochloride. 231 mg of hyaluronic acid was obtained. The introduction rate was 4%.

[Example 13]
Except that the amine was O- (2-aminoethyl) -O ′-(2-azidoethyl) heptaethylene glycol hydrochloride 23.7 mg (0.100 eq., 50.0 μmol), the same procedure as in Example 11 was performed. As a result, 231 mg of azide group-introduced hyaluronic acid was obtained. The introduction rate was 4%.

Example 14: Introduction of DIMAC-amine hydrochloride into chondroitin sulfate 500 mg of chondroitin sulfate (CS: 36 kDa, carboxy group 1.00 ea, derived from shark cartilage, Seikagaku Corporation) 5 ml of water for injection (WFI), and Dissolved in 5 mL of ethanol (EtOH), 103 mg (0.400 eq., 391 μmol) of DIMAC-amine hydrochloride 11 prepared in Step 8 of Example 1 was added. Further, 184 mg (0.678 eq., 666 μmol) of DMT-MM was added and stirred overnight. Thereafter, 5 mL of 10% aqueous sodium carbonate solution was added and stirred for 4 hours, and then 1 mL of 50% aqueous acetic acid solution and 0.5 g of sodium chloride were sequentially added to form a precipitate using ethanol and 85% ethanol / water for injection. The supernatant was removed and washed twice with 90% ethanol / water for injection, twice with ethanol, and once with diethyl ether. The obtained precipitate was dried under reduced pressure overnight to obtain 388 mg of the target DIMAC-introduced chondroitin sulfate. ^The introduction rate was calculated by ¹ H-NMR (in D ₂ O) to be 42%.

[Example 15]
The same method as in Example 14 except that the addition amount of DIMAC-amine hydrochloride 11 was 77.1 mg (0.300 eq., 279 μmol) and the addition amount of DMT-MM was 138 mg (0.509 eq., 500 μmol). Thus, 487 mg of the target DIMAC-introduced chondroitin sulfate was obtained. The introduction rate was 33%.

[Example 16]
Example 14 except that the amount of DIMAC-amine hydrochloride 11 added was 51.4 mg (0.200 eq., 196 μmol) and the amount of DMT-MM added was 91.8 mg (0.339 eq., 331 μmol). Thus, 487 mg of the target DIMAC-introduced chondroitin sulfate was obtained. The introduction rate was 21%.

[Example 17]: Introduction of DBCO-C2-amine hydrochloride into chondroitin sulfate 3 g of chondroitin sulfate (36 kDa, carboxy group 1.00 ea, derived from shark cartilage, Seikagaku Corporation) in 30 mL of water for injection and 30 mL of ethanol After dissolution, 161 mg (0.100 eq., 583 μmol) of DBCO-C2-amine and 583 μL of 1N hydrochloric acid (0.100 eq., 583 μmol) were added. Further, 276 mg (0.169 eq., 996 μmol) of DMT-MM was added and stirred overnight. Thereafter, 30 g of a 10% aqueous sodium carbonate solution was added and stirred for 2 hours, and then 6 mL of a 50% aqueous acetic acid solution and 3.0 g of sodium chloride were successively added to form a precipitate using ethanol and 85% ethanol / water for injection. The supernatant was removed and washed twice with 90% ethanol / water for injection, twice with ethanol, and once with diethyl ether. The resulting precipitate was dried overnight under reduced pressure to obtain 3.14 g of the target DBCO-introduced chondroitin sulfate. ^When the introduction rate was calculated by ¹ H-NMR (in D ₂ O), it was 11%.

[Example 18]
DBCO-C2-amine was added in an amount of 81.4 mg (0.100 eq., 295 μmol), 1N hydrochloric acid was added in an amount of 295 μL (0.0500 eq., 295 μmol), and DMT-MM was added in an amount of 137 mg (0.0848 eq., 496 μmol). The target DBCO-introduced chondroitin sulfate (2.95 g) was obtained in the same manner as in Example 17, except for the above. The introduction rate was 6%.

[Example 19]: Introduction of azido-amine hydrochloride into chondroitin sulfate 3.00 g of chondroitin sulfate (36 kDa, carboxy group 1.00 ea, derived from shark cartilage, Seikagaku Corporation) in 30 mL of water for injection and 30 mL of ethanol After dissolution, 73.1 mg (0.100 eq., 596 μmol) of 2- (2-azidoethoxy) ethanamine hydrochloride was added. Further, 275 mg (0.169 eq., 994 μmol) of DMT-MM was added and stirred overnight. 3.00 g of sodium chloride was added and a precipitate was formed using ethanol. The supernatant was removed, and washing with 90% ethanol / water for injection was performed twice, and washing with ethanol was performed three times. The resulting precipitate was dried overnight under reduced pressure to obtain 2.66 g of the desired azide-introduced chondroitin sulfate. The introduction rate was calculated by analyzing a derivative in which the azide group was coumarin-labeled with CuAAC by GPC-HPLC. The introduction rate was 11%.

[Example 20]
Example 2 except that the addition amount of 2- (2-azidoethoxy) ethanamine hydrochloride was 36.0 mg (0.0500 eq., 294 μmol) and the addition amount of DMT-MM was 139 mg (0.0848 eq., 501 μmol). In the same manner as in No. 19, 2.74 g of the target azide-introduced chondroitin sulfate was obtained. The introduction rate was 5%.

[Example 21]: Thickening property after mixing (CS + CS)
According to the following experimental procedure, the thickening characteristics after mixing of azide-introduced chondroitin sulfate (introduction rate 11%, derived from Example 19) solution A and DBCO-introduced chondroitin sulfate (introduction rate 11%, derived from Example 17) solution B were investigated. did.
(1) A solution A and a solution B having the following concentrations were prepared in 20 mM phosphate buffered saline (Na ₂ HPO ₄ + NaH ₂ PO ₄ + NaCl) (pH 6.5), respectively.
1.10 wt%, 1.75 wt%, 2.20 wt%, 3.50 wt%, 4.40 wt%
(2) The solutions A and B were uniformly mixed at 1: 1 (v / v) by Voltex (manufactured by Scientific Industries).
(3) E-type rotational viscometer TV-L (Cone: CORD-1, 1 ° 34'xR24) (manufactured by Toki Sangyo Co., Ltd.), tracking changes over time in viscosity value (Pa · s) at 25 ° C, 5 rpm did.
The results are shown in FIG. It was confirmed that the viscosity value increasing efficiency was improved depending on the concentration.

[Example 22]
The introduction rate of azido introduced chondroitin sulfate is 5% (derived from Example 20), the introduction rate of DBCO introduced chondroitin sulfate is 6% (derived from Example 18), and the concentrations are 1.75 wt%, 2.20 wt%, 3.50 wt%. %, 4.40 wt%, and 7.00 wt%, the thickening properties after mixing were investigated in the same manner as in Example 21.
The results are shown in FIG. As in Example 22, an improvement in the viscosity value increasing efficiency depending on the concentration was confirmed.

[Example 23]: Relationship between concentration after mixing and physical properties (CS + CS)
According to the following experimental procedure, mixing of azide-introduced chondroitin sulfate (introduction rate 11%, derived from Example 19) solution A and DBCO-introduced chondroitin sulfate (introduction rate 11%, derived from Example 17) solution B (1: 1 (v / The physical properties of the gel solid obtained by v)) were measured.
(1) A solution A and a solution B having the following concentrations were prepared in 20 mM phosphate buffered saline (Na ₂ HPO ₄ + NaH ₂ PO ₄ + NaCl) (pH 6.5), respectively.
1.10 wt%, 1.75 wt%, 2.20 wt%, 3.50 wt%, 4.40 wt%, 7.00 wt%
(2) The solutions A and B were uniformly mixed at 1: 1 (v / v) by Voltex (manufactured by Scientific Industries).
(3) 1.8 g of the mixed solution was poured into a 25 mL centrifuge tube and allowed to stand overnight at room temperature.
(4) The relationship between strain and stress was investigated using a texture analyzer (TA.XT plus) (manufactured by Stable Micro Systems), a P / 0.5 HS probe, and a 5 kg load cell.
The results are shown in FIG. 3 and Table 1 below. Table 2 shows the results of measurement with rat muscle or rat fat as a control. In correlation with Example 21, the higher the concentration, the higher the strain was observed. It can be said that the stress is significantly higher than that of the rat muscle. This stress can be said to be an important index related to the shape maintenance performance.

[Example 24]: Rat abdominal subcutaneous shape maintenance performance evaluation test (in vivo) HA, CS
The shape maintenance performance in the rat abdomen subcutaneous was evaluated by the following method.
(1) A male Wistar rat (6 weeks old) was injected subcutaneously into the abdomen at 400 μL / site under isoflurane anesthesia.
(2) The height of the bulging portion was measured over time using a height gauge and used as an index of shape maintenance ability.
<Shape maintenance performance test 1: Injection 4 weeks>
The test substances are as follows. Each substance was mixed uniformly and then injected subcutaneously into the abdomen of the rat, and the change in the height of the bulge was observed.
(1) 1.75 wt% azide-introduced chondroitin sulfate (introduction rate 11%, derived from Example 19) physiological saline aqueous solution and 1.75 wt% DBCO-introduced chondroitin sulfate (introduction rate 11%, derived from Example 17) physiological saline aqueous solution 1: 1 (v / v) homogeneous mixture [1.75% CS (N ₃ + DBCO)].
(2) 2.20 wt% azide-introduced hyaluronic acid (170 kDa) (introduction rate 10%, derived from Example 11) physiological saline solution and 2.20 wt% DIMAC-introduced hyaluronic acid (170 kDa) (introduction rate 10%, Example 5) Origin) 1: 1 (v / v) homogeneous mixture of physiological saline solution [2.20 wt% HA (N ₃ + DIMAC)]
(3) 1.00 wt% hyaluronic acid aqueous solution The results are shown in FIG. Compared with the 1.00 wt% hyaluronic acid aqueous solution of (3), the test substances of (1) and (2) showed a remarkably high shape maintenance performance even 28 days after administration.

[Example 25]
<Shape maintenance performance test 2: injection 24 weeks>
In the same manner as in Example 25, the shape maintenance performance in the rat abdomen was evaluated. The test substances are as follows. Each substance was injected after being uniformly mixed at 1: 1 (v / v).
(1) 2.20 wt% azide-introduced hyaluronic acid (170 kDa) (introduction rate 10%, derived from Example 11) physiological saline solution and 2.20 wt% DIMAC-introduced hyaluronic acid (introduction rate 10%, from Example 5) physiology 1: 1 (v / v) homogeneous mixture of saline solution [2.20 wt% HA (N ₃ + DIMAC)]
(2) 1.00 wt% hyaluronic acid aqueous solution The results are shown in FIG. Compared with the 1.00 wt% hyaluronic acid aqueous solution of (2), the test substance of (1) showed a remarkably high shape maintenance performance even for a long period of 24 weeks.

[Example 26]: Results of biocompatibility evaluation in rat abdominal subcutaneous shape maintenance performance evaluation test (in vivo) In the rat abdominal subcutaneous shape maintenance performance evaluation described in shape maintenance performance test 2 of Example 25, 2 days after injection, After 4 weeks and 24 weeks, the skin at the injection site of 2.2 wt% HA (N ₃ + DIMAC) was collected, immersed and fixed in 10% neutral buffered formalin solution, embedded in paraffin, and HE-stained specimens were prepared. Thereafter, histopathological examination was performed with an optical microscope.
As a result, only 2 days after the injection, only a slight acute inflammatory reaction was observed as seen in the injection of physiological saline. At 4 weeks after injection and 24 weeks after injection, the injection was encapsulated with fibers, and neither foreign body reaction nor tissue reaction such as inflammatory reaction was observed (FIGS. 6 and 7). From the above, it can be said that 2.2% wt HA (N ₃ + DIMAC) has good biocompatibility both in the acute phase and in the chronic phase.

The disclosure of Japanese Patent Application No. 2013-165116 (filing date: August 8, 2013) is incorporated herein by reference in its entirety.
All documents, patent applications, and technical standards mentioned in this specification are to the same extent as if each individual document, patent application, and technical standard were specifically and individually stated to be incorporated by reference, Incorporated herein by reference.

Claims

The first GAG molecule and the second GAG molecule have the following crosslinking group:
—CONH—R 1 —XR 2 —NHCO—, or —CONH—R 3 —XR 4 —X′—R 5 —NHCO—
[Where:
-CONH and NHCO- at both ends mean an amide bond derived from the carboxy group of the GAG molecule,
R 1 to R 5 are the same or different and each represents an alkylene group, an alkenylene group, or an alkynylene group, and —CH 2 — in the group represents> C═O, —CONH—, arylene, —O—, or May be replaced by -S-;
X and X ′ are the same or different and have the formula:

Represents the structure shown by;
Here, A and B each represent a binding site with any of R 1 to R 5 to which X or X ′ is bound;
Y 1 ~ Y 6 are the same or different, -CR 6 R 6 '-, - C (-R 6) =, - NR 7 -, = N -, - O-, or -S- represents, - NR 7 -, = N -, - O-, and -S- are never adjacent;
R 6 and R 6 ′ are the same or different and are a hydrogen atom, a halogen atom, a hydroxy group, an amino group, an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group or a carboxy group, which may be mono- or di-substituted with alkyl. Or together formed an oxo group, and —CH 2 — in the alkyl group, alkenyl group, alkynyl group or alkoxy group represents> C═O, —CONH—, arylene, —O—. Or may be replaced by -S-;
R 7 represents an alkyl group, an alkenyl group or an alkynyl group, and —CH 2 — in the alkyl group, alkenyl group or alkynyl group represents> C═O, —CONH—, arylene, —O— or —S—. May be replaced by; or
In two neighboring groups of Y 1 - Y 6, R 6 in R 6 and the other group of one group is a saturated or 3-unsaturated together with the ring atoms to which each is attached A 6-membered ring can be formed, and the bond B can be bonded to the 3- to 6-membered ring]
A GAG cross-linked product bound via a (the first GAG molecule and the second GAG molecule may be the same molecule).
The GAG molecule has the formula:

[Where:
R 8 and R 9 represent a hydrogen atom or a hydroxy group; provided that when R 8 is a hydroxy group, R 9 is a hydrogen atom, and when R 8 is a hydrogen atom, R 9 is a hydroxy group;
R 10 is ONa or OH],
In the case where R 8 is a hydroxy group, at least one of the hydroxy groups in the structural unit is —OSO 3 Na or —OSO 3 H. The cross-linked GAG according to claim 1.
A composition comprising the following (1) GAG derivative A and (2) GAG derivative B or (3) compound C:
(1) GAG derivative A in which a SPAAC type reactive group is introduced into a GAG molecule via an amide bond derived from a carboxy group of the GAG molecule and a divalent spacer group;
(2) GAG derivative B in which a reactive group complementary to the reactive group of GAG derivative A is introduced into the GAG molecule via an amide bond derived from a carboxy group of the GAG molecule and a divalent spacer group;
(3) Compound C defined by the following structure having at least two reactive groups complementary to the reactive group of GAG derivative A;

[Where:
Y is the same or different and is a reactive group complementary to the reactive group of GAG derivative A;
Z is an n-valent spacer group,
n is an integer of 2 or more].
the n-valent spacer group is an n-valent group derived from an alkyl group, an alkenyl group or an alkynyl group, and —CH 2 — in the spacer group is> C═O, —CONH—, arylene, — The composition according to claim 3, which may be replaced by O- or -S-.
The composition according to claim 4, wherein n in compound C is 2.
Either the SPAAC type reactive group or the reactive group complementary to the reactive group is an azide group, and the other is represented by the formula:

[Where:
Here, B represents a binding site with a divalent or n-valent spacer group;
Y 1 ~ Y 6 are the same or different, -CR 6 R 6 '-, - C (-R 6) =, - NR 7 -, = N -, - O-, or -S- represents, - NR 7 -, = N -, - O-, and -S- are never adjacent;
R 6 and R 6 ′ are the same or different and are a hydrogen atom, a halogen atom, a hydroxy group, an amino group, an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group or a carboxy group, which may be mono- or di-substituted with alkyl. Or together formed an oxo group, and —CH 2 — in the alkyl group, alkenyl group, alkynyl group or alkoxy group represents> C═O, —CONH—, arylene, —O—. Or may be replaced by -S-;
R 7 represents an alkyl group, an alkenyl group, or an alkynyl group, and —CH 2 — in the alkyl group, alkenyl group, or alkynyl group represents> C═O, —CONH—, arylene, —O—, or —S May be replaced by; or
In two neighboring groups of Y 1 - Y 6, R 6 in R 6 and the other group of one group is a saturated or 3-unsaturated together with the ring atoms to which each is attached A 6-membered ring can be formed, and the bond B can be bonded to the 3- to 6-membered ring]
The composition of Claim 4 or 5 which is a cyclic group shown by these.
A SPAAC type reactive group or a reactive group complementary to the reactive group,

The composition according to any one of claims 3 to 6, which is introduced into the GAG molecule by an amide bond between an amino group of a cyclooctyne derivative amine selected from the group consisting of and a carboxy group of the GAG molecule.
The composition according to any one of claims 3 to 7, wherein the GAG molecule is hyaluronic acid or a salt thereof, or chondroitin sulfate or a salt thereof.
The composition according to any one of claims 6 to 8, wherein the substituent introduction ratio of the azide group to the number of moles of the disaccharide repeating unit of the GAG derivative is 0.1 to 80%.
The composition according to any one of claims 6 to 8, wherein the substituent introduction ratio of the azide group to the number of moles of the disaccharide repeating unit of the GAG derivative is 0.5 to 20%.
The composition according to any one of claims 6 to 10, wherein the cyclooctynyl group has a substituent introduction rate of 0.1 to 80% with respect to the number of moles of the disaccharide repeating unit of the GAG derivative.
The composition according to any one of claims 6 to 10, wherein the cyclooctynyl group has a substituent introduction rate of 0.5 to 20% with respect to the number of moles of the disaccharide repeating unit of the GAG derivative.
The composition according to any one of claims 3 to 12, wherein the molecular weight of the GAG molecule is 5 to 2,000 kDa.
The composition according to any one of claims 3 to 12, wherein the GAG molecule has a molecular weight of 10 to 500 kDa.
A tissue swelling material comprising the composition according to any one of claims 3 to 14.
The tissue swelling material according to claim 15, wherein the composition is liquid at the time of administration.
The tissue swelling material according to claim 15, which is a treatment material for treatment of vesicoureteral reflux, stress urinary incontinence or fecal incontinence in the urological field.
The tissue swelling material according to claim 15, which is a swelling material for tissue reconstruction after radical prostatectomy.
The tissue swelling material according to claim 15, which is a tissue swelling material for tissue reconstruction in a surgical, orthopedic or plastic surgery region.
The tissue swelling material according to any one of claims 15 to 19, which is crosslinked in vivo.
The time until the viscosity value reaches 1 Pa · s at the E-type rotational viscometer, standard cone (1 ° 34'xR24), 25 ° C, 5 rpm after the start of crosslinking is 12 hours or less. Item 20. A tissue swelling material according to Item 20.
following:

A GAG derivative in which the amino group of the amine selected from the cyclooctyne derivative amine and the carboxy group of GAG are amide-bonded.
following:

A cyclooctyne derivative amine selected from:
A tissue swelling material kit comprising the following (1) GAG derivative A and (2) GAG derivative B or (3) compound C:
(1) GAG derivative A in which a SPAAC type reactive group is introduced into a GAG molecule via an amide bond derived from a carboxy group of the GAG molecule and a divalent spacer group;
(2) GAG derivative B in which a reactive group complementary to the reactive group of GAG derivative A is introduced into the GAG molecule via an amide bond derived from a carboxy group of the GAG molecule and a divalent spacer group;
(3) Compound C defined by the following structure having at least two reactive groups complementary to the reactive group of GAG derivative A;

[Where:
Y is the same or different and is a reactive group complementary to the reactive group of GAG derivative A;
Z is an n-valent spacer group,
n is an integer of 2 or more].
the n-valent spacer group is an n-valent group derived from an alkyl group, an alkenyl group or an alkynyl group, and —CH 2 — in the spacer group is> C═O, —CONH—, arylene, — The kit according to claim 24, which may be replaced by O- or -S-.