Hyaluronic acid derivative, gel of crosslinked hyaluronic acid derivative, and pharmaceutical composition

Abstract

The present invention provides: a hyaluronic acid derivative which, after having been gelled, is highly inhibited from swelling in the living body; a gel of a crosslinked hyaluronic acid derivative; and a pharmaceutical composition including the hyaluronic acid derivative or the gel of a crosslinked hyaluronic acid derivative. This hyaluronic acid derivative comprises hyaluronic acid having steryl groups introduced into at least some of the carboxyl groups contained in the glucuronic acid moieties of the hyaluronic acid and having maleimide groups introduced into at least some of either the carboxyl groups contained in the glucuronic acid moieties of the hyaluronic acid or the hydroxyl groups contained in the N-acetylglucosamine moieties thereof. This gel of a crosslinked hyaluronic acid derivative is a gel-like material obtained by chemically crosslinking the hyaluronic acid derivative with a crosslinking agent having two or more crosslinking groups.

Description

Hyaluronic acid derivative, hyaluronic acid derivative cross-linked gel, and pharmaceutical composition

[0001] The present invention relates to a hyaluronic acid derivative, a hyaluronic acid derivative crosslinked gel obtained by crosslinking the hyaluronic acid derivative, and a pharmaceutical composition containing the same. This application claims priority based on Japanese Patent Application No. 2023-166346 filed on September 27, 2023, and Japanese Patent Application No. 2023-204796 filed on December 4, 2023, the contents of which are incorporated herein by reference.

[0002] In recent years, biopharmaceuticals, which are pharmaceuticals containing proteins, peptides, or nucleic acids as active ingredients, have been put into practical use, and their number continues to increase year by year. Biopharmaceuticals can fulfill unmet medical needs that traditional small molecule drugs could not. However, they have challenges, such as poor absorption from the digestive tract or mucous membranes, instability in the body, and a short half-life in the blood. Therefore, biopharmaceuticals require frequent injections, which places a significant burden on both patients and medical professionals. Therefore, there is a need for a drug matrix (sustained-release drug delivery system matrix) that can encapsulate biopharmaceuticals without impairing their pharmacological activity and gradually release the active ingredient in the body.

[0003] Under these circumstances, Patent Document 1 and Patent Document 2 propose a sustained-release drug delivery system substrate made of hyaluronic acid derivatives with excellent safety.This hyaluronic acid derivative spontaneously aggregates in aqueous solution, can efficiently encapsulate drugs, especially biopharmaceuticals, while maintaining their biological activity, aggregates under physiological salt concentration (or disperses under physiological salt concentration), and has good blood retention.This hyaluronic acid derivative can be used as a carrier that can efficiently encapsulate many drugs while maintaining pharmacological activity, and as a blood sustained-release carrier and targeting carrier with excellent blood retention, especially when biopharmaceuticals are used as active ingredients, and can also be used as a local (for example, subcutaneous) sustained-release carrier that can sustainably release drugs.

[0004] In addition, hyaluronic acid is an important component of joints, and there is a treatment method of injecting hyaluronic acid into the joint cavity to treat joint diseases.For this treatment, it is desired that it is easy to inject, but that it remains in the joint cavity for a longer period of time.Therefore, Patent Document 3 proposes hyaluronic acid derivatives modified with various block polymers in order to achieve both ease of injection and long-term retention.

[0005] International Publication No. WO 2010 / 053140 International Publication No. WO 2004 / 046200 International Publication No. WO 2003 / 087019

[0006] When used to treat joint diseases, injected hyaluronic acid or its derivatives may gel within the joint cavity, but it is preferable that the volume does not change significantly before and after gelation. Furthermore, when used as a sustained-release drug delivery system substrate, it is preferable that the swelling of the hyaluronic acid crosslinked gel is as small as possible from the perspective of product design. Furthermore, crosslinked hydrogels that allow for the encapsulation of large amounts of growth factors and proteins are preferred. However, hyaluronic acid cannot encapsulate proteins, and being a polysaccharide, it has a high degree of swelling due to its high hydrophilicity. Furthermore, the hyaluronic acid derivatives described in Patent Documents 1 to 3 also had insufficient swelling suppression due to gelation.

[0007] The present invention has been made in view of the above circumstances, and provides a hyaluronic acid derivative and a hyaluronic acid derivative cross-linked gel that are excellent in suppressing swelling after gelation, as well as a pharmaceutical composition using these.

[0008] That is, the present invention includes the following aspects. [1] A hyaluronic acid derivative in which steryl groups have been introduced into at least a portion of the carboxyl groups in the glucuronic acid moiety of hyaluronic acid, and maleimide groups have been introduced into at least a portion of the carboxyl groups in the glucuronic acid moiety or the hydroxyl groups in the N-acetylglucosamine moiety of the hyaluronic acid. [2] The hyaluronic acid derivative of [1] above, in which maleimide groups have been introduced into at least a portion of the carboxyl groups in the glucuronic acid moiety of the hyaluronic acid. [3] The hyaluronic acid derivative of [1] or [2] above, in which the sum of the introduction rate of the steryl groups and the introduction rate of the maleimide groups in the hyaluronic acid derivative is less than 45%. [4] The hyaluronic acid derivative of any of [1] to [3] above, in which the introduction rate of the steryl groups in the hyaluronic acid derivative is 0.5% or more and 30% or less. [5] The hyaluronic acid derivative according to any one of [1] to [4], wherein the introduction rate of the maleimide group to the hyaluronic acid derivative is 1.0% or more and 25% or less. [6] A hyaluronic acid derivative represented by the following general formula (I):

[0009]

[0010] [In general formula (I), R １ , R ２ , R ３ , and R ４ are each independently a hydrogen atom, C １－６ Alkyl, formyl and C １－６ alkylcarbonyl; Z represents a direct bond or a peptide linker consisting of any amino acid residues of 2 to 30; X １ is -NR ｂ -R, -NR ｂ -COO-R, -NR ｂ -CO-R, -NR ｂ -CO-NR ｃ -R, -COO-R, -O-COO-R, -SR, -CO-Y ａ -SR, -O-CO-Y ｂ -S-R, -NR ｂ -CO-Y ｂ is a group selected from the group consisting of groups represented by -SR and -SSR; Rａ , R ｂ and R ｃ are each independently a hydrogen atom, C １－２０ Alkyl, Amino C ２－２０ Alkyl and hydroxy C ２－２０ alkyl, and R ａ , the R ｂ and the R ｃ The alkyl portion of the ｆ - may be inserted with a group selected from the group consisting of; ｆ is a hydrogen atom, C １－１２ Alkyl, Amino C ２－１２ Alkyl and hydroxy C ２－１２ alkyl, and R ｆ wherein the alkyl portion may be interrupted by a group selected from the group consisting of —O— and —NH—; R is a steryl group; Y is C ２－３０ Alkylene, or -(CH ２ CH ２ O) ｍ -CH ２ CH ２ -, and the alkylene portion of Y is -O-, -NR ｇ - and -S-S- may be inserted; R ｇ is a hydrogen atom, C １－２０ Alkyl, Amino C ２－２０ Alkyl and hydroxy C ２－２０ alkyl, and R ｇ The alkyl portion of Y may be inserted with a group selected from the group consisting of —O— and —NH—; ａ is C １－５ is alkylene; Y ｂ is C ２－８ Alkylene or C ２－８ and m is an integer of 1 to 100.] and has one or more repeating units represented by the following general formula (II):

[0011]

[0012] [In general formula (II), R１ , R ２ , R ３ , and R ４ are each independently a hydrogen atom, C １－６ Alkyl, formyl and C １－６ alkylcarbonyl; Z represents a direct bond or a peptide linker consisting of any amino acid residues of 2 to 30; X ２ is a maleimide group; R ａ is a hydrogen atom, C １－２０ Alkyl, Amino C ２－２０ Alkyl and hydroxy C ２－２０ alkyl, and R ａ The alkyl portion of the ｆ - may be inserted with a group selected from the group consisting of; ２－３０ Alkylene, or -(CH ２ CH ２ O) ｍ -CH ２ CH ２ -, and the alkylene portion of Y is -O-, -NR ｇ - and -S-S- may be inserted; R ｇ is a hydrogen atom, C １－２０ Alkyl, Amino C ２－２０ Alkyl and hydroxy C ２－２０ alkyl, and R ｇwherein the alkyl portion may have a group selected from the group consisting of -O- and -NH- inserted therein; and m is an integer of 1 to 100.] The hyaluronic acid derivative according to any one of [1] to [5] above, having one or more repeating units represented by the formula: [7] The hyaluronic acid derivative according to any one of [1] to [6] above, wherein the steryl group is a cholesteryl group. [8] The hyaluronic acid derivative according to any one of [1] to [7] above, wherein the hyaluronic acid derivative dissolved in phosphate buffer (10 mM) at a concentration of 1 mg / mL has an average particle size of 250 nm or less as calculated by dynamic light scattering. [9] A method for producing a hyaluronic acid derivative crosslinked gel, comprising reacting the hyaluronic acid derivative according to any one of [1] to [8] above with a crosslinking agent having two or more crosslinkable groups to gel it.

[10] A method for producing a hyaluronic acid derivative crosslinked gel according to [9] above, wherein the crosslinkable group is a thiol group.

[11] A hyaluronic acid derivative crosslinked gel, which is a gel-like substance obtained by chemically crosslinking the hyaluronic acid derivative of any one of [1] to [8] above with a crosslinking agent having two or more crosslinkable groups.

[12] The hyaluronic acid derivative crosslinked gel of

[11] above, in which the crosslinkable group is a thiol group.

[13] The hyaluronic acid derivative crosslinked gel of

[11] or

[12] above, having a swelling degree of 115% or less.

[14] A dried hyaluronic acid derivative crosslinked gel, which is a dried gel-like substance obtained by chemically crosslinking the hyaluronic acid derivative of any one of [1] to [8] above with a crosslinking agent having two or more crosslinkable groups.

[15] The dried hyaluronic acid derivative crosslinked gel of

[14] above, which has a porous structure.

[16] A pharmaceutical composition comprising the hyaluronic acid derivative of any one of [1] to [8] above.

[17] A pharmaceutical composition comprising a hyaluronic acid derivative crosslinked gel, in which the hyaluronic acid derivative according to any one of [1] to [8] above is chemically crosslinked with a crosslinking agent having two or more crosslinkable groups.

[18] The pharmaceutical composition according to

[17] above, further comprising an active ingredient.

[0013] According to the hyaluronic acid derivative of the above aspect, it is possible to provide a hyaluronic acid derivative and a hyaluronic acid derivative cross-linked gel that are excellent in suppressing swelling after gelation, as well as a pharmaceutical composition using these.

[0014] 1 is an NMR chart of the hyaluronic acid derivative crosslinked gel (HA-C6-Chol-0%-Male) synthesized in Example 2. 2 is an NMR chart of the hyaluronic acid derivative crosslinked gel (HA-C6-Chol-1%-Male) synthesized in Example 2. 3 is an NMR chart of the hyaluronic acid derivative crosslinked gel (HA-C6-Chol-5%-Male) synthesized in Example 2. 4 is an NMR chart of the hyaluronic acid derivative crosslinked gel (HA-C6-Chol-10%-Male) synthesized in Example 2. 5 is an NMR chart of the hyaluronic acid derivative crosslinked gel (HA-C6-Chol-15%-Male) synthesized in Example 2. 6 is an NMR chart of the hyaluronic acid derivative crosslinked gel (HA-C6-Chol-20%-Male) synthesized in Example 2. 2(A) shows the results of a HAMICH crosslinked gel prepared at a hyaluronic acid concentration of 7.0 mg / mL, and FIG. 2(B) shows the results of a HAMICH crosslinked gel prepared at a hyaluronic acid concentration of 16.7 mg / mL. This figure shows the results of measuring the encapsulation rate (%) of FITC-insulin in a HAMICH crosslinked gel prepared at a hyaluronic acid concentration of 16.7 mg / mL in Example 3. This figure shows the results of measuring the encapsulation rate (%) of FITC-insulin in a HAMICH crosslinked gel prepared at a hyaluronic acid concentration of 16.7 mg / mL in Example 3. 5 is a visible light photograph of the appearance of a HAMICH cross-linked gel in which FITC-insulin has been encapsulated in Example 3.

[0034] In Example 4, these are fluorescence microscope images of a Cy5-HAMICH cross-linked gel with a cholesteryl group introduction rate of 19% (upper row of FIG. 5) and a Cy5-HAMICH cross-linked gel with a cholesteryl group introduction rate of 0% (lower row of FIG. 5) after freeze-drying and after freeze-thawing.

[0035] In Example 8, these are figures showing the results of measuring the release rate (%) of FITC-insulin from a HAMICH cross-linked gel in which FITC-insulin has been encapsulated.

[0036] In Example 10, these are figures showing the change over time in the degree of swelling (%) from the start of gelation of a HAMICH cross-linked gel.Figure 7(A) shows the results of the cross-linked gel of HAMICH (HA-C6-Chol-1%-Male 8.1%) in Test Area 9-1, and Figure 7(B) shows the results of the cross-linked gel of HAMICH (HA-C6-Chol-17%-Male 4.6%) in Test Area 9-1.

[0015] An embodiment of the present invention (hereinafter referred to as "the present embodiment") will be described in detail below, but the present invention is not limited to this and various modifications are possible within the scope of the gist thereof.

[0016] The terms used in this specification will be explained below.

[0017] As used herein, "C １－２０ The term "alkyl" means a straight or branched chain alkyl group having from 1 to 20 carbon atoms, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, etc. １－４ alkyl," and further includes n-pentyl, 3-methylbutyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, n-hexyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3-ethylbutyl, 2-ethylbutyl, and the like. １－２０ Alkyl has 1 to 12 carbon atoms. １－１２ Alkyl, C having 1 to 6 carbon atoms １－６ Alkyl groups are also included.

[0018] As used herein, "C １－６ The term "alkylcarbonyl" refers to the alkyl moiety of the aforementioned C １－６ It means an alkylcarbonyl group, for example, acetyl, propionyl, n-propylcarbonyl, isopropylcarbonyl, n-butylcarbonyl, sec-butylcarbonyl, isobutylcarbonyl, tert-butylcarbonyl, etc. １－４ alkylcarbonyl".

[0019] As used herein, "amino C ２－２０The term "alkyl" means a linear or branched alkyl having from 2 to 20 carbon atoms and having an amino group as a substituent, and for example, the amino group may be located on the terminal carbon atom of the alkyl group. ２－２０ Alkyl includes amino C having 2 to 12 carbon atoms. ２－１２ Alkyl is also included.

[0020] As used herein, "hydroxy C ２－２０ The term "alkyl" means a linear or branched alkyl group having from 2 to 20 carbon atoms and having a hydroxy group as a substituent, for example, the hydroxy group may be located on the terminal carbon atom of the alkyl group. ２－２０ Alkyl includes hydroxy groups having 2 to 12 carbon atoms. ２－１２ Alkyl is also included.

[0021] As used herein, "C ２－３０ The term "alkylene" means a linear or branched divalent saturated hydrocarbon group having from 2 to 30 carbon atoms, and includes, for example, ethylene, propylene, etc., and has from 2 to 20 carbon atoms. ２－２０ Alkylene, C having 2 to 8 carbon atoms ２－８ Alkylene, group "-(CH ２ ) ｎ -" (where n is 2 or more and 30 or less, preferably 2 or more and 20 or less, and more preferably 2 or more and 15 or less).

[0022] As used herein, "C １－５ The term "alkylene" means a straight or branched chain saturated divalent hydrocarbon radical having from 1 to 5 carbon atoms and includes, for example, methylene, ethylene, propylene, and the like.

[0023] The term "C" referred to herein ２－８ The term "alkenylene" means a linear or branched, divalent saturated hydrocarbon group having from 2 to 8 carbon atoms and containing one or more double bonds, and examples thereof include -CH=CH-, -C(CH ３)=CH-, 2-butene-1,4-diyl, hepta-2,4-diene-1,6-diyl, octa-2,4,6-triene-1,8-diyl, etc. When geometric isomers exist, each isomer and a mixture thereof are also included.

[0024] In the present specification, "swelling degree" refers to the degree of volume increase of a hyaluronic acid derivative crosslinked gel obtained by crosslinking a crosslinked hyaluronic acid derivative under physiological salt concentration or in PBS(-) (in vivo) based on the relationship between osmotic pressure and elastic pressure. Specifically, it is calculated using the following formula. A hyaluronic acid derivative crosslinked gel with a negative swelling degree is a gel whose volume decreases (shrinks) when it reaches an equilibrium state in PBS(-) compared to the volume before gelation (corresponding to the volume immediately after gelation). A hyaluronic acid derivative crosslinked gel with a positive swelling degree is a gel whose volume increases (swells) when it reaches an equilibrium state in PBS(-) compared to the volume immediately after gelation. In a hyaluronic acid derivative crosslinked gel, the smaller the absolute value of the swelling degree, the smaller the volume change of the crosslinked gel, and therefore is preferable.

[0025] [Swelling degree (%)] = ([Volume (mm ３ ) )] / [volume of hyaluronic acid-containing composition before gelation (mm ３ ) )]) × 100 (%)

[0026] <Hyaluronic Acid Derivative> In the hyaluronic acid derivative of the present embodiment, steryl groups have been introduced into at least a portion of the carboxyl groups in the glucuronic acid moiety of hyaluronic acid, and maleimide groups have been introduced into at least a portion of the carboxyl groups in the glucuronic acid moiety or the hydroxyl groups in the N-acetylglucosamine moiety of the hyaluronic acid.

[0027] The reason why the hyaluronic acid derivative of this embodiment exhibits suppressed swelling after gelation is unclear, but is speculated as follows. It is speculated that the introduction of hydrophobic groups, steryl and maleimide groups, into the hydrophilic carboxyl and hydroxyl groups of hyaluronic acid reduces the hydrophilicity of the entire molecule, thereby suppressing swelling. Furthermore, the use of a combination of steryl and maleimide groups, in particular, as the hydrophobic groups introduced into hyaluronic acid allows gelation to proceed in a relatively short time, from 1 second to 30 minutes, which is also speculated to contribute to swelling suppression. In particular, since hyaluronic acid derivatives modified only with maleimide groups, with no steryl groups modified, exhibit poor swelling suppression, it is thought that the swelling suppression effect is due to the elastic pressure generated by the strong hydrophobic interaction of the steryl groups. Hyaluronic acid derivatives modified with functional groups that exhibit strong interactions due to π-π stacking, such as aromatic rings, may also exhibit excellent swelling suppression after gelation, similar to the hyaluronic acid derivative of this embodiment.

[0028] The swelling degree of the hyaluronic acid derivative of this embodiment is preferably 115% or less, more preferably 110% or less, and even more preferably 105% or less.The swelling degree of the hyaluronic acid derivative of this embodiment is preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more.The swelling degree of the hyaluronic acid derivative of this embodiment is particularly preferably 85% or more and 115% or less.

[0029] [Steryl Group] The term "steryl group" used herein is not particularly limited as long as it is a group having a steroid skeleton. Specific examples of steroids include cholesterol, cholestanol, campestanol, ergostanol, stigmastanol, coprostanol, stigmasterol, sitosterol, lanosterol, ergosterol, cimialenol, bile acids, testosterone, estradiol, progesterone, cortisol, cortisone, aldosterone, corticosterone, and deoxycortisterone. Examples of steryl groups include cholesteryl, stigmasteryl, lanosteryl, and ergosteryl groups, with cholesteryl groups (particularly cholest-5-en-3β-yl groups) being preferred.

[0030] In the hyaluronic acid derivative, the steryl group may be directly bonded to the hyaluronic acid, or may be bonded via a linker.The linkers that are connected to the steryl groups contained in one molecule of the hyaluronic acid derivative may all be the same linker, or may be linkers of different lengths or types.

[0031] The "linker" referred to here is a group in which a group reactive with a carboxy group and a group reactive with a steryl group are linked by a chain group (spacer). Examples of the group reactive with a carboxy group and the group reactive with a steryl group include an amino group and a hydroxyl group. As the chain group, for example, a chain hydrocarbon group, a polyethylene glycol (PEG) chain, any peptide linker that can be introduced by genetic engineering, or a synthetic compound linker can be used. The length of the linker is not particularly limited, and can be appropriately selected by those skilled in the art depending on the purpose. Examples of the chain hydrocarbon group include C １－３０ Alkylene is preferred, C １－１０ Alkylene is more preferred, C １－６Alkylene is more preferred.As the PEG group, a group in which the number of linked ethylene glycol groups is 1 to 15 is preferred, a group in which the number is 1 to 10 is more preferred, and a group in which the number is 1 to 5 is even more preferred.As the peptide linker, two or more amino acids (the upper limit is not particularly limited, but usually 30 amino acids or less, preferably 20 amino acids or less), particularly preferably 15 amino acids.As the hyaluronic acid derivative of this embodiment, it is preferred that at least a part of the carboxyl groups in the glucuronic acid moiety is linked to a steryl group by a linker having a chain hydrocarbon group.

[0032] [Steryl group introduction rate] The introduction rate of steryl group in the hyaluronic acid derivative of this embodiment (hereinafter sometimes simply referred to as "steryl group introduction rate") is preferably 0.5% or more, more preferably 1.0% or more, more preferably 5.0% or more, and even more preferably 10% or more.The introduction rate of steryl group in the hyaluronic acid derivative of this embodiment is preferably 30% or less, more preferably 25% or less, even more preferably 20% or less, and even more preferably 15% or less.By being within the above-mentioned range, the swelling degree can be sufficiently reduced, and when forming crosslinked gel, can contain a sufficient amount of drug.

[0033] The steryl group introduction rate is １ It can be measured by H-NMR measurement. １ The integral value of the peak derived from the steryl group of the hyaluronic acid derivative in the H-NMR spectrum and the peak derived from the acetyl group of N-acetyl-D-glucosamine contained in the hyaluronic acid derivative (COCH ３ , 1.6 ppm or more and 2.0 ppm or less, 3H) and the integral value of Ｈ represents the number of hydrogen atoms corresponding to the peak. Specifically, it can be measured, for example, according to the method described in the Examples below.

[0034] [Steryl group introduction rate] (%) = ([integrated value of peaks derived from steryl groups] × 3 / n Ｈ) / [peak integral value derived from the acetyl group of N-acetyl-D-glucosamine]×100

[0035] [Maleimide group] In the hyaluronic acid derivative, the maleimide group (N-maleimide group) may be directly bonded to the hyaluronic acid or may be bonded via a linker. The linkers that link the maleimide groups contained in one molecule of the hyaluronic acid derivative may all be the same linker, or may be linkers of different lengths or types.

[0036] The "linker" referred to here is a group in which a group reactive with a carboxy group and a group reactive with an NH group in a maleimide group are linked by a chain group. Examples of the group reactive with a carboxy group include an amino group and a hydroxyl group, and examples of the group reactive with an NH group in a maleimide group include a carboxy group and a hydroxyl group. Examples of the chain group include the same groups as the chain group in the linker that links the hyaluronic acid derivative and the steryl group. In the hyaluronic acid derivative of this embodiment, it is preferred that at least a portion of the carboxyl groups in the glucuronic acid moiety and the hydroxyl groups in the N-acetylglucosamine moiety are linked to the NH group of the maleimide group by a linker having a chain hydrocarbon group.

[0037] [Maleimide group introduction rate] The introduction rate of maleimide group in the hyaluronic acid derivative of this embodiment (hereinafter sometimes referred to simply as "maleimide group introduction rate") is preferably 1.0% or more, more preferably 3.0% or more, even more preferably 4.5% or more, even more preferably 5.0% or more, even more preferably 8.0% or more, particularly preferably 10% or more.The introduction rate of maleimide group in the hyaluronic acid derivative of this embodiment is preferably 25% or less, more preferably 20% or less.The introduction rate of maleimide group in the hyaluronic acid derivative of this embodiment, especially the introduction rate of steryl group, is within the above-mentioned range, so that the swelling degree can be sufficiently reduced.

[0038] The maleimide group introduction rate is １ It can be measured by H-NMR measurement. １The H-NMR spectrum is obtained by measuring a solution in which the hyaluronic acid derivative of this embodiment is dissolved in a heavy solvent 0.02N DC1 DMSO-d6 / D2O mixed solution (2N DC1 D2O:DMSO-d6 = 1:99). １ The peak (COCH) derived from the acetyl group of the glucosamine moiety of the hyaluronic acid derivative in the H-NMR spectrum ３ , 1.6 to 2.0 ppm; 3H) and the peak derived from the methyl group in the cholesteryl group (CH ３ The introduction rate of cholesteryl groups and maleimide groups into hyaluronic acid units is calculated using the integral value of the peak (-CH=CH-, 6.9 ppm; 2H) derived from the maleimide group (-CH=CH-, 6.9 ppm; 2H) and the integral value of the peak derived from the maleimide group, according to the formula shown below. Note that, since the peak derived from the cholesteryl group (5H) overlaps with the peak near 1.6 to 2.0 ppm, which includes the peak derived from the acetyl group of the glucosamine moiety, the value calculated by subtracting the integral value of the peak derived from the cholesteryl group methyl (0.7 ppm) multiplied by 5 / 3 from the integral value of the peak near 1.6 to 2.0 ppm (i.e., [[peak integral value (1.6-2.0 ppm)] - [peak integral value (0.7 ppm)] x 5 / 3] is used as the integral value (corrected value) of the acetyl group derived from hyaluronic acid to calculate the introduction rate. First, the cholesteryl group introduction rate (%) is calculated using the following formula:

[0039] [Cholesteryl group introduction rate (%)] = [Integrated value of peak derived from cholesteryl group (0.7 ppm)] / [Integrated value of peak derived from acetyl group of N-acetyl-D-glucosamine moiety (1.6 to 2.0 ppm, corrected value)] × 100 (%)

[0040] Furthermore, the maleimide group introduction rate (%) was calculated by multiplying the integral value of the peak derived from the maleimide group by the integral value of the peak derived from the cholesteryl group (COCH ３ , 1.6 ppm or more and 2.0 ppm or less, 3H) and the integrated value of the ion concentration, based on the following formula: Specifically, it can be measured according to the method described in the examples below.

[0041] [Maleimide group introduction rate (%)] = ([Peak integral value attributable to maleimide group] × 3) / ([Peak integral value attributable to cholesteryl group] × 2) × [Cholesteryl group introduction rate (%)]

[0042] The total of the steryl group introduction rate and maleimide group introduction rate in the hyaluronic acid derivative of this embodiment is preferably 55% or less, more preferably 45% or less, even more preferably less than 45%, even more preferably 40% or less, particularly preferably 35% or less.The total of the steryl group introduction rate and maleimide group introduction rate in the hyaluronic acid derivative of this embodiment is preferably 1.0% or more, more preferably 2.5% or more, even more preferably 5.0% or more.By the total unit number of the steryl group introduction rate and maleimide group introduction rate in the hyaluronic acid derivative being within the above-mentioned range, the volume change under physiological salt concentration (in vivo) after gelation can be made smaller, and when used as a DDS carrier, the crosslinking density can be increased, so that the sustained release period of the drug can be extended.

[0043] Specific examples of preferred hyaluronic acid derivatives include hyaluronic acid derivatives having one or more repeating units represented by the following general formula (I) (hereinafter, sometimes referred to as "repeating unit (I)") and one or more repeating units represented by the following general formula (II) (hereinafter, sometimes referred to as "repeating unit (II)"):

[0044]

[0045] (In general formula (I), R １ , R ２ , R ３ , and R ４ are each independently a hydrogen atom, C １－６ Alkyl, formyl and C １－６ alkylcarbonyl; Z represents a direct bond or a peptide linker consisting of any amino acid residues of 2 to 30; X １ is represented by the following formula: ｂ -R, -NR ｂ -COO-R, -NR ｂ -CO-R, -NR ｂ -CO-NRｃ -R, -COO-R, -O-COO-R, -SR, -CO-Y ａ -SR, -O-CO-Y ｂ -SR, -NR ｂ -CO-Y ｂ -SR, and -S-SR, a group selected from the group consisting of groups represented by R ａ , R ｂ and R ｃ are each independently a hydrogen atom, C １－２０ Alkyl, Amino C ２－２０ Alkyl and hydroxy C ２－２０ alkyl, where the alkyl portion of the group is selected from the group consisting of —O— and —NR ｆ - may be inserted with a group selected from the group consisting of; R ｆ is a hydrogen atom, C １－１２ Alkyl, Amino C ２－１２ Alkyl and hydroxy C ２－１２ alkyl, the alkyl portion of which may be interrupted by a group selected from the group consisting of -O- and -NH-; R is a steryl group; Y is C ２－３０ Alkylene, or -(CH ２ CH ２ O) ｍ -CH ２ CH ２ -, where the alkylene is -O-, -NR ｇ - and -S-S- may be inserted; R ｇ is a hydrogen atom, C １－２０ Alkyl, Amino C ２－２０ Alkyl and hydroxy C ２－２０ alkyl, the alkyl portion of which may be inserted with a group selected from the group consisting of -O- and -NH-; Y ａ is C １－５ is alkylene; Y ｂ is C ２－８ Alkylene or C ２－８ alkenylene; and m is an integer of 1 or more and 100 or less.

[0046]

[0047] (In general formula (II), R １ , R ２ , R ３ , and R ４ are each independently a hydrogen atom, C １－６ Alkyl, formyl and C １－６ alkylcarbonyl; Z represents a direct bond or a peptide linker consisting of any amino acid residues of 2 to 30; R ａ is a hydrogen atom, C １－２０ Alkyl, Amino C ２－２０ Alkyl and hydroxy C ２－２０ alkyl, where the alkyl portion of the group is selected from the group consisting of —O— and —NR ｆ - may be inserted with a group selected from the group consisting of; R ｆ is a hydrogen atom, C １－１２ Alkyl, Amino C ２－１２ Alkyl and hydroxy C ２－１２ alkyl, the alkyl portion of which may be inserted with a group selected from the group consisting of -O- and -NH-; X ２ is a maleimide group; Y is C ２－３０ Alkylene, or -(CH ２ CH ２ O) ｍ -CH ２ CH ２ -, where the alkylene is -O-, -NR ｇ - and -S-S- may be inserted; R ｇ is a hydrogen atom, C １－２０ Alkyl, Amino C ２－２０ Alkyl and hydroxy C ２－２０ alkyl, wherein the alkyl portion of the group may have inserted therein a group selected from the group consisting of -O- and -NH-; and m is an integer of 1 or more and 100 or less.

[0048] [Repeating unit (I)] The group "-Z-N(R ａ ) Y-X １」 is the following formula: -NH-(CH ２ ) ｍｚ -NH-R; -NH-(CH ２ ) ｍｚ -NH-COO-R; -NH-(CH ２ CH ２ O) ｍ -CH ２ CH ２ -NH-COO-R; -NH-(CH ２ ) ｍｚ -COO-R; -NH-(CH ２ CH ２ O) ｍ -CH ２ CH ２ -COO-R, -NH-(CH ２ ) ｍｚ -O-COO-R; -NH-(CH ２ CH ２ O) ｍ -CH ２ CH ２ [[ID=第46]]-O-COO-R, -NH-(CH<e000207>) ｍｚ -S-R; -NH-(CH ２ CH<00e0210>O) ｍ -CH ２ CH ２ -S-R; -NH-(CH ２ ) ｍｚ -O-CO-CH(R ８ )-CH ２ -S-R; -NH-(CH ２ ) ｍｚ -NHCO-CH(R ８ )-CH ２ -S-R; -NH-(CH ２ CH ２ ​​​​​​​​​​​​​​​​​​​​​​​​-SR; -NH-(CH ２ ) ｍｚ -S-S-R; and -Z-NR ａ -Y-NR ｂ -COO-R (where mz is an integer of 2 or more and 30 or less, and R ８ is a hydrogen atom or a methyl group, and R and m are as defined above in this specification.) The group includes a group selected from the group consisting of groups represented by: -NH-(CH ２ ) ｍｚ -NH-COO-R; -NH-(CH ２ CH ２ O) ｍ -CH ２ CH ２ -NH-COO-R; and -NH-(CH ２ ) ｍｚ Preferred are groups selected from the group consisting of -S-S-R 2 , where mz, R, and m are as previously defined herein.

[0049] (Z) In general formula (I), Z is preferably a direct bond. In another embodiment, when Z is a peptide linker, X １ Ha-NR ｂ In another embodiment, Z is preferably —NH—[CH(-Z ａ )-CONH] ｎ－１ -CH(-Z ａ )-CO-, where n is an integer of 2 to 30, and Z ａ are each independently H ２ N-CH(-Z ａ The peptide linker is attached to the carboxy group of the glucuronic acid moiety at the N-terminus and to the group —N(—R )—COOH at the C-terminus. ａ )-Y-X １Examples of amino acids that can be used as amino acid residues of the peptide linker include α-amino acids, such as natural (L-form) amino acids such as alanine, arginine, asparagine (Asn), aspartic acid, cysteine, glutamine, glutamic acid, glycine (Gly), histidine, isoleucine, leucine (Leu), lysine, methionine, phenylalanine (Phe), proline, serine, threonine, tryptophan, tyrosine, and valine, as well as D-forms thereof, and all α-amino acids, including synthetic amino acids, can be used. ａ Examples of the group include -CH ３ , H ２ NC(NH)NH(CH ２ ) ３ -, H ２ NCOCH ２ -, etc. Furthermore, n Z's may be the same or different. n is an integer of 2 or more and 30 or less, preferably 2 or more and 10 or less, and more preferably 2 or more and 4 or less. Preferred examples of peptide linkers include -Gly-Phe-Leu-Gly-, -Asn-Phe-Phe-, -Phe-Phe-, Phe-Gly-, etc.

[0050] (Y) In the general formula (I), Y is —(CH ２ ) ｎ１ - and - (CH ２ CH ２ O) ｍ１ -CH ２ CH ２ - (where n1 is an integer of 2 or more and 20 or less, preferably an integer of 2 or more and 15 or less, more preferably an integer of 2 or more and 12 or less, and even more preferably an integer of 2 or more and 6 or less; m1 is an integer of 1 or more and 4 or less) is preferred. Specifically, -(CH ２ ) ２ -, -(CH ２ ) ６ -, -(CH ２ ) ８ -, -(CH ２ ) １２ - or -(CH ２ CH ２ O) ２ -CH２ CH ２ In addition, from the viewpoint of realizing high solubility in pure water or at low salt concentrations and exhibiting high precipitate-forming ability at physiological salt concentrations, Y is preferably -(CH ２ ) ２ -, -(CH ２ ) ６ -, -(CH ２ ) ８ - and - (CH ２ ) １２ - is preferably a group selected from the group consisting of -(CH ２ ) ６ - is more preferable.

[0051] Y is, for example, —CH ２ CH ２ O-CH ２ CH ２ -S-S-CH ２ CH ２ O-CH ２ CH ２ -, -(CH ２ CH ２ O) ２ -CH ２ CH ２ -S-S-CH ２ CH ２ O-CH ２ CH ２ -, -CH ２ CH ２ O-CH ２ CH ２ -S-S-(CH ２ CH ２ O) ２ -CH ２ CH ２ -, -(CH ２ CH ２ O) ２ -CH ２ CH ２ -S-S-(CH ２ CH ２ O) ２ -CH ２ CH ２ - etc.

[0052] (Y ａ ) Y ａ As the group, -CH ２ - or -CH２ -CH ２ - is preferred.

[0053] (Y ｂ ) Y ｂ As the group, -CH ２ -CH ２ -, -CH(CH ３ ) CH ２ -, 2-butene-1,4-diyl, hepta-2,4-diene-1,6-diyl or octa-2,4,6-triene-1,8-diyl is preferred, and -CH ２ -CH ２ - or -CH(CH ３ ) CH ２ - is more preferable.

[0054] Group “-Z-N(R ａ ) Y-X １ " is exemplified by -NH-(CH ２ ) ２ -NH-CO-cholesteryl, -NH-(CH ２ ) ４ -NH-(CH ２ ) ３ -NH-(CH ２ ) ３ -NH-COO-cholesteryl, -NH-(CH ２ ) ３ -NH-(CH ２ ) ４ -NH-(CH ２ ) ３ -NH-COO-cholesteryl, -NH-(CH ２ ) ４ -NH-(CH ２ ) ３ -NH-COO-cholesteryl, -NH-(CH ２ ) ４ -N(-(CH ２ ) ３ -NH ２ )-COO-cholesteryl, -NH-(CH ２ ) ３ -NH-(CH ２ ) ４ -N(-(CH ２ ) ３ -NH ２ )-COO-cholesteryl, -NH-(CH ２ )３ -NH-(CH ２ ) ４ -N(-(CH ２ ) ３ -NH-(CH ２ ) ３ -NH ２ )-COO-cholesteryl, -NH-(CH ２ ) ３ -NH-(CH ２ ) ４ -N(-(CH ２ ) ３ -NH ２ )-CO-NH-cholesteryl, -NH-(CH ２ ) ３ -NH-(CH ２ ) ４ -N(-(CH ２ ) ３ -NH ２ )-CO-cholesteryl, -NH-(CH ２ ) ３ -NH-(CH ２ ) ４ -N(-(CH ２ ) ３ -NH ２ )-cholesteryl, etc. ａ ) Y-X １ " as R ａ , R ｂ and R ｃ is a hydrogen atom, and Y is a linear C ２－３０ Alkylene or -(CH ２ CH ２ O) ｍ -CH ２ CH ２ - and Y ａ But linear C １－５ alkylene, or Y ｂ But linear C ２－８ Alkylene or linear C ２－８ It is alkenylene.

[0055] The repeating unit (I) is particularly a repeating unit represented by the general formula (I), in which Z is a direct bond and R ａ is a hydrogen atom or C １－６ alkyl and Y is C ２－３０Alkylene or -(CH ２ CH ２ O) ｍ -CH ２ CH ２ - and X １ Ga-NR ｂ１ is a group represented by —COO—R, and R ｂ１ is a hydrogen atom or C １－６ Preferably, the repeating unit is an alkyl group and R is a steryl group; ａ is a hydrogen atom, and Y is C ２－３０ Alkylene or -(CH ２ CH ２ O) ｍ -CH ２ CH ２ - and X １ Ga-NR ｂ１ is a group represented by —COO—R, and R ｂ１ is a hydrogen atom or C １－６ More preferably, the repeat unit is an alkyl group and R is a steryl group; ａ is a hydrogen atom, and Y is C ２－３０ Alkylene or -(CH ２ CH ２ O) ｍ -CH ２ CH ２ - and X １ Ga-NR ｂ１ is a group represented by —COO—R, and R ｂ１ is a hydrogen atom or C １－６ More preferably, the repeat unit is an alkyl group and R is a cholesteryl group; ａ is a hydrogen atom, and —Y—X １ -ga- (CH ２ ) ２ -NH-COO-cholesteryl, -(CH ２ ) ４ -NH-COO-cholesteryl, -(CH ２ ) ５ -NH-COO-cholesteryl, -(CH ２ ) ６ -NH-COO-cholesteryl, -(CH ２ ) １２-NH-COO-cholesteryl, or -(CH ２ CH ２ O) ２ -CH ２ CH ２ Even more preferably, the repeat unit is —NH—COO-cholesteryl; is a direct bond, and R ａ is a hydrogen atom, and —Y—X １ -ga- (CH ２ ) ２ -NH-COO-cholesteryl, -(CH ２ ) ４ -NH-COO-cholesteryl, -(CH ２ ) ５ -NH-COO-cholesteryl, or -(CH ２ ) ６ The repeating unit is particularly preferably --NH--COO-cholesteryl.

[0056] The repeating unit (II) is particularly a repeating unit represented by the general formula (II), in which Z is a direct bond and R ａ is a hydrogen atom or C １－６ alkyl and Y is C ２－３０ Alkylene or -(CH ２ CH ２ O) ｍ -CH ２ CH ２ - and X ２ is a maleimide group; Z is a direct bond, and R ａ is a hydrogen atom or C １－６ alkyl and Y is C ２－３０ is alkylene, and X ２ is a maleimide group; Z is a direct bond, and R ａ is a hydrogen atom, and Y is C ２－３０ is alkylene, and X ２ is a maleimide group; Z is a direct bond, and R ａ is a hydrogen atom, and Y is C ２－１０ is alkylene, and X ２ It is more preferable that the repeating unit is a maleimide group.

[0057] In the hyaluronic acid derivative of this embodiment, the two or more repeating units (I) contained in one molecule of the hyaluronic acid derivative may all be the same repeating unit, or may be two or more different repeating units.

[0058] In the hyaluronic acid derivative of this embodiment, the two or more repeating units (II) contained in one molecule of the hyaluronic acid derivative may all be the same repeating unit, or may be two or more different repeating units.

[0059] The total number of repeating units of the hyaluronic acid derivative of this embodiment is preferably 40% or less, more preferably 35% or less, and even more preferably 30% or less.The total number of repeating units of the hyaluronic acid derivative of this embodiment is preferably 1.0% or more, more preferably 2.5% or more, and even more preferably 5.0% or more ... 1.0% or more, more preferably 2.5% or more, and even more preferably 5.0% or more.The total number of repeating units of the hyaluronic acid derivative of this embodiment is 1.0% or more, more preferably 2.5% or more, and even more preferably 5.0% or more.By making the total number of repeating units (I) and repeating units (II) within the above-mentioned range, the volume change after gelation can be reduced, and when used as DDS carrier, the encapsulation rate of drug can be increased.

[0060] [Weight-average molecular weight Mw] The weight-average molecular weight of the hyaluronic acid derivative of the present embodiment is not particularly limited, and is, for example, preferably 1,000 (1k) or more and 1,000,000 (1,000k) or less, more preferably 5k or more and 300k or less, even more preferably 6k or more and 200k or less, and particularly preferably 30k or more and 130k or less.

[0061] The weight-average molecular weight of the hyaluronic acid derivative of this embodiment is preferably below 250kDa, more preferably below 200kDa, and even more preferably below 150kDa, because it can easily reduce the volume change after gelation and the particle size of the hyaluronic acid crosslinked gel obtained by crosslinking can be easily adjusted within a suitable range.In addition, from the viewpoint of the gelation of hyaluronic acid derivative, it is preferably above 4kDa, more preferably above 20kDa, more preferably above 30kDa, and even more preferably above 40kDa.The weight-average molecular weight of the hyaluronic acid derivative of this embodiment is particularly preferably above 50kDa, and more preferably above 90kDa.The molecular weight of hyaluronic acid derivative can generally be adjusted by using the raw material with corresponding molecular weight.

[0062] The weight-average molecular weight Mw and number-average molecular weight Mn of a hyaluronic acid derivative can generally be adjusted by using raw materials having the corresponding molecular weights. The weight-average molecular weight Mw and number-average molecular weight Mn of a hyaluronic acid derivative can be measured, for example, by size exclusion chromatography multi-angle light scattering detector (SEC-MALS). Specifically, the weight-average molecular weight Mw and number-average molecular weight Mn of a hyaluronic acid derivative can be measured according to the method described in the examples below.

[0063] [Mean particle diameter (nm) of nanoparticles] The hyaluronic acid derivative of this embodiment forms nanoparticles in aqueous solution.The size of the nanoparticles that the hyaluronic acid derivative of this embodiment forms is not particularly limited, but for example, the mean particle diameter is preferably below 500nm, more preferably below 450nm, more preferably below 400nm, more preferably below 300nm, particularly preferably below 250nm.The mean particle diameter of the nanoparticles that the hyaluronic acid derivative of this embodiment forms is preferably above 10nm, more preferably above 20nm, more preferably above 50nm.

[0064] In the present specification, "the average particle size of nanoparticles of hyaluronic acid derivative" refers to the z-average particle size measured by dynamic light scattering.The z-average particle size measured by dynamic light scattering can be measured, for example, by dissolving hyaluronic acid derivative in phosphate buffer solution (10 mM) to a concentration of 1 mg / mL.

[0065] The nanoparticles formed by the hyaluronic acid derivative of this embodiment are hollow, and can contain various substances inside.For example, by dissolving the target substance to be contained together with hyaluronic acid derivative in aqueous solution, the nanoparticles are formed with the target substance contained inside, and the shell is made of hyaluronic acid derivative.The nanoparticles of the hyaluronic acid derivative of this embodiment can well contain even relatively large substances or poorly soluble substances in water.From these points, the hyaluronic acid derivative of this embodiment is good as a drug delivery system substrate, and is particularly preferred as a sustained-release drug delivery system substrate.

[0066] The hyaluronic acid derivative of this embodiment can obtain a crosslinked gel by reacting with a crosslinking agent while encapsulating a drug and without denaturing the drug.

[0067] [Production method of hyaluronic acid derivative] The hyaluronic acid derivative of the present embodiment can be obtained by, for example, converting the carboxyl group of glucuronic acid into amide, introducing steryl group directly or via linker into at least part of it, and introducing maleimide group directly or via linker into other part of it.By adjusting the amount of the compound having steryl group that is reacted with raw material hyaluronic acid or its derivative, it can control the introduction rate of steryl group, and by adjusting the amount of the compound having maleimide group that is reacted, it can control the introduction rate of maleimide group.It can be made to react raw material hyaluronic acid with a compound having steryl group, and the resulting reactant can be reacted with a compound having maleimide group, or it can be made to react raw material hyaluronic acid with a compound having maleimide group, and the resulting reactant can be reacted with a compound having steryl group, or it can be made to add raw material hyaluronic acid with a compound having maleimide group and a compound having steryl group together into the reaction system and react. Methods for introducing maleimide groups or steryl groups into the raw material hyaluronic acid include, for example, the methods described in JP-A-2021-123597, JP-A-2022-013861, JP-A-2022-044579, etc., which can be carried out by appropriately modifying them.

[0068] A specific example of a method for converting the carboxy group of glucuronic acid to an amide and introducing a steryl group or maleimide group is to ion-exchange the starting material hyaluronic acid or a derivative thereof with a tetraalkylammonium salt (e.g., tetrabutylammonium (TBA) salt), and then react the hyaluronic acid salt with an amine having a steryl group (particularly a cholesteryl group) introduced therein in a solvent in the presence of a suitable condensing agent.

[0069] The condensing agent that can be used in the above reaction is not particularly limited, and examples thereof include 4-(4,6-dimethoxy-1,3,5-triazine)-4-methylmorpholinium (DMT-MM), N,N'-carbonyldiimidazole (CDI), N,N'-dicyclohexylcarbodiimide (DCC), N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), 2-benzotriazole-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HODhbt), benzotriazole-1-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBOP), and benzotriazole-1-yl-oxy-tris(dimethylamino)phosphonium. Examples include hexafluorophosphate (BOP), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and N-hydroxysuccinimide (NHS).

[0070] In particular, although not limited to, DMT-MM is preferred in that the reaction proceeds highly efficiently even in a mixed solvent of water and an organic solvent. Furthermore, by using DMT-MM as a condensing agent, in a system in which a large number of hydroxy groups coexist, it is possible to highly selectively form an amide bond between an amino group and a carboxy group while suppressing the formation of an ester bond. The use of this condensing agent can, for example, prevent the reaction of the alcohol solvent with the carboxy group of the hyaluronic acid moiety, or prevent the formation of an undesired crosslink due to intramolecular or intermolecular bonding between the carboxy group and the hydroxy group simultaneously present in the hyaluronic acid moiety.

[0071] Examples of solvents used in the steryl group introduction reaction include water, DMSO, methanol, ethanol, propanol, butanol, isopropanol, polyhydric alcohols, acetonitrile, DMF, THF, dichloromethane, chloroform, hexane, diethyl ether, ethyl acetate, and mixed solvents thereof. The polyhydric alcohol may be a dihydric alcohol or a trihydric alcohol. Examples of dihydric alcohols include ethylene glycol, diethylene glycol, propylene glycol, dipropylene glycol, neopentyl glycol, 1,4-butanediol, and 1,6-hexanediol. Examples of trihydric alcohols include glycerin and trimethylolpropane.

[0072] In the steryl group introduction reaction, the pH of the reaction system is preferably under acidic conditions. Under basic conditions, succinimide groups may be generated by deactivation of the maleimide groups.

[0073] Alternatively, the starting hyaluronic acid or a derivative thereof may be ion-exchanged with a tetraalkylammonium salt (e.g., tetrabutylammonium (TBA) salt), and the hyaluronic acid salt may be reacted with a spacer moiety in a solvent in the presence of a suitable condensing agent (protection and deprotection reactions may be carried out as necessary), converting the carboxy group (-COOH) of the starting hyaluronic acid or a derivative thereof, followed by reaction with a suitable reagent. Examples of combinations of groups derived from carboxy groups and reaction reagents are shown below. -CONR ａ -Y-NR ｂ H + Hal-R; -CONR ａ -Y-NR ｂ H + Hal-COOR; -CONR ａ -Y-NR ｂ H + HOCO-R; -CONR ａ -Y-NR ｂ H + Hal-CO-R; -CONR ａ -Y-NR ｂ -COOH + HNR ｃ -R; -CONR ａ -Y-NR ｂ -CO-NR ｃH + Hal-R; -CONR ａ -Y-NR ｂ H + HOCO-NR ｃ -R; -CONR ａ -Y-NR ｂ H + Hal-CO-NR ｃ -R; -CONR ａ -Y-COOH + HO-R; -CONR ａ -Y-OH + Hal-COO-R; -CONR ａ -Y-OCOOH + HO-R; -CONR ａ -Y-OCOOH + Hal-R; -CONR ａ -Y-OCO-Hal + HO-R; -CONR ａ -Y-SH + Hal-R; -CONR ａ -Y-Hal + HS-R; -CONR ａ -Y-CO-Y ａ -Hal + HS-R; -CONR ａ -Y-CO-Y ａ -SH + Hal-R; -CONR ａ -Y-O-CO-CH=CH ２ + HS-R; -CONR ａ -Y-NR ｂ -CO-CH(CH ３ ) = CH ２ + HS-R; -CONR ａ -Y-SH + HS-R; -COZ-OH + HNR ａ -Y-NR ｂ -COO-R; -COZ-NR ａ -Y-NR ｂ H + Hal-COO-R (wherein, R ａ , R ｂ , R ｃ , Y, Y ａ , Y ｂ and Z are as previously defined herein, and Hal represents a halogen atom selected from the group consisting of fluorine, chlorine, bromine and iodine.

[0074] Examples of reaction modes include dehydrohalogenation reactions, condensation reactions, dehydration reactions, nucleophilic addition reactions such as Michael addition, and oxidative disulfide formation reactions. These are well-known reactions, and can be appropriately selected by those skilled in the art and carried out under preferred reaction conditions. When the conversion product or reactant has a carboxy group, it may be converted into an N-hydroxysuccinimide (hereinafter also referred to as "NHS") ester and then reacted.

[0075] Another method is to react 2-aminoethyl 2-pyridyl disulfide with the carboxyl group of the starting hyaluronic acid or its derivative to prepare a hyaluronic acid derivative into which a spacer having a mercapto group modified with a leaving group has been introduced at the end, and then to subject this to a nucleophilic substitution reaction with thiocholesterol to form a disulfide bond.

[0076] Another example is a method in which a hyaluronic acid or derivative thereof in which a part of a spacer is introduced into the carboxy group and a part of a spacer is introduced into the steryl group are prepared and then reacted. Some specific examples have been described above, but when -S-S- is inserted into Y, another example is a method in which a hyaluronic acid derivative in which a spacer having a terminal mercapto group is introduced into the carboxy group of hyaluronic acid and a steryl group in which a spacer having a terminal mercapto group is introduced are prepared, and then these are oxidatively reacted to form a disulfide bond. In this case, one mercapto group can be reacted with 2-mercaptopyridine to form a disulfide, and then substituted with the other mercapto group.

[0077] After preparing the hyaluronic acid derivative, other substituents may be further introduced. For example, 0.1% to 95.0%, preferably 10% to 60%, of the carboxy groups in the hyaluronic acid derivative may be replaced with -CO-X ｚ , [where X ｚ is the following group: -NH-(CH ２ ) ｐ１ —O—CO—C(R １７ ) = CH ２ -NH-(CH ２ ) ｐ１ -O-CO-CH(R １７ )-CH２ -S-CH ２ -CH(OH)-CH(OH)-CH ２ -SH; -NH-(CH ２ ) ｐ１ -SH; -NH-(CH ２ ) ｐ１ -NH-CO-C(R １７ )=CH ２ ; -NH-(CH ２ ) ｐ１ -NH-C(=NH)-(CH ２ ) ３ -SH; -NH-(CH ２ ) ｐ１ -NH-CO-(CH ２ ) ｒ -SH; -NH-(CH ２ ) ｐ１ -NH-CO-CH(R １７ )-CH ２ -S-CH ２ -CH(OH)-CH(OH)-CH ２ -SH; -NH-(CH ２ ) ｐ１ -NH-CO-CH(NH ２ )-CH ２ -SH; -NH-(CH ２ ) ｐ１ -NH-CO-CH(NH ２ )-(CH ２ ) ２ -SH; -NH-NH-CO-(CH ２ ) ４ -CO-NH-NH-C(=NH)-(CH ２ ) ３ -SH; -NH-(CH ２ -CH ２ -O) ｑ -CH ２ -CH ２ -O-CO-C(R １７ )=CH ２ ; -NH-(CH ２ -CH ２ -O) ｑ -CH ２ -CH ２ -O-CO-CH(R １７ )-CH ２ -S-CH２ -CH(OH)-CH(OH)-CH ２ -SH; -NH-(CH ２ -CH ２ -O) ｑ -CH ２ -CH ２ -SH; -NH-(CH ２ -CH ２ -O) ｑ -CH ２ -CH ２ -NH-CO-C(R １７ )=CH ２ ; -NH-(CH ２ -CH ２ -O) ｑ -CH ２ -CH ２ -NH-C(=NH)-(CH ２ ) ３ -SH; -NH-(CH ２ -CH ２ -O) ｑ -CH ２ -CH ２ -NH-CO-(CH ２ ) ｒ -SH; -NH-(CH ２ -CH ２ -O) ｑ -CH ２ -CH ２ -NH-CO-CH(R １７ )-CH ２ -S-CH ２ -CH(OH)-CH(OH)-CH ２ -SH; -NH-(CH ２ -CH ２ -O) ｑ -CH ２ -CH ２ -NH-CO-CH(NH ２ )-CH ２ -SH; -NH-(CH ２ -CH ２ -O) ｑ -CH ２ -CH ２ -NH-CO-CH(NH ２ )-(CH ２ ) ２ -SH; -NH-CH(CO ２H)-(CH ２ )-SH; -NH-CH(CO ２ H)-(CH ２ ) ２ -SH; and -NH-CH(CO ２ H)-(CH ２ ) ２ -CONH-CH(CONH-CH ２ -CO ２ H) —CH ２ -SH (where R １７ is a hydrogen atom or C １－６ wherein p1 is an alkyl group selected from the group consisting of: p1 is an integer of 2 or more and 10 or less; q is an integer of 1 or more and 200 or less; and r is an integer of 1 or more and 3 or less. By converting the hydroxyl group into a hydroxyl group selected from the group consisting of: p1 is an alkyl group selected from the group consisting of: p1 is an integer of 2 or more and 10 or less; q is an integer of 1 or more and 200 or less; and r is an integer of 1 or more and 3 or less.

[0078] <<Hyaluronic acid crosslinked gel>> The hyaluronic acid derivative of the present embodiment can be gelled by chemical crosslinking.That is, the hyaluronic acid crosslinked gel of the present embodiment is the gel obtained by gelling the hyaluronic acid derivative of the present embodiment by chemical crosslinking using a crosslinking agent that has two or more crosslinking groups per molecule.

[0079] The hyaluronic acid crosslinked gel of the present embodiment can be obtained by dissolving the hyaluronic acid derivative of the present embodiment and crosslinking agent in solvent, and then gelling the liquid composition by chemical crosslinking reaction.In chemical crosslinking reaction, reaction conditions such as hyaluronic acid derivative concentration, type of crosslinking agent, crosslinking agent concentration, type of solvent, solvent pH, salt concentration, temperature, time, etc. can be appropriately determined.For example, by increasing the concentration of crosslinking agent during chemical crosslinking and the introduction rate of the group that can form crosslinks into hyaluronic acid derivative or crosslinking agent, the crosslinking density of the gel that is produced can be increased.

[0080] In the process of gelling the hyaluronic acid derivative of the present embodiment, when using the one that has the group (crosslinkable group) that can form crosslinks at both ends, it is preferable to add the concentration that this group can participate in crosslinking reaction quickly without excess or deficiency.For example, because maleimide group and thiol group are bonded by condensation reaction, when the compound that has two or more thiol (SH) groups per molecule is used as crosslinking agent to crosslink by Michael addition reaction, maleimide group:SH group=preferably 3:1~1:3, particularly preferably 2:1~1:2.

[0081] As the crosslinking agent, there is no particular limitation as long as it is a compound that has two or more crosslinkable groups per molecule.Preferably, the hyaluronic acid crosslinked gel of this embodiment is a gel-like substance that is chemically crosslinked with the hyaluronic acid derivative of this embodiment, using a compound that has two or more thiol groups per molecule as a crosslinking agent.Examples of the compound that has two or more thiol groups per molecule include DTT (dithiothreitol), SH-containing PEG compounds that have thiol groups at both ends of single-chain PEG, and SH-containing PEG compounds that are branched PEG chains with 3 to 8 arms and have thiol groups at the end of each arm.In these SH-containing PEG compounds, the molecular weight can be adjusted by adjusting the polymerization degree of PEG.

[0082] In gelation, the higher the solid content concentration of the liquid composition that hyaluronic acid derivative and crosslinking agent are dissolved in solvent, the higher the osmotic pressure becomes, and the more easily swells during gelation.Therefore, under the same other reaction conditions, the higher the concentration of hyaluronic acid derivative, the higher the swelling degree of the crosslinked gel obtained tends to be.Similarly, under the same other reaction conditions, the higher the concentration of crosslinking agent, the higher the swelling degree of the crosslinked gel obtained tends to be.In addition, under the same number of crosslinking groups per molecule, the crosslinking agent with a larger molecular weight increases the mass of the crosslinking agent that is required for crosslinking to add the molar amount of thiol group to hyaluronic acid derivative, and therefore the swelling degree tends to be higher.

[0083] The crosslinking agent used in the production of the hyaluronic acid crosslinked gel of this embodiment includes a compound containing two or more mercapto groups that react with unsaturated bonds by nucleophilic addition in the same molecule, such as polyethylene glycol dithiol and a peptide containing two or more cysteines.

[0084] The crosslinking agent used in the production of the hyaluronic acid crosslinked gel of this embodiment is preferably a 2- to 8-armed PEG compound having an SH group at the end of each arm, more preferably a 3- to 6-armed PEG compound having an SH group at the end of each arm, even more preferably a 4-armed PEG compound having an SH group at the end of each arm, even more preferably a 4-armed PEG compound having an SH group at the end of each arm and a weight-average molecular weight of 2k to 40k, and particularly preferably a 4-armed PEG compound having an SH group at the end of each arm and a weight-average molecular weight of 5k to 20k.

[0085] The solvent used in the step of gelling the hyaluronic acid derivative is preferably one that can sufficiently dissolve the hyaluronic acid derivative and the crosslinking agent, and is not particularly limited, but it is preferable to use water, dimethyl sulfoxide (DMSO), dimethylacetamide (DMAc), dimethylformamide (DMF), N-methylpyrrolidone (NMP), and a mixed solvent selected from these. It is also possible to use a mixture of an organic solvent that is miscible with these solvents. Although not particularly limited, examples of miscible organic solvents include methanol, ethanol, propanol, isopropanol, butanol, polyhydric alcohols, acetone, acetonitrile, etc. Examples of polyhydric alcohols include the same as those exemplified above, and among them, ethylene glycol is preferred.

[0086] Furthermore, it is preferable to add a basic compound to improve the stability of the protein or peptide during the crosslinking reaction and to increase the reaction rate. The basic compound to be used is not particularly limited, but examples thereof include carbonates such as sodium carbonate and sodium bicarbonate, hydroxides such as sodium hydroxide and sodium hydroxide, aqueous ammonia, and amines such as pyridine, triethylamine, ethylenediamine, ethanolamine, diethanolamine, and triethanolamine, preferably amines, and more preferably triethanolamine.

[0087] Hyaluronic acid derivatives form nanoparticles in aqueous solution as targeting carriers, so that by crosslinking under dilute conditions, they can form nano-sized particle gel, and can be used as blood sustained release carriers.Dilute conditions are 10mg / mL or less, preferably 5mg / mL or less, more preferably 1mg / mL or less.On the other hand, by crosslinking under high concentration conditions, they can form bulky gel, in which particles are crosslinked with each other.This is useful as a subcutaneous sustained release carrier.High concentration conditions are 5mg / mL or more, preferably 20mg / mL or more, more preferably 30mg / mL.

[0088] The gelation process of the hyaluronic acid derivative may be carried out in bulk, or in a discontinuous phase such as emulsion or spray droplets. For example, when carried out in a W / O emulsion, the aqueous phase containing the hyaluronic acid derivative, crosslinking agent, etc., is emulsified in a water-immiscible solvent, and the gelation reaction is carried out. The water-immiscible solvent is not particularly limited, but examples thereof include hexane, chloroform, dichloromethane, ethyl acetate, medium-chain triglyceride (MCT), liquid paraffin, soybean oil, etc. A surfactant may be added to stabilize the emulsion. Alternatively, the process may be carried out in a solvent capable of desolvation, such as supercritical carbon dioxide or PEG. In this case, the aqueous phase or organic solvent phase containing the hyaluronic acid derivative, crosslinking agent, etc., is emulsified and dispersed in the aforementioned solvent, thereby concentrating the polymer due to desolvation (solvent diffusion), thereby obtaining a gel with a higher crosslink density.

[0089] The process of gelling hyaluronic acid derivative and thereafter, can be carried out the operation of stopping crosslinking reaction and the operation of deactivating or washing remaining crosslinking group.The crosslinking group that is not involved in reaction, the group that only one end of crosslinking agent is bonded to, remaining crosslinking agent, etc. are preferably removed from the viewpoint of safety, stability during storage, side reaction with encapsulated drug, etc.Not particularly limited, for example, when there is remaining unreacted crosslinking agent, it can be removed by washing with excess water, etc.

[0090] A pulverization step may be carried out after the step of gelling the hyaluronic acid derivative. Pulverization methods include pulverization using a pestle and mortar and pulverization using a mill, with pulverization using a mill being preferred. Examples of mill pulverization devices include rotating disk-type pulverizers such as a centrifugal pulverizer (manufactured by Nippon Seiki Seisakusho) and an impact mill (manufactured by Dalton Co., Ltd.), screen mill pulverizers such as an atomizer (manufactured by Tokyo Atomizer Mfg. Co., Ltd.), a sample mill (manufactured by Tokyo Atomizer Mfg. Co., Ltd.), a bantam mill (manufactured by Tokyo Atomizer Mfg. Co., Ltd.), and an SK mill (manufactured by Tokken Co., Ltd.), jet pulverizers such as an ultra-micro laboratory jet mill (A-O jet mill, Seishin Enterprise Co., Ltd.), and a Rinrex mill (Liquid Gas Co., Ltd.) capable of pulverization at ultra-low temperatures, with SK mill and Rinrex mill being preferred.

[0091] The hyaluronic acid crosslinked gel of the present embodiment can be the gel-like substance obtained by chemically crosslinking the hyaluronic acid derivative of the present embodiment with crosslinking agent, and can also be the dried substance obtained by drying it.Drying method can be, for example, freeze-drying, ventilation drying, drying in thermostatic oven, drying under reduced pressure, hot air circulation drying etc.Air velocity, drying time, temperature, pressure etc. are suitably selected within the range that the gel of hyaluronic acid derivative does not decompose or change in quality.

[0092] The dried product of the hyaluronic acid crosslinked gel of this embodiment and the hyaluronic acid crosslinked gel of this embodiment have a porous structure in both the dry and swollen states. For example, this allows relatively large components such as cells to be supported, and the dried product can be suitably used as a substrate for cells and tissues used in regenerative medicine, etc. For example, cells for transplantation can be attached and cultured within the dried product, and then transplanted into the target tissue. Furthermore, the dried product and crosslinked gel also function as a scaffold for cell growth in in situ regeneration therapy. For example, by embedding the dried product in tissue within a living body, cells that have migrated from surrounding tissues can use the dried product as a scaffold to attach.

[0093] The dried product of the hyaluronic acid crosslinked gel of this embodiment and the hyaluronic acid crosslinked gel of this embodiment can control the adhesiveness of cells by changing the introduction rate of the cholesteryl derivative.

[0094] <<Pharmaceutical composition>> The pharmaceutical composition of this embodiment comprises the hyaluronic acid derivative.In the pharmaceutical composition of this embodiment, the hyaluronic acid derivative mainly functions as the carrier of active ingredient.When this pharmaceutical composition is administered to living body, the active ingredient such as drug is gradually released from the carrier that comprises hyaluronic acid derivative, and can be expected to have good sustained release.

[0095] <Active Ingredient> The active ingredient contained in the pharmaceutical composition of this embodiment is not particularly limited as long as it is a drug for humans or animals (a substance administered for the diagnosis, treatment, or prevention of a disease) or a substance used as an active ingredient thereof. The active ingredient may be a water-soluble substance or a poorly soluble substance. Examples of the active ingredient include proteins, peptides, polysaccharides, oligosaccharides, nucleic acids, oligonucleotides, low-molecular-weight compounds, cells, etc. It may also be a protein-nucleic acid complex, such as a Cas9 / gRNA complex.

[0096] Examples of the pharmaceutical composition of this embodiment include nanoparticles formed from the hyaluronic acid of this embodiment and encapsulating an active ingredient, a dried product of the hyaluronic acid cross-linked gel of this embodiment and a hyaluronic acid cross-linked gel to which transplant cells have been engrafted, a hyaluronic acid cross-linked gel containing a poorly soluble drug or the like, and a dried product thereof.

[0097] <Form> The pharmaceutical composition of this embodiment may be a dispersible microparticle solution, a precipitated suspension, or a freeze-dried product. In addition, in the case of a freeze-dried product, it may be a precipitated sustained-release preparation in which a physician prepares the administration solution immediately before administration by adding an isotonic solution such as physiological saline. In this case, it is considered to be suitable for pharmaceutical compositions containing an active ingredient that is unstable in a solution state.

[0098] When the pharmaceutical composition of this embodiment is a dispersible microparticle solution or a precipitated suspension, the concentration of the hyaluronic acid derivative in the pharmaceutical composition is preferably 1 mg / mL or more and 200 mg / mL or less, more preferably 4 mg / mL or more and 100 mg / mL or less, even more preferably 4 mg / mL or more and 50 mg / mL or less, and particularly preferably 4 mg / mL or more and 12 mg / mL or less.

[0099] The pharmaceutical composition of this embodiment is not limited to the forms already mentioned, and may be in the form of nanoparticles, microparticles, solutions, emulsions, suspensions, gels, micelles, implants, powders, or films. Powders may be produced by grinding solids obtained by freeze-drying or spray-drying, or by drying precipitates.

[0100] The pharmaceutical composition of this embodiment may be administered orally, parenterally, intranasally, intravaginally, intraocularly, subcutaneously, intravenously, intramuscularly, intradermally, intraperitoneally, intracerebrally, or intraoral. Furthermore, the pharmaceutical composition of this embodiment is not limited to an injection, and may also be a patch preparation, a microneedle preparation, a topical ointment, an eye drop, a spray, an inhalant, or the like.

[0101] The present invention will be described in detail below with reference to examples, but it is not intended that the scope of the present invention be limited to these examples.

[0102] The methods for measuring and evaluating the physical properties of the hyaluronic acid derivatives prepared in the Examples and Comparative Examples are as follows: Unless otherwise specified, all experiments were carried out in triplicate.

[0103] < １ H NMR measurement> １ H NMR measurements were carried out using a JEOL JNM-A400 spectrometer (JEOL) equipped with a 400 MHz-NMR instrument (JNM-ECS400, JEOL) using 0.02 N DC1 / d-DMSO as a solvent unless otherwise specified.

[0104] Size-exclusion chromatography combined with multi-angle laser scattering. HA derivatives were analyzed by SEC-MALS using a Wyatt Dawn NEON multi-angle light scattering detector and an Optilab refractive index monitor equipped with an isocratic HPLC system (Waters). The HA derivatives were first diluted to 1.0 mg / mL in SEC-MALS buffer (10 mM phosphate buffer, pH 7.4). The separation step was performed in SEC-MALS buffer using a G4000SWXL column (Tosoh) at a flow rate of 0.5 mL / min. Data analysis was performed using the refractive index concentration coefficient dn / dc of hyaluronic acid (0.153, literature value) and Astra software (Wyatt Technology).

[0105] <Measuring average particle size by dynamic light scattering (DLS)> The HA derivatives were characterized by DLS analysis using an ELSZ-2000 (Otsuka Electronics Co., Ltd.) in a solution of the same solvent as SEC (10 mM phosphate buffer, pH 7.4). The measured autocorrelation function was performed by the cumulant method. The hydrodynamic diameter of the HA derivatives was analyzed using the Stokes-Einstein equation.

[0106] <Inverted Vial Test (HA Derivative Cross-Linking Gelling Test)> HA derivative cross-linked gels were prepared by cross-linking via a Michael addition reaction between maleimide and thiol groups. Unless otherwise specified, the cross-linking agent used for gelation was 4arm-PEGSH (molecular weight = 1 × 10), a PEG compound with SH groups at the ends of four arms. ４ g / mol, NOF Corporation). The gelling ability of the HA derivatives was analyzed based on the state of the vials when inverted. Specifically, the HA derivatives and PEGSH were completely dissolved separately in a solvent (10 mM phosphate buffer, pH 7.4), then both were poured into a single vial while cooling to 5°C and incubated at 37°C for 30 minutes. The vials were inverted over time from the start of incubation to check whether the samples flowed or dropped. Samples that did not flow within 5 or 30 minutes of incubation were classified as gels, and samples that continued to flow even after 30 minutes of incubation were classified as sols. All samples were classified as either gels or sols.

[0107] <Preparation of HA derivative crosslinked gel> Two precursor solutions (HA derivative phosphate buffer solution and PEGSH phosphate buffer solution) were poured into a disk-shaped silicone rubber mold (diameter 6 mm, depth 1 mm) placed on a polytetrafluoroethylene membrane while cooling to 5°C. Next, the mold was covered with a silicone cover sheet and incubated at 37°C for 30 minutes. After incubation, the sample was transferred to a glass vial. The molar ratio of maleimide groups to thiol groups was 1.1:1.

[0108] <Swelling of Hydrogels> HA derivative cross-linked gels (φ = 6 mm, thickness = 1.0 mm) were placed at the bottom of pre-weighed replicate glass vials (n = 4). The initial weight of the vial containing the HA derivative cross-linked gel was measured as the initial mass. Then, 1 mL of PBS was added and incubated at 37°C. At 1, 12, and 24 hours, the buffer solution was carefully removed from each vial, and the vial was weighed to measure the swollen mass. Fresh PBS was then added to replace the removed solution. The mass of the original hydrogel (W0) and the mass of the swollen hydrogel (WS) were calculated by subtracting the mass of the empty vial from the total mass. The mass swelling ratio (Q) of the HA derivative cross-linked gel was calculated by dividing the swollen mass by the initial mass.

[0109]

[0110] <Experiment for Encapsulating and Released Proteins from HA Derivative Cross-Linked Gels> HA derivative cross-linked gels (φ = 6 mm, thickness = 1.0 mm) were prepared in the same manner as described above and immersed in PBS. After reaching equilibrium swelling, the HA derivative cross-linked gels were immersed in a PBS solution of FITC-insulin (100 μg / mL) at 37°C. Subsequently, samples (200 μL of solution) were collected from specific points on the gel, and their fluorescence intensity was measured using a DeNovix DS-11 FX+ spectrophotometer fluorometer ("Fluoroprotein" module). The loading efficiency of FITC-insulin was estimated from the decrease in fluorescence intensity.

[0111] In vitro release of FITC-insulin from HA derivatives in the presence of serum was also evaluated. FITC-insulin-complexed HA derivative cross-linked gels (φ = 6 mm, thickness = 1 mm) were immersed in 1 mL of 10% fetal bovine serum (FBS)-containing PBS (pH 7.4) at 37°C. Samples (200 μL of the gel supernatant) were then collected at specific times, and the fluorescence intensity and FITC-insulin loading efficiency were measured as described above.

[0112] Example 1 An HA derivative (HA-C6-Chol-Male, sometimes referred to as "HAMICH") was prepared in which a steryl group and a maleimide group were introduced into at least some of the carboxyl groups in the glucuronic acid moiety of hyaluronic acid.

[0113]

[0114] Cholesteryl 6-aminohexylcarbamate hydrochloride (Chol-C6 hydrochloride) and tetrabutylammonium salt of hyaluronic acid (HA-TBA) were synthesized as described in Examples 1 and 2 of Patent Document 1. Specifically, HA-TBA was obtained by converting HA from the starting material, sodium hyaluronic acid (HA-Na) (average molecular weight = 120 kDa, purchased from Bloomage Biotechnology Japan), using a cation exchange resin (Dowex (registered trademark) 50WX-8-400, manufactured by Sigma-Aldrich), and then reacting the resulting HA with N-(5-aminopentyl)maleimide hydrochloride using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (manufactured by Kokusan Chemical Co., Ltd.) as a condensing agent. HA-TBA was completely dissolved in DMSO (1% w / v), and then N-(5-aminopentyl)maleimide dissolved in DMSO was added and stirred at room temperature for 5 minutes. Subsequently, DMT-MM was added to the mixture and stirred overnight at room temperature. The feed molar ratio was 100:20:24 (glucuronic acid of HA:DMT-MM:N-(5-aminopentyl)maleimide hydrochloride, x = 20). Cholesteryl-6-aminohexylcarbamate was then reacted using the same procedure. The feed molar ratio was 100:x:1.2x (glucuronic acid of HA:DMT-MM:cholesteryl-6-aminohexylcarbamate, x = 1.2, 5, 10, 16, 22, 31, or 44). The resulting reaction solution was dialyzed (Spectrapore 4, molecular weight cutoff (MWCO): 12 kDa to 14 kDa, manufactured by Spectrum Laboratories) against a DMSO solution, a 0.15 M NaCl aqueous solution, and ultrapure water in that order, and the resulting dialyzed solution was freeze-dried to obtain the target products (HA-C6-Chol-1%-Male 15% to HA-C6-Chol-40%-Male 15%) listed in Table 1 as white solids.

[0115] As a control, a hyaluronic acid derivative modified with maleimide groups but without Chol hydrochloride was prepared in the same manner as above. The target product (HA-C6-Chol-0%-Male) listed in Table 1 was obtained as a white solid.

[0116]

[0117] Each compound is １ The structure was confirmed by H NMR measurement. For NMR analysis, HAMICH was dissolved in 0.02 N DCI / DMSO. １ As a result of H NMR measurement, the degree of maleimide (maleimide group introduction rate) of each HA derivative was 15%. NMR charts of each synthesized product are shown in Figure 1A (Chol-0%) to Figure 1H (Chol-40%).

[0118] The z-average particle size (nm) of each HAMICH was measured. The measurement results are shown in Table 2. In addition, the degree of association of each HAMICH was calculated from the absolute molecular weight measured by GPC-MALS analysis and the theoretical average molecular weight per unit. The results are shown in Table 2.

[0119]

[0120] In HAMICH, the particle size decreased depending on the introduction rate of cholesteryl groups up to 20%, but increased to 40%. This indicates that the introduction rate of cholesteryl groups affects the particle size of HA derivative nanoparticles. Furthermore, in HAMICH without cholesteryl modification (0%), the hydrophobic interaction of the steryl groups does not work, and there was almost no association.

[0121] Example 2 The HAMICH prepared in Example 1 was chemically cross-linked using a cross-linking agent, 4-arm-PEGSH, to prepare a HAMICH cross-linked gel.

[0122] First, each HAMICH prepared in Example 1 was dissolved in 10 mM phosphate buffer (pH 7.4) to the concentrations listed in Table 3, to prepare a HAMICH solution. In a separate container, 4arm-PEGSH (Mw: 10,000) was dissolved in 10 mM phosphate buffer (pH 7.4) at a concentration of 20 mg / mL to prepare a PEGSH solution. After cooling both solutions to 5 ° C, the two solutions were mixed in a 1 mm thick, 6 mm diameter silicone mold so that the molar ratio of maleimide groups modified with hyaluronic acid to SH groups derived from 4arm-PEGSH was 1.1:1.0, and a HAMICH crosslinked gel was produced at 37 ° C. In both cases, the final concentration of the hyaluronic acid derivative was adjusted to 7 mg / mL.

[0123]

[0124] All eight types of HAMICH into which cholesteryl groups and maleimide groups had been introduced were sufficiently gelled and crosslinked gels were formed within 1 to 30 minutes from the start of gelation (mixing of the HAMICH solution and the PEGSH solution), regardless of the introduction rate of the cholesteryl groups. As shown in Comparative Example 2 below, in the case of HAMA into which methacryl groups had been introduced (HA-C6-Chol-3%-MA, HA-C6-Chol-19%-MA), gelation was difficult with hyaluronic acid derivatives into which steryl groups and methacryl groups had been introduced. These results demonstrate that the introduction of a combination of steryl groups and maleimide groups into hyaluronic acid enables rapid gelation that is difficult with hyaluronic acid derivatives into which steryl groups and methacryl groups have been introduced.

[0125] Furthermore, the volume change in PBS after gelation was measured to examine the degree of swelling. The measurement results are shown in Figure 2(A). Furthermore, for HA-C6-Chol-0%-Male and HAMICH with a 20% cholesteryl group introduction rate (HA-C6-Chol-20%-Male), the gelation reaction was carried out under the same conditions except that the final concentration of the hyaluronic acid derivative was 16.7 mg / mL. The volume change from the start of gelation was measured to examine the degree of swelling. The measurement results are shown in Figure 2(B). As shown in Figure 2, HA-C6-Chol-0%-Male, which is unmodified with cholesteryl groups, exhibited gel swelling over time. In contrast, HAMICH modified with both maleimide and steryl groups exhibited a smaller degree of swelling than HAMICH modified only with maleimide groups, demonstrating the characteristic property of suppressing volume increase in vivo after gelation.

[0126] Comparative Example 1 An HA derivative (HA-C6-Chol-MA, sometimes referred to as "HAMA") was prepared in which steryl and methacryl groups were introduced into at least some of the carboxyl groups in the glucuronic acid moiety of hyaluronic acid.

[0127]

[0128] Cholesteryl 6-aminohexylcarbamate hydrochloride (Chol-C6 hydrochloride) and tetrabutylammonium salt of hyaluronic acid (HA-TBA) were synthesized as described in Examples 1 and 2 of Patent Document 1. Specifically, HA-TBA was obtained by converting HA from the starting material, sodium hyaluronic acid (HA-Na) (average molecular weight = 120 kDa, purchased from Bloomage Biotechnology Japan), using a cation exchange resin (Dowex (registered trademark) 50WX-8-400, manufactured by Sigma-Aldrich), and then reacting it with 2-aminoethyl methacrylate hydrochloride (manufactured by Fuji Film Wako Co., Ltd., product number 510-51961) using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (manufactured by Kokusan Chemical Co., Ltd.) as a condensing agent. HA-TBA was completely dissolved in DMSO (1% w / v), and then 2-aminoethyl methacrylate hydrochloride (Fujifilm Wako Co., Ltd., product number 510-51961) dissolved in DMSO was added and stirred at room temperature for 5 minutes. DMT-MM was then added to the mixture, which was then stirred overnight at room temperature. The feed molar ratio was 100:20:24 (glucuronic acid of HA:DMT-MM:2-aminoethyl methacrylate hydrochloride, x = 20). Cholesteryl-6-aminohexyl carbamate was then reacted using the same procedure. The feed molar ratio was 100:x:1.2x (glucuronic acid of HA:DMT-MM:cholesteryl-6-aminohexyl carbamate, x = 2.0, 19). The HA product was purified by dialysis (MWCO: 12-14 kDa) against DMSO, then against 0.150 M NaCl, and finally against distilled water. The purified HA solution was filtered through a 0.22 μm membrane filter and lyophilized to dryness.

[0129] Thereafter, 2-aminoethyl methacrylate hydrochloride was added to each solution in the ratio relative to the HA-TBA units shown in Table 4. Next, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (manufactured by Kokusan Chemical Co., Ltd.) was added to each solution in the ratio relative to the HA-TBA units shown in Table 4, and the mixture was stirred overnight. Thereafter, Chol-C6 hydrochloride was added to each solution in the ratio relative to the HA-TBA units shown in Table 4.

[0130] Next, DMT-MM was added to the HA-TBA units in the ratio shown in Table 4, and the mixture was further stirred overnight at room temperature. Finally, the reaction solution was dialyzed (Spectrapore 4, molecular weight cutoff (MWCO): 12 kDa to 14 kDa, Spectrum Laboratories) against a DMSO solution, a 0.15 M NaCl aqueous solution, and ultrapure water in that order. The resulting dialyzed solution was lyophilized to obtain the target product (HA-C6-Chol-MA) shown in Table 4 as a white solid.

[0131]

[0132] Comparative Example 2 The HAMA prepared in Comparative Example 1 was chemically crosslinked using a crosslinking agent, 4arm-PEGSH, to prepare a HAMA crosslinked gel.

[0133] First, each HAMA prepared in Comparative Example 1 was dissolved in 10 mM phosphate buffer (pH 7.4) to the concentrations listed in Table 5 to prepare a HAMA solution. In a separate container, 4arm-PEGSH (NOF Corporation, Mw: 10,000) was dissolved in 10 mM phosphate buffer (pH 7.4) at a concentration of 50 mg / mL to prepare a PEGSH solution. After cooling both solutions to 5°C, the two solutions were mixed in a 1 mm thick, 6 mm diameter silicone mold so that the molar ratio of methacrylic groups modified on hyaluronic acid to SH groups derived from 4arm-PEGSH was 1.1:1.0, and a HAMA crosslinked gel was produced at 37°C. In both cases, the final concentration of the hyaluronic acid derivative was adjusted to 7 mg / mL.

[0134]

[0135] In both cases of HAMA into which a methacryl group had been introduced (HA-C6-Chol-3%-MA, HA-C6-Chol-19%-MA), gelation was still insufficient 30 minutes after the start of gelation, and gelation was not complete even 24 hours after the start of gelation.

[0136] Comparative Example 3 Furthermore, crosslinking was attempted using 4arm-PEGSH (NOF Corporation, Mw: 10,000) dissolved at a concentration of 100 mg / mL under the same conditions as in Comparative Example 2, except that the final concentration of the hyaluronic acid derivative was 25 mg / mL, for HAMA (HA-C6-Chol-19%-MA) into which a methacrylic group had been introduced. As a result, the HAMA did not crosslink even at high concentrations.

[0137] Example 3 Fluorescently labeled insulin (FITC-insulin) was encapsulated in the HAMICH cross-linked gel prepared in Example 2, and the encapsulation rate was examined.

[0138] In Example 2, a HAMICH crosslinked gel (6 mmφ, 1 mm thick) produced under conditions in which the final concentration of the hyaluronic acid derivative was 7 mg / mL was immersed in 1 mL of FITC-insulin solution (a solution prepared by dissolving FITC-insulin in PBS to give 250 μg / mL FITC-insulin) and incubated at 25°C, and the fluorescence intensity of the supernatant was quantified over time to calculate the encapsulation rate (%) of FITC-insulin within the gel. The encapsulation rates (%) measured 1 hour, 8 hours, and 24 hours after the start of incubation are shown in Table 6 and Figure 3. Figure 4 also shows the appearance of each gel after encapsulation.

[0139]

[0140] As shown in Table 6, the HAMICH cross-linked gels into which cholesteryl groups had been introduced had a higher encapsulation rate of FITC-insulin than the HAMICH cross-linked gels into which cholesteryl groups had not been introduced. Furthermore, the encapsulation rate of FITC-insulin was affected by the cholesteryl group introduction rate, with the best encapsulation rate after 8 and 24 hours of incubation being achieved at cholesteryl group introduction rates of 5-10%. Figure 4 clearly shows, even visually, that the cross-linked gels into which chol has not been introduced exhibit weak FITC-derived coloring. These results demonstrate that the hyaluronic acid derivative of this embodiment has excellent encapsulation efficiency for active ingredients such as proteins, and that optimizing the steryl group introduction rate allows for the encapsulation of larger amounts of active ingredients.

[0141] The results of Examples 2 and 3 show that by using the hyaluronic acid derivative of this embodiment, rapid gelation can be achieved, and furthermore, it is possible to obtain a novel crosslinked gel that can both suppress volume changes in the body and encapsulate large amounts of protein.

[0142] In view of the above, the crosslinked gel obtained from the hyaluronic acid derivative of this embodiment can encapsulate large amounts of cytokines necessary for regeneration or cell differentiation, and is expected to be applied to regenerative medicine applications, such as controlling cell adhesion and differentiation by releasing these cytokines.

[0143] [Example 4] A crosslinked gel of a hyaluronic acid derivative modified with both maleimide groups and steryl groups was produced, and it was examined whether the crosslinked gel would become porous even in a swelling equilibrium state by freeze-drying and freeze-thawing.

[0144] First, a fluorescent Cy5-modified hyaluronic acid derivative (Cy5-HAMICH) was prepared in the same manner as in Example 1, with a cholesteryl group introduction rate of 19% and a maleimide group introduction rate of 11%. Cy5 modification was performed using Cy5-amine (Funakoshi Co., Ltd., product number: BP-22559) by adding 0.02 equivalents relative to the HA-TBA unit and 0.03 equivalents of DMT-MM as a condensing agent. Furthermore, a fluorescent Cy5-modified hyaluronic acid derivative (Cy5-HAMICH) without introducing a cholesteryl group (introduction rate 0%) and with a maleimide group introduction rate of 7% was also prepared in the same manner as above. Next, a crosslinker was added to each Cy5-HAMICH in a ratio of maleimide group:SH group = 1.1:1.0 (molar ratio) in the same manner as in Example 3, and gelation was performed to prepare a crosslinked gel (diameter 8 mmφ, thickness 1 mm). After preparation, the mixture was left to stand in PBS for 1 hour and then subjected to a freeze-thaw treatment.

[0145] The freeze-thaw treatment was carried out as follows: First, the Cy5-modified HA derivative crosslinked gel was left to stand at room temperature (25°C) or 5°C for 30 minutes, then left to stand at -20°C for 1 hour, and then left to stand at -78°C for 24 hours to carry out a freeze-drying treatment. Then, it was thawed by incubation in a water bath at 25°C.

[0146] Fluorescence microscopy images of each crosslinked gel were obtained after freeze-drying and thawing using a two-photon laser microscope (Zeiss). Fluorescence microscopy images of each crosslinked gel are shown in Figure 5. It was confirmed that the Cy5-HAMICH crosslinked gel with a cholesteryl group introduction rate of 19% (top row of Figure 5) had a porous structure with numerous pores with diameters of several tens of micrometers formed on the surface like a network structure. In contrast, no such pores were observed in the Cy5-HAMICH crosslinked gel with a cholesteryl group introduction rate of 0% (bottom row of Figure 5). From these results, it was considered that the hyaluronic acid derivative crosslinked gel of this embodiment is useful as a scaffold for cell infiltration and regeneration.

[0147] Example 5 In the same manner as in Example 2, the HAMICH prepared in Example 1 was chemically crosslinked using a crosslinking agent, 4arm-PEGSH, to prepare a HAMICH crosslinked gel.

[0148] First, each HAMICH prepared in Example 1 was dissolved in 10 mM phosphate buffer (pH 7.4) to a concentration of 10 mg / mL to prepare a HAMICH solution. Only HA-C6-Chol-20%-Male15% was dissolved to a concentration of 25 mg / mL. In a separate container, 4arm-PEGSH (Mw: 10,000) was dissolved in 10 mM phosphate buffer (pH 7.4) to a concentration of 20 mg / mL to prepare a PEGSH solution. After cooling both solutions to 5°C, they were poured into a single vial and mixed. An inverted vial test was performed to verify the gel or sol. At this time, 10 mM phosphate buffer (pH 7.4) was appropriately added to the 4arm-PEGSH solution before mixing the hyaluronic acid derivative and 4arm-PEGSH solutions so that the final concentrations of the hyaluronic acid derivative and 4arm-PEGSH were as shown in Table 7.

[0149]

[0150] HAMICH, into which cholesteryl groups and maleimide groups had been introduced, gelled sufficiently and formed a crosslinked gel within 1 to 30 minutes from the start of gelation (mixing of HAMICH solution and PEGSH solution) in all of the test plots 5-1 to 5-30, regardless of the rate of cholesteryl group introduction. These results demonstrate that hyaluronic acid derivatives in which steryl groups and maleimide groups have been introduced into hyaluronic acid in combination can gel at various concentrations. It also suggests that the amount of drug encapsulated in the HAMICH crosslinked gel formed can be adjusted by changing the gel concentration.

[0151] Example 6 In the same manner as in Example 5, the HAMICH prepared in Example 1 was chemically crosslinked using a crosslinking agent, 4arm-PEGSH, to prepare a HAMICH crosslinked gel.

[0152] First, HAMICH (HA-C6-Chol-15%-Male15%) with a cholesteryl group introduction rate of 15% prepared in Example 1 was dissolved in 10 mM phosphate buffer (pH 7.4) to a concentration of 10 mg / mL to prepare a HAMICH solution. In a separate container, 4arm-PEGSH (Mw: 10,000) was dissolved in 10 mM phosphate buffer (pH 7.4) to a concentration of 70 mg / mL to prepare a PEGSH solution. After cooling both solutions to 5°C, they were poured into a single vial and mixed, and gel or sol verification was performed using an inverted vial test. At this time, 10 mM phosphate buffer (pH 7.4) was appropriately added to the 4arm-PEGSH solution before mixing the hyaluronic acid derivative and 4arm-PEGSH solution so that the final concentrations of the hyaluronic acid derivative and 4arm-PEGSH were as shown in Table 8.

[0153]

[0154] HAMICH, which contained 15% cholesteryl groups and 15% maleimide groups, was fully gelled and a crosslinked gel was formed within 30 minutes of the start of gelation (mixing of the HAMICH solution and the PEGSH solution) in all of Tests 6-1 and 6-2. These results demonstrate that hyaluronic acid derivatives, in which steryl groups and maleimide groups are combined and introduced into hyaluronic acid, can gel at various concentrations. It also suggests that the amount of drug encapsulated in the HAMICH crosslinked gel formed can be adjusted by changing the gel concentration.

[0155] [Example 7] The hyaluronic acid derivative modified with both maleimide and steryl groups was analyzed by gel permeation chromatography using multi-angle light scattering (GPC-MALS) to calculate the absolute molecular weight of the aggregate in 10 mM phosphate buffer (pH 7.4).

[0156] A MALS detector measures scattering signals from polymers or particles in a sample at different scattering angles θ. The basic light scattering equation (Anderson et al., Analytical Chemistry, 2003, vol. 75, pp. 4279-4291) is shown below: where Rθ is the excess Rayleigh ratio, K is an optical constant that depends, among other things, on the specific refractive index increment (dn / dc), c is the solute concentration, M is the molecular weight, Rg is the radius of gyration, and λ is the wavelength of the incident light. Calculation of molecular weight and radius of gyration from light scattering data requires extrapolation to zero angle (Wyatt, Analytica Chimica Acta, 1993, vol. 272(1), pp. 1-40). This is done by plotting (Kc / Rθ)½ as a function of sin²(θ / ²) in a so-called Debye plot. Generally, the molecular weight can be calculated from the intercept of the ordinate and the radius of gyration can be calculated from the initial slope of the curve.

[0157]

[0158] In this study, signals from the RI detector and MALS detector were collected and analyzed using ASTRA8 (Wyatt Technology) software for calculations. The calculated molecular weight, for example, the absolute weight-average molecular weight Mw (absolute value), was derived using a refractive index concentration coefficient dn / dc of 0.153, which is the literature value for hyaluronic acid. The measurement conditions and the calculation results are shown below.

[0159] (Measurement conditions) Apparatus: SEC-Multi Detector system Column: G4000SWXL (manufactured by Tosoh) MALS: DWAN NEON (manufactured by Wyatt Technology) Laser wavelength: 661 nm RI: Optilab (manufactured by NEON) Eluent: 10 mM PB, pH 7.4 Flow rate: 0.5 mL / min Injection volume: 50 μL Concentration: 1.0 mg / mL Temperature: 25°C Analysis software: ASTRA8 (manufactured by Wyatt)

[0160]

[0161] These results suggest that cholesterol is the driving force behind aggregate formation, and demonstrate that aggregates of cholesterol and maleimide-modified hyaluronic acid derivatives can be formed even in the absence of a crosslinker. By pre-encapsulating a drug in nanoparticles composed of hyaluronic acid derivative aggregates and then carrying out a crosslinking reaction to form a gel, it is expected that the effects of gelation and crosslinking on the drug can be reduced. This method allows for the production of crosslinked gels containing encapsulated drugs without compromising their stability, even for drugs highly reactive with crosslinkers. This drug encapsulation in hyaluronic acid derivative aggregates is thought to be applicable to a wide range of drugs, from small molecular weight compounds such as insulin to large molecular weight cytokines such as IL-4.

[0162] Example 8 An in vitro insulin release test was carried out on the HAMICH cross-linked gel obtained in Example 3, in which fluorescently labeled insulin (FITC-insulin) was encapsulated.

[0163] Each of the five FITC-insulin-encapsulated HAMICH crosslinked gels obtained in Example 3 was immersed in 1 mL of 10% FBS-containing PBS (pH 7.4) at 37°C and incubated. The fluorescence intensity of the supernatant was quantified over time to calculate the release rate (%) of FITC-insulin from each gel. Figure 6 shows the release rates (%) for the cholesteryl-modified HAMICH crosslinked gels (HA-C6-Chol-1%-Male 15%, HA-C6-Chol-5%-Male 15%, HA-C6-Chol-10%-Male 15%, and HA-C6-Chol-20%-Male 15%) up to 21 days after immersion.

[0164] In the case of a crosslinked gel unmodified with cholesterol (HA-C6-Chol-0%-Male 15%), the entire amount of encapsulated FITC-insulin was released into the supernatant one day after the start of immersion (100% release rate). In contrast, as shown in Figure 6, the HAMICH crosslinked gel modified with cholesteryl groups did not reach 100% release rate even 21 days after the start of immersion, demonstrating a prolonged sustained release period of FITC-insulin. These results demonstrate that the crosslinked gel obtained from the hyaluronic acid derivative of this embodiment not only has excellent encapsulation efficiency for active ingredients such as proteins, but also has excellent sustained release performance.

[0165] As shown by the above examples, the crosslinked gel obtained from the hyaluronic acid derivative of this embodiment has rapid gelation, and the volume change in vivo is suppressed, in addition, it can encapsulate a large amount of active ingredients such as protein, and also has excellent long-term sustained release of encapsulated substances.In view of these, the hyaluronic acid derivative of this embodiment and the crosslinked gel obtained from it are expected to be applied to regenerative medicine.The crosslinked gel obtained from the hyaluronic acid derivative of this embodiment can encapsulate a large amount of cytokines that are necessary for regeneration or cell differentiation, and then release these cytokines, thereby controlling cell adhesion and differentiation.

[0166] [Example 9] The target product (HA-C6-Chol-1%-Male 8.1%) of Test Plot 9-1 listed in Table 10 was obtained as a white solid in the same manner as in Example 1, except that hyaluronic acid sodium salt (HA-Na) with a weight-average molecular weight of 35 kDa (purchased from Bloomage Biotechnology Japan) was used as the starting material. The resulting synthetic product was １ The structure was confirmed by H NMR measurement. As a result, the HA derivative in test plot 9-1 had a Chol introduction rate of 1.3% and a maleimide group introduction rate of 8.1%.

[0167] In addition, using hyaluronic acid sodium salt (HA-Na) with a weight average molecular weight of 35 kDa as a raw material, cholesteryl groups and methacryl groups were introduced in the same manner as in Comparative Example 1, and then maleimide groups were modified in the same manner as in Example 1, to obtain the target product (HA-C6-Chol-17%-Male 4.6%) of Test Plot 9-2 shown in Table 10 as a white solid. １ The structure was confirmed by H NMR measurement. As a result, the HA derivative in test area 9-2 had a Chol introduction rate of 17.4%, a maleimide group introduction rate of 4.6%, and a methacryl group introduction rate of 9.7%.

[0168]

[0169] Example 10 The HAMICH prepared in Example 9 was chemically cross-linked using a cross-linking agent, 4arm-PEGSH, to prepare a HAMICH cross-linked gel.

[0170] First, HAMICH from test area 9-1 was dissolved in 10 mM phosphate buffer (pH 7.4) to a concentration of 14 mg / mL to prepare a HAMICH solution. In a separate container, 4arm-PEGSH (Mw: 10,000) was dissolved in 10 mM phosphate buffer (pH 7.4) at a concentration of 7 mg / mL to prepare a PEGSH solution. After cooling both solutions to 5 ° C, in a 1 mm thick, 6 mm diameter silicone mold, the two solutions were mixed so that the molar ratio of maleimide groups modified with hyaluronic acid and SH groups derived from 4arm-PEGSH was 1.1: 1.0, and a HAMICH crosslinked gel was produced at 37 ° C. At this time, the final concentration of the hyaluronic acid derivative was 7 mg / mL.

[0171] Similarly, HAMICH from test area 9-2 was dissolved in 10 mM phosphate buffer (pH 7.4) to a concentration of 30 mg / mL to prepare a HAMICH solution. In a separate container, 4arm-PEGSH (Mw: 10,000) was dissolved in 10 mM phosphate buffer (pH 7.4) at a concentration of 13 mg / mL to prepare a PEGSH solution. After cooling both solutions to 5 ° C, in a 1 mm thick, 6 mm diameter silicone mold, the molar ratio of maleimide groups modified with hyaluronic acid and SH groups derived from 4arm-PEGSH was 1.1: 1.0, and the two solutions were mixed to produce a HAMICH crosslinked gel at 37 ° C. At this time, the final concentration of the hyaluronic acid derivative was set to 20 mg / mL.

[0172] HAMICH, into which cholesteryl groups and maleimide groups had been introduced, fully gelled and formed a crosslinked gel within 1 to 30 minutes from the start of gelation (mixing of HAMICH solution with PEGSH solution), regardless of the ratio of maleimide groups introduced. It was predicted that a decrease in the ratio of maleimide groups would result in a decrease in the number of crosslinking points, making gelation more difficult. However, these results demonstrate that by introducing a combination of steryl groups and maleimide groups into hyaluronic acid, it is possible to achieve rapid gelation that is difficult with hyaluronic acid derivatives into which steryl groups and methacrylic groups have been introduced, regardless of the range of the ratio of maleimide groups introduced.

[0173] Furthermore, the volume change in PBS from the start of gelation was measured to examine the degree of swelling. The measurement results for HAMICH in Test Plot 9-1 are shown in Figure 7(A), and the measurement results for HAMICH in Test Plot 9-2 are shown in Figure 7(B). As shown in Figure 7, HAMICH modified with both maleimide groups and steryl groups exhibited the characteristic property of having a small degree of swelling, regardless of the introduction rate of maleimide groups, and suppressing volume increase in vivo after gelation.

[0174] Comparative Example 4: The gelation of a hyaluronic acid derivative not modified with maleimide was investigated. First, a hyaluronic acid derivative not modified with maleimide was synthesized. Specifically, cholesteryl groups were introduced into the hyaluronic acid derivatives in the compositions shown in Table 11 using sodium hyaluronate (HA-Na) with a weight-average molecular weight of 35 kDa or 100 kDa as the starting material in the same manner as in Example 1. The target products of Comparative Test 4-1 (HA-C6-Chol-19%) and Comparative Test 4-2 (HA-C6-Chol-40%) were obtained as white solids, respectively. Cholesteryl 6-aminohexylcarbamate hydrochloride (Chol-C6 hydrochloride) and tetrabutylammonium salt of hyaluronic acid (HA-TBA) were synthesized as described in Examples 1 and 2 of Patent Document 1. As a result, the target products (HA-C6-Chol-19% and HA-C6-Chol-40%) shown in Table 11 were obtained as white solids.

[0175]

[0176] Next, the HA derivative (HA-C6-Chol-19%) of Comparative Test 4-1 and the HA derivative of Comparative Test 4-2 were each chemically crosslinked using the crosslinker 4arm-PEGSH to prepare a HA derivative (maleimide-unmodified) crosslinked gel. First, each HA derivative (maleimide-unmodified) produced in Comparative Tests 4-1 and 4-2 was dissolved in 10 mM phosphate buffer (pH 7.4) to a concentration of 14 mg / mL to prepare an HA derivative solution. In a separate container, 4arm-PEGSH (Mw: 10,000) was dissolved in 10 mM phosphate buffer (pH 7.4) at a concentration of 7 mg / mL to prepare a PEGSH solution. After cooling both solutions to 5°C, the two solutions were mixed in a 1mm thick, 6mm diameter silicone mold so that the molar ratio of the molecular weight of the hyaluronic acid derivative to the SH group derived from 4arm-PEGSH was 1.1:1.0, and a crosslinked gel was prepared at 37°C. This was carried out under conditions such that the final concentration of the hyaluronic acid derivative was 7mg / mL. As a result, no gelation occurred even after 24 hours. The same results were obtained in the inverted vial test for both of these comparative test groups, confirming that no gelation occurred.

[0177] According to the hyaluronic acid derivative of this embodiment, it is possible to provide a hyaluronic acid derivative that undergoes little volume change after gelation and is capable of sustained release of an active ingredient.

Claims

1. A hyaluronic acid derivative in which a steryl group has been introduced to at least a portion of the carboxyl groups in the glucuronic acid portion of hyaluronic acid, and a maleimide group has been introduced to at least a portion of the carboxyl groups in the glucuronic acid portion of the hyaluronic acid or the hydroxyl groups in the N-acetylglucosamine portion of the hyaluronic acid.

2. The hyaluronic acid derivative according to claim 1, wherein maleimide groups are introduced into at least a portion of the carboxyl groups in the glucuronic acid portion of the hyaluronic acid.

3. The hyaluronic acid derivative according to claim 1, wherein the sum of the introduction rate of the steryl group and the introduction rate of the maleimide group to the hyaluronic acid derivative is less than 45%.

4. The hyaluronic acid derivative according to claim 1, wherein the introduction rate of the steryl group to the hyaluronic acid derivative is 0.5% or more and 30% or less.

5. The hyaluronic acid derivative according to claim 1, wherein the introduction rate of the maleimide group to the hyaluronic acid derivative is 1.0% or more and 25% or less.

6. A compound represented by the following general formula (I): [In general formula (I), R １ , R ２ , R ３ , and R ４ Each independently represents a hydrogen atom, C １－６ Alkyl, formyl and C １－６ alkylcarbonyl; Z represents a direct bond or a peptide linker consisting of 2 to 30 amino acid residues; X １ is -NR ｂ -R, -NR ｂ -COO-R, -NR ｂ -CO-R, -NR ｂ -CO-NR ｃ -R, -COO-R, -O-COO-R, -SR, -CO-Y ａ -SR, -O-CO-Y ｂ -S-R, -NR ｂ -CO-Y ｂ is a group selected from the group consisting of groups represented by -S-R and -S-S-R; R ａ , R ｂ and R ｃ Each independently represents a hydrogen atom, C １－２０ Alkyl, Amino C ２－２０ Alkyl and Hydroxy C ２－２０ alkyl, wherein R ａ , the R ｂ and the R ｃ The alkyl portion is —O— and —NR ｆ - may be inserted into the group consisting of; ｆ is a hydrogen atom, C １－１２ Alkyl, Amino C ２－１２ Alkyl and Hydroxy C ２－１２ alkyl, wherein R ｆ The alkyl portion of may be interrupted by a group selected from the group consisting of -O- and -NH-; R is a steryl group; Y is C ２－３０ Alkylene, or -(CH ２ CH ２ O) ｍ -CH ２ CH ２ -, and the alkylene portion of Y is -O-, -NR ｇ R may have inserted therein a group selected from the group consisting of - and -S-S-; ｇ is a hydrogen atom, C １－２０ Alkyl, Amino C ２－２０ Alkyl and Hydroxy C ２－２０ alkyl, wherein R ｇ The alkyl portion of Y may have an inserted group selected from the group consisting of -O- and -NH-; ａ is C １－５ is alkylene; ｂ is C ２－８ Alkylene or C ２－８ and m is an integer of 1 to 100.] and has one or more repeating units represented by the following general formula (II): [In general formula (II), R １ , R ２ , R ３ , and R ４ Each independently represents a hydrogen atom, C １－６ Alkyl, formyl and C １－６ alkylcarbonyl; Z represents a direct bond or a peptide linker consisting of 2 to 30 amino acid residues; X ２ is a maleimide group; R ａ is a hydrogen atom, C １－２０ Alkyl, Amino C ２－２０ Alkyl and Hydroxy C ２－２０ alkyl, wherein R ａ The alkyl portion is —O— and —NR ｆ - may be inserted; Y is C ２－３０ Alkylene, or -(CH ２ CH ２ O) ｍ -CH ２ CH ２ -, and the alkylene portion of Y is -O-, -NR ｇ R may have inserted therein a group selected from the group consisting of - and -S-S-; ｇ is a hydrogen atom, C １－２０ Alkyl, Amino C ２－２０ Alkyl and Hydroxy C ２－２０ alkyl, wherein R ｇ The hyaluronic acid derivative according to claim 1, which has one or more repeating units represented by the following formula: wherein the alkyl portion may have a group selected from the group consisting of -O- and -NH- inserted therein; and m is an integer of 1 or more and 100 or less.

7. The hyaluronic acid derivative according to claim 1, wherein the steryl group is a cholesteryl group.

8. The hyaluronic acid derivative according to claim 1, wherein the z-average particle diameter of the hyaluronic acid derivative dissolved in phosphate buffer (10 mM) at a concentration of 1 mg / mL, as calculated by dynamic light scattering, is 250 nm or less.

9. A method for producing a hyaluronic acid derivative crosslinked gel, comprising reacting the hyaluronic acid derivative according to any one of claims 1 to 8 with a crosslinking agent having two or more crosslinkable groups to form a gel.

10. A method for producing a hyaluronic acid derivative crosslinked gel according to claim 9, wherein the crosslinkable group is a thiol group.

11. A hyaluronic acid derivative crosslinked gel, in which the hyaluronic acid derivative according to any one of claims 1 to 8 is a gel-like substance chemically crosslinked by a crosslinking agent having two or more crosslinkable groups.

12. The hyaluronic acid derivative crosslinked gel according to claim 11, wherein the crosslinkable group is a thiol group.

13. The hyaluronic acid derivative crosslinked gel according to claim 11, having a swelling degree of 115% or less.

14. A dried hyaluronic acid derivative crosslinked gel, which is a dried gel-like material obtained by chemically crosslinking the hyaluronic acid derivative according to any one of claims 1 to 8 with a crosslinking agent having two or more crosslinkable groups.

15. A dried hyaluronic acid derivative crosslinked gel according to claim 14, which has a porous structure.

16. A pharmaceutical composition comprising a hyaluronic acid derivative according to any one of claims 1 to 8.

17. A pharmaceutical composition comprising a hyaluronic acid derivative crosslinked gel, which is a gel-like substance in which the hyaluronic acid derivative according to any one of claims 1 to 8 is chemically crosslinked with a crosslinking agent having two or more crosslinkable groups.

18. The pharmaceutical composition of claim 17, further comprising an active ingredient.

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