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

Abstract

Provided are a hyaluronic acid derivative and a hyaluronic acid derivative cross-linked gel which have excellent in vivo swelling inhibition performance after gelling, and a pharmaceutical composition using the hyaluronic acid derivative and the hyaluronic acid derivative cross-linked gel. The present invention relates to: a hyaluronic acid derivative in which a steroid group is introduced to at least some of the carboxyl groups in the glucuronic acid moiety of hyaluronic acid, and a maleimide group is introduced to at least some of the hydroxyl groups in the N-acetylglucosamine moiety or the carboxyl groups in the glucuronic acid moiety of hyaluronic acid; and a hyaluronic acid derivative cross-linked gel which is a gel-like substance in which the hyaluronic acid derivative is chemically cross-linked by a cross-linking agent having two or more cross-linkable groups.

Description

Technical Field

[0001] This 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 thereof.

[0002] This application claims priority based on Japanese Patent Application No. 2023-166346 filed in Japan on September 27, 2023 and Japanese Patent Application No. 2023-204796 filed in Japan on December 4, 2023, the contents of which are incorporated herein by reference. Background Technology

[0003] In recent years, biopharmaceuticals, with proteins, peptides, and nucleic acids as active ingredients, have been put into practical use, and their numbers have been increasing year by year. Biopharmaceuticals can meet unmet medical needs that traditional small-molecule drugs cannot address. However, they suffer from several problems: they are difficult to absorb from the digestive tract or mucous membranes, and they are unstable in vivo with short blood half-lives. Therefore, biopharmaceuticals require multiple injections, placing a significant burden on both patients and healthcare professionals. Thus, there is a need for a drug substrate (a sustained-release drug delivery system substrate) that can encapsulate biopharmaceuticals without impairing their pharmacological activity and slowly release the active ingredient in vivo.

[0004] Against this backdrop, Patent Documents 1 and 2 propose a substrate for a sustained-release drug delivery system composed of a hyaluronic acid derivative with excellent safety profile. This hyaluronic acid derivative spontaneously associates in aqueous solution, efficiently encapsulating drugs, particularly biological agents, while maintaining its biological activity. It aggregates at physiological saline concentrations (or remains dispersed at physiological saline concentrations) and exhibits good blood retention. This hyaluronic acid derivative, especially when using biological agents as the active ingredient, can be used as a carrier capable of efficiently encapsulating large amounts of drug while maintaining pharmacological activity, as well as a blood-based sustained-release carrier and a targeted carrier with excellent blood retention. It can also be used as a local (e.g., subcutaneous) sustained-release carrier capable of continuously releasing drugs.

[0005] Furthermore, hyaluronic acid is an important component of joints, and as a treatment for joint diseases, there is a method of injecting hyaluronic acid into the joint cavity. For this therapeutic purpose, although injection is easy, a longer retention time within the joint cavity is desired. Therefore, in Patent Document 3, to achieve both ease of injection and long-term retention, hyaluronic acid derivatives modified with various block polymers are proposed.

[0006] Existing technical documents

[0007] Patent documents

[0008] Patent Document 1: International Publication No. 2010 / 053140

[0009] Patent Document 2: International Publication No. 2004 / 046200

[0010] Patent Document 3: International Publication No. 2003 / 087019 Summary of the Invention

[0011] The technical problem that the invention aims to solve

[0012] When used to treat joint diseases, injected hyaluronic acid or its derivatives can 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 substrate for sustained-release drug delivery systems, from a product design perspective, it is preferable that the swelling of the hyaluronic acid cross-linked gel is as small as possible. Additionally, cross-linked hydrogels capable of encapsulating large amounts of growth factors or proteins are preferred. However, besides not being able to encapsulate proteins, hyaluronic acid, being a polysaccharide with high hydrophilicity, exhibits high swelling. Furthermore, the hyaluronic acid derivatives described in Patent Documents 1-3 do not sufficiently inhibit the swelling associated with gelation.

[0013] The present invention was made in view of the above circumstances, and provides a hyaluronic acid derivative with excellent swelling inhibition after gelation, a hyaluronic acid derivative crosslinked gel, and a pharmaceutical composition using the thereof.

[0014] Methods for solving problems

[0015] That is, the present invention includes the following methods.

[0016] [1] A hyaluronic acid derivative having a steroid group introduced on at least a portion of the carboxyl group of the glucuronic acid moiety of hyaluronic acid, and having a maleimide group introduced on at least a portion of the carboxyl group of the glucuronic acid moiety of hyaluronic acid or the hydroxyl group of the N-acetylglucosamine moiety.

[0017] [2] The hyaluronic acid derivative according to [1], wherein a maleimide group is introduced onto at least a portion of the carboxyl groups in the glucuronic acid portion of the hyaluronic acid.

[0018] [3] The hyaluronic acid derivative according to [1] or [2], wherein the sum of the introduction rate of the steroid group and the introduction rate of the maleimide group relative to the hyaluronic acid derivative is less than 45%.

[0019] [4] The hyaluronic acid derivative according to any one of [1] to [3], wherein the introduction rate of the steroid group relative to the hyaluronic acid derivative is 0.5% or more and 30% or less.

[0020] [5] The hyaluronic acid derivative according to any one of [1] to [4], wherein the introduction rate of the maleimide group relative to the hyaluronic acid derivative is 1.0% or more and 25% or less.

[0021] [6] The hyaluronic acid derivative according to any one of [1] to [5] has one or more repeating units represented by the following general formula (I) and one or more repeating units represented by the following general formula (II).

[0022]

Chemistry 1

[0023]

[0024] In general formula (I), R 1 R 2 R 3 and R 4 Each is independently selected from hydrogen atoms, C 1-6 Alkyl, formyl and C 1-6 The group in alkyl carbonyl;

[0025] Z represents a peptide linker that is directly bonded or consists of two or more but less than 30 amino acid residues;

[0026] X 1 Selected from -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 -SR、-NR b -CO-Y b -SR and -SSR represent groups in the group;

[0027] R a R b and R c Each is independently selected from hydrogen atoms, C 1-20 Alkyl, amino C 2-20 Alkyl and hydroxy C 2-20 The group in the alkyl group, said R a The R b and the R c The alkyl portion can be inserted with a selection from -O- and -NR. f - groups in;

[0028] The R f Selected from hydrogen atoms, C1-12 Alkyl, amino C 2-12 Alkyl and hydroxy C 2-12 The group in the alkyl group, said R f The alkyl portion may be inserted with a group selected from -O- and -NH-;

[0029] R is a steroidal group;

[0030] Y is C 2-30 Alkylene or -(CH2CH2O) m -CH2CH2-, where the alkylene portion of Y can be inserted from -O-, -NR. g - and -SS- groups;

[0031] R g Selected from hydrogen atoms, C 1-20 Alkyl, amino C 2-20 Alkyl and hydroxy C 2-20 The group in the alkyl group, said R g The alkyl portion may be inserted with a group selected from -O- and -NH-;

[0032] Y a It is C 1-5 Alkylene;

[0033] Y b It is C 2-8 Alkylene or C 2-8 alkenyl;

[0034] m is an integer greater than 1 and less than 100.

[0035]

Chemistry 2

[0036]

[0037] In general formula (II), R 1 R 2 R 3 and R 4 Each is independently selected from hydrogen atoms, C 1-6 Alkyl, formyl and C 1-6 The group in alkyl carbonyl;

[0038] Z represents a peptide linker that is directly bonded or consists of two or more but less than 30 amino acid residues;

[0039] X 2 It is maleimide group;

[0040] R a Selected from hydrogen atoms, C 1-20 Alkyl, amino C 2-20 Alkyl and hydroxy C 2-20The group in the alkyl group, said R a The alkyl portion can be inserted with a selection from -O- and -NR. f - groups in;

[0041] Y is C 2-30 Alkylene or -(CH2CH2O) m -CH2CH2-, where the alkylene portion of Y can be inserted from -O-, -NR. g - and -SS- groups;

[0042] R g Selected from hydrogen atoms, C 1-20 Alkyl, amino C 2-20 Alkyl and hydroxy C 2-20 The group in the alkyl group, said R g The alkyl portion may be inserted with a group selected from -O- and -NH-;

[0043] m is an integer greater than 1 and less than 100.

[0044] [7] The hyaluronic acid derivative according to any one of [1] to [6], wherein the steroid group is a cholesterol group.

[0045] [8] The hyaluronic acid derivative according to any one of [1] to [7], wherein the average particle size of the hyaluronic acid derivative dissolved in 10 mM phosphate buffer at a concentration of 1 mg / mL is less than 250 nm by dynamic light scattering.

[0046] [9] A method for manufacturing a hyaluronic acid derivative crosslinked gel, comprising: reacting the hyaluronic acid derivative described in any one of [1] to [8] with a crosslinking agent having two or more crosslinking groups to gel it.

[0047]

[10] The method for manufacturing hyaluronic acid derivative crosslinked gel according to [9], wherein the crosslinking group is a thiol group.

[0048]

[11] A hyaluronic acid derivative crosslinked gel, which is a gel formed by chemically crosslinking a hyaluronic acid derivative as described in any one of [1] to [8] with a crosslinking agent having two or more crosslinking groups.

[0049]

[12] The hyaluronic acid derivative crosslinked gel according to

[11] , wherein the crosslinking group is a thiol group.

[0050]

[13] A hyaluronic acid derivative crosslinked gel according to

[11] or

[12] , wherein the swelling degree is less than 115%.

[0051]

[14] A dried hyaluronic acid derivative cross-linked gel, which is a dried gel formed by chemically cross-linking a hyaluronic acid derivative having two or more cross-linking groups through any one of [1] to [8].

[0052]

[15] The hyaluronic acid derivative cross-linked gel dried product according to

[14] is a porous structure.

[0053]

[16] A pharmaceutical composition comprising any one of the hyaluronic acid derivatives described in [1] to [8].

[0054]

[17] A pharmaceutical composition comprising a hyaluronic acid derivative crosslinked gel, wherein the hyaluronic acid derivative crosslinked gel is a gel formed by chemically crosslinking a hyaluronic acid derivative as described in any one of [1] to [8] with a crosslinking agent having two or more crosslinking groups.

[0055]

[18] The pharmaceutical composition according to

[17] further contains an active ingredient.

[0056] The effects of the invention

[0057] Based on the hyaluronic acid derivatives described above, a hyaluronic acid derivative with excellent swelling inhibition after gelation, a hyaluronic acid derivative crosslinked gel, and a pharmaceutical composition using the above can be provided. Attached Figure Description

[0058] Figure 1A This is the NMR spectrum of the hyaluronic acid derivative crosslinked gel (HA-C6-Chol-0%-Male) synthesized in Example 2.

[0059] Figure 1B This is the NMR spectrum of the hyaluronic acid derivative crosslinked gel (HA-C6-Chol-1%-Male) synthesized in Example 2.

[0060] Figure 1C This is the NMR spectrum of the hyaluronic acid derivative crosslinked gel (HA-C6-Chol-5%-Male) synthesized in Example 2.

[0061] Figure 1D This is the NMR spectrum of the hyaluronic acid derivative crosslinked gel (HA-C6-Chol-10%-Male) synthesized in Example 2.

[0062] Figure 1E This is the NMR spectrum of the hyaluronic acid derivative crosslinked gel (HA-C6-Chol-15%-Male) synthesized in Example 2.

[0063] Figure 1FThis is the NMR spectrum of the hyaluronic acid derivative crosslinked gel (HA-C6-Chol-20%-Male) synthesized in Example 2.

[0064] Figure 1G This is the NMR spectrum of the hyaluronic acid derivative crosslinked gel (HA-C6-Chol-30%-Male) synthesized in Example 2.

[0065] Figure 1H This is the NMR spectrum of the hyaluronic acid derivative crosslinked gel (HA-C6-Chol-40%-Male) synthesized in Example 2.

[0066] Figure 2 This is a graph showing the change in swelling degree (%) over time from the start of gelation for HAMICH crosslinked gels with different cholesterol introduction rates in Example 2. Figure 2 (A) shows the results of HAMICH cross-linked gels prepared at a hyaluronic acid concentration of 7.0 mg / mL. Figure 2 (B) is the result of HAMICH crosslinked gel prepared at a hyaluronic acid concentration of 16.7 mg / mL.

[0067] Figure 3 This is a graph showing the results of measuring the FITC-insulin encapsulation rate (%) of the HAMICH cross-linked gel encapsulating FITC-insulin in Example 3.

[0068] Figure 4 This is a visible light photograph of the appearance of the HAMICH cross-linked gel encapsulating FITC-insulin in Example 3.

[0069] Figure 5 It is the Cy5-HAMICH crosslinked gel with a cholesterol introduction rate of 19% in Example 4. Figure 5 The upper part) and Cy5-HAMICH crosslinked gel with a cholesterol introduction rate of 0% ( Figure 5 Fluorescence microscopy images of the lower section after freeze-drying and freeze-thaw treatment.

[0070] Figure 6 This is a graph showing the results of measuring the FITC-insulin release rate (%) from the HAMICH cross-linked gel encapsulating FITC-insulin in Example 8.

[0071] Figure 7 This is a graph showing the change in swelling degree (%) of the HAMICH crosslinked gel from the start of gelation over time in Example 10. Figure 7 (A) shows the results of the HAMICH (HA-C6-Chol-1%-Male8.1%) crosslinked gel in experimental group 9-1. Figure 7(B) is the result of the HAMICH (HA-C6-Chol-17%-Male4.6%) crosslinked gel of experimental group 9-2. Detailed Implementation

[0072] Hereinafter, embodiments of the present invention (hereinafter referred to as "this embodiment") will be described in detail, but the present invention is not limited thereto and various modifications can be made without departing from its spirit.

[0073] The following is an explanation of the terminology used in this specification.

[0074] The "C" used in this instruction manual 1-20 The term "alkyl" refers to straight-chain or branched alkyl groups with 1 to 20 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, etc. 1-4 "alkyl" 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, etc. 1-20 Alkyl groups also include those with 1 or more but less than 12 carbon atoms. 1-12 Alkyl groups, C atoms with 1 or more but less than 6 carbon atoms 1-6 alkyl.

[0075] The "C" used in this instruction manual 1-6 The term "alkyl carbonyl" refers to an alkyl moiety that is already mentioned as a C10 group. 1-6 Alkyl carbonyl groups, such as acetyl, propionyl, n-propyl carbonyl, isopropyl carbonyl, n-butyl carbonyl, sec-butyl carbonyl, isobutyl carbonyl, tert-butyl carbonyl, etc., are classified as "C". 1-4 Alkyl carbonyl group.

[0076] The term "amino C" as used in this instruction manual 2-20 The term "alkyl" refers to a straight-chain or branched alkyl group having 2 or more but less than 20 carbon atoms as an amino group. For example, the amino group may also be located on the terminal carbon atom of the alkyl group. Amino C 2-20 Alkyl groups also include amino groups with 2 or more but less than 12 carbon atoms. 2-12 alkyl.

[0077] The term "hydroxyl C" as used in this instruction manual 2-20 The term "alkyl" refers to a straight-chain or branched alkyl group having 2 or more but less than 20 carbon atoms as a hydroxyl group. For example, the hydroxyl group may also be located on the terminal carbon atom of the alkyl group. (Hydroxy group C) 2-20 Alkyl groups also include hydroxyl groups with 2 or more but less than 12 carbon atoms. 2-12 alkyl.

[0078] The "C" used in this instruction manual 2-30 The term "alkylene" refers to a straight-chain or branched divalent saturated hydrocarbon group with 2 or more but less than 30 carbon atoms, such as ethylene and propylene, and includes C64 groups with 2 or more but less than 20 carbon atoms. 2-20 Alkylene, C with 2 or more but less than 8 carbon atoms 2-8 Alkylene group, "-(CH2)" n - (Here, n is 2 or more and 30 or less, preferably 2 or more and 20 or less, more preferably 2 or more and 15 or less).

[0079] The "C" used in this instruction manual 1-5 The term "alkylene" refers to a straight-chain or branched divalent saturated hydrocarbon group with 1 or more but less than 5 carbon atoms, such as methylene, ethylene, and propylene.

[0080] The term "C" mentioned in this specification 2-8 "Alkenyl" refers to a straight-chain or branched divalent saturated hydrocarbon group containing one or more double bonds, with 2 to 8 carbon atoms. Examples include -CH=CH-, -C(CH3)=CH-, 2-buten-1,4-diyl, 2,4-heptadien-1,6-diyl, and 2,4,6-octtrien-1,8-diyl. In the presence of geometric isomers, it also includes individual isomers and mixtures thereof.

[0081] In this application specification, "swelling degree" refers to the degree of volume increase of a cross-linked hyaluronic acid derivative gel formed by cross-linking hyaluronic acid derivatives in physiological saline concentration or in PBS (-) (in vivo), based on the relationship between osmotic pressure and elastic pressure. Specifically, it is calculated by the following formula. A hyaluronic acid derivative cross-linked gel with a negative swelling degree refers to a gel whose volume decreases (shrinks) when reaching equilibrium in PBS (-) compared to its volume before gelation (equivalent to the volume immediately after gelation). A positive swelling degree refers to a gel whose volume increases (swells) when reaching equilibrium in PBS (-) compared to its volume immediately after gelation. In hyaluronic acid derivative cross-linked gels, the smaller the absolute value of the swelling degree, the smaller the volume change of the cross-linked gel, which is preferred.

[0082] [Swelling degree (%)] = ([Volume (mm³) of the hyaluronic acid-containing composition after gelation and immersion in PBS (-) to reach equilibrium]) 3 [Volume of the hyaluronic acid-containing composition before gelation (mm)] 3 )])×100 (%)

[0083] Hyaluronic acid derivatives

[0084] For the hyaluronic acid derivative of this embodiment, a steroid group is introduced onto at least a portion of the carboxyl groups in the glucuronic acid portion of hyaluronic acid, and a maleimide group is introduced onto at least a portion of the carboxyl groups in the glucuronic acid portion of hyaluronic acid or the hydroxyl groups in the N-acetylglucosamine portion.

[0085] While the reason why the hyaluronic acid derivative of this embodiment inhibits swelling after gelation is unclear, it is speculated as follows: It is speculated that by combining steroidal and maleimide groups, which are hydrophobic groups, into the hydrophilic groups of hyaluronic acid (i.e., carboxyl and hydroxyl groups), the overall hydrophilicity of the molecule is reduced, thus inhibiting swelling. Furthermore, it is speculated that the relatively short gelation time of 1 second to 30 minutes, particularly through the combination of steroidal and maleimide groups, as the hydrophobic groups introduced into hyaluronic acid, is also believed to contribute to inhibiting swelling. In particular, since the swelling inhibition effect is poor in hyaluronic acid derivatives modified only with maleimide groups and not with steroidal groups, the elastic pressure generated by the strong hydrophobic interaction of the steroidal group may also contribute to the swelling inhibition effect. Even hyaluronic acid derivatives modified with functional groups that have strong interactions based on π-π stacking such as aromatic rings may, like the hyaluronic acid derivative of this embodiment, exhibit excellent swelling inhibition after gelation.

[0086] The swelling degree of the hyaluronic acid derivative in 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 in 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 in this embodiment is particularly preferably 85% or more and 115% or less.

[0087] [steroid]

[0088] As used in this specification, the term "steroid" refers to any group that has a steroid skeleton without particular limitation. Specifically, examples of steroids include: cholesterol, cholesterol, campesterol, ergosterol, stigmasterol, coccosterol, stigmasterol, sitosterol, lanosterol, ergosterol, cimetidine, bile acids, testosterone, estradiol, progesterone, cortisol, cortisone, aldosterone, corticosterone, deoxycorticosterone, etc. Examples of steroid groups include: cholesterol, stigmasterol, lanosterol, ergosterol, etc., with cholesterol (especially cholester-5-en-3β-yl) being preferred.

[0089] In hyaluronic acid derivatives, the steroid group can be directly bonded to hyaluronic acid or linked via a linker. The linkers attached to the steroid group in a molecule of hyaluronic acid derivative can be all identical or of different lengths or types.

[0090] Here, "connector" refers to a group formed by linking a carboxyl-reactive group and a steroidal-reactive group through a chain-like group (spacer). Examples of carboxyl-reactive and steroidal-reactive groups include amino and hydroxyl groups. The chain-like group can be, for example, a chain-like hydrocarbon group, a polyethylene glycol (PEG) chain, an arbitrary peptide connector that can be introduced through genetic engineering, or a synthetic compound connector. The length of the connector is not particularly limited and can be appropriately selected by those skilled in the art according to the purpose. A C-type chain-like hydrocarbon group is preferred. 1-30 Alkylene, more preferably C 1-10 Alkylene, more preferably C 1-6 Alkylene group. As a PEG group, it is preferably a group with 1 or more but less than 15 ethylene glycol groups, more preferably 1 or more but less than 10 groups, and even more preferably 1 or more but less than 5 groups. As a peptide linker, it has 2 or more amino acids (the upper limit is not particularly limited, but is generally 30 or less amino acids, preferably 20 or less amino acids), and particularly preferably 15 amino acids. As the hyaluronic acid derivative of this embodiment, it is preferable that at least a portion of the carboxyl groups in the glucuronic acid moiety are linked to the steroid group via a linker having a chain-like hydrocarbon group.

[0091] [Steroyl introduction rate]

[0092] The steroid introduction rate (hereinafter sometimes simply referred to as "steroid introduction rate") in the hyaluronic acid derivative of this embodiment 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 steroid introduction rate 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 keeping the steroid introduction rate within the above range, the swelling degree can be sufficiently reduced, and sufficient amount of drug can be encapsulated when forming a cross-linked gel.

[0093] The steroid introduction rate can be obtained through 1 The determination is performed using H-NMR spectroscopy. That is, it can be performed using... 1 The integral values ​​of the peak from the steroid group of the hyaluronic acid derivative and the integral values ​​of the peak from the acetyl group of N-acetyl-D-glucosamine contained in the hyaluronic acid derivative (COCH3, ≥1.6ppm and ≤2.0ppm, 3H) in the ¹H-NMR spectrum are calculated according to the following formula. Where, n H This indicates the number of hydrogen atoms corresponding to the peak. Specifically, it can be determined according to the method described in, for example, the examples described later.

[0094] [Steroyl introduction rate] (%)

[0095] = ([peak integral value derived from steroid] × 3 / n)H ) / [Peak integral value of acetyl groups derived from N-acetyl-D-glucosamine] × 100

[0096] [Maleimide group]

[0097] In hyaluronic acid derivatives, the maleimide group (N-maleimide group) can be directly bonded to hyaluronic acid or linked via a linker. The linkers attached to the maleimide group in a molecule of hyaluronic acid derivative can be all the same or different in length or type.

[0098] Here, "connector" refers to a group formed by linking a group that reacts with a carboxyl group and a group that reacts with the NH group in the maleimide group through a chain-like group. Examples of groups that react with a carboxyl group include amino groups and hydroxyl groups; examples of groups that react with the NH group in the maleimide group include carboxyl groups and hydroxyl groups. Examples of the chain-like group are the same as the chain-like group in the connector that links the aforementioned hyaluronic acid derivative and the steroid group. As the hyaluronic acid derivative of this embodiment, it is preferable that at least a portion of the carboxyl group in the glucuronic acid moiety and at least a portion of the hydroxyl group in the N-acetylglucosamine moiety are linked to the NH group in the maleimide group through a connector having a chain-like hydrocarbon group.

[0099] [Maleimide group introduction rate]

[0100] The maleimide group introduction rate (hereinafter sometimes simply referred to as "maleimide group introduction rate") in the hyaluronic acid derivative of this embodiment is preferably 1.0% or more, more preferably 3.0% or more, further preferably 4.5% or more, even more preferably 5.0% or more, still even more preferably 8.0% or more, and particularly preferably 10% or more. The maleimide group introduction rate in the hyaluronic acid derivative of this embodiment is preferably 25% or less, more preferably 20% or less. Regarding the maleimide group introduction rate in the hyaluronic acid derivative of this embodiment, when the steroid group introduction rate is within the above range, the swelling degree can be sufficiently reduced.

[0101] The maleimide group introduction rate can be determined by... 1 The measurements were performed using H-NMR. 1 The H-NMR spectrum was obtained by dissolving the hyaluronic acid derivative of this embodiment in a 0.02N DCl DMSO-d6 / D2O mixture (2N DCl D2O:DMSO-d6 = 1:99). 1The integral values ​​of the acetyl peak (COCH3, 1.6~2.0ppm; 3H) from the glucosamine moiety of the hyaluronic acid derivative, the integral values ​​of the methyl peak (CH3, 0.7ppm; 3H) from the cholesteric group, and the integral values ​​of the maleimide peak (-CH=CH-, 6.9ppm; 2H) from the H-NMR spectrum were used to calculate the introduction rate of the cholesteric and maleimide groups relative to the hyaluronic acid unit using the formula shown below. It should be noted that since the peak near 1.6–2.0 ppm of the acetyl source peak containing the glucosamine moiety overlaps with the peak (5H) from the cholesterol group, the value calculated is obtained by subtracting the integral value of the peak from the cholesterol methyl group (0.7 ppm) from the integral value of the peak near 1.6–2.0 ppm, multiplied by 5 / 3. (That is, [[peak integral value (1.6–2.0 ppm)] - [peak integral value (0.7 ppm)] × 5 / 3] is used as the integral value of the acetyl group from the hyaluronic acid source (the corrected value) for calculating the introduction rate). First, the cholesterol introduction rate (%) is calculated using the following formula.

[0102] [Cholesterol introduction rate (%)]

[0103] =[Peak integral value derived from cholesterol (0.7 ppm)] / [Peak integral value derived from acetyl groups of the N-acetyl-D-glucosamine moiety (1.6~2.0 ppm, corrected value)] × 100 (%)

[0104] Furthermore, the maleimide group introduction rate (%) can be calculated using the integral value of the peak derived from the maleimide group and the integral value of the peak derived from the cholesteric group (COCH3, ≥1.6ppm and ≤2.0ppm, 3H) according to the following formula. Specifically, it can be determined according to the method described in the examples below.

[0105] [Maleimide group introduction rate (%)]

[0106] = ([Peak integral value derived from maleimide group] × 3) / ([Peak integral value derived from cholesterol group] × 2) × [Cholesterol group introduction rate (%)]

[0107] The sum of the steroidal and maleimide group introgression rates in the hyaluronic acid derivative of this embodiment is preferably 55% or less, more preferably 45% or less, further preferably less than 45%, even more preferably 40% or less, and particularly preferably 35% or less. The sum of the steroidal and maleimide group introgression rates in 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. By keeping the total number of units in the steroidal and maleimide group introgression rates of the hyaluronic acid derivative within the above range, the volume change after gelation at physiological saline concentrations (in vivo) can be further reduced, and when used as a DDS carrier, the crosslinking density can be increased, thus prolonging the sustained-release period of the drug.

[0108] As preferred hyaluronic acid derivatives, examples include hyaluronic acid derivatives having one or more repeating units represented by the following general formula (I) (hereinafter sometimes referred to as "repeat unit (I)") and one or more repeating units represented by the following general formula (II) (hereinafter sometimes referred to as "repeat unit (II)").

[0109]

Transformation 3

[0110]

[0111] (In general formula (I), R) 1 R 2 R 3 and R 4 Each is independently selected from hydrogen atoms, C atoms 1-6 Alkyl, formyl and C 1-6 Alkyl carbonyl;

[0112] Z represents a peptide linker that is directly bonded or consists of two or more but less than 30 amino acid residues;

[0113] X 1 Choose from the groups represented by the following formulas:

[0114] -NR b -R、

[0115] -NR b -COO-R、

[0116] -NR b -CO-R、

[0117] -NR b -CO-NR c -R、

[0118] -COO-R、

[0119] -O-COO-R、

[0120] -SR、

[0121] -CO-Y a -SR、

[0122] -O-CO-Y b -SR、

[0123] -NR b -CO-Y b -SR and

[0124] -SSR;

[0125] R a R b and R c Each is independently selected from hydrogen atoms, C atoms 1-20 Alkyl, amino C 2-20 Alkyl and hydroxyl C 2-20 Alkyl group, where the alkyl portion of the group may be inserted from -O- and -NR. f - group;

[0126] R f Selected from hydrogen atoms, C 1-12 Alkyl, amino C 2-12 Alkyl and hydroxyl C 2-12 Alkyl group, wherein the alkyl portion of the group may be inserted with a group selected from -O- and -NH-;

[0127] R is a steroidal group;

[0128] Y is C 2-30 Alkylene or -(CH2CH2O) m -CH2CH2-, where the alkylene group can be inserted from -O-, -NR. g - and -SS- groups;

[0129] R g Selected from hydrogen atoms, C 1-20 Alkyl, amino C 2-20 Alkyl and hydroxy C 2-20 Alkyl group, wherein the alkyl portion of the group may be inserted with a group selected from -O- and -NH-;

[0130] Y a It is C 1-5 Alkylene;

[0131] Y b It is C 2-8 Alkylene or C 2-8 alkenyl;

[0132] m is an integer greater than 1 and less than 100.

[0133]

Chemistry 4

[0134]

[0135] (In general formula (II), R) 1 R 2 R 3 and R 4 Each is independently selected from hydrogen atoms, C atoms 1-6 Alkyl, formyl and C 1-6 Alkyl carbonyl;

[0136] Z represents a peptide linker that is directly bonded or consists of two or more but less than 30 amino acid residues;

[0137] R a Selected from hydrogen atoms, C 1-20 Alkyl, amino C 2-20 Alkyl and hydroxy C 2-20 Alkyl group, where the alkyl portion of the group may be inserted from -O- and -NR. f - group;

[0138] R f Selected from hydrogen atoms, C 1-12 Alkyl, amino C 2-12 Alkyl and hydroxy C 2-12 Alkyl group, wherein the alkyl portion of the group may be inserted with a group selected from -O- and -NH-;

[0139] X 2 It is maleimide group;

[0140] Y is C 2-30 Alkylene or -(CH2CH2O) m -CH2CH2-, where the alkylene group can be inserted from -O-, -NR. g - and -SS- groups;

[0141] R g Selected from hydrogen atoms, C 1-20 Alkyl, amino C 2-20 Alkyl and hydroxy C 2-20 Alkyl group, wherein the alkyl portion of the group may be inserted into a group selected from -O- and -NH-;

[0142] m is an integer greater than 1 and less than 100.

[0143] [Repeating Unit (I)]

[0144] The group "-ZN(R)" in general formula (I) a YX 1 "Contains groups selected from the following formulas:"

[0145] -NH-(CH2) mz -NH-R;

[0146] -NH-(CH2) mz -NH-COO-R;

[0147] -NH-(CH2CH2O) m -CH2CH2-NH-COO-R;

[0148] -NH-(CH2) mz -COO-R;

[0149] -NH-(CH2CH2O) m -CH2CH2-COO-R、

[0150] -NH-(CH2) mz -O-COO-R;

[0151] -NH-(CH2CH2O) m -CH2CH2-O-COO-R、

[0152] -NH-(CH2) mz -S-R; <\(0000677\)>-NH-(CH2CH2O) m -CH²CH²-S-R;

[0154] -NH-(CH2) mz -O-CO-CH(R 8 )-CH2-S-R;

[0155] -NH-(CH2) mz -NHCO-CH(R 8 )-CH2-S-R;

[0156] -NH-(CH2CH2O) m -CH2CH2-NHCO-CH(R 8 )-CH2-S-R;

[0157] -NH-(CH2CH2O) m -CH2CH2-O-CO-CH(R 8 )-CH2-S-R;

[0158] -NH-(CH2) mz -S-S-R;以及

[0159] -Z-NR a -Y-NR Note: There seems to be a formatting issue in the original text where the superscript in ID=44 should be corrected as shown in the translation. Also, the "<\(0000677\)>" in the original text might be a typo, and it's presented as "

[0153] " in the translation for consistency.b -COO-R

[0160] (Here, mz is an integer greater than 2 and less than 30, R) 8 (R is a hydrogen atom or a methyl group, and R and m are as defined in this specification.)

[0161] The preferred group is selected from the following groups:

[0162] -NH-(CH2) mz -NH-COO-R;

[0163] -NH-(CH2CH2O) m -CH2CH2-NH-COO-R; and

[0164] -NH-(CH2) mz -SSR

[0165] (Here, mz, R, and m are as defined in this specification.)

[0166] (Z)

[0167] In general formula (I), Z is preferably directly bonded. Additionally, in other embodiments, when Z is a peptide linker, X... 1 Preferred is -NR b -COO-R. Furthermore, in other embodiments, Z can be derived from -NH-[CH(-Z)]. a )-CONH] n-1 -CH (-Z) a The peptide linker is represented by )-CO-, where n is an integer greater than 2 and less than 30, and Z a Each can be independently represented as H2N-CH(-Z) a The -COOH group represents a substituent in the α-amino acid. This peptide linker binds to the carboxyl group of the glucuronic acid moiety at the N-terminus and to the -N(-R) group at the C-terminus. a -YX 1 Combination. Examples of amino acids that can be used as amino acid residues for this peptide linker include α-amino acids, such as naturally occurring (L-type) amino acids called 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 their D-type forms. All α-amino acids, including synthetic amino acids, can be used. That is, as Z... aExamples of preferred peptide linkers include: -CH3, H2NC(NH)NH(CH2)3-, H2NCOCH2-, etc. Furthermore, the n Zs can 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.

[0168] (Y)

[0169] In general formula (I), Y is preferably selected from -(CH2). n1 - and - (CH2CH2O) m1 The group is -CH2CH2- (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). Specifically, it is preferably -(CH2)2-, -(CH2)6-, -(CH2)8-, or -(CH2) 12 - or -(CH2CH2O)2-CH2CH2-. Furthermore, from the perspective of achieving high solubility in pure water and even at low salt concentrations, while exhibiting high precipitate-forming ability at physiological saline concentrations, Y is preferably selected from -(CH2)2-, -(CH2)6-, -(CH2)8-, and -(CH2). 12 -The group is more preferably -(CH2)6-.

[0170] Y can also be -CH2CH2O-CH2CH2-SS-CH2CH2O-CH2CH2-, -(CH2CH2O)2-CH2CH2-SS-CH2CH2O-CH2CH2-, -CH2CH2O-CH2CH2-SS-(CH2CH2O)2-CH2CH2-, -(CH2CH2O)2-CH2CH2-SS-(CH2CH2O)2-CH2CH2-, etc.

[0171] (Y) a )

[0172] As Y a Preferably, it is -CH2- or -CH2-CH2-.

[0173] (Y) b )

[0174] As Y bPreferably, it is -CH2-CH2-, -CH(CH3)CH2-, 2-butene-1,4-diyl, 2,4-heptadiene-1,6-diyl or 2,4,6-octtriene-1,8-diyl, more preferably -CH2-CH2- or -CH(CH3)CH2-.

[0175] As a group "-ZN(R a YX 1 Specific examples of this can be found in: -NH-(CH2)2-NH-CO-cholestylosinyl, -NH-(CH2)4-NH-(CH2)3-NH-(CH2)3-NH-COO-cholestylosinyl, -NH-(CH2)3-NH-(CH2)4-NH-(CH2)3-NH-COO-cholestylosinyl, -NH-(CH2) 4- NH-(CH2)3-NH-COO-cholestylosin, -NH-(CH2)4-N(-(CH2)3-NH2)-COO-cholestylosin, -NH-(CH2)3-NH-(CH2)4-N(-(CH2)3-NH2)-COO-cholestylosin, -NH-(CH2)3-NH-(CH2)4-N(-(CH2)3-NH-(CH2)3-NH2)-COO-cholestylosin, -NH-(CH2)3-NH-(CH2)4-N(-(CH2)3-NH2)-CO-NH-cholestylosin, -NH-(CH2)3-NH-(CH2)4-N(-(CH2)3-NH2)-CO-cholestylosin, -NH-(CH2)3-NH-(CH2)4-N(-(CH2)3-NH2)-cholestylosin, etc. The preferred group is "-ZN(R)". a YX 1 "R" a R b and R c Y is a hydrogen atom, and C is a straight-chain carbon atom. 2-30 Alkylene or -(CH2CH2O) m -CH2CH2-, Y a C is a linear chain 1-5 Alkylene, or Y b C is a linear chain 2-8 alkylene or straight-chain C 2-8 Alkenyl group.

[0176] As the repeating unit (I), it is particularly preferred that in the general formula (I), Z is direct bonding, and R a It is a hydrogen atom or a carbon atom. 1-6 Alkyl group, where Y is C 2-30 Alkylene or -(CH2CH2O) m -CH2CH2-, X 1 For -NRb1 -COO-R represents a group, R b1 It is a hydrogen atom or a carbon atom. 1-6 Alkyl group, where R is a repeating steroidal unit; more preferably, Z is a direct bond, R a Y is a hydrogen atom, and C is a carbon atom. 2-30 Alkylene or -(CH2CH2O) m -CH2CH2-, X 1 For -NR b1 -COO-R represents a group, R b1 It is a hydrogen atom or a carbon atom. 1-6 Alkyl group, where R is a repeating steroidal unit; more preferably: Z is a direct bond, R a Y is a hydrogen atom and C is a carbon atom. 2-30 Alkylene or -(CH2CH2O) m -CH2CH2-, X 1 For -NR b1 -COO-R represents a group, R b1 It is a hydrogen atom or a carbon atom. 1-6 Alkyl group, where R is a repeating cholesteric unit; more preferably, Z is a direct bond, R a For hydrogen atoms, -YX 1 - is -(CH2)2-NH-COO-cholestylosinyl, -(CH2)4-NH-COO-cholestylosinyl, -(CH2)5-NH-COO-cholestylosinyl, -(CH2)6-NH-COO-cholestylosinyl, -(CH2) 12 -NH-COO-cholesterol or -(CH2CH2O)2-CH2CH2-NH-COO-cholesterol repeating unit; particularly preferred: Z is a direct bond, R a For hydrogen atoms, -YX 1 - is a repeating unit of -(CH2)2-NH-COO-cholestyl, -(CH2)4-NH-COO-cholestyl, -(CH2)5-NH-COO-cholestyl or -(CH2)6-NH-COO-cholestyl.

[0177] As the repeating unit (II), it is particularly preferred that in the general formula (II), Z is direct bonding, and R... a It is a hydrogen atom or a carbon atom. 1-6 Alkyl group, where Y is C 2-30 Alkylene or -(CH2CH2O) m -CH2CH2-, X 2 The repeating unit is maleimide; more preferably, Z is directly bonded, and R is... a It is a hydrogen atom or a carbon atom. 1-6 Alkyl group, where Y is C 2-30 Alkylene, X 2The repeating unit is maleimide; more preferably, Z is directly bonded, and R is... a Y is a hydrogen atom and C is a carbon atom. 2-30 Alkylene, X 2 The repeating unit is maleimide; more preferably, Z is directly bonded, and R is... a Y is a hydrogen atom and C is a carbon atom. 2-10 Alkylene, X 2 It is a repeating unit of maleimide.

[0178] In the hyaluronic acid derivative of this embodiment, the two or more repeating units (I) contained in one molecule of hyaluronic acid derivative may all be the same repeating unit, or they may be two or more different repeating units.

[0179] In the hyaluronic acid derivative of this embodiment, the two or more repeating units (II) contained in one molecule of hyaluronic acid derivative may all be the same repeating unit or may be two or more different repeating units.

[0180] The ratio of the total number of repeating units (I) and repeating units (II) to the total number of repeating units in 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 ratio of the total number of repeating units (I) and repeating units (II) to the total number of repeating units in 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. By keeping the total number of repeating units (I) and repeating units (II) in the hyaluronic acid derivative within the above range, the volume change after gelation can be further reduced, and when used as a DDS carrier, the encapsulation efficiency of the drug can be improved.

[0181] [Weight-average molecular weight Mw]

[0182] The weight-average molecular weight of the hyaluronic acid derivative in this embodiment is not particularly limited. For example, it is preferably 1,000 (1k) or more and 1,000,000 (1,000k) or less, more preferably 5k or more and 300k or less, further preferably 6k or more and 200k or less, and particularly preferably 30k or more and 130k or less.

[0183] The weight-average molecular weight of the hyaluronic acid derivative in this embodiment is preferably 250 kDa or less, more preferably 200 kDa or less, and even more preferably 150 kDa or less, from the perspective of more easily and sufficiently reducing the volume change after gelation and easily adjusting the particle size of the cross-linked hyaluronic acid gel obtained by cross-linking within an appropriate range. Furthermore, from the viewpoint of hyaluronic acid derivative gelation, it is preferably 4 kDa or more, more preferably 20 kDa or more, even more preferably 30 kDa or more, and even more preferably 40 kDa or more. The weight-average molecular weight of the hyaluronic acid derivative in this embodiment is particularly preferably 50 kDa or more, and more preferably 90 kDa or more. The molecular weight of the hyaluronic acid derivative can generally be adjusted by using raw materials with corresponding molecular weights.

[0184] The weight-average molecular weight (Mw) and number-average molecular weight (Mn) of hyaluronic acid derivatives can usually be adjusted by using raw materials with the corresponding molecular weights.

[0185] The weight-average molecular weight (Mw) and number-average molecular weight (Mn) of hyaluronic acid derivatives can be determined, for example, by size exclusion chromatography combined with a multi-angle light scattering detector (SEC-MALS). Specifically, the weight-average molecular weight (Mw) and number-average molecular weight (Mn) of hyaluronic acid derivatives can be determined according to the methods described in the examples below.

[0186] [Average particle size (nm) of nanoparticles]

[0187] The hyaluronic acid derivative of this embodiment forms nanoparticles in an aqueous solution. The size of the nanoparticles formed by the hyaluronic acid derivative of this embodiment is not particularly limited; for example, the average particle size is preferably 500 nm or less, more preferably 450 nm or less, further preferably 400 nm or less, even more preferably 300 nm or less, and particularly preferably 250 nm or less. The average particle size of the nanoparticles formed by the hyaluronic acid derivative of this embodiment is preferably 10 nm or more, more preferably 20 nm or more, and even more preferably 50 nm or more.

[0188] In this application specification, the "average particle size of the hyaluronic acid derivative nanoparticles" is the z-average particle size (z-average particle size) measured by dynamic light scattering. The z-average particle size can be determined by, for example, by using a solution prepared by dissolving the hyaluronic acid derivative in phosphate buffer (10 mM) at a concentration of 1 mg / mL.

[0189] The nanoparticles formed by the hyaluronic acid derivative of this embodiment are hollow, enabling them to encapsulate various substances. For example, by dissolving the target substance to be encapsulated together with the hyaluronic acid derivative in an aqueous solution beforehand, nanoparticles can be formed with the target substance encapsulated internally and the outer shell composed of the hyaluronic acid derivative. The hyaluronic acid derivative nanoparticles of this embodiment can effectively encapsulate even relatively large substances or substances that are poorly soluble in water. Considering these aspects, the hyaluronic acid derivative of this embodiment is good as a substrate for drug delivery systems, and is particularly preferred as a substrate for sustained-release drug delivery systems.

[0190] The hyaluronic acid derivative of this embodiment can react with a cross-linking agent to obtain a cross-linked gel while maintaining the drug encapsulation state and without denaturing the drug.

[0191] [Manufacturing methods for hyaluronic acid derivatives]

[0192] The hyaluronic acid derivative of this embodiment can be obtained, for example, by converting the carboxyl group of glucuronic acid to an amide, introducing a steroid group directly or via a linker in at least one portion, and introducing a maleimide group directly or via a linker in another portion. The steroid introduction rate can be controlled by adjusting the amount of the compound containing the steroid group reacting with the raw material hyaluronic acid or its derivative; the maleimide introduction rate can also be controlled by adjusting the amount of the compound containing the maleimide group reacting with the raw material hyaluronic acid or its derivative. Alternatively, the compound containing the steroid group may be reacted with hyaluronic acid as a raw material, and then the compound containing the maleimide group may be reacted with the resulting reactant; or the compound containing the maleimide group may be reacted with hyaluronic acid as a raw material, and then the compound containing the steroid group may be reacted with the resulting reactant; or the compound containing the maleimide group and the compound containing the steroid group may be added together to the reaction system and reacted with the raw material hyaluronic acid. The method of introducing maleimide or steroid groups into hyaluronic acid as a raw material can be implemented, for example, by appropriately modifying the methods described in Japanese Patent Application Publication No. 2021-123597, Japanese Patent Application Publication No. 2022-013861, and Japanese Patent Application Publication No. 2022-044579.

[0193] As a method for converting the carboxyl group of glucuronic acid into an amide and introducing a steroid or maleimide group, specific examples include: ion-exchanging raw hyaluronic acid or a derivative thereof with a tetraalkylammonium salt (e.g., tetrabutylammonium (TBA) salt), and reacting the hyaluronic acid salt with an amine with a steroid (especially a cholesteric group) introduced in a solvent in the presence of a suitable condensing agent.

[0194] There are no particular limitations on the condensing agents that can be used in the above reactions, for example: 4-(4,6-dimethoxy-1,3,5-triazine)-4-methylmorpholinonium salt (DMT-MM), N,N'-carbonyldiimidazole (CDI), N,N'-dicyclohexylcarbodiimide (DCC), N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), 2-benzotriazole-1,1,3,3-tetramethylureatetrafluoroethylene Borate (TBTU), 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HODhbt), benzotriazine-1-oxy-tripyrrolidine-phosphine hexafluorophosphate (PyBOP), benzotriazine-1-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), etc.

[0195] In particular, while not limited, DMT-MM is preferred based on its high efficiency in reacting even in mixed solvents of water and organic solvents. Furthermore, by using DMT-MM as a condensing agent, amide bonds can be formed between amino and carboxyl groups with high selectivity in systems where a large number of hydroxyl groups coexist, while simultaneously inhibiting ester bond formation. By using this condensing agent, for example, it is possible to prevent the reaction between the alcohol as a solvent and the carboxyl group of the hyaluronic acid moiety, or to prevent the carboxyl and hydroxyl groups coexisting in the hyaluronic acid moiety from bonding intramolecularly or intermolecularly, leading to the formation of unwanted crosslinks.

[0196] Examples of solvents used in steroid introduction reactions include: water, DMSO, methanol, ethanol, propanol, butanol, isopropanol, polyols, acetonitrile, DMF, THF, dichloromethane, chloroform, hexane, diethyl ether, ethyl acetate, and mixtures thereof. Polyols can be diols or triols. Examples of diols include: ethylene glycol, diethylene glycol, propylene glycol, dipropylene glycol, neopentyl glycol, 1,4-butanediol, and 1,6-hexanediol. Examples of triols include: glycerol and trimethylolpropane.

[0197] In the steroid introduction reaction, the pH of the reaction system is preferably acidic. Under alkaline conditions, the maleimide group may be deactivated and generate a succinimide group.

[0198] Alternatively, the raw material hyaluronic acid or its derivative can be ion-exchanged with a tetraalkylammonium salt (e.g., tetrabutylammonium (TBA) salt), and the hyaluronic acid salt can be reacted with the spacer group in a solvent in the presence of a suitable condensing agent (protection and deprotection reactions can also be performed as needed) to convert the carboxyl group (-COOH) of the raw material hyaluronic acid or its derivative, and then reacted with a suitable reagent. Examples of combinations of carboxyl-derived groups and reaction reagents are shown below.

[0199] -CONR a -Y-NR b H + Hal-R;

[0200] -CONR a -Y-NR b H + Hal-COOR;

[0201] -CONR a -Y-NR b H + HOCO-R;

[0202] -CONR a -Y-NR b H + Hal-CO-R;

[0203] -CONR a -Y-NR b -COOH + HNR c -R;

[0204] -CONR a -Y-NR b -CO-NR c H + Hal-R;

[0205] -CONR a -Y-NR b H + HOCO-NR c -R;

[0206] -CONR a -Y-NR b H + Hal-CO-NR c -R;

[0207] -CONR a -Y-COOH + HO-R;

[0208] -CONR a -Y-OH + Hal-COO-R;

[0209] -CONR a -Y-OCOOH + HO-R;

[0210] -CONR a -Y-OCOOH + Hal-R;

[0211] -CONR a -Y-OCO-Hal + HO-R;

[0212] -CONR a-Y-SH + Hal-R;

[0213] -CONR a -Y-Hal + HS-R;

[0214] -CONR a -Y-CO-Y a -Hal + HS-R;

[0215] -CONR a -Y-CO-Y a -SH + Hal-R;

[0216] -CONR a -YO-CO-CH=CH2 + HS-R;

[0217] -CONR a -Y-NR b -CO-CH(CH3)=CH2 + HS-R;

[0218] -CONR a -Y-SH + HS-R;

[0219] -COZ-OH + HNR a -Y-NR b -COO-R;

[0220] -COZ-NR a -Y-NR b H + Hal-COO-R

[0221] (where R) a R b R c Y, Y a Y b And Z as defined in this specification, Hal represents a halogen atom selected from fluorine, chlorine, bromine and iodine.

[0222] Examples of reaction methods include: dehydrohalogenation reaction, condensation reaction, dehydration reaction, nucleophilic addition reactions such as Michael addition, and oxidized disulfide bond formation reaction. These are well-known reactions, and those skilled in the art can appropriately select and discover preferred reaction conditions. When the transformant or reactant has a carboxyl group, it can also be prepared as an N-hydroxysuccinimide (hereinafter also referred to as "NHS") ester for reaction.

[0223] Alternatively, the following method can be used: reacting 2-aminoethyl-2-pyridyl disulfide with the carboxyl group of hyaluronic acid or its derivative to prepare a hyaluronic acid derivative with a thiol group modified at the end by a leaving group, and then subjecting thiol cholesterol to a nucleophilic substitution reaction to form a disulfide bond.

[0224] Furthermore, the following method can be used: preparing a product by introducing a spacer group onto the carboxyl group of hyaluronic acid or its derivative and a product by introducing a spacer group onto the steroidal group, and then reacting them. As described above in some specific examples, further, in the case of inserting -SS- into Y, the following method can be used: preparing a hyaluronic acid derivative by introducing a spacer group terminally with a thiol group onto the carboxyl group of hyaluronic acid and a steroidal group by introducing a spacer group terminally with a thiol group, and then subjecting them to an oxidation reaction to form a disulfide bond. In this case, one of the thiol groups can also be reacted with 2-mercaptopyridine to form a disulfide, and then substituted with the other thiol group.

[0225] Furthermore, after preparing the hyaluronic acid derivative, other substituents can be introduced. For example, 0.1% to 95.0%, preferably 10% to 60%, of the carboxyl group in the hyaluronic acid derivative can be converted to -CO-X. z This allows for chemical cross-linking within the molecule or between molecules containing other molecules, resulting in gelation.

[0226] [Here, X] z Selected from the following groups:

[0227] -NH-(CH2) p1 -O-CO-C(R) 17 =CH2;

[0228] -NH-(CH2) p1 -O-CO-CH(R) 17 )-CH2-S-CH2-CH(OH)-CH(OH)-CH2-SH;

[0229] -NH-(CH2) p1 -SH;

[0230] -NH-(CH2) p1 -NH-CO-C(R) 17 =CH2;

[0231] -NH-(CH2) p1 -NH-C (=NH)-(CH2)3-SH;

[0232] -NH-(CH2) p1 -NH-CO-(CH2) r -SH;

[0233] -NH-(CH2) p1 -NH-CO-CH(R) 17 )-CH2-S-CH2-CH(OH)-CH(OH)-CH2-SH;

[0234] -NH-(CH2) p1 -NH-CO-CH(NH2)-CH2-SH;

[0235] -NH-(CH2) p1 -NH-CO-CH(NH2)-(CH2)2-SH;

[0236] -NH-NH-CO-(CH2)4-CO-NH-NH-C(=NH)-(CH2)3-SH;

[0237] -NH-(CH2-CH2-O) q -CH2-CH2-O-CO-C(R) 17 )=CH2;

[0238] -NH-(CH2-CH2-O) q -CH2-CH2-O-CO-CH(R) 17 )-CH2-S-CH2-CH(OH)-CH(OH)-CH2-SH;

[0239] -NH-(CH2-CH2-O) q -CH2-CH2-SH;

[0240] -NH-(CH2-CH2-O) q -CH2-CH2-NH-CO-C(R) 17 )=CH2;

[0241] -NH-(CH2-CH2-O) q -CH2-CH2-NH-C(=NH)-(CH2)3-SH;

[0242] -NH-(CH2-CH2-O) q -CH2-CH2-NH-CO-(CH2) r -SH;

[0243] -NH-(CH2-CH2-O) q -CH2-CH2-NH-CO-CH(R) 17 )-CH2-S-CH2-CH(OH)-CH(OH)-CH2-SH;

[0244] -NH-(CH2-CH2-O) q -CH2-CH2-NH-CO-CH(NH2)-CH2-SH;

[0245] -NH-(CH2-CH2-O) q -CH2-CH2-NH-CO-CH(NH2)-(CH2)2-SH;

[0246] -NH-CH(CO2H)-(CH2)-SH;

[0247] -NH-CH(CO2H)-(CH2)2-SH; and

[0248] -NH-CH(CO2H)-(CH2)2-CONH-CH(CONH-CH2-CO2H)-CH2-SH

[0249] (Here, R) 17 Represents a hydrogen atom or C 1-6 Alkyl group, p1 represents an integer of 2 or more and less than 10, q represents an integer of 1 or more and less than 200, r represents an integer of 1 or more and less than 3).

[0250] Hyaluronic Acid Crosslinking Gel

[0251] The hyaluronic acid derivative of this embodiment can be gelled through chemical crosslinking. That is, the hyaluronic acid crosslinked gel of this embodiment is a gel obtained by chemically crosslinking the hyaluronic acid derivative of this embodiment with a crosslinking agent having two or more crosslinking groups per molecule.

[0252] The hyaluronic acid crosslinked gel of this embodiment can be obtained by gelling a liquid composition obtained by dissolving the hyaluronic acid derivative and crosslinking agent of this embodiment in a solvent through a chemical crosslinking reaction. In the chemical crosslinking reaction, reaction conditions such as the concentration of the hyaluronic acid derivative, the type and concentration of the crosslinking agent, the type of solvent, solvent pH, salt concentration, temperature, and time can be appropriately determined. For example, by increasing the concentration of the crosslinking agent during chemical crosslinking and the introduction rate of crosslinking-forming groups onto the hyaluronic acid derivative and crosslinking agent, the crosslinking density of the resulting gel can be increased.

[0253] In the gelation step of the hyaluronic acid derivative in this embodiment, when using a crosslinking agent with crosslinking groups (crosslinking groups) at both ends, it is preferable to add the agent at a concentration in which the group rapidly participates in the crosslinking reaction without excess or deficiency. For example, since maleimide groups bind to thiol groups through a condensation reaction, when using a compound having two or more thiol groups (SH) per molecule as a crosslinking agent and performing crosslinking via Michael addition reaction, the ratio of maleimide groups to SH groups is preferably 3:1 to 1:3, and particularly preferably 2:1 to 1:2.

[0254] As a crosslinking agent, there is no particular limitation as long as it is a compound having two or more crosslinking groups per molecule. As the hyaluronic acid crosslinking gel of this embodiment, it is preferable to use a compound having two or more thiol groups per molecule as a crosslinking agent to chemically crosslink the hyaluronic acid derivative of this embodiment into a gel-like substance. Examples of compounds having two or more thiol groups per molecule include: DTT (dithiothreitol), SH-containing PEG compounds with thiol groups at both ends of a single-chain PEG, and SH-containing PEG compounds having branched PEG chains with three to eight arms and thiol groups at the end of each arm. The molecular weight of these SH-containing PEG compounds can be adjusted by regulating the degree of polymerization of PEG.

[0255] In gelation, the higher the solid fraction concentration of the liquid composition in the solvent containing the hyaluronic acid derivative and the crosslinking agent, the higher the osmotic pressure, and the easier it is to swell during gelation. Therefore, under the same reaction conditions, the higher the concentration of the hyaluronic acid derivative, the greater the swelling degree of the resulting crosslinked gel. Similarly, under the same reaction conditions, the higher the concentration of the crosslinking agent, the greater the swelling degree of the resulting crosslinked gel. Furthermore, with the same number of crosslinking groups per molecule, the larger the molecular weight of the crosslinking agent, the greater the mass of crosslinking agent required to add the required molar amount of thiol groups to the hyaluronic acid derivative, and therefore the greater the swelling degree.

[0256] Examples of crosslinking agents used in the manufacture of the hyaluronic acid crosslinked gel according to this embodiment include compounds containing two or more thiol groups in the same molecule that react with unsaturated bonds through nucleophilic addition reactions. Examples of such crosslinking agents include polyethylene glycol dithiol and peptides containing two or more cysteine ​​residues.

[0257] As the crosslinking agent used in the manufacture of the hyaluronic acid crosslinking gel of this embodiment, it is preferably a PEG compound with 2 to 8 arms having SH groups at the end of the arms, more preferably a PEG compound with 3 to 6 arms having SH groups at the end of the arms, even more preferably a PEG compound with 4 arms having SH groups at the end of the arms, even more preferably a PEG compound with 4 arms having SH groups at the end of the arms and a weight-average molecular weight of 2k to 40k, and particularly preferably a PEG compound with 4 arms having SH groups at the end of the arms and a weight-average molecular weight of 5k to 20k.

[0258] In the step of gelling the hyaluronic acid derivative, the solvent is preferably one that can sufficiently dissolve the hyaluronic acid derivative and the crosslinking agent. While not particularly limited, water, dimethyl sulfoxide (DMSO), dimethylacetamide (DMAc), dimethylformamide (DMF), N-methylpyrrolidone (NMP), and mixtures selected from these solvents are preferred. Alternatively, an organic solvent mixed with these solvents may also be used. While not particularly limited, examples of mixed organic solvents include, for example, methanol, ethanol, propanol, isopropanol, butanol, polyols, acetone, acetonitrile, etc. As polyols, the same polyols exemplified above can be used, with ethylene glycol being preferred.

[0259] Furthermore, to improve the stability of proteins or peptides during the cross-linking reaction and to increase the reaction rate, it is preferable to add a basic compound. There are no particular limitations on the basic compound used; examples include carbonates such as sodium carbonate and sodium bicarbonate, hydroxides such as sodium hydroxide and sodium hydroxide, and amines such as ammonia, pyridine, triethylamine, ethylenediamine, ethanolamine, diethanolamine, and triethanolamine. Amines are preferred, and triethanolamine is more preferred.

[0260] Hyaluronic acid derivatives, by forming nanoparticles in aqueous solution as a targeting carrier, can be cross-linked under dilute conditions to form nano-sized microparticle gels, which can be used as sustained-release carriers in blood. Dilute conditions refer to concentrations of 10 mg / mL or less, preferably 5 mg / mL or less, and more preferably 1 mg / mL or less. On the other hand, by cross-linking under high concentration conditions, a blocky gel formed by cross-linking microparticles can be formed. This is useful as a subcutaneous sustained-release carrier. High concentration conditions refer to concentrations of 5 mg / mL or more, preferably 20 mg / mL or more, and more preferably 30 mg / mL.

[0261] The gelation of hyaluronic acid derivatives can be carried out in bulk form or in a discontinuous phase such as an emulsion or spray droplet. For example, in the case of a W / O emulsion, the gelation reaction can be carried out by emulsifying an aqueous phase containing dissolved hyaluronic acid derivatives and crosslinking agents in a solvent that is immiscible with water. While there are no particular limitations on the immiscible solvent, examples include hexane, chloroform, dichloromethane, ethyl acetate, medium-chain triglycerides (MCT), liquid paraffin, and soybean oil. Surfactants can also be added to stabilize the emulsion. Furthermore, the gelation can be carried out in a desolvable solvent such as supercritical carbon dioxide or PEG. In this case, by emulsifying and dispersing the aqueous or organic solvent phase containing dissolved hyaluronic acid derivatives and crosslinking agents in the aforementioned solvent, polymer concentration is achieved along with desolvation (solvent diffusion), thus resulting in a gel with a higher crosslinking density.

[0262] After the gelling step of the hyaluronic acid derivative, operations such as stopping the cross-linking reaction and deactivating or rinsing residual cross-linking functional groups can also be performed. From the perspectives of safety, stability during storage, and side reactions with the encapsulated drug, it is preferable to remove unreacted cross-linking functional groups, groups that only bind to one side of the cross-linking agent, and residual cross-linking agents. Although there are no particular limitations, for example, in the case of residual unreacted cross-linking agent, it can be removed by rinsing with excess water or the like.

[0263] After the step of gelling the hyaluronic acid derivative, a pulverization step may also be performed. Examples of pulverization methods include pulverization using a mortar and pestle or pulverization using a grinder, with pulverization using a grinder being preferred. Examples of grinder pulverization devices include: rotary disc pulverizers such as centrifugal grinders (manufactured by Nippon Seiki Co., Ltd.) and impact grinders (manufactured by Dalton Corporation); pulverization devices using sieve grinders such as atomizers (manufactured by Tokyo Atomizer Co., Ltd.), sample grinders (manufactured by Tokyo Atomizer Co., Ltd.), bantum grinders (manufactured by Tokyo Atomizer Co., Ltd.), and SK grinders (manufactured by Tokken Co., Ltd.); jet pulverization devices such as ultra-small batch laboratory jet grinders (AO jet grinders, manufactured by Seishin Corporation); and linrex grinders (manufactured by Liquid Gas Co., Ltd.) capable of pulverization at ultra-low temperatures, with SK grinders and linrex grinders being preferred.

[0264] The hyaluronic acid crosslinked gel of this embodiment can be a gel obtained by chemically crosslinking the hyaluronic acid derivative of this embodiment with a crosslinking agent, or it can be a dried product obtained by drying it. Examples of drying methods include freeze drying, ventilation drying, drying in a constant temperature bath, vacuum drying, and hot air circulation drying. The air velocity, drying time, temperature, and pressure can be appropriately selected within a range that prevents the hyaluronic acid derivative gel from decomposing or deteriorating.

[0265] The hyaluronic acid crosslinked gel desiccant and the hyaluronic acid crosslinked gel of this embodiment are porous structures in either the dried or swollen state. For example, this allows them to support larger components such as cells, and the desiccant is also suitable as a substrate for cells or tissues used in regenerative medicine. For example, cells intended for transplantation can be cultured and adhered within the desiccant, and then transplanted into the target tissue. Furthermore, the desiccant and crosslinked gel can also function as scaffolds for cell growth in in situ regeneration therapy. For example, by embedding the desiccant into tissue within a living organism, cells migrating from surrounding tissues can colonize using the desiccant as a scaffold.

[0266] The dried hyaluronic acid cross-linked gel of this embodiment and the hyaluronic acid cross-linked gel of this embodiment can control cell adhesion by changing the introduction rate of cholesterol derivatives.

[0267] ≪Pharmaceutical Compositions≫

[0268] The pharmaceutical composition of this embodiment contains the hyaluronic acid derivative. In the pharmaceutical composition of this embodiment, the hyaluronic acid derivative mainly functions as a carrier of the active ingredient. When this pharmaceutical composition is administered into a living organism, the active ingredient, such as the drug, is slowly released from the carrier containing the hyaluronic acid derivative, and good sustained-release properties can be expected.

[0269] <Active Ingredients>

[0270] The active ingredient contained in the pharmaceutical composition of this embodiment is not particularly limited as long as it is a medicine for human or animal use (a substance administered for the diagnosis, treatment, or prevention of a disease) or a substance used as its active ingredient. This active ingredient can be a water-soluble substance or a poorly soluble substance. Examples of such active ingredients include: proteins, peptides, polysaccharides, oligosaccharides, nucleic acids, oligonucleotides, low-molecular-weight compounds, cells, etc. Furthermore, it can also be a protein-nucleic acid complex such as the Cas9 / gRNA complex.

[0271] Examples of pharmaceutical compositions according to this embodiment include: nanoparticles formed from hyaluronic acid of this embodiment and encapsulating active ingredients; substances formed by colonizing transplantation cells with dried hyaluronic acid crosslinked gel and hyaluronic acid crosslinked gel of this embodiment; and hyaluronic acid crosslinked gel and dried hyaluronic acid crosslinked gel containing poorly soluble drugs.

[0272] <morphology>

[0273] The pharmaceutical composition of this embodiment can be a dispersible particulate solution, a precipitated suspension, or a lyophilized product. Furthermore, in the case of a lyophilized product, it can be a precipitated sustained-release formulation that a physician can pre-prepare by adding saline or other osmotic fluid to prepare an administration solution before application. In this case, it is considered suitable for pharmaceutical compositions containing unstable active ingredients in solution form.

[0274] When the pharmaceutical composition of this embodiment is a dispersible particulate solution or a precipitating 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.

[0275] The pharmaceutical composition of this embodiment is not limited to the described form and may also be in the form of nanoparticles, microparticles, solutions, emulsions, suspensions, gels, micelles, implants, powders, or films. Powders may be manufactured by pulverizing solids obtained by freeze-drying or spray-drying, or by drying precipitates.

[0276] The pharmaceutical composition of this embodiment can be administered via oral, parenteral, nasal, vaginal, intraocular, subcutaneous, intravenous, intramuscular, intradermal, intraperitoneal, intracerebral, or oral routes. Furthermore, the pharmaceutical composition of this embodiment is not limited to injections, but may also be a patch or microneedle formulation, topical medication, eye drops, spray, inhalation medication, etc.

[0277] Example

[0278] The present invention will now be described in detail with reference to embodiments, but these are not intended to limit the scope of the invention to the embodiments.

[0279] The methods for determining and evaluating the physical properties of the hyaluronic acid derivatives manufactured in the Examples and Comparative Examples are described below. Unless otherwise stated, all experiments were repeated three times.

[0280] < 1 H NMR measurement>

[0281] 1 Unless otherwise specified, 1H NMR measurements were performed using 0.02N DCl / d6-DMSO as the solvent and a JEOL JNM-A400 spectrophotometer (manufactured by JEOL) equipped with a 400MHz NMR instrument (JNM-ECS400, manufactured by JEOL).

[0282] <Determination by Multi-Angle Laser Scattering Combined with Size Exclusion Chromatography>

[0283] HA derivatives were analyzed using a Wyatt Dawn NEON multi-angle light scattering detector and an Optilab refractive index monitor equipped with an isocratic HPLC system (Waters Corporation) for SEC-MALS analysis. First, the HA derivatives were diluted to 1.0 mg / mL in SEC-MALS buffer (10 mM phosphate buffer, pH 7.4). Separation was performed using a G4000SWXL column (Tosoh Corporation) at a flow rate of 0.5 mL / min in SEC-MALS buffer. Data analysis was performed using the refractive index concentration coefficient dn / dc of hyaluronic acid (0.153, literature value) and Astra software (Wyatt Technology Corporation).

[0284] <Mean particle size determined by dynamic light scattering (DLS)>

[0285] The performance of the HA derivatives was evaluated using DLS analysis with an ELSZ-2000 (manufactured by Otsuka Electronics) in solutions of the same solvent as SEC (10 mM phosphate buffer, pH 7.4). The measured autocorrelation functions were processed using the cumulant method. The hydrodynamic diameter of the HA derivatives was analyzed using the Stokes-Einstein equation.

[0286] <Inverted vial test (HA derivative cross-linking and gelation test)>

[0287] The HA derivative crosslinked gel was prepared by crosslinking based on a Michael addition reaction between maleimide and thiol groups. Unless otherwise specified, a PEG compound with SH groups at the ends of the four arms, namely 4arm-PEGSH (molecular weight = 1 × 10⁻⁶), was used as the crosslinking agent for gelation. 4 g / mol, manufactured by Nikkei Oil Company.

[0288] The gelation ability of the HA derivative was analyzed based on the state when the vial was inverted. Specifically, after completely dissolving the HA derivative and PEGSH in a solvent (10 mM phosphate buffer, pH 7.4) respectively, it was cooled to 5 °C. Then the two were injected into a vial and incubated at 37 °C for 30 minutes. The vial was inverted at regular intervals starting from the beginning of incubation to confirm whether the sample flowed and dripped. Samples that did not show flow within 5 minutes or 30 minutes after the start of incubation were classified as gels, and samples that were still flowing 30 minutes after the start of incubation were classified as sols. All samples were classified into either gels or sols.

[0289] <Preparation of HA Derivative Crosslinked Gel>

[0290] While cooling to 5 °C, two precursor solutions (phosphate buffer solution of HA derivative and phosphate buffer solution of PEGSH) were injected into a disc-shaped silicone rubber mold (diameter 6 mm, depth 1 mm) placed on a polytetrafluoroethylene membrane. Then, the mold was covered with a silicone cover plate and incubated at 37 °C for 30 minutes. After incubation, the sample was transferred to a glass vial. The molar ratio of maleimide group to thiol group was 1.1:1.

[0291] <Swelling of Hydrogel>

[0292] The HA derivative crosslinked gel (φ = 6 mm, thickness = 1.0 mm) was placed at the bottom of a pre-weighed replica glass vial (n = 4). The initial weight of the vial containing the HA derivative crosslinked gel was measured as the initial mass. Then, 1 mL of PBS was added and incubated at 37 °C. At the time points of 1, 12, and 24 hours, the buffer solution was carefully removed from each vial, and the weight of the vial was measured to determine the mass after swelling. Then, fresh PBS was 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 crosslinked gel was calculated by dividing the mass after swelling by the initial mass.

[0293]

Equation 1

[0294]

[0295] <Experiment on Encapsulation and Release of Protein in HA Derivative Crosslinked Gel>

[0296] A cross-linked gel containing the HA derivative (φ=6 mm, thickness=1.0 mm) was prepared in the same manner as described above and immersed in PBS. After reaching equilibrium swelling, the HA derivative cross-linked gel was immersed in a FITC-insulin (100 μg / mL) PBS solution at 37°C. Then, a sample (200 μL solution) from a specific spot on the gel was recovered, and its fluorescence intensity was measured using a DeNovix DS-11 FX+ spectrophotometer fluorometer (“Fluorescent Protein” module). The encapsulation efficiency of FITC-insulin was estimated by the decrease in fluorescence intensity.

[0297] Simultaneously, an in vitro release assay of FITC-insulin derived from HA derivatives in the presence of serum was evaluated. A cross-linked gel (φ=6 mm, thickness=1 mm) of HA derivatives complexed with FITC-insulin was immersed in 1 mL of PBS (pH 7.4) containing 10% fetal bovine serum (FBS) at 37°C. Subsequently, samples (200 μL of gel supernatant) were collected at specific intervals, and fluorescence intensity and FITC-insulin encapsulation efficiency were measured in the same manner as described above.

[0298] [Example 1]

[0299] A HA derivative (HA-C6-Chol-Male, sometimes referred to as "HAMICH") was prepared by introducing a steroid and a maleimide group onto at least a portion of the carboxyl group in the glucuronic acid moiety of hyaluronic acid.

[0300]

Transformation 5

[0301]

[0302] The preparation of cholesterol 6-aminohexylcarbamate hydrochloride (Chol-C6 hydrochloride) and tetrabutylammonium salt of hyaluronic acid (HA-TBA) was carried out according to the methods described in Examples 1 and 2 of Patent Document 1.

[0303] Specifically, HA-TBA is obtained by converting sodium hyaluronate (HA-Na) (average molecular weight = 120 kDa, purchased from Bloomage Biotechnology Japan) as a starting material into HA using a cation exchange resin (Dowex 50WX-8-400, manufactured by Sigma-Aldrich), and then reacting it with N-(5-aminopentyl)maleimide hydrochloride using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (manufactured by a domestic chemical company) as a condensing agent.

[0304] Completely dissolve HA-TBA in DMSO (1% w / v), then add N-(5-aminopentyl)maleimide dissolved in DMSO and stir at room temperature for 5 minutes. Next, add DMT-MM to the mixture and stir overnight at room temperature. The molar ratio of the feed is 100:20:24 (HA glucuronic acid: DMT-MM: N-(5-aminopentyl)maleimide hydrochloride, x=20).

[0305] Next, cholesterol-6-aminohexylcarbamate is reacted in the same manner. The molar ratio of the feed is 100:x:1.2x (HA glucuronic acid: DMT-MM: cholesterol-6-aminohexylcarbamate, x = 1.2, 5, 10, 16, 22, 31 or 44).

[0306] The resulting reaction solution was dialyzed sequentially with DMSO solution, 0.15M NaCl aqueous solution, and ultrapure water (SpectrPore 4, molecular weight cutoff (MWCO): 12kDa~14kDa, manufactured by Spectrum Laboratories). The dialysate was freeze-dried to obtain the target compounds (HA-C6-Chol-1%-Male15%~HA-C6-Chol-40%-Male15%) listed in Table 1, which were white solids.

[0307] As a control, a hyaluronic acid derivative modified with maleimide groups but without reacting with Chol hydrochloride was prepared using the same method as described above. The target compound (HA-C6-Chol-0%-Male) listed in Table 1 was obtained as a white solid.

[0308] Table 1

[0309]

[0310] Each compound is synthesized through 1 The structure was confirmed by 1H NMR. The NMR analysis used a sample in which HAMICH was dissolved in 0.02 NDCl / DMSO. 1 The 1H NMR results showed that the degree of maleimideization (maleimide group incorporation) of each HA derivative was 15%. The NMR spectra of each synthesis are shown below. Figure 1A (Chol-0%)~ Figure 1H (Chol-40%).

[0311] The z-average particle size (nm) of each HAMICH was determined. The results are shown in Table 2. Furthermore, the degree of association of each HAMICH was calculated based on the absolute molecular weight determined by GPC-MALS analysis and the theoretical average molecular weight of each unit. The results are shown in Table 2.

[0312] Table 2

[0313]

[0314] In HAMICH, the particle size decreased in a rate-dependent manner up to 20% cholesterol introduction. However, when the cholesterol introduction rate increased to 40%, the particle size increased, indicating that the cholesterol introduction rate affects the particle size of HA derivative nanoparticles. Furthermore, in HAMICH without cholesterol modification (0%), almost no association occurred because the hydrophobic interaction of the steroid groups was not utilized.

[0315] [Example 2]

[0316] Using the HAMICH manufactured in Example 1, a HAMICH crosslinked gel was prepared by chemical crosslinking with the crosslinking agent 4arm-PEGSH.

[0317] First, each HAMICH prepared in Example 1 was dissolved in 10 mM phosphate buffer (pH 7.4) to achieve the concentrations described in Table 3, thus preparing HAMICH solutions. In another 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 PEGSH solutions. After cooling both solutions to 5°C, the two solutions were mixed in a 1 mm thick, 6 mm diameter silicone mold, with the molar ratio of maleimide groups modified on the hyaluronic acid to SH groups derived from 4arm-PEGSH being 1.1:1.0, and HAMICH crosslinking gels were manufactured at 37°C. All procedures were carried out under the condition that the final concentration of the hyaluronic acid derivative reached 7 mg / mL.

[0318] Table 3

[0319]

[0320] Eight HAMICHs with introduced cholesteric and maleimide groups, regardless of the cholesteric group introduction rate, all gelled fully within 1–30 minutes after the start of gelation (mixing HAMICH and PEGSH solutions), forming cross-linked gels. As shown in Comparative Example 2 below, in HAMAs (HA-C6-Chol-3%-MA, HA-C6-Chol-19%-MA) with introduced methacrylyl groups, hyaluronic acid derivatives with introduced steroidal and methacrylyl groups were difficult to gel. These results indicate that by combining steroidal and maleimide groups in the introduction of hyaluronic acid, rapid gelation, which is difficult to achieve with hyaluronic acid derivatives with introduced steroidal and methacrylyl groups, can be achieved.

[0321] Furthermore, the volume change after gelation in PBS was measured, and the swelling degree was investigated. The results are shown in... Figure 2 (A). Additionally, for HA-C6-Chol-0%-Male and HAMICH (HA-C6-Chol-20%-Male) with a cholesterol introduction rate of 20%, gelation reactions were performed under the same conditions, except that the final concentration of the hyaluronic acid derivative was set to 16.7 mg / mL. The volume change from the start of gelation was measured, and the degree of swelling was investigated. The results are shown in... Figure 2 (B). For example Figure 2 As shown, swelling of the gel was observed over time in unmodified cholesteric HA-C6-Chol-0%-Male. In contrast, HAMICH modified with both maleimide and steroid groups exhibited less swelling compared to HAMICH modified with only maleimide, thus suppressing the characteristic property of increased volume in vivo after gelation.

[0322] [Comparative Example 1]

[0323] A HA derivative (HA-C6-Chol-MA, sometimes referred to as "HAMA") was prepared by introducing a steroid and a methacryloyl group onto at least a portion of the carboxyl group in the glucuronic acid moiety of hyaluronic acid.

[0324]

Transformation 6

[0325]

[0326] The preparation of cholesterol 6-aminohexylcarbamate hydrochloride (Chol-C6 hydrochloride) and tetrabutylammonium salt of hyaluronic acid (HA-TBA) was carried out according to the methods described in Examples 1 and 2 of Patent Document 1.

[0327] Specifically, HA-TBA is obtained by converting sodium hyaluronate (HA-Na) (average molecular weight = 120kDa, purchased from Bloomage Biotechnology Japan) as a starting material into HA using a cation exchange resin (Dowex 50WX-8-400, manufactured by Sigma-Aldrich), and then reacting it with 2-aminoethyl methacrylate hydrochloride (manufactured by Fujifilm and Koko, product number 510-51961) as a condensing agent using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (manufactured by Kokusai Chemical Co., Ltd.) as a condensing agent.

[0328] Completely dissolve HA-TBA in DMSO (1% w / v), then add 2-aminoethyl methacrylate hydrochloride (manufactured by Fujifilm and Kogaku: product number 510-51961) dissolved in DMSO, and stir at room temperature for 5 minutes. Next, add DMT-MM to the mixture and stir overnight at room temperature. The molar ratio of the reactants is 100:20:24 (HA glucuronic acid: DMT-MM: 2-aminoethyl methacrylate hydrochloride, x=20).

[0329] Next, cholesterol-6-aminohexylcarbamate was reacted in the same manner. The molar ratio of the feed was 100:x:1.2x (HA glucuronic acid: DMT-MM: cholesterol-6-aminohexylcarbamate, x=2.0, 19).

[0330] The HA product was purified by sequentially dialysis with DMSO, then with 0.150 M NaCl, and finally with distilled water (MWCO: 12–14 kDa). The purified HA solution was filtered through a 0.22 μm membrane filter and freeze-dried until dry.

[0331] Subsequently, 2-aminoethyl methacrylate hydrochloride was added to each solution in proportions relative to the HA-TBA units shown in Table 4. Next, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine hydrochloride (DMT-MM) (manufactured by a domestic chemical company) was added to each solution in proportions shown in Table 4, relative to the HA-TBA units, and the mixture was stirred overnight. Then, Chol-C6 hydrochloride was added to each solution in proportions shown in Table 4, relative to the HA-TBA units.

[0332] Next, DMT-MM was added relative to the HA-TBA unit at the ratio shown in Table 4, and the mixture was stirred overnight at room temperature. Finally, the reaction solution was dialyzed sequentially with DMSO solution, 0.15M NaCl aqueous solution, and ultrapure water (SpectrPore 4, molecular weight cutoff (MWCO): 12kDa~14kDa, manufactured by Spectrum Laboratories). The resulting dialysate was freeze-dried to obtain the target compound (HA-C6-Chol-MA) listed in Table 4, which was a white solid.

[0333] Table 4

[0334]

[0335] [Comparative Example 2]

[0336] Using the HAMA manufactured in Comparative Example 1, a cross-linked HAMA gel was prepared by chemical cross-linking with the cross-linking agent 4arm-PEGSH.

[0337] First, each HAMA prepared in Comparative Example 1 was dissolved in 10 mM phosphate buffer (pH 7.4) to achieve the concentrations listed in Table 5, thus preparing HAMA solutions. In another container, 4arm-PEGSH (manufactured by Nippon Oil Co., Mw: 10,000) was dissolved in 10 mM phosphate buffer (pH 7.4) at a concentration of 50 mg / mL to prepare PEGSH solutions. After cooling both solutions to 5°C, the two solutions were mixed in a 1 mm thick, 6 mm diameter silicone mold, with the molar ratio of the methacryloyl groups modified on the hyaluronic acid to the SH groups derived from 4arm-PEGSH being 1.1:1.0, and HAMA crosslinking gels were manufactured at 37°C. All procedures were carried out under the condition that the final concentration of the hyaluronic acid derivative reached 7 mg / mL.

[0338] Table 5

[0339]

[0340] HAMA (HA-C6-Chol-3%-MA, HA-C6-Chol-19%-MA) with introduced methacryl groups were not fully gelled 30 minutes after the start of gelation, and were not fully gelled 24 hours after the start of gelation.

[0341] [Comparative Example 3]

[0342] Furthermore, using the same method as Comparative Example 2, crosslinking was attempted on the HAMA (HA-C6-Chol-19%-MA) with introduced methacryl groups under the same conditions, except that the final concentration of the hyaluronic acid derivative was 25 mg / mL, using 4arm-PEGSH (manufactured by Nippon Oil Co., Mw: 10,000) dissolved at a concentration of 100 mg / mL. As a result, the HAMA did not crosslink even under higher concentration conditions.

[0343] [Example 3]

[0344] Fluorescently labeled insulin (FITC-insulin) was encapsulated in the HAMICH crosslinked gel prepared in Example 2, and the encapsulation efficiency was investigated.

[0345] The HAMICH crosslinked gel (6 mm φ, 1 mm thick) prepared in Example 2 with a final concentration of 7 mg / mL of hyaluronic acid derivative was immersed in 1 mL of FITC-insulin solution (250 μg / mL FITC-insulin solution dissolved in PBS) and incubated at 25°C. The encapsulation efficiency (%) of FITC-insulin in the gel was calculated by quantitatively analyzing the fluorescence intensity of the supernatant over time. The encapsulation efficiency (%) after 1 hour, 8 hours, and 24 hours after the start of incubation is shown in Table 6. Figure 3 .in addition, Figure 4 The state of each gel after encapsulation is shown.

[0346] Table 6

[0347]

[0348] As shown in Table 6, the FITC insulin encapsulation efficiency of the HAMICH cross-linked gel with introduced cholesteric groups was higher than that of the unintroduced cholesteric group cross-linked gel. Furthermore, the encapsulation efficiency of FITC-insulin was affected by the cholesteric group introduction rate; the best encapsulation efficiency was achieved when the cholesteric group introduction rate was 5–10% after 8 and 24 hours of incubation. Figure 4 As shown, even visual inspection clearly confirms that the FITC-derived color of the cross-linked gel without the introduction of chol is weaker. These results demonstrate that the hyaluronic acid derivative of this embodiment exhibits excellent encapsulation efficiency for active ingredients such as proteins, and that by optimizing the introduction rate of steroidal groups, a larger amount of active ingredients can be encapsulated.

[0349] As can be seen from the results of Examples 2-3, by using the hyaluronic acid derivative of this embodiment, rapid gelation can be achieved, and a novel cross-linked gel that can simultaneously inhibit volume changes in organisms and encapsulate a large number of proteins can be obtained.

[0350] In summary, the cross-linked gel obtained from the hyaluronic acid derivative of this embodiment can encapsulate a large number of cytokines necessary for regeneration or cell differentiation, and control cell adhesion or differentiation by releasing them, and is expected to be applied to regenerative medicine.

[0351] [Example 4]

[0352] Crosslinked gels of hyaluronic acid derivatives modified with both maleimide and steroid groups were fabricated. The crosslinked gels were verified to be porous even in a swelling equilibrium state by freeze-drying and freeze-thaw.

[0353] First, using the same method as in Example 1, a hyaluronic acid derivative (Cy5-HAMICH) modified with fluorescent Cy5 was prepared, exhibiting a cholesterol group introduction rate of 19% and a maleimide group introduction rate of 11%. Cy5 modification was performed using Cy5-amine (manufactured by Funakoshi Co., Ltd., product number: BP-22559), with 0.02 equivalents of HA-TBA unit added and 0.03 equivalents of DMT-MM added as a condensing agent. Additionally, a hyaluronic acid derivative (Cy5-HAMICH) modified with fluorescent Cy5, exhibiting no cholesterol group introduction (0% introduction rate) and a maleimide group introduction rate of 7%, was also prepared using the same method as described above. Next, for each Cy5-HAMICH, as in Example 3, a crosslinking agent was added at a maleimide group:SH group ratio of 1.1:1.0 (molar ratio) to gel, producing a crosslinked gel (8 mm diameter, 1 mm thickness). After manufacturing, the samples were left to stand in PBS for 1 hour and then subjected to freeze-thaw treatment.

[0354] The freeze-thaw process was performed as follows. First, the Cy5-modified HA derivative crosslinked gel was freeze-dried by standing at 5°C for 30 minutes at room temperature (25°C), then at -20°C for 1 hour, and finally at -78°C for 24 hours. Then, it was thawed by incubation in a 25°C water bath.

[0355] For each cross-linked gel, fluorescence microscopy images were acquired using two-photon laser microscopy (Zeiss) after freeze-drying and thawing. The fluorescence microscopy images of each cross-linked gel are shown below. Figure 5 In Cy5-HAMICH crosslinked gels with a cholesterol introduction rate of 19% ( Figure 5 In the upper section, a porous structure with a large number of fine pores with a diameter of tens of μm forming a network structure on the surface was confirmed. In contrast, in the Cy5-HAMICH crosslinked gel with a cholesterol introduction rate of 0%, Figure 5 No such pores were observed in the lower section. Based on these results, the hyaluronic acid derivative crosslinked gel of this embodiment is considered useful as a scaffold for cell infiltration and promoting regeneration.

[0356] [Example 5]

[0357] Using the same method as in Example 2, HAMICH crosslinked gel was manufactured by chemically crosslinking the HAMICH produced in Example 1 with the crosslinking agent 4arm-PEGSH.

[0358] 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 HAMICH solutions. Only HA-C6-Chol-20%-Male15% was dissolved to a concentration of 25 mg / mL. In another 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 PEGSH solutions. After cooling both solutions to 5°C, they were mixed in a vial and an inverted vial test was performed to verify gel or sol formation. At this time, before mixing the hyaluronic acid derivative and the 4arm-PEGSH solution, 10 mM phosphate buffer (pH 7.4) was added appropriately to the 4arm-PEGSH solution to achieve the final concentrations of the hyaluronic acid derivative and 4arm-PEGSH as shown in Table 7.

[0359] Table 7

[0360]

[0361] HAMICH, with the introduction of cholesteric and maleimide groups, fully gelled within 1–30 minutes after the start of gelation (mixing HAMICH and PEGSH solutions) in any of the experimental groups 5–5–30, regardless of the cholesteric group introduction rate, forming a cross-linked gel. These results indicate that hyaluronic acid derivatives obtained by incorporating steroidal and maleimide groups into hyaluronic acid can gel at various concentrations. Furthermore, it suggests that the encapsulation amount of the drug in the formed HAMICH cross-linked gel can be adjusted according to the gel concentration.

[0362] [Example 6]

[0363] Using the same method as in Example 5, HAMICH crosslinked gel was manufactured by chemically crosslinking the HAMICH produced in Example 1 with the crosslinking agent 4arm-PEGSH.

[0364] First, HAMICH (HA-C6-Chol-15%-Male15%) with a cholesterol introduction rate of 15% prepared in Example 1 was dissolved in 10 mM phosphate buffer (pH 7.4) to achieve a concentration of 10 mg / mL, thus preparing a HAMICH solution. In another container, 4arm-PEGSH (Mw: 10,000) was dissolved in 10 mM phosphate buffer (pH 7.4) at a concentration of 70 mg / mL to prepare a PEGSH solution. After cooling both solutions to 5°C, they were injected into a vial and mixed. The gel or sol was verified by an inverted vial test. At this point, before mixing the hyaluronic acid derivative with the 4arm-PEGSH solution, 10 mM phosphate buffer (pH 7.4) was appropriately added to the 4arm-PEGSH solution to achieve the final concentrations of the hyaluronic acid derivative and 4arm-PEGSH as shown in Table 8.

[0365] Table 8

[0366]

[0367] HAMICH, with 15% cholesterol and 15% maleimide groups introduced, fully gelled within 30 minutes after the start of gelation (mixing HAMICH and PEGSH solutions) in any of experimental groups 6-1 to 6-2, forming cross-linked gels. These results indicate that hyaluronic acid derivatives obtained by introducing a combination of steroidal and maleimide groups into hyaluronic acid can gel at various concentrations. Furthermore, it suggests that the encapsulation amount of the drug in the formed HAMICH cross-linked gel can be adjusted according to the gel concentration.

[0368] [Example 7]

[0369] For hyaluronic acid derivatives modified with both maleimide and steroid groups, the absolute molecular weight of the conjugates in 10 mM phosphate buffer (pH 7.4) was calculated by analysis using gel permeation chromatography (GPC-MALS) with multi-angle light scattering.

[0370] The MALS detector measures the scattering signal of polymers or particles in a sample at different scattering angles θ. The basic light scattering formula (Anderson et al, Analytical Chemistry, 2003, vol.75, p.4279-4291) is shown below. In the formula, Rθ is the excess Rayleigh ratio, K is the optical constant, which depends in particular 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 incident light wavelength. The molecular weight and radius of gyration need to be extrapolated to zero angle from the light scattering data (Wyatt, Analytica Chimica Acta, 1993, vol.272(1), p.1-40). This is done by plotting a so-called Debye plot as a function of (Kc / Rθ)¹ / ². Typically, the molecular weight can be calculated from the intercept on the ordinate, and the radius of gyration can be calculated from the initial slope of the curve.

[0371]

Number 2

[0372]

[0373] In this study, ASTRA8 (manufactured by Wyatt Technology) software was used to collect and analyze signals from the RI and MALS detectors. The calculated molecular weights, such as the absolute weight-average molecular weight Mw (absolute value), were derived using the literature value of 0.153 for hyaluronic acid as the refractive index concentration coefficient dn / dc. The measurement conditions and calculation results are shown below.

[0374] (Measurement conditions)

[0375] Device: SEC-Multi-Detector System

[0376] Column: G4000SWXL (manufactured by Tosoh)

[0377] MALS: DWAN NEON (made by Wyatt Technology)

[0378] Laser wavelength: 661nm

[0379] RI: Optilab (manufactured by NEON Corporation)

[0380] Elution buffer: 10mM PB, pH 7.4

[0381] Flow rate: 0.5 mL / min

[0382] Injection volume: 50 μL

[0383] Concentration: 1.0 mg / mL

[0384] Temperature: 25℃

[0385] Analysis software: ASTRA8 (made by Wyatt)

[0386] Table 9

[0387]

[0388] These results suggest the formation of cholesterol-driven associative compounds, indicating that even in the absence of cross-linking agents, associative compounds of hyaluronic acid derivatives modified with cholesterol and maleimide groups are formed. By pre-encapsulating drugs in nanoparticles composed of hyaluronic acid derivative associative compounds and then performing a cross-linking reaction to gel, it is expected that the effects of gelation or cross-linking reactions on the drug can be inhibited. This method allows for the production of cross-linked gels encapsulating drugs, even for drugs highly reactive to cross-linking agents, without compromising drug stability. It is believed that drug encapsulation using these hyaluronic acid derivative associative compounds is applicable to a wide range of drugs, from low molecular weight substances like insulin to high molecular weight cytokines like IL-4.

[0389] [Example 8]

[0390] An in vitro insulin release assay was performed on the HAMICH crosslinked gel encapsulating fluorescently labeled insulin (FITC-insulin) obtained in Example 3.

[0391] Five FITC-insulin-encapsulated HAMICH crosslinked gels obtained in Example 3 were each immersed in 1 mL of PBS (pH 7.4) containing 10% FBS and incubated at 37°C. The release rate (%) of FITC-insulin from each gel was calculated by quantifying the fluorescence intensity of the supernatant over time. For the cholesteryl-modified HAMICH crosslinked gels (HA-C6-Chol-1%-Male15%, HA-C6-Chol-5%-Male15%, HA-C6-Chol-10%-Male15%, and HA-C6-Chol-20%-Male15%), the release rate (%) up to 21 days after the start of immersion is shown below. Figure 6 .

[0392] In an unmodified cholesterol-crosslinked gel (HA-C6-Chol-0%-Male 15%), the entire amount of encapsulated FITC-insulin was released into the supernatant one day after the start of impregnation (release rate 100%). In contrast, as... Figure 6 As shown, even on day 21 after the start of impregnation, the release rate of FITC-insulin using the cholesteryl-modified HAMICH crosslinked gel did not reach 100%, and the sustained-release period of FITC-insulin remained for a long time. These results indicate 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 excellent sustained-release performance.

[0393] As shown in the above embodiments, the cross-linked gel obtained from the hyaluronic acid derivative of this embodiment not only gels rapidly and inhibits volume changes in vivo, but also encapsulates a large amount of active ingredients such as proteins, and exhibits excellent long-term sustained-release properties of the encapsulated material. In summary, it is anticipated that the hyaluronic acid derivative of this embodiment and the cross-linked gel obtained therefrom will be applied to regenerative medicine. The cross-linked gel obtained from the hyaluronic acid derivative of this embodiment can encapsulate a large amount of cytokines necessary for regeneration or cell differentiation, and subsequently, by releasing them, cell adhesion or differentiation can be controlled.

[0394] [Example 9]

[0395] Except for sodium hyaluronate (HA-Na) with a weight-average molecular weight of 35 kDa (purchased from Bloomage Biotechnology Japan) as the starting material, the target compound (HA-C6-Chol-1%-Male 8.1%) of experimental group 9-1, as shown in Table 10, was obtained using the same method as in Example 1. The resulting compound was a white solid. 1 The structure was confirmed by 1H NMR determination. The results showed that the HA derivative of experimental group 9-1 had a Chol incorporation rate of 1.3% and a maleimide incorporation rate of 8.1%.

[0396] In addition, using sodium hyaluronate (HA-Na) with a weight-average molecular weight of 35 kDa as the starting material, and following the composition in Table 10, cholesteryl and methacryloyl groups were introduced in the same manner as in Comparative Example 1, and maleimide groups were modified in the same manner as in Example 1 to obtain the target compound (HA-C6-Chol-17%-Male4.6%) of experimental group 9-2 as recorded in Table 10, which is a white solid. The obtained synthetic compound was obtained through... 1 The structure was confirmed by 1H NMR determination. The results showed that the HA derivatives in experimental group 9-2 had a Chol incorporation rate of 17.4%, a maleimide incorporation rate of 4.6%, and a methacryloyl incorporation rate of 9.7%.

[0397] Table 10

[0398]

[0399] [Example 10]

[0400] Using the HAMICH manufactured in Example 9, a HAMICH crosslinked gel was prepared by chemical crosslinking with the crosslinking agent 4arm-PEGSH.

[0401] First, HAMICH from experimental group 9-1 was dissolved in 10 mM phosphate buffer (pH 7.4) to achieve a concentration of 14 mg / mL, preparing a HAMICH solution. In another container, 4arm-PEGSH (Mw: 10,000) was dissolved in 10 mM phosphate buffer (pH 7.4) at a concentration of 7 mg / mL, preparing a PEGSH solution. After cooling both solutions to 5°C, they were mixed in a 1 mm thick, 6 mm diameter silicone mold, with the molar ratio of maleimide groups modified on the hyaluronic acid to SH groups derived from 4arm-PEGSH being 1.1:1.0. A HAMICH crosslinking gel was then fabricated at 37°C. This was done under conditions where the final concentration of the hyaluronic acid derivative reached 7 mg / mL.

[0402] Similarly, HAMICH from experimental group 9-2 was dissolved in 10 mM phosphate buffer (pH 7.4) to achieve a concentration of 30 mg / mL, preparing a HAMICH solution. In another 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, they were mixed in a 1 mm thick, 6 mm diameter silicone mold, with the molar ratio of maleimide groups modified on the hyaluronic acid to SH groups derived from 4arm-PEGSH being 1.1:1.0, to fabricate a HAMICH crosslinking gel at 37°C. This was carried out under conditions where the final concentration of the hyaluronic acid derivative reached 20 mg / mL.

[0403] HAMICH, with the introduction of cholesteric and maleimide groups, gelled fully within 1–30 minutes after the onset of gelation (mixing HAMICH and PEGSH solutions), forming a cross-linked gel, regardless of the maleimide group introduction rate. It was expected that a decrease in the number of cross-linking points would hinder gelation as the maleimide group introduction rate decreased; however, these results demonstrate that by combining steroidal and maleimide groups in hyaluronic acid, rapid gelation, which is difficult to achieve with hyaluronic acid derivatives incorporating steroidal and methacryloyl groups, can be achieved regardless of the maleimide group introduction rate.

[0404] Furthermore, the volume change in PBS from the onset of gelation was measured, and the degree of swelling was investigated. The results of the HAMICH determination in experimental group 9-1 are shown below. Figure 7 (A), The results of HAMICH measurement in experimental group 9-2 are shown in Figure 7 (B). For example Figure 7 As shown, HAMICH modified with both maleimide and steroid groups exhibits the characteristic property of low swelling degree and inhibition of volume increase in vivo after gelation, regardless of the introduction rate of maleimide groups.

[0405] [Comparative Example 4]

[0406] The gelation of unmodified maleimide-based hyaluronic acid derivatives was investigated.

[0407] First, unmodified maleimide-based hyaluronic acid derivatives were synthesized. Specifically, using sodium hyaluronate (HA-Na) with a weight-average molecular weight of 35 kDa or 100 kDa as the starting material, cholesteryl groups were introduced according to the composition in Table 11, similar to Example 1, to obtain the target compound (HA-C6-Chol-19%) in Comparative Experiment Group 4-1 and the target compound (HA-C6-Chol-40%) in Comparative Experiment Group 4-2, both of which were white solids. Cholesteryl 6-aminohexylcarbamate hydrochloride (Chol-C6 hydrochloride) and tetrabutylammonium salt of hyaluronic acid (HA-TBA) were synthesized according to the methods described in Examples 1 and 2 of Patent Document 1. As a result, the target compounds (HA-C6-Chol-19% and HA-C6-Chol-40%) listed in Table 11 were obtained, both of which were white solids.

[0408] Table 11

[0409]

[0410] Next, the HA derivatives (HA-C6-Chol-19%) from comparative experimental group 4-1 and HA derivatives from comparative experimental group 4-2 were chemically cross-linked using the cross-linking agent 4arm-PEGSH to prepare HA derivative (unmodified maleimide) cross-linked gels.

[0411] First, the HA derivatives (unmodified maleimide) prepared in comparative experimental groups 4-1 and 4-2 were dissolved in 10 mM phosphate buffer (pH 7.4) to achieve a concentration of 14 mg / mL, preparing HA derivative solutions. In another 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, they were mixed in a 1 mm thick, 6 mm diameter silicone mold to ensure a molar ratio of the molecular weight of the hyaluronic acid derivative to the SH group derived from 4arm-PEGSH of 1.1:1.0, and a cross-linking gel was prepared at 37°C. This was carried out under the condition that the final concentration of the hyaluronic acid derivative reached 7 mg / mL. As a result, no gelation occurred even after 24 hours. The same results were obtained in the inverted vial test in this comparative experimental group, confirming that no gelation occurred.

[0412] Industrial availability

[0413] According to this embodiment, a hyaluronic acid derivative can be provided that exhibits small volume change after gelation and can sustainably release the active ingredient.

Claims

1. A hyaluronic acid derivative having a steroid group introduced on at least a portion of the carboxyl group of the glucuronic acid moiety, and having a maleimide group introduced on at least a portion of the carboxyl group of the glucuronic acid moiety or the hydroxyl group of the N-acetylglucosamine moiety.

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

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

4. The hyaluronic acid derivative according to claim 1, wherein, The steroidal group introduced relative 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 incorporation rate of the maleimide group relative to the hyaluronic acid derivative is 1.0% or more and 25% or less.

6. The hyaluronic acid derivative according to claim 1, having one or more repeating units represented by the following general formula (I) and having one or more repeating units represented by the following general formula (II); 【Chemistry 1】 In general formula (I), R 1 R 2 R 3 and R 4 Each is independently selected from hydrogen atoms, C 1-6 Alkyl, formyl and C 1-6 The group in alkyl carbonyl, Z represents a peptide linker that is directly bonded or consists of two or more but less than 30 amino acid residues. X 1 Selected from -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 -SR、-NR b -CO-Y b The groups represented by -SR and -SSR, R a R b and R c Each is independently selected from hydrogen atoms, C 1-20 Alkyl, amino C 2-20 Alkyl and hydroxy C 2-20 The group in the alkyl group, said R a The R b and the R c The alkyl portion may be optionally inserted from -O- and -NR. f - groups in The R f Selected from hydrogen atoms, C 1-12 Alkyl, amino C 2-12 Alkyl and hydroxy C 2-12 The group in the alkyl group, said R f The alkyl moiety may optionally be inserted with a group selected from -O- and -NH-. R is a steroidal group. Y is C 2-30 Alkylene or -(CH2CH2O) m -CH2CH2-, wherein the alkylene moiety of Y is optionally inserted from -O-, -NR. g - and the groups in -SS-, R g Selected from hydrogen atoms, C 1-20 Alkyl, amino C 2-20 Alkyl and hydroxy C 2-20 The group in the alkyl group, said R g The alkyl moiety may optionally be inserted with a group selected from -O- and -NH-. Y a It is C 1-5 Alkylene Y b It is C 2-8 Alkylene or C 2-8 alkenyl, m is an integer greater than 1 and less than 100; 【Chemistry 2】 In general formula (II), R 1 R 2 R 3 and R 4 Each is independently selected from hydrogen atoms, C 1-6 Alkyl, formyl and C 1-6 The group in alkyl carbonyl, Z represents a peptide linker that is directly bonded or consists of two or more but less than 30 amino acid residues. X 2 It is maleimide group. R a Selected from hydrogen atoms, C 1-20 Alkyl, amino C 2-20 Alkyl and hydroxy C 2-20 The group in the alkyl group, said R a The alkyl portion may be optionally inserted from -O- and -NR. f - groups in Y is C 2-30 Alkylene or -(CH2CH2O) m -CH2CH2-, wherein the alkylene moiety of Y is optionally inserted from -O-, -NR. g - and the groups in -SS-, R g Selected from hydrogen atoms, C 1-20 Alkyl, amino C 2-20 Alkyl and hydroxy C 2-20 The group in the alkyl group, said R g The alkyl moiety may optionally be inserted with a group selected from -O- and -NH-. m is an integer greater than 1 and less than 100.

7. The hyaluronic acid derivative according to claim 1, wherein, The steroid group is a cholesterol.

8. The hyaluronic acid derivative according to claim 1, wherein, The z-average particle size of hyaluronic acid derivatives dissolved in 10mM phosphate buffer at a concentration of 1 mg / mL was less than 250 nm, as calculated by dynamic light scattering.

9. A method for manufacturing a hyaluronic acid derivative crosslinked gel, comprising: The hyaluronic acid derivative according to any one of claims 1 to 8 is reacted with a crosslinking agent having two or more crosslinking groups to gel it.

10. The method for manufacturing the hyaluronic acid derivative crosslinked gel according to claim 9, wherein, The crosslinking group is a thiol group.

11. A hyaluronic acid derivative crosslinked gel, which is a gel formed by chemically crosslinking the hyaluronic acid derivative of any one of claims 1 to 8 with a crosslinking agent having two or more crosslinking groups.

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

13. The hyaluronic acid derivative crosslinked gel according to claim 11, wherein, The swelling degree is below 115%.

14. A dried cross-linked gel of a hyaluronic acid derivative, which is a dried gel formed by chemically cross-linking a hyaluronic acid derivative according to any one of claims 1 to 8 with a cross-linking agent having two or more cross-linking groups.

15. The hyaluronic acid derivative cross-linked gel dried product according to claim 14, which is a porous structure.

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

17. A pharmaceutical composition comprising a hyaluronic acid derivative crosslinked gel, wherein the hyaluronic acid derivative crosslinked gel is a gel formed by chemically crosslinking the hyaluronic acid derivative of any one of claims 1 to 8 with a crosslinking agent having two or more crosslinking groups.

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

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